Ferrous-Citrate Complex and Nigral Degeneration: Evidence for Free-radical Formation and Lipid Peroxidation" K. P. MOHANAKUMARFc A. DE BARTOLOMEIS! R.-M. WU,b,e K. J. YEH! L. M. STERNBERGER! S.-Y. PENG! D. L. MURPHY! AND C. C. CHIUEHbf bLaboratory of Clinical Sciences National Institute of Mental Health National Institutes of Health 10l3D-41 Bethesda, Maryland 20892-1264 cDivision of Pharmacology and Experimental Therapeutics Indian Institute of Chemical Biology Calcutta 700032, India dExperimental Therapeutics Branch National Institute of Mental Health National Institutes of Health 1014N-212 Bethesda, Maryland 20892 eDepartMent of Neurology National Taiwan University Hospital College of Medicine National Taiwan University Taipei, Taiwan INTRODUCTION
There is a progressive increase in nonheme iron in the primate basal ganglia with age.' In addition to iron deposits, low molecular mass iron complexes (Fez+-citrate and Fez+-isocitratecomplexes)2are found in cytosolic ~ 0 0 1 sIt. ~has been shown that the neuronal loss in Parkinson's disease4 or in experimental parkinsonisms is accompanied by an accumulation of iron in the substantia nigra compacta neurons. We have recently demonstrated that in the presence of Fez+and oxygen, dopamine (DA) can be nonenzymatically oxidized to form melanin pigments and generate reactive hydroxyl free radicals (*OH).6Therefore, it is hypothesized that the progressive accumulation of iron complexes may cause oxidation of DA, resulting in excessive generation of free radicals, followed by lipid peroxidation and calcium overload, which in turn may cause nigral injury and parkin~onism.~ 'Dr. K.P. Mohanakumar is supported by the Indo-US STF Program of the United States Agency for International Development, and the Ministry of Science and Technology, Government of India. Mr. K. J. Yeh is a summer student from Brown University. Ms. L. Sternberger is a student intern from Springbrook High School and is sponsored by Howard Hu hes Medical Institute. Ms. A. Peng is a student fellow from Blair High School. &o whom correspondence should be addressed (e-mail:
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Recently, we have accumulated substantial evidence in support of the role of .OH generation and DA autoxidation in 1-methyl-4-phenyl pyridinium (MPP+)-induced nigral cell death.M In the present study, we investigated the effects of oxidative injury to rat nigral neurons caused by the intranigral infusion of Fez+-citrate complex. This report summarizes the preliminary results of the effects of Fez+-citrate complex on the in vitro formation of .OH and the in vivo neurotoxic effects in the dopaminergic nigrostriatal system. The results show that Fez+-citratecomplex is as potent in causing oxidative injury in nigral neurons and DA depletion as the dopaminergic neurotoxin, MPP+. MATERIALS AND METHODS
Male Sprague-Dawley rats (250-300 gm) were anesthetized with chloral hydrate (400 mg/kg i.p.) and prepared for intranigral infusion of the drug.9 Body temperature of the animals was maintained at 37°C using homeothermic blanket. Fez+-citrate complex was prepared by mixing 1:l solutions of ferrous ammonium sulfate and citric acid in filtered (0.4 pm; Millipore) Kreb's Ringer solution. Fe3+-citrate complex was also prepared similarly using Fe3+CI2..OH generation was monitored in vitro by a salicylate hydroxylation procedure using sodium