Assessment of the genotoxicity of imidacloprid and metalaxyl in cultured human lymphocytes and rat bone-marrow

Imidacloprid and metalaxyl are two pesticides that are widely used in agriculture, either separately, or in combination. These agents were studied for their possible genotoxic effects with respect to the following cytogenetic end-points: (1) in vitro micronucleus (MN) formation and sister-chromatid exchange (SCE) induction in human lymphocytes and (2) in vivo micronucleus induction in polychromatic erythrocytes (PCEs) of the rat bone-marrow. The results of the MN analysis indicate that MN frequencies after treatment with both pesticides, separately or as a mixture, do not significantly differ from those in the controls except after treatment with metalaxyl alone at 50 microg/ml (p<0.05). The results of the SCE analysis show that SCE frequencies after treatment with imidacloprid do not differ significantly from those in the controls. A statistically significant increase (p<0.05) in SCE frequency resulted from treatments with metalaxyl at 5, 10 and 100 microg/ml and with the combination of imidacloprid and metalaxyl at 100 and 200 microg/ml. Finally, the in vivo micronucleus assay with rat bone-marrow polychromatic erythrocytes showed a statistically significant effect upon separate treatments with imidacloprid and metalaxyl at doses of 300 mg/kg body weight (b.w.) (p<0.01) or upon combined treatment with 200 mg/Kg b.w. (p<0.001) and 400 mg/kg b.w. (p<0.05).     

Induction of sister-chromatid exchanges by pesticides in primary rat tracheal epithelial cells and Chinese hamster ovary cells

Possible induction of sister-chromatid exchanges by butachlor, paraquat, phorate and monocrotophos was examined in primary rat tracheal epithelial (RTE) and Chinese hamster ovary (CHO) cells. At dose levels that killed less than 50% of the cell population, monocrotophos induced SCEs positively in CHO and RTE cells, while paraquat was positive only in RTE cells. In two trials of the same experiment, paraquat and butachlor in CHO cells, and phorate in either RTE or CHO cells failed to induce a significant number of SCEs at any dose level within the ranges assayed. On the other hand, in RTE cells, butachlor induced a significant number of SCEs at a dose level of 5 micrograms/ml in one trial, but was insignificant in another. The inductions in these assays were, however, dose-dependent. The addition of S9 mixture did not alter the results of SCE induction by these 4 pesticides in CHO cells. RTE cells were more vulnerable to paraquat in cytotoxicity and SCE assays than CHO cells. Cytotoxicities were ranked as butachlor greater than phorate greater than paraquat greater than monocrotophos to CHO cells and paraquat greater than butachlor greater than phorate greater than monocrotophos to RTE cells. Significant cell cycle delays were only found in the treatments with the highest dose levels of butachlor, paraquat and phorate in CHO cells. In addition, this is the first report on SCE induction in RTE cells.     

Assessment of the genotoxicity of imidacloprid and metalaxyl in cultured human lymphocytes and rat bone-marrow

Imidacloprid and metalaxyl are two pesticides that are widely used in agriculture, either separately, or in combination. These agents were studied for their possible genotoxic effects with respect to the following cytogenetic end-points: (1) in vitro micronucleus (MN) formation and sister-chromatid exchange (SCE) induction in human lymphocytes and (2) in vivo micronucleus induction in polychromatic erythrocytes (PCEs) of the rat bone-marrow. The results of the MN analysis indicate that MN frequencies after treatment with both pesticides, separately or as a mixture, do not significantly differ from those in the controls except after treatment with metalaxyl alone at 50 μg/ml (p < 0.05). The results of the SCE analysis show that SCE frequencies after treatment with imidacloprid do not differ significantly from those in the controls. A statistically significant increase (p < 0.05) in SCE frequency resulted from treatments with metalaxyl at 5, 10 and 100 μg/ml and with the combination of imidacloprid and metalaxyl at 100 and 200 μg/ml. Finally, the in vivo micronucleus assay with rat bone-marrow polychromatic erythrocytes showed a statistically significant effect upon separate treatments with imidacloprid and metalaxyl at doses of 300 mg/kg body weight (b.w.) (p < 0.01) or upon combined treatment with 200 mg/Kg b.w. (p < 0.001) and 400 mg/kg b.w. (p < 0.05).     

The micronucleus test of methyl methanesulfonate with mouse peripheral blood reticulocytes using acridine orange-coated slides.

