Cadmium is a known contributing factor for cardiovascular diseases, such as atherosclerosis and hypertension. Although zinc protects against cadmium cytotoxicity in vascular endothelial cells, the detailed mechanisms, especially the differences in the roles of a metal transporter Zrt- and Irt-like protein 8 (ZIP8) and metallothionein (MT), remain unclear. ZIP8 is involved in the uptake of cadmium, whereas MT is induced by and sequesters it. Zinc protects bovine aortic endothelial cells from cadmium cytotoxicity in a culture system in a concentration-dependent manner. Zinc significantly decreased intracellular cadmium accumulation. Cadmium elevated the expression of ZIP8 mRNA, and zinc significantly suppressed this increase in a concentration-dependent manner. However, the expression of MT was not induced by zinc alone but by cadmium; the induction of MT by cadmium was suppressed by zinc at lower concentrations, but intensified by zinc at higher concentrations. However, even when MT induction was strongly suppressed by siRNA-mediated knockdown, cadmium cytotoxicity was reduced by zinc at both lower and higher concentrations. In the absence of zinc, cadmium cytotoxicity was increased by metal response element-binding transcription factor-1 (MTF-1) siRNA-mediated knockdown of MT-1/2. Consequently, it has been suggested that zinc protects the vascular endothelial cells through a concentration-dependent mechanism. Specifically, the decrease in ZIP8 expression is crucially important for the protective effect of zinc at low concentrations against cadmium cytotoxicity in vascular endothelial cells, whereas both the decrease in ZIP8 and the induction of MT contribute to the protection by zinc at high concentrations.

Aristolochic acids (AAs) with strong bio-toxicity are the natural compounds that consist in Aristolochiaceae plants. There are the governing health issues regarding toxicities of AAs although Aristolochiaceae plants have been used as herbal medicines. For example, AAs are known as significant risk factors for nephropathy, urological cancer, liver cancer and so on. However, the understandings about the molecular mechanisms of toxicity of each AA derivative have been still poor and insufficiently studied. In this study, we investigated the effects of four AA derivatives (AA-A, AA-B, AA-C and AA-D) on the viability and the all-trans retinoic acid (ATRA)-induced superoxide anion (O₂^-)-generating ability of human leukemia U937 cells. AA-A and AA-C remarkably reduced cell viability when co-treated with ATRA while AA-B and AA-D had little effect on viability of U937 cells. On the other hand, only AA-C among the four AAs dramatically up-regulated the ATRA-induced O₂^--generating ability. Quantitative RT-PCR and immunoblotting analyses showed that AA-C significantly enhances the ATRA-induced O₂^--generating ability via up-regulating gene expression levels of gp91-phox, which is an essential factor for the O₂^--generating ability of phagocytes. These findings revealed that AA-C has not only the ATRA-enhanced cytotoxic effect but also the remarkable enhancing effect on the ATRA-induced O₂^--generating ability via up-regulating transcription of gp91-phox gene.

Tomatidine is an aglycone of α-tomatine, a glycoalkaloid present in tomato plants, and has muscle atrophy inhibitory effect and anticancer activity. Tomatidine-Rich Tomato Leaf Extract powder (TRTLE) contains 60% tomatidine, which is converted to tomatidine by acid hydrolysis after extracting α-tomatine from tomato leaves. The purpose of this study was to evaluate the safety of TRTLE by conducting a series of toxicity studies in mice and bacteria to support its safe food use. Single-dose and 90-day repeated-dose toxicity study were conducted in 6-week-old ICR male and female mice to calculate the LD₅₀ and non-toxic dose (NOAEL) of TRTLE. In the single-dose toxicity study, a single oral dose of 667, 2,000, and 5,000 mg TRTLE/kg body weight (bw) was administered, and in the 90-day repeated-dose toxicity study, 133 mg TRTLE/kg bw was administered orally daily. In addition, the Ames test was performed with Salmonella Typhimurium and Escherichia coli to determine the genotoxic activity. The single-dose toxicity study indicated the LD₅₀ was 833 mg TRTLE/kg bw (tomatidine equivalent: 500 mg/kg bw). In the 90-day repeated-dose toxicity study, no abnormalities due to TRTLE were observed in each laboratory test, including general symptoms, body weight changes, hematology, urinalysis, and histopathological examination. In the Ames test, TRTLE was confirmed not to be mutagenic with or without metabolic activation. Based on these data, the NOAEL in mice was determined to be 133 mg TRTLE/kg bw (tomatidine equivalent: 80 mg/kg bw).

