Identification of a genotoxic mechanism for 2-nitroanisole carcinogenicity and of its carcinogenic potential for humans

Abstract

2-Nitroanisole (2-NA) is an important industrial pollutant and a potent bladder carcinogen for rodents. The mechanism of its carcinogenicity was investigated in this study. Here we have used two independent methods, (32)P-post-labeling and (3)H-labeled 2-NA, to show that 2-NA binds covalently to DNA in vitro after reductive activation by human hepatic cytosol and xanthine oxidase (XO). We also investigated the capacity of 2-NA to form DNA adducts in vivo. Male Wistar rats were treated i.p. with 2-NA (0.15 mg/kg body wt daily for 5 days) and DNA from several organs was analyzed by (32)P-post-labeling. Two 2-NA-specific DNA adducts, identical to those found in DNA incubated with 2-NA and human hepatic cytosol or XO in vitro, were detected in the urinary bladder (3.4 adducts/10(7) nt), the target organ, and, to a lesser extent, in liver, kidney and spleen. The two DNA adducts found in rat tissues in vivo were identified as deoxyguanosine adducts derived from a 2-NA reductive metabolite, N-(2-methoxyphenyl)hydroxylamine. This reactive metabolite of 2-NA was identified in incubations with human hepatic cytosol, besides 2-methoxyaniline (o-anisidine). The results of our study, the first report on the potential of human cytosolic enzymes to contribute to the activation of 2-NA by nitroreduction, strongly suggest a carcinogenic potency of this rodent carcinogen for humans.

Analysis of covalent ellipticine- and doxorubicin-derived adducts in DNA of neuroblastoma cells by the ³²P-postlabeling technique

“…(ii) 32 P-labeled adducts were also resolved by a modification described by Reddy et al 32 . This procedure has been shown to be suitable for resolution of DNA adducts formed by oanisidine 36 or o-nitroanisole 37 . The solvents used in this case were: D1, 2.3 M sodium phosphate (pH 5.77); D2 was omitted; D3, 2.7 M lithium formate, 5.1 M urea (pH 3.5); D4, 0.36 M sodium phosphate, 0.23 M Tris-HCl, 3.8 M urea (pH 8.0).…”

“…After D4 development and brief water wash, the sheets were developed (along D4) in 1.7 M sodium phosphate (pH 6.0) (D5), to the top of the plate, followed by an additional 30-40 min development with the TLC tank partially opened, to allow the radioactive impurities to concentrate in a band close to the top edge (method B)(ref. 32,36,37 ). Adduct levels were calculated in units of relative adduct labeling (RAL), which is the ratio of c.p.m.…”

“…3D-G). Likewise, modifications of all versions that are appropriate for resolution of more polar adducts on thin layer of PEI cellulose such as the adducts generated in DNA by o-anisidine 36 or o-nitroanisole 37 (see method B in Material and Methods), were tested to determine doxorubicin-derived DNA adducts. Using all these methods, no adducts were again detected (data not shown).…”

CARCINOGENIC POLLUTANTS o-NITROANISOLE AND o-ANISIDINE ARE SUBSTRATES AND INDUCERS OF CYTOCHROMES P450

“…Xanthine oxidase is the principal enzyme responsible for the reduction metabolism of o-nitroanisole, catalyzing formation of N-(2-methoxyphenyl)hydroxylamine and o-anisidine 4,5 . Deoxyguanosine adducts derived from N- (2-methoxyphenyl)hydroxylamine were found in vivo in tissues, mainly urinary bladder, of rats treated with o-nitroanisole as well as in vitro after incubation of o-nitroanisole with human hepatic cytosols or purified buttermilk xanthine oxidase 4 .…”

“…Deoxyguanosine adducts derived from N- (2-methoxyphenyl)hydroxylamine were found in vivo in tissues, mainly urinary bladder, of rats treated with o-nitroanisole as well as in vitro after incubation of o-nitroanisole with human hepatic cytosols or purified buttermilk xanthine oxidase 4 . In contrast, human hepatic microsomal cytochrome P450 (CYP) enzymes as well as these of experimental animals participate in the detoxification metabolism of o-nitroanisole, leading to its demethylation, which enables its excretion from the organism 6 .…”