Chemico-Biological Interactions 199 (2012) 74–86

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Chemico-Biological Interactions

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Protective role of glutathione reductase in paraquat induced neurotoxicity

Mirjana M. Djukic a,⇑, Marina D. Jovanovic b, Milica Ninkovic b, Ivana Stevanovic b, Katarina Ilic c,Marijana Curcic a, Jelena Vekic d

a Department of Toxicology, Faculty of Pharmacy at the University of Belgrade, Vojvode Stepe 450, 11221 Belgrade, Serbiab Institute for Medical Research, Military Medical Academy, Crnotravska 17, 11000 Belgrade, Serbiac Department of Pharmacology, Faculty of Pharmacy at the University of Belgrade, Vojvode Stepe 450, 11221 Belgrade, Serbiad Department of Medical Biochemistry, Faculty of Pharmacy at the University of Belgrade, Vojvode Stepe 450, 11221 Belgrade, Serbia

a r t i c l e i n f o

Article history:Received 6 February 2012Received in revised form 19 May 2012Accepted 21 May 2012Available online 18 June 2012

Keywords:ParaquatNeurotoxicityGlutathioneGlutathione reductase

0009-2797/$ - see front matter � 2012 Elsevier Irelanhttp://dx.doi.org/10.1016/j.cbi.2012.05.008

Abbreviations: ATP, adenosine-triphosphate; GPx,glutathione reductase; GSH, glutathione (reduced fodized form); HMPS, hexose monophosphate shunt; LPnicotinamide adenine dinucleotide; NADPH, nicotinphosphate; NAC, N-acetyl-cysteine; NO3

�, nitrate;superoxide anion radical; OS, oxidative stress; PQ,dismutase; MDA, malondialdehyde; VBRs, vulnerableatally; i.p., intraperitoneally.⇑ Corresponding author. Tel.: +381 11 3951 308; fa

E-mail address: mirjana.djukic@pharmacy.bg.ac.rs

a b s t r a c t

Paraquat (PQ), a widely used herbicide is a well-known free radical producing agent. The mechanistic path-ways of PQ neurotoxicity were examined by assessing oxidative/nitrosative stress markers. Focus was onthe role of glutathione (GSH) cycle and to examine whether the pre-treatment with enzyme glutathionereductase (GR) could protect the vulnerable brain regions (VBRs) against harmful oxidative effect of PQ.The study was conducted on Wistar rats, randomly divided in five groups: intact-control group, (n = 8)and four experimental groups (n = 24). All tested compounds were administered intrastriatally (i.s.) inone single dose. The following parameters of oxidative status were measured in the striatum, hippocampusand cortex, at 30 min, 24 h and 7 days post treatment: superoxide anion radical (O2

��), nitrate (NO3�),

malondialdehyde (MDA), superoxide dismutase (SOD), total GSH (tGSH) and its oxidized, disulfide form(GSSG) and glutathione peroxidase (GPx).

Results obtained from the intact and the sham operated groups were not statistically different, confirm-ing that invasive i.s. route of administration would not influence the reliability of results. Also, similar pat-tern of changes were observed between ipsi- and contra- lateral side of examined VBRs, indicating rapidspatial spreading of oxidative stress. Mortality of the animals (10%), within 24 h, along with symptoms ofParkinsonism, after awakening from anesthesia for 2–3 h, were observed in the PQ group, only. Increasedlevels of O2

��, NO3� and MDA, increased ratio of GSSG/GSH and considerably high activity of GPx were mea-

sured at 30 min after the treatment. Cytotoxic effect of PQ was documented by drastic drop of all measuredparameters and extremely high peak of the ratio GSSG/GSH at 24th hrs after the PQ i.s. injection. In theGR + PQ group, markedly low activity of GPx and low content of NO3

� (in striatum and cortex) were mea-sured during whole experiment, while increase value was observed only for O2

��, at 7th days.We concluded that oxidative/nitrosative stress and excitotoxicity are the most important events since the

early stage of PQ induced neurotoxicity. Based on the ratio GSSG/GSH, the oxidation of GSH to GSSG is prob-ably dominant way of GHS depletion and main reason for reduced antioxidative defense against PQ harmfuloxidative effect. The GR pre-treatment resulted in the absence of Parkinson’s disease-like symptoms andmortality of the rats. Additionally, oxidative/nitrosative stress did not developed, as well as almost dimin-ished metabolism of the VBRs at 24th hours (as has been documented in the PQ group) did not occurred inthe GR + PQ, suggesting a neuroprotective role for the GR in PQ induced neurotoxicity.

� 2012 Elsevier Ireland Ltd. All rights reserved.

d Ltd. All rights reserved.

glutathione peroxidase; GR,rm); GSSG, glutathione (oxi-O, lipid peroxidation; NADH,amide adenine dinucleotideNS, nitrosative stress; O2

��,paraquat; SOD, superoxidebrain regions; i.s., intrastri-

x: +381 11 3972 840.(M.M. Djukic).

1. Introduction

Misuse of pesticides, such as paraquat (PQ), can lead to excessivecontamination of the environment. Paraquat (1,10-dimethyl-4,40-bipyridinium), a fast-acting non-selective contact herbicide, iswidely used to control broad-leaved weeds in growing corn, fruittrees, vegetables, and as a desiccant before harvesting and fordestroying marijuana plants. Accidental and occupational exposuresof agriculture workers are the primary hazards for human health.Occupational and professional exposure to PQ is addressed to inha-lation or dermal route of administration and is considered as an eti-

M.M. Djukic et al. / Chemico-Biological Interactions 199 (2012) 74–86 75

ological factor of Parkinson’s disease [1,2]. Suicidal poisonings are re-ferred to ingestion. Ingestion of concentrated solutions (12–20%) ofPQ leads to early death due to organs failure, whereby the kidneysand lungs are main target organs. In the past few decades PQ hasbeen a popular agent for intentional (suicidal) poisonings. Paraquatis extremely toxic to humans (LD50 35 mg/kg) and animals (rats:LD50 is 110–150 mg/kg), by all routes of exposure. Since recently,usage of the PQ has been restricted [3–5]. There is no specific treat-ment for PQ poisoning, hence management of poisonings is to relievesymptoms and treat complications (supportive care) [6].

Red-ox metabolism is essential for PQ cytotoxic effect [7–10].Several enzymes may be involved in PQ metabolism, such as: thecytochrome P-450 reductase, ferricytochrome oxidoreductase andthe cytosolic enzyme nitric oxide synthase (NOS) [2,7,11]. In thepresence of electron donors, PQ2+ (commercial product: a salt ofa dication) readily undergoes the one-electron reduction to its rad-ical form PQ�+. Unpaired electron of PQ�+ is stabilized by the conju-gated double bond in the pyridine ring and quaternary nitrogen inanother ring (Scheme 1). In aerobic conditions, PQ�+ conveys its un-paired electron to molecular O2, generating superoxide anion rad-ical (O2

��), whilst PQ�+ becomes oxidized, to PQ2+. This triggers achain of free radicals reactions, which are essential for the PQ cyto-toxic effect [10].

Brain tissue is particularly vulnerable to oxidative injury in-duced by PQ, documented with manifestation of Parkinsonism likesymptoms (development of extrapyramidal behavior) [12]. Someparts of the brain [including: pyramidal neurons of CA1 and CA3sectors of the hippocampus, a small pyramidal neurons and thirdlayer of the cerebral cortex and striatum (Nucleus caudatus andputamen)] are more sensitive to oxidative/nitrosative stress [13].Given systemically PQ produces hyper-excitability, convulsions,hyperkinesis and incoordination in the early stages whilst intracer-bral administration leads to neurodegenerative, morphological andbehavioral changes in the central nervous (CNS) system, in a dose-dependent manner [14–19].

Glutathione (GSH) acts as a neuromodulator, neurotransmitterand neurohormone in the CNS. Its antioxidative role is recognizedin several reactions including glutathione peroxidase (GPx) cata-lyzed reduction of lipid hydro peroxides and conjugation withthiol, nitroso and metal ions [20–28].

According to literature, several approaches have been under-taken to retain GSH at the level to achieve its antioxidative effect.One of them is systemic administration of L-cysteine and/or N-acetyl-cysteine (NAC) which are substrates for the biosynthesisof GSH. L-cysteine passes across biological membranes and servesas a good source of SH groups, whereas GSH is ineffective when gi-ven orally because of its poor absorption from the digestive tractand/or poor ability to permeate through the membrane. However,therapeutic use of cysteine is limited by its excitotoxicity since itinteracts with glutamate causing acidification and neurotoxicity[29–31].

