Structure-activity relationships of epoxides: induction of sister-chromatid exchanges in Chinese hamster V79 cells

Analysis of SCE frequencies in Chinese hamster V79 cells was used to investigate structure-activity relationships of epoxides in mammalian cells. For this purpose the SCE-inducing potency of 58 epoxides was determined. Of these, 16 failed to induce SCE in V79 cells. According to the substitution of the oxirane ring the results show general agreement with results obtained in the Ames test. Mono-substituted epoxides had the highest genotoxic potency compared to di- and tri-substituted epoxides. In detail, there are differences in genotoxic potency between bacteria and mammalian cells which can be explained by differences in the cellular uptake of the compounds and by detoxification reactions.     

Induction of sister-chromatid exchanges in Chinese hamster ovary cells by the biotic ketoaldehyde methylglyoxal

The number of sister-chromatid exchanges (SCEs) per metaphase was determined in Chinese hamster ovary cells after 16 h exposure to methylglyoxal (MG) concentrations ranging from 0.1 to 0.75 mM. MG produced an increase of SCE frequency that proved to be dose-dependent, and to reach a maximum of 2 X baseline at the highest nontoxic concentration (0.5 mM).     

Induction of sister-chromatid exchanges in Chinese hamster ovary cells by thiol and hydrazine compoudns

Cysteine, cysteamine and glutathione all induce sister-chromatid exchanges (SCEs) in Chinese hamster ovary (CHO) cells when applied to cell cultures at concentrations between 10(-4) and 10(-2) M. Acute exposure of cells th thiol compound for a period of 2--3 h resulted in a unique dose--response relationship in each instance. This consisted of two peak SCE frequencies, one at either extreme of the concentration range. Each peak corresponded to a 2--3-fold increase over the spontaneous level. A chronic exposure of 24 h, in contrast, resulted in a dose--response relationship consisting of a single peak SCE frequency (representing a 4--5-fold increase over the spontaneous level) at a concentration of approx. 4 x 10(-4) M. The effect of Cu2+ ions included in the medium at a concentration of 10(-5) M was to increase the toxicity and, at some concentrations, the SCE levels occurring after either acute or chronic exposure to thiols. Hydrazine and its derivatives, dimethylhydrazine and isonicotinic acid hydrazide (isoniazid), as well as hydrogen peroxide, also induce SCEs in CHO cells. A 2--3-fold increase over the spontaneous level was observed, depending upon the particular treatment protocol applied. SCE yields after 3 h treatment with dimethylhydrazine and isoniazid were increased if Mn2+, but not Cu2+, was included in the tissue culture medium at a concentration of 10(-5) M. SCE yields after a 24-h treatment with dimethylhydrazine in which Mn2+ was present in, and absent from, the medium were similar. Catalase was observed to reduce the SCE levels resulting from treatment with hydrogen peroxide, dimethylhydrazine and isoniazid. The effect of catalase upon SCEs induced by dimethylhydrazine and isoniazid in the presence of Mn2+ was more evident than when Mn2+ was not included in the culture medium. The significance of these results with respect to the possible active chemical species produced and the mutagenic/carcinogenic risk associated with thiol and hydraizine compounds is discussed.     

Induction of sister-chromatid exchanges by procarcinogens in metabolically competent Chinese hamster epithelial liver cells

An epithelial cell strain has been established from the livers of male Chinese hamsters (CHEL cells). These cells, which proliferate in culture and retain their metabolic enzymatic activities during several subcultures, were used in a sister-chromatid exchange assay to evaluate the effectiveness of polycyclic aromatic hydrocarbons (PAHs), aflatoxin B1 (AFB1) and cyclophosphamide (CP). The results obtained demonstrate that CHEL cells are metabolically competent to activate different classes of procarcinogens into biologically active metabolites. Moreover, they showed a selective capacity to discriminate chemical carcinogens from noncarcinogens. Thus, the CHEL cell system appears to be a promising alternative to the short-term tests that include cell-free rodent liver homogenate to evaluate new promutagens and/or procarcinogens.     

