Arsenic content in the femur head of the residents of southern and central Poland

Arsenic content was assayed in the samples of the femur head of the people living in southern and central Poland (Kraków, n=13; Silesian region, n=13; Łódź, n=12). The average age being 68.7±8.7 yr. Arsenic content in the femur head was determined applying the hydride generation atomic absorption spectrometry (HG-AAS) method after microwave mineralization. The average arsenic contents in the femur head of the residents of the Łódź, Kraków, and Silesian regions were 0.41 μg/g, 0.37 μg/g, and 0.18 μg/g, respectively. No correlation has been found between arsenic content in the femur head and the content of other metals. Neither the age nor sex of the people tested affected the arsenic content in the femur head.     

三氧化二砷对人脐静脉内皮细胞凋亡及VCAM-1/ICAM-1 表达的影响

目的:观察三氧化二砷(As2O3)对血管内皮细胞增殖、凋亡及VCAM-1/ICAM-1表达的影响,探讨As2O3对血管内皮细胞增殖生长以及炎症反应的影响。方法:人脐静脉内皮细胞(HUVEC)体外培养,以不同As2O3浓度及时间对其进行干预。采用CCK-8测定细胞增殖活性,流式细胞仪AnnexinⅤ/PI双染法检测细胞的凋亡率,实时荧光定量PCR检测VCAM-1mRNA表达,酶联免疫吸附试验(ELISA)检测细胞间黏附分子(VCAM-1)及血管细胞黏附分子(ICAM-1)的表达情况。结果:当As2O3浓度在3μmol.L-1时HUVEC培养24 h的的凋亡率为(0.134±0.03)%,48 h为(3.305±0.53)%,72 h为(3.748±0.84)%(P<0.05),凋亡率均在一较低水平。当As2O3浓度>3μmol.L-1时HUVEC凋亡率明显增加(P<0.01)。不同浓度As2O3作用HUVEC48 h后检测上清液中ICAM-1与VCAM-1浓度时发现1μmol.L-1时VCAM-1表达即开始增加(123.32±3.78 mmol.L-1,P<0.01),而HUVEC表达ICAM-1含量与对照组相比差异并不明显(38.94±2.59 mmol.L-1,P>0.05),随着As2O3浓度的增加,HUVEC表达ICAM-1/VCAM-1的量均增加但敏感性不同。对照组及(1.0、2.0、3.0、4.0、5.0)μmol.L-1As2O3作用于HUVEC 48 h实时荧光定量PCR法检测VCAM-1mRNA表达量明显增加,与对照组相比实验组的表达量分别为(1.657±0.287,1.858±0.241,2.321±0.280,3.012±0.235,3.508±0.342)(P<0.01)。结论:As2O3可直接降低细胞活性,诱导细胞凋亡,并且呈一定的时间-浓度依赖性。在较低浓度时VCAM-1/ICAM-1的表达在一个相对较低的水平,随着As2O3浓度的逐渐升高,内皮细胞凋亡率增高,VCAM-1/ICAM-1表达增加,并且VCAM-1/ICAM-1对As2O3的敏感性呈现一定的差异性。     

Kinetics of arsenic methylation by freshly isolated B6C3F1 mouse hepatocytes

The toxic and carcinogenic effects of arsenic may be mediated by both inorganic and methylated arsenic species. The methylation of arsenic(III) is thought to take place via sequential oxidative methylation and reduction steps to form monomethylarsenic (MMA) and dimethylarsenic (DMA) species, but recent evidence indicates that glutathione complexes of arsenic(III) can be methylated without oxidation. The kinetics of arsenic methylation were determined in freshly isolated hepatocytes from male B6C3F1 mice. Hepatocytes (>90% viability) were isolated by collagenase perfusion and suspended in Williams' Medium E with various concentrations of arsenic(III) (sodium m-arsenite). Aliquots of the lysed cell suspension were analyzed for arsenic species by hydride generation-atomic absorption spectrometry. The formation of MMA(III) from sodium arsenite (1 microM) was linear with respect to time for >90 min. DMA(III) formation did not become significant until 60 min. MMA(V) and DMA(V) were not consistently observed in the incubations. These results suggest that the glutathione complex mechanism of methylation plays an important role in arsenic biotransformation in mouse hepatocytes. Metabolism of arsenic(V) was not observed in mouse hepatocytes, consistent with inhibition of arsenic(V) active cellular uptake by phosphate in the medium. The formation of MMA(III) increased with increasing arsenic(III) concentrations up to approximately 2 microM and declined thereafter. The concentration dependence is consistent with a saturable methylation reaction accompanied by uncompetitive substrate inhibition of the reaction by arsenic(III). Kinetic analysis of the data suggested an apparent K(M) of approximately 3.6 microM arsenic(III), an apparent V(max) of approximately 38.9 microg MMA(III) formed/L/h/million cells, and an apparent K(I) of approximately 1.3 microM arsenic(III). The results of this study can be used in the physiologically based pharmacokinetic model for arsenic disposition in mice to predict the concentration of MMA(III) in liver and other tissues.     

