Protein vicinal thiols as intrinsic probes of brain redox states in health, aging, and ischemia.

The nature of brain redox metabolism in health, aging, and disease remains to be fully established. Reversible oxidations, to disulfide bonds, of closely spaced (vicinal) protein thiols underlie the catalytic maintenance of redox homeostasis by redoxin enzymes, including thioredoxin peroxidases (peroxiredoxins), and have been implicated in redox buffering and regulation. We propose that non-peroxidase proteins containing vicinal thiols that are responsive to physiological redox perturbations may serve as intrinsic probes of brain redox metabolism. Using redox phenylarsine oxide (PAO)-affinity chromatography, we report that PAO-binding vicinal thiols on creatine kinase B and alpha-enolase from healthy rat brains were preferentially oxidized compared to other selected proteins, including neuron-specific (gamma) enolase, under conditions designed to trap in vivo protein thiol redox states. Moreover, measures of the extents of oxidations of vicinal thiols on total protein, and on creatine kinase B and alpha-enolase, showed that vicinal thiol-linked redox states were stable over the lifespan of rats and revealed a transient reductive shift in these redox couples following decapitation-induced global ischemia. Finally, formation of disulfide-linked complexes between peroxiredoxin-2 and brain proteins was demonstrated on redox blots, supporting a link between protein vicinal thiol redox states and the peroxidase activities of peroxiredoxins. The implications of these findings with respect to underappreciated aspects of brain redox metabolism in health, aging, and ischemia are discussed.

Toxic arsenolipids bioaccumulate in the developing brain of pilot whales.

Arsenic-containing hydrocarbons (AsHC), a subclass of arsenolipids (AsL), have been proven to exert neuro- and cytotoxic effects in in-vitro and in-vivo studies and were shown to pass through biological barriers like the blood-brain barrier. However, there has been no connection as to the environmental relevance of these findings, meaning there is no study based on samples from free living animals that are exposed to these compounds. Here, we report the identification of two AsHC as well as 3 arsenosugar phospholipids (AsPL) in the brains of a pod of stranded long-finned pilot whales (Globicephala melas) as well as the absence of arsenobetaine (AsB) which is often found to be a dominant As species in fish. We show data which suggests that there is an age-dependent accumulation of AsL in the brains of the animals. The results show that, in contrast to other organs, total arsenic as well as arsenolipids accumulate in an asymptotic pattern in the brains of the animals. Total As concentrations were found to range from 87 to 260 µg As/kg wet weight and between 0.6 and 27.6 µg As/kg was present in the form of AsPL958 in the brains of stranded pilot whales which was the most dominant lipophilic species present. The asymptotic relationship between total As, as well as AsPL, concentration in the brain and whale age may suggest that the accumulation of these species takes place prior to the full development of the blood-brain barrier in young whales. Finally, comparison between the organs of local squid, a common source of food for pilot whales, highlighted a comparable AsL profile which indicates a likely bioaccumulation pathway through the food chain.

Speciation of arsenic in milk from cows fed seaweed.

BACKGROUND: Including seaweed in cattle feed has gained increased interest, but it is important to take into account that the concentration of toxic metals, especially arsenic, is high in seaweed. This study investigated the arsenic species in milk from seaweed-fed cows. RESULTS: Total arsenic in milk of control diets (9.3 ± 1.0 µg As kg-1, n = 4, dry mass) was significantly higher than seaweed-based diet (high-seaweed diet: 7.8 ± 0.4 µg As kg-1, P < 0.05, n = 4, dry mass; low-seaweed diet: 6.2 ± 1.0 µg As kg-1, P < 0.01, n = 4, dry mass). Arsenic speciation showed that the main species present were arsenobetaine (AB) and arsenate (As(V)) (37% and 24% of the total arsenic, respectively). Trace amounts of dimethylarsinic acid (DMA) and arsenocholine (AC) have also been detected in milk. Apart from arsenate being significantly lower (P < 0.001) in milk from seaweed-fed cows than in milk from the control group, other arsenic species showed no significant differences between groups. CONCLUSION: The lower total arsenic and arsenate in seaweed diet groups indicates a possible competition of uptake between arsenate and phosphate, and the presence of AC indicates that a reduction of AB occurred in the digestive tract. Feeding a seaweed blend (91% Ascophyllum nodosum and 9% Laminaria digitata) does not raise As-related safety concerns for milk. © 2024 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Gestational α-ketoglutarate supplementation ameliorates arsenic-induced hepatic lipid deposition via epigenetic reprogramming of ß-oxidation process in female offspring.

