trichloroethylene and parkinson’s disease: risk assessment · trichloroethylene and parkinson’s...

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Trichloroethylene and Parkinsons Disease: Risk Assessment Mei Liu 1 & Eun-Joo Shin 2 & Duy-Khanh Dang 2 & Chun-Hui Jin 3 & Phil Ho Lee 4 & Ji Hoon Jeong 5 & Seok-Joo Park 2,6 & Yong-Sun Kim 6 & Bin Xing 1 & Tao Xin 7 & Guoying Bing 1 & Hyoung-Chun Kim 2 Received: 13 June 2017 /Accepted: 8 December 2017 /Published online: 22 December 2017 # Springer Science+Business Media, LLC, part of Springer Nature 2017 Abstract This study was conducted to investigate the mechanism of action and extent of selective dopaminergic neurodegeneration caused by exposure to trichloroethylene (TCE) leading to the endogenous formation of the neurotoxin 1-trichloromethyl-1,2,3,4-tetrahydro-β- carboline (TaClo) in rodents. Beginning at 3 months of age, male C57BL/6 mice received oral TCE dissolved in vehicle for 8 months. Dopaminergic neuronal loss was assessed by nigral tyrosine hydroxylase (TH) immunoreactivity. Selective dopaminergic neurode- generation was determined based on histological analysis of non-dopaminergic neurons in the brain. Behavioral assays were evaluated using open field activity and rotarod tests. Mitochondrial complex I activity, oxidative stress markers, and microglial activation were also examined in the substantia nigra. The level of TaClo was detected using HPLC-electrospray ionization tandem mass spectrometry. Dopaminergic neurotoxicity of TaClo was determined in midbrain organotypic cultures from rat pups. Following 8 months of TCE treatment, there was a progressive and selective loss of 50% of the dopaminergic neurons in mouse substantia nigra (SN) and about 50% loss of dopamine and 72% loss of 3,4-dihydroxyphenylacetic acid in the striatum, respectively. In addition, motor deficits, mitochondrial impairment, oxidative stress, and inflammation were measured. TaClo content was quantified in the brain after TCE treatment. In organotypic cultures, TaClo rather than TCE induced dopaminergic neuronal loss, similar to MPP + . TCE exposure may stimulate the endogenous formation of TaClo, which is responsible for dopaminergic neurodegeneration. However, even prolonged administration of TCE was insufficient for producing a greater than 50% loss of nigral dopamine neurons, indicating that additional co- morbid factors would be needed for mimicking the profound loss of dopamine neurons seen in Parkinsons disease. Keywords Trichloroethylene . 1-Trichloromethyl-1,2,3,4-tetrahydro-β-carboline . Parkinsons disease . Substantia nigra . Mitochondria . Oxidative stress Abbreviations PD Parkinsons disease TCE Trichloroethylene TaClo 1-Trichloromethyl-1,2,3,4-tetrahydro-β-carboline TH Tyrosine hydroxylase Mei Liu and Eun-Joo Shin contributed equally to this work. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s12035-017-0830-x) contains supplementary material, which is available to authorized users. * Guoying Bing [email protected] * Hyoung-Chun Kim [email protected] 1 Department of Neuroscience, College of Medicine, University of Kentucky, Lexington, KY 40536, USA 2 Neuropsychopharmacology and Toxicology Program, College of Pharmacy, Kangwon National University, Chunchon 24341, Republic of Korea 3 Department of Geriatrics, Wuxi Mental Health Center, Nanjing Medical University, Wuxi 214151, China 4 National Creative Research Initiative Center for Catalytic Organic Reactions, Department of Chemistry, Kangwon National University, Chunchon 24341, Republic of Korea 5 Department of Pharmacology, College of Medicine, Chung-Ang University, Seoul 06974, Republic of Korea 6 Ilsong Institute of Life Science, Hallym University, Anyang 14066, Republic of Korea 7 Department of Neurosurgery, Provincial Hospital Affiliated to Shandong University, Jinan, Shandong 250021, Peoples Republic of China Molecular Neurobiology (2018) 55:62016214 https://doi.org/10.1007/s12035-017-0830-x

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Page 1: Trichloroethylene and Parkinson’s Disease: Risk Assessment · Trichloroethylene and Parkinson’s Disease: ... HNE) in the substantia ... Trichloroethylene and Parkinson’s Disease:

Trichloroethylene and Parkinson’s Disease: Risk Assessment

Mei Liu1& Eun-Joo Shin2

& Duy-Khanh Dang2& Chun-Hui Jin3

& Phil Ho Lee4& Ji Hoon Jeong5

& Seok-Joo Park2,6 &

Yong-Sun Kim6& Bin Xing1

& Tao Xin7& Guoying Bing1

& Hyoung-Chun Kim2

Received: 13 June 2017 /Accepted: 8 December 2017 /Published online: 22 December 2017# Springer Science+Business Media, LLC, part of Springer Nature 2017

