Investigation of the protective effect of chitosan against arsenic-induced nephrotoxicity and oxidative damage in rat kidney tissue.

Arsenic is an important metalloid that can cause poisoning in humans and domestic animals. Exposure to arsenic causes cell damage, increasing the production of reactive oxygen species. Chitosan is a biopolymer obtained by deacetylation of chitin with antioxidant and metal ion chelating properties. In this study, the protective effect of chitosan on arsenic-induced nephrotoxicity and oxidative damage was investigated. 32 male Wistar-albino rats were divided into 4 groups of 8 rats each as control group (C), chitosan group (CS group), arsenic group (AS group), and arsenic+chitosan group (AS+CS group). The C group was given distilled water by oral gavage, the AS group was given 100 ppm/day Na-arsenite ad libitum with drinking water, the CS group was given 200 mg/kg/day chitosan dissolved in saline by oral gavage, the AS+CS group was given 100 ppm/day Na-arsenite ad libitum with drinking water and 200 mg/kg/day chitosan dissolved in saline by oral gavage for 30 days. At the end of the 30-day experimental period, 90 mg/kg ketamine was administered intraperitoneally to all rats, and blood samples and kidney tissues were collected. Urea, uric acid, creatinine, P, Mg, K, Ca, Na, Cystatin C (CYS-C), Neutrophil Gelatinase Associated Lipocalin (NGAL) and Kidney Injury Molecule 1 (KIM-1) levels were measured in serum samples. Malondialdehyde (MDA), Glutathione (GSH), Catalase (CAT) and Superoxide dismutase (SOD) levels in the supernatant obtained from kidney tissue were analyzed by ELISA method. Compared with AS group, uric acid and creatinine levels of the AS+CS group were significantly decreased (p<0.001), urea, KIM-1, CYS-C, NGAL, and MDA levels were numerically decreased and CAT, GSH, and SOD levels were numerically increased (p>0.05). In conclusion, based on both biochemical and histopathological-immunohistochemical- immunofluorescence findings, it can be concluded that chitosan attenuates kidney injury and protects the kidney.

Arsenite S-Adenosylmethionine Methyltransferase Is Responsible for Antimony Biomethylation in Nostoc sp. PCC7120.

Antimony (Sb) biomethylation is an important but uninformed process in Sb biogeochemical cycling. Methylated Sb species have been widely detected in the environment, but the gene and enzyme for Sb methylation remain unknown. Here, we found that arsenite S-adenosylmethionine methyltransferase (ArsM) is able to catalyze Sb(III) methylation. The stepwise methylation by ArsM forms mono-, di-, and trimethylated Sb species. Sb(III) is readily coordinated with glutathione, forming the preferred ArsM substrate which is anchored on three conserved cysteines. Overexpressing arsM in Escherichia coli AW3110 conferred resistance to Sb(III) by converting intracellular Sb(III) into gaseous methylated species, serving as a detoxification process. Methylated Sb species were detected in paddy soil cultures, and phylogenetic analysis of ArsM showed its great diversity in ecosystems, suggesting a high metabolic potential for Sb(III) methylation in the environment. This study shows an undiscovered microbial process methylating aqueous Sb(III) into the gaseous phase, mobilizing Sb on a regional and even global scale as a re-emerging contaminant.

Rice tonoplast intrinsic protein member OsTIP1;2 confers tolerance to arsenite stress.

The International Agency for Research on Cancer categorizes arsenic (As) as a group I carcinogen. Arsenic exposure significantly reduces growth, development, metabolism, and crop yield. Tonoplast intrinsic proteins (TIPs) belong to the major intrinsic protein (MIP) superfamily and transport various substrates, including metals/metalloids. Our study aimed to characterize rice OsTIP1;2 in As[III] stress response. The gene expression analysis showed that the OsTIP1;2 expression was enhanced in roots on exposure to As[III] treatment. The heterologous expression of OsTIP1;2 in S. cerevisiae mutant lacking YCF1 (ycf1∆) complemented the As[III] transport function of the YCF1 transporter but not for boron (B) and arsenate As[V], indicating its substrate selective nature. The ycf1∆ mutant expressing OsTIP1;2 accumulated more As than the wild type (W303-1A) and ycf1∆ mutant strain carrying the pYES2.1 vector. OsTIP1;2 activity was partially inhibited in the presence of the aquaporin (AQP) inhibitors. The subcellular localization studies confirmed that OsTIP1;2 is localized to the tonoplast. The transient overexpression of OsTIP1;2 in Nicotiana benthamiana leaves resulted in increased activities of enzymatic and non-enzymatic antioxidants, suggesting a potential role in mitigating oxidative stress induced by As[III]. The transgenic N. tabacum overexpressing OsTIP1;2 displayed an As[III]-tolerant phenotype, with increased fresh weight and root length than the wild-type (WT) and empty vector (EV line). The As translocation factor (TF) for WT and EV was around 0.8, while that of OE lines was around 0.4. Moreover, the OE line bioconcentration factor (BCF) was more than 1. Notably, the reduced TF and increased BCF in the OE line imply the potential of OsTIP1;2 for phytostabilization.

