Deniz Yildiz

l-Cysteine influx and efflux: A possible role for red blood cells in regulation of redox status of the plasma

The objective of this study was to investigate if erythrocytes play a role in the maintenance of redox homeostasis of the plasma. Thus, we studied l-cysteine efflux and influx in vitro in human erythrocytes. In the present study, we exposed the erythrocytes to different concentrations of l-cysteine and then measured the intracellular free –SH concentrations. Erythrocytes treated in the same manner were later utilized for the cysteine efflux studies. The effect of temperature on the influx and the efflux processes were also evaluated. Change in the free –SH content of the buffer was evaluated as a measure for the presence of an efflux process. The effects of free –SH depletion on l-cysteine transport is also investigated. We also determined the rate of l-cysteine efflux in the presence and absence of buthionine sulfoximine (BSO) in erythrocytes that are pretreated with 1-chloro-2,4-dinitro benzene, a glutathione (GSH) depletory. Our l-cysteine influx studies demonstrated that erythrocytes can respond to increases in l-cysteine concentration in the extracellular media and influx l-cysteine in a concentration-dependent manner. Free –SH concentrations in erythrocytes treated with 1 mM l-cysteine reached to 1.64 ± 0.06 mM in 1 h whereas this concentration reached to 4.30 ± 0.01 mM in 10 mM l-cysteine treated erythrocytes. The l-cysteine efflux is also determined to be time-and concentration-dependent. Erythrocytes that are pretreated with higher l-cysteine concentrations displayed a higher efflux process. Outside concentration of free –SH in 1 mM l-cysteine pretreated erythrocytes reached to 0.200 ± 0.005 mM in 1 h whereas this concentration reached to 1.014 ± 0.002 with 10 mM l-cysteine pretreated erythrocytes. Our results also indicate that the rate of inward and outward transport of l-cysteine is affected by the oxidative status of the erythrocytes. When GSH is depleted and GSH synthesis is blocked, the l-cysteine uptake and the efflux processes are significantly decreased. Depending on our results, it could be concluded that erythrocytes play a role in the regulation of the plasma redox status and intracellular level of GSH determines the rate of the l-cysteine efflux.

Arsenate V induced glutathione efflux from human erythrocytes.

OBJECTIVE The objective of the present study was to investigate if arsenate V exposure results in glutathione efflux from human erythrocytes. PROCEDURE The changes in intracellular and extracellular nonprotein sulfhydryl and glutathione levels were determined in arsenate (V) exposed erythrocytes. Presence of any cellular membrane damage was assessed by lactate dehydrogenase activity measurement in the supernatant. RESULTS When erythrocytes were exposed to 10 mM of arsenate (V) for 4 h, the intracellular NPSH level decreased to 0.28±0025 μmol/ml erythrocyte. In contrast, extracellular nonprotein thiol level was increased to 0.180±0.010 μmol/ml erythrocyte in 4 h. Extracellular glutathione levels reached to 0.028±0.001, 0.052±0.002, and 0.054±0.004 μmol/ml erythrocyte with 1, 5, and 10 mM of arsenate (V), respectively. Utilization of MK571 a multi drug resistance-associated protein 1 inhibitor decreased the rate of glutathione efflux from erythrocytes suggesting a role for this membrane transporter in the process. CONCLUSION The results of the present study indicate that erythrocytes efflux glutathione when exposed to arsenate (V).

L-Cysteine Uptake is Stimulated by 1-Chloro-2,4-Dinitrobenzene in vitro in Human Erythrocytes