Inorganic trivalent arsenic (iAsⅢ) at environmentally relevant levels has been found to cause developmental toxicity. Maternal exposure to iAsⅢ leads to enduring hepatic lipid deposition in later adult life. However, the exact mechanism in iAsⅢ induced hepatic developmental hazards is still unclear. In this study, we initially found that gestational exposure to iAsⅢ at an environmentally relevant concentration disturbs lipid metabolism and reduces levels of alpha-ketoglutaric acid (α-KG), an important mitochondrial metabolite during the citric acid cycle, in fetal livers. Further, gestational supplementation of α-KG alleviated hepatic lipid deposition caused by early-life exposure to iAsⅢ. This beneficial effect was particularly pronounced in female offspring. α-KG partially restored the ß-oxidation process in hepatic tissues by hydroxymethylation modifications of carnitine palmitoyltransferase 1a (Cpt1a) gene during fetal development. Insufficient ß-oxidation capacities probably play a crucial role in hepatic lipid deposition in adulthood following in utero arsenite exposure, which can be efficiently counterbalanced by replenishing α-KG. These results suggest that gestational administration of α-KG can ameliorate hepatic lipid deposition caused by iAsⅢ in female adult offspring partially through epigenetic reprogramming of the ß-oxidation pathway. Furthermore, α-KG shows potential as an interventive target to mitigate the harmful effects of arsenic-induced hepatic developmental toxicity.

Translocation, enzymatic reduction and toxicity of dimethylarsenate in rice.

Dimethylarsenate [DMAs(V)] can be produced by some soil microorganisms through methylation of inorganic arsenic (As), especially in anoxic paddy soils. DMAs(V) is more phytotoxic than inorganic As and can cause the physiological disorder straighthead disease in rice. Rice cultivars vary widely in the resistance to DMAs(V), but the mechanism remains elusive. Here, we investigated the differences in DMAs(V) uptake, translocation, and reduction to dimethylarsenite [DMAs(III)], as well as the effects on the metabolome, between two rice cultivars Mars and Zhe733. We found that Mars was 11-times more resistant to DMAs(V) than Zhe733. Mars accumulated more DMAs(V) in the roots, whereas Zhe733 translocated more DMAs(V) to the shoots and reduced more DMAs(V) to DMAs(III). DMAs(III) was more toxic than DMAs(V). Using heterologous expression and in vitro enzyme assays, we showed that the glutathione-S-transferases OsGSTU17 and OsGSTU50 were able to reduce DMAs(V) to DMAs(III). The expression levels of OsGSTU17 and OsGSTU50 were higher in the shoot of Zhe733 compared to Mars. Metabolomic analysis in rice shoots showed that glutathione (GSH) metabolism was perturbed by DMAs(V) toxicity in Zhe733. Application of exogenous GSH significantly alleviated the toxicity of DMAs(V) in Zhe733. Taken together, the results suggest that Mars is more resistant to DMAs(V) than Zhe733 because of a lower root-to-shoot translocation and a smaller capacity to reduce DMAs(V) to DMAs(III).

HSP90, a Common Therapeutic Target for Suppressing Skin Injury Caused by Exposure to Chemically Diverse Classes of Blistering Agents.

