S9 Preparations Research Articles

The metabolic activation of 4,4′‐methylene‐bis‐(2‐chlorobenzeneamine) to a bacterial mutagen by hepatic postmitochondrial supernatant from human and other species

4,4'-Methylene-bis-(2-chlorobenzeneamine) (MbOCA) is a commercially important industrial chemical that is carcinogenic in three animal species and mutagenic in the Ames test. The ability of hepatic postmitochondrial supernatant from humans, dogs, mice, and rats to activate MbOCA to a bacterial mutagen has been investigated using the Ames plate incorporation test and a bacterial fluctuation test. In the Ames plate test, hepatic S9 preparations from mice and Aroclor 1254-induced rats only were sufficiently active to produce a significant mutagenic response. Preincubation of MbOCA with S9 from human liver produced a slight increase in the number of revertants but not a doubling as compared to controls. However, using the more sensitive bacterial fluctuation test, liver S9 from all species activated MbOCA to a bacterial mutagen. The responses produced were dose-related and, for at least part of the dose range, were double the background levels observed in controls. The increases in the mutagenicity of MbOCA produced by liver S9 from humans, dogs, and rats were significant at the 0.1% level of probability. Liver S9 preparations from all species in which MbOCA is carcinogenic have now been demonstrated to be capable of activating this compound to a bacterial mutagen. The finding that S9 from human liver can also activate MbOCA to a mutagen increases the concern that it may be a human carcinogen.

Assay of phenacetin genotoxicity using in vitro and in vivo test systems.

Phenacetin was assayed in a battery of five short-term tests. (1) In a DNA-repair test using various Escherichia coli strains, the drug was not directly genotoxic nor did it induce nonreparable DNA damage in the presence of rat liver S9 fractions, while it was weakly active following activation with hamster liver S9. (2) In the Ames reversion test (strains TA97, TA98, TA100, and TA102 of Salmonella typhimurium, phenacetin reverted only TA100, and only in the presence of hamster liver S9. Mutagenicity was related to the concentration both of the drug and of the above metabolic system. There was no activation with hamster kidney S9, uninduced chicken liver S9, or with a variety of liver S9 preparations from rats treated with enzyme inducers (Aroclor 1254, phenobarbital, or 3-methylcholanthrene) and/or glutathione depletors (diethyl maleate or buthionine sulfoximine). Hamster liver S9 compared favorably to rat and even more to chicken liver S9 fractions also in activating various promutagens [3-amino-1-methyl-SH-pyrido (4,3-b)-indole, 2-aminofluorene, aflatoxin B1, benzo[a]pyrene, and benzo[a]pyrene-trans-7,8-diol] and in decreasing the mutagenicity of direct-acting compounds (4-nitroquinoline N-oxide and sodium dichromate). (3) Phenacetin was borderline positive in a forward mutation test (6-thioguanine resistance) in V79 cells, only in the presence of hamster liver S9, and gave negative results in the presence of rat liver S9 or without any metabolic system. (4) Following in vivo treatment, the alkaline elution assay did not reveal any DNA fragmentation in bone-marrow cells of ip-treated mice or in liver cells of rats treated by gavage. Apparent DNA damage was instead observed in the kidneys of rats receiving the drug by gavage or in the liver following ip administration. However, the effect was prevented (liver) or reduced (kidney) by preliminary perfusion of the organs, which discards (liver) or makes uncertain (kidney) the hypothesis of a true in vivo DNA damage. (5) Phenacetin ip induced in mouse bone-marrow cells a poor yet statistically significant increase in sister chromatid exchanges.

