Preventive Effect Of N-acetylcysteine Research Articles

Cigarette smoke increases susceptibility of alveolar macrophages to SARS-CoV-2 infection through inducing reactive oxygen species-upregulated angiotensin-converting enzyme 2 expression

Alveolar macrophages (AMs) are the drivers of pulmonary cytokine storm in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. This study aimed to investigate clinical–regulatory factors for the entrance protein of SARS-CoV-2, angiotensin–converting enzyme 2 (ACE2) in AMs. Human AMs were collected from 56 patients using bronchoalveolar lavage. ACE2 expression in AMs was positively correlated with smoking pack-year (Spearman’s r = 0.347, P = 0.038). In multivariate analysis, current smoking was associated with increased ACE2 in AMs (β-coefficient: 0.791, 95% CI 0.019–1.562, P = 0.045). In vitro study, ex-vivo human AMs with higher ACE2 were more susceptible to SARS-CoV-2 pseudovirus (CoV-2 PsV). Treating human AMs using cigarette smoking extract (CSE) increases the ACE2 and susceptibility to CoV-2 PsV. CSE did not significantly increase the ACE2 in AMs of reactive oxygen species (ROS) deficient Cybb–/– mice; however, exogenous ROS increased the ACE2 in Cybb–/– AMs. N-acetylcysteine (NAC) decreases ACE2 by suppressing intracellular ROS in human AMs. In conclusion, cigarette smoking increases the susceptibility to SARS-CoV-2 by increasing ROS–induced ACE2 expression of AMs. Further investigation into the preventive effect of NAC on the pulmonary complications of COVID-19 is required.

Epibrassinolide Triggers Apoptotic Cell Death in SK-N-AS Neuroblastoma Cells by Targeting GSK3β in a ROS Generation-Dependent Way

Objective: Epibrassinolide (EBR), a biologically active member of the brassinosteroids plant hormone family, has been recently indicated as an apoptotic inducer in various cancer cells without affecting non-tumor cell proliferation. Glycogen synthase kinase 3β (GSK3β) was the first identified molecule that acts as a critical mediator of glycogen metabolism and insulin signaling mechanism. GSK3β has been described as an essential factor for tumor progression by phosphorylating and inactivating the pro-apoptotic family member of the Bcl-2 family, Bax. It was recently shown to regulate cell division, differentiation, and adhesion. Materials and Methods: To investigate the relative cell viability affected by EBR treatment and the preventive effect of N-acetyl cysteine (NAC) we performed MTT assay and FACS analysis, respectively. Colony formation and soft agar techniques were used to understand the inhibitory effect of EBR on colony formation and diameters. Annexin V-PI analysis by flow cytometry was performed for the measurement of the apoptotic cell percentages. Fluorescence microscopy was performed for the determination of mitochondria membrane potential following DiOC6 staining. The expression profiles of apoptotic proteins, as well as GSK3β and β-catenin were investigated by immunoblotting. Results: Our results indicated that EBR induced mitochodria-mediated apoptosis by inducing ROS generation which can be prevented by NAC, a reactive oxgen species scavenger. EBR-induced apoptosis can influence the inhibitory phosphorylation of GSK3β by Ser9 and prevents the translocation of the down-stream target, β-catenin. Conclusion: This study evaluated EBR as a potential apoptotic inducer in neuroblastoma cell line SK-N-AS and investigated the GSK3β involvement.

Immunomodulatory and anti-inflammatory effects of N-acetylcysteine in ovalbumin-sensitized rats

BackgroundPro-inflammatory cytokines such as interleukin-5 (IL-5) and tumor necrosis factor-alpha (TNF-α) as well as immunoglobulin-E (IgE) appear to play a role in asthma. N-acetylcysteine (NAC), an antioxidant, might have clinical benefits in asthma prevention. The possible preventive effects of NAC against experimentally induced asthma in rats are investigated. The rats were allocated into five groups: a normal control, asthma control, a standard dexamethasone (DEXA, 1 mg/kg, orally) group, and two NAC groups (300 and 500 mg/kg, orally, respectively). Ovalbumin (OVA) sensitization was used to trigger asthma, which was then followed by an intra-nasal challenge. Test gents were administrated for 14 days before the challenge and during the three challenge days (20, 21, and 22). The tidal volume (TV) and peak expiratory flow rate (PEFR) as respiratory functions were determined. The pro-inflammatory cytokines as IL-5 and TNF-α were evaluated in lung homogenate. Serum IgE and absolute eosinophil count (AEC) in bronchoalveolar lavage fluid (BALF) were measured. In addition, the oxidative markers in lung tissue and nitrosative marker in BALF were assessed; finally, lungs were isolated for histopathological study.ResultsNAC restored lung functions, inhibited the asthma-dependent increase in TNF-α, IL-5, IgE, AEC, nitric oxide, and malondialdehyde levels. NAC further re-established lung glutathione content and superoxide dismutase activity, resulting in milder overall lung pathology.ConclusionsExperimental bronchial asthma may be protected by NAC. The anti-asthmatic potential of NAC may be explained by its suppressant influence on IgE antibody formation, pro-inflammatory cytokines production, eosinophil infiltration, and oxidative stress.

