EPA-6QG/1-78-089
January 1978
Environmental Health Effects ieseareii Serie
EFFECT OF LEAD ON GAMMA AMINO
BUTYRIC ACID SYNTHESIS
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, North Carolina 27711
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
6. Scientific and Technical Assessment Reports (STAR)
7. Interagency Energy-Environment Research and Development
8. "Special" Reports
9. Miscellaneous Reports
This report has been assigned to the ENVIRONMENTAL HEALTH EFFECTS RE-
SEARCH series. This series describes projects and studies relating to the toler-
ances of man for unhealthful substances or conditions. This work is generally
assessed from a medical viewpoint, including physiological or psychological
studies. In addition to toxicology and other medical specialities, study areas in-
clude biomedical instrumentation and health research techniques utilizing ani-
mals — 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.
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EPA-600/1-78-009
January 1978
EFFECT OF LEAD ON GAMMA AMINO BUTYRIC ACID SYNTHESIS
by
Henry Archie Moses
Department of Biochemistry and Nutrition
School of Medicine
Meharry Medical College
Nashville, Tennessee 37208
Grant No. R-802370
Project Officer
Larry L. Hall
Environmental Toxicology Division
Health Effects Research Laboratory
Research Triangle Park, N.C. 27711
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
HEALTH EFFECTS RESEARCH LABORATORY
RESEARCH TRIANGLE PARK, N.C. 27711
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DISCLAIMER
This report has been reviewed by the Health Effects Research
Laboratory, U.S. Environmental Protection Agency, and approved
for publication. Approval does not signify that the contents
necessarily reflect the views and policies of the U.S. Environmental
Protection Agency, nor does mention of trade names or commercial
products consitute endorsement or recommendation for use.
ii
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FOREWORD
The many benefits of our modern, developing, industrial society are
accompanied by certain hazards. Careful assessment of the relative risk of
existing and new man-made environmental hazards is necessary for the estab-
lishment of sound regulatory policy. These regulations serve to enhance
the quality of our environment in order to promote the public health and
welfare and the productive capacity of our Nation's population.
The Health Effects Research Laboratory, Research Triangle Park,
conducts a coordinated environmental health research program in toxicology,
epidemiology, and clinical studies using human volunteer subjects. These
studies address problems in air pollution, non-ionizing radiation, environ-
mental carcinogenesis and the toxicology of pesticides as well as other
chemical pollutants. The Laboratory develops and revises air quality
criteria documents on pollutants for which national ambient air quality
standards exist or are proposed, provides the data for registration of new
pesticides or proposed suspension of those already in use, conducts research
on hazardous and toxic materials, and is preparing the health basis for
non-ionizing radiation standards. Direct support to the regulatory function
of the Agency is provided in the form of expert testimony and preparation
of affidavits as well as expert advice to the Administrator to assure the
adequacy of health care and surveillance of persons having suffered imminent
and substantial endangerment of their health.
Research pertaining to gamma amino butyric acid and glutamic amino
acid decarboxylase is especially important because there is yet a paucity
of information concerning the specific neurochemical function of this
enzyme system and how it may be affected by, environmental insults. The
results presented in this report showing inhibition of GADC activity by
lead is significant to our further understanding of its neurotoxic effects
in chronic conditions at the molecular level.
John H. Knelson, M.D.
Director,
Health Effects Research Laboratory
iii
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ABSTRACT
This project was a study of the inhibitory effect of lead on the
enzymatic activity of brain glutamic ajnino acid decarboxylase (GADC). The
enzyme is responsible for the catalytic formation of gamma amino butyric
acid (GABA) in inhibitory neurons,which is believed to be involved with the
transmission of inhibitory impulses in the brain.
Lead nitrate solution was available to Sprague-Dawley female rats ad
libitum and the quantity of lead ingested was determined by the volumes con-
sumed. During the course of these experiments, animals were examined for
weight loss, locomotor activity, excitability and other behavioral manifesta-
tions of lead toxicity. At autopsy, the tissues selected for lead deter-
minations were brain, liver, bone (femur) and kidney. In another series of
experiments, GADC was isolated from fresh bovine brain tissue and iri vitro
studies were performed to determine the nature of lead inhibition of the
enzyme. Subsequently, Se and Cd inhibitions of the enzyme were studied and
compared to lead inhibition. Light microscopic studies of liver, brain
and kidney tissues were performed.
The activity of the enzyme GADC in brain tissue homogenates of rats
drinking lead nitrate solutions was less than the activity determined in
control rats. The Vmax of bovine brain GADC using glutamate as a substrate
was 938 counts of ^CC^ per minute in a system which contained 1.6 x 10~^
moles glutamate and tracer glutamate-l-ll*C, (880,000 dpm) and the Km was
3.6 x 10"1* moles/liter. When the inhibiting effect of Pb, Cd, and Se on
GADC activity were compared, Pb was the most potent inhibitor, Cd showed
less inhibition and Se showed no inhibition of enzyme activity.
