Non-protein Sulphydryl Levels Research Articles

Therapeutic efficacy of Picroliv in chronic cadmium toxicity

Cadmium (Cd), an industrial and environmental pollutant, is toxic to several tissues, most notably causing hepatotoxicity on acute administration and nephrotoxicity following chronic exposure. The therapeutic efficacy of Picroliv – a standardized fraction of Picrorhiza kurroa, was investigated in male rats treated with Cd as CdCl 2 (0.5 mg/kg, sc) 5 days/week for 24 weeks and Picroliv at two doses (6 and 12 mg/kg, p.o.) was given during the last 4 weeks. The Cd induced levels of malondialdehyde and membrane fluidity and decreased levels of non protein sulphydryls and Na +K +ATPase activity of hepatic tissue, along with liver function serum enzymes were restored to near normalcy on treatment with the higher dose of Picroliv. Enhanced excretion of urinary proteins, Cd, Ca and enzymes (lactate dehydrogenase and N-acetyl-β- d-glucosaminidase) evident at 24 weeks of Cd exposure, indicated severe renal damage. Picroliv appeared less effective in causing restoration of these urinary parameters as well as oxidative stress indices in the renal tissue. Picroliv not only reduced the accumulated levels of Cd, Zn and Ca and Cd-metallothionein in liver, but also enhanced the bile flow and biliary Cd. The morphological alterations in liver caused by Cd appeared less marked on Picroliv treatment. However, the renal morphology remained uninfluenced. Our earlier data on 18 weeks of Cd and 4 weeks of Picroliv co-treatment showed significant amelioration of both hepatic and renal manifestations of Cd. The hepatic protection by Picrilov is clearly demonstrated in this study, while marginal lowering of urinary proteins and enzymes is a positive signal of renal protective efficacy of Picroliv, which could be augmented by adopting higher doses and extended regimen.

Effect of Picroliv on cadmium induced testicular damage in rat

Ameliorative potential of Picroliv, a standardized extract of Picrorhiza kurroa on Cd induced early and advanced testicular damage was investigated in male rats. In the former experiment, the rats were administered Cd as CdCl 2 (0.5 mg/kg, s.c.) 5 days/week for 18 weeks and Picroliv at two doses (6 and 12 mg/kg, p.o.) was given for the last 4 weeks i.e. from week 15 to 18, to the Cd administered group. In the latter experiment, the Cd administration continued for 24 weeks and Picroliv was given from week 21 to 24. At 18 weeks, Cd caused alterations in oxidative stress indices like increased lipid peroxidation (MDA) and reduced levels of non protein sulphydryls (NPSH). They were found close to the control values by Picroliv treatment, suggesting its antioxidant potential. The increased levels of Zn and Ca were reduced by Picroliv, the Cd levels remained unaltered. The Cd induced testicular damage was also mitigated by Picroliv. The higher dose (12 mg/kg) being more effective than the lower dose. However, at 24 weeks of Cd exposure, the oxidative stress indicators in testis were more pronounced along with the morphological alterations. These parameters remained unaffected by Picroliv treatment. On comparative evaluation of the two studies, 18 weeks Cd exposure caused moderate testicular damage, which could be reversed significantly by Picroliv administration and correlated well with oxidative stress markers. Our results clearly demonstrate the ameliorative potential of Picroliv in Cd induced early testicular damage.

