Effect of pretreatment with some mixed-function oxidase enzyme inducers on the acute hepatotoxicity of coumarin in the rat

Fd Chem. Toxic. Vol. 31, No. 12, pp. 963-970, 1993 Printed in Great Britain

0278-6915/93 $6.00+ 0.00 Pergamon Press Ltd

EFFECT OF PRETREATMENT WITH SOME MIXED-FUNCTION OXIDASE ENZYME INDUCERS ON THE ACUTE HEPATOTOXICITY OF C O U M A R I N IN THE RAT B. G. LAKE* and J. G. EVANSt BIBRA Toxicology International, Woodmansterne Road, Carshalton, Surrey SM5 4DS, UK (Accepted 16 July 1993)

Abstract--Male Sprague--Dawley rats were pretreated with saline, corn oil, sodium phenobarbitone (PB) (100 mg/kg body weight/day), 20-methylcholanthrene (20 MC) (20 mg/kg body weight/day) or Aroclor 1254 (ARO) (100 mg/kg body weight/day) by daily ip injections for 5 days. Animals were then given single oral doses of either 250 or 500 mg coumarin/kg body weight and hepatotoxicity was assessed after 24 hr. Coumarin produced hepatotoxicity, which comprised hepatocyte necrosis and elevation of plasma alanine aminotransferase and aspartate aminotransferase activities, in all pretreated groups. Hepatic microsomal cytochrome P-450 levels were reduced after coumarin administration, In rats pretreated with saline, corn oil or PB, coumarin produced centrilobular hepatic necrosis, whereas in rats pretreated with 20 MC or ARO, coumarin produced periportal hepatic necrosis. These results demonstrate that mixed-function oxidase enzyme inducers can modulate acute coumarin-induced hepatotoxicity in the rat. As coumarin is known to be bioactivated by cytochrome P-450-dependent enzymes, the change in the lobular distribution of toxicity after pretreatment with 20 MC or ARO is presumably due to the induction of particular cytochrome P-450 isoenzymes in periportal hepatocytes.

INTRODUCTION

Coumarin (l,2-benzopyrone; cis-o-coumarinic acid lactone) is a natural plant product (Feuer, 1974; Soine, 1964), which is used in various soaps, detergents and cosmetics preparations (Opdyke, 1974). It is also present in certain alcoholic beverages and tobaccos (Cohen, 1979). The toxicology of coumarin merits attention since it has been shown to exhibit interspecies differences in both hepatotoxicity and metabolism (Cohen, 1979). Coumarin has been shown to produce liver damage in the rat and dog (Hagan et al., 1967; Hazelton et al., 1956), but not in the Syrian hamster (Ueno and Hirono, 1981) and baboon (Evans et al., 1979). In the rat, single doses of coumarin produce centtilobular hepatic necrosis (Fentem et al., 1992a; Lake, 1984; Lake et al., 1989) whereas prolonged administration results in bile duct lesions (Cohen, 1979; Evans et al., 1989; Hagan et al., 1967). As humans and the rat metabolize coumatin differently, the significance of these lesions in predicting potential hepatotoxicity for humans has been questioned (Cohen, 1979). In humans coumarin is extensively metabolized to 7-hydroxycoumarin (Shilling et al., *To whom correspondence should be addressed. tPresent address: Fisons Pharmaceuticals, Fisons pie, Bakewell Road, Loughborough, Leics. LEII 0RH, UK. Abbreviations: ALT=alanine aminotransferase; ARO= Aroclor 1254; AST = aspartate aminotransferase; 20 MC = 20-methylcholanthrene; PB ---sodium phenobarbitone.

1969), whereas this metabolic route represents only a very minor pathway in several other species, including the rat (Cohen, 1979; Gangolli et al., 1974). The major pathway of coumarin metabolism in the rat involves an initial 3-hydroxylation reaction, followed by ring opening and further metabolism to o-hydroxyphenylacetic acid (Cohen, 1979; Fentem and Fry, 1992; Kaighen and Williams, 1961; Lake et al., 1992). Although some studies have suggested that coumatin-induced hepatotoxicity is caused by the parent compound per se, more recent investigations have suggested that toxicity is due to one or more coumatin metabolites (Cohen, 1979; Gibbs et al., 1971; Lake, 1984; Lake et al., 1989). The aim of the present study was to investigate the effect of induction of hepatic xenobiotic-metabolizing enzymes on acute coumarin-induced hepatotoxicity in the rat. Rats were pretreated with either sodium phenobarbitone (PB), 20-methylcholanthrene (20 MC) or the polychlorinated biphenyi mixture Aroclor 1254 (ARO). While 20 MC induces cytochrome isoenzymes primarily in the CYP1A subfamily, PB induces cytochrome P-450 isoenzyme in the CYP2B, CYP3A and other subfamilies, and ARO induces isoenzymes in several subfamilies including the CYPIA, CYP2B and CYP3A subfamilies (Nebert et al., 1991; Okey, 1990; Ryan and Levin, 1990). MATERIALS AND METHODS

