Ferroptosis is involved in cisplatin sensitivity of the S3 segment of immortalized proximal tubule cells.

Cisplatin (CDDP) is administered as an anticancer drug across a broad spectrum of cancer treatments, but it causes severe renal damage. Several studies have attempted to elucidate the cause of CDDP-induced renal injury, but the detailed mechanism remains unclear. We previously found that S3 cells are more sensitive to CDDP than S1 and S2 cells by using immortalized cells derived from S1, S2, and S3 segments of proximal tubules. In this study, we investigated the potential contribution of reactive oxygen species (ROS) to the sensitivity of S3 cells to CDDP. The results showed that S3 cells have high sensitivity to CDDP, paraquat (PQ) and three ROS substances. To examine the mechanisms underlying the sensitivity to ROS in S3 cells, we compared the cellular responses of CDDP- and PQ-exposed S3 cells. The results indicated that the levels of intracellular ROS and lipid peroxides were increased in S3 cells after CDDP and PQ exposure. The intracellular levels of antioxidant proteins such as thioredoxin, thioredoxin reductase 1 and glutathione peroxidase 4 were also increased by exposure to PQ, but these proteins were decreased by CDDP exposure in S3 cells. Furthermore, the levels of intracellular free Fe2+ were increased by CDDP exposure only in S3 cells but not S1 or S2 cells, and cytotoxicity by exposure to CDDP in S3 cells was suppressed by ferroptosis inhibitors. These results suggested that the induction of ferroptosis due to the ROS production through attenuation of the antioxidant system and elevated free Fe2+ is partly responsible for the sensitivity of S3 cells to CDDP.

Arsenic Exposure-Related Hypertension in Bangladesh and Reduced Circulating Nitric Oxide Bioavailability.

BACKGROUND: Hypertension is a major cause of death worldwide. Although arsenic exposure has been associated with the risk of hypertension, this association appears nonuniform due to inconsistent results from studies conducted in different populations. Moreover, hypertension is a complex condition with multiple underlying mechanisms and factors. One factor is impaired production and bioavailability of vascular nitric oxide (NO). However, the implications of the effects of arsenic exposure on circulating NO and its association with hypertension in humans are largely unknown. OBJECTIVE: We investigated the dose-response relationship between arsenic exposure and hypertension with vascular NO levels as a potential mediator of arsenic-related hypertension in individuals exposed to a broad range of arsenic. METHODS: A total of 828 participants were recruited from low- and high-arsenic exposure areas in Bangladesh. Participants' drinking water, hair, and nail arsenic concentrations were measured by inductively coupled plasma mass spectroscopy. Hypertension was defined as a systolic blood pressure (SBP) value of ≥140 and a diastolic (DBP) value of ≥90 mmHg. Serum NO levels reflected by total serum nitrite concentrations were measured by immunoassay. A formal causal mediation analysis was used to assess NO as a mediator of the association between arsenic level and hypertension. RESULTS: Increasing concentrations of arsenic measured in drinking water, hair, and nails were associated with the increasing levels of SBP and DBP. The odds of hypertension were dose-dependently increased by arsenic even in participants exposed to relatively low to moderate levels (10-50µg/L) of water arsenic [odds ratios (ORs) and 95% confidence intervals (CIs): 2.87 (95% CI: 1.28, 6.44), 2.67 (95% CI: 1.27, 5.60), and 5.04 (95% CI: 2.71, 9.35) for the 10-50µg/L, 50.01-150µg/L, and >150µg/L groups, respectively]. Causal mediation analysis showed a significant mediating effect of NO on arsenic-related SBP, DBP, and hypertension. CONCLUSION: Increasing exposure to arsenic was associated with increasing odds of hypertension. The association was mediated through the reduction of vascular NO bioavailability, suggesting that impaired NO bioavailability was a plausible underlying mechanism of arsenic-induced hypertension in this Bangladeshi population. https://doi.org/10.1289/EHP13018.