salicylate (1 mM) as the .OH trapping reagent.'J The reaction mixture contained 210 nmol Fez+-citrate in 1 ml filtered Ringer, and 20 pl (4.2 nmol) of the sample was injected into the HPLC. Fez+-citrate,Fe3+-citrate,or citrate (4.2 nmol) was infused (1 pI) unilaterally into the substantia nigra. Stereotaxic coordinates were A-P: 3.2 mm, Lat: 2.1 mm, and D-V: 2 mm as per Paxinos and Watson rat atlas. Unilateral turning behavior was counted daily following intranigral infusion of the iron complexes. Animals were sacrificed by decapitation at different intervals following intranigral infusion. Their brains were removed and the substantia nigra and nucleus caudatus were dissected out from both sides. The fluorescence assay procedure for lipid peroxidation'" was modified to suit a microassay. The freshly dissected substantia nigra (6-8 mg) or the caudate nucleus (25-30 mg) were placed in microfuge tubes with 750 pI of chloroform and methanol (2:l). Each tube was capped, agitated gently, and placed on ice. The tissue was homogenized for 2 seconds in an ultrasonic tissue disruptor (Heat Systems, New York). Water (750 pl) was added to this and the mixture was vortexed for 1 minute and left on ice for 15 minutes. After centrifugation, 100 pl of methanol was added to 400 p1 of the chloroform layer. After thorough mixing, 200 pI of the sample were scanned for fluorescence in a luminescence spectrometer (Perkin-Elmer, LS50). A maximum fluorescence due to oxidized products of lipid was obtained at 356 nm excitation and 426 nm emission. For the measurement of DA, serotonin, 2,3-dihydroxyphenylacetic acid (DOPAC), and 5-hydroxyindole acetic acid, the tissue was homogenized in cold 0.1 N perchloric acid containing 0.01% EDTA and analyzed employing an HPLC equipped with an electrochemical detector.II For in situ hybridization, rat brains were quickly frozen in isopentane at -15°C. Serial 15 pm sections of the frozen midbrain were cut in a cryostat in the coronal plane. Sections were mounted onto gelatin-coated slides and stored at -70°C. Sections were processed for in situ hybridization of tyrosine hydroxylase.12The probe was 48 base oligodeoxyribonucleotides complementary to 1441-1448 of the rat mRNA. The probe was labeled at 3' using 3sS dATP (approximately 1400 mCi/ mmol), terminal deoxynucleotydyl transferase (15 units/ml), and tailing buffer. Sections were hybridized with 0.5 x lo6 cpm of the labeled oligonucleotide and incubated for 18 hours at 37°C. After being washed at stringent conditions, the
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sections were dried and exposed to X-ray film (p-max Hyperfilm, Amersham) for 48 hours. I4Cstandards with several known amounts of radioactivity and 3sSbrain paste standards were simultaneously exposed and cross calibrated. Relative transmittance of the autoradiogram in the substantia nigra region was measured using a computerassisted analyzer system and the McIntosh-based program, Image 1.42 (W. Rasband, NIMH). Optical density values were converted to relative radioactivity in tissue using the known values of the I4C standards. The average mRNA levels in the control side were set to 100% and the lesioned sides were expressed as percentage of controls. Results were expressed as mean ? SEM. Statistical analysis was conducted using ANOVA and Student’s t-test. A p value less than 0.05 was considered significant.
