ToxSci Advance Access originally published online on June 9, 2005
Toxicological Sciences 2005 87(1):46-56; doi:10.1093/toxsci/kfi218
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Forced Uptake of Trivalent and Pentavalent Methylated and Inorganic Arsenic and Its Cyto-/genotoxicity in Fibroblasts and Hepatoma Cells
* Institute of Hygiene and Occupational Medicine, University Hospital, Hufelandstrasse 55, 45122 Essen, Germany; Institute of Environmental Analysis, University of Duisburg-Essen, Universitätsstrasse 3-5, 45141 Essen, Germany; and Industrial Toxicology Research Centre, 226 001 Lucknow, India
Received March 17, 2005; accepted May 7, 2005
Mammals are able to convert inorganic arsenic to mono-, di-, and trimethylated metabolites. In previous studies we have shown that the trivalent organoarsenic compounds are more toxic than their inorganic counterparts and that the toxicity is associated with the cellular uptake of the arsenicals. In the present study, we investigated cyto-/genotoxic effects of the arsenic compounds arsenate [Asi(V)], arsenite [Asi(III)], monomethylarsonic acid [MMA(V)], monomethylarsonous acid [MMA(III)], dimethylarsinic acid [DMA(V)], dimethylarsinous acid [DMA(III)], and trimethylarsine oxide [TMAO(V)] after an extended exposure time (24 h) and compared the uptake capabilities of fibroblasts (CHO-9 cells: Chinese hamster ovary) used for genotoxicity studies, with those of hepatic cells (Hep G2: hepatoma cell-line). To find out whether the arsenic compounds are bound to membranes or if they are present in the cytosol, the amount of arsenic was measured in whole-cell extracts and in membrane-removed cell extracts by inductively coupled plasma-mass spectrometry (ICP-MS). In addition, we forced the cellular uptake of the arsenic compounds into CHO-9 cells by electroporation and measured the intracellular arsenic concentrations before and after this procedure. Our results show that organic and inorganic arsenicals are taken up to a higher degree by fibroblasts compared to hepatoma cells. The arsenic metabolite DMA(III) was the most membrane permeable species in both cell lines and induced strong genotoxic effects in CHO-9 cells after an exposure time of 24 h. The uptake of all other arsenic species was relatively low (<1% by Hep G2 and <4% by CHO cells), but was dose-dependent. Electroporation increased the intracellular arsenic levels as well as the number of induced MN in CHO-9 cells. With the exception of Asi(III) and DMA(III) in CHO-9 cells, the tested arsenic compounds were not bound to cell membranes, but were present in the cytosol. This may indicate the existence of DMA(III)-specific exporter proteins as are known for Asi(III). Our results indicate that the uptake capabilities of arsenic compounds are highly dependent upon the cell type. It may be hypothesized that the arsenic-induced genotoxic effects observed in fibroblasts are due to the high uptake of arsenicals into this cell type. This may explain the high susceptibility of skin fibroblasts to arsenic exposure.
Key Words: DNA damage; arsenic; dimethylarsinous acid; monomethylarsonous acid; cellular uptake; electroporation; cytotoxicity; micronuclei.
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