The cytotoxic effect of unconjugated bilirubin in human neuroblastoma SH-SY5Y cells is modulated by the expression level of MRP1 but not MDR1

Abstract

In vitro and in vivo studies have demonstrated that UCB (unconjugated bilirubin) is neurotoxic. Although previous studies suggested that both MRP1 (multidrug resistance-associated protein 1) and MDR1 (multidrug resistance protein 1) may protect cells against accumulation of UCB, direct comparison of their role in UCB transport was never performed. To this end, we used an inducible siRNA (small interfering RNA) expression system to silence the expression of MRP1 and MDR1 in human neuroblastoma SH-SY5Y cells. The effects of in vitro exposure to clinically-relevant levels of unbound UCB were compared between unsilenced (control) cells and cells with similar reductions in the expression of MRP1 or MDR1, documented by RT-PCR (reverse transcription-PCR) (mRNA), immunoblotting (protein), and for MDR1, the enhanced net uptake of a specific fluorescent substrate. Cytotoxicity was assessed by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] test. MRP1-deficient cells accumulated significantly more UCB and suffered greater cytotoxicity than controls. By contrast, MDR1-deficient cells exhibited UCB uptake and cytotoxicity comparable with controls. At intermediate levels of silencing, the increased susceptibility to UCB toxicity closely correlated with the decrease in the expression of MRP1, but not of MDR1. These data support the concept that limitation of cellular UCB accumulation, due to UCB export mediated by MRP1, but not MDR1, plays an important role in preventing bilirubin encephalopathy in the newborn.

Modulation of Mrp1 (ABCc1) and Pgp (ABCb1) by Bilirubin at the Blood-CSF and Blood-Brain Barriers in the Gunn Rat

“…At the same time, the Mrp1 protein level in the choroid plexuses of jj animals is significantly reduced, which may further impair the ability of the brain to protect itself from UCB accumulation and toxicity. This has been shown in vitro where silencing of Mrp1, but not Pgp, is associated with a greater cellular toxicity induced by UCB exposure [58].…”

Global changes in gene regulation demonstrate that unconjugated bilirubin is able to upregulate and activate select components of the endoplasmic reticulum stress response pathway

“…It has also been shown that these transporters may be able to transport UCB [43]. Interestingly, it was demonstrated that UCB was able to protect neuronal cells from its own toxicity by mediating upregulation of Mrp1 [44][45][46]. It has also been suggested that UCB may be a substrate for multidrug resistance protein 1 (Mdr1) and that this transporter may protect neuronal cells from toxicity by limiting its accumulation [47][48][49].…”

A transcriptome analysis identifies molecular effectors of unconjugated bilirubin in human neuroblastoma SH-SY5Y cells

“…To assess UCB effects, we have taken advantage of previous studies where the outcome of clinically relevant UCB concentrations was tested and the cytotoxic effects revealed [15,16]. The sensitivity of SH-SY5Y cells to UCB treatment was thus evaluated by measuring cell viability after exposing cells to a concentration of free bilirubin of 140 nM (Bf 140 nM) for 1, 4 and 24 h. As shown in Figure 1, cell viability was reduced to 60% already after 1 h of UCB treatment and the extent of reduction in cell viability never exceeded the 40% of the cell population, even with longer treatments.…”

“…This has been linked to a higher content of MRP1, a transporter with high affinity to UCB [41] able to keep the intracellular UCB concentration low. Importantly, the level of MRP1 expression in human neuroblastoma SH-SY5Y cells is inversely and linearly correlated with UCB toxic effects [16]. …”