BIOCHEMICAL EFFECTS
OF
ENVIRONMENTAL POLLUTANTS:
AN EPA SYMPOSIUM
ENVIRONMENTAL RESEARCH CENTER
(Adjacent to University of Cincinnati Campus)
MAY 31 - JUNE 3,1976
Sponsored By:
Health Effects Research Laboratory
Environmental Research Center
U.S. Environmental Protection Agency
Cincinnati. Ohio 45268
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PURPOSE OF THE SYMPOSIUM
This symposium is being organized to emphasize
the value of understanding the biochemical effects of
environmental pollutants. Detection of early biochemical
lesions that are related to subsequent changes in
structure and physiology associated with disease would
be useful for early signs of hazards to health in
human populations. More importantly In the long term,
this approach appears as the only rational basis for
making concrete decisions and predicting the hazards
of the mixtures o.f chemicals encountered in the real
world environment. Testing of all possible pollutant
combinations empirically is decidedly unachievable
because of the sheer volume and expense of such an
enterprise. Consequently, prediction based upon detailed
knowledge of the biochemical and pharmacodynamic
properties of individual chemicals appears as the only
viable alternative. The topics of the present symposium
deal with two areas in which a large volume of research
has been done. Perhaps a framework for prediction
Interactions may be discernible at this-time. If not,
It is hoped that this meeting will stimulate the
participants and others concerned with the effects of
environmental chemicals on health to focus future
research in the direction of developing the Information
required for predictive models.
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SYMPOSIUM COMMITTEE
Dr. R. John Garner General Chairman
Organizing Committee
S. D. Lee, Chairman
R. Bull
H. Petering
Program Committee
J. F. Stara, Chairman
V. F. Finelli
D. Tierney - •' ^ L ft
R. Bhatnager -
MONDAY. MAY 31. 1976
1:00 - 7:00 p.m.
Registration
(Stouffer Inn)
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TUESDAY, June 1, 1976
8:30 am Welcome and Introduci-ory Remarks
Dr. R. John Garner
8:45 am Keynote
Dr. Delbert Barth
SESSION I: BIOCHEMICAL EFFECTS OF GASEOUS POLLUTANTS
Co-chairman: Dr. Tierney
Co—chairman: Dr. Garner
9:00 — 9:30 am Biochemical Effects of Oxygen Toxicity,
Dr. D. Tierney
9:30 — 10:00 am Cell Renewal and Adaptation Tn Rat Lungs
Exposed to Ozone, Dr. Michael Evane —i’ A,- d2vvL R.r.
10:00 - 10:15 am COFFEE BREAK (CAFETERIA)
10:15 — 10:45 am Nucleic Acid Metabolism in Normal and Damaged
Lung, Dr. 11. Witachi
10:45 — 11:15 am Role of Superoxide in Oxidant Induced Pulmonary
Fibrosis, Dr. R. Bhatnager
11:15 — 11:45 am Open Discussion
11:45 — 1:00 pm LUNCH
1:00 — 1:30 pm Biochemical Effects of Environmental Oxidant
Pollutants in Animal Lungs, Dr. M. M4etafa LiCLl
1:30 — 2:00 pm Effects of Air Pollutant Oxidants on Biological
Membranes, Dr J. B. Mudd LiC. R.
2:00 - 2:30 pm Membrane Structure, Bulk Phase Lipid Properties
and the Effects of Small Molecules on Membrane
Functions, Dr. G. RouBer C .-4
2:30 - 2:45 pm COFFEE BREAK (CAFETERIA)
2:45 — 3:15 pm Ozone and Airway, Hyperirritability, Dr. Lu—Yuan Lee JCSF
3:15 — 3:45 pm Cellular Events in Inflammation, Dr. Yi Han Chang ‘cLt
3:45 — 4:15 pm Human Biochemical Effects of Gaseous Pollutants,
Dr. R. Buckley
4:15 — 4:45 pm Open Discussion
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WEDNESDAY, JUNE 2, 1976
SESSION I (continued )
9:00 — 9:30 am Effects of Sulfuric Acid Aerosol on Respiratory
System Morphology, Dr. W. Tyler .,c
9:30 — 10:00 am In Vivo and In Vitro Effects on Sulfur Dioxide
Upon Biochemical and Immunological Parameters,
Dr. E’. GauBe
10:00 — 10:15 am COFFEE BREAK (CAFETERIA)
10:15 — 10:45 am Sulfur Dioxide: A Review of Its Reactions with
Biomolecules, Dr. D. Petering
10:45 — 11:15 am Biological Origin and Metabolism of So ,
Dr. K. Rccjogopalan 2
11:15 — 11:45 am Open Discussion
11:45 - 1:30 pm LUNCH
SESSION II: BIOCHEMICAL EFFECTS OF HEAVY METALS
Co—chairman: Dr. Chvapil
Co—chairman: Dr. Petering
1:30 — 2:00 pm Cellular Mechanisms of Lung Fibrosis, Dr. M. Chvapil
2:00 - 2:30 pm Cellular Approaches to the Study of Environmental
Pollutants, Dr. R. Crystal
2:30 - 2:45 pm COFFEE BREAK (CAFETERIA)
2:45 3:15 pm Biochemical Interactions of Environmental Metal
Contaminants with Lung Connective Tissues,
Dr. M. Hussain j c. S F
3:15 — 3:45 pm Environmental Effects on the Biosythesis of Lung
Elastic Tissue, Dr. J. Foster
3:45 — 4:15 pm Open Discussion
6:30 pm SOCIAL HOUR
7:30 pm BANQUET
(University of Cincinnati Faculty Club)
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THURSDAY, JUNE 3, 1976
SESSION II (continued )
8:30 — 9:00 am Role of Nutrition in Heavy Metal Toxicity,
Dr. H. Petering
9:00 - 9:30 am ALA-D in Lead Exposure, Dr. V. Finelli
9:30 — 9:45 am COFFEE BREAK (CAFETERIA)
9:45 — 10:15 am Bioinorganic Chemical Reactions in the
Environment, Dr. J. Wood
10:15 0 10:45 am Effects of Heavy Metals on Isolated Mitochondria,
Dr. G. Brierley
10:45 — 11:15 am Metabolic Interactions of Selenium with Heavy
Meta I s, Dr. R. Rimerinan t - .,-,44 0 j 4 J..
11:15 — 11:45 am Open Discussion
11:45 - 1:00 pm LUNCH
1:00 — 1:30 pm Platelet Metabolism in the Study of Molybdenum and
Other Trace Substances, Dr. C. Solomone
1:30 — 2:00 pm Neurofransmit-t-er Mechanisms and Inorganic Lead
Poisoning, Dr. A Goldberg
2:00 — 2:15 pm COFFEE BREAK (CAFETERIA)
2:15 — 2:45 pm Evaluation of Animal Models Used to Study Lead
Effects on Neurochemistry and Behavior,
Dr. I. Michael8on
2:45 — 3:15 pm Effects of Trace Metals and Their Derivatives on
the Control of Brain Energy Metabolism, Dr. R. Bull
3:15 — 3:45 pm Open Discussion
3:45 pm Epi log, Dr. R. John Garner
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BIOCHEMICAL EFFECTS OF ENVIRONMENTAL POLLUTANTS
KEYNOTE ADDRESS
by D. S. Barth
I. INTRODUCTION
La order to put the subject of this Symposium into perspective, I
deem it necessary to spend some time discussing the objective of EPA,
how this objective is being attained, and how outputs from health effects
research contribute to the attainment of this objective. I will then
discuss the importance of biochemical effects research in relation to
health effects research in general.
II. EPA’S OBJECTIVES
The subjects dealt with by EPA include air pollution, water pollution,
pesticides, solid waste management, radiation, noise and toxic
substances. In brief, the objective of EPA is to abate or control environ-
mental pollution to socially acceptable levels. The role of research and
development then is to provide a body of research information sufficient
to enable an informed judgement to be made with regard to acceptable
levels for various environmental pollutants.
The laws which EPA must implement are many and varied.
However, the intent of Congress in each law is aimed at the protection
and enhancement of the environment. This implies that required
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controls are generally designed to abate adverse effects on health or
welfare to acceptable levels or to prevent the occurrence of new adverse
effects. In all instances health effects are deemed to be of primary
importance with welfare effects being secondary büt still of major concern.
III. EPA’S ORD PROGRAMS TO MEET OBJECTIVES
The research and development program of EPA is generally
concerned with the following subject areas:
• Effects
• Environmental exposure levels
• Predictive models linking source emissions to exposure
levels
• Control technology.
