Pablo Giraudi

Kinetics and Specificity of Feline Leukemia Virus Subgroup C Receptor (FLVCR) Export Function and Its Dependence on Hemopexin

The feline leukemia virus subgroup C receptor (FLVCR) is a heme export protein that is required for proerythroblast survival and facilitates macrophage heme iron recycling. However, its mechanism of heme export and substrate specificity are uncharacterized. Using [55Fe]heme and the fluorescent heme analog zinc mesoporphyrin, we investigated whether export by FLVCR depends on the availability and avidity of extracellular heme-binding proteins. Export was 100-fold more efficient when the medium contained hemopexin (Kd < 1 pm) compared with albumin (Kd = 5 nm) at the same concentration and was not detectable when the medium lacked heme-binding proteins. Besides heme, FLVCR could export other cyclic planar porphyrins, such as protoporphyrin IX and coproporphyrin. However, FLVCR has a narrow substrate range because unconjugated bilirubin, the primary breakdown product of heme, was not transported. As neither protoporphyrin IX nor coproporphyrin export improved with extracellular hemopexin (versus albumin), our observations further suggest that hemopexin, an abundant protein with a serum concentration (6.7–25 μm) equivalent to that of the iron transport protein transferrin (22–31 μm), by accepting heme from FLVCR and targeting it to the liver, might regulate macrophage heme export and heme iron recycling in vivo. Final studies show that hemopexin directly interacts with FLVCR, which also helps explain why FLVCR, in contrast to some major facilitator superfamily members, does not function as a bidirectional gradient-dependent transporter. Together, these data argue that hemopexin has a role in assuring systemic iron balance during homeostasis in addition to its established role as a scavenger during internal bleeding or hemolysis.

Cytotoxicity Is Predicted by Unbound and Not Total Bilirubin Concentration

Although it has been suggested that the unbound, free, (Bf) rather than total (BT) bilirubin level correlates with cell toxicity, direct experimental evidence supporting this conclusion is limited. In addition, previous studies never included a direct measurement of Bf, using newer, accurate methods. To test “the free bilirubin hypothesis”, in vitro cytotoxicity was assessed in four cell lines exposed to different Bf concentrations obtained by varying BT/Albumin ratio, using serum albumins with different binding affinities, and/or displacing unconjugated bilirubin (UCB) from albumin with a sulphonamide. Bf was assessed by the modified, minimally diluted peroxidase method. Cytotoxicity varied among cell lines but was invariably related to Bf and not BT. Light exposure decreased toxicity parallel to a decrease in Bf. In the absence of albumin, no cytotoxicity was found at a Bf of 150 nM whereas in the presence of albumin a similar Bf resulted in a 40% reduction of viability indicating the importance of total cellular uptake of UCB in eliciting toxic effect. In the presence of albumin-bound UCB, bilirubin-induced cytotoxicity in a given cell line is accurately predicted by Bf irrespective of the source and concentration of albumin, or total bilirubin level.

Functional induction of the cystine-glutamate exchanger system Xc(-) activity in SH-SY5Y cells by unconjugated bilirubin.

We have previously reported that exposure of SH-SY5Y neuroblastoma cells to unconjugated bilirubin (UCB) resulted in a marked up-regulation of the mRNA encoding for the Na+ -independent cystine∶glutamate exchanger System Xc − (SLC7A11 and SLC3A2 genes). In this study we demonstrate that SH-SY5Y cells treated with UCB showed a higher cystine uptake due to a significant and specific increase in the activity of System Xc −, without the contribution of the others two cystine transporters (XAG − and GGT) reported in neurons. The total intracellular glutathione content was 2 folds higher in the cells exposed to bilirubin as compared to controls, suggesting that the internalized cystine is used for gluthathione synthesis. Interestingly, these cells were significantly less sensitive to an oxidative insult induced by hydrogen peroxide. If System Xc − is silenced the protection is lost. In conclusion, these results suggest that bilirubin can modulate the gluthathione levels in neuroblastoma cells through the induction of the System Xc −, and this renders the cell less prone to oxidative damage.

