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Cu2+ toxicity inhibition of mitochondrial dehydrogenases in vitro and in vivo.

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Cu2⫹ Toxicity Inhibition of Mitochondrial
Dehydrogenases In Vitro and In Vivo
Christian T. Sheline, PhD, and Dennis W. Choi, MD, PhD
Wilson’s disease results from mutations in the P-type Cu2ⴙ-ATPase causing Cu2ⴙ toxicity. We previously demonstrated that
exposure of mixed neuronal/glial cultures to 20␮M Cu2ⴙ induced ATP loss and death that were attenuated by mitochondrial substrates, activators, and cofactors. Here, we show differential cellular sensitivity to Cu2ⴙ that was equalized to 5 ␮M
in the presence of the copper exchanger/ionophore, disulfiram. Because Cu2ⴙ facilitates formation of oxygen radicals (ROS)
which inhibit pyruvate dehydrogenase (PDH) and alpha-ketoglutarate dehydrogenase (KGDH), we hypothesized that their
inhibition contributed to Cu2ⴙ-induced death. Toxic CU2ⴙ exposure was accompanied by early inhibition of neuronal and
hepatocellular PDH and KGDH activities, followed by reduced mitochondrial transmembrane potential, ⌬⌿M. Thiamine
(1– 6mM), and dihydrolipoic acid (LA, 50␮M), required cofactors for PDH and KGDH, attenuated this enzymatic inhibition and subsequent death in all cell types. Furthermore, liver PDH and KGDH activities were reduced in the Atp7b
mouse model of Wilson’s disease prior to liver damage, and were partially restored by oral thiamine supplementation. These
data support our hypothesis that Cu2ⴙ-induced ROS may inhibit PDH and KGDH resulting in neuronal and hepatocellular death. Therefore, thiamine or lipoic acid may constitute potential therapeutic agents for Wilson’s disease.
Ann Neurol 2004;55:645– 653
The essential trace metal copper is highly redox active,
which allows for facile electron transfer by key Cu2⫹containing proteins: cytochrome c oxidase for electron
transport, Cu2⫹/Zn2⫹ superoxide dismutase for defense
against free radicals, and ceruloplasmin for iron homeostasis. Although these redox properties of Cu2⫹ are
essential for life, they can also cause deleterious oxidation of lipids and proteins by formation of free radical
species.1 Cu2⫹ preferentially facilitates the formation of
hydroxyl free radicals via the Fenton reaction, and copper redox cycling through glutathione, cysteine, or ascorbic acid can potentiate radical generation.2– 4
Disturbances in copper homeostasis are responsible
for cell death in the liver and central nervous system in
Wilson’s disease, a rare autosomal recessive disorder
(affecting approximately 1 in 50,000 people) caused by
loss-of-function mutations in a P-type copper ATPase,
ATP7B. This protein is localized primarily in the Golgi
of hepatocytes where it inserts Cu2⫹ into ceruloplasmin or allows for its excretion into bile. Upon liver
failure, copper overflows into other tissues, damaging
in particular basal ganglia as well as cerebral cortex,
white matter, and thalamus.5 Copper toxicity also may
contribute to the pathogenesis of other neurodegenera-
tive disorders6 including familial mutations in the superoxide dismutase-1 (SOD-1) gene.7–9 Copper also
has been speculatively implicated in the conformational
change of the prion protein to the infective form,10 –12
to potentiate ␤-amyloid toxicity,13,14 and to facilitate
fibril formation of ␣-synuclein.15–17
It is possible that mitochondria are preferentially
damaged by copper/free radical toxicity18 –20; copper
overload in rats or Bedlington terriers induces lipid
peroxidation, formation of 4-hydroxy nonenal (HNE),
and mitochondrial dysfunction.21 Increased free radical
generation, lipid peroxidation, and mitochondrial dysfunction occur in the livers of Wilson’s disease patients,22 and in Atp7b mice, which have a spontaneous
autosomal recessive mutation in atp7b, and develop
hepatocellular carcinoma due to copper overload.23
HNE inhibits pyruvate dehydrogenase (PDH) and
␣-keto glutarate dehydrogenase (KGDH), perhaps by
covalently modifying the lipoic acid moiety of these
enzymes.24,25 PDH also has been demonstrated to be
preferentially sensitive to other insults that induce oxygen free radicals, such as ischemia, or exposure to hydrogen peroxide.26 –29
We hypothesize that free radical–induced mitochon-
From the Department of Neurology and Center for the Study of
Nervous System Injury Washington University School of Medicine,
St. Louis, MO.