Effects research includes development of exposure- ffect relationships
for selected environmental pollutants, acting singly or in combination,
on selected populations of receptors for both health and welfare effects.
The documentation of environmental exposure levels is essentially a
monitoring task. Such exposure monitoring data are necessary to determine
where and to what extent environmental exposure levels exceed acceptable
values and to measure the efficacy of control programs as they are
implemented.
Validated predictive models linking source emissions to exposure
levels are required to design the most cost-effective control plans for
source emissions to reduce exposure levels to acceptable values.
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Control technology must be available to control major emission
sources adequately. In many cases this requires extensive research,
development and demonstration programs.
Let us now consider in somewhat more detail what outputs are
required from our health effects research programs and what research
methods and approaches are used to obtain those outputs. As I already
mentioned, we seek exposure-effect relationships for selected pollutants,
acting singly or in combination, on selected populations at risk.
Principal factors to be considered in the selection of pollutants for
study include:
• Our present state of knowledge for both regulated and
unregulated pollutants.
• Known or suspected seriousness of adverse
effects from over-exposure.
Availability of adequate measurement methods
Size of the populations at risk and estimates of exposure
levels
• Occupational health experience with polluta its under
con side ration
Principal factors to be considered in the selection of populations for
study include:
• Most sensitive populations at risk
• Higher exposure levels for most sensitive populations
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Likelihood of the presence of contributing factors not
related to environmental pollutant(s).
Approaches used to perform helath effects research in EPA include
epidemiologic, toxicologic and clinical studies. Whenever possible,
all three approaches are used in a coordinated fashion. Biochemical
studies may be included in any or all of the three approaches.
Our recently .rnplernented program to assess the contribution of
environmental carcinogens to cancer incidence in the general population
is an example of the meshing of these areas in a comprehensive study.
The initial thrust of this program will include media transport assessment,
inter - and intra-media tran sformation, measur ernent methodology,
exposure monitoring, dose assessment, and retrospective estimation
of exposure, all conducted under a rigorous quality assurance program
inareas of high and low cancer incidence. After this initial phase, the
coordinated data base will generate a requirement for targeted
epidemiological and toxicologic studies. Finally, the information will
provide values for the construction and validation of a predictive model.
IV. ROLE OF BIOCHEMICAL EFFECTS RESEARCH IN ORD PROGRAM
A distinction can be made, in biochemical effects research,
between “effects monitoring” and “health effects.” The former is a
requirement for the final stages of environmental exposure monitoring,
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i.e. exposure/dose assessment. The latter involves establishing
a meaningful relationship between a biochemical change and the health
or well being of the exposed population.
Toxicological lethality studies have long been used to evaluate
the hazards of various chemicals, however, such methods a e relatively
gross because of the comparatively large doses required to produce
bbservable effects within the short life spans of the experimental animals.
It is here that the study of biochemical effects may offer an advantage.
Such effects undoubtedly precede such end-points as the LD5O and, if
thoroughly understood, not only may explain the mechanism of the
hazard but also may indicate methods of control or reversal. These
considerations suggest that possible hazards may be identified and their
effects estimated long before the results qf chronic toxicity or
epidemiological studies are available. This would be particularly true
for carcinogenic chemicals which produce their end effect only after a
long latent period and for those, such as lead, which may accumulate
slowly to an end-effect level with continued exposure.
Some results along this line” are already appearing, for example,
•the relationship among blood-lead levels, ALA-D, and urinary homovanillic
acid concentration and the somewhat tenuous relationship between
mutagenesis and carcinogenesis which is th ’ .i...sis for the proposed use
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of the bacterial rnutagenesis test for detection of carcinogens. To
expand on these somewhat, if the indication of nerve damage suggested
by the biochemical detection of increased homovanillic acid excretion
can be confirmed, then an early indication of harmful effects may
be possible so that control can be established before permanent harm
ensues. Erythrocyte ALA-D, on the other hand serves as a convenient
“effect” for relating environmental exposure to dose assessment.
The subject of mutagenesis was addressed in the December 1975 Nobel
lecture by Dulbecco who advocated widespread use of bacterial
mutagenicity tests before releasing any new compound to the public.
The feasibility of such a program is strengthened by the finding that
most of the commonly available substances are not promutagens.
The foregoing are examples of 1 iochemical effects used in different
applications. The sensitivity, and even specificity, of such tests hold
great promise for the future. If developed to full potential, biochemical
changes related to the assessment of human health and welfare effects
would significantly aid the EPA in fulfilling its mandate to protect and
enhance the environment.
V. FUTURE CHALLENGES
Some current and future problems of major concern to EPA’s
health effects research programs include:
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• Development of suitable animal models for extrapolation to
humans
• Development of adequate screening tests, in vitro or in vivo , to
estimate toxic properties of environmental pollutants
• Development of methods for determining effects in humans or
experimental animals of long-term, low-level exposures
• Development of methods for determining varying effects of
different averaging times for different exposures
• Development of methods for measuring and interpreting
physiological or biochemical changes occurring as precursors
to disease
• Development of methods for biological monitoring to quantitate
exposure levels
• Development of personal exposure meters to improve our ability
to assess exposure to air pollutants
• Development of l iochemical exposure indicators to assess
exposure by any route
VI. EPILOGUE
This symposium was convened to address some of the problems I
have enumerated, and to assess current progress in the area of
biochemical change as related to effects of environmentalpollutarits. We
can expect much mutual education from the discussions scheduled during
the next three days.
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ABSTRACTS
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Ti erney
OXYGEN TOXICITY AND LUNG BIOCHEMISTRY
The phenomenon of adaptation or tolerance of lungs to increased
partial pressures of oxygen led us to consider that a metabolic
adaptation may have occurred. We therefore, compared some
aspects of lung metabolism in control and adapted animals.
initially we searched for changes in pathways which might be
mechanisms to protect against oxidants. We found, for instance,
changes in activity of glucose-6-phosphate dehydrogeriase and
Crapo and I observed increases of superoxide—dismutase. We
were unable to detect similar changes in a strain of mice which
did not adapt but did have inflammatory changes from the oxygen
exposure. Although these observations supported the concept
that these enzymes might play an important role in the adaptation,
the increased activity of these enzymes ruay be coincidental and
evidence from other investigators indicate that the change of
enzyme activity may be a nonspecific response to injury.
Two chemicals alter the sensitivity to oxygen toxicity.
Alpha-napthyi-thiourea (ANTLJ) when given intraperitoneally
to rats leads to relative tolerance for oxygen one week later.
ANTU also produces changes of glucose-6-phosphate dehydrogenase
similar to those from 85 02 for 7-days. ANTU is reported to
have a primary effect upon lung capillaries with little involve—
mont of epithelial cells. The pesti’.icle paraquat, when injected
into rats. increases the sensitivity to oxygen toxicity as
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demonstrated by Fisher and Clements. Paraquat also leads to a
markedly (4-6 fold) increased activity of the pentose pathway
as determined by production from glucose labelled in the
first or sixth position. Paraquat is thought to generate
superoxide and its effect may, therefore, be similar to oxygen
toxicity. The increased activity of the pentose oathway with
paraouat may be related to reduction of oxidants or peroxides
through glutathione. However, the toxic effects of paraquat
as determined by decreasing Q0 2 of tissue slices are not different
when glucose is present or absent.
Lipid synthesis in rat lungs is altered by oxygen toxicity
and during the recovery phase in air the content of saturated
phosphatidyichol me nearly doubles. This increased content may
relate to pulmonary surfactant and increased numbers of type II
cells.
Oxygen injury to the lung cells may have a similar mechanism
to other oxidant injuries includinq ozone and nitrogen dioxide.
However, the high P0 2 with oxygen breathing extends to all parts
of the lung and the distribution of injury may be very different
from inhalation of low concentrations of gases (ozone and nitrogen
dioxide) which may be lost from the inhaled gas as they impact
against the wall of the airway.
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Abstract
C 1 L RENEWAL MID ADAPTATION IN
RAT LUNGS POSED 10 OZONE
by
Michael J. Evans, Ph.D.
