Response of arsenic-induced oxidative stress,DNA damage,and metal imbalance to combined administration of DMSA and monoisoamyl-DMSA during chronic arsenic poisoning in rats

Arsenic and its compounds cause adverse health effects in humans. Current treatment employs administration of thiol chelators, such as meso-2,3-dimercaptosuccinic acid (DMSA) and sodium 2,3-dimercaptopropane 1-sulfonate (DMPS), which facilitate its excretion from the body. However, these chelating agents are compromised by number of limitations due to their lipophobic nature, particularly in case of chronic poisoning. Combination therapy is a new approach to ensure enhanced removal of metal from the body, reduced doses of potentially toxic chelators, and no redistribution of metal from one organ to another, following chronic metal exposure. The present study attempts to investigate dose-related effects of two thiol chelators, DMSA and one of its new analogues, monoisoamyl dimercaptosuccinic acid (MiADMSA), when administered in combination with the aim of achieving normalization of altered biochemical parameters suggestive of oxidative stress and depletion of inorganic arsenic following chronic arsenic exposure. Twenty-five adult male Wistar rats were given 25 ppm arsenic for 10 weeks followed by chelation therapy with the above chelating agents at a dose of 0.3 mmol/kg (orally) when administered individually or 0.15 mmol/kg and 0.3 mmol/kg (once daily for 5 consecutive days), respectively, when administered in combination. Arsenic exposure led to the inhibition of blood δ-aminolevulinic acid dehydratase (ALAD) activity and depletion of glutathione (GSH) level. These changes were accompanied by significant depletion of hemoglobin, RBC and Hct as well as blood superoxide dismutase (SOD) acitivity. There was an increase in hepatic and renal levels of thiobarbituric acid-reactive substances, while GSH:GSSG ratio decreased significantly, accompanied by a significant increase in metallothionein (MT) in hepatocytes. DNA damage based on denaturing polyacrylamide gel electrophoresis revealed significant loss in the integrity of DNA extracted from the liver of arsenic-exposed rats compared to that of normal animals. These changes were accompanied by a significant elevation in blood and soft-tissue arsenic concentration. Co-administration of DMSA and MiADMSA at lower dose (0.15 mmol/kg) was most effective not only in reducing arsenic-induced oxidative stress but also in depleting arsenic from blood and soft tissues compared to other treatments. This combination was also able to repair DNA damage caused following arsenic exposure. We thus recommend combined administration of DMSA and MiADMSA for achieving optimum effects of chelation therapy.     

长期砷胁迫下大屯海浮游植物群落的季节性特征及其驱动因子

重金属是影响湖泊水质和生态健康的重要胁迫因子,系统识别生物对长期污染胁迫的响应模式是开展污染湖泊生态修复的重要基础。本研究以经历持续砷污染的大屯海为研究对象,于2017年6月—2018年3月对水体浮游植物和环境因子开展季节性调查。结果显示: 大屯海的浮游植物群落主要由蓝藻门组成,与已有研究反映的长期砷胁迫下浮游植物组成以蓝藻门等耐受属种为主的特征一致。相似性和方差分析结果表明,浮游植物群落结构和生物量存在显著的时间差异而空间差异不显著。Pearson相关分析表明,浮游植物总生物量与溶解性正磷酸盐和砷呈显著正相关,与砷对藻类生长产生的低促高抑效应一致,同时磷酸盐的增加可能降低了砷对藻类的毒性效应。冗余分析显示,溶解性营养盐和砷是影响浮游植物群落变化的显著因子。方差分解结果表明,营养盐和水温分别单独解释了群落结构变化的17.6%和3.8%,且与砷产生了较强的相互作用(15.1%);而砷对浮游植物的群落构建无显著的独立作用,反映了现有优势藻类具有对砷较强的耐受性从而对砷浓度的变化不敏感。因此,大屯海的优势浮游植物以耐砷藻类为主,砷对藻类产生的低促效应是污染湖泊修复中需要重点关注的生态效应之一。     

IRE1α/NOX4 signaling pathway mediates ROS‐dependent activation of hepatic stellate cells in NaAsO2‐induced liver fibrosis

