[Purpose]

cDNA-uPA/SCID mouse with a humanized-liver (PXB-mouse®) presents a fatty liver because of species specificity of growth hormone (GH) that suppress lipid synthesis in human hepatocytes. In this study, we aimed for determining an appropriate dosage of human-GH (hGH) for evaluation of human drug-induced fatty livers.

[Methods]

hGH was continuously administered for 14 days with osmotic pumps at predetermined dosages. Based on the results, TO901317 (TO), one of LXR agonists, was orally administered into hGH-administered PXB-mice. Long acting hGH drugs (Sogroya and Ngenla) were also subcutaneously injected for a minimally invasive treatment.

[Results and Discussion]

The improvement in hepatic steatosis was observed at 0.25 mg/kg or more hGH dosing. Especially, serum hGH levels in PXB-mice administered with 0.25 mg/kg were comparable to physiological levels in human. Based on these results, TO was orally co-administrated (0.25 mg/kg hGH for 14 days, and 10, 30, 100 mg/kg TO for 4 days), presenting TO-dosage-dependent excitation of lipid accumulation in humanized livers. Besides, more sensitive evaluation of liver steatosis was constructed by a long-term co-administration of hGH (0.25 mg/kg, 4 weeks) and TO (30 mg/kg, 7 days). These results suggested that the hGH-administered PXB-mouse would be a valid model animal for the study of drug-induced fatty livers. Sogroya and Ngenla also improved fatty livers respectively.

[Conclusion]

We determined the appropriate hGH dose to PXB-mice that improves fatty livers and achieves blood hGH concentration comparable to the human physiological level. This hGH-administered PXB-mouse could be a valid evaluation model for drug-induced fatty livers. In addition, we are analyzing the expression levels of drug-metabolizing enzymes and transporters.

Purpose: Phosphoric acid has been widely used as an acid for crystallization of pharmaceuticals. It has been reported that bolus intravenous administration of phosphoric acid to rats causes glomerulopathy as a nephrotoxicity phenotype due to calcium phosphate crystallization during filtration through glomerular capillaries (Tsuchiya et al., 2004). In this study, we conducted an intravenous toxicity study of phosphoric acid in rats to investigate whether this nephrotoxicity can be avoided by decreasing the transient high phosphoric acid concentration in plasma by reducing the rate of administration.

Methods: Phosphate buffer was administered to female rats for 11 days by intravenous bolus administration or 30-minute infusion. Parameters evaluated in this study included mortality, clinical observations, body weights, clinical pathology (hematology, clinical chemistry and urinalysis), gross pathology, organ weights and histopathology.

Results: In the bolus administration group, increase of cystatin-C and albumin (nephrotoxicity biomarkers) in the urinalysis and glomerulopathy in the kidney histopathological examination were observed. On the other hand, 30-minute infusion group showed only transient increase of inorganic phosphorus and decrease of calcium in the clinical chemistry. These transient abnormal findings were milder than in the bolus administration group.

Conclusion: Infusion administration may have lower risk for phosphoric acid induced glomerulopathy or abnormal findings for nephrotoxicity biomarkers than bolus administration in rats.

Gastrointestinal (GI) toxicity (e.g., diarrhea) is one of the most serious adverse reactions leading to the discontinuation of oral drug development. However, drug-induced GI toxicity is not easy to assess because its mechanism is complicated by multiple factors. For instance, serotonin (5-HT), a major GI hormone, is associated with gut microbiota, which plays a role in various GI physiology, but its interaction with oral drugs has not been fully clarified yet. In the present study, we examined the hypothesis that changes in GI disposition of 5-HT due to orally administered drugs can influence gut microbiota dynamics, leading to disorders of the GI fluid regulation, using metformin which clinically causes diarrhea as a side effect. Studies using Caco-2 cells and transporter-expressed Xenopus oocytes suggested that metformin inhibits 5-HT uptake mediated by transporters (e.g., SERT). Administration of metformin into the intestinal lumen significantly increased the GI fluid volume and 5-HT levels. A similar tendency was observed after the long-term administration of metformin. Further analysis for fecal microbiota indicated that metformin influences gut microbiota dynamics (mainly Turicibacter) as well as GI 5-HT levels. When transferring the feces of these rats into the intestinal lumen of another untreated rat, the recipient rat showed the same diarrheal symptoms as the donor rat. Our findings indicate that drug-induced GI toxicity is caused by the changes in gut microbiota via an increase of the luminal 5-HT levels by inhibition of transporter-mediated GI 5-HT uptake.

Safety issue for cardiovascular system is one of the main reasons for termination of drug development. Accordingly, it is important to accurately understand the risk of drug candidates on the cardiovascular system from the early stage of drug development. The key function of heart is periodic contraction and relaxation of myocardial cells, which is controlled by ionic currents flowing through the myocardial cells. Safety issue such as prolongation of QT interval and proarrhythmia are induced by the effects of drugs on ionic currents, so accurate understanding of these effects will enable us to design safer drug candidates on heart.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) express major ion channels in human cardiomyocytes, and the effect of drug candidates on ionic currents can be evaluated by measuring action potentials using the patch clamp method. On the other hand, it is difficult to assess the effects of drug candidates on each ionic currents from extracellular field potential measured by multi-electrode array (MEA).

In this study, we constructed a machine learning model that performs feature value analysis on extracellular field potential measured by MEA, and estimates the main inhibition channel. This model was evaluated by experimental data of several drugs known to have effects on specific ionic currents, including hERG inhibitors. However, predictability was low when data obtained by drugs with similar inhibition properties were not available within the training data. To solve this problem, we created a simulation system of extracellular field potentials using the myocardial action potential model (ORd model) and incorporated in the machine learning model. It enabled us to estimate inhibitory mechanisms even if drugs with similar properties were not in the training data.

Background and purpose: In a rat 26-week percutaneous dose toxicity study of compound A, lens opacity (posterior cortex and posterior capsule) was observed only in the 10% and 20% formulation groups, despite almost the same plasma compound A levels in all groups. In this study, to clarify the mechanism of lens opacity and the effect of different administration methods on the lens, ex vivo and in vivo studies were conducted.

Methods: In ex vivo study, the effect on cholesterol biosynthesis was investigated using rat lens explants. In in vivo study, to demonstrate that contamination of compound A into the eye was responsible for lens opacity, a 4-week percutaneous dose study for determination of compound A levels in the eye, and an 8-week ocular dose study were conducted. To evaluate the effects on the lens in the conditions without ocular contamination, a 26-week subcutaneous dose study was conducted. To investigate the effect on the lens by suppressing the dose volume, a 2-year percutaneous dose study was conducted.

Results: From each study, data on the mechanism of onset of lens opacity caused by compound A and data on the effect of different administration methods on the lens were obtained. In this meeting, we plan to report on the more detailed results of each study, and we would like to discuss the challenges and perspectives in conducting a percutaneous dose toxicity study in rats.

Active metabolites of chemicals by enzymes including cytochrome P450 (CYP) are important in the pharmacological and toxicological effects in some cases. While it has been believed for a long time that thalidomide causes characteristic limb malformation only in rabbits and primates including human, the involvement of their CYP3A subtypes (CYP3As) has been suggested. Recently, it was reported that thalidomide caused defects of zebrafish pectoral fins, homologous organs of limbs in mammals, as well as other deformities. On the other hand, however, it was also reported that conventional aqueous exposure to thalidomide has only little effect in developing zebrafish. In this study, we generated human CYP3A7 (hCYP3A7) -, human CYP3A4 (hCYP3A4)- and human CYP1A1 (hCYP1A1) - expressing zebrafish (F0) using transposon system. Thalidomide caused pectoral fin defects and other malformation including pericardium edema and otic vesicles in both hCYP3A7- and hCYP3A4-expressing zebrafish but not in wild-type and hCYP1A1-expressing embryos/larvae. Thalidomide also reduced expression of fibroblast growth factor 8 in the pectoral fin bud in hCYP3A7-expressing embryos/larvae. The results suggest the involvement of human CYP3As but not by hCYP1A1 in augmentation of thalidomide teratogenicity. hCYPs-expressing zebrafish is an useful model for developmental toxicology.

Zebrafish embryotoxicity test (ZET) is useful for the evaluation of teratogenic potential at the drug discovery stage, but it is limited to qualitative evaluation. Concentration-response relationship is ususually observed for survival and morphology scores of larvae in ZET; therefore, the present study was conducted with the hypothesis that an evaluation of teratogenic risk based on exposure levels would be possible.

