EPA-600/1-76-013
March 1976
Environmental Health Effect
METHYL MERCURY AND THE
METABOLIC RESPONSES OF BRAIN TISSUE
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
-------
RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environ-
mental Protection Agency, have been grouped into five series. These five broad
categories were established to facilitate further development and application
of environmental technology. Elimination of traditional grouping was con-
sciously planned to foster technology transfer and a maximum interface in
related fields. The five series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
This report has been assigned to the ENVIRONMENTAL HEALTH EFFECTS
RESEARCH series. This series describes projects and studies relating to the
tolerances of man for unhealthful substances or conditions. This work is gener-
ally assessed from a medical viewpoint, including physiological or psycho-
logical studies. In addition to toxicology and other medical specialities, study
areas include biomedical instrumentation and health research techniques uti-
lizing animals—but always with intended application to human health measures.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
-------
EPA-600/1-76-013
March 1976
METHYL MERCURY AND THE
METABOLIC RESPONSES OF BRAIN TISSUE
by
Richard J. Bull
Water Quality Division
Health Effects Research Laboratory
Cincinnati, Ohio 45268
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
HEALTH EFFECTS RESEARCH LABORATORY
CINCINNATI, OHIO 45268
-------
DISCLAIMER
This report has been reviewed by the Health Effects Research Labora-
tory, U. S. Environmental Protection Agency, and approved for publication.
Mention of trade names or commercial products does not constitute en-
dorsement or recommendation for use.
ii
-------
FOREWORD
Man and his environment must be protected from the adverse effects of
pesticides, radiation, noise and other forms of pollution, and the unwise
management of solid waste. Efforts to protect the environment require a
focus that recognizes the interplay between the components of our physical
environment—air, water, and land. In Cincinnati, the Environmental
Research Center possesses this inultidisciplinary focus through programs
engaged in
• studies on the effects of environmental contaminants on man and
the biosphere, and
• a search for ways to prevent contamination and to recycle valuable
resources.
The Health Effects Research Laboratory conducts studies to identify
environmental contaminants singly or in combination, discern their re-
lationships, and to detect, define, and quantify their health and economic
effects utilizing appropriate clinical, epidemiological, toxicological,
and socio-economic assessment methodologies.
Some years ago, methyl mercury was found to be responsible for the
Minimata Bay incident and, more recently, a similar tragedy in Iraq.
Damage to the central nervous system typifies the effects of methyl mercury,
somewhat in contrast to the effects produced by inorganic salts of mercury.
This study was initiated to determine the minimal ingestion of methyl
mercury required to produce subtle alterations in brain metabolism.
R. J. Garner
Director
Health Effects Research Laboratory
iii
-------
ABSTRACT
Weanling, Sprague-Dawley rats have been exposed to methyl mercuric
chloride (concentrations from 0.01 to 10.0 mg/liter) in their drinking
water. At 10 mg/liter the animals exhibited neurological symptoms typical
of methyl mercury. Also, in this group a considerable decrease in growth
occurred which was associated with a decreased consumption of food.
Responses of the respiratory intermediates to stimulation were found
to be altered in cerebral cortex slices taken from exposed animals.
Effects on tissue pyridine nucleotides predominated. An enhancement of
the rate of pyridine nucleotide reduction by electrical stimulation was
observed at 0.1 mg/liter. This rate progressively decreased at higher
dose levels. Reoxidation of reduced pyridine nucleotide was also inhibited
at 0.1 mg/liter at both 90 and 180 days of exposure.
Potassium stimulated aerobic glycolysis was found to be enhanced in
its initial stages at 0.10 mg/liter of methyl mercuric chloride in the
drinking water but progressively declined at 1.0 and 10 mg/liter. A
close parallel was observed between the time constant of pyridine nucleotide
oxidation following electrical stimulation and the "responsiveness" of the
aerobic glycolytic rate to stimulation by potassium. These results suggest
an initial defect in the oxidation of cytoplasmic NADH which progresses
to loss of metabolic control of cytoplasmic oxidations.
iv
-------
CONTENTS
Page No,
ABSTRACT iv
LIST OF FIGURES vi
ACKNOWLEDGEMENTS vii
SECTIONS
INTRODUCTION 1
CONCLUSIONS 3
RECOMMENDATIONS 5
METHODS 6
RESULTS 9
DISCUSSION 17
REFERENCES 21
-------
LIST OF FIGURES
Page No.
Fig. 1. Dosage levels of methyl mercuric chloride with age
of animals 7
Fig. 2. Effect of methyl mercury on body weight gain 10
Fig. 3. Methyl mercury effect on consumption of food 11
Fig. 4. Methyl mercury effect on pyridine nucleotide reoxi-
dation in electrically-stimulated brain slices 12
Fig. 5. Effect of methyl mercury on pyridine nucleotide
reduction by electrical stimulation 13
Fig. 6. Effect of increasing doses of methyl mercury on the
characteristics of the electrically-induced metabolic
response of tissue pyridine nucleotides 15
Fig. 7. Effect of methyl mercury on potassium-stimulated
glycolytic rate of brain tissue 16
vl
-------
ACKNOWLEDGMENTS
The excellent technical assistance of Mr, S. D. Lutkenhoff is acknow-
ledged. Care of the experimental animals by Ms. M. Whitsell and Mr. N.
Scroggins was very much appreciated.
