Cloning metallothionein gene in Zacco platypus and its potential as an exposure biomarker against cadmium.

Zacco platypus, pale chub, is an indigenous freshwater fish of East Asia including Korea and has many useful characteristics as indicator species for water pollution. While utility of Z. platypus as an experimental species has been recognized, genetic-level information is very limited and warrants extensive research. Metallothionein (MT) is widely used and well-known biomarker for heavy metal exposure in many experimental species. In the present study, we cloned MT in Z. platypus and evaluated its utility as a biomarker for metal exposure. For this purpose, we sequenced complete complementary DNA (cDNA) of MT in Z. platypus and carried out phylogenetic analysis with its sequences. The transcription-level responses of MT gene following the exposure to CdCl2 were also assessed to validate the utility of this gene as an exposure biomarker. Analysis of cDNA sequence of MT gene demonstrated high conformity with those of other fish. MT messenger RNA (mRNA) expression and enzymatic MT content significantly increased following CdCl2 exposure in a concentration-dependent manner. The level of CdCl2 that resulted in significant MT changes in Z. platypus was within the range that was reported from other fish. The MT gene of Z. platypus sequenced in the present study can be used as a useful biomarker for heavy metal exposure in the aquatic environment of Korea and other countries where this freshwater fish species represents the ecosystem.

Tissue-specific antioxidant responses in pale chub (Zacco platypus) exposed to copper and benzo[a]pyrene.

In this study, antioxidant responses including lipid peroxidation (LPO), superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST), were evaluated in the liver, gill and muscle tissues of pale chub (Zacco platypus) exposed to copper (Cu) and benzo[a]pyrene (BaP). Cu exposure induced significant antioxidant responses in Z. platypus, particularly in the liver, whereas BaP exposure had a negligible effect. Following Cu exposure, both SOD and CAT activity increased in a concentration-dependent manner, showing significant correlations with malondialdehyde (MDA) levels as a measure of LPO (r = 0.646 and 0.663, respectively). SOD, CAT and GST mRNA levels were also enhanced following Cu exposure, except at 20 µg L(-1), although significant correlations with antioxidant enzyme activities were not found. The results of this study suggest that combined information on SOD and CAT activities together with LPO levels in the liver could be a useful indicator for assessing oxidative stress in freshwater fish.

Proteomic analysis in Daphnia magna exposed to As(III), As(V) and Cd heavy metals and their binary mixtures for screening potential biomarkers.

In this study, the effects of three widespread heavy metals, As(III), As(V) and Cd, and their binary mixtures on the proteomic profile in D. magna were examined to screen novel protein biomarkers using the two-dimensional gel electrophoresis method (2DE). Ten 20d daphnia were exposed to the LC20 concentrations for each of a total of 8 treatments, including the control, As(III), As(V), Cd, [As(III)+As(V)], [As(III)+Cd], [As(V)+Cd], and [As(III), As(V), Cd], for 24h before protein isolation. Three replicates were performed for each treatment. These protein samples were employed for 2DE experiments with a pH gradient gel strip from pH 3 to pH 10. The protein spots were detected by a silver staining process and their intensities were analyzed by Progenesis software to discover the differentially expressed proteins (DEPs) in response to each heavy metal. A total of 117 differentially expressed proteins (DEPs) were found in daphnia responding to the 8 treatments and mapped onto a 2D proteome map, which provides some information of the molecular weight (MW) and pI value for each protein. All of these DEPs are considered as potential candidates for protein biomarkers in D. magna for detecting heavy metals in the aquatic ecosystem. Comparing the proteomic results among these treatments suggested that exposing D. magna to binary mixtures of heavy metals may result in some complex interactive molecular responses within them, rather than just the simple sum of the proteomic profiles of the individual chemicals, (As(III), As(V), and Cd).

Development of an Enzyme-linked Immunosorbent Assay Using Vitellin for Vitellogenin Measurement in the Pale Chub, Zacco platypus.

