v>EPA
United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Research Triangle Park NC 27711
Research and Development
EPA-600/S1-82-008 Nov. 1982
Project Summary
Effects of Heavy Metals on the
Differentiation of Metabolic
Pathways in the CNS
John J. O'Neill, George van Rossum, and Leon Salganicoff
The effects of lead and organotin on
brain and spinal cord, liver or kidney in
the developing or adult Sprague-
Dawley and Wistar rat strains were
investigated. The effects were pri-
marily ascertained in vitro following
lead exposure. Two concentrations of
lead were present in drinking water:
200 ppm or 600 ppm as lead in lead
acetate. Blood and tissue lead levels
were either measured by anodic
stripping voltammetry or by atomic
absorption spectrophotometry (Per-
kin Elmer Model 303) following
digestion in nitric acid.
Tissue slices prepared from the
kidney cortex of animals exposed to
200 or 600 ppm lead showed that
respiration rates were 15% less and
ATP contents 30% less than those for
control animals. Lead treatment in
vivo had no significant effect on the
ion and water contents of kidney
cortex, in contrast with in vitro results.
The ability of kidney cortex slices to
restore ion and water contents after
incubation at 1 °C was also unim-
paired after in vivo treatment. Longer
in vivo exposure might be necessary
for effects to become evident.
Lead sensitivity of carbonic anhy-
drase depended upon the organ from
which it was derived. At a lead
concentration of 10 ppm, there was a
50% fall (5.11± 1.46 units to 2.54 ±
0.75 units) in myelin carbonic anhy-
draseanda 44% fall in kidney carbonic
anhydrase. The enzyme present in
nerve endings was particularly sen-
sitive and showed a drop from 20 units
± 4 to 2.3 ± 1.7 units (to 12% of
normal). The nerve ending enzyme
was sensitive even at 1 ppm. In vivo
administration of lead (600 ppm) was
carried out over a 3-month period.
Brain myelin, synaptosomal and kid-
ney microsomal carbonic anhydrases
were measured. Consistent with in
vitro measurements, myelin and sy-
naptosomal activity were decreased
67% and 57%, respectively, whereas
kidney microsomal carbonic anhy-
drase showed a relatively small change
(19%) from 9.46 units to 7.6 units
following 12 weeks exposure.
This Project Summary was devel-
oped by EPA's Health Effects Research
Laboratory, Research Triangle Park.
NC, to announce key findings of the
research project that is fully docu-
mented in a separate report of the
same title (see Project Report ordering
information at back).
Introduction
The neurotoxic effects of lead, mer-
cury, and organotin are well established
at high dose levels. The controversy and
source of concern is not with high
concentrations but rather with levels
that do not cause overt symptoms This
appears to be especially true in the case
of lead intoxication where the symptons
are not well established. The effects on
adults are qualitatively different from
those on young children and the
developing nervous system.
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This report describes research con-
ducted on central and peripheral effects
of controlled exposure of pregnant and
nonpregnant albino rats to lead and
tributyl-tin at levels of 200 or 600 ppm.
The research was divided into three
group projects. Project 1 focused on the
influence of lead on the developing and
adult central nervous system. Consid-
erable emphasis was placed upon
monitoring the development in the
spinal cord morphologically, by electron
microscopy, and neurochemically.
Although the fetal brain, especially the
cerebellum, appears to be most sus-
ceptible to lead exposure, the spinal
cord, which undergoes development
earlier, might be the first fetal organ to
manifest an effect as a result of
exposure in utereo.
Current literature suggests that lead
and organotm compounds might have
toxic effects on the energy production
of cells and on the regulation of
cellular ionic composition and tissue
volume. Since nephrotoxicity is a
prominent manifestation of lead poisoning,
the use of kidney as well as hepatic
tissue in these studies is especially
relevant in project 2
Project 3 dealt with the effects of lead
on the zinc metallo enzyme, carbonic
anhydrase, which appears to assist in
maintaining I-T ion and electrolyte
balance in kidney tissue. Although
carbonic anhydrase is present in the
intralamina of myelin in all nerve tissue
and thought to be involved in controlling
cerebral and spinal tissue edema, there
is no general agreement about this.
Because lead and some organotm
compounds, especially triethyl-tin,
cause cerebral edema, some attention
was devoted to the forms of the
enzyme which occur in brain and kidney.
Tissue lead has routinely been
measured by flame absorption spec-
trophotometry. This may requirethe use
of the major portion of a sample by
standard instrumentation or miniatur-
ization of the system The lack of
portability of such equipment further
limits its applicability to field conditions.