During oxidative stress, GSH becomes oxidized to glutathionedisulfide (GSSG). Recycling of glutathione (reduction of GSSG backto GSH) by glutathione reductase (GR) is particularly important tomaintain GSH concentrations at levels necessary to achieve itsantioxidative role. Leading by the fact that GR does not pass theblood–brain barrier, we decided to administer GR intrastriataly(i.s.). In this toxicological-experimental mechanistic study weexamined whether neuroprotection of vulnerable brain regions

++NNCH3 CH3

+ e

- e

Scheme 1. One-electron reduction of PQ2+ results in the formation of its stabile radical fand quaternary nitrogen in another ring.

(VBRs) (striatum, hippocampus and cortex) against harmful oxida-tive injury induced by i.s. administered PQ would be achieved bythe pre-treatment with GR intrastriatally (i.s.) applied. A positiveoutcome would have confirmed that the oxidative/nitrosativedamage of VBRs was the consequence of the reduced antioxidantdefense, primarily due to GSH oxidation to GSSG, thus minimizingthe significance of the other GSH depletion pathways in this partic-ular case of PQ neurotoxicity.

2. Materials and methods

2.1. Animals

The experimental animals were treated according to Guidelinesfor Animal Study, No. 282-12/2002 (Ethics Committee of the Mili-tary Medical Academy, Belgrade, Serbia and Montenegro). Theexperiments were performed on adult male Wistar rats weighingapproximately 220 g, randomly divided into two control groups(the intact group n = 8, and the sham-operated, n = 24) and threeexperimental groups (n = 24, each) which were further subdividedinto three subgroups (n = 8) according to the time of sacrificing.The rats were housed in cages under standardized housing condi-tions (ambient temperature of 23 ± 2 �C, relative humidity of55 ± 3% and a light/dark cycle of 13/11 h) and had free access tostandard laboratory pellet food and tap water. All the experimentswere performed after 7 days period of adaptation to laboratoryconditions, and were carried out between 9 a.m. and 1 p.m.

2.2. Experimental design

Rats were anesthetized by sodium pentobarbital (45 mg/kg perbody weight) given intraperitoneally (i.p.). In this study we usedyeast GR, according to: (a) literature evidence of its homology withGR sequences of rats and humans, substrate specificity, kineticscharacteristic and substrate affinity; and (b) our positive previousexperience in different animal models (unpublished data) [32–35]. Testing substances were administrated as single doses, i.s. infinal volumes of 10 lL, which is insufficient to burden nerve tissue.For this purposes we used Hamilton syringe, which was accuratelycoordinated by using a stereotaxic instrument for small laboratoryanimals (coordinates: 8.4 mm behind the bregma, 2.6 mm leftfrom the midline suture and 4.8 mm ventral from dura) [36]. Theexperiment was accomplished with the following (five) experi-mental groups, which received different testing substances: the in-tact group (not treated, n = 8), the sham-operated rats (10 lL ofsaline), n = 24; the GR group(GR, 15.63 U/10 lL), n = 24; the PQgroup (2.5 lg PQ/10 lL, i.e. 0.01 lM/10 lL) n = 24; and the GR + PQgroup (GR, 15.63 U/5 lL, immediately before PQ administration,0.01 lM/5 lL), n = 24. The animals were sacrificed by decapitationat 30 min, 24 h and 7 days after the treatments. Biochemicalparameters of OS were measured in the ipsi- and contra- lateralside of the VBRs. To exclude the possibility that mechanical injurycaused OS in the VBRs, we compared OS parameters between thesham-operated and the intact groups.

2.3. Reagents

All chemicals were of analytical grade. The following com-pounds were used in this study: Paraquat–Galokson� (200 g/L)

+NNCH3 CH3

.

orm (PQ�+) Stability is achieved by the conjugated double bond in the pyridine ring

Table 1Concentrations of parameters of oxidative status in the contralateral vulnerable brain regions of Wistar rats.

OS

parameters

VBRs Groups

Control Sham-operated GR PQ GR + PQ

0 min 30 min 24 h 7 days 30 min 24 h 7 days 30 min 24 h 7 days 30 min 24 h 7 days

(n = 8) (n = 8) (n = 8) (n = 8) (n = 8) (n = 8) (n = 8) (n = 8) (n = 8) (n = 8) (n = 8) (n = 8) (n = 8)

O2�

lmol

red

NBT/

mg

prot

S 4.0 ± 0.7 4.0 ± 0.8 3.8 ± 0.8 3.5 ± 0.4 4.3 ± 0.6 5.1 ± 0.2 4.2 ± 1.8 3.9 ± 0.9 1.1 ± 0.5 c⁄⁄⁄,�,⁄ 7.0 ± 0.6c⁄⁄⁄,�,⁄,£,⁄⁄⁄ 4.1 ± 0.1 2.4 ± 1.4 8.2 ± 2.9 c⁄⁄,�,⁄,£,⁄⁄⁄

H 4.8 ± 0.5 4.2 ± 0.6 4.3 ± 1.2 4.4 ± 1.0 5.3 ± 0.9 5.2 ± 1.2 3.6 ± 0.6 3.1 ± 0.8 3.0 ± 1.8 4.9 ± 1.6 4.3 ± 0.9 2.5 ± 1.2 c⁄⁄ 5.0 ± 2.3

C 3.9 ± 0.4 4.2 ± 1.0 4.0 ± 1.0 4.3 ± 0.4 4.2 ± 0.7 6.7 ± 1.3 4.2 ± 1.9 2.9 ± 1.1 0.7 ± 0.1 c⁄⁄⁄ 5.8 ± 0.8�,⁄,£,⁄⁄⁄ 3.9 ± 0.3 2.7 ± 1.4 7.8 ± 1.1 c⁄⁄,£,⁄

NO3�

nmol

NO3�/

mg

prot

S 20.0 ± 2.4 19.0 ± 3.9 18.0 ± 4.9 18.0 ± 2.0 22.0 ± 3.4 26.5 ± 1.1 17.3 ± 4.2 18.2 ± 4.2 4.1 ± 2.3 c⁄ 12.6 ± 4.7 c⁄ 5.8 ± 1.2 c⁄ 4.4 ± 2.7 c⁄ 3.6 ± 1.4 c⁄

H 20.0 ± 6.3 17.8 ± 5.2 16.3 ± 5.3 18.3 ± 5.3 21.1 ± 4.5 26.1 ± 1.7 25.7 ± 4.5 18.8 ± 8.7 2.5 ± 1.7 c⁄⁄⁄,�,⁄ 18.7 ± 7.0£,⁄⁄ 19.7 ± 11.7 9.2 ± 4.7 22.5 ± 4.3

C 13.8 ± 3.1 17.2 ± 4.0 16.7 ± 8.2 18.7 ± 3.8 15.4 ± 1.3 9.2 ± 2.3 12.1 ± 2.1 22.0 ± 15.0 2.5 ± 1.4 c⁄⁄⁄,�,⁄⁄ 8.4 ± 4.6�,⁄,£,⁄ 15.3 ± 6.6 5.6 ± 4.7�,⁄⁄ 7.2 ± 4.9�,⁄⁄

MDA

pmol

MDA/

mg

prot

S 50.6 ± 8.2 48.9 ± 14.1 47.7 ± 10.2 48.7 ± 9.1 55.6 ± 11.6 73.1 ± 6.9 68.0 ± 15.8 103.4 ± 25.6 55.7 ± 13.0 95.2 ± 12.6 52.0 ± 24.3 47.7 ± 36.8 97.7 ± 58.0

H 72.6 ± 12.7 48.6 ± 11.9 45.5 ± 7.9 46.7 ± 10.99 61.6 ± 17.6 81.3 ± 17.9 74.1 ± 14.3 103.8 ± 37.2 46.7 ± 10.5�,⁄ 135.3 ± 58.6£,⁄⁄⁄ 48.6 ± 3.1 21.4 ± 5.2 90.0 ± 29.8�,⁄,£,⁄⁄⁄