Mrs. D. White is thanked for her usual outstanding assistance in
preparing this report.
vii
-------
INTRODUCTION
The occurrence of trace elements in drinking water supplies has been
well documented (McCABE, et al., 1970). Many of the metals are present
in raw water and others are,to a greater or lesser extent,from the dis-
tribution systems. Since several toxic metals have been associated with
the development of disease states which take several months to many years
to become clinically evident, it is imperative that their concentrations
be limited to safe levels in drinking water. Although drinking water
standards have been established for many of these chemicals, there are
many questions as to the adequacy of the data available for such purposes,
particularly in relationship to the long-term development of certain types
of disease (INDRAPRASIT, et al., 1974),
Some metals are known to produce alterations in nervous system func-
tion when chronically ingested. The clinical syndromes which arise are
poorly defined and often confused with infectious disease (JAIN, et.al.,
1970). Metals which are known to produce central nervous system toxicity
include lead, manganese, thallium, copper, lithium, organic mercury and
tin. Other metals known to affect the central nervous system when locally
applied, but for which little data exists in terms of the oral route of
exposure are cobalt, cadmium, aluminum and inorganic mercury. Some of
these elements have been implicated or suggested as possible etiological
factors in the development of, or their chronic toxicity resembles,
certain chronic diseases of the nervous system, most notably Parkinson's
disease (COTZIAS, et al., 1971), multiple sclerosis (CAMPBELL, et al.,
1950), amyotrophic lateral sclerosis (PETKAU, et al,, 1974), and hyper-
kinesis (DAVID, et al., 1972).
Detection of marginal damage to the central nervous system is ex-
tremely difficult because of the organ1s diverse function. There are
four basic ways of approaching the problem experimentally: by using the
basic tenets and techniques of neurophysiology, neurochemistry, behavioral
research or neuropathology. The approach of each of these disciplines
has basic advantages and disadvantages. Neuropathology is perhaps the
most widely applicable, but suffers from the fact that it is primarily a
study of structure and lacks sensitivity in terms of function. Behavioral
research addresses itself in a very general way to function, but is most
often criticized on the basis of species specificity of much animal be-
havior. Neurophysiology and neurochemistry both address themselves
specifically, at function (at different levels of biological organization),
but often suffer from being too specific in their approach for wide ap-
plication. Our own efforts have combined certain elements of both neuro-
chemistry and neurophysiology. We have attempted to avoid falling into
-------
the trap of randomly examining very specific functions of the nervous
system and have developed an approach we feel has quite general application
to the study of chemically-induced alterations in the central nervous
system. All questions concerning the effects of heavy metals on brain
function will not be answered with our techniques; it would be presumptious
for us to pretend that they will. However, their application will yield
sensitive measurement of the alterations produced and form a framework
for more specific pursuit of a particular chemicals effect within the
central nervous system.
Because of the central role energy plays in the operation of any
biological system, we have chosen to use parameters of energy and inter-
mediary metabolism to assess damage to brain. The functional activities
of tissues are universally dependent upon a constant availability of
cellular energy. Conversely, each functional activity of a tissue will
have a discrete kinetic input to the overall energy metabolism of that
tissue. The functionalities of a given tissue can be activated by various
means. If the stimulus were selective enough, the intactness of the
function could be indirectly judged by its kinetic impact upon energy
metabolism. Brain tissue is excitable and functions by utilizing ionic
movements to conduct electrical signals. For this reason we employ elec-
trical pulses and ionic stimulation to evoke changes in "functional"
state within the tissue. The change in state is accompanied by increases
in the rate of energy metabolism (McILWAIN, 1966). Of course, in making
measurements of energy metabolism, direct effects of a chemical will also
become evident.
The present report represents the results of our first application
of these techniques. We have examined the effects of methyl mercury,
administered in vivo, on the metabolic responses of brain tissue to in
vitro stimulation. Several presentations and publications have been made
concerning the background data validating the experimental techniques
used (CUMMINS, 1971; CUMMINS and BULL, 1971; BULL and LUTKENHOFF, 1973;
BULL and CUMMINS, 1973; BULL and O'NEILL, 1975; BULL, 1975). The basic
form of the responses measured has also been confirmed independently
(LIPTON, 1973).
Short-term experiments have shown that metabolic responses of cerebral
cortex slices to stimulation were altered when animals were exposed to
low intraperitoneal doses of methyl mercuric chloride (BULL and LUTKENHOFF,
1975). The observed changes centered around the oxidation and reduction
of cellular electron transport intermediates in response to electrical
pulses or to elevated potassium concentrations. Cerebral function is
known to be highly dependent upon the provision of adequate energy in the
form of ATP (RIDGE, 1971). High doses of methyl mercury have been pre-
viously shown to decrease the ATP/ADP ratio, in vivo (PATERSON and USHER,
1971). Consequently, we decided to determine if similar changes in the
relationship of energy metabolism to function could be observed with long-
term oral exposures to methyl mercury.
-------
CONCLUSIONS
Alkyl mercurials differ in their effects on animals from other forms
of mercury in that neurological changes predominate the clinical picture.
Mercury contamination of drinking water supplies appears to be quite
uncommon. However, in considering the possibility of a mercury-contaminated
water supply, one would have to assume that some considerable portion of
the mercury in solution would be in the methylated form unless shown
otherwise. Inorganic mercury tends to be tightly complexed in bottom
sediments of reservoirs (GAVIS and FERGUSON, 1972). Under appropriate
conditions, bacterial conversion of inorganic mercury to methyl mercury
has been shown to occur and serves as a means of mobilizing mercury (JENSEN
and JERNELOV, 1969). By virtue of their almost complete absorption in the
gastrointestinal tract, the alkyl mercurials also present the greatest
danger in terms of their toxicity. It is appropriate, therefore, that
the drinking water standard for mercury be based upon the toxic effects
of methyl mercury.
