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Cited by 141Drug-Induced Hepatotoxicity and ProtectionLiver physiology and pathologyAdvanced Proteomics Techniques and Applications

Mechanistic evaluation of primary human hepatocyte culture using global proteomic analysis reveals a selective dedifferentiation profile

Open access
Heslop, James A., Rowe, Cliff, Walsh, Joanne, Sison-Young, Rowena, Jenkins, Roz, Kamalian, Laleh, Kia, Richard, Hay, David +8 more·2017·Archives of Toxicology
DOI 10.1007/s00204-016-1694-yPMC PMC5225178
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Abstract

The application of primary human hepatocytes following isolation from human tissue is well accepted to be compromised by the process of dedifferentiation. This phenomenon reduces many unique hepatocyte functions, limiting their use in drug disposition and toxicity assessment. The aetiology of dedifferentiation has not been well defined, and further understanding of the process would allow the development of novel strategies for sustaining the hepatocyte phenotype in culture or for improving protocols for maturation of hepatocytes generated from stem cells. We have therefore carried out the first proteomic comparison of primary human hepatocyte differentiation. Cells were cultured for 0, 24, 72 and 168 h as a monolayer in order to permit unrestricted hepatocyte dedifferentiation, so as to reveal the causative signalling pathways and factors in this process, by pathway analysis. A total of 3430 proteins were identified with a false detection rate of <1 %, of which 1117 were quantified at every time point. Increasing numbers of significantly differentially expressed proteins compared with the freshly isolated cells were observed at 24 h (40 proteins), 72 h (118 proteins) and 168 h (272 proteins) (p < 0.05). In particular, cytochromes P450 and mitochondrial proteins underwent major changes, confirmed by functional studies and investigated by pathway analysis. We report the key factors and pathways which underlie the loss of hepatic phenotype in vitro, particularly those driving the large-scale and selective remodelling of the mitochondrial and metabolic proteomes. In summary, these findings expand the current understanding of dedifferentiation should facilitate further development of simple and complex hepatic culture systems.

AI snapshot

Written from the paper’s full text.

TL;DR

This study used global iTRAQ proteomics to track primary human hepatocyte dedifferentiation over 168 h in monolayer culture, identifying an escalating and selective loss of hepatic proteins rather than a wholesale collapse of all hepatocyte functions. Donor identity dominated early proteomic clustering, while culture time drove convergence toward a dedifferentiated phenotype.

Key findings

  • Across three iTRAQ runs, 3430 unique proteins were detected with FDR <1%, and 1117 were quantified at every time point; significantly differentially expressed proteins increased from 40 at 24 h to 118 at 72 h and 272 at 168 h versus freshly isolated cells (p < 0.05).
  • Dedifferentiation was selective: cytochromes P450 were largely down-regulated, with CYP2A6, CYP2C8, and CYP4A11 among the top down-regulated proteins at 168 h, while some CYPs such as CYP2C18 and CYP2J2 remained unaltered; only 2 of 9 detected transporters, both influx transporters SLCO1B1 and SLCO1B3, were significantly down-regulated at 168 h (p < 0.05).
  • Mitochondrial and metabolic proteomes underwent major remodeling, but oxidative phosphorylation parameters remained remarkably stable between 24 and 168 h, and PHHs showed significantly increased resistance to rotenone toxicity at 168 h in all donors.
  • Hierarchical clustering showed donor source was the dominant factor over the first 72 h, with convergence toward a culture-time-dependent dedifferentiated phenotype by 168 h; pathway analysis implicated proliferative/inflammatory activation and inhibition of metabolic regulators including PPARα, insulin receptor, KLF15, and TFAM.

Methods

  • Primary human hepatocytes were isolated by a 2-step collagenase method from surgical waste with consent and cultured as monolayers on collagen-I-coated plates; samples were collected at 0, 24, 72, and 168 h.
  • Five donors were analyzed by iTRAQ-based quantitative proteomics across three 8-plex runs, with proteins identified by mass spectrometry and statistically analyzed using LIMMA and t test modules in R; pathway, upstream regulator, and transcription factor binding analyses used IPA, PANTHER, and Mapper2.
  • Functional validation included western blotting for CYP2D6, CYP2E1, CYP1A2, and actin; LC–MS–MS metabolism assays for CYP3A4 (testosterone) and CYP2D6 (dextromethorphan); Seahorse OCR/ECAR mitochondrial and glycolytic assays; and rotenone toxicity ATP assays.

