Effect of fasting, diethyl maleate, and alcohols on carbon tetrachloride-induced hepatotoxicity

rOXICOL.OGY

AND

Effect

APPLIED

PHARMACOLOGY

56, 19l-

198 (1980)

of Fasting, Diethyl Maleate, and Alcohols Tetrachloride-Induced Hepatotoxicity

on Carbon

R. N. HARRIS AND M. W. ANDERS Department

~fPharmaco/o~~.

Unil~ersit?,ofMinnesota.

435 Delaaure

Street.

SE. Minneclpolis.

Minncsotrr

5.54’5

Effect of Fasting, Diethyl Maleate, and Alcohols on Carbon Tetrachloride-Induced Hepatotoxicity. HARRIS, R. N.. AND ANDERS. M. W. (1980). Tuxicol. App. Pharmacol. 56, l9l- 198. The effect of fasting, diethyl maleate treatment, and methanol, ethanol, isopropanol. or tert-butanol treatment on carbon tetrachloride (CCl,)-induced hepatotoxicity in male Sprague-Dawley rats was studied. A 24-hr fast and the administration of diethyl maleate were found to be equipotent and not additive in potentiating Ccl,-induced hepatotoxicity: this potentiation appeared to be due to a depletion of hepatic glutathione (GSH) levels. Concomitant diurnal variations in hepatic GSH content and in CCL,-induced hepatotoxicity also suggested hepatic GSH involvement in CC],-induced hepatic damage. Whereas ethanol has been reported to potentiate Ccl,-induced hepdtotoxicity and to lower hepatic GSH levels, the present study suggested that these effects are due to an ethanolinduced loss of body weight. Methanol, iso-propanol. and tert-butanol, on the other hand. show the same maximal potentiation of CC&-induced hepatotoxicity and do so without inducing a depletion of hepatic GSH content or producing a loss of body weight. In contrast to a previous report, however, methanol was found to be markedly less effective on an equimoiar basis than either iso-propanol or tert-butanol in potentiating Ccl,-induced hepatotoxicity. The study suggests that GSH is an important modulator of CC],-induced hepatotoxicity and also suggests that methanol, i$o-propanol. and tcrt-butanol. but not ethanol. potentiate CC],-induced hepatotoxicity by a mechanism that does not involve altered hepatic GSH levels.

The potentiation of carbon tetrachloride (CCL&induced hepatotoxicity by starvation (Kirshnan and Stenger, 1966; Highman et al., 1973; Diaz Gomez et al., 1975) and by treatment with various short-chain alcohols (Cornish and Adefuin, 1967; Traiger and Plaa, 1971: Strubelt et al., 1978; Strubelt, 1980) is well known, but the mechanisms of this potentiation have not been clarified. Carbon tetrachloride-induced hepatotoxicity results from bioactivation of Ccl, to toxic metabolites (Slater, 1966; Reynolds, 1967; Recknagel, 1967). Although an increase in Ccl, bioactivation has been suggested as being involved in the alcohol potentiation of Ccl,-induced hepatotoxicity

(Cote rt al., 1974; Traiger and Plaa, 1973: Maling et al., 1974, 1975; Sipes (‘1 ul.. 1973: Strubelt et (I/., 1978), evidence on this point is largely indirect. Reduced glutathione (GSH), a cellular nucleophile. serves to detoxify reactive intermediates formed by the bioactivation of many compounds (Orrenius and Jones. 1978). However, the role of GSH in Ccl,induced hepatotoxicity is poorly understood. Since both fasting (Tateishi rt tri.. 1974; Maruyama er (11.. 1968) and ethanol (Macdonald et al., 1977) have been reported to lower hepatic GSH levels, the objective of the present study was to investigate the interrelationships between starvation.