Effects of methylation of arginine residue 83 on the enzymatic activity of human arsenic (+3 oxidation state) methyltransferase.

Arsenic (+3 oxidation state) methyltransferase is an enzyme responsible for arsenic methylation, and it requires S-adenosyl-methionine (SAM) as a coenzyme. We here generated two mutants to clarify the role of the highly conserved 83rd arginine residue (Arg83) in Motif I, the SAM-binding domain, of human AS3MT. When the AS3MT activity was compared between the mutants and the wild type (WT) recombinant protein, little activity was detected in the glycine mutant (Arg83Gly) or lysine mutant (Arg83Lys). When we examined the ability of transfected HEK293 cells exposed to arsenite to methylate arsenic, the methylation ability was significantly reduced in Arg83Gly compared to the WT, but was not significantly different between Arg83Lys and WT. Western blot analysis of the recombinant WT and Arg83Gly with an antibody that recognizes methylated Arg showed that an Arg residue in the WT was mono- and di-methylated, but not in Arg83Gly. Furthermore, a peptide containing dimethylated Arg83 was detected by MALDI-TOF/MS of the WT digested with chymotrypsin. These results indicate that AS3MT maintains its enzymatic activity through the methyl modification of Arg83.

CHAC1 exacerbates arsenite cytotoxicity by lowering intracellular glutathione levels.

We here examined whether CHAC1 is implicated in arsenite (As(III))-induced cytotoxicity in HaCaT cells. We found that HaCaT cells in which the intracellular GSH levels were elevated by transfection with CHAC1 siRNA showed decreased sensitivity to As(III) compared to the control cells. Treatment with BSO (an inhibitor of GSH biosynthesis) abolished the decrease in sensitivity to As(III), suggesting that an increase in intracellular GSH levels was involved in the decrease in sensitivity to As(III) due to the decrease in the levels of CHAC1 expression. When we examined the expression of CHAC1 after exposure of HaCaT cells to As(III), the levels of CHAC1 were increased. Since CHAC1 is a proapoptotic factor, we examined appearance of apoptotic cells and cleavage of caspase-3 after exposure to As(III) to determine whether As(III)-induced CHAC1 up-regulation was involved in apoptosis induction. The results showed that induction of apoptosis by As(III) exposure was not detected in CHAC1 siRNA-transfected cells. Together, our findings indicate that CHAC1 is involved in the sensitivity of HaCaT cells to As(III) by regulating the intracellular GSH levels, and in particular, CHAC1 is involved in As(III)-induced apoptosis.

Association between serum periostin levels and the severity of arsenic-induced skin lesions.

Arsenic is a potent environmental toxicant and human carcinogen. Skin lesions are the most common manifestations of chronic exposure to arsenic. Advanced-stage skin lesions, particularly hyperkeratosis have been recognized as precancerous diseases. However, the underlying mechanism of arsenic-induced skin lesions remains unknown. Periostin, a matricellular protein, is implicated in the pathogenesis of many forms of skin lesions. The objective of this study was to examine whether periostin is associated with arsenic-induced skin lesions. A total of 442 individuals from low- (n = 123) and high-arsenic exposure areas (n = 319) in rural Bangladesh were evaluated for the presence of arsenic-induced skin lesions (Yes/No). Participants with skin lesions were further categorized into two groups: early-stage skin lesions (melanosis and keratosis) and advanced-stage skin lesions (hyperkeratosis). Drinking water, hair, and nail arsenic concentrations were considered as the participants' exposure levels. The higher levels of arsenic and serum periostin were significantly associated with skin lesions. Causal mediation analysis revealed the significant effect of arsenic on skin lesions through the mediator, periostin, suggesting that periostin contributes to the development of skin lesions. When skin lesion was used as a three-category outcome (none, early-stage, and advanced-stage skin lesions), higher serum periostin levels were significantly associated with both early-stage and advanced-stage skin lesions. Median (IQR) periostin levels were progressively increased with the increasing severity of skin lesions. Furthermore, there were general trends in increasing serum type 2 cytokines (IL-4, IL-5, IL-13, and eotaxin) and immunoglobulin E (IgE) levels with the progression of the disease. The median (IQR) of IL-4, IL-5, IL-13, eotaxin, and IgE levels were significantly higher in the early-and advanced-stage skin lesions compared to the group of participants without skin lesions. The results of this study suggest that periostin is implicated in the pathogenesis and progression of arsenic-induced skin lesions through the dysregulation of type 2 immune response.

