The performance of two computer programs, DEREK and TOPKAT, was examined with regard to predicting the outcome of the Ames bacterial mutagenicity assay. The results of over 400 Ames tests conducted at Glaxo Wellcome (now GlaxoSmithKline) during the last 15 years on a wide variety of chemical classes were compared with the mutagenicity predictions of both computer programs. DEREK was considered concordant with the Ames assay if (i) the Ames assay was negative (not mutagenic) and no structural alerts for mutagenicity were identified or (ii) the Ames assay was positive (mutagenic) and at least one structural alert was identified. Conversely, the DEREK output was considered discordant if (i) the Ames assay was negative and any structural alert was identified or (ii) the Ames assay was positive and no structural alert was identified. The overall concordance of the DEREK program with the Ames results was 65% and the overall discordance was 35%, based on over 400 compounds. About 23% of the test molecules were outside the permissible limits of the optimum prediction space of TOPKAT. Another 4% of the compounds were either not processable or had indeterminate mutagenicity predictions; these molecules were excluded from the TOPKAT analysis. If the TOPKAT probability was (i) > or =0.7 the molecule was predicted to be mutagenic, (ii) < or =0.3 the compound was predicted to be non-mutagenic and (iii) between 0.3 and 0.7 the prediction was considered indeterminate. From over 300 acceptable predictions, the overall TOPKAT concordance was 73% and the overall discordance was 27%. While the overall concordance of the TOPKAT program was higher than DEREK, TOPKAT fared more poorly than DEREK in the critical Ames-positive category, where 60% of the compounds were incorrectly predicted by TOPKAT as negative but were mutagenic in the Ames test. For DEREK, 54% of the Ames-positive molecules had no structural alerts and were predicted to be non-mutagenic. Alternative methods of analyzing the output of the programs to increase the accuracy with Ames-positive compounds are discussed.
In 1982, Levin et al. published a paper describing a new Salmonella typhimurium strain, TA102, for detecting mutagenic agents that react preferentially with AT base pairs. This strain has an AT base pair at the critical mutation site within the hisG gene, which is located on a multicopy plasmid, pAQ1; the chromosomal copy of the hisG gene has been deleted. It also has an intact excision repair system, thus facilitating the detection of cross-linking agents, and carries the mutator plasmid, pKM101. Although TA102 has been shown to be reverted by certain mutagenic agents that are not detected in the usual battery of strains (TA1535, TA1537, TA1538, TA98 and TA100), there has been a general reluctance within the field to include TA102 as one of the standard screening strains. This may in part result from the difficulties which have been experienced in many laboratories in maintaining the strain, and in obtaining reproducible spontaneous and induced revertant counts. At Glaxo we routinely include certain Escherichia coli strains in our microbial test battery, and were aware that some of the genetic features offered by TA102 were already being covered by these strains. For example, E.coli WP2 (pKM101) has an AT base pair at the critical mutation site within the trpE gene, is excision proficient (and thus will detect cross-linking agents) and carries the pKM101 plasmid to enhance error-prone repair. From the published literature it was apparent that a number of the 'TA102 specific' mutagens could be detected in E.coli e.g. neocarzinostatin, UV and 8-MOP plus UV.(ABSTRACT TRUNCATED AT 250 WORDS)
In vitro assays for mutagenicity are an important feature of pre-clinical testing and form part of the current regulatory testing conducted early in drug development. They can also play a part in compound selection since mutagenic compounds can be eliminated from a range of potential candidates. Bacterial tests are particularly useful in this area because they generate results quickly, though their use may be limited because they can require up to 4 g of material. A scaled-down version of the Ames test has been developed which requires only approximately 20 mg of material. Initial experiences with this assay using a range of known mutagens and novel compounds have shown that the Miniscreen has similar sensitivity to the Ames test. The major exception is for those mutagens preferentially detected with strains TA1537 and TA1535, which, because of their low spontaneous counts, are not employed in the Miniscreen.
A major constitutive enzyme in the liver of the uninduced rat is cytochrome P450-2E1. This isozyme has been shown to metabolize a number of carcinogens, including low molecular weight nitrosamines and a number of compounds normally regarded as non-mutagenic in the Ames test, e.g. aniline, urethane and benzene. Using the standard induction procedures [Aroclor 1254 or a combination of phenobarbitone (PB) and beta-naphthoflavone (beta-NF)] the level of CYP2E1 in rat liver is actually suppressed and it has been suggested that this may account for the negative findings with these compounds in the Ames test. S9 fractions were prepared from rats pre-treated with pyrazole or ethanol (inducers of CYP2E1) and then used in the Ames test (or pre-incubation modification) with urethane, acetaminophen, aniline, benzene, procarbazine and N-nitrosopyrrolidine. Both pyrazole and ethanol induced S9 were superior to PB/beta-NF-S9 and uninduced-S9 for the activation of N-nitrosopyrrolidine, a known CYP2E1 substrate. However, there was no evidence of mutagenic activity with urethane, aniline, benzene, procarbazine or acetaminophen. As these compounds have demonstrated genotoxicity in vivo, additional important metabolic pathways must be required which are not present in rat liver S9 fraction.
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