Search

Clostridium perfringens Beta-1 toxin (CPB1) is a lethal toxin, which can lead to necrotic enteritis, but the pathological mechanism has not been elucidated. We investigated whether reactive oxygen species (ROS) participated in CPB1-induced pyroptosis and ferroptosis, and investigated the effects of calpain on CPB1-induced oxidative stress and inflammation. Scavenging ROS by N-Acetyl-L cysteine (NAC) led to the reduction of ROS, inhibited the death of macrophages, cytoplasmic swelling and membrane rupture, the expression of pyroptosis-related proteins and proinflammatory factor, while increased the expression of anti-inflammatory factors in cells treated with rCPB1. Adenosine triphosphate (ATP) synthase, H + transporting, mitochondrial F1 complex, alpha subunit 1 (ATP5A1) was identified specifically interact with rCPB1. Silencing ATP5A1 inhibited accumulation of ATP and ROS, leaded to less cytoplasmic swelling and membrane rupture, attenuated pyroptosis and inflammation in rCPB1-treated cells. We also found that rCPB1 induces ferroptosis in macrophages, and the level of ferroptosis was similar with H 2 O 2 . Of note, H 2 O 2 is a major ROS source, indicated that ROS production may play a major role in the regulation of ferroptosis in macrophages treated with rCPB1. This finding was further corroborated in rCPB1- induced human acute monocytic leukemia cells, which were treated with NAC. In addition, the inhibition of ferroptosis using liproxstatin-1 inhibited the shriveled mitochondrial morphology, increased the expression of glutathione peroxidase 4, nicotinamide adenine dinucleotide (phosphate) hydrogen: quinone oxidoreductase 1 and cysteine/glutamic acid reverse transport solute carrier family 7 members 11, decreased the expression of heme oxygenase 1, nuclear receptor coactivator 4 and transferrin receptor proteins, reduced malondialdehyde and lipid peroxidation levels, and increased intracellular L-glutathione levels in cells treated with rCPB1. Furthermore, calpain inhibitor PD151746 was used to investigate how pyroptosis and ferroptosis were involved simultaneously in rCPB1-treated macrophages. We showed that PD151746 inhibited ATP and ROS production, reversed the representative pyroptosis/ferroptosis indicators and subsequently reduced inflammation. The above findings indicate that rCPB1 might lead to macrophage pyroptosis and ferroptosis through the large and sustained increase in intracellular calpain and oxidative stress, further lead to inflammation., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

Fish exposed to xenobiotics like petroleum-derived polycyclic aromatic hydrocarbons (PAHs) will immediately initiate detoxification systems through effective biotransformation reactions. Yet, there is a discrepancy between recognized metabolic pathways and the actual metabolites detected in fish following PAH exposure like oil pollution. To deepen our understanding of PAH detoxification, we conducted experiments exposing Atlantic haddock ( Melanogrammus aeglefinus ) to individual PAHs or complex oil mixtures. Bile extracts, analyzed by using an ion mobility quadrupole time-of-flight mass spectrometer, revealed novel metabolites associated with the mercapturic acid pathway. A dominant spectral feature recognized as PAH thiols set the basis for a screening strategy targeting (i) glutathione-, (ii) cysteinylglycine-, (iii) cysteine-, and (iv) mercapturic acid S-conjugates. Based on controlled single-exposure experiments, we constructed an interactive library of 33 metabolites originating from 8 PAHs (anthracene, phenanthrene, 1-methylphenanthrene, 1,4-dimethylphenanthrene, chrysene, benz[ a ]anthracene, benzo[ a ]pyrene, and dibenz[ a , h ]anthracene). By incorporation of the library in the analysis of samples from crude oil exposed fish, PAHs conjugated with glutathione and cysteinylglycine were uncovered. This qualitative study offers an exclusive glimpse into the rarely acknowledged mercapturic acid detoxification pathway in fish. Furthermore, this furnishes evidence that this metabolic pathway also succeeds for PAHs in complex pollution sources, a notable discovery not previously reported.

A wide variety of stresses have been proposed to exert killing effects upon bacteria by stimulating the intracellular formation of reactive oxygen species (ROS). A key part of the supporting evidence has often been the ability of antioxidant compounds to protect the cells. In this study, some of the most-used antioxidants-thiourea, glutathione, N-acetylcysteine, and ascorbate-have been examined. Their ability to quench superoxide and hydrogen peroxide was verified in vitro, but the rate constants were orders of magnitude too slow for them to have an impact upon superoxide and peroxide concentrations in vivo, where these species are already scavenged by highly active enzymes. Indeed, the antioxidants were unable to protect the growth and ROS-sensitive enzymes of E. coli strains experiencing authentic oxidative stress. Similar logic posits that antioxidants cannot substantially quench hydroxyl radicals inside cells, which contain abundant biomolecules that react with them at diffusion-limited rates. Indeed, antioxidants were able to protect cells from DNA damage only if they were applied at concentrations that slow metabolism and growth. This protective effect was apparent even under anoxic conditions, when ROS could not possibly be involved, and it was replicated when growth was similarly slowed by other means. Experimenters should discard the use of antioxidants as a way of detecting intracellular oxidative stress and should revisit conclusions that have been based upon such experiments. The notable exception is that these compounds can effectively degrade hydrogen peroxide from environmental sources before it enters cells., (© 2024 The Author(s). Molecular Microbiology published by John Wiley & Sons Ltd.)

Cu-thiosemicarbazones have been intensively investigated for their application in cancer therapy or as antimicrobials. Copper(II)-di-2-pyridylketone-4,4-dimethyl-thiosemicarbazone (Cu II -Dp44mT) showed anticancer activity in the submicromolar concentration range in cell culture. The interaction of Cu II -Dp44mT with thiols leading to their depletion or inhibition was proposed to be involved in this activity. Indeed, Cu II -Dp44mT can catalyze the oxidation of thiols although with slow kinetics. The present work aims to obtain insights into the catalytic activity and selectivity of Cu II -Dp44mT toward the oxidation of different biologically relevant thiols. Reduced glutathione (GSH), L-cysteine (Cys), N-acetylcysteine (NAC), D-penicillamine (D-Pen), and the two model proteins glutaredoxin (Grx) and thioredoxin (Trx) were investigated. Cu II -Dp44mT catalyzed the oxidation of these thiols with different kinetics, with rates in the following order D-Pen>Cys≫NAC>GSH and Trx>Grx. Cu II -Dp44mT was more efficient than Cu II chloride for the oxidation of NAC and GSH, but not D-Pen and Cys. In mixtures of biologically relevant concentrations of GSH and either Cys, Trx, or Grx, the oxidation kinetics and spectral properties were similar to that of GSH alone, indicating that the interaction of these thiols with Cu II -Dp44mT is dominated by GSH. Hence GSH could protect other thiols against potential deleterious oxidation by Cu II -Dp44mT., (© 2024 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

Iron accumulation in the brain causes oxidative stress, blood-brain barrier (BBB) breakdown, and neurodegeneration. We examined the preventive effects of acetylated oligopeptides (AOP) from whey protein on iron-induced hippocampal damage compared to N-acetyl cysteine (NAC). This 5-week study used 40 male albino rats. At the start, all rats received 150 mg/kg/day of oral NAC for a week. The 40 animals were then randomly divided into four groups: Group I (control) received a normal diet; Group II (iron overload) received 60 mg/kg/day intraperitoneal iron dextran 5 days a week for 4 weeks; Group III (NAC group) received 150 mg/kg/day NAC and iron dextran; and Group IV (AOP group) received 150 mg/kg/day AOP and iron dextran. Enzyme-linked immunosorbent assay, spectrophotometry, and qRT-PCR were used to measure MMP-9, tissue inhibitor metalloproteinase-1 (TIMP-1), MDA, reduced glutathione (GSH) levels, and nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) gene expression. Histopathological and immunohistochemical detection of nestin, claudin, caspase, and GFAP was also done. MMP-9, TIMP-1, MDA, caspase, and GFAP rose in the iron overload group, while GSH, Nrf2, HO-1, nestin, and claudin decreased. The NAC and AOP administrations improved iron overload-induced biochemical and histological alterations. We found that AOP and NAC can protect the brain hippocampus from iron overload, improve BBB disruption, and provide neuroprotection with mostly no significant difference from healthy controls., (© 2024 John Wiley & Sons Ltd.)

Fetal growth restriction (FGR) seriously threatens perinatal health. The main cause of FGR is placental malperfusion, but the specific mechanism is still unclear, and there is no effective treatment for FGR. We constructed a FGR mouse model by adding exogenous asymmetric dimethylarginine (ADMA) through in vivo experiments and found that ADMA could cause placental dysplasia and induce the occurrence of FGR. Compared with the control group, reactive oxygen species (ROS) production in the placenta was increased in mice with FGR, and the expression of autophagy-related proteins p-AKT/AKT, p-mTOR/mTOR, and P62 was significantly decreased, while the expression of Beclin-1 and LC3-II was significantly increased in the FGR group. Furthermore, ADMA had a favorable effect in promoting the formation of autophagosomes. Hydroxychloroquine (HCQ) and N-acetylcysteine (NAC) improved ADMA-induced disorders of placental development and alleviated ADMA-induced FGR. This study found that ADMA could cause excessive autophagy of trophoblasts by increasing the level of oxidative stress, ultimately leading to the occurrence of FGR, and HCQ and NAC had therapeutic effects on ADMA-induced FGR., (© 2023. The Author(s), under exclusive licence to Society for Reproductive Investigation.)

