In this study, we developed screening-level hepatotoxicity prediction models using test data on in vitro assays, which measure key events at molecular levels that are possibly linked to hepatotoxicity. Hepatotoxic chemicals were retrieved from repeated-dose toxicity databases of the Hazard Evaluation Support System Integrated Platform and the Toxicogenomics Project. In vitro assay data with specified protein targets likely leading to hepatotoxicity were selected using the hepatotoxic chemicals. In total, 47 in vitro assays were selected for constructing the hepatotoxicity prediction models. Then, two predictive models were constructed. Model A returns “Hepatotoxic” if the query chemical is tested, and the test result is “Active” in any of the selected in vitro assays. Model B returns “Hepatotoxic” if an analog of the query chemical is tested, and the test result is “Active” in any of the selected in vitro assays. External validation of the two models was performed using repeated-dose toxicity test data from the Toxicity Reference Database. Model A and Model B had sensitivity values of 0.67 and 0.72 and specificity values of 0.74 and 0.72, respectively. Our models could predict the hepatotoxic chemicals underlying the toxic mechanisms that are not established by the existing knowledge base model. On the other hand, false negatives were found to involve mechanisms requiring metabolic activation. Because our hepatotoxicity prediction model is based on the biological activity of key molecular events leading to the toxicity endpoint, scientific justification would be more acceptable as adverse outcome pathway information becomes more available.

Sulforaphane [1-isothiocyanato-4-(methyl-sulfinyl)butane] is an isothiocyanate derivative from cruciferous vegetables, with anti-proliferative actions on various cancer and tumor cells. In this paper, we envisaged the effects of sulforaphane on various functions (growth inhibition, cytotoxicity and enhancement of O₂^--generating activity) of human monoblastic leukemia U937 cells. Sulforaphane showed strong cytotoxicity, resulting in inhibition of proliferation in a dose-dependent manner. In addition, cell differentiation induced by 1 μM all-trans retionic acid (RA) remarkably caused the enhanced resistance against cytotoxicity of sulforaphane. Moreover, the RA-induced O₂^--generating activity was also enhanced by sulforaphane in a dose dependent manner. When U937 cells were cultured in the presence of 1 μM RA and 2 μM sulforaphane, the O₂^--generating activity increased more than 2.5-fold compared with that in the absence of the latter. Semiquantitative RT-PCR showed that co-treatment with RA and sulforaphane slightly enhanced transcription of only p47-phox gene among five essential components (p22-phox, gp91-phox, p40-phox, p47-phox and p67-phox) for the O₂^--generating system in phagocytes. On the other hand, immunoblot analysis revealed that co-treatment with RA and sulforaphane caused accumulation of protein levels of p47-phox (upto ~130%) and p67-phox (upto ~240%) compared with those of the RA-treatment alone. These results indicated that sulforaphane may enhance the RA-induced O₂^--generating activity in U937 cells via accumulation of p47-phox and p67-phox proteins. These data suggested that sulforaphane may serve as an effective drug for leukemia treatment.

Hesperidin is a flavonoid with many nutritional benefits including antioxidant activity in food formulations; however, hesperidin is practically insoluble in water. A commercial enzymatic process has been developed in which a glucose molecule is attached to hesperidin increasing the solubility by approximately 100,000 times. The substance is called glucosyl hesperidin (GH) with the main component being monoglucosyl hesperidin (MGH; 75 to 85%). This paper presents results of OECD-compliant toxicity studies with GH, including 4-week and 13-week sub-chronic toxicity, and teratogenicity studies in rats, and chromosomal aberration and mouse micronucleus formation tests. There were no deaths and no treatment-related adverse effects in the 4-week (highest dose 15,000 ppm) or the 13-week sub-chronic (highest dose 50,000 ppm) studies. There were no statistically significant treatment-related adverse effects on any parameter evaluated. The NOEL in the 4-week study was calculated as 1,280 mg/kg/day in females and 1,206 mg/kg/day for males, and in the 13-week study, the NOEL was 3,428 and 3,084 mg/kg/day, for females and males, respectively. In the teratogenicity study, the NOAEL was 1,000 mg/kg/day of treatment for both dams and fetuses. No genotoxicity was observed in the chromosomal study at 5,000 μg/mL and no micronuclei at 2,000 mg/kg, respectively. The results of these OECD-compliant studies support the safe use of GH as a food and beverage ingredient.

The diabetic state is considered to be one of the risk factors of drug-induced liver injury (DILI) because of the lower levels of glutathione for detoxification by conjugation with drugs. Carbamazepine (CBZ) -induced hepatotoxicity in humans is rare and unpredictable with the present state of knowledge, but it is somehow related to disturbance of glutathione metabolism, although data in this regard are limited. In order to estimate the potential risk of DILI in patients with type 2 diabetes mellitus (T2DM), we investigated the liver injury from CBZ, which is often used in the treatment of painful diabetic neuropathy in diabetic patients, using SD rats and Spontaneously Diabetic Torii-Lepr^fa (SDT fatty) rats as a model for human T2DM. The SDT fatty rats appropriately mimic the diabetic state in humans and have similar profiles of glucose metabolism, hepatic function tests and glutathione synthesis to those in patients with T2DM. Short-term oral dosing with CBZ to the SDT fatty rats revealed that liver injury was detected in the SDT fatty rats but not in the SD rats and the difference was considered to be due to lower hepatic detoxification of the metabolites of CBZ by depleted hepatic glutathione synthesis. In conclusion, the potential for CBZ to induce liver injury is considered to be higher in diabetic patients than in non-diabetic humans.

Drug-induced mitochondrial dysfunction can lead to severe adverse effects. Accordingly, new in vitro assay systems for assessing mitochondrial-related toxicity are required. Current systems evaluate drug-induced mitochondrial dysfunction based on cell death. However, if mitochondria are damaged without cell death, these methods run the risk of overlooking toxic or dangerous compounds. To solve this problem, we attempted to measure morphological changes semi-quantitatively by transmission electron microscopy and to detect subtle changes in mitochondrial function. To this end, we exposed HepG2 cells cultured in galactose-containing medium to benzbromarone (BBR), which impairs mitochondrial function. After 24 hr of BBR exposure, we compared the rate of cell death between galactose and glucose cultures. Before the onset of cell death, BBR increased the ratio of damaged mitochondria to a greater extent in galactose-cultured than glucose-cultured HepG2 cells. Our results suggested that this new in vitro assay system could detect mitochondrial-related toxicity before the onset of cell death.