The role of metabolism in chloroform hepatotoxicity

The role of metabolism in chloroform hepatotoxicity

TOXICOLOGY AND The APPLIED Role PHARMACOLOGY of Metabolism 29, 312-326 (1974) in Chloroform Hepatotoxicity JEAN-GUY LAVIGNE AND CLAUDE MARC...

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TOXICOLOGY

AND

The

APPLIED

Role

PHARMACOLOGY

of Metabolism

29, 312-326

(1974)

in Chloroform

Hepatotoxicity

JEAN-GUY LAVIGNE AND CLAUDE MARCHAND De’partement de pharmacologic, Faculte’ de r&de&e, UniversitP de Montre’al, MontrPal 101, Qukbec, Canada Received October 18, 1973; accepted January 22,1974

The Role of Metabolism in Chloroform Hepatotoxicity. LAVIGNE, J.-G. MARCHAND, C. (1974).Toxicol. Appl. Pharmacol. 29, 312-326. Since there is indirect evidence that a metabolite may be responsiblefor the hepatotoxicity of CC&, the possibilitythat chloroform may alsoact through a similar mechanismwas investigated.Pretreatment with stimulators of drug metabolizing enzymeslike phenobarbital, 3-methylcholanthreneor 3,4benzopyreneincreasedthe toxicity of chlorform in rats, asreflectedby increasedserumglutamic-pyruvic transaminaseand a decreasein liver glucose-6-phosphatase activity. This enhancementof the toxicity of CHC13 was associatedwith an increasein 14CHC13 metabolism,as measuredby pulmonary excretion of 14C02.An inhibitor of drug metabolizingenzymes, SKF 525-A, wasfound to decreasepulmonary excretion of 14C02.If these observationsmay be taken as indirect evidencethat the hepatotoxic effect of CHC& is dueto a metabolite,other data do not seemto support suchan hypothesis. No metabolite of CHC& could be detected by gas-liquid chromatography; therewasno quantitative correlation betweenthe increase in toxicity and the increasein CHC13metabolism.Finally, SKF 525-A did not decreaseCHC& hepatotoxicity. It is concluded that metabolismof CHC& may beinvolved in the hepatotoxiceffect of CHC13,but other factors may alsoplay a role in determiningthis response. AND

Many hypotheses have been proposed to explain the hepatotoxicity of halogenated hydrocarbons. Of these compounds, carbon tetrachloride has been the most widely studied (Recknagel, 1967). There is indirect evidence that Ccl, may exert its hepatotoxicity through a metabolite (Butler, 1961; Glende and Recknagel, 1969).The purpose of the present investigation was to study the possibility that chloroform, another chloromethane, may also act through a hepatotoxic metabolite. METHODS Sprague-Dawley male rats (Canadian Breeding Farm, St-Constant, Quebec), weighing between 150and 200 g, were usedthroughout theseexperiments. They were subjected to one of the following treatments: 3,4-benzopyrere (3,4-BP), 20 mg/kg/day, ip, for 2 days; 3-methylcholanthrene (3-MC), 40 mg/kg/day, ip, for 2 days; phenobarbital, 50 mg/kg/day, ip, for 3 days; 2 diethyl aminoethyl-2,2-diphenylvalerate hydrochloride (SKF 525-A), 40 mg/kg, ip. Control animals were given the vehicle, either corn oil or saline, 1.0 ml/kg. Copyright 0 1974 by Academic Press. Inc. All rights of reproduction in any form reserved. Printed in Great Britain