Hypothermic liquid storage at 4-5 °C has emerged as a novel approach for preserving boar semen, offering innovative possibilities for semen preservation. However, this method also presents challenges, including cold shock and excessive reactive oxygen species (ROS) production. Therefore, reducing oxidative damage induced by low temperatures becomes essential while supplementing appropriate protectants. In this study, we investigated the efficacy of Bovine Serum Albumin (BSA) compared to Polyvinylpyrrolidone (PVP) and Skim Milk Powder (SMP) in maintaining boar sperm motility and progressive motility using computer-assisted sperm analysis (CASA). Among the tested concentrations, 4 g/L of BSA exhibited the best protective effect. Subsequently, we supplemented different concentrations of l-cysteine (LC) and N-acetyl-l-cysteine (NAC) as additives in the presence of BSA as a protectant. Our results demonstrated that 1 mmol/L of LC and 0.5 mmol/L of NAC exhibited superior protection of sperm quality compared to other concentrations. Furthermore, the 1 mmol/L LC and 0.5 mmol/L NAC groups showed significantly improved plasma membrane integrity and acrosome integrity compared to the control group. These groups also exhibited enhanced antioxidant capacity, evidenced by increased mitochondrial membrane potential (MMP), ATP production, total superoxide dismutase (T-SOD) activity, total antioxidant capacity (T-AOC), glutathione (GSH), glutathione peroxidase (GSH-PX), and GPX-4 levels. Additionally, they demonstrated decreased reactive oxygen species (ROS) and malondialdehyde (MDA) levels, as well as reduced oxidized glutathione (GSSG) and glutathione reductase (GR) levels. Furthermore, LC and NAC treatment enhanced AMP-activated protein kinase (AMPK) phosphorylation. However, inhibiting AMPK using compound C did not inhibit the protective effects of LC and NAC on low-temperature preserved boar sperm. These findings suggest that 4 g/L BSA can serve as an effective protectant for hypothermic liquid storage of boar semen. Additionally, LC and NAC supplementation reduces oxidative damage by enhancing antioxidant capacity rather than through AMPK-mediated ATP supplementation. These results contribute to advancing the application of LC and NAC in hypothermic liquid storage of boar semen., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Approximately 10% of smokers will develop lung cancer. Sensitive predictive biomarkers are needed to identify susceptible individuals. 1,3-Butadiene (BD) is among the most abundant tobacco smoke carcinogens. BD is metabolically activated to 3,4-epoxy-1-butene (EB), which is detoxified via the glutathione conjugation/mercapturic acid pathway to form monohydroxybutenyl mercapturic acid (MHBMA) and dihydroxybutyl mercapturic acid (DHBMA). Alternatively, EB can react with guanine nucleobases of DNA to form N7-(1-hydroxyl-3-buten-1-yl) guanine (EB-GII) adducts. We employed isotope dilution LC/ESI-HRMS/MS methodologies to quantify MHBMA, DHBMA, and EB-GII in urine of smokers who developed lung cancer ( N = 260) and matched smoking controls ( N = 259) from the Southern Community Cohort (white and African American). The concentrations of all three biomarkers were significantly higher in smokers that subsequently developed lung cancer as compared to matched smoker controls after adjusting for age, sex, and race/ethnicity ( p < 0.0001 for EB-GII, p < 0.0001 for MHBMA, and p = 0.0007 for DHBMA). The odds ratio (OR) for lung cancer development was 1.63 for MHBMA, 1.37 for DHBMA, and 1.97 for EB-GII, with a higher OR in African American subjects than in whites. The association of urinary EB-GII, MHBMA, and DHBMA with lung cancer status did not remain upon adjustment for total nicotine equivalents. These findings reveal that urinary MHBMA, DHBMA, and EB-GII are directly correlated with the BD dose delivered via smoking and are associated with lung cancer risk.

Background: Increased reactive oxygen species (ROS) and oxidative stress response lead to cardiomyocyte hypertrophy and apoptosis, which play crucial roles in the pathogenesis of heart failure. The purpose of current research was to explore the role of antioxidant N-acetylcysteine (NAC) on cardiomyocyte dysfunction and the underlying molecular mechanisms., Methods and Results: Compared with control group without NAC treatment, NAC dramatically inhibited the cell size of primary cultured neonatal rat cardiomyocytes (NRCMs) tested by immunofluorescence staining and reduced the expression of representative markers associated with hypertrophic, fibrosis and apoptosis subjected to phenylephrine administration examined by reverse transcription-polymerase chain reaction (RT-PCR) and western blot. Moreover, enhanced ROS expression was attenuated, whereas activities of makers related to oxidative stress response examined by individual assay Kits, including total antioxidation capacity (T-AOC), glutathione peroxidase (GSH-Px), and primary antioxidant enzyme Superoxide dismutase (SOD) were induced by NAC treatment in NRCMs previously treated with phenylephrine. Mechanistically, we noticed that the protein expression levels of phosphorylated phosphatidylinositol 3-kinase (PI3K) and AKT were increased by NAC stimulation. More importantly, we identified that the negative regulation of NAC in cardiomyocyte dysfunction was contributed by PI3K/AKT signaling pathway through further utilization of PI3K/AKT inhibitor (LY294002) or agonist (SC79)., Conclusions: Collected, NAC could attenuate cardiomyocyte dysfunction subjected to phenylephrine, partially by regulating the ROS-induced PI3K/AKT-dependent signaling pathway., (© The Author(s) 2023. Published by Oxford University Press on behalf of American Journal of Hypertension, Ltd. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Pyroptosis is a critical form of cell death during the development of chronic kidney disease (CKD). Tripartite motif 6 (TRIM6) is an E3-ubiquitin ligase that participates in the progression renal fibrosis (RF). The aim of this study was to investigate the roles of TRIM6 and Glutathione peroxidase 3 (GPX3) in oxidative stress-induced inflammasome activation and pyroptosis in Ang-II treated renal tubular epithelial cells., Methods: To study its role in RF, TRIM6 expression was either reduced or increased in human kidney-2 (HK2) cells using lentivirus, and Ang-II, NAC and BMS-986299 were served as reactive oxygen species (ROS) inducer, ROS scavenger and NLRP3 agonist respectively. Pyroptosis and mitochondrial ROS were measured by flow cytometry. The levels of malondialdehyde (MDA), glutathione (GSH), and superoxide dismutase (SOD) were determined using commercial kits, while the levels of IL-1β, IL-18, IL-6, and tumor necrosis factor-α (TNF-α) were determined by Enzyme-Linked Immunosorbent Assay (ELISA). Co-immunoprecipitation (Co-IP) assay was used to evaluate the interaction between TRIM6 and GPX3. Reverse transcription-polymerase chain reaction (RT-PCR) and western blot were used to measure mRNA and protein expression, respectively., Results: Treatment with Angiotensin II (Ang II) increased the protein and mRNA levels of TRIM6 in HK2 cells. Ang II also increased mitochondrial ROS production and the malondialdehyde (MDA) level, but decreased the levels of GSH and SOD. In addition, Ang II enhanced HK2 cell pyroptosis, increased the levels of IL-1β, IL-18, IL-6, and TNF-α, and promoted the expression of active IL-1β, NLRP3, caspase-1, and GSDMD-N proteins. These effects were reversed by knockdown of TRIM6 and by treatment with N-acetyl-L-cysteine (NAC), a ROS scavenger. BMS-986299, an NLRP3 agonist treatment, did not affect ROS production in HK2 cells exposed to Ang II combined with NAC, but cell pyroptosis and inflammation were aggravated. Moreover, the overexpression of TRIM6 in HK2 cells resulted in similar effects to Ang II. NAC and GPX3 overexpression in HK2 cells could reverse ROS production, inflammation, and pyroptosis induced by TRIM6 overexpression. TRIM6 overexpression decreased the GPX3 protein level by promoting its ubiquitination, without affecting the GPX3 mRNA level. Thus, TRIM6 facilitates GPX3 ubiquitination, contributing to increased ROS levels and pyroptosis in HK2 cells., Conclusions: TRIM6 increases oxidative stress and promotes the pyroptosis of HK2 cells by regulating GPX3 ubiquitination. These findings could contribute to the development of novel drugs for the treatment of RF., Competing Interests: The authors declare no conflict of interest., (© 2024 The Author(s). Published by IMR Press.)