Stanford Research Institute
Menlo Park, California 94025
This research was undertaken to study the early effects of low
levels of ozone on cell renewal in thGr lungs of rats • ‘lb accomplish
this, male rats were exposed to ozone for up to 8 days. Dividing cells
were labeled with tritiated thymidine ( 3 ff-TdR) and studied with auto-
radiographic techniques in the light microscope. The results showed
that all labeled cells increased and then decreased to near control
levels within 4 days. Type 2 cells showed the largest change in
labeling index. Because the labeling indexes decreased by the fourth
day despite continuous exposure and because no further injury occurred,
it was assumed that the tissue had become tolerant to that concentra-
tion of 03. ‘lb test the degree of tolerance, groups of animals adapted
to O were exposed to higher concentrations of 03, and the labeling
indexes of Type 2 cells were studied. These studies showed that
tolerance to the initial concentration of 03 did not ensure total
protection to rats against reexposure to higher concentrations of 03.
lb determine which cells were being injured on’ reexpos are to 03,
another experiment was performed. Rats were exposed to 0 for 2 days,
labeled with 3 E-TdR, and allowed to recover for 4 days. Because
Type 2 cells transform into Type 1 cells Within 3 days under these
conditions, this treateent left labeled Type 2 and Type 1 cells in
the alveoli • These “adapted” rats were then reexposed to higher
concentrations of 03 and the number of labeled Type 2 and Type 1
cells were determined. Preliminary results show that labeled Type 1
cells were not rein.jured on reexposuro to 039 indicating that they
are adapted cells in the alveolar epithelium.
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NUCLEIC ACID NETABOLISM IN NORM&L AND DAMAGED LUNG
Ranapeter Witschi
D6partement de Pharmacologie
UnLversit de Nontr al
Nontr6al, Canada
Pulmonary RNA. synthesis can be evaluated in vivo by injecting ra—
dio].abelled uridine or orotic acid and determining the specific activity of
total pulmonary RN&. Zn rats, pulmonary RN& synthesis proceeds at about
the same rate as in liver, whereas in hamster lung it is less. The kinetic
constants êf enzymes involved in pyrimidine biosynthesis are comparable be-
tween lung and liver. In comparing ENA synthesis in normal and chemically
damaged lung, variability in precursor pool size has to be taken into account.
It may be possible to overcome this problem by measuring incorporation of
uridi.ne or orotic acid into RNL in vitro in lung slices or minced lung tissue;
incorporation rates are linear up to 40 mm. incubation time.
Pulmonary DNA synthesis may be measured in vivo by following the
incorporation of thymidine—’ 4 C into DNA. Zn normal lung, incorporation rates
are very low. However, in lungs injured by several chemicals an intense pro-
liferation of type II alveolar cells repairs the damage. This repair process
can easily be followed by measuring in vivo DNA synthesis. Thyinidine lr{nase,
an enzyme found in a high—speed supernatant prepared from lung homogenates
may serve as another biochemical indicator of cell proliferation in lung,
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—2—
whereas DNA polymerase is much less suitable.
Proliferation of type II alveolar cells may be produced by treating
mice with the antioxidant butylated hydroxytoluene (BHT; 250-500 mg/kg i.p.).
Dose—effect and time-effect relationships have been quantitated by measuring
in vivo DNA, synthesis or thymidine kinase. Exposure to 100Z oxygen inhibits
DNA synthesis in lung. Oxidants do therefore not only produce lung damage
per se, but will also compromise the following repair of the alveolar epithe-
hum.
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ROLE OF SUPEROXIDE IN OXIDANT INDUCED PULMJNARY FIBROSIS
Rajendra S. Bhatnagar
Laboratory of Connective Tissue Biochemistry
School of Dentistry 630 Sciences
University of California, San Francisco
San Francisco, California 94143
Oxidants, e.g. nitrogen oxides and 0 3 ,are a health hazard
of increasing significance. Their primary target is the lungs
which undergo functional and morphological deterioration, the
severity of which depends on the extent of exposure.. A major
outcome of oxidant injury is interstitial thickening in the
lungs involving incTeased connective tissue components.
Exposure to oxidants results in chemical injury which elicits-
a reparative response. Inflammation and repair culminate in
tissue alterations which hamper function and lead to prolifera-
tion of injury and repair resulting in progressively increasing
deposition of connective tissue. Exposure to oxidants results
in inflaniinat ion and marked increases in parameters of collagen
synthesis. Interaction of oxidants (02,03) with physiological
systems generates the superoxide free radical (0 ) and 02 is
also a major reactive species in inflanunation. Symptoms of
pulmonary injury are alleviated by the enzyme superoxide dis-
niutase (SOD) which is also induced in exposed tissues as a•
protective response and by several anti -oxidant a. Recent
studies in our laboratory have shown that 0 is a component
of the crucial hydioxylation steps ‘in collagen synthesis and
it also induces enhanced collagen polypeptide synthesis as
well as prolyl hydroxylase in cells in culture. Thus it
appears that the fibrogenic effect of oxidant gases in lungs
may be mediated through O radicals. Because 02 is consl2ned
during collagen synthesis, this may be a prctective mechanism
against O toxicity, although excess collagen deposition may
have deleterious effects on pulmonary function. Supported by
EPA Contract 68-03-2005 and NIH Grant De-03861.
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, ,T
BIO CAL E( ’1’S OF 4V L CZ R POW7 S Th1 AN]NAL U S.
M.G. Mustafa, A.D. Hacker, J.J. Ospital and S.D. Lee. University of C 1ffornia,
Abs, California 90024 and &wirorn ta]. Protection Agency, Cinclrnati,
to ozone or nitrogen d{ f de arising . f, ia photochand.cal g or fr in
frwlii jal or occupational envfrctmits may cause lung ceiliilm injury and mata-
bolic alterations. Although several laboratories have studied enzymatic or other
biodi f eel changes in the lung due to oz or nitrogen dL de exposure, the data
rated are rather sparse. The purpose of this study is to establish a general
pattern of matabolic changes In the lung that result £ u exposure to orkl nt
pollutants. Aninial osures. have been conducted with ozone at concentrations below
1 ppn (a nilx ting ethieit ctiiditions in phototh l?1cal ixg ) or to nitrogen dio cLde
at 5_ppn (the esho1d T.41n4t Value). The matabolic p ters st iel4ed fr t!livIp
wiy i consunptlixi, gl1v ose uPi 11 tion , pyruvate and lactate production, and
protein and lipid biosynthesis In lung slices, substrate uttl ti.on in lung ham-
gaiate, and marker enzyme activities in subcelliilm- fractious (viz., nd.tocIxx ia].
succinate oxie , s’ 4 nnte-cytochr c reductase; iwl i’i geii 1 NADH- and NADPH-
cytochz c reductases; and cytosolic jbicose-6-phosphate dehydrogenase. gliitii-
thione rethictase, disulfide reductase, glutathicne peraxidase and glutathione-
disulfide shydA gm1R e). Results shown below are £iun ozone exposures, but
g{tn(l ir trends obtMm d £ 1 f ted exposures conducted using nitrog i
d( r14 . As judged £LuIL enzymatic activLr( ( essed as units per lung) lung
metabol 4 ’ may exhibit at init4 *1 - depression (usually wirIth, 24 hours of exposure,
reflecting an injury phase), but a subsequent elevation (coincident with the
initiatian of a repair phase) which peaks between 3 and 4 days of exposure. For
exposures itwolvlng 0.8 ppn ozone, a 50-l007 elevation of metabolic par ters
has been observed. For lower levels of ozone (viz., 0.5 end 0.2 ppm) the changes
In metabolic p ci ter8 are relatively small, e th1bit1ng a fairly dose-dependent
response. Ordinarily, no substanr4i 1 changes in lung metabol{ mi are observed for
o te exposure at 0.1 ppm. However, in anhi 1 g “ fritained on a vit nfrt E-deficiant
diet,_significant biochead.cal changes in the lung may result £i exposure to 0.1
ppm ozone , and the changes may be considerably magnified for higher levels of ozone .
Prolonged qosures to ozone (viz., 0.8 ppn) show that the biochauLcal changes in
the lung, which reach the peak level between 3 and 4 days, r m 4 n elevated far as
long as 50 days of exposure, but than decline 1iyr gt” to control level after 90 days
of exposure. For lower levels of ozone (viz., 0.5 and 0.2 ppm) the changes are
found to revert to control levels within 20 days of exposure. If the exposure
is discontinued after a few days, the biochanical changes in recovering aIthi l s
return to control levels within a week. It may be concluded that low-level axidant
exposures biocheutcal changes in the lung, the magnitude of which is dependent
upon d.dant concentration as weU as the nutritional status of In l a with respect
to dietary suppl niRr1tatim of antin f ts. These changes in enzymatic activities
appear to be t siant, i.e., they rapidly disappear after t nation of_an exposure,
or slowly dfndnish chirix*g a prolonged qosure. (Supported In part by US PA contract
68-03-2221 and USPLIS HLl77l9; a part of the animal exposure was conducted at
the California Primate Research Center, Davis, C 1ifornia).