Limitations

  • The paper does not include a dedicated limitations section.
  • The authors state that some results may be specific to monolayer culture, which was chosen deliberately to exacerbate dedifferentiation and may not fully represent complex culture systems.
  • Evident from the text: proteomic analysis used five donors, functional assays used varying donor numbers (e.g., metabolism n = 3; Seahorse and rotenone n = 5), and some ADME heatmap data were derived from n = 2 or n = 1 donors, limiting generalizability and statistical power.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1007/s00204-016-1694-y

Cites

46 of 46 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

5 of 16 cited sources checked · 2 supported · 0 flagged · accuracy not yet measured

  • Premisecitation

    Only approximately 40% of the variation in cell protein levels can be explained by differences in the corresponding mRNA levels.

    “only ~40 % of the variation in cell protein levels can be explained by differences in the corresponding mRNA levels (Schwanhausser et al. 2011).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Schwanhausser et al. 2011 source
  • Findingnone given

    The study carried out the first proteomic comparison of primary human hepatocyte differentiation.

    “We have therefore carried out the first proteomic comparison of primary human hepatocyte differentiation.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The aetiology of dedifferentiation has not been well defined.
    • Rests on an assumption: The exact mechanisms governing hepatocyte dedifferentiation remain incompletely understood.
  • Findingown data

    Many energy production-associated factors are down-regulated at 168 h, including PPARalpha, insulin receptor, KLF15 and the mitochondrial transcription factor TFAM.

    “Moreover, many energy production-associated factors are down-regulated at 168 h including PPARα, insulin receptor, KLF15 and the mitochondrial transcription factor TFAM.”
    Quote in the paperSubstantial claim
  • Findingown data

    Hierarchical clustering of the entire dataset shows that the dominant factor determining how samples are related is the original source of the cells, rather than time in culture.

    “Hierarchical clustering of the entire dataset shows that the dominant factor in determining how the samples are related is in fact the original source of the cells, rather than time in culture (Fig. 1c).”
    Quote in the paperSubstantial claim
  • Findingown data

    Coefficient of variance analysis identified proteins with the greatest variability across the five donors, including CYP2C9 and glutamine synthetase.

    “This criterion highlights a number of proteins, including CYP2C9, which is known to vary in expression between individuals (Sistonen et al. 2009), and glutamine synthetase, which is associated with differential expression according to liver zonation (Jungermann and Kietzmann 1996).”
    Quote in the paperSubstantial claim
  • May combine two claims. 26
  • Classified as probably not a substantial claim. 8

  • May combine two claims.

    Premisecitation

    Primary human hepatocytes undergo rapid dedifferentiation following isolation, losing many unique hepatocyte characteristics and reducing the physiological relevance of the model.

    “The application of primary human hepatocytes following isolation from human tissue is well accepted to be compromised by the process of dedifferentiation.”
    • Also stated in: § Introduction · plain
      “following isolation, primary human hepatocytes undergo a rapid dedifferentiation process in which many unique hepatocyte characteristics are lost, reducing the physiological relevance of the model (Fraczek et al. 2013; Godoy et al. 2013).”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites Fraczek et al. 2013 source
    • cites Godoy et al. 2013 source

  • May combine two claims.

    Interpretationown data

    Zonation appears to be a large influencing factor on variation during culture, as pathway analysis of the most variable proteins revealed significant association with ketogenesis, cholesterol biosynthesis, fatty acid beta oxidation and glutamine synthesis, all related to the portocentral axis gradient of the lobule.

    “In fact, zonation appears to be a large influencing factor on variation during culture as our pathway analysis of the most variable proteins revealed significant association with ketogenesis, cholesterol biosynthesis, fatty acid β oxidation and glutamine synthesis (table S4), all of these are related to the portocentral axis gradient of the lobule (Godoy et al. 2013).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Godoy et al. 2013 source

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

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Primary human hepatocytes are an important tool for studying human liver disease and drug-induced liver injury, and other models such as rat hepatocytes, hepatic cell lines and stem cell-derived hepatocyte-like cells fail to offer metabolically competent and species-relevant alternatives.

