RESUMEN
Cinnabar has been used for treatment of various disorders for thousands of years. The medical use of cinnabar, however, has been controversial because of its heavy metal mercury content. A large quantity of studies indicate that the toxicity of cinnabar is far below other inorganic or organic mercury-containing compounds. Yet, the underlying molecular basis has remained unresolved. Here, we investigated the beneficial effects of cinnabar on serum-nutrient starvation-elicited cell injury. Our findings showed that treatment of human renal proximal tubular cells (HK-2) with 4 nM cinnabar effectively inhibited nutrient deprivation induced apoptosis, reduced intracellular reactive oxygen species generation and increased GSH content, which was contrary to the exacerbated apoptotic cell death and oxidative stress in cells treated with HgCl2 at equal mercury concentration. In addition, cinnabar exerted robust antioxidative and antiapoptotic effects in cells under dual challenges of nutrient deprivation and treatment of H2O2. The protein expression levels of both CHOP and PERK were remarkably down-regulated in the cells treated with cinnabar compared to the control cells or cells treated with HgCl2. Overall, our data indicates that cinnabar at low concentration exerts anti-oxidative stress and anti-apoptosis effects by inhibiting the expression of the endoplasmic reticulum stress pathway proteins CHOP and PERK.
RESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Xian-Ling-Gu-Bao (XLGB) Fufang is herbal formula widely used to treat osteoporosis and other bone disorders. Because of its commonality in the clinical use, there is a safety concern over the use of XLGB combined with other androgen deprivation therapy (ADT) drugs such as flutamide (FLU) that is associated with reduced bone density. To date, there have been no evaluations on the side effects of the drug-drug interaction between XLGB and FLU. AIM OF THE STUDY: The present study was designed to investigate the hepatotoxicity in the context of the combined treatment of XLGB and FLU in a mouse model, and to determine whether the metabolic activation of FLU through induction of CYP1A2 plays a role in the increased hepatoxicity caused by the combination of XLGB and FLU. MATERIALS AND METHODS: C57 mice were administered with either XLGB (6,160 mg/kg), FLU (300 mg/kg), or with the combination of the two drugs. Animals were treated with XLGB for 5 days before the combined administration of XLGB and FLU for another 4 days. The serum of mice from single or the combined administration groups was collected for biochemical analysis. The mouse liver was collected to examine liver morphological changes, evaluate liver coefficient, as well as determine the mRNA expression of P450 isozymes (Cyp1a2, Cyp3a11 and Cyp2c37). For metabolism analysis, mice were treated with XLGB, FLU, or the combination of XLGB and FLU for 24 h. The urine samples were collected for the analysis of FLU-NAC conjugate by UPLC-Q-Orbitrap MS. The liver microsomes were prepared from fresh livers to determine the activity of metabolizing enzyme CYP1A2. RESULTS: The combined treatment of XLGB and FLU caused loss of mice body weight and elicited significant liver toxicity as evidenced by an increased liver coefficient and serum lactate dehydrogenase (LDH) activity as well as pathological changes of fatty lesion of liver tissue. FLU increased hepatic expression of Cyp1a2 mRNA that was further elevated in the liver of mice when administered with both FLU and XLGB. Treatment of FLU resulted in an increase in the expression of Cyp3a11 mRNA that was negated when mice were co-treated with FLU and XLGB. No significant difference in Cyp2c37 mRNA expression was observed among the different treatment groups as compared to the control. Analysis of metabolic activity showed that the combined administration caused a synergic effect in elevating the activity of the CYP1A2 enzyme. Mass spectrometry analysis identified the presence of FLU reactive metabolite derived FLU-NAC conjugate in the urine of mice treated with FLU. Strikingly, about a two-fold increase of the FLU-NAC conjugate was detected when treated with both FLU and XLGB, indicating an elevated amount of toxic metabolite produced from FLU in the present of XLGB. CONCLUSION: FLU and XLGB co-treatment potentiated FLU-induced hepatoxicity. This increased hepatoxicity was mediated through the induction of CYP1A2 activity which in turn enhanced bioactivation of FLU leading to over production of FLU-NAC conjugate and oxidative stress. These results offer warnings about serious side effects of the FLU-XLGB interaction in the clinical practice.