l-Glutamate Seed Priming Enhances 2-Acetyl-1-pyrroline Formation in Fragrant Rice Seedlings in Response to Arsenite Stress.

2-Acetyl-1-pyrroline (2-AP) is a fragrance compound and flavor in fragrant rice whose precursors are generally glutamate (Glu) and proline (Pro). Our previous study revealed that exogenous Glu enhanced the arsenic (As) tolerance in fragrant rice by improving the ascorbic acid-glutathione cycle and the Pro content in roots. However, less is known about how Glu is involved in 2-AP biosynthesis in fragrant rice under As stress. Herein, a hydroponic experiment of L-Glu seed priming with 0, 100, and 500 µM l-glutamic acid solutions was conducted with two fragrant rice varieties. After that, the 10-day-old seedlings were cultured under 0 and 100 µM arsenite stress for 10 d. Results showed that the 2-AP and Pro contents were increased by 18-30% and 21-78% under As100 µM-Glu100 µM treatment in comparison to the control As100 µM to Glu0 µM, while the activities of pyrroline-5-carboxylate synthetase (P5CS) and proline dehydrogenase (ProDH) were increased by 19-46% and 3-19%, respectively. Furthermore, the 2-AP, Pro contents, and P5CS activity were correlated positively. Correspondingly, a significant abundance of differential expressed metabolites (18) and differential expressed genes (26) was observed in amino acid metabolism and glutathione metabolism pathways. In addition, several essential genes were verified and grouped into the pathways of glutathione metabolism, proline, and arginine metabolism with antioxidant defense system to comodulate 2-AP biosynthesis and stress detoxification. Therefore, the Glu seed priming treatment had a positive impact on the 2-AP biosynthesis of fragrant rice under 100 µM arsenite toxicity.

Arsenite Methyltransferase Diversity and Optimization of Methylation Efficiency.

Arsenic is methylated by arsenite (As(III)) S-adenosylmethionine (SAM) methyltransferases (ArsMs). ArsM crystal structures show three domains (an N-terminal SAM binding domain (A domain), a central arsenic binding domain (B domain), and a C-terminal domain of unknown function (C domain)). In this study, we performed a comparative analysis of ArsMs and found a broad diversity in structural domains. The differences in the ArsM structure enable ArsMs to have a range of methylation efficiencies and substrate selectivities. Many small ArsMs with 240-300 amino acid residues have only A and B domains, represented by RpArsM from Rhodopseudomonas palustris. These small ArsMs have higher methylation activity than larger ArsMs with 320-400 residues such as Chlamydomonas reinhardtii CrArsM, which has A, B, and C domains. To examine the role of the C domain, the last 102 residues in CrArsM were deleted. This CrArsM truncation exhibited higher As(III) methylation activity than the wild-type enzyme, suggesting that the C-terminal domain has a role in modulating the rate of catalysis. In addition, the relationship of arsenite efflux systems and methylation was examined. Lower rates of efflux led to higher rates of methylation. Thus, the rate of methylation can be modulated in multiple ways.

Reductive stress induced by NRF2/G6PD through glucose metabolic reprogramming promotes malignant transformation in Arsenite-exposed human keratinocytes.

