EPA - 560/6-76-<
UPTAKE, EXCRETION, AND PHYSIOLOGICAL
EFFECTS OF HEXACHLOROBENZENE IN
GROWING LAMBS
ENVIRONMENTAL PROTECTION AGENCY
Office of Toxic Substances
Washington, D.C. 20460
June, 1976
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Document is available to the
public through the National
Technical Information Service,
Springfield, Virginia 22151
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EPA -560/6-76-013
UPTAKE, EXCRETION, AND PHYSIOLOGICAL
EFFECTS OF HEXACHLOROBENZENE IN
GROWING LAMBS
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EPA-560/6-76-013
UPTAKE, EXCRETION, AND PHYSIOLOGICAL
EFFECTS OF HEXACHLOROBENZENE
IN GROWING LAMBS
By
Dr. Ronald L. Mull
Mr. Wray L. Winterlin
Dr. S. A. Peoples
Contract No. 68-01-2254
Project Officer
William A. Coniglio
Prepared for
Environmental Protection Agency
Office of Toxic Substances
Washington, D.C. 2046.0
June 1976
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This report has been reviewed by the Office of Toxic Substances, EPA,
and approved for publication. Approval does not signify that the contents
necessarily reflect the views and policies of the Environmental Protection
Agency, nor does mention of trade names or commercial products constitute
endorsement or recommendation for use.
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Table of Contents
I. Introduction 1
Reports of HOB in Humans 2
Reports of HCB In Animals 2
Experiments In Animals 3
II. Methods and Materials - General 9
A. Source and care of the animals 9
B. Preparation and administration of HCB 9
C. Blood parameters determination 11
D. Plasma Enzyme Analysis 12
E. In vivo metabolism of antipyrine 12
F. In vitro liver metabolism studies 13
G. Determination of HCB residues in various tissues 13
1. Biopsy procedure 13
2. Extraction procedure 14
3. GLC analysis 14
H. Histopathological procedures 15
III. Results and Discussion 16
A. Effect of Chronic HCB Feeding on Body Growth Rate of Lambs 16
1. Specific Materials and Methods 16
2. Results 16
3. Discussion 16
B. Toxic Effects of HCB Feeding 20
1. Specific Materials and Methods 20
2. Results 20
3. Discussion 20
C. Determination of Clinical Blood Parameters 20
1. Hematocrit 20
a. Results 20
b. Discussion 23
2. Plasma Protein 23
a. Results 23
b. Discussion 23
3. Other Clinical Blood Parameters 26
a. Specific Methods 26
b. Results 26
c. Discussion 26
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D. Plasma enzyme analysis after chronic and acute HCB
administration . 27
1. Alkaline Phosphatase 27
a. Results 27
2. Glutamic Oxaloacetic Transaminase 27
a. Results 27
3. Glucose-6-Phosphate Dehydrogenase 27
a. Results 27
4. Succiriic Dehydrogenase 33
a. Results 33
5. General Discussion ~ .33
E. In VJ.VQ antipyrine metabolism 38
1. Specific Materials and Methods 38
2. Results 38
3. Discussion 38
F. In vitro liver enzyme studies 43
1. Specific Materials and Methods 43
2. Results ' 43
3. Discussion . 43
G. Uptake and Decay Characteristics of HCB in Omental Fat and
Other Tissues 49
1. Specific Materials and Methods 49
2. Results 49
3. Discussion 53
H. Gross and Microscopic Pathological Changes seen after HCB.
Administration . 54
1. Specific Materials and Methods 54
2. Results .54
3. Discussion 57
IV. Bibliography . .58
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List of Tables
Table 1. Effects of HCB feeding at different dose levels on lamb weight ... 17
Table 2. Effect of HCB feeding for 90 days at different dosages on
Hematocrit 21
Table 3. Effect of HCB feeding for 19 days at 100 ppm on Hematocrit 22
Table 4. Effect of HCB feeding for 90 days at different dosages on
Plasma Protein 24
Table 5. Effect of HCB feeding for 19 days at 100 ppm on Plasma Protein ... 25
Table 6. Effect of HCB at different dose levels of feeding for 90 days on
Plasma Alkaline Phosphatase Activity 28
Table 7. Effect of HCB feeding at 100 ppm for 19 days on Plasma Alkaline
Phosphatase Activity 29
Table 8. Effect of HCB at different dose levels of feeding for 90 days on
Plasma GOT activity 30
Table 9. Effect of HCB feeding at 100 ppm for 19 days on Plasma GOT
Activity 31
Table 10. Effect of HCB at different dose levels of feeding for 90 days on
Plasma Glucose-6-Phosphate Activity 32
Table 11. Effect of HCB feeding at 100 ppm for 19 days on Plasma G-6-PDH
Activity 34
Table 12. Effect of HCB at different dose levels of feeding for 90 days on
Plasma Succlnic Dehydrogenase Activity 35
Table 13. Effect of HCB feeding at 100 ppm for 19 days on Plasma SDH
Activity 36
Table 14. Effect of HCB feeding on Antipyrine Half-life 39
Table 15. Average plasma levels of HCB in lambs fed HCB at 1.0 ppm during
days 0-90 42
Table 16. Effect of HCB feeding on N- and 0-demethylase activity and micro-
somal protein ..... 44
Table 17. HCB concentration in omental fat after HCB feeding at different
dose levels 50
Table 18. HCB residues in various tissues sampled immediately after 90 days
feeding at 0.00, 0.01, 0.1, and 1.0 ppm HCB 52
Table 19. HCB residues in various tissues sampled 210 days after termination
of 90 days feeding at 0.00, 0.01, 0.1, and 1.0 ppm HCB 55
Table 20. HCB residues in various tissues sampled immediately after 19 days
feeding at 100 ppm HCB 56
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List of Figures
Figure 1. Effect of chronic HCB feeding on lamb body weight gain. HCB
was fed days 0-90 19
Figure 2. Effect of 90 days feeding of HCB at 1.0 ppm, and 19 days feeding of
HCB at 100 ppm, on in vivo antipyrine metabolism 40
Figure 3. Effect of 90 days feeding of HCB at 1.0 ppm, and 19 days feeding of
HCB at 100 ppm, on hepatic N-demethylase activity 45
Figure 4. Effect of 90 days feeding of HCB at 1.0 ppm, and 19 days feeding of
HCB at 100 ppm, on hepatic 0-demethylase activity 46
Figure 5. Effect of 19 days feeding of HCB at 100 ppm on hepatic microsomal
protein concentration . 47
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Summary and. Conclusions
A 90 day feeding trial was performed in male crossbred (Targee/whiteface)
lambs with hexachlorobenzene at 0.01, 0.1, and 1.0 ppm daily. The results
showed:
1. There was no significant difference in the growth rates of any
group of lambs compared with control over a 306 day observation
period (HCB fed days 0-90);
2. The blood and blood forming tissues remained unaffected by the HCB
at these dosages, as assessed by standard clinical blood parameters;
3. Plasma alkaline phosphatase, glutamic oxaloacetic transaminase,
glucose-6-phosphate dehydrogenase, and succinic dehydrogenase ac-
tivities were not elevated by the exposure regimen;
4. Antipyrine metabolims measured in vivo was not significantly in-
creased from control after the 90 day trial;
5. In vitro N- and 0-demethylase activities were significantly in-
creased over control;
6. HCB accumulated in omental fat to a peak level approximately 10-12
times the intake level over 90 days feeding, and this fat concen-
tration is 50-100 times the level seen in other tissues analyzed;
7. HCB decayed from fat after cessation of administration with a half-
time of approximately 95 days;
8. No gross or microscopically visible lesions were caused by these
levels of intake.
A parallel 19 day feeding trial at 100 ppm HCB for 19 days (daily)
showed similar results, with the following exceptions:
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1. Growth rates were not measured;
2. In vivo antipyrlne metabolism was significantly increased over
control; and
3. In vitro 0-demethylase activity was not significantly increased
over control.
We concluded that HCB given daily at 0.01, 0.1, or 1.0 ppm for 90 days,
or at 100 ppm for 19 days, causes no detectable harmful effects to growing
male lambs.
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I. Introduction
Hexachlorobenzene (RGB) has become of concern because of its wide-
spread distribution as an environmental contaminant and a contaminant of
food products used for human consumption (1). This compound was initially
synthesized in France in 1946 (2). Since then, it has been used in many
countries to control bunt fungi (Tilletia spp.) in cereal crops (3), seed-
borne inoculum, soil-borne spores (4), and seed-borne flag smut (Urocystis
agropyri) (5), perhaps through inhibition of spore germination (6).
HCB is an halogenated benzene. The chemical structure of this compound
is shown below:
It is a white crystalline substance, water insoluble, easily sublimable
and nearly odorless. Its melting point range is 226-230°C; synthesis is
effected by direct catalytic halogenation of C.H..
o o
In order to investigate the metabolism, distribution and excretion of
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HCB, Mehendale and Matthews (7) administered C-labeled HCB to male rats
by the oral route. They found that less than 20% of the administered dose
had been excreted by 7 days. Over 90% of the stored HCB was retained in
fat, muscle, liver, and small intestine. These tissues each contained at
least one dechlorinated metabolite. Urinary excretion accounted for less
than 1Z of the total dose administered, and the urine contained at least 7
metabolites, including pentachlorophenol, pentachlorobenzene and tetra-
chlorohydroquinone (7). It was also demonstrated that microsomal prepara-
tions of liver, lung, small intestine, and kidney metabolized HCB to
dechlorinated products. These microsomal preparations produced penta-
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chlorophenol in the presence of nicotinamlde adenine dlnucleotide phosphate,
reduced form (NADPH). In the presence of uridine diphosphoglyceric acid
(UDPGA) and NADPH, the liver microsomal preparations resulted in disappear-
ance of pentachlorophenol. It was also found that a slight amount of HCB
was excreted in the feces (7). Parke and Williams (8) reported as early
as 1922 that HCB does not conjugate to glucuronic acids, ethereal sulphates,
or mercapturic acids.
Reports of HCB in Humans
There have been many reports of significant levels of HCB in the
human body (fat, milk and blood), including serious cases of human poisoning.
In the years 1955-1959, more than 3000 persons ate HCB treated wheat.
These people developed a "Porphyria Cutanea Tarda Syndrome" with symptomatic
photosensitization, porphyrinuria, hyperpigmentation and hypertrichosis,
hepatomegaly, weight loss, osteoporosis and enlargement of the thyroid
gland and lymph nodes (9,10,11 p. 463).
Significant levels of HCB have been found in human breast milk, as
reported by investigators in Australia (12,13), the Netherlands (14),
Germany (15), and Switzerland ((16). HCB has also been found in human
blood (17), and in perirenal and other adipose tissue (15,18,19,20). In
all of these studies HCB levels ranged from trace amounts to 8.2 ppm. The
sources of HCB residues in the aforementioned cases were water, cereals
(16), milk (21), milk products (22), eggs (18), wild birds (23), meat and
poultry fat (24), and beans and potatoes (16).
Reports of HCB in Animals
The most serious cases of HCB residues in animal fat in the U.S. were
reported in Central Louisiana (cattle) (25), and in Western Texas and
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Eastern California, where the United States Department of Agriculture
(USDA) detected the residue in slaughtered sheep (26). Appreciable residues
of HCB have also been found in wild and domestic animals from all over
North America (27). Because of- these findings, the USDA asked the Environ-
mental Protection Agency (EPA) to set an interim tolerance limit for HCB,
which was set at 0.5 ppm for cattle, sheep, swine, goats and horses (26).
Experiments in Animals
Because of the importance of HCB, various experiments have been done
to investigate its distribution, metabolism, excretion, toxicity, rate of
accumulation, and placental transfer.
