PB82-108184
Mechanisms of Cadmium Absorption in Rats
E. C. Foulkes, et al
University of Cincinnati
Cincinnati, Ohio
U.S. Environmental Protection Agency
Publication EPA-600/1-81-063, 1981
September 1981
U.S. DEPARTMENT OF COMMERCE
National Technical Information Service
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EPA-600/1-81-063
PS82-103184
MECHANISMS OF CADMIUM ABSORPTION
IN RATS
by
E. C. Foulkes, in collaboration with
D.R. Johnson, N. Sugawara,
R.F. Bonewitz and C. Voner
Institute of Environmental Health
University of Cincinnati
Cincinnati, Ohio 45267
Grant R805840
Project Officer
N. E. Kowal
Epidemiology Division
Health Effects Research Laboratory
Cincinnati, Ohio 45268
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45229
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/1-81-063
ORD Report
3. RECIPIENTS ACCESSION NO.
PB32 103133*
4. TITLE AND SUBTITLE
5. REPORT DATE
September 1981
Mechanisms of Cadmium Absorption in Rats
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
8. PERFORMING ORGANIZATION REPORT NO.
E.G. Foulkes, D.R. Johnson, N. Sugawara
R.F. Bonewitz and C. Voner
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Institute of Environmental Health
University of Cincinnati
Cincinnati, Ohio 45267
10. PROGRAM ELEMENT NO.
CAYB1B
11. CONTRACT/GRANT NO.
R805840
12. SPONSORING AGENCY NAME AND ADDRESS
Same as box 9-
13. TYPE OF REPORT AND PERIOD COVERED
14. SPONSORING AGENCY CODE
15. SUPPLEMENTARY NOTES
16. ABSTRACT This study was undertaken in order to help clarify the factors which determine
the fractional absorption of an oral load of cadmium (Cd) from the intestine of the rat.
The experiments utilized intact segments of intestine, perfused or incubated in situ with
their blood supply intact. Absorption of Cd from the jejunal lumen can be ascribed to a
saturable membrane system; that is, after short periods of exposure essentially all the
metal removed from the lumen was recovered in mucosal tissue (Step I). The second step
in Cd absorption, i.e., transfer of the metal from mucosa into blood, proceeded at only
1-2% of the rate of uptake from the lumen (Step I). No evidence was obtained for a role
of metallothionein in the mucosal retention of Cd. Step I of Cd absorption was inhibited
by a variety of exogenous and endogenous factors. Thus, zinc was found to depress Cd
transport in an apparently competitive manner. Addition of milk to the lumen also
inhibited Cd uptake, an effect entirely due to the Ca content. Bile salts act as
endogenous modulators of Cd absorption; their effect may be related to micelle formation.
The research also included studies of duodenal and ileal Cd transport. Heal Cd
absorption differed from that in the jejunum by a relatively much faster Step II. Unlike
the low ratio of Steps II/I for the toxic metal in the jejunum, the ratio for the essential
metals Cu and Zn was much higher (about 50%) . Absorption of Cd by the gut in neonatal rats
proceeded much faster than in adults; reasons for this difference have not yet been
clarified. Another question remaining under study is the extent to which different
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS
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18. DISTRIBUTION STATEMENT
Release to the Public
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Unclassified
21. NO. OF PAGES
20. SECURITY CLASS (This page)
Unclassified
22. PRICE
EPA Form 2220-1 (Rev. 4-77)
PREVIOUS EDITION IS OBSOLETE /
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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. Environ-
mental Protection Agency, nor does mention of trade names or com-
mercial products constitute endorsement or recommendation for
use.
i i
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FOREWORD
The U.S. Environmental Protection Agency was created because
of increasing public and government concern about the dangers of
pollution to the health and welfare of the American people. Nox-
ious air, foul water, and spoiled land are tragic testimony to
the deterioration of our natural environment. The complexity of
that environment and the interplay between its components require
a concentrated and integrated attack on the problem.
Research and development is that necessary first step in
problem solution and it involves defining the problem, measuring
its impact, and searching for solutions. The primary mission of
the Health Effects Research Laboratory in Cincinnati (HERL) is to
provide a sound health effects data base in support of the regu-
latory activities of the EPA. To this end, HERL conducts a re-
search program to identify, characterize, and quantitate harmful
effects of pollutants that may result from exposure to chemical,
physical, or biological agents found in the environment. In ad-
dition to valuable health information generated by these activi-
ties, new research techniques and methods are being developed
that contribute to a better understanding of human biochemical
and physiological functions, and how these functions are altered
by low-level insults.
Cadmium is a highly toxic heavy metal, to which man is be-
coming increasingly exposed. This report discusses the mechan-
isms of intestinal absorption of cadmium, and the factors which
reduce the net fractional absorption to only a few percent of the
oral load, in the hope that the information can be used to re-
duce cadmium's toxic effect.
J. B. Lucas
Acting Director
Health Effects Research Laboratory
111
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ABSTRACT
This study was undertaken in order to help clarify the fac-
tors which determine the fractional absorption of an oral load of
cadmium from the intestine of the rat. The experiments utilized
intact segments of intestine, perfused or incubated in situ with
their blood supply intact. Absorption of Cd from the jejunal lu-
men can be ascribed to a saturable membrane system; after short
periods of exposure essentially all the metal removed from the
lumen is recovered in mucosal tissue. The second step in Cd ab-
sorption, i.e. transfer of the metal from mucosa into blood, pro-
ceeds at only 1-2% of the rate of uptake from the lumen (Step I).
No evidence could be obtained for a role of metallothionein in
the mucosal retention of Cd. Step I of Cd absorption is inhib-
ited by a variety of exogenous and endogenous factors. Thus, Zn
was found to depress Cd transport in an apparently competitive
manner. Addition of milk to the lumen also inhibits Cd uptake,
an effect entirely due to its Ca content. Bile salts act as en-
dogenous modulators of Cd absorption; their effect may be related
to micelle formation. The work also included studies of duodenal
and ileal Cd transport. Heal Cd absorption differs from that
in jejunum by a relatively much faster Step II. Unlike the low
ratio of Steps I/II for the toxic metal in the jejunum, that for
the essential metals Cu and Zn is much higher (^50%). Absorp-
tion of Cd by the gut in neonatal rats proceeds much faster than
in adults; reasons for this difference have not yet been clari-
fied. Another question remaining under study is the extent to
which different metals such as Cd and Zn share common absorptive
mechanisms.
This report was submitted in fulfillment of Grant R805840,
awarded by the U.S. Environmental Protection Agency to Dr. E.G.
Foulkes in the Department of Environmental Health at the Univer-
sity of Cincinnati. The work was carried out during the period
of June, 1978 to April, 1981.
IV
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CONTENTS
Foreword iii
Abstract iv
Figures vii
Tables vii
1. Introduction 1
2. Conclusions 2
3. Results and Discussion 4
a) Activity gradients along jejunum 4
b) Kinetic studies of Cd transport
out of the lumen 4
c) Interaction between Cd and Zn 5
d) Action of bile salts 8
e) Steps I and II of metal absorption 8
f) Role of metallothionein 12
g) Specificity of metal absorption systems ... 12
h) Cd absorption in duodenum and ileum 12
i) Cd absorption in the newborn 14
v
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Appendices
Paper A.
Paper B.
Foulkes, E.G. Some determinants of in-
testinal cadmium transport in the rat.
J. Environ. Path, and Toxicol. 3:471-
481, 1980
17
Kello, D. , Sugawara, N. ,. Voner, C. and
Foulkes, E.G. On the role of metallo-
thionein in cadmium absorption by rat
jejunum in situ. Toxicology, 14:199-
208, 1979
Paper C.
Sugawara, N. and Foulkes, E.G. Relation-
ship between Cd and Zn absorption by rat
jejunum. Submitted for publication. . .
Abstract A.
Abstract B.
Abstract C.
Abstract D.
Abstract E.
Foulkes, E.G. and Voner, C. Steps in
Cd transport by rat jejunum in situ.
Fed. Proc. 39:1183, 1980
28
38
46
Bonewitz, R.F. and Foulkes, E.G. Modi-
fication of jejunal Zn and Cd transport
in rats by glucocorticoid not correlated
with metallothionein synthesis. Soc.
Toxicol. Meeting, San Diego, 1981 . . . ,
Voner, C. and Foulkes, E.G. Inhibition
of jejunal Cd absorption in the rat by
bile salts. Fed. Proc. 40:1071, 1981.
Johnson, D.R., Foulkes, E.G. and Leon,
L. Intestinal transport of Cd in new-
born rats. Fed. Proc. 40:1073, 1981 .
Bonewitz, R.F., Voner, C. and Foulkes,
E.G. Comparison of mucosal uptake and
transmural transport of Zn, Cu, and Cd
in rat jejunum. Fed. Proc. 40:1395,
1981
47
48
49
50
VI
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FIGURES
Number Page
1 Effect of Zn on Cd uptake 6
2 Effect of Cd on Zn uptake 7
3 Reversible inhibition of Cd transport by bile .... 9
4 Dose-effect relationship between glycocholate
and Cd absorption 10
5 Model of Cd absorption 11
6 Comparison of Cd, Zn and Cu absorption 13
TABLES
I Ratio of Steps II/I as function of Step I 12
II Cd transport in ileum 14
III Cadmium transport in duodenum of newborn rat . . . „ 15
IV Body weight and tissue iron levels 16
V Iron supplementation and Cd transport in jejunum. . „ 16
VII
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SECTION 1
INTRODUCTION
The human environment contains a variety of heavy metals,
originating from both natural and anthropogenic sources. Some
of these metals are clearly essential for life, while for some
others a biological function has been suggested. However, many
of these elements, in the form of organic or inorganic compounds,
are highly toxic. The level of such compounds in the environment
may therefore bear directly on human health. Analysis of their
effects assumes additional urgency because of the likelihood that
ambient concentrations of these metals may be increasing.
A special problem in this regard is posed by cadmium. Sig-
nificant amounts of this element are being added to the environ-
ment through use of sewage sludges and other fertilizers on agri-
cultural land, from combustion of fossil fuels, and by other pro-
cesses. The problem is exacerbated by the fact that the half
life of Cd in the body, and especially the kidneys, is very long;
in effect, Cd acts as cumulative poison. Its prime target organ
is the kidney, and significant nephropathy has frequently been
reported in exposed human populations.
Main sources of the human body burden of Cd outside of occu-
pational environments are Cd in food, water and tobacco smoke.
The non-smoker derives most of his Cd through gastrointestinal
absorption. In spite of this fact, relatively little is known
about mechanisms of intestinal Cd absorption, and about the fac-
tors which on the whole reduce net fractional absorption to only
a few percent of the oral load. This consideration defined the
objective of research work described in this report. A scien-
tific interest in the basic mechanism of metal transport, and
the possibility of applying knowledge gained to the control of
metal absorption, formed the starting points for the present
inve stigation.
Throughout the work attempts were made to follow absorption
under as physiological conditions as possible, using isolated
segments of intestine in situ in the living animal. Results ob-
tained are described under appropriate subheadings, but no at-
tempt is made to repeat in detail material already published.
Copies of papers and abstracts based on these results are ap-
pended to this report. These papers should be consulted for
details.
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SECTION 2
CONCLUSIONS
The results reported here extend earlier work by other in-
vestigators. However, unlike several earlier investigators, our
findings were made with intact intestinal segments in situ at Cd
concentrations one might conceivably encounter in heavily pol-
luted areas. Previous work in many cases had used excessively
high Cd concentrations; in addition, the preferred techniques of-
ten were the analysis of absorption in the intact animal, or the
measurement of transport by everted sacs of intestine in vitro.