C 60.2 ± 8.9 53.5 ± 18.1 53.9 ± 12.9 53.4 ± 12.1 55.2 ± 8.7 40.3 ± 6.4 66.3 ± 10.5 110.3 ± 32.3 c⁄ 45.2 ± 9.9�,⁄⁄ 125.1 ± 44.1£,⁄⁄⁄ 46.1 ± 4.5 29.8 ± 1 8.4 116.0 ± 42.9£,⁄⁄⁄

t GSH

nmol/

mg

prot

S 16.4 ± 3.9 14.2 ± 2.0 14.4 ± 3.0 14.1 ± 2.0 17.5 ± 3.1 18. ± 3.6 22.2 ± 5.9 16.1 5.8 6.3 ± 2.1 c⁄⁄⁄ 18.8 ± 2.7£,⁄ 13.3 ± 1.9 17.1 ± 11.5 18.1 ± 8.6

H 18.0 ± 2.4 16.7 ± 1.9 17.1 ± 1.7 17.2 ± 1.9 16.0 ± 1.9 16. ± 1.9 22.0 ± 3.8 16.5 ± 4.4 4.4 ± 1.1 c⁄⁄⁄ 23.0 ± 6.0£,⁄⁄⁄ 15. ± 0.1 11.1 ± 6.2 23.9 ± 14.1

C 17.9 ± 2.0 14.9 ± 3.01 14.6 ± 3.0 15.0 ± 2.0 22.4 ± 2.9 22.0 ± 4.4 22.7 ± 4.2 12.2 ± 2.5 4.7 ± 1.8c⁄⁄⁄ 13.5 ± 3.3 12.1 ± 2.4 6.8 ± 1.3 22.5 ± 9.5£,⁄⁄

GSSG

nmol/

mg

prot

S 1.0 ± 0.1 1.1 ± 0.1 1.0 ± 0.2 1.0 ± 0.1 0.8 ± 0.2 0.8 ± 0.2 1.0 ± 0.2 2.5 ± 0.3 c⁄⁄⁄ 1.8 ± 0.1 c⁄⁄⁄ 1.2 ± 0.4�,⁄ 1.6 ± 0.4 1.3 ± 0.1 1.3 ± 0.3

H 0.9 ± 0.1 0.8 ± 0.1 1.0 ± 0.1 1.0 ± 0.1 0.9 ± 0.2 0.8 ± 0.1 1.0 ± 0.1 2.6 ± 0.1 c⁄⁄⁄ 1.8 ± 0.1 c⁄⁄⁄ 1.6 ± 0.3�,⁄ 1.7 ± 0.2 1.7 ± 0.2 1.2 ± 0.3

C 1.2 ± 0.2 0.9 ± 0.1 0.9 ± 0.1 0.9 ± 0.1 0.9 ± 0.1 0.9 ± 0.1 1.1 ± 0.2 3.1 ± 0.2 c⁄⁄⁄ 2.2 ± 0.1 c⁄⁄⁄ 1.6 ± 0.3�,⁄ 1.9 ± 0.3 c⁄ 1.9 ± 0.2 c⁄ 1.3 ± 0.1

SOD

U SOD/

mg

prot

S 740.1 ± 218.3 818.5 ± 150.3 781.3 ± 54.7 789.2 ± 138.9 720.2 ± 327.3 770.6 ± 350.2 936.3 ± 425.4 639.8 ± 142.0 149.5 ± 46.2 c⁄⁄,�,⁄ 835.8 ± 343.7£,⁄ 553.6 ± 254.5 632.8 ± 410.5 1043.4 ± 425.2

H 860.0 ± 204.4 953.876 ± 186.9 933.5 ± 201.2 905.6 ± 268.9 790.0 ± 238.4 1169.2 ± 352.8 608.3 ± 183.5 648.8 ± 239.1 191.6 ± 36.0 c⁄⁄⁄ 804.0 ± 390.5£,⁄ 401.3 ± 150.7 427.1 ± 145.3 913.1 ± 335.1

C 833.3 ± 208.2 844.33 ± 157.4 841.3 ± 69.9 864.6 ± 200.8 814.3 ± 278.1 675.9 ± 230.8 838.8 ± 286.4 854.2 ± 294.8 108.1 ± 70.3 c⁄⁄⁄,�,⁄ 618.3 ± 265.2£,⁄ 438.4 ± 40.3 262.5 ± 65.8 1165.9 ± 439.6�,⁄,£⁄⁄

GPx

mU

GPx/

mg

prot

S 94.8 ± 9.3 84.8 ± 24.8 90.8 ± 38.5 86.8 ± 20.1 88.8 ± 10.3 83.6 ± 10.9 89.1 ± 23.9 249.7 ± 57.9 c⁄⁄⁄ 123.9 ± 30.0 232.8 ± 153.3 11.5 ± 9.0 c⁄⁄⁄ P⁄⁄ 30.0 ± 12.8 c⁄⁄⁄ P⁄⁄ 74.0 ± 35.0 P⁄

H 80.4 ± 12.9 94.6 ± 35.9 96.2 ± 30.1 98.0 ± 35.9 79.0 ± 12.4 66.7 ± 15.0 67.0 ± 9.5 269.1 ± 107.0 c⁄ 115.1 ± 51.5�,⁄ 176.2 ± 89.5 31.8 ± 0.2 c⁄⁄⁄ P⁄⁄ 11.7 ± 7.3 c⁄⁄⁄ P⁄⁄ 87.4 ± 76.9

C 115.3 ± 11.9 139.0 ± 20.9 125.0 ± 19.2 133.0 ± 18.2 112.9 ± 31.6 99.1 ± 35.4 105.4 ± 69.1 316.4 ± 154.0 c⁄ 106.4 ± 34.8�,⁄⁄⁄ 133.2 ± 90.8 27.8 ± 6.5 c⁄⁄⁄ P⁄⁄⁄ 20.7 ± 14.2 c⁄⁄⁄ P⁄⁄ 101.4 ± 61.1�⁄

Note: The capture of Table 1 is the same as for Graphs 1–6, with only one difference that it was for contralateral side of the VBRs, thus I have doubt, so please help me about that.There are two options (please make choice, or maybe you have better solution?).(1) The experimental conditions, parameters of oxidative/nitrosative stress and antioxidative defense and labeling of significance are the same as for Graphs 1–6.(2) In the contralateral side of vulnerable brain regions (striatum, hippocampus and cortex) following parameters are presented: a) parameters of oxidative stress/nitrosative stress [Graphs (1–3)]: Graph 1 - superoxide anionradicals (O2

��: lmol red NBT/mg proteins); Graph 2 - nitrates (nmol NO3�/mg proteins); Graph 3 - lipid peroxidation (pmol MDA/mg proteins); b) parameters of antioxidative defense [Graphs (4–6)]: Graph 4 - (A) total glutathione

content (tGSH); (B) oxidized glutathione (GSSG) (nmol GSSG/mg proteins) and (C) the relationship between oxidized and reduced glutathione is presented as the ratio GSSG/GSH, Graph 5 - Superoxide dismutase activity (U SOD/mg proteins); Graph 6 - Glutathione peroxidase activity (mU GPx/mg proteins).See the experimental conditions presented in the Section 2.2 Experimental design.Values are presented as means ± S.D. Statistical significance was considered at: ⁄p < 0.05; ⁄⁄p < 0.01; ⁄⁄⁄p < 0.001. Labels of statistical significance: c - compared to the control group (intact animals); p – results from the GR + PQgroup compared to the PQ group (refers to the same time point); � - compared to 30 min; £ - compared to 24 h (within the certain group).

76M

.M.D

jukicet

al./Chemico-Biological

Interactions199

(2012)74–

86

M.M. Djukic et al. / Chemico-Biological Interactions 199 (2012) 74–86 77

(Galenika - Zemun, Serbia); Sodium pentobarbital – Vetanarcol�

(0.162 g/mL) (Werfft - Chemie,Vienna, Austria); Glutathione reduc-tase (EC 1.6.4.2), Type III, from yeast [9001-48-3], Sigma ChemicalCo (St Luis, MO, USA) - highly refined suspension in 3.6 M(NH4)2SO4, at pH 7.0; 2500 U/1.6 mL (9.2 mg prot/mL – biuret)170 U/mg proteins (Note: 1 units reduces 1 lmol GSSG/min, pH7.6 at 25 �C); saline solution (0.9% w/v) (Hospital Pharmacy Mili-tary Medical Academy, Belgrade, Serbia); glutathione, glutathionedisulfide and NADPH (Boehringer Corp. - London, UK); sodium ni-trate (NaNO3) (Mallinckrodt Chemical Works - St. Louis, MO, USA);sodium gluconate, ethylenediaminetetraacetic acid – EDTA, 2-vinylpyridine, nitrobluetetrazolium – NBT, epinephrine (Sigma–Al-drich - Sr. Louis, USA); sodium phosphate -Na2HPO4, potassiumdihydrogen phosphate - KH2PO4, glycerol, methanol, acetonitrile,trichloroacetic acid, thiobarbituric acid, triethanol amine, sulfosal-icylic acid (Merck - Darmstadt, Germany); sodium tetraborate andboric acid (Zorka - Sabac, Serbia); TRIS–HCl buffer (0.4 M, pH 8.9),carbonate buffer (50 mM, pH 10.2), (Serva, Feinbiochemica - Hei-delberg, New York). Deionised water was prepared by the Milliporemilli-Q water purification system (Waters - Millipore, Milford, MA,USA).

