Elucidating Key Characteristics of PFAS Binding to Human Peroxisome Proliferator-Activated Receptor Alpha: An Explainable Machine Learning Approach.

Per- and polyfluoroalkyl substances (PFAS) are widely employed anthropogenic fluorinated chemicals known to disrupt hepatic lipid metabolism by binding to human peroxisome proliferator-activated receptor alpha (PPARα). Therefore, screening for PFAS that bind to PPARα is of critical importance. Machine learning approaches are promising techniques for rapid screening of PFAS. However, traditional machine learning approaches lack interpretability, posing challenges in investigating the relationship between molecular descriptors and PPARα binding. In this study, we aimed to develop a novel, explainable machine learning approach to rapidly screen for PFAS that bind to PPARα. We calculated the PPARα-PFAS binding score and 206 molecular descriptors for PFAS. Through systematic and objective selection of important molecular descriptors, we developed a machine learning model with good predictive performance using only three descriptors. The molecular size (b_single) and electrostatic properties (BCUT_PEOE_3 and PEOE_VSA_PPOS) are important for PPARα-PFAS binding. Alternative PFAS are considered safer than their legacy predecessors. However, we found that alternative PFAS with many carbon atoms and ether groups exhibited a higher affinity for PPARα. Therefore, confirming the toxicity of these alternative PFAS compounds with such characteristics through biological experiments is important.

Molecular Insights into Structural and Ligand Binding Features of Methoprene-Tolerant in Daphnids.

Juvenile hormone (JH) is an important endocrine factor regulating many biological activities in arthropods. In daphnids, methoprene-tolerant (Met) belongs to a basic helix-loop-helix/Per-Arnt-Sim (bHLH/PAS) family protein which has recently been confirmed as a JH receptor and can bind and be activated by JHs and JH agonists. Although the activation of the JH signaling pathway causes many physiological effects, the molecular basis for the structural feature and ligand binding properties of Daphnia Met are not fully understood. To study the ligand preference in terms of structural features of Daphnia Met, we built in silico homology models of the PAS-B domain of Daphnia Mets from cladoceran crustaceans, Daphnia pulex and D. magna. Structural comparison of two Daphnia Met PAS-B domain models revealed that the volume in the main cavity of D. magna Met was larger than that of D. pulex Met. Compared with insect Met, Daphnia Met had a less hydrophobic cavity due to polar residues in the core-binding site. Molecular docking simulations of JH and its analogs with Daphnia Met indicated that the interaction energies were correlated with each of the experimental values of in vivo JH activities based on male induction and in vitro Met-mediated transactivation potencies. Furthermore, in silico site-directed mutagenesis supported experimental findings that Thr292 in D. pulex Met and Thr296 in D. magna Met substitution to valine contribute to JH selectivity and differential species response. This study demonstrates that in silico simulations of Daphnia Met and its ligands may be a tool for predicting the ligand profile and cross species sensitivity.

Choriogenin transcription in medaka embryos and larvae as an alternative model for screening estrogenic endocrine-disrupting chemicals.

This study assessed the transcription levels of estrogen-responsive genes, such as vitellogenins (Vtg1 and Vtg2), choriogenins (ChgL, ChgH, and ChgHm), cytochrome P450 aromatase (CYP19a1b), and ER subtypes (ERα, ERß1, and ERß2), in 7 days-post-fertilization (dpf) embryos and 9 and 12 dpf larvae of medaka (Oryzias latipes) exposed to estrogenic endocrine-disrupting chemicals (EDCs). The <5 h-post-fertilization embryos were exposed to EDCs such as 17ß-estradiol (E2), p-n-nonylphenol (NP), and bisphenol A (BPA). In E2 (0.10-222 nM)-treated 7 dpf embryos and 9 or 12 dpf larvae, ChgL, ChgH, and ChgHm expression was up-regulated in a concentration-dependent manner. By contrast, interestingly, Vtg1 and Vtg2 expression was not induced in E2-treated 7 dpf embryos but was significantly induced in 9 and 12 dpf larvae, suggesting a developmental-stage-specific regulatory mechanism underlying Vtg expression. The maximum concentrations of NP (0.09-1.5 µM) and BPA (1.8-30 µM) up-regulated Chg expression in 9 or 12 dpf larvae, and the relative estrogenic potencies (REPs) of E2, NP, and BPA were 1, 2.1 × 10-4, and 1.0 × 10-5, respectively. Chg messenger RNA (mRNA) in medaka embryos and larvae can be used as a sensitive biomarker for screening potential estrogenic EDCs. Our assay system using embryos and larvae can be used as an in vivo alternative model because independent feeding stages (e.g., embryonic and early larval stages) are suitable alternatives.

