Abstract
In mammalian cells, heme is degraded by heme oxygenase to biliverdin, which is then reduced to bilirubin by biliverdin reductase (BVR). Both bile pigments have reducing properties, and bilirubin is now generally considered to be a potent antioxidant, yet it remains unclear how it protects cells against oxidative damage. A presently popular explanation for the antioxidant function of bilirubin is a redox cycle in which bilirubin is oxidized to biliverdin and then recycled by BVR. Here, we reexamined this putative BVR-mediated redox cycle. We observed that lipid peroxidation-mediated oxidation of bilirubin in chloroform, a model of cell membrane-bound bilirubin, did not yield biliverdin, a prerequisite for the putative redox cycle. Similarly, H(2)O(2) did not oxidize albumin-bound bilirubin to biliverdin, and in vitro oxidation of albumin or ligandin-bound bilirubin by peroxyl radicals gave modest yields of biliverdin. In addition, decreasing cellular BVR protein and activity in HeLa cells using RNA interference did not alter H(2)O(2)-mediated cell death, just as BVR overexpression failed to enhance protection of these cells against H(2)O(2)-mediated damage, irrespective of whether bilirubin or biliverdin were added to the cells as substrate for the putative redox cycle. Similarly, transformation of human BVR into hmx1 (heme oxygenase) mutant yeast did not provide protection against H(2)O(2) toxicity above that seen in hmx1 mutant yeast expressing human heme oxygenase-1. Together, these results argue against the BVR-mediated redox cycle playing a general or important role as cellular antioxidant defense mechanism.
Highlights
Biliverdin reductase (BVR)3 forms part of the major pathway for the disposition of cellular heme in mammalian cells
We examined the yield of biliverdin formation using bilirubin bound to albumin or glutathione S-transferase (GST) and aqueous peroxyl radicals derived from thermolabile AAPH as the oxidant
In sharp contrast to peroxyl radicals, albumin-bound bilirubin was essentially resistant to H2O2, a non-radical oxidant that was generated at constant rate using glucose oxidase plus glucose, with negligible biliverdin being detected (Fig. 1D)
Summary
Materials—HPLC grade methanol and glacial acetic acid were obtained from Scharlau (Barcelona, Spain). 2,2Ј-Azobis(2amidinopropane)hydrochloride (AAPH) and 2,2Ј-azobis(2,4dimethylvaleronitrite) (AMVN) were obtained from Wako Pure Chemical Industries (Japan). An aliquot of the resulting supernatant corresponding to 200 g of protein (determined by the bicinchoninic acid assay) was added to an NADPH-generating system in a plastic cuvette at the following final concentrations: 200 g of cellular protein, 5–50 M biliverdin added as DMSO solution, 2 mM glucose 6-phosphate, 1 unit of glucose 6-phosphate dehydrogenase, 1 mM NADPH in Buffer A (PBS containing 250 mM D-glucose, 20 mM Tris, and one protease inhibitor mixture tablet, pH 7.3). Reverse Transcription-PCR of Yeast cDNA for BVR and HO-1— Yeast cells were grown to exponential phase (A600 ϭ 1), cells were broken in TRIzol reagent (Invitrogen) on a vortex in the presence of acid-washed glass beads for 45 s followed by placement on ice for 30 s; and the RNA was extracted according to the manufacturer’s instructions.
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