Spatial localization of cadmium and metallothionein in the kidneys of mice at the early phase of cadmium accumulation.

Chronic exposure to cadmium (Cd) leads to an accumulation of Cd in the kidneys. Metallothionein (MT) is a low-molecular-weight protein having a high affinity for Cd. Cd bound to MT in serum is filtered through the glomeruli of kidney nephrons and reabsorbed by endocytosis into the proximal tubules from the luminal side. Accumulation of Cd in renal cells induces MT synthesis, leading to long-term deposition of Cd and the suppression of Cd toxicity. Recently, many studies have investigated the tissue distribution of metals using laser ablation ICP-MS (LA-ICP-MS). However, little information has been available regarding renal Cd distribution. Hence, we dually investigated the renal distribution of Cd by LA-ICP-MS and that of MT by immunohistochemistry to clarify the dose- and time-dependent changes in the distributions of Cd and MT in mice exposed to Cd from drinking water for 1, 2, and 4 months. Both Cd and MT exhibited characteristic heterogeneous distribution patterns in the renal cortex. The accumulation of Cd and MT near the surface of the cortex suggests a preferential accumulation of Cd in the surface nephrons. MT distribution was more pronounced in the proximal tubules than in the distal tubules, and there were clear differences in MT immunostaining even among the proximal tubules. The heterogeneous localization of MT may reflect the nephron-specific accumulation of Cd. Combining elemental imaging of Cd with immunostaining of MT proved a successful strategy to reveal the characteristic renal Cd distribution, especially in the early stages of Cd accumulation.

Effects of individual amino acid mutations of zinc transporter ZIP8 on manganese- and cadmium-transporting activity.

Zinc (Zn) transporter ZIP8, encoded by SLC39A8, is a unique transporter that can transport divalent manganese (Mn) and cadmium (Cd) in addition to Zn. Recently, associations between various human diseases and variant forms of ZIP8 have been reported. Four amino acid residues, V33, G38, S335, and I340, of human ZIP8 (hZIP8) are mutated in patients with congenital disorders of glycosylation (CDG), whose blood Mn levels are extremely low. Many genome-wide association studies have reported that the A391T mutation of hZIP8 caused by rs13107325 is associated with a wide range of diseases. However, the roles of individual mutations of hZIP8 on metal-transporting activity remain elusive. We established DT40 cells respectively expressing the four mutant hZIP8s and compared the Mn- and Cd-transporting activity between the mutants and wild-type hZIP8. Among the four mutations observed in the ZIP8-mutated CDG patients, the S335T and I340 N mutations in the predicted transmembrane domain 5 (TMD5) completely abolished Mn- and Cd-transporting activity, while V33 M or G35R mutations at the N-terminus did not. We also examined the A391T mutation, which slightly reduced metal transporting activity. Finally, we examined the effects of artificial mutations in the metal-binding motif EEXXH in the TMD5. Replacing EEXXH with HEXXH, which exists in most ZIP transporters, abolished the Mn- and Cd-transporting activity of hZIP8, indicating that glutamic acid in this motif plays a critical role in the unique affinity of ZIP8 for Mn and Cd. Thus, the utilization of DT40 cells enabled us to clarify the different functions of each residue of hZIP8 on metal transport.

Elevated serum periostin levels among arsenic-exposed individuals and their associations with the features of asthma.