    “Primary human hepatocytes (PHHs) are an important tool for studying human liver disease and drug-induced liver injury (DILI), with other models, such as rat hepatocytes, hepatic cell lines and stem cell-derived hepatocyte-like cells failing to offer metabolically competent and species-relevant alternatives (Godoy et al. 2013).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Godoy et al. 2013 source

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

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim.

    Premisecitation

    CYP2A6 has been reported to be the largest discriminant between foetal and adult hepatocytes.

    “CYP2A6 has been reported to be the largest discriminant between foetal and adult hepatocytes (Rowe et al. 2013)”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Rowe et al. 2013 source

      The Discussion reports that CYP2A6 was 5.75-fold more abundant in adult hepatocytes than in fetal liver, and the Results (Fig. 8A) show strong detection in most adult samples but weak or absent detection in fetal samples. The paper identifies multiple proteins that discriminate the groups; it does not identify CYP2A6 as the largest discriminant. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingown data

    Dedifferentiation and loss of characteristic hepatocyte morphology were confirmed by microscopy.

    “Dedifferentiation and loss of characteristic hepatocyte morphology were confirmed by microscopy.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    At 24 h, superoxide dismutase increased and histone proteins decreased.

    “Interestingly, the first noticeable effects at 24 h are an increase in stress-related mediator superoxide dismutase and a decrease in histone proteins.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Metabolic pathways display a trend of increasing significance during culture, while acute phase response signalling and ROS/NOS production are significantly affected at 24 h but not at 168 h.

    “Whilst metabolic pathways display a trend of increasing significance during culture, acute phase response signalling and ROS/NOS production are significantly affected at 24 h, but not at 168 h.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    A total of 3430 proteins were identified with a false detection rate of less than 1%, of which 1117 were quantified at every time point.

    “A total of 3430 proteins were identified with a false detection rate of <1 %, of which 1117 were quantified at every time point.”
    • Also stated in: § Global proteomics reveals donor-dependent variation as a major influence on dedifferentiation · plain
      “A total of 3430 unique proteins were detected across three iTRAQ runs (table S1), with 1117 identified across all samples.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Many pathways follow a linear pattern of increasing activation or inhibition, but cell survival pathways are predicted to be activated at 72 h and not at 24 or 168 h.

    “Many of the pathways follow a linear pattern of increasing activation or inhibition; however, some, such as the cell survival pathways, display different dynamics, as they are predicted to be activated at 72 h, but not at 24 or 168 h.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    At 168 h, donors 2 and 5 remain separated from the other donors, while donors 1, 3 and 4 cluster together, demonstrating convergence towards a dedifferentiated phenotype more reflective of time in culture than cell source.

    “The separation from the other donors is still evident at 168 h for donors 2 and 5, although these samples do cluster away from the earlier time points, whereas by this time the other three donors (1, 3 and 4) cluster together, demonstrating a convergence towards a dedifferentiated phenotype which is more reflective of time in culture than the cell source.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Upstream regulation analysis predicts activation of proliferative and inflammatory regulators and inhibition of metabolic-associated factors.

    “The results suggest an activation of proliferative and inflammatory regulators and inhibition of metabolic-associated factors.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    OCR analysis demonstrated clear variation in individual bioenergetic profiles at 24 h, with the 168 h profile showing greater dependence on the 24 h profile rather than time in culture.

    “The analysis demonstrated a clear variation in individual bioenergetic OCR profiles at 24 h (Fig. 3b (i)), with the 168-h profile showing greater dependence on the 24 h profile, rather than time in culture.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Over 168 h of monolayer culture, hepatocytes maintained confluency but lost typical polygonal morphology by 24 h, becoming flatter and less defined epithelial cells at 72 and 168 h.

    “Figure 1a shows images of the cells taken at 24, 72 and 168 h and clearly demonstrates the maintenance of confluency and the loss of the typical hepatocyte polygonal morphology (24 h), towards a culture with flatter and less defined epithelial characteristics at 72 and 168 h.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Hepatic-associated proteins are not universally down-regulated; alpha-1-antitrypsin and cytokeratins 8 and 18 are up-regulated throughout the analysis.

    “Of note, hepatic-associated proteins are not universally down-regulated, as exemplified by alpha-1-antitrypsin and cytokeratins 8 and 18, which are up-regulated throughout the analysis (Table S2 and S6).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Basal glycolysis shows an increase during culture in donors with lower basal glycolysis, whereas those with higher basal levels at 24 h decrease.

