Maria Sokołowska

Alteration in plasma levels of nonprotein sulfhydryl compounds and S-nitrosothiols in chronic renal failure patients

BACKGROUND Uremia is accompanied by the elevated nitric oxide (NO) synthesis, and it has not yet been established how this influences the levels of nonprotein sulfhydryl compounds (NPSH) and formation of S-nitrosothiols (SNT). METHODS Our study was designed to determine plasma levels of SNT and NPSH in chronic renal failure (CRF) patients, who were hemodialysed (HD) or were not on hemodialysis treatment (ND), and in the control group. RESULTS In ND patients, the plasma levels of SNT were significantly increased (11.25+/-2.08 nmol/ml, p<0.01), while NPSH levels were simultaneously decreased (66.67+/-15.0 nmol/ml, p<0.05) in comparison with the control subjects (SNT: 8.75+/-2.08 nmol/ml, NPSH: 86.66 nmol/ml). In HD patients, plasma concentration of SNT before hemodialysis was significantly lower than in the control group (0.150+/-0.042 nmol/mg protein vs. control: 0.175+/-0.075 nmol/mg protein), and no significant change was observed after dialysis (0.142+/-0.058 nmol/mg protein, p<0.05). The level of NPSH in HD patients before dialysis was significantly decreased in comparison with the control subjects, both, when the results were calculated per 1 ml of plasma (45.96+/-17.87 nmol/ml) and per 1 mg of protein (0.70+/-0.25 nmol/mg protein). In the postdialysis samples, NPSH rose (79.15+/-22.9 nmol/ml, p<0.001 which corresponds to 1.30+/-0.55 nmol/mg protein, p<0.001) as compared to the level before dialysis. CONCLUSIONS Firstly, plasma SNT level was found to be increased in CRF patients who were not treated with hemodialysis, while in HD patients, it dropped below the control values. It indicates that hemodialysis disturbs an equilibrium of reactions involved in S-nitrosothiols formation most probably by removing low molecular weight S-nitrosylating compounds. Secondly, the increased level of NPSH after each hemodialysis session indicates reestablished antioxidant capacity of plasma and suggests the existence of dialysable compounds, which via unknown mechanism become responsible for the decreased level of thiols.

The effect of modulation of γ-glutamyl transpeptidase and nitric oxide synthase activity on GSH homeostasis in HepG2 cells

High glutathione (GSH) level and elevated γ‐glutamyl transpeptidase (γGT) activity are hallmarks of tumor cells. Toxicity of drugs and radiation to the cells is largely dependent on the level of thiols. In the present studies, we attempted to inhibit γGT activity in human hepatoblastoma (HepG2) cells to examine whether the administration of γGT inhibitors, acivicin (AC) and 1,2,3,4‐tetrahydroisoquinoline (TIQ) influences cell proliferation and enhances cytostatic action of doxorubicin (DOX) and cisplatin (CP) on HepG2 cells. The effects of these inhibitors were determined by 1‐(4,5‐dimethylthiazol‐2‐yl)‐3,5‐diphenylformazan (MTT), BrdU and lactate dehydrogenase (LDH) tests and by estimation of GSH level. Additionally, we investigated the changes in caspase‐3 activity, which is a marker of apoptosis. The obtained results showed that the γGT inhibitors introduced to the medium alone elicited cytotoxic effect, which was accompanied by an increase in GSH level in the cells. TIQ concomitantly increased caspase‐3 activity. Doxorubicin and CP proved to be cytotoxic, and both inhibitors augmented this effect. As well DOX as CP radically decreased GSH levels, whereas γGT inhibitors had diverse effects. Therefore, the obtained results confirm that γGT inhibitors can enhance pharmacological action of DOX and CP, which may permit clinicians to decrease their doses thereby alleviating side effects. Aminoguanidine (nitric oxide synthase inhibitor) given alone was little cytotoxic to HepG2 cells, while its introduction to the medium together with DOX and CP significantly increased their cytotoxicity. Aminoguanidine on its own did not show any effect on GSH level in HepG2 cells, but markedly and significantly elevated its concentration when added in combination with CP but not with DOX. This indicates that when CP was used as a cytostatic, GSH level rose after treatment with its combination with both AC and aminoguanidine.

The effect of the uremic toxin cyanate (CNO−) on anaerobic cysteine metabolism and oxidative processes in the rat liver: a protective effect of lipoate

Chronic renal failure (CRF) patients have an increased plasma level of urea, which can be a source of cyanate. This compound can cause protein carbamoylation thereby changing biological activity of proteins. Therefore, in renal failure patients, cyanate can disturb metabolism and functioning of the liver. This work presents studies demonstrating that the treatment of rats with cyanate alone causes the following changes in the liver: (1) inhibition of rhodanese (TST), cystathionase (CST) and 3-mercaptopyruvate sulfotransferase (MPST) activities, (2) decrease in sulfane sulfur level (S*), (3) lowering of nonprotein sulfhydryl groups (NPSH) group level, and (4) enhancement of prooxidant processes (rise in reactive oxygen species (ROS) and malondialdehyde (MDA) level). This indicates that cyanate inhibits anaerobic cysteine metabolism and shows prooxidant action in the liver. Out of the above-mentioned changes, lipoate administered with cyanate jointly was able to correct MDA, ROS and NPSH levels, and TST activity. It had no significant effect on MPST and CST activities. It indicates that lipoate can prevent prooxidant cyanate action and cyanate-induced TST inhibition. These observations can be promising for CRF patients since lipoate can play a dual role in these patients as an efficient antioxidant defense and a protection against cyanate and cyanide toxicity.