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

BackgroundThe deposition of unconjugated bilirubin (UCB) in selected regions of the brain results in irreversible neuronal damage, or Bilirubin Encephalopathy (BE). Although UCB impairs a large number of cellular functions in other tissues, the basic mechanisms of neurotoxicity have not yet been fully clarified. While cells can accumulate UCB by passive diffusion, cell protection may involve multiple mechanisms including the extrusion of the pigment as well as pro-survival homeostatic responses that are still unknown.ResultsTranscriptome changes induced by UCB exposure in SH-SY5Y neuroblastoma cell line were examined by high density oligonucleotide microarrays. Two-hundred and thirty genes were induced after 24 hours. A Gene Ontology (GO) analysis showed that at least 50 genes were directly involved in the endoplasmic reticulum (ER) stress response. Validation of selected ER stress genes is shown by quantitative RT-PCR. Analysis of XBP1 splicing and DDIT3/CHOP subcellular localization is presented.ConclusionThese results show for the first time that UCB exposure induces ER stress response as major intracellular homeostasis in surviving neuroblastoma cells in vitro.

Effects of oral administration of silymarin in a juvenile murine model of non-alcoholic steatohepatitis

The increasing prevalence of non-alcoholic fatty liver disease (NAFLD) in adolescents is challenging the global care system. No therapeutic strategies have been defined so far, and changes in the lifestyle remain the only alternative. In this study, we assessed the protective effects of silymarin in a juvenile non-alcoholic steatohepatitis (NASH) model and the in vitro effects on fat-laden human hepatocytes. C57Bl/6 mice were exposed to HFHC diet immediately after weaning. After eight weeks, animals showed histological signs of NASH. Silymarin was added to the HFHC diet, the treatment continued for additional 12 weeks and the effects on BMI, hepatomegaly, visceral fat, lipid profile, transaminases, HOMA-IR, steatosis, inflammation, fibrosis, oxidative stress, and apoptosis were determined. The switch from HFHC to control diet was used to mimic lifestyle changes. In vitro experiments were performed in parallel in human hepatocytes. HFHC diet supplemented with silymarin showed a significant improvement in glycemia, visceral fat, lipid profile, and liver fibrosis. Moreover, it reduced (both in vitro and in vivo) ALT, hepatic inflammation, oxidative stress, and apoptosis. Lifestyle changes restored the control group parameters. The data presented show the beneficial effects of the oral administration of silymarin in the absence of changes in the dietary habits in a juvenile model of NASH.

A simple in silico strategy identifies candidate biomarkers for the diagnosis of liver fibrosis in morbidly obese subjects

Non‐alcoholic fatty liver disease (NAFLD) is a chronic liver disorder, tightly associated with obesity.

579 Bilirubin Upregulates Gene Expression of Slc7A11 and Increases Cystine Uptake Mediated by System Xc- in Sh-Sy5Y Neuroblastoma Cells

Background and aims: Severe neonatal hyperbilirubinemia can produce encephalopathy. We have previously demonstrated that exposure of SHSY5Y cells to unconjugated free bilirubin (Bf) for 24 h induces several genes as the both components of a glutamate-cystine antiporter (System Xc -). Cystine is implicated in glutathione (GSH) formation. Present study was performed to analyze if the upregulation of System excluded induced by Bf offers protection from oxidative stress. Methods: Cell viability was assessed by MTT test in SH-SY5Y cells exposed to 140 nM Bf for 24 h (T0). [14C]-cystine uptake was measured with and without l-quisqualate (an specific System Xc - inhibitor). Cells were then growth in medium for 48h (T48) and 156h (T156) in the absence of Bf. GSH level and viability were measured after H2O2 exposure. Results: Cell viability was reduced 30-40% after 24 h of Bf exposure. Cystine uptake was increased in surviving cells after 24 h Bf exposure. Further incubation with l-quisqualate for 4 h reduced cystine uptake to normal levels. GSH levels were 2.5 times that in control cell both at T0 and T48 but returned to normal levels at T156. Cells were protected from H2O2 exposure when GSH levels were high, but cells death was identical to controls at T156 when GSH levels returned to basal levels. Conclusions: SH-SY5Y cells respond to Bf exposure by an increased gene expression and activity of the System Xc -. This was associated with a higher incorporation of cystine and GSH content which protected the cell from oxidative stress H2O2 induced.