Indirect mutagen assay in human lymphocyte in the presence of a metabolic activation system

Chromosome aberrations in cultured human lymphocytes were examined after exposures to various concentrations (from 1 X 10(-6) to 1 X 10(-3) mol X l-1) of cyclophosphamide (CP) in the presence or absence of a metabolic activation system (S9 mix). With metabolic activation, increases in the frequency of aberrant cells (AB. C.) produced by CP were significant and dose-dependent. At a concentration of 5 X 10(-4) mol X l-1, activated CP induced 29% AB. C. versus 6% AB. C. detected after exposures to CP without metabolic activation. The freshly prepared S9 mix did not virtually differ in its activation potency from the S9 mix stored for 3 weeks at -20 degrees C. CP preincubated for 100 min with S9 mix caused little or no increase in AB. C. frequency above the control level.     

Genotoxic effects of o-phenylphenol metabolites in CHO-K1 cells

The effects of microsomal activation and/or deactivation on the induction of chromosomal aberrations and sister-chromatid exchanges (SCEs) in cultured Chinese hamster ovary cells (CHO-K1 cells) by o-phenylphenol (OPP) were studied, and concurrently the metabolites were determined. After a 3-h incubation in the presence of 15% S9 mix (45 microliters/ml of S9), OPP (25-150 micrograms/ml) dose-independent SCEs and chromosomal aberrations were induced, while the amount of phenylhydroquinone (PHQ) metabolite produced from OPP did not increase linearly in the higher doses. The maximum induction of chromosomal aberrations was 18% at the 150 micrograms/ml dose, and of SCEs 13.8/cell at 75 micrograms/ml. The corresponding control values were 3% and 5.8/cell. The lowest dose required to induce SCEs in the presence of S9 mix was 25 micrograms/ml. Changing the percent of S9 mix (0-50%) while holding the OPP dose constant (100 micrograms/ml) produced a correlation between SCEs and the production of PHQ. PHQ caused cytogenetic effects both with and without S9 mix, however, in the absence of S9 mix it was more lethal and was oxidized to phenylbenzoquinone (PBQ). These results suggest that the enhanced cytogenetic effects of OPP by the addition of S9 mix correlated with the amount of PHQ produced or with the further oxides of PHQ such as phenylsemiquinone and/or PBQ which are capable of being produced from PHQ spontaneously or by the mixed-function oxidase system.     

Mutagenic properties of 2-amino-N6-hydroxyadenine in Salmonella and in Chinese hamster lung cells in culture

The mutagenicity of the base analogue, 2-amino-N6-hydroxyadenine (AHA), was tested in Salmonella typhimurium TA100 and TA98 and in Chinese hamster lung (CHL) cells. AHA showed very potent mutagenicity in TA100 without S9 mix, inducing 25,000 revertants/micrograms. The mutagenicity increased about 2-fold upon addition of S9 mix containing 10 microliters S9. AHA was found to be one of the strongest mutagens for TA100. Addition of S9 mix containing 100 microliters S9 induced no significant increase of revertants with AHA at amounts up to 50 ng per plate. AHA was also mutagenic for the frameshift mutant, TA98, without S9 mix, the mutagenicity for TA98 being about 1/1000 of that for TA100. When the mutagenicity of AHA was tested in CHL cells, with diphtheria toxin resistance (DTr) as a selective marker in the absence of S9 mix with a 3-h treatment of cells, DTr mutants increased dose-dependently at concentrations of 2.5-15 micrograms/ml. When cells were incubated with AHA for 24 h, a 200-fold increase in the number of DTr mutants was observed; the mutagenicity was 500-fold higher than that of ethyl methanesulfonate. This marked increase of mutagenicity by prolonged incubation may indicate that AHA induces mutations mainly after incorporation into DNA. The addition of a small amount of S9 increased the mutagenicity obtained with a 3-h treatment 2-fold, but a larger amount of S9 decreased the mutagenicity as was found with S. typhimurium TA100.     