In search of alternative methods we
elected to study blood, brain, kidney,
and liver tissue lead levels by Anodic
Stripping Voltammetry. The basic unit is
relatively inexpensive, portable, and
requires a relatively small (100 micro-
liter) sample size. The method is highly
versatile and can be employed in
measuring such metal ions as lead, tin,
cadmium, iron, or its chelates.
Conclusions
The results of this study show that
lead exhibits energy metabolism in
kidney cortex and specifically alters K+-
ion transport and CA*2-ion distribution
in the cells, suggesting that effects on
cellular energetics and/or ion transport
may play a role in the nephrotoxic
manifestations of lead poisoning
In contrast to findings of others
regarding delayed synaptic develop-
ment in the cerebral cortex of rat pups
exposed to lead in utereo, no significant
effect was observed on synaptic devel-
opment in the thoracolumbar region of
the developing spinal cord. Inability to
demonstrate a gross morphologic effect
may be due to the lack of specificity of
the method. Although some features of
the presynaptic and postsynaptic ap-
paratus can be discerned, the method
does not permit a study of the receptors
known to be present. When the potent
muscarinic binding drug, Quinuclidinyl
benzilate-3H (QNB-3), which has high
affinity for muscarinic receptors (Katt = 10~
13M), was employed, lead (600 ppm) in
drinking water consumed by the preg-
nant nursing dam caused a slight delay
in the appearance of muscarinic binding
sites in spinal cord during fetal andearly
neonatal development
Lead poisoning can produce a devas-
tating encephalopathy in children. The
symptoms are characterized by in-
creased mtracranial pressure (ICP),
convulsion and coma The metabolic
basis is not understood but the disrup-
tion of fluid and electrolyte metabolism
undoubtedly is involved in the develop-
ment of edema responsible for the ICP.
In studies in which liver tissue was
exposed to tributyl-tm, this compound,
which has a weak inhibitory effect on
energy metabolism, was discovered to
possess a hitherto unknown inhibiting
effect on cell volume regulation in the
liver. We suggest that this latter effect
may underlie the previously observed
edematous changes in the central
nervous system of animals poisoned by
trialkyl-tm compounds and possibly by
lead as well.
Experiments in which rats were
administered lead (600 ppm) in drinking
water for several weeks showed that
exposure to lead reducedthe respiratory
activity by 15%. When intact cells of
kidney cortex were incubated as a slice
preparation, it was determined that lead
had reduced the ability of the kidney to
maintain ATP levels by 30% This is
consistent with other findings on
mitochondria isolated from kidneys, and
our results show these findings to
relevant to intact cells. Further, wl
more lead was added in vitro to
slices, an extra decrease occur
in respiration and ATP levels. The mini
levels, however, did not differ fr
those obtained by directly treating
slices from control rats with leac
vitro. This finding indicates that in \
and in vitro effects of lead expos
were not additive and were probe
acting at the same site In view of
rapidity of action in vitro, we believe!
lead acted directly as an inhibitor
uncoupler of mitochondnal oxida
phosphorylation Similar findings
brain slices from rats treated for 20 c
at 200 and 600 ppm lead also occur
help to explain effects on acetylcho
synthesis.
The marked inhibitory effect of I
on oxidative phosphorylation v
observed through in vitro studies
mitochondria isolated from kidi
cortices of control animals. Sim
results obtained using rat brain m
chondna from untreated anim
showed brain mitochondria to be hie
sensitive to lead ions. These result
adult animals make it improbable 1
lead exposure in vivo reduces the s
thesis of cytochromes (through esl
hshed effects on heme synthesis) si
ciently to produce a marked effect
cell respiration. Rather, we believe 1
lead had a direct effect on respirai
metabolism, the precise nature of wl-
is being further investigated.
Lead administered in vivo affec
neither the water and ionic (K+, Na+,
Ca2+, Mg2+) composition of the kid
cortex nor the ability of slices of
tissue to transport any of these ion
water Thus, the reduction in ent
metabolism through lead treatmen
vivo was insufficient to limit the su[
of ATP required for these homeost
mechanisms. Further, the levels of I
attained in the tissue were msuffic
to inhibit the transport mechani;
directly
Currently, rats are being treated v
lead for longer periods (up to six mon
to determine whether any additic
effects are produced.
Lead added to kidney-cortex slice
vitro at 50-200 /jM had two effects
ion exchange in vitro, lead reduced
active accumulation of K+ by a m
mum of 30% and had no signific
effect on the net extrusion of Na+,
Ca2+ or water or on the content of N
in the tissue The partial inhibition c
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accumulation becomes apparent only
after 30 mm. of incubation.
