Hepatoprotective effects of zingerone on sodium arsenite-induced hepatotoxicity in rats: Modulating the levels of caspase-3/Bax/Bcl-2, NLRP3/NF-κB/TNF-α and ATF6/IRE1/PERK/GRP78 signaling pathways.

OBJECTIVE: Long-term exposure to arsenic has been linked to several illnesses, including hypertension, diabetes, hepatic and renal diseases and cardiovascular malfunction. The aim of the current investigation was to determine whether zingerone (ZN) could shield rats against the hepatotoxicity that sodium arsenite (SA) causes. METHODS: The following five groups of thirty-five male Sprague Dawley rats were created: I) Control; received normal saline, II) ZN; received ZN, III) SA; received SA, IV) SA + ZN 25; received 10 mg/kg body weight SA + 25 mg/kg body weight ZN, and V) SA + ZN 50; received 10 mg/kg body weight SA + 50 mg/kg body weight ZN. The experiment lasted 14 days, and the rats were sacrificed on the 15th day. While oxidative stress parameters were studied by spectrophotometric method, apoptosis, inflammation and endoplasmic reticulum stress parameters were measured by RT-PCR method. RESULTS: The SA disrupted the histological architecture and integrity of the liver and enhanced oxidative damage by lowering antioxidant enzyme activity, such as those of glutathione peroxidase (GPx), catalase (CAT), superoxide dismutase (SOD), glutathione (GSH) level and increasing malondialdehyde (MDA) level in the liver tissue. Additionally, SA increased the mRNA transcript levels of Bcl2 associated x (Bax), caspases (-3, -6, -9), apoptotic protease-activating factor 1 (Apaf-1), p53, tumor necrosis factor-α (TNF-α), nuclear factor kappa B (NF-κB), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), c-Jun NH2-terminal kinase (JNK), mitogen-activated protein kinase 14 (MAPK14), MAPK15, receptor for advanced glycation endproducts (RAGE) and nod-like receptor family pyrin domain-containing 3 (NLRP3) in the liver tissue. Also produced endoplasmic reticulum stress by raising the mRNA transcript levels of activating transcription factor 6 (ATF-6), protein kinase RNA-like ER kinase (PERK), inositol-requiring enzyme 1 (IRE1), and glucose-regulated protein 78 (GRP-78). These factors together led to inflammation, apoptosis, and endoplasmic reticulum stress. On the other hand, liver tissue treated with ZN at doses of 25 and 50 mg/kg showed significant improvement in oxidative stress, inflammation, apoptosis and endoplasmic reticulum stress. CONCLUSIONS: Overall, the study's data suggest that administering ZN may be able to lessen the liver damage caused by SA toxicity.

The ameliorative effect of carvacrol on sodium arsenite-induced hepatotoxicity in rats: Possible role of Nrf2/HO-1, RAGE/NLRP3, Bax/Bcl-2/Caspase-3, and Beclin-1 pathways.

Arsenic is a toxic environmental pollutant heavy metal, and one of its critical target tissues in the body is the liver. Carvacrol is a natural phytocompound that stands out with its antioxidant, anti-inflammatory, and antiapoptotic properties. The current study aims to investigate the protective feature of carvacrol against sodium arsenite-induced liver toxicity. Thirty-five Sprague-Dawley male rats were divided into five groups: Control, Sodium arsenite (SA), CRV, SA + CRV25, and SA + CRV50. Sodium arsenite was administered via oral gavage at a dose of 10 mg/kg for 14 days, and 30 min later, CRV 25 or 50 mg/kg was administered via oral gavage. Oxidative stress, inflammation, apoptosis, autophagy damage pathways parameters, and liver tissue integrity were analyzed using biochemical, molecular, western blot, histological, and immunohistological methods. Carvacrol decreased sodium arsenite-induced oxidative stress by suppressing malondialdehyde levels and increasing superoxide dismutase, catalase, glutathione peroxidase activities, and glutathione levels. Carvacrol reduced inflammation damage by reducing sodium arsenite-induced increased levels of NF-κB and the cytokines (TNF-α, IL-1ß, IL-6, RAGE, and NLRP3) it stimulates. Carvacrol also reduced sodium arsenite-induced autophagic (Beclin-1, LC3A, and LC3B) and apoptotic (P53, Apaf-1, Casp-3, Casp-6, Casp-9, and Bax) parameters. Carvacrol preserved sodium arsenite-induced impaired liver tissue structure. Carvacrol alleviated toxic damage by reducing sodium arsenite-induced increases in oxidative stress, inflammation, apoptosis, and autophagic damage parameters in rat liver tissues. Carvacrol was also beneficial in preserving liver tissue integrity.

