Immunohistochemical localization and semi-quantitation of hepatic tamoxifen-DNA adducts in rats exposed orally to tamoxifen

Administration of tamoxifen (TAM) has been shown to induce hepatocellular c arcinogenesis and TAM-DNA adduct formation in rat liver. Here we present TA M-DNA adduct localization and semi-quantitation in hepatic tissue of rats b y immunohistochemical staining followed by image analysis. We have also use d a quantitative immunoassay to provide a validation for the immunohistoche mical values. Rats were fed diets containing 0, 5, 50, 150 or 500 p.p.m. TA M for 45 weeks. Serial sections of paraffin-embedded liver were stained for TAM-DNA adducts using a polyclonal TAM-DNA antiserum. Subsequently, visual ization of TAM-DNA adducts was performed by peroxidase-conjugated secondary antibody-mediated signal amplification using biotinyl tyramide followed by streptavidin-alkaline phosphatase and fast red. Semi-quantitation of nucle ar color intensity was achieved with an Automated Cellular Imaging System ( ACIS), with a detection limit of 1 TAM-DNA adduct per 10(7) nt for these ex periments. In parenchymal cells of liver sections from TAM-exposed animals a dose-dependent increase in nuclear staining was observed by ACIS and the TAM-DNA adduct levels determined by ACIS were validated in liver DNA by qua ntitative chemiluminescence immunoassay (CIA). Comparison of semi-quantitat ive values determined by ACIS with quantitative values determined by CIA sh owed a strong correlation (r = 0.924) between the two methods. At 45 weeks of TAM exposure the liver cytoplasm contained placental glutathione S-trans ferase (GST-p)-positive foci, as indicated by new fuchsin staining. Stainin g of serial sections revealed a relative lack of TAM-DNA adducts within the se enzyme-altered foci. In addition, some GSTp foci contained islands of ce lls that did not stain for GSTp but were positive for TAM-DNA adduct format ion. This study validates the use of ACIS for TAM-DNA adduct formation and demonstrates that steady-state TAM-DNA adduct levels observed in livers of rats chronically fed TAM for several months increase in relation to dose. I n addition, unlike the normal surrounding liver, preneoplastic GST-p-positi ve foci have virtually no TAM-DNA adducts.