Plasmid DNA damage caused by methylated arsenicals, ascorbic acid and human liver ferritin
Introduction
Arsenic is one of the most important global environmental toxicants. Arsenic commonly exists in +3 and +5 valence states and in a number of inorganic and organic forms. There is clear evidence from studies in humans that arsenic exposures are associated with cancer of the skin, lungs, liver, kidney, and bladder. Although the metabolism of inorganic arsenic is fairly well understood, the precise biochemical mechanism of arsenic-induced carcinogenicity in humans is not known. Both in vivo and in vitro studies show that trivalent arsenical forms are more toxic than the corresponding pentavalent forms (Ahmad et al., 2000a, Styblo et al., 2000, Petrik et al., 2000). In biological systems pentavalent arsenicals are reduced to trivalency in the presence of endogenous reducing agents. Earlier it was believed that methylation of the inorganic form of arsenic (iAs(V) or iAs(III)) is a detoxification pathway. But studies from our laboratory and others have shown that some methylated arsenicals are even more cytotoxic and genotoxic than inorganic arsenic (Brown et al., 1997, Ahmad et al., 1999, Petrik et al., 2000, Mass et al., 2001).
The free radical theory of arsenic carcinogenesis has been gaining acceptance (Yamanaka et al., 1991, Yamanaka and Okada, 1994, Ahmad et al., 2000a, Kitchin, 2001). Exposure to arsenic may generate reactive oxygen species (ROS) in vivo (such as dimethylarsenic radical ((CH3)2As), dimethylarsenic peroxyl radicals ((CH3)2AsOO), superoxide anion, singlet oxygen and hydroxyl radicals) and be responsible for cellular toxicity and/or carcinogenicity (Yamanaka and Okada, 1994, Barchowsky et al., 1996, Lynn et al., 1998). Earlier we reported that acute exposure to methylated arsenicals led to a significant decrease in cellular glutathione in rats (Brown et al., 1997) and mice (Ahmad et al., 1999). Glutathione depletion may be correlated with ROS-mediated oxidative stress. We also have reported varying degrees of DNA damage caused by arsenicals—in rat lungs by DMA(V) (Brown et al., 1997), in mouse liver by DMA(V) (Ahmad et al., 1999) and in an in vitro system containing calf thymus DNA and bleomycin by DMA(III) (Ahmad et al., 2000a). Iron is both an essential element and the most abundant transition metal found in biological systems because of its important role in major biological reactions such as the oxygen transport, energy production, toxification and detoxification reactions (Thiel, 1987). The iron storage protein, ferritin, plays a key role in iron storage, utilization and metabolism. Despite the essential nature of iron, it is toxic when present in excess concentrations in cells. Free iron causes redox cycling, production of ROS and oxidative stress.
In this study we further demonstrate the interaction between DMA(V) or DMA(III) with HLF. We also investigated the role of iron released from HLF by arsenic species in generating ROS and inducing significant plasmid DNA damage.
Section snippets
Materials
pBR322 plasmid DNA was initially purchased from three different suppliers and evaluated in order to select a plasmid DNA source containing as few nicks (open circular (OC) form) as possible. ICN Biochemicals (Aurora, OH) human liver ferritin (purified) and pBR322 plasmid DNA were then purchased and utilized. Agarose, ferrozine (3-(2-pyridyl)-5,6-bis(4-phenyl-sulfonic acid)-1,2,4-triazine), ascorbic acid, superoxide dismutase (SOD) (bovine erythrocyte), catalase (bovine liver), ferrous sulfate,
Ferritin iron release
In the present aerobic in vitro study, we observed that both DMA(V) and DMA(III) released iron from HLF with or without ascorbic acid (Table 1). The rate of ferritin iron release by DMA(V), DMA(III) or ascorbic acid was low. However, in the presence of ascorbic acid (a strong synergistic agent), the rate of iron release from HLF by DMA(V) was increased to 3.37 nM/min (P<0.05) and was much higher in the presence of DMA(III) (16.3 nM/min, P<0.001). The rate of iron mobilization from HLF by DMA(V)
Discussion
Arsenic is commonly found in the environment and is a known human carcinogen (NRC, 1999, IARC, 1987). Generally, biomethylation of inorganic arsenicals has been considered a major detoxification pathway. However, recent biochemical studies showed that some methylated arsenicals (MMA(III) and DMA(III)) are even more toxic than the parent iAs(V) and iAs(III) (Styblo et al., 2000, Petrik et al., 2000). Our lab studies also indicate that DMA may induce oxidative stress in Sprague–Dawley rats (Brown
Disclaimer
This manuscript has been reviewed in accordance with the policy of the National Health and Environmental Effects Research Laboratory, US Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency, nor does mention of trade names of commercial products constitute endorsement or recommendation for use.
Acknowledgements
This work was partially supported by a grant from the National Research Council, Washington, DC, to a postdoctoral fellow (Sarfaraz Ahmad) at the US Environmental Protection Agency, North Carolina.
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