We used already known rat teratogens to compare exposures (rCmax and rAUC) at the lowest-observed-adverse-effect-level in rats to the lowest-effect-concentration of formulations (zCOF) or the teratogen concentration in larvae (zCIL) in ZET. Fertilized eggs of zebrafish were continuously exposed to teratogen formulations from 5 hours post fertilization (hpf) to 120 hpf. On 120 hpf, morphological abnormalities of the larvae were scored and determined the positive concentrations. zCIL was measured by a high-performance liquid chromatography. zCOF or zCIL as zCmax was compared to rCmax, respectively. In addition, zAUC calculated from zCmax was also compared to rAUC.

As a result, the ratios of zCmax (zCOF or zCIL) to rCmax were almost ranging from10^-1 to 10¹-folds. Meanwhile, zAUC calculated from zCIL was almost equivalent to rAUC, indicating that the rAUC could be estimated with high predictability from the ZET results. This study suggests that ZET is a useful evaluation system that can accurately and quantitatively assess teratogenic risk at the drug discovery stage.

Genotoxicity of chemicals is evaluated using methods such as Ames test that detect mutations in indicator genes. Error-corrected sequencing (ECS) is a newly developed method that uses complementary strand-derived DNA sequences; it can reduce sequencing error frequency to ca. 1/10⁷ bp. ECS can directly detect mutations induced by mutagens; this could help overcome the limitations in genotoxicity evaluation. The Hawk-Seq^TM, an ECS developed by Kao, could sensitively detect the mutations induced by various mutagens conventional test models. Evaluating the utility of ECS-based assays in terms of sensitivity and reproducibility is important for their promotion. Therefore, we initiated a collaborative study in the Mammalian Mutagenicity Study Group in the Japanese Environmental Mutagen and Genome Society to evaluate the ECS methods. We aimed to evaluate the sensitivity and reproducibility of Hawk-Seq^TM in terms of mutation frequency and pattern. We evaluated the effects of the experimental device and sequencers on mutation analysis to set a common protocol. The DNA electrophoresis tools, Agilent 4200TapeStation and 2100Bioanalyzer, were both applicable to a quality control of Hawk-Seq^TM library preparation. The background error (e.g. C>G) frequencies obtained in Hawk-Seq^TM analysis varied up to two-fold between sequencers, while the mutation frequency and pattern induced by mutagens were similar. Therefore, we decided the protocol and initiated technical transfer. We will evaluate inter-laboratory reproducibility using mouse DNA samples, thereby evaluate utility of ECS-based methods.

Homologous recombination (HR) is one of major DNA repair pathways and utilizes homologous DNA as a template to accomplish acute, error-less DNA repair. Defects in HR cause chromosome instability, and mutations in genes involved in HR are related to cancer (e.g. Brca2) and genetic disorders (e.g. Blm) in humans. Thus, understanding the mechanisms of HR is critical for therapies for cancer and diseases. Acetaldehyde is one of DNA inter-strand crosslink (ICL)-causing chemicals produced in vivo, and induces DNA damage and cancer in humans. While studies using mice have uncovered critical roles of fanconi-anemia (FA) pathway in ICL repair, roles of HR in ICL repair remain elusive, mostly due to early embryonic lethality of mice with mutations in genes involved in HR. SWSAP1 is a recently identified RAD51 paralog required for efficient HR in mice and humans, and Swsap1 knock-out mice develop normally, though infertile, allowing us to investigate its roles as an HR factor in ICL repair. Here we study Swsap1 Aldh2 double knock-out mice where acetaldehyde accumulates in vivo by ethanol exposure, and revealed that the frequency of micronucleus formation is increased in mouse embryonic fibroblasts (MEFs) and the number of hematopoietic cells in born marrow is reduced. Our results suggest essential roles of SWSAP1, likely HR-mediated repair pathway, in ICL repair even when FA-pathway is active, and provide new insight into mechanisms by which HR maintains chromosome stability and suppress cancer.

Object: Cytokine release syndrome (CRS) has a variety of systemic effects. However, the detailed mechanisms are unclear. In this study, we focused on cardiotoxicity and assessed the mechanism of toxicity using mouse and monkey models of CRS. Materials and Methods: Male mice were once administered (i.p.) LPS (Exp. 1), and cynomolgus monkeys were infused LPS for 2 hours (Exp. 2). Clinical pathology, necropsy and histopathology were examined. In addition, male cynomolgus monkeys were once administered at doses of 100 and 1000 µg/kg of antibody X, a CRS-inducer. Clinical pathology was examined (Exp. 3). Results: Exp. 1) Hypercytokinemia was observed with a peak at 3 hours, leading to severely decreased body temperature at 7 to 24 hours. Degeneration/necrosis of cardiomyocytes and increased serum troponin T (TnT) were observed at 24 hours. Exp. 2) Hypercytokinemia was observed at 2 to 6 hours, followed by hypotension and increased neutrophils/CRP. A transient and slight increase in serum TnT was observed in the animal with the most severe hypercytokinemia, but no histopathological abnormality was observed. Exp. 3): Hypercytokinemia was observed at 2 to 4 hours followed by severe hypotension and a marked increase in serum TnT at 24 hours in the monkey with 1000 µg/kg. Discussion and Conclusion: All mice and monkeys with cardiac injury or significant increases in serum TnT were accompanied by severe hypothermia or hypotension. These results suggest CRS-induced cardiotoxicity was mainly due to ischemic changes associated with deterioration of systemic circulation.

Small molecular pharmaceuticals (SMPs) have been developed for years and used widely in therapy for various diseases. Although it’s recognized that the immunogenicity of SMPs is weak, excessive immunity activated by SMPs is usually occurred in non-clinical and clinical tests, leading to severe consequences such as toxic epidermal necrolysis, drug-induced hypersensitivity syndrome and anaphylaxis. Therefore, it’s critical to assess immunogenicity of systemic-exposed small molecular pharmaceuticals with evaluation system having high throughput and extrapolation. Human Cell Line Activation Test (h-CLAT) is an in vitro method for the assessment of skin sensitization potential of chemicals. We considered that whether h-CLAT can also be used in assessment of systemic response potential of chemicals. Thus, the purpose of this study is to clarify whether there is a relationship between in vitro h-CLAT results and in vivo systemic immune activation induced by SMPs. Compound A induced drug rush when treated to monkeys. Compound B resulted in severe inflammation when treated to rats. It was found they were positive for h-CLAT within 2-fold safety margin. Next, we used anti-bacterial and anti-viral drugs that is reported to induce immune risk in clinical application. Our results showed that short-administration drugs including clindamycin, rifampicin, colistin and spectinomycin are positive for h-CLAT with safety margin less than 10-15-fold, long-administration drugs including raltegravir, ritonavir, nelfinavir and efavirenz are positive for h-CLAT with safety margin less than 4-17-fold. Conclusively, our study indicated h-CLAT results have a good extrapolation into non-clinical/ clinical consequences. And we recommended h-CLAT for screening immunity induction of systemic-exposed small molecular pharmaceuticals in early stage.

Understanding the interface between microplastics and biological systems will provide new insights into the impacts of microplastics on living organisms. When microplastics enter the body, they are engulfed preferentially by phagocytes such as macrophages. However, it is not fully understood how phagocytes recognize microplastics and how microplastics impact phagocyte functions. In this study, we demonstrate that T cell immunoglobulin mucin 4 (Tim4), a macrophage receptor for phosphatidylserine (PtdSer) on apoptotic cells, binds polystyrene (PS) microparticles as well as multi-walled carbon nanotubes (MWCNTs) through the extracellular aromatic cluster, revealing a novel interface between microplastics and biological systems via aromatic-aromatic interactions. Genetic deletion of Tim4 demonstrated that Tim4 is involved in macrophage engulfment of PS microplastics as well as of MWCNTs. While Tim4-mediated engulfment of MWCNTs causes NLRP3-dependent IL-1β secretion, that of PS microparticles does not. PS microparticles neither induce TNF-α, reactive oxygen species, nor nitric oxide production. These data indicate that PS microparticles are not inflammatory. The PtdSer-binding site of Tim4 contains an aromatic cluster that binds PS, and Tim4-mediated macrophage engulfment of apoptotic cells, a process called efferocytosis, was competitively blocked by PS microparticles. These data suggest that PS microplastics do not directly cause acute inflammation but perturb efferocytosis, raising concerns that chronic exposure to large amounts of PS microplastics may cause chronic inflammation leading to autoimmune diseases (Kuroiwa, M. et al., Sci. Total. Environ., 2023 published online).

