Xenobiotica the fate of foreign compounds in biological systems

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The metabolism of the carcinogen dibenz[a,j]acridine in isolated rat hepatocytes and in vivo in rats H. K. Robinson, C. C. Duke, G. M. Holder & A. J. Ryan To cite this article: H. K. Robinson, C. C. Duke, G. M. Holder & A. J. Ryan (1990) The metabolism of the carcinogen dibenz[a,j]acridine in isolated rat hepatocytes and in vivo in rats, Xenobiotica, 20:5, 457-470 To link to this article: http://dx.doi.org/10.3109/00498259009046861

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XENOBIOTICA,1990, VOL.

20, NO. 5, 457-470

The metabolism of the carcinogen dibenz[a, jlacridine in isolated rat hepatocytes and in vivo in rats H. K. ROBINSON, C. C. DUKE, G. M. HOLDER? and A. J. RYAN

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Department of Pharmacy, University of Sydney, NSW 2006, Australia Received 20 July 1989; accepted 10 December 1989 1. ’H-Dibenz[a, jlacridine (DBAJAC) metabolism occurred readily in uitro in incubations with hepatocytes from phenobarbital-pretreated, 3-methylcholanthrene-pretreated and untreated rats, with the formation of water-soluble conjugates and unconjugated metabolites. 2. For incubations of ’HH-DBAJAC with hepatocytes the major organic solvent-soluble metabolites found with and without j-glucuronidase/arylsulphatasehydrolysis were the phenols, 3-hydroxy-DBAJAC, and 4-hydroxy-DBAJAC, and the proposed proximate carcinogen, trns-3,4-dihydroxy-3,4-dihydro-DBAJAC. The latter comprised 3446% of the total organic solvent-soluble metabolites.

3. In contrast to results previously reported for rat hepatic microsomes, the K-region 5,6-oxide, and its dihydrodiol were minor metabolites detected after hepatocyte incubations. 4. Faecal excretion accounted for the bulk of radioactivity after i.p. doses of ’HDBAJAC (0.5 mg/kg), and i.v. doses (0.5 mg/kg) were rapidly excreted into the 6 h bile. The organic solvent-soluble fraction obtained after enzymic hydrolysis of bile (-25% of excreted radioactivity) was subjected to h.p.1.c. It contained polar secondary oxidation products and virtually no 3,4-dihydrodiol. 5. Experiments conducted with greater hepatocyte densities (10’ cells/ml) and longer incubation times showed increased extents of metabolism, DNA and protein binding of radioactivity which paralleled the extent of metabolism. Very considerable metabolism of the 3,4-dihydrodiol occurred by the end of the incubation period.

Introduction Investigations of chemical carcinogenesis amongst polycyclic aromatic compounds have probed for differences in the metabolism and bioactivation between compounds regarded unquestionably as animal carcinogens and those with questionable carcinogenicity. Benzo[a]pyrene (BP) is activated through its trans7,8-dihydrodiol (all diols mentioned are trans) to its ultimate tumorigen, the anti7,8-diol9,10-oxide (Thakker et al. 1985 a) while benzo[e]pyrene is not carcinogenic. The 9,lO-dihydrodiol and related diol epoxides of benzo[e]pyrene are weakly active (IARC 1983, Buening et al., 1980, Slaga et al. 1980, Chang et al. 1981) but little dihydrodiol (MacLeod et al. 1980)and virtually no diol epoxide (Wood et al. 1979) are produced metabolically using rat liver microsomes. Amongst other pentacyclic systems dibenz[a,h]anthracene is tumorigenic with ‘sufficient’ evidence of its carcinogenic properties while dibenz[a, jlanthracene and dibenz[a,c]anthracene are ‘questionable’ carcinogens with limited evidence of tumorigenicity (IARC 1983, Chouroulinkov et al. 1983). A study of the comparative metabolism of these three

t T o whom correspondence should be addressed. 0049-8254/90

0 1990 Taylor & Francis Ltd.

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Figure 1. The structure of dibenz[a, jlacridine (DBAJAC).