HCB has a relatively low toxicity to animals (3). In one study pigs
fed HCB treated wheat for more than 12 weeks showed no apparent harmful
effects (28). However, in these animals HCB was shown to accumulate with
time (29).
Avrahami and Steele (30) carried out a study in sheep dosed orally
for 18 weeks with 0.1, 1.0, 10, and 100 mg HCB per sheep per day. They
found accumulation of the drug in fat to a maximum level of 0.9, 7.5, 75,
and 650 ppm, respectively.
The same investigators reported that laying hens and growing chickens
exposed to the same dietary levels of HCB (0.01, 1.0, 10, and 100 ppm)
accumulated residues of HCB in the tissues according to the tissue fat
content (body fat, egg yolk, liver, and muscle). However, feeding HCB up
to 100 ppm did not effect the general health of the animals (31,32).
Studies have been carried out in the Netherlands involving broiler
chickens and Japanese quail. The chickens were dosed with 0.05 to 0.3 ppm
HCB for 7 weeks. This study demonstrated that the residue level in the
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fat was directly proportional to the level of HCB fed in the ration,
and that the concentration of HCB in fat leveled off by the end of the
fourth week. The Japanese quail received dietary concentrations of HCB of
0, 1, 5, 20, and 80 ppm for 90 days. Results showed that 0 and 1 ppm HCB
did not cause any effect, 5 ppm produced slight liver damage and caused
excretion of porphyrins in feces, while 20 and 80 ppm caused extensive
liver damage and death (33).
In feeding experiments with rats, death occurred when the HCB concen-
tration in brain reached 300 ppm (3.4). Liver damage with intracytoplasmic
inclusions was seen in rats fed a diet containing 0.2% HCB (35), and
severe porphyria accompanied with liver damage was induced in rats fed
(36) or injected (37) with HCB at 0.8 to 1.0 g/kg/day, and 20 mg/ml,
respectively.
In 1973 placental transfer of HCB was demonstrated in pigs (38). It
was shown that sows dosed with HCB transferred a considerable quantity of
residue to their offspring before birth. In addition, sows secreted HCB
in the milk in sufficient amount as to make the piglets accumulate signi-
ficant HCB residue in their bodies (38). These findings were later
confirmed by Villenueve et al. (39). These investigators dosed pregnant
rabbits orally with subtoxic doses of HCB of 0, 0.1, 1.0, and 10 mg/kg
over a period of 27 days. The fetuses showed HCB accumulation in fat,
liver, heart, kidneys, brain, lung, spleen, and plasma. It was noted that
no toxic effects were observed in these fetuses at any of these dose
levels (39).
Since HCB is a commonly used fungicide, and has been demonstrated to
localize and accumulate in mammals, it was felt advisable to conduct a
controlled experiment to evaluate quantitatively the kinetics of HCB
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uptake and excretion, and physiological effects. For this reason, we
chose to study the effects of HCB on growing lambs. To accomplish this
purpose, several characteristics were chosen for study. These included
growth rate, plasma enzyme activities and associated blood parameters,
liver drug metabolising enzyme activity, and HCB residue levels in animal
fat. Associated with these determinations were gross and microscopic
pathological examinations. It was felt that using this battery of analyses,
we could best determine the effects of chronic HCB administration to
growing lambs.
Some plasma enzymes have been used as indicators of organ integrity.
A high level of activity of an enzyme in plasma not normally found in
plasma is generally thought to be due to the release of intracellular
enzyme from damaged tissue (40). Four plasma enzymes were chosen in this
study as indicators in detecting tissue damage caused by HCB. These
plasma enzymes were alkaline phosphatase (AF), glutamic oxalacetic trans-
aminase (GOT), glucose-6-phosphate dehydrogenase (G6PDH), and succinic
dehydrogenase (SD).
Phosphatases are enzymes which hydrolyse phosphoric esters, releasing
inorganic phosphate. Two principal types of phosphatases are known in
blood, alkaline phosphatase, which has a pH optimum range between 9 and
/
10, and acid phosphatase, with a pH optimum of approximately 5 (41).
These are normally contained in the white blood cells. A second kind of
alkaline phosphatase has been reported recently by Neuman et al. (42) in
the serum of patients with lymphatic leukemia and infectious mononucleosis.
Plasma alkaline phosphatase is thought to be of liver origin, but is
is widely distributed in high concentrations in bone, intestinal mucosa,
and renal tubular cells as well. This enzyme is useful in the study of
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hepatic diseases. An elevated AP activity has been associated with nucleic
acid synthesis in reparative processes (43). AP also shows increasing
levels of activity in the blood of apparently health persons as they age
(44). This agrees with other studies carried out in different species (40).
GOT is found in high concentration in straited muscle. Therefore,
elevated levels in plasma can be a valuable tool to confirm diagnosis of
muscular degeneration (41,45). Additionally, serum GOT activity has been
reported in sheep with liver flukes (46,47) and in some cases of copper
poisoning (48). Thus, GOT activity can be used as a measure the level of
liver damage if no evidence of other organ damage exists. Like AP, GOT
also shows differences in activity associated with the age of sheep (49).
One characteristic of sheep is the absence of erythrocytic glucose-6-
phosphate dehydrogenase. This enzyme, synthesized in the liver, can
therefore also be used to assess liver integrity.
The last chosen plasma enzyme was succinic dehydrogenase, a mito-
chondria! enzyme found in the red or dark granular muscle fibers (50). It
was felt that plasma succinic dehydrogenase could be used as an indicator
of skeletal muscle degradation, since SDH would be released into the
plasma with cellular lysis.
In addition to these plasma enzymes, the hematocrit and plasma proteins
were periodically checked to assess any gross hematological effect of HCB.
The chronic administration of many drugs is characterized by gradual
decline of the steady state level of the drug because of its ability to
stimulate its own metabolism via enhanced enzyme activity (51). This can
have special significance in chronic toxicity studies. Signs of toxicity
present at the beginning of a drug administration regimen may disappear
after repeated dosage because the drug might stimulate its own metabolism
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and thus result in a decreased circulating level of the drug. This has
been demonstrated in growing rats, by evaluation of microsomal cytochrome
P-450 (52,53,54), and in dogs and monkeys by measuring antipyrine metabolism
in vivo (55).
As this project was concerned with chronic effects of HCB administra-
tion, a concept we explored was whether HCB could act as an inducer of
microsomal enzyme activity in growing lambs. To evaluate this, the activity
of two microsomal enzymes, N- and 0-demethylase, were measured in vitro.
The plasma half-life of antipyrine, metabolised via N-demethylation, was
also determined in these lambs in order to correlate in vitro findings
with the in vivo ability of these animals to metabolize a drug.
In summary, the specific alms of the present investigation were as
follows:
1. To evaluate the effect, if any, of HCB on the growth rate of
lambs;
2. To observe the lambs closely for any signs of systemic toxicity
from the drug;
3. To monitor standard clinical hematology parameters to determine
any gross effects of HCB on the blood or hematopoietic systems;
4. To monitor the plasma activities of the enzymes alkaline phos-
phatase, glutamic oxaloacetic transaminase, succinic dehydro-
genase, and glucose-6-phosphate dehydrogenase, and use these
activities as indicators of any biochemical lesions that the
drug might produce;
5. To measure the in vivo metabolism of antipyrine at various
times;
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6- To measure in vitro N- and 0-demethylase activities, and to
correlate any changes seen in vitro after chronic HCB admini-
stration to the in vivo metabolic studies:
7. To determine the rate of increase of HCB residue in omental fat
in the growing lambs, and to also measure the maximal resulting
concentration at each of several dosage levels;
8. To determine the decay characteristics of HCB residue levels
after abrupt cessation of exposure; and
9. To examine for any gross or microscopic pathological changes in
the lamb tissues by necropsy at various stages during and after
the HCB administration.
Although we were interested in all the above aspects of HCB toxicity,
our major emphasis was on the determination of uptake and elimination
kinetics of HCB in the growing lambs. We felt that it was important to
determine these kinetics at doses approximating what one might expect to
find in an environmental contamination situation, as from pesticide misuse.
It was also hoped that we could characterize any toxic effects of the HCB
administration by careful biochemical and pathological examinations.
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II. Methods and Materials - General
A. Source and care of the animals
50 neutered male cross-bred (Targee rams out of white-face ewes)
weanling lambs (4-5-months old), representative of those grown in
California's winter lamb industry, were purchased from the U.C. Hopland
Field Station and housed during the initial 90 days of the project in
runs which were divided between an indoor and an outdoor area. The
indoor portion of the area was swept clean five times a week. During
the final 210 days the sheep were housed in all indoor runs. These
runs were divided in half. One part was bedded with sawdust, which
was changed every other day; the imbedded portion was washed down
everyday.
B. Preparation and administration of HCB
HCB technical grade (BDH Chemicals Ltd.) was recrystallized four
times before dissolving it in Mazola corn oil previously analyzed to
be free of HCB (detection limit 4 ppb), at three concentrations:
0.04, 0.4, and 4.0 rag/ml. The purity was calculated to be greater
than 99.5 percent. The solution was placed in 000 size Lilly gelatin
capsules.
.All lambs were biopsed for HCB levels in omental fat (detection
limit 4 ppb) prior to being included in the experiment. They were
then randomly divided into 6 groups as follows:
1. Ten sheep in the control group, which was kept isolated from the
others, and was given a daily dose of corn oil of equivalent
volume to that received by the treated sheep;
2. Ten sheep which were given a calculated daily oral dose of HCB
at 0.01 ppm of diet;
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1U
3. Ten sheep which were given a calculated daily oral dose of 0.1
ppm HCB;
4. An associate group of 3 sheep.was placed with the 0.1 ppm group
but did not receive any treatment except for a daily dose of corn
oil of equivalent volume to that given the treated sheep. This
was done because contamination of control animals from association
with treated animals has been reported (30);
5. Ten sheep which were given a calculated daily oral dose of 1.0
ppm HCB; and
6. A second associate group of 5 sheep which was placed with the
1.0 ppm group. They were handled as was the first associate
group described.
The groups were placed in four separate runs:
1) control group alone; 2) 0.01 ppm group alone; 3) 0.1 ppm group and
associated 0.1 ppm group; 4) 1.0 ppm group and associated 1.0 ppm
group.
All sheep were fed alfalfa pellets shown to be free of HCB
(detection limit 4 ppb) by gas liquid chromatographic (GLC) analysis
prior to use. The daily feed given to each group was calculated to
increase the average weight 4.54 kg/lamb/month. The calculation was
based on the following equation derived by Garret et al. (56):
TON - 0.029 W3/4 (1 + 5.072 g)
TDN « Total Digestible Nutrients, kg/day
w - Average herd weight, kg
g Desired weight gain, kg/day. In our experiment, g « 0.151
kg/day
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TDN was then divided by 0.52, which was considered to be the
average metabolisable energy content of the feed.
The amount of HCB administered was based on the calculated
average weight of feed each sheep consumed per day. The HCB and/or
the corn oil were given daily during the first 90 days of the experiment.
After 90 days, 20 sheep were slaughtered for necropsy and
determination of HCB residues levels in various tissues. Liver samples
were also obtained for N- and 0-demethylation assays. Five sheep were
sacrificed from the control group, 3 from the 0.01 ppm group (one
lamb had died from infection in this group), 4 each from the 0.1 and
1.0 ppm groups, and 2 each from the control associates of the 0.1 and
1.0 ppm groups. HCB administeration on the remainder was stopped to
determine the temporal characteristics of the disappearance of HCB.