The usefulness of the micronucleus assay using mouse peripheral blood erythrocytes and acridine orange (AO)-coated slides was evaluated with methyl methanesulfonate (MMS). The micronucleus test was carried out at doses ranging from 20 to 80 mg/kg body weight in CD-1 mice by intraperitoneal injection. Peripheral blood cells were examined from 0 to 72 h after treatment at 12- or 24-h intervals. Bone marrow cells from other mice treated with 80 mg/kg MMS were also sampled at the same times. The frequency of micronucleated reticulocytes (MNRETs) increased dose-dependently at every sampling time except 72 h, and the maximum frequency of MNRETs was observed at about 36 h after treatment. Micronucleated polychromatic erythrocytes (MNPCEs) in bone marrow after a dose of 80 mg/kg were significantly induced at 12 h to 36 h, and the maximum frequency of MNPCEs was observed at 24 h after treatment. The induction of MNRETs was delayed by about 12 h compared to that of MNPCEs in bone marrow, and the maximum frequencies of MNRETs were lower than those of MNPCEs, but the induction of MNRETs by MMS was significant and dose-dependent. It is concluded, therefore, that bone marrow cells could be replaced by peripheral blood cells as material for the micronucleus assay using AO-coated slides.     

Genotoxicity of the cancer chemopreventive drug candidates CP-31398, SHetA2, and phospho-ibuprofen

The genotoxic activities of three cancer chemopreventive drug candidates, CP-31398 (a cell permeable styrylquinazoline p53 modulator), SHetA2 (a flexible heteroarotinoid), and phospho-ibuprofen (PI, a derivative of ibuprofen) were tested. None of the compounds were mutagenic in the Salmonella/Escherichia coli/microsome plate incorporation test. CP-31398 and SHetA2 did not induce chromosomal aberrations (CA) in Chinese hamster ovary (CHO) cells, either in the presence or absence of rat hepatic S9 (S9). PI induced CA in CHO cells, but only in the presence of S9. PI, its parent compound ibuprofen, and its moiety diethoxyphosphoryloxybutyl alcohol (DEPBA) were tested for CA and micronuclei (MN) in CHO cells in the presence of S9. PI induced CA as well as MN, both kinetochore-positive (Kin+) and -negative (Kin-), in the presence of S9 at ≤100μg/ml. Ibuprofen was negative for CA, positive for MN with Kin+ at 250μg/ml, and positive for MN with Kin- at 125 and 250μg/ml. DEPBA induced neither CA nor MN at ≤5000μg/ml. The induction of chromosomal damage in PI-treated CHO cells in the presence of S9 may be due to its metabolites. None of the compounds were genotoxic, in the presence or absence of S9, in the GADD45α-GFP Human GreenScreen assay and none induced MN in mouse bone marrow erythrocytes.     

Vitamin C is positive in the DNA synthesis inhibition and sister-chromatid exchange tests

Ascorbate caused a dose-dependent increase in sister-chromatid exchanges (SCEs) in Chinese hamster ovary (CHO) cells and in human lymphocytes. Moreover, in the DNA synthesis inhibition test with HeLa cells, ascorbate gave results typical of DNA-damaging chemicals. Catalase reduced SCE induction by ascorbate, prevented its cytotoxicity in CHO cells, and prevented its effect on HeLa DNA synthesis. Ascorbate reduced induction of SCE in CHO cells by N-methylN′-nitrosoguanidine (MNNG) by direct inactivation of MNNG.     

Genetic toxicology evaluation of commercial beers, III. SCE, chromosome aberrations, and forward mutation (HGPRT) of commercial beer products in CHO cells.

Concentrated organic residues extracted from 5 blended aliquots of commercial beers were evaluated for their ability to induce sister chromatid exchange (SCE), chromosomal aberrations and forward mutation in Chinese hamster ovary (CHO) cells. Each extract was prepared by blending 4 commercial beers of similar ingredients and brewing method, passing the beer pool over XAD-2 resin, extracting the resin and concentrating the extract. Studies were performed both with and without metabolic activation using variable amounts of reconstituted residues from 225-fold concentrates of the blended samples. CHO cultures were treated with 0.75 microliters/ml through 10.0 microliters/ml of the concentrates in the SCE assays, 1.0 microliters/ml through 10.0 microliters/ml of the extracts in the aberration assays and 2.5 microliters/ml up to 20 microliters/ml for forward mutation assays. In preliminary screening for SCE as an indicator of potential DNA damage, a significant increase was observed for 3 of 5 concentrated samples; however, no increase in SCE was induced by any of the 5 samples when S9 was added as a source of exogenous metabolic activation. More definitive tests for induction of genetic events, i.e., chromosome aberrations and forward HGPRT mutations, were negative for all 5 extracts whether or not S9 mix was present. Since SCE were not induced in tests with metabolic activation and since there was no concordant aberration or point mutation induction, the preliminary indication of potential DNA damage shown by elevated SCE under conditions without metabolic activation appears to have little biological significance.     

