Glutathione peroxidase inhibitory assay for electrophilic pollutants in diesel exhaust and tobacco smoke

Abstract

We developed a rapid kinetic bioassay demonstrating the inhibition of glutathione peroxidase 1 (GPx-1) by organic electrophilic pollutants such as acrolein, crotonaldehyde, and p-benzoquinone that are frequently found as components of tobacco smoke, diesel exhaust, and other combustion sources. In a complementary approach, we applied a high-resolution proton-transfer reaction time-of-flight mass spectrometer (PTR-ToF-MS) to monitor in real-time the generation of electrophilic volatile carbonyls in cigarette smoke. The new bioassay uses the important antioxidant selenoenzyme GPx-1, immobilized to 96-well microtiter plates, as a probe. The selenocysteine bearing subunits of the enzyme's catalytic site are viewed as cysteine analogues and are vulnerable to electrophilic attack by compounds with conjugated carbonyl systems. The immobilization of GPx-1 to microtiter plate wells enabled facile removal of excess reactive inhibitory compounds after incubation with electrophilic chemicals or aqueous extracts of air samples derived from different sources. The inhibitory response of cigarette smoke and diesel exhaust particle extracts were compared to chemical standards of a group of electrophilic carbonyls and the arylating p-benzoquinone. GPx-1 activity was directly inactivated by millimolar concentrations of highly reactive electrophilic chemicals (including acrolein, glyoxal, methylglyoxal, and p-benzoquinone) and extracts of diesel and cigarette smoke. We conclude that the potential of air pollutant components to generate oxidative stress may be, in part, a result of electrophile-derived covalent modifications of enzymes involved in the cytosolic antioxidant defense.

Electrophiles modulate glutathione reductase activity via alkylation and upregulation of glutathione biosynthesis

“…In contrast to GRX, expression of GPX1 has been found to be dependent on Nrf2 activity and is responsive to electrophiles [58]. In addition, electrophilic pollutants from diesel exhaust and tobacco smoke have previously been found to inhibit GPX activity [67]. While expression of GPX is known to be dependent on Nrf2, in our experiments NO 2 -OA did not affect cell sensitivity to peroxide exposure, suggesting that activity of GPX is not impacted by this electrophile.…”

Predicting the Cytotoxic Potency of Cigarette Smoke by Assessing the Thioredoxin Reductase Inhibitory Capacity of Cigarette Smoke Extract

“…Harmful low molecular–weight carbonyl compounds, such as dicarbonyls including glyoxal and methylglyoxal, are formed in cigarette smoke [ 34 ]. Glyoxal and methylglyoxal have been demonstrated to inactivate GPx activity through the electrophilic attack of Sec residue [ 35 ]; therefore, it is believed that glyoxal and methylglyoxal would preferentially inhibit TrxR at low concentrations according to the present results ( Figure 2 ). Glutathione-crotonaldehyde adducts have been identified from cells exposed to cigarette smoke [ 36 ]; thus, it is conceivable that crotonaldehyde, a highly reactive α,β-unsaturated aldehyde and a major harmful compound found in CSE, would also inactivate TrxR because Sec residue in selenoproteins can react 10 7 times faster with electrophiles than free cysteine [ 37 ].…”

Decreased photosynthetic rate under high temperature in wheat is due to lipid desaturation, oxidation, acylation, and damage of organelles

“…The absorbance by background was subtracted from the sample to get the actual change in absorbance due to the enzyme in the sample. One unit enzyme will oxidize 1 nmol of NADPH to NADP + min − 1 on leaf fresh weight basis [ 59 ].…”