Cytochrome P450 1B1 expression in rat esophageal tumorigenesis promoted by gastric and duodenal reflux.

Cytochrome P450 1B1 (CYP1B1) mRNA is constitutively expressed in most normal extra-hepatic tissues; however the protein is not detectable in these tissues but is expressed in a wide variety of tumors. CYP1B1 is responsible for the activation of a number of carcinogens present in tobacco smoke and food. A surgical model of rat esophageal tumorigenesis, promoted by gastric or duodenal reflux was used to determine CYP1B1 expression in premalignant esophageal tissue. Immunohistochemistry was performed using a modified amplified fluorescein tyramide protocol. CYP1B1 was not observed in normal esophageal mucosa, submucosa, or muscularis mucosa. Animals exposed to gastric reflux developed mild hyperplasia. Varying degrees of hyperplasia were observed in the duodenal reflux group. All regions of hyperplasia showed moderate or strong CYP1B1 immunoreactivity. Duodenal reflux induced a small number of premalignant changes: immunoreactivity was absent from the epithelium of squamous dysplasia (0/10), Barrett's esophagus (0/7), and majority of dysplastic Barrett's esophagus (1/4). Moderate or strong immunoreactivity was observed in the majority (7/8) of squamous cell carcinomas (SCCs) in situ. Immunoreactivity was also observed in the lamina propria and submucosa in association with inflammation, regardless of the severity of inflammation. The expression of CYP1B1 in hyperplasia, SCCs in situ, or in association with inflammation may increase the production of carcinogenic metabolites, which may promote esophageal tumorigenesis.

Inhibition of cytochrome P450-mediated metabolism enhances ex vivo susceptibility of Fasciola hepatica to triclabendazole

A study has been carried out to investigate whether the action of triclabendazole (TCBZ) against Fasciola hepatica is altered by inhibition of drug metabolism. The cytochrome P450 (CYP P450) system was inhibited using piperonyl butoxide (PB). The Oberon TCBZ-resistant and Cullompton TCBZ-susceptible isolates were used for these experiments. The CYP P450 system was inhibited by a 2 h pre-incubation in PB (100 mu M). Flukes were then incubated for a further 22 h in NCTC medium containing either PB; PB + nicotinamide adenine dinucleotide phosphate (NADPH) (1 nM); PB + NADPH + TCBZ (15 mu g/ml); or PB + NADPH + TCBZ.SO (15 mu g/ml). Morphological changes resulting from drug treatment and following metabolic inhibition were assessed using scanning electron microscopy. After treatment with either TCBZ or TCBZ.SO alone, there was greater disruption to the TCBZ-susceptible than the resistant isolate. However, co-incubation with PB and TCBZ/TCBZ.SO lead to more severe surface changes to the TCBZ-resistant Oberon isolate than with each drug on its own. With the TCBZ-susceptible Cullompton isolate, there was limited potentiation of drug action, and only with TCBZ.SO. The results support the concept of altered drug metabolism in TCBZ-resistant flukes and this process may play a role in the development of drug resistance.

Molecular Modeling on Inhibitor Complexes and Active-Site Dynamics of Cytochrome P450 C17, a Target for Prostate Cancer Therapy

A molecular model for the P450 enzyme cytochrome P450 C17 (CYP17) is presented based on sequence alignments of multiple template structures and homology modeling. This enzyme plays a central role in the biosynthesis of testosterone and is emerging as a major target in prostate cancer, with the recently developed inhibitor abiraterone currently in advanced clinical trials. The model is described in detail, together with its validation, by providing structural explanations to available site-directed mutagenesis data. The CYP17 molecule in this model is in the form of a triangular prism, with an edge of similar to 55 angstrom and a thickness of similar to 37 angstrom. It is predominantly helical, comprising 13 alpha helices interspersed by six 3(10) helices and 11 beta-sheets. Multinanosecond molecular dynamics simulations in explicit solvent have been carried out, and principal components analysis has been used to reveal the details of dynamics around the active site. Coarse-grained methods have also been used to verify low-frequency motions, which have been correlated with active-site gating. The work also describes the results of docking synthetic inhibitors, including the drug abiraterone and the natural substrate pregnenolone, in the CYP17 active site together with molecular dynamics simulations on the complexes. (C) 2010 Elsevier Ltd. All rights reserved.