Sister-chromatid exchanges in human lymphocytes induced by dimethoate, omethoate, deltamethrin, benomyl and their mixture.

Dimethoate and omethoate, two common organophosphorus insecticides, induced a dose-related increase in the frequency of sister-chromatid exchanges (SCEs) in human lymphocytes in vitro (P of the regression lines less than 0.01). Two other common pesticides, the pyrethroid insecticide deltamethrin and the systemic fungicide benomyl, induced a modest increase in SCEs which bordered on statistical significance (P = 0.053 and 0.055, respectively). Mixtures of the four pesticides at total concentrations of 41.5 and 83 micrograms/ml (composed of 43% dimethoate, 43% omethoate, 12% deltamethrin and 1.2% benomyl) induced a dose-dependent increase in SCEs (P less than 0.01). The effects of these mixtures of pesticides were variable using lymphocytes from different individuals, although these differences did not attain statistical significance. Moreover, low concentrations of the four pesticides that did not increase SCEs significantly when tested alone, were positive for SCE induction when tested as a mixture. The experiments show that sub-threshold doses of pesticides may increase SCEs when present in a mixture.     

Genetic toxicity of the benzene metabolite trans, trans-muconaldehyde in mammalian and bacterial cells

Previous studies in our laboratory identified trans,trans-muconaldehyde (MUC), a six-carbon diene dialdehyde, as a microsomal metabolite of benzene. This ring-opened metabolite of benzene was also shown to be hematotoxic in mice in a manner similar to benzene. To further explore the role of MUC in relation to benzene toxicity, a number of test systems were utilized to determine its genotoxic potential. In B6C3F1 mice, MUC induced a highly significant increase in sister-chromatid exchange (SCE), the lowest effective dose being 3 mg/kg, but failed to induce any micronuclei (MN). In Chinese hamster ovary (CHO) cells, MUC at concentrations up to 0.8 micrograms/ml was negative in the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) assay. Dose-related increases in the percentage of cells with MN were observed in CHO cells treated with 0.4-0.8 micrograms/ml MUC. MUC did not-cause unscheduled DNA synthesis in rat primary hepatocytes. Treatment of Salmonella typhimurium TA97 with MUC induced a low level of mutations at concentrations ranging from 10 to 70 micrograms/ml with or without S9 activation. MUC was inactive in strains TA1535, TA100, TA1538 and TA98. In CHO cells and rat primary hepatocytes, MUC was cytotoxic at 0.4 and 4.0 micrograms/ml, respectively. Concentrations of 100 micrograms/plate MUC were toxic for bacterial cells. The present findings indicate that MUC is nonmutagenic or minimally mutagenic in bacterial and mammalian in vitro systems. In mammalian cells, MUC is highly cytotoxic and genotoxic.     

Cytogenetic studies of mice exposed to styrene by inhalation.

The data for the in vivo genotoxicity of styrene (STY) are equivocal. To evaluate the clastogenicity and sister-chromatid exchange (SCE)-inducing potential of STY in vivo under carefully controlled conditions, B6C3F1 female mice were exposed by inhalation for 6 h/day for 14 consecutive days to either 0, 125, 250 or 500 ppm STY. One day after the final exposure, peripheral blood, spleen, and lungs were removed and cells were cultured for the analysis of micronucleus (MN) induction using the cytochalasin B-block method, chromosome breakage, and SCE induction. Peripheral blood smears were also made for scoring MN in erythrocytes. There was a significant concentration-related elevation of SCE frequency in lymphocytes from the spleen and the peripheral blood as well as in cells from the lung. However, no statistically significant concentration-related increases were found in the frequency of chromosome aberrations in the cultured splenocytes or lung cells, and no significant increases in MN frequencies were observed in binucleated splenocytes or normochromatic erythrocytes in peripheral blood smears.     

