Maternal exposure effect of glyphosate (GlyP) and glyphosate-based herbicide (GBH) on hippocampal neurogenesis was examined in rats. Pregnant rats were treated with 0, 15,000, or 30,000 ppm GlyP via diet and 1% GBH via drinking water from gestational day 6 to postnatal day (PND) 21, and offspring were examined on PND 21 and PND 77. As a result, GlyP and GBH decreased proliferation of neurogenic niche and increased synaptic plasticity of granule cells transiently on PND 21. However, GBH further induced oxidative stress in the dentate gyrus on PND 77.

Attention-deficit/hyperactivity disorder (ADHD) is a common neurodevelopmental disorder characterized by inattention, hyperactivity, and impulsivity. The number of prescriptions for ADHD patients is increasing, suggesting that the number of fertile women use such medication might also be increasing. However, the effects of MPD, the first-line drug for ADHD, on offspring have not been well examined. The purpose of this study was to clarify the effects of MPD exposure during the fetal period.

Dams were administered either water or MPD during pregnancy. Obtained male offspring were subjected to measurement of the locomotor activity and the elevated plus-maze test at 6 weeks old. Approximately 1/3 of the offspring were subjected to locomotor activity test after therapeutic treatment with MPD. The other 1/3 of the offspring were subjected to elevated plus-maze test after atomoxetine (ATX) treatment. The brains were collected from remaining offspring, gene expressions were determined by quantitative RT-PCR.

MPD-offspring showed an increase in the locomotor activity, which was suppressed by MPD treatment. The number of open arm entries, the time spent in open arms, and distance traveled in open arms were significantly increased in the MPD-offspring. This impulsivity was suppressed by ATX, another medication for ADHD, which a different action of the mechanism. MPD-offspring showed decreased Drd2 and Slc6a3 expression levels, which are often observed in ADHD model animals. Our results suggest that continuous use of MPD during pregnancy induces ADHD phenotypes in the offspring.

This study examined 28-day repeated oral exposure effect of ethanol (EtOH) on hippocampal neurogenesis in rats. Five-week-old male SD rats were administered 0, 10 or 16% (v/v) EtOH via drinking water for 28 days. EtOH at the high dose decreased the number of type-1 and type-2a cells in the subgranular zone, increased PVALB+ interneurons in the dentate gyrus hilus, and decreased c-FOS+ granule cells, suggesting a prevention of neural stem cell proliferation by activating PVALB+ interneuron, as well as suppressed synaptic plasticity.

A developmental toxicity test system for intravaginal exposures via semen should be newly considered. The teratogenicity of chemotherapeutic drug thalidomide is species-specific in primates and rabbits. Seminal plasma concentrations of thalidomide and its 5‑hydroxylated metabolite were detected after oral doses (2, 250, and 500 mg/kg) of thalidomide in male rabbits (NZW) by liquid chromatography-mass spectrometry, in a similar time-dependent manner to the blood plasma. Based on this toxicokinetic information, an intravaginal administration of thalidomide to female rabbits will be conducted.

CRISPR/Cas9 mediated genome-editing (GE) technology is a very powerful tool for the easy and rapid development of genetically modified animals. Although the off-target effects of GE were primarily of concern, diverse and frequent unintentional mutations were found in the so-called “on-target” site in generating knock-in mice with a single nucleotide substitution at the H1 enhancer Hox binding site (HBS) of Myf5 locus. Here, we report on the transition of these mutations to the next generation.

In zygotic GE, ribonucleoprotein complexes of Cas9 nuclease and 2 guide RNAs flanking HBS, alongside with a donor single-stranded DNA were electroporated at one-cell. After the 2-cell eggs were transplanted into the oviduct of pseudo pregnant mice, 15 offspring (N0) was obtained. Furthermore, N1 was obtained by mating N0 with the wild type. The target region was amplified by PCR using the tail DNA as a template, and TA cloning was performed to analyze the sequence.

The number of mutant allele types detected in most N1 mice was lower than that in N0 mice corresponding to each N1 mouse. However, not only the knock-in type but also unintentional mutations such as large deletion type and tandem knock-in type were detected in N1. Therefore, under the conditions of this study, most of the various on-target mutations were transmitted to the next generation without exclusion. These potential features of GE technology strongly suggest that proper selection for the purpose is essential for ensuring the safety of GE foods and medicines.

This study investigated the effect of continuous exposure to α-glycosyl isoquercitrin (AGIQ) as an antioxidant on a rat valproic acid (VPA)-induced autism model on hippocampal adult neurogenesis. Developmental VPA exposure suppressed hippocampal neurogenesis in male offspring targeting type-2a and type-3 neural progenitor cells in early postnatal life. AGIQ alleviated VPA-induced disruptive neurogenesis through amplification of neural stem/progenitor cells through increased reelin and neurotransmitter signals at the infant stage and increased synaptic plasticity at the adult stage.