192

HARRIS AND ANDERS tion. Serum glutamate-pyruvate transaminase activity in aortic blood samples collected under light ether anesthesia was determined using a Beckman System TR enzyme analyzer. Reduced glutathione was quantified according to the spectrofluorometric method of Hissin and Hilf (1976) except that rat livers were perfused with ice-cold 1.15% KCI and blotted dry prior to weighing. Nonperfused livers showed about 50% higher GSH concentrations than perfused livers; this difference is attributable to blood GSH. Results were analyzed using Student’s t test or a one-way analysis of variance followed by the Duncan’s new multiple-range test. A log transformation of SGPT and hepatic GSH concentrations yielded an equivariant data population and was used for statistical analysis. 0

207

413 Ccl. .mmoies/kg

620

826

FIG. I. Effect of varying doses of Ccl, on SGF’T levels. Ccl, was administered as described under Methods; control animals were given corn oil. SGFT levels were determined 24 hr after administration of CCL, or corn oil. Data represent the mean ? SE of four animals.

and modulation of alcohol treatment, hepatic GSH levels as they relate to Ccl,induced hepatotoxicity. METHODS Male Sprague-Dawley rats (190-250 g) were used. Animals were maintained on a 12-hr light and l2-hr dark cycle (6 AM--~ PM). Animals were fasted by withholding food for 24 hr; water was available ad libitum. Ccl, was administered ip as a 10% corn oil solution between the hours of 10 AM and 11 AM, unless otherwise specified. Alcohols were given ip or orally 18 hr prior to giving Ccl, or measuring GSH levels. Ethanol, iso-propanol, and tert-butanol were administered ip as 25% aqueous solutions and methanol as a 50% aqueous solution. In some experiments, high doses of alcohols [5 (84 mmol) or 6 ml (100 mmol) ethanol/kg; 7 ml (173 mmol) methanol/kg; 2.5 ml (33 mmol) iso-propanol/kg] were administered orally as 10 ml of the appropriate aqueous dilution/kg. In these experiments, animals were weighed and the alcohols administered at 4 PM. Eighteen hours later the animals were again weighed and either sacrificed for the determination of hepatic GSH levels or given 4.13 mmol CClJkg and sacrificed for serum glutamatepyruvate

transaminase

(SGPT)

determination

24 hr

later. Diethyl maleate (DEM) was administered ip, undiluted, at a dose of 0.6 ml (3.7 mmol)/kg, 30 min prior to Ccl, administration or GSH determina-

RESULTS Serum glutamate-pyruvate transaminase levels were used to evaluate CC&-induced hepatotoxicity. Dose-response studies were used to establish a dose of CCL which produced a SGFT level against which either protection from or potentiation of hepatotoxicity could be evaluated (Fig. 1). A dose of 4.13 mmol CC&/kg, which caused an 11-fold elevation in SGFT levels, was chosen for future experiments. The effects of four short-chain alcohols on CC&-induced SGPT release were investigated (Fig. 2). The administration of the alcohols alone did not alter SGPT levels (data not shown). On an equimolar basis, tert-butanol was slightly more potent than iso-propanol in potentiating CC&-induced SGFT release (significantly different at 5.15 mmol/kg); methanol was considerably less potent than iso-propanol at low doses and ethanol showed no potentiating ability. The maximum level of enzyme release was not significantly different for tert-butanol, iso-propanol, or methanol. It should be noted that, at the highest doses used in these studies, the alcohols produced obvious toxic effects. For example, one of four rats died after receiving 20.6 mmol tert-butanoll kg and all animals given 41.2 mmol tertbutanol/kg died; one of four rats died after receiving 26.8 mmol iso-propanollkg and three of four rats died after 82.4 mmol

C&INDUCED

i93

HEPATOTOXICITY

&&BUTANOL

ALCOHOL.mmoles/kg

FIG. 2. Effect of varying doses of methanol, ethanol, iso-propanol, and fert-butanol on CC&-induced elevations of SGPT levels. Alcohols were administered ip as 25 or 50% aqueous solutions as described under Methods. All animals received 4.13 mmol CC&/kg ip as a 10% corn oil solution. SGPT levels were determined 24 hr after administration of Ccl,. Data represent the mean + SE of four animals unless indicated otherwise under Results.