Clastogenicity of the fungicide afugan in cultured human lymphocytes

The genotoxic effects of the fungicide afugan were analysed by measuring chromosomal aberrations (CAs), sister chromatid exchange (SCE) and micronuclei (MN) in cultured human peripheral lymphocytes. Concentrations of 2.5, 5, 10 and 20 microg/ml of afugan were used during 24 and 48 h. Afugan significantly increased the frequency of CAs at 5, 10 and 20 microg/ml concentrations during a 48 h treatment period. A significant increase was observed for induction of SCE and MN at all treatments compared with the negative control. A significant dose-response correlation was found in all tests. Afugan did not affect the replicative index (RI), however it significantly decreased the mitotic index (MI) at all treatment concentrations except 2.5 microg/ml, and at both treatment times. The present results indicate that afugan is clastogenic and cytotoxic to cultured human lymphocytes.     

Resveratrol, a naturally occurring polyphenol, induces sister chromatid exchanges in a Chinese hamster lung (CHL) cell line.

We tested the genotoxicity of 3,5,4'-trihydroxystilbene (resveratrol), a polyphenolic phytoalexin found in grapes, in a bacterial reverse mutation assay, in vitro chromosome aberration (CA) test, in vitro micronucleus (MN) test, and sister chromatid exchange (SCE) test. Resveratrol was negative in the strains we used in the bacterial reverse mutation assay (S. typhimurium TA98 and TA100 and E. coli WP2uvrA) in the absence and presence of a microsomal metabolizing system. It induced structural CAs at 2.5-20 microg/ml and showed weak aneuploidy induction in a Chinese hamster lung (CHL) cell line. It induced MN cells and polynuclear and karyorrhectic cells after 48h treatments in the in vitro MN test. In the SCE test, resveratrol caused a clear cell-cycle delay; at 10 microg/ml, the cell cycle took twice as long as it did in the control. Resveratrol induced SCEs dose-dependently at up to 10 microg/ml, at which it increased SCE six-fold, and the number was almost as large as mitomycin C, a strong SCE inducer. No second mitoses were observed at 20 microg/ml even after 54h. Cell cycle analysis by FACScan indicated that resveratrol caused S phase arrest, and 48h treatment induced apoptosis. Our results suggest that resveratrol may preferentially induce SCE but not CA, that is, it may cause S phase arrest only when SCEs are induced.     

Genotoxic effects of potassium bromate on human peripheral lymphocytes in vitro

The aim of this study was to investigate the genotoxic effects of potassium bromate, which is used as a bleaching agent in flour, on human peripheral blood lymphocytes in vitro by sister chromatid exchange (SCE), chromosomal aberrations (CA) and micronucleus (MN) tests, and also to determine whether it has any genotoxic potential for humans. Cells were treated with 400, 450, 500, 550 microg/ml concentrations of potassium bromate for 24 and 48 h. The SCE frequencies showed an increase after both treatment periods, however, the differences between the treated cells and the control groups were found to be statistically significant only for the 48-h treatment. In addition, potassium bromate statistically significantly induced CA after the 24-h and 48-h treatment periods. Strikingly, potassium bromate induced CA as much as the positive control, mitomycin-C (MMC). Furthermore, potassium bromate decreased both the cell proliferation index (PI) and the mitotic index (MI). Although micronucleus formation was induced by potassium bromate during the 24-h treatment period in a dose-dependent manner, only the doses 500 and 550 microg/ml yielded statistically significant results. In contrast, MN formation was significantly induced at all doses during the 48-h treatment period. These in vitro results provide important evidence about genotoxicity of potassium bromate on a human cell culture system.     

Genotoxicity testing of fluconazole in vivo and in vitro

The genotoxic effects of the antifungal drug fluconazole (trade name triflucan) were assessed in the chromosome aberration (CA) test in mouse bone-marrow cells in vivo and in the chromosome aberration, sister chromatid exchange (SCE) and micronucleus (MN) tests in human lymphocytes. Fluconazole was used at concentrations of 12.5, 25.0 and 50.0 mg/kg for the in vivo assay and 12.5, 25.0 and 50.0 microg/ml were used for the in vitro assay. In both test systems, a negative and a positive control (MMC) were also included. Six types of structural aberration were observed: chromatid and chromosome breaks, sister chromatid union, chromatid exchange, fragments and dicentric chromosomes. Polyploidy was observed in both the in vivo and in vitro systems. In the in vivo test, fluconazole did not significantly increase the frequency of CA. In the in vitro assays, CA, SCE and MN frequencies were significantly increased in a dose-dependent manner compared with the negative control. The mitotic, replication and cytokinesis-block proliferation indices (CBPI) were not affected by treatments with fluconazole. According to these results, fluconazole is clastogenic and aneugenic in human lymphocytes, but these effects could not be observed in mice. Further studies should be conducted in other test systems to evaluate the full genotoxic potential of fluconazole.     

