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.

In silico simulations and molecular descriptors to predict in vitro transactivation potencies of Baikal seal estrogen receptors by environmental contaminants.

Baikal seals (Pusa sibirica) are vulnerable to high levels of organic pollutants. Here, we evaluated the transactivation potencies of bisphenols (BPs) and hydroxylated polychlorinated biphenyls (OH-PCBs) via the Baikal seal estrogen receptor α and ß (bsERα and bsERß) using in vitro and in silico approaches. In vitro reporter gene assays showed that most BPs and OH-PCBs exhibited estrogenic activity with bsER sub-type-specific potency. Among the BPs tested, bisphenol AF showed the lowest EC50 for both bsERs. 4'-OH-CB50 and 4'-OH-CB30 showed the lowest EC50 among OH-PCBs tested for bsERα and bsERß, respectively. 4-((4-Isopropoxyphenyl)-sulfonyl)phenol, 4'-OH-CB72, and 4'-OH-CB121 showed weak bsERα-specific transactivation. Only 4-OH-CB107 did not affect both bsERs. In silico docking simulations revealed the binding affinities of these chemicals to bsERs and partially explained the in vitro results. Using the in silico simulations and molecular descriptors as explanatory variables and the in vitro results as objective variables, the quantitative structure-activity relationship (QSAR) models constructed for classification and regression accurately separated bsER-active compounds from non-active compounds and predicted the in vitro bsERα- and bsERß-transactivation potencies, respectively. The QSAR models also suggested that chemical polarity, van der Waals surface area, bridging atom structure, position of the phenolic-OH group, and ligand interactions with key residues of the ligand binding pocket are critical variables to account for the bsER transactivation potency of the test compounds. We also succeeded in constructing computational models for predicting in vitro transactivation potencies of mouse ERs in the same manner, demonstrating the applicability of our approach independent of species-specific responses.

Olfactory Bulbs in Arthritis Model Mouse Persistently Express Interleukin-6 before the Onset of Arthritis: Relationship to Food Intake.

INTRODUCTION: Rheumatoid arthritis (RA) can be comorbid with psychiatric symptoms. Brain abnormalities in RA patients and in arthritis models have been reported. However, it remains unclear when these abnormalities occur and where they are distributed. In this study, we analyzed spatiotemporal changes in gene expression in the brains of mice with collagen-induced arthritis (CIA). METHODS: Mice were divided into three groups: (i) CIA (all mice developed arthritis on day 35): complete Freund's adjuvant (CFA) and type II collagen at initial immunization, and incomplete Freund's adjuvant (IFA) and type II collagen at booster immunization; (ii) C(+/-) (50% mice developed arthritis on day 35): only IFA at booster immunization; and (iii) C(-/-) (no arthritis): only CFA at initial immunization and only IFA at booster immunization. Whole brains were collected at ten stages of arthritis and divided into six sections. Real-time polymerase chain reaction was performed using RNA extracted from the brain, and the expression of proinflammatory cytokines and glial markers was semi-quantified. Arthritis score, body weight, and food and water intakes were recorded and analyzed for correlations with brain gene expression. We also investigated the effect of interleukin-6 (IL-6) injection in the olfactory bulbs (OBs) on the food intake. RESULTS: After booster immunization, a transient increase in Integrin subunit α-M and IL-1ß was observed in multiple areas in CIA. IL-6 is persistently expressed in the OB before the onset of arthritis, which is correlated with body weight loss and decreased food intake. This change in the OB was observed in the C(+/-) but not in the C(-/-) groups. In the C(+/-) group, non-arthritic mice showed the same changes in the OB as the arthritic mice. This elevation in IL-6 levels persisted throughout the chronic phase until day 84. In addition, IL-6 injection into the OB reduced food intake. CONCLUSION: Persistent elevation of IL-6 in the OB from the early stage of arthritis may be an important finding that might explain the neuropsychiatric pathophysiology of RA, including appetite loss, which is present in the early stages of the disease and manifests as a variety of symptoms over time.

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.

In vivo and in silico assessments of estrogenic potencies of bisphenol A and its analogs in zebrafish (Danio rerio): Validity of in silico approaches to predict in vivo effects.

