N-acetyl-L-cysteine Pretreatment Research Articles

Protective effect of N-acetyl-L-cysteine against maneb induced oxidative and apoptotic injury in Chinese hamster V79 cells

The role of antioxidant N-acetyl-L-cysteine (NAC) in protection against cellular changes triggered by maneb during in vitro exposure was investigated in cultured Chinese hamster V79 cells. We observed high apoptotic activity and high oxidative stress induced by exposure to maneb evidenced by a statistically significant increase in lipid peroxidation (measured as TBARS – thiobarbituric acid reactive substances) as well as a decrease of glutathione (GSH) and glutathione disulfide (GSSG) ratio (GSH/GSSG). Maneb did not exhibit any effect on protein oxidation (measured by protein carbonyls content). NAC suppressed cellular changes induced by maneb in V79 cells. NAC pre-treatment prevented TBARS production and significantly decreased the number of apoptotic cells. However, protective effect of NAC on GSH and GSSG levels has been shown only in cells exposed to lower concentration of maneb (100 μM).

Fluoride‐induced genotoxicity in mouse bone marrow cells: effect of buthionine sulfoximine and N‐acetyl‐ l‐cysteine

A significant level of reactive oxygen species generation was observed in sodium fluoride (NaF) treated mouse bone marrow cells (BMCs). Reduced glutathione (GSH) as a free radical scavenger could be an important determining factor in F-induced genotoxicity. We therefore attempted to monitor GSH to understand the mechanism of NaF-induced genotoxicity. NaF was injected intra-peritoneally in normal, buthionine sulfoximine (BSO) or N-acetyl-L-cysteine (NAC) treated mice (n = 5). After 13 h of NaF-treatment BMCs were collected to harvest them at the same divisional cycle and processed for analysis of cell cycle, induction of apoptosis and chromosomal aberrations (CAs). Level of GSH was also measured concomitantly. NaF induced significant CAs in all treatment groups except at 2.5 mg NaF kg(-1) body weight. BSO-treatment alone induced significantly high frequency of CAs. BSO treatment prior to injection of 2.5-7.5 mg NaF kg(-1) b.w. was found to increase the frequency of CAs, significantly when compared with the positive control group, but the level was not significant in case of higher doses of NaF treatment (15 and 30 mg kg(-1) b.w.). NaF-treated cells also showed a higher population of Annexin-V positive cells. NAC pre-treatment significantly reduced the extent of NaF-induced CAs, which clearly indicates the involvement of GSH in the NaF response. However, further study is warranted to evaluate the low synergistic effect of BSO on higher doses of NaF-induced CAs.

Roles of reactive oxygen species and mitochondria in cadmium-induced injury of liver cells

The roles of reactive oxygen species (ROS) and mitochondrial damage in the cadmium (Cd)-induced injury of liver cells were studied by using N-acetyl-L-cysteine (NAC) and acetyl-L-carnitine hydrochloride (ALCAR). After exposure of experimental rats to cadmium (Cd) for 16 h, mitochondrial membrane potential (MMP), ROS production, glutathione peroxidase (GSH-Px) activity, glutathione (GSH) content, malondialdehyde (MDA) content and DNA single-strand break (DNA-SSB) were analyzed. Loss of MMP, increase of ROS production, inhibition of GSH-Px activity, elevation of GSH content, rise of MDA content and DNA-SSB level suggest the participation of ROS and mitochondrion in Cd-induced injury of liver cell. NAC pretreatment attenuated oxidative stress, reversed the decline in GSH-Px activity and reduced GSH and MDA levels significantly. However, Cd-induced loss in MMP was significantly exacerbated by NAC. For another, ALCAR did not perform as well as NAC in terms of reducing ROS production, restoring GSH-Px activity and reducing GSH content. Nevertheless, it significantly improved the recovery of MMP and reduction of MDA content. In addition, conspicuous DNA damage was observed in the samples treated with NAC or ALCAR, indicating Cd could attack DNA through other pathways. These results suggest that oxidative stress or mitochondrial impairment plays a main role in different injuries respectively.