Induction of DNA double-strand breaks in Chinese hamster V79 cells by 2-chlorodeoxyadenosine

2-Chlorodeoxyadenosine was found to induce DNA double-strand breaks as well as cell death in log-phase Chinese hamster V79 cells. The induction of DNA double-strand breaks, measured by a neutral elution technique, was observed after a 2-h incubation of the cells in the presence of 5 microM of 2-chlorodeoxyadenosine, but these breaks were almost rejoined by a subsequent 1-h incubation, even though this drug was present in the medium during incubation. This repair was prevented by the addition of nicotinamide, which is known to inhibit poly(ADP-ribose) synthesis that is strongly associated with the DNA ligation, but not prevented by the addition of 9-beta-D-arabinofuranosyladenine (araA), which is known to inhibit DNA polymerization. These results suggest that the repair of CdA-induced double-strand breaks is achieved by ligation alone without DNA polymerization. When 35 microM of cycloheximide and 1.3 mM of dibutyryl cAMP were added to the medium, it was found that the induction of double-strand breaks by 2-chlorodeoxyadenosine was suppressed, while the cytotoxicity of 2-chlorodeoxyadenosine measured by colony-forming ability was not interfered with. These results suggest that the induction of DNA double-strand breaks is not associated with the cytotoxicity of this drug.     

Induction of aneuploidy by nickel sulfate in V79 Chinese hamster cells

The ability of nickel sulfate (NiSO(4)) to induce chromosome aneuploidy was investigated in vitro using the V79 Chinese hamster cell line. V79 cells were treated with 100-400 microM NiSO(4) for 24h, and monitored up to 72 h following treatment with a chromosome aberration assay, a micronuclei assay using antikinetochore antibodies (CREST assay) and an anaphase/telophase assay.Aneuploid cells were induced in a significant fraction of the cell population 24-48 h following treatment with nickel sulfate. The majority of these cells were hyperdiploid. In addition, nickel sulfate caused increased frequency of cells with kinetochore-positive micronuclei as well as kinetochore-negative micronuclei. Abnormal chromosome segregation such as lagging chromosomes, chromosome bridges and asymmetric segregation were also observed in more than 50% of anaphase or telophase cells following treatment with NiSO(4). The incidences of these abnormalities were dose-dependent in general, although the effects were prominent in a sublethal dose.These results indicate that NiSO(4) has the ability to induce aneuploidy in V79 cells. In addition, the results in anaphase/telophase assay suggest that the compound may have an effect on spindle apparatus, which could result in aneuploidy following abnormal chromosome segregation.     

Induction of sister-chromatid exchanges by pesticides in primary rat tracheal epithelial cells and Chinese hamster ovary cells

Possible induction of sister-chromatid exchanges by butachlor, paraquat, phorate and monocrotophos was examined in primary rat tracheal epithelial (RTE) and Chinese hamster ovary (CHO) cells. At dose levels that killed less than 50% of the cell population, monocrotophos induced SCEs positively in CHO and RTE cells, while paraquat was positive only in RTE cells. In two trials of the same experiment, paraquat and butachlor in CHO cells, and phorate in either RTE or CHO cells failed to induce a significant number of SCEs at any dose level within the ranges assayed. On the other hand, in RTE cells, butachlor induced a significant number of SCEs at a dose level of 5 micrograms/ml in one trial, but was insignificant in another. The inductions in these assays were, however, dose-dependent. The addition of S9 mixture did not alter the results of SCE induction by these 4 pesticides in CHO cells. RTE cells were more vulnerable to paraquat in cytotoxicity and SCE assays than CHO cells. Cytotoxicities were ranked as butachlor greater than phorate greater than paraquat greater than monocrotophos to CHO cells and paraquat greater than butachlor greater than phorate greater than monocrotophos to RTE cells. Significant cell cycle delays were only found in the treatments with the highest dose levels of butachlor, paraquat and phorate in CHO cells. In addition, this is the first report on SCE induction in RTE cells.     

Use of the photo-micronucleus assay in Chinese hamster V79 cells to study photochemical genotoxicity

2-Methoxyaniline (o-anisidine) is a urinary bladder carcinogen in both mice and rats. Since the urinary bladder contains substantial peroxidase activity, we investigated the metabolism of this carcinogen by prostaglandin H synthase (PHS), a prominent enzyme in the urinary bladder, and lactoperoxidase as model mammalian peroxidases. Horseradish peroxidase (HRP)-mediated oxidation of o-anisidine was also determined and compared with the reactions catalyzed by mammalian peroxidases. All three peroxidases oxidized o-anisidine via a radical mechanism. Using HPLC combined with electrospray tandem mass spectrometry, we determined that peroxidases oxidized o-anisidine to a diimine metabolite, which subsequently hydrolyzed to form a quinone imine. Two additional metabolites were identified as a dimer linked by an azo bond and another metabolite consisting of three methoxybenzene rings, which exact structure has not been identified as yet. Using [¹⁴C]-labeled o-anisidine, we observed substantial peroxidase-dependent covalent binding of o-anisidine to DNA, tRNA and polydeoxynucleotides [poly(dX)]. The ³²P-postlabeling assay (a standard procedure and enrichment of adducts by digestion with nuclease P1 or by extraction into 1-butanol prior to ³²P-labeling) was employed as the second method to detect and quantitate binding of o-anisidine to DNA. Using these versions of the ³²P-postlabeling technique we did not observe any DNA adducts derived from o-anisidine. The o-anisidine-DNA adducts became detectable only when DNA modified by o-anisidine was digested using three times higher concentrations of micrococcal nuclease and spleen phosphodiesterase (MN/SPD). We found deoxyguanosine to be the target for o-anisidine binding in DNA using poly(dX) and deoxyguanosine 3′-monophosphate (dGp). A diimine metabolite of o-anisidine is the reactive species forming adducts in dGp. The results strongly indicate that peroxidases play an important role in o-anisidine metabolism to reactive species, which might be responsible for its genotoxicity, and its carcinogenicity to the urinary bladder in rodents. The limitation of the ³²P-postlabeling technique to analyze DNA adducts derived from o-anisidine as a means to estimate its genotoxicity is discussed.     