Cytogenetic damage induced in human lymphocytes by sodium bisulfite.

The frequencies of chromosomal aberrations (CA), sister-chromatid exchanges (SCE), and micronuclei (MN) in human blood lymphocytes exposed to sodium bisulfite (sulfur dioxide) at various concentrations ranging from 5 x 10(-5) M to 2 x 10(-3) M in vitro were studied. It was shown that sodium bisulfite (NaHSO3 and Na2SO3, 1:3 M/M) caused an increase in SCE and MN in human blood lymphocytes in a dose-dependent manner, and also induced mitotic delays and decreased mitotic index. For CA, our results indicated that sodium bisulfite induced an increase of chromatid-type aberrations in lymphocytes from three of four donors in a dose-dependent manner. The chemical at low concentrations induced chromatid-type aberrations, but not chromosome-type aberrations; high concentrations induced both chromatid- and chromosome-type aberrations. No cytogenetic damage in human lymphocytes was induced by sodium sulfate. The results have confirmed that sulfur dioxide is a clastogenic and genotoxic agent.     

The chlorophenoxy herbicide dicamba and its commercial formulation banvel induce genotoxicity and cytotoxicity in Chinese hamster ovary (CHO) cells

The sister chromatid exchange (SCE) frequency, the cell-cycle progression analysis, and the single cell gel electrophoresis technique (SCGE, comet assay) were employed as genetic end-points to investigate the geno- and citotoxicity exerted by dicamba and one of its commercial formulation banvel (dicamba 57.71%) on Chinese hamster ovary (CHO) cells. Log-phase cells were treated with 1.0-500.0 microg/ml of the herbicides and harvested 24 h later for SCE and cell-cycle progression analyses. All concentrations assessed of both test compounds induced higher SCE frequencies over control values. SCEs increased in a non-dose-dependent manner neither for the pure compound (r=0.48; P>0.05) nor for the commercial formulation (r=0.58, P>0.05). For the 200.0 microg/ml and 500.0 microg/ml dicamba doses and the 500.0 microg/ml banvel dose, a significant delay in the cell-cycle progression was found. A regression test showed that the proliferation rate index decreased as a function of either the concentration of dicamba (r=-0.98, P<0.05) or banvel (r=-0.88, P<0.01) titrated into cultures in the 1.0-500.0 microg/ml dose-range. SCGE performed on CHO cells after a 90 min pulse-treatment of dicamba and banvel within a 50.0-500.0 microg/ml dose-range revealed a clear increase in dicamba-induced DNA damage as an enhancement of the proportion of slightly damaged and damaged cells for all concentrations used (P<0.01); concomitantly, a decrease of undamaged cells was found over control values (P<0.01). In banvel-treated cells, a similar overall result was registered. Dicamba induced a significant increase both in comet length and width over control values (P<0.01) regardless of its concentration whereas banvel induced the same effect only within 100.0-500.0 microg/ml dose range (P<0.01). As detected by three highly sensitive bioassays, the present results clearly showed the capability of dicamba and banvel to induce DNA and cellular damage on CHO cells.     

Cytogenetic monitoring of a group of Italian floriculturists: no evidence of DNA damage related to pesticide exposure

The induction of sister chromatid exchanges (SCE), structural chromosome aberrations (CA) or micronuclei (MN) was investigated in peripheral lymphocytes of a group of Italian floriculturists exposed to a mixture of pesticides. No statistically significant difference in the frequencies of cytogenetic damage was detected between exposed and control subjects. Assessment of the effect of confounding factors indicated that smoking affected both SCE and CA frequencies. Multiple regression analysis showed that in heavy smokers (≥ 20 cigarettes/day), SCE and CA levels increased significantly by 17% and 54%, respectively, as compared to non-smokers.     

