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Microfluorometric Determination of DNA Adducts in Immunofluorescent-stained Liver Tissue from Rats Fed 2-Acetylaminofluorene¹

Laboratory for Immunohistochemistry and Immunopathology, Institute of Pathology, The National Hospital, University of Oslo, Norway [H. S. H.]; Microbiological Associates, Bethesda, Maryland 20816 [E. F. S.]; Department of Pharmacology, College of Medicine, University of Iowa, Iowa City, Iowa, 52242 [J. B.]; and the Laboratory of Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute, NIH, Bethesda, Maryland 20892 [M. C. P.]

The intensities of immunofluorescence in nuclei stained by an antiserum specific for the DNA adduct N-deoxyguanosin(8-yl)aminofluorene (dG-8-AF), were quantified by microfluorometry in frozen liver sections from male Fischer rats fed 2-acetylaminofluorene (AAF). Results of previous studies demonstrated that dG-8-AF is the predominant adduct (80–100%) formed in livers of rats fed AAF continuously, and that nuclei of hepatocytes and bile duct epithelial cells in rats fed AAF exhibit an adduct-specific immunofluorescence. In the present investigation, nuclear staining for dG-8-AF was quantified by microfluorometry in liver sections from male Fischer rats fed 0.02% AAF continuously for 2, 4, 8, 12, 16, 20, and 28 days. Microfluorometric determinations of the intensities of nuclear immunofluorescence staining within periportal, midzonal, and centrilobular hepatocytes and bile duct epithelial cells revealed that levels of the dG-8-AF adduct increased in these cells during AAF feeding, reaching a plateau by 12 days. However, significant differences were detected in dG-8-AF levels within cells of each lobular area. Nuclei of periportal hepatocytes exhibited the most intense immunofluorescence, nuclei of centrilobular hepatocytes and bile duct epithelial cells emitted the least intense fluorescence, and nuclei of midzonal hepatocytes exhibited an intermediate fluorescence intensity. Quantitation of whole-liver levels of the dG-8-AF adduct by RIA, after extraction of DNA, also revealed that adduct accumulation reached a plateau by 12 days of AAF feeding. Thus, similar profiles of adduct accumulation were obtained by microfluorometric analysis of immunofluorescence staining within frozen liver sections, and by RIA analysis of DNA extracted from whole livers. The periportal concentration of DNA adducts in livers of rats continuously fed a carcinogenic dose of AAF may be an important early event in AAF-induced liver tumorigenesis.

¹ This investigation was supported in part by USPHS Grant GM-33253 to J. B.

² Norwegian Cancer Society Research Fellow (Landsforenigen mot Kreft).

³ To whom requests for reprints should be addressed, at National Cancer Institute, NIH, Laboratory of Cellular Carcinogenesis and Tumor Promotion, Building 37, Room 3B25, Bethesda, MD 20892.

Received 9/29/86. Revised 1/20/87. Accepted 1/23/87.