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Studies on the disposition, metabolism and hepatotoxicity of coumarin in the rat and Syrian hamster
Food and Chemical Toxicology 40 (2002) 809–823 www.elsevier.com/locate/foodchemtox
Research section
Studies on the disposition, metabolism and hepatotoxicity of coumarin in the rat and Syrian hamster B.G. Lakea,*, J.G. Evansa, F. Chapuisb, D.G. Waltersa, R.J. Pricea a TNO BIBRA International Ltd, Woodmansterne Road, Carshalton, Surrey SM5 4DS, UK ENS BANA, Universite´ de Bourgogne, Campus Universitaire, 1 Esplanade Erasme, F21100 Dijon, France
b
Abstract The hepatotoxicity, metabolism and disposition of coumarin has been compared in male SpragueDawley rats and Syrian hamsters. The treatment of rats for 12, 24 and 42 weeks with diets containing 0.2 and 0.5% coumarin resulted in hepatotoxicity and increased relative liver weights. While levels of cytochrome P450 (CYP) and CYP-dependent enzymes were decreased, levels of reduced glutathione (GSH) and activities of UDP glucuronosyltransferase, g-glutamyltransferase and GSH S-transferase were increased. In contrast, coumarin produced few hepatic changes in the Syrian hamster. Following a single oral dose of 25 mg/kg [3-14C]coumarin, radioactivity was rapidly excreted by the rat and Syrian hamster with the urine containing 63.5 and 89.9%, respectively, and the faeces 38.0 and 12.4%, respectively, of the administered dose after 96 h. The biliary excretion of radioactivity was greater in the rat than in the Syrian hamster. Analysis of 024-h urine samples revealed that both species were poor 7-hydroxylators of coumarin. In the rat, treatment with 0.5% coumarin in the diet for 24 weeks was found to increase the urinary excretion of single oral gavage doses of 25 and 300 mg/kg [3-14C]coumarin. The marked species difference in hepatotoxicity between the rat and Syrian hamster observed in this study may be at least partially attributable to differences in coumarin disposition. However, additional studies are required to elucidate the metabolic pathways of coumarin in both species. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Coumarin; Hepatotoxicity; Metabolism; Rat; Species differences; Syrian hamster
1. Introduction Coumarin (1,2-benzopyrene, cis-o-coumarinic acid lactone) occurs naturally in several plant families and essential oils (Soine, 1964; Opdyke, 1974; Cohen, 1979; Lake, 1999). Although no longer used itself as an added food flavouring, it is present in some natural flavouring source materials added to food, and coumarin is employed as a fixative and enhancing agent in perfumes and is added to toilet soap and detergents, toothpaste, tobacco products and some alcoholic beverages (Opdyke, 1974; Cohen, 1979). Coumarin has also been used as a therapeutic agent for the treatment of various cancers (reviewed in Lake, 1999). The toxicology of coumarin merits attention, since coumarin exhibits marked species differences in both
Abbreviations: CDNB, 1-chloro-2,4-dinitrobenzene; CYP, cytochrome P450; ENPP, 1,2–epoxy-3-(p-nitrophenoxy)propane; GSH, reduced glutathione; GSSG, oxidised glutathione. * Corresponding author. Tel.: +44-20-8652-1006; fax: +44-208661-7029. E-mail address: [email protected] (B.G. Lake).
metabolism and hepatotoxicity (Cohen, 1979; Lake, 1999). In humans, coumarin 7-hydroxylation, catalysed by hepatic CYP2A6, is the major route of coumarin biotransformation in most subjects, whereas this is only a minor route of coumarin metabolism in the rat (Kaighen and Williams, 1961; Shilling et al., 1969; Cohen, 1979; Pelkonen et al., 1993; Lake, 1999). The major route of coumarin biotransformation in the rat involves an initial 3,4-epoxidation reaction (Fig. 1), which is followed by the loss of carbon dioxide to form o-hydroxyphenylacetaldehyde (Lake et al., 1989a, 1992a,b; Fentem et al., 1991; Born et al., 1997, 2000a; Lake, 1999). While sex and strain differences in the extent of coumarin 7-hydroxylation have been reported in the mouse, like the rat, the 3,4-epoxidation pathway predominates in this species (Lush and Andrews, 1978; van Iersel et al., 1994; Lovell et al., 1999). Other studies have demonstrated that Old- but not New-World primates (such as the baboon and Cynomolgus monkey) are also extensive 7-hydroxylators of coumarin (reviewed in Lake, 1999). In the rat, the acute administration of high doses of coumarin have been reported to result in centrilobular
0278-6915/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0278-6915(02)00036-4
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Fig. 1. Some pathways of coumarin metabolism (Cohen, 1979; Lake et al., 1989a; Born et al., 1997, 2000a; Lake, 1999). In the present study, coumarin administration to the rat results in decreased cytochrome P450 (CYP) enzyme activities, but enhanced levels of hepatic reduced glutathione (GSH) and activities of UDPglucuronosyltransferase, GSH S-transferase and g-glutamyltransferase.
hepatic necrosis, whereas chronic administration results in bile duct lesions (Hagan et al., 1967; Lake, 1984; Evans et al., 1989; Lake et al., 1989a, 1994; Carlton et al., 1996; Lake and Grasso, 1996). Moreover, the chronic administration of high doses of coumarin has been reported to result in both parenchymal cell tumours and in cholangiocarcinoma (Carlton et al., 1996). Coumarin has also been reported to produce liver tumours in female, but not in male, B6C3F1 mice (NTP, 1993); whereas coumarin did not produce any liver lesions or liver tumours in male and female Syrian hamsters after 2 years’ treatment (Ueno and Hirono, 1981). The aim of the present study was to obtain further information on species differences in coumarin-induced hepatotoxicity. Studies were conducted with male SpragueDawley rats and male Syrian hamsters as these species have been previously reported to be susceptible and resistant, respectively, to coumarin-induced liver injury. Apart from investigating the time course of the hepatic effects of coumarin in the rat and Syrian hamster, some limited studies on coumarin metabolism and the effect of prior coumarin treatment on the disposition of coumarin in both species have been investigated.
2. Materials and methods 2.1. Materials Coumarin, enzyme substrates and cofactors were obtained from Sigma-Aldrich Co. Ltd (Poole, Dorset, UK). [3-14C]Coumarin (sp. act. 5.3 mCi/mmol) was purchased from ICI Chemicals and Polymers Group (Billingham, Cleveland, UK) and purified to > 99.5% radiochemical purity by chromatography on thin layer plates of silica gel G developed in toluenechloroform (1:1, v/v). Unlabelled coumarin metabolites were obtained from the sources described previously (Lake et al., 1989a). 2.2. Animals and treatment Male SpragueDawley rats were obtained from Harlan Olac (Bicester, Oxon, UK) and male DSN strain Syrian hamsters from Intersimian Ltd (Milton Trading Estate, Milton, Oxon, UK) and allowed free access to R and M No.3 laboratory animal diet (Special Diets Services, Witham, Essex, UK) and water. The animals were housed in polypropylene cages with stainless-steel grid
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tops and floors in rooms maintained at 22 3 C with a relative humidity of 40–70% and a 12-h light cycle. After being allowed to acclimatise to these conditions for 2 weeks, rats (6 weeks old) and Syrian hamsters (8 weeks old) were fed diets containing no test compound (0% coumarin) or diets containing 0.2, 0.5 and 1.0% coumarin for periods of up to 42 weeks. Animals were killed by exsanguination under diethyl ether and the livers immediately removed for biochemical and morphological studies. 2.3. Biochemical investigations Whole liver homogenates (rat 0.25 and Syrian hamster 0.1 g fresh tissue/ml) were prepared in ice-cold 0.154 m KCl containing 50 mm TrisHCl, pH 7.4, using a Potter-type, teflonglass, motor-driven homogeniser (A.H. Thomas Co., Philadelphia, PA, USA). Liver whole homogenates were assayed for reduced (GSH) and oxidised (GSSG) glutathione by an enzymatic recycling assay employing GSH reductase and 5,50 dithio-bis(2-nitrobenzoic acid) as described previously (Adams et al., 1983; Lake et al., 1989b). The activity of whole homogenate and serum g-glutamyltransferase was determined by the method of Smith et al. (1979) employing l-g-glutamyl-7-amino-4-methylcoumarin as substrate. Washed microsomal and cytosolic fractions were prepared by differential centrifugation (Lake, 1987). Microsomal fractions were assayed for total cytochrome P450 (CYP) content and for activities of 7-ethoxycoumarin O-deethylase and ethylmorphine N-demethylase (Lake, 1987). Liver microsomal fractions were also assayed for UDPglucuronosyltransferase activity employing 4-hydroxybiphenyl as substrate (Bock et al., 1980). Cytosolic fractions were assayed for activities of GSH Stransferase employing 1-chloro-2,4-dinitrobenzene (CD NB) and 1,2-epoxy-3-(p-nitrophenoxy)propane (ENPP) as substrates (Habig et al., 1974). Whole homogenate, microsomal and cytosolic fraction protein content was determined by the method of Lowry et al. (1951) employing bovine serum albumin as standard.
singly in all-glass metabolism cages (Jencons Scientific Ltd, Hemel Hempstead, Herts, UK) with provision for collection of urine and faeces. Radioactivity present in urine was determined directly by scintillation counting, whereas faeces samples were first homogenised in methanol and as with tissue samples radioactivity was measured after sample oxidation. The profile of coumarin metabolites in 024-h urine samples was determined by HPLC. Urine samples were centrifuged at 10,000 g average for 15 min at 4 C and a 1-ml aliquot incubated with 10 mg b-glucuronidase and 10 mg sulphatase in 0.4 ml of 0.1 m acetate buffer pH 5.0 for 24 h at 37 C. After centrifugation at 10,000 g average for 15 min, an 80-ml aliquot was injected onto a Partisil 10 ODS-2 column and coumarin and coumarin metabolites separated by gradient elution with tetrahydrofuran/distilled water/acetic acid as described previously (Walters et al., 1980). For the studies on the biliary excretion of coumarin, the animals were maintained under sodium pentobarbitone anaesthesia (60 mg/kg, ip). After the bile duct was cannulated, the animals were dosed orally with 25 mg/kg [3-14C]coumarin and bile samples collected every 30 min for determination of radioactivity by scintillation counting. In other studies rats and Syrian hamsters were fed diets containing 01.0% coumarin for 24 weeks prior to being given single oral doses of either 25 or 300 mg/kg of [3-14C]coumarin (approximately 10 mCi/animal). Urine, faeces and tissue samples were collected and processed as described above. The macromolecular binding of [3-14C]coumarin metabolites to liver proteins was determined by exhaustive extraction of whole homogenates with 5% (w/v) trichloroacetic acid and 80% (v/v) methanol as described previously (Lake, 1984). 2.6. Statistical analysis Statistical evaluation of data was performed by oneway analysis of variance. Comparisons between means were made using the least significant difference test.
2.4. Morphological investigations
3. Results
Liver slices were fixed in neutral buffered formalin. Paraffin sections of about 5 mm thickness were cut, stained with haematoxylin and eosin and examined by light microscopy,
3.1. Chronic effects of coumarin in the rat and Syrian hamster
2.5. Metabolic studies Untreated male SpragueDawley rats (7 weeks old) and male Syrian hamsters (8 weeks old) were given a single oral 25 mg/kg dose of [3-14C]coumarin (approximately 13.5 and 8.0 mCi/animal, respectively) in corn oil (5 ml/kg body weight) by gastric intubation and caged
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Male SpragueDawley rats and male Syrian hamsters were fed diets containing 0 (control), 0.2 and 0.5% coumarin for periods of 12, 24 and 42 weeks. Calculated daily intakes of coumarin based on body weight and food consumption data for rats fed 0.2 and 0.5% coumarin diets were calculated to be 113 and 298 mg/kg/ day, respectively. Corresponding values for Syrian hamsters were 92 and 253 mg/kg/day, respectively (data not shown).
