title: Predominance of human versus rat phenotype in the metabolic pathways for bile acid synthesis by hybrid WIF-B9 cells.
abstract: The rat hepatoma-human fibroblast hybrid cell line WIF-B9 stably exhibits the structural and functional characteristics of normal differentiated hepatocytes. The abilities of these cells to synthesize bile acids and amidate them with glycine and taurine were investigated. The release of bile acids into the culture media over 72 h was assessed by gas chromatography-mass spectrometry. WIF-B9 cells were able to synthesize bile acids (1.10+/-0.17 nmol/mg protein) but less efficiently than rat hepatocytes in primary culture (2.19+/-0.19 nmol/mg protein; P<0.01). The patterns of major bile acid species produced by both types of cells were also different. Cholic acid (CA; 72%) and beta-muricholic acid (19%) were the major bile acids produced by rat hepatocytes, while chenodeoxycholic acid (CDCA) accounted for only 4.5% of total bile acids. In contrast, muricholic acids were absent, while CA (62%) and CDCA (34%) were the most abundant bile acids synthesized by WIF-B9 cells. Using reverse transcription-polymerase chain reaction and gene- and species-specific primers for key enzymes involved in bile acid synthesis, the expression of human, but not rat, orthologues of CYP7A1, CYP27, CYP8B and CYP7B1 was found in WIF-B9 cells. Induction of cell stress by serum deprivation did not change the amount of total bile acids synthesized by these cells, but an inversion of the CA-to-CDCA ratio from 1.8 to 0.3 together with a marked increase in the proportion of intermediate metabolites related to the acidic pathway was found. Using 500 microM radiolabeled CA and 2 mM of taurine or glycine, the ability to amidate CA over 48 h was determined by high performance liquid chromatography. Rat hepatocytes conjugated more than 90% CA with either amino acid, whereas this ability was very poor (< 2%) in WIF-B9 cells. Regarding the expression of enzymes and the products of bile acid synthesis, it may be concluded that the human phenotype predominates over that of the rat in WIF-B9 cells. Moreover, these cells are almost completely unable to further conjugate primary bile acids, which facilitates the manipulation of these steroids in analytical procedures. These characteristics make WIF-B9 cells a suitable in vitro model to carry out studies on bile acid synthesis by 'human-like' metabolic pathways.
authors.forename: M J
authors.initials: MJ
authors.lastname: Monte
authors.forename: M D
authors.initials: MD
authors.lastname: Badia
authors.forename: M A
authors.initials: MA
authors.lastname: Serrano
authors.forename: M P
authors.initials: MP
authors.lastname: Sacristan
authors.forename: D
authors.initials: D
authors.lastname: Cassio
authors.forename: J J
authors.initials: JJ
authors.lastname: Marin
mesh_terms: D000818:Animals; D001647:Bile Acids and Salts / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D002460:Cell Line; D002478:Cells, Cultured; D002784:Cholesterol / Q000378:metabolism; D004798:Enzymes / Q000235:genetics; D022781:Hepatocytes / Q000378:metabolism; D006801:Humans; D006822:Hybrid Cells / Q000378:metabolism*; D008297:Male; D023421:Models, Animal; D010641:Phenotype; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D001647:Bile Acids and Salts; D004798:Enzymes; D002784:Cholesterol