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.     

Induction of chromosome aberrations and sister-chromatid exchanges by caprolactam in vitro

Caprolactam (CAP) induced chromosome aberrations in whole-blood cultures of human lymphocytes at 50 mM without metabolic activation (24-h treatment) and at 200 mM in the presence of rat liver S9 mix (1-h treatment). CAP also produced a dose-dependent increase in polyploid cells, the effect being statistically significant at 25 and 50 mM without S9 mix and at 100 and 200 mM with S9 mix. Without metabolic activation, there was an increase in hypodiploid cells at 50 mM and hyperdiploid cells at 12.5 mM. In Chinese hamster ovary cells, CAP produced a marginal elevation of sister-chromatid exchanges at 125 mM in the presence of S9 mix (4-h treatment). The results show that CAP is able to induce cytogenetic changes in vitro at very high toxic concentrations.     

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.     

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.     

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.     

Effects of exposure to diesel exhaust particles (DEP) on pulmonary metabolic activation of mutagenic agents

Exposure of rats to diesel exhaust particles (DEP) or carbon black (CB) has been shown to induce time-dependent changes in CYP1A1and CYP2B1 in the lung. The present study evaluated the role of these metabolic enzymes on the pulmonary bioactivation of mutagens. Male Sprague-Dawley rats were intratracheally instilled with saline (control), DEP or CB (35 mg/kg body weight) and sacrificed at 1, 3, or 7 days post-exposure. Both control and exposed lung S9 increased the mutagenic activity of 2-aminoanthracene (2-AA), 2-aminofluorene (2-AF), 1-nitropyrene (1-NP), and the organic extract of DEP (DEPE) in Ames tests with Salmonella typhimurium YG1024 in a dose-dependent manner. Lung microsomes prepared form control or particle-exposed S9, but not cytosolic protein, activated 2-AA mutagenicity. Compared to saline controls, CB-exposed S9 was a less potent inducer of 2-AA mutagenicity at all time points, whereas DEP-exposed S9 was less potent than control saline at 3 and 7 days but not 1 day post-exposure. At 3 days post-exposure, DEP- or CB-exposed lung S9 did not significantly affect the mutagenicity of DEPE or 1-NP, when compared to the controls. The mutgenicity of 2-AA, 2-AF, 1-NP, and DEPE were significantly decreased in the presence of inhibitors for CYP1A1 (alpha-naphthoflavone) or CYP2B (metyrapone), but markedly enhanced by CYP1A1 or CYP2B1 supersomes with all the cofactors, suggesting that both CYP1A1 and CYP2B1 were responsible for mutagen activation. These results demonstrated that exposure of rats to DEP or CB altered metabolic activity of lung S9 and S9 metabolic activity dependent mutagen activation. The bioactivation of mutagens are metabolic enzyme- and substrate-specific, and both CYP1A1 and CYP2B1 play important roles in pulmonary mutagen activation.     

Induction of sister-chromatid exchanges in human lymphocytes by microsomal activation of benzene metabolites

Metabolic activation of the benzene metabolites, catechol, hydroquinone, and phenol, by rat-liver microsomes and an NADPH-generating system (S9 mix) caused an increased induction of sister-chromatid exchanges (SCEs) in cultured human lymphocytes. There were different optimal concentrations of S9 mix for converting each benzene metabolite into further reactive forms that could induce SCE-forming lesions. The data indicate that catechol and hydroquinone can be optimally metabolized to produce reactive species, presumably benzo(semi)quinones, under conditions of lower metabolic activity than those necessary for phenol and benzene.     

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.     

Genotoxic effects of estradiol-17beta on human lymphocyte chromosomes

The cytogenetic effect of a hormonal steroid, estradiol-17beta, was assessed in peripheral blood human lymphocyte culture. Sister chromatid exchanges (SCE) and chromosome aberrations (CA) were scored as genetic end points. Significant induction of CA was observed at 25 microg/ml and 50 microg/ml concentrations of estradiol-17beta in the absence of microsomal activation. The drug was effective in all treatments in the presence of rat liver S(9) microsomal fraction (S(9) mix) and exhibited increased frequency of chromosomal aberrations. The drug was effective in increasing the SCE frequency which was found to be maximum at the dose of 50 microg/ml concentration (i.e., 4.34+/-1.22) both with and without metabolic activation. It was found that estradiol-17beta itself and possibly its metabolites are potent mutagens beyond a particular dose in human lymphocytes.     

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.     

Chromosomal damage induced by caprolactam in human lymphocytes

Caprolactam was tested in the in vitro human lymphocyte cytogenetic assay both in the presence and absence of S9 mix at dose levels up to 5500 micrograms/ml using lymphocytes obtained from a male donor and in the presence of S9 mix using lymphocytes obtained from a female donor. Statistically significant increases in chromosomal damage were observed at 5500 micrograms/ml dose level in cells from both donors. This positive response was enhanced by the inclusion of chromosomal gaps in the calculations. It was concluded that caprolactam induces chromosomal damage in human lymphocytes in vitro albeit at comparatively high dose levels.     

Induction of sister-chromatid exchanges in cultured human lymphocytes by Aldicarb, a carbamate pesticide

The aim of this work was to investigate the effects of Aldicarb on human lymphocytes in vitro in the presence of an exogenous metabolic activation system. This was done by means of an analysis of SCE and mitotic delay. CP was used to compare the chromosomal effects of Aldicarb with a known genotoxic agent. Our experiments showed that Aldicarb as well as CP induced a significant increase of SCE values in the absence of S9 mix. In vitro metabolic activation of both chemicals increased the SCE values. The addition of a metabolic system slightly decreased the successive mitotic progression of cells in culture.     

Further investigations on the clastogenicity of paracetamol and acetylsalicylic acid in vitro

Paracetamol (PCM) and acetylsalicylic acid (ASA), both widely used analgesics, were tested for their clastogenicity in V79 cells in vitro. Rat liver S9 mix and primary rat hepatocytes (PRH) were used as external activation systems. ASA was found to be negative with and without activation system in concentrations up to 10(-2) M. In contrast PCM induced concentration-dependent chromosomal aberrations with and without activation system within the range of 3 x 10(-3) and 10(-2) M. The greatest effects were observed following continuous treatment with PRH activation and without external metabolization. Pulse treatments without external metabolization, with S9 mix and PRH were less effective. The clastogenic potency of PCM seems to be partly independent of metabolic activation. Although clastogenic effects in vitro were observed only in very high concentrations pharmacokinetic data and other published mutagenicity data indicate that there might be a risk for human use. Peak plasma levels of more than 10(-4) M have been reported (Forrest et al., 1982) and 2 groups of investigators (Kocisova et al., 1988; Hongslo et al., 1990) found PCM to be weakly clastogenic in human lymphocytes in vivo in the maximum human therapeutic dose range.     

Mutagenicity of some substituted 1-phenyl-3,3-dimethyltriazenes. II. Chromosomal aberrations in human peripheral lymphocytes in vitro

The chromosome-breaking activity of four 1-(phenyl)-3,3-dimethyltriazenes was tested in vitro on human peripheral blood lymphocytes using S9 mix as a metabolic activation system. 1-(4-Nitrophenyl)-3,3-dimethyltriazene was the most active compound. The difference in the frequency of chromosomal aberrations in a test with and without metabolic activation was significant at the 1% level of significance. The lowest frequency of chromosomal aberrations was induced by 1-(4-methylphenyl)-3,3-dimethyltriazene which, under the conditions of this experiment, is the least stable and probably rapidly degraded to non-active compounds. The chromosomal aberrations were also induced by 1-(4-chlorophenyl)-3,3-dimethyltriazene and 1-(4-bromophenyl)-3,3-dimethyltriazene, this activity was unrelated to metabolic activation.     

Mutagenicity of some substituted 1-phenyl-3,3-dimethyltriazenes: II. Chromosomal aberrations in human peripheral lymphocytes in vitro

The chromosome-breaking activity of four 1-(phenyl)-3,3-dimethyltriazenes was tested in vitro on human peripheral blood lymphocytes using S9 mix as a metabolic activation system. 1-(4-Nitrophenyl)-3,3-dimethyltriazene was the most active compound. The difference in the frequency of chromosomal aberrations in a test with and without metabolic activation was significant at the 1% level of significance. The lowest frequency of chromosomal aberrations was induced by 1-(4-methylphenyl)-3,3-dimethyltriazene which, under the conditions of this experiment, is the least stable and probably rapidly degraded to non-active compounds. The chromosomal aberrations were also induced by 1-(4-chlorophenyl)-3,3-dimethyltriazene and 1-(4-bromophenyl)-3,3-dimethyltriazene, this activity was unrelated to metabolic activation.     