Analysis of cytogenetic effect in human lymphocytes induced by metabolically activated 2-nitropropane

Chromosome analyses were carried out in human lymphocytes treated in vitro with 2-nitropropane (2-NP) in the presence and absence of the mammalian metabolic activation system, S9 mix. Without S9 mix, only the frequency of gaps was significantly increased at 80 mM 2-NP as compared to controls. With S9 mix, the incidences of gaps and chromatid-type aberrations were significantly increased at 60 mM and 80 mM. Sister-chromatid exchanges (SCE) have been induced at concentrations as low as 7.5 mM. The present findings demonstrate that in human lymphocytes, 2-NP requires metabolic activation to express clastogenicity and SCEs.     

Clastogenicity of 1-nitropyrene, dinitropyrenes, fluorene and mononitrofluorenes in cultured Chinese hamster cells

The chromosomal aberration test using a Chinese hamster lung cell line (CHL) was carried out on 1-nitropyrene (NP), 3 dinitropyrenes (DNPs), fluorene and 4 mononitrofluorenes with and without metabolic activation (rat S9 mix). The 3 DNPs (1,3-, 1,6- and 1,8-DNP) induced chromosomal aberrations in the absence of S9 mix. The frequencies of cells with aberrations after treatment for 48 h were 43% at 2 micrograms/ml of 1,3-DNP, 55% at 0.1 microgram/ml of 1,6-DNP and 45% at 0.025 microgram/ml of 1,8-DNP, indicating the order of clastogenic potency as 1,8- greater than 1,6- greater than 1,3-DNP. On the other hand, 1-NP, which is known to be a direct-acting mutagen in bacteria, was negative in the chromosomal aberration test without S9 mix, but clearly positive with S9 mix. This effect was dependent on the concentration of the S9 fraction in the reaction mixture. High-pressure liquid chromatography analysis showed that 1-NP was converted by S9 mix to several metabolites, including 1-aminopyrene (AP). The clastogenic activity of 1-AP, however, was equivocal without S9 mix, suggesting that active clastogens other than 1-AP exist. Fluorene induced chromosomal aberrations only in the presence of S9 mix (61.8% at 25 micrograms/ml). 1-, 2-, 3- and 4-nitrofluorene (NF) were more clastogenic in the presence of S9 mix than in the absence of S9 mix, suggesting that NFs were converted to more active clastogens by S9 mix.     

In vitro genotoxicity of danthron and its potential mechanism

To ascertain the in vitro genotoxicity of danthron and its potential mechanism of action, we performed an Ames test, a cytokinesis-block micronucleus assay and a comet assay in Balb/c 3T3 cells. The Ames test revealed that danthron was mutagenic only toward Salmonella typhimurium strain TA102 in the presence of an exogenous metabolic activation system (S9 mix). Danthron (25, 50 and 100μg/ml) increased the frequencies of micronuclear cells with or without S9 mix, and the comet length, tail length and Olive tail moment in comet assays without S9 mix in a dose-dependent manner. These results demonstrated the in vitro genotoxicity of danthron and that 3T3 cells are capable of activating danthron. When NADP was replaced by NAD in the S9 mix, danthron remained mutagenic toward strain TA102. The addition of dicoumarol, a DT-diaphorase inhibitor, decreased the number of danthron-induced histidine revertants by 35-39%, indicating that DT-diaphorase is involved in the metabolic activation of danthron in the presence of NADH as an electron donor. In 3T3 cells, increases in reactive oxygen species (ROS) formation and 8-hydroxydeoxyguanosine levels as well as a reduction in GSH levels were induced by danthron in a dose-dependent manner, indicating that oxidative stress may be a major contributing pathway in the genotoxicity of danthron.     

