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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPO
1EPOHTNO. I
EPA|600/l-76f018
2.
3. RECIPIENT'S ACCESSION-NO.
4. TITLE AND SUBTITLE
TOXICOLOGY OF METALS - Volume I
5. REPORT DATE
March 1976
6. PERFORMING ORGANIZATION CODE
on the Toxicology of Metals, Lara
Friberg, Chairman
•. PSRPORMINQ ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Permanent Commission and International Association
of Occupational Health v.
1O. PROGRAM ELEMENT NO.
1AA601
11. CONTRACT/GRANT NO.
68-02-1287
T2. SPONSORING AGENCY NAME AND ADDRESS
U.S. Environmental Protection Agency
Office of Research and Development, Health Effects
Research Laboratory
Research Triangle Park, North Carolina 27711
13. TYPE OF REPORT AND PERIOD COVERED
Progress report
14. SPONSORING AGENCY CODE
EPA-ORD
15. SUPPLEMENTARY NOTES
Volume 1 of three annual volumes.
16.
fe Areport consists of three main sections. The first (36 p. ) is the transcript
of a panel discussion of a meeting of the Subcommittee on the Toxicology of
Metals. It covers the general principles and mechanisms of absorption and
excretion of all metals, but of heavy metals in particular. The second section,
by a task group from the Subcommittee, consists of additional inf ormatibn ^bn
accumulation and retention ot toxic metals, with special emphasis on absorption,
excretion, and biological half-times, particularly of cadmium, lead, and mercury.
The third section (182 p.) , a consensus report of a Subcommittee meeting, covers
the dose-effects and dose-response relationships of toxic metals, specifically
cadmium, lead, and mercury. The second and third sections contain information
on critical organs, effects, and concentrations, as well as on models of retention
and excretion and homeostatic mechanisms in general.
PRICES SUBJECT TO CHANGE
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b. IDENTIFIERS/OPEN ENDED TERMS
COSATI Field/Group
Absorption
Excretion
Homeostasis
Metals
Cadmium
Lead
Mercury
Toxicity
Toxicology
Pulmonary clearance
Particle deposition
Distribution/transport
Body burden
06, T, A
11. F
3. DISTRIBUTION STATEMENT
KbLEASE TO PUBLIC
19. SECURITY CLASS (This Report!
UNCLASSIFIED
20. SECURITY CLASS (Thispage)
UNCLASSIFIED
EPA Form 2225-• ._-
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EPAf600/1-761018
March 1976 '
TOXICOLOGY OF METALS - Volume I
By
Subcommittee on the Toxicology of Metals
Permanent Commission and International Association of
Occupational Health
Prof. Lars Friberg, Chairman
in cooperation with
The Swedish Environmental Protection Board, and
The Karolinska Institute
Contract No. 68-02-1287
Project Officer
Robert J. M. Horton
Criteria and Special Studies Office
Health Effects Research Laboratory
Research Triangle Park, N.C. 27711
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
HEALTH EFFECTS RESEARCH LABORATORY
RESEARCH TRIANGLE PARK, N.C. 27711
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DISCLAIMER
This report has been reviewed by the Health Effects Research
Laboratory, U.S. Environmental Protection Agency, and approved for
publication. Approval does not signify that the contents necessarily
reflect the views and policies of the U.S. Environmental Protection
Agency, nor does mention of trade names or commercial products
constitute endorsement or recommendation for use.
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. TABLE OF CONTENTS
Page
LIST OF FIGURES v
LIST OF TABLES v
LIST OF ABBREVIATIONS vi
SECTION I - INTRODUCTION 1
SECTION II - ABSORPTION AND EXCRETION OF TOXIC METALS '. 5
II.l BACKGROUND 7
II.2 PANEL DISCUSSION 8
II.3 REFERENCES FOR SECTION II 39
SECTION III - ACCUMULATION OF TOXIC METALS WITH SPECIAL REFERENCE
TO THEIR ABSORPTION, EXCRETION, AND BIOLOGICAL HALF-TIMES 43
III.l BACKGROUND 45
III.2 FUNDAMENTAL ASPECTS OF METAL TOXICITY 46
III.2.1 Conceptual Considerations 46
III.2.2 Biochemical Considerations -^-^~ ^- 46
III.2.3 Critical Organ Concept and Critical Concentrations in Cells and Organs 46
III.3 ABSORPTION 48
III.3.1 Absorption by Inhalation 48
III.3.2 Absorption by Ingestion 53
III.3.3 Placental Transfer 55
III.4 TRANSPORT AND DISTRIBUTION. 56
III.4.1 Transport and Binding in Blood 56
III.4.2 Distribution 58
III.5 EXCRETION 60
III.5.1 Gastrointestinal Excretion 60
III.5.2 Renal Excretion , ... 63
HI.5.3 Mammary Gland Excretion '. 65
III.6 ACCUMULATION AND RETENTION IN CRITICAL ORGANS 66
III.6.1. General Aspects 66
III.6.2. Specific Data 68
III.7 METAL CONCENTRATIONS IN BIOLOGICAL MATERIALS AS INDICES OF EXPOSURE
AND CONCENTRATION IN CRITICAL ORGANS 71
III.7.1 General Aspects 71
III.7.2 Specific Data 71
III.8 GENERAL DISCUSSION AND NEED FOR FURTHER RESEARCH 74
III.9 ACKNOWLEDGMENTS AND LISTS OF WORKING PAPERS AND AUTHORS; :;„' 76
111.10 REFERENCES FOR SECTION III.'.: C. 77
SECTION IV - EFFECTS AND DOSE-RESPONSE RELATIONSHIPS OF TOXIC METALS 87
IV.l BACKGROUND 89
IV.2 CONCEPTUAL CONSIDERATIONS: CRITICAL ORGAN, CRITICAL CONCENTRATION
IN CELLS AND ORGANS, CRITICAL EFFECT, DOSE-EFFECT AND DOSE-RESPONSE
RELATIONSHIPS 93
IV.3 DOSE 98 '
IV.3.1 General Considerations on Dose and Exposure; Possibilities for Their Quantitation 98
IV.3.2 Metabolic Model and Its Relation to Metal Concentrations in BiologjcaTMedia
as Indicators (or Indices) of Exposure and of Concentrations in Critical Organs '103
IV.3.3 Metal Concentrations in Biological Media as Indicators (or Indices) of Exposure
and of Concentrations in Critical Organs '121
IV.4 EFFECTS 127
IV.4.1 General Considerations 127
IV.4.2 Effects of Cadmium and Its Compounds 135
IV.4.3 Effects of Lead and Its Compounds. 149
IV.4.4 Effects of Mercury and Its Compounds 160
IV.5 DOSE-RESPONSE AND DOSE-EFFECT RELATIONSHIPS 169
IV.5.1 General Aspects 169
IV.5.2 Cadmium: Dose-Effect and Dose-Response Relationships 174
IV.5.3 Lead (Inorganic Lead Compounds): Dose-Effect and Dose-Response Relationships 187 /
IV.5.4 Mercury:!. Dose-Effect and Dose-Response Relationships 196 ,
111
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IV 6 FACTORS INFLUENCING EFFECTS AND DOSE-RESPONSE RELATIONSHIPS
FOR TOXIC METALS: EFFECTS OF SELENIUM AND METAL-METAL OR METAL-
MINERAL INTERACTIONS 212
IV.6.1 Introduction 212
IV.A.2 Interaction of Selenium with Cadmium and Mercury Compounds 213
IV.6.3 Mercury 216
IV.6.4 Cadmium 216
1V.6.5 Lead 220
IV 7 ACKNOWLEDGMENTS AND LISTS OF WORKING PAPERS AND PARTICIPANTS 223
IV.8 REFERENCES FOR SECTION IV. 233
ABSTRACT AND TECHNICAL REPORT DATA 270
IV
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LIST OF FIGURES page
1 Patterns of Particle Deposition in Various Portions of the Respiratory System 9
2 Clearance Curves of Iron Oxide Particles from the Lungs of a 21-Year-Old Male 10
3 A Compartmental Model describing the Input-output Relationship during the Absorption of
Metals from Food 12
4 Schematic Presentation of the Arrangement for Disappearance Experiments ifljay^ 15
5 The Relative Absorption at Different Concentrations of Sodium Chromate, Cadmium Chloride,
Mercuric Chloride and Potassium Mercuric Iodide 16
6 The Absolute Absorption of Sodium Chromate, Mercuric Chloride, and Potassium Mercuric
Iodide (mean) correlated to the Applied Amounts -17
7 Number of Surviving Animals at Different Times after Percutaneous Application of Metal Compounds. .18
8 Chemical Structures of Resins Added to Food to Test Reduction of Gastro-Intestinal Absorption of
Mercury 21
9 The Elinination of Mercury via Urine after Intravenous and Skin Application 22
10 Biliary Excretion of Mercury after Intravenous Administration of HgCl2 ..„„ 29
11 Accumulation of '"" Cd in Stomach Mucosa after Intravenous Injection of *®™CdCl2 to a Mouse 31
12 Accumulation of 109 QJ jn Colonic Mucosa after Intravenous Injection of '"9 Cd&2 to a Mouse 31
13 Respiratory Tract Clearance Model 48
14 Deposition of Particles in the Lungs of 'the Standard Man' 49
15 Principal Routes for Metals Introduced into the Gastrointestinal Tract 53
16 Model for the Exchange of Metal between Blood and Other Tissues 58
1 7 The Relationship between the Frequency of Signs and Symptoms and the Estimated Body Burden
of MeHg+. (A) at the Time of Onset of Symptoms, (B) at the Time of the Cessation of Ingestion
of MeHg+ in Bread 173
18 Relation between Blood Lead Levels and the Onset of a Number of Effects 194
LIST OF TABLES Page
1 Lead Intake and Output of a Normal Subject during a 2-Year Period of Oral Administration of Lead ... 13
2 Absorption of Orally Administered^ 12pb Calculated by Comparing the Urinary Excretion with
that after Intravenous Administration 14
3 Mean Relative Absorption from Aqueous Solutions of 12 Metal Compounds 17
4 Elimination of Metals by Various Ways of Absorption (the Ratio Urine/Feces) 23
5 Turnover Time of Intestinal Epithelial Cells 32
6 Organs Critical in Metal Intoxication 47
7 Calculation of Total Absorption into the Body as a Function of Two Different Rates of Alveolar
Absorption and Different Particle Sizes for a Specific Deposition and Clearance Model (Gastro-
intestinal Absorption Presumed to be 5%) 51
8 Cadmium Exposure Necessary for Reaching a Kidney Cortex Concentration of 200/^g Cd/g
Using Different Alternatives for Biological Half-time in Kidney Cortex and Different Exposure
Times 178
9 Epidemiological Data from Industrial Exposure Situations Useful for Dose-Response Evaluations 181
10 Estimated Minimum Concentrations in Brain Associated with Signs and Symptoms and Death
in Poisoning by Methylmercuric Compounds. 202
-------
LIST OF ABBREVIATIONS
(Used in Section IV)
ALA-6-amino levulinic acid
ALA-D-5-amino levulinic acid dehydrase
ALA--S-6—amino levulinic acid synthetase
ALA-U-6—amino levulinic acid in urine
AMD-aerodynamic mass diameter
cAMP-adenosine-3':5'-cyc-monophosphate (Cyc 3':5'-AMP)
CNS—central nervous system
CPG—coproporphyrinogen
CP-U—coproporphyrin in urine
EDTA-ethylenediamine tetraacetic acid
FEP—free erythrocyte protoporphyrin IX or fluorescent porphyrin
Heme-S—heme synthetase
ICMACMC—International Committee on Maximum Allowable Concentrations
of Mercury Compounds
L-chains—light chains, example: gamma globulin L-chains
LDso-lethal concentration for 50% of a population
MeHg+—methyl mercuric radical = CH3Hg+
MMAD—mass median aerodynamic diameter
m.w.—molecular weight
Pb-B-lead in blood
Pb-U—lead in urine
PAH—paraaminohippurate
PBG—proporphobilinogen
PP-protoporphyrin (always followed by a Roman numeral which designates
the type of protoporphyrin)
RBC-red blood cells
RBP—retinol binding protein
TGMA—see Task Group on Metal Accumulation in Section IV. 8
-SH group-sulfhydryl group
1,25-OH-cholecalciferol-a vitamin D metabolite
w.w.—wet weight
VI
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SECTION I
INTRODUCTION
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SECTION I
INTRODUCTION
At the time of the XVIth International Congress on Occupational Health in Tokyo in
1969 the,£ermanent Commission and Internati6nal Association of Occupational Health de-
cided to form a subcommittee on the toxicology of metals. The need for such a group had
been recognized during an international meeting on mercury toxicology in 1968. The sub-
committee was formed with Prof. Lars Friberg of Stockholm as chairman. The program de-
veloped by this group consisted of exploration and documentation of the general princi-
ples of metal toxicology by means of symposia and workshops of experts in the field. A
broad approach was selected including general environmental exposures as well as those re-
lated to occupation. Emphasis uras placed on greater depth and precision of understand-
ing in terms of metabolic processes and dose-effect relationships. The Permanent Com-
mission, the Swedish Environmental Protection Board, and the Karolinska Institute have
supported the committee in carrying out this program. Two workshops were conducted,
one in conjunction with the meeting of the Permanent Commission in 1971, and another in
conjunction with the XVIIth International Congress on Occupation Health in 1972.
----, ( ,
Recently the subcommittee has decided to broaden its work by preparing a handbook
on metal toxicology which will contain the general material being developed in its work-
shops and also specific information on a large number of metals for use by lexicologists
and occupational and environmental health workers. The Environmental Protection Agency
is cooperating with the other sponsors in supporting this phase of the subcommittee's work.
As information is developed on the subject it will be collected in three annual progress re-
ports. The first of these is contained in this volume. It consists of the conclusions of the sub-
committee's third workshop which was held in Tokyo in 1974.
Since the three workshops which have been held constitute a cumulative series, with
frequent reference back to previous definitions and statements, it was thought best on this
occasion to reproduce all of them together as a unit. Therefore, the body of this report con-
tains three sections, the first two being reproductions of the already published conclusions
of the first two workshops, and the third being the additional material produced last fall
Preceding page blank
-------
at the meeting in Japan. We are grateful to the scientists and institutions concerned, and
also to the journals, Nordisk Hygienisk Tidskrift and Environmental Physiology and Biochem-
istry, for permission to reproduce these earlier publications. We are grateful, as well, to
Elsevier Scientific Publishing Company for permission to preprint the information that com-
prises Section IV, which, along with working papers from the meeting, they will publish
subsequently.
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SECTION II
ABSORPTION AND EXCRETION OF TOXIC METALS
Report from a panel discussion at the meeting
of the Subcommittee on the Toxicology of Metals held in
Slanchev Briag, Bulgaria, September 24, 1971.
Edited by
Kersti Dukes, B.A. and Lars Friberg, M.D.
(Reprinted from Nordisk Hygienisk Tidskrift,
53: 70-104, 1971.)
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SECTION II
ABSORPTION AND EXCRETION OF TOXIC METALS
Report front a panel discussion at the meeting of the
Subcommittee on the Toxicology of Metals, *)
held in Slanchev Brian. Bulgaria, September 24, 1971.
edited by
Kersti Dukes, B.A. and Lars Friberj>, M.D.
II.1. BACKGROUND
At the meeting of the Permanent Commission and International Associa-
tion on Occupational Health in Slanchev Briag, Bulgaria, September 20—
24, 1971 the various subcommittees had been asked to arrange their own
programs. The Subcommittee on the Toxicology of Metals had chosen as
its main theme "Metal Absorption and Toxicity by Oral, Inhalatory and
Cutaneous Routes". The last session of its meetings was devoted to a panel
discussion aimed at summing up the general principles and basic data on
absorption, excretion and biological half-lives of toxic metals.
In the panel participated Lars Friberg, M.D., The Karolinska Institute,
Stockholm, Sweden (Chairman), Maths Berlin, M.D., The University of
Lund, Lund, Sweden, Thomas Clarkson, Ph.D., The University of Roches-
ter, Rochester, New York, USA, Lennart Danielson, Ph.D., Environment
Protection Board, Stockholm, Sweden, Robert Goyer, M.D., The University
of North Carolina, Chapel Hill, N.C., USA, Norton Nelson, M.D., New
York University Medical Center, New York, N.Y., USA, Magnus Piscator,
M.D., The Karolinska Institute, Stockholm, Sweden, Kenzaburo Tsuchiya,
M.D., Keio University, Tokyo, Japan, and Jan Wahlberg, M.D., The Karo-
linska Institute, Stockholm, Sweden.
From the audience participated the following persons, Sven Yllner, M.D.,
Swedish Employers Association, Stockholm, Sweden, Tadeush Dutkiewicz,
M.D., Medical Academy of Lodz, Poland, Tor Norseth, M.D., Institute
of Occupational Health, Oslo, Norway, Miroslav Cikrt, M.D., Institute of
Hygiene and Epidemiology, Prague, Czechoslovakia, Dennis Malcolm,
M.D., Wilmslow, England, George P. Lewis, M.D., Veterans Administra-
tion Hospital, Boston, Mass., USA.
The chairman opened the session and it was decided to consider first,
*) The meeting was financially sponsored by the Permanent Commission and In-
ternational Association of Occupational Health and the Swedish Environment Pro-
tection Board.
Preceding page blank
-------
questions on absorption of metals anil thereafter questions on excretion
and biological half-lives. Dr. Nelson was asked to start out with absorp-
tion via inhalation.
11.2. PANEL DISCUSSION
NEI.SON: I believe that absorption and uptake by inhalation are pre-
sently capable of more reliable generalizations than uptake by the cutaneous
route or gastro-intestinal absorption. I say this because I believe the system
can be described more accurately in mechanical terms and with fewer bio-
logical variants than is possible with the intestinal or the cutaneous system.
Basically, in the pulmonary tract we are dealing with a relatively straight-
forward system for the uptake of inhaled materials.
For the purpose of the present discussion, I will use the term "absorp-
tion" to apply to that portion of the inhaled substance which has entered
or crossed one of the surface membranes of the respiratory tract; under
this definition the subsequent fate of the substance is irrelevant to its "ab-
sorption".
The major systemic absorption of inhaled materials occurs in the deep
pulmonary tract, the alveolar part of the lung, which has a very thin
diffusible membrane with apparently relatively few selective properties.
This contrasts with the highly selective system found especially in the
intestinal tract and perhaps to a lesser extent in the cutaneous system.
Accordingly, I am going to review those patterns of inhalation and de-
position which are the basis for respiratory absorption.
The patterns of respiratory deposition have undergone extensive study
over the last 30 years. It is one of the triumphs of the physiological
modeling that a prediction, developed by Findeisen, 1935, on the charac-
teristic patterns of respiratory deposition, has been confirmed in general
and indeed in some detailed features.
Findeisen analyzed the deposition from purely engineering and mechani-
cal standpoints in which he defined the several respiratory segments, deve-
loped patterns of airflow, taking into account the three major means by
which dust reaches the respiratory surfaces. These three are, inertia with
the very large particles, settling or gravity with the large and intermediate
particles and Brownian motion (molecular bombardment) with very fine
particles.
Findeisen's predications have now been verified in several studies in
humans, including work in our own laboratories (Altshuler, Yarmus and
Palmes, 1957). The general pattern is that the total deposition is high
at the large particle sizes and falls through a minimum at around 0.4 mic-
rons for unit density particles. It tends to rise again with smaller particles
where their mobility permits them to be moved by the molecular movement
of the gases. It has become a dictum of this particular field that a "hit"
-------
Q.Q] 0.05 0.\ 0.5 1.0 5 K> 50 KX>
MASS MEDIAN DIAMETER-MICRONS
/. I'.illerus «/ Panicle Deposition in Various Portions of the Ri'sl'iratory
i'j.fA'w (Irum Task Gron\i on LHn% Dynamics,
is a "trap" meaning that a particle once reaching the respiratory surface
is there trapped and is no longer airborne.
The subsequent fate of particles is then determined by what portion
of the lung that they are deposited in and the chemical and physical
characteristics of the particle.
There is a critical size dependency of particle deposition within the
respiratory tract. The large particles are mostly filtered out in the nasal
passages while there is heavy deposition in the deep pulmonary system of
particles in the one to two micron range and again in the fractional micron
sizes, see Figure 1.
Generally speaking, relatively insoluble particles, which aie deposited
in the upper airways, those with ciliated surfaces, will be moved on up via
the mucous flow under the ciliary motion. They will then be either ex-
-------
IUIJIC! K«
Figure 2. Clearance' Curves of Iron Oxide Panicles from the Lungs of a 21 Year
Main (from Alhett, Lippmann and Briscoe, 1969).
pectorated or swallowed and thereby subject to gastro-intestinal absorption
depending on their chemical characteristics.
On the other hand those materials that reach the deeper lungs have se-
veral fates in store for them. One is that they may be immediately dissolved
and distributed systemically, or they may be phagocytized and enter the
parenchyma of the lung. The fate of phagocytized particles again depends
on the physical and chemical properties which will alter their residence
time and determine the extent of their contribution to body burden. Both
extent and locus of deposition are subject to a variety of factors; the respi-
ratory rate and volume are particularly important.
Figure 2 illustrates a series of more recent examinations of particle
behavior in humans carried out at our Institute based on the use of sphe-
rical iron oxide particles (Albert, I.ippmann and Briscoe, 1969). The tech-
nique, which measures both deposition and clearance, is based on the in-
halation of monodisperse particles so generated that they will be uniform
in particle size over a range of sizes. The particles are spiked with a radio-
nuclide and the rate of clearance is measured externally by a scintillation
detector. As shown in Figure 2, the total amount deposited in the deep
lung, which corresponds to the slow portion of the clearance curve, is least
in the 4.9 microns particle size and greatest in the smallest particle size.
The initial clearance rates (at the left hand side of the figure) which are
very rapid correspond to the ciliary clearance patterns and show how rapidly
10
-------
the particles are mobilized from the ciliated airways. The slow phase (to
the right in the figure) represents clearance of particles deposited in the
deeper airways. The rate of migration from the lower airways is a complex
one—partially phagocytosis and subsequent mobilization into the ciliary
tract, partially passing into the lung parenchyma with slow disappearance
from that region.
One can use techniques of this sort to estimate the amount of material
entering the deep lung where it is most readily absorbed and where it will
be most available for contributing to the systemic body burden. These tech-
niques could be extended to materials other than iron dusts used in these
particular experiments. Thus, I think we do have tools for a systematic
examination of the rate of uptake of inhaled metals and other materials.
The employment of the procedures I have described represents a relatively
sophisticated and so far, little used approach. Meanwhile a cruder and more
generalized technique for estimating respiratory absorption has been pub-
lished by the International Commission on Radiologic Protection (Task
Group on Lung Dynamics, 1966). They characterize particles according to
three classes of solubility. The classes correspond roughly to the half-times
of solubilization in the lung, one day, one week and one year. Using this
model it is mathematically possible to make crude estimates of body uptake
and absorption.
CHAIRMAN: Is it possible to predict in a systematic way how different
metals are absorbed from the lungs into the body, e.g. cadmium versus lead
versus mercury versus manganese?
NELSON: One very direct way to do it is in tracer studies through the
use of radio tags in which one can measure the compound in the appro-
priate organ, be it blood or other tissue. One can also use the classical
techniques of examining distribution and excretion.
CHAIRMAN: Are such data lacking for the time being? My own feeling
is that it is not possible to give data for different metals and particle sizes
whether the absorption rate is e.g. 10 percent, 25 percent or 40 percent.
NELSON: Yes, mostly they are lacking. The ICRP approach does give a
basis for very crude estimates. To refine this, one should go to the actual
measurement of specific materials; this is a quite feasible way.
The chairman then asked Dr. Clarkson to proceed with absorption via
the intestinal tract.
CLARKSON: You will notice the marked contrast between the rather
sophisticated, well-studied situation in the respiratory tract versus the re-
markable lack of data on gastro-intestinal (G.I.) absorption. I should like
11
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INTAKE
G.I. TRACT
BODY TISSUES
FECAL
EXCRETION
OTHER EXCRETION
Figure .5. A Comparlmental Mattel describing the Input-output Relationship during
tht' Absorption o\ Melals from Food.
to summarize the results of previous attempts at the measurements of
gastro-intestinal absorption of metals in human subjects. Figure 3 simply
defines diagramatically the input-output relationship with regard to the in-
gestion of metals with food. The G.I. tract is represented by the upper com-
partment. Metals absorbed from the G.I. tract enter the lower compartment.
Excretion is assumed to be by way of urine and via feces. The real in vivo
situation is obviously a great deal more complicated than this. For example
methyl mercury compounds are probably absorbed in the stomach, excreted
in the bile and reabsorbed again in the lower intestine. However, in this
diagram, the two vertical arrows represent G.I. absorption and excretion
and must be defined in terms permitting experimental measurements.
In their attempts to measure these processes previous workers have made
the following explicit or implicit definitions. The G.I. absorption is the
amount of metal absorbed from food when the body burden is zero. G.I.
excretion is equal to the fecal excretion when the intake of metal in food is
zero. Thus, the two arrows depicted in this diagram for absorption and ex-
cretion in the G.I. tract do not take into account the enterohepatic recircula-
tion where the excretion of the metal in the bile is cancelled by the subse-
quent reabsorption in the lower intestine.
G.I. absorption cannot usually be directly measured in human subjects.
For example, the administration of an oral dose of a metal labelled with
a gamma-emitting isotope will permit measurements of retention by whole
body counting techniques. The whole body count will include of course
the radioactivity of unabsorbed metal in the G.I. tract and correction must
be made for this. Furthermore, the whole body count will decline due to
excretion and we need to correct for this also. Nevertheless, whole body
counting methods have been used successfully in the case of compounds
12
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Table 1. Lead Intake and Output of a Normal Subject During a Two Year Period
of Oral Administration of Lead (from Kehoe el al., 1943).
Ingested percent total
In drinking water
In food and beverages
Administered in solution
Total
Excreted
In feces
In urine
Total
Retained
0.8
8.)
90.7
100.0
87.1
5.1
92.2
7.8
of mercury and cadmium. Miettinen, 1971, in press, has reviewed observa-
tions on volunteers given single doses of compounds of methyl mercury and
inorganic mercury. Similar studies on methyl mercury by Aberg et al., 1969,
have also been reported. The metal has been administered in food, bound
to fish or liver protein or in aqueous solution. Mercury was labelled with
the gamma-emitting isotope 203jjg having a half-time of 47 days. Whole
body counts were made at least daily for the first few days. A graph was
plotted on semilogarithmic paper for whole body counts against time.
In the study on inorganic mercury the whole body count declined rapidly
during the first two or three days corresponding to fecal excretion of
unabsorbed mercury. Subsequently the whole body activity declined at a
much slower rate equivalent to a biological half-time of approximately 50
days. The intercept obtained by extrapolating the second line back to zero
time gave the amount of oral dose absorbed. A similar procedure was
followed for methyl mercury and cadmium salts. In the case of all these
compounds, excretion of the metal was very slow, allowing the rapid
decline in the whole body count due to clearance of unabsorbed metal to
be distinguished from the much slower decline due to excretion of the
absorbed metal.
Miettinen, 1971, in press, has reported the following values for absorp-
tion from single oral doses. Approximately 15 percent inorganic di-valent
mercury is absorbed from food. Methyl mercury compounds are absorbed to
90—100 percent and cadmium salts to approximately 5 percent.
Lead does not have a gamma-emitting isotope suitable for whole body
counting techniques. It does, however, offer the advantage that the gastro-
intestinal excretion is negligible. Two experimental approaches have been
taken to the measurements of the gastro-intestinal absorption of lead in
13
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Table 2. Absorption of Orally Administered tltPb calculated by comparing the
Urinary Excretion with that after Intravenous Administration (from Hursh and
Suomela,
Subject
A
D
A
B
C
Dose
/*c
1.08
1.17
5.05
5.01
4.80
Mode
i.v.
i.v.
oral
oral
oral
24 hr urinary
loss
/*c
.053
.040
.00)
.017
.032
Absorption
/«c
.067
.404
.768
% dose
1.3
8.1
26.0
48 hr fecal
excretion
/K
.003
.003
humans. Kehoe et al., 1943, reported on the results of lead balance in
individuals given lead in food for a period of two years. Table 1 gives
the results on one subject taking 2 mg of lead salts for two years. Essen-
tially, a balance study was carried out in which the daily intake of lead
from food, beverages and water was compared to the fecal and urinary
excretion. Kehoe et al., 1943, have presented evidence that the clearance
of unabsorbed lead from the gastro-intestinal tract accounts for all of the
fecal excretion. Measurements of fecal excretion of lead following cessa-
tion of chronic oral doses revealed that within a few days fecal lead ex-
cretion was indistinguishable from fecal excretion prior to exposure despite
the fact that the body burden was still elevated. Hursh and Suomela, 1968,
demonstrated that fecal excretion of lead was very low as compared to
urinary excretion in two individuals receiving an intravenous dose of 2i2pt
as PbClo (Table 2). Thus, in the balance studies reported by Kehoe et al.,
1943, the amount retained over two years plus the amount excreted in the
urine will be equal to tlie amount absorbed. The amount absorbed, accord-
ing to Table 1, was approximately 13 % of the ingested lead.
Hursh and Suomela, 1968, reported observations on human volunteers
given lead salts labelled with the isotope 212. The half-time of this gamma-
emitting isotope, 10.6 hours, is too small to permit measurement of ab-
sorption by whole body counting techniques. It will take too long, 70 hours
or more (about 7 half-times), before the G.I. tract is cleared of unabsorbed
lead. Hursh and his colleagues made the assumption that the amount of ra-
dioactive lead excreted in urine for the first 24 hours was proportional to the
amount of absorbed lead in the body. They gave a known amount of radio-
active lead chloride intravenously to two subjects and measured the first
24 hours of urinary excretion, which was 5 percent of the injected dose
(Table 2). They gave 3 other subjects an oral dose in beer (lead-212 is
generated from radon and the gases trapped in beer) and also measured
14
-------
Scintillation
dettctor .
Cryttal
Radioactive
^solution
Skin
Figure 4. Schematic Presentation of the Arrangement for Disappearance Experiments
in vivo. Unit of Measurement: Millimeter (from Wahlberg, 196) a).
Amount of oral dose absorbed = ]
urinary excretion over the first 24 hours. The amount of lead absorbed
in the first 24 hours was calculated from the relationship:
24 hr.-urinary lead (oral dose) X(i.v. dose)
: 24 hr. urinary lead (i.v. dose)
The values obtained on three subjects ranged from 1.3 percent to 16
percent (Table 2).
Factdrs influencing gastro-intestinal absorption of metals are poorly
understood. Miler, Sattler and Menden, 1970, have shown that the reten-
tion of 2i2pb may be increased by 7 to 49 % by increasing the protein
content of isocaloric diets of experimental animals. On the other hand,
methyl mercury compounds undergo virtually complete G.I. absorption
whether given as the chloride salt or complexed with fish protein (Aberg
et al., 1969, Miettinen, 1971, in press). Friberg, Piscator and Nordberg
1971, have reviewed literature reports indicating that low dietary calcium
leads to enhanced absorption of cadmium in experimental animals. One ex-
planation offered is that low dietary calcium slows the passage of cadmium
along the digestive tract. The rate of transit along the G.I. tract may be an
important consideration for all metals that undergo slow absorption.
15
-------
ijion
min."1
20
HgCI2
K2Hgl4
IS "
le-
Sensitivity limit
Log Cone.
»Cr, Cd.HgM
-4 -3 -2 -I 0 I (Molarily)
Figure 5. The Relative Absorption at Dijjerent Concentrations of Sodium Chromate,
Cadmium Chloride, Mercuric Chloride and Potassium Mercuric Iodide (mean) (from
Stag and Wahlkerg, 1964).
The chairman then asked Dr. Wahlberg to continue with skin absorp-
tion.
WAHLBERG: Percutaneous absorption is defined as the penetration of
substances from the outside into the skin and thereafter through the skin
and into the blood and lymph vessels. Percutaneous toxicity is defined as
the systemic poisoning following penetration of toxic materials through
the cutaneous barriers and their distribution throughout the whole body.
Figure 4 shows a schematic arrangement for the disappearance measure-
ments in vivo, where the radioactivity above a deposit of an isotope labelled
metal on the skin is continuously recorded by a scintillation detector. The
decrease in radioactivity is an indication of absorption. The mean relative
absorption for different substances and at different concentrations is seen
in Figure 5. It increases with increasing concentrations up to a maximum
which seems to be characteristic for each substance. If the concentration
is increased still further, the relative absorption decreases gradually. In
Figure 6 the absolute absorption is correlated to the applied amount. At
lower concentrations the absorption increases with rising concentrations.
However, despite further increase in concentration and hence also an
increase in the total amount applied, absorption does not continue to rise,
16
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Absorption
(nwM.cm-Z.hr-1)
900-
800-
700-
600
500-
4OO-
300-
200
100-
back skin
-— HgCfc belly skin
..... K2Hgl4 back skin
.—. Na2Crp4
back skin /
0.100 0200 0.300 0.400 0.500
Concentration.-
' Cr.Hg,
Molority
Figure 6. The Absolute Absorption of Sodium Chromate, Mercuric Chloride and
Potassium Mercuric Iodide (mean) correlated to the Applied Amounts (from Stag
and Vahlbfrg, 1964).
but a level is reached for sodium chromate and potassium mercuric iodide.
The absorption conditions for 12 metal compounds are summarized in
Table 3. Relative absorption varies from less than 1 percent to 4.5 percent
during a *> hour period.
Table 3. Mean Relative Absorption from Aqueous Solutions of 12 Mttal Compound!.
Exposure Area: 3-1 cm*.
Compound
NajCrjOi
CoCIs
ZnClj
AgNOj
CdCI2
CrCU
NaCl
SrCl2
HgC!2
NazCrO)
K»HgI«
CHjHgQHjN,
Isotope
"Cr
680)
"Zn
"""Ag
H5»Cd
"Cr
MNa
89Sr
«aHg
"Cr
""Kg
aoHg
Relative Absorption
percent / 5 h
<1.0
<1.0
<1.0
<1.0
1.8
2.2
2.9
3.1
3.2
4.0
4.1
4.5
17
-------
X.. of
TTM'4' 5'6 7' 1' 9'lo'in2 13 14 IS'l6'l7 18 19 W'imn 24'li'26 2/21
Figure 7, Number of Surviving Animals at Different Times after Percutaneous
Application of Metal Compounds (2.0 ml of 0.239 M Aqueous Solutions) (from
Wahlberg, 196} b).
The differences in absorption rate cannot solely explain the variations in
the potency to induce hypersensitivity. Cobaltous chloride and bichromate,
which are well-known sensitizers, have comparatively low absorption rates.
In the percutaneous toxicity tests 2 ml of an aqueous solution of a metal
compound were applied. The skin depot had the same exposure area as
in previous absorption experiments, i.e. 3.1 cm2. The mortality increased
with increasing concentrations and most of the animals died between the
7th and the 15th day. The final comparison among nine metal compounds
was made at a concentration of 0.239 molar. Figure 7 shows that all ani-
mals exposed to zinc chloride and silver nitrate survived, whereas only 2
animals exposed to potassium mercuric iodide survived. With few excep-
tions there was a good agreement between observed and expected mortality
on the basis of iknown absorption rates and intraperitoneal toxicity. The
animals exposed to chromic chloride manifested a parallel weight increase
with that of animals exposed to distilled water and controls, whereas the
mean weight increase for animals treated with zinc chloride and silver
nitrate ceased by the first week. When a small amount of mercuric chloride
was applied, the mean weight decreased during the first week of the ob-
servation period.
CHAIRMAN: These three presentations are now open for discussion and
18
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1 invite both members of the- panel and the audience to take part in the
discussion.
CLARKSON: This is a question to Dr. Goyer about the fecal excretion
of lead. Data reported in the literature indicate that fecal excretion is very
small, but, in an earlier session, you presented evidence that lead is found
in the bile. Is there an enterohepatic recirculation of lead as observed in
the case of methyl mercury?
GOYER: I presume it is very similar. The data 1 presented about lead
being excreted in bile were from a work by Castellino and Aloj, 1964, in
which they injected labelled lead and then recovered about 80 percent of
the label in bile. Similar studies have been done by Black, 1962, with dogs.
When talking about an absorption of lead with regard to a value for the
gastrointestinal absorption, we are really talking about a net absorption of
10—15 percent.
I would like to make one further comment with regard to this. We are
talking about people without excessive body burden of metals and I am
not sure that we can extrapolate this degree of absorption truthfully in
persons who have greater or increased body burdens. From our studies in
rats fed lead from HO ppm to Ml,000 ppm, we had a variation of absorp-
tion from 1—2 percent with a minimal dose, i.e. 80 ppm to a very small
fraction of one percent (<().! %) with an oral intake of about 10,000 ppm
lead (Goyer et al., 1970). These numbers should not be translated directly
to what would apply to man, but I think that the principle can be applied,
that is, if we have someone with excessive exposure, the efficiency of ab-
sorption may not be expected to equal the 10 to 15 percent that we would
see in a non-exposed or slightly exposed person. These kinds of data, that
is, with several times usual environmental exposure, are not available from
human observations. I think it is difficult to extrapolate what we know
about non-exposed people to heavily exposed people.
CLARKSON: In Kehoe's study the subjects were given 2 mg of lead a
day for 2 years, thus giving a substantial body burden. Absorption from
the gastro-intestinal tract was 13 percent. The evidence that there is a small
fecal excretion of lead is from Kehoe's work where subjects would inhale
lead and increase their body burden in this way. Although the urinary
excretion increased in parallel to the retention, there is very little change
in the fecal excretion. Once the subjects were taken off exposure and when
the unabsorbed lead had been cleared from the gastro-intestinal tract, the
fecal excretion hardly changed at all, even though their body burden and
urinary excretion had greatly increased. Hursh and Suomela, 1968, studying
19
-------
much lower doses of lead in human volunteers came to the same conclusion.
In their study, fecal excretion of lead was measured in individuals given
a single intravenous dose of 2'2Rb in the form of PbG2.
BERLIN: One important question concerning absorption via inhalation
is to what extent the general model can be adapted to humans in real life
in industry. There is a number of factors that should be taken into consi-
deration. Liquids of different kinds easily penetrate down to the peripheral
part of the lung. Irritating gases often accompany metal fumes or particles
and may give rise to effects, e.g. secretion of mucous and bronchial construc-
tion. Mucous secretion may occur to such an extent that reflux of secretion
in the bronchial tree and even down to the peripheral part of the lung
may occur and thus change the model concerning retention and absorption
considerably.
NELSON: I accept totally the suggestion that in "real life", as found in
community air pollution or occupational exposures, the situation is different
from that in carefully controlled laboratory studies. The latter are different
in a variety of ways; among others, the particle size is known and uniform.
This is a pretty rare circumstance in a natural situation. What I did not
go into at all are the physiological variables that alter host responses. For
example, clearance patterns are very much subject to a whole series of
physiological factors including disease and presence of irritants. Such fac-
tors will influence patterns of respiration and patterns of clearance. I have
presented only general principles; where they have been applied, even in
a crude way, for example to airborne lead experimentally, these principles
were verified within expected limits.
DANIELSON: I have a general question to all the experts here on absorp-
tion. To what extent is absorption a passive or an active (e.g. energy re-
quiring) process? This question may have some bearing on what was
mentioned today by Dr. Goyer, that the more lead you ingest, the less
percentage you absorb. If this reflects a toxic effect on an active absorption
process, it is possible that other compounds, which e.g. are absorbed from
the gastro-intestinal tract, behave in the same way.
GOYER: Bingham et al., 1968, studied the effect of continued inhalation
of small particle size (99 % less than 0.1 /j. in diameter) of lead and found
that the number of alveolar macrophages actually cleared from the lung
was decreased. It is interesting to speculate what influence this finding has
on the ability of the lung to absorb and excrete lead. The amount of lead
being excreted with the alveolar cells has not been determined. This type
of study perhaps represents the level where further work should be done.
20
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DOWEX 1x8 (CD I-$V.CH2N (CH3)3
/CHjCOO9 No*
DOWEX A-l CHELATING RESIN ' ' '
DOW-SH RESIN
Figure 8. Chemical Structure! of Reiias Added to Food to Ten Reduction of
Gastro-Inttstinal Absorption of Mercury (from Clarkson, Small and Nonetb, 1971).
I think a lot of the clearance studies done today give us information about
deposition in the alveolar cells, but I do not think they tell us very clearly
how much really gets into the blood.
YLLNER: In view of recent knowledge on absorption could you comment
upon the old question of giving, for example lead workers, certain amounts
of milk every day? Is there anything speaking in favor of this? I believe
this is being done in some countries.
CLARKSON: Kehoe et al., 1943, studied a variety of compounds added
to food, including ascorbic acid. Ingestion of varying amounts of milk and
diets containing high and low calcium and phosphate were also studied.
They were unable to detect any direct effects of these compounds or of the
different diets. In the German lead study I referred to (Miler, Sattler and
Menden, 1970) the protein concentration in the food was varied and differ-
ences in absorption rate were found. It is difficult to generalize because
there are tremendous differences among different types of metals.
GOYER: From experimental studies on animals, similar to Kehoe's human
studies, we were not able to show that increase in calcium above recom-
mended daily intake did anything in terms of decreasing lead absorption.
On the other hand we did learn that, if you reduce calcium in the diet
to less than recommended amounts, there is an enhancement of lead ab-
sorption or at least increased body stores (Six and Goyer, 1970). In answer
to Dr. Yllner's question I would say that one should be certain that the
daily calcium intake is adequate in workers but not necessarily expect any
beneficial effect from excessive intake.
CLARKSON: Figure 8 is taken from a study by Clarkson, Small and Nor-
21
-------
% o(thedose/h
1.000-
Wayofapplic
i.v.
s.r.
Dose
40 /ugHg
11336
Elimination in
% of dose
23.56
13.59
0.100-
0.010
0.001
skin resorption 0__0
1 23456789 10 days
Figure 9. The Elimination of Mercury via Urine after Intravenous and Skin
Application.
seth, .1971, and indicates three types of polystyrene resin added to food
to test if the gastro-intestinal absorption of mercury could be reduced. The
resins tested contained the following active groupings: 1) a quaternary
nitrogen group, 2) carboxylate groups and 3) SH groups. The resin con-
taining the thiol groups markedly inhibited the absorption of methyl mer-
cury compounds. It also increased the fecal excretion by methyl mercury
by trapping the metal secreted in the bile and thus preventing reabsorption.
22
-------
Table 4. Elimination oj Metals by Various Ways of Absorption, The Ratio Urine]
Peces '
Substance
Arsenic
Beryllium
Chromium
Mercury
Selenium
Application
Intravenous
Skin
Intravenous
Skin
Intravenous
Skin
Intravenous
Skin
Intravenous
Skin
Dose in mg
0.200
0.203
0.0043
0.568
0.050
0.125
0.04(1
1 1.366
0.116
0.069
Ratio urine/fetes
8:1
1:1
3:2
1:9
1:1
3:7
4:3
2:5
4:1
2:1
Synthetic resins may prove useful in reducing the gastrointestinal absorp-
tion of other metals and may increase fecal excretion of those metals under-
going enterohepatic recirculation.
DUTKIEWICZ: I think that more attention should be placed on skin
absorption as a cause of metal poisoning. There are several examples from
laboratory investigations as well as from industrial conditions where skin
absorption of metals is very essential in evaluating exposure. Animal ex-
periments show that the rate of metal absorption via the skin could be
quite important. Our data show that the rate could reach 10 to 1000 micro-
grams per cm- per hour or more, depending on the nature of the metal
and on the concentration. Data in humans show similar values. I mean
that in real life the cutaneous absorption could be equal or higher than
that of the respiratory tract. For substances with a significant skin absorp-
tion, evaluating exposure based on concentration of the substances in air
only is insufficient. In this case toxicological exposure tests should be
elaborated, based on for instance the excretion of metals in urine, which
could show the exposure independently of the route of absorption. Un-
fortunately there are great differences in the kinetics of elimination and
in the ratio urine/feces excretion at various ways of application. Figure 9
indicates the varied character of excretion of mercury in urine after intra-
venous skin resorption. In Table 4 the ratio urine/feces excretion is de-
monstrated at various ways of application: intravenous and percutaneous,
and there are great differences in the ratio. For arsenic, e.g. the ratio is
one to one after skin absorption and after intravenous and iritratracheal
23
-------
application 8 to I. If urine analysis is used for evaluating exposure due-
attention must be- paid to route of absorption.
NORSETH: I also have tested the effect of some macromolecules on ab-
sorption of methyl mercury from the gastro-intestinal tract. These are a
thiolated sephadex (Eldjarn and Jellum, 1963) and thiolated dextran (Jel-
lum, Aaseth and Eldjarn, 1971) which affect absorption, but which un-
fortunately are broken down to a great extent in the G.I. tract.
We have heard that heavy metals are probably bound to proteins in
food. We have also heard that mercury molecules of certain kinds, that
are probably not broken down and not reabsorbed, inhibit absorption. I
would like to force the panel to answer how metals are absorbed, as free
metals, as peptide bound metals or as amino acid bound metals?
CLARKSON: We do not know the mechanisms of absorption and we cannot
therefore make any predictions. There are certain metals probably like the
essential trace elements which have special mechanisms. In the case of iron
there are special mechanisms. As far as lead, mercury and cadmium are
concerned, the mechanisms are not known.
NORSETH: If we go back to the methyl mercury example again, this is
interesting because there are at least four different forms of methyl mercury
bindings in the intestinal tract. Methyl mercury in food is bound to pro-
teins and is reabsorbed to 100 percent. Methyl mercury in bile is probably
excreted as some kind of peptide and reabsorbed very efficiently. Some
methyl mercury in bile is protein-bound and not reabsorbed that efficiently,
but to a certain extent. I do not think that methyl mercury bound to pro-
teins which are released in the intestinal tract by cell shed is reabsorbed
at all.
CLARKSON: The explanation usually given for the efficient gastro-in-
testinal absorption of methyl mercury is that methyl mercury chloride is
lipid soluble. This explanation is at least plausible in the case of absorp-
tion from the gastric contents where the concentration of hydrochloric acid
may be high. However, methyl mercury in plasma, bile and other biological
fluids is probably in the form of a complex with sulfhydryl containing
proteins, peptides or amino acids. Methyl mercury in bile has been shown to
be complexed to protein and to small organic molecules (Norseth and Clark-
son, 1971, Norseth, 1971, in press). Such complexes are water soluble. The
high chemical affinity of the mercury cation for the sulfhydryl group
suggests that mercury chloride will be present in very small concentrations
in tissues and biological fluids other than in the gastric contents. Thus, it is
24
-------
possible that special transport mechanisms, related to specific complexes of
mercury, may be important in the transport of mercury across the intestinal,
bloodbrain and placental barriers. Levels of mercury in brain and fetal tissue
are lower after doses of phenyl mercury compounds than after doses of
methyl mercury salts. The difference cannot be explained by differences
in lipid solubility since both methyl mercuric chloride and phenyl mercuric
chloride are soluble in lipids.
NELSON: I would like to make some points here in relationship to the
question of active versus passive absorption in the two contrasting systems,
the lung and the G.I. tract. I would expect that active absorption against
a concentration gradient may be obtained fairly frequently in G.I. tract
absorption. On the other hand I tend to relegate to a very secondary posi-
tion the role of active transport in the respiratory tract.
WAHLBERG: I have two questions to answer. The first is about the
mechanism of skin absorption, which we believe is passive diffusion in
rodents and in humans. But an active absorption has been described through
frog skin for sodium.
The second question was from Dr. Dutkiewicz. The figures I presented
here concerned skin that was normal the seconds before I applied the
metals. We think that the metals precipitate proteins in different ways
and create new barriers and that is why the absorption is not so much
increased as could be expected. Most workers in industry have small
wounds. If the skin has been damaged by water, solvents, detergents and
so on, we cannot speak of normal skin. This might explain the higher
absorption that was described in the cases of poisoning. I think that
measurements in urine are very rough measurements of skin absorption.
We can see from the data presented by Dr. Dutkiewicz that the amounts
applied on the skin were very much higher than those given intravenously,
which explains the difference that was observed.
DUTKIEWICZ: The doses by intravenous application and by skin absorp-
tion of arsenic were equal and in spite of this fact, the differences in the
ratio urine/feces were very important. According to the method of measur-
ing skin absorption that Dr. Wahlberg uses, there is a disappearance of
activity in the solution during for instance one hour. I think that there
could be changes in the geometry of the measurement and there is a risk
that Dr. Wahlberg's results may be wrong. I use another method in which
the rats' tails are immersed in the solution for one hour whereupon the
animals are put in metabolitic cages where urine and feces samples are
collected every day for instance for 10 days. The absorbed dose is then
25
-------
calculated on the basis of excretion and on the content of elements in or-
gans and tissues. In my opinion this method is better because the absorbed
dose is measured here directly.
CHAIRMAN: With the exception of Dr. Wahlberg's presentation, so far
we have discussed only mercury, cadmium and lead. That goes for the inhala-
tion and intestinal absorption. Can I conclude that we know nothing about
other metals or do you have some information you could give us?
NELSON: We, for example, have examined cobalt uptake in inhalation
toxicity studies (Palmes et al., 1959). Uptake of cobalt fitted to a good
approximation what could be anticipated from physical characteristics of
the inhaled particles. In the few studies that have been done, I think that
the model has been generally supported.
CHAIRMAN: Which model? Do you have an absorption model?
NELSON: The absorption model I am talking about is that most of the
material reaching the alveolar part of the respiratory tract is "absorbed"
according to the definition I gave earlier, while the amount of material
reaching the alveoli can be estimated from particle deposition theory.
CHAIRMAN: I think that it would be a very useful conclusion if it really
is true that, independently of the nature of the substance, if it reaches the
alveoli, then it is absorbed to 100 percent.
NELSON: Although not necessarily 100 percent, it is generally high and
in many ways equivalent to intraperitoneal or intravenous injection. Mobili-
zation from that point is going to .be dependent on the physical and
chemical properties of the particles. Some materials, for example beryllium,
have a high degree of tissue affinity and will be localized in the lung.
Others will be less localized and will become systemically available. If one
starts with the assumption that the material which reaches the alveolar part
of the lung is absorbed in the sense I have defined, then one must go from
this point and describe, in appropriately simple or complex ways, (depend-
ing on the material) what happens as it leaves the lung parenchyma. If
it is very soluble, it may be immediately distributed and accessible to all
tissues. If it is very insoluble, it will be localized. If it is reactive with
local tissue, it may be sequestered, as in the case of beryllium. Materials
that are relatively insoluble but not intensely bound may serve as depots for
long-term discharge to other parts of the body.
CHAIRMAN: This means that certain substances may have biological half-
26
-------
lives in the lung for years. Others would have half-lives of weeks or
months ?
NELSON: Yes, this is dependent not on the mechanical properties, but
on the chemical and biochemical behavior of inhaled dust. Some particles
may end up in the lymph nodes where they may stay for a lifetime.
