Document xwOp0Lm8Q1r61b7BbqOxvdxQ

jf halothane xuamne test c of sisterc tes and B, and N- ' thase of ' proper- - I i 4 i I SAFROLE: ITS METABOLISM, CARCINOGENICITY - AND INTERACTIONS WITH CYTOCHROME P-450 C. Ioannides, M. Delaforge and D. V. Parke Department of Biochemistry. University of Surrey, Guildford, Surrey GU2 SXH, England (Received IJ April IVK1) Seminary--A review of studies on safrole metabolism shows that the compound gives rise to a large number of metabolites by two major pathways, oxidation of the allyl side chain and oxidation of the methylenedioxy group with subsequent cleavage to form a catechol. The mechanism by which safrole exerts the weak hepatocarcinogenicity that has been demonstrated in rats and mice is considered on the basis of published work and recent studies by the authors. Metabolic conversion of the allyl group gives rise so intermediates capable of covalent binding with DNA and protein, and recent findings are compatible with conversion of the methylenedioxy group to a carbene, which forms ligand complexes with the haem moiety of cytochromes P-450 and P-448. It is suggested that while the allyl group is responsible for the mutagenic potential of safrole. the methylenedioxy moiety may be associated with epigenetic aspects of carcinogenicity. Introduction Oxidation of the allyl chain I Safrole (4-allyl-1,2-methylenedioxybcnzene) is a Safrole undergoes oxidation of the allylic group to natural plant constituent, found in oil of sassafras and yield the 2',3'-epoxide (safrole epoxide) in both rat f certain other essential oils (Arctander, 1960; Fishbein and guinea-pig (Janiaud et al. 1977; Stillwell et al. & Falk. 1969; Friedman A Shibko. 1969; Guenther. 1974). This epoxide is only a minor metabolite, poss ( 1948-1952). It is a member of the methyienedioxyben- ibly because of its slow rate of formation, or because I zene group of compounds, many of which (e.g. pipero- of its further metabolism by epoxide hydratase to the nyl butoxide) are extensively used as insecticide syner corresponding dihydrodiol (Delaforge, Janiaud. Ches- gists. sebeuf. Padieu A Maume, 1976; Delaforge, Janiaud, f A major source of human exposure to safrole is Levi A Morizot, 1980ck The dihydrodiol is one of the t through consumption of spices, such as nutmeg, cin metabolites of safrole in rats and guinea-pigs, and % namon and black pepper, in which safrole is a con presumably arises from the hydration of the stituent (FenarolCs Handbook of Flavor Ingredients. 2',3'-epoxide. as administration of the epoxide to these I 1971; Friedman A Shibko, 1969; Synerholm A Hart- species resulted in excretion of the dihydrodiol in the zell. 1945; Weil, 1965). Safrole is also present in root urine (Borchert et al. 1973b; Delaforge et al. 1980c; beer, and has been used as an additive ia chewing Stillwell er al 1974); the unchanged epoxide was also gum, toothpaste, soaps and certain pharmaceutical excreted in the urine, demonstrating its relative stab preparations (Fishbein A Falk, 1969k ility. Furthermore, the epoxide was detected in the j Safrole is a weak hepatocarcinogen (Hornburger, liver microsomes of rats pretreated with safrole and in Kelley, Friedler A Russfield, 1961; Long. Nelson, rat hepatocytes incubated with safrole (Delaforge, Fitzhugh A Hansen, 1963) and it is a matter of con Janiaud. Maume A Padieu, 1978k siderable interest whether the allyl moiety or the It is believed that the carcinogenicity of safrole is at methytenedioxy group, or both, are involved in the least partly mediated through its metabolite, l'-hy- mechanism of its carcinogenesis. droxysafrole. This metabolite has been detected in the liver, unne and bile of animals, and is also found in Metabolism the urine conjugated with glucuronic add (Borchert. Miller. Miller A Shires, 1973a; Borchert et al. 1973b; The metabolism of safrole both in vim and in vitro, Janiaud et al. 1977; Stillwell et al. 1974k The I'-hy- using hepatic homogenates and cell cultures, has been droxysafrote forms an ester, when incubated with the subject of many studies (Borchert Wislocki, cytosolic fractions from mouse and rat liver in the Miller A Miller. 1973b; Janiaud. Delaforge. Levi, presence of 3'-pbosphoadenosine-3'-phosphosulphate Maume A Padieu. 1977; Stillwell. Carman, Bell A (Wislocki, Borchert. Miller A Miller. 