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|Title: ||Impact of unconjugated bilirubin on brain parenchyma of the Gunn rat|
|Authors: ||Berengeno, Andrea Lorena|
|Supervisor/Tutor: ||Ghersi Egea, Jean Francois|
|Co-Advisor: ||Gazzin, Silvia|
|Issue Date: ||28-Apr-2011|
|Publisher: ||Università degli studi di Trieste|
|Abstract: ||In infants and in the Crigler-Najjar syndrome type I patients, severe hyperbilirubinemia due to high levels of unconjugated bilirubin (UCB) may cause Kernicterus, leading to an irreversible and selective brain damage. The Gunn rat is the animal model for the study of these pathologies.
It has been suggested that different enzymes of the phase I (cytochrome P-450-dependent mixed function oxygenases 1A1, 1A2, 2A3), phase II (glutathione-S-transferases α2, α3, µ3, µ4, π) and phase III transporters (particularly, Mrp1) seems to be involved in UCB detoxification pathways. However, to date, their in vivo brain expression has been evidenced only at the blood-brain interfaces, while remains largely unexplored in brain parenchyma.
Particularly for Mrp1, in vitro evidence reported a role of this transporter in protection of neural primary cultures from dissected cortex, by extruding bilirubin out of the cell.
The aim of this study is establish the developmental profile of these genes in brain parenchyma, and assess their alteration in hyperbilirubinemic jj animals. Due to the high regional selectivity of UCB-induced neurotoxicity, cerebellum (Cll), striatum (St), hippocampus (Hip) and cerebral cortex (Cx) were chosen for this study.
Our results regard the Mrp1 protein in cerebral cortex of normobilirubinemic (JJ) rats showed that its expression varied during the post-natal age, reaching the highest levels at 9 days after birth. No changes were found between JJ and Jj (having a temporary hyperbilirubinemia in the first week of life) rat for all ages analyzed. Similarly, no differences were detected among JJ/Jj and jj (hyperbilirubinemic) rats at P2, P17 and P60, while a significant increase (p < 0.005) was evidenced in P9 jj rats as compared to age-matched JJ animals.
Our Mrp1 mRNA analysis in four regions of P9 animals by Real Time-qPCR revealed the absence of differences among Cx, Cll, St and Hip of P9 normobilirubinemic JJ rats. Moreover, no variations between jj and JJ control animals were detected.
Regarding the Mrp1 protein expression in the same four regions by Western blot analysis, our results showed that the levels of this transporter in normobilirubinemic JJ rats were lower in Cx, similar in Cll, St and Hip (p < 0.05 vs Cx). Comparing genotypes, a reduction on Mrp1 in jj animals (compared to Mrp1 amount in the same region of JJ pups) was detected in Cll, St, but reached the statistical significance only in Hip (p < 0.05 vs Hip JJ).
The analysis of CYPs gene expression in P9 Gunn rats indicate that CYP1A1, 1A2 and 2A3 mRNA were differently expressed among Cx, Cll, St and Hip of JJ rat. Similarly a region-specific modulation of CYPs expression in jj Gunn rats (compared to JJ) was pointed-out. Surprisingly, UCB seems to generate a plateau effect on CYPs mRNA levels among brain regions of jj rats.
In P60 JJ Gunn rats the CYPs expression is higher than in P9 animals, with the following pattern among regions: Cx CllSt Hip. A down-regulation (p < 0.05) in St of P60 jj compared to normal animals was observed.
Analyzing the GSTs expression in P9 animals, higher variability in the GSTs expression among the four brain areas was evidenced. In hyperbilirubinemic (jj) rats (compared to JJ), statistically relevant down-regulations were detected for GSTα2 (in St;p < 0.05), GSTα3 (in Hip;p < 0.05), µ3 (in Cx;p < 0.01), µ4 (in Cx; p < 0.05) and π (in Cll: p < 0.05); while GSTµ4 was up-regulated in St (p < 0.05). From P9 to P60, in JJ animals: GSTα3 expression increased (13-75-fold depending on the region); while GSTα2 (5-fold), µ3 (p < 0.05), µ4 (2-fold) and π (2-fold) mRNA amounts decreased. In P60 jj Gunn rats, compared to controls (JJ):a relevant up-regulation of GSTα3 was observed in Cll (p < 0.005) and Hip (p < 0.05), while GSTµ3 in jj was down-regulated (p < 0.05).
The Mrp1 results obtained in the present in vivo study seems not to be in agreement with the in vitro data reported, to date. Thus, the Mrp1 expression is low in brain parenchyma and bilirubin affect (up-regulation) only marginally the protein amounts in cortex of P9 animals, while in other regions Mrp1 is not modulated, indicating a marginal role in vivo in bilirubin clearance.
Similarly, while in liver GSTα2 and α3 act together as ligandin, this seems not happens in brain where the two subunit are expressed at very low levels (P9: α2 77000- α3 1500 fold difference; P60: α2 112000-α3 2200 fold difference with respect to age matched livers).
For all genes under analysis, a very complex and variable pattern of expression among brain areas was evidenced. Consequently, no general rules concerning bilirubin-induced modulation could be drawn, as both up and down-regulation were observed. Additionally, in Cx of P9 jj animals, a translational control of Mrp1 might be hypothesized due to a significant increase in Mrp1 protein, without changes in mRNA level.
Therefore, the genomic screening made in this work provides the first general overview on the mRNA developmental profiles of several CYPs and GSTs genes in brain parenchyma (specifically Cx, Cll, St and Hip) of normal rats, and of animals suffering from hyperbilirubinemia, underlying the necessity to find functional evidence to finally understand the role of these enzymes associated with the kernicterus and Crigler-Najjar type I syndrome pathologies.|
|PhD cycle: ||XXIII Ciclo|
|PhD programme: ||SCUOLA DI DOTTORATO DI RICERCA IN BIOMEDICINA MOLECOLARE|
|Main language of document: ||en|
|Type: ||Tesi di dottorato|
|Scientific-educational field: ||BIO/11 BIOLOGIA MOLECOLARE|
|Appears in Collections:||Scienze biologiche|
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