compounds indicated little conversion to tetraols derived from bay-region diol epoxides, although dibenz[a,c]anthracene and dibenz[a,h]anthracene are converted to dihydrodiols with bay-region double bonds, in significant amounts for the latter (Lecoq et al. 1989, Nordqvist et al. 1979). Dibenzo[c,h]carbazole is a local and systemic tumorigen converted mainly to phenols in incubations with liver microsomes and cultured hepatocytes (Perin et al. 1981, Stong et al. 1989). The isomeric dibenz[a,h]acridine and dibenz[a, jlacridine (DBAJAC, figure 1) both show ‘sufficient’ evidence of carcinogenicity (IARC 1983) while dibenz[c,h]acridine has not been reviewed. The latter was thought to be more tumorigenic than its isomers in a skin painting study (Lacassagne et al. 1956). All three compounds are converted by hepatic microsomal enzymes to dihydrodiols possessing a vicinal double bond in the bay-region in relative yields of 1 7 4 2 % (Thakker et al. 1985a, Gill et al. 1986, Steward et al. 1987). The 3,4-dihydrodiol formed metabolically from dibenz[c,h]acridine and DBAJAC in liver microsomes possessed an excess of 3R,4R-enantiomer (Thakker et al. 1985b, Duke et al. 1988). The present study shows that metabolism of DBAJAC in vitro with rat hepatocytes, and in vivo in rats, exhibits considerable differences in the extent of production of the 3,4-dihyrodiol, a putative proximate carcinogen.

Methods 14-,H-DBAJAC (Rosario et al. 1987a) was purified by reverse-phaseh.p.l.c., dissolved in toluene and the solution extracted with 0075 M-NaOH in 425% v/v dimethylsulphoxide (DMSO). The resulting material was >97% radiochemically pure by h.p.1.c. with specific radioactivity of 22.1 Cilmmol (818 GBqlmmol). A methanol-water gradient of 6040% over 20min, followed by 8CtlOO% methanol over lOmin using a 1Opm Brownlee RP-8 column (250 x 4 6 m m int. dim.) and a flow rate of 1.2mllmin was used. For all studies, except the DNA and protein binding experiments, it was diluted with unlabelled DBAJAC before use. Chromatographic standards, the isomeric tram-dihydrodiols of DBAJAC, the phenols 3-hydroxy-DBAJAC and 4-hydroxy-DBAJAC, DBAJAC-5,6-oxide, DBAJAC-N-oxide and the bis-5,6,8,9-oxides (Rosario et ul.1987 b) were availabie through syntheses and other chemicals were of the highest grade commercially available. fi-Glucuronidase (100 OOO U/ml) with arylsuphatase activity from Helix pomatia was obtained from Boehringer-Mannheim. Animals Male Wistar rats (150 g) were either untreated (controls) or dosed i.p. with 3-methylcholanthrene (MC) (20mg/kg) dissolved in corn oil daily for 2 days or with phenobarbital (PB) (60.80, and 100mg/kg) in isotonic saline daily for 3 consecutive days. The last doses of mixed-function oxidase inducer were given 24h before surgery. Viable hepatocytes were prepared as described by Stacey et al. (1980) and assessed for viability by Trypan blue exclusion (>85%). For in wiwo disposition studies, DBAJAC (0.5mg/kg) was given i.p. as a soh. in DMSO or by slow i.v. injection, to anaesthetized rats bearing biliary cannulae, and bile was collected hourly for 6 h. Rat urine and faeces were also collected daily for 7 days following 0 5 mg/kg i.p. administration of DBAJAC in DMSO (0.3 ml) from animals housed in all-glass Metabowls (Jencons,UK). Radioactivity was monitored by liquid scintillation counting of aliquots of bile, urine and HN0,-digested faeces (Wright et al. 1985).

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Hepatocyte dibenz[a,jlacridine metabolism

