AoE 7b (Revised 8/05)
UNIVERSITY GRANTS COMMITTEE
AREAS OF EXCELLENCE SCHEME
Annual Progress Report on Funded Projects
Stage:______Two______
Reporting Period: April 2005 – *March 2006
(* report on financial information up to end of February 2006)
- Project title:Centre for Marine Environmental Research and Innovative Technology
2.Investigator(s) and Academic Department(s) / Unit(s) Involved#
Investigation Team
/ Name / Post / Institution / Department / UnitPrincipal Investigator / Prof. Rudolf Wu
/ CityU
Director, Centre for Coastal Pollution and Conservation; Chair Professor, Dept. of Biology and Chemistry
Co-investigator(s) / Prof. Paul Harrison
(Leader, Field Studies and Validation Team) / HKUST
Director and Chair Professor, Atmospheric, Marine and Coastal Environmental Program, School of Science
Prof. Joseph Lee
(Leader, Impact and Risk Assessment Team) / HKU
Pro-Vice-Chancellor;
Redmond Chair of Civil Engineering, Dept. of Civil Engineering
Prof. M.H. Wong
(Leader, Mitigatory, Control and Bioremediation Technology Team) / HKBU
Chair Professor, Dept. of Biology;
Director of Croucher Institute for Environmental Sciences
Prof. David Randall
(Leader, Novel Technologies for Environmental Diagnosis Team) / CityU
Head and Chair Professor, Dept. of Biology and Chemistry
Prof. Paul Lam
(Deputy Leader, Field Studies and Validation Team) / CityU
Chair Professor, Dept. of Biology and Chemistry
Dr. Wen Wang
(Deputy Leader, Impact and Risk Assessment Team) / HKUST
Associate Professor, Dept. of Biology
Prof. Nora Tam
(Deputy Leader, Mitigatory, Control and Bioremediation Technology Team) / CityU
Chair Professor, Dept. of Biology and Chemistry
Prof. Norman Woo
(Deputy Leader, Novel Technologies for Environmental Diagnosis Team) / CUHK
Professor, Dept. of Biology;
Ex-Director, Marine Science Laboratory
Prof. W.K. Li / HKU
Chair Professor, Dept. of Statistics & Actuarial Science
Dr. X.Y. Li / HKU
Associate Professor, Dept. of Civil Engineering
Dr. Kenneth Leung / HKU
Assistant Professor, Dept. of Ecology & Biodiversity
Prof.Pei Yuan Qian / HKUST
Director, Coastal Marine Laboratory;
Professor, Dept. of Biology
Dr. Chris Wong / HKBU
Associate Professor, Dept. of Biology
Dr. Xiangdong Li / PolyU
AssociateProfessor, Dept. of Civil and Structural Engineering
Dr. Put O Ang / CUHK
Associate Professor, Dept of Biology
Prof. Peter Yu / CityU
Professor, Dept. of Physics and Materials Science
Prof. T.C. Lau / CityU
Professor, Dept. of Biology and Chemistry
Prof. Michael Yang / CityU
Professor, Dept. of Biology and Chemistry
Dr. S.H. Cheng / CityU
Associate Professor, Dept. of Biology and Chemistry
Dr. Bruce Richardson / CityU
Associate Professor, Dept. of Biology and Chemistry
Dr. Doris Au / CityU
Associate Professor, Dept. of Biology and Chemistry
Dr. Michael Lam / CityU
Associate Professor, Dept. of Biology and Chemistry
Dr. Richard Kong / CityU
Associate Professor, Dept. of Biology and Chemistry
Overseas Collaborators / Prof. Xiang Jianhai / Chinese Academy of Sciences Director and Professor, Institute of Oceanology
Prof. Des Connell / GriffithUniversity
Head, School of Public Health and Professor of Environmental Toxicology and Chemistry
Prof. John Giesy / MichiganStateUniversity
Professor
Prof. John Gray / OsloUniversity
Professor
Prof. Bruce Hammock / University of CaliforniaDavis
Professor
Prof. H.R. Lin / ZhongshanUniversity
Professor
Dr. Don Anderson / Woods Hole Oceanographic Institution
Senior Scientist
# Please highlight and provide reasons for any changes in the composition of the project team. The CV of the new project team member should be provided.
3.Project Objectives
Summary of objectives addressed / achieved to-date:
RevisionΩ / ObjectivesΓ / Percentageachieved / Remarks
To develop innovative chemical, biological and engineering technologies to provide an early warning system and monitor the health of marine environments / 50%
To identify sensitive receivers in Hong Kong coastal waters / 100%+ / Further field studies were carried out to identify sensitive receivers, and data were incorporated into the existing database for use by all team members.