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S. B. MUDD AND B. A. FRE AN
DEPARThENT OF BIOCH fISTRT
UNivi SIT! OF AT.TO 14 V RIVERSIDE
RIVERSIDE, CALIFORNIA 92502
EFFECT OF- AIR POLLUTANT OTTflMITS ON BIOLOGICAL M IBRANE8
Ozone has been shown to affect the permeability properties of
plant cells, and its effect on alveolar macrophage may also be
interpreted as an effect on the cell”1 membrane. Other conse-
quences of ozone exposure show that there are responses distal
to the membrane, but it is not clear that these are direct responses
of ozone rather Fhni - responses to chemit 1 s produced by primary
reactions of ozone.
In model systems, it can be shown that ozone reacts with phos—
pholipid vesiclea- ends that the point of attack is at the double
bonds of unsaturated fatty acids. Model systems also indicate that
peroxyacetyl nitrate attacks at double bonds. As far as proteins
are concerned, ozone attacks cyst 4 i l , tryptophan, methionine,
histidine, tyrosine, pheny1 1an(ne and cystine. Peroxyacetyl nitrate
attacks only cyateine and methionine.
When ozone and peroxyacetyl nitrate in polluted air reach the
plasma m ivhrane of a lung alveolar cell or the meeophyli. cell of a
leaf, is the protein or the lipid component of the ‘ ‘ 1 ’raae affected
first? Can the oxidants penetrate the i nhrane unchmiged and affect
the cell contents directly? Our recent evidence shows that the ex-
ternal. proteins of the membrane are readily attacked by ozone, and
that there are distinct effects on proteins at the inner surface of
the i.iihrane before any effects on lipid can be detected.
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Membrane Structure, Bulk Phase Lipid Properties,
and the Effects of Sinai I Molecules on Membrane Functions
by George Rouser
In membranes, studies with spin labels. and other methods have shown that some
of the lipid binds directly to protein on one side and to other lipid molecules
on the’ other to form a lipid comparthent in which most of the lipid has bulk
phase properties closely similar to those of lipid extracted from the membrane.
Many of the membrane proteins have enzymatic activities, or transport functions
that are entirely dependent upon their being associated with phosphoilpid
having a particular type of polar group and acyl chains of the proper
length and degree of unsaturatlon. These specific requirements are dictated
by the specificity 0 f lipid-protein interaction, and the requirement that
lipids be in the fluid state at body temperature. This delicate balance of’
factors can be upset by the entrance’ of small molecules Into the lipid phase.
A striking example Is provided by the inert gas Xenon that is a potent general
anesthetic that acts by entering membranes and blocking conduction. Xenon
enters “free” space In membranes but does not interact chemically. Entrance
Into the “free” space appears to be relatively specific for molecules of the
proper size since other inert gases are much less effective. A comparison of
the types of molecules that are effective general anesthetics indicates the
existence of both polar and non—polar “free” or potentially free spaces In
membranes. Despite its. Importance, systematic exploration of the effects
on enzyme activites and transport processes of small molecules that do not
react with membranes to form covalent bonds. has not been undertaken.
The way in which small molecules that can react with doutile bonds of
lipid carbon chains may influence membrane functions can be understood, at
least In part, from the bulk phase lipid properties with change in temperature.
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Arrhenius plots of enzyme and transport functions show abrupt changes at
characteristic temperatures determined by the type of lipid.. Some of the
abrupt changes In rate are due to transition of fluid lipid to- a. more visious
fluid rather than the solid state. It Is thus possible that small, reactive
molecules that can link lipid molecules may cause a change in activity similar
to that produced by lowering temperature to force cooperative movement. The
type of change can be expected to depend upon the size and shape of the
perturbing molecule. A careful study of the “free” spaces in membranes
can be expected to disclose the types of monoreactive molecules. that can enter
membranes and reduce the harmful effects of the more toxic,reactlve molecules.
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AIRWAY HYPERIRRITABILI1Y INDUCED BY OZCNE
by Lu—Yuan Lee, Eugene Bleocker, and Jay A. Nadel
front the Cardiovascular Research Institut, and Department of Medicine
UMversily of California San. Francisco Medical Center
San Francisco, Caflfomia 94143
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A characteristic Feature of asthma is the extreme sensitivity of
airways to stimuli such as histamine asiosols. We propose that damage of
the airway epithel ium is a cause of airway hyperirritabil ity. Thus, we had
shown that respiratory viral infections (which damage airway epithelium) cause
transient airway hyperirritabilily in otherwise healthy subjects. Since ozone
damages airway epithelium, we are studying its effect on airway irritability
in clogs anesthetized with pentobarbital (2 mg/kg, iv), intubated, and
ventilated with a Harvard respirator. Transpulmanauy pressur. was measured with
an esophageal balloon, airflow with. a pneumotachograph; total pulmonary resistance
(R 1 ) was obtained by a method of electrical subtraction. Prior to ozone exposure
inhalation of histamine dphosphate aerosol (5 breaths; 2% solutIon) increased R 1
a mean of 335%. Sham exposure to filtered dry air did not change the bronchomotor
response to inhaled histamine (p, 0.5). The dogs then breathed spontaneously From a
free stream of - one mixture (0.7—1.0 ppm) fcr two hours. In 8 experiments an
four dogs, twenty—four hours after ozone, baseline R 1 was not significantly changed
(p>O.05), but the response to histamine aerosol was greater than prior to ozone
exposure (mean increase in R 1 following histamine; 576%; p <0.01). The increased
bronchomotor response was maximum on the first day after ozone and returned to
control levels in 7—28 days. Inhalation of isoproterenol aerosol (10 breaths; 0.5%
solution) abolished the usual increase in R 1 after histamine aerosol, suggesting
that contraction of airway smooth muscle was responsible for the increase in RL.
We conclude that inhalation of relatively low concentrations of ozone causes
transient airway hyperirritability in otherwise healthy dogs. These studies take
on added importance in lieu of the fact that ozone has reached ambient levels
as high as 0.54 ppm in Los Angeles and 0.4 ppm in other cities. Ozone may
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play a role in the- iniflatI of a thmu and in the deterioration in the
ci mica 1 stat, of asthmatic and branchitic patients exposed to iow levels
of ozone in the ambient- atmosphere.
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Yi-Ha;n Chang
CELLULAR ASPECTS. OF INFLAI’W TION
In the sequence of events that constitutes the tnflamatlon process,.
the cellular response appears to be central and other events such as
vasodfl$lon, permeability augmentation. complenent activation, mediator
release 1 ; etc... facilitate the imbflizatton and localization- of Teukocytes.
at the tnflamation site which involvesLa complex sequence of events
1nciuding adherence to endothil lum, d!apedesis into the extravascular
co parthtent,. directional movenent. in response to chenotactic gradients,
and phagocytos1s of Injurious material a t the site of InflaninatIon . The
current undérstandIng of inflatanation fs reviewed with eaphasts on the
cellular aspects. Experimental, approaches and methodologies that proved
useful ‘tfl the éTucldatlon. of ‘the mechantsm of Inflamatory response are
discussed with a view toward possible application to the study of inflam.
matton in the lung.
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ABSTRACT
HIJP tI BIOCHEMICAL EFFECTS OF GASEOUS POLLUTANTS
R. 0. Buckley,. J. 0. Hackney, C. Posin, K. Clark
Healthy adult human subjects were exposed to low levels of selected
air pollutants during a 2.5—3.0 hour regimen of exercise and pulmonary
function tests. They breathed either filtered clean air (sham) only,
or filtered air to which pollutants had been added. The temperature
was maintained at 86 0 F and the relative humidIty 43-47 percent. Several
levels of pollutants were employed, although generally one at a time.
The two most studied pollutants were ozone (03) and nitrogen diOxide
whose concentrations did not exceed 0.5 ppm and 2 ppm respectively.
Monitori ng of pollutant level s. was done continuously. Blood samples
were taken at the end of each sham and exposure period and analyzed
imediately. Paired group t-dependent statistical analyses were
performed.
Statistically significant differences (P <0.05) were detected in
selected biochemical parameters of human blood foil owl ng inhalation of
both 03 and NO,. Two si gnl fi cant changes In. the erythrocyte membrane
(membrane fragility [ RBC Frag.] and acetylchollnesterase [ AcChase]
activity) showed a dose—related response to 03. Other changes did not
show this relationship but behaved more like all -or-none responses.
