Unconjugated bilirubin differentially affects the redox status of neuronal and astroglial cells

Abstract

We investigated whether nerve cell damage by unconjugated bilirubin (UCB) is mediated by oxidative stress and ascertained the neuronal and astroglial susceptibility to injury. Several oxidative stress biomarkers and cell death were determined following incubation of neurons and astrocytes isolated from rat cortical cerebrum with UCB (0.01–1.0 μM). We show that UCB induces a dose-dependent increase in neuronal death in parallel with the oxidation of cell components and a decrease in the intracellular glutathione content. Comparison of the results obtained in both cell types demonstrates that neurons are more vulnerable than astrocytes to oxidative injury by UCB, for which accounts the lower glutathione stores in neuronal cells. Moreover, neuronal oxidative injury is prevented by supplementation with N-acetylcysteine, a glutathione precursor, whereas astroglial sensitivity to UCB is enhanced by inhibition of glutathione synthesis, using buthionine sulfoximine. Collectively, we demonstrate that oxidative stress is involved in UCB neurotoxicity and depict a new therapeutic approach for UCB-induced oxidative damage.

Introduction

Unconjugated bilirubin (UCB) is the principal end product of heme catabolism. Increased levels of UCB are responsible for the clinical manifestation of jaundice, a common condition in the neonatal period usually referred as physiologic jaundice of the newborn.

UCB has been regarded as a natural antioxidant since the pioneer studies of Stocker et al. (1987). Because of the antioxidant properties of low UCB concentrations, it is nowadays believed that physiologic jaundice may have inherent benefits (Sedlak and Snyder, 2004). However, in some newborn infants, plasma UCB levels can increase dramatically owing to impaired postnatal maturation of hepatic transport or conjugation of UCB, and/or enhanced entero-hepatic circulation of UCB, or augmented hemolysis (Brito et al., 2006b). If untreated, and depending on the level of severity, hyperbilirubinemia can lead to minor brain deficits, acute bilirubin encephalopathy, kernicterus, or even death (American Academy of Pediatrics, 2004, Harris et al., 2001, Kaplan and Hammerman, 2005, Soorani-Lunsing et al., 2001).

Different brain cells, as neurons and astrocytes, have their own functions and specialized machinery, and tend to have unique responses towards a stimulus (Brito et al., in press). Comparative data on the cell susceptibility to UCB showed that neurons are more sensitive than astrocytes to cytoskeleton disruption and to necrotic or apoptotic cell death, while astrocytes are more disturbed than neurons regarding both MTT [(3-(4,5-dymethylthiazol, 2-yl)-2,5-diphenyltetrazolium bromide] metabolism and glutamate uptake (Silva et al., 2002). In addition, astrocytes are more competent in releasing glutamate and display a more marked inflammatory response than neurons, as indicated by the higher secretion of pro-inflammatory cytokines and the highest activation of the nuclear factor-κB (Falcão et al., 2006).

Although the primary concern with respect to hyperbilirubinemia is the potential for neurotoxic effects, general cellular injury also occurs. Our previous studies conducted in erythrocytes, isolated brain mitochondria and synaptosomal membrane systems indicated that UCB-induced cytotoxicity is mediated, at least in part, by perturbation of cell membranes (Brito et al., 2000, Brito et al., 2001, Brito et al., 2002, Brito et al., 2004, Rodrigues et al., 2002a). In fact, morphological changes, alterations in membrane composition and assembly, with the accompanying calcium intrusion and cytochrome c release, are among the UCB effects. An increase in protein mobility, and an elevation of lipid polarity and fluidity, as well as a disorder of the redox status also result from the physical interaction of UCB with membranes. In addition, reactive oxygen species (ROS) production, protein oxidation and lipid peroxidation, together with a disruption of glutathione homeostasis occur by UCB exposure. When taken together, these observations point to oxidative injury as a relevant component of UCB toxicity in several experimental systems. However, the contribution of oxidative stress in the mechanisms of neurotoxicity by UCB, namely the spectrum of the oxidative effects in different nerve cell types, remains to be characterized.

Oxidative stress occurs when the physiological balance between radical-generating and radical-scavenging is disrupted in favour of the former (Sies, 1997). In this condition, oxidized nucleic acids, proteins and lipids accumulate as a result of the ROS attack to cell components. Protein carbonyls and 4-hydroxy-2-nonenal (HNE) are widely used as reliable markers of protein oxidation and lipid peroxidation, respectively (Butterfield et al., 2002, Butterfield and Stadtman, 1997, Stadtman, 1992). The antioxidant capacity of tissues can also be assessed by quantification of oxidative stress markers, as glutathione, which is one of the major cell antioxidants and reflects the chain-breaking thiol antioxidant capacity (Dringen, 2000). Glutathione is a tripeptide, γ-l-glutamyl-l-cysteinylglycine, synthesized by the action of two enzymes, γ-glutamylcysteine (γGluCys) synthetase and glutathione synthetase, at expenses of ATP. Although this two-steps synthesis takes place in both neurons and astrocytes, neurons cannot use cysteine as a cysteine donor, relying on astrocytes as a cysteine donor in vivo. Moreover, intracellular glutathione stores are greater in astroglial cells than in neuronal ones, as well as in neurons co-cultures with astrocytes, as compared to isolate neuron cultures. This fact, together with the greater antioxidant enzymatic machinery of astrocytes, renders astroglial cells more resistant than neuronal ones to free radicals and oxidant insults and, thus, to stressful situations (Brito et al., in press).

In this study, we evaluate several oxidative stress biomarkers, as well as cell death, following incubation of primary culture of rat neurons and astrocytes obtained from cerebrum cortex with free UCB (0.01–1.0 μM). We show that the UCB-induced loss of cell viability is associated with a disruption of the redox status, which involves oxidative damage of cell components, as well as impairment of the cellular antioxidant defense system provided by glutathione. Comparison of the results obtained in neurons and astrocytes demonstrate that neurons are more vulnerable than astrocytes to oxidative injury by UCB, to which contributes the lower intracellular levels of glutathione in neuronal cells. Finally, by demonstrating that oxidative injury to neurons is prevented by supplementation with a glutathione precursor, whereas astroglial sensitivity to the pro-oxidant effects of UCB is enhanced by inhibition of the thiol synthesis, the present results point to the restoration of glutathione homeostasis as a new potential therapeutical approach for UCB-induced oxidative damage.