iv
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CONTENTS
Abstract iv
List of Figures vi
List of Tables vii
Abbreviations and Symbols viii
Acknowledgements ix
1. Introduction 1
2. Conclusions 2
3. Recommendations 3
4. Materials and Methods 4
5, Experimental Procedures 6
6. Results and Discussion 10
References 16
Bibliography 17
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List of Figures
1. Average Lead Consumption During Experimental Period
2. GADC Isolation and Purification
3. Electrophoretic Pattern of GADC Preparation Compared to
A Versatol-N Control
vi
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LIST OF TABLES
Table 1. Effect of Lead on The Weight of Rats
Table 2. GADAC Activity in Rat Brain Homogenates Measured
as cpm 1<+C02 Released
Table 3. Attrition Schedule of Animals Receiving Lead as Lead
Nitrate
Table 4. The Effect of Lead The Weight of Rats
Table 5. Effect of Lead on Animals Tissue Weight
Table 6. Concentration of Lead in Blood and Tissues
Table 7. GADC Activity In Rat Brain Homogenates Measured As
cmp I1+C0o Released
Table 8. Kinetic Studies on Calf Brain GADC In Vitro
Table 9. Effect of Lead on Calf Brain GADC Activity In Vitro
Table 10. Comparative Inhibition Effect of Lead, Cadmium and
Selenium on GADC Activity
vii
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LIST OF ABBREVIATIONS AND SYMBOLS
AAS - atomic absorption spectrophotometry
B W - body weight
ll*C - an isotope of carbon possessing radioactivity
Cd - cadmium
CM - centimeters
CNS - central nervous system
C02 - carbon dioxide
CPM - counts per minute
dpm - disintergrations per minute
g - gram
GABA- gamma amino butyric acid
GADC- glutamic acid decarboxylase
gms - grams
KOH - Potassium hydroxide
K2HP04- potassium phosphate, dibasic
Km - Michaelis Constant
L - liter
M - molar concentration
m - mole
mis - milliliters
MSG - monosodium glutamate
Pb++- divalent lead ion
Pb - lead
POPOP- 1,4 bis -2-{5-phenyloxazoyl)-Benzene
PPO - 2,5 diphenyloxazole
S - substrate concentration
/ - per
u - micro
!_
v - reciporical of velocity or activity
!_
s - reciporical of substrate concentration
Vmax- maximum velocity
viii
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ACKNOWLEDGEMENTS
The cooperation of the Morrissey Meat Company, Nashville, Tennessee
for providing fresh bovine brain is gratefully acknowledged. Assistance in
the care of the experimental animals by the Animal Facilities Staff at
Meharry Medical College is also appreciated.
The assistance given by Dr. Edward J. Faeder, former project officer,
and Dr. Larry Hall, current project officer in the development and com-
pletion of this project is kindly acknowledged.
ix
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Section 1
INTRODUCTION
The occurrence of leading poisoning is perhaps one of the oldest
occupational diseases known, and lead probably causes more poisonings than
any other metal in the environment. Poisoning is not uncommon in children
who reside in poor housing areas while adult lead poisoning is caused
primarily by occupational exposure, by consumption of lead-contaminated
whiskey or food,by beverages stored or served in improperly glazed earthen-
ware, or by inhalation of the fumes resulting from battery factories.
When a small amount of lead is taken into the body from the enviro-
ment, it is excreted via the kidney; however, when the intake is greatly
increased, lead may accumulate to toxic levels in soft tissues and affect
many organs of the body. The systemic effects may be appropriately placed
under three headings; the CNS, the hematopoietic and renal systems.
Researchers who have studied lead toxicity differ on the lead plasma
level that constitutes a toxic concentration, and there is considerable
controversy with regard to whether the amount of lead with which modern man
comes into contact is greater than that of his ancestors. It is a recent
recognition that the absorption of lead by inhalation may be 10-20 times as
great as .absorption via the digestion tract or skin, and although lead
poisoning has not disappeared, the clinical symptoms may not be immediately
evident since absorption and accumulation to toxic levels in tissues may
occur over an extended period of time.
This study has determined the level of orally administered lead
sufficient to cause a measurable decrease in GADC activity in rat brains.
It has also shown the effect of orally administered lead on the tissue
absorption and the nature of the lead inhibiting of bovine brain GADC.
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Section 2
CONCLUSIONS
There was no difference in the attrition rate of experimental animals
which received up to one percent lead compared to control animals which
received up to a one percent sodium nitrate solution ad libitum for sixteen
weeks.
The average final weights of the experimental groups were slightly
less than the control group, however the lead treatment had little effect
on tissue or organ size except for the femur. The femur weighed less per
one hundred grams of animal body weight after exposure to lead at both the
0.5 and 1.0 percent concentrations.
In animals exposed to 0.5 percent lead,no accumulation occurs in the
liver and femur? however,there was a significant accumulation in the kidney,
brain and whole blood, indicating selective tissue uptake of the heavy
metal. At the one percent level the tissue with the greatest uptake per
unit weight was the kidney, this tissue having levels nearly ten times higher
than the brain, liver, femur and whole blood.
Assay of GADC activity of brain homogenates showed that as larger
quantities of lead were consumed by the animals, correspondingly lower GADC
activity was observed in homogenates prepared from their brains.
GADC isolated and purified from bovine brain can be used for model
kinetic studies on competitive inhibition by lead.
Lead is the most potent inhibitor of GADC in comparison to Cd and
Se.
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Section 3
RECOMMENDATIONS
It is recommended that the pathophysiology at the molecular level of
lead metabolism continue to be investigated and that the research efforts
along these lines more definitively explain the relation of lead concen-
trations in various tissues with associated damage to the tissues.
Furthermore/it is recommended that there be continued basic research
to determine how the presence of lead affects the metabolic response of
animals to other toxic elements, and how the presence of disease states
(sickle-cell anemia, emphysema, infection, and hypertension) may influence
the response of the animal to various lead burdens.