1976k These Horning. 1974), and a single study dealing with its workers also reported that alkaline digestion of hepa metabolism in humans has also been reported (Bene- tic protein, isolated from animals treated with l'-hy- detti. Malnoe A Broillet, 1977)l Safrole is extensively droxysafroie. released a metabolite which appeared to metabolized, giving rise to a large number of metab be 3-methylmercapiotsosafrole, indicating the further olites. Metabolism involves essentially two major reaction of the hydroxylated metabolite with tissue routes, oxidation of the allyl side chain, and oxidation 5-proteins or with glutathione. l'-Hydroxysafrole. like of the methylenedioxy group with subsequent cleav its parent compound, safrole. undergoes oxidation of age to form the catechol (Fig. Ik the allyl group to yield r-hydroxy-2',3'-epoxide (Wis- k? 921001 RowVerK W-V:< 658 C Ioannides. M. Delaforge and D V Parke Fig. I. Major routes of safrole metabolism: la) propen-I'-ylphenol: (b) 1.2 -epoiypropylphenol: |c) allylphenol: Id) 2'_3'-epoypropylphenol; |e) allylcatechol: (f) 2',3-eposypropylcatechol; (g) 2'.3'-dihydroxypropylcalechol: (h) 2'-hydroxy-3'-(3.*-dihydroxyphenyl)propanoic acid; (t) eugenol--<4-allyl-2methoxyphenol): (j) safrole--(4-allyl-U-tnethylenedioxybenzenel: (It) safrole epoxide: II) dihydroxysafrole:|m) l-methoxy-2-hydroxy-4-ailylbcnzetw:(n) I'-hydroxysafrole; (o) 1 -hydroxy-2'J'-poxysafrole: (pi tnhydroxysafrolc: (q) 3'-hydroxysafrolc; (r) IJ-methylenedioxybenzene-4'-acstic acid: (s) I'-oxosafrole: (t) 3'-dialltylamino-2'.3'-dihydro-r-oxosafrolc. lock) et al. 1976), and incuhaiion of l'-hydroxysafrole with mouse- and rat-liver microsomal preparations and NADPH generated the hydroxy-epoxide. How ever. free l'-hydroxyepoxysafrole has not been detected in rico. although the glucuronic acid conju gate has been detected by gas-liquid chromatogra phy-mass spectrometry of its trimethylsilyl derivative in the urine of animals dosed with safrole (Levi. Janiaud. Delaforge. Moraot, Maume & Padieu. 1977). This hydroxy-epoxide also serves as a substrate of epoxide hydratase. being converted to the trihydroxysafrok (Delaforge et at. 1976; Stillwell et al. 1974). Administration of safrole or l'-hydroxysafrole to animals or man pve rise to the excretion of its isomer. 3-hydroxysafrok (Benedetti et at. 1977; Janiaud et at. 1977: Peek St Oswald. 1978), which is believed to result from rearrangement of the l'-hydroxysafrok during enzymic hydrolysis with 0-glu curonidase iBorchen. Miller St Miller. 1971). Other metabolites of l'-hydroxysafrole include 3.4-methylenedioxyphenyl vinyl ketone, also known as l'-oxosafrok (Peek & Oswald. 1978k and the formation of this metabolite was also indicated by the excretion of small amounts of adducts of oxosafrole with second- VI ary amines (McKinney, Oswald. Fishbein & Walker. 1972: Oswald. Fishbein Sl Corbett, 1969; Oswald. Fishbein. Corbett St Walker. 1971). Oxidation of the methvienedioxy group The principal route of metabolism of safrole is through cleavage of the methyknedioxy group, the major metabolites being allylcatechol and its isomer. propenykatechoL Eugenol and its isomer 1-methoxy2-hydroxy-4-allylbenzene have been detected as minor metabolites in the rat, mouse and man (Benedetti et al. 1977; Janiaud et at. 1977; Stillwell et al. 1974). The intact allyl side chain of allylcatechol may be oxidized to yield ZJ'-epoxypropykatechol, which serves as a substrate for epoxide hydratase and is hydrated to 2'.3`-dihydroxypropylcatechoi; this in turn is oxidized to the corresponding propanoic arid (Delaforge et al. 1976: Stillwell et al. 1974). The epoxide of allylcatechol may also be generated from the cleavage of the methyknedioxy group of the safrok epoxide. Using sensitive techniques, such as high-resolution capillary columns, T.2`-epoxypropylphenol and its isomer TJ'-epoxypropytphenoL as well as 2\J'-epoxypropylcatcchol have been detected as minor metabolites in rat urine (Delaforge er al. 1976). These epoxides are ^21001 RowVf - L) >. Walker. Oswald, -a/role is oup. (he ts isomer, -methoxyas minor icdetti ci 1974). The e oxidized ves as a rated to ' oxidized orge rr al. illylcate* of Ihe --e. Using i capillary ' isomer ypropylolites in oxides are It * j 4 i 4 < ? Safrole metabolism and