Protective effect of vitamin E against chromosomal aberrations and mutation induced by sodium chromate in Chinese hamster V79 cells

The effect of vitamin E on chromosomal aberrations and mutation caused by Na2CrO4 was investigated in Chinese hamster V79 cells. Pretreatment with 25 microM alpha-tocopherol succinate (vitamin E) for 24 h prior to chromate exposure (2.5-5 microM) resulted in a decrease of metal-induced chromosomal aberrations. Na2CrO4 (2.5-7.5 microM) induced mutations at the HGPRT locus, but only within a very limited concentration range. This mutagenic response could also be suppressed by pretreatment with vitamin E. These results suggest that vitamin E can protect cells from the clastogenic and mutagenic action of chromate compounds, possibly through its ability to scavenge chromium(V) and/or free radicals.     

Naphthofurans induced chromosomal aberrations detected in metaphase, anaphase and telophase V79 Chinese hamster cells

The mutagenic activities of 5 newly synthesized naphthofurans were analysed in two in vitro cytogenetic assays: the metaphase chromosomal aberration assay and the anaphase telophase bridge-fragment assay. Both assays were conducted using V79 Chinese hamster cells. The compounds included: 2-nitro-7-methoxynaphtho[2,1-b]furan (A), 2-nitro-8-methoxynaphtho[2,1-b]furan (B), 2-nitro-naphtho[2,1-b]furan (C), 2-nitro-7-bromonaphtho[2,1-b]furan (D) and 7-methoxynaphtho[2,1-b]furan (E). The cells were treated with 3 concentrations (0.1, 0.2 and 0.4 microgram/ml) of each compound, in the dose range already tested in studies on the mutagenic properties of the same compounds realised with other systems. The highest concentration, only, was used in the anaphase-telophase assay. In the first approach, compounds A, B and C were active while compounds D and E did not increase significantly the aberration frequency above that of the DMSO controls. The results were confirmed in the second approach. They demonstrated that the two studies were complementary. Based on their genotoxic activities, the 5 compounds were ranked in the following decreasing order of potency: A congruent to B much greater than C greater than D congruent to E congruent to DMSO; which is comparable to the ranking order obtained in different in vitro mutagenic and carcinogenic assays. All these activities are closely related to the highly specific molecular structure of each compound, particularly to the nature and position of the different substituents introduced on the skeleton.     

Chromosome aberrations induced by pyrolysates of carbohydrates in Chinese hamster V79 cells

The clastogenic activity of some pyrolysates of carbohydrates was examined in cultured Chinese hamster V79 cells. These pyrolysates include levoglucosan (LG-I), levoglucosenone (LG-II), furfural (FF), 5-(hydroxymethyl)-2-furfural (HMF), glyoxal (GL), methylglyoxal (MGL), 3-deoxy-D-glucosone (DG) and thiazolidine (TZ). LG-I did not induce a significant number of chromosome aberrations at doses up to 8000 micrograms/ml. In contrast, the related compound LG-II induced aberrations and reduced mitosis in a dose-dependent fashion at around 1/2000 of the LG-I doses. Both furan derivatives, FF and HMF, and both glyoxal derivatives, GL and MGL, induced a significant number of chromosome aberrations and a significant lowering of mitotic activity. Among these compounds, FF and MGL showed stronger clastogenic activity than HMF and GL, respectively. DG slightly but positively induced chromosome aberrations. TZ was one of the most potent clastogens among the compounds examined in this study, showing the highest incidence of aberrant cells with many exchanges at doses inducing a significant lowering of mitotic activity. The results of this study indicate the need for a re-evaluation of the thermal decomposition of carbohydrates as a source of genotoxic contaminants.     

Influence of ammonium sulfate and sodium chloride on ethyl methanesulfonate-induced chromosomal aberrations and HPRT mutations in V79 hamster cells

V79 hamster cells were mutagenized with ethyl methanesulfonate (EMS) and immediately afterwards posttreated with hypertonic solutions of sodium chloride or ammonium sulfate. The posttreatment had a clear effect on chromosomal aberrations, but TGr mutations were only enhanced by ammonium sulfate, but not by sodium chloride posttreatment. It is suggested that hypertonic salt posttreatment leads to conformational changes in the DNA, resulting in an increase in TGr mutations and chromosomal aberrations.     

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.     

