Enhanced electrophysiological activity and neurotoxicity screening of environmental chemicals using 3D neurons from human neural precursor cells purified with PSA-NCAM.

The assessment of neurotoxicity for environmental chemicals is of utmost importance in ensuring public health and environmental safety. Multielectrode array (MEA) technology has emerged as a powerful tool for assessing disturbances in the electrophysiological activity. Although human embryonic stem cell (hESC)-derived neurons have been used in MEA for neurotoxicity screening, obtaining a substantial and sufficiently active population of neurons from hESCs remains challenging. In this study, we successfully differentiated neurons from a large population of human neuronal precursor cells (hNPC) purified using a polysialylated neural cell adhesion molecule (PSA-NCAM), referred to as hNPCPSA-NCAM+. The functional characterization demonstrated that hNPCPSA-NCAM+-derived neurons improve functionality by enhancing electrophysiological activity compared to total hNPC-derived neurons. Furthermore, three-dimensional (3D) neurons derived from hNPCPSA-NCAM+ exhibited reduced maturation time and enhanced electrophysiological activity on MEA. We employed subdivided population analysis of active mean firing rate (MFR) based on electrophysiological intensity to characterize the electrophysiological properties of hNPCPSA-NCAM+-3D neurons. Based on electrophysiological activity including MFR and burst parameters, we evaluated the sensitivity of hNPCPSA-NCAM+-3D neurons on MEA to screen both inhibitory and excitatory neuroactive environmental chemicals. Intriguingly, electrophysiologically active hNPCPSA-NCAM+-3D neurons demonstrated good sensitivity to evaluate neuroactive chemicals, particularly in discriminating excitatory chemicals. Our findings highlight the effectiveness of MEA approaches using hNPCPSA-NCAM+-3D neurons in the assessment of neurotoxicity associated with environmental chemicals. Furthermore, we emphasize the importance of selecting appropriate signal intensity thresholds to enhance neurotoxicity prediction and screening of environmental chemicals.

Safety assessment and gastrointestinal retention of orally administered cerium oxide nanoparticles in rats.

Cerium oxide nanoparticles (CeO2 NPs, NM-212) are well-known for their catalytic properties and antioxidant potential, and have many applications in various industries, drug delivery, and cosmetic formulations. CeO2 NPs exhibit strong antimicrobial activity and can be used to efficiently remove pathogens from different environments. However, knowledge of the toxicological evaluation of CeO2 NPs is too limited to support their safe use. In this study, CeO2 NPs were orally administered to Sprague Dawley rats for 13 weeks at the doses of 0, 10, 100, and 1000 mg/kg bw/day, followed by a four week recovery period. The hematology values for the absolute and relative reticulocyte counts in male rats treated with 1000 mg/kg bw/day CeO2 NPs were lower than those in control rats. The clinical chemistry values for sodium and chloride in the treated male rat groups (100 and 1000 mg/kg/day) and total protein and calcium in the treated female rat groups (100 mg/kg/day) were higher than those in the control groups. However, these changes were not consistent in both sexes, and no abnormalities were found in the corresponding pathological findings. The results showed no adverse effects on any of the parameters assessed. CeO2 NPs accumulated in the jejunum, colon, and stomach wall of rats administered 1000 mg/kg CeO2 NPs for 90 days. However, these changes were not abnormal in the corresponding histopathological and immunohistochemical examinations. Therefore, 1000 mg/kg bw/day may be considered the "no observed adverse effect level" of CeO2 NPs (NM-212) in male and female SD rats under the present experimental conditions.

Hepa-ToxMOA: a pathway-screening method for evaluating cellular stress and hepatic metabolic-dependent toxicity of natural products.