It is recommended that a definitive study be done on a selected inner
city population of children in which the total lead consumed from all
sources (food, air) be monitored along with the total lead excreted. The
subjects of this type of study should be evaluated during the course of
the study using typical physical examination and evaluation procedures for
determining behavioral changes. Families should be selected carefully so
that attrition rates may be kept at a minimum and the community in which
the study is to be made should be thoroughly apprised of the significance
of the study. Utilizing schools, churches and community, civic, and social
clubs could be of high value in this aspect of such a study.
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Section 4
MATERIALS AND METHODS
Experimental animals, rats of the Sprague-Dawley strain, were obtained
from the O'Fallion Laboratories, St. Louis, Missouri, at 21-23 days of
age. They were housed individually in wire cages in the animal facility
of Meharry Medical College during the entire course of the experiment,
which began after a 25 day adaption and adjustment period. Hence, the
rats were approximately 50 days old when the experiment started. During
the course of the experiment, the rats were observed for physiological and
behavioral symptoms of lead toxicity and the quantity of food and liquid
consumption available ad libitum were recorded daily.
When the rats were killed, liver, blood, brain, kidney, and bone
(femur) tissues were removed, weighed and a section placed in buffered
formalin solution or immediately frozen for subsequent protein and lead
analyses. GADC activity was assayed immediately in the brain of the
animals using one hemisphere of the cerebral cortex for preparation of the
homogenate (1,2). Protein was determined in all tissues using the Folin
phenol protein nitrogen procedure (3). Lead was determined in samples that
had been wet-ashed with HN03:HC104 using a Perkin Elmer Atomic Absorption
Spectrophotometer Model 303 (4).
Fresh calf brains were contributed by Morrisey Meat Company,
Nashville, Tennessee. GADC was isolated from calf brain following the
procedure of Susz (2) with minor modifications. All centrifugations
requiring a force greater than 600xg were done using a Beckman Model
L-5-50 Ultra centrifuge and fractions from the Sephadex Columns were
collected using Buchler Fractomette 200. Studies of the isolated enzyme
were performed in vitro. Warburg Flasks were placed into a Bench Scale
metabolic shaker and held at 37°C. The metal solutions were prepared
from reagent grade pure metals by converting them to nitrate salts using
HNC>3 and buffering with phosphate buffer to pH 6.5. For the assay of GADC
activity the procedure of Lupein was followed (1) . The liberated ltfC as
C02 from the alpha carbon lifC-labeled sodium glutamate (New England Nuclear)
which was added to the homogenate was counted using a Beckman LS-150
Scintillation Counter. A PPO-POPOP soultion in Toluene (42 mis of
Liquiflor in 1000 mis of toluene) was used as the scintillation fluid.
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Section 5
EXPERIMENTAL PROCEDURES
GENERAL PROCEDURES
Experiment 1
Three different sets of rats were utilized in the study. In the first
set, fifty day old rats of the Wistar strain were grouped into five groups
with each group consisting of ten animals individually housed in wire cages.
The lead was administered as the acetate salt in water which was available
ad libitum. The concentrations of lead ranged from one to four percent
with the control group receiving sodium acetate titrated with hydrochloric
acid to pH of approximately 7.0. Many of these animals developed a
respiratory problem, and the poor solubility of lead acetate in water
encouraged us to switch to another strain and source of rats as well as
lead nitrate which is more soluble in water. Remaining animals in this
set were used by the research team for practice of the procedures that
were to be subsequently carried out.
Experiment 2
The second set of animals (of the Sprague-Dawley strain) were divided
into five groups, housed in a fashion identical to the first set but in
improved animal quarters and observed for a period of six weeks. The five
experimental groups had as the only source of liquid 1 percent Na N03
(control), 0.5 percent lead, 1 percent lead, 2 percent lead and 3 percent
lead respectively, all as the nitrate salt. The average liquid consumed
per week was tabulated from records of daily consumption, and the average
amount of lead consumed per week was calculated per one hundred grams of
body weight. During the six-week period, at weekly intervals, animals
from each of the experimental groups Were tested for glucosuria, righting
reflex, locomotor activity, awareness and observed for skin ruptures,
alopecia and hind leg paralysis.
Of all of these tests, only the observations of glucosuria, measured
using dextrostix (Ames Laboratories, Elkhart, Indiana), skin rupture and
alopecia were relatively free of bias. Awareness was assessed by counting
the movements within a three minute time span across a central line drawn
in the animal's cage. This parameter was measured only during the sixth
week of the study. -
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Experiment 3
The third set of animals consisted of sixty albino rats of the Sprague-
Dawley strain fifty days of age. They were divided into three groups
consisting of twenty animals each. One group of animals received one per
cent sodium nitrate as the only liquid source and served as the control
group. A second group received a 0.5 percent lead solution as the nitrate
as the only liquid source and the third group received a one percent lead
solution as the nitrate as the only liquid source. Previous and pre-
liminary studies had shown that nitrate administered to rats at the level
comparable to the level of nitrate being consumed in the one per cent
lead group was well tolerated. These animals were maintained for sixteen
weeks.