AbstractThis study was conducted to investigate the mechanism of action and extent of selective dopaminergic neurodegeneration caused byexposure to trichloroethylene (TCE) leading to the endogenous formation of the neurotoxin 1-trichloromethyl-1,2,3,4-tetrahydro-β-carboline (TaClo) in rodents. Beginning at 3 months of age, male C57BL/6 mice received oral TCE dissolved in vehicle for 8 months.Dopaminergic neuronal loss was assessed by nigral tyrosine hydroxylase (TH) immunoreactivity. Selective dopaminergic neurode-generation was determined based on histological analysis of non-dopaminergic neurons in the brain. Behavioral assays were evaluatedusing open field activity and rotarod tests. Mitochondrial complex I activity, oxidative stress markers, and microglial activation werealso examined in the substantia nigra. The level of TaClo was detected using HPLC-electrospray ionization tandemmass spectrometry.Dopaminergic neurotoxicity of TaClo was determined in midbrain organotypic cultures from rat pups. Following 8 months of TCEtreatment, there was a progressive and selective loss of 50% of the dopaminergic neurons in mouse substantia nigra (SN) and about50% loss of dopamine and 72% loss of 3,4-dihydroxyphenylacetic acid in the striatum, respectively. In addition, motor deficits,mitochondrial impairment, oxidative stress, and inflammation were measured. TaClo content was quantified in the brain after TCEtreatment. In organotypic cultures, TaClo rather than TCE induced dopaminergic neuronal loss, similar to MPP+. TCE exposure maystimulate the endogenous formation of TaClo, which is responsible for dopaminergic neurodegeneration. However, even prolongedadministration of TCEwas insufficient for producing a greater than 50% loss of nigral dopamine neurons, indicating that additional co-morbid factors would be needed for mimicking the profound loss of dopamine neurons seen in Parkinson’s disease.

Keywords Trichloroethylene . 1-Trichloromethyl-1,2,3,4-tetrahydro-β-carboline . Parkinson’s disease . Substantia nigra .

Mitochondria . Oxidative stress

AbbreviationsPD Parkinson’s diseaseTCE Trichloroethylene

TaClo 1-Trichloromethyl-1,2,3,4-tetrahydro-β-carbolineTH Tyrosine hydroxylase

Mei Liu and Eun-Joo Shin contributed equally to this work.

Electronic supplementary material The online version of this article(https://doi.org/10.1007/s12035-017-0830-x) contains supplementarymaterial, which is available to authorized users.

* Guoying [email protected]

* Hyoung-Chun [email protected]

1 Department of Neuroscience, College of Medicine, University ofKentucky, Lexington, KY 40536, USA

2 Neuropsychopharmacology and Toxicology Program, College ofPharmacy, Kangwon National University, Chunchon 24341,Republic of Korea

3 Department of Geriatrics, Wuxi Mental Health Center, NanjingMedical University, Wuxi 214151, China

4 National Creative Research Initiative Center for Catalytic OrganicReactions, Department of Chemistry, Kangwon National University,Chunchon 24341, Republic of Korea

5 Department of Pharmacology, College of Medicine, Chung-AngUniversity, Seoul 06974, Republic of Korea

6 Ilsong Institute of Life Science, Hallym University, Anyang 14066,Republic of Korea

7 Department of Neurosurgery, Provincial Hospital Affiliated toShandong University, Jinan, Shandong 250021, People’s Republic ofChina

Molecular Neurobiology (2018) 55:6201–6214https://doi.org/10.1007/s12035-017-0830-x

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Introduction

One of the principal pathophysiological features ofParkinson’s disease (PD) is the degeneration and profoundloss of dopamine neurons in the substantia nigra. Studies havesuggested a dominant role of environmental toxins in promot-ing the development of late-onset sporadic PD. Important in-sights were gained when an intravenously injected syntheticheroin elicited severe Parkinsonism in addicts [1]. The initiat-ing agent was a contaminant, 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP) [2], which is metabolized intoMPP+ and can be taken up specifically by the dopamine neu-rons, where it inhibits mitochondrial complex I function.Subsequent epidemiological studies have found an increasedincidence of PD associated with rural living as well as expo-sure to herbicides, pesticides, and heavy metals [3–5], whichwere reported to directly or indirectly inhibit mitochondrialcomplex I activity [6]. We recently identified a cluster of 30industrial co-workers who developed PD and Parkinsonismafter long-term (8–33 years) exposure to a widely used indus-trial solvent, trichloroethylene (TCE) [7]. A recent epidemiol-ogy study have identified that risk of Parkinson’s disease wasincreased 6-fold in people who worked with the solvent TCE[8]. Several studies have demonstrated that TCE instigatedselective complex I mitochondrial impairment with a concom-itant loss of dopaminergic neurons in the substantia nigra ofFischer 344 rats after oral administration of TCE for 6 weeks[7, 9]. For many years, TCE was extensively used in industryand government facilities as a chlorinated solvent [10].Despite decades of contamination, data for understandingthe effects of TCE exposure on neurotoxicity is lacking [10].Case reports suggest a potential link between chronic TCEexposure and PD. In addition to our report, others have foundthe onset of PD in an adult female exposed to TCE for 7 yearsdue to her profession and in three individuals chronically ex-posed to TCE [11, 12]. Thus, further studies are needed toelucidate the underlying mechanisms of TCE exposure in do-paminergic neurodegeneration.

Inhalation, oral, and dermal routes are possible avenues ofexposure to TCE in humans and laboratory animals. AbsorbedTCE is distributed to different target organs via the circulatorysystem. Most TCE taken into the body is metabolized, and themetabolites are considered responsible for the toxicity andcarcinogenicity observed in various organ systems [10]. Ithas been postulated that chloral, a major metabolite producedin the oxidative pathway, can spontaneously form 1-trichloromethyl-1,2,3,4-tetrahydro-β-carboline (TaClo),which has a structural similarity to the dopaminergic neuro-toxin MPTP through Pictet-Spenger condensation of the en-dogenously present biogenic amine tryptamine [13, 14].

