Fd Chem. Toxic. Vol. 30, No. 6, pp. 467-473, 1992 Printed in Great Britain. All rights reserved

0278-6915/92 $5.00 + 0.00 Copyright © 1992 Pergamon Press Ltd

I N F L U E N C E OF M O D U L A T O R S OF E P O X I D E M E T A B O L I S M ON T H E C Y T O T O X I C I T Y OF trans-ANETHOLE IN F R E S H L Y I S O L A T E D RAT HEPATOCYTES A. D. MARSHALL and J. CALDWELL* Department of Pharmacology and Toxicology, St Mary's Hospital Medical School, Imperial College of Science, Technology and Medicine, London W2 IPG, UK (Accepted 13 January 1992)

Abstract--The effect of modulating epoxide metabolism by inhibiting microsomal and cytosolic epoxide hydrolases and depleting glutathione, on the cytotoxicity of trans-anethole has been examined in freshly isolated rat hepatocytes in suspension. Hepatocytes derived from female Sprague-Dawley CD rats by collagenase perfusion were incubated in suspension and sampled at intervals over a 6-hr period. Cytotoxicity was assessed by the leakage of lactate dehydrogenase into the culture medium and in the cells after lysis. Glutathione was determined by fluorimetry. Anethole showed a dose-dependent cytotoxicity at concentrations ranging from 5 x 10 -4 to 5 x 10 -3 M, with concentrations of 10 -3 M and above causing greater than 63% leakage of lactate dehydrogenase in 6 hr. Microsomal epoxide hydrolase was inhibited by trichloropropene oxide (10 -4 M) and cyclohexene oxide (10 -3 M), and cytosolic epoxide hydrolase by 4-fluorochalcone oxide (5 × 10 -4 M). Cellular glutathione was depleted by diethyl maleate (5 × 10-4 M), and its synthesis inhibited by 2.5 x 10 -3 M-L-buthionine (S,R)-sulphoximine. Suspensions treated with a sub-cytotoxic concentration of anethole (5 x l0 -4 M) showed a rapid increase in cytotoxicity when 4-ftuorochalcone oxide was present (complete loss of viability within 2 hr), while pretreatment of hepatocytes with diethyl maleate in combination with buthionine sulphoximine, to deplete glutathione, slowly increased the cytotoxic response at later times (after 4 hr of incubation). The association of the effects of 4-fluorochalcone oxide with the inhibition of cytosolic epoxide hydrolase is strengthened by the inability of chalcone oxide, a close structural analogue of 4-fluorochalcone oxide, which has no effect on epoxide hydrolase or glutathione conjugation, to influence the effects of anethole on hepatocytes. These data are discussed in terms of the role of anethole epoxide in the cytotoxicity of trans-anethole.

INTRODUCTION t r a n s - A n e t h o l e (p-methoxypropenylbenzene) is responsible for the popular aniseed flavouring used worldwide in confectionery, savouries and anise beverages. It occurs naturally as the major component of the essential oils of fennel and Chinese star anise, and is also present in dill, basil and tarragon. The oral LDs0 of t r a n s - a n e t h o l e in rats is very low (900 mg/kg body weight; Boissier et al., 1967), and a number of investigations of its chronic toxicity in rats have shown a reduction in body fat (Le Bourhis, 1973) and slight hepatic changes (Hagan et al., 1967). Anethole is not tumorigenic in mice (Miller et al., 1983), but a recent study (Truhaut et al., 1989) showed it to be a weak hepatocarcinogen in female C D rats receiving the highest dose of 1% in the diet

*To whom correspondence and reprint requests should be addressed. Abbreviations: BSO = L-buthionine S,R-sulphoximine; cEH = cytosolic epoxide hydrolase; CHO = cyclohexene oxide; DEM =dimethyl maleate; GSH = glutathione; HBSS = Hanks' balanced salt solution; LDH = lactate dehydrogenase; mEH = microsomal epoxide hydrolase; TCPO = 1,1, l-trichloropropene-2,3-oxide. 467

