Redox cycling and increased oxygen utilization contribute to diquat-induced oxidative stress and cytotoxicity in Chinese hamster ovary cells overexpressing NADPH-cytochrome P450 reductase

Abstract

Diquat and paraquat are non-specific defoliants that induce toxicity in many organs including the lung, liver, kidney and brain. This toxicity is thought to be due to the generation of reactive oxygen species (ROS). An important pathway leading to ROS production by these compounds is redox cycling. In the present studies, diquat and paraquat redox cycling was characterized using human recombinant NADPH-cytochrome P450 reductase, rat liver microsomes, and Chinese Hamster Ovary (CHO) cells constructed to overexpress cytochrome P450 reductase (CHO-OR) and wild type control cells (CHO-WT). In redox cycling assays with recombinant cytochrome P450 reductase and microsomes, diquat was 10-40 times more effective in generating ROS when compared to paraquat (KM = 1.0 and 44.2 μM, respectively for H2O2 generation by diquat and paraquat using recombinant enzyme, and 15.1 and 178.5 μM, respectively for microsomes). In contrast, at saturating concentrations, these compounds showed similar redox cycling activity (Vmax ≈ 6.0 nmoles H2O2/min/mg protein) for recombinant enzyme and microsomes. Diquat and paraquat also redox cycle in CHO cells. Significantly more activity was evident in CHO-OR cells than CHO-WT cells. Diquat redox cycling in CHO cells was associated with marked increases in protein carbonyl formation, a marker of protein oxidation, as well as cellular oxygen consumption, measured using oxygen microsensors; greater activity was detected in CHO-OR cells than CHO-WT cells. These data demonstrate that ROS formation during diquat redox cycling can generate oxidative stress. Enhanced oxygen utilization during redox cycling may reduce intracellular oxygen available for metabolic reactions and contribute to toxicity.

CYP3A4 overexpression enhances apoptosis induced by anticancer agent imidazoacridinone C-1311, but does not change the metabolism of C-1311 in CHO cells

“…The higher sensitivity to C-1311 and increased susceptibility to apoptosis observed in CHO-HR cells compared to CHO-WT cells following drug treatment could be explained by the fact that overexpression of cytochrome P450 reductase itself increased the level of free radicals (ROS) in the cell [30] . We showed here that C-1311 induced ROS in CHO-HR cells but not in wild-type CHO cells.…”

“…There is some evidence indicating that overexpression of cytochrome P450 reductase itself induces a higher level of reactive oxygen species (ROS) [30] . Stimulation of the formation of ROS can lead to oxidative stress, which is involved in the induction of apoptosis [31] .…”

Sepiapterin Reductase Mediates Chemical Redox Cycling in Lung Epithelial Cells

“…In these assays, 4.5 g of recombinant sepiapterin reductase/100 l of reaction mix were used. In some experiments, oxygen utilization in enzyme reactions during redox cycling was measured using a Clark-type oxygen electrode as described previously (27). In these assays, 16 g of recombinant sepiapterin reductase/0.8 ml of reaction mix were used.…”

On the methodological limitations of detecting oxidative stress: effects of paraquat on measures of oxidative status in greenfinches

“…Redox cycling and consequent ROS generation are thought to be key cytotoxic mechanisms induced by these bipyridyl herbicides (reviewed by Fussell et al, 2011). During redox cycling, diquat and PQ undergo an enzymatic one-electron reduction, forming radical cations.…”