Here, we have evaluated the risks posed by repeated expo-sure for to 400 mg/kg for 8 months in C57BL/6 mice.Following chronic TCE oral administration, we detected

progressive and selective dopaminergic neurodegeneration inthe substantia nigra using immunohistochemistry. TCEcaused impairment of mitochondrial complex I activity, in-creased levels of oxidative stress markers, and microglial ac-tivation. The endogenous formation of TaClo in the brains ofmice and the dopaminergic neurotoxicity of TaClo inorganotypic cultures were also detected for the first time inour study.

Material and Methods

Animals and Treatment

All procedures involving animals were performed in strictaccordance with the National Institutes of Health guidelinesand approved by the Institutional Animal Care and UseCommittee of Kangwon National University and Universityof Kentucky. Male C57BL/6 mice (Bio Genomic Inc., CharlesRiver Technology, Gapyung-Gun, Gyeonggi-Do, SouthKorea) were maintained under a 12 h:12 h light/dark cycleand fed ad libitum. The mice at 3 months of age receivedeither 400 mg/kg/day TCE (Sigma, St Louis, MO, USA) invehicle (olive oil) or an equal volume of vehicle by oral ga-vage, 5 days a week, for 8 months. In addition, postnatal day1–3 Sprague-Dawley rat pups (Harlan Laboratories,Indianapolis, IN, USA) were prepared for organotypic brainslice cultures.

Histological Analysis

At the desired end point, mice (n = 8 per group) were deeplyanesthetized with CO2 prior to transcardial perfusion withPBS followed by 4% paraformaldehyde. Perfused brains weresectioned on a sliding microtome into 30-μm free-floatingtissue sections. For immunohistochemical staining of tyrosinehydroxylase (TH), DARPP-32, ChAT, 3-NT, phosphorylatedα-synuclein, or Iba1, brain sections were incubated overnightat 4 °C with a primary antibody against TH (polyclonal,1:2000, Pel Freez, Rogers, AR, USA), GAD67 (monoclonal,1:1000, Chemicon, Temecula, CA, USA), DARPP-32 (poly-clonal, 1:2000, Chemicon), ChAT (monoclonal, 1:500,Chemicon), 3-NT (polyclonal, 1:500; Chemicon), α-synuclein phosphorylation at Ser129 (monoclonal, 1:1000;Wako, Osaks, Japan), Iba1 (polyclonal, 1:2000, Wako), orGFAP (monoclonal, 1:250, Chemicon). After washes and in-cubation with the appropriate secondary antibody (1:1000,Vector Laboratories, Inc., Burlingame, CA, USA), immuno-reactive cells were visualized using the avidin-biotinimmunoperoxidase method (ABC kits, Vector Laboratories,Inc.) with chromogen 3,3′-diaminobenzidine tetrahydrochlo-ride (Sigma). Moreover, sections immunostained with 3-NTand phosphorylated α-synuclein were counterstained with

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Harris hematoxylin to reveal the locations of positive signalsin the neurons. For Giemsa staining, slides mounted withbrain sections were stained in Giemsa’s solution (EMScience, Lawrence, KS, USA) at 60 °C for 10 min. For luxolfast blue staining, brain sections were incubated in 0.1% luxolfast blue solution overnight and counterstained in 1% neutralred. Additionally, TH-immunoreactive cells were counted in aseries composed of every sixth section of the substantia nigra,while the adjacent sections were subjected to conventionalNissl staining, and Nissl-stained neurons were counted.Stereological estimation of the total number of TH or Nissl-stained neurons was performed by visually counting accord-ing to the manufacturer’s instructions (Bioquant, Nashville,TN, USA) using the optical fractionator method [15].Likewise, the total number of 3-NT- and pSer129 α-synuclein-positive neurons as well as Iba1-stained microgliawere respectively estimated in a series composed of everysixth section of the substantia nigra.

Mitochondrial Enzyme Activity

Following CO2 anesthesia, mice were quickly decapitated andthe nigral regions promptly isolated using a mouse brain ma-trix for mitochondrial complex I activity measurement andslot blot analyses. The mitochondria were isolated fromsynaptoneurosomes using Ficoll density gradient ultracentri-fugation and nitrogen decompression. Mitochondrial complexI activity in rotenone-sensitive conditions was assayed bymeasuring the decrease in fluorescence of NADH as substrate(340 nm excitation, > 450 nm emission) using a BioTekSynergy HT plate reader (BioTek Instruments, Winooski,VT, USA).

Slot Blot Analysis

The levels of protein carbonyl and 4-hydroxynonenal (4-HNE) in the substantia nigra were determined with slot blotanalysis with the Oxyblot protein oxidation detection kit(Chemicon) and 4-HNE antibody (Calbiochem, San Diego,CA, USA) as previously described [16].

Behavioral Tests

Behavioral tests were conducted on mice treated with TCE orvehicle as previously described [17]. Locomotor activity wasmeasured for 30 min using an automated video tracking sys-tem (Noldus Information Technology, Wageningen,Netherlands) before treatment and once every 2 weeks aftertreatment. The horizontal distance traveled (cm) by animalswas analyzed at each time point. Additionally, motor perfor-mance and coordination were assessed with the acceleratedrotarod test using the procedural apparatus (Ugo Basile model7650, Comerio, VA, Italy). The test was performed before

treatment and once every 2 weeks after treatment. The dura-tion time (s) on the rotating drum was recorded and analyzed.