Protective effect of N-acetyl- l-cysteine against disulfiram-induced oxidative stress and apoptosis in V79 cells

This work investigated the effect of N-acetyl- l-cysteine (NAC) on disulfiram (DSF) induced oxidative stress in Chinese hamster fibroblast cells (V79). An increase in oxidative stress induced by DSF was observed up to a 200 μM concentration. It was evidenced by a statistically significant increase of both GSH t and GSSG levels, as well as elevated protein carbonyl (PC) content. There was no increase in lipid peroxidation (measured as TBARS). DSF increased CAT activity, but did not change SOD1 and SOD2 activities. Analysis of GSH related enzymes showed that DSF significantly increased GR activity, did not change Se-dependent GPx, but statistically significantly decreased non-Se-dependent GPx activity. DSF showed also pro-apoptotic activity. NAC alone did not produce any significant changes, besides an increase of GSH t level, in any of the variables measured. However, pre-treatment of cells with NAC ameliorated DSF-induced changes. NAC pre-treatment restored the viability of DSF-treated cells evaluated by Trypan blue exclusion assay and MTT test, GSSG level, and protein carbonyl content to the control values as well as it reduced pro-apoptotic activity of DSF. The increase of CAT and GR activity was not reversed. Activity of both GPx was significantly increased compared to their values after DSF treatment. In conclusion, oxidative properties are at least partially attributable to the cellular effects of disulfiram and mechanisms induced by NAC pre-treatment may lower or even abolish the observed effects. These observations illustrate the importance of the initial cellular redox state in terms of cell response to disulfiram exposure.

Pretreatment with N‐Acetyl‐L‐Cysteine Increases Viability of C6 Glioma cells Following DNB Exposure

In previous studies, N‐acetyl‐L‐cysteine (NAC), which increases intracellular concentrations of GSH was shown to protect oligodendrocytes in culture against exposure to toxic stimuli such as organo‐ mercury. The aim of the present in vitro study was to determine if pretreatment with NAC would protect C6 Glioma cells from exposure to the three individual DNB isomers. Cytotoxicity was determined using MTT reduction for mitochondrial integrity and neutral red (NR) uptake for lysosomal integrity. C6 Glioma cells were pretreated with NAC (2μM) for 24 hours, and then exposed to one of the DNB isomers (10–250 μM) for up to 6 hours. The protective effect of NAC (2μM) on C6 Glioma cells exposed to DNB isomers was in the following order 1,2‐<1,3‐<1,4‐DNB using the MTT assay. On the other hand, using the NR assay, an equal protective effect by NAC was observed when C6 Glioma cells were exposed to the DNB isomers. Based on the results obtained with these two assays, MTT was the most sensitive in detecting changes in cell viability in this model. Our results indicate that NAC pretreatment has a concentration‐ and isomer‐dependent effect on cell viability. Studies are underway to explore the cellular mechanism(s) and DNB intermediates involved in NAC mediated protection of C6 Glioma cells from DNB toxicity with regard to, and also separate from, the glutathione detoxification process. This project was supported in‐part by 5G12‐RR008124 from NCRR to UTEP and by NIEHS/NIH grant ES011982 to RTM.

Differential Metabolomics for Quantitative Assessment of Oxidative Stress with Strenuous Exercise and Nutritional Intervention: Thiol-Specific Regulation of Cellular Metabolism with N-Acetyl-l-Cysteine Pretreatment

Despite several decades of active research, the success of large-scale clinical trials involving antioxidants remains equivocal given the complex biological interactions of reactive oxygen/nitrogen species in human health. Herein, we outline a differential metabolomics strategy by capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) to assess the efficacy of nutritional intervention to attenuate oxidative stress induced by strenuous exercise. A healthy volunteer was recruited to perform a submaximal prolonged ergometer cycling trial until volitional exhaustion with frequent blood collection over a 6 h time interval, which included pre-, during, and postexercise periods while at rest. A follow-up study was subsequently performed by the same subject after high-dose oral intake of N-acetyl-L-cysteine (NAC) prior to performing the same exercise protocol under standardized conditions. Time-dependent changes in global metabolism of filtered red blood cell lysates by CE-ESI-MS were measured to reveal a significant attenuation of cellular oxidation associated with high-dose oral NAC intake relative to a control. Untargeted metabolite profiling allowed for the identification and quantification of several putative early- and late-stage biomarkers that reflected oxidative stress inhibition due to nutritional intervention, including oxidized glutathione (GSSG), reduced glutathione (GSH), 3-methylhistidine (3-MeHis), L-carnitine (C0), O-acetyl-L-carnitine (C2), and creatine (Cre). Our work demonstrates the proof-of-principle that NAC pretreatment is effective at dampening acute episodes of oxidative stress by reversible perturbations in global metabolism that can provide deeper insight into the mechanisms of thiol-specific protein inhibition relevant to its successful translation as a prophylaxis in clinical medicine.