This study examined 28-day repeated oral exposure effect of aluminum chloride (AlCl₃) on hippocampal neurogenesis in rats. Immunohistochemically, AlCl₃ showed a decreasing trend in type-2a neural progenitor cells and an increase of GFAP⁺ astrocytes in the dentate gyrus hilus. Gene expression analysis suggested ameliorating responses of interneurons to suppressed neurogenesis. Enhancement of oxidative stress caused by suppression of antioxidant defense mechanisms and nitric oxide production may have induced the progenitor cell apoptosis and the increased reactivity of astrocytes.

This study examined 28-day repeated oral exposure effect of lead acetate (PbAc) on hippocampal neurogenesis in rats. PbAc decreased TBR2+ cells but increased NeuN+ and ARC+ granule cells. PbAc also increased GFAP+ astrocytes and CALB2+ interneurons in the hilus. Mt1, Gsta5, Hmox1 and Il6 upregulated the expression. Thus, PbAc induced oxidative stress and neuroinflammation to inhibit differentiation into type-2b neural progenitor cells. Increases in granule cells and their ARC-mediated synaptic plasticity were suggested to be due to compensatory increased CALB2+ interneuron signaling.

Lipopolysaccharide (LPS) exposure induces neuroinflammation in animals. We examined the ameliorating effect of α-glycosyl isoquercitrin (AGIQ), an antioxidant, from the developmental stage on neural functions in an autism model produced by fetal LPS exposure in rats. On the weaning and/or adult stage, LPS-exposed offspring revealed abnormal behaviors and progressive disruption of hippocampal neurogenesis without accompanying neuroinflammation. AGIQ ameliorated the LPS-induced defects in neural functions in terms of behaviors and hippocampal neurogenesis.

Antibiotics are widely used in veterinary drugs and feeding additives, which causing the accumulation of antibiotics in animals, and excreted in the urine and feces at high concentrations in a biologically active form, and causing environmental pollution. In this study, zebrafish were used as an animal model to research the reproductive toxicity of tetracycline hydrochloride (TCH) at environmental concentrations. Four-month-old parental zebrafish (F0) were exposed to TCH (0.1 μg·L-1, 1 μg·L-1, and 100 μg·L-1) for 30 d and then mated to obtain F1 generation zebrafish. The results showed that low-dose TCH exposure caused a significant reduction in zebrafish spawning and a significant decrease in the overall quality of embryos (p < 0.05). TCH also delayed the development of ovaries and testes in the F0 generation and inhibited the maturation of follicles (p<0.05). TCH significantly inhibits the expression of reproduction-associated genes (eg. cyp19a, vtg1, and vasa mRNA) in a dose-dependent manner (p<0.05). Morphological changes such as pericardial edema and curvature of the spinal cord were observed in the F1 offspring. Further detection of genes related to bone development revealed that TCH inhibited the expression of runx1, sox9a, sox10, and col2α1a mRNA in juvenile fish. The above findings indicated that low-dose of TCH affects the reproductive function and embryonic development of zebrafish.

Keywords: Tetracycline hydrochloride; Zebrafish; Reproductive toxicity; Embryonic development

Evaluation of Ames test with small amount of sample is requested not only for pharmaceutical impurities and early stage drugs, but also for compounds that are difficult to synthesize or expensive. In this study, we used 24 well plate as a miniaturized Ames test and compared it with the existing test methods for four strains of TA100, TA98, TA97 and WP2 uvrA/pKM101. We report on the optimal test design for miniaturized Ames tests, the consistency of miniaturized Ames tests with existing test methods, evaluation methods, and usefulness for pre-incubation methods for miniaturized Ames tests.

The purpose of this study is to evaluate γH2AX as an in vivo genotoxic marker of ocular surface. The 0.5% doxorubicin solution was instilled once onto the eyes in male F344 rats, and immunohistochemical staining of the corneal epithelium was performed. As a result, γH2AX-positive corneal epithelial cells were observed at wide area similarly in the doxorubicin-treated and the negative control groups. Expression of γH2AX was constitutive and not increased under exposure of a genotoxicant. This implies a caution of utilizing γH2AX as a genotoxic marker on the ocular surface.

Thio-dimethylarsinic acid (thio-DMA^V), which is a pentavalent methyl arsenic compound in which the oxygen atom of dimethylarsinic acid (DMA^V) is substituted with a sulfur atom, has been identified in the human urine. Previously, we have showed 75% of clones have abnormal chromosome number in which isolated from the V79 cells exposed with 2.5 μM thio-DMA^V for 40 hours. In this study, we investigated the effects of long-term culture on chromosome number of each clones. Then we showed that some clones had indicated the different abnormal chromosome number after long-term culture.