2.4. The tissue preparation

Homogenates of the VBRs were prepared from individual rats asdescribed earlier [37]. The ipsi- and contra- lateral side of the VBRswere removed from brain tissue and kept on ice during the wholeprocedure. Slices of the VBRs were transferred separately into coldbuffered sucrose (0.25 mol/L sucrose, 0.1 mmol/L EDTA in sodium–potassium phosphate buffer, pH 7). Aliquots (1 mL) were placedinto a glass tube homogeniser (Tehnica Zelezniki Manufacturing,Slovenia). Homogenization was performed twice with a Teflon pes-tle at 800 rpm (1000g) for 15 min at 4 �C. The supernatant was cen-trifuged at 2500g for 30 min at 4 �C. The resulting precipitate wassuspended in 1.5 mL of deionised water. Solubilisation of subcellu-lar membranes in hypotonic solution was performed by constantmixing for 1 h using a Pasteur pipette. Thereafter, homogenateswere centrifuged at 2000g for 15 min at 4 �C and the resultingsupernatant was used for analysis. Total protein concentrationwas estimated with bovine serum albumin as a standard [38].

2.5. Measurements

Parameters of oxidative status (superoxide anion radicals, ni-trates and malondialdehyde, superoxide dismutase, glutathioneperoxidase and gluthatione- total and oxidized) were measuredin both side of the VBRs (striatum, hippocampus and cortex), theispilateral and contralateral, after the treatments at 30 min, 24 hand 7 days.

2.5.1. Superoxide anion radical (O2��)

Content of O2�� was quantified by the method based on the

reduction of nitrobluetetrazolium – NBT to monoformazan byO2��. The yellow color of the reduced product was measured spec-

trophotometrically at 550 nm [39]. The results were expressed aslmol reduced NBT per mg of proteins.

2.5.2. Nitrate (NO3�)

Deproteination of brain homogenates was performed using ace-tonitrile (sample: acetonitrile, 2:1, v/v), then centrifuged andsupernatant was filtered (0.45 lm) before the chromatographicanalysis (ion-exchange HPLC) [40]. A mobile phase composed ofborate buffer/gluconate concentrate, methanol, acetonitrile anddeionised water in a ratio 2:12:12:74 (v/v/v/v) (pH 8.5) was usedfor isocratic elution at a flow rate of 1.3 mL/min at room tempera-ture. Spectroscopic detection was performed at a single wave-

length of 214 nm. For NO3� determination, 50 lL of filtrate was

injected into the HPLC system. The results were expressed as nmolNO3

� per mg of proteins.

2.5.3. Malondialdehyde (MDA)Malondialdehyde (MDA) was determined by the spectrophoto-

metric method of Ohkawa and coworkers [41]. MDA, a secondaryproduct of lipid peroxidation, gives a red colored pigment afterincubation with thiobarbituric acid-TBA reagent (15% trichloroace-tic acid and 0.375% TBA, water solution), at 95 �C at pH 3.5. Absor-bance was measured at 532 nm. The results were expressed aspmol MDA per mg of proteins.

2.5.4. Total glutathione (tGSH) and oxidized glutathione (GSSG)For determination of total glutathione (tGSH) and GSSG, brain

tissue was prepared in 10% sulfosalicylic acid. The tGSH and GSSGwere then determined before and after masking of the GSH contentof the samples with 2-vinylpyridine [homogenates were incubatedwith 1 M 2-vinylpyridine solution (10 lL/mL of homogenate) fortwo hours, at room temperature,] and 3.4% triethanol amine tomask GSH) followed by the enzymatic recycling assay by using5,5-dithiobis-2-nitrobenzoic acid (DTNB) (36.9 mg DTNB in 10 mLof methanol) which reacts with aliphatic thiol compounds inTRIS–HCl buffer (0.4 M, pH 8.9) and produces yellow color of p-nitrophenol anion. Intensity of color was measured spectrophoto-metrically at 412 nm [42–46]. The results of tGSH and GSSG wereexpressed as nmol per mg of proteins. The ratio GSSG/GSH wasconsidered for the interpretation of the results.

2.5.5. Superoxide dismutase (SOD)Activity of SOD was measured spectrophotometrically, as the

inhibition of spontaneous autooxidation of epinephrine at480 nm. The kinetics of sample enzyme activity was followed ina carbonate buffer (50 mM, pH 10.2, containing 0.1 mM EDTA),after the addition of 10 mM epinephrine [47]. The results were ex-pressed as U SOD per mg of proteins.

2.5.6. Glutathione peroxidase (GPx)This method is based on indirect determination of the GPx

activity by spectrophotometric measurement of NADPH consump-tion at 340 nm. Briefly, enzyme GPx catalyzes the reduction of (li-pid) hydroperoxides to (alcohols)/H2O using reducing equivalentsof GSH, which itself then becomes oxidized. Furthermore, regener-ation of depleted GSH occurs throughout the reduction of GSSG toGSH, catalyzed by the enzyme GR, which consumes NADPH as adonor of reducing equivalents. Reduction of every mole of GSSG re-quires one mole of NADPH [48]. The results were expressed as UGPx per mg of proteins.

2.6. Statistical analysis

Data analysis was performed using Statistica software version7.0 (Stat Soft, Inc.). Parameters of OS were presented graphicallyfor the ispilateral and in tabular form for the contralateral side ofthe VBRs. Data are shown as mean ± standard deviation. Parame-ters of OS measured at different time points within each groupwere compared by the independent Student’s t-test. OS parametersfor the same time point, but between the groups, were comparedusing ANOVA with the Tukey’s post hoc test. Differences were con-sidered statistically significant when the p < 0.05. Spearman corre-lation analysis was used to assess association between parametersof OS within PQ-intoxicated rats. Multiple linear regression analy-sis was used to estimate the association between parameters ofantioxidative defense (SOD, GPx, GSH) and MDA concentration inthe VBRs in PQ and GR + PQ groups.

Graph 1: Superoxide anion radical

0

2

4

6

8

10

12

0 minControl

30 minSham-

operated

24 h 7 day 30 minGR

24 h 7 day 30 minPQ

24 h 7 day 30 minGR+PQ

24 h 7 day

µmol

red

NB

T/m

g pr

otei

nStriatum Hipp Cortex

c***

c***c**

c***

c**£***

†***

**

***

£**

£***

£***

£***£**

Graph 2: Nitrates

0

10

20

30

40

50

60

0 minControl

30 minSham-

operated

24 h 7 day 30 minGR

24 h 7 day 30 minPQ

24 h 7 day 30 minGR+PQ

24 h 7 day

nmol

NO

3 – /m

g pr

otei

ns

Striatum Hippocampus Cortex

†***c**

c*†** †**

p***c*

Graph 3. Lipid peroxidation

0

50

100

150

200

0 minControl

30 minSham-

operated

24 h 7 day 30 minGR

24 h 7 day 30 minPQ

24 h 7 day 30 minGR+PQ

24 h 7 day

pmol

MD

A/m

g pr

otei

ns

Striatum Hipp Cortex

†*

c***c**

†*

£***

£*

£* £*

Graphs 1–6. In the ipsilateral side of the vulnerable brain regions (striatum, hippocampus and cortex) following parameters are presented: (a) parameters of oxidative stress/nitrosative stress [Graphs (1–3)]: Graph 1 - superoxide anion radicals (O2

��: lmol red NBT/mg proteins); Graph 2 - nitrates (nmol NO3�/mg proteins); Graph 3 - lipid

peroxidation (pmol MDA/mg proteins); (b) parameters of antioxidative defense [Graphs (4–6)]: Graph 4 – (A) total glutathione content (tGSH); (B) oxidized glutathione(GSSG) (nmol GSSG/mg proteins) and (C) the relationship between oxidized and reduced glutathione is presented as the ratio GSSG/GSH, Graph 5 - Superoxide dismutaseactivity (U SOD/mg proteins); Graph 6 - Glutathione peroxidase activity (mU GPx/mg proteins). See the experimental conditions presented in the Section 2.2 Experimentaldesign. Values are presented as means ± S.D. Statistical significance was considered at: ⁄p < 0.05; ⁄⁄p < 0.01; ⁄⁄⁄p < 0.001. Labels of statistical significance: c - compared to thecontrol group (intact animals); p – results from the GR + PQ group compared to the PQ group (refers to the same time point); � - compared to 30 min; £ - compared to 24 h(within the certain group). Values are presented as means ± S.D. Statistical significance was considered at: ⁄p < 0.05; ⁄⁄p < 0.01; ⁄⁄⁄p < 0.001. Labels of statistical significance: c- compared to the control group (intact animals). p – results from the GR + PQ group compared to the PQ group (refers to the same time point); � - compared to 30 min; £ -compared to 24 h (within the certain group).