To study recovery of a marine ecosystem after pollution abatement / 60% / In addition to studies on recovery of plankton and benthic communities, we have capitalized on the unique opportunity of pollution abatement in VictoriaHarbour and conducted additional work to investigate recovery of intertidal community, microbial community, as well as changes in biomarkers and sediment chemistry.
To develop models to assess carrying capacity, kinetic transfers and risk assessment / 40%
To develop cost-effective pollution control and bioremediation technologies / 40%
GoalTo provide relevant training / 40% /
- Two postgraduate workshops were organized: one on “Research Philosophy and Methodology”, attended by some 90 RA/RS/PDF of AoE members from the six collaborating institutions, and another one on “Hong Kong’s waters: how transport processes influence distribution patterns”, attended by over 60 RS/PDF and professionals from government agencies.
- Admitted 30 graduate students and partially funded 26 graduate students for the various AoE projects; provided training to 2 undergraduate students, 25 Research Assistants, 1 Research Fellow, 2 Research Associates, 3 Senior Research Associates, and 19 Post Doctoral Fellows.
To develop innovative technologies and marketable products for assessing environmental risk / 0% / This As mentioned in indicative time-table, this objective will be addressed in Stage 4-5 (Year 4-5)
ΩIn case of revision, the numbering system should be 0, 1, 2, etc. in order to reflect the sequence of revision.
ΓAs itemized in the original proposal. In case of revision, the project team should formally and separately write to the UGC for approval. Both the original objectives and all subsequently revised objectives should be listed.
Please provide reasons for major under-achievement. In case of revision, please quote the date when the UGC granted the approval.
4.Deliverables
Summary of deliverables addressed / achieved to-date:
Percentage achieved refers to that for the deliverables promised for 2nd year.
Date for target completion refers to the project completion date.
StageΩ/Revision / DeliverablesΓ / Percentage
achieved / Remarks / Date for target completion
Stage 2
(Year 2 ) / Program 1: Novel Technologies for Environmental Diagnosis
Project #* / 1.1 Chemical Technologies
1a
1b / 1.1.1 Solid Phase Microextraction (SPME)
Deliverable: Development of molecularly imprinted SPME coating on optical fibres / 100%
80% / Development of advanced microextraction technologies for the determination of algal toxins in natural waters and biota tissues
- Successful development of a SPME-HPLC analytical method for the direct determination of saxitoxin
- Detail investigation of the photodegradation mechanism of Irgarol-1051 in seawater and the identification of another new degradation product of Irgarol-1051.
- We have already developed a very simple and effective technique to imprint target analytes onto the surface of an optical fibre. Works on the fabrication and performance evaluation of molecularly imprinted SPME devices for 17-estradiol and polybrominated diphenyl ethers are in progress.
2007
1c
1d / 1.1.2 Molecular Imprinting Chemical Sensing (MICS)
Deliverable: -Development of molecular imprinting technique for surface imprinting on nanoparticles
-Development of chemosensors for PSP toxins / 80%
85% / Development of surface imprinting technique on nano-particles for the fabrication of MIP-based nano-particle chemosensors for algal toxins and other trace organic pollutants
- Two special ligand systems have been developed for the incorporation of luminescent metal complexes onto the surface of nano-silica particles.
- An amorphous titanium oxide material for the selective adsorption of domoic acid (a potent red tide toxin that can cause diarrheic shellfish poisoning) has been successfully developed. Works on using this material for the molecular imprinting of domoic acid (for more specific determination of the toxin) and other algal toxins are in progress.
- An azobenzene-based photoswitchable MIP material that is able to regulate the release and uptake of caffeine by light irradiation has been developed.
- Pure fluoro-tagged estradiol adduct for the fabrication of molecularly imprinted chemosensing materials for estradiol determination has been synthesized.
2007
2 / 1.1.3 Biosensors – Immunosensors for direct detection of genotoxicants and algal toxins
Deliverable: Development of protocols for detection of toxins and genotoxicants based on direct binding assay, competitive binding assay or fluorescent reporters / 100% / We have demonstrated the technical feasibility of using piezoelectric ceramic resonators as a core component of biosensor devices. Experiments are in progress, to demonstratethe technical feasibility of applying the ceramic-based biosensor in detecting environmental toxins, andthe practical use of the ceramic-based biosensor as a competitive alternative to existing products. / 2008
16a
16b / 1.1.4 Semi-Permeable Membrane Devices (SPMDs) & Artificial Mussels (AMs)
Deliverables:
-Comparative studies with mussels on uptake and depuration
-Confounding factors of metal uptakes in AMs / 100%
100% / SPMDs
- Comparative studies of mussel and SPMD contaminant uptake and depuration under laboratory conditions have been completed, and a paper published in the international literature.