Chemicals. Coumarin and 20 MC were obtained

from Aldrich Chemical Co. Ltd (Gillingham, Dorset, 963

B.G. LAKEand J.G. EVANS

964

UK) and PB from BDH Chemicals (Poole, Dorset, UK). ARO was a generous gift from Monsanto Chemical Co. (St Louis, MO, USA). Animals and treatment. Male Sprague-Dawley rats were obtained from Charles River U K Ltd (Margate, Kent, UK) and allowed free access to R and M No. 1 diet (Special Diets Services, Witham, Essex, UK) and water. The animals were housed in mesh-floored cages at 2 2 + 3 ° C with a relative humidity of 40-70%, and were allowed to acclimatize to these conditions for at least 7 days before use. Rats (6 wk old) received five daily ip doses of PB (100mg/kg body weight/day), 20 MC (20 mg/kg body weight/day) or ARO (100 mg/kg body weight/day). PB was administered in 0.9% (w/v) saline, and 20 MC and ARO in corn oil. Control animals received corresponding quantities (5 ml/kg body weight) of the appropriate vehicle. Coumarin was administered to rats pretreated with saline, corn oil, PB, 20 MC or ARO 24 hr after the last pretreatment at single oral doses of either 0, 250 or 500 mg/kg body weight in corn oil (5 ml/kg body weight). Rats were killed after 24 hr by exsanguination under diethyl ether anaesthesia, and the livers were excised immediately for biochemical and morphological studies. Biochemical investigations. Whole liver homogenates (0.25 g fresh tissue/ml) were prepared in ice-cold 0.154 M KC1 containing 50 m i Tris-HCl (pH 7.4), using a Potter-type teflon-glass, motor-driven homogenizer (A.H. Thomas Co., Philadelphia, PA, USA). Washed microsomal fractions were prepared and assayed for cytochrome P-450 content (Lake, 1987). Protein content was determined by the method of Lowry et al. (1951), using bovine serum albumin as standard. Plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined with a Roche (Roche Products Ltd, Welwyn Garden City, Herts., UK) COBAS Mira reaction rate analyser, using standard diagnostic test kits. Morphological investigations. Liver slices were fixed in neutral buffered formalin. Paraffin sections of about 5/~m thickness were cut and stained with haematoxylin and eosin. Five sections of the liver from each animal (i.e. two from the left lobe and one each from the right, median, right caudal and caudate lobes) were examined by light microscopy. For each animal the extent of hepatic necrosis was assessed by scoring the extent of liver injury in all five sections on an arbitrary scale, and averaged to provide an overall value as defined in the legend to Table 2. Statistical analysis. Statistical evaluation of the data was performed by one-way analysis of variance. Comparisons between means were made using the least-significant difference test. RESULTS

Treatment with PB, 20 MC or ARO resulted in a stimulation of rat hepatic xenobiotic-metabolizing enzymes as demonstrated by an induction of

microsomal cytochrome P-450 content (Table 1). Cytochrome P-450 levels were increased to 206-316% of either the saline- or corn oil-treated controls (Table 1); there was no significant difference in cytochrome P-450 levels in animals given either of the injection vehicles (data not shown). In keeping with previous studies (Fentem et al., 1992a; Lake, 1984; Lake et al., 1989), a single dose of coumarin resulted in a significant reduction of microsomal cytochrome P-450 content within 24 hr (Table 1). Coumarin administration also reduced the cytochrome P-450 content in liver microsomes from rats pretreated with PB, 20 MC or ARO to 71, 68 and 75% of control levels, respectively (Table 1). Coumarin-induced hepatotoxicity was also assessed after 24 hr by measurement of plasma ALT and AST activities, and by morphological examination of liver sections. The administration of 250 and 500 mg coumarin/kg body weight to either saline- or corn oil-pretreated rats produced marked dose-related increases in plasma ALT (Figs 1A and 2A) and AST (Figs 1B and 2B) activities. Coumarin also produced dose-related increases in plasma transaminase activities in rats pretreated with PB, 20 MC or ARO (Figs. 1 and 2). No significant differences in the magnitude of elevation of plasma transaminase activities were observed between saline- and PB-pretreated rats given 250 or 500 mg coumarin/kg body weight (Fig. 1). However, in control rats given corn oil (i.e. 0 mg coumarin/kg body weight) plasma ALT levels (Fig. IA) were slightly higher in PB-pretreated than in saline-pretreated animals. Coumarin administration at the two dose levels examined resulted in significantly greater increases in plasma ALT levels in both 20MC- and ARO-pretreated rats compared with corn oil-pretreated animals (Fig. 2A). With the exception of 20 MC-pretreated animals given 500 mg coumarin/kg body weight, plasma AST levels were significantly higher in rats pretreated with 20 MC or ARO than in rats pretreated with corn oil in response to coumarin (Fig. 2B). In keeping with previous studies (Fentem et al., 1992a; Lake, 1984; Lake et al., 1989), the administration of coumarin to rats pretreated with saline or Table 1. Effect of coumarin administration on cytochrome P-450 levels in liver microsomes from control and sodium phenobarbitone (PB)-20-methylcholanthrene (20 MC)- or Aroclor 1254 (ARO)-pretreated rats Pretreatment (mg/kg body weight/day) Saline or corn oil PB (100) 20 MC (20) A R O (100)