[Analysis and Health Risk Assessment of Cadmium and Arsenic in Japanese, Vietnamese, and Indonesian Rice].

Herein, we quantified the concentrations of cadmium (Cd) and arsenic (As) in 63 milled rice (Oryza sativa L.) cultivated in Japan, Vietnam, and Indonesia. We estimated the daily intake of Cd and As by adults and children consuming this rice by inductively coupled plasma-mass spectrometer (ICP-MS). Cd and As were detected in all milled rice samples. No significant differences were observed in Cd concentrations between Japanese (50th percentile concentration: 0.036 mg/kg), Vietnamese (0.035 mg/kg), and Indonesian rice (0.022 mg/kg). However, As concentrations in Vietnamese rice (50th percentile concentration: 0.142 mg/kg) were significantly higher than those in Japanese (0.101 mg/kg, p<0.001) and Indonesian rice (0.038 mg/kg, p<0.0001). Target hazard quotients (THQs) were then calculated to evaluate the non-carcinogenic health risk from ingestion of individual heavy metals (Cd and As) by rice consumption. Results revealed that THQs of individual heavy metals for Japanese, Vietnamese, and Indonesian adults and children consuming this rice were all less than one, suggesting that no health risk is associated with the intake of a single heavy metal via rice consumption.

In Vitro and In Silico Evaluations of Binding Affinities of Perfluoroalkyl Substances to Baikal Seal and Human Peroxisome Proliferator-Activated Receptor α.

In this study, we assessed the binding affinities of perfluoroalkyl substances (PFASs), including perfluoroalkyl carboxylates (PFCAs) and perfluoroalkyl sulfonates (PFSAs), to the ligand-binding domains (LBDs) of Baikal seal ( Pusa sibirica; bs) and human (h) peroxisome proliferator-activated receptor alpha (PPARα). An in vitro competitive binding assay showed that six PFCAs and two PFSAs could bind to recombinant bs and hPPARα LBD proteins in a dose-dependent manner. The relative binding affinities (RBAs) of PFASs to bsPPARα were as follows: PFOS > PFDA > PFNA > PFUnDA > PFOA > PFHxS > PFHpA > PFHxA. The RBAs to bsPPARα showed a significant positive correlation with those to hPPARα. In silico PPARα homology modeling predicted that there were two ligand-binding pockets (LBPs) in the bsPPARα and hPPARα LBDs. Structure-activity relationship analyses suggested that the binding potencies of PFASs to PPARα might depend on LBP binding cavity volume, hydrogen bond interactions, the number of perfluorinated carbons, and the hydrophobicity of PFASs. Interspecies comparison of the in vitro binding affinities revealed that bsPPARα had higher preference for PFASs with long carbon chains than hPPARα. The in silico docking simulations suggested that the first LBP of bsPPARα had higher affinities than that of hPPARα; however, the second LBP of bsPPARα had lower affinities than that of hPPARα. To our knowledge, this is the first evidence showing interspecies differences in the binding of PFASs to PPARαs and their structure-activity relationships.

In Vitro Assessment of Activation of Baikal Seal ( Pusa sibirica) Peroxisome Proliferator-Activated Receptor α by Polybrominated Diphenyl Ethers.

We investigated the Baikal seal ( Pusa sibirica) peroxisome proliferator-activated receptor α (bsPPARα) transactivation potencies of polybrominated diphenyl ethers (PBDEs) using an in vitro bsPPARα reporter gene assay. BDE47, BDE99, and BDE153 induced bsPPARα-mediated transcriptional activities in a dose-dependent manner. To compare bsPPARα transactivation potencies of PBDEs, perfluorooctanoic acid (PFOA)-based relative potencies (REPs), a ratio of 50% effective concentration of PFOA to the test chemical, were determined. The order of REPs of PBDEs was BDE153 (13) > BDE99 (8.1) > BDE47 (6.6) > PFOA (1.0) > BDE100, BDE154, and BDE183 (not activated). PBDEs with two bromine atoms at the ortho position showed higher bsPPARα transactivation potencies than those with three bromine atoms. Comparison of the lowest-observed-effect concentration in bsPPARα reporter gene assays revealed that BDE99 was 7-fold more potent than CB99, a polychlorinated biphenyl congener with the same IUPAC number, indicating that brominated congeners could more efficiently activate bsPPARα than chlorinated congeners. The REPs of PBDEs for bsPPARα transactivation were approximately 7- to 13-fold higher than those of perfluorochemicals (PFCs), suggesting that the effects of PBDEs on the bsPPARα signaling pathway may be superior to those of PFCs. This study provides the first evidence that PBDE congeners activate PPARα in vitro.