- Experiments were carried out to examine the uptake of five metals (Cd, Cr, Cu, Pb and Zn) by the AM under various environmentally realistic concentrations, times, salinities, and temperatures. Results indicated that the AM was able to accumulate the bioavailable and toxic fractions of metals. Compared with the green lipped mussels, uptake and release of metals by the AM are less affected by salinity and temperature changes. The results have been written up and accepted for publication in “Environmental Pollution”.
2008
1.2 Genomic Technologies
5 / 1.2.1 Quantification of waterborne pathogens
Deliverables:
-Identification and validation of species-
specific gene probes for development of DNA-based assays
-Development of quantitative multiplex-
PCR (Q-mPCR) for pathogens / 100% /
- Identified and analyzed a number of virulence and/or virulence-associated genes (for probe design) for multiplex detection of specific waterborne pathogens. The specificity of all probes has been experimentally validated.
- We have successfully developed a quantitative multiplex PCR (Q-mPCR) assay that can simultaneously quantify 4 bacterial pathogens − E. coli, Salmonella, Shigella and V. cholerae − in a single test.
- Identified two previously unrecognized (novel) “virulence-associated” gene clusters in a pandemic O3:K6 strain of V. parahaemolyticus.
3 / 1.2.2 Toxicogenomic studies
Deliverable: Gene expression analysis of effects of hypoxia using high-density and pathway-specific mouse cDNA and fish microarrays / 100% / Fabricateda high-density mouse cDNA microarrays containing 20,000 genes from a cDNA library obtained from NIA and a cDNA microarray containing 200 genes derived from marine medaka. We have used the cDNA microarrays to study the gene expression profiles of murine RAW264.7 macrophage cells under the treatment of H2O2and hypoxia (published in Apoptosis). / 2008
1.3 Biomarker Technologies
4
7 / 1.3.1 Molecular Markers
Deliverables:
-In vitro and in vivo studies of relationships between molecular
biomarkers and levels of hypoxic and/or xenobiotic stress using H295R and marine medaka
-Generation of founder transgenic line / 80%
100%
100%
90% / In vivo studies in fish under hypoxia
In vivo expression and response pattern of a number of hypoxia-responsive genes (e.g. leptin, leptin receptor, VEGF-A, VEGF-R, erythropoietin and CITED cotransactivators) have been studied by quantitative real-time PCR and/or in situ hybridization and immunohistochemistry in several fish species including common carp, grass carp, garoupa and marine medaka. Functional studies on the effects of HIF-1, HIF-2 and HIF-4 transcription factors have also been carried out by gene transfection assays. Our findings indicated differential responses of all of the above genes in a tissue-specificmanner, suggesting differential/ protective roles of these genes in hypoxia signaling.
Based on in-house cloning of numerous medaka-specific cDNAs, we have raised medaka-specific antibodies for a number of proteins (LH, LHR, FSH, FSHR, mGnRH, GnRHR2, omTERT1, omTERT2, CYP19a, omHIF1, omHIF2 and omHIF4) to develop and/or carry out Western blot, immunohistochemistry, chromatin immunoprecipitation (ChIP) and ELISA assays for functional studies. These probes have also been made available for other AoE members in different projects.
In vitro studies in H295R cells under hypoxia
Following incubation of H295R cells under either normoxic or hypoxic conditions for 6, 12 and 24 h, changes in the expression levels of 11 steroidogenic genes − CYP11A (desmolase), CYP11B1 (steroid 11β-hydroxylase), CYP11B2 (aldosterone synthetase), CYP17 (steroid 17α-hydroxylase), CYP19 (aromatase), CYP21 (21 α-hydroxy lase), 3βHSD-1 and -2 (3β-hydroxy steroid dehydrogenase), 17βHSD (17β-hydroxysteroid dehydrogenase), HMGR (HMG-CoA reductase), and StAR (steroidogenic acute regulatory protein), were quantified by a molecular beacon-based real-time RT-PCR method. Ten out of the 11 steroidogenic genes were suppressed after the first 6 h of hypoxic exposure. More than 2-fold suppression was exhibited by CYP11B1, CYP19, CYP21, 3β HSD-1 and -2, 17βHSD and HMGR. Exposure time was also found to affect the dynamics of differential gene expression. For example, suppression of CYP19 at 24 h was about double when compared to that at 6 and 12 h. In contrast, CYP11B1 was suppressed by about 3-fold at 6 h and upregulated by 1.7- and 4.5-fold at 12 and 24 h, respectively. Our results show that hypoxia decreases the transcription of most of the genes regulating the steroidogenesis pathway and this might in turn alter the production rates of steroid hormones, thereby causing potential endocrine disruption.