78 M.M. Djukic et al. / Chemico-Biological Interactions 199 (2012) 74–86

Graph 4 A: Total glutathione

0

5

10

15

20

25

30

0 minControl

30 minSham-

operated

24 h 7 day 30 minGR

24 h 7 day 30 minPQ

24 h 7 day 30 minGR+PQ

24 h 7 day

tGS

H (n

mol

/mg

prot

eins

Striatum Hippocampus Cortex

c*

c***c*c***†**

†**

£***

£***

£*

Graph 4 B: Oxidized glutathione

0

1

2

3

4

5

0 minControl

30 minSham-

operated

24 h 7 day 30 minGR

24 h 7 day 30 minPQ

24 h 7 day 30 minGR+PQ

24 h 7 day

GS

SG

(nm

ol/m

g pr

otei

ns

Striatum Hippocampus Cortex

c***

†**

c**

c*c*

Graph 4 C: GSSG/GSH

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

0 minControl

30 minSham-

operated

7 day 30 minGR

7 day 30 minPQ

7 day 30 minGR+PQ

7 day

GS

SG

/GS

H

Striatum Hippocampus Cortex

c***

c**

p***

Fig. 1 (continued)

M.M. Djukic et al. / Chemico-Biological Interactions 199 (2012) 74–86 79

3. Results

Differences between the control (the intact group) and thesham-operated groups were not significant. As well, results ob-tained from the ipsilateral side of the VBRs (Graphs 1–6) show

almost identical trend of changes as in the contralateral side ofthe VBRs (Table 1).

Mortality (10%) within 24 h and Parkinson’s disease-like symp-toms such as rigidity of the limbs and tail, tremor of the head androtational motion, were observed only in the PQ group (0.01 lM of

Graph 5: Superoxide dismutase

0

500

1000

1500

2000

2500

0 minControl

30 minSham-

operated

24 h 7 day 30 minGR

24 h 7 day 30 minPQ

24 h 7 day 30 minGR+PQ

24 h 7 day

U S

OD

/mg

prot

eins

Striatum Hippocampus Cortex

c***c**c**

c**

†*†*

£***

£*

Graph 6: Glutathione peroxidase

0

100

200

300

400

500

0 minControl

30 minSham-

operated

24 h 7 day 30 minGR

24 h 7 day 30 minPQ

24 h 7 day 30 minGR+PQ

24 h 7 day

mU

GP

x/m

g pr

otei

ns

Striatum Hipp Cortex

†*

c***c***c*

c*

c***

†**

†*

†*

p***p**

†***£***

Fig. 1 (continued)

80 M.M. Djukic et al. / Chemico-Biological Interactions 199 (2012) 74–86

PQ were i.s. administered), after awakening from anesthesia. Thesesymptoms disappeared 2–3 h after poisoning, indicating theirreversible character.

3.1. Superoxide anion radical (O2��)

In the PQ group, O2�� content was significantly reduced in the

ipsilateral side of the VBRs (p < 0.001) and in the contralateral stri-atum and cortex (p < 0.001) at 24 h; and significantly higher con-tent was measured bilaterally in striatum (p < 0.001) at 7 daysafter the treatment, compared to the controls.

In the GR + PQ group, significantly reduced O2�� content was

found bilaterally in the hippocampus (p < 0.01) at 24 h; while, sig-nificantly higher values were found bilaterally in striatum (ipsi:p < 0.001 and cotralateral: p < 0.01) and in cortex (p < 0.01), at7 days after the treatment compared to the controls (Graph 1,Table 1).

The trend of O2�� content changes in the VBRs was similar in the

PQ and the GR + PQ groups, thus pretreatment with GR did notinfluence significantly on O2

�� content, except that no noticeabledrop in the O2

�� content was observed at 24th hours, as was thecase in the PQ group.

3.2. Nitrate (NO3�)

In the PQ group, at 24 h the NO3� level was significantly lower

in the ipsilateral striatum (p < 0.01) and hippocampus (p < 0.05)and in the contralateral side of the VBRs [(striatum (p < 0.05), hip-pocampus (p < 0.001) and cortex (p < 0.001)]; and also significantlylower (p < 0.05) in contralateral striatum at 7th days of the treat-ment, compared to the controls.

In the GR + PQ group, NO3� content was significantly reduced in

ipsilateral cortex (p < 0.05) at 30 min and ipsilateral striatum(p < 0.05) during whole experiment (24 h–7 days); and in the con-tralateral side of the VBRs (p < 0.05), compared to the controls(Graph 2, Table 1). Content of NO3

� was not changed in hippocam-pus by GR pre-treatment, compared to the controls.

Comparing to the PQ group, nitrates were lower at 30 min in theGR + PQ group, with statistically lower value, in the cortex.

3.3. Malondialdehyde (MDA)

In the PQ group, at 30 min, MDA levels were significantly ele-vated in the ipsilateral striatum (p < 0.01) and in the contralateralcortex (p < 0.05); and at 7 days, significantly elevated in the ipsilat-

M.M. Djukic et al. / Chemico-Biological Interactions 199 (2012) 74–86 81

eral cortex (p < 0.001), compared to the controls. (Graph 3, Table1).

Lipid peroxidation did not develop in the GR + PQ, contrary tothe PQ group, where MDA values were elevated at 30 min and at7 days of the treatment.

3.4. Total glutathione (tGSH), oxidized glutathione GSSG, reducedglutathione (GSH) and ratio GSSG/GSH

The concentration of the reduced glutathione was estimated bysubtracting its oxidized form, GSSG, from the total glutathione(tGSH = GSH + GSSG) in each sample. Mainly, tGSH is calculatedas we did in this study, but some authors calculate it differently,such as: tGSH = GSH + 1/2 GSSG, although rarely; and/or even as:tGSH = GSH + 2 GSSG [42–46]. The ratio GSSG/GSH is presentedgraphically (Graph 4C).

In the PQ group, at 24 h, tGSH was significantly lower in both,ipsi- and contra- lateral VBRs: ipsilaterally: striatum (p < 0.001),hippocampus (p < 0.05) and cortex (p < 0.001); and contralaterally,in all VBRs (p < 0.001), compared to the controls. In the GR + PQgroup, at 24 h, tGSH concentration was significantly lower in theipsilateral striatum (p < 0.05), compared to that of the controlgroup (Graph 4A, Table 1). Contrary to the PQ group, statisticaldepletion of tGSH did not occurred in the GR + PQ at 24th hours,although levels within 24 h were slightly lower compared to thecontrols.

In the PQ group, in all VBRs, bilaterally, GSSG was significantlyhigher (p < 0.001) at 30 min and (p < 0.01) at 24 h, whilst in theGR + PQ group, level of GSSG were significantly higher in bilateralcortex at 30 min–24 h (p < 0.05), compared to the controls (Graph4B Table 1). The pre-treatment with GR resulted in reduced GSSGcontent, compared to the PQ group. Having on mind that slightconsumption of tGSH has been observed in the GR + PQ groupand probability that great part of it was oxidized, obtained resultsindicates prompt and intense recycling of glutathione, i.e. reduc-tion of GSSG.

