Current risk assessment of DNT has been immature, yet. We here generated transgenic mouse in which the luciferase (Luc) gene expressed under the control of a promoter of neuronal differentiation marker for DNT evaluation. The Luc expression pattern was consistent with the synaptic formation pattern during developmental periods, suggesting that Luc expression reflects the state of neuronal differentiation. Our reporter mouse could be a potential tool in DNT studies and “visualization” of neuronal differentiation using in vivo imaging may be applied to a novel DNT in vivo evaluation system.

Neurotoxicity of environmental electrophiles such as 1-bromopropane (1-BP) and acrylamide (ACR) has been reported in human and experimental animals. Our previous studies found that exposure to 1-BP or ACR can cause degeneration of noradrenergic axons in murine brain, while the mechanism remain obscure. To further investigate the underlying mechanism, 1C11 mouse neuroectodermal stem cell line and BV2 microglia cell line were used in this study. 1C11 stem cells were differentiated into noradrenergic neural cells (1C11NE), and then exposed to 0~1mM ACR or to conditioned media (CM) of ACR-treated BV2 microglia. Evaluation of cytotoxicity and plasma membrane integrity by MTS and LDH assay showed that exposure to ACR alone did not decrease cell viability, nor increase LDH release in 1C11NE cells. Exposure to BV2 CM did not decrease 1C11NE cell viability but increased LDH release. Quantification of neurite length showed that treatment with ACR alone did not decrease neurite length of 1C11NE, but treatment with CM of ACR-exposed BV2 microglia significantly decreased neurite length. The addition of ACR into CM of PBS-exposed BV2 also significantly decreased neurite length of 1C11NE. Moreover, transcriptomic RNA-seq analysis identified 2544 differentially expressed genes in 1C11NE after exposure to ACR or CM of BV2. WGCNA clustering and gene ontology analysis revealed different gene expression profiles and enriched different GO items/pathways in different treatment groups. The above results suggest that microglia play a critical role in ACR-induced retraction of noradrenergic axons.

We investigated the usefulness of echocardiography for evaluation of cardiotoxicity due to anticancer drugs in rodents. Echocardiography was performed after 2-week treatment with doxorubicin, resulted in no change in left ventricular ejection fraction, but significant decreases in diastolic function and strain. These decreases tended to be consistent with subsequent mortality, suggesting that impaired diastolic function and strain might be potential prognostic factors. In the clinical settings, it is said that changes in strain precedes the decrease in left ventricular ejection fraction, and our results were in good agreement with this.

Renal toxicity remains a major issue in clinical trials, and stresses the need for more predictive models fit for implementation in early drug development1. Here, we describe the use of a high throughput, microfluidic platform for the detection of drug-induced nephrotoxicity. A microfluidic platform (Mimetas’ OrganoPlate®)2 was combined with renal proximal tubule epithelial cell lines (PTEC) and exposed to fluid shear stress. A 12-compound nephrotoxicity screen across multiple laboratories was performed in collaboration with sponsors and the NC3Rs3. ciPTEC-OAT1 or RPTECs (Sigma) seeded against an ECM gel under perfusion flow were used to establish a proximal tubule-on-a-chip. Tubules with polarized epithelium containing functional transporter expression were obtained. Drug-induced toxicity was assessed by exposing the tubules to 4 benchmark compounds with known clinical effect and 8 blinded compounds supplied by the sponsors for 24 and 48h. Epithelial barrier tightness and drug-transporter interactions were evaluated. Parallel to this, cellular damage and stress were assessed using various read-outs. Finally, gene expression analysis was performed to assess AKI markers. The Nephroscreen revealed that a combination of cell viability, LDH and miRNA release were the most predictive readouts in determining nephrotoxicity. Most of the blinded compounds resulted in toxicity detected by at least one of the functional read-outs. Nephroscreen provides a reliable standardized and automatable system for efficacious identifying nephrotoxicants and revealing their mode of action.

The present study searched for signal factors in the mechanism underlying our mouse model of NASH-hepatocarcinogenesis using CDAA-HF-T(-). In the liver of mice fed CDAA-HF-T(-) for 52 or 63 weeks, hepatocellular adenomas and carcinomas, and hemangiosarcomas were observed, and the Rho Family GTPase and IL-8 signaling were upregulated. IL-8 and its receptors were upregulated in the non-tumor liver tissue, while in the tumor, IL-8 was abundantly expressed, but its receptors were downregulated. It is suggested that IL-8 complicatedly regulates NASH-hepatocarcinogenesis.

