Sayaka Eguchi

Development of an in vitro binding assay for ecdysone receptor of mysid shrimp (Americamysis bahia)

A global effort has been made to establish screening and testing methods that can identify the effects of endocrine-disrupting chemicals (EDCs) on invertebrates. The purpose of our study was to develop an in vitro receptor binding assay for ecdysone receptor (EcR) in mysid shrimp (Americamysis bahia). We cloned mysid shrimp EcR cDNA (2888 nucleotides) and ultraspiracle (USP) cDNA (2116 nucleotides), and determined that they encode predicted proteins of length 570 and 410 amino acids, respectively. The deduced amino acid sequences of these proteins shared 36-71% homology for EcR and 44-65% for USP with those of other arthropods. Phylogenetic analysis revealed that mysid shrimp EcR was classified into an independent cluster together with the EcRs of another mysid species, Neomysis integer and the cluster diverged early from those of the other taxonomic orders of crustaceans. We then expressed the ligand-binding domains (DEF regions) of mysid shrimp EcR (abEcRdef) and USP (abUSPdef) as glutathione S-transferase (GST)-fusion peptides in Escherichia coli. After purifying the fusion peptides by affinity chromatography and removing the GST labels, we subjected the peptides to a ligand-receptor binding assay. [(3)H]-ponasterone A did not bind to abEcRdef or abUSPdef peptides alone but bound strongly to the abEcRdef/abUSPdef mixture with dissociation constant (K(d))=2.14 nM. Competitive binding assays showed that the IC(50) values for ponasterone A, muristerone A, 20-hydroxyecdysone, and α-ecdysone were 1.2, 1.9, 35, and 1200 nM, respectively. In contrast, the IC(50) values for two dibenzoylhydrazine ligands (tebufenozide and chromafenozide) were >1.0 × 10(5)nM. The intra- and inter-assay coefficient of variation values for the IC(50) values of 20-hydroxyecdysone were 14.7% (n=5) and 16.1% (n=8), respectively. Our results indicate that the binding assay with a mixture of abEcRdef and abUSPdef can be used to screen compounds with a broad range of binding affinities for crustacean EcRs.

Assessment of in vitro antiovulatory activities of nonsteroidal anti-inflammatory drugs and comparison with in vivo reproductive toxicities of medaka (Oryzias latipes)

Nonsteroidal anti‐inflammatory drugs (NSAIDs) are widely used therapeutic agents; however, their pharmacological actions raise concerns about potential risks to the reproductive health of aquatic vertebrates. In the present study, a medaka ovulation assay was applied as an in vitro model to evaluate NSAID‐induced antiovulatory activity. We first tested five NSAIDs, including diclofenac sodium (DCF), ketoprofen (KP), salicylic acid (SA), mefenamic acid (MA), and acetylsalicylic acid (ASA) for their antiovulatory activities toward the follicles isolated from the ovaries of spawning females. Of all the chemicals tested, DCF had the highest antiovulatory activity, with the concentration that caused 50% inhibition (IC50) (101 µM). MA was the second most potent inhibitor following DCF, but KP, SA, or ASA had little inhibitory effect on the ovulation of the follicles. The in vitro antiovulatory activity of five NSAIDs showed good correlation with data published on the inhibitory activity on human COX‐2. Second, we selected DCF and SA as the most and least potent NSAIDs, respectively, and examined the effects on reproduction of intact fish in order to evaluate whether the ovulation assay was a reasonable predictor of potential reproductive effects in fish. Females exposed to DCF showed a concentration‐dependent decrease in the number of spawned eggs and an increment in the gonadosomatic index (GSI), possibly due to an anovulation in the females. In contrast, neither fecundity nor the GSI of females decreased at up to 20 mg/L of SA, at which acute lethality to medaka was induced. In conclusion, the medaka ovulation assay reflected the potency of NSAID‐induced antiovulatory activity and may thus serve as an in vitro model for the prediction of NSAID‐induced reproductive toxicity.

Recovery from reproductive and morphological abnormalities in medaka (Oryzias latipes) following a 14‐day exposure to diclofenac

Mating pairs of medaka (Oryzias latipes) were exposed to diclofenac at measured concentrations of 0 (control), 7.1, 37, and 78 μg/L for 14 d under static-renewal conditions. Effects on reproductive success, as well as morphological abnormalities, of the fish were assessed. During the exposure period, both fecundity and fertility were significantly decreased in the 37- and 78-μg/L treatment groups, and swollen abdomens in females were observed in all exposure groups. Notably, a defect of the lower jaw was also observed in 4 male fish: 2 at 37 μg/L and 2 at 78 μg/L of diclofenac. Subsequently, we investigated whether the reproductive and morphological abnormalities induced by diclofenac would be permanent or reversible once the medaka were returned to clean water. The reproductive ability of paired medaka was gradually restored to fish that were cultured in clean water for 14 d. After this period in clean water, we also observed a noticeable decrease in swollen abdomens in females; however, mandibular defects in the males remained, even after the 14-d recovery period. Radiographic and histochemical examinations revealed that diclofenac might affect bone remodeling in the lower jaw of male medaka because of a disruption in osteoclast function. These results suggest that reproductive impairments in pairs of medaka exposed to diclofenac may be reversible but that skeletal deformities (i.e., mandibular defect) in males may be persistent. Environ Toxicol Chem 2017;36:3277-3283.

Chronic exposure to diclofenac induces delayed mandibular defects in medaka (Oryzias latipes) in a sex-dependent manner

Diclofenac is widely distributed in freshwater environments. To support a robust aquatic risk assessment, medaka (Oryzias latipes) were exposed to diclofenac at sublethal concentrations of 0.608, 2.15, 7.29, 26.5, and 94.8 μg/L (as mean measured concentrations) from fertilized eggs to 90-day posthatch. Except for the induction of mandibular defects, no deleterious effects were observed on hatching success and time to hatching at the embryonic stage, or on posthatch mortality, growth in hatched larvae and juveniles, and no abnormal behavior was observed. After 40-day posthatch, mandibular defects in the fish were observed at a concentration of 7.29 μg/L and above. Cumulatively, a morphological examination showed that 4% of the fish in the 7.29 μg/L treatment, 20% in the 26.5 μg/L treatment, and 38% in the 94.8 μg/L treatment exhibited mandibular defects, and the sex ratio of fish with mandibular defects was skewed toward males. These results suggest that diclofenac affects bone remodeling in the lower jaw of medaka after puberty in a sex-dependent manner. The lowest observed-effect concentration and no observed-effect concentration of diclofenac for mandibular dysmorphism through the partial life cycle exposure of the medaka were 26.5 and 7.29 μg/L, respectively.