The decline in male fertility caused by environmental pollutants has attracted worldwide attention nowadays. Tris(2-chloroisopropyl) phosphate (TCPP) is a chlorine-containing organophosphorus flame retardant applied in many consumer products and has multiple side effects on health. However, whether TCPP impairs spermatogenesis remains unclear. In this study, we found that TCPP reduced the sperm motility and blastocyst formation, inhibited proliferation and induced apoptosis in mice testes and spermatocyte cell line GC-2. Moreover, TCPP induced imbalance of oxidant and anti-oxidant, DNA damage and mitochondrial dysfunction, thus induced abnormal spermatogenesis. In this process, p53 signaling pathway was activated and N-acetylcysteine treatment partially alleviated the side effects of TCPP, including decrease of sperm motility, activation of p53 signaling pathway and DNA damage. Finally, our study verified that TCPP elevated reactive oxygen species (ROS), decreased mitochondrial membrane potential and induced apoptosis in human semen samples. Overall, ROS mediated TCPP-induced germ cell proliferation inhibition and apoptosis, which finally led to the decline of sperm motility., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Silver nanoparticles (AgNPs) are used increasingly often in the biomedical field, but their potential deleterious effects on the cardiovascular system remain to be elucidated. The primary aim of this study was to evaluate the toxic effects, and the underlying mechanisms of these effects, of AgNPs on human umbilical vein endothelial cells (HUVECs), as well as the protective role of N-acetylcysteine (NAC) against cytotoxicity induced by AgNPs. In this study, we found that exposure to AgNPs affects the morphology and function of endothelial cells which manifests as decreased cell proliferation, migration, and angiogenesis ability. Mechanistically, AgNPs can induce excessive cellular production of reactive oxygen species (ROS), leading to damage to cellular sub-organs such as mitochondria and lysosomes. More importantly, our data suggest that AgNPs causes autophagy defect, inhibits mitophagy, and finally activates the mitochondria-mediated apoptosis signaling pathway and evokes cell death. Interestingly, treatment with ROS scavenger-NAC can effectively suppress AgNP-induced endothelial damage.Our results indicate that ROS-mediated mitochondria-lysosome injury and autophagy dysfunction are potential factors of endothelial toxicity induced by AgNPs. This study may provide new evidence for the cardiovascular toxicity of AgNPs and serve as a reference for the safe use of nanoparticles(NPs) in the future., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The aim of this work was to evaluate possible mechanisms involved in the protective effect of N-acetyl-L-cysteine (NAC) on hepatic endocrine-metabolic, oxidative stress, and inflammatory changes in prediabetic rats. For that, normal male Wistar rats (60 days old) were fed for 21 days with 10% sucrose in their drinking water and 5 days of NAC administration (50 mg/kg, i.p.) and thereafter, we determined: serum glucose, insulin, transaminases, uric acid, and triglyceride levels; hepatic fructokinase and glucokinase activities, glycogen content, lipogenic gene expression; enzymatic and non-enzymatic oxidative stress, insulin signaling pathway, and inflammatory markers. Results showed that alterations evinced in sucrose-fed rats (hypertriglyceridemia, hyperinsulinemia, and high liver fructokinase activity together with increased liver lipogenic gene expression and oxidative stress and inflammatory markers) were prevented by NAC administration. P-endothelial nitric oxide synthase (P-eNOS)/eNOS and pAKT/AKT ratios, decreased by sucrose ingestion, were restored after NAC treatment. In conclusion, the results suggest that NAC administration improves glucose homeostasis, oxidative stress, and inflammation in prediabetic rats probably mediated by modulation of the AKT/NOS pathway. Administration of NAC may be an effective complementary strategy to alleviate or prevent oxidative stress and inflammatory responses observed in type 2 diabetes at early stages of its development (prediabetes).

Introduction: Chronic lung infection due to bacterial biofilms is one of the leading causes of mortality in cystic fibrosis (CF) patients. Among many species colonizing the lung airways, Pseudomonas aeruginosa and Staphylococcus aureus are two virulent pathogens involved in mechanically robust biofilms that are difficult to eradicate using airway clearance techniques like lung lavage. To remove such biological materials, glycoside hydrolase-based compounds are commonly employed for targeting and breaking down the biofilm matrix, and subsequently increasing cell susceptibility to antibiotics., Materials and Methods: In this study, we evaluate the effects of N-acetyl cysteine (NAC) and Cysteamine (CYST) in disrupting interfacial bacterial films, targeting different components of the extracellular polymeric substances (EPS). We characterize the mechanics and structural integrity of the interfacial bacterial films using pendant drop elastometry and scanning electron microscopy., Results and Discussion: Our results show that the film architectures are compromised by treatment with disrupting agents for 6 h, which reduces film elasticity significantly. These effects are profound in the wild type and mucoid P. aeruginosa , compared to S. aureus . We further assess the effects of competition and cooperation between S. aureus and P. aeruginosa on the mechanics of composite interfacial films. Films of S. aureus and wild-type P. aeruginosa cocultures lose mechanical strength while those of S. aureus and mucoid P. aeruginosa exhibit improved storage modulus. Treatment with NAC and CYST reduces the elastic property of both composite films, owing to the drugs' ability to disintegrate their EPS matrix. Overall, our results provide new insights into methods for assessing the efficacy of mucolytic agents against interfacial biofilms relevant to cystic fibrosis infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Balmuri, Noaman, Usman and Niepa.)

Backgrounds: Infection during pregnancy is a significant public health concern due to the increased risk of adverse birth outcomes. Group B Streptococcus or Streptococcus agalactiae (GBS) stands out as a major bacterial cause of neonatal morbidity and mortality. We aimed to explore the involvement of reactive oxygen species (ROS) and oxidative stress pathways in pro-inflammatory responses within human fetal membrane tissue, the target tissue of acute bacterial chorioamnionitis., Methods: We reanalyzed transcriptomic data from fetal membrane explants inoculated with GBS to assess the impact of GBS on oxidative stress and ROS genes/pathways. We conducted pathway enrichment analysis of transcriptomic data using the Database for Annotation, Visualization and Integrated Discovery (DAVID), a web-based functional annotation/pathway enrichment tool. Subsequently, we conducted ex vivo experiments to test the hypothesis that antioxidant treatment could inhibit pathogen-stimulated inflammatory responses in fetal membranes., Results: Using DAVID analysis, we found significant enrichment of pathways related to oxidative stress or ROS in GBS-inoculated human fetal membranes, for example, "Response to Oxidative Stress" (FDR = 0.02) and "Positive Regulation of Reactive Oxygen Species Metabolic Process" (FDR = 2.6*10 -4 ). There were 31 significantly changed genes associated with these pathways, most of which were upregulated after GBS inoculation. In ex vivo experiments with choriodecidual membrane explants, our study showed that co-treatment with N-acetylcysteine (NAC) effectively suppressed the release of pro-inflammatory cytokines (IL-6, IL-8, TNF-α) and prostaglandin PGE2, compared to GBS-treated explants (p < .05 compared to GBS-treated samples without NAC co-treatment). Furthermore, NAC treatment inhibited the release of cytokines and PGE2 stimulated by lipoteichoic acid (LTA) and lipopolysaccharide (LPS) in whole membrane explants (p < .05 compared to LTA or LPS-treated samples without NAC co-treatment)., Conclusions: Our study sheds light on the potential roles of ROS in governing the innate immune response to GBS infection, offering insights for developing strategies to mitigate GBS-related adverse outcomes., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Thiol molecules have been recently re-considered as drug candidates in viral infections because of their ability to induce redox changes which interfere with virus life cycle and modulate the host immune response. Little is known about the molecular mechanisms of their immunomodulatory properties. Here we show that I-152, a thiol molecule metabolized to release N-acetyl-l-cysteine and cysteamine and acting as a pro-glutathione agent, causes early up-regulation of immunoproteasome subunits in the lymph nodes of murine leukemia virus infected mice. This evidence suggests that the immunoproteasome may be modulated by thiol-based compounds with important implications in understanding redox-controlled immunoregulation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Acetaminophen (APAP) overdose causes liver injury and acute liver failure, as well as acute kidney injury, which is not prevented by the clinical antidote N-acetyl-L-cysteine (NAC). The absence of therapeutics targeting APAP-induced nephrotoxicity is due to gaps in understanding the mechanisms of renal injury. APAP metabolism through Cyp2E1 drives cell death in both the liver and kidney. We demonstrate that Cyp2E1 is localized to the proximal tubular cells in mouse and human kidneys. Virtually all the Cyp2E1 in kidney cells is in the endoplasmic reticulum (ER), not in mitochondria. By contrast, hepatic Cyp2E1 is in both the ER and mitochondria of hepatocytes. Consistent with this subcellular localization, a dose of 600 mg/kg APAP in fasted C57BL/6J mice induced the formation of APAP protein adducts predominantly in mitochondria of hepatocytes, but the ER of the proximal tubular cells of the kidney. We found that reactive metabolite formation triggered ER stress-mediated activation of caspase-12 and apoptotic cell death in the kidney. While co-treatment with 4-methylpyrazole (4MP; fomepizole) or the caspase inhibitor Ac-DEVD-CHO prevented APAP-induced cleavage of procaspase-12 and apoptosis in the kidney, treatment with NAC had no effect. These mechanisms are clinically relevant because 4MP but not NAC also significantly attenuated APAP-induced apoptotic cell death in primary human kidney cells. We conclude that reactive metabolite formation by Cyp2E1 in the ER results in sustained ER stress that causes activation of procaspase-12, triggering apoptosis of proximal tubular cells, and that 4MP but not NAC may be an effective antidote against APAP-induced kidney injury., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Hartmut Jaeschke reports financial support was provided by National Institutes of Health. Anup Ramachandran reports financial support was provided by National Institutes of Health. Darren Wallace reports financial support was provided by National Institutes of Health. Hartmut Jaeschke reports a relationship with Johnson & Johnson Consumer Companies Inc that includes: consulting or advisory and funding grants., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

