3-Nitrobenzanthrone (3-NBA) is one of the most potent mutagens in the Ames Salmonella typhimurium assay and a suspected human carcinogen recently identified in diesel exhaust and in airborne particulate matter. 3-Aminobenzanthrone (3-ABA), 3-acetylaminobenzanthrone (3-Ac-ABA) and N-acetyl-N-hydroxy-3-aminobenzanthrone (N-Ac-N-OH-ABA) have been identified as 3-NBA metabolites. In the present study we investigated the genotoxic effects of 3-NBA and its metabolites in the human B lymphoblastoid cell line MCL-5. DNA strand breaks were measured using the Comet assay, chromosomal damage was assessed using the micronucleus assay and DNA adduct formation was determined by 32P-post-labelling analysis. DNA strand-breaking activity was observed with each compound in a concentration-dependent manner (1-50 microM, 2 h incubation time). At 50 microM median comet tail lengths (CTLs) were 25.0 microm for 3-NBA, 48.0 microm for 3-ABA, 54.5 microm for 3-Ac-ABA and 65.0 microm for N-Ac-N-OH-ABA. Median CTLs in control incubations were in the range 7.7-13.1 micro m. Moreover, the strand-breaking activity of 3-NBA was more pronounced in the presence of a DNA repair inhibitor, hydroxyurea. Depending on the concentration used (1-20 microM, 24 h incubation time), 3-NBA and its metabolites also showed clastogenic activity in the micronucleus assay. 3-NBA and N-Ac-N-OH-ABA were the most active at low concentrations; at 1 microM the total number of micronuclei per 500 binucleate cells was 4.7 +/- 0.6 in both cases, compared with 1.7-3.0 for controls (P < 0.05). Furthermore, multiple DNA adducts were detected with each compound (1 microM, 24 h incubation time), essentially similar to those found recently in vivo in rats treated with 3-NBA or its metabolites. DNA adduct levels ranged from 1.3 to 42.8 and from 2.0 to 39.8 adducts/10(8) nt using the nuclease P1 and butanol enrichment procedures, respectively. DNA binding was highest for N-Ac-N-OH-ABA, followed by 3-NBA, and much lower for 3-ABA and 3-Ac-ABA. All major 3-NBA-derived DNA adducts produced in MCL-5 cells were found to be formed from reductive metabolites bound to purine bases and lacked an N-acetyl group. These results demonstrate that 3-NBA and its metabolites are effectively activated to DNA-damaging species in human MCL-5 cells, which may reflect the genotoxic potential of 3-NBA in humans. Environmental exposure to 3-NBA may be a health hazard for large sections of the population and the risks associated with such exposure require further assessment.