The handling of all animals at the kill time and subsequent treatment
of tissues were identical. Animals were weighed, placed under terminal
ether anesthesia and a blood sample was drawn via cardiac puncture with a
heparinized syringe. The liver, kidney, bone (femur) and brain were
removed and weighed. One hemisphere of the brain was placed into buffered
formalin. The other hemisphere was immediately homogenized, diluted to a
volume of 10mis with phosphate buffer, pH 7.4, and assayed for GADC
activity. Lead and protein analyses were performed on aliquots of this
homogenate. Representative samples of the livers, and kidneys, were placed
into buffered formalin, and the remainder of the tissues frozen until lead
and protein analyses could be performed.
The lead in all tissues was determined by atomic absorption spectro-
scopy using a Perkin-Elmer Model 303 with an air-acetylene flame. In this
procedure, the kngwn weights of tissue were wet-ashed using nitric acid,
and after an overnight digest in Brlenmeyer flasks, perchloric acid was
added to the samples as gentle heat was applied until a clear solution was
obtained. Digested samples were quantitatively transferred to volumetric
flasks and diluted to volume. When the lead level was too low to give
reliable absorbance on the AAS 303, an aliquot of the sample solution was
buffered to form a stable complex with ammonium pyrrolidine dithio-
carbamate and the stable complex was extracted with methyl isbutylketone.
This enhances the sensitivity of AAS for heavy metals.
Known aliquots of brain tissue homogenates were solubilized in hot
KOH and protein determined. Brain hemispheres were removed intact by
cutting the skull,weighed,and immediately placed into ice cold 0.02M
phosphate buffer, pH 7,4, They were homogenized with a Potter-Elvejhem
all glass homogenizer after the concentration was adjusted to one gram of
tissue per 8.6 mis of buffer.
From this homogenate, 5 mis were pipetted into the outside chamber
of Warburg Flask into which 20 ul of glutamic acid-l-^C (880,000 dpm) and
0.7 ml of 0,4 per cent non radioactive sodium glutamate had been previously
introduced. They flasks were gently agitated to assure mixing of the
contents, and 0.24 ml of 16N H2S04 were placed into the side arm of the
flask.
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A multifolded strip of Whatman No. 1 filter paper (3x5 cm) which
had been saturated with Hymine hydroxide were placed into the center well
of the flasks, the filasks gently flushed with nitrogen,capped with a glass
stopper and placed into the metabolic shaker which contained water at 37°C.
At the end of a thirty minute incubation period the H2S04 was emptied
from the side arm into the reaction chamber of the flask in order to stop
the reaction. The flasks were than reincubated for an additional fifteen
minutes at 37°C. This enabled the HCO^ in the solution which formed upon the
acidification by the 1*2804,to be converted to:.CO2. The C02 was trapped
onto the Hymine hydroxide impregnated filter paper. The filter paper was
then placed into a scintillation counting vial which contained 15 mis of
scintillation counting solution. The vials were counted for radioactivity
after standing for a minimum of eight hours.
Histologic slides were prepared using the routine hematoxylin and
eosin stains and observed under the light microscope for evidence of
nuclear damage and cellular necrosis.
The calf brains form which the GADC was isolated were obtained from
a local supplier minutes after the animals was killed. They were
immediately brought to the laboratory and the isolation procedure initiated
as detailed by the flow digram in Figure 2.
The Supernatant IV (Figure 2) was placed on a 45 x 2.5 cm column and
fractions of eluant collected in three ml aliquots. Absorbance was
measured at 280 nM using a Beckman Model 25 spectrophotometer, and assays
for GADC carried out on selected tubes. The GADC rich fractions were
pooled, lyophylized, and stored in the forzen state in a buffer containing
glutatione and pyridoxal phosphate each at a concentration of 10 moles/
liter. An aliquot of this preparation was subjected to electrophoresis
using a Beckman Microzone System with a cellulose acetate membrane, pH 8.6.
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Section 6
RESULTS AND DISCUSSION
Animal Studies
Data collected from rats in experiment two is summarized in Tables
1 and 2. In Table 1, information is presented showing the toxicity of lead
nitrate. When the lead as the nitrate was available in the drinking water
at 2 grams percent or above, death occurred within two weeks for twenty-
eight of thirty female rats of the Sprague-Dawley strain. The consumption
of lead solution was less by the experimental groups when compared to the
nitrate control group over the six week experimental period. Rats receiving
0.5 percent lead solution and those receiving one percent lead solution
gained weight. Those receiving 2 percent lead lost nearly half of their
initial weight before dying, and those receiving 3 percent lead died with-
in three days without any weight loss. Only two of fifteen animals
receiving the 2 percent lead survived for the six week period.
With reference to the growth data, the averages of the initial weights
of the nitrate controls, the 0.5 percent test group and the one percent
test group, suggest that the lead might abate the slight growth supression
effect of the nitrate. The environmental implication of this observation
merits further inquiry. Heavy metals presented to the atmosphere
are generally accompained by the release of nitrates and sulfates or the
oxides which may be converted to various nitrogen and sulfur oxygen con-
taining anions. Such anion production might have the overall effect or
reducing environmental lead toxicity. To be sure, this is highly
speculative and does not negate the environmental impact of these anions
as bio-hazardous substances.
The parameters observed in the animals were glucosuria, presence of
righting reflex, skin rupture, alopecia, locomotor activity and awareness.
The observations were made on the animals at weekly intervals and no
significance could be attached to any of the data except the awareness.
Animals receiving the one percent lead were less aware than the nitrate
control groups. This parameter was measured by counting the number of
times the animals crossed a line drawn on the bottom center of the cage.