Our previous research found that the nuclear factor-E2-related factor 2 (NRF2) protein was sustained activated in malignant transformation of human keratinocyte (HaCaT cells) caused by NaAsO2, but the role of NRF2 in it remains unknown. In this study, malignant transformation of HaCaT cells and labeled HaCaT cells used to detect mitochondrial glutathione levels (Mito-Grx1-roGFP2 HaCaT cells) were induced by 1.0 µM NaAsO2. Redox levels were measured at passages 0, early stage (passages 1, 7, 14), later stage (passages 21, 28 and 35) of arsenite-treated HaCaT cells. Oxidative stress levels increased at early stage. The NRF2 pathway was sustained activated. Cells and mitochondrial reductive stress levels (GSH/GSSG and NADPH/NADP+) increased. The mitochondrial GSH/GSSG levels of Mito-Grx1-roGFP2 HaCaT cells also increased. The indicators of glucose metabolism glucose-6-phosphate, lactate and the glucose-6-phosphate dehydrogenase (G6PD) levels increased, however Acetyl-CoA level decreased. Expression levels of glucose metabolic enzymes increased. After transfection with NRF2 siRNA, the indicators of glucose metabolism were reversed. After transfection with NRF2 or G6PD siRNA, cells and mitochondrial reductive stress levels decreased and the malignant phenotype was reversed. In conclusion, oxidative stress occurred in the early stage and the NRF2 was sustained high expression. In the later stage, increased NRF2/G6PD through glucose metabolic reprogramming induced reductive stress, thereby leading to malignant transformation.

Bifunctional protein ArsRM contributes to arsenite methylation and resistance in Brevundimonas sp. M20.

BACKGROUND: Arsenic (As) with various chemical forms, including inorganic arsenic and organic arsenic, is the most prevalent water and environmental toxin. This metalloid occurs worldwide and many of its forms, especially arsenite [As(III)], cause various diseases including cancer. Organification of arsenite is an effective way for organisms to cope with arsenic toxicity. Microbial communities are vital contributors to the global arsenic biocycle and represent a promising way to reduce arsenite toxicity. METHODS: Brevundimonas sp. M20 with arsenite and roxarsone resistance was isolated from aquaculture sewage. The arsHRNBC cluster and the metRFHH operon of M20 were identified by sequencing. The gene encoding ArsR/methyltransferase fusion protein, arsRM, was amplified and expressed in Escherichia coli BL21 (DE3), and this strain showed resistance to arsenic in the present of 0.25-6 mM As(III), aresenate, or pentavalent roxarsone. The methylation activity and regulatory action of ArsRM were analyzed using Discovery Studio 2.0, and its functions were confirmed by methyltransferase activity analysis and electrophoretic mobility shift assays. RESULTS: The minimum inhibitory concentration of the roxarsone resistant strain Brevundimonas sp. M20 to arsenite was 4.5 mM. A 3,011-bp arsenite resistance ars cluster arsHRNBC and a 5649-bp methionine biosynthesis met operon were found on the 3.315-Mb chromosome. Functional prediction analyses suggested that ArsRM is a difunctional protein with transcriptional regulation and methyltransferase activities. Expression of ArsRM in E. coli increased its arsenite resistance to 1.5 mM. The arsenite methylation activity of ArsRM and its ability to bind to its own gene promoter were confirmed. The As(III)-binding site (ABS) and S-adenosylmethionine-binding motif are responsible for the difunctional characteristic of ArsRM. CONCLUSIONS: We conclude that ArsRM promotes arsenite methylation and is able to bind to its own promoter region to regulate transcription. This difunctional characteristic directly connects methionine and arsenic metabolism. Our findings contribute important new knowledge about microbial arsenic resistance and detoxification. Future work should further explore how ArsRM regulates the met operon and the ars cluster.

ISG20L1 acts as a co-activator of DAPK1 in the activation of the p53-dependent cell death pathway.

Our previous studies have revealed that GADD45α is a liable proapoptotic protein, which undergoes MDM2-dependent constitutive ubiquitylation and degradation in resting cancer cells. Under chemotherapeutic agent (such as arsenite, 5-Fu and VP-16) exposure, DAPK1 functions as a novel p53 (also known as TP53) kinase, which induces phosphorylation of p53 at Ser15 and transactivates the p53 target Ets-1, to synergistically repress IKKß-dependent MDM2 stability, and ultimately removes the inhibitory effect of MDM2 on GADD45α, resulting in GADD45α accumulation and cell apoptosis. In the current study, we show that there is a strong induction of ISG20L1 (also known as AEN) expression in several cancer cell lines under exposure of arsenite and other chemotherapeutic agents. Surprisingly, although originally identified as a transcriptional target of p53, ISG20L1 induction was not controlled by p53. Instead, ISG20L1 functioned as upstream activator of p53 by interacting with DAPK1, and plays an essential role in promoting DAPK1-p53 complex formation and the subsequent activation of Ets-1/IKKß/MDM2/GADD45α cascade. Therefore, our findings have revealed novel function of ISG20L1 in mediating cancer cell apoptosis induced by chemotherapeutic agents via modulating activation of the DAPK1- and p53-dependent cell death pathway.