The ratio GSSG/GSH was significantly higher (p < 0.001) in allVBRs, bilaterally, in the PQ group, at 30 min and emphasizing ex-tremely high values at 24 h (for cortex, in particular) comparedto the controls. In the GR + PQ group, values of the ratio GSSG/GSH are significantly higher in all VBRs, bilaterally (p < 0.01) at30 min and at 24 h for cortex, bilaterally (p < 0.01), compared tothat of the control group. The graphical presentation of the ratioGSSG/GSH is addressed to both, ipsi- and contra- lateral sides ofthe examined VBRs (Graph 4C).

Significant difference of the ratio GSSG/GSH between the PQand the GR + PQ group (p < 0.001) at 24th hours clearly indicatedpowerful effect of the GR pre-treatment on glutathione recyclingin the direction of GSH regenerating, although values of the ratioGSSG/GSH were statistically higher during 24 h in that group.

3.5. Superoxide dismutase activity (SOD)

In the PQ group, SOD activity was significantly reduced in bothsides of the VBRs [ipsilateral side: striatum (p < 0.01), hippocam-pus (p < 0.001) and cortex (p < 0.01); and the contralateral side:striatum (p < 0.01), hippocampus (p < 0.001) and cortex(p < 0.001)], at 24 h, compared to that of the control group.

In the GR + PQ group, SOD activity was significantly elevatedonly in the ipsilateral cortex (p < 0.01) at 7 days, compared to thatof the control group (Graph 5, Table 1). Although, SOD activity wasslightly lower within 24 h in the GR + PQ group, drop of its activitydid not occur at 24th hours, as has been observed in the PQ group.

Taking into the consideration that O2�� (the substrate for SOD)

content was elevated in both, the PQ and the GR + PQ groups at7th days and that higher SOD activity was noticed in the GR + PQ

group at 7th days if compared to the PQ group, it means that betterantioxidative defense was accomplished in the GR + PQ group, at7th days, what is also confirmed with undeveloped lipid peroxida-tion at the time, comparing to the PQ group.

3.6. Glutathione peroxidase (GPx)

In the PQ group, GPx activity was exceedingly increased in bilat-eral striatum (p < 0.001), ipsi- and contra- lateral hippocampus(p < 0.001, p < 0.05, respectively) and bilateral cortex (p < 0.05), at30 min, compared to the controls.

In the GR + PQ group, significantly reduced GPx activity wasmeasured in ipsilateral striatum and hippocampus (p < 0.05) andcortex (p < 0.001) and in all contralateral VBRs (p < 0.001) at30 min, compared to controls. Significantly lower GPx activities(p < 0.001) were measured in all VBRs, bilaterally, at 24 h, com-pared to the controls (Graph 6, Table 1).

GPx activity is statistically lower within 24 h in the GR + PQgroup (p < 0.001 at 30 min and p < 0.01 at 24th hours), comparedto the PQ group. Obtained result confirmed huge impact of theGR pre-treatment on glutathione cycle, including GPx, which usesGSH as a donor of reducing equivalents for reactions of lipid hydro-peroxides conversion to alcohols and H2O2 to H2O.

3.7. Statistical analysis

3.7.1. Spearman correlation analysis3.7.1.1. Within the PQ group. Correlation between MDA and OSparameters in the ispilateral striatum of PQ intoxicated Wistar ratswere depicted in Table 2. MDA concentrations were significantly pos-itively correlated with O2

�� levels 30 min (r = 0.943, p < 0.01) and 24 h(r = 0.886, p < 0.05) after PQ administration. Also, after PQ adminis-tration, MDA concentrations were significantly positively correlatedwith NO3

� levels at 24 h (r = 0.90, p < 0.05) and GSH levels at30 min (r = 0.881, p < 0.05), while after 7 days was significantly neg-atively correlated with SOD activity (r = �0.80, p < 0.05) (Table 2).

In the ispilateral cortex of the PQ group, MDA levels were signif-icantly positively correlated only with GSH concentrations after7 days (r = 0.901; p < 0.05), while in ispilateral hippocampus nosignificant correlation was found between MDA and OS parameters(data not shown).

3.7.1.2. Within the GR+PQ group. In the ispilateral side of the VBRsof the GR + PQ group MDA were highly positively correlated withGSH in the ispilateral striatum (r = 0.92; p < 0.01) after 24 h, hippo-campus and cortex (r = 0.95; p < 0.01 and r = 0.93; p < 0.01, respec-tively) after 7 days and with GPx activity in the ispilateral cortex(r = 0.94; p < 0.05) after 24 h.

3.7.2. Multiple regression analysis3.7.2.1. In the PQ and GR+ PQ group. In the PQ group, the significantpredictor of MDA concentration in the ispilateral striatum and cor-tex was GSH, while in hippocampus predictors were GSH and GPx.

In the GR + PQ group, at 24 h, MDA concentration was signifi-cantly associated only with GSH concentrations in all the ispilater-al VBRs (Table 3).

4. Discussion

Based on our results, neuroprotective role of GSH could beessential in an early stage of PQ-induced neurotoxicity mediatedby oxidative and/or nitrosative stress, which is in agreement withdata from other laboratories [49]. Markedly increase of GPx activ-ity and oxidized glutathione (GSSG) were accompanied withslightly increased lipid peroxidation and NO3

� level in the VBRs(striatum, hippocampus and cortex) at 30 min of i.s. administration

Table 2Spearman correlation between malondialdehyde (MDA) and other parameters ofoxidative status in the ispilateral striatum of the Wistar rats is. intoxicated withparaquat.

Time Parameters of oxidative status (n = 8/time point)

O2�� NO3

� GSH SOD GPx

30 min MDA 0.943⁄⁄ �0.174 0.881⁄ 0.174 0.71424 h MDA 0.886⁄ 0.900⁄ �0.562 0.128 0.6007 days MDA �0.143 �0.142 0.666 �0.750⁄ 0.200

Wistar rats (n = 24) i.s. treated with PQ (is. 2.5 lg PQ/10 lL, i.e. 0.01 lM/10 lL);n = 8/subgroup related to time of sacrificing. Correlated parameters of oxidativestatus (within ipsilateral striatum of the PQ group): O2

�� content (expressed aslmol red NBT/mg proteins), NO3

� concentration (nmol NO3�/mg proteins), lipid

peroxidation (expressed as nmol MDA/mg proteins), reduced glutathione (nmolGSH/mg proteins), SOD activity (U SOD/mg proteins) and GPx activity (mU GPx/mgproteins). Statistical significance was considered at: ⁄p < 0.05; ⁄⁄p < 0.01.

82 M.M. Djukic et al. / Chemico-Biological Interactions 199 (2012) 74–86

of PQ. Extreme collapse of all biochemical parameters of oxidative/nitrosative status was measured at 24th hours in the all VBRs,which could be explained by PQ inhibitory effect on complex I ofmitochondrial respiratory chain and consequent diminished en-ergy and decreased metabolic activities [56]. Concurrently, mortal-ity of the animals (10%) and Parkinson’s disease-like symptoms(extrapyramidal behavior: rigidity of the limbs and tail, tremor ofthe head and rotational motion) were observed after awakeningfrom anesthesia for 2–3 h, within 24 h since PQ was i.s. injected.Oxidative/nitrosative stress was markedly decreased after 7 days.Almost all measured parameters were within the range of controls,except moderately increased concentration of O2

�� (in striatum)and lipid peroxidation (in cortex), and low content of NO3

� level(in stratum and cortex). Activity of GPx along with GSH depletion(due to its oxidation to GSSG) were recognized as the most respon-sive biomarkers of the early stage of PQ induced neurotoxicity.

Parkinson’s disease symptoms and lethality of the animals didnot observe in the GR + PQ group and oxidative stress was weakercompared to the PQ group. Furthermore, decrease of metabolicactivity did not occur (as it was measured in the PQ group at24th hours) and lipid peroxidation did not develop during thewhole experiment, although, red-ox imbalance was documentedby: significantly increased GSGG/GSH at 24 h (but statistically low-er if comparing to the PQ group, p < 0.001); then GPx activity andNO3

- level (in striatum and cortex) were significantly decreasedduring the whole experiment; and O2

�� was increased (in ipsilat-eral striatum and cortex) at 7th days (Graphs 1–6, Table 1). Intra-striatal administration of GR itself did not induce any harmful

Table 3The association of antioxidative defense parameters (reduced glutathione and gluta-thione peroxidase) with malondialdehyde in the ipsilateral vulnerable brain regions ofWistar rats in the PQ and GR + PQ groups, at 24 h.