Genotoxic potential of medroxyprogesterone acetate in cultured human peripheral blood lymphocytes

Medroxyprogesterone acetate was studied at three different concentrations (1, 5 and 10 microM), for its genotoxic effects in human peripheral blood lymphocyte culture using chromosomal aberrations and sister chromatid exchanges as parameters. Duplicate peripheral blood cultures were treated with three different concentrations (1, 5 and 10 microM) of medroxyprogesterone acetate. The study was carried out both in the absence as well as in the presence of metabolic activation (S9 mix) with and without NADP. Medroxyprogesterone acetate was found genotoxic at 5 and 10 microM in the presence of S9 mix with NADP. To study the possible mechanism of the genotoxicity of medroxyprogesterone acetate, superoxide dismutase and catalase at different doses were used separately and in combination with 10 microM of medroxyprogesterone at different doses in the presence of S9 mix with NADP. Superoxide dismutase treatment results in an increase of the genotoxic damage but catalase treatment reduce the genotoxic damage of medroxyprogesterone acetate. Catalase treatment in combination with superoxide dismutase also results in the further reduction of the genotoxic damage. The results of the present study reveal that medroxyprogesterone acetate is genotoxic only in the presence of metabolic activation (S9 mix) with NADP. Treatments with superoxide dismutase and catalase suggests the possible generation of reactive oxygen species by redox cycling of various forms of quinones, similar to estrogens, that are the results of aromatic hydroxylation by cytochrome P450s.     

Hypersensitive character of Bloom syndrome B-lymphoblastoid cell lines usable for sensitive carcinogen detection

Various carcinogens were tested with regard to the induction of sister-chromatid exchanges (SCEs) and chromosome aberrations using 3 types of Bloom syndrome (BS) B-lymphoblastoid cell lines (LCLs) (type I with normal frequency of SCEs and normal karyotype; type II with high frequency of SCEs and normal karyotype; type III with high frequency of SCEs and abnormal karyotypes) in the presence and absence of S9 mix. Three types of BS B-LCLs and normal cells showed different responses to the various carcinogens in the level of SCE induction. BS type I cells had the same SCE response as normal cells to carcinogens. Some carcinogens that require metabolic activation (S9 mix) had little effect on type II cells without S9 mix but had high SCE levels with S9 mix. BS type III cells were highly susceptible to both direct and indirect carcinogens with respect to high SCE increase without S9 mix (ca. 140 SCEs/cell), though some carcinogens produced SCEs rated in the medium (ca. 120 SCEs/cell) range, and had a high rate (more than 10%) of centromere spreading (CS), in addition to quadriradials. Therefore BS type III is a unique cell line which can be used to detect carcinogens.     

1,3-Butadiene and its epoxides induce sister-chromatid exchanges in human lymphocytes in vitro

Sister-chromatid exchanges (SCEs) were induced in human lymphocytes by 1,3-butadiene and its epoxides 3,4-epoxy-1-butene and 1,2:3,4-diepoxybutane. After a pulse treatment of 2 h, 1,3-butadiene produced a weak but reproducible increase in SCEs both with and without S9 mix. The response was similar in cultures of whole blood and of isolated lymphocytes. The 2 epoxide metabolites of butadiene, studied in whole-blood lymphocyte cultures without exogenous metabolic activation, were highly active SCE inducers. The lowest effective concentrations of butadiene, monoepoxybutene, and diepoxybutane were 2000 microM, 25 microM and 0.5 microM, respectively. A slight but dose-dependent increase in SCEs was also observed without an exogenous metabolic system after a 48-h treatment with 1,3-butadiene. Already the lowest concentration tested (500 microM) was effective. Again, the response was similar in cultures of whole blood and isolated lymphocytes, suggesting that the lymphocytes are capable of metabolically activating 1,3-butadiene.     