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The treatment of male SpragueDawley rats with 0.2 and 0.5% coumarin for 12 weeks and with 0.5% coumarin for 24 and 42 weeks significantly reduced body weight in the rat (Fig. 2A). In contrast, coumarin produced little effect on body weight in the Syrian hamster, with only a small decrease being observed after treatment with 0.2% coumarin for 12 weeks (Fig. 2B). The treatment of rats with coumarin for 12, 24 and 42 weeks resulted in dose-dependent increases in relative liver
weight (Fig. 2C). In contrast, relative liver weight was only significantly increased in the Syrian hamster after treatment with 0.5% coumarin for 42 weeks (Fig. 2D). The administration of 0.5% coumarin for 12 and 24 weeks and 0.2 and 0.5% coumarin for 42 weeks resulted in significant increases in rat serum g-glutamyltransferase activity (Fig. 2E). In contrast, coumarin treatment had no significant effect on serum g-glutamyltransferase activity in the Syrian hamster (Fig. 2F).
Fig. 2. The effect of treatment of rats (A, C, E) and Syrian hamsters (B, D, F) for 12, 24 and 42 weeks with diets containing 0 (control, &), 0.2 ( ) and 0.5 (&) % coumarin on body weight (A, B), relative liver weight (C, D) and serum g-glutamyltransferase activity (E, F). Results are expressed as meanS.E. for groups of 4 to 6 animals. Values significantly different from control are: *P <0.05; **P <0.01; ***P<0.001.
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Liver whole homogenates were assayed for g-glutamyltransferase activity and content of reduced (GSH) and oxidised (GSSG) glutathione. In the rat coumarin treatment for 12, 24 and 42 weeks produced a marked dose-dependent induction of hepatic g-glutamyltransferase activity (Fig. 3A). The treatment of rats with 0.5% coumarin also produced significant increases in hepatic GSH content, whereas hepatic GSSG content
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was not affected by coumarin administration (Fig. 3C,E). In the Syrian hamster coumarin treatment had no significant effect on hepatic g-glutamyltransferase activity (Fig. 3B) and produced only small changes in levels of GSH and GSSG (Fig. 3D and F). Microsomal fractions were assayed for total cytochrome P450 (CYP) content and activities of ethylmorphine N-demethylase, 7-ethoxycoumarin O-deethylase
Fig. 3. The effect of treatment of rats (A, C, E) and Syrian hamsters (B, D, F) for 12, 24 and 42 weeks with diets containing 0 (control, &), 0.2 ( ) and 0.5 (&) % coumarin on hepatic whole homogenate g-glutamyltransferase activity (A, B) and content of reduced (C, D) and oxidised (E, F) glutathione. Results are presented as meanS.E. for groups of 4 to 6 animals. Values significantly different from control are: *P<0.05; **P<0.01; ***P <0.001.
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and 4-hydroxybiphenyl UDP glucuronosyltransferase. In the rat coumarin treatment for 12, 24 and 42 weeks produced a significant dose-dependent decrease in total CYP content (Fig. 4A) and ethylmorphine N-demethylase activity (Fig. 4C). Rat hepatic 7-ethoxycoumarin Odeethylase activity was significantly reduced by treatment with 0.2 and 0.5% coumarin for 12 weeks and
with 0.5% coumarin for 24 and 42 weeks (Fig. 4E). In contrast, coumarin treatment had no significant effect on total CYP content and ethylmorphine N-demethylase activity in the Syrian hamster and produced only a small decrease in 7-ethoxycoumarin O-deethylase activity in Syrian hamsters given 0.2%, but not 0.5%, coumarin for 42 weeks (Fig. 4B, D and F). Coumarin
Fig. 4. The effect of treatment of rats (A, C, E) and Syrian hamsters (B, D, F) for 12, 24 and 42 weeks with diets containing 0 (control, &), 0.2 ( ) and 0.5 (&) % coumarin on hepatic microsomal cytochrome P450 (CYP) content (A, B) and activities of ethylmorphine N-demethylase (C, D) and 7-ethoxycoumarin O-deethylase (E, F). Results are presented as meanS.E. for groups of 4 to 6 animals. Values significantly different from control are: *P <0.05; **P<0.01; ***P< 0.001.
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treatment for 12, 24 and 42 weeks produced dosedependent increases in microsomal UDP glucuronosyltransferase activity in the rat, whereas no changes in UDP glucuronosyltransferase activity were observed in the Syrian hamster (Fig. 5A and B). Liver cytosols were assayed for GSH S-transferase activity employing 1-chloro-2,4-dinitrobenzene (CDNB) and 1,2-epoxy-3-(p-nitrophenoxy)propane (ENPP) as
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substrates. In the rat, treatment with 0.2 and 0.5% coumarin for 12 and 24 weeks and with 0.5% coumarin for 42 weeks resulted in significant increases in GSH Stransferase activities towards CDNB (Fig. 5C) and ENPP (Fig. 5E) as substrates. The treatment of Syrian hamsters with 0.5% coumarin for 12, 24 and 42 weeks significantly increased GSH S-transferase activity towards ENPP as substrate (Fig. 5F), with GSH
Fig. 5. The effect of treatment of rats (A, C, E) and Syrian hamsters (B, D, F) for 12, 24 and 42 weeks with diets containing 0 (control, &), 0.2 ( ) and 0.5 (&) % coumarin on hepatic microsomal 4-hydroxybiphenyl UDP glucuronosyltransferase activity (A, B) and cytosolic GSH S-transferase activity towards CDNB (C, D) and ENPP (E, F) as substrates. Results are presented as meanS.E. for groups of 4 to 6 animals. Values significantly different from control are: *P<0.05; **P<0.01; ***P <0.001.
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S-transferase activity towards CDNB as substrate also being significantly increased after 42 weeks treatment with 0.5% coumarin (Fig. 5D). Morphological examination of liver sections from control rats and Syrian hamsters revealed no abnormalities. In contrast, histological examination of liver sections from rats treated for 12, 24 and 42 weeks with 0.5% coumarin revealed extensive vacuolation and numerous eosinophilic inclusions in midzonal and centrilobular hepatocytes. Bile duct hyperplasia was also observed, which consisted of five ductules extending from the portal areas often reaching the region of the central vein. These changes were less marked in rats receiving 0.2% coumarin and no histological abnormalities were observed in Syrian hamsters treated with 0.2 and 0.5% coumarin. 3.2. Disposition and metabolism of [3-14C]coumarin in the rat and Syrian hamster Untreated male SpragueDawley rats and Syrian hamsters were given a single oral dose of 25 mg/kg [3-14C]coumarin. This coumarin dose level was selected as this represents a non-toxic dose in the rat (Lake, 1999 and unpublished observations). Following oral administration of [3-14C]coumarin radioactivity was rapidly excreted by both species, with the bulk of the dose being excreted within 24 h (Fig. 6). The urine was the major route of elimination with 60.7 0.9 (mean S.E., n=6) and 86.4 2.4% of the dose being excreted within 24 h by the rat and Syrian hamster, respectively. After 96 h the excretion of radioactivity in the urine and faeces of the rat was 63.5 2.3 and 38.0 1.6% of the dose,
respectively. Corresponding values for the Syrian hamster were 89.9 1.7 and 12.4 0.6% of the dose, respectively. The overall recoveries of administered radioactivity in the urine and faeces of the rat and Syrian hamster after 96 h were 101.5 3.6 and 102.3 2.1% of the dose, respectively. The 024-h urine samples from rats and Syrian hamsters given 25 mg/kg [3-14C]coumarin were subjected to enzymatic hydrolysis with b-glucuronidase and sulphatase and analysed by HPLC. Coumarin was extensively metabolised in both the rat and Syrian hamster, with unchanged coumarin representing only 0.4 and 1.3% of the administered dose, respectively, in 024-h urines (Table 1). The formation of 7-hydroxycoumarin accounted for only 0.56 and 1.3% of the administered dose in the rat and Syrian hamster, respectively. Coumarin was also metabolised to 3- and 5-hydroxycoumarin (these metabolites not being completely resolved by the acid HPLC system employed) and to o-hydroxyphenyllactic acid and o-hydroxyphenylacetic acid by both species (Table 1). In the rat, o-hydroxyphenylacetic acid was the major identified urinary metabolite, comprising 19.0% of the administered dose, whereas this metabolite and o-hydroxyphenyllactic acid were the major identified 024-h urinary coumarin metabolites in the Syrian hamster (Table 1). The biliary excretion of radioactivity was studied in rats and Syrian hamsters given a 25 mg/kg oral dose of [3-14C]coumarin. Over the 6-h collection period there was a time-dependent excretion of radioactivity in the bile of both species (Fig. 7). After 6 h the biliary excretion of radioactivity accounted for 32.8 2.8 (mean S.E., n=5) and 13.4 2.4% of the administered dose
Fig. 6. Cumulative excretion of radioactivity in urine (*, &) and faeces (*, &) following administration of a single oral 25 mg/kg dose of [3-14C]coumarin to rats (*, *) and Syrian hamsters (&, &). Results are presented as mean S.E. for groups of 6 animals.
B.G. Lake et al. / Food and Chemical Toxicology 40 (2002) 809–823 Table 1 Urinary metabolites of [3-14C]coumarin in the rat and Syrian hamster Compound
Urinary excretion (% of administered dose)a Rat
Coumarin 3- and 5-Hydroxycoumarin 7-Hydroxycoumarin o-Hydroxyphenyllactic acid o-Hydroxyphenylacetic acid
Syrian hamster b
0.40 0.01 0.53 0.02 0.56 0.02 3.9 0.2 19.0 0.4
1.3 0.2 6.2 0.5 1.3 0.2 16.4 2.0 10.9 1.2
a Rats (n=6) and Syrian hamsters (n=3) were given a single oral 25 mg/kg dose of [3-14C]coumarin and 024-h urine samples collected and subjected to b-glucuronidase and sulphatase hydrolysis prior to HPLC analysis. b Results are presented as mean S.E. for groups of 6 rats and 3 Syrian hamsters.
in the rat and Syrian hamster, respectively. Examination of pooled 06-h rat bile samples, following enzymatic hydrolysis with b-glucuronidase and sulphatase, revealed small amounts of 3-hydroxycoumarin, o-hydroxyphenyllactic acid and o-hydroxyphenylacetic acid. The majority of the radioactivity present in pooled 06h rat bile samples appeared to comprise unknown coumarin metabolites (data not shown). 3.3. Effect of coumarin treatment on the disposition of [3-14C]coumarin in the rat and Syrian hamster Male Sprague–Dawley rats were fed diets containing 0 (control), 0.2 and 0.5% coumarin and male Syrian
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hamsters were fed diets containing 0.5 and 1.0% coumarin for 24 weeks. Syrian hamsters were fed diets containing 0.5 and 1.0% coumarin, rather than 0.2 and 0.5% coumarin, for these studies as the 0.2% dose level produced only minor hepatic effects (Figs. 2–5) and in a previous 13-week study the administration of a 1.0% coumarin diet also produced no marked hepatic effects (Lake and Grasso, 1996). Calculated daily intakes of coumarin based on body weight and food consumption data for rats fed 0.2 and 0.5% coumarin for 24 weeks were 127 and 330 mg/kg/day, respectively, and for Syrian hamsters fed 0.5 and 1.0% coumarin for 24 weeks were 306 and 613 mg/kg/day, respectively. Rats and Syrian hamsters were then given single oral doses of either 25 or 300 mg/kg [3-14C]coumarin. The 25 mg/kg dose level was selected to represent a non-toxic dose in the rat (Lake, 1999 and unpublished observations), whereas the 300 mg/kg dose level was selected as an hepatotoxic dose in the rat, being similar to the daily intake of animals fed a 0.5% coumarin diet. In these studies the recovery of radioactivity after 96 h ranged from 98.4 to 105.1% of the administered dose in both species (Tables 2 and 3). Following treatment with single oral doses of either 25 or 300 mg/kg [3-14C]coumarin to rats and Syrian hamsters urine and faeces were collected for periods of 24, 48, 72 and 96 h. The time course of urinary and faecal excretion of radioactivity was similar in rats fed diets containing 0 (control) and 0.2% coumarin for 24 weeks and subsequently given single oral doses of 25 and 300 mg/kg [3-14C]coumarin (Fig. 8A and B). In contrast, the disposition of single oral doses of both 25
Fig. 7. Biliary excretion of radioactivity following administration of a single oral dose of 25 mg/kg [3-14C]coumarin to rats (*) and Syrian hamsters (*). Results are presented as mean S.E. for groups of 5 animals.