This study assessed the estrogen-like potencies of bisphenol A (BPA) and its analogs (BPs) using in vivo and in silico approaches in zebrafish. Zebrafish embryos were exposed to 16 BPs, most of which concentration-dependently induced cytochrome P450 19A1b (CYP19A1b) expression. BPs-induced CYP19A1b expression was suppressed by fulvestrant, a nonselective high affinity antagonist for estrogen receptor (Esr) subtypes. For BPs that concentration-dependently induced CYP19A1b expression, we estimated their 50 % effective concentration (EC50) and relative potencies (REPs) with respect to the potency of BPA for inducing CYP19A1b expression. BP C2, Bis-MP, and BPAF showed lower EC50 than BPA, BPE, and BPF, while BPZ and BPB showed moderate EC50. The REP order of the BPs was BP C2 (26) > Bis-MP (24) > BPAF (21) > BPZ (5.8) > BPB (2.7) > BPE (1.5) > BPF (0.63) > 2,4'-BPF (0.22), indicating that some BPs showed greater estrogenic potencies than BPA in our system. We also constructed in silico homology models of ligand binding domains for zebrafish Esr subtypes, including Esr1, Esr2a, and Esr2b. Molecular docking simulations of ligands with the Esr subtypes revealed the interaction energies of some BPs were lower than that of BPA. The interaction energies showed significant positive correlations with their EC50 values for inducing CYP19A1b expression in vivo. This study showed that some BPA analogs have greater estrogenic potencies than BPA and that in silico simulations of interactions between ligands and Esr subtypes may help predict in vivo estrogenic potencies of untested chemicals.

Developmental toxicity and transcriptome analysis of equine estrogens in developing medaka (Oryzias latipes) using nanosecond pulsed electric field incorporation.

Equine estrogens (EQs) are steroidal hormones isolated from the urine of pregnant mares and are used in the formulation of human medications. This study initially investigated the embryonic developmental toxicity of equilin (Eq) and equilenin (Eqn) in medaka (Oryzias latipes). Malformations were observed in embryos exposed to nominal concentrations of 1 and 10 mg/L of Eq and Eqn. Delayed hatching was observed at 1 mg/L of Eq. To further investigate the molecular mechanism of developmental toxicity caused by Eq and Eqn, transcriptome and bioinformatics analyses were performed. Among 2016 and 3855 total differentially expressed genes (DEGs), 1117 DEGs overlapped between Eq. (55.4 % of total DEGs) and Eq. (29.0 % of total DEGs). Gene ontology indicated effects in terms related to blood circulation and cell junctions. Pathway analyses using DEGs revealed that both Eq and Eqn treatments at 10 mg/L affected various KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways, such as neuroactive ligand-receptor interaction, mitogen-activated protein kinase signaling, retinol metabolism, and cytokine-cytokine receptor interaction. These results suggest that the disruption of these KEGG pathways is involved in the developmental toxicity of EQs in medaka embryos.

Inhibitory effect of baricitinib on microglia and STAT3 in a region with a weak blood-brain barrier in a mouse model of rheumatoid arthritis.

OBJECTIVES: In patients with RA, baricitinib not only improves arthritis symptom severity, but also patients' neuropsychological symptoms, such as depression and fatigue. However, the cellular mechanisms through which baricitinib can affect neural activity is unexplored. While the blood-brain barrier (BBB) permeability of this drug remains unclear, Janus kinase inhibitors (JAKi) might reach the area postrema, which is a unique brain region with a weak BBB function. Our recent study demonstrated microglial activation during experimental arthritis in the area postrema. Therefore, we sought to assess the effect of baricitinib on microglia in the area postrema using the CIA mouse model. METHODS: Microglia number and morphology in the area postrema were assessed by immunostaining for ionized calcium-binding adaptor molecule-1 (Iba-1). Data were collected on post-immunization day 35 (early phase) and 84 (late phase), and compared between baricitinib- and vehicle-treated mice. The effect on signal transducers and activators of transcription (STAT3) in the area postrema was also immunohistochemically examined. Behavioural outcomes were assessed by examining feeding behaviours and sucrose preference tests. RESULTS: In the early phase, activated microglial levels in the area postrema were decreased by baricitinib, accompanied by the inhibition of phosphorylated-STAT3 and recovery of food intake and sucrose preference. On the other hand, baricitinib did not affect microglial morphology in the late phase. CONCLUSION: Our results demonstrate that baricitinib can affect brain cells, specifically microglia, in the brain region with a weak BBB and mitigate aberrant behaviours during autoimmune arthritis, pointing to the potential therapeutic effect of JAKi on brain pathologies underpinning RA.

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.

Assessment of binding potencies of polychlorinated biphenyls and polybrominated diphenyl ethers with Baikal seal and mouse constitutive androstane receptors: Comparisons across species and congeners.