Intestinal homeostasis disruption in mice chronically exposed to arsenite-contaminated drinking water.

Chronic exposure to inorganic arsenic [As(III) and As(V)] affects about 200 million people, and is linked to a greater incidence of certain types of cancer. Drinking water is the main route of exposure, so, in endemic areas, the intestinal mucosa is constantly exposed to the metalloid. However, studies on the intestinal toxicity of inorganic As are scarce. The objective of this study was to evaluate the toxicity of a chronic exposure to As(III) on the intestinal mucosa and its associated microbiota. For this purpose, BALB/c mice were exposed during 6 months through drinking water to As(III) (15 and 30 mg/L). Treatment with As(III) increased reactive oxygen species (43-64%) and lipid peroxidation (8-51%). A pro-inflammatory response was also observed, evidenced by an increase in fecal lactoferrin (23-29%) and mucosal neutrophil infiltration. As(III) also induced an increase in the colonic levels of pro-inflammatory cytokines (24-201%) and the activation of some pro-inflammatory signaling pathways. Reductions in the number of goblet cells and mucus production were also observed. Moreover, As(III) exposure resulted in changes in gut microbial alpha diversity but no differences in beta diversity. This suggested that the abundance of some taxa was significantly affected by As(III), although the composition of the population did not show significant alterations. Analysis of differential taxa agreed with this, 21 ASVs were affected in abundance or variability, especially ASVs from the family Muribaculaceae. Intestinal microbiota metabolism was also affected, as reductions in fecal concentration of short-chain fatty acids were observed. The effects observed on different components of the intestinal barrier may be responsible of the increased permeability in As(III) treated mice, evidenced by an increase in fecal albumin (48-66%). Moreover, serum levels of Lipopolysaccharide binding proteins and TNF-α were increased in animals treated with 30 mg/L of As(III), suggesting a low-level systemic inflammation.

Potential of methyltransferase containing Pseudomonas oleovorans for abatement of arsenic toxicity in rice.

Arsenic (As) has become natural health hazard for millions of people across the world due to its distribution in the food chain. Naturally, it is present in different oxidative states of inorganic [As(V) and As(III)] and organic (DMA, MMA and TMA) forms. Among different mitigation approaches, microbe mediated mitigation of As toxicity is an effective and eco-friendly approach. The present study involves the characterization of bacterial strains containing arsenite methyltransferase (Pseudomonas oleovorans, B4.10); arsenate reductase (Sphingobacterium puteale, B4.22) and arsenite oxidase (Citrobacter sp., B5.12) activity with plant growth promoting (PGP) traits. Efficient reduction of grain As content by 61 % was observed due to inoculation of methyltransferase containing B4.10 as compared to B4.22 (47 %) and B5.12 (49 %). Reduced bioaccumulation of As in root (0.339) and shoot (0.166) in presence of B4.10 was found to be inversely related with translocation factor for Mn (3.28), Fe (0.073), and Se (1.82). Bioaccumulation of these micro elements was found to be associated with the modulated expression of different mineral transporters (OsIRT2, OsFRO2, OsTOM1, OsSultr4;1, and OsZIP2) in rice shoot. Improved dehydrogenase (407 %), and ß-glucosidase (97 %) activity in presence of P. oleovorans (B4.10) as compared to arsenate reductase (198 and 50 %), and arsenite oxidase (134 and 69 %) containing bacteria was also observed. Our finding confers the potential of methyltransferase positive P. oleovorans (B4.10) for As stress amelioration. Reduced grain As uptake was found to be mediated by improved plant growth and nutrient uptake associated with enhanced soil microbial activity.

Arsenic biotransformation genes and As transportation in soil-rice system affected by iron-oxidizing strain (Ochrobactrum sp.).

