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PubMed1996

Effect of vitamin E and selenium on hypothermic restraint stress and chemically-induced ulcers.

The present study was undertaken to determine the effect of a combination of selenium and vitamin E on stress and chemical-induced gastric ulcers in rats. The gastric mucosal lesions were produced by hypothermic restraint stress, indomethacin, reserpine, and mucosal damaging agents including 80% ethanol, 0.6 M HCl, 25% NaCl, and 0.2 M NaOH. The gastric secretion studies were undertaken using Shay's pylorus ligation model. The results of this study demonstrated that the treatment of rats with selenium or vitamin E significantly reduced the basal gastric acid secretions when given individually; however, the combination of these agents produced a better inhibition of gastric acid secretions as compared to their individual effect. Both selenium and vitamin E were found to protect gastric mucosa against the lesions produced by hypothermic restraint stress and chemicals, but a highly significant protection was observed when they were used concomitantly. Vitamin E alone and with selenium significantly inhibited ethanol-induced depletion of gastric nonprotein sulfhydryl compounds. Our findings also showed that the combination of selenium and vitamin E provided better protection to gastric mucosa against hypothermic restraint-induced gastric wall mucus depletion. This study clearly suggests the feasibility of using selenium and vitamin E concurrently to achieve better gastroprotective effects.

Animals↗
PubMed1981

Reactivity of sulphydryl groups of cytosolic and mitochondrial bovine aspartate aminotransferases.

Reactivity of sulphydryl groups of cytosolic and mitochondrial aspartate aminotransferases from ox heart has been studied. A total of 5 and 7 cysteine residues per monomer are present in cAATo and mAATo, respectively. In native conditions only a single sulphydryl group can be titrated by Nbs2 while the catalytic activity remains unchanged, however in the mitochondrial isozyme the reactivity depends on the functional state of the enzyme. Reactivity toward NEM reveals the existence of a syncatalytic sulphydryl group in the cytosolic isozyme. Titration of cAATo with pMB at pH 8 and pH 5 confirms the existence of two exposed sulphydryl groups with a different reactivity. The results compared with those reported on the corresponding isozymes from pig and chicken heart show that syncatalytic sulphydryl groups are of general occurrence in these enzymes.

Animals↗
PubMed1995

Kinetic analysis of 75selenium uptake by mitochondria of germinating Vigna radiata of different selenium status.

Earlier studies in our laboratory demonstrated the beneficial role of Se in Vigna radiata, a Se-deficient legume, during germination, as reflected in growth-related parameters and specific uptake of 75Se. Uptake of Na2(75)SeO3, added in vitro by mitochondria isolated from seedlings germinated in control (without Se), and Se-supplemented groups (0.5, 1.0, and 2.0 ppm Se) indicated a proportional increase in the uptake with added Na2(75)SeO3, in concentrations up to 25 microM. The uptake of 75Se, increased linearly with time up to 15 min and a definite efflux followed at 30 min. The results were indicative of cooperative effects during Se transport. Kinetic analyses of the uptake of 75Se during time intervals of 15 and 30 min were carried out both in the whole mitochondria and the mitochondrial protein fractions. Graphical analyses using Lineweaver-Burk plot, Hill plot, log [v] vs log [A] and Scatchard plot confirmed the existence of negative cooperativity during 75Se uptake. Hill coefficient (nH) values were estimated to be around 0.7-0.8. Scatchard plots for 75Se uptake were biphasic, suggesting the probable presence of two classes of binding sites. The number of high and low affinity binding sites were estimated to be around 4-7 and 26-30 nmol/mg protein, respectively. Studies with mitochondrial respiratory inhibitors indicated about 10-20% of the total 75Se uptake to be energy dependent. Inhibition of 75Se uptake by about 60-70% by sulfate and sulfite (5-25 microM) implies the involvement of dicarboxylate port in Se transport. A decrease in the uptake of 75Se by 40-60% effected by CdCl2, HgCl2, mersalyl, and NEM confirmed the interaction of thiols in the process. Evidence for the regulatory nature of 75Se uptake by mitochondria of V. radiata emerges from the present study.

2,4-Dinitrophenol↗
PubMed1991

Temporal changes in tissue glutathione in response to chemical form, dose, and duration of selenium treatment. Relevance to cancer chemoprevention by selenium.