Effect of Picroliv on cadmium-induced hepatic and renal damage in the rat

The therapeutic efficacy of Picroliv--a standardized extract of Picrorhiza kurroa--was investigated in male rats exposed to CdCl2 (0.5 mg/kg, sc), 5 days/week for 18 weeks. Picroliv at two doses (6 and 12 mg/kg, po) was given to the cadmium (Cd)-administered group for the last 4 weeks (i.e., weeks 15-18). The Cd altered oxidative stress indices, such as increased lipid peroxidation and membrane fluidity, reduced levels of non-protein sulphydryls (NPSHs), and Na+K+ATPase activity in the liver and kidney were found close to the control values by Picroliv treatment, suggesting its antioxidant potential. The hepatoprotective action of Picroliv was evident by its ability to lower the Cd-induced liver function parameters--the serum enzymes, such as alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transpeptidase (GGT) and lactate dehydrogenase (LDH). Bile flow and biliary Cd also increased as a result of Picroliv's choleretic property. The Cd-induced serum urea and urinary excretion of proteins, calcium (Ca), Cd and enzymes, such as N-acetyl-beta-D-glucosaminidase (NAG) and LDH, were less marked on Picroliv treatment, indicating recovery from nephrotoxicity. Organ uptake of Cd and essential metals by Cd exposure was reduced on Picroliv treatment. Cd-induced hepatic metallothionein (MT) was lowered by Picroliv, whereas renal MT was unaltered. Cd-induced hepatic damage was also minimized. However, the renal morphological changes were marginally protected by Picroliv. The 12-mg Picroliv dose was more effective than the 6-mg dose in causing amelioration of the above parameters. This study has provided clear evidence for the hepato- and renal protective efficacy of Picroliv against experimental Cd toxicity.

Prevention of acute cadmium toxicity by Picroliv

The potential of Picroliv, a herbal extract against acute cadmium (Cd) intoxication, was evaluated in male rats. Biochemical and histopathological profile in rats pretreated with Picroliv (12 mg/kg, oral) followed by a single dose of Cd as cadmium chloride (CdCl2) (3 mg/kg, ip) revealed marked suppression of oxidative stress in liver and testes. The Cd-induced enhanced levels of lipid peroxidation, membrane fluidity and reduced levels of nonprotein sulphydryls and Na(+)K(+)ATPase were significantly restored to near normal by Picroliv pretreatment. In addition, the Cd-induced serum levels of glutamate oxaloacetate transaminase, glutamate pyruvate transaminase, gamma glutamyl transpeptidase and lactate dehydrogenase were restored to near basal levels. Hepatic and testicular histopathological damage was also minimized. The results strongly suggest definite hepato- and testicular protection by Picroliv. The antioxidant potential of the herbal extract in the major part, and not its chelating property, seems to be responsible for its ameliorative action.

The role of glutathione S-transferase- and cytochrome P450-dependent metabolism in the olfactory toxicity of methyl iodide in the rat

The aim of this study was to investigate the role of metabolic activation in the olfactory toxicity of methyl iodide (MeI). Adult male rats were exposed via nose-only inhalation to 100 ppm MeI for 0-6 h, and non-protein sulphydryl (NP-SH) concentrations determined in selected tissues. Depletion of NP-SH occurred in all tissues, but was most marked and rapid in the respiratory epithelium of the nasal cavity and the kidney. Olfactory, lung and liver NP-SH levels were affected to a lesser extent, and those of the brain declined by only 20-30% over the whole time course. In order to modulate glutathione (GSH) status, animals were pre-treated with (1) phorone plus L-buthionine sulphoximine (BSO), which depleted NP-SH levels in all the tissues examined, or (2) the isopropyl ester of GSH (IP-GSH), which was shown to replenish NP-SH concentrations in all tissues except the liver of animals previously administered phorone. When animals were pre-treated with phorone plus BSO and then exposed to 100 ppm MeI for 2 h, there was a potentiation of the toxicity of MeI as judged by the clinical observations on the animals. In contrast, treatment with IP-GSH prior to and during exposure to MeI for 4 h afforded a marked protection to the olfactory epithelium. In order to inhibit cytochromes P450, animals were pre-treated with cobalt protoporphyrin IX. This decreased hepatic cytochrome P450 concentrations by > 90%, but when animals were then exposed to 100 ppm MeI for 4 h there was no effect on the severity of the olfactory lesion. These results indicate that conjugation of MeI with GSH is a detoxification rather than an activation pathway. Also, there is no major role for cytochrome P450-dependent oxidation in the development of the olfactory lesion.

