Multiple-endpoint mutagenesis with Chinese hamster ovary (CHO) cells: evaluation with eight carcinogenic and non-carcinogenic compounds.

Previously, we have shown that Chinese hamster ovary (CHO) cells are useful for quantifying chemical-induced gene mutations. We have defined the conditions of a Multiplex CHO System which permits determination of mutagen-induced chromosome aberration, and sister chromatid exchange (SCE) in addition to cytotoxicity and gene mutation in the same treated culture. This allows us to extend the spectrum of quantitative mutagenesis to include clastogenic endpoints. In the present study, we used four carcinogenic/noncarcinogenic pairs to validate the relative utility and sensitivity of each endpoint, and to study the interrelationship of these four distinct biological effects. These compounds include the direct-acting carcinogens N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ICR 170 and their noncarcinogenic analogue N-methyl-N'-nitroguanidine (MNG) and ICR 170-OH, and the procarcinogens benzo[a]pyrene (B[a]P) and dimethylnitrosamine (DMN) and their noncarcinogenic analogues pyrene and dimethylamine (DMA) respectively. A rat liver homogenate preparation (S9) was used to assay for the biological activities of procarcinogens. Under our experimental conditions, we observed that carcinogens DMN, B[a]P, MNNG and ICR 170, but not their noncarcinogenic counterparts, showed all four biological effects. Our studies with these chemicals showed that cytotoxicity does not necessarily correlate with any of the genetic endpoints. On a molar basis, noncarcinogens, pyrene and ICR 170-OH show similar toxicity to carcinogens B[a]P and ICR 170, respectively. The other two non-carcinogenic analogues, DMA and MNG, exhibit minimal toxicity at concentrations 10-1,000 times higher than cytotoxic concentrations of the corresponding carcinogens, DMN and MNNG. In general, gene mutation and SCE are more sensitive than chromosome aberration assay. The gene mutation assay is more specific than SCE and chromosome aberration assays since none of the noncarcinogens exhibit a detectable response in the gene mutational assay. ICR 170 and MNNG are much more active than B[a]P and DMN as ranked on a molar basis. These results indicate that the Multiplex CHO System is capable of discriminating divergent structural classes of carcinogenic and noncarcinogenic compounds, such as the eight chemicals chosen for our study.

Genotoxic activities of 2-nitronaphthofurans and related molecules.

The genotoxic activities of 63, 2-nitronaphthofurans and related molecules were examined using two bacterial short-term tests, the Salmonella mammalian microsome assay test or Mutatest, a mutagenesis assay, and/or the SOS Chromotest, an assay for induction of an SOS function in Escherichia coli. Seven compounds were also investigated in the Chinese hamster ovary cells/hypoxanthine-guanine phosphoribosyl transferase (CHO/HGPRT) test, a mammalian gene mutation assay. Our main conclusions are the following: (a) Simple empirical rules relating structure to mutagenic activity in the Mutatest can be derived for some of the compounds. In particular, they account for the extremely high Mutagenic Potency of 7-methoxy-1-methyl-2-nitronaphtho[2,1-b]furan (R7372), approximately 2 X 10(6) mutants/nmol on strain TA100. (b) There is a good quantitative correlation between the Mutagenic Potency in the Salmonella/mammalian microsomes assay and the SOS-inducing potency in the SOS Chromotest. This, and previous evidence, suggests strongly that the 2-nitronaphthofurans derivatives are essentially recA and thus probably umuDC-dependent mutagens. (c) Four out of seven compounds tested in the CHO/HGPRT assay gave responses correlated with the bacterial responses. One of them, 7-methoxy-2-nitronaphtho[2,1-b]furan (R7000), is among, or is, the strongest mutagen described for mammalian cells. We briefly discuss the practical and theoretical implications of these results.

The toxicity of sixteen metallic compounds in Chinese hamster ovary cells.

The toxicity of 16 metal salts to Chinese hamster ovary (CHO) cells was determined by measuring the cloning efficiency (CE) of CHO cells after exposure to the metals. CHO cells differed by a factor of 10(5) to 10(6) in their toxic response to these metal salts. While Cd(II) was the most toxic ion, Mg(II) exhibited the least toxicity based on either CE50 (concentration required to reduce the CE to 50%) or D0 (concentration increment which reduced the CE by 63%). On the basis of CE50, the toxicity ranking was Ag greater than Tl for monovalent metals, Cd greater than Zn greater than Hg greater than Co greater Cu greater than Mn greater than Ni greater than Be greater than Pd greater than Sr greater than Mg for divalent metals, and In greater than Rh greater than Y for trivalent metals. A similar ranking was found for D0. For the 11 divalent metals, correlations of CE50 and D0 in the CHO cell assay and the Pearson-Mawby softness parameter for metals (sigma p) were reasonably strong. A good correlation exists between the results of this study on the toxic response in CHO cells and published data on toxicity in mice and Drosophila. It appears that the CHO cell cloning assay may be useful in preliminary screening of metallic compounds as an indicator of their predicted toxicity in higher organisms.

Mutation induction in Haemophilus influenzae by ICR-191. I. Development of a detection system for frameshift mutations.