Short-term experiments (BULL and LUTKENHOFF, 1975) revealed complex
changes in the metabolic responses of cerebral cortex slices taken from
animals exposed to methyl mercury. Effects of methyl mercury tended to
primarily involve tissue pyridine nucleotide (NAD(P)) responses to stimu-
lation. Changes were also observed in the cytochrome chain, but these
appeared secondary to the NAD(P)H responses. Very low doses of methyl
mercury (as the chloride, 0.01 to 0.1 mg/Kg per day for 14 days) substan-
tially increased the activation of substrate oxidation as measured by
an enhancement of NAD(P) reduction with electrical and/or potassium ion
stimulation. In this dose range, small but measurable increases in the
mercury content of the cerebral cortex could be measured (0.026 i 0.004
ug/g at 0.02 mg/Kg/day vs. 0.005 ± 0.002 ug/g in control animals). Pro-
gressive increases in methyl mercury dosage lead to increased mercury
content of the cerebral cortex (0.78 ± .06 and 9.9 ± 0.9 ug/g with 0.2
and 2.0 mg/Kg/day, respectively), but began to progressively inhibit the
activation of substrate oxidation produced by electrical stimulation.
At the highest dose (2.0 mg/Kg/day), this could be correlated with an
inhibition of potassium-stimulated aerobic glycolysis. In addition, at
doses exceeding 0.05 mg/Kg/day, measurable increases in the duration of
the NAD(P) response indicated some inhibition of NAD(P)H reoxidation.
Only at the highest dose were there any substantial losses in body weight
or signs of definite neurological impairment.
Long-term oral exposures to methyl mercury confirmed the results seen
with short-term exposures. Weanling rats were exposed to concentrations
of methyl mercuric chloride of 0.01, 0.1, 1.0 and 10 mg/liter in their
-------
drinking water for six months. Animals in the highest dose range showed
substantial inhibition of growth (measured as body weight gained over the
experimental period) which was associated with a decrease in the consumption
of food. Again this was the only dose at which substantial neurological
impairment was observed. Enhancement of the stimulated reduction of tissue
(NAD(P)) was observed at low doses (0.1 and 1.0 mg/liter) followed by
inhibition at higher doses (10 mg/liter) with 90 days of exposure. At
180 days, the enhancement of the responses was no longer observed and
significant inhibition of NAD(P) reduction was observed at both 1.0 and
10 mg/liter methyl mercuric chloride. These results also coincided with
an inhibition of potassium^stimulated aerobic glycolysis which had been
reported by earlier investigators (YOSHINO, et al., 1966). Inhibition
of NAD(P)H reoxidation following electrical stimulation was apparent at
0.1 mg/liter at both 90 and 180 days, but appeared to become less prominent
at higher doses, perhaps as a result of the inhibition of NAD(P) reduction.
These results can all be reconciled with previous observations (PATERSON
and USHER, 1971; SALVATERRA, et al., 1973) dealing with in vivo changes
in the concentrations of the adenine nucleotides and glycolytic inter'-
mediates produced by low acute intraperitoneal doses of methyl mercury.
From this work it can be concluded that methyl mercury induces sub-
stantial changes in the organization and control of energy metabolism of
brain as it relates to changes in functional activity. This occurs at
exposure levels far below those required to produce overt toxicity. If
sufficient time and personnel had been available, it may have been possible
to apply more sophisticated behavioral parameters and functional deficits
in the whole animal may have been apparent at lower dosage levels. However,
the deficits in metabolism are more than sufficient to explain the neuro-
logical changes seen with the highest doses.
-------
RECOMMENDATIONS
In terms of the drinking water standard for mercury, we can make the
following generalizations:
1. Intake of mercury was not constant with time over the duration
of the long-term experiments, because animals drank less than
half the amount of water/unit of body weight after they matured.
Taking, however, the minimum intake/unit of body weight, the
doses of methyl mercuric chloride to the animal consuming 0.01,
0.1, 1.0 and 10 mg/liter correspond closely to 0.001, 0.01, 0.1
and 1 mg/Kg/day.
2. Inhibition of NAD(P)H reoxidation was observed at a dose of 0.01
mg/Kg/day. Reduction of NAD(P) by electrical stimulation was
enhanced at this dose in 90 days and slightly, but not signifi-
cantly, inhibited at 180 days of exposure.
3. No significant differences were observed with doses in the
drinking water corresponding to 0.001 mg/Kg/day. To this, however,
must be added approximately another 0.001 mg/Kg/day as being
derived from the standard lab chow.
4. Extrapolating these data to man would yield a minimum effect level
of:
70 Kg x 0.01 mg/Kg/day =0.7 mg/day
and a "no-effect" level of:
70 Kg x 0.002 mg/Kg/day =0.14 mg/day.
FRIBERG (1971) estimated a "no-effect" dose rate in man of 0.3 mg/day.
This figure is somewhat less than a previous estimate of BERGLUND and
BERLIN (1969) of 0.6 to 1.0 mg/day. To the extent that our data may be
extrapolated to man, it is clear that our estimate is more compatible with
that of FRIBERG. It was on this estimate that the proposed drinking water
standard was established. The 0.002 mg/liter figure proposed would lead
to a maximum intake of 4 ug/day, assuming a 2-liter daily consumption of
water. If water were the sole source of mercury, this allows a more than
adequate safety factor of 75. This safety factor is lowered if dietary
sources are considered. If the maximum "safe" dose recommended by FRIBERG,
of 30 ug is not exceeded from dietary sources, it is doubtful that an
additional contribution of 4 ug from drinking water would significantly
add to the overall risk.