carcinogenicity 659 poor substrates for epoxide hydratase. Finally the et al. 19761 and when injected into newborn mice this acids. methylene-3.4-dioxyphenylacetic acid and compound gave rise to liver tumours, similar to those 3.4-dihydroxyphenylacetic acid, have been detected in induced by l'-hydroxysafrole (Borchert et al. 1973a). the urine and bile of animals treated with safrole. However, this metabolite could not be detected fol The cleavage of the methylenedioxy ring and the lowing incubation, of l'-hydroxysafrole with micro metabolism of the allyl group involve the hepatic tomes or cytosol from rat or mouse liver in the pres microsomal mixed-function oxidases (Casida. 1970; ence of acetyl-CoA (Wislocki et al. 1976). Another Hodgson St Philpot, 1974). Administration to rats of possible ultimate carcinogen, l'-oxosafrole. did not the typical inducers of the mixed-function oxidases, result in carcinogenicity when administered orally to phenobarbital and 3-methylcholanthrene. resulted in rats (Wislocki er al. 1977). increased urinary excretion of epoxypropylphenol The bacterial system devised by Ames (Ames, epoxysafrole and t'-hydroxysafirole (Borchert el al. McCann St Yamasaki 1973) has been used exten 1973b; Janiaud et al. 1977), and phenobarbital stimu sively in studies to establish the identity of the ulti lated the production of other epoxy and hydroxy de mate carcinogens) of safrole. With the standard test rivatives and the conjugation of metabolites with glu system, safrole does not give a positive mutagenic re curonic acid (Janiaud et al. 1977). sponse (McCann, Choi Yamasaki St Ames. 1973; Swanson, Chambliss, Blomquisi Miller St Miller. 1979; Wislocki et al. 1977). However, when safrole Carcinogenicity of safrole was pre-incubated with liver microsomes from 3-tnethylcholanthrene-treated animals, a positive The carcinogenic properties of safrole have been mutagenic response was obtained (Dorange, Janiaud. the subject of several studies and the compound is Delaforge, Levi St Padieu. 19781 although these find described as a weak hepatocardnogen in both mice ings could not be reproduced by other workers and rats (Borchert er al. 1973a; Epstein, Fujii, Andrea (Swanson et al. 1979). Green St Savage (1978) & Mantel, 1970; Hagan, Jenner, Jones, Fitzhugh. obtained a positive mutagenic response for safrole in Long, Brouwer St Webb, 1963; Homburger, Kelley, the Ames test using other bacterial strains and a Baker St Russfield, 1962; Homburger et al. 1961; mouse-liver microsomal preparation, and also in a Long, Hansen St Nelson, 1961; Parke St Gray, 1978). host-mediated system in vivo. When fed to rats for 2 yr, a diet containing less than I'-Hydroxysafrole is more mutagenic than safrole 1000 ppm safrole caused minimal hepatic damage, but is still a relatively weak mutagen. Its mutagenicity malignant changes being evident only at higher doses was increased in the presence of an activating system, (Hagan er at. 1963; Long et al. 1963). The isomer, indicating that it is metabolized, at least partly, to isosafrole, and dihydrosafrole exhibited even weaker more potent mutagen(s) (Swanson et al. 19791 How hepatocarcinogenicity (Hagan er ai 1963); however, ever, other workers reported no positive mutagenic the latter compound fed for long periods to rats at a response either in the presence or absence of an acti dose of 5000 ppm in the diet produced benign and vation system (Dorange, Delaforge, Janiaud St malignant oesophageal tumours. Short-term adminis Padieu, 1977; McCann et aL 1975; Wislocki er al. tration of high doses of all three compounds resulted 1977). The low mutagenicities of safrole and its in extensive gross pathological damage (Taylor, 1'-hydroxy derivative probably reflect the low rates of Jenner St Jones. 1964). Liver is not the only tissue metabolism observed in vitro with hepatic micro damaged by safrole; lymphomas and adenomas of the somes (Wislocki er aL 1976). In contrast, all of the lung have also been described (Taylor et al. 1964). epoxides of safrole that were investigated were found Neither allylbenzene nor methylenedioxybenzene to be mutagenic in the Ames test, in the absence of an show any significant toxicity when compared with activation system (Dorange et aL 1977; Swanson et al. safrole (Hagan et ai. 1963), indicating that both the 1979; Wislocki et aL 1977). I'-Acetoxysafrole, a postu methylenedioxybenzene group and the allyl side chain lated ultimate carcinogen, was also directly mutagenic are concerned in the manifestation of safrole toxicity. in the Ames test (McCann et al. 1975; Wislocki et al. The monohydroxylated derivative of safrole, 1977) but no significant mutagenic response was r-hydroxysafrote. is more hepatotoxic and hepatocar- obtained from dihydrosafrole. 