Xenobiotics
The effects of forskolin, an activator of cAMP-dependent protein kinase, or metyrapone, ketoconazole and aminoglutethimide, either singly or in binary mixtures with forskolin on the expression of steroidogenic genes and on the production of progesterone, testosterone and estradiol were evaluated in H295R cells. The expression of genes for 10 steroidogenic enzymes was determined by quantitative Q-RT-PCR. Changes in gene expression of cells exposed to chemical mixtures ranged from no interaction to interactive, including both antagonistic and super-additive effects. Forskolin preferentially increased production of estradiol, which was in agreement with the observed significant increase in CYP19 gene expression. In contrast, treatment of H295R cells with aminoglutethimide and ketoconazole resulted in decreased production of all three hormones, progesterone testosterone and estradiol, compared to solvent controls. Our results showed that H295R cells provide a valuable test system with which to examine effects of xenobiotics on steroidogenesis.
Generation of founder transgenic lineThe choriogenin H and choriogenin L genes have been cloned from two medaka (Oryzias javanicus and O. melastigma). Exposure studies showed that the lowest-observed -effect concentration (LOEC) of E2 on induction of hepatic ojChgH mRNA is 1 ng/L in adult fish. In addition, effects of 4 other endocrine disruptors were also tested in O. melastigma and showed that omChgHexpression is induced in the liver of male adult fish.Construction of transgenic O. melastigma carrying the GFP reporter gene under control of the omChgH promoter is now underway. In addition,we have also completed a baseline study on the embryonic development of O. melastigma and correlated the stages to Oryzias latipes. The expression pattern of omChgH during embryonic development has been examined. / 2008
2008
2008
2008
9, 10, 15 / 1.3.2 Biochemical markers
Deliverable: Laboratory experiments to develop biochemical markers for ROS, hypoxia and xenobiotics in mussels and fish / 100%+
100%+
90% / Establishment of biochemical, endocrinological and molecular markers for exposure to red tide toxins and xenobiotics in fish
- Our results indicate three possible indices for Chattonella exposure, i.e., serum cortisol level, thyroid hormone (both T3 and T4) levels, and hepatic RNA/DNA ratio.Hepatic RNA/DNA ratio can be used to indicate exposure to either Chattonella whole cells or its extract. The overall change in hormonal profile can be used to differentiate between exposure to Chattonella cells or Chattonella toxic extract since there is a differential hormonal profile when fish are exposed to either cells or extract. Thus, hypercortisolemia is only observed with exposure to Chattonella cells, decline in serum thyroid hormones is only observed following exposure to Chattonella extract. Despite the fact that underlying mechanisms remain unclear, these differential effects may serve as specific biomarkers for differentiatingexposure to Chattonella cells or its extracted toxin.
- Our experiments showed that mussels exposed to xenobiotics or biological toxins experienced significant oxidative stress (e.g., lipid peroxidation, DNA adduct), as well as antioxidant responses (e.g., CAT, SOD, GPx, GR, GSSG). There is some potential to use these biological responses as biomarkers.
- Additional work has been carried out to investigate cell surface protein of Alexendrium affine (a toxic red tide species). This work indicated great potential inemploying these proteomic techniques to identify and characterizespecific proteins as biomarkers for red tide species.
- Our results support the vital role of TERT in controlling cell proliferation in fishin vivo.
- Significant reduction of muscle telomerase activity occurred prior to growth impairment in grouper fish under chronic foodborne BaP exposure, suggesting that telomerase plays a key role in fish growth and can serve as an effective growth marker.
- This marine medaka whole mount fish model will be widely applied to study in vivo expression and regulation of other key genes and proteins of interest in fish under hypoxia.
2009
2008
14
14
8 / 1.3.3 Cytological markers
Deliverables:
-Stereological studies on cytological responses of fish intestines to xenobiotics
-Stereological studies on fish gills in relation to red tide toxin
-Refine prototypical software of 3-D reconstruction / 100%
100%+ / Development of novel cytological, immunological and physiological markers in gills and intestines of fish exposed to hypoxia, xenobiotics and toxins
Physiological analysis and stereological studies on cytological responses of fish gills to toxic red tide algae
- The rapid fish kill mechanisms via direct contact with C. marina cells could be explained by concomitant development of osmotic distress and decreased blood pO2 level in exposed goldlined seabream. Cell-cell interaction between C. marina and fish led to impairment of osmotic homeostasis in fish. The water soluble toxin(s) produced by C. marina are likely to be responsible for a lowering of blood pO2 level in fish, resulting in fish kills. Our earlier and present data confirm that osmotic impairment and blood pO2 reduction are two independent causes of fish kills by this harmful algal species.
For the first time, we demonstrated the chronic (4 weeks of exposure and 4 weeks of recovery) effects of dietary B[a]P on EROD induction, histopathological and physiological/tissue homeostatic responses in fish intestines. The present study has not only opened the possibility of using intestinal EROD activity and selected histo-physiological changes in the intestine as novel biomarkers of B[a]P/PAH exposure in fish, and more importantly, enables us to differentiate aqueous versus dietary uptake of BaP/PAHs