Induction of genotoxic damage is not correlated with the ability to methylate arsenite in vitro in the leukocytes of four mammalian species

Arsenic is a natural drinking water contaminant that impacts the health of large populations of people throughout the world; however, the mode or mechanism by which arsenic induces cancer is unclear. In a series of in vitro studies, we exposed leukocytes from humans, mice, rats, and guinea pigs to a range of sodium arsenite concentrations to determine whether the lymphocytes from these species showed differential sensitivity to the induction of micronuclei (MN) assessed in cytochalasin B-induced binucleate cells. We also determined the capacity of the leukocytes to methylate arsenic by measuring the production of MMA [monomethylarsinic acid (MMA(V)) and monomethylarsonous acid (MMA(III))] and DMA [dimethylarsinic acid (DMA(V)) and dimethylarsonous acid (DMA(III))]. The results indicate that cells treated for 2 hr at the G(0) stage of the cell cycle with sodium arsenite showed only very small to negligible increases in MN after mitogenic stimulation. Treatment of actively cycling cells produced induction of MN with increasing arsenite concentration, with the human, rat, and mouse lymphocytes being much more sensitive to MN induction than those of the guinea pig. These data gave an excellent fit to a linear model. The leukocytes of all four species, including the guinea pig (a species previously thought not to methylate arsenic), were able to methylate arsenic, but there was no clear correlation between the ability to methylate arsenic and the induction of MN.     

Comparison of the urinary excretion of arsenic metabolites after a single oral dose of sodium arsenite,monomethylarsonate, or dimethylarsinate in man

Summary The urinary elimination of the metabolites of arsenic has been followed up as a function of time in volunteers who ingested a single oral dose of arsenic (500 g As) either as sodium arsenite (As_i), monomethylarsonate (MMA), or cacodylate (DMA). The excretion rate increased in the order As_i < DMA < MMA. After 4 days, the amount of arsenic excreted in urine represents 46, 78, and 75% of the ingested dose in the case of As_i, MMA and DMA, respectively. With regard to the in vivo biotransformations, it is concluded that DMA is excreted unchanged; MMA is slightly (13%) methylated into DMA while roughly 75% of the arsenic excreted after ingestion of As_i is methylated arsenic (about 1/3 as MMA and about 2/3 as DMA).This study was supported by a grant from the Commission of the European Communities     

Protective effect of curcumin against arsenic-induced apoptosis in murine splenocytes in vitro

Arsenic is a potent environmental pollutant and immunotoxic agent. Curcumin is a natural anti-oxidant used to treat a broad variety of diseases. Here, the effects were investigated of curcumin on sodium arsenite-induced apoptosis in murine splenocytes in vitro. Cells were exposed to sodium arsenite (NaAsO₂, 5 µM) with and without curcumin (5 and 10 µg/ml) and incubated at 37°C for 12?h. NaAsO₂ caused a decrease in cell viability and induction of apoptosis. These outcomes were concurrent with increases in the numbers of cells with reactive oxygen species generation, loss of mitochondrial transmembrane potential, an increase in the frequency of cells with sub-G₁ DNA content, and DNA fragmentation. Co-administration of curcumin with the NaAsO₂ caused significant recoveries in cell viability values and mitigation of the induced apoptosis-related molecular changes. A significant protection against apoptosis parameters in murine splenocytes simultaneously treated with NaAsO₂ and curcumin suggested a protective efficacy of curcumin. From the results it is concluded that the immuno-modulation exerted by curcumin might be attributed to its multifaceted effects including its anti-oxidative and anti-apoptotic properties. These findings have implications not only for the under-standing of the toxicity of arsenic to murine splenocytes in vitro but are also potentially important for developing preventive and/or corrective strategies against/during chronic arsenicosis.     

