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Title: Physiological, biochemical and molecular responses of the marine diatom skeletonema costatum to 2,4-dichlorophenol
Other Titles: Gu tiao zao dui 2,4-er lü fen de sheng li, sheng hua he fen zi fan ying
骨條藻對 2,4-二氯酚的生理, 生化和分子反應
Authors: Yang, Shao (楊劭)
Department: Dept. of Biology and Chemistry
Degree: Doctor of Philosophy
Issue Date: 2001
Publisher: City University of Hong Kong
Subjects: Chlorophenols
Toxicity testing
Notes: CityU Call Number: QK569.D54 Y36 2001
Includes bibliographical references (leaves 123-145)
Thesis (Ph.D.)--City University of Hong Kong, 2001
xiv, 147 leaves : ill. ; 30 cm.
Type: Thesis
Abstract: Synthetic organic contaminants have posed a significant threat to the marine ecosystems over large areas, and contamination/pollution of marine environment by synthetic organic chemicals will continue to be a pressing global problem. The diatom, Skeletonema costatum, is not only abundant in marine waters worldwide, but also an important producer in the marine food chain. Thus, any deleterious effects caused by xenobiotics on this important species may lead to major ecological consequences on marine ecosystems. In this thesis, a synthetic organic chemical. 2,4-dichlorophenol (2.4-DCP) u-as used as a model toxicant, to study the sublethal effects on the diatom Skeletonema costatum. Experiments were also designed to elucidate the physiological, biochemical and molecular responses of this diatom upon exposure to sublethal concentrations of 2,4-DCP. The ability of this diatom to degrade or biotransform 2.4-DCP, and the pathway involved, were also investigated. Molecular studies were carried out to investigate the response of the gene expression of cytochrome P450 to exposure by 2,4-DCP, in order to test whether P450 is involved in detoxification. Growth rate of the diatom was significantly inhibited by 2,4-DCP at and above 3.0 mg/L (96h-ECjo on growth rate = 8.03 mg/L), while other biological parameters such as photosynthetic and respiration rates, carotenoids and protein content, ATP level and adenylate energy charge were unaffected. Exposure to 6.0 mg-11, 2,4-DCP for 96 h resulted in the total lipid content being increased to 304 % (ANOVA, p4.O 1 ). while the RN.A/DNA ratio was reduced to 31 % (ANOVA, p<0.01) of the control values. A parallel study by transmission electron microscopy further confirmed the increase in cellular lipid content, as evidenced by the accumulation of lipid droplets within diatom cells. A slight increase in carbohydrate (+37.9 %, ANOVA, p<0.05) and decrease in Chlorophyll a (-20.4 %, ANOVA, p<0.05) and total Chlorophyll c (-14.4 %. ANOVA, p<0.01) were also found after exposure to 6 mg/L 2,4-DCP. Although 2,4-DCP is known to uncouple oxidative phosphorylation, our results show that energy production was not inhibited at sublethal concentrations of 2,4-DCP. The observed growth inhibition in S. costatum caused by 2,4-DCP was mainly attributable to an increase in energy storage and inhibition of protein synthesis. The deposition of lipid droplets may be viewed as a protective strategy that allows the cells to sequester lipophilic xenobiotics to reduce their bioavailability. 2,4-DCP was readily metabolised by the diatom, but bioaccumulation and adsorption tvere negligible, and hence the possibility of food chain transfer via diatoms is unlikely to be important. Glutathione S-transferase, ascorbate peroxidase and superoxide dismutase activities were increased markedly after exposure to 6 mg/L 2.4-DCP for 96 h. while no appreciable change in peroxidase activity was observed. The addition of exogeneous glutathione to diatom cultures enhanced the degradation of 2.1-DCP, and promoted diatom growth. The inhibition of glutathione synthesis enhanced the toxicity of 2.4-DCP. These results suggest that glutathione conjugation was one of the principal mechanisms involved in the detoxification of 2,4-DCP in the diatom. The full-length cDNA of a novel cytochrome P450 isoform, termed CYP97E1, was isolated from S. costaturn by using DD-PCR strategy. CYP97EI codes a protein of 659 amino acids, with a molecular weight of 74.2KD, pI of 5.47 and a hqdrophilic N-temlinus. It has the closest phylogenetic relationship with the members in CYP97B subfamily. and also closely relates to the members in CYP4F7 CYP72. CYP46 subfamilies. CYP97E1 is the first full-length sequence of cytochrome P450 from eukarxotic algae, and its appearance in this diatom indicates that CYP97 family is the second distantly-distributed P450 family in addition to CYP51. The expression of CYP97E1 was not affected significantly upon exposure to 2,4-DCP at 1.0 - 6.0 mg/L for 96 11. This result is consistent with the result of a test with P450 inhibitors, confirming that cytochrome P450 did not play n significant role in the detoxification of 2,4-DCP by the diatom. Among the various physiological, biochemical and cytological responses investigated in this study, the RNA/DNA ratio, deposition of lipid droplet and GST show some promise as potential biomarkers either for growth of the diatom or exposure of 2,4-DCP, although there are clear limitations on their specificity and sensitivity. This thesis represents the first investigation on biotransformation of 2,4-DCP, and on cytological, physiological, biochemical and molecular responses to 2.4-DCP in the marine diatom Skeletonema costatum. This is also the first investigation on cytochrome P450 gene sequence of eukaryotic algae. Information gained in this study does not only shed light on toxicity of other chlorinated phenols on phytoplankton, but also on biological responses of algae to toxic organic compounds in general.
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