Decrease In NADPH Research Articles

Pyridine nucleotide flux and glutathione oxidation in the cultured rat conceptus

It is proposed that protection of the developing embryo from chemical and environmental insults that produces oxidative stress requires a proper glutathione (GSH) and pyridine nucleotide status in both the embryo and extra-embryonic membranes. Modulation of pyridine nucleotide flux {NAD(H) and NAD(P)H} in the visceral yolk sac (VYS) by the thiol oxidants diamide and tert-butyl hydroperoxide (tBH) was studied in real time using microfiberoptic sensors in GD 10 rat conceptuses. Consecutive 5-min exposures to 125- and 250-μM diamide resulted in a fluorescence decrease of 14 and 32 Arbitrary Fluorescence Units (AFU). An additional consecutive exposure to 500-μM diamide caused an attenuated decrease followed by a rebound increase of 22 AFU. Consecutive 5-min exposures to tBH at 250 and 500 μM produced fluorescence decreases similar to that of 500 μM diamide, but the decreases were attenuated at 1000 μM. However, there was variability in the rebound increase. A 5-min exposure to tBH (500 μM) alone caused a fluorescence decrease of 14 AFU followed by a rebound increase of 8 AFU. The rate of fluorescence decrease was attenuated by 50% with pretreatment with the glutathione reductase (GSSG-Rd) inhibitor, BCNU (1,3, bis(2 chloroethyl)-1-nitrosourea), indicating that the decrease in surface fluorescence was probably attributable to a decrease in NADPH. Decreases in fluorescence, observed from the surface of the VYS, correlated with decreases in GSH/GSSG ratios in the embryos and the VYS. After exposure to tBH, GSH levels in conceptuses decreased at the end of 5 and 15 min, with a corresponding increase in oxidized glutathione (GSSG) at the end of 3, 5, and 15 min. Our results demonstrate that the increased production of GSSG on exposure to thiol oxidants correlates with a decrease in the reduced pyridine nucleotide, implying the presence of an active GSSG-Rd pathway in the conceptus during organogenesis, and implicating an important role of the pyridine nucleotides in the restoration of GSH homeostasis in the developing rat conceptus during organogenesis.

Prevention of N-nitrosodiethylamine-induced lung tumorigenesis by ellagic acid and quercetin in mice

The polyphenolic antioxidants, consumed as an integral part of vegetables, fruits and beverages, are suggested as possessing anticarcinogenic properties. In the present study we have looked into the anticarcinogenic potential of plant polyphenols ellagic acid (EA) and quercetin against N-nitrosodiethylamine-induced lung tumorigenesis in mice. Ellagic acid was able to significantly reduce tumour incidence to 20% from the control value of 72.2%. Similarly, tumour burden was also decreased, although not significantly, from 3.15 to 2.5. Quercetin (QR) caused the tumour incidence to decrease from 76.4% to 44.4% when fed until the third dose of carcinogen. Both of the polyphenols suppressed the tumour incidence mainly by acting at the initiation phase of the carcinogenesis, since continuing the feeding of polyphenols until the termination of the experiment did not cause any apparent change in tumour incidence or tumour burden. Besides this, ellagic acid was found to be a better chemopreventor than quercetin. In order to search for their mechanism of action, the effect of feeding of these compounds on reduced glutathione (GSH), an important endogenous antioxidant, and on lipid peroxidation was investigated. Both ellagic acid and QR caused a significant increase in GSH and decrease in NADPH- and ascorbate-dependent lipid peroxidation. Ellagic acid was found to be more effective in decreasing the lipid peroxidation and increasing the GSH. This may be one of the reasons for its observed better anticarcinogenic property as compared to quercetin.