In both cases, the avid retention of Cd in the intestinal wall is
a source of difficulty.
Thus, in sacs, cadmium is not likely to diffuse across the
submucosal tissues into serosal fluid as readily as, for in-
stance, sugars or amino acids. Purely a priori, therefore, the
release of Cd from mucosa in sacs may differ quantitatively from
that occurring under physiological conditions. In the intact
animal the additional difficulty arises that enterohepatic re-
circulation makes it impossible to obtain absolute values for
the unidirectional movement of Cd from lumen into mucosa and
blood. We submit therefore the method employed in the present
experiments as a more appropriate procedure for the detailed
analysis of Cd absorption under reasonably physiological con-
ditions.
If we may accept then the results obtained as approximately
reflecting the normal process of Cd absorption the following
main conclusions may be drawn:
1) Cd is removed from the lumen of the rat jejunum by a mem-
brane-related process which exhibits saturation kinetics (Step
I). After short periods, essentially all Cd thus removed can be
recovered from the mucosa. An activity gradient exists along
the jejunum.
2) Step I of Cd transport is modulated by bile salts as well
as by a variety of food constituents.
3) Zinc interacts in an apparently competitive manner with
Cd for transport by Step I. Unlike Cd, however, Zn is not ap-
preciably retained in the mucosa; in spite of the competition
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for Step I, the transmural movement of Zn and Cd is not mediated
by identical mechanisms.
4) Step II in the absorption of Cd, i.e., its movement from
mucosa into blood, proceeds at only 1-2% of the rate of Step I.
Step II is in series with Step I, and under present conditions
determines the rate of Cd absorption into the body.
5) Metallothionein, the low molecular weight protein able
to bind 7 moles Cd/mole, could not be shown to play any role in
the absorption of Cd.
6) Cd absorption in duodenum resembles qualitatively that
described for the jejunum; in contrast, Step II of Cd transport
is relatively much faster in ileum.
In summary, this investigation has contributed to a better
definition of factors which may be responsible for the control
of Cd absorption in vivo. In addition, it has confirmed the
possibility of altering fractional absorption of an oral load of
Cd by dietary manipulations. Work along these lines is con-
tinuing.
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SECTION 3
RESULTS AND DISCUSSION
a) Activity gradients along jejunum; Preliminary studies,
in which dilute solutions of -LoycdCl.2 in saline were placed into,
or perfused through segments of intestine, had confirmed that
isotope is readily removed from the lumen of various segments of
the small intestine. Because of its relatively short length, and
in order to avoid complications arising from bile secretion (see
below), duodenum proved less convenient than jejunum for further
detailed studies. Some experiments were carried out with ileal
segments but their activity in general was somewhat lower than
that of jejunum. Most of the studies reported here were there-
fore based on jejunum. The proximal portion only was used be-
cause of the strong activity gradient along this tissue, with
maximum Cd uptake occurring in the first 12 cm distal to the lig-
ament of Treitz (see Figure 1, paper A). Note that in these
studies the activity gradient revealed by measurement of Cd dis-
appearance from the lumen agreed closely with the accumulation
of the metal in the intestinal wall. This fact will be further
considered in section e).
b) Kinetic studies of Cd transport Out of the lumen: Be-
cause of the tissue heterogeneity, and in order to permit collec-
tion of accurately timed samples, a perfusion technique was de-
vised in which a small volume (1.6 ml) of solution is recircu-
lated through the jejunum at 0.4-0.8 ml/min. Details of this
technique are described in paper A. When measured in this man-
ner the disappearance of Cd from the perfusate follows first
order kinetics (see Figure 2, paper A). Two further points stand
out: 1) The rate of exponential disappearance of Cd follows sat-
uration kinetics; in spite of wide variability between animals
some approximate values for maximal velocity and affinity con-
stant could be obtained (see Fig. 4, paper A). It is important
to emphasize that the reaction studied is only that of accumula-
tion of Cd in the intestinal wall, (Step I of Cd transport), not
its absorption into the body (Step II, see sections a and e).
It could be further shown that the saturation is at least par-
tially reversible (Table 2, paper A). The apparent saturating
effects of relatively high Cd concentrations (0.2 mM) in the
lumen cannot be ascribed to general toxic effects of the metal,
as volume and glucose uptake remained within normal limits.
2) Step I of Cd transport can be inhibited by Ca. It is
-------
interesting to note that addition of milk to the lumen inhibits
Cd transport, and that this effect is fully accounted for by the
Ca content of the milk (Table 1, paper A). This finding empha-
sizes the well-known fact that the composition of the luminal
fluid strongly influences metal absorption, i.e. that Cd from a
food digest or in presence of normal constituents of luminal
fluids is not likely to be absorbed at the same rate as seen here
in absence of organic ligands or transport inhibitors (competi-
tors?, see sections c and d).
The finding that Cd and other metals such as Zn are readily
removed from the glucose-saline perfusate leads to a further con-
clusion. Thus, it has been claimed that metals are absorbed as
compounds of biological chelators (see e.g. Evans, G.W., Nutr.
Rev. 38:137-141, 1980). Under the conditions of the present ex-
periments, however, metal was equally readily absorbed from per-
fusate free of exogenous ligands, whether this perfusate was re-
circulated through the intestine so that endogenous chelators
might have accumulated, or whether single-pass perfusions were
employed. It is clear, therefore, that absorption can proceed
without the necessary involvement of endogenous chelators in the
lumen. In presence of food constituents etc. the postulated che-
lators might, of course, contribute to absorption, in competition
with non-absorbable metal-ligand complexes.
c) Interaction between Cd and Zn: An interaction between
Cd and Zn, possibly competitive in nature, has often been re-
ported and is not surprising in light of the chemical similari-
ties between the two metals. We tested therefore the ability of
Zn and Cd to interact at the level of Step I of their transport
out of the lumen. Details of these experiments are described in
manuscript C. In particular, a double inverse plot (Fig. 1,
manuscript C), suggests that the Zn inhibition of Cd uptake may
be competitive in nature; similarly, Zn absorption is inhibited
by Cd in what also appears to be a competitive manner (Fig. 2,
manuscript C). The inhibitory effect of Zn on Cd uptake, and
that of Cd on Zn uptake, are also illustrated in Figures 1 and 2
below. The interpretation of these results is somewhat obscured
by the fact that Cd appears to have a higher affinity for the
transport system than does Zn in Figure 1, whereas the inverse
appears true in Figure 2. If, nevertheless, the two metals are
competing for a common site then they share at least in part, a
common absorption mechanism. At the same time, the overall
transport into the blood is different for Zn and Cd. This con-
clusion is based on the contrast between Steps II for Zn and for
Cd: unlike Cd, Zn is retained in the jejunal mucosa to a rela-
tively small extent; the significance of this fact is further
considered in section e). A practical implication of the find-
ing of interaction between Cd and Zn is that addition of Zn, as
that of Ca (see section b), might conceivably be used to depress
the extent of Cd absorption from contaminated food.
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Direct plot of the data from Figure 1, paper C.
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Effect of Cd on Zn uptake
Direct plot of the data from Figure 2, paper C.
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d) Action of bile salts; As pointed out in section b) up-
take of Cd out of the lumen of the intestine is sensitive to the
action of metal ligands and transport inhibitors. Among such
compounds are possible endogenous modulators of Cd absorption.
Thus, we observed that fresh rat bile contained inhibitor (s) of
Step I of Cd absorption, and further work showed this to be due
to the presence of bile salts (see abstract C). The effect is
freely reversible, as illustrated in Figure 3.
Figure 4 shows a dose-effect relationship for the action of
glycocholate on Cd absorption. A close relationship between the
critical micellar concentration of the bile salt, and its inhib-
itory concentrations is apparent. The present hypothesis is
that Cd is bound to micelles and thus rendered unavailable for
further transport. Preliminary experiments have further indi-
cated that these micelles also interfere with Cd uptake in the
ileum. Further work on this question is continuing, but in any
case endogenous factors clearly can strongly influence the ab-
sorption of Cd. This represents an important conclusion in at-
tempts to explain the low fractional Cd absorption in the intact
adult animal.
e) Steps I and II of metal absorption; The results pre-
sented so far refer mostly to the removal of Cd (and other met-
als) from a perfusion solution in the jejunal lumen. As pointed
out in section a) there was found close agreement between the
rate of this removal and the accumulation of Cd in the tissue.
Further work showed that essentially all the Cd thus transported
could be recovered in the mucosa. These findings are detailed
in paper B. Thus, Table II, paper B shows that after 5 minutes'
perfusion, 25.1 nmol Cd had disappeared from the perfusate; 24.5
nmol were recovered from mucosal scrapings. Clearly, therefore,
analysis of luminal Cd concentration can yield information only
on Step I in Cd absorption. The second step in this process,
the release of retained Cd from the mucosa and its further move-
ment into the body, determines the overall rate of Cd uptake into
the body.
Determination of Step II of Cd is difficult (see also sec-
tion g). This fact is due primarily to the small fraction of Cd
appearing in blood and tissues. Thus, while it is perfectly fea-
sible to measure Step II of Zn transport in our preparation by
serial assays on portal blood, this technique cannot be applied
to study of Cd absorption. A new method was therefore devised
(see paper B, abstract A): It is based on the fact that, as
pointed out above, essentially all Cd transported out of the
lumen can be recovered from the intestinal mucosa immediately
after the end of perfusion. It is only at significantly later
times that some of the mucosal Cd is seen to have moved further
into the body. This is illustrated in Figure 1, paper B. The
difference between the Cd removed from the intestinal lumen and
that recovered from the intestinal wall, e.g. 5 hours later,
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Figure 3
Reversible inhibition of Cd transport by bile
Fresh rat bile (20% v/v) was added to perfusate in period
II in left panel, and period I in right panel.
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100
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provides a measure of Step II.
A necessary conclusion following upon this kinetic analysis
is that Steps I and II represent two processes in series, and
that no significant parallel absorption pathway, such as a para-
cellular shunt, is operating under present conditions. The over-
all process of Cd absorption under our experimental conditions
can then be represented schematically as shown in Fig. 5. Be-
cause no significant backflux from mucosa into lumen could be ob-
served, Step I is represented as a unidirectional process. Sim-
ilarly, under our conditions, blcod levels of Cd are very low,.
so that Step II also can be represented as a unidirectional pro-
cess. Step II amounted to only 1-2% of Step I (see Table 4?
paper B, and Table 1 of this report), In absence from the lumen
of ligands and other absorption modulators, Step II therefore
determines the rate of overall Cd absorption.
Cd bound to
Cd in
LUMEN ——•—————-> macromolecules in — •—-> blood and
tissues
Step I MUCOSA Step II
Figure 5
Jgj_QJ. Cd absorption
The model illustrated visualizes a large mucosal accumula-
tion of Cd, presumably due to binding of the metal to various
cell constituents. Table ~i, paper B, indeed, shows that only
about 5% of total Cd taken up by the mucosa is present in low
molecular weight fractions. Provided the total binding capacity
for Cd in the mucosa has not been exceeded, it then follows from
Figure 5 that the rate of overall absorption should be determined
by release of Cd from its mucosal ligands. To test this predic-
tion the ratio of Steps II/I was measured over a 10-fold range of
luminal Cd concentration, i.e. of absolute amounts of Cd trans-
ferred into the mucosa by Step I. This ratio should be indepen-
dent of the absolute value of Step I. In contrast, one would ex-
pect Steps I and II to vary independently if Cd absorption were
better represented by a parallel model. Table I summarizes the
results of this study, and clearly confirms the prediction of the
model shown in Figure 5. Mote in this table that over the lumi-
nal concentration range studies (20-200 yM), Step I approached
saturation; at the same time the ratio of Steps II/I did not
change appreciably. Either, therefore, Step II is independent
of Step I but exhibits the same saturation kinetics, or more
likely, Cd absorption is adequately described by Figure 5.