The data suggests that two basic kinds of responses to Inhaled oxidant
are seen in- erythrocytes. The first is the initial Injury or irritation
and the second the respo ise of the cell ‘s biochemistry to that injury.
Indirect evidence suggests that lipid peroxidatlon might be one Of the
primary causes of 03 and P lO 2 toxicity.
Additional studies compared. the response of Los Angeles residents
with subjects living in cleaner atmospheres and found that nonresidents
showed responses that were similar in kind but greater in magnitude
than those of local residents. This lower reactivity of Los Angeles
residents suggests that adaptation to oxidant toxicity occurs upon pro-
longed breathing of the ambient local atmosphere, which contains
appreciable 03 and NO 2 , as well as other irritant substances.
From the Specialized Center of Research in Environmental Lung
Disease, National Heart and Lung Institute, Grant No. HL-15098-05.
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Short—term effects of sulfuric acid aerosols.
A merphological study in guinea pigs, mice, and rats.
V. S. Tyler, L. V. Schwartz, and D. L. Dungworth
California Primate Research Center
and School of Veterinary Medicine
University of C l-ffornia, Davis, California 95616
This. study was. designed to ev l”ate the merphological effects
of sulfuric acid aerosols on the respiratory tracts of anfinnie free of
spontaneous respiratory disease and to establish an order of species
susceptibility. Hartley a.lbin.o guinea pigs, Sprague Dawley rats, and
Swiss—Webster mice were continuously exposed for periods ranging from 4
to 14 days to aerosols of H 2 S0 4 . Exposure details are included in Table
Table I. Exposure Siim nnry
Aerosol Size Mass Conce!trat ion Exposure Length
_______ ( urn) ( us/rn ) ( days )
Rats 0.4 (NMAD 68 6
Rats 0.45 (CI1D) 2 172 7
Rats 0.52 11
Guinea Pigs 0.52 (CND) 71 4
Guinea Pigs 0.31*0.05 (NMAD) 1 , Ggl.6 30 . 7
Guinea Pigs 0.31*0.03 (IIMAD) 1 , Gg’l.5 38 7
Nice 0.62+0.04 (I4MAD) , Gg1.7 170 10
Mice 0.32+0.03 (NMAD) , Gg l.4 140 14
Mass Median Mrodynni (c DI tin t
Count Median D ’ ter
Anfmnlg were confined within winners—type st*Inless steel exposure
hainhers, and acid aerosols were generated from- vaporized SO 3 entering a
humidified airs tream.
Following exposure, n{ina1 s were deeply anesthetized with pento—
barbital sodium and euthiiwfzed by exsang”fn tion. The lungs were excised
as rapidly as possible, inspected for gross lesions, and perfused with
Karnovsky’ & fixative via the airway at 30 cm of water pressure. For optiin l
perfusion, guinea pig lungs were submerged and perfused tn fixative warmed
to 37°C. Nasal septa were fixed by submersion. Blocks of tissue were
selected for histiologic, scanning and transmission electron microscopic
v* f IAtion from nasal septa, trachea, and right middle and caudal lung
lobes.
Results indicate a striking variability in species susceptibility
to acid aerosol Induced respiratory system ‘l m ge. Guinea pigs were much
n ve sensitive than the meuse or rat. Microscopic areas of erosion and
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ulceration of bronchi and bronchioles, were frequently observed in large
airways. Coagulation necrosis of bronithint epitheliuia, sn oth muscle,
and cartilage was present. S{i r1 l r chmiges were observed in exposed mice,
but the di.stribution. differed in that lesions were confined to the larynx
and adjacent trachea. The focal necrotizing laryngitis was associated
with a prominent infiltration of ede fluid and neutrophila. The
respiratory tracts of exposed rats ware indistinguishable from those of
controls.
Results suggest that large conducting airways of guinea pigs and
the larynx of mice are sensitive regions of H 2 SO, induced damage. The rat
respiratory tract was remarkably resistant to ef!ects of H 2 S0 4 aerosols.
Supported by EPA contract number 68-02—1732.
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In Vivo wtd In Vitro Effects of Sulfur Dioxide
Upon Biochemical and Inmuinological Parameters
E.M Gause, ND. Greene,
M.L. Meltz, and J.R. Rowlands
Southwest Foundation for Research and Education
P.O. Box 28147 (8848 West Coninerce Street)
San Antonio, Texas 78284
A broad-spectrum,. multi-disciplinary study of the effects of sulfur
dioxide- upon biological systems has been undertaken. Studies to date have
encompassed various levels of biological organization. Results of these
studies and the implications for human. populations will be discussed.
The effects of inhaled sulfur dioxide upon. alveolar macrophage func-
tional capacity have been studied In the rat. These in vivo studies have
been paralleled by in vitro exposures of baboon alveolar macrophages to sul-
fur dioxide. Parameters studied include cell physiological function by
phagocytosi s and respiration; bi ochend cal studies of al veol ar macrophage
enzymes from various subcellular localizations (plasma membrane, cytoplasmic,
lysosomal, secretory and microsomal) and RNA,. DNA, and protein metabolism,
ininunological mechanisms (effects of in vitro SO 2 exposure upon macrophage
migration and inhibition of migration in response to MIF); nxwphology, and
dynamic physical measurements (spin labeling) of plasma membrane perturbation.
In addition, the effects of sulfur dioxide upon human lymphocyte mem-
branes have been exanrtned by the spin label technique and are consistent with
an instantaneous denaturati on of membrane protein resul ti ng in rapid clear-
ance from the membrane of the àl tered proteins.
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Interactions of sulfur dioxide with biological systen and with
atmospheric conponents have also been explored at the molecular level
by a combi nation of spectroscopic . Evidence for heretofore
unreported reactions of sulfur dioxide with: 1) protein nitrogen, and
2) TIght of visible region wavelengths will be presented.
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SULFUR DIOXIDE: A’ VIEW OF ITS REACTIONS WITH BIOMOLECULES
David if. Petering
Department of Chemistry
University of Wisconsin-Milwaukee
Milwaukee, Wisconsin 53201
Sulfur dioxide and related materials are present in the
atmosphere of industrial centers. Together with other effluents,
this complex of air pollutants can have adverse effects on health
as many epidemio].ogical studies indicate. In the attempt to
understand the role and importance of various pollutants in
producing such effects, exposure of test animals to single
agents under controlled conditions has been frequently used as
a model for study. The ambivalent results in the case of sulfur
dioxide point out the problems inherent in this approach. It is
argued that a knowledge of the potential biochemical effects of
sulfur dioxide would greatly aid an examination of its possible
influence on the health of organisms. Interestingly, the infor-
mation about its biochemical reactivity comes largely from’ source
which have not been concerned with its environmental impact.
Sulfur dioxide is a highly reactive molecule. It reacts
readily with water, aldehydes, ketones, disulfides, nicotinamide
adenine dinucleotide, flavin adenine dinuci.eotide, and mononucleo—
tides, vitaiii4i K 3 , thiainitie pyrophosphate, cytosine, and epine—
phrine. In order to provide a perspective on the relative
importance of these reactions, they are compared in terms of
their thermodyn wtcs and kinetics. Further depth is given to
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the comparison by considering the plausible biotransforaations
of sulfur dioxide, a principal one being its conversion into
cysteine—S-thio uIf ate. It is possible that thiosulfate is
generated from this species in the liver and that both S-thio-
sulfates and the thiosuif ate anion serve as the direct precursors
for the sulfite oxidase detoxification reaction which produces
sulfate. Some chemical and biochemical properties of S—thio—
sulfates and thiosulfates are provided. Finally, these findings
are organized in terms of the question of threshold of response
of an organism to sulfur dioxide. The thermodynamic and- kinetic
properties of the various reactions described above are used in
this. analysis.
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Biological Origin and Metabolism of SO 2
K. V. Raj’agopalan
Because of the chemical reactivity of’ SO 2 (or bisulfite) it is of
importance to determine whether intracellular HSO 3 could attain concen-
trations at which it could be considered a threat to physiological con-
stituents. Particularly, the finding that 1 HSO 3 can react with nucleic
acid components raises the spectre of’ possible genetic hazards from at-
mospheric 502.
In animals HSOf is generated endogenously from the amino acids
inethionine and cysteine. However, the presence of sulfite oxidase in
animal tissues ensures that the HS0 3 is rapidly oxidized to SOI and
excreted. In studies on the effect of respired SO 2 and injected HS0 3
on control and sulfite oxidase deficient rats it has been demonstrated that
tissue sulfite oxidase is indeed capable of affording protection against
the lethal effects of’ acute doses of these compounds.. The extent of pro-
tection afforded by the enzyme suggests that the maximum in vivo activity
of the enzyme approaches the value predicted from in vitro activity measure-
ment.