Vesicants such as arsenicals and mustards produce highly painful cutaneous inflammatory and blistering responses, hence developed as chemical weapons during World War I/II. Here, using lewisite and sulfur mustard surrogates, namely phenylarsine oxide (PAO) and 2-chloroethyl ethyl sulfide (CEES), respectively, we defined a common underlying mechanism of toxic action by these two distinct classes of vesicants. Murine skin exposure to these chemicals causes tissue destruction characterized by increase in skin bifold thickness, Draize score, infiltration of inflammatory cells, and apoptosis of epidermal and dermal cells. RNA sequencing analysis identified ∼346 inflammatory genes that were commonly altered by both PAO and CEES, along with the identification of cytokine signaling activation as the top canonical pathway. Activation of several proinflammatory genes and pathways is associated with phosphorylation-dependent activation of heat shock protein 90α (p-HSP90α). Topical treatment with known HSP90 inhibitors SNX-5422 and IPI-504 post PAO or CEES skin challenge significantly attenuated skin damage including reduction in overall skin injury and clinical scores. In addition, highly upregulated inflammatory genes Saa3, Cxcl1, Ccl7, IL-6, Nlrp3, Csf3, Chil3, etc. by both PAO and CEES were significantly diminished by treatment with HSP90 inhibitors. These drugs not only reduced PAO- or CEES-induced p-HSP90α expression but also its client proteins NLRP3 and pP38 and the expression of their target inflammatory genes. Our data confirm a critical role of HSP90 as a shared underlying molecular target of toxicity by these two distinct vesicants and provide an effective and novel medical countermeasure to suppress vesicant-induced skin injury. SIGNIFICANCE STATEMENT: Development of effective and novel mechanism-based antidotes that can simultaneously block cutaneous toxic manifestations of distinct vesicants is important and urgently needed. Due to difficulties in determining the exact nature of onsite chemical exposure, a potent drug that can suppress widespread cutaneous damage may find great utility. Thus, this study identified HSP90 as a common molecular regulator of cutaneous inflammation and injury by two distinct warfare vesicants, arsenicals and mustards, and HSP90 inhibitors afford significant protection against skin damage.

Cutaneous Arsenical Exposure Induces Distinct Metabolic Transcriptional Alterations of Kidney Cells.

Arsenicals are deadly chemical warfare agents that primarily cause death through systemic capillary fluid leakage and hypovolemic shock. Arsenical exposure is also known to cause acute kidney injury, a condition that contributes to arsenical-associated death due to the necessity of the kidney in maintaining whole-body fluid homeostasis. Because of the global health risk that arsenicals pose, a nuanced understanding of how arsenical exposure can lead to kidney injury is needed. We used a nontargeted transcriptional approach to evaluate the effects of cutaneous exposure to phenylarsine oxide, a common arsenical, in a murine model. Here we identified an upregulation of metabolic pathways such as fatty acid oxidation, fatty acid biosynthesis, and peroxisome proliferator-activated receptor (PPAR)-α signaling in proximal tubule epithelial cell and endothelial cell clusters. We also revealed highly upregulated genes such as Zbtb16, Cyp4a14, and Pdk4, which are involved in metabolism and metabolic switching and may serve as future therapeutic targets. The ability of arsenicals to inhibit enzymes such as pyruvate dehydrogenase has been previously described in vitro. This, along with our own data, led us to conclude that arsenical-induced acute kidney injury may be due to a metabolic impairment in proximal tubule and endothelial cells and that ameliorating these metabolic effects may lead to the development of life-saving therapies. SIGNIFICANCE STATEMENT: In this study, we demonstrate that cutaneous arsenical exposure leads to a transcriptional shift enhancing fatty acid metabolism in kidney cells, indicating that metabolic alterations might mechanistically link topical arsenical exposure to acute kidney injury. Targeting metabolic pathways may generate promising novel therapeutic approaches in combating arsenical-induced acute kidney injury.

Dapagliflozin alleviates arsenic trioxide-induced hepatic injury in rats via modulating PI3K/AkT/mTOR, STAT3/SOCS3/p53/MDM2 signaling pathways and miRNA-21, miRNA-122 expression.