Liver fibrosis is a severe health problem worldwide, and it is characterized by the activation of hepatic stellate cells (HSCs) and excessive deposition of collagen. Prolonged arsenic exposure can induce HSCs activation and liver fibrosis. In the present study, the results showed that chronic NaAsO₂ ingestion could result in liver fibrosis and oxidative stress in Sprague–Dawley rats, along with representative collagen deposition and HSCs activation. In addition, the inositol‐requiring enzyme 1α (IRE1α)–endoplasmic reticulum (ER)‐stress pathway was activated, and the activity of nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) was upregulated in rat livers. Simultaneously, the excessive production of reactive oxygen species (ROS) could induce HSCs activation, and NOX4 played an important role in generating ROS in vitro. Moreover, ER stress occurred with HSCs activation at the same time under NaAsO₂ exposure, and during ER stress, the IRE1α pathway was responsible for NOX4 activation. Therefore, inhibition of IRE1α activation could attenuate the HSCs activation induced by NaAsO₂. In conclusion, the present study manifested that inorganic arsenic exposure could activate HSCs through IRE1α/NOX4‐mediated ROS generation.     

Trichoderma spp. alleviate phytotoxicity in lettuce plants (Lactuca sativa L.) irrigated with arsenic-contaminated water

The influence of two strains of Trichoderma (T. harzianum strain T22 and T. atroviride strain P1) on the growth of lettuce plants (Lactuca sativa L.) irrigated with As-contaminated water, and their effect on the uptake and accumulation of the contaminant in the plant roots and leaves, were studied. Accumulation of this non-essential element occurred mainly into the root system and reduced both biomass development and net photosynthesis rate (while altering the plant P status). Plant growth-promoting fungi (PGPF) of both Trichoderma species alleviated, at least in part, the phytotoxicity of As, essentially by decreasing its accumulation in the tissues and enhancing plant growth, P status and net photosynthesis rate. Our results indicate that inoculation of lettuce with selected Trichoderma strains may be helpful, beside the classical biocontrol application, in alleviating abiotic stresses such as that caused by irrigation with As-contaminated water, and in reducing the concentration of this metalloid in the edible part of the plant.     

Organic anion transporting polypeptide-C mediates arsenic uptake in HEK-293 cells

Summary Arsenic is an established human carcinogen. The role of aquaglyroporins (AQPs) in arsenic disposition was recently identified. In order to examine whether organic anion transporting polypeptide-C (OATP-C) also plays a role in arsenic transport, OATP-C cDNA was transfected into cells of a human embryonic kidney cell line (HEK-293). Transfection increased uptake of the model OATP-C substrate, estradiol-17β-D-glucuronide, by 10-fold. In addition, we measured uptake and cytotoxicity of arsenate, arsenite, monomethylarsonate(MMA^V), and dimethylarsinate (DMA^V). Transfection of OATP-C increased uptake and cytotoxicity of arsenate and arsenite, but not of MMA^V or DMA^V. Rifampin and taurocholic acid (a substrate of OATP-C) reversed the increased toxicity of arsenate and arsenite seen in OATP-C-transfected cells. The increase in uptake of inorganic arsenic was not as great as that of estradiol-17β-D-glucuronide. Our results suggest that OATP-C can transport inorganic arsenic in a (GSH)-dependent manner. However, this may not be the major pathway for arsenic transport.     

外源磷对苗期小麦和水稻根际砷形态及其生物有效性的影响

采用土(中度砷污染土)-土根袋培养的方法研究了两个浓度的外源磷(P)对苗期小麦和水稻根际砷(As)形态分布及其生物有效性的影响.结果表明:(1)两种作物生长的土壤中各砷形态的分配比例依次为:结晶铁锰或铁铝水化氧化物结合态(45%～52%)＞无定形和弱结晶铁铝或铁锰水化氧化物结合态(26%～34%)＞专性吸附态(12%～14%)＞残渣态(4%～7%)＞非专性吸附态(0.09%～0.25%).(2)添加外源磷浓度为100 mg·kg-1与不施磷处理相比显著提高了两种作物地上部的生物量(p＜0.01).(3)苗期小麦在添加100 mg·kg-1外源磷时,不仅促进了作物生长而且抑制根中砷向地上部的转运.(4)任何磷处理下,水稻对砷的吸收能力以及由根系向地上部转移能力均高于小麦.因此,在轻中度砷污染土壤上与水稻相比更适宜种植小麦(或其他旱作植物);而在水稻种植季,可以通过添加适量磷肥(100 mg·kg-1)来减弱砷在水稻体内的累积.     