The investigation of mutagenic mechanisms in Haemophilus influenzae has been confined until now to mutagens that normally produce mainly base pair substitutions. This paper describes the development of a system suitable for detecting frameshift mutations induced by ICR-191. The system involves reversions from thymidine dependence to thymidine independence. Evidence is presented from a comparison of the responses to ICR-191 and to N-methyl-N'-nitro-N-nitrosoguanidine that the system is specific for frameshift mutations. The genetic recombination involved in transformation leads to a marked increase in "spontaneous" reversion of the frameshift mutations but not of the base substitution mutations. Presumably, this is a consequence of mispairing, with consequent change in the number of bases, during the recombination.

Mutation induction in Haemophilus influenzae by ICR-191. II. Role of DNA replication and repair.

Evidence is presented to show that presumptive frameshift mutations induced in Haemophilus influenzae by ICR-191 are fixed very rapidly, essentially at the time of treatment. DNA synthesis during treatment is essential for fixation, but DNA synthesis after treatment has no effect. The conclusion is drawn that the mutagen acts at the replication fork, possibly to stabilize misannealings arising in association with the discontinuities in the newly synthesized DNA. These results agree with earlier results on Escherichia coli showing that ICR-191 produces peak mutation frequencies in synchronized cultures at times when the replication fork has reached the locus being studied. They are in sharp contrast to the earlier results in H. influenzae with nitroso compounds and hydrazine that suggest these agents produce randomly distributed, reparable pre-mutational damage that still can be fixed (converted to final mutation) for some time after treatment when the replication fork reaches them. No evidence for such persistent pre-mutational lesions was found with ICR-191. A defect in incision appeared to have very little influence on mutation induction by ICR-191 though it caused much more lethality. The interpretation of the mutation data was made somewhat uncertain, however, by an unexplained plating-density effect on the expression of the mutants in this strain. In contrast, incision deficiencies in E. coli and Salmonella typhimurium have been reported to cause a large increase in mutation induction and to allow lesions at some distance from the replication fork to produce mutations.

The role of pre-replication and post-replication processes in mutation induction in Haemophilus influenzae by N-methyl-N'-nitro-N-nitrosoguanidine.

Studies were carried out on the repair and fixation of premutational damage induced in Haemophilus influenzae by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The studies employed a temperature-sensitive DNA elongation mutant (dna9) and its combinations with mutants defective in pyrimidine dimer excision (uvr1, uvr2) and in recombination (rec1). The dna9 mutant is shown to be leaky, allowing about 1% of the normal rate of DNA synthesis at the restrictive temperature. Repair of premutational lesions was detected by a decline in mutation frequency with increasing delay in DNA replication in dna9 at the restrictive temperature. This repair is unaffected by the pyrimidine dimer excision system. Mutation fixation was detected by the ability of DNA from treated and then lysed cells to transfer mutants to recipient cells by transformation. Some fixation occurred at the restrictive temperature but much less than at the non-restrictive temperature suggesting that an appreciable minority of the mutations resulted from lesions introduced near the replication fork but that the majority of mutations arise from lesions introduced at some distance from the fork, perhaps randomly. The DNA synthesized immediately after MNNG treatment is of lower molecular weight than normal and returns to normal with time. This return is blocked in the rec1 mutant, suggesting that recombination is involved. The possible role of this process in MNNG mutagenesis is discussed.

Evidence that UV-inducible error-prone repair is absent in Haemophilus influenzae Rd, with a discussion of the relation to error-prone repair of alkylating-agent damage.

Haemophilus influenzae Rd and its derivatives are mutated either not at all or to only a very small extent by ultraviolet (UV) radiation, X-rays, methyl methanesulfonate, and nitrogen mustard, though they are readily mutated by such agents as N-methyl-N'-nitro-N-nitrosoguanidine, ethyl methanesulfonate, and nitrosocarbaryl. In these respects H. influenzae Rd resembles the lexA mutants of Escherichia coli that lack the SOS or reclex UV-inducible error-prone repair system. This similarity is further brought out by the observation that chloramphenicol has little or no effect on post-replication repair after UV irradiation. In E. coli, chloramphenicol has been reported to considerably inhibit post-replication repair in the wild type but not in the lexA mutant. Earlier work has suggested that most or all the mutations induced in H. influenzae by NC result from error-prone repair. Combined treatment with NC and either X-rays or UV shows that the NC error-prone repair system does not produce mutations from the lesions induced by these radiations even while it is producing them from its own lesions. It is concluded that the NC error-prone repair system or systems and the reclex error-prone system are different.

Attempts to induce mutations in Haemophilus influenzae with the base analogues 5-bromodeoxyuridine and 2-aminopurine.

Attempts were made to induce mutations in Haemophilus influenzae with the base analogues 5-bromodeoxyuridine and 2-aminopurine. These attempts were unsuccessful. Incorporation studies with BrdUrd showed, in agreement with earlier studies on Escherichia coli, that BrdUrd was discriminated against when dThd was also present but was incorporated to essentially the same extent as dThd when only BrdUrd was present. In this latter case, strands fully substituted with BrdUrd was produced, but survival data suggest that bacteria deriving their DNA by replication on such fully substituted templates were inviable. However, bacteria with about 20% of the thymine substituted with bromouracil were usually viable. No mutations could be detected in the descendants of such bacteria. The reasons for this are discussed and it is concluded that in all probability the replication system in species rarely if every treats incorporated bromouracil as anything except a thymine analogue. The alternative possibility, that the negative results are a consequence of the absence of the reclex (SOS) error-prone repair system in this species, is considered much less likely.