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Glycine Formation Drives Bile Acid Toxicity in Sandwich‐Cultured Human Hepatocyes
Author(s) -
Deferm Neel,
Chatterjee Sagnik,
Richert Lysiane,
Witte Peter,
Bouillon Thomas,
Annaert Pieter
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.lb647
Subject(s) - chenodeoxycholic acid , toxicity , bile acid , glycine , chemistry , taurocholic acid , in vitro , cell culture , pharmacology , medicine , biochemistry , biology , amino acid , genetics
Disturbances in bile acid (BA) disposition and consequent intracellular accumulation of bile acids (BAs) are hypothesized to be associated with toxicity observed in cholestatic disease. The aim of the current study was to assess the link between toxicity and disposition of both chenodeoxycholic acid (CDCA) and its glycine conjugate glycochenodeoxycholic acid (GCDCA) in sandwich‐cultured human hepatocytes (SCHH). Biochemical integrity of the hepatocytes was assessed on multiple days of culture (day‐2 and day‐6) by determining the urea formation capacity of the cultured cells. CDCA exerted culture‐time dependent toxicity, as reflected by a more prominent reduction in relative urea formation in day‐2 SCHH, compared to day‐6 SCHH. To further explore the observed culture time‐dependent toxicity, in vitro disposition data were obtained by treating day‐2 and day‐6 SCHH with either 2.5, 10 or 25 μM CDCA. Media containing CDCA and GCDCA were collected, after which cells were lysed and intracellular accumulation was determined. Additionally, amounts excreted into the bile canaliculi were calculated. Concentration‐time profiles were fitted to a mechanistic ten‐compartment model using NONMEM v7.3.0. The model includes compartments representing the media, cells, bile and cells+bile with compound distributed among them through linear processes. Culture time (day‐2 and day‐6) was implemented as a covariate in the dataset and its effect on kinetic parameter estimations was assessed by the difference in successive objective function values. Covariate analysis showed a significant effect (p < 0.01) of culture time on both sinusoidal efflux clearance of GCDCA (day‐2: 0.6 (± 1%) μL/min/10 6 cells, day‐6: 1.7 (± 2%) μL/min/10 6 cells) and metabolic clearance (day‐2: 17.7 (± 3%) μL/min/10 6 cells, day‐6: 7.2 (± 11%) μL/min/10 6 cells), indicating that the lower toxicity of CDCA in day‐6 SCHH can be explained by decreased intracellular levels of GCDCA due to reduced formation and increased sinusoidal efflux of GCDCA. Interestingly, culture time‐dependent changes in mRNA and protein levels of the conjugating enzyme bile acid‐coenzyme A:amino acid N‐acetyltransferase (BAAT) did not correlate with the observed culture time‐dependent decrease in GCDCA formation. In conclusion, our results indicate that CDCA exerts culture‐time dependent toxicity in SCHH which seems to be related to changes in both sinusoidal efflux clearance of GCDCA and metabolic clearance of CDCA. Support or Funding Information Financial support for this research was provided by the Research Foundation ‐ Flanders (FWO) This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .