In vitro screening for population variability in toxicity of pesticide-containing mixtures

Abstract

Population-based human in vitro models offer exceptional opportunities for evaluating the potential hazard and mode of action of chemicals, as well as variability in responses to toxic insults among individuals. This study was designed to test the hypothesis that comparative population genomics with efficient in vitro experimental design can be used for evaluation of the potential for hazard, mode of action, and the extent of population variability in responses to chemical mixtures. We selected 146 lymphoblast cell lines from 4 ancestrally and geographically diverse human populations based on the availability of genome sequence and basal RNA-seq data. Cells were exposed to two pesticide mixtures - an environmental surface water sample comprised primarily of organochlorine pesticides and a laboratory-prepared mixture of 36 currently used pesticides - in concentration response and evaluated for cytotoxicity. On average, the two mixtures exhibited a similar range of in vitro cytotoxicity and showed considerable inter-individual variability across screened cell lines. However, when in vitro-to-in vivo extrapolation (IVIVE) coupled with reverse dosimetry was employed to convert the in vitro cytotoxic concentrations to oral equivalent doses and compared to the upper bound of predicted human exposure, we found that a nominally more cytotoxic chlorinated pesticide mixture is expected to have greater margin of safety (more than 5 orders of magnitude) as compared to the current use pesticide mixture (less than 2 orders of magnitude) due primarily to differences in exposure predictions. Multivariate genome-wide association mapping revealed an association between the toxicity of current use pesticide mixture and a polymorphism in rs1947825 in C17orf54. We conclude that a combination of in vitro human population-based cytotoxicity screening followed by dosimetric adjustment and comparative population genomics analyses enables quantitative evaluation of human health hazard from complex environmental mixtures. Additionally, such an approach yields testable hypotheses regarding potential toxicity mechanisms.

Keywords: Mixture; Pesticide; Population.

Fig. 1

Inter-individual and population variability and reproducibility of the cytotoxicity of pesticide-containing mixtures in human lymphoblast cell lines. (a) A population concentration response was modeled using in vitro cytotoxicity of the chlorinated pesticide mixture (top) and the current use pesticide mixture (bottom). Logistic dose–response modeling was applied to each individual cell line, with individual data shown by thin gray lines. Bars represent a histogram of the individual EC₁₀ values, and the dashed curve represents the fit of the logistic model to the pooled data. A histogram in each graph depicts a frequency distribution (y-axis) for the cell lines with a corresponding EC₁₀ (x-axis is identical to that already displayed). (b) Intra-experimental reproducibility of EC₁₀ values for within-batch replicate plates for cell lines for the chlorinated pesticide mixture (top) and the current use pesticide mixture (bottom). Spearman and Pearson's correlation coefficients are shown.

Fig. 2

Distribution of EC₁₀s across 146 cell lines for each mixture. (a) A density plot for the distribution and mean of EC₁₀ of each pesticide mixture (red: chlorinated pesticide mixture, blue: current use pesticide mixture) across 146 cell lines. (b) Box plots (box represents first and third quartiles; vertical line inside the box, the median; whiskers are the 1.5 inter-quantile range; circles are outliers with >1.5 IQR above minimum or maximum). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Comparative analysis of the mixtures. (a) Scatter plot comparison of EC₁₀ values of each cell line between pesticide mixtures. Symbols represent populations as shown in the inset. Pearson and Spearman correlations are also shown. (b) Scatter plot of 1st and 2nd principal components of the molecular descriptors of the individual chemicals in each pesticide mixture. (c) Frequency histogram of 15,931 pair-wise correlation values (Spearman) among 179 chemicals screened in (Abdo et al., 2015). The green dashed line represents a median ρ value for all correlations, and the red dashed line represents pairwise correlation of pesticide mixtures. Blue shading represents non-significant correlations after correction for false discoveries. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

In vitro-to-in vivo extrapolation of cytotoxicity EC₁₀ values. Box plots (box represents first and third quartiles; horizontal line inside the box is the median; whiskers are the 1.5 inter-quantile range; circles are outliers with >1.5 IQR above minimum or maximum) of the cumulative oral doses for each pesticide mixture (red: chlorinated pesticide mixture, blue: current use pesticide mixture) across 146 cell lines in four different scenarios for handling missing data, weighted by chemical percentage in the mixture or not (“equi-weighted”), and assuming the “worst case scenario” (WCS) vs median for missing values. Red and blue dotted horizontal lines indicate the estimated cumulative human oral exposure levels to each pesticide mixture. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

Genome-wide association analysis of population variability in cytotoxicity of the current use pesticide mixture. (a) Manhattan plot of MAGWAS −log10(p) vs. genomic position for association of genotype and cytotoxicity to current use pesticide mixture. The dashed blue line indicates suggestive association (expected once per genome scan). A LocusZoom plot of the most significant (p = 6.5 × 10⁻⁸) region at SNP rs1947825. (b) Average concentration–response profiles of cytotoxicity of current use pesticide mixture plotted separately for each genotype at rs1947825. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)