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The ubiquity of arsenic in the environment has led to the evolution of arsenic defense mechanisms in almost every organism studied from Escherichia coli to man Bhattacharjee et al.
Microorganisms take up As i V in the form of arsenate via phosphate transporters Bun-ya et al. In contrast, glycerol channels appear to play a key role in the cellular uptake of As i III , e.
As i III is extruded from cells or sequestered in intracellular compartments, either as free arsenite or as a conjugate with GSH or other thiols Ghosh et al.
In addition, arsenic can be methylated Aposhian, , although this process may increase arsenic toxicity rather than contributing toward detoxification since certain metabolic intermediates are more toxic than the inorganic arsenic Styblo et al.
Dopp et al. The authors concluded that a defense mechanism exists involving arsenic extrusion and that uptake at higher arsenic concentrations is inhibited.
The higher uptake of the trivalent methylated arsenicals may be responsible for the reported greater genotoxic effects of these compounds e.
In the present study, we compare the uptake capabilities of fibroblasts CHO-9 cells used for genotoxicity studies with those of hepatoma cells Hep G2.
These cells were chosen because the liver is the primary site of arsenic metabolism within the body and skin, along with the bladder, is a target organ for arsenic carcinogenicity.
The cellular uptake of the arsenic compounds was forced by electroporation to test the hypothesis that arsenic-induced genotoxic effects are related to the intracellular arsenic concentration and dependent upon uptake.
Also, we investigated whether the arsenic compounds are bound to membranes or whether they are present in the cytosol.
For these investigations we removed the cell membranes by osmotic lysis and subsequent centrifugation before the intracellular arsenic concentrations were measured by ICP-MS.
CHO-9 cells obtained from A. Yamanashi, Japan. Preparation of dilute solutions of these iodide precursors results in the formation of the corresponding acids, monomethylarsonous acid MeAs OH 2 and dimethylarsinous acid Me 2 AsOH Gong et al.
All chemicals were of analytical grade or of the highest quality obtainable. Due to the high volatility and sensitivity to oxidation of dimethylarsinous acid, solutions of this arsenical were always prepared immediately before each experiment and were discarded if not used within a two-day period.
All other arsenicals tested, including the trivalent compounds sodium arsenite and monomethylarsonous acid, were found to be stable over a minimum two month period.
All arsenicals used in the cytotoxicity, genotoxicity, uptake, and electroporation experiments are listed in Table 1. Trypan blue, Cytochalasin B, May-Grünwald and Giemsa solutions were purchased from Sigma, and the hypoosmotic buffer for electroporation from Eppendorf Germany.
CHO-9 and Hep G2 cells were treated with the arsenicals at different concentrations for 24 h, and all experiments were performed in duplicate. Cell viability was evaluated immediately after exposure.
Treated and untreated cells were harvested by trypsin treatment Sigma. Cell counting was performed following trypan blue staining. The cell suspension was mixed with an equivalent volume of 0.
Cell viability is expressed as percentage of surviving cells compared to the total number of cells. Significance was tested by using the Student's t -test.
Then the arsenic compounds were applied for 24 h at different concentrations 0. The frequency of MN formation was expressed as percentage of binucleated bn cells with MN.
Using the cytokinesis-blocked MN assay, the extent and progression of nuclear division could be assessed by analysing the NDI. A number of effects, including a decreased optimal pulse voltage, ensure that the plasma membrane can be permeated more easily.
The tolerance of CHO-9 cells to hypoosmolar conditions was tested in preliminary experiments. A suspension of cells 0.
The cells were transferred to electroporation cuvettes with a 4 mm gap. Following an incubation period of 24 h recovery time , the CBMN-assay was carried out.
The experiments were set up as follows: a unexposed control, b unexposed control and electroporation, c exposed with arsenicals, and d exposed with arsenicals and electroporation.
All experiments were performed in triplicate. To assess the membrane permeability of the arsenic compounds under normal and forced conditions, CHO-9 cells were incubated with the arsenic compounds at different concentrations 0.
Since the results obtained from experiments with exposure periods of 1 h and 24 h did not differ significantly, all uptake experiments were performed over a period of 1 h.
For comparison of arsenic uptake in different cell lines, 8. Following incubation, cells were washed with PBS and subsequently resuspended in 10 ml fresh culture medium.
The absence of intact cells was confirmed by microscopic examination. Whole-cell and cell-free extract samples were prepared as described above.
Solutions up to 1 in dilutions were delivered at 0. The signals 75 As ms , 77 Cl ms , and In ms were monitored. The chi-square-test was used for the statistical analysis of the results of the micronucleus assay and the two-tailed Student's t -test for evaluating the data from the cytotoxicity test and the NDI.
A concentration-dependency was observed for these species with a maximum uptake at the highest external concentration applied. Cells were exposed to arsenicals for 1 h.
The uptake of the organoarsenic compounds by Hep G2 cells was tested using concentrations from 0. The difference between the arsenic concentration in whole-cell extract and the cell-free extract was not significant in all cases.
The intracellular concentration of arsenic was in most cases lower for Hep G2 cells than in CHO-9 cells.
The cellular uptake of the arsenic species by CHO-9 cells increased after treatment of cells using electroporation.
Uptake of arsenic compounds by CHO-9 cells under forced uptake electroporation condition. Cytotoxicity of pentavalent and trivalent arsenic compounds in CHO-9 cells.
The percentage of decreased cell viability is shown in relation to the untreated control. The cytotoxicity was determined by trypan blue-staining.
Cytotoxicity of pentavalent and trivalent arsenic compounds in Hep G2 cells. All experiments were performed in duplicate. The number of micronucleated cells was not significantly increased when cells were exposed to MMA V for 24 h Fig.
The formation of MN rose significantly at a tested concentration of 7. Micronucleus induction after exposure of CHO-9 cells to different concentrations of arsenite and arsenate for 24 h.
Only incubation of cells with 0. Using longer recovery periods 28 h and 35 h , this delay was not statistically significant anymore data not shown.
These compounds were chosen for electroporation experiments because they were negative in the MN assay under normal exposure conditions Figs.
No significant differences in MN induction were observed between the unexposed control and the unexposed control after electroporation.
MN induction after electroporation of untreated and treated CHO cells. After exposure and electroporation the cells were treated with cytochalasin B for additional 24 h recovery time.
The experiments were repeated four times. Collaboration with Bastinelli Knives. Weight of g. Made in Italy. Kydex case with Tek-Lock. Filming V.
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