Association of NQO2 With UDP-Glucuronosyltransferases Reduces Menadione Toxicity in Neuroblastoma Cells

Abstract

The balance between detoxification and toxicity is linked to enzymes of the drug metabolism Phase I (cytochrome P450 or oxidoreductases) and phase II conjugating enzymes (such as the UGTs). After the reduction of quinones, the product of the reaction, the quinols—if not conjugated—re-oxidizes spontaneously to form the substrate quinone with the concomitant production of the toxic reactive oxygen species (ROS). Herein, we documented the modulation of the toxicity of the quinone menadione on a genetically modified neuroblastoma model cell line that expresses both the quinone oxidoreductase 2 (NQO2, E.C. 1.10.5.1) alone or together with the conjugation enzyme UDP-glucuronosyltransferase (UGT1A6, E.C. 2.4.1.17), one of the two UGT isoenzymes capable to conjugate menadione. As previously shown, NQO2 enzymatic activity is concomitant to massive ROS production, as previously shown. The quantification of ROS produced by the menadione metabolism was probed by electron-paramagnetic resonance (EPR) on cell homogenates, while the production of superoxide was measured by liquid chromatography coupled to mass spectrometry (LC-MS) on intact cells. In addition, the dysregulation of the redox homeostasis upon the cell exposure to menadione was studied by fluorescence measurements. Both EPR and LCMS studies confirmed a significant increase in the ROS production in the NQO2 overexpressing cells due to the fast reduction of quinone into quinol that can re-oxidize to form superoxide radicals. However, the effect of NQO2 inhibition was drastically different between cells overexpressing only NQO2 vs. both NQO2 and UGT. Whereas NQO2 inhibition decreases the amount of superoxide in the first case by decreasing the amount of quinol formed, it increased the toxicity of menadione in the cells co-expressing both enzymes. Moreover, for the cells co-expressing QR2 and UGT the homeostasis dysregulation was lower in presence of menadione than for the its counterpart expressing only QR2. Those results confirmed that the cooperation of the two enzymes plays a fundamental role during the cells’ detoxification process. The fluorescence measurements of the variation of redox homeostasis of each cell line and the detection of a glucuronide form of menadiol in the cells co-expressing NQO2 and UGT1A6 enzymes further confirmed our findings.

Polymorphisms and Pharmacogenomics of NQO2: The Past and the Future

“…To rule out this possibility, one has to recall that NQO2 activity can be recorded in the presence of added NRH and towards a large series of quinone substrates [10,37]. More importantly, expressing both NQO2 and the main Phase II enzyme, the conjugating UDP-glucuronosyltransferase (UGT) in SH-SY5Y cells, it was possible to demonstrate the appearance of menadione glucuronides if NQO2 was expressed and incubated in the presence of its co-substrate, while in the absence of NRH, menadione glucuronide remained undetectable, strongly suggesting that the reduction of menadione to its diol was enough to permit UGT to conjugate it [9]. We also hypothesized such a role in the UGT/NQO2 activity balance [38].…”

“…This peculiar role of NQO2 was observed in astrocytes treated with neurotoxins such as paraquat, 6-OH-dopamine and MPTP, but not in dopaminergic SH-SY5Y cells [11][12][13][14]. Thus, the current view is that NQO2 has a dual nature, either detoxifying or toxifying depending on the substrate, cell type, expression and activity of other detoxifying enzymes such as UDP-glucuronosyltransferase [1,9]. In addition, the detoxifying or toxic effect of the NQO2 activity might be determined by amino acid sequence differences in polymorphic variants and orthologs as discussed below.…”

“…In fact, the chemical reactions carried by NQO2 involve a "ping-pong" two-electron transfer to quinone substrates [6,7]. Such a reduction of quinones produces variably stable hydroquinones that can be eliminated by conjugation with sulfates and glucuronides by different II-phase drug metabolic enzymes and excreted as conjugates [8,9]. This mechanism was initially proposed as a safe way of reducing quinones without the generation of semiquinones and ROS, but challenged by more recent findings suggesting that NQO2 can produce free radicals as a by-product of spontaneous autooxidation of hydroquinones [10].…”

Bioactive compounds of Curvularia species as a source of various biological activities and biotechnological applications

“…Quinones are a ubiquitous class of compounds that are commonly found in various natural products and synthesized through hydroquinones and/or catechols metabolism (Chhour et al, 2021 ). Quinones also act as toxicological intermediates which have many hazardous effects including acute cytotoxicity, carcinogenesis, and immunotoxicity (Bolton et al, 2000 ).…”