for up to 121 wk. The genotoxic potential of anethole is equivocal in the Ames test, with four out of nine reports showing weakly positive effects; however, these positive findings were only seen with protocols that departed significantly from the standard procedures. Anethole does not induce unscheduled D N A synthesis in rat hepatocytes (Howes et al., 1990; Marshall et al., 1990). A detailed examination (Newberne et al., 1989) of the hepatic pathology in the rats from the study of Truhaut et al. (1989) showed marked cytotoxicity and evidence for hepatic enzyme induction, both of which would be of importance in secondary (non-genotoxic) mechanisms of carcinogenicity. Anethole is metabolized along three pathways, namely O-demethylation, o-hydroxylation followed by side-chain oxidation, and epoxidation of the 1,2double bond as shown in Fig. 1 (Sangster et al., 1984a). The occurrence of an epoxide intermediate is indicated by the excretion of diastereoisomeric 1,2-diols formed by hydration of the epoxide, sidechain degradation products deriving from these diols and thioethers presumably derived from glutathione (GSH) conjugation of the epoxide. This pathway is especially prevalent in the rat, becoming

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Modulation of trans-anethole cytotoxicity quantitatively more important at higher doses (Sangster et al., 1984b). In view of the fact that epoxides can be reactive metabolic intermediates responsible for a number of toxic manifestations, it is important to have information about the possible significance of anethole 1,2-epoxide. The present report describes studies on aspects of the cytotoxicity of trans-anethole in freshly isolated hepatocytes from CD rats, the strain used by Truhaut et al. (1989) in their long-term carcinogenicity/ toxicity study of this compound. The significance of the putative epoxide metabolic intermediate of anethole for the competing reactions of detoxication and cytotoxicity has been investigated by the use of compounds modulating the activities of enzyme systems that regulate the levels of epoxides in the cell. These include microsomal epoxide hydrolase (mEH), inhibited by cyclohexene oxide and 1,1,1trichloropropene 2,3-oxide (Guest and Dent, 1980), and cytosolic epoxide hydrolase (cEH), inhibited by 4-fluorochalcone oxide (Mullin and Hammock, 1982). A structural analogue of 4-fluorochalcone oxide, chalcone oxide, which is not an EH inhibitor, has also been used to investigate the specificity of the observed effects. The important pathway of GSH conjugation has been modulated by depleting GSH with either diethyl maleate, a non-cytotoxic compound that is extensively conjugated with GSH (Hrgberg and Kristofferson, 1977), or buthionine sulphoximine, an inhibitor of GSH synthesis (Gritiith and Meister, 1979).

Hepatocyte isolation and determination of lactate dehydrogenase leakage from cells. These procedures were as described by Howes et al. (1990). Assay of reduced glutathione. The procedure used was a modification of that of Hissin and Hilf (1976). Reduced glutathione was extracted from 106 hepatocytes in suspension by centrifugation at 150g for 3 min, the medium was aspirated and 0.5 ml perchloric acid (I M) was added to the pellet. The perchloric acid solutions were snap-frozen in liquid nitrogen and stored at - 7 0 ° C until assayed. Thawed samples were centrifuged at 3010g for 15 min at 4°C, and a 100-/~1 aliquot of the supernatant was adjusted to pH 8.0 with KOH (1 ra), diluted to 2ml with phosphate buffer (0.1 M, pH 8.0) containing 0.005 M-EDTA. The entire procedure was carried out at 4°C. o-Phthalaldehyde (100/al of a 1 mg/ml solution in methanol) was added; after 15 min of incubation at 22°C, the fluorescence of the solution was measured (excitation 350 nm, emission 420 nm) and glutathione concentration determined by reference to a standard curve, which was established each time the assay was used and was linear over the range 0~t x 10-rM-gluta thione. Control glutathione levels in untreated cells were 22.6 ( + 7 . 0 ) x 10-9mol/106 cells ( m e a n _ SD, n = 14), which fell by an average of 38.3 I O0

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Chemicals. trans-Anethole was obtained as a gift from Centre de Recherche Pernod-Ricard (Crrteil, France) and purchased from Sigma Chemical Co. Ltd (Poole, Dorset, UK). Chalcone oxide and 4fluorochalcone oxide were the kind gifts of Dr B. D. Hammock (Department of Entomology, University of California, Davis, CA, USA). Culture medium (RPMI 1640) and NuSerum were purchased from Flow Laboratories (Irvine, Scotland) and Hanks' balanced salt solution (HBSS) from Gibco BRL (Paisley, Scotland). Collagenase was obtained from Boehringer Co. Ltd (Worthing, Sussex, UK), ophthalaldehyde from Fluka A G (Buchs, Switzerland), diethyl maleate (DEM) from Aldrich Chemical Co. Ltd (Gillingham, Dorset, UK), 1,l,l-trichloropropene-2,3-oxide (TCPO), cyclohexene oxide (CHO) and L-buthionine S,R-sulphoximine (BSO) from Sigma. All other chemicals were of the highest grade commercially available from Sigma or Aldrich Chemical Co. Ltd (Gillingham, Dorset, UK). Animals. Female Sprague-Dawley CD rats (150-260 g) were purchased from Charles River Ltd (Kent, UK) and were fed on LabSure CRM rat pellets from Special Diet Services (Witham, Essex, UK). Rats were kept in the animal house for at least 7 days before use.

