Preself-Feeding Medaka Fry Provides a Suitable Screening System for in Vivo Assessment of Thyroid Hormone-Disrupting Potential.

Endocrine-disrupting chemicals are assessed based on their physiological potential and their potential associated adverse effects. However, suitable end points for detection of chemicals that interfere with the thyroid hormone (TH) system have not been established in nonmammals, with the exception of amphibian metamorphosis. The aims of the current study were to develop an in vivo screening system using preself-feeding medaka fry (Oryzias latipes) for the detection of TH-disrupting chemicals and elucidate the underlying molecular mechanism. 17α-Ethinylestradiol (EE2: <100 ng/L) did not induce mRNA expression of estrogen-responsive genes, vitellogenins (vtgs) mRNA. Meanwhile, coexposure with thyroxin (T4) induced an increase of vtg expression. TH-disrupting chemicals (thiourea (TU), perfluorooctanoic acid (PFOA), and tetrabromobisphenol A (TBBPA)) significantly suppressed EE2 (1,000 ng/L)-induced vtg1 expression, while T4 rescued their expression as well as that of thyroid hormone receptor α (tRα) and estrogen receptors (esrs). These results were supported by in silico analysis of the 5'-transcriptional regulatory region of these genes. Furthermore, the esr1 null mutant revealed that EE2-induced vtg1 expression requires mainly esr2a and esr2b in a TH-dependent manner in preself-feeding fry. Application of preself-feeding medaka fry as a screening system might help decipher the in vivo mechanisms of action of TH-disrupting molecules, while providing an alternative to the traditional animal model.

Toxicity assessment of pollutants sorbed on environmental microplastics collected on beaches: Part II-adverse effects on Japanese medaka early life stages.

While microplastics are present in great abundance across all seas and oceans, little is known about their effects on marine life. In the aquatic environment, they can accumulate a variety of chemicals and can be ingested by many marine organisms including fish, with chronic physical and chemical effects. The purpose of this paper is to evaluate the toxic effects of pollutants sorbed at the surface of environmental microplastics (MPs), collected on various beaches from three islands of the Pacific Ocean. Developmental toxicity of virgin MPs or artificially coated with B[a]P and environmental MPs from Easter Island, Guam and Hawaii was evaluated on embryos and prolarvae of Japanese medaka. Mortality, hatching success, biometry, malformations, EROD activity and DNA damage were analyzed after exposure to DMSO extracts. No toxicity was observed for extracts of virgin MPs whatever the endpoint considered. Extracts of virgin MPs coated with 250 µg.g-1 of B(a)P induced lethal effects with high embryo mortality (+81%) and low hatching rate (-28%) and sublethal effects including biometry and swimming behavior changes, increase of EROD activity (+94%) and DNA damage (+60%). Environmental MPs collected on the three selected islands exhibited different polymer, pollutant and toxicity patterns. The highest toxicity was detected for MPs extract from Hawaï with head/body length and swimming speed decreases and induction of EROD activity and DNA stand breaks. This study reports the possible sublethal toxicity of organic pollutants sorbed on MPs to fish early life stages.

Medaka (Oryzias latipes) Multigeneration Test with Triclosan.

The medaka extended one-generation reproduction test (MEOGRT) is a tier-2 study in the US Environmental Protection Agency's Endocrine Disruptor Screening Program and a level-5 study in the Organisation for Economic Co-operation and Development's conceptual framework. Integrating nonspecific apical and endocrine-specific mechanistic endpoints, results of a MEOGRT can be used, with other data, in a weight-of-evidence evaluation to establish a dose-response relationship for risk assessment and identify potential causal relationships between an endocrine mode of action and adverse effects. The MEOGRT test design was used to evaluate the multigenerational effects of the antimicrobial agent triclosan. Japanese medaka were exposed to nominal concentrations of 1.4, 2.8, 5.6, 11, and 23 µg/L triclosan and a dilution water control starting with adult medaka (F0) through hatch in the second generation (F2). No consistent or concentration-related responses occurred in the 182-d test that suggested an endocrine-mediated effect. There were no impacts on hepatic vitellogenin, secondary sex characteristics, or sex ratio that were linked to an adverse reproductive outcome. Histopathological responses were consistent with a toxic or stress effect, particularly when considered in context with observed reductions in growth. The overall population-relevant no-observed-effect concentration was 11 µg/L based on effects on growth. The results of the present study support a previously conducted weight-of-evidence evaluation concluding that triclosan does not act as an agonist or antagonist within estrogen, androgen, thyroid, or steroidogenic pathways. Environ Toxicol Chem 2019;38:1770-1783. © 2019 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

Sharing the Roles: An Assessment of Japanese Medaka Estrogen Receptors in Vitellogenin Induction.