BERLIN: I would like to point to the fact that a small fraction of inhaled
particles will be deposited in the upper respiratory tract, i.e. the nasal and
the tracheal part. Very little is known about to what extent an absorption
takes place for different particles and gases in the mucosa of the upper
respiratory tract.
CHAIRMAN: Dr. Clarkson, would you be kind to comment on if you
have any data on G.I. absorption for metals other than lead, cadmium and
different mercury compounds?
CLARKSON: There is a large number of papers concerning the absorp-
tion of physiologically important metals such as sodium, potassium and
calcium. Iron absorption is carefully controlled by complex mechanisms
acting in the mucosal epithelial cells. The exfoliation of epithelial cells
plays an important role in the control of iron absorption (Crosby, 1968).
In the normal individual, iron is absorbed from food into the mucosal
cell. A substantial fraction is retained in the mucosal cells and re-excreted
when the mucosal cells are lost by the exfoliation. A substantial fraction of
the fecal excretion of mercury in animals dosed with methyl mercury salts
originated from exfoliation of cells from the small intestine (Norseth and
Clarkson, 1971). Complex interactions and interdependencies are known
to occur among iron, copper and cobalt with regard to intestinal absorp-
tion. I understand that small molecular weight proteins similar to metal-
lothionein are present in the intestinal cells, but perhaps Dr. Piscator
would comment on this?
PISCATOR: We really have two problems here because, when talking
about the lung, any essential or non-essential metal can be absorbed. For
example, exposure via the air to manganese can be a serious health hazard.
However, when manganese is in food we have a homeostatic mechanism
which quite efficiently regulates the uptake. A worker exposed to copper
will absorb the copper which is excreted via the bile, for instance. Under
ordinary circumstances there is a reabsorption. In workers exposed to copper
via inhalation the reabsorption of copper from the intestines will decrease,
because of a homeostatic mechanism. In the intestinal walls of several
27
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species specific carriers have been found which are proteins of low mole-
(ular weight, very similar lo melullolliionein. 'lhe.se low mnleiuhir weight
proteins. I think espctially topper binding proteins, inayhc iil.so at I as
carriers for zinc and cadmium. We do not know that yet, but it is quite
conceivable. From the intestines we know that iron lias specific carriers.
I do not think we know which carriers we have in the lungs.
CI.ARKSON: Miss Barbara Davis, a graduate student in our department,
has been studying the homeostatic control of iron absorption. The control
mechanism is effective in concentrations of iron normally encountered in
food. However, when the oral dose is sharply increased, the homeostatic
mechanism breaks down thus allowing a large increase in iron absorption.
Fatalities in children who have taken overdoses of iron tablets are probably
the result of the loss of homeostatic control. Thus we cannot expect that
the normal physiological control mechanism will be life-saving when toxic
doses of metals are ingested.
CIKRT: I have a short comment with regard to the excretion of metals
via bile and their reabsorption in the intestinal tract. In our laboratory
the biliary excretion of r'-Mn, '^Cu, 2o:(Hg and -'i«Pb after intravenous
administration of •"•aMnCI.,,, «-»CuG2, -'"«HgCI.,, and iM<>Pb/NO:!/.,
was studied in rats. Cumulative biliary excretion 24 hours after administra-
tion reached in case of (i4Cu 31 percent, of ">-Mn 28 percent, of ao:iHg 4
percent and of -HiRb 7 percent (each in percent of the administered dose).
The maximum rate of excretion in the bile was reached in the individual
metals during different periods after administration. In manganese the
excretion was quickest with a maximum rate of excretion about 10 minutes
after administration. '-'":!Hg differed significantly by the character of the
excretion curve from three other metals (see Figure 10). On the B curve
(percentage of excreted -o:!Hg per mg of bile) you can see two peaks.
The first peak is about 2—3 hours after administration and the second
peak 17—20 hours after administration.
The bile was separated using the gel electrophoresis and we found that
metals excreted via bile in high amounts (Cu, Mn) were located in the
front of the electrophoreogram where the bile pigments and the proteins
of low molecular weight are found, whereas Pb and Hg were located in
the start of the electrotrophoreogram where the high molecular weight
proteins are found. We obtained very similar results at separation of bile
on Sephadex G-100. We also studied the reabsorption of biliary excreted
manganese, copper and mercury and we found the following results:
r>'-'Mn is reabsorbed to about 10 percent, "-iCu to about 5 percent and
'-":'Hg to about 1 percent only.
28
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15
10
5
Hg
203
B
i
-+-
2 L 6 5 10 12 U 16 18 20 22 24 TIME (HOURS)
A. Cumulative biliary excretion of mHg in percent of the administered dose during
a 24 hour experiment.
B. Percentage of excreted 2MHg per mg of bile (ValuesXlO3)-
C. Excretion of anHg in percentage of the administered dose per minute (ValuesX
10*).
D. Bile flow in mg per minute. Solid lines—results in the individual experimental
animals. Open circles—mean values of excretion.
Figure 10. Biliary Excretion of Mercury after Intravenous Administration of HgClt-
29
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TSUCHIYA: I will refer to the absorption through the intestinal tract.
In my experience, some workers exposed to lead sulphide did not show
any clinical symptoms although the environmental lead concentration was
rather high, exceeding 0.5 mg/m3. Therefore, I think that not only the
type of metal, but also the type of compounds are very important in ab-
sorption. For cadmium oxide fumes we have lower TLV-values than for
cadmium dusts.
MALCOLM: I would like also to comment on this as not only the type
of compound, but also the particle size are essential. In industry it is well
recognized that cadmium fumes and lead fumes are absorbed much more
readily than the ordinary dust. I think perhaps the TLV should be related
to some extent to the profile of the particle size. On the other hand in
practical work we find that lead or cadmium dust in the theoretical absorp-
tion range is absorbed only to about 20 percent. When you have it in fume
form, in a 100 percent theoretical absorption range, the absorption is just
about twice the level for a given concentration in the atmosphere. There is
something missing here. I do think it is important to get more information
on this, to be able to define TLV's in the particle size profiles. So far as lead
sulphide is concerned, I have never been quite sure whether this is because
of the nature of the compound or because very frequently the lead sulphide
is in very large particle sizes and therefore not absorbed in the alveoli.
CHAIRMAN: ]) The next topic for our discussion is excretion and bio-
logical half-lives.
BERLIN: There are three excretion routes which should be considered the
main routes, i.e. the alimentary tract, the urogenital route and the skin. In
the alimentary tract there is a number of possible routes: salivary glands,
stomach mucosa, small intestines, the liver, pancreas, the bile and the
colonic mucosa. Although these routes are all possible, they may not be
important in all instances. The excretion from the salivary glands, for
example, is not very often significant but could be used for diagnostic
purposes. Mercury is excreted that way and it may be used for evaluating
the blood concentration. For such metals as cadmium, for example, which
are retained for very long periods of time, this route could also be of
importance. It is commonly observed in autoradiograms on distribution
after injection of metals, and even other substances, that there is an ex-
cretion via the stomach mucosa. In Figure 11 you see the accumulation in
the epithelium after intravenous injection of loocd to a mouse and you
') At this point in the discussion, Drs. Clarkson and Nelson had to leave to catch
their flights out of Slanchev Briag.
30
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Reproduced from
best available copy.
Figure 11. Accumulation of tmCd in Stomach Mucosa after Intravenous Injection
of tiS>Cd(:i< 10 a Mouse (from Rcrlin and Ulll/erK. I9(>.1).
Figure 12. Accumulation uj l'*Cd in Co&iiic Miicosa after Inlrafi'tiuus lajeclion of
""CJCI-j to a Mouse (from Berlin and Uilberg, 196.1).
31
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t',rll\
Site Species Method
Turnover
lime
(days)
References
Mouse Labelling
Duodenum
Rat
Cat
Man
Man
Mitotic counting
Mitotic counting
Mitotic counting
Labelling
2
1.6
2.3
2
5—6
Creamer, Shorter &
Bamforth, 1961
Leblond & Stevens, 1948
McMinn, 1954
Bertalanffy & Nagy, 1961
MacDonald, Trier & Everett,
1964
Jejunum
Mouse Labelling
Mouse Labelling
Rat Mitotic counting
Man Labelling
3 Leblond & Messier, 1958
2 Creamer t-t a!., 1961
1.3 Bertalanffy, I960
5 Shorter et al., 1964.
Mouse Labelling
Mouse Labelling
Mouse Labelling
Rat Mitotic counting
Cat Mitotic counting
Man Labelling
3
2
1
1.4
2.8
3
Leblond & Messier, 1958
Quastler & Sherman, 1959
Creamer et al., 1961
Leblond & Stevens, 1948
McMinn, 1954
Liplcin, Sherlock & Bell, 1963
sec a halo of an isotope just outside, indicating excretion. Similar results
for mercury compounds have been observed.
The colon is a route which is overlooked to a large extent probably due
to our limited possibilities, so far, to study this. It has been shown by
several workers that mercury, for example, is excreted via the colonic mu-
cosa. Cadmium is also excreted this way as you can see in Figure 12. It is
rapidly accumulated in the mucous membrane and the free isotope can be
seen in the lumen on the top of the epithelium. Of course, since these
observations are made mainly shortly after the intravenous injection, the
form of the metal in blood may be of importance. Later it may be in a
form which is not excreted in the colon.
Another important route is the shedding of epithelium in the small
intestines, where there is a tremendous turnover of cells. Table 5 shows
the turnover time in different animals and in man as brought together by
Creamer, 1967. The daily turnover time in man is about three times. As
many metals are actually accumulating in the epithelium of the small in-
testines, the amount, which is liberated this way and probably excreted,
32
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cannot be unimportant* especially for metals like cadmium and others which
may be retained over very long periods of time.
We have already discussed the excretion in bile, which also is an impor-
tant route. The hepato-intestinal circulation which has been observed ex-
perimentally for several metals, such as .lead, cadmium, mercury and
manganese, may be a significant factor in considering our therapeutic
possibilities. If you have such a circulation, it is logical to try to break that
to get more excreted. This has been successfully done with methyl mercury
experimentally as Dr. Clarkson has shown and I am sure it may be a
possibility also for other metals.
In the skin, there is a number of important glands. The mammary glands
are of course essential in pregnancy and especially for the new-borns. If
tlie women are exposed to metals in industry, the excretion through the
mammary glands could be an essential risk to the new-born. Since this
is the only food intake, it would be a considerable daily intake. The other
routes, like sweat glands and so on, are certainly of less importance, but
can be used for diagnostic purposes. The epithelial shedding from the skin
on the other hand should be of some importance for metals, such as cad-
mium where there also is this kind of accumulation in the skin.
Hair can often be a good indicator of body burden for metals and has
been used as such successfully in epidemiological studies. In animal experi-
ments a large part of the body burden can be found in the fur. In our
studies on methyl mercury on squirrel monkeys for example the fur was
found to contain more than 50 percent of the body burden after 2 months
(Berlin, Nordberg and Hellberg, 1971, in press).
Kidneys are one of the most important organs for the excretion of
metals. Maybe this is the best studied route. Epithelial shedding in the
kidneys is another possible route for metal excretion, which has to be
considered in cases where we have very low output. Perhaps semen could
be of interest, especially from the point of view of genetic effects. The
fetus is another important route of excretion in experimental animals and
even in man this could be a way to lose a large part of the body burden
of accumulated metals. In methyl mercury studies, for example in the hen,
a considerable part of the body burden is lost via the egg.
I would like to turn to the question of biological half-lives and would
like to stimulate a discussion here by saying that I do not personally believe
that the biological half-life is really a useful concept in terms of evaluating
risks. Only in very few exceptions where the excretion is so slow that any
other turnover rate in the body is rapid in comparison to excretion, can
we use this concept practically. In other cases I feel it is better to discuss
the turnover in-various organs, especially of course the turnover in critical
organs. Since the effect is a matter of the dose at the site of action, it is
33
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therefore much hotter l<> discuss the turnover in the slowest organ. Whidi
organs would show a slow turnover generally? The testicles have several
times been shown to have a very slow turnover for cadmium, but not only
for cadmium, but the same is true for mercury and polonium. Polonium
is an alpha-emitter which is distributed in the testicles. We get a lot of
polonium by cigarettes, for example. This could be of much importance
from the point of view of genetical risks.
The brain has some parts with a very slow physiological turnover and
it shows a slow turnover even for some of the metals deposited in the
brain. This has been found for mercury and lots of facts indicate that
there are other metals that have similarly slow turnovers, like manganese
for example. Of course, the deposition in bone is related to the turnover
of the bone tissue which again varies according to different physiological
and pathological conditions. This was just to point out some of the impor-
tant questions. I am sure there is a lot here that can be discussed.
TSUCHIYA: 1 will add a few comments on the relationship between ab-
sorption and biological half-life of cadmium. As you know there is a great
disagreement with regard to biological half-lives of metals. For example,
for cadmium, some animal experiments have shown values of only around
50 to 100 days. Of course, absorption and excretion may differ in different
animal species and result in a great discrepancy in biological half-life.
Nor have we reached an agreement in human observations. Kitamura
(personal communication) made an observation on autopsy materials (on
humans) and estimated the biological half-life of cadmium as 4 to 5 years.
He also made an experiment on a human subject, and observed 5.3 percent
absorption of cadmium from ingested water when starved, and 1.5 percent
of absorption through the gastro-intestinal tract when rice was added with
CdNO;,. As I have already presented in another session, we derived the
biological half-life for cadmium as 16 years, based on the information about
the total body burden, daily intake, and absorption via the G.I. tract as
3 fug, via respiratory tract as 1 ^g.
When calculating the total body burden or biological half-life, it is most
important to evaluate the methodology, including absorption rate. Usually
in deriving the biological half-life we use short-term experiments over a
period of some clays, at most one year or so. For cadmium or lead how-
ever, we ingest very low amounts of cadmium daily over a period of many
years, 50—80 years. Long-term ingestion with a low dose of cadmium may
be quite different from short-term experimentation. Therefore I want to
point out again that we have to appraise very carefully the methodology of
how to calculate or derive a biological half-life of metals. We should collect
more information in the future about the absorption and excretion which
34
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may .vary according to the dose or concentration and the length of exposure
as well as to host factors.
Dr. Berlin mentioned that the biological half-life may not be very im-
portant, but I do not agree, because from the point of view of total body
burden we have to know the biological half-life in order to set adequate
standards for metals in food and air in the future. I agree with Dr. Berlin
on the point that there is a target organ. For example, lead is mostly
accumulated in the bones and the biological half-life in bones and the
total body burden might be different. Nevertheless I still think it is very
important to know the biological half-life of many toxic substances.
CHAIRMAN: 1 do not think there is any disagreement between Dr. Berlin
and .Dr. Tsuchiya concerning a very long biological half-life. What Dr.
Berlin mentioned was that he did not think that the measurements of the
biological half-life were useful for those elements for which the turnover
within the body is relatively slow in relation to the excretion. I am also in
complete agreement with Dr. Berlin that the biological half-life in critical
organs is of great importance. The question is how to measure the bio-
logical half-life in such organs. One could always do it in animal experi-
ments, of course, but how do we approach that for humans, since we know
that one cannot just extrapolate from animal experiments to humans ?
BERLIN: If you know what organ you want to study, I think, there are
several types of methods which could be applied. Some organs are reach-
able from the point of view of blood supply. One can measure output and
input. Generally biopsies are not very realistic to use clinically, but for
experimental studies on animals one can certainly use them.
One interesting organ is bone tissue. We have not exhausted our possi-
bilities to find methods to determine different kinds of metals in bone. One
method is to use X-rays with specific absorption characteristics for different
metals. One of the most difficult organs to study is the brain. We do not
really know the relationship between metal concentration for example in
cerebro:spinal fluid and in brain tissue. The probable reason for that is
that the amount in the cerebro-spinal fluid generally is very low and our
analytical possibilities have been very limited. Rapid improvements have
brought modern methods by which very low concentrations can be ana-
lyzed. As the cerebro-spinal fluid is accessible for measurements, we have
some possibilities.
TSUCHIYA: But Dr. Berlin, if there is a close relationship between the
concentration of methyl mercury in the brain and the total body burden
35
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of methyl mercury, you may know the biological half-life in a target organ
by estimating the total body burden of mercury.
BERLIN: I guess methyl mercury is one of the metals for which we believe
there is a relationship between body burden and the brain concentration.
However, recent experimental evidences show that the transport and distri-
bution of methyl mercury in brain are very slow processes. So far the crude
clinical and experimental data still support the idea which is a good work-
ing hypothesis, that there is a correlation between brain concentration and
body burden. It still remains to be proven however.
CHAIRMAN: If you take, e.g. inorganic mercury, what is the biological
half-life in the brain? Would it be a couple of months, a couple of years
or perhaps 20 years?
BERLIN: I would like to point out that the brain is not really one organ.
It cannot really be considered as one type of cells or one entity metabolic-
ally. It covers a number of functions with different biochemical organiza-
tions, having been put together in a mass. We have to consider the brain
as different kinds of centers, different metabolic activities which certainly
are not morphologically organized from the point of view that you have
one center here and one there. Since they are all mixed cells, we have to
find a way to classify different kinds of biochemical systems in the brain.
What system is affected and where do we have the metal ? Some years ago
in our laboratories we had some interesting results in guinea pigs showing
tremendous accumulation in a few types of neurons in the brain stem as
compared to the rest of the brain. It is very important to know what the
half-life is in these neurons compared to the rest of the brain. I believe that
one could easily have rather inert mercury deposited somewhere in the brain
with no effect whatsoever. On the other hand the concentration in some
areas may be very important where you may have another kind of half-life.
We cannot really simplify it in saying that the brain is one entity with
one common half-life.
CHAIRMAN: 1 appreciate that, but could you give any figures for any
part of the brain or of total brain ? Make a guess between half a year, two
years or 30 years or something like that.
BERLIN: No, we do not know that much at the present, but we can say
that we have some clinical cases which actually suggest half-lives, exceeding
years. The question remains open.
TSUCH1YA: In my experience I have seen many cases of inorganic mer-
36
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cury poisoning, most of them, I think, reversible in clinical symptoms, i.e.
the clinical symptoms will sooner or later disappear after removal from
exposure. If you remove a worker with mercury poisoning for 4—5 months,
his clinical symptoms such as tremor etc, may mostly disappear. At an
earlier session-I showed a figure in which I estimated the biological half-
life of inorganic mercury. The half-life for total body burden would be
around one month. But I do not know of course the biological half-life in
the brain. This remains to be solved. But as long as organic changes in the
brain do not develop, the decrease of excretion of mercury parallel the
improvement of clinical symptoms.
NORSETH: It is unrealistic to talk about methods to determine the critical
organ body burden in humans. I think that the last part of this discussion
shows very clearly that we have not only to determine the critical organ
burden, but also the critical cell burden, which is an impossible task. There-
fore, let us look at it from a practical point of view. I would suggest that
we do some animal experiments with doses, organ distribution, eventually
site of action, distribution, if we can, and excretion, whereafter we go to
humans to try to determine dose and excretion. Based on the kinetic studies
in animal experiments and what we know about mechanisms, we make
some kind of model for organ content in humans at different times. Then
we take someone accidentally poisoned and study the organs to see if they
fit the curves. This has been done for methyl mercury and the results
combined with a few human experiments are the basis for our toxicological
considerations related to this compound.
MALCOLM: From a clinical point of view there are certain practical
difficulties with regard to methyl mercury, but also to lead. A lead para-
lyses can last a very long time. If it is due to the chelation of lead in the
neuromuscular junction, there is some evidence that even quite long lasting
paralyses has been improved by chelation. I am never quite convinced by
this work, or have we not an accumulation of lead but an actually patholo-
gical damage to the cells which does not recover? I think we must differen-
tiate between these two effects, either the presence of metal or pathology
and brain damage. With methyl mercury you may get a permanent damage
to brain cells which will not recover, even after the metal has disappeared.
GOYER: In the nervous system I am not sure that we really know which
cells are involved in the partitioning of lead. In our laboratory Dr. Krig-
man and his group are working with suckling rats given a large amount
of lead. He has found that there is an increased number of astrocytes and
microglial cells. He has been able to give enough lead to form an intra-
37
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nuclear inclusion body in astrocytes at the periphery of the brain where
it is in contact with cerebro-spinal fluid, suggesting that these are prob-
ably the cells that take up the largest burden of lead. But as far as clinical
symptomatology and long-term effect are concerned, it is the neuron that
is important. Studies on children have shown that even of those children
that do recover from clinical encephalopathies a large percentage suffers
to some degree from permanent damage, in some instances minor, but in
others very severe (Perlstein and Attala, 1966).
With regard to Dr Malcolm's comment, I agree that there are probably
two types of lead effects on the nervous system, one functional and re-
versible, the second resulting in permanent nerve cell damage. Experimental
studies have shown that lead may interfere with transmission of impulses
at preganglionic nerve endings by reducing output of acetylcholine output
(Kostial and Vouk, 1957) so that this is a reversible type of lead effect.
On the other hand, Schlaepfer, 1969, has suggested from his experimental
studies that demyelination and Wallerian degeneration of peripheral nerves
in lead nephropathy may result from a lead effect on the Schwann cell,
the supporting cell of peripheral nerves.
DUTKIEWICZ: In the excretion of metals the kinetics is essential because
when we know the character of the excretion and its mathematical model,
one can calculate the absorbed dose and period of time necessary to reach
the steady state. By various ways of absorption the kinetics of elimination
changes according to the way of absorption. The problem is not that simple.
Selenium elimination in urine after both intravenous and intratracheal app-
lication can be shown as a straight line, that is, it has a single phase eli-
mination, whereas after skin application, the elimination is two-phased.
It is similar for many other metals such as, mercury, chromium, arsenic,
etc.
LEWIS: One phenomenon that has been touched on this morning, but not
discussed in detail, is the proportional redistribution of trace metals within
the body with time. Bonnell, Ross and King, I960, give tissue concentra-
tions of cadmium in liver and kidneys obtained at necropsy from a small
series of men who had died of chronic cadmium poisoning, together with
years of exposure and interval between last exposure and death. The longer
the survival of a patient following exposure the smaller the liver/kidney
cadmium ratio observed, indicating that cadmium is removed more rapidly
from the liver than from the kidneys. Similarly, the data I presented yester-
day, relating cadmium accumulation lo cigarette smoking, showed that the
mean cadmium content of lungs derived from ex-smokers was similar to
that of non-smokers. In contrast, when mean kidney values were considered
38
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ex-smokers identified themselves with smokers. It is therefore, extremely
important to define the distribution dynamics within specific organs, when
considering the kinetics of trace metal distribution and elimination parti-
cularly when possible associations with disease, social habits and occupation
are being sought.
PISCATOR: In another session here we showed how the kidney was
accumulating cadmium for months with a simultaneous slow decrease in
the liver after just one single injection. Dr Berlin mentioned earlier with
regard to excretion routes that it is quite conceivable that after injection,
cadmium will at first be in a low molecular form and excreted in some
part of the intestines. In a later phase the main part of cadmium is stored
in the liver. We have found that in mice after several months the concen-
tration is higher in the pancreas than in the liver, meaning that accumula-
tion and excretion are taking place also in the pancreas.
U.3. REFERENCES FOR SECTION II
References
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of methylmercury (203Hg) compounds in man (excretion and distribution). Arch.
Environ. Health 19: 478—484, 1969.
Albert, R. E., Lippmann, M., and Briscoe, W. The characteristics of bronchial
clearance in humans and the effects of cigarette smoking. Arch. Environ. Health
18: 738—755, 1969.
Altshuler, B., Yarmus, L., and Palmes, E. D. Aerosol deposition in the human
respiratory tract: I. Experimental procedures and total deposition. A.M.A. Arch.
Indust. Health 15: 29}—303, 1957.
Berlin, M., Nordberg, G., and Hellberg, J. The uptake and distribution of methyl
mercury in the brain of saimiri sciureus in relation to behavioral and morpholo-
gical changes, in: Mercury, mercurials and mercaptans, Miller, M. W., and Clark-
son, T.W. (eds.) (from the 4th Rochester conference on environmental toxicity,
University of Rochester, Rochester, New York, June 17—19, 1971). Charles C.
Thomas Publ., Springfield, III., in press.
Berlin, M., and Ullberg, S. The fate of Cd109 in the mouse. An autoradiographic
study after a single intravenous injection of Cd1MClj. Arch. Environ. Health 7:
686—693, 1963.
Bingham, E., Pfitzer, E. A., Barkley, W., and Radford, E. P. Areolar macrophages:
Reduced number in rats after prolonged inhalation of lead sesquioxide. Science
162: 1297, 1968.
Black, S. C. Storage and excretion of lead210 in dogs. Arch. Environ. Health 5:
423, 1962.
Bonnell, J. A., Ross, J. H., and King, E. Renal lesions in experimental cadmium
poisoning. Brit. J. Indust. Med. 17: 69—80, I960.
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Castellino, N., and Aloj, S. Kinetics of the distribution and excretion of lead in the
rat. Brit. J. Indust. Med. 21: 308, 1964.
Clarkson, T. W., Small, H., and Norseth, T. The effect of a thiol containing resin
on the gastro-intestinal absorption and fecal excretion of methylmercury compounds
in experimental animals. 55th annual meeting Fed. Amer. Soc. Exp. Biol., 1971.
Creamer, B. Table 1—Turnover time of intestinal epithelial cells. Brit. Med. Bull.
23: 227, 1967.
Crosby, W. H. Iron absorption in alimentary canal, in: Handbook of physiology,
Codie, Charles E. (ed.). Amer. Physiol. Soc. Washington, D. C, Vol. Ill, Section
6. 1968, pp. 1553—1570.
Eldjarn, L., and Jellum, E. Organomercurial-polysaccharide, a chromatographic ma-
terial for the separation and isolation of SH-proteins. Acta Chem. Scand. 17:
2610—2621, 1963.
Findeisen, W. Ueber das Absetzen kleiner, in der Luft suspendierter Teilchen in der
menschlichen Lunge bei der Atmung, Arch. ges. Physiol. 236: 367, 1935.
Friberg, L., Piscator, M., and Nordberg, G. Cadmium in the Environment. The Che-
mical Rubber Co., Cleveland, 1971, pp. 31—32.
Goyer, R. A., Leonard, D. L., Moore, J. F., Rhyne, B., and Krigman, M. R. Lead
dosage and the role of the intranuclear inclusion body. Arch. Environ. Health
20: 705, 1970.
Hursh, J. B., and Suomela, J. Absorption of 218Pb from the gastro-intestinal tract of
man. Acta Radiol. 7: 108—120, 1968.
Jellum, E., Aaseth, J., and Eldjarn, L. Mercaptodextran. A new thiolprotecting and
metal chelating agent, in preparation, 1971.
Kehoe, R. A., Cholak, J., Hubbard, D. M., Bamback, K., and McNary, R. R. Ex-
perimental studies on lead absorption and excretion and their relation to the
diagnosis and treatment of lead poisoning. J. Indust. Hyg. Toxicol. 25: 71—79,
1943.
Kostial, K., and Vouk, V. B.: Lead ions and synaptic transmission in the superior
cervical ganglion of the cat. Brit. J. Pharmacol. 12: 219, 1957.
Krigman, M. R. Unpublished observations.
Miettinen, J. K. Absorption and elimination of dietary mercury (Hg+ + ) and methyl-
mercury in man, in: Ibid. Berlin, Nordberg, and Hellberg, 1971, in press.
Miler Von, N., Saltier, E. L., and Menden, E. Aufnahme und Einlagerung von Blei
in Korper unter verschiedenen Ernahrungsbedingungen. Med. Ernahrung 2: 2—11,
1970.
Norseth, T. Biliary complexes of methylmercury. A possible role in organ distribu-
tion, in: Ibid. Berlin, Nordberg, and Hellberg, 1971, in press.
Norseth, T., and Clarkson, T. W. Intestinal transport of 2MHg-labelled methyl-
mercury chloride. Arch. Environ. Health 22: 568—577, 1971.
Palmes, E. D., Nelson, N., Laskin, S., and Kuschner, M. Inhalation toxicity of cobalt
hydrocarbonyl. Amer. Indust. Hyg. Ass. J. 20: 453—468, 1959.
40
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Perlstein, M. A., and Attala, R. Neurologic sequelae of plumbism in children. Clin.
Fed. 3: 292, 1966.
Schlaepfer, W. W. Experimental lead neuropathy: A disease of the supporting cells
in the peripheral nervous system. J. Neuropath. Exp. Neurol. 28: 401, 1969.
Six, K. M., and Goyer, R. A. Experimental enhancement of lead toxicity by low
dietary calcium. J. Lab. Clin. Med. 76: 93$, 1970.
Skog, E., and Wahlberg, J. E. A comparative investigation of the percutaneous ab-
sorption of metal compounds in the guinea pig by means of the radioactive
isotopes: 61Cr, "Co, 65Zn, 110mAg, '"""Cd, 203Hg. J. Invest. Derm. 43: 187—192,
1964.
Task Group on Lung Dynamics, International Commission on Radiologic Protection,
Deposition and retention models for international dosimetry of the human respira-
tory tract. Health Phys. 12: 173—207, 1966.
Wahlberg, J. E. "Disappearance measurements", a method for studying percutaneous
absorption of isotope-labelled compounds emitting gammarays. Ada Dermatovener.
45: 397, 1965 (a).
Wahlberg, J. E. Percutaneous toxicity of metal compounds. A comparative investiga-
tion in guinea pigs. Arch. Environ. Health 11: 201—204, 1965 (b).
41
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SECTION III
ACCUMULATION OF TOXIC METALS WITH SPECIAL REFERENCE
TO THEIR ABSORPTION, EXCRETION, AND BIOLOGICAL HALF-TIMES
Prepared by the
Task Group on Metal Accumulation
(Reprinted from Environmental Physiology and Biochemistry,
3:65-107, 1973.)
43
Preceding page blank
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SECTION III
ACCUMULATION OF TOXIC METALS WITH SPECIAL REFERENCE
TO THEIR ABSORPTION, EXCRETION, AND BIOLOGICAL
HALF-TIMES
in.l. BACKGROUND
At an international symposium on maximum
allowable concentrations of mercury compounds
in Stockholm, 1968 (MAC 1969), it was considered
advantageous to form a group experienced in
metal toxicology. At the meeting of the Permanent
Commission and International Association on
Occupational Health in Tokyo, 1969, it was decided
to form a subcommittee on the toxicology of
metals under this organization. This subcommittee
(chairman, L. Friberg, Stockholm) organized a
panel discussion dealing with absorption and
excretion of toxic metals (Dukes & Friberg 1971)
at the congress of the Permanent Commission and
International Association on Occupational Health
in Slunchev Bryag, Bulgaria, September, 1971.
At the meeting in Bulgaria, certain principles
concerning absorption of metals via the pulmonary,
gastrointestinal and cutaneous routes were dis-
cussed, as well as certain characteristics concerning
the excretion of metals. It appeared to the sub-
committee from these discussions that it would
be worthwhile to examine the possibility of es-
tablishing models for some forms of absorption
which would have general validity for several
metals. As a result of these considerations, u
working group convened in Buenos Aires, Sep-
tember. 1972, prior to the XVIIih International
Congress on Occupational Health.
All participants had prepared working papers
dealing with specific topics to he discussed, and
these working papers had been circulated among
the participants in advance of the meeting.
The meeting, which took place at the Ministry of
Social Welfare, Public Health Area, Buenos Aires.
Argentina, was officially opened by Dr. Adolfo
Antoni, President of the Organizing Committee of
the XVI Ith International Congress on Occupational
Health together with Dr. Lars Friberg, Chairman
of the Subcommittee on the Toxicology of Metals
under the Permanent Commission and International
Association on Occupational Health. Dr. Lars
Friberg of Sweden was elected Chairman of the
meeting. Dr. Ana Singerman of Argentina was
elected Vice-Chairman and Dr. Gunnar Nordberg
of Sweden was elected Rapporteur.
The discussion at the meeting was centered on a
"skeleton report" prepared by Dr. Nordberg
mainly from the working papers (section lf».
Some sections in the present report emerged from
a detailed scrutiny of sections in the "skeleton
report", and other sections were introduced and
written during the meeting. The complete report
was read, discussed and approved by the "Task
Group" as a whole, but the detailed preparatory
work for each section was performed by four
working groups gi\en responsibility for the specific
sections, i.e. "Fundamental aspects of metal
loxicity: gastrointestinal absorption and excretion"
wasdeall with by a group chaired by Dr. T. Norselh:
"Absorption by inhalation" by a group chaired by
Dr. K. Albert: "Transport, distribution, placenta!
transfer and renal excretion" by a group chaired
by Dr. J. Vosial: and "Accumulation and retention
in critical organs and indices of exposure and
retention" b> a group chaired by Dr. M.
Ik-din.
45
Preceding page blank
-------
III.2. FUNDAMENTAL ASPECTS OF
METAL TOXICITY
III.2.1. CONCEPTUAL CONSIDERATIONS
Ccrlain metallic elements arc necessary Tor the
normal biological development and function
of human beings and arc known as "essential
metals". Others may not be identifiable as
serving a beneficial biological function and are
known as ."non-essential". However, the
concept of essentiality is under constant review
and an unequivocal classification of metals
according to it cannot yet be made. All metals
have the potential to cause adverse effects in
human beings at certain levels of exposure and
absorption. Mechanisms exist for regulating
the body levels of some metals, mainly essential
ones, but for certain unusual routes of exposure
such protective mechanisms may be inadequate
and for other metals they do not exist at all.
This report will be concerned mainly with
mechanisms of absorption, distribution and
excretion, with particular reference to biological
half-times and accumulation. Primary con-
sideration will be given to cadmium, lead,
mercury and their compounds. Although the
term "heavy metals" is frequently encountered
in literature on metal toxicology, it will not be
used in this report. Elements included by this
term vary among authors, and the principles
of absorption, excretion and accumulation
dealt with in this report may apply also to
elements not included in a particular definition
of heavy metals.
III.2.2. BIOCHEMICAL CONSIDERATIONS
A toxic effect arises because certain bio-
chemical reactions in the body become altered
by the metal in question. Other mechanisms
may operate but these will not be discussed.
Metals may produce structural changes in
the cell membrane, alfccting its permeability.
When the metal reaches the inside of I he cell,
a number of en/ymalic activities can become
inhibited. Such interference of metals with
en/yino -.•.•'• i-.-nv may iiivc rise to functional
impairment. i\>c cn/yrnasic inhibition may
involve an interactional the active center of an
cn/yme, but (he inhibition may also rcsuli from
interactions between metals and ligamls noi
directly involved in the aciive center. Changes
in electrostatic charge may occur along with a
shift in the ionization constant of ihe active
center. Also conformational changes in a pro-
tein or the combination of the metal with a
coenzymc may provide the basis for impair-
ment of the enzymatic reaction. ,
Another way by which exposure to a
potentially toxic metal may cause adverseeffects
is by changing the metabolism of other metals.
Some tissues may be depleted of ligands or
metals necessary for enzymatic function even
if excessive amounts of the toxic metal are
not stored in that tissue.
Metal binding occurs not only at sensitive
sites in cnzymalic systems of the cell, but also
at sites in the cell where the metal is less active
from a toxicological point of view. If there is
a sufficient reserve of enzymes in a cell or
additional enzymatic activity can be induced,
inhibition of a portion of a particular enzyme
wiihin a cell will usually not impair cell
function seriously. If an interference occurs
with enzymes necessary for the build-up of ATP
or other high energy nucleotides, some of the
cell functions requiring little energy may con-
tinue as normal whereas functions requiring
stored energy will be impaired. For several
biochemical reactions in the body, important
functions might continue by means of an
alternative pathway. Usually, such pathways
arc less favorable and might produce an extra
load on other systems in the cell.
III.2.3. CRITICAL ORGAN CONCEPT AND
CRITICAL CONCENTRATIONS IN CELLS
AND ORGANS
With the accumulation of metal, when un-
desirable functional changes, reversible or
46
-------
irreversible, occur in the cell, it can be said
that the "critical concentration" has been
reached. Any increase beyond the "critical
concentration" is accompanied in most cases
by increasing impairment of cellular function.
The cellular concentration sufficient to cause
cell death is the "lethal concentration" for
that cell.
Analogously, the "critical organ concentra-
tion" is defined as the mean concentration in
the organ at the time any of its cells reaches
critical concentration. The critical organ
concentration may be considerably higher or
lower than the critical concentration for a
particular cell.
The concept of a "critical organ" refers to
that particular organ which first attains its
critical concentration of metal. The organ of
greatest accumulation or retention is not
necessarily the critical organ.
Metabolic factors and sensitivity of organs
show interindividual variability that is so
great as to make one organ critical for one
person and another organ critical for another.
Epidemiological considerations pertaining to
variations in the "sensitivity" of individuals
to a particular metal at a certain level of ex-
posure are therefore of interest.
It is not the intention in this report to discuss
which specific concentrations of various metals
are critical for various organs. However, in
general, the information contained in Table 6
cites the organs which are critical following
exposure to cadmium, lead and mercury.
These considerations show that it is of
importance to determine which organs and
which groups of cells within them arc most
susceptible to damage when metals accumulate
there. Subccllular partitioning of metals
among various organcllcs and iniraccllulur
fluid may also be of considerable importance.
It is now known for some metals that specific
proteins or classes of proteins which have
preferential allinily for the metal arc present
in cells. These proteins may hind a particular
Table 6
Orfunx critical in inclal intoxication
Cadmium (Cdat)
Lead(Pb2*)
Tetracthyl lead
(C,H,)4Pb
Triethyllead(C2H5)jPb
Mercury vapor (Hg°)
Mercury (Hg2+)
Methoxyethyl mercury
(CH3-0-CH,CH2Hg+)
Phenyl mercury (G6H5Hg
Methyl mercury (CH3Hg+
Lung
. Kidney
Liver
Hcmutopoictic tissue
Central and peripheral
nervous systems
Kidney
Central nervous system
Central nervous system
Central and peripheral
nervous systems
Kidney
Kidney
Kidney
;*) Kidney
) Central nervous system
metal so that it is not available to interact with
normal cellular functions or enzyme activity.
Such protein binding might detoxify the metal.
It becomes apparent that not only the cellular
concentration of a particular metal, but also
its intracellular location, form, and whether
it is free to react in the cell, are critical. Other
factors which may determine the efficacy of
the protein-metal complexing reaction in any
particular cell at a specific time are age of cell,
functional demands, nutritional state, rate of
exposure to metal, chemical form of metal, etc.
The proteins which bind metals to detoxify
them may not be present in all cells of the
body at all times. It has been shown that such
proteins (e.g. metallothionein) may be in-
duciblc so that availability to bind with metals
may vary. Similarly the fate of such metal-
protein complexes may vary, that is, the rate
at which they are cataboli/ed or excreted.
These considerations suggest, therefore, that
critical concentrations of a metal might vary
depending 0:1 the extent of formation of in-
active chemical forms of metals within cells.
It is not within the scope of this report to
consider the accumulation of metals in relation
47
-------
to mutagenesis or (eratogenesis ut subccllular.
cellular or orgun levels. Furthermore, (his
report will not deal with (he effects of radiation
emanating from metals.
IH.3. ABSORPTION
Absorption is defined as entry into the body
by passage of the metal or its compound across
a membrane. The metal compounds may be
retained at the Jocal site of absorption for long
periods of time or they may be transported
elsewhere.
The two principal routes for absorption are
inhalation and ingestion and these will be dis-
cussed in detail. Under certain circumstances,
the skin is an important route by which metals
enter the body. This type of absorption was
dealt with to some extent at the previous
meeting of the subcommittee on the toxicology
of metals (Wahlberg, in Dukes & Friberg
1971), but will not be discussed in this report.
Placenta! transfer is of importance for effects
on the fetus. Knowledge of this form of ab-
sorption makes it possible to evaluate the
contribution to the body burden at birth.
Since the placenta! route of absorption has
not been dealt with previously at the sub-
committee's meetings, it was decided to include
a short discussion in the present report.
HI.3.1. ABSORPTION BY INHALATION
III.3.1.1. Introduction
Pulmonary absorption of metals is usually the
most important route for their entry into the
human body in industry. This absorption route
is also of importance in exposure from (he
general environment, including exposure (o
tobacco smoke. Metals may be taken into the
respiratory system in the form of particles,
gases or vapors.
///. 3.1.2. A bsorption of metals in particle form
III. 3.1.2.1. General principles
Absorption of metals and mclul compounds
iVi particle form is influenced by (hrcc processes
in the lung, namely, deposition, mucociliary
clearance and alveolar clearance. A model of
these three processes is depicted in Fig. 13.
After deposition in the nasopharyngcul,
tracheobronchial or alveolar (pulmonary)
B
L
0
0
D
t ]t
(a)
(c)
lli)
1 LYMPH
1
N-P
A°4
T - R
-|D5
P
ibi
(d) •
(f>
li)
G.
I.
T
R
A
C
T
1
Figure 13. Respiratory tract clearance model
(From Task Group on Lung Dynamics 1966).
N-P = Nasopharyngeal compartment; T-B -
Tracheobronchial compartment; P = Alveolar
(Pulmonary) compartment
D, = total dust inhaled.
D2 = dust in the exhaled air.
D} =- amount of dust deposited in the N-P.
D* = amount of dust deposited in the T-B.
D9 =- amount of dust deposited in the P.
a •-- rapid uptake of material deposited in the N-P
directly into the blood; b - mucociliary clearance
from N-P to G-l tract; c rapid uptake of material
deposited in T-B directly into the blood; d ••»
mucociliary clearance: c - direct absorption of
dust from the alveolar compartment to blood;
f inclusion of alveola ily deposited particles by
macrophagcs and their transport by the mucociliary
escalator to the G-l tract; g «• same as f, but slower
process; h - slow removal of particles by the lymph
system: i - transport of dusl cleared by (h) into
(he blood; j - G-l absorption of particles cleared
to the G-l tract by processes b, d, f, and g.
48
-------
compartments, the metal may be transported
by mucociliary action (o (he gastrointestinal
tract or absorbed into the blood. The fraction
deposited in the non-alveolar compartments of
the lung participates mainly in the first named
type of translocation. The portion deposited
in the alveolar compartment is more likely to be
absorbed, but may take different routes.
III.3.1.2.1.1. Deposition of particles in pulmonary
contparrineiits. Deposition occurs by three
physical mechanisms: inerlial impuction. gravi-
tational sedimentation anddifTusion.ini paction
and sedimentation depend on particle mass(size
and density): diffusion is important only at
particle diameters of less than a few tenths of a
micrometer. Impaction is the dominant mech-
anism for unit-density particles larger than a
few micrometers: it is also dependent on the
velocity of the airstream entering the res-
piratory tract. Impaction occurs at sites where
the airstream bends sharply and is the most
important deposition mechanism in the nose,
mouth, pharynx and upper bronchial tree.
Gravitational sedimentation also depends on
particle mass as well as the residence time of
airborne; particles in the lung. Sedimentation
is an important mechanism in the smaller
bronchi and alveoli for unit-density particles
in the range of a few tenths to a few micro-
meters.
It is evident that deposition of inhaled
metal particles (as is true for particles of all
types) depends principally on their physical
characteristics as well as on the patterns of
respiration including the route of inhalation,
whether nose or mouth, and the tidal volume
and respiratory rate.
Deposition in the three compartments for
particles with various mass median aerodyna-
mic diameters has been estimated by the Task
(iroup on Lung Dynamics, 1966, and is given
in Kig. 14 for a tidal volume of 1450 ml, 15
respirations per minute, which represents
moderate physical activity. The estimations
in Fig. 14 were made on (he basis of certain
0.01 0.05 O.I 0.5 1.0 5 IO SO ICO
MASS MEDIAN DIAMETER-MICRONS
Figure 14. Deposition of particles in the lungs of
"the Standard man" (from Task Group on Lung
Dynamics 1966).
assumptions about the "standard" human lung
and were only partly based on experimental
data. The shaded areas in this figure indicate
the calculated ranges of deposition that would
result from variation of the physical character-
istics of an aerosol in the lungs of the ICRP
"standard" man. Observations show that in
the particle size range 2-5 x/m the average
deposition pattern fits the ICRP model, but
that individual variation is large, of the order
of two to threefold (Lippmann et al. 1971).
Individual differences in aerosol deposition
have also been shown for animals (Holma 1967.
Albert et al. 1968) and these individual differ-
ences appear to be stable (Albert et ul. 1968.
Tomenius 1973).
111.3.1.2.1.2. Mucociliary clearance. Mucous flow
and ciliary activity in the tracheobronchial
and nasopharyngcal systems constitute the
process of particle transport designated as
mucociliary clearance. The ultimate result is
the iranslocation of material into the gastro-
intestinal tract or its expectoration. Because
of t he relative rapidity of mucociliary clearance.
usually completed in less than S hours (Albert
el al. 1971). the portion of the total deposition
49
-------
that falls within the tracheobronchial and
nasal compartments is generally not absorbed
into the body to any considerable extent, unless
the aerosol particles consist of very easily
soluble salts. Data to substantiate absorption
of metalcompounds by these routesare lacking.
Measurement of the amount of radioactivity
in the lung at various times after the inhalation
of an aerosol tagged with a radionuclide has
been used for -studies on "lung clearance".
As a rule, curves obtained by such measure-
ments may be simplified iVito at least two phases,
an initial fast phase usually taking between
one and 24 hours (usually less than 8 hours)
and a second, slower phase. The first phase is
considered to reflect "tracheobronchial clear-
ance", a result of mucociliary activity. The
second phase is considered dependent upon
clearance from the alveolar compartment.
The first phase of lung clearance, "tracheo-
bronchial clearance", is dependent upon
particle size in that larger particles appear
to be cleared more quickly than smaller ones
due to their differing deposition and con-
sequently different distances to travel up the
tracheobronchial tree. Another factor that
increases the difference is the higher speed of
the mucous flow in the upper parts of the
tracheobronchial tree compared to that in the
lower regions.
Considerable differences among individuals
in the tracheobronchial clearance of particles
of the same size have been demonstrated in
persons inhaling the aerosol under standardized
conditions(Camneretal. 1971, Lippmannetal.
1971). These differences seem to be genetically
dependent since monozygotic twins have clear-
ance patterns very similar to each other, whereas
dizygotic twins do not display more similar
clearance rates than persons in general
(Camncr et al. I972d). The Iracheobronchial
deposition and clearance studies show mat
certain persons have higher alveolar deposition
and thus potentially higher absorption than
others.
///. 3.1.2.1.3. Alveolar clearance. Alveolar clear-
ance consists principally of three different
processes:
I. Transport of material from the alveolar
compartment onto the mucociliary escalator
and its subsequent translocation to the
gastrointestinal tract or its expectoration.
2. Passage of the metal through one of the
membranes into pulmonary tissues (where
it will remain for a long time).
3. Passage of the metal through the pulmonary
tissues into • lymph and blood, and its
translocation to other tissues by the sys-
temic circulation.
Possible pathways for these processes include
penetration through the junctions of the
alveolar pneumocyte, interstitial space and the
terminal lymphatics. Material which does not
follow the interstitial space may be phago-
cytized by the interstitial macrophages, or
may penetrate the endothelium of the blood
capillary. It is not known which of these path-
ways is the most important. Phagocytosis by
the alveolar macrophages is probably the most
important mechanism for the transfer of
particles to the mucociliary escalator (Morrow
1972).
It is not known for particles with low solu-
bility, neither in human beings nor animals,
how large a part of the alveolar clearance
results from transfer of particles to the muco-
ciliary escalator and how much is absorbed
through the lung. Soluble metal compounds
will be absorbed quickly, which will make them
less available for mucociliary transport.
However, it should be appreciated that with
a long residence in the lung, even substances
usually considered insoluble may undergo
dissolution. Furthermore, solubility in lung
fluids may be quite dilfcrent than in water.
For the part of the dust retained in the alveoli,
a solubility model may be (he most accurate
means tocvaluate the ratcof absorption! Mercer
1967, Morrow 1970).
The Task Group on Lung Dynamics (I9M>)
50
-------
categorized inorganic substances according
to their predicted retention in the lungs. It is
difficult to know the accuracy of this approach
and to judge the reliability of the classification,
since it was documented by experimental data
to only a limited extent.
Where a substance is poorly absorbed from
the gastrointestinal tract, the translocation of
particles from the alveoli to the gut will
minimize absorption. To illustrate this point, a
hypothetical example is shown in Table 7,'
comparing the absorption for the case of no
translocation but complete alveolar absorption
with the case of 50% alveolar absorption,
SO"/, translocation from the alveoli to the gut
and S°0 intestinal absorption. In both cases
the entire tracheobronchial deposition is
translocated to the gut and the alveolar and
tracheobronchial deposition behave according
to the 1CRP model (Morrow 1970). With no
translocation, the total absorption ranges from
9-50% with decreasing particle size. With 50%
translocation there is substantially lower
absorption, with a range of 7-27% at all
particle sizes. This illustrates the importance
of defining the extent of translocation from the
alveoli particularly for metals which have a
low intestinal absorption.
III.3.1.2.2. Specific experimental data on abtorp-
' lion of metals after aerosol exposure
For cadmium, lead and mercury, the metals
to be treated specifically in this report, data on
transpulmonary absorption are scarce. Fri-
berg et al. (1971) reviewed observations on
cadmium uptake in humans exposed to this
metal and concluded that quantitative data
were not obtainable. From various acute and
chronic animal exposures, absorption amount-
ing to 10-40% of inhaled cadmium could be
estimated. As the gastrointestinal absorption
of cadmium is relatively small, it was concluded
that the major part of this absorption was trans-
pulmonary.
For lead, a study by Hursh et aJ. (1969)
provides data on the absorption in human
beings of a 212Pb-aerosol obtained by the
decay of thoron to 212Pb absorbed onto the
natural aerosols of room air. The particle
size of the inhaled aerosol was unknown, but
the authors assumed a mass median diameter
of 0.2 micrometers. Deposition of lead in the
lung varied from 14 to 45% of the amount
inhaled, and less than 8 % was deposited on the
tracheobronchial tree. The authors calculated
a half-time of 6.5 hours for the absorption of
lead from lung to blood. However, these data
Table 7
Calculation of total absorption into the body as a function of two different rates of alveolar absorption and
different particle sizes for a specific deposition and clearance model. Gastrointestinal absorption
presumed to be 5 %
Total absorption (%)
Particle
size
(MMAD)*
lim
O.I
0.5
2.0
5.0
10.0
Alveolar
. (%)
50
30
20
10
5
Tracheobronchial-
nasopharyngeal
i«/\
\ /or
9
16
43
68
83
into body when alveolar
absorption is
^
100%
50.4
30.8
22.2
13.4
9.2
50%
26.7
16.6
12.6
8.6
6.8
' mass median aerodynamic diameter.
51
-------
urc nol necessarily applicable to ordinary
industrial lead exposure due to the unusual
physical and possibly chemical properties of
the inhaled 212Pb.
Specific data on transpulmonary absorption
of inorganic and organic mercury compounds
arc lacking, although it is known from both
animal experiments and human exposures that
amounts of several of the compounds which are
sufficient to cause poisoning can be absorbed.
///. 3.1.3. A bsorption of metals in gas and vapor
form
The only general principle known for the
deposition of gases or vapors in the respiratory
tract is the dependence upon water solubility.
