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Metyrapone, an inhibitor of cytochrome oxidases, does not affect viability in a neuroblastoma cell model of bilirubin toxicity

View Article: PubMed Central - PubMed

ABSTRACT

Background: Unconjugated hyperbilirubinemia may cause brain damage in infants, and globally remains a source of neonatal morbidity and mortality. A significant inter-individual variability in vulnerability to bilirubin toxicity remains largely unexplained. An enzyme located in mitochondria oxidizes bilirubin. We hypothesized that inhibiting bilirubin oxidation in human neuronal cell cultures exposed to bilirubin would increase cell death.

Methods: The ability of mitochondrial membranes from CHP-212 human neuroblastoma cells to oxidize bilirubin was verified by spectrophotometry. Intact cells in culture were exposed to bilirubin (75 μM) with or without metyrapone (250 μM) for 24 h, stained with Annexin-V and Propidium iodide and analyzed for apoptosis and necrosis by flow cytometry.

Results: Bilirubin caused a significant reduction of viability, from 84 ± 2.0% (mean ± SEM) vs 67 ± 2.7% (p < 0.05), but adding metyrapone to the bilirubin-exposed cells did not further impact cell viability. Metyrapone alone did not influence cell viability.

Conclusion: Herein we have shown that metyrapone does not increase cell death in neuroblastoma cells in culture exposed to bilirubin. Our results question the relationship between the oxidative mechanism evaluated by spectrophotometry and cell viability. Our findings add to the discussion on whether bilirubin oxidation represents a potentially important protective mechanism in neurons challenged by hyperbilirubinemia.

No MeSH data available.


Cells incubated with 75 μM bilirubin showed significant reduction in viability compared to controls (p < 0.05). However, adding 250 μM metyrapone, did not significantly change the viability in cells exposed to bilirubin (p > 0.05). Adding 250 μM metyrapone did not have any significant effect on the viability of control cells (p > 0.05). Staurosporine was used as positive control in the viability assay and caused massive cell death (p < 0.05).
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f0005: Cells incubated with 75 μM bilirubin showed significant reduction in viability compared to controls (p < 0.05). However, adding 250 μM metyrapone, did not significantly change the viability in cells exposed to bilirubin (p > 0.05). Adding 250 μM metyrapone did not have any significant effect on the viability of control cells (p > 0.05). Staurosporine was used as positive control in the viability assay and caused massive cell death (p < 0.05).

Mentions: The statistical analysis was performed using independent sample T-tests between groups using PASW 18 (IBM Corporation, Armonk, NY, USA). The mean viability of the CHP2-12 cells in the metyrapone group remained unchanged compared to the control group, thus metyrapone alone had no effect on cell viability. Bilirubin 75 μM caused a significant (p < 0.05) reduction of viability, from 84.1 (SEM 2.0) to 66.6% (SEM 2.7) (Fig. 1). However, adding 250 μM metyrapone to the bilirubin-exposed cells did not alter cell viability.


Metyrapone, an inhibitor of cytochrome oxidases, does not affect viability in a neuroblastoma cell model of bilirubin toxicity
Cells incubated with 75 μM bilirubin showed significant reduction in viability compared to controls (p < 0.05). However, adding 250 μM metyrapone, did not significantly change the viability in cells exposed to bilirubin (p > 0.05). Adding 250 μM metyrapone did not have any significant effect on the viability of control cells (p > 0.05). Staurosporine was used as positive control in the viability assay and caused massive cell death (p < 0.05).
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5121316&req=5

f0005: Cells incubated with 75 μM bilirubin showed significant reduction in viability compared to controls (p < 0.05). However, adding 250 μM metyrapone, did not significantly change the viability in cells exposed to bilirubin (p > 0.05). Adding 250 μM metyrapone did not have any significant effect on the viability of control cells (p > 0.05). Staurosporine was used as positive control in the viability assay and caused massive cell death (p < 0.05).
Mentions: The statistical analysis was performed using independent sample T-tests between groups using PASW 18 (IBM Corporation, Armonk, NY, USA). The mean viability of the CHP2-12 cells in the metyrapone group remained unchanged compared to the control group, thus metyrapone alone had no effect on cell viability. Bilirubin 75 μM caused a significant (p < 0.05) reduction of viability, from 84.1 (SEM 2.0) to 66.6% (SEM 2.7) (Fig. 1). However, adding 250 μM metyrapone to the bilirubin-exposed cells did not alter cell viability.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Unconjugated hyperbilirubinemia may cause brain damage in infants, and globally remains a source of neonatal morbidity and mortality. A significant inter-individual variability in vulnerability to bilirubin toxicity remains largely unexplained. An enzyme located in mitochondria oxidizes bilirubin. We hypothesized that inhibiting bilirubin oxidation in human neuronal cell cultures exposed to bilirubin would increase cell death.

Methods: The ability of mitochondrial membranes from CHP-212 human neuroblastoma cells to oxidize bilirubin was verified by spectrophotometry. Intact cells in culture were exposed to bilirubin (75&nbsp;&mu;M) with or without metyrapone (250&nbsp;&mu;M) for 24&nbsp;h, stained with Annexin-V and Propidium iodide and analyzed for apoptosis and necrosis by flow cytometry.

Results: Bilirubin caused a significant reduction of viability, from 84&nbsp;&plusmn;&nbsp;2.0% (mean&nbsp;&plusmn;&nbsp;SEM) vs 67&nbsp;&plusmn;&nbsp;2.7% (p&nbsp;&lt;&nbsp;0.05), but adding metyrapone to the bilirubin-exposed cells did not further impact cell viability. Metyrapone alone did not influence cell viability.

Conclusion: Herein we have shown that metyrapone does not increase cell death in neuroblastoma cells in culture exposed to bilirubin. Our results question the relationship between the oxidative mechanism evaluated by spectrophotometry and cell viability. Our findings add to the discussion on whether bilirubin oxidation represents a potentially important protective mechanism in neurons challenged by hyperbilirubinemia.

No MeSH data available.