-------
METHODS
Sixty, weanling, Sprague-Dawley rats were divided into 5 groups of
12 animals each. Methyl mercuric chloride was added to the drinking water
at levels of 0.01, 0.10, 1.0 and 10 mg/liter in the first four groups,
respectively, and the fifth group served as control. Double-distilled
(glass) water was utilized for all groups. The rats were housed three
to a cage and water consumption was taken daily on a cage basis. Correc-
tions for spillage were made for the first 80 days of the study by catching
spilled water in a screened jar containing a film of paraffin oil over the
collected water to prevent evaporation. These jars were periodically
weighed and the accumulated water subtracted from the water consumption
of the particular cage of rats. This exercise corrected an error of only
2-3% and was discontinued for the remainder of the study. Food consumption
was measured twice weekly and the animals were weighed on a weekly basis
for the first 60 days of the study and at longer intervals (usually 2
weeks) for the remainder of the experiment. On the basis of the periodic
measurements of body weight and the daily water consumption an average
dose per unit of body weight was derived for each group exposed to methyl
mercury. These data are found in Fig, 1. As can be seen, the dose per
unit weight decreased with time. This was the result of a decrease in
water consumption per unit of body weight as the animals increased in
size. Because the nature of this curve precludes the use of any meaningful
average dose for the entire experimental period we will refer to doses in
terms of the concentration added to the drinking water. Food was supplied
ad lib. and was the standard laboratory chow used in our laboratory (Rock-
land) . The chow was assayed for mercury by atomic absorption spectro^-
photometry and was found to contain <0.006 ± 0.002 ug/g (SEM). Using
this figure, it was determined that this source of mercury contributed
significantly to the total intake of mercury only at the 0.01 mg/liter
level. This amounted to a dose 30% greater than indicated for this group
in Figure 1. This group may then be calculated to be receiving a total
mercury dose averaging approximately four times the dose of mercury that
the control group was receiving from their diet.
Six animals from each group were sacrificed after 90 days of exposure
and the remainder were sacrificed at 180 days. As only 3 animals could
be examined on a given day the sacrifice period was in fact 10 successive
days. To avoid prejudicing the results, the animals were sacrificed in
rotation rather than by group. Some instrument problems required a pro-
longation of this period at 180 days and it was extended to 20 days.
Spectrophotometric changes and lactic acid production of brain slices
taken from exposed animals were measured basically as described earlier
-------
UJ
O
o: 2.0
o
o ^
MERCURIC
mg/Kg/da:
b bi
_j
> 0.5
H
iLl
O^ 0 X 10°
o • x icr1
" o o x l0"2
S * x l0"3
.8
•o •
°° o g § * t
O
-
1 1 1 1
40 80 120 160
DAYS OF EXPOSURE
Figure 1. Dosage levels of methyl mercuric chloride in differing
experimental groups with age. • 0.01 mg/liter; O0.10
mg/liter; •1.0 mg/liter; O 10 mg/liter.
-------
(BULL and LUTKENHOFF, 1974). Two specific changes must be noted. Since
the aerobic glycolytic response to addition of potassium proved more sen-
sitive than the respiratory response the perfusion method was modified
slightly in hopes of increasing the time resolution of the measurements
of changing lactic acid production. This involved primarily an increase
in the perfusion rate to 1.3 ml/min, roughly twice that utilized in the
short term studies. At this flow rate measurements of changes in oxygen
consumption were not reliable and consequently will not be presented.
Statistical analysis of the results was done by analysis of variance
and t-test. Unless otherwise noted a P of <0.05 is reported as signifi-
cant.
-------
RESULTS
Only in the 10 mg/liter group was evidence of gross toxicity apparent
which could be attributed to methyl mercury. One measure of this toxicity
was a decrease in growth rate over the experimental period. The body
weight of animals in this group failed to keep pace with that of the
control group (Fig. 2). Lower levels of methyl mercury in the drinking
water had no measurable effect on body weight (data not shown). The
lessened weight gain of the 10 mg/liter group was associated with signifi-
cantly decreased consumption of lab chow (Fig. 3), suggesting that anorexia
may have been responsible. At this dose level two deaths resulted in only
four animals being available for sacrifice following 180 days of exposure.
Additional signs of toxicity were noted in this group which were similar
to observations of other investigators (e.g., KLEIN, et al., 1972), and
thus will not be dealt with here.
The effects of methyl mercury on electrically stimulated responses
of cerebral cortex slices were similar, but not identical, to those observed
in short-term experiments (BULL and LUTKENHOFF, 1975). As was seen in
the short-term experiments methyl mercury did produce an inhibition of
pyridine nucleotide reoxidation following electrical stimulation at an
exposure level of 0.1 mg/liter of drinking water (Fig. 4). Some hint of
inhibition was observed at 0.01 mg/liter at 90 days (P <0.05) of exposure,
but this was not confirmed at 180 days of exposure. Possibly the falling
dose with increasing body weight could account for this difference. At
0.1 mg/liter the time constants for reoxidation were greater on the average
than the maximum observed in the short term studies (130 sec vs. 100 sec,
respectively). This appeared as a maxima, however, and at 1.0 and 10
mg/liter methyl mercuric chloride this effect of methyl mercury appeared
to decrease. Little difference was observed in this result in animals
sacrificed at 90 or 180 days of exposure.
Methyl mercury also reproduced the biphasic effect on the initial
rate of pyridine nucleotide reduction with electrical stimulation seen in
the short-term studies at 90 days of exposure, but not at 180 days. A
stimulatory effect of methyl mercury was clearly evident at 0.1 mg/liter
for 90 days but this effect appeared to progressively decline until at
10 mg/liter, it again approached control levels (Fig. 5). At 180 days
of exposure only a progressive decline in the phase II response was
noted at 1.0 and 10 mg/liter. At this time, the values obtained at both
dose levels were significantly decreased relative to the control responses.
