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Metabolism of [3-14C]coumarin to polar and covalently bound products by hepatic microsomes from the rat, syrian hamster, gerbil and humans
Fd Chem. Toxic. Vol. 30, No. 2, pp. 105-115, 1992 Printed in Great Britain
0278-6915/92 $5.00 + 0.00 Pergamon Press pie
METABOLISM OF [3-t4C]COUMARIN TO POLAR A N D COVALENTLY B O U N D PRODUCTS BY HEPATIC MICROSOMES FROM THE RAT, SYRIAN HAMSTER, GERBIL A N D H U M A N S B. G. LAKE*,H. GAUDIN~',R. J. PRICE and D. G. WALTERS BIBRA Toxicology International, Woodmanstcrne Road, Carshalton, Surrey SM5 4DS, UK and tENSBANA, Universit6 de Bourgogne, Campus Universitaire Montmuzard, 21000 Dijon, France (Accepted 15 November 1991)
Abstract--The metabolism of 0.19 and 2.0 mM-[3J4C]coumarinto polar products and covalently bound metabolites has been studied with hepatic microcomes from the rat, Syrian hamster, Mongolian gerbil and humans. [3-t4C]Coumarinwas metabolized by liver microsomes from all species to a number of polar products and to metabolite(s) that became covalently bound to microsomal proteins. The polar products included 3-, 5- and 7-hydroxycoumarins, o-hydroxyphenylacetaldehyde and o-hydroxyphenylacetic acid. Coumarin 7-hydroxylation was observed in all species except the rat. With 0.19 mM-[3-14C]coumarin, 7-hydroxycoumarin was the major metabolite in human liver microsomes, whereas in the other species with 0.19 mM substrate and in all species with 2.0 mM substrate o-hydroxyphenylacetaldehyde was the major metabolite. Of the three animal species studied the gerbil most resembled humans as this species also had a high coumarin 7-hydroxylaseactivity. The administration of Aroclor 1254 to the rat and Syrian hamster induced both microsomal cytochrome P-450 content and [3-14C]coumarinmetabolism. With liver microsomes from all species a good correlation between rates of [3J4C]coumarin metabolism and covalent binding was observed at both substrate concentrations. However, in view of the known species difference between the rat and Syrian hamster in coumarin-induced hepatotoxicity, the present data are not consistent with microsomal coumarin metabolite covalent binding being an indicator of potential liver damage.
INTRODUCTION
Coumarin (1,2-benzopyrone, c i s - o - c o u m a r i n i c acid lactone) occurs naturally in several plant families and essential oils (Cohen, 1979; Soine, 1964). Although no longer used as a food flavouring, coumarin is present in certain tobaccos and alcoholic beverages and is used in various soap, detergent and cosmetic preparations (Cohen, 1979; Opdyke, 1974). Some coumarin derivatives are of economic importance; these include 3,4-dihydrocoumarin used mainly in the perfume industry and 6-methylcoumarin used as a flavour enhancer (Egan et al., 1990). The toxicity of coumarin merits attention because it has been shown to exhibit interspecies differences in both hepatotoxicity and metabolism (Cohen, 1979). In the rat, single doses of coumarin produce centrilobular hepatic necrosis (Lake, 1984; Lake et al., 1989b), whereas chronic administration results in bile duct lesions (Cohen, 1979; Evans et al., 1989; Hagan et al., 1967). However, no significant hepatotoxic effects were observed in chronic feeding studies con*To whom correspondence should be addressed. Abbreviations: DHC = dihydrocoumarin;3°HC = 3-hydroxy-
coumarin; 5-HC= 5-hydroxycoumarin; 7-HC = 7hydroxycoumarin; o-HPA = o-hydroxyphenylacetaldehyde; o-HPAA = o-hydroxyphenylaceticacid; o-HPPA = o -hydroxyphenylpropionicacid. FCT 30i2 B
ducted either in the Syrian hamster (Lake et al., 1990; Ueno and Hirono, 1981) or baboon (Evans et al., 1979). Studies on the metabolism of coumarin have demonstrated that in humans (Egan et al., 1990; Shilling et al., 1969) and in the baboon (Gangolli et al., 1974; Waller and Chasseaud, 1981) coumarin is extensively metabolized to 7-hydroxycoumarin (Fig. 1). In contrast, 7-hydroxylation represents only a minor pathway in several other species including the rat, most strains of mouse, Syrian hamster, dog, rabbit and marmoset (Cohen, 1979; Gangolli et al., 1974; Kaighen and Williams, 1961; Lake et al., 1989a). The major metabolic pathway of coumarin biotransformation in the rat appears to involve an initial 3-hydroxylation reaction with subsequent ring opening and further metabolism to o-hydroxyphenylacetic acid (Cohen, 1979; Kaighen and Williams, 1961). Studies with rat hepatic microsomes have demonstrated that coumarin is metabolized by cytochrome P-450-dependent mixed-function oxidase enzymes to various polar products and to metabolite(s) that bind covalently to microsomal proteins (Feuer, 1970; Gibbs et al., 1971; Lake, 1984; Lake et al., 1992; Peters et al., 1991; Walters et al., 1980). In agreement with in vivo findings, coumarin 7-hydroxylase activity is either low or absent in rat liver microsomes, but
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Fig. 1, Some pathways of coumarin metabolism (Cohen, 1979; Lake et al., 1992: Norman and Wood, 1984). The coumarin metabolites shown are 3-, 4-, 5-. 6-, 7- and 8-hydroxycoumarins(3-HC, 4-HC. 5-HC, 6-HC, 7-HC and 8-HC), 6,7-dihydroxycoumarin(6,7-DiHC), dihydrocoumarin (DHC), o-coumaric acid (o-CA), o-hydroxyphenyllactic acid (o-HPLA/ o-hydroxyphenylpropionic acid (o-HPPA), u-hydroxyphenylacetaldehyde (o-HPA), o-hydroxyphenylethanol (o-HPE) and o-hydroxyphenylacetic acid (o-HPAA).
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[3-~4C]Coumarin metabolism in different species can be readily detected in gerbil and human liver microsomes (Dominguez et al., 1990; Feuer, 1970; Gibbs et al., 1971; Lake et al., 1989a; Miles et al., 1990; Pelkonen et al., 1985; Peters et al., 1991; Raunio et al., 1988). Recent studies have shown that a member of the cytochrome P4501IA subfamily is responsible for the majority of coumarin 7-hydroxylase activity in human liver microsomes (Miles et al., 1990), whereas in rat liver microsomes coumarin is a substrate for isoenzymes in both the cytochrome P450IA and P450IIB subfamilies (Peters et al., 1991). The aim of the present investigations was to obtain further data on species differences in coumarin metabolism by studying the metabolism of [3-~4C]coumarin in liver microsomes from the rat, Syrian hamster, Mongolian gerbil and humans. The gerbil was selected because like humans it is known to metabolize coumarin to 7-hydroxycoumarin (Dominguez et al., 1990), whereas the rat and Syrian hamster were selected because of the known species difference in both acute (B. G. Lake and J. G. Evans, unpublished observations) and chronic (Lake et al., 1990; Ueno and Hirono, 1981) coumarin-induced hepatotoxicity. With the rat and Syrian hamster, the effect of Aroclor 1254 treatment on hepatic microsomal [3-~4C]coumarin metabolism was also studied. This polychlorinated biphenyl mixture, which induces several cytochrome P-450 isoenzymes (Ryan et al., 1979) is known to induce markedly [3-z4C]coumarin metabolism in rat liver microsomes (Peters et al., 1991). MATERIALS
AND METHODS
Chemicals. [3-14C]Coumarin (sp. act. 5.3mCi/ mmol) was obtained 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 toluene-chloroform (1:1, v/v). Unlabelled coumarin and coumarin metabolites were obtained from the sources described previously (Lake eta!., 1989b). Enzymes, cofactors etc. were purchased from Sigma Chemical Co. Ltd (Poole, Dorset, UK) and HPLCgrade methanol and tetrahydrofuran (unstabilized) from Rathburn Chemicals (Walkerburn, UK). Animals and treatment. Male Sprague-Dawley rats were obtained from Harlan Olac (Bicester, Oxon, UK) and male Golden Syrian hamsters from Bantin and Kingman (The Field Station, Grimston, Aldbrough, Hull, UK) and allowed free access to R and M No. 1 and No. 3 diets (Special Diets Services, Witham, Essex, UK), respectively, and water. The animals were housed in accommodation maintained at 22_+ Y'C with a relative humidity of 40-70% and were allowed to acclimatize to these conditions for at least 7 days before use. Rats and hamsters, both 6 wk old, were given five daily ip doses of 100mg Aroclor 1254/kg body weight. Control
107
animals received corresponding quantities (5 ml/kg body weight) of the corn-oil vehicle. Animals were killed by cervical dislocation. Livers from untreated male Mongolian gerbils (approximately 50g body weight) were kindly provided by Dr A. G. Smith (MRC Toxicology Unit, Carshalton, Surrey, UK). Samples of human liver (seven subjects, five male aged 10, 20, 23, 25 and 64 yr and two female aged 16 and 38 yr) were obtained from various sources and stored at - 8 0 ° C until use. Preparation o f microsomes. Whole liver homogenates (0.25 g fresh tissue/ml) were prepared in ice-cold 0.154M-KC1, containing 50mM-Tris-HC1 pH 7.4, using a Potter-type, teflon-glass, motor-driven homogenizer (A. H. Thomas Co., Philadelphia, PA, USA). The homogenates were centrifuged at 10,000 g av. for 20 min to obtain the postmitochondrial supernatants and subsequently at 158,000g av. for 40 min to separate the microsomal fraction from the cytosol. The pellets were resuspended in fresh homogenizing medium and again centrifuged at 158,000g av. for 40 rain. Washed microsomal fractions were stored at - 8 0 ° C for up to 6 wk until use. Assays. Microsomes were assayed for content of cytochrome P-450 (Omura and Sato, 1964) and protein (Lowry et al., 1951). The activity of 7-ethoxycoumarin O-deethylase was determined as described previously (Lake, 1987). For studies of [ 3 - 1 4 C ] coumarin metabolism standard incubation mixtures contained 2 m g microsomal protein, either 0.19 or 2.0mM-[3-14C]coumarin (1/~Ci/tube, added in 5/~1 dimethylsulphoxide), 1 mN-NADPH, 5 mM-MgSO4 and 0.1 mM-phosphate buffer pH 7.6 in a final volume of 1 ml. After 2min preincubation at 3TC in a shaking water-bath, the reaction was started by adding NADPH. Blank tubes were incubated with [3-14C]coumarin but in the absence of NADPH. After 10 rain, incubations were terminated with l ml icecold methanol. Following centrifugation for 15 min at 1200g at 43C, the supernatant was taken for H P L C analysis of [3-14C]coumarin and [3-~4C]coumarin polar metabolites (Peters et al., 1991) and the pellet for determination of covalent binding of [3-~4C]coumarin metabolites to microsomal proteins. Polar [3-~4C]coumarin metabolites were identified by comparison with the retention times of known standards and quantified by measurement of the radioactivity present in the appropriate HPLC fractions, whereas values for total polar products were calculated from the radioactivity present in all HPLC fractions excluding unmetabolized [3-~4C]coumarin substrate. Macromolecular binding of [ 3 - 1 4 C ] coumarin metabolites was determined by exhaustive solvent extraction with 5% (w/v) trichloroacetic acid and 80% (v/v) methanol in water as described previously (Lake, 1984; Lake et al., 1981). Statistical analysis. Statistical evaluation of data was performed by one-way analysis of variance. Comparisons between means were made using the least significant difference test.