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Metabolism of DBAJAC by heflatocytes 3H-DBAJAC in acetone (finalconcn. in incubation mixture was 2% v/v) was added to suspensions of hepatocytes in incubation buffer at viable cell densities of 1 x 106/ml unless otherwise indicated. 3HDBAJAC concentrations were 5 - 5 0 ~ ~ The . incubation medium consisted of Leibovitz L-15 medium, pH 7.4, (Flows Labs, Sydney, 38.5 ml; Nuzzolo and Vellucci 1983) with foetal calf serum ( 5 ml, Flow) tryptose phosphate broth (5 ml, Flow), penicillin (100 U/ml) and streptomycin solution (100pglrnl; 1.0 ml) and 2 0 m glutamine ~ (0.5 ml). Incubations (4-10ml) were effected in 50ml conical flasks at 37°C beneath air or carbogen gas with gentle shaking. Incubation times were 20,lO and 3 min for cells prepared from untreated, PB-pretreated and MC-pretreated rats respectively. Total metabolism was measured by a hexane extraction technique of unchanged substrate (Gill et al. 1987). Organic solvent-soluble metabolites were determined after homogenization of the cells with a Potter-Elvejhem homogenizerand ethyl acetate extraction. Usually the homogenatewas treated in 005 M acetate buffer pH 5.0 with /?-glucuronidase/arylsulphatase(500 U /?-glucuronidase/mL)for 2 h at 37°C. The aqueous and organic solvent phases were assayed for radioactivity to determine the partitioning of metabolites; the bulk of the ethyl acetate was evaporated to dryness and metabolites were dissolved in dimethylformamide (5-15 pl) before reverse-phase h.p.1.c. In some experiments intracellular glutathione levels were decreased by a lOmin preincubation with diethyl maleate (1.16m~)at 37°C. High-performance liquid chromatography Metabolite distributions were determined by reverse-phase separation of components on a Hibar Lichrosorb RP-8 column (25 x0.4cm). For separation a water-methanol gradient and flow rate of 1.0ml/min were used. An initial linear 2842% methanol ramp over 40rnin was followed 15 min later by a second 10min linear increase to 60% methanol. After a further 20min isocratic elution a final ramp to 100% methanol over 5 min was employed. Detection was by U.V. absorption using a Hewlett-Packard HP 1040Ahigh-speed spectrophotometric detector (linear diode array) or by liquid scintillation counting. Protein and D N A binding of DBAJAC metabolites Undiluted 3H-DBAJAC (11 /AM, 5 x lO*dpm/ml) was incubated with hepatocytes (1 x lO’cells/ml) from untreated or MC-pretreated rats for up to 60 and 40min respectively. At various time points two samples of the mixture were withdrawn, one for the extent of metabolism determined by hexane extraction, and the other for DNA (Kirby and Cook 1967, Eastman et al. 1978) and protein (Stowers and Anderson 1984)isolation. The DNA was extracted twice with ethyl acetate before the h a l precipitation and washing with 70% ethanol, and the protein, which was isolated from the phenolic extracts obtained in the DNA isolation, was washed with methanol. DNA was determined using the fluorescent dye Hoechst 33258 (Cesaroneet at. 1979), protein was assayed by the method of Lowry et 01. (1951) and ’H-DBAJAC binding determined by liquid scintillation assay.

Results 3H-DBAJAC metabolism by hepatocytes DBAJAC metabolism was maximal in the three hepatocyte suspension preparations studied at substrate concentrations of 30-50 FM, and was linear with time to at least 20 rnin at 50 PM DBAJAC. The metabolic activities for hepatocytes from control, and PB- and MC-pretreated rats, were 038,069, and 1.60nmol substrate oxidized/l O6 cells per min, respectively, and displayed the expected increases of activity due to enzyme induction. Using values of the cytochrome P-450 content of control and MC-induced hepatocytes of 0-23 and 82 nmol/106 cells (Moldeus et al. 1978) the activities expressed per nmol of cytochrome P-450 are 1.5 and 1.8 nmol metabolized per min, and are similar to rat hepatic microsomal results which were 1-7and 2-5nmol metabolized/nmol P-450 per min (Gill et al. 1987), obtained under saturating conditions for substrate concentration. Water-soluble metabolites comprised about 25-30% of the total metabolites formed by control hepatocytes when about 60% of the substrate was metabolized, and this reached more than 50% of total metabolites when 80% of substrate was consumed by hepatocytes from MC-pretreated rats. Only about a 10% reduction (in absolute terms) in the water-soluble radioactivity occurred when the cell homogenates were treated with B-glucuronidaselarylsulphatasebefore ethyl acetate

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Hepatocyte dibenz[a,jlacridine metabolism

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Figure 2. The U.V. spectra of metabolites of DBAJAC formed by hepatocytes from livers of MC-pretreated rats (A) eluting at lOmin and (B) eluting at 12min on h.p.1.c. Metabolites (-), and authentic DBAJAC 4-hydroxy-DBAJAC (---).

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extraction, indicating that the bulk of the water-soluble metabolite fraction was thioether conjugates. When hepatocytes from MC-pretreated animals were preincubated with diethyl maleate to deplete glutathione the fraction of ethyl acetate-soluble metabolites reached 75%. Metabolites formed by hepatocytes from MC-pretreated animals which were ethyl acetate-soluble were identified by cochromatography with standards or by their U.V. spectra before quantification (table 1). In the quantitative data, obtained under substrate saturation conditions, some metabolites were pooled in metabolite fractions 3, 4, 5 and 6 because of their relatively low amounts. The U.V. spectrum of the metabolite eluting at 10min from the reverse-phase column (included in fraction no. 3) was similar to that of DBAJAC, while the spectrum of the 12 min metabolite was similar to the spectrum of 3-hydroxy-DBAJAC (but not the 4-phenol, figure 2). These were tentatively

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Table 2. Metabolites of DBAJAC-3,4-dihydrodiol formed by hepatocytes prepared from MCpretreated rats.

Fraction'

Percentage of ethyl acetate-soluble metabolitesb 29 3.0 11.9 41.1 0.7 41.3

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