Hydrogen sulfide regulates arsenic-induced cell death in yeast cells by modulating the antioxidative system.

Arsenic (As) is a metal with potentially toxic effects on different organisms. Hydrogen sulfide (H2S) plays a vital role in mitigating heavy metal toxicity by reducing oxidative stress in plants and animals. However, the role of H2S in alleviating arsenic toxicity in yeast cells remains unclear. In this study, the role of NaHS (exogenous physiological H2S) in alleviating As-induced yeast cell death was investigated. Yeast cells in the logarithmic phase were pretreated with 0.05 mmol/L NaHS for 6 h, and then incubated in the YPD medium with or without 1 mmol/L As. After 12 h of treatment, relative survival rate, H2S content, oxidative stress biomarkers, and antioxidant machinery were measured. Our results showed that sodium arsenite-induced yeast cell death and pretreatment with 0.05 mmol/L NaHS significantly alleviated sodium arsenite-induced cell death. Under sodium arsenite conditions, the levels of intracellular reactive oxygen species (ROS) and malondialdehyde (MDA) increased, accompanied by the inhibition of the catalase (CAT) activity and the downregulation of CTT1 expression. However, the activities of the superoxide dismutase (SOD) and glutathion peroxidase (GPX) increased, and the expression of SOD1 and GPX2 was markedly upregulated in the group treated with sodium arsenite. When yeast cells were pretreated with NaHS, the intracellular ROS and MDA levels decreased significantly, and the activities of SOD, CAT, and GPX increased significantly. This was associated with a significant increase in relative survival rate and H2S content compared to the arsenic treatment alone. Our findings indicate that NaHS alleviates sodium arsenite-induced yeast cell death, mainly by enhancing the antioxidant defense system.

Sodium arsenite induces hepatic stellate cells activation by m6A modification of TGF-ß1 during liver fibrosis.

The compound known as Sodium arsenite (NaAsO2), which is a prevalent type of inorganic arsenic found in the environment, has been strongly associated with liver fibrosis (LF), a key characteristic of nonalcoholic fatty liver disease (NAFLD), which has been demonstrated in our previous study. Our previous research has shown that exposure to NaAsO2 triggers the activation of hepatic stellate cells (HSCs), a crucial event in the development of LF. However, the molecular mechanism is still unknown. N6-methyladenosine (m6A) modification is the most crucial post-transcriptional modification in liver disease. Nevertheless, the precise function of m6A alteration in triggering HSCs and initiating LF caused by NaAsO2 remains unknown. Here, we found that NaAsO2 induced LF and HSCs activation through TGF-ß/Smad signaling, which could be reversed by TGF-ß1 knockdown. Furthermore, NaAsO2 treatment enhanced the m6A modification level both in vivo and in vitro. Significantly, NaAsO2 promoted the specific interaction of METTL14 and IGF2BP2 with TGF-ß1 and enhanced the TGF-ß1 mRNA stability. Notably, NaAsO2-induced TGF-ß/Smad pathway and HSC-t6 cells activation might be avoided by limiting METTL14/IGF2BP2-mediated m6A modification. Our findings showed that the NaAsO2-induced activation of HSCs and LF is made possible by the METTL14/IGF2BP2-mediated m6A methylation of TGF-ß1, which may open up new therapeutic options for LF brought on by environmental hazards.

Nod-like receptor protein 3 inflammasome-mediated pyroptosis contributes to chronic NaAsO2 exposure-induced fibrotic changes and dysfunction in the liver of SD rats.

The metalloid arsenic, known for its toxic properties, is widespread presence in the environment. Our previous research has confirmed that prolonged exposure to arsenic can lead to liver fibrosis injury in rats, while the precise pathogenic mechanism still requires further investigation. In the past few years, the Nod-like receptor protein 3 (NLRP3) inflammasome has been found to play a pivotal role in the occurrence and development of liver injury. In this study, we administered varying doses of sodium arsenite (NaAsO2) and 10 mg/kg.bw MCC950 (a particular tiny molecular inhibitor targeting NLRP3) to Sprague-Dawley (SD) rats for 36 weeks to explore the involvement of NLRP3 inflammasome in NaAsO2-induced liver injury. The findings suggested that prolonged exposure to NaAsO2 resulted in pyroptosis in liver tissue of SD rats, accompanied by the fibrotic injury, extracellular matrix (ECM) deposition and liver dysfunction. Moreover, long-term NaAsO2 exposure activated NLRP3 inflammasome, leading to the release of pro-inflammatory cytokines in liver tissue. After treatment with MCC950, the induction of NLRP3-mediated pyroptosis and release of pro-inflammatory cytokines were significantly attenuated, leading to a decrease in the severity of liver fibrosis and an improvement in liver function. To summarize, those results clearly indicate that hepatic fibrosis and liver dysfunction induced by NaAsO2 occur through the activation of NLRP3 inflammasome-mediated pyroptosis, shedding new light on the potential mechanisms underlying arsenic-induced liver damage.

