Effect of methacrylonitrile on membrane bound enzymes of rat brain

Methacrylonitrile (MeAN) is a plastic monomer. Its effect on membrane bound enzymes like Na+K+ -ATPase, Ca2+ -ATPase, Mg2+ -ATPase, NADH dehydrogenase, alkaline phosphatase (ALP) and various elements like sodium (Na+), potassium (K+), and calcium (Ca2+) in rat brain were studied. Administration of 50 mg/kg body weight/day (0.25 LD50) and 100 mg/kg body weight/day (0.5 LD50) by gavage to rats for 7 days resulted in a significant decrease in activities of Na+K+ -ATPase, Ca2+ -ATPase, Mg2+ -ATPase, and NADH dehydrogenase. A significant reduction in calcium content, potassium content and a significant increase in sodium content and alkaline phosphatase activity in MeAN treated animals were observed. Inhibition of membrane bound enzymes occurred due to either direct effect of MeAN or indirect effect of changes in ionic homeostasis in MeAN treated animals.     

Direct demonstration of an antiinflammatory effect of simvastatin in subjects with the metabolic syndrome

CONTEXT: Metabolic syndrome (MS) is characterized by low-grade inflammation and confers an increased risk for cardiovascular disease. Hydroxymethylglutaryl coenzyme A reductase inhibitors (statins) reduce cardiovascular events in MS patients. There is a paucity of data examining the effect of statins on inflammation in MS. OBJECTIVE: We aimed to test the effect of simvastatin (40 mg/d) compared with placebo on biomarkers of inflammation [high-sensitivity C-reactive protein (hsCRP) and monocytic cytokines TNF, IL-6, and IL-1] in MS subjects. DESIGN AND PATIENTS: We conducted a randomized, double-blind, placebo-controlled study at the University of California, Davis, Medical Center. PARTICIPANTS: Participants were subjects with MS. INTERVENTION: Simvastatin (40 mg/d) or placebo was administered for 8 wk. METHODS AND RESULTS: The hsCRP levels were assayed using a high-sensitivity immunoassay. Monocyte cytokines were assayed by ELISA after activation with lipopolysaccharide. Simvastatin therapy significantly decreased hsCRP levels in MS subjects compared with placebo (P < 0.0005) and resulted in a significant reduction in plasma and lipopolysaccharide-activated monocytic release of IL-6 and TNF (P < 0.025). Simvastatin therapy significantly decreased nuclear factor-kappaB and increased Akt activity in MS subjects compared with placebo. To gain mechanistic insights, human monocytes were pretreated with lovastatin with and without mevalonate or a phosphatidyl-3-kinase inhibitor or Rho kinase inhibitor. Lovastatin significantly decreased Rho kinase and nuclear factor-kappaB activity, significantly increased Akt activity, and resulted in decreased monocyte IL-6 levels; these effects were reversed with mevalonate and geranylgeranyl pyrophosphate, indicating direct effects of statins on protein prenylation. CONCLUSIONS: Thus, we show a direct antiinflammatory effect of simvastatin therapy in MS. These findings could partly explain the benefit of statin therapy in these patients.     

Native pentameric C-reactive protein displays more potent pro-atherogenic activities in human aortic endothelial cells than modified C-reactive protein

Inflammation is pivotal in atherogenesis. Numerous prospective studies have shown that levels of high sensitive C-reactive protein (CRP) predict cardiovascular events. Recently, data suggests that CRP could be a mediator in atherothrombosis. Loss of pentameric symmetry in CRP has been shown to result in the formation of modified CRP (mCRP). The main aim of this study was to examine the biological effects of the native, pentameric form of CRP compared to a modified form in human aortic endothelial cells. Human pentameric native CRP (n-CRP) from two different sources (recombinant and serum) was purified and used. It was then subjected to EDTA chelation and urea treatment to prepare modified CRP (m-CRP). Purity of both n-CRP and m-CRP preparations was checked by gel electrophoresis. Both n-CRP and m-CRP were incubated with human aortic endothelial cells (HAEC) and biological activities was tested by assaying for interleukin-8 (IL-8), plasminogen activator inhibitor-1(PAI-1), cyclic GMP and prostaglandin F1-alpha. n-CRP significantly upregulated IL-8 at concentrations > or = 10 microg/mL while m-CRP upregulated IL-8 only at concentrations of 50 microg/mL (p < 0.05). PAI-1 levels were significantly increased to a greater extent with native compared to m-CRP (p < 0.05). While both decreased PGF1-alpha at concentrations of 50 microg/mL, the effect of native CRP was more pronounced and was evident at 10 microg/mL (p < 0.05). The most pronounced difference was observed with regard to inhibition of eNOS activity as assessed by cGMP which was observed at 10 microg/ml of native CRP but only at 50 microg/mL for m-CRP (native CRP versus mCRP: p < 0.001). Thus, native pentameric CRP compared to modified CRP exerts more potent atherogenic effects in human aortic endothelial cells.     