Chronic exposure to arsenic via drinking water is a serious public health issue in many countries. Arsenic causes not only cancers but also non-malignant diseases, including asthma. We have previously reported that arsenic exposure increases the risk of Th2-mediated allergic asthma. The serum level of periostin, an extracellular matrix protein activated by Th2 cytokines, is recognized as a biomarker for Th2-mediated eosinophilic asthma and contributes to enhanced airway inflammation and remodeling. However, the role of periostin in arsenic-related asthma is unknown. Therefore, this study was designed to explore the associations of serum periostin levels with arsenic exposure and the features of asthma in 442 individuals in Bangladesh who participated in our previous study. Exposure levels of the participants were determined by measuring the arsenic concentrations in drinking water, hair, and nails through inductively coupled plasma mass spectroscopy. Periostin levels in serum were assessed by immunoassay. In this study, we found that serum periostin levels of the participants were increased with increasing exposure to arsenic. Notably, even the participants with 10.1-50 µg/L arsenic in drinking water had significantly higher levels of periostin than participants with <10 µg/L of water arsenic. Elevated serum periostin levels were positively associated with serum levels of Th2 mediators, such as interleukin (IL)-4, IL-5, IL-13, and eotaxin. Each log increase in periostin levels was associated with approximately eight- and three-fold increases in the odds ratios (ORs) for reversible airway obstruction (RAO) and asthma symptoms, respectively. Additionally, causal mediation analyses revealed that arsenic exposure metrics had both direct and indirect (periostin-mediated) effects on the risk of RAO and asthma symptoms. Thus, the results suggested that periostin may be involved in the arsenic-related pathogenesis of Th2-mediated asthma. The elevated serum periostin levels may predict the greater risk of asthma among the people living in arsenic-endemic areas.

Arsenic Secondary Methylation Capacity Is Inversely Associated with Arsenic Exposure-Related Muscle Mass Reduction.

Skeletal muscle mass reduction has been implicated in insulin resistance (IR) that promotes cardiometabolic diseases. We have previously reported that arsenic exposure increases IR concomitantly with the reduction of skeletal muscle mass among individuals exposed to arsenic. The arsenic methylation capacity is linked to the susceptibility to some arsenic exposure-related diseases. However, it remains unknown whether the arsenic methylation capacity affects the arsenic-induced reduction of muscle mass and elevation of IR. Therefore, this study examined the associations between the arsenic methylation status and skeletal muscle mass measures with regard to IR by recruiting 437 participants from low- and high-arsenic exposure areas in Bangladesh. The subjects' skeletal muscle mass was estimated by their lean body mass (LBM) and serum creatinine levels. Subjects' drinking water arsenic concentrations were positively associated with total urinary arsenic concentrations and the percentages of MMA, as well as inversely associated with the percentages of DMA and the secondary methylation index (SMI). Subjects' LBM and serum creatinine levels were positively associated with the percentage of DMA and SMI, as well as inversely associated with the percentage of MMA. HOMA-IR showed an inverse association with SMI, with a confounding effect of sex. Our results suggest that reduced secondary methylation capacity is involved in the arsenic-induced skeletal muscle loss that may be implicated in arsenic-induced IR and cardiometabolic diseases.

Comparisons of segment-specific toxicity of platinum-based agents and cadmium using S1, S2, and S3 cells derived from mouse kidney proximal tubules.