Comparisons of mutation induction by six monocyclic aromatic amines in salmonella typhimurium tester strains TA97, TA1537, and TA1538

The mutagenicity of six monocyclic aromatic amines (2,4-diaminoanisole, 2,4-diaminoethoxybenzene, 2,4-diaminopropoxybenzene, m-phenylenediamine, 2,4-diaminotoluene, and nitro-p-phenylenediamine) was investigated in Salmonella typhimurium strains TA97, TA1537, and TA1538 in the presence of two different amounts of Aroclor 1254-induced S9 preparations. Strain TA1538 was found to be the most responsive of the three strains with this group of compounds. Regarding the other strains, TA1537 responded to three of the compounds better than strain TA97, if one calculates responsiveness as the fold-increase in numbers of revertants per plate. However, if one calculates the number of revertants per nanomole or compares the number of induced revertants per plate, TA97 was more responsive than TA1537 for all six compounds. Comparisons of mutagenesis from tests involving strain TA97 are complicated by the large variations in spontaneous mutation frequencies in this strain. The amount of S9 per plate is another important variable in tests of monocyclic aromatic amines; in general, more revertants are detected when the S9 mix contains 10% S9 than when it contains 4% S9. Nevertheless, in all our tests of 2,4-diaminoanisole, 2,4-diaminoethoxybenzene, and 2,4-diaminopropoxybenzene, the same relationship between chemical structure and mutagenic activity was observed. In all three strains, the mutagenic responses become less when the alkoxy substituent on the molecule becomes larger.

Mutagenicity of derivatives and metabolites of 1-nitropyrene: activation by rat liver S9 and bacterial enzymes

The mutagenicity and activation requirements of purified synthetic derivatives and potential metabolites of 1-nitropyrene have been characterized in the Ames plate incorporation assay with the Salmonella tester strains TA98, TA98NR and TA98/1,8-DNP 6, in the presence or absence of exogenous metabolic activation provided by Aroclor-induced rat liver S9. All the compounds tested (1-aminopyrene, N-acetyl-1-aminopyrene, N-hydroxy- N-acetyl-1-aminopyrene, 3-hydroxy-1-nitropyrene, 6-hydroxy-1-nitropyrene, and 8-hydroxy-1-nitropyrene) exhibited mutagenic activity under one or more assay conditions. 1-Nitropyrene was metabolized to 3-hydroxy-1-nitropyrene, 6- or 8-hydroxy-1-nitropyrene, 1-aminopyrene, N-acetyl-1-aminopyrene and other unidentified products (including some bound to protein) by an S9 preparation analogous to that used for exogenous metabolic activation in the Ames assay. 1-Nitropyrene and 3-hydroxy-1-nitropyrene were activated primarily by the ‘classical’ nitroreductase, while the other compounds, particularly in the presence of S9 metabolic activation, were dependent on transesterification for expression of their mutagenicity.

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.

Effect of liver enzymes on the mutagenicity of nitroheterocyclic compounds: activation of 3a,4,5,6,7,7a-hexahydro-3-(1-methyl-5-nitro-1 H-imidazol-2-yl)- 1,2-benzisoxazole and deactivation of nitrofurans and nitroimidazoles in the Ames test

The effect of liver enzymes (S9) on the mutagenic response of nitroimidazoles and nitrofurans in the Ames test was evaluated with strain TA100. A diminished response was observed with a 5-nitroimidazole and 5-nitrofurans when the S9 preparation was incorporated in the agar layer. Preincubation with S9 under anaerobic conditions prior to adding the bacteria resulted in a greater and sometimes complete loss of the mutagenic effect. The loss of mutagenic potency was dependent on both incubation time and quantity of the S9 preparation. These results suggest that metabolites formed after reductive metabolism are neither mutagenic (presumably due to the loss of the nitro group) nor capable of activation to mutagenic metabilites. One 5-nitroimidazole, 3a,4,5,6,7,7a-hexahydro-3-(1-methyl-5-nitro-1 H-imidazol-2-yl)-1,2-benzisoxazole (MK-0436), gave an increased response in the presence of S9 in both the plate test and when preincubated under aerobic conditions. 7 metabolties were produced by the incubation. 4 monooxygenated metabolites were isolated and found to possess significant mutagenic activity. 2 synthetic dihydroxy analogs were more mutagenic than MK-0436. Similar results were obtained with S9 preparations from human liver and the livers of control, phenobarbital and Aroclor-1254 pretreated rats.

