Toxicology, 6 (1975) 09—77
© EUcvicr/North-HoHmul, Amsterdam

Printed in The Netherlands

POTKN'I'IATION OK CARBON TETRACHLORIDE HEPATOTOXICITY
IN RATS BY PRETREATMENT WITH POLYCHLORINATED BIPHENYLS

GARY P. CARLSON
Depor/mcnf of Pharmacology and Toxicology, College of Pharmacy, University of llhodc
Itland, Kingston, H.l. 02881 (U.S.A.)
(Received Miirch 16th, 1975)
(Accepted May 6th, 1975)
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SUMMARY

Pretreatment of male rats with Aroclor 1254 at a dose of 25 mg/kg i.p. for
6 days resulted in potentiation of the hepatotoxicity of inhaled carbon
tetrachloride (CCI4) as evidenced by a decrease in liver glucose-6-phospha­
tase and elevations of serum glutamic oxalacetic transaminase (SCOT), se­
rum glutamic pyruvic transaminase (SGPT), isocitrate dehydrogenase, and
sorbitol dehydrogenase. Aroclor 1254 alone did not demonstrate hepatotox­
icity. Aroclor 1254 administration resulted in large increases in cytochrome
c reductase, cytochrome P-450 (448) and p-nitroanisole demethylation. Sub­
sequent exposure to CCU vapor resulted in over 70% decreases in the latter
two parameters. The potentiation was dose-dependent with a dose of 5
mg/kg or higher being effective. Aroclor 1260 administration gave results
similar to those of Aroclor 1254, but Aroclor 1221 enhanced CCI4 toxicity
to a lesser extent.

INTRODUCTION

Since McLean and McLean (1) first demonstrated that the enzyme-induc­
ing agent DDT was capable of potentiating the toxicity of CC14, many
investigators have found this to be true of other enzyme-inducing agents
such as phenobarbitai (2~4), In addition to phenobarbitai and DDT, other
investigators have looked at the influence of the polycyclic hydrocarbon
type inducers which differ in the microsomal carbon monoxide binding pig­
ment induced, i.e. P-448 rather than P-450, and in the spectrum of enzymes
induced (51 Pitchumoni et a). [6] demonstrated that benzofa] pyrene was
similar to phenobarbitai in enhancing the toxicity of CC14. However, Suarez
Abbreviation*: DDT, 1,1 ,Ttrichloro-2,2-bis(p-chlorophcnyl)ethanc; I’CB, polychlorinated
biphenyl; SOOT. 6erum glutamic oxaiacetic transaminase; SGPT, serum glutamic pyruvic
transaminase.

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el *1. [3] demonstrated that 3-methylcholanthrene protected against the
hepalotoxicity of CC14, It thus appears that enhancement versus protection
is not simply a matter of cytochrome P-450 versus P-448.
Recently Alvaros and coworkers |7] have shown that the polychlorinated
biphenyls are unique as enzyme-inducing agents in that they induce the
formation of cytochrome P-448 as does 3-methyichoianthrene, but they
resemble phenobarbital in that they bring about a more general type of
induction. In view of this uniqueness, it was of interest to ascertain what
effects these compounds have on CCI4 toxicity.
Although the PCB’s as commercially used both in the past and in the
present are complex mixtures, they are numbered according to the percent
chlorine by weight, i.e., Aroclor 1264 contains 54% chlorine. Since the
chlorine content appears to have an influence on the inducing potential of
the PCB’s [8,9], it was also important to determine if percent chlorine
would be a factor in alteration of CCi4 toxicity.
MRTHODS