The toxicity of acrylamide (AA) has continuously attracted wide concerns as its extensive presence from both environmental and dietary sources. However, its hepatic metabolic transformation and metabolic fate still remain unclear. This study aims to unravel the metabolic profile and glutathione (GSH) mediated metabolic fate of AA in liver of rats under the dose-dependent exposure. We found that exposure to AA dose-dependently alters the binding of AA and GSH and the generation of mercapturic acid adducts, while liver as a target tissue bears the metabolic transformation of AA via regulating GSH synthesis and consumption pathways, in which glutamine synthase (GSS), cytochrome P450 2E1 (CYP2E1), and glutathione S-transferase P1 (GSTP1) play a key role. In response to high- and low-dose exposures to AA, there were significant differences in liver of rats, including the changes in GSH and cysteine (CYS) activities and the conversion ratio of AA to glycidamide (GA), and liver can affect the transformation of AA by regulating the GSH-mediated metabolic pathway. Low-dose exposure to AA activates GSH synthesis pathway in liver and upregulates GSS activity and CYS content with no change in γ-glutamyl transpeptidase 1 (GGT1) activity. High-dose exposure to AA activates the detoxification pathway of GSH and increases GSH consumption by upregulating GSTP1 activity. In addition, molecular docking results showed that most of the metabolic molecules transformed by AA and GA other than themselves can closely bind to GSTP1, GSS, GGT1, N-acetyltransferase 8, and dimethyl sulfide dehydrogenase 1. The binding of AA-GSH and GA-GSH to GSTP1 and CYP2E1 enzymes determine the tendentiousness between toxicity and detoxification of AA, which exerts a prospective avenue for targeting protective role of hepatic enzymes against in vivo toxicity of AA., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The toxic heavy metal antimony (Sb) is ubiquitous in our daily lives. Various models have shown that Sb induces neuronal and reproductive toxicity. However, little is known about the developmental toxicity of Sb exposure during gestation and the underlying mechanisms. To study its effects on growth and development, Drosophila stages from eggs to pupae were exposed to different Sb concentrations (0, 0.3, 0.6 and 1.2 mg/mL Sb); RNA sequencing was used to identify the underlying mechanism. The model revealed that prenatal Sb exposure significantly reduced larval body size and weight, the pupation and eclosion rates, and the number of flies at all stages. With 1.2 mg/mL Sb exposure in 3rd instar larvae, 484 genes were upregulated and 694 downregulated compared to controls. Biological analysis showed that the disrupted transcripts were related to the oxidative stress pathway, as verified by reactive oxygen species (ROS) scavenger N-acetylcysteine (NAC) and glutathione (GSH) intervention experiments. Sb exposure induced oxidative stress imbalance could be rectified by chelation and antioxidant effects of NAC/GSH. The Drosophila Schneider 2 (S2) model further demonstrated that NAC and GSH greatly ameliorated cell death induced by Sb exposure. In conclusion, gestational Sb exposure disrupted oxidative stress homeostasis, thereby impairing growth and development., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

Adriamycin (ADR) is an important chemotherapeutic drug, but it has serious side effects such as hepatotoxicity. This study aimed to evaluate whether N-acetylcysteine (NAC) has hepatoprotective effects against ADR-induced hepatotoxicity in rats. In addition, it was aimed to determine how Meteorin-Like (MtrnL), which has pleiotropic effects on immunology, inflammation, and metabolism, is affected by ADR and/or NAC applications in liver tissue. 28 rats were randomly assigned to one of four equal groups in the study: control (no treatment), NAC (150 mg/kg/day of NAC intraperitoneally (i.p), ADR (15 mg/kg only on the first day of the experiment), and ADR + NAC (ADR 15 mg/kg on the first day of the experiment + 150 mg/kg/day NAC i.p). After 15 days, liver enzyme levels in serum, oxidant/antioxidant parameters in liver tissue, histopathological changes, caspase 3 (Casp3) and heat shock protein 70 (HSP-70) immunoreactivities, and MtrnL levels were examined. Histopathological changes, liver enzyme levels, as well as HSP-70, and Casp3 immunoreactivities increased due to ADR application. Additionally, MtrnL levels in liver tissue were significantly increased as a result of ADR application. However, it was detected that the NAC application significantly regulated the ADR-induced changes. Furthermore, it was determined that NAC administration regulated the changes in ADR-induced oxidative stress parameters. We propose that NAC may exert a hepatoprotective effect by regulating ADR-induced altered oxidative stress parameters, MtrnL levels, Casp3, and HSP-70 immunoreactivities in the liver., (© 2023. The Author(s), under exclusive licence to Cell Stress Society International.)

Glycation is a non-enzymatic reaction wherein sugars or dicarbonyls such as methylglyoxal (MGO) and glyoxal (GO) react with proteins, leading to protein inactivation. The hydrolysing enzyme human deglycase-1 (hDJ-1) is reported to decrease glycative stress by deglycating the modified proteins, specifically at cysteine, lysine, and arginine sites. This specificity of hDJ-1 is thought to be regulated by its active site cysteine residue (Cys106). Structural analysis of hDJ-1 by molecular docking and simulation studies, however, indicates a possible role of glutamate (Glu18) in determining its substrate specificity. To elucidate this, Glu18 present at the catalytic site of hDJ-1 was modified to aspartate (Asp18) by SDM, and the resultant mutant was termed mutant DJ-1 (mDJ-1). Both hDJ-1 and mDJ-1 were heterologously expressed in Escherichia coli BL21 (DE3) strain and purified to homogeneity. The hDJ-1 showed k cat values of 1.45 × 10 3 s -1 , 3.6 × 10 2 s -1 , and 3.1 × 10 2 s -1 , and K m values 0.181 mM, 18.18 mM, and 12.5 mM for N-acetylcysteine (NacCys), N-acetyllysine (NacLys), and N-acetylarginine (NacArg), respectively. The mDJ-1 showed altered k cat values (8 × 10 2 s -1 , 3.8 × 10 2 s -1 , 4.9 × 10 2 s -1 ) and K m values of 0.14 mM, 6.25 mM, 5.88 mM for NacCys, NacLys and NacArg, respectively. A single amino acid change (Glu18 to Asp18) improved the substrate specificity of mDJ-1 toward NacLys and NacArg. Understanding hDJ-1's structure and enhanced functionality will facilitate further exploration of its therapeutic potential for the treatment of glycation-induced diabetic complications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Skeletal muscle atrophy is a frequent complication after spinal cord injury (SCI) and can influence the recovery of motor function and metabolism in affected patients. Delaying skeletal muscle atrophy can promote functional recovery in SCI rats. In the present study, we investigated whether a combination of body weight support treadmill training (BWSTT) and glycine and N-acetylcysteine (GlyNAC) could exert neuroprotective effects, promote motor function recovery, and delay skeletal muscle atrophy in rats with SCI, and we assessed the therapeutic effects of the double intervention from both a structural and functional viewpoint. We found that, after SCI, rats given GlyNAC alone showed an improvement in Basso-Beattie-Bresnahan (BBB) scores, gait symmetry, and results in the open field test, indicative of improved motor function, while GlyNAC combined with BWSTT was more effective than either treatment alone at ameliorating voluntary motor function in injured rats. Meanwhile, the results of the skeletal muscle myofiber cross-sectional area (CSA), hindlimb grip strength, and acetylcholinesterase (AChE) immunostaining analysis demonstrated that GlyNAC improved the structure and function of the skeletal muscle in rats with SCI and delayed the atrophication of skeletal muscle.

Intracellular nutrient metabolism, particularly the metabolism of essential amino acids (EAAs), is crucial for cellular functions, including energy production and redox homeostasis. An EAA deficiency can lead to cellular dysfunction and oxidative stress. This study explores the mechanisms underlying cellular responses to EAA starvation, focusing on ROS-induced DNA damage and apoptosis. MC3T3-E1 cells were subjected to EAA starvation, and various assays were conducted to assess cell proliferation, survival, DNA damage, and apoptosis. The antioxidant N-acetylcysteine (NAC) was employed to block ROS formation and mitigate cellular damage. Gene expression and Western blot analyses were performed to elucidate molecular pathways. EAA starvation-induced ROS generation, DNA damage, and apoptosis in MC3T3-E1 cells. NAC administration effectively reduced DNA damage and apoptosis, highlighting the pivotal role of ROS in mediating these cellular responses during EAA deficiency. This study demonstrates that EAA starvation triggers ROS-mediated DNA damage and apoptosis, offering insights into the intricate interplay between nutrient deficiency, oxidative stress, and programmed cell death. NAC emerges as a potential therapeutic intervention to counteract these adverse effects.

Acetaminophen (APAP) hepatotoxicity is one of the biggest drawbacks of this relatively safe and widely used drug. In addition to its hepatotoxicity, APAP also cause comparable levels of toxicity on human hepatoma cells. Here we show activation of the intrinsic caspase-9/3 pathway of apoptosis followed by gasdermin E (GSDME) cleavage and subsequent ballooning in APAP (10 mM, 72 h)-treated Huh-7 human hepatocarcinoma cells. N-acetylcysteine (NAC), an antioxidant currently used as an antidote for APAP overdose, does not alleviate APAP toxicity in Huh-7 cells; NAC overdose (10 mM) rather aggravates APAP toxicity. NAC overdose not only aggravates cell death, but also decreases the cellular GSH/GSSG ratio, an indicator of redox homeostasis of glutathione. These results show for the first time that APAP-induced apoptosis in hepatoma cells is followed by secondary necrosis via the caspase-3/GSDME pathway. NAC overdose (10 mM) not only worsens the glutathione redox status, but also accelerates this pathway., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests.Toshihiko Aki reports financial support was provided by Tokyo Medical and Dental University. Toshihiko Aki reports financial support was provided by Tokyo Medical and Dental University. Toshihiko Aki reports a relationship with MEXT KAKENHI that includes: funding grants., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Periodontitis is a type of chronic inflammatory oral disease characterized by the destruction of periodontal connective tissue and progressive alveolar bone resorption. As oxidative stress is the key cause of periodontitis in the early periodontal microenvironment, antioxidative therapy has been considered a viable treatment for periodontitis. However, more stable and effective reactive oxygen species (ROS)-scavenging nanomedicines are still highly needed due to the instability of traditional antioxidants. Herein, a new type of N-acetyl-l-cysteine (NAC)-derived red fluorescent carbonized polymer dots (CPDs) has been synthesized with excellent biocompatibility, which can serve as an extracellular antioxidant to scavenge ROS effectively. Moreover, NAC-CPDs can promote osteogenic differentiation in human periodontal ligament cells (hPDLCs) under H 2 O 2 stimulation. In addition, NAC-CPDs are capable of targeted accumulation in alveolar bone in vivo, reducing the level of alveolar bone resorption in periodontitis mice, as well as performing fluorescence imaging in vitro and in vivo. In terms of mechanism, NAC-CPDs may regulate redox homeostasis and promote bone formation in the periodontitis microenvironment by modulating the kelch-like ECH-associated protein l (Keap1)/nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. This study provides a new strategy for the application of CPDs theranostic nanoplatform for periodontitis., (© 2023 Wiley-VCH GmbH.)