In the field of drug discovery, natural products have emerged as therapeutic agents for diseases such as cancer. However, their potential toxicity poses significant obstacles in the developing effective drug candidates. To overcome this limitation, we propose a pathway-screening method based on imaging analysis to evaluate cellular stress caused by natural products. We have established a cellular stress sensing system, named Hepa-ToxMOA, which utilizes HepG2 cells expressing green fluorescent protein (GFP) fluorescence under the control of transcription factor response elements (TREs) for transcription factors (AP1, P53, Nrf2, and NF-κB). Additionally, to augment the drug metabolic activity of the HepG2 cell line, we evaluated the cytotoxicity of 40 natural products with and without S9 fraction-based metabolic activity. Our finding revealed different activities of Hepa-ToxMOA depending on metabolic or non-metabolic activity, highlighting the involvement of specific cellular stress pathways. Our results suggest that developing a Hepa-ToxMOA system based on activity of drug metabolizing enzyme provides crucial insights into the molecular mechanisms initiating cellular stress during liver toxicity screening for natural products. The pathway-screening method addresses challenges related to the potential toxicity of natural products, advancing their translation into viable therapeutic agents.

Long-chain perfluoroalkyl carboxylates induce cytoskeletal abnormalities and activate epithelial-mesenchymal transition in both renal cell carcinoma 3D cultures and Caki-1 xenografted mouse model.

Exposure to perfluorooctanoate (PFOA; a type of perfluoroalkyl carboxylates [PFACs]) may be correlated with the incidence of kidney cancer in individuals exposed to high levels of PFOA. However, mechanistic studies on the influence of PFACs on renal cell carcinoma (RCC) development are lacking. We explored the effects of five types of PFACs on RCC using in vitro and in vivo models to fill this knowledge gap and provide information for environmental/usage regulations. Using 2D/3D cultures of Caki-1 cells, a human clear cell RCC line, we examined the effects of short-chain (SC) PFACs and long-chain (LC) PFACs on RCC physio/pathological markers, including the cytoskeleton, epithelial-mesenchymal transition (EMT)-related proteins, and Na+/K+-ATPase. We also administered three different PFACs orally to mice harboring Caki-1 xenografts to assess the impact of these compounds on engrafted RCC in vivo. Compared with the effects of SCPFACs, mice with Caki-1 xenografts treated with LCPFACs showed increased EMT-related protein expression and exhibited liver toxicity. Therefore, LCPFACs induced EMT, influencing cancer metastasis activity, and displayed higher toxicity in vivo compared with SCPFACs. These findings improve our understanding of the effects of PFACs on RCC development and their corresponding in vivo toxicity, which is crucial for regulating these substances to protect public health.

Integrated transcriptomic analysis of liver and kidney after 28 days of thioacetamide treatment in rats.

Thioacetamide (TAA) was developed as a pesticide; however, it was soon found to cause hepatic and renal toxicity. To evaluate target organ interactions during hepatotoxicity, we compared gene expression profiles in the liver and kidney after TAA treatment. Sprague-Dawley rats were treated daily with oral TAA and then sacrificed, and their tissues were evaluated for acute toxicity (30 and 100 mg/kg bw/day), 7-day (15 and 50 mg/kg bw/day), and 4-week repeated-dose toxicity (10 and 30 mg/kg). After the 4-week repeated toxicity study, total RNA was extracted from the liver and kidneys, and microarray analysis was performed. Differentially expressed genes were selected based on fold change and significance, and gene functions were analyzed using ingenuity pathway analysis. Microarray analysis showed that significantly regulated genes were involved in liver hyperplasia, renal tubule injury, and kidney failure in the TAA-treated group. Commonly regulated genes in the liver or kidney were associated with xenobiotic metabolism, lipid metabolism, and oxidative stress. We revealed changes in the molecular pathways of the target organs in response to TAA and provided information on candidate genes that can indicate TAA-induced toxicity. These results may help elucidate the underlying mechanisms of target organ interactions during TAA-induced hepatotoxicity. Supplementary Information: The online version contains supplementary material available at 10.1007/s43188-022-00156-y.

Sublethal pulmonary toxicity screening of silica nanoparticles in rats after direct intratracheal instillation.