Alternative splicing variants of human arsenic (+3 oxidation state) methyltransferase

Arsenic (+3 oxidation state) methyltransferase (As3MT) catalyzes the methylation of trivalent arsenic (As(III)) to monomethylarsonate (MMA(V)) and dimethylarsinic acid (DMA(V)), and plays an important role in the detoxification of arsenicals. Here, we report the identification of two splicing variants of the human As3MT gene. One splicing variant was an exon-3 skipping (Δ3) form which produced a premature stop codon, and the other was an exon-4 and -5 skipping (Δ4,5) form which produced a 31.1 kDa As3MT protein. In addition to the full-length mRNA of As3MT, Δ4,5 mRNAs were detected in HepG2, A549, HL60, K562, and HEK293 cells. The methyltransferase activity of the recombinant Δ4,5 As3MT and wild-type As3MT proteins purified from Escherichia coli was determined. Speciation analysis by HPLC–ICP-MS showed a clear peak of MMA(V) after incubation of As(III) with the wild-type As3MT protein, but not with the Δ4,5 As3MT protein. In addition, COS-7 cells transfected with Δ4,5 As3MT cDNA did not convert As(III) to MMA(V) or DMA(V). The lack of methyltransferase activity of Δ4,5 As3MT seems to be related to the deletion of an S-adenosylmethionine-binding site and a critical cysteine residue. These data suggest that the expression pattern of splicing variants of the As3MT gene may affect the capacity for arsenic methylation in cells.     

Arsenic trioxide induces gallbladder carcinoma cell apoptosis via downregulation of Bcl-2

Gallbladder carcinoma (GBC), an aggressive and mostly lethal malignancy, is known to be resistant to a number of apoptotic stimuli. Here, we report for the first time the pro-apoptosis role of arsenic trioxide (As2O3) in gallbladder carcinoma and identify the contribution of Bcl-2 in the As2O3-induced apoptosis. The treatment of As2O3 in gallbladder carcinoma cells could induce apoptosis in a dose-dependent manner and downregulate the expression of anti-apoptotic protein Bcl-2 at mRNA level. Moreover, Bcl-2 overexpression could protect gallbladder carcinoma cells from As2O3-induced apoptosis, indicating the contribution of Bcl-2 in As2O3-induced apoptosis. Taken together, these results suggest that arsenic trioxide induces gallbladder carcinoma cell apoptosis via downregulation of Bcl-2, which may have important therapeutic implications in gallbladder carcinoma patients.     

芯片杂交中荧光标记样品定量与非定量的比较研究

比较在芯片杂交中,荧光标记样品定量与非定量对杂交结果的影响。其方法是,提取经As2O3作用K562细胞前后的总RNA,逆转录成cDNA第一链,并分别用Cy3／Cy5标记。标记后的样品再次定量或不定量,但均取相同体积上样与K562芯片杂交,用扫描仪扫描并分析。其结果,标记后样品定量与不定量杂交的结果都与理论推测一致,但以样品定量进行杂交的效果更好,标记样品杂交前再次定量的,分析发现2个基因表达下调;杂交前不定量仅取相同体积进行杂交的,发现6个基因片段表达下调,其中只有2个基因与细胞凋亡通路密切相关。认为在芯片的杂交检测中,对荧光标记样品杂交前再次定量可大大提高杂交结果的可靠性。     

砷污染土壤中毛球菌的分离及其砷还原特性

生物异化还原作用是地球化学循环中的重要部分,其对环境污染物具有良好的治理作用。从湖南省石门县的土壤中分离出1株厌氧砷还原菌,通过16S rRNA分析为毛球菌属,命名为Trichococcus sp.CC328。菌落圆形,无色透明,短杆状革兰阴性菌,最适生长条件为p H 9,Na Cl浓度12-16 g/L。当As(V)的初始浓度为63mg/L时,24 h时细胞数目最多(18×108个/m L),48 h时砷还原率为83%;当Fe(III)的初始浓度为30 mg/L,72 h时还原率为37%。本研究是首次发现具有砷和铁还原能力的毛球菌,为土壤中砷的生物转化提供参考。     

Arsenic toxicity is not due to a direct effect on the oxidation of reduced inorganic sulfur compounds by Thiobacillus caldus

Abstract Thiobacillus caldus is a moderately thermophilic acidophile which has been implicated in the biooxidation of arsenic containing mineral Sulfides. The toxic effects of arsenic on this bacterium are presented here. Addition of arsenite to a growing culture of T. caldus caused a transient increase in the optical density of the culture while causing a simultaneous decrease in cell viability. The increase in optical density was shown to be due to the formation of extracellular sulfur. The oxidation rates of tetrathionate and thiosulfate were decreased by increasing concentrations of arsenite, while in a culture induced to arsenic resistance the rates were not as adversely effected. Sulfur oxidation was also inhibited to the same extent as tetrathionate oxidation, with the oxidation of solid sulfur being slightly more effected than the oxidation of sulfur dissolved in acetone. Thus, bactericidal arsenite causes a transient formation of extracellular sulfur in the culture supernatant of T. caldus yet the toxicity of arsenite is not due to direct inhibitory effects on reduced inorganic sulfur compound oxidation by these bacteria.