The sulfite oxidase activity of adult human liver has the capacity
for the oxidation of 14,000_8,000 mmoles of HSO to SO1 per day. In
comparison the average daily endogenais production of HSO 3 in man is
about 25 xmnoles. Dietary consumption of HS0 3 as food additive is only
about 0.2 inmole per day. In human lung there is sufficient sulfite oxi—
dase to convert 150 mmoles of HS0 3 to SOi per day. In contrast, maximum
H60 3 respired per day at 5 ppm 502 corresponds to only 1.3 nunoles. From
a consideration of’ these data it may be concluded that significant ac-
cumulation of’ HSOç in human tissues, at prevalent atmospheric concentrations
of SO 2 or at the present levels of dietary intake of HS0 3 , is extremely
unlikely. These compounds therefore may not be classified as environmental
genetic hazards. The possibility of damage to lung tissue from respired
SO 2 needs to be evaluated.
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CELLUIJR MECHANISMS OF LUNG FIBROSIS
Milos Chvapil, M.D., Fh.D.
The process of lung fibrosis could be beet controlled by affecting the
early cellular stages of Nacrophages seem to play an active
role in activation of fibroblasts - only activated macrophagea stimulated
collagen synthesis by fibroblasta. In the silica injured lung tissue, sub-
stances are formed at a certain time, which also specifically activate fibro—
blasts . This period coincides with macrophage accumulation in the lesion.
Among various trace elements, zinc administered in vitro to incubation
medium or in vivo to intact animals exerts an inhibitory effect on various
functions of cells, like mast cell, granulocytes, macrophages and platelets.
A working hypothesis will, be presented implicating trace elements in homeo-
stasis of inflRimui*tory cells. The importance of this mechanism to lung
patholo will be discussed.
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CELLULAR APPROACHES To THE STUDY OF ENVIRONMENTAL POLLUTANTS. N.A. Elson
and R.G. Crystal, NIH, Bethesda, Maryland. 20014.
Exposure to environmental pollutants produce lung ’ pathology with complex
features Including destructive lesions resmubi ing emphysema, bronchiolar lesions,
vascUlar lesions and proliferative lesions, of the- lnterstitium and. epithellum.
It Is apparent that multiple cell types are involved and the changes seen
undoubtedly represent a montage of the primary toxic effects of the noxious agent,
secondary destructive effects of the Inflamatory process,, plus repair processes
and fibrosis.. The sorting out of these different processes Is made more difficult
by repeated exposure to- the- agent, resulting in superimposition of multiple itages
of the process. However, to help evaluate the effects of environmental pollutants
on the lung, several experimental approaches are now available to examine these
complexities at the biochemical level.
One such approach Is the analysis of the cellular and protein content of
lung lavage. This technique ‘Is valuable because- studies in the experimental model
can readily be applied, to human material through the use of fiberoptic segmental
lavage. Preliminary human studies have shown.. that in idiopathic pulmonary fibrosis
there Is an increase In alveolar polymorphonuclear ‘leukocytes, while In chronic
hypersensi ti vi ty pneimionttl s there is a striking appearance of lymphocytes, pre-
dominantly T-cel is. Other studies have suggested that patients with pulmonary
fibrosis have circulating T-lymphocytes which are sensitized to collagen. This
raises the possibility of screening alveolar lymphocytes.- obtained for sensitization-
to. specific extrinsic or intrinsic antigens which may be’ part of the disease
process. Other studies indicate that alveolar amcrophages and lavage fluid super-
natant can be screened for specific enzyme activities (collagenase, neutral protease,
elastase, B-g’lucuronidase, lysozyme) and- for inmiunologic components (Inmiunoglobul Ins,
complement).
A second experimental approach is the short term lung explant. In this
method, animal or human lung. fragments are incubated in the presence’ of a
specific radioactive precursor in. order’ to quantitate- rates and types of
macromolecules being synthesized and degraded. Data to’ date indicates that
Important changes occur In’ rates of collagen- and- glycosaminoglycan synthesis
with age- in the developing animal, and that these rates can be al tered I ri response
to external stimuli e.g.. post-pneumonectomy lung growth-or radiation induced
pulmonary fibrosis. These methods. could easily be, expanded to evaluate the
effect of environmental pollutants.
A third approach is that of separating the explant model Into Its component
cell- constituents. Our laboratory has utilized’ several Isolated cell types
- found In the lung,, including the lung fibroblast, alveolar type II cell, alveolar
macrophage, polymorphonuclear leukocyte and monocyte. Additional work -Is In
progress on the Isolation of a vascular endothel ial and smooth muscle cell.
These cells are-maintained -In short or long term culture,,: during which time
they can be exposed to- various environmental manipulations. The results of these
manipulations are monitored by observing quanti tati ye and qualitative alterations
In the pattern of Intracellular and secreted cellular proteins; this pattern
serves as a “biochemical fingerprint” to detect the effects. of Injury at the
cellular level.
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BIOCHFIIICAL MECHANISMS OF INTERACTION OF ENVIROM4ENtAL
MEtAL CONtAMINANTS WITh LUNG CONNECTIVE TISSUE
M. Zamirul Hussain and Rajendra S. Bhatnagar
Laboratory of Connective Tissue Biochemistry.
School of Dentistry 630 Sciences
University of California, San Francisco
San Francisco, California 94143
Heavy- metals are becoming increasingly significant as
a potent hazard in the environment. The ions of these metals
are involved in cmi ing a wide variety of biological effects.
As for example, Cd 2 decreases lucose metaolisin, and affects
respiration of pulmonary inacrophages, uncouples oxidative -
phosphorylation and inhibits mitochondrial oxygen uptake.
Cd 2 ’, Hgt and Pd 2 all interact with a large number of
enzymes through -SH groups and destabilizes lysosomal membranes
resulting in the release of lysosomal hydroxylases and impairing
ion transport through membranes. These effects directly con-
tribute to the biochemical injury in the lung which then evoke
a repair response. Chronic exposure to the metal and con-
tinuous repair processes culminate in “fibrosis”. We have
examined this possibility in one of the steps in collagen
synthesis. Hydra].azine, shown by us to be a potent inhibitor
of prolyl hydroxylase, a critical enzyme in collagen synthesis,
elicits a fibrotic response in lungs, in uivo. We examined
the effect of this drug on collagen synthesis in organ cultures
of lung. Hydralazine stimulated the incorporation of labeled
proline into the collagen sequence. The possibility that
heavy metals may also act in the sante manner was examined in
terms of their interactions with purified prolyl hydroxylase
in vitro. All these metals prove to be potent inhibitors of
prolyl hydroxylase. The mecnanisms involved a competition
between the metal and Fe 2 , the prosthetic metal required for
activating the enzyme. Our studies suggest that interference
with prolyl hydroxylase ray be an improtant part of biochemical
injury leading to proliferat .on of conncctive tissues in lungs.
Supported by EPA Contract #68-03-2005 and NIH Grant DE-03861.
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Environmental Effects on the Biosynthesis of Lung Elastic Tissue
Judith Ann Foster and Carl Franzblau, Boston University School of
Medicine, Department of Biochemistry, Boston, Mass. 02118
The involvement of elastic tissue in the pathogenesis of various
pulmonary diseases has recently gained the attention of many
investigatiors. Lung elastic tissue has not been extensively
studied or characterized since much of the work thus far has
centered on the elastic tissue of aorta and ligament. For the
last year our laboratory has been investigating the chemistry
of the major protein component of elastic tissue, namely elastin.
We have been particularly interested in comparing the chemistry
and biosynthesis of lung elastin to elastins isolated from different
tissues. Our initial experiments have centered on young chicks
as a source of elastin since we have already accumulated much
data on elastin synthesis, structure and degradation from these
anim is. To date we have been able to isolate soluble precursor
forms of elastin from lung, aorta and gizzard tissues. Amino
acid composition and molecular weights of the precursor molecules
from each tissue are very similar if not identical. Antibody
generated to aortic tropoelastin crossreacts and exhibits identity
to the lung and gizzard samples. We have also been examining
the effect of various toxic chemicals, such as lathrogens and
metal chelators on the synthesis of lung elastin. Such studies
should shed some light on the disease mechanism occuring in lung
elastic tissue aberrations.
