A.Ruth Steward

Metabolism of benzo[a]pyrene and (−)-trans-benzo[a]pyrene-7,8-dihydrodiol by freshly isolated hepatocytes of brown bullheads

The metabolism of [3H]benzo[a]pyrene (BP) and (-)-trans-[14C]7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP-7,8-diol) was studied in freshly isolated hepatocytes of the wild benthic fish, brown bullhead (Ictalurus nebulosus). Bullhead hepatocytes incubated with 40 microM [3H]BP for 1 h metabolized BP to water soluble metabolites which were separated on silica gel t.l.c. plates to reveal conjugates with glucuronic acid, glutathione, and sulfate (51%, 14% and 4% of total metabolites, respectively). Additional metabolites that were extractable with ethyl acetate were separated by reversed phase HPLC to reveal only two major metabolites: BP-9,10-dihydrodiol and BP-7,8-diol (13% and 2.6% of total metabolites, respectively). Hepatocytes isolated from individual fish displayed an 11-fold variability in the rates at which they metabolized BP (756 +/- 167 pmol x mg dry wt-1 x h-1), which correlated negatively (r = -0.7, P less than 0.01) with an 18-fold variability in the glycogen content of the cells. Hepatocytes isolated from the same fish, in parallel incubations under the same optimum conditions, metabolized BP-7,8-diol 4.5-fold faster than they metabolized BP. The variability in the rate of BP-7,8-diol metabolism was about 7-fold. Major metabolites included glutathione conjugates, glucuronides and sulfates (35%, 25% and 30% of total metabolites, respectively). These conjugates, like those formed from BP, were degradable with gamma-glutamyltransferase, beta-glucuronidase and arylsulfatase, respectively. Ethyl acetate extractable metabolites were predominantly isomeric benzo-ring tetrahydrotetrols (9% of total metabolites). In summary, this study indicates that during short-term incubations bull-head hepatocytes metabolize BP and BP-7,8-diol primarily to conjugated derivatives. The usefulness of thin-layer chromatography for the convenient determination of the rate of BP-7,8-diol metabolism is demonstrated.

Disposition and metabolic fate of benzo[a]pyrene in the common carp

The biological fate of intraperitoneally administered benzo[a]pyrene (BP) was investigated in the wild strain of common carp (Cyprinus carpio) maintained in flowing water. The tissue distribution of BP-derived radioactivity was determined at 24 and 72 h after treatment. Bile contained high concentrations of BP-derived radioactivity (7.9 μg BP equivalentsg bile at 72 h), which amounted to 25% of the radioactivity found in the fish at this time point. The major components of bile collected at 72 h were glucuronides (54%), sulfates (12%) and unmetabolized BP (14%). The potentially genotoxic metabolite BP-7,8-dihydrodiol and its glucuronide represented 0.7 and 20%, respectively, of the radioactivity of the bile at 72 h. High concentrations of BP-derived radioactivity were also present in liver and gonads (1,060 and 843 ng equivalents of BPg tissue at 24 h, respectively). Muscle contained 10% of the BP-derived radioactivity found in the fish (46 ng eqg at 24 h). No significant change was found between 24 and 72 h in the concentrations or proportional amounts of BP-derived radioactivity except in liver, in which the proportional amount decreased from 19 to 10% of the total found in the fish. In muscle, unmetabolized BP represented 95% of the radioactivity that could be extracted with ethyl acetate and characterized by HPLC. At 24 h BP-7,8-diol represented 0.6% of this extractable radioactivity in muscle and 1.0% of the extractable (0.4% of the total) radioactivity in liver. These data indicate that carp tissues produce significant amounts of BP-7,8-diol and its glucuronide, potentially capable of being converted (after hydrolysis of the glucuronide) to active metabolites capable of alkylating DNA and other macromolecules. However, only relatively small amounts of covalently bound radioactivity were found (amounting to 4% and 7% of the total radioactivity present in muscle and liver, respectively).

Comparative metabolism of benzo[a]pyrene and (−)benzo[a]pyrene-7,8-dihydrodiol by hepatocytes isolated from two species of bottom-dwelling fish

Abstract In order to elucidate the biochemical bases of species differences in susceptibility to hepatotumorigenesis induced by polycyclic aromatic hydrocarbons (PAHs) such as benzo[a]pyrene (BP), we investigated the metabolism of [ 3 H]BP and its proximate carcinogenic metabolite (−)BP-7,8-dihydrodiol (BP-7,8-diol) by two benthic fish: brown bullheads ( Ictalurus nebulosus ) (a susceptible species) and mirror carp ( Cyprinus carpio ) (not known to be susceptible). Freshly isolated hepatocytes prepared from both species efficiently converted both BP and BP-7,8-diol (the proximate carcinogenic metabolite of BP) to glutathione conjugates, glucuronides and sulfates and polyhydroxylated compounds. Nevertheless, carp hepatocytes (which metabolized both BP and BP-7,8-diol about 2-fold faster than did bullhead hepatocytes) produced several-fold larger amounts of DNA adducts from both substrates, suggesting the formation by carp liver of significantly larger amounts of reactive intermediates that bind to DNA. The apparently greater susceptibility of brown bullheads to PAH-induced carcinogenesis thus awaits further clarification.

