Utilization of piperonyl butoxide and 1-aminobenzotriazole for metabolic studies of toxic chemicals in Daphnia magna and Chironomus yoshimatsui.

Daphnids and chironomids have been used to assess the ecological effects of chemicals released into water bodies; however, the toxicity mechanisms in organisms are generally difficult to identify. Here, we developed a system capable of estimating the contribution of cytochrome P450 (CYP) to the metabolism of test substances in Daphnia magna and Chironomus yoshimatsui based on toxicity differences in the absence and presence of the CYP inhibitors piperonyl butoxide (PBO) and 1-aminobenzotriazole (ABT). The optimum concentrations of PBO and ABT that could effectively reduce the toxicity of diazinon, which is toxic after oxidative metabolism in vivo, were determined as 0.5 and 0.6 mg/L for D. magna, and 2.0 and 40.0 mg/L for C. yoshimatsui, respectively. Acute immobilization tests of 15 insecticides were conducted for D. magna and C. yoshimatsui, with and without the optimum concentrations of PBO or ABT. In the presence of either inhibitor, chlorpyrifos and chlorfenapyr toxicity was reduced in both organisms, whereas those of thiocyclam, nereistoxin, and silafluofen were enhanced in C. yoshimatsui. Liquid chromatography-mass spectrometry analysis of D. magna and C. yoshimatsui samples exposed to chlorfenapyr confirmed that the level of the active metabolite produced by CYP was decreased by PBO or ABT in both organisms. The system to which the test substance was co-exposed to PBO or ABT will be valuable for estimating the contribution of CYPs to metabolism and elucidating the toxicity mechanism in daphnids and chironomids.

The effect of using light emitting diodes and fluorescent lamps as different light sources in growth inhibition tests of green alga, diatom, and cyanobacteria.

Aquatic organisms have been used to investigate the safety of chemicals worldwide. One such assessment is an algal growth inhibition test. Algal growth inhibition tests are commonly performed using a growth chamber with fluorescent lamps as the lighting source, as test guidelines require continuous uniform fluorescent illumination. However, fluorescent lamps contain mercury, which has been identified as hazardous to humans and other organisms. The Minamata Convention (adopted in 2013) requires reduction or prohibition of products containing mercury. On the other hand, light-emitting diodes do not contain mercury and provide a photosynthetically effective wavelength range of 400-700 nm which is an adequate light intensity for algal growth. Light-emitting diodes are thus preferable to fluorescent lamps as a potential light source in algal growth inhibition tests. In this study, we investigated if light-emitting diodes could be substituted for fluorescent lamps in growth inhibition studies with green alga (Pseudokirchneriella subcapitata), diatom (Navicula pelliculosa), and cyanobacteria (Anabaena flos-aquae). Algal growth inhibition tests were performed using five different chemicals known to have different modes of action and are assigned as reference substances in the test guidelines. The results of each algal test showed similar values between light-emitting diodes and fluorescent lamps in terms of conditions for the growth inhibition rate and percent inhibition in yield of each chemical. It was therefore concluded that using light-emitting diodes instead of fluorescent lamps as a lighting source had no effect on the algal growth inhibition test results.

Production of genome-edited Daphnia for heavy metal detection by fluorescence.

Aquatic heavy metal pollution is a growing concern. To facilitate heavy metal monitoring in water, we developed transgenic Daphnia that are highly sensitive to heavy metals and respond to them rapidly. Metallothionein A, which was a metal response gene, and its promoter region was obtained from Daphnia magna. A chimeric gene fusing the promoter region with a green fluorescent protein (GFP) gene was integrated into D. magna using the TALEN technique and transgenic Daphnia named D. magna MetalloG were produced. When D. magna MetalloG was exposed to heavy metal solutions for 1 h, GFP expression was induced only in their midgut and hepatopancreas. The lowest concentrations of heavy metals that activated GFP expression were 1.2 µM Zn2+, 130 nM Cu2+, and 70 nM Cd2+. Heavy metal exposure for 24 h could lower the thresholds even further. D. magna MetalloG facilitates aqueous heavy metal detection and might enhance water quality monitoring.

Is lipid peroxidation induced by the aqueous suspension of fullerene C60 nanoparticles in the brains of Cyprinus carpio?

This study aimed to clarify whether fullerene C60 nanoparticles induced lipid peroxidation in Cyprinus carpio brains. A stable well-characterized aqueous suspension of C60 nanoparticle (diameter: 50th and 95th percentiles, 36 and 95 nm respectively) with 0.1% Tween80 solution was prepared by bead milling. Lipid hydroperoxides (LPO) were measured in vitro in homogenized fish brain tissues containing 33 microg/mg-protein dispersed C60 nanoparticles under light and dark conditions to verify the lipid peroxidation ability of C60 and the interference of light exposure by using a commercial assay kit The LPO concentration significantly increased under the light condition but not under the dark condition. This suggests that C60 has the lipid peroxidation ability under light condition, and the light exposure that occurs during the dissection and preparation of fish brain samples containing C60 for the LPO assay interferes with the measurements of the in vivo LPO levels. Therefore, dissection and assay in the in vivo experiment were conducted under a yellow lamp or dark condition to avoid the interference of light. Moreover, the result of the in vitro test suggests that the LPO assay with irradiation might be a good method for detecting C60 in brain tissues. In the in vivo experiment, C. carpio was exposed to 4.5 mg/L nano C60 suspension for 48 h, following which the brain LPO concentration was measured. In the in vivo experiment, no fish died or exhibited abnormal symptoms during exposure. LPO assay of the C. carpio brain samples confirmed the absence of lipid peroxidation after exposure to 4.5 mg/L aqueous C60 nanoparticle suspension for 48 h. Additional LPO assay under irradiation showed that C60 did not reach the brain.