Selenium has been reported to affect glutathione (GSH) concentrations in short-term animal-feeding experiments. Given the central role that this tripeptide plays in maintaining cellular homeostasis, it was hypothesized that perturbations in glutathione metabolism induced by selenium might account for its cancer chemopreventive activity. In the present study, four experiments were conducted in which the effect of acute, short-, or long-term exposure to selenium was assessed. Selenium was provided as either sodium selenite or D,L-selenomethionine. Selenite was observed to induce a biphasic response in total liver GSH. Injected selenium caused an acute reduction in GSH, whereas short-term feeding (up to 8 wk) increased both total GSH and oxidized glutathione (GSSH), an effect that gradually diminished in magnitude with prolonged feeding. Our data suggest that such changes are unlikely to account for the chemopreventive activity of selenium for the following reasons: Perturbations in glutathione metabolism occurred only at doses of selenite that approached toxicity. These doses are higher than what would be required for producing cancer chemoprevention. The transient nature of these changes also contrasts with the need for a continuous supplementation of selenite in suppression of tumorigenesis. Furthermore, selenomethionine was found to have little activity in altering glutathione metabolism, even though it compares favorably with selenite as a cancer chemopreventive agent. Nonetheless, these findings do not discount the possibility that sulfhydryl compounds, such as glutathione, might be used to modify the toxicity and/or enhance the cancer prophylactic activity of selenium compounds.

Animals↗
PubMed2002

In vitro activation of dibromoacetonitrile to cyanide: role of xanthine oxidase.

Dibromoacetonitrile (DBAN) is a disinfection byproduct of chlorination of drinking water. Epidemiological studies indicate that it might present a potential hazard to human health. The present work provides evidence for DBAN activation to cyanide (CN(-)) by the hypoxanthine (HX)/xanthine oxidase (XO)/iron (Fe) system in vitro. Optimum conditions for the oxidation of DBAN to CN(-)were characterized. Addition of the sulfhydryl compounds glutathione, N-acetyl- L-cysteine or dithiothreitol significantly enhanced the rate of CN(-)release. A high positive correlation existed between hydroxyl free radical ((*)OH) generation and CN(-) formation. Addition of the (*)OH scavengers mannitol or dimethylthiourea to the reaction mixtures resulted in a significant decrease in the rate of DBAN oxidation. Addition of the antioxidant enzymes catalase or superoxide dismutase resulted in a significant decrease in the rate of DBAN oxidation. The iron chelator desferrioxamine significantly decreased CN(-) formation. The maximum velocity (V(max)) and Michaelis-Menten constant (K(m)) of the reaction were assessed. Allopurinol competitively inhibited the reaction, while folic acid uncompetitively inhibited the reaction. In conclusion, (*)OH generated by the HX/XO/Fe system are implicated in DBAN oxidation. The present results represent a novel pathway for DBAN activation and might be important in explaining DBAN-induced toxicity.

Acetonitriles↗
PubMed2004

Glutathione-conjugated toluene diisocyanate causes airway inflammation in sensitised mice.

Toluene diisocyanate (TDI) is a highly volatile compound that reacts readily with nucleophilic compounds, sulfhydryl groups in particular. Since the epithelial lining fluid of the airways contains high levels of the sulfhydryl, glutathione (GSH), inhalation of TDI is likely to result in the formation of GS-TDI conjugates. We therefore investigated whether GS-TDI is capable of provoking irritant and/or allergic reactions. Irritant effects of GS-TDI were studied after intratracheal administration of a range of doses of GS-TDI in saline to naive BALB/c mice. GS-TDI caused a dose-dependent increase in neutrophils in the lungs 24 h after instillation. A dose equivalent to 150 microg of TDI or lower had no effect. For provocation of allergic reactions, mice were sensitised by application of 1% TDI onto the skin on days 0 and 1, and challenged intratracheally with a sub-irritant dose of GS-TDI on day 8. GS-TDI did not induce non-specific tracheal hyperreactivity to carbachol 24 and 48 h after challenge in TDI-sensitised mice. However, it increased the numbers of neutrophils in the lungs as compared with the control mice. These findings suggest that GSH conjugation does not diminish the capacity of TDI to elicit irritant-induced inflammation in the lungs of mice at doses above 150 microg of TDI in the conjugate. Moreover, the capacity to induce allergic-specific inflammation was retained at concentrations of GS-TDI being devoid of irritant activity. However, the GS-TDI conjugate failed to induce non-specific tracheal hyperreactivity. This may be the consequence of the deposition of excess of GSH upon local dissociation of the conjugate.