Essentially the same result was observed with the cytochrome intermediates
(data not shown).
-------
800
600
2400
Ul
200
O
m
8
o
-------
35
30
h-
Q.
O
O
O
15
10
H
*
Control
Methyl mercury
20 40 60 80
DAYS OF EXPOSURE
Figure 3. Methyl mercuric chloride (10 mg/liter) effect upon
consumption of laboratory chow. Vertical bars represent
± SEM.
11
-------
isor
AN
0 0.01 0.10 1.0 10
METHYL MERCURIC CHLORIDE
mg./l. drinking water
Figure 4. Pyridine nucleotide reoxidation in electrically
stimulated brain slices derived from rats
exposed to methyl mercury, O 90 days; •180 days.
Vertical bars represent - SEM.
12
-------
O 2.0
I-
O
o
LU
o:
6
Q.
1.5
U. o
O E
U ^
o:
0.5
\\
0 0.01
0.10 1.0 10
METHYL MERCURIC CHLORIDE
mg./l. drinking water
Figure 5. Initial rate of pyridine nucleotide reduction
following electrical stimulation of brain slices
taken from animals exposed to methyl mercury,
O 90 days; • 180 days. Vertical bars represent
± SEM.
13
-------
The data in Figure 6 illustrate more completely the changes in the
pyridine nucleotide responses to electrical stimulation. These responses
were not necessarily representative of the particular dose, but are used
to illustrate what appears to be the progression of alterations produced
by methyl mercury. The typical control response, measured at 340-374 nm,
distinctly shows the characteristic changes in absorbance observed following
10 sec of electrical pulses. Little change in these responses was observed
at 0.01 mg/liter. At 0.1 mg/liter, the example illustrates the considerable
prolongation of the response typical of this dose, although the one pictured
is among the longest recorded. At 1.0 mg/liter, the response to electrical
stimulation tends to assume a somewhat different configuration involving
both a decrease in size and apparent duration of the response. In both
slices from which the two examples of this dose were drawn, responses
recorded in the cytochrome chain appeared very little changed, in either
magnitude or duration. In the example shown taken from the 10 mg/liter
group, the pyridine nucleotide response to electrical stimulation was
purely oxidative in direction. This type of response has never been ob-
served at this wavelength pair under the same conditions of incubation
in a control animal (total of 29 in this and the short-term study and 12
in preliminary experiments). Although only 1 of 6 displayed this response
at 90 days and 1 of 3 at 180 days, results from the short-term study confirm
such an alteration in the response to electrical stimulation at high doses
of methyl mercury.
Methyl mercury produced an alteration in the aerobic glycolytic re-
sponse to an increase of media potassium concentration from 3 to 30 mM
not observed in shorter term experimentation. At 180 days of exposure,
the increase in lactic acid produced by tissues was markedly augmented,
particularly during the initial stages of the response, at 0.1 mg/liter
in the drinking water (Fig. 7). At higher doses a definite decrease in
the rate at which the response developed was observed, consistent with the
inhibition observed at 2.0 mg/Kg/day for 14 days in the short-term study.
The lactic acid response was not measured at 90 days of exposure because
of a shortage of technical personnel.
14
-------
I
CONTROL, 90 days
UJ
O
CD
a:
o
0)
CD
TIME (min.)
Figure 6. Effect of increasing doses of methyl mercuric
chloride on the characteristics of the electrically-
induced metabolic responses of tissue pyridine
nucleotides. Taken at 180 days of exposure.
15
-------
Oi
2.0
Z
2 1.8
I-
o
O .!
1.6
°- s
Q
-------
DISCUSSION
In appraising the results of the present paper it is well to keep in
mind that the results obtained in our experimental system cannot necessarily
be attributed exclusively to direct effects on the parameters measured.
The measurements were not made with the limited view of measuring direct
effects on these individual components of the tissue. If that had been
the case it would have been much simpler to use isolated mitochrondrial
preparations as the experimental system. However, our efforts have been
directed towards measurement of methyl mercury induced alterations in the
relationship of energy metabolism to function. This can only be accomp-
lished when some functional capability is retained by the test preparation.
Tissue slices retain such properties (e.g., electrical excitability and
neurosecretory properties) but isolated mitochondrial preparations do not.
There are certain advantages to be gained from such an approach as opposed
to measurements directed towards specific enzymes or enzyme systems. Most
obvious of these was the measurement of the respiratory intermediates
under conditions which more closely approximate their in vivo environment.
More important, however, is that such measurements allow an integrated
approach to the effects of toxic chemicals on brain function. Inhibition
of a specific enzyme by a toxic chemical necessitates adaptations in
cellular metabolism. The adaptations are not merely functions of affinity
constants and maximum velocities of various enzymes and metabolic pathways,
but involve a complex array of feedback mechanisms of which only the barest
outlines are clearly understood at present (ATKINSON, 1971). The possi-
bility of this type of interference with tissue function has been demon-
strated by methyl mercury's ability to reverse the effects of allosteric
effectors on glutamate dehydrogenase (BITENSKY, et al., 1965) and the
dissociation of subunits of glyceraldehyde-3-phosphate dehydrogenase by
mercurials (SMITH and SCHACHMAN, 1971), in vitro. For these reasons we
have made our first effort in the study of the central nervous system
effects of methyl mercury at the tissue level of organization where many
of the control mechanisms alluded to were still present. In addition, we
have perturbed the tissue in such a way as to increase the functional
activity of the tissue and thus activate these control mechanisms. Analysis
of the kinetics of the responses allow an evaluation based upon alterations
in the integrative aspects of function and metabolism. The result has not
been a complete explanation of the central nervous system effects of methyl
mercury. However, changes in the biochemistry and physiology of brain
tissue taken from animals exposed to quite low doses of methyl mercury
have been documented.