Drug-induced liver injury is one of the leading causes of market withdrawal in the pharmaceutical industry and poses a serious health risk to affected patients. Here, we developed a 3D in vitro model of the human liver, the OrganoPlate LiverTox compatible with automated liquid handling and validated for hepatotoxicity screening. To build the model, up to 96 independent 3D perfused cultures were established on MIMETAS’ OrganoPlate 2-lane. Induced pluripotent stem cell-derived hepatocytes (iHep) in extracellular matrix were added to a microfluidic channel, following which endothelial and Kupffer cells were added to an adjacent channel. Characterization of the model revealed hepatocyte function including CYP3A4 activity and albumin production for up to 14 days. Fetal hepatocyte marker alpha-fetoprotein (AFP) declined over the 14 day culture, supporting iHep maturation. Assay validation studies using troglitazone as a positive hepatotoxic control revealed robust Z-factors ≥ 0.2 for albumin, urea, iHep viability (propidium iodide staining), and iHep nuclear size (Hoechst 33342 staining) assay readouts. Using these assays, 159 compounds of known hepatotoxicity were screened in the OrganoPlate LiverTox (50 μM, 72 h) and ranked by a composite score by combining the assay readouts. A follow-up dose response evaluation of select hits suggested the albumin assay to be the most sensitive readout in calculating TC50 values. Together, the OrganoPlate LiverTox is a promising platform for hepatotoxicity detection and has the potential to be used in a high throughput screening capacity.

Various research methods and concepts (QSAR, AOP, WoE, MoA, IATA, etc.) have been proposed and developed to evaluate the toxicity of compounds and elucidate the mechanism. Rapidly evolving data science and artificial intelligence are becoming increasingly important on applying these diverse and complex studies. However, when applying data science or artificial intelligence in the field of handling chemical compounds, it is important to apply it correctly according to the purpose of analysis. In this presentation, we will discuss on these issues.

Molecular signaling pathway networks are regulated in epithelial-mesenchymal transition (EMT) and cancer stem cells (CSCs), which exhibit anti-cancer drug resistant features. To reveal the mechanism of human treatment-resistant gastric cancer (GC) and the relationship between oxidative stress response and GC, analysis on oxidative stress response and molecular networks in diffuse- and intestinal-type GC has been performed. The network pathways of GC were analyzed by Ingenuity Pathway Analysis (IPA). NRF2-mediated oxidative stress response network included molecules related to Regulation of EMT by growth factors pathway and Production of nitric oxide and reactive oxygen species in macrophages such as PI3K and AKT. We have developed the Adverse Outcome Pathway (AOP) on chronic reactive oxygen species (ROS) leading to human treatment-resistant gastric cancer consists of Molecular Initiating Event (MIE) as chronic ROS, followed by Key Event (KE)1 as sustained tissue damage / macrophage activation / porcupine-induced Wnt secretion, KE2 as proliferation / beta-catenin activation, KE3 as EMT, and AO as human treatment-resistant GC. Updates on the AOP development will be introduced.

Epidemiological studies have suggested that more than 75% of individuals in the developed countries routinely ingest more than the recommended dietary allowance for vitamin A (VA), most of it from fortified foods and supplements. The current recommended daily allowance of VA in adults is 3000 IU/day in males while the upper tolerable limit is 10,000 IU/day. Although it has been reported that long-term VA deficiency (VAD) results in spermatogenic arrest at the early stage of spermatogenesis, the effects of VA excess (VAE) on spermatogenesis have not been well investigated. The present study was designed to determine the effects of chronic VAE on mouse spermatogenesis and to explore potential molecular toxicological mechanisms of VAE. The VA content of 250 IU/g in the diet used in this study is 3.75 times the upper tolerable limit for VA. As a result, body weights were not significantly different between the control and VAE groups. Testicular cross-sections from the control and VAE mice contained a normal array of germ cells, and the daily sperm production was similar between the two groups. However, the percentage of seminiferous tubules in stages VII and VIII was significantly lower in the VAE mice than in the control. In addition, significant changes in the expression of genes involved in retinoid metabolism, spermatogenesis, and spermiogenesis were detected in the testes of the VAE mice. Consistently, sperm motility and head morphology were significantly impaired in the VAE mice. Our findings suggest that long-term dietary intake of VAE were able to influence both pre- and post-meiotic spermatogenesis.

While thyroid hormones (THs) are essential for brain development, effects of mild suppression of maternal blood THs by hepatic enzyme inducers on the infant brain developments is not fully understood. A simple screening test for investigating whether maternal chemical exposure reduces brain THs in fetal and neonatal rats would be valuable. To verify reliability of the Comparative Thyroid Assay with additional examination of the brain, THs and histology, propylthiouracil (PTU) and phenobarbital (PB) were dosed. Clearly suppressed THs in rat fetuses were noted with PTU but not with PB.

While thyroid hormones (THs) are essential for brain development, effects of mild suppression of maternal blood THs by hepatic enzyme inducers on the infant brain developments is not fully understood. A simple screening test for investigating whether maternal chemical exposure reduces brain THs in fetal and neonatal rats would be valuable. To verify reliability of the Comparative Thyroid Assay with additional examination of the brain, THs and histology, propylthiouracil (PTU) and phenobarbital (PB) were dosed. Clearly suppressed THs in rat pups were noted with PTU but not with PB.

Nucleic acid analogs are highly effective therapeutic agents for viral infections. Teratogenicity is often observed in this class, and its avoidance is important. We evaluated usefulness of Whole Embryo Culture for screening teratogenicity of nucleic acid analogues. We applied analogs which were teratogenic in rats to the WEC and confirmed abnormal embryogenesis except for purine base analogs. In conclusion, the WEC is useful for assess teratogenicity of nucleic acid analogs other than purine base analogs. Further studies are required to avoid false negatives of purine base analogs.

On January 29, 2021, PMDA published the DETECTION OF REPRODUCTIVE AND DEVELOPMENTAL TOXICITY FOR HUMAN PHARMACEUTICAL S5(R3) by ICH. And zebrafish developmental toxicity test as an alternative method is now in full swing in Japan. In this study, we have established a zebrafish quality control protocol, which is the most important basis for this zebrafish developmental toxicity test. We found that it was possible to remove these low-quality fertilized eggs, thereby reducing false positive frequency and enhancing the accuracy of zebrafish developmental toxicity testing.

Thoracolumbar supernumerary ribs (TSR, 14th rib) are skeletal findings observed in fetuses in teratogenicity studies. We have reported the mechanism of 5-FC induced TSR (Kumamoto et al., FTS, 2020; Kuwagata et al., CGA, 2018), but the spontaneous development remains unclear. In this study, we conducted to elucidate by sequence analysis rat strains (Wistar / SD). Although no significant differences were observed in most sites of homeobox genes, a significant difference was shown in a certain region, which overlaps with a miRNA that negatively regulate the expression of multiple Hox genes and control posteriorization of axial skeleton.

Prenatal exposure to the antiepileptic drug valproic acid (VPA) has been reported to increase the risk of a teratogenic and developmental disorder. In this study, VPA (400 mg/kg) was subcutaneously administered to ICR mice on the 12th day of gestation. Increased neural stem cell proliferation and abnormal activation of microglia were observed in the VPA-treated group at E15 embryos. Behavioral analysis observed increased motor activity and abnormal social behavior. These data showed that prenatal exposure of VPA might be induced the proliferation of neural stem cells by abnormal activity of microglia.

Transcriptome data of cultured cells treated with small compounds are profile data and their analysis enables us to evaluate toxicity of compounds. The effects of compounds are complex, and it is difficult to detect relatively weak secondary effects by general methods that lump all the characteristics of compounds together. To address this issue, we have developed a method to separate and understand the effects of compounds. In this study, we applied this method to two compounds of natural origin with similar structures, and evaluated their ability to detect unreported effects and to discriminate between compounds with similar structures.

We investigated the mechanisms of neurotoxicity of a pyrethroid pesticide deltamethrin (DM) focusing on proteolytic systems. DM dose-dependently increased expression of autophagy markers LC3-II and p62 and induced apoptosis in the Neuro2a cells. The expression of mitochondria markers was decreased by DM and co-localized with LC3-II, which suggests the accumulation of autolysosomes containing damaged mitochondria. The amount of ubiquitinated protein was increased by DM while proteasome activities were decreased. These results revealed that DM induced neurotoxicity caused by mitophagy activation and proteasome inhibition.

Selenoprotein P (SeP) is a plasma protein that possesses 10 selenocysteine residues and supplies selenium to brain. Recently we’ve shown that methylmercury covalently modifies selenocysteines in SeP (Se-mercuration) by using our original methodologies. We’ve also found that Se-mercurations of SeP are robust and irreversible, although its biological and toxicological significances are not clear. Briefly, our present study suggested Se-mercuration of SeP causes inhibition of its selenium supplies, and besides resulting in induction of vulnerability against oxidative stresses.

Our previous study demonstrated that Cd inhibited the activities of RAR and PPARD transcription factors. In this study, the pretreatment of HK-2 cells with retinoic acid (RA) and its precursor, retinol, conferred resistance to Cd. In addition, PPARD knockdown showed resistance to Cd in HK-2 cells but not RARG (one of RAR subtype) knockdown. On the other hand, treatment of PPARD knockdown cells with RA strengthened resistance to Cd compared with each alone group. These results suggested that RA and PPARD knockdown play protective rolls against Cd toxicity.

We recently identified 40S ribosomal protein SA (RPSA) as a protein that binds to the intracellular domain of TNFR3, which is involved in methylmercury toxicity. Knockdown of RPSA suppressed methylmercury-induced caspase-3 activation, but there was little suppression of caspase-3 activation by knockdown of TNFR3 in RPSA-knockdown cells. Methylmercury was observed to localize RPSA to specific sites on the cell membranes, and TNFR3 and RPSA were bound to each other even at steady state. These results suggest that methylmercury may induce apoptosis through activating the TNFR3/RPSA complex by assembling it at specific sites on cell membranes.