Groups Brain structures Predictors b (SE b) p

PQ Striatum GSH 0.720 (0.212) <0.05Hippocampus GSH

GPx0.602 (0.200)0.425 (0.180)

<0.05<0.05

Cortex GSH 0.795 (0.211) <0.001

GR + PQ Striatum GSH 0.803 (0.921) <0.01Hippocampus GSH 0.792 (0.99) <0.01Cortex GSH 0.705 (1.17) <0.05

Abbreviations: GSH – reduced glutathione, GPx- glutathione peroxidase. ⁄See theexperimental conditions presented in the Section 2.2 Experimental design. Multiplelinear regression analysis: Variables included in the statistical analyses wereparameters of antioixidative defense measured in ipsilateral vulnerable brainregions (striatum, hippocampus and cortex): reduced GSH content (nmol GSH/mgproteins), SOD activity (U SOD/mg proteins) and GPx activity (mU GPx/mg pro-teins). Insignificant association was found for SOD and MDA. Values are presentedas as b (SE b). Statistical significance was considered at: p < 0.05; p < 0.01; p < 0.001.

effect based on the biochemical parameters and observation ofthe animals, as shown from the results (Graphs 1–6, Table 1).

Taking into the consideration the above facts, it becomes obvi-ous that tissues of the VBRs of Wistar rats were protected by GRpre-treatment (Graphs 1–6) implicating importance of antioxida-tive role of GSH against oxidative/nitrosative injury induced byPQ induced neurotoxicity.

Our observations are consistent with findings that PQ is a poten-tial environmental factor contributing to Parkinson’s disease[2,12,50]. Corsanti, Beggeta and co-workers showed that a singlesubcutaneous (s.c.) dose of PQ 20 mg/kg, to animals can cause excit-ability and lung congestion, which resulted in epileptogenic and/orlimbic seizures in 33% of treated rats. Neuronal cell death was ob-served only in piroform cortex, while the lowest concentrationwas measured in the Caudate nucleus, prefrontal cortex and hippo-campus. A lower s.c. dose of Pq (5 mg/kg/day, during 14 days) isineffective [51]. Intracerebrally (i.c.) administered doses of PQ(0.02–0.4 lM) to rats evoked excitotoxicity (manifested with neu-rodegenerative, morphological and behavioral changes in the brainregions rich in noradrenergic, dopaminergic and serotonergic neu-rons) in dose-dependent manner [24–31,36]. Lower dose of PQ(0.01 lmol) did not induce above mentioned changes on CNS. Inour experiment, we injected i.s. dose of PQ (0.01 lmol/10 lL) toWistar rats and observed reversible extrapiramidal symptoms,whilst 10% of rats died. Apparently, applied dose was satisfactoryto study mechanism of PQ neurotoxicity. Seven days is sufficientperiod for recovery to be achieved, if reversible tissue damage oc-curred [49–51].

An early onset of Parkinson’s disease-symptoms and mortalityof animals seen in the PQ group, in our experimental study, as wellas considerable low values of examined parameters of oxidativestatus in the VBRs, including immensely high GSSG/GSH ratio, at24 h of PQ administration (Graphs 1–6, Table 1), strongly supportliterature data about PQ cytotoxic effect (PQ inhibitory effect oncomplex I of mitochondrial respiratory chain) and energetically ex-hausted cells due to diminished ATP synthesis [12,52–55,57,58].Our results and observations showed that remarkable high GPxactivity (within 30 min of PQ i.s. administration) coincided withseizures (within 2–3 h from awakening form anesthesia, in thePQ group) and is associated with significant oxidation of GSH(Graph 4A–C) [59]. According to our results, massively developedoxidative/nitrosative stress which culminated at 24 h after PQ i.s.administration coincided with animals’ deaths (10% of animalsdied in the PQ group).

Extremely increased peak of the GSSG/GSH at 24 h of PQ i.s. poi-soning (Graph 4C) confirms that the GSH oxidation to GSSG isdominant way of GSH depletion in PQ neurotoxicity comparingto the other pathways (reactions of conjugation: with proteins,NO, metals, etc.) [60]. Steady state concentrations of O2

�� andPQ2+ interfere metabolism of glutathione forms such as: thiyl rad-ical (GS�), thiolate anion (GS�) and conjugate of thiyl radical andthiolate [GSSG]��. Redox compounds, which radical forms are sta-bilized by the conjugated double bond, such as semiquinones(Q2

��/Q2�] and bipyridilium compounds (PQ2+/PQ�+), could beinvolved in the oxidation of GS� oxidation to GS� [10,61,62]. Inthe presence of O2

�� and PQ2+, oxidation of GS� to GS� could taketwo possible pathways: (a) when [PQ2+]ss or [Q2

��]ss� [O2��]ss, en-

zyme SOD favors oxidation of GS� to GS�; (b) if [PQ2+]ss or [Q2��]ss <

[O2��]ss, then GS� formation is suppressed by SOD. Also, when

[PQ2+]ss or [Q2��]ss � [O2

��]ss, enzyme SOD favors conjugation ofthiyl radical with thiolate (RS� + RS� = [GSSG]��) and oxidation of[GSSG]�� to GSSG ([GSSG]�� + O2 = GSSG + O2

��). Therefore, relativesteady state concentrations of O2.and PQ2+ have an influence oncell redox state.

Thiyl radical enhances the LP, because it is a strong oxidant (E�’RS�

/RSH = +0.9 V) and easily reacts with free fatty acids: (a) abstrac-

M.M. Djukic et al. / Chemico-Biological Interactions 199 (2012) 74–86 83

tion of hydrogen (rate of reaction is �107 M�1 s�1), (b) addition todouble bonds (rate of reaction is �108 M�1 s�1), when formed alkylradical may react with O2 to form corresponding peroxyl radicals[63]. Obviously, depleted GSH and consequently reduced antioxi-dative defense is crucial for the development of Parkinsonismand mortality of rats i.s. poisoned with PQ [64]. Importance of glu-tathione cycle in PQ neurotoxicity and its neuroprotective role isconfirmed in our study by i.s. applying of GR.

Mammalian cells can deal with an increased GSSG/GSH ratio inseveral ways: synthesis of more GSH (using g-GCS), convertingGSSG into GSH (using GR) or exporting GSSG [63]. It is not knownwhat is the proportion between ‘‘free’’ (GSH and GSSG), and‘‘bound’’ glutathione forms (bound to proteins and/or NO-boundfractions) [60]. Positive outcome of i.s. administered GR in ourstudy, suggests the possibility that GSSG reduction occurs extracel-lularly and within this context, reduction of ‘‘free’’ GSSG back toGSH by GR deserves noteworthy attention, in sense that reductionby GR could prevent its conjugation with proteins, lipids, NO, met-als, etc., and thus avoid/minimize damage of cells/tissues [65].

Cellular synthesis and consumption of GSH are balanced by itssynthesizes (ATP dependent two-steps synthesis: (a) the gGluCyssynthetase catalysis formation of dipeptide gGluCys from gluta-mate and cysteine; and (b) glutathione synthetase combined gGlu-Cys with glycine to generate GSH. Synthesis of gGluCys is balancedby feedback mechanism of GSH intracellular level [20,65–67]. Forexample, in mitochondria, GSH concentration is maintained con-stantly via its uptake from the cytosol (transport systems, whichare stimulated by ATP and and ADP), because mitochondria lackthe enzymes for GSH synthesis [60]. Red-ox turnover of GSH ismore intense mitochondria than in cytosol (the ratio of GSH/GSSGin mitochondria is 10:1) indicating a powerful oxidative metabo-lism in this organelle [66].

Extracellular GSH and glutathione conjugates are substrates forthe ectoenzyme g-glutamyl transpeptidase (gGT), which catalyzesthe transfer of the g-glutamyl moiety from GSH or a glutathioneconjugate onto an acceptor molecule, thereby generating thedipeptide CysGly or the CysGly conjugate, respectively [68–70].CysGly is cleaved by dipeptidase to form cysteine and glycine.Formed g-glutamyl amino acids undergo cyclization by intracellu-lar enzyme g-glutamyl cyclotransferase to form 5-oxo-L-proline(by decyclization, 5-oxo-L-proline is transformed to glutamate)which is considered to be an activator of the amino acid transportsystems B0+ and A at the albuminal membrane of brain endothelialcells [71].

