An investigation of the genotoxic effects of N-nitrosomorpholine in mammalian cells.

N-nitrosomorpholine (NMOR) is a well-known hepatocarcinogen. Since this compound is representative of the group of indirect-acting N-nitrosamines, its metabolic activation should be essential. However, the mechanism of NMOR-induced carcinogenesis is still not completely clear. In this paper we tried to further our understanding of the genotoxic effects of NMOR. The central aim of this study was to elucidate to what extent NMOR requires metabolic activation. For evaluation of the mutagenicity of NMOR, V79 cells were used either in the presence or absence of the microsomal S9 fraction in the mutation assay and formation of reactive oxygen/nitrogen species (ROS/RNS) in Caco-2 cells treated with NMOR was measured by a fluorescent assay. A very weak rise of 6-thioguanine resistant mutations was observed in both NMOR-treated model cells, V79/-S9 and V79/+S9. A significant difference between the level of mutations in V79/-S9 and V79/+S9 cells was recorded on the 7th day of expression only. Data obtained by the fluorescent assay confirmed that NMOR caused generation of ROS/RNS. In summary, the presented results showed that NMOR might induce DNA damage not only indirectly by its activation by drug-metabolizing enzymes but also via direct formation of ROS/RNS.

DNA lesions and cytogenetic changes induced by N-nitrosomorpholine in HepG2, V79 and VH10 cells: the protective effects of Vitamins A, C and E.

INTRODUCTION: N-Nitrosomorpholine (NMOR), present in the workplace of tyre chemical factories, is a known hepatocarcinogen. This compound belongs to the group of N-nitrosamines, which are indirect-acting and require metabolic activation. However, the mechanism of its carcinogenic effect is not completely clear. AIMS: The objective of this study was (i) to compare the DNA-damaging and clastogenic effects of NMOR in three cell lines (HepG2, V79 and VH10) with different levels of metabolizing enzymes and (ii) to determine the protective effects of Vitamins A, C and E against deleterious effects of NMOR. METHODS: The exponentially growing cells were pre-treated with Vitamins A, C and E and treated with NMOR. Genotoxic effects of NMOR were evaluated by single-cell gel electrophoresis (SCGE, comet assay), while the chromosomal aberration assay was used for the study of clastogenic effects. KEY RESULTS: NMOR-induced a significant dose-dependent increase of DNA damage as analyzed by SCGE, but the extent of DNA migration in the electric field was unequal in the different cell lines. Although the results obtained by SCGE confirmed the genotoxicity of NMOR in all cell lines studied, the number of chromosomal aberrations was significantly increased only in HepG2 and V79 cells, while no changes were observed in VH10 cells. In HepG2 cells pre-treated with Vitamins A, C and E we found a significant decrease of the percentage of tail DNA induced by NMOR. The reduction of the clastogenic effects of NMOR was observed only after pretreatment with Vitamins A and E; Vitamin C did not alter the frequency of NMOR-induced chromosomal aberrations under the experimental conditions of this study. CONCLUSIONS: The fat-soluble Vitamins A and E, which are dietary constituents, reduce the harmful effects of N-nitrosomorpholine in human hepatoma cells HepG2, which are endowed with the maximal capacity for metabolic activation of several drugs.

Molecular and cellular influences of butylated hydroxyanisole on Chinese hamster V79 cells treated with N-methyl-N'-nitro-N-nitrosoguanidine: antimutagenicity of butylated hydroxyanisole.

The antioxidant butylated hydroxyanisole (BHA) is a rodent carcinogen that also reduces the mutagenicity and carcinogenicity of other agents. In this study, we have evaluated possible mechanisms for the antimutagenicity of BHA by investigating its effects on N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-treated Chinese hamster V79 cells. Mutant frequency was determined using the hprt/V79 assay, while plating efficiency was used to measure cytotoxicity, and apoptosis was measured by flow immunofluorocytometry. In addition, DNA strand breaks and the kinetics of strand-break rejoining were investigated by the alkaline elution of DNA and by single-cell gel electrophoresis (SCGE). Although the higher concentration of BHA (0.5 mM) increased the cytotoxicity of MNNG and the lower concentration of BHA (0.25 mM) did not change it, both concentrations were antimutagenic in MNNG-treated cells, with the greater effect occurring at the lower BHA concentration. Neither BHA nor MNNG nor BHA + MNNG increased the level of apoptotic nuclei, and BHA did not change the level of MNNG-induced DNA strand breaks, though it did inhibit their rejoining. Determination of O(6)-methylguanine-DNA-methyltransferase (MGMT) activity confirmed that V79 cells do not synthesize active MGMT protein; MGMT activity was also undetectable after MNNG and BHA + MNNG treatment. The ability of BHA to reduce the level of MNNG-induced mutations did not correlate with cytotoxicity, induction of apoptosis, the level of DNA strand break induction, or MGMT activity. A modified SCGE assay showed that BHA significantly reduced the level of formamidopyrimidine-DNA-glycosylase + endonucleaseIII-sensitive sites, which at least partially are caused by oxidative DNA lesions. The results suggest that the protective effect of BHA on MNNG-induced mutagenicity is best explained by the antioxidative activity of BHA, which may scavenge free radicals that participate in MNNG-induced mutagenicity.

Effect of dietary intake of vitamin A or E on the level of DNA damage, chromosomal aberrations, and micronuclei induced in freshly isolated rat hepatocytes by different carcinogens.