Highly water soluble gases and vapors are
dissolved in the mucous membranes of the
nasopharyngeal and trachcobronchial systems
and never reach the alveoli. For example,
Strandberg (1964) showed that only a relatively
small fraction was present in the inspired air
at the tracheal level in rabbits exposed to
sulfur dioxide. Those gases and vapors which
are less soluble in water may not be dissolved
in this way and therefore reach the alveoli.
Depending on their ability to penetrate the
alveolar structure, they are absorbed to varying
degrees. Since the alveolar cells arc covered by
a phospholipid layer, lipid solubility of the gases
and vapors will probably be of importance for
their penetration of the alveolar membrane.
Most of the evidence available for metals
concerns mercury vapor, probably the only
case in which vapor exposure to a metal in
elemental form is of any practical importance.
Other metals occur in the form of vapors at
very high temperatures. These vapors may cause
certain occupational problems, but at the time
they are inhaled the temperature is much lower
and they have then become particles. It may
well be that the particles are very small; never-
theless, this type of exposure falls within Ihe
above section on paniculate matter. However,
for various compounds of metals, especially
some orgunomclullic compounds, the vapor
pressure at body temperature is high so as to
make possible the inhalation of toxic con-
centrations of vapors.
Mercury vapor is nonpolar and easily
penetrates the alveolar membrane into the
blood (Magos 1967, Berlin et al. I969a).
Vapors behaving like mercury which are nol
dissolved in mucous membranes of the naso-
pharyngeal and trachcobronchial tracts, but
are quickly transferred through the alveolar
membrane, will be absorbed to an extent of
about 80% of the inhaled amount. This per-
centage is a result of the quantitative relations
between inspiratory volume and the.physio-
logical dead space of the lung (Nielsen-Kudsk
1965). Specific data for the transpulmonary
absorption of organometallic compounds of
lead and mercury are lacking, but it is generally
believed that the absorption is high, i.e. about
80% of the inhaled amount.
III.3.1.4. Effects of age and pathological
conditions
Information in this area is limited. No
systematic data exist on the effects of age,
particularly whetherchildren differ significantly
from adults with regard to deposition and
clearance.
Nasal obstruction increases mouth breath-
ing, which tends to increase the total pulmonary
deposition of inhaled particles. Bronchial
deposition in chronic bronchitics with airway
obstruction is markedly increased probably
due to the narrowing of the airways (Albert
ct al. 1973). This is probably also true for
asthmatics. Such an effect would tend to
decrease alveolar deposition.
Mucociliary clearance in cigarette smokers
is slower than normal, which might facilitate
the absorption of soluble aerosols deposited
in (he bronchi (Albert ct al. 1969. Cannier &
Philipson 1972). Chronic obstructive lung
discusctclironic bronchitis) also has been slum n
to decrease the rale of trachcobronchiul
52
-------
clearance (Toigo et al. 1963. Camner et al.
1972a). Acute infectious bronchitis markedly
slows bronchial mucociliary clearance (Camner
el al. I972b, c).
Little information exists on the influence of
pathological conditions of the lung on alveolar
clearance. There is inconclusive experimental
evidence on the effects of emphysema on
alveolar clearance (Ferin 1971). On the basis
of dust burdens in autopsied miners' lungs,
Davies (1963) has suggested that there is
progressive impairment of the clearance of
dust from the alveoli with increasingly severe
pneumoconiosis.
Hl.3.2. ABSORPTION BY INGESTION
IH.3.2.1. General aspects
A major source of metals entering the gastro-
intestinal tract is by the ingestion of food
and beverages, but a substantial part of metals
in particle form deposited in the lung may be
transferred to the gastrointestinal tract by
mucociliary clearance. A scheme illustrating
the principal routes taken by'metals introduced
in the gastrointestinal tract is shown in Fig.15.
The uptake of metals originating from the
mucociliary clearance of the lung is governed
by the same mechanisms as for the metals
ingested, but the rates of absorption may some-
times differ. The transfer of metals from the
intestinal lumen into the cells of the epithelial
lining of the gastrointestinal tract (absorption)
may not lead to further transport into the
organism. The presence of metals as soluble
salts or complexes is usually a prerequisite for
absorption, but cndocytosisofmacromolecules
or particles may be of some importance, as
demonstrated in young animals (Sunders &
Ashworlh 1961).
The rate of absorption of metals from the
gastrointestinal tract is dependent upon (heir
chemical form. Some organomclallic com-
pounds are absorbed to a greater extent than
Body
O
Figure 15.Principal routes for metals introduced
into the gastrointestinal tract.
a = metal absorbed into the body.
b = metal introduced in g.i. tract but passing
unabsorbed through the g.i. tract.
c = G-I excretion of metal.
d = metal G-l excreted and reabsorbed.
e = net gastrointestinal excretion.
b + e = fecal content of metal.
their corresponding inorganic compounds
(Clarkson 1971). Anions are important for
the degree of absorption only to the extent
that they influence the solubility of metal
compounds in the acid medium of the stomach
(Magos 1972b, Pfitzer 1972). After transfer to
the small intestine, metals are usually bound to
various organic molecules and the anions
become of minor importance.
Gastrointestinal secretions and digestive
enzymes are important for converting the
metal to a form available for absorption.
Shifts in pH may change the valenceform of the
metal. It may also increase its solubility or
ionization which can promote absorption after
the formation of a complex more suitable for
absorption. The absorption of metals varies de-
pending upon location in the gastrointestinal
tract, on functional variations, including
differences in passage time, and on the patterns
of intestinal microbiological flora.
Metal interaction may influence absorption
processes as demonstrated by in vitro experi-
53
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men Is with manganese, zinc, mercury and
cadmium (Sahagian el al. 1966, 1967). Re-
duction of dietary calcium increases the
absorption of lead and cadmium (Flcischman
et al. 1968, Six & Coyer 1970. Larsson &
Piscator 1971. Kobayashietal. !97l,Piscator&
Larsson 1972). The great demand for calcium
in children, pregnant and luctating women will
cause high absorption rates for calcium, which
may increase the absorption of cadmium and
lead. Similarly, lowering dietary iron increases
lead absorption (Six AGoyer 1972). Difference
in lead absorption between the newborn and
adult may also reflect differences in gastro-
intestinal function such as capacity to absorb
molecules of certain size, and quality and
quantity of digestive enzymes and biliary
excretion.
The protein content in food may influence
metal absorption. Low protein in an otherwise
isocaloric diet increased lead uptake (Milev
et al. 1970).
III.3.2.2. Specific data
Data on gastrointestinal absorption (transfer
of metals from the gastrointestinal tract to
blood) have been listed for all metals by 1CRP
(ICRP publication 2, 1959). With regard to
radiation protection, these values have served
a good purpose. However, many entries were
not based on experimental observations in
human beings and sometimes only on limited
animal data. For some of the metals which are
of specific toxicological interest, including
those metals chosen for particular focus here,
the ICRP data are unreliable. They will not be
discussed further in this report.
Cadmium. Studies on the uptake of inorganic
cadmium compounds in animals (reviewed by
Friberget al. 1971) have shown retention after
about I week of up to 3 "/„ of the ingested dose.
Balance studies on "normal" intake of in-
organic cadmium compounds via food in
human beings theoretically give figures on
whole-body retention of the metal, but to
derive a precise figure is difficult for cadmium
because of the low and variable intake of this
element and t he lowgastrointestinal absorption.
Using "SmCd, Rahola et al. (1971) deter-
mined the retention of cadmium in five male
volunteer subjects (ages 19-50) after adminis-
tration of """Cd mixed with calf kidney
proteins. Whole-body counting as well as
analysis of fecal and urine samples revealed that
approximately 6% was retained after 10-15
days. It may well be that the gastrointestinal
absorption as defined above is higher than 6%
because some of the cadmium excreted within
10-15 days was absorbed into a body compart-
ment with a very short half-time (a few days),
as discussed below for mercury. The final
interpretation of the data by Rahola etal. must
await further data.
Lead. Gastrointestinal absorption of lead in
humans, measured by determining the dif-
ference between lead ingested and lead elimina-
ted in feces and urine, is between 5 and 15%.
These data, although carefully derived, were
only from a few normal adults and give no
information about variation related to sex,
age, and the large number of other factors
known to influence metal absorption (see
section 3.2.1) (Kehoe 1961). That such aspects
should be taken into consideration is shown by
an experiment made by Hursh & Suomela
(1967). They measured lead absorption in
three human volunteers exposed to 2l2Pb
and found a range of 1.3 to 16 %.
Experimental studies have shown that
absorption of lead is age dependent. Newborn,
rats have a higher absorption of lead when
compared to adult ruts (Kostial et al. I97la);
this may be related to the higher absorption
of calcium in young rats. Greater retention of
trace amounts of dietary -l2Pb occurred in
5- to 7-day-old rats fed cow's milk only than
in ruts fed cow's milk supplemented with
calcium and phosphate (Kostial et ul. I97lb).
Mercury. Inorganic mercuric '"-'Hg2* was
administered orally cither in the free or in the
54
-------
protein-bound form to ten volunteers of both
soxQR und body retention measured by whole-
body counting (Ruholu ct ul. 1971, 1973).
No difference in absorption between free and
protein-bound mercury was found. The whole-
body radioactivity measurements revealed
three elimination phases with varying half-
times. The first phase, dominating during the
first 1-2 days after dosing, had a biological
half-time of less than 2 days. The second phase,
dominating up to 6 days after dosing, had a
biological half-time of 2.5 days and constituted
13-38 °/0 of the dose. The remaining part of the
dose. Ij4-15.6% (mean 7.0), was eliminated
according to a biological half-time of about
42 days. The interpretation of these data with
regard to absorption is difficult. What is clear
is that the 7% (eliminated according to the
long biological half-time) had been absorbed.
Whether, in addition, a part of the amount
eliminated according to the 2.5 days half-time
had been absorbed cannot be established with
certainty. Part of this phase, as well as the
mercury eliminated according to the first phase,
undoubtedly represents mercury passing un-
absorbed through the gastrointestinal tract.
Elemental mercury introduced into the gas-
trointestinal tract is absorbed to a very limited
extent. Animal experiments (Bornmann et al.
1970) indicate that less than 0.01 % of the
administered dose is absorbed. An increased
blood level of mercury was found in persons
who had accidentally ingested large doses
(several grams) of metallic mercury (Suzuki &
Tanaka 1971).
Specific data on the absorption in human
beings of organic mercury compounds other
than methyl mercury are not available. For
the latter compound the absorption has been
shown to be about 95% of the administered
dose irrespective of whether the radioactive
methyl mercury compound was administered
as a salt in water solution (Abcrg ct al. 1969)
or in protein-bound form (Kaholn et al.
M97I).
IH.3.3. PLACENTAL TRANSFER
111.3.3.1. General aspect!
In principle, trunsplacental passage of metal*
must be distinguished from placenta! binding
as the latter may involve both maternal and
fetal tissues. In turn, species differences in
placental structure and^mplantation are likely
to influence both the binding and the passage
of toxic metals. The hemochorial placental
barrier of primates and rats is more easily
traversed than the more complex placental
barriers found in other species of experimental
animals. Placental binding and passage are
influenced by gestational age and the availa-
bility of the particular metal in maternal blood.
Although it is generally recognized that the
diffusable fraction of metals in plasma would
seem easily transported across the placenta,
one must remember that macromolecutes can
also cross the placental barrier.
Transplacental transfer gives an initial body
burden which will be augmented by later
environmental exposures. It is prudent to
consider fetuses as an especially susceptible
population group. If transplacental transfer
of a particular metal is high, then fetal ex-
posure may prove a limiting factor upon which
exposure standards must be based.
In the USA a systematic collection of cord
blood, maternal and fetal tissues has been
commenced through the CHESS (Community
Health and Environmental Surveillance
/
System) program utilizing standardized tissue
collection procedures (Riggan et al. 1972).
•
IU.3.3.2. Specific data
Cadmium. Laboratory studies involving
experimental animals have shown a limited
passage of cadmium across the placental
barrier (literature reviewed by Friberg et al.
1971). The variable penetration observed may
be explained by differences in experimental
species, challenge compound and dose, route
of administration, and gestational age.
55
-------
Four human studies have shown that cad-
mium is bound to and crosses the placenta.
Dawson et al. (1968, 1969). Piscator (1971)
and Finklea ct ul. (1972) found that cadmium
was bound to placenta! tissue. The passage of
cadmium over the placenta! membrane takes
place only toa limited degree, sinceHenkeet .il.
(1970) found that the liver and kidr.ey of tin
newborn contained much smaller amounts ul
cadmium than would be expected in the corres-
ponding organs of the mother. The concentra-
tion difference between the maternal and fetal
liver can be estimated to amount to a factor
exceeding 1,000.
Lead. Placenta! transfer of lead has been
shown (Baumann 1933, Kehoe et al. 1933,
Thompsett & Anderson 1935, Horiuchi et al.
1966, Holtzman 1966,Blanchard 1966,Barltrop
1969, Scanlen 1971, Harris & Holley 1972,
Haasetal. 1972, Finklea etal. 1972). In a com-
prehensive survey of human fetal tissues Barl-
trop (1969) found that transplacental passage
became detectable at 12-14 weeks of gestation
and that whole body lead increased from that
point until parturition. Fetal bone was found
to have the highest tissue lead level (80 x/g/g).
Lower lead levels were found in placenta, heart,
kidney, liver and blood.
Haas et al. (1972) reported on 294 sets of
maternal and fetal blood samples. Maternal
blood samples were 16.9 ± 8.6 j/g/100 ml and
fetal levels were 15.0 ± 7.9 //g/100 ml. Blood
levels in the newborn correlated reasonably
well (coefficient of correlation, r = .57) with
levels in their mothers. Recently four groups of
investigators in the United States (Finklea
et al. 1972) reported lead levels in maternal -
fetal sets of placenta, maternal blood and
cord blood. They also found that lead is bound
to and passes across the placenta. Fetal blood
levels in these studies were generally lower than
maternal levels, with maternal erythrocyte
levels exhibiting a greater excess than whole
blood. Significant regional differences may
have been associated with differences in the
mothers' environmental lead exposure before
the birth of the children.
Mercury. The placenta! barrier is more
effective in preventing the passage of mercuric
ion than of elemental mercury. Recently
Clarkson et al. (1972) found that after equal
exposures of pregnant rats to metallic mercury
vapor or mercuric ion, respectively, the fetal
uptake of elemental mercury was 10-40 times
higher than that of inorganic mercuric mercury,
whereas the placenta! content of mercury after
exposure to metallic mercury was only about
40 % of that after exposure to mercuric mercury.
Among 44 mothers who gave no history of
occupational exposure or high fish intake,
Finklea et al. (1972) reported that maternal
and cord blood levels were similar to each other,
both with a range of 2-20 ng/g.
Alky! mercury compounds penetrate the
placenta easily and intrauterine intoxications
have been reported from several nations
(Engleson & Herner 1952, Sweden; Harada
1968, Japan; Bakulina 1968, USSR; Snyder
1971, USA; Bakir et al. 1973, Iraq). In a study
of women with normal pregnancies and a low
or moderate fish intake, methyl mercury levels
in fetal blood cells were 30% higher than in
maternal erythrocytes (Tejning 1970). This
relative excess in fetal blood was confirmed in
another study (Suzuki et al. 1971). Mercury
levels in the blood cells from the umbilical
cord were 31 ng/g (S.D. 22) which were higher
than concentrations in maternal blood cells
(23 ng/g with a S.D. of 12) but plasma levels
were similar (II and 12 ng/g respectively).
Differences in hematocrit and in binding
characteristics between fetal and adult hemo-
globins may account for the higher blood
concentrations found in fetal blood.
IH.4. TRANSPORT AND DISTRIBUTION
HI.4.1. TRANSPORT AND BINDING IN BLOOD
Both plasma and blood cells can take part in.
the transport of metal compounds, but
generally cell-bound metals do not exchange
56
-------
with other tissues as easily as metals bound to
plasma constituents. Partition of a metal
between erythrocytes and plasma, which thus
may limit its availability for exchange with
tissues, varies extensively with different metals
and their organometallic compounds as well
as with the dose and time after dosing. Thus,
e.g. plasma-to-blood cell ratio in man varies
from 1.0 (i.e. equal concentration of metal in
erythrocytes as in plasma) for normal intake
of cadmium, copper, chromium and nickel
toonlyO.OI for lead (Vostal 1972a). An example
of a ratio range among different forms of a
metal is shown by mercury: 2.5 for inorganic
mercuric mercury after oral absorption
(Miettinen 1972), 1.0 for inorganic mercury
after industrial exposure to mercury vapor
(Lundgren et al. 1967) down to 0.1-0.2 for
methyl mercury in man (Birke et al. 1972).
For the same chemical form of metal the ratio
changes also in various animal species, e.g.
data for methyl mercury range from 0.002-
0.004 in rat and chicken to 0.03-0.05 in guinea
pig, dog and Rhesus monkey, and 0.1-0.2 in
swine, rabbit, mouse (Vostal I972b) and man
(Birke et al. 1972).
Many constituents of erythrocytes may
participate in metal binding. Metals may thus
be bound to hemoglobin and other specific
metalloproteins or to erythrocyte membrane.
A specific chemical form of a metal usually
displays a specific distribution pattern among
these constituents.
Although a role of erythrocyies for a direct
exchange of metals between circulating metal
in blood and between tissues cannot be ex-
cluded, plasma is generally considered as the
main mediating pathway for transport of
metals. Erythrocyies should therefore be
looked upon mainly as one of many compart-
ments in the body from which there is a con-
tinuous exchange of metals with plasma.
The binding of mclals 10 plasma proteins is
related to the occurrence of ligands and to the
magnitude of the stability constants for the
metal ligand bond. The stability of the metal-
protein bond varies with the type of protein
as well as the metal involved, and in general,
three different forms of existence of metal in
plasma can be assumed:
(I) Metal firmly bound to a specific plasma
metalloprotein demonstrated so far to
exist only for a few metals; the metal-
protein bond is so firm that the metal is
not removed by usual isolation and purifi-
cation procedures. Protein can be isolated
and the structure of the metal-protein
bond studied in detail. Release of the
metal from the bond in vivo can therefore
occur only in the presence of an excess of
other ligands with high affinity for metal,
by exchange with metal with higher affinity,
or when the protein is catabolized.
(2) Metals bound to various fractions of
plasma-proteins, e.g. albumin. Metal is
bound loosely to protein and dissociates
readily.
(3) Metal bound to low-molecular weight
plasma components; this fraction will
probably behave differently for individual
metals in regard to diffusibility or ultra-
filtrability, but is usually included in what
is called the "diffusible" fraction, con-
sisting of other fractions of metal bound
to various forms of diffusible organic
ligands as e.g. amino acids, etc. Theoreti-
cally, based on the mass action law, a
minute amount of metal should be in
ionized form to sustain the equilibrium
with "bound" fractions of metal.
The low-molecular weight or "diffusible"
fraction of metal in plasma is of fundamental
interest for transfer of metal lo and from various
organs and for excretory mechanisms. This
fraction has not yet been quantitatcd for many
metals, partly because of methodological
difficulties in separating the very small metal
concentrations at the steady state dominated
largely by the protein-bound fraction and
57
-------
partly because detection limits of analytical
methods are such that it is often impossible
to detect these low concentrations under
conditions of "normal exposure".
Small amounts of elemental mercury havei
been found to exist physically dissolved in
plasma for a short time after exposure to
metallic mercury vapor (Magos 1967).
III.4.2. DISTRIBUTION
• HI.4.2.1. General aspects
Different organs manifest striking variations
in the fraction of the total body burden of any
metal that each contains. Factors that deter-
mine the transfer of a metal from plasma to a
particular organ are complex and their quan-
titative aspects are not fully understood.
However, the following factors may be
considered to be of significance:
(I) Fraction of the metal in plasma that is in
the "diffusible" form and the rate of organ
perfusion (/'/; vivo).
(2) The permeability of cell membranes for
the metal in the forms that occur in plasma.
(3) The availability and turnover rate of suit-
able membrane and intracellular ligands
for the metal.
A simplified model for the exchange of metal
between the circulating plasma and organs,
shown in Fig. 16, considers the relationships
among five compartments: the "non-diffusible"
plasma protein-bound fraction, the plasma
"diffusible" fraction, the interstitial (extra-
cellular) "diffusible" fraction, the intracellular
"diffusible" fraction, and the inlracellular
"non-diffusible" protein-bound fraction.
As staled above, the plasma "diffusible"
fraction is thought to be of prime importance
in the transfer of metals between intravascular
extracellular and intracellular compartments.
The protein-bound fractions in plasma and in
the cells constitute depots which arc supposed
to equilibrate continuously with the more
"diffusible" fractions.
Interstitial (extra- .
cellular) fluid
"diffusible" fraction
Intncellulir
Protein- bound
fraction
fl
. Intracellular
"diffusible"
fraction
Figure 16.Model for the exchange of metal between
blood and other tissues.
Binding of a metal with an intravascular
or intracellular ligand disturbs the equilibrium
and creates a concentration gradient that
favors transfer of metal to that depot. Any
change in the availability of ligands in the
plasma or in the cells may similarly shift the
equilibration in either direction and result
in the movement of metal.
This simplified scheme for the general type
of exchange between plasma and tissue
certainly does not consider the specific prop-
erties of diversified cell systems within an
organ, as e.g. the reticulo-endothelial system
in the liver, or of organs with diversified
functions such as e.g. the kidney. In the kidney,
tubular cells may accumulate metals from
tubular fluid in addition to direct uptake from
plasma. It must be furthermore assumed
that many other factors such as e.g. catabolism
of plasma proteins, may also contribute to the
deposition of metals in some organs. The
distribution of the metal may be influenced
by the possibility that-metals can get a "free
ride" on intra- and extracellular carriers of
other metals. Organs such as the brain and the
tesles may be protected by "barriers" through
which some metals can be transferred only to a
limited extent.
The amount of metal that is accumulated
in the cells of an organ is obviously the
difference between the amount that has been
transferred into the cells of the organ and the
amount that has been transferred out. The
58
-------
same amount can accumulate in (he cells of an
organ when the rates of transfer in and out
are high as when the rates of transfer in and
out are low.
The level of exchange rate is, however, a
limiting factor in achieving the steady state.
Steady state may be delayed with low levels
of exchange rates for a long period of time.
This should be considered especially in cases
in which the levels in the organ are extrap-
olated from stationary levels in plasma,
although steady state might not yet have been
achieved.
III.4.2.2. Consideration on the penetration of
metals into the brain
UI.4.2.2.1. General aspects
Although it is evident from the foregoing
discussion that specific transfer systems for
metals may exist for several organs in the
body,- the brain has been chosen for special
consideration here; accumulation of metals in
the kidney will be discussed to some extent in
the section on renal excretion.
The central nervous system, usually con-
sidered as a special compartment separated
from blood by the so-called blood-brain
barrier, often shows a slow uptake of metals,
probably due to a low transfer rate through
this barrier when met by a metal compound
circulating in the intravascular compartment
of the brain.
Penetration rates depend on the form in
which the metal or organometallic compound
enters the brain. Thus e.g. it has been shown
experimentally that the uptake of mercuric ion
in the mouse brain is facilitated by complcxing
with dimercaptopropanol (Berlin & Lcwander
1965). In general, little is known about metal
transport and deposition in brain. On the
basis of limited data, the turnover of metal
deposits in brain appears to be slow for a
number of metals.
As there are differences in structure and
metabolism between adult and infant brain.
differences in metal uptake and turnover rate
can be postulated. Epidemiological data on
incidence of intoxication and signs of poisoning
in relation to exposure to methyl mercury give
some support to such a postulate.
III.4.2.2.2. Specific data
Cadmium. Cadmium does not easily pene-
trate into the brain (Berlin & Ullberg I963b,
Nordberg & Nishiyama 1972) and only traces
were found in the brain of mice after a single
intravenous injection of '°*Cd. Human data
are scarce, but it can be mentioned that in a
case of excessive exposure to cadmium where
the concentration of cadmium in liver was
94 ppm wet weight, the concentration in brain
was 0.6 ppm (Ishizaki et al. 1970).
Lead. Lead passes into the brain but does not
accumulate there to any great extent (Schroe-
der & Tipton 1968, Barry & Mossman 1970).
The local distribution of lead in the brain may
be of more importance than the mere passage
from blood to brain. The concentration of
lead has been found to be higher in cortical
grey matter and basal ganglia than it is in
cortical white matter in cases of lead intoxica-
tion (Klein et al. 1970). Horiuchi (1970)
found 2.1 x/g/100 g (wet weight) as the average
concentration in the cerebrospinal fluid of 6
normal Japanese adults. These quantities are
similar to those found in the cerebrum and
cerebellum and are consistent with brain lead
levels reported from various countries (Schroe-
der & Tipton 1968).
Nonpolar organolead compounds pene-
trate more rapidly into brain, as well as other
membranes, than do inorganic lead compounds.
This penetration will often be as the partially
metabolized material, i.e. triethyl lead com-
pounds following administration of tetraethyl
lead (Cremcr 1965). There arc some differences
in brain levels for different organolead com-
pounds (Schcpcrs 1964). Although the brain
is I he critical organ for organolead compounds,
the concentrations of lead in the brain do not
59
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highly exceed those in other tissues (Cussels
& Dodds 1946).
Mercury. Animal data published by Fribcrg
(1956) showed thai the hrain-to-hlood ratio
increased during live weeks of exposure to in-
organic mercury, and Berlin & Ullbcrg (I963a)
found the same phenomenon after a single
injection in mice. Similarly, Swcnsson &
Ulfvarson (1968) found that brain levels of
mercury were several times higher than those
in the blood 10 or 20 days after single injection
of mercuric chloride in poultry. Moreover,
after exposures to elemental mercury, the brain
reaches levels several fold higher llian after
similar doses of mercuric salts (Berlin ct al.
1966. Magos 1967, 1968, Nordbcrg & Serenius
1969). Mercury levels in brain, several times
higher than those in liver and oilier organs ex-
cept kidney, were reported in miners with
long-term exposures to high concentrations of
mercury vapor even several years after cessa-
tion of exposure (Takahata et al. 1970,
Watanabe 1971). Large dillercnccs among
various parts of the brain have been demon-
strated with regard to mercury concentration
after exposure to mercury vapor as well as after
injection of mercury salt (Nordbcrg & Serenius
1969, Berlin et al. I969H, Cassanoel al. I9(>9).
After administration of methyl mercury
compounds, mercury levels in the bruin are
higher than after administration of inorganic
mercury compounds or aryl mercury com-
pounds (Berlin 1963). Abcrg ct ul. (1969)
found approximately 10",, of total body
burden of pcrorally administered melhyl
mercury in the posterior region of the head
of human volunteers.
Data on the distribution of mercury in
various species of animals were recently
collected and considerable differences in
distribution ratios between blood ami brain
discovered (Bcrglundct al. 1971). It was shown
that the ratio between the levels in blood and
brain is approximately 10 20 in rais, 1.0 in
mice, 0.5 in dogs and suine and O.I 0.2 in
primates. Wide intcrspecies deferences in
plasma to red blood cell ratios (Vostal I972b)
may account for these variations in the blood-
lo-brain ratios since the plasma-to-brain
ratios are very similar in the various species.
The distribution of mercury among different
parts of the central nervous system after
administration of methyl mercury has been
studied in animal species. The data show
differences in mercury .concentrations among
different parts and structures of the brain
(Berlin 1963, Nordberget al. I97lb, review by
Berglundetal. J97I).
III.5. EXCRETION
Hl.5.1. GASTROINTESTINAL EXCRETION
///. 5.1.1. General aspects
The amount of metal found in feces derives
from the fraction of ingested material that
passes unabsorbed through the gastrointestinal
tract and the net gastrointestinal excretion.
Net gastrointestinal excretion consists of metal
which has been excreted into the lumen of the
gut from the gastrointestinal tract (total
gastrointestinal excretion) minus the amount
of metal reabsorbed. A scheme illustrating
the various routes is seen in Fig. 3.
The relative importance of gastrointestinal
excretion varies from, metal to metal and is,
furthermore, dependent on several factors,
such as the route of absorption (Dutkiewicz,
in Dukes & Friberg 1971, Nordberg &
Skcrfving 1972), type of exposure (acute or
prolonged), and a time dependent organ dis-
tribution.
Gastrointestinal excretion takes place by a
number of routes. The most important of these
are (I) active secretion or simple passive
loss of metal with the secretion from various
glands into the alimentary tract, i.e. salivary
glands, pancreas, and glands located in the
intestinal epithelium (Berlin, in Dukes &
Friberg 1971), (2) loss by the shedding of
epithelial cells, and (3) biliary excretion.
60
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Different parts of the gastrointestinal tract
have different excretory capacities depending
on (lie cellular structure of the intestinal wall.
Excretion, furthermore, depends on the con-
centration of the metal in plasma and the organs
excreting their contents into the gut. Present
data do not allow definite conclusions as to the
mechanism(s) of gastrointestinal secretion
of metals, i.e. whether mediated by specific
excretory mechanisms or transferred passively.
There are some data suggesting a specific
excretion process for metals in the cells of the
gastrointestinal mucosa. Berlin & UIIberg
(I963a, b) found high amounts of mercury
and cadmium in various parts of the gastro-
intestinal mucous lining, both within the cells
and outside them as a halo adjacent to the
luminul border. Although these data can be
explained by passive transfer, they could also
indicate a specific secretory process for metals
in those cells. Such a secretory function has
been postulated for the cells of Paneth
(Halbhuber et al. 1970), but their relative
importance for total gastrointestinal excretion
of metals is likely to be low.
The excretion by intestinal cell shed is often
of major importance for the total gastro-
intestinal excretion. The cells in the gastro-
intestinal mucosa have a high turnover. Since
part of their metal content is derived from
circulation (the rest being taken from the
intestinal content), significant amounts of
metal are removed by this mechanism. The
mechanism is particularly important when
plasma concentrations are high, and thus the
major source of metal for the cell will be
circulatory and not uptake from intestinal
contents. This excretion route appears to be
particularly important for methyl mercury
(Norseth & Clarkson 1971).
Elimination of lead and mercury has also
been related to gastrointestinal catabolism of
proteins (Witschi 1964, 1965). This mecha-
nism may, however, account for only a small
amount of the excretion because of the low
amount of plasma proteins normally catab-
olized in the intestinal tract (Kerr et al. 1967).
The part accounted for by this mechanism
would also have to pass through the gastro-
intestinal mucous lining and thus will be
included in one of the above-mentioned
mechanisms.
Biliary excretion may be the most important
pathway contributing to gastrointestinal ex-
cretion. Plasma fractions of several metals
bound to proteins have been shown to be
associated with excretion of the metal into the
bile (Holmbcrg 1961, Gaballah et al. 1965).
This is in accordance with the hypothesis that
only compounds with a molecular weight higher
than about 400 are preferentially excreted in
bile (Abdel Aziz et al. 1971). In accord with
this, metals and organometallic compounds in
the bile are bound to proteins or organic
ligands with relatively high molecular weight
(Norseth & Clarkson 1971, Cikrt 1972).
However, it has not been possible to assess
whether this binding is a true part of the
excretion mechanism or a result of a secondary
complexing to some substance in the bile.
The nature of the complexing agent has been
shown to be important for the degree of
rcabsorption (Norseth & Clarkson 1971) of
metal primarily excreted to the gut. In this way
the nature of the complex formed will be of
great importance for net gastrointestinal
excretion of metals. Reabsorption, presumably
in the form of such complexes, has been shown
to take place for methyl mercury and cadmium
(Norseth & Clarkson 1971, Caujolle et al.
1971).
Differences among species have been de-
scribed for the extent of biliary excretion in
relation to other routes of excretion of metals
(Vostal I972b). Caution should be exercised
when applying conclusions from animal data
to man, especially concerning the quantitative
importance of biliary excretion. Several
metals which have been demonstrated in the
bile of experimental animals may not necessarily
61
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be found 10 ihe same extent in human bile. The
role of biliary excretion and reabsorption in
gastrointestinal transport in man therefore
remains uncertain.
III. 5.1.2. Specific data
No human data on total gastrointestinal
excretion exist. Studies have been made only
on net gastrointestinal excretion or content
in feces of the metal in question.
Cadmium. Raholaet al.(!97l (studied 5 male
volunteer subjects (ages 19-50) who had
ingested "SmCd mixed with calf kidney pro-
teins (there are data from this study in section
3.2.2). They estimated that the net gastro-
intestinal excretion of the amount retained in
the body was less than 0.1 % per day.
By animal experiments cadmium has been
demonstrated in the intestinal mucosal lining
and in pancreas, indicating a role of these
organs in the slow elimination of this metal
(Berlin & Ullberg 1963b, Nordberg & Nishi-
yama 1972).
Biliary excretion of cadmium is found in
the rat (Caujolle et al. 1971) and cadmium has
also been found in the bile of human autopsy
cases (Smith etal. 1955, Tsuchiyaetal. 1972b).
Lead. Available evidence suggests that
gastrointestinal excretion of lead may not be
as quantitatively important as thoseof cadmium
and mercury (Clarkson in Dukes & Friberg
1971). A review of balance studies by Kehoe
et al. (1943) suggests that clearance of un-
absorbed lead from the gastrointestinal tract
accounts for most of the fecal excretion of lead.
Hursh & Suomela (l"967) measured gastro-
intestinal excretion of lead in two human
volunteers following a single intravenous
administratioh of 2l2Pb. Less than 0.5% was
found in feccs during the first 48 hours after
the injection.
Excretion of lead in bile was postulated
many years ago by Canlarow & Trumper
(1944) but supporting data arc scarce. Castcll-
ino et al. (1966) recovered 8.4% of intra-
venously injected 2lnPb in (he bile of rats in
50 hours, and Cikrl (1972) reported 6.7% in
24 hours. In a similar experiment using sheep,
Blaxter & Cowie (1946) recovered about 6.0%
of an intravenously injected dose of lead salt
in bile over a 6-day period. More recently
Gage & Litchfield (1968) found in rats that
slight increases in dietary lead(2; 6and20ppm)
were not associated with detectable increase
of urinary lead, but as little as 6 ppm increased
the bile content of lead.
Mercury. After a single perc-Ml dose of
mercuric mercury was given to human volun-
teers, less than 1 % of the dose per day was
recorded as net gastrointestinal excretion after
the initial passage of unabsorbed material
(Rahola et al. 1971, in press).
Animal experiments indicate that excretion
of inorganic mercury by the gastrointestinal
tract is dependent on dose and time of the
exposure. The fecal route of excretion domi-
nates soon after exposure. This excretion route
is also favored when high doses are given.
(Prickettetal. 1950, Friberg 1956, Rothstein &
Hayes I960, Ulfvarson 1962, Cember 1962,
Phillips & Cember 1969, review by Nordberg
& Skerfving 1972). After such inhalation ex-
posure, the oxidation of metallic mercury to
mercuric mercury is fast enough to prevent
the diffusion of appreciable quantities of
mercury into the gastrointestinal tract. Ac-
cordingly, such diffusion will be of no con-
sequence for total excretion.
In human beings, methyl mercury is mainly
excreted in feces and the proportion does not
vary with the dose levels or exposure time,
90% of the total excretion taking place in
feces after a single dose (Aberg et al. 1969,
Rahola et al. 1971). Mercury was also found in
the mucosal lining of mice after methyl mercury
exposure (Berlin & Ullberg I963a) and in-
testinal cell shed was suggested to be a major
source of fecal metal in rats (Norseth &
Clarkson 1971).
62
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No experimental data on gastrointestinal
excretion from humans exist for other organic
mercury compounds. Animal experiments
indicate that gastrointestinal excretion varies
with the exposure pattern and levels of ex-
posure (review by Nordberg & Skerfving
1972).
Biliary excretion of both inorganic and
organic mercury compounds has been demon-
strated in the rat (Norseth & Clarkson 1971,
Cikrt 1972). The significance of this excretion
for net gastrointestinal excretion is uncertain
because of a possible reabsorption (Norseth &
Clarkson 1971). Furthermore, conclusions
with regard to other species are impossible
because of interspecies differences in biliary
excretion of both inorganic and organic forms
of mercury (Vostal 1972b).
III.5.2. RENAL EXCRETION
III.5.2.1. General aspects
The highly complex physicochemical state
of metals in blood makes it difficult to use
classical physiological methods for exploring
how the metals are handled by the kidney.
Routine methods for the calculation of renal
clearance may be used only for the portion of
metal which is present in plasma in "diffusible"
form and can pass through the glomcrular
membrane or tubular cells.
The glomerular ultrafiltraic contains metals
bound to compounds of varying molecular
sizes and properties, from low molecular
weight proteins to simple anions. Glomerular
capillaries behave as though they were per-
forated by pores of a size just adequate to
permit the passage even of plasma albumin
molecules (Pappenheimer 1955). However, it
is known that only a minute fraction of plasma
albumin passes the membrane. The combined
effects of steric and electric hindrance and of
viscous drag (Wallenius 1954) restrict filtration
rates of the major amounts of plusma proteins.
The proportion of such proteins with large
molecular weight that passes the glomerular
membrane is only a small fraction of the
filtration of plasma water. Substances of
relatively low molecular weight, e.g. purified
tuberculin protein (mol. wt. 14,500) or inulin
(mol. wt. 5,000) are excreted into the tubular
lumen more readily (Bott & Richards 1941).
Metals bound to low molecular weight pro-
teins may consequently be cleared from the
plasma by glomerular filtration mechanisms.
The plasma filtrable fraction and the renal
clearance have been determined for copper
(Walshe 1961), nickel (Hendel & Sunderman
1972), radium (Hursh et al. 1960), zinc
(Prasad 1966) and chromium (Collins et al.
1961, review of all metals: Vostal I972a).
Values for their renal clearance were lower
than the glomerular filtration rate and in-
dicated that tubular reabsorption was in-
volved in the renal excretory mechanisms of
all studied metals. Analytical inaccessibility
of plasma filtrable fractions of lead and
mercury prevented similar direct analysis of
renal excretion of these metals. Calculations
by indirect methods (Vostal 1972a) suggest
that tubular mechanisms may also participate
in the renal excretion of these metals.
The plasma filtrable fraction may be in-
fluenced by the presence of increased con-
cent rations of some normal plasma components
with the ability to bind metal ions. As examples
may be mentioned that sodium bicarbonate
increases the filtrable fraction and excretion
of uranium (Neuman 1949). Cysteine en-
hances the passage of mercury and organo-
mercurials into the tubular urine (Clarkson
& Vostal 1972), and histidine and other amino
acids may influence the amount of copper and
nickel in plasma ultrafiltrate (Sarkar &
Kruck 1966, von Soestbergen & Sunderman
1972).
Since many of these compounds have their
own cellular transport system involving
tubular secretion or reabsorption, their par-
ticipation in renal handling of metals cannot
63
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be excluded. Metal ions can gel a "free ride"
and follow Ihe renal pathways of the organic
moiety. This obviously would modify the
mechanism of renal handling and clearance
of metals, as discussed for high exposures to
lead (Vostal 1963).
Changes in urinary pH are followed by
acute effects on the urinary excretion of
.uranium (Neuman 1949) and lead (Webster
1941, Vostal 1966), which suggests the existence
of tubular.reabsorption of ihese metals.
Any reabsorptive process for a metal during
its passage through the renal tubule results in
its entrance into renal tubular cells and may
lead to accumulation of the metal in renal
tissue if the metal is not transported further
into the peritubular capillary blood. It is not
known in any detail to what extent the exis-
tence and effectiveness of these tubular re-
absorptive processes may depend on factors
such as changes in urine pH, metal charge,
competition with other compounds, etc.
That fraction of metal which has not been
reabsorbed is excreted and usually represents
a major part of the urinary excretion of metal.
In addition, shedding of tubular cells con-
taining the metal may contribute to excretion.
Thus, specific avidity of the renal tissue for a
metal may introduce a variable factor in the
process of renal excretion of metals. The
possibility exists that metals may be added to or
extracted from the urine during their further
passage along the urinary tract, but little is
known about such a process.
The mechanisms for accumulation of metals
in the renal tissue are largely unknown. Some
metals are present in the kidney tubules in the
form of specific tissue-metalloproleins (e.g.
metallothionein) with binding ability for
cadmium, zinc, copper and mercury (Ka'gi &
Vallee I960, 1961, reviews by Friberg el al.
1971, Piotrowski et al. 1972). A lead protein
complex has also been identified in the intra-
nuclear inclusion bodies in renal tubular
lining cells (Richter ct al. 1968, Coyer et al.
1970, Goyer 1972). These proteins mainly
constitute a storage pool for metals; when thr
capacity of available mtraccllular metalio-
proteins is exceeded, redistribution of the
metal may occur.
III.5.2.2. Specific data
Cadmium. The low levels of cadmium in
plasma (review by Friberg et al. 1971) and its
binding mainly to proteins with a molecular
size similar to or larger than albumin (Nord-
berg et al. 197la) make it difficult to study the
renal excretory mechanisms for cadmium,
especially under conditions of low or moderate
exposure. Piscator (1964) and Friberg et al.
(1971) introduced a model for renal accumula-
tion and excretion of cadmium implying a
role of metallothionein. The participation of
metallothionein-bound cadmium in urinary
excretion has been supported by animal experi-
ments with exposures sufficient to induce
proteinuria (Nordberg & Piscator 1972).
Lead. The analytical inaccessibility of the
small plasma concentrations is the reason for
the lack of knowledge on the quantitative
aspects of the mechanisms responsible for
urinary lead excretion under conditions of
"normal" exposure. By an indirect method,
Vostal (1963) postulated the existence of
tubular reabsorption mechanisms for lead in
acutely exposed subjects with high blood
levels of lead (more than 100 //g/100 ml),
whereas the role of these mechanisms in
persons and animals with lower levels in the
blood was considered- insignificant. Further
evidence for the participation of tubular
reabsorption has been discussed by Vostal
(1972a).
Mercury. Recent reviews on the renal ex-
cretory mechanisms of mercury concluded that
despite the presence of a minute diffusible
fraction in plasma, glomerular mechanisms
do not completely explain urinary excretion of
mercury and its compounds (Vostal 1969,
Nordberg & Skerfving 1972, Clarkson 1972).
64
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Experiments in rats (Clurkson & Magos 1970)
indicate a peritubular transfer of mercuric ion
from blood into renal tubules. Transport via
the tubular wail into urine has been shown in
avian kidney (Vostal & Heller 1968). This
excretion route might also be important in
humans (Vostal I972a). There might be an
exposure-dependent change in the mechanism
for mercury excretion, judging from variations
in the proportion of different forms of mercury
in workers' urine (Henderson et al. 1972).
IH.5.2.3. Renal excretion in relation to functional
impairment of organs
As the renal handling of metals is dependent
on glomerular function, tubular function and
the state of the renal tissue, any change in one
of these functions may cause changes in
distribution or excretion of metals. Generally,
any renal disease with high proteinuria may
lead to increased excretion of metals bound to
high molecular weight proteins and eventually
lead to depletion of body stores of metals.
Renal disease with decreased filtration may
lead to higher blood levels of certain metals
bound to low molecular weight protein or
other low molecular weight compounds.
Congenital or acquired tubular dysfunction
will cause a decreased reabsorption and in-
creased excretion of those metals ordinarily
reabsorbed at different sites in the tubules.
Furthermore, there may be an excretion of
metal through the exfoliation of tubular cells.
A change in excretion of metals may also be
due to metabolic changes, as shown for mer-
cury (Clarkson & Magos 1967, Magos &
Stoytchev 1969). Similarly, changes in the
acid-base balance are likely to change ex-
cretion and reabsorption rates. 11 is known that
alcohol will cause an increased excretion of
some essential metals, possibly due to liver
impairment (review by Piscalor I972b).
There is reason to believe (hat accumulation
of some of the metals in the kidney can give
rise to changes in renal function and thus in
the renal excretion of the metal itself. Only in
the case of cadmium, however, has this phenom-
enon - been investigated in sufficient depth.
Since normally the amounts of cadmium
excreted in urine are ver>jlow, there is probably
a very efficient tubular reabsorption of the
cadmium-containing protein (Friberg et al.
1971). Animal! experiments (Friberg 1952,
Axelsson & Piscator 1966, Nordberg &
Piscator 1972)'have shown that when renal
damage occurs,';:there is a sudden increase in
cadmium excretion simultaneously with the
appearance of proteinuria. Results from in-
vestigations on, workers exposed to cadmium
indicate similar effects in man (Piscator
1972a). The changes in the turnover of cad-
mium may lead to changes in distribution so
that in advanced renal damage, urinary losses
of cadmium will, increase and eventually the
•£
renal concentration of cadmium will approach
the "normal" range (review by Friberg et al.
1971). «i-"
Hemolysis may increase the amount of
ftltrable plasma cadmium as indicated by
experiments on'irabbits (Piscator 1963). Other
extra-renal factors that could be of importance
for the excretion of cadmium are infection or
trauma. It was found by Bonnell et al. (19S9)
that cadmium excretion was increased in
workers with pneumonia or pneumothorax.
Possible mechanisms responsible for the
mobilization of cadmium probably involve
sudden release of accumulated metal in the
lung tissue.
#
III.5.3. MAMMARY GLAND EXCRETION
HI.53.1. General aspects
Exposure of the newborn through milk will
constitute a hazard, especially in cases in
which the initial.body burden is already raised
due to transplucental transfer. This hazard
sometimes exists even when the initial body
burden is negligible (Bakir et al. 1973). The
processes through which the metals under
65
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consideration are excreted through the mam-
mary glands are not known. The apocrinic
type of secretion typical of mammary glands
will, however, provide a possibility forjtransfer
to the milk of ull metals with a tendency to
accumulate in mammary gland cells. \:
III.5.3.2. Specific data ; T
Cadmium. Berlin & Ullberg (I963b), de-
tected cadmium in the mammary glands of •
mice injected with lo<>Cd. Miller et a^ ((1967)
exposed cows to cadmium by daily oral:doses
of 3 g for 2 weeks. The cadmium concentration
in milk never exceeded O.I ppm, and it was
calculated that less than 0.02 % of the dose was
excreted via milk. Lucis et al. (1971) gave
nursing rats injections of l09Cd and found that
cadmium was retained in mammary /tissue
and that low quantities were found in milk.
Lucis et al. (1972) studied the soluble proteins
from the mammary gland of cadmium-exposed
pregnant rats and found that cadmium was
bound to high molecular weight proteins.
Murthy & Rhea (1971) found that'human
milk in the USA contained on the Average
0.019 ppm of cadmium, which is higher than
the range of 0.001-0.010 found in cow's; milk
in different countries (Friberg et al. 1971;).
The animal data indicate that although
cadmium is taken up in the mammary gland
tissue, it is excreted only in small amounts via
milk. There is a lack of human data. • • • >.
Lead. Excretion via milk seems to be of
importance, and as excretion will increase
when blood levels increase, exposure via milk
may be of importance in children of lead-
exposed women, especially if there has also
been prenatal exposure.
Hammond &. Aronson (1964) found that
the lead concentration in milk from normal
cows was 0.009 ppm on the average, whereas
in cows exposed to lead, concentrations of
0.05-0.27 ppm were found. The lead con-
centrations in. milk were correlated to. Icud
concentrations in blood. A relationship hc-
7
tween lead concentrations in blood and milk
of cows can also be found from the data of
Donovan et al. (1969).
Lead poisoning can be produced in suckling
rats or mice by exposing (he mothers to lead
(Pcntschew & Garro 1966, Roseoblum &
Johnson 1968).
The average lead concentration in human
milk was found to be 0.012 ppm in the USA
(Murthy & Rhea 1971) and from trace to
0.12 ppm in three samples from Japan
(Horiuchi 1970). There are data that indicate
that in industrialized areas human milk may
have higher concentrations than cow's milk
or formula milk (Noirfalise et al. 1967).
Mercury. After injection of radioactive
mercury chloride in mice, mercury was found
in the mammary glands (Berlin & Ullberg
I963a). In lactating guinea pigs given a single
intraperitoneal injection of methyl mercury,
the mercury levels in milk were generally below
2% of the whole blood levels (Trenholm et al.
1971). Investigations on lactating women who
had been exposed to methyl mercury in fish
(Skerfving & Westoo 1972) or in bread
(Bakir et al. 1973) revealed that the total
mercury concentrations in milk averaged
about 5% of the total mercury concentrations
in blood (Skerfving & Westoo 1972) or the
organic mercury concentrations in milk
averaged about 3% of the mean organic
mercury concentrations in blood (Bakir
etal. 1973).
III.6. ACCUMULATION AND
RETENTION IN CRITICAL ORGANS
IH.6.1. GENERAL ASPECTS
///. 6.1.1. Uptake and retention
The critical organ accumulates metal when
uptake exceeds elimination. A steady state
is reached when uptake and elimination are
equal. Provided that the metal movement is
determined by concentration gradients, a
slow uptake will result in a longer time for
66
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reaching steady state. With a low elimination
rate the metal will be retained for a long time
even if uptake is stopped.
IIL6.1.2. Biological half-time
A common way to express the elimination rate
is in terms of biological half-time, which is a
measurement of the elimination rate when an
elimination phase succeeds the steady-state
condition. '-'Biological half-time" is only
meaningful when the elimination can be
approximated with reasonable accuracy to a
single exponential first order function. This
is fulfilled when the metal movement from the
organ is mainly dependent on concentration
gradient. The concentration in the organ at
any time then can be expressed by the following
equation:
C = C0 x e ~
(I)
C = concentration in the organ at time I.
C0 •= concentration in the organ at time 0.
b = elimination constant
/ = time
The relation between the elimination constant
and the biological half-time is as follows:
tration, in addition to the exposure, is the
rate of elimination of the metal from the body
and in particular from the critical organ. A
low elimination rate will cause accumulation
of metal in the critical organ even at low ex-
posure, whereas at rapid elimination rate the
exposure level will be the stronger factor.
To predict the concentration in the critical
organ at constant exposure and at a certain
time requires a mathematical model based on
intake level and elimination rate. A simple
model based on the assumptions that {I)
metal movement is linearly dependent on
concentration gradient, (2) the rate of uptake
of the metal is faster than the elimination rate,
and (3) a constant fraction of the intake is
taken up by the organ—gives the following
expression for the concentration in the critical
organ:
(3)
A = accumulated amount
a = fraction of daily intake taken up by the
organ
b ----- elimination constant
I —• time of exposure
At steady state:
T
In 2
(2)
T - biological half-time
In 2 - logarithm for 2 0.693
The exponential function is not always
representative of the elimination of toxic
metals; therefore the equations should be used
with caution.
III.6.1.3. Concentration in critical organ at
continuous exposure
Of importance for toxicological evaluation is
the highest concentration reached in the
critical organ at continuous exposure. The
dominant factor determining that concen-
This simple model has been extensively used
by ICRP for calculation of accumulation of
rudionuclides. These calculations have con-
stituted a major step fofward for predicting
risks at exposure.
Recently the simple model has also found use
for calculations of risks of chemical toxicity.