Aromatic amines (AA) are widely used as dyes. Several decades ago, an increased risk for bladder cancer among male hairdressers using hair dyes containing AA was reported. While, the recent epidemiological studies have suggested the association between hairdressers and increased risk for skin cancer at the scalp and neck, sites of contact for hair dyes. In this study, we investigated the skin accumulation and genotoxicity of ortho-phenylenediamine (OPDA) as a model substance for AA used in hair dye to see if the experimental results support the epidemiological data. The skin absorption of OPDA was examined using C-14 labeled OPDA and the modified Franz diffusion cell method with a 3D reconstructed human epidermis model (3D-Skin), a cultured cell model similar to the human epidermis. We found that OPDA enters the 3D-Skin easily and time-dependently accumulates in it (about 60% of the added amount of OPDA entered the 3D-skin by 24 h, and almost no passed through the 3D-skin). In comparison to OPDA, aniline, the simplest structure of the AA, did not accumulate in the 3D-skin, and almost passed through the 3D-skin. The genotoxicity of OPDA is being investigated using 3D-Skin and HaCaT cells with γH2AX as an DNA damage indicator. Although the results are preliminary, the induction of γH2AX by OPDA has been confirmed in the concentration range that does not affect cell viability. As shown in this study, the high accumulation of OPDA, which has DNA-damaging properties, in the epidermis may cause the occurrence of skin cancer.

Nuclear receptors AHR, CAR, PPARα and PXR are involved in hepatocyte proliferation and chemical hepatocarcinogenesis, but the precise mechanisms are unknown. In this study, we investigated the ability of liver cancer-inducing agrochemicals on the activation of those nuclear receptors. Among the 35 agrochemicals that were selected as non-genotoxic carcinogens based on rat carcinogenicity test results, 6, 18, 4 and 20 chemicals activated AHR, CAR, PPARα and PXR, suggesting that CAR significantly contributes to liver tumor formation via non-genotoxic mechanisms.

The phosphorylated form of histone variant H2AX at Ser 139, called γ-H2AX, is strongly induced by DNA double-strand breaks (DSB) and is widely used as a biomarker of DNA damage. We have found that the increase of γ-H2AX-positive cells could be a useful marker of urinary bladder carcinogenicity of chemicals. On the other hand, hepatocarcinogens that do not exhibit hepatocyte proliferative stimuli were not efficiently detected by the increase of γ-H2AX-positive hepatocytes as an index. In this study, we sought novel biomarkers that can detect liver carcinogenicity of test chemicals, and found the increases of EpCAM- and cytoplasmic CD13 positive hepatocytes in the liver specimen of male F344 rats administrated with hepatocarcinogens for 4 weeks. Hepatocytes expressing EpCAM and CD13 were increased in the rats treated with hepatocarcinogens o-aminoazotoluene, 2-nitropropane, 3,3′-dimethylbenzidine, N-nitrosomorpholine, and thioacetamide. Moreover, EpCAM-positive hepatocytes was also increased in N-bis(2-hydroxypropyl)nitrosamine, p-cresidine, and di(2-ethylhexyl)phthalate treated groups. On the other hand, there were no increase of neither EpCAM- nor CD13-positive hepatocytes in N-nitrosodiethylamine and 1,4-dioxane, which increases γ-H2AX-positive hepatocytes. For non-hepatocarcinogenic carcinogens, neither EpCAM, CD13 nor γ-H2AX positive cells were increased in 4-chloro-o-phenylendiamine and N-ethyl-N-nitrosourea treated groups. These results suggest that EpCAM and/or CD13 could be useful biomarkers for the early detection of hepatocarcinogenicity in combination with γ-H2AX.

Our previous study revealed that acetamide (AA) induced chromosome aberrations (CAs) specifically in rat liver. However, involvement of CAs in its hepatocarcinogenicity remains unclear. In this study, we focused on the difference in tumor incidences between F344 and Wistar rats in carcinogenicity studies and examined inducibility of micronucleus and preneoplastic lesion of the liver in these strains after AA administration. As a result, the number of micronucleated hepatocytes was positively correlated with preneoplastic lesion, suggesting that CAs contributed to AA carcinogenesis.

Male rats were treated with aniline (AL), p-toluidine (PT), acetoaceto-o-toluidide (AAOT) or o-toluidine (OTD) for 4 weeks. Simple hyperplasia and increased cell proliferation were observed in urinary bladder of rats treated with AAOT and OTD, but not in rats treated with AL or PT. A larger amount of OTD were detected in the urine of rats treated with AAOT and OTD. In contrast, OTD was no or less detected in urine of rats treated with AL and PT. There results suggested that urinary OTD is a useful metabolite marker for predicting bladder carcinogenicity of aromatic amines in rats.