Uranium is an internal radionuclide that is exposed daily through food and water. Renal toxicity is the primary health effect of uranium. The chronic ingestion of naturally occurring uranium in contaminated groundwater resulted in decreased renal function related to renal tubular injury. The uses of uranium in the nuclear industry and the decommissioning work of nuclear reactors following the nuclear accident have led to growing public concern over the health effects. Details of renal uranium transport, however, is not well understood. The renal proximal tubules play pivotal roles in the reabsorption of uranium from the urine. The proximal tubules are divided into three regions (S1, S2, and S3) and the regions exhibit region-specific material transport systems. In the present study, the uranium uptake and the sensitivity to uranium were examined in cell lines derived from the three regions of mouse proximal tubules. The intracellular distribution of uranium was analyzed by quantum beam based elemental analyses, such as μPIXE (particle induced X-ray emission with microbeam) and SR-XRF (synchrotron radiation X-ray fluorescence spectrometry) with micro and nanobeam. The chemical form of the localized uranium in cells was assessed by X-ray absorption fine-structure with microprobe (μXAFS). The data including the region characteristics of uranium uptake, localized quantity of uranium, its oxidative condition, and colocalized elemental composition after uranium exposure will be presented.

Epidemiological studies have indicated that cadmium is a risk factor for atherosclerosis. Previously, we reported that CHSY1, an elongation enzyme of dermatan sulfate chains that contributes to the formation of lipid plaques, is induced by cadmium exposure in cultured vascular endothelial cells. The purpose of this study is to investigate the involvement of CHSY1 on the cytotoxicity of cadmium in vascular endothelial cells. Confluent cultures of bovine aortic endothelial cells were transfected with CHSY1 overexpression vector or siRNA and then exposed to cadmium. The cytotoxicity of cadmium was inversely correlated with the expression level of CHSY1. Since cadmium cytotoxicity in cultured endothelial cells is characterized by the formation of de-endothelialized space associated with cell detachment and CHSY1 has been reported to be involved in epithelial-mesenchymal transition (EMT), a process that epithelial cells lose cell-cell adhesion and gain migratory properties, we analyzed the gene expression of EMT marker molecules that affect cell adhesion. A mesenchymal marker N-cadherin and an epithelial marker VE-cadherin were both induced by cadmium. Furthermore, the induction of N-cadherin was markedly suppressed in CHSY1 knock-downed cells, and cadmium cytotoxicity was enhanced by repression of N-cadherin expression. Although the direct interrelationship between cadmium cytotoxicity and EMT is not certain, this study showed that cadmium-induced CHSY1 exacerbates cadmium cytotoxicity and that N-cadherin, whose expression is upregulated with increased CHSY1, reduces the cytotoxicity by cadmium in vascular endothelial cells.

Arsenic is an environmental pollutant that causes atherosclerosis, although the pathogenic mechanisms are not fully elucidated. Tissue factor (TF) is the initiator of the coagulation and it has been shown to be involved in the development of atherosclerosis. In this study, we found that arsenite stimulates the expression of TF via activation of the Nrf2 pathway in both human aortic smooth muscle cells and macrophage-like human THP-1 cells. It is postulated that arsenite induces a procoagulant state of blood to progress atherosclerosis through induction of TF expression.

We have recently found that methylmercury induces neuronal cell death via induction of OSM expression and its extracellular release in microglia in the mouse brain. Induction of OSM expression by methylmercury was attenuated by knockdown of JAK1 or ASK1/JNK kinase expression. In addition, knockdown of STAT3 or cJun, transcription factors activated by these kinases, attenuated the induction of OSM expression, but simultaneous repression of both factors did not result in further attenuation, suggesting that they may be involved in the induction of OSM expression by cooperating with each other.

Uranium is considered to be a chemical and radiological toxic metal that accumulates in the kidneys and bones after the incorporation. The practical therapy to reduce the health effects by the internal exposure is to treat with decorporation agents such as chelating agents. X-ray absorption spectroscopy (XAS) can provide detailed information about the electronic state and local structure around a specific atom, e.g. bond lengths, coordination number. In the present study, uranium chemical form in serum was analyzed by XAS in the presence and absence of chelating agents to study decorporation effectiveness of human body exposed to uranium. XAS spectra of uranium L3 edge were measured at BL14B1 beamline of SPring-8. Chelating agents used in this study were ethane-1-hydroxy-1,1-bisphosphonic acid and diethylenetriaminepentaacetic acid, respectively. About 150 μL of the serum sample containing uranium and chelating agent was placed in a hole on an acryl cell with 10 mm thickness, and sealed by Kapton poly imide tape. XAS spectra of uranium in serum changed depended on the concentration of the chelating agents, resulting that XAS spectra in the presence of chelating agents were distinguished with those in the absence of the chelating agents. Coordination structure of uranium in serum in the presence of chelating agents was estimated based on the bond distance between uranium and oxygen atom of the ligands.

Diphenylarsinic acid (DPAA) is one of organic arsenic compounds detected in the well water in Kamisu, Ibaraki, Japan. People exposed to DPAA developed cerebellar symptoms. Here, we evaluated cytokines secreted from cultured normal human cerebellar astrocytes (NHA) exposed to DPAA. NHA exposed to DPAA at 50 μM for 96 h or 10 μM for 288 h increased secretion of MCP-1, adrenomedullin, FGF-2, CXCL1, IL-6, and IL-8. These cytokines secreted by astrocytes exposed to DPAA could disturb surrounding neurons, microglia, blood vessels, or astrocytes, which might lead to cerebellar symptoms.