Cytochrome P-450 (nmol/mg protein) Corn oil

Coumarin

1.16 4- 0.08 2.55_+0.11 2.39 _+ 0.08 3.67 + 0.11

0.64 _+ 0.04** 1.82_+0.12"* 1.62 _+0.17"* 2.75 _+ 0.15"*

Rats were pretreated with either saline, corn oil, PB, 20 MC or ARO by daily ip injections for 5 days. Animals were then given a single oral dose of either 0 (corn oil) or 250 mg coumarin/kg body weight and killed 2 4 h r later. Results are expressed as m e a n s _ SEM for groups of four animals. Asterisks indicate values significantly different from corresponding controls (**P < 0.01).

965

Modulation of coumarin hepatotoxicity

1000

B

5_.#e

]Saline

-;

5_ [ ] $.,,n.

~ 1000

[]

PB

!

w

7/,

/// 10

0

250

Control

Coumarin

500 Coumarin

0 Control

250

500

Coumarin

Coumarln

Fig. I. Effect of pretreatment with saline or PB (100 mg/kg body weight/day by daily ip injections for 5 days on coumarin-induced hepatotoxicity in the rat. Animals were then given a single oral dose of either 0 (control), 250 or 500 mg coumarin/kg body weight, and plasma alanine aminotransferase (A) and aspartate aminotransferase (B) activities determined after 24 hr. Results are expressed as means + SEM for groups of eight animals, Note the use of a logarithmic scale for the enzyme activities shown on the y axis. Significant differences from controls (i.e. 0 mg coumarin/kg body weight) (*P < 0.05; ***P < 0.001 and those between saline and PB pretreatments (+P < 0.05) are indicated. corn oil resulted in centrilobular hepatic necrosis (Plate 1A). Coumarin treatment also produced centrilobular hepatic necrosis in PB-pretreated rats (data not shown), whereas in 20 MC-pretreated (Plate 1B) or ARO-pretreated (Plate IC) rats periportal hepatic necrosis was observed. Coumarin-induced liver necrosis was scored on an arbitrary scale as defined in the legend to Table 2. N o necrosis was observed in either saline-pretreated (Table 2) or corn oilpretreated (Table 3) rats in response to a single oral dose o f corn oil (i.e. 0 mg coumarin/kg body weight). However, in rats pretreated with saline, corn oil, PB, 20 M C or A R O coumarin administration produced dose-related increases in the severity of hepatic necrosis (Tables 2 and 3). Compared with salinepretreated rats, coumarin-induced hepatic necrosis in PB-pretreated rats was somewhat less marked, especially in response to 500 mg coumarin/kg body weight (Table 2). While coumarin-induced liver ne-

was in periportal rather than in centrilobular hepatocytes (Plate 1). DISCUSSION

Previous studies have demonstrated that acute coumarin-induced hepatotoxicity in the rat is associated with centrilobular hepatic necrosis, elevated plasma transaminase activities, and a depression of hepatic cytochrome P-450 content and associated mixed-function oxidase enzyme activities (Fentem et al., 1992a; Lake, 1984; Lake et aL, 1989). In the present study, coumarin also produced hepatotoxic effects within 24 hr in rats pretreated with three known inducers of the cytochrome P-450 namely, PB, 20 M C and A R O .

