Abstract 3461: DNA adducts of ortho-toluidine in human bladder

Abstract

Abstract Recently, ortho-toluidine (o-Tol) has been added to the list of aromatic amines in group 1 of human carcinogens by the International Agency for Research on Cancer. High levels of o-Tol hemoglobin adducts have been found in workers of a chemical factory with high excess of bladder cancer (Ward et al., JNCI 88:1046-52, 1996) as well as in patients treated with the local anesthetic prilocaine (Gaber et al., Toxicology 229:157-64, 2007). In contrast to 4-aminobiphenyl (4-ABP), the presence of DNA adducts of o-Tol has not yet been proven in humans. For the present study we obtained bladder tissue from 46 sudden death victims (SDV) and 12 samples from human bladder tumors. DNA was isolated from the epithelial and a submucosal layer of the SDV tissue samples and from tumor samples. For analysis of DNA adducts releasing o-Tol and 4-ABP, the aromatic amines were hydrolyzed from DNA by addition of 100 μL 4 N HCl. As internal standards d9-o-Tol and d5-4-ABP were added prior to hydrolysis. The acidic DNA solution was washed with dichloromethane and the amines were extracted with hexane after alkalinization. Determination of o-Tol and 4-ABP was achieved after derivatization with heptafluorobutyric anhydride by capillary gas chromatography/mass spectrometry with negative chemical ionization. Water samples were analyzed with each batch of samples to control for background contamination. Values less than 2-fold higher than background values were designated as not detectable and included as zero values in calculation of mean±standard error. Adducts of o-Tol well above background levels could be detected in 11 of 12 tumor samples (8.72±1.30 fmol/µg DNA). In contrast, only 4 of 12 tumor samples were positive for ABP (0.057±0.036 fmol/µg; p<0.0001 vs o-Tol). In 46 samples of bladder tissue from SDV, o-Tol DNA adduct levels were significantly lower compared to tumor samples but still detectable in 13 samples from the epithelial (0.24±0.09 fmol/µg DNA; p<0.0001) and 10 samples from the submucosal layer (0.27±0.10 fmol/µg DNA; p<0.0001), respectively. 4-ABP was detectable at significantly lower levels compared to tumor samples in 32 samples from the epithelial (0.011±0.003 fmol/µg DNA; p<0.0001) and 28 samples from the submucosal layer (0.019±0.007 fmol/µg DNA; p<0.005) of the 46 samples of SDV bladder tissue. Further characterization of the data concerning active smoking and polymorphism of N-acetyltransferase 2 genotype is underway. In conclusion, for the first time DNA adducts releasing o-Tol were detected in human urinary bladder. The presence of DNA adducts in the human target tissue of o-Tol strongly support a carcinogenic risk which should be taken into account in preventive hazard minimization in both occupational medicine and drug therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3461.