ethanol/kg. In contrast, all rats survived high doses of alcohols when given orally (Figs. 4 and 5). The effects of fasting and DEM administration on CC&-induced elevations in SGFT were also studied (Table 1). In these studies, Ccl, was given either ip or po to compare the two routes of administration. In fed rats, the ip route of administration resulted in higher SGPT levels than did the oral route (Table 1, I vs V). Fasting, DEM treatment and the combination of these treatments resulted in SGFT levels that were significantly greater than those seen in controls (Table 1, I and V), but not significantly different from each other. Fasting did not alter the iso-propanol potentiation of CC&-induced SGPT release (data not shown). The effect of time of administration of Ccl, was investigated (Fig. 3). A significant (p < 0.05) increase in SGPT levels was seen only at a Ccl, dose of 4.13 mmol/kg; no difference was seen at a lower (2.07 mmol/kg) or a higher (8.26 mmol/kg) dose. Fasting (Tateishi e? al., 1974; Maruyama et ul., 1968), DEM treatment (Boyland and Chasseaud, 1970) and diurnal variation (Beck ef nl., 1958) each produce changes in hepatic GSH levels. The changes in GSH

levels produced by these treatments as well as by alcohol treatment and Ccl., administration were investigated (Table 2). Treatment of rats with DEM lowered hepatic TABLE EFFECT ANDDIETHYL

I

OF ROUTE OF ADMINISTRATION. MALEATEOKCARBONTEIRACHLORIDF-

INDUCED CHANGES IN SERUM TRANSAMINASE LEVELS”

GLUTAMATE-P\

FAST IN{,. RW

41 I-

” DEM wab given ip, undiluted. 0.6 ml (3.7 mmc~lvhg 30 mitt prior to Ccl, administration. Fasted animal\ h,td food withheld 24 hr prior to Ccl, administration. ,411 animal\ received 4. I3 mmol CCl.,/kg ip between IO ,\\t and I i \‘.t as a 10% corn oil solution. SGPT levels were determined 24 htafter Ccl, administration and are expressed a> mean : \E. ‘j Significantly different from all other treatment\ I ,’ . 0.0 Duncan’s new multiple-range test). ’ Significantly different from treatments 1 and V I/’ . . 0.0’. Duncan‘s new multiple range testl. hut not from any ,>ther treatment.

194

HARRIS AND ANDERS

3500

UAM 1 EaPM

-8

FIG. 3. Effect of time of administration on Ccl,induced elevations in SGPT levels. Ccl, was administered ip as a 10% corn oil solution at 10 AM or 10 PM. SGPT levels were determined 24 hr after Ccl, administration. Corn oil was given to control animals; control SGPT levels were 29 + 2 IUiliter. Data represent the mean ? SE of four animals. The asterisk indicates a value significantly different from the corresponding 10 AM value (p < 0.05).

GSH levels by 83% in 30 min and fasting produced a 56% decrease. Fasting plus DEM treatment led to a 90% reduction in

1,

-15

I,

I,

-10 -5 0 5 CHANGE IN BODY WEIGHT;%)

I

+I0

FIG. 4. Correlation between alcohol-induced loss of body weight and hepatic GSH levels. Alcohols (see Methods) were administered orally at 4 PM and the change in body weight and hepatic GSH concentration was determined 18 hr later. Correlation analysis yielded a sample correlation coefficient (r) of 0.74 (p < 0.001). The 95% confidence interval for the population coefficient (p) is 0.57 5 p 5 0.85. Water (10 ml/kg), 0; ethanol (84 mmol/kg), 0; ethanol (100 mmol/kg), n ; iso-propanol (33 mmol/kg), & methanol (173 mmol/kg), r).

t4 -4 0 CHANGE IN BODY WEIGHT

1%)

FIG. 5. Correlation between ethanol-induced loss of body weight and ethanol potentiation of CC&-induced elevation of SGPT levels. Ethanol [84 (0) or 100 (w) mmoYkg] was given orally 18 hr prior to the administration of 4.13 mmol CC&/kg. SGFT levels were determined 24 hr after CCI, administration. Correlation analysis yielded a sample correlation coefficient (r) of -0.83 (p < 0.001). The 95% confidence interval for the poptdation coefficient (p) is -0.94

5 p 5 -0.58.