The number of movements across the line per three minute periods were
recorded and the average calculated. Seven animals from each group were
utilized. The number of movements for the nitrate control group was 4.8
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per three minute time period, while the group receiving 0.5 per cent lead
crossed the line an average of 5 times and the group receiving one per cent
lead crossed the line an average of 2.6 times. This implies that the group
receiving one per cent lead was less aware than the other groups.
The GADC activity observed in brain homogenates v/as somewhat inversely
related to the quantity of lead in the solutions that the animals had
received. The findings are summarized in Table 2, The evidence shows
that lead accumulated in the brain tissue when given at the 0.5 per cent
level or any level.
In the third experiment, rats were placed on the experiment at fifty
days: of age. The average amount of lead consumed (recorded in grams of
lead per 100 grams of animal weight over a sixteen week period as calculated
from the volume of lead solution consumed) fluctuated in a much more pro-
nounced manner for the one per cent lead group than for the 0.5 per cent
lead group. The pattern is presented in Figure 1. Also, as the time of
the experiment progressed, the lead intake for the one per cent group
generally decreased such that after sixteen weeks on the experiment the
average lead consumption was down to approximately thirty-five per cent
(0.31 g Pb/week/100 gms) of the high of 0.86 gm Pb/week/100 grams.
The attrition schedule was acceptable as deaths were not limited to
any single experimental group and the deaths of the rats occurred throughout
the entire course of the study. Table 3 gives the attrition schedule.
The growth rate depression effect of lead is seen in Table 4. While
there was wide variation in the final weights of animals within the epxeri-
mental groups, the trend observed was that the group receiving the one per
cent lead weighed less than the other two group.
In Table 5, the results were presented showing the effect of lead on
the weights of the liver, kidney, femur and brain. The weights of these
tissues are expressed as grams per one hundred grams of animal weight. No
significant differences were observed for any of the tissues studied,
except the general trend suggests that a smaller femur resulted from the
lead consumption.
Tables 6 shows the distribution of lead in these tissues. As
expected, the blood lead levels were higher in the one per cent lead group.
Whiles the lead concentration was only twice as high in the liquid of this
group as compared to the 0.5 per cent group, the lead level in the blood
in the one per cent lead group was three times as high 477 ug%) as the level
in the 0.5 per cent group (23 ug%). Some lead was found in the blood and
all other tissues of the control group. This lead is no doubt of dietary
orgin. (Purina Laboratory Chow which the animals were fed contained 34 ug
Pb per gram). The femur of the control group contained as much lead as
did the 0.5 per cent lead test group and only slightly less than the one
per cent lead test group. (28, 28 and 33 ugs Pb/gram dry weight,
respectively). This finding suggests that there was adequate lead in the
animal feed to saturated the capacity of the bone to store lead. Further
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support of the finding is the increased levels of lead found in the brain
and kidney tissues of both test groups. Only the one per cent lead group
showed accumulated lead in the liver. The lead level in the
kidney probably represents an accumulation associated with attempts to
excrete as well as store it, but once the renal capacity is surpassed, the
storage and excretion via the liver occurs. Of the three soft tissues
analyzed for lead, brain and kidney accumulated significant amounts of
lead in the 0.5 per cent grams but liver did not. In the one per cent
group, the kidney contained more lead than did the kidney of animals from
the 0.5 percent lead group. These findings suggest that brain and kidney
are among the soft tissues capable of accumulating lead at low consumption
levels if the rate of consumption is high enough to elevate blood lead
significantly.
When the brain homogenates of the rats were assayed for GADC activity
and the protein content of the homogenate determined, it was observed that
the activity of the enzyme was less in homogenates of rats given the higher
lead solutions. These findings are presented in Table 7.
Enzyme Studies
The procedure of Susz et al, (2) was used for isolation of the enzyme
from calf brain in a 500-fold purification. This value was determined by
measuring the activity of the enzyme at different stages of isolation and
purification beginning with the brain homogenates and expressing the
enzyme activity as the radioactive llfC liberated per unit weight of protein.
The homogeneity of the isolated protein was assessed using Microzone
electrophoresis, pH 8.6, with cellulose acetate membranes as the support
system. The homogeneity, it was concluded, was sufficient such that the
protein could be used for enzyme studies. Figure 3 shows the electro-
phoretic pattern obtained using this system and comparing it with a control
specimen. Using the data in Table 8 the Vmax and Km of the enzyme pre-
paration was claculated. Based on data presented in Table 8 the Vmax was
1034 counts per minute per microgram of protein and the Km obtained by
extrapolation was 4.5 x 10~ moles of glutamic acid per milliliter. When
the first seven data points were placed on the Sigma 7 Computer and the
Vmax and Km determined by use of a program first adapted by W. W. Cleland,
the Vmax was determined to be 938 CPM per microgram of protein and the Km
was 3.6 x 10"^ moles of glutamic acid per ml. Susz (2) reported that the
Km of the GADC isolated from rat brain to be 3.9 x 10 moles of glutamic
acid per liter. The difference may be due to species, since bovine brain
was used in this study, and/or the differences may be due to different
degrees of purity of the enzyme preparation.
In Table 9 the results showing the effect of lead on calf brain
GADC activity in vitro is given. It is noted that substrate inhibition of
the enzyme occurs as suggested by the data in column I; however>at all
substrate concentrations below 2.01 x 10"^ moles per liter, 10 mole of
Pb of lead inhibited the enzyme. It may be that the higher lead concen-
trations complex with the substrate making less lead and substrate avail-
able to inhibit the enzyme.