 Abacavir (ABC) hypersensitivity (AHS) is restricted to patients carrying HLA-B*57:01. AHS is characterized with presentation of fever, rash, malaise, and various organ dysfunction. Rechallenge with ABC in patients who have experienced AHS should be avoided because it might even precipitate a life-threatening reaction. Previously, we constructed HLA-B*57:01 transgenic mice and observed skin rash, fever, and inflammatory cell infiltration in the dermis and liver in the mice treated with 20 mg/body/day ABC p.o. for five days in combination with PD-1 knockout and CD4 T cell removal. Here, we examined the outcome of rechallenge with ABC in the mice.

 ABC rechallenge (20 mg/body p.o.) was conducted after a washout period of 15-18 weeks, and serum, skin, and liver samples were collected at 24 hours after the ABC administration. In the rechallenged mice, skin rash and clear infiltration of lymphocytes were observed. More infiltration of lymphocytes into the skin could be seen than in mice which experienced only the first course of ABC. In the mice treated with ABC for five days without rechallenge, the infiltrated lymphocytes formed a cluster in the liver marginal lobules. On the other hand, with ABC rechallenge, lymphocytes aggregation was often observed in the liver sinusoids, and the serum ALT level was increased.

 In conclusion, ABC-rechallenge exhibited more immunological outcomes and toxic symptoms in the examined tissues of HLA-B*57:01 transgenic mice. This mice model can be utilized to elucidate the onset mechanism of severe clinical recurrence by rechallenge with ABC.

Cardiovascular diseases are the leading cause of global mortality; with endothelial dysfunction as the initiating stage and air pollution as a substantial environmental risk factor. Several lines of evidence have linked air pollution to cardiovascular morbidity and mortality. However, there is dearth of evidence on the health effects of air quality in enclosed subways with high metal composition due to wear of rail and brake. Therefore, the aim of this study was to investigate the effects of iron oxide particles on the health and physiological function of the vascular endothelium. Human Umbilical Vein Endothelial cells (HUVECs) were cultured as monolayers before exposure to 0, 25, 75 and 200 μg/mL of iron oxide particles for 6, 24 and 48 h. Cell viability, Lactate dehydrogenase (LDH) release, reactive oxygen species (ROS), Interleukins 6 and 8 (IL-6 and IL-8), Endothelin 1 (ET-1) and Plasminogen Activator Inhibitor 1 (PAI-1) production were assayed. Iron oxide caused significant dose-dependent loss in HUVECs’ cell viability with corresponding substantial increased LDH release. In addition, increased production of ROS and pro-inflammatory cytokines such as IL-6 and IL-8 were observed. Furthermore, production of PAI-1, a fibrinolytic modulator and ET-1, a vasoconstrictor significantly decreased compared to control with accompanying cell death and loss of membrane integrity. Overall, these results suggest that iron oxide-rich subway particulate matter can significantly impair endothelial function and cause coagulation dysfunction especially through gross cellular damage.

Carbon nanotubes (CNTs) are the highly representative products of nanotechnology. Some multi-walled CNTs (MWCNTs) reportedly induce macrophage cell death and NLRP3 inflammasome activation leading to granuloma and mesothelioma in rodents; however, it remains unknown how macrophages recognize MWCNTs on their cell surface. Recently we identified mouse T cell mucin immunoglobulin 4 (Tim4) as a CNT-recognizing receptor (Omori et al., Cell Rep., 2021). In human, however, we found that macrophages lacking Tim4 also recognize MWCNTs, indicating the existence of unknown human CNT-recognizing receptor. Here we by in silico screen identify sialic acid-binding lectin (Siglec)-5 and Siglec-14 as human CNT-recognizing receptor. Ectopic expression of Siglec-5 or Siglec-14 on THP-1 cells, a human monocytic leukemia cell line, enabled these cells to recognize MWCNTs. Importantly, Siglec-14 mediated phagocytosis of MWCNTs and induced the subsequent IL-1β secretion. R406, a spleen tyrosin kinase (Syk) inhibitor, or genetic deletion of Syk attenuated the Siglec-14-mediated phagocytosis and IL-1β secretion. Taken together, these results suggest that Siglec-14 mediates Syk-dependent inflammatory responses to MWCNTs (Yamaguchi et al., Nat. Nanotechnol., 2023).

Ferroptosis, a recently identified form of atypical programmed cell death, is triggered by iron-dependent accumulation of lipid peroxides. Because cancer cells contain higher concentrations of iron than normal cells, ferroptosis is considered as an effective approach to eliminate cancer cells specifically. However, there are no anti-cancer drugs that induce ferroptosis. In this study, we screened novel ferroptosis inducers from among existing drugs, and found that polymyxin B (PMB), a polypeptide antibiotic agent, can induce ferroptosis in cancer cell lines. Mechanistically, PMB induced ferroptosis by increasing intracellular free iron through ferritinophagy, a type of autophagy that specifically degrades the intracellular iron-storage protein ferritin. Moreover, it turned out that PMB upregulates the expression of NCOA4, an inducer of ferritinophagy. Interestingly, PMB translationally upregulated the NCOA4 expression under iron-rich conditions, even though, in general, NCOA4 is upregulated in a transcription-dependent manner when iron is depleted. Together, these findings suggest that PMB induces ferritinophagy-dependent ferroptosis by targeting NCOA4, and works as an inducer of ferroptosis. Considering that PMB is an already-approved drug, our study raised the possibility that PMB can be applied to an anti-cancer drug through drug repositioning approaches.

To elucidate the immediate effects (pharmacological action or toxicity) of drugs on cells, it is effective to capture the spatiotemporal dynamics of multiple signaling proteins as a network. Our novel analysis method, the PLOM-CON (Protein localization and modification-based covariation network) analysis, allows for the visualization of the changes in protein quantity, localization, and quality (such as post-translational modifications), in response to specific stimuli, as a network. This is achieved by quantification of immunofluorescence images and by generating a network that consists of edges that connects the proteins having a strong temporal correlation in feature quantities. In this study, we obtained covariation networks for ~50 proteins in insulin-stimulated rat liver H4IIEC3 cells. We discovered that Akt, a central molecule in insulin signaling, and its phosphorylated form p-Akt (Ser473) served as the hub of the network. Our analysis also revealed that the proteins involved in glycogen synthesis such as p-GSK3β showed temporal correlation mainly at the actin domain, a transiently formed structure in response to insulin. By inhibiting actin domain formation, we found that glycogen synthesis was also inhibited, which suggested that the actin domain is the site in which proteins accumulate to regulate glycogen synthesis in response to insulin signaling. In addition, the networks generated under conditions in which actin domain formation was inhibited by CK666 treatment (which recapitulates diabetic hepatocyte conditions) showed a significant change compared to the network in control conditions. Thus, PLOM-CON analysis not only aids in uncovering location-dependent protein function but also provides a means of verifying cellular state and drug toxicity by sensitively capturing cellular changes in response to various stimuli.

Proteoglycans (PGs) have sugar chains consisting of amino sugars and uronic acids repeated on a tetrasaccharide linker of xylose, galactose, galactose, and glucuronic acid attached to specific serine residue on the protein. Owing to these features, PGs are high-cost molecules requiring large amounts of amino acids, sugars, and ATP for synthesis. Nevertheless, PGs are synthesized in various species and continually secreted out of the cells. Therefore, it is expected that PGs synthesis has the physiological significance beyond organisms and molecular species.

Advanced glycation endproducts (AGEs) are produced by the non-enzymatic reaction of reducing sugars with amino groups of proteins. AGEs are known to increase reactive oxygen species (ROS) and cause cellular aging and vascular dysfunction. Although the above sugars involved in PGs synthesis are all reducing sugars, their reducing ability is lost during sugar chain elongation. For this reason, the reducing ability of PGs is much smaller than that of the source sugars. Therefore, we hypothesized that PGs synthesis would reduce the AGEs-mediated cellular injury by decreasing reactivity of reducing sugars and releasing PGs to the extracellular space.