Airway Resistance↗
PubMed1994

Allyl alcohol cytotoxicity in isolated rat hepatocytes: mechanism of cell death does not involve an early rise in cytosolic free calcium.

We examined the effect of a toxic concentration of allyl alcohol (0.5 mM) on intracellular calcium concentrations in isolated rat hepatocytes. An increase in phosphorylase a activity was evident in the hepatocytes after 30 min of incubation with allyl alcohol, suggesting that the toxicant may produce an early rise in cytosolic free calcium. The increase in phosphorylase a activity was not reversed by the addition of dithiothreitol (DTT), a sulfhydryl compound that reverses the events that initiate cell killing by allyl alcohol. When intracellular calcium concentrations were measured directly, using fura-2 as the calcium indicator, there was no effect of allyl alcohol on cytosolic free calcium during the first 60 min of exposure, a critical period for development of irreversible damage. Incubation with allyl alcohol did not interfere with the measurement of intracellular calcium. The increases in cytosolic free calcium produced by phenylephrine or ATP were similar to those reported by others and not affected by the presence of allyl alcohol. The results from this study demonstrate that increased cytosolic free calcium is not essential for allyl alcohol-induced cytotoxicity to isolated rat hepatocytes.

1-Propanol↗
PubMed2002

Quick measurement of protein sulfhydryls with Ellman's reagent and with 4,4'-dithiodipyridine.

Since its introduction in 1959, Ellman's reagent (5,5'-dithio-bis(2-nitrobenzoic acid)) has been the favorite reagent for spectrophotometric measurement of protein sulfhydryls. Meanwhile however, evidence has accumulated that many protein sulfhydryls give an incomplete reaction with Ellman's reagent, even during prolonged assay times. In the present study, the kinetic problem was solved by including cystamine as a "mediator" between the protein sulfhydryl and Ellman's reagent, as previously applied in an enzymatic thiol assay [9]. As an alternative, 4,4'-dithiodipyridine (DTDP) was used in place of Ellman's reagent. Due to its small size, amphiphilic nature, and lack of charge, DTDP quickly reacts with poorly accessible protein sulfhydryls, without any catalysis by cystamine. The DTDP method and the Ellman/cystamine method were both optimized for maximal sensitivity, minimal sample consumption (detection limit 0.2 nmol mL(-1), determination limit 0.6 nmol mL(-1)), and minimal assay time (5 min). In validation experiments, both methods gave identical results and the measured sulfhydryls/protein matched the expected values. Electronic supplementary material to this paper can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00216-002-1347-2.

Animals↗
PubMed2001

HPLC-based method using sample pre-column clean-up for the determination of methanethiol and ethanethiol in parenteral amino acid solutions.

A method has been developed for the chromatographic determination of methanethiol (MT) and ethanethiol (ET) as contaminants in amino acid parenteral nutrition (PN) solutions. The clean-up of the samples before chromatographic analysis was investigated by solid-phase extraction (SPE) on pre-columns filled with polyethylene powder (PE), aluminium oxide (AlOx), silica (SiOx), or polyurethane foam (PUF) as adsorbents. The thiols were more efficiently separated from the matrices by SPE on PUF pre-columns. Simultaneous derivatization and elution with DTNB (5,5'-dithiobis(2-nitrobenzoic acid)) enabled further discrimination between MT and ET by reversed-phase HPLC with spectrophotometric detection. The retention times for the derivatized MT and ET species were 12.5 and 23.0 min, respectively. Recoveries from spiked PN samples were calculated to be approximately 90%, and the MT and ET content of commercial PN solutions was determined using the methodology described. Detection limits of 15 and 10 microg L(-1) were calculated for MT and ET, respectively.

Aluminum Oxide↗
PubMed1997

Effects of thiol-modifying agents on a K(Ca2+) channel of intermediate conductance in bovine aortic endothelial cells.

