Methylated Arsenicals: The Implications of Metabolism and Carcinogenicity Studies in Rodents to Human Risk Assessment

Monomethylarsonic acid (MMA^V) and dimethylarsinic acid (DMA^V) are active ingredients in pesticidal products used mainly for weed control. MMA^V and DMA^V are also metabolites of inorganic arsenic, formed intracellularly, primarily in liver cells in a metabolic process of repeated reductions and oxidative methylations. Inorganic arsenic is a known human carcinogen, inducing tumors of the skin, urinary bladder, and lung. However, a good animal model has not yet been found. Although the metabolic process of inorganic arsenic appears to enhance the excretion of arsenic from the body, it also involves formation of methylated compounds of trivalent arsenic as intermediates. Trivalent arsenicals (whether inorganic or organic) are highly reactive compounds that can cause cytotoxicity and indirect genotoxicity in vitro. DMA^V was found to be a bladder carcinogen only in rats and only when administered in the diet or drinking water at high doses. It was negative in a two-year bioassay in mice. MMA^V was negative in 2-year bioassays in rats and mice. The mode of action for DMA^V-induced bladder cancer in rats appears to not involve DNA reactivity, but rather involves cytotoxicity with consequent regenerative proliferation, ultimately leading to the formation of carcinoma. This critical review responds to the question of whether DMA^V-induced bladder cancer in rats can be extrapolated to humans, based on detailed comparisons between inorganic and organic arsenicals, including their metabolism and disposition in various animal species. The further metabolism and disposition of MMA^V and DMA^V formed endogenously during the metabolism of inorganic arsenic is different from the metabolism and disposition of MMA^V and DMA^V from exogenous exposure. The trivalent arsenicals that are cytotoxic and indirectly genotoxic in vitro are hardly formed in an organism exposed to MMA^V or DMA^V because of poor cellular uptake and limited metabolism of the ingested compounds. Furthermore, the evidence strongly supports a nonlinear dose-response relationship for the biologic processes involved in the carcinogenicity of arsenicals. Based on an overall review of the evidence, using a margin-of-exposure approach for MMA^V and DMA^V risk assessment is appropriate. At anticipated environmental exposures to MMA^V and DMA^V, there is not likely to be a carcinogenic risk to humans.     

全反式维甲酸联合三氧化二砷抗膀胱癌作用及其毒副作用的实验研究

目的观察全反式维甲酸（ATRA）联合三氧化二砷（As2O3）对膀胱癌细胞株BIU-87裸鼠移植瘤生长的抑制作用,及其毒副作用。方法建立人膀胱癌裸鼠皮下移植瘤模型40只,随机等分为4组：对照组,ATRA组,As2O3组,ATRA和As2O3联合用药组;裸鼠瘤体内连续注射用药14d。停药后48h检测血常规和肝、肾功能;处死裸鼠,测量移植瘤体积、重量,计算抑瘤率;移植瘤及心、肝、肾等组织HE染色,观察其病理变化;瘤组织免疫组化S-P法检测血管内皮生长因子（VEGF）表达、CD43标记的微血管密度MVD的表达。结果与对照组比较,As2O3组及ATRA组移植瘤生长明显受到抑制（质量抑瘤率分别为43．77％,41．82％）,两者联合用药后,抑制作用显著增强（质量抑瘤率为68．55％）,抑瘤率差异具有统计学意义（X2=26．81,P〈0．01）;As2O3组、ATRA组、联合用药组均不同程度下调VEGF表达（OD值分别为27．33±2．17,20．72±2．01,19．23±2．32,17．16±1．59）及MVD的表达（OD值分别为141．12±8．38,43．39±7．41,44．77±8．25,30．56±7．71）,联合用药组下调最显著;各用药组均出现轻度白细胞抑制（t=3．16,3．08,3．37,P〈0．01）,联合用药组抑制程度与单一用药组比较差异无统计学意义（P〉0．05）,肝、肾功能各指标比较差异均无统计学意义（P〉0．05）。结论ATRA联合As2O3在体内能够协同抑制膀胱癌BIU-87细胞移植瘤的生长和血管生成,仅有白细胞轻度抑制,对肝肾功能无毒副作用。     