The lead concentration (200/uM) that
inhibited K+ accumulation by 30% also
inhibited respiration and reduced ATP
content by 25 to 30%. ATP reduction
and K+ transport inhibition occurred in
close succession. Thus, circumstantial
evidence suggests that by reducing the
availability of ATP, lead caused the
inhibition of ion transport. However, the
fact that lead had no inhibiting effect on
Na+ extrusion was rather unusual The
known inhibitory effect of lead on Na+
and K+- dependent adenosme tnphos-
phatase (Na-K-ATPase) of the kidney
and other cells could have accounted
directly for the transport inhibition To
distinguish between these two possibil-
ities, kidney cortex slices were titrated
with a specific inhibitor of mitrochon-
drial respiration, antimycm A, and with
an inhibitor of the Na-K-ATPase,
ouabam This permitted us to compare
the effects of these agents when K*
transport was inhibited by 30% with the
effects of Pb2+ given the same reduction
of K+ transport. However, the results of
the comparison were equivocal. When
antimycm A was the inhibitor, 30%
inhibition of K+ transport was accom-
panied by a similar decrease in respir-
ation and ATP contents but, unlike the
results obtained with lead, Na+ extru-
sion was also inhibited by antimycin H
When ouabam inhibited K+transport by
30%, the extrusion of Na+ was not
inhibited, whereas respiration de-
creased by 25%, results similar to those
with Pb2+. ATP was unchanged by
ouabam. The biggest difficulty using
this comparison to explain the results
with Pb2+ is that a 30% reduction of ATP
levels produced by lead failed to inhibit
Na+ extrusion The temporal coinci-
dence of inhibited K+ transport and
depressed ATP levels suggests that the
latter accounts for the former, but it is
clear that further experiments are
required to elucidate the mechanism by
which Pb2+inhibits K+ accumulation
The second effect of lead in vitro on
ion exchange was a reduction in entry of
Ca2+ into the slices while metabolic
activity was greatly reduced, that is, to
1 °C. Examination of the Ca2+ content of
mitochondria isolated from the slices
revealed that lead prevented the entry of
Ca2+ into these organelles, apparently
by inhibiting a residual, calcium-
accumulating activity of the mitochon-
dria which persisted at 1°C However,
this did not account for the entire
reduction in the entry of Ca2+ into the
whole slice at this temperature. Studies
of 45Ca influx and efflux in the slices
were performed in order to obtain more
information on possible fractions of
tissue Ca2+ that might be affected by
lead We found that lead reduced the
quantity of Ca2+ which was inexchange-
able with 45Ca in the medium and the
quantity of Ca2+ in the most slowly
exchanging component of the three into
which the exchangeable Ca2+ could be
divided, probably the mitochondnal
Ca2+ This result complements our
findings with the directly measured Ca2+
on mitochondria The mexchangeable
portion may represent Ca2+ which is so
tightly bound to nonmitochondnal
tissue structures that it cannot be
displaced by 45Ca but which may be
displaceable by Pb2+. This latter fraction
would then account for the nonmit-
ochondnal portion of the Ca2+ uptake
that was prevented by lead in the other
experiments
Initially, the laboratory investigated
the pathway of metabolism leading from
glucose to acetylcholine syntheses. It is
well established that brain and nervous
tissue cannot synthesize cholme and
must depend on an exogenous source
for supply. Equally well established is
the fact that glycolysis supplies pyru-
vate as a precusor for Acetyl-CoA and
ultimately acetylcholine formation. The
problem which has confronted neur-
ochemists for two decades is that
pyruvate forms Acetyl-CoA mtramito-
chondnally. As such it remains within
the mitochondrion, because no known
mechanism exists for its transport to the
cytoplasm, the site of acetylcholine
synthesis
We recently demonstrated that citric
acid formed intramitochondnally leaves
and enters the cytosol There it is
cleaved by an enzyme, ATP. citrate lyase
to reform Acetyl-CoA and thence to
form acetylcholine We have deter-
mined that lead at either 200 ppm or
600 ppm m drinking water results in the
inhibition of acetylcholine synthesis
We find that at 600 ppm, the conversion
of glucose to lactate and citrate de-
creases markedly Hence, lead interfer-
ence with glycolysis and the tn-carboxy-
lic cycle (TCA) may interfere with energy
metabolism and affect neurotransmitter
function.
The mechanism by which acetylcho-
line is derived from glucose and pyruvate
has been intimately linked to brain
mitochondria, since the enzyme which
converts pyruvate to acetyl-CoA (Pyruvate
dehydrogenase E.G. 1.2 4.1) exists
exclusively mtramitochondrially. Acetyl-
CoA thus formed must be converted to a
permeate anion in order to cross the
mitochondnal membrane to the cytosol.