Renal toxicants such as cisplatin and cadmium cause segment-specific damages in kidney proximal tubules. Recently, we established an in vitro experimental system for evaluating segment-specific toxicity and transport of chemicals using immortalized S1, S2, and S3 cells derived from the S1, S2, and S3 regions of mouse kidney proximal tubules. In the present study, we examined the toxicity and accumulation of cisplatin, carboplatin, oxaliplatin, and cadmium in S1, S2, and S3 cells. We found that not only cisplatin but also carboplatin and oxaliplatin exhibited higher lethal toxicity in S3 cells than in S1 and S2 cells. At sublethal doses, cisplatin showed delayed induction of Kim-1 and clusterin on days 3 and 6, which may reflect the latent renal toxicity of cisplatin in vivo. The high sensitivities of S3 cells to the platinum-based agents were not due to the high accumulation of Pt in S3 cells. Exposure to cadmium resulted in similar toxicity among these cells, suggesting that S3 cells were not sensitive to any renal toxicants. Thus, the utilization of S1, S2, and S3 cells may provide a useful tool for the in vitro evaluation of the proximal tubule segment-specific toxicity of chemicals.

T helper 2-driven immune dysfunction in chronic arsenic-exposed individuals and its link to the features of allergic asthma.

Limited information is available regarding the effects of arsenic exposure on immune function. We have recently reported that chronic exposure to As was associated asthma, as determined by spirometry and respiratory symptoms. Because T helper 2 (Th2)-driven immune responses are implicated in the pathogenesis of allergic diseases, including asthma, we studied the associations of serum Th1 and Th2 mediators with the As exposure markers and the features of asthma among individuals exposed to As. A total of 553 blood samples were selected from the same study subjects recruited in our previous asthma study. Serum levels of Th1 and Th2 cytokines were analyzed by immunoassay. Subjects' arsenic exposure levels (drinking water, hair and nail arsenic concentrations) were determined by inductively coupled plasma mass spectroscopy. Arsenic exposure levels of the subjects showed significant positive associations with serum Th2-mediators- interleukin (IL)-4, IL-5, IL-13, and eotaxin without any significant changes in Th1 mediators- interferon-γ and tumor necrosis factor-α. The ratios of Th2 to Th1 mediators were significantly increased with increasing exposure to As. Notably, most of the Th2 mediators were positively associated with serum levels of total immunoglobulin E and eotaxin. The serum levels of Th2 mediators were significantly higher in the subjects with asthma than those without asthma. The results of our study suggest that the exacerbated Th2-driven immune responses are involved in the increased susceptibility to allergic asthma among individuals chronically exposed to As.

Arsenite suppresses IL-2-dependent tumoricidal activities of natural killer cells.

Chronic exposure to arsenic causes cancers in various organs including the skin, liver, lung, and bladder in humans, but the mechanisms of the multi-organ carcinogenicity of arsenic remain unknown. Natural killer (NK) cells play important roles in the immune surveillance and elimination of tumor cells. Although accumulating evidence has indicated that arsenic has immunosuppressive properties, little is known about the effects of arsenic on the tumoricidal functions of NK cells. We examined the effects of arsenite on the cytotoxic activities of human and mouse NK cells toward target tumor cells. Exposure of human NK-92 cells and primary mouse NK cells to sublethal doses of arsenite reduced the IL-2-activated cytotoxic activities toward human K562 cells and murine YAC-1 cells, respectively. NK cells recognize target cells via integrated signals from both activating and inhibitory receptors and induce apoptosis of target cells via a granzyme/perforin system. We found that exposure of NK-92 cells to arsenite diminished the IL-2-activated down-regulation of the inhibitory receptors, KIR2DL2 and KIR2DL3, and the up-regulation of granzyme B and lymphotoxin-α. The IL-2-activated increases in secretion of interferon-γ and IL-10 were also slightly reduced by arsenite. Thus, arsenite suppressed the IL-2-activated cytotoxic activity of NK cells by disrupting multiple pathways required for the recognition and killing of target tumor cells. Our findings provide new insights into the roles of NK cell-mediated tumor immunity in cancer development by arsenic.

Arsenite suppresses the transcriptional activity of EVI1 through the binding to CCHC-type Zn finger domain.