B. G. LAKE et al.
108
Table 1. Cytochrome P-450 content and 7-ethoxycoumarin O-deethylase activity in hepatic microsomes from various species 7-Ethoxycoumarin Cytochrome P-450 O-deethylase Species (nmol/mg p r o t e i n ) (nmol/min/mg protein) Rat Control (5) Aroclor 1254 treated (5)
1.05 ± 0.07 3.18 ± 0.09***
1.44 ± 0.11 37.3 ± 3.1'**
Syrian hamster Control (5) Aroclor 1254 treated (5)
1.22 ± 0.05 2.83 ± 0.14"**
7.45t 17.4 _+ 0.3
Gerbil (4)
0.80 ± 0.03
4.99 ± 0.24
H u m a n (7)
0.31 ± 0.02
0.32 +_ 0.03
fValue shown is for a single determination only. Values are means ± SEM with number of samples shown in parentheses. For Aroclor 1254treated rats and hamsters values significantly different from corresponding controls are: ***P < 0.001. RESULTS
Microsomal enzyme activities
Levels o f c y t o c h r o m e P-450 and 7-ethoxycoumarin O-deethylase in hepatic microsomes from the various species are shown in Table 1. Aroclor 1254 treatment induced both cytochrome P-450 content and mixedfanction oxidase enzyme activity in both the rat and Syrian hamster, with a more marked effect being observed in the rat. Cytochrome P-450 content and 7-ethoxycoumarin O-deethylase activity in human liver microsomes were only 25-39% and 4-22%, respectively, of levels found in the other three species (i.e. in non-Aroclor 1254-treated animals). Metabolism
q[" [3- J4C]coumarin
An initial study was performed with control and Aroclor 1254 rat liver microsomes over a coumarin substrate concentration range of 0.34 to 2.6mM. [3-14C]Coumarin metabolism to total polar products and covalently bound metabolites is shown in Fig. 2. Pretreatment with Aroclor 1254 markedly enhanced the rate of [3-14C]coumarin metabolism to polar products and covalently bound metabolites at all substrate concentrations examined. A double reciprocal plot of [3J4C]coumarin metabolism to polar products against coumarin substrate concentration yielded apparent affinities (K m values) of 3.01 and 0.34 mM and maximal velocities (Vmax) of 6.84 and 23.5 nmol/min/mg protein for microsomes from control and Aroclor 1254-treated rats, respectively (data not shown). Corresponding kinetic constants for covalently bound metabolites were 1.40 and 0.23 mM, and 156 and 910 pmol/min/mg protein, respectively. Coumarin substrate concentrations of 0.19 and 2.0 mM were selected for all subsequent studies, the former representing the minimum amount of radioactivity that could be added, the latter representing a near saturating substrate concentration for rat liver microsomes. In hepatic microsomes from all species [3J4C]coumarin was metabolized to both polar products (Fig. 3A) and to metabolite(s) that became covalently bound to microsomal proteins (Fig. 3B). With all
microsomal preparations, the rate of [3-t4C]coumarin metabolism to total polar products and covalently bound metabolites was greater at 2.0 than 0.19 mM[3J4C]coumarin substrate concentration. At the 0.19 mM-[3J4C]coumarin substrate concentration and with microsomes from control (i.e. non-Aroclor 1254-treated) animals, the highest rates of metabolism to polar products and covalent binding were observed for the gerbil and the least with the rat and humans. For 2.0 mM-coumarin, the highest and 24
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Fig. 2. Effect of substrate concentration on [3-~4C]coumarin metabolism to total polar products (A) and covalently bound metabolite(s) (B) by hepatic microsomes from control (A) and Aroclor 1254-treated (&) rats. Each point represents the mean of duplicate incubations and the results of a typical experiment are shown.
[3J4C]Coumarin metabolism in different species
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Fig. 3. [3J4C]Coumarin metabolism to total polar products (A) and covalently bound metabolites (B) by hepatic microsomes from various species. Results are expressed as means +_ SEM for groups of 4-7 control (RC) and Aroclor 1254-treated (RA) rat, control (HC) and Aroclor 1254treated (HA) Syrian hamster, gerbil (G) and human (HU) liver microsomes. For Aroclor 1254-treated rats and hamsters values significantly different from corresponding controls are: **P < 0.0l; ***P < 0.001.
lowest rates of metabolism and covalent binding were observed with gerbil and human microsomes, respectively. Treatment with Aroclor 1254 significantly induced [3J4C]coumarin metabolism and covalent binding at both substrate concentrations in the rat and Syrian hamster. Induction was more marked in the rat liver microsomes than in the hamster, particularly at the lower coumarin substrate concentration. Rates of covalent binding expressed as a percentage of the metabolism of 0.19 mM-[3-14C]coumarin to total polar metabolites were 5.5, 4.6, 4.0, 5.4, 2.6 and 1.5% for control and Aroclor 1254-treated rat, control and Aroclor 1254-treated Syrian hamster, gerbil and human liver microsomes, respectively. Corresponding values for a 2 mM-[3J4C]coumarin substrate concentration were 1.8, 3.7, 1.9, 1.9, 2.1 and 1.6%, respectively. Linear regression analysis of the data for all species and treatments revealed good correlations between [3J4C]coumarin metabolism to polar products and covalent binding at both the 0.19mM (r = 0.948; P < 0.001) and 2.00 mM (r = 0.894; P < 0.001) substrate concentrations (Fig. 4, A and B).
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Fig. 4. Relationship between metabolism of 0.19ram (A) and 2.0 mM (B) [3J4C]coumarin to total polar products and covalently bound metabolite(s). Points represent individual control (•) and Aroclor 1254-treated (A) rat, control (O) and Aroclor 1254-treated ( 0 ) Syrian hamster, gerbil ( I ) and human (,~') liver microsomes. Note that for the human liver samples covalent binding was determined for seven and four subjects at [3-14C]coumarin substrate concentrations of 0.19 and 2.0raM, respectively.