As current experimental methods used to evaluate carcinogenicity are often time-consuming and expensive, there is a great need to develop short-term and comprehensive animal models for risk assessment. We have successfully developed ultra-short-term rat model for detecting genotoxic hepatocarcinogen using expression data in livers 24 hours after single administration of chemicals. Ten genes were identified as marker genes and a SVM classification model was developed. We showed that our gene expression-based classification model can discriminate genotoxic hepatocarcinogens from other chemicals with high sensitivity and specificity.

Because detecting psychiatric adverse effects are difficult in conventional nonclinical toxicity studies, we attempted to detect them by evaluating social behavior of pair-housing dog living in EURO Guide-compliant cages. Diazepam was intravenously administered to beagle dogs and they were observed with a focus on their social behavior, either directly or by video. As a result, diazepam induced increased activity and sniff with following the other dog in behavioral patterns, which we considered suggesting psychological adverse effects.

Acephate (Ace), an organophosphate insecticide, is also toxic to mammals. In this study, we examined the effects of chronic Ace exposure in early life on the brain functions and gut microbiota in adult male and female mice, including acceptable daily intake (ADI) level concentrations. These results suggested that chronic exposure to Ace during the early developmental stages, even at ADI level concentrations, leads to neurobehavioral diseases and disruption of the gut microbiota. The correlation between Ace-induced behavioral effects and an altered microbiome will be further investigated.

Cefixime is the third generation cephalosporins, which is famous on high efficiency and low toxicity with widely used in clinic. However, the embryonic toxicity of cefixime needs to be further studied. In this study, zebrafish embryos were used as an animal model to evaluate the embryotoxicity of cefixime. Zebrafish embryos were exposed to cefixime (0-100 μ g/mL) from 4 hours after fertilization (4hpf), until observation. Cefixime had no significant effects on the mortality of zebrafish embryos, but the hatching rate was decreased significantly (P < 0.05) at 96 hpf. The mortality rate was significant raised (P < 0.05), swimming distance were deceased and tilted to one side (P < 0.05) at 120 hpf after 100 μ g / mL cefixime exposure. Also, the fluorescent spots of hair cells on the side of zebrafish larvae were decreased significantly with the increase of concentration by DASPEI fluorescence staining. Compare with control group, the otolith area were significantly decreased about 50% after Cefixime exposure (P < 0.01 or 0.05). Cefixime also down-regulated eya1 mRNA expression and increased stat3 mRNA expressions. Our results show that cefixime has a toxic effect on zebrafish development, especially on ear development. It is recommended to use with caution for pregnant women and children.

Keywords：Cefixime; Ototoxicity; Otoliths; Hair cells; Zebrafish embryos

A mycotoxin citrinin produced by Penicillium citrinum and P. verrucosum, mainly contaminates cereals. Previous studies demonstrated that citrinin exposure mainly exerted kidney toxicity but other adverse effects particularly against the immune system have to be examined. Therefore, this study was aimed to investigate the novel toxic effect of citrinin using a mouse model of psoriasis. A mouse model of psoriasis was generated by repetitive topical application of 5% imiquimod cream in female BALB/c mice. Standard rodent diet and rice samples with 3 ppm of citrinin were mixed to obtain a final citrinin concentration of 0.3 ppm, and citrinin contaminated diet was fed to mice daily. Skin thickness, scratching behavior, and trans epidermal water loss (TEWL) were monitored continuously during the imiquimod application. Immediately after the final imiquimod application, ear skin and auricular lymph node (LN) were sampled for further analysis. Only a slight increase was seen in skin thickness in the citrinin exposure group, however, citrinin exposure significantly exacerbated hyperkeratinization and inflammatory cell infiltration in histological evaluation. TEWL, which represents a condition of cutaneous barrier function, was significantly increased by citrinin exposure. In terms of immune function, the number of immune cells in LN (T cells and dendritic cells) and gene expression of IL-17 in skin tissue were significantly increased by citrinin exposure. Taken together, our results imply that oral exposure to citrinin exacerbates the symptoms of a mouse model of psoriasis.

We investigated the intervention of inorganic arsenic (AS, 10 ppm) exposure with allergic diseases. Atopic dermatitis (AD) and asthma models were developed by repetitive administration of 2.4-Toluenediisocyanate and mite allergen, respectively. In the AD model, trans epidermal water loss and AD symptoms were significantly aggravated by AS exposure. AS exposure also significantly increased the amount of Th2 cytokines, IL-33, TSLP, IgE positive B cells and total serum IgE in both models. Our findings imply that AS exposure exacerbated the pathology of allergy in mouse models.