    “Despite this variation, a trend emerges: basal glycolysis shows an increase during culture in donors with lower basal glycolysis, whereas those with higher basal levels at 24 h decrease”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Both AMPK and mitochondrial fusion pathways are associated with the bioenergetic adaptation to a monolayer culture system.

    “Here we have additionally shown both AMPK and mitochondrial fusion pathways to be associated with the bioenergetic adaption to a monolayer culture system.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The iTRAQ proteomic quantification accurately reflects relative protein expression changes in PHH culture.

  • May combine two claims.

    Findingown data

    Despite an increase in CYP3A4 protein at 168 h, no corresponding increase in CYP3A activity was detected.

    “despite the increase in CYP3A4 protein at 168 h, no corresponding increase in CYP3A activity was detected”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The iTRAQ proteomic quantification accurately reflects relative protein expression changes in PHH culture.

  • May combine two claims.

    Findingown data

    The early stages of dedifferentiation are predominantly donor-dependent, with inter-individual variation in the starting donor phenotype maintained throughout the first 72 h of culture.

    “Our analysis revealed that the early stages of dedifferentiation are predominantly donor-dependent with the inter-individual variation that exists in the starting donor phenotype being maintained throughout the first 72 h of culture.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The iTRAQ proteomic quantification accurately reflects relative protein expression changes in PHH culture.

  • May combine two claims.

    Findingown data

    In the proteomic data, three complex I subunits were significantly down-regulated, whereas the remaining nine detected subunits were not significantly differentially expressed.

    “Further investigation of the proteomic data revealed a significant down-regulation of three complex I subunits, whereas the remaining nine detected subunits were not significantly differentially expressed.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The iTRAQ proteomic quantification accurately reflects relative protein expression changes in PHH culture.

  • May combine two claims.

    Findingown data

    ECAR measurements showed similar stability, but there was a general trend of increased glycolytic parameters, including the only significantly altered factor, glycolytic reserve.

    “ECAR measurements showed similar stability; however, there was a general trend of increased glycolytic parameters, including the only significantly altered factor, namely glycolytic reserve (Fig. 3b (iv)).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    PANTHER analysis revealed similar percentages of proteins in each class, with the greatest proportion of proteins associated with metabolism in all subsets.

    “This analysis revealed similar percentages of proteins in each class, with the greatest proportion of proteins associated with metabolism in all subsets”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The iTRAQ proteomic quantification accurately reflects relative protein expression changes in PHH culture.

  • May combine two claims.

    Findingown data

    Targeted analysis revealed specific transcription factors that are significantly perturbed and associated with ADME DEPs.

    “Targeted analysis of the transcription factors that are associated with the ADME DEPs revealed the specific regulators, which are significantly perturbed and may cause the loss of ADME proteins during dedifferentiation (table S9).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Predicted transcription factor binding sites are functionally relevant to CYP expression.

  • May combine two claims.

    Interpretationown data

    A subset of proteins remains relatively stable during dedifferentiation and may retain in vivo-like functionality and serve as useful normalisers for protein expression changes.

    “Thus, while many proteins show conserved differential expression or high levels of inter-donor variability, a subset of proteins remains relatively stable and consequently may retain in vivo-like functionality and also serve as useful normalisers for protein expression changes.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Glutamine synthetase is associated with differential expression according to liver zonation.

    “glutamine synthetase, which is associated with differential expression according to liver zonation (Jungermann and Kietzmann 1996)”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Jungermann and Kietzmann 1996 source

  • May combine two claims.

    Interpretationown data

    Using iTRAQ-based stable isotope labelling and pathway analysis, the key changes in the PHH proteome during dedifferentiation and the inter-donor variability were identified, including variability in donor phenotype and in the subsequent dedifferentiation profile.

    “Using iTRAQ-based stable isotope labelling and pathway analysis, we have identified the key changes in the PHH proteome during dedifferentiation and the inter-donor variability, which exists not only in donor phenotype, but also in the subsequent dedifferentiation profile.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The aetiology of dedifferentiation has not been well defined.
    • Rests on an assumption: The exact mechanisms governing hepatocyte dedifferentiation remain incompletely understood.

  • May combine two claims.

    Interpretationreasoning

    The donor-dependent variation in ECAR may be attributable to the known gradient of glycolysis across the portocentral axis and is consistent with the findings regarding the most variable proteins.