The role of non-protein sulphydryls in the protective effects of antioxidants against ethanol-induced vascular permeability changes in the rat stomach

Increased vascular permeability has been reported to preceed the development of ethanol-induced gastric lesions. Both generation of oxygen-derived free radicals and depletion of non-protein sulphydryls may be involved in the ethanol-induced vascular permeability. Thus, this study aimed to examine the effect of antioxidants, allopurinol and dimethylsulphoxide, and a sulphydryl blocker, N-ethylmaleimide, on ethanol-induced vascular permeability changes and to evaluate the possible interactions between antioxidants and endogenous sulphydryls. Extravasation of intravenously administered Evans blue into the stomach of rats following 30 min exposure to ethanol was used as an indicator of vascular permeability. The glandular non-protein sulphydryl and extravasated Evans blue were determined spectrophotometrically. Increased vascular permeability and a significant depletion of non-protein sulphydryl contents of the gastric mucosa were observed following 30 min exposure to 50% ethanol. Treatment with N-ethylmaleimide (50 mg/kg subcutaneously) caused enhancement of ethanol-induced vascular permeability and further depletion of non-protein sulphydryls. Intraperitoneal pretreatment with either allopurinol (12.5-50 mg/kg) or dimethylsulphoxide (20-40 mg/kg) attenuated ethanol-induced vascular permeability changes and restored the non-protein sulphydryl levels towards control. In contrast, treatment with N-ethylmaleimide before allopurinol (50 mg/kg) or dimethylsulphoxide (40 mg/kg) reduced the protective effect of both and are also associated with corresponding depletion of non-protein sulphydryl contents. These results suggest that oxygen-derived free radicals may be involved in the pathogenesis of ethanol-induced vascular permeability changes and endogenous sulphydryls may facilitate and mediate beneficial effects of antioxidants.

Extracellular biotransformation potential in mouse airways

The aims of this study were to determine if freshly isolated bronchiolar Clara cells retained biotransformation potential and whether components of phase 1 and/or phase 2 metabolism were present in the extracellular lining fluid of the lung airways. Approximately 550 μl of lavage fluid was obtained from the mouse lung, which had been totally perfused of blood in order to facilitate the isolation of Clara cells from the same animal. Samples of acellular lavage fluid and aliquots of purified Clara cells were frozen at — 20°C prior to analysis. Minimum numbers of cells and lavage fluid volumes, pooled from 2 CD-1 mice, were assayed for ethoxycoumarin- O-deethylase, NADPH-dependent cytochrome c reductase, glutathione- S-transferase(s) and non-protein sulphydryls (mostly reduced glutathione). Isolated Clara cells retained a high activity/level of all these parameters confirming their functional status for subsequent studies of xenobiotic metabolism in vitro. Acellular lavage fluid, from all the healthy animals, also contained mono-oxygenase, reductase and transferase activities and high non-protein sulphydryl levels suggesting that phase 1 and 2 reactions with xenobiotics could take place in the extracellular environment of the lung. Clara cells are known to undergo apocrine secretion and this removal of the apical cap containing secretory granules and smooth endoplasmic reticulum could account for the airway biotransformation potential that was observed.

Changes in the Nasal Epithelium of Rats Exposed by Inhalation to Mixtures of Formaldehyde, Acetaldehyde, and Acrolein

Formaldehyde, acetaldehyde, and acrolein are well-known upper respiratory tract irritants and occur simultaneously as pollutants in many indoor and outdoor environments. The upper respiratory tract, and especially the nose, is the prime target for inhaled aldehydes. To study possible additive or interactive effects on the nasal epithelium we carried out 1- and 3-day inhalation studies (6 hr/day) with formaldehyde (1.0, 3.2, and 6.4 ppm), acetaldehyde (750 and 1500 ppm), acrolein (0.25, 0.67, and 1.40 ppm), or mixtures of these aldehydes, using male Wistar rats and exposure concentrations varying from clearly nontoxic to toxic. The (mixtures of) aldehydes were studied for histopathological and biochemical changes in the respiratory and olfactory epithelium of the nose. In addition, cell proliferation was determined by incorporation of bromodeoxyuridine and proliferating cell nuclear antigen expression. Effects were primarily observed after 3 days of exposure. Histopathological changes and cell proliferation of the nasal epithelium induced by mixtures of the three aldehydes appeared to be more severe and more extensive in both the respiratory and the olfactory part of the nose than those observed after exposure to the individual aldehydes at comparable exposure levels. As far as nasal histopathological changes and cell proliferation are concerned neither dose addition nor potentiating interactions occurred at no-toxic-effect levels, except for a possible potentiating effect of acetaldehyde at noneffect levels. The results did not indicate a major role for aldehyde dehydrogenases in the biotransformation of the aldehydes studied. Activities of glutathione S-transferase and glutathione reductase after 3 days of exposure to acrolein, alone or in combination with formaldehyde and acetaldehyde, were depressed whereas the glutathione peroxidase activity was elevated. No decrease of nonprotein sulphydryl levels were observed. These findings suggest that, for no-toxic-effect levels, combined exposure to these aldehydes with the same target organ (nose) and exerting the same type of adverse effect (nasal cytotoxicity), but partly with different target sites (different regions of the nasal mucosa), is not associated with a greater hazard than that associated with exposure to the individual chemicals.