The H P L C system used (Lake et al,, 1992; Peters et al., 1991) could separate coumarin from 14 of its known metabolites (Fig. 1). Coumarin metabolites were identified by comparison with the retention times of known standards and quantified by measurement of the radioactivity present in the appropriate fractions (Table 2). Typical metabolite profiles for each source of hepatic microsomes incubated with 0.19- and 2.0 mM-[3-~4C]coumarin are shown in Figs 5 and 6, respectively. With the exception of human liver microsomes incubated with 0.19 mM-[3J4C]coumarin (Fig. 5), the major metabolite identified was ohydroxyphenylacetaldehyde (o-HPA). The formation of o - H P A was greater at the higher than the lower substrate concentrations in all species and was induced by Aroclor 1254 administration in the rat and Syrian hamster. The formation of 7-hydroxycoumarin (7-HC) was not observed in either control or Aroclor 1254-treated rat liver microsomes, whereas this metabolite was present in control and Aroclor 1254-treated Syrian hamster microsomes and
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in gerbil and human liver microsomes (Figs 5 and 6: Table 2). Although the formation of 7-HC was increased at the higher substrate concentration in Syrian hamster liver microsomes, only a small increase was observed in gerbil liver microsomes and no increase was observed in human liver microsomes (Table 2). [3-~4C]Coumarin was also metabolized in rat liver microsomes to o-hydroxyphenylacetic acid ( o - H P A A ) and to 3- and/or 5-hydroxycoumarin (3-HC/5-HC) and with 2.0 mM substrate to dihydrocoumarin and/or o-hydroxyphenylpropionic acid ( D H C / o - H P P A ) . These two sets of coumarin metabolites were quantified as pairs as the individual metabolites were not separated from one another by the H P L C system used in these studies. The formation of o - H P A A and 3-HC/5-HC was induced by Aroclor 1254 treatment, The formation of 3-HC/5HC and induction by the polychlorinated biphenyl mixture was also observed in hamster liver microsomes at both substrate concentrations (Table 2). Apart t¥om o - H P A and 7-HC, the Formation of 3-HC/5-HC and D H C / o - H P P A was observed in gerbil liver microsomes, whereas the formation of o - H P A A and o - H P E was observed in human liver microsomes. With human liver microsomes only the formation of o - H P A was greatly increased at the higher [3-~4C]coumarin substrate concentration (Table 2).
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The purpose of this in ritro study was to obtain more information on species differences in hepatic coumarin metabolism. In keeping with previous studies, levels of cytochrome P-450 in rat, Syrian hamster and gerbil liver microsomes were higher than those found in human liver (Dominguez et al., 1990: Sesardic et al., 1990:Souhaili-E1 Amri e t a / . , 1986). The cytochrome P-450 levels of 0.80 and 0.31 nmol/mg protein found in gerbil and human liver microsomes, respectively, are in agreement with values reported in other studies (Dominguez et al,. 1990: Sesardic et al., 1990:Souhaili-E1 Amri et ell.. 1986). Both qualitative and quantitative differences in [3-~4C]coumarin metabolism to polar products by liver microsomes from the four species were observed in this study. For example, coumarin metabolism to 7-hydroxycoumarin was observed in the Syrian hamster, gerbil, and human microsomes but not with rat liver microsomes. Previous studies have shown that rat liver microsomes contain either no or very low levels of coumarin 7-hydroxylase activity unless very high substrate concentrations arc used (Feuer, 1970: Gibbs et al.. 1971: Lake et al., 1989a: Pelkonen et al., 1985; Raunio et al., 1988). The observation that Syrian hamster liver microsomes contain detectable coumarin 7-hydroxylase activity is in agreement with the results of Gangol[i et al. (1974), who observed that 5% of an orally administered dose of coumarin was excreted in the urine as 7-hydroxycoumarin,
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Fig. 5. Typical profiles for the metabolism of 0.19 mM-[3J4C]coumarin to polar products by liver microsomes from various species. Coumarin (C) was separated from the coumarin metabolites (for abbreviations see Fig. 1) by HPLC.
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Fig 6. Typical preiiles For the metabolism of 2.0 mM-[3-~a('lcoumarm to polar products b~ li~er microsomes l'rom ~arious species. ('oumarin ((') ,.,,as separated from the coumarin metabolites d'or abbreviations see Fig, 1) by HPLC.
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45000
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[3-~4C]Coumarin metabolism in different species Previous studies on microsomal coumarin 7hydroxylase have used a spectrofluorimetric assay for determining enzyme activity, as against the radiometric assay used in the present study. However, the enzyme activities observed in this study were similar to those obtained in some previous studies. For example, in this study with human liver microsomes, enzyme activities of 0.26 and 0.28 nmol/min/mg protein were obtained at coumarin substrate concentration of 0.19 and 2.0mM, respectively. Previous studies have reported values of 0.33, 0.31 and 0.08 nmol/min/mg protein at coumarin substrate concentrations of 0.1, 0.1 and 5 mM, respectively (Kratz, 1976; Raunio et al., 1988; Sesardic et al., 1990). It should be noted that a considerable inter-individual variation in human liver coumarin 7-hydroxylase activity has been reported (Kratz, 1976; Miles et al., 1990; Pelkonen et al., 1985). Liver microsomes from all species formed o-HPA. which has been recently identified as a novel coumarin metabolite in rat liver microsomes (Lake et al., 1992). This was the major metabolite in rat, Syrian hamster and gerbil liver microsomes. With human liver microsomes, 7-HC was the major metabolite at the 0.19 mM substrate concentration, whereas o-HPA was the major metabolite with 2 mg substrate. As coumarin 7-hydroxylase activity was not increased at the higher substrate concentration in human liver microsomes, the 7-hydroxylation pathway appears to be saturated at lower substrate concentrations than do other pathways (e.g. o-HPA formation) of coumarin metabolism. Thus, at low coumarin doses, the isoenzymes ofcytochrome P-450 that metabolize coumarin in human liver microsomes (Miles et al., 1990; Pelkonen et al., 1985; Raunio et al., 1988) produce mainly 7-hydroxycoumarin, which accounts for this being the major metabolite observed in vi~,o in humans (Egan et al., 1990; Shilling et al., 1969). Gerbil liver microsomes appear to be similar to human liver rnicrosomes in this respect as there was very little increase in coumarin 7-hydroxylase activity at the 2.0 mM compared with the 0.19 mg substrate concentration (Table 2). This study demonstrates that several pathways of coumarin metabolism exist in liver microsomes from all four species studied. In the rat, Kaighen and Williams (1961) demonstrated that o-HPAA was produced by further metabolism of 3-HC (Fig. 1), whereas Norman and Wood (1984) have suggested that both o-HPE and o-HPAA were likely to be formed from an intermediate other than 3-HC. With respect to o-HPA, the major coumarin metabolite in all species at high substrate concentration, it is not clear whether this is derived from 3-HC or other intermediate(s) (Lake et al., 1992). Further research is thus required to elucidate all the pathways of coumarin metabolism in liver microsomes from the rat, humans and other species. A major focus of this study was the comparison of the rat and Syrian hamster, as the former species is
113
susceptible and the latter species resistant to both acute (B. G. Lake and J. G. Evans, unpublished observations) and chronic (Lake et al., 1990~ Ueno and Hirono, 1981) coumarin-induced liver injury. In agreement with our previous observations (Peters et al., 1991), Aroclor 1254 induced [3-~4C]coumarin metabolism to both polar products and covalently bound metabolites in rat liver microsomes. A kinetic study with rat liver microsomes revealed changes in both Km and Vmax values after Aroclor 1254 treatment. The change in K mpresumably reflects the change in microsomal cytochrome P - 4 5 0 isoenzyme composition produced by Aroclor 1254 induction (Peters et al., 1991; Ryan et a l , 1979). Aroclor 1254 also induced [3-~4C]coumarin metabolism in Syrian hamster liver microsomes but the effect was less marked than in the rat. With both the rat and Syrian hamster the pattern of [3-~4C]coumarin metabolites after Aroclor 1254 treatment was similar to that of control microsomes. The major difference between these two species observed in this study was that the Syrian hamster, unlike the rat, had detectable coumarin 7-hydroxylase activity. Although it has been suggested that coumarin metabolism to 7-HC represents a detoxification pathway (Cohen, 1979), the comparatively small difference in 7-HC [brmation between these two species, either in this study in t,itro or in eiro (Gangolli et al., 1974), is unlikely to explain the known species difference in susceptibility to coumarin-induced hepatotoxicity. Previous studies with rat liver microsomes and isolated hepatocytes have shown that coumarin is metabolized by cytochrome P-450 dependent enzymes to metabolite(s) that bind covalently to proteins (den Besten et aL, 1990; Lake, 1984; Peters et al., 1991). The covalent binding of reactive coumarin metabolites could represent a possible mechanism of hepatotoxicity of this compound (den Besten et al., 1990; Lake, 1984). In this study with microsomes from control and Aroclor 1254-treated species, good correlations between [3-J4C]coumarin metabolism and covalent binding were observed at both substrate concentrations (Fig. 4, A and B). If coumarin metabolite covalent binding was related to subsequent toxicity then the lowest rates of covalent binding were observed with human liver microsomes (Fig. 3B). However, because of the known species difference between the rat and Syrian hamster in coumarin-induced liver injury, the present data are not consistent with microsomal coumarin metabolite binding being an indicator of potential hepatotoxicity. For example, with 0.19 mM-[3-~C]coumarin the rate of covalent binding was greater in Syrian hamster than in rat hepatic microsomes, whereas at 2.0 mM substrate concentrate the rate of covalent binding was similar in both species (Fig. 3B). In summary, [3-~4C]coumarin was metabolized by hepatic microsomes from the four species studied. Of the three animal species, the Mongolian gerbil most resembled humans, as this species also had high
B. G. LAKE et al.
114
c o u m a r i n 7 - h y d r o x y l a s e activity. A d d i t i o n a l investig a t i o n s w o u l d be r e q u i r e d to e s t a b l i s h w h e t h e r t h e gerbil w o u l d be a useful a n i m a l m o d e l for f u r t h e r studies on coumarin metabolism and hepatotoxicity, to assist in t h e e v a l u a t i o n o f the r e l e v a n c e o f e x i s t i n g rat toxicity d a t a for h u m a n h a z a r d a s s e s s m e n t . F r o m t h e p r e s e n t s t u d i e s w i t h the rat a n d S y r i a n hamster, the measurement of the covalent binding of reactive c o u m a r i n m e t a b o l i t e ( s ) to m i c r o s o m a l p r o teins d o e s n o t a p p e a r to c o r r e l a t e with s u b s e q u e n t hepatotoxicity.