Methylmercury (MeHg) induces neuronal degeneration in the brain, but mechanisms are unclear, which underlie cell selectivity and developmental dependency. It is hard to analyze them at an appropriate stage by monitoring Hg of the dissected brain. To address this issue, we developed sensor vectors for MeHg toxicity.

The study of selenoprotein showed that MeHg inhibits the insertion of selenocysteine (Sec) during translation. By utilizing this new toxic model, vectors were designed to show MeHg-dependent signals and evaluated for sensors. We will present and discuss these results.

Methylmercury (MeHg) binds to the cysteine residues of proteins in organs causing cytotoxicity. However, the relationship between MeHg and gut bacteria remains poorly understood. In this study, we showed that MeHg binds covalently to gut bacterial proteins via cysteine residues and MeHg inhibits the growth of Lactobacillus. We found that gut bacteria contribute to the production of reactive sulfur species such as hydrogen sulfide and hydrogen persulfide in the gut. We also discovered that the removal of gut bacteria accelerated accumulation of mercury in the cerebellum, liver, and lungs of mice subsequent to MeHg exposure.

Glioblastoma is the most aggressive type of solid cancer that begins within the brain. We previously demonstrated that photodynamic therapy using talaporfin sodium (TS-PDT) treatment exhibits anti-tumor activity against glioblastoma. In this study, we found that sodium arsenite selectively decreased the cell viability of U251 glioblastoma cell line. Moreover, arsenite enhanced the anti-tumor activity of TS-PDT in the cells. These results suggest that arsenite has the potential to exhibit anti-tumor activity against U251 glioblastoma cell line and enhance the anti-tumor effect of TS-PDT.

Cadmium (Cd) is a toxic environmental pollutant. Recently, it was reported that maternal blood Cd levels during pregnancy are positively associated with the risk of early preterm birth. It might be caused by Cd-induced hypertensive disorders of pregnancy (HDP) via inhibition of trophoblast differentiation. Here, we showed that Cd inhibited the induction of several cell differentiation markers in human trophoblast stem cell models and BeWo choriocarcinoma cells. These results suggest that Cd causes HDP through the disorder of the placental formation process.

We performed immunohistochemistry for γ-H2AX in the urinary bladder of rats treated with aromatic amines to develop a method for early detection of bladder carcinogenicity. Nine of 11 bladder carcinogens increased γ-H2AX formation in the urothelium (sensitivity 82%), whereas 12 of 14 non-bladder carcinogens were negative (specificity 86%). Three aromatic amines of unknown toxicity increased γ-H2AX formation, suggesting the potential bladder carcinogenicity. Our results suggest that the bladder carcinogenicity of aromatic amines can be detected by γ-H2AX immunostaining in the short-term study.

Fumonisins are one of the fungal toxins produced by Fusarium species. Fumonisin B3 (FB3), a member of the fumonisin B group, is known to inhibit the biosynthesis of sphingolipids by inhibiting ceramide synthesis, but the toxic effects caused by FB3 remain unclear. We herein investigated the effects of FB3 on allergy using an atopic dermatitis (AD) mouse model. The AD model was generated by topical application of 2,4-tolylene diisocyanate (TDI) in female NC/Nga mice. FB3 (0.1%) was administered topically before each TDI application. AD score and trans epidermal water loss (TEWL) were measured weekly. One day after the last TDI application, auricular lymph nodes (LN), serum and skin samples were collected for analysis of the number of immunocompetent cells, cytokine level, total IgE level, and related gene expression. FB3 treatment significantly increased AD score compared to the AD control group. The numbers of helper T cells and IgE-producing B cells in LN were significantly increased by FB3 treatment, whereas there were no significant differences in cytokine production and serum IgE level. The gene expression of IL-33, which is involved in the exacerbation of AD, was significantly increased in the FB3 group. Significant upregulation of TEWL in FB3 group reveals that FB3 breakdowns the cutaneous barrier function, corroborated by a significant influence of the related genes expression (Cers3 and Degs1) in FB3 group. Our findings suggest that FB3 exacerbates AD by breakdown the cutaneous barrier function and activating IL-33 expression.

To investigate the membrane permeability and drug metabolic activities of human iPS cell-derived small intestinal epithelial cells, we compared these characteristics with Caco-2 cells or human primary enterocytes. Human iPS cell-derived intestinal epithelial cells showed a better correlation between intestinal absorption rates and membrane permeation rates than Caco-2 cells and showed close metabolic rates with human primary enterocytes for UGT substrates. Therefore, human iPS cell-derived intestinal epithelial cells might be a useful tool for predicting human intestinal absorption.

