Coenzyme Q10 ameliorates the reduction in GLUT4 transporter expression induced by simvastatin in 3T3-L1 adipocytes.

BACKGROUND: Statins significantly reduce cardiovascular events in a broad population of patients with hyperlipidemia. However, a small, but significant risk of new-onset diabetes has been reported in patients treated with statins. The mechanism by which statins cause diabetes has not been elucidated and therefore preventive strategies have yet to be defined. METHOD: Our goal was to study the differing effects of a lipophilic (simvastatin) statin, hydrophilic (pravastatin) statin, and ezetimibe on glucose transporter-4 (GLUT4) protein expression in 3T3-L1 adipocytes. We hypothesized that the reductions in GLUT4 protein secondary to statin treatment would be prevented when cells were co-incubated with coenzyme Q10 (CoQ10). GLUT4 protein expression was determined using the In-Cell Western technique. Confluent adipocytes were differentiated using a hormonal cocktail for 3 days; followed by treatment with simvastatin, pravastatin, ezetimibe and CoQ10. Cell morphology was observed after treatment using phase-contrast microscopy. RESULTS: Treatment with simvastatin (P<0.001) and simvastatin plus ezetimibe (P<0.001) significantly decreased GLUT4 protein expression in the adipocytes compared to control conditions. GLUT4 protein levels were similar to control after treatment with ezetimibe alone (P=0.52) or pravastatin (P=0.32). There was no significant difference (P=0.098) in GLUT4 protein levels after co-treatment with CoQ10 between any of the treatments and control conditions. CONCLUSION: Our studies have shown that lipophilic statins (simvastatin) reduce the GLUT4 protein levels in adipocytes, whereas hydrophilic statins (pravastatin) or ezetimibe do not. Co-treatment with CoQ10 appears to prevent the reduction in GLUT4 protein levels caused by simvastatin.

Clopidogrel variability: role of plasma protein binding alterations.

AIM: The large inter-individual variability in clopidogrel response is attributed to pharmacokinetics. Although, it has been used since the late 1990s the pharmacokinetic fate of clopidogrel and its metabolites are poorly explained. The variable response to clopidogrel is believed to be multi-factorial, caused both by genetic and non-genetic factors. In this study, we examined whether the inactive metabolite can alter the plasma protein binding of the active metabolite, thus explaining the large inter-individual variability associated with clopidogrel response. METHODS: Female subjects (n = 28) with stable coronary disease who were not taking clopidogrel were recruited. Serial blood samples were collected following 300 mg oral dose of clopidogrel, plasma was isolated and quantified for total and free concentrations of active and inactive metabolites. Inhibition of platelet aggregation was measured using the phosphorylated vasodilator stimulated phosphoprotein (VASP) assay. RESULTS: A significant correlation was observed between VASP and both free (r = 0.49, P < 0.05) and total (r = 0.49, P < 0.05) concentrations of the active metabolite. Surprisingly, we observed a significant correlation with both free (r = 0.42, P < 0.05) and total (r = 0.67, P < 0.001) concentrations of the inactive metabolite as well. Free fractions of the active metabolite rose with increasing protein binding of the inactive metabolite (P < 0.05). CONCLUSIONS: The above in vivo data suggest that the inactive metabolite displaces the active metabolite from binding sites. Thus, the inactive metabolite might increase the free concentration of the active metabolite leading to enhanced inhibition of platelet aggregation. The plasma protein binding mechanism would offer an additional therapeutic strategy to optimize clopidogrel pharmacotherapy.

Understanding the mechanisms for metabolism-linked hemolytic toxicity of primaquine against glucose 6-phosphate dehydrogenase deficient human erythrocytes: evaluation of eryptotic pathway.

Therapeutic utility of primaquine, an 8-aminoquinoline antimalarial drug, has been limited due to its hemolytic toxicity in population with glucose 6-phosphate dehydrogenase deficiency. Recent investigations at our lab have shown that the metabolites generated through cytochrome P(450)-dependent metabolic reactions are responsible for hemotoxic effects of primaquine, which could be monitored with accumulation of methemoglobin and increased oxidative stress. The molecular markers for succeeding cascade of events associated with early clearance of the erythrocytes from the circulation were evaluated for understanding the mechanism for hemolytic toxicity of primaquine. Primaquine alone though did not induce noticeable methemoglobin accumulation, but produced significant oxidative stress, which was higher in G6PD-deficient than in normal erythrocytes. Primaquine, presumably through redox active hemotoxic metabolites generated in situ in human liver microsomal metabolism-linked assay, induced a dose-dependent methemoglobin accumulation and oxidative stress, which were almost similar in normal and G6PD-deficient erythrocytes. Primaquine alone or in presence of pooled human liver microsomes neither produced significant effect on intraerythrocytic calcium levels nor affected the phosphatidyl serine asymmetry of the normal and G6PD-deficient human erythrocytes as monitored flowcytometrically with Annexin V binding assay. The studies suggest that eryptosis mechanisms are not involved in accelerated removal of erythrocytes due to hemolytic toxicity of primaquine.

Inhibition of human monoamine oxidase A and B by 5-phenoxy 8-aminoquinoline analogs.

