An examination of the weak mutagenic response of 1-nitropyrene in Chinese hamster ovary cells

Incubation of suspension cultures of Chinese hamster ovary (CHO) cells with 1-nitropyrene for as long as 2.5 h failed to induce mutations at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus, while incubation with 1-nitrosopyrene, a reduced derivative of 1-nitropyrene, resulted in a strong mutagenic response. Examination of the metabolites produced during these incubations indicated that 1-nitrosopyrene was rapidly reduced to 1-aminopyrene while 1-nitropyrene was not detectably metabolized. Both compounds produced a single major DNA adduct, N-(deoxyguanosin-8-yl)-1-aminopyrene, in the CHO cells and a strong linear relationship was found between mutation induction and the extent of DNA binding. The low level of adducts produced by 1-nitropyrene was consistent with the weak mutagenic response produced by this compound. These results indicate that both 1-nitropyrene and 1-nitrosopyrene are reduced to a reactive electrophile, presumably N-hydroxy-1-aminopyrene, which produces potentially mutagenic DNA damage in CHO cells. Comparison of the relationship between N-(deoxyguanosin-8-yl)-1-aminopyrene formation and mutation induction in CHO cells with the levels of 1-nitropyrene-induced DNA damage associated with positive responses in other assays of genetic toxicity and with the number of mutations associated with the DNA adducts produced by other agents in CHO cells suggests that the CHO/HGPRT assay may be relatively insensitive to 1-nitropyrene-induced DNA damage. The poor capability of CHO cells in reducing 1-nitropyrene and the relative insensitivity of the assay to the DNA damage produced by this compound may contribute to the weak mutagenic response of 1-nitropyrene in CHO cells.     

Clastogenicity of lead chromate particles in hamster and human cells.

Several insoluble compounds of chromium, such as lead chromate, are respiratory carcinogens in experimental animals and suspected to be so in humans. Lead chromate induces neoplastic transformation in cultured cells but the mechanism of genotoxicity is unknown. We examined the effect of lead chromate on the integrity of chromosomes of Chinese hamster ovary (CHO) and human foreskin fibroblasts (HFF) after a 24-h exposure. At 0.4 microgram/cm2, 0.8 microgram/cm2, 2 microgram/cm2 and 8 microgram/cm2 lead chromate particles reduced survival of CHO cells to 86%, 62%, 2% and less than 1% respectively. These concentrations induced a dose-dependent 4-19-fold increase in the percent metaphases with damage. The HFF cells exhibited higher sensitivity in both cytotoxicity and clastogenicity. The spectrum of damage observed for both cell types was primarily achromatic lesions affecting one or both chromatids. To test for particle dissolution effects, CHO cells were treated for 24 h with either clarified medium that had been incubated for 24 h with lead chromate particles, or clarified medium that had been pre-conditioned by CHO cells treated with lead chromate particles for 24 h. No damage was detected in these cells, indicating that extracellular dissolution into ionic lead and chromate did not contribute to the genotoxicity. This is consistent with a previous study in which scanning electron micrographs illustrated internalization of the particles. These results suggest that clastogenesis may be a mechanism for lead chromate induced carcinogenesis.     

Clastogenicity of 2-chlorobenzylidene malonitrile (CS) in V79 Chinese hamster cells.

The clastogenicity of the sensory irritant 2-chlorobenzylidene malonitrile (CS) to V79 Chinese hamster cells was investigated at various exposure conditions. CS efficiently induced chromatid-type aberrations in a dose-dependent manner provided the cells could run through at least one or two S-phases during a 20-h exposure over a 3-h exposure followed by a 20-h recovery period (cell cycle time 8-10 h). The induction of SCEs indicates an S-dependent mechanism. The hydrolysis products o-chlorobenzaldehyde and malonitrile were inactive in these experiments.     

Rotenone induces aneuploidy, polyploidy and endoreduplication in cultured Chinese hamster cells

The clastogenic potential of rotenone, an insecticide, was investigated in cultured Chinese hamster cells. Rotenone induced aneuploidy (hypodiploidy and hyperdiploidy), polyploidy, and endoreduplication, but not structural chromosome aberrations. The highest frequency of polyploidy and endoreduplication was 58.8% and 3.0%, respectively, when cells were treated with rotenone at 1.0 microgram/ml for 30 h.     