Acknowledgements We are grateful to Dr S. D. Gangolli for support and encouragement and to Dr A. R. Boobis ( H u m a n Tissue Bank, Royal Postgraduate Hospital Medical School, London) and Dr J. M. Tredger (The Liver Unit, Kings College Hospital School of Medicine and Dentistry, London) for supplying h u m a n liver samples. This work torms part of a research project sponsored by the U K Ministry of Agriculture, Fisheries and Food. to whom our thanks are due. The results of the research are the property of the Ministry of Agriculture, Fisheries and Food and are Crown Copyright. REFERENCES
den Besten C., K6r6si S. A., Beamand J. A.+ Waiters D. G. and Lake B. G. (1990) Studies on the mechanism of coumarin-indueed toxicity in rat hepatocytes. Toxicology in Vitro 4, 518- 521. 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. Dominguez K., Fentem J. H., Garle M. J. and Fry J. R. (1990) Comparison of Mongolian gerbil and rat hepatic microsomal monooxygenase activities: high coumarin 7-hydroxylase activity in the gerbil. Biochemical Pharmacology 39, 1629 1631. Egan D , O ' K e n n e d y R.. Moran E., Cox D., Prosser E. and Thornes R D. (1990) The pharmacology, metabolism, analysis and applications of coumarin and coumarin-related compounds. Drug Metabolism Reviews 22, 503 529. Evans J. G., Appleby E. C., Lake B. G. and Conning D. M. (1989) Studies on the induction of cholangiofibrosis by coumarin in the rat. To.vieology 55, 207 224. Evans J. G., (}aunt 1. F. and Lake B. G. (1979) Two-year toxicity study on coumarin in the baboon. Food and Chemical Toxicology 17, 187 193. Feuer G. (1970) 3-ttydroxylation of coumarin or 4-methylcoumarin by rat-liver microsomes and its induction by 4-methylcoumarin given orally. Chemieo-Biologieal luteracli(ms 2, 203 216. Gangolli S. D.. Shilling W. H., Grasso P. and G a u n t I. F. (1974) Studies on the metabolism and hepatotoxicity of coumarin in the baboon. Biochemical Society Transactions 2, 310 312. Gibbs P. A., Janakidevi K. and Feuer G. ( 1971 ) Metabolism of coumarin and 4-methylcoumarin by rat-liver microsomes. Canadian Journal qf Biochemisto, 49, 177 184. Hagan E. C+. Hansen W. H., Fitzhugh O. G., Jenner P. M., Jones W. I., Taylor J. M., Long E. L., Nelson A. A. and Brouwer J. B. (1967) Food flavourings and compounds of related structure. II. Subacute and chronic toxicity. Food and Chemical Toxicology 5, 141 157. Kaighen M. and Williams R. T. (19611 The metabolism of [3-~4C]coumarin. Journal of Medicinal Chemistry 3, 25M.3. Kratz F. (1976) Coumarin 7-hydroxylase activity in microsomes from needle biopsies of normal and diseased
h u m a n liver. European Journal of Clinical Pharmacology 10, 131 137. Lake B. G. (1984) Investigations into the mechanism of coumarin-induced hepatotoxicity in the rat. 4rchives Ol Toxicology (Suppl. 7), 16 29. Lake B. G. (1987) Preparation and characterisation of microsomal fractions for studies of xenobiotic metabolism. In Biochemical To.vicology: A Praelical Approach Edited by K. Snell and B. Mullock. pp. 183 215. IRL Press, Oxford. Lake B. G., Evans J. G., Waiters D. G. and Price R. J (I990) Studies on the hepatotoxicity and metabolism of coumarin in the rat and Syrian h a m s t e r ttuman ami Experimental Toxicology 9, 356. Lake B. G., Gray T. J. B., Evans J. G., Lewis D. |~. V., Beamand J. A. and Hue K. t_.. (1989b) Studies on the mechanism of coumarin-induced toxicity in rat hepatocytes: comparison with dihydrocoumarin and other coumarin metabolites. 7'r;.vicologv and .4pplied Pharmacology 97, 311 323. Lake B. G., Harris R. A., Phillips J. C. and Gangolli S. D. (1981) Studies on the effects of t.-ascorbic acid on acetaminophen-induced hepatotoxicity. I. Inhibition of the covalent binding of acetaminophen to hepatic microsomes in vitro. Toxieology and Applied Pharmacology 60, 229 240. Lake B. G., Osborne D. J., Waiters D. G. and Price R. 3. (1992) Identification of o-hydroxyphenylacetaldehyde as a major metabolite of coumarin m rat hepatic microsomes. Food and Chemical Toxicology 30, 99 104. Lake B. G., Waiters D. G. and Gangolli S. D. (1989a~ Comparison of the metabolism and disposition of [3-~C] coumarin in the rat and marmoset (Callithrir /a~chz+.~i Toxicology Letter.s 45, 299 306. Lowry O. H., Rosebrough N. J.. t:arr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193, 265 275 Miles J. S.+ McLaren A. W., Forrester L. M., Glancey M J . Lang M. A. and Wolf C. R. (1990) Identification of the h u m a n liver cytochrome P-450 responsible [\~r coumarin 7-hydroxylase activity. Biochemical Jaurmzl 267, 365 371. N o r m a n R. L. and Wood A. W. (1984) o-Hydruxyphenylethanol, a novel lactone ring-opened metabolite of coumarin. Drug MetabolLs'm and Disposition 12, 543 54t1. Omura T. and Sato R. (l 964) The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. Journal o[ Biolo2ical Chemistry 239, 2370 2378. Opdyke D. L. J. (1974) Monographs ol fragrance raw materials: coumarin. Food and Chemical 1)?.\'i~oloev 12, 385 388. Pelkonen O., Sotaniemi E. A. and Ahokas J. T (1985} Coumarin 7-hydroxylase activit~ in h u m a n liver micro+ somes. Properties of the enzyme and interspecies comparisons. British Journal (71 Clinical Pharmacologt 19, 59 66. Peters M. M. C. G., Waiters D. G.. Van O m m e n B.. Van Bladeren P. J. and Lake B. G. (1991) Effect of inducers of cytochrome P-450 on the metabolism of [3-HC] coumarin b3 rat hepatic microsomes. ,~'enobioti(a 21, 499 514. Raunio H., Syngelm'a T., Pasanen M., Juvonen R , Honkakoski P., Kairaluoma M. A., Sotaniemi E., Lang M. A. and Pelkonen O. (1988) lmmunochemical and catalytical studies on hepatic coumarin 7-hydroxylasc in man, rat and mouse. Biochemical Pharnzac~doev 37, 3889 3895. Ryan D. E., T h o m a s P. E., Korzeniowski D. and Lcvtn W. (1979) Separation and characterization of highly purified forms of liver microsomal cytochrome P-450 from rats treated with polychlorinated biphenyls, phenobarbital, and 3-methylcholanthrene. Journal O! Biological Chemistry 254, 1365 1374.
[3-~4C]Coumarin metabolism in different species Sesardic D., Boobis A. R., Murray B. P., Murray S., Segura J., De La Torre R. and Davies D. S. (1990) Furafylline is a potent and selective inhibitor of cytochrome P450IA2 in man. British Journal of Clinical Pharmacology 29, 651~63. Shilling W. H., Crampton R. F. and Longland R. C. (1969) Metabolism of cournarin in man. Nature, London 221, 664~565. Soine T. O. (1964) Naturally occurring coumarins and related physiological activities. Journal of Pharmaceutical Sciences 53, 231. 264. Souhaili-El Amri H., Batt A. M. and Siest G. (1986) Comparison of cytocbrome P-450 content and activities
115
in liver microsomes of seven animal species including man. Xenobiotica 16, 351-358. Ueno I. and Hirono I. (1981) Non-carcinogenic response to coumarin in Syrian golden hamsters. Food and Chemical Toxicology 19, 353-355. Waller A. R. and Chasseaud L. F. (1981) The metabolic fate of [14C]coumarin in baboons. Food and Chemical Toxicology 19, 1~. Waiters D. G., Lake B. G. and Cottrell R. C. (1980) High-performance liquid chromatography of coumarin and its metabolites. Journal of Chromatography 196, 501-505.