RESULTS AND DISCUSSION
In Vitro Generation of .OH in Fe2+-CitrateComplex System The .OH adducts of salicylate, 2,5- and 2,3-dihydroxybenzoic acid (DHBA) were measured as indices of .OH generation. An in vitro cell-free system containing either Fez+ or citrate alone produced little or no .OH. Addition of Fez+-citrate (4.2 nmol/20 PI) to the .OH trapping solution (1 mM sodium salicylate in Ringer’s solution; final volume, 1 ml), caused a linear increase in the production of the 2,3and 2,5-DHBA with time (FIG.1). 2,3-DHBA, the more reliable indicator of .OH reaction with salicylic acid, was two times more pronounced (4.1 nmol/ml/6 hr) as compared to 2,5-DHBA (2 nmol/ml/6 hr). Fez+-citrate-complexproduced a significant 6-fold increase in the .OH as compared to that of Fez+. Catechol, the breakdown product of 2,3-DHBA was also detectable by the HPLC procedure (data not shown). In contrast to the profound ability of Fez+-citratecomplex to generate .OH, Fe3+-citratecomplex was relatively inactive in the absence of reducing agents, such as ascorbate. Surprisingly, ascorbate acts like a prooxidant in this .OHgenerating system. The production of *OHelicited by the Fez+-citratecomplex was significantly reduced when the samples were kept in the dark and at 4°C. Therefore, the Fez+-citratecomplex can promote the generation of cytotoxic .OH which is time and temperature dependent. Interestingly, this Fez+-citrate-induced *OHformation can be blocked by reducing temperature. These in vitro findings suggest that hypothermia may become an alterative neuroprotective therapy which may protect brain neurons against iron-mediated oxidative injury. It also indicates that prolonged hyperthermia may possibly trigger .OH formation and brain injury, especially in the iron-rich basal ganglia. Fez+-citratecomplex resembles closely the intracellular free “labile iron pool” or “transit ~ O O I . ” ~ ,Fez+-citrate ~ or Fez+-ammoniumcitrate, are endogenous cofactors in the DA synthesisI3pathway, and are the forms suggested to be present in cases of external iron 10ading.l~The molecular mechanisms concerning the generation of .OH by the Fez+-citrate complex is not clear. Fez+,when reacted with an equimolar amount of citric acid, forms tridentate mononuclear complex, [ Fe citratel-, which would undergo oxidation and hydrolysis to form tridentate [Fe(OH)citrate]- and bidentate [Fe(OH)zcitrate]z- ferric ~omplex.’~ During this process, oxygen may be reduced to form superoxide anion. Furthermore, Fez+ itself may take part in an electron transfer reaction with molecular oxygen or with citrateI6 to produce unstable perferryl radicals, forming Fe3+and superoxide, leading to the formation of .OH by the Haber-Weiss and/or Fenton reaction as demonstrated in the in vitro
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study. The proposed iron-catalyzed redox coupling of Fez+-citrate and oxygen is likely since (1) the presence of oxygen and Fez+-citratecomplex were found to be necessary, as citrate alone failed to generate .OH; and (2) the amount of .OH
T
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4
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2
4
6
a
rn I n
4 ol 6
Time (hr) FIGURE 1. Effects of citrate, ferrous ion, and ferrous citrate complex on .OH generation in vifru. Hydroxyl radical generation was measured by the salicylate hydroxylation proced~re',~ using sodium salicylate as the trapping reagent. The reaction mixture contained in 1 ml, a final concentration of 1 mM sodium salicylate, and 0.21 mM of Fe?+-citratewas kept at 32°C under a heating lamp. Samples of 20 GI (4.2 nmol of Fez+-citratecomplex) were injected into an HPLC equipped with electrochemical detector at different intervals. 2,3- and 2,5-DHBA, the .OH adducts of salicylic acid, were oxidized at +0.74 V and the peaks were measured in relation to known standards of 2,3- and 2,5-DHBA.
generated was negligible when the mixture was kept in the dark at 4"C, or when the Fez+was not chelated with citrate, or when the Fez+ was replaced with Fe3+ (data not shown).
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Dopamine Metabolism and Lipid Peroxidation One Hour Following Unilateral Intranigral Infusion of Fez+-CitrateComplex (4.2 nrnol) (N) Control Side Infused Side
TABLE I.
Dopamine (pmol/mg) DOPAC (pmol/mg) DOPAC/dopamine Lipid peroxidation (fluorescence unit)
7 7 7
63.3 f 3.4 12.9 2 0.9 0.21 f 0.02
67.1 2 2.1 17.5 f 1.1* 0.26 f 0.02*
3
0.54 f 0.04
0.83 f 0.09*
*p < 0.05.