Adult male albino rats (Charles River Breeding Laboratories) were used.
They were allowed food and water ad lib. and were housed in temperatureand light-controlled rooms. They were injected i.p. with Aroclor 1254 dis­
solved in com oil at a dose of 25 mg/kg unless otherwise indicated. Controls
received com oil alone. Injections were made daily for 6 days and 24 h after
the last dose the rats were exposed to CC14 vapor in a dynamic inhalation
chamber [10] for a period of 2 h. Chamber concentrations were determined
using a Packard gas chromatograph with a flame ionization detector. 22 h
after exposure the rata were lightly anesthetized with ether, liver and tail
vein blood samples taken and various parameters of hepatotoxicity were
assessed.
Liver and body weights were recorded. Liver giucose-6-phosphatase was
measured using the procedure of Harper [11] with-maleate buffer, pH 6.25.
p-Nitroanisole demethylation was determined according to the method of
Netter and Seidel [12] as modified by Kinoshita et al. [13]. SGOT and
SGPT transaminases were measured utilizing the method of Reitman and
Frankel [14]. Isocitrate dehydrogenase was determined .ccording to the
procedure of Ellis and Goldberg [16] and sorbitol dehydrogenase by the
method of Gerlach (16).
In the microsomal cytochrome experiments, the livers were perfused with
cold isotonic KC1, removed, and homogenized ' i cold isotonic KC1. The
homogenate was centrifuged at 90(V0g for 20 min in a Sorvall Model RC2B
refrigerated centrifuge and the supernatant further centrifuged at 105 000g
for 1 h in an International Mode) 11-00 UHraccntrifuge. Cytochrome c reduc­
tase activity and cytochrome P-450 content in the resulting microsomal
fraction were measured according to the methods of Dallner [17]. Protein
concentrations of the microsomes were determined by the method of Lowry
et al. 118).

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The value expressed is Ihe mean t standard error. Student's t test was used
to compare means at a chosen level of significance of P = 0.05.
RESULTS

The effects of exposing the rats to 3600 ppm of CCI4 for 2 h on 3
parameters are presented in Table I. Pretreatment with Aroclor 1254 re­
sulted in an increase in the liver to body weight ratio. This increase was still
greater in those animals exposed to C'C14, although CCI4 exposure by itself
did not alter the ratio. At this level of exposure there was a small decrease in
liver glucosc-6-pho8phatase activity due to the CCl4 alone. However, in those
animaU pretreated with Aroclor 1254 there was a large decrease in activity
to one-third of the air-exposed level. As a further indication of damage to
the functioning of the liver, drug metabolism was measured since this is
known to be decreased by CC14 and the effect potentiated by phenobnrbital
but protected against by 3-mcthylcholanthrenc [4]. As indicated in Table 1,
a decrease in activity was seen with CC14 inhalation. Aroclor 1254 adminis­
tration resulted in an 8-fold increase in activity which decreased dramatically
following CCI4 inhalation.
To further study the potentiation by Aroclor 1254 on the decrease in
microsomal enzyme activity due to CCI4, animals were exposed to 4200
ppm of CCI4 and cytochrome c reductase activity and cytochrome P-450
content were measured. As indicated in Table 11, cytochrome c reductase
activity was elevated 2-fold by Aroclor 1254. In neither the controls nor the
Aroclor-pretreated rats was the activity altered by CCI4. In the case of the
P-460 (P-448) content, Aroclor 1254 pretreatment resulted in a 3-fold in­
crease in this cytochrome. CC14 inhalation resulted in a decrease in both the

TABLE 1
EFFECT OF AROCLOR 1264 AND CCI4 ON LIVER WEIGHT/BODY WEIGHT,
GLUCOSE-6PIIOSPHATASE, AND pNITKOANISOLE DEM ETHYLATION
Treatment

N*

yr«rwi. x
Body wt.

Corn oil—Air
Corn oll~CCl4tf
Aroclor 1264*—air
Aroclor 1264—CCI4

5
6
6
6

4.28 ±0.07
4.02*0.16
4.96 ± 0.26*
6.91 ±

jqq

Glucose-6phosphatase11

/>-Nitroam»ole
demeihylation*

12.0 t 0.66
10.2 X 0.58f
10.2 ± 0.34
3.6 1 0.37f-B

5.1 ± 0.39
2.9 t 0.17*
43.1 J. 0.42*
12.0 i 1.57f«

* Number of rata
t vmolec P04/g/mln.
e vg/60 mg/30 min.
4 36OO ppm for 2 h.
* 26 mg/kg i.p. for i days.
* Significantly different (P < 0.06) from group receiving same pretreatment.
* Significantly different (P < 0.05) from group receiving same exposure.