Immunotoxicity due to innate immune activation is one of the potential adverse reactions of nucleic acid drug candidates. There are currently no guidelines and standard test systems for determining the potential toxicity of these compounds. In this study, we investigated the basic test conditions for developing an innate immune activation assay using human peripheral blood mononuclear cells.

We examined the suitability of TLR ligands, analytes, culture time, and donors. ODN 2216 and R 848 ligands responded well and were selected as positive controls for this study. There were no major differences in cytokine responses at 24, 36, and 48 h of exposure; we selected the 24-h time point as it was the shortest and the least influenced by acquired immunity. Donors with high expression levels of several CD markers were found to respond well to TLR ligands. If appropriate donor selection becomes possible, the number of donors required may be reduced.

In this study, we found that data variability and outlier evaluation were problematic; therefore, we also examined the method of data analysis. By taking the geometric mean of donors using the median of the data of one donor as the representative value, it was possible to obtain data with small variations between studies.

Although the conditions investigated in this study were very useful for the construction of the test systems, many issues are yet to be clarified, including the selection of appropriate reference compounds and in vitro-in vivo correlation; therefore, further studies are needed.

To aid preclinical prediction of immune reactions associated with cytokine release and caused by LMW pharmaceutical drugs and biologics, Alcyomics developed an in vitro human skin explant assay Skimune® involving activation of dendritic cells and T cells. Activated cells are added to a skin biopsy taken from the same healthy volunteer and incubated for 3 days and then routinely fixed and stained with haematoxylin and eosin and graded from Grade I-IV in order of severity. The activated cells are also assessed for T cell proliferation and the supernatants for IFNƴ analysis. Twelve Low Molecular Weight (LMW) drugs associated with T cell-mediated hypersensitivity reactions in the clinic (including abacavir and amoxicillin,) were compared with 5 drugs with rare or no clinical reports including (acetaminophen and cimetidine). Results for T cell proliferation (r=0.6 p<0.01), IFNγ release (r=0.51 p<0.04) and Skimune® (r=0.77, p<0.001) were statistically significant. The Skimune® method was modified for the testing of biologics and 17 biologics were tested. Biologics known to cause adverse reactions such as Campath and OKT3 as well as a TGN1412 analogue gave a Grade II or III histopathological skin response in 70% to 90% of 10 individual tests. There was a positive correlation between adverse clinical outcome and the response in Skimune® (r=0.815, p<0.0001). The present results demonstrate that Skimune® is a highly sensitive assay for predicting drug-induced adverse immune reactions involving acute cytokine release.

Animals must learn self-administration behavior before drugs can be tested. The learning process was compared using cocaine, a stimulant used in such studies, and ketamine, a depressant.

SD rats self-administered cocaine at 0.25 mg/kg/infusion (n=8) and ketamine at 3 mg/kg/infusion (n=9) in a fixed-ratio (FR) schedule. FR values increased in order from FR1, 3, 5, 7 to 10.

Five and 7 rats learned self-administration behavior at FR10 in approx. 24.6 and 29.9 days using cocaine and ketamine, respectively. While the number of animals was similar, cocaine was faster than ketamine, notably during FR1.

Polymyxin B (PMB), a last-line antibiotic used against multidrug-resistant bacteria, causes undesirable cytotoxic side effects, including nephrotoxicity and neurotoxicity. However, its mechanisms remain unknown. In this study, we found that the E3 ubiquitin ligase MKRN1 strongly protects cells from the toxicity of PMB by suppressing PMB-induced ferroptosis, a newly discovered iron-dependent cell death. Thus, our study identified MKRN1 as a key determinant of the toxicity of PMB, which may provide a new strategy to overcome the cytotoxic side effects of PMB.

In HepG2 cells, we analyzed the interaction between erythropoietin (EPO) production and autophagy induction by arsenate. 24-hour treatment of 100 μM arsenate promoted EPO production and induction of autophagy, but did not affect survival rate. The addition of arsenate and autophagy inhibitor SBI-0206965 (SBI) did not suppress EPO mRNA expression, and no decrease in survival rate was observed. Under siEPO treatment, treatment of arsenate and SBI decreased the survival rate. These results suggest that the promotion of EPO production and autophagy induction by arsenate are independent actions.

The purpose of this study was to evaluate the cytotoxicity of titanium dioxide nanoparticles using NHEK and LabCyte EPI. In NHEK, after 24 h exposure, three of seven titanium dioxide nanoparticles reduced cell viability at more than 500 μ g/ml. After 72 h exposure, all of them showed cytotoxicity. LabCyte EPI in 6 and 13 day cultures showed no cytotoxicity after 24 hours of exposure at concentrations up to 20 mg/ml. It was concluded that the 3D human skin reconstitution system is resistant to the cytotoxicity of titanium dioxide nanoparticles due to the epidermal barrier function, even in the immature layer.