Toxicity and DNA damage induced by 1-nitropyrene and its derivatives in Chinese hamster lung fibroblasts

1-Nitropyrene and its chemically synthesised derivatives were investigated for their cytotoxicity and ability to induce DNA-strand breaks in Chinese hamster lung fibroblasts. Both 1-nitrosopyrene (0.25-60 micrograms/ml) and 1-aminopyrene (0.25-25 micrograms/ml) were cytotoxic, and induced the formation of DNA lesions, which were measured as DNA single-strand breaks after sedimentation in alkaline sucrose-density gradients. Higher doses of 1-aminopyrene (25-60 micrograms/ml) inhibited the formation of DNA single-strand breaks. 1-Nitropyrene was not toxic (0.25-60 micrograms/ml) and induced low levels of detectable DNA strand breaks, whilst N-acetyl-1-aminopyrene was inactive. The post-mitochondrial supernatant fraction of Aroclor-induced rat-liver containing 4 mM NADPH (S9 mix) did not promote the activation of 1-nitropyrene. In fact DNA strand breaks induced by either 1-nitropyrene or 1-nitrosopyrene was abolished in the presence of S9 mix. The 1-nitropyrene reduced intermediate, N-hydroxy-1-aminopyrene was synthesised by the reduction of 1-nitrosopyrene with ascorbic acid. In the presence of ascorbic acid, 1-nitrosopyrene caused a 5-fold increase in the number of DNA single-strand breaks when compared to cells treated with 1-nitrosopyrene alone. The results are discussed in terms of the metabolic activation of 1-nitropyrene and 1-aminopyrene in Chinese hamster lung cells.     

Clastogenicity of low pH to various cultured mammalian cells.

It has been reported that low pH itself can be clastogenic to Chinese hamster ovary cells or mouse lymphoma L5178Y cells. On the other hand, there was no indication that low pH is clastogenic to rat or human lymphocytes. Therefore, in order to evaluate the generality of clastogenicity of low pH conditions, chromosomal aberration tests were carried out on Chinese hamster cell line cells (CHO-K1, CHL, Don and V79 379A) and human cells (HeLa and peripheral lymphocytes used as whole-blood cultures). The cytotoxicity of low pH to each cell line was also evaluated by counting surviving cells. The treatment medium used was Eagle's MEM containing 15 mM MES or Bis-Tris as an organic buffer to maintain the acidity of the medium for the 6-h or 24-h treatment period, and pH adjustment was done with NaOH or HCl. Chromosomal aberrations were induced at pH 6.5 or below in CHO or CHL cells, and the maximum frequency was 24.7% at pH 6.0 or 34% at pH 6.3, respectively. About 5-10% of Don or HeLa cells had aberrations over the range of pH 6.6-6.0 or pH 6.6-6.3, respectively. In V79 379A cells or human lymphocytes, however, aberrant cells amounted to about 8% at near pH 6.0, where cell survival was low (less than 20%). About 90% of aberrations induced in each cell line examined were chromatid-type gaps and breaks. When CHO or CHL cells were treated with acidic medium for 6 h plus 18 h recovery in fresh medium, about 20% of cells had aberrations including chromatid exchanges at pH 5.5 or pH 5.7, respectively. These results indicate that clastogenicity of low pH is a general finding, although the extent of it varies with cell type, and that the clastogenicity is associated with varying extents of cytotoxicity. The mechanisms of clastogenesis at low pH are not known, but might involve inhibition of DNA or protein synthesis or DNA-repair enzymes.     

Rate of spontaneous chromosome aberration occurrence in cultured Chinese hamster cells

The spontaneous chromosome mutation rate was studied in cultured aneuploid Chinese hamster cells (clone 237(1)) using the method of slowing down the rate of cell division in a limiting medium containing 0.1% of serum. It was shown that during one cell generation (which lasted 14 days in limiting medium) the accumulation of chromosome aberrations with time took place. The data obtained are in keeping with the assumption of a linear dependence of this accumulation on time. The spontaneous chromosome rearrangement rate was 1.2 X 10(-2) mutations per cell per 24 hours. Proceeding from this value the spontaneous chromosome aberration rate in cells with a normal duration of the cell cycle was 0.6 X 10(-2) per cell per generation.     

Mutagenicity of instant coffee on cultured Chinese hamster lung cells

Coffee showed mutagenic activity in cultured Chinese hamster lung (CHL) cells as assessed by using diphtheria toxin resistance as a selective marker. Most of the mutagenicity was suppressed in the presence of sodium bisulfite. The contribution of methylglyoxal to the total mutagenicity of coffee was less than 3%.     