Sex influences in the preventive effects of N-acetylcysteine in a two-hit animal model of schizophrenia.

Schizophrenia (SCZ) is a neurodevelopmental disorder influenced by patient sex. Mechanisms underlying sex differences in SCZ remain unknown. A two-hit model of SCZ combines the exposure to perinatal infection (first-hit) with peripubertal unpredictable stress (PUS, second-hit). N-acetylcysteine (NAC) has been tested in SCZ because of the involvement of glutathione mechanisms in its neurobiology. We aim to investigate whether NAC administration to peripubertal rats of both sexes could prevent behavioral and neurochemical changes induced by the two-hit model. Wistar rats were exposed to polyinosinic:polycytidylic acid (a viral mimetic) or saline on postnatal days (PND) 5-7. On PND30-59 they received saline or NAC 220 mg/kg and between PND40-48 were subjected to PUS or left undisturbed. On PND60 behavioral and oxidative alterations were evaluated in the prefrontal cortex (PFC) and striatum. Mechanisms of hippocampal memory regulation such as immune expression of G protein-coupled estrogen receptor 1 (GPER), α7-nAChR and parvalbumin were also evaluated. NAC prevented sensorimotor gating deficits only in females, while it prevented alterations in social interaction, working memory and locomotor activity in both sexes. Again, in rats of both sexes, NAC prevented the following neurochemical alterations: glutathione (GSH) and nitrite levels in the PFC and lipid peroxidation in the PFC and striatum. Striatal oxidative alterations in GSH and nitrite were observed in females and prevented by NAC. Two-hit induced hippocampal alterations in females, namely expression of GPER-1, α7-nAChR and parvalbumin, were prevented by NAC. Our results highlights the influences of sex in NAC preventive effects in rats exposed to a two-hit schizophrenia model.

Protective effect of N-acetylcysteine against cisplatin ototoxicity in rats: a study with hearing tests and scanning electron microscopy

IntroductionOtotoxicity is a health problem appearing after powerful treatments in serious health conditions. It is sometimes inevitable when treatment of the serious disease is required. Cisplatin is an antineoplastic agent which was investigated previously to reveal increased nitrogen and reactive oxygen radicals that damages hair cells, resulting in ototoxicity. N-acetylcysteine, previously shown to decrease ototoxicity caused by different agents, is known to be a powerful in vitro antioxidant. Probably N-acetylcysteine, in addition to its antioxidant effect, blocks a cascade where reactive oxygen species result in apoptosis in the cochlea. ObjectivesThe possible preventive effect of N-acetylcysteine in cisplatin ototoxicity was studied with auditory brain stem responses, otoacoustic emissions, and histopathological investigation of the cochlea in a scanning electron microscopy. MethodsThis study was conducted on 21 Wistar Albino rats in four groups. 1mL/kg/day three times in total intraperitoneal (i.p.) Saline (n=5), 500mg/kg/day i.p. three times in total N-acetylcysteine (n=5), i.p. 15mg/kg cisplatin alone (single dose) (n=5) and i.p. 15mg/kg cisplatin plus 500mg/kg/day N-acetylcysteine (n=6) were administered. The rats were anesthetized to study the hearing tests before and after the experiment. The rats were sacrificed to investigate the cochleas by scanning electron microscopy. ResultsAuditory brain stem responses and otoacoustic emissions values were attenuated in the cisplatin group. The group that received N-acetylcysteine in addition to cisplatin had better auditory brain stem responses thresholds and otoacoustic emissions. The samples obtained from the cisplatin group showed surface irregularities, degeneration areas, and total or partial severe stereocilia losses. The changes were milder in the cisplatin+N-acetylcysteine group. ConclusionCisplatin ototoxicity can be detected by auditory brain stem responses and otoacoustic emissions testing in rats. N-acetylcysteine may protect the cochlear cells from histopathological changes. We concluded that N-acetylcysteine given 4h after cisplatin injection has a potential otoprotective effect against cisplatin ototoxicity. which suggests it could be used in clinical trials.

