ROLE OF MULTIDRUG RESISTANCE-ASSOCIATED PROTEIN 1 EXPRESSION IN THE IN VITRO SUSCEPTIBILITY OF RAT NERVE CELL TO UNCONJUGATED BILIRUBIN A. S. FALCÃO, a C. BELLAROSA, b A. FERNANDES, a M. A. BRITO, a R. F. M. SILVA, a C. TIRIBELLI b AND D. BRITES a * a Centro de Patogénese Molecular–UBMBE, Faculdade de Farmácia, University of Lisbon, Avenida Professor Gama Pinto, 1649-003 Lis- bon, Portugal b Centro Studi Fegato, Bld Q, AREA Science Park, Basovizza Campus, and Department of BBCM, University of Trieste, 34012 Trieste, Italy Abstract—Nerve cell injury by unconjugated bilirubin (UCB) has been implicated in brain damage during neonatal hyper- bilirubinemia, particularly in the preterm newborn. Recently, it was shown that UCB is a substrate for the multidrug resis- tance-associated protein 1 (Mrp1), an ATP-dependent efflux pump, which may decrease UCB intracellular levels. To ob- tain a further insight into the role of Mrp1 in the increased vulnerability of immature cells to UCB, we evaluated the mRNA and the protein levels of Mrp1 throughout differentia- tion in primary cultures of rat neurons and astrocytes. Fur- thermore, in order to provide supportive evidence for the role of Mrp1 in the protection of nerve cells from UCB-induced effects, we evaluated cell susceptibility to UCB when Mrp1 was inhibited with MK571 ((E)-3-[[[3-[2-(7-chloro-2-quinolinyl) ethenyl]phenyl]-[[3-dimethylamino)-3-oxopropyl]thio]methyl]- thio]-propanoic acid). The results are the first to demonstrate that Mrp1 is expressed in neurons and that both mRNA and protein levels of Mrp1 increase with cell differentiation. Addi- tionally, inhibition of Mrp1 was associated with an increase in UCB toxic effects, namely cell death, cell dysfunction, and se- cretion of interleukin (IL)-1, tumor necrosis factor (TNF)-, as well as of glutamate. These results point to a novel role of Mrp1 in the susceptibility of premature babies to UCB encephalopa- thy, and provide a startup point for the development of a new therapeutic strategy. © 2006 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: astrocytes, multidrug resistance-associated pro- tein 1, neural cell development, neurons, prematurity, uncon- jugated bilirubin. High concentrations of unconjugated bilirubin (UCB) are commonly observed in newborn infants, due to a shorter life span of the erythrocytes and a reduced hepatic clear- ance of UCB (Rubaltelli and Griffith, 1992). This condition may lead to the deposition of UCB in the CNS causing transient bilirubin encephalopathy, with minimal neurolog- ical injury, or a more severe and permanent condition, called kernicterus (Porter and Dennis, 2002; Oh et al., 2003), by mechanisms that are still not completely clari- fied. Interest in bilirubin encephalopathy has been reawak- ened by the re-emergence of kernicterus as a clinically relevant phenomenon (Weinberger, 1987; Hansen, 2000; Ebbesen, 2000; Wennberg et al., 2006). Recent observations indicate that UCB induces the disruption of several vital functions rather than a single death pathway (Ostrow et al., 2004). In fact, we demon- strated that exposure of nerve cells to UCB increases the apoptotic and necrotic cell death (Silva et al., 2002), as well as the extracellular concentration of glutamate by decreased uptake (Silva et al., 1999) and/or enhanced secretion (Fernandes et al., 2004). These UCB toxic ef- fects vary with nerve cell type, since astrocytes are func- tionally more affected, revealing increased glutamate se- cretion and higher inhibition of glutamate uptake, whereas neurons are more sensitive to cell death by both necrosis and apoptosis (Silva et al., 2002). The levels of the proin- flammatory cytokines tumor necrosis factor (TNF)- and interleukin (IL)-1 detected following interaction of astro- cytes with UCB (Fernandes et al., 2004; Falcão et al., 2005), may further contribute for the pigment neurotoxicity (Fernandes et al., 2006). Interestingly, neurons can also participate in this UCB-induced immunological response by increasing TNF- secretion, although with levels signif- icantly lower than glial cells. The UCB-induced immunos- timulant effects and neural toxicity also vary with the mat- uration state, since immature nerve cells are more reactive to UCB than the most differentiated ones (Falcão et al., 2006). This fact may provide an additional supportive ev- idence for the higher susceptibility of premature babies to UCB neurotoxicity during moderate to severe neonatal jaundice (Gourley, 1997; Dennery et al., 2001; Kaplan and Hammerman, 2004). Multidrug resistance-associated protein 1 (Mrp1) is an ATP-dependent plasma membrane efflux pump, member of the superfamily of ATP-binding cassette (ABC) trans- porters (Borst et al., 1999). Mrp1 has been shown to be expressed in rat brain endothelial cells and/or the choroid plexus epithelium of the blood– cerebrospinal fluid barrier and plays an important role in the cellular export of a wide *Corresponding author. Tel: +351-217946450; fax: +351-217946491. E-mail address: dbrites@ff.ul.pt (D. Brites). Abbreviations: C T , cycle number at which the fluorescence passes the threshold level of detection; DIV, days in vitro; DMEM, Dulbecco’s modified Eagle’s medium; FCS, fetal calf serum; HBSS-1, Hanks’ balanced salt solution; HBSS-2, Hanks’ balanced salt solution without Ca 2+ and Mg 2+ ; HSA, human serum albumin; IL, interleukin; LDH, lactate dehydrogenase; MK571, (E)-3-[[[3-[2-(7-chloro-2-quinolinyl) ethenyl]phenyl]-[[3-dimethylamino)-3-oxopropyl] thio]methyl]thio]-pro- panoic acid; Mrp1, multidrug resistance-associated protein 1; MTT, (3-(4,5-dimethylthiazol, 2-yl)-2,5-diphenyltetrazolium bromide, NF-B, nuclear factor-B; P-gp, P-glycoprotein; TNF-, tumor necrosis fac- tor-; TTBS, 0.2% Tween 20, 20 mM Tris-HCl (pH 7.5), 500 mM NaCl; UCB, unconjugated bilirubin. Neuroscience 144 (2007) 878 – 888 0306-4522/07$30.00+0.00 © 2006 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2006.10.026 878