The present aimed to characterize the toxicity of silica nanoparticles in Sprague Dawley rats and determine the dose levels for a repeated-dose toxicity study. Silica nanoparticles (SiO2, 20 nm and 50 nm) were administered as a single intratracheal instillation of standardized SiO2 20 nm (low dose, 200 µg/mL; high dose, 400 µg/mL) and 50 nm (low dose, 200 µg/mL; high dose, 400 µg/mL). Each group consisted of five male rats. We documented the mortality rate, clinical signs, body weight, bronchoalveolar lavage fluid analysis, hematological values, serum chemistry values, organ weight, gross findings at necropsy, and histopathological assessments. Rats treated with 200 µg/mL and 400 µg/mL SiO2 50 nm exhibited a decreased mean corpuscular volume, while those treated with 400 µg/mL of SiO2 50 nm showed increases in absolute monocyte and absolute lymphocyte count as well as prothrombin time. In addition, rats treated with 400 µg/mL SiO2 20 nm and 50 nm presented reduced creatinine, alanine aminotransferase, and sodium levels. Therefore, a single intratracheal instillation of SiO2 20 nm and 50 nm elicited no toxicity up to a dose of 400 µg/mL, and the approximate lethal dose of this test substance exceeded 400 µg/mL in male Sprague Dawley rats under the present experimental conditions.

ToxSTAR: drug-induced liver injury prediction tool for the web environment.

SUMMARY: Drug-induced liver injury (DILI) is a challenging endpoint in predictive toxicology because of the complex reactive metabolites that cause liver damage and the wide range of mechanisms involved in the development of the disease. ToxSTAR provides structural similarity-based DILI analysis and in-house DILI prediction models that predict four DILI subtypes (cholestasis, cirrhosis, hepatitis and steatosis) based on drug and drug metabolite molecules. AVAILABILITY AND IMPLEMENTATION: ToxSTAR is freely available at https://toxstar.kitox.re.kr/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Pulmonary inflammation and cellular responses following exposure to benzalkonium chloride: Potential impact of disrupted pulmonary surfactant homeostasis.

Benzalkonium chloride (BKC) is a prototypical quaternary ammonium disinfectant. Previously, we suggested a no lethal dose level (0.005%) and an LD50 range (0.5-0.05%) of BKC following a single pharyngeal aspiration. Herein, we exposed BKC repeatedly by pharyngeal aspiration for 14 days (0.005 and 0.01%, female mice, total five times with interval of two days, 5 mice/group) and 28 days (0, 0.001, 0.005, and 0.01%, male and female mice, weekly, 16 mice/sex/group). Death following 14 days-repeated exposure did not occur. Meanwhile, chronic pathological lesions were observed in the lung tissues of mice exposed to BKC for 28 days. The total number of bronchial alveolar lavage cells increased, and pulmonary homeostasis of immunologic messenger molecules was disturbed. Following, we investigated BKC-induced cellular responses using human bronchial epithelial cells. The cytotoxicity increased rapidly with concentration. Lysosomal volume, NO production, and lipid peroxidation increased in BKC-treated cells, whereas intracellular ROS level decreased accompanying structural and functional damage of mitochondria. We also found that BKC affected the expression level of immune response, DNA damage, and amino acid biosynthesis-related molecules. More interestingly, lamellar body- and autophagosome-like structures were notably observed in cells exposed to BKC, and necrotic and apoptotic cell death were identified accompanying cell accumulation in the G2/M phase. Therefore, we suggest that repeated respiratory exposure of BKC causes pulmonary inflammation and lung tissue damage and that dead and damaged cells may contribute to the inflammatory response. In addition, the formation process of lamellar body-like structures may function as a key toxicity mechanism.

Comparison of toxicity and cellular responses following pulmonary exposure to different types of nanofibers.

Pulmonary effects of inhaled microfibers are an emerging public health concern. In this study, we investigated toxicity following pulmonary exposure to synthetic polyethylene oxide fibroin (PEONF) and silk fibroin (SFNF) nanofibers and the cellular responses. When instilled intratracheally weekly for four weeks, body weight gain was significantly reduced in female mice exposed to the higher dose of SFNF when compared with the control group. The total number of cells in the lungs was more significant in all treated groups than in the control, whereas the relative portion of neutrophils and eosinophils increased significantly only in female mice exposed to SFNF. Both types of nanofibers induced notable pathological changes and increased pulmonary expression of MCP-1α, CXCL1, and TGF-ß. More importantly, blood calcium, creatinine kinase, sodium, and chloride concentration were affected significantly, showing sex- and material-dependent differences. The relative portion of eosinophils increased only in SFNF-treated mice. In addition, both types of nanofibers induced necrotic and late apoptotic cell death in alveolar macrophages after 24 h of exposure, with accompanying oxidative stress, increased NO production, cell membrane rupture, intracellular organelle damage, and intracellular calcium accumulation. Additionally, multinucleated giant cells were formed in cells exposed to PEONF or SFNF. Taken together, the findings indicate that inhaled PEONF and SFNF may cause systemic adverse health effects with lung tissue damage, showing differences by sex- and material. Furthermore, PEONF- and SFNF-induced inflammatory response may be partly due to the low clearance of dead (or damaged) pulmonary cells and the excellent durability of PEONF and SFNF.