Testing natural indigo for genotoxicity

The genotoxicity of indigo has been assessed by two short-term tests. The mutagenicity of natural indigo was compared with that of synthetic indigo. Both chemicals were tested using the standard procedure of the Salmonella/microsome mutagenicity test as described by Ames. The substance exhibits mutagenicity towards strains TA1538 and TA98 when S9 preparations of rat liver induced with Aroclor 1254 were present in the medium. The clastogenic potential was evaluated by the micronucleus test in the bone marrow of male mice. The test compound was administered twice with an interval of 24 h, the animals were killed 30 h and 54 h after the first treatment. When the test compound was given by oral gavage as two equal dosages of 0.1, 1 and 1.2 g/kg body weight, no statistically significant increase in the percentage of polychromatic erythrocytes with micronuclei was observed for any group treated with natural indigo.

Potential of 2,4-dichlorophenoxyacetic acid conjugates as promutagens in the Salmonella/microsome mutagenicity test.

Hepatic S9 preparations from Aroclor 1254 induced rats and 3-methylcholanthrene induced woodchucks were used to investigate, in vitro, the mutagenic potential of five amino acid conjugates of 2,4-Dichlorophenoxyacetic acid (alanine, aspartic acid, leucine, methionine and tryptophan). Five strains of Salmonella typhimurium (TA97, TA98, TA100, TA1535, TA1538) were utilized for this purpose. Dose-response effects producing a two-fold increase of revertants over spontaneous levels were not observed with either S9 preparation indicating that the amino acid conjugates are not promutagens in these assays.

The influence of 18 environmentally relevant polycyclic aromatic hydrocarbons and Clophen A50, as liver monooxygenase inducers, on the mutagenic activity of benz[a]anthracene in the Ames test.

In the presence of various liver S9 preparations induced by different polycyclic aromatic hydrocarbons, the weak carcinogen, benz[a]anthracene, exhibited mutagenic activities comparable to those of benzo[a]pyrene in the Ames test. A series of positive correlations were found between mutagenic activity and the hydroxylation products of benz[a]anthracene at the 3,4-, 5,6- and 8,9-positions, of which 5,6-position was the most pronounced one (p less than 0.01). Our results suggest that the observed mutagenic activity is due mainly to the generation of K-region epoxides.

Rat liver S9 preparations contain comutagenic activity

Rat liver S9 preparations contain material which causes enhancement of UV mutagenesis in Escherichia coli WP2. This comutagenic activity is present in S9 preparations from both uninduced and Aroclor-induced rats. Strains of E. coli which are defective in the uvr-dependent excision repair pathway fail to show comutagenic action by S9. The comutagenic material is heat-labile and non-dialyzable, suggesting that it might be protein. This differs from the small amount of mutagenic material present in rat liver S9, as the latter is dialyzable and can be demonstrated in the repair-deficient strain E. coli WP2 s ( uvrA).

Absence of mutagenic activity of fodder proteins in the Ames Salmonella/microsome test

The mutagenicities of fodder proteins (Pekilo, l-lysine and Orsan) were tested towards Salmonella typhimurium in the plate-incorporation assay in the presence or absence of metabolic activation with a rat-liver S9 preparation. Filtrates and 2-, 5- and 10-fold-concentrated filtrates of saline- or ethanol-soluble fodder proteins were tested. No mutagenic activity was observed.