Vascular endothelial cells were cultured in serum-free low D-glucose (5.55 mM) or D-glucose-addeed (40.55 mM) DMEM, after suppressing GlcAT-I involved in tetrasaccharide linker synthesis by RNA interference. After 48 h incubation, the amount of ROS was increased, and cell viability was decreased by GlcAT-I suppression. In addition, although intracellular AGEs and ROS levels were increased in the GlcAT-I-suppressed group under D-glucose supplemented condition, cell viability was not affected. These results suggest that PGs synthesis works as a defense system against AGEs formation and glycation stress accelerated by carbohydrate loading.

Vascular endothelial cells that cover the luminal surface of blood vessels control several vascular functions, in which heparan sulfate proteoglycans (HSPGs) are involved. Although the amount of HSPGs decreases in association with the progression of atherosclerosis, the mechanisms underlying the regulation of endothelial HPSG synthesis remain unclear. Adenine metabolites (ATP, ADP, and adenosine) are released from cytosol to extracellular space by extracellular-derived stimuli including inflammatory response, which bind to purinergic receptors and regulate cellular functions. In this study, we elucidate the influence of the adenine metabolites on the transcriptional regulation of endothelial HSPG synthesis and the purinergic receptors involved in the transcriptional regulation. The expression of endothelial perlecan mRNA was decreased by ATP, but not ADP and adenosine. The expression of syndecan-1 mRNA was decreased by not only ATP but also ADP and adenosine. The expression of syndecan-4 mRNA was first increased and then decreased by ATP, ADP, and adenosine. A2BR, P2X4R, P2X7R, P2Y1R, and P2Y2R were expressed in vascular endothelial cells. siRNA-mediated knockdown indicated that P2X4R and P2Y2R were involved in the transcriptional repression of perlecan by ATP; P2X4R and P2Y1R were involved in the transcriptional repression of syndecan-1 by ATP and ADP. Additionally, A2BR and P2X4R were involved in the early transcriptional induction of syndecan-4 by ATP and ADP, and P2Y2R in the subsequent suppression.

Drug-induced liver injury (DILI) is a major cause of drug development discontinuation. The application of metabolomics and proteomics-based multi-omics analysis for DILI evaluation is expected to further elucidate the toxicity mechanisms. However, primary human hepatocyte (PHH), suitable for DILI evaluation, have not been applied to multi-omics studies because PHHs is expensive and limited in availability. In this study, we established a 96-well plate sample preparation method that allows for multi-omics analysis using 5 × 10⁴ cells. The sample preparation protocol was optimized so that drug metabolites, metabolomes, and proteomes could be measured using the same PHH sample. All steps in this method, from pretreatment on a 96-well culture plate to introduction of the sample into the LC/MS, were completed on a 96-well plate. The method was applied to PHHs exposed to acetaminophen (APAP) at a 10% inhibitory concentration. The results showed that APAP and its metabolites were detected only in the APAP-exposed group, and the expression levels of some enzymes involved in CYP and conjugation reactions also significantly increased compared to those in the control group. Furthermore, pathway analysis using the in vitro multi-omics information reproduced endogenous metabolic changes observed in vivo (e.g. GSH depletion). Future evaluation of various drugs using this method is expected to lead to a more detailed elucidation of toxicity mechanisms.

Benzophenones (BPs) are a category of chemicals with similar structures, commonly used as UV filters (e.g., BP itself and BP1-BP12) in personal care products such as sunscreens, skincare products, and body washes. Their derivatives such as 2-methyl benzophenone (2MBP), 3-methyl benzophenone (3MBP), 4-methyl benzophenone (4MBP), 4-phenyl benzophenone (4PBP), and 4,4'-diethylamino-benzophenone (DEAB) are often used as photoinitiators, binders, or additives in printing inks of food contact materials. Because of the high usage of BPs and their potential for environmental persistence, residues of these chemicals are frequently detected in various environmental matrices and organisms. Studies have shown that BP3 increases coral bleaching rates and exhibits the estrogenic activity in aquatic life. However, much fewer studies report on the ecotoxicity of BP derivatives. Hence, the objective of this study is to systematically analyze in vivo toxic effects of specific BPs and their derivatives on medaka (Oryzias latipes) fish. The LC50 value from 96 hr-acute mortality of 7-day post-hatching medaka larvae showed that DEAB was the most toxic compound and BP was the least toxic compound. With the 7-day-sublethal exposure, only 4PBP and DEAB dose-dependently increased larval mobility at tested concentrations. The altered larval locomotion by 4PBP may be associated with chemical-induced oxidative stress. We will further investigate the toxic mechanism of 4PBP and DEAB in medaka fish regarding fish behavior under environmentally relevant exposure conditions.

As one of the alternative methods to animal tests for evaluating the safety of chemical substances, in silico methods have attracted a great deal of attention due to recent advances in machine learning technology. ITSv2 is a reliable skin sensitization hazard and multi-step risk assessment method listed in OECD Guideline No. 497, but does not support quantitative risk assessment such as LLNA EC3 values. In this study, we developed an in silico evaluation system that quantitatively predicts EC3 of chemical substances by combining ITSv2 hazard identification and machine learning models. This system used in chemico/in vitro test data, molecular descriptors and distance information based on read-across concepts. Performance of the prediction system was 0.862 for training data, 0.550 for internal-validation data, and 0.617 for external-validation data as R² value. Moreover, substances that were misclassified in ITSv2 hazard identification are considered to have properties that don't match the correspondence between tests expressing the AOP assumed in ITSv2 and skin sensitization, therefore, the correctness of ITSv2 hazard identification was determined as the threshold of the applicability limitation of this prediction system. When the performance was re-evaluated by excluding the substances misclassified in ITSv2 hazard identification, R² value was 0.963, 0.681 and 0.815 for each data. In addition, by reconstructing the CatBoost models using only substances within the applicability limitation, 0.995, 0.787 and 0.824 for each data, further improving the prediction performance.

In 2020, International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) revised the guideline for reproductive and developmental toxicity testing (S5) to allow alternative methods for embryo-fetal development studies (EFD studies). In Europe, zebrafish (ZF) embryos up to 120 hours post-fertilization (hpf) are not treated as protected animals. Thus, developmental toxicity tests using ZF embryos are advantageous in terms of animal welfare. Furthermore, due to its advantages such as histological and genetic similarity to humans/mammals, it is attracting attention as an alternative to EFD studies. However, the relationship between drug concentrations in aqueous solution (Cw) and embryonic concentrations (Ce), and the relationship with humans/mammals in terms of drug exposure related to toxicity, are unclear. In this study, we investigated the relationship between the drug exposures in ZF embryos/larvae and in humans/mammals for 21 drugs of ICH S5 positive control compounds (PCs). Analyzed with the PCs judged to be positive in ZF embryos/larvae, the area under the concentration-time curve in ZF (zAUC) based on the estimated drug concentrations in embryos/larvae showed a good correlation with the area under the blood concentration-time curve (AUC) in humans, rats, and rabbits. This result indicates that the exposure level causing developmental toxicity by each drug was similar between ZF embryos and humans/mammals.

An ideal vascular toxicity testing model would be the development of a vascular-like tissue constructs that can be used to evaluate changes over time in their structure at specific focal locations without shielding by gel adsorption of test drugs in extracellular matrix gels during induction of three dimensional vascular lumen formation. In this study, we have successfully prepared fiber-shaped cell aggregates composed of human umbilical vein endothelial cells and human mesenchymal stem cells as vascular pericytes anchored on the bottom of culture dishes at a defined location using our developed cell self-aggregation technique and a specific dumbbell-shaped culture chamber. The fiber-shaped cell aggregates maintained their shape for at least two weeks without rupture, and histological analysis interestingly revealed that they formed a unique tissue structure with multiple capillary-like structures oriented in the same direction as the long axis of the fiber in the medial side and a gapless monolayer of endothelial cells on the outer side, respectively. Then, it was shown that exposure of cadmium chloride to such vascular endothelialized fiber-shaped tissues elicited a toxic response in which only their outer endothelial layer disintegrated while maintaining their fiber shape. These results suggest that our vascular endothelialized fiber-shaped tissue could have the potential to be used as a model for vascular toxicity testing that enable us to evaluate toxicity over time without shielding by gel adsorption of test drugs, and we are continuing to analyze the results in detail.

[Introduction] Intratracheal administration of compounds have been used widely to establish models of lung injury. Recently, selective administration into the left or right lungs has become possible due to the development of the bronchoscope for rodents. In this study, to clarify the characterization of selective bronchi administration, bleomycin (BLM) was chosen as a model compound, and distribution of pulmonary lesions induced by selective exposure were analyzed.