Pyridine nucleotide pool size changes in iron‐deficient Plantago lanceolata roots during reduction of external oxidants

The involvement of pyridine nucleotides in the reduction of extracytoplasmatic electron acceptors by iron‐deficient Plantago lanceolata L. roots has been examined by measuring the changes in NAD(P)H and NAD(P)− induced by various external acceptors. Exposure of the plants to FeEDTA, ferricyanide, ferric citrate or hexachloroiri‐date resulted in a transient decrease in NADPH and an increase in NAD−. No major differences in this pattern were observed between acceptors which were assumed to be reduced by different enzymes. The application of the membrane‐permeable oxidant nitro blue tetrazolium led to similar changes in reduced and oxidized pyridine nucleotides and decreased the reduction of external acceptors. The amino acid analog p‐fluorophenylalanine caused a transient decline in both NADPH level and NADPH/ NADP− ratio and a decrease in the ratio of NADH to NAD− without affecting the level of NADH. Exposure of the plants to the translation inhibitor cycloheximide increased both NADH and NADPH concentrations. A comparison of the redox activities and pyridine nucleotide fractions after inhibitor treatment revealed that the constitutive, but not iron stress‐induced redox activity correlates with NADPH levels. These results are interpreted as confirming that the redox systems on the root plasma membrane are separately regulated. Possible metabolic reactions during the reduction processes are discussed.

Control of sorbitol metabolism in renal inner medulla of diabetic rats: regulation by substrate, cosubstrate and products of the aldose reductase reaction

Streptozotocin diabetes induces a 4-fold increase in the maximal velocity of inner medullary aldose reductase as determined in vitro but increases sorbital synthesis in intact inner medullary collecting duct (IMCD) cells only 1.3-fold [1]. In order to resolve this discrepancy we investigated the importance of intracellular factors in controlling the role of cellular sorbitol synthesis. These factors include glucose concentration, sorbitol concentration, the activity of the NADPH-regenerating pentose phosphate pathway, intracellular NADP and NADPH content, and intracellular reduced (GSH) and oxidized glutathione (GSSG). It was found that the apparent K m of cellular sorbitol production for glucose was identical in control and diabetic rats ( 56 ± 18 vs. 59 ± 14 mmol/l d-glucose ), whereas V max increased by 31% in diabetes. In inner medullary collecting duct cells of diabetic rats containing 146 ± 5 μmol sorbitol/g protein, sorbitol synthesis slightly lower (−15%), compared to cells which had been sorbitol-depleted prior to the experiment (87 ± 4 μmol sorbitol/g protein). However, no inhibitory effect of sorbitol (up to 200 mmol/l) was observed on aldose reductase activity in vitro. In diabetic rats the content of NADPH was about 32% lower than in the control rats (3.8 ± 0.3 vs. 5.6 ± 0.4 μmol/g protein) and the ratio of NADPH/NADP was decreased from 25.6 ± 5.1 to 8.6 ± 1.7. In homogenates of the inner medulla the activity of 6-phospho-gluconate dehydrogenase (EC 1.1.1.43) was identical in both experimental groups, so the pentose phosphate shunt seems to be unaltered. GSH content in diabetic rats was also diminished (4.2 ± 0.67 μmol/g protein vs. 7.41 ± 0.5 μmol/g protein) and the GSH/GSSG ratio fell from 92.6 to 57.4. In enzyme tests in vitro an apparent K m of 7.3 ± 1.9 μmol/l of the aldose reductase for NADPH was found; NADP acted as competitive inhibitor with a apparent K i of 183 ± 31 μmol/l. Aldose reductase activity was also found to be strongly inhibited by the SH-group reagent p-chloromercurybenzoesulfonate (apparent K i = 0.85 · 10 −6 mol/l). Combining the results obtained on the properties of the aldose reductase in vitro and the observation made in the intact cells, the investigators suggest that the decrease in NADPH/ NADP ratio, as well as changes in the redox state in the cells of diabetic animals, can play a significant role in the control of sorbitol synthesis.

Metabolism of pyridine nucleotides in cultured rat hepatocytes intoxicated with tert-butyl hydroperoxide