0278-6915/92 $5.00 + 0.00 Pergamon Press pie
METABOLISM OF [3-t4C]COUMARIN TO POLAR A N D COVALENTLY B O U N D PRODUCTS BY HEPATIC MICROSOMES FROM THE RAT, SYRIAN HAMSTER, GERBIL A N D H U M A N S B. G. LAKE*,H. GAUDIN~',R. J. PRICE and D. G. WALTERS BIBRA Toxicology International, Woodmanstcrne Road, Carshalton, Surrey SM5 4DS, UK and tENSBANA, Universit6 de Bourgogne, Campus Universitaire Montmuzard, 21000 Dijon, France (Accepted 15 November 1991)
Abstract--The metabolism of 0.19 and 2.0 mM-[3J4C]coumarinto polar products and covalently bound metabolites has been studied with hepatic microcomes from the rat, Syrian hamster, Mongolian gerbil and humans. [3-t4C]Coumarinwas metabolized by liver microsomes from all species to a number of polar products and to metabolite(s) that became covalently bound to microsomal proteins. The polar products included 3-, 5- and 7-hydroxycoumarins, o-hydroxyphenylacetaldehyde and o-hydroxyphenylacetic acid. Coumarin 7-hydroxylation was observed in all species except the rat. With 0.19 mM-[3-14C]coumarin, 7-hydroxycoumarin was the major metabolite in human liver microsomes, whereas in the other species with 0.19 mM substrate and in all species with 2.0 mM substrate o-hydroxyphenylacetaldehyde was the major metabolite. Of the three animal species studied the gerbil most resembled humans as this species also had a high coumarin 7-hydroxylaseactivity. The administration of Aroclor 1254 to the rat and Syrian hamster induced both microsomal cytochrome P-450 content and [3-14C]coumarinmetabolism. With liver microsomes from all species a good correlation between rates of [3J4C]coumarin metabolism and covalent binding was observed at both substrate concentrations. However, in view of the known species difference between the rat and Syrian hamster in coumarin-induced hepatotoxicity, the present data are not consistent with microsomal coumarin metabolite covalent binding being an indicator of potential liver damage.
INTRODUCTION
Coumarin (1,2-benzopyrone, c i s - o - c o u m a r i n i c acid lactone) occurs naturally in several plant families and essential oils (Cohen, 1979; Soine, 1964). Although no longer used as a food flavouring, coumarin is present in certain tobaccos and alcoholic beverages and is used in various soap, detergent and cosmetic preparations (Cohen, 1979; Opdyke, 1974). Some coumarin derivatives are of economic importance; these include 3,4-dihydrocoumarin used mainly in the perfume industry and 6-methylcoumarin used as a flavour enhancer (Egan et al., 1990). The toxicity of coumarin merits attention because it has been shown to exhibit interspecies differences in both hepatotoxicity and metabolism (Cohen, 1979). In the rat, single doses of coumarin produce centrilobular hepatic necrosis (Lake, 1984; Lake et al., 1989b), whereas chronic administration results in bile duct lesions (Cohen, 1979; Evans et al., 1989; Hagan et al., 1967). However, no significant hepatotoxic effects were observed in chronic feeding studies con*To whom correspondence should be addressed. Abbreviations: DHC = dihydrocoumarin;3°HC = 3-hydroxy-
coumarin; 5-HC= 5-hydroxycoumarin; 7-HC = 7hydroxycoumarin; o-HPA = o-hydroxyphenylacetaldehyde; o-HPAA = o-hydroxyphenylaceticacid; o-HPPA = o -hydroxyphenylpropionicacid. FCT 30i2 B
ducted either in the Syrian hamster (Lake et al., 1990; Ueno and Hirono, 1981) or baboon (Evans et al., 1979). Studies on the metabolism of coumarin have demonstrated that in humans (Egan et al., 1990; Shilling et al., 1969) and in the baboon (Gangolli et al., 1974; Waller and Chasseaud, 1981) coumarin is extensively metabolized to 7-hydroxycoumarin (Fig. 1). In contrast, 7-hydroxylation represents only a minor pathway in several other species including the rat, most strains of mouse, Syrian hamster, dog, rabbit and marmoset (Cohen, 1979; Gangolli et al., 1974; Kaighen and Williams, 1961; Lake et al., 1989a). The major metabolic pathway of coumarin biotransformation in the rat appears to involve an initial 3-hydroxylation reaction with subsequent ring opening and further metabolism to o-hydroxyphenylacetic acid (Cohen, 1979; Kaighen and Williams, 1961). Studies with rat hepatic microsomes have demonstrated that coumarin is metabolized by cytochrome P-450-dependent mixed-function oxidase enzymes to various polar products and to metabolite(s) that bind covalently to microsomal proteins (Feuer, 1970; Gibbs et al., 1971; Lake, 1984; Lake et al., 1992; Peters et al., 1991; Walters et al., 1980). In agreement with in vivo findings, coumarin 7-hydroxylase activity is either low or absent in rat liver microsomes, but
105
~
/
0
7-HC
[
0 0 Coumarin
o-HPPA
O
OH
~ o-HPE
CH 2CH2 OH
//
3,4-epoxide
~
Coumarin
DHC
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OH
/
_ _
\
~
~
o-HPA
CH2CHO
3-HC
O
_ _°. "
OH
o-HPLA
o-HPAA
CH2COOH
-OH
HzCHOHCOOH
Fig. 1, Some pathways of coumarin metabolism (Cohen, 1979; Lake et al., 1992: Norman and Wood, 1984). The coumarin metabolites shown are 3-, 4-, 5-. 6-, 7- and 8-hydroxycoumarins(3-HC, 4-HC. 5-HC, 6-HC, 7-HC and 8-HC), 6,7-dihydroxycoumarin(6,7-DiHC), dihydrocoumarin (DHC), o-coumaric acid (o-CA), o-hydroxyphenyllactic acid (o-HPLA/ o-hydroxyphenylpropionic acid (o-HPPA), u-hydroxyphenylacetaldehyde (o-HPA), o-hydroxyphenylethanol (o-HPE) and o-hydroxyphenylacetic acid (o-HPAA).
HO
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HO
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OH
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~ 0 0 4-HC OH
~
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[3-~4C]Coumarin metabolism in different species can be readily detected in gerbil and human liver microsomes (Dominguez et al., 1990; Feuer, 1970; Gibbs et al., 1971; Lake et al., 1989a; Miles et al., 1990; Pelkonen et al., 1985; Peters et al., 1991; Raunio et al., 1988). Recent studies have shown that a member of the cytochrome P4501IA subfamily is responsible for the majority of coumarin 7-hydroxylase activity in human liver microsomes (Miles et al., 1990), whereas in rat liver microsomes coumarin is a substrate for isoenzymes in both the cytochrome P450IA and P450IIB subfamilies (Peters et al., 1991). The aim of the present investigations was to obtain further data on species differences in coumarin metabolism by studying the metabolism of [3-~4C]coumarin in liver microsomes from the rat, Syrian hamster, Mongolian gerbil and humans. The gerbil was selected because like humans it is known to metabolize coumarin to 7-hydroxycoumarin (Dominguez et al., 1990), whereas the rat and Syrian hamster were selected because of the known species difference in both acute (B. G. Lake and J. G. Evans, unpublished observations) and chronic (Lake et al., 1990; Ueno and Hirono, 1981) coumarin-induced hepatotoxicity. With the rat and Syrian hamster, the effect of Aroclor 1254 treatment on hepatic microsomal [3-~4C]coumarin metabolism was also studied. This polychlorinated biphenyl mixture, which induces several cytochrome P-450 isoenzymes (Ryan et al., 1979) is known to induce markedly [3-z4C]coumarin metabolism in rat liver microsomes (Peters et al., 1991). MATERIALS
AND METHODS
Chemicals. [3-14C]Coumarin (sp. act. 5.3mCi/ mmol) was obtained 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 toluene-chloroform (1:1, v/v). Unlabelled coumarin and coumarin metabolites were obtained from the sources described previously (Lake eta!., 1989b). Enzymes, cofactors etc. were purchased from Sigma Chemical Co. Ltd (Poole, Dorset, UK) and HPLCgrade methanol and tetrahydrofuran (unstabilized) from Rathburn Chemicals (Walkerburn, UK). Animals and treatment. Male Sprague-Dawley rats were obtained from Harlan Olac (Bicester, Oxon, UK) and male Golden Syrian hamsters from Bantin and Kingman (The Field Station, Grimston, Aldbrough, Hull, UK) and allowed free access to R and M No. 1 and No. 3 diets (Special Diets Services, Witham, Essex, UK), respectively, and water. The animals were housed in accommodation maintained at 22_+ Y'C with a relative humidity of 40-70% and were allowed to acclimatize to these conditions for at least 7 days before use. Rats and hamsters, both 6 wk old, were given five daily ip doses of 100mg Aroclor 1254/kg body weight. Control
107