10
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An experiment designed to show the comparative inhibitory effects of
Pb,, Cd and Se on GADC activity in_ vitro showed that Pb is the most potent
inhibitor of the three metals and Se the least potent^ Table 10 give the
per cent inhibition of the three metals on the enzyme.
The data presented indicates that iri vitro lead inhibits GADC and
that this inhibition may be overcome by addition of substrate. Such
reversal of inhibition is expected for competitive inhibitors.
This study has shown that the levels of the activity of the enzyme,
GADC which may be detected in rat brain homogenates given sub-lethal
levels of lead in their drinking water is decreased. This may be
associated with decreased synthesis of GABA; however, the quantitation of
brain GABA in the brains of animals so treated was not done.
Many researchers have shown associations of lead with disturbed
CNS function and amine metabolism (7,8,9, 10), and the role of GABA in
synaptic transmission in the CNS seems to be clearly established (11, 12).
One of the clinical symptoms of acute lead toxicity is the onset
of convulsions. These convulsions may be terminated by the use of com-
plexing agents as penicillamine and/or versene (13). We speculate that
the removal of the lead by these complexing agents results in reactivated
GADC and as GABA is endogenously formed, the convulsions cease.
Apparently in gradual depletion of GADC activity, there are no
convulsions. Such has been the observation of Bayoumi et. al^ (14). They
showed that in the brains of rats placed on Bg deficient diets and a Bg
antagonist (4-deoxypyridoxine), while both groups of animals
had lower GADC activity, no convulsions occurred. This may be explained
by the fact that GABA did not get to some critically depletedlevel,or that
other neuroinhibitory transmitter substances may have been produced which
protected the animals from convulsions.
11
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REFERENCES
1. Lupein, P. J., C.M. Hinse and L. Berlinguet. Determination of Glutamic
Acid Decarboxylase Activity in Rat Brain. Analytical Biochemistry 24;
1-8, 1968.
2. Susz, Jean P., Bernard Haber and Eugene Roberts. Purification and Some
Properties of Mouse Brain L-Glutamic Acid Decarboxylase. Biochemistry
5_: 2870-2877, 1966.
3. Lowry, O.K., N.J. Rosebrough, A.L. Farr and R. J. Randall: Protein
Measurement with the Folin Phenol Reagent. J_. Biol. Chem. 193; 265-
275, 1951.
4. The Perkin-Elmer Corporation. Analytical Methods for Atomic Absorption
Spectrophotometry. Lead Standard Conditions and Lead in Blood. 1968.
5. Proskey, Leon and R. G. O'Dell. Effect of Dietary Monosodium L-Glutamate
on Some Brain and Liver Metabolites in Rats. (35930). P.S.E.B.M; 138
517-522, 1971.
6. Roberts, Eugene, and Sam Frankel. -Amino Butyric in Brain: its
Formation from Glutamic Acid. J_. Biol. Chem; 187, 55-63, 1950.
7. Goyer, R. A. and B.C. Rhyne. Pathological Effects of Lead. Int. Rev.
>. Path 12:1-77, 1973.
8. Thomas, J. A., F.D. Dallenbach, and M. Thomas. Considerations on the
Development of Experimental Lead Encephalopathy Virchows Arch. (Pathol.
Anat): 352; 61-74, 1971.
9. Souerphoff, Mitchell W. and I. A. Michaelson. Hyperactivity and Brain
Catecholamines in Lead-Exposed Developing Rats. Science 182; 1022-1024,
1973.
10. Goiter, M. and I. A. Michaelson. Growth Behavior and Brain Catechol-
amines in Lead-Exposed Neonatal Rats: A Reappraisal. Science 187;
359-361, 1975.
11. Roberts, Eugene and K. Kurimaya. Biochemical-Physiological Cor-
relations in Studies of the Gamma-Aminobutyric Acid System. Brain
Research 8; 1-35, 1968.
12. Krnjevic, K. Micro-Ionophoretic Studies on Cortical Neurons. Int.
Rev. Neurobiol; 7:41-98, 1964.
12
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13. Chisolm, J. J. and E. Kaplan. Lead Poisoning in childhood. Compre-
hensive Management and Prevention. J. Pediatr. 73: 942-950, 1968.
14. Bayoumi, R.A., J. R. Kirwan and W.R. Smith. Some Effects of Dietary
Vitamin Bg Deficiency and 4-Deoxypyridoxine on -Amino butyric Acid
Metabolism in Rat Brain. Journal of_ Neurochemistry; 19; 569-576,
1972.
13
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BIBLIOGRAPHY
1. Carson, T.L., Gary A. Van Gelder, George C. Karas, and William B. Buck.
Slowed Learning in Lambs Prenatally Exposed to Lead. Arch, of_ Environ
Health 29; 154-156, 1974.
2. Haber, Bernard, K. Kyriyama, and E. Roberts. Mitochondrial Loca-
lization of a New L-Glutamic Acil Decarboxylase in Mouse and Human
Brain, Brain Research 22; 105-112, 1970.
3. Patel, A.J., I.A. Michaelson, J.E. Cremer and R. Balazs. Changes
within Metabolic Compartments in the Brains of Young Rats Ingesting
Lead, Journal of Neurochemistry. 22; 591-598, 1974.
4. Sauer, R.M., B.C. Zook, and P.M. Garner. Demyelinating Encephalo-
myelopathy Associated with Lead Poisoning in Nonhuman Primates.