HPLC Assay

Striatal levels of dopamine, dopamine metabolite 3,4-dihydroxyphenylacetic acid (DOPAC), serotonin, serotoninmetabolite 5-hydroxyindoleacetic acid (5-HIAA), and norepi-nephrine were determined using HPLC with electrochemicaldetection as previously described [17, 18]. Results wereexpressed as ng/mg wet weight of tissue from vehicle andTCE-treated mice.

Acetylcholine Measurement

Striatal tissues were homogenized in ice-cold 20 mM sodiumphosphate buffer (pH 7.4) and centrifuged at 12,000×g for30 min at 4 °C. The supernatant samples were assayed foracetylcholine level using Amplex® Red Acetylcholine/Acetylcholinesterase Assay Kit (A-12217) (Invitrogen,Eugene, OR, USA) [19].

HPLC with Electrospray Ionization Tandem MassSpectrometry

The methods used were previously described [20–22]. Mousebrain, liver, kidney, and blood tissues were quickly frozen andstored at − 80 °C prior to analysis. TaClo was analyzed on aTSQQuantumUltra® triple-stage quadrupole mass spectrom-eter (Thermo Scientific). 1-Trichloromethyl-1,2,3,4-tetrahydro-β-carboline (TaClo) was synthesized [23];deuterium-labeled [3,3,4,4-2H4]-1-trichloromethyl-1,2,3,4-tetrahydro-β-carboline ([D4]TaClo) was prepared through asimilar synthetic procedure. Synthetic [D4]TaClo was appliedas an internal standard. TaClo was detected and reliablyquantified.

Real-Time Reverse Transcription-Polymerase ChainReaction

Real-time RT-PCR was performed as described previously[19]. Total RNA was isolated from the hippocampus usingthe RNeasy Mini kit (Qiagen, Valencia, CA, USA).Extracted RNA (1 μg) was reverse transcribed into cDNAby RNA to cDNA EcoDry Premix (Clontech, Palo Alto,CA, USA). Equal amount of cDNA was added to 50 μL oftotal PCR reactionmixture, containing 25 pmol of each primerand QuantiTect SYBR Green PCR Master Mix (Qiagen).They were amplified in duplicate with CFX96 Touch real-time PCR system (Bio-Rad Laboratories, Hercules, CA,USA). The reference gene (GAPDH) and target gene fromeach sample were run in parallel in the same plate with thesame amount of cDNA. The applied primers were as

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followed: preprodynorphin (GenBank accession number,AF026537.1), 5′- GCG TGG TCC AGG CTG ATG -3′(forward) and 5′- AGG CAG TCC GCC ATA ACA TT ′(reverse) [24]; preproenkephalin (GenBank accession num-ber, M13227.1), 5′- AGA AGC GAA CGG AGG AGAGAT -3′ (forward) and 5′- TTC AGC AGATCG GAG GAGTTG -3′ (reverse) [24]; GAPDH, 5′- ACC ACA GTC CATGCC ATC AC -3′ (forward) and 5′- TCC ACC ACC CTGTTGCTG TA -3′ (reverse) [19]. Real-time cycling parameterswere as follows: activation of HotStarTaq DNA polymerase at95 °C for 15 min, 40 cycles of denaturation at 94 °C for 45 s,annealing at 60 °C for 1 min, and extension at 72 °C for 1 min.The relative mRNA expression level was quantified using the2−ΔΔCt method [25].

Western Blot Analysis

Striatal tissues were lysed in buffer containing 200 mM Tris-HCl (pH 6.8), 1% SDS, 5 mM ethylene glycol-bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, 5 mM ethylene-diaminetetraacetic acid, 10% glycerol, and 1× protease inhib-itor cocktail (Sigma-Aldrich, St. Louis, MO, USA). Lysatewas centrifuged at 12,000×g for 30 min and the supernatantwas used for Western blot analysis as described previously[17, 19]. Proteins (20–50 μg/lane) were separated by 8% so-dium dodecyl sulfate-polyacrylamide gel electrophoresis andtransferred onto the PVDF membranes. Following transfer,the membranes were preincubated with 5% non-fat milk for30 min and incubated overnight at 4 °C with primary antibodyagainst dopaminergic D1 receptor (1:200; Santa CruzBiotechnology, Inc., Santa Cruz, CA, USA), dopaminergicD2 receptor (1:200; Santa Cruz Biotechnology, Inc., SantaCruz, CA, USA), or β-actin (1:50,000, Sigma-Aldrich). Andthen, membranes were incubated with HRP-conjugated sec-ondary anti-rabbit IgG (1:1000, GE Healthcare, Piscataway,NJ, USA) or anti-mouse IgG (1:1000, Sigma) for 2 h.Subsequent visualization was performed using enhancedchemiluminescence system (ECL plus®, GE Healthcare).Relative intensities of the bands were quantified byPhotoCapt MW (version 10.01 for Windows; VilberLourmat, Marne la Vallée, France) and then normalized tothe intensity of β-actin.