312

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AND

CHLOROFORM

HEPATOTOXICITY

313

Before administration of chloroform, the animals were fasted overnight with water ad libitum. Twenty hr after the last injection of phenobarbital, 3,4-BP or 3-MC, or 40 min after injection of SKF 525-A, the animals received, ip, 14CHC13, 0.5 ml (0.83 pCi)/kg (NEN, Canada). Chloroform excretion by the lungs. After 14CHCI, administration, rats were placed in a sealed desiccator into which filtered, humidified air was flowing at constant pressure. Air leaving the desiccator passed through 2 consecutive toluene towers. With the help of a needle, a small aliquot of toluene was taken 1, 2 and 4 hr after 14CHCl, administration. The radioactivity was then measured by liquid scintillation spectroscopy (Packard Instrument Co.). ‘“CHCI, measurement in tissue. 14CHCI, determinations in different tissues were made according to a method previously described for 14CC14 (Marchand et al., 1970, 197 1). At different times after injection of 14CHC13, animals were decapitated and tissues rapidly taken out. Two milliliters of blood were shaken mechanically with 10 ml of toluene for 10 min and the radioactivity, extracted into toluene, was measured by liquid scintillation spectrophotometry. Specimens of the following tissues were also taken: 1 lobe of liver (0.8-1.0 g); the entire brain (1.6-1.8 g); striated muscle from the hind leg (0.6-1.0 g); fat tissue from the epididymal region (0.4-0.6 g) and the 2 lungs (0.8-1.0 g). These tissues were rapidly weighed and placed in 10 ml of refrigerated toluene. 14CHCI, was allowed to diffuse in toluene for 3 days. Throughout these experiments we made the assumption that toluene-soluble 14C is 14CHCl,. Chloroform biotransformation. For the measurement of 14C02, a third tower, containing 2 N NaOH was added to the system previously described. The measurement of CO2 was carried out according to the method of Ugazio et al. (1972) except that the carbonate precipitate was suspended in 10 ml of Aquasol (NEN, Canada) for the measurement for the radioactivity. Results were expressed in terms of 14CHCl, converted to expired COa. A gas chromatograph (Varian Aerograph, series 2100) with electron capture detector, fitted with 250 mCi of 3H, was used for determination of the chloromethanes. The stainless steel 5’ x l/8” column was filled with 10% silicone DC 550 on Chromosorb P, acid washed, mesh 45/60. The injector temperature was maintained at 80°C; the column temperature was 45°C and the detector temperature was maintained at 150°C. The carrier gas flow (NJ was delivered at 20 ml/min. Chloroform, obtained from American Chemical, was used for these experiments with Fisher Scientific pesticide grade benzene as the carrier solvent. Measurement of chloroform and other possible chloromethanes in the blood and the liver by gas-liquid chromatography was made according to the protocol described for 14CHC13, except that 2.0 ml of benzene was used for extraction purpose. Enzymatic determinations. Serum glutamic-pyruvic transaminase (SGPT) activity was measured according to the method of Reitman and Frankel(l957); 1 Sigma-Frankel unit will form 4.82 x 10e4 prnol of glutamate/min at pH 7.5 and 25°C. Glucose-B phosphatase (G-6-Pase) activity was determined as previously described (Mar&and et al., 1971). Statistics. Significance of the difference between control and treated rats was assessed by Student’s t test and a p value of 0.05 or less was considered significant.

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RESULTS

As illustrated in Fig. 1, rats pretreated with phenobarbital excreted much more 14C02 through the lungs than did control animals, 2 and 4 hr after 14CHCl, injection. During the first 4 hr, these last animals excreted, in the form of 14C02, 0.39 % of the administered dose of 14CHCl,; this compares with about 1.3 % found by Paul and

IPI 1

2 Ttme

4

1

2

Chr)

:lmr

4 Chrl

FIG. 1. Effect of sodium phenobarbital (50 mg/kg/day, ip for 3 days) on the pulmonary excretion of r4CHCls (panel A) and “TO2 (panel B) at different times after ip injection of “THQ, 0.5 ml (0.83 ,Ki)/kg. Each point represents the mean of &8 rats + SE. * p < 0.05. x 10,ooor

1,000

A

i L

~~ -1 1

2 Ttme thr:

4



1

2

4 T,TP

(hrl

FIG. 2. Effect of 3,4-benzopyrene (20 mg/kg/day, ip for 2 days) on the pulmonary excretion of “CHC13 (panel A) and “TO2 (panel B) at different times after ip injection of 0.5 ml (0.83 pCi)/kg. Each point represents the mean of 68 rats + SE. * p < 0.05.