Treatment with 1,2,3,4-tetrahydroisoquinolone affects the levels of nitric oxide, S-nitrosothiols, glutathione and the enzymatic activity of γ-glutamyl transpeptidase in the dopaminergic structures of rat brain

Depletion of glutathione (GSH), nitrosative stress and chronic intoxication with some neurotoxins have been postulated to play a major role in the pathogenesis of Parkinsons disease. This study aimed to examine the effects of acute and chronic treatments with 1,2,3,4-tetrahydroisoquinoline (TIQ), an endo-/exogenous substance suspected of producing Parkinsonism in human, on the levels of nitric oxide (NO), S-nitrosothiols and glutathione (GSH) in the whole rat brain and in its dopaminergic structures. TIQ administered at a dose of 50 mg/kg i.p. significantly increased the tissue concentrations of NO and GSH in the substantia nigra (SN), striatum (STR) and cortex (CTX) of rats receiving this compound both acutely and chronically. Moreover, it decreased the level of oxidized glutathione (GSSG) and enhanced GSH:GSSG ratio affecting in this way the redox state of brain cells. TIQ also increased the level of S-nitrosothiols when measured in the whole rat brain and CTX, although it markedly decreased their level in the STR after both treatments. Inhibition of the constitutive NO synthase by l-NAME in the presence of TIQ caused decreases in GSH and S-nitrosothiol levels in the brain. The latter effect shows that the TIQ-mediated increases in GSH and S-nitrosothiol concentrations were dependent on the enhanced NO level. The above-described results suggest that TIQ can act as a modulator of GSH, NO and S-nitrosothiol levels but not as a parkinsonism-inducing agent in the rat brain.

Inhibition of the catalytic activity of rhodanese by S-nitrosylation using nitric oxide donors

Rhodanese (EC 2.8.1.1.) from bovine liver contains four reduced cysteine groups. The -SH group of cysteine 247, located in a rhodanese active centre, transfers sulfane sulfur in a form of hydrosulfide (-S-SH) from appropriate donors to nucleophilic acceptors. We aimed to discover whether S-nitrosylation of critical cysteine groups in rhodanese can inhibit activity of the enzyme by covalent modification of -SH groups. The inhibition of rhodanese activity was studied with the use of a number of nitric oxide (NO) donors. We have successfully confirmed using several methods that the inhibition of rhodanese activity is a result of the formation of stable S-nitrosorhodanese. Low molecular weight NO donors, such as S-nitroso-N-acetylpenicillamine (SNAP) and S-nitrosoglutathione (GSNO), inactivate rhodanese and are much more effective in this regard (100% inhibition at 2.5mM) than such known inhibitors of this enzyme, as N-ethylmaleimide (NEM) (25 mM < 50%) or sulfates(IV) (90% inhibition at 5mM). On the other hand, sodium nitroprusside (SNP) and nitrites inhibit rhodanese activity only in the presence of thiols, which suggests that S-nitrosothiols (RSNO) also have to participate in this reaction in this case. A demonstration that rhodanese activity can be inhibited as a result of S-nitrosylation suggests the possible mechanism by which nitric oxide may regulate sulfane sulfur transport to different acceptors.

Bioactivation of nitroglycerin to nitric oxide (NO) and S‐nitrosothiols in the rat liver and evaluation of the coexisting hypotensive effect

The aim of the present study was to investigate nitroglycerin (NTG) bioactivation pathways in the liver after various periods of its administration. We also attempted to elucidate the relationship between nitric oxide (NO) and S‐nitrosothiol (SNT) levels, and concentration of nonprotein thiols (NPSH) and intensity of peroxidative processes. Intravenous injections of NTG cause an increase in NO and SNT levels in the rat liver. The same intravenous NTG injections in the rats pretreated with 5‐day i.p. NTG administrations lead to a drop in the levels of the biologically active NO, SNT and NPSH, with no concomitant changes in the rate of lipid peroxidation. This indicates that after such period of nitroglycerin pretreatment, levels of pharmacologically active NO and SNT decrease. However, during longer periods of NTG administration (for 10 and 17 days) NO, SNT and NPSH concentrations remain at the control level in spite of a considerably enhanced lipid peroxidation, which indicates that tolerance did not develop. Effects of NTG bioactivation in the liver, i.e. the levels of NO and SNT released from it, after different periods of drug administration correspond with hypotensive effects, which are known to be dependent on NTG biodegradation in vascular endothelial cells. The changes in NO and SNT levels observed in the rat liver after different periods of NTG administration parallel alterations in the hypotensive effect. In conclusion, NTG treatment for 10 and 17 days does not lead to tolerance, however, a transient loss of its pharmacological activity occurs after 5‐day NTG pretreatment.