Transcription factor EVI1 is essential for normal hematopoiesis in embryos but is aberrantly elevated in bone marrow cells of myelodysplastic syndrome (MDS) patients. EVI1 and its downstream GATA-2 appear to be a possible therapeutic target of MDS. Here we found that treatment of EVI1-expressing K562 cells with arsenite (As(III)) reduced the mRNA and protein levels of EVI1 and GATA-2. A gel shift assay using the nuclear extract of K562 cells showed that As(III) suppressed the DNA-binding activity of EVI1. The DNA-binding activity of the recombinant EVI1 protein was also suppressed by As(III) but was recovered by excess amounts of dithiothreitol, suggesting the involvement of cysteine residues of EVI1. Since the 7th Zn finger domain of EVI1, having a motif of CCHC, is known to be involved in DNA-binding, the synthetic peptide of 7th Zn finger domain was reacted with As(III) and subjected to MALDI-TOF-MS analysis. The results showed that As(III) binds to this peptide via three cysteine residues. As(III)-induced reduction of the DNA-binding activity of the recombinant EVI1 was abolished by the mutations of each of three cysteine residues to alanine in the 7th Zn finger domain. These results demonstrate that As(III) causes the down-regulation of EVI1 and GATA-2 by inhibiting the transcriptional activity of EVI1 through the binding to the cysteine residues of CCHC-type Zn finger domain.

Toxicometallomics of Cadmium, Manganese and Arsenic with Special Reference to the Roles of Metal Transporters.

The transport systems for metals play crucial roles in both the physiological functions of essential metals and the toxic effects of hazardous metals in mammals and plants. In mammalian cells, Zn transporters such as ZIP8 and ZIP14 have been found to function as the transporters for Mn(II) and Cd(II), contributing to the maintenance of Mn homeostasis and metallothionein-independent transports of Cd, respectively. In rice, the Mn transporter OsNramp5 expressed in the root is used for the uptake of Cd from the soil. Japan began to cultivate OsNramp5 mutant rice, which was found to accumulate little Cd, to prevent Cd accumulation. Inorganic trivalent arsenic (As(III)) is absorbed into mammalian cells via aquaglyceroporin, a water and glycerol channel. The ortholog of aquaporin in rice, OsLsi1, was found to be an Si transporter expressed in rice root, and is responsible for the absorption of soil As(III) into the root. Since rice is a hyperaccumulator of Si, higher amounts of As(III) are incorporated into rice compared to other plants. Thus, the transporters of essential metals are also utilized to incorporate toxic metals in both mammals and plants, and understanding the mechanisms of metal transports is important for the development of mitigation strategies against food contamination.

Chronic exposure to submicromolar arsenite promotes the migration of human esophageal Het1A cells induced by heparin-binding EGF-like growth factor.

Chronic arsenic exposure causes cancers in multiple organs in humans. However, the mechanisms underlying arsenic-induced carcinogenesis remain obscure. Here, we examined whether chronic arsenite (As(III)) exposure promotes cell migration induced by heparin-binding EGF-like growth factor (HB-EGF) in human esophageal immortalized Het1A cells. When Het1A cells were exposed to 0.5 µM As(III) for 4 months, HB-EGF-induced migration was enhanced in As(III)-exposed Het1A cells compared to controls. To elucidate the mechanisms underlying the promotion of HB-EGF-induced migration by chronic exposure to As(III), we compared ERK phosphorylation between As(III)-exposed and control Het1A cells and found that HB-EGF-induced ERK phosphorylation was enhanced in the As(III)-exposed cells. We next measured mRNA levels of 88 genes related to cell cycle regulation. The results showed elevated cyclin D1 mRNA levels in As(III)-exposed Het1A cells. The inhibitors of ERK and cyclin D/Cdk4 markedly suppressed HB-EGF-induced upregulation of cyclin D1 and the migration of Het1A cells, respectively, suggesting that cyclin D1 is located downstream of ERK and is required for HB-EGF-induced migration of Het1A cells. Collectively, these findings indicate that the promotion of HB-EGF-induced migration of Het1A cells chronically exposed to submicromolar As(III) might be caused by increased expression of cyclin D1 mediated by enhanced activation of the ERK pathway.