In previous studies, the transport of dinitrophenyl-glutathione from erythrocytes has been extensively investigated. However, the effect of treatment of erythrocytes with such xenobiotics on free-SH status of cells has not been well documented. Also, the effects of N-acetyl-L-cysteine or other-SH containing compounds on glutathione conjugate transport have not been investigated. The objectives of the present study were to investigate how the presence N-acetyl-L-cysteine and L-cysteine affect the free-SH status of 1-chloro-2,4-dinitrobenzene treated erythrocytes and how N-acetyl-L-cysteine or L-cysteine affects the rate of dinitrophenyl-glutathione conjugate transport form erythrocytes. Our results indicated that L-cysteine is more efficient than N-acetyl-L-cysteine in increasing the free-SH content of erythrocytes in the presence of 1-chloro-2,4-dinitrobenzene. At the end of 20 min of exposure, free-SH levels remained at 5.3 μmol/ml erythrocyte in the presence of L-cysteine. However, in the presence of N-acetyl-L-cysteine the free-SH level was 2 μmol/ml erythrocyte. In the absence of 1-chloro-2,4-dinitrobenze, L-cysteine uptake by erythrocytes was not efficient compared to N-acetyl-L-cysteine. The free-SH concentrations in the presence of N-acetyl-L-cysteine and L-cysteine, in this case were, 9 ± 1 and 1.5 ± 0.1 μmol/ml erythrocytes respectively. These results clearly suggest that 1-chloro-2,4-dinitrobenzene stimulates the L-cysteine uptake in eryhtrocytes by a mechanism not described before. Our results also indicated that 1-chloro-2,4-dinitrobenzene induced L-cysteine uptake is a Na+ and ATP dependent process. Replacement of NaCl with LiCl decreased the L-cysteine uptake by about 5-fold and in the presence of NaF decrease in L-cysteine uptake was about 2 fold. Our results conclude the presence of an in vitro L-cysteine uptake mechanism in erythrocytes stimulated by 1-chloro-2,4-dinitrobenzene.

Efflux of glutathione and glutathione complexes from human erythrocytes in response to vanadate

The main objective of the present study was to investigate if vanadate is extruded from the cells in a glutathione dependent manner resulting in the appearance of extracellular glutathione and complexes of glutathione with vanadium. Vanadate significantly depleted intracellular non-protein sulfhydryl (NPSH) levels in a time- and concentration-dependent manner. The intracellular NPSH level was decreased to 0.0 ± 0.0 μmol/ml erythrocyte when exposed to 10 mM of vanadate for 4h. Extracellular NPSH level was increased concomitantly with the intracellular decrease and reached to 0.1410 ± 0.005 μmol/ml erythrocyte in 4h. Intracellular decrease and extracellular increase in NPSH levels were significantly inhibited in the presence of DIDS, a chloride-bicarbonate exchanger which also mediates phosphate and arsenate transport in erythrocytes. In parallel with the increase in extracellular NPSH levels, significant increases in extracellular glutathione levels were detected following exposure to vanadate. Extracellular glutathione levels reached to 0.0150 ± 0.0.001, 0.0330 ± 0.001, and 0.0576 ± 0.002 μmol/ml erythrocyte with 1, 5, and 10 mM of vanadate respectively. Dimercaptosuccinic acid treatment of supernatants significantly increased the glutathione levels measured in the extracellular media. Utilization of MK571 an MRP inhibitor decreased the rate of glutathione efflux from erythrocytes suggesting a role for this membrane transporter in the process. A known methylation inhibitor periodate oxidized adenosine decreased the rate of glutathione efflux from erythrocytes. This observed decrease in extracellular GSH levels suggests that GSH release partly requires a proper cellular methylation process and that part of GSH detected in the extracellular media may arise from GSH-vandium complexes. The results of the present study indicate that human erythrocyte efflux glutathione in reduced free form and in conjugated form/s that can be recovered with dimercaptosuccinic acid when exposed to vanadate.

Methyl Parathion-Induced Changes in Free and Protein-Bound SH Levels in Rat Tissues.

The main objective of this study was to investigate the changes in free and protein-bound SH contents in methyl parathion-exposed rat tissues. The free and protein-bound SH levels are usually affected and depleted by oxidative stress-inducing agents. Results would indicate if methyl parathion toxicity partly results from depletion of sulfhydryl content of tissues. Six-week-old male Wistar albino rats were used in this study. Following exposure to methyl parathion for 3 months, the liver, the brain, and the kidney tissues were removed from the rats. The free and protein-bound SH contents were determined in these tissues. In addition, plasma lactate dehydrogenase levels were determined. Our results showed that methyl parathion exposure significantly lowers the free and protein-bound SH levels in rat tissues. However, lactate dehydrogenase activity in the blood plasma did not display any differences compared to the control group. The free SH concentrations in the control rat liver, brain, and kidney tissues were 3.78 ± 0.1 μmol/100 mg tissue, 1.56 ± 0.08 μmol/100 mg tissue, and 2.16 ± 0.08 μmol/100 mg tissue, respectively, whereas the free SH concentrations in rats exposed to methyl parathion were determined as 0.536 ± 0.1 μmol/100 mg tissue in the liver, 1.06 ± 0.1 μmol/100 mg tissue in the brain, and 0.108 ± 0.03 μmol/100 mg tissue in the kidney. The protein-bound SH concentrations in the liver and in the kidney in rats exposed to methyl parathion displayed a significant decrease also. However, the protein-bound SH level in the brain did not change significantly. These results indicate that methyl parathion exposure partially depletes the free and protein-bound SH levels. Thus, it was concluded that methyl parathion toxicity may partly result from oxidative stress.

Comparision of Pure Nicotine and Smokeless Tobacco Extract Induced Formation of 8-OH-dG

Nicotine and smokeless tobacco extract have been shown to induce oxidative stress in different experimental systems. However, the effect of nicotine and smokeless tobacco extract containing equal amounts of nicotine on 8-OH-dG formation has not been investigated. 8-OH-dG is a DNA adduct formed by free radical attack and is elevated in tumor cells. The objective of the present study was to evaluate the formation of 8-OH-dG following exposure to different concentrations of nicotine and smokeless tobacco extract containing equal amounts of nicotine. Exposure of Chinese Hamster Ovary cells to 5 and 10 mM of nicotine resulted in generation of 8-OH-dG. The observed 8-OH-dG levels at these concantrations of nicotine were 5.1 ± 1.7 and 9.4 ± 2.3 8-OH-dG/dG × 100.000, respectively. Smokeless tobacco extract containing 1 and 5 mM nicotine also induced generation of 8-OH-dG. The measured 8-OH-dG levels at these concentrations were 4.7 ± 0.6 and 20.6 ± 2.2 8-OH-dG/dG × 100.000 respectively. Exposure of cells to smokeless tobacco extract containing 10 mM nicotine resulted in cell death. Coaddition of superoxide dismutase and catalase along with nicotine reversed the 8-OH-dG generation. However, superoxide dismutase and catalase did not inhibit the 8-OH-dG generation in smokeless tobacco extract treated cells.

Comparison of N-acetyl-L-cysteine and L-cysteine in respect to their transmembrane fluxes

The objective of the present study was to compare cysteine and N-acetyl-L-cysteine in respect to their transmembrane fluxes and find out which one is a better available precursor for the cells and thus better supports the intracellular glutathione synthesis. Cysteine can directly participate in glutathione synthesis, whereas N-acetyl-L-cysteine must be first deacetylated before its incorporation to glutathione. In the present study we investigated and compared the efficiencies of cysteine and N-acetyl-L-cysteine influx and efflux through the erythrocyte membrane. Erythrocytes transported both cysteine and N-acetyl-L-cysteine in a concentration-dependent manner. However, our results demonstrated that cysteine crosses the erythrocyte membranes more efficiently as compared to N-acetyl-L-cysteine. Treatment of erythrocytes with 5 mM of cysteine or N-acetyl-L-cysteine for 1 hr raised the intracellular free sulfhydryl group (free-SH) levels to 3.37 ± 0.006 or 2.23 ± 0.08 μ mol/ml erythrocyte, respectively. Cysteine more effectively than N-acetyl-L-cysteine restored the intracellular free-SH level depleted beforehand. In erythrocytes previously depleted of free-SH, 5 mM cysteine raised the free-SH level to 1.45 ± 0.075 μ mol/ml within 1 hr, whereas N-acetyl-L-cysteine at the same concentration raised this level to 0.377 ± 0.034 μmol/ml only. The results of our study also revealed that both cysteine and N-acetyl-L-cysteine influx and efflux processes are temperature dependent indicating that their transport requires biological activity. Our results demonstrate that cysteine is a better thiol precursor for the erythrocytes. Availability of cysteine for the cells is higher than that of N-acetyl-L-cysteine.

Inhibitory effects of acetylsalicylic acid on exocrine pancreatic carcinogenesis

Abstract We investigated short (6 months) and long (12 months) term inhibitory effects of low (200 ppm) and high (400 ppm) dosages of acetylsalicylic acid (aspirin) on exocrine pancreatic carcinogenesis. It is known that exocrine pancreatic carcinogenesis can be detected by the presence of atypical acinar cell foci (AACF) in pancreas. We investigated possible inhibitory effects of acetylsalicylic acid in an azaserine-treated rat model. AACF were produced in rats by injection with azaserine according to previous studies. Our findings showed that the number, volume and diameter of pancreatic AACF were reduced after acetylsalicylic acid application. These observations suggest that acetylsalicylic acid may exert a protective effect against neoplastic development of pancreatic acinar cells in azaserine injected rats. Our findings corroborate reports in the literature concerning the effects of aspirin in reducing neoplastic development.