Nanosecond pulsed electric field incorporation technique to predict molecular mechanisms of teratogenicity and developmental toxicity of estradiol-17ß on medaka embryos.

Herein, we propose using a nanosecond pulsed electric field (nsPEF) technique to assess teratogenicity and embryonic developmental toxicity of estradiol-17ß (E2 ) and predict the molecular mechanisms of teratogenicity and embryonic developmental defects caused by E2 on medaka (Oryzias latipes). The 5 hour post-fertilization embryos were exposed to co-treatment with 10 µm E2 and nsPEF for 2 hours and then continuously cultured under non-E2 and nsPEF conditions until hatching. Results documented that the time to hatching of embryos was significantly delayed in comparison to the control group and that typical abnormal embryo development, such as the delay of blood vessel formation, was observed. For DNA microarray analysis, 6 day post-fertilization embryos that had been continuously cultured under the non-E2 and nsPEF condition after 2 hour co-treatments were used. DNA microarray analysis identified 542 upregulated genes and one downregulated gene in the 6 day post-fertilization embryos. Furthermore, bioinformatic analyses using differentially expressed genes revealed that E2 exposure affected various gene ontology terms, such as response to hormone stimulus. The network analysis also documented that the estrogen receptor α in the mitogen-activated protein kinase signaling pathway may be involved in regulating several transcription factors, such as FOX, AKT1 and epidermal growth factor receptor. These results suggest that our nsPEF technique is a powerful tool for assessing teratogenicity and embryonic developmental toxicity of E2 and predict their molecular mechanisms in medaka embryos.

Neofunctionalization of Androgen Receptor by Gain-of-Function Mutations in Teleost Fish Lineage.

Steroid hormone receptor family provides an example of evolution of diverse transcription factors through whole-genome duplication (WGD). However, little is known about how their functions have been evolved after the duplication. Teleosts present a good model to investigate an accurate evolutionary history of protein function after WGD, because a teleost-specific WGD (TSGD) resulted in a variety of duplicated genes in modern fishes. This study focused on the evolution of androgen receptor (AR) gene, as two distinct paralogs, ARα and ARß, have evolved in teleost lineage after TSGD. ARα showed a unique intracellular localization with a higher transactivation response than that of ARß. Using site-directed mutagenesis and computational prediction of protein-ligand interactions, we identified two key substitutions generating a new functionality of euteleost ARα. The substitution in the hinge region contributes to the unique intracellular localization of ARα. The substitution on helices 10/11 in the ligand-binding domain possibly modulates hydrogen bonds that stabilize the receptor-ligand complex leading to the higher transactivation response of ARα. These substitutions were conserved in Acanthomorpha (spiny-rayed fish) ARαs, but not in an earlier branching lineage among teleosts, Japanese eel. Insertion of these substitutions into ARs from Japanese eel recapitulates the evolutionary novelty of euteleost ARα. These findings together indicate that the substitutions generating a new functionality of teleost ARα were fixed in teleost genome after the divergence of the Elopomorpha lineage. Our findings provide a molecular explanation for an adaptation process leading to generation of the hyperactive AR subtype after TSGD.

Gene expression analyses of vitellogenin, choriogenin and estrogen receptor subtypes in the livers of male medaka (Oryzias latipes) exposed to equine estrogens.