Arsenic (As) biotransformation in soil affects As biogeochemical cycling and is associated with As accumulation in rice. After inoculation with 1% iron-oxidizing bacteria (FeOB) in paddy soil, As speciation, As biotransformation genes in soil, As/Fe in Fe plaques, and As accumulation in rice were characterized. Compared with the control, the available As concentrations in soils decreased while amorphous and poorly crystalline Fe-Al oxidized As and crystalline Fe-Al oxidized As fractions increased of F (FeOB) and RF (rice and FeOB) treatments. Fe concentrations increased and positively correlated with As concentrations in Fe plaques on the rice root surface (***P < 0.001). Compared with R (rice), Monomethyl As (MMA), dimethyl As (DMA), arsenate (As(V)), and arsenite (As(III)) concentrations in rice plants showed a downwards trend of RF treatment. The As concentration in grains was below the National Standard for Food Safety (GB 2762-2017). A total of 16 As biotransformation genes in rhizosphere soils of different treatments (CK, F, R and RF were quantified by high-throughput qPCR (HT-qPCR). Compared with the control, the As(V) reduction and As transport genes abundance in other treatments increased respectively by 54.54%-69.17% and 54.63%-73.71%; the As(III) oxidation and As (de) methylation genes did not change significantly; however, several As(III) oxidation genes (aoxA, aoxB, aoxS, and arsH) increased. These results revealed that FeOB could reduce, transport As, and maybe also oxidize As. In addition, As(III) oxidation gene (aoxC) in rhizosphere soil was more abundant than in non-rhizosphere soil. It indicated that radial oxygen loss (ROL) promoted As(III) oxidation in rhizosphere soils. The results provide evidence for As biotransformation by ROL and FeOB in soil-rice system. ROL affects As oxidation and immobilization, and FeOB affects As reduction, transportation and may also affect As oxidation.

Ex vivo exposures to arsenite and its methylated trivalent metabolites alter gene transcription in mouse sperm cells.

We have previously reported that preconception exposure to iAs may contribute to the development of diabetes in mouse offspring by altering gene expressions in paternal sperm. However, the individual contributions of iAs and its methylated metabolites, monomethylated arsenic (MAs) and dimethylated arsenic (DMAs), to changes in the sperm transcriptome could not be determined because all three As species are present in sperm after in vivo iAs exposure. The goal of the present study was to assess As species-specific effects using an ex vivo model. We exposed freshly isolated mouse sperm to either 0.1 or 1 µM arsenite (iAsIII) or the methylated trivalent arsenicals, MAsIII and DMAsIII, and used RNA-sequencing to identify differentially expressed genes, enriched pathways, and associated protein networks. For all arsenicals tested, the exposures to 0.1 µM concentrations had greater effects on gene expression than 1 µM exposures. Transcription factor AP-1 and B cell receptor complexes were the most significantly enriched pathways in sperm exposed to 0.1 µM iAsIII. The Mre11 complex and Antigen processing were top pathways targeted by exposure to 0.1 µM MAsIII and DMAsIII, respectively. While there was no overlap between gene transcripts altered by ex vivo exposures in the present study and those altered by in vivo exposure in our prior work, several pathways were shared, including PI3K-Akt signaling, Focal adhesion, and Extracellular matrix receptor interaction pathways. Notably, the protein networks associated with these pathways included those with known roles in diabetes. This study is the first to assess the As species-specific effects on sperm transcriptome, linking these effects to the diabetogenic effects of iAs exposure.

Selective Methylation by an ArsM S-Adenosylmethionine Methyltransferase from Burkholderia gladioli GSRB05 Enhances Antibiotic Production.