Science; 169, 1091-1093, 1970.
5. Tapai, R., Pasantes Herminia, and G. Massieu. Some Properties of
Glutamic Acid Decarboxylase and the Content of Pyridoxal Phosphate
in Brains of Three Vertebrate Species. Journal of_ Neurochemistry; 17,
921-925, 1970.
6. The Medical Clinics of North America. Trace Elements. 1976. Vol.
60, No. 4 W. B. Saunders Company, Philadelphia, Pa. 19105. Albert
M. Meier, Editor.
14
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TABLE 1. EFFECT OF LEAD ON THE WEIGHT OF RATS
Group
Control
0.5% Pb
1.0% Pb
2.0% Pb
3.0% Pb
Number of
Animals
10
15
10
15
15
Average Volume Liquid Average Pb
Consumed/week (ml) Consumed *
157 0
145 0.42
137 1.29
#
##
Average g
Beginning Wt
181
202
189
182
163
Average g
Terminal Wt
186
228
243
103
163
* In grams per week per 100 g body weight
# Thirteen animals expired within two weeks.
## All animals expired within one week.
- No data collected.
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TABLE 2. GADAC ACTIVITY IN RAT BRAIN HOMOGENATES
MEASURED AS cpm lf+C02 RELEASED
Group CPM/gram Brain
1.0% (14)* 3114
0.5% (13)* 2225
1.0% (11)* 1915
2.0% (3)* 947
* Number of animals in each group. Animals sacrificed after
six weeks.
16
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TABLE 3. ATTRITION SCHEDULE OF ANIMALS RECEIVING LEAD AS LEAD NITRATE
Treatment 1
0.0%Pb 0
0.5%Pb 2
1.0%Pb 1
WEEK
2 3 45 6 7 8 9 10 11 12 13 14 15 16 Total
0 00 0 1 001 0 011000 4
0 0 0 0 0 200 0 000010 5
0 0 0 0 0 000 1 000200 4
TOTAL 3000012001 001210 13
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TABLE 4. THE EFFECT OF LEAD THE WEIGHT OF RATS
Group
Volume Liquid
Consumed/Week
(mis)
Average Lead
Consumed*
Initial
Weight
(g)
Terminal
Weight-16 wks
(g)
00
1.0% NO,
as
133
71 + 6
166 + 24
0.5%Pb
1.0%PB
85
83
0.18 + .003
0.47 + .22
79 + 5
80 + 5
155 + 27
146 + 37
In grains per week per lOOg body weight.
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TABLE 5. EFFECT OF LEAD ON ANIMAL TISSUE WEIGHT
v£>
Sacrifice
Weight (Cms)* LIVER^ KEDNEY# FEMUR (DRY)# BRAIN*
Group
1% NaN03 166+24 3.4 + 0.7 1.1+0.2 0.56 +_ .02 1.2 + 0.4
0.5% Pb 155 +_ 27 2,8+_0.6 1.3+_0.2 0.46 +_ .04 1.1+_0.2
1.0% Pb 146 + 37 3.0 + 0.6 1.2 + 0.4 0.44 + .03 1.1 + 0.3
* After 16 weeks
# Expressed as gms/100 gms animal weight
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TABLE 6. CONCENTRATION OF LEAD IN BLOOD AND TISSUES
to
o
Group Whole Blood*
1.0% NaNO3 9 + 0.01
0.5% Pb 23 + 0,02
1.0% Pb 74 H^ 0.03
TISSUE (ug Pb/g)
Femur Brain Kidney
28 +_ 1.2 0.36 0.4 +_ 1.4
28 +_ 5.6 2.0 18 +_ 5.0
33 +_ 6.8 1.7 25 +_ 2.0
Liver
0.07 + 0.01
0.08 + 0.06
0.52 + 0.07
*Micrograms Pb per 100 mis blood.
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TABLE 7. GADC ACTIVITY IN RAT BRAIN HOMOGENATES
MEASURED AS CPM ^CC RELEASED
Group CPM/gram Brain
1.0% NaN03 3403
0.5% Pb as N03 2177
1.0% Pb as N03 1750
2.0% Pb as N03* 1169
Pb ug/gram Brain CPM/ug Pb
0.36 9453
2.0 1089
1.7 1029
1.3 899
* Only two animals. All other data represent the average of six animals.
21
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TABLE 8. KINETIC STUDIES ON CALF BRAIN GADC
IN VITRO
x 10~4 M/Liter
.200 =
.400 =
.900 =
1.900 =
3.800 =
5.700 =
9.500 =
11.400 =
13.300 =
1
s
5.0
2.5
1.1
0.5
0.3
0.2
.14
.08
.08
ACTIVITY I
CPM*
304.52 =
495.58 =
707.81 =
801.09 =
822.31 =
869.68 =
919.06 =
661.86 =
541.15 =
( 1 )
V
1/CPM
0.003.0
0.002.0
0.001.4
0.001.2
0.001.2
0.001.1
0.001.0
0.001.5
0.001.8
* CPM per microgram of enzyme protein
22
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TABLE 9. EFFECT OF LEAD ON CALF BRAIN GADC ACTIVITY IN VITRO
to
u>
(S) x 10~b mole/liter
0.134
0.330
0.670
1.340
2.010
2.680
CONTROL
CPM/mg Protein x 102
12.70
11.74
5.70
3.58
0.30
Background 16 cpm
10~4M Pb CPM/mg Protein x 102
1.77
0.32
0.60
0.62
6.15
19.23
Background 16 cpm
* 1
Each reaction chamber contained 26 micrograms of purified enzyme protein preparation.