Influence of beta-myrcene on sister-chromatid exchanges induced by mutagens in V79 and HTC cells

The influence of beta-myrcene (MC) on sister-chromatid exchanges (SCE) in V79 cells induced by 4 S9 mix-activated indirect mutagens was studied. The mutagens used were cyclophosphamide (CP), benzo[a]pyrene (BP), aflatoxin B1 (AFB) and 9,10-dimethyl-1,2-benz[a]anthracene (DMBA). MC effectively inhibited SCEs induced by CP and AFB in a dose-dependent manner, but it had no effect on SCE induction by BP and DMBA. MC also reduced CP-induced SCE frequencies in a hepatic tumor cell line (HTC). These cells are metabolically competent and activate CP into its biologically active metabolites. Our results support the suggestion that MC modulates the genotoxicity of indirect-acting mutagens by inhibiting certain forms of the cytochrome P-450 enzymes required for activation of premutagens like CP and AFB.     

Induction of endoreduplication by hydrazine in Chinese hamster V 79 cells and reduced incidence of sister chromatid exchanges in endoreduplicated mitoses

Hydrazine in high concentrations very effectively induces endoreduplication in Chinese hamster V 79 cells. The addition of 5-bromodeoxyuridine (BrdU) for the duration of one cell cycle prior to the induction of endoreduplication produces diplochromosomes with sister chromatid differentiation (SCD) after differential chromatid staining. The fact that diplochromosomes with complete SCD are obtained shows that endoreduplication was induced in cells that were in G2-phase. The analysis of sister chromatid exchanges (SCEs) showed that hydrazine treatment rarely led to increased SCE frequencies in mitoses after endoreduplication, but that it caused a strong SCE induction in diploid second division metaphases in the same culture. Neither catalase nor cysteine had an effect on the induction of endoreduplication or the incidence of SCEs. Treatment of the cells with mitomycin C prior to addition of BrdU led to increased SCE frequencies. Compared with the normal mitoses from the same preparation, the mitoses after endoreduplication showed a significantly reduced induction of SCEs. In contrast to these findings, SCE induction was not reduced in the common tetraploid V 79 cells after colcemid-induced polyploidization.     

Hypoxanthine enhances hydroxyurea-induced sister-chromatid exchanges in Chinese hamster ovary cells

Treatment with 1 mM hydroxyurea (HU) for 12 h induced sister-chromatid exchange (SCE) in CHO-K1 cells. The induced SCE frequency was always higher in cells grown in Ham's F12 medium than in those grown in RPMI 1640 medium. It was shown that hypoxanthine (Hyp), a component of Ham's F12, was to a great extent responsible for producing a higher level of HU-induced SCEs were synergistically enhanced when Hyp was added to RPMI 1640 medium supplemented with dialyzed fetal bovine serum at a concentration of 30 μM, which is the concentration in Ham's F12 medium.

The radioactivity of [¹⁴C]Hyp was readily incorporated into DNA in either the presence or the absence of HU. The greater part was in the forms of dGMP and dAMP. It was not clear whether Hyp was incorported in the form of dIMP or not. Deoxyguanosine (dGuo), but not deoxyadenosine (dAdo) reversed both the incorporation of radioactivity into DNA and the SCE-enhancing effect of Hyp. Our results indicate that incorporation of Hyp into the dNTP pools and into the DNA, together with perturbation of dGuo metabolism under abnormal conditions during and after HU treatment, is involved in the enhancement by Hyp of HU-induced SCEs.     

