Mutations to azaguanine resistance induced in cultured diploid human fibroblasts by the carcinogen, N-acetoxy-2-Acetylaminofluorene

The ability of the carcinogen, N-acetoxy-2-acetylaminofluorene (N-AcO-AAF), to induce mutations to azaguanine resistance in diploid human cells was quantitatively investigated and shown to be dose-dependent. The 8-azaguanine (AG) resistance was shown to be heritable in the absence of mutagen or selective agent and the cells of the mutant clones were shown to retain normal sensitivity to N-AcO-AAF.     

Mutagenic activity of selected aromatic amines and polycyclic hydrocarbons in Drosophila melanogaster

With the use of a series of wild-type and repair-deficient strains and appropriate application procedures, it is possible to demonstrate that carcinogenic aromatic amines and polycyclic hydrocarbons are mutagens in Drosophila. We have shown evidence that AAF, N-OH-AAF, AcO-AAF, BP, DAS and DMBA produce recessive lethals when fed to or injected into adult males. Mutagenic activity was also observed when male larvae were exposed to AAF, BP, DMBA, 3-MC or NA. DA was not mutagenic in the recessive lethal assay under the conditions of the test. DMBA can now be considered as a potent mutagen for Drosophila, although demonstration of its activity depends upon the choice of the treatment procedure and the strain selected. One of the questions concerning the action of aromatic amines and polycyclic hydrocarbons is how their genetic effectiveness in Drosophila can be enhanced. The observation that none of several enzyme inducers (PB, BF, AC, 3-MC) increased their mutagenicity may be interpreted in terms of a more efficient metabolic activation or deactivation. This assumes that active metabolite(s) did not reach the testis in doses sufficient for mutation induction. It also appears that, since the problems pertaining to mutagenicity in Drosophila of aromatic hydrocarbons are obviously a matter of metabolism, the use of repair-deficient strains is no longer an attractive proposal for their elucidation. The present investigation shows that, with weak mutagens, usage of strains mei-9Li or y mei-9a mei-4lD5 does not improve the sensitivity of the recessive lethal method or the test for chromosomal loss. As an alternative, in our opinion more attention should be devoted to possible differences in metabolism between somatic and gonadal tissue. We feel strongly that somatic assay systems might be particularly valuable as a complement to recessive lethal tests on the germ line.     

Effects of DNA damaging agents on cultured fibroblasts derived from patients with Cockayne syndrome.

The cytotoxic action of physical and chemical agents on 10 skin fibroblast strains in culture derived from individuals with Cockayne's syndrome was measured in terms of colony-forming ability. As compared to fibroblasts from normal donors, all Cockayne cell strains tested exhibited a significantly increased sensitivity to UV light and a normal sensitivity to X-rays. Cells from two sets of parents of unrelated Cockayne children showed an intermediate level of UV sensitivity. There was no effect of 0.5 mM caffeine on UV survival in normal and two Cockayne strains tested, indicating that postreplicational repair in Cockayne cells as measured by caffeine sensitivity was probably normal. Sensitivity of normal and Cockayne cells to the chemical carcinogens and mutagens 4NQO, N-AcO-AAF, ICR-170 and EMS was also compared. An increased sensitivity of Cockayne cells to 4NQO or N-AcO-AAF, but not the ICR-170 or EMS, was observed. However, unlike the intermediate UV sensitivity, the cell strains from two parents of Cockayne patients showed the same sensitivity to N-AcO-AAF or 4NQO as fibroblasts from normal individuals. Quantiation of damage to the DNA after 20 J . m-2 UV irradiation indicates normal levels of [3H] thymidine incorporation in the Cockayne cells, in contrast to UV-irradiated xeroderma pigmentosum cells (XP 12BE) in which there was a very low level of repari synthesis. Moreover, we have shown previously that excision of UV-induced pyrimidine dimers in 2 of the 10 Cockayne cell strains was normal.     

Comparison of the frequency of diphtheria toxin and thioguanine resistance induced by a series of carcinogens to analyze their mutational specificities in diploid human fibroblasts

The mutagenic specificities of ethylnitrosourea (ENU), X-rays (+/-)7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7, 8,9,10-tetrahydrobenzo[a]pyrene (BPDE), ICR-191, and N-acetoxy-2-acetylaminofluorene (N-AcO-AAF) were analyzed and compared in diploid human fibroblasts and Salmonella typhimurium. In the human fibroblasts, we compared the frequency of diphtheria toxin (DT)-resistant mutants, presumably induced in the gene coding for elongation factor-2, with the frequency of 6-thioguanine (TG) resistance induced by mutations in the gene coding for hypoxanthine(guanine)phosphoribosyltransferase (HPRT). Recovery of DT-resistant (DTr) cells requires that the mutant EF-2 retain the ability to carry on protein synthesis since the normal EF-2 will be inactivated by DT selection. Therefore, the DTr mutation cannot involve major changes in the gene. In contrast, cells can acquire TG resistance by any mechanism which eliminates HPRT activity, e.g., base substitution, frameshift, deletion, loss of chromosomes. Each agent was assessed by calculating the ratio of the slopes of the dose-response plots (induced variant frequency as a function of dose of the agent used) for the two markers (DTr/TGr variants.). In S. typhimurium we examined the reversion frequency in four histidine-requiring strains bearing forward mutations of the frameshift (TA1538, TA98) or missense (TA1535, TA100) type. ENU, which was predominantly a base substitution mutagen in the bacteria, gave a ratio of DTr to TGr variants of 1.5. As expected of an agent inducing gross chromosomal changes, X-rays induced no revertants in bacteria and in human cells gave a ratio of 0.1. ICR-191 which was predominantly a frameshift mutagen in bacteria gave a ratio of 0.15. In the set of bacterial strains containing the plasmid pKM101, BPDE reverted both frameshift and base substitution mutations. It did not cause reversions in the other set of strains. In human cells BPDE gave a response similar to ENU, i.e., a ratio of DTr/TGr variants of 1.5. As reported by others, N-AcO-AAF was predominantly a frameshift mutagen in bacteria. However, in the human cells it gave a ratio of DTr/TGr variants of 1.5, similar to ENU and BPDE. These results suggest that in human cells, BPDE and N-AcO-AAF, like ENU, yield predominantly base substitutions, while ICR-191 and X-rays largely produce mutations by mechanisms which result in more extensive alterations in the gene.