Organotypic Midbrain Culture

Organotypic brain slice cultures were prepared as previouslydescribed [26, 27]. Coronal slices (350 mm thick) of the mes-encephalon were dissected from rat pups and placed ontoMillicell-CM insert membranes in contact with a striatal slice.Slices were maintained in a 5%CO2 humidified atmosphere at34 °C. After 3 weeks in vitro, the slices were treated withTaClo, TCE, or MPP+ at 10, 50, or 100 μM in neurobasalmedium with B27 supplement. Loss of dopaminergic neurons

was confirmed with TH-immunoreactive cell counting after48 h of treatment.

Statistical Analysis

Data were analyzed with one-way ANOVA, with treatment asthe independent factor, using SigmaPlot 12.3 (SPSS Inc.,Chicago, IL, USA). Repeated measures ANOVA wasemployed to analyze the data from the open field test androtarod test. Post hoc comparisons were made using Fisher’sLSD test, with statistical significance set at p < 0.05. Data arepresented as mean ± standard error of the mean (SEM).

Results

TCE Selectively Induces Nigrostriatal DopaminergicDamage

TCE chronic oral administration led to a progressive and sig-nificant loss of dopaminergic neurons in the substantia nigra,dramatic decreases of dopamine and its metabolites in thestriatum, andmotor behavior deficits (Fig. 1). A representativephotomicrograph showed gradually decreased TH immunore-activity, the rate-limiting process in dopamine synthesis, in theSNc of mouse brain sections after a 3- to 8-month treatmentwith TCE; likewise, the decreased expression of Nissl stainingwas correspondingly exhibited in the SNc of their adjacentsections (Fig. 1a). Statistical analysis of cell counting indicat-ed a significant loss of TH-positive neurons by 32.7% at3 months and 52.3% after an 8-month treatment with TCEcompared to vehicle, and significant losses of Nissl-stainedneurons in the SNc by 34.1% after a 3-month and 48.1% afteran 8-month treatment with TCE compared to vehicle (Fig.1b), respectively. Thus, TCE-induced dopaminergic neuronalloss was not due to downregulation of the TH marker, but tocell destruction or degeneration. Furthermore, dopamine andits metabolite content were examined in the striatum after TCEtreatment. The dopamine and DOPAC levels were significant-ly decreased by 50.2 and 72.1% after an 8-month treatmentwith TCE, respectively, compared to vehicle (Fig. 1c).However, serotonin, 5-HIAA, norepinephrine, and acetylcho-line levels were not significantly changed in the striatum afteran 8-month treatment with TCE (Supplementary Fig. S1).Additionally, behavioral studies using open field and rotarodperformance were tested before and after TCE treatment.These behavioral tests were mainly designed to measure mo-tor abilities such as locomotion, exploration, coordination, andbalance. A progressive and significant decrease was foundafter TCE treatment compared to vehicle (Fig. 1d).

Chronic TCE treatment did not show any significant dif-ference on expression of dopaminergic D1 and D2 receptorsin the striatum as compared to vehicle (Fig. 2a). However,

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preproenkephalin mRNA expression was significantly in-creased after an 8-month treatment with TCE (Fig. 2b).

Systemic TCE treatment did not significantly induce non-dopaminergic neuronal damage in the nigrostriatal system,motor neuronal damage in the cerebellum, andmyelin damagein the whole brain after an 8-month treatment with TCE(Fig. 3). Histological analysis showed slight differences in

Nissl staining for neurons in the hippocampus and cortex(Fig. 3a), immunostaining of GAD67 and DARPP-32 forGABAergic neurons and immunostaining of ChAT for cho-linergic neurons in the striatum (Fig. 3b), Giemsa staining forPurkinje cells in the cerebellum (Fig. 3c), and luxol fast bluestaining for myelin distribution in the brainstem (Fig. 3d) be-tween vehicle and TCE-treated mice.

Fig. 1 Chronic oraladministration of TCE inducedneurodegeneration of the mousenigrostriatal dopaminergicsystem. a Representativephotomicrograph of THimmunostaining and Nisslstaining shown in the substantianigra pars compacta (SNc) after a3- and 8-month treatment withTCE or vehicle control. bUnbiased stereological cellcounting indicated progressiveand significant loss of bothTH-positive and Nissl-positiveneurons in SNc of TCE-treatedmice as compared with vehicle. cLevels of dopamine and itsmetabolite DOPAC weresignificantly decreased in thestriatum after an 8-monthtreatment with TCE as comparedwith vehicle. d Behavioraldeficits displayed in TCE-treatedmice. Compared with vehicle,distance traveled in the open fieldwas significantly reduced in TCE-treated mice. Similarly, TCE-treated mice showed a reducedduration on the rotating drum ascompared with vehicle. *p < 0.05,**p < 0.01 vs. vehicle. Thenumber of animals in each groupis indicated in parentheses. Scalebar = 500 μm

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TCE Causes Mitochondrial Dysfunction, OxidativeStress, α-Synuclein Misfolding, and Inflammationin the Substantia Nigra

Neurotoxicity of TCE may be mediated via mitochondrialdysfunction, oxidative stress, α-synuclein toxicity, and in-flammation in the nigrostriatal system (Fig. 4). Eight monthsfollowing oral administration, NADH, a direct measure ofcomplex I enzyme activity, was significantly reduced in theSN of TCE-treated mice compared with vehicle (Fig. 4a).Immunostaining for 3-NT and counterstaining with hematox-ylin showed that protein tyrosine nitration was localized pre-dominantly to large, dopaminergic neuron-like cells in theSNc of TCE-treated mice (Fig. 4b). Moreover, immunostain-ing for phosphorylated α-synuclein and counterstaining withhematoxylin showed that phosphorylated α-synuclein on