[NRF2 mediated redox stress in arsenic induced human keratinocytes malignant transformation].

OBJECTIVE: To explore the role of nuclear transcription factor E2-related factor 2(NRF2)-mediated reductive stress in arsenite induced malignant transformation in human keratinocytes. METHODS: HaCaT cells and fluorescent labeled mitochondrial glutathione HaCaT cells(Mito-Grx1-roGFP2 HaCaT) were cultured to 35 passages in medium containing 0.0 and 1.0 µmol/L NaAsO_2 to establish a model of malignant transformation of cells. Cellular and mitochondrial reduced glutathione/oxidized glutathione(GSH/GSSG) and reduced coenzyme II/oxidized coenzyme II(NADPH/NADP~+) ratios were measured in HaCaT cells. Cell doubling time, cell migration ability, soft agar clone formation ability and GSH/GSSG at different times in the 0 passage, the early stage(1st, 7th and 14th passages) and later stage(21st, 28th and 35th passages) were measured in Mito-Grx1-roGFP2 HaCaT cells. NaAsO_2 induced malignant transformation cells were transfected with NRF2 siRNA, and detected the expression level of NRF2 and the redox-related indexes and malignant transformation indexes. RESULTS: Compared with the control group, the GSH/GSSG ratio in 1.0 µmol/L NaAsO_2 treated HaCaT cells significantly decreased in the 1st and 7th generations, but significantly increased after the 21st generation, and the NADPH/NADP~+ ratio significantly increased in the 1st, 14th, 21st, 28th and 35th generations; The levels of GSH/GSSG in mitochondria significantly increased from 1st to 35th generation, and the levels of NADPH/NADP~+ in mitochondria significantly increased at 1st, 7th, 21st, 28th and 35th generation. After continuous treatment of Mito-Grx1-roGFP2 HaCaT cells with 0.0 or 1.0 µmol/L NaAsO_2 to 35 passages, the doubling time of cells treated with 1.0 µmol/L NaAsO_2 was significantly shortened, the cell migration rate was increased greatly, and more clones with larger volumes than the control cells formed. The GSH/GSSG ratio in mitochondria of Mito-Grx1-roGFP2 HaCaT cells showed a significant decrease in the 1st generation and increased from the 7th generation onwards(all P<0.05). After transfection of NaAsO_2 treated cells with NRF2 siRNA, the levels of hydrogen peroxide and superoxide increased compared with the siRNA controls. The levels of cell and mitochondrial NADPH/NADP~+ and GSH/GSSG decreased and the level of mitochondrial GSH/GSSG in Mito-Grx1-roGFP2 HaCaT cells decreased. Cell doubling time increased, cell migration rate and soft agar clone formation ability decreased(all P<0.05). The malignant phenotype was reversed. CONCLUSION: In the early stage(1st, 7th and 14th passages) of NaAsO_2 treated HaCaT cells, oxidative stress occurred with continuous high NRF2 expression. Later(21st, 28th and 35th passages), NRF2 induced reductive stress, leading to malignant transformation.

Supplementation with sesame oil suppresses genotoxicity, hepatotoxicity and enterotoxicity induced by sodium arsenite in rats.

BACKGROUND: Sesame oil, an edible essential oil, is known to be rich in unsaturated fatty acids, vitamins and lignans with several reported health-promoting benefits. Acute arsenic poisoning produces toxic hepatitis, bone marrow depression and adverse gastrointestinal responses. In this study, we investigated the protective effect of sesame seed oil (SSO) against genotoxicity, hepatotoxicity and colonic toxicity induced by sodium arsenite (SA) in Wistar rats. METHODS: Twenty-eight male Wistar albino rats were randomly allocated into four groups: control, SA only (2.5 mg/kg), SA + SSO (4 ml/kg) and SSO alone for eight consecutive days. Liver function and morphology, bone marrow micronuclei induction, colonic histopathology, mucus production and immune expression of Bcl-2, carcinoembryonic antigen (CEA), MUC1 and cytokeratins AE1/AE3 were evaluated. RESULTS: SA provoked increased serum activities of liver enzymes, including alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and caused severely altered morphology of hepatic and colonic tissues with increased frequency of micronucleated polychromatic erythrocytes (MnPCEs/1000PCE) in the bone marrow. In addition, SA triggered increased expression of colonic CEA and MUC1 but weak Bcl-2 immunoexpression. However, cotreatment with SSO demonstrated protective activities against SA-induced damage, as indicated by significantly reduced serum ALT and AST, fewer micronucleated bone marrow erythrocytes and well-preserved hepatic and colonic morphologies compared to the SA-treated rats. Furthermore, SSO protected the colonic mucosa by boosting mucus production, elevating anti-apoptotic Bcl-2 expression and reducing CEA expression. GC-MS analysis of SSO revealed that it was predominated by linoleic acid, an omega-3 fatty acid, and tocopherols. CONCLUSIONS: Our data indicated that SSO protected the liver, colon and bone marrow potentially via anti-inflammatory and anti-apoptotic activities. The data suggest that sesame oil has potential therapeutic applications against chemical toxicities induced by arsenic.