1,3-Dichloro-2-propanol (1,3-DCP), a food contaminant, is carcinogenic in rats. To understand the mechanisms of 1,3-DCP-induced hepatocarcinogenesis, we examined histopathology, in vivo mutagenicity and preneoplastic lesions in the livers of gpt delta rats treated with 1,3-DCP under carcinogenic conditions. gpt mutant frequencies were significantly increased in 1,3-DCP-treated rats, suggesting the involvement of genotoxic mechanisms in hepatocarcinogenecity of 1,3-DCP. On the other hand, lack of increases in GST-P+ foci was observed and will be discussed along with further investigations.

Environmental electrophiles modify the thiol groups of sensor proteins, leading to activation of redox signaling such as the Keap1/Nrf2 pathway. While activation of Nrf2 caused by single exposure has been established well, effect of combined exposure to electrophiles on the activation has not been evaluated. Here, we examined whether or not atmospheric electrophiles such as 1,2-naphthoquinone and 1,4-naphthoquinone could enhance a phytochemical (E)-2-hexenal-mediated Nrf2 activation in HepG2 cells (Abiko and Aoki, 2021).

Airborne quinones are produced during incomplete combustion of gasoline and photooxidation in the atmosphere. While such reactive species are found to covalently bind protein nucleophiles in cells, the extracellular regulation is not understood. Here we found that airborne quinones such as 1,2-naphthoquinone are readily trapped by cysteine released from A431 cells, leading to the cysteine adducts formation, thereby repressing covalent modification of the cellular proteins. Our finding therefore provides a concept that there is existence of “the Phase-zero reaction” for airborne electrophiles prior to the uptake of cells.

Methyl vinyl ketone (MVK), an environmental electrophile, is known to interact with proteins via Michael reaction. In this study, we investigated the effect of MVK on phosphatidyl inositol-3 kinase (PI3K) –Akt signaling. We found that MVK attenuated PI3K–Akt pathway in a dose-dependent manner. Interestingly, MVK affected PI3K p85 subunit and inhibited to bind to epidermal growth factor receptor. These results indicated that modification of PI3K by MVK was involved in the regulation of PI3K–Akt signaling.

The arylhydrocarbon receptor (AhR) binds strongly to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the most potent dioxin, to induce a variety of toxic effects, including fatty liver. One of the downstream mediator genes of AhR activation is thought to be Pla2g4a, which codes a member of phospholipase A2 (PLA2) family, because genetic ablation of Pla2g4a ameliorates TCDD-induced fatty liver. The present study focused on two questions; (1) Do ones of other PLA2 genes have the same expression property as Pla2g4a in TCDD-induced fatty liver? (2) Is there a PLA2 gene that compensates the role of Pla2g4a in the absence of Pla2g4a? In order to examine these questions, Pla2g4a wt and ko mice were exposed to TCDD or vehicle, and were examined for the expression levels of PLA2 genes in the liver. Among 14 genes of PLA2 family examined, Pnpla7, Pnpla2, and Pla2g7 had the expression property similar to Pla2g4a, implying a possible role of these PLA2 genes in the TCDD-induced fatty liver. On the other hand, no PLA2 gene had the expression property which support the potential role for compensating the absence of Pla2g4a.

The cerebellum of CSE-knockout mice was more susceptible to methylmercury (MeHg) than that of wild type mice. These CSE-deficient mice exhibited a higher level of mercury accumulation in the brain. Co-treatment with Na₂S₄, an RSS able to capture MeHg, leading to the formation of its sulfur adduct (MeHg)₂S, blocked the increased accumulation of mercury, motor dysfunction caused by CSE deficiency. Our findings suggest that capturing MeHg by RSS in association with its sulfur adduct formation is involved in the repression of the brain distribution and deleterious effects of MeHg.

The discharge of industrial wastewater in the Bohai Bay area causes potential pollution to the Bohai Sea. To evaluate the pollution of polycyclic aromatic hydrocarbons (PAH), sludge samples were collected from a factory in Bohai Bay, and samples were extracted by ethanol for biological monitoring and chemical detection. The zebrafish embryos/larvae were exposed to sludge extract samples (SE) and PHAs (fluorene, phenanthrene, and pyrene), which used as positive controls from 4 hours post-fertilization (hpf) until observations. Similar to PAHs, SE increased the mortality and inhibited the hatching rate of zebrafish embryos/larvae at 72 hpf (P < 0.05). Interestingly, we also found that SE caused a dose-dependent inhibition of pigmentation intensity in zebrafish eyes at 36 hpf (P < 0.05). TPO, Dio2, and Thrβ mRNA expression were down-regulated and Dio3 mRNA was induced by SE at 120 hpf. The results of chemical analysis showed that the main pollutants were fluorene, phenanthrene, fluoranthene, pyrene, and other PAHs, which were consistent with biological assessment methods. The above results suggest that the pigment intensity of zebrafish eyes can also be used as an initial biological assessment of PAH pollution in Bohai bay.

