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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. To address this challenge, the ten most prevalent human liver cytochrome P450 (CYP) enzymes were introduced into a human cell line (HEK293T) with low endogenous metabolic capacity. The CYP enzymes were introduced via transfection of modified mRNAs as either singlets or as a mixture in relative proportions as expressed in human liver. Initial experiments using luminogenic substrates demonstrate that CYP enzyme activities are significantly increased when co-transfected with an mRNA encoding a CYP accessory protein, P450 oxidoreductase (POR). Transfected HEK293T cells demonstrate the ability to produce predicted metabolites following treatment with well-studied CYP substrates for at least 18 h 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 some of the tested chemicals such as aflatoxin B1 (bioactivation) and fenazaquin (detoxification). Overall, transfection of CYP-encoding mRNAs is an effective and portable solution for retrofitting existing cell-based HTS assays with metabolic competence.