The use of short-term tests to measure the preventive action of reducing agents on formation and activation of carcinogenic nitroso compounds

The effect of reducing agents on the nitrosation of methylguanidine (MG) and on the in vitro activation of dimethylnitrosamine (DMN) was examined by measuring DNA-repair synthesis (unscheduled incorporation of [³H]TdR), shifts in alkaline sucrose gradients, frequency of chromosome aberrations, and clone-forming capacity of cultured human fibroblasts. The reducing agents examined were sodium ascorbate, cysteine, cysteamine, and propyl gallate. Since the short-term bioassays used can be quantitated, it has become relatively easy to detect the inhibitory action of reducing compounds on the nitrosation reaction of MG and metabolic activation (with S-9 preparation) of the precarcinogen DMN, to measure their effective dose range, and to establish the most effective ratios between inhibitory agent and reactant. The results indicate that DNA-repair synthesis is a suitable short-term test for studying the numerous combinations and permutations between several carcinogenic or non-carcinogenic agents, and for estimating the capacity of inhibitory agents to affect formation and activation of chemical carcinogens.     

Mutagenic action of nitroso compounds on Escherichia coli cells]

An attempt to induce some forward and back mutations in two Escherichia coli strains (his- and HfrH requiring thiamine) under the action of the carcinogenic nitrosamines--dimethylnitrosamine (DMN) and diethylnitrosamine (DEN)--is described. For this purpose the cells of E. coli were treated with 5% DMN or 1% DEN for 1 hour at 37 degrees C in 0.14 M NaCl. It was shown that the sensitivity of both strains to both nitrose compounds was not the same. DEN was 5-fold as toxic as DMN for the E. coli cells. DMN and DEN induced neither mutations of resistance to 10(-3) M valine, nor reversions in histidine-dependent strain. These mutations were obtained after the cells were treated with 0.1 M NaNO2. Lethal effects of DMN increased more than in 5 times and the toxicity of DEN did not change in hydroxylating mixture, in which nitrosamines derived to active compounds. Under these conditions both carcinogenes showed a mutagenic activity. DEN proved to be about twice as strong mutagenically as DMN. Thus, in our experiments we could see that DMN and DEN could induce both forward and back mutations in E. coli.     

Diet-induced endogenous formation of nitroso compounds in the GI tract

Red or processed meat, but not white meat or fish, is associated with colorectal cancer. The endogenous formation of nitroso compounds is a possible explanation, as red or processed meat--but not white meat or fish--causes a dose-dependent increase in fecal apparent total N-nitroso compounds (ATNC) and the formation of nitroso-compound-specific DNA adducts. Red meat is particularly rich in heme and heme has also been found to promote the formation of ATNC. To investigate the underlying mechanism of ATNC formation, fecal and ileal samples of volunteers fed a high red meat or a vegetarian diet were analyzed for nitrosyl iron, nitrosothiols, and heme. To simulate the processes in the stomach, food homogenates and hemoglobin were incubated under simulated gastric conditions. Nitrosyl iron and nitrosothiols were significantly (p < 0.0001) increased in ileal and fecal samples after a high red meat diet compared with a vegetarian diet; significantly more nitrosyl iron than nitrosothiols was detectable in ileal (p < 0.0001) and fecal (p < 0.001) samples. The strong correlation between fecal nitrosyl iron and heme (0.776; p < 0.0001) suggested that nitrosyl heme is the main source of nitrosyl iron, and ESR confirmed the presence of nitrosyl heme in fecal samples after a high red meat diet. Under simulated gastric conditions, mainly nitrosothiols were formed, suggesting that acid-catalyzed thionitrosation is the initial step in the endogenous formation of nitroso compounds. Nitrosyl heme and other nitroso compounds can then form under the alkaline and reductive conditions of the small and large bowel.     

The specificity of the genotoxic action of carcinogenic aromatic compounds on Drosophila mus mutants]

Hypersensitivity to the toxic effect of benzo(a)pyrene (B(a)P) was determined in homozygous larvae of two D. melanogaster mus strains (mus 208B2 and mus210). The two others (mus205B1 and mus208B1) were found to be less sensitive and the parent strain was resistant. The lack of correlation between the sensitivity in larvae and the activity of aryl hydrocarbon hydroxylase in S15 fractions from adult flies whole body homogenates of the same strains was demonstrated. The hypertoxic effect of B(a)P and 2-acethylaminofluorene in strain mus210 seems to be rather specific because noncarcinogenic pyrene, benzo(e)pyrene and fluorene did not affect the survival of this most sensitive strain. Perspectives of the strain mus210 use for the environmental genotoxic pollutants screening were discussed.     

Effects of reducing and oxidizing agents on the action of bleomycin.

The effects of reducing agents, such as 2-mercaptoethanol, dithiothreitol, L-ascorbic acid, or sodium borohydride, and oxidizing agents, such as hydrogen peroxide or dehydroascorbic acid, on the in vitro action of bleomycin were investigated. After the incubation of DNA with a low concentration of bleomycin and a reducing or oxidizing agent, single strand breaks were mainly caused in the DNA molecules. The degradation of DNA was largely prevented by the removal of oxygen, or by the addition of divalent cations or of S-(2-aminoethyl)isothiuronium bromide hydrobromide, a radical scavenger, to the incubation mixture. Preincubation of bleomycin with these reducing or oxidizing agents reduced the DNA-degrading activity of the antibiotic. However, this reduction in activity was observed even in the absence of oxygen, or in preincubation mixture supplemented with radical scavenger.     

Effects of beta-carotene on genotoxic lesions in the liver under formation and action of carcinogenic N-nitrosodimethylamine

beta-carotene pretreatment of rats decreased the methylation and formation of single-strand breaks in DNA and also decreased activity of alanine-aminotransferase, sorbitol dehydrogenase, gamma-glutamyl transpeptidase activities, caused by action of N-nitrosodimethylamine or synthesis of this carcinogen from precursors.