Penicillamine Increases Free Copper and Enhances Oxidative Stress in the Brain of Toxic Milk Mice

Ding-Bang Chen,Li Feng,Xiao-Pu Lin,Xiu-Ling Liang,Fu-Rong Li,Wei Zhang,Xun-Hua Li,Thomas Langmann

doi:10.1371/journal.pone.0037709

Abstract

Wilson disease (WD) is characterized by the accumulation of copper arising from a mutation in the ATP7B gene. Penicillamine (PA) makes 10–50% of the patients with neurologic symptoms neurologically worse at the early stage of administration. The aim of this study was to determine how the copper metabolism changes and whether the change impairs the brain of toxic milk (tx) mice, an animal model of WD, during the PA administration. The free copper and protein-bound copper concentrations in the serum, cortex and basal ganglia of tx mice with PA administration for 3 days, 10 days and 14 days, respectively, were investigated. The expression of copper transporters, ATP7A and CTR1,was analyzed by real-time quantitative PCR, immunofluorescence and Western blot. Then SOD, MDA and GSH/GSSG were detected to determine whether the oxidative stress changed correspondingly. The results revealed the elevated free copper concentrations in the serum and brain, and declined protein-bound copper concentrations in the brain of tx mice during PA administration. Meanwhile, transiently increased expression of ATP7A and CTR1 was observed generally in the brain parenchyma by immunofluorescence, real-time quantitative PCR and Western blot. Additionally, ATP7A and CTR1 were observed to locate mainly at Golgi apparatus and cellular membrane respectively. Intense staining of ATP7A in the choroid plexus was found in tx mice on the 3rd and 10th day of PA treatment, but rare staining of ATP7A and CTR1 in the blood-brain barrier (BBB). Decreased GSH/GSSG and increased MDA concentrations were also viewed in the cortex and basal ganglia. Our results suggested the elevated free copper concentrations in the brain might lead to the enhanced oxidative stress during PA administration. The increased free copper in the brain might come from the copper mobilized from brain parenchyma cells but not from the serum according to the ATP7A and CTR1 expression analysis.

Highlights

Wilson disease (WD) is an autosomal recessive disease arising from a mutation in the ATP7B gene, which results in the failure of hepatocytes to excrete copper into bile, leading to the hepatic copper accumulation and cell injury
The pivotal role of penicillamine in the initial treatment for WD patients with neurologic symptoms has been a matter of debate for the risk of neurological worsening for the past three decades. 10%–50% of patients neurologically worsened by PA and 50% of them never recovered to the level before treatment [2]
Copper concentrations The average serum free copper concentration detected by ultrafiltration method was about 8.9% of serum copper concentration, the sum of free copper and protein-bound copper, in control group of tx mice,while in derived from the wild-type (DL) mice it was 1.9%

Summary

Introduction

Wilson disease (WD) is an autosomal recessive disease arising from a mutation in the ATP7B gene, which results in the failure of hepatocytes to excrete copper into bile, leading to the hepatic copper accumulation and cell injury. Copper incorporation into the protein ceruloplasmin (Cp) is impaired by dysfunction of ATP7B, leading to the reduced circulating holoceruloplasmin. The treatment for WD patients involves reversing the positive copper balance. The pivotal role of penicillamine in the initial treatment for WD patients with neurologic symptoms has been a matter of debate for the risk of neurological worsening for the past three decades. 10%–50% of patients neurologically worsened by PA and 50% of them never recovered to the level before treatment [2]. Trientine shares the propensity of penicillamine to deteriorate neurologic symptoms (about 25%) [3]

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