In the final 19 days of the project the 6 remaining associate
*
sheep (3 from each of the 1.0 and 0.1 ppm associate groups) were given
HCB at 100 ppm. Due to the large volume needed (9-12 ml), it was
necessary that the suspension of HCB be placed in the back of the
mouth using a syringe. The 5 remaining control sheep were given 10.0
ml of corn oil in the same manner.
C. Blood parameters determination (57)
1. Hematocrit was determined by use of standard microhematocrit
procedure on whole heparinized venous blood.
2. Plasma proteins were determined by measurement of total solids
of plasma using a Goldberg refractometer (American Optical
Company). This method has been reported to produce good agree-
ment with the Biuret reaction for nitrogen (58).
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3. The following additional blood parameters were determined according
to standard procedures (57): total RBC, total WBC, differential
WBC, total (Hb) and mean corpuscular (MCHC) hemoglobin, mean
corpuscular volume (MCV), erythrocytic sedimentation rate (ESR),
and clotting time.
D. Plasma Enzyme Analysis
Following venipuncture and collection of blood samples from the
jugular vein in a heparinized vacutainer, the following assays were
performed on plasma:
1. Alkaline phosphatase was determined using the method of Bessey,
et al. (59).
2. Glutamic oxalocetic transaminase was determined spectrophoto-
metrically using the method of Freedland, et al,(60).
3. Glucose-6-phosphate dehydrogenase was determined using the method
of Lohr and Waller (61).
4. Succinic dehydrogenase was assayed using the method of Freedland
(62).
E. In vivo metabolism of antipyrine
Following i.v. injection of 100 rag/kg antipyrine, blood samples
were obtained by venipuncture at 15, 30, .60, 120 and 180 minutes.
Plasma concentration was determined by the methods of Brodie, et al.
(63,64) as modified by Welch, et. al. (55).
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F. In vitro liver metabolism studies
N- and 0-demethylase activities were determined on the 10,000 xg
supernatant of 12.5% whole liver homogenate. Assays were by the
methods of Mazel (65, ch. 27). Microsomes were isolated according to
the scheme of Mazel (65, ch. 27).
G. Determination of HCB residues in various tissues
1. Biopsy procedure
The group of sheep to be biopsed, fasted overnight, was
placed in a holding pen and given 1 ml promazine HCl (i.m.) each
as a tranquilizer. The sheep were then secured on their backs
to a surgery board by tying their legs.
/©
The abdominal area was shaved and washed with Septisol**
(ft
and Betadine** scrubs. An area near the midline was chosen for
the incision and 3.5 ml of lidocaine HCl was injected subcut-
aneously. An oblique incision 4-5 cm long was made through the
skin and muscle layers. An incision 3 cm long was made through
the peritoneum, and using a spay hook, the omental fat was pulled
out through the incision and 10-20 grams excised. The peritoneum
and muscle were then sutured, and the skin sutured separately.
.The area was sprayed with buffered iodine and the sheep were then
returned to their runs.
For 3 days following surgery the temperature of each sheep
was monitored. If temperature rose above 408C, 4 ml of procaine
penicillin G was administered TM. Six to eight days after surgery
the skin sutures were removed and operated area cleaned and
inspected.
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Blood samples were taken every time fat samples were collected.
In addition, at the termination of the chronic feeding, samples
of brain, liver, and kidney were taken for HCB residue analysis.
2. Extraction procedure
HCB was extracted from fat according to the procedure of
Collet and Harrison (66). Extraction from plasma was according
to the method of Siyali (17).
Extraction from tissue was performed using the following
method:
10 g of tissue was homogenized with 50 g of anhydrous sodium
sulfate in 100 ml of hexane in a Polytron homogenizer. This
homogenate was filtered (liver and kidney) or centrifuged (brain)
and the filtrate/supernatant was hydrolyzed with 15-20 ml of
concentrated sulfuric acid. Brain samples required a second
hydrolysis due to the formation of emulsions. The mixture was
allowed to sit overnight to permit separation of layers. A 50
ml (5 g) aliquot of the hexane phase was concentrated to approxi-
mately 5 ml and transferred to a 1.5 cm x 10 cm PR grade Florisil
column prewashed with hexane. The column containing the sample
.was eluted with 30 ml of hexane into a round bottom flask. The
sample was then diluted or concentrated depending on the amount
of residue in the sample in preparation for GLC-EC analysis.
3. GLC Analysis
The samples were chromatographed on a Aerograph 204 gas
chromatograph equipped with an electron capture (tritium foil)
detector and a Honeywell strip chart recorder. The fat and tissue
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15
samples were chromatographed on a 1.8 m glass column, 3.2 mm i.d.,
packed with 10% QF-1 on 80-100 mesh Gas Chrom Q. The injector
and detector temperatures were 210°C and the column temperature
was 175°C. The plasma samples were chromatographed on a 1.8 m
glass column, 3.2 mm i.d., packed with 5% OV-210 on 80-100 mesh
Gas Chrom Q. The injector, detector, and column temperatures
were 200°C, 207°C, and 180°C, respectively. The reason for this
change in columns was due to interferences associated with the
plasma samples. The chosen plasma column was adequate for
separating these extranous peaks from the desired peaks. The
flow rate for the nitrogen carrier gas for both columns was 16
cm /min. HOB was quantitated using peak height calibrated from
a standard curve. Control and fortified control samples were
analyzed daily with the treated samples.
f
H. Histopathological procedures
Sheep were killed for necropsy by captive bolt or electrocution.
Samples of the following tissues were taken for histopathological
examination: brain, lung, myocardium, small and large intestine,
liver, kidney, adrenal, and mesenteric lymph node.
Tissues were fixed by immersion in 10% formalin. After mounting
in paraffin, sections 8-10 y were taken. Stain was hematoxylin and
eosin. The slides were examined by a veterinary pathologist.
-------
16
III. Results and Discussion
Included in each section is a brief statement of specific proce-
dures employed.
A. Effect of Chronic HCB Feeding'on Body Growth Rate of Lambs.
1. Specific Materials and Methods
The lambs were fed HCB as described earlier.
During the initial 90 days the sheep were weighed on
a commercial scale every week. Following this period the
sheep were weighed every other week. The average weight
of each group was then calculated.
2. Results
The group means + standard deviation are presented
tabularly in Table 1, and graphically in Figure 1. No
significant difference (p>0.05) was found between growth
rates of the experimental groups as compared to control.
3. Discussion
In the present study doses of HCB had no significant
effect on the lambs' growth rate. Other investigators (31)
have reported that the growth rate in sheep was affected
by as much as one-third at a dose of 100 mg HCB per day for
18 weeks. This difference was reported as significant
(p<0.05). However, when weight differences between their
sheep and ours are taken into account, this dose of 100 mg
HCB per day works out to approximately 110 ppm HCB per day
for 18 weeks - a dose about 100 times as concentrated, and
administered 130% as long, as our highest dosage level. In
our experiment the lamb growth rate was not affected by
dietary levels of HCB up to 1.0 ppm during a 90 day (13
week) feeding trial.
-------
TABLE 1
Effects of HCB feeding at different dose levels on lamb weight
Body weight for each group9 of animals expressed in kilograms as x + s.d.
HCB fed (ppm)
Time in days
after
beginning HCB
feedingb Control 0.01 0.1 0.1 Assoc. 1.0 1.0 Assoc.
0
17
24
31
38
45
52
58
67
80
87
94
109
122
136
151
165
175
193
208
41.7 + 5.1
44.3 + 5.1
45.7 + 5.1
47.1 +4.9
49.0 + 5.1
46.0 + 5.0
46.9 + 4.9
47.7 + 5.1
48.4 + 4.6
50.3 + 5.4
51.0 + 5.5
52.9 + 6.1
54.7 -1- 6.1
60.4 + 7.0
61.8 + 6.4
62.5 + 6.2
64.0 + 7.1
66.5 + 7.0
69.3 + 7.5
71.5 + 7.7
40.6 + 1.9
42.9 + 1.6
44.8 + 1.4
44.4 + 1.8
46.0 + 2.0
45.9 + 1.9
45.8 + 1.9
47.1 + 2.8
50.8 + 2.0
50.4 +2.1
51.3 + 1.7
53.7 + 2.0
55.2 + 3.2
57.4 + 2.7
58.2 + 3.6
59.0 + 3.6
59.5 + 4.1
62.3 +4.0
64.0 + 3.8
68.0 + 3.5
43.7 + 0.6
45.2 + 1.4
46.0 + 1.6
46.5 + 1.9
48.0 + 2.3
47.7 + 2.0
49.3 + 2.9
49.9 + 2.9
50.2 + 2.8
53.2 + 3.5
54.1 + 4.0
57.2 + 5.0
58.7 + 3.3
61.3 + 4.0
63.5 + 3.6
63.5 + 3.9
63.7 + 3.8
66.4 + 3.9
67.5 + 3.0
72.8 + 4.8
44.7 + 2.0
45.1 + 1.5
47.5 + 2:8
48.4 + 2.4
51.1 + 4.0
50.1 + 3.1
51.7 + 5.2
51.9 + 4.9
53.5 + 4.5
54.7 + 5.5
56.6 + 5.2
59.2 + 4.9
60.7 + 5.6
62.6 + 6.6
64.6 + 7.1
65.9 + 7.0
65.8 + 6.6
67.4 + 7.4
70.2 + 6.9
73.6 + 6.6
39.6 + 1.2
41.6 + 1.7
42.9 + 1.4
42.9 + 1.7
43.9 + 2.0
45.0 + 1.8
45.1 + 1.7
46.0 + 2.0
46.5 + 1.5
48.5 + 2.3
50.3 + 3.2
52.0 + 3.4
52.3 + 3.6
55.5 + 4.8
56.6 + 4.6
58.2 + 4.6
59.6 + 5.1
60.2 + 5.3
61.8 + 5.9
65.4 + 6.0
40.6 + 1.5
42.5 + 1.6
44.7 + 1.3
43.6 + 0.8
44.8 + 0.9
45.8 + 0.7
44.5 + 0.6
47.7 + 0.8
48.0 + 2.3
48.8 + 1.6
50.4 + 2.6
51.6 + 3.1
52.2 + 2.3
55.1 + 3.0
56.8 + 4.7
58.8 + 4.6
60.0 + 5.6
60.9 + 5.7
62.2 + 6.0
66.4 + 6.0
(continued)
-------
00
tH
TABLE 1 (continued)
Effects of HCB feeding at different dose levels on lamb weight
Body weight for each groupa of animals expressed in kilograms as x + s.d.
HCB fed (ppm)
Time in days
after
beginning HCB
feeding5
Control
0.01
0.1
0.1 Assoc.
1.0
1.0 Assoc.
222
237
251
264
280
292
306
73.3 + 8.4
74.8 + 9.0
77.3 + 9.4
78.6 + 10.1
76.3 + 9.2
82.4 + 10.2
86.5 + 10.4
70.0 + 3.6
71.1 + 4.4
73.4 + 4.6
75.6 + 4.9
73.8 + 4.6
77.9 + 4.5
81.9 ± 4.7
73.0 + 5.0
74.8 + 5.5
79.2 + 6.0
80.1 + 6.9
81.8 + 6.7
83.0 + 7.5
85.8 + 6.5
74.5 + 6.8
75.2 + 7.8
79.2 + 7.6
80.3 + 8.1
83.0 + 8.4
84.6 + 8.7
85.4 + 8.8
66.4 4- 5.9
66.0 + 5.4
69.1 + 5.4
70.4 + 5.9
72.0 + 5.3
73.1 + 6.2
76.8 + 6.8
66.3 + 7.9
66.0 + 7.2
67.9 + 7.9
69.2 + 6.0
71.2 ± 8.4
73.6 + 8.7
74.6 ± 6.8
a. Before day 90:
After day 90:
10 animals in control and each exposed group, 5 in each associate group,
6 animals in control and each exposed group, 3 in each associate group,
b. HCB was fed for 90 days,
-------
o\
H
U.U
0.16
0.15
]? QI3
8 0.12
r*i
6 0.11
|QIO
£ 0.09
2 0.08
§ 0.07
§ O-06
S 0.05
g 0.04
5 0.03
0.02
0.01
nnn
CONTROL
-
0.01
*
mmm
O.I
ppm
ppm
~ 1.0 ppm
mm
FIGURE I. Effect of chronic HCB feeding on lomb body weight (join. HCB wos fed doys 0-90
-------
20
B. Toxic Effects of HCB Feeding.
1. Specific Materials and Methods
During the course of this experiment the lambs were ob-
served closely for any gross signs of toxicity i.e., list-
lessness, vomiting, anorexia, etc.