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Table 2 Excretion and tissue distribution of [3-14C]coumarin after oral administration to male Sprague–Dawley rats In
Percentage of administered radioactivity found after 96 ha 25 mg/kg [3-14C]coumarin Control
Urine Faeces Total gut and contentsb Liver Total Covalently bound Kidney Percentage recovery of administered dose
300 mg/kg [3-14C]coumarin
0.2% Coumarin
0.5% Coumarin
Control
0.2% Coumarin
0.5% Coumarin
55.52.5 43.90.5 0.0550.007
59.84.2 39.23.3 0.0430.010
81.0 2.3*** 23.2 2.2*** 0.0470.017
60.0 1.9 37.7 2.6 0.149 0.089
66.24.9 32.75.9 0.1080.038
88.51.8*** 11.81.1*** 0.0460.011
0.970.03 0.540.03 0.0130.001 100.42.5
0.770.02** 0.320.02*** 0.0120.002 98.91.7
0.85 0.05* 0.25 0.02*** 0.0080.001* 99.8 1.3
0.79 0.12 0.31 0.03 0.027 0.003 100.6 2.6
0.880.09 0.260.03 0.0170.003* 105.10.8
0.930.06 0.320.03 0.0150.002** 101.30.9
c
a Rats were fed diets containing 0 (control), 0.2 and 0.5% coumarin for 24 weeks and were then given single oral doses of either 25 or 300 mg/kg [3-14C]coumarin. Urine and faeces were collected for 96 h prior to determination of tissue levels of radioactivity. b Radioactivity present after 96 h in the entire gastrointestinal tract and contents. c Results are presented as mean S.E. for groups of 4 animals. Values significantly different from control (0% coumarin diet) are: *P<0.05; **P< 0.01; ***P <0.001.
Table 3 Excretion and tissue distribution of [3-14C]coumarin after oral administration to male Syrian hamsters In
Percentage of administered radioactivity found after 96 ha 25 mg/kg [3-14C]coumarin
Urine Faeces Total gut and contentsb Liver Total Covalently bound Kidney Percentage recovery of administered dose
300 mg/kg [3-14C]coumarin
Control
0.5% Coumarin
1.0% Coumarin
Control
0.5% Coumarin
1.0% Coumarin
84.61.8c 13.31.7 0.0270.003
96.6 0.8** 6.20.5** 0.0320.004
96.9 3.9** 6.8 1.5** 0.1140.060
90.44.7 8.81.6 0.0480.013
93.44.0 5.80.8 0.0350.002
95.92.7 4.90.8* 0.0280.004
0.510.07 0.450.06 0.0130.001 98.41.8
0.16 0.02*** 0.15 0.01*** 0.0080.001 102.90.4
0.14 0.02*** 0.08 0.01*** 0.0110.004 103.93.6
0.340.03 0.330.33 0.0110.001 99.64.8
0.210.01 0.150.003*** 0.0110.003 99.44.3
0.300.07 0.140.02*** 0.0060.001 101.12.6
a Syrian hamsters were fed diets containing 0 (control), 0.5 and 1.0% coumarin for 24 weeks and were then given single oral doses of either 25 or 300 mg/kg [3-14C]coumarin. Urine and faeces were collected for 96 h prior to determination of tissue levels of radioactivity. b Radioactivity present after 96 h in the entire gastrointestinal tract and contents. c Results are presented as mean S.E. for groups of 4 animals. Values significantly different from control (0% coumarin diet) are: *P<0.05; **P< 0.01; ***P <0.001.
and 300 mg/kg [3-14C]coumarin was significantly different in rats fed a 0.5% coumarin diet for 24 weeks. The treatment of rats with 0.5% coumarin resulted in a significantly greater excretion of radioactivity in the urine and a significantly decreased excretion of radioactivity in the faeces after 24, 48, 72 and 96 h following oral administration of either 25 (Fig. 8A) or 300 (Fig. 8B) mg/kg [3-14C]coumarin. After 96 h, rats fed 0 (control), 0.2 and 0.5% coumarin diets and subsequently given an oral 25 mg/kg dose of [3-14C]coumarin excreted 55.5, 59.8 and 81.0% of the dose, respectively, in the urine and 43.9, 39.2 and 23.2% of the dose, respectively, in the faeces (Table 2). Corresponding values for rats fed 0 (control), 0.2 and 0.5% coumarin diets and subsequently given an oral 300 mg/kg dose of [3-14C]coumarin were 60.0, 66.2 and 88.5% of the dose excreted in
the urine, respectively, and 37.7, 32.7 and 11.8% of the dose excreted in the faeces, respectively (Table 2). Prior coumarin treatment had no significant effect on the amount of radioactivity present in the entire gastrointestinal tract and contents 96 h after oral administration of either 25 or 300 mg/kg [3-14C]coumarin (Table 2). Liver levels of coumarin were determined both as total radioactivity and as radioactivity covalently bound to liver proteins 96 h after administration of [3-14C]coumarin. Compared to control (0% coumarin diet) animals, treatment with 0.2 and 0.5% coumarin diets significantly reduced the levels of total and covalently bound radioactivity after oral administration of 25 mg/kg, but not 300 mg/kg, [3-14C]coumarin (Table 2). Kidney levels of radioactivity were also significantly reduced in rats fed a 0.5% coumarin diet and given 25 mg/kg [3-14C]coumarin
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but had no effect on levels of radioactivity present in the kidney and entire gastrointestinal tract and contents after 96 h (Table 3). The treatment of Syrian hamsters with 0.5 and 1.0% coumarin diets had little effect on the subsequent disposition of a single oral 300 mg/kg dose of [3-14C]coumarin (Fig. 9B and Table 3). While urinary excretion of radioactivity was somewhat increased, faecal excretion was decreased, this being statistically significant in Syrian hamsters fed a 1.0% coumarin diet (Table 3). Prior treatment with 0.5 and 1.0% coumarin diets also significantly decreased liver levels of covalently bound, but not total, radioactivity 96 h after a single oral 300 mg/kg dose of [3-14C]coumarin (Table 3).
4. Discussion
Fig. 8. The effect of treatment of rats for 24 weeks with control diet (*, *) or diets containing 0.2 (~, ~) and 0.5 (&, &) % coumarin on the disposition of [3-14C]coumarin. Rats were given single oral doses of either 25 (A) or 300 (B) mg/kg [3-14C]coumarin and radioactivity present in urine (*, ~, &) and faeces (*, ~, &) determined for up to 96 h. Results are presented as meanS.E. for groups of 4 animals.
and in animals fed 0.2 and 0.5% coumarin diets and given 300 mg/kg [3-14C]coumarin (Table 2). Coumarin disposition was significantly changed in Syrian hamsters fed 0.5 and 1.0% coumarin diets compared to controls (0% coumarin diet) prior to administration of a single oral dose of 25 mg/kg [3-14C] coumarin (Fig. 9A). Prior coumarin treatment resulted in a significantly greater excretion of radioactivity in the urine and a significantly decreased excretion of radioactivity in the faeces 24, 48, 72 and 96 h following an oral 25 mg/kg dose of [3-14C]coumarin (Fig. 9A). After 96 h, Syrian hamsters fed 0 (control), 0.5 and 1.0% coumarin diets and subsequently given an oral 25 mg/kg dose of [3-14C]coumarin excreted 84.6, 96.6 and 96.9% of the dose, respectively, in the urine and 13.3, 6.2 and 6.8% of the dose, respectively, in the faeces (Table 3). The treatment of Syrian hamsters with 0.5 and 1.0% coumarin diets also significantly decreased total and covalently bound liver levels of radioactivity after 96 h,
Previous studies have demonstrated that coumarin exhibits marked species differences in both metabolism and hepatotoxicity (Cohen, 1979; Fentem and Fry, 1993; Lake, 1999). The aim of the present study was to compare the disposition, metabolism and hepatotoxicity of coumarin in the rat and Syrian hamster. Previous studies have demonstrated that these two species appear to be susceptible and resistant, respectively, to coumarin-induced liver injury. Several studies have demonstrated that the acute administration of coumarin to rats results in centrilobular hepatic necrosis, whereas prolonged administration results in bile duct lesions (Hagan et al., 1967; Cohen, 1979, Lake, 1984; Evans et al., 1989; Lake et al., 1989a, 1994; Carlton et al., 1996; Lake and Grasso, 1996). In addition, the chronic administration of high doses of coumarin can result in the formation of both parenchymal cell tumours and cholangiocarcinoma (Carlton et al., 1996). Unlike the rat, the administration of coumarin to the Syrian hamster for 13 weeks has been reported not to result in any significant hepatotoxicity (Lake and Grasso, 1996) and after 2 years’ treatment with 0.1 and 0.5% coumarin diets, no liver lesions or liver tumours were observed (Ueno and Hirono, 1981). As observed in previous studies, the administration of high (e.g. 0.5% in the diet) doses of coumarin to the rat was shown to increase relative liver weight and produce histological evidence of hepatotoxicity including bile duct proliferation. These effects were less marked in rats treated with 0.2% coumarin and no morphological changes were observed in the Syrian hamster. In addition, coumarin treatment had no marked effect on relative liver weight in the Syrian hamster. Coumarin administration also resulted in a number of hepatic biochemical changes. For example, coumarin treatment produced a sustained reduction in total CYP content and CYP-dependent enzyme activities. In contrast, levels of GSH were enhanced in rats given 0.5% coumarin and at one or both of the two coumarin dose
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Fig. 9. The effect of treatment of Syrian hamsters for 24 weeks with control diet (*, *) or diets containing 0.5 (~, ~) and 1.0 (&, &) % coumarin on the disposition of [3-14C]coumarin. Syrian hamsters were given single oral doses of either 25 (A) or 300 (B) mg/kg [3-14C]coumarin and radioactivity present in urine (*, ~, &) and faeces (*, ~, &) determined for up to 96 h. Results are presented as mean S.E. for groups of 4 animals.