An example of a metal compound for which the
model is applicable is methyl mercury. However
as staled above, this model is based on the
assumption that intake and absorption are
constant. If either one of these changes sys-
tematically by age, period in time, etc., the
equation must be adapted, as attempted by
67
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Tsuchiya & Sugila (1971). Kjcllstrdm et al.
(1971), and Kjellstiom & Friberg (1972).
For most metals, the assumption (hat all of
their movement in the body is dependent
only on concentration gradient is an over-
simplification. Active transport or metabolic
processes are often involved in metal turnovers.
Consequently it has been recognized and
also demonstrated experimentally that gener-
ajly this simple model does not give a sufficient-
ly accurate description of the concentration
in the critical organ.
The application of the model requires
knowledge about intake (a) and the excretion
constant (b) or biological half-time In 2/b. For
metals with long biological half-times, the
concentration in critical organs (allowing
calculation of the amount (A) accumulated
in the organ) has been determined at autopsy
in a cross-sectional sample of human beings
and the intake (a) during lifetime for each case
has been calculated. Though this method is
often the only way to obtain human data, it
is subject to difficulties and sources of error.
The fundamental prerequisites (outlined above
in connection with equation (3)) for such
calculations must be fulfilled. Such is not the
case: (I) if there is time-dependent variation
in absorption and distribution or in exposure
(discussed above and by Kjellstrom & Fri-
berg( 1972)); (2) if the biological half-time in the
population is not constant. There is reason to
believe that the biological half-times of par-
ticular metals in the body may vary depending
on dose, sex, age and physiological character-
istics of a person. One obvious difficulty when
using autopsy material is that the cause of
death may influence the concentration in the
critical organ at autopsy.
In cases of biological half-times lusting over
decades, changes in exposure resulting from
changes in the general environment (due to
altered technology), in food consumption or
smoking habits of the population may be of
great importance. If such changes in exposure
cannot be ruled out or corrected for, it will
not be possible to calculate biological half-
times (Kjellstrom & Friberg 1972).
A possible way to get information concern-
ing the importance of past changes in exposure,
and to some extent other influencing factors
as well, is by periodic replication of autopsy
studies which measure metal residues in
critical organs. Such studies will allow an
empirical description of how metal levels
change over time in each cohort of the popu-
lation. That is, each cross-sectional replicate
will add another point to all but the oldest
cohort curve. One can then compare the
resulting cohort curves and make inferences
which could be helpful in constructing models
to assess biological half-time and in estimating
secular trends in human exposure.
III.6.2. SPECIFIC DATA
In spite of the difficulties in adequately
describing the accumulation in critical organs
of some metals, an attempt will here be made
to provide information on the use of models
for cadmium, lead and mercury accumulation
in tissues and numerical values for fractions
to critical organs and biological half-times
(data on absorption will be found in section
3.2.2).
Cadmium. A simple model implying that a
constant fraction of absorbed cadmium is
directly transferred to the kidney may not be
adequate to define renal accumulation of cad-
mium (Nordberg I972c) since a part of the
transfer probably occurs slowly via the liver.
However, at present no other models have been
used.
, Using the simple model, biological half-times
for the kidney between 17.6 years (Tsuchiya
& Sugila 1971) and 33 years (Kjellstrom et al.
1971) have been estimated using available
information on the accumulation of cadmium
in cross-sectional samples of Japanese. US and
Swedish populations.
68
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The fraction of whole body cadmium that
will be deposited in the kidney varies depending
on the time after administration (according to
animal data compiled by Friberg et al. (1971).
Autopsy material from u normal population
at age 50 (Friberg et al. 1971) indicates that i
of the body burden is located in the kidneys.
However, the fraction of the body burden that .
is found in (he kidney is smaller than j at
higher exposure (industrial) (Friberg et al.
!97l,Nordbtrg I972c).
Using model (3) and correcting it for sys-
tematic changes in caloric intake and kidney
weight during lifetime, it has been calculated
(Kjellslrom et ai. 1971, Kjellstrom & Friberg
1972) that the cadmium concentration in
kidney cortex at 50 years of age would be 50
ppm following a daily intake of 24 ng/calorle
(65 //g/day for an adult) assuming about 1.7%
(i of 5%) deposition of body intake in the
kidney.
Lead. The accumulation of lead has been
evaluated by analysis of human tissues ob-
tained at autopsy (Nusbaum et al. 1965,
Schroeder & Tipton 1968, Barry & Mossman
1970, Horiuchi 1970). The studies agree as to
the general increase of lead in bone and aorta
with age. However, the lead in bone was found
to (I) increase linearly throughout life (2) reach
a plateau after the third decade or reach a
plateau from the fourth through the seventh
decades but decrease thereafter. A complete
analysis of the statistical significance of these
various trends has not been performed and a
definite interpretation at the moment is
impossible.
All of these autopsy studies lack information
about previous exposure and excretion rates.
It is not clear if the scatter of the data repre-
sents mostly biological or environmental
variation. These data, therefore, cannot be
used to confirm theoretical models of reten-
tion of lead.
Indirect estimates of retention of lead have
been made by balance studies in human sub-
jects (Kehoc 1961, Horiuchi 1970). Although
these studies have been made on relatively
few individuals, they have been conducted
for prolonged periods of time. They are limited
to healthy adults receiving different doses of
lead during their adult lives. These studies
provide the best estimates of total body re-
tention for specific levels of intake as well as
the rate of elimination from the body after the
administration of lead was terminated. Only
limited attempts have been made to fit the
data from these studies to a mathematical
model (Sterling et al. 1964).
Knclson et al. (1972) studied human beings
continuously exposed to a mean level of
atmospheric lead oxide of 10.9 //g/m3 or 3.5
tig/m1 for a period of 4 months. Data derived
from their studies, namely blood and urine
lead, were used to develop a two-compartment
mathematical model describing lead uptake
and excretion kinetics for humans exposed to
atmospheric lead.
The major problem with establishing a
mathematical model for lead absorption or
lead retention is the strong affinity of lead
for bone. The estimates of half-times for lead
in bone have varied from 64 days in the spine
of rats (Torvik et al. 1972) to 7,500 days for
the skeleton of a dog (Fisher 1969). The half-
time varies with different bones, presumably
due to the relative proportion of cortical to
trabecular bone. The 1CRP has used 10 years
as the biological half-tiir.e for lead in bones of
humans (1CRP publication 2, 1959).
«
Prcrovska & Teisinger (1970) have also indi-
cated that there are at least two compartments
for lead in bone. Hammond & Aronson (1967)
also proposed an additional compartment con-
sisting of lead firmly bound to soft tissue. This
compartment is consistent with the observa-
tions by Goyer (1972) that lead binds firmly
with nuclear proteins in ruts.
Studies in animals by Bolanowska et al.
(1967) and Bolanowska & Piotrowski (1968,
1969.), have provided mathematical models
69
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for excretion of lead based upon an exponential
model with a four-compartment system:
(I) a rapid-exchange compartment (blood
and internal organs),
(2) the skin and muscles,
(3) the exchangeable part of lead in bones,
and
(4) a very slowly exchangeable part of lead
in bone.
The retention of lead was treated as a power
function model rather than an exponential
model:
R, -=A-t—
where R, = retention of lead at a specified
time(/)asa fraction of the given
dose
A = value of the retention at t = I
// = coefficient characterizing the rate
of loss from the body
Piotrowski (1971) summarized these animal
studies by affirming that lead requires a
particularly complex metabolic model, which
has not yet been satisfactorily solved.
Mercury. Accumulation of mercury after
the administration of inorganic mercuric
mercury and alkyl mercury compounds follows
different patterns. The rapid conversion of
phenyl mercury (Gage 1964) and methoxyethyl
mercury (Daniel et al. 1971) results in a dis-
tribution pattern which, after a preliminary
period, resembles the distribution of inorganic
mercury (Gage 1964, Ellis & Fang 1967).
Inorganic mercury—Mercuric mercury. A
multicompartment model for mercury accu-
mulation in the kidney has been used for
calculations (Nordberg & Skerfving 1972) in
animals. With all probability a similar lype of
model would be necessary if precise calcu-
lations of kidney accumulation in human
beings were to be made.
The biological half-lime of mercury in
whole body of humans administered inorganic
mercuric mercury orally has been found to be
29-41 days for 5 women and 32-60 days for
5 men (Rahola et al. 1971, Miettinen 1972).
About 7% of the total administered dose was
excreted according to this half-time (Rahola
et al. 1971, 1973). For further data from this
experiment, see section 3.2.2. Studies with
experimental animals (reviewed by Nordberg
& Skerfving 1972) indicate that the half-time
in the kidney is somewhat longer than that
in the whole body. For humans, almost no
data are available on the fraction of the ab-
sorbed dose transferred to the kidney. Limited
data from acute poisoning cases (Sollman &
Schreiber 1936, Lomholt 1928) suggest that
10-20% of the body burden of mercury was
in the kidney. However, since many of these
patients died from mercury-induced kidney
disease, it is likely that normal individuals
would have a considerably higher proportion
of the body load in the kidney. This assumption
is supported by studies by Magos (1972a)
in rats. When given non-toxic doses of mercury,
animals with kidney damage had a smaller
proportion of the body burden in the kidney
than animals without such damage.
Mercury vapor. Quantitative data are not
available for use in a model for mercury
accumulation in the human brain after vapor
exposure. The biological half-time in brain
has been shown in animal experiments to be
longer than in other organs (Berlin 1963,
Berlin et al. 1966, Magos 1967, Berlin et al.
I969b). This is especially true for certain
brain structures (Berlin 1963, Nordberg &
Serenius 1969, Berlin et al. I969b, Cassano
et al. 1969, review by Nordberg & Skerfving
1972). Data from Takahata et al. (1970) and
Wutanabe (1971) indicate a long biological
half-time, al least some years, for mercury in
the brain of human beings after vapor ex-
posure.
Methyl mercury. The data on the metabolism
of methyl mercury are adequate for calcula-
tions of accumulation. Data from studies in
70
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animals and man (Bcrglund el al. 1971) justify
the assumption that the distribution and turn-
over of mercury in different tissues of the body
are faster Ifyin the elimination from the body
as a whole. This means that a relationship
exists between the concentration in each organ
and the total body burden of mercury, and
that a definite relationship exists among the
levels of mercury in the different organs. It
can also be assumed that elimination is corre-
lated with the total body burden, i.e. that a
definite fraction of this is eliminated per unit
of time. If this is the case, then the course of
elimination in continuous exposure can be
expressed according toequat ion (3). Reasonably
accurate calculations should be possible for
the brain content at non-toxic levels.
Data on the biological half-time in brain
(Aberg et al. 1969) do not support a definitely
different half-time there compared to that in
the body as a whole. The latter has been
estimated to about 70 days on the basis of
both experimental and epidemiological data
on human beings (Berglund et al. 1971).
In studies by Aberg et al. (1969) the body
burden in the head was measured at 10% of
the total body burden. Since it is known from
studies in primates other than human beings
(Nordberg et al. 1971 b) that the brain con-
centration substantially exceeds that in other
tissues of the head, it can be assumed that the
amount present in brain is about 90% of the
total head burden.
Using the formula (3) and the above in-
formation, calculations of mercury concen-
tration at steady slate in brain have been made
which show a concentration of 5 //g Hg/g brain
tissue at a daily absorption of 4 //g Hg/kg
body weight as methyl mercury (Berglund el al.
1971).
Other organic mercury compounds have
nol been investigated as extensively as methyl
mercury. It is not possible at present to stale
any numerical values for the parameters
necessary for calculations of accumulation.
HI.7. METAL CONCENTRATIONS DM
BIOLOGICAL MATERIAL AS INDICES
OF EXPOSURE AND"CONCENTRATION
IN CRITICAL ORGANS
III.7.1. GENERAL ASPECTS
. ' v- ^
In assessing the \risks connected with the
accumulation of metals, .especially those with
long biological half-times, it is often desirable
to use an index of accumulation, such as
blood, urine or hair concentrations of the
metal. When steady state is reached, there is a
constant ratio between blood and tissue
w
concentrations of the metal and, except for
metabolic variations, there is also a relation-
ship to the urinary concentration of the metal.
However, for a biological material to be a
good indicator of accumulation, especially
for substances with long biological half-times,
it is not enough that the relation should be
constant at steady-state conditions it should
also be reasonably constant during the period
of accumulation. BJood, which transports
most absorbed metals', is not necessarily a
good indicator of accumulation in the critical
organ. In the following subsections, data on
the usefulness of blo'od, urine and hair values
as indices of exposure or accumulation will be
considered. Saliva,'teeth, nails, meconium,
placenta and biopsy material may also be
useful material for studies of the body burden
of metals.
HI.7.2. SPECIFIC DATA
Cadmium. The usefulness of various bio-
logical materials as indices of exposure and
retention has recently been discussed by
Friberget al. (I97l)and by Nordberg(I972a).
Available evidence from animal experiments
shows that the low levels of cadmium in urine
during continuous-exposure increase sharply
with the advent of renal tubular impairment.
In view of the analytical problems imposed
by these low urinary cadmium levels in animals
und man at the early phase prior to tubular
impairment, difficulties have been met in
71
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using this us un index of body accumulation.
However. Nordberg (I972a, b), by means of
radioactive isotope' experiments in animals.
found a correlation on a group basis between
body burden and urinary concentration
measured before tubular protcinuria appears.
On an individual basis, there was a wide
scattering of data. Piscator (I972a), in a study
of cadmium workers, could not find a clear (
relationship between cadmium concentration
in urine of workers (without proteinuria) and
duration of exposure (assumed to be an index
of kidney accumulation). In recent studies
using "'mCd on human beings, Miettincn
(1972), and Raholaet al. (1973) found that the
cadmium concentration in urine was corre-
lated to body burden during two months
followingadministration. Inanepidemiological
study, Tsuchiya et al. (I972a, b) estimated the
biological half-time for the body compartment
excreting into the urine at 13 years, not widely
different from the estimated biological half-
time in the liver (7 years) and kidney (17 years)
in the same study. It can be concluded that
urinary cadmium, excretion in certain exposure
situations might be useful as an indicator of
accumulation of cadmium in the body pro-
vided that proteinuria is not present. However,
further evidence is needed, especially con-
cerning its relation to cadmium concentration
in the critical organ.
Conclusive evidence on the relation of
blood values of cadmium to the concentration
in the kidneys has not been presented. Available
evidence from animal experiments and human
data has been compiled and discussed by
Fribergetal.(l97l)andbyNordberg(l972a,b).
It was concluded that blood values probably
do not reflect kidney accumulation of cadmium
but may reflect the most recent exposure. This
is also supported by data from studies in
human beings reported by Piscalor (I972a).
He measured blood concentrations of cad-
mium in 20 cadmium workers who had been
exposed for 1-20 years to concentrations of
cadmium in air (about 0.05 mg Cd/m3). Some
showed proteinuria. There was no coirelation
between blood concentrations of cadmium and
duration of exposure (which was assumed to be
an index of kidney accumulation) in these
workers.
Regarding hair values. Hammer et al. (1971)
reported a correlation between the extent of
exposure to cadmium and hair levels in US
citizens, whereas data by Tsuchiya et al. (1971)
showed random variation in hair levels among
different groups of people. However, the last-
mentioned study is difficult to interpret since
no information on exposure was given. A
correlation in animals between hair and whole
body values has been reported by Nordberg &
Nishiyama (1972). However, concerning ex-
periments with human hair, Nishiyama &
Nordberg (1972) showed that it was virtually
impossible to remove external contamination
of cadmium by various washing procedures.
When there is a risk of external contamination
of the hair it is evident that scalp hair values
of cadmium are probably not useful for evalua-
tion of body burden or levels in the critical
organ.
Lead. The lexicologically important par-
tition of lead between bone (passive lead) and
tissue (active lead) makes it difficult to evaluate
the relationshipamongconcentrations in blood,
urine and critical organs.
Westerman et al. (1965) found a rough
relationship between lead in blood and lead
in bone (as obtained from bone marrow
biopsy specimens) for 12 lead workers. How-
ever, in biopsy samples, the ratios between
cortical bone, trabecular bone and bone
marrow are unknown. Such samples do not
accurately reflect lead content of bone marrow,
which is the critical organ.
For estimating total body burden, tissue
samples, preferably the cortical bones, would
be the best material. Such specimens are not
easily available during life and therefore
other material has been used. Turnover of
72
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lead in teeth following exposure is low and
may be u good indicator Tor bone levels and
body burden. Recent data from studies in
schoolchildren show thai the lead concen-
tration in teeth of exposed schoolchildren was
as much as rive times greater than in those of
children not so exposed (Necdlcman & Shapiro
1972). Because of the problem of external
contamination, hair levels of lead may not be
a good indicator of body burden.
One method which might be useful is the
mobilization of lead from stores by EDTA.
This method has been successfully used for
diagnostic purposes to detect past exposures,
but a quantitative relationship between dif-
ferences in pre- and post-treatment blood and
urine values and bone lead concentration has
not been established.
In contrast to the lack of studies relating
lead concentrations in blood, urine and other
biological samples with those in the critical
organs, there is an abundance of data relating
the first two of these to current exposure (e.g.
Goldsmith & Hexter 1967, Williams et al.
1969, Committee on Biological Effects of
Atmospheric Pollutants 1972). Some data
showing the relationship between lead in
blood and current exposure have been sum-
marized by Hern berg (1972). It was concluded
that particularly lead in blood but also urinary
lead reflect exposure well. In general, blood
lead is more reliable than urinary lead, which
fluctuates according to metabolic factors in
renal handling. Since at least 90% of lead in
Wood is-foound to theeryihrocyles, a correction
for hematocrit should be made in cases of
anemia (Hernberg 1972).
x Mercury. On the basis of experimental and
epidemiological data, it has been shown that
blood concentration would be a good indicator
of brain accumulation of methyl mercury in
chronic low exposure (Bcrglund el al. 1971).
It was also discussed whether whole blood,
plasma or blood cell values should be used as
an index of exposure lo mclhyl mercury and of
retention of methyl mercury in the body and
in the nervous system. It was concluded that
the blood cell value was the best indicator,
but that whole blood values, though less
reliable, could also be used successfully. If
exposure to other mercury compounds can be
excluded, total mercury level in blood cells
is a good index. The ratio between levels in
blood cells and plasma is about 10 at methyl
mercury exposure in man.
Available evidence indicates a rectilinear
relation between total mercury levels in blood
and hair, the hair levels being about 300 times
higher than the whole blood levels for methyl
mercury (Berglund et al. 1971). These con-
ditions make hair levels a good indicator of
body or brain accumulation of mercury. Since
hair from different sites grows at different rates
and since hair may take up some metals ex-
ternally, great care must be taken when samp-
ling hair to provide (1) freedom from external
contamination and (2) standard type and
length of the hair sample. Possible external
contamination should be remembered, es-
pecially with regard to industrial exposure,
since it is likely to involve airborne mercury.
Urinary excretion of methyl mercury is
correlated to blood levels. Because of the low
levels of methyl mercury in urine and the
possible presence of inorganic mercury, total
mercury in urine is not a reliable measure of
body burden of methyl mercury. For organo-
mercury compounds other than the alkyl group,
it is probable that both urinary excretion data
and blood values give some'information about
recent exposure (Nordberg & Skerfving
1972).
For mercuric mercury, there is not a con-
stant ratio between mercury concentrations
in blood and critical organs according to
animal experiments (Berlin 1963, Nordberg &
Skcrfving 1972). Studies in man by Miettinen
(1972), following a single oral dose, showed
lhat the biological half-lime in blood (20
days) is half of,lhul in whole body (40 days).
73
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It seems probable that blood is not a good
index for evaluation of mercury accumulation
in kidney.
Animal experiments and epideiniological
data do not support urinary mercury as a good
index of kidney concentration (Friberg &
Nordberg 1972).
With regard to mercury vapor, a correlation
on a group basis between blood values and
exposure has been reported (Goldwater 1964,
Smith et al. 1970). In individual cases urinary
values may give conflicting conclusions about
the.risks for poisoning, whereas in larger
groups of people urinary-mercury levels might
serve to some extent as an indication of ex-
posure (Smith et al. 1970).
Ill .8. GENERAL DISCUSSION AND NEED
FOR FURTHER RESEARCH
The purpose of the meeting was to examine the
available information on models for the
absorption, excretion and accumulation in
critical organs of toxic metals with special
emphasis on general principles. Although such
models were available for some parameters,
the differences were evident among the three
metals especially examined, cadmium, lead
and mercury. Furthermore, although data
exist, it is obvious that there are gaps in what
is known of the behaviour of metals both
during and following absorption within the
organism. Some of these gaps are such that a
critical evaluation of risks related to different
exposure situations is difficult.
The terms to describe the processes leading
to accumulation were scrutinized. The con-
cept of critical concentration in a particularly
susceptible organ or tissue was defined as the
earliest stage at which functional damage
exists there. Models of accumulation were
attempted, based on the biological half-time
of the metal in the critical organ, but these
were limited by lack of data on whether a
single or a number of exponential functions
would provide the better description or.
indeed, whether an exponential function would
be appropriate at all. More complicated
niuliicompartmental models, in fact, might
better describe the processes involved. How-
ever, the simple exponential equation and the
concept of biological half-time implicit to it
were found, in some cases, to be useful in
interpreting experimental data, as for example
in the case of methyl mercury.
With regard to absorption by inhalation,
there arc fairly good data on average dep-
osition of different particle sizes. The gap
here concerns the limited understanding of
the wide fluctuations among individuals.
Furthermore, the influences of age, physical
activity, particle concentrations in inspired air,
and respiratory disease require further defi-
nition. Internal redistribution of particles
deposited in the alveoli is poorly understood
with respect to the dose of toxic materials
to specific structures in this part of the lung.
It is not known to what extent metal par-
ticles, once they are deposited in the alveoli,
are subject to transpulmonary absorption or to
translocation via the mucociliary escalator.
Data on total alveolar clearance are extremely
scarce, particularly with regard to particle
size and chemical form of the metal. For
example, differences among very soluble metal
chlorides, modcrably soluble metal oxides and
relatively insoluble metal su I fides are not
sufficiently understood. Interindividual health
and constitutional factors have been shown to
be significant for clearance rates and should be
better quantified.
Absorption following ingestion and the
excretion of metals are important functions
of the gastrointestinal tract. However, fewer
data arc available on the basic processes
involved than is the case with absorption by
inhalation. Most current .experimental work
concerns local elimination of metal or net
gastrointestinal excretion. The mechanisms
of absorption and excretion arc not fully
understood, nor have kinetic intcrrelation-
74
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ships been quantitatively determined. Al-
though some balance studies in humans have
been reported, more duta arc needed to
quantify total gastrointestinal absorption in
relation to variation with age. diet and disease
stales. Especially urgent arc investigations into
physiological differences between children and
adults that appear to be of significance to
health as fur as human exposure to metals is
concerned.
Experimental studies in animals are needed
to determine the direct and interrelated roles
of-various factors influencing gastrointestinal
absorption. Such studies should differentiate
total from net excretion as may result from
pancreatic juice, bile, secretions from intestinal
glands and the shedding of epithelial cells into
the intestinal lumen. Models for gastroin-
testinal absorption and excretion valid for
humans can be designed only by the combined
use of human balance studies and experimental
animal data.
With regard to the transport of these metals
within the body and their distribution to
organs and tissues, the important role of the
small, diffusible plasma' fraction for the
exchange of metals between the larger fraction
bound to plasma protein and among the
vascular, interstitial and intracellular com-
partments was stressed. Metals are distributed
non-uniformly and preferential organ accu-
mulation occurs in a pattern that is specific
for a given metal, but may be different for its
organic compounds. However, the metal
,may be released from its initial binding sites
to be taken upxby organs with more stable
metal binding. Basic research on the nature of
metal binding by various compounds of body
fluids in man and experimental animals is
needed to identify factors responsible for trans-
port of metals within the body. Physiological
' and pathological conditions that may have an
effect on the uptake, binding and release of
metals by tissues and organs should he studied
to enable belter understanding of relations
between concentrations in body fluids and
in critical organs.
Potentially toxic metals exhibit marked
differences'among them with regard to binding.
to and passage through the human placenta)
barrier. There is a need for more detailed
studies on metal levels in cord blood, tissues
from infants, and placenta! tissues from
mothers. Experimental studies involving pri-
mates should complement the human studies
so that a broad range of metal doses related
to gestational stages can be evaluated.
Although the brain is a critical organ for
metals such as lead and mercury, little is known
about mechanisms involved in the uptake
and turnover of metals in nervous tissue.
Available data indicate a complex inter-
relationship between metal turnover and the
nervous system compared to other organs of
the body. Animal studies are required to
elucidate the basic mechanisms involved.
With regard to excretion, understanding of
the mechanisms involved in the renal excretion
of metals should be improved. The complex
process of urine formation, together with the
high variability among individuals in many
cases prevents more efficient use of urinary
levels for evaluation of body burdens of metals.
The identification of nutritional and other
physiological factors which may influence the
renal handling of metals may help inter-
pretation of metal concentrations in urine.
Metals in milk constitute both an excretory
and an exposure route and mammary ex-
cretion processes in man and-domestic animals
need to be defined and quantified.
Indices of metal accumulation and exposure
arc urgently needed. It has been shown that
mclal levels in available biological material
(blood, urine, hair, elc.) might give an approx-
imate indication on recent exposure but with
fewcxceptions cannot be used to calculate mclal
concentration in critical organs or in whole
body. Consequently, in many cases, it is
impossible lo tell on the basisof samplcanalysis
75
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whether accumulation in (he critical organ is
progressing and how far metal concentration
in (he critical organ is from the critical level.
The reason for this lack of knowledge is two-
fold. One is the proper numerical values which
could beset up in an equation; the other is the
lack of a proper mathematical model which
is adequate to describe with approximate
accuracy the complexity of metal accumulation
in the body.
Available data for metal retention in organs
are mainly derived from experimental animals
or from adult humans in the medium age
range. There is a need to obtain additional
data from both young and old normal in-
dividuals.
It would be valuable to establish the total
exposure for defined populations so that
changes in tissue levels can be related to
changes in the environment. In this connection
it would be of value to organize systematic
collection of data on metal concentrations in
critical organs from subjects with known
exposure, together with indices of exposure
such as blood, urine, hair, teeth and placenta!
tissue. Storage of samples in tissue banks will
facilitate the evaluation of time trends in
relation to accumulation and concentration.
Further estimates may be performed with
improved and new analytical methods. In
order to make such data collection useful,
collaboration among institutes for the stan-
dardization of analytical and other relevant
procedures on an international level is of
paramount importance.
There is a lack of epidemiological data on
the parameters discussed above in populations
with industrial exposure. This lack is even
more striking in relation to the growing
number of such studies on populations with
general environmental exposure, which is
usually at a much lower level. There is a need
in industrially exposed populations for stan-
dardised, wherever possible collaborative,
epidemiological studies, where cohorts can
, be followed in time and where groups can be
related to each other. With some occupational
exposures to the less common metak, only
small groups may be available for study in any
one country, so that international collabora-
tion in epidemiological studies would again be
of value.
ffl.9. ACKNOWLEDGMENTS AND LIST
OF WORKING PAPERS !••
The Symposium was arranged by the Sub-
committee on the Toxicology of Metals in
collaboration with the Organizing Committee
of the XVI 1th International Congress on
Occupational Health and the Department of
Environmental Hygiene of the Karolinska
Institute.
The Subcommittee is financially supported
by the Research Committee of the Swedish
National Environmental Protection Board,
The Swedish Petroleum Institute, The Jungner
Co., The Granges Co., The Permanent Com-
mission and International Association on
Occupational Health.
The Symposium took place at the Argentine
Ministry of Social Welfare, which also pro-
vided technical facilities for the meeting.
Dr. Ana Singerman was in charge of the pre-
symposium arrangements.
During the meeting in Buenos Aires the task
group received assistance from Dr. N. Cozzi;
Mrs. Kersti Dukes supervised the secretarial
work. Editorial assistance for this report was
given by Pamela Boston.
(Referre(f lo at the present as Author, 1972. All
of the working papers listed below will be pub-
lished in the Proceedings of the XVllth Inter-
national Congress on Occupational Health).
Roy t. Albert (in collaboration with M. Lippmann,
D. Bohning & N. Nelson): Gastrointestinal
translocation of inhaled particles.
Maths IJcrlin: Transport, distribution and re-
tention of toxic metals in animals and man with
76
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special reference lo inorganic compounds of
mercury und lead.
John F. Finklcu (in collahoralio'n with J. C.
Crcason, V. Hasselblad, IX I. Hammer, L. F..
Priester. S. H. Sandiler & J. E. Kcil): Trans-
placcntal transfer of toxic meials.
Lars Frihcrg (in collahoralinn with Tord Kjcll-
strom): Intcrprclation of empirically docu-
'memed body burdens by age of incliils with
long biological half-lives with special reference
to past changes in exposure.
Robert A. Goyer: Cellular and subccllular effects
of lead.
Richard Henderson (in collaboration with H. P.
Shotwell & L. A. Krausc): Significance of total,
ionic, and elemental mercury in urine for
establishing biological threshold limit values.
Sven Hernbere: The value of lead analyses in
blood and urine as indices of body burden,
accumulation in critical organs and exposure.
Albert C. Kolbye, Jr. (in collaboration with R. E.
Shapiro & S. 1. Shibko): Analysis of parameters
affecting regulation of heavy metals in foods in
the United Stales.
Laszlo Magos: Information on accumulation of
metals in critical organs in relation to the chemical
structure of the substance.
Jorma K.. Miettinen: Gastrointestinal absorption
and whole-body retention of toxic heavy metal
compounds (methyl mercury, ionic mercury,
cadmium) in man.
Paul E. Morrow: Pulmonary absorption of toxic
metals.
Gunnar Nordberg: Models used for calculation of
accumulation of toxic metals.
Tor Norseth: Gastrointestinal absorption and
excretion of toxic metals in animals and man.
Emit A. Pfilzer: Absorption, distribution and
retention of toxic metals with special reference
to inorganic and organic compounds of lead.
Magnus Piscator: Excretion of metals in relation
to functional impairment of organs (referred
toasI972b).
Samuel Shibko: Sec Albert Kolhyc, Jr. above.
Ana Singerman: Biochemical background for
functional impairment induced by toxic meials.
Kenzahuro Tsuchiya (in collaboration with M.
Sugila & Y. Scki): A mathematical approach to
deriving biological half-lime of cadmium in
some organs—calculation from observed ac-
cumulation of the metal in organs- (rcfeircd
was I972b).
Jarosluv Vostal: General mechanisms of renal
and biliary excretion of toxic metals.
III.10. REFERENCES FOR SECTION III
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18, 97
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LIST OF AUTHORS
Roy Albert, M. D.
Institute of Environmental Medicine
New York University Medical Center
550 First Avenue
New York, N.Y. 10016
USA
Maths Berlin. M.D.
Department of Environmental Health
University of Lund
Box 2009
S-220 02 Lund 2
Sweden
John Fink lea. M.D.
National Environmental Research Center
Environmental Protection Agency
Research Triangle Park
North Carolina 27711
USA
Lars Fribcrg, M.D.
Department of Environmental Hygiene
The Karolinska Institute and
The National Environmental Protection Board
S-104 01 Stockholm 60
Sweden
Robert A. Coyer, M.D.
Department of Pathology
School of Medicine
University of North Carolina
Chapel Hill, North Carolina 27514
USA
Richard Henderson, Ph.D.
Environmental Hygiene and Toxicology
Department,
Olin Research Center
275 Winchester Ave.
New Haven, Conn. 06504 USA
Si-en Hernherg, M.D.
Institute of Occupational Health
Haartmaninkatu I
SF00290 Helsinki 29, Finland
George Kuzanl:is, M. D.
Department of Community Medicine
The Middlesex Hospital
London WIN KAA, England
R. A. Kfltoe, M.D.
Department of Environmental Health
University of Cincinnati
Cincinnati, Ohio 45129, USA
Albert C Kolbyt,Jr.,M.D.
Bureau of Foods
Food and Drug Administration
200CSI.S.W.
Washington, D.C. 20204, USA
Laszlo Mugos, M.I).
Medical Research Council Laboratories
MRC Toxicology Unit
Woodmansterne Road
Carshalton/Surrey, England
Jormu K. Micllmen, M.D.
Department of Radiochemistry
University of Helsinki
Unioninkatu 35
SF00170 Helsinki 17, Finland
Cnnnar Nordberg, M.D.
Department of Environmental Hygiene
The Karolinska Institute
S-104 01 Stockholm 60, Sweden
Tor Norseth, M.D.
Institute of Occupational Health
Gydas Vei 8
Oslo 3, Norway
Emil A. PJitzer, Sc.D.
Hoffman Laroche Inc.
Nutley, N.J.07I10, USA
Magnus Piscator, M.D.
Department of Environmental Hygiene
The Karolinska Institute
S-104 01 Stockholm 60, Sweden
Samuel I. Shibko, Ph.D.
Division of Toxicology
Bureau of Foods
Food and Drug Administration
Washington, D.C. 20204, USA
Ann Singerniaii, Ph.D.
Junin 1032
Buenos Aires, Argentina
Kcnzaburo Tsuchiya, M.D.
Department of Preventive Medicine and Public
Health
School of Medicine
Keio University
Shinanomnchi, Shinjuku-ku
Tokyo, Japan
Jaroslao I'oxlal. M.D.
Department of Pharmacology and Toxicology
School of Medicine and Dentistry
The Universit) of Rochester
Rochester, N.Y. 14620, USA
85
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v a /
SECTION IV
EFFECTS AND DOSE-RESPONSE RELATIONSHIPS
OF TOXIC METALS
(To be published in conjunction with the working papers from the meeting by flsevier Scientific Publishing
Company.)
Preceding page blank
87
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Editor' H Preface
to
Effects and Dose-Response Relationships of Toxic Metals
The field of environmental toxicology of metals has attracted considerable
interest in the general and scientific communities during recent years.
Although it is well recognized that health effects of metals in the general
environment in some instances have been demonstrated to be closely re-
lated to events in the overall ecosystem, the present volume is focused on
human toxicology, always an important part of the total evaluation of
environmental toxicology.
The book constitutes the proceedings from a meeting of thirty-six experts
on metal toxicology and epidemiology from twelve countries, which was
organized by the Subcommittee on the Toxicology of Metals under the
Permanent Commission and International Association of Occupational Health
and held in Tokyo, November 18-23. 1974. The first part of the volume
(Chapters 1-7) is the consensus report, worked out by the participants and
circulated among them prior to the meeting. Contributions of original reports
of experimental and epidemiological data, as well as reviews on various
topics within metal toxicology, made up the working papers which are pre-
sented in the other parts of this book (Parts A1-C4). While it is evident that
the contributions vary considerably in length and style of presentation, and
a certain overlap sometimes occurs, the Editorial Committee has con-
sidered it valuable to include most of the contributions in the present volume
so as to give the reader a comprehensive review of what written information
was available for the discussions in Tokyo. This does not imply that the
Editorial Committee and its reviewers have always completely agreed on
the scientific approach and interpretation of data by individual authors. The
working papers (referred to in the table of contents as contributed articles),
though they appear after the consensus report in this volume, were written
prior to the meeting, meaning that the terminology and concepts defined
in the report have not been consistently used in them.
.Farther details concerning how the meeting was organized are given in
the introduction to the consensus report. The final version of the report
and the bcmk were reviewed by the Editorial Committee dxiring a meeting
in North Carolina, U.S.A., in March 1975.
It is believed that this volume, and the consensus report in particular,
constitutes a unique document in defining concepts in metal toxicology. I(
also provides an up-to-date review of the toxicology of cadmium, lea'.;.
mercury, and their compounds, as well as an insight into interactions
among them and with other metals. This volume thus represents a con-
siderable total effort on the part of all of tho participants. The Editor and
the Editorial Committee wish to thank each contributor for his work and
cooperation in completing the. document.
On behalf of the Editorial Committee,
Gunnar Nordberg
'•'Proceedings from International Meeting, Subcommittee on Toxicology
of Motals, Permanent Commission and International Association of
Occupational Health, Tokyo, November 18-23, 1974. To be published
by Elsovior Scientific Publishing Company.
88
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SECTION IV
EFFECTS AND DOSE RESPONSE RELATIONSHIPS OF TOXIC METALS'
IV. 1 BACKGROUND
The Subcommittee on the Toxicology of Metals under the Permanent
Commission and International Association on Occupational Health,
at meetings in Slanchev Bryag, Bulgaria, 1971, and in Buenos
Aires, 1972, discussed questions on accumulation of toxic metals
with special reference to their absorption, excretion and bio-
logical half-times. As a result of the meetings, two reports have
been published (Dukes and Friberg, 1971, and Task Group on Metal
Accumulation, 1973). At the last"mentioned meeting it was decided
to continue the work on metal toxicology by arranging a third
meeting where questions concerning effects and dose-response
relationships of toxic metals would be discussed in particular.
As a result of this decision a working group, the participants
of which were invited by the subcommittee, convened in Tokyo,
November 18.-23, 1974. In addition to the participants about
30 observers attended the plenary sessions and occasionally
took part in small group discussions. The participants are listed
separately. The meeting, which took place at the Research Institute
of Industrial Safety, Tokyo, was officially opened in welcoming
speeches by Dr. Yoshio Ohtaki, Executive Director of the Japan
Industrial Safety Association and also Secretary General of the
meeting; Mr. Kinnosuke Tohmura, Director, Department of Labor
Standards of the Ministry of Labor; Dr. Velimir Vouk representing
the World Health Organization, Geneva; and Dr. Juko Kubota,
Director, Japan Industrial Health Association and Honorary "
Chairman of the meeting. Opening remarks were given by Dr. Lars
Friberg, Chairman of the Subcommittee.
Dr. Lars Friberg was elected Chairman of the meeting, Dr. Kenzaburo
Tsuchiya was elected Vice-chairman, and Dr. Gunnar Nordberg, Sec-
retary of the Subcommittee, was elected Rapporteur.
i
The participants of the meeting had prepared working papers dealing
with specific.topics in the area of effects and dose-response
89 .
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relationships and these were circulated in advance to all
participants. Based on the working papers, Dr. Gunnar Nordberg
had prepared a draft report which was sent to participants and
observers in advance. The discussions at the meeting were centered
on this draft report, which was revised during the meeting.
The entire report was read, discussed, and approved by the
group of participants as a whole. The detailed preparatory
work was performed in five subgroups: one general group (Chair-
man: Dr. Vouk), one group on cadmium (Chairman: Dr. Vostal),
one on lead (Chairman: Dr. Goyer), one on mercury (Chairman:
Dr. Suzuki), and one on interactions (Chairman: Dr. Parizek).
Final editing of this report was done by the Rapporteur (Dr.
Nordberg), with assistance of an editorial committee consisting
of Drs. Friberg, Horton, Pfitzer, Tsuchiya, Vostal and Vouk.
Administrative editorial.assistance was provided by Ms. Eamela
Boston.
As was the case during the two previous meetings arranged by the
Subcommittee it was decided to take into consideration not only
experience from occupational exposure, but also that from expo-
sure through other media, including ambient air, water, and
food. One reason for this was that effects would be related
to the total exposure from the environment. Further, much evi-
dence from.experience related to .the general environment was
available for certain evaluations concerning relationships be-
tween exposure to metals and effects.
The term "toxic metals" is used in this report to include met-
allic elements. It also sometimes includes nonmetallic elements,
which, under certain conditions, may exhibit properties of metals
to some degree, e.g. selenium. In a strictly scientific sense,
all metals, in fact all chemicals, are toxic when too much
reaches vulnerable sites in the body. This report centers on
metals whose toxic potentials have been recognized as serious
problems for humans. Thus, primary emphasis has been given to
_lead, mercury, and cadmium, but a few other elements are also
90
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discussed. The term "metals" may occasionally be used broadly
to include metals in elemental form, metals as inorganic or
organic salts, or organometallic compounds. Detailed data in
the report will include, when possible, the description of
the specific form of the metal or metal compound.
Because the present meeting to a large extent is a continuation
of the meeting held in Buenos Aires, 1972, dealing with "Accumu-
lation of toxic metals with special reference to their absorp-
tion, excretion and biological half-times", the conclusions
reached there have been taken advantage of here. Reference is
therefore made repeatedly in this paper to the published re-
port from that earlier meeting (i.e. TGMA, 1973).
A summary of the most important conclusions from the earlier
meeting will be incorporated here for easy reference, since
this information constitutes a substantial part of the con-
siderations on the "dose" in the dose-response relationships
which were discussed at the present meeting. The main focus .
of this meeting has been on the effects of certain doses of
metals as well as associations between such doses and effects.
The earlier meeting outlined the general philosophy behind met-
abolic models and critical organ concentrations for evaluation
of metal toxicology (TGMA, 1973). This philosophy is based on
the concept of a threshold dose for the occurrence of adverse
effects in the organ first displaying such effects during the
exposure to toxic metals. The basis for a threshold dose con-
cept lies in the interference of metals with certain biochemical
processes and structural components, e.g. membranes, organelles
and the enzyme systems within the cells of the body. Usually a
certain reserve capacity on the enzymatic level.allows the cell
to absorb certain amounts of metal without undergoing evident
functional changes. By the use of the critical-organ-concentration
—metabolic model approach, certain features of dose-effect re-
lationships for toxic metals may be explained, for example effects
91
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resulting from accumulation due to long-term exposure to substances
with long biological half-times. It is felt that the approach
is helpful in many cases to interpret and to establish dose-
response relationships.
The group was aware that much important work was under way on an
international level under the auspices of other international or-
ganizations; particular reference was made to the intensive proj-
ect of the World Health Organization environmental health cri-
teria documents, which even now, but more so in the future, will
be of great merit for the endeavors within the Subcommittee on
the Toxicology of Metals, whose work could be of mutual importance
for the work within WHO. it was therefore clear that a close col-
laboration, as was the case during this meeting, should be es-
tablished between the Subcommittee and the World Health Organi-
zation. Also, the work within other organizations, as, for
example the International Union for Pure and Applied Chemistry
(IUPAC), and the International Council of Scientific Unions (ICSU)
and its Scientific Committee on the Problems of the Environment
(SCOPE) was referred to as being of great relevance for the work
within the Subcommittee.
92
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IV. 2 CONCEPTUAL CONSIDERATIONS: CRITICAL ORGAN, CRITICAL CONCENTRATION
IN CELLS AND ORGANS, CRITICAL EFFECT, SUBCRITICAL EFFECT, DOSE-EFFECT AND
DOSE-RESPONSE RELATIONSHIPS
The definitions to be given in this section of the report were
much discussed during the meeting, and it was pointed out that
the resulting concepts might clarify some problems involved in
the early identification and prevention of adverse effects of
exposure to toxic metals. Since1 the prevention of adverse health
effects is the primary objective of environmental health, it was
hoped that the concepts defined here and used in the other chap-
ters of the report could be considered by other workers in the
field of toxicology and environmental health.
The critical concentration for a cell was defined by the Task
Group on Metal Accumulation, 1973, as the concentration at which
undesirable functional changes, reversible or irreversible, oc-
cur in the cell. The lethal concentration for a cell was defined
as the cellular concentration sufficient to cause death of the
cell.
The present meeting suggested that "adverse" might be a more
appropriate term than "undesirable". It also stressed that
critical concentration should always be related to a defined
effect.
Critical organ concentration was defined .as the mean concen-
tration in the organ at the time any of its cells reaches crit-
ical concentration (TGMA, 1973). This meeting noted that in
most practical cases a certain number of the most sensitive type
of cells in the organ will be affected in order for the crit-
ical organ concentration to be reached. The critical organ con-
centration may be considerably higher or lower than the crit-
ical concentration for a particular cell. This is possible since
the type of cell that first attains the critical concentration
93
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is not necessarily the type of cell with the highest concen-
tration. The usefulness of ^he concept "critical organ con-
centration" stems from the lesser likelihood, generally, of
this concentration's displaying fluctuation in relation to the
appearance of effects, compared to the whole body concentration
or other "dose" or exposure estimates.
Since there exists biological variation in sensitivity among
individuals, a certain interindividual variation is to be
expected also in critical organ concentration. The critical
organ concentration may even be subject to variation both in
an individual and in a population. This has recently been
pointed cut for this meeting by Tsuchiya et al. (1975) and
Sakabe et al. (1975) for cadmium and by Suzuki and Shishido
(1975) for methylmercury compounds. Further detailed consid-
erations for the various metal compounds will be given in
Chapters 3-6. '..
H
Critical organ is defined as that particular organ which
first attains the critical concentration of a metal under
specified circumstances of exposure and for a given popula-
tion.
"The definition of the term "critical organ" is consistent
with earlier definitions given in this report (and TGMA,
1973), but differs from the definition given by, e.g., the
International Commission on Radiological Protection: "Critical
organ is defined as the organ of the body whose damage by ra-
diation results in the greatest injury to the individual (or
his descendants). The injury may result from inherent radio-
sensitivity or indispensability of the organ, or from high
dose, or from a combination of all three." (ICRP, 1959). A
different approach has been chosen here because it has been
considered advantageous from the point of view of preventive
medicine. The present definition implies that if critical effects
in critical organs are identified and prevented, no adverse
effects for the whole organism shall appear. Certainly effects
in organs which might be "critical" from the clinical point
of view (i.e. signalling a grave danger for the patient)
would be prevented. Other definitions of "critical organ"
relating the term to clinical disease or damage to the in-
dividual as a whole, may not necessarily achieve the goal
of preventing.all types of adverse effects. Under certain cir-
cumstances the term "oigan" may not be adequate and one could
talk about critical system or critical tissue. In this report
we have not striven after such exact usage in this case and
the term organ is used broadly.
94
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The organ (system, tissue) of greatest accumulation is not
necessarily the critical organ, e.g., in lead exposure the
highest concentrations may be reached in bone without any
identifiable effect; the bone is therefore not the critical
organ. Sensitivity of organs may show interindividual vari-
ability due to metabolic and other factors that in some cases
may make one organ critical for one person and another organ
critical for another person. It is also evident that the organ
that becomes critical may vary with the type or route of ex-
posure (single, repeated, short- or long-term exposure; oral,
inhalation, etc.).
For these reasons several organs may be classified as critical
for a defined metal or metal compound. For example, for short-
term exposure to high concentrations of cadmium in air, the
lung may be the critical organ, whereas for long-term exposure
to lower concentrations by the same route, the kidney may be
the critical organ.
The organ that becomes critical may also vary depending on
the characteristics of the population exposed. There is some
evidence, for example, that in children exposed to lead, the
brain may be the critical organ, whereas this is not necessarily
the case for adults.
The above given definition of critical concentration in the
critical organ represents a defined point in the relationships
between dose and effects in the individual, namely the point
at which an adverse effect is present. This is the critical
effect. This critical effect may or may not be of immediate
importance for the health of the whole organism. It is of
interest to discuss doses higher than those corresponding
to the critical concentration, and particularly to discuss
the relationship between adverse effects at the cellular level
and significant hazards to the health of the organism.
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At an exposure Ieve3. that is lower than the one giving criti-
cal concentration of metal in the critical organ, some effects
might occur that do not impair cellular function, but still
are evident by means of biochemical or other tests. These
effects are defined as subcritical effects. The concentra-
tions producing such effects are defined as subcritical con-
centrations, and each must be related to a defined effect.
The biological meaning of a subcritical effect is sometimes
not known; in some case? it only indicates that an exposure
has taken place; in some cases it may be a sign of an adapta-
tion; in other cases it may be a precursor of a critical ef-
fect. The last mentioned type of subcritical effect may
be especially useful in preventive medicine, e.g., when it
is possible by a biochemical test to determine a graded sub-
critical effect that precedes the development of the critical
effect. For example, in lead exposure, an inhibition of the
enzyme ALA-dehydrase in the cells of the bone marrow is a
subcritical effect which precedes an increased level of ALA
in blood and urine and the occurrence of anemia (critical
effects). A decrease in ALA-dchydrase activity in blood is
an example of an indicator of a subcrit.ical effect of ] ead
exposure. A general discussion on types of effects that should
be taken into consideration when defining critical organ con-
centration and the possibilities for their detection will
be elaborated upon in Section IV. 4 of this report.
The term "effect" is used to mean a biological change caused
by an exposure. Sometimes this effect can be measured on a
graded scale of severity, although at other times one may
only be able to describe a qualitative effect that occurs
within some range of exposure levels. When data are available
for the graded effect, it is apparent that one may establish
a relationship between dose (usually an estimate of dose)
and the gradation of the effect in the population; this
is the dose-effect relationship. An example may be the
relationship between lead concentration in industrial air
and the concentration of ALA in urine samples from workers.
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Ideally a dose-effect relationship is established with
measurements.on many individuals over a range from minimum
to maximum effect. The curvilinear relationship that represents
the best fit to all of the data points should be expressed
in terms of mean values and their standard deviations at
various doses. In other words the scatter of data will usually
lead to confidence limits around the mean values, and these
expressions of degree of uncertainty should be evaluated
wherever possible.
The term "response'1 is used to mean the proportion of a
population that demonstrates a specific effect, and its
correlation with estimations of dose provides the dose-
response relationship. For example, a dose-response re-
lationship might compare different lead concentrations in
industrial air (estimates of dose) with the percent of
the exposed workers that have greater than 5 mg ALA/liter
of urine.
At times one measures a change (in terms of frequency or
degree) that is not a biological change, e.g., lead in the
blood or cadmium in the kidney. When such data are correlated
graphically with the degree of exposure, they may have the
same form as a dose-effect or dose-response relationship;
however, such relationships simply define the correlation
between two different estimates of dose, according to the
principles and purposes of this report.
To establish dose-response and dose-effect relationships requires
much data that are often lacking for human beings with
regard to many metals. In such cases, in which only limited
data are available, knowledge of general principles governing
the action of metals may sometimes permit approximations
of such relationships.
In addition to these definitions of the words response and -. I ^ , ,
effect, which will be further elaborated upon in section IV.5.1, the
term dose has been given a specific meaning and usage (section IV. 3.1.1).
•/*•,,.«>.
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" IV. 3. DOSE
IV.3.1 General considerations on dose and exposure;
possibilities for their quantitation
Occupational exposure to toxic metals or their compounds occurs
mainly through inhalation of dusts or vapors, although ingestion
and skin absorption may also play a substantial role. The
general population may be exposed via air, water, and food.
Smoking may also contribute to the intake of some metals.
The total exposure of the occupationally exposed population
may include a significant contribution from food and water
in the general environment as well. Hence, the relation"
ship of these two routes of exposure to other routes must
be considered.
Difficulties may arise if all of ths different routes of ex-
posure are not taken into account. In general environment
studies, for instance, it may not be useful to correlate ex-
posure from airborne lead with blood lead levels if the ex-
posure of the population to lead via food is not simultaneous-
ly measured (review: Fiscator, 1975). When dealing with the
exposure to metals via food, the chemical form of the metal
is a primary consideration. In many instances, merely analyzing
for the metal as an element is not adequate. There is little
information on the forms of metals in food, with the exception
of mercury in fish. Mercury in food may be in the form of
methylmercury and/or inorganic mercury. Lead in plants may
exist as lead phosphate and may also be complexed with or-
ganic acids. In soft tissues of animals, complexes are formed
with proteins and/or lipoproteins. Cadmium may be found com-
plexed with low-molecular-weight proteins, as in metallothionein,
and also with other types of proteins.