animals received corresponding quantities (5 ml/kg body weight) of the corn-oil vehicle. Animals were killed by cervical dislocation. Livers from untreated male Mongolian gerbils (approximately 50g body weight) were kindly provided by Dr A. G. Smith (MRC Toxicology Unit, Carshalton, Surrey, UK). Samples of human liver (seven subjects, five male aged 10, 20, 23, 25 and 64 yr and two female aged 16 and 38 yr) were obtained from various sources and stored at - 8 0 ° C until use. Preparation o f microsomes. Whole liver homogenates (0.25 g fresh tissue/ml) were prepared in ice-cold 0.154M-KC1, containing 50mM-Tris-HC1 pH 7.4, using a Potter-type, teflon-glass, motor-driven homogenizer (A. H. Thomas Co., Philadelphia, PA, USA). The homogenates were centrifuged at 10,000 g av. for 20 min to obtain the postmitochondrial supernatants and subsequently at 158,000g av. for 40 min to separate the microsomal fraction from the cytosol. The pellets were resuspended in fresh homogenizing medium and again centrifuged at 158,000g av. for 40 rain. Washed microsomal fractions were stored at - 8 0 ° C for up to 6 wk until use. Assays. Microsomes were assayed for content of cytochrome P-450 (Omura and Sato, 1964) and protein (Lowry et al., 1951). The activity of 7-ethoxycoumarin O-deethylase was determined as described previously (Lake, 1987). For studies of [ 3 - 1 4 C ] coumarin metabolism standard incubation mixtures contained 2 m g microsomal protein, either 0.19 or 2.0mM-[3-14C]coumarin (1/~Ci/tube, added in 5/~1 dimethylsulphoxide), 1 mN-NADPH, 5 mM-MgSO4 and 0.1 mM-phosphate buffer pH 7.6 in a final volume of 1 ml. After 2min preincubation at 3TC in a shaking water-bath, the reaction was started by adding NADPH. Blank tubes were incubated with [3-14C]coumarin but in the absence of NADPH. After 10 rain, incubations were terminated with l ml icecold methanol. Following centrifugation for 15 min at 1200g at 43C, the supernatant was taken for H P L C analysis of [3-14C]coumarin and [3-~4C]coumarin polar metabolites (Peters et al., 1991) and the pellet for determination of covalent binding of [3-~4C]coumarin metabolites to microsomal proteins. Polar [3-~4C]coumarin metabolites were identified by comparison with the retention times of known standards and quantified by measurement of the radioactivity present in the appropriate HPLC fractions, whereas values for total polar products were calculated from the radioactivity present in all HPLC fractions excluding unmetabolized [3-~4C]coumarin substrate. Macromolecular binding of [ 3 - 1 4 C ] coumarin metabolites was determined by exhaustive solvent extraction with 5% (w/v) trichloroacetic acid and 80% (v/v) methanol in water as described previously (Lake, 1984; Lake et al., 1981). Statistical analysis. Statistical evaluation of data was performed by one-way analysis of variance. Comparisons between means were made using the least significant difference test.
B. G. LAKE et al.
108
Table 1. Cytochrome P-450 content and 7-ethoxycoumarin O-deethylase activity in hepatic microsomes from various species 7-Ethoxycoumarin Cytochrome P-450 O-deethylase Species (nmol/mg p r o t e i n ) (nmol/min/mg protein) Rat Control (5) Aroclor 1254 treated (5)
1.05 ± 0.07 3.18 ± 0.09***
1.44 ± 0.11 37.3 ± 3.1'**
Syrian hamster Control (5) Aroclor 1254 treated (5)
1.22 ± 0.05 2.83 ± 0.14"**
7.45t 17.4 _+ 0.3
Gerbil (4)
0.80 ± 0.03
4.99 ± 0.24
H u m a n (7)
0.31 ± 0.02
0.32 +_ 0.03
fValue shown is for a single determination only. Values are means ± SEM with number of samples shown in parentheses. For Aroclor 1254treated rats and hamsters values significantly different from corresponding controls are: ***P < 0.001. RESULTS
Microsomal enzyme activities
Levels o f c y t o c h r o m e P-450 and 7-ethoxycoumarin O-deethylase in hepatic microsomes from the various species are shown in Table 1. Aroclor 1254 treatment induced both cytochrome P-450 content and mixedfanction oxidase enzyme activity in both the rat and Syrian hamster, with a more marked effect being observed in the rat. Cytochrome P-450 content and 7-ethoxycoumarin O-deethylase activity in human liver microsomes were only 25-39% and 4-22%, respectively, of levels found in the other three species (i.e. in non-Aroclor 1254-treated animals). Metabolism
q[" [3- J4C]coumarin
An initial study was performed with control and Aroclor 1254 rat liver microsomes over a coumarin substrate concentration range of 0.34 to 2.6mM. [3-14C]Coumarin metabolism to total polar products and covalently bound metabolites is shown in Fig. 2. Pretreatment with Aroclor 1254 markedly enhanced the rate of [3-14C]coumarin metabolism to polar products and covalently bound metabolites at all substrate concentrations examined. A double reciprocal plot of [3J4C]coumarin metabolism to polar products against coumarin substrate concentration yielded apparent affinities (K m values) of 3.01 and 0.34 mM and maximal velocities (Vmax) of 6.84 and 23.5 nmol/min/mg protein for microsomes from control and Aroclor 1254-treated rats, respectively (data not shown). Corresponding kinetic constants for covalently bound metabolites were 1.40 and 0.23 mM, and 156 and 910 pmol/min/mg protein, respectively. Coumarin substrate concentrations of 0.19 and 2.0 mM were selected for all subsequent studies, the former representing the minimum amount of radioactivity that could be added, the latter representing a near saturating substrate concentration for rat liver microsomes. In hepatic microsomes from all species [3J4C]coumarin was metabolized to both polar products (Fig. 3A) and to metabolite(s) that became covalently bound to microsomal proteins (Fig. 3B). With all
microsomal preparations, the rate of [3-t4C]coumarin metabolism to total polar products and covalently bound metabolites was greater at 2.0 than 0.19 mM[3J4C]coumarin substrate concentration. At the 0.19 mM-[3J4C]coumarin substrate concentration and with microsomes from control (i.e. non-Aroclor 1254-treated) animals, the highest rates of metabolism to polar products and covalent binding were observed for the gerbil and the least with the rat and humans. For 2.0 mM-coumarin, the highest and 24
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Fig. 2. Effect of substrate concentration on [3-~4C]coumarin metabolism to total polar products (A) and covalently bound metabolite(s) (B) by hepatic microsomes from control (A) and Aroclor 1254-treated (&) rats. Each point represents the mean of duplicate incubations and the results of a typical experiment are shown.
[3J4C]Coumarin metabolism in different species
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Fig. 3. [3J4C]Coumarin metabolism to total polar products (A) and covalently bound metabolites (B) by hepatic microsomes from various species. Results are expressed as means +_ SEM for groups of 4-7 control (RC) and Aroclor 1254-treated (RA) rat, control (HC) and Aroclor 1254treated (HA) Syrian hamster, gerbil (G) and human (HU) liver microsomes. For Aroclor 1254-treated rats and hamsters values significantly different from corresponding controls are: **P < 0.0l; ***P < 0.001.
lowest rates of metabolism and covalent binding were observed with gerbil and human microsomes, respectively. Treatment with Aroclor 1254 significantly induced [3J4C]coumarin metabolism and covalent binding at both substrate concentrations in the rat and Syrian hamster. Induction was more marked in the rat liver microsomes than in the hamster, particularly at the lower coumarin substrate concentration. Rates of covalent binding expressed as a percentage of the metabolism of 0.19 mM-[3-14C]coumarin to total polar metabolites were 5.5, 4.6, 4.0, 5.4, 2.6 and 1.5% for control and Aroclor 1254-treated rat, control and Aroclor 1254-treated Syrian hamster, gerbil and human liver microsomes, respectively. Corresponding values for a 2 mM-[3J4C]coumarin substrate concentration were 1.8, 3.7, 1.9, 1.9, 2.1 and 1.6%, respectively. Linear regression analysis of the data for all species and treatments revealed good correlations between [3J4C]coumarin metabolism to polar products and covalent binding at both the 0.19mM (r = 0.948; P < 0.001) and 2.00 mM (r = 0.894; P < 0.001) substrate concentrations (Fig. 4, A and B).
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Fig. 4. Relationship between metabolism of 0.19ram (A) and 2.0 mM (B) [3J4C]coumarin to total polar products and covalently bound metabolite(s). Points represent individual control (•) and Aroclor 1254-treated (A) rat, control (O) and Aroclor 1254-treated ( 0 ) Syrian hamster, gerbil ( I ) and human (,~') liver microsomes. Note that for the human liver samples covalent binding was determined for seven and four subjects at [3-14C]coumarin substrate concentrations of 0.19 and 2.0raM, respectively.