Difference in the sensitivity to chemical compounds between female and male neonates of Daphnia magna.

Daphnia magna usually produce female offspring by parthenogenesis, and thus only female neonates are used to evaluate the environmental toxicity to chemicals. Additionally, it is known that male daphnids are induced by exposure to a juvenile hormone, methyl farnesoate, during late ovarian development. In this study, we investigated the concentration of methyl farnesoate in a 24-h exposure producing 100% males, and the difference in sensitivity to chemical compounds, potassium dichromate, pentachlorophenol, and paraquat, between females and males, referring to OECD Test Guideline 202. The results show that the minimum concentration for 100%-male induction of methyl farnesoate in adult females was 50 microg/L. In addition, acute toxicity tests (immobility test) with the other chemicals showed that male neonates have higher tolerance to potassium dichromate and pentachlorophenol than females for at least 24 h after birth, while no sex difference was observed in the sensitivity to paraquat. The differences in the median effective concentrations in these compounds between female and male neonates suggest two different overall modes of action. Using female daphnids for environmentally toxicity testing seems reasonable, since the females are more sensitive to chemicals than males. Furthermore, the method of male induction established in this study could be used for screening of endocrine disruptors.

Chemical sensitivity of the male daphnid, Daphnia magna, induced by exposure to juvenile hormone and its analogs.

It was reported that males daphnid Daphnia magna that have been induced by methyl farnesoate exposure exhibit higher tolerance to chemical compounds such as potassium dichromate and pentachlorophenol than females. Male neonates are also known to be induced by exposure to juvenile hormone analogs, such as fenoxycarb and pyriproxyfen. If these analogs can be used to produce male progeny, the biological and physiological studies of daphnid male would be progressed since the effects of these analogs were several hundred times higher than that of methyl farnesoate. Therefore, in the present study, it was investigated that the chemical sensitivity of male neonates induced by exposure to juvenile hormone (methyl farnesoate) and its analogs. The minimum concentrations of methyl farnesoate, fenoxycarb and pyriproxyfen to induce 100% male-reproduction were 200nM (50microg/l), 0.23nM (70ng/l) and 0.31nM (100ng/l), respectively. In addition, no reduction of relative reproduction was observed at the juvenoid concentrations in 24h exposure producing 100% male progeny. The median effective concentrations (EC50) of potassium dichromate for immobility of male neonates, established by a standardized method for investigating sensitivity to chemicals, were significantly higher (12-29%) than that of females at least after 24h exposure in all the male neonates induced by juvenoids used in this study (P<0.05). This study demonstrated that the male daphnids induced by exposure to juvenile hormone and its analogs exhibit similar chemical tolerance.

Production of low-estrogen goldfish diet for in vivo endocrine disrupter test.

A low-estrogenic diet for goldfish Carassius auatus was produced for an in vivo estrogen activity test, because commercial fish feed has estrogenic activity and may affect the results of estrogen assays. The newly produced diet (FD5) was formulated with defatted rice bran and casein, and did not contain any soybean meal or fish meal. Phytoestrogen contents (genistein, daidzein, equol, and coumestrol) of FD5 were measured by liquid chromatography-mass spectroscopy/mass spectroscopy (LC-MS/MS) and compared with those of the commercial trout diet (TD) and carp diet (CD). The genistein, daidzein, and coumestrol contents of TD and CD were much higher (5-2000 times) than those of FD5, but equol was detected only in FD5. Estrogenic activity of the fish diets was estimated in vitro by the yeast estrogen-screen assay (YES assay). The estrogenic activity was detected in TD and CD, but not in FD5. The in vivo estrogenic activity of the diets was examined by determining the production of vitellogenin in male goldfish. When male goldfish were fed TD or CD, plasma vitellogenin levels increased, but fish that were fed FD5 maintained low vitellogenin levels. These results indicate that FD5 produced in the present study has a low estrogenic activity, and FD5 would be suitable for the in vivo estrogen activity test using goldfish.

Survey of estrogenic activity in fish feed by yeast estrogen-screen assay.

Fishes have been used as laboratory animal for research of estrogenic endocrine disrupters by many researchers. However, much less attention was paid to the possibility that compounds with estrogenic activity are present in fish diets. In order to examine this possibility, we measured the estrogenic activity in commercial fish feed by in vitro yeast estrogen-screen (YES) assay based on the binding ability of tested compounds to estrogen receptors. Estrogenic activity was detected in all the commercial fish feed examined (0.2-6.2 ng estradiol equivalent/g fish feed), some phytoestrogens (genistein, formononetin, equol and coumestrol; relative activity to estradiol, 8.6 x 10(-6)-1.1 x 10(-4) by giving a value of 1.0 to estradiol) and some androgens (testosterone, 11-ketotestosterone and 5 alpha-dihydrotestosterone; relative activity to estradiol, 3.0 x 10(-6)-1.2 x 10(-4)). Therefore, it is possible that these compounds could affect the results of in vivo estrogen assay, such as vitellogenin production in male fish, especially when fish are fed commercial feed.