Determination of 4,4'-methylene-bis(2-chloroaniline)-DNA adduct formation in rat liver and human uroepithelial cells by the 32P postlabeling assay.
The probable human carcinogen 4,4'-methylene-bis(2-chloroaniline) (MOCA) was utilized to develop biomarkers of exposure to occupational carcinogens. The 32P postlabeling assay, utilizing the nuclease P1 enhancement procedure, was used to evaluate MOCA-DNA adduct formation in target tissues. Male Sprague-Dawley rats were treated with different dosing regimens of MOCA, and DNA was isolated from the liver. Additionally, a human uroepithelial cell (HUC) line was treated with N-hydroxy-MOCA for 24 hr, cells were harvested, and DNA was isolated. DNA was analyzed for MOCA-DNA adduct formation by the 32P postlabeling assay. Five MOCA adducts were detected in rat liver DNA. Adduct A, which corresponded to N-(deoxyadenosin-8-yl)-4-amino-3-chlorobenzyl alcohol, was the major adduct in rat liver DNA appearing in all treatment groups. Levels 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 concentrations used: 2.5, 5, and 10 microM. However, an additional adduct (E) was observed at the 10 microM treatment level only. A major DNA adduct was detected in the target tissue of rats and target human cells for MOCA-induced carcinogenesis, thus making it useful as a biomarker of exposure. Other DNA adducts were also observed with the different doses and routes of exposure investigated.
DeBord
D G
DG
Department of Health and Human Services, National Institute for Occupational Safety and Health, Cincinnati, Ohio 45226, USA.
Cheever
K L
KL
Werren
D M
DM
Reid
T M
TM
Swearengin
T F
TF
Savage
R E
RE
D000818:Animals; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism*; D018736:DNA Adducts / Q000032:analysis*; D004848:Epithelium / Q000187:drug effects; D006801:Humans; D008099:Liver / Q000187:drug effects*; D008297:Male; D008753:Methylenebis(chloroaniline) / Q000378:metabolism*; D008940:Mitotic Index / Q000187:drug effects*; D010761:Phosphorus Radioisotopes; D051381:Rats; D017207:Rats, Sprague-Dawley; D014551:Urinary Tract / Q000187:drug effects*
D018736:DNA Adducts; D010761:Phosphorus Radioisotopes; D008753:Methylenebis(chloroaniline); D004247:DNA