Effects of particulate matter from straw burning on lung fibrosis in mice

ObjectiveTo investigate the impacts of particulate matter 2.5 (PM2.5) from straw burning on the acute exacerbation of lung fibrosis in mice and the preventive effects of N-acetylcysteine (NAC). MethodsThe composition, particle size, and 30-min concentration change in an exposure system of the PM2.5 from straw-burning were determined. Forty C57BL male mice were equally randomized to two groups: bleomycin (BLM)-induced lung fibrosis with an exposure to air (BLM+air) and BLM+PM2.5 groups. On day 7 after receiving intratracheal injection of BLM, mice were exposed to air or PM2.5 in an exposure system for 30min twice daily and then sacrificed after one-week or four-week exposure (10 mice/group). Mouse survival, lung histopathology, macrophage accumulation in the lung, and pro-inflammatory cytokine levels in alveolar lavage fluid (ALF) were determined. ResultsPM2.5 from straw burning were mainly composed of organic matter (74.1%); 10.92% of the inorganic matter of the PM2.5 were chloride ion; 4.64% were potassium ion; other components were sulfate, nitrate, and nitrite. Particle size was 10nm–2μm. Histopathology revealed a greater extent of inflammatory cell infiltration in the lung, widened alveolar septum, and lung fibrosis in the BLM+PM2.5 group than in the BLM+air group and a greater extent of those adverse effects after four-week than after one-week exposure to PM2.5. The BLM+PM2.5 group also showed macrophages containing particular matter and increased pulmonary collagen deposition as the exposure to PM2.5 increased. Interleukin (IL)-6 and TNF-α levels in ALF were significantly higher in the BLM+PM2.5 group than in the BLM+air group (P<0.05) and significantly higher after four-week exposure than after one-week exposure to PM2.5 (P<0.05). TGF-β levels in ALF after four-week exposure were significantly higher in the BLM+PM2.5 group than in the BLM+air group (P<0.05). The levels of IL-6, TNF-α, and TGF-β in peripheral serum were not significantly different in the BLM+PM2.5 and BLM+air groups. Lung hydroxyproline contents increased as the exposure to PM2.5 increased and were significantly higher after four-week than after one-week exposure (P=0.019). Exposure to PM2.5 did not affect the survival of normal mice (100%) but reduced the survival of mice with BLM-induced IPF (30%), whereas NAC extended the survival (70%, vs. BLM+PM2.5, P=0.032). ConclusionExposure of mice with BLM-induced IPF to PM2.5 from straw burning exacerbated lung inflammation and fibrosis and increased mortality; NAC increased the mouse survival, indicating protective effects.

Potential protective effect of N-acetyl cysteine in acoustic trauma: An experimental study using scanning electron microscopy.

Oxidative stress has been associated with pathological processes involved in acoustic trauma. In this prospective experimental study, we investigated the potential preventive effect of N-acetyl cysteine (NAC) in rats exposed to acoustic trauma (AT). Light microscopic and scanning electron microscopic (SEM) evaluations were performed. Healthy Wistar albino rats (n = 18) were divided into 3 groups: group 1 (control group, n = 6), group 2 (acoustic trauma group, n = 6), and group 3 (AT+NAC group, n = 6). The rats in group 2 were exposed to AT. The rats in group 3 received NAC at a dose of 100 mg/kg/day by gavage for 7 days, and then 10 min after the 7th-day dose, they were exposed to AT. From light and scanning electron microscopy evaluations in the control group, the cochlear structure and epithelium were normal. In group 2 (AT group), extensive hair cell loss was observed in the cochlea by light microscopy evaluation. In the SEM evaluation, various epithelial damage and loss of stereocilia were also observed. In group 3 (AT+NAC group), decreased damage with preserved cochlear structures was seen by light microscopy. In the SEM evaluation, although stereocilia loss was also seen, nearly normal cell structures and vertical and symmetrical alignment of stereocilia structures were observed compared to the AT group. NAC reduced cochlear damage due to acoustic trauma. Because NAC has antioxidant capacity, AT mat have caused an increase in free radicals and death of outer hair cells. NAC is an antioxidant agent and it prevented cochlear damage due to AT in rats.