Ser129 mainly accumulated in the cytoplasm of dopaminergicneuron-like cells in the SNc of TCE-treated mice (Fig. 4c).Furthermore, Iba1 immunostaining exhibited increasedmicroglial activation in the SN of TCE-treated mice (Fig.4d). GFAP immunostaining revealed an increase of astrocyteactivation in the SN after a 2-week treatment with TCE ascompared with vehicle. However, the GFAP immunoreactiv-ity was returned to near control level after a 6-week(Supplementary Fig. S2) and an 8-month treatment withTCE (Fig. 4e). Stereological cell counting indicated a signif-icant increase of 3-NT-positive neurons by 421.9%, phosphor-ylated α-synuclein-positive neurons by 347.6%, and Iba1-labeled microglia by 554.9% compared to vehicle, butGFAP immunoreactivity in the SN did not show significantdifference between TCE- and vehicle-treated mice (Fig. 4f).Consistently, protein oxidation and lipid peroxidation in thenigral area, as evaluated using slot blot analyzes, were signif-icantly induced after a 3- and an 8-month treatment with TCE,respectively, compared to vehicle (Fig. 4g).

TaClo Detection After Chronic TCE Administration

TCE metabolites are considered responsible for the toxicityobserved in various organ systems after TCE exposure [28,29]. Studies have reported that TaClo can be produced endog-enously in rats and humans following exposure to chloralhydrate (in equilibrium with chloral during TCE metabolicprocesses) [21, 30]; however, the endogenous formation ofTaClo following TCE exposure is currently unknown andunderstudied. To our knowledge, this is the first animal studythat experimentally detected TaClo in mouse tissues afterchronic oral administration of TCE. Using HPLC-electrospray ionization tandem mass spectrometry methods,we identified TaClo concentration at 30.94 ± 4.21 pg/g in thebrain, 36.52 ± 3.94 pg/g in the liver, and 45.27 ± 8.62 pg/g inthe kidney tissues of mice treated with TCE for 8 months(Fig. 5 and Supplementary Fig. S3). Our results demonstratedthat TaClo can be formed endogenously following TCE expo-sure, which may advance our understanding of the mecha-nisms of TCE pathogenesis.

TaClo Elicits MPP+-Like Dopaminergic Neurotoxicity

To determine any direct neurotoxic effects of TCE and TaClo,as compared with a well-known dopaminergic toxin MPP+,we applied TCE, TaClo, or MPP+ to nigrostriatal organotypiccultures (Fig. 6). Forty-eight hours following treatment, bothTaClo- and MPP+-treated slices exhibited a dramatic loss ofTH immunostaining in dopaminergic neurons compared withboth vehicle control and TCE treatment (Fig. 6a, b). Cellcounting confirmed a significant loss of TH-positive neuronsin 10, 50, and 100 μM TaClo and 50 to 100 μM MPP+ treat-ment as compared with vehicle control, whereas almost no

Fig. 2 Chronic oral administration of TCE insignificantly affected D1and D2 receptors and preprodynorphin mRNA level, but significantlyincreased preproenkephalin mRNA level. a Changes dopaminergic D1and D2 receptor protein expression in the striatum after an 8-monthtreatment with TCE or vehicle control. b Changes in preprodynorphinand preproenkephalinmRNA expression in the striatum after an 8-monthtreatment with TCE or vehicle control. *p < 0.05 vs. vehicle. The numberof animals in each group is indicated in parentheses

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loss of TH-positive neurons was shown in 10 to 100 μMTCEtreatment (Fig. 6c). In this study, TaClo appeared to be moretoxic than MPP+, as it induced dopaminergic neuron damage

at the lowest dose of 10 μM. Our results strongly suggest thatendogenous formation of TaClo may be responsible for TCE-induced dopaminergic neurotoxicity in mice.

Fig. 3 Chronic oraladministration of TCE hardlyinduced non-dopaminergicneuron damage in the centralnervous system of mice. a Nisslstaining rarely showed neuronaldamage (arrow) in thehippocampus and cortex afterTCE compared with vehicle. bLittle change was found inGABAergic neurons (arrow)based on DARPP-32immunostaining and cholinergicneurons (arrow) based on ChATimmunostaining in the striatumafter TCE treatment comparedwith vehicle. However,GABAergic neurons based onGAD67 immunostaining werehardly identified in all subjectsdue to the poor signal to noiseratio. c Giemsa staining revealedan intact Purkinje cell monolayer(arrow) in the cerebellum of bothTCE treatment and vehicle. dLuxol fast blue staining exhibitslittle myelin disruption in thebrain after TCE compared withvehicle. Scale bar = 100 μm in a–c and 1500 μm in d

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Discussion

The present study demonstrated that oral TCE administrationfor 8 months induced selective and progressive loss of dopa-minergic neurons in the SNc of C57BL/6 mice. Loss of neu-rons was accompanied by decreases in the neurotransmitterdopamine and its metabolite DOPAC in the striatum.Accordingly, mice treated with TCE experienced significantdeficits in locomotor activity and rotarod performance.Similar to MPTP case [31–33] and postmortem PD patients[34], TCE treatment significantly induced preproenkephalinmRNA expression in the striatum. In addition, TCE treatmentcaused impairment of mitochondrial complex I activity, in-creased 3-NT immunoreactivity, intracellular accumulationof phosphorylated α-synuclein, and activated microglia inthe SNc, which implied that the cascade of events of mito-chondrial dysfunction, oxidative stress, protein mishandling,and inflammation induced by TCE lead to nigral dopaminer-gic neurodegeneration. Most notably, we detected the endog-enous formation of TaClo in the brain following TCE treat-ment. Further study confirmed that TaClo rather than TCEinduced dopaminergic neuronal death in the nigrostriatalorganotypic cultures, similar to the specific neurotoxinMPP+, indicating that TaClo formation has a similar affect toMPP+ cytotoxicity on dopaminergic neurons. Taken together,these results indicate that chronic systemic TCE exposure re-produces the neuropathological, neurochemical, and behav-ioral features of Parkinsonism, which may establish a novelprogressive neurodegenerative rodent model of PD.

