THE EFFECTS OF PHENOBARBITAL AND CARBON

TETRACHLORIDE ON THE RATE OF DECLINE

OF BODY BURDENS OF HEXACHLOROBENZENE
IN THE RAT
D. C. VILLENEUVE, W. E. J. PHILLIPS, L. G. PANOPIO, C. E. MENDOZA,
G. V. HATINA, and D. L. GRANT
F o o d Research Laboratories, Foods Directorate
Health Protection Branch Tunney's Pasture
Ottawa, Canada

Male and female rats were raised from weaning on diets containing 0, 20, and
40 ppm of hexachlorobenzene (HCB) for three months. The animals were then
placed on an HCB-free diet and dosed with either sodium phenobarbital or carbon
tetrachloride. Animals were killed at 0, 15, and 30 days on the HCB-free regimen
and tissue residues determined. Plasma cholinesterase activity was depressed by
carbon tetrachloride treatment as were liver aniline hydroxylase and N-demethylase
activities. Phenobarbital treatment increased liver aniline hydroxylase and N-
demethylase activities. HCB decay profiles were established for plasma, fat, and
liver. No direct correlation was observed between induction of enzyme activity and
rate of disappearance of HCB residues in liver tissue.

One o f the most important properties of a toxicant is the degree to which it is stored
in the various tissues of the body. It is also o f considerable interest to study the rates of
decline o f such toxicants and how such rates are affected by certain stress conditions. A
number o f studies have demonstrated that certain drugs that induce microsomal enzymes
will also increase the rate of depletion of b o d y burdens of organochlorine compounds
(Davies e t al. 1969, Alary e t al. 1971). Conversely, reports on the levels o f pesticide
residues in human tissues have shown that organochlorine levels were often higher in
patients with abnormal livers (Radomski e t al. I968, Casarett e t al. 1968). Grant and
Phillips (1972)have observed higher residue levels of DDT in the liver and fat o f rats when
the liver was damaged by carbon tetrachloride. In similar studies Laug and Kunze (1951)
noted 10 to 100 times the concentration of methoxychlor in the fat and livers o f carbon
tetrachloride-treated rats compared to controls. Pre-treatment with carbon tetrachloride
is also known to increase tissue storage of PCB's in the rat (Grant e t al. 1971).

The present study was designed to investigate the role o f the liver in the depletion of
body burdens of hexachlorobenzene (HCB). Animals having known b o d y burdens of
HCB were treated either with phenobarbital to induce microsomal enzymes or with
carbon tetrachloride to damage the liver. Body burdens o f HCB were then determined
after 15 and 30 days. Microsomal enzyme activities were also determined at these times
and compared with control animals.

Archives of Environmental Contamination 243

244 D.C. Villeneuve et al.

Methods
Male and female rats were raised from weaning on diets containing 0, 20, and 40 ppm
of HCB for approximately three months. The animals were then placed on an HCB-free
diet and dosed with either sodium phenobarbital or carbon tetrachloride according to
the scheme outlined in Table I. Groups of animals were killed at 0, 15, and 30 days after
removal from their HCB diets. After killing the animals, tissues were excised and weighed.
Plasma cholinesterase activity was determined by an automated procedure based on the
method of Gary and Routh (1965). Aniline hydroxylase and N-demethylase activities
were determined in fresh liver homogenates according to the methods described earlier
(Villeneuve et al. I971). HCB was determined on plasma, liver, kidney, spleen, brain, fat,
and heart. The HCB was extracted by the method of Kuchar e t al. (1969). The final solu-
tions were subjected to GLC-EC analyses on a Varian Model 1400 gas chromatograph
fitted with a 6-ft by I/8-inch OD glass column containing four percent SE-30 and six
percent QF-1 on 80-100 mesh Supelcoport. The nitrogen flow rate was 100 ml/min, with
column and injection port temperatures of 200°C. Enzyme activities and tissue levels of
HCB are presented as the mean value of five animals -+ S.E. Statistical significance
(P < 0.05) was determined by the Students t-test.

Results
The effects of HCB, phenobarbital, and carbon tetrachloride treatments on liver
weights is shown in Tables II and III.

With male rats the liver weight was increased at 0 and 30 days at 40 ppm of HCB.
Phenobarbital increased the liver weights of both 20 and 40 ppm HCB groups at 30 days.
Carbon tetrachloride increased liver weights after 15 and 30 days on the 20 ppm HCB
group. With females, the effects on liver weights were more consistent. Feeding of HCB
alone did not cause an increase in liver weight in 90 days. However, both phenobarbital
and carbon tetrachloride increased liver weights at both HCB levels and at both time
intervals.