Further studies on tetracycline-induced mutation in V79 Chinese hamster cells

The interaction between ultraviolet light and tetracycline in producing cell killing and mutation has been studied in V79 Chinese hamster cells. It has been established that these agents act independently of each other. Cycloheximide altered the response to tetracycline in the fractionation experiment: when cycloheximide was not present, fractionation of TC treatment resulted in a higher mutation yield but no change in survival level; in the presence of cycloheximide, however, mutation was greatly reduced but survival increased. The results were taken to indicate that for tetracycline action to take place, de novo protein synthesis during tetracycline treatment was necessary. Caffeine had no influence on tetracycline-induced lethality or mutagenicity. This observation was considered to suggest that tetracycline did not affect cellular repair processes.     

Reduction of chromium(VI) in Chinese hamster V-79 cells

The cellular reduction of chromate(VI) was studied by electron spin resonance spectrometry. Incubation of Chinese hamster V-79 cells with Na2CrO4 resulted in the formation of both chromium(V) and chromium(III) complex in a manner dependent on time (30 min-2 h) and concentration (50-500 microM). Following removal of extracellular chromate, the level of chromium(V) complex decreased quickly during the first hour but more slowly for the next hour, whereas the level of chromium(III) remained unchanged, indicating that chromium(III) is the ultimate ion of this metal in cells. Alkaline elution studies demonstrated that treatment of cells with Na2CrO4 induced DNA single-strand breaks that decreased quickly and DNA-protein crosslinks that persisted for 2 h after removal of this metal. These results suggest that the cellular levels of chromium(V) and chromium(III) may be associated with the formation of DNA damage induced by chromium (VI).     

1,6-Dinitropyrene causes spindle disturbances and chromosomal damage in V79 Chinese hamster cells

We have investigated the cytogenetic effect of 1,6-dinitropyrene (1,6-DNP) in Chinese hamster V79 cells. The chemical caused a dose-dependent increase in the incidence of initial and full C-mitoses, polyploid mitoses, ana-telophases with lagging chromosomes, non-disjunction and multipolar configurations, in a range of 0.05-5 microM. These findings indicate that 1,6-DNP interferes with the functioning of the spindle apparatus in V79 cells. Early signs of spindle disturbances were seen at 1,6-DNP concentrations which only moderately reduced cell growth and division. Analysis of structural chromosomal aberrations revealed the appearance of chromatid-type aberrations with open breaks and exchanges accompanied by gaps. The results indicate that 1,6-DNP is both a spindle-disturbing and a clastogenic agent in V79 cells.     

Chromosomal aberrations in V79 hamster cells induced by hyperosmotic solutions of NaCl

In this study V79 hamster cells were treated with hypertonic NaCl solutions. A pulse treatment of 30 min made it possible to use hypertonic solutions of up to 1500 mM. Hypertonic treatment induced high frequencies of chromosomal aberrations and the aberration pattern led to the conclusion that hypertonicity acts similarly to S-phase-independent mutagens. We also tested the influence of several parameters on aberration induction and tried to standardize the experimental protocol. The mechanisms involved in aberration production after hypertornic treatment are unknown, we discuss a change in chromatin structure, inhibition of repair processes or protein damage responsible for chromosomal aberrations.     

Induction of chromosomal aberrations in Chinese hamster ovary cells by triethyllead acetate.

Organolead compounds enter the environment primarily through the combustion of leaded gasoline and industrial discharge. Lead and lead-containing compounds have been shown to induce a broad spectrum of toxic effects, including hematopoietic, renal, neurologic, and carcinogenic effects. In this study, the mutagenic activity of triethyllead acetate (Et3PbAc) was determined by measuring the induction of chromosomal aberrations in Chinese hamster ovary cells. The results indicate that Et3PbAc is very cytotoxic and a potent clastogen. In preliminary cytotoxicity studies used to determine appropriate test concentrations for chromosomal aberration analysis, the LC50 of Et3PbAc was approximately 10 microM in the absence of metabolic activation, and 80 microM in the presence of metabolic activation. The maximal response was greater with metabolic activation than without. However, a much higher dose was required to elicit a significant response in the presence of metabolic activation than in its absence.     

Comutagenesis of sodium arsenite with ultraviolet radiation in Chinese hamster V79 cells

Summary Solar ultraviolet radiation has been associated with the induction of skin cancer. Recent studies have indicated that near-ultraviolet, especially UVB, is mutagenic. Exposure to trivalent inorganic arsenic compounds has also been associated with increased skin cancer prevalence. Trivalent arsenic compounds are not mutagenicper se, but are comutagenic with a number of cancer agents. Here, we test the hypothesis that arsenite enhances skin cancer via its comutagenic action with solar ultraviolet radiation. Irradiation of Chinese hamster V79 cells with UVA (360 nm), UVB (310 nm) and UVC (254 nm) caused a fluence-dependent increase in mutations at thehprt locus. On an energy basis, UVC was the most mutagenic and UVA the least. However, when expressed as a function of toxicity, UVB was more mutagenic than UVC. Nontoxic concentrations of arsenite increased the toxicity of UVA, UVB and UVC. Arsenite acted as a comutagen at the three wavelengths; however, higher concentrations of arsenite were required to produce a significant (P < 0.05) comutagenic response with UVB. The increased mutagenicity of UVB and UVA by arsenite may play a role in arsenite-related skin cancers.     