The H P L C system used (Lake et al,, 1992; Peters et al., 1991) could separate coumarin from 14 of its known metabolites (Fig. 1). Coumarin metabolites were identified by comparison with the retention times of known standards and quantified by measurement of the radioactivity present in the appropriate fractions (Table 2). Typical metabolite profiles for each source of hepatic microsomes incubated with 0.19- and 2.0 mM-[3-~4C]coumarin are shown in Figs 5 and 6, respectively. With the exception of human liver microsomes incubated with 0.19 mM-[3J4C]coumarin (Fig. 5), the major metabolite identified was ohydroxyphenylacetaldehyde (o-HPA). The formation of o - H P A was greater at the higher than the lower substrate concentrations in all species and was induced by Aroclor 1254 administration in the rat and Syrian hamster. The formation of 7-hydroxycoumarin (7-HC) was not observed in either control or Aroclor 1254-treated rat liver microsomes, whereas this metabolite was present in control and Aroclor 1254-treated Syrian hamster microsomes and
ll0
B . G . LaK~ et al.
in gerbil and human liver microsomes (Figs 5 and 6: Table 2). Although the formation of 7-HC was increased at the higher substrate concentration in Syrian hamster liver microsomes, only a small increase was observed in gerbil liver microsomes and no increase was observed in human liver microsomes (Table 2). [3-~4C]Coumarin was also metabolized in rat liver microsomes to o-hydroxyphenylacetic acid ( o - H P A A ) and to 3- and/or 5-hydroxycoumarin (3-HC/5-HC) and with 2.0 mM substrate to dihydrocoumarin and/or o-hydroxyphenylpropionic acid ( D H C / o - H P P A ) . These two sets of coumarin metabolites were quantified as pairs as the individual metabolites were not separated from one another by the H P L C system used in these studies. The formation of o - H P A A and 3-HC/5-HC was induced by Aroclor 1254 treatment, The formation of 3-HC/5HC and induction by the polychlorinated biphenyl mixture was also observed in hamster liver microsomes at both substrate concentrations (Table 2). Apart t¥om o - H P A and 7-HC, the Formation of 3-HC/5-HC and D H C / o - H P P A was observed in gerbil liver microsomes, whereas the formation of o - H P A A and o - H P E was observed in human liver microsomes. With human liver microsomes only the formation of o - H P A was greatly increased at the higher [3-~4C]coumarin substrate concentration (Table 2).
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The purpose of this in ritro study was to obtain more information on species differences in hepatic coumarin metabolism. In keeping with previous studies, levels of cytochrome P-450 in rat, Syrian hamster and gerbil liver microsomes were higher than those found in human liver (Dominguez et al., 1990: Sesardic et al., 1990:Souhaili-E1 Amri e t a / . , 1986). The cytochrome P-450 levels of 0.80 and 0.31 nmol/mg protein found in gerbil and human liver microsomes, respectively, are in agreement with values reported in other studies (Dominguez et al,. 1990: Sesardic et al., 1990:Souhaili-E1 Amri et ell.. 1986). Both qualitative and quantitative differences in [3-~4C]coumarin metabolism to polar products by liver microsomes from the four species were observed in this study. For example, coumarin metabolism to 7-hydroxycoumarin was observed in the Syrian hamster, gerbil, and human microsomes but not with rat liver microsomes. Previous studies have shown that rat liver microsomes contain either no or very low levels of coumarin 7-hydroxylase activity unless very high substrate concentrations arc used (Feuer, 1970: Gibbs et al.. 1971: Lake et al., 1989a: Pelkonen et al., 1985; Raunio et al., 1988). The observation that Syrian hamster liver microsomes contain detectable coumarin 7-hydroxylase activity is in agreement with the results of Gangol[i et al. (1974), who observed that 5% of an orally administered dose of coumarin was excreted in the urine as 7-hydroxycoumarin,
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Fig 6. Typical preiiles For the metabolism of 2.0 mM-[3-~a('lcoumarm to polar products b~ li~er microsomes l'rom ~arious species. ('oumarin ((') ,.,,as separated from the coumarin metabolites d'or abbreviations see Fig, 1) by HPLC.
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[3-~4C]Coumarin metabolism in different species Previous studies on microsomal coumarin 7hydroxylase have used a spectrofluorimetric assay for determining enzyme activity, as against the radiometric assay used in the present study. However, the enzyme activities observed in this study were similar to those obtained in some previous studies. For example, in this study with human liver microsomes, enzyme activities of 0.26 and 0.28 nmol/min/mg protein were obtained at coumarin substrate concentration of 0.19 and 2.0mM, respectively. Previous studies have reported values of 0.33, 0.31 and 0.08 nmol/min/mg protein at coumarin substrate concentrations of 0.1, 0.1 and 5 mM, respectively (Kratz, 1976; Raunio et al., 1988; Sesardic et al., 1990). It should be noted that a considerable inter-individual variation in human liver coumarin 7-hydroxylase activity has been reported (Kratz, 1976; Miles et al., 1990; Pelkonen et al., 1985). Liver microsomes from all species formed o-HPA. which has been recently identified as a novel coumarin metabolite in rat liver microsomes (Lake et al., 1992). This was the major metabolite in rat, Syrian hamster and gerbil liver microsomes. With human liver microsomes, 7-HC was the major metabolite at the 0.19 mM substrate concentration, whereas o-HPA was the major metabolite with 2 mg substrate. As coumarin 7-hydroxylase activity was not increased at the higher substrate concentration in human liver microsomes, the 7-hydroxylation pathway appears to be saturated at lower substrate concentrations than do other pathways (e.g. o-HPA formation) of coumarin metabolism. Thus, at low coumarin doses, the isoenzymes ofcytochrome P-450 that metabolize coumarin in human liver microsomes (Miles et al., 1990; Pelkonen et al., 1985; Raunio et al., 1988) produce mainly 7-hydroxycoumarin, which accounts for this being the major metabolite observed in vi~,o in humans (Egan et al., 1990; Shilling et al., 1969). Gerbil liver microsomes appear to be similar to human liver rnicrosomes in this respect as there was very little increase in coumarin 7-hydroxylase activity at the 2.0 mM compared with the 0.19 mg substrate concentration (Table 2). This study demonstrates that several pathways of coumarin metabolism exist in liver microsomes from all four species studied. In the rat, Kaighen and Williams (1961) demonstrated that o-HPAA was produced by further metabolism of 3-HC (Fig. 1), whereas Norman and Wood (1984) have suggested that both o-HPE and o-HPAA were likely to be formed from an intermediate other than 3-HC. With respect to o-HPA, the major coumarin metabolite in all species at high substrate concentration, it is not clear whether this is derived from 3-HC or other intermediate(s) (Lake et al., 1992). Further research is thus required to elucidate all the pathways of coumarin metabolism in liver microsomes from the rat, humans and other species. A major focus of this study was the comparison of the rat and Syrian hamster, as the former species is
113
susceptible and the latter species resistant to both acute (B. G. Lake and J. G. Evans, unpublished observations) and chronic (Lake et al., 1990~ Ueno and Hirono, 1981) coumarin-induced liver injury. In agreement with our previous observations (Peters et al., 1991), Aroclor 1254 induced [3-~4C]coumarin metabolism to both polar products and covalently bound metabolites in rat liver microsomes. A kinetic study with rat liver microsomes revealed changes in both Km and Vmax values after Aroclor 1254 treatment. The change in K mpresumably reflects the change in microsomal cytochrome P - 4 5 0 isoenzyme composition produced by Aroclor 1254 induction (Peters et al., 1991; Ryan et a l , 1979). Aroclor 1254 also induced [3-~4C]coumarin metabolism in Syrian hamster liver microsomes but the effect was less marked than in the rat. With both the rat and Syrian hamster the pattern of [3-~4C]coumarin metabolites after Aroclor 1254 treatment was similar to that of control microsomes. The major difference between these two species observed in this study was that the Syrian hamster, unlike the rat, had detectable coumarin 7-hydroxylase activity. Although it has been suggested that coumarin metabolism to 7-HC represents a detoxification pathway (Cohen, 1979), the comparatively small difference in 7-HC [brmation between these two species, either in this study in t,itro or in eiro (Gangolli et al., 1974), is unlikely to explain the known species difference in susceptibility to coumarin-induced hepatotoxicity. Previous studies with rat liver microsomes and isolated hepatocytes have shown that coumarin is metabolized by cytochrome P-450 dependent enzymes to metabolite(s) that bind covalently to proteins (den Besten et aL, 1990; Lake, 1984; Peters et al., 1991). The covalent binding of reactive coumarin metabolites could represent a possible mechanism of hepatotoxicity of this compound (den Besten et al., 1990; Lake, 1984). In this study with microsomes from control and Aroclor 1254-treated species, good correlations between [3-J4C]coumarin metabolism and covalent binding were observed at both substrate concentrations (Fig. 4, A and B). If coumarin metabolite covalent binding was related to subsequent toxicity then the lowest rates of covalent binding were observed with human liver microsomes (Fig. 3B). However, because of the known species difference between the rat and Syrian hamster in coumarin-induced liver injury, the present data are not consistent with microsomal coumarin metabolite binding being an indicator of potential hepatotoxicity. For example, with 0.19 mM-[3-~C]coumarin the rate of covalent binding was greater in Syrian hamster than in rat hepatic microsomes, whereas at 2.0 mM substrate concentrate the rate of covalent binding was similar in both species (Fig. 3B). In summary, [3-~4C]coumarin was metabolized by hepatic microsomes from the four species studied. Of the three animal species, the Mongolian gerbil most resembled humans, as this species also had high
B. G. LAKE et al.
114
c o u m a r i n 7 - h y d r o x y l a s e activity. A d d i t i o n a l investig a t i o n s w o u l d be r e q u i r e d to e s t a b l i s h w h e t h e r t h e gerbil w o u l d be a useful a n i m a l m o d e l for f u r t h e r studies on coumarin metabolism and hepatotoxicity, to assist in t h e e v a l u a t i o n o f the r e l e v a n c e o f e x i s t i n g rat toxicity d a t a for h u m a n h a z a r d a s s e s s m e n t . F r o m t h e p r e s e n t s t u d i e s w i t h the rat a n d S y r i a n hamster, the measurement of the covalent binding of reactive c o u m a r i n m e t a b o l i t e ( s ) to m i c r o s o m a l p r o teins d o e s n o t a p p e a r to c o r r e l a t e with s u b s e q u e n t hepatotoxicity.