3'-hydroxyisosafrole. anogexuc to animals than is the parent compound 3 -acetoxyisosafrole and l'-oxosafrole. with or without when fed at the same dietary level indicating that it activation (Wislocki er at. 1977). may act as a proximate carcinogen of safrole (Borchert er al. I973a.b; Wislocki et al. 1976; Wis- lockl Miller. Miller. McCoy St Rosenkranz. 1977). Interactions of safrole with cytochrome P-450 When administered to rats. I'-hydroxysafrole labelled with tritium gives (ise to tritium-labelled DNA. RNA and protein, indicating the covalent binding of the compound or of a further metabolite. Furthermore, Safrofe. as a substrate of the hepatic microsomal mixed-function oxidases, interacts with cytochrome P-450, giving rise to a type l spectral change (Frank application of the electrophilic l'-hydroxysafrole epoxide, derived from l'-hydroxysafrole. to mouse skin followed by repeated applications of the tumour lin, 1971). In the presence of either NADPH and oxy gen. or cumene hydroperoxide, it is transformed into a reactive intermediate which interacts with the sixth promoter, croton oil. resulted in the formation of skin papillomas (Wislocki et aL 19771 The reactivity of I'-acetoxysafrole. with nucleosides and with methio ligand of fern- and ferrocytochrome P-450 to yield characteristic complexes (Ekombe, Bridges, Gray, Nimmo-Smith St Netter. 1975: Franklin. 1971 St nine led to speculation that this might also act as an 1976; Kulkarni St Hodgson, 1978; Parke & Rahman. ultimate carcinogen (Borchert et aL 1973b; Wislocki 1971k The complex formed with reduced cytochrome iter, I*-5--t 921001 RowVern 0H8,1 660 C Ioannioes. M. Delaforge and D. V Parke P-450 exhibits absorption maxima at 427 and 455 nm. while only one absorption maximum at 437 nm is ob served with the oxidized cytochrome (Elcombe et ai. 1975; Gray A Parke, 1973; Hodgson & Philpot, 1974; Lake A Parke. 1972). Safrole does not form the complex when incubated with microsomes plus dithionite or NADH, indicating that the ligand is formed by a reactive intermediate of safrole and not by safrole itself (Elcombe et al. 1975). The formation of the complex in vivo has also been shown to occur in rats and mice following administration of safrole (Delaforge. loannides A Parke. 1980a; Fennell Sweatman A Bridges, 1980; Parke A Rahman, 1971). The nature of the reactive safrole intermediates remains unclear, but it is generally believed that the complex is formed between the cytochrome and a carbene generated from the hydroxylation of the methylenedioxy group with subsequent loss of water (Mansuy. Battioni, Chottard A Ullrich, 1979; Nastainczyk, Ullrich A Sies. 1978; Ullrich. Nastainczyk A RuH 1975). Carbenes are known to have a high affinity for reduced cytochrome P-450 (Mansuy, Nastainczyk Sc. Ullrich, 1974). Other possible reactive intermediates for safrole have been suggested, such as carbanions formed by removal of a proton from the methylene group (Ullrich St Schnabel 19731 radicals generated following the homolytic scission of the methylenedioxy group (Hansch. 1968X and benzodioxolium ions produced following the loss of a hydride ion from the methylenedioxy bridge (Hennessy, 1965). Experimental evidence for the involvement of hydroxyl radicals in the formation of the ligand complex of safrole and cytochrome P-450 The formation of the reactive intermediate that gives rise to the formation of the safrole-cytochrome P-450 ligand complex may involve hydroxylation by cytochrome P-450 or interaction with hydroxyl or other radicals. Recent work has demonstrated that NADPHcytochrome P-450 reductase can generate hydroxyl radicals (Lai. Grover St Piette, 1979) To investigate the role of hydroxyl radicals in the formation of the safrole ligand complex, we incubated hepatic micro somes with NADPH and safrole in the presence of scavengers of the active forms of oxygen. In addition to the study of the effects on safrole, the microsomal oxidation of methiona! to ethylene, a reaction effected by hydroxyl radicals, was also investigated. Male Wistar albino rats (150-200 g) received a daily intraperitoneal administration of phenobarbital (80 mg/kg) for 3 days and were killed 24 hr after the last administration. Hepatic microsomal preparations were prepared as previously described (loannides A Parke. 