Plasma glutathione peroxidase activity is reduced in haemodialysis patients

Cardiovascular disease is the major cause of morbidity and mortality in patients with end-stage renal failure. Increased free radical production and antioxidant depletion may contribute to the greatly increased risk of atherosclerosis in these patients. Glutathione peroxidase (GPX) is an important antioxidant, the plasma form of which is synthesized mainly in the kidney (eGPX). The aim of this study was to assess the activity of eGPX in patients with end-stage renal failure on haemodialysis. Venous blood was collected from 87 haemodialysis patients immediately prior to and after dialysis and from 70 healthy controls. Serum eGPX activity was measured using hydrogen peroxide as substrate and immunoreactivity determined by ELISA. eGPX activity was significantly reduced in dialysis patients when compared to controls (106 +/- 2.7 and 281 +/- 3.6 U/l respectively, p <0.001). Following haemodialysis, eGPX activity rose significantly to 146 +/- 3.8 U/l, p <0.001, although remaining below control values (p <0.005). Immunoreactive eGPX, however, was similar in all groups (pre-dialysis 14.10 +/- 1.26 microg/ml, post-dialysis 14.58 +/- 1.35 microg/ml, controls 15.20 +/- 1.62 microg/ml, p = NS). A decrease was observed in the specific activity of eGPX in patients when compared to controls (8.81 +/- 1.14, 10.71 +/- 1.54 and 21.97 +/- 1.68 U/mg respectively, p <0.0001). eGPX activity is impaired in patients undergoing haemodialysis and so may contribute to atherogenesis in renal failure.

Glutathione peroxidase 1 genotype is associated with an increased risk of coronary artery disease

Objective Oxidative stress is implicated in the pathogenesis of many human diseases including atherosclerosis. Human glutathione peroxidase 1 (hgpx1) participates in limiting cellular damage caused by oxidation. A characteristic polyalanine sequence polymorphism in exon 1 of hgpx1 produces three alleles with five, six or seven alanine (ALA) repeats in this sequence. The objective of this study was to determine whether hgpx1 genotype is associated with an altered risk of coronary artery disease (CAD).

Methods The frequency of the ALA6 allele was determined in 207 men with angiographic evidence of significant CAD compared to a control group (n = 146), by analysing the lengths of polymerase chain reaction fragments containing the ALA repeat polymorphism. Additional information was collected on severity of CAD, presence or absence of a prior acute myocardial infarction (AMI), smoking status, body mass index (BMI) and other clinical data.

Results There was a significant association between individuals with at least one ALA6 allele and an increased risk of CAD after adjustment for age, BMI and smoking status (odds ratio, 2.07, 95% confidence interval, 1.08-3.99, P = 0.029). However, there was no association between hgpx1 genotype and a previous history of AMI or hgpx1 genotype and severity of CAD.

Conclusion We conclude that individuals possessing one or two ALA6 alleles appear to be at a modest increased risk of CAD. This observation merits further investigation in other patient populations.

Active site labeling of cysteine cathepsins by a straightforward diazomethylketone probe derived from the N-terminus of human cystatin C

We designed a straightforward biotinylated probe using the N-terminal substrate-like region of the inhibitory site of human cystatin C as a scaffold, linked to the thiol-specific reagent diazomethylketone group as a covalent warhead (i.e. Biot-(PEG)2-Ahx-LeuValGly-DMK). The irreversible activity-based probe bound readily to cysteine cathepsins B, L, S and K. Moreover affinity labeling is sensitive since active cathepsins were detected in the nM range using an ExtrAvidin®-peroxidase conjugate for disclosure. Biot-(PEG)2-Ahx-LeuValGly-DMK allowed a slightly more pronounced labeling for cathepsin S with a compelling second-order rate constant for association (kass = 2,320,000 M−1 s−1). Labeling of the active site is dose-dependent as observed using 6-cyclohexylamine-4-piperazinyl-1,3,5-triazine-2-carbonitrile, as competitive inhibitor of cathepsins. Finally we showed that Biot-(PEG)2-Ahx-LeuValGly-DMK may be a simple and convenient tool to label secreted and intracellular active cathepsins using a myelomonocytic cell line (THP-1 cells) as model.

Placental antioxidant enzyme status and lipid peroxidation in pregnant women with type 1 diabetes: The effect of vitamin C and E supplementation

AIM: In view of the increased rates of pre-eclampsia observed in diabetic pregnancy and the lack of ex vivo data on placental biomarkers of oxidative stress in T1 diabetic pregnancy, the aim of the current investigation was to examine placental antioxidant enzyme status and lipid peroxidation in pregnant women with type 1 diabetes. A further objective of the study was to investigate the putative impact of vitamin C and E supplementation on antioxidant enzyme activity and lipid peroxidation in type 1 diabetic placentae.

METHODS: The current study measured levels of antioxidant enzyme [glutathione peroxidase (Gpx), glutathione reductase (Gred), superoxide dismutase (SOD) and catalase] activity and degree of lipid peroxidation (aqueous phase hydroperoxides and 8-iso-prostaglandin F2α) in matched central and peripheral samples from placentae of DAPIT (n=57) participants. Levels of vitamin C and E were assessed in placentae and cord blood.

RESULTS: Peripheral placentae demonstrated significant increases in Gpx and Gred activities in pre-eclamptic in comparison to non-pre-eclamptic women. Vitamin C and E supplementation had no significant effect on cord blood or placental levels of these vitamins, nor on placental antioxidant enzyme activity or degree of lipid peroxidation in comparison to placebo-supplementation.

CONCLUSION: The finding that maternal supplementation with vitamin C/E does not augment cord or placental levels of these vitamins is likely to explain the lack of effect of such supplementation on placental indices including antioxidant enzymes or markers of lipid peroxidation.