NEOPLASTIC TRANSFORMATION AND DNA-BINDING OF 4,4'-METHYLENEBIS(2-CHLOROANILINE) IN SV40-IMMORTALIZED HUMAN UROEPITHELIAL CELL-LINES

The tumorigenic transformation of certain occupationally significant c hemicals, such as N-hydroxy-4,4'-methylenebis[2-chloroaniline] (N-OH-M OCA), N-hydroxy-orthotoluidine (N-OH-OT), 2-phenyl-1,4-benzoquinone (P BQ) and N-hydroxy-4-aminobiphenyl (N-OH-ABP) were tested in vitro usin g the well established SV40-immortalized human uroepithelial cell line SV-HUC.PC. SV-HUC cells were exposed in vitro to varying concentratio ns of N-OH-MOCA, N-OH-OT, N-OH-ABP and PBQ that caused approximately 2 5% and 75% cytotoxicity. The carcinogen treated cells were propagated in culture for about six weeks and subsequently injected subcutaneousl y into athymic nude mice, Two of the fourteen different groups of SV-H UC.PC treated with different concentrations of N-OH-MOCA, and one of t he three groups exposed to N-OH-ABP, formed carcinomas in athymic nude mice, P-32-postlabeling analyses of DNA isolated from SV-HUC.PC after exposure to N-OH-MOCA revealed one major and one minor adduct, The ma jor adduct has been identified as the N-(deoxyadenosin-3' ,5'-bisphosp ho-8-yl)-4-amino-3-chlorobenzyl alcohol (pdAp-ACBA) and the minor addu ct as sin-3',5'-bisphospho-8-yl)-4-amino-3-chlorotoluene (pdApACT). Fu rthermore, SV-HUC.PC cytosols catalyzed the binding of N-OH-MOCA to DN A, in the presence of acetyl-CoA, to yield similar adducts. The same a dducts were also formed by chemical interaction of N-OH-MOCA with calf thymus DNA, suggesting that the aryl nitrenium ion may be the ultimat e reactive species responsible for DNA binding. The tumorigenic activi ty 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 reactiv e electrophilic species that bound to DNA, strongly suggest that MOCA could be a human bladder carcinogen. These findings are consistent wit h the International Agency for Research on Cancer's classification of MOCA as a probable human carcinogen.