Aims: Type 1 diabetes mellitus (T1DM) has been linked to the occurrence of skeletal muscle atrophy. Insulin monotherapy may lead to excessive blood glucose fluctuations. N-acetylcysteine (NAC), a clinically employed antioxidant, possesses cytoprotective, anti-inflammatory, and antioxidant properties. The objective of our study was to evaluate the viability of NAC as a supplementary treatment for T1DM, specifically regarding its therapeutic and preventative impacts on skeletal muscle., Main Methods: Here, we used beagles as T1DM model for 120d to explore the mechanism of NRF2/HO-1-mediated skeletal muscle oxidative stress and apoptosis and the therapeutic effects of NAC. Oxidative stress and apoptosis related factors were analyzed by immunohistochemistry, immunofluorescence, western blotting, and RT-qPCR assay., Key Findings: The findings indicated that the co-administration of NAC and insulin led to a reduction in creatine kinase levels, preventing weight loss and skeletal muscle atrophy. Improvement in the reduction of muscle fiber cross-sectional area. The expression of Atrogin-1, MuRF-1 and MyoD1 was downregulated, while Myh2 and MyoG were upregulated. In addition, CAT and GSH-Px levels were increased, MDA levels were decreased, and redox was maintained at a steady state. The decreased of key factors in the NRF2/HO-1 pathway, including NRF2, HO-1, NQO1, and SOD1, while KEAP1 increased. In addition, the apoptosis key factors Caspase-3, Bax, and Bak1 were found to be downregulated, while Bcl-2, Bcl-2/Bax, and CytC were upregulated., Significance: Our findings demonstrated that NAC and insulin mitigate oxidative stress and apoptosis in T1DM skeletal muscle and prevent skeletal muscle atrophy by activating the NRF2/HO-1 pathway., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Intervertebral disc degeneration (IVDD) is associated with oxidative stress induced reactive oxygen species (ROS) dynamic equilibrium disturbance. Nanozymes, as nanomaterials with enzyme-like activity, can regulate intro-cellular ROS levels. In this study, a new carbon dots nanozyme, N-acetylcysteine-derived carbon dots (NAC-CDs), is developed and proved to be an ideal antioxidant and anti-senescent agent in IVDD management. The results confirmed the NAC-CDs have satisfactory biocompatibility and strong superoxide dismutase (250 U mg -1 ), catalase, glutathioneperoxidase-like activity, and total antioxidant capacity. Then, the powerful free radical scavenging and antioxidant ability of NAC-CDs are demonstrated in vitro as observing the reduced ROS in H 2 O 2 induced senescent nucleus pulposus cells (NPCs), in which the elimination efficiency of toxic ROS is more than 90%. NAC-CDs also maintained mitochondrial homeostasis and suppressed cellular senescence, subsequently inhibited the expression of inflammatory factors in NPCs. In vivo, evaluations of imaging and tissue morphology assessments suggested that disc height index, magnetic resonance imaging grade and histological score are significantly improved from the degenerative models when NAC-CDs is applied. In conclusion, the study developed a novel carbon dots nanozyme, which efficiently rescues IVDD from ROS induced NPCs senescence and provides a potential strategy in management of IVDD in clinic., (© 2023 Wiley-VCH GmbH.)

Mitochondrial Complex I dysfunction and oxidative stress have been part of the pathophysiology of several diseases ranging from mitochondrial disease to chronic diseases such as diabetes, mood disorders and Parkinson's Disease. Nonetheless, to investigate the potential of mitochondria-targeted therapeutic strategies for these conditions, there is a need further our understanding on how cells respond and adapt in the presence of Complex I dysfunction. In this study, we used low doses of rotenone, a classical inhibitor of mitochondrial complex I, to mimic peripheral mitochondrial dysfunction in THP-1 cells, a human monocytic cell line, and explored the effects of N-acetylcysteine on preventing this rotenone-induced mitochondrial dysfunction. Our results show that in THP-1 cells, rotenone exposure led to increases in mitochondrial superoxide, levels of cell-free mitochondrial DNA, and protein levels of the NDUFS7 subunit. N-acetylcysteine (NAC) pre-treatment ameliorated the rotenone-induced increase of cell-free mitochondrial DNA and NDUFS7 protein levels, but not mitochondrial superoxide. Furthermore, rotenone exposure did not affect protein levels of the NDUFV1 subunit but induced NDUFV1 glutathionylation. In summary, NAC may help to mitigate the effects of rotenone on Complex I and preserve the normal function of mitochondria in THP-1 cells., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. and Mitochondria Research Society. All rights reserved.)

Deferasirox (DFS) is used for the treatment of iron accumulation caused by the need for long-term blood transfusions, such as thalassemia or other rare anemia. Liver injury due to exposure to DFS has been documented, and the toxic mechanisms of DFS are unknown. The present study aimed to investigate the reactive metabolites of DFS in vitro and in vivo to help us understand the mechanisms of DFS hepatotoxicity. Two hydroxylated metabolites (5-OH and 5'-OH) were identified during incubation of DFS-supplemented rat liver microsomes. Such microsomal incubations fortified with glutathione (GSH) or N -acetylcysteine (NAC) as capture agents offered two GSH conjugates and two NAC conjugates. These GSH conjugates and NAC conjugates were also detected in bile and urine of rats given DFS. CYP1A2 and CYP3A4 were found to dominate the metabolic activation of DFS. Administration of DFS induced decreased cell survival in cultured primary hepatocytes. Pretreatment with ketoconazole and 1-aminobenzotrizole made hepatocytes less susceptible to the cytotoxicity of DFS.