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ABSTRACT FOR E • P • A. SYMPOSIUM
“Biochemical Effects of Environmental Pollutants:
E.P.A. Symposium”
ROLE OF NUTRITION IN HEAVY !‘ TAL TOXICITY
H. C. PETERING, L. MURTHY, AND F. L.. CERXLEWSXI
The nutritional status of experimental animals must be’ considered
of just as great importance in evaluating the toxicity of any agent, as
is the health of the animals with respect to the presence of infection.
Thus it has been shown that composition of the diet may modify the
responses of n finals to carcinogens and many heavy metals • A review
of the literature shows that. the dietary concentrations of protein,
calcium , phosphorus., vitamins, and some essential transition metals
will, alter the toxic’ responses of several species of experi “ “ tal
n in ls to the oral ingestion of cadmium or lead. The toxicity’ of
these metals when they are aiiiniiiistered by inhalation has not been
extensively studied.
In this presentation the roles of dietary concentrations of zinc,
copper, and iron in affecting the toxicity of cadmium or lead will be
emphasized. It will be shown that the toxicity of lead or cadmium is
increased wh ’n the intake of one or more of these essential metals is
reduced. It is known that females and children are more susceptible
to lead toxicity than are males, and it is also known that these sane
portions of our population have at times a greater need for dietary
zinc, copper, and iron; therefore, the possibility exists that better
nutrition of the population as a whole with respect to essential
transition metals may reduce the health hazards of cadmium and lead
which are now dissipated in our environment and which may continue
to be increased.
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AL -D N LEAD EXPOSURE
By
V. N. Finelli, University of Cincinnati and
S. D. Lee, U.S. Environmental Protection Agency
Lead at very low concentratiors is known to inhibit s—Amino-
levulinate dehydratase (ALA—D) which catalyses the formation of
porphobilinogen, a precursor of h.eme. At least one more enzyme
involved in the pathway of heine synthesis is inhibited by lead,
that is ferrochelatase which catalyses the insertion of iron in the
protoporphyrin IX to form heme. Due to the lead inhibition of
these enzymes, the elevation of heme precursors in blood arid urine
can be detected in lead exposed men and animals.
Among the many indices utilized for the assessment of lead
absorption such as blood lead levels, urinary lead, urinary ALA,
zinc protophorphyrIns (ZPP) etc., erythrocyte ALA—D activity is the
method of choice for low levels of exposure since its inhibition by
lead is highly specific and sensitive.
Numerous investigators have reported that a slight increase in
lead absorption is associated with a significant depression of
ALA—D activity. However, some other factors may affect the activity
of this enzyme: Acute alcoholism, tyrosinemia and zinc deficiency
could significantly decrease MA—D.
This is a sulfhydryl containing enzyme and recently we have
found it to be zinc dependent. In rats under controlled nutritional
intake of zinc ALA—D is positively correlated with dietary zinc
levels. Furthermore, in vitro addition of ZnC1, to heinolysates of
blood from lead exposed animal brings ALA-D acttvity back to control
values.
A study on one occupationally lead—exposed worker confirmed
the above findings. Two important questions must be raised in
consideration of these findings: a) should chelation therapy, which
drastically increases urinary zinc excretion, be accompanied with
administration of zinc and b) should the nutritional state of the
exposed population, at least in regard to zinc, be considered in
order to determine the biological importance of exposure levels.
Current studies in our laboratories are aimed at answering these
questions.
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BIOINORGANIC CHEMICAL REACTIONS
IN THE ENVIRONMENT
J. M. Wood, Director
Freshwater Biological Institute
University of Minnesota
Navarre, Minnesota 55392
The chemical behavior of any element in the environment depends on its
form. For natural elements. biogeochemical cycles have evolved which lead to
steady- state concentrations of Individual species In metabolism. For the heavy
metals and metal bids, coordination complexes and organometallic complexes are
of prime’ Importance’, especially the latter because metal-alkyls have a tendency
to bioaccumulate as a result of their solubility In lipids.
During, the’ last ten years my research group. has delineated metabolic
pathways leading to. the biosynthesis of methyl-mercury, methyl—arsenic,
methyl—sebeniuna-and methyl-tin compounds. Recently, we have turned our attention’
to enzyme systems which dealkylate metal alkyls. From our study it is clear
that the kinetics for the biosynthesis of metal-alkyls In the environment, and
the kinetics for the uptake of metal-alkyls in specific ecosystems represent
fundamental problems which must be faced. Monitoring steady state concentrations
of elements which enter natural biological cycles yields information which is of
limited value, since the kinetics of uptake by living systems will always determine
environmental impact.
In this lecture I shall deal with the kinetics and mechanisms for the bio-
synthesis of metal-alkyls. In addition I shall discuss recent work on enzyme
systems which dealkybate metal-alkyls, followed by reduction of inorganic salts
of heavy metals to their lowest oxidation states. Preliminary work on the
possible alkylation of lead In well—defined biochemical systems will be discussed.
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Effects of Heavy Metal s on Isolated Ml tochondria
Gerald P. Brlerley
Dept. of Physiological Chemistry, College of Medicine
Ohio State- University, Columbus, Ohio 43210
Under appropriate experimental conditions, a number of heavy metals,
Including Pb. Cu, Hg. Cd, Ag and Zn, have been shown to Inhibit mitochondrial
enzyme and transporter activities and to alter the permeability of the- inner
membrane to cations and anions. In several cases, there Is reason to Infer
that such reactions are directly related to the toxicity of the metal j
vivo . The observed effect in vitro of a given concentration of a metal Ion-,
such as Pb. will reflect an Interplay between the following factors: (a) The
composition of the suspending medium - In the presence of P1, for example,
Pb 2 Is effectively removed from. solution and has little effect on mitochon-
dna whereas, Zn 2 becomes more reactive when adsorbed on the membrane as
a phosphate complex. (b) The extend of reaction of the metal with membrane
binding sites - There are over 100 potentIal binding sites per mg of protein
for a divalent cation In Isolated heart mitochondria. Reaction Of 5-10 øf
these sites with Cu’ 2 , for Instance, produces an increased passive permeability
to -P and markedly stimulates the energy-dependent uptake of this cation.
As more Cu+ 2 Is reacted with the membrane, permeability to anions and loss
of energy-coupling are noted. the energized and non-energized membrane
often show a different reactivity to a given amount of- heavy metal, suggesting
that different ligands are available to the metal under these two conditions.
(c) The ability of the metal ion to penetrate Into the matrix compartment —
Zn ’ 2 and Pb’ 2 have been shown to be accumulated by an energy-dependent process
and an extensively reacted membrane will probably permit passive inward
diffusion of the metal ions. (d) The intrinsic susceptibility of primary
dehydrogenases and other matrix enzymes to Inhibition by the metal ion in
question.
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ABSTRACT
Metabolic Interactions of Selenium with Heavy Metals
H.. A. Rimerman, D. H.. Buhier and P. 1). WhAnger
DeparP’ nt of Agricultural Ch miI try and Environmental Health
Sciences Center, Oregon State University
Corv l1ts, Oregon
Mortality and other toxic symptoms of cadmium, mercury, silver and thal-
lium in lanlfiwl a- are imach reduced by ib i4 n atration of selenium. Even though
sel vi4um protects against heavy metal toxicity, higher concentrations of
these metals acc”qilate in tissues of selenium treated i 1 1 than Lu um—
treated ones. Studies on the nzei .hRv.4sin(s) for the protective role of
selenium Lu heavy metal toxicity have shown that selenium alters tissue
and subcellular distributions, of *dmf um- and mercury and changes the bind-
ing of these metals. to constituents of the cell cytosol. The physiological
significance of some of these results. is questionable, however, since in
many cases the mti j were injected with selenium and the heavy metal.
For mnple, redistribution of cadmium or mercury from low to high mole—
c iii ar weight proteins in the cytosol occurs when selenium and the metals
are injected but not when these elements are given orally.
The me hen{am for the protective effect of selenium apparently differs,
depending on the chemical form of the metal. Treatment of Aaihn*ls with in-
organic mercury or cadmium and selenium causes both tissue and subcellular
redistribution of the metals whereas administration of inethylmercury and
selenium alters only the tissue distribution of methylmercury. Selenium
shifts much of a dose of methylmercury into muscle but causes inorganic
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mercury to concentrate primarily in liver and kidney. The relative effective-.
ness of selenium compounds in n difying the distribution of inorganic mer-
cury in tissues. is in the decreasing order: selenomethioneine> selenocy—
atine > selenate> selenite.
