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Previous studies indicate that gentian violet (GV), a triphenylmethane dye used in agriculture and human medicine, is a clastogen in vitro and a carcinogen in chronically exposed mice and rats. Data on its genotoxic activity, however, have been incomplete and partly contradictory. Mutagenesis and DNA damage experiments were conducted to re-evaluate the genotoxic potential of GV in both bacterial and mammalian cell systems. GV was mutagenic in Salmonella typhimurium tester strains TA97 and TA104, but there was little mutagenic activity detected in strains TA98 and TA100. A rat liver homogenate fraction (S9) tended to increase mutagenicity. The major microsomal metabolites of GV, pentamethylpararosaniline and N,N,N',N'-tetramethylpararosaniline were less mutagenic in TA97 and TA104, while N,N,N',N"-tetramethylpararosaniline was a weak mutagen in Salmonella. GV was not mutagenic in Chinese hamster ovary (CHO) cell strain CHO-K1-BH4, and was a questionable mutagen in CHO-AS52 cells. While GV produced DNA damage as measured by sedimentation of nucleotids derived from B6C3F1 mouse lymphocytes treated in vitro, no damage was found in lymphocytes isolated from mice dosed with GV. GV was also a weak producer of gene amplification in an SV40-transformed Chinese hamster cell line. The results indicate that GV is a point mutagen in bacteria; however, since similar exposure conditions produced weak mutagenic activity in mammalian cells, GV may be carcinogenic by virtue of its clastogenic activity.

With acridine orange (AO) and monochromatic 460-nm light, the mutation rate of the wild-type strain of Escherichia coli (WP2) was 3-fold higher than that of the endonuclease-deficient strain WP2 (uvrA). In addition, the mutation rates of the recombination-deficient strains WP10 (recA) and Bs-1 (uvrB lexA) were also about 3-fold less than that of wild-type strain. This observation is in striking contrast to the earlier results with AO and 500-nm light in which strains WP10 and Bs-1 yielded mutation rates that were 12-fold and 5-fold greater, respectively, than the wild-type response. The relatively large decrease in mutation rate when the uvrA endonuclease was absent together with structural considerations in the binding of AO to DNA lead us to propose that the major lesions leading to mutations produced by 460-nm light in the presence of AO may be true DNa single-strand breaks and occur before DNA replication.

Binary mixtures of benzo(a)pyrene (B(a)P) and benzo(e)pyrene (B(e)P) produce synergistic mutagenic (comutagenic) responses in Salmonella typhimurium strain TA98 (a frameshift detector). The optimum enhancement (25 X) was found at B(a)P concentration of 0.3 microgram/plate and B(e)P concentration of 1.5 microgram/plate. The response of strain TA100 (mostly a base-substitution detector) is opposite that of TA98, showing antagonism and additivity in similar concentration ranges.

Acridine orange (AO) and methylene blue (MB) in the dark were shown to be weak to moderate mutagens (induction of resistance to T5 phage) in repair-deficient strains of Escherichia coli B/r. However, strain WP2 (wild-type) was not mutated by AO in the dark, in confirmation of earlier data. The presence of 2 microM AO reduced by 41% the spontaneous mutation rate in strain WP2, from 4.1 to 2.4 mutants/10(8) cells/generation. In the polymerase I-deficient strain WP6 (polA1), 2 microM AO increased the mutation rate in the dark 14-fold. We propose that both spontaneous and AO-induced mutagenesis in the absence of light occur at the site of semiconservative DNA replication. If the intercalation mechanism for the effects in the absence of light is valid, the wild-type strain (WP2) may be resistant to frameshift mutagenesis induced by intercalated compounds, while the polymerase I-deficient strain (WP6) may be highly suceptible to the presence of an intercalated dye such as AO at the DNA-replication fork. MB and AO likely act through different mechanisms since MB is only a moderate mutagen in strain WP6 and the other repair-deficient strains tested.

Previous studies have shown that 1- and 3-nitrobenzo[a]pyrene (NBaP) were mutagenic in the Salmonella reversion assay without exogenous activation and that 1-, 3- and 6-NBaP were mutagenic in the presence of hepatocytes or liver homogenate (S9). In the present study, 1-, 3- and 6-NBaP were tested for mutagenicity in Chinese hamster ovary (CHO) cells under activation conditions similar to those used in the bacterial studies. None of the NBaPs was mutagenic without exogenous activation and none was mutagenically activated by hepatocytes from unpretreated rats or rats pretreated with Aroclor 1254 or 3-methylcholanthrene. Benzo-[a]pyrene (BaP), the parent compound, induced a strong mutagenic response under all hepatocyte mediation conditions. The NBaPs did produce positive mutagenic responses with S9 activation (3- = 1- greater than 6-NBaP), but these moderate responses were less than those of BaP. The difference between the bacterial and CHO results under the variety of activation conditions suggests the importance of the endogenous metabolism of the target cell as well as the source and the type of exogenous metabolic activation.

The mononitro-substituted isomers of benzo[a]pyrene (B[a]P), 1-, 3- and 6-nitrobenzo[a]pyrene (NB[a]P), are environmental pollutants and are metabolized to mutagens in Salmonella by rat-liver homogenate postmitochondrial supernatant (S9) fractions. In this study, activation of these compounds to mutagens was investigated using the hepatocyte-mediated Salmonella mutagenicity assay. Hepatocytes from rats treated with Aroclor 1254 activated both 3-NB[a]P and 1-NB[a]P to mutagens, while 6-NB[a]P was not mutagenic. The positive mutagenicity responses were functions of both the chemical dose and the hepatocyte concentration. By using a nitroreductase-deficient strain (TA98NR) and a transesterificase-deficient strain (TA98/1,8-DNP6), it was verified that the direct-acting mutagenicities of 1- and 3-NB[a]P primarily were due to metabolic processes involving nitroreduction while the S9- and hepatocyte-mediated mutagenicity responses were also dependent on transesterification. When compared with the mutagenic responses produced with S9, the mutations induced by 1- and 3-NB[a]P in the presence of hepatocytes were relatively more dependent upon nitroreductase metabolism and less on transesterification. Thus, intact hepatocytes were capable of activating 1- and 3-NB[a]P to mutagenic metabolites and some of these metabolites appeared to be different from those produced by S9.