Rubinstein (1963) in the same perod and 4-5 % reported by Van Dyke et al. (1964) over a 12-hr period. Similar changes in 14C0, excretion were observed in rats pretreated with 3,4-BP (Fig. 2). Total excretion of CO, in rats pretreated with 3-MC was about twice that observed with the two other pretreatments (Fig. 3). SKF 525-A, an inhibitor of drug-metabolizing enzymes, was found to decrease 14C0, excretion by the lungs (Fig. 4).

METABOLISM

AND

CHLOROFORM

HEPATOTOXICITY

2,500

10 L

L-. 1

4

2 Time

1

4 T,me

cn0

hrl

FIG. 3. Effect of 3-methylcholanthrene (40 mg/kg/day, ip for 2 days) on the pulmonary excretion of WHCl, (panel A) and %Oz (panel B) at different times after ip injection of WHC13, 0.5 ml (0.83 &)/kg. Each point represents the mean of 6-8 rats + SE. * p < 0.05.

TIME (hr)

FIG. 4. Effect of SKF 525-A (40 mg/kg, ip) on the pulmonary excretion of r4COz at different times after ip injection of “CHC13, 0.5 ml (0.83 &i)/kg. Each point represents the mean of 6 rats + SE. * p < 0.05.

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The increased excretion of CO, in rats pretreated with inducers of drug metabolizing enzymes was associated with a parallel decrease in chloroform excretion by the lungs (Figs. l-3). During the 4-hr study, rats pretreated with phenobarbital excreted 7.5 % of the administered CHCI,, compared to 10.4% for control rats (Fig. 1). However, it does not seem that a quantitative relationship exists between CHCl, conversion to CO, and the excretion of the parent compound, as reflected by animals pretreated with 3-MC (Fig. 3). In the rats pretreated with SKF 525-A, no difference from control animals was found in 14CHC1, pulmonary excretion 1 and 2 hr following its administration; a relatively small difference was observed 4 hr after injection of the halogenated hydrocarbon (Fig. 5). 20,000 I

TIME (hr)

FIG. 5. Effect of SKF 525-A (40 mg/kg, ip) on the pulmonary excretion of 14CHC13, at different times after ip injection of 14CHCla, 0.5 ml (0.83 ,uCi)/kg. Each point represents the mean of 6 rats + SE. * p < 0.05.

In order to establish a possible relationship between CHCI, pulmonary excretion and tissue distribution, the halogenated hydrocarbon was measured in different tissues. In rats pretreated with phenobarbital, there was a decrease in CHCl, concentration in the blood, brain and muscle, whereas an increase was observed in the liver 1 hr after administration of CHCl, (Table 1). However, 4 hr after CHCl, injection, a significant increase in 14CHCl, fat concentration was observed in rats pretreated with phenobarbital. Pretreatment with 3-MC caused a slight increase in the halogenated hydrocarbon concentration in the blood and fat tissues 4 hr after administration of chloroform. Little change was observed in animals pretreated with 3,4-BP (Table 2). On the other hand, pretreatment with SKF 525-A was associated with a decrease in CHCl, concentration in the muscle and the brain tissue 1 hr after injection of the halogenated hydrocarbon but an increase in concentration was found 3 hr later in all tissues except the lungs and muscle (Table 3).

1 1 2 2 4 4

Time after administration (W 285 fr 234k 203 k 185f 172+ 188 *

Blood 8 6” 14 7 7 9

8143 + 10209 f 6079 + 6724 + 3494+ 4442 rt

Fat 1708 1291 1101 1556 131 356’

642 + 881 + 367 f 394 & 170 + 18Ok

Liver 56 71” 65 57 12 8

208 + 8 178 f 7” 136+6 127?4 116 + 3 122+4

Brain

14CHC13Ocglml or ,a/g) -

’ Each value represents the mean of 6-8 rats + SE. b Phenobarbital was given at a dose of 50 mg/kg/day, ip for 3 days before ip injection of WZHC13, 0.5 ml (0.83 &i)/kg. cp < 0.05.