Carvacrol reduces abnormal and dead sperm counts by attenuating sodium arsenite-induced oxidative stress, inflammation, apoptosis, and autophagy in the testicular tissues of rats.

Arsenic (As) is a highly toxic metalloid. Carvacrol (CAR) is the active ingredient of Lamiaceae plants and has various biological and pharmacological properties. The present study investigated the protective effects of carvacrol (CAR) against testicular toxicity induced by sodium arsenite (SA). Rats were given SA (10 mg/kg) and/or CAR (25 or 50 mg/kg) for 14 days. Semen analyzes showed that CAR increased sperm motility and decreased the percentage of abnormal and dead sperm. It was determined that the oxidative stress induced by SA decreased with the increase of Nrf-2 and HO-1 expressions, SOD, CAT, GPx, and GSH levels, and MDA levels decreased after CAR treatment. It was observed that autophagy and inflammation triggered by SA in testicular tissue were alleviated by suppressing the expressions of LC3A, LC3B, MAPK-14, NF-κB, TNF-α, IL-1ß, iNOS, and COX-2 biomarkers in rats given CAR. Also, CAR treatment suppressed SA-induced apoptosis by inhibiting Bax and Caspase-3 expressions in testicles and up-regulating Bcl-2 expression. Histopathological analyzes showed that rats given SA had deterioration in tubule structure and spermatogenesis cell line, especially a serious loss of spermatogonia cells, atrophy of seminiferous tubules, and deterioration of germinal epithelium. In the group given CAR, the germinal epithelium and connective tissue were in normal morphological structure and an increase in seminiferous tubule diameters was observed. As a result, it was determined that oxidative stress, inflammation, autophagy, and apoptosis induced by SA were suppressed by CAR, thus protecting the testicular tissue from damage and increasing semen quality.

A Novel Quinazoline Derivative Prevents and Treats Arsenic-Induced Liver Injury by Regulating the Expression of RecQ Family Helicase.

Arsenic is a carcinogenic metalloid toxicant widely found in the natural environment. Acute or prolonged exposure to arsenic causes a series of damages to the organs, mainly the liver, such as hepatomegaly, liver fibrosis, cirrhosis, and even hepatocellular carcinoma. Therefore, it is imperative to seek drugs to prevent arsenic-induced liver injury. Quinazolines are a class of nitrogen heterocyclic compounds with biological and pharmacological effects in vivo and in vitro. This study was designed to investigate the ameliorating effects of quinazoline derivatives on arsenic-induced liver injury and its molecular mechanism. We investigated the mechanism of the quinazoline derivative KZL-047 in preventing and ameliorating arsenic-induced liver injury in vitro by cell cycle and apoptosis. We performed real-time fluorescence quantitative polymerase chain reaction (qPCR) and Western blotting combined with molecular docking. In vivo, the experiments were performed to investigate the mechanism of KZL-047 in preventing and ameliorating arsenic-induced liver injury using arsenic-infected mice. Physiological and biochemical indices of liver function in mouse serum were measured, histopathological changes in liver tissue were observed, and immunohistochemical staining was used to detect changes in the expression of RecQ-family helicases in mouse liver tissue. The results of in vitro experiments showed that sodium arsenite (SA) inhibited the proliferation of L-02 cells, induced apoptosis, blocked the cell cycle at the G1 phase, and decreased the expression of RecQ family helicase; after KZL-047 treatment in arsenic-induced L-02 cells, the expression of RecQ family helicase was upregulated, and the apoptosis rate was slowed, leading to the restoration of the cell viability level. KZL-047 inhibited arsenic-induced oxidative stress, alleviated oxidative damage and lipid peroxidation in vivo, and ameliorated arsenic toxicity-induced liver injury. KZL-047 restored the expression of RecQ family helicase proteins, which is consistent with the results of in vitro studies. In summary, KZL-047 can be considered a potential candidate for the treatment of arsenic-induced liver injury.