11
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Table I
Ratio of Steps II/I as function of Step I
Luminal Step II
Cd Step I Step I
(yiM) n (nmol/g/min) (%)
20 5 2.8 - 1.6 1.9 ± 1.1
100 6 9.3 ± 4.3 2.2 ± 1.2
200 7 14.7 i 5.1 2.4 ± 1.9
Results are given as mean - SD.
f) Role of metallothionein; It has repeatedly been sug-
gested that the low molecular weight metal-binding protein metal-
lothionein (MT) may be involved in the metabolism of heavy met-
als, and in particular in their absorption. Such a role might
consist of increased mucosal metal retention under conditions
where presence of heavy metals has previously induced synthesis
of MT. This attractive hypothesis was put to the test with Cd.
Obviously, the hypothesis predicts that presence of excess MT in
the cell would lead to depression of Step II of Cd transport as
opposed to Step I. Paper B shows that rats whose Cd binding
capacity in the MT fraction of the mucosa had been increased al-
most 4-fold (Table I, paper B), and who accordingly retained in-
creased amounts of freshly absorbed Cd in their MT (Table III,
paper B), showed the same ratio of Steps II/I as did control ani-
mals (Table IV, paper B). There is little support here for the
suggestion that MT might be involved in the control of Cd ab-
sorption.
g) Specificity of metal absorption systems; As mentioned
under subheading c), there is evidence that Zn and Cd share at
least in part a common mechanism of absorption. Further tests
of this hypothesis have been initiated with Zn-deficient rats.
In such animals the rate of overall Zn absorption is homeostat-
ically increased. If, now, Zn is transported by the same system
as is Cd one would predict that Cd uptake in such animals would
also be accelerated. Whatever the outcome of these studies,
there are clear differences, however, between the overall pro-
cesses of Cd and Zn absorption. These experiments have also been
extended to the study of Cu absorption, and are illustrated in
Figure 6 (see abstract B; paper in preparation).
h) Cd absorption in duodenum and ileum; A complete evalua-
tion of the mechanism of intestinal Cd absorption requires exten-
sion of the work so far reported to other sections of the small
12
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Figure 6
Comparison of Cd, Zn and Cu absorption
13
-------
intestine. Accordingly, studies were carried out with isolated
segments of ileum and duodenum, similar to those employed in the
analysis of metal transport in the jejunum. Characteristics of
Cd absorption in duodenum resemble those observed in the jejunum,
with a Step I transporting Cd out of a 20 yM Cd solution at a
rate of 1.8 ± 0.9 nmol/g/min, and Step II being very slow. In
such studies it is essential to tie off the common bile duct;
otherwise, especially in experiments employing recirculation of
the perfusate, the accumulation of bile depresses Cd absorption,
as it does in the jejunum.
Heal Cd absorption, in contrast, differs significantly from
the process in jejunum. This fact can be seen in Table II: note
first the somewhat lower rate of Step I, and secondly the rela-
tively and absolutely much greater rate of Step II (about 50% of
that of Step I). Under suitable conditions a bile salt inhibi-
tion of ileal Cd uptake can be demonstrated, but this work is
still in progress.
Table II
Cd Transport in Ileum
Animal Step I Step II/I
Number (nmol/g/min) (%)
1165 1.3 86
1166 1.0 56
1169 1.5 72
1170 0.9 61
1259 0.2 54
1260 1.3 63
1261 0.6 28
1262 1.5 20
1.0 - 0.5 55 - 22
i) Cd absorption in the newborn; This work was begun be-
cause of the well-documented observation that Cd, like many other
metals, is absorbed more readily in the newborn than in the
adult. This raises the following basic questions: a) Is the
transport mechanism for Cd in the newborn similar to that in the
adult although functioning at a greater rate; alternatively, are
one or two distinct mechanisms involved? b) If two distinct
mechanisms exist, what factors influence the developmental change
in absorption of Cd in the newborn? c) Are Cd and Zn transported
14
-------
by the same mechanisms at the level of Step I? If so, develop-
mental changes should be the same for Cd and Zn absorption.
Seventeen-day pregnant Sprague-Dawley rats were housed in
individual breeding cages. Upon birth of the pups, litters were
culled to between 8 and 12 animals each. The majority of experi-
ments were performed between day 12-16 after birth. Rat pups
were weighed and anesthetized with Inactin (100 mg/kg body wt,
i.p.). The animals were placed on a heated surgical table and
the intestine was exposed. Initial attempts to measure Cd trans-
port using the recirculating system used in adult studies were
unsuccessful because of lack of an adequate volume marker. Con-
sequently stationary segmental incubation in situ was employed
as in section a). Briefly, the duodenum from the pylorus to the
ligament of Treitz or a segment of proximal jejunum were iso-
lated. The segments were washed out with saline followed by air;
0.05-0.50 ml 20 yM CdCl2 glucose-saline was then placed into the
segment. The segments were tied off and replaced into the abdo-
men for 30 minutes. The animals were then killed, the remaining
intestinal fluid collected, and the lumen washed with 10 mM Na2
EDTA. The wash and remaining luminal fluid were combined. Fluid
and tissue were counted for l°9Cd activity. Percent Cd uptake was
calculated as 100% - % dose remaining in the intestinal lumen.
Percent Cd absorption was calculated as 100% - % dose remaining in
lumen + % dose found in intestinal wall.
Results of experiments to determine the effect of time on Cd
transport in the neonatal duodenum are shown in Table III. Re-
moval of Cd from the lumen is rapid, but as in the adult, the
major portion of the removed Cd can be recovered in the intesti-
nal wall.
Table III
Cadmium Transport in Duodenum of Newborn Rat
Incubation Recovery Recovery
Time in Tissue in Lumen Absorbed
min 11 1
1 85 ± 4 19 - 5 0
5 83 1 6 15 t 2 2
10 81 ± 9 11 t 4 8
20 88 ± 4 6 t 1 6
30 81 ± 4 6 t 2 13
Values represent mean - SEM of 3-4 animals, and are ex-
pressed as % of original amount of Cd placed into the lumen.
15
-------
The factors determining the high Cd uptake in the newborn
intestine remain unknown. One factor suggested is the low iron
concentration in mothers' milk. Iron deficiency, at least in
the adult, increases intestinal transport of both Fe and Cd. If
Fe status influences intestinal Cd transport similarly in the
newborn, correction of the Fe deficiency resulting from low iron
intake would reduce Cd uptake. To test this hypothesis, rat pups
from day 7 were fed 0.5 ml cow's milk/day, with or without sup-
plementary Fe (200 ppm FeSO4). After one week, Cd absorption
was measured as usual and the Fe concent of intestine and liver
determined by atomic absorption photometry.
Table IV shows intestine and liver iron levels in control
and iron-supplemented rat pups. Clearly, there is an increase
in iron concentrations in both tissues as a result of iron sup-
plementation. Table V presents data on intestinal Cd transport
in newborn rats. No difference between control and Fe supple-
mented rats could be found in the percent of l°9Cd dose removed
from the intestinal lumen or retained in the tissue.
The results of these experiments indicate that increased
iron levels of intestinal mucosal cells do not reduce Cd uptake
in newborn rats. Thus, unlike in the adult, Fe status does not
appear to be a critical influence on Cd uptake in the newborn.
This finding suggests the possibility that intestinal Cd trans-
port in the newborn differs qualitatively from that in the adult.
Table IV
Body Weight and Tissue Iron Levels
Body Weight Intestine Liver
g ug Fe/g wet wt.
Control (5) 35 ± 3 27 ± 4 42 ± 9
Fe Supplemented (5) 35 ± 3 41 ± 6 260 ± 58
The number of animals used in each group is in parentheses.
Values are presented as the mean ± SEM.
Table V
Iron Supplementation and Cd transport in Jejunum
% Remaining % Dose in In- % Dose
in Sac testinal Mucosa Absorbed
Control (5) 8.4±2.5 77.4±1.4 14.211.3
Fe Supplemented (4) 9.4±1.6 79.813.9 11.1±3.6
The number of animals used in each group is in parentheses.
Values are presented as the mean ± SEM.
16
-------
APPENDIX
Paper A
SOME DETERMINANTS OF INTESTINAL CADMIUM TRANSPORT
IN THE RAT1-3
E.G. Foulkes
Departments of Environmental Health and Physiology, Uni-
versity of Cincinnati, Colleget>f Medicine, Cincinnati, Ohio
45267
The hypothesis was tested that Cd absorption from the
intestinal lumen is mediated by cellular transport systems. Cd is
readily extracted from glucose-saline during perfusion of je-
juna! segments in the living rat. Over periods as long as 40
minutes, essentially all extracted Cd is recovered in the wall of
the intestine. Cd uptake by the tissue obeys saturation kinetics
with KM values of the order ofO. 1 mM, and Vmax approximately
0.01 fjjnol/g/min. Although washing after exposure to ""Cd
removes only little radioactivity from the tissue, it reverses at
least partly the saturating effects of higher Cd concentrations.
Unidirectional flux of Cd into the tissue is inhibited by 10 mM
Ca; no effect on backflux of Cd is seen. In contrast, Zn and
EDTA both accelerate washout of Cd. The Ca content of skim-
med milk fully accounts for the depressing effect of dried milk
on Cd uptake. These results point to the presence in mucosal
cell membranes of a saturable process responsible for Cd up-
take and sensitive to inhibition by certain solutes in the lumen.
INTRODUCTION
Outside the occupational environment, ingestion represents the major
route of human exposure to Cd. Net fractional absorption of the metal from the
gut, however, amounts to only a few percent of the oral load (Moore et a/.,
1973); the factors restricting absorption to such low values are not well
understood. One important variable undoubtedly is diet and the presence in
the intestinal lumen of dietary constituents and other compounds which might
either directly affect systems involved in Cd transport, or indirectly exert their
effect by reacting with Cd, thus altering its diffusibility and transport
characteristics. Thus, chronic milk feeding was reported to increase Cd absorp-
tion in young rats (Kello and Kostial, 1977). A direct influence of intraluminal
'The work was supported by EPA grant #R805840010, and by NIH grant ES-00159.
2A preliminary report appeared in the Physiologist, Vol. 21, p. 38, 1978.
Journal of Environmental Pathology and Toxicology* 3:471-481
Copyright (c~ 1980 by Pathotox Publishers, Inc.
17
-------
reactions of Cd is seen, for instance, in the work of Kojima and Kiyozumi (1974)
and of Cherian et a/. (1978). Effects of Cd chelators on epithelial Cd uptake
have also been noted in the renal tubule (Foulkes, 1974).
Although it has been reported that movement of Cd out of the intestinal
lumen obeys first order kinetics (Kojima and Kiyozumi, 1974), results of the
present work suggest a different conclusion. Saturation of a hypothetical
transport system, then, or its low affinity f^r Cd, could constitute additional
determinants of Cd uptake. A suggestion that proteins similar to metallothio-
nein may be involved in Cd absorption (Evans et a/., 1970) is attractive but
unproven. Sugawara and Sugawara (1977) proposed a role for metallothionein
in the well-documented prolonged retention of Cd in the intestinal wall;
sloughing of mucosal cells with their metal content presumably also contri-
butes to the low netCd absorption into the body (Richards and Cousins, 1974).
