DNA lesions and carcinogenicity from the urban air pollutants 2- and 3-nitrobenzanthrone

Abstract: In the early 1990's, Japanese doctors in urban areas discovered an increasing incidence of lung cancer among women, which was not attributed to a change in diet or smoking. This led to a growing investigation of urban air, and subsequently the discovery of 3-nitrobenzanthrone (3-NBA), a pollutant originating from diesel emissions. 3-NBA was isolated from the organic fraction of particulate matter and immediately qualified to be among the most mutagenic substances known. More over, it has the ability to rearrange into its isomer 2-NBA once it is emitted into the atmosphere. Although 2-NBA iis not as genotoxic as 3-NBA it exists in a 70-fold higher concentration, which urges for continuing investigations on its contribution to human health hazard. Extensive in vitro tests have been conducted to gain information about the mechanisms behind the activation of 3-NBA and recently of 2-NBA as well. The results revealed that reactive intermediates of 3-NBA are formed through nitro-reduction, which are potent DNA damaging agents by covalently attaching to the nucleotides - the building blocks of DNA. This covalent binding between the nueleotides and a foreign substance is referred to as DNA addition products, or DNA adducts. Different methods can be used to examine DNA adducts, and the one used in the studies in this thesis is 32P-HPLC. This highly sensitive and reproducible method employs radioactive postlabelling to detect DNA adducts down to about 0.5 DNA adducts/108 normal nueleotides. In addition, since 3-NBA and its isomer 2-NBA belong to a group of substances called quinones, they also possess the potential to induce oxidative damage in cells. Oxidative lesions in DNA can be measured by a very sensitive method called Single cell gel electrophoresis (SCGE or Comet assay), which has been used to measure the level of damage and compared them between these substances and some of their metabolites, in vitro as well as in vivo. The results in paper I and II, presented in this thesis, show that 3-NBA in cell cultures is more genotoxic compared to its isomer 2-NBA metabolite 3amin obenzan throne (3-ABA), and parent compound benzanthrone (BA), regarding DNA adduct formation. On the other hand, 3-ABA and BA were equally potent in inducing oxidative damage, whereas 2-NBA did so the least. This was then also investigated in vivo. Paper 11 showed that DNA adduct data for 3-NBA was comparable in vitro and in vivo, but this was not true for 2-NBA Further, the genotoxic potential of 2-NBA was not as low in vivo as in vitro compared to 3-NBA. In paper III, 3-NBA was also shown to induce massive acute toxic effects, unusual for nitro-PAHs at the levels used in these experiments. The most prominent DNA adducts formed in vivo were characterised using synthesised standards. Finally, the carcinogenic effect of 3-NBA was shown in paper IV, where female rats developed squamous cell carcinomas in the lungs after intratracheal administration. The conclusions are that both 3-NBA and its isomer 2-NBA are health hazardous substances, and it is worth devoting more effort into evaluating their potential to harm humans, especially since the metabolite 3-ABA has been detected in humans occupationally exposed to diesel emission, such as mining workers.