三氧化二砷对肝癌细胞内活性氧水平的影响

目的探讨三氧化二砷（As2O3）对肝癌细胞增殖及细胞内活性氧（ROS）水平的影响。方法用As2O3作用于体外培养的人肝癌SMMC-7721细胞株，分别用MTT法和流式细胞仪观察SMMC-7721细胞增殖情况并检测ROS。结果MTT结果显示As2O3能明显抑制SMMC-7721细胞的增殖．并呈时间和浓度依赖性。流式细胞仪分析显示As2O3作用后的人肝癌SMMC-7721细胞内ROS水平明显增高（P〈0．01）．并也呈时间和浓度依赖性。结论As2O3可通过抑制人肝癌SMMC-7721细胞的增殖和提高细胞内ROS水平诱导细胞凋亡而发挥抗肿瘤作用，这可能也是As2O3抗肝癌的主要途径之一。     

两种尿砷检测方法的比较

目的 对比两种尿砷检测方法的可靠性与可行性.方法 2006年11月采集内蒙古托县砷病区15名高砷暴露者的尿样,以氢化物发生-冷阱捕集-原子吸收法(HG-cold trap-AAS法)检测尿中各种形态砷,以高效液相色谱-氢化物发生-原子荧光法(HPLC-HG-AFS法)检测尿中各种形态与价态的砷.对检测结果进行配对t检验.结果 HG-cold trap-AAS和HPLC-HG-AFS测定的尿总砷的波动范围分别为180.75～1 644.98μg/L和179.32～1 579.24μg/L;对于全部尿样,所测总砷结果差异无统计学意义(t=0.56,P>0.5),对于总砷含量大于500μg/L的尿样,所测总砷结果差异有统计学意义(f=2.70,P<0.05);两法所测无机砷结果差异无统计学意义(t=-0.66,P>0.05);两法所测甲基砷结果差异有统计学意义(t=3.85,P<0.05);两法所测二甲基砷结果差异无统计学意义(t=-0.21,P>0.05);全部尿样均未检出三甲基砷氧化物.结论 HG-coldtrap-AAS法和HPLC-HG-AFS法均能快速有效地检测尿中不同砷化合物,前者更适合高尿砷的测定.     

Effect of As2O3 on gluconeogenesis

1) The effect of As₂O₃ and As₂O₅ on gluconeogenesis from various substrates in the liver and kidney of rats was investigated. 2) A concentration-dependent inhibition by As₂O₃ was found. The effect was not dependent on the amount of investigated material (hepatocytes or kidney tubules). For either hepatocytes or kidney tubules the extent of inhibition depended strongly on the substrate used. The highest degree of inhibition was observed in incubations with pyruvate. The inhibition of glucose formation was accompanied to a lesser extent by a diminution in O₂ consumption and ATP content. The effect was also dependent on the substrate used. Maximum effect was found in incubations with pyruvate. 3) Oleate, 0.5 mmol/l, increased gluconeogenesis from pyruvate. The effect was not abolished by As₂O₃. 4) A decrease in the content of acetyl-CoA, 3-hydroxybutyrate, and reduced glutathione was found in suspensions of isolated rat kidney tubules or hepatocytes incubated with As₂O₃. 5) About 10 times higher concentrations of As₂O₅ were necessary to induce a similar extent of inhibition of gluconeogenesis, decrease in O₂ consumption, and in ATP content as compared with As₂O₃. The extent of the As₂O₅ effect depended on the concentration of the toxicant and on the substrate used. Gluconeogenesis from pyruvate exhibited the highest sensitivity to As₂O₅. 6) All findings can be largely explained by inhibition of pyruvate dehydrogenase as the central target for arsenicals. The subsequent depletion of acetyl CoA results in impaired formation of reducing equivalents in the citric acid cycle, decrease in high energy phosphates and, acetyl CoA being a strong positive modulator of pyruvate carboxylase, in gluconeogenesis inhibition. Carbohydrate depletion, resulting mainly from gluconeogenesis inhibition, is proposed to be a major problem in poisoning with trivalent arsenicals. In accordance with this proposal, starved rats were shown to be much more sensitive to As₂O₃ than animals with free access to food.     