Ca2+-activated K+ channels (K(Ca2+)) constitute key regulators of the endothelial cell electrophysiological response to InsP3-mobilizing agonists. Inside-out and outside-out patch clamp experiments were thus undertaken to determine if the gating properties of a voltage-insensitive K(Ca2+) channel of intermediate conductance present in bovine aortic endothelial (BAE) cells could be modified by specific sulfhydryl (SH) oxidative and/or reducing reagents. The results obtained first indicate that cytosolic application of hydrophilic oxidative reagents such as 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) (0.2 to 5 mM) or [(O-carboxyphenyl)thio]ethyl mercury sodium salt (thimerosal) (0.5 to 5 mM) reduces gradually the K(Ca2+) channel activity with no modification of the channel unitary conductance. The inhibitory action of DTNB (1 to 5 mM) or thimerosal (1 to 5 mM) was not reserved following withdrawal of the oxidative agents, but channel activity could partly be restored by the addition of the SH group reducing agents dithiothreitol (DTT) (5 mM) or reduced glutathione (GSH) (5 mM) in 53% and 50% of the inside-out experiments performed with DTNB and thimerosal respectively. Similar results were obtained using H2O2 at concentrations ranging from 500 microM to 10 mm as oxidative reagent. In contrast, the lipid soluble oxidative agent 4,4'-dithiodipyridine (4-PDS) (1 mM) appeared in inside-out experiments less potent than DTNB and thimerosal at inhibiting the K(Ca2+) channel activity, suggesting that the critical SH groups involved in channel gating are localized at the inner face of the cell membrane. This conclusion was further substantiated by a series of outside-out patch clamp experiments which showed that DTNB (5 mM) and thimerosal (5 mM) were unable to inhibit the K(Ca2+) channel activity when applied to the external surface of the excised membrane. Finally, no significant changes of the gating properties of the K(Ca2+) channel were observed in inside-out experiments where the SH group reducing agents DTT and GSH were applied immediately following membrane excision. However, the application of either GSH or DTT was found to partly restore channel activity in experiments where the K(Ca2+) channels showed significant rundown.

Animals↗
PubMed2001

A comparison of the effects of potassium citrate and sodium bicarbonate in the alkalinization of urine in homozygous cystinuria.

For many years, urine alkalinization has been one of the cornerstones in the treatment of homozygous cystinuria. Because of the relationship found between the excretion of urinary sodium and cystine, potassium citrate has emerged as the preferred sodium-free alkalizing agent. To evaluate the usefulness of potassium citrate for urine alkalization in cystinuric patients, sodium bicarbonate and potassium citrate were compared in 14 patients (10 on tiopronin treatment and four without treatment with sulfhydryl compounds). The study started with 1 week without the use of any alkalizing agents (Period 0) followed by 2 weeks with sodium bicarbonate (Period 1) and 2 weeks with potassium citrate (Period 2). Urinary pH, volume, excretion of sodium, potassium, citrate and free cystine, as well as the plasma potassium concentration, were recorded. Potassium citrate was shown to be effective as an alkalizing agent and, in this respect, not significantly different from sodium bicarbonate. Even though a normal diet was used, a significant increase in urinary sodium excretion was observed with sodium bicarbonate (Period 1). Urinary potassium and citrate excretion increased with potassium citrate (Period 2). A significant correlation was found between urinary sodium and cystine in the tio-pronin-treated patients. No significant differences in cystine excretion were recorded in Periods 0, 1 and 2. Plasma potassium was significantly higher during Period 2, but only one patient developed a mild hyperkalemia (5.0 mmol/l). The use of potassium citrate for urine alkalization in homozygous cystinuria is effective and can be recommended in the absence of severe renal impairment.

Adult↗
PubMed1997

Mercurochrom can be used for the histochemical demonstration and microphotometric quantitation of both protein thiols and protein (mixed) disulfides.

Mercurochrom [2,7-dibromo-4-(hydroxymercuri)-fluorescein disodium salt] used for staining of protein thiols in addition binds to other groups of proteins. Experimental evidence is provided that mercurochrom bound to non-thiol groups forms a 1:1 adduct with protein (mixed) disulfides. The disulfide contents of three different types of cells determined biochemically correlated with the corresponding mean integrated optical densities determined microphotometrically after mercurochrom staining of groups other than thiols. Intracellular disulfide exchange has been studied, leading to a transformation of protein mixed disulfides to protein disulfides and an equimolar loss of protein thiols. Protein mixed disulfides were generated from protein thiols using both methyl methanethiosulfonate (MMTS) and 2,2'-dihydroxy-6,6'-dinaphthyldisulfide (DDD). Loss of thiols as well as the equimolar increase of protein mixed disulfides were followed using both mercurochrom staining for thiols and for disulfides. Generation of protein mixed disulfides due to the DDD reaction was also followed by azocoupling with Fast blue B. On the basis of the observed stoichiometry between the loss of protein thiols and the quantity, increase or conversion of protein disulfides determined microphotometrically using both mercurochrom staining and DDD Fast blue B staining, we conclude that: (1) 1 mol of mercurochrom is bound per mol of protein (mixed) disulfide; and (2) the molar absorptivity of mercurochrom bound to disulfides is epsilon 520 = 34940. This study demonstrates that mercurochrom can be used for the quantitative determination of the oxidative status of protein thiols in cells.

