King Ming Chan

Metallothionein: Potential biomarker for monitoring heavy metal pollution in fish around Hong Kong

Metallothioneins (MTs) are a family of low-molecular-weight, cysteine-rich proteins that have a high affinity for divalent ions. Recently, MT level has become a major biomarker for monitoring metal pollution in fish. As a first step towards the development of a reliable and standard assay method for the detection of fish MTs in Hong Kong waters, reverse transcription-polymerase chain reaction (RT-PCR) was employed to obtain complementary DNAs (cDNAs) coding for goldfish (Carrasius auratus) and tilapia (Tilapia mossambica) MTs. Based on the cloned tilapia MT cDNA sequence, specific primers were designed to detect MT mRNA levels in tilapia tissues using RT-PCR. Screening of a common carp liver cDNA library also yielded MT cDNAs. For the development of an immuno-assay for monitoring MT levels in fish tissues, synthetic peptide, made according to the conserved teleost MT N-terminal amino acid sequences, was employed to raise anti-fish MT antibodies. These molecular and immunological probes will be useful in 1. testing the feasibility of using tilapia MT levels as a biomarker for monitoring metal pollution in Hong Kong fish and 2. determining the basic physiological significance of MT in metal tolerance in tilapia.

PCR-cloning and gene expression studies in common carp (Cyprinus carpio) insulin-like growth factor-II

Insulin-like growth factor-II (IGF-II) is a member of a growth factor family related to fetal growth in mammals but its physiological role has not been clearly identified in fish. In teleosts, the basic mechanism of the growth hormone (GH)-IGF axis is known to be operative but in a different manner. For instance, IGF-I exhibits GH dependence whereas for IGF-II, its GH dependence varies in different fish species. In this study, we used polymerase chain reaction (PCR) to obtain a common carp IGF-II (ccIGF-II) cDNA fragment and methods of rapid amplification of cDNA ends (RACEs) to obtain a full-length ccIGF-II sequence. The ccIGF-II encodes for a predicted amino acid sequence showing identities of 70.6%, 68.7%, 63.4% and 35% in comparison with salmon, barramundi, tilapia and human IGF-II, respectively. The nucleotide identity between the open reading frame (ORF) of the ccIGF-II and ccIGF-I cDNA sequence is only 36.2%. Distribution of ccIGF-II mRNA levels in common carp tissues was also studied; ccIGF-II expressed in hepatopancreas, heart, and many other tissues in adult carps are similar to the levels of ccIGF-I except in gills and testis. ccIGF-II levels were significantly higher than that of ccIGF-I in most juvenile tissues except in hepatopancreas, where ccIGF-I was higher (threefold) than that of ccIGF-II. The levels of ccIGF-I were also higher than ccIGF-II in carp larvae, from pre-hatched stage to day 30 post-hatching. Injection of porcine GH (pGH) increased the IGF-I and IGF-II mRNA levels in the hepatopancreas and brain of juvenile carps. However, hepatic IGF-I mRNA levels were induced more than IGF-II by pGH, whereas ccIGF-II levels gave a higher response than IGF-I in the brain in response to GH induction.

Concentrations of copper, zinc, cadmium and lead in rabbitfish (Siganus oramin) collected in Victoria Harbour, Hong Kong

The urban area of Hong Kong supports a population of about 4 million people, with an annual production of several hundred million tonnes of untreated domestic and industrial effluent. This causes significant marine pollution problems in Victoria Harbour. Moreover, urban runoff also brings pollutants to Victoria Harbour. This paper presents preliminary data on the concentrations of heavy metals from different parts of the rabbitfish (Siganus oramin) taken from Hung Hom, Wan Chai and the Central Ferry Pier, close to urban and sewage outfalls in Victoria Harbour, from June to September 1983. Metal concentrations in the seawaters and from the gills, viscera, vertebrae and muscle were determined by atomic absorption spectrophotometry. Concentrations of copper, zinc and lead in the muscle were found to be the lowest in the muscle as compared with other parts of the body (cadmium concentrations were similar in muscle and viscera), but concentrations were nevertheless still high (mean±SD, dry weight): 5.7±4.0 ppm for copper, 66.6±31.8 ppm for zinc, 3.3±1.1 ppm for cadmium and 19.1±6.3 ppm for lead. Cadmium and lead concentrations in the rabbitfish samples of this study were significantly higher than those previously reported in fish from the nearby Pearl River estuary.