++

+++

1000

crosis appeared to be potentiated in 2 0 M C - or ARO-pretreated rats, compared with corn oil-pretreated rats (Table 3), the observed hepatotoxicity

+++

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Corn oil 20MC

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100

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Control

i 250

Coumarln

,

///

10

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i

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0

250

Coumerin

Control

Coumarin

=

500

Coumarin

Fig. 2. Effect ofpretreatment with corn oil, 20 MC (20 mg/kg body weight/day) or ARO (100 mg/kg body weight/day) by daily ip injections for 5 days on coumarin-induced hepatotoxicity in the rat. Animals were then given a single oral dose of either 0 (control), 250 or 500 mg coumarin/kg body weight and plasma alanine aminotransferase (A) and aspartate aminotransferase (B) activities determined after 24 hr. Results are expressed as means +SEM for groups of six to eight animals. Note the use of a logarithmic scale for the enzyme activities shown on the y axis. Significant differences from controls (i.e. 0 mg coumarin/kg body weight) (*P <0.05; ***P <0.001) and those between corn oil and either 20MC or ARO pretreatments (+P < 0.05; ++P < 0.01; +++P < 0.001) are indicated.

966

B. G. LAKE and J. G. EVANS Table 2. Effect of sodium phenobarbitone (PB) pretreatment on coumarin-induced hepatotoxicity in the rat Pretreatment (mg/kg body weight/day)

Extent of hepatic necrosis* Coumarin (mg/kg body weight) No. of rats

0

+

++

+++

Control (saline)

0 250 500

8 8 8

8 ---

-6 2

-2 5

-1

PB (100)

0 250 500

8 8 8

8 ---

-7 7

-1 I

----

++++

*Hepatic necrosis was assessed in five sections from each liver and averaged to provide an overall value. Figures refer to the number o f rats from each group showing liver lesions classified as: 0, no necrosis; + , subcapsular or occasional single cell necrosis; + + , necrosis of 5-20% of hepatocytes; + + + , necrosis of 20~I0% o f hepatocytes; and + + + + , necrosis of more than 40% of hepatocytes. Rats were pretreated with either saline or PB by daily ip injections for 5 days. Animals were then given a single oral dose of coumarin and killed 24 hr later.

Both in vitro and in vivo studies have demonstrated that coumarin-induced toxicity in rat hepatocytes is the result of bioactivation by cytochrome P-450dependent enzymes to toxic metabolite(s) (den Besten et al., 1990; Fentem et al., 1992b; Lake, 1984; Lake et al., 1989; Peters et al., 1991). Several inducers of hepatic xenobiotic metabolism, including PB, 20 MC and ARO, are known to stimulate coumarin metabolism in rat liver microsomes, and coumarin is also a substrate for the purified rat liver cytochrome P-450 isoenzymes CYP1AI and CYP2B1 (Fentem and Fry, 1992; Lake et aL, 1992; Peters et al., 1991). As with a number of other cytochrome P-450-bioactivated hepatotoxins, including bromobenzene, carbon tetrachloride and paracetarnol (Zimmerman, 1978), coumatin produces centrilobular hepatic necrosis in untreated and vehicle (saline and corn oil)-pretreated rats. These observations correlate with the finding that in control rats the levels of total cytochrome P-450 are higher in centrilobular than in periportal hepatocytes (Baron et al., 1978 and 1981; Gooding et al., 1978). Coumarin administration also resulted in centrilobular hepatic necrosis in PB-pretreated rats, where both the total cytochrome P-450 content and levels of particular PB-inducible cytochrome P-450 isoenzymes are greater in centrilobular than in periportal hepatocytes (Baron et al., 1978; Gooding

et al., 1978; Wolf et al., 1984). In contrast to PB, the

administration of coumarin to either 20MC- or ARO-pretreated rats resulted in periportal hepatic necrosis. Although the levels of CYPIA subfamily isoenzymes have been reported to be marginally higher in centrilobular than in periportal hepatocytes in control animals, these and other cytochrome P-450 isoenzymes can be markedly induced in periportal hepatocytes (Baron et al., 1978 and 1982; Wolfet al., 1984). The induction of CYPIA, and possibly other cytochrome P-450 isoenzymes, in periportal hepatocytes by 20 MC and ARO may thus account for the periportal liver injury observed in the present study, and perhaps the apparent sparing of toxicity in centrilobular hepatocytes of animals pretreated with these two agents. However, it should be noted that the zonal induction of specific cytochrome P-450 isoenzymes in the rat liver is dependent on the particular inducer studied and on the dose level administered (Bars and Elcombe, 1991; Bell et al., 1991; van Sliedregt and van Bezooijen, 1990). Further studies are required to determine which particular cytochrome P-450 isoenzymes are responsible for coumarin bioactivation to toxic metabolite(s) in the rat liver and to confirm why centrilobular hepatotoxicity is not observed in 20 MC- or ARO-pretreated rats given coumarin.

