Evaluation of Metabolism of a Defined Pesticide Mixture through Multiple In Vitro Liver Models

Abstract

The evaluation of exposure to multiple contaminants in a mixture presents a number of challenges. For example, the characterization of chemical metabolism in a mixture setting remains a research area with critical knowledge gaps. Studies of chemical metabolism typically utilize suspension cultures of primary human hepatocytes; however, this model is not suitable for studies of more extended exposures and donor-to-donor variability in a metabolic capacity is unavoidable. To address this issue, we utilized several in vitro models based on human-induced pluripotent stem cell (iPSC)-derived hepatocytes (iHep) to characterize the metabolism of an equimolar (1 or 5 µM) mixture of 20 pesticides. We used iHep suspensions and 2D sandwich cultures, and a microphysiological system OrganoPlate® 2-lane 96 (MimetasTM) that also included endothelial cells and THP-1 cell-derived macrophages. When cell culture media were evaluated using gas and liquid chromatography coupled to tandem mass spectrometry methods, we found that the parent molecule concentrations diminished, consistent with metabolic activity. This effect was most pronounced in iHep suspensions with a 1 µM mixture, and was lowest in OrganoPlate® 2-lane 96 for both mixtures. Additionally, we used ion mobility spectrometry–mass spectrometry (IMS-MS) to screen for metabolite formation in these cultures. These analyses revealed the presence of five primary metabolites that allowed for a more comprehensive evaluation of chemical metabolism in vitro. These findings suggest that iHep-based suspension assays maintain higher metabolic activity compared to 2D sandwich and OrganoPlate® 2-lane 96 model. Moreover, this study illustrates that IMS-MS can characterize in vitro metabolite formation following exposure to mixtures of environmental contaminants.

Reanalysis of Trichloroethylene and Tetrachloroethylene Metabolism to Glutathione Conjugates Using Human, Rat, and Mouse Liver in Vitro Models to Improve Precision in Risk Characterization

“… 58 Although the opportunities are many, the challenges are also formidable, including the cost, complexity, and low throughput of most of the available models. 38 , 59 , 60 …”

“…These models also improved our ability to collect and interpret pharmacokinetic data. 35 – 38 One liver model that showed great potential for in vitro metabolism studies is a micropatterned coculture (MPCC) system. 39 The MPCC platform has been shown to maintain the viability and metabolic activity of hepatocytes from rodents and humans for several weeks.…”

Reproducibility and Robustness of a Liver Microphysiological System PhysioMimix LC12 under Varying Culture Conditions and Cell Type Combinations

“…The collected media was processed using high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) to determine the concentration of both the remaining parent compound (MDZ) and metabolites formed (1 -OH MDZ and 1 -OH MDZ glucuronide). We also tested the metabolism of equimolar mixtures (1 or 5 µM) of 20 pesticides that were previously used to evaluate the metabolic competence of different in vitro liver models [44]. Cells were treated on day 6 of culture, and media were collected for analysis 48 h thereafter.…”

“…We also examined the metabolism of a mixture of 20 pesticides from different chemical classes and of different hydrophobicity with the HU8373 PHH mono-culture condition in the PhysioMimix LC12 and compared this to the published results of experiments with iHeps suspensions, 2D cultures, and OrganoPlate ® 2-lane 96 experiments [44]. We examined metabolism in a mixture setting because previous studies using suspensions of PHHs showed that hepatocyte metabolic clearance was slower under mixture conditions when compared to the individual incubations with these pesticides [47].…”

Hepatocellular Metabolic Abnormalities Induced by Long-Term Exposure to Novel Brominated Flame Retardant, Hexabromobenzene

“…Combining metabolomics with in vitro experiments has the advantage of substituting animal testing and enabling a rapid identification of the toxicological properties of new compounds [ 26 , 27 ]. In particular, hepatic in vitro studies are widely conducted to evaluate the metabolic mechanisms related to toxicity [ 28 ] because the human hepatoma cell line maintains the activity of various enzymes that play an important role in activation and detoxification [ 29 ]. In a nutshell, in vitro metabolomics of hepatic cells have the potential to explain the effect of exposure to toxic substances [ 26 , 30 ].…”