Arsenic methylation contributes to the formation and diversity of environmental organoarsenicals, an important process in the arsenic biogeochemical cycle. The arsM gene encoding an arsenite (As(III)) S-adenosylmethionine (SAM) methyltransferase is widely distributed in members of every kingdom. A number of ArsM enzymes have been shown to have different patterns of methylation. When incubated with inorganic As(III), Burkholderia gladioli GSRB05 has been shown to synthesize the organoarsenical antibiotic arsinothricin (AST) but does not produce either methylarsenate (MAs(V)) or dimethylarsenate (DMAs(V)). Here, we show that cells of B. gladioli GSRB05 synthesize DMAs(V) when cultured with either MAs(III) or MAs(V). Heterologous expression of the BgarsM gene in Escherichia coli conferred resistance to MAs(III) but not As(III). The cells methylate MAs(III) and the AST precursor, reduced trivalent hydroxyarsinothricin (R-AST-OH) but do not methylate inorganic As(III). Similar results were obtained with purified BgArsM. Compared with ArsM orthologs, BgArsM has an additional 37 amino acid residues in a linker region between domains. Deletion of the additional 37 residues restored As(III) methylation activity. Cells of E. coli co-expressing the BgarsL gene encoding the noncanonical radical SAM enzyme that catalyzes the synthesis of R-AST-OH together with the BgarsM gene produce much more of the antibiotic AST compared with E. coli cells co-expressing BgarsL together with the CrarsM gene from Chlamydomonas reinhardtii, which lacks the sequence for additional 37 residues. We propose that the presence of the insertion reduces the fitness of B. gladioli because it cannot detoxify inorganic arsenic but concomitantly confers an evolutionary advantage by increasing the ability to produce AST.

Iron-based passivator mitigates the coupling process of anaerobic methane oxidation and arsenate reduction in paddy soils.

Arsenic (As) is a toxic metalloid that is ubiquitous in paddy soils, where passivation is the most widely used method for remediating As contamination. Recently, anaerobic methane oxidation coupled with arsenate (As(V)) reduction (AOM-AsR) has been shown to act as a critical driver for As release in paddy fields. However, the effect and mechanism of the passivators on the AOM-AsR process remain unclear. In this study, we incubated arsenate-contaminated paddy soils under anaerobic conditions. Using isotopically labelled methane and different passivators, we found that an iron-based passivator containing calcium sulfate and iron oxide (9:1, m/m) named IBP showed a much better performance than the other passivators. Adding IBP decreased the arsenite (As(III)) concentration in the soil solution by 78% and increased the AOM rate by 55%. Furthermore, we employed high-throughput sequencing and real-time quantitative polymerase chain reaction (qPCR) to investigate the ability of IBP to control As release mediated by AOM-AsR in paddy fields, as well as its underlying mechanism. Our results showed that IBP addition significantly increased anaerobic methanotrophic (ANME) archaea (ANME-2a-c, ANME-2d, and ANME-3) by 91%, and increased the methane-oxidizing bacterium Methylobacter by 262%. Similarly, IBP addition significantly increased the Fe(III) concentration in soil solution by 39% and increased the absolute abundance of Fe(III)-reducing bacteria (Geobacteraceae) by 21 times in soil. Adding IBP may significantly promote AOM coupled with Fe(III) reduction, significantly reducing electron transfer from AOM to As(V) reduction. Hence, IBP may be used as an efficient passivator to remediate As-contaminated soil using an active AOM-AsR process. These results provide a novel insight into controlling soil As release by regulating an active and critical As mobilization pathway in the environment.

Impacts of arsenic on Rad18 and translesion synthesis.

Arsenite interferes with DNA repair protein function resulting in the retention of UV-induced DNA damage. Accumulated DNA damage promotes replication stress which is bypassed by DNA damage tolerance pathways such as translesion synthesis (TLS). Rad18 is an essential factor in initiating TLS through PCNA monoubiquitination and contains two functionally and structurally distinct zinc fingers that are potential targets for arsenite binding. Arsenite treatment displaced zinc from endogenous Rad18 protein and mass spectrometry analysis revealed arsenite binding to both the Rad18 RING finger and UBZ domains. Consequently, arsenite inhibited Rad18 RING finger dependent PCNA monoubiquitination and polymerase eta recruitment to DNA damage in UV exposed keratinocytes, both of which enhance the bypass of cyclobutane pyrimidine dimers during replication. Further analysis demonstrated multiple effects of arsenite, including the reduction in nuclear localization and UV-induced chromatin recruitment of Rad18 and its binding partner Rad6, which may also negatively impact TLS initiation. Arsenite and Rad18 knockdown in UV exposed keratinocytes significantly increased markers of replication stress and DNA strand breaks to a similar degree, suggesting arsenite mediates its effects through Rad18. Comet assay analysis confirmed an increase in both UV-induced single-stranded DNA and DNA double-strand breaks in arsenite treated keratinocytes compared to UV alone. Altogether, this study supports a mechanism by which arsenite inhibits TLS through the altered activity and regulation of Rad18. Arsenite elevated the levels of UV-induced replication stress and consequently, single-stranded DNA gaps and DNA double-strand breaks. These potentially mutagenic outcomes support a role for TLS in the cocarcinogenicity of arsenite.