Thiol levels in rat bronchio-alveolar lavage fluid after administration of cysteine esters.

1. The intraperitoneal administration of cysteine, N-acetylcysteine, the methyl, isopropyl, cyclo pentyl, neo pentyl, cyclo hexyl and tertiary butyl esters of cysteine and of cystine dimethyl ester increased the levels of total non-protein sulphydryls and cysteine in the bronchioalveolar lavage fluid and plasma of rats. In all cases the non-protein sulphydryl levels reflected the increased cysteine levels. 2. Cysteine, N-acetylcysteine, the cysteine esters and cystine dimethyl ester raised the levels of non-protein sulphydryls and hence cysteine in the bronchioalveolar lining fluid as follows: CIPE > CCPE > CME > CDME > CneoPE > CCHE > Nac > CySH > CTBE. 3. Plasma levels of NPSH were increased as follows: Nac > CySH > CCPE > CCHE > CneoPE > CIPE > CME > CDME > CTBE. 4. All except CTBE have been shown to protect against the lethal effects of inhaled perfluoroisobutene, a pyrolysis product of polytetrafluoroethene which induces a fulminating pulmonary oedema. 5. This study showed that by raising the levels of thiols in the bronchioalveolar lavage fluid (BALF), the epithelial cells lining the bronchiolar, alveolar regions of the lung could be protected against inhaled toxicants. 6. It is proposed that increased thiol levels in the BALF may contribute to the overall protection induced by these compounds by reacting with inhaled electrophiles to prevent or reduce damage to tissue in close proximity to the airways.

The effect of 2-substituted thiazolidine-4( R)-carboxylic acids on non-protein sulphydryl levels and sulphurtransferase activities in mouse liver and brain

2-Substituted thiazolidine-4( R)-carboxylic acids (TD) were found to increase the concentration of non-protein sulphydryls (NPSH) and the activity of rhodanese (thiosulphate sulphurtransferase, EC 2.8.1.1) and 3-mercaptopyruvate sulphurtransferase (EC 2.8.1.2) in mouse liver. These properties suggest TDs are potentially hepatoprotective compounds. However TDs also cause depletion of NPSH in the mouse brain and this may be the reason for their toxic side effects on the central nervous system.

Cysteine isopropylester protects against paracetamol-induced toxicity

Cysteine isopropylester (CIPE), a novel ester of cysteine, has been synthesized in order to evaluate its potential as a chemoprotectant. The increased lipophilicity of the ester relative to cysteine should facilitate its entry into cells where, following hydrolysis, it should act as an intracellular source of cysteine or be utilized for the synthesis of glutathione so protecting the cell against various types of chemical insult. In this study, we evaluate the ability of CIPE to protect against paracetamol-induced hepatotoxicity in mice. When administered to mice, CIPE produced a rapid but transient elevation of levels of non-protein sulphydryls (NPSH) in liver, lung, kidney and spleen. The greatest increase in NPSH was seen in the lung, but after 60 min all NPSH values had returned to control levels, demonstrating the capacity of the mouse to rapidly metabolize both CIPE and cysteine. In mice pretreated with benzo( a)pyrene, CIPE protected against paracetamol-induced toxicity as measured by the prevention of mortality, the fall in hepatic NPSH and the decreased elevation of serum transaminases. CIPE (1.5mmol/kg) appeared as effective as N-acetylcysteine (1.5 mmol/kg). Higher doses of CIPE (3.0 mmol/kg) alone were toxic to mice induced with benzo( a)pyrene but not to control or phenobarbitone-induced mice. The mechanism of this increased toxicity is unclear. CIPE has a short in vivo half life but was capable of protecting against paracetamol-induced toxicity. The potential of CIPE and other related cysteine esters to act as chemoprotectants merits further investigation.