1975). To study the generation of the ligand complex in vitro, microsomal fractions (1 mg pro tein/ml) were incubated with safrole (004-05 mM| and various oxygen scavengers at concentrations shown in Table 1 (with the data obtained with 03 mM-safroiel Following addition of NADPH (05 mMl the forma tion of the ligand complex was monitored for 2 min by measuring the optical difference between 455 and 490 nm using a double-beam dual-wavelength record ing spectrophotometer. The antioxidants butylated hydroxytoluene (BHT) and ascorbic acid inhibited the formation of the safrole complex with cytochrome P-45Q, suggesting the requirement for oxidation in the formation of the complex. The hydroxyl-radical scavengers mannitol dimcthylsulphoxide and methional inhibited the for mation of the ligand complex by 30% (Table II Lineweaver-Burk presentations, such as that for methional in Fig. 2. showed that with these four com pounds inhibition was likely to be of the competitive type. In contrast, the inhibition by catalase and super oxide dismutase was uncompetitive, indicating that these do not compete directly with safrole for hydro gen peroxide. It is possible that catalase, by removing HjOj. prevents its further conversion to hydroxyl radicals in the presence of ferrous ions (Walling. Partch A Weil 19751 The involvement of the OH- radical in the forma tion of the ligand complex was further indicated by the decreased evolution of ethylene from methional in the presence of safrole. Aliquots of a liver-microsomal preparation from phenobarbital-treated rats (3 mg protein/ml 0-1 m-phosphate buffer. pH 7-4) were incu bated with an NADPH-generating system (2 janol NADP, 20 fimol glucose 6-phosphate and 2 units glu cose 6-phosphate dehydrogenase) in rubber-sealed tubes and the reaction was started by -2 `.iipa of methional (0-04 mui Ethylene evolution was deter mined by removing 1-ml aliquots and injecting them into a Packard 409 gas chromatograph equipped with Table I. Effect of oxygen scarengers on the formation of the safrole complex with cytochrome P--SJO in rats pretreated with phenobarbital Oxygen scavenger Concentration Complex formation 1% of control) Butylated hydroxytoluene Ascorbic acid Oimethylsulphoxide Mannitol Methional Catalase Superoxide dismutase 60 pm 600 fim 10 mis 6mm <00 pm 3000IU 5001U 3S 68 70 71 <6 72 80 Incubations were earned out at a safrole concentration of 03 am. Reactions were started by addition of NADPH (OS mul Control incubations contained O) mu safrole and no oxygen scavenger. 921001 RowVer'k ion effected ived a daily enobarbital tr after the reparations annides &. the ligand I mg pro5 mM) and s shown in iM-safrolek the forma- for 2 min -'n 4S5 and gth record- tne (BHT) on of the suggesting ion of the mannitol *d the for(Table Ik that for four com* impetitive and superating that or hydroremoving > hydroxyl (Walling. ic formaJicated by rthional in crosomal ts (3 mg ere incuti (2 janol intts gluer-sealed -Jmon of as deterng them bed with 4 ! Safrole metabolism and carcinogenicity 661 Safrole (eM) Fig. 2. Liver microtomes from phenobarbital-treated rats (I mg protein/ml) were incubated with safrole (004--0-5 mu). The reaction was commenced by addition of NADPH (0-5 mM| and the initial velocity of the ligand complex formation was monitored for 2 min. a 2 m x 2 mm glass column packed with Carbosteve B (60-80 meshk Analysis was carried out isothermally at I70'C using a flame ionization detector and helium or oxygen-free nitrogen as carrier gas (25 ml/min), air and hydrogen flows were 300 and 30 mi/min respect ively. and the chromatograph was operated with an injection temperature of 200CC and detection tem perature of 250C; under these conditions ethylene had a retention time of 2-2 min, and the response was linear from OS pmol to at least 200 pmol/ml. Safrole at concentrations of 2 and 