DETERMINATION OF 4,4'-METHYLENE-BIS(2-CHLOROANIIINE)-DNA ADDUCT FORMATION IN RAT-LIVER AND HUMAN UROEPITHELIAL CELLS BY THE P-32 POSTLABELING ASSAY

The probable human carcinogen 4,4'-methylene-bis(2-chloroaniline) (MOC A) was utilized to develop biomarkers of exposure to occupational carc inogens. The P-32 postlabeling assay, utilizing the nuclease P1 enhanc ement procedure, was used to evaluate MOCA-DNA adduct formation in tar get tissues. Male Sprague-Dawley rats were treated with different dosi ng regimens of MOCA, and DNA was isolated from the liver. Additionally , a human uroepithelial cell (HUC) line was treated with N-hydroxy-MOC A for 24 hr, cells were harvested, and DNA was isolated. DNA was analy zed for MOCA-DNA adduct formation by the P-32 postlabeling assay. Five MOCA adducts were detected in rat liver DNA. Adduct A, which correspo nded to N-(deoxyadenosin-8-yl)-4-amino-3-chlorobenzyl alcohol, was the major adduct in rat liver DNA appearing in all treatment groups. Leve ls of adduct A were higher when MOCA was administered by ip injection versus oral gavage. Phenobarbital pretreatment increased the amount of adduct A approximately 12-fold. The pathway leading to the formation of adduct A in DNA from HUC appeared to be saturated at the concentrat ions used: 2.5, 5, and 10 mu M. However, an additional adduct (E) was observed at the 10 mu M treatment level only. A major DNA adduct was d etected in the target tissue of rats and target human cells for MOCA-i nduced carcinogenesis, thus making it useful as a biomarker of exposur e. Other DNA adducts were also observed with the different doses and r outes of exposure investigated.