Saline Phenobarbital Saline Phenobarbital Saline Phenobarbital

Treatmentb

1

184f 6 142+ 7c 166 f 17 135 + 20 105 + 4 124f 11

Muscle

EFFECT OF PHENOBARBITAL PRETREATMENT, ON WHC& TISSUE CONCENTRATION AT DIFFERENT TIMES AFTER INJECTION OF WHCI 3 ’

TABLE

77 + 3 72 + 7

-

Lungs

2 3 2

54 s tf 8 E 31 Y 5 0

5

x 3 $ $ E $

318

LAVIGNE

AND

TABLE EFFECTOF 3,4-BENZOPYRENEOR TISSUE DISTRIBUTION,

4

MARCHAND

2

%METHYLCHOLANTHRENEON HR AFTER INJECTION OF

CHClj 14CHClJ

14CHC13 (&ml or ,&g) TreatmenP Corn oil 3-MC Corn oil 3,4-BP

Blood 141+ 183 + 179 + 203 i-

Fat 7 11” 11 17

2650 3289 3164 3191

+ ) rt *

Liver 141 254’ 161 221

Brain

167 + 10 171+ 7 196f 11 209+ 6

Muscle

99 It 5 114+6 1271- 5 139f 6

93 92 116 118

5 f k +

Lungs 5 5 5 5

70+ 4 70+ 8 97+ 10 98+ 4

0 Each value represents the mean of 9 rats + SE. * 3,4-Benzopyrene was given at a dose of 20 mg/kg/day, ip for 2 days; 3-methylcholanthrene was given at a dose of 40 mg/kg/day, ip for 2 days. The dose of ‘*CHC& was 0.5 ml (0.83 &i)/kg, ip. = p < 0.05.

r

-

-

2

& 3

b FIG. 6. Effect of phenobarbital sodium (50 mg/kg/day, ip for 3 days) on liver G&Pa se and SGPT 4 hr after ip injection of CH& 0.5 ml/kg. Each value represents the mean of 6 rats + SE. SGPT G-6-Pase

1 vs2 3 vs 4 1 vs 3 2 vs 4

p < 0.05 p < 0.05 NS p < 0.05

1 vs 2

NS

3 vs 4

p < 0.05 p < 0.05 p < 0.05

1 vs 3 2 vs 4

Blood 236-c 8 171* 31 136+ 10 172 + 13’ 124+ 8 150+ 8’

Time after administration (hr)

1 1 2 2 4 4

6351+ 1018 4165 +_1426 4125+ 924 3345& 530 2462+ 125 3534+ 300’

Fat 524+ 74 423 +_123 187k 11 216+ 22 130&- 6 159* 4”

Liver 19.5+ 15 135+ 23” 104f 7 124f 9 84+ 4 104* 4’

Brain

r4CHCIJ bg/ml or g)

’ Eachvaluerepresents the meanof 6-8 rats+ SE. * SKF 525-Awasgivenat a doseof 40 mg/kg,ip, beforeip injectionof WHCIJ, 0.5 ml (0.83&i)/kg. cp < 0.05.

Treatment” --Saline SKF 525-A Saline SKF 525-A Saline SKF 525-A

3

228 f 120+ 129f 155f 79+ 92k

12 24c 11 12 6 4

Muscle

EFFECT OF SKF 525-A, ON CHC& TISSUE DISTRIBUTION AT DIFFERENT TIMES AFTER INJECTION OF 14CHC13’