密闭囊袋冲洗系统模拟抑制人晶状体上皮细胞的研究

目的 利用密闭囊袋冲洗系统和人后发性白内障(PCO)囊袋模型,研究三氧化二砷(As2O3)在短时间内对PCO的防治作用.方法 实验研究.严格将时间控制在2 min时,在细胞培养条件下,四甲基偶氮唑盐比色法观察As2O3对人晶状体上皮细胞(LEC)系FHL124细胞的抑制作用;乳酸脱氢酶释放实验观察As2O3对FHL124细胞的损伤作用;荧光显微镜动态检测细胞内Ca2+浓度的变化.建立人PCO囊袋模型,应用密闭囊袋冲洗系统观察As2O3对原代人LEC的作用.浓度反应曲线用Hill公式求IC50值.所有实验数据结果用均数±标准差表示,采用SPSS 13.0软件处理,组间差异用单因素方差分析,组间两两比较用Dunnett检验.结果 As2O3在作用仅2 min的情况下,就可以抑制人LEC和清除残留于囊袋内的人原代LEC.As2O3(10、30、100、300、1000 μmol/L)对FHL124细胞的作用呈剂量依赖性,100 μmol/L以上浓度的As2O3对FHL124细胞具有明显的毒性作用.与对照组比较,差异有统计学意义(t=5.217,P＜0.01),半效抑制量(IC50)=130 μmol/L.乳酸脱氢酶检测显示,As2O3可影响细胞膜结构的完整性.细胞内动态Ca2+检测显示,As2O3对细胞内Ca2+作用呈剂量依赖性.高浓度As203可引起细胞内Ca2+库释放,同时抑制细胞的Ca2+内流,最终导致细胞内Ca2+稳态的破坏而引起细胞的死亡.1×104 μmol/L As2O3作用2 min后,使Ca2+内流的幅值及速度分别降低了(43.24±2.98)%(t=3.134,P＜0.01)和(46.27±6.01)%(t=3.521,P＜0.01).人PCO囊袋模型显示,联合应用As2O3和密闭囊袋冲洗系统可以在2 min内清除残留于囊袋内的原代人LEC.结论 As2O3可以在短时间内彻底清除白内障手术中存留于囊袋内的人LEC,与密闭囊袋冲洗系统结合可能抑制PCO的发生.     

硫化砷对白血病细胞的生长抑制及作用机制

 目的 研究硫化砷（As4S4）通过体外培养对白血病细胞HL-60细胞的生长抑制作用及其分子机制。方法 以不同浓度（7.5 ~ 60 mmol／L）的As4S4作用于体外培养HL-60细胞12 ~ 48 h,四甲基偶氮唑蓝（MTT）法检测细胞生长抑制率,流式细胞仪观察细胞凋亡率,免疫组化法检测bcl-2、p53蛋白表达,应用RT-PCR法检测HL-60细胞中p53 mRNA表达。结果 不同浓度的As4S4作用12 ~ 48 h后,可显著抑制HL-60细胞的生长,呈时间浓度依赖性,并诱导细胞发生凋亡,凋亡率为30.18 % ~ 70.98 %,不同作用时间、不同浓度间凋亡率差异有统计学意义（P＜0.01）,RT-PCR结果显示As4S4作用24 h后,下调p53 mRNA的表达。免疫组化结果显示,bcl-2、p53蛋白表达逐渐降低,并呈浓度依赖性。结论 As4S4能够显著抑制HL-60细胞的生长。诱导其凋亡,并下调bcl-2、p53的表达,可能是其重要机制。     

三氧化二砷抑制耐药bcr-abl突变细胞株生长的机制

 目的　研究三氧化二砷（ATO）在体外对bcr-abl突变细胞株的生长抑制作用,并探讨其作用机制。方法　采用锥虫蓝拒染法观察ATO及ATO与丁硫氨酸亚矾胺（BSO）共同作用对bcr-abl野生型细胞株（K562、KBM5、32Dp210）和伊马替尼（IM）耐药bcr-abl突变细胞株（K562R、KBM5R、32Dp210T315I、32Dp210Q252H、32Dp210Y253H、32Dp210M351T和32Dp210E255K）的生长抑制作用;Annexin V和PI染色检测细胞凋亡,以5,5'-双硫代对硝基苯甲酸直接比色法（DTNB比色法）测定细胞内谷胱甘肽含量（GSH）。结果　ATO对bcr-abl野生型和IM耐药细胞株均呈现剂量依赖性的生长抑制作用。且含bcr-abl点突变的耐药细胞株对ATO格外敏感,其ATO的IC50值均比其对应的bcr-abl野生型细胞株低,但无点突变的K562R细胞株的IC50值与野生型K562细胞无差异。对上述细胞株细胞内还原型GSH的含量进行检测,结果显示：含有bcr-abl点突变的细胞株内GSH含量显著低于其对应的野生型细胞（均P＜0.05）,而K562和K562R细胞中GSH含量差异无统计学意义（P＝0.315）。选用GSH生物合成抑制剂BSO处理各细胞株后,细胞对ATO的敏感性均显著增加。结论　与bcr-abl野生型细胞株相比ATO对含有点突变的细胞株生长抑制作用更为显著,这可能与细胞内GSH的含量有关,提示ATO有可能成为治疗包括T315I在内的bcr-abl突变慢性粒细胞白血病患者的新选择。     

