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mRNA transfection retrofits cell-based assays with xenobiotic metabolism
Citation:
DeGroot, D., A. Swank, R. Thomas, M. Strynar, M. Lee, P. Carmichael, AND Steve Simmons. mRNA transfection retrofits cell-based assays with xenobiotic metabolism. JOURNAL OF PHARMACOLOGICAL & TOXICOLOGICAL METHODS. Elsevier Science Ltd, New York, NY, 92:77-94, (2018). https://doi.org/10.1016/j.vascn.2018.03.002
Impact/Purpose:
Many of the 700 cell-based and cell-free HTS assays currently in use in the EPA’s ToxCast program do not possess endogenous xenobiotic metabolic activity. Thus, the current screening assays only evaluate the biological activity of the parent chemical and not those of potential metabolites generated in metabolically-competent models or in vivo. This lack of metabolic capability can lead to the mischaracterization of chemical hazards, overestimating the toxicity of readily-metabolized parent chemicals and underestimating the hazards posed by toxic metabolites, and is a common criticism of using such in vitro approaches to predict in vivo toxicities. The work presented herein describes a novel approach to retrofit existing cell-based assays with metabolic competence using transfection of chemically-modified mRNAs.
Description:
The US EPA’s ToxCast program is designed to assess chemical perturbations of molecular and cellular endpoints using a variety of high-throughput screening (HTS) assays. However, existing HTS assays have limited or no xenobiotic metabolism which could lead to false positive (chemical is detoxified in vivo) as well as false negative results (chemical is bioactivated in vivo) and thus potential mischaracterization of chemical hazard. We have addressed this challenge by introducing the ten most prevalent human liver cytochrome P450 (CYP) enzymes into a human cell line (HEK293T) with low endogenous metabolic capacity. The CYP enzymes were introduced via transfection of modified mRNAs as singlets or as a mixture in relative proportions expressed in the liver. Initial experiments using luminogenic CYP450 substrates demonstrate that cell models express metabolic enzymes from the transfected mRNAs and activities are significantly increased when co-transfected with a CYP accessory protein, P450 oxidoreductase (POR). Transfected HEK293T cells demonstrate the ability to produce predicted metabolites following treatment with well-studied CYP substrates, with metabolite formation occurring through 18 hours post-treatment. As a demonstration of how this method can be used to retrofit existing HTS assays, a proof-of-concept screen for cytotoxicity in HEK293T cells was conducted using 56 test compounds. The results demonstrate that the xenobiotic metabolism conferred by transfection of CYP-encoding mRNAs shifts the dose-response relationship for certain test chemicals such as aflatoxin B1 (bioactivation) and fenazaquin (detoxification). Overall, transfection of CYP-encoding mRNAs is an effective and portable solution for retrofitting metabolic competence to existing cell-based HTS assays.
