Immunoaffinity-Based Mass Spectrometry ― a Method for the Analysis of Combinatorial Pesticide Effects on Liver Proteins in HepaRG Cells

Abstract

Pesticides are used worldwide to protect plants and fields as it is a necessity to provide sufficient and safe food. When these substances shall be approved, their toxicological effects must be investigated first. However, as today, not only individual pesticides but also pesticide mixtures are widely applied, consumers come into contact with a variety of pesticide residues. Therefore, it is essential to investigate potential mixture effects. If all potential mixtures were tested with the standard, mandatory toxicological tests that must be carried out for approval, the number of animal tests would increase dramatically. Hence, this work aimed to develop an in vitro test system to investigate the influence of potential mixing effects on toxicologically relevant liver proteins. Method development was initially started for 36 proteins. A total of six multiplex assays with 16 different proteins were successfully developed. In addition, a CYP 17-plex assay method already developed in targeted selected ion monitoring (tSIM) was transferred to the parallel reaction monitoring (PRM) like the six other multiplex assays. This resulted in the total number of 32 proteins used for the analysis of potential mixing effects in HepaRG cells. During method development, it was shown that immune precipitation and the use of targeted mass spectrometric methods could significantly increase the number of analytes detected (especially for the CYPs from 5/17 to 14/17) compared to full-scan analysis. These assays were used to investigate the effect of 30 individual pesticides on 32 proteins in HepaRG cells. These pesticides were also investigated at the mRNA level by a collaboration partner. Similarities have been observed between the two molecule species, but the effect was more pronounced at the mRNA level after 24 hours of treatment. By a similarity analysis using Pearson correlation, the pesticides were classified into four groups, and then four mixtures of five single substances were prepared. Based on the mRNA/protein expression result comparison, it was additionally decided that the treatment periods should be extended to 24, 48, and 72 hours. A novel mathematical model was developed to determine whether combinatorial effects such as antagonism, additivity, or synergism were observed after mixture treatment. For the very first time, multiple linear regression analysis was used to identify combinatorial effects after pesticide treatment. The results of the individual treatments were used to obtain theoretical model values of the respective mixture. These proposed values were then compared with the measured values from the mixture experiments. According to this model, the results showed that most mixtures had an additive effect on the different proteins analyzed. However, in some cases, synergistic or antagonistic effects were observed. Most model deviations that indicate synergistic/antagonistic effects were obtained for the three analytes S100P, followed by CYP2B6, and CYP1A2, suggesting them as the most sensitive sensors for detecting these effects.