Dapagliflozin (DPG) is a sodium-glucose co-transporter 2 inhibitor that is commonly used in the treatment of type 2 diabetes. However, studies have shown that DPG has a protective effect under a variety of experimental conditions through its antioxidative and anti-inflammatory properties. DPG's effect on experimental hepatotoxicity caused by arsenic trioxide (ATO) has yet to be investigated. The purpose of this study was to investigate the protective effect of DPG in preventing hepatic damage caused by ATO and discover the underlying mechanisms. The effect of DPG (1 mg/kg, orally) on ATO (5 mg/kg, i.p.)-induced hepatic injury was evaluated in rats. Serum liver function parameters, as well as oxidative stress biomarkers and inflammatory cytokine levels were assessed. Histopathological changes in the liver were detected using H&E staining. Using Western blotting and PCR techniques, the molecular mechanisms of DPG in ameliorating hepatic injury were investigated. DPG improved liver function by inhibiting histopathological changes, decreasing levels of hepatic function and toxicity parameters measured in both serum and tissues, and exhibiting antioxidant and anti-inflammatory effects, according to the findings. Consistent with the PCR results, DPG also decreased the expression of LC3-II, micro-RNA-122, and micro-RNA-21 while increased the expression of SOCS3. Furthermore, according to western blotting results, DPG was able to reduce the protein expression of AKT, mTOR, PI3K, and STAT3. Although further clinical research is necessary, this study highlights the potential of DPG in preventing liver damage in a rat model of hepatotoxicity induced by ATO.

Study on arsenic speciation, bioaccessibility, and gut microbiota in realgar-containing medicines by DGT technique and artificial gastrointestinal extraction (PBET) combine with simulated human intestinal microbial ecosystem (SHIME).

It is well-known that several Chinese patent medicines use realgar as a specific component. People are more aware of the health dangers associated with realgar since it includes arsenic. Previous research overstated the arsenic toxicity of realgar-containing Chinese prescription medications because little thought was given to the influence of arsenic bioaccessibility by gut microbiota. In light of this, this study examined the total content, bioaccessibility and speciation of targeted medications while also examining intestinal epithelial transit utilizing the diffusive gradients in thin-films (DGT). All samples contained arsenic, and the bioaccessibilities of the colon, intestine and gastric regions ranged from 0.19% to 1.73%, 0.25-1.88% and 0.21-1.70% respectively. The range of DGT-bioaccessibility is 0.01-0.0018%. Three steps of analysis were conducted on inorganic As(III) and As(V). In health risk assessment, the ADDs and HQs of DGT-bioaccessibility were below the threshold levels when compared to computing average daily intake dose (ADD) and hazard quotient (HQ) by bioaccessibility of gastric, intestinal and colon. Additionally, Proteobacteria and Firmicutes were discovered to be the two predominant kinds of gut microbes in this study. Under arsenic exposure, the abundance of Christensenellaceae, Desulfovibrionaceae and Akkermansiaceae increased, but the quantity of Rikenellaceae decreased. These findings revealed that alterations in gut microbiota had an impact on host metabolism.

Metabolomics for identifying pathways involved in vesicating agent lewisite-induced corneal injury.

