| ObjectiveArsenic is a trace element that can be found broadly in the crust. The two main forms of arsenicals are inorganic arsenic (trivalent and pentavalent) and organic arsenic ( monomethyl and dimethyl). The biotransformation of arsenic mainly involves a progress including two times of reduction and two times of methylation. Arsenate reductase, monomethylarsonic acid reductase, dimethylarsonic acid reductase, arsenite methyltransferase, monomethylarsonic acid methyltransferase, reduced glutathione ( GSH) and S - adenosylmethionine ( SAM ) were involved in the biotransformation. It was believed that the one among all the arsenicals caused the most serous damage on organism was inorganic trivalent arsenic, and that the biotransformation of arsenic was the detoxification progress of it. However, according to the recent research, it has been pointed that the metabolites induced in the biotransformation of arsenic are also toxic to organism. Trivalent monomethylarsonic acid ( MMA ) , one of the metabolites, has been demonstrated to be the most toxic one. It is supposed that the methylation progress of arsenic is relevant to the activation of protooncogene and inactivation of anti - oncogerie, and that reactive oxygen species (ROS) may be induced during the biotransformation. ROS has the a-bilities to attack DNA, resulting in DNA strand break and DNA - protein crosslink. It has been revealed by epidemiological investigation that the incidence rates of lung cancer and skin cancer of the population exposed to arsenic are much higher than those of unexposed popu-lation. The relationship between arsenic exposure and DNA damage has been the top spot in the arsenic research field.The single cell gel electrophoresis (SCGE) , also known as comet assay, is a method that broadly used in the detection of DNA damage in mammalian cells. In SCGE, the membrane of cells is destroyed by the lysing solution. Due to relatively higher molecular weight, DNA remains in the initial place while the other components diffuse to the solution. DNA will unwind at high PH resulting in the release of break fragments, which will run out of the nucleus during electrophoresis. Stained by fluorescence dye, the cells will demonstrate images just like comets. The comet length or fluorescence intensity is correlated with the DNA damage in certain range. The technique of SCGE is rapid, simple, visual and sensitive for the measuring and analyzing of DNA damage.The purpose of this study is to investigate whether differential DNA damage expression will be induced by the three arsenicals in human lymphocytes, using the SCGE as the detection method. The mechanism of DNA breakage induced by arsenic exposure will also be discussed.Method1. Separation of lymphocyte:20 ml heparinized blood was sterilely collected from healthy donor, mixed with 20 ml RPMI1640, and then put slowly on the top of 20 ml lymphocyte separation solution. After centrifugation (3000r/ min,30min,4C ) , lymphocytes were drawn from the mixture, washed two times with RPMI1640 (1000r/min,10min,4C ) , and then mixedwith RPMI1640 containing fetal bovine serum (10% ) , adjusting the volume to 16 ml. The single cell suspension was incubated in 24 -well plate (31 C , 5%CO2) , each well containing 1 ml. 30 minutes later, cells were exposed to trivalent sodium arsenite (AsIII) , penta-valent sodium arsenate ( As ) arid pentavalent sodium monomethyl ar-senate (MAsv) by 1, 5, 10, 20 and 50umol/L. Arsenicals (20ul) were added in each of the 16 wells. 20 ul saline were loaded into the control well. At the end of the 24 - h incubation with the arsenicals, cell viability of each well was measured by trypan blue method. Then the single cell suspension in each well was removed and rinsed twice with PBS by centrifugation ( 1000r/min,10Omin,4C). Cell suspension volume of each well was finally adjusted to 200 ul by PBS.2. SCGE:Pre - preparation of the microscope slide; 20 ul of 1% normal melting point agarose ( NMA) were smeared on the slide and dried on the alcohol burner...
|
PDF Full Text Request