TABLE

54+ 3 55 2 2

-

Lungs

5 =i <

3

8 E 31 4 * 3

0

5 n P %

z z 5 0 c B

320

LAVICNE

AND

MARCHAND

In order to establish a possible relationship between CHCl, metabolism and its toxicity, we measured SGPT activity and hepatic G-6-Pase activity. As shown in Fig. 6, the dose of CHCl, used in these experiments significantly increased SGPT activity; a marked potentiation of this effect occurred in rats pretreated with phenobarbital. This same dose of CHCl, when given alone had no effect on G-6-Pase. This confirms previous reports (Reynolds and Yee, 1967; Klaassen and Plaa, 1969; Reynolds, 1972). However, in animals pretreated with phenobarbital, a marked decrease in G-6-Pase activity was observed.

a

b

FIG. 7. Effect of 3,4-benzopyrene (20 mg/kg/day, ip for 2 days) on G-6-Pase and SGPT, 4 hr after ip injection of CHC&, 0.5 ml/kg. Each value represents the mean of 6 rats + SE. G-6-Pase SGPT 1 vs 2 3 vs 4

p < 0.05 p < 0.05

1 vs 3

NS

2 vs 4

p < 0.05

1 vs2

NS

3 vs4 I vs 3 2 vs 4

p < 0.05 p < 0.05 p < 0.05

Pretreatment with either 3,4-BP or 3-MC increased the toxicity of chloroform (Figs. 7 and 8). In terms of the SGPT values, pretreatment with phenobarbital potentiated the hepatotoxicity of CHCl, to a much greater extent than did pretreatment with 3-MC or 3,4-BP. It must also be pointed out that, although CHCI, had no effect on G-6-Pase when given alone, 3-MC and 3,4-BP triggered a decrease in this microsomal enzyme in rats treated with CHCl,. As to the pretreatment with SKF 525-A, it did not seem to

METABOLISM

AND

CHLOROFORM

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HEPATOTOXICITY

i a

I -

cm, 2

-

,-MI

-

3

b

FIG. 8. Effect of 3-methylcholanthrene (40 mg/kg/day, ip, for 2 days) on liver G-6-Pase and SGPT, 4 hr after ip injection of CHCL, 0.5 ml/kg. Each value represents the mean of 6 rats f SE. SGPT G-6-Pase 1 vs 2 3 vs 4 1 vs 3 2 vs 4

p < 0.05 p < 0.05 NS p < 0.05

1 vs 2 3 vs 4 1 vs 3 2 vs 4

NS p < 0.05 NS p < 0.05

protect against CHCl, hepatotoxicity (Fig. 9). As previously reported (Marchand et al., 1971; Koff and Fitts, 1972), SKF 525-A alone caused a slight but significant increase in SGPT activity. Since it was conceivable that pretreatment with these different enzyme inducers could cause the formation of otherwise undetectable metabolites, we looked for such metabolites in our gas chromatographic system. No toluene-soluble metabolite in the blood or the liver could be detected under our experimental conditions. Although Butler (1961) reported that chloroform was reduced to methylene chloride in vitro by mouse liver, no metabolite other than CO, has ever been found in vivo (Paul and Rubinstein, 1963; Van Dyke et al., 1964; Fry et af., 1972). DISCUSSION

There is indirect evidence that Ccl, may exert its hepatotoxic effect through a metabo1961; Glende and Recknagel, 1969). Some of this evidence comes from the

lite (Butler,

322

LAVIGNE

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MARCHAND

l-l

2

a

-z +

cm,

3

r

CHCI, 4

b

FIG. 9. Effect of SKF 525-A (40 mg/kg; ip) on liver G-dPase and SGPT, 4 hr after ip injection of CHCl,, 0.5 ml/kg. SKF 525-A was administered 40 min before CHC13. Each value represents the mean of 6 rats + SE. SGF’T G-6-Pase __.____1 vs 2 p < 0.05 1 vs 2 NS 3vs4 p < 0.05 3 vs 4 NS 1 vs 3 p < 0.05 1 vs 3 NS 2 vs 4 NS 2 vs 4 NS