Several animal models of PD have been developed to sharemany of the clinical and neuropathological characteristics ofPD. The most notable is the MPTP model because MPTPreplicates most of the biochemical and pathological featuresobserved under clinical conditions [35]. MPTP is converted to

MPP+, which enters dopamine-producing neurons and in-hibits the activity of mitochondrial complex I, resulting inthe generation of free radicals and oxidative damage to thecell [36]. However, the MPTP model does not replicate anumber of important characteristic features of PD. For exam-ple, MPTP administration does not show the progressive na-ture of dopaminergic neuronal loss orα-synuclein inclusion inthe nigral dopaminergic neurons [37]. Another PD animalmodel was developed by injecting 6-hydroxydopamine (6-OHDA) into different parts of the nigrostriatal pathway, me-dial forebrain bundle, SNc, or striatum, which led to specificdopamine depletions [38–41]. An essential difference be-tween the 6-OHDA model of PD and human idiopathic PDis the opposing development of PD symptoms. Human PD hasa progressive nature, whereas the 6-OHDAmodel is subject tocompensatory mechanisms [42]. Moreover, the herbicideparaquat induces dopaminergic neurodegeneration accompa-nied by α-synuclein inclusions in the SN of mice [43]. Thetoxicity of paraquat apparently is mediated by the formation ofsuperoxide radicals [44]. However, paraquat does not easilypenetrate the blood-brain barrier [45]. Additionally, rotenone,a widely used insecticide and fish poison, has been used tocreate a rodent model of PD. Greenamyre and colleagues re-ported that intravenous rotenone administration in rats pro-duced a selective degeneration of dopaminergic neurons inthe SN, accompanied by α-synuclein-positive Lewy body in-clusions and Parkinsonism [46]. However, rotenone has lim-ited uses, poor oral bioavailability, and a short half-life in theenvironment. Therefore, the TCE animal model of PD repli-cates several of the chronic and progressive characteristics ofPD and improves our understanding of the interaction mech-anisms between endogenous molecules and environmentaltoxins. TCE is likely to contribute significantly to the occur-rence of PD in the general population.

It has been widely accepted that the functional imbalancebetween direct and indirect pathways of the basal ganglia ac-counts for the development of PD symptoms [47, 48].Electrophysiological studies have showed the higher neuronalactivity in the internal segment than in the external segment ofglobus pallidus in PD patients [49, 50]. Consistently, postmor-tem analyses have indicated that the level and gene expressionof substance P or dynorphin are lower [51, 52], while those ofenkephalin are higher in the basal ganglia of PD patients ascompared with age-matched control subjects [37]. These phe-nomena have been reproduced in rodent PD models treatedwith MPTP or 6-OHDA [31–33, 53–59]. In these studies, 6-OHDA treatment resulted in the increased gene expressionand binding activity of dopaminergic D1 receptors and thedecreased gene expression and binding activity of dopaminer-gic D2 receptors in the striatum [54, 59]. However, MPTPappeared to insignificantly affect the binding activity or geneexpression of dopaminergic D1 and D2 receptors [58]. Inanimal model using MPTP or 6-OHDA, the level and gene

�Fig. 4 Chronic oral administration of TCE caused mitochondrialdysfunction, oxidative stress, and inflammation in the substantia nigraof mice at 8 months. a Mitochondrial complex I enzyme activitymeasured based on NADH substrate oxidation was significantlyreduced in the substantia nigra of TCE-treated mice as compared withvehicle. b 3-NT immunostaining and counterstaining with hematoxylinshow that tyrosine nitration was mostly localized in neuron-like cells(arrows) in SNc of TCE-treated mice. c Phosphorylated α-synuclein atSer129 immunostaining and counterstaining with hematoxylin showedthat phosphorylated α-synuclein aggregation was mainly localized inthe cytoplasm of neuron-like cells (arrowheads) in SNc of TCE-treatedmice. d Iba1 immunostaining showed more activated microglia in SNc ofTCE-treated mice as compared with vehicle. e TCE treatment did notsignificantly alter GFAP immunoreactivity as compared with vehicle. fStereological cell counting confirmed significant increases of 3-NT,phosphoryla ted α -synucle in at Ser129, Iba1, and GFAPimmunoreactivity in SNc of TCE-treated mice as compared withvehicle control. g Significant increases in the protein oxidation and 4-hydroxynonenal levels were found in the substantia nigra of TCE-treatedmice as compared with vehicle control. *p < 0.05, **p < 0.01 vs. vehicle.The number of animals in each group is indicated in parentheses. Scalebar = 100 μm

