Potentiation of thioacetamide-induced hepatotoxicity in alloxan- and streptozotocin-diabetic rats

Toxicology Letters, 17 (1983) 293-300 Elsevier

293

POTENTIATION OF THIOACETAMIDE-INDUCED HEPATOTOXICITY IN ALLOXAN- AND STREPTOZOTOCIN-DIABETIC RATS (Fasting on toxicity; ketosis; inducers on toxicity)

A. MONAEM EL-HAWARI*

and GABRIEL L. PLAA**

De’partement de Pharmacologic, FacultP de Mdecine, 3J7 (Canada)

Universitk de MontrPal, Mont&al, QuPbec H3C

(Received January 27th, 1983) (Accepted February 4th, 1983)

SUMMARY Thioacetamide-induced hepatoxicity was potentiated in male Sprague-Dawley rats rendered diabetic by alloxan or streptozotocin. The response was more striking in alloxan-diabetic rats. Insulin administration prevented the potentiation following alloxan pretreatment. Fasting also resulted in an enhanced hepatotoxic response to thioacetamide, but the increase was much less than that observed in rats given the diabetogenic agents. The ketosis produced by alloxan was more severe than that induced by streptozotocin, but was unlike that caused by fasting. Pretreatment with phenobarbital, 3-methylcholanthrene or 3,4-benzpyrene did not enhance thioacetamide liver injury.

INTRODUCTION

Hanasono et al. [l] demonstrated that the hepatotoxic response to a challenging dose of CCL was potentiated in rats rendered diabetic by pretreatment with alloxan or streptozotocin. The hepatotoxic properties of CHCI3 and 1,1,3-trichloroethane were also enhanced by alloxan [2]. These studies were performed to see if a metabolic disease state involving the generation of abnormal amounts of ketonic substances could affect the course of haloalkane hepatotoxicity, since several ketones * Fellow of the Medical Research Council of Canada. Present address: Midwest Research Institute, Kansas City, MO (U.S.A.) ** To whom requests for reprints should be sent. Abbreviations:

ICD, isocitric dehydrogenase;

GPT, glutamic-pyruvic transaminase.

0378-4274/83/0000-OOOO/$ 03.00 0 Elsevier Science Publishers

294

or ketogenic chemicals can potentiate haloalkane-induced hepatotoxicity [3, 41. The results obtained with alloxan and streptozotocin are consistent with the hypothesis that the induction of a metabolic ketosis increases the susceptibility of the liver to the toxic effects of haloalkanes in much the same fashion as does the administration of ketonic solvents. Recently, Price and Jollow [5] demonstrated that acetaminophen liver injury was not potentiated in streptozotocin-pretreated rats. In their experiments, diabetic rats were less susceptible to acetaminophen toxicity than normal animals. This prompted us to report our findings with thioacetamide-induced hepatotoxicity; we find that alloxan and streptozotocin pretreatment enhances the hepatotoxic response. MATERIALS AND METHODS

The diabetic state was produced in male Sprague-Dawley rats (180-240 g) obtained from Bio-Breeding Laboratories as previously described [ 11. Alloxan (40, 60 or 80 mg/kg, i.v.) dissolved in saline, was administered 4 days and streptozotocin (65 mg/kg, i.v.), dissolved in citrate, 5 days before challenging the animals with thioacetamide (50 or 100 mg/kg, i.p.). Some alloxan-treated groups were given protamine zinc insulin (30 units/kg/day, s.c.). The rats were killed 24 h after the administration of thioacetamide. Blood samples were obtained for the following analyses: glucose [6] and bilirubin [7] content; GPT [8] and ICD [9] activity. In a second experiment, blood and liver ketone bodies (acetoacetate and phydroxybutyrate) were determined [lo, 1l] 2 and 4 days after alloxan or 2 and 5 days after streptozotocin treatment. In a third experiment, thioacetamide hepatotoxicity was assessed in rats pretreated with phenobarbital (75 mg/kg/day, i.p., 3 days), 3-methylcholanthrene (20 mg/kg/day, i.p., 3 days) or 3,4-benzpyrene (20 mg/kg/day, i.p., 1 day); thioacetamide (100 mg/kg, i.p.) was administered 24 h after the pretreatment and the rats killed 24 h later. The data obtained in the experiments were subjected to an analysis of variance before effecting the appropriate intergroup comparisons. RESULTS