[Methods] C57BL/6JJcl mice were intrabronchially administered 20 µL of BLM (6 mg/mL) using a bronchoscope. Each mouse was injected BLM into only right or left lung. Animals were sacrificed by exsanguination under anesthesia at 7, 14, and 28 days after the administration, and the lungs were evaluated histopathologically.

[Results and Discussion] In the BLM-treated animals, pulmonary lesions composed by inflammation including fibrosis were observed. In the right lung exposed group, there were individual differences in the distribution of pulmonary lesions, and the distribution of lesions were different among 4 lobes of the right lung. Anatomical features such as lobe structure and bronchial branching resulted in heterogeneous exposure to each lobe in the right lung. On the other hand, in the left lung exposed group, the pulmonary lesion was observed uniformly throughout the lobe and no individual difference was noted. In order to establish a highly homogeneous lung injury models, it is recommended to select the left lung, which is a single lobe, as the selective administration site.

[Background and purpose] LLNA: BrdU-ELISA (LLNA-BrdU) is widely used to evaluate the skin sensitization potential of chemicals as well as LLNA-RI. In LLNA, estimated concentration (EC) that chemicals induce positive response is used as indicator of skin sensitization potency, and EC1.6 in LLNA-BrdU and EC3 in LLNA-RI can be used for GHS sub-categorization and quantitative risk-assessment. Since there have been no reports of quantitative comparison for EC1.6 and EC3, relationship between these two parameters was statistically analyzed based on existing data, and regression equation was constructed. Additionally, practicality of the equation was examined by comparing predicted EC3 with the actual value.

[Materials and Methods] Regression analyses were performed on the existing data of EC3 and EC1.6 (15 sensitizers) listed in the performance standard of LLNA, and the optimal regression equation was determined based on R² values. We estimated the 95% confidence interval of existing EC3 data (34 sensitizers). In addition, practicality was examined by comparing predicted EC3 with the actual EC3 using 32 sensitizers, in which the actual EC1.6 and EC3 were known, listed in ICCVAM LLNA: BrdU-ELISA evaluation report.

[Results] The established regression equation (LogEC3=0.972LogEC1.6+0.118(±0.618)) showed good predictive accuracy for EC3 and it was confirmed to be practical: 27/32 sensitizers were within the estimated variation range and deviations of estimated EC3 for remaining 5 sensitizers from the estimated range were minimal.

The Ames test is a typical test that evaluates the effects (mutagenicity) of chemical substances contained in pharmaceuticals and industrial products on DNA. The test evaluates the mutagenicity of both the chemical itself and metabolites of the compound using rat metabolic enzymes. To evaluate toxicity concerns quickly and inexpensively, in silico (QSAR) technology that predicts toxicity from compound structures is actively being developed. The use of QSAR for evaluation is progressing, such as the guideline (ICH-M7). Methods is roughly classified into knowledge-based judgments based on structures of concern for compounds, and statistically-based judgments that use computational scientific methods such as machine learning. ICH-M7 requires the use of both Ames QSAR assessments. Knowledge-based method makes judgments by establishing rules for chemical reaction mechanisms with DNA based on chemical reaction knowledge. Whereas, the compound structure input in silico is the structure before the metabolic reaction by the enzyme, and if the compound structure changes due to the metabolic reaction, the reactivity with DNA cannot be determined. Therefore, we constructed a function that simulates how the target compound is metabolized in the Ames test environment and affects the test results, lead to improvement in performance of knowledge-based prediction. Furthermore, we developed a prediction models trained on public and in-house Ames test data using GNN, a type of machine learning method, and combine it with the above knowledge-based model to make complementary judgments.

Drug-induced convulsive seizures are a serious adverse event and drug evaluation is usually carried out using behavioural indicators in laboratory animals such as rodents. However, these evaluation methods use large numbers of animals and require large amounts of test substance, resulting in low throughput. While tests using laboratory animals provide a lot of useful information, they are also considered to be an area for improvement in terms of animal welfare, speed of research and development and cost. To address this issue, a test method using zebrafish, which can be evaluated with a small amount of test substance (-µg) and can contribute to the promotion of the 3Rs, has been reported using the amount of behaviour immediately after drug addition as an indicator. However, in the previously reported evaluation system, the presence or absence of convulsions is finally determined by visual observation by researchers, which causes problems such as inconsistency in evaluation, reduced throughput and difficulty in multi-tracking. Therefore, in this study, we aimed to solve the above issues by introducing AI into the evaluation of seizure-like behaviour using zebrafish and automating some of the tasks.

In this presentation, an overview of automated tracking for each designated site using DeepLabCut, extraction of behavioural parameters that change in relation to compound administration, and evaluation of a seizure-like behaviour classification model using the behavioural parameter dataset will be presented.

Keywords: Toxicity prediction, Deep Learning, Multi-animal tracking, Central Nervous System

Drug-induced behavior changes in nonclinical safety studies could correlate to serious side effects in humans and hence must be detected prior to clinical trials. We investigated a video analysis using deep learning to assess locomotor activity (LA) and convulsions in conscious non-human primates (NHPs). The training data was created by annotating the waist landmarks for pose estimation based on the video data from each cage. The time series of 2D coordinates was calculated with the deep learning-based pose estimation model. Study 1. LA evaluation: Apomorphine hydrochloride (APO, 0 or 1 mg/kg, sc) was dosed (n=4). A momentum was calculated from the difference between the frames of 2D coordinates, and a trajectory was created from transition of 2D coordinates for each 1 hour epoch. Study 2. Convulsion detection: Pentylenetetrazole (PTZ, 60 to 70 mg/kg, sc) was dosed (n=4). A convulsion was determined when the number of frames was greater than the threshold for ten seconds using 2D coordinates and vibration intensity calculated with wavelet transformation. In Study 1, behavioral changes characteristic of APO (circling, jumping, cage licking, etc.) were observed differently between animals. The LA increased in two NHPs, showing different trajectories correlated with circling or jumping. In Study 2, PTZ induced convulsions in all NHPs and the algorithm could detect the convulsions in all animals while this algorithm did not falsely detect any convulsions for animals in study 1. Our deep learning model using video data is considered useful to assess LA and convulsion in NHPs.

Rationale: Drug-induced liver injury (DILI) is known to be influenced by diverse factors, resulting in a difficulty of predicting the development of DILI. To study factors influencing DILI, the ability to generate potential hypotheses is required. With the power of processing huge data sets comprehensively and objectively as well as the ability of adding and subtracting language data, natural language processing (NLP) has recently been utilized in generating new hypotheses. In the study, we evaluated the applicability of NLP to search the factors influencing DILI, focusing on human genes in this case, aiming to generate new hypothesis.

Method: Liver Toxicity Knowledge Base DILIrank dataset including 1036 U.S. FDA-approved drugs was vectorized. Bootstrapping logistic regression models were generated to obtain DILI scores. The sum of vectors calculated by 141 drugs with higher DILI scores and approximately 20,000 genes was used to generate DILI scores. To determine potential reduction factors in hepatoxicity, common genes among the drugs were systematically assessed by looking at lower DILI scores in the sum of vectors compared to the drug alone.

Results and discussion: Over 0.5 was defined as a DILI positive. In most-DILI concerned drugs, DILI scores were clearly lowered in 141 cases (89%) when the sum of the vectors were determined unlike the observation in the drug alone. Intriguingly, 219 common genes including ones that never have been associated with DILI, were uncovered. Following cluster analysis of the genes lowering DILI scores, the association with xenobiotic metabolism, protein dephosphorylation, response to ER stress, miRNA-mediating gene silencing but not limited were shown. Together NLP could be a useful tool to facilitate novel hypothesis generation to determine the factors influencing DILI.

Serotonin (5-HT) secreted from enterochromaffin cells (EC cells) stimulates the vomiting center via afferent neurotransmission, developing drug-induced nausea and vomiting. In this study, we aimed to construct in vitro assay system which can predict the risk of drug-induced nausea and vomiting by efficiently differentiating crypt-derived human jejunal stem cell spheroids into EC cells and evaluating their drug exposure-dependent 5-HT release.