Activation of nitrosamines and other carcinogens by mouse-liver S9, mouse hepatocytes and in the host-mediated assay produces different mutagenic responses in Salmonella TA1535

5 indirect alkylating carcinogens, namely, dimethylnitrosamine (DMNA), methyl-ethylnitrosamine (MENA), diethylnitrosamine (DENA), 1,2-dimethylhydrazine (DMH) and cyclophosphamide (CP), were tested in liquid incubation assays for their mutagenic activity towards Salmonella TA1535 in the presence of mouse-liver homogenate (S9) or freshly isolated, single liver-cell preparations. The capacity of these mouse-liver preparations to activate the compounds to mutagens for TA1535 was compared with the mutagenic effect of low doses of the carcinogens in intrasanguineous host-mediated assays, with the same strain of mice as host. Although the mouse hepatocytes retained their activating capacity longer than S9 preparations did during incubation at 37°C, the latter gave much higher yields of mutants with 10 mM (DMNA, MENA, DMH) and 5 mM (CP) of 4 out of the 5 compounds. DENA was not mutagenic in either assay. These differences between whole cell and disrupted cell preparations were reduced or absent when the concentrations of the test compounds were reduced by a factor of 10. It was concluded that hepatocytes at the maximal concentration of cells have a limited capacity to metabolize the mutagens. On the basis of protein concentration, hepatocytes are more effective (nitrosamines) or equally effective (CP and DMH) in activating the compounds. Compared with the host-mediated assays, both liver fractions have only a marginal potential to activate equal low amounts of the carcinogens. The present results do not indicate that hepatocytes take an ‘intermediate’ position between existing in vitro and in vivo activation systems, although they do suggest that these mouse hepatocyte preparations activate the nitrosamines DMNA and MENA in a quantitatively or qualitatively different way than do mouse-liver homogenates.

Increased Mutagenicity of Chloroethylnitrosoureas in the Presence of a Rat Liver S9 Microsome Mixture<xref ref-type="fn" rid="fn2">2</xref>

The mechanism for an enhanced effect of Aroclor 1254-induced Sprague-Dawley rat liver 9,000 x g supernatant (S9) microsome preparation on the mutagenicity of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), 1-(2-chloroethyl)-3-cyclohexyl-N-nitrosourea (CCNU), and 2-[3-(2-chloroethyl)-3-nitrosoureido]-2-deoxy-D-glucopyranose (chlorozotocin) for Salmonella typhimurium strain TA1535 was studied. Although all three compounds were direct-acting mutagens, rat liver S9 increased the mutagenic response to BCNU, CCNU, and chlorozotocin. The enhanced mutagenic effect was independent of NADPH. Heat-denatured S9 enhanced the mutagenicity of BCNU and CCNU, but not that of chlorozotocin. Mutagenic enhancement, however, was less than that observed with untreated S9. The substitution of extractable S9 lipid and bovine serum albumin for S9 in the reaction mixture resulted in an enhanced mutagenicity of CCNU with little or no effect on BCNU or chlorozotocin mutagenicity. These results suggest that the enhanced mutagenicity of CCNU, and possibly that of BCNU, in the presence of S9 was due in part to nonspecific factors that are present in the S9 preparation.

The biological activity of 4-chloromethylbiphenyl, benzyl chloride and 4-hydroxymethylbiphenyl in 4 short-term tests for carcinogenicity: A report of an individual study in the UKEMS genotoxity trial 1981

4-Chloromethylbiphenyl (4CMB), benzyl chloride (BC) and 4-hydroxymethyl-biphenyl (4HMB) were tested for biological activity in the following assays: (i) the Salmonella/microsome assay; (ii) a bacterial ‘fluctuation’ assays; (iii) a DNA repair assay in Hela cells, and (iv) a mouse lymphoma mutation assay. 4CMB was active in assays (i), (ii) and (iii) but not in (iv); BC was active in assays (i), (ii), (iii) but not in (iv) while 4HMB was inactive in all assays. Where biological activity was seen this did not require addition of a liver S9 preparation. 4CMB was more active than BC in all the test systems in which a positive response was obtained. The implication of these results for a test battery approach to in vitro testing is discussed.