Published online Mar 21, 2004, in Wiley InterScience
( DOI: 10.1002/ana.20047
Received Nov 26, 2003, and in revised form Dec 31. Accepted for
publication Jan 2, 2004.
Current address for Dr Choi: Merck Research Laboratories, WP 142500, P.O. Box 4, 770 Sumneytown Pike, West Point, PA 194860004.
Address correspondence to Dr Sheline, Department of Neurology
and Center for the Study of Nervous System Injury Washington
University School of Medicine, 660 S. Euclid Avenue, St. Louis,
MO 63110. E-mail:
© 2004 American Neurological Association
Published by Wiley-Liss, Inc., through Wiley Subscription Services
drial dehydrogenase inactivation is a prominent mechanism of Cu2⫹-induced neurodegeneration. The purpose of this study was to test the hypothesis that
neurons and HepG2 cells exposed to excess extracellular copper would develop impairment of energy metabolism associated with inhibition of PDH, and KGDH,
and that the administration of the PDH (and KGDH)
cofactors, thiamine or dihydro-lipoic acid,30 might restore dehydrogenase activity levels and reduce cell
death. An abstract has appeared.31
Materials and Methods
Cell Culture and Toxicity Studies
Near-pure cortical neuronal, glial, and mixed neuronal-glial
cell cultures were prepared from E15 mice as previously described.32 HepG2 cultures were grown in minimum essential
medium (MEM) ⫹10% fetal bovine serum, glutamine, and
nonessential amino acids and passaged every 2 to 3 days by
trypsin/EDTA digestion. Cultures were grown to 50 to 75%
confluence before exposure to Cu2⫹.
Toxicity was initiated by exposure to CuCl2 in MEMdefined media after thorough washing in the same medium.
Cu2⫹ exposures in near-pure neuronal or HepG2 cultures
were also performed in the presence of 300nM disulfiram, a
copper exchanger/ionophore, to facilitate entry. Cell death
was assayed at varying times later. Cell death was assessed by
lactate dehydrogenase efflux to the bathing medium, Trypan
blue staining, or staining with propidium iodide,33 and confirmed visually.
The specificity of the reaction was demonstrated by leaving
out CoA or the substrate, and the absorbance of sister cultures,
or slices exposed in the absence of substrate, was subtracted
from each sample. PDH was assayed in a similar fashion, except that pyruvate was substituted for ␣-ketoglutarate. The
formazan was extracted in 10% Igepal CA-630 detergent by
sonication, and the absorbance measured at 595nm for quantitation. In liver sections, the signal was normalized to slice
area, and only noncancerous liver sections were utilized.
Colony Maintenance and Trials
The Atp7b inbred mouse strain (Jackson Labs, Bar Harbor,
ME) was maintained in our barrier facility and treated in accordance with our animal protocol. Pups of the inbred strain
were foster parented by pseudopregnant C57/Bl6 mothers to
provide Cu2⫹-proficient milk. Atp7b and wild-type C57/Bl6
controls were used in a double-blind trial, where the animals
in each group were age and sex matched. Treatment of the
Atp7b animals began at 5 weeks of age and continued for the
duration of the study by ad libitum oral administration in the
drinking water of 2% thiamine. Water and food ingestion
were monitored weekly and did not vary among the different
groups. At the time of death, the liver was weighed, photographed, and sequential slices were processed for paraffin embedding or freezing for activity assays. Liver tissue that was
noncancerous by gross morphological microscopic examination was frozen, and 20␮m sections were obtained using a
cryostat. These sections were traced to determine total liver
volume and then used in the assays for PDH and KGDH.