Inorganic arsenic exposure-induced premature senescence and senescence-associated secretory phenotype (SASP) in human hepatic stellate cells.

Exposure to inorganic arsenic has been known to induce cancers in various organs, however, the underlying mechanisms remain unclear. Premature senescence refers to the irreversible growth arrest induced by stress stimuli. The senescence-associated secretory phenotype (SASP), particularly in fibroblasts, has been shown to promote cancer development. In this study, we examined whether arsenite exposure causes premature senescence and induction of SASP in liver fibroblasts using the human hepatic stellate cell line, LX-2. Exposure of LX-2 cells to 5 or 7.5 µM of sodium arsenite for 144 h induced the features of senescence in the cells, including morphological changes, growth inhibition, increased senescence-associated ß-galactosidase activity, increased P21 gene expression, and decreased LAMINB1 gene expression. The mRNA expressions of SASP factors, such as MMP1, MMP3, IL-8, IL-1ß, and CXCL1, were also highly upregulated. The wound healing assay revealed that the conditioned medium from LX-2 cells with arsenite-induced senescence increased the migration activity of cells of the human hepatoma cell line, Huh-7. Gene expression data of liver cancer samples from the Human Protein Atlas showed that high expression levels of the SASP factors that were upregulated in the cells with arsenite-induced senescence were strongly associated with a poor prognosis. In addition, the cellular levels of γ-H2AX, a DNA double-strand break marker, were increased by arsenite exposure, suggesting that DNA damage could contribute to premature senescence induction. These results show that arsenite exposure induces premature senescence in hepatic stellate cells and suggest that the SASP factors from the senescent cells promote hepatic carcinogenesis.

[Rat serum containing oxymatrine inhibits the proliferation of LX2 human hepatic stellate cells induced by sodium arsenite via blocking PI3K/AKT pathway].

Objective To investigate the effect of rat serum containing oxymatrine (OM) on the activation of LX2 human hepatic stellate cells induced by sodium arsenite and its mechanism. Methods SD rats were gavaged with 100 mg/kg OM or equal volume of normal saline to prepare OM-containing serum and blank serum. LO2 human embryonic liver cell line was treated with 100 µmol/L sodium arsenite for 24 hours, and then the supernatant was collected. LX2 cells were incubated with the mixture of the supernatant and normal medium at the ratio of 1:4 for 24 hours to establish the cell model of indirect arsenic exposure. Blank serum group (160 mL/L blank serum), indirect arsenic exposure group (160 mL/L blank serum with arsenic exposure), low-dose OM-containing serum group (80 mL/L blank serum and 80 mL/L OM-containing serum with arsenic exposure), high-dose OM-containing serum group (160 mL/L medicated serum with arsenic exposure) were set up. MTT assay and flow cytometry were used to detect cell proliferation and cell cycle, respectively. Western blot analysis was performed to detect the protein expressions of α-SMA, Bcl2, BAX, cyclin D1, PI3K, and phospho-AKT (p-AKT) in LX2 cells. Results After indirect arsenic treatment, the proliferation rate of LX2 cells increased, the proportion of G1 phase decreased, the proportion of apoptosis decreased, the expression of α-SMA, PI3K, p-AKT, cyclin D1, Bcl2 were significantly up-regulated, and the expression of BAX decreased. After OM-containing serum treatment, the proportion of cells in G1 phase increased, the proportion of apoptosis increased, the expression of BAX protein increased significantly, and the expression of other proteins were significantly down-regulated, especially in the high-dose group. Conclusion OM-containing serum can effectively inhibit the proliferation of LX2 hepatic stellate cells induced by arsenite and promote their apoptosis, which may be related to the blocking of PI3K/AKT signaling pathway.

Arsenite inhibits M2a polarization of macrophages through downregulation of peroxisome proliferator-activated receptor gamma.