In the present study, we investigated transcriptional profiles of estrogen-responsive genes, such as vitellogenins (Vtg1 and Vtg2), choriogenins (ChgL and ChgH) and estrogen receptor subtypes (ERα, ERß1, and ERß2), in the liver of male medaka fish (Oryzias latipes) that were exposed to six equine estrogens (1-300 ng l(-1) ) for 3 days. Our quantitative reverse transcription-polymerase chain reaction (RT-PCR) analyses revealed that the expression levels of hepatic Vtg, Chg and ERα genes in male medaka responded to various types and concentrations of equine estrogens. The estrogenic potentials of the tested chemicals were in the order of equilin > 17ß-estradiol > equilenin > 17ß-dihydroequilin > 17ß-dihydroequilenin > 17α-dihydroequilin > 17α-dihydroequilenin, showing the higher estrogenic potential of equilin than that of 17ß-estradiol. Our results also showed that the estrogenicities of 17ß-dihydroequilin and 17ß-dihydroequilenin were more potent than that of 17α-dihydroequilin and 17α-dihydroequilenin. Furthermore, in gene expression analyses of hepatic ER subtypes, observations were made to note that 17ß-estradiol and equilin induced ERα transcription in male medaka, and the ERα transcription level had significantly positive correlations with the expression of Vtg and Chg genes. In contrast, in the same 17ß-estradiol and equilin treatment groups, it was shown that the transcription levels of hepatic ERß1 and/or ERß2 had significantly negative correlations with the expression of Vtg and Chg genes. These results suggested some potential involvement of the ER subtypes in the regulation of Vtg and Chg gene expressions in the liver. This is the first report describing the comprehensive analyses of in vivo estrogenicity of the equine estrogens in male medaka. Copyright © 2016 John Wiley & Sons, Ltd.

Transcriptional response of mysid crustacean, Americamysis bahia, is affected by subchronic exposure to nonylphenol.

Nonylphenol (NP) has been classified as an endocrine-disrupting chemical. In this study, we conducted mysid DNA microarray analysis with which has 2240 oligo DNA probes to observe differential gene expressions in mysid crustacean (Americamysis bahia) exposed to 1, 3, 10 and 30 µg/l of NP for 14 days. As a result, we found 31, 27, 39 and 68 genes were differentially expressed in the respective concentrations. Among these genes, the expressions of five particular genes were regulated in a similar manner at all concentrations of the NP exposure. So, we focused on one gene encoding cuticle protein, and another encoding cuticular protein analogous to peritrophins 1-H precursor. These genes were down-regulated by NP exposure in a dose-dependent manner, and it suggested that they were related in a reduction of the number of molting in mysids. Thus, they might become useful molecular biomarker candidates to evaluate molting inhibition in mysids.

Synergistic antioxidative effect of astaxanthin and tocotrienol by co-encapsulated in liposomes.

Astaxanthin and vitamin E are both effective antioxidants that are frequently used in cosmetics, as food additives, and in to prevent oxidative damage. A combination of astaxanthin and vitamin E would be expected to show an additive anntioxidative effect. In this study, liposomes co-encapsulating astaxanthin and the vitamin E derivatives α-tocopherol (α-T) or tocotrienols (T3) were prepared, and the antioxidative activity of these liposomes toward singlet oxygen and hydroxyl radical was evaluated in vitro. Liposomes co-encapsulating astaxanthin and α-T showed no additive anntioxidative effect, while the actual scavenging activity of liposomes co-encapsulating astaxanthin and T3 was higher than the calculated additive activity. To clarify why this synergistic effect occurs, the most stable structure of astaxanthin in the presence of α-T or α-T3 was calculated. Only α-T3 was predicted to form hydrogen bonding with astaxanthin, and the astaxanthin polyene chain would partially interact with the α-T3 triene chain, which could explain why there was a synergistic effect between astaxanthin and T3 but not α-T. In conclusion, co-encapsulation of astaxanthin and T3 induces synergistic scavenging activity by intermolecular interactions between the two antioxidants.

Understanding the molecular basis for differences in responses of fish estrogen receptor subtypes to environmental estrogens.

Exposure to endocrine disrupting chemicals (EDCs) can elicit adverse effects on development, sexual differentiation, and reproduction in fish. Teleost species exhibit at least three subtypes of estrogen receptor (ESR), ESR1, ESR2a, and ESR2b; thus, estrogenic signaling pathways are complex. We applied in vitro reporter gene assays for ESRs in five fish species to investigate the ESR subtype-specificity for better understanding the signaling pathway of estrogenic EDCs. Responses to bisphenol A, 4-nonylphenol, and o,p'-DDT varied among ESR subtypes, and the response pattern of ESRs was basically common among the different fish species. Using a computational in silico docking model and through assays quantifying transactivation of the LBD (using GAL-LBD fusion proteins and chimera proteins for the ESR2s), we found that the LBD of the different ESR subtypes generally plays a key role in conferring responsiveness of the ESR subtypes to EDCs. These results also indicate that responses of ESR2s to EDCs cannot necessarily be predicted from the LBD sequence alone, and an additional region is required for full transactivation of these receptors. Our data thus provide advancing understanding on receptor functioning for both basic and applied research.