Sulfhydryl compounds inhibit the cyto- and geno-toxicity of o-phenylphenol metabolites in CHO-K1 cells

The effects of cysteine and reduced glutathione (GSH) on the genotoxicity of o-phenylphenol (OPP) and its metabolites, phenylhydroquinone (PHQ) and phenylbenzoquinone (PBQ), were examined using the frequency of sister-chromatid exchanges (SCEs) and chromosome aberrations in CHO-K1 cells as parameters. Cytotoxic (cell-progression delay) and cytogenetic effects induced by a 3-h treatment with OPP, PHQ (100 micrograms/ml) or PBQ (50 micrograms/ml) with S9 mix after a 27-h expression time were inhibited by cysteine or GSH (3-10 mM). Materials corresponding to the cysteine or GSH adducts were found by HPLC in each incubation mixture. In the culture without S9 mix, PHQ and PBQ showed severe cytotoxicity since no metaphases could be obtained at doses over 25 and 5 micrograms/ml, respectively, and the sulfhydryl compounds inhibited the toxicity by the formation of adducts with PBQ and by inhibiting the formation of PBQ in the case of PHQ. With PHQ, the sulfhydryl compounds appeared to inhibit autooxidation. However, the sulfhydryl compounds did not inhibit the cytotoxic and cytogenetic effects caused by OPP in the cell mixture without S9 mix, but on the contrary intensified them. No adduct formation was detected in the incubation solution. On the basis of these results, it is considered that electrophilic quinone (PBQ) and/or semiquinone (phenylsemiquinone, PSQ) radicals, capable of binding to nucleophilic small molecules (such as cysteine and GSH) or (biological) macromolecules, are produced from metabolite PHQ in metabolic oxidation of OPP, and induce cyto- and geno-toxic effects in the cells. The cyto- and geno-toxic effects of OPP itself to the cells are clearly independent of any electrophilic radical reaction.     

Chromosomal aberrations induced in vitro by 3,7- and 3,9-dinitrofluoranthene

The chromosomal aberration test using a Chinese hamster cell line (CHL) was carried out with 3,7- and 3,9-dinitrofluoranthene (DNF) with and without exogenous metabolic activation (rat liver S9 mix). The highest dose tested was limited to 20 μg/ml because of the compounds' insolubility in dimethyl sulfoxide. Both DNFs induced chromosomal aberrations in the absence of S9 mix; the frequency was not very high. Results were reproducible, but without clear dose-response relationships. Neither DNF induced chromosomal aberrations in the presence of S9 mix. Both DNFs did not induce polyploid cells under any conditions.     

Urinary mutagenicity tests in lead-exposed workers

Urinary mutagenic activity detected by the bacterial fluctuation assay, using Salmonella typhimurium TA98 and Escherichia coli WP2 uvrA with and without metabolic activation (S9 mix), was studied in a group of 21 workers exposed to inorganic lead and a control group of 22 non-occupationally exposed subjects. Occupational exposure to inorganic lead had no effect on urinary mutagenicity in the strains considered, with or without metabolic activation. In smokers (exposed and non-exposed), urinary mutagenic activity appeared to increase compared to non-smokers (exposed and non-exposed), only with Salmonella typhimurium TA98 in the presence of S9 mix.     

Chromosome damage by dothistromin in human peripheral blood lymphocyte cultures: a comparison with aflatoxin B1

The clastogenic potential of the pine tree fungal toxin dothistromin was studied by metaphase chromosome analysis of stimulated human peripheral blood lymphocytes exposed in vitro. The frequency of gaps, breaks, deletions and exchanges was scored in a series of cultures from 3 different donors. 50 cells were analysed for each dose level on coded slides. Testing was performed with and without added metabolic activation (as S9 mix) and aflatoxin B1 was used as a positive control in all experiments. Dothistromin caused a dose-dependent increase in the frequency of gaps and deletions which was not dependent on added metabolic activation. Even at high doses of dothistromin only a very small number of complex exchange-type aberrations were seen. This is in contrast to aflatoxin B1 where such aberrations were seen at low dose levels and especially in cultures to which S9 mix was added. High doses of dothistromin caused culture toxicity manifesting as haemolysis of the donor red blood cells and reduction of mitotic index. Culture toxicity occurred without a marked increase in aberration frequency. This toxicity may be masking any major potential for clastogenicity by dothistromin.     

