PXD071083

PXD071083 is an original dataset announced via ProteomeXchange.

Title	Development of an In Vitro Metabolic Dysfunction-Associated Steatohepatitis Model to Investigate Altered Drug Metabolizing Enzymes, Transport Proteins, and Hepatobiliary Disposition
Description	Metabolic dysfunction-associated steatotic liver disease (MASLD) is estimated to affect approximately 30% of adults worldwide. The progressive form of MASLD, namely metabolic-dysfunction steatohepatitis (MASH), is a leading cause of chronic liver disease. MASH is marked by hepatocellular fat accumulation (steatosis), ballooning, and inflammation. Although many in vitro and in vivo models replicate MASH pathophysiology, no in vitro hepatocyte MASH model has been evaluated for its ability to reflect clinically observed changes in drug metabolizing enzyme (DME) and transporter characteristics such as abundance and activity. This study addressed this knowledge gap by developing a model using sandwich-cultured human hepatocytes (SCHH) that mimics both MASH pathophysiology and alterations in DME and transporter concentrations and function. Lipid-cytokine treatments were first optimized using differentiated HuH-7 cells based on cellular toxicity profiles and their ability to induce a MASH-like phenotype. Three final treatments, all including TNF-α (1 ng/mL) and IL-6 (1.2 ng/mL), were selected for evaluation in SCHH: (1) oleic acid (OA):palmitic acid (PA) (1:2, 0.5 mM), (2) a lipid mix (lysophospholipids mixture + OA:PA), and (3) lipid mix+0.01 mM cholesterol. Treatments were incubated for 72 h with SCHH from three donors. Quantitative targeted absolute proteomics (QTAP), employing stable isotope labeled (SIL) peptide standards and microLC-MS/MS measurement, was used to quantify transporter and DME concentrations in the SCHH total membrane fraction. Concentrations of ten transporters, ten CYP450s, seven UGTs and five other DMEs could be measured above the lower limit of quantification of 0.1 pmol/mg membrane protein. B-CLEAR® technology was used, also, to evaluate transporter function with [3H]-taurocholate (TCA) and [3H]-estradiol-17β-glucuronide (E217G) employed as probe substrates of function. All three treatments significantly increased lipid droplet formation and peroxidation, characteristics of MASH, in SCHH with minimal toxicity. The treatments also altered DME and transporter concentrations in a similar manner to changes observed in liver tissue from patients with MASH. Across treatments, concentrations of bile salt export pump (BSEP), sodium taurocholate co-transporting polypeptide (NTCP), organic anion transporting polypeptide (OATP) 1B1, OATP1B3, and multidrug resistance-associated protein (MRP) 2 were reduced by 0.66–0.57-fold, 0.71–0.52-fold, 0.74–0.63-fold, 0.82–0.80-fold, and 0.71–0.48-fold, respectively. Correspondingly, TCA apparent uptake clearance and biliary clearance were reduced by 0.70–0.26-fold and 0.61–0.27-fold, respectively. E217G apparent uptake clearance was reduced by 0.67–0.35-fold while biliary excretion index values for E217G were reduced to negligible levels. Overall, the findings demonstrate that lipid–cytokine treatments induce MASH-like changes in SCHH, including clinically relevant reductions in DME and transporter concentrations and transporter function. This model may serve as a valuable tool for predicting altered hepatobiliary drug disposition in MASH.
HostingRepository	PRIDE
AnnounceDate	2025-12-12
AnnouncementXML	Submission_2025-12-12_08:43:21.606.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	John Fallon
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: NEWT:9606;
ModificationList	No PTMs are included in the dataset
Instrument	Q TRAP

Revision	Datetime	Status	ChangeLog Entry
0	2025-11-22 17:14:25	ID requested
⏵ 1	2025-12-12 08:43:22	announced

10.3389/FPHAR.2025.1664808;

submitter keyword: Human, LC-MSMS, SCHH, MASH

Kim Brouwer
contact affiliation	Division of Pharmacotherapy and Experimental Therapeutics, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, USA
contact email	kbrouwer@unc.edu
lab head
John Fallon
contact affiliation	Eshelman School of Pharmacy, University of North Carolina at Chapel Hill
contact email	jfallon@email.unc.edu
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD071083
     1. Label: PRIDE project
     2. Name: Development of an In Vitro Metabolic Dysfunction-Associated Steatohepatitis Model to Investigate Altered Drug Metabolizing Enzymes, Transport Proteins, and Hepatobiliary Disposition