EFFECTS OF INTRANIGRAL ADMINISTRATION OF FEZ+-CITRATEON THE NIGROSTRLATAL NEURONS 1. Acute Lipid Peroxidation In Vivo. The above in vitro data indicate that Fez+-citratecomplex can generate .OH. This highly reactive .OH can cause hydrogen abstraction from polyunsaturated fatty acids. The oxidized phospholipids, phosphatidylethanolamine, phosphatidylserine, and malondialdehyde and dihydropyridines could yield fluorescence. This fluorescent product can be detected at 370 nm and 430 nm excitation and emission, respectively. This method is more specific than the conventional thiobarbituric acid procedure, since thiobarbituric acid can react not only with malondialdehyde but also with deoxyribonucleic acid, and amino acids. In this study, we used the chloroform methanol extraction procedure for the measurement of lipid peroxidation products in the brain. Unilateral infusion of the Fez+-citratecomplex (4.2 nmol) elicited a significantly high amount of lipid peroxidation products in the substantia nigra. Lipid peroxidation was progressive and doubled the basal value one hour following the intranigral infusion of Fez+-citratecomplex (TABLE1). An increase in Fez+-citratecomplex in the substantia nigra must have caused the generation of .OH, as seen with the in vitro study (FIG. 1). Once .OH is formed, it can abstract a hydrogen atom from a methylene carbon of an unsaturated fatty acid in the biological membrane to initiate lipid peroxidation.” However, this Fez+-citrate-inducedoxidation of iron and associated lipid peroxidation cannot be blocked by catalase.I8 The availability of Fez+citrate in the vicinity allows the free radical reaction to propagate, resulting in lipid peroxidation products as detected in the present study. In the iron-rich substantia nigra, excessive free-radical-induced lipid peroxidation will result in membrane blebbing and increased permeability, leading to the rupture of cell organelles and probably cell death.
2. Circling Behavior and Dopamine Turnover. Rats treated with 4.2 nmol of Fez+-citrateexhibited spontaneous rotations contralateral to the side of infusion. This behavior was prominent for 24 hours, and diminished by the third day of treatment. After seven days, only a few animals showed any spontaneous rotations. Acutely, neither the caudate nucleus nor the substantia nigra showed any changes in the content of DA one to 24 hours following the intranigral infusion of Fez+-citrate complex. Only DOPAC showed a significant increase in content at the nucleus caudatus (TABLE1). Other metabolites were not changed in these nuclei. Thus, the initial contralateral turning response to the administration of the Fe2+-citrate complex may be due to an increased release of DA at the nerve terminal region of the nigrostriatal neurons as indicated by a significant increase in DOPAC levels and the DOPAC to DA ratio (TABLE1). Thus, this iron-induced increased DA turnover in
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the striatum may explain the initial spontaneous contralateral rotation in the animals. 3. Chronic and Progressive Injuy of the Nigrostnutul Neurons. One week following the intranigral administration of Fe2+-or Fe3+-citrate (4.2 nmol), there was a significant decrease of DA in the ipsilateral caudate nucleus (TABLE2). DOPAC was also significantly decreased (from 12.6 2 1.3 to 8.4 2 0.5 pmol/mg). However, there was no change in the content of serotonin or 5-hydroxyindole acetic acid. There was a significant decrease in the amount of translatable mRNA of tyrosine hydroxylase in the substantia nigra seven days following the infusion of Fez+-citrate complex (TABLE2). The severe DA depletion in the caudate nucleus and the decreased expression of tyrosine hydroxylase in the substantia nigra seven days postinfusion indicate a relatively selective dopaminergic toxicity of the small mass iron complex in the substantia nigra. The relatively selective dopaminergic toxicity may be due to the fact that the nigrostriatal dopaminergic system can take up iron' following intranigral infusion of Fez+-citrate.The mixture of iron, oxygen, and DA may be a potential free-radical generating system, resulting in the accumulation of melanin pigments that can be blocked by .OH scavengers.6 This