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TABLE n
EFFECT OF AROCLOR 1254 AND CC14 ON SERUM TRANSAMINASES AND MICROSOMAL CYTOCHROME c
REDUCTASE AND CYTOCHROME P-450
Treatment

Na

Corn oil—air
Corn oil—CCI4*
Aroclor 12541—air
Aroclor 1254-CC14

5
5
5

Cytochrome e
j-educta»eh

Cytochrome
P-450*

SGPT*

SGOT*

76 ± 4.9
7S * 8.1
144 ± 11.4*
132 ± 7.2*

161 t 13.3
95 ± 18.7h
522 ± 37.3*
63 ± 20.2*

13 ±
2.3*
338 ± 43.1**
11 ±
1.4
2213 ± 193.4‘hJ

r,.6*
40 ±
446 ± 140.2*
33 ±
2.3
2316 ± 297.71*'*

* Number of rats.
b nmoles of cytochrome e reduced/mg protein,'min.
* Difference in absorbance between 450 and 500 nm/mg protein x 10*.
d Reitman-Frankel units
* 4200 ppm for 2 h.
f 25 mg/kg «.p. for 6 days.
* 4 animals in this group.
h Significantly different (P < 0.05) from group receiving same pretreatment.
1 Significantly different (P < 0.05) from group receiving same exposure.

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controls and in the Aroclor-pretreated groups although the magnitude of the
change in the latter case (88%) was much greater than that seen in the
controls (41%).
Serum enzyme measurements were utilized as additional indicators of
increased CCI4 toxicity. As indicated in Table I), CCj4 exposure increased
SGPT and SCOT 26-fold and ll-foid, respectively. However, in the Aroclorpretreated rats these transaminases were elevated 200-fold and 70-fold, re­
spectively. Aroclor 1254 itself was without effect.
To test the influence of the chlorine content on hepatotoxicity, Aroclor
1254 was compared with Aroclor 1221 and Aroclor 1260 in separate experi­
ments. Two other serum enzymes, sorbitol dehydrogenase and isocitrate
dehydrogenase were used in hopes of increasing the sensitivity. Preliminary
experiments indicated no alteration of theue enzymes due to Aroclor 1254
alone. Therefore, Aroclor-pietreated rats exposed to air were not included in
these studies. As indicated in Table 111, exposure to 2900 ppm of CC14 for
2 h did not result in a significant change in gtucose-6-phosphatasc activity in
the corn oil-pretreated rats. Activity was decreased by 35% in the Aroclor
1221-prctreatcd animals. In the animals pretreated with Aroclor 1254 the
decrease in the enzyme activity due to CC14 was 70%. In the case of the
sorbitol dehydrogenase, no activity was seen in the air controls, but, meaTABLE III
COMPARISON OP EFFECTS OF AROCLORS 1221, 1254 AND 1260 ON CCI4
HEPATOTOXICITY
Treatment

NB

Glucose-6
phosphatase1*

Sorbitol
dehydrogenase6

Corn oil—air
Corn oil—CC14*
1221*—CC14C
1264*—CC14e

5
5
5
6

16.9 4 0.75
13.5 4 0.90
10.4 ± 0.78*J
4.7 + 0.40*44*

f*5 1 22.3*
62 ± 10.7*
495 ±

Corn oil—air
Corn oil—CC14«
1264*-CC14"
12C01—CCl4*

6
6
5
6

10.4 4 0.91
16.0 10.83*
7.1 1 0.99*4
8.7 + 0.90*4

40± 1.1*
J09 + 86.3*4
424 4 6B.7,J

Isocitrate
dehyrirogcnntted

0

0
.

9+
0 7**4
lf>i
1.6*4
1324 * 465.]U'k

0

0
13+
1.0*
670 « 220.3*4
11 10 1 349.5*4

“ Number of rata.
b pmolea P04/g/min.
! 4
m * 10s/0.2 ml scrum. iv/i.
* 2600 ppm for 2 h.
* 26 mg/kg for 6 days.
1 2900 ppm for 2 h.
h 4 rat* in this group.
* Significantly different (P < 0.06) from corn oil—air group.
1 Significantly different (/' < 0.06) from com oil—CC14 group.
** Significantly different (P < 0.05) from other Aroclor pretreated group.