Induction of superoxide dismutase, chromosomal aberrations and sister-chromatid exchanges by paraquat in Chinese hamster fibroblasts

We have recently shown that Bloom syndrome fibroblasts have elevated levels of superoxide dismutase activity compared to those of normal fibroblasts. Based on this observation we decided to test whether an increased rate of superoxide radical production could be responsible for the induction of superoxide dismutase and of chromosomal aberrations and sister-chromatid exchanges characteristic of Bloom syndrome. Utilizing the superoxide-generating herbicide paraquat in Chinese hamster fibroblasts, we assayed the cells for dismutase activity, chromosomal aberrations and sister-chromatid exchanges. All 3 parameters investigated demonstrated a dose-dependent increase with paraquat and, consequently, with the superoxide produced. Since the induction of the enzyme is mediated by its substrate, the superoxide anion radical, we concluded that the increased dismutase activity (in Bloom syndrome and paraquat-treated cells) may be a secondary manifestation of an overall imbalance in oxygen metabolism and that this elevated enzymatic activity is insufficient to detoxify the high superoxide levels, which results in elevated levels of chromosomal damage.     

Induction of sister-chromatid exchanges in human lymphocytes and Chinese hamster cells exposed to aflatoxin B1 and N-methyl-N-nitrosourea.

The effect of a 1-h exposure to aflatoxin B1 (AFB1) in inducing sister-chromatid exchange in Chinese hamster ovary (CHO) cells and human lymphocytes in the presence or absence of mixed function oxidase ("S9 mix") was compared. CHO cells were also exposed to a graded series of doses of N-methyl-N-nitrosourea, a powerful inducer of SCE whose action was independent of the presence or absence of S9 mix. CHO and human cells showed a close correlation in response to SCE induction by AFB1 and in both cell systems the additon of mixed function oxidases in the S9 mix resulted in a marked enhancement of action of AFB1.     

Restriction endonucleases do not induce sister-chromatid exchanges in Chinese hamster ovary cells

Bacterial restriction enzymes offer the unique opportunity to determine the biological and cytogenetic consequences of DNA double-strand breakage. To examine the role of various types of breaks in sister-chromatid exchange (SCE) formation, we used restriction enzymes with different recognition sequences and different cutting frequencies to generate DNA double-strand breaks in Chinese hamster ovary (CHO) cells. The restriction enzymes were introduced by electroporation into exponentially growing cells during the second replication cycle in bromodeoxyuridine, and SCEs were analyzed at mitosis. Contrary to results reported by others, we found no increase in SCE frequency in cells exposed to restriction enzymes despite the presence of numerous cells with chromatid aberrations. These data suggest that DNA double-strand breaks do not lead to SCE formation.     

Structure-activity relationships of epoxides: induction of sister-chromatid exchanges in V79 cells by enantiomeric epoxides.

Analysis of SCE frequencies in Chinese hamster V79 cells was used to investigate the influence of the stereoisomeric forms of epoxides in mammalian genotoxicity tests. The SCE-inducing potency of 12 pairs of (R)- and (S)-enantiomeric epoxides which differed in the degree of substitution of the oxirane ring was determined. Of these, 2 pairs of epoxides failed to induce SCE. Different SCE-inducing potencies between the (R)- and (S)-enantiomers were shown for 5 epoxides. This study demonstrates that stereoselectivity might play an important role in genotoxicity testing of chemicals with asymmetric C atoms.     

Induction of mutation in V79 Chinese hamster cells by tetrachlorohydroquinone, a metabolite of pentachlorophenol

Tetrachlorohydroquinone (TCHQ) and tetrachlorocatechol (TCC), two metabolites of the environmental mutagen and carcinogen pentachlorophenol, were tested without exogenous activation in V79 Chinese hamster cells for the induction of mutation at the hypoxanthine phosphoribosyl transferase (HPRT) locus to 6-thioguanine resistance (TGr) and at the Na/K-ATPase locus to ouabain resistance (OuaR). Treatment was for 24 h at 37 degrees C. TCHQ produced statistically significant increases in the frequency of TGr mutants. The lowest observed effective dose (LOED) was 20 microM, where the relative cloning efficiency was 63%. The relationship between the dose of TCHQ and the frequency of TGr mutants was approximately linear over the range of 0-60 microM with an estimated slope (+/- 95% confidence limits) of 1.1 +/- 0.3 mutants per 10(6) clonable cells per microM. At the highest tested dose of TCHQ, 60 microM, the relative cloning efficiency was reduced to 7%. In contrast to TCHQ, TCC was unable to induce TGr mutants at doses up to 120 microM. The relative cloning efficiency at this dose was 5%. Both TCHQ and TCC were unable to induce OuaR mutants. The results suggest that TCHQ is at least partly responsible for the genotoxic activity of pentachlorophenol. TCHQ can produce reactive oxygen species, which may cause large genetic damage such as deletions, resulting in mutation to TGr but not to OuaR.