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Increasing Intracellular Levels of Iron with Ferric Ammonium Citrate Leads to Reduced P-glycoprotein Expression in Human Immortalised Brain Microvascular Endothelial Cells

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Abstract

Purpose

P-glycoprotein (P-gp) at the blood-brain barrier (BBB) precludes the brain penetration of many xenobiotics and mediates brain-to-blood clearance of β-amyloid, which accumulates in the Alzheimer’s disease (AD) brain. Zinc and copper are reported to modulate BBB expression and function of P-gp; however, the impact of exogenous iron, which accumulates in AD, on P-gp dynamics remains unknown.

Methods

P-gp protein and MDR1 transcript levels were assessed in immortalised human cerebral microvascular endothelial (hCMEC/D3) cells treated with ferric ammonium citrate (FAC; 250 μM, 72 h), by Western blotting and RT-qPCR, respectively. P-gp function was assessed using rhodamine-123 and [3H]-digoxin accumulation. Intracellular reactive oxygen species (ROS) levels were determined using 2′,7′-dichlorofluorescin diacetate and intracellular iron levels quantified using a ferrozine assay.

Results

FAC treatment significantly reduced P-gp protein (36%) and MDR1 mRNA (16%) levels, with no significant change in rhodamine-123 or [3H]-digoxin accumulation. While P-gp/MDR1 downregulation was associated with elevated ROS and intracellular iron, MDR1 downregulation was not attenuated with the antioxidant N-acetylcysteine nor the iron chelators desferrioxamine and deferiprone, suggesting the involvement of a ROS-independent mechanism or incomplete iron chelation.

Conclusions

These studies demonstrate that iron negatively regulates P-gp expression at the BBB, potentially impacting CNS drug delivery and brain β-amyloid clearance.

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Abbreviations

Aβ:

β-amyloid

AD:

Alzheimer’s disease

BBB:

Blood-brain barrier

BCRP/ABCG2:

Breast cancer resistance protein

DCFH-DA:

2′,7′-dichlorofluorescin diacetate

DFP:

Deferiprone

DFO:

Desferrioxamine

FAC:

Ferric ammonium citrate

hCMEC/D3:

Immortalised human cerebral microvascular endothelial cell line

NAC:

N-acetylcysteine

P-gp:

P-glycoprotein

ROS:

Reactive oxygen species

R123:

Rhodamine-123

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Acknowledgments and Disclosures

The work presented in this manuscript was funded by the Mason Foundation National Medical Program (MAS2017F035). Stephanie Newman was supported by a ‘Research Training Program’ stipend, provided by the Australian Government.

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Authors and Affiliations

  1. Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, Victoria, 3052, Australia

    Stephanie A Newman, Yijun Pan & Joseph A Nicolazzo

  2. Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, Victoria, 3052, Australia

    Jennifer L Short

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  1. Stephanie A Newman
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Correspondence to Joseph A Nicolazzo.

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Newman, S.A., Pan, Y., Short, J.L. et al. Increasing Intracellular Levels of Iron with Ferric Ammonium Citrate Leads to Reduced P-glycoprotein Expression in Human Immortalised Brain Microvascular Endothelial Cells. Pharm Res 38, 97–111 (2021). https://doi.org/10.1007/s11095-021-03006-y

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