S9 System Research Articles

Benzene and Benzene Metabolites as Embryotoxic Agents: Effects on Cultured Rat Embryos

Benzene and several of its metabolites were investigated for dysmorphogenic and embryotoxic effects after direct exposures of cultured whole rat conceptuses. Benzene produced no statistically significant effects at concentrations up to 1.6 mM. Inclusion with 1.6 mM benzene of an hepatic xenobiotic-biotransforming system (S9) resulted in only minor decreases in embryonic growth parameters and no detectable dysmorphogenesis. Phenol, a major benzene metabolite, also elicited only minimal embryotoxicity at 1.6 mM concentrations. However, inclusion of an S9 system with phenol resulted in significant dysmorphogenic and embryotoxic effects at concentrations as low as 0.01 mM. For phenol bioactivation, S9 from phenobarbital-induced rats was the most effective, with induction by pregnenolone-16 alpha-carbonitrile, isopropanol, Aroclor 1254, no inducer, and 3-methylcholanthrene following in order of effectiveness. Bioactivating activity resided solely in the microsomal fraction. Metabolites coeluting on HPLC with hydroquinone and catechol were the major metabolites generated from phenol by each S9 system, but no significant correlation between specific metabolite generation and embryotoxicity was apparent. Of the benzene metabolites studied, trans, trans-muconaldehyde exhibited the highest embryotoxic potency but was not detectably generated by any of the S9 systems. Hydroquinone, catechol, and benzoquinone were approximately equipotent, each producing 100% lethality at 0.1 mM. Combined additions to the culture medium of hydroquinone together with phenol resulted in greater than additive effects, indicating a possible synergistic interaction between these metabolites and suggesting that peroxidase activity may be important to the mechanism of phenol-elicited embryotoxicity.

Development of short-term mutagenicity test systems in vitro: metabolic activation of indirectly acting mutagens by three immortal rat hepatocyte lines.

The metabolic capacity to activate the indirectly acting promutagens aflatoxin B1, cyclophosphamide, benzo[a]-pyrene, 7,12-dimethylbenz[a]anthracene and dimethylnitrosamine into DNA-reactive metabolites was investigated in three immortalized rat hepatocyte cell lines (NRL cl-B, NRL cl-C and ARL) by analysing chromosome aberrations and sister chromatid exchange (SCE). In all three cell lines a significant clastogenic and SCE inducing response was observed after exposure to each test compound. Furthermore, activities of the two enzymes aryl hydrocarbon hydroxylase and aldrin-epoxidase, which play major roles in the cytochrome P450-dependent metabolism, could be determined in all cell lines. In contrast to the hepatocyte lines in V79 Chinese hamster cells, which were used as a reference cell line without any cytochrome P450 metabolizing capacity, no arylhydrocarbon hydroxylase or aldrinepoxidase activities were detected. A cytogenetic response to the test compounds was only observed in the presence of the exogenous activating system S9 mix. Due to the wide, efficient and stable spectrum of their metabolizing capacities, the tested rat hepatocyte lines offer promising perspectives as alternative assay systems for the detection of indirectly acting mutagens.

Effects of norharman on the mutagenicity of chlorophenylhydroxylamine and its metabolism with rat liver S9

o,p-Chlorophenylhydroxylamines (CPHAs) (10468-16-3, 823-86-9) only demonstrated mutagenicity in the presence of S9 mix and norharman (NOH) (244-63-3), as well as chloronitrobenzenes. The mutagenic activity of o-CPHA was 30 times higher than that of p-CPHA. When o-CPHA was preincubated with S9 mix without NOH, the mutagenic activity disappeared rapidly. The decrease in activity during the preincubation was suppressed by addition of NOH. HPLC analysis revealed that o-CPHA was metabolized to o-chloroaniline ( o-CA) (95-51-2) and that the metabolic reduction was inhibited by NOH. When microsomes containign NADPH were used instead of S9 mix, o-CPHA exhibited only very weak mutagenicity. The activity in the microsome system, however, was greatly enhanced by adding cytosol or ascorbic acid (50-81-7). These phenomena were only observed in the conventional plate incorporation method. In the case of the liquid incubation assay, in which test compound metabolism and tester strain mutation only occur in the liquid incubation medium, the mutagenic activity of o-CPHA in the microsome system with NOH was comparable to that in the S9 system, indicating that o-CPHA was activated by an enzyme in microsomes in the presence of NOH. Consequently, it was concluded that NOH not only affects the metabolic inactivation of o-CPHA to o-CA by S9, but also the metabolic activation of o-CPHA by microsomes. No appreciable enhancing effects of cytosol and ascorbic acid were observed in the liquid incubation assay, suggesting that these factors affect the stability of CPHA or an active metabolite. The microsome activation of o-CPHA was dependent on NADPH and oxygen; SKF-525A (62-68-0), metyrapone (54-36-4) and α-naphthoflavone (604-59-1) inhibited the mutagenic activity by about 50%, suggesting that cytochrome P-450 was involved in the metabolic activation.