Acrylamide (AA), a chemical compound currently classified as "reasonably anticipated to be a human carcinogen", is formed through the Maillard reaction in processed carbohydrate-rich foods and is also present in tobacco smoke. The primary sources of AA exposure in the general population are dietary intake and inhalation. Within a 24-h period, humans eliminate approximately 50% of AA in the urine, predominantly in the form of mercapturic acid conjugates such as N-acetyl-S-(2-carbamoylethyl)-L-cysteine (AAMA), N-acetyl-S-(2-carbamoyl-2- hydroxyethyl)-L-cysteine (GAMA3), and N-acetyl-3-[(3-amino-3-oxopropyl)sulfinyl]-L-alanine (AAMA-Sul). These metabolites serve as short-term biomarkers for AA exposure in human biomonitoring studies. In this study, we analysed first-morning urine samples from the adult population (aged 18-65 years) residing in the Valencian Region, Spain, (n = 505). AAMA, GAMA-3 and AAMA-Sul were quantified in 100% of the analysed samples, with geometric means (GM) of 84, 11 and 26 μg L -1 , respectively, while the estimated daily intake of AA in the studied population ranged from 1.33 to 2.13 μg·kg-bw -1 ·day -1 (GM). Statistical analysis of the data indicated that the most significant predictors of AA exposure were smoking and the amount of potato fried products and, biscuits and pastries consumed last 24 h. Based on risk assessment approaches conducted, the findings suggest that exposure to AA could pose a potential health risk. Therefore, it is crucial to closely monitor and continuously evaluate AA exposure to ensure the well-being of the population., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Borja Peris Camarasa reports financial support was provided by Government of Valencia., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Trichloroethylene (TCE) is a known human carcinogen with toxicity attributed to its metabolism. S-(1,2-Dichlorovinyl)-L-cysteine (DCVC) is a metabolite of TCE formed downstream in TCE glutathione (GSH) conjugation and is upstream of several toxic metabolites. Despite knowledge that DCVC stimulates reactive oxygen species (ROS) generation and apoptosis in placental cells, the extent to which these outcomes are attributable to DCVC metabolism is unknown. The current study used N-acetyl-L-cysteine (NAC) at 5 mM and aminooxyacetic acid (AOAA) at 1 mM as pharmacological modifiers of DCVC metabolism to investigate DCVC toxicity at concentrations of 5-50 µM in the human placental trophoblast BeWo cell model capable of forskolin-stimulated syncytialization. Exposures of unsyncytialized BeWo cells, BeWo cells undergoing syncytialization, and syncytialized BeWo cells were studied. NAC pre/co-treatment with DCVC either failed to inhibit or exacerbated DCVC-induced H 2 O 2 abundance, PRDX2 mRNA expression, and BCL2 mRNA expression. Although NAC increased mRNA expression of CYP3A4, which would be consistent with increased generation of the toxic metabolite N-acetyl-DCVC sulfoxide (NAcDCVCS), a CYP3A4 inhibitor ketoconazole did not significantly alter BeWo cell responses. Moreover, AOAA failed to inhibit cysteine conjugate β-lyase (CCBL), which bioactivates DCVC, and did not affect the percentage of nuclei condensed or fragmented, a measure of apoptosis, in all BeWo cell models. However, syncytialized cells had higher CCBL activity compared to unsyncytialized cells, suggesting that the former may be more sensitive to DCVC toxicity. Together, although neither NAC nor AOAA mitigated DCVC toxicity, differences in CCBL activity and potentially CYP3A4 expression dictated the differential toxicity derived from DCVC., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Ulcerative colitis (UC) is a chronic and recurrent inflammatory bowel disease (IBD) characterized by continuous inflammation in the colonic mucosa. Extraintestinal manifestations (EIM) occur due to the disruption of the intestinal barrier and increased permeability caused by redox imbalance, dysbiosis, and inflammation originating from the intestine and contribute to morbidity and mortality. The aim of this study is to investigate the effects of oral N -acetylcysteine (NAC) on colonic, hepatic, and renal tissues in mice with colitis induced by dextran sulfate sodium (DSS). Male Swiss mice received NAC (150 mg/kg/day) in the drinking water for 30 days before and during (DSS 5% v/v; for 7 days) colitis induction. On the 38 th day, colon, liver, and kidney were collected and adequately prepared for the analysis of oxidative stress (superoxide dismutase (SOD), catalase (CAT), glutathione reduced (GSH), glutathione oxidized (GSSG), malondialdehyde (MDA), and hydrogen peroxide (H 2 O 2 )) and inflammatory biomarkers (myeloperoxidase (MPO) -, tumor necrosis factor alpha - (TNF- α , and interleukin-10 (IL-10)). In colon, NAC protected the histological architecture. However, NAC did not level up SOD, in contrast, it increased MDA and pro-inflammatory effect (increased of TNF- α and decreased of IL-10). In liver, colitis caused both oxidative (MDA, SOD, and GSH) and inflammatory damage (IL-10). NAC was able only to increase GSH and GSH/GSSG ratio. Kidney was not affected by colitis; however, NAC despite increasing CAT, GSH, and GSH/GSSG ratio promoted lipid peroxidation (increased MDA) and pro-inflammatory action (decreased IL-10). Despite some beneficial antioxidant effects of NAC, the negative outcomes concerning irreversible oxidative and inflammatory damage in the colon, liver, and kidney confirm the nonsafety of the prophylactic use of this antioxidant in models of induced colitis, suggesting that additional studies are needed, and its use in humans not yet recommended for the therapeutic routine of this disease., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2023 Amylly Sanuelly da Paz Martins et al.)

Growing evidence suggests that the depletion of plasma NAD + and glutathione (GSH) may play an important role in the development of metabolic disorders. The administration of Combined Metabolic Activators (CMA), consisting of GSH and NAD + precursors, has been explored as a promising therapeutic strategy to target multiple altered pathways associated with the pathogenesis of the diseases. Although studies have examined the therapeutic effect of CMA that contains N-acetyl-l-cysteine (NAC) as a metabolic activator, a system-wide comparison of the metabolic response to the administration of CMA with NAC and cysteine remains lacking. In this placebo-controlled study, we studied the acute effect of the CMA administration with different metabolic activators, including NAC or cysteine with/without nicotinamide or flush free niacin, and performed longitudinal untargeted-metabolomics profiling of plasma obtained from 70 well-characterized healthy volunteers. The time-series metabolomics data revealed the metabolic pathways affected after the administration of CMAs showed high similarity between CMA containing nicotinamide and NAC or cysteine as metabolic activators. Our analysis also showed that CMA with cysteine is well-tolerated and safe in healthy individuals throughout the study. Last, our study systematically provided insights into a complex and dynamics landscape involved in amino acid, lipid and nicotinamide metabolism, reflecting the metabolic responses to CMA administration containing different metabolic activators., Competing Interests: Declaration of competing interest A.M., J.B., and M.U. are the founder and shareholders of ScandiBio Therapeutics and they filed a patent application on the use of CMA to treat patients with different diseases. The other authors declare no conflict of interest., (Crown Copyright © 2023. Published by Elsevier Inc. All rights reserved.)

Multiple organs, including the testes, are damaged by iron overload. It has been shown that N-acetyl cysteine (NAC) influences oxidative stress in iron overload. The present study aimed to evaluate the roles of acetylated peptide (AOP) and NAC in the inhibition of iron-overload induced-testicular damage. At the beginning of the experiment, NAC (150 mg /kg) was given for a week to all 40 rats. Then, four groups were formed by dividing the animals (10 rats/group). Group I included healthy control rats. Group II (iron overload) was given intraperitoneal iron dextran (60 mg/kg/day) 5 days a week for 4 weeks. Group III (NAC) was given NAC orally at a dose of 150 mg/kg/day for 4 weeks in addition to iron dextran. Group IV (AOP) was given AOP orally at a dose of 150 mg/kg/day for 4 weeks besides iron dextran. When the experiment time was over, testosterone serum level, testicular B cell lymphoma-2 (BCL-2) and protein kinase B (PKB) protein levels, nuclear factor kappa-B (NF-κB), and Beclin1 mRNA expression levels, and malondialdehyde (MDA), and reduced glutathione (GSH) were determined by ELISA, quantitative reverse transcription-PCR, and chemical methods. Finally, histopathological examinations and immunohistochemical detection of claudin-1 and CD68 were performed. The iron overload group exhibited decreased testosterone, BCL-2, PKB, claudin-1, and GSH and increased MDA, NF-κB, Beclin1, and CD68, while both NAC and AOP treatments protected against the biochemical and histopathological disturbances occurring in the iron overload model. We concluded that NAC and AOP can protect against testes damage by iron overload via their antioxidant, anti-inflammatory, antiapoptotic, and ant-autophagic properties. The NAC and AOP may be used as preventative measures against iron overload-induced testicular damage., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

In previous work, we showed that cancer cells do not depend on glycolysis for ATP production, but they do on fatty acid oxidation. However, we found some cancer cells induced cell death after glucose deprivation along with a decrease of ATP production. We investigated the different response of glucose deprivation with two types of cancer cells including glucose insensitive cancer cells (GIC) which do not change ATP levels, and glucose sensitive cancer cells (GSC) which decrease ATP production in 24 h. Glucose deprivation-induced cell death in GSC by more than twofold after 12 h and by up to tenfold after 24 h accompanied by decreased ATP production to compare to the control (cultured in glucose). Glucose deprivation decreased the levels of metabolic intermediates of the pentose phosphate pathway (PPP) and the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) in both GSC and GIC. However, glucose deprivation increased reactive oxygen species (ROS) only in GSC, suggesting that GIC have a higher tolerance for decreased NADPH than GSC. The twofold higher ratio of reduced/oxidized glutathione (GSH/GSSG) in GIS than in GSC correlates closely with the twofold lower ROS levels under glucose starvation conditions. Treatment with N-acetylcysteine (NAC) as a precursor to the biologic antioxidant glutathione restored ATP production by 70% and reversed cell death caused by glucose deprivation in GSC. The present findings suggest that glucose deprivation-induced cancer cell death is not caused by decreased ATP levels, but rather triggered by a failure of ROS regulation by the antioxidant system. Conclusion is clear that glucose deprivation-induced cell death is independent from ATP depletion-induced cell death.

Objective: Periostin is important for the maintenance of periodontal tissue, but its role in periodontitis is controversial. This research investigated the effect of periostin in periodontitis and the underlying mechanism., Design: Mouse periodontitis models in vivo and inflammation model in vitro which were induced by Porphyromonas gingivalis lipopolysaccharide were established to evaluate periostin expression. Human periodontal ligament fibroblasts (PDLFs) were treated with lipopolysaccharide and N-acetylcysteine, fluorescence staining, flow cytometry, Western blot, and qRT-PCR were used to detect reactive oxygen species (ROS), periostin expression, and apoptosis-related makers. The periostin gene was successfully transfected into PDLFs to verify the effect of periostin on apoptosis. Then, the Nrf2 inhibitor was added to clarify the mechanism., Results: Periostin expression decreased in the periodontal ligaments of mouse periodontitis models and lipopolysaccharide-induced PDLFs. Lipopolysaccharide promoted the activation of ROS and apoptosis in PDLFs, whereas N-acetylcysteine reversed this condition. Overexpression of periostin suppressed apoptosis of PDLFs and reversed the inhibitory effect of lipopolysaccharide on nuclear Nrf2 expression. Moreover, the Nrf2 inhibitor attenuated the protective effect of periostin on lipopolysaccharide-induced apoptosis., Conclusions: Lipopolysaccharide induced apoptosis in PDLFs by inhibiting periostin expression and thus Nrf2/HO-1 pathway, indicating that periostin could be a potential therapeutic target for periodontitis., (© 2022 Wiley Periodicals LLC.)