A source of error when dose estimates are to be made concerning
exposure via food is the fact that the levels of metals in
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food may be unpredictable and not easily controllable (review:
Shibko et al., 1975). The vastly different dietary habits
of exposed individuals lead to differences not only in expo-
sure but also in interactions between dietary components,
in turn possibly leading to differences in metabolism and
effects.
A quantitative assessment of the dose or even of accumulated
exposure is especially difficult as regards long-term exposure.
Assessment of dose by analysis of biological samples, blood
for example, may be possible, but may be burdened with several
difficulties. The following sections of this report will put
special emphasis on discussion of possibilities and difficulties
involved in using metal determination in biological materials
as dose estimates.
IV.3.1.1 Definition of "dose"; general considerations on its assessment
Ideally, the dose should be defined as the amount or concentra-
tion of a given chemical at the site of effect, i.e., where
~ K
its presence leads to a given effect. Unfortunately, the
determination of this amount is often not possible in practice.
It is therefore clear that the dose may have to be estimated
in various ways, some common ones of which are -
a) From experimental exposure: injection; ingestion,
including feeding experiments; inhalation; and
dermal or other topical applications. In all these
cases the dose is estimated from the amount or con-
centration applied, the time and, when available,
relevant deposition, retention, and absorption factors
This definition of the concept "dose" was considered during
the present meeting to be useful for establishing dose-response
and dose-effect relationships at various structural levels.
However, there was no objection to the term "dose" in its
classical sense, meaning amount administered to, e.g., an
experimental animal. When the term "dose" is used in this
classical sense in this report it will always be specified
as "administered dose", "oral dose", "injected dose", etc.
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b) From occupational exposure: This usually involves
the concentration in air, rate of inhalation, time,
and the appropriate deposition, retention and ab-
sorption factors. In addition, dermal exposure
and ingestion during work time should be consid-
ered, noting that the worker is also exposed
to ambient air, drinking water, and food, as a
member of the general population.
c) From general environmental exposure: This involves
inhalation of air, ingestion of food and water, a.»d
exposure from other sources including drugs, consumer
products, tobacco smoke, pica in children, variouc
beverages, etc. The same considerations as to amount
or concentration, time, and deposition, retention,
and absorption factors apply.
d) From measurement in bodily compartments: This in-
volves indicator (or index) media such as blood.
urine, feces, sweat or hair. In addition, compartments
such as organs, tissues, specific groups.of cells,
and subcellular components may be useful. This is
discussed in detail in subsequent sections
of this chapter.
The accuracy and precision of the various estimates of doso
will depend upon the validity of sampling and analytical meth-
odology. The estimates must also consider, the interactions
and biotransformations of the chemical in the organism (see
sections IV. 3.3 and IV. 3.4.
It is evident from this discussion that the term dose as used
in this report will necessarily be an estimate of the dose
as defined at the beginning of this section. Therefore, when-
ever the term dose is used it should be made as clear as pos-
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sible how the dose was estimated, including the specific units,
the physical form and the chemical species involved.
IV.3.1.2 Quantitative estimations of dose
IV.3.1.2.1 General Aspects
In the practical situation, when trying to establish the ex-
tent of health hazards in industry or in the general environment,
chemical analysis is necessary of metal concentrations in
air, drinking water, food products or indicator (or index)
iuedia, like blood, urine, and hair. The problems ralated to
such analyses are often considerable and are partly due to
the difficulties in performing accurate chemical determina-
tions of netai concentrations in the various biological mat-
erials mentioned. However, problems related to santpling may
be of equal or greater weight, particularly in relation to
the variations in concentrations of the materials sampled.
In addition, variations in consumption of food, inhalation
of air, the nature of the population studied, and uncertainties
in relationships between concentrations in the indicator (or
index) media and the organs will contribute to the overall
errors. Further, as regards matals with long biological half-
times in the critical organ, estimations of the history of
exposure are necessary in order to calculate the dose from
analyses of environmental samples.
The difficulties can be exernplified as follows. Blood con-
centration is sometimes taken as a measure of the dose in
spite of the absence of adequate knowledge as to the rela-
tionship between the blood concentration and the exposure,
or the blood concentration and the concentration in the crit-
ical organs. When, in such coses, no correlation is found
between the blood concentration and a certain effect, it can
be assumed that the blood concentration is not a good indi-
cator (or index) of the concentration in tho critical organ,
rendering the approach useless. Datta^ and discussions given
elsewhere (TGMA, 1973), which can serve to illustrate the
101
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best available copy.
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usefulness of metal concentrations in indicator (or index)
madia for the estimation of the dose and their relation to
the concentration in the critical organ, will bo summarized
in section IV.3.4.
IV.3.1.2.2 Chemical analysis of metal concentrations in food,
air, water, and various biological indicator (or
indexj media
Problems related to analysis of metals in biological materials
are substantial. In addition to the problems in connection
with elemental analysis, there are those with identification
of the chemical species and its physicochemical properties,
e.g., organomercury compounds. It is not the intention of
the present report to give a detailed discussion of the
analytical chemistry of ir.etals, but the following comments
are meant to give an appropriate point cf departure for later
topics.
The main problem in the determination of metals in industrial
air is generally not the final chemical analysis, but the
sampling technique, which might be inadequate. The sampling
site may be a distance away from the actual place of exposure,
resulting in data that do not reflect the true exposure. Whenever
possible, personal sampling equipment should be distributed.
This may be feasible in occupational exposure studies, but
very difficult in epidemiological studies involving general
population groups. With regard to analysis of drinking water,
metal concentrations are often low and may necessitate con-
centration of the sample, which may introduce errors. For
other media such as blood, urine, and certain food products,
concentrations of metals are often low and interfering substances
may be present. Analytical errors thus may become serious
stumbling blocks (for further discussion see Piscator, 1975).
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Blood lead concentrations are often not accurately determined
in spite of the relatively long experience with such analyses.
In a recent interlaboratory comparison among 66 European labor-
atories employing varying analytical techniques, differences
with a factor up to/ and sometimes more than, 10 were noted
in the results (Berlin, A. et al., 1974). Similar examples
are available from other studies on metals and media. This
reveals the need for inter- and intralaboratory quality control
programs on a more systematic basis.
In conclusion, it should be pointed out that a critical approach
toward sampling and evaluative techniques is a prerequisite
for assessing published results. Even a theoretically appropriate,
precise, and accurate method will not automatically ensure
reliable results. Obviously, reliable dose-response relation-
ships cannot be obtained from erroneous exposure data.
IV.3.2 The metabolic model and its relation to metal concentrations
'in biological media as indicators (indices) of exposure
gnd of concentrations in critical organs
A metabolic model describes,in qualitative and quantitative
terms, the processes (i.e., type and rate) cf absorption,
H
distribution, deposition, biot.ransformation , retention, accu-
mulation, and excretion. It also includes other chemical and
physical interactions (e .gv protein binding) that a metal
or its compounds may undergo in their passage through the
organism. Other terms have been used to describe such relation-
ships, the most common being "pharmacokinetics". The present
meeting has decided upon the term "metabolic model".
The metabolic model includes data concerning absorption of
metals after inhalation, ingestion, or contact with the skin, as well
as transplacental transfer of metals. The relationship
H ' *""
The term "biotransformation" when applied to metals will
be understood to refer to changes in the oxidation state of
the metal and the formation and cleavage of covalent organo-
inctallic bonds.
103
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between metal concentrations in biological materials, as in-
dicators (or indices) of exposure, and concentration in critical
organs is of special interest.
The various components of the metabolic model have been dis-
cussed in detail and specific data on lead, mercury, and cad-
mium and their compounds given by TGMA (1973). The following
summary provides the most important data brought forth and
conclusions reached at that time, in ad-
dition, more recent data have been included as a result
of discussions during the present meeting.
It is obvious that it would be extremely valuable if precise
data on the various parameters included in the metabolic model
for a certain substance could be deduced from theoretical
considerations based on generally valid principles governing
these parameters. Unfortunately, it became equally as obvious
from discussions at the Buenos Aires meeting that such generally
valid principles could not be put forth to any notable extent,
except for certain pulmonary absorption parameters. It was
thus evident that specific data concerning absorption, distri-
bution, and excretion for every individual metal and metal
compound must be determined experimentally, in order to con-
struct a metabolic model that would be sufficiently precise
for practical purposes. Such data can be obtained in various
ways, e.g. from studies of substances labelled with radio-
active or stable isotopes and administered by the various
exposure routes, to experimental animals, and even to human
beings.
IV. 3.2.1 Absorption
IV.3.2.1.1 Absorption by inhalation
Though usually of a more obvious nature for human exposures
in the industrial environment, pulmonary absorption of metals
may also be of importance in the general environment, including
exposures from tobacco smoking. Metals may be taken into the
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respiratory system in the form of particles, gases or vapors.
General principles of the absorption of metals in these various
airborne forms were presented by TGMA (1973). With regard
to absorption of metals and metal compounds in particle form,
it was concluded that absorption was influenced by three processes
in the lung, namely: deposition, mucociliary clearance, and
alveolar clearance. For an explanatory model of these different
processes, the model by the Task Group on Lung Dynamics (1966)
was used. After deposition in the nasopharyngeal, tracheobron-
chial or alveolar (pulmonary) compartments, the metal may
be transported by mucociliary action to the gastrointestinal
tract or absorbed into the blood. The fraction deposited in
the nonalveolar compartments of the lung participates mainly
in the first-named type of translocation. The portion deposited
in the alveolar compartment is more likely to be absorbed
but may take different routes.
Deposition of particles in alveolar and tracheobronchial-naso-
pharyngeal compartments of the lung was considered to be dep-
endent on the physical characteristics of the aerosol (such
as the size and density of the particles) and also on the
parameters of respiration (nose or mouth breathing, tidal
volume and respiratory rate). The anatomical variability of
the lung may have major relevance for deposition. Variation
depending on such interindividual variability may amount to
a factor of 2-to-3. The portion of the total deposition that
falls within the tracheobron.chial and nasal compartments is
generally not absorbed into the body unless the aerosol particles
consist of easily soluble salts. This proportion of the metal
is translocated into the gastrointestinal tract.
The proportion deposited in the alveolar compartment will
be cleared from this compartment by (1) transport onto the
mucociliary escalator and translocation into the gastrointes-
tinal tract, (2) deposition for a long time in the pulmonary
105
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tissue or (3) passage through the pulmonary tissues into lymph
and blood. As concerns particles with low solubility, it is
not known either in human beings or in animals, how large
a part of the alveolar clearance results from transfer of
particles to the mucociliary escalator and how large a part
from their absorption through the lung. The proportion absorbed
was, however, estimated to range between 50 and 100% of the
material deposited in the alveoli. Taking this variation into
consideration, the total bodily absorption of particles with
different sizes between 0.1 and 10 pm MMAD and with a gastro-
intestinal absorpLion of 5%, was calculated to range from
about 5 to 50% of the inhaled amount (TGMA, 1973).
Deposition of gases or vapors of metals in the respiratory
tract depends on water solubility. Highly water-soluble gases
are dissolved in the mucous membranes of the nasopharyngeal
and tracheobronchial systems and never reach the alveoli.
Less soluble gases, on the other hand, do reach the alveoli.
Depending on their ability to penetrate the alveolar structure,
they are absorbed to varying degrees. An example of a metal
which occurs in vapor form is mercury. Its vapor reaches the
alveoli and easily penetrates the alveolar membrane, resulting
in a high extent of absorption (about 80%) .
Insufficient data are at hand concerning what influence age
and pathological conditions would have upon the pulmonary
absorption of metals. It is known that such conditions as
chronic bronchitis and cigarette smoking may affect bronchial
deposition and mucociliary clearance and thus also pulmonary
absorption.
IV.3.2.1.2 Absorption by ingestion
Absorption of metal compounds after ingestion via food and
beverages is a major source of exposure, but a substantial part of
metals in particle form deposited in the lung may be transferred
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to the gastrointestinal tract by mucociliary clearance. Gastro-
intestinal absorption of metals has been discussed by TGMA
(1973) and has also been subject to some discussion during
the present meeting (reviews: Goyer and Rhyne, 1973; Shibko
et al., 1975), especially with regard to the recently obtained
data on influence of age and the interactions of various es-
sential as well as non-nutritive constituents of food in re-
lation to the absorption of toxic metals.
The vastly different absorption rate between organic and in-
organic compounds of mercury is well known (TGMA, 1973) , but
reasons also exist to believe that there could be differences
in absorption depending on the type of inorganic compound oc-
curring in food as well as differences depending on other
components in the diet.
Studies with experimental animals and wan have shown that
other substances in the diet, such as dietary fiber, phytate,
protein, and lactose, may have significant effects on the
absorption of metals. Reviews of those relationships have
been contributed to the present'. meeting by Goyer and Rhyne
(1973) and Shibko et al. (1975) and other literature includes
Averill and King (1926), McCance and Widdowson (1935), Maley
and Mellor (1950), Vohra et al. (1965), Oberleas et al. (1966),
Evans (1973), Trowell (1973), Davis and Nightingale (1974),
Klevay (1975a, 1975b), Reinhold (1975), and Dacre and Ter
Haar (1975).
Factors to be considered in evaluating the bioavailability
ol an element in a diet also include the physico-chemical
.form and properties of the compound, the site of absorption,
the surface area involved, the blood supply to the site of
absorption, and other membrane transport factors. Although
these factors may "be tested only in model systems, they may
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be important in the evaluation of the absorption of particulate
inorganic matter following inhalation and subsequent swallowing,
such as might occur in an industrial situation. In addition,
as in the case of toxic elements normally present in food,
key factors affecting bioavailability are mineral interaction
and dietary components.
IV.3.2.1.2.1 Cadmium
On the basis of data on human beings (Rahola et al., 1972)
it may be concluded (TGMA, 1973) that approximately 6% of
an oral dose of cadmium is retained after 10-15 days. Data
by Kitamura (1972) and Yamagata et al. (1974) as well as by
Rahola et al. (1973) are in accord with this conclusion.
Observations discussed at the present meeting make it reason-
able to conclude that this figure may vary depending on such
factors as calcium and protein content of the diet, etc.,
between 3 and 10% (Friberg et al., 1974).
Recent data from experiments on rats (Stowe et al. , 1974)
suggest that the dietary concentration of pyridoxine (vitamin
Bg) will influence cadmium absorption. In young mice the absorp-
tion rate of cadmium was found to be higher than in adult
mice (Matsusaka et al., 1972), the whole-body retention being
about 10 and 1%, respectively, 2 weeks after an oral dose
of radioactive cadmium. Age might also be meaningful for the
gastrointestinal absorption of cadmium in human beings.
IV.3.2.1.2.2 Lead
TGMA (1973) concluded that the gastrointestinal absorption
of lead compounds in adult human beings is between 5 and
£ •
It is well recognized that there are both inorganic and or-
ganic (e.g. tetraethyl- ) lead compounds of toxicological
importance. However, because of the considerably greater
ubiquity of the inorganic compounds, these were the only ones
considered during the previous meeting in Buenos Aires (TGMA,
1973). In the present report data concerning lead will refer
to inorganic lead compounds unless otherwise explicitly stated.
108
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15%. This figure may be greatly altered by components in the
diet, age and whether the subject ingesting lead is in the
fasting state or not. The absorption of lead was shown to
be as high as 50% in fasting human subjects while in non-
fasting subjects it was 6-14% (Wetherill et al., 1974). Alexander
et al. (1973) reported that a group of children whose ages
ranged from 9 months to eight and a half years absorbed
50% of .the dietary lead intake. Studies on experimental
animals have shown that other dietary compounds, e.g. phytate,
protein and lactose may have significant effects on the
absorption of lead (see section IV. 3.3.1.2).
H
IV.3.2.1.2.3 Mercury
TGMA (1973) considered that the absorption of inorganic
mercury was at least 7% on the average. This figure will
be valid for inorganic salts of mercury (II). As for the
absorption of elemental mercury introduced into the gastro--
intestinal tract, it is extremely limited, probably amounting
to less than 0.01% of the administered dose. The absorption
of methylmercuric compounds is about 95% of the administered
dose in human beings, irrespective of whether the methylmercuric
radical is bound to protein or administered as a water
solution of a salt-.
IV.3.2.1.3 Percutaneous absorption
The skin can be an important route by which metals enter
the body, especially for organomctallic compounds and metal
K "*
The different forms of mercury will be classified as ele-
mental, inorganic and organic. Elemental mercury is the
unoxidized element which usually exists as liquid or vapor.
Inorganic compounds include both monovalent (mercury (I)
or mercurcus) and divalent (nercury (II) or mercuric) mercury
salts and include also complexes and chelates with organic
ligands in which mercury is reversibly bound. For the sake
of simplicity the term inorganic will refer to mercuric
compounds unless specified as mercurous. Those compounds
in which mercury is directly linked to a carbon atom by
a covalent bond will be classified as organomercurial com-
pounds or organic ruercury compounds.
109
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salts of fatty acids. This type of absorption has been
dealt with at a meeting of the Subcommittee on the Toxicology
of Metals (Wahlberg, in Dukes and Friberg, 1971) and will not
be discussed further in this report.
y, IV.3.2.1.4 Transfer of metals via placenta and maternal milk
Another route of absorption is the transplacental transfer,
which gives an initial body burden. Fetuses should be con-
sidered as an especially susceptible group. If transplacental
transfer of a particular metal is high, fetal exposure
may be a significant factor for the assessment of the total
exposure. Yet another route of absorption which may be of
consequence in infants is transmission via mother's milk.
These types of exposure were discussed previously (TGMA,
1973).
There is evidence that the human placenta is freely permeable
to certain metal compounds and that some metals are secreted
in maternal milk during the perinatal period. It is during
this period that growth, especially of the central nervous
system, is most rapid. Direct assimilation of metal by
the fetus and neonate may be especially important from
the viewpoint of age-related susceptibility factors. For
example, in the case of methylmercury compounds, transplacen-
tal transfer results in higher blood levels in the infant
at birth than in the mother (Tejning, 1970; Suzuki et al.,
1971; Bakir et al., 1973). Transmission via milk has been
shown to contribute significantly to infant blood levels
of methylmercury (Amin Zaki et al., 1974).
' IV.3.2.2 Transport, distribution and excretion
'.'.j. IV.3.2.2.1 Transport and distribution
An outline of general principles of transport and distribu-
tion of metals in the body has been discussed previously
(TGMA, 1973). It was concluded that the binding of a metal
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to plasma proteins is of great importance for its distribution.
The portion of the metal bound to the "diffusible" fraction
plays an especially prominent role in the exchange of metals
through biological membranes and in the subsequent transloca-
tion to critical body organs. Special comment was made
. on metal accumulation in organ systems with specialized
functions, such as the kidney and the brain. Quantitative
data on the proportion of metal transferred to the critical
organ will be given in section IV.3.4.
J IV.3.2.2.2 Excretion
General aspects of the gastrointestinal excretion as well
as the renal excretion of metals have been reviewed, in-
cluding such topics as the mechanism of these two types .
of excretion and the factors governing them. Specific in-
formation included the relative participation of various
mechanisms in the excretion of different metals, and where
possible, also the quantitative relations to the total.
body burden (TGMA, 1973).
In addition to the mentioned aspects, the renal excretion was
discussed in relation to the functional impairment of organs
as well as the excretion of metals via the mammary gland.
These data are basic to the understanding of the metabolic
models for the various metals and to the understanding
of the use of excretion media as indicators (or indices) of
exposure and/or accumulation. Since such data are not in-
dispensable for dose-response evaluations and calculations,
they will not be given in detail in this report.
''. IV.3.2.3 Accumulation and retention in critical organs
'•. IV.3.2.3.1 General principles
An organ accumulates metal when uptake exceeds elimination;
a steady state is reached when uptake equals elimination.
A common way to express the elimination rate is in terms
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of biological half-time, a concept which is only meaningful
when the elimination can be approximated with a reasonable
accuracy to a single exponential first order function.
The concentration in the organ at any time then can be
expressed by the following equation:
C = concentration in the organ at time t
C0= concentration in the organ at time 0
b = elimination constant
t — time
(1)
The relation between the elimination constant and the bio
logical half-time is as follows:
T = biological half-time
In2 = natural logarithm of 2 = 0.693.
The exponential function is not always representative of the
elimination of toxic metals; therefore the equations should be
treated with caution.
Of pertinence for toxicological evaluation is the highest con-
centration reached in the critical organ during continuous ex-
posure. A simple model, based on the assumption that metal
turnover occurs according to the above mentioned retention
equation (1), that the rate of uptake of the metal is faster
than the elimination rate, and that a constant fraction
of the intake is taken up by the organ, will give the fol-
lowing expression for the amount accumulated in the critical
organ during continuous exposure:
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» a /i ~btv ,-»
b ^ ^ '
A = accumulated amount
a «= fraction of daily intake taken up by the organ
b = elimination constant
t = time of exposure
At steady state:
The above mentioned mathematical expressions were worked out
by the TGMA (1973), and to reiterate, they are based on
the assumption that intake and absorption are constant.
If either of these changes systematically with age, type
of exposure, period in time, etc., the equation must be
modified.
If the elimination rate represents elimination from more than
one compartment, it will be necessary to have a multifunctional
mathematical expression. In such cases, multiple biological
half-times or multiple elimination rates must be calculated
from available data.
For most metals, the assumption that all of their movement:
in the body is dependent only on the concentration gradient
is an oversimplification. Active transport or metabolic
processes are often involved in metal turnovers. Consequently,
it has been recognized that this simple model generally does
not give a sufficiently accurate description of the concentration
in the critical organ. Specific data as to the accumulation of
various metals in tissues as well as numerical values for
fractions- in critical organs and biological half-times must
be presented to broaden the possibilities of the model, as
was done by TGMA (1973).
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IV.3.2.3.2 Cadmium
A simple model implying that a constant fraction of absorbed
cadmium is directly transferred to the kidney may not be adequate
to arrive at the renal accumulation of cadmium, since»a
part of the transfer probably occurs slowly via the liver
(Nordberg, 1972b). Since to date no other models have-been
introduced, the simple model might at least provide an
approximation. Interpretations of such approximations of
course must be made with caution.
Using the simple model, biological half-times for the'kidney
and kidney cortex between 17.6 years (Tsuchiya and Sugita,
1971) and 38 years (Kjellstrom, 1971; Kjellstrom et ai.,
1971; Friberg et al., 1974) have been estimated, based
on available information on the accumulation of cadmium
in cross-sectional samples of Japanese and U.S. populations.,
The fraction of whole-body cadmium that will be deposited
in the kidney varies depending on the time after administra-
tion. Autopsy material from a normal population at age
50 suggests that 1/3 of the body burden is located in'the
kidneys. On higher exposure (industrial) the fraction of
the body burden that is found in the kidney is smaller
than 1/3.(Friberg et al., 1974).
t
Using the model described by equation (3) and correcting
it for systematic changes in caloric intake and kidney
weight during lifetime, it has been calculated that the
cadmium concentration in kidney cortex at 50 years of age
would be 50 yug/g following a daily intake of 24 ng/calorie
(65 ^ig/day for an adult) assuming about 1.7% (1/3 of 5%)
deposition of body intake in the kidney (Friberg et al.,
1974). Further data from calculations based on these assump^
tions will be found in section IV.5.2.2.
/"'
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IV.3.2.3.3 Lead
The major complication in establishing a mathematical model
for lead absorption or retention is the strong affinity
of lead for bone. The estimates of half-time for lead in
bone have varied from 64 days in the spine of rats to 7,500
days in the skeleton of a dog. The biological half-time
*
for lead in bones of humans has been estimated to be 10 years
(TGMA, 1973). 4
It has been thought that there are at least four bodily com-
partments for lead. The retention of lead has been treated
as a power function model rather than an exponential model
in some publications. It was concluded during the Buenos
Aires meeting that lead required a particularly complex
metabolic model.
Since that time, Rabinowitz et al. (1973) have introduced
a new technique for measuring lead metabolism, by administra-
tion of a stable isotope to human subjects and determination
of isotope ratio and lead concentration in indicator (or index)
media. The results fit a 3-compartment mathematical model.
Compartment one is blood lead, possibly having a rapidly
exchanging soft tissue component, and has an estimated
X
mean life of 27 days (corresponding to a biological half-
time (T) of 18.7 days). A second compartment consists of
a more stable soft tissue component and the rapidly exchange-
able fraction in bone with an estimated mean life of 30
days (corresponding to T = 20.8 days). The third compartment
is the stable fraction in bone with a much longer mean
life of almost 30 years (T = 20.8 years). This is compatible
with earlier estimates of half-time of about 10 years.
These data are derived from studies of two normal male
adults without abnormal exposure to lead.
lean life =^^r or = , •£
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.15 IV.3.2.3.4 Mercury \
The different forms of mercury exhibit^ different kinetics as
regards distribution in the body. Distinction will be made
between elemental mercury, inorganic mercury compounds,
and various organomercury compounds. ^
I /
").i IV.3.2.3.4.1 Mercury vapor
TGMA (1973) concluded that quantitative data were not suf-
ficient to be entered into a model for mercury accumulation
in the human brain after vapor exposure. The biological
half-time in the brain has been shown to be longer than
that in other organs in animal experiments, but it is not
possible to give exact figures. Certain areas of the brain
especially retain mercury (TGMA, 1973). Data from Takahata
et al. (1970) indicate a long biological half-time, at
least some years, for mercury in the brain vof human being?
after vapor exposure. A discussion of the biological half-
time of mercury in the brain, including how to interpret
animal data when modelling the human situation is available
(Berlin, M. 1975); in rats approximately one percent of
the absorbed dose was present in the brain.in a short-term
experiment. No corresponding percentage has been established
for human beings.
Mercury vapor (Hg°) is oxidized to Hg in blood (Clarkson
et al., 1961). However, the delay after inhalation allows
some mercury vapor to remain dissolved in the blood stream
for sufficient time to reach the blood-brain barrier (Magos,
1967, 1968). This explains why, after .vapor exposure, the
brain takes up more mercury than after^ the injection of
equivalent doses of inorganic mercury vsalts (Berlin, M.
et al., 1966, 1969; Nordberg and Serenius, ig.69). Approx-
imately 30% of the inhaled dose is retained in man after a
brief exposure (Teisinger and Fiserova-Bergerova, 1964j
Nielsen-Kudsk, 1965a). The uptake of mercury in man (Nielsen-
'*'' 116
-------
Kudsk, 1965b) and rats (Magos et al., 1973) is decreased
by ethanol. Amino-triazole has the same effect on rats
(Magos et al., 1974a). Because these agents increase the
exhalation of Hg° from rats injected i.v. with elemental
mercury, it is clear that this effect is due to the inhi-
bition of the oxidatii
Magos et al., 1974a).
bition of the oxidation of Hg° to Hg+* (Magos et al., 1973
Differences in the distribution of mercury between pretreated
and control groups indicated that oxidation takes place
not only in the blood but also in other tissues. Studies
in vitro using blood as a model system showed that catalasa
is the enzyme mainly responsible for the oxidation of Hg°
(Nielsen-Kudsk, 1973; Magos et al., 1974a). Experiments
carried out with acatalasemic mice confirmed this finding
(Sugata and Clarkson, unpublished data).
IV.3.2.3.4.2 Inorganic mercury compounds
The biological half-time of mercury in the whole body of
humans given mercury (II) orally has been found to be 29-
41 days for 5 women and 32-60 days for 5 men (Rahola et
al., 1972; Miettinen, 1973). About 7% of the total administered
dose was excreted with this half-time. This figure involved
-a miniirmm absorption figure (TGMA, 1973). According to
data from animal experiments it may be concluded that the
half-time in the kidney is somewhat longer than that in
the whole body (Rothstein and Hayes, 1960, and others;
review: Nordberg and Skerfving, 1972). The retention of
mercury in the kidney of animals is shorter than in the
brain (Berlin, M. and Ullberg, 1963; review: Nordberg and
Skerfving, 1972). The 'rather complicated, multicompartmen-
tal behavior of the kidney on different types of exposure
to mercury is still poorly understood. At low dose levels
a rather large amount of total mercury elimination occurs
-via bile and feces. With increasing administered dose,
a larger part of the absorbed amount is eliminated by urine,
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as shown for rats (Rothstein and Hayes, 1960) and man (Bflrgi,
1906) .
IV.3.2.3.4.3 Organomercury compounds
The short-chain alkylmercurials (methyl, ethyl) possess
kinetics markedly different from those of other organomer-
curials. They will therefore be treated separately.
IV.3.2.3.4.3.1 Methylmercuric compounds
The metabolism of methylmercuric compounds is sufficiently
well known to allow adequate calculations of accumulation.
Data from studies in animals and man justify the assumption
that a relationship exists between the concentration in
each organ and the total body burden of rnethylmercury,
and that a relationship exists among the levels of methyl-
mercury in the different organs (Berglund et al., 1971;
Nordberg and Skerfving, 1972). Elimination is correlated
with the total body burden, i.e. a definite fraction of
this is eliminated per unit of time. The course of elimination
in continuous exposure thus can be expressed according
to equation (3). Reasonably accurate calculations should
be possible for the brain content as long as no signs of
toxicity occur. A higher brain/blood quotient in monkeys
after neurological damage than in monkeys not exhibiting
signs of toxicity has bean shown by Berlin, M. (1975).
This implies that at the time neurological damage takes
place, the distribution pattern of methyImercury .changes.
This point will be discussed further in the section on
dose-response relationships.
Data on the biological half-time in the brain of humans given
tracer doses do not suggest that it would be different from
that in the body as a whole (Aberg et al. , 1969; Berglund
et al., 1971). The latter has been estimated to be about
70 days on the basis of both human experimental and epi-
demiological.data. In nine male and six female volunteers
118
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given tracer amounts of methylmercury, the half-time for
whole-body clearance ranged from 52 to 93 days, with a
mean value of 76 days (Miettinen, 1973). An even more sub-
stantial variation in biological half-time was found in
blood by Skerfving (1973), ranging from 58 to 164 days
in 5 persons consuming MeHg from fish. One person had
164 days and significantly differed from the others (58-
87 days). Bakir et al. (1973) found a range of 45-105 days
and a mean of 65 days for mercury in the blood of 16 persons
in the Iraqi outbreak.
Recent data from Al-Shahristani and Shihab (1974), based
on sequential analysis of mercury in hair from 48 persons
poisoned by methylmercury compounds in Iraq, show a vari-
ation in the half-time from 35 to 189 days, with a mean
value of 72 days. The distribution curve for the biological
half-time in these 48 persons had two distinct peaks, one
ranging from 35 to 100 days which represented about 90%
of the group and the other ranging from 110 to 120 days.
This finding and that of Skerfving (1973) suggest that
a deviant population might exist who have a longer elimina-
tion time for MeHg .
Using tracer doses of a radioactive methylmercuric compound (Aberg et
al., 1969), it was found that the amount of radioactivity
in the head was 10% of the total body burden. Since the
brain concentration substantially exceeds that in other
tissues of the head, it can be assumed that the amount
present in the brain is about 90% of the total head burden.
Taking the formula (3) and a biological half-time of 70
days, calculations at steady state in the brain have been
made which show a concentration of approximately 1.5 ug
Hg/g brain tissue at a daily absorption of 4 pg Hg/kg
body.weight in the form of a methylmercuric compound (Figures on
steady state brain concentrations at a certain intake level were
119
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erroneously stated in the report by TGMA (1973).
Biotransformation of methylmercuric compounds, resulting
in the release of inorganic mercury, has been demonstrated
in different animal species (review: Nordberg and Skerfving,
1972) and also in man (Sumino, 1973; Bakir et al., 1973).
The mechanism of this biotransformation is unknown. The
concentration of inorganic mercury in the brain is low,
that is, less than 10%, relative to methylmercury. In
whole blood the relative amount of inorganic mercury is
low, which is due to the difference in red-cell-to-plasma
distribution of inorganic mercury and methylmercury. The
relative amount of inorganic mercury in plasma is high
while almost all mercury in the red cells is in the organic
form. From 25-85% of the mercury in bile was found to
be inorganic in the squirrel monkey (Berlin, M. et al.,
1975a) and 40% was inorganic in human mothers' milk (Bakir
et al.,1973). The urine also contains a relatively high
proportion of inorganic mercury.
IV.3.2.3.4.3.2 Ethylmercuric compounds
Suzuki et al. (1973) have reported on the metabolic fate
of an ethylmercuric compound. Five patients accidentally
received human plasma infusion containing ethylmercurithio-
salicylate. The mercury in red cells from these patients
was mainly in an ethylmercuric form; in contrast that in
plasma and urine was inorganic. Only the former showed
a rapid decrease with time (half-time: about 10 days).
Observations on autopsy tissue from one patient revealed
that 8.6% of the administered dose was recovered from
the brain.
IV.3.2.4.3.3 Other organomercury compounds
The other organomercurials of practical importance are
the aryl and alkoxyalkyl mercurials such as phenyl- and .
methoxyethylmercury compounds. Studies on experimental
animals indicate that these compounds are rapidly con-
120
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verted to inorganic mercury in the body tissues. Within a
few days after a single administered dose, the organmarcuri-
al has disappeared and the distribution in tissues closely
approaches that of inorganic mercury (review: Nordberg
and Skerfving, 1972).
IV.3.3 Metal concentrations in biological media as indicators
(or indices) of exposure and of concentrations in critical
organs
The following general aspects have been presented previously
(TGMA, 1973). In assessing the effects and dose-response
relationships connected with the accumulation of metals,
especially those with long biological half-times, it is
often desirable to use concentrations of metals in blood,
urine or hair. When steady state is reached, a constant
ratio is held between blood and tissue concentrations of •
the metal, and except for metabolic variations, there is
also a relationship to the urinary concentration of the
metal. For a biological material to be a good indicator
of accumulation, especially for substances with long bio-
logical half-times, it is not enough that the relation
should be constant at steady state conditions; it should
also be reasonably constant during the period of accumulation.
Blood, which transports most absorbed metals, is not necessarily
a good indicator .of accumulation in the critical organ.
V, IV.3.3.1 Cadmium
The usefulness of various biological materials as indicators
(or indices) of exposure and retention has recently been
discussed (Friberg et al. , 1974; Nomiyama et "al. ,-' 1975a) .
TGMA (1973) and Friberg et al. (1974) considered that cadmium
concentrations in urine during continuous exposure would in-
crease sharply with the advent of renal tubular impairment.
Nomiyama et al. (1974) and Nomiyama and Nomiyama (1975)
found an increase after long-term administration of high
levels of cadmium to experimental animals per os.
121
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A sharp increase in urinary concentrations of cadmium in
connection with the appearance of morphological changes
in the renal tubules of rats has been reported (Kawai et
al., 1975). In mice, Nordberg (19723, 1972c) showed that
during the early phase of exposure, before renal tubular
impairment occurred, a correlation existed between the
body burden and urinary concentration of cadmium. Tsuchiya
et al. (1972a, 1972b), in studies on normal Japanese not
excessively exposed to cadmium, estimated a biological
half-time of 13 years for the body compartment reflected
in urinary excretion, which is not widely different from
the estimated biological half-time in the liver (7 years)
and kidney (17.6 years) in the same study. These results
support the evidence of a relationship on a group basis
between urinary excretion and accumulation in the main
storage organs for cadmium in the body during the phase
when there is no renal tubular impairment (TGMA, 1973).
Due to a substantial interindividual variation, individual
values may not always lend themselves to estimates of body
burdens of cadmium.
Conclusive evidence on the relation of blood values of
cadmium to the concentration in the kidneys has not been
presented. Available evidence from animal experiments and
human data has been compiled and discussed elsewhere (Friberg
et al., 1974). It was concluded that blood values probably
do not reflect kidney accumulation of cadmium but may reflect
the most recent exposure.
IV.3.3.2 Lead
Except under controlled experimental conditions the dose
cannot be measured exactly, but lead concentrations in
various biological media may be used as an estimate of
dose. These are indirect measures, or in other words in-
dicators of dose.
122
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Blood lead levels are the most reliable measurement of recent
exposure and particularly useful for epidemiological studies.
Interpretation of blood lead in the individual must take into
account the following considerations. This indicator reflects
a dynamic balance between exposure, amounts in tissue, and
excretion. Blood lead level, however, reflects only one
point in time and it may reflect a dose which is steady,
increasing, or decreasing. Lead levels of critical organs
such as brain, bone marrow and kidney probably relate closely
to effects in these organs, but they cannot be measured readily,
For this reason, studies relating lead concentrations in
blood, urine, and other biological samples with those in
the critical organs are lacking while data relating the
blood lead levels to current exposure are abundant. There
is a long-standing debate as to whether blood lead levels
should be corrected for hematocrit values. About 90% or
more of blood lead is in red blood cells; therefore care
should be taken to assure that the sample is homogeneous.•
Urinary lead may also reflect recent exposure but is more
highly variable than blood lead. One method for estimating
mobile tissue lead is the measurement of urinary excretion
following EDTA administration. In studies on children and
adolescents (Chisolm et al., 1975) a statistically signi-
ficant linear relationship was found between blood lead
concentration and the logarithm of the quantity of lead
excreted in the 24-hour period immediately following ad-
ministration of calcium EDTA in a dose of 25 mg/kg. Thus,
the mobile fraction of the body lead burden increases ex-
ponentially as blood lead concentration increases arithmet-
ically in the range of 8-75 pg Pb/100 g blood.
Further investigation with regard to dose and rate of ad-
ministration of calcium EDTA may provide better standardi-
zation of this test for estimating the chelatable or mobile
portion cf the body lead. This may serve as a more reliable
"chemical biopsy" of the concentration of lead in critical
i23
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organs in individual humans than single measurements of
lead in blood or urine.
Lead levels in hair may reflect long-term exposure but
because of the problem of external contamination, these
may not be a good indicator of body burden (TGMA, 1973) .
'Increased concentration of lead in teeth persists even
after blood lead levels have decreased (Albert et al.,
1974).
It has been suggested on the basis of studies in humans
(Needleman and Shapiro, 1974) and baboons (Albert et al. ,
1974) that the lead concentration in dentine of shed deciduous
teeth is a good indicator of past exposure to lead during
infancy and early childhood. Factors such as time of minerali-
zation of the tooth, its status in regard to caries, resorp-
tion of the root, localization of the dentine analyzed,
and the method of sampling have to be taken into considera-
tion when interpreting data about dentine lead content.
For estimating total body burden, bone lead concentration
is most accurate but is largely inaccessible except at •
autopsy If bone is biopsied, care should be taken to
distinguish between membranous (cortical) and cancellous
(trabecular) bone.
IV.3.3.3 Mercury
IV.3.3.3.1 Mercury vapor
Correlations have been established between exposure and blood
or urine values in studies of groups of workers (Smith et al.,
1970). Urine values for individuals are useful as an indi-
cator (or index) of exposure only when serial monitoring
is carried out (Suzuki et al., 1968).
No evidence has been obtained on the relation between blood
and brain (critical organ for prolonged exposures) values
in humans. In individual cases, blood or urine values may
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give conflicting conclusions about the risks for poisoning.
'. IV.3.3.3.2 Inorganic mercury compounds
There is not a constant ratio between mercury concentration
in blood and kidney (critical organ) according to animal
experiments (Berlin, 1963; review: Nordberg and Skerfving,
1972). Studies in man by Miettinen (1972, 1973) following
a single oral tracer dose showed that the biological half-
time in blood (20 days) is half of that in the entire body
(40 days). It seems probable that blood is not a good index
for evaluation of mercury accumulation in kidney. Animal
experiments do not support urine mercury as a good index
of kidney concentration (TGMA, 1973).
IV.3.3.3.3 Organomercury compounds
IV.3.3.3.3.1 Methylmercuric compounds
Animal experiments have shown that blood concentrations of
mercury correlate with mercury concentrations in the brain
after exposure to methylmercuric compounds, and that these
parameters are linearly correlated with the absorbed amount
of MeHg up to dose levels at which clinical signs occur
(review: Nordberg and Skerfving, 1972). In epidemiological
studies, a linear correlation between dose measured as
daily intake of MeHg , and mercury level in blood and hair
(Bakir et al., 1973; Skerfving, 1974; Kazantzis, 1975;
Suzuki, 1975) has been observed. Thus, at dose levels of
MeHg not associated with signs or symptoms of poisoning,
there is good evidence that blood concentrations of mercury
reflect the MeHg concentration in the brain. At dose levels
of MeHg causing toxic manifestations, animal experiments
have shown that mercury concentrations in blood and brain
rise more rapidly with cumulative daily dose than at lower
body burdens of MeHg . Epidemiological data from Iraq (Bakir
et al., 1973) suggest that this may also be the case in
man.
125
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Available evidence indicates a rectilinear relation between
total mercury levels in blood and hair, the hair levels being
about 300 times higher than the whole blood levels for MeHg
(Berglund et al. , 1971; Nordberg and Skerfving, 1972). These
conditions make hair levels a good indicator of body or brain
accumulation of mercury. Standards for type and length of
hair to be clipped must be set up. The delay in the appearance
of mercury in hair can be a drawback, but it can also be useful.
With knowledge about the growth rate of hair, the course of
past exposures can be evaluated (Tsubaki, 1968; Al-Shahristani
and Shihab, 1974). Several factors may lead to errors in the
interpretation of mercury levels in hair:
(1) Hair collected from different sites of the body
has different growth rates.
(2) External contamination may result from occupational
exposure and from the use of cosmetic and phar-
maceutical preparations. The possibility
of external contamination from mercury in sweat
needs investigation.
(3) When hair samples are used to recapitulate brief,
high level exposures such as in the Iraqi outbreak,
corrections must be made for differences in growth
rates of individual strands of hair and for accident-
al displacement of the strands during collection
and transportation (Giovanoli and Berg, 1974).
Urinary values of methylmercuric compounds do not have practi-
cal significance because of the low levels of MeHg in urine,
and the concomitant presence of inorganic mercury. Total
mercury in urine therefore is not a reliable measurement
of body burden of methylmercuric compounds .
IV.3.3.3.3.2 EthyMercuric compounds
Because of the limitations of available data, concentrations
in blood, hair, and urine relevant to exposure cannot be fully
126
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discussed. Except for the elevated rate of release of in-
organic mercury which has been shown in the mouse, rat and
man (Takeda and Ukita, 1970; Suzuki et al., 1973), ethyl-
mercuric compounds display a similarity to methylmercuric
compounds in their kinetics. For this reason, the remarks
on indicator (or index) media for methylmercuric compounds
probably apply to ethylmercuric compounds as well.
IV.3.3.3.3.3 Other organomercury compounds
Concerning organomercurial compounds other than the alkyl-
mercuric ones, it is assumed that both urinary excretion data
and blood values, when used on a group basis,might provide
information about the exposure (review:,Nordberg and Skerfving,
1972).
IV.4 EFFECTS
IV.4.1 General considerations
When a toxic metal compound contacts a cell or tissue, binding
occurs to various ligands in that tissue. This binding may
be defined as an effect, but may or may not harm the function
of the cell. A small number of metal-containing molecules usual-
ly do not influence the function of the cell, or there may
exist a subcritical effect, i.e. an influence which is not
adverse. When larger numbers of molecules are bound, the
degree of effect depends on the function of the binding li-
gands .
A functional change may occur if the response at the molecular
level involves a certain number of binding sites. AS defined
earlier, the critical concentration in a cell is reached when
the functional change becomes adverse. Only a few studies on
critical cell concentrations have been reported probably begause
of the difficulties in conforming to adequate model systems
and the extreme difficulties in studying critical effects on
individual cells in a living animal.
127
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Theoretically, individual cells can be observed in microscopic
investigations, but if cellular injury does not include speci-
fic morphological features, it is often difficult to assess
whether changes in individual cells are induced by metals.
A small number of cells normally undergo degeneration.
It is not easy, at present, to correlate morphological find-
ings with concentration of metals in individual cells. A further
discussion will be given in sections IV.4.1.2 and IV.5.1. /;,.• ,
Granted the present state of knowledge concerning metal toxic-
ity, a more viable entity for discussion is the critical or-
gan concentration. Specific quantitative data for critical
organ concentrations for various metals will be discussed in
Section IV.5; also, relationships between concentrations of metals
in indicator (or index) media and effects will be given there.
In this section, a general outline of the types of changes
that may be induced by metals will be given, and it will also
be discussed how they can be assessed by clinical observation,
functional tests, and morphological and biochemical techniques.
It will be attempted to draw general conclusions concerning
the pathogenetic mechanisms by which metals exert their ef-
fects in biological tissues. Types of effects evidenced for
various aetals, especially cadmium, mercury, and lead, in
experimental animals and exposed human beings will also be
reviewed. An appraisal as to which of these effects may be re-
garded as the critical effect in human beings during different
types of exposure situations will be made.
IV.4.1.1 Genera] aspects of symptoms, signs, and other changes
assessable by functional tests
Two kinds of effects are symptoms and signs. By symptoms are
meant here complaints of individuals; signs are observed by
examinations and tests. Both symptoms and signs can be in-
duced by exposures to metals. Metals may interact with other
128
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substances to give a certain clinical syndrome. They may also
interact with each other, thus modifying their per se effects.
Standardized classification of functional disturbances at the
physiological and clinical levels is a prerequisite for quan-
titative evaluation of such effects. This is particularly true
for case studies and epidemiological work, which constitute
the foremost approaches for evaluating clinical manifestations
of effect.
Some metals give rise to an immediate, local effect at site
of absorption, whereas others give systemic effects a consi-
derable time after absorption has taken place. A clinical effect
after repeated exposures may be a result of accumulation of
metal, reaching a certain critical concentration, or sometimes
a result of repeated subclinical damage. In the specific part
of section IV.4 to follow (sections IV.4.2, IV.4.3, and IV.4.4), the patho-
genic mechanisms for clinical manifestations of metal toxicities
will be delved into. An attempt will be made to place the types .
of damage in the mentioned categories.
The clinical effect may be followed by complete restitution,
but chronic functional changes may also result. The relation
between biochemical alterations and clinical observations is
of great interest, especially when latency periods and secondary
effects enter into the picture.
IV.4.1.2 General aspects of morphological changes at various
structural levels
Metals may give rise to gross structural macroscopioally evi-
dent organ damage, light microscopical morphological changes
as well as to ultrastructural ones. These various types of damage
may be observed both in laboratory animals and human beings.
A compelling topic is how morphological observations relate
.to biochemical and functional ones. It may be argued that any
129
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biochemical change involves changes in molecular configuration
and therefore structural change. Thus, the debate on which
comes first/ the biochemical or the morphological change, is
artificial; the decisive factor is actually the resolving power
of biochemical analysis versus morphological analysis. For
some types of injury such as permeability changes in membranes,
there is often ultrastructural change discernible by transmission
electronmicroscopy (e.g., for metals like lead, mercury and
cadmium). Interaction with mitochondrial membranes usually
results in change in mitochondrial configurations. Combinations
of data concerning clinical observations, light microscopical
and ultrastructural, as well as biochemical changes may give ,
interesting indication on the mechanism by which metals exert1
their effects on tissues.
Conceptually, there may be two mechanisms for producing a
change observable by morphological techniques. One mechanism is
metal interaction with membranes in the cell in such a way
as to bring changes in the function and integrity of intra-
cellular organelles such as mitochondria. Such changes, when
known to reflect cellular degeneration, may be regarded as
adverse effects. The other mechanism behind morphological change
is the binding of the metal to certain ligands in the cell,
these tissue-metal complexes becoming visible by morpho-
logical techniques. Such metal-tissue binding may or may not
interfere with the structural and functional integrity of the
cell. Depending on its nature, such an effect may or may not
be regarded as an adverse effect in the cell. Sometimes one
morphological change alone (e.g., lead inclusion bodies).enables
a specific diagnosis of the kind of metal exposure. In most
cases a combination of observations is necessary to ascertain
that one given change has indeed been induced by metals. This
is especially true of the type of morphological change that
is nonspecific.
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Single or repeated exposures may give quite different types
of morphological changes. Usually more subtle morphologi-
cal changes are reversible, whereas necrotic tissues represent
nonreversible changes. Some individual necrotic cells may be
replaced by regeneration. Direct and delayed effects may have
specific morphological features and their relation to biochemical
modifications could be a fruitful topic for discussion and research.
Morphological aspects of changes arising as a result of accu-
mulation of damage versus damage from accumulation of metals
are noteworthy. Sometimes interactions of various effects may
be explainable on the basis of morphological features.
A specific type of structural effect, chromosomal aberrations,
can be induced by metals and metal compounds (e.g. methylmer-
cury). These aberrations reflect interference with the genetic
material of the cells, but their relation to somatic effects
in the individual or his offspring is difficult to assess.,
Carcinogenic and cocarcinogenic effects can also be induced
by metals. Such effects are usually assessed by long-term
experiments on animals or by statistical evaluation of obser-
vations on man. Much attention has recently been drawn to
structural '.effects of various chemicals, including metals and
metal compounds, that are induced during embryogenesis and
referred to as teratogenic effects.
IV.4.1.3 General aspects of biochemical changes
Biochemical approaches are indispensable for our understanding
of the effects of toxic metals and other toxic substances.
Many avenues of approach have opened with modern technology
and new knowledge, especially in molecular biology, e.g., the
role of cAMP. These avenues remain to be explored in greater
depth. This review cannot give an exhaustive treatment on
biochemical effects, but has been limited to some examples.
Specific data on biochemical modifications that may constitute
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the basis for signs of symptoms of adverse effects will be
given in later sections. It should be pointed out that the
knowledge of biochemical changes is limited, and most of the
results have been obtained with in vitro experiments.
IV.4.1.3.1 Binding with proteins and other ligands '
Binding of different metals with differentligands may constitute
the primary effect at the biochemical level, accounting for the
many effects of metals on cell membranes, enzymes, and other
cellular constituents. For example, lead, mercury, and cadmium
show a strong affinity for ligands such as sulfhydryl groups,
phosphates, cysteinyl and histidyl side-chains of proteins,
purines, pteridines, and porphyrins, and can therefore act at
a large number of biochemical sites. In addition, metals may
interact with many other ligands, such as hydroxyl, carboxyl,
amino and other groups. The interference with specific bio-
chemical activities will depend on the type of ligand and .the
structural and functional organization of these ligands, e.g.,
whether active binding sites are affected or not. General aspects
on the biochemical background for functional impairment induced by
toxic metals have been.given elsewhere (Singerman, 1972; Vallee
and Ulmer, 1972; Sigel, 1974).
IV.4.1.3.2 Effects on the cell membrane
The affinity of metals for cell membranes results in a
particular susceptibility of membrane functions. The cell
membrane is considered to be an active part of the cell, con-
trolling the transfer of substances into and out of the cell
by means of exchange mechanisms for active transport or car-
rier mechanisms for passive transport. The essential elements
of the carrier mechanism are the reactions of a transport
substrate with a membrane component to form a complex, which
then moves from one side of the membrane to the other where
the substrate is released. The structure of cell membranes
contains large quantities of lipids, and,since metals may
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bind strongly to the cell membrane, this may lead to changes
of the surface active properties of the lipid. These and
other changes may further lead to alterations of membrane
permeability and metabolic activities of surface enzymes.
