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Consumption of fossil fuels has increased indoor and outdoor concentrations of polycyclic aromatic hydrocarbons (PAHs) and nitrogen dioxide (NO2). To study the combined effect of PAH administration and NO2exposure on mutagenicity of urine from animals we injected 400 mg/kg body wt i.p. one of five kinds of PAH (pyrene, fluoranthene, fluorene, anthracene and chrysene) into ICR mice, Wistar rats, Syrian golden hamsters or Hartley guinea pigs after exposure to 20 p.p.m. NO2gas for 24 h and then exposed the animals to NO2gas for an additional 24 h. During the latter 24 h we collected the urine and assayed its mutagenicity with the AmesSalmonellastrains after treatment with β-glucuronidase and arylsulfatase and extraction with dichloromethane. The urine from mice treated with both PAH and NO2showed high mutagenicity for Salmonellatyphimuriumstrains TA98 and TA100, whereas the urine from mice treated with PAH and air showed almost no mutagenic activity. The mutagenicity was decreased in nitroreductase- and acetyltransferase-deficient strains TA98NR and TA98/1, 8-DNP6 respectively. Treatment with a mixture of 20% of each of the five kinds of PAH and NO2augmented the urinary mutagenicity of mice 1.5-fold. The urine from hamsters treated with pyrene or fluoranthene and NO2was also highly mutagenic, but that from rats or guinea pigs was not very mutagenic. The mutagenicity was also decreased in strains TA98NR and TA98/1, 8-DNP6. These results suggest that the urine contains nitro compounds and that the nitration of PAHs occurs in the body of animals under exposure to NO2gas. Actually, the nitrated metabolites of pyrene, l-nitro-6/8-hydroxypyrene and l-nitro-3-hydroxypyrene, were detected in the urine from mice treated with pyrene under exposure to NO2gas. To elucidate the mechanism ofin vivonitration, NO2(20 p.p.m.) was bubbled through 50 mM TVis-HCl buffer (pH 7.4) or dichloromethane solution containing pyrene or 1-hydroxypyrene (10 μg/ml). Pyrene was not nitrated by NO2in either aqueous or organic solutions. However, 1-hydroxypyrene was changed to nitrohydroxy-pyrenes by NO2in the Tris-HCl buffer, but not in the organic solution. Ascorbic acid, α-tocopneroi, giutatmone, oleic acid and hemoglobin were found to inhibit the nitration of 1-hydroxypyrene in aqueous solution. The urinary mutagenicity of mice treated with both pyrene and NO2was also decreased by oral administration of ascorbic acid and α-tocopherol. These results suggest that 1-hydroxypyrene is nitrated by an ionic reaction in the animal body after hydroxylation of pyrene in the liver.

carcinogenesis

CARCINOGENESIS:              In vivo               formation of mutagens by intraperitoneal administration of polycyclic aromatic hydrocarbons in animals during exposure to nitrogen dioxide