Arsenite suppresses NO production evoked by lipopolysaccharide and poly(I:C) via the suppression of interferon-ß expression in RAW264.7 cells.

Immunological functions are disturbed in humans who have been chronically exposed to arsenic via contaminated groundwater. Little is known about the specific mechanisms underlying the impairment of immunological defense system caused by arsenic. The activation of macrophage cells upon infection with bacteria and viruses plays important roles in the defense against these pathogens. Here we show that exposure to arsenite (As(III)) suppresses nitric oxide (NO) production in murine RAW264.7 macrophage cells stimulated with lipopolysaccharide (LPS) and poly(I:C), the compounds mimicking bacterial and viral infection, respectively. As(III) suppressed the LPS- or poly(I:C)-evoked induction of inducible NO synthase (iNOS) without affecting the transactivation of NF-κB. As the interferon (IFN)-ß/STAT1 pathway is also involved in the induction of iNOS in addition to NF-κB, we examined the effects of As(III) on the expression and secretion of IFN-ß, the expression of the components of IFN-α/ß receptor, the phosphorylation of STAT1, and the levels of cytokines involved in STAT1 activation. The results showed that the expression and secretion of IFN-ß were specifically suppressed by As(III) treatment in RAW264.7 cells stimulated with LPS or poly(I:C). These results suggest that As(III) suppresses the expression and secretion of IFN-ß, leading to the reduced STAT1 activation and consequently the reduced iNOS induction in macrophage cells. Our data suggest an important role of the arsenic-induced suppression of IFN-ß on the disturbances in immunological defense against both bacteria and viruses.

Hydrogen peroxide triggers a novel alternative splicing of arsenic (+3 oxidation state) methyltransferase gene.

We previously reported that two splicing variants of human AS3MT mRNA, exon-3 skipping form (Δ3) and exons-4 and -5 skipping form (Δ4,5), were detected in HepG2 cells and that both variants lacked arsenic methylation activity (Sumi et al., 2011). Here we studied whether hydrogen peroxide (H2O2) triggers alternative splicing of AS3MT mRNA. The results showed that exposure of HepG2 cells to H2O2 resulted in increased levels of a novel spliced form skipping exon-3 to exon-10 (Δ3-10) in an H2O2-concentration-dependent manner, although no change was detected in the mRNA levels of Δ3 AS3MT. We found decreased protein levels of serine/arginine-rich 40 (SRp40), which we determined to be a candidate splice factor for controlling the splicing of AS3MT mRNA. We next compared the amounts of methylated arsenic metabolites between control and H2O2-exposed HepG2 cells after the addition of arsenite as a substance. The results showed lower levels of methylated arsenic metabolites in HepG2 cells exposed to H2O2. These data suggest that the splicing of AS3MT pre-mRNA was disconcerted by oxidative stress and that abnormal alternative splicing of AS3MT mRNA may affect arsenic methylation ability.

Arsenic trioxide augments all-trans retinoic acid-induced differentiation of HL-60 cells.

AIMS: All-trans retinoic acid (ATRA) is used to treat patients with acute promyelocytic leukemia (APL) due to its ability to resume the differentiation of APL cells. Recently, clinical trials have been started to evaluate ATRA plus arsenic trioxide (ATO) as a combination treatment for APL patients. However, little is known about the detailed mechanisms underlying its efficacy. We therefore investigated the effects of this combination on the differentiation and differentiation-related gene expression. MAIN METHODS: Human leukemia HL-60 cells differentiation was examined using nitro blue tetrazolium and CD11b. The levels of mRNA and protein were determined by RT-qPCR, microarray, western blot and ELISA, respectively. The promoter activity was assessed by luciferase activity. The arsenic concentration was determined by ICP-MS. KEY FINDINGS: ATRA-induced HL-60 differentiation was augmented by co-treatment with ATO. A microarray analysis showed that ATRA plus ATO treatment markedly down-regulated the expression of proteinase 3 (PRTN3), which is involved in the differentiation arrest of leukemia cells, compared with treatment with ATRA alone. The PRTN3 mRNA level was suppressed by treatment with ATRA alone, and then further suppressed by co-treatment with ATO, accompanied by a concomitant increase in Sp1 protein, which is known to facilitate differentiation. The expression levels of azurocidin, telomerase reverse transcriptase, ferritin, and interleukin-1ß were also altered by co-treatment with ATO. SIGNIFICANCE: Co-treatment with ATO enhances ATRA-induced HL-60 differentiation by altering the expression of genes involved in cell differentiation, providing the molecular basis for a combination therapy using ATO plus ATRA to treat leukemia patients.