Relationship of EMAST and Microsatellite Instability Among Patients with Rectal Cancer

Background  

Elevated microsatellite instability at selected tetranucleotide repeats (EMAST) is a genetic signature identified in 60% of sporadic colon cancers and may be linked with heterogeneous expression of the DNA mismatch repair (MMR) protein hMSH3. Unlike microsatellite instability-high (MSI-H) in which hypermethylation of hMLH1 occurs followed by multiple susceptible gene mutations, EMAST may be associated with inflammation and subsequent relaxation of MMR function with the biological consequences not known. We evaluated the prevalence of EMAST and MSI in a population-based cohort of rectal cancers, as EMAST has not been previously determined in rectal cancers.     

Differential expression and activities of cytochrome P450 3A in the rat brain microsomes and mitochondria

Midazolam (MDZ), a benzodiazepine derivative, is metabolized to 1′- and 4-hydroxylated metabolites (1′-OH-MDZ and 4-OH-MDZ, respectively) by cytochrome P450 3A (CYP3A). The purpose of this study was to investigate the CYP3A-mediated hydroxylation of MDZ in the rat brain mitochondria (MT). Brain microsomes (MC) and MT fractions were prepared from rats (n = 8) using differential and density gradient centrifugations, and the purity of the fractions was evaluated using VDAC1 and calreticulin as markers of MT and MC, respectively. The formation rates of 1′-OH-MDZ and 4-OH-MDZ in the rat brain MC and MT samples were determined using an LC–MS/MS method after validation. Subsequently, Michaelis–Menten kinetics of 1′- and 4-hydroxylation of MDZ were estimated. Western blot (WB) analysis was used to determine the protein expression of CYP3A in the rat brain MC and MT. The MC fractions had 5.93% ± 3.01% mitochondrial impurity, and the MT fractions had 19.3% ± 7.8% microsomal impurity (mean ± SD). The maximum velocity (V_max) values of the formation of the hydroxylated metabolites in the brain MT were 2.4–9-fold higher than those in MC. Further, the V_max values of 4-OH-MDZ in both MC and MT fractions were substantially higher than those of 1′-OH-MDZ. The WB analysis showed that the intensity of the CYP3A immunoreactive band in MT was more than twofold higher than that in MC. It is concluded that compared with MC, rat brain MT contains substantial CYP3A, which may affect the pharmacology or toxicology of centrally acting xenobiotic and endogenous substrates of this enzyme.     

Effects of chronic cirrhosis induced by intraperitoneal thioacetamide injection on the protein content and Michaelis–Menten kinetics of cytochrome P450 enzymes in the rat liver microsomes

Chronic intraperitoneal injection of thioacetamide (TAA) in rats has been used as an animal model of human cirrhosis to study the effects of the disease on drug metabolism. However, TAA inhibits P450 enzymes directly and independently of cirrhosis. We investigated the effects of chronic cirrhosis in rats, induced by 10 weeks of intraperitoneal TAA, on the P450 enzymes after a 10-day washout period to eliminate TAA. Liver histology and serum biomarkers of hepatic function confirmed cirrhosis in all animals. Microsomal total P450 content, P450 reductase activity and ethoxycoumarin O-deethylase activity, a general marker of P450 activity, were significantly reduced by 30%–50% in cirrhotic animals. Additionally, the protein content and Michaelis–Menten kinetics of the activities of CYP2D, CYP2E1 and CYP3A were investigated. Whereas cirrhosis reduced the microsomal protein contents of CYP2D and CYP3A by 70% and 30%, respectively, the protein contents of CYP2E1 were not affected. However, the activities of all the tested isoenzymes were substantially lower in the cirrhotic livers. It is concluded that the TAA model of cirrhosis that incorporates a 10-day washout period after intraperitoneal injection of the chemical to rats produces isoenzyme-selective reductions in the P450 proteins or activities, which are independent of the direct inhibitory effects of TAA.