Protection of rats against the effects of α-naphthylthiourea (ANTU) by elevation of non-protein sulphydryl levels

We have investigated the influence of the elevation of pulmonary glutathione (GSH) levels on the toxicity of the rodenticide α-naphthylthiourea (ANTU) to rat lung. Administration of phorone (diisopropylidene acetone; 200 mg/kg i.p.) caused an initial depletion of both pulmonary and hepatic GSH followed after 48 hr by a marked elevation in both tissues, due most probably to a compensatory rebound synthesis. In control rats, ANTU produced a dose-dependent lethality, hydrothorax and loss of ability of lung tissue to accumulate adenosine and spermidine (markers of endothelial and epithelial cell function, respectively). These effects were prevented or markedly ameliorated when ANTU was given 48 hr after pretreatment with phorone. The mechanism of the protection by phorone pretreatment against ANTU-induced pulmonary toxicity is unclear. It may be due, in part, to elevated GSH levels in pulmonary endothelial cells and, in addition, to increased detoxification of ANTU in the liver, resulting in a decreased availability to the lung.

The Oxygen Effect: Variation of theK-value and Lifetimes of O2-dependent Damage in Some Glutathione-deficient Mutants ofEscherichia Coli

Radiosensitization by various concentrations of O2 has been studied in an Escherichia coli K-12 wild-type strain and some derived glutathione (GSH)-deficient mutants using 60Co gamma-irradiation. The maximum oxygen enhancement ratio (OER) and the K-value, the O2 concentration that produced half the maximum O2 effect, were found to depend on the GSH biosynthetic capacity of the strains. For the GSH+ wild-type strain, AB1157, and the GSH- mutant, 830, which is deficient in glutathione synthetase, the final enzyme in the GSH biosynthetic pathway, the maximum OERs were both about 3.9 and the K-values were 0.53% and 0.24% O2, respectively. On the other hand, the maximum OERs for two GSH- mutants, 7 and 821, both deficient in gamma-glutamylcysteine synthetase, the penultimate enzyme in the GSH biosynthetic pathway, were about 2.7 and the K-values were about 0.06% O2 for both. The fast chemical repair of O2-dependent damage in these strains was measured using a fast mixing and irradiation method, the gas explosion technique. The chemical repair rates in the various E. coli strains varied approximately in proportion to the O2 K-values, and both the rates of chemical repair and the K-values correlated approximately with the levels of non-protein sulphydryls in the various strains.

Radiosensitization by Misonidazole, Pimonidazole and Azomycin and Intracellular Uptake in Human Tumour Cell Lines

The radiosensitization of two human tumour in vitro cell lines, HT-1080 and LoVo, has been compared with that of the Chinese hamster cell line V79-379A. Although the two human tumour cell lines were more radiosensitive than the V79 cell line sensitizer, enhancement ratios for misonidazole, pimonidazole and azomycin were similar (relative to extracellular concentration) for all three cell lines. Average intracellular concentrations of radiosensitizer were measured by high-performance liquid chromatography. In all three cell lines the uptake of misonidazole and azomycin was extremely rapid whereas that of pimonidazole was initially much slower before reaching a plateau. The ratios of intracellular concentration of radiosensitizer to extracellular concentration (Ci to Ce) for misonidazole were 0.8 (HT-1080) and 0.7 (LoVo and V79); for azomycin 0.9 (HT-1080 and LoVo) and 0.8 (V79). In contrast Ci/Ce for pimonidazole varied with cell line, the values being 1.8 (LoVo), 2.6 (HT-1080) and 3.3 (V79). Intracellular amounts of non-protein sulphydryl (NPSH) varied between cell lines by about a factor of three. However, when the average cell volume was taken into consideration the concentrations of NPSH were very similar, being 4.2 (HT-1080), 5.6 (LoVo) and 5.7 (V79) mmol dm-3. NPSH levels expressed as nmol per mg protein were also similar.