Differences in the induction of SCEs between human whole blood cultures and purified lymphocyte cultures and the effect of an S9 mix

Studies for SCE induction are frequently performed on human blood cultures. Either whole blood cultures (WBC) or purified lymphocyte cultures (PLC) are employed. However, it has been shown that fundamental differences with respect to metabolic activity exist between these two systems. In order to further characterize the whole blood culture and the purified lymphocyte culture, differently acting substances were studied comparatively with and without an Aroclor-1254-induced S9 mix. Treatment with ethyl methanesulfonate (EMS), a direct mutagen, produced distinct SCE induction in both systems. Cyclophosphamide (CP) and benzo[a]pyrene (BP), two indirect mutagens, also led to a significant increase of SCEs both in WBC and PLC without S9 mix. Only with CP was this effect more pronounced after addition of S9 mix. Sodium selenite (Na2SeO3), which induced SCEs in WBC, did not show this effect in the PLC. After S9 mix was added to purified lymphocytes, an increase of SCEs by sodium selenite was observed as in WBC. H2O2, a radical former, led to SCE induction in purified lymphocytes but not in the whole blood culture. By adding S9 mix, a distinct reduction of the SCEs induced by H2O2 was established. These results show that human lymphocytes can metabolize indirect mutagens and that it should be kept in mind when using S9 mix that, besides mixed-function oxygenases, it also contains enzymes which influence the SCE-inducing effects of substances.     

In vitro genotoxic perspective of Tamiflu

The aim of this study was to investigate the genotoxic and/or cytotoxic effects of Tamiflu, commercial form of the oseltamivir antiviral and most frequently prescribed for the treatment of influenza infections, on cultured human peripheral lymphocytes by using sister chromatid exchange (SCE), chromosomal aberration (CA), and cytokinesis-blocked micronucleus (CBMN) assays. Cells were treated with 0.5, 1, 2 μg/mL oseltamivir, the Tamiflu capsule ingredient, for 24 or 48 h in the absence or presence of an exogenous metabolic activation system (S9 mix). The test chemical did not demonstrate any genotoxic effect dose-dependently but it showed a weak cytotoxicity on cells in this study. On the other hand, some concentrations of Tamiflu (2 μg/mL without S9 mix for 48 h and 1 μg/mL with S9 mix) induced SCE and also decreased significantly the proliferation index (PI) (48 h period) and the nuclear division index (NDI) (24 h period) (P < 0.05) in the absence of S9 mix. Considering the results, Tamiflu did not induce significant increases of CA or micronucleated cells in vitro in cultured peripheral blood lymphocytes under the treatment conditions used but weak SCE induction was observed. On the other hand, the weak cytotoxic effects observed disappeared in the cultures treated in presence of the S9 mix.     

Genotoxic activity of potassium permanganate in acidic solutions

Potassium permanganate (KMnO4) combined with sulfuric acid is a strongly oxidizing mixture which has been recommended for the destruction and the decontamination of various mutagens/carcinogens in the publication series of the International Agency for Research on Cancer. Evaluation of the genotoxicity of 4 potassium permanganate solutions was performed using a microtechnique of the Ames test with the tester strains TA97, TA98, TA100 and TA102 with and without metabolic activation. Presence of direct-acting mutagens was detected in all the samples with the tester strain TA102 without S9 mix (163-357 revertants/microliters of the solutions). Three samples containing either acetone or ethanol as an organic solvent also induced a mutagenic response on tester strain TA100 without S9 mix (167-337 revertants/microliters). In addition, DNA damage in human peripheral blood lymphocytes was also measured for one of the mixtures by a new technique: the single-cell gel assay (SCGA). A sample with no organic solvent induced DNA damage in human lymphocytes with a dose-response relationship as determined by SCGA. The major mutagenic agent generated by the permanganate solutions was found to be manganese ion (Mn2+). Both manganese sulfate (MnSO4) and manganese chloride (MnCl2) gave mutagenic dose-response relationships on tester strain TA102 without S9 mix. The mutagenic potencies were 2.8 and 2.4 revertant/nmole for MnSO4 and MnCl2 respectively. MnCl2 also induced DNA damage in human lymphocytes as determined by the SCGA. The genotoxic effects of KMnO4 in acidic conditions were probably mediated by the conversion of MnO4- to Mn2+. KMnO4 in alkaline solutions did not produce mutagenic species and may offer an alternative for the degradation of genotoxic compounds.     

In vivo and in vitro promutagen activation by Vicia faba of thiocarbamate herbicides molinate and butylate to products inducing sister chromatid exchanges in human lymphocyte cultures

Molinate and butylate treatments for 4 h of Vicia faba root tip meristems, showed that both thiocarbamate herbicides increased significantly SCE frequency. Direct treatments of molinate and butylate on human lymphocytes applied 24 h after the beginning of culture did not induce SCE. When S10 extracts of the Vicia roots, treated for 4 h with molinate and butylate (in vivo activation) were added to lymphocytes (24 h after of the beginning of culture), SCE were induced in a concentration-response manner. The in vitro assays, in which molinate and butylate was added at 48 h lymphocyte cultures for 4 h, showed a negative response, however, in the treatment where the S10 metabolic mix was added the SCE frequencies were significantly different to the control, and the concentration-response relationship was not observed with molinate, but it was obtained with butylate. The results showed that both herbicides needed the V. faba metabolism to produce SCE in human lymphocyte culture.     

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 results, 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.