In vitro effects of imatinib on glucose-6-phosphate dehydrogenase and glutathione reductase.

Imatinib (Gleevec, STI571) is a drug used to treat certain types of cancer. Glucose-6-phosphate dehydrogenase and glutathione reductase are enzymes important for redox homeostasis and play key roles in many cellular processes. The purpose of the present work is to evaluate the in vitro effects of imatinib on sheep brain cortex glucose-6-phosphate dehydrogenase, and on bovine kidney cortex, bovine liver and yeast glutathione reductase. Kinetic studies on the inhibition of enzymes by imatinib have been investigated by using Lineweaver-Burk double reciprocal plot and values summarized with graphs by plotting the data using Linewear-Burk diagrams of 1/v against 1/[S] at each [I]. Imatinib inhibits glucose- 6-phosphate dehydrogenase with an IC50 value of 0.7 mM. It inhibits bovine kidney cortex, liver and yeast glutathione reductase in a concentration-dependent manner with IC50 values of 0.8, 0.92, 1 mM, respectively. We have investigated the kinetic characteristics, inhibition types and constants (Ki). Inhibition of the glucose-6-phosphate dehydrogenase and glutathione reductase represents an attractive approach to the development of anticancer agents. This study shows the molecular effectiveness of the drug on purified enzymes of various sources. Understanding the kinetic mechanism of the drug and enzyme relationship may be a powerful approach to the future drug studies concerning new cancer drugs, drug resistance and new aspects in cancer therapy.

Purification and kinetics of bovine kidney cortex glutathione reductase.

Glutathione reductase was purified 34806-fold with a final yield of 85 % from the bovine kidney cortex. Some molecular and kinetic properties of purified enzyme are investigated. Product inhibition studies showed that the enzyme obeys 'branched" mechanism: Km(NADPH) 18 +/- 3 microM and Km(GSSG) 65 +/- 5 microM were determined.

Angiotensin II receptor blockage prevents diabetes-induced oxidative damage in rat heart.

Current findings suggest a role for the angiotensin II (Ang II) signalling pathway in generation of reactive oxygen species and diabetes-induced cardiac complications. In this study we aimed to investigate the effect of angiotensin II type 1 (AT1) receptor blockage on some antioxidant enzymes such as glucose- 6-phosphate dehydrogenase (G6PD), 6-phoshogluconate dehydrogenase (6PGD), glutathione reductase (GR), glutathione-S-transferase (GST), glutathione peroxidase (GSH-Px), and catalase (CAT) in the heart of streptozotocin (STZ)-induced diabetic rats. The effect of AT1 receptor blocker, candesartan-cilexetil (5 mg/kg/day for 4 weeks) was studied. Diabetes caused hyperglycaemia (4-fold of control) with significant increases in G6PD, 6PGD, GR, GSH-PX, CAT and no effect on GST in heart tissues as compared to normal control rats. Treatment of STZ-induced diabetic rats with candesartan-cilexetil had significant beneficial effects on these parameters without any side effect on control rats. These results suggest that Ang II can take part in induction of oxidative stress in diabetic rat heart and that blockage of its activity by AT1 receptor blocker is potentially protective against diabetes-induced cellular damage.

Sarco(endo)plasmic reticulum and plasmalemmal Ca(2+)-ATPase activities in cremaster muscles and sacs differ according to the associated inguinal pathology.

Sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) and plasmalemmal Ca(2+)-ATPase (PMCA) activities in cremaster muscles and sacs, which have been subjected to different autonomic tonuses, were determined and compared. Samples of cremaster muscles and sacs associated with male or female inguinal hernia, hydrocele or undescended testis were obtained from children during operations and activities of SERCA and PMCA were determined. While highest SERCA and PMCA activities were encountered among cremaster muscles and sacs associated with undescended testis, least activities were encountered among structures associated with hydrocele. The alterations in SERCA and PMCA activities in cremaster muscles associated with undescended testis appear to reflect the attempts at maintaining the levels of cytosolic calcium. Despite similar total calcium contents, lower SERCA and PMCA activities were found in sacs associated with hydrocele compared to those associated with undescended testis suggest a difference among the levels of cytosolic calcium.