The role of highly purified cytochrome P-450 isozymes in the activation of 4-aminobiphenyl to mutagenic products in the Ames test.

The role of cytochromes P-450 and P-447 in the activation of 4-aminobiphenyl to mutagens in the Ames test was studied using S9 preparations and highly purified isozymes. S9 preparations from beta-naphthoflavone-pretreated rats were more efficient in converting 4-aminobiphenyl to mutagens than the corresponding preparations from phenobarbitone-pretreated animals. Similarly, reconstituted systems comprising purified cytochrome P-447 were twice as efficient as cytochrome P-450 in activating the carcinogen. Of all the known Phase I metabolites of 4-aminobiphenyl, only the N-hydroxy-derivative was mutagenic in the Ames test. These findings indicate that arylamine N-hydroxylase is a cytochrome P-450 dependent enzyme, and the nature of the isozyme of the cytochrome is an important determinant of its mutagenicity.

Mutagenic activity of 2-(2′,4′-diaminophenoxy)ethanol in strains TA1538 and TA98 of Salmonella typhimurium

The mutagenicity of 2-(2′,4′-diaminophenoxy)ethanol (2,4-DAPE) was compared with that of 2,4-diaminoanisole (2,4-DAA), a chemically related compound previously used in hair-dye formulations. Both chemicals were tested in standard procedures with the Salmonella/microsome mutagenicity test as described by Ames and colleagues. In several experiments, which extended over a total period of 2 years, 2,4-DAA exhibited definite, but variable mutagenicity toward strain TA1538 when S9 preparations of rat liver induced with Aroclor 1254 were present in the incubation mixtures. The compound 2,4-DAPE did not exhibit detectable mutagenic activity when tested concomitantly under the same experimental conditions. We conclude that 2,4-DAPE is not mutagenic for Salmonella under conditions of the standard mammalian microsome assay with strain TA1538 and TA98 as indicators.

Differences in liver homogenates from Donryu, Fischer, Sprague-Dawley and Wistar strains of rat in the drug-metabolizing enzyme assay and the salmonella/hepatic S9 activation test

Comparison studies for detecting differences between liver microsome and S9 preparations from 4 strains (Donryu, Fischer, Sprague-Dawley, Wistar) of young male rats were carried out with pretreatment of the animals by inducers such as PCBs and PB plus 5,6-BF. Each microsome fraction was assayed for the enzymic activity of metabolism of model substrates such as aniline, benzophetamine, BP, DMN and 7-ethoxycoumarin. The hepatic S9 sample was also compared, as regards its metabolizing ability to activate 9 pre-mutagens (2AA, AAF, o-AAT, BP, DAB, DMBA, DMN, m-PDA, quinoline) to directly acting mutagens in the Salmonella/hepatic S9 activation test by using TA98, TA100 and TA1537 strains with or without cytochrome P450 inhibitors (SKF-525A, metyrapone, 7,8-benzoflavone). In the enzymic assay with PCBs-induced microsomes, BP hydroxylation revealed a strain-specific difference: the microsomes from Fischer and Wistar rats were more effective for metabolizing BP than those from the other strains of rat. The effect of induction by PB plus 5,6-BF for Fischer rats showed relatively higher enzymic activity in the same induction group. Other microsomes prepared from rats with and without induction by PB plus 5,6-BF did not show a clear-cut strain dependency in the enzymic activities assayed. In the mutation experiments with hepatic S9 samples, the examination of DAB and quinoline revealed a marked strain difference when S9 samples prepared from PCBs-pretreated and PB-plus-5,6-BF-induced rats were used: the S9 sample from Fischer rats was available for activating the two pre-mutagens to directly acting mutagens. No marked difference in the metabolic activation of the remaining 7-pre-mutagens was observed on other S9 preparations. In examinations of mutagenicity activities with the use of three inhibitors, the two S9 preparations made with the two induction methods showed inhibition profiles closely similar to each other. However, there were minor differences in the profiles by these inhibitors. From these findings it was concluded that Fischer rat-liver S9 is useful for detecting mutagens in the metabolic activation test, when induction by PB plus 5,6-BF was used in the Ames Salmonella test.