    “which may be attributable to the known gradient of glycolysis across the portocentral axis, and is consistent with our findings regarding the most variable proteins.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    CYP2C9 is known to vary in expression between individuals.

    “CYP2C9, which is known to vary in expression between individuals (Sistonen et al. 2009)”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Sistonen et al. 2009 source

  • May combine two claims.

    Interpretationcitation

    Simple in vitro culture systems may support similar expression patterns during both hepatic dedifferentiation and differentiation, because proteins up-regulated in dedifferentiation are readily detectable in stem cell-derived hepatocyte-like cells whereas many down-regulated proteins are not.

    “Interestingly, these proteins are readily detectable in hepatocyte-like cells derived from stem cells (Rambhatla et al. 2003), whereas many of the down-regulated proteins are not, demonstrating that simple in vitro culture systems may support similar expression patterns during both hepatic dedifferentiation and differentiation.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Rambhatla et al. 2003 source

  • Classified as probably not a substantial claim.

    Premisecitation

    A global proteomic approach to phenotype rat hepatocytes can provide novel information on dedifferentiation.

    “We have previously shown that the use of a global proteomic approach to phenotype rat hepatocytes can provide novel information on dedifferentiation (Rowe et al. 2010)”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Rowe et al. 2010 source

  • Probably not a substantial claim: may not pass S2 (checkable).

    Interpretationreasoning

    Studies of more rudimentary models that result in more pronounced dedifferentiation may provide greater insight into the partially understood and currently unknown causes of dedifferentiation.

    “studies of more rudimentary models which result in more pronounced dedifferentiation may consequently provide a greater insight into the partially understood and currently unknown causes of dedifferentiation.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: The aetiology of dedifferentiation has not been well defined.
    • Rests on an assumption: The exact mechanisms governing hepatocyte dedifferentiation remain incompletely understood.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Western blot was used to validate iTRAQ results.

    “Samples collected in iTRAQ buffer were quantified by Bradford assay and assessed by western blot to validate iTRAQ results.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    Dedifferentiation is not solely a change in expression of whole protein classes, but is a complex, differentially controlled and active process, in which selected pathways and functions from many classes are up- or down-regulated.

    “Grouping of these data showed that dedifferentiation is not solely a change in expression of whole protein classes, but that it is a complex, differentially controlled and active process, in which selected pathways and functions from many classes are up- or down-regulated.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The iTRAQ proteomic quantification accurately reflects relative protein expression changes in PHH culture.

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    Only proteins present in all samples, identified with 95% confidence (2 or more peptides) or 99% confidence (single peptide) with a false detection rate of less than 1%, were statistically analysed.

    “Following iTRAQ analysis, only proteins, which were present in all samples, identified with 95 % confidence (2 or more peptides) or 99 % confidence (single peptide) with a false detection rate (FDR) of less than 1 % were statistically analysed using R open-source software”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    iTRAQ results were obtained relative to each donor's fresh sample to control for inter-donor variation.

    “Samples were run across three 8-plex iTRAQ runs (table S1), and results obtained relative to each donor’s fresh sample to control for inter-donor variation.”
    Quote in the paperSubstantial claim
34 of 34

  • The paper relies on this without support.

    The aetiology of dedifferentiation has not been well defined.

    “The aetiology of dedifferentiation has not been well defined, and further understanding of the process would allow the development of novel strategies for sustaining the hepatocyte phenotype in culture or for improving protocols for maturation of hepatocytes generated from stem cells.”

  • The paper relies on this without support.

    The exact mechanisms governing hepatocyte dedifferentiation remain incompletely understood.

    “however, the exact mechanisms which govern these changes remain incompletely understood.”

  • The paper relies on this without support.

    The full in vivo phenotype has not yet been achieved in culture.

    “Despite these continued improvements and advances towards complex culture conditions which can maintain an improved phenotype over a period of weeks, the full in vivo phenotype has not yet been achieved.”

  • The paper relies on this without support.

    The iTRAQ proteomic quantification accurately reflects relative protein expression changes in PHH culture.

    “The general trend of expression relative to freshly isolated PHHs is downward; however, the majority of the detected CYP proteins are maintained in expression at 24 h in relation to the freshly isolated cells”

  • The paper relies on this without support.