Present understanding of reactions involved in intestinal Cd transport does
not permit a full evaluation of all these possibilities. The work reported here
was undertaken in order to explore further the contribution of various factors to
Cd uptake from the lumen of the rat jejunum.
MATERIALS AND METHODS
Male Sprague-Dawley rats, weighing 250-300 g, were maintained on
commercial chow (Purina) and tap water ad lib for at least one week before
study. Twenty-four hours before the experiment, food was removed.
Anesthesia was induced with Inactin (100mg/kg IP); body temperature was
maintained close to 37°C by means of a rectal probe and a thermostatically-
controlled heat pad. The trachea was cannulated. In some studies mean arterial
blood pressure was determined in the femoral artery with a Harvard Instrument
Co. transducer; in general, blood pressures remained normal throughout the
studies. A 15 cm length of jejunum, starting at the ligament of Treitz, was
cannulated at both ends and perfused at varying rates from a reservoir kept at
37°C. The problem of mixing and therefore of long periods (>20 minutes)
required before effluent concentrations reached steady values interfered with
use of physiologically reasonable flow rates (<0.1 ml/min). With a somewhat
higher flow (0.4 ml/min, average perfusion pressure 2 cm H2O) 5 minutes
sufficed to attain a steady state which could thereupon be maintained for at
least 30 minutes. As many as 4 different solutions could thus be tested sequen-
tially over a period short enough to assure the stability of the preparation. In
some studies, perfusion solutions were recirculated through the intestine.
Inclusion of the reservoir in the dead-space volume necessitated further in-
creases in perfusion rate. This procedure permits convenient sequential samp-
ling for the determination of transport kinetics. Transit time of perfusate through
the intestine was estimated with a small bolus of Dextran Blue in saline, and
luminal volume was calculated as the product of perfusion rate and transit
time.
Under no conditions did it prove practicable to perfuse more than 2 or 3
segments of intestine simultaneously, making difficult simultaneous compari-
son of effects of several variables. In one series of experiments, therefore, the
18
-------
cannulated intestine was filled as usual; 3 cm sections were then tied off and
the abdomen closed for 30 minutes (stationary incubation). Mixing of luminal
contents in such short isolated segments proved inadequate for sequential
sampling. After stationary incubation, therefore, the intestine was removed for
analysis of contents and tissue in each segment, as well as for evaluation of Cd
uptake by the remaining carcass in a Packard whole body counter.
The standard intestinal solution contained 5 mM glucose in saline, to-
gether with 0.1 /xCi/ml 3H-polyethylene glycol (New England Nuclear) as
volume marker. Glucose was added because of its tendency, observed early in
this study, to support constant Cd transport. CdCI2 labelled with 109Cd (0.02
/iCi/ml) was added to the desired concentration, and removal of Cd from the
lumen was calculated from changes in the 109Cd/3H ratio as determined on a
Packard liquid scintillation spectrometer with automatic external standard.
Rates are expressed as //.mol Cd removed/unit weight or length tissue/minute.
Tissues to be analyzed were first cut open, then rinsed for 10 seconds in saline,
blotted on the serosal side, and finally weighed and measured. Cadmium
content of the tissue was determined on a Packard well-type scintillation
spectrometer. Knowledge of the relative counting efficiencies made it possible
directly to relate disappearance of Cd from the lumen with its accumulation in
the tissue. Glucose absorption from recirculating perfusate was measured with
a glucose oxidase procedure.
RESULTS
Activity gradient along jejunum
Ideally one would wish to compare simultaneously effects of several
variables on Cd removal from the lumen. To determine the feasibility of such a
proceduVe, changes in the luminal 109Cd/3H ratio were measured in a series of
contiguous 3 cm segments after a period of 30 minutes (stationary incubation).
Fig. 1 shows results of 1 of 3 such studies.
An activity gradient along the intestine is clearly apparent, with the ability
to remove Cd from the lumen mirrored by its accumulation in the tissue.
Existence of this gradient makes it impossible to compare simultaneous ac-
tivities in more than 2 contiguous segments. Study of 2 segments proved useful,
provided each comprised about 1/2 of the average length employed for experi-
ments similar to that shown in Fig. 1 and provided the order of addition of
control or experimental solution was alternated. On this basis consistent results
could be obtained in the study of a single variable, each animal serving as its
own control (cf. Table 1).
Extent of Cd transport
The ability of the intestinal wall to accumulate and retain Cd is well
documented. Under present conditions also, as seen in Fig. 1, Cd is ac-
cumulated in the tissue. In two studies the absolute loss of Cd from the lumen
(perfused with 0.02 mM Cd) was calculated from the combined volume of
19
-------
Tissue Accumulation
90
80
b
x 70
o>
Tissue Accumulation (cpm
_ ro 01 -f> tn a>
o o o o o o
.
.
-
%
W.
w
I
1
^
^
6
\
| | Fractional Abs(
i
1
I
jrp
tion
-
1.2
1.0
0.8
0.6
0.4
0.2
3 6 9 12 15
LENGTH (cm)
B
FIGURE 1. Stationary incubation (30 mm) in contiguous jejuna! segments. For details, see text.
TABLE 1. Effect of Milk on Cd Removal
Solution
Saline
10mMCaCl2
in saline
4%(w/v)SMr*
in saline
Fractional
Removal'
n (%)
15 21
(range 10-48)
5 12
10 10
Activity
(% of control* SE)
100 ,
55 ±6
50 ± 10
'Removal of Cd was measured during 30 minutes' stationary incubation in 2 contiguous segments, each
animal serving as its own control; removal was normalized for tissue weight. bSMP: skimmed milk powder,
containing 0.25 nmol Ca/g, i.e. final Ca concentration 10 mM. Concentration of ""Cd: 0.02 mM.
20
-------
luminal fluid recovered and saline washings, together with the 109Cd/3H ratio.
Tissue recovery of Cd lost from the lumen amounted to 85 percent (experiment
1, 12 minute perfusion) and 79 percent (experiment 2, 40 minutes). In two
further studies on 2 animals each, 1 ml of the usual 0.02 mM Cd solution was
introduced into the jejunum. One animal in each pair was killed immediately
and the gut removed; stationary incubation in the second animal continued for
30 minutes. As counted with the whole body counter, the ratio of carcass
activity in animal 2/animal 1 never exceeded 1.0. In other words, no significant
transmural movement of Cd occurred under present conditions.
Effects of milk
The stationary incubation technique described above was used to de-
termine effects of some soluble dietary constituents (milk) on Cd removal from
the lumen. Each segment contained 1 ml saline (glucose was omitted in these
experiments); dried skimmed milk powder (40 mg/ml) was added randomly to
one of the 2 contiguous segments under study. Table 1 illustrates the strong
inhibition exerted by milk constituents. In each case, activity in the control
segment simultaneously determined was equated to 100 percent. Fractiona-
tion of the milk powder showed no activity in the protein fraction isolated on
Sephadex G75. Instead, full inhibitory power was retained upon wet ashing of
the powder. Finally, as also shown in Table 1, the Ca content of the ash
adequately accounted for the ability of milk acutely to interfere with Cd
removal.
Ca inhibition of Cd flux
More detailed understanding of Cd translocation and its inhibition by Ca
requires information on kinetics of the process. Fig. 2 illustrates the rapid
removal of Cd from a recirculating perfusate; also shown are the Ca inhibition
previously observed during stationary incubation and the saturating effect of
highCd concentration.
Ca inhibition of net Cd removal out of the lumen involves depression of
flux from lumen to tissue, not accelerated washout of Cd from the tissue. This is
illustrated in Fig. 3 by results of one of 3 similar studies in which intestines had
been preloaded by perfusion for 20 minutes with 0.02 mM 109Cd in saline-
glucose as usual. The perfusate was then replaced with a Cd-free solution, and
the ratio of residual' 09Cd to 3H was equated to 100 percent. During the next 15
minutes only little 109Cd was washed out of the tissue, as deduced from the slow
rise in the isotope ratio. This control washout rate was not altered by addition of
10 mM Ca. The inactivity of Ca may be contrasted with the effects of Zn and
EDTA, as determined in separate studies; both substances strongly accelerated
washout. Zn not only accelerates washout of Cd, but also depresses the
forward flux of Cd, as shown in experiments (not further detailed here) in which
initial Cd removal from the lumen was measured in intestines not previously
exposed to Cd.
21
-------
or
I
UJ
or
2
g
LU
o
z
o
o
T3
O
100
90
80
70
60
50
high Cd
lowCd
+ IOmMCa
A low Cd
I
10
MINUTES
15
FIGURE 2. Time course of Cd uptake from lumen of perfused jejunum. Initial concentrations of Cd were
0.02 mM and 0.20 mM; total volume perlusate 3 ml, pertusion rate 0.6 ml/min, length tissue 22 cm.
Kinetics of Cd removal
Although reduced fractional Cd removal at higher Cd concentrations
could be readily demonstrated in recirculation experiments (Fig. 2), somewhat
more consistent results were obtained with steady state perfusion at 0.4
ml/min. Even with this technique, however, great individual variation was
found, as shown in Fig. 4 by the results of three consecutive studies. Similar
results were repeatedly obtained (Table 2). Although these further experiments
all confirmed the saturability of Cd transport, only two Cd concentrations were
studied in each, and they have, therefore, not been included in Fig. 4. What is
very apparent in both Fig. 4 and Table 1 is the great variability between
animals; this renders difficult attempts to define accurately the kinetic con-
stants of Cd transport.
22
-------
EDTA
800
700
:s 600
_3
H^
1
5 500
£
X400
^
•o
o
fl»
300
200
100
3 6 9 12 15 18 21 24 27 30
MINUTES
FIGURE 3. Reversal of Cd uptake from lumen. Tissue was preloaded with ""Cd as described in text;
perfusate was then diluted 20-fold with Cd-free solution, and the ratio of residual "™Cd to JH equated to 100.
After 15 minutes 10 mMCa, Znor 5 mM EDTA were added as shown.
To test the hypothesis that the effect of high Cd concentrations represents a
non-specific intoxication of the system, two experimental approaches were
used. In two studies removal of glucose from the perfusate was followed over a
period of 25 minutes and found not to be reduced by 0.5 mM Cd. In another
two animals, jejunum (28 cm) was filled with glucose-saline as usual. The
outflow cannula was closed and the lumen connected to a horizontal pipette
23
-------
whose emptying provided a direct measure of volume absorption. Control
values of fluid absorption were 0.17 and 0.27 ml/minute compared to 0.21 and
0.25 ml/min after addition of 0.5 mM Cd. In other words, no acute effects of 0.5
mM Cd on intestinal function could be observed. Substitution of 5 percent
mannitol for the saline completely abolished volume absorption and provided
a positive control.
ISOOr-
^ IOOO
c1
'E
500
10
20
30 40
[mM]"1
50
60
FIGURE 4. Kinetics of Cd absorption from lumen. Steady-state perfusion at 0.4 ml/min; results of 3 different
animals are shown.