nigrostriatal degenerative effects of Fez+-citratccomplex are comparable to the oxidative effects of MPP+,9the toxic metabolite of the parkinsonism-producing neurotoxin, MPTP. Significant increases of iron in the degenerating neurons of the substantia nigra of monkeys treated with MPTP" suggests that iron mediated oxidative stress could be the primary cause of cell death in this model. The pigmented substantia nigra is particularly vulnerable to oxidative stress during aging and MPTP intoxication since this nucleus is metabolically active and contains high levels of DA, oxygen, and iron. Decreased activity of the antioxidative enzyme system, including peroxidase, catalase, glutathione peroxidase, and an Increased conceninhibition of glutathione are reported in Parkinson's di~ease.l~-~I trations of lipid peroxidation products,22and iron: accompanied by decreased iron sequestering protein (ferritin),23 have been detected in the substantia nigra of postmortem parkinsonian brains. Based on the postmortem parkinsonian brain findings, attempts have been made to analyze the possible effects of Fe3+loading into the substantia nigra. These studies used intranigral infusion of FeCI3 (50 pg) in very high dosesz4or Fe3+-citratecomplex (6.3 to 12.6 nm01)~~ to produce DA depletion in the striata. Thus Fe3+-citrate complex is more toxic than Fe3+ in causing oxidative injury to the nigral neurons. Moreover Fez+-citratecomplex is more potent than Fe.I+-citrate complex. While 4.2 nmol of Fez+-citratecaused 66% DA depletion in the caudate nucleus, Fe3+-citrate complex induced only 44% reduction (TABLE2). Interestingly, when the same dose of MPP+ was intranigrally administered, the depletion was 47%. Apparently, Fez+-citratecomplex in the substantia nigra might
Intranigral Effects of Iron-Citrate Complexes on the Nigral Degeneration as Reflected by Dopamine Depletion and Tyrosine Hydroxylase mRNA Expression in the Nigrostriatal Neurons Seven Days Following Infusion
TABLE 2.
lntranigral Infusion (4.2 nmol) Citrate Fez+-citrate Fe3+-citrate * p < 0.05.
(N) 6 10 6
Nigral TH mRNA (%Iof control) 99.8 2 3.1 35.8 2 4.4; Not done
Striatal Dopamine (%of Control) 100.3 2 7 34.3 2 6* 56.0 2 9*
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promote the generation of .OH and initiate peroxidation of brain lipids, thus causing oxidative injury as demonstrated in this study.
SUMMARY AND CONCLUSION Increased nigral iron content in the parkinsonian brain is now well documented and is implicated in the pathogenesis of this movement disorder. Free iron in the pigmented DA-containing neurons catalyze DA autoxidation and Fenton reaction to produce cytotoxic .OH, initiating lipid peroxidation and consequent cell damage. The present results clearly demonstrate that a regional increase in the levels of the “labile iron pool” can result in the degeneration of dopaminergic nigral neurons as reflected by a significant inhibition in the expression of tyrosine hydroxylase mRNA and DA depletion. Iron-complex-induced damage of dopaminergic neurons in the substantia nigra, might have resulted from a sequence of cytotoxic events including the .OH generation and lipid peroxidation as demonstrated in this study. This free-radical-induced oxidative nigral injury may be a reliable free-radical model for studying parkinsonism and may be relevant to idiopathic Parkinson’s disease. This apparent nigral injury stimulated by Fez+-citrateis more severe than that produced by ferric ironz4and its citrate complex.z Moreover, these data indicate that Fez+citrate is as potent as MPP+ in causing oxidative injury to the substantia nigral neurons. However, the nigral toxicity of MPTP and its congeners are not progressive, while Fez+-citrate complex may produce a progressivez degeneration of the nigrostriatal neurons which is similar to the progression of ideopathic Parkinson’s disease. Thus, this unique Fez+-citrate complex animal model could be used for studying neuroprotective treatments for retarding or halting the progressive nigrostriatal degeneration caused by free radicals in the iron-rich basal ganglia. REFERENCES 1. HILL,J. A. 1990. Iron and proteins of iron metabolism in the central nervous system. In: In
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