Investigation of Ifosfamide Toxicity Induces Common Upstream Regulator in Liver and Kidney.

Ifosfamide is an alkylating agent, a synthetic analogue of cyclophosphamide, used to treat various solid cancers. In this study, the toxicity of ifosfamide was evaluated using single-and multiple-dose intraperitoneal administration in rats under Good Laboratory Practice guidelines, and an additional microarray experiment was followed to support toxicological findings. A single dose of ifosfamide (50 mg/kg) did not induce any pathological changes. Meanwhile, severe renal toxicity was observed in the 7 and 28 days consecutively administered groups, with significant increases in blood urea nitrogen and creatinine levels. In the tox-list analysis, cholesterol synthesis-related genes were mostly affected in the liver and renal failure-related genes were affected in the kidney after ifosfamide administration. Moreover, interferon regulatory factor 7 was selected as the main upstream regulator that changed in both the liver and kidney, and was found to interact with other target genes, such as ubiquitin specific peptidase 18, radical S-adenosyl methionine domain containing 2, and interferon-stimulated gene 15, which was further confirmed by real-time RT-PCR analysis. In conclusion, we confirmed kidney-biased ifosfamide organ toxicity and identified identically altered genes in both the liver and kidney. Further comprehensive toxicogenomic studies are required to reveal the exact relationship between ifosfamide-induced genes and organ toxicity.

Surgical removal of a telemetry system in a cynomolgus monkey (Macaca fascicularis): a 12-month observation study.

BACKGROUND: Telemetry is a wireless implanted device that measures biological signals in conscious animals and usually requires surgery for its removal when the study is finished. After removing the device, the animals are either used for other studies or euthanatized. CASE PRESENTATION: Herein, we report the case of a living cynomolgus monkey (Macaca fascicularis) that was used for the entire experimental period, instead of euthanasia, after surgical removal of an implanted telemetry system. Radiography was used to determine the status of the implanted telemetry, following which, a repair surgery was performed for removing the system; clinical signs were used to preserve the life of the cynomolgus monkey. Postoperative clinical signs, food consumption, hematology, and serum biochemistry were examined during the 12-month observational period. No abnormal readings or conditions were observed in the subject after implant removal. CONCLUSIONS: This study may be a useful case report for living cynomolgus monkeys in telemetry implantations used throughout the study period. We suggest minimizing the suffering and improving the welfare of these animals.

Impedance Measurement System for Assessing the Barrier Integrity of Three-Dimensional Human Intestinal Organoids.

Human pluripotent stem cell (hPSC)-derived intestinal organoids (HIOs) hold unprecedented promise for basic biology and translational applications. However, developing a quantitative method to evaluate the epithelial cell membrane integrity of HIOs as an in vitro intestinal barrier model is a major challenge because of their complex three-dimensional (3D) structure. In this study, we developed an impedance system to measure the change in electrical resistance of 3D HIOs depending on the integrity of the intestinal epithelial cell membrane, which can reflect functionality and maturity. The expression of intestinal maturation- and tight junction-related markers was significantly higher in HIOs matured in vitro by treatment with IL-2 than in control HIOs. Analysis of gap junction size indicated that mature HIOs have greater integrity, with approximately 30% more compact gaps than immature HIOs. We designed a multi-microchannel system controlled by the inhalation pressure where the HIO is loaded, which enhances the stability and sensitivity of the impedance signal. We demonstrated the applicability of the impedance system by showing the difference in resistance between control and mature HIOs, reflecting the expression of tight junction proteins and their maturation status. We also validated the impedance system by monitoring its resistance in real time during junctional damage to HIOs induced by a digestive agent. In summary, we suggest a quantitative method to directly quantify the physiological changes in complex 3D organoid structures based on impedance spectroscopy, which can be applied to noninvasively monitor live cells and therefore enable their use in subsequent experiments.