24
-------
TABLE 2. Reversible Saturation of Cd Transport
Cadmium Transport
Experiment
#
1
2
3
4
5
5
Mean
Period 1
(.02 mM)
A 8
3.5
1.8
2.2
5.5
3.4
2.9
100
100
100
100
100
100
100
Period II
(nM)
A B
0
9.6
4.0
17.6
4.0
0
0
53
18
32
12
0
19
Period III
(.02 mM)
A B
1.2
1.3
2.0
1.9
1.1
1.1
34
72
91
35
32
38
50
Perfusion rate 0.4 ml/min. Cd transport is expressed in Column A in nmol/g/min, in B as fractional
absorption in percent of control. The initial Cd concentration for each period is also shown.
Reversibility of saturation effects
In nine experiments, jejunum was first perfused as usual at 0.4 ml/min
with glucose-saline containing 0.02 mM 109Cd, and the rate of Cd removal was
determined. The perfusion solution was then replaced with glucose-saline
containing 0.40 mM unlabelled Cd, a concentration adequate to severely
depress fractional Cd transport. The tissue was allowed to accumulate Cd for
20 minutes before the perfusate was replaced with the original 0.02 mM Cd
solution. Cadmium transport was now measured again and found to equal 51
percent (SD 29 percent) of the original control. In 4 further studies, the 0.4 mM
Cd was omitted in period 2 in order to evaluate the influence of experimental
procedures* on the stability of Cd transport; in this case the mean activity in
period 2 equalled 52 percent of that in period 1. Although these results were
highly variable, they nevertheless suggest that the specific action of Cd in
depressing, at higher concentrations, the fractional Cd absorption from the
intestine, is largely reversible. Further evidence for reversibility was sought in 6
consecutive studies in which intestines were perfused at constant tracer con-
centration, first in presence of 0.02 mM Cd, then at 0.20 mM Cd, and finally
again with the low Cd concentration. Results of these studies are collected in
Table 2 and show that simple dilution can at least partially reverse the inhibi-
tory effects of higher Cd concentrations.
DISCUSSION AND CONCLUSIONS
It is important to emphasize that results described in this paper do not refer
to the transmural movement of Cd, but only to its transport from lumen into
intestinal wall. The term transport is used advisedly, as simple physical diffu-
sion cannot readily explain the saturation kinetics observed here. The rate of
this transport is relatively fast, and it obviously does not limit transmural Cd
uptake. Such a conclusion in turn implies that transfer of Cd into the body is
determined by the rate of its release from the intestinal wall. Prolonged reten-
25
-------
tion in the mucosal cells, as pointed out by others (e.g. Richards and Cousins,
1974) could lead to return of Cd to the lumen upon sloughing of cells. This fact
presumably contributes to the generally low net absorption of Cd.
Characteristics of the process of Cd transport out of the lumen remain
poorly defined. Under present conditions the process appears to obey satura-
tion kinetics, with an approximate KM of 0.1 - 0.2 mM (Fig. 4), and a Vmax of the
order of 0.01 ^mol/g intestine/minute. Such a saturable process would contri-
bute a major portion of total Cd absorption only at relatively low Cd concentra-
tions, such as might be encountered under natural conditions. In contrast to
present results, Kojimaand Kiyozumi (1974) did not observe saturation kinetics
and concluded thatCd outflux from the lumen obeys first order kinetics. These
experiments were carried out at higher Cd concentrations than used here.
Although our results provide no basis for such an assumption, it is possible that
a second component of Cd translocation exists which does not become satur-
ated in the concentration range studied. Evidence for two mechanisms of Cd
uptake by duodenal tissue in vitro was also provided by Hamilton and Smith
(1978). Kooeta/., (1978), in their work on transmuralCd movement out of the
chicken intestine, studied Cd concentrations as high as 1 mM. Their conclu-
sion that such Cd uptake could not be saturated is at variance with their
observation of partial saturation over the concentration range of 0.01 to 0.10
mM. In addition, fractional Cd accumulation in duodenal tissue was clearly
depressed by higher Cd concentrations. In any case, the saturable component
studied here appears to be associated with mucosal cell membranes. This
conclusion is based on the reversibility of saturation by simple dilution, a
procedure which does not lead to significant washout of Cd from the tissue (Fig.
3). Work is in progress to determine whether the Cd accumulated in the tissue is
concentrated in mucosal cells.
The great variability encountered in the kinetic analysis of Cd movement
has so far precluded attempts to distinguish between competitive and non-
competitive inhibition of Cd uptake by Ca. Calcium uptake by duodenal
mucosa in vitro was reported to be non-competitively inhibited by Cd
(Hamilton and Smith, 1978). Inversely, Ca exerted no effect on Cd uptake in
this study, but, as in the work of Kojima and Kiyozumi quoted above, relatively
very high Cd levels were employed.
Inhibition of Cd transport by milk illustrates the expected critical impor-
tance of the composition of luminal fluid. An influence of diet on Cd uptake
into the body has often been reported, but it must be emphasized that changes
in net, long-term Cd retention may not be related to acute effects of dietary
constituents on unidirectional Cd flux into the gut wall. Thus, present results on
the inhibition of Cd uptake by milk do not contradict the increased net Cd
retention seen in young rats on a high milk diet by Kelloand Kostial (1977).
Ability to transported varies markedly along the jejunum (Fig. 1). Similar
activity gradients for other solutes are well known. Further work will be
necessary to explore the contributions of duodenum and ileum to total Cd
transport.
26
-------
it gives me much pleasure to thank Ms. Cathleen Voner for her help and
collaboration. Mr. William Powers contributed to this research during a sum-
mer student fellowship.
REFERENCES
Cnenan, M.G., Coyer, R.A., and Valbarg, L.S.: Gastrointestinal absotptioii and organ distribution! of ora!
cadmium chloride and cadmium-metallothionein in mice. !. I oxicol. Environ. Health 4:861 -86tJ, 1478.
Evans, G.W., Majors, P.F , and Comatzer, W E.: Mechanism for cadmium and zinc antagonism of copp;?!
metabolism. Biochem. Biophys. Res. Com. 40: ! 1 42-1 148, 1970.
Foulkes, E.G.: Excretion and retention on' cadmium, zint,
21
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Paper B
Toxicology, 14 (1979) 199-208
© Elsevier/North-Holland Scientific Publishers Ltd.
ON THE ROLE OF METALLOTHIONEIN IN CADMIUM ABSORPTION
BY RAT JEJUNUM IN SITU
DINKO KELLO*. NAOKI SUGAWARA**, CATHLEEN VONER and
ERNEST C. FOULKES***
Departments of Environmental Health and Physiology, University of Cincinnati, College
of Medicine, Cincinnati, Ohio 45267 (U.S.A.)
(Received September 25th, 1979)
(Accepted November 7th, 1979)
SUMMARY
The rcle of metallothionein (MT) in the mechanism of cadmium absorp-
tion from the jejunum was studied in 7—9-week-old-male rats exposed to
50 ppm of cadmium in drinking water for 9 days. Exposed animals contained
an average of 144 /ig MT/g of mucosal tissue, compared to 40 Mg in control
animals. During jejuna! perfusion in situ with 5 mM glucose-saline contain-
ing 10—20 nM CdClj the increased MT content of mucosa exerted no effect
either on cadmium absorption from the lumen (step I), or on its further
transport into the body (step II). Immediately after perfusion, essentially all
cadmium removed from the lumen was fully recovered in the intestinal
mucosa. About 50% of the mucosal cadmium was found in the sediment
after homogenization and centrifugation; a large portion of this cadmium
may be assigned to the membrane fraction. The binding of freshly absorbed
cadmium in the mucosal cytosol was not restricted to low molecular weight
protein, although cadmium binding capacity in the MT fraction of controls
as well as of exposed animals greatly exceeded actual binding of newly
absorbed cadmium. Our results offer no support for the view that MT in the
jejunal mucosa serves as determinant of cadmium absorption.
INTRODUCTION
Sufficient evidence has been accumulated in recent years suggesting that
*Fogarty International Fellow. Institute for Medical Research and Occupational Health,
Mose Pijade 158, 41000 Zagreb, Yugoslavia.
** Department of Public Health, Sapporo Medical College, S-l W-17, Sapporo, Japan 060.
***To whom correspondence and reprint requests should be addressed.
Abbreviation: MT, metallothionein.
28
-------
oral intake represents the primary source of the cadmium accumulated in the
human body [1—4}. Although cadmium absorption from the gastrointestinal
tract has been investigated in a number of studies, the exact mechanism of
cadmium transport from the intestinal lumen to blood is still unknown [3,5,6].
It has been suggested that MT, the low molecular weight Cd-binding protein
found in mucosa [7—10], plays an active role in cadmium absorption from
the gastrointestinal tract [6,11 J. Although it is generally believed that intes-
tinal MT is capable of sequestering intracellular cadmium, thus preventing
transfer of the metal to the basolateral membrane, evidence for such a con-
clusion remains incomplete. It has even been proposed that the intestinal
absorption of cadmium might possibly be increased in animals pretreated
with cadmium [12].
The purpose of the present experiments was to study the role of mucosal
MT in cadmium transport by rat intestine. Both uptake of cadmium from the
lumen [13] (step I), and its further movement into the body (step II) were
measured in animals as a function of intestinal MT levels. Results obtained
permit the conclusion to be drawn that under present experimental condi-
tions, MT does not influence Cd transport by rat jejunum.
MATERIALS AND METHODS
Animals and diets
Male rats of the Sprague—Dawley strain aged 7—9 weeks were obtained
from Charles River Co. and maintained on Purina rat chow. This commercial
diet contained an average of 0.11 ppm of cadmium, 65 ppm of zinc and 355
ppm of iron (supplier's analysis). Half the animals were exposed for 9 days
to 50 ppm cadmium (as CdQ2) in deionized water, offered ad lib; control
animals received regular tap water.
Intestinal uptake and transport of cadmium
The rats were fasted for 24 h prior to the experiment. Anesthesia was
induced by intraperitoneal injection of pentobarbital sodium (50 mg/kg).
The abdomen was opened by a midline incision, and the proximal jejunum
was identified, starting at the ligament of Treitz. A segment approx. 15 cm
in length was selected and ligatures were loosely applied proximally and
distally, care being taken not to compromise the mesenteric circulation to
the bowel. Inflow and outflow catheters were inserted and sutures were
tightened; the abdominal incision was closed with surgical clamps. Body
temperature was maintained with a heating pad and was monitored with a
rectal thermometer. The lumen of the jejuna! segment was perfused with
10 ml of solution (saline with 5 mM glucose) containing 200 nmol cadmium
chloride labeled with 0.5 ;uCi 109Cd (New England Nuclear, Boston, MA),
at a flow rate of 0.4 ml/min for 25 min. The concentration of Cd of 0.02 mM
is lower than that used by many other investigators. It was chosen because
it does not saturate the mechanism responsible for Cd transport at levels
which might be encountered in polluted environments [13]. Immediately
29
-------
following the perfusion, the lumen was rapidly flushed with 10 ml of ice-
cold 0.15 M NaCl followed by 10 ml of air. Blood (6—8 ml) was collected
from the abdominal aorta. The jejuna! segment, liver and kidneys were
removed, weighed and their radioactivity counted in an automatic well-type
scintillation counter (Packard, Model A-5921). Mucosa was scraped from the
serosal layers of the entire length of perfused jejuna! segment with a glass
slide, weighed, counted, and stored at —€5°C. The 109Cd activity of the
remaining carcass of each rat was determined in a whole-body counter using
a 5 X 5 inch Nal (TL) crystal connected to a Packard analyzer (Packard,
Model A-5921). Results are expressed as nmol CdCl2, as calculated from the
specific activity of the solution.