Plasma cholinesterase activity was not affected by hexachlorobenzene or phenobarbital

treatment in male rats. With carbon tetrachloride a significant decrease was observed after
30 days with the animals receiving 20 ppm of HCB. The enzyme activities expressed as
mMoles of SH groups released per three min per ml of plasma were 0.52 -+ 0.04 for the no-
treatment group and 0.07 -+ 0.05 for the 20 ppm HCB + CC14 group. With female rats
carbon tetrachloride caused a significant reduction in plasma cholinesterase at both HCB
levels at 15 and 30 days while an increased level of cholinesterase activity was observed
after 15 days in the 20 ppm HCB + phenobarbital group. The enzyme activities for the
15-day interval were 2.10 + 0.13 for the no-treatment group, 2.70 -+0.21 for the 20 ppm
HCB + phenobarbital group, 1.54 -+ 0.16 for the 20 ppm HCB + CC14 group, and
t.48 -+ 0.14 for the 40 ppm HCB + CCI4 group. At 30 days the activities were 1.56 -+0.06
for the no-treatment group, and 0.90 -+0.02 for the 20 ppm HCB + CC14 group.

Phenobarbital induced aniline hydroxylase activity in male rats at both time intervals
and in both the 20 ppm and 40 ppm HCB groups. The rates expressed as mMoles of p-
 o~
Table 1. Phenobarbital and Carbon Tetrachloride Dosing Schemes for Male and Female Rats
O
Group a
m"
Treatment 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
O

Level of HCB in diet (ppm) 0 0 0 20 20 20 40 40 40 20 20 40 40 20 20 40

Treatment received
after removal from diets b,c 0 0 0 0 0 0 0 0 0 P P P P CC14 CC14 CC14 e~

Day animals were killed ¢3

after removal from their
O
respective diets 0 15 30 0 15 30 0 15 30 15 30 15 30 15 30 15

C~

aFive animals per group. t~

bSodium phenobarbital administered i.p. at a rate of 25 mg/kg body wt 5 days/week.

cCCI4 administered orally at a rate of 0.5 cc/kg body wt in corn oil on the 1st, 4th, 8th, 12th days when animals were killed on O
15th day. Animals killed after 30 days were also administered CC14 at 16 and 24 days.
¢3

bO

L/I
246 D.C. Villeneuve et al.

aminophenol formed per hour per mg o f liver for the 15-day interval were 1.48 +- 0.13
for the no-treatment group, 2.45 +- 0.11 for the 20 ppm HCB + phenobarbital group, and
2.36 -+ 0.22 for 40 ppm HCB + phenobarbital group. The rates for the 30-day interval
were 1.14 +- 0.11 for the no-treatment group, 2.12 -+ 0.24 for the 20 ppm HCB + pheno-
barbital group, 0.44 -+ 0.14 for the 20 ppm HCB + CC14 group, and 2.05 +- 0.02 for the
40 ppm HCB + phenobarbital group. With females, aniline hydroxylase activity increased

Table II. Male Rat Liver Weights (% Body Weight) a

Days after removal from HCB diet

Treatment 0 15 30

None 3.90 -+0.11 3.99 +0.09 3.32 -+0.08

20 ppm HCB 3.66 +-0.11 3.86 +0.12 3.57 +0.09

20 ppm HCB + phenobarbital 4.35 -+0.18 4.23 b +0.08

20 ppm HCB + CC14 4.63 b + 0.10 5.06 b + 0.24

40 ppm HCB 4.29b -+ 0.09 3.82 -+ 0.11 3.61b + 0.08

40 ppm HCB + phenobarbital 4.15 +0.17 4.24b-+0.06

40 ppm HCB + CCI4 4.06 + 0.22

aMean weight of 5 animals -+ S. E.

b Significantly different from no-treatment animals (P = 0.05).

Table III. Female Rat Liver Weights (% Body Weight)a

Days after removal from HCB diet

Treatment 0 15 30

None 3.84 -+ 0.12 3.61 +-0.11 3.37 .+ 0.04

20ppmHCB 3.84 -+ 0.07 3.90 -+0.17 3.28 +- 0.01

20 ppm HCB + phenobarbital 4.19 b + 0.09 4.36 b -+ 0.20

20 ppm HCB + CCt 4 3.98 b + 0.05 4.93 b -+ 0.20

40ppmHCB 3.99 -+ 0.05 3.91 .+0.14 3.54 -+ 0.07

40 ppm HCB + phenobarbital 4.47 b +- 0.06 4.29 b +- 0.08

40 ppm HCB + CC14 4.32 b + 0.14

aMean weight of 5 animals +- S. E.

b Significantly different from no-treatment animals (P = 0.05).
 Effects of Phenobarbital and CC14 on Decline Rate of HCB 247

at 15 days in the 40 ppm HCB + phenobarbital group, and at 30 days in both groups
treated with phenobarbital. Carbon tetrachloride caused a reduced activity after 30 days
in the 20 ppm HCB group. The rates for the 15-day interval were 0.93 ± 0.11 for the no-
treatment group and 1.77 ± 0.21 for 40 ppm HCB + phenobarbital. At 30 days the rates
were 0.80 ± 0.12 for no treatment, 1.22 ± 0.11 for 20 ppm HCB + phenobarbital,
0.26 ± 0.07 for 20 ppm HCB + CC14, and 1.30 + 0.12 for 40 ppm + phenobarbital.