levels examined, hepatic UDP glucuronosyltransferase (towards 4-hydroxybiphenyl), g-glutamyltransferase and GSH S-transferase activities (towards both CDNB and ENPP as substrates) were significantly increased. The hepatic biochemical effects of 0.5% coumarin observed in this study after 12, 24 and 42 weeks of treatment are similar to previous studies of up to 13 weeks’ duration (Lake et al., 1994; Lake and Grasso, 1996) and one study of up to 18 months’ duration (Evans et al., 1989). Compared to the rat, the hepatic biochemical effects of coumarin treatment were less marked in the Syrian hamster. The only consistent change noted after 12, 24 and 42 weeks’ treatment with 0.5%, but not 0.2%, coumarin was an induction of GSH S-transferase activity towards ENPP as substrate. Overall, the hepatic biochemical effects of coumarin in the rat involve an inhibition of phase I CYP-dependent pathways of xenobiotic metabolism, together with
a stimulation of phase II pathways of xenobiotic metabolism. Previous studies have shown that coumarin can be metabolised in the rat to a number of hydroxycoumarins which can be conjugated with d-glucuronic acid and sulphate (Mead et al., 1958; Kaighen and Williams, 1961; Cohen, 1979; Lake, 1999). In addition, coumarin is known to enhance urinary mercapturic acid excretion in the rat and a mercapturic acid, derived from the conjugation of coumarin 3,4-epoxide with GSH, has been identified (Lake, 1984; Huwer et al., 1991). The hepatic biochemical changes in response to sustained coumarin treatment may be interpreted as an enhancement of pathways of coumarin detoxification and elimination. As shown in Fig. 1, coumarin is known to be converted to a coumarin 3,4-epoxide intermediate by CYP-dependent enzymes which can be conjugated with GSH by GSH S-transferase enzymes and subsequently converted by g-glutamyltransferase and other enzymes to a urinary mercapturic acid. In addition, the stimulation of UDPglucuronosyltransferase enzymes would favour the elimination of hydroxycoumarin metabolites. Investigations into the mechanisms of coumarininduced hepatotoxicity in the rat have demonstrated the importance of the 3,4-double bond in coumarin toxicity and the role of GSH in protecting against coumarininduced liver injury (reviewed in Lake, 1999). In vitro studies have demonstrated that coumarin is more toxic to rat hepatocytes than a number of coumarin metabolites, including various hydroxycoumarins and o-hydroxyphenylacetic acid (Lake et al., 1989a). One exception is o-hydroxyphenylacetaldehyde, which is more toxic to rat hepatocytes than either coumarin or 3-hydroxycoumarin (Born et al., 2000b), suggesting that this metabolite together with coumarin 3,4-epoxide may have a role in coumarin-induced toxicity (Fig. 1). While the metabolism of coumarin in the rat has been investigated in a number of studies (Cohen, 1979; Lake, 1999), the profile of coumarin metabolism in this species has yet to be fully elucidated. Moreover, the metabolism of coumarin in the Syrian hamster has been examined in only one in vivo and a few in vitro studies (Gangolli et al., 1974; Cohen, 1979; Lake et al., 1992a; Pearce et al., 1992; Lake, 1999). The aim of the limited metabolic studies conducted in these investigations was to obtain more information on the metabolism and disposition of coumarin in the Syrian hamster. In the present study the disposition and metabolism of [3-14C]coumarin was examined in untreated rats and Syrian hamsters given a single oral low dose of 25 mg/ kg [3-14C]coumarin. Radioactivity was rapidly excreted by both species, with the bulk of the administered dose being eliminated in either the urine or faeces within 24 h. However, while the urine was the major route of excretion of radioactivity in the Syrian hamster, some 38% of the administered dose was excreted after 96 h in
B.G. Lake et al. / Food and Chemical Toxicology 40 (2002) 809–823
the faeces by the rat. Additional studies with bile duct cannulated animals revealed that the biliary excretion of radioactivity was much greater in the rat than the Syrian hamster. These observations are in agreement with previous studies on the disposition of coumarin in the rat (Kaighen and Williams, 1961; van Sumere and Teuchy, 1971; Lake et al., 1989c). In addition, Williams et al. (1965) reported that following an oral or ip dose of 50 mg/kg of coumarin to rats, some 50% of the dose was excreted in the bile as unknown metabolites within 24 h. While coumarin metabolites may undergo extensive enterohepatic circulation in the rat, as with the rabbit, baboon and humans (Lake, 1999), the urine appears to be the major route of coumarin metabolite excretion in the Syrian hamster. The examination of 024-h hydrolysed urine samples from rats given a single oral 25 mg/kg dose of [3-14C] coumarin revealed the presence of small amounts of 3-, 5- and 7-hydroxycoumarin, together with larger quantities of o-hydroxyphenyllactic acid and o-hydroxyphenylacetic acid. Such urinary metabolites have also been detected in previous studies after the administration of coumarin to rats (Mead et al., 1958; Booth et al., 1959; Kaighen and Williams, 1961; van Sumere and Teuchy, 1971; Lake et al., 1989c). In agreement with the study of Kaighen and Williams (1961), the major identified urinary coumarin metabolite was o-hydroxyphenyl acetic acid, this metabolite comprising 19.0% of the administered dose in the present study and 19.4% (range 12.527.2%, n=3) of the dose following oral administration of a 100 mg/kg dose of [3-14C]coumarin in the study performed by Kaighen and Williams (1961). Coumarin metabolism to 7-hydroxycoumarin only accounted for 0.56% of the administered dose in the 024-h urine. This value is in agreement with other in vivo and many in vitro studies in confirming that the rat is a poor 7-hydroxylator of coumarin (Kaighen and Williams, 1961; van Sumere and Teuchy, 1971; Lake et al., 1989c; Lake, 1999). In the present study 1.3% of the dose was excreted as 7-hydroxycoumarin in the 024-h urine of Syrian hamsters following the administration of a single oral 25 mg/kg dose of [3-14C]coumarin to Syrian hamsters. This value is similar to the figure of 5% reported after an oral 200 mg/kg dose by Gangolli et al. (1974). Moreover, in the present study all of the coumarin metabolites detected in rat urine were also present in Syrian hamster urine (Table 1). These metabolites included ohydroxyphenylacetic acid, which is known to be an endpoint of coumarin metabolism by the 3,4-expoxidation pathway (Born et al., 1997; Lake, 1999). The present in vivo data, suggesting that the Syrian hamster is a poor 7-hydroxylator of coumarin, is also in agreement with results of in vitro studies. As with rat liver microsomes, the 3,4-epoxidation pathway of coumarin metabolism was found to predominate over the 7-
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hydroxylation pathway in Syrian hamster liver microsomes, with o-hydroxyphenylacetaldehyde being the major identified product formed by both species (Lake et al., 1992a). In another study, 7-hydroxycoumarin was found to account for only 0.3 and 9% of total coumarin metabolism in rat and Syrian hamster liver microsomes, respectively (Pearce et al., 1992). The effect of prior coumarin treatment on the disposition of oral 25 and 300 mg/kg (namely toxic and non-toxic doses in the rat, respectively) doses of [3-14C]coumarin was also investigated. In the rat, treatment with 0.5% coumarin enhanced the urinary excretion of radioactivity and reduced the faecal excretion of radioactivity after single oral doses of both 25 and 300 mg/kg [3-14C]coumarin. Prior coumarin treatment thus favoured the elimination of coumarin in the urine. Additional studies would be required to ascertain whether prior coumarin treatment reduces the biliary excretion and subsequent enterohepatic circulation of coumarin metabolites. Previous studies with hepatic microsomes have demonstrated that coumarin can be converted by CYP-dependent enzymes to metabolite(s) that bind covalently to liver proteins (Lake, 1984; Lake et al., 1992a). In the present study, covalent binding of radioactivity to liver proteins was observed after the administration of single oral doses of 25 and 300 mg/kg [3-14C]coumarin (Table 2). Prior coumarin treatment at dose levels of 0.2 and 0.5% significantly reduced liver levels of both total and covalently bound radioactivity following an oral dose of 25, but not 300, mg/kg [3-14C]coumarin. These results suggest that prior coumarin treatment favours coumarin metabolism to nontoxic products and more rapid elimination from the liver. Prior coumarin treatment at dietary levels of 0.5 and 1.0% also produced some changes in coumarin disposition in the Syrian hamster. These effects comprised the enhanced urinary excretion of radioactivity after an oral 25 mg/kg dose of [3-14C]coumarin and a reduction in liver levels of either total and/or covalently bound radioactivity (Table 3). The results of the present study, together with those of other investigations (Lake and Grasso, 1996), demonstrate a marked species difference in coumarininduced hepatotoxicity. While high doses of coumarin are clearly hepatotoxic in the rat, the Syrian hamster is resistant to coumarin-induced liver injury. From the limited metabolic studies performed, it is clear that, like the rat, the Syrian hamster is a poor 7-hydroxylator of coumarin. The present results are in agreement with those of previous in vivo and in vitro studies on the metabolism of coumarin in the Syrian hamster (Gangolli et al., 1974; Lake et al., 1992a; Pearce et al., 1992). However, while the metabolism of coumarin appears to favour the 3,4-epoxidation, rather than the 7-hydroxylation, pathway in both the rat and Syrian hamster,
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marked quantitative species differences were observed in coumarin disposition. In agreement with the majority of studies (reviewed in Lake, 1999), a significant proportion of an oral dose of coumarin to the rat was excreted in the faeces. As observed by Williams et al. (1965), a large amount of an oral dose of coumarin to the rat was excreted in the bile as unidentified metabolites. Unlike the rat, the urine was the predominant route of excretion of radioactivity following administration of oral doses of [3-14C]coumarin to the Syrian hamster. In addition, the biliary excretion of radioactivity was lower in the Syrian hamster than the rat, suggesting that enterohepatic circulation of coumarin metabolites may not occur to any great extent in this species. In summary, the present study demonstrates a marked species difference between the rat and Syrian hamster in coumarin-induced hepatotoxicity. Clearly, the resistance of the Syrian hamster to coumarininduced liver injury is not attributable to coumarin 7hydroxylation status. The observed species difference may be at least partially attributable to differences in coumarin disposition. However, additional studies are required to elucidate the pathways of coumarin metabolism in both species. Such studies should include the identification of coumarin metabolites present in the bile as well as in the urine and faeces.
Acknowledgements The authors wish to acknowledge the great contribution made by Dr. F.J.C. Roe to the science of toxicology. In addition, we are grateful to Dr. Roe and to Professors S.D. Gangolli and P. Grasso (two former members of BIBRA staff) for many valuable discussions on the metabolism and toxicity of coumarin.
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1997. Synthesis reactivity of coumarin 34-epoxide. Drug Metabolism and Disposition 25, 1318–1323. Carlton, B.D., Aubrun, J.-C., Simon, G.S., 1996. Effects of coumarin following perinatal and chronic exposure in Sprague-Dawley rats, CD-1 mice. Fundamental and Applied Toxicology 30, 145–151. Cohen, A.J., 1979. Critical review of the toxicology of coumarin with special reference to interspecies differences in metabolism and hepatotoxic response and their significance to man. Food and Chemical Toxicology 17, 277–289. Evans, J.G., Appleby, E.C., Lake, B.G., Conning, D.M., 1989. Studies on the induction of cholangiofibrosis by coumarin in the rat. Toxicology 55, 207–224. Fentem, J.H., Fry, J.R., 1993. Species differences in the metabolism and hepatotoxicity of coumarin. Comparative Biochemistry and Physiology 104C, 1–8. Fentem, J.H., Fry, J.R., Whiting, D.H., 1991. O-Hydroxyphenylacetaldehyde: a major novel metabolite of coumarin formed by rat, gerbil and human liver microsomes. Biochemical and Biophysical Research Communications 179, 197–203. Gangolli, S.D., Shilling, W.H., Grasso, P., Gaunt, I.F., 1974. Studies on the metabolism and hepatotoxicity of coumarin in the baboon. Biochemical Society Transactions 2, 310–312. Habig, W.H., Prabst, M.J., Jakoby, W.B., 1974. Glutathione S-transferases. The first step in mercapturic acid formation. Journal of Biological Chemistry 249, 7130–7139. Hagan, E.C., Hansen, W.H., Fitzhugh, O.G., Jenner, P.M., Jones, W.I., Taylor, J.M., Long, E.L., Nelson, A.A., Brouwer, J.B., 1967. Food flavourings compounds of related structure. II. Subacute and chronic toxicity. Food and Cosmetics Toxicology 5, 141–157. Huwer, T., Altmann, H.-J., Grunow, W., Lenhardt, S., Przybylski, M., Eisenbrand, C., 1991. Coumarin mercapturic acid isolated from rat urine indicates metabolic formation of coumarin 3,4-epoxide. Chemical Research in Toxicology 4, 586–590. Kaighen, M., Williams, R.T., 1961. The metabolism of [3-14C]coumarin. Journal of Medicinal Chemistry 3, 25–43. Lake, B.G., 1984. Investigations into the mechanism of coumarininduced hepatotoxicity in the rat. Archives of Toxicology. 16 29 (Suppl. 7). Lake, B.G., 1987. Preparation and characterisation of microsomal fractions for studies of xenobiotic metabolism. In: Snell, K., Mullock, B. (Eds.), Biochemical Toxicology: A Practical Approach. IRL Press, Oxford, pp. 183–215. Lake, B.G., 1999. Coumarin metabolism, toxicity and carcinogenicity: relevance for human risk assessment. Food and Chemical Toxicology 37, 423–453. Lake, B.G., Grasso, P., 1996. Comparison of the hepatotoxicity of coumarin in the rat, mouse Syrian hamster: a dose and time response study. Fundamental and Applied Toxicology 34, 105–117. Lake, B.G., Evans, J.G., Lewis, D.F.V., Price, R.J., 1994. Comparison of the hepatic effects of coumarin, 3,4-dimethylcoumarin, dihydrocoumarin and 6-methylcoumarin in the rat. Food and Chemical Toxicology 32, 743–751. Lake, B.G., Gaudin, H., Price, R.J., Walters, D.G., 1992a. Metabolism of [3-14C]coumarin to polar and covalently bound products by hepatic microsomes from the rat, Syrian hamster, gerbil and humans. Food and Chemical Toxicology 30, 105–115. Lake, B.G., Gray, T.J.B., Evans, J.G., Lewis, D.F.V., Beamand, J.A., Hue, K.L., 1989a. Studies on the mechanism of coumarin-induced toxicity in rat hepatocytes: comparison with dihydrocoumarin and other coumarin metabolites. Toxicology and Applied Pharmacology 97, 311–323. Lake, B.G., Gray, T.J.B., Ko¨ro¨si, S.A., Walters, D.G., 1989b. Nafenopin a peroxisome proliferator depletes hepatic vitamin, E content and elevates plasma oxidised glutathione levels in rats. Toxicology Letters 45, 221–229. Lake, B.G., Osborne, D.J., Walters, D.G., Price, R.J., 1992b. Indentification of o-hydroxyphenylacetaldehyde as a major metabolite of