5mM inhibited, possibly competitively, the release of ethylene from methionai, confirming the involvement of the OH- radical in the metabolism of safrole (Fig. 3k This involvement of the OH- radical in the formation of a ligand complex between safrole and cytochrome P-450 is compatible with Use formation of a carbene (Fig. 4). IriaVtiT- of miwil f--rrWm oxidase activity by safrole The formation of the safrole carbene complex ts accompanied by a decrease in the concentration of free cytochrome P-450 and by a concomitant loss of mixed-function oxidase activity (Anders, 1968; Frank lin. 1972; Parke & Rahman, 1971k The characteristic ligand complex of reduced cytochrome P-450 and car bon monoxide, with the absorption maximum at 450 nm. is decreased, showing that carbon monoxide cannot displace the carbene from the sixth ligand of the haem iron. An inverse linear relationship (r a 096) exists between the ratio of free and total cytochrome P-4S0 concentrations, and the ratio of carbene-bound and total cytochrome P-450 concen trations in vivo when safrole was administered to the rat (Delaforge al. 1980ak A similar relationship was reported by the same workers for the formation of the complex in vitro. In the presence of some type I and reverse type I substrates, but not of type II substrates (which ligand to the haem moiety), the ferricytochrome carbene complex dissociates, resulting in removal of the 455-nm peak and restoration of the catalytic activity or the cytochrome (Delaforge. Ioannides & Parke. 1980b; Dickins, Elcombe, Moloney, Netter & Bridges, 1979; Elcombe er a/. 1975; Elcombe, Bridges & Nimmo-Smith, 1976; Gray & Parke, 1973; Ullrich, 1977k presumably because of removal of the haem-bound safrole metabolite. The extent of the increase in mixed-function oxidase following displacement is dependent on the amount of complex formed and the displacing conditions (Elcombe Dickins, Sweatman Sl Bridges, 1977k Furthermore, when the substrates that act as displacers are incubated with microsomal preparations isolated from animals pretreated with safrole or isosafrole, only very weak binding spectra with oxidized cytochrome P-450 are exhibited, but these undergo a time-dependent intensification result ing from the displacement of the safrole complex (Gray & Parke, 1973k Displacement occurs anaerobi cally and does not involve competition for the type I binding site, since the carbene ligands to the haem iron and is not bound to the apoprotein. However, as no type II substrates act as displacers, the dissociation of the complex may be initiated by the breaking of some additional linkage of the carbene with the apo protein. possibly involving the ally! side chain. Alternatively, the interaction of the displacing sub- ^2iuul tfoVVerK. ohh.i 66: C. Ioan.nides. M. Delatorge and D V Parke Fig. .V Incubation mixtures compnsed 10 ml liver mtcrosomes from phenobarbilaMreatcd rats t.tmgmli. and methional (0-002-04 mu l. The reaction was commenced by addition of an NADPHgencriiting system (2 jimol NAOP. 20/imol glucose 6-phosphate and 2 units glucose-6-phosphale de hydrogenase). Evolution of ethylene as determined by GLC as described. strate with the type I site may lead to a conformatio nal change of the cytochrome, resulting in the loss of the 'safrole' ligand and conversion of the cytochrome to the htgh-spin state. In our studies (unpublished data. 19811 we have found that the carcinogen benzo[a]pyrene can also act as a displacer to remove the carbene and restore mixed-function oxidase activity. The ability of benzo(u]pyrene to displace the carbene. and then of the free cytochrome to convert the benzo(a]pvrene to mutagenic intermediates was investigated in safrole- treated rats. Mutagenicity was determined by the Ames test (Ames et at. 1975) using Salmonella typhimurium strain TA98. The activation system was pre pared with microsomcs from safrole-pretreated rats (single daily intraperiionea! injections of 150 mg/kg for 3 days, the animals being killed 24 hr after the last administration). Displacement of the carbene was achieved by pre-incubation of the microsomes with benzo[a]pyrene (2/ig) for 15 min prior to addition of the NADPH-generating system to initiate metab olism. Following pre-incubation, the number of his- Safrets Safral* carton Safral* carton