Metabolic activation of organic extracts from diesel, coke oven, roofing tar, and cigarette smoke emissions in the ames assay

Four environmental emissions samples were ranked by their genotoxic potency in several bioassays. Although the relative potency of a series of automotive emissions (diesel and gasoline) in the Ames assay correlated well with the relative potency in mammalian cell and mouse skin, this was not the case for the coke oven, roofing tar, and cigarette smoke condensate (CSC) emissions. This study examines the role of metabolic activation in determining the difference between a microbial and a mammalian bioassay in ranking the genotoxic potency of these environmental emissions. Uninduced and Aroclor 1254-induced S9 from both rat and hamster liver were compared as the metabolic activator in the Ames assay with Salmonella typhimurium TA98. The diesel emissions sample was direct-acting while the other samples required activation. The standard S9 concentration (only Aroclor-induced rat, approximately 1.25 mg protein/plate) also produced the maximum mutagenic activity. Induced S9s produced higher mutagenic activity than uninduced. The hamster S9 gave significantly higher mutagenic activities than rat S9 for the coke oven and CSC. The relative potency of these four samples was not significantly different between the microbial (Ames), mammalian cell (mouse lymphoma), and tumor initiation (mouse skin) assays. These results suggest that the differences observed between the relative mutagenic activity of these emissions in the mammalian cell and microbial assays was not due to a lack of optimization of the S9 system but may be inherent in the different response of the indicator cells to different chemical classes.

In vivo and in vitro metabolism of the new anticancer drug bisantrene

The metabolism of bisantrene, a new anthracene anticancer agent active in the treatment of disseminated breast cancer, was studied in vitro using rat liver S9 preparations and in vivo in patients receiving the drug as treatment for their cancers. 14C-ring labeled bisantrene (248 mCi/40 mg) plus cold bisantrene were administered IV to cancer patients (260-340 mg/m2). Fractional urine samples were collected at various time intervals up to 120 h after drug administration and analyzed by HPLC. The percent of total 14C excreted as unchanged parent drug per ml urine ranged from 37 to 79% in the 0 to 24 h samples. The remainder of the radioactivity appeared chromatographically just prior to the bisantrene peak, indicating that compounds more polar than the parent were present as transformation products. Metabolism of bisantrene was also studied in vitro under oxic (O2) and hypoxic (N2) conditions, using commercially available Aroclor 1254 induced rat liver S9 preparations. Following N2 incubation at 37 degrees C for 1 h there was no evidence of metabolism, whereas there was more than 50% decrease in parent drug within 1 h following O2 incubation in the presence of NADPH generating system, suggesting that the metabolic process involves an oxidative reduction. HPLC chromatogram profiles of the mixtures exposed to the activated S9 system indicated that there were at least 3 polar metabolites. In vitro human tumor clonogenic assay showed that the biological activity of bisantrene decreased greater than 4-fold when the drug was incubated with S9 preparations in the presence of NADPH and O2, indicating that the transformation process leads to relatively inactive bisantrene metabolites.

