City University of Hong Kong

CityU Institutional Repository >
3_CityU Electronic Theses and Dissertations >
ETD - Dept. of Biology and Chemistry  >
BCH - Doctor of Philosophy  >

Please use this identifier to cite or link to this item:

Title: Production of reactive oxygen species and antioxidant responses in fish exposed to xenobiotics, chattonella marina and hypoxia
Other Titles: Wai yuan wu ran wu, chi chao hai yang he bao zao ji que yang you dao yu chan sheng zhi huo xing yang ji kang yang hua fan ying
外源污染物, 赤潮海洋褐胞藻及缺氧誘導魚產生之活性氧及抗氧化反應
Authors: Liu, Wenhua (劉文華)
Department: Dept. of Biology and Chemistry
Degree: Doctor of Philosophy
Issue Date: 2005
Publisher: City University of Hong Kong
Subjects: Active oxygen -- Physiological effect
Antioxidants -- Physiological effect
Fishes -- Effect of water pollution on
Notes: CityU Call Number: QP535.O1 L58 2005
Includes bibliographical references (leaves 135-176)
Thesis (Ph.D.)--City University of Hong Kong, 2005
xviii, 182 leaves : ill. (some col.) ; 30 cm.
Type: Thesis
Abstract: Numerous studies have been carried out to determine antioxidant responses and oxidative damages in aquatic organisms, and subsequently relating the observed changes to pollution. However, no direct scientific evidence has been provided thus far to show that the observed responses and damages are mediated through production of reactive oxygen species (ROS). Meanwhile, studies in mammalian models have provided substantial experimental evidence that ROS production is the major contributing factor for the toxicity of different stressors including xenobiotics, hypoxia and hydrogen peroxide. In this study, I hypothesize that xenobiotics (i.e. menadione, duroquinone and B[a]P), hypoxia and Chattonella marina (a red tide alga known to kill fish and capable of producing about 100 times higher concentration of hydrogen peroxide than most other algal species) share a common toxic mechanism by inducing the production of ROS, which will then cause antioxidant responses in fish. In case these antioxidant responses are not sufficient to combat the ROS produced, the excessive ROS will result in lipid peroxidation, protein oxidation and DNA damage. In order to test this hypothesis, the present thesis sets out to determine ROS production in fish exposed to these three different stressors, and to relate ROS levels to antioxidant responses and oxidative damages A single i.p. injection of menadione, duroquinone and B[a]P (1.0 mg/kg body wt.) caused a significant increase of ROS level in both hepatic mitochondria and microsomes in the grouper (Epinephelus tauvina) (Tukey test, P<0.05). While significant decreases of hepatic total oxidant scavenging capability (TOSC) were observed at 24, 48 and 96 h upon menadione treatment, significant decrease of hepatic TOSC was only found at 96 h. upon B[a]P treatment (Tukey test, P<0.05). Protein carbonyl and lipid peroxide (LPO) in mitochondria and microsomes, as well as hepatic 8-OHdG level also markedly increased following enhanced ROS production. However, only B[a]P, but not menadione and duroquinone, could cause a significant induction of ethoxyresorufin-O-deethylase (EROD) activity, suggesting that the toxic mechanisms of menadione, duroquinone and B[a]P were different. For B[a]P, production of ROS (and hence toxicity) is mediated through activation of CYP1A1, and dependent on binding with AhR. In contrast, toxic mechanisms of menadione and duroquinone are AhR independent. Menadione causes oxidative damages possibly through inducing ROS production during redox cycling, and affecting thiol levels via direct arylating to the thiol groups (protein and glutathione), while toxic action of duroquinone is mainly mediated through ROS production during redox cycling. Experiments were further carried out to establish the dose response relationship between a variety of antioxidant responses and duroquinone. ROS, antioxidant capacity, oxidation of protein and lipid in liver, as well as DNA damage (measured by Comet assay) in red blood cells, were measured at 12, 24, 72 and 168 h. after a single i.p. injection of 0.1, 1.0 and 10 mg/kg body wt. duroquinone to the grouper (Epinephelus tauvina). A good dose-response was demonstrated for ROS production, protein carbonyl and LPO in both hepatic mitochondria and microsomes (Two-way ANOVA, P<0.05), and significant correlations were also found between levels of ROS and oxidative damages (e.g. mitochondrial ROS and protein carbonyl) upon treatment of 10 mg/kg body wt. duroquinone (r=0.979 and 0.974 at 12 and 24 h respectively). Temporal changes in ROS production and oxidative damage were also evident, showing that oxidative damages were responsive to ROS production. The results therefore clearly demonstrated that oxidative damages induced by duroquinone are mainly mediated through ROS production, and levels of ROS can serve as a good predictor for subsequent oxidative damages. Increase in TOSC was only found at the highest dose (i.e. 10 mg/kg body wt.), whereas mitochondrial and microsomal ROS, protein carbonyl and LPO were inducible at both 1.0 and 10 mg/kg body wt. of duroquinone (Tukey test, P<0.05). DNA damages in red blood cells appeared to be most sensitive, and readily observable even at the lowest dose administered (viz. 0.1 mg/kg body wt.) after 24 hour (Tukey test, P<0.05). Further studies however, are required to examine the tissue-specific response and the threshold value for induction of TOSC. Grouper (Epinephelus tauvina) exposed to hypoxia (2.0 mg O2/l) for 24, 72 and 120 h showed no significant changes in their hepatic mitochondrial and microsomal ROS and LPO, as compared with the normoxic control (One-way ANOVA, P<0.05). However, microsomal ROS level in the hypoxic group was significantly lower than that of the normoxic control at 24 h (t test, P<0.05). The observed decrease may be due to the decrease in CYP1A1 enzyme induced by hypoxia. However, since antioxidant and oxidative responses to hypoxia may be tissue-specific, the absence of oxidative response in liver may not necessarily rule out the possibility of enhanced ROS production under hypoxic condition. It would be instructive to conduct further experiments to determine ROS levels, antioxidant responses and oxidative damages in other tissues. Despite ROS levels measured in Chattonella marina culture (20 μM H2O2) being some 20 times higher than those in the seawater and the algal (Dunaliella tertiolecta) controls, ROS in mitochondria, as well as TOSC and LPO in the gill of gold-lined sea bream (Rhabdosargus sarba) did not change upon exposure to both low and high (blooming) cell density of C. marina. The results therefore showed that fish exposed to high ROS level in the external medium (at least at the level used in the present experiment) is not able to induce ROS formation, antioxidant responses and oxidative damages in fish gill. Results of this experiment therefore refute the current postulation that ROS is the culprit of fish kills in C. marina blooms. This study presents, for the first time, direct evidence to demonstrate in vivo ROS production and their correlation with oxidative responses in fishes upon treatment of xenobiotics (i.e. menadione, duroquinone and B[a]P). Hypoxia can neither induce ROS production nor cause oxidative damages in fish liver. Enhanced ROS production is also unlikely to be the main cause of oxidative damage and mortality when fish are exposed to C. marina. Overall, this thesis refutes the hypothesis that xenobiotics, hypoxia and Chattonella marina share a common mechanism mediated through ROS production.
Online Catalog Link:
Appears in Collections:BCH - Doctor of Philosophy

Files in This Item:

File Description SizeFormat
fulltext.html158 BHTMLView/Open
abstract.html158 BHTMLView/Open

Items in CityU IR are protected by copyright, with all rights reserved, unless otherwise indicated.


Valid XHTML 1.0!
DSpace Software © 2013 CityU Library - Send feedback to Library Systems
Privacy Policy · Copyright · Disclaimer