Mlti tfi cytodtrw* J-4S0 Fig 4. Possible route of formation of a safrole carbene-cytochrome P-4J0 complex. RowVerK $ I .4* f * . I i S f i i t ? s Hle- d by the la typhiwas pre dated rats 150 mg, leg the last ne was ntes with Idition of metabof his- i I fe:* t i M : ii -4S0 ! i I i I -1 Safrole metabolism and carcinogenicity 66) tidine revertants was more than doubled (from 128 to 288/plate, the number in the absence of any activation system being 47/platek demonstrating that the func tion of the cytochrome was restored when the ligand complex was dissociated. When biphenyl, a non mutagen. was`used as the displacing agent and no benzo[a]pyrene was added, no mutagenic response was observed, demonstrating that the displaced carbene itself did not elicit the mutagenic response. Pre vious workers have failed to displace the safrole carbene with benzol[a]pyrene (Elcombe et al. 1977k Dissociation of the safrole carbene ligand complex results spontaneously in the replacement of the 455 nm absorption maximum with a 450 nm peak, re sembling the CO-difTerence spectrum with cyto chrome P-450 (Delaforge St Coon, 1981; Hodgson & Philpot, 2974; Hodgson, Philpot, Baker St Mailman. 1973; Kulkarni St Hodgson. 1978). The presence of this CO was initially attributed to endogenous break down of haem (Schmid. 1973k but recent evidence suggests that it is generated from the methylenic car bon during the metabolism of methylenedioxyphenyl compounds by the cytochrome P-450 enzyme system (Yu. Wilkinson St Anders, 1980). Certain methylenedioxy compounds that generate CO do not elicit a 455 nm absorption maximum, showing that the car bene and CO are formed via different pathways, per haps from a common unstable intermediate (Yu et al. 1980). The formation of CO subsequent to displace ment of the safrole moiety from the ligand complex, indicates that the C atom of the methylenedioxy moiety is still intact, confirming, the involvement of a carbene and indicating that the product of the dis placement is likely to be an allylcatechol. The dis placed product may then bind covalently to cyto chrome P-450, inhibiting further formation of the ligand complex (Delaforge St Coon. 1981k Ligand complex formation with cytochromes P-450 and P-448 Cytochrome P-450 exists in several forms, differing in their spectral, immunological and electrophoretic properties as well as in their structures and substrate specificity (Guengrnch, 1979k One of these forms, namely, cytochrome P-448, predominates in foetal and neonatal rat liver and in malignant tissue, and is formed by the treatment of animals with hepatocarcinogens. induduig safrole and isosafrole (Parke, 1981k Cytochromes P-450 and P-448 form ligand complexes with safrole both in cioo and in citro, showing that they can convert safrole to the carbene and then inter act with it (Delaforge St Coon. 1981; Delaforge et at. 1980ak However, the safrole carbene complexes formed show different stability. The absorption maxi mum at 455 nm is formed less readily with cyto chrome P-448, indicating that either the rate of gener ation of the carbene, or its interaction to form the complex, is slower with cytochrome P-448 than with cytochrome P-450. Furthermore, dissociation by biphenyl of the safrole complex formed in nro in ani mals pretreated with the cytochrome P-450 inducer, phenobarbital resulted in a large increase (150%) in the cytochrome P-450-mediated 4-hydroxylation of biphenyl but in only a small increase (20*4) in the cytochrome P-448-mediated 2-hydroxylation of biphenyl (Delaforge et at. 1980b). In contrast, dissoci ation of the safrole complex formed in animals pre treated with the cytochrome P-448 inducer, 3-methylcholanthrene. resulted in a small increase (10%) in the 4-hydroxylation of biphenyl with no significant in crease in the 2-hydroxylation. indicating that dis placement of the ligand from the cytochrome P-448 form is less facile than displacement from the cytochrome P-450 complex. Inhibition of the mixedfunction oxidases by piperonyl butoxide, another methylenedioxy compound, was more marked in ani mals pretreated with phenobarbital than in those treated with 3-methylcholanthrenc. indicating that the piperonyl butoxide carbene also interacted more readily with cytochrome P-450 to form a complex (Anders, 1968; Franklin. 