The alterations in the metabolism of pyridine nucleotides, as well as the role such changes play in the genesis of lethal cell injury, were explored in cultured rat hepatocytes intoxicated with tert-butyl hydroperoxide (TBHP). The loss of NADPH, NADH, and NAD equalled the increase in NADP, with little if any change in the total content of pyridine nucleotides. Identical alterations occurred in the presence of N, N′-diphenyl- p-phenylenediamine, an antioxidant that prevented the death of the cells. Inhibition of glutathione reductase by l, 3-bis(2-chloroethyl)-1-nitrosourea (BCNU) reduced the extent of the increase in NADP and the decrease in NADPH. At the same time, BCNU increased the cell killing. Depletion of ATP with oligomycin reduced the loss of NAD and the accumulation of NADP. Treatment of the hepatocytes with the poly(ADP-ribose) polymerase inhibitor 3-aminobenzamide had no effect on the depletion of NAD. Thus, all of the alterations in pyridine nucleotides that accompany the exposure of cultured hepatocytes to TBHP can be dissociated from the development of lethal cell injury. The changes do suggest, however, a rapid interconversion of the respective species. The initial response reflects activation of glutathione reductase with the consequent oxidation of NADPH to NADP. The conversion of NADH to NAD and then NAD to NADP, the latter by nicotinamide adenine dinucleotide kinase, can account for the increase in NADP over that resulting from the oxidation of NADPH by glutathione reductase. Finally, there was no evidence in cultured hepatocytes treated with TBHP for changes in NAD that reflect the activation of poly(ADP-ribose) polymerase.

Inhibition of rat hepatic microsomal cytochrome P-450 system by cobaltous chloride and reversal of inhibition by iron in vivo.

The effects of preexposure of rats to cobaltous chloride (CO) mixed in iron-sufficient (I.S) and -deficient (I.D) diets on hepatic microsomal electron transfer system was investigated. Male Sprague-Dawley rats were fed for 4 weeks on I.D diets mixed with 0, 100 and 200 ppm CO. At the end of 4 weeks three rats from each group were transferred to I.S diets mixed with the same amount of CO. Liver microsomal NADPH - Cytochrome C reductase, NADPH - dehydrogenase, cytochrome P-450 and aniline binding were determined in both batches of rats. The rats receiving CO in the I.D diets showed a 35, 60, 75 and 40% decrease in NADPH - Cyt. C reductase, dehydrogenase, Cyt. P-450 and aniline binding respectively. The rats transferred from I.D diet to I.S diet showed a complete recovery of the inhibition of the microsomal electron transfer system. The rats receiving 200 ppm mixed with I.S diets did not show any changes in any biochemical parameter measured.

Cerebral oxidized and reduced nicotinamide-adenine dinucleotide phosphate and glucose 6-phosphate dehydrogenase in mice during exposure to high oxygen pressure.

NADP+, NADPH and glucose 6-phosphate dehydrogenase were determined in the cerebral cortex of mice exposed to high O2 pressure for 0, 8 and 16 min. These time intervals corresponded to 0, 50 and 100% of the CT50 (the time taken for 50% of the mice to convulse). Cerebral NADP+, NADPH and glucose 6-phosphate dehydrogenase also were determined in O2-exposed mice exhibiting hyperactivity, convulsions, and in mice killed 10s after convulsions. Similar increases in cortical NADP+ and decreases in NADPH were found in mice exposed to 610kPa (6 atm.) of 100% O2 for 0, 50 and 100% of the CT50, during hyperactivity, onset of seizure and 10s after convulsions. The NADP+/NADPH ratio increased approx. 25% at 0% of the CT50, and remained at this increased value at all O2-exposure periods including the hyperactive state, onset of seizure and 10s after convulsions. Identical changes in cerebral NADP+ , NADPH and the NADP+/NADPH ratio were found in mice exposed for 16min to 100% O2 at 100, 350 or 610kPa. No change in cerebral glucose 6-phosphate dehydrogenase was found in mice exposed to 610kPa of 100% O2 during the various stages of O2 toxicity. Only in the 10s post-convulsive group was a statistically significant decrease in glucose 6-phosphate dehydrogenase observed. Disulfiram [bis(diethylthiocarbamoyl) disulphide], an effective O2-protective agent, did not prevent the O2-induced increase in cerebral NADP+ and the NADP+/NADPH ratio, or decrease in NADPH.

The induction of sister chromatid exchanges by cyclophosphamide in the presence of differently induced microsomal fractions of rat liver

The induction of sister chromatid exchanges can be monitored by a test that incorporates the factor of metabolic activation in a simple manner. The results with cyclophosphamide show that in this test the induction of the metabolizing enzymes of the rat liver homogenates used is very important. 3-Methylcholanthrene induces little if any extra conversion of cyclophosphamide to SCE-inducing metabolites, compared with no induction. Aroclor 1254 and phenobarbital however, were very good inducers. The difference found between the liver homogenates concerning SCE induction corresponded with the differences in cyclophosphamide metabolism, which was measured as the decrease in NADPH induced by cyclophosphamide.

Effect of tolbutamide on aminophylline-, 3,5-AMP-dibutyrate-or glucagon-induced insulin release from pancreatic islets after impairment of pyridine nucleotide metabolism caused by 6-aminonicotinamide (6-AN)

The effect of tolbutamide on pyridine nucleotides and insulin secretion stimulated by aminophylline, 3,5-AMP-dibutyrate or glucagon was studied in pancreatic islets of rats previously treated with 6-aminonicotinamide (6-AN), an inhibitor of pyridine nucleotide synthesis. After being incubated for 60 min in a Krebs-Ringer-Bicarbonate-Buffer in the absence of glucose, pancreatic islets of rats i.p. injected with 35 mg/kg of 6-AN 6 hrs before pancreas removal contained about 30% less NADP and NADPH than did islets of control rats. No changes of NDA or NADH were observed in islets of 6-AN-treated animals. Addition of 16.5 mM glucose led to an increase of NADH, NADPH and a decrease of NADP in islets of both groups of animals; NAD levels remained unchanged. In vitro addition of tolbutamide to islets of control rats did not affect the levels of NADPH or NADP in the presence of 5.5 mM glucose. When 16.5 mM glucose were present, a decrease of NADPH and an increase of NADP was obvious. No effect of tolbutamide on insular NADPH or NADP was observed in islets of rats previously treated with 6-AN be it in the presence of 5.5 or 16.5 mM glucose. In islets of 6-AN-treated rats insulin release in response to aminophylline or 3,5-AMP-dibutyrate in the presence of 5.5 mM glucose was significantly depressed, when compared to islets of untreated controls. Addition of tolbutamide increased insulin release due to aminophylline, 3,5-AMP-dibutyrate or glucagon islets of controls. Tolbutamide alone was without effect. In islets of 6-AN-treated rats aminophylline, 3,5-AMP-dibutyrate or glucagon stimulated insulin release only when tolbutamide was present. Our data suggest that there is no direct interference of tolbutamide with pyridine nucleotides of pancreatic islets, and that tolbutamide increases the secretory response of the beta-cell to aminophylline, 3,5-AMP-dibutyrate or glucagon when insulin release due to these agents is inhibited during decrease of insular NADP and NADPH, caused by 6-AN.

Intracellular restraint: a new basis for the limitation in response to oxidative stress in human erythrocytes containing low-activity variants of glucose-6-phosphate dehydrogenase.

Several mechanisms recently proposed for regulation of the hexose monophosphate shunt require the concentration of NADP to be low or that of NADPH to be high. The present study indicates that the first enzyme of the hexose monophosphate shunt of human erythrocytes is under severe restraint even when these conditions do not exist. In human erythrocytes containing low-activity variants of this enzyme, glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate:NADP(+) 1-oxidoreductase; EC 1.1.1.49), measurements of the rate of oxidation of C-1 labeled glucose show that the enzyme is operating at a rate much closer to its maximum than in normal cells. This requires that the ratio of inhibitory NADPH to NADP be much lower in the variant cells than in normal cells. A small increase in oxidative rate, induced by naphthol, then causes a disappearance in reduced glutathione in the variant cells, presumably because a significant further decrease in NADPH occurs in these cells, whereas the same oxidative stress in normal cells would not lower the NADPH level appreciably. A low NADPH/NADP ratio in unstressed cells deficient in glucose-6-phosphate dehydrogenase is confirmed by direct measurement. The maximum activity of the variant enzyme in the cell, as measured with methylene blue to keep most of the NADP in the oxidized form, is only about 1/60 of that found in hemolysates, thus accounting for the failure to compensate for a relatively small oxidative stress in vivo in spite of an apparent sufficiency of enzyme. The reason for the limitation on maximum intracellular activity is unknown. A similar limitation is seen with normal cells incubated with methylene blue, where the maximum intracellular rate is also only about 1/60 of that found in hemolysates.