Arsenite (As+3) is a group one human carcinogen, which has been associated with many diseases. Previous studies indicated that As+3 could inhibit wound healing and repair. M2a cells are known as tissue remodeling macrophages, which play an important role in wound repair process. Peroxisome proliferator-activated receptor gamma (PPAR-γ), a key regulator of lipid and glucose metabolism, was found to mediate the IL-4-dependent M2a polarization of macrophages. In the present study, As+3 induced dose-dependent inhibition of M2a polarization starting from 0.1 µM in THP-1-derived macrophages stimulated with 20 ng/mL IL-4. Increased lipid accumulation and decreased PPAR-γ expression were also observed in As+3-treated M2a macrophages. Rosiglitazone (RSG), a potent PPAR-γ agonist, alleviated the suppressions of PPAR-γ and M2a polarization induced by 2 µM As+3. Collectively, these results not only demonstrated that As+3 was able to inhibit polarization of M2a cells through PPAR-γ suppression, but also indicated that PPAR-γ could be utilized as a target for the prevention and treatment of As+3-induced immunotoxicity.

26S proteasomes become stably activated upon heat shock when ubiquitination and protein degradation increase.

Heat shock (HS) promotes protein unfolding, and cells respond by stimulating HS gene expression, ubiquitination of cell proteins, and proteolysis by the proteasome. Exposing HeLa and other cells to 43 °C for 2 h caused a twofold increase in the 26S proteasomes' peptidase activity assayed at 37 °C. This increase in activity occurred without any change in proteasome amount and did not require new protein synthesis. After affinity-purification from HS cells, 26S proteasomes still hydrolyzed peptides, adenosine 5'-triphosphate, and ubiquitinated substrates more rapidly without any evident change in subunit composition, postsynthetic modification, or association with reported proteasome-activating proteins. After returning HS cells to 37 °C, ubiquitin conjugates and proteolysis fell rapidly, but proteasome activity remained high for at least 16 h. Exposure to arsenite, which also causes proteotoxic stress in the cytosol, but not tunicamycin, which causes endoplasmic reticulum stress, also increased ubiquitin conjugate levels and 26S proteasome activity. Although the molecular basis for the enhanced proteasomal activity remains elusive, we studied possible signaling mechanisms. Proteasome activation upon proteotoxic stress required the accumulation of ubiquitinated proteins since blocking ubiquitination by E1 inhibition during HS or arsenite exposure prevented the stimulation of 26S activity. Furthermore, increasing cellular content of ubiquitin conjugates at 37 °C by inhibiting deubiquitinating enzymes with RA190 or b-AP15 also caused proteasome activation. Thus, cells respond to proteotoxic stresses, apparently in response to the accumulation of ubiquitinated proteins, by activating 26S proteasomes, which should help promote the clearance of damaged cell proteins.

As(III) Exposure Induces a Zinc Scarcity Response and Restricts Iron Uptake in High-Level Arsenic-Resistant Paenibacillus taichungensis Strain NC1.

The Gram-positive bacterium Paenibacillus taichungensis NC1 was isolated from the Zijin gold-copper mine and shown to display high resistance to arsenic (MICs of 10 mM for arsenite in minimal medium). Genome sequencing indicated the presence of a number of potential arsenic resistance determinants in NC1. Global transcriptomic analysis under arsenic stress showed that NC1 not only directly upregulated genes in an arsenic resistance operon but also responded to arsenic toxicity by increasing the expression of genes encoding antioxidant functions, such as cat, perR, and gpx. In addition, two highly expressed genes, marR and arsV, encoding a putative flavin-dependent monooxygenase and located adjacent to the ars resistance operon, were highly induced by As(III) exposure and conferred resistance to arsenic and antimony compounds. Interestingly, the zinc scarcity response was induced under exposure to high concentrations of arsenite, and genes responsible for iron uptake were downregulated, possibly to cope with oxidative stress associated with As toxicity. IMPORTANCE Microbes have the ability to adapt and respond to a variety of conditions. To better understand these processes, we isolated the arsenic-resistant Gram-positive bacterium Paenibacillus taichungensis NC1 from a gold-copper mine. The transcriptome responding to arsenite exposure showed induction of not only genes encoding arsenic resistance determinants but also genes involved in the zinc scarcity response. In addition, many genes encoding functions involved in iron uptake were downregulated. These results help to understand how bacteria integrate specific responses to arsenite exposure with broader physiological responses.