observation that phenobarbital, a compound which stimulates drug metabolizing enzymes, increases the toxicity of Ccl., (Garner and McLean, 1969; Stenger et al., 1970; Reynolds et al., 1972), whereas SKF 525-A, an inhibitor of the same enzymes, protects against the hepatotoxicity of the halogenated hydrocarbon (Slater, 1966; Castro et al., 1968). Some of our observations are compatible with the hypothesis that CHCIJ, like CCL, acts through a metabolite. Like Scholler et nl. (1968), we found that phenobarbital increases the toxicity of chloroform as reflected by SGPT and G-6-Pase activities. According to these same criteria, 3,4-BP and 3-MC would also potentiate CHC& hepatotoxicity. Since elevation of SGPT activity is a fairly quantitative measure of certain forms of liver toxicity (Balazs et al., 1961; Zimmerman et al., 1965; Klaassen and Plaa, 1969), 3,4-BP and 3MC appear to be less effective than phenobarbital for potentiating the hepatotoxicity of CHC13. This is not surprising since compounds that stimulate drug-metabolizing enzymes may differ in the spectrum of enzymes induced, the character of CO-binding

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323

and possibly their mechanism of action (Fouts and Rogers, 1965; Sladek and Mannering, 1969). In this respect it is interesting that whereas 3-MC was found to potentiate the hepatotoxic effect of chloroform, it protects against the hepatotoxicity of Ccl, (Reid et aE., 1971; Suarez et al., 1972). Failure of CHCl, alone to decrease liver G-6-Pase in our studies is not surprising since others have reported similar findings (Klaassen and Plaa, 1969; Reynolds, 1972). In this context, the decrease in liver G-6-Pase by CHCl, in animals pretreated with 3-MC, 3,4-BP and phenobarbital raises interesting questions. These compounds could possibly condition the liver cell to make it more sensitive to CHCI,. Also, one cannot readily rule out a latent hepatotoxic potential of these drug-metabolizing enzyme inducers that could be responsible for an additive or potentiating effect on CHCI, hepatotoxicity. Phenobarbital, by itself, decreased liver G-6-Pase activity. Although it is premature to draw definite conclusions from this isolated observation, a possible mild hepatotoxic response to phenobarbital cannot be ruled out. Ariyoshi and Remmer (1968) have shown that phenobarbital increases hepatic triglycerides; Sorrel1 et al. (1973) have observed fatty liver after administration of slightly higher doses of phenobarbital. Since SGPT and liver G-6-Pase activities are measurements of specific enzymatic systems that can be taken only as indicators of certain forms of toxicity, different liver function and morphologic studies need be undertaken before a generalization can be made. If pretreatment with enzyme inducers enhances the toxicity of CHCIJ, it is interesting to note that no decrease in the hepatotoxic effect of the halogenated hydrocarbon was observed in animals pretreated with an inhibitor of drug metabolizing enzymes like SKF 525-A. The protective effect of SKF 525-A on hepatotoxicity after oral administration of CCL, has been observed by different investigators (Slater, 1966; Castro et al., 1968). These last investigators interpreted the protective effect of SKF 525-A as an inhibition of the metabolism of CC&, whereas Marchand et al, (1970,197l) explained the protection by a decrease in CCI, liver concentration. Contrary to these last observations, no significant decrease in CHCI, liver concentration was observed in rats pretreated with SKF 525-A 1 hr after administration of the halogenated hydrocarbon, although a significant diminution in concentration was found in the muscle and the brain. However, 4 hr after injection of 14CHC13, there was significant increase in the halogenated hydrocarbon concentration in all tissues except lungs and muscle. We have no explanation for this apparent biphasic effect of SKF 525-A although changes in blood flow to different organs or tissues may play a role in these changes in chloroform concentrations (Marchand and Brodeur, 1970; Brodeur and Marchand, 1971). In this regard, it should be noted that SKF 525-A has been found to have a biphasic effect on drug metabolizing enzyme activity (Serrone and Fujimoto, 1962; Rogers and Fouts, 1964).