Human lymphocytes assay: Cyclophosphamide metabolic activation by S9 system with low cytotoxicity

The general suitability of exposing human lymphocytes directly to prolonged contact with an Ames-type microsomal (S9) activation system has been examined, for testing the effect of the indirect chemical mutagen, cyclophosphamide (CPA), on induction of chromosomal aberrations. Direct exposure of lymphocytes to only S9 mix produced a decrease in the mitotic index within 30–60 min, whereafter it stabilized at acceptable values. Further toxic effects following treatment with different doses of CPA and S9 mix, for the longest times of exposure were due to production of clastogenic metabolites. On the basis of these result, the low cytotoxicity of S9 mix in our conditions allows extension of the application of the test to the study of metabolites which require prolonged contact with the target cells.

A comparison of the effect of selenium on the mutagenicity and metabolism of benzo[ a]pyrene in rat and hamster liver S9 activation systems

When selenium (Na 2SeO 3) was included in the incubation mix containing rat or hamster liver S9 preparations both the metabolism and mutagenicity of benzo[ a]pyrene (BaP) and several of its metabolites were altered. At non-toxic concentrations selenium inhibited the S9 dependent mutagenicity of BaP and number of its metabolites on Salmonella typhimurium strain TA100 as indicated by the number of histidine independent revertants observed. High performance liquid chromatographic analysis of S9 generated metabolites of BaP from rat and hamster liver indicated that selenium caused quantitative differences in the amounts of the metabolic products. In hamster liver S9 differences were reflected in decreased amounts of strongly mutagenic BaP-7,8-dihydrodiol and increased amounts of 4,5- and 9,10-dihydrodiols that were weakly mutagenic to TA100 in that system. In rat liver S9 selenium caused quantitatively similar decreases in BaP-7,8- and 9,10-dihydrodiol and 3-hydroxy-BaP. When used as substrate 3-hydroxy-BaP was the most mutagenic to TA100 in the rat activation system whereas BaP-7,8-dihydrodiol was most mutagenic in the hamster S9 system. Assays that measured the formation of water-soluble conjugates of BaP indicated that selenium did not significantly alter the formation of sulfate ester or glutathione conjugates although a 12–17% reduction of labeled metabolites bound to the glucuronide fraction was observed. Results described in this report suggest that selenium modified the metabolism and hence the mutagenicity of BaP to TA100 by affecting mixed-function oxidase and/or epoxide hydratase activity in both the rat and hamster liver S9 activation systems.

Human lymphocyte assay: CPA-metabolic activation by S9 system with low cytotoxicity

Human lymphocyte assay: CPA-metabolic activation by S9 system with low cytotoxicity

Inhibition of retinoid toxicity by rat liver S9 fraction in cultured mammalian cells

The effects of retinol (Rol), retinyl acetate (RAc) or retinoic acid (RA) on growth of Chinese hamster V79 cells were studied. In addition, the effect of Rol on cell growth in the human lymphoid cell line RPMI no. 1788 was examined. Cultures were treated with retinoid alone, with retinoid plus S9 mix and with retinoid plus the supernatant of S9 fraction. Treatment of Rol, RAc or RA alone inhibited the growth of both cell lines as evidenced by a dose-dependent decrease of viable cell counts. Concurrent treatment of a retinoid with the well-known metabolic activation system S9 mix containing the rat liver S9 fraction plus enzyme cofactors resulted in a total elimination of the toxic effect of a retinoid. For instance, V79 cells treated with Rol, RAc or RA alone at the highest dose of 32 μg/ml, and human lymphoid cells treated with Rol at the highest dose of 24μg/ml resulted in killing of over 90% of the cells, while addition of S9 mix to the cultures treated with such high doses of a retinoid showed no reduction of viable cells as compared with the controls. The supernatant of S9 fraction after high speed centrifugation also had dose-dependent protective effects against the toxicity of retinol in V79 cells. Finally, experiments using sucrose gradient centrifugation and [ 3H] Rol suggest that binding of a retinoid to its specific binding protein in the S9 mixture and supernatant greatly decreases or abolished the toxicity of free retinoid in cultured mammalian cells.