Clastogenicity, photo-clastogenicity or pseudo-photo-clastogenicity: Genotoxic effects of zinc oxide in the dark, in pre-irradiated or simultaneously irradiated Chinese hamster ovary cells

Zinc oxide (ZnO), a widely used ingredient in dermatological preparations and sunscreens, is clastogenic in vitro, but not in vivo. Given that ZnO has an approximately four-fold greater clastogenic potency in the presence of UV light when compared with that in the dark, it has been suggested to be photo-clastogenic. In order to clarify whether this increased potency is a genuine photo-genotoxic effect, we investigated the clastogenicity of ZnO (mean particle size, 100 nm) in Chinese hamster ovary (CHO) cells in the dark (D), in pre-irradiated (PI, i.e. UV irradiation of cells followed by treatment with ZnO) and in simultaneously irradiated (SI, i.e. ZnO treatment concurrent with UV irradiation) CHO cells at UV doses of 350 and 700 mJ/cm(2). The cytotoxicity of ZnO to CHO cells under the different irradiation conditions was as follows: SI>PI>D. In the dark, ZnO produced a concentration-related increase in chromosome aberrations (CA). In PI or SI CHO cells, ZnO was clastogenic at significantly lower concentrations (approximately two- to four-fold) when compared with effective concentrations in the dark, indicating an increased susceptibility of CHO cells to ZnO-mediated clastogenic effects due to UV irradiation per se. The incidence of CA in SI or PI cells was generally higher than that in the dark. At similar ZnO concentrations, SI conditions generally produced higher CA incidence than PI conditions. However, when ZnO concentrations producing similar cytotoxicity were compared, CA incidences under PI or SI conditions were nearly identical. The modest increase in the clastogenic potency of ZnO following UV irradiation contrasts with the results observed with genuine photo-clastogenic agents, such as 8-MOP, which may produce an increase in clastogenic potency of >15,000-fold under SI conditions. Our results provide evidence that, under conditions of in vitro photo-clastogenicity tests, UV irradiation of the cellular test system per se may produce a slight increase in the genotoxic potency of compounds that are clastogenic in the dark. In conclusion, our data suggest that minor increases in clastogenic potency under conditions of photo-genotoxicity testing do not necessarily represent a photo-genotoxic effect, but may occur due to an increased sensitivity of the test system subsequent to UV irradiation.     

Decreased clastogenicity of dinitropyrenes in Chinese hamster lung (CHL) subclone cells with low NADPH-cytochrome P-450 reductase activity

Clastogenic potentials of 1,3-, 1,6- and 1,8-dinitropyrenes (DNPs) were compared between Chinese hamster lung (CHL) cells and its subclone MM1 cells, which were recently isolated as menadione-resistant cells after treatment with MNNG. NADPH-cytochrome P-450 reductase activity of the MM1 cells decreased to 50% of that in the parental CHL cells. All 3 DNPs induced chromosomal aberrations without exogenous metabolic activation systems in the CHL cells. 1,6- and 1,8-DNP showed equivalent clastogenic potency: the maximum frequency of cells with chromosomal aberrations was about 50% for both chemicals. The clastogenic potential of 1,3-DNP was lower than that of 1,6- and 1,8-DNP: the maximum frequency of aberrant cells was 10%. In the MM1 cells, in contrast, the frequencies of aberrant cells decreased to about 30% of those observed for the parental CHL cells after treatment with 1,6- and 1,8-DNP, and to the same level as that of the concurrent control after treatment with 1,3-DNP. These results suggest a possibility that the reduced clastogenic effect of 3 DNPs in MM1 cells may correlate with the reduced activity of NADPH-cytochrome P-450 reductase which is thought to contribute to the metabolic conversion of these DNPs to their clastogenic forms in the CHL cells.     

Condensed tannins induce micronuclei in cultured V79 Chinese hamster cells

The tannins, delphinidin and procyanidin were isolated from flowers of white clover (Trifolium repens) and the leaves of Arnot Bristly Locust (Robina fertilis) respectively, and tested for mutagenic properties in a range of systems. There was no evidence for either compound causing significant levels of frameshift or base-pair mutagenesis in bacterial mutagenicity assays, although both were weakly positive in a bacterial DNA-repair test. Both compounds very slightly increased the frequency of petite mutagenesis in Saccharomyces cerevisiae strain D5. In V79 Chinese hamster cells, both were efficient inducers of micronuclei. In each of these test systems, increasing the potential of the compound for metabolic activation by addition of 'S9' mix had little effect on toxicity or mutagenicity of either tannin. It would seem that potential chromosome-breaking activity of condensed tannins could represent a carcinogenic hazard for animals grazing on pastures of white clover in flower. It may also have wider implications for human carcinogenesis by some, if not all, condensed tannins.     

The effect of ploidy on chemical mutagenesis in cultured Chinese hamster cells

The frequency of mutations induced by ethyl methane sulfonate was compared in a pseudodiploid Chinese hamster cell strain and in a tetraploid substrain derived from it. The frequency of reverse mutations from glycine auxotrophy to glycine independence was similar in the two strains, as expected for a dominant phenotype. Forward mutation to 6-thioguanine-resistance was 25 fold lower in the tetraploid as compared to the diploid strain. The resistant mutants lack hypoxanthine phosphoribosyl transferase activity and their resistant phenotype is recessive in somatic cell hybrids. A combination of chromosomal segregation and mutation could account for the frequency of these recessive drug-resistant mutants in the tetraploid population.