Cytokinetic and cytogenetic effects of some agricultural chemicals on human lymphoid cells in vitro: organophosphates

In the present study, cytotoxic, cytostatic and cytogenetic effects of a number of organophosphate pesticides on human lymphoid cells (LAZ-007) in culture have been examined. Cytotoxic effects were dose related and often led to extensive cell kill. The pronounced effects of various organophosphates on the cell-cycle traverse were shown in data based on the enumeration of M1 and M3 metaphases after incubation of cells with BrdU. In cells incubated with 20 μg/ml of the various chemicals, the number of M1 metaphases ranged from 6% (R-1303) to 18% (Azodrin) compared to no M1 metaphases in control cultures. The number of M3s in cultures treated with 20gmg/ml of the various chemicals tested varied from 0% (Phosdrin) to 7% (parathion) as compared to 17% in control cultures. 11 out of the 14 organophosphates tested, significantly increased the SCE frequency. Of the 9 chemicals tested after metabolic activation by liver microsomal S9 preparation, significant increases in SCE frequency were seen in diazinon-, dimethoate-, Dursban- and Phosdrin-treated cells.

Induction of mitotic gene conversion by browning reaction products and its modulation by naturally occurring agents

Mitotic gene conversion in the D7 strain of Saccharomyces cerevisiae was significantly enhanced by exposure to non-enzymatic browning reaction products. These products were formed during the heating of sugar (caramelization reaction) or sugar-amino acid mixtures (Maillard reaction) at temperatures normally used during the cooking of food. Several modulating factors of this convertogenic activity were identified. These factors included two main groups: (1) trace metals which are widely distributed in the environment; and (2) several cellular enzymatic systems. The convertogenic activities of a heated glucose-lysine mixture and a commercial caramel powder were completely suppressed when yeast were concurrently exposed to these products and to either FeIII or CuII. Equimolar concentrations of MnII or sodium selenite had no effect on the convertogenic activity of the products of either model system. Horse-radish peroxidase, beef liver catalase and rat liver S9 preparations each decreased the frequency of gene conversion induced by the caramel powder and the heated glucose-lysine products. This modulating activity of the enzymes was lost if they were heat-inactivated. These studies indicate the presence of a variety of protective mechanisms which can modify genotoxic components in complex food mixtures.

The effects of dietary selenium on the biotransformation of 7,12-dimethylbenz[ a]anthracene

The influence of dietary selenium on the mutagenic activation of 7,12-dimethylbenz[ a]anthracene (DMBA) by rat liver S9 was studied using the Ames test. Rats received supplemental selenium, as sodium selenite, in the drinking water or in the diet. All rats additionally received 0, 20, 50, 100, or 500 mg Aroclor 1254 per kg body weight. Revertant counts decreased 72 and 31% at the 20- and 100-mg/kg induction levels, respectively, with S9 preparations from rats given selenium supplementation, compared to controls. No significant effect of selenium on S9 preparations was observed in rats treated with 500 mg/kg Aroclor. Preparations of S9 from rats receiving 2.5 ppm Se in their diet produced 46, 84 and 70% less revertants than controls at the 20-, 50- and 100-mg/kg induction levels. Increasing the selenium concentration in the diet to 5 ppm reduced the revertant counts by 71, 68 and 65%, at the 20-, 50- and 100-mg/kg induction level of Aroclor, respectively. Dietary selenium supplementation was shown to decrease the mutagenic activation of DMBA by liver microsomes. These studies indicate that in vivo selenium supplementation may reduce susceptibility to the action of various carcinogens.