B.G. Lake et al. / Food and Chemical Toxicology 40 (2002) 809–823 coumarin in rat hepatic microsomes. Food and Chemical Toxicology 30, 99–104. Lake, B.G., Walters, D.G., Gangolli, S.D., 1989c. Comparison of the metabolism, disposition of [3-14C] coumarin in the rat and marmoset (Callithrix jacchus). Toxicology Letters 45, 299–306. Lovell, D.P., van, Iersel, M., Walters, D.G., Price, R.J., Lake, B.G., 1999. Genetic variation in the metabolism of coumarin in mouse liver. Pharmacogenetics 9, 239–250. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193, 265–275. Lush, I.E., Andrews, K.M., 1978. Genetic variation between mice in their metabolism of coumarin and its derivatives. Genetics Research 31, 177–186. Mead, J.A.R., Smith, J.N., Williams, R.T., 1958. Studies in detoxication. 72. The metabolism of coumarin and of o-coumaric acid. Biochemical Journal 68, 67–74. National Toxicology Program (NTP), 1993. Toxicology and carcinogenesis studies of coumarin (CAS No. 91-64-5) in F344/N rats and B6C3F1 mice (gavage studies). Technical Report No. NTP TR 422. NIH Publication No. 93-3153. US Department of Health and Human Services. NIH, Research Triangle Park, NC. Opdyke, D.L.J., 1974. Monographs on fragrance raw materials: coumarin. Food and Chemical Toxicology 12, 385–388. Pearce, R., Greenway, D., Parkinson, A., 1992. Species differences and interindividual variation in liver microsomal cytochrome P450 2A
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enzymes: effects on coumarin dicumarol, and testosterone oxidation. Archives of Biochemistry and Biophysics 298, 211–225. Pelkonen, O., Raunio, H., Rautio, A., Ma¨enpa¨a¨, J., Lang, M.A., 1993. Coumarin 7-hydroxylase: characteristics regulation in mouse man. Journal of the Irish Colleges of Physicians and Surgeons 22 (Suppl. 1), 24–28. Shilling, W.H., Crampton, R.F., Longland, R.C., 1969. Metabolism of coumarin in man. Nature 221, 664–665. Smith, D.J., Ding, J.L., Peters, D.J., 1979. A sensitive fluorimetric assay for g-glutamyltransferase. Analytical Biochemistry 100, 136–139. Soine, T.O., 1964. Naturally occurring coumarins and related physiological activities. Journal of Pharmaceutical Sciences 53, 231–264. Ueno, I., Hirono, I., 1981. Non-carcinogenic response to coumarin in Syrian golden hamsters. Food and Chemical Toxicology 19, 353–355. van, Iersel, M., Walters, D.G., Price, R.J., Lovell, D.P., Lake, B.G., 1994. Sex and strain differences in mouse hepatic coumarin 7hydroxylase activity. Food and Chemical Toxicology 32, 387–390. van Sumere, C.F., Teuchy, H., 1971. The metabolism of [2-14C]coumarin, [2-14C]-7-hydroxycoumarin in the rat. Archives Internationales de Physiologie et de Biochimie 79, 665–679. Walters, D.G., Lake, B.G., Cottrell, R.C., 1980. High-performance liquid chromatography of coumarin and its metabolites. Journal of Chromatography 196, 501–505. Williams, R.T., Milburn, P., Smith, R.L., 1965. The influence of enterohepatic circulation on toxicity of drugs. Annals of the New York Academy of Sciences 123, 110–124.
Research section
Studies on the disposition, metabolism and hepatotoxicity of coumarin in the rat and Syrian hamster B.G. Lakea,*, J.G. Evansa, F. Chapuisb, D.G. Waltersa, R.J. Pricea a TNO BIBRA International Ltd, Woodmansterne Road, Carshalton, Surrey SM5 4DS, UK ENS BANA, Universite´ de Bourgogne, Campus Universitaire, 1 Esplanade Erasme, F21100 Dijon, France
b
Abstract The hepatotoxicity, metabolism and disposition of coumarin has been compared in male SpragueDawley rats and Syrian hamsters. The treatment of rats for 12, 24 and 42 weeks with diets containing 0.2 and 0.5% coumarin resulted in hepatotoxicity and increased relative liver weights. While levels of cytochrome P450 (CYP) and CYP-dependent enzymes were decreased, levels of reduced glutathione (GSH) and activities of UDP glucuronosyltransferase, g-glutamyltransferase and GSH S-transferase were increased. In contrast, coumarin produced few hepatic changes in the Syrian hamster. Following a single oral dose of 25 mg/kg [3-14C]coumarin, radioactivity was rapidly excreted by the rat and Syrian hamster with the urine containing 63.5 and 89.9%, respectively, and the faeces 38.0 and 12.4%, respectively, of the administered dose after 96 h. The biliary excretion of radioactivity was greater in the rat than in the Syrian hamster. Analysis of 024-h urine samples revealed that both species were poor 7-hydroxylators of coumarin. In the rat, treatment with 0.5% coumarin in the diet for 24 weeks was found to increase the urinary excretion of single oral gavage doses of 25 and 300 mg/kg [3-14C]coumarin. The marked species difference in hepatotoxicity between the rat and Syrian hamster observed in this study may be at least partially attributable to differences in coumarin disposition. However, additional studies are required to elucidate the metabolic pathways of coumarin in both species. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Coumarin; Hepatotoxicity; Metabolism; Rat; Species differences; Syrian hamster
1. Introduction Coumarin (1,2-benzopyrene, cis-o-coumarinic acid lactone) occurs naturally in several plant families and essential oils (Soine, 1964; Opdyke, 1974; Cohen, 1979; Lake, 1999). Although no longer used itself as an added food flavouring, it is present in some natural flavouring source materials added to food, and coumarin is employed as a fixative and enhancing agent in perfumes and is added to toilet soap and detergents, toothpaste, tobacco products and some alcoholic beverages (Opdyke, 1974; Cohen, 1979). Coumarin has also been used as a therapeutic agent for the treatment of various cancers (reviewed in Lake, 1999). The toxicology of coumarin merits attention, since coumarin exhibits marked species differences in both
Abbreviations: CDNB, 1-chloro-2,4-dinitrobenzene; CYP, cytochrome P450; ENPP, 1,2–epoxy-3-(p-nitrophenoxy)propane; GSH, reduced glutathione; GSSG, oxidised glutathione. * Corresponding author. Tel.: +44-20-8652-1006; fax: +44-208661-7029. E-mail address: [email protected] (B.G. Lake).
metabolism and hepatotoxicity (Cohen, 1979; Lake, 1999). In humans, coumarin 7-hydroxylation, catalysed by hepatic CYP2A6, is the major route of coumarin biotransformation in most subjects, whereas this is only a minor route of coumarin metabolism in the rat (Kaighen and Williams, 1961; Shilling et al., 1969; Cohen, 1979; Pelkonen et al., 1993; Lake, 1999). The major route of coumarin biotransformation in the rat involves an initial 3,4-epoxidation reaction (Fig. 1), which is followed by the loss of carbon dioxide to form o-hydroxyphenylacetaldehyde (Lake et al., 1989a, 1992a,b; Fentem et al., 1991; Born et al., 1997, 2000a; Lake, 1999). While sex and strain differences in the extent of coumarin 7-hydroxylation have been reported in the mouse, like the rat, the 3,4-epoxidation pathway predominates in this species (Lush and Andrews, 1978; van Iersel et al., 1994; Lovell et al., 1999). Other studies have demonstrated that Old- but not New-World primates (such as the baboon and Cynomolgus monkey) are also extensive 7-hydroxylators of coumarin (reviewed in Lake, 1999). In the rat, the acute administration of high doses of coumarin have been reported to result in centrilobular
0278-6915/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0278-6915(02)00036-4
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Fig. 1. Some pathways of coumarin metabolism (Cohen, 1979; Lake et al., 1989a; Born et al., 1997, 2000a; Lake, 1999). In the present study, coumarin administration to the rat results in decreased cytochrome P450 (CYP) enzyme activities, but enhanced levels of hepatic reduced glutathione (GSH) and activities of UDPglucuronosyltransferase, GSH S-transferase and g-glutamyltransferase.
hepatic necrosis, whereas chronic administration results in bile duct lesions (Hagan et al., 1967; Lake, 1984; Evans et al., 1989; Lake et al., 1989a, 1994; Carlton et al., 1996; Lake and Grasso, 1996). Moreover, the chronic administration of high doses of coumarin has been reported to result in both parenchymal cell tumours and in cholangiocarcinoma (Carlton et al., 1996). Coumarin has also been reported to produce liver tumours in female, but not in male, B6C3F1 mice (NTP, 1993); whereas coumarin did not produce any liver lesions or liver tumours in male and female Syrian hamsters after 2 years’ treatment (Ueno and Hirono, 1981). The aim of the present study was to obtain further information on species differences in coumarin-induced hepatotoxicity. Studies were conducted with male SpragueDawley rats and male Syrian hamsters as these species have been previously reported to be susceptible and resistant, respectively, to coumarin-induced liver injury. Apart from investigating the time course of the hepatic effects of coumarin in the rat and Syrian hamster, some limited studies on coumarin metabolism and the effect of prior coumarin treatment on the disposition of coumarin in both species have been investigated.
2. Materials and methods 2.1. Materials Coumarin, enzyme substrates and cofactors were obtained from Sigma-Aldrich Co. Ltd (Poole, Dorset, UK). [3-14C]Coumarin (sp. act. 5.3 mCi/mmol) was purchased from ICI Chemicals and Polymers Group (Billingham, Cleveland, UK) and purified to > 99.5% radiochemical purity by chromatography on thin layer plates of silica gel G developed in toluenechloroform (1:1, v/v). Unlabelled coumarin metabolites were obtained from the sources described previously (Lake et al., 1989a). 2.2. Animals and treatment Male SpragueDawley rats were obtained from Harlan Olac (Bicester, Oxon, UK) and male DSN strain Syrian hamsters from Intersimian Ltd (Milton Trading Estate, Milton, Oxon, UK) and allowed free access to R and M No.3 laboratory animal diet (Special Diets Services, Witham, Essex, UK) and water. The animals were housed in polypropylene cages with stainless-steel grid
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tops and floors in rooms maintained at 22 3 C with a relative humidity of 40–70% and a 12-h light cycle. After being allowed to acclimatise to these conditions for 2 weeks, rats (6 weeks old) and Syrian hamsters (8 weeks old) were fed diets containing no test compound (0% coumarin) or diets containing 0.2, 0.5 and 1.0% coumarin for periods of up to 42 weeks. Animals were killed by exsanguination under diethyl ether and the livers immediately removed for biochemical and morphological studies. 2.3. Biochemical investigations Whole liver homogenates (rat 0.25 and Syrian hamster 0.1 g fresh tissue/ml) were prepared in ice-cold 0.154 m KCl containing 50 mm TrisHCl, pH 7.4, using a Potter-type, teflonglass, motor-driven homogeniser (A.H. Thomas Co., Philadelphia, PA, USA). Liver whole homogenates were assayed for reduced (GSH) and oxidised (GSSG) glutathione by an enzymatic recycling assay employing GSH reductase and 5,50 dithio-bis(2-nitrobenzoic acid) as described previously (Adams et al., 1983; Lake et al., 1989b). The activity of whole homogenate and serum g-glutamyltransferase was determined by the method of Smith et al. (1979) employing l-g-glutamyl-7-amino-4-methylcoumarin as substrate. Washed microsomal and cytosolic fractions were prepared by differential centrifugation (Lake, 1987). Microsomal fractions were assayed for total cytochrome P450 (CYP) content and for activities of 7-ethoxycoumarin O-deethylase and ethylmorphine N-demethylase (Lake, 1987). Liver microsomal fractions were also assayed for UDPglucuronosyltransferase activity employing 4-hydroxybiphenyl as substrate (Bock et al., 1980). Cytosolic fractions were assayed for activities of GSH Stransferase employing 1-chloro-2,4-dinitrobenzene (CD NB) and 1,2-epoxy-3-(p-nitrophenoxy)propane (ENPP) as substrates (Habig et al., 1974). Whole homogenate, microsomal and cytosolic fraction protein content was determined by the method of Lowry et al. (1951) employing bovine serum albumin as standard.