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V

suitable levels were found alter CC14 inhalation. These were not significantly
altered by Aroclor 1221 pretreatment but were elevated 9-fold by Aroclor
1251 pretreatment, Similar findings were seen with the isocitrate dehydro­
genase. Only slight activity was seen with CC14 exposure in the com oil
controls. This was increased to a small degree (G6%) in the Aroclor 1221
pretreated rats but much elevated in the Aroclor 1254-pretreated animals
(147-fold).
Aroclor 1260 presentment was very similar to Aroclor 1264 (Table III).
In none of the three parameters was there a statistical difference between the
two Aroclors although they both showed remarkable potentiation of the
changes due to CC14.
Because of the impressive potentiating ability of Aroclor 1264 of the
hepalotoxicity of CC14, it was of interest to determine if even lower doses of
the Aroclor would enhance CC14 toxicity. Therefore, groups of rats were
injected with 6, 10 or 25 mg/kg of Aroclor i.p. for 6 days. The results as
presented in Table IV indicate that even at the dose of 6 mg/kg Aroclor
1264 potentiated the toxicity of CCi4 measured in terms of alterations In
liver glucose-6-pho8phatase and the serum enzymes sorbitol dehydrogenase
and isocitrate dehydrogenase although in the case of the latter enzyme, the
large standard errors prevented the nearly 8-fold potentiation from being
statistically significant. It should also be noted that the potentiating ability
appeared to be dose-related.
Of further interest in view of the ability of Aroclor 1254 to potentiate
CC14 hepatotoxicity was determining if it would also potentiate the toxicity
TABLE IV
DOSE RESPONSE OF AROCLOR 1254 ON HEPATOTOXICITY
Treatment

N*

Glucose-6phosphatase*

Sorbitol
dehydrogenase'

Corn oil—air
Corn oil—CCJ4*
Aroclor 1254r~OCl4

8
8

18.6 4 0.61
13.2 ± 0.47ft

1 t
46?

6 mg/kg

8

0.6 4 0.51**h

10 nig/kg
25 mg/kg

8
8

8.3 i. 0.41
5.8 i 0.16*’h,(

132 1 27.0*,h
240 1 53.7*-h
340 1 40.8*,h'1

laocitrate
dehydrogenase*

0

0.7
9.6*

12 ±

3.1*

01 1 37.16
819 i 140.0***
488± 119.7fch,‘

• Number of rot*.
b jimolrt P04/g/min.
c
108/0.2 ml

* HJ/f
* 4200 ppm for 2 h.
1 i.p. daily for 0 days.
• Significantly different (P < 0.06) from corn oil -air.
h Significantly different (P < 0.05) from corn oil- CCJ4.
1 Significantly different (P < 0.05) from Aroclor 1254—5 mg/kg.
’ Significantly different (P < 0.05) from Aroclor 1204-10 mg/k".

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TABLE V
INFLUENCE OF CONCENTUATTON OF CCl, ON POTENTIATION OF HEVATOTOXJCITY BY AROCLOR I 284
Pretreatment

CCI4*
(ppm)

Nb

Olucoftr-6phosphnts*ec

Sorbitol
dehydrogenase*1

IsocitraU*
dehydrogenase'

Corn oil
Corn oil
Aroclor 1264*
Corn oil
Aroclor 1264*

0
690
590
2350
2360

3
3
3
3
3

19.7 i 1.06
17.6 i,0.36
10.4 i 0.24*b
Ifi.l ± 0.821
7.1 1 1.56*b

0
0
31 1 3.8**
15* 6.0*’1
148 1 15.9*"J

1 ±
0.7
21
0.6
42 i ll.l*h
8 *
7.3
2R8 1 113.6*

* 2 h exposure.
Number of ril*.

* jimo)eftP04/8/rni».
4

* 10»/0.2mlw»rum.

