Regulation of intracellular glutathione.

It is well recognized that the cellular effects of reactive electrophiles and reactive oxygen species (ROS) are inter-related, and that glutathione plays a central role in the regulation and detoxification of both. For example, a number of processes including lipid peroxidation involve ROS and generate electrophilic products, thiol groups are favored targets of both classes of reactant, and one of the downstream effects of electrophiles is to upregulate expression of antioxidant defenses. How electrophiles affect the cellular glutathione system is addressed in an article by Schopfer and colleagues in this issue of Redox Biology, which focuses on the lipid-based electrophile, nitro oleic acid (NOA; Fig. 1). NOA is one of the nitroalkenes formed from the reaction of nitric oxide metabolites with unsaturated fatty acids. It is formed during inflammation and in the acidic environment of the gut, and is reported to have anti-inflammatory properties [1].

Fig. 1

Reaction of 9-nitro oleic acid with a thiol protein (Pr-SH).

In their mechanistic study, Schopfer and colleagues treated RAW macrophages with NOA and observed an increase in cellular GSH content after an initial drop, as well as marked increases in GSSG and GSSG: GSH ratio. The GSH changes are explicable in terms of conjugation of NOA to GSH causing partial depletion, plus the activation of the Nrf2 system and an increase in expression of γ-glutamyl-cysteine ligase, the rate limiting enzyme of GSH synthesis. These effects of NOA have been observed [2] and are common electrophile responses [3]. The increase in GSSG was more surprising and was substantially greater than with the other electrophiles they tested. It appeared unrelated to oxidative stress, as the redox state of peroxiredoxins 1 and 3 (present in the cytoplasm and mitochondria respectively) were unaffected. Therefore, they explored other possible explanations for the NOA-induced increase in cellular GSSG. For this they studied the interaction of electrophiles with isolated glutathione-metabolizing enzymes. Essentially what they found was that NOA has a selective affinity for glutathione reductase, forming a covalent adduct with the active site cysteine and inhibiting the enzyme. They also followed up on early work showing that glutathione reductase is finely regulated by product inhibition by GSH [4], to demonstrate that a rise in GSH concentration such as observed in cells after Nrf2 induction can attenuate the ability of the enzyme to reduce GSSG. They then incorporated all these mechanisms for increasing GSH production and inhibiting GSSG reduction into a theoretical kinetic model and showed that with a complex interplay between them it is possible to simulate the effects of NOA on cellular glutathione.

In addition to demonstrating an apparently selective ability of NOA to inhibit glutathione reductase, this study is instructive in showing that glutathione homeostasis can be modulated in a complex way by electrophile exposure. It is also an example where different electrophiles may have a similar ability to activate the Nrf2 pathway, yet not necessarily giving the same responses. For example, whereas NOA readily inhibited glutathione reductase and caused excessive cellular GSSG accumulation, the other electrophiles tested, including dimethyl fumarate, caused a more modest increase in GSSG and were not potent glutathione reductase inhibitors. However, there is still much to be learned about electrophile-glutathione interactions, including how widely each regulatory mechanism is applicable. Induction of glutathione synthesis through the activation of Nrf2 is a common electrophile response, and associated product inhibition of glutathione reductase should therefore be a likely consequence. However, the selectively of NOA for glutathione reductase compared with other potential reactive proteins needs to be more widely tested before concluding that it is a favored cellular target. Also, the explanation for glutathione reductase inhibition being responsible for the large increase in GSSG in NOA-treated cells is indirectly based on simulations, and needs direct confirmation with cellular measurements. The proposal that NOA selectively targets glutathione reductase also raises the possibility that this may be a feature of a subgroup of electrophiles and this warrants further investigation.

The glutathione system has long been recognized as important for removal of hydrogen peroxide and other reactive oxidants, and for regulating redox processes. However, how the system is viewed has evolved as we learn more about redox regulation. For a long time glutathione was considered as a redox buffer, equilibrating with other thiol disulfide couples and thus controlling their oxidation status. It is now appreciated that cellular redox changes are driven much more by kinetics than thermodynamics, so although different redox systems interact, they are not in equilibrium with one another [5], [6]. This is apparent in the present study where a more oxidized glutathione system was not paralleled by an increase in peroxiredoxin oxidation. Another important consideration is what influences the GSSG:GSH ratio. It is common to interpret an increase as being due to oxidative stress but, as seen with NOA, this is not necessarily the case and can occur without any need for oxidant generation.