The effect of taurine on renal ischemia/reperfusion injury.

Ischemia-reperfusion (I/R) injury is one of the most common causes of renal dysfunction. Taurine is an endogenous antioxidant and a membrane-stabilizing, intracellular, free beta-amino acid. It has been demonstrated to have protective effects against I/R injuries to tissues other than kidney. The aim of this study was to determine whether taurine has a beneficial role in renal I/R injury. Forty Wistar-Albino rats were allocated into four groups as follows: sham, taurine, I/R, and I/R+taurine. Taurine 7.5 mg/kg was given intra-peritoneally to rats in the groups taurine and I/R+taurine. Renal I/R was achieved by occluding the renal arteries bilaterally for 40 min, followed by 6 h of reperfusion. Immediately thereafter, blood was drawn and tissue samples were harvested to measure 1) serum levels of BUN and creatinine; 2) serum and/or tissue levels of malondialdehyde (MDA), glutathione (GSH), glucose 6-phosphate dehydrogenase (G-6PD), 6-phosphogluconate dehydrogenase (6-PGD) and glutathione reductase (GSH-red); 3) renal morphology; and 4) immunohistochemical staining for P-selectin. Taurine administration reduced I/R-induced increases in serum BUN and creatinine, and serum and tissue MDA levels (p<0.05). Additionally, taurine lessened the reductions in serum and tissue glutathione levels secondary to I/R (p<0.05). Taurine also attenuated histopathologic evidence of renal injury, and reduced I/R-induced P-selectin immunoreactivity (p<0.05). Overall, then, taurine administration appears to reduce the injurious effects of I/R on kidney.

Circadian variations in the activities of 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase in the liver of control and streptozotocin-induced diabetic rats.

The aim of this study was to examine: the 24 h variation of 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase activities, key enzymes for the maintenance of intracellular NADPH concentration, in rat liver in control and streptozotocin-induced diabetic animals. Adult male rats were fed ad libitum and synchronized on a 12:12 h light-dark cycle (lights on 08:00 h). One group of animals was treated with streptozotocin (STZ, 55 mg/kg, intraperitoneal) to induce experimental diabetes. Eight weeks after STZ injection, the animals were sacrificed at six different times of day--1, 5, 9, 13, 17 and 21 Hours After Lights On (HALO)--and livers were obtained. Enzyme activities were determined spectrophotometrically in triplicate in liver homogenates and expressed as units per mg protein. 6-phosphogluconate dehydrogenase activity was measured by substituting 6-phosphogluconate as substrate. Glucose-6-phosphate dehydrogenase activity was determined by monitoring NADPH production. Treatment, circadian time, and interaction between treatment and circadian time factors were tested by either one or two way analysis of variance (ANOVA). Two-way ANOVA revealed that 6-phosphogluconate dehydrogenase activity significantly depended on both the treatment and time of sacrifice. 6-phosphogluconate dehydrogenase activity was higher in control than diabetic animals; whereas, glucose-6-phosphate dehydrogenase activity did not vary over the 24 h in animals made diabetic by STZ treatment. Circadian variation in the activity of 6-phosphogluconate dehydrogenase was also detected in both the control and STZ treatment groups (one-way ANOVA). Time-dependent variation in glucose-6-phosphate dehydrogenase activity during the 24 h was detected in control but not in diabetic rats. No significant interaction was detected between STZ-treatment and time of sacrifice for both hepatic enzyme activities. These results suggest that the activities of NADPH-generating enzymes exhibit 24 h variation, which is not influenced by diabetes.

Adenosine triphosphatase activity of streptozotocin-induced diabetic rat brain microsomes. Effect of vitamin E.