c-jun末端激酶在亚砷酸钠致人胚胎肝细胞损伤中的表达

目的探讨亚砷酸钠染毒对人胚胎肝(L-02)细胞中c-jun末端激酶(JNK)的变化。方法将处于对数生长期的L-02细胞分别暴露于终浓度为0(对照)、50、100、150μmol/L的亚砷酸钠溶液中培养24 h,采用四甲基偶氮唑蓝(MTT)法检测细胞生长情况,采用流式细胞术检测染毒24 h时的细胞周期及细胞凋亡率;采用蛋白杂交(Western-blot)法检测JNK及p-JNK的蛋白表达水平。结果与对照组相比,各浓度亚砷酸钠染毒组L-02细胞的存活率及G0-G1期构成比均较低,JNK、p-JNK的蛋白表达水平和S期构成比及凋亡率均较高;而G2-M期构成比在50μmol/L亚砷酸钠染毒组较低,在100、150μmol/L亚砷酸钠染毒组均较高,差异均有统计学意义(P0.05)。且随着亚砷酸钠染毒浓度的升高,L-02细胞凋亡率及JNK和p-JNK蛋白的表达水平均呈上升趋势,细胞存活率呈下降趋势。结论亚砷酸钠诱导L-02细胞凋亡可能与JNK及p-JNK表达的增加有关。     

Arsenite downregulates H3K4 trimethylation and H3K9 dimethylation during transformation of human bronchial epithelial cells

Arsenic is an established human carcinogen but with weak mutagenic activity. The mechanisms of arsenic‐induced carcinogenesis are not well understood. In the present study, we investigated the role of histone methylation in transformation of human bronchial epithelial (BEAS‐2B) cells. After 16 weeks’ exposure, cells were transformed by 0.1, 0.5 and 1 μm arsenite. Global trimethylated H3K4 (H3K4me3) was decreased by 0.1 μm arsenite at 12 weeks, and 0.5 and 1 μm arsenite at 8, 12 and 16 weeks, which could be attributed to reduced histone methyltransferase activities, increased histone demethylase (HDM) activities as well as increased protein levels of H3K4 demethylase KDM5A. Global dimethylated H3K9 (H3K9me2) was also decreased after exposure to 0.5 μm arsenite for 4, 8, 12 and 16 weeks and 1.0 μm arsenite for 8 and 12 weeks, which was associated with an increase of HDM activities. Our findings indicated that arsenite decreased global H3K4me3 and H3K9me2 levels during cell transformation by modulating the enzymatic activities of histone methyltransferases and/or HDMs, and by upregulation of KDM5A protein levels for H3K4me3.     

3,4‐Dihydroxybenzaldehyde lowers ROS generation and protects human red blood cells from arsenic(III) induced oxidative damage

Arsenic (As) is a potent environmental toxicant and chronic exposure to it results in various malignancies in humans. Oxidative stress has been implicated in the etiopathogenesis of As‐induced toxicity. This investigated the protective effect of plant antioxidant 3,4‐dihydroxybenzaldehyde (DHB) on sodium meta‐arsenite (SA), an As‐(III) compound, induced oxidative damage in human red blood cells (RBC). The RBC were first incubated with different concentrations of DHB and then treated with SA at 37°C. Hemolysates were prepared and assayed for various biochemical parameters. Treatment of RBC with SA alone enhanced the generation of reactive oxygen species and increased lipid and protein oxidation. Reduced glutathione levels, total sulfhydryl content and cellular antioxidant power were significantly decreased in SA alone treated RBC, compared to the untreated control cells. This was accompanied by membrane damage, alterations in activities of antioxidant enzymes and deranged glucose metabolism. Incubation of RBC with DHB, prior to treatment with SA, significantly and dose‐dependently attenuated the SA‐induced changes in all these parameters. Scanning electron microscopy of RBC confirmed these biochemical results. Treatment of RBC with SA alone converted the biconcave discoids to echinocytes but the presence of DHB inhibited this conversion and the RBC retained their normal shape. These results show that DHB protects human RBC from SA‐induced oxidative damage, most probably due to its antioxidant character.     