Dioxin pollutants are environmental persistent organic pollutants (POPs), which remain in trace amounts in rivers and silts. Dioxin poses a potential threat to aquatic animals from a raised temperature. In this study, Tg (CYP1A: mCherry) transgenic zebrafish was used as a model animal to compare the toxicity caused by tetrachlorodibenzo-p-dioxin (TCDD) in zebrafish embryos at 26 ℃ and 30 ℃. Zebrafish embryos were exposed to TCDD (0.1, 03, and 1 ppb) or DMSO (control) from 2 to 4 hours after fertilization (4 hpf), until the observation. Comparing the mortality of zebrafish embryos caused by TCDD under the two temperature conditions, there was no significant difference. Whether it is 26 ℃ or 30 ℃, 1ppbTCDD caused pericardial edema in zebrafish embryos at 96 hpf, the ratio of pericardial edema area to body length was 0.3 and 0.48, respectively (P < 0.05). At 96 hpf, the fluorescence intensity of Tg (CYP1A: mCherry) transgenic zebrafish induced by TCDD at 30 ℃ was significantly higher that than induced by TCDD at 26 ℃. Furthermore, we also quantified CYP1A mRNA expression by RT-qPCR, the results showed that the TCDD induced CYP1A mRNA expression at 30 ℃ was significantly higher than TCDD caused expression at 26 ℃. At the same time, the expression of lipid metabolism relative genes ppar- α, ppar- γ, acadm, fasn and slc2a2 also increased by 1ppb TCDD exposure. The results show that the increase of temperature enhances the toxic effect of TCDD on zebrafish embryos, and we will further study its mechanism.

Keywords：TCDD; Ambient temperature; cyp1a; Zebrafish embryo; Lipid metabolism

The present it is known that exposure to hazardous chemical can adversely affect the health of the underlying disease, but scientific verification and research on the mechanisms of an exacerbation are insufficient. In this study investigated the exacerbating effect of lung injury in a monocrotaline (MCT)-induced pulmonary arterial hypertension (PHA) rat model by formaldehyde (FA). We constructed an animal disease model by inducing PHA by single subcutaneous injection with MCT, and then treated FA (2.5 and 5 mg/kg) using the intratracheal instillation (ITI) method. After 4 weeks of experiment, the rat were euthanized and weighed, and their tissues were subjected to histopathological examination, ELISA assay, western blotting, and RNA sequencing assay. As a result, all the MCT+FA groups decreased weight compared to the other groups, and the weight of heart or lung tissue increased. The right ventricular hypertrophy (RVH) and neutrophil changes in bronchoalveolar lavage fluid (BALF) were significantly increased in all MCT + FA groups. Finally, in all MCT + FA groups, exacerbation of lung lesions such as granulomatous inflammation, mucous cells, and epithelial hyperplasia were observed. Moreover, it was observed that the expression of genes related to lung injury and hypertension such as apoptosis, inflammatory response, angiogenesis, and oxidative stress increased in all MCT + FA groups compared to FA and MCT groups. Therefore, it is suggested that the FA, which is known as a hazardous chemical in the indoor environment, has the potential to exacerbate the underlying disease.

In vitro reporter gene assays are widely used to detect endocrine disruptors. Previously, we established the novel yeast bioassay for progesterone receptor (PR) and conducted the validation assay. This study demonstrates that 2,2-Bis(4-hydroxyphenyl)propane (Bisphenol A; BPA) and BPA-related compounds have antagonistic activity using the yeast-based and human cell-based bioassays.

BPA decreased progesterone-induced reporter activity in yeast-based PR bioassay. Several bisphenol-related compounds also exhibited PR antagonist activity. These antagonistic effects of bisphenols were also observed in human-based PR bioassay.

Particulate air pollution causes abnormal development of immune system of neonates. Previous studies have revealed that maternal exposure to carbon black nanoparticle (CB-NP) disturbs development of the lymphoid tissues. The immune-activation elicited by CB-NP were dependent on the stage of gestation in which they were exposed. Thus, the present study aimed to investigate the effect of CB-NP on neonatal lymphoid tissues depending on the time of exposure during gestation in mice.

Pregnant ICR mice were treated with CB-NP (95 μg/kg/time) by intranasal instillation, in gestational days 4 and 5 (preimplantation period), 8 and 9 (organogenesis period), or 15 and 16 (fetal developmental period). Spleen and thymus were collected from offspring mice at 1-, 3-, and 5-days post-partum. Lymphoid phenotypes were examined by flow cytometry. Gene expression was examined by quantitative RT-PCR.

The numbers of total splenocytes and splenic CD3−B220− phenotype (non-B/non-T cell) of offspring in the postnatal day 5 were significantly increased by the exposure to CB-NP during organogenesis period comparing with no exposure (control), pre-implantation period exposure, and fetal development period exposure. In contrast, expression levels of mRNA associated with chemotaxis and differentiation for immune cells in spleen were not affected by the CB-NP exposure during any gestational period. Overall, the organogenesis period was the most sensitive to CB-NP exposure with regard to lymphoid tissue development. Moreover, repetitive CB-NP exposure across the several gestational periods may be critical causes resulting in the neonatal immunotoxicity.