The present study evaluated the binding potencies (equilibrium dissociation constant: KD) of polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) with the constitutive androstane receptor (CAR)_ligand binding domain (LBD) of the Baikal seal (bsCAR_LBD) and mouse (mCAR_LBD) using a surface plasmon resonance (SPR) biosensor. The binding affinities of individual congeners with mCAR_LBD tended to be higher than those with bsCAR_LBD but the differences were within the same order of magnitude. Notably, PBDE congeners showed higher binding affinities for both CAR_LBDs than PCB congeners. In silico docking simulations demonstrated that PBDEs had more non-covalent interactions with specific amino acid residues in both CAR_LBDs than PCBs, supporting the results of their binding affinities. Binding affinity comparisons among congeners revealed the structural requirements for higher binding; mono or di ortho-, tri meta-, and di para­chlorine substitutions for PCBs, and di or tri ortho-, mono meta-, and di para­bromine substitutions for PBDEs. The binding potencies of these congeners unlikely accounted for their previously reported CAR-mediated transactivation potencies, implying that their transactivation is regulated in a ligand-dependent, but a distinct manner from ligand binding. Risk assessment analysis showed that the KD values of individual PCB and PBDE congeners were 1-4 orders of magnitude higher than their respective hepatic concentrations in wild Baikal seal population.

Juvenile hormone synthesis and signaling disruption triggering male offspring induction and population decline in cladocerans (water flea): Review and adverse outcome pathway development.

Juvenile hormone (JH) are a family of multifunctional hormones regulating larval development, molting, metamorphosis, reproduction, and phenotypic plasticity in arthropods. Based on its importance in arthropod life histories, many insect growth regulators (IGRs) mimicking JH have been designed to control harmful insects in agriculture and aquaculture. These JH analogs (JHAs) may also pose hazards to nontarget species by causing unexpected endocrine-disrupting (ED) effects such as molting and metamorphosis defects, larval lethality, and disruption of the sexual identity. This critical review summarizes the current knowledge of the JH-mediated effects in the freshwater cladoceran crustaceans such as Daphnia species on JHA-triggered endocrine disruptive outputs to establish a systematic understanding of JHA effects. Based on the current knowledge, adverse outcome pathways (AOPs) addressing the JHA-mediated ED effects in cladoceran leading to male offspring production and subsequent population decline were developed. The weight of evidence (WoE) of AOPs was assessed according to established guidelines. The review and AOP development aim to present the current scientific understanding of the JH pathway and provide a robust reference for the development of tiered testing strategies and new risk assessment approaches for JHAs in future ecotoxicological research and regulatory processes.

Directly Reprogrammed Neurons as a Tool to Assess Neurotoxicity of the Contaminant 4-Hydroxy-2',3,5,5'-tetrachlorobiphenyl (4'OH-CB72) in Melon-Headed Whales.

Whales accumulate high levels of environmental pollutants. Exposure to polychlorinated biphenyls (PCBs) and their metabolites (OH-PCBs) could be linked to abnormal behavior, which may lead to mass stranding of marine mammals. Whales may thus suffer from adverse effects such as neuronal dysfunction, yet testing the neurotoxicity of these compounds has never been feasible for these species. This study established neurons chemically reprogrammed from fibroblasts of mass stranded melon-headed whales (Peponocephala electra) and used them for in vitro neurotoxicity assays. Exposure to 4-hydroxy-2',3,5,5'-tetrachlorobiphenyl (4'OH-CB72), a metabolite of PCBs, caused apoptosis in the reprogrammed neurons. Transcriptome analysis of 4'OH-CB72-treated whale neurons showed altered expressions of genes associated with oxidative phosphorylation, chromatin degradation, axonal transport, and neurodegenerative diseases. These results suggest that 4'OH-CB72 exposure may induce neurodegeneration through disrupted apoptotic processes. A comparison of the results with human reprogrammed neurons revealed the specific effects on the whale neurons. Our noninvasive approach using fibroblast-derived neurons is useful for hazard and risk assessments of neurotoxicity in whales.

In vivo and in silico analyses of estrogenic potential of equine estrogens in medaka (Oryzias latipes).