Cytotoxicity and Sister Chromatid Exchanges Induced In Vitro by Six Anticancer Drugs Developed in the People's Republic of China<xref ref-type="fn" rid="FN2">2</xref>

Growth inhibition in the Chinese hamster cell line V79 and in the human lymphoid cell line Raji and induction of sister chromatid exchange(s) (SCE) in V79 cells after treatment with six anticancer drugs [harringtonine (HRT), homoharringtonine (HHRT), camptothecin (CPT), hydroxycamptothecin (HCPT), lycobetaine (LBT), and oxalysine (OXL)] developed in the People's Republic of China were studied. OXL is a new antibiotic; all other drugs are plant extracts. All drugs caused a dose-dependent growth inhibition in both cell types, as evidenced by decreases in plating efficiencies of V79 cells and in viable cell counts of Raji. However, the degree of inhibition differed widely among the drugs. HRT, HHRT, CPT, and HCPT were the most potent growth inhibitors, LBT was next, and OXL was the least effective inhibitor. SCE analyses were made in V79 cells treated with a drug in the presence or absence of the metabolic activation system S9 mixture (S9 mix), except for the HRT assay in which the S9 mix was not used. CPT, HCPT, and LBT induced a dose-dependent increase in SCE frequencies, while HRT, HHRT, and OXL caused no SCE induction at any dose level used. CPT was the most powerful SCE inducer. HCPT induced SCE but at a much reduced rate when compared to that of CPT. LBT was a weak SCE inducer; SCE induction was seen only in cultures treated with 40 micrograms or more LBT/ml. Addition of the S9 mix did not alter SCE frequencies, indicating that the drugs were direct-acting agents. HRT and HHRT were highly toxic, but they induced no increases in SCE frequency, indicating that cytotoxicity of a compound does not necessarily correlate with SCE induction.

Modulation of aromatic amine mutagenicity in Salmonella typhimurium with rat-liver 9000 g supernatant or monolayers of rat hepatocytes as an activation system

2-Aminofluorene (AF), 2-acetylaminofluorene (AAF) and N-hydroxy-2-acetylaminofluorene (N-OH-AAF) were studied for mutagenic activity in S. typhimurium and either liver 9000 f supernatant fractions (S9) or monolayer cultures of hepatocytes isolated from Wistar rats were used as an activation system. All 3 compounds were converted into mutagens excreted into the incubation medium by the cell-culture system, with N-OH-AAF > AF > AAF. Cultures used 24 h after plating were less efficient in promutagen conversion than were cultures used after 2 h. Phenobarbital, but not 3-methylcholanthrene, pretreatment of the rats caused similar effects on AF, AAF and N-OH-AAF mutagenicity with both S9 and hepatocyte cultures. The mutagenicities of AF and AAF were reduced by the cytochrome-P-450 inhibitors metyrapone and α-naphthoflavone, whereas the mutagenicity of N-OH-AAF was increased by using both inhibitors. Further, the microsomal deacetylase inhibitor paraoxon caused only a moderate reduction in N-OH-AAF mutagenicity, but a total inhibition of AAF mutagenicity. No significant effect of paraoxon on AF mutagenicity was seen. With the S9 system, no effect of ascorbate on the mutagenicity of AF, AAF or N-OH-AAF was observed. In contrast, the mutagenicity of all 3 compounds was increased by ascorbate when hepatocyte cultures were used as activation system. Incubation of hepatocyte monolayers in a sulfate-free medium did not change the mutagenicity of AF, AAF or N-OH-AAF. Galactosamine, an inhibitor of glucuronidation in cells, increased the mutagenicity of AF, AAF and N-OH-AAF with hepatocyte cultures. The addition of cofactor for glucuronidation in the S9 system, however, had no effect. A reduction in mutagenicity of AF and AAF, but not that of N-OH-AAF, was observed with the addition of glutathione (GSH) in both the S9 and the hepatocyte systems. On the other hand, no effect of cellular GSH depletion was seen on aromatic-amine mutagenicity in the hepatocyte system. The data indicate that the hepatocyte culture system offers advantages over the conventional liver-sub-fraction activation system as a model, in vivo, for the metabolism of the aromatic amine mutagens/carcinogens.