Hepatocytes freshly isolated from male Wistar rats fed a common diet or a vitamin A- or vitamin E-supplemented diet (each for 21, 28, or 41 days) were assayed for sensitivity to DNA breakage and cytogenetic changes induced by carcinogens. Different indirectly acting carcinogens were assayed. N-nitrosomorpholine (NMOR) was the only agent that induced DNA breaks, chromosomal aberrations, and micronuclei in all experiments. Benzo[a]pyrene (B[a]p) and dimethyldibenzo [c,g]carbazole (diMeDBC) induced only DNA breaks in all experiments. Occasionally, B[a]P induced chromosomal aberrations and micronuclei, and diMeDBC induced micronuclei, but not chromosomal aberrations. These results demonstrated that the tested carcinogens assayed at concentrations highly effective in a hypoxanthine phosphoribosyltransferase/V79 system significantly increased DNA damage, while cytogenetic changes were less frequent. In hepatocytes from rats fed vitamin A, a reduction in the severity of all three end points was observed after NMOR treatment. After B[a]P treatment, we found a reduction in DNA breaks and chromosomal aberrations; after treatment with diMeDBC, we observed a reduction in DNA breaks. Treatment with vitamin E was less effective: it reduced DNA strand breaks induced by B[a]P and partially reduced those induced by diMeDBC and NMOR and the level of micronuclei induced by NMOR and B[a]P. Both vitamins reduced the level of DNA strand breaks induced by the oxidative effect of a visible light-excited photosensitizer.

Effects of vitamins C and E on cytotoxicity induced by N-nitroso compounds, N-nitrosomorpholine and N-methyl-N'-nitro-N-nitrosoguanidine in Caco-2 and V79 cell lines.

Since N-nitroso compounds as strong carcinogens are closely related to food and nutrition, the cytotoxic effects of N-nitrosomorpholine (NMOR) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and their reduction by vitamins C and E were investigated in hamster V79 cells and human colon carcinoma Caco-2 cells. Cytotoxicity was evaluated by the trypan blue exclusion technique in Caco-2 cells and by the plating efficiency assay in V79 cells. NMOR caused a dose-dependent decline of viable cells in both cell lines; MNNG induced a dose-dependent cytotoxic effect only in V79 cells. Pretreatment of cells with vitamin C and vitamin E significantly reduced the cytotoxicity of NMOR, however, both vitamins had not effect on cytotoxicity induced by MNNG. These results suggest that different N-nitroso compounds react differently with cellular macromolecules. Measurement of the level of NMOR-induced DNA strand breaks and alkali-labile sites in both cell types using the alkaline comet assay also indicates a protective effect of both vitamins against the genotoxic effects of NMOR.

Mutagenicity of 7H-dibenzo[c,g]carbazole and its tissue specific derivatives in genetically engineered Chinese hamster V79 cell lines stably expressing cytochrome P450.

Genetically engineered Chinese hamster V79 cell lines with stable expression of human cytochrome P4501A1 and 1A2 were used to characterize the particular form of P450 enzymes capable of activating 7H-dibenzo[c,g]carbazole (DBC) and its tissue- and organ-specific derivatives, N-methylDBC (N-MeDBC) and 5,9-dimethylDBC (diMeDBC). In addition, a V79 cell line with co-expression of CYP1A2 together with a phase II enzyme, N-acetyltransferase was utilized to study the role of an entire metabolic activation system in biotransformation of these carbazoles. The rise of 6-thioguanine resistant (6-TG(r)) mutations was followed as a marker of biological activity of these agents. None of the carbazoles elevated significantly the frequency of mutations in the parental V79MZ cell line lacking any cytochrome P450 (CYP) activity or in the V79NH cells expressing N-acetyltransferase activity. A variable, however, increase of mutations was found in the cell lines expressing CYP activity. Both DBC, a potent liver and skin carcinogen, and N-MeDBC, a specific sarcomagen, increased significantly (P<0.001) the frequency of 6-TG(r) mutations in V79MZh1A1 cells, expressing the human CYP1A1; in contrast, a strict hepatocarcinogen diMeDBC was devoid of any activity. All carbazoles elevated significantly the level of mutations in the V79MZh1A2 cell line expressing the human CYP1A2, N-MeDBC was most efficient. Co-expression of CYP1A2 together with NAT activity significantly reduced or totally eliminated the mutagenicity of all carbazoles. These data confirm that CYP1A1 is explicitly involved in the activation of sarcomagenic DBC derivatives, whereas CYP1A2 is included in biotransformation of all DBC derivatives. Reactive intermediates formed due to CYP1A2 activation are substrate for conjugation reactions mediated by N-acetyltransferase.

Rosemary-stimulated reduction of DNA strand breaks and FPG-sensitive sites in mammalian cells treated with H2O2 or visible light-excited Methylene Blue.

In this study possible protective effects of rosemary against oxidative DNA damage induced by H2O2- and visible light-excited Methylene Blue in colon cancer cells CaCo-2 and hamster lung cells V79 were investigated. The level of DNA damage (DNA strand breaks) was measured using the classical and modified single cell gel electrophoresis, so-called comet assay. Our findings showed that an ethanol extract from rosemary reduced the genotoxic activity of both agents after a long-term (24 h; 0.3 microg/ml) or short-term (2 h; 30 microg/ml) pre-incubation of cells. We suggest that the extract of rosemary exhibits a protective effect against oxidative damage to DNA as a consequence of scavenging of both *OH radicals and singlet oxygen ((1)O2).