8-Aminoquinolines (8-AQs) are important class of anti-infective therapeutics. 5-Phenoxy 8-aminoquinoline analogs have shown improved metabolic stability compared to primaquine. In view or predictive role of monoamine oxidases (MAO) in metabolism of 8-aminoquinolines the 5-phenoxy analogs were evaluated in vitro for the inhibition of recombinant human MAO-A and MAO-B. The analogs were several folds more potent inhibitors of MAO-A and MAO-B compared to primaquine, the parent drug, with selectivity for MAO-B. 5-(4-Trifluoromethylphenoxy)-4-methylprimaquine (6) Inhibited MAO-B with IC(50) value of 150 nM (626-fold more potent than primaquine). These results will have important implications in optimizing metabolic stability of 8-AQs to improve therapeutic value and also indicate scope for development of 8-AQs as selective MAO inhibitors.

Cytochrome P450-dependent toxicity of dapsone in human erythrocytes.

The most prominent adverse effects seen during treatment with dapsone, an antibacterial and antiprotozoal agent, are hemolysis and methemoglobinemia. An in vitro microsomal/cytochrome P(450) (CYP)-linked assay, which allows reactive metabolites generated in situ to react with the co-incubated human erythrocytes, was employed to profile CYP isoforms responsible for hemotoxicity of dapsone. Dapsone caused a robust generation of methemoglobin in human erythrocytes in the presence of human/mouse liver microsomes, which indicates contribution of CYP-mediated metabolism for hemotoxicity. The highest methemoglobin formation with dapsone was observed with CYP2C19, with minor contributions from CYP2B6, CYP2D6 and CYP3A4. Cimetidine and chloramphenicol completely abrogated methemoglobin generation by dapsone, thus confirming a predominant contribution of CYP2C19. The results provide useful insights into CYP-dependent hemotoxicity of dapsone in human erythrocytes.

Cytochrome P(450)-dependent toxic effects of primaquine on human erythrocytes.

Primaquine, an 8-aminoquinoline, is the drug of choice for radical cure of relapsing malaria. Use of primaquine is limited due to its hemotoxicity, particularly in populations with glucose-6-phosphate dehydrogenase deficiency [G6PD(-)]. Biotransformation appears to be central to the anti-infective and hematological toxicities of primaquine, but the mechanisms are still not well understood. Metabolic studies with primaquine have been hampered due to the reactive nature of potential hemotoxic metabolites. An in vitro metabolism-linked hemotoxicity assay has been developed. Co-incubation of the drug with normal or G6PD(-) erythrocytes, microsomes or recombinant cytochrome P(450) (CYP) isoforms has allowed in situ generation of potential hemotoxic metabolite(s), which interact with the erythrocytes to generate hemotoxicity. Methemoglobin formation, real-time generation of reactive oxygen intermediates (ROIs) and depletion of reactive thiols were monitored as multiple biochemical end points for hemotoxicity. Primaquine alone did not produce any hemotoxicity, while a robust increase was observed in methemoglobin formation and generation of ROIs by primaquine in the presence of human or mouse liver microsomes. Multiple CYP isoforms (CYP2E1, CYP2B6, CYP1A2, CYP2D6 and CYP3A4) variably contributed to the hemotoxicity of primaquine. This was further confirmed by significant inhibition of primaquine hemotoxicity by the selective CYP inhibitors, namely thiotepa (CYP2B6), fluoxetine (CYP2D6) and troleandomycin (CYP3A4). Primaquine caused similar methemoglobin formation in G6PD(-) and normal human erythrocytes. However, G6PD(-) erythrocytes suffered higher oxidative stress and depletion of thiols than normal erythrocytes due to primaquine toxicity. The results provide significant insights regarding CYP isoforms contributing to hemotoxicity and may be useful in controlling toxicity of primaquine to increase its therapeutic utility.

In vivo and in vitro CYP1B mRNA expression in channel catfish.

Our goal was to study the induction of CYP1B mRNA expression in channel catfish (Ictalurus punctatus). CYP1B belongs to the cytochrome P450 superfamily of genes, is involved in the oxidation of endogenous and exogenous compounds, and could potentially be a useful biomarker in fish for exposure to AhR ligands. The full-length catfish CYP1B cDNA is 2417 nt to the polyA tail and encodes a putative protein of 536 amino acids. It has 67% amino acid similarity to carp and zebrafish CYP1B and 68% similarity to carp CYP1B2. Male channel catfish were collected from three Mississippi Delta sites: Lake Roebuck, Itta Bena; Bee Lake, Thornton; and Sunflower River, Indianola. Total RNA was isolated from wild-caught catfish gill, blood, gonad and liver tissues. Quantitative real-time reverse transcriptase PCR was used to determine relative induction of CYP1B in wild catfish compared to laboratory control and BaP-exposed catfish (20mg/kg i.p. after 4 days). BaP exposure significantly induced CYP1B message in blood, gonad, and liver of laboratory catfish. In these same tissues of wild catfish from sites with relatively low sediment contaminants, CYP1B message was not statistically increased relative to laboratory control catfish. CYP1B transcript abundance was higher in gills compared to other tissues in both laboratory and wild catfish. When primary cultured gill cells were treated with increasing concentrations of BaP, TCDD, and PCBs 77, 126 and 169, CYP1B mRNA was induced more than 10-fold while PCB153 and 4,4'DDT did not cause significant CYP1B induction. Our results suggest that catfish CYP1B is induced by the classic AhR ligands.