Cadmiumt,, silver and mercury also reduce the tonicity and the availability
of selenium as well as hatig1ng the tissue distribution of this el nt. In
other work, silver and mercury markedly decreased the activity of glutathione
peroxidase, a selenoezizyine, whereas cadmium had no effect.
It thus appears that selenium does not counteract heavy metal toxicity
by a single, co n mechanism. Furthermore, the metal and its ch mical
form, the route and tine sequence of administration and the relative dos—
I
ages of selenium and metal all influence the observed response.
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Platelet and RBC Metabolism
In The Study of Mo1ybd nu’. and Other Trace Substances.
C1.ive C. Solosons, Ph.D.
Blood cells from rats and hiiIR I were used as models for measuring
the. effects of molybdenum. Changes in platelet purine metabolism of
industrial workers and students correlated well with their plasma uric
acid concentrations (ruiO. 77). Less significant correlations of uric
aci& and xanthIn oxidase activities of red blood cells were also seen
(r0.44). Thimai blood cells were found to differ significantly from
those of rats- in the No—dependent uric acid pathway and the stress of
obt 4n(v g blood from the rats could also alter the measur m nt of platelet
function and metabolism in an uncontrolled fashion.
It was concluded ( L) that enzymatically—controlled reactions of
bu i platelets appeared to be n 1!411gfully correlated with Mo metabolism
and uric acid levels. This syaten appears to be capable of further development
for detecting exposures which could result in blood Mo levels in the 5—50 ppb
range. (ii) that the rat is not a suitable model for evaluating Mo effects
on hi,mna , populations, although the opportunity to study rats was a key factor
in optimizing the “ lytical methods. (iii) that further work should proceed
relating Mo effects in the 0-100 ppb range on huiwi platelets in order to
define the basic mechawfsm of Mo in AlP metabolism and develop a simple
field test for detecting exposure to Mo. The effects due to Mo deficiency
and 14o excess could be evaluated in hnm*ii populations living in different
geographical areas or receiving different occupational exposures to Mo.
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Neurotranazuitter Mechanisms in Inorganic Lead Poisoning
Alan M. Goldberg
The Johns Hopkins University
Department of Environmental Medicine
School of Hygiene and Public Health
615 North Wolfe Street
Baltimore, Maryland 21.205
Mice, rats and monkeys chronically exposed to. lead during critical
periods of development present with increased levels of spontaneous motor
activity, (Silbergeld and Goldberg, 1973; Sauerhoff and Michaelson, 1973;
Allen, McWey and Suomi, 1974). In the mouse, lead induced hyperactivity
is suppressed by drugs that enhance central cholinergic function (e.g.
physostigmine) and is exacerbated by anticholinergic drugs (e.g. benz —
tropine) and miriergic agonists (e.g. 1-dopa) (Silbergeid and Goldberq,
1975). This experimental hyperactivity, however, presents a paradoxical
pharmacology in that it is suppressed by methylphenidate and amphetamine,
dr igs classically thought of as antinergic agonists (Silbergeld and Gold-
berg, 1974).
Animals chronically exposed to lead appear to have enhanced aminergic
function. The turnover (Goiter and Michae].son, 1975) and steady-state
levels of norepinephrine. (Goiter and Michaeison, 1975; Silbergeld and
Goldberg, 1974) are increased. In addition, the excretion of vanillyl—
mandelic acid and homovanillic 1 metaboliteg of norepinephrine and
dopantine, are increased in lead exposed mice and children (Silbergeld
and Chisoim, 1976). Lead has also been shown to impair cholinargic
function by blocking acetyicholine release in the superior cervical
ganglion (Kostial and Vouk, 1957), nerve-muscle preparations (Mannelis
and Cooper, 1974; Silbergeld, Fales and Goldberg, 1974) and in brain
preparations (Carroll, Silbergeld and Goldberg, 1976).
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This paper will. snmlnarize the data dealing with neurotransmitter
function in lead induced hyperactivity. The emphasis will be on cho-
linergic aspects and the interaction between cholinergic and. aminergic
function.. Supported in part by grants from the National Institute of
Health S0O034 and 00454.
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EVALUATION OF ANIMAL MODELS USED TO STUDY
EFFECTS ON NEUROCHEMISTRY AND BEHAVIOR
I. A. Michaelson, R. L. Bornschein
D. A. Fox and R. Loch
Laboratories of Behaviorial and Neurochemical Toxicology
Division of Toxicology
Department of Environmental Health
University of Cincinnati College of Medicine
Cincinnati, Ohio 45267 U.S.A.
ABSTRACT
Inorganic lead produces cerebral dysfunction and
clinically definable encephalopathy. It is not known,
whether chronic low level lead exposure has an adverse
effect on brain maturation of children. Studies attempting
to define behavioral and/or neurochemical changes following
lead exposure have been primarily conducted in the neonatal
rat or mouse.
Newborns are frequently exposed to lead via milk from
dams consuming lead in food or drinking water. Sucklings
are often weaned to and maintained on the dam’s lead source.
Young adult rats and mice exposed in this manner have been
reported to be hyperactive, exhibit disruptions in normal
brain neurotransmitter metabolism and display “paradoxical”
responses to stimulants and depressants. These lead exposed
animals show growth retardation.
Control subjects are matched for age but seldom are
they matched for growth rate. Undernutrition, wherein
growth retardation is comparable to that seen in lead
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exposed mice can lead to enhanced spontaneous locomotor
activity, apparent t paradoxicalI response to d—axnphetainine
and enhanced activity following phenobarbital. These data
suggest that some of the behavioral and pharmacological
responses seen in lead exposed rodents can be explained
on the basis of early undernutrition.
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EFFECTS OF TRACE METALS AND ThEIR DERIVATIVES ON ThE CONTROL OF
BRAIN ENERGY METABOLISM -
R. J. Bull,’ Water Quality Division, Health Effects Research Lab.
U. S. Environmental Protection Agency, Cincinnati, Ohio 45268
A means has been devised whereby changes induced In the redox
state of the mitochondrial electron transport intermediates can be
measured within slices of brain tissue (Bull and Lutkenhoff, J.
Neurochem. 21,913—22 1973) Spectral changes induced by elevated
media potassium concentrations (3 to 30 mM) parallel closely changes
induced in more conventional measures of metabolism such as oxygen
consumption, aerobic glycolytic rate, adenine nucleotides and
phosphocreatine. This provides a technique which can be used to
detect subtle changes in the relationship of function to metabolism
in small samples- of brain tissue taken from animals exposed to
environmental ch ni cals.
PbCI 9 , administered I .P to Charles River CD strain rats over
a two week period to; brain levels of lead approximating 0.41 ug/
g fresh weight or higher, results In alterations in the metabolic
responses of cerebral cortical slices to potassium. The effect Is
observed as an inhibition of NAD(P)H oxidation which Is accompanied
by an inhibition of potassium -induced respiration. This inhibition
is paralleled by a decrease in the rate of potassium—Induced calcium
turnayer in- the tissues that Is closely associated in time with the
respiratory burst produced by elevated potassium. Previous work
has Implicated a calcium component in the respiratory response’ to
potassium (Bull and Cumins, J. Neurochem. 21,923.37 1973),
Nethyl mercury administered daily for a- two week period in
doses greater than 0.05 mg/Kg per day gave rise to cortical Hg con
centratlons of 0.1 ug/g fresh weight. At these levels, changes In
the spectral responses of cortical slices were seen to be altered
In a manner similar to that observed with lead. The accumulation
of NAD(P)H produced by low doses of methyl mercury gradually de
creased towards control levels with higher doses. This latter ef-
fect was associated with a decrease In the tissues’ capabilities
for aerobic glycolysis.
Dialkyltin compounds, used as stabilizers In PVC ptpe, have
been compared, in vitro , with triethylti n, a known C. N S toxin,
Triethyltin hal max1mally inhibits NAD(P) reduction induced by po
tasslum at a concentration of 1,1 uM (0.12 mg Sn/liter), This ef
fect Is opposite to what Is observed with lead and methyl mercury
either in vivo or in vitro . Dimethyltin and dibutyltin were observ. .
ed to produce the same effect as triethyltin at concentrations of
66 and 45 uM, respectively (7.9 and 5.3 mg Sn/liter).
The present results suggest that the trace metals studied
interfere with the control of energy metabolism wtthln brain ttssue.
These effects may be direct or Indirect, but It is clear that coupe-
l ng of funct o to metaboflsm is affected at doses well below
those which give riseto overt toxIcity ,
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