Human jejunal spheroids supplemented with Notch inhibitor, DAPT, showed increased gene expression levels of EC cell markers, CHGA and TPH1, and the increase of 5-HT positive cell population was confirmed by immunostaining, suggesting that differentiation into EC cells was promoted. Next, forskolin stimulation was performed as a positive control for 5-HT release, and the amount of 5-HT release into the supernatant was quantified using LC-MS/MS. The amount of 5-HT release was greatly increased under the culture condition supplemented with DAPT, suggesting that the 5-HT release activity from EC cells can be evaluated in this system.

We confirmed the increase in 5-HT release by the exposure of drugs for which the clinical risk of nausea and vomiting was reported, such as cisplatin, crizotinib, and metformin in our assay system. We are currently investigating the usefulness of 5-HT release assessment as a surrogate marker of emesis by evaluating the relation between drug potency of accelerated 5-HT release and the incidence of nausea and vomiting in clinical situations. We are also exploring the mechanism of the regulation of 5-HT release from EC cells upon drug exposure by evaluating direct cytotoxicity to EC cells and functional changes.

Target safety assessment (TSA), which identify toxicity hazards as drug targets and consider countermeasures, in the early stage of drug development is important to efficiently generate development candidates and increase the success rate of drug development. However, in the early stage, there are often no tool compounds, and TSA is limited to in silico TSA using databases and existing search tools. The creation of gene-knockout mice (KO mice) or conditional KO mice (cKO mice) is a possible option, but the former involves the concern of fetal lethality, while the latter issue requires a very long time using the common Cre/loxP system. Therefore, we have developed a technology that can delete target gene easily in adult mice by combining adeno-associated virus (AAV) and genome editing technology. In experiments targeting blood coagulation factor 9 (F9), whose function in vivo is known, the prolongation of APTT, which is a phenotype observed in F9 gene-deficient mice, was observed about one month after induction of gene deletion by AAV infection. Although there remain the challenges that KO cannot be expected in organs and tissues that cannot be effectively infected with AAV, it was considered that the technology can generate cKO mice for any gene in a short term.

Complex molecules are highly versatile synthetic and catalytic reagents that have led to significant advancements in chemical research. Despite their usefulness, effective utilization methods for these molecules have not been fully developed in some research fields. However, optimizing the outreach directionality of complex molecules can transform them into highly effective utilization methods. In this regard, two examples are discussed: the creation of zinc complexes to promote cell proliferation, and the use of tea waste as a low-cost base for adsorption removal of harmful metals. The results demonstrate the replaceability of complex molecules as functional molecules in the body and the highly practical nature of tea waste as a biomass for worldwide use. The analysis highlights the importance of complex molecules and their potential for interdisciplinary research.

Background: Chemotherapy induced neutropenia and febrile neutropenia is one of frequent side effects in anticancer drug treatment and could be a cause to reduce relative dose intensity and therapeutic effects of the drugs. TK / TD simulation of bone marrow toxicity provide important information to minimize the risk of ineffectiveness and to increase the chances of prescribing a pharmacologically effective dose to patients / subjects. MTT (Mean transit time: the time required for metamyelocytes in bone marrow to mature and migrate to the peripheral blood) in rats was calculated to establish a TK / TD model reflected the dynamics of neutrophils in vivo. Based on the calculated MTT, species differences in bone marrow toxicity observed in previous studies will be discussed.

Methods: BrdU was dosed intraperitoneally once to rats. Peripheral blood was collected over time, and the ratio of BrdU⁺ cells in CD11b/c⁺RP-1⁺ cells was measured by FACS Celesta to calculate MTT.

Results: The percentage of BrdU⁺ cells in CD11b/c⁺RP-1⁺ cells showed a maximum value of 30-50% at 72-96 hours after administration. From these results, the MTT in rats was calculated to be about 64 hours. In the future, we will utilize the MTT calculated in the TK/TD model to improve the prediction accuracy of bone marrow toxicity in humans. At this meeting, the results of comparison of MTT in rats with those of other animal species will be presented.

Selenoprotein P (SeP) is a selenium-containing plasma protein, which is secreted from the liver into plasma. SeP levels increase in response to hyperglycemia and induce insulin resistance. Thus, excess SeP is an aggravating factor in diabetes and a promising new therapeutic target. However, little progress has been made in the study of its inhibitors. Recently, we found that sulforaphane (SFN), a phytochemical contained in broccoli sprouts, inhibits SeP expression. In this study, we aimed to elucidate the inhibitory mechanism of SFN on SeP expression. A cultured hepatocyte HepG2 cell is a model that produces SeP and secretes it into the culture medium. Since we found that SFN markedly reduced the SeP protein levels without affecting the mRNA levels, we hypothesized that increased degradation was involved, and examined the effect of lysosome inhibitors. As a result, the decrease in SeP by SFN was completely canceled by the lysosomal inhibitor. Furthermore, the lysosomal pH indicator, Lysotracker, revealed that SFN enhances lysosomal acidification. SFN is a well-known activator of the transcription factor Nrf2. SFN activated Nrf2 under the above conditions, but Nrf2 siRNA had no effect on lysosomal acidification or SeP reduction. These results indicate that the decrease in SeP by SFN is an Nrf2-independent effect. Although it is well known that SFN is involved in detoxification of various toxicants via Nrf2 activation, their Nrf2-independent actions are not well understood. This study suggests that multi-targeting effect of SFN and this may exert their health-promoting effects through combined actions.

Warfarin is an anticoagulant that suppresses vitamin K-dependent coagulation factor production by competitively inhibiting VKOR and has been used as rodenticide. Abuse of warfarin-based rodenticides led to the emergence of resistant rodents, which became difficult to control. Second-generation rodenticides were developed based on warfarin with improved toxicity, but its cumulative effect has caused death of wild animals. Therefore, a new rodenticide that is effective against resistant rodents and has high environmental safety is needed. The objective of this study was to compare the properties of geometric isomers of second-generation rodenticides since it was reported that the half-lives in vivo differ significantly among these geometric isomers. In this study, geometric isomers of five major second-generation rodenticides were isolated and subjected to VKOR inhibition assays using cultured cells and in silico VKOR-rodenticide molecular docking. The results showed that bromadiolone and difethialone showed lower IC50 values for their trans form, while the other three substances showed lower IC50 values for their cis form. However, all substances and isomers showed more potent VKOR inhibitory activity than warfarin, and there were no significant differences in binding and inhibitory activity against VKOR between the cis and trans isomers. The differences in pharmacokinetics of the two isomers may be due to albumin binding and P450 metabolism rather than VKOR binding.

Chronic arsenic exposure is known to cause diabetes, but the mechanism is unknown. We focused on the muscle responsible for glucose metabolism, and investigated the effect of arsenic compounds on differentiation into muscle cells using mouse myoblast C2C12 cells. In order to investigate the effect of arsenite (As(III)) on differentiation of C2C12 cells, As(III) was added to the differentiation medium. The results showed that the appearance of multinucleated cells was markedly reduced by As(III) and the levels of MHC as known for differentiation marker was decreased in an As(III) concentration-dependent manner. Next, C2C12 cells that had been exposed to As(III) for 6 days were seeded, and differentiation was induced in the absence of As(III) for 8 days. The results indicate that As(III) inhibits the differentiation of C2C12 cells into multinucleated cells, regardless, there was no change in the MHC expression level by Western blot. These results indicate that As(III) inhibits the differentiation of C2C12 cells into multinucleated cells, but the inhibitory mechanism differs depending on the timing of As(III) exposure. We plan to clarify the inhibitory mechanism of C2C12 cells differentiation by As(III) exposure by investigating the factors involved in induction of differentiation.

Cadmium is a toxic heavy metal widely present in the environment, and we are exposed to cadmium through diet and smoking unintentionally. Epidemiologically, cadmium is known as a risk factor for atherosclerosis and causes dysfunction of the endothelial cells covering the lumen of blood vessels. At the surface of endothelial cells, hyaluronan (HA) which is composed of N-acetylglucosamine and glucuronic acids, is synthesized as large HA of 1000-2000 kDa and small HA of about 100 kDa by three types of HA synthases (HAS1-3) and regulates cell growth and inflammatory responses. Since HA accumulates in atherosclerotic lesions, it is considered that HA contribute to the development and progression of cadmium-induced atherosclerosis. However, the effect of cadmium on HA synthesis in vascular endothelial cells remains unclear. In this study, we found that cadmium enhances HA synthesis and specifically upregulates HAS3 expression via activation of JNK-c-Jun pathway in vascular endothelial cells.