Differences in naphthalene-induced toxicity in the mouse and rat

Following the intraperitoneal administration of naphthalene (200 mg/kg) to mice, the lung, in comparison with other organs, was selectively damaged. Histological examination of the lungs showed that it was the non-ciliated, bronchiolar epithelial cells (Clara cells) which were damaged. At higher doses (400 mg/kg and 600 mg/kg, i.p.), there was also damage to the cells in the proximal tubules of the kidney. In contrast to the effect in mice, doses of naphthalene as high as 1600 mg/kg (i.p.) caused no detectable pulmonary or renal damage in the rat. This difference in toxicity between the mouse and rat was reflected by the ability of naphthalene to more severely deplete the non-protein sulphydryls in the mouse lung and kidney than in those organs in the rat. In order to investigate the species difference in toxicity, the metabolism of naphthalene by lung and liver microsomes of the mouse and rat was studied. In all cases, naphthalene was metabolised to a covalently bound product(s) and to two major methanol-soluble products, which co-chromatographed with 1-naphthol and 1,2-dihydro-1,2-dihydroxynaphthalene. However, both the covalent binding and metabolism were approximately 10-fold greater in microsomes prepared from mouse lung compared with those from the rat. This observation may in part explain the difference in toxicity of naphthalene to the mouse and rat lung. As 1-naphthol is a major metabolite of naphthalene and previous work had suggested that most of the microsomal catalysed binding of naphthalene was due to further oxidation of 1-naphthol, the role of 1-naphthol in mediating the naphthalene-induced toxicity was investigated. In neither the mouse nor the rat did 1-naphthol cause a depletion of non-protein sulphydryl levels or tissue damage in the liver, lung or kidney. Thus the toxicity of naphthalene does not appear to be mediated via 1-naphthol.

The effect of reducing intracellular non-protein sulphydryl levels on the toxicity and sensitization of platinum co-ordination complexes in vitro

The effect of reducing intracellular non-protein sulphydryl levels on the toxicity and sensitization of platinum co-ordination complexes in vitro

Investigations into the mechanism of coumarin-induced hepatotoxicity in the rat.

The administration of single doses of coumarin to the rat was found to produce hepatic centrilobular necrosis and also to depress a number of hepatic enzyme activities within 24 h. Coumarin-induced liver damage was diminished by pretreatment with cobaltous chloride but potentiated by the administration of diethyl maleate. Hepatic reduced non-protein sulphydryl levels were rapidly depleted following coumarin treatment whereas urinary mercapturic acid excretion was enhanced suggesting the formation of a coumarin metabolite or metabolites hitherto undetected in this species. In in vitro studies [3-14C]coumarin was converted by rat hepatic microsomes to reactive intermediates which became bound covalently to microsomal proteins. Additional studies established that the formation of reactive metabolites was a cytochrome P-450 dependent process and that macromolecular binding could be inhibited by sulphydryl compounds (including reduced glutathione) and hepatic cytosol fractions. These results demonstrate that coumarin-induced hepatotoxicity in the rat is likely to be mediated via one or more reactive metabolites generated by cytochrome P-450 dependent enzymes and that reduced glutathione and other thiol agents constitute a detoxification pathway.

The state of sulphydryl groups in normal and cataractous human lenses

The levels of non-protein sulphydryls, protein sulphydryls and protein-bound sulphydryls were measured in the separated nuclei and cortices from individual normal and cataractous lenses. As the colour of the lens nucleus increases, there is a progressive decrease in the levels of non-protein sulphydryl and protein sulphydryl. In the nuclei of advanced cataractous lenses, these decrease to 10% and 3%, respectively, of the levels found in the normal nucleus. Similar, but smaller, changes take place in the cortex. The level of protein-bound sulphydryl increases in the early stages of cataract formation but thereafter remains constant in both the cortex and nucleus. In the advanced cataractous lens, the protein bound sulphydryl accounts for only about 2% of the loss of protein sulphydryl. There appear to be no intermolecular disulphide bonds in the urea-soluble proteins of the lens but some may be present in the urea-insoluble proteins.