Haem oxygenase shows pro-oxidant activity in microsomal and cellular systems: implications for the release of low-molecular-mass iron

Haem oxygenase-1 (HO-1) is a highly inducible stress protein that removes h aem from cells with the release of biliverdin, carbon monoxide and low-mole cular-mass iron (LMrFe). Several antioxidant functions have been ascribed t o HO; its induction is considered to be a protective event. However, LMrFe produced during haem catabolism might elicit a pro-oxidant response, with d eleterious consequences. We therefore investigated the delicate balance bet ween pro-oxidant and antioxidant events with the use of a microsomal lipid peroxidation (LPO) system. By using microsomal-bound HO in an NADPH-depende nt LPO system, we assessed the pro-oxidant nature of the released LMrFe and the antioxidant effect of the released bilirubin. Hb, a biologically relev ant substrate for HO, was included with the microsomes to supplement the so urce of haem iron and to promote LPO. We found significant increases in mic rosomal LPO, by using the thiobarbituric acid (TBA) test, after incubation with Hb. This Hb-stimulated peroxidation was inhibited by HO inhibitors and by iron chelators, suggesting a MO-driven, iron-dependent mechanism. GLC-M S was employed to measure the specific LPO product 4-hydroxy-2-nonenal and to confirm our TEA test results. A HO inhibitor attenuated an increase in i ntracellular LMrFe that occurred after treatment of rat pulmonary artery sm ooth-muscle cells with Hb. Additionally, exogenously added bilirubin at an equimolar concentration to the LMrFe present in both microsomal and liposom al systems was unable to prevent the pro-oxidant effect of the iron. Under certain circumstances HO can act as a pro-oxidant and seems to have a role in stimulating microsomal LPO.