Thioacetamide given alone (Table I, Groups I, V, VII, XII) resulted in only a slight increase in plasma GPT and ICD activities, indicating that the challenging doses (50 or 100 mg/kg) selected were only slightly hepatotoxic in non-treated animals. Bilirubin content was not affected. However, pretreatment with alloxan (Groups II, III, IV, VIII, IX, XI) resulted in a marked potentiation of thioacetamide liver injury in a dose-related manner. Hyperbilirubinemia, as well as severely evelated plasma GPT and ICD activities, were observed in the alloxanpretreated animals. Streptozotocin pretreatment also enhanced the hepatotoxic response, but the potentiation was similar to that observed with the lowest dosage

295

TABLE I EFFECT OF ALLOXAN OR STREPTOZOTOCIN INDUCED HEPATOTOXICITY IN MALE RATS’ Group

n

Treatment (mglkg)

Thioacetamide dosage: 50 mg/kg I Vehicle (Saline)

GPT activity (units/ml)

8 +

II

Alloxan (40)

6

III

Alloxan (60)

6

IV

Alloxan (80)

6

V

Vehicle (Citrate)

6

VI

Streptozotocin

6

f f f f (65)

f Thioacetamide dosage: 100 mg/kg VII Vehicle (Saline)

8 +

VIII

Alloxan (40)

6

IX

Alloxan (60)

6

X

Alloxan (60 + insulin’ Alloxan (80)

6

2 f

XI

* 5 2

XII

Vehicle (Citrate)

6

XIII

Streptozotocin

6

* (65)

PRETREATMENT

f

91 1 570b 103 898b 99 1024b 157 138 9 640’ 110

244 21 1710* 314 5421* 1097 93’ 12 6150* 1655 195 13 2050r 442

ON THIOACETAMIDE-

ICD activity (units/mI/min)

Bilirubin concentration (mg/lOO ml)

12 1 204b 27 268b 20 307b 53 11 2 195’ 27

0.19 f 0.01 0.24 * 0.02 0.62b + 0.09 1.20b + 0.28 0.20 + 0.01 0.38 + 0.04

73 5 422* f 52 1403* + 201 ND

0.25 + 0.01 0.89* * O.-l2 4.7* r 1.13 0.36’ + 0.05 5.9* rt 1.27 0.22 + 0.01 0.82E f 0.20

f f. + + f +

*

1570* 264 41 4 * 860r f 102 f

aAlloxan (40, 60 or 80 mg/kg, i.v.) was administered 4 days and streptozotocin (65 mg/kg, i.v.) 5 days before thioacetamide (50 or 100 mg/kg, i.p.). Rats were killed 24 h after thioacetamide. ND, not determined. bSignificantly larger than Group I (PcO.05). Significantly larger than Group V (P
of alloxan. Both diabetogenic regimens failed to alter GPT, ICD and bilirubin in rats not treated with thioacetamide (data not shown).

296

TABLE

II

EFFECT

OF

REDUCED

THIOACETAMIDE-INDUCED

FOOD

n

Treatment

INTAKE

Mean change in body

Vehicle (saline)

OR

HEPATOTOXICITY

8

in

weight

ALLOXAN

IN MALE

GPT activity

10.5

6

-

14.9

5

-

11.0

244

0.25

21

* 0.01

(24 h)

5

-

9.8

f

678bC

(48 h)

5

-

112

* 0.03 0.36bC + 0.05

9%

0.42bC

131 800bc

+ 0.05 0.63bC

156

* 0.11

* Fasted

(72 h)

5

-

17.6 *

“Alloxan fasted

(60 mg/kg, rats received

bSignificantly

larger

‘Significantly

smaller

iv.) was administered no alloxan.

ml)

1.13

429b

13.5

concen(mg/lOO

0.48bC

+ Fasted

ON

4.7b

1097

* Fasted

tration

5421b f

Pair-fed

Bilirubin

(070) (units/ml)

* Alloxan

PRETREATMENT

RATS=

4 days before thioacetamide

(100 mg/kg,

i.p.). Pair-fed

and

Rats were killed 24 h after thioacetamide.

than vehicle-treated

group

than alloxan-treated

(P
group

(P< 0.05).

