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(WEH042) Study of time- and concentration-dependent CYP1A expression in hepatic and extrahepatic tissues of gilthead-seabream Sparus aurata, following exposure to waterborne and dietary benzo(a)pyrene and TCDD. Ortiz Delgado, Juan Bosco1, Sarasquete, Carmen2, González de Canales, María Luisa1, Behrens, Anya3, Segner, Helmut3, 1 2 3 ABSTRACT- The objectives of the present study were to compare time and concentration-dependent induction of CYP1A in different tissues of Sparus aurata and to identify possible route-specific patterns of cellular CYP1A expression following waterborne and dietary exposure to: TCDD (waterborne exposure 4 and 6 pg/l; dietary exposure 1pg/gr dry weight)and benzo(a)pyrene (BaP) (waterborne exposure 300 and 500 ug/l; dietary exposure 100 ug/g dry weight).Comparative examination of CYP1A was performed in liver, gills, intestine, kidney and heart. CYP1A expression was assessed at the level of enzyme catalytic activity, specific protein concentration and mRNA expression. The catalytic activity was measured as 7-ethoxyresorufin-O-deethylase activity, CYP1A protein levels were determined by means of ELISA assays and immunohistochemistry and mRNA levels were measured by means of RT-PCR. In the liver, EROD activity was rapidly induced (within 5 days) following waterborne exposure while with dietary exposure, EROD activity was induced only in TCDD treated specimens and only after 10 days of treatment. In kidney, gills and heart, EROD induction patterns were similar to those observed for the liver, but the levels of activity were much lower. Intestinal EROD activity was strongly induced following dietary exposure to the contaminants, leading to a plateau of EROD activity after 15 days of Bap exposure, whereas with TCDD, no plateau was reached within the experimental period. RT-PCR results showed strong bands of mRNA from day 5 of exposure onwards, mainly in liver and kidney tissues. Immunohistochemical examinations indicated an induction of CYP1A by waterborne exposure in specific cells of liver (hepatocytes and vascular endothelia), gills(pillar cells), kidney (epithelial cells), heart(endocardial cells of the ventricle and atrium) and in the gut (mainly vascular system and enterocytes). After dietary exposure, however, only the liver (hepatocytes near vascular system) and the gut (mainly enterocytes) showed strong immunostaining. Key words: CYP1A, immunohistochemistry, EROD/ELISA/PCR, Sparus aurata |