Systematic engineering of phytochelatin synthesis and arsenic transport for enhanced arsenic accumulation in E. coli

Phytochelatin (PC) is a naturally occurring peptide with high affinity towards arsenic (As). In this article, we demonstrated the systematic engineering of PC‐producing E. coli for As accumulation by addressing different bottlenecks in PC synthesis as well as As transport. Phytochelatin synthase from Schizosaccharomyces pombe (SpPCS) was expressed in E. coli resulting in 18 times higher As accumulation. PC production was further increased by co‐expressing a feedback desensitized γ‐glutamylcysteine synthetase (GshI*), resulting in 30‐fold higher PC levels and additional 2‐fold higher As accumulation. The significantly increased PC levels were exploited further by co‐expressing an arsenic transporter GlpF, leading to an additional 1.5‐fold higher As accumulation. These engineering steps were finally combined in an arsenic efflux deletion E. coli strain to achieve an arsenic accumulation level of 16.8 µmol/g DCW, a 80‐fold improvement when compared to a control strain not producing phytochelatins. Biotechnol. Bioeng. 2010. 105: 780–785. © 2009 Wiley Periodicals, Inc.     

Transition metal ions and selenite modulate the methylation of arsenite by the recombinant human arsenic (+3 oxidation state) methyltransferase (hAS3MT)

This report demonstrates that transition metal ions and selenite affect the arsenite methylation by the recombinant human arsenic (+3 oxidation state) methyltransferase (hAS3MT) in vitro. Co²⁺, Mn²⁺, and Zn²⁺ inhibited the arsenite methylation by hAS3MT in a concentration-dependent manner and the kinetics indicated Co²⁺ and Mn²⁺ to be mixed (competitive and non-competitive) inhibitors while Zn²⁺ to be a competitive inhibitor. However, only a high concentration of Fe²⁺ could restrain the methylation. UV-visible, CD and fluorescence spectroscopy were used to study the interactions between the metal ions above and hAS3MT. Further studies showed that neither superoxide anion nor hydrogen peroxide was involved in the transition metal ion or selenite inhibition of hAS3MT activity. The inhibition of arsenite methylating activity of hAS3MT by selenite was reversed by 2 mM DTT (dithiothreitol) but neither by cysteine nor by β-mercaptoethanol. Whereas, besides DTT, cysteine can also prevent the inhibition of hAS3MT activity by Co²⁺, Mn²⁺, and Zn²⁺. Free Cys residues were involved in the interactions of transition metal ions or selenite with hAS3MT. It is proposed that the inhibitory effect of the ions (Co²⁺, Mn²⁺, and Zn²⁺) or selenite on hAS3MT activity might be via the interactions of them with free Cys residues in hAS3MT to form inactive protein adducts.     

A redox‐linked novel pathway for arsenic‐mediated RET tyrosine kinase activation

We examined the biochemical effects of arsenic on the activities of RET proto‐oncogene (c‐RET protein tyrosine kinases) and RET oncogene (RET‐MEN2A and RET‐PTC1 protein tyrosine kinases) products. Arsenic activated c‐RET kinase with promotion of disulfide bond‐mediated dimerization of c‐RET protein. Arsenic further activated RET‐MEN2A kinase, which was already 3‐ to 10‐fold augmented by genetic mutation compared with c‐RET kinase activity, with promotion of disulfide bond‐mediated dimerization of RET‐MEN2A protein (superactivation). Arsenic also increased extracellular domain‐deleted RET‐PTC1 kinase activity with promotion of disulfide bond‐mediated dimerization of RET‐PTC1 protein. Arsenic increased RET‐PTC1 kinase activity with cysteine 365 (C365) replaced by alanine with promotion of dimer formation but not with cysteine 376 (C376) replaced by alanine. Our results suggest that arsenic‐mediated regulation of RET kinase activity is dependent on conformational change of RET protein through modulation of a special cysteine sited at the intracellular domain in RET protein (relevant cysteine of C376 in RET‐PTC1 protein). Moreover, arsenic enhanced the activity of immunoprecipitated RET protein with increase in thiol‐dependent dimer formation. As arsenic (14.2 µM) was detected in the cells cultured with arsenic (100 µM), direct association between arsenic and RET in the cells might modulate dimer formation. Thus, we demonstrated a novel redox‐linked mechanism of activation of arsenic‐mediated RET proto‐oncogene and oncogene products. J. Cell. Biochem. 110: 399–407, 2010. © 2010 Wiley‐Liss, Inc.