Effect of metabolic activation on the cytotoxicity and mutagenicity of 1,2-dibromoethane in the CHO/HGPRT system

When ethylene dibromide (EtBr 2) was assayed with the Chinese hamster ovary/hypoxanthine-guanine phosphoribosyl transferase (CHO/HGPRT) system coupled with a rat liver metabolic activation system (S9), which contains Ca 2+ (Ca, Mg−S9), the cytotoxicity of EtBr 2 was greatly increased over that obtained when NADP was omitted from the Ca, Mg−S9 or when EtBr 2 was assayed as a direct-acting agent. However, on a molar basis, the mutagenicity of EtBr 2 remained unaffected. The omission of Ca 2+ from the Ca, Mg−S9 metabolic activation system (Mg−S9), with either the addition or omission of NADP, caused approximately a 2-fold decrease in the mutagenicity of EtBr 2 when compared to the results obtained by using the Ca, Mg−S9 system. The cytotoxicity of EtBr 2 was further increased when a purified microsomal fraction, prepared from the S9 fraction, was used in the presence of Ca 2+. In the absence of this calcium ion, this metabolic activation system was extremely cytotoxic Chinese hamster ovary cells even without the presence of a mutagen or promutagen. The cytotoxicity of EtBr 2 in the following assay systems decreased in this order: Ca, Mg-microsomes > Ca, Mg−S9 > Mg−S9 > direct-acting agent ⩾ Ca, Mg−S9 without NADP ⩾ Mg−S9 without NADP. Cytotoxicity appears to be NADP-dependent. Although the mutagenic activity of EtBr 2 (mutants/10 6 cells/mM) was not dependent on the presence of NADP in the S9 system, the mutant yield (number of mutants that could be induced) was higher in its absence. Addition of reduced glutathione to Mg−S9 without NADP increased the mutagenicity of EtBr 2 to values that did not exceed those obtained when EtBr 2 was tested as a direct-acting agent. On a molar basis, ethyl methanesulfonate (EMS) is less cytotoxic but equally as mutagenic as EtBr 2. However the mutant yield of EMS was higher than that of EtBr 2. Inclusion of Ca, Mg−S9 in the assay system had no effect on the biological activities of EMS.

Detection of sister chromatid exchanges in the newly hatched chick

2-Aminofluorene (AF), 2-acetylaminofluorene (AAF) and N-hydroxy-2-acetylaminofluorene (N-OH-AAF) were studied for mutagenic activity in S. typhimurium and either liver 9000 f supernatant fractions (S9) or monolayer cultures of hepatocytes isolated from Wistar rats were used as an activation system.All 3 compounds were converted into mutagens excreted into the incubation medium by the cell-culture system, with N-OH-AAF > AF > AAF. Cultures used 24 h after plating were less efficient in promutagen conversion than were cultures used after 2 h. Phenobarbital, but not 3-methylcholanthrene, pretreatment of the rats caused similar effects on AF, AAF and N-OH-AAF mutagenicity with both S9 and hepatocyte cultures. The mutagenicities of AF and AAF were reduced by the cytochrome-P-450 inhibitors metyrapone and α-naphthoflavone, whereas the mutagenicity of N-OH-AAF was increased by using both inhibitors. Further, the microsomal deacetylase inhibitor paraoxon caused only a moderate reduction in N-OH-AAF mutagenicity, but a total inhibition of AAF mutagenicity. No significant effect of paraoxon on AF mutagenicity was seen. With the S9 system, no effect of ascorbate on the mutagenicity of AF, AAF or N-OH-AAF was observed. In contrast, the mutagenicity of all 3 compounds was increased by ascorbate when hepatocyte cultures were used as activation system.Incubation of hepatocyte monolayers in a sulfate-free medium did not change the mutagenicity of AF, AAF or N-OH-AAF. Galactosamine, an inhibitor of glucuronidation in cells, increased the mutagenicity of AF, AAF and N-OH-AAF with hepatocyte cultures. The addition of cofactor for glucuronidation in the S9 system, however, had no effect. A reduction in mutagenicity of AF and AAF, but not that of N-OH-AAF, was observed with the addition of glutathione (GSH) in both the S9 and the hepatocyte systems. On the other hand, no effect of cellular GSH depletion was seen on aromatic-amine mutagenicity in the hepatocyte system.The data indicate that the hepatocyte culture system offers advantages over the conventional liver-sub-fraction activation system as a model, in vivo, for the metabolism of the aromatic amine mutagens/carcinogens.