Cellulose nanofibrils (CNFs), also known as cellulose nanofibers, there is a concern about risks of carcinogenicity or mesothelioma. A battery of three commonly used assays, namely Ames test, an in vitro chromosomal aberration test, and a mammalian erythrocyte micronucleus test illustrated that the phosphorylated CNFs examined in this study did not induce genotoxicity. We believe that these results will be useful for predicting the human carcinogenic risk of CNFs and accelerate their industrial application. This study was supported by project grants from the New Energy and Industrial Technology Development Organization (NEDO).

The present study was conducted to assess whether and how the use of different devices affect on the outcome of a 2-year toxicity study of MWCNT administered by intermittent intratracheal instillations. Male Fischer 344 rats (9 weeks old) were intratracheally administered MWNT-7 using a feeding cannula or a micro-sprayer, at a dose of 0.5 mg/kg BW every 4 weeks and examined at the ends of years 0.5 and 1 as interim sacrifices. While MWCNTs tended to be located in the caudal position of the lung lobes in cannula group, overall, no apparent difference was noted by the use of 2 different devices.

This study investigated pulmonary effects of exposure to Fe₃O₄-PEG-PLGA nanoparticles (NPs) and role of Nrf2. Exposure to Fe₃O₄-PEG-PLGA NPs increased total cells and macrophages in wild-type (WT) mice, lymphocytes, neutrophils and eosinophils in Nrf2 KO mice, and basophils in both genotypes. Exposure to Fe₃O₄-PEG-PLGA NPs increased pulmonary expression of TNF-alpha, KC and MIP-2 only in Nrf2 KO mice, and SOD-1, GcLc, GcLm, MMP-2, and TGF-beta only in WT mice. The results suggest that Nrf2 plays a role in regulation on leucocyte migration and inflammatory response induced by Fe₃O₄-PEG-PLGA NPs.

Very little is known about the in vivo toxicity of inhaled double-walled carbon nanotubes (DWCNTs). In the present study, we compared the pulmonary toxicity of DWCNT to MWCNT-7, a well-known multi-walled carbon nanotube. Rats were divided into six groups: untreated, vehicle, low dose DWCNT, middle dose DWCNT, high dose DWCNT, and MWCNT-7. The test materials were administered by intra-tracheal intra-pulmonary spraying (TIPS) every other day for 15 days: the low dose, middle dose, and high-dose DWCNT groups were administered final total doses of 0.125, 0.25, and 0.50 mg/rat DWCNT and the MWCNT-7 group was administered 0.50 mg/rat MWCNT-7. The 1-year result showed DWCNT was internalized in granulation tissue. The granulation tissue was higher in the rats administered 0.5 mg DWCNT compared to the rats administered 0.50 mg MWCNT-7. Importantly, the number of macrophages was significantly increased in DWCNT treated rats compared to the control rats, and the number of macrophages in the 0.5 mg DWCNT treated rats was also significantly higher than in the 0.5 mg MWCNT-7 treated rats. However, the alveolar and mesothelial PCNA index were significantly higher in MWCNT-7 treated rats compared to the vehicle and DWCNT treated groups. Rats treated with DWCNT developed hyperplasia that was dose-dependent, but no adenomas or carcinomas were found in the lung or pleura of these rats. One rat administered MWCNT-7 developed mesothelioma. Conclusion: DWCNT was not shown to be carcinogenic to rats after 1 year, however, long-term studies are needed to predict the risk and safety of respirable DWCNT.

Convulsion causes significant impact on drug development, therefore, appropriate evaluation is important for the development of compounds with convulsion potential. The aim of this study was to evaluate the convulsion potential after drug administration by using motor evoked potentials (MEP) in rats. Rats were dosed with 3 convulsants or 1 non-convulsant, and then, MEP was measured under anesthesia. As a result, convulsant-related effects were observed in MEP. It suggests MEP is useful for evaluating drug-induced convulsions in rats.

Central nervous system (CNS) toxicity is a critical factor in the possible adverse effects in drug development. Seizures and convulsions are frequent manifestations of CNS toxicity and are the leading cause of drug development attrition. Conventional behavioral batteries such as the Irwin test and the functional observational battery (FOB) are used to examine toxicity. While they are useful to evaluate animals' locomotor and overall health conditions, they overlook specific CNS toxicity. Thus, assay systems that can assess neural mechanisms' underlying CNS toxicity are critical. Recently, several laboratories have developed in vitro electrophysiological assay systems using hippocampal brain slices from various animal species (rats, dogs, monkeys, and mini pigs) for seizure liability assessment. Here, we report a novel assay system using VSD imaging in the mouse hippocampus. We applied electrical stimulation to the Schaffer collateral pathway from a stimulation electrode placed near the CA1/CA3 border. CA1 responses were recorded every thirty seconds with an imaging system (MiCAM02, Brainvision Ltd.) and field potential recordings from the stratum radiatum. Four positive control substances of picrotoxin, SR95331 (Gabazine), 4-aminopyridine, and pilocarpine were tested. All of them induce seizures or convulsions in animals. The VSD responses were significantly different in these substances. The various effects on CA1 activity indicate that the system can evaluate neural mechanisms, thus examining CNS toxicity.