1972). Similar findings have been reported for SKF-525A and amphetamine ligand complexes (Buening St Franklin. 1974; Franklin. 1974). The ligand complex formed with cytochrome P-450 is metastable, 50% being destroyed within 6 hr follow ing safrole administration to rats (Delaforge et al. I980ak In contrast, the complex with cytochrome P-448, although more slowly formed, is more stable, no degradation being evident in the first 6 hr. if indeed CO generation is directly related to the ligand complex formation (Yu et al. 1980) these findings may expltun why rat-liver mkrosomes induced with phe nobarbital, but not those induced with 3-methykholanthrene, lead to CO production on dissociation of the safrole carbene complex. A novel baemoproteia formed by safrole and tsosafroie Administration of safrole to animals leads initially to a marked inhibition of the mixed-function oxidases (Anders, 1968; Fujii, Jafle, Bishop, Amok), Mackin tosh St Epstein. 1970; Hodgson St Casida, 1961; Nakatsugawa St Dahtn, 1967k The inhibition is achieved through two different mechanisms, on the one hand competitive inhibition by safrole itself oT the substrate for metabolism, and on the other formation of the safrole carbene ligand complex, preventing the ready hydroxylation of the substrate. However, like many inhibitors, safrole and isosafrole also stimulate the synthesis of new enzymic protein and act as potent inducers of the mixed-func tion oxidase system (Lotlikar & Wasserman. 1972: Parke St Rahman. 1970k The pattern of induction has characteristics of both the barbiturate and the poly cyclic aromatic hydrocarbon inducers (Fennell et at. 1980; Gray, Parke. Grasso St Crampton. 1972: Lake St Parke, 1972: Wagstaff St Short. 1971k Administra tion of a safrole-containing diet (025%) to rats resulted in induction of hepatic cytochrome P-450 and cytochrome P-448 activities within 7 days I Parke St Gray. 1978k Extrabepatic mixed-function oxidase activities, such as those of the small intestine and kid ney, were also induced by isosafrole (Lake. Hopkins. Chakraborty. Bridges St Parke, 1973k The full extent of the inducibility of the mixed-function oxidases is only evident when the carbene ligand complex with cytochrome P-450 is dissociated to release the free cytochrome. In rats pretreated with safrole. there were 0981, 664 C. Ioannides. M. DelaTORge and D. V. Parke increases in both cytochrome P-450 and P-448 activi ties following dissociation of the ligand complexes with biphenyl (Delaforge et of. 1980b). These obser vations indicate that safrole induces a haemoprotein having overlapping activities of cytochrome P-450 and .cytochrome P-448, or may induce a mixture of the two. Indeed, using SDS-disc gel electrophoresis, it has been demonstrated that isosafirole induces a novel haemoprotein. which is distinct from those induced by phenobarbital or by 3-methylcholanthrene but which may be similar to the minor protein band induced by 3-methylcholanthrene (Fennell. Dickins & Bridges. 1979). Further work has led to the isolation from isosafrole-pretreated rats, and the purification of a unique form of hepatic cytochrome P-4S0 existing in the form of an isosafrole metabolite complex (Ryao. Thomas & Levin. 1980). The cytochrome P-448 induced by 3-methylcholanthrene has also been shown to contain'a bound moiety of the inducing agent (Schenkman. Greim. Zange ft Remmer. 1969). Conclusions This ligand complexing and covalent binding of reactive intermediates with the haem and protein moieties, respectively, of cytochrome P-450, may be fundamental to the mechanism of carcinogenesis. The structural and functional properties of the cyto chrome are substantially changed by this binding which, since ribosomes are attached to the endoplas mic reticulum at cytochrome P-450, may be associ ated with the loss of ribosomes known to occur fol lowing treatment with safrole or other carcinogens (Parke. 1981). Chemical carcinogenesis is known to be associated with increases in hepatic cytochrome P-448 and simultaneous loss of cytochrome P-450 activity (Parke. 1981) and could, through loss of ribosomes, result in impairment of glycoprotein synthesis and so contribute to the process of malignant transformation by epigenetic mechanisms (Parke. 1981). 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