The effect on activity at the cell membrane may be propor-
tionately low compared to the amount of metal present, in that
the metal may bind in large proportion to inactive sites of
the membrane.
"• IV.4.1.3.3 Effects on enzymes
Metals may inhibit the enzymes of the membrane, resulting in
modifications of the utilization of nutritionally-required
external substrates, the active transport mechanism and the
synthesis of membrane constituents. Obviously, the effect on,
enzymes may also occur wherever they are present, including
intra- and extracellular locations. Reduction in enzyme
activity depends upon the accessibility of metal-binding
groups. For example, it has been shown in vitro that lead,'
mercury, and cadmium inhibit a large number of enzymes having
functional sulfhydryl groups. Additional important binding
sites on enzymes include the carboxyl groups of glutamic and
aspartic acid and the epsilon aminoacid group of lysine. Some
enzymes, the so-called metalloenzymes, contain a metal which
is bound in such a specific manner that it cannot be removed
without loss of enzyme activity. Toxic metals may displace
the essential metals from such metalloenzymes with resulting
loss in enzyme activity. In some cases the new metalloenzyme
may retain activity, e.g., cadmium and lead carboxypeptidases
have been shoWn to have appreciable esterase activity (Vallee
and Ulmer, 1972). The kinetics of enzyme inhibition has provided
basic information on the probable nature of the action of metals
at the molecular level. In addition to the interaction of metals
with enzymes per se, the rate of enzymic reactions may be re-
duced by binding of metals with coenzymes or substrates. The
-------
observable effects of enzyme inhibition may be on such functions
as synthesis of molecules such as heme, or basic bioenergetics.
When the metal has affected the cell permeability or cellular
structures, such as lysosomes, there may be an increase of en-
zymes in intracellular or extracellular fluids, but metal in-
teractions may also directly "activate" some enzymes.
IV.4.1.3.4 Effects an the energy metabolism system
The chemical reactions providing the energy for the various
functions of the cell are mediated by enzyme systems, but also
require high energy nucleotides. Lead, mercury, and cadmium
disrupt pathways of oxidative phosphorylation, a process as-
sociated with the integrity of the mitoahondrial membrane;
the precise effect depends upon the individual properties of
the metal. Lead forms complexes with the phosphate groups of
nucleotides and catalyzes a nonenzymatic hydrolysis of
nucleosidetriphosphates, particularly ATP (Rosenthal et al,.,
1966a, 1966b). It may also interfere with ATP synthesis when
an energy dependent mitochondrial lead uptake occurs (Walton,
1973).
IV.4.1.3.5 Effects en nonenzymic molecules
Metals may cause structural changes in proteins resulting in
denaturation, alteration of bioelectric properties, impairment
of the transmission of nerve impulses, and loss of transport
or other vital functions. The activity of very large molecules,
such as hemoglobin, intermediate-sized molecules such as
metallothionein, and small molecules important for their
redox potential, such as ascorbic acid or glutathione, may
all be affected by toxic metals. Lead, mercury and cadmium
all bind to nucleic acids affecting the configuration, of these
essential molecules. Those metals which are transition
elements may form many different complexes in biological
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systems on account of their changes in oxidative state. While
lead, mercury, and cadmium are not transition metals, they
do have strong tendencies to complex formations.
IV.4.1.3.6 Other biochemical considerations
It should be clear that metals may interact with cells in many
ways, including the membranes, organelles, metabolic and
mitotic systems. The cell is a complex organism with its own
internal homeostatic mechanisms, and its function as a whole
should not be forgotten when investigating biochemical
mechanisms at the molecular level. Similarly, the whole
human being must be kept in focus when examining cells, organs
or functional system. For example, if metals interfere with
biochemical mechanisms for the production of hormones, the -
functional and morphological damage caused by hormone im-
balance may be remote from the cell affected by the metal.
Trace elements are usually considered as those required by
man in microgram to milligram quantities. These include iron,
zinc, copper, and manganese in larger amounts; selenium,
chromium, cobalt, and, possibly, molybdenum are included
in lesser amounts. Certain animals on deficient diets have
been shown to require tin, nickel and vanadium in order
to perform essential functions (WHO, 1973) . At present
there is no known essential role for lead, cadmium and
mercury in biological systems; rather, they may interfere
with the role of essential metals by competing for binding
sites. (Such metal interactions will be elaborated upon
in Chapter 6). Some effects of toxic metals may involve
a mechanism in which the metals act as haptens, and protein-
bound metals act as antigens, resulting in an allergic
type of effect, e.g., metal fume fever and skin sensitization
due to nickel and chromium. It is also possible that •
toxic metals interfere with immunological reactions.
IV.4.2 Effects of cadmium and its compounds
Acute cadmium intoxication usually occurs accidentally after
inhalation of cadmium dust or fume in industry and following
135
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ingestion of food, water and beverages contaminated with
high levels of cadmium. Long-term inhalatory and ingestion
exposures may occur via the same respective media in the
industrial and general environments and may affect relatively
large groups of people. In this section some comments on acute
effects will first be given. Later on, more emphasis will be
placed upon effects observed in connection with the long-term
type of exposure.
IV.4.2.1 Acute effects; general aspects of cadmium toxicity
Acute effects of cadmium have been recognized for a long time
and, in man, include pneumonitis resulting from inhalation of high
concentrations of cadmium dust or fume. With ingestion of fluids
with concentrations of cadmium soluble salts exceeding about
15 mg/liter, vomiting occurs in man. In animals such as rats
or mice this reaction does not occur (Schwartze and Alsberg,
1923) and exposure to much higher peroral concentrations is
possible. This explains why experimental poisoning by inges-
tion may be rather easily achieved in rodents, whereas poi-
soning showing systemic effects requires very long times in
man and other primates. Acute effects in experimental animals
upon injection of cadmium salts include, e.g., effects on .
testicles, sensory ganglia, nonovulating ovaries, placenta,
and fetuses. Although these latter effects have not been observed
in human beings and therefore are of seemingly limited pract-
ical relevance, the studies on them have revealed relationships
of fundamental interest to metal toxicology, e.g. the interaction
between various trace elements, or the influence of tissue
binding upon the type of toxic manifestation.
A specific, low molecular weight protein, metallothionein,
was detected in equine (Margoshes and Vallee, 1957; KMgi
and Vallee, 1960, 1961), and human renal tissue (Pulido et
al., 1966). Its role as a sequestering and detoxifying protein
136
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for cadmium was subsequently suggested by its isolation.
from cadmium-exposed animals (Piscator, 1964) . The synthesis
of metallothionein is inducible by cadmium exposure (Shaikh
and Lucis, 1970, 1971; Nordberg et al., 1971b, 1972; Squibb
and Cousins, 1974), and such induction may play a telling
role in the modification of the toxicological response to
cadmium. It was observed that pretreatment with smaller
doses of cadmium protected against the development of tes-
ticular necrosis (Terhaar et al., 1965; Ito and Sawauchi,
1966; Gunn and Gould, 1970; Nordberg, 1971), lesions in
sensory ganglia (Gabbiani et al., 1967a) as well as the
lethality of injected cadmium (Gabbiani et al., 1967b;
Yoshikawa, 1970). The potential role of metallothionein
in the protection against cadmium toxicity has been docu-
mented, at least for the testicular effects (Nordberg, 1971,
1972a). The inducible synthesis of metallothionein leads
to differences in retention and distribution of cadmium
as well as changes in biological effects, and is of basic
importance for critical organ concentrations of cadmium
and other metals (Nordberg, 1971; Cherian and Vostal, 1974;
Vostal, 1975; Nordberg et al., 1975).
Changes in blood pressure have been reported in experimental
animals after a single injection of very low doses of cadmium
(Dalhamn and Friberg, 1954; Perry et al., 1970). After injec-
tion of relatively low doses, morphologically evident tissue
damage has taken place in testicles (Parizek, 1957, 1960;
Gunn and Gould, 1970; Nordberg, 1971, 1972a), in the non-
ovulating ovaries of prepubertal rats (Kar et al., 1960) and
of adult rats in persistent estrus (Parizek et al., >1968a) ,
and in the placenta (Parizek, 1964). In the last third of
pregnancy in the rat, cadmium salts induce a specific and
highly lethal syndrome affecting not only fetuses but also
mothers (Parizek, 1965); this effect is strictly dependent
on the presence of placenta in the maternal organism (Parizek
et al., 1969a). Cadmium salts given to pregnant hamsters
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were shown to be teratogenic (Ferm and Carpenter, 1968).
With higher injected doses, changes have been observed in
sensory ganglia (Gabbiani, 1966; Gabbiani et al., 1967a).
In addition, several visceral organs including the pancreas,
kidney, gastrointestinal tract, salivary glands, and spleen
display morphological alterations (Kawai et al., 1975).
Vascular injury, which has been shown to be involved in
the morphogenesis of testicular lesions, may also be the
mechanism for the development of the alterations in such
organs as the kidney in cases of acute intoxication. The
type of lesion seen in, for example, the kidney after
acute intoxication differs morphologically from the lesions
seen in chronic intoxication (Kawai et al., 1975). Care
should therefore be taken in interpreting morphological
changes in the parenchymal cells of sensitive organs since
these might be evoked, at least partly, by a coexisting
circulatory disturbance.
The two categories of renal tissue changes (acute and chronic)
have the following morphological features (Kawai et al., 1975)
In the acute category, hydropic swelling and acidophilic
necrosis prevail; the chronic category is characterized by
tubular atrophy. Edema in the surrounding interstitium is
conspicuous, and may eventually proceed into interstitial
fibrosis and nephrosclerosis at a later stage. The chronic
alterations also include mild changes in the walls of blood
vessels and marked thickening of the basement membrane of
the renal tubules. Acute types of lesions in the tubular
epithelium may be seen along with the chronic effect during
repeated administration of large doses. The extent and
severity of acute epithelial changes correspond.roughly
to the daily administered dose (review: Kawai et al. ,,
1975).
Nomiyama (1972) and Nomiyama et al. CL973a, 1973b) demon-
strated that cadmium can depress vital renal functions,
tubular maximum for PAH-transport and tubular reabsorption
of low-molecular-weight proteins (myoglobin).
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IV.4.2.2 Effects of long-term cadmium exposure
Upon long-term exposure via inhalation, cadmium gives rise
to a syndrome including emphysema and effects on the kidney
reflected by the appearance of proteinuria (Friberg, 1948,
1950), and increased formation of renal stones (Friberg 1950;
Ahlmark et al., 1961; Kazantzis, 1970). Slight to modest
anemia also accompanies the clinical picture. Bone mani-
festations of cadmium poisoning have been observed upon oral
exposure in situations in which nutritional conditions have
not been optimal or upon inhalation of high concentrations
of cadmium in industry (review: Friberg et al., 1974).
It is not known with certainty whether this effect can occur
in the absence of evident renal effects of cadmium. A further
discussion on the mentioned effects will be given below. At •
low-level long-term inhalation or oral exposures to cadmium,
the renal changes are usually the critical effect.
IV.4.2.2.1 Respiratory effects >
Friberg (1950) showed that industrial workers exposed to
cadmium dust s'uffered from emphysema with increased residual
capacity. These findings have been confirmed by Baader (1951).
Similar findings, but also including bronchitis, have been
reported (Vorobjeva, 1957; Kazantzis, 1963; Potts, 1965;
Adams et al., 1969; Lauwerys et al., 1974) after exposure
to cadmium dust. After exposure to cadmium oxide fumes,
emphysema (Lane and Campbell, 1954; Bonnell, 1955) and
increased residual volume (Buxton, 1956) have been reported.
An association in epidemiological studies between cadmium
concentrations in tissues and pulmonary disease has been
reported (Lewis et al., 1969; Morgan, 1971. Hirst et al., 1973)
This may be explained by the fact that cigarette smoke contains
cadmium, and cigarette smoking alone can give rise to chronic
respiratory disease (Friberg et al., 1974).
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with age-matched controls. However, the emphysematous group
contained more heavy smokers than the control group.
Pulmonary changes are inducible in animals upon long-term in-
halation of cadmium. Both emphysema and inflammatory changes
were reported by Friberg (1950) after exposure of rabbits to
cadmium dust. Vorobjeva (1957) found similar changes after
intratracheal instillation of cadmium oxide dust in rats.
Snider et al. (1973) reported changes resembling human centri-
lobular emphysema in rats dosed with a 0.1% polydisperse aero-
sol of cadmium chloride in physiological saline.
IV.4.2.2.2 Renal effects; histopathological and biochemical
features
Morphological observations on the nature of the renal damage'
after long-term exposure to cadmium in human beings have dis-
closed variable patterns from isolated slight tubular altera-
tions to severe renal lesions. The earliest change seems to be
the proximal tubular involvement (Friberg et al., 1974).
In experimental animals, additional morphological changes have
been reported. When moderate daily doses of cadmium had been
administered, degenerative changes in the proximal tubules were
the earliest observable alteration (Axelsson et al., 1968).
With prolonged exposure, edema of the interstitium, thickening
of the glomerular capsule, and mild or pronounced fibrosis of
the basement membrane have also been observed (Bonnell et
al., 1960; Axelsson et al., 1968; Stowe et al. , 1972).
Pronounced fibrotic and edematous interstitial changes reported
in long-term studies, in which relatively high peroral doses
of cadmium had been employed, may have been at least partly
caused by primary effects on the blood vessels (Fowler and
Jones, 1973; Kawai et al., 1975) whereas tubular changes
may be elicited by a direct action of cadmium on proximal
tubule cells (Kawai et al., 1975). Further aspects of the
mechanism behind this latter effect will be given later.
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Friberg (1950) described clinically detectable proteinuria in
more than 80% of a group of 43 Swedish workers exposed for about
20 years to high air concentrations of cadmium oxide dust. More
than 20% of exposed workers also had decreased ability to con-
centrate the urine. Only slight decrease in glomerular function
was observed in several cases. Studies on the character of
urinary proteins from these workers (Olhagen, 1950) disclosed
that the molecular weight of the main fraction was only 20,000-
30,000. In electrophoresis, the albumin fraction was relatively
small and an a-globulin predominated in globulin proteins.
Cadmium proteinuria was confirmed by further industrial studies
(Friberg et al., 1974} and reports on the increased prevalence
of proteinuria in groups from the general population endemi-
cally exposed to cadmium were published fron several locations
in later years (Ishizaki, 1969, 1971; Fukushima Prefecture,
1971; Hyogo Prefecture, 1972; Fukuyama and Kubota, 1972).
Total protein in urine as measured by a biuret method showed
no significant differences between polluted and nonpolluted
areas according to Watanabe et al., 1973, 1974. The precipi-
tating agent was not given in the report.
Molecular weight of urinary proteins in chronic cadmium poi-
soning varies from 10,000 to 200,000, but more than half of
the total protein has a molecular size smaller than albumin,
the globulin fraction being represented predominantly by
alpha-2, beta-2, and gamma globulins; B2-microglobulin, ret-
inol binding protein, and gamma globulin L-chains are typi-
cal representatives for this group. This type of proteinuria
is not specific for cadmium poisoning, being also found in
other renal tubular dysfunctions, congenital or acquired
(Butler and Flynn, 1958).
Increased urinary excretion of low molecular weight protein
has been reported among persons in general populations exposed
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to cadmium and other metals in the food, both in areas without
Itai-itai disease patients (Watanabe and Murayama, 1974)
and in areas with high prevalence of Itai-itai disease (Fukushima
and Sugita, 1970? Fukuyama and Kubota, 1972). In these studies
methods for detection of low-molecular-weight proteinuria
were applied primarily in order to evaluate the response
to cadmium exposure in population groups.
Since proteinuria is not pathognomonic for cadmium poisoning,
proper control groups must be set up.
According to Friberg (1957) and Bonnell et al. (1959) the pro-
teinuria can appear long after cessation of exposure. Piscator
(1962b) showed that 24 workers with proteinuria from the group
of workers studied by Friberg (1950) retained proteinuria even
10 years after cessation of exposure. On the other hand,
Tsuchiya (1974) presented some evidence that proteinuria may
be reversible in workers whose exposure to cadmium ceased or
diminished considerably.
It has been shown (Friberg, 1952; Axelsson and Piscator, 1966)
that urinary cadmium excretion in rabbits is rather low at
the beginning of exposure, a sudden increase then occurring
concomitantly with the appearance of proteinuria. Nordberg
and Piscator (1972) observed a similar phenomenon in mice,and
Suzuki (1974) observed it in rats.
Friberg et al. (1974) reviewed the observations obtained in
man. Exposed workers without proteinuria excrete small amounts
of cadmium, while high excretions up to over 100 /ag/24 hours
are found in those individuals with proteinuria. Lauwerys
et al. (1974), as well as Singerman (1975) , have presented
results which confirm these findings. Harada, A. (1975)
did not find a continuous relationship between exposure level
and urinary cadmium among workers with from 6 months to 5
years of exposure; the average cadmium excretion increased
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suddenly after a certain level of exposure had been achieved.
Tubular proteinuria was not found among these workers. In
mice with tubular proteinuria, cadmium was found in the urine
partly bound to a low-molecular-weight (#10,000) protein
(Nordberg and Piscator, 1972).
Tubular type proteinuria and increased urinary cadmium excre-
tion thus occur relatively early in long-term cadmium exposure.
In more advanced cases, multiple tubular defects, such as,
e.g. slight glucosuria, may appear before aminoaciduria and
phosphaturia develop (Piscator, 1966). Kazantzis et al. (1963)
and Adams et al. (1969) found changes in the renal handling
of uric acid and calcium. The disturbances in bone mineral
metabolism may cause renal stones (Ahlmark et al., 1961;
Adams et al., 1969) or osteomalacia (Nicaud et al. , 1942).
The significant increase in total excretion of amino acids
is considered as a relatively late and unspecific sign. Tpyoshima
et al. (1973) reported that particularly citrulline and ar-
ginine were excreted in large amounts in cadmium-exposed workers.
Telling changes in the excretion of these amino acids may
occur earlier than changes in the total amino-nitrogen excre-
tion. Patients with Itai-itai disease and other cadmium-ex-
posed persons in the general population have disclosed similar
patterns in amino acid excretion. In addition, increased
excretion of proline and hydroxyproline has been reported
(Sano and Iguchi, 1974). The role of cadmium exposure in
inducing proteinuria, glucosuria, and aminoaciduria has been
confirmed in long-term exposure through respiratory, parenteral,
or peroral administration routes on animals (Friberg, 1950;
Axelsson and Piscator, 1966; Nomiyama et al., 1975).
Biochemical mechanisms responsible for renal tubular effects
of cadmium have not yet been elucidated. Cadmium may either
activate or inhibit a large number of enzymes in.vitro as
well as iri vivo (Vallee and Ulmer, 1972).
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IV.4.2.2.3 Methods for detection of tubular dysfunction in
chronic cadmium intoxication; possible indicators
of effects
The methodology applied for the detection and evaluation of the
tubular proteinuria consists of determining the concentration
and electrophoretic distribution of proteins in urine (Butler
and Flynn, 1958; Piscator, 1962a, 19.62b, 1966). Recent quan-
titative immunological methods for determination of certain
low molecular weight proteins may offer even better possibi-
lities of detecting early changes in urinary excretion of
proteins in the future.
The determination of total urinary protein can be accomplished
by complete precipitation (as with a reagent containing phos^-
photungstic acid) and quantitative protein determination with
a biuret method (Piscator, 1962b). Complete precipitation is
not obtained by trichloracetic acid or sulfosalicylic acid.
Another adequate method utilizes complete fixation of urinary
protein in a filter paper or cellulose membrane and subsequent
staining (Piscator, 1962b).
Electrophoretic separations can be performed in different
media, e.g. paper, cellulose acetate, starch gel, agar, agarose,
or polyacrylamide. All these methods will give different
patterns, and great difficulties obviously arise when patterns
obtained by different methods are to be compared.
Among the low molecular weight proteins, ribonuclease and mura-
midase have earlier been determined in some studies (Piscator,
1966), but &2 -microglobulin now seems to be the most promising
protein since a radioimmunochemical method has been developed
(Evrin and Wibell, 1972; Evrin, 1973) which allows its quan-
titative determination even at its normal levels. This method seems
to be the most fruitful for specific determination of an
increase in the excretion of a low-molecular-weight protein.
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Retinol-binding proteins (RBP) can be quantitatively deter-
mined by ixnmunodif fusion techniques (Peterson and Berggird,
1971; Kanai et al., 1971). Peterson and Berggard (1971) and
Nomiyama et al. (1973.O) demonstrated increased amounts of RBP
and &2-microglobulin in occupationally exposed workers and
with Itai-itai disease, respectively.
By combining total protein determination, paper electropho-
retic separations, and determination of 6? -microglobulin by
the above mentioned method of Evrin, it was possible to de-
tect very small changes in the renal handling of low-molecular
weight-proteins by cadmium-exposed workers (Piscator, 1972) .
The above mentioned methods are for small-scale studies of
selected groups. For large-scale epidemiological studies,
which may involve thousands of urine examinations, qualitat-
ive methods may by necessity be used. It is of utmost im-
portance in such studies that urine from both controlled
and exposed groups are examined by the same person, since
a bias may otherwise be introduced. The specific gravity
of the urines should also be measured, since different degrees
of concentration of the urines may influence the results.
In the future, large-scale determinations of &2 -microglobulin
may also be feasible, which would augment the possibilities
for detection of increased prevalence of tubular proteinuria
in a population.
Increased excretion of these low-molecular-weight proteins
(retinol-binding protein, 62 -microglobulin) may be the most
common sign in early stages of cadmium intoxication and their
quantitative determination may be an early and specific method
of detecting renal tubular impairment in cadmium exposure.
As noted in the previous section (IV. 4. 2. 2. 2) glucosuria and
aminoaciduria may also appear as a result of the tubular de-
fect. Glucosuria is generally determined by test-tapes,
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originally intended for finding diabetes. By quantitative
determination it is possible to show slight increases in
the excretion of glucose, which do not show up with test-
tape (Piscator, 1966) . Total excretion of amino acids is
determined by total amino-nitrogen. Chromatographic deter-
mination of specific amino acids may be more sensitive. It
should be remembered that the excretion of amino acids and
glucose is also dependent on dietary habits.
IV.4.2.3 Other effects of cadmium
It has been stated above that renal tubular impairment is
usually the critical effect in long-term, low-level cadmium
exposure and that the pulmonary effects >are critical in more,
intensive exposures. However, a number of other effects have
also been reported in both human beings and experimental ani-
mals as a result of intensive cadmium exposure. The said effects
are anemia, hypertension, liver disturbance, and effects on,
bone tissue, as well as effects on endocrine functions, and
genetic, carcinogenic, and teratogenic effects (review: Friberg
et al., 1974).
Available data are inconclusive with regard to several of
these effects, and more research is needed. Anemia, hyper-
tension, and effects on bone tissue deserve more specific
attention here.
IV.4.2.3.1 Anemia
Anemia has been reported both in human beings after industrial
exposures to cadmium (Nicaud et al., 1942; Friberg, 1950) and
in animals after oral exposures (Wilson et al. , 1941). Anemia
is usually moderate and includes low haptoglobin levels.
This indicates that a hemolytic component is partly responsible
for the effect of cadmium. Some animal experiments by Berlin, M.
et al. (1961) indicated that an inhibition of iron incorpora-
tion into hemoglobin may occur. Other animal experiments have
shown decreased availability of iron for the synthesis of
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hemoglobin in bone marrow. Parenterally administered iron
in cadmium-exposed animals prevented the anemia (Friberg,
1955). In the experiments and industrial report in which
anemia was induced, it can be postulated to have stemmed
from, decreased absorption of iron from food. Data are not
sufficient to allow a detailed dose-response relationship
for anemia in human beings. The occurrence of anemia is probably
dependent on the dietary content of vitamin C and iron, since
both of these dietary components, if supplied in high amounts,
have been shown to prevent anemia in animal experiments (review:
Friberg et al., 1974).
IV.4.2.3.2 Hypertension
Animal experiments show that hypertension can be induced by ..
oral and parenteral administration of cadmium. Studies
indicating higher body burdens of cadmium in people with
cardiovascular disease have come to light (Schroeder, 1967;
Friberg et al., 1974). This effect of cadmium has been
thought to arise at low concentrations of cadmium in the
kidney, at concentrations similar to those found in normal
subjects. Systematic studies for hypertension in human
beings exposed to excessive amounts of cadmium in the general
environment or in industry have not been reported. (See
also section IV.6).
IV.4.2.3.3 Effects on bone tissue
After the Itai-itai disease (osteomalacia) in Japan had been
shown to be associated with cadmium exposure, interes't in
effects of cadmium on osseous tissue was stimulated. The effect
of cadmium on bone can be secondary to an effect on the renal
tubules, since the kidneys have key functions in the regula-
tion of the calcium and phosphorus balance in the body. Osteo-
malacia and severe osteoporosis have been reported among
cadmium exposed workers (Nicaud et al., 1942; Gervais and
Delpech, 1963; Adams et al., 1969; Kazantzis, 197s).. as
well as among persons in general populations with high
cadmium exposure and probable calcium deficiency (Itai-
itai disease, see Friberg et al., 1974).
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Kobayashi (1973) reported that cadmium exposure caused a neg-
ative calcium balance in rats. A decreased calcium absorption
and histopathological changes in the duodenum were observed
in rats receiving 50 ug Cd/g drinking water (Sugawara
and Sugawara, 1974). Kawai et al. (1975) described decalci-
fication and cortical atrophy in the skeletal tissue during
long-term peroral exposure of experimental animals. These
alterations were observed earlier than the morphological
changes in the renal tissue.
Feldman and Cousins (1973) and Kimura et al. (1974) demonstrated
that cadmium may decrease the production of the active vitamin-D
metabolite, 1,25-OH-cholecalciferol, in,viyo.
Osteomalacia can be induced by a combination of cadmium exposure
and calcium deficiency, but the mechanism is still unclear
(Itokawa et al., 1974). Since nutritional factors such as vita-
min D and calcium are highly influential, the effect on the
skeletal tissue may possibly be a critical one in cadmium ex-
posed populations for whom intakes of these constituents are
marginal.
With regard to the etiology of Itai-itai disease, it was defined
as renal osteomalacia, for which the main etiological factor
was cadmium, but host factors such as malnutrition, pregnancy
and aging, "are of large consequence. However, Kajikawa et
al. (1973) reviewed 11 autopsies of Itai-itai patients and came
forth with a speculative statement that Itai-itai disease is
a nutritional osteomalacia caused by the insufficient intake
of vitamin D, as well as other malnutritional factors. Kajikawa
et al. (1973) referred to studies from 1906 showing that osteo-
malacia and rickets had been prevalent in Toyama Prefecture
as early as that time. These authors used this as supporting
evidence for their hypothesis.
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IV.4.2.4 Critical effects and critical organs at exposure to
cadmium and its compounds
In long-term exposures to cadmium, the kidney appears to be
the critical organ and the most commonly studied critical effect
is manifestation of proteinuria. Increased urinary excretion
of low molecular weight proteins, such as RBP and /? -micro-
globulin, may be considered as an early and specific renal effect
of cadmium. Fully developed tubular proteinuria, characterized
by the prevailing presence of proteins with molecular weight
up to that of plasma albumin and higher, is a typical sign
of chronic cadmium poisoningj this type of proteinuria is,
however, not specific for cadmium effects, having been
found in other renal tubular dysfunctions, congenital or ac-
quired. The same conclusion regarding nonspecificity can be •
applied to glucosuria and aminoaciduria, frequently reported . . ,"..
in chronic cadmium poisoning. Bone manifestations, such as osteomalacia,
can be induced by experimental cadmium exposures or may appear in popula-
tion groups with high exposures and non-optimal nutritional conditions;
mechanisms of their origin may, however, be secondary to the effects of
cadmium on renal tubules and subsequent changes in calcium and phosphorus
balance.
After inhalation of high concentrations of cadmium fumes or
dusts, the respiratory system becomes the critical organ
and acute pneumonitis or chronic emphysema with increased '
residual capacity and bronchitis represent critical effects
of cadmium, particularly in exposures such as may occur
in industry.
Acute effects of injected cadmium on testicles, sensory
ganglia, nonovulating ovaries, placenta and fetuses were
demonstrated in experimental animals but have not been observed
in human exposures so far.
IV.4.3 Effects of lead and its compounds
Lead effects may occur in a number of organs and systems and
may follow exposure to either inorganic or organic compounds
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of lead. The comments in this document relate principally
to inorganic lead exposure except for one section dealing
with consequences of exposure to organic forms (tetraethyl
lead). The major effects of lead concern three systems,
namely, hematological, neurological and renal. Effects on
other organs are also discussed briefly, but are not considered
for establishing dose-response relationships .
IV.4.3.1 Effects of inorganic lead compounds
IV.4.3.1.1 Hematological effects
Lead exposure may give rise to microcytic anemia, shown by bio-
chemical methods to be caused by a combination of inhibition of
hemoglobin synthesis and shortened life'span of circulating ,
erythrocytes. From the biochemical point of view, the most
impressive effects of lead are on the biosynthesis of hemo-
globin. According to numerous studies, it may be concluded
that lead can affect many steps in the pathway of heme syn-
thesis (Griggs, 1964). Since several recent reviews give
detailed accounts of this matter (Waldron, 1966; Airborne
Lead in Perspective, 1972; Goyer and Rhyne, 1973), only
a brief recapitulation will be given here. In vitro exper-
iments using avian erythrocytes have been particularly fruitful
because these cells contain the complete heme biosynthetic
system. Such studies show that aminolevulinic acid dehydrase
(ALA-D), catalyzing the formation of porphobilinogen (PEG)
from ALA, and heme synthetase (heme-S), incorporating iron
into protoporphyrin IX (PP IX), are the enzymes most sensitive
to the action of lead (Eriksen, 1952; Dresel and Falk, 1956a,
1956b). In addition it has been demonstrated by in vitro
experiments that high lead concentrations inhibit ALA-syn-
thetase (ALA-S), cataly2ing the formation of ALA from glycine
and succinate, and coproporphyrinogen decarboxylase (CPG
decarboxylase), converting CPG into PP (Goldberg et al.,
1956; Kreimer-Birnbaum and Grinstein, 1965; Wada et al.,
1972; Wada, 1975).
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Recent in vivo studies on heme biosynthesis in erythroid cells
of bone marrow from workers exposed to lead show a remarkable
reduction of the activities of ALA-D and heme-S, a slight eleva-
14
tion of ALA-S activity, and a parallel reduction of C-glycine
incorporation into heme and globin (Wada et al., 1972; Wada,
1975). Increased activity of ALA-S, in contrast to the results
of the in vitro study by Goldberg et al. (1956), is not
likely to imply the activation of the enzyme by lead but
may represent a positive feedback mechanism in response
to the decreased heme content in erythroid cells which has
resulted from the inhibited heme biosynthesis by lead.
Depression of the activity of ALA-D in circulating erythrocytes
occurs without fail in lead poisoning (Lichtman and Feldman,
1963; Bonsignore et al., 1965; Hernberg and Nikkanen, 1972).
Partial inhibition has been demonstrated at lead levels
far below those commonly thought to be toxic (De Bruin and
Hoolboom, 1967; De Bruin, 1968; Hernberg et al., 1970;
Hernberg and Nikkanen, 1970; Miller et al., 1970; Weissberg
et al., 1971; Hernberg and Nikkanen, 1972; Tola et al.,
1973; Wada et al., 1973; Sakurai et al., 1974; Singerman,
1975) . In fact, the inhibition of erythrocyte ALA-D activity
is the most sensitive effect known for the action of lead.
The blocking effects of lead upon various steps in the heme
synthesis are manifested as increased excretion of ALA-U,
CP-U, elevation of ALA in serum, and increased free erythro-
cyte protoporphyrin IX (FEP). These abnormalities can be at-
tributed to the combined effect of the inhibition of heme bio-
synthetic enzymes other than ALA-S, and a positive feedback
mechanism upon certain steps, induced by reduced heme content
in the heme forming cells.
It can be concluded that the combination of (1) .inhibition of
ALA-D activity in circulating erythrocytes, (2) increased ex-
_cretion of ALA-U and (3) increased excretion of type III
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CP-U, and (4) accumulation of FEP, is quite pathognomonic
for lead; thus, lead poisoning can be distinguished from
all other disorders by keeping this in mind.
Shortening the life span of circulating erythrocytes is the
other main mechanism by which lead causes anemia. In chronic
poisoning the shortening is moderate, and thus slighter than
in most hemolytic anemias (Rubino, 1962; Sroczynski et al.,
1965; Hernberg et al., 1967). According to several studies,
the survival time is rarely reduced below 60 days. Using in
vivo labelling with H-di-isopropylfluorophosphonate, it has
been possible to show slight shortening (to about 100 days)
of the erythrocyte life span in a group of lead workers with
blood lead levels between 59 and 162 jug/100 ml (Hernberg et,
al., 1967).
The mechanism by which lead shortens the erythrocyte life span
is not well understood (Griggs, 1964; Waldron, 1966; Harris
and Elsea, 1967). It might be that the microcytic anemia
i.e. the size and shape of the red blood cells explains the
increased vulnerability. A number of observations show that
the osmotic resistance of erythrocytes from patients with
lead poisoning is increased '(Griggs, 1964; Waldron, 1966; Harris
and Elsea, 1967)• Increased mechanical fragility has also
been reported. However, studies dealing with these phenomena
are equivocal (Hasan and Hernberg, 1966; Waldron, 1966).
The biochemical changes at the erythrocyte membranes responsible
for changes in the mechanical fragility and the osmotic re-
sistance are not well known. Loss of potassium and water oc-
curred when lead salts were added to erythrocytes in vitro
(Passow et al., 1961), and when whole blood from lead workers
was incubated at 37 , a potassium loss occurred as well (Hasan
et al., 1967a). This phenomenon may or may not be related to
the fact that erythrocyte membrane Na /K -ATPase activity is
inhibited by lead (Hasan et al., 1967b; Hernberg et al., 1967).
Red cell glutathione has also been shown to be diminished due
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to the action of lead (Roels et al., 1974). The phenomena re--
capitulated above represent only a few possible explanations
for the lead-induced shortening of red blood cell life span.
A consequence of all these effects of lead on hematopoiesis is
that the production of red blood cells increases, which is mani-
fested by reticulocytosis. Basophilic stippling of the erythro-
cytes is another feature of lead-induced anemia, and has earlier
been used for the diagnosis of lead poisoning. However, this
nonspecific phenomenon correlates poorly — if at all — with
the intensity of lead poisoning.
IV.4.3.1.2 Neurological effects
Lead exposure may have serious effects on the central and peri-
pheral nervous systems. Central nervous system effects are most
frequent in childhood, from pica or other forms of childhood
lead exposure. Peripheral nervous system effects are more char-
acteristic of long-term lead exposure in adults. However, chil- .
dren may also manifest a peripheral neuropathy and adults may
have central nervous effects particularly if exposure is short-
term and very heavy, e.g. paint chippers in missile silos (Lambie,
1967) . Tetraethyl lead exposure in the adult may be associated with
central nervous system effects.
IV.4.3.1.2.1 Central nervous system effects (encephalopathy)
The classical signs and symptoms of lead encephalopathy are
ataxia, coma and convulsions. Acute encephalopathy may be
fatal. Survivors of lead encephalopathy may sustain residual
brain damage which is manifested by mental and/or neurologi-
cal impairment.
The most prominent morphological changes noted in the brain are
cerebral edema, proliferation and swelling of endothelial cells
accompanied by dilatation of capillaries and artefioles, pro-
liferation of glial cells and focal necrosis, and neural degenera-
tion. Another common feature of lead encephalopathy is a diffuse
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astrocytic proliferation in the grey and white matter. The
extent of neuronal loss in cortical grey matter is variable,
but provides a morphological basis for the permanent sequelae
of lead encephalopathy (Pentschew and Garro, 1966) .
From the few autopsied cases of lead poisoning in which brain
tissue has been analyzed, it has been found that lead concen-
centrations in this organ are not as high as in other organs
such as liver and kidney. In one case in which lead was mea-
sured in more than one part of the brain, the highest concen-
trations were found in the cortical grey matter and basal
ganglia (Klein et al., 1970). A more detailed mapping of var-
iation in lead concentration in different parts of the brain '
of lead poisoned dogs showed similar results (Stowe et al.,
1972). These studies support a relationship between areas
of the most marked histological change and high lead concen-
trations .
The effects of lead on the central nervous system may not
be completely reversible (Chisolm and Harrison, 1956; Byers,
1959). Sequelae include mental retardation, seizures, paralys-
is and optic atrophy (Perlstein and Attala, 1966). Likeli-
hood of sequelae is related in some degree to severity of
symptoms prior to treatment. The risk of permanent neurologi-
cal complications increases with repeated episodes of acute
intoxication or with chronic excessive exposure to lead
(Chisolm and Harrison, 1956; Byers, 1959).
Whether less severe or subclinical effects of lead on the
central nervous system are recognizable in children is a
topic of current debate. A relationship has been suggested
between slight loss of intelligence (de la Burde and Choate,
1972) and hyperactivity (David et al., 1972) on the basis
of studies carried out primarily in school age children with
epidemiological and clinical histories suggestive of increased
lead absorption during the preschool years. Behavioral and
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psychological changes and loss of fine motor function (Pueschel
et al., 1972; Albert et al., 1974) have been reported in young chil-
dren with blood lead levels > 50-60 vg Pb/100 ml blood during the
preschool years.
At the present time, only partial experimental confirmation of
such lead effects is on hand. Silbergeld and Goldberg (1974)
have induced hyperactivity in mice exposed to lead through
their mother's milk, and, like humans with hyperactivity, the
lead treated mice responded paradoxically to the central ner-
vous system stimulants. Also, Carson and coworkers (1974)
have shown a slowness in learning in lambs prenatally exposed
.in utero to maternal blood lead levels of 34 jig/100 ml during
gestation. . .
IV.4.3.1.2.2 Peripheral nervous system manifestations
Peripheral neuropathy is seen only sporadically nowadays and
almost exclusively under conditions of uncontrolled exposure.
The neuropathy is characterized by selective involvement of
motor neurons with little sensory involvement. Mild forms
exist too, which can be assessed by electrophysiological tech-
niques (Catton et al. , 1970). Experiments with isolated perfused
ganglia (Kostial and Vouk, 1957) or isolated sciatic nerves
(Manalis and Cooper, 1973) have shown that a reduction of the
end-plate potential by a presynaptic block may be the mechanism
of the lead effect on nerves. Pathological changes in experi-
mental lead-induced peripheral neuropathy include segmental
degeneration of myelin sheaths and axonal degeneration
(Schlaepfer, 1969). (See also section IV. 5.3.2.2.2).
/ •
IV.4.3.1.3 Renal effects
In children with short-term but heavy exposure to lead, func-
tional and morphological changes occur in proximal renal tu-
bular cells (Marsden and Wilson, 1955; Chisolm and' Leahy, 1962).
The functional effects include the Fanconi syndrome manifested by
aminoaciduria, glucosuria, and hyperphosphaturia in the presence
of hypophosphatemia.
]55
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Morphologically there are nonspecific degenerative changes
in proximal tubular lining cells, including mitochondrial
swelling, and a more specific change, eosinophilic, dense
staining nuclear inclusion bodies. Electron microscopy of
the inclusion bodies shows that they have a characteristic
outer fibrillar margin useful for distinguishing them from
other nonspecific bodies. A number of experimental studies
using autoradiography, direct chemical analysis, and X-ray
microanalysis show that the bodies are a lead-protein com-
plex and may have a role in the excretion of lead. A re-
cent study shows that the bodies are excreted during EDTA
chelation and may represent one source of EDTA mobilizable
lead (Goyer et al., 1974). The functional and morphological
renal effects of relatively short-term lead exposure (prob-
ably less than 1 to 5 years) are reversible following treat-
ment with chelating agents (Chisolm and Leahy, 1962).
The effect of long-term exposure to lead on the kidney differs
from the short-term effects just described and is seen in adults
with heavy occupational or other exposures to lead. It consists
of a nonspecific nephropathy characterized morphologically by
intense interstitial fibrosis, tubular atrophy and dilatation
with relatively late involvement of glomeruli. Tubular dysfunc-
tion is manifested by a defect in uric acid excretion often
resulting in hyperuricemia, and sometimes in gout. The typical
Fanconi syndrome has not been documented in the adult form of
lead nephropathy. Reduced glomerular filtration is a late ef-
fect of lead nephropathy but renal failure may occur. There
have been many studies of lead nephropathy in occupationally
exposed individuals from different parts of the world. These
have been reviewed and their associated morphological changes
summarized (Goyer, 1971) .
A weighty question is whether or not children with short-term
exposure to lead are more susceptible to a form of chronic
nephropathy later in life. Ten to twenty year follow-up stud-
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ies of children in the United States known to have had lead
poisoning in childhood showed no increase in nephropathy in
later life (Tepper, 1963} Chisolm, 1970). However, studies
from Australia (Henderson, 1954; Emmerson, 1968) suggested
that long-term exposures to lead in childhood or adolescence
may progress to a chronic nephropathy with the same charac-
teristics as in chronic lead nephropathy in occupational exposure,
that is, a high incidence of hyperuricemia and severe intersti-
tial fibrosis. It may be that decisive differences existed
between the form of the lead nephropathy seen in Australia
and that seen in young people in the U.S.A., perhaps with re-
gard to age and length of exposure to lead.
IV.4.3.1.4 Gastrointestinal effects
.The gastrointestinal system is invariably involved in obvious
lead poisoning, and symptoms from the alimentary tract occur
frequently even in milder forms. Acute lead colic may result
from massive short exposure to lead, but it may also develop
quite suddenly in subjects with long-term exposure. It is
characterized by attacks of crampy diffuse abdominal pain,
generalized muscle aches and constipation. The skin of the
patient is pallid and the blood pressure may be elevated.
Lead lolic may be preceded by more diffuse symptoms. Pro-
longed, severe constipation is considered a warning of in-
cipient colic. The mechanism for the gastrointestinal ef-
fects caused by lead is unknown (Airborne Leatd in Perspective,
1972).
IV.4.3.1.5 Teratogenic and other reproductive effects
Although effects of lead on reproductive fitness have been
suspected to occur in individuals with increased body burden
of lead, this effect has received limited study in man.
From experimental studies, lead has been found to be toxic
to gametes of both sexes, resulting in reduced numbers and
size of offspring from lead-poisoned rats (Stowe and Goyer,
1971). These studies imply that lead is transported via sperm
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or ova to the zygote, but actual transport of lead in this
manner has not been demonstrated. Histopathological changes
in ovaries in lead-poisoned Rhesus monkeys have been demon-
strated (Vermande-van Eck and Meigs, 1960), but such changes
have not been seen in man.
Reports of sterility, stillbirths, and miscarriages among
women working in the lead industry have been summarized in
the older literature (Oliver, 1914; Lund, 1936; Cantarow
and Trumper, 1944). There are no recent data on occupational-
ly exposed women in the childbearing age range concerning
possible effects of increased body burden ,of lead on re-
productive fitness or on the fetus. Likewise, there arc
few experimental studies demonstrating a possible terato-
genic effect of lead. Ferm and Carpenter (1967) showed that
the intravenous administration of lead salts to the golden
hamster at a specific time during gestation results in
skeletal malformations. Of greater concern, particularly
with regard to occupational low-level lead exposure of
women in the childbearing age range, is the risk of central
nervous system effects in the fetuses.
IV.4.3.1.6 Endocrine effects
An effect of lead on various endocrine glands has been reported.
The observations have been made primarily in individuals who
had consumed "moonshine" whiskey for many years (Sandstead,
1973). Functional tests of the pituitary-adrenal axis (Sandstead,
1973), the pituitary-thyroid axis (Sandstead et al., 1969),
the pituitary-gonadal axis (Sandstead, 1973) and the renin-
aldosterone system (Sandstead et al., 1970; McAllister et al. ,
1970) have been found abnormal. The impairment of I uptake
by the thyroid has been confirmed in rats chronically in-
toxicated with lead (Sandstead, 1967). The impairment of the
renin-aldosterone response to sodium deprivation has been
reversed by treatment with Ca-Na2~EDTA (McAllister et al.,
1970) . Therefore it seems that these latter two adverse ef-
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fects are indeed due to lead. It is possible that other un-
defined substances in the "moonshine" contributed to some
of the abnormalities. The practical importance of these
findings is at present unknown.
Others have also reported an adverse effect of lead on the
pituitary-gonadal axis (Raule and Morra, 1952) and other organs
of the endocrine system (Pines, 1965j Muraslov, 1966). These
reports are generally supportive of the above interpretation
of the studies cited above.
IV.4.3.2 Effect of alkyl lead compounds
Alkyl lead compounds, in contrast to inorganic .lead, are
readily absorbed by inhalation. This may be followed by central
nervous system effects and may occur without hematological,
gastrointestinal, or renal effects. Concentrations of lead
in the brain of persons dying from tetraethyl lead intoxica-
tion are much higher than those in the same organ of persons
with inorganic lead poisoning (Cassells and Dodds, 1946;
Cummings, 1967). Other soft tissues and bone may contain less
lead than is found in persons with inorganic lead toxicity
but this depends on length of exposure. Alkyl lead compounds
are lipid soluble which is thought to explain the tissue
distribution. The lipid soluble tetraethyl lead, though it
might interfere with membranes, is not responsible for the
toxic effect at the biochemical level. The toxic metabolite
is triethyl lead which is formed mainly in the liver (Cremer,
1959). Although there are some data on poisoning by tetraethyl
lead compounds, they were considered too scanty to establish
dose-effect or dose-reponse relationships.
IV.4.3.3 Critical effects and critical organs at exposure to
inorganic and organic lead compounds
The critical effect of lead is adverse interference with heme
synthesis. Increased concentrations of heme intermediates in
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blood or urine (ALA-S, ALA-U, CP-U, FEP) are evidence of
critical effects of lead on heme synthesis. Decreased ALA-
D activity in red blood cells, is regarded as a subcritical
effect.
According to the knowledge at hand, the critical organ for
lead effect is the hematopoietic bone marrow. Continuing re-
search may develop methods for measuring neurochemical or
neurophysiological disturbances which underlie nervous system
manifestations occurring prior to the known heme synthesis
effects. It is difficult now to state whether or not nervous
disorders may precede hematological disturbances in particular
circumstances.
For tetraethyl lead exposure, particularly following short-
term inhalation, the critical organ is the central nervous
system.
IV.4.4 Effects of mercury and its compounds
It is evident that the biological effects of mercury and its
compounds are determined by the different physical and chemical
forms of mercury. These have been referred to in an earlier
section. In some parts of the following review, representing
more general knowledge on mercury toxicology, references have
not been given. Recent reviews where such references may be
found are ICMACMC (1969) , Friberg and Vostal (1972) and
Clarkson (1972a, 1972b).
IV.4.4.1 General biochemical aspects of the toxicity of mercury
and its compounds
The action of mercury and its compounds at the biochemical
level is poorly understood. Biochemical studies and mechanisms
of action are complicated by the fact that mercury cations
such as Hg , CgH5Hg and CH3Hg are nonspecific enzyme in-
hibitors. Mercuric cation possesses a high chemical affinity
for -SH groups (Rothstein, 1973). Since the -SH group is found
in most protein molecules, mercury cations can inhibit most
160
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enzymes with which they come into contact in sufficient con-
centrations. Webb (1966) has listed hundreds of enzymes in-
hibited by mercury in vitro. The -SH group is also essential
to the integrity of cell membranes and mercury has been
demonstrated to disrupt most membrane functions (active
transport, permeability properties) in vitro (Rothstein,
1973) .
Despite the lack of specificity in biochemical systems in
vitro, mercury and its compounds nevertheless exhibit remark-
able selectivity throughout the whole organism. Many studies
have been reported testing the hypothesis that the selective
distribution in the body may be responsible for selective toxi-
city (Berlin, M., 1963; Clarkson, 1972a, 1972b). Recently,
investigations have been made with the various complexes and
chelates (biocomplexes) formed by mercuric cations in vivo
in various tissues and body fluids in order to see if this
information might help in understanding the selective distri-
bution and toxicity. No general conclusion can be made from
the results obtained to date, but certain findings are worth
noting. Norseth (1968) reported that Hg accumulates sel-
ectively in lysosomes and suggested that cellular toxicity may
be produced by the release of hydrolytic enzymes. That mercury
is taken up by lysosomes has also been demonstrated morpho-
logically (Fowler, 1972; Fowler et al., 1974). Alterations
in lysosomal and microsomal enzymes have been discovered in
renal tissue of animals that accumulated Hg as well as MeHg+
in renal tissue upon MeHg exposure (Fowler et al., 1975).
Biochemical studies on the binding of Hg in tissues have
provided some insight into renal toxicity and into the dev-
elopment of kidney tolerance. Observations by Kessler et al.
(1957), Weiner et al. (1962), Miller and Farah (1962), and
Stoytchev et al. (1969) indicate that Hg++ forms a two-point
attachment to a receptor (active site) in kidney tissue. One
point of attachment is a thiol group and the other is a non-
thiol ligand.
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Clarkson and Magos (1966) identified three classes of binding
sites in kidney tissue distinguished by the affinity for Hg
Kidney levels of Hg as observed in not excessively exposed
human beings (KSgi and Vallee, 1960, 1961) correspond to
saturation of the highest affinity class of sites. Subsequently
Wisniewska et al. (1970) demonstrated that Hg + may attach
to metallothionein in kidney cells under conditions of ex-
perimental exposure of rats to Hg . The levels of a metall-
othionein-like protein, as measured by Piotrowski et al.,
1973) corresponded roughly to the tight binding sites described
by Clarkson and Magos (1966).
It has been claimed that metallothione.in plays an impoi ,_cint
and protective role, and that once the binding capacity of
this protein has been saturated, toxic effects appear in the
kidney. This organ is known to be able to accumulate much
higher levels of mercury during chronic exposure than the toxic
level observed after a single dose, without any detectable
deleterious effect. Mercury is able to induce the synthesis
of a metallothionein-like protein in kidney tissue (Wisniewska
et al., 1970; Piotrowski et aL, 1973; Nordberg, M. et al.,
1974) but not in liver. Whereas kidney levels of mercury rise
on repeated dosing, the liver levels are stable (Piotrowski
et al., 1973, 1974).
The biochemical lesion underlying MeHg toxicity has not yet
been elucidated. Yoshino et al. (1966) and Cavanagh et al.
(1970) studied animals given high single doses of methyl-
mercuric compounds. They claimed that the decrease of protein
synthesis was the first detectable change in cell function
and was accompanied by morphological abnormalities in the
ribosomes. Bull (1974) reported interference with enzymes of
the respiratory chain in the CNS of rats exposed to 0.05 mg
per kg daily for 14 days. The relevance of these findings to
poisonings due to long-term exposures in man has not yet
been established.
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The kidney toxicity of phenylmercuric compounds as reported
in animal experiments (Fitzhugh et al., 1950) is probably
due to rapid biotransformation to inorganic mercury (review:
Clarkson, 1972a, 1972b).