Metal toxicity and metallothionein gene expression studies in common carp and tilapia

Abstract In Hong Kong, carp and tilapia are common in ponds and rivers. Tilapia is the dominant fish species found in contaminated rivers and estuarine regions. Heavy metal pollution is one of the major problems. Here we report the use of juvenile common carp and tilapia to study: (i) lethal concentrations of copper and zinc; and (ii) metallothionein (MT) mRNA levels in liver and gills of these fish after exposure to different concentrations of metals. For tilapia, the 96-h LC 50 values for copper and zinc were determined to be 1.52 and 24.3 ppm, respectively. However, for common carp, the 96-h LC 50 values of copper and zinc were found to be 50 and 17 ppm, respectively. This indicated that tilapia is a copper-resistant species while carp is sensitive to copper ions. Juvenile carp and tilapia were exposed to different sub-lethal concentrations of copper, zinc and cadmium for 3 weeks in the laboratory, to study MT gene expression. Our results showed that exposure of common carp to these metals did not induce MT mRNA in gill and liver tissues. However, exposure of tilapia to metal ions showed significant induction of MT mRNA in the same tissues. In conclusion, carp MT level is not a sensitive biomarker for monitoring metal pollution, and tilapia MT levels may be a better biomarker for monitoring metal contamination in local waters.

Magnetic Nanochains of FeNi3 Prepared by a Template-Free Microwave-Hydrothermal Method

Magnetic FeNi3 nanochains were synthesized by reducing iron(III) acetylacetonate and nickel(II) acetylacetonate with hydrazine in ethylene glycol solution without any template under a rapid and economical microwave irradiation. The morphology and composition of the as-prepared products were characterized by field-emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), and elemental mapping. The size of the aligned nanospheres in the magnetic FeNi3 chains could be adjusted from 150 to 550 nm by increasing the amounts of the precursors. The length of the nanochain is about several tens of micrometers. The ratio of the precursors plays an important role in the formation of FeNi3 nanostructures. Magnetic measurement reveals that the FeNi3 nanochains show enhanced coercivity and saturation magnetization. The formation mechanism of the product is discussed. Toxicity tests of FeNi3 nanochains show that the as-prepared nanochains are nontoxic to zebrafish larvae. In vitro magnetic resonance imaging (MRI) confirms the effectiveness of the FeNi3 nanochains as sensitive MRI probes.

Toxicity assessment and vitellogenin expression in zebrafish (Danio rerio) embryos and larvae acutely exposed to bisphenol A, endosulfan, heptachlor, methoxychlor and tetrabromobisphenol A.

Organochlorine pesticides and brominated flame retardants, such as tetrabromobisphenol A and polybrominated diphenyl ethers, pose an environmental hazard owing to their persistence, low solubility and estrogenic effects, and concerns have been raised regarding their effects on aquatic biota. In the present study, zebrafish embryos and larvae were used as a model to investigate the sublethal and lethal effects of three different organochlorine pesticides, namely methoxychlor, endosulfan and heptachlor, as well as the flame retardant tetrabromobisphenol A, and its precursor compound bisphenol A. Preliminary data for chemical exposure tests were obtained by determining the 96 h median effective concentration EC50 (hatching rate) and 96 h median lethal concentration LC50. Quantitative polymerase chain reaction was used to investigate the gene expression levels of the biomarker vitellogenin (vtg1) after 96 h exposures to 10, 25, 50 and 75% of the 96 h EC50 value for embryos and 96 h LC50 value for larvae. The use of vtg1 mRNA induction in zebrafish embryos and larvae was found to be a sensitive biomarker of exposure to these organic compounds, and was helpful in elucidating their adverse effects and setting water quality guidelines. Copyright

Organochlorines in Hong Kong fish

Abstract Muscle samples from 15 species of fish ( n =1) purchased from markets in Hong Kong and 10 liver samples of tilapia ( Tilapia mossambica ) collected from the Shing Mun River were analysed for organochlorines (polychlorinated biphenyls, ΣDDTs, hexachlorobenzene, hexachlorocyclohexanes, chlordanes, mirex and dieldrin). Polychlorinated biphenyls (PCBs) were detected in 15 market fish samples but the levels were very low (around 1 ng g −1 ). PCB levels in tilapia livers collected from Tai Wai (29.3–65.1 ng g −1 ) were higher than those from Fo Tan (3.5–23.2 ng g −1 ) suggesting that there may be some local point soucres. ΣDDTs were detected in all samples, ranging from 3.3 to 75.6 ng g −1 in the market fish and from 7.1 to 88.8 ng g −1 in tilapia. The DDE/DDT ratios in the market fish (0.12–0.75) showed higher variability than those of tilapia (0.30–0.46), suggesting that some of the market fish may have been collected from areas where DDT was recently used. Results of this preliminary study show that organochlorine levels in Hong Kong market fish are low and do not cause any concern for human consumption. An on-going monitoring program, however, is recommended.

Cytotoxicities and induction of metallothionein (MT) and metal regulatory element (MRE)-binding transcription factor-1 (MTF-1) messenger RNA levels in the zebrafish (Danio rerio) ZFL and SJD cell lines after exposure to various metal ions.