singly in all-glass metabolism cages (Jencons Scientific Ltd, Hemel Hempstead, Herts, UK) with provision for collection of urine and faeces. Radioactivity present in urine was determined directly by scintillation counting, whereas faeces samples were first homogenised in methanol and as with tissue samples radioactivity was measured after sample oxidation. The profile of coumarin metabolites in 024-h urine samples was determined by HPLC. Urine samples were centrifuged at 10,000 g average for 15 min at 4 C and a 1-ml aliquot incubated with 10 mg b-glucuronidase and 10 mg sulphatase in 0.4 ml of 0.1 m acetate buffer pH 5.0 for 24 h at 37 C. After centrifugation at 10,000 g average for 15 min, an 80-ml aliquot was injected onto a Partisil 10 ODS-2 column and coumarin and coumarin metabolites separated by gradient elution with tetrahydrofuran/distilled water/acetic acid as described previously (Walters et al., 1980). For the studies on the biliary excretion of coumarin, the animals were maintained under sodium pentobarbitone anaesthesia (60 mg/kg, ip). After the bile duct was cannulated, the animals were dosed orally with 25 mg/kg [3-14C]coumarin and bile samples collected every 30 min for determination of radioactivity by scintillation counting. In other studies rats and Syrian hamsters were fed diets containing 01.0% coumarin for 24 weeks prior to being given single oral doses of either 25 or 300 mg/kg of [3-14C]coumarin (approximately 10 mCi/animal). Urine, faeces and tissue samples were collected and processed as described above. The macromolecular binding of [3-14C]coumarin metabolites to liver proteins was determined by exhaustive extraction of whole homogenates with 5% (w/v) trichloroacetic acid and 80% (v/v) methanol as described previously (Lake, 1984). 2.6. Statistical analysis Statistical evaluation of data was performed by oneway analysis of variance. Comparisons between means were made using the least significant difference test.
2.4. Morphological investigations
3. Results
Liver slices were fixed in neutral buffered formalin. Paraffin sections of about 5 mm thickness were cut, stained with haematoxylin and eosin and examined by light microscopy,
3.1. Chronic effects of coumarin in the rat and Syrian hamster
2.5. Metabolic studies Untreated male SpragueDawley rats (7 weeks old) and male Syrian hamsters (8 weeks old) were given a single oral 25 mg/kg dose of [3-14C]coumarin (approximately 13.5 and 8.0 mCi/animal, respectively) in corn oil (5 ml/kg body weight) by gastric intubation and caged
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Male SpragueDawley rats and male Syrian hamsters were fed diets containing 0 (control), 0.2 and 0.5% coumarin for periods of 12, 24 and 42 weeks. Calculated daily intakes of coumarin based on body weight and food consumption data for rats fed 0.2 and 0.5% coumarin diets were calculated to be 113 and 298 mg/kg/ day, respectively. Corresponding values for Syrian hamsters were 92 and 253 mg/kg/day, respectively (data not shown).
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The treatment of male SpragueDawley rats with 0.2 and 0.5% coumarin for 12 weeks and with 0.5% coumarin for 24 and 42 weeks significantly reduced body weight in the rat (Fig. 2A). In contrast, coumarin produced little effect on body weight in the Syrian hamster, with only a small decrease being observed after treatment with 0.2% coumarin for 12 weeks (Fig. 2B). The treatment of rats with coumarin for 12, 24 and 42 weeks resulted in dose-dependent increases in relative liver
weight (Fig. 2C). In contrast, relative liver weight was only significantly increased in the Syrian hamster after treatment with 0.5% coumarin for 42 weeks (Fig. 2D). The administration of 0.5% coumarin for 12 and 24 weeks and 0.2 and 0.5% coumarin for 42 weeks resulted in significant increases in rat serum g-glutamyltransferase activity (Fig. 2E). In contrast, coumarin treatment had no significant effect on serum g-glutamyltransferase activity in the Syrian hamster (Fig. 2F).
Fig. 2. The effect of treatment of rats (A, C, E) and Syrian hamsters (B, D, F) for 12, 24 and 42 weeks with diets containing 0 (control, &), 0.2 ( ) and 0.5 (&) % coumarin on body weight (A, B), relative liver weight (C, D) and serum g-glutamyltransferase activity (E, F). Results are expressed as meanS.E. for groups of 4 to 6 animals. Values significantly different from control are: *P <0.05; **P <0.01; ***P<0.001.
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Liver whole homogenates were assayed for g-glutamyltransferase activity and content of reduced (GSH) and oxidised (GSSG) glutathione. In the rat coumarin treatment for 12, 24 and 42 weeks produced a marked dose-dependent induction of hepatic g-glutamyltransferase activity (Fig. 3A). The treatment of rats with 0.5% coumarin also produced significant increases in hepatic GSH content, whereas hepatic GSSG content
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was not affected by coumarin administration (Fig. 3C,E). In the Syrian hamster coumarin treatment had no significant effect on hepatic g-glutamyltransferase activity (Fig. 3B) and produced only small changes in levels of GSH and GSSG (Fig. 3D and F). Microsomal fractions were assayed for total cytochrome P450 (CYP) content and activities of ethylmorphine N-demethylase, 7-ethoxycoumarin O-deethylase
Fig. 3. The effect of treatment of rats (A, C, E) and Syrian hamsters (B, D, F) for 12, 24 and 42 weeks with diets containing 0 (control, &), 0.2 ( ) and 0.5 (&) % coumarin on hepatic whole homogenate g-glutamyltransferase activity (A, B) and content of reduced (C, D) and oxidised (E, F) glutathione. Results are presented as meanS.E. for groups of 4 to 6 animals. Values significantly different from control are: *P<0.05; **P<0.01; ***P <0.001.
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and 4-hydroxybiphenyl UDP glucuronosyltransferase. In the rat coumarin treatment for 12, 24 and 42 weeks produced a significant dose-dependent decrease in total CYP content (Fig. 4A) and ethylmorphine N-demethylase activity (Fig. 4C). Rat hepatic 7-ethoxycoumarin Odeethylase activity was significantly reduced by treatment with 0.2 and 0.5% coumarin for 12 weeks and
with 0.5% coumarin for 24 and 42 weeks (Fig. 4E). In contrast, coumarin treatment had no significant effect on total CYP content and ethylmorphine N-demethylase activity in the Syrian hamster and produced only a small decrease in 7-ethoxycoumarin O-deethylase activity in Syrian hamsters given 0.2%, but not 0.5%, coumarin for 42 weeks (Fig. 4B, D and F). Coumarin
Fig. 4. The effect of treatment of rats (A, C, E) and Syrian hamsters (B, D, F) for 12, 24 and 42 weeks with diets containing 0 (control, &), 0.2 ( ) and 0.5 (&) % coumarin on hepatic microsomal cytochrome P450 (CYP) content (A, B) and activities of ethylmorphine N-demethylase (C, D) and 7-ethoxycoumarin O-deethylase (E, F). Results are presented as meanS.E. for groups of 4 to 6 animals. Values significantly different from control are: *P <0.05; **P<0.01; ***P< 0.001.
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treatment for 12, 24 and 42 weeks produced dosedependent increases in microsomal UDP glucuronosyltransferase activity in the rat, whereas no changes in UDP glucuronosyltransferase activity were observed in the Syrian hamster (Fig. 5A and B). Liver cytosols were assayed for GSH S-transferase activity employing 1-chloro-2,4-dinitrobenzene (CDNB) and 1,2-epoxy-3-(p-nitrophenoxy)propane (ENPP) as
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substrates. In the rat, treatment with 0.2 and 0.5% coumarin for 12 and 24 weeks and with 0.5% coumarin for 42 weeks resulted in significant increases in GSH Stransferase activities towards CDNB (Fig. 5C) and ENPP (Fig. 5E) as substrates. The treatment of Syrian hamsters with 0.5% coumarin for 12, 24 and 42 weeks significantly increased GSH S-transferase activity towards ENPP as substrate (Fig. 5F), with GSH
Fig. 5. The effect of treatment of rats (A, C, E) and Syrian hamsters (B, D, F) for 12, 24 and 42 weeks with diets containing 0 (control, &), 0.2 ( ) and 0.5 (&) % coumarin on hepatic microsomal 4-hydroxybiphenyl UDP glucuronosyltransferase activity (A, B) and cytosolic GSH S-transferase activity towards CDNB (C, D) and ENPP (E, F) as substrates. Results are presented as meanS.E. for groups of 4 to 6 animals. Values significantly different from control are: *P<0.05; **P<0.01; ***P <0.001.
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S-transferase activity towards CDNB as substrate also being significantly increased after 42 weeks treatment with 0.5% coumarin (Fig. 5D). Morphological examination of liver sections from control rats and Syrian hamsters revealed no abnormalities. In contrast, histological examination of liver sections from rats treated for 12, 24 and 42 weeks with 0.5% coumarin revealed extensive vacuolation and numerous eosinophilic inclusions in midzonal and centrilobular hepatocytes. Bile duct hyperplasia was also observed, which consisted of five ductules extending from the portal areas often reaching the region of the central vein. These changes were less marked in rats receiving 0.2% coumarin and no histological abnormalities were observed in Syrian hamsters treated with 0.2 and 0.5% coumarin. 3.2. Disposition and metabolism of [3-14C]coumarin in the rat and Syrian hamster Untreated male SpragueDawley rats and Syrian hamsters were given a single oral dose of 25 mg/kg [3-14C]coumarin. This coumarin dose level was selected as this represents a non-toxic dose in the rat (Lake, 1999 and unpublished observations). Following oral administration of [3-14C]coumarin radioactivity was rapidly excreted by both species, with the bulk of the dose being excreted within 24 h (Fig. 6). The urine was the major route of elimination with 60.7 0.9 (mean S.E., n=6) and 86.4 2.4% of the dose being excreted within 24 h by the rat and Syrian hamster, respectively. After 96 h the excretion of radioactivity in the urine and faeces of the rat was 63.5 2.3 and 38.0 1.6% of the dose,
respectively. Corresponding values for the Syrian hamster were 89.9 1.7 and 12.4 0.6% of the dose, respectively. The overall recoveries of administered radioactivity in the urine and faeces of the rat and Syrian hamster after 96 h were 101.5 3.6 and 102.3 2.1% of the dose, respectively. The 024-h urine samples from rats and Syrian hamsters given 25 mg/kg [3-14C]coumarin were subjected to enzymatic hydrolysis with b-glucuronidase and sulphatase and analysed by HPLC. Coumarin was extensively metabolised in both the rat and Syrian hamster, with unchanged coumarin representing only 0.4 and 1.3% of the administered dose, respectively, in 024-h urines (Table 1). The formation of 7-hydroxycoumarin accounted for only 0.56 and 1.3% of the administered dose in the rat and Syrian hamster, respectively. Coumarin was also metabolised to 3- and 5-hydroxycoumarin (these metabolites not being completely resolved by the acid HPLC system employed) and to o-hydroxyphenyllactic acid and o-hydroxyphenylacetic acid by both species (Table 1). In the rat, o-hydroxyphenylacetic acid was the major identified urinary metabolite, comprising 19.0% of the administered dose, whereas this metabolite and o-hydroxyphenyllactic acid were the major identified 024-h urinary coumarin metabolites in the Syrian hamster (Table 1). The biliary excretion of radioactivity was studied in rats and Syrian hamsters given a 25 mg/kg oral dose of [3-14C]coumarin. Over the 6-h collection period there was a time-dependent excretion of radioactivity in the bile of both species (Fig. 7). After 6 h the biliary excretion of radioactivity accounted for 32.8 2.8 (mean S.E., n=5) and 13.4 2.4% of the administered dose
Fig. 6. Cumulative excretion of radioactivity in urine (*, &) and faeces (*, &) following administration of a single oral 25 mg/kg dose of [3-14C]coumarin to rats (*, *) and Syrian hamsters (&, &). Results are presented as mean S.E. for groups of 6 animals.