HAS3 is induced in early stage in atherosclerosis, and small HA promotes angiogenesis and inflammatory responses. Macrophages and T-lymphocytes accumulate in atherosclerotic lesions, and enhanced HA synthesis in vascular endothelial cells leads to recruitment of macrophages and T lymphocytes to the site of inflammation in a HA receptor CD44-dependent manner. Therefore, cadmium mediated HAS3 induction in vascular endothelial cells would contribute to the development and progression of atherosclerotic lesions by enhancing inflammation.

Vascular endothelial cells cover the luminal surface of blood vessels and the dysfunction is crucially associated with cardiovascular diseases, which are progressed by cadmium exposure. Morphologically, in vascular endothelial cell layer exposed to cadmium, increases of the area of de-endothelialization occured. Integrins (ITGs) are heterodimer of α and β subunits, transmembrane, and cellular adhesion molecule to basement membrane. In the present study, we investigated the influence on ITGs of cadmium-induced cytotoxicity and the expression by cadmium. The study revealed that: (1) ITGA1, ITGA2, ITGA3, ITGA5, ITGA6, ITGAV, ITGB1, ITGB3, ITGB4, and ITGB5 was constitutionally expressed in vascular endothelial cells; (2) the knockdown of ITGB4 or ITGB5 exhibited the cytotoxicity of vascular endothelial cells, but those of the other subunits did not; (3) cadmium reduced the expression of not only ITGB4 and ITGB5 and but also the other subunits. These results suggested that ITGB4 and ITGB5 were suppressed by cadmium and the suppression was involved in cadmium-induced cytotoxicity in vascular endothelial cells.

Cadmium (Cd) is accumulated in rice and other crops. Chronic exposure to Cd causes itai-itai disease and is characterized by kidney damage and osteomalacia. However, the detailed pathogenic mechanisms are not well understood. It is known that proximal renal tubular damage is induced in mice treated with Cd-metallothionein complex (Cd-MT) intravenously. Therefore, we thought that bone disorders could also be triggered by this model. In this study, we investigated the effects of Cd-MT administration on the bone in a postmenopausal model of ovariectomized (OVX) mice. 8-weeks-old female mice were performed OVX and treated with 0.3 mg Cd/kg Cd-MT intravenously at one week after OVX. The kidney and femur were harvested for histological analysis at 12 or 24 weeks after Cd-MT administration. The bone density and bone mass of the femur was measured by X-ray μCT. We observed kidney damage in mice by Cd-MT administration, as enlarged Bowman's capsules. The femur bone mineral density and bone mass showed no significant change at 12 weeks after Cd-MT administration, but at 24 weeks after administration, bone mass in the Cd-MT group was significantly lower than that in the control group. No additive effects of OVX and Cd-MT administration were observed on bone tissue in this experiment. These results suggested that Cd-MT administration may reproduce pathology similar to chronic Cd toxicity in humans.

Methylmercury (MeHg) causes severe CNS disorders such as Minamata disease. However, the molecular mechanisms underlying MeHg-induced neurotoxicity remain unclear. We previously reported that MeHg induces endoplasmic reticulum (ER) stress and activates the unfolded protein response (UPR). Here, to identify the negative role of ER stress in MeHg toxicity, we investigated the signaling alternations of ER stress-mediated apoptosis pathway by MeHg exposure in vivo and tested the effects of 4-phenylbutyric acid (4-PBA), a chemical chaperone, against MeHg toxicity.

Mice carrying an ER stress activation indicator (ERAI), whose expression is induced under ER stress, were exposed to MeHg via drinking water. We performed immunofluorescence and TUNEL staining of the brain sections and observed ER stress/UPR activation and apoptosis, respectively.

Simultaneously with an increase of ERAI-positive cells, the phosphorylation of IRE1α and PERK was enhanced by MeHg exposure. These increases in ER-stress indicators were followed by an increase in CHOP-positive cells, a key driver of ER stress-mediated apoptosis. By contrast, the administration of 4-PBA significantly suppressed ERAI-positive cells as well as p-IRE1α and p-PERK-positive cells. Most importantly, 4-PBA also significantly suppressed CHOP-positive cells and TUNEL-positive cells.

The ER stress-mediated apoptosis pathway may play a critical role in neuronal cell death by MeHg exposure in vivo. Chemical chaperones such as 4-PBA may be a potential therapeutic approach for MeHg poisoning.

Benzophenones (BPs) are a class of ultraviolet filters with similar chemical structures, commonly used in personal care products such as sunscreen to protect the skin from excessive sun exposure. Some BPs are also added to plastic products to prevent their photo-degradation. Certain BPs (e.g., BP-3) can induce coral bleaching and estrogenic effects on aquatic life, making BPs an environmental concern. However, little literature analyzes both the toxicity and beneficial function possessed by BP-type UV filters. The purpose of this study is to assess the cytotoxicity of commercially available BPs and their additional function on melanin inhibition in the mouse skin melanoma cell line (B16F10). According to the cytotoxicity rank (e.g., 48-hr LC₅₀ values) of BPs by the MTT assay, BP-2 had the lowest toxicity among the six tested BPs (BP-1, BP-2, BP-3, BP-4, BP-7, BP-8), while BP-8 and BP-7 had higher cytotoxicity. In addition, BP-2 caused the dose-dependent inhibition of melanin content in B16F10 cells, as compared to the control. Lastly, we will discuss the molecular mechanism of how BPs (particularly BP-2) regulate tyrosinase-related melanogenesis. We will suggest a better BP alternative that is environmentally friendly, low toxic and multiple functional for sunscreen-related product manufacturing.

Recently, plastic food contact materials such as tea bags can release large amounts of nanoplastics when exposed to hot water. These particles may enter the human body through ingestion and have the potential for absorption and bioaccumulation. The size of nanoplastics is one of the crucial factors affecting the efficiency of absorption. Therefore, it is important to understand how size of nanoplastics change during digestion. In the study, we selected the commercial fluorescent polystyrene (PS) with two diameters (200 nm and 750 nm) and the fluorescent 366 nm melamine-formaldehyde resin (MF) as models. Nanoparticle Tracking Analysis (NTA) operated in the fluorescence model was used to measure the three types of nanoplastics in artificial digestive fluids with and without food matrix. All nanoplastics formed larger agglomerates in gastric juice, but the agglomeration was reduced after intestinal digestion. In gastric juice with pH adjusted while no enzyme added, 200 nm nano-PS agglomeration did not decrease after intestinal digestion. On the other hand, 750 nm PS and 366 nm MF showed similar size of agglomerates throughout the digestion process. The size changes were all associated with zeta potential of the nanoplastics. However, when pH was not adjusted, all particles retained their original secondary size as observed in saliva. Hence, we suggested that in the absence of food matrix, the pH affects differently between various nanoplastics in determining the agglomeration states. Considering the presence of food matrix, all nanoplastics have a larger size in saliva and intestinal fluids, but in gastric juice we observed a lower degree of agglomeration. We thus believed that the formation of protein or lipid corona on the surface of the nanoplastics contributed the size of nanoplastics (e.g. thicker shell or stereo barrier) in the presence of food matrix.

Arsenic is a toxic pollutant in environment. Chronic arsenic exposure is known to be related to the progression of atherosclerosis. The molecular mechanism underlying the development of arsenic toxicity include the inhibition of enzyme activity and increased production of reactive oxygen species (ROS). Recent reports indicate that reactive sulfur species (RSS) have protective effects against ROS. We have previously found that arsenite induces the expression of cystathionine gamma-lyase (CSE), a RSS-producing enzyme, in vascular endothelial cells. In this study, we investigated the involvement of induction of CSE expression in arsenite toxicity in vascular endothelial cells. We found that knockdown of CSE by siRNA enhances the sensitivity of vascular endothelial cells to arsenite. This suggests that CSE acts as a protective factor against arsenite-induced cytotoxicity. Nrf2, NF-κB, HIF-1, and ATF4 have been reported to be transcription factors involved in the regulation of CSE expression. Therefore, we investigated the involvement of these transcription factors in arsenite-induced CSE expression. siRNA-mediated knockdown of Nrf2 did not affect the induction of CSE expression, while NF-κB inhibitor (SC-514) conversely promoted the induction of CSE expression. On the other hand, siRNA-mediated knockdown of HIF-1 partially suppressed the induction of CSE expression by arsenite. These findings suggest that activation of the HIF-1 pathway is partially involved in the mechanism of CSE expression induction by arsenite. The involvement of ATF4 in this induction mechanism is currently under investigation.