N-acetylcysteine Prevents Gentamicin Ototoxicity in a Rat Model.

The possible preventive effect of N-acetylcysteine (NAC) in gentamicin ototoxicity was studied with auditory brain stem responses (ABRs), otoacoustic emissions (OAEs), and histopathological investigation of the cochlea. This study is conducted on 36 rats in three groups. Gentamicin, gentamicin plus NAC, and NAC alone were intraperitoneally administered for 15 days. The rats were sacrificed to study the cochleas after testing hearing levels. ABR thresholds and OAEs were attenuated in the gentamicin group, in which apoptosis was detected with histopathological investigation. The group that received NAC in addition to gentamicin had better ABR thresholds and better OAEs. The histopathological evidence of apoptosis in was considerably less in this group. Gentamicin ototoxicity can be detected by ABR and OAE testing in rats, and NAC may protect the cochlear cells from apoptosis.

Preventive effect of N-acetylcysteine in a mouse model of steroid resistant acute exacerbation of asthma.

Oxidative stress appears to have an important role in glucocorticoid insensitivity, as a crucial problem in asthma therapy. We studied the preventive effect of antioxidant N-acetylcysteine (NAC) on the airway hyper-responsiveness (AHR) and the accumulation of inflammatory cells in the airways in an animal model of steroid resistant acute exacerbation of asthma. Systemically sensitized Balb/C mice were exposed to Ovalbumin aerosol on days 13, 14, 15 and 16, followed by intratracheal lipopolysaccharide (LPS) to induce acute exacerbation. NAC (intraperitoneal, 320 mg/kg 30 min before and 12 hours after each challenge) reduced hyper-responsiveness with/out dexamethasone. LPS application caused neutrophilia in bronchoalveolar lavage fluid (BALF) and eosinophil count was higher than respective control in BALF as well as neutrophils after dexamethasone treatment. NAC significantly decreased neutrophil and eosinophil count in BALF as well as inflammatory cytokines (IL-13 and IL-5).We concluded that addition of NAC to asthma therapy has beneficial preventive effects in an animal model of steroid resistant acute exacerbation of asthma.

The effect of N-acetylcysteine on growth and biofilm formation in Staphylococcus epidermidis strains

Aim: To examine the possible preventive effect of N-acetylcysteine (NAC) on biofilm formation in Staphylococcus epidermidis strains, which cause very important problems such as implant-associated infections in hospitalized and immunocompromised patients worldwide. Materials and methods: The effects of varying concentrations of NAC on the formation of biofilm by 28 strains of Staphylococcus epidermidis was investigated spectrophotometrically by micro-ELISA assay. Results: Significant differences in biofilm formation in the presence of various NAC concentrations were found in 15 methicillin-resistant Staphylococcus epidermidis and 13 methicillin-sensitive Staphylococcus epidermidis strains (P < 0.05). Conclusion: It was found that slime formation was decreased due to the increased concentrations of the NAC treatments. Based on these findings, it is possible to use NAC as an antibiofilm agent on medical devices or in catheter lock solutions to prevent colonization and implant-associated infections.

Preventive effects of N-acetyl cysteine on lipids, lipoproteins and myocardial infarct size in isoproterenol induced myocardial infarcted rats: An in vivo and in vitro study

The present study evaluated the preventive effects of N-acetyl cysteine in isoproterenol induced myocardial infarcted rats. Rats were pretreated with N-acetyl cysteine (10mg/kg body weight) daily for a period of 14days. After pretreatment, rats were injected with isoproterenol (100mg/kg body weight) at an interval of 24h for two days to induce myocardial infarction. Isoproterenol induced myocardial infarction was indicated by increased activity of creatine kinase-MB and levels of cardiac troponins in the serum. The weight of heart and the levels of serum and heart cholesterol, triglycerides, free fatty acids were increased in isoproterenol induced myocardial infarcted rats. Isoproterenol also increased the levels of serum low density and very low density lipoprotein cholesterol and decreased high density lipoprotein cholesterol. It enhanced the activity of liver 3-hydroxy-3 methyl glutaryl-Coenzyme-A reductase and the levels of lipid peroxidation products. Pretreatment with N-acetyl cysteine showed significant preventive effects in all the biochemical parameters studied in myocardial infarcted rats. Also, N-acetyl cysteine reduced myocardial infarct size. Histopathological findings of N-acetyl cysteine pretreated myocardial infarcted heart correlated with these biochemical findings. The in vitro study confirmed the strong antioxidant action of N-acetyl cysteine. Thus, the present study revealed that N-acetyl cysteine prevented increased heart weight, accumulation of lipids, altered levels of lipoproteins thereby reducing myocardial infarct size due to its antilipidemic and antioxidant effects in isoproterenol induced myocardial infarcted rats. This study may have a significant impact on myocardial infarction.