2. Results
No signs of systemic toxicity were observed in lambs
fed 0, 0.01, 0.1, and 1.0 ppm HCB for 90 days, and observed
for 210 days after cessation of exposure. In the acute
feeding of 100 ppm for 19 days there were no apparent signs
of toxicity like those described by Sweeny (37) and
Nigogosyam (9) and San Martin de Viale, e£ al. (36).
3. Discussion
Mo signs of systemic toxicity were noted at any dose
level we administered. These observations, coupled with
the lack of histopathological abnormalities (see below),
led us to conclude the HCB in these doses was not appre-
ciably toxic to these sheep.
C. Determination of Clinical Blood Parameters.
1. Hematocrit
a. Results
The results of the chronic feeding study, expressed
as percent packed-cell volume, are shown in Table 2.
There was no significant difference in hematocrit
between control sheep and sheep fed HCB at different
levels (0.01, 0.1, 1.0 ppm) for 90 days.
Results of a similar nature were obtained after
feeding 100 ppm HCB to a group of sheep for 19 days
(Table 3).
-------
21
TABLE 2
Effect of HCB feeding for 90 days
at different dosages on Hematocrlt.
Days
0
6
13
20
27
34
41
48
56
64
69
75
82
Control
37.7 + 0.5
36.0 + 1.3
35.2 + 1.0
35.8 + 2.5
36.0 + 1.7
38.0 + 1.4
38.2 + 2.0
38.0 + 1.4
38.0 + 0.9
38.7 + 1.2
38.2 + 1.8
37.5 + 2.3
37.8 -I- 1.6
0.01 ppm
36.8 + 2.5
37.2 + 1.6
35.2 + 1.8
35.5 + 2.3
35.5 + 1.5
37.2 + 2.4
38.0 + 1.3
35.8 + 3.1
35.5 + 2.2
38.2 + 1.8
38.2 + 1.2
38.3 + 1.4
38.2 + 1.6
0.1 ppm
37.0 + 2.7
36.0 + 1.4
34.0 + 2.4
34.8 + 3.6
35.5 + 3.0
37.2 + 2.8
36.0 + 2.0
37.0 + 2.4
37.7 + 1.2
34.5 + 4.9
36.2 + 3.1
36.2 +3.3
36.7 + 2.6
1.0 ppm
38.8 + 2.0
36.7 + 1.2
36.2 + 4.0
36.7 + 1.0
36.8 + 3.2
36.5 + 3.9
37.0 + 5.8
36.8 + 2.6
39.5 + 4.1
36.3 + 3.9
37.8 + 3.8
39.0 + 2.8
38.8 + 3.1
Hematocrit expressed as % red blood cells. Each value represents the mean +
s.d. of 6 sheep.
-------
TABLE 3
Effect of HCB Feeding for 19 days at
100 ppm on Hematocrit.
Days Control 100 ppm
0
5
13
19
35.5 + 3.7
35.6 + 3.3
34.2 + 2.3
36.5 + 3.3
36.8 + 3.0
35.3 + 2.5
35.9 + 2.0
36.6 + 4.3
Hematocrit expressed as % red blood cells.
Each value represents the mean + s.d. of 6
sheep.
-------
23
b. Discussion
HCB did not influence hematocrit values in any of
the dosed groups. A decrease in hematocrit value has
been reported in Japanese quail fed HCB (33). In view
of this, it is notable that we failed to observe any
detrimental effect of HCB on the hematocrit even after
100 ppm. This discrepancy might be explained by species
difference.
2. Plasma Protein
a. Results
The effects of HCB feeding on plasma protein are
summarized in Table 4. Plasma proteins are expressed as
grams per 100 ml plasma.
During the 90 day period of HCB feeding at differ-
ent dose levels no significant change in the level of
plasma protein between control and HCB fed groups was
found. A similar result was also obtained when HCB was
fed 100 ppm to a group of sheep for 19 days (Table 5).
b. Discussion
The total plasma protein levels are normally main-
tained at a relatively constant level but may be de-
ranged in some cases due to excessive loss of protein,
as in liver disfunction due to the liver's inability to
synthesize protein. The protein concentration in plasma
also rises when water is lost and is decreased when
water is returned to the vascular compartment.
In our study the plasma protein was determined by
use of a refractometer. By this means, slight changes
in hydration can be detected. During the course of the
-------
24
TABLE 4
Effect of HCB feeding for 90 days at
different dosages on Plasma Protein
Days
0
6
13
20
27
34
41
48
56
69
75
82
Control
6.55 + 0.19
6.88 + 0.48
6.82 + 0.90
7.10 + 1.09
6.98 + 0.69
7.15 + 0.89
7.32 + 0.64
7.12 + 0.63
7.17 + 0.52
7.30 + 0.28
7.53 + 0.31
7.40 + 0.54
0.01 ppm
6.88 + 0.32
6.97 + 0.29
6.78 + 0.23
6.98 + 0.38
6.77 + 0.16
6.83 + 0.22
7. 02 + 0.20
6.98 + 0.22
6.85 + 0.35
6.95 + 0.05
6.98 + 0.12
6.82 + 0.04
0.1 ppm
6.79 + 0.22
6.48 + 0.41
6.80 + 0.27
6.85 + 0.48
6.90 + 0.45
7.02 + 0.37
7.20 + 0.43
7.05 + 0.35
7.00 + 0.28
7.07 + 0.58
6.97 + 0.54
7.03 + 0.31
1.0 ppm
6.70 + 0.48
6.67 + 0.43
6.45 + 0.47
6.75 + 0.27
6.73 + 0.20
6.88 + 0.31
6.95 + 0.38
6.88 + 0.45
6.88 + 0.40
6.80 + 0.23
6.78 + 0.27
6.95 + 0.27
Plasma protein expressed as gm per 100 ml. Each value represents the mean + s.d.
of 6 sheep.
-------
25
TABLE 5
Effect of HCB feeding for 19 days
at 100 ppm on Plasma Protein.
Days Control 100 ppm
0
5
13
19
6.82 + 0.19
6.56 + 0.20
6.45 + 0.21
6.62 + 0.32
6.65 + 0.18
6.48 + 0.20
6.41 + 0.50
6.52 + 0.38
Plasma protein expressed as gm per 100
ml. Each value represents the mean +
s.d. of 6 sheep.
-------
26
experiment we did not find great changes in hydration
levels or note clinical signs to indicate alteration in
these levels. Our results for plasma protein were con-
sistantly within the normal range of 5.5 to 7.5 grams/
100 ml (57). Therefore, we can say that HCB does not
alter the normal plasma protein level in growing lambs
after 90 days of treatment at 1.0 ppm, or after 19 days
at 100 ppm.
If Table 4 is examined closely, the sheep seem to
increase plasma protein with age. This trend is not
felt to be important here; perhaps this reflects a
natural maturation process. When again subjected to
daily analyses during the last 3 weeks of the study, the
percent protein was somewhat less than at 90 days (Table
5).
3. Other Clinical Blood Parameters.
a. Specific Methods
After 90 days of HCB administration blood samples
were taken for complete analysis by the Clinical
Pathology Laboratory. This was repeated at 300 days.
The following parameters were analyzed: total RBC,
total WBC, differential WBC, total (Hb) and mean
copuscular volume (MCV), erythrocyte sedimentation
rate (ESR), and clotting time (CT).
b. Results
No difference were observed between control and
experimental groups with regards to any of the measured
parameters at either 90 or 300 days.
c. Discussion
The lack of any observable difference between
groups would imply that HCB in doses up to 1.0 ppm for
-------
27
90 days, and 100 ppm for 19 days, has little or no ef-
fect on the blood forming tissues.
D. Plasma enzyme analysis after chronic and acute HCB administration.
Examination of the data in tables 6-13 will reveal an appar-
ent fluctuation in the activities of the serum enzymes in the con-
trol group from one sampling time to another. These same patterns
of fluctuation may be seen in the treated groups as well. These
changes were no doubt largely or entirely laboratory induced by
unrecognized differences in reaction conditions and by different
technicians doing the assays. The reader should therefore compare
only the data shown for any particular day of sampling with that
day's control values.
1. Alkaline Fhosphatase
a. Results
The effects of chronic HCB feeding on the activity
of plasma alkaline phosphatase are summarized'in Table
6.
No significant difference in alkaline phosphatase
activity were found between the control and HCB treated
groups. This was true at all levels of exposure for
the entire period of the 90 day treatment.
A similar result following 100 ppm HCB feeding for
3 weeks was obtained. The results of this study are
summarized in Table 7.
2. Glutamic Oxaloacetic Transaminase
a. Results
The effects of chronic HCB feeding on the plasma
GOT activity are summarised in Table 8. No significant
difference in plasma GOT activity was found between con-
trol sheep and-the sheep fed HCB (0.01, 0.1, 1 ppm) for
-------
28
TABLE 6
Effect of HCB at different dose levels of feeding for 90 days on
Plasma Alkaline Phosphatase Activity.*
Days
0
6
13
20
27
34
41
48
56
64
69
75
82
Control
24.03 + 9.34
17.51 + 4.94
21.08 + 10.21
16.87 + 9.26
21.78 + 11.54
14.53 + 6.46
18.10 + 5.08
16.47 + 6.31
24.46 + 11.25
27.99 + 14.51
17.12 + 5.34
5.00 + 1.87
5.66 + 2.24
0.01 ppm
28.50 + 10.83
20.79 + 8.51
22.02 + 9.01
14.58 + 6.77
23.05 + 7.82
18.76 + 9.16
25.45 + 10.63
18.47 + 8.61
24.78 + 7.28
34.82 + 4.98
32.16 + 7.01
9.57 + 2.59
10.49 + 2.39
0.1 ppm
27.58 + 11.01
25.59 + 11.68
19.45 + 12.85
15.67 + 7.79
20.15 + 13.45
16.08 + 5.90
19.69 + 13.31
15.27 + 4.25
24.03 + 6.92
21.80 + 5.13
23.26 + 6.14
5.98 + 3.18
6.71 + 4.11
1.0 ppm
16.23 + 3.66
21.66 + 2.77
15.54 + 4.81
14.77 + 4.03
18.10 + 4.38
14 .'09 -1- 6.58
17.24 + 6.59
13.40 + 4.06
20.03 + 6.22
21.81 + 5.21
22.16 + 4.55
5.33 + 1.54
6.34 + 2.37
*Alkaline phosphatase activity expressed in nmoles of product formed/min/ml
plasma. Each value represents the mean + s.d. of 6 animals.
-------
29
TABLE 7
Effect on HCB feeding at 100 ppm for 19 days on
Plasma Alkaline Phosphatase Activity.