Teleost fish express at least three estrogen receptor (ER) subtypes. To date, however, the individual role of these ER subtypes in regulating expression of estrogen responsive genes remains ambiguous. Here, we investigate putative roles of three ER subtypes in Japanese medaka (Oryzias latipes), using vitellogenin (VTG) I and II as model genes. We identify specific ligand/receptor/promoter dynamics, using transient transactivation assays that incorporate luciferase reporters comprising 3kb promoter/enhancer regions of medaka VTGI and VTGII genes. Four steroidal estrogens (17ß-estradiol, estrone, estriol, and 17α-estradiol) were tested in these assays. Results indicate that all three medaka ERs (mERs) are capable of initiating transactivation of both VTG I and II, with ERß2 exhibiting greatest activity. Promoter deletion analysis suggests that ligand-specific receptor transactivation and utilization of regional-specific estrogen response elements may be associated with differential activities of each medaka ER. Further, cluster analysis of in vivo gene expression and in vitro transactivation suggests that all three ER subtypes putatively play a role in up-regulation of VTG. Results illustrate that preferential ligand/receptor/promoter interactions may have direct implications for VTG gene expression and other ER-mediated regulatory functions that are relevant to the risk assessment of estrogenic compounds.

Criteria for deviation from predictions by the concentration addition model.

Loewe's additivity (concentration addition) is a well-known model for predicting the toxic effects of chemical mixtures under the additivity assumption of toxicity. However, from the perspective of chemical risk assessment and/or management, it is important to identify chemicals whose toxicities are additive when present concurrently, that is, it should be established whether there are chemical mixtures to which the concentration addition predictive model can be applied. The objective of the present study was to develop criteria for judging test results that deviated from the predictions by the concentration addition chemical mixture model. These criteria were based on the confidence interval of the concentration addition model's prediction and on estimation of errors of the predicted concentration-effect curves by toxicity tests after exposure to single chemicals. A log-logit model with 2 parameters was assumed for the concentration-effect curve of each individual chemical. These parameters were determined by the maximum-likelihood method, and the criteria were defined using the variances and the covariance of the parameters. In addition, the criteria were applied to a toxicity test of a binary mixture of p-n-nonylphenol and p-n-octylphenol using the Japanese killifish, medaka (Oryzias latipes). Consequently, the concentration addition model using confidence interval was capable of predicting the test results at any level, and no reason for rejecting the concentration addition was found. Environ Toxicol Chem 2016;35:1806-1814. © 2015 SETAC.

Assessment of the role of cortisol and corticosteroid receptors in epidermal ionocyte development in the medaka (Oryzias latipes) embryos.

Cortisol is a pleiotropic glucocorticoid hormone that acts through the intracellular glucocorticoid receptors (GR). Cortisol affects many important biological functions in mammals, including immune function, behavior, stress, metabolism, growth and organogenesis. In fishes, cortisol has an additional function in the osmoregulatory activity of ionocytes (ICs). Although much progress has been made toward understanding cortisol action at the levels of adult osmoregulatory tissues, the developmental functions of cortisol and its receptors in ICs remain to be clarified. We first analyzed the total contents of both cortisol and corticosteroid receptor mRNAs (GR1, GR2 and MR) during medaka development. Although low levels of cortisol were detected during development of the medaka embryo, maternal GR1, GR2 and MR transcripts were detected at higher levels than zygotic transcripts. We investigated the effect of exogenous cortisol on IC number during medaka embryogenesis. We observed that cortisol treatment induced an earlier expansion of the IC population but did not modify the final IC number. Using functional genomic approaches, we also tested the involvement of GR1, GR2 and mineralocorticoid receptor (MR) in IC development by systematic knock-down with translation-blocking morpholinos. Only GR2 knock-down led to a reduction of the total number of ICs in the epidermis. In addition, a GR2 splice-blocking morpholino did not have any effect on the biogenesis of ICs, underscoring the importance of maternally inherited GR2 mRNAs. We propose that maternal GR2, but not GR1 or MR, is a major pathway in the IC biogenesis in medaka most likely through cortisol activation, and that cortisol exposition fine-tunes their developmental timing. These findings provide a framework for future research on the regulatory functions of corticosteroids in euryhaline fishes and provide medaka as an advantageous model to further elucidate the underlying molecular regulatory mechanisms of IC development.

Aquatic toxicity and ecological risk assessment of seven parabens: Individual and additive approach.

