title: Stable overexpression of arginase I and ornithine transcarbamylase in HepG2 cells improves its ammonia detoxification.
abstract: HepG2 is an immortalized human hepatoma cell line that has been used for research into bioartificial liver systems. However, a low level of ammonia detoxification is its biggest drawback. In this work, a recombinant HepG2 cell line with stable overexpression of human arginase I (hArgI) and human ornithine transcarbamylase (hOTC), HepG2/(hArgI + hOTC)4, was developed using a eukaryotic dual gene expression vector pBudCE4.1. (1) The hArgI and hOTC enzymatic activity in HepG2/(hArgI + hOTC)4 cells were higher than in the control cells. (2) The ammonia tolerance capacity of HepG2/(hArgI + hOTC)4 cells was three times that of HepG2 cells and 37.5% of that of primary human hepatocytes in cultivation. In the experiment of ammonia detoxification, HepG2/(hArgI + hOTC)4 cells produced 3.1 times more urea (at 180 mM NH(4) Cl) and 3.1 times more glutamine (at 120 mM NH(4) Cl and 15 mM glutamate) than HepG2 cells, reaching 63.1% and 36.0% that of primary human hepatocytes, respectively. (3) The hArgI and hOTC overexpression did not influence the growth of HepG2 cells and also promoted the expression of other ammonia detoxification associated proteins including glutamine synthetase (GS), arginase II (ArgII), arginosuccinate synthase (ASS) and arginosuccinate lyase (ASL) in HepG2 cells. This work illustrates that the modification reported here made significant progress in the improvement of HepG2 cell function and the HepG2/(hArgI + hOTC)4 cells will provide a better selection for the application of bioartificial liver system.
authors.affiliation: Fujian Institute of Hepatobiliary Surgery, Union Hospital, Fujian Medical University, Fuzhou, PR China.
authors.forename: Nanhong
authors.initials: N
authors.lastname: Tang
authors.forename: Yan
authors.initials: Y
authors.lastname: Wang
authors.forename: Xiaoqian
authors.initials: X
authors.lastname: Wang
authors.forename: Liangyi
authors.initials: L
authors.lastname: Zhou
authors.forename: Feiyuan
authors.initials: F
authors.lastname: Zhang
authors.forename: Xiujin
authors.initials: X
authors.lastname: Li
authors.forename: Yanlin
authors.initials: Y
authors.lastname: Chen
mesh_terms: D000641:Ammonia / Q000378:metabolism* / Q000494:pharmacology; D001119:Arginase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D001123:Argininosuccinate Lyase / Q000235:genetics / Q000378:metabolism; D001124:Argininosuccinate Synthase / Q000235:genetics / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D015971:Gene Expression Regulation, Enzymologic; D005974:Glutamate-Ammonia Ligase / Q000235:genetics / Q000378:metabolism; D005973:Glutamine / Q000096:biosynthesis; D056945:Hep G2 Cells / Q000201:enzymology*; D006801:Humans; D019164:Liver, Artificial; D060847:Metabolic Engineering; D009954:Ornithine Carbamoyltransferase / Q000096:biosynthesis* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D014508:Urea / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D005973:Glutamine; D000641:Ammonia; D014508:Urea; D009954:Ornithine Carbamoyltransferase; C477631:ARG2 protein, human; D001119:Arginase; D001123:Argininosuccinate Lyase; D005974:Glutamate-Ammonia Ligase; D001124:Argininosuccinate Synthase