Days Control 100 ppm
0
5
12
19
28.43 + 10.98
31.60 + 12.04
29.88 + 11.38
24.98 + 6.42
31.62 + 5.02
28.76 + 4.06
28.46 + 6.59
27.48 + 8.33
Alkaline phosphatase activity expressed in
nmoles of product formed/min/ml plasma. Each
value represents the mean + s.d. of 6 animals.
-------
TABLE 8
Effect of HCB at different dose levels of feeding for 90 days
on Plasma GOT Activity.
Days
0
6
13
20
27
34
41
48
56
64
69
75
82
Control
27.01 + 16.98
39.74 + 18.09
72.35 + 15.48
35.59 + 13.17
77.41 + 17.07
58.60 + 8.20
34.77 + 14.76
67.86 + 55.90
56.53 + 17.41
90.19 + 28.84
59.08 + 55.56
46.06 + 6.95
45.72 + 7.81
0.01 ppm
31.54 + 5.45
62.07 + 18.04
80.88 + 14.85
46.30 + 21.32
75.38 + 11.96
72.01 + 16.59
57.49 + 12.68
62.84 + 11.67
73.41 + 39.84
123.81 ± 70.27
41.38 + 4.63
52.23 + 7.52
55.85 + 13.84
0.1 ppm
30.87 + 7.77
43.36 + 28.41
58.02 + 12.06
48.57 + 8.73
70.90 + 11.86
66.56 -1- 19.34
39.84 + 10.03
44.71 + 6.90
35.11 + 15.58
70.03 + 5.93
57.54 + 52.81
39.93 + 7.86
50.98 + 16.30
1.0 ppm
34.15 + 15.96
23.49 + 8.01
85.90 + 31.64
29.08 + 20.74
76.30 ± 13.65
60.77 + 16.16
40.66 + 18.09
49.58 + 21.22
42.10 + 10.95
71.24 + 15.00
41.57 + 15.34
48.23 + 9.79
56.91 + 26.96
GOT activity expressed in nmoles of product formed/min/ml plasma.
represents the mean + s.d. of 6 animals.
Each value
-------
31
TABLE 9
Effect of HCB feeding at 100 ppm
for 19 days on Plasma GOT Activity.
Days Control 100 ppm
0
5
12
19
67.52 + 18.81
41.53 + 11.24
104.27 + 81.75
90.67 + 38.01
58.84 + 7.23
50.93 + 11.33
63.90 + 14.76
153.37 + 77.75
GOT activity expressed in nmoles of product
formed/min/ml plasma. Each value represents
the mean + s.d. of 6 animals.
-------
CM
CO
TABLE 10
Effect of HCB at different dose levels of feeding for 90 days
on Plasma Glucose-6-Phosphate Dehydrogenase Activity.
Days
0
6
13
20
27
34
41
48
56
64
69
75
82
Control
71.10 + 28.80
57.70 + 23.50
69.70 + 19.00
57.70 + 19.10
69.70 + 21.50
23.30 + 6.80
20.10 + 9.50
28.10 + 2.70
27.70 + 8.80
39.80 + 11.50
43.40 + 10.10
30.00 + 7.10
43.40 + 11.50
0.01 ppm
54.90 + 12.80
67.90 + 15.90
54.50 + 17.90
48.00 + 17.50
60.50 + 10.10
18.70 + 3.70
21.10 + 5.50
22.70 + 2.70
32.40 + 15.70
28.40 + 4.60
37.60 + 11.60
22.10 + 4.30
29.10 + 5.40
0.1 ppm
50.00 + 22.90
62.10 + 23.20
71.80 + 24.40
74.50 + 12.70
58.50 + 10.60
21.90 + 8.30
23.30 + 5.30
33.20 + 10.40
21.10 + 4.10
40.80 + 12.30
41.80 + 8.50
35.20 + 5.40
34.80 + 17.20
1.0 ppm
55.90 + 29.70
43.80 + 14.00
51.70 + 8.90
55.10 + 11.70
64.70 + 18.70
18.67 + 8.30
23.90 + 11.80
19.50 + 6.10
26.00 + 11.90
32.30 + 7.60
33.40 + 16.20
29.60 + 12.10
31.70 + 8.90
G-6-P.D. activity expressed in nomoles of NADPH formed/min/ml plasma each value
represents the mean + S.D. of 6 animals.
-------
33
90 days or 100 ppm for 19 days (Table 9).
3. Glucose-6-Phosphate Dehydrogsenase
a. Results
The effects of chronic HCB feeding at 0, 0.01, 0.1,
and 1.0 ppm on the plasma glucose-6-phosphate dehydro-
genase activity are summarized in Table 10. No signi-
ficant difference in plasma G-6-PDH activity was found
between control sheep and the sheep fed HCB (0.01, 0.1,
1 ppm) for 90 days or 100 ppm for 19 days (Table 11).
4. Succinic Dehydrogenase
a. Results
The effects of chronic HCB feeding on the plasma
succinic dehydrogenase activity are summarized in Table
12. No significant difference in plasma succinic de-
hydrogenase activity was found between control sheep and
the sheep fed HCB (0.01, 0.1, 1 ppm) for 90 days or 100
ppm for 19 days (Table 13).
5. General Discussion
Changes in the activities of a number of enzymes in the
plasma have been studied in man and animals suffering from
various diseases. An increase in the activity of an enzyme
in plasma not normally found in high levels is generally
thought to be due to the release of intracellular enzymes
from damaged tissue (40). Changes in plasma enzyme activity
due to liver malfunction can occur in 3 ways:
a. Disruption of hepatic cells resulting from necrosis or
altered membrane permeability can cause an elevation of
enzyme levels. GOT is a marker enzyme for this type of
liver disfunction.
-------
34
TABLE 11
Effect of HCB feeding at 100 ppm
for 19 days on Plasma G-6-PDH Activity
Days Control 100 ppm
0
5
12
19
36.58 + 6.93
26.13 + 4.42
32.56 + 8.83
41.40 + 9.42
23.71 + 7.88
25.92 + 12.48
34.77 + 12.19
34.57 + 5.04
G-6-PDH activity expressed in nmoles of NADPH
formed/min/ml plasma. Each value represents the
mean + s.d. of 6 animals.
-------
TABLE 12
Effect of HCB at different dose levels of feeding for 90 days on
Plasma Succinic Dehydrogenase Activity
Days
0
6
13
20
27
34
41
48
56
64
69
75
82
Control
3.45 + 1.13
3.39 + 0.70
4.64 + 1.60
3.10 + 1.12
5.93 + 1.27
8.51 + 0.68
5.48 + 1.23
6.40 + 1.16
7.56 + 1.28
4.67 + 1.32
3.93 + 0.81
6.13 + 1.29
6.01 + 0.92
0.01 ppm
2.71 + 0.62
3.15 + 0.55
4.35 + 1.01
3.10 + 0.40
6.49 + 1.97
8.96 + 1.55
6.19 + 1.27
5.42 + 1.75
5.51 + 1.03
5.51 + 0.83
4.85 + 0.69
7.17 + 1.09
5.24 + 0.66
0.1 ppm
3.24 + 1.44
4.08 + 0.89
4.05 + 1.08
3.04 + 0.69
5.51 + 0.51
7.32 + 1.99
5.12 + 0.62
5.48 + 0.65
8.42 + 1.84
4.52 + 0.66
3.69 + 0.57
5.86 + 0.80
5.03 + 0.97
1.0 ppm
2.83 + 1.30
3.57 + 0.76
4.11 + 1.18
4.05 + 0.52
4.85 + 0.62
6.99 + 2.07
4.91 + 1.19
5.95 + 1.38
9.29 + 2.99
5.80 + 0.62
5.12 + 1.57
6.46 + 2.14
5.92 + 1.03
Enzyme activity expressed in nmoles of 2,6 dichlorophenol indolphenol reduced/
min/ml of plasma. Each value represents the mean + s.d. of 6 animals.
-------
36
TABLE 13
Effect of HCB feeding at 100 ppm
for 19 days on Plasma SDH Activity
Days Control 100 ppm
0
5
12
19
12.95 + 1.12
10.71 + 1.60
9.97 + 1.31
11.01 + 3.51
13.39 + 0.67
10.42 + 1.08
8.33 + 0.87
11.16 + 2.09
Enzyme activity expressed in nnnoles of 2,6
dichlorophenol indolphenol reduced/min/ml
plasma. Each value represents the mean +
s.d. of 6 animals.
-------
Ti
b. Elevated enzyme levels due to the lack of biliary excre-
tion as seen in obstructive icterus, exemplified by
changes in alkaline phosphatase activity.
c. Lowered plasma enzyme activity can be a result of im-
paried synthesis by the liver.
As mentioned above, alkaline phosphatase levels in the
blood are elevated when tissues rich in this enzyme are dam-
aged. Our findings of no significant changes in the plasma
enzyme activity of HCB fed sheep are consistent with no ap-
preciable degree of liver damage as found in pathological
studies (see below).
GOT is not a liver specific enzyme, but it can be used
diagnostically to measure the level of liver necrosis if no
disease exists in other tissues in which this enzyme is found
in high concentration. Therefore, as the enzyme appears in
extremely high concentration in muscle, both skeletal and
cardiac, it is of value in confirming a diagnosis of muscular
degeneration was well (41).
No significant difference between GOT activity in the
plasma of control and experimental sheep was found, which
agrees with pathological studies where no evidence of muscu-
lar degeneration was found. This enzyme activity data is
also consistant with the absence of hepatic necrosis.
Mountain (67) has mentioned that an elevated glucose-6-
phosphate dehydrogenase activity is found in erythrocytes of
young infants and also in some adults where abnormal hemoglo-
bins are present in the cells. It is also thought that G-6-
PDH could be depressed by lead poisoning. It is theorized
that G-6-PDH, as well as other enzymes and cofactors of the
-------
38
pentose pathway, are probably involved in stabilizing reduced
glutathione (GSH) and membrane sulfhydryl in the lung (67).
Here again our findings with regard to G-6-PDH did not demon-
strate any significant alteration in activity during the ex-
periment at any of the dosage levels of HOB administered.
Based on the aforementioned lack of enzyme changes, we can
conclude that HCB did not lead to any detectable lung, blood,
or liver damage at chronic dosage levels up to 1.0 ppm, and
acute (19 days) dosage levels of 100 ppm.
With succinic dehydrogenase, a. mitochondrial sulfhydryl
enzyme, we did not observe any significant change in activity
with respect to the controls. This again is in agreement
with no significant histopathological changes; i.e., no ob-
servable tissue lesion or damage is produced by HCB at the
dose levels administered.
E. In vivo antipyrine metabolism
1. Specific Materials and Methods.
At 90 days, six lambs (3 control, 3 from 1.0 ppm group)
were assessed for in vivo antipyrine metabolism. The same
experiment was performed after feeding of HCB at 100 ppm for
19 days.
2. Results
The effects of chronic and acute feeding of HCB to lambs
at 1.0 and 100 ppm on the plasma half life of antipyrine are
summarized in Table 14 and Figure 2. The half life of anti-
pyrine in both cases was shorter than the control group;
however, statistical significance (p<0.01) was obtained only
between control and sheep fed HCB at 100 ppm for 19 days.
There was no statistically significant difference (p>0.05)
-------
39
in antipyrine half life between controls and lambs fed HCB
1 ppm for 90 days.
3. Discussion
Sobennan et al. (68) have reported that antipyrine is
distributed evenly in body water. Antipyrine is completely
metabolized via microsomal N-demethylase. This suggests that
by estimating changes in plasma half-life of this compound
before and after chronic exposure to drugs, information may
be obtained regarding the effects of the drug metabolizing
enzymes.
