Characterisation of bilirubin metabolic pathway in hepatic mitochondria

Abstract

Bilirubin (BR), a toxic waste product of degraded haem, is a potent antioxidant at physiological concentrations. To achieve the maximum benefit of BR, its intracellular level needs to be carefully regulated. A system comprising of two enzymes, haem oxygenase-1 (HMOX1) and cytochrome P450 2A5 (CYP2A5) exists in the endoplasmic reticulum (ER), responsible for regulating BR homeostasis. This system is induced in response to oxidative stress. In this thesis, oxidative stress caused accumulation of these enzymes in mitochondria — major producers and targets of reactive oxygen species (ROS) — is demonstrated. To understand the significance of this intracellular targeting, properties of microsomal and mitochondrial BR metabolising enzymes were compared and the capacity of mitochondrial CYP2A5 to oxidise BR in response to oxidative stress is reported. Microsomes and mitochondrial fractions were isolated from liver homogenates of DBA/2J mice, administered with sub-toxic dose of pyrazole, an oxidant stressor. The purity of extracted organelles was determined by analysing the expressions and activities of their respective marker enzymes. HMOX1 and CYP2A5 were significantly increased in microsomes and even more so in mitochondria in response to pyrazole-induced oxidative stress. By contrast, the treatment did not increase either microsomes or mitochondrial Uridine-diphosphate-glucuronosyltransferase 1A1 (UGT1A1), the sole enzyme that catalyses BR elimination through glucuronidation. In response to pyrazole-induced oxidative stress, BR oxidation was enhanced not only in microsomes but also in mitochondria. The reaction in both control and pyrazole treated microsomes were blocked by up to 70% with anti-CYP2A5 antibody. By contrast, no CYP2A5 antibody inhibition on BR oxidation was observed in control mitochondria but was 55% inhibited in treated mitochondria. Antibody against adrenodoxin reductase (AdxR) (a component of mitochondrial monooxygenase system) had no effect on the microsomal BR oxidation, but the reaction was inhibited by up to 50% in mitochondria after pyrazole treatment. As in microsomes, the antibody had no effect in control mitochondria. Inhibition study with ascorbic acid — a scavenger of ROS — was undertaken to determine the contribution of ROS to BR oxidation. In control microsomes, a slight inhibition of 5% in BR degradation was noted which was augmented to 22% after pyrazole treatment. The inhibition was 100% in control mitochondria but limited to 50% after pyrazole treatment. Mass spectrometry analysis reveals that in microsomes, biliverdin (BV), the main metabolite of CYP2A5-dependant BR oxidation was formed. Meanwhile, mitochondria produced predominantly dipyrroles, the products of BR scavenging ROS. Pyrazole treatment caused mitochondria to yield relatively similar amount of BV and dipyrroles. Enzyme kinetic analysis showed that mitochondrial and microsomal CYP2A5 have an equally strong affinity towards BR. Additionally, no initiation of apoptosis, as indicated by the absence of cytosolic cytochrome C was observed in treated liver. Collectively, these observations suggest that: i) both HMOX1 and CYP2A5 are recruited into mitochondria to regulate BR metabolism during oxidative stress; (ii) CYP2A5-dependent BR oxidation in mitochondria is driven by the mitochondria-specific electron transfer chain; (iii) constitutive mitochondrial BR oxidation is driven un-enzymatically yet equally driven by ROS and CYP2A5 in response to oxidative stress; (iv) BR affinity to mitochondrial CYP2A5 is in the same range to that of microsomal CYP2A5; (v) mitochondrial BR is not eliminated by glucuronidation but recycled by CYP2A5 oxidation to BV; and (vi) mitochondrial targeting of enzymes crucial for BR homeostasis is not associated with initiation of mitochondria specific apoptosis. It is thus submitted that targeting of key BR regulatory enzymes to mitochondria is an adaptive response to oxidative stress, aimed at mitochondrial protection against oxidative damage.