Protective effect of N-acetyl- l-cysteine on amyloid β-peptide-induced learning and memory deficits in mice

This study aimed to examine the effects of N-acetyl- l-cysteine (NAC) on protecting neurons function and improving learning and memory deficits in mice. Mice were intracerebroventricularly (icv) injected with the aggregated amyloid β-peptide (Aβ) to produce Alzheimer's disease (AD). Learning and memory functions in mice were examined by the step through test and the water maze performance. The results showed that the mice pretreated with NAC had significantly greater retention in the step through test and shorter latencies in the water maze performance. Biochemical studies showed the potential role of free radical toxicity and the damage of cholinergic neurons in the Aβ-treated mice. There was an increased lipid peroxidation as indicated by elevated malondehyde (MDA) and decrease of glutathione (GSH) levels. There was also an increase in acetylcholinesterase (AChE) activity and a reduction in the choline acetyltransferase (ChAT) activity and acetylcholine (ACh) levels. NAC pretreatment significantly reversed the elevated MDA, AChE and the reduced GSH, ChAT and ACh in the Aβ-model mice. The results of the present study suggest the potential usage of the neuroprotective action of NAC on AD.

N-acetylcysteine and endothelial cell injury by sulfur mustard

Understanding the underlying mechanisms of cell injury and death induced by the chemical warfare vesicant sulfur mustard (HD) will be extremely helpful in the development of effective countermeasures to this weapon of terror. We have found recently that HD induces both apoptosis and necrosis in endothelial cells (Toxicol. Appl. Pharmacol. 1996; 141: 568-583). Pretreatment of the endothelial cells for 20 h with the redox-active agent N-acetyl-L-cysteine (NAC) selectively prevented apoptotic death induced by HD. In this study, we tested the hypotheses that pretreatment with NAC acts through two different pathways to minimize endothelial injury by HD: NAC pretreatment acts via a glutathione (GSH)-dependent pathway; and NAC pretreatment acts to suppress HD-induced activation of the nuclear transcription factor NFkappaB. We used a fluorescence microscopic assay of apoptotic nuclear features to assess viability and electrophoretic mobility shift assays (EMSAs) to assess the activity of NFkappaB following exposure to HD. The cells were treated with 0-10 mM GSH for 1 h prior to and during exposure to 0 or 500 microM HD for 5-6 h. Cells were also treated with 50 mM NAC or 200 microM buthionine sulfoximine (BSO), an inhibitor of GSH synthesis, alone or in combination overnight prior to exposure to 0 or 500 microM HD for 5-6 h. Externally applied GSH up to a concentration of 5 mM had no toxic effect on the cells. Mild toxicity was associated with 10 mM GSH alone. There was a dose-related enhancement of viability when 2.5 and 5 mM GSH were present during the HD exposure. Pretreatment with BSO alone had no discernible toxicity. However, pretreatment with this inhibitor of GSH synthesis potentiated the toxicity of HD. Pretreatment with 50 mM NAC, as previously reported, provided substantial protection. Combining pretreatment with both BSO and NAC eliminated the protective effect of NAC pretreatment alone on HD injury. These observations are highly suggestive that NAC enhances endothelial survival via GSH-dependent effects and confirms and extends the work of others with different models that externally supplied GSH alone may be a fairly effective countermeasure against HD injury of endothelium. We next examined the hypothesis that HD may activate the nuclear transcription factor NFkappaB by performing EMSAs with nuclear extracts of endothelial cells following exposure to 0, 250 or 500 microM HD. This demonstrated an up to 2.5-fold increase (scanning densitometry) in activation of NFkappaB binding to its consensus sequence induced by 500 microM HD after 5 h of HD exposure. Paradoxically, treatment of the endothelial cells alone with 50 mM NAC activated NFkappaB, although HD-induced activation of NFkappaB was partially suppressed by NAC at 5 h. Factor NFkappaB is an important transcription factor for a number of cytokine genes (e.g. tumor necrosis factor, TNF), which can be activated following stress in endothelial cells. Taken together, these observations suggest that the protective effects of NAC may be mediated by enhanced GSH synthesis. The increased GSH may act to scavenge HD and also prevent oxidative activation of NFkappaB. Under some conditions, NAC may act as an oxidizing agent and thus increase NFkappaB activity. The NFkappaB-dependent gene expression may be important in inducing endothelial cell death as well as in generating a local inflammatory reaction associated with the release of endothelial-derived cytokines.

N-acetyl-L-cysteine protects SHSY5Y neuroblastoma cells from oxidative stress and cell cytotoxicity: effects on beta-amyloid secretion and tau phosphorylation.