In male rats, N-demethylase activity was increased by both phenobarbital and HCB
treatment at 15 days while carbon tetrachtoride reduced activity in the 20 ppm HCB
group. At 30 days, phenobarbital treatment resulted in an increased activity while carbon
tetrachloride caused a decreased activity in the 20 ppm HCB group. The enzyme activities
expressed as mMoles of 4-aminoantipyrene formed per hour per g of liver for the
15-day interval were 0.60 -+ 0.10 for the no-treatment group, 1.13 ± 0.13 for the 20 ppm
HCB group, 1.82 ± 0.20 for the 20 ppm HCB + phenobarbital group, 0.19 ± 0.02 for the
20 ppm HCB + CC14 group, 1.t4 -+ 0.17 for the 40 ppm HCB group, and 1.67 ± 0.32 for
the 40 ppm + phenobarbital group. At 30 days the activities are 0.62 ± 0.15 for the no-
treatment group, 1.65 ± 0.14 for the 20 ppm HCB + phenobarbital group, 0.15 -+ 0.06 for
the 20 ppm HCB + CC14 group, and 1.80 ± 0.16 for the 40 ppm HCB + phenobarbital
group. With female rats, only phenobarbital had significant effect. This treatment resulted
in elevated enzyme activities at 20 and 40 ppm of HCB at both time intervals. The rates
at 15 days were 0.12 -+ 0.03 for the no-treatment group, 0.38 -+ 0.09 for the 20 ppm
HCB + phenobarbital group, and 0.67 ± 0.16 for the 40 ppm HCB + phenobarbital
group. At 30 days the enzyme activities were 0.23 + 0.04 for no treatment, 0.51 -+ 0.04
for the 20 ppm HCB + phenobarbital group, and 0.50 ± 0.06 for the 40 ppm HCB +
phenobarbital group.

Table IV. Residue Levels o f Hexachlorobenzene in Tissues o f Male and Female

Rats Fed 20 or 40 PPM tlexaehlorobenzene a

HCB in diet (ppm)

20 40 20 40
Tissue Male Male Female Female

Plasma 0.86 ± 0.01 2.1 ± 0,1 0.88 ± 0.01 2.3 +- 0.1

Fat 239 -+ 19 503 ± 40 265 ± 27 554 ± 30
Liver 10.9 ± 0.8 17.2 ± 0.9 t 1.8 ± 1.2 20.4 ± 1.2
Kidney 9.6 ± 0.5 24.0 ± 2 7.3 -+ 1.2 11.0 ± 0.9
Spleen 4.42 ± 0.4 10.5 ± 1.3 4.6 ± 0.5 t8.4 ± 2.8
Heart 5.25 ± 0.4 10.6 ± 0.7 9.0 ± 0.3 13.3 ± 1.4
Brain 5.34 ± 0.3 12.4 ± 1.4 6.5 ± 0.2 14.0 ± 0.6

aMean + S. E. of 5 animals.
248 D.C. Villeneuve et al.

A comparison of HCB levels in different tissues of rats fed HCB in their diets at 20
and 40 ppm is shown in Table IV. In general, females tended to have higher residues in
their tissues than males, and the general order of magnitude was as follows: fat > liver >
kidney > spleen = heart = brain > plasma.

The decay profiles established for plasma are shown in Figure 1. The only significant
differences were obtained with males at 15 days fed 20 ppm of HCB. Phenobarbital
caused a decreased level in the plasma, while carbon tetrachloride had the reverse effect.
These effects were not observed after 30 days. The decay profiles for fat are shown in
Figure 2. With females fed 20 ppm of HCB, both phenobarbital and carbon tetrachloride
caused a significant reduction of HCB in adipose tissue. With female rats fed 40 ppm of

2.4 20 ppm HCB 40 ppm HCB

2.2
[] No treatment
2.0
[] Phenobarbital treated
1.8
[] CCI 4 treated
1.6
* Significantly different
1.4
1.2
from no treatment
groups P=0.05 ©
1.0
0.8
uJ 0.6
+1 0.4
0.2
0
CD
2.4
-r

E
r~
o 2.0

1.6
1.4
1.2 U
1.0
0.8
0.6
0.4
0.2
0
15 30 0 15 30
Days after removal from HCB diet

Fig. 1. Hexachlorobenzene decay profiles in plasma.

