As2O3 oxidation by vitamin C: cell culture studies

Abstract

The ability of As(2)O(3) to induce apoptosis in various malignant cell lines has made it a potential treatment agent for several malignancies. In this study the chemical stability of As(2)O(3) (As(III)) in cell-free growth media with various compositions was studied (MEM with different amount of amino acids and DMEM). Special attention was given to evaluate the influence of serum (FBS; fetal bovine serum) absence and vitamin C addition on the oxidation of As(III) to As(V) in cell-free growth media. FBS is an important source of antioxidants and vitamin C (ascorbic acid) is acting as a prooxidant in millimolar concentrations. Media were incubated with As(III) (0.6, 2 and 7 μmol l(-1)) up to 72 h. Experiments were performed at 37°C in light or/and in the dark, with or without added serum (10%) or vitamin C (1.4, 0.14 mM). Metabolites were followed with high-performance liquid chromatography directly coupled to a hydride generation-atomic fluorescence spectrometry system. After 72 h up to 30% of As(III) was transformed into As(V) in MEMs and up to 35% in DMEM when exposed in dark. Light had no influence on transformations in MEMs, but changed the situation dramatically in DMEM where almost all As(III) was oxidized to As(V) after 72 h when exposed to light. Except for some faster oxidation rate the absence of FBS had little effect on the transformation rate in all media. The most visible impact on As(III) oxidation was observed by addition of vitamin C. Addition of vitamin C (1.4 mM) transformed almost all As(III) to As(V) within 72 h. In lower concentrations (0.14 mM) a pro-oxidative effect was still observed reaching approximately 60% oxidation of As(III) during 72 h. All oxidation processes could be explained by pseudo first order reaction kinetics, yielding reaction rates increasing with initial As(III) concentration and vitamin C concentration whereas the FBS content additionally increased the As(III) oxidation rate in the DMEM (light). The temporal oxidation of As(III) to As(V) in various cell-free growth media necessitates routine checking of the valence state of arsenic during cell culture experiments and the results of biological effects attributed to As(III) should be interpreted with caution. Special attention is needed particularly in cases with vitamin C which was acting pro-oxidatively in all conditions examined.

ATM directs DNA damage responses and proteostasis via genetically separable pathways

“…Arsenite is thought to induce ROS through activation of NADPH oxidase (Nox) isoforms as well as through the mitochondria (28, 29) and can produce both superoxide as well as hydrogen peroxide in the cytoplasm. Arsenite is stable in serum-containing media over long periods unlike exogenously added hydrogen peroxide (30, 31) and has been reported to induce checkpoint responses mediated by caffeine-sensitive protein kinases (26). As expected, ATM depletion resulted in a complete defect in the phosphorylation of known ATM substrates including KAP1 and CHK2 after each treatment we used (Fig.…”

Radical Model of Arsenic(III) Toxicity: Theoretical and EPR Spin Trapping Studies

“…Moreover, the use of common exogenous antioxidants such as resveratrol can indirectly attenuate As 2 O 3 -induced hepatoxicity by increasing antioxidant enzyme activities and GSH/GSSG ratio or through direct chelation in the case of α-lipoic acid to reduce As 2 O 3 -induced cardiotoxicity . However, studies in cell-free growth media show vitamin C to be prooxidative by increasing the rate of As III oxidation to As V , while in rats vitamin E showed a protective effect by reducing DNA damage and the TNF-α level, and inhibiting caspase cascade activation . In consideration of recent studies that link arsenic exposure to oxidative stress and ROS generation, our findings further show that organic arsenicals generate various ROS when exposed to common oxidants in solution.…”

Metronomic chemotherapy and anti‐angiogenesis: can upgraded pre‐clinical assays improve clinical trials aimed at controlling tumor growth?

“…In general, basic cell growth media are deficient in antioxidants. The amount of ROS produced during growth in culture medium depends not only on the cell type but also on the composition of the medium and whether the culture is incubated in light or (to a lesser extent) in the dark 41 – 43 . The buffering capacity and composition of the medium are important with regard to the behavior of the cultured cells and may affect significantly the results 44 .…”

“…The buffering capacity and composition of the medium are important with regard to the behavior of the cultured cells and may affect significantly the results 44 . In fact, it has been suggested that cell studies using different cell growth media are scarcely comparable 43 .…”

“…Modification in simple experimental variables, such as the choice of medium buffer, the control of exposure to light, the pH of the medium, and the amount of ROS in the medium 40 , 43 , as well as oxygen-controlled cultures 55 could arguably improve the outcomes from a biological point of view. If all of these factors were controlled, the results obtained from in vitro studies on angiogenesis would be more reproducible and thereby more useful for the design and interpretation of animal experiments and clinical trials.…”