GSH levels. A diurnal variation in hepatic GSH levels was seen, the value at 10 PM being 38% lower than that found at 10 AM. Treatment of rats with 6.44 mmol/kg of iso-propanol, ethanol or .fert-butano1 did not alter hepatic GSH levels. The administration of either Ccl, alone or CCI, plus iso-propanol did not alter hepatic GSH levels (Table 2, IX and XIII vs I). The hepatic GSH levels seen 12 hr after giving CCL, (measured at 10 PM) were not significantly different than those seen at 10 PM in the absence of Ccl, (Table 2, XI vs II). Twenty-four hours after giving Ccl,, hepatic GSH levels increased to 149% of those seen in controls (Table 2, XII vs I). High doses of alcohols (see Methods) were administered to determine their effects on weight loss, hepatic GSH levels, and Ccl,induced hepatotoxicity. A direct correlation was found between weight loss and the lowering of hepatic GSH levels after giving methanol, ethanol, or iso-propranol (Fig. 4). A direct correlation was aIso found between the weight loss following ethanol administration and the potentiation of Ccl,induced hepatotoxicity (Fig. 5). In contrast, a potentiation of CC&-induced hepatotoxic-

CC&-INDUCED

195

HEPATOTOXICITY

TABLE 2 EFFECT OF FASTING, DIETHYL MALEATE, DIURNAL VARIATION, ALCOHOL TREATMENT. AND CARBON TETRACHLORIDE ON HEPATIC GLUTATHIONE LEVELS AND BODY WEIGHT”

N

Treatment I Control

38

Fasting, DEM and diurnal variation 11 Diurnal variation III 24-hr fast IV 0.6 ml (3.7 mmol) DEM/kg V 24-hr fast plus 0.6 ml DEMikg Prior (18 hr) Vl 6.44 VI1 6.44 VIII 6.44

5 5 7 4

alcohol treatment mmol ethanol/kg mmol iso-propanol/kg mmol /err-butanolikg

Time after 4.13 mmol CCl,/kg, ip IX I hr X 6 hr XI 12 hr XII 24 hr Xl11 1 hr (rats treated 18 hr previously with 6.44 mmol iso-propanolikg,

2.99 t- 0.06 1.86 1.33 0.52 0.36

k 2 -t +

Change in body weight”,” (%) +4.h + 0 7

0.11 (10 PM) 0.09 0.05 0.05

Not done -11.8 -c 07 Not done - 12.2 _i 0 9

3.14 + 0.15 2.90 2 0.17 2.91 lr 0.21

-7.0 i- 0.2 1-4.7 i. 0.9 il.95 1.4

I 3 3 3

2.78 2.53 2.27 4.47

Not done -4.3 -1- 0,s ---3.7 ‘I 0.4 -m4.0 2- I.2

4

2.59 2 0.13 ( 1 I AM)

4 12 4

ip)

pmol GSHig liver wet weight”.’

2 0.16(11 AM) + 0.41 (4 PM) t 0.11 (10 PM) -c O.l6(10AM)

Not done

” DEM was administered undiluted, ip, 30 min prior to GSH determination. Alcohols were administered ip as 2S% aqueous solutions. Ccl, was administered as a 10% corn oil solution. Data are expressed as mean f SE. h GSH levels determined as 10 AM unless otherwise specified in parentheses. ’ Results analyzed by Duncan’s new multiple-range test [treatment numbers appearing on the same line are not significantly different from each other (p > 0.05); treatment numbers appearing on different lines arc significantly different from each other (p < 0.05)]: pmol GSH/g liver wet wt

Change in body weight

IV V XII III II. XI I, VI. VII, VIII. IX, x, XI. XIII

III, v I. VI. VII, 1, VII. VIII. x. XI. XII

‘I Change in body weight determined from time of treatment to time of tissue sampling.

ity, occurred following methanol or isopropanol administration whether or not the animals showed a loss of body weight.

DISCUSSION The potentiation of Ccl,-induced hepatotoxicity by treatment with various shortchain alcohols is well known (Comish and

Adefuin, 1967; Traiger and Plaa, 1971; Strubelt et al., 1978). No dose-response studies of this phenomenon have been reported and, therefore, it is not known whether the doses used in previous studies may be producing secondary effects which prevent causal interpretation of the data. A 24-hr fast results in a decrease in body weight, a lowering of hepatic GSH levels and a potentiation of CCL,-induced hepa--