Using zebrafish liver (ZFL) and caudal fin (SJD) cell-line models, the induction of metallothionein (MT) and metal regulatory element-binding transcription factor-1 (MTF-1) mRNA levels by various metal ions (Zn(2+)(,) Cd(2+), Cu(2+), Hg(+), As(3+), As(5+), Cr(3+) and Cr(6+)) were studied using the real-time PCR. The LC(50)-24h values of the metal ions were determined for the two cell lines prior to their exposure to different concentrations (10%, 25%, 50%, 75% and 100% LC(50) values) of the heavy metal ions for RNA assay. The two cell lines were sensitive to As(3+), Cd(2+) and Hg(+). Zn(2+)and Cu(2+) were moderately toxic, and As(5+) and Cr(3+) were less toxic to both cell lines. Each of the metal ions tested was found to cause significant induction of the mRNA levels in the SJD cells. Only Zn(2+), Cd(2+), Cu(2+), Hg(2+) and As(3+) caused significant induction of MT mRNA levels in ZFL cells. Zn(2+)and Cd(2+) were efficient MT inducers in the two cell lines, but As(3+) and As(5+) are strong inducers only in the SJD cell line. In both cell lines, Cu(2+) and Hg(2+) are moderate inducers, and Cr(3+) and Cr(6+) were weak inducers of MT mRNA. MTF-1 induction is believed to be insufficient to cause MT gene induction, but As(3+) and Cd(2+) induced MTF-1 in ZFL cell line. Cd(2+) was the most efficient inducer of MT in the cell lines, and also induced MTF-1 levels with clear dose-responsiveness in SJD cell line. These results indicated that MT induction can occur without MTF-1 induction for many metal ions such as Zn(2+) and Cu(2+).

Evaluation of the toxic effects of brominated compounds (BDE-47, 99, 209, TBBPA) and bisphenol A (BPA) using a zebrafish liver cell line, ZFL.

The toxic effects of three polybrominated diphenyl ether (PBDE) congeners (BDE-47, -99, and -209), tetrabromobisphenol A (TBBPA) and bisphenol A (BPA), were evaluated by determining their 24h and 96 h median lethal concentrations using a zebrafish liver cell line, ZFL. It was found that BDE-47, BDE-99 and TBBPA showed comparative cytotoxicity within the range of 1.2-4.2 μM, and were more toxic than BPA (367.1 μM at 24 h and 357.6 μM at 96 h). However, BDE-209 induced only 15% lethality with exposures up to 25 μM. The molecular stresses of BDE-47, -99, TBBPA and BPA involved in thyroid hormone (TH) homeostasis and hepatic metabolism were also investigated. Using a reporter gene system to detect zebrafish thyroid hormone receptor β (zfTRβ) transcriptional activity, the median effective concentration of triiodothyronine (T3) was determined to be 9.2×10(-11) M. BDE-47, BDE-99, TBBPA and BPA alone, however, did not exhibit zfTRβ agonistic activity. BPA displayed T3 (0.1 nM) induced zfTRβ antagonistic activity with a median inhibitory concentration of 19.3 μM. BDE-47, BDE-99 and TBBPA displayed no antagonistic effects of T3-induced zfTRβ activity. Target gene expressions were also examined under acute exposures. The significant inhibition of different types of deiodinases by all of the test chemicals indicated TH circulation disruption. All four chemicals, especially BPA, were able to affect transcripts of phase II hepatic metabolizing enzymes (UGT2A1, SULT1) in vitro. In conclusion, the zfTRβ reporter gene system developed here helps delineate an in vitro model to enable the analysis of the TH disruption effects of environmental pollutants in fish. BPA and the brominated compounds tested were able to disrupt the TH system at the gene expression level, probably through the deiodination pathways.

Changes in the protein expression profiles of the Hepa-T1 cell line when exposed to Cu2+

Copper is an essential element in a variety of biological processes, but it can be toxic when present in excessive amounts. The central regulators of cellular copper metabolism include copper-binding proteins, copper transporters, metal membrane active transporters and copper-dependent enzymes. However, the way in which cupric ions (Cu(2+)) cause cellular changes in proteins and lead to toxic effects is less well-known. The aim of this study is to identify the proteins related to Cu(2+) toxicity or detoxification mechanisms in tilapia (Oreochromis niloticus) using a proteomic approach. A cell line derived from the liver of tilapia, Hepa-T1, was used as a model and exposed to two sub-lethal concentrations of waterborne copper for 96 h. The proteins expressed in Hepa-T1 were investigated by differential protein profiling using two-dimensional gel electrophoresis (2DE). It was found that Cu(2+) (120 and 300 microM) caused the differential expression of 93 different proteins, 18 of which were further verified by real-time quantitative polymerase chain reaction (PCR) analysis. Following analysis with ingenuity pathway software, several proteins were found to be involved in lipid metabolism, tissue connective development and cell cycle control, thus indicating that copper toxicity affects these cellular functions.