PubMed · 5013040
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K Y Zee-Cheng, C C Cheng. 1972-01-15. 4-Hydroxyurazole.. https://doi.org/10.1007/bf01928230
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15-Oxosteviol, postulated to be the mutagenic metabolite of steviol, was observed to be non-mutagenic in preliminary experiments using a number of different systems. Repetition of the original experiment in Salmonella TM677 failed to show any significant induction of 8-azaguanine resistant mutants by 15-oxosteviol even when the number of bacteria tested was greatly increased. Examination of the earlier positive result showed that it could not be justified from the data and revealed a commonly applied way of mishandling data obtained with the TM677 system.
Steviol is the aglycone of stevioside, which is a non-caloric sugar substitute commonly used in Japan. Our previous studies and Pezzuto et al. have demonstrated that steviol is mutagenic after metabolic activation in the forward mutation assay using Salmonella typhimurium TM677 (TM677), whereas it is non-mutagenic in the reverse mutation assay (Ames test) using S. typhimurium TA 100, TA98, TA102 and TA97. There is the possibility, therefore, that activated steviol selectively induces a deletion or insertion of more than one base pair which cannot be detected by strains commonly used in the Ames test. In this study, we confirmed first that the 8-azaguanine (8-AG) resistance of the TM677 mutants appears to reside in the chromosomal gpt gene, since it can be complemented by the gpt gene of E. coli (Ecogpt). The chromosomal DNA of TM677 and TM677 mutants were digested by several restriction enzymes: BamHI, Sau3AI, AluI, TaqI, HaeIII, HpaII and RsaI, and analyzed by the Southern blot hybridization technique with a probe to the gpt gene DNA of E. coli. No significant differences in DNA fragment length, however, were formed between the wild type and spontaneous or steviol-induced mutants.
Singlet oxygen (1 delta gO2) is the lowest energy-excited state of molecular oxygen, and more reactive than the triplet ground-state molecule. Although singlet oxygen has been implicated in a variety of biological effects, including reactions with DNA or some of its components, evidence for mutagenesis by singlet oxygen has remained unclear. We have previously described a system for bacterial exposure to pure exogenous singlet oxygen that eliminates ambiguity regarding the identity of the reactive species responsible for observed results. Despite the potent toxicity of pure singlet oxygen for several different strains of bacteria, we have found no evidence for mutagenicity of singlet oxygen in 26 Salmonella typhimurium histidine-auxotrophic strains killed to 35% survival. These strains included a variety of base-pair substitution or frameshift target sequences for reversion, including targets responsive to oxidative damage and targets rich in GC base pairs. Some strains combined histidine mutations with one or more mutations affecting DNA-repair capacity. 4 strains possessing the hisG46 mutation also were not mutated when exposed to dose ranges killing less than 28% and up to 99% of the bacteria. The relative frequency of small inphase deletions was assayed in hisG428 bacteria exposed to single oxygen and found to be the same as the spontaneous level. In addition to lack of induction of mutation in these strains, the 8-azaguanine forward mutation assay yielded no evidence of mutagenesis by singlet oxygen in strains killed to 15% survival. No induction of genetic changes by singlet oxygen was seen in an assay for duplication of approximately 1/3 of the bacterial chromosome. Tests for the ability of singlet oxygen to induce lambda prophage in E. coli K12 also proved negative. These studies collectively indicate that pure singlet oxygen generated outside the bacterial cell does not react significantly with the bacterial chromosome in ways leading to base-pair substitutions, frameshift mutations, small or large deletions, large duplications, or damage that interferes with DNA replication and induces the SOS system.