IV.4.4.2 Effects of elemental mercury
Short-term exposure to high concentrations of mercury vapor
may give rise to symptoms of pulmonary irritation, and central
nervous system involvement. Occupational exposure to mercury
vapor is usually long-term. Sometimes there is a combination
of exposure to vapor and dust of inorganic and/or organic
salts of mercury. In long-term exposure to mercury vapor at
higher concentrations, CNS symptoms and signs are most common-
ly found, with tremor and psychological disturbances (erethismus
mercurialis) being most prominent. Proteinuria may also occur,
possibly followed by the fcephrotic syndrome in rare cases, but
definitive evidence concerning the latter is not available in
humans.
Gingivitis, stomatitis and excessive salivation may occur in
mercury-exposed workers, who may sometimes develop dermatitis
as well. Mercurialentis (a discoloration of the anterior lens
capsule) is seen following long-term exposure to mercury, but
does not indicate intoxication (ICMACMC, 1969). Anorexia,
weight loss, anemia, and muscular weakness have been reported.
Smith et al. (1970) showed that loss of appetite and weight
correlated well with exposure.
A syndrome that is characterized by decreased productivity,
increased fatigue, loss of self-confidence, muscular weakness,
vivid dreams, depression etc., has been subject to much discus-
sion in the literature from USSR. This syndrome has been cal-
led the asthenic-vegetative syndrome or, when connected with
mercury exposure, micromercurialism (Trachtenberg, 1969;
review: Friberg and Nordberg, 1972).
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Singerman and Catalina (1969) demonstrated inhibition of red
cell membrane Na K ATP-ase in industrial workers exposed to
mercury vapor. They also reported an increase in the total
activity of serum and urinary lactic dehydrogenase and an in-
crease in the cathodic molecular fractions (isoenzymes) No.
4 and No. 5. No significant changes were found in other en-
zymes studied which included transaminases and cholinesterase.
IV.4.4.3 Effects of inorganic mercury compounds
Single and short-term exposure to high doses of inorganic
mercury is now a relatively rare occurrence, but can be found
in accidental or suicidal intake or during occupational
exposure. The clinical manifestations of oral intake of mercuric
salts include gastroenteritis with abdominal pain, nausea,
vomiting, bloody diarrhea, and severe renal disorder leading
to anuria and uremia.
The effects of easily soluble mercuric salts upon the kidney
have been confirmed in a number of animal experiments (review:
Skerfving and Vostal, 1972). Acute total or segmental necrosis of
the terminal portions of the renal tubules has been reported
in rabbits, guinea pigs, and frogs after injection of mercuric
chloride. At higher doses the initial and middle portions of
the proximal tubules are also affected. Electron microscopical
changes in the mitochondria of the proximal tubules in the rat
kidney have been reported after repeated subcutaneous administra-
tion (Bergstrand et al., 1959). At injection of high doses of
mercuric chloride, on the order of 10 mg/kg or higher, there
is an evident ischemia in the renal cortex.
Disturbances of blood flow within the cortical tissue are
thought to be of importance for elicitation of the tubular
changes at these high doses. It has been postulated that
a primary decrease in glomerular filtration rate due to
afferent arteriolar constriction takes place at such doses.
At doses lower than 5 mg of mercury/kg body weight, vascular
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disturbance do.es not complicate the picture according to Biber
et al. (19,68), who did find necrosis of tubular epithelium in
the distal three-fourths of the length of the proximal tubules
and no anuria.
Concerning long-term exposures, there have not been many reports
on morphological changes in the tissues of patients or experimental
animals, ppwever, Kazantzis et al. (1962) performed renal biopsy
in two c^ses of workers exposed to mercury vapor and inorganic
mercury salts. The workers had high urinary losses of protein,
hyaline ca.sts in urinary sediment and normal glomerular filtra-
tion rates. Renal biopsy showed abundance of lipids and vacuoli-
zation in the epithelium of the proximal tubules. No abnormali-
ties were seen in the glomeruli. Renal concentrations of mer-
cury ranged between 10 and 20 ;ug/g fresh tissue.
IV.4.4.4 Effects of organomercury compounds
Organomercurials fall into classes with different stability
in biological systems, as mentioned earlier in this report.
A subdivision of organomercurials into more stable compounds
like alkylmercury salts and less stable compounds like
phenyl- and alkoxylalkylmercury compounds is necessary. It
is also evident that the symptomatology and the type of bio-
logical effects differ between these two types of compounds.
IV.4.4.4.1 Methylmercuric compounds
Symptoms observed in man after exposure to methylmercuric
compounds are dominated by neurological disturbances. Symptoms
of methylmercury poisoning may occur weeks to months after an
acute exposure to toxic concentrations (ICMACMC, 1969). The
symptoms and signs of poisoning after short- and long-term
exposure include numbness and tingling of the lips, mouth,
hands and feet, dysarthria, ataxia, concentric constriction
of the visual fields, blurred, vision, blindness, deafness
and an impaired level of consciousness. With severe intoxi-
_cation the symptoms and signs were partially reversible
165
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in the relatively short-term exposure in Iraq, in contrast
to irreversibility after much longer exposure in Minamata.
Infants who were exposed prenatally to large amounts of
MeHg"*" had signs of cerebral palsy, convulsions and in some
cases, blindness (Clarkson and Marsh, 1975).
Morphological findings in the brain in fatal cases of methyl-
mercury poisoning include severe changes in the cerebral
cortex, especially of the visual cortex, including degenera-
tion of nerve cells, and changes in the granular layer of the
cerebellum. All or some of these sites may be involved
(Hunter and Russell, 1954; Takeuchi, 1968; Nordberg et al.,
1971a; Grant, 1973). Most of these morphological changes
have been recorded in both human beings and in animals,
e.g. cats and monkeys. There are marked species differences,
especially when comparing the metabolism of MeHg in rodents
to that in primates. Observations on the CNS effects of
MeHg in rats therefore cannot be applied to humans.
The alterations in the nerve cells seen in the microscope
after experimental MeHg intoxication have had a topographical
distribution which has corresponded to the topographical
localization of subcortical accumulation of mercury. A
direct pathogenic relationship between this accumulation
and the elicitation of neuronal damage thus seems likely.
It is not yet clear how this mechanism comes about, but
it may be that a primary lesion occurs in the function of
the glia cells which secondarily gives rise to the neuronal
necrosis (Nordberg et al., 1971a; Grant, 1973). This mechanism
may also be related to the latency time clinically observed
in MeHg intoxication.
MeHg also causes brain damage to the human fetus as was
initially observed in the Minamata area of Japan in 22 in-
fants who were born between 1953 and 1959. They had clinical
features of cerebral palsy. A few of the mothers experienced
166
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paresthesias during pregnancy but most of them' did not have
other features of Minamata disease. One of those 22 infants
had a retinal anomaly and another had malformation of an
external ear (Harada, Y., 1968).
Apart from cerebral palsy, the existence of teratogenic effects
is not clear. Data on the relationship between the maternal and
fetal toxicity of MeHg have been given by Clarkson and
Marsh (19^75) . They concluded that fetal toxicity occurs
at approximately the same exposure levels that cause early
maternal toxicity. It may be that early effects of MeHg
can result from the impact of MeHg on cell division
and chromosome segregation. Indeed, chromosomal alterations
have been witnessed (Skerfving et al., 1970; Ramel, 1972;
Skerfving et al., 1974). Litter size was reduced in untreated
female rats allowed to mate with male rats treated with methylmer-
curic compounds. These results have been interpreted as evid-
ence for a dominant lethal mutation (Khera, 1973).
IV.4.4.4.2 Ethylmercuric compounds
Most clinical data about poisoning by ethylmercuric compounds
are derived from patients who had consumed seed grain that had
been treated with ethylmercury preparations. It is probable,
however, that other mercurials were partially responsible for
the symptoms and signs that arose. The neurological features
were less marked than in MeHg poisoning, but gastro-
intestinal and renal effects were more prominent (Jalili
and Abbasi, 1961; Damluji, 1964).
IV.4.4.4.3 Phenyl- and alkoxyalkylmercuric compounds
Methoxyethylmercuric compounds have come to replace methylmer-
curic ones as fungicides after the recognition of the latter
compounds' toxicity. Methoxyethylmercuric salts have only given
rise to a few reported cases of poisoning. The symptoms have been
loss of appetite, diarrhea, weight loss, and fatigue. The symp-
toms probably are ascribable to the action of inorganic mer-
167
-------
cury, resulting from the degradation of the mothoxycthyl-
mercuric radical in the body. Kidney damage with albuminuria
and occasionally a nephrotic syndrome also have been reported
after exposure to methoxyethylmercuric compounds. (For ref-
erences see ICMACMC, 1969).
In spite of the rather extensive use to which phenylmercuric
compounds have been subject, very few cases of intoxica-
tion have been reported. An irritant effect on the skin has
been observed, but systemic effects are rare. In an acute case
in a worker reported by Goldwater et al. (1964) mild evidence
of kidney damage occurred. Long-term exposure to phenylmercuric
salts has not given any conclusive evidence of toxic
effects in industrial exposure situations.
IV.4.4.5 Summary and definition of critical organs in ir^rcury
exposure
The critical organ in exposure to mercury and mercurials varies
with the type of compound, with dose, with route of absorption,
with duration of exposure, and with stage of development of the
organism.
After severe short-term exposure to mercury vapor, the critical
organ is the lung. With several repeated exposures, the critical
organ is likely to be the brain, and after prolonged exposure
the critical organ is the brain.
After exposure to mercuric salts, whether absorbed by ingestion
or by inhalation, the proximal tubule of the kidney is the
critical organ.
The critical organ after exposure to ethyl- and methylmercuric
compounds is the brain in man. In pregnant womun, the fetal
brain may be regarded as the critical organ. For phenylmercuric
and methoxyethylmcrcury compounds, the critical organ is likely
to be the kidney.
168
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IV.5 DOSE-RESPONSE AND DOSE-EFFECT RELATIONSHIPS
IV.5.1 General aspects
The concept of dose-response relationship is central in toxi-
cology. In its simplest form it is usually displayed graphically
as an g-shaped curve with dose on the x-axis and response on the
y-axis. It can be visualized that"a complete graphical summariza-
tion of the relationship between dose and response, if different
effects are taken into consideration, would require at least a
family of curves and a three-dimensional model.
There will be no attempt to reduce the complexities of the
dose-response relationships and dose-effect relationships to
a single model,. but rather to point out some of these com-
plexities. The limitations as well as the utility of simple
expressions may then be better understood and communicated.
The distinction between effect and response has been elaborated
upon in earlier sections. Effects are usually quantifiable and
graded (as, to degree) , so that with, for example, the urinary
excretion of ALA (an effect) after lead exposure a dose of one
unit (here arbitrarily chosen) may produce no measurable increase
in excretion. A dose of 2 units may produce an increase of y
above the. background levels, and a dose of 3 units may produce
another defined increase in excretion of ALA.
Death, as a biological effect, is not usually considered to be
graded, but instead an all-or-none-condition. Such an effect
is called a quantal effect. To deal with these, one takes a
population given a specific dose and measures the percentage
showing this specific effect? this percentage value has been
defined previously as the response. Using this definition, an
LD5Q value may be described as the dose expected to cause a
50% response among the population tested for the lethal effect.
169
-------
The distinction between "all-or-none" and "graded" effects is
not an absolute one. In a physiological sense, individual
fibers or cell components may function only on an all-or-none
basis, but when a very large number of these units react
to a chemical together, the summation of their individual ac-
tions may yield measurements that one observes as graded effects
Many graded effects can be expressed as a percentage of the
maximum effect observable.
The important distinction is not dependent upon the observation
being made in percentage units. The important distinction is
that response designates the enumeration of "reactors" or
"non-reactors" within a population, all given the same dose
and demonstrating the same degree of a specific biological
effect, while effect designates a defined biological reaction,
which in some cases may be measurable on a continuous scale.
These concepts are most easily visualized by considering ef-
fect as the change (which in some cases may be graded) due
to a dose within an individual and response as the number of
different individuals showing a specific effect. However, ef-
fect does not have to be limited to observations on individuals.
Just as the effect value plotted graphically against dose may
be the average value of replicates on an individual, it may
also be the average value for the different individuals of
the population given that dose. Further considerations are
taken up by Pfitzer (1975).
When one wishes to enumerate all types of effects at each of
several specific doses, a single curvilinear expression is not
appropriate. In such cases there must be a dose-response curve
for each effect. If this is not feasible, a tabular representa-
tion listing the effects and, when possible, their magnitude,
at various dose levels may be useful.
170
-------
Much of the experimental data pertaining to the toxicology of
metals will be obtained as measurements of graded effects on
individuals within a test population. Nevertheless, these data
may often be converted to expressions of response for the
population as a whole. For example, one may measure the in-
crease in urinary ALA excretion in individuals receiving cer-
tain doses of lead but one may express the results as the in-
creasing percent of the population showing above normal values
with increasing lead dose or period of lead exposure. This
practice for expressing response is justified especially by
the recognition of the variation, often very wide, of the re-
action of individuals within any population to a specific
dose of a chemical. Such variations depend not only on well
recognized characteristics as age, sex, race, health status,
body weight, and diet but also an interaction of metals or other substances
in the environment. It will often be impossible to separate populations
into small groups based upon these characteristics, but their potential influ-
ence must not be forgotten.
When one deals with a human population, the deviation in response
from the normal occurrence of a certain symptom or sign1 will
strongly help to ascertain the possibility of detecting an ef-
fect by epidemiological methods. The specificity of the methods
and the type of statistical analysis as well as a normal occur-
rence of the symptom in question, will determine the quantitative
percentage that will be relevant in this context. It is also
important to decide whether a deviation from the normal value .
which is statistically significant is also biologically so.
As mentioned earlier in this section it is generally believed
that the dose-response curves displaying the frequency of a
certain effect in relation to dose will have a sigmoid shape
when the background frequency of the effect in question in the
population has been subtracted. This classical dose-response
171
-------
curve is the cumulative form of a normal, distribution. This
curve is based on the "law of biological random variation"
and it has been shown to be valid for e.g. the distribution
of lethal doses of various compounds. In empirical studies,
depending on which dose interval is under investigation, the
resulting dose-response relationship may imitate a semi-
logarithmic or linear relation.
Generally, dose-response relationships for metals have not been
reported as dose-response curves. Data have usually not been
collected in a way suitable for statistical analysis, or even
when they were so collected such analysis has not been at-
tempted. In Figure 1 dose-response curves are displayed for
various effects of MeHg from the epidemic in Iraq (Bakir
et al., 1973; Clarkson and Marsh, 1975)i A logarithmic dose
scale was used in those reports. Whenever possible, such
distributions should be given for each effect. The determina-
tion of the type of distribution is of importance, since,
if the distribution is log-normal, it will be possible to
make a more accurate estimate of the doses which give rise
to low level response.
A metal or metal compound, may cause effects of increasing
severity as well as an increasing response for each effect
when the level of exposure increases. Clarkson and Marsh
(1975) have illustrated this well, giving a separate dose-
response curve for each effect (Figure 1). Their data also
provide an example of the difficulties in assessing a low
response by clinical observations. Under the assumption
that the dose-response curve is correct, it also depicts
another essential)point. It is evident from the figure that
if one takes, for example, parasthesia as the effect and 15%
as the response, a dose corresponding to a body burden of about
30 mg would give rise to this response. This of course does not
mean that one could not find individuals with a higher dose
who do not display paresthesia. In 85 persons out of 100 ran-
172
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• PARESTHESIA
• ATAXIA
A DYSARTHRIA
O DEAFNESS
DEATH
10
200
6 16 40 78 156 312
Estimated body burden of mercury
(mg)
Figure 17. The relationship between the frequency of signs and symptoms
and the estimated body burden of MeHg (a) at the time of onset of symp-
toms and (b) at the time of the cessation of ingestion of MeHg in bread.
The two scales on the abscissa are for body burden of MeHg calculated
as described by Bakir et al. (1973) from linear regression lines (upper
scale) and from the Miettinen line (lower-scale). (From Bakir et al. ,
1973.)
173
-------
domly selected, this would be the case. In ord.er to prove that
30 mg is too low an estimate of the given response rate of 15%,
it would be necessary to show the existence of a statistically
significant higher percentage than 85% that are nonaffected at
the dose corresponding to a body burden of 30 mg. (Further
considerations by Nordberg and Norseth, 1975; Kjellstrom,
1975a).
Sufficient epidemiological observations are often not avail-
able for an assessment of a dose-response curve. This may be
true especially in long-term exposure, for which the quan-
titative assessment of exposure during different time periods
is particularly difficult. In such cases, provided data on the
metabolic model as well as on the threshold for critical effects
are available, a mathematical approach may serve to derive a
dose-response relationship. Such an approach has been used
in connection with the evaluation of health hazards from
long-term exposure to cadmium (Kjellstrom, 1975b) and from
varying exposures to me thyImereuric compounds (Berglund et al.,
1971). Such calculations should take into consideration, if
possible, the interindividual variability in the metabolic
model and in the critical concentration in the critical organ
(Nordberg and Strangert, 1975).
IV. 5.2 Cadmium; dose-effect and dose-response relationships
Critical organs have been discussed in IV.4 where the lung was
concluded to be the critical organ for short-term inhalation
exposure and the kidney for prolonged low-level exposure.
IV.5.2.1 Dose-effect and dose-response relationships in critical
organs; critical concentrations in critical organs in
cadmium exposures
Pulmonary effects may be critical in inhalation exposures to
cadmium. Data on the relationship between lung concentration of
cadmium and its effects are not adequate for a meaningful dose-
effect or dose-response evaluation. The only reasonably well de-
174
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fined evaluation of this sort that can be made is based upon
the dose estimated from air concentrations; this evaluation
will be given in the paragraph about dose-response relation-
ships based on other types of data.
Renal effects are critical in low-level long-term exposure to
cadmium. There is evidence from animal data that at kidney cortex
concentrations above 200 ^g Cd/g wet weight, tubular dysfunction
and/or histopathological kidney changes occur (Bonnell et al.,
1960; Axelsson et al., 1968; Stowe et al., 1972; Suzuki, 1974;
Nomiyama et al., 1975; Kawai et al., 1975). Some data
support the occurrence of an effect already at lower concen-
trations (Piscator and Larsson, 1972; Kawai et al., 1975).
it
Kawai et al. (1975) found a correlation between renal cad-
mium concentration and severity of lesions under a constant
exposure situation. However, when the exposure varied, the.
threshold concentration in the kidney for renal tubular damage
also varied. Kawai and associates also pointed out that the
time of exposure and the duration after exposure are important
for the development of chronic lesions in the renal'tubules.
Nordberg (1972a) and Suzuki (1974) reported data for different
animals indicating that at the time at which cadmium-induced
tubular changes appear, cadmium accumulation in kidney cortex
levels off. Kawai and Fukuda (1974) presented data showing the
same tendency. At the breaking point, the rate of urinary ex-
cretion of cadmium changes and cadmium concentration in renal
cortex decreases. On the other hand, if exposure continues,
liver cadmium accumulation may continue even after kidney
cortex cadmium concentration has decreased (Kawai and Fukuda,
1974; Suzuki, 1974; Friberg et al., 1974). Bonnell et al. (1960)
and Nomiyama et al. (1973a, 1974) showed that at high dose lev-
els liver cadmium concentration may even decrease. From the
animal data, it may be concluded that cadmium concentration
in kidney cortex, at the time at which more pronounced renal
damage has already occurred, is not a good indicator of the
175
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individual critical concentration or total accumulated ex-
posure. If, in humans suffering from renal cadmium injury,
the total absorbed dose cannot be calculated from data on
exposure, concentration of cadmium in liver is probably a
better indicator of total absorbed amount of cadmium than is
renal cortex concentration.
Information about morphological changes, proteinuria, and
cadmium levels in kidneys and liver of human beings are
on hand from about 30 autopsies and biopsies of persons
exposed to cadmium. A detailed account of these data has
been given by Friberg et al. (1974) . It is evident that a
detailed dose-effect or dose-response curve cannot be established
with this limited amount of data. In persons with pronounced
kidney damage the renal concentrations of cadmium were low,
whereas in persons with no or slight renal changes, cadmium
levels in kidney cortex with one exception varied between
180 and 450 ug/g wet weight.
Nomiyama (1973) reported data from 5 persons in an area
with cadmium exposure. Three persons showed liver values
higher than those commonly found in Japan (Tsuchiya et
al., 1972a). Of those three, one (without proteinuria)
had a kidney cortex concentration of 264 jag Cd/g wet
weight, whereas another (with proteinuria) had 29 pg Cd/g
wet weight. Proteinuria was not studied in the third person,
who was highly exposed to cadmium for only 7 months and
whose kidney cortex level was 134 pg Cd/g wet weight.
Friberg et al. (1974) concluded on the basis of available
animal and human data that "it is considered justified to
start out from a value of 200 pg/g wet weight in renal
cortex, when discussing which exposures can bring about
cadmium-induced, detectable renal dysfunction in man. This
of course does not mean that 200 Jig/g would give rise to
renal tubular dysfunction in all persons exposed." It is
176
-------
thus understood that this concentration, refers to a response
of a. part of the population displaying average or more than
average sensitivity to cadmium. The magnitude of the inter-
individual variation in sensitivity is not possible to es-
tablish more precisely at present.
This meeting agreed that 200 >ig/g may be considered as a
tentative critical concentration estimate in human kidney
cortex, the validity of which will have to be asseesed
subsequent to further, greatly heeded studies in humans.
Animal data indicate that renal effects may be seen even
at lower concentrations.
IV.5.2.2 Estimation, based on metabolic models, of necessary
cadmium exposure in order to reach' 200 yg/g in renal
cortex
The shortcomings of the metabolic models for cadmium have
been pointed out in section IV. 3, and the uncertainties in
defining a critical concentration for detectable renal
tubular effects were alluded to in the foregoing paragraph.
Even so, it can be worthwhile to illustrate how the models
work..
Calculations of exposure situations giving rise to the
assumed critical concentration of 200 ug/g wet weight in
the critical organ, the kidney cortex, have been presented
by Friberg et al. (1974), Nordberg (1974) and KjellstrSm
(1975b). A summary of calculations made from entering data,
assumptions and two different biological half-times into
these models is presented in Table 8. The biological half-
time has been estimated to be over 38 years (Kjellstrom
et al., 1971; Kjellstrom, 1971) and about 18 years (Tsuchiya
and Sugita, 1971). These calculations are admittedly tentative
but are a good foundation for future research. They need
to be verified by direct observations or re-evaluated if
better metabolic models are found.
177
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Table 8
Cadmium exposure necessary for reaching a kidney cortex concentration
of 200 pg Cd/g using different alternatives for biological half-time in
kidney cortex and exposure time (compiled from data by Friberg et al. ,
1974.)
Estimated biological half-time
in kidney cortex
Basis of calculation
XXX
38 years
xx
18 years
Constant daily
retention during
whole exposure
time
Exposure time
(years)
10
25
50
Daily retention (ug)
36 39
16 20
10 13
25% pulmonary absorp-
tion, 10 m3 inhaled
per work day, 225
work days/year
10
25
Industrial air concentration
(yug/in )
23
11
25
13
Food exposure for 50
year old person (2500
cal/day) (4.5% reten-
tion)
(changing caloric intake
by age accounted for)
50
Daily cadmium intake (ug)
250
360
Corresponding average concentra-
Total
amount
of food/day
(w.w.) 300
600
1000
g 50
g
g
tion
0
0
0
in foodstuffs (ug/g)
.8
,4
,25
1
0
0
.2
,6
,35
KjellstrQm, 1971
xx
Tsuchiya and Sugita, 1971
XXX
Assumptions: one third of whole body retention reaches kidney
and kidney cortex concentration 50% higher than average kidnev
concentration.
178
-------
Based on a suggestion from Professor Kitamura, some members
of the present group. expressed a different 'opinion. They
considered that in order to calculate the time necessary
for accumulation of the level 200 ;ug Cd/g in the renal cortex
(Table 8) , it was necessary that the biological half-time
of that organ section be known. Specific figures such as
18 years or 38 years used in Table 8 were only apparent
biological half-times calculated from kidney concentration
of autopsy samples from the U.S.A. and Japan and could not
therefore be considered applicable in the calculation of
accumulated levels, if inter-organ redistribution of cadmium
occurred in the body .
IV. 5.2.3 Dose-effect and dose- response relationships for cadmium
based on direct observations of exposure and effects
IV. 5. 2. 3.1 Pulmonary effects
Acute effects in the form of pneumonitis as well as chronic
effects in the form of emphysema have occurred in industrial
exposures. When it comes to evaluating dose-response relation-
ships, considerable difficulties exist, primarily depending
upon the paucity of data concerning exposure. In an inhalation
study on cadmium fume (0.2 ju AMD) , LC-Q values on rats were
about
1974).
about 25 mg/m for 30 minutes' exposure (Yoshikawa et al. ,
Friberg et al. (1974) reviewed the existing data and concluded
that for human beings , fatal exposure to cadmium oxide fumes
is not higher, but probably lower than about 2,500 mg/m
x min (corresponding to about 5 mg/m for 8 hours' exposure).
Interindividual variability has not been possible to evaluate.
For associations between long-term exposure and emphysema,
the data are even more limited. Friberg et al. (1974) con-
cluded that a prolonged industrial exposure to about 0.1
mg/m of cadmium oxide fumes might well be considered hazard-
179
-------
ous with reference to emphysema. They based their conclusions
primarily on reports by Bonnell (1955), Buxton (1955), King
(1955) and Kazantzis (1956). Recent data from Lauwerys et
al. (1974) suggest effects on the lungs of tobacco smoking
workers at even lower occupational exposure to cadmium dust
(66 jig Cd/m ) . The average tobacco consumption among these
workers was 13 ± 1.5 cigarettes per day.
IV. 5.2.3.2 Renal effects
In Table 9 data from different epidemiological studies on
cadmium-exposed workers are summarized. Most of the reported
air concentrations are based on very short sampling periods
although the exposure times are very long. By combining the
data from Bonnell (1955) and Bonnell et al. (1959) it was
possible to compare exposure time and response for about 150
factory workers. A higher prevalence of proteinuria was seen
after about 10-20 years of an estimated exposure to cadmium
fume of 50 jig/m air.
Harada, A. (1973, 1974) studied 19 workers in a cadmium pigment
factory,and reported proteinuria (trichloracetic acid method)
among three of five workers with more than 10 years of exposure.
The average exposure is difficult to estimate but Harada, A.
(1973), after having made determinations in the air, calculated
114 jug Cd/m for the worker with the longest exposure and
50 pg/m for a group of newly employed workers (Harada, A.,
pers. comm.). When a more sensitive.electrophoretic method
for the analysis of tubular proteinuria was used, seven
out of eight workers with an exposure time over 5 years
had positive findings.
Lauwerys et al. (1974) did not find any difference in the
prevalence of abnormal urinary electrophoretic patterns
between controls and workers exposed to 134 tig CdO dust
per m at an average 8.6 years of exposure. Significantly
180
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Table 9
Epidemiological data from industrial exposure situations useful for
dose-response evaluations (data compiled from Kjellstrom, 1975b)
Reference
Bonnell (l955)
and
Bonnell et al.
(J.959")
Harada (1973}
(1974) and
personal
communication
Effect
studied
Proteinuria
(SA or TCA
method)
Increased
globulins
in elect ro-
ph ores is
Proteinu-
ria (TCA
method)
Estimated air
concent rat ion
3
40-50 ug/m
CdO fume
Average among
some workers—
5O ug/m3. (Time
weighted average)
Average for Worker
with longest ex-
posure time=
114 pg/m3
( range among
air samples
24 - 1220 >ig/m3) .
C
Exposure time
Cont rol s
< 9 years
9-13 years
14-18 years
19-23 years
>23 years
•^5 years
>5 years
< 10 years
>10 years
Calculated
response or
jffect
1/60
6/22
7/15
9/19
8/14
7/30
1/11
7/8
0/14
.'i/5 .
I.auwerys Abnormal
et al. (1974) urinary
elect ro-
phoretic
pattern
(as defined
by the
authors)
-
Total CdO dust
JL34 pg/m3
respirable
fraction 88 ug/m3
Tr>t->1 TrlO Huot
Controls 0/22
Average 8.6 0/27
years ( range
0.6-19.6)
Avoi-nnro 97 8 IR/OT
66 pg/m3 respir-
able fraction
21 ug/m3
years (range
21-40 years)
181
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Table 9 (continued)
Epidemiological data from industrial exposure situations useful for
dose-response evaluations (data compiled from Kjellstrom, 1975b)
Reference
Horstowa,
Sikorski and
Tyborski ;
(1966")
Friberg 0.950)
Hscator,
1
Adams et al. ,
(1969)
Effect
studied
Proteinuria
(method not
given)
Proteinuria
(Heller
method)
Estimated air
concentration
Total dust 130 -
1170 ug/m
Total CdO dust
400-15000 ug/m .
Total quan 40 workers from
tative deter- the study by
mination of Friberg, 1950
urinary pro- who were not
tein (biuret exposed since
method, using that study
Tsuchiya' s
reagent as
precipitating
agent)
Proteinuria
(SA-method)
Total dust
500 ug/m
Exposure time
1-12 years
1- 5 years
6-10 years
11-15 years
16-20 years
21-25 years
>26 years
1-40 years
>5 years
time for develop- •
ment of proteinuria=
7-24 years
Calculated
response
or effect
7/26
0/26
1/4
3/9
6/12
3/9
6/9
100 mg/24 hours
at average 3 years
exposure and 1000
mg/24 hours at
34 years
20 %
182
-------
higher prevalence was found among workers exposed to 66 iig
3
CdO dust/m for 27.8 years (average).
Other studies (Table 9) have shown renal effects at higher
exposure levels. A dose-response curve based on a combination
of all data cannot be constructed because of the different
methods used for analysis of both exposure and effect.
An increased response as well as effect (increased concentration
of protein in urine) is seen with increasing exposure time in
some studies, and proteinuria may persist after exposure has
ceased (Piscator, 1962b, 1966). When sensitive methods are
employed, tubular proteinuria can be detected already after
a few years at estimated exposure levels of 50 yug/m (Harada,
A., 1973, 1974). These data support Tsuchiya (1967) who
found that industrial air concentrations of cadmium dust
and fumes must be kept below 50 ug/m in order to prevent
cadmium-induced tubular proteinuria after prolonged exposure.
Reports used for dose-effect relationships among occupa-
tionally exposed workers have been collected to some extent
in terms of the concentrations in the working environment.
However, one must be careful when these results from occupa-
tionally exposed workers are applied to the general population
since the sensitivity to cadmium may be different in different
population groups.
Friberg et al. (1974) reviewed the epidemiological studies
made in areas of Japan where the general population has
been exposed to cadmium via food. The same data and some
additional data were discussed by Kjellstrom (1975b) from
the dose-response point of view. Friberg et al. (1974) con-
cluded "considering all the data together, there can be
no doubt that cadmium plays a role in the etiology of ele-
vated prevalence of proteinuria in heavily exposed areas."
The present daily intake in areas where health effects have
been seen was estimated by Friberg et al. (1974) to be 200
to 300 ^ug or higher. The studies in Japan were not performed
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for dose-response evaluation of renal effects but for de-
tection of Itai-itai patients. Therefore, the dose evaluations,
based mainly on cadmium concentrations in rice, are only
approximate. Analysis of cadmium in feces may be a promising
method for cadmium intake estimates in epidemiological studies
(Tati et al., 1975).
When Watanabe et al. (1974)., in their studies in Japan, give
a value of, for instance, 0.3 or 0.4 yug'Cd/g in rice, this
does not of necessity reflect the actual exposure levels
over a period of many years. Hence, such values cannot be
readily applied to dose-response relationships. The inten-
tion was to use cadmium level in rice only as an "indicator"
for evaluating cadmium intake; it is recognized that the
rice levels documentionly the present level of exposure.
Kjellstrb'm (1975b) estimated on the basis of epidemio-
logical data and his metabolic model that somewhere between
0.4 and 0.6 ^g Cd/g average concentration in basic food would
be high enough to induce tubular proteinuria in a significant
part of the population. He assumed that the intake of rice was
300 g/day and that half the cadmium intake came from rice.
Tati et al. (1975) presented data showing that the average
daily cadmium intake in a non-exposed Japanese population
was about 60 /ug. Kjellstrom1s estimation implies that the
necessary average intake for causing tubular proteinuria
in a population is 240-360 yug.
Friberg et al. (1974) concluded that the data from autopsies,
animal experiments, theoretical accumulation models, and
epidemiological evidence from the industrial and general
environment agree well as to the relationship between cadmium
and renal effects.
IV. 5.2.4 Factors influencing dose-effect and dose-response re-
lationships for cadmium
Metals suspected to have an influence on dose-response relation-
ships for cadmium in human beings include zinc, sel-
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enium, and possibly copper. As mentioned earlier, protein
and calcium content of the diet will influence gastroin-
testinal absorption of cadmium. An interaction between cad-
mium and lead has been suspected, but not confirmed. Some
experimental data on influence of selenium, copper* and some
other metals will be presented in section IV.6. The following
discussion will be devoted mainly to the influence
of zinc on cadmium toxicity.
It has been postulated that the renal dysfunction occurring
at higher renal levels of cadmium may be caused by interference
of cadmium with zinc-metalloenzymes (Friberg et al., 1974),
and the renal content of zinc can thus be an important factor
when discussing dose-response relationships. Leucine amino-
peptidase is a zinc metalloenzyme, which has been isolated
from renal cortex of pigs (Himmelhoch, 1969; Lisowski et
al., 1970). Cousins et al. (1973) found that in cadmium-ex-
posed pigs the activity of this enzyme was decreased in the
renal cortex.
In human beings it has been shown that the "normal" accumu-
lation of cadmium in the renal cortex is accompanied by an
equimolar increase in zinc (Piscator and Lind, 1972) , whereas
no change in copper concentrations takes place. There are
few data on both cadmium and zinc in renal cortex from human
beings with excessive exposure. Piscator and Lind (1972)
found that a 24-year-old man with occupational exposure to
cadmium had a cadmium concentration in renal cortex of 195
ug/g wet weight and a zinc concentration of 67 ug/g wet weight.
The averages in a "normal" group, mean age 44 years, were
30 and 52 yg/g respectively.
In normal horses without excessive exposure, an equimolar
increase in zinc was observed in the renal cortex at cadmium
concentrations in the same range as those found in human
beings, e.g. <75 pg/g wet weight. At higher cadmium levels
185
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up to about 250 yg/g wet weight, zinc die} hot increase to
the same extent. In fact, at concentrations of cadmium around
200 yg/g, e.g. about 3 times higher than the upper "normal"
limit in human beings, zinc concentrations had increased by
only 50% (Piscator, 1974). This indicates that cadmium affects
zinc kinetics in the kidney at concentrations below the critical
concentration in renal cortex.
The equimolar relationship between cadmium and zinc in equine
renal metalflothionein under normal conditions (Kagi et al.,
1974) may explain the "normal" equimolar relationship in the
renal cortex. In experimental animals, the ratio between cadmium
and zinc in metallothionein will increase under exposure to
cadmium. This may be related to the changed relationship between
zinc and cadmium found at higher renal cadmium concentrations;
IV.5.2.5 Summary of dose-effect and dose-response relationships
for cadmium and its compounds
Only solitary data exist on renal concentrations of cadmium
in human exposures and even dose-response relationship data
in animals are scarce. Various metabolic models have been
proposed to estimate the critical levels of cadmium in the
renal cortex. On the basis of available studies, a level of
200 yg Cd/g wet weight has been suggested as a tentative crit-
ical concentration in the renal cortex for human beings. At
this concentration there is a response of a part of the popu-
lation that displays average or more than average sensitivity
to cadmium. The final validity of this value needs to be ver-
ified by further epidemiological studies on occupationally
exposed workers and residents of cadmium-polluted areas, par-
ticularly regarding the relationship of cadmium to coexisting
kidney levels of zinc and metallothionein.
The limited data base, built upon observed human exposures
up to this date, indicates that in long-term inhalation
exposures, air concentrations lower than 50 ug Cd/m
are necessary to prevent cadmium-induced proteinuria. in
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peroral exposures, the corresponding figure for daily intake
has been suggested at the level of 200-300 :Vg Cd/day in adult
persons living under satisfactory nutritional conditions.
IV. 5.3 Dose-effect and dose-response relationships for
'.lead (inorganic lead compounds)
IV.5.3.1 Introductory remarks
It has been concluded earlier (section IV.3.3) that a useful
metabolic model for lead describing the relationships between
exposure, body burden, and concentrations in critical organs
and index media, is not available, nor are there many data
on concentration-effect relationships in the organs considered
critical in lead exposure (bone marrow, nervous system,
kidney - see discussion in Section IV.4). For these reasons the
discussion in this section will be mainly devoted to the
relationships between the blood lead concentration (Pb-B) and
hematological, nervous, renal and other effects, under the
assumption that the blood lead concentration reflects soft
tissue concentrations.
Valid conclusions require valid measurements. Even disregarding
that Pb-B is only an indirect measure of dose, or of exposure,
it is well known that lead measurements are extremely vulnerable
to methodological error. This vulnerability has created great
confusion regarding both dose-effect and dose-response relation-
ships . In general, older data tend to be higher than those
obtained in the 1960 s and 1970 s, but considerable variations
in accuracy and precision remain, even between experienced
laboratories, as shown by some recent comparative studies
(Donovan et al., 1971; Berlin, A., et al., 1973, 1974). Under
these circumstances, it is small wonder that discrepancies
are found when all literature to date is examined. In the fol-
lowing review only data from laboratories or research in-
stitutes with a good methodological level are considered; yet,
variations of 10 to 20 yg Pb/100 ml blood will become apparent.
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Quite naturally, the validity (precision and accuracy) of
the measurements of effects is as important as that of the
dose. Interlaboratory comparisons have shown different
degrees of validity for different responses, e.g. the
validity of ALA-D measurement is relatively high, and that
of ALA moderate to high as compared to lead in blood measure-
ments (Berlin, A., et al., 1973). Quantitative comparative
data for other parameters of response are scanty.
IV.5.3.2 Dose-effect and dose-response relationships in
critical organs and indicator (or index) media
IV.5.3.2.1 Hematological effects
The hematological effects of lead are the only ones extensively
studied by biochemical methods, and for which the mechanism. . .
of a lead action is known in reasonable detail (see Chapter 4).
These effects are also the only ones for which relationships
to dose have been established with any satisfactory accuracy
and, even so, the only relationships defined are those to lead
in blood. Direct relations between lead concentrations in the
bone marrow and effects are unknown. However, Wada and Ohi (1972)
have observed depressed ALA-D and heme-S activities and re-
duced heme synthesis in erythroid cells from the bone marrow
of lead workers whose blood lead concentrations were in range
of 40-90 yg/100 ml.
The earliest effect related to hematopoietic tissue is inhibi-
tion of ALA-D which becomes measurable at about 10 to 20 vg
Pb/100 ml blood (Hernberg et al., 1970). Erythrocyte ALA-D
is also inhibited in vitro by a number of substances, e.g.,
EDTA,mercury, copper, silver, manganese and cobalt (Hernberg
and Nikkanen, 1972). There are data on in vivo inhibition
by ethyl alcohol (Millar, 1973), but the inhibitory action
of all these agents is less than that of lead. There exists
a highly significant semilogarithmic inverse relationship
between lead in blood and ALA-D activity. When high lead
levels are included, the correlation coefficients are as
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high as 0.8-0.9. In a large study by Tola (1973) the level
for detection of a slight inhibitory effect upon ALA-D was
10-20 yg Pb/100 ml blood. Similar results have been reported
by Haeger-Aronsen et al. (1971) and by Sakurai et al. (1974).
However, another study (Hernberg et al., 1970) has not demon-
strated such a threshold. It is not yet clear if a linear re-
lationship persists below the 10-20 yg Pb/100 ml level in
blood, or if a plateau is formed. Sassa et al. (1973) have
proposed, on the basis of kinetic-studies, that inhibition
of ALA-D activity would not occur below 15 yg Pb/100 ml of
blood.
The activity of ALA-D is almost totally inhibited at blood
lead levels of 70-90 yg/100 ml. For adults in industry, the
relationship between lead in blood and ALA-D is roughly the
same regardless of whether the exposure is long-term steady
state, acute, or has been terminated (Tola et al., 1973).
A partial ALA-D inhibition cannot be regarded as a deleterious
effect in itself.
A minor depression (approx. 30%) of the ALA-D activity occurs
in 50 percent of the population at approximately 30 yg/100 ml
lead in blood. At a blood_lead level of 50 yg/100 ml, 50 percent
of the population shows about 65 percent reduction in the ALA-D
activity. A 90 percent reduction occurs in 50 percent of the
population at blood lead levels around 75 to 80 yg/100 ml
(Tola, 1973).
A threshold of about 35 to 40 yg Pb/100 ml of blood for an
increase in the excretion of ALA-U has been reported by some
authors (Selander and Cramfir, 1970; Haeger-Aronsen et al.,
1971; Tola et al., 1973), and one of about 50 by others
(Sakurai et al., 1974; Chisolm et al., 1975). Such slight
differences probably stem from methodological discrepancies.
Above this threshold the concentration tends to increase
rapidly. However, the correlation of ALA-U with Pb-B is
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not as close as that for the ALA-D activity. Usually, the
correlation coefficients have been between 0.5 to 0.7 for
blood lead values in the range of 40 yg/100 ml and above
(Selander and Cramer, 1970} Tola, 1973). Approximately 50
percent of a population with a blood lead level of 50 yg/100
ml exhibited ALA-U values greater than 5 mg/1 urine, and
at 65 yg Pb/100 ml blood 50% of ALA-U values exceeded 10 mg/1 urine
(Zielhuis, 1973a).
Urinary coproporphyrin (CP-U) excretion also starts increasing
at approximately 35 to 40 yg lead/100 ml blood, and it is thus
almost as sensitive as ALA-U to the action of lead (Tola et
al., 1973). However, increase of CP-U may also be due to other
factors than lead, e.g. alcohol abuse, liver disease, porphyrias,
etc. This parameter is thus less specific than ALA-U.
The concentration of PP IX in the erythrocytes (FEP) starts to
increase at Pb-B levels of about 25 to 35 yg/100 ml in children
and women, and at about 30 to 40 yg/100 ml in men (Stuik, 19*74;
Zielhuis, 1975a) . The difference between women and men may
be due to the fact that PP IX is sensitive to iron deficiency;
ii) general, women have smaller stores of iron. Elevated PP IX
levels persist long after^the cessation of exposure; thus
the correlation to Pb-B cannot always be expected to be as
close as those for the parameters discussed above. However,
in steady state, coefficients of up to +0.91 have been demon-
strated (Sassa et al., 1973). Chisolm et al. (1974) also
found a relationship between blood-lead concentration and
protoporphyrin concentration in children. The increase oc-
curred at somewhat lower blood lead concentrations in
children with mild anemia and was interpreted as an effect
of low iron intake.
No studies of suitable epidemiological design have as yet come
forth which would allow a close approximation of the dose-
response relationship for FEP in men, women and children. For
a further discussion on this topic, see Zielhuis (1975a).
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Reticulocytosis becomosmeasurable at Pb-B's between 60 and 80
yg/100 ml. In this range a shortening of erythrocyte life
span begins as well (Hernberg et al., 1967a). The degree of
anemia correlates poorly, if at all, with Pb-B. However, a
slight drop in the hemoglobin level has been shown at Pb-B
levels of about 50 to 80 yg/100 ml in new lead workers
during their first three to four months of employment
(Tola et al., 1973). Obvious anemia usually does not develop
when Pb-B is below 80 yg/100 ml. At higher Pb-B levels all
abnormalities become more and more pronounced, but inter-
individual variation is large.
IV.5.3.2.2 Neurological effects
IV.5.3.2.2.1 Central nervous system effects (Encephalopathy)
As discussed in section IV. 5.3.1.2.1, encephalopathy resulting from
lead exposure usually requires more intensive and more prolonged
exposure than that giving rise to hematological effects. .
The exact mechanism behind lead-elicited acute encephalopathy
is not known. It is difficult to ascertain whether the effect
is usually a result of repeated acute incidents of lead exposure
or a result of continuous exposure, causing a high level
of lead in the CNS. The onset and course of acute lead en-
cephalopathy are always unpredictable. Although high blood
lead concentrations -usually much above 100 yg/100 ml - are
required, other severe symptoms such as gastrointestinal
colic are not always present. It is hence obvious that '.neither
clear-cut dose-effect nor dose-response relationships for
encephalopathy may be stated at the present time.
It may be that children suffer other CNS effects at levels
even lower than 100 yg/100 ml. It has especially been dis-
cussed whether long-term low level exposure to lead in children
can give rise to an increase in frequency of mental retardation
(Perlstein and Attala, 1966) or hyperactivity (David et al.,
1972j David, 1974). Albert et al. (1974) found an increased
191
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frequency of deleterious effects in a follow-up of children
without diagnosed lead poisoning, but with a. history of blood
lead levels above 60 ug/100 ml. Data on concentrations of
lead in the CNS in human beings with various types of lead-
induced CNS-effects are not available. Such data are lacking
for experimental animals as well.
Lead readily crosses the placenta, and the correlation between
cord and maternal blood lead levels is good. Ratio of maternal
and fetal blood lead is near unity regardless of differences in
hematocrit. Therefore although no data are available, it
must be said that an increased body burden of lead in women
of child-bearing age may mean a possibility of effects on the
central nervous system in the fetus. This may hold true even
in the absence of evidence of lead intoxication. This question
needs systematic study.
IV.5.3.2.2.2 Peripheral nervous system manifestations
While dose-response relationships for the clinical manifesta-
tions of lead effects on the peripheral nervous system are
incompletely established, there are better data available
concerning subtle effects, which have been assessed by elec-
trophysiological techniques. It has been possible to demonstrate
effects on the peripheral nervous system in lead workers
whose blood lead levels were in the order of 80 to 120 ug/100
ml and who had varying degrees of lead poisoning, but who
were completely without any clinical neurological symptoms
or signs (Sessa et al., 1965; Catton et al., 1970; Seppalainen
and Hernberg, 1972) . The abnormalities consisted of a slowing
of the conduction velocity of the nerves of the upper limbs,
particularly in the slower fibers and in the distal portion,
and electromyographic abnormalities such as fibrillations
and diminished numbers of motor units upon maximal contraction.
Similar abnormalities, although of a slighter degree, have
been demonstrated in a group of lead workers who were exposed
between 1 and 23 years, whose blood lead values had never
-------
exceeded 70 pig/100 ml, and in whom no signs of lead poisoning
had been noticed. At examination the mean blood lead level was
40 t 9 ug/100 ml with a maximum of 65 ug/100 ml (Seppalainen
1974; Seppalainen et al., 1975).
In Figure 18 it is proposed that.slight peripheral neuropathy
may occur in a small percentage of the population already
at blood lead levels of about 50 ug/100 ml. About 50% of
the population would be expected to show this effect at lead
levels of about 120 ug/100 ml and 90% would probably experience
at least slight damage at lead levels of about 150 ug/100
ml. At this uppermost level, a few percent of the population
could be expected to show severe damage. However, more exact
dose-response data are required to confirm these assumptions
(Zielhuis, 1975b). Data relating lead concentrations in nerves
or nerve cells to effects are not available for human beings
or animals.
IV.5.3.2.3 Renal effects
Data on the renal effect of lead are incomplete, and the dose-
response relationships are even less well-established than those
for the neurological effects. Neither human nor animal data
have been interpreted in such a way as to show a critical organ
concentration for renal effects of lead. When more extensive
information is available, the older data can perhaps be rein-
terpreted with that purpose in mind. Goyer et al. (1970)
found intranuclear inclusion bodies in the kidneys at a
renal lead concentration of 10 vg/g in rats. About 60 yg Pb/g
kidney will be required for aminoaciduria and renal edema
to appear in rats given lead in drinking water.
Renal effects of lead have also been related to blood lead lev-
els, but this relationship is incompletely documented. Goyer
and Rhyne (1973) suggested that blood lead levels in the
range 40-80 \ig/10Q ml will be associated with inclusion bodies
in the renal tubular epithelium.. At blood lead levels above
150 yg/100 ml Fanconi's syndrome may develop among exposed
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HEMATOLOGICAL SYMPTOMS
ALA-D inhibition in RBC
PP elevation in RBC
ALA excr. increased in urine
CP excr. increased in urine
Shortening of RBC life span
Reticulocytosis
Anemia
OTHER SYMPTOMS
Subjective symptoms
Peripheral neuropathy
Encephalopathy
Colic
Kidney function impairment
I
I
50
100 150
Pb,ji g/100 ml
200
250
Figure 18. Relation between blood lead levels and the onset of a number of effects. (From Hernberg, 1975.)
-------
children (Chi^olm and Leahy, 1962)f but the exact dose-response
relationship is not known. In occupationally exposed workers,
kidney function impairment, as shown by increased blood urea
levels/may occur at blood lead levels of about 120 Ug/100
ml in a small portion of the population (a few percent);
about 50% of the population would suffer such impairment
at a blood lead level of about 160 Ug/100 ml, while more
than 90% at 180 ]ig Pb/100 ml (Hernberg, 1975). However, these
dose-response relationships for kidney function impairment
are uncertain.
IV. 5.3.3 Summary of dose-effect and dose-response relationships
for inorganic lead compounds
Dose-effect relationships in critical organs of humans cannot
be given because no substantial body of data exists on the
concentrations of lead in the organs which it affects (i.e.
bone marrow, nervous system, kidney) in conjunction with .
measurements of effects in these organs. There are substantial
data relating the various effects to several ranges in blood
lead concentration. The critical effects on heme synthesis
(increased ALA-U and CP-U) begin to appear in the range of
30-50 pg Pb/100 ml whole blood but the range may be slightly
lower for increased PP IX (FEP) to appear. Depressed ALA-
D activity, depressed heme-S activity and reduced heme synthesis
in erythroid cells from the bone marrow have been observed
in lead workers with blood lead concentration in the 40-
90 ug/100 ml range. Renal effects and acute clinical effects
have generally been observed only at higher ranges of blood
lead concentration. Limited data in humans strongly suggest
that the CaEDTA mobilization test, which provides a measure
of the biologically active lead, may be a better indicator
in intact man of the concentration of lead in affected organs.
It should also be pointed out that the CNS may be the critical
organ under certain circumstances, particularly in the very
young child or the fetus. A discussion of this possibility
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has been undertaken (IV.4.3.1.2.1 and IV.4.3.3) but no precise
dose-response relationships can be set up. .
Recent studies suggest that a nerve conduction deficit detect-
able by electromyography might be the critical effect in
certain adults with industrial exposure to lead. A preliminary
estimation of dose-response relationships for this effect
has been presented (IV.5.3.2.2.2).