Analysis of the cytogenetic effect in human lymphocytes induced by metabolically activated 1- and 2-methylnaphthalene

Chromosome analyses were carried out in human lymphocytes treated in vitro with 1- and 2-methylnaphthalene (1-MN, 2-MN) in the presence and absence of the mammalian metabolic activation system, S9 mix. Without S9 mix there was no indication of induction of any significant cytogenetic effect by either compound. With S9 mix a weak clastogenic effect was apparent at 4 mM 2-MN only and sister-chromatid exchange frequencies were significantly increased at each dose of 1- and 2-MN, yet always less than twice the control level. The present observations do not indicate that 1- and 2-MN must be classified as potential genotoxic substances.     

Mutagenicity of rat-liver S9 to L5178Y mouse lymphoma cells

Rat-liver S9 preparations became highly mutagenic to cultured L5178Y mouse lymphoma cells when the exposure period was increased to 18-24 h or when S9 mix was preincubated in Fischer's medium at 37 degrees C for 19 h and then used to treat the cells for 4 h. Five different S9 preparations (from untreated and Aroclor 1254-treated Fischer 344 or Sprague-Dawley male rats) behaved similarly. S9 mix, which contained 1 mM NADP and 5 mM isocitrate as cofactors, was more mutagenic than S9 alone. Heat treatment of S9 did not destroy its mutagenic activity, but the addition of cofactors no longer stimulated an increase in mutagenicity, as observed with native S9. Treatment with cofactors was not mutagenic. These results implied the involvement of both energy-independent and NADPH-dependent enzymatic changes in S9 mix in producing mutagenic substances. The mutagenic treatments with S9 or S9 mix induced predominantly small TFT-resistant mutant colonies, which suggested that these treatments should be clastogenic to cultured mammalian cells. A warning was given that test chemicals evaluated as mutagenic only in the presence of S9 mix may instead be accelerating the decomposition of S9 mix into mutagens, and it may become necessary to experimentally distinguish between these two mechanisms before a chemical can be regarded as mutagenic.     

Genotoxic potential of ethinylestradiol in cultured mammalian cells

Ethinylestradiol, a steroidal estrogen, is widely used with various progestogens in oral contraceptives formulations. There are sufficient evidences for the carcinogenicity of ethinylestradiol in experimental animals. The reports on the genotoxic potential of ethinylestradiol are contradictory. Here in the present study we have tested the genotoxicity of ethinylestradiol in human lymphocytes using chromosomal aberrations (CAs), mitotic index (MI) and sister chromatid exchanges (SCEs) as a parameter. The study was carried out in the absence, as well as in the presence, of rat liver microsomal fraction, with and without NADP. Ethinylestradiol was studied at three different concentrations (1, 5 and 10 microM) and was found non-genotoxic in the absence of metabolic activation (S9 mix) and in S9 mix without NADP. Ethinylestradiol was found to be genotoxic at 5 and 10 microM in the presence of S9 mix with NADP. To study the possible mechanism of the genotoxicity of ethinylestradiol, superoxide dismutase (SOD) and catalase (CAT) were used separately and in combination along with 10 microM of ethinylestradiol at different doses. SOD treatment increased CAs and SCEs and decreases MI as compared with treatment with 10 microM of ethinylestradiol alone in the presence of S9 mix with NADP at both of the tested doses. CAT treatment decreased the frequencies of CAs and SCEs and increased MI, as compared with treatment with 10 microM of ethinylestradiol alone in the presence of S9 mix with NADP. CAT treatment in combination with SOD also decreased the frequencies of CAs and SCEs and increased MI suggesting a possible role of reactive oxygen species for the genotoxic damage.     

The clastogenic potential in vitro of pyrrolizidine alkaloids employing hepatocyte metabolism.