Equine estrogens (EEs) are widely used in hormone replacement therapy pharmaceuticals for postmenopausal women. Previous studies have shown that EEs occur in the aquatic environment; however, the potential estrogenicity and risk of EEs in aquatic organisms, including fish, have yet to be studied in detail. Therefore, we evaluated the estrogenic potential of major EEs, namely equilin (Eq), 17α-dihydroequilin (17α-Eq), 17ß-dihydroequilin (17ß-Eq), equilenin (Eqn), 17α-dihydroequilenin (17α-Eqn), and 17ß-dihydroequilenin (17ß-Eqn), on medaka (Oryzias latipes) using in vivo and in silico assays. Quantitative real-time RT-PCR analyses revealed that expression levels of choriogenin L (ChgL) and choriogenin H (ChgH) in medaka embryos responded to various types and concentrations of EEs in a concentration-dependent manner, whereas transcription levels of vitellogenin 1 were not significantly affected by any of the EEs in the concentration range tested. The order of the in vivo estrogenic potencies of EEs was as follows: 17ß-Eq > Eq > 17ß-Eqn > Eqn > 17α-Eqn > 17α-Eq. Additionally, the 50% effective concentrations (EC50) of 17ß-Eq was lower than that of 17ß-estradiol. We also investigated the interaction potential of EEs with medaka estrogen receptor (ER) subtypes in silico using a three-dimensional model of the ligand-binding domain (LBD) for each ER and docking simulations. All six EEs were found to interact with the LBDs of ERα, ERß1, and ERß2. The order of the in silico interaction potentials of EEs with each ER LBD was as follows: 17ß-Eq > 17α-Eq > Eq > 17ß-Eqn > 17α-Eqn > Eqn. Furthermore, we identified the key amino acids that interact with EEs in each ER LBD; our findings suggest that amino acids and/or their hydrogen bonding may be responsible for the ligand-specific interactions with each ER. This study is the first to comprehensively analyze the estrogenic potential of EEs in medaka both in vivo and in silico.

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.

In Vitro Cytotoxicity and Risk Assessments of Environmental Pollutants Using Fibroblasts of a Stranded Finless Porpoise (Neophocaena asiaeorientalis).

Cetaceans accumulate high levels of environmental pollutants, yet their toxicological studies have been difficult due to technical and ethical issues. It is essential to identify and fill the current knowledge gaps in the in vitro assays available for cetaceans. The present study establishes a novel in vitro assay that uses the fibroblasts of a finless porpoise (Neophocaena asiaeorientalis) (FF) stranded in the Seto Inland Sea (SIS) to answer questions about the cytotoxicity and risks of environmental pollutants. FF were treated with 17 compounds including polychlorinated biphenyls (PCBs) and dichlorodiphenyltrichloroethane and their metabolites (DDTs) and evaluated for cytotoxicity, viability, and apoptosis. The results of FF were compared with those of human fibroblasts (HF). The relative potencies of the test compounds were comparable between the two species, as EC50 of these compounds significantly correlated for FF and HF. Exposure-activity ratios (EARs) revealed that accumulation of PCBs and DDTs are likely to pose adverse effects at the cellular level in the SIS finless porpoises, as their tissue concentrations exceeded EC50 values obtained in this study. This study successfully evaluated the risks of environmental pollutants using cetacean fibroblasts isolated by a non-invasive method that may be applied to various cetacean species and compounds.

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.

Potential mechanisms underlying embryonic developmental toxicity caused by benzo[a]pyrene in Japanese medaka (Oryzias latipes).

Polycyclic aromatic hydrocarbons (PAHs), such as benzo[a]pyrene (BaP), are widely distributed in air, water, and sediments; however, limited data are available regarding their potential adverse effects on the early life stages of fish. In this study, we evaluated the embryonic teratogenicity and developmental toxicity of BaP in Japanese medaka (Oryzias latipes) using a nanosecond pulsed electric field (nsPEF) technique and predicted their molecular mechanisms via transcriptome analysis. The gas chromatography/mass spectrometry analyses revealed that the BaP was efficiently incorporated into the embryos by nsPEF treatment. The embryos incorporating BaP presented typical teratogenic and developmental effects, such as cardiovascular abnormalities, developmental abnormalities, and curvature of backbone. DNA microarray analysis revealed several unique upregulated genes, such as those involved in cardiovascular diseases, various cellular processes, and neural development. Furthermore, the gene set enrichment and network analyses found several genes and hub proteins involved in the developmental effects of BaP on the embryos. These findings suggest a potential mechanism of teratogenicity and developmental toxicity caused by exposure to BaP. The nsPEF and transcriptome analyses in combination can be effective for evaluating the potential effects of chemical substances on medaka embryos.

In vitro assessment of effects of persistent organic pollutants on the transactivation of estrogen receptor α and ß (ERα and ERß) from the Baikal seal (Pusa sibirica).