Genotoxicity studies on the antimicrobial drug sulfamethoxazole in cultured human lymphocytes

The genotoxicity of the antimicrobial drug sulfamethoxazole was evaluated in cultured human peripheral blood lymphocytes. The frequencies of sister-chromatid exchange (SCE) and micronuclei (MN) were scored as genetic endpoints. Both tests cover a wide range of induced genetic damage such as primary DNA damage, clastogenicity and aneugenicity. Cultures were set up with blood samples from two healthy donors and the treatment was done with different sulfamethoxazole concentrations ranging from 10 to 500 microg/ml. From the results obtained it appears that this drug is able to induce weak genotoxic effects, as revealed by the slight increase in the SCE and MN frequencies, at least at one of the two highest concentrations tested. However, the results of the SCE assay should be interpreted with caution because the increase is just significant. In addition, cyotoxic/cytostatic effects of sulfamethoxazole were revealed by a decrease in the proliferative rate index (PRI) and in the cytokinesis block proliferation index (CBPI).     

Mechanistic investigations of low dose exposures to the genotoxic compounds bisphenol-A and rotenone

A complete hazard and risk assessment of any known genotoxin requires the evaluation of the mutagenic, clastogenic and aneugenic potential of the compound. In the case of aneugenic chemicals, mechanism of action (MOA) and quantitative responses may be investigated by studying their effects upon the fidelity of functioning of components of the cell cycle. These present studies have demonstrated that the plastics component bisphenol-A (BPA) and the natural pesticide rotenone induce micronuclei and modify the functioning of the microtubule organising centres (MTOCs) of the mitotic spindles of cultured mammalian cells in a dose-dependent manner. BPA and rotenone were used as model compounds in an investigation of dose response relationships for the hazard/risk assessment of aneugens. Thresholds of action for the induction of aneuploidy have been predicted for spindle poisons on the basis of the multiple targets, which may need disabling before a quantitative response can be detected. The cytokinesis blocked micronucleus assay (CBMA) methodology was utilised in the human lymphoblastoid cell lines AHH-1, MCL-5 and Chinese hamster V79 cell lines. A no observable effect level (NOEL) at 10.8 microg/ml BPA was observed for MN induction. Rotenone showed a small increase in MN induction with the first significant effect at 0.25 ng/ml in V79 cells but there was no significant effect in the metabolically competent cell line, MCL-5. For a mechanistic evaluation of the aneugenic effects of BPA and rotenone, fluorescently labelled antibodies were used to visualise microtubules (alpha-tubulin) and MTOCs (gamma-tubulin). The NOELs for tripolar mitotic spindle induction in V79 cells were 7 microg/ml for BPA and 80 pg/ml for rotenone (concentrations which produced similar changes to mitotic index (M.I.)). Interestingly there was close proximity to the NOEL of 10.8 microg/ml BPA for micronucleus (MN) induction in the human lymphoblastoid AHH-1 cell. Multiple MTOCs can therefore be predicted as a possible mechanism for MN induction. The similarity in concentration inducing tripolar mitosis, M.I. and MN changes suggests immunofluorescence analysis to be a useful dose setting assay with emphasis on the mechanism.     

Induction of sister-chromatid exchanges by 2-aminofluorene in cultured human lymphocytes with and without erythrocytes.

2-Aminofluorene (2-AF), an indirect mutagen reported to be metabolically activated by erythrocytes in the Salmonella mutagenicity test, was studied for the induction of sister-chromatid exchanges (SCEs) in human lymphocytes in vitro with (whole-blood cultures) and without erythrocytes (isolated lymphocyte cultures). 2-AF (0.025-0.8 mM) was present in the cultures for the last 48 h of 72-h cultures. In both types of culture, SCEs increased in a dose-dependent manner, with a statistically significant elevation already at the lowest concentration of 2-AF tested and maximum responses of 2.4-fold (whole blood) and 2.1-fold (isolated lymphocytes), in comparison with mean SCEs/cell in control cultures, at 0.4 and 0.2 mM concentrations (respectively). Thus, the induction of SCEs by 2-AF was not dependent on the presence of erythrocytes. Styrene (2 mM), a positive control chemical known to require erythrocytes for efficient SCE induction in vitro, was shown to produce a 4.9-fold increase in SCEs in whole-blood cultures, but only a slight (1.3-fold) effect in isolated lymphocyte cultures. The results suggest that leukocytes, but not erythrocytes, are important in the metabolic activation of 2-AF in the human lymphocyte SCE assay.