Acknowledgements We are grateful to Dr S. D. Gangolli for support and encouragement and to Dr A. R. Boobis ( H u m a n Tissue Bank, Royal Postgraduate Hospital Medical School, London) and Dr J. M. Tredger (The Liver Unit, Kings College Hospital School of Medicine and Dentistry, London) for supplying h u m a n liver samples. This work torms part of a research project sponsored by the U K Ministry of Agriculture, Fisheries and Food. to whom our thanks are due. The results of the research are the property of the Ministry of Agriculture, Fisheries and Food and are Crown Copyright. REFERENCES
den Besten C., K6r6si S. A., Beamand J. A.+ Waiters D. G. and Lake B. G. (1990) Studies on the mechanism of coumarin-indueed toxicity in rat hepatocytes. Toxicology in Vitro 4, 518- 521. 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. Dominguez K., Fentem J. H., Garle M. J. and Fry J. R. (1990) Comparison of Mongolian gerbil and rat hepatic microsomal monooxygenase activities: high coumarin 7-hydroxylase activity in the gerbil. Biochemical Pharmacology 39, 1629 1631. Egan D , O ' K e n n e d y R.. Moran E., Cox D., Prosser E. and Thornes R D. (1990) The pharmacology, metabolism, analysis and applications of coumarin and coumarin-related compounds. Drug Metabolism Reviews 22, 503 529. Evans J. G., Appleby E. C., Lake B. G. and Conning D. M. (1989) Studies on the induction of cholangiofibrosis by coumarin in the rat. To.vieology 55, 207 224. Evans J. G., (}aunt 1. F. and Lake B. G. (1979) Two-year toxicity study on coumarin in the baboon. Food and Chemical Toxicology 17, 187 193. Feuer G. (1970) 3-ttydroxylation of coumarin or 4-methylcoumarin by rat-liver microsomes and its induction by 4-methylcoumarin given orally. Chemieo-Biologieal luteracli(ms 2, 203 216. Gangolli S. D.. Shilling W. H., Grasso P. and G a u n t I. F. (1974) Studies on the metabolism and hepatotoxicity of coumarin in the baboon. Biochemical Society Transactions 2, 310 312. Gibbs P. A., Janakidevi K. and Feuer G. ( 1971 ) Metabolism of coumarin and 4-methylcoumarin by rat-liver microsomes. Canadian Journal qf Biochemisto, 49, 177 184. Hagan E. C+. Hansen W. H., Fitzhugh O. G., Jenner P. M., Jones W. I., Taylor J. M., Long E. L., Nelson A. A. and Brouwer J. B. (1967) Food flavourings and compounds of related structure. II. Subacute and chronic toxicity. Food and Chemical Toxicology 5, 141 157. Kaighen M. and Williams R. T. (19611 The metabolism of [3-~4C]coumarin. Journal of Medicinal Chemistry 3, 25M.3. Kratz F. (1976) Coumarin 7-hydroxylase activity in microsomes from needle biopsies of normal and diseased
h u m a n liver. European Journal of Clinical Pharmacology 10, 131 137. Lake B. G. (1984) Investigations into the mechanism of coumarin-induced hepatotoxicity in the rat. 4rchives Ol Toxicology (Suppl. 7), 16 29. Lake B. G. (1987) Preparation and characterisation of microsomal fractions for studies of xenobiotic metabolism. In Biochemical To.vicology: A Praelical Approach Edited by K. Snell and B. Mullock. pp. 183 215. IRL Press, Oxford. Lake B. G., Evans J. G., Waiters D. G. and Price R. J (I990) Studies on the hepatotoxicity and metabolism of coumarin in the rat and Syrian h a m s t e r ttuman ami Experimental Toxicology 9, 356. Lake B. G., Gray T. J. B., Evans J. G., Lewis D. |~. V., Beamand J. A. and Hue K. t_.. (1989b) Studies on the mechanism of coumarin-induced toxicity in rat hepatocytes: comparison with dihydrocoumarin and other coumarin metabolites. 7'r;.vicologv and .4pplied Pharmacology 97, 311 323. Lake B. G., Harris R. A., Phillips J. C. and Gangolli S. D. (1981) Studies on the effects of t.-ascorbic acid on acetaminophen-induced hepatotoxicity. I. Inhibition of the covalent binding of acetaminophen to hepatic microsomes in vitro. Toxieology and Applied Pharmacology 60, 229 240. Lake B. G., Osborne D. J., Waiters D. G. and Price R. 3. (1992) Identification of o-hydroxyphenylacetaldehyde as a major metabolite of coumarin m rat hepatic microsomes. Food and Chemical Toxicology 30, 99 104. Lake B. G., Waiters D. G. and Gangolli S. D. (1989a~ Comparison of the metabolism and disposition of [3-~C] coumarin in the rat and marmoset (Callithrir /a~chz+.~i Toxicology Letter.s 45, 299 306. Lowry O. H., Rosebrough N. J.. t:arr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193, 265 275 Miles J. S.+ McLaren A. W., Forrester L. M., Glancey M J . Lang M. A. and Wolf C. R. (1990) Identification of the h u m a n liver cytochrome P-450 responsible [\~r coumarin 7-hydroxylase activity. Biochemical Jaurmzl 267, 365 371. N o r m a n R. L. and Wood A. W. (1984) o-Hydruxyphenylethanol, a novel lactone ring-opened metabolite of coumarin. Drug MetabolLs'm and Disposition 12, 543 54t1. Omura T. and Sato R. (l 964) The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. Journal o[ Biolo2ical Chemistry 239, 2370 2378. Opdyke D. L. J. (1974) Monographs ol fragrance raw materials: coumarin. Food and Chemical 1)?.\'i~oloev 12, 385 388. Pelkonen O., Sotaniemi E. A. and Ahokas J. T (1985} Coumarin 7-hydroxylase activit~ in h u m a n liver micro+ somes. Properties of the enzyme and interspecies comparisons. British Journal (71 Clinical Pharmacologt 19, 59 66. Peters M. M. C. G., Waiters D. G.. Van O m m e n B.. Van Bladeren P. J. and Lake B. G. (1991) Effect of inducers of cytochrome P-450 on the metabolism of [3-HC] coumarin b3 rat hepatic microsomes. ,~'enobioti(a 21, 499 514. Raunio H., Syngelm'a T., Pasanen M., Juvonen R , Honkakoski P., Kairaluoma M. A., Sotaniemi E., Lang M. A. and Pelkonen O. (1988) lmmunochemical and catalytical studies on hepatic coumarin 7-hydroxylasc in man, rat and mouse. Biochemical Pharnzac~doev 37, 3889 3895. Ryan D. E., T h o m a s P. E., Korzeniowski D. and Lcvtn W. (1979) Separation and characterization of highly purified forms of liver microsomal cytochrome P-450 from rats treated with polychlorinated biphenyls, phenobarbital, and 3-methylcholanthrene. Journal O! Biological Chemistry 254, 1365 1374.
[3-~4C]Coumarin metabolism in different species Sesardic D., Boobis A. R., Murray B. P., Murray S., Segura J., De La Torre R. and Davies D. S. (1990) Furafylline is a potent and selective inhibitor of cytochrome P450IA2 in man. British Journal of Clinical Pharmacology 29, 651~63. Shilling W. H., Crampton R. F. and Longland R. C. (1969) Metabolism of cournarin in man. Nature, London 221, 664~565. Soine T. O. (1964) Naturally occurring coumarins and related physiological activities. Journal of Pharmaceutical Sciences 53, 231. 264. Souhaili-El Amri H., Batt A. M. and Siest G. (1986) Comparison of cytocbrome P-450 content and activities
115
in liver microsomes of seven animal species including man. Xenobiotica 16, 351-358. Ueno I. and Hirono I. (1981) Non-carcinogenic response to coumarin in Syrian golden hamsters. Food and Chemical Toxicology 19, 353-355. Waller A. R. and Chasseaud L. F. (1981) The metabolic fate of [14C]coumarin in baboons. Food and Chemical Toxicology 19, 1~. Waiters D. G., Lake B. G. and Cottrell R. C. (1980) High-performance liquid chromatography of coumarin and its metabolites. Journal of Chromatography 196, 501-505.