-------
TABLE 10. COMPARATIVE INHIBITION EFFECT OF LEAD, CADMIUM
AND SELENIUM ON GADC ACTIVITY
• • . ••.••.-.•.• .v -..-. .• - • • • ^ . • . - •
Concentration
2 x 10~2M
2 x 10~3M
2 x 10~4M
2 x 10"5M
0
Percent inhibition
Pb* Cd* Se*
61 45 3
42 27 10
31 17 8
24 02
- _ _
* Prepared as Nitrates and Buffered to pH 7.4 with 0.02 M Phosphate
24
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Figure 1. AVERAGE LEAD CONSUMPTION DURING EXPERIMENTAL PERIOD
1.0-
0.9-
0.8-
0.7-
o
o
0.6H
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Figure 2. GADC ISOLATION AND PURIFICATION
I. FRESH CALF BRAIN
1. Prepare Brain Homogenate in 0.25M Sucrose
0.02M K2HP04 buffer at pH 7.4
2, Centrifuge at 900 x g for 15 minutes
1
II. SUPERNATANT LIP ID
(discard)
Centrifuge at 55,000 x g for 60 minutes
i ' i
III. MITOCHONDRIAL PELLET SUPERNATANT
(discard)
1. Osmotic Rupture in 0.02M K2HP04 Buffer pH 7.4
2 . Homogenize
3, Centrifuge at 100,000 x g for 60 minutes
- , - - ^
IV. SUPERNATANT PERCIPITATE
(discard)
1. Add pyridoxal Phosphate (10'%) and Gluta-
thione (10~4M)
2. Run through Sephadex Gel Filtration Column
-upper 1/3, G-200: lower 2/3, G-100 using
0.02M K2HPO4 pH 6.5 with pyridoxal phosphate
and Glutathione (at 10"^M) .
3. Pool active fractions and concentrate by lyo-
philization
26
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Figure 3. ELECTROPHORETIC PATTERN OF GADC PREPARATION COMPARED TO A
VERSATOL-N CONTROL
- GADC PREPARATION
- VERSATOL-N
ORGIN
RELATIVE MIGRATION TOWARD POSITIVE ELECTRODE
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO. |2.
EPA-600/1-78-009 1 .._
4. TITLE AND SUBTITLE
Effect of Lead on Gamma Ami no Butyric Acid
7. AUTHOR(S)
Henry Archie Moses
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Department of Biochemistry and Nutrition
School of Medicine
Meharry Medical College
Nashville. TN 37205
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory RTF
Office of Research and Development
U.S. Environmental Protection Agency
Research Trianale Park. N.C. 27711
15. SUPPLEMENTARY NOTES
3. RECIPIENT'S ACCESSION- NO.
p. RFPom OATF
Synthesis .l.-4im;it-.y .-IM/O
•' h. r'tHhOhMIISM OhiiANIlA'l ION I . .lit
8. PERFORMING ORGANIZATION REPORT NO.
10. PROGRAM ELEMENT NO.
1EA615
11. CONTRACT/GRANT NO.
R-802370
13. TYPE OF REPORT AND PERIOD COVERED
NC
'"" 14. SPONSORING AGENCY CODE
FPA eon/n
16. ABSTRACT - ' —
This project was a study of the inhibitory effect of lead on the
enzymatic activity of brain Glutamic Amino Acid Decarboxylase (GADC) . The
enzyme is responsible for the catalytic formation of gamma amino butyric
acid (GAB A) inhibitory neurons which is believed to be involved with the
transmission of inhibitory impluses in the brain.
Lead nitrate solution was available to Sprague-Dawley female rats
ad libitum and the quantity of lead ingested was determined by the volumes
consumed. During the course of these experiments, animals were examined
for weight loss, activity, and excitability and other behavioral manifestat-
ions of lead toxicity. At autopsy, the tissues selected for lead determin-
ations were brain, liver, bone (femur) . In another series of experiments
GADC was isolated from fresh bovine brain tissue, and in vitro studies were
performed to determine the nature of lead inhibition of the enzyme. Subse-
quently, Se and Cd inhibition of the enzyme were studied and compared to
lead inhibition. Light microscopic studies of liver, brain and kidney tis-
• sues were performed.
The activity of the enzyme GADC in brain tissue homogenates of rats
drinking lead nitrate solutions was less than the activity determined in
control rats. The Vmax of bovine brain GADC using Glutamate as a substrate
was 1.54 x 10s and the Km was 3.2 x 10~4 moles/liter. When the inhibiting
effect of Pb, Cd, and Se on GADC activity were compared, Pb proved to be
the most potent inhibitor, while Cd showed less inhibition, showed no inhi-
bition of enzyme activity.
17. KEY WORDS AND DOCUMENT ANALYSIS
a. DESCRIPTORS
lead (metal)
enzymes
inhibitors
brain
18. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
b.lDENTIFIERS/OPEN ENDED TERMS C. COSATI Held/Group
06 A, T
19. SECURITY CLASS (This Report) 21. NO. OF PAGES
UNCLASSIFIED 37
20. SECURITY CLASS (This page) 22. PRICE
UNCLASSIFIED
EPA Form 2220-1 (9-73)
28
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