Differential developmental toxicity of naphthoic acid isomers in medaka (Oryzias latipes) embryos

Polycyclic aromatic hydrocarbons (PAHs) are widespread persistent pollutants that readily undergo biotic and abiotic conversion to numerous transformation products in rivers, lakes and estuarine sediments. Here we characterize the developmental toxicity of four PAH transformation products each structural isomers of hydroxynaphthoic acid: 1H2NA, 2H1NA, 2H3NA, and 6H2NA. Medaka fish (Oryzias latipes) embryos and eleutheroembryos were used to determine toxicity. A 96-well micro-plate format was used to establish a robust, statistically significant platform for assessment of early life stages. Individual naphthoic acid isomers demonstrated a rank order of toxicity with 1H2NA > 2H1NA > 2H3NA > 6H2NA being more toxic. Abnormalities of circulatory system were most pronounced including pericardial edema and tube heart. To determine if HNA isomers were AhR ligands, spatial-temporal expression and activity of CYP1A was measured via in vivo EROD assessments. qPCR measurement of CYP1A induction proved different between isomers dosed at respective concentrations affecting 50% of exposed individuals (EC50s). In vitro, all ANH isomers transactivated mouse AhR using a medaka CYP1A promoter specific reporter assay. Circulatory abnormalities followed P450 induction and response was consistent with PAH toxicity. A 96-well micro-plates proved suitable as exposure chambers and provided statistically sound evaluations as well as efficient toxicity screens. Our results demonstrate the use of medaka embryos for toxicity analysis thereby achieving REACH objectives for the reduction of adult animal testing in toxicity evaluations.     

Utilizing in vivo nuclear magnetic resonance spectroscopy to study sublethal stress in aquatic organisms

In vivo nuclear magnetic resonance spectroscopy (NMR) is a powerful technique for characterizing the sublethal actions of physical and chemical stressors in live, intact organisms. In particular, 31P NMR is ideal for observing perturbations to cellular energetics since critical metabolite concentrations, including phosphagens, ATP and inorganic phosphate (Pi), can be measured non-invasively and in real time. This technique's versatility is demonstrated not only in the diversity of organisms that can be studied, but also in its broad-ranging applicability to environmental research. Illustrative studies include the actions of copper in adult red abalone (Haliotis rufescens) and changes in energetically important metabolites in developing medaka embryos (Oryzias latipes). Advantages and disadvantages of in vivo NMR will be discussed.     

Environmentally relevant exposure of 17α-ethinylestradiol impairs spawning and reproductive behavior in the brackish medaka Oryzias melastigma

We assessed the effects of 17α-ethinylestradiol (EE2) on spawning and reproductive behavior of the brackish medaka Oryzias melastigma at environmental concentrations. Breeding pairs were exposed to EE2 at either lower concentrations (0, 1, and 10 ng/L) or higher concentrations (0, 50, and 100 ng/L) for 14 days. Swimming performance and reproductive behavior (following, dancing, and copulation) of the males were analyzed at the end of exposure. Additionally, gonad histology was conducted in the males in the higher EE2 experiment. We found that spawning was significantly inhibited in the 50–100 ng/L EE2 groups. Swimming performance was not affected in all groups. Dancing and copulation were significantly suppressed in the 50–100 ng/L groups. No effect was observed in testis histology in the 50–100 ng/L groups. Our data suggests that reproductive behavior of O. melastigma can be utilized as a behavioral endpoint for ecotoxicology studies of endocrine disruptors in estuarine/marine environments.     

Cloning of cytochrome P450 1A (CYP1A) genes from the hermaphrodite fish Rivulus marmoratus and the Japanese medaka Oryzias latipes

To use two small fish Rivulus marmoratus (Cyprinodontiformes, Rivulidae) and the Japanese medaka Oryzias latipes (Belloniformes) as testing models in molecular ecotoxicology, we have cloned the cytochrome P450 1A (CYP1A) gene after screening of both genomic DNA libraries, and sequenced 11,863 and 7,243 bp including all the exons and introns with promoter regions, respectively. The Rivulus and the medaka CYP1A gene consisted of seven exons (including non-coding exons) with high homology to mammals. In the promoter region, Rivulus CYP1A gene has seven xenobiotic response elements (XREs) and two metal response elements (MREs), while the Japanese medaka CYP1A gene has six XREs and four MREs. Interestingly, medaka CYP1A gene has a number of MREs at the promoter, which may affect its response on metal exposure. We describe here the gene structure of both fish CYP1A genes.     

Characteristics of ChgH–GFP transgenic medaka lines, an in vivo estrogenic compound detection system

We previously reported the characteristics of a ChgH–GFP transgenic medaka line that indicates estrogenic compound pollution in environmental water by the green fluorescence of their liver. Recently, we established four more lines. In this study, the characteristics of the five transgenic medaka lines were investigated. The intensity of reporter gene expression varied among transgenic lines and generally correlated well with the amount of integrated transgene in each line. Line-specific ectopic expression was also observed. However, the sensitivity to 17-β estradiol did not differ among transgenic lines. Three transgenic lines are considered to be suitable as bio-indicators of estrogenic activity, due to the ease of observing green fluorescence in their livers. The transgenic lines can also detect the estrogenic activity of testosterone and 17-β trenbolone at the nominal concentration of 30 and 100 μg/l, respectively.