Lewisite (LEW) is an arsenical vesicant that can be a potentially dangerous chemical warfare agent (CWA). Eyes are particularly susceptible to vesicant induced injuries and ocular LEW exposure can act swiftly, causing burning of eyes, edema, inflammation, cell death and even blindness. In our previous studies, we developed a LEW exposure-induced corneal injury model in rabbit and showed increased inflammation, neovascularization, cell death, and structural damage to rabbit corneas upon LEW exposure. In the present study, we further assessed the metabolomic changes to delineate the possible mechanisms underlying the LEW-induced corneal injuries. This information is vital and could help in the development of effective targeted therapies against ocular LEW injuries. Thus, the metabolomic changes associated with LEW exposures in rabbit corneas were assessed as a function of time, to delineate pathways from molecular perturbations at the genomic and proteomic levels. New Zealand white rabbit corneas (n = 3-6) were exposed to LEW vapor (0.2 mg/L; flow rate: 300 ml/min) for 2.5 min (short exposure; low dose) or 7.5 min (long-exposure; high dose) and then collected at 1, 3, 7, or 14 days post LEW exposure. Samples were prepared using the automated MicroLab STAR® system, and proteins precipitated to recover the chemically diverse metabolites. Metabolomic analysis was carried out by reverse phase UPLC-MS/MS and gas chromatography (GC)-MS. The data obtained were analyzed using Metabolon's software. The results showed that LEW exposures at high doses were more toxic, particularly at the day 7 post exposure time point. LEW exposure was shown to dysregulate metabolites associated with all the integral functions of the cornea and cause increased inflammation and immune response, as well as generate oxidative stress. Additionally, all important metabolic functions of the cells were also affected: lipid and nucleotide metabolism, and energetics. The high dose LEW exposures were more toxic, particularly at day 7 post LEW exposure (>10-fold increased levels of histamine, quinolinate, N-acetyl-ß-alanine, GMP, and UPM). LEW exposure dysregulated integral functions of the cornea, caused inflammation and heightened immune response, and generated oxidative stress. Lipid and nucleotide metabolism, and energetics were also affected. The novel information about altered metabolic profile of rabbit cornea following LEW exposure could assist in delineating complex molecular events; thus, aid in identifying therapeutic targets to effectively ameliorate ocular trauma.

Differential modulation of cytochrome P450 enzymes by arsenicals in non-human experimental models.

Arsenic is a hazardous heavy metalloid that imposes threats to human health globally. It is widely spread throughout the environment in various forms. Arsenic-based compounds are either inorganic compounds (iAs) or organoarsenicals (oAs), where the latter are biotically generated from the former. Exposure to arsenic-based compounds results in varying biochemical derangements in living systems, leading eventually to toxic consequences. One important target for arsenic in biosystems is the network of metabolic enzymes, especially the superfamily of cytochrome P450 enzymes (CYPs) because of their prominent role in both endobiotic and xenobiotic metabolism. Therefore, the alteration of the CYPs by different arsenicals has been actively studied in the last few decades. We have previously summarized the findings of former studies investigating arsenic associated modulation of different CYPs in human experimental models. In this review, we focus on non-human models to get a complete picture about possible CYPs alterations in response to arsenic exposure.

Prevalence of Methylated Arsenic and Microbial Arsenic Methylation Genes in Paddy Soils of the Mekong Delta.

Microbially mediated inorganic-methylated arsenic (As) transformation in paddy soil is crucial to rice safety; however, the linkages between the microbial As methylation process and methylated As species remain elusive. Here, 62 paddy soils were collected from the Mekong River delta of Cambodia to profile As-related functional gene composition involved in the As cycle. The soil As concentration ranged from <1 to 16.6 mg kg-1, with average As contents of approximately 81% as methylated As and 54% as monomethylarsenate (MMAs(V)) in the phosphate- and oxalate-extractable fractions based on As sequential extraction analysis. Quantitative PCR revealed high arsenite-methylating gene (arsM) copy numbers, and metagenomics identified consistently high arsM gene abundance. The abundance of As-related genes was the highest in bacteria, followed by archaea and fungi. Pseudomonas, Bradyrhizobium, Burkholderia, and Anaeromyxobacter were identified as bacteria harboring the most genes related to As biotransformation. Moreover, arsM and arsI (As demethylation) gene-containing operons were identified in the metagenome-assembled genomes (MAGs), implying that arsM and arsI could be transcribed together. The prevalence of methylated As and arsM genes may have been overlooked in tropical paddy fields. The As methylation-demethylation cycle should be considered when manipulating the methylated As pool in paddy fields for rice safety.

