Carcinogen-induced alterations in rat liver DNA adduct formation determined by computerized fluorescent image analysis.
These studies employed continuous feeding of a carcinogenic level of N-2-acetylaminofluorene to male rats for 28 days. Under these conditions normal hepatocytes are known to be inhibited from proliferation, whereas xenobiotic-resistant putative preneoplastic hepatocytes with altered liver enzyme phenotypic expression appear to have a growth advantage. A novel technique using computerized fluorescent image analysis of triple-stained frozen liver sections was developed and used to visualize three different molecular markers in individual hepatic cells. Proliferating liver cells were identified by anti-5-bromodeoxyuridine immunostaining in livers of rats injected with 5-bromodeoxyuridine 1 hour before sacrifice. Anti-cytokeratin immunostaining was used to identify bile ducts and putative oval cells. Characterization of DNA adduct formation was achieved with an antiserum specific for N-(deoxyguanosine-8-yl)-2-aminofluorene, the major DNA adduct of 2-acetylaminofluorene. The image analysis demonstrated low but distinct DNA adduct concentrations in putative oval cells identified by anti-cytokeratin staining and in scattered, replicating liver cells recognized by anti-5-bromodeoxyuridine. Adducts were not detected in replicating foci consisting of 3 to 11 nuclei. It is possible that proliferating liver cells that have low N-2-acetylaminofluorene-DNA adduct levels may clonally expand to become foci protected from further adduct accumulation and preneoplastic liver lesions. Thus, the computerized fluorescent image analysis demonstrated here may provide a novel procedure for identification of carcinogen-induced liver cell alterations.
Huitfeldt
H S
HS
Laboratory for Immunohistochemistry and Immunopathology, National Hospital, University of Oslo, Norway.
Brandtzaeg
P
P
Poirier
M C
MC
D015073:2-Acetylaminofluorene / Q000032:analysis / Q000633:toxicity* / Q000031:analogs & derivatives; D000818:Animals; D002455:Cell Division / Q000187:drug effects; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D003201:Computers; D004247:DNA / Q000187:drug effects* / Q000378:metabolism; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000032:analysis; D005455:Fluorescent Antibody Technique; D008099:Liver / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D008297:Male; D008856:Microscopy, Fluorescence / Q000379:methods; D051381:Rats; D011916:Rats, Inbred F344
C018776:N-(deoxyguanosin-8-yl)acetylaminofluorene; D004247:DNA; D015073:2-Acetylaminofluorene; D003849:Deoxyguanosine