Mild Oxidative Stress Induced by Sodium Arsenite Reduces Lipocalin-2 Expression Levels in Cortical Glial Cells.

Astrocytes and microglia, the most abundant glial cells in the central nervous system, are involved in maintaining homeostasis in the brain microenvironment and in the progression of various neurological disorders. Lipocalin-2 (LCN2) is a small secretory protein that can be transcriptionally upregulated via nuclear factor kappa B (NF-κB) signaling. It is synthesized and secreted by glial cells, resulting in either the restoration of damaged neural tissues or the induction of neuronal apoptosis in a context-dependent manner. It has recently been reported that when glial cells are under lipopolysaccharide-induced inflammatory stress, either reduced production or accelerated degradation of LCN2 can alleviate neurotoxicity. However, the regulatory mechanisms of LCN2 in glial cells are not yet fully understood. In this study, we used primary astroglial-enriched cells which produce LCN2 and found that the production of LCN2 could be reduced by sodium arsenite treatment. Surprisingly, the reduced LCN2 production was not due to the suppression of NF-κB signaling. Mild oxidative stress induced by sodium arsenite treatment activated antioxidant responses and downregulated Lcn2 expression without reducing the viability of astroglial-enriched cells. Intriguingly, reduced LCN2 production could not be achieved by simple activation of the nuclear factor erythroid-2-related factor 2 (Nrf2)-Kelch-like ECH-associated protein 1 (Keap1) pathway in astroglial-enriched cells. Thus, it appears that mild oxidative stress, occurring in an Nrf2-independent manner, is required for the downregulation of Lcn2 expression. Taken together, our findings provide new insights into the regulatory mechanisms of LCN2 and suggest that mild oxidative stress may alter LCN2 homeostasis, even under neuroinflammatory conditions.

Arsenic induces bronchial epithelial carcinogenesis with mitochondrial dysfunction through AKAP95-mediated cell cycle alterations.

Arsenic is a widely existing pollutant in the environment, but the mechanism of occurrence and development of lung cancer by long-term arsenic exposure needs to be elucidated further. How the high and low doses of arsenic induce human bronchial epithelial cell transformation is yet to be elucidated. In the present study, human bronchial epithelial cells were exposed to varying high-dose sodium arsenite (NaAsO2) for the short-term or treated with low dose for long-term. The data showed that both short- and long-term treatment promoted G1/S transition of Beas-2B cells, inducing a significant increase in the expression of AKAP95, cyclin D1, cyclin D2, and cyclin E1. However, silencing AKAP95 by treating cells with siAKAP95 exerted a protective function that inhibited G1/S transition, suggesting a regulatory mechanism of AKAP95 on the cell cycle during cell malignant transformation induced by NaAsO2. In addition, mitochondrial dysfunctions occurred during NaAsO2 exposure. Beas-2B cells exposed to low-dose NaAsO2 for long-term were subcultured for 20 generations, and the exposure time was positively proportional to the growth and migration rate of the cells. The exposed cells were used in a tumor-bearing transplantation experiment (mice), and the results showed that the longer the exposure time, the faster the tumor volume growth rate of As-Beas-2B cells. Tumor tissues were excised for hematoxylin-eosin staining, which showed altered cell morphology and increased volume.

Glutathione protects against the meiotic defects of ovine oocytes induced by arsenic exposure via the inhibition of mitochondrial dysfunctions.

Accumulating evidences revealed the connections between arsenic exposure and mitochondrial dysfunctions induced reproductive toxicology. Meanwhile, production declines were found in livestock suffering from arsenic exposure. However, the connections between arsenic exposure and livestock meiotic defects remain unclear. In this study, the effects of sodium arsenite (NaAsO2) exposure during the in vitro maturation (IVM) on the meiotic potentials of ovine oocytes were analyzed. Furthermore, the effects of glutathione (GSH) supplementation on the meiotic defects of NaAsO2 exposed ovine oocytes were investigated by the assay of nuclear maturation, spindle organization, chromosome alignment, cytoskeleton assembly, cortical granule (CGs) dynamics, mitochondrial dysfunctions, reactive oxygen species (ROS) accumulation, oxidative DNA damages, cellular apoptosis, epigenetic modifications and fertilization capacities. The results showed that the meiotic defects of NaAsO2 exposed ovine oocytes were effectively ameliorated by the GSH supplementation via the inhibition of mitochondrial dysfunctions, which not only promoted the nuclear maturation, spindle organization, chromosome alignment, cytoskeleton assembly, CGs dynamic and fertilization capacities, but also inhibited the ROS accumulation, oxidative DNA damages and apoptosis of ovine MII oocytes. The abnormal expressions of 5mC, H3K4me3 and H3K9me3 in NaAsO2 exposed ovine oocytes, indicating the abnormal epimutations of DNA methylation and histone methylation, were also effectively ameliorated by the GSH supplementation. Taken together, this study confirmed the connections between arsenic exposure and meiotic defects of ovine oocytes. Meanwhile, the effects of GSH supplementation on the developmental competence of livestock oocytes, especially for these suffering from arsenic exposure were also founded, benefiting the extended researches for the GSH applications.