MDM2-mediated Inhibitory Effect of Arsenic Trioxide on Small Cell Lung Cancer Cell Line by Degrading Mutant p53.

INTRODUCTION: Small cell lung cancer (SCLC) is featured by a high TP53 mutant rate. Our previous research found that arsenic trioxide (As2O3) could significantly inhibit the growth and metastasis of SCLC. Studies have shown that the degradation of mutant p53 mediated by murine double minute 2 (MDM2) can be induced by As2O3, which probably contributes to the inhibition of SCLC, but the detailed mechanism is still unclear. We aimed to testify that As2O3 can inhibit the growth of SCLC cells by degrading mutant p53 protein via binding to MDM2. METHODS: CCK-8 assay, cell cycle analysis, and western blot of apoptosis markers were used to evaluate the inhibitory effect of As2O3 on NCI-H446 cells (containing mutant p53) and NCI-H1299 cells (p53 null). The effects of As2O3 on p53 and its downstream proteins were identified by western blot using mut-p53-knockdown and overexpressed cell models. MDM2-knockdown cell models were constructed, and western blot, co-IP of mut-p53, and ubiquitin were carried out to explore the mediating effect of MDM2 in As2O3 induced mut-p53 degradation. RESULTS: As2O3 inhibited proliferation and induced cell cycle arrest and apoptosis of SCLC cells in a dose- and timedependent manner. After mut-p53 knockdown or overexpressed, the inhibitory effect of As2O3 was dampened or enhanced. Additionally, As2O3-induced mut-p53 ubiquitination was significantly weakened after MDM2 knockdown. CONCLUSION: As2O3 could inhibit SCLC cells by inhibiting proliferation and inducing cell cycle arrest and apoptosis. These inhibitory effects were achieved at least in part by upregulating MDM2, which, in turn, promotes ubiquitination and degradation of mut-p53.

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.

Urinary microbiota and metabolic signatures associated with inorganic arsenic-induced early bladder lesions.

Inorganic arsenic (iAs) contamination in drinking water is a global public health problem, and exposure to iAs is a known risk factor for bladder cancer. Perturbation of urinary microbiome and metabolome induced by iAs exposure may have a more direct effect on the development of bladder cancer. The aim of this study was to determine the impact of iAs exposure on urinary microbiome and metabolome, and to identify microbiota and metabolic signatures that are associated with iAs-induced bladder lesions. We evaluated and quantified the pathological changes of bladder, and performed 16S rDNA sequencing and mass spectrometry-based metabolomics profiling on urine samples from rats exposed to low (30 mg/L NaAsO2) or high (100 mg/L NaAsO2) iAs from early life (in utero and childhood) to puberty. Our results showed that iAs induced pathological bladder lesions, and more severe effects were noticed in the high-iAs group and male rats. Furthermore, six and seven featured urinary bacteria genera were identified in female and male offspring rats, respectively. Several characteristic urinary metabolites, including Menadione, Pilocarpine, N-Acetylornithine, Prostaglandin B1, Deoxyinosine, Biopterin, and 1-Methyluric acid, were identified significantly higher in the high-iAs groups. In addition, the correlation analysis demonstrated that the differential bacteria genera were highly correlated with the featured urinary metabolites. Collectively, these results suggest that exposure to iAs in early life not only causes bladder lesions, but also perturbs urinary microbiome composition and associated metabolic profiles, which shows a strong correlation. Those differential urinary genera and metabolites may contribute to bladder lesions, suggesting a potential for development of urinary biomarkers for iAs-induced bladder cancer.

Monomethylarsonous acid binds to Cys-104α and Cys-112ß of hemoglobin in acute promyelocytic leukemia patients treated with arsenic trioxide.

