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Cited by 49Pharmacogenetics and Drug MetabolismDrug-Induced Hepatotoxicity and ProtectionAnimal testing and alternatives

Application of In Vitro Metabolism Activation in High-Throughput Screening

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Ooka, Masato, Lynch, Caitlin, Xia, Menghang·2020·International Journal of Molecular Sciences
DOI 10.3390/ijms21218182PMC PMC7663506
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Abstract

In vitro methods which incorporate metabolic capability into the assays allow us to assess the activity of metabolites from their parent compounds. These methods can be applied into high-throughput screening (HTS) platforms, thereby increasing the speed to identify compounds that become active via the metabolism process. HTS was originally used in the pharmaceutical industry and now is also used in academic settings to evaluate biological activity and/or toxicity of chemicals. Although most chemicals are metabolized in our body, many HTS assays lack the capability to determine compound activity via metabolism. To overcome this problem, several in vitro metabolic methods have been applied to an HTS format. In this review, we describe in vitro metabolism methods and their application in HTS assays, as well as discuss the future perspectives of HTS with metabolic activity. Each in vitro metabolism method has advantages and disadvantages. For instance, the S9 mix has a full set of liver metabolic enzymes, but it displays high cytotoxicity in cell-based assays. In vitro metabolism requires liver fractions or the use of other metabolically capable systems, including primary hepatocytes or recombinant enzymes. Several newly developed in vitro metabolic methods, including HepaRG cells, three-dimensional (3D) cell models, and organ-on-a-chip technology, will also be discussed. These newly developed in vitro metabolism approaches offer significant progress in dissecting biological processes, developing drugs, and making toxicology studies quicker and more efficient.

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  • HepG2 is suitable for studying general mechanisms of cytotoxicity and cellular stress response but limited for quantitative prediction of metabolism and biotransformation of many compounds.

    “Recent reviews and experimental studies directly interpret HepG2 as a model suitable for solving certain toxicological problems, in particular for studying general mechanisms of cytotoxicity and cellular stress response (e.g., activation of transcription factors activator protein-1 (AP1), p53, nuclear factor-erythroid 2 related factor 2 (Nrf2), and nuclear factor kappa B (NF-κB)) [109,110,111],”

    From The HepG2 Cell Line as a Model for Studying Metabolic Dysfunction-Associated Steatotic Liver Disease (2026) · cites this paper as [109]

    Section 3.3 says HepG2 cells are used in viability and high-content screening for hepatotoxicity, while their low CYP activity makes them poor detectors of toxicity caused by reactive metabolites. Section 2.5 also documents enzyme-expression differences from primary hepatocytes. These passages support the broad use-and-metabolism distinction, but the paper does not establish HepG2’s suitability for studying the specified AP1, p53, Nrf2, and NF-κB stress responses. (LLM, conf=0.85)

    Experimental — accuracy not yet measured

  • Strategies for optimizing HepG2 can be divided into genetic modification to express key biotransformation enzymes, epigenetic and cultural maturation, external metabolic activation using subcellular liver fractions (S9 or microsomes), and transitioning from monolayer culture to 3D or hybrid formats.

    “In practice, strategies for optimizing HepG2 can be divided into several approaches. The first approach is based on genetic modification of cells to express key biotransformation enzymes, in particular cytochrome P450 isoforms [109]. The second approach involves epigenetic and cultural maturation of cells, which allows for increased expression of nuclear receptors and, consequently, of xenobiotic”

    From The HepG2 Cell Line as a Model for Studying Metabolic Dysfunction-Associated Steatotic Liver Disease (2026) · cites this paper as [109]

    Split into 2 atoms — 1 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

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  • S9 fractions contain both microsomal (including cytochromes P450) and cytosolic (glutathione-S-transferases, sulfotransferases) enzymes, which provides a more complete metabolic profile in vitro; however, S9 fractions exhibit pronounced cytotoxicity toward cultured cells, requiring additional washing steps, and do not replicate key aspects of living cell physiology such as membrane transport of

    “In contrast, S9 fractions contain both microsomal (including cytochromes P450) and cytosolic (glutathione-S-transferases, sulfotransferases) enzymes, which provides a more complete metabolic profile in vitro. However, as noted in the literature, S9 fractions exhibit pronounced cytotoxicity toward cultured cells, which requires additional steps to wash the cells after metabolic activation. Further”

    From The HepG2 Cell Line as a Model for Studying Metabolic Dysfunction-Associated Steatotic Liver Disease (2026) · cites this paper as [109]

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  • Microsomes are convenient for CYP450-dependent biotransformation, are relatively low-toxicity, and are well-suited for HTS; however, they are deficient in Phase II enzymes and lack cytosolic conjugation enzymes (sulfotransferases, glutathione-S-transferases, etc.), which makes them less suitable for studying the full spectrum of detoxification reactions.

    “Microsomes are convenient for CYP450-dependent biotransformation, are relatively low-toxicity, and are well-suited for HTS; however, they are deficient in Phase II enzymes and lack cytosolic conjugation enzymes (sulfotransferases, glutathione-S-transferases, etc.), which makes them less suitable for studying the full spectrum of detoxification reactions [109].”

    From The HepG2 Cell Line as a Model for Studying Metabolic Dysfunction-Associated Steatotic Liver Disease (2026) · cites this paper as [109]

    Split into 2 atoms — 1 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

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Cited by

Sorted by citations

Authors
Ooka, M., Lynch, C., & Xia, M.
Affiliations
National Institutes of Health (US)
Corresponding author
Xia, Menghang
ORCID
Venue
International Journal of Molecular Sciences
Publisher
MDPI AG
Published
2020-10-31
Volume
21
Issue
21
Pages
8182
ISSN
1422-0067 (ISSN-L)
DOI
10.3390/ijms21218182
PMC
PMC7663506
OpenAlex
W3095776327
Cited by
49
References
7
Citations by year
2026: 7 · 2025: 11 · 2024: 9 · 2023: 7 · 2022: 9 · 2021: 6
FWCI
0.96
Type
journal article
Language
en
Open access
Licence
Locations
  • International Journal of Molecular Sciences (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • National Institutes of Health
  • National Center for Advancing Translational Sciences
Topics
  • Life Sciences › Pharmacology, Toxicology and Pharmaceutics › Pharmacology › Pharmacogenetics and Drug Metabolism
  • Life Sciences › Pharmacology, Toxicology and Pharmaceutics › Pharmacology › Drug-Induced Hepatotoxicity and Protection
  • Health Sciences › Veterinary › Small Animals › Animal testing and alternatives
Keywords
in vitro metabolism, high-throughput screening, metabolic activation, S9 mix, primary hepatocytes, recombinant enzyme, HepaRG cells, three-dimensional cell models, organ-on-a-chip, drug metabolism, toxicity testing
MeSH terms
Cells, Cultured, Drug Evaluation, Preclinical, Humans, Inactivation, Metabolic, Hepatocytes, High-Throughput Screening Assays

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