Many halogenated compounds are metabolized by the mixed
function oxidase system of liver (69, 70). The administra-
tion of some drugs that are metabolised by the mixed function
oxidase system of liver is known to induce the membrane com-
ponents of the endoplasmic reticulum involved in the drug
metabolism (71, 72). Proliferation of the endoplasmic reti-
culum can result in the increased metabolism of the drug.
During the chronic administration of many drugs this reticu-
lar proliferation results in a gradual decline in the plasma
concentration of the drug due to the ability of the compound
to stimulate its own metabolism by liver microsomes (53).
This did not occur in our study (Table 15). The plasma lev-
els of HCB showed a gradual increase up to a few days after
the day of cessation of drug administration, then declined
(see Table 15). This is undoubtably because of the seques-
tration of HCB in the fat. Sequestration of a drug in fat
has the effect of "smoothing" the fluctuations of plasma lev-
el. If one examines the data in Table 15, it can be seen
that as time increases towards day 120, the relative (to fat)
-------
TABLE 14
Effect of HCB feeding on
Antipyrine Half-life
AC!
Treatment
Minutes
Range
Control
HCB 1.0 ppma
Control
HCB 100 ppmb
66.00 + 23.60 (3)
38.60 + 6.30 (3)c
88.50 + 33.90 (6)
34.70 + 7.60 (6)d
43 - 90
35 - 45
Antipyrine half-life expressed in minutes.
Each value represents the mean + s.d. (N).
a. HCB treatment for 90 days.
b. HCB treatment for 19 days.
c. Statistically not significant as compared
to its own control (p>0.05).
d. Statistically significant at p<0.01 level.
-------
41
i
UJ
a.
120
110
100
90
80
70
60
50
40
30
20
10
CONTROL
CONTROL
1.0 ppm
100 ppm
FIGURE 2. Effect of 90 days feeding of HCB at 1.0 ppm,and
19 days feeding of HCB at 100 oom. on in vivo
ontipyrine metabolism
-------
42
TABLE 15
Average plasma levels of HCB In lambs fed
HCB at 1.0 ppm during days 0-90
Day
0
7
15
30
45
60
90
120
150
ISO
210
240
270
300
plasma HCB
(ppb)
<4a
<4
6.7 +
15.9 +
12.2 +
15.7 +
20.8 +
35.8 +
24.3 +
16.9 ±
15.3 +
17.0 +
11.3 +
6.6 +
1.8 (10)b
2.3 (10)
3.3 (10)
4.4 ( 9)c
4.8 ( 9)c
14.2 ( 6)
5.9 ( 5)c
5.2 ( 6)
4.6 ( 6)
7.2 ( 6)
3.8 ( 6)
2.1 ( 6)
Z of
fat HCB
_
0.3
0.4
0.2
0.2
0.2
0.4
0.3
0.3
0.4
0.5
0.4
0.3
a. Detection limit 4 ppb
b. x + s.d. (N)
c. Values below the detection limit were not included in this
statistical determination.
-------
42
level of HCB fluctuates somewhat, but is relatively constant.
The continued high blood levels after exposure was stopped no
doubt reflects movement of HCB between lipid compartments.
This indicates that the blood concentration is a reliable in-
dicator of recent chronic exposure (i.e., greater than 30
days) at the relatively low-level 1.0 ppm exposure.
Our findings in lambs are in agreement with the find-
ings of other investigators that HCB stimulates the activity
of the microsomal enzyme system of the rat and the pig (54).
In v*tro liver enzyme studies
1. Specific materials and methods
Lambs from control and 1.0 ppm groups were sacrificed
at day 90, and from control and 100 ppm groups at day 300
after the acute feeding (100 ppm for 19 days) experiment. N-
and 0-demethylase activities were determined as described.
Microsomal protein was determined after the method of Lowry,
as modified by Miller (73).
2. Results
The effect of 1.0 ppm and 100 ppm HCB feeding to lambs
for 90 and 19 days respectively on the N- and 0-demethylase
activity and microsomal protein are shown in Table 16 and
Figures 3, 4, and 5, respectively. A statistically signifi-
cant increase in the activity of both enzymes occurred in
sheep fed HCB at 1.0 ppm for 90 days as compared to control
sheep. At 100 ppm the significant increase was found only in
activity of N-demethylase. At this dose, the activity of
0-demethylase remained unaffected. A marked increase in the
hepatic microsomal protein occurred following 100 ppm HCB
feeding; microsomal protein was not measured following the
1.0 ppm regimen.
-------
TABLE 16
Effect of HCB feeding on N- and 0-demethylase
activity and microsomal protein
Control group
1.0 ppm groupc
p
-------
45
UJ
4.00
350
3.00
2.50
100
1.50
i 1.00
0.50
a
g 0.00
1.0 ppm
CONTROL
n
100 ppm
CONTROL
FIGURE 3. Effect of 90 doys feedina of HCB o» 1.0 ppm,
and 19 doys feeding of HCB at 100 ppm, on
hepatic N-demethylase activity
-------
46
c/i
en
I
1.75
1.50
2 1.25
!
1.00
OT,
SLQ75
6"
5 Q50
ttZS
UJ
6 QOO
LOppm
CONTROL
CONTROL
100 ppm
RGURE 4. Effect of 90 days feeding of HC8 at 1.0 ppm, and
19 days feed of HC8 at 100 ppm, on hepatic 0-
demethylase activity
-------
60.0
50.0
en
CO
«= 40.0
t
0»
=L
5 30.0
20.0
10.0
0.0
CONTROL
FIGURE 5. Effect of 19 days feeding of HCB at
100 ppm on hepatic microsoma!
protein concentration
-------
4<
3. Discussion
It has been demonstrated that chronic feeding of HCB to
rats causes an increase in microsomal protein (54). An abun-
dance of evidence indicates that the stimulation of micro-
somal enzyme activity involves new protein synthesis which
can be measured in vitro using the whole microsomal fraction
from liver (65, chapter 14). Many investigators feel that
the level of microsomal enzyme activity is a steady-state
which is determined by the rate of synthesis and degradation.
The increase in microsomal protein found in the present
study after 19 days of HCB feeding at 100 ppm could represent
a change in this steady-state which led to an increase in the
rate of new microsomal protein synthesis and/or a decrease in
.the rate of degradation. Stodnard and Nenow (54) feel that
HCB microsomal induction can be best thought of as qualita-
tively similar to the induction caused by chronic phenobar-
bital administration. This, then, would indicate that the
induction seen is primarily the result of an increased syn-
thesis of new microsomal protein, as opposed to a decrease in
degradation (65, chapter 14).
The chronic feeding of HCB also led to an increase in
the hepatic tf- and 0-demethylase activity. This is consis-
tent with an increased amount of.microsomal protein in lambs
chronically treated with HCB. It is also consistent with
the decreased half-life of antipyrine, metabolised via
hepatic N-demethylase, noted in vivo.
The effects of HCB at the two dose schedules was not
consistent with respect to 0-demethylase activity. The
enzyme activity was greatly increased following treatment
-------
with 1 ppm for 90 days but no significant increase in the
activity of this enzyme was observed following treatment with
100 ppm of HCB for 19 days. This inconsistency is difficult
to explain but might be attributed to a basal level of 0-
demethylase activity lower than that of N-demethylase (74).
G. Uptake and Decay Characteristics of HCB in Omental Fat and Other
Tissues
1. Specific Materials and Methods
Biopsies were performed at day 0 and Initial HCB fat
concentration (detection limit 4 ppb) determined. Feeding
was started in the chronic studies at day 0, and continued
through day 90 as per the schedule given previously (see sec-
tion II B, this report). Omental fat samples were taken at
days 0, 7, 15, 30, 45, 60, 90, 120, 150, 180, 210, 240, 270,
and 3QO. Blood samples were taken at the same times.
a. Samples of brain, liver, kidney, and perirenal fat were
taken at day 90, in addition to the omental fat, for HCB
residue analysis. Samples of the same five tissues were
taken at day 300, 210 days after cessation of HCB feed-
ing.
In the acute feeding experiment, six former associate
control sheep (three 0.1 ppm associate control, three
1.0 ppm associate control) were fed 100 ppm HCB for 19
days. Their tissues were analyzed for HCB residues im-
mediately after cessation of feeding; thus, only peak
concentration was determined in this group. All deter-
minations of plasma, fat, and tissue HCB residue levels
were determined as per the methods of Section II G, this
report.
-------
2. Results
The uptake of HCB in omental fat was directly propor-
tional to the dose given. The peak group mean concentration
was reached after 90 days of exposure (Table 17). In the
0.01 ppm group the peak concentration was 115.8 ppb. In the
0.1 ppm group it was 1245 ppb. The associate control group
showed slight contamination, having an HCB fat concentration
of 24.1 ppb at the end of the 90 days. The 1.0 ppm group
showed a peak concentration of 10,186 ppb, and its associate
group was also slightly contaminated with HCB. The free
control group had an initial HCB concentration of 23.7 ppb,
which decreased toward the end of the experiment to a. final
concentration of 12.2 ppb (see Table 17).
After peak concentration was realized at day 90, the
levels of HCB residues in the omental fat decayed with half
times of 100, 82, and 96 days in the 0.01 ppm, 0.1 ppm, and
1.0 ppm groups, respectively.
Undosed control sheep did not accumulate appreciable
amounts of HCB (Table 17), but the associated control groups
(0.1 and 1.0 ppm) accumulated significant amounts, probably
due to fecal contamination from the treated sheep. Maximum
concentrations in their fat were approximately 24.1 ppb and
28.5 ppb, respectively. One lamb in the 1.0 associate con-
trol group almost assurredly received an HCB capsule by ac-
cident, as his omental fat concentration at day 90 reached
308 ppb. This lamb was omitted from calculations.
Table 18 indicated HCB residue levels immediately after
90 days feeding of HCB at the various levels. It can be seen
that the predominant sequestration of the drug occurs in the
-------
TABLE 17
HCB concentration in omental fat after HCBa feeding at different dose levels
HCB concentration in omental fat expressed in ppba
Days after 0.00 ppm
feeding Control
0
7
15
30
45
60
90
120
150
180
210
240
270
300
23.7 +
15.5 +
16.4 +
12.2 +
12.6 +
10.3 +
9.9 +
11.3 +
9.1 +
10.3 +
11.3 +
20.8
,___
3.1
2.5
1.3
2.1
1.9
0.9
0.9
0.8
2.5
1.7
0.01 ppm
HCB
15.6
27.9
37.8
49.8
59.2
89.5
115.8
80.2
70.1
69.0
51.2
29.5
36.2
26.8
+ 2.4
+ 4.6
+ 7.5
+ 11.7
+ 14.8
+ 13.2
+ 32.4
+ 10.1
+ 11.3
+ 11.6
+ 10.0
+ 3.5
+ 4.0
+ 6.1
0.1 ppm
HCB
17.5
177.0
349.0
537.6
647.4
1024.0
1245.0
747.7
716.6
596.0
449.0
289.0
295.0
213.0
+ 5.1
+ 76.7
+ 58.1
+ 99.7
+ 126
+ 213
+ 253
+ 141
+ 99.5
+ 86.0
+ 61.7
+ 50.3
+ 46.3
+ 46.8
0.1 ppm
associate
18.7
16.5
21.8
24.1
14.2
13.5
14.7
13.1
6.8
11.5
+ 11.2
+ 8.6
+ 10.2
+ 12.5
+ 1.5
+ 1.0
+ 2.3
+ 3.3
+ 1.9
+ 1.1
£
1.0 ppm
HCB
990
2387
4085
5801
8394
10186
8124
7293
5832
4167
3525
2578
2218
+ 174
+ 448
+. 418
+ 937
+ 1053
+ 1364
+ 1325
+ 1680
+ 1137
+ 801
+ 803
+ 711
+ 331
1.0 ppm
associate
16.7 + 4.3
24.9 + 3.1
28.5 + 7.0
27.1 + 1.9
27.7 + 8.8
30.1 + 10.7
26.7 + 7.4
22.6 + 9.8
_ _ c
a. HCB was given everyday for a period of 90 days.
b. Each value represents the mean + s.d. of ten sheep in each treatment group and five sheep in each associate
group for the first 90 days.