It is perhaps not surprising that long term exposures to methyl mercury
produced a different gradation of responses that observed in shorter term
17
-------
experiments. The basic difference in the long-term data is that the
inhibition of pyridine nucleotide reoxidation, indicated by the increased
time constant following electrical stimulation, appeared to be reversed
at higher dosage levels. The qualitative nature of these changes were
described in Figure 6 and the probable meaning of these changes is dis-
cussed later in this paper. In terms of a difference between long- and
short-term exposures, however, it would seem reasonable to assume that
longer term accumulations of methyl mercury gave brain tissue a chance
to adapt to a given situation that it is not capable of or is unnecessary
in the short-term. It was interesting to observe that the average time
constant of pyridine nucleotide reoxidation following electrical stimulation
remained little changed at 180 days relative to that observed at 90 days
of exposure. This was despite an apparent loss of the enhancement in
the reductive response to electrical stimulation at 0.1 mg/liter observed
at 90 days. The enhancement of responses to electrical stimulation was
also observed in shorter term exposures (BULL and LUTKENHOFF, 1975).
Consequently, its apparent absence at 180 days of exposure requires
explanation. It was possible that this effect of methyl mercury was
confined to a dose between 0.01 and 0.1 mg/liter with the longer exposure
time. The alternative explanation would be that this apparent adaptation
to the inhibition of pyridine nucleotide oxidation cannot be sustained
indefinitely. In either case the rather large shift in the dose-response
curve relative to the doubling of the exposure time appears to indicate
a cumulative effect of methyl mercury in depressing the reductive phase
of the response to electrical stimulation.
There appeared to be a parallel between the rapidity of the aerobic
glycolytic response to addition of potassium and the time constant of
pyridine nucleotide reoxidation following electrical stimulation. This
result strongly suggests that inhibition of the oxidation of cytoplasmic
NAD(P)H may be involved in the observed effects of methyl mercury on
electrically stimulated responses. This was another point of departure
from the results obtained from short-term experiments, where little
tendency for such an increased responsiveness to potassium was observed
in lactic acid production by the tissue. On the other hand, higher
levels of reduced pyridine nucleotide were achieved with addition of
potassium at low doses of methyl mercury relative to controls in these
short-term exposures. Consequently, the observation of a more "responsive"
glycolytic pathway could be associated with the higher average time
constant of pyridine nucleotide oxidation observed in the long-term
results. One cannot, however, neglect the possibility that the increased
time resolution afforded by the higher perfusion rate in the present
study may have influenced the results.
The change in character of the response to electrical stimulation
at the higher doses of methyl mercury (i.e., 10 mg/liter in the long-term
study, with some indication of such a change at 1.0 mg/liter, and at
2.0 mg/Kg/day in the 14-day experiments) tends to fit well with the idea
that the reducing equivalents for mitochrondrial oxidations may be derived
from different pools. Differing pools of reduced pyridine nucleotides
would be expected to display differing kinetic relationships with the
18
-------
process of oxidative phosphorylation depending primarily on how direct
the access to the cytochrome chain was (VANDAM and MEYER, 1971). We have
previously shown that the reductive response to addition of potassium
is glucose dependent (BULL and LUTKENHOFF, 1973) and that the pyridine
nucleotide response to electrical stimulation is considerably attenuated
with pyruvate as the sole substrate (unpublished results) relative to
responses in the cytochrome chain. This may be taken as evidence of
compartmentation of pyridine nucleotides in the tissue.
Experiments performed subsequent to the present work has shown that
the form of the NAD(P) response is dependent upon the cytochrome redox
potential (BULL, unpublished results). If the reduction of cytochrome
c_ is reduced to somewhat less than 35% in the steady-state, the NAD(P)
response becomes strictly oxidative in direction, similar to the observation
made here with high doses of methyl mercury. Maintenance of a high steady-
state level of reduction in isolated brain slices appears to be a peculiar
property of glucose—presumably accounted for by its donation of a cyto-
plasmic reducing equivalent (BULL, 1975). The present data suggest,
therefore, that delivery of cytoplasmic reducing equivalents to mitochron-
drial oxidations in brain is severely impaired in rats exposed to high
levels of methyl mercury.
The alterations in the metabolic response of rat cerebral cortex
slices is consistent with observations made by PATERSON and USHER (1971)
on in vivo levels of the adenine nucleotides of rat brain. These authors
found an elevation of the ATP/ADP + AMP ratio in rats administered low
doses of methyl mercury, but a decrease in this ratio at higher doses.
High ATP levels would tend to inhibit oxidative phosphorylation and thus
reduce the oxidation of reduced pyridine nucleotides. The increased
levels of glycolytic intermediates noted by these authors may also account
for the enhancement of reductive responses to stimulation noted throughout
the respiratory chain (BULL and LUTKENHOFF, 1975) at the lower doses of
methyl mercury. Lowered ATP levels accompanied by increases in ADP and P
would act to stimulate oxidative phosphorylation and thus reduce the
reductive response at higher doses of methyl mercury. SALVATERRA and
co-workers (1973) documented the increase in ATP levels with low doses
of methyl mercury in the mouse, but failed to show a decrease in these
levels at higher doses. These authors observed certain behavioural changes
which coincided with ATP elevations. The absence of a decrease in ATP
with higher doses of methyl mercury may represent a species difference
or may indicate a requirement for higher or repeated exposures in mice
to produce the effect.