Our previous study showed 1-Nitropyrene (1-NP) exposure disrupted testicular testosterone synthesis in mouse, but the exact mechanism needs further investigation. The present research found 4-phenylbutyric acid (4-PBA), an endoplasmic reticulum (ER) stress inhibitor, recovered 1-NP-induced ER stress and testosterone synthases reduction in TM3 cells. GSK2606414, a protein kinase-like ER kinase (PERK) kinase inhibitor, attenuated 1-NP-induced PERK-eukaryotic translation initiation factor 2α (eIF2α) signaling activation and downregulation of steroidogenic proteins in TM3 cells. Both 4-PBA and GSK2606414 attenuated 1-NP-induced steroidogenesis disruption in TM3 cells. Further studies used N-Acetyl-L-cysteine (NAC) as a classical antioxidant to explore whether oxidative stress-activated ER stress mediated 1-NP-induced testosterone synthases reduction and steroidogenesis disruption in TM3 cells and mouse testes. The results showed NAC pretreatment mitigated oxidative stress, and subsequently attenuated ER stress, particularly PERK-eIF2α signaling activation, and downregulation of testosterone synthases in 1-NP-treated TM3 cells. More importantly, NAC extenuated 1-NP-induced testosterone synthesis in vitro and in vivo. The current work indicated that oxidative stress-caused ER stress, particularly PERK-eIF2α pathway activation, mediates 1-NP-downregulated steroidogenic proteins and steroidogenesis disruption in TM3 cells and mouse testes. Significantly, the current study provides a theoretical basis and demonstrates the experimental evidence for the potential application of antioxidant, such as NAC, in public health prevention, particularly in 1-NP-induced endocrine disorder., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Cryopreservation can cause mechanical and chemical stress, ultimately leading to the formation of reactive oxygen species (ROS) and oxidative stress. ROS inhibits the expression of antioxidant enzymes in cells, resulting in increased DNA fragmentation and apoptosis. In this paper, we used a vitrification method that has the advantage of producing less ice crystal formation, cost-effectiveness, and time efficiency during cryopreservation. The objective of this paper is to evaluate the degree of protection of ovarian tissue against oxidative stress when N-acetylcysteine (NAC) and Klotho proteins are treated in the vitrification process of ovarian tissue.  METHODS: The control group and the cryopreservation groups were randomly assigned, and treated NAC, Klotho, or the combination (NAC + Klotho). The cell morphological change, DNA damage, senescence, and apoptosis of each group after the freeze-thaw process were compared using transmission electron microscopy, immunohistochemistry, and western blot analysis., Results: Both NAC and Klotho were found to be more effective at protecting against DNA damage than the control; however, DNA damage was greater in the NAC + Klotho group than in the group treated with NAC and Klotho, respectively. DNA damage and cellular senescence were also reduced during the vitrification process when cells were treated with NAC, Klotho, or the combination (NAC + Klotho). NAC increased apoptosis during cryopreservation, whereas Klotho inhibited apoptosis and NAC-induced apoptosis., Conclusion: This study highlights Klotho's benefits in inhibiting DNA damage, cell senescence, and apoptosis, including NAC-induced apoptosis, despite its unclear role in vitrification., (© 2023. Korean Tissue Engineering and Regenerative Medicine Society.)

Introduction: Platelet function, rather than platelet count, plays a crucial role in thrombosis in essential thrombocythemia (ET). However, little is known about the abnormal function of platelets in ET. Here, we investigated the functional characteristics of platelets in ET hemostasis to explore the causes of ET platelet dysfunction and new therapeutic strategies for ET., Materials and Methods: We analyzed platelet aggregation, activation, apoptosis, and reactive oxygen species (ROS) in ET patients and JAK2V617F-positive ET-like mice. The effects of ROS on platelet function and the underlying mechanism were investigated by inhibiting ROS using N-acetylcysteine (NAC)., Results: Platelet aggregation, activation, apoptosis, ROS, and clot retraction were elevated in ET. No significant differences were observed between ET patients with JAK2V617F or CALR mutations. Increased ROS activated the JAK-STAT pathway, which may further influence platelet function. Inhibition of platelet ROS by NAC reduced platelet aggregation, activation, and apoptosis, and prolonged bleeding time. Furthermore, NAC treatment reduced platelet count in ET-like mice by inhibiting platelet production from megakaryocytes., Conclusions: Elevated ROS in ET platelets resulted in enhanced platelet activation, function and increased risk of thrombosis. NAC offers a potential therapeutic strategy for reducing platelet count., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Gestational diabetes mellitus (GDM) is a common pregnant disorder that needs careful medical attention. N-acetyl-L-cysteine (NAC) is a well-recognized antioxidant to treat oxidative stress-induced diseases. Although its role in GDM has been reported, the mechanism remains largely unknown. Therefore, our current study further explored its protective role against GDM. An 8-week-old, db/+, female mice were injected with a single dose of 100 mg/kg streptozotocin (STZ) to induce diabetes. Pregnant mice were randomly treated with 1200 mg/kg NAC or water daily. On gestational day 19, oral glucose tolerance test was performed, and visceral fat tissue and blood samples were collected. After delivery, litter size and weight were recorded. NAC could effectively improve GDM-induced glucose intolerance, hyperlipidemia, activated inflammation, and oxidative stress in GDM mice. Moreover, NAC reduced the litter size and weight of GDM mice. NAC also activated the nuclear factor-erythroid factor 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway in the liver of GDM mice. NAC effectively ameliorated GDM symptoms and the reproductive outcome of GDM mice through inhibiting oxidative stress, inflammation, and hyperlipidemia. Therefore, NAC might serve as a potential candidate drug against GDM., (© 2022 International Union of Biochemistry and Molecular Biology, Inc.)

Engineered nanosystems offer a promising strategy for macrophage-targeted therapies for various diseases, and their physicochemical parameters including surface-active ligands, size and shape are widely investigated for improving their therapeutic efficacy. However, little is known about the synergistic effect of elasticity and surface-active ligands. Here, two kinds of anti-inflammatory N-acetylcysteine (NAC)-loaded macrophage-targeting apoptotic-cell-inspired phosphatidylserine (PS)-containing nano-liposomes (PSLipos) were constructed, which had similar size and morphology but different Young's modulus (E) (H, ~ 100 kPa > E macrophage vs. L, ~ 2 kPa < E macrophage ). Interestingly, these PSLipos-NAC showed similar drug loading and encapsulation efficiency, and in vitro slow-release behavior of NAC, but modulus-dependent interactions with macrophages. Softer PSLipos-L-NAC could resist macrophage capture, but remarkably prolong their targeting effect period on macrophages via durable binding to macrophage surface, and subsequently more effectively suppress inflammatory response in macrophages and then hasten inflammatory lung epithelial cell wound healing. Especially, pulmonary administration of PSLipos-L-NAC could significantly reduce the inflammatory response of M1-like macrophages in lung tissue and promote lung injury repair in a bleomycin-induced acute lung injury (ALI) mouse model, providing a potential therapeutic approach for ALI. The results strongly suggest that softness may enhance ligand-directed macrophage-mediated therapeutic efficacy of nanosystems, which will shed new light on the design of engineered nanotherapeutics., (© 2023. The Author(s).)

Bone marrow endothelial cells (BMECs) play a crucial role in the maintenance of bone homeostasis. The decline in BMECs is associated with abnormal bone development and loss. At present, the mechanism of age-related oxidative stress enhancement in BMEC dysfunction remains unclear. Our experiment explored injury caused by oxidative stress enhancement in BMECs both in vivo and in vitro. The BMECs, indicators of oxidative stress, bone mass, and apoptosis-related proteins were analyzed in different age groups. We also evaluated the ability of N-Acetyl-L-cysteine (NAC) attenuate oxidative stress injury in BMECs. NAC treatment attenuated reactive oxygen species (ROS) overgeneration and apoptosis in BMECs in vitro and alleviated the loss of BMECs and bone mass in vivo. In conclusion, this study could improve our understanding of the mechanism of oxidative stress-induced BMECs injury and whether NAC has therapeutic potential in senile osteoporosis., Competing Interests: Declaration of competing interest Authors declare no conflict of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Sows are highly sensitive to deoxynivalenol (DON) and susceptible to reproductive toxicity caused by oxidative stress, but the potential mechanisms and effective interventions remain unclear. Here, we investigated the role of two antioxidants (cysteamine and N -acetyl-cysteine) in regulating the reproductive performance, redox status, and placental barrier function of sows and their potential mechanisms under DON exposure. Maternal dietary supply of antioxidants from day 85 of gestation to parturition reduced the incidence of stillbirths and low-birth-weight piglets under DON exposure. Moreover, the alleviation of DON-induced reproductive toxicity by dietary antioxidants was associated with the alleviation of placental oxidative stress, the enhancement of the placental barrier, and the vascular function of sows. Furthermore, in vivo and in vitro vascularized placental barrier modeling further demonstrated that antioxidants could reverse both DON transport across the placenta and DON-induced increase of placental barrier permeability. The molecular mechanism of antioxidant resistance to DON toxicity may be related to the signal transducer and activator of the transcription-3-occludin/zonula occludens-1 signaling pathway. Collectively, these results demonstrate the potential of antioxidants to protect the mother from DON-induced reproductive toxicity by alleviating placental oxidative stress and enhancing the placental barrier.

