Arsenite and monomethylarsonous acid generate oxidative stress response in human bladder cell culture.
Arsenicals have commonly been seen to induce reactive oxygen species (ROS) which can lead to DNA damage and oxidative stress. At low levels, arsenicals still induce the formation of ROS, leading to DNA damage and protein alterations. UROtsa cells, an immortalized human urothelial cell line, were used to study the effects of arsenicals on the human bladder, a site of arsenical bioconcentration and carcinogenesis. Biotransformation of As(III) by UROtsa cells has been shown to produce methylated species, namely monomethylarsonous acid [MMA(III)], which has been shown to be 20 times more cytotoxic. Confocal fluorescence images of UROtsa cells treated with arsenicals and the ROS sensing probe, DCFDA, showed an increase of intracellular ROS within five min after 1 microM and 10 microM As(III) treatments. In contrast, 50 and 500 nM MMA(III) required pretreatment for 30 min before inducing ROS. The increase in ROS was ameliorated by preincubation with either SOD or catalase. An interesting aspect of these ROS detection studies is the noticeable difference between concentrations of As(III) and MMA(III) used, further supporting the increased cytotoxicity of MMA(III), as well as the increased amount of time required for MMA(III) to cause oxidative stress. These arsenical-induced ROS produced oxidative DNA damage as evidenced by an increase in 8-hydroxyl-2'-deoxyguanosine (8-oxo-dG) with either 50 nM or 5 microM MMA(III) exposure. These findings provide support that MMA(III) cause a genotoxic response upon generation of ROS. Both As(III) and MMA(III) were also able to induce Hsp70 and MT protein levels above control, showing that the cells recognize the ROS and respond. As(III) rapidly induces the formation of ROS, possibly through it oxidation to As(V) and further metabolism to MMA(III)/(V). These studies provide evidence for a different mechanism of MMA(III) toxicity, one that MMA(III) first interacts with cellular components before an ROS response is generated, taking longer to produce the effect, but with more substantial harm to the cell.
Eblin
K E
KE
Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, 1703 E. Mabel, PO Box 210207, Tucson, AZ 85721, USA. eblin@pharmacy.arizona.edu <eblin@pharmacy.arizona.edu>
Bowen
M E
ME
Cromey
D W
DW
Bredfeldt
T G
TG
Mash
E A
EA
Lau
S S
SS
Gandolfi
A J
AJ
D000080242:8-Hydroxy-2'-Deoxyguanosine; D018053:Arsenites / Q000633:toxicity*; D001711:Biotransformation; D002274:Carcinogens, Environmental / Q000378:metabolism / Q000633:toxicity*; D002374:Catalase / Q000378:metabolism; D002460:Cell Line; D004247:DNA / Q000187:drug effects / Q000378:metabolism; D004249:DNA Damage; D003849:Deoxyguanosine / Q000031:analogs & derivatives / Q000032:analysis; D004305:Dose-Response Relationship, Drug; D018840:HSP70 Heat-Shock Proteins / Q000096:biosynthesis; D006801:Humans; D008668:Metallothionein / Q000096:biosynthesis; D009942:Organometallic Compounds / Q000378:metabolism / Q000633:toxicity*; D018384:Oxidative Stress / Q000187:drug effects*; D017382:Reactive Oxygen Species / Q000378:metabolism; D013482:Superoxide Dismutase / Q000378:metabolism; D013997:Time Factors; D015854:Up-Regulation / Q000187:drug effects; D001743:Urinary Bladder / Q000187:drug effects* / Q000378:metabolism; D019459:Urothelium / Q000187:drug effects / Q000378:metabolism
D018053:Arsenites; D002274:Carcinogens, Environmental; D018840:HSP70 Heat-Shock Proteins; D009942:Organometallic Compounds; D017382:Reactive Oxygen Species; C406082:monomethylarsonous acid; D000080242:8-Hydroxy-2'-Deoxyguanosine; D004247:DNA; D008668:Metallothionein; D002374:Catalase; D013482:Superoxide Dismutase; D003849:Deoxyguanosine; C015001:arsenite