HepG2 cells do not express xanthine oxidoreductase (XOR): Implications for XOR and uric acid-related research

Although the relative extent of xanthine oxidoreductase (XOR) varies considerably in human tissues, the greatest specific activity is reported in the liver and intestines. Unlike murine models, where primary hepatocytes are readily available, human counterparts are not. As such, investigators often utilize the human carcinoma cell line HepG2 for in vitro experimentation as these cells proliferate well in culture medium. Some of the studies using HepG2 cells for proof-of-principal experimentation focus on uric acid (UA) and/or XOR activity. However, it has been reported that hepatocellular carcinoma diminishes XOR expression to nearly unmeasurable levels when compared to normal cell counterparts which posits the question of validity in the context of using HepG2 cells for XOR/UA assessments. As such, we closely examined XOR expression, protein abundance, and enzymatic activity in HepG2 cells and compared these results to an immortalized murine hepatocyte line (AML12) as well as murine liver homogenate. We report the absence of detectable XOR message, protein, and enzymatic activity in HepG2 cells. Since cellular XOR expression has been reported to be stimulated by hypoxia and serum starvation, we then exposed both AML12 and HepG2 cells to hypoxia (1% O₂) for 24 h or serum starvation which impacted XOR activity in AML12 cells, but not in HepG2 cells. Knowing that several studies report XOR activity in HepG2 cells, we compared a commonly used non-selective XOR assay kit to our sensitive and selective HPLC/electrochemical assay. The kit assay indicated XOR activity in HepG2 cells; however, XOR inhibition did not diminish this activity. To account for alternative enzymatic oxidization of hypo/xanthine to UA, we investigated HepG2 cells for aldehyde oxidase (AO) abundance and found immunodetectable AO protein as well as AO-inhibitable conversion of hypoxanthine to UA; albeit, at a much slower rate when compared to XOR. In toto, these data confirm that HepG2 cells do not demonstrate XOR activity and thus commonly used kit assays for XOR, in the absence of XOR inhibitor controls, may indicate a positive response induced by an alternative source of peroxide and UA such as AO.