NADH Oxidase Activation Is Involved in Arsenite-Induced Oxidative DNA Damage in Human Vascular Smooth Muscle Cells

Abstract

Abstract-Arsenic is atherogenic, carcinogenic, and genotoxic. Because atherosclerotic plaque has been considered a benign smooth muscle cell tumor, we have studied the effects of arsenite on DNA integrity of human vascular smooth muscle cells. By using single-cell alkaline electrophoresis, apparent DNA strand breaks were detected in a 4-hour treatment with arsenite at a concentration above 1 mol/L. DNA strand breaks of arsenite-treated cells were increased by Escherichia coli formamidopyrimidine-DNA glycosylase and decreased by diphenylene iodinium, superoxide dismutase, catalase, pyruvate, DMSO, or D-mannitol. Extract from arsenite-treated cells showed increased capacity for producing superoxide when NADH was included in the reaction mixture; however, addition of arsenite to extract from untreated cells did not increase superoxide production. The superoxide-producing ability of arsenite-treated cells was also suppressed by diphenylene iodinium, 4,5-dihydroxy-1,2-benzenedisulfonic acid disodium salt (Tiron), or superoxide dismutase. Superoxide production and DNA strand breaks in arsenite-treated cells were also suppressed by transfecting antisense oligonucleotides of p22phox, an essential component of NADH oxidase. Treatment with arsenite also increased the mRNA level of p22phox. These results suggest that arsenite activates NADH oxidase to produce superoxide, which then causes oxidative DNA damage. The result that arsenite at low concentrations increases oxidant levels and causes oxidative DNA damage in vascular smooth muscle cells may be important in arsenic-induced atherosclerosis. (Circ Res. 2000;86:514-519.)Key Words: arsenite Ⅲ oxidative DNA damage Ⅲ NADH oxidase Ⅲ atherosclerosis A rsenic, an element present in the earth's crust, comes to the surface through mining and utilization of groundwater. Serious contamination by inorganic arsenic occurs through long-term ingestion of high concentrations of arsenic in drinking water. Chronic exposure to arsenic has been related to increased incidences of skin, lung, bladder, liver, and kidney cancers. 1,2 Arsenic exposure is also associated with various vascular disorders, including angiosarcomas, 3 atherosclerotic plaques, 4 and hypertension in humans. 5 The epidemiological evidence for shared risk factors for cancer and atherosclerosis has been reviewed by Hansen. 6 Exposure to carcinogenic environmental agents is associated with an increased risk of atherosclerosis. Therefore, somatic mutation and cell proliferation may play a role in the pathogenesis of atherosclerotic plaques. The predominant cell type in plaques is the vascular smooth muscle cell (VSMC). Proliferation of VSMCs is essential for plaque formation and development. By examining the isoenzymes of glucose-6-phosphate dehydrogenase, human atherosclerotic plaques were shown to be monoclonal in origin. 7 Furthermore, DNA samples from human coronary artery plaques were demonstrated to transform NIH3T3 cells. 8 These observations suggest that atherosclerotic plaques are presumably benign smooth muscle...

Arsenic-induced dysfunction in relaxation of blood vessels.

“…As 3+ reduced eNOS activity in a concentration-dependent manner (Figure 2A), suggesting that As 3+ can suppress vascular NO production in endothelial cells. Others have reported that As 3+ can generate superoxide anions in cultured endothelial cells and smooth muscle cells (Lynn et al 2000;Smith et al 2001). Superoxide anions interact with NO to form peroxynitrite, resulting in suppression of vascular relaxation by blocking the NO pathway (Pryor and Squadrito 1995).…”

“…As 3+ is reported to stimulate the formation of reactive oxygen species (ROS) in vascular endothelial and smooth muscle cells, mainly via NADH/NADPH oxidase (Lynn et al 2000;Smith et al 2001); thus, the generation of superoxide could elicit the reduced relaxation due to elimination of NO. Therefore, we considered the possibility that superoxide production is involved in suppression of vasorelaxation by As 3+ .…”

Inorganic Arsenic Activates Reduced NADPH Oxidase in Human Primary Macrophages through a Rho Kinase/p38 Kinase Pathway

“…Besides phagocytic NADPH oxidase, iAs can also stimulate nonphagocytic NADPH oxidase in endothelial cells (16,34), vascular smooth muscle cells (35), and in the human promyelocytic NB4 cell line (15); however, molecular mechanisms mediating its activation remain poorly understood. Chou et al (15) have shown that prolonged treatment of NB4 cells (9 days) with 0.75 M As 2 O 3 markedly up-regulate mRNA levels and membrane expression of p47 phox .…”

Prevalence of non-insulin-dependent diabetes mellitus and related vascular diseases in southwestern arseniasis-endemic and nonendemic areas in Taiwan.

“…The present study demonstrated the odds ratios of 1.2 (95% CI, 1.1-1.4) in diabetics and 12.5 (95% CI, 9.5-16.5) in nondiabetics. Arsenic, mainly trivalent arsenicals (e.g., arsenite), may induce atherosclerosis through damage of endothelial cells or smooth muscle cells by intracellular-reduced glutathione or through oxidative DNA damage (Chang et al 1991;Chiou et al 1997a;Lynn et al 2000;Wu et al 2001). Further studies are necessary to test the hypothesis that arsenic induces renal (Mitchell et al 2000) and neural (Brouwer et al 1992;Mahajan et al 1992) damage directly or through angiopathy.…”