A CHROMATOGRAPHIC ASSAY FOR HEME OXYGENASE ACTIVITY IN CULTURED HUMAN-CELLS - APPLICATION TO ARTIFICIAL HEME OXYGENASE OVEREXPRESSION

Heme oxygenase (HO) activity oxidizes heme, releasing carbon monoxide; heme iron; and biliverdin, which is converted to bilirubin by biliver din reductase. Inducible HO-I expression is a marker of oxidative stre ss in mammalian cells, while noninducible HO-II contributes to basal H O activity. HO-I and HO-II activities are implicated in cellular antio xidant defense mechanisms. We describe a microassay for HO activity in cultured human cells, using highperformance liquid chromatography of biliverdin and bilirubin. The assay is sufficiently sensitive to quant ify basal and inducible HO activity in various human cell types. We ha ve established human cell lines overexpressing heme oxygenase-II activ ity in microsomes using a metallothionein promoter-regulated expressio n system. Stable transformants treated with ZnCl2 express up to ninefo ld induction of HO activity. We have constructed human cell lines over expressing HO-LI protein and activity (5-15-fold) in the absence of te tracycline, using the HtTA-1 cell line transfected with tetracycline-r egulated expression vectors (Gossen et al., Proc. Natl. Acad. Sci. USA 89, 1992). Functional HO-II overexpressing clones will be useful in i nvestigating anti-or pro-oxidant effects of HO activity during cellula r oxidative stress. (C) 1998 Elsevier Science Inc.