    Rotenone resistance is a valid functional readout of complex I mitochondrial function in PHH.

    “To assess the functional consequence of these changes, rotenone was used as a model of complex I mitochondrial perturbation.”

  • The paper relies on this without support.

    Predicted transcription factor binding sites are functionally relevant to CYP expression.

    “Investigation into the predicted transcription factor binding sites of differentially expressed and non-differentially expressed CYPs revealed the factors that may underlie the selectivity found in the loss of xenobiotic CYPs (Table S10).”

  • The paper relies on this without support.

    The majority of toxicological endpoints are assessed within 72 h of isolation.

    “the majority of toxicological endpoints are assessed within 72 h of isolation”

  • The paper relies on this without support.

    Ferrochelatase catalyses the final step of the heme production pathway.

    “Ferrochelatase catalyses the final step of the heme production pathway”

  • The paper relies on this without support.

    CYPs are dependent on heme for function.

    “CYPs are dependent on heme for function”

  • The paper relies on this without support.

    The mRNA data from Richert et al. 2006 in monolayer culture are comparable to the protein data in this study.

    “Comparison of the trend in the CYP and transporter proteins with previously published hepatocyte monolayer gene expression data (Richert et al. 2006) allows further investigation of the loss of metabolic competence (table S8).”

  • The paper relies on this without support.

    The culture system's conditions are strictly defined and consistent across donors.

    “Taken together, these data suggest that the strictly defined culture system promotes a specific protein expression profile”
11 of 11

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Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Figure 2 caption reverses the activation-score threshold

    Minor

    The methods define Z-scores of ≥2 as activated and ≤−2 as inhibited. Figure 2 instead writes the activation threshold as "2 ≥ Z-score," which reverses the inequality; the same expression appears again for the upstream-regulator panel.

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Authors
Heslop, J. A., Rowe, C., Walsh, J., Sison-Young, R., Jenkins, R., Kamalian, L., Kia, R., Hay, D., Jones, R. P., Malik, H. Z., Fenwick, S., Chadwick, A. E., Mills, J., Kitteringham, N. R., Goldring, C. E. P., & Kevin Park, B.
Affiliations
  • University of Liverpool (GB)
  • University of Oxford (GB)
  • Aintree University Hospital (GB)
  • AstraZeneca (United Kingdom) (GB)
ORCID
Venue
Archives of Toxicology
Publisher
Springer Science and Business Media LLC
Published
2016-04-02
Volume
91
Issue
1
Pages
439-452
ISSN
  • 0340-5761 (ISSN-L)
  • 1432-0738
DOI
10.1007/s00204-016-1694-y
PMC
PMC5225178
OpenAlex
W2321561488
Semantic Scholar
738c624a59802860e61440bd9f6ab427176b529f
Cited by
141
References
46
Citations by year
2026: 16 · 2025: 14 · 2024: 10 · 2023: 15 · 2022: 16 · 2021: 12 · 2020: 17 · 2019: 10 · 2018: 18 · 2017: 7 · 2016: 6
FWCI
15.18
Type
journal article
Language
en
Open access
Licence
Locations
  • Archives of Toxicology (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • PubMed Central (submittedVersion, cc-by)
  • Edinburgh Research Explorer (University of Edinburgh) (submittedVersion, other-oa)
Funders
  • Biotechnology and Biological Sciences Research Council (GB) (BB/J500756/1)
  • Medical Research Council (GB) (MR/L006758/1 and G0700654)
  • European Parliament (BE) (115336)
Topics
  • Life Sciences › Pharmacology, Toxicology and Pharmaceutics › Pharmacology › Drug-Induced Hepatotoxicity and Protection
  • Health Sciences › Medicine › Hepatology › Liver physiology and pathology
  • Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
Keywords
primary human hepatocytes, hepatocyte dedifferentiation, hepatocyte culture, proteomic profiling, cytochrome P450, mitochondrial proteome, drug metabolism, hepatotoxicity assessment, stem cell-derived hepatocytes
MeSH terms
Cells, Cultured, Humans, Kinetics, Mitochondria, Liver, Pharmacology, Rotenone, Toxicology, Uncoupling Agents, Reproducibility of Results, Gene Expression Regulation, Developmental, Proteome, Gene Expression Profiling, Hepatocytes, Electron Transport Complex I, Cell Dedifferentiation, Protein Stability

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