In the present study, aquatic concentrations of seven parabens were determined in urban streams highly affected by treated or untreated domestic sewage in Tokushima and Osaka, Japan. The detected highest concentrations were 670, 207, and 163ngl(-1) for methylparaben, n-propylparaben, and n-butylparaben, respectively in sampling sites with watershed area of no sewer system in Tokushima. Conventional acute/chronic toxicity tests were conducted using medaka (Oryzias latipes), Daphnia magna, and Psuedokirchneriella subcapitata for four parabens, which was consistent with our previous study on three parabens, n-butylparaben, i-butylparaben, and benzylparaben. The aquatic toxicity on fish, daphnia, and algae was weaker for the parabens with a shorter alkyl chain than those with a longer alkyl chain as predicted by their hydrophobicity. Medaka vitellogenin assays and DNA microarray analysis were carried out for methylparaben and found induction of significant vitellogenin in male medaka at 630µgl(-1) of methylparaben, while the expression levels of genes encoding proteins such as choriogenin and vitellogenin increased for concentrations at 10µgl(-1) of methylparaben. Measured environmental concentrations (MECs) of seven parabens in Tokushima and Osaka were divided by predicted no effect concentrations (PNECs) and hazard quotient (MEC/PNEC) was determined for individual parabens. The MEC/PNEC was highest for n-propylparaben and was 0.010 followed by n-butylparaben (max. of 0.0086) and methylparaben (max. of 0.0042). The sum of the MEC/PNEC for the seven parabens was 0.0049. Equivalence factors were assigned for each paraben on the basis of the toxicity of n-butylparaben for each species, and n-butylparaben equivalence was calculated for the measured environmental concentrations. The MEC/PNEC approach was also conducted for the n-butylparaben-based equivalence values. The maximum MEC/PNEC was 0.018, which is lower than the trigger level for further detailed study such as large-scale monitoring for chronic toxicity tests including full-life cycle tests for fish.

Initial ecological risk assessment of eight selected human pharmaceuticals in Japan.

Eight pharmaceuticals were selected on the basis of their domestic consumption in Japan, the excretion ratio of the parent compound and the frequency of detection in the aquatic environment or wastewater treatment plant effluent. Toxicity tests on these pharmaceuticals were conducted using Japanese medaka (Oryzias latipes), daphnia (Daphnia magna), and green algae (Psuedokirchneriella subcapitata). Predicted no effect concentration (PNEC) was calculated using lethal or effect concentration 50 (LC50 or EC50) values and no effect concentration (NOEC) obtained in the toxicity tests for these compounds. Predicted environmental concentration (PEC) was also calculated from annual consumption, the excretion rate of the parent compound, and removal rate in the preliminary batch activated sludge treatment performed in this study. Maximum concentrations found in the aquatic environment or sewage effluent in Japan or foreign countries were also used for another calculation of PEC. Initial risk assessment on the selected pharmaceuticals was performed using the PEC/PNEC ratio. The results of initial risk assessment on the eight selected pharmaceuticals suggest neither urgent nor severe concern for the ecological risk of these compounds, but further study needs to be conducted using chronic toxicity tests, including reproduction inhibition and endocrine disruption assessments.

Toxicity of o,p'-DDE to medaka d-rR strain after a one-time embryonic exposure by in ovo nanoinjection: an early through juvenile life cycle assessment.

The toxicity of o,p'-DDE (1,1-dichloro-2-(p-chlorophenyl)-2-(o-chlorophenyl) ethylene) was evaluated in embryos of medaka (Oryzias latipes) following a one time exposure via nanoinjection. Medaka eggs (early gastrula) were injected with 0.5 nl of triolein (vehicle control) or 0.5 nl of 4 graded doses (0.0005-0.5 ng/egg) of o,p'-DDE in triolein. Embryos were allowed to develop, and fry were reared. Embryonic survival was monitored daily during the first 10 d until hatching and thereafter, on a weekly basis until day 59, at which time the fish were monitored for sexual maturity until day 107. In general, o,p'-DDE caused a dose- and time-dependent mortality. No changes in mortality were observed between the last two time points (day 38 and 59, respectively), and hence a 59 day-LD50 of 346 ng o,p'-DDE/egg was derived from the linear dose-response relationship. Prior to late stage death, only isolated cases of cardiovascular lesions and spinal deformities were observed, but were not dose-dependent. The lowest observable adverse effect level (LOAEL), based on upper 95% CI for regression line=0.0018 mg/kg, and the LOAEL based on exposure doses=0.5 mg/kg. Likewise, the no observable adverse effect level (NOAEL) based on linear extrapolation to 100% survival=0.0000388 mg/kg, while the NOAEL based on exposure doses=0.05 mg/kg. The nanoinjection medaka model has potential in the study of hormonally active compounds in the environment.