B.G. Lake et al. / Food and Chemical Toxicology 40 (2002) 809–823 Table 1 Urinary metabolites of [3-14C]coumarin in the rat and Syrian hamster Compound
Urinary excretion (% of administered dose)a Rat
Coumarin 3- and 5-Hydroxycoumarin 7-Hydroxycoumarin o-Hydroxyphenyllactic acid o-Hydroxyphenylacetic acid
Syrian hamster b
0.40 0.01 0.53 0.02 0.56 0.02 3.9 0.2 19.0 0.4
1.3 0.2 6.2 0.5 1.3 0.2 16.4 2.0 10.9 1.2
a Rats (n=6) and Syrian hamsters (n=3) were given a single oral 25 mg/kg dose of [3-14C]coumarin and 024-h urine samples collected and subjected to b-glucuronidase and sulphatase hydrolysis prior to HPLC analysis. b Results are presented as mean S.E. for groups of 6 rats and 3 Syrian hamsters.
in the rat and Syrian hamster, respectively. Examination of pooled 06-h rat bile samples, following enzymatic hydrolysis with b-glucuronidase and sulphatase, revealed small amounts of 3-hydroxycoumarin, o-hydroxyphenyllactic acid and o-hydroxyphenylacetic acid. The majority of the radioactivity present in pooled 06h rat bile samples appeared to comprise unknown coumarin metabolites (data not shown). 3.3. Effect of coumarin treatment on the disposition of [3-14C]coumarin in the rat and Syrian hamster Male Sprague–Dawley rats were fed diets containing 0 (control), 0.2 and 0.5% coumarin and male Syrian
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hamsters were fed diets containing 0.5 and 1.0% coumarin for 24 weeks. Syrian hamsters were fed diets containing 0.5 and 1.0% coumarin, rather than 0.2 and 0.5% coumarin, for these studies as the 0.2% dose level produced only minor hepatic effects (Figs. 2–5) and in a previous 13-week study the administration of a 1.0% coumarin diet also produced no marked hepatic effects (Lake and Grasso, 1996). Calculated daily intakes of coumarin based on body weight and food consumption data for rats fed 0.2 and 0.5% coumarin for 24 weeks were 127 and 330 mg/kg/day, respectively, and for Syrian hamsters fed 0.5 and 1.0% coumarin for 24 weeks were 306 and 613 mg/kg/day, respectively. Rats and Syrian hamsters were then given single oral doses of either 25 or 300 mg/kg [3-14C]coumarin. The 25 mg/kg dose level was selected to represent a non-toxic dose in the rat (Lake, 1999 and unpublished observations), whereas the 300 mg/kg dose level was selected as an hepatotoxic dose in the rat, being similar to the daily intake of animals fed a 0.5% coumarin diet. In these studies the recovery of radioactivity after 96 h ranged from 98.4 to 105.1% of the administered dose in both species (Tables 2 and 3). Following treatment with single oral doses of either 25 or 300 mg/kg [3-14C]coumarin to rats and Syrian hamsters urine and faeces were collected for periods of 24, 48, 72 and 96 h. The time course of urinary and faecal excretion of radioactivity was similar in rats fed diets containing 0 (control) and 0.2% coumarin for 24 weeks and subsequently given single oral doses of 25 and 300 mg/kg [3-14C]coumarin (Fig. 8A and B). In contrast, the disposition of single oral doses of both 25
Fig. 7. Biliary excretion of radioactivity following administration of a single oral dose of 25 mg/kg [3-14C]coumarin to rats (*) and Syrian hamsters (*). Results are presented as mean S.E. for groups of 5 animals.
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Table 2 Excretion and tissue distribution of [3-14C]coumarin after oral administration to male Sprague–Dawley rats In
Percentage of administered radioactivity found after 96 ha 25 mg/kg [3-14C]coumarin Control
Urine Faeces Total gut and contentsb Liver Total Covalently bound Kidney Percentage recovery of administered dose
300 mg/kg [3-14C]coumarin
0.2% Coumarin
0.5% Coumarin
Control
0.2% Coumarin
0.5% Coumarin
55.52.5 43.90.5 0.0550.007
59.84.2 39.23.3 0.0430.010
81.0 2.3*** 23.2 2.2*** 0.0470.017
60.0 1.9 37.7 2.6 0.149 0.089
66.24.9 32.75.9 0.1080.038
88.51.8*** 11.81.1*** 0.0460.011
0.970.03 0.540.03 0.0130.001 100.42.5
0.770.02** 0.320.02*** 0.0120.002 98.91.7
0.85 0.05* 0.25 0.02*** 0.0080.001* 99.8 1.3
0.79 0.12 0.31 0.03 0.027 0.003 100.6 2.6
0.880.09 0.260.03 0.0170.003* 105.10.8
0.930.06 0.320.03 0.0150.002** 101.30.9
c
a Rats were fed diets containing 0 (control), 0.2 and 0.5% coumarin for 24 weeks and were then given single oral doses of either 25 or 300 mg/kg [3-14C]coumarin. Urine and faeces were collected for 96 h prior to determination of tissue levels of radioactivity. b Radioactivity present after 96 h in the entire gastrointestinal tract and contents. c Results are presented as mean S.E. for groups of 4 animals. Values significantly different from control (0% coumarin diet) are: *P<0.05; **P< 0.01; ***P <0.001.
Table 3 Excretion and tissue distribution of [3-14C]coumarin after oral administration to male Syrian hamsters In
Percentage of administered radioactivity found after 96 ha 25 mg/kg [3-14C]coumarin
Urine Faeces Total gut and contentsb Liver Total Covalently bound Kidney Percentage recovery of administered dose
300 mg/kg [3-14C]coumarin
Control
0.5% Coumarin
1.0% Coumarin
Control
0.5% Coumarin
1.0% Coumarin
84.61.8c 13.31.7 0.0270.003
96.6 0.8** 6.20.5** 0.0320.004
96.9 3.9** 6.8 1.5** 0.1140.060
90.44.7 8.81.6 0.0480.013
93.44.0 5.80.8 0.0350.002
95.92.7 4.90.8* 0.0280.004
0.510.07 0.450.06 0.0130.001 98.41.8
0.16 0.02*** 0.15 0.01*** 0.0080.001 102.90.4
0.14 0.02*** 0.08 0.01*** 0.0110.004 103.93.6
0.340.03 0.330.33 0.0110.001 99.64.8
0.210.01 0.150.003*** 0.0110.003 99.44.3
0.300.07 0.140.02*** 0.0060.001 101.12.6
a Syrian hamsters were fed diets containing 0 (control), 0.5 and 1.0% coumarin for 24 weeks and were then given single oral doses of either 25 or 300 mg/kg [3-14C]coumarin. Urine and faeces were collected for 96 h prior to determination of tissue levels of radioactivity. b Radioactivity present after 96 h in the entire gastrointestinal tract and contents. c Results are presented as mean S.E. for groups of 4 animals. Values significantly different from control (0% coumarin diet) are: *P<0.05; **P< 0.01; ***P <0.001.
and 300 mg/kg [3-14C]coumarin was significantly different in rats fed a 0.5% coumarin diet for 24 weeks. The treatment of rats with 0.5% coumarin resulted in a significantly greater excretion of radioactivity in the urine and a significantly decreased excretion of radioactivity in the faeces after 24, 48, 72 and 96 h following oral administration of either 25 (Fig. 8A) or 300 (Fig. 8B) mg/kg [3-14C]coumarin. After 96 h, rats fed 0 (control), 0.2 and 0.5% coumarin diets and subsequently given an oral 25 mg/kg dose of [3-14C]coumarin excreted 55.5, 59.8 and 81.0% of the dose, respectively, in the urine and 43.9, 39.2 and 23.2% of the dose, respectively, in the faeces (Table 2). Corresponding values for rats fed 0 (control), 0.2 and 0.5% coumarin diets and subsequently given an oral 300 mg/kg dose of [3-14C]coumarin were 60.0, 66.2 and 88.5% of the dose excreted in
the urine, respectively, and 37.7, 32.7 and 11.8% of the dose excreted in the faeces, respectively (Table 2). Prior coumarin treatment had no significant effect on the amount of radioactivity present in the entire gastrointestinal tract and contents 96 h after oral administration of either 25 or 300 mg/kg [3-14C]coumarin (Table 2). Liver levels of coumarin were determined both as total radioactivity and as radioactivity covalently bound to liver proteins 96 h after administration of [3-14C]coumarin. Compared to control (0% coumarin diet) animals, treatment with 0.2 and 0.5% coumarin diets significantly reduced the levels of total and covalently bound radioactivity after oral administration of 25 mg/kg, but not 300 mg/kg, [3-14C]coumarin (Table 2). Kidney levels of radioactivity were also significantly reduced in rats fed a 0.5% coumarin diet and given 25 mg/kg [3-14C]coumarin
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but had no effect on levels of radioactivity present in the kidney and entire gastrointestinal tract and contents after 96 h (Table 3). The treatment of Syrian hamsters with 0.5 and 1.0% coumarin diets had little effect on the subsequent disposition of a single oral 300 mg/kg dose of [3-14C]coumarin (Fig. 9B and Table 3). While urinary excretion of radioactivity was somewhat increased, faecal excretion was decreased, this being statistically significant in Syrian hamsters fed a 1.0% coumarin diet (Table 3). Prior treatment with 0.5 and 1.0% coumarin diets also significantly decreased liver levels of covalently bound, but not total, radioactivity 96 h after a single oral 300 mg/kg dose of [3-14C]coumarin (Table 3).
4. Discussion
Fig. 8. The effect of treatment of rats for 24 weeks with control diet (*, *) or diets containing 0.2 (~, ~) and 0.5 (&, &) % coumarin on the disposition of [3-14C]coumarin. Rats were given single oral doses of either 25 (A) or 300 (B) mg/kg [3-14C]coumarin and radioactivity present in urine (*, ~, &) and faeces (*, ~, &) determined for up to 96 h. Results are presented as meanS.E. for groups of 4 animals.
and in animals fed 0.2 and 0.5% coumarin diets and given 300 mg/kg [3-14C]coumarin (Table 2). Coumarin disposition was significantly changed in Syrian hamsters fed 0.5 and 1.0% coumarin diets compared to controls (0% coumarin diet) prior to administration of a single oral dose of 25 mg/kg [3-14C] coumarin (Fig. 9A). Prior coumarin treatment resulted in a significantly greater excretion of radioactivity in the urine and a significantly decreased excretion of radioactivity in the faeces 24, 48, 72 and 96 h following an oral 25 mg/kg dose of [3-14C]coumarin (Fig. 9A). After 96 h, Syrian hamsters fed 0 (control), 0.5 and 1.0% coumarin diets and subsequently given an oral 25 mg/kg dose of [3-14C]coumarin excreted 84.6, 96.6 and 96.9% of the dose, respectively, in the urine and 13.3, 6.2 and 6.8% of the dose, respectively, in the faeces (Table 3). The treatment of Syrian hamsters with 0.5 and 1.0% coumarin diets also significantly decreased total and covalently bound liver levels of radioactivity after 96 h,
Previous studies have demonstrated that coumarin exhibits marked species differences in both metabolism and hepatotoxicity (Cohen, 1979; Fentem and Fry, 1993; Lake, 1999). The aim of the present study was to compare the disposition, metabolism and hepatotoxicity of coumarin in the rat and Syrian hamster. Previous studies have demonstrated that these two species appear to be susceptible and resistant, respectively, to coumarin-induced liver injury. Several studies have demonstrated that the acute administration of coumarin to rats results in centrilobular hepatic necrosis, whereas prolonged administration results in bile duct lesions (Hagan et al., 1967; Cohen, 1979, Lake, 1984; Evans et al., 1989; Lake et al., 1989a, 1994; Carlton et al., 1996; Lake and Grasso, 1996). In addition, the chronic administration of high doses of coumarin can result in the formation of both parenchymal cell tumours and cholangiocarcinoma (Carlton et al., 1996). Unlike the rat, the administration of coumarin to the Syrian hamster for 13 weeks has been reported not to result in any significant hepatotoxicity (Lake and Grasso, 1996) and after 2 years’ treatment with 0.1 and 0.5% coumarin diets, no liver lesions or liver tumours were observed (Ueno and Hirono, 1981). As observed in previous studies, the administration of high (e.g. 0.5% in the diet) doses of coumarin to the rat was shown to increase relative liver weight and produce histological evidence of hepatotoxicity including bile duct proliferation. These effects were less marked in rats treated with 0.2% coumarin and no morphological changes were observed in the Syrian hamster. In addition, coumarin treatment had no marked effect on relative liver weight in the Syrian hamster. Coumarin administration also resulted in a number of hepatic biochemical changes. For example, coumarin treatment produced a sustained reduction in total CYP content and CYP-dependent enzyme activities. In contrast, levels of GSH were enhanced in rats given 0.5% coumarin and at one or both of the two coumarin dose
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Fig. 9. The effect of treatment of Syrian hamsters for 24 weeks with control diet (*, *) or diets containing 0.5 (~, ~) and 1.0 (&, &) % coumarin on the disposition of [3-14C]coumarin. Syrian hamsters were given single oral doses of either 25 (A) or 300 (B) mg/kg [3-14C]coumarin and radioactivity present in urine (*, ~, &) and faeces (*, ~, &) determined for up to 96 h. Results are presented as mean S.E. for groups of 4 animals.
