Identification and Enzymatic Activity Evaluation of a Novel CYP2C9 Allelic Variant Discovered in a Patient.

Warfarin is a widely prescribed anticoagulant but the doses required to attain the optimum therapeutic effect exhibit dramatic inter-individual variability. Pharmacogenomics-guided warfarin dosing has been recommended to improve safety and effectiveness. We analyzed the cytochrome P450 2C9 (CYP2C9) and vitamin K epoxide reductase complex subunit 1 (VKORC1) genes among 120 patients taking warfarin. A new coding variant was identified by sequencing CYP2C9. The novel A > G mutation at nucleotide position 14,277 led to an amino acid substitution of isoleucine with valine at position 213 (I213V). The functional consequence of the variant was subsequently evaluated in vitro. cDNA of the novel variant was constructed by site-directed mutagenesis and the recombinant protein was expressed in vitro using a baculovirus-insect cell expression system. The recombinant protein expression was quantified at apoprotein and holoprotein levels. Its enzymatic activities toward tolbutamide, warfarin and losartan were then assessed. It exhibited changed apparent Km values and increases of 148%, 84% and 67% in the intrinsic clearance of tolbutamide, warfarin and losartan, respectively, compared to wild-type CYP2C9*1, indicating dramatically enhanced in vitro enzymatic activity. Our study suggests that the amino acid at position 213 in wild-type CYP2C9*1 may be important for the enzymatic activity of CYP2C9 toward tolbutamide, warfarin and losartan. In summary, a patient taking high-dose warfarin (6.0 mg/day) in order to achieve the target international normalized ratio was found to have a mutation in the CYP2C9 gene.

Alpha-Lipoic Acid Attenuates Cadmium- and Lead-Induced Neurotoxicity by Inhibiting Both Endoplasmic-Reticulum Stress and Activation of Fas/FasL and Mitochondrial Apoptotic Pathways in Rat Cerebral Cortex.

Although many studies have reported toxic effects of cadmium (Cd) and lead (Pb) in the central nervous system, few studies have investigated the combined toxicity of Cd and Pb. The mechanisms by which these combined heavy metals induce toxicity, as well as effective means to exert neuroprotection from these agents, remain poorly understood. To investigate the protective effects of alpha-lipoic acid (α-LA) on Cd- and/or Pb-induced cortical damage in rats, 48 Sprague-Dawley rats were exposed to drinking water containing 50 mg/L of Cd and/or 300 mg/L of Pb for 12 weeks, in the presence or absence of α-LA co-treatment (50 mg/kg) via gavage. We observed that exposure to Cd and/or Pb decreased the brain weight/body weight ratio and increased Cd and/or Pb contents as well as ultrastructural damage to the cerebral cortex. Cd and/or Pb also induced endoplasmic-reticulum (ER) stress and activated Fas (CD95/APO-1)/Fas ligand (FasL) and mitochondrial apoptotic pathways. Furthermore, co-treatment of Cd and Pb further exacerbated part of these phenotypes than treatment of Cd or Pb alone. However, simultaneous supplementation with α-LA attenuated Cd and/or Pb-induced neurotoxicity by increasing the brain weight/body weight ratio, reducing Cd and/or Pb contents, ameliorating both nuclear/mitochondrial damage and ER stress, and attenuating activation of Fas/FasL and mitochondrial apoptotic pathways. Collectively, our results indicate that the accumulation of Cd and/or Pb causes cortical damage and that α-LA exerts protection against Cd- and/or Pb-induced neurotoxicity. These findings highlight that α-LA may be exploited for the treatment and prevention of Cd- and/or Pb-induced neurotoxicity.