Metabolism of benzo[a]pyrene and persistence of DNA adducts in the brown bullhead (Ictalurus nebulosus)

1. The in vitro metabolism of [3H]benzo[a]pyrene (BP) and [14C]benzo[a]pyrene-7,8-dihydrodiol (BP-7,8-diol) by liver of brown bullhead (Ictalurus nebulosus) was characterized, as was the formation and persistence of BP-DNA adducts in vivo. 2. Compared to rat liver microsomes, bullhead liver microsomes produced relatively larger amounts of BP-7,8-diol (predominantly the [-] enantiomer) and smaller amounts of of BP-7,8-diol (predominantly the [-] enantiomer) and smaller amounts of BP-4,5-diol. 3. BP phase I metabolites were efficiently converted by freshly isolated bullhead hepatocytes to conjugates, predominantly glucuronides. 4. BP-7,8-diol was metabolized by hepatocytes 4-fold more rapidly than was BP and was converted to approximately equal amounts of glucuronides, glutathione conjugates and sulfates. 5. BP-DNA adducts formed in bullhead liver with a lag time of several days and maximum adduct formation at 25-30 days. The major adduct was anti-BPDE-deoxyguanosine.

Metabolic fate and disposition of 2-acetylaminofluorene in rainbow trout, Oncorhynchus mykiss

Abstract Rainbow trout, Shasta strain, in contrast to several mammalian species, are highly resistant to hepatocarcinogenesis by 2-acetylaminofluorene (AAF). In order to examine factors that may contribute to this resistance, we investigated the metabolic fate of AAF. Trout treated orally with [14C]AAF rapidly eliminated AAF derivatives via the liver (in which the concentration of AAF-derived radioactivity decreased 4-fold between 8 and 24 h) into the bile which, at 24 h, contained 83% of the recovered radioactivity. At 24 h only 3.5% of the AAF-derived radioactivity retained by the liver represented unmetabolized AAF. In the bile, unmetabolized AAF and 2-aminofluorene accounted for 3.3% and 4.8%, respectively, of total biliary radioactivity, while glucuronide and sulfate conjugates accounted for 63% and 12%, respectively. Analysis of extracts of the deconjugated metabolites revealed that conjugates of 7-OH-AAF and 5-/8-/9-OH-AAF accounted for 49% and 14%, respectively, of total radioactivity. The potentially carcinogenic metabolite N-OH-AAF occurred predominately as the glucuronide conjugate which amounted to only 2% of total metabolites. These data demonstrate that the liver of Shasta trout is highly efficient in the detoxification and elimination of AAF and its derivatives.

Metabolism of 2,3,7,8-tetrachlorodibenzofuran by rainbow trout (Oncorhynchus mykiss)

Abstract Bile was collected 7 days after treating rainbow trout with 1 ppb [ 3 H]2,3,7,8-tetrachlorodibenzofuran (TCDF). HPLC analysis of methylated extracts of this bile, before and after hydrolysis with β-glucuronidase and arylsulfatase, revealed that the major metabolite was the glucuronide conjugate of 4-OH-2,3,7,8-TCDF, which amounted to 38% of the TCDF-derived radioactivity in bile. In order to examine the inherent capability of trout liver to metabolize this compound under defined conditions, [ 3 H]TCDF was incubated with freshly isolated hepatocytes prepared from rainbow trout pretreated with 3-methylcholanthrene (an inducer of liver monooxygenase enzymes). The maximum (initial) metabolism observed was 20 pmol TCDF equiv/mg dry wt in 30 min. Extraction and HPLC analysis of cells and medium revealed one major metabolite: 4-OH-2,3,7,8-TCDF.

Metabolism of 2-acetylaminofluorene by rainbow trout

In order to examine factors that may contribute to the reported resistance of rainbow trout, Shasta strain, to the well-known hepatocarcinogenic effects of 2-acetylaminofluorene (AAF), the in-vitro and in-vivo metabolism of [14C]AAF in trout has been examined. Trout (compared to rat) liver microsomes metabolized AAF more efficiently, producing 3-fold larger amounts of ring-hydroxylated metabolites (7-hydroxy-AAF and 5-hydroxy-AAF), but 5-fold less N-hydroxy-AAF. Freshly isolated trout hepatocytes extensively metabolized AAF to form the same ring-hydroxylated metabolites and their respective glucuronide and sulfate conjugates. N-OH-AAF (plus its conjugates) and covalently-bound AAF derivatives amounted, respectively, to < 1% and 1.4–1.6% of total metabolites. Liver DNA of trout treated with AAF contained a single AAF-DNA adduct identified as N-(deoxyguanosin-8-yl)-2-aminofluorene (the major persistent AAF-DNA adduct found in rat liver). The level of this adduct (12 attomoles/μg DNA) was about 1000-fold lower than the level of AAF-DNA adduct previously reported in rat liver. The data show that trout liver, compared to rat liver, is considerably less efficient in metabolizing AAF to carcinogenic metabolites, and more efficient in forming nontoxic products, thus possibly explaining, in part, the resistance of trout to AAF-induced hepatocarcinogenesis.