Organophosphorus flame retardants (OPFRs) are increasingly used as a replacement for polybrominated diphenyl ethers, and thus, human exposure to OPFRs has been increasing. Epidemiological studies indicate possible effects of OPFRs on children’s health status such as rhinoconjunctivitis. The objective of the present study was to assess the developmental effects of OPFRs and their metabolites, and possible mechanisms underlying developmental toxicity using zebrafish as a model. Zebrafish embryos were exposed to OPFRs or their metabolites observations. Significant increases in circulatory failure including blood flow reduction and pericardial edema were detected in embryos exposed to TPHP, TDCIPP, EHDPHP, and some of their metabolites (HO-m/p-TPHP and 5-HO-EHDPHP). The chemicals with higher logKow induced greater severity of the circulatory failure. RNA-Seq results of embryos exposed to EHDPHP, 5-HO-EHDPHP, and EHPHP at the highest concentrations where no significant circulatory failure was observed showed that genes involved in glucose homeostasis and retinol metabolism, as well as GUCY-cGMP and calcineurin signalings, were significantly altered by EHDPHP and/or 5-HO-EHDPHP, while immunity-related genes were significantly altered by EHPHP. These results clearly indicate distinct potency for developmental toxicity among OPFRs and their metabolites. Transcriptomic data suggest differences in possible molecular mechanisms underlying the developmental effects of these compounds.

2,3,7,8-tetrachroldibenzo-p-dioxin (TCDD) is one of most persistent and bioaccumulative among the Dioxins, so its chronic toxicity is a great concern. However, it has been reported that TCDD is excreted in urine, although only in small amounts. This report suggests that the mammalians might have a metabolic machinery of TCDD, but it remains unclear for a long time. In this study, we newly found that thyroid hormones (TH) promote urinary excretion of TCDD. We here investigated the effects of TH on the urinary excretion of TCDD and its distribution in various tissues, as well as the mechanism by which TH promote its excretion. In order to trace excretion and tissue distribution levels of TCDD, male ddY mice and C57BL/6J mice were intraperitoneally administrated [³H]-labeled TCDD and measured its radioactivity in the liver, feces, and urine. In both species, ddY and C57BL/6J mice, TH did not significantly change fecal excretion of [³H]TCDD, but significantly increased its urinary excretion with a decrease in its accumulation in the liver, the major organ of TCDD accumulation. Similar experiments were conducted using TH receptor (TR) α-knockout (KO) or TRβKO mice. In TRαKO mice, urinary excretion of [³H]TCDD was enhanced with decreased accumulation in the liver, as in wild-type mice, but such effects were not observed in TRβKO mice. These results suggest that the mice have a machinery for urinary excretion of TCDD which may be regulated by TRβ.

Surface charges of a nanomedicine is one of the multiple factors that would affect its therapeutic and toxicologic outcomes. Positively charged nanomaterials have been shown to have high tendency to interact with cells. Accordingly, positively charged nanoparticles are usually more toxic and have poor pharmacokinetic properties, compared with negatively or neutrally charged particles. Previously, we developed a method to prepare organosilica nanoparticles, which are positively charged at physiological pH and carry two functional groups (thiol and amine) for drug conjugation and delivery. This study aimed to control the cellular interaction and uptake of positively charged organosilica nanoparticles by modifying the particle surface with adsorbed serum proteins. Protein adsorption and binding of nanoparticles were fully explored by adjusting various factors, including pH, temperature, time, etc. Particle size and surface charges were measured by DLS, and TEM was used to confirm protein adsorption. The amount of protein adsorbed were further quantified by a protein assay. Cellular uptake were evaluated by fluorescence microscopy and cytotoxicity of nanoparticles were measured by MTT assays. The results demonstrated that, after protein adsorption, particle size increases from 91.87±0.88 nm to 149.57±1.82 nm. The surface charge will switch from positive to negative (from 53.13±3.35 mV to -17.07±0.65 mV), confirming successful protein adsorption on the surface of nanoparticles. Protein adsorption efficiency ranges from 0.01% to 1.98%, depending on preparation condition. The quantitative cellular uptake and cytotoxicity data will be presented in the meeting.

In recent years, the application of nanomaterials (NMs) is expanding. Although NM-based drug delivery carriers are expected to enhance drug transfer to the brain via the nose-to-brain pathway, the detailed pathway and hazards have not been understood. Therefore, in order to develop safe and effective drug delivery carriers to the brain, it is important to evaluate the kinetics of NMs. In this context, we focused on amorphous silica nanoparticle (nSP) which has been reported to be effective as a drug carrier. We investigated the intracellular uptake pathway in vitro and kinetics of nSP after the intranasal administration in vivo. The mouse microglial cell line MG6 were used in this study. Cells were exposed to fluorescent nSP with a particle size of 10 nm, 50 nm and 100 nm in diameter (nSP-10, -50 and -100). Uptake of nanoparticles were analyzed by confocal high content imaging system and flow cytometry. nSP10 was administered intranasally to mice, and sagittal sections of heads were observed by fluorescent microscopy. The fluorescence intensity of nSP10 taken up into MG6 was measured by flow cytometry, and results indicated that intracellular uptake of nSP10 was increased in a concentration-dependent manner. We further observed intracellular uptake of nSP10, 50 and 100 in MG6 by time-lapse analysis. Results indicated that nSP with smaller particle size was taken up into cells in early time point. Furthermore, when cells were treated with endocytosis inhibitors, the amount of nSP uptake was reduced. These results suggest that nSP might be taken up by endocytosis. In addition, in sagittal sections of heads, the fluorescence of nSP10 was observed in olfactory bulb, suggesting that nSP10 could reach the brain. Currently, we are analyzing protein expression changes in the mice brain after intranasal administration of nSP10, and try to evaluate the hazard of nSP10.

Nanomaterials are particles with 100 nm or less diameter. There have been concerns about their unexpected biological responses due to their use in daily or medical supplies. Hence, the risk analysis of nanomaterials to the people who are vulnerable to exposure to chemicals, like pregnant women, is in need. From this viewpoint, based on our research, which shows that silver nanoparticles with a diameter of 10 nm (nAg10) suppressed the trophoblast syncytialization during the pregnancy process, we attempted to elucidate their mechanism. Based on the report that nanomaterials can cause epigenetic changes, and the report that histone acetylation regulates syncytializaiton, we aim to address the mechanism of suppression on the syncytialization by nAg10 from the epigenetic point of view. First, we analyzed the effect of nAg10 on the activity of HDAC1 and HDAC2. Results derived that nAg10 might decrease the activity of HDAC1 and HDAC2. Then, BeWo, a human choriocarcinoma cell line, was co-treated with forskolin, syncytialization-inducing reagent, and nAg10. Western blotting suggested that no significant changes in HADC1 expression. Currently, we are evaluating the effect of nAg10 on HDAC activity in vitro.

Silica nanoparticles as drug carriers have drawn much attention in recent years. They have the advantages of good biocompatibility and high surface modification. Triple-negative breast cancer (TNBC) is a breast cancer that lacks estrogen receptors, progesterone receptors and human epidermal growth factor receptor 2. Current standard treatment strategies for TNBC include chemotherapy, surgery, and radiation. Paclitaxel (PTX) is the first-line drug for the treatment of breast cancer. Due to its high hydrophobicity, a special excipient, Cremophor EL, is needed to dissolve PTX for clinical use, which, however, may cause various solvent induced toxicity. Histone deacetylase inhibitor (HDACi), such as vorinostat (SAHA), is a new class of agent with the potential to treat multiple cancers via several mechanisms. Combination use of PTX and HDACi may lead to higher efficacy and lower toxicity of cancer therapy. In this study, silica nanoparticles with trifunctional groups (-SH, -NH₂, -COOH) were developed and used as a carrier to deliver PTX and SAHA. The particle size, zeta potential, the appearance, encapsulation efficiency and in vitro drug release were measured. Moreover, combined therapy of PTX and SAHA was evaluated in MCF-7 cells and MDA-MB-231 cells. The results demonstrated that only PTX can be effectively encapsulated in multi-functionalized silica nanoparticles. Cytotoxicity of PTX or SAHA encapsulated in silica nanoparticles were also observed. In conclusion, this study demonstrated that drug-loaded trifunctional silica nanoparticles have potential to be a good carrier for breast cancer therapy.