 Effects of Phenobarbital and CC14 on Decline Rate of HCB 249

HCB, only carbon tetrachloride caused a significant reduction. With males fed 20 ppm of
HCB, carbon tetrachloride caused an increase in the tissue level of HCB at both time inter-
vals. With males receiving 40 ppm of HCB, phenobarbital caused an increase in the level
of HCB at the 30-day interval. The decay profiles for liver are shown in Figure 3. Carbon
tetrachloride caused an accumulation of HCB in the livers of both males and females.
Phenobarbital caused a decrease in the residues of hexachlorobenzene in the liver of
female rats, but had the reverse effect with male rats.

Discussion
The present study was designed primarily to investigate the role of the liver in the
metabolism of body burdens of HCB. Phenobarbital, a known inducer of microsomal

600 20 ppm HCB 40 ppm HCB

[-] No treatment
500
[] Phenobarbital treated
[] CCI 4 treated
400
* Significantly different
from no treatment

300
groups P-0.05
q)
200
uJ
+l 100

*" 0
m

-r
E
& so0

400

300 d

n
2ooi

100

o 15 30 0 15 30
Days after removal from HCB diet

Fig. 2. Hexachlorobenzene decay profiles in fat.

250 D.C. Villeneuve et al.

enzymes, was used to stimulate liver function while carbon tetrachloride, a hepatotoxin,
is known to decrease microsomal enzyme activity (Glende 1972). Liver weights, expressed
as percent body weight, indicated that phenobarbital caused liver hypertrophy in both
male and female rats. Increased liver weights were also observed in both male and female
rats after carbon tetrachloride treatment. Both aniline hydroxylase and N-demethylase
enzyme activities were used to monitor changes in microsomal enzyme activity. As ex-
pected, phenobarbital increased these activities while carbon tetrachloride decreased the
activities. Of the two microsomal enzymes, N-demethylase seemed to be the more

[] No treatment *Significantly different

[] Phenobarbital treated from no treatment
groups P=0,05
ITN CCI 4 treated
20 ppm HCB 40 ppm HCB

~ '62.2

18
16
14
12
10
v~

8 I v~

fZ

6
"13
4
I v,
v~
0 i v~
I
E
0
2
0
I F,

g *30.2
E
o~
L

18
16
14
12i
10
I

! U
I
8
6
4
2
JI
0
0 15 30 0 15 3O
ppm HCB in tissue +- S,E.

Fig. 3. Hexachlorobenzene decay profiles in liver.

 Effects of Phenobarbital and CCI 4 on Decline Rate of HCB 251

sensitive indicator. Plasma cholinesterase activities were decreased only in male and female
rats dosed with carbon tetrachloride. This enzyme was monitored because it is an im-
portant factor in organophosphate and carbamate intoxication and any lowered
cholinesterase might lead to an increased susceptibility to poisoning by these compounds.

Rates of decline of HCB residues in liver were in general increased by phenobarbital in

females but decreased in males; carbon tetrachloride caused an accumulation of HCB in
both males and females. In adipose tissue a sex difference was also observed. With females,
both phenobarbital and carbon tetrachloride increased the rate of decline of HCB. In
males, phenobarbital had no consistent effect on the decline of HCB while carbon
tetrachloride caused a slower rate of decline. Plasma levels of HCB showed no consistent
effect and did not reflect liver HCB values.

In summary then, tissue residue data for male and female rats fed 20 and 40 ppm of
HCB for three months have been presented. In general females tended to have higher
residues in their tissues than males. The relative concentrations of residues in the tissues
were: F a t > liver > kidney > spleen = heart = brain > plasma. HCB decay profiles for
male and female rats fed 20 and 40 ppm of HCB and treated with phenobarbital or
carbon tetrachloride were established. Enzyme data indicated that microsomal enzymes
were induced by phenobarbital and decreased by carbon tetrachloride. No direct correla-
tion could be made between the induction of enzyme activity and an increased rate of
disappearance of HCB residues in liver tissue. Plasma cholinesterase values were decreased
in male and female rats treated with carbon tetrachloride.

References

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metabolism of DDT in the rat and the bovine. Toxicol. Appl. Pharmacol. 18,457
(1971).
Casarett, L. J., G. C. Fryer, W. L. Yauger, and H. W. Klemmer: Organochlorine pesticide
residues in human tissue. Arch. Environ. Health, 17,306 (1968).
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Glende, E. A.: On the mechanism of carbon tetrachloride toxicity: Coincidence of loss of
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6, 102 (!971).
252 D.C. Villeneuve et al.

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metabolism of Terraclor in beagle dogs, rats and plants. J. Agr. Food Chem., 17,
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Manuscript received October 29, 1973; accepted December 15, 1973