Comparison of the effects of N-acetyl cysteine and erdosteine in rats with renal injury caused by paracetamol intoxication

The aim of the present study was to investigate the therapeutic and preventive effects of N-acetyl cysteine and erdosteine on renal injury associated with paracetamol (acetaminophen) intoxication. Female albino Wistar rats were divided into six groups: control; paracetamol (1 g/kg, oral); paracetamol (1 g/kg, oral) + erdosteine (150 mg/kg/day, oral); paracetamol (1 g/kg, oral) + N-acetyl cysteine (140 mg/kg bolus, followed by 70 mg/kg, oral); N-acetyl cysteine control (140 mg/kg bolus, followed by 70 mg/kg, oral); and erdosteine control (150 mg/kg/day, oral). Potential renal injury was assessed using biochemical analyses, radionuclide imaging, and histopathological parameters. In the paracetamol group, blood urea nitrogen and creatinine levels were significantly increased compared with controls. Histopathological examination showed tubular vacuolization, tubular necrosis, and remarkable interstitial inflammation. The excretion function was observed to be insufficient on radionuclide imaging. However, in the groups treated with erdosteine or N-acetyl cysteine after paracetamol, biochemical analyses, radionuclide imaging, and histopathological parameters showed significantly less evidence of renal toxicity than that observed in the group receiving paracetamol alone. Less renal toxicity was detected in rats receiving N-acetyl cysteine than in those receiving erdosteine. Renal injury may develop after paracetamol overdose. Erdosteine and N-acetyl cysteine are both effective in the prevention of renal injury when given in the early phase of paracetamol nephrotoxicity. N-acetyl cysteine is more protective than erdosteine.

Protective effects of N-acetyl cysteine on lipid peroxide metabolism on isoproterenol-induced myocardial infarcted rats.

The present study was designed to evaluate the preventive effects of N-acetyl cysteine on lipid peroxide metabolism in isoproterenol (ISO) induced myocardial infarcted rats. Male albino Wistar rats were pretreated with N-acetyl cysteine (5 and 10 mg/kg) daily for a period of 14 days. After the pretreatment period, ISO (100 mg/kg) was subcutaneously injected to rats twice at an interval of 24 h. Increased activities of serum creatine kinase, creatine kinase-MB, lactate dehydrogenase, and increased intensities of serum lactate dehydrogenase-isoenzyme bands (LDH-1, LDH-2) were observed in ISO-induced rats. The heart lipid peroxidation products were significantly increased, and the antioxidant system was significantly reduced in ISO-induced rats. Pretreatment with N-acetyl cysteine (5 and 10 mg/kg) to ISO-induced rats showed significant effects on all the biochemical parameters studied. Histopathological findings of the myocardium also showed the protective role of N-acetyl cysteine in ISO-induced rats. Furthermore, in vitro study confirmed the potent-free radical scavenging activity of N-acetyl cysteine. The effect at a dose of 10 mg/kg of N-acetyl cysteine was more pronounced than the dose, 5 mg/kg. The results of our study show that N-acetyl cysteine protects the heart against ISO-induced myocardial infarction by its free radical scavenging effect.

AS-256: Preventive Effect of N-Acetylcysteine in Contrast-Induced Nephropathy during Elective Coronary Intervention

Contrast-induced nephropathy is a major cause of morbidity and mortality associated with percutaneous coronary intervention. Recently, several reports have shown that N-acetylcysteine is effective to reduce the occurrence of contrast-induced nephropathy, but some reports could not confirm this. Different dosages of N-acetylcysteine may lead the conflicting results, and thus we examined the effect of N-acetylcysteine at >3600 mg in total dosage on serum creatinine levels after coronary intervention.

