Neoplastic transformation and DNA-binding of 4, 4'-methylenebis (2-chloroaniline) in SV40-immortalized human uroepithelial cell lines

• Published in: Carcinogenesis (New York) Vol. 17; no. 4; pp. 857 - 864
• Main Authors: Swaminathan, Santhanam, Frederickson, Susan M., Hatcher, James F., Reznikoff, Catherine A., Butler, Mary A., Cheever, Kenneth L., Savage, Russell E.
• Format: Journal Article
• Language: English
• Published: Oxford Oxford University Press 01.04.1996
• Subjects: Animals; Binding Sites; Biological and medical sciences; Carcinogenesis, carcinogens and anticarcinogens; Carcinogens - metabolism; Carcinogens - toxicity; Cell Line, Transformed; Cell Transformation, Neoplastic; Chemical agents; DNA - drug effects; DNA - metabolism; Female; Humans; Medical sciences; Methylenebis(chloroaniline) - metabolism; Methylenebis(chloroaniline) - toxicity; Mice; Mice, Nude; Simian virus 40 - physiology; Tumors; Urinary Bladder Neoplasms - chemically induced; Urogenital System - cytology; Urogenital System - drug effects; Human; Urinary system disease; Tumorigenicity; Urinary tract disease; Malignant tumor; Occupational exposure; In vitro; Carcinogen; Urinary bladder; DNA; Malignant transformation; Established cell line; Bladder disease; Arylamine; Molecular adduct
• ISSN: 0143-3334; 1460-2180
• DOI: 10.1093/carcin/17.4.857

The tumorigenic transformation of certain occupationally significant chemicals, such as N-hydroxy-4, 4'-methyl-enebis[2-chloroaniline] (N-OH-MOCA), N-hydroxy-ortho-toluidine (N-OH-OT), 2-phenyl-1, 4-benzoquinone (PBQ) and N-hydroxy-4-aminobiphenyl (N-OH-ABP) were tested in vitro using the well established SV40-immortalized human uroepithelial cell line SV-HUC.PC. SV-HUC cells were exposed in vitro to varying concentrations of N-OH-MOCA, N-OH-OT, N-OH-ABP and PBQ that caused approximately 25% and 75% cytotoxicity. The carcinogen treated cells were propagated in culture for about six weeks and subsequently injected subcutaneously into athymic nude mice. Two of the fourteen different groups of SV-HUC.PC treated with different concentrations of N-OH-MOCA, and one of the three groups exposed to N-OH-ABP, formed carcinomas in athymic nude mice. 32P-postlabeling analyses of DNA isolated fromSV-HUC.PC after exposure to N-OH-MOCA revealed one major and one minor adduct. The major adduct has been identified as theN-(deoxyadenosin-3',5' -bisphospho-8-yl)-4-amino-3-chlorobenzyl alcohol (pdAp-ACBA) and the minor adduct as N-(deoxyadenosin-3', 5'-bisphospho-8-yl)-4-amino-3-chlorotoluene (pdAp-ACT). Furthermore, SV-HUC.PC cytosols catalyzed the binding of N-OH-MOCA to DNA, in the presence of acetyl-CoA, to yield similar adducts. The same adducts were also formed by chemical interaction of N-OH-MOCA with calf thymus DNA, suggesting that the aryl nitrenium ion may be the ultimate reactive species responsible for DNA binding. The tumorigenic activity of N-OH-MOCA in this highly relevant in vitro transformation model, coupled with the findings that SV-HUC.PC cells formed DNA-adducts in vitro and contained enzyme systems that activated N-OH-MOCA to reactive electrophilic species that bound to DNA, strongly suggest that MOCA could be a human bladder carcinogen. These findings are consistent with the International Agency for Research on Cancer's classification of MOCA as a probable human carcinogen.