T. Kada

In vitro and host-mediated "Rec-assay" procedures for screening chemical mutagens ; and phloxine, a mutagenic red dye detected

Summary Recombinationless mutant cells of Bacillus subtilis are more sensitive to the cell-killing action of typical chemical mutagens, such as ethyl methanesulfonate, hydroxylamine, mitomycin C , N -methyl- N ′-nitrosoguanidine or4-nitroquinoline- N -oxide than are the wild-type bacteria. Based on this relation, a simple method was devised for screening new chemical mutagens by examining their increased lethal action on Rec − over Rec + bacteria instead of assessing their mutagenic capacities directly. We call this method “rec-assay”. When it was applied to a number of dyes, phloxine was found to be positive. Though this rec-assay procedure did not show the mutagenicity of phloxine directly, successive experiments using E. coli cells showed that this dye really was mutagenic. Thus a correlation between the rec-assay result and the genetic data was confirmed. The rec-assay was also carried out in the peritoneal cavity of mice that had received intramuscular injections of phloxine.

Mutagenicity screening of pesticides in the microbial system

A survey on the mutation induction capacity was made in the microbial system on 166 pesticides including 57 fungicides, 63 herbicides and 46 insecticides. The screening methods consisted of the rec-assay procedure, a sensitivity test utilizing H17 Rec+ and M45 Rec- strains of Bacillus subtilis, as well as the reversion assays on plates utilizing auxotrophic strains of Escherichia coli (WP2) and Salmonella typhimurium (Ames series). Chemicals inducing reversions were detected only among those showing positive effects in the rec-assay but not among negative samples. In addition to Captafol, Captan, Dexon and NBT of which mutagenicities have been previously reported, Dichlorvos, Folpet, 2-hydrazinoethanol (HEH), 5-nitro-1-naphthonitrile (NNN) and Vamidothion were found to be mutagens in our systems.

Inhibitory effects of flavourings on mutagenesis induced by chemicals in bacteria

The antimutagenic potential of twenty-five flavourings was tested against the activity of several kinds of chemical mutagens in Escherichia coli and Salmonella typhimurium. Anisaldehyde, ethylvanillin and vanillin showed marked antimutagenic effects on mutagenesis induced by 4-nitroquinoline 1-oxide, furylfuramide (AF-2), captan or methylglyoxal in E. coli WP2s. However, they were not effective against mutations provoked by 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) or 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) in S. typhimurium TA98. Despite the decrease in the number of mutants, a remarkable increase was observed in the survival of mutagen-treated WP2s cells after exposure to these flavourings. We assume that these compounds may act as bio-antimutagens by enhancing an error-free recombinational repair system, because this reactivation of survival was strictly dependent on the recA gene function but not on the lexA and uvrA gene functions.

Antimutagenic effects of 5-fluorouracil and 5-fluorodeoxyuridine on UV-induced mutagenesis in Escherichia coli

Inhibitors of UV induction of the SOS function were screened. A log phase culture of E. coli PQ37 (sulA::lacZ, rfa, uvrA, Phoc) was irradiated with UV and then immediately subjected to culture for 2 h in a liquid LB medium containing each test compound. Expression of the SOS gene (sulA) was assayed by monitoring the levels of beta-galactosidase. In order to examine the inhibitory effects of test compounds on protein synthesis, the levels of the constitutive alkaline phosphatase were assayed in parallel. The total number of compounds tested was 233, including 44 food and feed additives, 23 naturally occurring compounds and derivatives, 21 antibiotics, 61 pesticides, 33 inorganics and 51 other chemicals. As a result, 5-fluorouracil and 5-fluorodeoxyuridine were found to inhibit considerably the UV induction of the SOS gene without any inhibition of protein synthesis. Mutagenesis induced by UV irradiation was depressed by the addition of either compound at non-toxic concentrations.

Differences in liver homogenates from Donryu, Fischer, Sprague-Dawley and Wistar strains of rat in the drug-metabolizing enzyme assay and the salmonella/hepatic S9 activation test