Table 3. Effect o f 20-methylcholanthrene (20 MC) and Aroclor 1254 (ARO) pretreatment on coumarin-induced hepatotoxicity in the rat Pretreatment (mg/kg body weight/day) Control (corn oil)

20 M C (20)

A R O (100)

Extent of hepatic necrosis* Coumarin (mg/kg body weight) No. of rats

0

+

0 250 500

8 8 7

8 2 --

.

0 250 500

8 8 8

8 ---

.

0 250 500

8 8 6

8 ---

.

+ + .

.

6 1

.

-3 .

3 --

1 --

+ + +

. 5 7

.

. 6 4

+ + + +

. -2

-1

. -I

--

.

. I 2

---

*Hepatic necrosis was assessed in five sections from each liver and averaged to provide an overall value. Figures refer to the number o f rats from each group showing liver lesions classified as described in the legend to Table 2. Rats were pretreated with corn oil, 20 M C or A R O by daily ip injections for 5 days. Animals were then given a single oral dose o f coumarin and killed 24 hr later.

Plate 1. Effect of prctreatment with corn oil, 20 MC (20 mg/kg body weight/day) or A R O (100 mg/kg body weight) by daily ip injections for 5 days on coumarin-induced hepatotoxicity in the rat. Animals were then given a single oral dose of 500 mg coumarin,kg body weight and killed 24 hr later. Liver sections from corn oil-pretreated rats given coumarin (A) revealed extensive necrosis of hepatocytes in the centrilobular area of the liver lobule, whereas liver sections from 20 MC-pretreated (B) or ARO-pretreated (C) rats given coumarin revealed extensive necrosis of hepatocytes in the periportal area of the liver lobule. Haematoxylin and eosin. Original magnification x 180.

Modulation of coumarin hepatotoxicity Based on the liver necrosis scores (Table 2) a n d the elevation o f plasma t r a n s a m i n a s e activities (Fig. 1), PB t r e a t m e n t did n o t increase acute c o u m a r i n induced hepatotoxicity in the rat liver. The present data r a t h e r suggest that PB p r e t r e a t m e n t m a y produce a slight reduction in c o u m a r i n - i n d u c e d liver necrosis. Because o f the different lobular location o f hepatic necrosis in corn oil-pretreated rats, as against 20 M C or A R O - p r e t r e a t e d animals, no conclusions can be d r a w n from the present study as to the effect o f either 20 M C or A R O p r e t r e a t m e n t on c o u m a r i n induced hepatotoxicity in the rat. A l t h o u g h the plasma A L T a n d A S T levels in 20 M C - or A R O - p r e treated rats given c o u m a r i n were generally higher than those in corn oil-pretreated rats given c o u m a r i n (Fig. 2), this m a y be associated with the change in the site of liver necrosis. F o r example, rat hepatic A L T activity is k n o w n to be higher in periportal t h a n in centrilobular hepatocytes ( G r r g e n s et al., 1988; J u n g e r m a n n a n d Katz, 1982), the f o r m e r being preferentially d a m a g e d by c o u m a r i n in 20 M C - or A R O pretreated rats. The m o d u l a t i o n o f c o u m a r i n - i n d u c e d hepatotoxicity by variation of hepatic c y t o c h r o m e P-450d e p e n d e n t enzyme activities bears some resemblance to the studies o f Seawright a n d co-workers o n the hepatotoxicity o f ngaione in the m o u s e (Lee et al., 1979; Seawright a n d Hrdlicka, 1972). In these studies ngaione p r o d u c e d a m i d z o n a l hepatic necrosis in control mice, whereas inhibition o f mixed-function oxidase enzyme activity resulted in a centrilobular necrosis a n d P B - p r e t r e a t m e n t resulted in a periportal necrosis. In conclusion, these results d e m o n s t r a t e that mixed-function oxidase enzyme inducers m a y m o d u late the zonal distribution of c o u m a r i n - i n d u c e d liver necrosis in the rat liver. F u r t h e r studies are required to ascertain which particular c y t o c h r o m e P-450 isoenzymes are responsible for the bioactivation o f c o u m a r i n to toxic metabolite(s) in this species.

Acknowledgements--This work forms part of a research project sponsored by the UK Ministry of Agriculture, Fisheries and Food, to whom our thanks are due. The results of the research are the property of the Ministry of Agriculture, Fisheries and Food and are Crown copyright.

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