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Cells for bioartificial liver devices: The human hepatoma‐derived cell line C3A produces urea but does not detoxify ammonia
Author(s) -
MavriDamelin Demetra,
Damelin Leonard H.,
Eaton Simon,
Rees Myrddin,
Selden Clare,
Hodgson Humphrey J.F.
Publication year - 2007
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.21599
Subject(s) - arginase , urea cycle , urea , ornithine transcarbamylase , bioartificial liver device , ammonia , ammonium chloride , biochemistry , chemistry , ornithine , ammonium , argininosuccinate synthase , carbamoyl phosphate synthetase , arginine , microbiology and biotechnology , enzyme , biology , hepatocyte , amino acid , in vitro , organic chemistry
Extrahepatic bioartificial liver devices should provide an intact urea cycle to detoxify ammonia. The C3A cell line, a subclone of the hepatoma‐derived HepG2 cell line, is currently used in this context as it produces urea, and this has been assumed to be reflective of ammonia detoxification via a functional urea cycle. However, based on our previous findings of perturbed urea‐cycle function in the non‐urea producing HepG2 cell line, we hypothesized that the urea produced by C3A cells was via a urea cycle‐independent mechanism, namely, due to arginase II activity, and therefore would not detoxify ammonia. Urea was quantified using 15 N‐ammonium chloride metabolic labelling with gas chromatography–mass spectrometry. Gene expression was determined by real‐time reverse transcriptase‐PCR, protein expression by western blotting, and functional activities with radiolabelling enzyme assays. Arginase inhibition studies used N ω ‐hydroxy‐nor‐ L ‐arginine. Urea was detected in C3A conditioned medium; however, 15 N‐ammonium chloride‐labelling indicated that 15 N‐ammonia was not incorporated into 15 N‐labelled urea. Further, gene expression of two urea cycle genes, ornithine transcarbamylase and arginase I, were completely absent. In contrast, arginase II mRNA and protein was expressed at high levels in C3A cells and was inhibited by N ω ‐hydroxy‐nor‐ L ‐arginine, which prevented urea production, thereby indicating a urea cycle‐independent pathway. The urea cycle is non‐functional in C3A cells, and their urea production is solely due to the presence of arginase II, which therefore cannot provide ammonia detoxification in a bioartificial liver system. This emphasizes the continued requirement for developing a component capable of a full repertoire of liver function. Biotechnol. Bioeng. 2008;99: 644–651. © 2007 Wiley Periodicals, Inc.