Activation of DNA biosynthesis in human hepatoblastoma HEPG2 cells by the nitric oxide donor, sodium nitroprusside

The role of nitric oxide (NO) in carcinogenesis is controversial as it has been shown to both stimulate and inhibit tumour growth. Also, there are contradictory opinions regarding the effects of NO on the proliferation of normal and tumour cells. The aim of our study was to use an in vitro model to determine the influence of exogenous NO donors on DNA biosynthesis by measuring [3H] thymidine incorporation in human hepatoblastoma cells (HepG2). The studies were conducted with the following NO precursors: sodium nitroprusside (SNP), S‐nitrosoglutathione, and nitroglycerine (NTG). Out of all three NO donors, SNP increased NO levels and strongly stimulated DNA biosynthesis. A SNP concentration of 150 μm induced optimal NO levels necessary for the activation of DNA biosynthesis. Lower levels of DNA biosynthesis (118% increase over the control) were observed in the presence of NTG, whereas S‐nitrosoglutathione had no effect. Antioxidants such as thiol‐containing drugs, N‐acetylcysteine and tocopherol, proved to be the most efficient co‐activators of SNP‐induced DNA synthesis. On the other hand, supplementing the SNP‐containing medium with compounds that induce oxidative stress and lower the level of –SH groups such as hydrogen peroxide, doxorubicin, and N‐ethylmaleimide, led to the inhibition of DNA synthesis. Therefore, our results firmly confirm the hypothesis that biological effects of exogenous NO donors depends on the redox status of the cell.

The effect of uremic toxin cyanate (OCN--) on anaerobic sulfur metabolism and prooxidative processes in the rat kidney: A protective role of lipoate

Cyanate and its active form isocyanate are formed mainly in the process of nonenzymatic urea biodegradation. Cyanate is capable of protein S- and N-carbamoylation, which can affect their activity. The present studies aimed to demonstrate the effect of cyanate on activity of the enzymes implicated in anaerobic cysteine metabolism and cyanide detoxification and on glutathione (GSH) level and peroxidative processes in the kidney. In addition, we examined whether a concomitant treatment with lipoate, a dithiol that may act as a target of S-carbamoylation, can prevent these changes. The studies were conducted in Wistar rats. The animals were assigned to four groups, which received injections of physiological saline, cyanate (200 mg/kg), cyanate (200 mg/kg) + lipoate (100 mg/kg) and lipoate alone (100 mg/kg). The animals were killed 2 h after the first injection, the kidneys were isolated and kept at −80°C until biochemical assays were performed. Cyanate inhibited rhodanese (TST) and mercaptopyruvate sulfotransferase (MPST) activity, decreased GSH level and enhanced peroxidative processes in the kidney. All these changes were abolished by cyanate treatment in combination with lipoate.

The effect of lipoic acid on cyanate toxicity in the rat heart

BACKGROUND Cyanate is a uremic toxin formed principally via spontaneous urea biodegradation. Its active isoform, isocyanate, is capable of reaction with proteins by N and S carbamoylation, which influences their structure and function. Sulfurtransferases implicated in anaerobic cysteine transformation and cyanide detoxification belong to the enzymes possessing SH groups in their active centers. The present studies aimed to demonstrate the effect of cyanate and lipoic acid on the activity of these enzymes as well as on the level of antioxidants and prooxidants in the rat heart. METHODS Wistar rats, which received intraperitoneal injections of cyanate and lipoic acid alone and in combination were sacrificed 2.5 h after the first injection. The hearts were isolated and homogenized in phosphate buffer and next biochemical assays were performed comprising determination of the level of glutathione, malondialdehyde and sulfane sulfur and the activity of antioxidant enzymes as well as glutathione S-transferase and gamma glutamyl transferase. RESULTS Sulfurtransferases and glutathione S-transferase were deactivated by cyanate treatment. It was accompanied by the decreased level of glutathione and sulfane sulfur and the increased level of reactive oxygen species and malondialdehyde. In parallel, antioxidant enzymes: catalase, glutathione peroxidase and gamma glutamyl transferase were activated under such circumstances. Lipoic acid, administered in combination with cyanate prevented the decrease in the level of glutathione and reduction of a pool of sulfane sulfur-containing compounds, concomitantly preserving the activity of antioxidant enzymes. CONCLUSIONS Since uremia, characterized by the elevated cyanate/isocyanate level, is accompanied by frequent cases of cardiovascular diseases, the addition of lipoic acid to the therapy seems promising in prophylaxis of heart diseases in uremic patients.