JNK信号通路对NaAsO2诱导NIH3T3细胞中hnRNP K蛋白聚集体形成的影响

 目的：观察核不均一核糖核蛋白(hnRNP)K的表达与定位以及亚砷酸钠（NaAsO2）刺激NIH3T3细胞的反应情况及氧化应激变化,探讨JNK信号通路在hnRNP K蛋白聚集体形成中的作用。方法：构建重组载体pcDNA3-hnRNP K-HA,转染NIH3T3细胞,荧光显微镜观察内源性hnRNP K在细胞中的表达与定位,以及在NaAsO2不同刺激时间该蛋白聚集体的形成情况,并利用活性氧（ROS）检测试剂盒测定胞内ROS水平;给予JNK、MEK、PI3K/Akt、NF-κB和核转运等5种信号通路的抑制剂预处理后,观察聚集体的变化情况。结果：重组质粒构建正确,荧光显微镜观察显示hnRNP K主要定位于胞核中,而重组质粒转染NIH3T3细胞后,可见胞内有蛋白聚集体形成并随NaAsO2刺激时间的延长而递增,且过表达hnRNP K可抑制NaAsO2刺激细胞生成ROS;JNK信号通路抑制剂SP600125明显抑制聚集体的形成。结论：hnRNP K主要定位在NIH3T3细胞的胞核中,胞浆少量分布;NaAsO2可诱导NIH3T3细胞形成hnRNP K蛋白聚集体,此聚集体可抑制胞内ROS生成,且该聚集体的形成有赖于JNK介导的信号通路。     

无机砷对人皮肤成纤维细胞增殖毒性的实验观察

目的观察亚砷酸钠诱导体外培养的人皮肤成纤维细胞的作用，探讨砷性皮肤损伤的分子机制。方法通过直接细胞计数法和噻唑蓝（MTT）还原法检测亚砷酸钠对皮肤成纤维细胞生长的影响。结果与对照组比较，皮肤成纤维细胞经不同剂量的亚砷酸钠诱导后，吸光度值均有改变，存在剂量效应关系（P〈0.01）。0．5～5．0μmol／L浓度的亚砷酸钠对皮肤成纤维细胞有明显的增殖作用，而10μmol／L浓度的亚砷酸钠产生明显的毒性作用。结论低浓度NaAsO2、NaAsO2刺激人皮肤成纤维细胞增殖，高浓度具有细胞毒性。     

Arsenite-induced thymus atrophy is mediated by cell cycle arrest: a characteristic downregulation of E2F-related genes revealed by a microarray approach.

Thymus atrophy is induced by a variety of chemicals, including environmental contaminants and is used as a sensitive index to detect their adverse effects on lymphocytes. In the present study we adopted a toxicogenomics approach to identify the pathways that mediate the atrophy induced by arsenite. We also analyzed gene expression changes observed in the course of thymus atrophy by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), dexamethasone (DEX), and estradiol (E2), to determine whether arsenite induces atrophy by activating an arsenite-specific pathway or the same pathways as other chemicals. These compounds were intraperitoneally administered to C57BL/6 mice at doses that reduce thymus weight by approximately 30% within 3 days, and gene expression changes in the thymus 24 h after the administration were analyzed by using microarrays and real-time PCR. The microarray analysis showed that arsenite specifically downregulates a variety of E2F target genes that are involved in cell cycle progression. The same genes were also downregulated when mouse B-cell lymphoma A20 cells were exposed to arsenite. Arsenite exposure of the A20 cells was confirmed to induce cell cycle arrest, mainly in the G(1) phase, and reduce cell number. Cell cycle arrest in the G(1) phase was also confirmed to occur in the thymocytes of the arsenite-exposed mice. These results indicate that arsenite induces thymus atrophy through E2F-dependent cell cycle arrest. The results of this study also show that analysis of gene expression in thymuses is a useful method of obtaining clues to the pathways that mediate the effects of atrophy-inducing chemicals.