TNFα enhances trovafloxacin-induced in vitro hepatotoxicity by inhibiting protective autophagy.

Trovafloxacin (TVX) is associated with idiosyncratic drug-induced liver injury (iDILI) and inflammation-mediated hepatotoxicity. However, the inflammatory stress-regulated mechanisms in iDILI remain unclear. Herein, we elucidated the novel role of tumor-necrosis factor alpha (TNFα), an inflammatory stress factor, in TVX-induced in vitro hepatotoxicity and synergistic toxicity. TVX specifically induced synergistic toxicity in HepG2 cells with TNFα, which inhibits autophagy. TVX-treated HepG2 cells induced protective autophagy by inhibiting the expression of mTOR signaling proteins, while ATG5 knockdown in HepG2 cells, responsible for the impairment of autophagy, enhanced TVX-induced toxicity due to the increase in cytochrome C release and JNK pathway activation. Interestingly, the expression of mTOR signal proteins, which were suppressed by TVX, disrupted the negative feedback of the PI3K/AKT pathway and TNFα rebounded p70S6K phosphorylation. Co-treatment with TVX and TNFα inhibited protective autophagy by maintaining p70S6K activity, which enhanced TVX-induced cytotoxicity. Phosphorylation of p70S6K was inhibited by siRNA knockdown and rapamycin to restore TNFα-inhibited autophagy, which prevented the synergistic effect on TVX-induced cytotoxicity. These results indicate that TVX activates protective autophagy in HepG2 cells exposed to toxicity and an imbalance in negative feedback regulation of autophagy by TNFα synergistically enhanced the toxicity. The finding from this study may contribute to a better understanding of the mechanisms underlying iDILI associated with inflammatory stress.

Preclinical safety assessment of a therapeutic human papillomavirus DNA vaccine combined with intravaginal interleukin-7 fused with hybrid Fc in female rats.

This study was conducted to establish the toxicological profile of combination treatment with therapeutic HPV DNA vaccines (GX-188E) and the long-acting form of recombinant human interleukin-7 fused with hybrid Fc (IL-7hyFc). GX-188E was administered intramuscularly by electroporation with or without IL-7hyFc intravaginally once per 2 weeks for 8 weeks (five times) in female Sprague-Dawley rats. Because up-regulation of immune responses and migration of antigen-specific T cells in cervicoviginal tissue were predicted as therapeutic effects, we distinguished adverse effects from therapeutic effects based on the severity of the systemic immune response, reversibility of lymphoid tissue changes, target tissue damage, and off-target immune responses. We observed that the number of neutrophils was increased, and the number of lymphocytes was decreased in the blood. Further, myofiber degeneration, necrosis, fibroplasia, and cell infiltration were observed at the GX-188E administration site. These changes were fully or partially recovered over a 4-week period. Analysis of lymphocytes in spleen revealed that CD4+ T cells and total T cells decreased in rats treated with GX-188E in combination with a high dose of IL-7hyFc (1.25 mg/animal). However, these changes were not considered adverse because they were transient and may have been related to electroporation-mediated DNA delivery or the local migration of lymphocytes induced by IL-7. Therefore, the potential toxicity of the combination of GX-188E and IL-7hyFc treatment was comparable to that of GX-188E treatment alone, and the no observed adverse effect level for GX-188E with IL-7hyFc was considered as 320 µg/animal for GX-188E and 1.25 mg/animal for IL-7hyFc.

Toxicity of orally administered food-grade titanium dioxide nanoparticles.

This year, France banned the application of titanium dioxide nanoparticles as a food additive (hereafter, E171) based on the insufficient oral toxicity data. Here, we investigated the subchronic toxic responses of E171 (0, 10, 100, and 1,000 mg/kg) and tried to elucidate the possible toxic mechanism using AGS cells, a human stomach epithelial cell line. There were no dose-related changes in the Organisation for Economic Cooperation and Development test guideline-related endpoints. Meanwhile, E171 deeply penetrated cells lining the stomach tissues of rats, and the IgM and granulocyte-macrophage colony-stimulating factor levels were significantly lower in the blood from rats exposed to E171 compared with the control. The colonic antioxidant protein level decreased with increasing Ti accumulation. Additionally, after 24-h exposure, E171 located in the perinuclear region of AGS cells and affected expression of endoplasmic reticulum stress-related proteins. However, cell death was not observed up to the used maximum concentration. A gene profile analysis also showed that immune response-related microRNAs were most strongly affected by E171 exposure. Collectively, we concluded that the NOAEL of E171 for 90 days repeated oral administration is between 100 and 1,000 mg/kg for both male and female rats. Additionally, further study is needed to clarify the possible carcinogenesis following the chronic accumulation in the colon.