OBJECTIVES: Fish vitellogenin (VTG) is produced in the female liver during oogenesis through the estradiol cycle and produced in the male liver by endocrine disrupting chemicals (EDCs) such as alkylphenols. In this study, we propose that the VTG concentration in the pale chub could be detected using monoclonal antibodies and polyclonal antibodies against vitellin (Vn) in a VTG enzyme-linked immunosorbent assay (ELISA) system. METHODS: Monoclonal antibodies and polyclonal antibodies were produced using the Vn extracted from the matured ovum of the ovary. The VTG was extracted from the plasma of the male pale chub. The Vn and VTG were confirmed by measuring the molecular weight of their proteins using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the specificity of the antibodies was checked through western blotting methods. The assay system was validated with respect to optimal assay concentrations, specificity, recovery, and intra- and inter-assay variations. RESULTS: The Vn consisted of two protein bands with apparent molecular weights of 64 and 37 kDa. The SDS-PAGE indicated protein weights of 146 and 77 kDa in the VTG. The assay range was 15.6 ng/mL to 2,000 ng/mL, and the value of the intra- and inter-assay variations were within 10.0% and 14.7%, respectively. The recovery rate was 99.5±5.5%. CONCLUSIONS: A sandwich ELISA was developed that could be used to qualify the VTG of pale chub in screening for EDCs. Pale chub is an ideal species for observing estrogen activity in the environment because of its extensive habitat and extensive food chain. The ELISA developed here would be more favorable than those for other species for determining the effect of long-term food chain accumulation of EDCs in aquatic environments.

Toxicity evaluation of verapamil and tramadol based on toxicity assay and expression patterns of Dhb, Vtg, Arnt, CYP4, and CYP314 in Daphnia magna.

In this study, the toxicities of two pharmaceuticals, verapamil and tramadol were evaluated in Daphnia magna using the conventional toxicity tests (acute and chronic test) and the expression patterns of five stress responsive genes. In the chronic toxicity test, several parameters, such as the survival percentage, the body length of D. magna, the time of first reproduction, and the number of offspring per female, were adversely affected during the exposure to 4.2 mg L(-1) verapamil and 34 mg L(-1) tramadol. During the 24-h short-term exposure, verapamil particularly caused a downregulated expression of the CYP4 and CYP314 genes, whereas tramadol upregulated the expression of the CYP314 gene. Neither pharmaceutical affected the expression of Dhb, Arnt, and Vtg. However, during the 21-day long-term exposure, both verapamil and tramadol significantly reduced the expression level of the Vtg gene, a biomarker of the reproduction ability in an oviparous animal, whereas neither affected the other genes.

Effects of glyphosate and methidathion on the expression of the Dhb, Vtg, Arnt, CYP4 and CYP314 in Daphnia magna.

In this study, the expression of five stress responsive genes was quantified and analyzed using a semi-quantitative RT-PCR to study the changes in their expression in Daphnia magna after exposure to known pesticides, glyphosate and methidathion. Hemoglobin (Dhb), which was used to show the effect of the oxygen level in the aquatic system, was significantly expressed in D. magna after exposure to glyphosate and methidathion. Additionally, aryl hydrocarbon receptor nuclear translocator (Arnt), a gene related to the metabolism of aryl hydrocarbons, had lower expression levels in D. magna than within the control. CYP4, which was used among cytochrome P450s (CYPs) to show the effects on the fatty acid and steroids metabolisms, was down-regulated in D. magna exposed to glyphosate. However, methidathion affected the expression of CYP314, which was used to show effects of ecdysis, not CYP4 in D. magna. Therefore, glyphosate and methidathion probably caused physiological effects with different patterns in D. magna, especially metabolisms related to CYPs. On the other hand, only vitellogenin (Vtg), which was responsive to the estrogenic potency, did not show any differences in D. magna after exposure to glyphosate or methidathion.

Biodegradation of a phthalate plasticizer, di-isononyl phthalate (DINP), by Sphingobium chungbukense.

In this study, di-isononyl phthalate (DINP) was efficiently degraded by Sphingobium chungbukense KCTC 2955. The optimal conditions for DINP (100 mg L(-1)) degradation by S. chungbukense in a mineral salts medium were found to be pH 7.0, 30 degrees C, and stirring at 200 rpm. The maximum specific rate of DINP degradation was found to be concentration dependent, with a maximum of 4.12 mg DINP L(-1) h(-1). DINP was transformed rapidly by S. chungbukense, with the formation of monoisononyl phthalate (MIP) and phthalic acid, which subsequently degraded further. These results highlight the potential of this bacterium for removing DINP-contaminated waste in the environment.

Degradation of diphenyl phthalate by Sphingomonas chungbukensis.

More than 80% of diphenyl phthalate (DPP) at 100 mg l(-1) was degraded by Sphingomonas chungbukensis KCTC 2955 in a mineral salts medium at pH 7.0 and 30 degrees C within 48 h. The maximum specific degradation rate was 5 mg DPP l(-1 )h(-1). It was rapidly converted to monophenyl phthalate and phthalic acid which were further degraded.