The evidence to date strongly supports
citrate as that anion; when citrate
transport is interfered with, acetoacetate
fulfills this role The approach used to
test this hypothesis is based on the
observation that glucose labeled in the
carbon-6 position with either tritium
(3H) or carbon-14 (14C) is converted to
pyruvate without loss of either isotope.
However, when acetyl-CoA is condensed
with oxaloacetate (OAA), a hydrogen is
lost to OAA which is exchanged and lost
to water in the formation of citrate The
probability that the hydrogen lost would
be tritium, barring isotope discrimination,
would be one-third and that would
reduce the 3H/14C ratio to 2/3 (0 67)
This ratio remains constant only in
citrate carbons 4 and 5 and is drastically
reduced in other molecules with each
turn of the cycle within mitochondria
Acetyl-CoA arising from citrate (C-4~5) in
the cytosol gives rise to acetylcholine
with the same 3H/14C ratio (0 67), direct
conversion from glucose through acetate
would require the ratio to be one.
Tnbutyl-tm was studied for its effects
in vitro on rat liver, since most bio-
chemical studies of the organotm
compounds have concentrated on the
mitochondria isolated from this tissue
Despite the reported high sensitivity of
several aspects of oxidative metabolism
in isolated mitochondria to tributyl-tm,
we needed a concentration of 1 mM to
produce a 20% decrease in respiration
and ATP content in si ices of liver/>? vitro.
At this concentration, the net transport
of K+, Na+, Cl~ and water declined
substantially.
Much lower concentrations of tn-
butyl-tin (1 -1 00 pM), however, signifi-
cantly reduced the extrusion of Cl~ from
the liver slices, an effect accompanied
by less marked inhibitions of Na* and
water extrusion. But K+ accumulation
was completely resistant to these
concentrations of the tin compound. We
conclude that tributyl-tin at 1-100/uM
was probably acting as a specific
inhibitor of the mechanism for net
extrusion of Na+ and Cl~, which is not
coupled to K+ uptake and which is partly
responsible for the control of cell
volume This effect maybe related tothe
known effect of tributyl-tm infacilitating
CI~/OH exchanges through biological
membranes. Higher concentrations (1
mM) also affect mitochondrial energy
provision and thus inhibit the ouabam-
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sensitive transport of Na+ and K+
through the paucity of ATP If tributyl-tm
is indeed a specific inhibitor of the Na+
and Cl~ transport not coupled to K+, it
may be a most useful agent with which
to study the properties of this system
and hence to study volume control Its
action as an inhibitor of volume control
may well account for the finding of
manifestations of edema in the central
nervous system of animals intoxicated
with trialkyl-tm compounds
Recommendations
The studies on lead have extended
knowledge of the mechanism of action
of this heavy metal and may be particu-
larly relevant to the means by which it
causes nephrotoxicity We have indi-
cated that lead acts on energy provision
in vitro by a mechanism similar to that
by which it acts in vivo Lead causes
alterations of K+ homeostasis in the
kidney cells and also affects the
distribution of Ca2+ in them, especially
with regard to the compartmentahza-
tion between mitochondria and other
cell components Such effects on lon-
homeostasis, especially that of Ca2+,
may well lead to far-reaching altera-
tions of metabolic balance and of
reabsorptive and excretory activities of
the kidney cortex.
In light of the findings on brain
metabolism by us and others and recent
clinical evidence that there is a strong
correlation between lead exposure and
minimal brain damage characterized by
hyperactivity that may interfere with
classroom performance, further studies
on this important aspect are warranted
The social implications of present drug
treatment of hyperactivity should be
reappraised and greater emphasis be
placed on monitoring of safe lead levels
in drinking water to prevent such
neurophysiologic damage in future
young.
The action of tributyl-tin as an
inhibitor of volume-controlling activity,
at concentrations substantially lower
than those at which it affects energy
metabolism in the intact cells, provides
a completely new indication of its
possible mode of action. Such an action,
if it also occurs in nervous tissue, could
readily provide an explanation of the
central nervous toxicity of the trialkyl-
tm compounds. This line of investiga-
tion may well prove to be more profit-
able in elucidating the mechanism of
toxicity of the trialkyl-tm compounds
than studies of energy metabolism and
other functions of isolated mitochondria.
John J. O'Neill, George van Possum, and Leon Salganicoff are with the Temple
University School of Medicine, Philadelphia, PA 19140.
Richard J. Bull is the EPA Project Officer (see below).
The complete report, entitled "Effects of Heavy Metals on the Differentiation of
Metabolic Pathways in the CNS," (Order No. PB 82-249 145; Cost: $9.00,
subject to change) will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield. VA22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
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