Nitrate and nitrite are products of the oxidation of nitrogen by microorganisms in plants, soil and water. The amount of nitrate in the environment has increased dramatically since the Industrial Revolution, primarily due to the use of fossil fuels and the production of artificial fertilizers. This increase has led to a number of environmental problems, including the accumulation of nitrate in both freshwater and coastal marine ecosystems and increased levels of stratospheric nitrous oxide (N2O), the most important ozone-depleting substance emitted in the 21st century. Human exposure to nitrate and nitrite occurs primarily through ingestion of food, particularly leafy vegetables, and water. Nitrate can be reduced in the body to nitrite, which can react under acidic conditions with nitrosatable amino compounds to form potentially carcinogenic N-nitroso compounds. Some epidemiological studies have indicated a possible association between various cancers and the ingestion of nitrate and nitrite in drinking water and food. Other recent studies have revealed that nitrate and nitrite can be reduced in vivo to nitric oxide (NO), especially during the stress of hypoxemia and ischemia. In a variety of animal models, the reduction of nitrite to NO suppresses apoptosis and cytotoxicity at reperfusion in the mammalian heart, liver, kidney, and brain. Because vegetables often contain large amounts of nitrate and have protective effects against coronary heart diseases, some investigators have claimed that nitrate and nitrite are responsible for their beneficial effects. However, this speculation should be interpreted cautiously, because the physiological and pathophysiological roles of ingested nitrate and nitrite at high doses remain unclear. We discuss the beneficial and hazardous aspects of ingested nitrate and nitrite in human health.
The effect of dimethyl sulfoxide (DMSO) on the mutagenicity of 14 promutagens belonging to several chemical classes and one direct mutagen as a reference compound was examined in the Ames preincubation test, to clarify how much the test results were affected by its inhibitory activity on metabolic enzymes. The mutagens were assayed by the preincubation method using the TA100 or WP2uvrA(pKM101) bacterial test strains in the presence of 1% and 14% DMSO (concentrations in treatment mixture) with S9 mix for 12 promutagens that are known to be activated by CYP enzymes or without S9 mix for 2 promutagens that are known to be activated by bacterial nitroreductase enzymes, and the direct-mutagen. The data indicate that the mutagenicity of 11 of the 14 promutagens was significantly reduced in the presence of 14% DMSO as compared with that in the presence of 1% DMSO, while the 3 remaining promutagens and the direct mutagen exhibited mutagenicity of equal degree at both concentrations. The largest inhibitory effect of DMSO was found on the nitrosamines in WP2uvrA(pKM101) and TA100, and no cytotoxicity was detected by survival test, with 14% DMSO in WP2uvrA(pKM101), at any amounts of dimethylnitrosamine. Further equivalent or slightly lower cytotoxicity in the presence of 14% DMSO than in the presence of 1% DMSO was detected by decrease in the bacterial background-lawn density, as a whole. These observations suggest that the reduction in the yield of revertant colonies in the presence of 14% DMSO with the 11 chemicals was not due to cytotoxicity of DMSO. The inhibitory effect of DMSO on the mutagenicity of the promutagens can be explained by its inhibitory effect on the drug-metabolizing enzymes involved in the activation/detoxification pathways. Use of DMSO at a low concentration, such as 1%, may be suggested for the Ames test, as for other in vitro genotoxicity tests.
The comet assay has been widely used as a genotoxicity test in vitro/vivo for detecting initial DNA damage in individual cells. One of the difficulties of the assay is slide preparation, for which agarose top and bottom layers and a cell-containing middle layer are needed to immobilize the cells. To establish a practical methodology while maintaining sensitivity and reproducibility, we assessed a simple comet assay method with a hydrophilic slide glass (MAS-coat type, Matsunami glass Ind., Ltd.) instead of an agarose bottom layer. Ethyl methanesulfonate (EMS), mitomycin C (MMC), and N-nitroso dimethylamine (DMN) as genotoxic chemicals and triton X-100 (TRX) as a non-genotoxic chemical were used for validation of this method. Chinese hamster lung (CHL) cells were used. The results showed that EMS and DMN induced a significant increase in tail intensity. However, MMC, a known interstrand cross-linker, did not increase tail intensity, and it was considered that this was because MMC-induced DNA-DNA crosslinks prevent separation of the DNA duplex. TRX did not increase tail intensity. These results are consistent with previous reports and demonstrate that the simple comet assay can clearly detect genotoxicity of chemicals other than interstrand cross-linkers.