Arsenic trioxide (As2O3) has prominent effect in treating acute promyelocytic leukemia (APL). Identification of arsenic-binding proteins has gained attention for their important biological functions. However, none has been published concerning the binding mechanism of arsenic with hemoglobin (Hb) in APL patients after treatment of As2O3. The present study discloses the binding sites of arsenic on Hb in APL patients. Concentrations of inorganic arsenic (iAs), monomethyl arsenic (MMA), and dimethyl arsenic (DMA) in erythrocytes of APL patients were quantified using HPLC-inductively coupled plasma-mass spectroscopy (HPLC-ICP-MS). Hb-bound arsenic was identified by size-exclusion chromatography ICP-MS. The binding sites of arsenic on Hb were determined by mass spectrometry (MS). The concentration trend of arsenic species in erythrocytes of 9 APL patients treated with As2O3 was iAs>MMA>DMA, and MMA was the predominant methylated arsenic metabolite. Size-exclusion chromatography separation of free and protein-bound arsenic by simultaneous monitoring of 57Fe and 75As demonstrated the presence of Hb-bound arsenic. MS information suggested monomethylarsonous (MMAIII) was the dominant arsenic bound to Hb, and further identified that Cys-104α and Cys-112ß were two binding sites of MMAIII in Hb. MMAIII binding to Cys-104α and Cys-112ß was responsible for arsenic accumulation in erythrocytes of APL patients. This interaction may contribute to understand the therapeutic effect of As2O3 as an anticancer drug and its toxicity on APL patients.

As3MT is related to relative RNAs and base modifications of p53 in workers exposed to arsenic.

As3MT is the key enzyme involved in the methylation metabolism of arsenic. It is associated with DNA methylation closely also. This study is to explore the relationships between As3MT and epigenetic changes, and how p53 and relative ncRNAs and mRNAs play roles in the process. In this study, workers from four arsenic plants and individuals who resided in villages far away from the four plants were recruited. Arsenic compounds, relative indices, 28 relative RNAs, and base modifications of exons 5-8 of p53 were detected separately. Several methods were used to analyze the associations between them. Results shown that As3MT RNA was closely associated with all selected lncRNAs, miRNAs, and mRNAs related to miRNA production and maturation, tumorigenesis, and base modifications of p53. There probably exists causal relationship. Base modifications of exons 7 and 8 of p53 had significant synergistic effects on the expression of As3MT RNA and a series of genetic indices. But miR-190, miR-548, and base modifications of exon 5 of p53 had substantial inhibitory effects. Arsenic compounds and relative indices of metabolic transformation may have limited roles. The main novel finding in the present study is that As3MT play special and significant roles in the genotoxicity and carcinogenesis which could be coordinated operation with p53, and influenced by epigenetic factors largely, such as lncRNAs and miRNAs. P53 and relative ncRNAs and mRNAs may regulate the process by interacting with As3MT. The changes may initiate by arsenic, but probability through indirect relationship.

Targeting HDAC3 to overcome the resistance to ATRA or arsenic in acute promyelocytic leukemia through ubiquitination and degradation of PML-RARα.

Acute promyelocytic leukemia (APL) is driven by the oncoprotein PML-RARα, which recruits corepressor complexes, including histone deacetylases (HDACs), to suppress cell differentiation and promote APL initiation. All-trans retinoic acid (ATRA) combined with arsenic trioxide (ATO) or chemotherapy highly improves the prognosis of APL patients. However, refractoriness to ATRA and ATO may occur, which leads to relapsed disease in a group of patients. Here, we report that HDAC3 was highly expressed in the APL subtype of AML, and the protein level of HDAC3 was positively associated with PML-RARα. Mechanistically, we found that HDAC3 deacetylated PML-RARα at lysine 394, which reduced PIAS1-mediated PML-RARα SUMOylation and subsequent RNF4-induced ubiquitylation. HDAC3 inhibition promoted PML-RARα ubiquitylation and degradation and reduced the expression of PML-RARα in both wild-type and ATRA- or ATO-resistant APL cells. Furthermore, genetic or pharmacological inhibition of HDAC3 induced differentiation, apoptosis, and decreased cellular self-renewal of APL cells, including primary leukemia cells from patients with resistant APL. Using both cell line- and patient-derived xenograft models, we demonstrated that treatment with an HDAC3 inhibitor or combination of ATRA/ATO reduced APL progression. In conclusion, our study identifies the role of HDAC3 as a positive regulator of the PML-RARα oncoprotein by deacetylating PML-RARα and suggests that targeting HDAC3 could be a promising strategy to treat relapsed/refractory APL.