N-acetyl cysteine (NAC) is a nutritional supplement and greatly applied as an antioxidant in vivo and in vitro . Therefore, this study aimed to assess the metabolic and antioxidant protective effect of NAC against selenium (Se) toxicity and gamma irradiation in rats by measuring biochemical and molecular parameters. This study was conducted on sixty rats divided into six equal different groups; control, NAC, Rad, Se, Rad + NAC, and Se + NAC groups. Oxidative/nitrosative makers (LPO, NO, and NOS), antioxidants status markers (GSH, GPx, and SOD), liver metabolic markers (LDH, SDH, and ATP), and plasma metabolic markers (Glucose, total cholesterol, and total proteins) were measured using commercial colorimetric kits while plasma corticosterone concentration was measured using commercial ELISA kit. Also, Levels of NR3C1 and Glut-2 genes expression using reverse transcription-quantitative polymerase chain reaction were done. Our results revealed that Se toxicity and gamma irradiation induced significant increases in oxidative/nitrosative stress markers and a significant decrease in antioxidant status markers in the liver and adrenal tissues. Moreover, metabolic disorders were recorded as manifested by elevation of plasma ALT, Albumin, glucose and cholesterol, and decrease in protein levels associated with a significant increase in corticosterone concentration. This was also accompanied by a significant decrease in SDH activity and ATP production in the hepatic tissue. Molecular analysis showed a marked increase in NR3C1 mRNA and decrease in Glut-2 mRNA in liver tissue. However, NAC supplementation attenuated the changes induced by these toxins. Finally, we could conclude that, oral supplementation of NAC can modulate the metabolic disturbances and has protective effects in rats exposed to Se toxicity and gamma irradiation.

Betel quid and areca nut are complex mixture carcinogens, but little is known about whether their derived single-agent arecoline or arecoline N -oxide (ANO) is carcinogenic, and the underlying mechanisms remain unclear. In this systematic review, we analyzed recent studies on the roles of arecoline and ANO in cancer and strategies to block carcinogenesis. In the oral cavity, flavin-containing monooxygenase 3 oxidizes arecoline to ANO, and both alkaloids conjugate with N -acetylcysteine to form mercapturic acid compounds, which are excreted in urine, reducing arecoline and ANO toxicity. However, detoxification may not be complete. Arecoline and ANO upregulated protein expression in oral cancer tissue from areca nut users compared to expression levels in adjacent normal tissue, suggesting a causal relationship between these compounds and oral cancer. Sublingual fibrosis, hyperplasia, and oral leukoplakia were diagnosed in mice subjected to oral mucosal smearing of ANO. ANO is more cytotoxic and genotoxic than arecoline. During carcinogenesis and metastasis, these compounds increase the expression of epithelial-mesenchymal transition (EMT) inducers such as reactive oxygen species, transforming growth factor-β1, Notch receptor-1, and inflammatory cytokines, and they activate EMT-related proteins. Arecoline-induced epigenetic markers such as sirtuin-1 hypermethylation, low protein expression of miR-22, and miR-886-3-p accelerate oral cancer progression. Antioxidants and targeted inhibitors of the EMT inducers used reduce the risk of oral cancer development and progression. Our review findings substantiate the association of arecoline and ANO with oral cancer. Both of these single compounds are likely carcinogenic to humans, and their mechanisms and pathways of carcinogenesis are useful indicators for cancer therapy and prognosis.

Carcinogen and toxicant uptake by e-cigarette users have not been fully evaluated. In the study reported here, we recruited 30 e-cigarette users, 63 nonsmokers, and 33 cigarette smokers who gave monthly urine samples over a period of 4-6 months. Their product use status was confirmed by measurements of exhaled CO, urinary total nicotine equivalents, cyanoethyl mercapturic acid (CEMA), and total 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol. Urinary biomarkers of exposure to the carcinogens acrolein (3-hydroxypropyl mercapturic acid, 3-HPMA), benzene ( S -phenyl mercapturic acid, SPMA), acrylonitrile (CEMA), and a combination of crotonaldehyde, methyl vinyl ketone, and methacrolein (3-hydroxy-1-methylpropyl mercapturic acid, HMPMA) were quantified at each visit. Data from subject visits with CEMA > 27 pmol/mL were excluded from the statistical analysis of the results because of possible unreported exposures to volatile combustion products such as secondhand cigarette smoke or marijuana smoke exposure; this left 22 e-cigarette users with 4 or more monthly visits and all 63 nonsmokers. Geometric mean levels of 3-HPMA (1249 versus 679.3 pmol/mL urine) were significantly higher ( P = 0.003) in e-cigarette users than in nonsmokers, whereas levels of SPMA, CEMA, and HMPMA did not differ between these two groups. All analytes were significantly higher in cigarette smokers than in either e-cigarette users or nonsmokers. The results of this unique multimonth longitudinal study demonstrate consistent significantly higher uptake of the carcinogen acrolein in e-cigarette users versus nonsmokers, presenting a warning signal regarding e-cigarette use.

Introduction: Respiratory syncytial virus (RSV) is one of the most common causes of acute lower respiratory infection in infants and young children. Mucolytic agents, such as acetylcysteine and carbocysteine have reported benefits in alleviating acute upper or lower respiratory infections. Among these, N-acetylcysteine (NAC) has cyto-protective effects when cells are infected with the RSV., Materials and Methods: Our study investigated primarily the dose-dependent effects of NAC on respiratory alveolar epithelial (A549) cells when co-cultured with RSV in vitro. Three different concentrations of NAC were used, 0.1 mM, 1 mM, and 10 mM. The cytotoxicity of RSV-infected cells was measured by lactate dehydrogenase and antiviral activity of NAC on cell cultures was evaluated by immunofluorescence., Results: Pre-treatment with the highest dose, 10 mM NAC, resulted in features of cell injury even without RSV infection. The proportion of cells infected by RSV and RSV-induced cell death decreased by more than 3-fold when cells were pre-treated with 1 mM NAC. Pre-treatment at the lowest dose, 0.1 mM, did not show any significant changes., Conclusion: A moderate dose of NAC (1 mM) appeared protective of RSV infection to lung alveolar epithelial cells. However, a higher dose of NAC (10 mM) may be relatively toxic and injurious to these cells.

Cultured meat is an efficient, safe and sustainable meat production technology. Adipose-derived stem cell (ADSC) is a promising cell type for cultured meat. In vitro, obtaining numerous of ADSCs is a pivotal step for cultured meat. In this research, we demonstrated that the proliferation and adipogenic differentiation of ADSCs significantly decreased during serial passage. Then, senescence β-galactosidase (SA-β-gal) staining showed that the positive rate of P9 ADSCs was 7.74-fold than P3 ADSCs. Subsequently, RNA sequencing (RNA-seq) was performed for P3 and P9 ADSCs and found that PI3K-AKT pathway was up-regulated, but cell cycle and DNA repair pathway were down-regulated in P9 ADSCs. Then, N-Acetylcysteine (NAC) was added during long-term expansion and showed that NAC enhanced the ADSCs proliferation and maintained adipogenic differentiation. Finally, RNA-seq was performed for P9 ADSCs cultured with or without NAC and showed that NAC restored the cell cycle and DNA repair pathway in P9 ADSCs. These results highlighted that NAC was an excellent supplement for large-scale expansion of porcine ADSCs for cultured meat., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Copper avidly binds thiols and is redox active, and it follows that one element of copper toxicity may be the generation of undesirable disulfide bonds in proteins. In the present study, copper oxidized the model thiol N-acetylcysteine in vitro. Alkaline phosphatase (AP) requires disulfide bonds for activity, and copper activated reduced AP both in vitro and when it was expressed in the periplasm of mutants lacking their native disulfide-generating system. However, AP was not activated when it was expressed in the cytoplasm of copper-overloaded cells. Similarly, this copper stress failed to activate OxyR, a transcription factor that responds to the creation of a disulfide bond. The elimination of cellular disulfide-reducing systems did not change these results. Nevertheless, in these cells, the cytoplasmic copper concentration was high enough to impair growth and completely inactivate enzymes with solvent-exposed [4Fe-4S] clusters. Experiments with N-acetylcysteine determined that the efficiency of thiol oxidation is limited by the sluggish pace at which oxygen regenerates copper(II) through oxidation of the thiyl radical-Cu(I) complex. We conclude that this slow step makes copper too inefficient a catalyst to create disulfide stress in the thiol-rich cytoplasm, but it can still impact the few thiol-containing proteins in the periplasm. It also ensures that copper accumulates intracellularly in the Cu(I) valence., (© 2023 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

Pichia pastoris is widely used for the production of recombinant proteins, but the low secretion efficiency hinders its wide application in biopharmaceuticals. Our previous study had shown that N -acetyl-l-cysteine (NAC) promotes human serum albumin and porcine follicle-stimulating hormone fusion protein (HSA-pFSHβ) secretion by increasing intracellular GSH levels, but the downstream impact mechanism is not clear. In this study, we investigated the roles of autophagy as well as cell phenotype in NAC promoting HSA-pFSHβ secretion. Our results showed that NAC slowed down the cell growth rate, and its effects were unaffected by Congo Red and Calcofluor White. Moreover, NAC affected cell wall composition by increasing chitin content and decreasing β-1,3-glucan content. In addition, the expressions of vesicular pathway and autophagy-related genes were significantly decreased after NAC treatment. Further studies revealed that autophagy, especially the cytoplasm-to-vacuole targeting (Cvt) pathway, mitophagy and pexophagy, was significantly increased with time, and NAC has a promoting effect on autophagy, especially at 48 h and 72 h of NAC treatment. However, the disruption of mitophagy receptor Atg32, but not pexophagy receptor Atg30, inhibited HSA-pFSHβ production, and neither of them inhibited the NAC-promoted effect of HSA-pFSHβ. In conclusion, vesicular transport, autophagy and cell wall are all involved in the NAC-promoted HSA-pFSHβ secretion and that disruption of the autophagy receptor alone does not inhibit the effect of NAC.