Toxicity and oxidative stress of acrylonitrile in rat primary glial cells: Preventive effects of N-acetylcysteine

Brain is a target organ for acrylonitrile (ACN) toxicity. The objective of the current work was to investigate ACN cytotoxicity in rat primary glial cells, using N-acetyl- l-cysteine (NAC) as a potential protective agent. Cells were exposed in vitro to different concentrations of ACN for different time intervals. Cell membrane integrity was assessed by trypan blue exclusion and lactate dehydrogenase (LDH) leakage. Approximately 50% membrane damage was observed in the incubations containing 1.0 mM ACN for 3 h. Therefore, these experimental conditions were used in subsequent studies. ACN enhanced lipid peroxidation, as indicated by malondialdehyde (MDA) accumulation, and depleted reduced glutathione (GSH) level with no change in total glutathione. Also, ACN was activated to cyanide (CN −) with dramatic decrease in ATP level. Cell treatment with NAC prior to exposure to ACN afforded some protection; as indicated by reducing MDA level and elevating level of both reduced and total glutathione. Further, pretreatment with NAC inhibited CN − formation and caused an increase in ATP level. Our results indicate that ACN is toxic to rat primary glial cells as evidenced by induction of oxidative stress and generation of CN − with subsequent energy depletion. NAC can play an important role against ACN-induced oxidative damage.

Contrast-induced nephropathy: a review

Contrast-induced nephropathy (CIN) is one of the leading causes of renal impairment in the United States and the third cause of hospital-acquired renal failure. Reduction in the incidence of CIN can lead to a decrease in the morbidity, mortality, and length of hospital stay. Although prophylactic hydration has been promising in decreasing the occurrence of CIN, other efforts such as diuretics, calcium channel blockers, theophylline, aminophylline, atrial natriuretic peptide, dopamine, and fenoldopam have been disappointing. The preventive effect of N-acetylcysteine on CIN has not been consistent in the literature. In a recent clinical trial, bicarbonate infusion was more effective than hydration in the prevention of CIN. Mechanical devices are in development to perfuse renal arteries with protective drugs during contrast exposure or for removal of contrast from coronary sinus during coronary angiography. In this article, we have reviewed available data in regards to CIN.

Oxidative Stress-mediated, Post-translational Loss of MafA Protein as a Contributing Mechanism to Loss of Insulin Gene Expression in Glucotoxic Beta Cells

Glucose toxicity in pancreatic islet beta cells causes loss of insulin gene expression, content, and secretion due to loss of binding of transcription factors, most notably PDX-1 and RIPE-3b1 activator, to the promoter region of the insulin gene. Recently, RIPE-3b1 activator was cloned and identified as the mammalian homologue of avian MafA/Maf-L (MafA). This enabled us to carry out more extensive studies of the role of MafA in glucotoxicity than were hitherto possible. Northern analysis of glucotoxic HIT-T15 cells revealed normal amounts of MafA mRNA, but Western analysis demonstrated a 97 +/- 1% reduction in MafA protein (p < 0.0001). The proteasome is a likely site for MafA degradation as lactacystin, an irreversible proteasome inhibitor, caused an accumulation of MafA protein. Antioxidants have previously been shown to prevent the adverse effects of glucose toxicity on beta cell function both in vivo and in vitro. In the current study, chronic culturing of HIT-T15 cells with the antioxidant N-acetylcysteine (NAC) prevented loss of MafA protein (late passage = 18.9 +/- 10.4% of early passage, p < 0.001; late passage with NAC = 68.7 +/- 19.7% of early passage, p = not significant) and loss of DNA binding (late passage = 63.7 +/- 9% of early passage, p < 0.02; late passage with NAC = 116 +/- 10% of early passage, p = not significant). Additionally, transient transfection of PDX-1 or MafA cDNA into glucotoxic cells increased PDX-1 and MafA protein levels and individually increased insulin promoter activity (untreated = 34%, PDX-1 = 70%, MafA = 78%; percentage of activity of early passage cells), whereas the combined transfection of MafA and PDX-1 completely restored insulin promoter activity. This recovery of promoter activity following transient transfection had no effect on endogenous insulin mRNA. However, adenoviral infection of MafA and PDX-1 significantly increased endogenous insulin mRNA levels by 93% (121 +/- 9 versus 233 +/- 18 density light units; n = 5, p < 0.001). We conclude that the absence of MafA protein from beta cells via chronic oxidative stress contributes importantly to the loss of endogenous insulin gene expression as glucose toxicity develops.