Intraperitoneal kisspeptin-10 administration ameliorates sodium arsenite-induced reproductive toxicity in adult male mice.

The current study investigated the protective ameliorative effect of intraperitoneally administered kisspeptin-10 (50 nmol/day) against reproductive toxicity in adult male mice challenged with 35 days of exposure to sodium arsenite in drinking water. Mice were divided into tap water control, sodium arsenite-alone (4 ppm and 10 ppm), kisspeptin-alone (intermittent and continuous) and combined (sodium arsenite +kisspeptin-10 intermittent and continuous) treatment groups. Results revealed protective effect of both intermittent and continuous kisspeptin doses on reproductive organs against sodium arsenite-induced toxicity. This was indicated by an increase (p < 0.001) in the activity of antioxidant enzymes and a decrease (p < 0.001) in the levels of oxidative stress biomarkers. Concomitant significant increase was noticeable in the relative organ weight (p < 0.01), and serum testosterone and seminal fructose (p < 0.001), and a significant improvement in sperm parameters was also observed. A significant downregulation of lactate dehydrogenase concentration demonstrated further the protective effect of kisspeptin against tissue damage. Histologically, both treatment regimens of kisspeptin combined with sodium arsenite exposure prevented massive germ cell loss and tissue damage, a condition prominent in sodium arsenite-alone-treated mice. The study demonstrates for the first time kisspeptin's potential to mitigate the biochemical and histotoxic effects of arsenic on male reproductive system.

Ameliorative role of inducible nitric oxide synthase inhibitors against sodium arsenite-induced renal and hepatic dysfunction in rats.

Arsenic exposure causes immense health distress by increasing risk of cardiovascular abnormalities, diabetes mellitus, neurotoxicity, and nephrotoxicity. The present study explored the role of inducible nitric oxide synthase (iNOS) inhibitors against sodium arsenite-induced renal and hepatic dysfunction in rats. Female Sprague Dawley rats were subjected to arsenic toxicity by administering sodium arsenite (5 mg/kg/day, oral) for 4 weeks. The iNOS inhibitors, S-methylisothiourea (10 mg/kg, i.p.) and aminoguanidine (100 mg/kg, i.p.) were given one hour before sodium arsenite administration in rats for 4 weeks. Sodium arsenite led rise in serum creatinine, urea, uric acid, electrolytes (potassium, fractional excretion of sodium), microproteinuria, and decreased creatinine clearance (p < 0.001) indicated renal dysfunction in rats. Arsenic-intoxication resulted in significant oxidative stress in rat kidneys, which was measured in terms of increase in lipid peroxides, superoxide anion generation and decrease in reduced glutathione (p < 0.001) levels. A threefold increase in renal hydroxyproline level in arsenic intoxicated rats indicated fibrosis. Hematoxylin-eosin staining indicated tubular damage, whereas picrosirius red staining highlighted collagen deposition in rat kidneys. S-methylisothiourea and aminoguanidine improved renal function and attenuated arsenic led renal oxidative stress, fibrosis, and decreased the kidney injury score. Additionally, arsenite-intoxication resulted in significant rise in hepatic parameters (serum aspartate aminotransferase, alanine transferase, alkaline phosphatase, and bilirubin (p < 0.001) along with multi-fold increase in oxidative stress, fibrosis and liver injury score in rats, which was significantly (p < 0.001) attenuated by concurrent administration of iNOS inhibitors). Hence, it is concluded that iNOS inhibitors attenuate sodium arsenite-induced renal and hepatic dysfunction in rats.

PUB22 and PUB23 U-box E3 ubiquitin ligases negatively regulate 26S proteasome activity under proteotoxic stress conditions.

The mechanism regulating proteasomal activity under proteotoxic stress conditions remains unclear. Here, we showed that arsenite-induced proteotoxic stress resulted in upregulation of Arabidopsis homologous PUB22 and PUB23 U-box E3 ubiquitin ligases and that pub22pub23 double mutants displayed arsenite-insensitive seed germination and root growth phenotypes. PUB22/PUB23 downregulated 26S proteasome activity by promoting the dissociation of the 19S regulatory particle from the holo-proteasome complex, resulting in intracellular accumulation of UbG76V -GFP, an artificial substrate of the proteasome complex, and insoluble poly-ubiquitinated proteins. These results suggest that PUB22/PUB23 play a critical role in arsenite-induced proteotoxic stress response via negative regulation of 26S proteasome integrity.