Insulin administration prevented the development of diabetes during the alloxan (60 mg/kg) treatment. The following plasma glucose concentrations (mg/lOO ml) were observed: vehicle + thioacetamide, 127 f 11; alloxan + thioacetamide, 431 * 68; alloxan + insulin + thioacetamide, 93 + 8. Insulin treatment also prevented the alloxan potentiation of thioacetamide liver injury (Group X). Rats treated with alloxan lost weight during the pretreatment period (Table II). Therefore, an experiment was performed to assess the effect of reduced food intake TABLE

III

CONCENTRATION

OF

KETONE

STREPTOZOTOCIN-DIABETIC Treatment

BODIES

MALE

IN

BLOOD

AND

LIVER

OF

ALLOXAN-

AND

RATSa

Blood

Blood

glucose

@mol/ml)

(amoljg)

AcAc

AcAc

(mg/ 100 ml)

ketones

Liver ketones

/?I-HB Total

/3-HB

Total

Vehicle (Saline)

146

0.08

0.11

0.19

0.10

0.19

0.29

Alloxan

(2 days)

328

1.93

4.41

6.34

2.32

4.70

7.02

Alloxan (4 days) Vehicle (Citrate)

490 155

2.76 0.11

5.70 0.13

8.46 0.24

3.10 0.08

6.91 0.14

10.01 0.22

Streptozotocin

(2 days)

413

0.51

1.06

1.57

0.64

1.40

2.04

Streptozotocin

(5 days)

506

0.23

0.38

0.61

0.30

0.77

1.07

88

0.49

1.38

0.87

0.53

1.74

2.27

Fasted

(2 days)

aAcetoacetate

(AcAc) and &hydroxybutyrate

treatment with alloxan (60 mg/kg, an or streptozotocin (n, 2-3).

(fl-HB) concentrations

i.v.) or streptozotocin

(65 mg/kg,

were determined

2, 4 or 5 days after

i.v.). Fasted rats received no allox-

297

on thioacetamide liver injury. The pair-fed (no alloxan treatment) animals lost an equivalent amount of weight and also exhibited an enhanced (about 3-fold) hepatotoxic response to thioacetamide. However, the response was far less than that observed with alloxan (about 20-fold). Fasting for 24-72 h also resulted in an enhanced response, but again it was not comparable to that observed in the alloxanpretreated group. Concentrations of acetoacetate and P-hydroxybutyrate were determined in rats pretreated with alloxan, streptozotocin or subjected to a prolonged fast. Alloxan markedly increased the concentrations of both ketone bodies in the blood and liver (Table III). A smaller increase was observed after streptozotocin, but the increase was comparable to that determined in rats subjected to a 2-day fasting period. To assess the role of mixed function oxidases, rats were also pretreated with phenobarbital, 3-methylcholanthrene or 3,4-benzpyrene before being challenged with thioacetamide. These inducers of mixed-function oxidase did not enhance the hepatotoxic response to thioacetamide (Table IV). DISCUSSION

Thioacetamide-induced hepatotoxicity was markedly potentiated in alloxandiabetic rats, and the potentiation was prevented when the diabetic state was conTABLE IV EFFECT OF PRETREATMENT WITH VARIOUS ENZYME INDUCERS ON THIOACETAMIDEINDUCED HEPATOTOXICITY IN MALE RATSa Treatment

n

GPT activity (units/ml)

ICD activity (units/ml/min)

Bilirubin concentration (mg/lOO ml)

Saline

6

Phenobarbital

6

340 +29 375 + 52

69 *6 81 *12

0.21 * 0.01 0.27 +O.Ol

Carboxymethylcellulose

4

3-Methylcholanthrene

6

290 +48 330 f31

83 * 10 75 +9

0.19 *0.01 0.24 + 0.01

Corn oil

6

3,CBenzpyrene

6

620 f76 571 f64

89 + 12 86 f 10

0.26 kO.01 0.20 f 0.01

“Phenobarbital (75 mg/kg/day, i.p., 3 days), 3-methylcholanthrene (20 mg/kg/day, i.p., 3 days), 3,4-benzpyrene (20 mg/kg/day, 1 day), or their respective vehicles (saline, carboxymethylcellulose, corn oil), was administered before the thioacetamide (100 mg/kg, i.p.) challenge. Rats were killed 24 h after the thioacetamide.