Endocrine-disrupting potentials of equine estrogens equilin, equilenin, and their metabolites, in the medaka Oryzias latipes: in silico and DNA microarray studies.

Although several previous studies have demonstrated the presence of equine estrogens in the aquatic environment, limited data are currently available on the endocrine-disrupting potentials in fish and the risks they pose to aquatic organisms. To investigate the interactions of major equine estrogens equilin (Eq) and equilenin (Eqn), as well as their metabolites 17α-dihydroequilin, 17ß-dihydroequilin, 17α-dihydroequilenin and 17ß-dihydroequilenin, with the estrogen receptor α (ERα) of medaka (Oryzias latipes), a three-dimensional model of the ligand-binding domain (LBD) of ERα was built in silico, and docking simulations were performed. The docking simulation analysis indicated that the interaction of 17ß-dihydroequilenin with the ERα LBD is the most potent, followed by those of 17α-dihydroequilin and 17ß-dihydroequilin, whereas those of Eq and Eqn were least potent. We further analyzed gene expression profiles in the livers of male medaka exposed to Eq and Eqn. A DNA microarray representing 6000 genes revealed that 24-h exposure to Eq and Eqn (100 ng/L) upregulated the expression of 6 and 34 genes in the livers of males, respectively. Genes upregulated by Eq included the estrogenic biomarker genes vitellogenins and choriogenins, suggesting the estrogenic potential of Eq. In contrast, Eqn exposure upregulated several cancer-related genes, such as mediator complex subunit 16 and RAS oncogene family members, suggesting a carcinogenic potential for Eqn. These results suggest that equine estrogens may have not only endocrine-disrupting potentials via the ERα signaling pathway but also carcinogenic potency in male medaka.

Effects of lithium on growth, maturation, reproduction and gene expression in the nematode Caenorhabditis elegans.

Lithium (Li) has been widely used to treat bipolar disorder, and industrial use of Li has been increasing; thus, environmental pollution and ecological impacts of Li have become a concern. This study was conducted to clarify the potential biological effects of LiCl and Li(2)CO(3) on a nematode, Caenorhabditis elegans as a model system for evaluating soil contaminated with Li. Exposure of C. elegans to LiCl and Li(2)CO(3) decreased growth/maturation and reproduction. The lowest observed effect concentrations for growth, maturation and reproduction were 1250, 313 and 10 000 µm, respectively, for LiCl and 750, 750 and 3000 µm, respectively, for Li(2)CO(3). We also investigated the physiological function of LiCl and LiCO(3) in C. elegans using DNA microarray analysis as an eco-toxicogenomic approach. Among approximately 300 unique genes, including metabolic genes, the exposure to 78 µm LiCl downregulated the expression of 36 cytochrome P450, 16 ABC transporter, 10 glutathione S-transferase, 16 lipid metabolism and two vitellogenin genes. On the other hand, exposure to 375 µm Li(2)CO(3) downregulated the expression of 11 cytochrome P450, 13 ABC transporter, 13 lipid metabolism and one vitellogenin genes. No gene was upregulated by LiCl or Li(2)CO(3). These results suggest that LiCl and Li(2)CO(3) potentially affect the biological and physiological function in C. elegans associated with alteration of the gene expression such as metabolic genes. Our data also provide experimental support for the utility of toxicogenomics by integrating gene expression profiling into a toxicological study of an environmentally important organism such as C. elegans.

In vivo and in silico analyses of estrogenic potential of bisphenol analogs in medaka (Oryzias latipes) and common carp (Cyprinus carpio).