Enhanced Transdermal Delivery of N-Acetylcysteine and 4-Phenylbutyric Acid for Potential Use as Antidotes to Lewisite.

Lewisite is a highly toxic chemical warfare agent that leads to cutaneous and systemic damage. N-acetylcysteine (NAC) and 4-phenylbutryic acid (4-PBA) are two novel antidotes developed to treat toxicity caused by lewisite and similar arsenicals. Our in vivo studies demonstrated safety and effectiveness of these agents against skin injury caused by surrogate lewisite (Phenylarsine oxide) proving their potential for the treatment of lewisite injury. We further focused on exploring various enhancement strategies for an enhanced delivery of these agents via skin. NAC did not permeate passively from propylene glycol (PG). Iontophoresis as a physical enhancement technique and chemical enhancers were investigated for transdermal delivery of NAC. Application of cathodal and anodal iontophoresis with the current density of 0.2 mA/cm2 for 4 h followed by passive diffusion till 24 h significantly enhanced the delivery of NAC with a total delivery of 65.16 ± 1.95 µg/cm2 and 87.23 ± 7.02 µg/cm2, respectively. Amongst chemical enhancers, screened oleic acid, oleyl alcohol, sodium lauryl ether sulfate, and dimethyl sulfoxide (DMSO) showed significantly enhanced delivery of NAC with DMSO showing highest delivery of 28,370.2 ± 2355.4 µg/cm2 in 24 h. Furthermore, 4-PBA permeated passively from PG with total delivery of 1745.8 ± 443.5 µg/cm2 in 24 h. Amongst the chemical enhancers screened for 4-PBA, oleic acid, oleyl alcohol, and isopropyl myristate showed significantly enhanced delivery with isopropyl myristate showing highest total delivery of 17,788.7 ± 790.2 µg/cm2. These studies demonstrate feasibility of delivering these antidotes via skin and will aid in selection of excipients for the development of topical/transdermal delivery systems of these agents.

The ArsQ permease and transport of the antibiotic arsinothricin.

The pentavalent organoarsenical arsinothricin (AST) is a natural product synthesized by the rhizosphere bacterium Burkholderia gladioli GSRB05. AST is a broad-spectrum antibiotic effective against human pathogens such as carbapenem-resistant Enterobacter cloacae. It is a non-proteogenic amino acid and glutamate mimetic that inhibits bacterial glutamine synthetase. The AST biosynthetic pathway is composed of a three-gene cluster, arsQML. ArsL catalyzes synthesis of reduced trivalent hydroxyarsinothricin (R-AST-OH), which is methylated by ArsM to the reduced trivalent form of AST (R-AST). In the culture medium of B. gladioli, both trivalent species appear as the corresponding pentavalent arsenicals, likely due to oxidation in air. ArsQ is an efflux permease that is proposed to transport AST or related species out of the cells, but the chemical nature of the actual transport substrate is unclear. In this study, B. gladioli arsQ was expressed in Escherichia coli and shown to confer resistance to AST and its derivatives. Cells of E. coli accumulate R-AST, and exponentially growing cells expressing arsQ take up less R-AST. The cells exhibit little transport of their pentavalent forms. Transport was independent of cellular energy and appears to be equilibrative. A homology model of ArsQ suggests that Ser320 is in the substrate binding site. A S320A mutant exhibits reduced R-AST-OH transport, suggesting that it plays a role in ArsQ function. The ArsQ permease is proposed to be an energy-independent uniporter responsible for downhill transport of the trivalent form of AST out of cells, which is oxidized extracellularly to the active form of the antibiotic.