長期食用源性多環芳香族碳氫化合物 (PAHs) 對魚類腸道的影響 : 7-乙氧基異吩惡唑正脫乙基酶 (EROD) 活性與形態功能效應之關聯

• Main Author: Yuen, Bonny Bun Ho (阮賓皓)
• Format: Dissertation
• Language: English
• Published: City University of Hong Kong 2005
• Subjects: Cytochrome P-450 > Physiological effect; Fishes > Effect of water pollution on; Polycyclic aromatic hydrocarbons > Physiological effect

Fish have been shown to readily take up polycyclic aromatic hydrocarbons (PAHs), a major persistent organic pollutant, from contaminated food. When exposure is via the dietary pathway, the gastrointestinal tract is the primary and major site of PAHs entry and exposure. Despite the intestine being a target organ for toxic chemicals, only sparse information is available on the long term effects of dietary intake of PAHs on intestine tissue homeostasis and fish health. Subsequently, few studies have explored the use of the cytochrome P450 enzyme (Ethoxy Resorufin-O-Deethylase or EROD), and histo-cytopathological changes of fish intestines as biomarkers for assessing exposure and effects of dietary PAHs. In this thesis, a comprehensive study was designed to investigate the chronic effects of dietary benzo[a]pyrene (B[a]P) on juvenile grouper (Epinephelus coioides) over a 4-week exposure and 4-week depuration periods. Fish were force-fed daily with pellets containing environmentally realistic concentrations of B[a]P (dissolved in corn oil) at 0.25 μg/g body weight (low-dose) and 12.5 μg/g body weight (high-dose), and responses were compared with appropriate controls. Changes in intestinal EROD activities were studied and related to (i) quantitative intestinal histo-cytopathological changes, as measured by mucosal integrity, de novo crypt formation, hyperplasia and lysosome accumulation; (ii) intestine tissue homeostasis as represented by epithelial turnover, BrdU proliferative index and apoptotic index; and (iii) absorption efficiency of intestine as presented by the expression of proton/peptide co-transporter (PepT-1). The temporal patterns of change (i.e. the time required for initial signal induction, maximum induction and adaptation) and reversibility of these biochemical and morphofunctional alternations were investigated. The sensitivity and persistence of EROD induction in intestines and livers of fish were also compared. Although a significant decrease in growth rate was observed in fish treated with high-dose B[a]P during the exposure period, no mortality was observed during either the exposure or depuration period. Intestinal EROD activity was highly responsive to dietary B[a]P intake and a good dose-response relationship was demonstrated between intestinal EROD induction and the levels of foodborne B[a]P. Conversely, hepatic EROD induction was weak and not persistent in the exposed fish over the study period. Mucosal integrity, as measured by the villous length and density, as well as the mucosal area, was unaffected by B[a]P, while significant hyperplasia of proliferating enterocytes at the bases mucosal folds was detected after 3-day exposure to the high-dose B[a]P. Moreover, the formation of de novo crypts, which showed high resemblance to the cancer predisposition syndrome "juvenile polyposis", was significantly higher in the proximal intestine of high-dose treated fish as compared to the control at exposure week 2 and onwards. A faster epithelial turnover (i.p. injection of BrdU at 100 µg/g body weight) was also measured in the high-dose B[a]P exposed fish at week 1 of the exposure period, as compared to the controls. This was followed by an increase in cell proliferation (i.e. BrdU proliferative index) at exposure week 2. No significant difference in apoptotic index was found throughout the exposure period, whereas severe cytoplasmic extrusions, especially at the basal regions of mucosal folds, were observed in fish treated with high-dose B[a]P for 4 weeks. Significant accumulation of 2n d1 3r d lysosomes in enterocytes, as measured by Vv (lysosome, mucosa), located at the basal regions of mucosal folds was observed in the high-dose group at exposure day 3, with Vv (lysosome, mucosa) doubled by the end of the 4-week exposure period. Furthermore, a drastic loss of microvilli along the brush border membrane was observed in the high-dose B[a]P-challenged fish. Expression of the key protein transporter, PepT-1, located predominantly on the mucosal brush border was reduced after exposure to dietary high-dose B[a]P for 7 days, and a significant reduction was also observed in the low-dose treatment group following 2 weeks of exposure, suggesting a decrease in the protein absorption efficiency of fish intestine. Unlike intestinal EROD activity which subsided immediately after cessation of B[a]P intake, the associated morphofunctional changes (e.g. hyperplasia, epithelial turnover, lysosome accumulation and reduced PepT-1 protein expression) persisted into depuration week 2, indicating longer period was required for intestine morphofunctional recovery. Nevertheless, the present findings clearly demonstrate that all these biochemical and morphofunctional changes in the fish intestines are reversible. Intestinal EROD induction is indicative of current exposure to dietary B[a]P, while morphofunctional changes of intestine are useful to indicate both current and recent exposure of fish to dietary B[a]P. Importantly, intestinal EROD activity is significantly related to intestinal morphofunctional effects, in a descending order: epithelial turnover > hyperplasia of basal proliferative enterocytes > PepT-1 protein expression > lysosome accumulation > BrdU proliferative index > de novo crypt formation (r = 0.678 - 0.376, p < 0.01). These provide strong evidence to support the notion that induction of EROD activities in fish intestine do not merely indicate current exposure to B[a]P/PAHs but also link to harmful biological effects. Therefore, the use of EROD induction should not be limited to a biomarker of exposure, but also a biomarker of effects. When evaluating the effects of B[a]P/PAH exposure, the use of any one of these biomarkers alone may result in a "false-positive" and/or "false-negative" interpretation of the pollution status. Using the time-integrated response information generated in this study, together with its ecological relevance, specificity, technical difficulties and cost-effectiveness of a biomarker, the following suite of biomarkers are recommended for future studies and routine monitoring activities: (i) intestinal EROD activity; (ii) 2nd I 3rd lysosome accumulation in enterocytes; and (iii) de novo crypt formation at bases of mucosal folds. A sensitive and reliable diagnostic tool for differentiating the route of B[a]P/PAH entry in fish (i.e. via water or food) is essential to provide a trustworthy interpretation of the pollution status and typifying the pollution scenario. Such information is also crucial for assessing the risk of xenobiotics in the marine environment. Includes bibliographical references (leaves 157-195) Thesis (Ph.D.)--City University of Hong Kong, 2005