After 90 days, each value represents six sheep in each treatment group and three from each associate control.
c. These sheep were used in the acute feeding experiment (100 ppm for 19 days).
-------
- 37 -
TABLE 18
HCB residues in various tissues sampled immediately after
90 days feeding at 0.00, 0.01, 0.1, and 1.0 ppm HCB
[results expressed as parts per billion (ppb)]
GROUP
Tissue
Brain
Liver
Kidney
Perirenal fat
Omental fat
0.00 ppm
Control
<4(4)a
<4(4)
<4(4)
-
12 + 1.7(4)
0.01 ppm
HCB
<4(3)
<4(3)
<4(3)
69 + 3.8(2)
119 + 23 (3)
0.10 ppm
HCB
29
11
8.0
856
1435
+ 1.9(4)b
+ 0.7(4)
-1- 3.0(4)
+ 142 (4)
+ 183 (4)
0.10 ppm
Control
<4(2)
<4(2)
<4(2)
_
36 + 11(2)
1.0 ppm
HCB
217 + 53) (4)
281 + 41(4)
112 + 41(4)
7160 -1- 1189(4)
9503 + 669(4)
1.0 ppm
Control
<4(2)
<4(2)
<4(2)
-
31 + 0.
9(2)
a. Detection limit 4 ppb
b. x + s.d. (N)
-------
s:
fat, particularly omental fat; the levels of HCB residues in
brain, liver and kidney were only about 0.01 - 0.02 times the
level in the omental fat.
3. Discussion
Hexaclorobenzene administered orally is sequestered in
the animal's body fat. This is consistant with known physi-
cal characteristics of the compound; i.e., low water solu-
bility, readily oil dissolvable. The difference noted in
the levels seen in omental versus perirenal fat does not seem
related to anything in particular; perhaps it has something
to do with the perfusion characteristics of the two fat loci.
In any event, it can be concluded that growing lambs store
HCB residues in omental fat at levels 10-12 times the intake
level (intake levels 0.01 ppm to 1.0 ppm) after 90 days,
and at 50-100 times the levels seen in the other tissues
studied.
The decay half times of 100, 82, and 96 days in the
0.01 ppm, 0.1 ppm, and 1.0 ppm groups are all of the same
order of magnitude. This is consistant with common pharmaco-
kinetic.s.
The primary origin of the HCB that contaminated the two
associate control groups (Table 17) would presumably have
been the feces of the dosed sheep living in the same run.
These dosed sheep would presumably have excreted unabsorbed
HCB during the 90 day dosing period. This has been previous-
ly reported by Avrahami and Steele (30), and confirmed later
by Mehendale and Mathews (7), who reported HCB-contaminated
14
fecal material after oral administration of labeled HCB C
It was also postulated that levels of HCB in the associated
-------
control groups were due to the continued ingestlon of bedding
containing HCB derived from the dosed sheep during the
post-dosing period. We attempted to control this by moving
the sheep to clean runs at the end of the exposure period,
by bedding only half of the runs, and changing the bedding
frequently (every other day).
The residue profile seen (Tables 18 and 19) indicates
that HCB might be accumulated in all tissues examined in a
dose-dependent manner. In this study the HCB residues were
higher in omental fat as compared with the other tissues.
H. Gross and Microscopic Pathological Changes seen after HCB
Administration
1. Specific Materials and Methods
Animals were slaughtered at day 90, Immediately after
the chronic HCB administration. The number of animals from
each group was as given in section II B, this report. Gross
and microscopic tissue examination was performed as per Sec-
tion II H, this report. The same procedure was performed at
the termination of the 19 day 100 ppm experiment.
2. Results
No grossly observable pathology was noted upon necropsy.
Some histopathological changes were observed in lung and
intestine of both control and treated groups. These were not
felt to be due to the HCB but rather due to parasitic in-
festation of the animals (i.e., lung worms, coccidia). Simi-
lar, parasite-induced changes were occasionally noted in the
kidneys of both the control and exposed groups.
Some histopathological changes occurred in the livers
of the exposed animals that might be attributable to the HCB.
-------
- 39 -
TABLE 19
HCB residues In various tissues sampled 210 days after
termination of 90 days feeding at 0.00, 0.01, 0.1, and 1.0 ppm HCB
(i.e., sampled at day 300) [results expressed as parts per billion (ppb)]
GROUP
Tissue
Brain
Liver
Kidney
Perlrenal fat
Omental fat
0.00 ppm
Control
<4(6)
<4(6)
<4(6)
-
11 ± 1.7(6)
0.01 ppm
HCB
<4(6)a
<4(6)
<4(6)
27 -1- 2.8(6)
27 + 6.1(6)
0.1 ppm
HCB
7.0 +
<4(5)
<4(5)
200 +
213 +
0.1 ppm
Control
1.7(5)
-
-
36 (5)
47 (5)
1.0 ppm
HCB
58 + 14
36 + 7
21 + 6
2113 + 529
2218 + 331
1.0 ppm
Control
(6)
.2(6)
.8(6)
(6)
(6)
__
-
-
-
a. Detection limit 4 ppb
b. x + s.d. (N)
-------
5f
TABLE 20
HCB residues in various tissues sampled immediately after
19 days feeding at 100 ppm HCB [results expressed
as parts per billion (ppb)]
organ
Brain
Liver
Kidney
Perirenal fat
Omental fat
Control
group
<4(6)a
<4(6)
<4(6)
-
11.3 + 11.7(6)
HCB- treated
group
2,333 + 449(6)b
2,197 -1- 553(6)
522 + 208(6)
108,500 + 23,880(6)
100,333 + 3,098(6)
a. Detection limit 4 ppb
b. :c + s.d. (N)
-------
These changes were minor and not uniformly seen In the ex-
posed animals;-they were not noted in the control group.
These changes included inflammatory cell infiltration into
the portal triad areas, generalized necrosis, and some
vacillation of cells.
3. Discussion
As a result of our histopathological findings, we con-
cluded that HCB does not appreciably affect the organs and
tissues when it is given in oral doses of 0.01, 0.1, 1.0,
and 100 ppm (daily) to growing lambs. This does not conflict
necessarily with work of other investigators who found tissue
damage (prlnicpally liver damage) at a daily dose of 2000 ppm
HCB in rats (35), and 100 mg/kg of body weight for 14 con-
secutive days in rats (34).
-------
Bibliography
1. Grant, D.L., Hatina, G.V., and Munro, I.e. .(1974): Hexachlorobenzene
accumulation and decline of tissue residues and relationship to some
toxicity criteria in rats. Food Research Laboratories, Health
Protection Branch, National Health and Welfare, Ottawa, Canada.
2. Yersin, H., Chomett, H., Bauman, G., and Lhoste, J. (1946): Hexa-
chlorobenzene, an organic synthetic used to combat wheat smut. Compt.
Rend. Acad. Agr. France 31:24.
3. Ben-Dyke, R., Sanderson, D.M., and Noakes, K.N. (1970): Acute toxicity
data for pesticides. World Review of Pest Control .9:119.
4. Holton, C.S. and Pardy, L.H. (1954): Control of soil-born common bunt
of winter wheat in the Pacific Northwest by seed treatment. Plant Dis.
Reptr. 38(11):753.
5. Purdy, L.H. (1966): Soil moisture and soil temperature, their influ-
ence on infection by the wheat flag smut fungus, and control of the
disease by three seed-treatment fungicides. Phytopathology 56(1):98.
6. Siang, W.N. and Holton, C.S. (1953): Mode of action of HCB on wheat
bunt fungi in vitro. Plant Dis. Reptr. 37:63.
7. Mehendale, H.M. and Mathews, H.B. (1973): Metabolism, excretion, and
storage of hexachlorobenzene in the male rat. Presented at the 165th
A.C.S. National Meeting, Dallas, Texas.
8. Park, D.V. and Williams, R.T. (1922): The metabolism of halogenoben-
zenes. (a) Penta and hexachlorobenzenes. (b) Further observations on
1, 3, 5, - trichlorobenzene. J. Chem. Soc. 121:1021.
9. Cam, C. and Nigogosyan, G. (1963): Acquired toxic porphyria cutanea
tarda due to hexachlorobenzene. JAMA 183:88.
10. Schmid, R. (1960): Cutaneous porphyria in turkeys. New Eng. J. Med.
263:397.
11. Goldstein, A., Aronow, L. and Kalman, S.M. (1974): Prinicples of Drug
Action: The Basis of Pharmacology. Second Edition, Wiley, New York.
12. Siyali, D.S. (1973): Polychlorinated biphenyls hexachlorobenzene and
other organochlorine pesticides in human milk. Med. J. Aust. 60;2(17) ;
815.
13. Miller, G.J. and Fox, J.A. (1973): Chlorinated hydrocarbon pesticide
residues in Queensland human milk. Med. J. Aust. 60:2:261.
14. Tuinstra, L.G.N. (1971): Organochlorine insecticide residues in human
milk in Leiden Neth. Milk Dairy J. 25_:24.
15. Acker, L. and Schulte, E. (1970): Uber das Vorkommen von chlorierten
Biphenylen and Hexachlorobienzol neben chlorierten Insecktiziden in
human milch med menschlicken tettgevelc. Naturwissenschaften. 57:497.
-------
59
16. Westoo, G. and Noren, K. (1968-72): Residues of organochlorlne and
certain organophosphorus pesticides in fruits, berries, vegetables and
roots. Var Foda, 25 Supp. 1:6 (Stockholm).
17. Siyali, D.S. (1972): Hexachlorobenzene and other organochlorine pesti-
cides in human blood. Med. J. Austr. 59:2(19);1063.
18. Brady, M.N. and Siyali, D.S. (1972): Hexachlorobenzene in human body.
fat. Med. J. Aust. 59:1(1);158.
19. Curley, A., Burse, V.W., Jennings, R.W., Villanueva, E.G., Tomatis, L.
and Akazaki. (1973): Chlorinated hydrocarbon pesticides and related
compounds in adipose tissue from people of Japan. Nature 242:338.
20. Abbott, B.C., Collins, G.B. and Goulding, R. (1972): Organochlorine
pesticide residues in human fat in the United Kingdom, 1967-71. Br.
Med. J. .2:553.
21. Goursaud, J., Luquet, F., Boudier, J.F. and Casalis, J. (1972):
Contamination of milk with hexachlorobenzene residues. Ind. Aliment.
Agr. 89(1):31.
22. National Health and Medical Research Council (1971): Report of Pesticide
residues in the total Austrailian diet. NHMRC, Canberra.
23. Gilbertson, M. and Reynolds, L.M. (1972X: Hexachlorobenzene (HCB) in
eggs of common terns in Hamilton Harbour, Ontario. Bull. Environ.
Contam. Toxicol. 7^(6), 371.
24. Swiss Fedreal Health Service (1973): Communication to FAO.
25. Burns, J.E. and Miller, F.M. (1975): Hexachlorobenzene Contamination:
Its effect in a Louisiana population. Arch. Environ. Health 30(1);44.
26. United States Environmental Protection Agency (1975): HCB review
report: Fifth 90-day HCB meeting and status of HCB studies.
27. Zitco, V. and Choi, P.M.K. (1972): PCB and DDE in eggs of cormorants/
gulls and ducks from the Bay of Fundy, Canada. Bull. Env. Contam.
