title: Immunohistochemical localization and semi-quantitation of hepatic tamoxifen-DNA adducts in rats exposed orally to tamoxifen.
abstract: 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 10(7) 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.
authors.affiliation: National Cancer Institute, NIH, Bethesda, MD 20892, USA.
authors.forename: R L
authors.initials: RL
authors.lastname: Divi
authors.forename: Y P
authors.initials: YP
authors.lastname: Dragan
authors.forename: H C
authors.initials: HC
authors.lastname: Pitot
authors.forename: M C
authors.initials: MC
authors.lastname: Poirier
mesh_terms: D000284:Administration, Oral; D000818:Animals; D004247:DNA / Q000378:metabolism*; D018736:DNA Adducts / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004965:Estrogen Antagonists / Q000494:pharmacology*; D005260:Female; D005455:Fluorescent Antibody Technique; D005982:Glutathione Transferase / Q000378:metabolism; D007091:Image Processing, Computer-Assisted; D007118:Immunoassay / Q000379:methods; D008099:Liver / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D008163:Luminescent Measurements; D010920:Placenta / Q000201:enzymology; D011247:Pregnancy; D051381:Rats; D017207:Rats, Sprague-Dawley; D013629:Tamoxifen / Q000494:pharmacology*
chemical_list: D018736:DNA Adducts; D004965:Estrogen Antagonists; D013629:Tamoxifen; D004247:DNA; D005982:Glutathione Transferase