Detailed evaluation of a cellular in vitro method to assess oxidative potential of atmospheric aerosols as performed by two independent laboratories

Abstract

Various methods have been applied to quantify oxidative stress induced by atmospheric particle matter (PM), which are often employing different principles, dissimilar positive controls, normalization techniques and reporting units, as well as displaying quite diverse sensitivities. To date, no universal method has been accepted and the need remains to identify and validate a method that could easily be utilized across varied study designs, different types of samples, and be reproducible between different laboratories. In this study we propose and demonstrate the utility of a measure of biological ROS response utilizing a broad ROS probe (DCFH-DA) in an in vitro rat alveolar macrophage system. A method comparison and validation experiments were conducted in two independent laboratories. Method performance was characterized using extracts of ambient PM as well as a variety of positive control materials. We demonstrate that our method can yield very good reproducibility, sensitivity and accuracy. The coefficient of variation (CV) of the untreated control group and positive controls ranged from 8% to 11% and 7%–13%, respectively, while the average signal to noise ratio (SNR) of untreated (negative) control group and positive controls for both labs was larger than 3. Comparing the dose response relationship for multiple positive controls between the two labs, the NIST reference material (Urban Dust) extract appeared to be the most consistent reference material for the ROS measurement (R2 = 0.996). The relative percent difference (RPD) of ROS activities as measured by two different labs and expressed as equivalents of Urban Dust was only 14%, which suggests that as the optimal normalization method. Our results indicate the macrophage-DCFH-DA method is effective in assessing the biological oxidative potential of PM exposure in different laboratories and should be utilized for cross-comparison of biological ROS response between different studies.