Measurement of the Mitochondrial Transmembrane
Potential, ⌬⌿M
Mitochondrial transmembrane potential was assessed using
the dyes tetra-methyl rhodamine methyl ester and confirmed
with the ratioable dye ApoAlert. Near-pure neuronal cultures
were washed three times into MEM containing 21mM glucose, loaded by bath application with 50nM tetra methyl
rhodamine methyl ester (TMRE; Sigma, St. Louis, MO; excitation ␭: 560nm, emission ␭: 615nm) for 30 minutes at
37°C and washed three times again before Cu2⫹ exposure.
Representative fluorescence photomicrographs were taken before and at various time points after Cu2⫹ exposure using a
Spot camera, and Nikon Diaphot 200 microscope.
␣-KGDH and Pyruvate Dehydrogenase Assays
In situ KGDH and PDH assays were performed on near-pure
cortical neuronal cells, or on 20␮m sections of livers from
Atp7b mice as described.34 This method relies on the formation of formazan from nitroblue tetrazolium upon electron
transfer from NADH that is the product of the enzyme reactions of PDH and KGDH. In brief, live cultures or sections of
fresh-frozen livers were incubated in buffer containing 50mM
Tris-HCl (pH 7.6), 1mM MgCl2, 0.1mM CaCl2, 0.05mM
EDTA, 0.3mM TPP, 0.5␮g/ml rotenone, 0.2% Triton
X-100, 3.5% polyvinyl alcohol, 3mM ␣-ketoglutarate, 3mM
NAD⫹, 0.75mg/ml coenzyme A (CoA), 0.75mM nitroblue
tetrazolium (NBT), and 0.05mM phenazine methosulfate for
up to 60 minutes at room temperature. The NBT and phenazine methosulfate were added immediately before the reaction.
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Unless otherwise stated, all reagents were from Sigma, and
Trypan blue was from Gibco (Grand Island, NY).
Induction of Toxicity in Near-Pure Neuronal or
HepG2 Cultures Required a Cu2⫹ Ionophore:
Thiamine and Lipoic Acid Attenuated Toxicity
We examined the susceptibility of near-pure neuronal
cultures (PNCs), glial cultures, mixed neuronal/glial, or
HepG2 cultures of varying age (days in vitro, DIV) to
copper exposure for 23 hours, after which the 50% lethal dose (LD50) was determined (Table).
We went on to examine the susceptibility of nearpure neuronal cultures and HepG2 cells to 5␮M Cu2⫹
and showed that the copper exchanger/ionophore, disulfiram,35 was necessary to induce cell death at this Cu2⫹
concentration. Thiamine and LA still attenuated death
in the presence of Cu2⫹ and disulfiram (Fig 1). Glia and
mixed cultures were similarly susceptible to 5␮M
Cu2⫹ ⫹ 300nM disulfiram and thiamine and LA attenuated death in theses cultures as well (see Table and
data not shown). These data are consistent with differential accumulation of Cu2⫹ causing differential susceptibility to Cu2⫹ toxicity in these cultures. When these
cultures were made permeable to Cu2⫹, they were then
equally sensitive. 64Cu2⫹ accumulation studies showed
that the most susceptible culture system (mixed DIV13)
Table. HepG2, Glia, and Near-Pure Cortical Neuronal Cultures Are Resistant to Cu2⫹ Exposure
Mixed neurons/glia DIV 13
Mixed DIV 8
Pure glia DIV 28
Pure neuronal DIV 13
Pure neuronal DIV 8
50% Lethal Dose
(LD50, ␮M Cu2⫹)
Percentage of Cell Death (⫾SEM)
(5␮M Cu2⫹ ⫹ 0.3␮M DiS)
⬎ 600b
⬎ 600b
77 ⫾ 7.9a
73 ⫾ 8.2a
49.2 ⫾ 6.4
61.9 ⫾ 6.8
59.1 ⫾ 7.9
41.8 ⫾ 9.7
Glial, near-pure neuronal, mixed neuronal, or HepG2 cultures of age DIV were exposed to copper for 23 hours, after which the LD50 was
determined by lactate dehydrogenase release from dose titrations. Sister cultures also were exposed to 5␮M Cu2⫹ in the presence of 0.3␮M
disulfiram for 23 hours, and death was determined. Cell death is determined by lactate dehydrogenase release from three experiments n ⫽
9 –12, expressed relative to the near-maximal death determined in sister cultures treated for 22 hours with 20␮M A23187 for glia, PNC, and
HepG2 cultures or with 300␮M N-methyl-D-aspartate for mixed cultures (⫽100).