196

HARRIS

AND

totoxicity. Previous studies have demonstrated that fasting lowers hepatic GSH levels (Tateishi et al., 1974; Maruyama et al., 1968) and that fasting potentiates CC&-induced hepatotoxicity (Krishnan and Stenger 1966; Highman et al., 1973; Diaz Gomez et al., 1975). Likewise, it was found that DEM treatment lowers hepatic GSH levels and potentiates Ccl,-induced hepatotoxicity, as previously reported (Gillette, 1973). The potentiation of drug toxicity by DEM treatment is often attributed to the depletion of hepatic GSH (Mitchell et al., 1973; Reid and Krishna, 1973; Brown et al., 1974; Siegers et al., 1977). The depletion of hepatic GSH levels by DEM has been reported to potentiate (Lindstrom et al., 1978; Gillette, 1973; Siegers et al., 1977) and to protect (Suarez and Bhonsle, 1977) against CC&-induced hepatotoxicity. The present investigation demonstrates that DEM treatment and fasting are equipotent in potentiating CC&-induced hepatotoxicity and suggests that lowered hepatic GSH levels are involved in the potentiation of CC&-induced hepatotoxicity seen after fasting and DEM administration even though fasting does not deplete hepatic GSH levels to the same extent as does DEM treatment. The lowering of hepatic GSH levels beyond a critical level may result in no further potentiation of CC&-induced hepatotoxicity or fasting may involve factors additional to GSH depletion. Ethanol treatment has been reported to lower hepatic GSH levels (Macdonald, 1973; Macdonald et al., 1977) and to potentiate CC&-induced hepatotoxicity (Comish and Adefuin, 1967; Traiger and Plaa, 1971; Strubelt et al., 1978; Strubelt, 1980). This suggests that the potentiation of CC&induced hepatotoxicity by ethanol, and perhaps other alcohols, may be caused by a lowering of hepatic GSH levels. The present investigation shows that doses of alcohols which cause a loss of body weight produce a decrease in hepatic GSH levels and potentiate Ccl,-induced hepatotoxicity. In con-

ANDERS

trast, doses of isa-propanol, tert-butanol, and methanol, but not ethanol, which do not cause a loss of body weight do not lower hepatic GSH levels, but still potentiate CC&-induced hepatotoxicity. On a millimole per kilogram basis, iso-propanol and tert-butanol are virtually equipotent in potentiating, while methanol is much weaker in potentiating Ccl,-induced hepatotoxicity. Ethanol does not appear to potentiate CC&-induced hepatotoxicity apart from that potentiation associated with loss of body weight and the concomitant hepatic GSH depletion. These results are in contrast to a previous report that methanol is equipotent with iso-propanol in potentiating CC&-induced hepatotoxicity (Cantilena et al., 1979). Cantilena et al. (1979) used a dose of 7.0 ml (173 mmol) methanol/kg; the present study shows that this is an excessively large dose of methanol and may cause a loss of body weight. This methanolinduced loss of body weight compromises the elucidation of the processes responsible for the potentiation of C&-induced hepatotoxicity, since fasting and the accompanying decrease in hepatic GSH levels themselves markedly potentiate Ccl,-induced hepatic damage. Dose-dependent diurnal changes in susceptibility to CC&-induced hepatotoxicity suggest the involvement of GSH in this phenomenon (Fig. 3). Hepatic GSH levels at 10 PM are significantly lower than those measured at 10 AM; this agrees with previous reports (Jaeger et al., 1973; Beck et al., 1958). The lack of diurnal variation in CC&-induced hepatotoxicity after administration of 2.07 and 8.26 mmol CCl,/kg suggests that these doses are, respectively, subthreshold and supramaximal challenges of Ccl, to the protective function of hepatic GSH. The diurnal variation seen after giving 4.13 moles CCl,/kg, however, suggests that, when the dose of Ccl, exceeds a critical threshold, the protection provided by hepatic GSH is overwhelmed. The steep

