Drug-induced liver injury (DILI), in rare instances, can become severe and lead to fatal outcomes; therefore, it is crucial to clarify the mechanisms underlying DILI. Antibiotics are well known to induce a high frequency of DILI. We hypothesized that alterations in the bile acid composition, owing to the destruction of enterobacteria, is one factor frequently associated with antibiotics-induced liver injury. To verify this hypothesis, we constructed and evaluated a mouse model of antibiotic-induced liver injury. Although no liver injury was observed upon administering three antibiotics (vancomycin, flucloxacillin, and ampicillin) alone, oral administration of antibiotics (vancomycin and flucloxacillin) with cholic acid (CA) increased levels of alanine transaminase (ALT). In the high ALT group, plasma taurocholic acid (TCA) levels were significantly increased, with a positive correlation detected between ALT and blood TCA levels (Spearman’s ρ = 0.839). Overall, we constructed a mouse model of antibiotic-induced livery injury and confirmed our hypothesis that elevated blood levels of conjugated bile acids (increased TCA levels in this mouse model) can induce DILI.

Protocatechuic acid (PCA) is a phenolic compound naturally occurring in various plants. Although numerous studies have reported on its various biological activities, information on the anti-carcinogenic potential and its molecular mechanisms in animal models has never been conclusively determined. Therefore, this study aimed to study the inhibitory effect of PCA against diethylnitrosamine (DEN)-induced rat hepatocarcinogenesis. Rats received three intraperitoneal injections of 100 mg kg^-1 body weight of DEN to initiate hepatic preneoplastic lesions, and glutathione S-transferase placental form (GST-P)-positive foci were used as the end-point marker. Rats were treated with PCA at 40 mg kg^-1 body weight by oral gavage administration for 15 weeks to study its chemopreventive effect on the early stages of hepatocarcinogenesis. PCA treatment decreased the number and the area of hepatic GST-P-positive foci in DEN-induced rats. It inhibits the activity of cytochrome P450 reductase and reduces the expression of cytochrome P450 2E1 protein. It also suppresses cell proliferation by down-regulation of Cyclin D1 expression. Additionally, it induces apoptosis, as indicated by the up-regulation of pro-apoptotic genes, Bax and Bad, in DEN-induced rats. These findings suggest that PCA is an anti-cancer agent that inhibits hepatocarcinogenesis in DEN-treated rats.

The aim of this work was to develop and validate a cell-based in vitro assay for predicting photoallergenicity by expanding the scope of our previously reported in vitro method, photo-KeratinoSens™, which is a luciferase-based assay dependent on activation of the Keap1-Nrf2-ARE pathway. First, we increased the maximum starting test concentration from a fixed 2000 µM in the original KeratinoSens™ to either 5000 μg/mL or 4 times the concentration providing a cell viability of 75% (under UV irradiation), depending on the cytotoxicity. Then, we established that 0.5% ethanol, 0.5% acetone and 0.1% tetrahydrofuran are available as solvents in addition to 1% DMSO, which is used in the standard KeratinoSens™ method (OECD TG442D). We confirmed that a representative photoallergen, 6-methylcoumarin, gave reproducible results. To validate the developed assay, we used it to evaluate a library of 90 chemicals consisting of 60 known photoallergens and 30 non-photoallergens. The accuracy, sensitivity, specificity and balanced accuracy of photo-KeratinoSens™ were 76.7% (69/90), 66.7% (40/60), 96.7% (29/30) and 81.7%, respectively. When we excluded chemicals with no UVA absorption, the accuracy, sensitivity, specificity and balanced accuracy were improved to 81.8% (45/55), 80.0% (36/45), 90.0% (9/10) and 85%, respectively. Our results suggest that photo-KeratinoSens™, which combines the OECD TG442D in vitro skin sensitization test detecting key event 2 in the adverse outcome pathway (AOP) of skin sensitization with exposure to UV iradiation, may be useful as a contributory input in a weight-of-evidence approach for evaluating photoallergenicity potential without animal testing.

Urinary biomarkers have been used widely in non-clinical toxicity studies to detect kidney dysfunction/injury caused by drugs under development. Although their usefulness to evaluate nephrotoxicity has been well studied, knowledge about sex differences in the urinary excretion levels of these biomarkers remains inadequate. We previously demonstrated the existence of sex differences in the excretion levels of urinary biomarkers and that these differences were associated with the endogenous testosterone levels. In this study, testosterone was repeatedly administered subcutaneously to female rats for 4 weeks along with male rats as a comparison control, to investigate how the blood levels of testosterone contribute to the sex differences in the urinary biomarker and renal cortical protein levels. The results showed that the urinary excretion of leucine aminopeptidase (LAP), gamma-glutamyltransferase (γ-GTP), cystatin C (Cys-C), liver-type fatty acid binding protein (L-FABP), and beta2-microglobulin (β2MG) were increased, and the urinary excretion of kidney injury molecule 1 (Kim-1) was decreased. The protein level of megalin, an endocytic receptor, in the renal cortex, was higher in female rats than in male rats, and testosterone treatment led to decrease in the level in the female rats. Our results suggest that the blood testosterone level might be responsible for the sex differences in the urinary excretion levels of low-molecular-weight proteins via regulating the expression level of megalin in the renal cortex.

The slow-releasing hydrogen sulfide (H₂S) donor GYY4137 and A549 human alveolar epithelial cells were used to investigate H₂S toxicity in pulmonary epithelial cells. The cells were exposed to 0, 0.3, 1, or 3 mM GYY4137 for 6 days. Morphological changes, including cell death and transformation into spindle-shaped mesenchymal-like cells, were observed in cells exposed to 1 or 3 mM GYY4137. Transcriptome analysis of cells exposed to 3 mM GYY4137 for 6 days indicated ER stress and epithelial-mesenchymal transition (EMT), the latter confirmed by qRT-PCR, immunoblot, and immunocytochemical analyses. These results show the possible roles of EMT in the homeostasis of pulmonary epithelial cells during exposure to H₂S.

Exposure of embryos or fetuses to harmful substances, such as teratogens, can result in embryonic or fetal death and a wide range of malformations. Zebrafish models have emerged as a valuable tool for assessing developmental toxicity and safety profiles of chemical compounds. Our previous research demonstrated that zebrafish larvae exhibit developmental abnormalities that mirror those observed in mammalian studies for more than 80% of the known Reference Compounds listed in the ICH S5 (R3) guideline. In this study, we presented high-resolution images depicting pharmaceutical-induced malformations across multiple anatomical regions, including the body axis, somites, notochord, fins, head, eyes, otoliths, jaw, heart, abdomen, and whole body. Frequent co-occurrence of specific defects, such as body axis and notochord malformations, was observed as described previously. Some physiological and morphological features, including heartbeat rate alterations and swim bladder inflation, were deemed dispensable for MEFL testing in zebrafish. Reproducibility was confirmed through inter-laboratory testing conducted both within our group and by other groups, supporting the reliability of zebrafish MEFL testing as an alternative approach in line with ICH S5 (R3).