This preliminary investigation of the effects of methyl mercury on
brain metabolism frankly arouses more questions than it answers. It is
clearly evident that very low doses of methyl mercury, on the order of
0.01 mg/Kg/day, produce measurable changes in the pattern of cerebral
metabolism following stimulation. It has been well established that the
stimulated state, in vitro, more closely approximates in vivo rates of
metabolic activity and that glucose serves as the primary substrate
19
-------
of brain in vivo (MCILWAIN, 1966), With these facts in mind and viewing
the preliminary nature of the findings, we find it advantageous to think
of the results as indicating a progression from no effect, to compensation,
to frank impairment of the coordination of the energy conserving mechanisms
to the functional needs of the tissue. It appears that compensation is
required for the decreased availability of one pyridine nucleotide pool
for mitochrondrial oxidations. The cellular location of this pool cannot
be stated for certain, but data from 180 days of exposure suggest that
it may reside in the cytoplasm. When the tissue is required to rely
exclusively on glucose as substrate, several problems of metabolic control
can be envisioned. The aerobic glycolytic rate must be maintained in
order to provide reducing equivalents for mitochondrial oxidations, either
as reduced pyridine nucleotide and/or pyruvate. If oxidation of pyruvate
or mitochrondrial oxidation of glycolytic NADH is impaired, a problem of
coordination is introduced. In order for glycolysis to continue NAD must
be regenerated either by conversion of pyruvate to lactate or by mito-
chrondrial oxidations through various shunts. If the tissue is required
to use the lactic dehydrogenase step for regeneration of NAD, the relative
amount of pyruvate available as a mitochondrial substrate is reduced,
compounding the problem associated with a reduced mitochondrial oxidation
of NADH. Consequently, one can see a need for an increase in the aerobic
glycolytic rate upon increasing the energy demand of the tissue. This
means of compensation, however, would eventually break down as inhibition
increases because of the need to regenerate the catalytic quantities of
NAD for glycolysis to remain in operation. This cannot be done without
depleting pyruvate as a mitochondrial substrate. We feel that this pro-
gression is eventually responsible for the conversion of the pyridine
nucleotide response to purely oxidative in direction. At this point, it
is necessary for the tissue to draw on NADH generated during the resting
state, a time at which energy demand is relatively low, in order to meet
the energy demands of a brief burst of activity. Further work is planned
to substantiate this hypothesis.
20
-------
REFERENCES
ATKINSON, D. E. (1971) Adenine nucleotides as stoichiometric coupling agents
in metabolism and as regulatory modifiers. The adenylate energy charge.
In: Metabolic Regulation, Metabolic Pathways. (Vogel, H. J., ed.) V_,
1-21, Academic Press, New York.
BITKENSKY, M. W., YIELDING, K. L. and TOMKINS, G. M. (1965) The reversal by
organic mercurials of "allosteric" changes in glutamate dehydrogenase. J_.
Biol. Chem. 240. 668-673.
BERGLUND, F. AND BERLIN, M. (1969) Risk of methyl mercury cumulation in man
and mammals and the relation between body burden of methyl mercury and
toxic effects. In: Chemical Fall-out. (Miller, M. W. and Berg, G. G.,
eds.) Current Research on Persistent Pesticides, C. C. Thomas, Springfield,
pp. 258-73.
BULL, R. J. (1975) Cytochrome redox potential dependence on substrate in
rat cerebral cortex slices. Importance of cytoplasmic NAD(P)H and potassium.
Accepted for publication.
BULL, R. J. and CUMMINS, J. T. (1973) Influence of potassium on the steady-
state redox potential of the electron transport chain in slices of rat
cerebral cortex and the effect of ouabain. ^J. Neurochem. 21, 923-37.
BULL, R. J. and LUTKENHOFF, S. D. (1973) Early changes in respiration,
aerobic glycolysis and cellular NAD(P)H in slices of rat cerebral cortex
exposed to elevated concentrations of potassium. J_. Neurochem. 21, 913-22,
BULL, R. J. and LUTKENHOFF, S. D. (1975) Changes in the metabolic responses
of brain tissue to stimulation, in vitro, produced by in vivo administration
of methyl mercury. Neuropharmacol. 14, 351-9.
BULL, R. J. and O'NEILL, J. J. (1975) Spectral changes in the respiratory
chain of cerebral cortex slices: Correlation with the energy status of
the tissue. Psychopharmacol. Comm. 1^, 109-15.
CAMPBELL, A. M. G., HERDAN, G., TATLOW, W. F. T. and WHITTLE, E. G. (1950)
Lead in relation to disseminated sclerosis. Brain 73, 52-70.
CHANCE, B. (1972) The nature of electron transfer and energy coupling
reactions. FEB.S Letters 23, 3-20.
21
-------
COTZIAS, G. C., PAPAVASILIOU, F. S., GINOS, J., STECK, A. and DUBY, S.
(1971) Metabolic modification of Parkinson's disease and of chronic
manganese poisoning. Ann. Rev. Med. 22, 305-^26.
CUMMINS, J. T. (1971) Spectral changes in respiratory intermediates of
brain cortex in response to depolarizing pulses. Biochjbn. Biophys. Acta
253. 39-45.
CUMMINS, J. T. and BULL, R. J. (1971) Spectrophotometric measurements of
metabolic responses in isolated rat brain cortex. Biochim. Biophys. Acta
253. 29-38.
DAVID, 0., CLARK, J, and VOELLER, K, (1972) Lead and hyperactivity, Lancet
I_, 900-3,
FRIBERG, L. (1971) Methyl mercury in fish. A toxicological-epidemiological
evaluation of risks. Nordisk Hygienisk Tidskrift, Suppl. 4 - National
Institute of Public Health, Stockholm, Sweden.