The cell death determinations shown here represent approximately 50% death of both the neurons and the glia in these mixed cultures as
determined by cell counting after Trypan blue staining (data not shown).
Cultures differ significantly from DIV13 mixed at p ⬍ 0.05 by one-way analysis of variance followed by a Bonferroni test.
LD50 ⫽ lethal dose; SEM ⫽ standard error of the mean; DiS ⫽ disufiram; DIV ⫽ days in vitro.
accumulated at least four times more 64Cu2⫹ than the
least sensitive culture system (PNC DIV8, data not
Cu2⫹ Exposure Induced an Early Inhibition of PDH
and KGDH Activities in Neuronal and HepG2
Cultures, Which Were Restored by Thiamine and
Lipoic Acid
Our previous studies suggested that the reactive oxygen
species–sensitive mitochondrial dehydrogenases, pyruvate dehydrogenase (PDH), and ␣-ketoglutarate dehydrogenase (KGDH) might be inhibited by Cu2⫹ exposure.36 We next directly tested their activity in the
presence or absence of the required cofactors, thiamine
pyrophosphate (TPP) and dihydro-lipoic acid (LA).
Neuronal PDH and KGDH activities were reduced after a 2-hour 5␮M Cu2⫹ ⫹ 0.3␮M disulfiram exposure, and the presence of thiamine and lipoic acid–restored activity. The presence of both disulfiram and
Cu2⫹ was required for enzyme inhibition consistent
with the requirement of both compounds for toxicity
(Figs 1 and 2, and data not shown).
A similar result was obtained in HepG2 cells exposed
to sham wash or 5␮M Cu2⫹ ⫹ 0.3␮M disulfiram in
the presence and absence of 6mM thiamine, 50␮M LA
or 300␮M trolox for 4 hours (Fig 3). Trolox was less
effective than thiamine or lipoic acid at preventing death
induced by Cu2⫹ ⫹ disulfiram or restoring PDH/
KGDH activity, though pretreatment increased its effectiveness (see Fig 3 and data not shown). The activity of
these two enzymes at time points before cell death was
quantitated by extracting the blue formazan product of
the reaction and assaying absorbance at 595nm.
Cu2⫹ Exposure Induced a Later Reduction in the
Mitochondrial Transmembrane Potential,⌬⌿M,,
Which Was Restored by Thiamine and Lipoic Acid
A deficiency in PDH, KGDH, and ATP production
by the mitochondria should reduce the mitochondrial
transmembrane potential, ⌬⌿M. We measured ⌬⌿M
Fig 1. Induction of toxicity requires a Cu2⫹ ionophore; thiamine and lipoic acid attenuate toxicity. HepG2 cells or nearpure neuronal cultures were exposed to 5␮M Cu2⫹ in the
presence or absence of 300nM disulfiram, 6mM thiamine, or
50␮M LA for 24 hours as indicated. Cell death is determined
by lactic acid dehydrogenase release from three experiments,
n ⫽ 9 –12, expressed relative to the near-maximal death determined in sister cultures treated for 22 hours with 20␮M
A23187 (⫽100). Pound signs signify cultures differ significantly from 5␮M Cu2⫹ exposure alone. Asterisks signify cultures differ significantly from Cu/DiS exposure at p ⬍ 0.05
by one-way ANOVA followed by a Bonferroni test.