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Modulation of trans-anethole cytotoxicity ( + 2.5)% over the 6-hr incubation to 13.9 (-t-2.5) x 10-gmol/106 cells (mean + SD, n = 14). The influence of the various treatments on cellular glutathione concentration is expressed as a percentage of control glutathione concentration (untreated cells) at each time point. Incubation o f hepatocytes in suspension. Hepatocytes were incubated at a density of 10 6 viable cells/ml in R P M I 1640 medium supplemented with 5% NuSerum, 10 -6 M-insulin, 10 -4 r,i-hydrocortisone 21-sodium succinate and 5 x 10-6M-gentamicin/ml (Howes et al., 1990), in an atmosphere of 95% 02/5% CO2 and at 37°C in a shaking water-bath for various times up to 6 hr. The different compounds were a d d e d to the cell suspensions in solution, either in dimethyl sulphoxide (DMSO) or medium as appropriate, so as to give final D M S O concentrations of 0.4% (v/v) or less. The cells were preincubated for 30 min with the epoxide hydrolase inhibitors, and for 60 min with D E M and/or BSO prior to the addition of anethole. RESULTS

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Sprague-Dawley CD rats in suspension are illustrated in Fig. 2, which shows the concentration dependence of these effects. 5 x l0 -3 M was a clearly cytotoxic dose with effects also seen at l0 -3 M. There was also considerable glutathione depletion at these doses. The epoxide modulators had different effects on the cytotoxicity of anethole. Each of the modulators was studied at the highest sub-cytotoxic concentration of anethole possible (5 x 10 -4 M) to maximize any effect these compounds might show. TCPO (Fig. 3a) and D E M (Fig. 3b) had comparable effects, showing a synergistic interaction with anethole in increasing GSH depletion; the maximal depletion was achieved quickly (within 1 hr). C H O caused a marked and sustained depletion of GSH, and the depletion of GSH, which is seen at later times after D E M or TCPO treatment, did not occur after C H O treatment (Fig. 3c). The effect of C H O was unaltered by the presence of anethole. The effects of the epoxide modulators on GSH depletion did not extend to the cytotoxicity of anethole, which was unaffected by any of these chemicals. 4-Fluorochalcone oxide markedly and rapidly increased the cytotoxicity of anethole (Fig. 4); however, since 4-fluorochalcone oxide itself depleted G S H

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substantially, the addition of anethole had no discernible further effect. In addition, the fact that 4-fluorochalcone oxide was cytotoxic at late times at the concentration tested indicated the difficulty of using such an inhibitor. The structural analogue of 4-fluorochalcone oxide, chalcone oxide, tested at the marginally cytotoxic concentration of 5 x 10 -4 M, depleted GSH to approximately the same extent as did CHO (Fig. 3c). Like CHO, chalcone oxide had no effect on the cytotoxicity or glutathione depletion caused by 5 x 1 0 - 4 i-anethole (Fig. 5). In a second set of experiments, before incubation with anethole, hepatocytes were treated with DEM to deplete GSH, in the presence of 2.5 x 10 -3 M-BSO to inhibit GSH resynthesis. The results are presented in Fig. 6. In these cells, GSH levels were maintained at less than 10% of control values throughout the 6-hr experiment. Under these conditions, where the de novo synthesis of glutathione was inhibited, the cytotoxicity of anethole was increased at later times (after 4 hr of incubation) in contrast with the result obtained with 4-fluorochalcone oxide.

