The induction of hepatic cytochrome P4501A1 and its monooxygenase activity 7-ethoxyresorufin O- deethylase, (EROD) in fish by PAHs, PCBs and dioxins has been suggested as an early warning system &ldquo / most sensitive biochemical response&rdquo / for assessing environmental contamination conditions. In this study, the degree of induction of cytochrome P4501A1 protein as determined immunochemically and CYP1A1 associated EROD activity in fish were utilized as biomarkers of exposure to PAHs, PCBs and related organic pollutants along the izmir Bay on the Aegean Sea Coast after the Great Canal Project. Three different fish species were used throughout this study, namely leaping mullet (Liza saliens), annular seabream (Diplodus annularis) and common sole (Solea vulgaris) which were representatives of pelagic, benthopelagic and benthic fish, respectively. Fish were sampled in November 2002 and October 2003 from different sites of the Bay. The mullet caught from Harbor, &Uuml / &ccedil / kuyular port site, and Pasaport region displayed highly elevated EROD activities which were 2258&plusmn / 840 (n=15), 2011&plusmn / 490 (n=4), 1813&plusmn / 287 (n=11) pmole /min/mg protein, respectively and were 104, 80 and 79 fold higher than that of fish obtained from the reference point (25&plusmn / 9 pmole/min/mg protein / n=4). Mullet caught along the pollutant gradient at three other sites (Hekim Island, inciralti, and Zeytinalani) exhibited less but highly significant induced EROD activity. EROD activities of common sole sampled from Fo&ccedil / a open sites (107&plusmn / 20 pmol/min/mg protein, n=5) and site16A (80&plusmn / 12 pmol/min/mg protein, n=9) were found to be very low and the latter was accepted as reference site. The highest EROD activity were seen in fish captured from inciralti which was about 6.3 times higher than those obtained from reference site. Common sole caught from the mouth of Gediz River and Hekim Island exhibited also highly elevated EROD activities. Annular seabream was tested to monitor CYP1A inducing chemicals for the first time in this study. The highest EROD activity (1376&plusmn / 279 pmol/min/mg protein, n=8) were detected in fish samples collected from Harbor region. An inverse relationship was found between distance to the harbor region and EROD activities of annular seabream captured from other sampling sites. In this study for the first time, major cytochrome P450 dependent mixed function oxidase activities such as benzphetamine N- demethylase, ethylmorphine N-demethylase and aniline 4- hydroxylase, were characterized in annular seabream. Changes in the P450 1A1 protein level were determined by immunochemical analysis to monitor the pollutant based induction in all fish species and good correlation was obtained between EROD activity and CYP1A protein content. Fish from polluted sites had both highly induced EROD activity and cytochrome P450 1A content. Chemical analysis of total PAH concentration in sediment and liver tissues of some fish sample were also carried out. Although, izmir Great Canal Project has been active since 2000 to treat and protect the izmir Bay from the contamination of domestic and industrial wastes this study clearly demonstrated that the level of PAH, PCB and dioxin type persistent organic contaminants are still very high especially in the Inner and Middle Bay. This has implications for human fish consumption from contaminated areas, as well as for the health status of aquatic organisms.

Cytochrome P4501A1 (CYP1A1) is a phase I bio-transformation enzyme involved in the metabolism of xenobiotics via the oxygenation of polycyclic aromatic hydrocarbons (PAHs) including the carcinogen, benzo(a)pyrene. Induction of the CYP1A1 gene is regulated at the transcriptional level and is ligand dependent with the prototypical 2,3,7,8,-tetrachlorodibenzo-p-dioxin (TCDD) being the most potent known inducer of CYP1A1 transcription. This process is mediated by the AHR/ARNT signaling pathway whereby ligand binds AHR in the cytoplasm allowing its translocation to the nucleus where it binds with its hertrodimerization partner, ARNT and subsequently binds DNA at cognate binding sites termed xenobiotic responsive elements (XREs) located in the 5' flanking region of the CYP1A1 and other genes. The zebrafish (Danio rerio) has recently become an important model system for the study of TCDD-mediated developmental toxicity due to their relative ease of maintaining and breeding, external fertilization, abundant transparent embryos, and sensitivity to TCDD similar to mammalian models. It is therefore essential to vii characterize the molecular mechanisms of AHR mediated gene regulation in this organism. The upstream flanking region of a putative CYP1A gene from zebrafish was identified by the screening of a PAC genomic library. Sequencing revealed a region which contains 8 putative core xenobiotic response elements (XREs) organized in two distinct clusters. The region between -580 to -187 contains XRE 1-3 while the region between -2608 to -2100 contains XRE 4-8. Only XRE 1, 3, 4, 7, and 8 exhibited TCDD-dependant association of AHR/ARNT complexes when evaluated by gel shift assays. The use of in vitro mutagenesis and Luciferase reporter assays further showed that only XRE's 4, 7, and 8 were capable of conveying TCDD-mediated gene induction. The role of nucleotides flanking the core XRE was investigated through the use of EMSA and reporter assays. Similar methods were employed on additional transcription factor binding sites identified by in silico analyses revealing two sites conforming to an HNF- 3α and CREB motif, respectively, which demonstrate importance to regulation of the gene.