Animals↗
PubMed1999

Effect of thiols exported by cancer cells on the stability and growth-inhibitory activity of Pt(IV) complexes.

PURPOSE: The role of thiols in the reduction of Pt(IV) antitumor agents to Pt(II) is well recognized and it is widely thought that this reaction is required for activity. The sources of extracellular thiols in cell culture have been less studied. The purpose of the present work was to determine whether the stability of Pt(IV) complexes in culture medium can be affected by thiols that are released by cancer cells. METHODS: A two-column HPLC assay with UV/visible detection was used to determine the stability of two Pt(IV) complexes in culture medium with and without cells. The kinetics of the thiol release from a human ovarian cancer cell line SK-OV-3 and a human glioblastoma cell line U-87 MG were determined by a modification of the Ellman's method. RESULTS: The stability of a Pt(IV) complex with equatorial iodo ligands, trans, cis-[Pt(en)(OAc)(2)I(2)], was dramatically lower in culture medium in the presence of cells than in fresh culture medium, whereas the half-life of the dichloro analog, trans,cis-[Pt(en)(OAc)(2)Cl(2)], was somewhat increased. Although both complexes showed similar in vitro cell-growth-inhibitory activity, trans, cis-[Pt(en)(OAc)(2)Cl(2)] required a longer incubation time than the iodo analog to reach its maximal effect. The thiol content of the culture medium in the presence of cells was measured after 2 days: the concentrations from cultures of U-87 MG and SK-OV-3 cells were 3. 6 +/- 0.1 microM and 9.3 +/- 0.1 microM respectively, compared to 0. 07 +/- 0.04 microM in fresh medium. During the rapid growth phase, the extracellular thiol content reached a maximum of 20.0 +/- 0.5 microM and 47.8 +/- 0.2 microM for U-87 MG and SK-OV-3 cells respectively. CONCLUSIONS: These findings show that the culture medium conditioned by cancer cells can influence the stabilities of certain Pt(IV) complexes in cytotoxicity studies.

Antineoplastic Agents↗
PubMed2000

Modulation of the chemical and biological properties of trans platinum complexes: monofunctional platinum complexes containing one nucleobase as potential antiviral chemotypes.

Replacement of one of the chloride leaving groups in trans-[PtCl2(NH3)(L)] by the nucleobase 9-ethylguanine gives the nucleobase cations [SP-4-2]-[PtCl(9-ethylguanine)(NH3)(L)]+ (L = NH3, 1; L = quinoline, 3), which are models for the monofunctional adduct on DNA. Displacement of Cl- in 1 and 3 by either 5'-guanosine monophosphate (5'-GMP) or N-acetyl-L-methionine (N-AcMet) showed clear kinetic preference for the sulfur (estimated half-lives of 1.5 and 4 h with N-AcMet against 7 and 17 h for 5'-GMP for 1 and 3, respectively). To further examine the kinetic preference, 1-methylcytosine (1-MeCyt) analogs were prepared, [SP-4-2]-[PtCl(1-Me-Cyt)(NH3)(L)]+ (L=NH3, 2; L=quinoline, 4). The -MeCyt compounds, 2 and 4, resulted in slower rates of substitution by both 5'-GMP and N-AcMet in comparison to 1 and 3 (estimated half-lives for N-AcMet of 5 and 13.5 h and for 5'-GMP of 6 and 14 h for 2 and 4, respectively). Interestingly in this case, however, no selectivity for the sulfur site was observed, a possible explanation being that molecular recognition across the square plane enhances the rate of reaction with 5'-GMP. The affinity of 3 towards S-donor ligands was exploited to remove zinc from the zinc-finger site of the C-terminal finger of the HIV-nucleocapsid protein, NCp7. The ability to eject zinc further suggested the biological antiviral application of [SP-4-2]-[PtCl(nucleobase)(NH3)(L)]+. A preliminary survey against HIV and herpes viruses indeed showed encouraging results with some antiviral specificity, dependent on the exact nature of the compound. The initial results suggest consideration of [SP-4-2]-[PtCl(nucleobase)(NH3)(L)]+ as a novel antiviral chemotype.

Anti-HIV Agents↗