298

trolled by the concomitant administration of insulin. These results are comparable to those observed when CCL was used as the hepatotoxicant [ 11. The potentiation was more severe as the dosage of alloxan was increased and was evident with both dosages of thioacetamide. Streptozotocin-induced diabetes also resulted in an enhanced response to thioacetamide, but the overall effect was iess than that observed with alloxan, a finding similar to that reported with CC4 [I]. The loss of weight associated with ailoxan-induced diabetes probably contributed in part to the potentiated hepatotoxic response, since pair-fed nondiabetic animals also exhibited enhanced liver injury. However, the elevations in plasma GPT activity and bilirubin content observed even after 3 days of complete food deprivation failed to reach the very large values obtained in alloxan-pretreated rats. This is unlike the effect of a l-day fast on CC4 hepatotoxicity observed by Nakajima et al. (121 and attributed to carbohydrate deficiency. Thus, we conclude that the potentiation of thioacetamide toxicity is due to the uncontrolled diabetic state in the rats treated with the diabetogenic agents. The ketotic state that occurs in diabetes could be the cause of the potentiation. Hanasono et al. [I] suggested this possibility for CC4, but ketone bodies were not measured in those experiments. In the present study, alloxan resulted in large increased in both blood and hepatic ketone bodies. The increases seen with streptozotocin were less marked and more comparable to those produced after 2 days of complete food deprivation. Recent studies have shown that the consumption of 1,3-butanediol in drinking water for 8 days produces a metabolic ketosis 1131. This treatment regimen potentiates the hepatotoxic effects of CC4 [13]; the cholestatic properties of taurolithocholate and manganese-bilirubin combination are increased as well [ 141. Thus, metabolic ketosis has been associated with enhanced hepatotoxic responses. Enhanced metabolic activation of thioacetamide, as a result of the chemically induced diabetes, could be offered as an explanation for the potentiation. Neal and coworkers have shown involvement of the mixed-function oxidase system in thioacetamide hepatotoxicity [15, 161. They also reported [ 151 that phenobarbitalpretreated animals exhibited enhanced toxicity, but the histologic data used to support this conclusion are not that striking. Castro et al. [17] observed no potentiation after phenobarbital, but the dose of the hepatotoxicant was much larger than those employed by Hunter et al. [15] or the ones we employed. Our experiments with inducers of mixed-function oxidase indicate that phenobarbital, 3-methylcholanthrene, and 3,4-benzpyrene were unable to potentiate thioacetamide under our experimental conditions. The effect of various diabetogenic agents on hepatic drug metabolism has been studied [ 18-221. Both increases and decreases in activity have been observed, depending on the substrate employed, the diabetogenic agent used, and the sex of the animal. Thus, a clear relationship does not exist. In the absence of more definitive experiments dealing with thioacetamide bioactivation, we feel that this question remains unresolved.

Price and Jollow [S] observed no potentiation of acetaminophen-induced hepatoxicity in streptozotocin-diabetic rats; instead, an increased resistance to the hepatotoxicant was observed. These authors did not use alloxan as a diabetogenic agent, so one cannot tell whether the lack of potentiation was due to the use of streptozotocin or to the use of acetaminophen. In the present study with thioacetamide and in the previous one with CC4 [1], alloxan-diabetic animals were much more responsive to the hepatoto~cant then were the streptozotocin-diabetic animals. The ketotic state in the latter animals was much less severe than that produced by alloxan in the present work; ketone bodies were said to be ‘only modestly elevated’ in the acetaminophen study [5]. Whether or not this can explain the difference observed is a matter of speculation. Further work is needed to unravel the various components (diabetogenic agent, hepatotoxicant, severity of ketosis) involved in these interaction studies. In any event, the present work demonstrates that alloxaninduced potentiation of liver injury observed after CC4 [1] CHCl3 and 1,1,2-trichloroethane [2] is not specific to haloalkane hepatotoxicants, but also occurs after thioacetamide. ACKNOWLEDGEMENT

This work was supported by the Medical Research Council of Canada.

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