三氧化二砷联合甲磺酸伊马替尼、咖啡因对K562白血病细胞的协同作用

 目的 探讨三氧化二砷（Arsenic trioxide,As2O3）与酪氨酸激酶抑制剂甲磺酸伊马替尼（实验药物代号STI571）以及细胞周期调节剂咖啡因联合诱导K562细胞凋亡的作用及机制,为寻找克服K562细胞对As2O3抵抗的有效手段提供实验依据。方法 以As2O3与STI571、咖啡因联合作用于K562细胞,采用MTT方法检测细胞增生活性,PI染色流式细胞仪检测细胞周期,PI和Annexin V双染色流式检测细胞凋亡,Western blot法检测细胞周期相关调节蛋白的表达。结果 5.0 μmol／L浓度的咖啡因对K562细胞增生无抑制作用,亦不能增强As2O3的抑制作用,单独以STI571 1.0 μmol／L处理,即可有效抑制K562细胞的生长,与As2O3联用能明显增加其抑制增生的效应;咖啡因与As2O3联合作用,不增加As2O3诱导的K562细胞凋亡率[（14.7±3.6）%vs（15.3±3.3）%,P＞0.05]; STI571具有轻度诱导K562细胞凋亡的作用[（18.3±4.5）%],与As2O3合用可显著增加诱导凋亡率[（14.7±3.6）%vs（42.8±4.2）%,P＜0.01],并明显降低As2O3诱导的G2／M期细胞比例。与单用As2O3比较,As2O3 + STI571明显抑制cdc2、cdc2-p及survivin蛋白表达,而As2O3与咖啡因合用不能诱导survivin蛋白表达下调,对cdc2、cdc2-p蛋白的表达无明显影响。结论 周期调节药物咖啡因对As2O3诱导K562细胞凋亡无增敏作用;酪氨酸激酶抑制剂STI571能协同As2O3诱导K562细胞凋亡,下调抗凋亡蛋白survivin的表达可能是其机制之一,值得深入研究其临床应用效果。     

三氧化二砷对尤文肉瘤细胞迁移和侵袭能力抑制作用的体外研究

 目的　研究三氧化二砷在体外对尤文肉瘤细胞系迁移和侵袭能力的抑制作用 。方法　通过噻唑蓝（MTT）实验选择对尤文肉瘤细胞系（A-673、RD-ES）无毒性的三氧化二砷浓度（＜2 μmol／L）后,Transwell迁移和侵袭实验检测三氧化二砷作用下尤文肉瘤细胞的迁移和侵袭能力。Western blot实验及明胶酶谱实验检测PI3K-AKT通路相关作用蛋白及基质金属蛋白酶-9（MMP-9）的表达变化。结果　迁移和侵袭穿过Transwell基底膜的尤文肉瘤细胞的数量随着三氧化二砷浓度的增加而逐渐降低,经不同浓度三氧化二砷处理过的A-673迁移平均数量分别占未处理组的54.3 %、49.0 %和17.0 %,差异有统计学意义（F＝112.78,P＜0.01）,在侵袭实验中分别为52.7 %、32.3 %和10.3 %,差异有统计学意义（F＝183.76,P＜0.01）;经过不同浓度三氧化二砷处理后的RD-ES细胞迁移平均数量分别占未处理组的46.0 %、39.0 %和8.0 %,差异有统计学意义（F＝408.25,P＜0.01）,在侵袭实验中分别为58.7 %、22.3 %和9.0 %,差异有统计学意义（F＝373.25,P＜0.01）。PI3K-AKT通路相关蛋白和MMP-9表达水平下降。结论　低浓度三氧化二砷可以通过下调PI3K-AKT信号通路的表达来抑制尤文肉瘤细胞系的转移能力。