Semiquantitation of Polycyclic Aromatic Hydrocarbon-DNA Adducts in Human Esophagus by Immunohistochemistry and the Automated Cellular Imaging System

It has been suggested that ingestion of polycyclic aromatic hydrocarbons (PAHs) may contribute to the high incidence and mortality of esophageal cancer in Linxian, China. To explore this relationship a semiquantitative immunohistochemical staining method was developed for localization of PAH-DNA adducts. Nuclear color intensity (bright field average pink intensity per nucleus for >1000 cells) was measured using the ChromaVision Automated Cellular Imaging System (ACIS). Paraffin-embedded sections of cultured human keratinocytes exposed to increasing concentrations of 7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydro-benzo[a]pyrene (BPDE) were incubated with BPDE-DNA antiserum and served as an internal positive control (standard curve). Values for nuclear staining intensity correlated directly with BPDE exposure concentration (r 2 = 0.99) and were reproducible. DNA adduct levels determined by BPDE-DNA chemiluminescence immunoassay in DNA from BPDE-exposed keratinocytes, correlated with BPDE exposure concentrations (r 2 = 0.99), showing that nuclear staining intensity determined by ACIS correlated directly with BPDE-DNA adduct levels determined by chemiluminescence immunoassay. The ACIS methodology was applied to 5 human samples from Linxian, and significantly positive nuclear PAH-DNA adduct staining was observed in this group when compared with esophageal tissue from 4 laboratory-housed monkey controls and 6 samples obtained at autopsy from smokers and nonsmokers in the United States. Nuclear PAH-DNA staining was absent from Linxian samples when serial sections were incubated with normal rabbit serum (negative control) and was significantly reduced on incubation with BPDE-DNA antiserum absorbed previously with the immunogen BPDE-DNA. These results appear to support the hypothesis that high PAH exposure levels may be etiologically associated with the development of esophageal cancer in Linxian.