Imaging and semiquantitation of polycyclic aromatic hydrocarbon (PAH)-DNA adducts in human cervix, vuvla, placenta and prostate using Immunohistochemistry (ICH) and the Automated Cellular Imaging System (ACIS).

Abstract

Epidemiological and laboratory studies have shown significant associations between PAH exposure and subsequent development of cancer in various organs. Molecular dosimetry of DNA adduct formation typically involves tissue DNA extraction followed by DNA adduct analysis using techniques incapable of observing specific cells within a complex tissue. Here we report localization and semi-quantitation of PAH-DNA adducts in paraffin-embedded sections of human cervix, vulva, placenta and prostate utilizing IHC with an antibody generated against DNA modified with (+/-)-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE). Serial sections were stained with hematoxylin (to identify nuclei), specific anti-BPDE-DNA antiserum (to visualize adducts) or immunogen-absorbed anti-BPDE-DNA serum (for background). Semi-quantitation was achieved by comparison of test sample nuclear color intensity with a standard curve of human keratinocytes exposed to increasing concentrations of BPDE and analyzed for BPDE-DNA adducts by both IHC/ACIS and quantitative BPDE-DNA immunoassay. A survey of cervix from 75 individuals, placenta from 14 individuals, vulva from 19 individuals and prostate from 5 individuals has revealed large inter-individual variability in PAH-DNA adduct levels in these reproductive organ tissues. The highest concentrations of PAH-DNA adducts were observed in the basal epithelial cells of human cervix and vulva, in the cytotrophoblast cells lining the chorionic villi of the placenta and in the endothelial cells lining the ducts of the prostate. Comparison with the standard curve revealed PAH-DNA adduct concentrations ranging from ∼12 (limit of detection) to approximately 190 adducts/108 nucleotides. The study shows that many if not most individuals have PAH-DNA adducts in cervical, vulvar and prostate tissue that could reflect short- or long-term PAH exposures, while the placental PAH-DNA adducts clearly represent short-term exposures. The non-destructive DNA adduct analysis method reported here may provide a novel approach to dissect genotoxic events occurring in cancer target tissues.