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2609657 
Journal Article 
Abstract 
A new assay to screen for the inhibitory capacity of air pollutant components on antioxidant enzyme activities 
Staimer, N; Cho, A; Delfino, RJ 
2010 
Yes 
American Journal of Respiratory and Critical Care Medicine
ISSN: 1073-449X
EISSN: 1535-4970 
181 
A1159 
English 
WOS:000208771000160 
is part of a larger document 3452678 Proceedings of the American Thoracic Society 2010 International Conference, May 14-19, 2010, New Orleans
Rationale: Toxicological evidence is mounting that air pollution induces inflammatory and oxidative stress responses by reactive chemicals present in particulate and volatile fractions. More specifically, air pollutant-derived electrophiles may irreversibly alter antioxidant enzymes. Diminished antioxidant response has been linked to aging and to chronic disorders including asthma likely resulting from oxidative damage of cellular components. Therefore, we developed a rapid kinetic bioassay to measure the enzyme inhibitory capacity of electrophilic components present in air and air particulate suspensions. We investigated the antioxidant selenoenzyme glutathione peroxidase (GPx-1), which is vulnerable to electrophilic attack. We previously reported that traffic-related air pollution was associated with decreased GPx-1 activity in the blood of elderly subjects followed in a longitudinal study.

Methods: GPx-1 was tested as a bioassay probe of the capacity of PM to inactivate enzyme activity under the following conditions: In a 96-well microtiter plate format, immobilized bovine erythrocyte GPx-1 was incubated (1 hr, at room temperature) with different electrophilic α,β-unsaturated aldehydes that are frequently found as components of tobacco smoke, diesel exhaust, and other combustion products. The excess of inhibitors was removed in each well with assay buffer. Enzyme activity was determined by adding a mixture of GPx-1 co-substrate and cumene hydroperoxide and measuring the decrease in absorbance at 340 nm. Additionally, aqueous extracts of 24 hr PM2.5 samples (collected on quartz filters using a Harvard Impactor in a heavily trafficked urban area of Los Angeles) were evaluated for their inhibitory effects on GPx-1 activity. Effects of PM2.5 on GPx-1 activity were expressed as μg/mL EDTA-phosphate buffer. The extracted particulate suspensions were spun at 18,000 g 15 min at 4°C before analysis.

Results: Figure 1 shows the inhibition of GPx-1 by three different electrophilic compounds (acrolein, crotonaldehyde, and p-benzoquinone) in the low mmolar range. GPx-1 results are the average of duplicate samples (variation between duplicates at 1mM: <25%, at 10mM: <16%, at 100mM: <28%). Figure 2 demonstrates the direct inhibitory effect of air particle extracts on the activity of the antioxidant enzyme GPx-1.

Conclusion: A high throughput assay has been developed to screen for the inhibitory capacity of air pollutant components. Moreover, the direct inhibition of GPx-1 activity by both highly reactive electrophilic chemicals and air particulate extracts suggests the potential of ambient air pollutants to generate oxidative stress by electrophile-derived covalent modifications of enzymes involved in the cytosolic defense against reactive oxygen and nitrogen species. 
American Thoracic Society 2010 International Conference 
New Orleans, LA 
May 14-19, 2010