Toxicol. 1(1):63.
28. Gardiner, M.R. and Armstrong, J. (1960): Feeding pickled wheat to pigs.
Res. Vet. Sci. ,1:237.
29. Wit, S.L. and Van de Kamp, C.G. (1973): Stapeling van persistente
bestrijdingsmildolden in varkers. Report No. 43/73. Tox. Report to
Veterinary Inspector, Netherlands, Ministry of Agriculture.
30. Avrahami, M. and Steele, R.T. (1972): Hexachlorobenzene. I.
Accumulation and elimination of HCB in sheep after oral dosing.
New Zealand J. Agr. Res. 15_(3):476.
31. Avrahami, M. and Steele, R.T. (1972): Hexachlorobenzene. II. Residues
in laying pullets fed HCB in their diet and the effects on egg produc-
tion, egg hatch-ability, and on chickens. New Zealand J. Agr. Res.
15(3):482.
-------
60
32. Avrahami, M. and Steele, R.T. (1972): Hexachlorobenzene. III. The
effects of feeding HCB to growing chickens. New Zealand J. Agr. Res.
15(3):489.
33. Vos. J.G. , Van Der Maas, H.L., Mu'sch, A. and Ram. E. (1971): Toxicity
of hexachlorobenzene in Japanese Quail with special reference to
prophyria, liver damage, reproduction, and tissue residues. Tox. Appl.
Pharmacol. 18 ; 944 .
34. Villenueve, D.C. (1975): The effect of food restriction on the redis-
tribution of hexachlorobenzene in the rat. Tox. Appl. Pharmacol. 31:
313.
35. Medline, A., Bain, E. , Menon, A.I. and Haberman, H.F. (1973): Hexa-
chlorobenzene and rat liver. Arch. Pathol. 96:61.
36. San Martin de Viale, L.C., Viale, A.A. and Grinstein, M. (1970):
Experimental porphyria induced in rats by hexachlorobenzene. A study
of the porphyrines excreted by urine. Clin. Chim. Acta 28jl3.
37. Sweeney, G.D. (1963): Patterns of porphyrin excretion in South African
prophyric patients. S. Afr. J. Lab. Clin. Med. jhl82.
38. McCray, C.W. (1973): Preliminary report on study of excretion of HCB
from pigs and cows. Report to Pesticides Coordinator, Australia.
39. Villenueve, B.C., Panopio, L.G. and Grant, D.L. (1974): Placental
transfer of hexachlorobenzene in the rabbit. Environ. Physiol. Biochem.
40. Harvey, D.G. and Obeid, H.M.A. (1974): The application of certain
liver function tests including serum alkaline phosphatase estimations
to domesticated animals in the Sudan. Br. Vet. J. 130:544.
41. Coles, E.H. 1974: Veterinary Clinical Pathology, Saunders, Philadelphia.
42. Neuman, H. , Moran, E.M. , Russell, R.M. and Rosenberg, I.H. (1974):
Distinct alkaline phosphatase in serum of patients with lymphatic
leukemia and infectious mononucleosis. Science 186:151.
43. Fairchild, E.J. (1967): Tolerance mechanisms. Arch. Environ. Health
14:111.
44. Sandlin, R.E. (1974): Persistently elevated serum alkaline phosphatase
levels in apparently healthy persons. JAMA 230(6) ;901.
45. Kuttler, K.L. and Marbel, D.W. (1968): Relationship of serum
transaminase to naturally occurring and artificially induced white
muscle disease in calves and lambs. Am. J. Vet. Res. 19 ;632.
46. Roberts, H.E. (1968): Observations on experimental acute fasciolasis
in sheep. Brit. Vet. J. 124:433.
47. Hansen, M.A. (1964): An outbreak of toxic liver injury in ruminants.
Nord. Vet. Med. 16:322.
-------
48. McPherson, A. and Henrlngway, R.G. (1969): The relative merit of
various blood analysis and liver function tests in giving an early
diagnosis of chronic copper poisoning in sheep. Br. Vet. J. 125;213.
49. Lagace, A.,-Bel, D.S., Moxon, A.L. and Pouden, W.B. (1961): Serum
transaminase in the blood of lambs given preventive treatments for
white muscle disease. Am. J. Vet. Res. 22;686.
50. Padykula, H.A. (1952) : The localization of succinic dehydrogenase in
tissue sections of the rat. Am. J. Anat. 91;1Q7.
51. Bums, J.J., Conney, A.H. and Roster, R. (1963): Stimulatory effect of
chronic drug administration on drug-metabolizing enzymes in liver
microsomes. Ann. N.Y. Acad. Sci. 104;881.
52. Rajamanickam, C., Amrutavalli, M.R.S. Rao and Padmanaban, C. (1972):
Effect of hexachlorobenzene on haem synthesis. Biochem. J. 12:381.
53. Rajamanickam, C. and Padmanaban, C. (1974): Biochemical effects of
hexachlorobenzene. Ind. J. Biochem. Biophys. 11:119.
54. Stonard, M.D. and Nenov, P.A. (1974): Effect of hexachlorobenzene on
hepatic microsomal enzymes in the rat. Biochem. Pharmacol. 23:2175.
55. Welch, R.M., Harrison, Y.E., and Bums, J.J. (1967): Implications of
enzyme induction in drug toxicity studies. Tox. Appl. Pharm. 10:340.
56. Garret, W.N., Meyer, J.H. and Lofgreen, G.P. (1959): The comparative
energy requirements of sheep and cattle for maintenance and gain.
J. An. Sci. 18:528.
57. Schalm, O.W., Jain N.C. and Carroll E.J. (1975): Veterinary Hematology,
Lea and Febiger, Philadelphia.
58. Schalm, O.W. (1965): The Goldberg refractometer or.T.S. meter. Calif.
Vet. 19(3).
59. Bessey, 0., Lowry, O.H. and Brock, M.J. (1946) : A method for the rapid
determination of alkaline phosphatase with five cubic millimeters of
serum. J. Biol. Chem. 164;321.
60. Freedland, R.A., Hjerpe, C.A. and Cornelius, E. (1965): Comparative
studies on plasma enzyme activities in experimental hepatic necrosis
in the horse. Res. Vet. Sci. .6:18.
61. LShr, G.W. and Waller, H.D. (1965): Glucose-6-Phosphate, Dehydrogenase,
in Methods of. Enzymatic Analysis, Academic Press, New York.
62. Freedland, R.A. (1965): Effects of thyroid homones on metabolism.
Effect of thyroxin and iodinated casein on liver enzyme activity.
Endocrinology 77:19.
63. Brodie, B.B., Axelrod, J., Soberman, R., and Levy, B.B. (1949): The
estimation of antipyrine in biological materials. J. Biol. Chem.
179:25.
-------
64. Brodie. B.B., and Axelrod, J. (1950): The fate of aminopyrlne
(Pyramidon) in man and methods for the estimation of aminopyrine and
its metabolites in biological material. J. Pharmacol. Exp. Ther. 99:
171.
65. LaDu, B.N., Mandel, H.G. and Way, E.L. (1971): Fundamentals of Drug
Metabolism and Distribution, Williams and Wilkins, Baltimore.
66. Collet, J.N. and Harrison, D.L. C1968): Lindane residues on pasture and
in the fat of sheep grazing treated with Lindane prills. New Zealand
J. Agr. Res. 11(3):589.
67. Mountain, J.T. (1963): Detecting hypersusceptibility to toxic sub-
stances. Arch. Environ. Health 63:357.
68. Soberman, R., Brodie, B.B. and Levy, B.B. (1949): The use of antipyrine
in the measurement of total body water in man. J. Biol. Chem. 179:31.
69. Kaufmann, R. (1961): The enzymic conversion of 4-flurophenyl alanine
to tyrosine. Biochem. Biophys. Acta 51:619.
70. Renson, J. and Bourdon, V. (1968) : Hydroxylation et deflourination des
de'rive's fluores de aniline. Arch. Int. Pharmacodyn. Ther. 171:240.
71. Coney, A.H. (1967) : Pharmacological implications of microsomal enzyme
induction. Pharmacol. Rev. 19:317-366.
72. Kuntzman, R.A. (1969): Drugs and enzyme induction. Ann. Rev. Pharmacol.
2:21.
73. Miller, G.L. (1959): Protein determination for large numbers of samples.
Anal. Chem. 31:964.
74. Giri, S.N. (1971): Stimulatory effects of phenobarbital pretreatment on
the metabolism of glyceryl guaiacolate ether. Arch. Int. Pharmacodyn.
191:147-
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TECHNICAL REPORT DATA
(Please read Inunctions on :he reverse before completing)
1. REPORT NO.
EPA 560/6-76-013
3. RECIPIENT'S ACCESSION«NO.
4. TITLE AND SUBTITLE
Uptake, Excretion, and Physiological Effects of
Hexachlorobenzene in Growing Lambs
5. REPORT DATE
Prepared 10 August 1976
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Ronald.L. Mull, Wray L. Winterlin (Dept. of
Environmental Toxicology); Stuart A. Peoples (Dept. of
g/*fir>n1 of Triai-oiHna-ry
?0«* ^^^^^ J
8. PERFORMING ORGANIZATION REPORT NO.
_ _,_ ., ** " J -" .*-
9. PfiRFORMlVlG ORGANIZATION NAME AND ADDRESS
Food Protection and Toxicology Center
University of California, Davis
Davis, CA 95616
10. PROGRAM ELEMENT NO.
11. CONTRACT/GRANT NO.
68-01-2254
12. SPONSORING AGENCY NAME AND ADDRESS
Environmental Protection Agency
Washington, D.C. 20460
13. TYPE OF REPORT AND PERIOD COVERED
Final
1*. SPONSORING AGENCY CODE
15. SUPPLEMENTARY NOTES
16. ABSTRACT "
Growing lambs were exposed to daily doses of hexachlorobenzene (HCB) per
os. The daily dose was calculated to provide 0.01, 0.10 or 1.00 ppm of the feed con-
sumed. Exposure was terminated at 90 days and 20 of the 50 lambs were slaughtered.
Remaining lambs were monitored for an additional 210-days. Biopsies of omental fat
were taken periodically. At slaughter, samples were collected for HCB analysis and
histological preparations. Blood samples were periodically collected for determina-
tion of HCB, plasma protein, and plasma enzyme activities. At the end of the 90 day
exposure, the ±n vivo metabolism of aminopyrine and the in vitro 0 and N-demethylation
of the liver microsomes were determined. Results of the analyses for HCB in omental
fat show that the peak concentration attained was 10.186, 1.024, 0.116 and 0.012 ppm
in the 1.00, 0.10, 0.01 ppm and control groups respectively. Following cessation of
the exposure, the HCB declined to less than one-half the peak concentration within
100 days. Histological examination of tissues from the slaughtered animals showed no
pathological changes attributable to the HCB. Marked changes were noted in the micro-
somal enzymes but changes were not significant in the plasma enzymes.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lOENTIFIERS/OPEN ENDED TERMS
c. COSATI Field/Group
chlorobenzenes1
pollutants
contaminants
pathology
pathophysiology
drug excretion
animal nutrition
chlorine aromatic cmpds
hexachlorobenzene toxi-
cology drug tissue dis-
tribution lamb growth
pharmacokinetics
06T/06F
13. DISTRIBUTION STATEMENT
Document is available to the public through
the National Technical Information Service,
Sorinzfield. VA 22151
19. SECURITY CLASS (This Report)
Unclassified
21. NO. OF PAGES
74
20. SECURITY CLASS (Thispagaj
Unclassified
22. PRICE
EPA Form 2220-1 (9-73)
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