Xanthine oxidase‐generated hydrogen peroxide is a consequence, not a mediator of cell death

Abstract

Oxidative stress has been associated with a wide range of diseases including atherosclerosis, cancer and Alzheimer’s disease. When present in excessive concentrations, reactive oxygen species (ROS) can cause deleterious effects. This has led to the notion that the anticancer effects of various chemotherapeutics may be mediated, at least in part, by an increase in ROS. To investigate the role of xanthine oxidase (XO), a source of hydrogen peroxide, in cell death, MCF7, HeLa and 293T cells were treated with various cell‐death‐inducing drugs in the presence and absence of allopurinol, a specific inhibitor of XO. In the absence of allopurinol, each drug led to a time‐ and concentration‐dependent increase in percent DNA fragmentation and ROS levels, regardless of the mechanism of cell death incurred, i.e. caspase dependent and caspase independent. By contrast, pretreatment with allopurinol led to dramatically lower ROS levels in all cases, suggesting that XO is a major contributor to oxidative stress. However, allopurinol did not exhibit a protective effect against cell death. Similarly, the administration of siRNA against XO also did not exhibit a protective effect against cell death. The level of oxidative stress was recorded using the ROS sensor CM‐H2DCFDA and a ratiometric bioluminescent assay that takes advantage of the increased sensitivity of Firefly luciferase to hydrogen peroxide, compared with a stable variant of Renilla luciferase (RLuc), RLuc8. Overall, these findings suggest that XO‐generated hydrogen peroxide, and perhaps hydrogen peroxide in general, is a consequence, but not a mediator of cell death.

Reactive Oxygen Species in Pathogenesis of Atherosclerosis

“…Moreover, XDH can also generate superoxide when NAD + is in deficit (for example, during inflammation and concomitant hypoxic microenvironment of the enzyme, though before XDH transforms to XO) [68,69]. According to recent data, XO is a major contributor to oxidative stress [70]. In the atherosclerotic plaque, XO-mediated ROS formation is pro-inflammatory and XOinhibition by febuxostat is a potential therapy for atherosclerosis [71].…”

“…In the atherosclerotic plaque, XO-mediated ROS formation is pro-inflammatory and XOinhibition by febuxostat is a potential therapy for atherosclerosis [71]. However, allopurinol or siRNA against XO does not exhibit a protective effect against cell death suggesting that XO-generated H2O2, and perhaps H2O2 in general, is a consequence but not a mediator of cell death [70].…”

Thiol-disulphide independent in-cell trapping for the identification of peroxiredoxin 2 interactors

“…As in this study we specifically aim to find H 2 O 2 -mediated Prdx2 interactors, we sought to establish a system that would lead to elevated H 2 O 2 levels in HEK293T cells over the 24 h required for maximum biotinylation [ 55 ]. We decided to test the xanthine/xanthine oxidase (X/XO) H 2 O 2 generating system [ 56 , 57 ], and auranofin, a TrxR inhibitor [ 58 ] which blocks the electron transfer from NADPH to thioredoxin, and as a consequence to all the thioredoxin-dependent peroxidases, including Prdx2. Since reduced Prdx2 is one of the most efficient and abundant intracellular H 2 O 2 scavengers [ 59 ], this is expected to lead to an increase in intracellular H 2 O 2 levels.…”

Unraveling the in vitro antitumor activity of Vismia baccifera against HepG2: role of hydrogen peroxide

“…baccifera . Recently, it has been reported that H 2 O 2 generated by xanthine oxidase did not cause, but was rather the result of cell death in various cancer cell types [26] .…”