trans-Anethole shows a dose-dependent cytotoxicity to rat hepatocytes in culture, and the depletion of cellular GSH is indicative of the participation of a reactive metabolic intermediate in this toxicity. Although neither the addition of inhibitors of mEH or the depletion of GSH by DEM influenced the cytotoxicity of anethole to hepatocytes in suspension, a role for anethole epoxide is suggested by the increased cytotoxicity of non-cytotoxic doses of anethole (a) markedly by the cEH inhibitor, 4-fluorochalcone oxide, and (b) to a lesser extent under conditions where cytoprotective mechanisms against epoxides are greatly impaired by depletion of GSH by DEM and BSO in combination. The association between the increased cytotoxicity of anethole caused by 4-fluorochalcone oxide and the inhibition of cEH is strengthened by the failure of the very close analogue of 4-fluorochalcone oxide, chalcone oxide, which has minimal effects on cEH and mEH (Mullin and Hammock, 1982), to influence anethole cytotoxicity. Although anethole epoxide is hydrated spontaneously very rapidly in aqueous media (S. V. J. Bounds, A. D. Marshall and J. CaldweU, unpublished data, 1992), the lack of a effect of chalcone oxide strongly suggests that this hydration in the hepatocyte would involve cEH. The effects of 4-fluorochalcone oxide on the cytotoxicity of anethole are of interest since they indicate the structural similarity between anethole and trans-fl-methylstyrene, the epoxide of which is a known marker substrate for cEH (Ota and Hammock, 1980). The relative contributions of GSH depletion and cEH inhibition to the observed increase in the cytotoxicity of anethole are worthy of discussion. In addition to its inhibitory effect on cEH, 4-fluorochaicone oxide depletes the cell of GSH; however, its GSH conjugate may inhibit GSH transferase (Mullin and Hammock, 1982), which would be expected to throw further emphasis on hydration for the removal of the epoxide. A consideration of the kinetics of these effects serves to discriminate between them. The increase in anethole cytotoxicity caused by 4fluorochalcone oxide was seen very rapidly, within the first hour of incubation, whereas the potentiation caused by the marked and sustained GSH depletion with DEM and BSO occurred at much later times, after 4 hr. When GSH was depleted by DEM alone, cEH and the synthesis of GSH de novo appeared to act together to guard against cytotoxicity, whereas only the latter mechanism provided defence in cells exposed to 4-fluorochalcone oxide. BSO and DEM in combination left the hepatocytes with only cEH as a protective mechanism. Thus, it is likely that cEH plays the major role in terminating the activity of anethole epoxide in the cell, and that the effects of 4-fluorochalcone oxide may be distinguished from those of compounds that simply deplete the cell of GSH.

Modulation of trans-anethole cytotoxicity Although m E H inhibitors have been used toxicological investigations in vitro (Guest and Dent, 1980), the present study would appear to be the first exploitation of the substituted chalcone oxide cEH inhibitors in an intact mammalian cell system. These cEH inhibitors are thought to act as alternative substrates for the enzyme and will therefore be slowly inactivated (Mullin and H a m m o c k , 1982). The constitutive levels of cEH are much lower in the rat than in the mouse or humans (Meijer et al., 1987; Ota and H a m m o c k , 1980). Thus, the rat would be expected to be relatively more susceptible to the effects of an epoxide that is a c E H substrate, and it appears that the lower amounts of cEH present are sufficient to offer protection under normal circumstances. A number of studies in vivo have shown that the hepatotoxicity of anethole is generally of a low order (Le Bourhis, 1973), but it is of interest to note that Newberne et al. (1989) reported that the administration of 0.5 and 1% anethole in the diet of rats for up to 121 wk resulted in a marked incidence of hepatic necrosis. The present data, suggesting that anethole epoxide is a cytotoxic intermediate in the metabolism of anethole, may be relevant to these observations at sustained very high dose levels. We may thus conclude that the metabolic intermediate anethole epoxide is responsible for the cytotoxicity of anethole in isolated hepatocytes in suspension. The principal routes for the detoxication of anethole epoxide are G S H conjugation and hydration by cEH, which normally have sufficient capacity for cytoprotection. These mechanisms must be severely compromised by metabolic inhibitors or very high dose levels of anethole before cytotoxicity can be evoked. The work reported here is now being extended to the investigation of the impact of these metabolic inhibitors on the response to anethole in the U D S assay and on the metabolism of anethole in isolated hepatocytes. Acknowledgements--This work was supported by a grant from the FEMA Anethole Task Force, Washington, DC, USA. We are grateful to Dr B. D. Hammock for the supplies of chalcone oxide and 4-fluorochalcone oxide and for helpful discussion.

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