levels examined, hepatic UDP glucuronosyltransferase (towards 4-hydroxybiphenyl), g-glutamyltransferase and GSH S-transferase activities (towards both CDNB and ENPP as substrates) were significantly increased. The hepatic biochemical effects of 0.5% coumarin observed in this study after 12, 24 and 42 weeks of treatment are similar to previous studies of up to 13 weeks’ duration (Lake et al., 1994; Lake and Grasso, 1996) and one study of up to 18 months’ duration (Evans et al., 1989). Compared to the rat, the hepatic biochemical effects of coumarin treatment were less marked in the Syrian hamster. The only consistent change noted after 12, 24 and 42 weeks’ treatment with 0.5%, but not 0.2%, coumarin was an induction of GSH S-transferase activity towards ENPP as substrate. Overall, the hepatic biochemical effects of coumarin in the rat involve an inhibition of phase I CYP-dependent pathways of xenobiotic metabolism, together with
a stimulation of phase II pathways of xenobiotic metabolism. Previous studies have shown that coumarin can be metabolised in the rat to a number of hydroxycoumarins which can be conjugated with d-glucuronic acid and sulphate (Mead et al., 1958; Kaighen and Williams, 1961; Cohen, 1979; Lake, 1999). In addition, coumarin is known to enhance urinary mercapturic acid excretion in the rat and a mercapturic acid, derived from the conjugation of coumarin 3,4-epoxide with GSH, has been identified (Lake, 1984; Huwer et al., 1991). The hepatic biochemical changes in response to sustained coumarin treatment may be interpreted as an enhancement of pathways of coumarin detoxification and elimination. As shown in Fig. 1, coumarin is known to be converted to a coumarin 3,4-epoxide intermediate by CYP-dependent enzymes which can be conjugated with GSH by GSH S-transferase enzymes and subsequently converted by g-glutamyltransferase and other enzymes to a urinary mercapturic acid. In addition, the stimulation of UDPglucuronosyltransferase enzymes would favour the elimination of hydroxycoumarin metabolites. Investigations into the mechanisms of coumarininduced hepatotoxicity in the rat have demonstrated the importance of the 3,4-double bond in coumarin toxicity and the role of GSH in protecting against coumarininduced liver injury (reviewed in Lake, 1999). In vitro studies have demonstrated that coumarin is more toxic to rat hepatocytes than a number of coumarin metabolites, including various hydroxycoumarins and o-hydroxyphenylacetic acid (Lake et al., 1989a). One exception is o-hydroxyphenylacetaldehyde, which is more toxic to rat hepatocytes than either coumarin or 3-hydroxycoumarin (Born et al., 2000b), suggesting that this metabolite together with coumarin 3,4-epoxide may have a role in coumarin-induced toxicity (Fig. 1). While the metabolism of coumarin in the rat has been investigated in a number of studies (Cohen, 1979; Lake, 1999), the profile of coumarin metabolism in this species has yet to be fully elucidated. Moreover, the metabolism of coumarin in the Syrian hamster has been examined in only one in vivo and a few in vitro studies (Gangolli et al., 1974; Cohen, 1979; Lake et al., 1992a; Pearce et al., 1992; Lake, 1999). The aim of the limited metabolic studies conducted in these investigations was to obtain more information on the metabolism and disposition of coumarin in the Syrian hamster. In the present study the disposition and metabolism of [3-14C]coumarin was examined in untreated rats and Syrian hamsters given a single oral low dose of 25 mg/ kg [3-14C]coumarin. Radioactivity was rapidly excreted by both species, with the bulk of the administered dose being eliminated in either the urine or faeces within 24 h. However, while the urine was the major route of excretion of radioactivity in the Syrian hamster, some 38% of the administered dose was excreted after 96 h in
B.G. Lake et al. / Food and Chemical Toxicology 40 (2002) 809–823
the faeces by the rat. Additional studies with bile duct cannulated animals revealed that the biliary excretion of radioactivity was much greater in the rat than the Syrian hamster. These observations are in agreement with previous studies on the disposition of coumarin in the rat (Kaighen and Williams, 1961; van Sumere and Teuchy, 1971; Lake et al., 1989c). In addition, Williams et al. (1965) reported that following an oral or ip dose of 50 mg/kg of coumarin to rats, some 50% of the dose was excreted in the bile as unknown metabolites within 24 h. While coumarin metabolites may undergo extensive enterohepatic circulation in the rat, as with the rabbit, baboon and humans (Lake, 1999), the urine appears to be the major route of coumarin metabolite excretion in the Syrian hamster. The examination of 024-h hydrolysed urine samples from rats given a single oral 25 mg/kg dose of [3-14C] coumarin revealed the presence of small amounts of 3-, 5- and 7-hydroxycoumarin, together with larger quantities of o-hydroxyphenyllactic acid and o-hydroxyphenylacetic acid. Such urinary metabolites have also been detected in previous studies after the administration of coumarin to rats (Mead et al., 1958; Booth et al., 1959; Kaighen and Williams, 1961; van Sumere and Teuchy, 1971; Lake et al., 1989c). In agreement with the study of Kaighen and Williams (1961), the major identified urinary coumarin metabolite was o-hydroxyphenyl acetic acid, this metabolite comprising 19.0% of the administered dose in the present study and 19.4% (range 12.527.2%, n=3) of the dose following oral administration of a 100 mg/kg dose of [3-14C]coumarin in the study performed by Kaighen and Williams (1961). Coumarin metabolism to 7-hydroxycoumarin only accounted for 0.56% of the administered dose in the 024-h urine. This value is in agreement with other in vivo and many in vitro studies in confirming that the rat is a poor 7-hydroxylator of coumarin (Kaighen and Williams, 1961; van Sumere and Teuchy, 1971; Lake et al., 1989c; Lake, 1999). In the present study 1.3% of the dose was excreted as 7-hydroxycoumarin in the 024-h urine of Syrian hamsters following the administration of a single oral 25 mg/kg dose of [3-14C]coumarin to Syrian hamsters. This value is similar to the figure of 5% reported after an oral 200 mg/kg dose by Gangolli et al. (1974). Moreover, in the present study all of the coumarin metabolites detected in rat urine were also present in Syrian hamster urine (Table 1). These metabolites included ohydroxyphenylacetic acid, which is known to be an endpoint of coumarin metabolism by the 3,4-expoxidation pathway (Born et al., 1997; Lake, 1999). The present in vivo data, suggesting that the Syrian hamster is a poor 7-hydroxylator of coumarin, is also in agreement with results of in vitro studies. As with rat liver microsomes, the 3,4-epoxidation pathway of coumarin metabolism was found to predominate over the 7-
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hydroxylation pathway in Syrian hamster liver microsomes, with o-hydroxyphenylacetaldehyde being the major identified product formed by both species (Lake et al., 1992a). In another study, 7-hydroxycoumarin was found to account for only 0.3 and 9% of total coumarin metabolism in rat and Syrian hamster liver microsomes, respectively (Pearce et al., 1992). The effect of prior coumarin treatment on the disposition of oral 25 and 300 mg/kg (namely toxic and non-toxic doses in the rat, respectively) doses of [3-14C]coumarin was also investigated. In the rat, treatment with 0.5% coumarin enhanced the urinary excretion of radioactivity and reduced the faecal excretion of radioactivity after single oral doses of both 25 and 300 mg/kg [3-14C]coumarin. Prior coumarin treatment thus favoured the elimination of coumarin in the urine. Additional studies would be required to ascertain whether prior coumarin treatment reduces the biliary excretion and subsequent enterohepatic circulation of coumarin metabolites. Previous studies with hepatic microsomes have demonstrated that coumarin can be converted by CYP-dependent enzymes to metabolite(s) that bind covalently to liver proteins (Lake, 1984; Lake et al., 1992a). In the present study, covalent binding of radioactivity to liver proteins was observed after the administration of single oral doses of 25 and 300 mg/kg [3-14C]coumarin (Table 2). Prior coumarin treatment at dose levels of 0.2 and 0.5% significantly reduced liver levels of both total and covalently bound radioactivity following an oral dose of 25, but not 300, mg/kg [3-14C]coumarin. These results suggest that prior coumarin treatment favours coumarin metabolism to nontoxic products and more rapid elimination from the liver. Prior coumarin treatment at dietary levels of 0.5 and 1.0% also produced some changes in coumarin disposition in the Syrian hamster. These effects comprised the enhanced urinary excretion of radioactivity after an oral 25 mg/kg dose of [3-14C]coumarin and a reduction in liver levels of either total and/or covalently bound radioactivity (Table 3). The results of the present study, together with those of other investigations (Lake and Grasso, 1996), demonstrate a marked species difference in coumarininduced hepatotoxicity. While high doses of coumarin are clearly hepatotoxic in the rat, the Syrian hamster is resistant to coumarin-induced liver injury. From the limited metabolic studies performed, it is clear that, like the rat, the Syrian hamster is a poor 7-hydroxylator of coumarin. The present results are in agreement with those of previous in vivo and in vitro studies on the metabolism of coumarin in the Syrian hamster (Gangolli et al., 1974; Lake et al., 1992a; Pearce et al., 1992). However, while the metabolism of coumarin appears to favour the 3,4-epoxidation, rather than the 7-hydroxylation, pathway in both the rat and Syrian hamster,
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marked quantitative species differences were observed in coumarin disposition. In agreement with the majority of studies (reviewed in Lake, 1999), a significant proportion of an oral dose of coumarin to the rat was excreted in the faeces. As observed by Williams et al. (1965), a large amount of an oral dose of coumarin to the rat was excreted in the bile as unidentified metabolites. Unlike the rat, the urine was the predominant route of excretion of radioactivity following administration of oral doses of [3-14C]coumarin to the Syrian hamster. In addition, the biliary excretion of radioactivity was lower in the Syrian hamster than the rat, suggesting that enterohepatic circulation of coumarin metabolites may not occur to any great extent in this species. In summary, the present study demonstrates a marked species difference between the rat and Syrian hamster in coumarin-induced hepatotoxicity. Clearly, the resistance of the Syrian hamster to coumarininduced liver injury is not attributable to coumarin 7hydroxylation status. The observed species difference may be at least partially attributable to differences in coumarin disposition. However, additional studies are required to elucidate the pathways of coumarin metabolism in both species. Such studies should include the identification of coumarin metabolites present in the bile as well as in the urine and faeces.
Acknowledgements The authors wish to acknowledge the great contribution made by Dr. F.J.C. Roe to the science of toxicology. In addition, we are grateful to Dr. Roe and to Professors S.D. Gangolli and P. Grasso (two former members of BIBRA staff) for many valuable discussions on the metabolism and toxicity of coumarin.
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