Ccl,-INDUCED

197

HEPATOTOXICITY

dose-response curve for Ccl,-induced increases in SGPT levels may be due to the small dose range over which Ccl, exceeds protection by hepatic GSH. Although the diurnal increase in hepatic cytochrome P-450 activity seen at night (Radzialowski and Bousquet, 1967) may be involved in the diurnal variation of Ccl,-induced hepatotoxicity, the lack of potentiation of 2.07 mmol CCl,/kg at 10 PM argues against such an interpretation. The concept of a threshold dose has been discussed by Gillette rt (11. (1974) in elaborating the mechanism of toxicity of bromobenzene and acetaminophen which deplete hepatic GSH during their metabolism. Siegers et al. (1977) have reported that Ccl, administration lowers hepatic GSH levels in mice. However, in the present study, the administration of Ccl, was not found to alter hepatic GSH levels, even though decreases in hepatic GSH levels paralleled increased sensitivity to Ccl,-induced hepatotoxicity. The apparent decrease in hepatic GSH levels seen 12 hr after Ccl, administration (at 10 PM) is not different from the level of hepatic GSH seen at 10 PM in control animals. Treatment withiso-propanol markedly potentiates CCL-induced hepatotoxicity. but does not cause a Ccl,-induced depletion of hepatic GSH at 1 hr after Ccl, administration. An increase in hepatic GSH levels to values greater than control levels is seen 24 hr after Ccl, administration. A less dramatic increase was reported 12 hr after administration of 1 ml (10.3 mmol) CCl,/kg (Priestly and Plaa, 1970). but such an increase was not reported 24 hr following administration of chloroform (Docks and Krishna, 1976). An increase in hepatic nonprotein sulfhydryl levels to values greater than control levels has been reported following protein refeeding after a depletion of hepatic nonprotein sulfhydryl by a 3-day dietary protein deprivation (Register et ~1.. 1959). The mechanism of the rebound in GSH levels seen after giving certain hepatotoxins is not understood.

ACKNOWLEDGMENTS The authors wish to thank Joan Sunram for techmcal assistance. This research was supported by National Institutes of Health Grant ES 00953. RNH was supported by Institutional National Research Serblice Award No. GM 07397.

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(1973). Diurnal variation of hepatic glutathione concentration and its correlation with l,l-dichloroethylene inhalation toxicity in rats. Res. Commun. Chem. Pathol. Pharmacol. 6, 465-471. KRISHNAN, N., AND STENGER, R. J. (1%6). Effects of starvation on the hepatotoxicity of carbon tetrachloride: A light and electron microscopic study. Amer. J. Pathol. 49, 239-246. LINDSTROM, T. D., ANDERS, M. W., AND REMMER, H. (1978). Effect of phenobarbital and diethyl maleate on carbon tetrachloride toxicity in isolated rat hepatocytes. Exp. Mol. Pathol. 28, 48-57. MACDONALD, C. M. (1973). The effects of ethanol on hepatic lipid peroxidation and on the activities of glutathione reductase and peroxidase. FEBS Lett. 35, 227-230. MACDONALD, C. M., Dow, J., AND MOORE, M. R. (1977). A possible protective role for sulfhydryl compounds in acute alcoholic liver injury. Biochem. Pharmacol. Xi, 1529- 153 1. MALING, H. M., EICHELBAUM, F. M., SAUL, W., SIPES, I. G.. BROWN, E. A. B., ANDGILLETTE, J. R. (1974). Nature of the protection against carbon tetrachloride-induced hepatotoxicity produced by pretreatment with dibenamine [N-(2-chloroethyl) dibenzylamine]. Biochem. Pharmacol. 23, 14791491. MALING, H. M.. STRIPP, B., SIPES, I. G., HIGHMAN, B., SAUL, W., AND WILLIAMS, M. A. (1975). Enhanced hepatotoxicity of carbon tetrachloride, thioacetamide. and dimethylnitrosamine by pretreatment of rats with ethanol and some comparisons with potentiation by isopropanol. Toxicol. Appl. Pharmacol. 33,291-308. MARUYAMA, E., KOJIMA. K., HIGASHI, T., AND SAKAMOTA, Y. (1968). Effect of diet on liver glutathione and glutathione reductase. J. Biochem. 63, 398-399. MITCHELL, J. R.. JOLLOW, D. J., POTTER, W. Z., GILLETTE, J. R., AND BRODIE, B. B. (1973). Acetaminophen-induced hepatic necrosis. IV. Protective role of glutathione. J. Pharmacol. Exp. Ther. 187, 211-217. ORRENIUS, S., AND JONES, D. P. (1978). Functions of glutathione in drug metabolism. In Functions of Glutathione in Liver and Kidney (H. Sies and A. Wendel. eds.), pp. 164-175. Springer-Verlag, New York. PRIESTLY, B. B., AND PLAA, G. L. (1970). Temporal aspects of carbon tetrachloride-induced alteration of sulfobromophthalein excretion and metabolism. Toxicol. Appl. Pharmacol. 17, 786-794.

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of J.