Effect of PM10 on pulmonary immune response and fetus development.

Despite numerous reports that ambient particulate matter is a key determinant for human health, toxicity data produced based on physicochemical properties of particulate matters is very lack, suggesting lack of scientific evidence for regulation. In this study, we sampled inhalable particulate matters (PM10) in northern Seoul, Korea. PM10 showed atypical- and fiber-type particles with the average size and the surface charge of 1,598.1 ± 128.7 nm and -27.5 ± 2.8, respectively, and various toxic elements were detected in the water extract. On day 90 after the first pulmonary exposure, total cell number dose-dependently increased in the lungs of both sexes of mice. PM10 induced Th1-dominant immune response with pathological changes in both sexes of mice. Meanwhile, composition of total cells and expression of proteins which functions in cell-to-cell communication showed different trends between sexes. Following, male and female mice were mated to identify effects of PM10 to the next generation. PM10 remained in the lung of dams until day 21 after birth, and the levels of IgA and IgE increased in the blood of dams exposed to the maximum dose compared to control. In addition, the interval between births of fetuses, the number of offspring, the neonatal survival rate (day 4 after birth) and the sex ratio seemed to be affected at the maximum dose, and particularly, all offspring from one dam were stillborn. In addition, expression of HIF-1α protein increased in the lung tissue of dams exposed to PM10, and level of hypoxia-related proteins was notably enhanced in PM10-exposed bronchial epithelial cells compared to control. Taken together, we suggest that inhaled PM10 may induce Th1-shifting immune response in the lung, and that it may affect reproduction (fetus development) by causing lung hypoxia. Additionally, we propose that further study is needed to identify particle-size-dependent effects on development of the next generation.

Formation of lamellar body-like structure may be an initiator of didecyldimethylammonium chloride-induced toxic response.

Due to the pandemic of coronavirus disease 2019, the use of disinfectants is rapidly increasing worldwide. Didecyldimethylammonium chloride (DDAC) is an EPA-registered disinfectant, it was also a component in humidifier disinfectants that had caused idiopathic pulmonary diseases in Korea. In this study, we identified the possible pulmonary toxic response and mechanism using human bronchial epithelial (BEAS-2B) cells and mice. First, cell viability decreased sharply at a 4 µg/mL of concentration. The volume of intracellular organelles and the ROS level reduced, leading to the formation of apoptotic bodies and an increase of the LDH release. Secretion of pro-inflammatory cytokines (IL-1ß, IL-6, and TNF-α) and matrix metalloproteinase-1 also significantly increased. More importantly, lamellar body-like structures were formed in both the cells and mice exposed to DDAC, and the expression of both the indicator proteins for lamellar body (ABCA3 and Rab11a) and surfactant proteins (A, B, and D) was clearly enhanced. In addition, chronic fibrotic pulmonary lesions were notably observed in mice instilled twice (weekly) with DDAC (500 µg), ultimately resulting in death. Taken together, we suggest that disruption of pulmonary surfactant homeostasis may contribute to DDAC-induced cell death and subsequent pathophysiology and that the formation of lamellar body-like structures may play a role as the trigger. In addition, we propose that the cause of sudden death of mice exposed to DDAC should be clearly elucidated for the safe application of DDAC.

Amorphous silica nanoparticle-induced pulmonary inflammatory response depends on particle size and is sex-specific in rats.