Redox changes within neurones are increasingly being implicated as an important causative agent in brain ageing and neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD) and Alzheimer's disease (AD). Cells have developed a number of defensive mechanisms to maintain intracellular redox homeostasis, including the glutathione (GSH) system and antioxidant enzymes. Here we examine the effects of N-acetyl-L-cysteine (NAC) on beta-amyloid (A beta) secretion and tau phosphorylation in SHSY5Y neuroblastoma cells after exposure to oxidative stress inducing/cytotoxic compounds (H(2)O(2), UV light and toxic A beta peptides). A beta and tau protein are hallmark molecules in the pathology of AD while the stress factors are implicated in the aetiology of AD. The results show that H(2)O(2), UV light, A beta 1-42 and toxic A beta 25-35, but not the inactive A beta 35-25, produce a significant induction of oxidative stress and cell cytotoxicity. The effects are reversed when cells are pre-treated with 30 mM NAC. Cells exposed to H(2)O(2), UV light and A beta 25-35, but not A beta 35-25, secrete significantly higher amounts of A beta 1-40 and A beta 1-42 into the culture medium. NAC pre-treatment increased the release of A beta 1-40 compared with controls and potentiated the release of both A beta 1-40 and A beta 1-42 in A beta 25-35-treated cells. Tau phosphorylation was markedly reduced by H(2)O(2) and UV light but increased by A beta 25-35. NAC strongly lowered phospho-tau levels in the presence or absence of stress treatment.

Effects of N‐acetyl‐L‐cysteine on Teratogenicity of 5‐Fluorouracil in Mice

ABSTRACT Embryotoxicity and teratogenicity of 5‐fluorouracil (5‐FU) and modulation of its effect by N‐acetyl‐L‐cysteine (NAC) andor phorone were evaluated in mice. Pregnant ICR mice were intraperitoneally injected with 25 mglkg of 5‐FU on day 11 of gestation (vaginal plug = day 0). Pregnant mice were pretreated with NAC at dose levels of 80, 160, and 320 mg/kg injected intravenously 2 hours before dosing with 5‐FU. Pregnant mice were killed on day 17 of gestation. Fetuses were examined for external malformations, especially limb malformations. Pretreatment with 160 mg/kg and 320 mg/ kg of NAC decreased the incidence and severity of oligodactyly induced by 5‐FU. There was little difference in maternal body weight gain, fetal mortality, and fetal weight between the 5‐FU group and the 5‐FU plus NAC groups. Pretreatment with phorone, a glutathione depleting agent, at dose levels of 160 and 320 mg/kg injected intraperitoneally, 4 hours before dosing with 5‐FU, increased the incidence and severity of oligodactyly induced by 5‐FU. Cotreatment with NAC at dose levels of 160 and 320 mg/kg decreased the incidence and severity of oligodactyly induced by 5‐FU and 80 mg/kg of phorone. Cotreatment with NAC 160 mg/kg could not suppress the augmentative effect of phorone on 5‐FU teratogenicity under the severe condition, that is, the excess amount of phorone such as 320 mg/kg. These results indicate that the teratogenicity of 5‐FU is mitigated with NAC pretreatment, and also the level of endogenous glutathione is one of the factors which significantly affects teratogenicity of 5‐FU.

N-acetyl- l-cysteine attenuates thrombin-induced increases of pulmonary and airway pressures and lung lymph flow in anesthetized sheep

We examined whether N-acetyl- l-Cysteine (NAC), an antioxidant which also inhibits fibrin deposition in animal models of the microembolism syndrome, would prevent the acute thrombin-induced increases of pulmonary artery pressure (PAP), airway pressure (AWP), lung lymph flow (Q I), and transvascular plasma protein clearance (Clp) in anesthetized sheep with a lung lymph fistula. Five control sheep were infused with both bovine thrombin (60 U/kg) and tranexamic acid (TXA), an inhibitor of fibrinolysis. Five other sheep were given NAC as well as thrombin and TXA. During the first hour after initiating the thrombin infusion, NAC treatment reduced by 40% to 60% the marked thrombin-induced increase of arterial plasma thromboxane B 2 levels (average increases, during the first hour, of 1.56 ± 0.41 versus 0.60 ± 0.18 ng/mL in control and NAC treated sheep, respectively, P < .05), the twofold to threefold increases of pulmonary vascular resistance, PAP, Q I, and the 30% increase of peak AWP. N-acetyl- l-Cysteine did not modify either the 2- to 2.5-fold increase of lung lymph Clp or the 40% decrease of plasma fibrinogen concentration. We conclude that NAC pretreatment attenuates thrombin-induced thromboxane release and the associated increases of PAP, AWP, and Q I, in sheep. The precise mechanism of NAC's protective effect is unknown.