Limited Role for the Bilirubin-Biliverdin Redox Amplification Cycle in the Cellular Antioxidant Protection by Biliverdin Reductase

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, H₂O₂ 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 H₂O₂-mediated cell death, just as BVR overexpression failed to enhance protection of these cells against H₂O₂-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 H₂O₂ 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.Biliverdin reductase (BVR)³ forms part of the major pathway for the disposition of cellular heme in mammalian cells. This pathway is initiated by heme oxygenase, which converts heme to carbon monoxide, iron, and biliverdin, which in turn is reduced to bilirubin by BVR at the expense of NADPH. Because of its intramolecular hydrogen bonding, the bilirubin produced is sparingly soluble in water at physiological pH and ionic strength (1). Hence, bilirubin is usually tightly bound to albumin in order to be transported within the blood circulation (2), from which it is removed mainly through uptake by hepatocytes. Once bilirubin is transferred across the cell membrane of hepatocytes, it binds glutathione S-transferases before being transformed to water-soluble derivatives by conjugation of one or both of its propionyl groups before its excretion into bile and then the intestine (3).Under physiological conditions, plasma bilirubin concentrations in humans range from ∼5 to 20 μm, practically all of which is unconjugated pigment bound to albumin (1). Abnormally high plasma concentrations are associated with the risk of developing neurologic dysfunction due to preferential deposition of bilirubin in brain and its toxic effects on cell functions. In fact for many years, biliverdin and bilirubin were generally regarded as waste products of heme metabolism in higher animals, although earlier work suggested that these bile pigments might play a role as natural antioxidants, since small quantities of the pigment stabilize vitamin A and β-carotene during intestinal uptake, and animals with low plasma bilirubin showed early signs of vitamin E deficiency (4, 5).In a series of in vitro studies, Stocker et al. (6–8) demonstrated that unconjugated bilirubin, at micromolar concentrations, efficiently scavenged peroxyl radicals in homogenous solution or multilamellar liposomes. At physiologically relevant oxygen tension, bilirubin surpassed α-tocopherol as an antioxidant in liposomes (8), and it is thought to protect plasma proteins and lipids from many but not all oxidants (9). However, it is less clear whether this antioxidant activity extends to in vivo situations or protection of cells from oxidative stress. Although produced in essentially all cells, the normal range of cellular bilirubin concentrations is unknown. However, it is probably in the low nanomolar range, well below that of established cellular antioxidants, such as glutathione and ascorbate, arguing against bilirubin being an important cellular antioxidant. Nonetheless, in vitro studies with rat neuronal cultures showed that the presence of 10 nm bilirubin in the culture medium protected cells against 10,000-fold higher concentrations of hydrogen peroxide (10). Later, Barañano et al. (11) confirmed such observations in HeLa cells and demonstrated that BVR depletion increased reactive oxygen species (ROS) and cell death. This led to the following proposal of the BVR-amplified redox cycle. While acting as an antioxidant, bilirubin is oxidized to biliverdin that is then reduced back to bilirubin by the ubiquitous and abundant BVR.An important underlying assumption of this amplification cycle is that ROS-mediated bilirubin oxidation in cells is specific and yields substantial if not stoichiometric amounts of biliverdin. Inconsistent with this assumption, however, earlier studies showed that high yields of biliverdin formation are limited to certain oxidants (i.e. peroxyl radicals) and albumin-bound bilirubin. In cells, bilirubin is probably present in membranes, bound to proteins other than albumin, or present in conjugated form. Therefore, we reexamined the putative redox amplification cycle. Our results show that reaction of these forms of bilirubin with 1e- or 2e-oxidants at best generates modest amounts of biliverdin. Furthermore, overexpression of BVR does not protect mammalian or yeast cells from hydrogen peroxide-mediated damage, thereby casting doubt on the importance of the putative BVR redox cycle for cellular antioxidant protection.