Synergistic augmentation of ATP-induced interleukin-6 production by arsenite in HaCaT cells.

Chronic arsenic exposure causes cutaneous diseases such as hyperkeratosis and skin cancer. However, little information has been available regarding the molecular mechanisms underlying these symptoms. Because extracellular ATP and interleukin-6 (IL-6) are involved in pathological aspects of cutaneous diseases, we examined whether sodium arsenite (As(III)) affects ATP-induced IL-6 production in human epidermal keratinocyte HaCaT cells. The results showed that the addition of As(III) into the medium of HaCaT cells dose dependently increased the production of IL-6 induced by extracellular ATP, although As(III) alone had no effect on IL-6 production. To elucidate the mechanism of the synergistic effect of As(III) on IL-6 production by extracellular ATP, we next examined the phosphorylation of p38, ERK and epidermal growth factor receptor (EGFR), since we found that these signaling molecules were stimulated by exposure to extracellular ATP. The results indicated that ATP-induced phosphorylation of p38, ERK and EGFR was synergistically enhanced by co-exposure to As(III). To clarify the mechanisms underlying the enhanced phosphorylation of p38, ERK and EGFR by As(III), we explored two possible mechanisms: the inhibition of extracellular ATP degradation and the inhibition of protein tyrosine phosphatases (PTPs) activity by As(III). The degradation of extracellular ATP was not changed by As(III), whereas the activity of PTPs was significantly inhibited by As(III). Our results suggest that As(III) augments ATP-induced IL-6 production in HaCaT cells through enhanced phosphorylation of the EGFR and p38/ERK pathways, which is associated with the inhibition of PTPs activity.

High accumulation of arsenic in the esophagus of mice after exposure to arsenite.

Arsenic-induced toxicity appears to be dependent on the tissue- or cell-specific accumulation of this metalloid. An early study showed that arsenic was retained in the esophagus as well as the liver, kidney cortex and skin of marmosets after intraperitoneal administration of (74)As-arsenite. However, there is little available information regarding the distribution of arsenic in the esophagus. Here, we compared the retention of arsenic in the esophagus, liver, lung, kidney and heart in mice intraperitoneally administered 1 or 5 mg/kg sodium arsenite (As(III)) daily for 3 or 7 days. The results showed that the arsenic concentration was highest in the esophagus. We compared the mRNA levels of aquaglyceroporin (AQP) 3, AQP7 and AQP9, which are responsible for arsenic influx, and those of multidrug-resistance protein (MRP) 1 and MRP2, which are responsible for arsenic efflux. The levels of AQP3 mRNA in the esophagus were much higher than those in liver, lung and heart, while the mRNA levels of MRP2 were very low in the esophagus. In addition, we found extremely low expression of Nrf2 in the esophagus at the basal and under the activated conditions, which might have resulted in low levels of glutamyl-cysteine ligase catalytic and modulatory subunits, and subsequently in the low levels of glutathione. Thus, the highest retention of arsenic was detected in the esophagus after intraperitoneal administration of As(III) to mice, and this appeared to result from multiple factors, including high expression of AQP3, low expression of MRP2, low capacity of glutathione synthesis and low activation of Nrf2.