Comparison studies for detecting differences between liver microsome and S9 preparations from 4 strains (Donryu, Fischer, Sprague-Dawley, Wistar) of young male rats were carried out with pretreatment of the animals by inducers such as PCBs and PB plus 5,6-BF. Each microsome fraction was assayed for the enzymic activity of metabolism of model substrates such as aniline, benzophetamine, BP, DMN and 7-ethoxycoumarin. The hepatic S9 sample was also compared, as regards its metabolizing ability to activate 9 pre-mutagens (2AA, AAF, o-AAT, BP, DAB, DMBA, DMN, m-PDA, quinoline) to directly acting mutagens in the Salmonella/hepatic S9 activation test by using TA98, TA100 and TA1537 strains with or without cytochrome P450 inhibitors (SKF-525A, metyrapone, 7,8-benzo-flavone). In the enzymic assay with PCBs-induced microsomes, BP hydroxylation a strain-specific difference: the microsomes from Fischer and Wistar rats were more effective for metabolizing BP than those from the other strains of rat. The effect of induction by BP plus 5,6-BF for Fischer rats showed relatively higher enzymic activity in the same induction group. Other microsomes prepared from rats with and without induction by PB plus, 5,6-BF did not show a clear-cut strain dependency in the enzymic activities assayed. In the mutation experiments with hepatic S9 samples, the examination of DAB and quinoline revealed a marked strain difference when S9 samples prepared from PCBs-pretreated and PB-plus-5,6-BF-induced rats were used: the S9 sample from Fischer rats was available for activating the two pre-mutagens to directly acting mutagens. No marked difference in the metabolic activation of the remaining 7-pre-mutagens was observed on other S9 preparations. In examinations of mutagenicity activities with the use of three inhibitors, the two S9 preparations made with the two induction methods showed inhibition profiles closely similar to each other. However, there were minor differences in the profiles by these inhibitors. From these findings it was concluded that Fischer rat-liver S9 is useful for detecting mutagens in the metabolic activation test, when induction by PB plus 5,6-BF was used in the Ames Salmonella test.

Radiosensitization of cultured mammalian cells by 5-iodouridine.

Radiosensitization of cultured mammalian cells was studied with halogenated pyrimidines, such as 5-iodouridine or 6-chloropurine, which have been shown to promote bacterial cell lethality when combined with gamma-irradiation. When Chinese hamster cells were exposed to gamma-rays to acidic pH values and the number of colonies was scored after 6 to 11 days of incubation, many more cells were inactivated in the presence of the drug than in its absence. This may be due to radiation-induced cytotoxic iodine radicals from the reagent in the case of 5-iodouridine, because the cells were inactivated efficiently only be contact with the previously-irradiated drug solution. The toxicity of the irradiated drug solution increased remarkably when the pH shifted to acidic side. The radiosensitization and the cytotoxic effects of gamma-irradiated drug solution were not found with 6-chloropurine. This may be the first observation on the lethal effect of chemical radicals on mammalian cells, and it is concluded that radiosensitization with 5-iodouridine does not require the drug incorporation into cellular DNA, at least under the conditions adopted in the present studies.

Studies on the mutability of Escherichia coli K12. II. Modification of radiation sensitivity by reversions in a threonine auxotroph.

Abstract Reversions in a threonine auxotroph of E. coli K12 TH 1014 were due to suppressions involving mod and fgr loci. Large numbers of the descendants from revertant colonis showed increased sensitivities to UV irradiation or γ-rays and formed filamentous cells in the course of post-irradiation incubation. Reversions caused no modification in the host-cell reactivation capacity. These revertants were as sensitive to irradiation as E. coli B, whereas the parent auxotroph had a radiation resistance similar to that of E. coli B/r. Studies on several F − strains carrying differently constructed mod and fgr genes indicated that the genetic character mod + frg + was required to manifest the marked increased radiation sensitivity accompanying filamentation/ The above observations showed an example in which common suppressor gene(s) function both in auxotrophy and filamentation.

Isolation and characterization of variant clones of Chinese hamster cells after treatment with irradiated 5-iodouridine.

Variant clones were isolated from cultured Chinese hamster Don cells after treatment with irradiated 5-iodouridine. The following characters of a primary variant clone, C-11 and a secondary variant clone, C-24 were compared with those of the original clone C-1: colony-forming activity, growth rate in the presence of irradiated and unirradiated 5-iodouridine, distribution of chromosome numbers and cell cohesion. The variant clones C-11 and C-24 were partially resistant to unirradiated 5-iodouridine at lower concentration and C-24 cells were slightly resistant to short-term treatment with irradiated 5-iodouridine. Unlike clines C-1 and C-11 the variant clone C-24 showed no lag phase on growth in 5-iodouridine medium. The modal numbers of the chromosomes of all three clones were 22, like that of normal Chinese hamster diploid cells. Of the three clones, the variant C-24 cells showed the least mutual cohesion and the original C-1 cells showed the most. The possibility that an alteration in cellular membrane might be related to an increase in the resistance to radiosensitizing agents are discussed.