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expression of substance P or dynorphin were decreased, whilethose of enkephalin were increased in the basal ganglia[31–33, 53, 55–57]. In this study, chronic TCE treatment in-significantly affected dopaminergic D1 and D2 receptor

expressions, suggesting that the neurotoxicological profileby TCE is, at least in part, in line with that by MPTP in termsof dopamine receptor modulation. Furthermore, TCE signifi-cantly induced preproenkephalin mRNA in the striatum,

Fig. 5 Chronic oral administration of TCE stimulated TaClo formation inmouse brain tissue. a TaClo level was detected in the brain, liver, andkidney tissues of TCE-treated mice, whereas no TaClo was detected in thevehicle control-treated mice. b, c Representative chromatograms showthe measurement of TaClo in the brains of vehicle- or TCE-treated mice

based on online coupling of HPLC with electrospray ionization tandemmass spectrometry. Synthetic [D4]TaClo was applied as an internalstandard (chromatogram top and bottom panels); TaClo was detectedand quantified (top and middle panel)

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whereas TCE insignificantly altered preprodynorphinmRNA,suggesting that chronic TCE treatment might produce thefunctional imbalance between direct and indirect striatal pro-jection neurons, as shown in MPTP case.

Neuroinflammation is one of the main pathologic events invarious neurodegenerative diseases, including PD [60]. Hongand colleagues [61–63] and others [60] proposed thatmicrogliosis plays a key role in neuroinflammation, and astro-cytes, as the main regulator of neuroinflammation, play a pro-tective or detrimental role depending on the condition [64,65]. In our study, microglial activation was noted in the SNafter chronic TCE treatment. However, the increased astroglialactivation was observed as early as 2 weeks treatment withTCE in the SN, and activation was returned to near controllevel after a 6-week treatment and remained near control levelby an 8-month treatment with TCE. Thus, we suggest thatmicroglia specifically contribute to neuroinflammation in-duced by chronic TCE treatment in our experimentalcondition.

Significant findings from the current study included thedetection of TaClo concentration in the brain following TCEoral administration and confirmation that the dopamine cyto-toxicity of TaClo was similar to MPP+ of in vitro testing.Evidence suggests that TaClo, produced endogenously in ratsand humans following exposure to chloral hydrate, may be apotent dopaminergic neurotoxin [21, 30]. Systemically ad-ministered TaClo induced immediate and transient release ofdopamine and serotonin, followed by a progressive increase inhydroxyl free radical formation in the striatum of rats [66].

Intranigral injection of TaClo and derivatives caused reductionin TH activity and loss of DOPAC content in the striatal tis-sues of rats [67]. Researchers further suggested that TaClo andits derivatives modulate dopamine activity by interacting withthe protein kinase TH-activating system [68]. Additionally,TaClo reportedly induced an apoptotic pathway in the humanneuroblastoma cell line, which involved the translocation ofmitochondrial cytochrome c to the cytosol and activation ofcaspase 3 [69]. In primary C57BL/6 mouse mesencephaloncell cultures, TaClo not only induced swollen dendrites andsoma and loss of axons and dendrites but also a 50% loss ofTH-immunoreactive neurons [70]. Notably, TaClo was shownto exhibit highly selective in vitro inhibition of complex I inthe mitochondrial respiratory chain, the required concentra-tions being much lower than that needed for MPP+ [13], andappeared 10-fold more effective in inhibiting mitochondrialcomplex I activity than MPP+ [71]. However, one caveat inthese previous studies is whether TaClo could be formedin vivo following TCE exposure [10]. The current study isthe first animal study that experimentally detected the endog-enous formation of TaClo in mouse kidney, liver, and wholebrain tissues after prolonged exposure to TCE. Based on thepublished and current studies, we concluded that chloral, me-tabolized from TCE through the oxidative pathway, can reactwith biogenic amine tryptamine to form TaClo in vivo, andTaClo inhibited mitochondrial complex I activity, generatedexcessive reactive oxygen/nitrogen species to induce oxida-tive stress, increased microglial activation, and caused α-synuclein misfolding, which may elevate the vulnerability of

Fig. 6 TaClo-induced dopaminergic neurotoxicity in comparison toMPP+ in the organotypic midbrain culture. a TH immunoreactivity wasexhibited in brain slices after vehicle, TCE, TaClo, or MPP+ treatment. bTH immunoreactivity was representatively revealed in the whole mount

organotypic culture slice of vehicle control. c Cell counting proceduresdemonstrated a significant reduction in the total number of TH-positiveneurons in 10, 50, or 100 μM TaClo-treated group and 50 or 100 μMMPP+-treated groups when compared with the vehicle control (*p < 0.01)

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nigral dopaminergic neurons to degenerative processes. TCE,a common industrial solvent, may be an environmental riskfactor of development of late-onset PD.

This novel TCE rodent model presents progressive dopa-minergic neurodegeneration and replicates characteristic fea-tures of PD, which may aid in a better understanding of envi-ronmental risk factors in the etiology of PD.

Acknowledgements This study was funded by the following grants: theBasic Science Research Program through the National ResearchFoundation of Korea (NRF) funded by the Ministry of Science, ICTand Future Planning (#NRF-2017R1A2B1003346 and #NRF-2016R1A1A1A05005201), Republic of Korea, and the NRF grantfunded by the Korea Government (MSIP) (2011-0018355), Republic ofKorea.

Compliance with Ethical Standards

Conflict of Interest The authors declare that they have no conflict ofinterests.

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