' IUfl.
1 26 mg/Vg for 6 days.
* Significantly different (P < 0.06) from com oil—air.
h Significantly different (P < 0.06) from group with tame exposure.
1 Significantly different (J* < 0.06) from group with same pretreatment.

of a lower level of CCI4 exposure. To determine this, animals were exposed
to 590 ppm CC14 for 2 h and compared with animals exposed to 2350 ppm.
The data in Table V indicate that at this lower level of exposure, no toxicity
due to CC14 alone was evidenced by any of the three parameters measured.
However, In all three cases there were significant changes in the case of the
Aroclor pretreated animals exposed to CC14. As expected, there were
changes due to CC14 alone when it was inhaled at the higher concentration
and these were further exacerbated in those* animals pretreated with Aroclor
1264.
DISCUSSION

Although Bruckner el a?. (19] reported that Aroctor 1242 administration
resulted in liver toxicity as evidenced by 1 .leveled SGOT values, the present
experimonte indicated lilUc if any hcpatotoxicity due to the Aroclor alone.
This discrepancy is probably due to the fact that Bruckner cl at. used larger
doses and/or longer time periods. CCt* alone produced toxicity similar to
other studies concerned with its .inhalation fl4J.
That Aroclor 12G4 potentiates the toxicity of CCl4 was demonstrated in
all of the studies reported hon.v As expected (It)] the administration of the
Aroclor increased the liver to body weight ratio. This was further increased
following CC14 administration, hiver I'lucofce-G-phospbuttisc activity was de­
creased. The rises in the four serum enzymes that were measured were all
greatly increased by the pretrenlment with Aroclor 1264.
lire effects of Aroclor 1254 on drop metabolism were us expected.

75

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ArocJor-indueed increases in cytochrome r reductase and cytochrome P-460
have been shown by other workers j 10] as has the increase in / -nitroanisole
demethylntion (9]. Ar with phcnobarbitel |4], neither the normal level of
cytochrome c reductase nor the elevated level following induction was al­
tered by CO 4 inhalation. However, similar to the case with phenobarbetal,
there was a dramatic decrease in l'-450 content following CCI4 exposure in
those animals pretreated with the Aroclor 1264 as compared with the con­
trols. Similar comparisons can bo made with the effect on drug metabolism
as evidenced by the large decrease in />-nitroanisole demcthylation.
The comparison of the Aroclors was important in view of the findings of
Chen and DuBois [9] that the ability of Aroclors 1221, 1264 and 1260 to
induce JV-demelhylation and also EPN detoxification increased in proportion
to the chlorine content of the Aroclor but. that the O-demethylation of
p-nilroanisoic was induced to a greater extent by Aroclor 1264 than by
Aroclor 1260. The results (Table 111) indicate that the two more chlorinated
mixtures increase the hcpatotoxicity of CC14 more than the less chlorinated
Aroclor 1221, although no difference was detected between the two more
chlorinated ones.
An indication that Aroclor 1254 is a potent potentiator of CC14 toxicity
was indicated by the finding that a dose of oniy 5 mg/kg for 6 days wot
enough to demonstrate potentiation. Because of the difficulty in showing
statistical significance due to the large variation in response from animal to
animal following CCl4 inhalation, it did not seem worthwhile to give lower
doses of the Aroclor. Another indication of its potency was the observation
that even at a lower level of CC14 (690 ppm) than is usually employed in
these studies there were significant elevations of the serum enzymes and
decrease in liver glucose-6-phosphatase in the induced animals.
A general conclusion that can be drawn from the results of these experi­
ments is that the Aroclors which induce P-448 rather than P-460 (7] arc
very potent enhancers of CCl4 hepatotoxicity so that care must be taken in
considering the suggestion that 3-methylcholanthrene protects against CC14
by virtue of the fact that it induces P-448 [20].
ACKNOWLEDGEMENTS

The author wishes to acknowledge the able technical assistance of Mrs.
Barbara Schulte and Mrs. b’adylis Wood. The Aroclors were n gift of the
Monsanto Company, St. T^ouis, Missouri. The work was supported by N1EHS
Grant No. 00596.
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