Rate of transmural transport of cadmium
Because the amount of Cd taken up by the jejunum very greatly exceeds
that further transported into the body, i.e. because the rate of step I greatly
exceeds that of step II, accurate comparison of the 2 steps and an appro-
priate mass-balance of transported Cd could not be readily achieved with the
whole body counter. A technique was therefore developed [14] for measure-
ment of steps I and II on the basis of mass-balance measurements. In a
manner similar to that previously described [13], a small volume (1.6—2 ml)
of the same solution as above was recirculated through the jejunal lumen at
0.8 ml/min; perfusion pressure did not exceed 2 cm H2O. The rate of step I
was computed from the fall in the ratio of I09Cd/3H, as determined with a
Packard liquid scintillation spectrometer. At the end of perfusion remaining
perfusate was collected and combined with 3 ml saline used to wash out the
lumen. Quantity A, the amount of Cd removed from the lumen, was calcu-
lated from the total 109Cd content of this solution, as measured on a Packard
well-type scintillation spectrometer. If, immediately after washing, the intes-
tine was cut out and its 109Cd content (quantity B) determined, essentially
all Cd removed from the lumen could be recovered in the tissue (Fig. 1). To
determine step II, the catheters were clamped and the abdominal incision
closed. Two hundred and seventy-five minutes later, the segment of jejunum
was removed for determination of its radioactivity, together with that of its
contents. At this time, recovery from the tissue fell below the amount
originally absorbed by the tissue, i.e. A > B. The deficit in recovery (C),
where A — B = C, provides a measure of step II. After it had been blotted
on the outside, the tissue was then weighed, so that the rates of steps I and
II could be calculated in nanomoles/g fresh wt/min.
Cadmium binding components in perfused jejunal mucosa
To determine which tissue components react with freshly absorbed cad-
mium, mucosa obtained from perfused jejunal segments was homogenized
in 0.25 M sucrose (1 : 4 w/v) and centrifuged at 100 000 g for 1 h. The
radioactivity in supernatant and precipitate was determined. The super-
natant was chromatographed on a Sephadex G-75 column (1.5 X 30 cm) in
20 mM Tris—HC1 buffer, pH 8.6 and the radioactivity of each fraction was
30
-------
40
35
30
23
20
15
10
5
0
C
-
Cd DEFICIT
( LUMINAL LOSS -
TISSUE SECOVEH't
n inol / gm
- (9
1.9*..
«
1 2 3
HOURS
Fig. 1. Quaatitatioa of step II by measurement of the difference (C) between Cd removed
from the lumen during 25 mia profusion (A) and that recovered from the intestinal wall
at the times shown (B), i.e. C - A — B. Time is measured froas the begraiiing
and mean deficits are indicated by dashed lines.
measured. The results am ex;
hofflogenats.
as perceai of total i0!*Cd radioactivit
Determination of intestinal metallothionein
The total amount of MT in the mucosa was determined -with the satura-
tion method described by Kotsonis and Klaassen [15]. Homogenized mucosa
(25% in 0.25 M sucrose) was centrifuged at 10 000 g for 15 min, 10% TCA
was added to the supernatant to a final pH of 2. The mixture was centrifuged
again at 15 000 g for 30 min. The TCA supernatant was mixed with cadmium
solution (300 nmol 109CdCl2/g tissue). The pH was adjusted to 8.8 with
dilute NaOH and the solution was fractionated on a Sephadas G-75 column
(1.5 X 30 ism) aad the radioactivity of each fraction was. determined. Thf-.
amount of cadmium in the MT peak was calculated from the specific activity
of the 109CdQU. The amount of Cd-binding protein of low molecular weight
was then derived from its Cd-binding capacity on the assumption that it
binds 7 mol Cd/mol of 10 000,
Determination of cadmium and zinc
The intestine was thoroughly washed with saline before the analysis.
Cadmium and zinc were determined in mucosa by atomic absorption spectrc-
31
-------
photometry (Perkin Elmer, Model 403) after wet digestion with nitric and
perchloric acid. In samples from control group of animals cadmium was
extracted and concentrated using the method described by Yeager et al. [16].
Statistics
All results are expressed as the arithmetic mean with the standard devia-
tion of the mean. The significance of the difference among the groups was
determined by Student's f-test for unpaired data.
RESULTS
Induction of metallothionein in intestinal mucosa
The daily intake of cadmium in animals exposed to 50 ppm cadmium in
drinking water has been estimated as 1.4 mg, i.e. total intake of cadmium
after 9 days was about 12.6 mg. Data presented in Table I show that such
treatment caused an increase in the amount of cadmium and metallothionein
in mucosa. Mucosal cadmium in pretreated rats was 68 times higher than in
control animals; at the same time the concentration of metallothionein in-
creased only 3.6 times. The concentration of zinc in these 2 groups of animals
did not differ significantly.
Intestinal uptake and transport of cadmium
The data presented in Table II demonstrate that after intestinal perfusion
with 0.02 mM l<39CdCl7. solution for 25 min, extensive accumulation of cad-
mium was found only in the intestinal wall. By contrast, the total intestinal
transfer of cadmium, expressed as the sum of cadmium recovered in carcass,
liver, kidneys and blood, represented less than 0.3 percent of the total per-
fused dose in both groups of animals. As further described below essentially
all cadmium accumulated by the intestine was confined to the mucosa.
TABLE I
CONCENTRATIONS OF CADMIUM, ZINC AND METALLOTHIONEIN IN MUCOSA
(/ug/g wet wt)
Results are expressed as arithmetic mean * S.D. Exposed animals had 50 ppm of cadmium
in drinking water for 9 days.
6 Control rats 6 Pretreated rats
Cadmium
Zinc
Cd -binding
capacity*
Metallothionein
0.06 ± 0.04
21.5 ± 3.4
3.2 ± 0.5
40 ±6
4.32 ± 0.86
22.2 ± 4.1
11.3 ± 2.8
144 ± 36
"Equivalent to total amount of Cd bound in MT fraction, as measured during MT assay.
32
-------
Although these results (Table II) show that the intestinal uptake of cadmium
was slightly higher in pretreated than in control animals (17% and 13% of
dose respectively, P < 0.05), the total transmural transport was the same in
both groups.
Cadmium binding components in perfused jejunal mucosa
In order to determine the distribution of cadmium in the intestinal wall,
the mucosa of perfused jejunal segments was harvested by scraping and
analyzed. The supernatant fraction of mucosal homogenates in both groups
of animals contained about one-half of total tissue 109Cd (Table III). Although
the supernatant from exposed animals contained a little more cadmium than
that from controls, major differences between these 2 groups appeared after
chromatography. As shown in Table HI there were 2 major cadmium peaks
in both groups. The second peak corresponds to a mol. wt of about 10 000;
the first peak contains high molecular weight compounds. In control rats
most of the 109Cd appeared near the void volume, indicating that most of the
cadmium was bound to high molecular weight proteins and very little to MT.
In contrast, in the pretreated rats little cadmium was bound to high molecular
weight proteins and more was associated with the low molecular weight
fraction, tentatively identified as MT. In neither group did significant amounts
of cadmium remain unbound.
Rate of transmural transport of cadmium
Because of the small amount of Cd transferred to the body during 25 min
perfusion (Table II), the transfer step (step II) was measured over a period
of 5 h as discussed in the Materials and Methods section. Table IV, based on
results similar to those shown in Fig. 1, demonstrates that the rate of trans-
TABLEH
INTESTINAL UPTAKE AND TRANSMURAL TRANSPORT OF CADMIUM
Perfusate contained 200 nmol CdCl,. Results are expressed as nmoles of cadmium (Mean ±
S.D.). Rats were killed for tissue analyses after 25 min perfusion.
15 Control rats 15 Pretreated rats
Weight of intestine (g) 1.4 ±0.3 1.4 ± 0.2
Intestinal uptake 25.1 ± 6.5 34.0 ± 13.3
Retained in mucosa 24.5 ± 6.4 33.1 ± 12.8
Carcass 0.211 ± 0.091 0.277 ± 0.192
Liver 0.186 ± 0.108 0.252 ± 0.180
Kidney 0.007 ± 0.003 0.012 ± 0.006
Blood 0.007 ± 0.003 0.016 ± 0.009
Total transfer 0.412 ± 0.136 0.555 ± 0.319
to body
33
-------
TABLE HI
DISTRIBUTION OF PERFUSED CADMIUM IN JEJUNAL MUCOSA
Results are expressed as percent of levels in original homogenate.
Homogenate
Sediment
Supernatant
High mol. wt fraction
(above 10 000)
Low mol. wt fraction
(appro*. 10 000)
Remaining fraction
(below 10 000)
aP< 0.01.
bP< 0.001.
5 Control rats
100.0
51.4 ± 2.5
48.6 ± 2.5
30.0 ± 3.7
13.4 ± 5.1
5.6 ± 1.0
5 Pretreated rats
100.0
40.4 ± 5.7
59.6 ± 5.7"
17.2 ± 1.8b
38.9 ± 7.2b
3.4 ± 0.5
mural transport as measured by disappearance of cadmium over a period of
5 h Tras low and did not exceed 1.3% of the rate of cadmium removal from
the lumen. The ratio of step II to step I was the same in both groups of ani-
mals.
DISCUSSION
While relatively little is known about the mechanism of cadmium absorp-
tion, it is clear that this process must consist of at least 2 steps: (1) uptake
TABLE IV
RATE OF UPTAKE AND TRANSMURAL TRANSPORT OF CADMIUM
Results are shown as mean ± S.D.
10 Control rats 13 Pretreated rats
Intestinal uptake* 1.9 ±0.8 1.3 ±0.4
(nmol/g/min)
Transmural transport11 0.023 ± 0.010 0.015 ± 0.009
(nmol/g/min)
Rate of transmural 1.3 ±0.7 1.3 ±0.8
transport as % of
rate of uptake
aJejunal segment was perfused with 0.02 mM ""CdCl, solution for 25 min.
bRate of transport was calculated from difference between initial uptake and final reten-
tion of ""Cd in tissue 275 min after the end of perfusion.
34
-------
of the metal by the mucosal cells; and (2) transmural movement of cadmium
into the body [6,14,17]. Despite differences in methodology and species,
there is considerable agreement between the results presented here and those
obtained by previous workers [5,6,17], namely that intestinal uptake of
cadmium is much higher than its transport into the body (Table II). Thus,
we found that even after 5 h, only a small portion of cadmium accumulated
in jejuna! mucosa is released into the animal; the rate of release amounted to
only 1—2% of the rate of uptake (Table IV).
It is important to emphasize that under present conditions, in the absence
of normal luminal contents, uptake of cadmium in all likelihood greatly
exceeds that to be expected in presence of normal intestinal contents. We may
assume that in rats on a normal diet most of the metal is bound to non-
absorbable food components and is therefore eliminated from the body [18];
there is evidence also for the presence in food of direct inhibitors of cadmium
absorption [3].
The prolonged retention of cadmium by the intestinal tract was attributed
to extracellular adsorption [17,19] or intracellular accumulation and binding
on different ligands, particularly MTs [12,20]. Sahagian et-al. [17] proposed
that in the process of mucosal uptake, cadmium probably reacts with the
cell membrane. This is fully compatible with the present study which shows
that about one-half of 109Cd present in mucosal homogenate is collected in
the precipitate after centrifugation. Since numerous studies have shown that
only a few percent of intracellular cadmium are bound to nuclei, mitochon-
dria and endoplasmic reticulum [21], a large portion of the cadmium in the
precipitate may be assigned to die membrane fraction. Similar findings by
Taguchi and Suzuki [22] support this conclusion. Detailed information
about the nature of binding sites or classes of ligands present in membranes'
for a given metal is not available.