Tributyltin (TBT), which has been widely used as an antifouling agent in marine paints, is a common environmental contaminant. Although neurotoxic effects of TBT have been reported in mammals, its mechanism remains unknown. In this study, we investigated the effect of TBT on autophagy, a lysosomal degradation pathway, in human neuroblastoma cell line SH-SY-5Y. We revealed that TBT inhibits autophagy and decreases protein levels of mature cathepsin B, a lysosomal cysteine protease. These results suggest that dysfunctional autophagy is involved in TBT-induced neurotoxicity.

In the cells, incorporated polystyrene particles (0.1 μm in diameter) (PP) accumulate in lysosome. To explore the possibility that PP-induced dysfunction of lysosome cause inhibition of autophagy and late endosome degradation, we evaluated the effects of PP on lysosomal membrane permeabilization (LMP) in Caco-2 cells. PP accumulated galectin-3 protein in the lumen of lysosomes, indicating induction of LMP. PP accumulated in lysosome involved with accumulation of autolysosome and late endosome. These results suggest that PP may induce lysosomal dysfunction via LMP.

Excess reactive oxygen species (ROS)-induced vascular smooth muscle cell (VSMC) hypermigration contributes to the development of arteriosclerosis. Here we show that intracellular sulfane sulfur donors inhibited the migration of VSMCs in response to PDGF. Specifically, PDGF increased the levels of cellular ROS, which were decreased by sulfane sulfur donors. Further, such molecules inhibited PDGF-induced activation of Akt and formation of focal adhesions. These findings suggest that sulfane sulfurs regulate ROS-dependent PDGF signaling, which may contribute to PDGF-induced migration of VSMCs.

Colistin, a class of polypeptide antibiotics that is effective against multidrug-resistant Gram-negative bacillus, is conceived as an essential drug for infectious diseases. Meanwhile, colistin frequently causes renal dysfunction that limits its clinical use. However, toxic mechanisms of colistin remain unknown. In this study, we investigated cellular responses to colistin in macrophages that play a major role in inflammation, and found that colistin stimulates IL-1β and HMGB1 release via two distinct mechanisms. These observations may provide insights into colistin-induced renal dysfunction.

Compelling epidemiological evidence has shown that trans-fatty acids (TFAs) consumption are associated with various diseases including atherosclerosis. However, the underlying mechanisms are largely unknown. Here, we show that elaidic acid, the most abundant TFA in foods, promotes apoptosis induced by cisplatin (a DNA crosslinking agent) in a p53-dependent manner, while that induced by doxorubicin (a DNA double-strand break-inducing agent) in a p53-independent manner. In this meeting, we discuss the difference of the underlying mechanisms between two types of apoptosis facilitated by TFAs.

We experimented with elucidation of the mechanisms of one of the more commonly seen adverse events, hepatitis, in the setting of combinatorial immune checkpoint inhibitor (ICI). PD-1^-/- mice were treated with anti-CTLA4 and/or Epacadostat. Necropsy was performed 2 weeks after initial treatment and single cell RNAseq was performed on CD45⁺ liver cells. Single cell transcriptomics revealed that the liver injury due to ICIs is associated with a population of CD8 T cells expressing activation/exhaustion markers; and ICIs combination treatment decreased T cell clonality, indicating a broadening of the immune repertoire.

We previously demonstrated that cannabidiolic acid, a major component of the fiber-type cannabis plant, down-regulates COX-2 expression and inhibits cell migration in human breast cancer MDA-MB-231 cells (Toxicol. Lett., 2012; J. Toxicol. Sci., 2014; J. Nat. Med., 2017; Nat. Prod. Commun., 2017; J. Toxicol. Sci., 2020). However, the details of down-regulation mechanisms of COX-2 expression by CBDA are still unknown. Here, we show that CBDA can down-regulate COX-2 expression through the abrogation of PPARβ/δ signaling.

Significant individual differences have been noted in “sick house syndrome”, while the mechanism has not been elucidated. To elucidate the mechanism of individual differences in hyper-responsiveness in this study, we investigated the expression levels of both matrix metalloproteinases and its endogenous inhibitor, TIMPSs, as key factors in airway remodeling. Real-time RT-PCR revealed the significant individual differences in the expression levels of MMP8 in lungs. The results obtained in this study may provide important information for discussing on individual differences in airway hyperresponsiveness.

[Purpose] Platelet activation is known to involve in liver injury. In addition to classical activation leading to blood coagulation, activation by mitochondrial permeability transition (MPT) is drawing attention from pathophysiological point of view. In the present study, we examined if MPT-mediated platelet activation is involved in acetaminophen (APAP)-induced liver injury in mice.