2-Chlorobenzylidene malonitrile (CS) causes spindle disturbances in V79 Chinese hamster cells

Exposure of V79 Chinese hamster cells to 2-chlorobenzylidene malonitrile (CS), a chemical used as a sensory irritant for riot control, caused a concentration-dependent increase in the incidence of spindle disturbances. A C-mitotic effect with the appearance of C-metaphases, a metaphase block and the concomitant disappearance of ana-telophase figures were observed after a 3-h treatment. The results indicate that CS might induce aneuploidy in mammalian cells by interacting with the mitotic apparatus.     

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.     

No induction of chromosomal aberrations in Chinese hamster ovary cells by chrysophanol

Chrysophanol is an anthraquinone which occurs in several herbal drugs, e.g. senna, a commonly used laxative. As there are only limited data on its clastogenic potential we have investigated its capability to cause chromosomal aberrations in the Chinese hamster ovary cell assay. There were no significant increases in chromosomal aberrations when chrysophanol was tested up to its limit of solubility with or without metabolic activation. We conclude that chrysophanol had no clastogenic activity under the conditions described.     

Genotoxicity of 2,4,6-trichlorophenol in V79 Chinese hamster cells.

The genotoxicity of the rodent carcinogen 2,4,6-trichlorophenol (TCP) was studied without exogenous metabolic activation in V79 Chinese hamster cells. TCP did not induce mutation at the hprt locus to 6-thioguanine resistance or structural chromosome aberrations. However, it produced statistically significant, dose-related increases in hyperdiploidy and micronuclei. From these results it appears that TCP causes chromosome malsegregation as its major mode of genotoxic action.     

Cytotoxicity and genotoxicity testing of sodium fluoride on Chinese hamster V79 cells and human EUE cells.

The cytotoxic effects of sodium fluoride (NaF) on hamster V79 cells and human EUE cells were studied by measuring the cloning efficiency and DNA, RNA and protein synthesis in cells cultured in the presence of NaF. Potential mutagenicity of NaF was followed on the basis of induced 6-thioguanine-resistant mutants in treated Chinese hamster V79 cells. The results showed that the addition of 10-150 micrograms of NaF per ml of culture medium induced 10-75% cytotoxic effect on hamster V79 cells but had no toxic effect on human EUE cells. NaF was cytotoxic to human EUE cells at considerably higher concentrations (200-600 micrograms/ml). Growth of both cell types with 100 and 200 micrograms of NaF per ml caused inhibition of 14C-thymidine, 14C-uridine and 14C-L-leucine incorporation. This means that NaF inhibits macromolecular synthesis whereby damaging effects were less drastic in human EUE cells. The results of detailed mutagenicity testing on hamster V79 cells showed that NaF did not show any mutagenic effect after long-term (24-h) incubation of hamster cells in the presence of 10-400 micrograms of NaF per ml of culture medium.     

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.     

The induction of chromosome exchange aberrations by carbon ultrasoft X-rays in V79 hamster cells

V79 hamster cells in plateau (extended G1) phase were irradiated with either 250 kV ('hard') X-rays or carbon K characteristic ultrasoft X-rays under conditions minimizing cell overlap. These cells were killed most effectively by the carbon X-rays, by a factor of about 3 relative to hard X-rays, in agreement with our previous findings with cells in exponential growth. Chromosome-type aberrations were measured at 3 fixation times within the first division cycle after irradiation, and an approximately uniform sensitivity to aberration induction was found for both radiations. The combined aberration data show that carbon X-rays are 2 or more times as effective as hard X-rays, depending on dose and/or data fit. Exchange aberrations require recombination between two separate chromosomes, but they are induced efficiently by carbon X-rays with a substantial linear component to the dose-response despite the very short electron tracks (approximately less than 7 nm) that they produce in the cell. This implies either that the participating DNA helices must be lying extremely close together at the time of radiation damage, so that one track can effectively damage both helices, or that only one radiation-damaged chromosome is needed to promote an exchange event.