IV.5.4 Mercury: dose-effect and dose-response relationships
IV.5.4.1 Mercury vapor
IV.5.4.1.1 Dose-effect relationships in critical organs and
indicator (or index) media; critical concentrations
in critical organs
On short-term exposure to high levels of mercury vapor, the
lung is the critical organ. Following repeated or prolonged
exposure to moderate levels of mercury vapor the brain will
be the critical organ. Reliable data on concentrations of'
mercury in the lungs on short exposure to mercury vapor have
not been found in the literature, nor is there any suitable
diagnostic indicator (or index) media reflecting the' concen-
tration in the lungs after exposure to mercury vapor. Therefore,
it is possible to provide ^neither dose-response nor dose-effect
relationships for mercury concentrations in the lung. There
are too few data about effects on the kidney after exposure
to mercury vapor to establish a dose-effect relationship.
t
In animal,studies, mercury concentrations found in the brain
after exposure to mercury vapor have been reported by Ashe
et al. (1953). They exposed rabbits to 6 mg Hg/m , 0.9 mg
Hg/m and 0.1 mg Hg/m during 7 hours per day, five days per
week during various periods. At mercury concentrations between
3 and 17 ug/g they found moderate histopathological changes
and at levels between 1 and 2 ug/g definite but mild histo- .
pathological changes. At levels up to 0.5 ug/g no changes
were observed. In studies by Berlin '. (1975), levels up to 8
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Vtg/g in the occipital part of the cortex, were observed without
any definite pathological changes in the squirrel monkey. In
Berlin's opinion, behavioral disturbances may have occurred
in those monkeys.
Studies from the USSR have reported behavioral changes at
exposures to mercury vapor of less than 0.1 mg/m , but
conclusive data on brain concentrations in relation to these
findings are not available. Friberg and Nordberg (1972) have
summarized the Russian literature in this respect, especially
studies by Trachtenberg (1969) in cats and by Kournossov
(1962) on rats. The Russian data indicate that behavioral
effects may occur at considerably lower concentrations in the
brain than those giving rise to clear-cut morphological
damage. This may be relevant when discussing dose-response
relationships for mercury vapor.
IV.5.4.1.2 Mercury vapor; dose-response relationships
Data on dose-response relationships on exposure to mercury
vapor are limited (review: Friberg and Nordberg, 1972;
NIOSH, 1973). Smith et al. (1970) studied a group of 567
workers in the chlorine industry exposed to mercury vapor.
They showed increasing prevalence of loss of appetite, loss
of body weight, tremor, insomnia, shyness, and history of
nervousness at exposure levels from 0.1 to 0.27 mg/m . Exposure
in chlorine industries, however, includes chlorine as well
as mercury.
IV.5.4.1.3 Use of metabolic models for establishing dose-effect
relationships., for mercury vapor
It is evident from the foregoing account that data, both with
regard to the metabolic model .and with regard to the critical
organ concentration for mercury in the brain and lungs after vapor
exposure, are too uncertain to allow any useful calculations
on dose-effect and dose-response relationships. Further
research is needed on this question and also on the rela-
197
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tionship between concentration of mercury in blood and urine versus that in brain.
IV. 5.4.2 inorganic mercury compounds '• dose-effect relation-
ships in critical organs and indicator , (or index)
media; critical concentrations in critical organs
On exposure to mercuric salts regardless of whether inhaled as
a dust or ingested, the kidney is the critical organ (section IV.4.3).
Available evidence concerning relationships between concentration in
the kidney and effects has been summarized by Berlin (1975). In case
of known poisoning due to short-term exposure to mercuric salts,
values between 10 and 70 yg Hg/g have been reported in human beings
(see also IV.4.4.3). It seems from evidence to date that levels of
mercury in the kidney between 10-40 yg/m give rise to slight to
moderate changes whereas concentrations of over 100 yg/g are asso-
ciated with severely damaged kidneys. However, the level at which
effects occur is determined not only by variation in individual
sensitivity but also by the rate of dosage, or, rate of accumulation
in kidney tissue.
As mentioned in earlier sections there is no medium that is
good for paralleling mercury concentration in the kidney; thus
relationships between mercury concentration in indicator (or
index) media and effects are not meaningful.'Even so, it cannot
be excluded that urinary excretion of mercury or blood mercury
levels during conditions of* no exposure may reflect mercury
concentrations in the kidney. This has to be studied.
IV. 5.4.3 Summary of dose-effect and dose-response relationships
for mercury vapor and inorganic mercury compounds
Data of possible relevance to the critical organ concentration
of inorganic mercury in the kidney and the critical organ concentration
of mercury vapor in the brain have been reviewed, but it has
been concluded that it is difficult to see any certain, exact
relationship between the concentration and the response. However,
the data at hand allow a very rough estimation of the approximate
concentration connected with effects. For mercury vapor,also,
effects on the brain displayed as behavioral changes have to
be taken into account. Their dose-response relationships are
difficult to evaluate on the basis of the critical organ concentra-
tion, because of the scarcity of data and because of the insecure
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experimental and analytical foundation on which they are built.
Data on direct exposure-effect relationships do suggest that
such changes may occur at considerably lower chronic exposures
than those giving rise to histopathologically evident changes.
IV.5.4.4 Organomercury compounds
IV.5.4.4.1 Methylmercuric compounds
IV.5.4.4.1.1 Dose-effect and dose-response relationships in
critical organs; critical concentrations in critical
organs (critical body concentration)
As mentioned earlier, the critical organ system in poisoning by
alkylmercuric compounds is the CNS in man and other primates. In
lower animals like rabbits or rats, the peripheral nervous system
or the kidney may be critical organs. Especially the rat, which suf-
fers renal damage from exposure to methylmercuric compounds, is not
a good model for studies on dose-effect relationships for MeHg .
Caution should be exercised when using data from this species for
evaluation of the dose-response relationships for other animals and
man. The fetal brain is the critical organ during pregnancy. It has
been stated above that MeHg may affect the chromosomes at a low
level, below that giving rise to detectable changes in the CNS. It
was also said that sufficiently documented data on this .possibility
are not yet available. It is not possible, at present, to talk about
a critical concentration for this type of effect.
*
An attempt will be made here to estimate the adult brain concentra-
tion associated with death and also the brain concentrations asso-
ciated with signs and symptoms of methylmercury poisoning. Special
attention will be paid to the more sensitive part of the population
and thus to the lowest brain concentrations that may ever have been
associated with symptoms. In these discussions about brain concen-
trations, data derived from studies on blood values, liver values,
total intakes and body burdens will be used. The concentrations
of MeHg in brain associated with death in the more sensitive
individuals of the population may be estimated from data
derived from various sources. Studies on limited numbers of
non-human primates (Nordberg et al., 1971a; Berlin, M. et al.,
1975a, 1975b) indicate that the minimum lethal concentration
is above 9 yg Hg/g wet weight and that the minimum concentration
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1 I
for signs is lower than 9 ug/g. Data on brain levels 'in ..
human beings poisoned in the Minamata outbreak, as inter-
preted by a Swedish expert committee (Berglunfl.et al., 1971),
indicate that the average brain level at the time of onset
of symptoms was 25 ug/g. In one patient, it may have been
as low as 6 iig/g. The levels at the time of onset of symptoms
probably correspond to maximum brain levels, since this
patient most likely entered the hospital at about this time
and therefore stopped consuming contaminated fish.
Magos et al., (1974c) reported on fifty autopsy specimens
of liver from the recent Iraqi outbreak. The average level
was 16.3 ug Hg/g wet weight, the lowest being 1.4 ug Hg/g
wet weight. This level is so low that it is difficult to
believe that me thy liner cur y compounds were the sole cause of
death. The highest recorded value was 75.5 wg/g wet weight. .
Taking the average liver levels and assuming a ratio of 2
for liver to brain concentrations (Berglund et al. , 1971) ,.
these data indicate an average brain level at death of ,
8 ug/g wet weight. Most of these patients died early in the
outbreak, approximately 45 days after the exposure to mercury.
Thus the maximum average brain level in fatal cases, probably
was about 16 ug/g wet weight. Examining the distribution
of liver levels in those cases, and taking into account other
t
possible causes of death, the lowest liver level in a fatal
case of methylmercury poisoning was 10 wg/g. Correcting for
a 45-day period after exposure, the maximum brain level in
this case would be about 10 ug/g.
Bakir et al. (1973) reported estimates of maximum body burden
of MeHg in fatal cases in Iraq. (Figure 17b). They noted that
the probability of a fatal outcome rose slightly above a
body burden of 200 mg in a 50 kg person, corresponding to
280 mg in a 70 kg person. Using the parameters of the metabolic
model for MeHg4" in man (section IV.3.3.3.4.3.1), this should
200
-------
correspond to a brain level of approximately 17 ug/g.
An alternative estimation of the body burden also
presented by Bakir et al. indicates a "threshold" lethal
body burden of 312 mg corresponding to a brain level of
18 ug/g. Data obtained from the outbreaks in Japan and
Iraq are in reasonable agreement on minimum lethal level
in the brain (Table 10) indicating that this level is above
6 ug/g and probably lies in a range from 6 to 12 ug/g.
Data from animal tests and from the outbreaks of poisoning
in Japan and Iraq allow estimates to be made of minimum
brain concentrations of MeHg associated with signs and
symptoms of poisoning. The studies on monkeys referred to
above indicate the minimum brain level for signs and
symptoms to be below 9 ug/g.
Analysis of data from the outbreak in Niigata (Berglund et
al. , 1971) indicates an estimated lowest blood level at
the time of onset of symptoms of 200 ng/g. This would corre-
spond to a brain level of about 1 ug/g. Most hair samples
collected during the Niigata epidemic had concentrations
above 200 ug/g corresponding to a blood level of abbut 670
ng/g. The hair data cast some doubt as to the minimum blood
level of 200 ng/g. In the case of mild poisoning, an exact
recollection by the patient concerning the time of onset
of symptoms is not to be expected. The value of 200 ng/g
estimated by back extrapolation may have been higher at the
time of onset of symptoms.
The information reported in Figure 17 of this report (quoted
from Bakir et al., 1973) indicates that the first effects
of MeHg become detectable at a body burden in the range
of 25 to 40 mg in a 50 kg individual. This would correspond
to brain levels of 2.1 to 3.4 ug/g in a 70 kg standard man.
New data reported by Kazantzis (1975) support the dose-
response data of Bakir et al. (1973). For example, Kazantzis
201
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Table 10
Estimated Minimum Concentrations in Brain Associated with Signs and
Symptoms and Death in Poisoning by Methylmercuric Compounds
Data Source
Squirrel Monkey
Minamata
(Brain autopsy)
Niigata
(blood levels)
:Iraq +
(liver autopsy)
Iraq
(body burden )+
Iraq t
(ingested dose)
Iraq
(hair levels)'''
Concentratio
Signs &
symptoms
<9
1
2-3
2-3
4
n (yg/g)
Death
> 9
> 6
> 10
>.17
n
16
12
17
51
125
470
93
Reference
Berlin et al. (1975b)
Berglund et al.
(1971)
n
Magos et al. (1974c)
Bakir et al. (1973)
Kazantzis (1975)
Al-Shahristani et al.
(197.4)
n = number of individual observations.
*
t
Brain concentrations calculated on the basis of the metabolic
model for MeHg in the standard man (see text) .
202
-------
reported on a population of 473 people for whom the average
dose was 150 mg Hg ingested over a period of 32 days: Assuming
a clearance half-time of 70 days for the whole body and using
equation (3) , it may be calculated that the 'average maximum
body burden in the population was 125 mg. The frequency of
symptoms in the population was 50%» In a study by Al-Mufti
et al. (1974) the frequency of paresthesia was 26% in a total
of 165 persons consuming 50-99 contaminated loaves (1.4 mg
per loaf). The Bakir et al. (1973) dose-response curve f
indicates that the frequency of paresthesia at this body
burden is between 50 and 60%. There is thus a close agreement
between these two independent estimates. The Kazantzis study
(Kazantzis, 1975) also points at a minimum brain level
associated with symptoms of from 2 to 3 ug/g using the
metabolic model.
Al-Shahristani et al. (1974) have compared signs and symptoms
of poisoning in the Iraqi outbreak with maximum hair con-
centrations of mercury. These results indicated that the
mildest cases of poisoning occurred at hair levels of 120 ug
per g and above. Al-Shahristani et al. were able to compare
hair concentrations to body burdens of MeHg estimated from
the ingested dose. These observations indicated that a hair
level of 60 lig/g in the Iraqi subjects was equivalent to an
average body burden of 0.8 rag/kg. According to the metabolic
model this would be equivalent to a brain level of 3.7 jjg/g.
In summary (Table 10), observations on the outbreaks in Japan
and Iraq are essentially in good agreement, indicating that
the minimum brain levels in adults associated with signs
and symptoms fall in the range of 1-4 ug Hg/g wet weight.
It was mentioned above that the fetal brain is the critical
organ in pregnant females, but there are at present no data
in the literature on concentration of MeHg in the fetal
brain of exposed human or primate fetuses. A quantitative
evaluation of this effect thus cannot be made.
203
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IV.5.4.4.1.2 Methylmercuric compounds; dose-effect relationships
based on observations of intake versus effects
Data have been presented in the previous section on critical
and lethal brain concentrations of MeHg in squirrel monkeys
and adult humans. Comparable data are not yet available on
the fetus.
V
The Iraqi studies on a human population exposed to methyl-
mercuric compounds by ingestion of contaminated, bread
demonstrated a correlation between intake and severity of
clinical manifestations of poisoning. Those who had consumed
65 mg MeHg (range 35-100 mg because of the varying content
of MeHg in flour) had symptoms but no signs. 80% of those
with signs had eaten more than approximately 130 mg, (range
70-200 mg) (Kazantzis, 1975). Correlation between intake ;
of methylmercuric compounds and clinical evidence of toxicity
was also reported (Bakir et al. 1973f Clarkson and Marsh,.
1975). Although it is not possible to quantitate the degree
of effect with the available data one may establish a rank
order of these effects baaed on the dose required to produce
50% response of each effect.
In the Iraqi studies relationships between intake levels and
blood and hair concentrations were also recorded. The relation-
ship between the concentration of total mercury in blood and
the amount of mercury ingested was established for a group
of children aged 10 to 15 years and for a group of adults
by Bakir et al. (1973). Correlation between intake of total
mercury and hair concentration of total mercury was demon-
strated by Kazantzis (1975).
In the latter study, the mean maximal hair concentration of
total mercury for those who had eaten less than 50 loaves
(approximately 1.3 mg/loaff but could have varied between
0.5 mg and 2.0 mg per loaf or even more widely), 50 to 100
loaves, 100 to 200 loaves, and more than 200 loaves was
respectively 90, 88, 194, and 259 /ug/g.
204
-------
Thus dose-effect relationships for methylmercury compounds
have been indicated for dose estimated variously, e.g.
as ingested amount, blood concentration, hair concentration,
and brain concentration. Unfortunately, despite the wealth
of data available on dose-effect and dose-response relation-
ship for methylmercury compounds, the critical effect has
to be defined as broadly as paresthesia. No other effect
has yet been recognized at lower doses.
IV.5.4.4.1.3 Methylmercuric compounds; dose-response
relationships (describing exposure-response)
Comparatively little information is available on the relation-
ships between intake and response in humans. However, measure-
ments of concentrations of total mercury or MeHg in various
indicator (or index) media in the outbreaks in Japan and
Iraq and in populations having high dietary intake of fish
have allowed an estimation of the dose, using a metabolic
model of MeHg in man.
The minimum concentrations in the brain (critical organ)
associated with signs and symptoms have been reported in
Table 10. The section on "critical concentration in critical
organ" (section IV. 5.4.4.1.1) discusses the sources of evidence
for these minimum critical concentrations. The evidence on
dose-response relationships comes from the same source.
The concentration of mercury in indicator (or index) media
(blood, hair) and the maximum body burden may be calculated
from the metabolic model. For example, the most consistent
figure for the minimum brain concentration associated with
signs and symptoms, i.e. 1 ug/g, corresponds to a blood
level of 200 ng per gram and a hair value of 60 ug/g. The
calculated body burden for a 70 kg person would be about
20 mg corresponding to approximately the same amount of
totally ingested MeHg in an acute exposure.
205
-------
Detailed dose-response relationships in which dose was
estimated as body burden of MeHg (corresponding to
approximately the same acute exposure) are gupted in
Figure 17. The general principles involved in the relation-
ship have been discussed elsewhere (IV. 5.1). The data
indicate that considerable individual variability exists
in human sensitivity to methylmercury compounds. The
data in Figure 17 point to a factor of 10 or so as measured
by the frequency of paresthesias. Variability is less
with signs of poisoning such as .ataxia and dysarthria.
Nordberg and Strangert (1975) have noted that, if dose is
to be expressed as a daily intake, another important vari-
able must be taken into account, i.e. the clearance half-
time for man of which the wide individual variability has
been indicated by studies on hair by Al-Shahristani and
Shihab (1974). The range was from 40 to more than 120 days.
In two individuals having the same daily intake of MeHg ,
but having clearance half-times of 120 versus 40 days, ,
the first would accumulate a body burden three times higher
than the second upon long-term exposure to MeHg . By takingf
into account the distribution of interindividual variation
in critical concentration (as reported by Bakir et al.,
1973; Figure 17) and the variation in biological half-time,
Nordberg and Strangert calculated a relationship between
daily dose and probability of poisoning at long-term exposure
to methylmercuric compounds.
In the fetal cases of methylmercury poisoning reported by
Amin-Zaki et al. (1974) (and discussed further by Clarkson
and Marsh, 1975), it was found that of 8 infants born to
mothers with blood MeHg concentrations of 50 to 500 ng/ml
only 2 were affected, whereas 3 out of 3 infants were affected
when the maternal blood concentration was above 1,000 ng
Hg/ml. Body burdens (= total short term exposure) corresponding
to these blood concentrations are 5 to over 100 mg. A more
precise dose-response evaluation cannot be made on basis of
these limited observations.
206
-------
IV. 5.4.4. 2 Ethylmercuric compounds
As was mentioned earlier/ no adequate data are' available to
discuss dose-effect relationships in critical organs and
indicator (or index) media, or critical concentrations in
critical organs and indicator (or index) media.
IV.5.4.4.3 Other organomercury compounds
AS said previously the critical organ and the critical effect
for phenylmercury exposure are not easily definable, but
the kidney is the probable critical organ and the critical
effect most likely is changes in the renal tubules. A detailed
evaluation of phenylmercury involving critical organ concent-
ration and metabolic models is an even more difficult task.
There are no conclusive data which permit definite assertions
about mercury concentrations in the kidney at exposure to
phenylmercury compounds. In animal experiments, levels of
mercury in the kidney have been found close to those seen,
at poisoning due to mercuric salts, but differences in
absorption between the two compounds on oral exposure make
total mercury concentrations higher for phenylmercury than
for mercuric salts. Still, concentrations which are connected
with signs of toxicity seem to be the same. Fitzhugh et al.
(1950) found a mean kidney level of 2.3 ug/g in rats showing
slight morphological changes, and 39 ug/g in rats showing
severe changes. Tryphonas and Nielsen (1970) reported between
160 and 370 pg/g in the kidneys of pigs with severe renal
damage. A summary of available data has been given by Skerfving
(1972), in which ingestion of 100 mg of mercury as a phenyl-
mercuric compound was described as having caused only slight
gastrointestinal symptoms. In another case, laboratory in-
vestigation and renal biopsies showed normal conditions after
a person had ingested as much as 1,250 mg Hg. The oral toxicity
of phenylmercuric compounds is thus obviously rather low.
207
-------
There is evidence for absorption of mercury from phenylmercuric
compounds applied onto the surface of the skin and into the
vagina. Mercury concentrations in urine in cpnnection with
albuminuria have been 8.5 mg/1 in one case; levels up to 6 mg
Hg/1 in spot samples of urine have been published for workers
exposed to phenylmercuric salts and considered to be free of
symptoms and signs.
Specific data about mercury concentrations in the kidney at
exposure to methoxyethylmercury compounds are not available.
Lehotzy and Bordas (1968) found histological changes similar
to those found after intoxication with mercuric chloride in
the kidney of rats exposed to methoxyethylmercury chloride
intraperitoneally. These authors have also found some neuro-
logical effects after exposure to this type of mercurial,
and it may therefore be questioned as to whether the kidney
is really the critical organ. On the other hand, the possi-
bility cannot be excluded that a primary kidney damage can
also give rise to symptoms of a neurological character as1 a
secondary manifestation of uremia.
IV.5.4.5 Factors affecting dose-response relationships for ,
mercury and its compounds
Many factors (host selected, other chemicals) could influence
the dose-response relationship. This text will be restricted
to examples for which evidence in experimental animals or
in man points to a reasonable probability of influencing the
dose-response relationship. Four general factors will be
discussed: (a) the development of tolerance, (b) sensitivity
to mercury as related to the life cycle, (c) other sensitivities
and idiosyncratic reactions, (d) interaction with other chemi-
cals.
(a) Pretreatment with a small dose of cadmium or mercury
protected against the subsequent toxic dose (Yoshikawa, 1970).
Tolerance can also develop in the course of daily injection
of 0.5 mgAg HgCl2 for 14 days: at the end of this period,
208
-------
virtually complete regeneration occurred and regeneration
was advanced even in rats receiving 2.0 mg/kg HgCl2
(Prescott and Ansari, 1969). Increased tolerance to cadmium
may be due to metallothionein formation.
,*•
(b) Studies on rats reported by Greenwood et al. (1972)
indicate transplacental passage to the fetus in exposures
to elemental mercury vapor. However, no information is
available on the effects of prenatal exposure to the vapor
in humans.
Evidence of prenatal poisoning in the Minamata outbreak has
been reviewed (Berglund et al., 1971). Twenty-two infants
whose mothers had been exposed to methylmercury compounds
were born suffering from cerebral palsy, but exhibited only
slight effects of methylmercury poisoning. Early data on
infant-mother pairs in whom prenatal exposure had occurred
in the Iraqi outbreak have been quoted by Clarkson and
Marsh (1975). No higher frequency of poisonings in the infants
as compared to the mothers was detectable. However, only
twelve infant-mother pairs were studied and the observations.
were made soon after the epidemics. A greatly expanded follow-
up study is now in progress.
(c) Certain clinical manifestations such as hypersensitivity,
idiosyncrasy and acrodynia (pink disease) can be caused by
inorganic or organic forms of mercury (Clarkson, 1972a, 1972b).
(d) Interactions of other chemicals with mercury are under
discussion in other sections (IV.6.3) of this report. It may
be mentioned here that the concomitant exposure to ethanol
decreases the pulmonary absorption and toxicity of mercury
vapor. Animal experiments have shown that exposure to amino-
triazole can modify distribution of mercury (section IV.3.3.3.4.1).
Protection by selenium against the toxic effects of inorganic
mercury has been demonstrated (section IV.6.2). Selenium protection
i -
has been witnessed only for extremely high dietary levels of
209
-------
MeHg+ (20 to 40 ug/g in the diet, Ganther et al., 1972).
The relevance of the studies on animals to actual MeHg
levels in human diet (less than 1 ug/g) remains to be
established. No information is available so far on
selenium-MeHg interaction in man.
IV.5.4.6 Summary of dose- response relationships for mercury
and its compounds
This summary will comprise the major .conclusions as to
critical organ, critical concentration and critical effect,
and the corresponding concentrations of mercury in indicator
(or index) media. It should be borne in mind that considerable
individual variability exists in susceptibility to mercury
in all of its forms. In the case of methylmercury compounds,
the variability in the human population has been indicated
to be on the order of a factor of ten.
Considerable information is at our disposal on critical Con-
centrations of methylmercury compounds. Still, there is a
clear need to improve tests and diagnostic procedures in
order to detect effects prior to the onset of paresthesia,
which is the slightest effect known at present to be associ-
ated with exposure to methylmercuric compounds.
With respect to Hg°, information on dose-response relation-
ships is available for long-term exposure. Nothing is known
about brain levels of mercury in humans at the time of onset
of the signs and symptoms of poisoning. The critical organ
is difficult to assign to a particular sign or symptom.
The dose-response relationships reported in one industrial
study indicate complaint of such symptoms as loss of appetite,
insomnia, and shyness, and signs, e.g. tremor, as related
to certain air levels of mercury.
210
-------
The acute effects of inorganic mercury are well described.
The concentrations in blood, hair and urine at the time of
onset of kidney failure have not been reported. The con-
sequences of long-term exposure to inorganic rtercury in man
are not known. Animal experiments indicate the kidney as the
critical organ. Dose and concentration relationships cannot
be specified in part because of lack of data and in part
because of the possibility of the development of tolerance.
Information on the toxicology of organomercurials in man
other than methylmercury compounds is meager. Based on their
known rapid conversion to inorganic mercury in animals, it
is possible that the effects are similar to those of inorganic
mercury.
Factors affecting the dose-response relationship were discussed.
Alcohol, aminotriazole, and probably other inhibitors of
catalase markedly affect the metabolism of mercury vapor.
The possible protection of humans by selenium against the,
toxicity of methylmercury compounds is a question of considerable
importance with respect to risks for oceanic fish ingesting
these substances.
211
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IV.6 FACTORS INFLUENCING THE EFFECTS AND DOSE-RESPONSE RELATION-
SHIPS FOR TOXIC METALS; THE EFFECTS OF SELENIUM AND METAL-
METAL OR METAL-MINERAL INTERACTIONS
IV. 6.1 Introduction
Definition; Interaction is a process by which metals in their
various forms or other factors change the critical concentra-
tion or a critical effect of a metal under consideration. In
this section the term interactions will also be used to describe
the effect of certain elements on the absorption, excretion/
retention, and toxicity of cadmium, mercury and lead.
Even though it has been shown that certain organic nutrients
such as proteins (Fitzhugh and Meiller, 1941; Suzuki et al.,
1969) and ascorbic acid (Evans, 1973; Fox, 1974), interact
with cadmium, that dietary protein, vitamins D and C, and
ethanol (Goyer and Mahaffey, 1972) interact with lead, and
that vitamin C (Magos, 1973) and ethanol (Magos et al., 1973)
interact with mercury, these types of interaction will not be
discussed in this report. Furthermore, the following text will
mainly rely on data from animal experiments, since there are
very few data on metal interactions in human beings.
The following interactions will be considered:
(a) Interaction of selenium with mercury and cadmium
Because selenium is known to affect the toxicity of both these
and some other metals in a similar way, and since this topic
has been extensively investigated, the Subcommittee decided
to deal with it separately.
(b) Some interactions of cadmium, mercury and lead with other
metals or minerals
212
-------
The molecular mechanisms underlying these interactions are not
well understood. Various aspects of the hypotheses and theories
of proposed mechanisms for these interactions can be found in
this volume (Magos, 1975; Parizek, 1975; Sandstead, 1975).
•i
IV.6.2 Interaction, of selenium with cadmium and mercury compounds
Selenium has been shown to protect against several of the toxic
effects of cadmium. This was first demonstrated for cadmium-induced
testicular necrosis (Kar et al., :1960; Mason et al., 1964; Mason
and Young, 1967; for review see Gunn and Gould, 1970; Friberg et
al., 1971 or 1974). Since then selenium has been shown to pro-
tect against cadmium-induced hemorrhagic necrosis of nonovulating
ovaries (Parizek et al., 1968a), necrosis of placenta (Parizek
et al., 1968b), a toxemia-like syndrome in the third period of
pregnancy in rats (Parizek et al., 1968b), teratogenic effects
in hamsters (Holmberg and Ferm, 1969), lethal effects in non-
pregnant rats (Parizek et al., 1968b) and in micre (Gunn et al.,
1968) and elevation of blood pressure in rats (Perry and
Erlanger, 1974).
Selenium protects against the lethal effect of inorganic mer-
cury salts in rats; at the same time the rats are protected
from renal tubular and intestinal necrosis (Parizek and
Ostadalova, 1967; Parizek, 1971a). Selenite added to food
will enhance the growth of rats receiving mercuric chloride
by mouth (Potter and Matrone, 1974). Dietary selenium as sodium
selenate partially protects from chronic nephritis induced by
peroral mercuric chloride in rats, even though there is an in-
crease in the mercury concentration in the kidney (Groth et
al., 1973; Groth et al., 1975).
Toxic effects induced by methyImercurie salts are also pre-
vented by inorganic selenium. Selenite was shown to prevent
growth inhibition as well as the lethal and neurotoxic effects
of methylmercury compounds when administered simultaneously
in the diet of rats and Japanese quail (Ganther et al., 1972;
213
-------
Stillings et al., 1972; Potter and Matrone, 1974; Ueda et al.,
•
1974; Ohi et al., 1975). In chronic studies with dietary exposure
to MeHg and selenite, control rats exposed to MeHg. died with
symptoms of neurotoxicity and concentrations of MeHg in the
brain well below those present in rats protected by selenite
(Ueda et al., 1974; Ueda et al., 1975). Selenite given par-
enterally also decreased the toxicity of dietary MeHg while
at the same time temporarily increasing the MeHg concentration
in rat brain (Iwata et al., 1973>.
Similar protective effects of selenium have been described
for the toxic effects of thallium (Hollo and Zlatarov, 1960;
Rusiecki and Brzezinski, 1966), silver (Diplock et al., 1967)
and arsenic (Levander and Argrett, 1969).
At .the same time that selenium .protects the critical organs
from the toxic effects of these metals, it can also increase
their concentrations in critical organs. (Critical organs as
discussed here sometimes refer to sites of effects seen af-
ter injection of relatively large single doses of metal salts,
a situation not given much attention in previous chapters)..
The best demonstration of this course of events is the ability
of selenium to prevent the necrotizing effect of cadmium on
the testicles, and at the same time to produce a several-fold
increase in the concentration of cadmium in this organ (Gunn
and Gould, 1970). A recent study indicates that this might
be related to a concomitantly occurring change in the binding
of cadmium to certain proteins in the testes (Chen et al.,
1974). Under certain conditions the oral administration of
selenium will increase the concentration of inorganic mer-
cury in the kidneys without an apparent increase of the chron-
ic toxic effect (Groth et al., 1975).
Selenium exposures that decrease cadmium or inorganic mercury
toxicity have been shown to increase markedly the concentra-
tion of the respective metals in blood (Parizek et al., 1969a,
214
-------
1969b, 1974). A recent paper has shown that mercury and selenium
are bound to a plasma protein in an atomic ratio of l:l.(Burk
et al., 1974). A binding of this type may explain alterations
in the biological activity of metals and in the biological
availability of selenium when selenium and the metals interact
in the organism, as referred to by Parizek (1975) and in
a recent review (Parizek et al., 1974). This could also ex-
plain the altered distribution of the metals, as well as the
decreased passage of mercury and .selenium across the placenta
and into the milk when inorganic salts of these elements are
given simultaneously (Parizek et al. , 1969a, 1969c, 197'la,
1971b). It remains to be shown if selenium can affect the
transplacental passage of MeHg and its subsequent fetal ef-
fect.
The report that MeHg in tunafish and large oceanic fish is
less toxic than MeHg given under other circumstances, and
the hypothesis that selenium could be one of the factors .in-
volved (Ganther et al., 1972$ Ueda et al., 1974) deserve to
be considered as issues of moment. High concentrations of
mercury and selenium have been found in livers and brains
of apparently healthy sea mammals. The atomic ratio of mer-
cury to selenium in these cases was again approximately 1:1
(Koeman et al., 1972, 1973).
An interrelation for the better between mercury and selenium
has also been reported recently in human subjects exposed to
inorganic mercury (Kosta et al., 1974). Inorganic compounds
of mercury and arsenic, in contrast, increase the toxicity
of dimethylselenide or trimethyl selenium salts by several
orders of magnitude (Parizek et al., 1971a, 1974; Palmer and
Halverson, 1974). Further research is needed to show whether
or not MeHg is capable of producing this effect. These
examples illustrate how interactions of a toxic metal with
selenium can have adverse effects, as well as beneficial ef-
fects, on the dose-response relationship.
215
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IV. 6.3 Mercury
Interactions of mercury with other minerals have received
little attention, with the exception of its interactions
with selenium discussed above. Cadmium pretreatment has been
shown to influence the distribution of mercury, resulting in
higher kidney levels of mercury in rats (Magos et al., 1974x).
In the same groups, cadmium protected the kidneys from the reno-
toxic effects of mercury. Long-term exposure to HgCl_ may bring
about some tolerance (Prescott arid Ansari, 1969). The mechanism'
for these effects might involve the binding of mercury to
metallothionein.
IV.6.4 Cadmium
One of the first interactions discovered for cadmium was the
protective effect of zinc against testicular necrosis induced.
by injection of cadmium (Parizek, 1957). The total dose of
zinc needed to offer complete protection against cadmium •
amounted to about 100 times the molar equivalent of cadmium
(Parizek, 1956, 1957, 1960; Gunn et al., 1961). The protective
effect of zinc is believed to be partly dependent on the in-
duction of an increased synthesis of metallothionein-like
proteins (Webb, 1972b; Davies et al., 1973). There is some
uncertainty as to whether zinc actually induces synthesis of
metallothionein (Shaikh and Lucis, 1970; Friberg et al., 1974).
As pointed out previously (sections IV.4.2.1 and IV.5.2.4), both
cadmium and zinc bind to metallothionein and induction of the
synthesis of this protein therefore is likely to influence
the turnover and toxicity of these two metals. it may be of
interest to mention in this context that Cotzias et al.
(1961, 1962) reported that administration of zinc salts in-
creased the cadmium retention in the body.
Tne term "metallothionein-like proteins" is used to indicate
that the proteins had some characteristics in common with
metallothionein (e.g. molecular size) but that a complete
identification has not been reported.
216
-------
The mechanism involving metallothionein seems to explain
some but not all of the observations on zincrcadmium inter-
actions. The possibility that the protective effect of pretreat-
ment by zinc on testicular cadmium necrosis is related to the
induction of metallothionein corresponds well with the time
interval necessary between the administration of zinc and
cadmium for development of resistance to cadmium (Webb, 1972;
Chen et al., 1974). On the other hand this mechanism alone
does not explain why simultaneous administration of zinc with
cadmium completely protects against cadmium-induced testicular
necrosis (Mason and Young, 1967). These latter observations
seem to indicate that a different interaction could be oper-
ating, involving competition for ligands involved in the trans-
port of cadmium, or involving metal binding ligands in the
target tissue itself.
The teratogenic effect of cadmium is prevented by the simul-
taneous injection of zinc (Ferm and Carpenter, 1968).
The interaction between zinc and cadmium appears to be import-
ant for human health. The levels of cadmium and zinc in kidneys
from human beings without known industrial exposure have been
measured in autopsy material (see section IV. 5.2.4). with age,
the levels of cadmium and zinc increase in parallel and in
equimolar amounts in normal people (Piscator and Lind, 1972).
In hypertensive patients, an increase in the ratio of cadmium
to zinc has been observed (Schroeder, 1967; Lener and Bibr,
1971).
Experiments in the rat have shown that cadmium will induce
increased blood pressure under carefully controlled conditions
(Schroeder and Vinton, 1962; Perry and Erlanger, 1974). These
studies have also shown that the levels of cadmium required
for such an effect are relatively low, and that when high doses
are given, increased blood pressure does not occur (Perry and
Erlanger, 1974). In addition, it appears that the level of zinc
217
-------
in the diet is also quite specific (Perry, 1974; Doyle et al.,
1975). When rats were given 2.5 yg of Cd/ml. plus 12.5 yg of
zinc/ml in the drinking water along with 23 pg of zinc/g of
the diet, their blood pressures were not significantly greater
than those of the controls (Perry, 1974; Sandstead, 1975).
When the zinc intake was increased to 50 yg/ml with 2.5 yg
Cd/ml of water and the dietary zinc concentrations were un-
changed, the animals developed hypertension. At a higher
level, 100 yg/ml of water, zinc was .antagonistic to cadmium
and increased blood pressure did not occur. Thus, zinc was
antagonistic to cadmium at some levels and "synergistic"
with cadmium at others.
Analysis of renal cortex from rats has shown that the ratio
of cadmium to zinc in the tissue is better correlated to the
occurrence of hypertension than to the absolute level of
cadmium. Thus, animals with a molar ratio of cadmium to zinc
of less than 0.37 were not hypertensive and rats with a ratio
of 0.46 were always hypertensive (Schroeder et al., 1966).
Additional evidence for the idea that cadmium is an etio-
logical factor in hypertension is the finding that its removal
from the above hypertensive rats by a zinc chelate resulted
in a cure of the hypertension (Schroeder, 1967). While the
mechanism of the toxicity of cadmium on the kidney has not
been defined, one effect which may relate to the induction
of hypertension is the sodium retention which is caused by
cadmium (Lener and Musil, 1971; Doyle et al., 1975. Presum-
ably the sodium retention is related to an increased level
of renin secretion by the kidney (Perry and Erlanger, 1971).
In addition to these studies in which hypertension has been
produced, there are also negative reports in this respect.
Part of the explanation may lie in the genetic variability
in susceptibility (review: Friberg et al., 1974). The findings
of hypertension in the rat are not in agreement with observa-
tions on industrial workers exposed to cadmium. Perhaps the
disparity is related to the levels of cadmium exposure as
218
-------
well as to the levels of zinc and/or selenium in the diets
of the subjects.
Studies in man which may relate to the observations in the
rat are those which suggest that the cadmium content of
drinking water (soft water) may be a factor in the occur-
rence of hypertensive cardiovascular disease. This hypoth-
esis has not been adequately tested. At present it can only
be stated with confidence that the softness or acidity of
water correlates with the incidence of cardiovascular dis-
ease. Such water is more likely to have a relatively high
concentration of cadmium than hard water (Perry, 1973).
An interaction between cadmium and copper has been shown in
the intestinal mucosa (Evans and Hahn, 1974; Hahn and Evans,
1974) . Both cadmium and copper complex with a metallothionein-
like protein present in the intestinal epithelial cells. It
is not known whether this protein is identical with metallo-
thionein or with a slightly different copper binding pro-
tein with a molecular weight almost identical with metallo-
thionein and recently isolated from the liver (Winge
et al. , 1974). The intestinal protein has also been
found within the intestinal lumen (Evans et al., 1974). It
appears to facilitate the intestinal absorption of copper and
to have a strong influence on the intracellular metabolism of
copper (Evans, 1973). It is unknown whether the binding of
cadmium to this protein in some way impairs the metabolism
of copper in the intestine.
A nutritional interaction between calcium and cadmium has
been observed (Larsson and Piscator, 1971; Piscator and
Larsson, 1972). Low levels of dietary calcium increase the
absorption of cadmium by the rat. Conversely, it is probable
that cadmium can interfere with calcium absorption through
its toxic effect on the renal tubules and inhibition of the
formation of the vitamin D metabolite 1-25(OH)_ cholecalci-
;219
-------
ferol (Feldman and Cousins, 1973) Kimura et al., 1974). Calcium
may also influence the toxicity of cadmium through its presence
in hard water (see above) .
Altered sensitivity can be achieved by pretreating "the animal
with zinc, selenium and certain other metals. In addition,
pretreatment with small doses of cadmium will protect from
a subsequent injection of a larger dose of cadmium (Terhaar,
1965; I to and Sawauchi, 1966} Gabbiani et al., 1967b;
Yoshikawa, 1970, 1973; Nordberg, 1971, 1972a). While the
mechanism of this phenomenon has not been established, it
seems probable that the induction of metallothionein and
subsequent sequestration of cadmium are at least partly
responsible (Nordberg, 1971, 1972a).
IV.6.5 Lead '
The most studied interactions of lead with essential metals
are those with iron and calcium. Lead probably interferes
with iron metabolism at two levels. It blocks the introduc-
tion of iron into the tetrapyrole ring by inhibiting heme-
ferrochelatase activity (Chisolm, 1964). Bessis and 'Jensen
(1965) have shown that iron in the form of apoferritin and
ferruginous micelles accumulates in mitochondria of bone
marrow reticulocytes in lead-poisoned rats.
There may also be some relationship between iron and lead
absorption in the gastrointestinal tract. Iron deficient
rats show an increased absorption of dietary lead (Six
and Goyer, 1972). This finding may be particularly signifi-
cant in terms of childhood lead effects, since many young
children with elevated blood lead levels also have iron defi-
ciency.
The interaction between calcium and lead has been shown most
dramatically in the rat. Rats fed low calcium diets took up
sufficient ambient lead to show an increase in blood lead
220
-------
and urinary ALA excretion compared to animals given.adequate
dietary calcium (Mahaffey and Goyer, 1973). Also, the lead
content of soft tissues as well as biochemical and morpho-
logical manifestations of lead toxicity were more severe
in rats fed low-calcium diets than in rats fed normal dietary
calcium, both groups having received lead in drinking water.
Kostial et al. (1971) found that retention of lead by weanling
rats is decreased when calcium and phosphate are added to
milk.
There may also be a relationship between zinc and lead metab-
olism, although such studies are relatively incomplete. The
feeding of toxic amounts of zinc to horses prevents mani-
festations of lead toxicity (Willoughby et al., 1973). At
the cellular level, it has recently been shown that the
fluorescent porphyrin, or so-called FEP, elevated in pa-
tients with lead poisoning, may indeed not be free but instead
chelated with zinc. Zinc protoporphyrin is also increased in
erythrocytes of children with iron deficiency anemia (Lamola
and Yamane, 1974). The influences of these relationships upon
manifestations of lead poisoning or blood lead levels are yet
unknown.
IV. 6.6 Conclusions
Though research on interactions affecting the metabolism and
action of those metals discussed in the present report (i.e.
cadmium, mercury, lead) is still undergoing a rapid development,
certain conclusions can already be drawn which are pertinent
to the effects of these metals and their respective dose-
response relationships.
Of particular interest are those interactions which increase
the body burden of the toxic metal, its concentration in the
critical organ, and/or in media used for quantitation of
its presence, and which at the same time influence the toxic
effects. This has been demonstrated in some of the experiments
*
221
-------
noted above. Studies of this type have identified some of
the factors which should be included in the protocol of studies
on the relation between the dose and the effect of toxic
metals. This conclusion pertains both to epidemiological
studies and prospective experimental work.
j»
In addition, a better understanding of interactions may open
new possibilities for the prevention and/or therapy of ad-
verse effects. From the point of view of public health, it
is of particular importance to study the interaction between
two chemicals (or metals) when there is a high probability
that populations will be simultaneously exposed to both.
222
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IV.7 ACKNOWLEDGMENTS AND LISTS OF WORKING PAPERS AND PARTICIPANTS
ACKNOWLEDGMENTS
The Symposium was organized by the Subcommittee on the
Toxicology of Metals under the permanent Commission
and International Association on Occupational Health.j
It took place at the Japan Industrial Safety Association,
which also provided technical facilities for the meeting.
The main sponsors of the meeting were the Japan Industrial
Safety Association, the Japanese Ministry of Labor, and
the Japanese Environment Agency. Some support was also
received from the U.S. Environmental Protection Agency.
In addition, the Subcommittee receives continuing financial
support from the Research Committee of the National Swedish
Environment Protection Board and the Swedish Petroleum
Institute.
During the meeting in Tokyo the administrative work was
supervised by Dr. Y. Seki and Ms. Cecilia Hamagami. Ms.
Birgitta Morin and Ms. Elisabeth Mueller supervised the
secretarial work.
The Editor, Editorial Committee, and the Administrative
Editorial Assistant met at the Environmental Protection
Agency, Research Triangle Park, N.C., USA, for final decisions
on editorial matters. The editorial work was performed in
part while Dr. Nordberg was a visiting professor at the
Department of Pathology, University of North Carolina,
Chapel Hill, N.C., USA under a grant from US. Environmental
Protection Agency.
223
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Administrative and technical support during preparation and
editing of the manuscript, provided by the Department of
Environmental Hygiene of the Karolinska Institute and of
the Swedish National Environment Protection Board, is
gratefully acknowledged.
LIST OF WORKING PAPERS NOT INCLUDED IN THIS VOLUME
The following reports were presented and treated as working
papers at the meeting but have been or will be published else-
where. They will be listed here in full.
Goyer, R.A. and Rhyne, B. (1973). Pathological effects of lead.
In: "International Review of Experimental Pathology." (G.W.
Richter and M.A. Epstein, eds) Vol. 1 pp 1-77, Academic Press,
New York.
Nordberg, G.F. (1974). Health hazards of environmental cadmium
pollution. Ambio 3_, 55-66.
Tsuchiya, K. (197?). Perspectives in the environmental toxicology
of trace metals.
Ueda, K., Yamanaka, S., Rawai, M. and Nakamura, Y. (1975). Signific-
ance of hair mercury levels in exposure to alkylmercury compounds.
Yamaguchi, S., Matsumoto, H., Kaku, S., Shiramizu, M., Hirota>
Y. and Shimojo, N. (197?). Factors affecting the amount of mercury
in the human scalp hair. Amer. J. Pub. Health.
Yamaguchi, S., Matsumoto, H., Kaku, S., Shiramizu, M., Hirota,
Y. and Shimojo, N. (1975). Effect of tunafish diet on the con-
centration of total- and methylmercury in hair and other tissues
from cats.
224
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LIST OF PARTICIPANTS
Maths Berlin, M.D.
Department of Environmental Health
University of Lund
Box 2009
S-220 02 Lund 2
Sweden
J. Julian Chisolm, Jr., M.D.
Baltimore City Hospitals
4940 Eastern Ave.
Baltimore, Maryland 21224
USA
Thomas W. Clarkson, Ph.D.
Department of Radiation Biology
and Biophysics
School of Medicine and Dentistry
The University of Rochester
Rochester, N.Y. 14627
USA
Lars Friberg, M.D. (Chairman)
Department of Environmental Hygiene
The Karolinska Institute and
The National Environment
Protection Board
S-104 01 Stockholm 60
Sweden
Robert A. .Goyer, M.D.
Department of Pathology
University of Western Ontario
London
Ontario N6 A5 Cl
Canada
225
-------
David H. Groth, M.D.
National Institute of Occupa-
tional Safety and Health
1014 Broadway
Cincinnati, Ohio 45202
USA
Akira Harada, M.D.
Sanyo Electric Co., Ltd.
18-2 Keihan-hondori
Moriguchi City, Osaka
Japan
Sven Hernberg, M.D.
Institute of Occupational Health
Haartmaninkatu 1
SF00290 Helsinki 29
Finland
Robert J.M. Horton, M.D.
National Environmental Research Center
U.S. Environmental Protection Agency
Research Triangle Park. N.C. 27711
USA
Noburu Ishinishi, M.D.
Department of Hygiene
Faculty of Medicine
Kyushu University
1276 Katakasu, Higashi-ku
Fukuoka
Japan
Kiyoyuki Kawai, M.D.
Department of Experimental Toxicology
Ministry of Labour
National Institute of Industrial Health
2051 Kitsukisumiyoshi-cho
Nakahara-ku, Kawasaki
Japan
226
-------
Shoji Kitamura, M.D.
Department of Public Health
Kobe University School of Medicine
12 Kusunoki-cho, 7-chome
Ikuta-ku
Kobe
Japan
lord Kjellstrom, M.B., M.Eng.
Department of Environmental Hygiene
The Karolinska Institute
S-104 01 Stockholm 60
Sweden
Theodore Kneip, Ph.D.
Institute of Environmental Medicine
New York University Medical Center
550 First Avenue
New York, N.Y. 10016
USA
Juko Kubota, M.D. (Honorary chairman)
Japan Industrial Safety Association
Occupational Health Service Center
35-4., Shiba 5-chome
Minato-ku
Tokyo
Japan
Laszlo Magos, M.D.
Medical Research Council
Laboratories
MRC Toxicology Unit
Woodmansterne Road
Carshalton, Surrey
England
227
-------
David Marsh, M.D.
Department of Neurology
School of Medicine and Dentistry
and Strong Memorial Hospital
The University of Rochester
Rochester, N.Y. 14627
USA
Kazuo Nomiyama, M.D.
Department of Environmental Health
Jichi Medical College
3311-1 Yakushiji,
Minamikawachi-machi
Kawachi-gun, Tochigi-ken 329-04
Japan
Gunnar Nordberg, M.D. (Rapporteur)
Department of Environmental Hygiene
The Karolinska Institute
S-104 01 Stockholm
Sweden
Tor Norseth, M.D.
Institute of Occupational Health
Gydas Vei 8
Box 81 49
Oslo 1
Norway
JJ . Parizek, M.D.
Institute of Physiology
Czechoslovak Academy of Sciences
Budejovicka 1083
CS-142 20 Prague 4
CSSR
228
-------
Emil A. Pfitzer, Sc.D.
Experimental Pathology and
Toxicology
Hoffmann-La Roche Inc.
Nutley, New Jersey 07110
USA
Magnus Piscator, M.D.
Department of Environmental Hygiene
The Karolinska Institute
S-104 01 Stockholm
Sweden
A.V. Roschin, M.D.
Institute of Industrial Hygiene and
Occupational Diseases
The Academy of Medical Sciences
31 Meyerovskii Proezd
Moscow E-275
105275 USSR
Hiroyuki Sakabe, M.D.
National Institute of Industrial Health
The Ministry of Labour
2015 Kitsukisumiyoshi-cho
Nakahara-ku
Kawasaki
Japan
Harold H. Sandstead, M.D.
United States Department
of Agriculture
Agricultural Research Service
North Central Region
Human Nutrition Laboratory
2420 Second Avenue North
Grand Forks. North Dakota 58201
USA
229
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Ana Singerman, Ph.D.
Professor of Occupational Biochemistry
Junin 1032
Buenos Aires
Argentina
Tsuguyoshi Suzuki, M.D.
Department of Public Health
School of Medicine
Tohoku University
2-1 Seiryo-cho
Sendai
Japan
Masatomo Tati, M.D.
Department of Public Health
School of Medicine
Gifu University
40 Tusukasa-cho
Gifu
Japan
Rene Truhaut, M.D.
Chaire de Toxicologie et d'Hygiene
Industrielle
Faculte de Pharmacie
4, avenue de 1'Observatoire
Paris VI
France
Kenzaburo Tsuchiya, M.D. (Vice-Chairman)
Pepartment of Preventive Medicine
' and Public Health
School of Medicine
Keio University
35 Shinanomachi, Shinjuku-ku
Tokyo
Japan
230
-------
Kiichi Ueda, M.D.
Department of Hygiene
Tokyo Dental College
2-9-18 Misaki-cho
Chiyoda-ku, Tokyo 101
Japan
Jaroslav Vostal, M.D.
Biomedical Science Department
General Motors Research
Laboratories
Warren, Michigan 48090
USAV
Velimir B. Vouk, M.D.
Environmental Pollution Division
of Environmental Health
World Health Organization
1211 Geneva 27
Switzerland
Osamu Wada, M.D.
Department of Hygiene
Faculty of Medicine
Tokyo University
1-3-7 Hongo, Bunkyo-ku
Tokyo
Japan
Seiya Yamaguchi, M.D.
Department of Public Health
School of Medicine
Kurume University
67 Asahi-machi, Kurume-shi
Fukuoka 830
Japan
231
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The following were invited to participate and,, contributed
working papers which were discussed at the meeting and are
published in.this volume. Unfortunately, they could not attend
the meeting.
George Kazantzis, M.D.
Department of Community Medicine
The Middlesex Hospital
London W I N 8 AA
England
S.I. Shibko, M.D.
Division of Toxicology
Bureau of Foods
Food and Drug Administration
200 C Street S.W.
Washington, D.C. 20204
USA
232
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