Due to mass production and extensive use, the potential adverse health effects of amorphous silica nanoparticles (ASiNPs) have received a significant attention from the public and researchers. However, the relationship between physicochemical properties of ASiNPs and their health effects is still unclear. In this study, we manufactured two types of ASiNPs of different diameters (20 and 50 nm) and compared the toxic response induced in rats after intratracheal instillation (75, 150 or 300 µg/rat). There were no dose-related differences in mortality, body weight gain or organ weight between the groups. However both types of ASiNPs significantly decreased the proportion of neutrophils in male rats, whereas the levels of hemoglobin and hematocrit were markedly reduced only in female rats instilled with 20 nm-ASiNPs. ASiNPs-induced lung tissue damage seemed to be more evident in the 20 nm ASiNP-treated group and in female rats than male rats. Similarly, expression of caveolin-1 and matrix metalloproteinase-9 seemed to be most notably enhanced in female rats treated with 20 nm-ASiNPs. The total number of bronchial alveolar lavage cells significantly increased in rats instilled with 20 nm-ASiNPs, accompanying a decrease in the proportion of macrophages and an increase in polymorphonuclear leukocytes. Moreover, secretion of inflammatory mediators clearly increased in human bronchial epithelial cells treated with 20 nm-ASiNPs, but not in those treated with 50 nm-ASiNPs. These results suggest that pulmonary effects of ASiNPs depend on particle size. Sex-dependent differences should also be carefully considered in understanding nanomaterial-induced adverse health effects.

Repeated-oral dose toxicity of polyethylene microplastics and the possible implications on reproduction and development of the next generation.

With the increased distribution of microplastics in the environment, the potential for harmful effects on human health and ecosystems have become a global concern. Considering that polyethylene microplastics (PE-MPs) are among the most produced plastics worldwide, we administered PE-MPs (0.125, 0.5, 2 mg/day/mouse) by gavage to mice (10 mice/sex/dose) for 90 days. Compared to control, the body weight gain was significantly reduced in the male mice, and the proportion of neutrophils in the blood stream clearly increased in both sexes of mice. Persistence of a PE-MPs-like material and migration of granules to the mast cell membrane and accumulation of damaged organelles were observed in the stomachs and the spleens from the treated dams, respectively. Additionally, the IgA level in the blood stream was significantly elevated in the dams administered with PE-MPs compared to control, and the subpopulation of lymphocytes within the spleen was altered. Following, we performed an additional study to screen the effects of PE-MPs on reproduction and development (5 mice/sex/dose). Importantly, number of live births per dam, the sex ratio of pups, and body weight of pups was notably altered in groups treated with PE-MPs compared to the control group. Additionally, PE-MPs affected the subpopulation of lymphocytes within the spleen of the offspring, as did in the dams. Therefore, we propose that reproductive and developmental toxicity testing is warranted to evaluate the safety of microplastics. Additionally, we suggest that the IgA level may be used as a biomarker for harmful effects following exposure on microplastics.

Preventive Effect of Citrus aurantium Peel Extract on High-Fat Diet-Induced Non-alcoholic Fatty Liver in Mice.

Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic lipid accumulation, which is the most common form of chronic liver disease. Multiple clinical studies using natural compounds such as flavonoids have been conducted to treat NAFLD. In the present study, the pharmacological effect of Citrus aurantium L. (Rutaceae) peel extract (CAE), which contains over 27% of polymethoxyflavone nobiletin, on NAFLD was evaluated using a high-fat diet (HFD) animal model susceptible to developing NAFLD. C57BL/6 mice were fed an HFD (60% kcal of energy derived from fat) for 8 weeks to induce obesity. Obese mice were randomly allocated to four groups of eight mice each (HFD alone, HFD with silymarin, HFD with 50 mg/kg CAE, and HFD with 100 mg/kg CAE). After 8 weeks of treatment, all mice were euthanized, and plasma and liver tissues were analyzed biochemically and histopathologically. The results indicate that CAE treatment significantly reduced HFD-induced NAFLD, as shown by decreased serum lipid index and prevented liver histopathology. The expression of genes involved in lipid synthesis including free fatty acid (FFA), peroxisome-proliferator-activated receptor γ (PPAR-γ), sterol receptor element binding protein 1c (SREBP-1c), and fatty acid synthesis enzyme was suppressed by CAE treatment. Moreover, compared to untreated mice, CAE-treated HFD mice showed decreased pro-inflammatory cytokine expression. These results demonstrated that CAE prevented HFD-induced NAFLD by reducing plasma levels of triglyceride and cholesterol and de novo lipid synthesis.