Comparison of Cell Lines and Culture Models to Evaluate Toxicity of Pesticides and Pesticide Mixtures

There is a current shift in toxicology toward using in vitro cell-based assessments to screen environmental chemicals, such as pesticides, for their potential organ-specific toxicity as well as explore potential mechanisms of action. Many mammalian-derived cells lines are used to evaluate the organ-specific toxicity of chemicals; however, the ultimate use of the data obtained from these models in terms of translation to human toxicity remains challenging. Cell-based assays present distinct advantages and limitations, necessitating careful consideration by toxicologists when designing tests to address specific questions or interpret results. The primary goal of this thesis was to deepen our comprehension of cell-specific toxicity and in vitro assays. It is anticipated that understanding how individual factors impact variability in toxicity responses will help improve the accuracy and applicability of toxicity data derived from cell-based studies.\nFirst, we assessed the cytotoxic effects of pesticide chlorpyrifos (CPF), and its active metabolite (chlorpyrifos oxon, CPFO), across two human cell lines representative of liver (HepG2) and kidney (HK-2). The cytotoxicity to CPF and CPFO differed between cell lines, which was attributed to lower basal expression and inducibility of metabolizing enzymes, efflux transporters, and nuclear receptors in HK-2 cells. Co-exposure of CPF with specific inhibitors of efflux transporters enhanced CPF and CPFO cytotoxicity in HepG2 cells, indicating the role of these transporters in eliminating either CPF or CPFO. Co-incubation with transporter inhibitors also increased CPF accumulation in HepG2 cells, supporting the role of efflux transporters in elimination of CPF. These results underscore the crucial role of efflux transporter expression levels in selected cell lines for assessing the potential toxicity of environmental pollutants, such as pesticides.\nUnderstanding the role of efflux transporters in cell-specific toxicity responses to individual pesticides in cell lines prompted questions about their potential influence on mixture toxicity in vitro. Many pesticides act as substrates or inhibitors of efflux transporters; thus, their inhibitory effects could impede the elimination of other pesticides in a mixture, resulting in chemosensitization and increased cellular toxicity. We examined the combined toxicity of CPF with two other known P-glycoprotein (P-gp) inhibitor pesticides, endosulfan-α and heptachlor, in HepG2 cells. Binary mixtures of CPF with either endosulfan-α or heptachlor at concentrations causing less than 20% cytotoxicity (<IC20) were tested to assess interactive effects on P-gp-mediated toxicity mechanisms. CPF with endosulfan exhibited additive cytotoxicity, while CPF and heptachlor resulted in synergistic cytotoxicity in HepG2 cells. Our results show that pesticide mixtures had a greater ability to inhibit the efflux capacity than individual chemicals, and that pesticide interactions at efflux transporters may influence toxicokinetic and toxicity thresholds. The key finding of this chapter underscores the importance of considering the efflux transporters-mediated interaction of pesticides, particularly P-gp, in cell-based toxicity studies, given the prevalence of such interactions and exposure occurrence of pesticide mixtures.\nDifferent cell models not only show distinct toxicity responses to chemicals due to variations in toxicokinetic factors, but they may also differ in cellular proliferation and molecular pathways, leading to varying sensitivities. We then examined the responses of porcine-origin (IPEC-J2) and human colorectal adenocarcinoma origin (Caco-2) intestinal epithelial cells to the pesticide CPF. At a functional level, CPF disrupted the epithelial monolayer barrier in differentiated IPEC-J2 cells but not Caco-2 cells. Our results suggest that disrupted barrier function in IPEC-J2 cells may involve the transcellular pathway, indicated by decreased transepithelial electrical resistance (TEER) values and increased dextran permeability at cytotoxic concentrations of chlorpyrifos, with no changes in tight junction protein expressions. However, CPF did not affect the epithelial barrier function in Caco-2 cells, possibly due to the cancerous nature of the Caco-2 cell line. Our main conclusion was that CPF disrupts the intestinal barrier function in IPEC-J2 cells by affecting transcellular permeability pathway instead of through targeting tight junction proteins. \nIn conclusion, the results this research highlight the different responses of cells to certain pesticides. The differences in response were observed between cell line types, as well in single cell lines when exposed to pesticides individually or in combination. Such differences in response may arise from differences of cells in expression of transporters affecting pesticide toxicokinetics and other intrinsic molecular features. Differing expression levels of enzymes, transporters, and nuclear receptors across cell lines can influence the detoxification, cellular concentration, and toxicity of individual pesticides and their interactions. Enhanced understanding of the biochemical and molecular characteristics of commonly used cell lines will increase their applicability in assessing the toxicity of environmental pollutants and improve their correlation with real-life scenarios. The findings of this thesis research directly impact the choice of cell lines for evaluating pesticides and other environmental chemicals, highlighting concerns about interpreting cell-based assays in environmental hazard assessment.

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