To assess the effect of exposure to persistent organic pollutants (POPs) on the estrogen receptor (ER) signaling pathway in Baikal seals (Pusa sibirica), we investigated the molecular characterizations and functions of two Baikal seal ER (bsER) isoforms, bsERα and bsERß. The bsERα and bsERß cDNA clones isolated have an open reading frame of 595 and 530 amino acid residues, respectively. The tissue distribution analyses of bsER mRNAs showed that bsERα transcripts were primarily found in the ovary and uterus, and bsERß in the muscle in wild Baikal seals. The immunofluorescence staining assay showed that 17ß-estradiol (E2) treatment promoted the nuclear translocation of in vitro-expressed bsERα. Transient transfection of bsERα in U2OS cells enhanced the transcription of pS2, an ER target gene of E2. We then measured bsER-mediated transactivation potencies of POPs in an in vitro reporter gene assay system, in which a bsERα or bsERß expression vector was transfected into COS-1 cells. For comparison, transactivation potencies of POPs on mouse ERs (mERα and mERß) were also evaluated in the same manner. Results showed significant dose-dependent responses of bsERs and mERs when treated with p,p'-dichlorodiphenyltrichloroethane (p,p'-DDT), and p,p'-dichlorodiphenyldichloroethylene (p,p'-DDE). bsERs and mERs showed no response when exposed to polychlorinated biphenyls (PCBs) or 2,3,7,8-tetrachlorodibenzo-p-dioxin. Comparison of the dose-response curves of DDTs across species (bsERs vs. mERs) showed that bsERα had a response similar to mERα, but bsERß was less sensitive than mERß. Comparing the lowest observable effective concentrations of p,p'-DDT (2.8 µM) and p,p'-DDE (10 µM) for in vitro bsERα-mediated transactivation with their hepatic concentrations in wild Baikal seals indicated that some individuals accumulated these compounds at levels comparable to the effective concentrations, suggesting the potential disruption of the bsERα signaling pathway in the wild population by these compounds. Co-transfection experiments with bsER and the aryl hydrocarbon receptor (AHR) suggested that high accumulation of estrogenic compounds exerts a synergistic effect with dioxin-like congeners on ER signaling through AHR activation in the wild seal population.

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.

Accumulation properties of polychlorinated biphenyl congeners in Yusho patients and prediction of their cytochrome P450-dependent metabolism by in silico analysis.

In what has become known as the Yusho incident, thousands of people in western Japan were poisoned by the accidental ingestion of rice bran oil contaminated with polychlorinated biphenyls (PCBs) and various dioxins and dioxin-like compounds. In this study, we investigated the accumulation patterns of 69 PCB congeners in the blood of Yusho patients in comparison with those of non-exposed controls. The blood samples were collected at medical check-ups in 2004 and 2005. To compare the patterns of PCB congeners, we calculated the concentration ratio of each congener relative to the 2,2',4,4',5,5'-hexaCB (CB153) concentration. The concentration ratios of tetra- and penta-chlorinated congeners in the blood of Yusho patients were significantly lower than those of controls. To examine the cytochrome P450 (CYP)-dependent metabolic potential of the 2,3',4,4'5-pentaCB (CB118), CB153, and 2,3,3',4,4'5-hexaCB (CB156) congeners, we conducted PCB-CYP (CYP1A1, CYP1A2, CYP2A6, and CYP2B6) docking simulation by in silico analysis. The docking models showed that human CYP1A1, CYP2A6, and CYP2B6 isozymes have the potential to metabolize CB118 and CB153. On the other hand, it was inferred that CB156 is difficult to be metabolized by these four CYP isozymes. These results indicate that CYP1 and CYP2 isozymes may be involved in the characteristic accumulation patterns of PCB congeners in the blood of Yusho patients.

Design of a 15N Molecular Unit to Achieve Long Retention of Hyperpolarized Spin State.

Nuclear hyperpolarization is a phenomenon that can be used to improve the sensitivity of magnetic resonance molecular sensors. However, such sensors typically suffer from short hyperpolarization lifetime. Herein we report that [15N, D14]trimethylphenylammonium (TMPA) has a remarkably long spin-lattice relaxation time (1128 s, 14.1 T, 30 °C, D2O) on its 15N nuclei and achieves a long retention of the hyperpolarized state. [15N, D14]TMPA-based hyperpolarized sensor for carboxylesterase allowed the highly sensitive analysis of enzymatic reaction by 15N NMR for over 40 min in phophate-buffered saline (H2O, pH 7.4, 37 °C).