[Effects of arsenic and its main metabolites on A549 cell apoptosis and the expression of pro-apoptotic genes Bad and Bik].

Objective: To investigate the effect of arsenic and its main metabolites on the apoptosis of human lung adenocarcinoma cell line A549 and the expression of pro-apoptotic genes Bad and Bik. Methods: In October 2020, A549 cells were recovered and cultured, and the cell viability was detected by the cell counting reagent CCK-8 to determine the concentration and time of sodium arsenite exposure to A549. The study was divided into NaAsO(2) exposure groups and metobol: le expoure groups: the metabolite comparison groups were subdivided into the control group, the monomethylarsinic acid exposure group (60 µmol/L) , and the dimethylarsinic acid exposure group (60 µmol/L) ; sodium arsenite dose groups were subdivided into 4 groups: control group (0) , 20, 40, 60 µmol/L sodium arsenite NaAsO(2). Hoechst 33342/propidium iodide double staining (Ho/PI) was used to observe cell apoptosis and real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression levels of Bad and Bik mRNA in cells after exposure. Western blotting was used to detect the protein expressions of Bad, P-Bad-S112, Bik, cleaved Bik and downstream proteins poly ADP-ribose polymerase PARP1 and cytochrome C (Cyt-C) , using spectrophotometry to detect the activity changes of caspase 3, 6, 8, 9. Results: Compared with the control group, the proportion of apoptotic cells in the 20, 40, and 60 µmol/L NaAsO(2) dose groups increased significantly (P<0.01) , and the expression levels of Bad, Bik mRNA, the protein expression levels of Bad, P-Bad-S112, Bik, cleaved Bik, PARP1, Cyt-C were increased (all P<0.05) , and the activities of Caspase 3, 6, 8, and 9 were significantly increased with significantly differences (P<0.05) . Compared with the control group, the expression level of Bad mRNA in the DMA exposure group (1.439±0.173) was increased with a significant difference (P=0.024) , but there was no significant difference in the expression level of Bik mRNA (P=0.788) . There was no significant differences in the expression levels of Bad and Bik mRNA in the poison groups (P=0.085, 0.063) . Compared with the control group, the gray values of proteins Bad, Bik, PARP1 and Cyt-C exposed to MMA were 0.696±0.023, 0.707±0.014, 0.907±0.031, 1.032±0.016, and there was no significant difference between the two groups (P=0.469, 0.669, 0.859, 0.771) ; the gray values of proteins Bad, Bik, PARP1 and Cyt-C exposed to DMA were 0.698±0.030, 0.705±0.022, 0.908±0.015, 1.029±0.010, and there was no difference between the two groups (P=0.479, 0.636, 0.803, 0.984) . Conclusion: Sodium arsenite induces the overexpression of Bad and Bik proteins, initiates the negative feedback regulation of phosphorylated Bad and the degradation of Bik, activates the downstream proteins PARP1, Cyt-C and Caspase pathways, and mediates the apoptosis of A549 cells.

Bivalve haemocyte adhesion, aggregation and phagocytosis: A tool to reckon arsenic induced threats to freshwater ecosystem.

The freshwater aquifers of the Indo-Gangetic plains support rich biodiversity which is under the threat of arsenic contamination. The filter feeding bivalve mollusc Lamellidens marginalis is a sessile and sentinel resident of these freshwater habitats. In the present study, the classical cell behaviours of adhesion and aggregation were monitored in the circulating haemocytes of the freshwater bivalve under the exposure of sodium arsenite (NaAsO2) at sublethal concentrations in controlled laboratory conditions for a maximum time-span of sixteen days. The toxic metalloid significantly inhibited non-self adhesion, inter-haemocyte interactions and haemocyte aggregation in a dose and time dependent manner. The natural occurrence of the filopods on the haemocytes was significantly diminished in the bivalves exposed to the inorganic arsenite. Moreover, a significant fall in the kinetics of phagocytosis index and haemocyte adhesion was observed under the in vitro exposure to NaAsO2. Compromised non-self adhesion, cell-cell aggregation and phagocytosis of non-self particles by the bivalve haemocytes probably indicate susceptible immunological status of the bivalve. Such vulnerable immunity of the bivalve probably signifies the nature of imminent threat to the freshwater ecosystem as a whole under inorganic arsenite exposure. The findings would be helpful to design bivalve haemocyte based inexpensive biomonitoring tool to assess the health of freshwater ecosystem under potential arsenic threat.