[Methods] Drug-induced MPT in isolated platelet was evaluated using flowcytometry. C57B6/J mice (8 week-old) were fasted overnight and treated with 300 mg/kg APAP. Platelet accumulation was assessed in frozen liver section. Ca2+-induced MPT was evaluated by decrease of the absorbance at 540 nm using isolated liver mitochondria.

[Result & Discussion] Isolated platelets were activated by well-known MPT-inducers, such as amiodarone and troglitazone in the presence of thrombin. These activations were not observed in platelets from cyclophilin D (regulator of MPT pore opening) knockout mice, indicating that platelet could be activated by drug compounds via MPT. Increased susceptibility to Ca2+-induced MPT and platelet accumulation in the liver was observed 2 hr after APAP administration prior to ALT increase. The enhancement of MPT sensitivity was suppressed by pre-administration of either heparin (anticoagulant) or cyclosporin A (MPT inhibitor), implying that APAP administration first induced blood coagulation and MPT in any cells, resulting in increased hepatic MPT sensitivity. Although not strictly proven, MPT-mediated platelet activation might occur in APAP-induced liver injury model mice.

Overdose when APAP is metabolized by CYP2E1, which consequently increases the reactive Acetaminophen (APAP)-induced liver injury (AILI) occurs as a consequence of APAP metabolite N-acetyl-p-benzoquinone imine (NAPQI). APAP overdose can cause a serious acute liver injury and acute hepatic failure that can result in need for liver transplantation or death. Although AILI is also reported to be immune related, the detail is unclear. In this study, the mechanism of AILI pathogenesis was evaluated in vivo and in vitro study. In AILI model rats (800 mg/kg, ip), the levels of AST and ALT levels were increased only at 12-24 h. Caspase (C)-3 and C-9 levels rose at 6-9 h, whereas C-8 level rose hours later, moreover, 24 h after; C-3/-8/-9 levels re-rose. In human hepatocyte cells, cytochrome c was released from the mitochondria which is promoted by oxidative stress due to drug metabolism (APAP 1.0 mM) and C-9 was activated. Thus, mitochondrial damage might be caused by NAPQI as early reaction. In the next stage, inflammasomes of human antigen presenting cells were activated by damage-associated molecular patterns (DAMPs), which were released from damaged hepatocyte caused by APAP (0.3 mM). It is confirmed that AILI is included in immune related adverse event, thereby, in case of N-acetylcysteine refractory, it is suggested that steroid hormones administration should be effective and recommended as a novel strategy for AILI with immune related adverse events.

Glycation products cause various inflammatory disorders and accelerate aging. In this study, we have focused on dihydropyrazines (DHPs)—a group of glycation products—and characterized their cellular effects. Recently, we reported that 3-hydro-2,2,5,6-tetramethylpyrazine (DHP-3) suppressed the TLR4-mediated inflammatory signaling pathway in lipopolysaccharide-stimulated HepG2 cells and showed toxicity in a dose-dependent manner. In the present study, to elucidate the molecular mechanism underlying the effects of DHP-3, we examined the role of the receptor for advanced glycation end products.

Vascular toxicity is important for understanding organ toxicity. Vascular endothelial cells, which cover the luminal surface of blood vessels, can be a target of the toxicity of chemical agents. In the present study, we investigated the effects of methylmercury on the expression of metal transporter ZIP8 in cultured vascular endothelial cells. It was found that methylmercury induces endothelial ZIP8 by activating the EGFR-p38 MAPK-PKA pathway. Since cadmium enters endothelial cells via ZIP8, methylmercury may intensify cadmium toxicity to the cells.

Drug-induced fatal liver injury is a serious concern, but its mechanism is unclear. We hypothesized that inhibition by drugs of inflammatory reaction and fibrosis essential for liver regeneration could finally induce fatal outcome. Then we focused on hepatic stellate cells, which is closely associated with fibrosis. Present study investigated the effects of benzbromarone (BBR), that induced fatal liver injury in clinical, on hepatic stellate cell line LX-2.

As a result, BBR suppressed inflammatory response and fibrosis, which may be due to a decrease in mitochondrial membrane potential.

Here we show that OBG, which was identified as a naturally occurring LXR ligand without causing hepatic steatosis, dramatically increases the expression of LXR-alpha in Fa2N-4 cells that overexpress LXR-beta. However, the expression level of SREBP-1c, a known target of LXR-alpha, remains marginal in OBG-treated Fa2N-4 cells, in which LXR-alpha expression is upregulated by LXR-beta. Furthermore, we show that OBG stimulates the expression of KLF15, a repressive factor of LXR, thereby reducing SREBP-1c expression.

