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Carcinogenesis, Vol. 22, No. 10, 1693-1699, October 2001
© 2001 Oxford University Press

CARCINOGENESIS

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

Rao L. Divi, Yvonne P. Dragan,1, Henry C. Pitot,2 and Miriam C. Poirier,3

National Cancer Institute, NIH, Bethesda, MD 20892,
1 Ohio State University, Columbus, OH 43210 and
2 University of Wisconsin, Madison, WI 53706, USA

Administration of tamoxifen (TAM) has been shown to induce hepatocellular carcinogenesis and TAM–DNA adduct formation in rat liver. Here we present TAM–DNA adduct localization and semi-quantitation in hepatic tissue of rats by immunohistochemical staining followed by image analysis. We have also used a quantitative immunoassay to provide a validation for the immunohistochemical values. Rats were fed diets containing 0, 5, 50, 150 or 500 p.p.m. TAM for 45 weeks. Serial sections of paraffin-embedded liver were stained for TAM–DNA adducts using a polyclonal TAM–DNA antiserum. Subsequently, visualization 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 nuclear color intensity was achieved with an Automated Cellular Imaging System (ACIS), with a detection limit of 1 TAM–DNA adduct per 107 nt for these experiments. 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 quantitative chemiluminescence immunoassay (CIA). Comparison of semi-quantitative values determined by ACIS with quantitative values determined by CIA showed a strong correlation (r = 0.924) between the two methods. At 45 weeks of TAM exposure the liver cytoplasm contained placental glutathione S-transferase (GST-p)-positive foci, as indicated by new fuchsin staining. Staining of serial sections revealed a relative lack of TAM–DNA adducts within these enzyme-altered foci. In addition, some GST-p foci contained islands of cells that did not stain for GST-p but were positive for TAM–DNA adduct formation. 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. In addition, unlike the normal surrounding liver, preneoplastic GST-p-positive foci have virtually no TAM–DNA adducts.


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