Recently several authors suggested that intestinal MT plays an important
role in cadmium absorption from the gastrointestinal tract [6,10,11]. It
was postulated that this protein would sequester intracellular cadmium and
prevent in this way its transport into the circulation [12,20]. This statement
is based on the assumption that the sequestering of cadmium in the mucosa
resembles the handling of iron [20] which, when taken up by the mucosa in
excess of bodily needs, is believed to bind to ferritin and subsequently to be
excreted upon desquamation of the epithelium. This hypothesis, though
attractive, is not fully supported by available evidence. Thus, Sasser and
Jarboe [19] and Foulkes and Stemmer (unpublished observation) observed
accumulation of cadmium in the deeper mucosal layers which do not readily
participate in the rapid turnover of epithelial cells. In addition, the binding
of cadmium observed in the present study was pronounced even in control
animals, although these contained only small levels of MT in the mucosa.
Clearly, binding of cadmium is not restricted to MT, a conclusion which
raises questions about the proposed primary role of this compound in the
sequestration of the metal.
A similar conclusion can be drawn from the fact that the great increase
35
-------
in MT seen in exposed animals was not accompanied by either significant
increases in cadmium uptake or increases in its retention in the mucosa.
Indeed, as can be deduced from Table I, an increase in the Cd-binding
capacity of the MT fraction from 28 to 100 nmol/g mucosa exerted no
effect on the transmural transport of cadmium (Tables II and IV). Even a
partial involvement of the relatively very large Cd-binding capacity in the
mucosal MT fraction should have been reflected in some change in the
very much smaller amounts of cadmium transported across the tissue. The
small increase in accumulation of 109Cd in mucosa of pretreated rats (Table II)
may perhaps be explained by alterations in the permeability of the mem-
branes [20,23].
Different authors found that cadmium in the cytosol of intestinal tissue
is mainly bound to MT [7—10]. We observed that cadmium, freshly absorbed
by control animals, reacts primarily with high molecular weight proteins
rather than with MT (Table III), although the mucosa actually contained
significant amounts of the protein (Table I). Presence of MT in normal
animals has previously been reported by other authors [8,20], and pre-
sumably reflects normal background levels induced by zinc in the diet.
Our calculations show that cadmium binding capacity of MT, even in control
animals (Table I), greatly exceeds actual binding of newly absorbed cadmium
(Tables II and III). Kotsonis and Klaassen [11] recently reported similar
findings in experiments involving measurement of Cd-binding capacity of
MT in liver and kidneys. Apparently, even though MT may not be fully
saturated with metal, a variety of ligands can and do compete for freshly
absorbed cadmium. Obviously, a short segment of jejunum in the surgically
prepared animal is not fully representative of the intestine in an intact rat.
Nevertheless, our results clearly show that MT in the mucosa does not serve
as a primary determinant of cadmium absorption, at least under present
experimental conditions.
ACKNOWLEDGEMENTS
This work was supported in part by NIH grant ES-01462 and by US EPA
grant R-805840010. The authors are grateful to Mrs. Sheila Blanck and
Mrs. Mirjana Kello for their valuable technical assistance.
REFERENCES
1 L. Friberg, M. Piscator, G.F. Nordberg and T. Kjellstrom, Cadmium in the Environ-
ment, CRC Press, Cleveland, Ohio, 1974.
2 G.F. Nordberg, Effects and Dose-Response Relationship of Toxic Metals, Elsevier,
Amsterdam, 1976.
3 I. Bremner, Cadmium toxicity, in G.H. Bourne, (Ed.), Human and Animal Nutrition.
World Review of Nutrition and Dietetics, Vo. 32, S. Karger Publ., Basel, 1978, p. 165.
4 B.A. Fowler, Environ. Health Perspect., 28 (1979) 297.
5 S.I. Koo, C.S. Fullmer and R.H. Wasserman, J. Nutr., 108 (1978) 1812.
6 J. McGivern and J. Mason, J. Comp. Pathol., 89 (1979) 293.
7 K. Tanaka, K. Nishiguchi and K. Okahara, J. Hyg. Chem., 19 (1973) 202.
36
-------
8 R.W. Chen and H.E. Ganther, Environ. Physiol. Biochem., 5 (1975) 378.
9 C. Sugawara and N. Sugawara, Bull. Environ. Con tarn. Toxicol., 14 (1975) 159.
10 M.G. Cherian, R.A. Goyer and L.S. Valberg, J. Toxicol. Environ. Health, 4 (1978)
861.
11 F.N. Kotsonis and C.D. Klaassen, Toxicol. Appl. Pharmacol., 46 (1978) 39.
12 K.S. Squibb, R.J. Cousins, B.L. Silbonand S. Levin,Exp.Mol.Pathol.,25 (1976) 163.
13 E.C. Foulkes, J. Environ. Pathol. Toxicol., 1979, in press.
14 E.C. Foulkes and C. Voner, Fed. Proc. 1980, in press.
15 F.N. Kotsonis and C.D. Klaassen, Toxicol. Appl. Pharmacol., 42 (1977) 583.
16 D.W. Yeager, J. Cholak and E.W. Henderson, Environ. Sci. Technol., 5 (1971) 1020.
17 B.M. Sahagian, I. Harding-Barlow and H.M. Perry Jr., J. Nutr., 93 (1967) 291.
18 D. Kello and K. Kostial, Toxicol. Appl. Pharmacol., 40 (1977) 277.
19 L.B. Sasser and G.E. Jarboe, Toxicol. Appl. Pharmacol., 41 (1977) 423.
20 L.S. Valberg, J. Sorbie and D.L. Hamilton, Am. J. Physiol., 231 (1976) 462.
21 D. Bhattacharjee, T.K. Shetty and K. Sundaram, Indian J. Exp. Biol., 17 (1979) 74.
22 T. Taguchi and S. Suzuki, Jpn. J. Hyg., 33 (1978) 467.
23 L.S. Valberg, J. Haist, M.G. Cherian, L. Delaquerriere-Richardson and R.A. Goyer,
J. Toxicol. Environ. Health, 2 (1977) 963.
37
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Paper C
(Submission Draft)
RELATIONSHIP BETWEEN CADMIUM AND ZINC ABSORPTION BY RAT JEJUNUM
Naoki Sugawara and Ernest C. Foulkes
Departments of Environmental Health and Physiology
University of Cincinnati Medical Center
Cincinnati, Ohio 45267 (U.S.A.)
•Present Address: Department of Public Health
Sapporo Medical College
S-l, W-17, Sapporo, Japan 060
Send Correspondence to: Dr. E. C. Foulkes
Department of Environmental Health
University of Cincinnati Medical Center
3223 Eden Avenue
Cincinnati, Ohio 45267 (U.S.A.)
38
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- 2 -
SUMMARY
Interaction between Cd and Zn during their absorption from
the intestinal lumen was studied in the rat jejunum perfused in
situ. Cadmium and Zn depress each other's transport out of the
lumen in an apparently competitive manner. The two metals presum-
ably share, in part, a common absorption mechanism. However, im-
portant quantitative differences are seen in the second step of
absorption, i.e. transfer from mucosa into the body. Overall ab-
sorption of Cd and Zn may thus be mediated by differing mechanisms.
INTRODUCTION
Interaction between Cd and Zn has been extensively documented
in many tissues (1) including the intestine (2-4). However, the
mechanism of absorption of heavy metals is complex, and the nature
of Cd-Zn interaction is not understood. The complexity derives in
part from the fact that more than one step is clearly involved in
heavy metal absorption. The first of these (step I) represents up-
take of metals from lumen into the mucosa, while step II consists
of their further transfer into the body (5). The observation that
Zn depresses step I of Cd transport (2) raises the question to what
extent these two metals share a common absorption mechanism. It is
the purpose of the present paper to explore this question.
MATERIALS AND METHODS
Male Sprague Dawley strain rats aged 7-9 weeks were obtained
from Charles River Co., and maintained on Purina rat chow. The
rats were fasted for 24 hours prior to the experiment. Anesthesia
and surgical procedures have been previously described in detail
(2, 5). Briefly, the lumen of a segment of proximal jejunum
39
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- 3 -
approximately 12 cm in length was perfused in situ. The perfusate
consisted of 2.4 ml saline solution containing 5 mM glucose and
was recirculated at a rate of 0.8 ml/min.
For measuring uptake of Cd, cadmium chloride labelled with
109Cd (0.13 uCi) and 0.63 vCi 3H-polyethylene glycol as volume
marker were added to the recirculating system. Disappearance of
Zn from the lumen was determined in presence of ZnCl- labelled
with 652n (0.625 yCi) and 0.02 uCi 14C-polyethylene glycol. Trans-
port of Cd and Zn from the lumen was calculated from changes in
109Cd/3H and 65Zn/14C ratios as determined on a Packard liquid
scintillation spectrometer with automatic external standards.
Measurement of step II in Zn absorption followed essentially
the technique previously described for Cd (5). Jejunum was per-
fused with 0.05 mM Zn solution for 20 min. At that time, final
perfusate and washings were collected to determine on a gamma
counter the amount of Zn removed by the intestine. This amount
was compared with the Zn retained in the intestinal wall. As in
the case of Cd, a deficit in tissue recovery was equated to trans-
fer from tissue into body, i.e. step II.
RESULTS
As previously shown (2), Zn inhibits step I of Cd absorption.
This is confirmed by the lines of best fit in Figure 1 which reports
results from studies at three different Cd concentrations in pre-
sence of three inhibitory levels of Zn. The large variance in the
results reflects the previously noted variance between animals (2).
In spite of this, the figure shows that with increasing concentra-
tion, Zn increases the slope of the double reciprocal lines without
significantly altering their small intercept on the ordinate. Such
40
-------
- 4 -
a result is compatible with competition between Cd and Zn.
Figure 2 shows similarly that Cd inhibits Zn transport, again
in a manner suggesting competitive interaction.
Table I summarizes experiments on the rates of steps I and
II of Zn transport. Unlike the essentially full recovery in the
tissue observed for Cd after 25 min perfusion (5), a large fraction
of the Zn removed from the lumen in 20 min was transported further
during that period. In 10 studies, a mean recovery of Zn in tis-
sue of only 36 ± 7% of that removed from the lumen could be obtained,
i.e. the rate of step II of Zn transport approximates 64% of that of
step I; the corresponding value for Cd, in contrast, is only 1-2%
(5). A relatively rapid movement of Zn from gut into body has re-
peatedly been reported in the past (6, 8).
DISCUSSION
The finding that Cd and Zn may compete with one another at
the level of step I in their absorption suggests that they share,
at least in part, a common uptake mechanism, and that a common
binding site may be involved in their transport. It is worth
recalling that common mechanisms for Cd and Zn transport have
been previously suggested (7).
While a common mechanism can therefore explain transport of
Cd and Zn out of the lumen, their further movement into the body
(step II) is quantitatively very different. Thus, Cd is tightly
bound in the mucosa (3), whereas Zn rapidly enters the body (see
Table I). The difference in step II indicates that the overall
absorption processes of the two metals are not identical.
The question whether metallothionein is involved in the
common pathway of Cd and Zn transport can be answered in the
41
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- 5 -
negative on the basis of our earlier results (5). Similarly,
if low molecular weight ligands are involved in Zn transport,
then the inability of Cd to react with such a compound, as re-
ported by Sugawara, et al. (9), suggests that the ligands also
cannot form a common element in the uptakes of Cd and Zn.