Methylmercury (MeHg) results in neuronal cell death and injures the specific areas of the brain. However, the detailed mechanism has yet to be fully understood. Here, we report a possible involvement of endoplasmic reticulum (ER) stress in MeHg-induced neuronal apoptosis in vivo. We found that ER stress was specifically observed in several specific areas in the cerebral cortex. In addition, we detected the signals derived from the unfolded protein response prior to neuronal apoptosis. These results indicated that ER stress is implicated in the neuronal loss by MeHg.

Vascular endothelial cells, which cover the luminal surface of blood vessels, can be a target of the toxicity of chemical agents. In the present study, we investigated the role of reactive sulfur species (RSS) in the proliferation of the cells in a culture system. The experiments indicated that RSS promote endothelial cell proliferation. It was also found that FGF-2 induces CSE, an RSS-producing enzyme, through activation of the ERK1/2 pathway and increases RSS to intensify the promotion of the proliferation by the growth factor.

We attempted to elucidate a role of PGRMC1, membrane-bound heme protein, on induction of ferroptosis. In this study, we showed that overexpression of PGRMC1 increased the sensitivity to ferroptosis promoters, and that ferroptosis promoters-treatment increased lipid peroxide, decreased intracellular free heme concentration, and increased intracellular free iron in PGRMC1 stable expression cell lines. These results suggest that overexpression of PGRMC1 might suppress the ability to resist ferroptosis promoters in lung cancer cells.

The Organ-on-a-Chip field provides better insight into healthy biology, but also disease, and drug development, through more physiologically relevant models. In drug development, Organs-on-Chips aim to improve the evaluation of compound efficacy. In addition, secondary effects and toxicity could be assessed in a more relevant context. Thereby, Organs-on-Chips contribute to multiple phases of the drug discovery and development pipeline.

Here, we report the screening of 1546 compounds on a 3D vascular endothelial sprouting model in the OrganoPlate® 3-lane 64. A plate comprises 64 chips, consisting of three channels each. In this model, the middle channel was filled with collagen-I, supporting a tube of HUVECs in the flanking channel which forms under gravity-driven perfusion. In addition, the ECM serves as scaffold for the angiogenic sprouting, induced through a gradient of angiogenic factors. Using a 1546 compound protein kinase inhibitor library, we screened for an effective inhibitor or enhancer of angiogenic sprouting. Through fluorescent staining and high content imaging, the effect of each compound on multiple sprouting parameters could be captured. In addition, potential off-target effects and toxicity were checked by assessing the morphology of the parent tube simultaneously. In conclusion, our compound screening using the OrganoPlate® 3-lane 64 yielded multiple non-toxic angiogenic modulators. To our knowledge, this effort is also the largest reported Organ-on-a-Chip screen, demonstrating the potential of the OrganoPlate® platform in drug discovery and development.

Drug delivery across the BBB represents a significant clinical challenge. In vitro models are widely used for prediction of permeability and toxicity of compounds. These are typically achieved via culture of brain endothelial cells on permeable membrane inserts. However, such models are limited in physiological relevance due to their 2D nature, lack of extracellular matrix, presence of an artificial membrane, and absence of perfusion. Microfluidic models are becoming increasingly adopted. The OrganoPlate is a microfluidic platform enabling perfused, 3D co-culture of up to 96 tissues in a membrane free manner. It allows growth of tubular endothelial structures and is based on a 384-well plate. To create a BBB model, brain endothelial cells are seeded against a collagen-I extracellular matrix. Under perfusion, endothelia form confluent tubule structures, accessible from both apical and basolateral sides. It comprises a perfused 3D microvessel of hBMECs, with option to include cell types such as astrocytes and pericytes. It shows expression of phenotypic and junctional proteins, transporters, and receptors. Using fluorescent assays, we demonstrate low permeability of primary hBMECs to sodium fluorescein, as well as functional P-glycoprotein and GLUT1 activity. Barrier and transport readouts are performed directly in-plate without the need for sampling. Time-dependent barrier disruption by toxic compounds is measured in 40 cultures in parallel with OrganoTEER. This BBB model serves as a versatile model for high-throughput applications including toxicity and transport studies.

Epigenetic toxicity is thought to be caused by the disruption of epigenetic regulation due to chemical exposure. We constructed a novel in vitro system for detection of alterations in DNA methylation status by chemicals. In Neuro-2a, we introduced a reporter system by selecting Agouti-IAP and Daz1 as promoters. Using 5azaC, we found an increase in luciferase activity, suggesting a decrease in methylation. Pyrosequencing confirmed the decrease in DNA methylation. Thus, we could develop a convenient cellular system for detection of modifying effect of chemicals on DNA methylation status.