Analytical and Bioanalytical Chemistry

, Volume 403, Issue 2, pp 431–441 | Cite as

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

  • Norbert Staimer
  • Tran B. Nguyen
  • Sergey A. Nizkorodov
  • Ralph J. Delfino
Original Paper


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 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 with 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.


Cu/Zn superoxide dismutase (SOD-1) and glutathione peroxidase (GPx) are linked together in the cytosolic defense against reactive oxygen and nitrogen species (RONS). Cu/Zn-SOD catalyzes the dismutation of superoxide to oxygen and hydrogen peroxide (H2O2). H2O2 and other hydroperoxides are subsequently reduced by the selenoenzyme GPx. The selenofunction is viewed as a cysteine analogue, and in comparison to other thiol enzymes, is even more vulnerable to electrophilic attack by chemicals such as acrolein at physiological conditions. Cu/Zn-SOD and GPx team up with a complex cellular antioxidant system that includes catalase, glutathione transferase and reduced glutathione (not shown). Environmental exposure to reactive electrophiles present in cigarette smoke and diesel exhaust emissions may add to the endogenous burden of oxidative stress by direct inactivation of GPx


Air pollution Electrophiles Antioxidant enzymes Oxidative stress 



This work was supported by the US Environmental Protection Agency (USEPA) STAR Grant No. RD83241301, National Institute of Environmental Health Sciences (grant no. R01 ES12243), and UCI Multi-Investigator Faculty Research Grant MI 7 2008–2009. We are grateful to Professor Arthur Cho, University of California, Los Angeles, for useful comments after reading this manuscript, helpful discussions, and for the kind gift of DEP samples.


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Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Norbert Staimer
    • 1
  • Tran B. Nguyen
    • 2
  • Sergey A. Nizkorodov
    • 2
  • Ralph J. Delfino
    • 1
  1. 1.Department of Epidemiology, School of MedicineUniversity of CaliforniaIrvineUSA
  2. 2.Department of ChemistryUniversity of CaliforniaIrvineUSA

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