ACKNOWLEDGMENTS
We are indebted to Mrs. Cathleen Voner for her valuable help.
This work was supported by EPA grant R-805840010 and NIH grant
ES-00159.
REFERENCES
1. M. Webb, Protection by zinc against cadmium toxicity, Biochem.
Pharmacol., 21 (1972) 2767.
2. E. C. Foulkes, Some determinants of intestinal cadmium trans-
port in the rat, J. Environ. Pathol. Toxicol., 3 (1979) 471.
3. K. R. Roberts, W. J. Miller, P. E. Stake, R. P. Grentry and
M. W. Neathery, High dietary cadmium on zinc absorption and
metabolism in calves fed for comparable nitrogen balances,
Proc. Soc. Exp. Biol. Med., 144 (1973) 906.
4. B. M. Sahagian, I. Harding-Barlow and H. M. Perry, Jr.,
Uptakes of zinc, manganese, cadmium and mercury by intact
strips of rat intestine. J. Nutr., 90 (1966) 259.
5. D. Kello, N. Sugawara, C. Voner and E. C. Foulkes, On the
role of metallothionein in cadmium absorption by rat jejunum
in situ, Toxicol., 14 (1979) 199.
6. D. M. Foster, R. L. Aamodt, R. I. Hekin and M. Herman, Zinc
metabolism in humans: a kinetic model, Am. J. Physiol., 237
(1979) R340.
42
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- 6 -
7. B. V. Kingsley and J. M. Frazier, Cadmium transport in iso-
lated perfused rat liver: Zn-Cd competition. Am. J. Physiol.,
236 (1979) C139.
8. K. T. Smith, R. J. Cousins, B. L. Silbon and M. L. Failla,
Zinc absorption and metabolism by isolated vascularly perfused
rat intestine, J. Nutr., 108 (1978) 1849.
9. N. Sugawara, C. Sugawara and H. Miyake, Effect of parenteral
cadmium on zinc in the liver and duodenum, Toxicol. letters,
2 (1978) 339.
TAB&E I
STEPS I AND II IN ZN TRANSPORT
% of initial Zn content
in perfusate (Mean ± SD)
Removed from perfusate (step I) 52.3-7.4
Recovered from intestinal wall 18.8 ± 2.4
Deficit (step II) 33.5 ± 7.4
Step II/I (%) 64.0 i 7.0
Data were obtained from 10 rats. Each jejunum was perfused for
20 minutes with 2.4 ml perfusate containing 120 nmoles Zn,
43
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Figure 1. Effect of Zn on Cd uptake. Cd was transported out
of the lumen at a constant exponential rate for 30 mln, from
which the mean initial rate (M * SB) of transport was calculated
in nmole Cd/g tissue/min for 4 to 6 rats.
Zn&omM
1.8
10 20 30 40 SO
44
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Figure 2. Effect of Cd on Zn uptake. Zn was transported out
of the lumen at a constant exponential rate for 6 min, from
which the mean initial rate (M i SE) of transport was calcu-
lated in nmole Zn/g tissue/man for 3 to 10 rats.
0.3-
0.2-
o
?
N
a
£
o
0.1-
Cd 1.0 mM
Cd0.5mM
NoCd
I i 1 1 1 1 1 1 1 1—
24 6 8 10 12 14 16 18 20
Zn (mM)"1
45
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Abstract A
PHYSIOLOGY
STEPS IN CADMIUM TRANSPORT BY RAT JEJUNUM IN SITU. E.G.
Foulkes and C. Voner, Depts. Environ. Health & Physio!.,
Univ. of Cincinnati Med. Center, Cincinnati, Oh. 45267.
The fact has been repeatedly observed that uptake of Cd
from the intestinal lumen (step #1 of Cd transport) is much
faster than its further movement into the body (step #2).
Less well documented is a quantitative comparison of steps 1
and 2 in vivo, at low Cd concentrations approaching those
which might be encountered in dietary exposure. The mechanism
mediating step #1 at these low levels becomes saturated at the
higher concentrations used in much of the earlier work (J.
Env. Path. Toxicol., in press). At a concentration in luminal
perfusate of 20 uM Cd, step #1 proceeds at 2.0 ± 0.7 (SD, n =
17) nmoles/g fresh weight jejunum/min. Immediately after 25
minutes' perfusion essentially all Cd removed from the lumen
could be recovered from the intestinal mucosa. If 5 hours
were allowed to elapse before excision of the intestine, a
significant fraction of Cd taken up during perfusion had moved
beyond the intestine. This deficit may be attributed to step
#2, and was incurred at a mean rate of 0.03 t .02 nmol/g/min.
The ratio of steps 2/1 equalled 1.7 i 1.0%. We have shown
elsewhere that the long retention of Cd in the intestine is
not a function of endogenous metallothionein levels (Toxicol.,
in press). These could be raised by 360% above control levels
during exposure to 0.5 mM Cd in drinking water, without sig-
nificantly altering steps 1 or II. The factors determining Cd
retention remain undefined. (Supported by EPA grant R-805840-
010).
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Abstract B
MODIFICATION OF JEJUNAL Zn AND Cd TRANSPORT IN
RATS BY GLUCOCORTICOID NOT CORRELATED WITH METAL-
LOTHIONEIN SYNTHESIS. R.F. Bonewitz and B.C.
Foulkes (Spon. P.B. Hammond) Dept. Env. Health,
Univ. Cincinnati Med. Ctr., Cincinnati, OH 45267.
Hepatic and intestinal metabolism of Zn, Cd,
and several other heavy metals may involve metal-
lothionein (MT). Synthesis of MT is induced in
many tissues by these metals. Furthermore, glu-
cocorticoids have been found to induce MT synthe-
sis in some cultured cells and in liver of adre-
nalectomized (ADX) rats. We investigated effects
of dexamethasone (dex) (2.0 mg/kg i.p.) on re-
moval of 65Zn (20-100 pM in 5 mM glucose-saline)
from the lumen of jejunal segments perfused in
situ, and on induction of mucosal MT. In con-
trols not given dex, uptake was first order (k =
2.18 ± 0.53 x 10~2 min-ignT1, 95% C.I.). In con-
trast, in animals given dex 7 h (but not 0, 1, 4,
or 12 h) before assay, uptake was biphasic with
a rapid component (k >6.84 ± 0.84 x 10~2 min"1
gm~l) which was completed in -3 min, and a slower
component not significantly different from con-
trols. Results were not affected by adrenalec-
tomy. Dex had a qualitatively similar effect on
first-order ^O^Cd uptake. Dex and 35g-cystine
(15.9 pCi, i.p.) were administered 7 and 3 h, re-
spectively, prior to sacrifice, and mucosal cell
cytosol was chromatographed on Sephadex G-75.
In neither dex-treated nor untreated animals was
significant 35S incorporated into MT. Control
studies confirmed ^s^^eHea MT synthesis in
mucosa of Zn2+_TREATED (150 pmol Zn2+/kg, i.p.)
rats and in liver of dex-treated ADX rats. The
results indicate that dex-modifies intestinal Zn
transport, apparently by creating a second com-
partment, possibly a sink, accessible to luminal
Zn2 . The results further show that MT is not
detectably induced by dex and is therefore un-
likely to be involved in this transport effect.
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Abstract C
1'MYSIOLOCiY
INHIBITION OF JEJUNAL Cd ABSORPTION IN THE RAT BY BILE SALTS.
C. Voner* and B.C. Foulkes, Depts. of Env. Health & Physiol.,
Univ. of Cincinnati Med. Cen., Cincinnati, OH 45267.
Composition of food is well-known to influence intestinal
absorption of various heavy metals; in addition, endogenous
factors also may play a role. We report here effects of bile
on removal of Cd from the lumen of the proximal jejunum of
the rat perfused in situ. In these experiments 1.6 ml saline,
containing 5 mM glucose, 20 pM CdCl2 labelled with 0.15 yCi
109c(jf an
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Abstract D
PHYSIOLOGY
INTESTINAL TRANSPORT OF CADMIUM IN NEWBORN RATS. D.R.
Johnson, E.G. Foulkes and L. Leon*, Depts. Env. Health and
Physiol., Univ. Cincinnati Med. Cen., Cincinnati, OH 45267.
Considerable attention has been given to absorption of Cd
from the small intestine of adult rats. However, absorption
of metals, including Cd, is greater in newborn rats than
adults. The present studies were conducted to investigate in-
testinal Cd transport in the newborn rat. Transport in situ
from duodenum and jejunum of 14 day old rats was measured
thirty minutes after placing 0.1 ml 20 pM CdCl2 labelled with
0.02yCi 109cd into the intestinal lumen. Duodenal uptake from
lumen to mucosal cell and absorption from cell to blood were
2.4 ± 0.6%/min/O.lg and 0.5 ± 0.4%/min/O.lg (S.D., n=5), re-
spectively. Uptake and absorption from jejunum were 1.0±0.3
and 0.20 i 0.05%/min/O.lg, respectively. Although duodenal
transport was greater than jejunal, the absorption to uptake
ratio in these two segments was equal. Both rate of trans-
port and ratio of absorption to uptake are greater in the new-
born than in the adult. Unlike in adults, neither chronic nor
acute administration of iron to pups altered Cd uptake from
jejunum. Cd uptake in the presence of 200 yM FeSO4 was 0.9 ±
0.2%/min/O.lg. Feeding of milk supplemented with 200 uM FeSO4
for 7 days (day 7-14) did not alter the rate of Cd uptake
even though mucosal iron level was doubled. These results
suggest that the mechanism of intestinal Cd transport in the
newborn rat differs quantitatively and qualitatively from
that in the adult. (Supported in part by EPA grant
R-805840010 and NIH grant ES-00159)
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Abstract E
COMPARISON OF MUCOSAL UPTAKE AND TRANSMURAL TRANSPORT OF Zn,
Cu, AND Cd IN RAT JEJUNUM. Roland F. Bonewitz, Jr.*, Cathleen
Voner* and E.G. Foulkes, Depts. Env. Health & Physiol., Univ.
Cincinnati Med. Ctr., Cincinnati, OH 45267.
Intestinal absorption is a major route for the assimilation
of both nutritionally essential and toxic metals by the body.
The low net fractional absorption of Cd suggests however that
the intestine may possess a mechanism for discriminating be-
tween Cd and essential metals. Mucosal (M) uptake and trans-
mural transport of Zn, Cu, and Cd were compared under identi-
cal conditions in segments of the adult rat jejunum in situ.
20 pM metal salt + tracer in 5mM glucose-O.ISM NaCl was re-
circulated through the lumen (L) . L-*M transfer (Step I) of
all metals was 1-2 nmol min~lg~l. However, the further trans-
port into the body (Step II) was higher for Zn and Cu (48%
and 47%, respectively, of Step I) than for Cd (<2%). Discrim-
ination against Cd thus occurs past the level of M uptake.
As the segment was isolated and perfused with a medium lacking
added ligands, and as tissue through which perfusate was not
recirculated suffered no decrease in the rate of Step I, it is
unlikely that specific metal binding ligands accumulated in L
to mediate metal absorption. Although some ligands present in
food or secreted into the intestine may enhance metal absorp-
tion through competition with food constituents for metal
binding, such ligands are apparently not specific and obliga-
tory components of the uptake mechanism. (Supported by NIH
grants ES00159 and ES07073 and EPA grant R805840)
50
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