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Updated: Nov 16, 2025
Mass Spectrometry and Luminogenic-based Approaches to Characterize Phase I Metabolic Competency of In Vitro Cell Cultures
Published on: March 28, 2017
Exploration of xenobiotic metabolism within cell lines used for Tox21 chemical screening
Wei Qu1, David M Crizer1, Michael J DeVito1
1Division of the National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA.
Abstract:
The Tox21 Program has investigated thousands of chemicals with high-throughput screening assays using cell-based assays to link thousands of chemicals to individual molecular targets/pathways. However, these systems have been widely criticized for their suspected lack of 'metabolic competence' to bioactivate or detoxify chemical exposures. In this study, 9 cell line backgrounds used in Tox21 assays (i.e., HepG2, HEK293, Hela, HCT116, ME180, CHO-K1, GH3.TRE-Luc, C3H10T1/2 and MCF7) were evaluated via metabolite formation rates, along with metabolic clearance and metabolite profiling for HepG2, HEK293, and MCF-7aroERE, in comparison to pooled donor (50) suspensions of primary human hepatocytes (PHHs). Using prototype clinical drug substrates for CYP1A2, CYP2B6, and CYP3A4/5, extremely low-to-undetectable CYP450 metabolism was observed (24 h), and consistent with their purported 'lack' of metabolic competence. However, for Phase II metabolizing enzymes and metabolic clearance, surprisingly proficient metabolism was observed for bisphenol AF, bisphenol S, and 7-hydroxycoumarin. Here, comparatively low glucuronidation relative to sulfation was observed in contrast to equivalent levels in PHHs. Overall, while a lack of CYP450 metabolism was confirmed in this benchmarking effort, Tox21 cell lines were not 'incompetent' for xenobiotic metabolism, and displayed surprisingly high proficiency for sulfation that rivaled PHHs. These findings have implications for the interpretation of Tox21 assay data, and establish a framework for evaluating of 'metabolic competence' with in vitro models.
Insights
Tox21 cell lines show limited CYP450 metabolism but surprisingly high sulfation capacity, challenging assumptions about their metabolic competence for chemical exposure assessments. This impacts interpreting high-throughput screening data.
Area of Science:
- Toxicology
- Pharmacology
- Biochemistry
Background:
- The Tox21 Program uses cell-based assays for chemical screening, but faces criticism regarding metabolic competence.
- Assessing metabolic capacity is crucial for accurate chemical risk evaluation.
Purpose of the Study:
- To evaluate the metabolic competence of nine cell line backgrounds used in Tox21 assays.
- To compare their metabolic activity, including Phase I and Phase II enzymes, with primary human hepatocytes (PHHs).
Main Methods:
- Metabolite formation rates, metabolic clearance, and metabolite profiling were assessed for selected cell lines (HepG2, HEK293, MCF-7aroERE) and PHHs.
- Prototype clinical drug substrates for CYP1A2, CYP2B6, and CYP3A4/5 were used to evaluate Phase I metabolism.
- Bisphenol AF, bisphenol S, and 7-hydroxycoumarin were used to assess Phase II metabolism (glucuronidation and sulfation).
Main Results:
- Extremely low-to-undetectable CYP450 (Phase I) metabolism was observed in Tox21 cell lines, confirming their limited bioactivation/detoxification capacity.
- Surprisingly proficient Phase II metabolism, particularly sulfation, was observed for bisphenol AF, bisphenol S, and 7-hydroxycoumarin, rivaling PHH activity.
- Glucuronidation levels were comparatively lower in Tox21 cell lines than in PHHs.
Conclusions:
- Tox21 cell lines are not entirely metabolically incompetent, demonstrating significant sulfation capabilities.
- Findings necessitate a re-evaluation of Tox21 assay data interpretation due to cell line-specific metabolic profiles.
- A framework for assessing in vitro model metabolic competence is established, crucial for chemical safety and toxicology studies.