Sheline and Choi: Cu2⫹ Toxicity and Dehydrogenases
Fig 2. Cu2⫹ exposure induced an early inhibition of neuronal pyruvate dehydrogenase (PDH) and ␣-ketoglutarate dehydrogenase
(KGDH) activities which were restored by thiamine and lipoic acid. Near-pure cortical neuronal cultures were exposed to the indicated conditions after which the activity of the PDH and KGDH complexes were assayed causing deposition of blue/black formazan
dye (Materials and Methods), and representative bright-field photomicrographs were taken (A, bar ⫽ 50␮m). Sham-wash control
(Ctrl); sham-wash control assayed without pyruvate (-sub); exposure to 5␮M Cu2⫹ ⫹ 0.3␮M disulfiram for 2 hours, (2h Cu);
exposure to 5␮M Cu2⫹ ⫹ 0.3␮M disulfiram for 2 hours in the presence of 50␮M lipoic acid and 4mM thiamine (2h ⫹ LA/T).
The formazan dye generated in the near-pure neuronal cultures shown in panel A was extracted and the absorbance quantified at
595nm (B, PDH; C, KGDH). The absorbance from samples assayed in the absence of substrate was subtracted from each condition, and the activity of the sham-wash control was set at 100% (three experiments, SEM, n ⫽ 9 –12 cultures per condition). Asterisks signify difference from copper only at p ⬍ 0.05 by one-way ANOVA followed by a Bonferroni test.
Annals of Neurology
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Fig 3. Cu2⫹ exposure induced an early inhibition of PDH and KGDH activities in HepG2 cells, which were restored by thiamine
and lipoic acid. A similar experiment was performed in HepG2 cells exposed to sham wash or 5␮M Cu2⫹ ⫹ 300nM disulfiram
in the presence and absence of 6mM thiamine, 50␮M LA, or 300␮M trolox for 4 hours. Pound signs signify treated cultures differ
significantly from copper treatment only at p ⬍ 0.05.
in near-pure neuronal cultures exposed to 5␮M
Cu2⫹ ⫹ 0.3␮M disulfiram for 6 hours. As shown in
Figure 4, ⌬⌿M was reduced after 6 hours in near-pure
cortical neuronal cultures only in the presence of both
Cu2⫹ (5␮M) and disulfiram (DiS, 0.3␮M), both of
which are required for Cu2⫹ toxicity and PDH/
KGDH inhibition in these cultures. The presence of
thiamine or LA restored ⌬⌿M.
KGDH and PDH Activities Were Reduced in the
Livers of Atp7b Mutant Mice Starting at 6 Months
of Age: Oral Thiamine Partially Restored
Their Activities
Atp7b mice were subjected to a trial of oral supplementation with 2% thiamine in the drinking water
where the caretaker and the pathologist were blinded
to treatment condition. Atp7b animals were split into
two groups at 5 weeks of age, the control group received water only, and the other group received 2%
thiamine in the drinking water. At 6, 9.5, 12, and 16
months, animals were killed, and the livers were removed, photographed, weighed, and processed for mitochondrial enzymology, or histology. The activities of
PDH and KGDH were decreased starting at 6 months,
and by 16 months only 50 to 60% of the activity remained. Thiamine supplementation restored activity to
80 to 90% (Fig 5).
We have previously demonstrated that in mixed neuronal/glial cultures Cu2⫹ exposure selectively induced neuronal death after ATP depletion, and a buildup of the
upstream substrates, pyruvate, PEP, and 2-P-glycerate.
