Expression of P4501A1 in a primary culture of rainbow trout hepatocytes exposed to beta-naphthoflavone or 2,3,7,8-tetrachlorodibenzo-p-dioxin.
Primary cultures of rainbow trout hepatocytes were used to study the expression of CYP1A1 mRNA, its protein product (P4501A1), and catalytic activities (7-ethoxy-resorufin-O-deethylase, EROD) during a 96-h period after exposure of cells to beta-naphthoflavone (BNF) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Hepatocytes were isolated from immature rainbow trout by a two-step perfusion method and incubated at 10 degrees C. Cells were exposed to the inducers for 48 h after 24 h of preculturing. The EROD activity of BNF-treated hepatocytes was higher than that of the control hepatocytes 12 h after addition of the inducer. Activities peaked after 48 h and had declined to control levels after 72 h. EROD activity in TCDD-treated cells was significantly elevated after 12 h, but in contrast to BNF-exposure, activities continued to increase during the experimental period. The content of P4501A1 protein, measured with an indirect ELISA technique (using anti-codP4501A1 IgG), increased linearly during the first 12 h and remained constant thereafter. In TCDD-exposed cells the immunochemically determined P4501A1 levels changed in parallel with EROD activity. CYP1A1 mRNA levels, determined by Northern blot and slot-blot analyses (using the trout P4501A1 cDNA pSg15 probe), were hardly detectable in control cells. In BNF- and TCDD-treated cells a 2.8-kb mRNA band was detected by the probe 6 h before the protein and catalytic activities became detectable. The elevated levels of CYP1A1 mRNA were sustained more effectively by TCDD than by BNF. In addition, a second mRNA band at 1.9 kb was seen. The results suggest that transcriptional activation is probably the prime factor even though post-transcriptional events may be involved in the regulation of P4501A1 induction by PAHs in rainbow trout hepatocytes.
Pesonen
M
M
Department of Zoophysiology, University of Göteborg, Sweden.
Goksøyr
A
A
Andersson
T
T
D000818:Animals; D001571:Benzoflavones / Q000494:pharmacology*; D002384:Catalysis; D002478:Cells, Cultured; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000235:genetics; D004790:Enzyme Induction; D004797:Enzyme-Linked Immunosorbent Assay; D015971:Gene Expression Regulation, Enzymologic; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D000072317:Polychlorinated Dibenzodioxins / Q000031:analogs & derivatives* / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D014337:Trout; D019324:beta-Naphthoflavone
D001571:Benzoflavones; D000072317:Polychlorinated Dibenzodioxins; D012333:RNA, Messenger; D019324:beta-Naphthoflavone; C066568:2-nitro-3,7,8-trichlorodibenzo-4-dioxin; D003577:Cytochrome P-450 Enzyme System