If a compound which stimulates drug metabolizing enzymes enhances the hepatotoxicity of CHCI, by increasing its metabolism, one would expect an increase in its main metabolite, namely CO, (Paul and Rubinstein, 1963; Van Dyke et al., 1964). Our observations indeed indicate an increase in metabolism of chloroform in rats pretreated with either of these compounds. However, quantitatively, there seems to be little correlation between the increase in hepatotoxicity and the augmentation in chloroform metabolism. This lack of correlation between CHCl, metabolism and toxicity may be

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observed in the excretion of CO, in rats pretreated with SKF 525-A. This compound did not protect against the hepatotoxicity of CHCI, whereas a decrease in CO, excretion was noted in animals pretreated with SKF 525-A. This apparent contradiction may be due to the fact that we measured i4C0, excretion by the lungs of rats and not 14C02 formation. Indeed, any effect of the drugs on the respiratory center of rats could change the meaning of 14C0, excretion in relation to 14CHC1, metabolism. Also, the respiratory rate, sensitive to different external factors, such as changes in temperature, humidity and stress may explain the marked differences in 14C0 and 14CHCI, excretion between control groups in experiments reported here. Large viriations in 14C02 excretion by the lungs after treatment with 14CHCl, have also been observed by Van Dyke et al. (1964). From these observations, it may be concluded that compounds which stimulate drugmetabolizing enzymes potentiate CHCl, liver hepatotoxicity and increase its metabolism. However, no cause and effect relationship could be established between these two phenomena. Furthermore, there are observations that are not easily reconciled with the hypothesis that CHCl, acts through a metabolite. No quantitative relationship between an increase in CHCl, metabolism and the hepatotoxic effects could be established. SKF 525-A, which decreased CHCl, metabolism, did not protect against CHCl, hepatotoxicity. Finally, no metabolite other than COZ could be detected under our experimental conditions. This obviously does not preclude the existence of such metabolites, especially if free radicals are formed or if chloroform is transformed into products other than chloromethanes.

ACKNOWLEDGMENTS This research was supported by the Medical Research Council of Canada. We wish to thank Smith Kline and French of Canada for the generous supply of SKF 525-A. The authors are grateful to Miss Claudette Lamoureux for her able technical assistance.

REFERENCES ARIYOSHI, T. AND REMMER,H. (1968). Die Wirkung von Phenobarbital und Diphenylhydantoin auf verschiedene. Naunyn-Schmiedeberg’s Arch. Pharmakol. 260, 90-91. BALAZS, T., MURRAY, T. K., MCLAUGHLAN, J. M. AND GRICE, H. C. (1961).Hepatic testsin toxicity studieson rats. Toxicol. Appl. Pharmacol. 3, 71-79. BRODEUR, J. AND MARCHAND, C. (1971). Effect of splenectomyon the activity of drugmetabolizingenzymesin the liver of rats. Can. J. Physiol. Pharmacol. 49, 161-166. BUTLER, T. C. (1961).Reduction of carbon tetrachloridein viuo and reduction of carbon tetrachloride and chloroform in vitro by tissuesand tissueconstituents.J. Pharmacol. Exp. Ther. 134,311-319. CASTRO, J. A., SASAME, H. A., SUSSMAN, H. AND GILLEI-TE, J. R. (1968).Diverse effects of SKF 525-A and antioxidants on carbon tetrachloride-inducedchangesin liver microsomalP-450 content and ethylmorphinemetabolism.Life Sci. Pt. 1 7, 129-136. FOUTS, J. R. AND ROGERS, L. A. (1965).Morphological changes in the liver accompanying any stimulation of microsomal drug metabolizing enzyme activity benzopyrene or methylcholanthrene in rats. J. Pharmacol. FRY, B. J., TAYLOR, J. AND HATHWAY, D. E. (1972).Pulmonary its metabolite in man. Arch. Znt. Pharmacodyn. Ther. l%,

by phenobarbital,

chlordane,

Exp. Ther. 147, 112-119. elimination

98-l 11.

of chloroform

and

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