Three pyrrolizidine alkaloids (PAs), monocrotaline, retrorsine and isatidine, were tested for their clastogenic activity under different conditions of metabolic activation in vitro. All three compounds exhibited a weak activity when V79 cells were treated at very high concentrations for 18 h in the absence of a metabolizing system. Short-term (2 h) treatment with rat liver S9 mix led to a strong and concentration-dependent increase in chromosomal aberrations for retrorsine. Isatidine was not mutagenic with S9 mix and monocrotaline was positive at high concentrations only. In contrast, a prolonged treatment (18 h) in vitro under activation conditions in the presence of primary hepatocytes led to clear concentration-dependent positive responses for all three PAs investigated. Particularly the results with isatidine demonstrate that in vitro tests using S9 mix for metabolization can generate misleading results. It is not clear whether the results could be attributed to a better activation of the test compounds by intact hepatocytes in comparison to S9 mix or if the fact that only hepatocytes allow a treatment for the whole culture period under activation conditions was more important. Owing to its strong cytotoxicity the exposure to S9 mix is generally limited to 2-4 h, limiting also the exposure of the target cells to a test chemical as well as its metabolites. The results presented show significant differences in mutagenic potency of PAs due to variations in the activation system. This underlines the usefulness of primary hepatocytes, e.g., for the detection of pre-mutagens. The PAs investigated are present in plants which are used for phytotherapeutic medicinal products. They do not contribute to their efficacy and are, therefore, not to be tolerated in amounts that may impose a risk for the user.     

Modes of genotoxicity of a macromolecular antibiotic, SN-07, a novel type of interstrand DNA cross-linker

The modes of genotoxicity of a novel macromolecular antitumor antibiotic (SN-07) were examined using both prokaryotic and eukaryotic cells in vitro. The antibiotic induced a frameshift-type reverse mutation in Ames Salmonella typhimurium TA98 at 1.6-400 ng/plate with and without S9 mix. SN-07 also induced chromosomal aberrations and a forward mutation (6-TGr) in Chinese hamster V79 cells after 1 h treatment at 12.5-100 ng/ml without metabolic activation. The alkaline elution technique revealed that SN-07 induced interstrand DNA cross-linking dose-dependently after treatment with 2.5-10 micrograms/ml for 1 h followed by elution at pH 12.1, but it did not induce the dose-dependent cross-linking after the same treatment followed by elution at pH 12.6. It was also found that SN-07 induced single-strand DNA breaks (pH 12.1) and alkali-labile (pH 12.6) sites after treatment with 0.1-10 micrograms/ml for 1 h followed by 24-h post-incubation.     

Author index

A technique using human lymphocytes together with an Ames-type microsomal (S9) activation system for testig indirect chemical mutagens has been developed and examined. The cytotoxic drug cyclophosphamide (CP), which only displays mutagenic properties after metabolic activation, was used as a test chemical and mutagenicity assessed in terms of sister-chromatid exchange (SCE) induction. Direct exposure of lymphocytes to CP and S9 mix produced increases in the yield of SCE for CP concentrations down to 10^?6 M. Exposure times of 30, 60 and 120 min commencing at the beginning of cell culture or after 48 h gave different ranges of detection and response with later treatment being more effective for SCE induction. Variations in relative proportions of the S9 mix culture medium constituents affected the lymphocytes' tolerance of toxic factors and modifiec the mutagen's effect. CP pre-incubated with S9 mix for 1 h before adding to the lymphocyte cultures also produced increases in SCE levels but the method was complicated and did not reduce toxicity. Direct addition of CP and S9 mix to the lymphocytes without prior pre-incubation showed maximum sensitivity, minimum toxocity and was a simpler, more reliable technique.     

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 containing 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 alpha-naphthoflavone (604-59-1) inhibited the mutagenic activity by about 50%, suggesting that cytochrome P-450 was involved in the metabolic activation.