Hyperglycemia causes protein glycosylation, oxidation and alterations in enzyme activities, which are the underlying causes of diabetic complications. This study was undertaken to test the role of vitamin E treatment on Ca2+-ATPase activity, protein glycosylation and lipid peroxidation in the brain of streptozotocin (STZ)-induced diabetic rats. Male rats weighing about 250-300 g were rendered diabetic by a single STZ injection of 50 mg/kg via the tail vein. Both the diabetic and non-diabetic rats were fed a vitamin E supplemented diet (500 IU/kg/day). Ca2+-ATPase activity was significantly reduced at week 10 of diabetes compared to the control group (p < 0.05), with 0.225+/-0.021 U/I (mean +/- S.E.M.) in the control group and 0.072 +/- 0.008 U/l (mean +/- S.E.M.) in the diabetic group. Vitamin E treatment prevented the enzyme activity from decreasing. The activities observed were 0.226 +/- 0.020 U/l and 0.172 +/- 0.011 U/I (mean +/- S.E.M.) in the vitamin E-treated control and diabetic group, respectively. STZ-induced diabetes resulted in an increased protein glycosylation and lipid peroxidation. Vitamin E treatment led to a significant inhibition in blood glucose, protein glycosylation and lipid peroxidation, which in turn prevented abnormal activity of the enzyme in the brain. This study indicates that vitamin E supplementation may reduce complications of diabetes in the brain.

A comparative study on effect of dietary selenium and vitamin E on some antioxidant enzyme activities of liver and brain tissues.

Since selenium and vitamin E have been increasingly recognized as an essential element in biology and medicine, current research activities in the field of human medicine and nutrition are devoted to the possibilities of using these antioxidants for the prevention or treatment of many diseases. The present study was aimed at investigating and comparing the effects of dietary antioxidants on glutathione reductase and glutathione peroxidase activities as well as free and protein-bound sulfhydryl contents of rat liver and brain tissues. For 12-14 wk, both sex of weanling rats were fed a standardized selenium-deficient and vitamin E-deficient diet, a selenium-excess diet, or a control diet. It is observed that glutathione reductase and glutathione peroxidase activities of both tissues of the rats fed with a selenium-deficient or excess diet were significantly lower than the values of the control group. It is also shown that free and bound sulfhydryl concentrations of these tissues of both experimental groups were significantly lower than the control group. The percentage of glutathione reductase and glutathione peroxidase activities of the deficient group with respect to the control were 50% and 47% in liver and 66% and 61% in the brain, respectively; while these values in excess group were 51% and 69% in liver and 55% and 80% in brain, respectively. Free sulfhydryl contents of the tissues in both experimental groups showed a parallel decrease. Furthermore, the decrease in protein-bound sulfhydryl values of brain tissues were more pronounced than the values found for liver. It seems that not only liver but also the brain is an important target organ to the alteration in antioxidant system through either a deficiency of both selenium and vitamin E or an excess of selenium alone in the diet.

A rapid method for the purification of glucose-6-phosphate dehydrogenase from bovine lens.

This paper describes a simple and rapid method for the purification of glucose-6-phosphate dehydrogenase from bovine lens, together with analysis of the kinetic behaviour and some properties of the enzyme. The purification consisted of two steps, 2',5'-ADP-Sepharose 4B affinity chromatography and DEAE Sepharose Fast Flow ion exchange chromatography in procedure which took two working days. The enzyme was obtained with a yield of 13.7% and had a specific activity of 2.64 U/mg protein. The overall purification was about 19,700-fold. The molecular weight of the enzyme was found to be 62 +/- 3 kDa by Sephadex G-200 gel filtration chromatography. A protein band corresponding to a molecular weight of 69.2 +/- 3.2 kDa was obtained on SDS polyacrylamide slab gel electrophoresis. On chromatofocusing, lens glucose-6-phosphate dehydrogenase gave a single peak at pI 5.14. The activation energy of the reaction catalyzed by the enzyme was calculated from Arrhenius plot as Ea = 5.88 kcal/mol. The pH versus velocity curve had two peaks at pH 7.7 and 9.6. By the double-reciprocal plots and the product inhibition studies, it was shown that the enzyme follows 'Ordered Bi Bi' sequential kinetics. From the graphical and statistical analyses, KmNADP+, KmG-6-P, KiNADPH, Ki6-PGA were estimated to be 0.008 +/- 0.002, 0.035 +/- 0.013, 0.173 +/- 0.007 and 1.771 +/- 0.160 mM, respectively. The observed kinetic behaviour of glucose-6-phosphate dehydrogenase from bovine lens was in accordance with the enzyme from other sources.