Gallic acid ameliorates sodium arsenite-induced renal and hepatic toxicity in rats.

Chronic exposure to toxic inorganic arsenic results in the adverse health effects including skin lesions, cardiovascular diseases, diabetes, neurological disorders, and liver and kidney diseases. Gallic acid (GA) is an important phenolic compound, which could protect different tissues from oxidative stress induced damage. The present study investigated effects of GA against sodium arsenite (SA)-induced renal and hepatic toxicity. Thirty-five rats were randomly divided in to five groups; group 1 was treated with normal saline (2 ml/kg/day, p.o.; for 21 days); group 2 was exposed to SA (10 mg/kg/day, p.o.; for 14 days); groups 3 and 4 were treated with GA (10 and 30 mg/kg/day, respectively; for 7 days) prior to exposure to SA, and treatment was continued up to 21 days in parallel with SA administration; group 5 was treated with GA (30 mg/kg/day, p.o.; for 21 days). The level of MDA, IL-1ß, NO and glutathione (GSH) and the activity of glutathione peroxidase (GPx), superoxide dismutase (SOD) and catalase (CAT) were evaluated in kidney and liver tissues. Histopathological parameters and serum levels of ALT, AST, ALP, Cr and BUN were also assessed. Treatment with GA remarkably improved SA-induced alteration of hematological and histopathological parameters; these protective effects were associated with the reduction of SA-induced elevation of MDA, IL-1ß and NO levels as well as reduction of GSH level and GPx, SOD and CAT activity. Our results suggest that GA may inhibit SA-induced kidney and liver toxicity through scavenging reactive free radicals and increasing intracellular antioxidant capacity.

Molecular Evidence of the Inhibitory Potential of Melatonin against NaAsO2-Induced Aging in Male Rats.

Arsenic (As) poisoning is widespread due to exposure to pollution. The toxic level of (As) causes oxidative stress-induced aging and tissue damage. Since melatonin (MLT) has anti-oxidant and anti-aging properties, we aimed to evaluate the protective effect of MLT against the toxicity of sodium arsenite (NaAsO2). Healthy male NMRI mice were divided into eight different groups. The control group received a standard regular diet. Other groups were treated with varying diets, including MLT alone, NaAsO2, and NaAsO2 plus MLT. After one month of treatment, biochemical and pathological tests were performed on blood, heart, and lung tissue samples. NaAsO2 increased the levels of TNF-α, 8-hydroxy-2-deoxy guanosine (8OHdG), malondialdehyde (MDA), reactive oxygen species (ROS), and high mobility group box 1 (HMGB1), increased the expression of TNF receptor type 1-associated death domain (TRADD) mRNA and telomerase reverse transcriptase, and decreased the expression of Klotho (KL) mRNA in both plasma and tissues. In contrast, MLT reduced MDA, ROS, HMGB1, lactate, and TNF-α enhanced the mRNA expression of KL, and suppressed the mRNA expression of the TERT and TRADD genes. Thus, MLT confers potent protection against NaAsO2- induced tissue injury and oxidative stress.

Cytoplasmic localization of amyotrophic lateral sclerosis-related TDP-43 proteins modulates stress granule formation.

TDP-43 is an RNA/DNA-binding protein associated with amyotrophic lateral sclerosis (ALS). Under pathological conditions, TDP-43 exported from the nucleus accumulates in the cytoplasm, forming inclusion bodies. However, the molecular mechanisms that contribute to such aggregation are unclear. The pathogenic processes that lead to aggregation in ALS were investigated by analysing the effects of wildtype human TDP-43 or with mutations in the nuclear localization sequence (NLS) or those associated with ALS in stress granule formation. TDP-43 (WT, ∆NLS or G348C), with or without a GFP-tag, was expressed in SH-SY5Y neuroblastoma or HeLa cells and stress granules induced by oxidative stress or heat shock. Stress granule formation was altered in cells strongly expressing GFP-TDP-∆NLS, or untagged TDP-43-∆NLS in the cytoplasm but not the negative controls, GFP or GFP-UtrCH. In contrast, there was no reduction in stress granule formation by cells that expressed untagged TDP-43 (WT or G348C) in the nucleus upon stress induction. GFP labelling of TDP-43 (WT or G348C) promotes high cytoplasmic expression and nuclear aggregation. Stress granule formation was impaired in cells expressing GFP-TDP-43 (WT or G348C) in the cytoplasm. Overall, these results suggest that stress granule formation may be inhibited by high levels of TDP-43 protein in the cytoplasm. As stress granules serve a protective function, their deregulation may promote neurodegeneration due to an aberrant stress response.