GAVIS, J. and FERGUSON, J. F, (1972) The cycling of mercury through the
environment. Water Res, 6^ 989-1008.
INDRAPRASIT, S., ALEXANDER, G. V., and GONICK, H. C. (1974) Tissue composi-
tion of major and trace elements in uremia and hypertension. J_. Chron. Pis.
27., 135-61.
JAIN, S., O'BRIEN, B., FOTHERGILL, R., MORGAN, H. V., and GEDDES, A. M.
(1970) Lead poisoning presenting as infectious disease. Practitioner 205,
784-6.
JENSEN, S. and JERNELOV, A. (1969) Biological methylation of mercury in
aquatic organisms. Nature 2^23, 753-4.
KLEIN, R., HERMAN, S. P., BRUBAKER, P- E., and LUCIER, G. W. (1972) A model
of acute methyl mercury intoxication in rats. Arch. Pathol. 93, 408-18.
LIPTON, P. (1973) Effects of membrane depolarization on nicotinamide
fluorescence in brain slices. Biochem. J_. 136, 999-1009,
McCABE, L. J., SYMONS, J. M., LEE, R. D. and ROBECK, G. G. (1970) Survey
of community water supply systems. J_. Amer. Water Works Assoc. 62, 670-87.
McILWAIN, H. (1966) Biochemistry and the Central Nervous System. Little,
Brown & Co., Boston.
PATERSON, R. A. and USHER, D. R. (1971) Acute toxicity of methyl mercury
on glycolytic intermediates and adenine nucleotides of rat brain. Life
Sciences 10, 121-8.
22
-------
PETKAU, A., SAWATZKY, A., HILLIER, C. Rt and HOOGSTRATEN, J. (1974) Lead
content of neuromuscular tissue in amyotrophic lateral sclerosis: Case
report and other considerations. Brit. ^J. Ind. Med. 31, 275-^37.
RIDGE, J. W. (1972) Hypoxia and the energy charge of the cerebral adenylate
pool. Biochem. £. 127. 351-55.
SALVATERRA, P., LOWN, B., MORGANTI, J. and MASSARO, E. J. (1973) Alterations
in neurochemical and behavioral parameters in the mouse induced by low
doses of methyl mercury. Acta pharmacol. toxicol. 33, 177-90.
SMITH, G. D. and SCHACHMAN, H. K, (1971) A disproportionate mechanism
for the all'-or-none dissociation of mercurial-treated glyceraldehyde
phosphate dehydrogenase. Biochemistry 10, 4576-88,
VANDAM, K. and MEYER, A. J. (1971) Oxidation and energy conservation by
mitochondria. Ann. Rev. Biochem. 40, 115^60.
YOSHINO, Y., MOZAI, T. and NAKAO, K. (1966) Biochemical changes in the
brain in rats poisoned with an alkylmercury compound, with special re-
ference to the inhibition of protein synthesis in brain cortex slices.
£. Neurochem. 13, 1223-30.
23
-------
TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
REPORT NO.
EPA-600/1-76-013
2.
3. RECIPIENT'S ACCESSION-NO.
TITLE AND SUBTITLE
Methyl Mercury and the Metabolic Responses of Brain
Tissue
5. REPORT DATE
March 1976 (Issuing Date)
6. PERFORMING ORGANIZATION CODE
. AUTHOR(S)
Richard J. Bull
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
10. PROGRAM ELEMENT NO.
1CC614
11. CONTRACT/GRANT NO.
12. SPONSORING AGENCY NAME AND ADDRESS
Same as above
13. TYPE OF REPORT AND PERIOD COVERED
Final Reoort
rinai Kept
.SPONSORING /
14. SPONSORING AGENCY CODE
EPA-ORD
15. SUPPLEMENTARY NOTES
16. ABSTRACT
Weanling, Sprague-Dawley rats have been exposed to methyl mercuric chloride (con-
centrations from 0.01 to 10.0 mg/liter) in their drinking water. At 10 mg/liter the
animals exhibited neurological symptoms typical of methyl mercury. Also, in this
group a considerable decrease in growth occurred which was associated with a decreased
consumption of food.
Responses of the respiratory intermediates to stimulation were found to be
altered in cerebral cortex slices taken from exposed animals. Effects on tissue pyri-
dine nucleotides predominated. An enhancement of the rate of pyridine nucleotide re-
duction by electrical stimulation was observed at 0.1 mg/liter. This rate progres-
sively decreased at higher dose levels. Reoxidation of reduced pyridine nucleotide
was also inhibited at 0.1 mg/liter at both 90 and 180 days of exposure.
Potassium stimulated aerobic glycolysis was found to be enhanced in its initial
stages at 0.10 mg/liter of methyl mercuric chloride in the drinking water but progres-
sively declined at 1.0 and 10 mg/liter. A close parallel was observed between the
time constant of pyridine nucleotide oxidation following electrical stimulation and
the "responsiveness" of the aerobic glycolytic rate to stimulation by potassium.
These results suggest an initial defect in the oxidation of cytoplasmic NADH which
progresses to loss of metabolic control of cytoplasmic oxidations.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS C. COSATI Field/GlOUp
Mercury organic compounds; Toxicity;
Cerebral cortex; Metabolism; Rats; Potable
water
Methyl mercury; Drinking
water; Brain energy
metabolism
6F
18. DISTRIBUTION STATEMENT
Release to Public
19. SECURITY CLASS (ThisReport)'
Unclassified
21. NO. OF PAGES
32
20. SECURITY CLASS (Thispage)
Unclassified
22. PRICE
EPA Form 2220-1 (9-73)
24
*USGPO: 1976 —657-69S/5396 Region 5-11
------- |