The PDH cofactors thiamine or dihydrolipoic acid, or
the PDH activator dichloroacetate, reduced copper neurotoxicity in vitro, and oral thiamine supplementation
reduced hepatocellular death and extended the life span
of the Long Evans Cinnamon (LEC) rat. In this study,
we showed (1) differential sensitivity of cell types to
Cu2⫹ exposure which was equalized by addition of the
Cu2⫹ ionophore, disulfiram, with thiamine and LA still
attenuating the toxicity; (2) that Cu2⫹ exposure induced
an early reduction in neuronal PDH and KGDH activities, which were restored by thiamine and LA; (3) that
reduced PDH and KGDH activities resulted in decreased ⌬⌿M, which could be restored by thiamine and
LA; and 4) that PDH and KGDH activities were progressively reduced in the livers of Atp7b mice starting at
6 months (before liver damage), and thiamine supplementation partially restored this activity.
Previous studies have examined copper toxicity, including in DIV 3-6 cultures of cortical, hippocampal,
glial, and cerebellar granule neurons, and the concentration of copper chloride determined to kill 50% of cells
was approximately 200␮M, 200␮M, 500␮M, and
Sheline and Choi: Cu2⫹ Toxicity and Dehydrogenases
Fig 4. Cu2⫹ exposure induced a later reduction in the mitochondrial transmembrane potential, ⌬⌿M,, which was restored by thiamine
and lipoic acid. Near-pure cortical neuronal cultures were loaded with 50nM TMRE for 30 minutes and exposed to the indicated
conditions for 6 hours, after which the mitochondrial transmembrane potential was assessed (Materials and Methods), and fluorescence
photomicrographs were taken. Sham-wash control (Ctrl); exposure to 0.3␮M disulfiram (DiS); exposure to 5␮M Cu2⫹ (Cu2⫹); exposure to 5␮M Cu2⫹ ⫹ 0.3␮M disulfiram (DiS⫹Cu2⫹); presence of 4mM thiamine (⫹ T); exposure to 5␮M Cu2⫹ ⫹ 0.3␮M disulfiram in the presence of 50␮M lipoic acid (⫹ LA); presence of both thiamine and lipoic acid (⫹T/LA).
150␮M, respectively.37 The qualitative sensitivity of mature neurons ⬍ glia ⬍ peripheral cells was similar to
that reported here, although our DIV 14 cortical neurons in the presence of glia had a substantially lower
LD50. Our observations of a relationship between
Cu2⫹ accumulation and toxicity and enhancement of
Cu2⫹ toxicity by the Cu2⫹ ionophore disulfiram are
consistent with the likely hypothesis that Cu2⫹ cytotoxicity is at least in part mediated by Cu2⫹ entry into cells.
An increase in Cu2⫹ has been shown to induce free
radical reactions resulting in hydroxyl radicals, lipid
peroxidation, and damage to DNA, and proteins (reviewed in Britton18 and Stohs and Bagchi38). The
metal-induced DNA damage 39 in the liver may have
consequences for malignant transformation, because
hepatocellular carcinomas are common in LEC rats,
atp7b mice, Wilson’s disease, and hemochromatosis.40,41 Lipid peroxidation and HNE generation have
been demonstrated to occur in these diseases,22,42 and
Cu2⫹ or Fe3⫹ induce lipid peroxidation-dependent
dysfunction of mitochondria in vitro or in vivo.43,44 In
the livers of Wilson’s disease patients and its animal
models, this lipid peroxidation is associated with impairment of membrane-dependent functions of mitochondria (oxidative metabolism),45 and copper chelation with D-penicillamine reverses the widespread
nuclear and mitochondrial swelling, vacuolization, and
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degeneration of hepatocytes seen in their livers.46 –51
Affected patients have liver mitochondrial lipid peroxidation, HNE accumulation, and decreased hepatic and
plasma levels of vitamin E.22 We previously suggested
that Cu2⫹ toxicity induced hydroxyl radicals, and a
dysfunction in PDH activity in neurons,36 and now
suggest that Cu2⫹ overload reduces mitochondrial
function, in general, and PDH and KGDH, in particular, both in vitro and in vivo. We previously demonstrated that the free radical scavengers vitamin E and