p-Cymene, is a monocyclic monoterpene hydrocarbon, commonly used as a flavoring agent in food. A 90-day repeated oral toxicological study of p-cymene was conducted to examine the toxicological properties and determine the no-observed-adverse-effect level (NOAEL) of p-cymene in Crl:CD (SD) rats at the following doses: 0 (corn oil), 2.4, 12, and 60 mg/kg/day. No mortality or abnormal clinical signs were observed in the treatment groups. The body weight, food consumption, ophthalmoscopy, and gross pathology of the rats were also not affected by p-cymene treatment. However, in the 60 mg/kg group, certain parameters decreased in males, including hemoglobin and hematocrit, red blood cell count, triglyceride, total protein, and albumin. In females, urine volume and total potassium excretion increased, whereas specific gravity, and sodium, potassium, and chlorine concentrations decreased. Increased liver weight was observed in both males and females. Histopathological observations revealed centrilobular hepatocellular hypertrophy. In the 12 mg/kg group, no adverse effects of p-cymene treatment were observed in both sexes. In conclusion, the NOAEL of p-cymene was 12 mg/kg/day for both sexes under the present experimental conditions, considering the alterations in urinalysis, hematology, clinical biochemistry, and histopathology.

Titin is a giant protein that is specifically expressed in striated muscle and essential for the maintenance of sarcomere structure and function. Recently, the N-terminal fragment of the Titin (N-titin) has been reported to show high levels in human urine in patients with muscular diseases and is expected to serve as a diagnostic biomarker for these diseases. In this study, we examined the utility of N-titin as a biomarker to detect muscle atrophy in mice. Male BALB/c mice (6 weeks of age, n=5 per group) were given 10 mg/L dexamethasone (DEX) dissolved in drinking water for 4 weeks. The gastrocnemius muscle (GAS) weight was significantly decreased and mRNA levels of muscle atrophy-related genes (Atrogin-1 and MuRF-1) were increased in the GAS after 4 weeks of DEX treatment. Although there were no degenerative/necrotic changes in the histopathological examination, the muscle fiber cross-sectional area significantly decreased in the GAS. On the other hand, there were no DEX treatment-related changes in the muscle weights and the muscle fiber cross-sectional area in the soleus muscle. These results suggest that 4-week of DEX treatment preferentially caused atrophy of fast-dominant muscle. Under the condition of this study, urinary N-titin/CRN ratio markedly increased from Week 2 of the DEX treatment. From the above results, the urinary N-titin/CRN ratio could be a biomarker for monitoring skeletal muscle atrophy in mice.

We investigated the characteristics of pharmaceutical concentrations and antimicrobial activities in river water from the Tone River system in Gunma Prefecture. The mean concentrations of diphenhydramine, clarithromycin, carbamazepine, and bezafibrate in the midstream of the Tone River were 8.6, 29, 3.8, and 8.1 ng/L, respectively. Their concentrations were nearly half of those in the midstream of the Ayase River, the main water source of which is wastewater. Seasonal variations in pharmaceutical concentrations were high in winter and low in late spring and autumn. This variation depended on the flow rate of the river water, which in turn depended on the rainfall in the upstream area. Except for bezafibrate, the pharmaceutical concentrations in river water did not change after 5 days of incubation at 30°C, indicating that biochemical degradation during the hot summer season was minimal. A comparison of the concentrations between the sampling locations revealed that the pharmaceutical load was proportional to basin population, and the annual fluxes of pharmaceuticals from Gunma Prefecture were estimated to be 98, 210, 28, and 53 kg/year, respectively. Disc diffusion assay of some samples of Tone River water extracts revealed inhibition zones owing to their antimicrobial activity. However, no relationship was observed between the diameter of the inhibition zone and clarithromycin concentration in the river water. These results suggest that the antimicrobial activities of the river samples were not dependent on clarithromycin. We are currently investigating the pollution and drug-resistant bacteria present in the Tone River in detail.