Various studies have demonstrated the estrogenic effect of bisphenol A (BPA), a member of bisphenol analogs (BPs), in in vitro and in vivo assays. However, limited data are available on the estrogenic potentials and risks of other BPs in aquatic organisms. In addition, the estrogenic effect of chemicals is known to have species-specific responses in teleost fish. The objective of this study was to evaluate the potential estrogenic effects of BPs on the medaka (Oryzias latipes) and common carp (Cyprinus carpio) using in vivo and in silico assays. Our quantitative real-time PCR analyses revealed that the expression levels of several hepatic estrogen-responsive biomarker genes in male medaka responded to various types and concentrations of BPs in a dose-response manner. The order of in vivo estrogenic potencies of BPs was as follows: BPC≈BPAF>BPB>BPA⋙BPP. To further investigate the interaction potential of BPs with medaka estrogen receptor α (ERα) in silico, a three-dimensional model of the ERα ligand-binding domain (LBD) was built and docking simulations were performed. The docking simulation analysis revealed that BPC interaction potential for medaka ERα LBD was the most potent, followed by BPAF and BPA. Comparing this with carp ERα LBD revealed that the interaction potentials of these BPs to medaka ERα LBD were more stable than to carp ERα LBD. Furthermore, we identified key amino acid residues in medaka ERα LBD that interacted with BPC (Glu356, Arg397, and Cys533), BPAF (Thr350 and Glu356), and BPA (Glu356 and Met424), and found some differences in these key amino acid residues between medaka and carp ERα LBDs. These results of in vivo and in silico analyses showed potential estrogenic effects of BPs in teleost fish, and they also indicated that the differences in interaction potentials and key amino acid residues between medaka and carp ERα LBDs may be due to the differences between the species and estrogenic potencies of the selected BPs.

Transcriptome analysis reveals limited toxic effects of the UV-filter benzophenone-3 (BP-3) on the hermatypic coral Acropora tenuis and its symbiotic dinoflagellates.

This study aimed to investigate the toxic and transcriptomic effects of the ultraviolet filter benzophenone-3 (BP-3) on Acropora tenuis and its symbiotic dinoflagellates while using acetone as a solvent. Seven-day exposure to 50 and 500 µg/L, which is higher than most BP-3 records from coastal waters, did not affect coral colour or dinoflagellate photosynthesis. Differentially expressed genes (DEGs) between seawater and solvent controls were <20 in both corals and dinoflagellates. Eleven coral DEGs were detected after treatment with 50 µg/L BP-3. Fourteen coral DEGs, including several fluorescent protein genes, were detected after treatment with 500 µg/L BP-3. In contrast, no dinoflagellate DEGs were detected in the BP-3 treatment group. These results suggest that the effects of 50-500 µg/L BP-3 on adult A. tenuis and its dinoflagellates are limited. Our experimental methods with lower acetone toxicity provide a basis for establishing standard ecotoxicity tests for corals.

Analysis of predictors of fever after aortic valve replacement: Diabetic patients are less likely to develop fever after aortic valve replacement, a single-centre retrospective study.

BACKGROUND: Postoperative temperature dysregulation affects the length of hospital stay and prognosis. This study evaluated the factors that influence the occurrence of fever in patients after aortic valve replacement surgery. METHODS: Eighty-seven consecutive patients who underwent aortic valve replacement surgery were included. Patients' age, sex and body mass index; presence of diabetes mellitus; operation time; blood loss; blood transfusion volume; preoperative and postoperative laboratory findings; presence or absence of oral function management; and fever >38°C were retrospectively analysed through univariate and multiple logistic regression analyses. RESULTS: Among the variables, only diabetes mellitus status was significantly associated with fever ⩾38°C. Postoperatively, patients with diabetes mellitus were significantly less likely to develop fever above 38°C and a fever rising to 38°C. CONCLUSIONS: This study shows that the presence of comorbid diabetes mellitus decreases the frequency of developing fever >38°C after aortic valve replacement surgery.

Expression analyses of stress-responsive genes in the hermatypic coral Acropora tenuis and its symbiotic dinoflagellates after exposure to the herbicide Diuron.

