STUDY ON THE ROLE OF CYTOCHROME P450 IN BILIRUBIN-INDUCED ENCEPHALOPATHY

SABRINA ELIANA GAMBARO; SILVIA GAZZIN; M. CELESTE ROBERT; CLAUDIO TIRIBELLI

Venecia

Simposio; International Symposium on Biology and Translational Aspects of Neurodegeneration; 2012

Istituto Veneto di Scienze, Lettere ed Arti (IVSLA), Italian Society of Neurology

STUDY ON THE ROLE OF CYTOCHROME P450 IN BILIRUBIN-INDUCED ENCEPHALOPATHY Gambaro, Sabrina Eliana; Gazzin, Silvia; Robert, Maria Celeste; Tiribelli, Claudio. Fondazione Italiana Fegato - Centro Studi Fegato, SS14 Km 163.5 Bldg. Q AREA Science Park Basovizza, Trieste, Italy Bilirubin encephalopathy is the neurological disorder caused by the toxicity of unconjugated bilirubin (UCB) to neuronal cells. There is a strong evidence of regional and cell specific neurotoxicity. The Gunn rat, unable to eliminate bilirubin due to a genetic absence of glucuronidation, is the animal model used to study this pathology. At birth the homozygote (jj) Gunn rat accumulates bilirubin in the blood rapidly, with a peak at 9 days after the birth (12 mg/dL). Interestingly, during the third week this levels decreases and reach a plateau (5 mg/dL) indicating the existence of alternative mechanism(s) of elimination. The hepatic cytochrome P450 enzymes (Cyps) have been shown to be involved in the oxidation of bilirubin leading to its elimination. The levels of Cyp1A1 and Cyp1A2 expression were markedly elevated in liver in the jaundice rats at the age of 10 days. The hepatic Cyp1A1 mRNA and protein levels increase between 10 days and 1 month and is undetectable in non-jaundiced littermates. In contrast the hepatic Cyp1A2 mRNA and protein are higher in jaundiced rather than in non-jaundiced immediately after the birth, reaching similar levels after a month. Their ability to oxidise bilirubin was demonstrated in vitro by De Matties et al. (1991). Another possible Cyp involved in bilirubin cleareance was CYP2A3 suggested by Abu-Bakar et al. (2005). It is known that the Cyps expression profile varies depending on the tissue with a expression lower in the brain than the liver. Specific expression of Cyps enzymes in certain parts of the brain may reduce accumulation of bilirubin and the resulting cell neurotoxicity. To elucidate the role of Cyps in bilirubin encephalopathy, we first analysed the Cyp/CYP expression in primary culture of cortex astrocytes and cerebellum and cerebellum granular cells of Wistar rat and two hepatoma cell line (Huh7 and HepG2). As expected, the expression of CYP1A1/Cyp1A1 was notably higher in the hepatoma cell lines than in the primary culture. Between the neuronal cultures, the level of Cyp1A1 was higher in cortex astrocytes than the astrocytes and granular cells from cerebellum. The CYP1A2/Cyp1A2 expression was low in all the cells analysed. CYP2A6 (Cyp2A3 in rat) it is important to make notice that was not expressed in the liver cell lines but it was found in all the neuronal cells. The expression in astrocytes from cortex was comparable with the expression in granular cells from cerebellum. Astrocytes and granular cells from cerebellum were exposed to UCB (140nM, in DMSO) for 2 and 24H. A significant increase in the mRNA expression of Cyp1A1 (3; P