trolox reduced chronic Cu2⫹ toxicity in mixed cultures
by 50 to 60%.36 In these studies, we demonstrated differential Cu2⫹ sensitivity of neurons and glia and
therefore utilized near-pure neuronal cultures which require the more acute Cu2⫹ exposure induced by disulfiram. Trolox was less effective in counteracting Cu2⫹mediated effects under these acute exposure conditions,
although pretreatment increased efficacy. KGDH52
and PDH have been shown to be preferentially inhibited after HNE, superoxide or hydrogen peroxide treatment,21,26,27,29 and after ischemia reperfusion.28,53
Thiamine, through its biologically active form, thiamine pyrophosphate (TPP), enhances PDH and
KGDH activities,54,55 perhaps by sterically limiting oxidation of critical histidine, or lysine residues,56 –58
TPP also inhibits pyruvate dehydrogenase kinase,59
which phosphorylates and inhibits PDH. Thiamine is
ameliorating this inhibition. Thiamine, in particular,
might be considered as an adjunct to chelator therapy
in Wilson’s disease or other diseases where links to
copper toxicity have been suggested.
This work was supported by NIH (National Institute of Neurological Disorders and Stroke, NS 30337, C.T.S.).
We acknowledge A.-L. Cai for her expert help in preparing cultures.
Fig 5. KGDH and PDH activities are reduced in the livers of
Atp7b mutant mice starting at 6 months of age: oral thiamine
partially restores their activities. Atp7b mice were subjected to a
double-blind trial of oral supplementation with 2% thiamine in
the drinking water. Atp7b animals were split into two groups
at 5 weeks of age; one group received water only, and the other
group received 2% thiamine in the drinking water. At the indicated time points, 20␮m sections of fresh-frozen liver tissue
from untreated Atp7b animals, thiamine-treated Atp7b animals,
and wild-type animals were assessed for PDH and KGDH activities as designated in the key; the formazan dye generated was
extracted and the absorbance was quantified at 595nm. The
absorbance from samples assayed in the absence of substrate was
subtracted from each condition, and the activity of the wild-type
control was set at 100% (SEM, n ⫽ 9 –12 from three to four
independent experiments). Asterisks signify difference from wildtype at p ⬍ 0.05 by two-way ANOVA followed by a Bonferroni test. Pound signs signify difference from Atp7b alone at p
⬍0.05 by two-way ANOVA followed by a Bonferroni test.
effective in treating several disorders associated with deficiencies in PDH and KGDH such as mitochondrial
encephalomyopathy, West syndrome, lactic acidemia,
and some forms of Leigh’s syndrome.60 – 63
The ␣-lipoic acid moiety of the PDH complex
moves pyruvate and acetyl CoA within the enzyme active site.30 LA has been demonstrated previously to attenuate neuronal death–induced by ␤-amyloid, H2O2,
glutamate, ischemia, and iron and to reduce reactive
oxygen species inhibition of PDH (reviewed in Packer
and colleagues64). One of the major products of lipid
peroxidation, 4-hydroxy-2-nonenal (HNE), may specifically inhibit PDH and KGDH by modifying the lipoic acid moiety of these enzymes.25 Excess unconjugated dihydrolipoic acid could act as a sink to keep
HNE from inactivating these enzymes, but other enhancing or inhibitory effects on PDH activity also have
been suggested.65,66
In summary, we propose as a working hypothesis
that copper neurotoxicity is substantially mediated by
radical-mediated inhibition of PDH and KGDH, and
that thiamine or LA reduce copper neurotoxicity by
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Sheline and Choi: Cu2⫹ Toxicity and Dehydrogenases
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cu2, toxicity, inhibition, dehydrogenase, vivo, vitro, mitochondria
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