Diuron is one of the most frequently applied herbicides in sugarcane farming in southern Japan, and Australia. In addition, it is used as a booster substance in copper-based antifouling paints. Due to these various uses, Diuron is released into the marine environment; however, little information is available on gene expression in corals and their symbiotic algae exposed to Diuron. We investigated the effects of Diuron on stress-responsive gene expression in the hermatypic coral Acropora tenuis and its symbiotic dinoflagellates. After seven days of exposure to 1 µg/L and 10 µg/L Diuron, no significant changes in the body colour of corals were observed. However, quantitative reverse transcription-polymerase chain reaction analyses revealed that the expression levels of stress-responsive genes, such as heat shock protein 90 (HSP90), HSP70, and calreticulin (CALR), were significantly downregulated in corals exposed to 10 µg/L of Diuron for seven days. Moreover, aquaglyceroporin was significantly downregulated in corals exposed to environmentally relevant concentrations of 1 µg/L Diuron. In contrast, no such effects were observed on the expression levels of other stress-responsive genes, such as oxidative stress-responsive proteins, methionine adenosyltransferase, and green/red fluorescent proteins. Diuron exposure had no significant effect on the expression levels of HSP90, HSP70, or HSP40 in the symbiotic dinoflagellates. These results suggest that stress-responsive genes, such as HSPs, respond differently to Diuron in corals and their symbiotic dinoflagellates and that A. tenuis HSPs and CALRs may be useful molecular biomarkers for predicting stress responses induced by the herbicide Diuron. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-023-00183-0.

Adverse effects of contamination by fipronil and its derivatives on growth, molting, and gene expression in the mysid crustacean, Americamysis bahia, in Japanese estuaries.

Concentrations of fipronil (Fip) and several of its derivatives were detected in samples from four rivers and four estuaries in Japan. LC-MS/MS analysis detected Fip and its derivatives, except for fipronil detrifluoromethylsulfinyl, in almost all samples. The total concentrations of the five compounds were approximately two-fold greater in river water (mean: 21.2, 14.1, and 9.95 ng/L in June, July, and September, respectively) compared to those in estuarine water (mean: 10.3, 8.67, and 6.71 ng/L, respectively). Fipronil, fipronil sulfone (Fip-S), and fipronil sulfide (Fip-Sf) represented more than 70 % of all compounds. This is the first report to demonstrate the contamination of estuarine waters of Japan by these compounds. We further investigated the potentially toxic effects of Fip, Fip-S, and Fip-Sf on the exotic mysid, Americamysis bahia (Crustacea: Mysidae). The lowest effective concentrations of Fip-S (10.9 ng/L) and Fip-Sf (19.2 ng/L) on mysid growth and molting was approximately 12.9- and 7.3-fold lower than Fip (140.3 ng/L), suggesting they had higher toxicity. Quantitative reverse transcription polymerase chain reaction analysis revealed that ecdysone receptor and ultraspiracle gene expression were not affected after 96-h of exposure to Fip, Fip-S, and Fip-Sf, suggesting that these genes may not be involved in the molting disruption induced by Fip, Fip-S, and Fip-Sf. Our findings suggest that environmentally relevant concentrations of Fip and its derivatives can disrupt the growth of A. bahia by promoting molting. However, further studies are required to elucidate its molecular mechanism.

Efficient in vivo and in silico assessments of antiandrogenic potential in zebrafish.

This study aimed to establish zebrafish-based in vivo and in silico assay systems to evaluate the antiandrogenic potential of environmental chemicals. Zebrafish embryos were exposed to 17α-methyltestosterone (TES) alone or coexposed to TES and representative antiandrogens including flutamide, p,p'-DDE, vinclozolin, fenitrothion, and linuron. We assessed the transcript expression of the androgen-responsive gene sulfotransferase family 2, cytosolic sulfotransferase 3 (sult2st3). The expression of sult2st3 was significantly induced by TES in the later stages of embryonic development. However, the TES-induced expression of sult2st3 was inhibited by flutamide in a concentration-dependent manner (IC50: 5.7 µM), suggesting that the androgen receptor (AR) plays a role in sult2st3 induction. Similarly, p,p'-DDE, vinclozolin, and linuron repressed the TES-induced expression of sult2st3 (IC50s: 0.35, 3.9, and 52 µM, respectively). At the highest concentration tested (100 µM), fenitrothion also suppressed sult2st3 expression almost completely. Notably, p,p'-DDE and linuron did not inhibit sult2st3 induction due to higher concentrations of TES; instead, they potentiated TES-induced sult2st3 expression. Fenitrothion and linuron, which had relatively low antiandrogenic potentials in terms of sult2st3 inhibition, induced broader toxicities in zebrafish embryos; thus, the relationship between developmental toxicities and antiandrogenic potency was unclear. Additionally, an in silico docking simulation showed that all five chemicals interact with the zebrafish AR at relatively low interaction energies and with Arg702 as a key amino acid in ligand binding. Our findings suggest that a combination of zebrafish-based in vivo and in silico assessments represents a promising tool to assess the antiandrogenic potentials of environmental chemicals.