The trafficking of GSH and/or its precursors or breakdown prod-ucts across the membranes and/or the blood–brain barrier is still asubject of controversy [72–77]. It is still not clear whether GSH istransported intact or whether GSH uptake depends on breakdownof GSH initiated by gGT on the luminal side of brain capillaries orextent sodium-dependent GSH transporter contributes to thedelivery of GSH from blood into the brain and to the glutathionehomeostasis of brain remains to be elucidated [78–81].

Insufficient synthesis of ATP is the consequence of complex Iinhibition by PQ and is linked to NADPH depletion and activationof the hexose monophosphate shunt (HMPS) [54]. NADPH deple-tion and energy exhaustion eventually led to cell death. Some re-dox-cycling compounds completely inhibit the activation ofHMPS by inhibiting GR [82]. There is however no data specificallyrelating to inhibition of GR with PQ. If we presume that it is possi-ble, we can speculate that the GR pre-treatment procedure adoptedby us could be one rationale more, for such experimental approach.Controversial reports about relationship between PQ cytotoxicityand GSH and/or GR were published [82–84].

As reported by Witschi and co-workers, NADPH-dependent cellpathways could be severely altered within a short period of timeafter PQ intoxication, resulting in a total collapse of the metabolic

system and sudden death of rats (lung model was used) [57]. Ourresults are in consitence with this study. In fact, MDA was posi-tively associated with O2

�� at 30 min (p < 0.01) and at 24 h(p < 0.05) and with NO3

� at 24 h (p < 0.05), indicating developmentof oxidative/nitrosative stress, what coincided with animals’deaths and Parkinsonism symptomatology in the PQ group (Table2). Additionally, MDA was positively associated with GSH depel-tion (p < 0.01), implying the importance of perpetuation the GSHlevel at the level requiring to achieve antioxidative role and to pre-vent oxidative tissue injury, including lipid peroxidation (Table 3).

Several reports have showed that peroxynitrite (ONOO�) effec-tively inhibits enzymes in the mitochondrial respiratory chain fol-lowed by reduced ATP synthesis [12,62,85–87]. Initially producedNO� (based on moderately increased of NO3

� content, at 30 minafter PQ i.s. injection) can react spontaneously with O2

�� to formONOO�. Upon NMDA receptor stimulation, glutamate release cor-relates with NO� production. Also, extracellular NO� movementmay provide signaling from activated NMDA receptors into sur-rounding synaptic terminals of the cerebral cortex [88]. DecreasedNO3

� content at 24 h and 7 days after PQ-triggered neurotoxicitycould be direct consequence of energetically exhausted and oxida-tively damaged neuronal cells, or additionally, as an outcome ofseveral pathways such as: formation of ONOO�� (reaction betweenNO� and O2

��); formation of GS-NO mediated by GSH-S-transferase[24,25,85,89]; and activation of (non) NMDA receptors [12]. Also,depletion of tGSH with a simultaneous decrease in NO3

� couldbe attributed to the formation of GS-NO, catalyzed by GSH-S-trans-ferase, which also may explain decrease of GPx activity (Graph 6)[90]. Berendji et al. found (1999) that NO-donors could decreaseintracellular glutathione (GSH) levels in lymphocytes by as muchas 75% [91]. Changed thiol-disulfide status of critical cystein on en-zymes, receptors, transport proteins, and transcription factors isrecognized as an important mechanism of signal transductionand an important consequence of oxidative stress associated dis-eases [26]. Signs of Parkinsonism, mortality of rats and compro-mised energy metabolism did not observed in the GR + PQ groupindicating the significance of maintaining the content of GSH atthe level necessary to achieve antioxidative role. We accomplishedto protect the animals by i.s. application of GR.

Additionally, GSSG directly activates NMDA receptors and/orenhances its response, by which excitotoxicity becomes part ofoverall respond to PQ i.s. exposure [64]. Thus both, increased GPxactivity followed by subsequent GSSG increase (significantly ele-vated GSSG/GSH within 24 h) and elevated NO� production possi-bly contribute to excitotoxicity via stimulation of NMDAreceptors and subsequent release of glutamate, which coincidedwith overt Parkinsonism and mortality of rats in the PQ group.Thus, contrary to the PQ group, in the GR + PQ group, low NO�

and GSSG concentrations (GPx activity was significantly low) didnot result in stimulation of NMDA receptors, based on the observa-tion of animals. Therefore, low GPx activity and NO� concentrationin the VBRs of the rats in the GR + PQ group might be attributed tothe formation of GS-NO (Graphs 3–5: Table 3). Acctually, i.s.applied GR sustained GSH levels by two synergistic effects: (1)extracellular reduction of GSSG; and (2) significantly decreasedGPx activity.

Almost equal values of OS parameters between ipsi- and contra-lateral side of the VBRs of treated rats (Graphs 1–6: ipsilateral side;Table 1: contralateral side) proved promptly and spatially spread-ing of oxidative brain injury triggered by PQ [10,54,92,93]. The GSHlevel varies between brain regions (cortex, hippocampus, striatum,midbrain, and cerebellum) of the small experimental animals (ratsvs. mice) due to many factors, but not significantly [94]. Based onour results, striatum and cortex responded to PQ neurotoxic effectmore intense, compared to hippocampus. Although, we targetedstriatum by i.s. injection of the testing substances, obtained results

84 M.M. Djukic et al. / Chemico-Biological Interactions 199 (2012) 74–86

showed that red-ox balance was not significantly changed in thisstructure (sham operated vs. intact group).

Guided by the fact that GR does not pass across the cell mem-brane and/or the blood–brain barrier and further, that the conver-sion of GSSG into the GSH in the extracellular/interstitialenvironment could be achieved by GR and followed by subsequentneuronal cells’ uptake of regenerated GSH and/or amino acids con-stituents (precursors or breakdown constituents of GSH) (docu-mented by positive outcome of the applied pre-treatment), welaunched the hypothesis that systemic administration of GR is likelyto be effective because of the ability of GSH/or its breakdown productsto pass cell membranes and barriers. There is growing evidence thatGSH plays an important role in the detoxification of reactive oxy-gen species in oxidative stress related poisoning/neurodegenera-tive diseases (Parkinson’s disease, Alzheimer’s disease andamyotrophic lateral sclerosis (ALS) [20,95].

Intrastriatal application is invasive route of administration forhumans, thus not acceptable. In no doubt, wide-ranging additionalin vivo experimental studies have to be done to test that hypothe-sis. Having on mind that GSH and/or its precursors or breakdownproducts can pass across cell membranes or the blood–brain bar-rier or other barriers and despite the fact that GR does not passthe blood–brain barrier, its systemic administration (subcutaneousor intravenous), or maybe via the liquor (!) might be consideredpromising for management of oxidative stress related poisonings/diseases. To prove this, a lot of additional experimentations arerequired.

5. Conclusion

In summary, oxidative and nitrosative stress, with an emphasison lipid peroxidation, GSH – mediated antioxidative response andexcitotoxicity are probably the most important and critical patho-physiological pathways in an early stage of PQ i.s. induced neuro-toxicity. Glutathione-based redox regulation and signaling wasproved to be the most important for antioxidative defense againstPQ harmful effect. Cytotoxicity of PQ, mediated by free radicalchain reactions and altered cellular respiration (complex I in mito-chondria) causing immense energy deficit, was documented by sig-nificant decrease of the oxidative/nitrosative stress biomarkers inthe VBSs, 24 h after i.s. poisoning of rats with PQ.

The absence of Parkinson’s disease - like symptoms and mortal-ity in the group of rats pre-treated with GR, altogether indicate aneuroprotective role of i.s. administered GR, probably achievedthrough several synergistic mechanisms such as: (a) extracellularreduction of GSSG was followed by subsequent neuronal cells’ up-take of GSH or its amino acid constituents; (b) significantly de-creased GPx activity; (c) insufficient level of GSSG and NO� toactivate NMDA receptors (lack of excitotoxicity).

Results of this study firstly indicate that i.s. pre-treatment withGR accomplished protective role against harmful i.s. PQ poisoning,probably due to extracellularly/interstitially glutathione recycling.Thus, our findings provide useful information for those who studythiol-based redox regulation and signaling and treatment manage-ment of oxidative/nitrosative stress related poisonings or neurode-generative diseases, such as Parkinson’s disease.

Ethics committee approval

The authors have complied with the institutional policies gov-erning the humane and ethical treatment of the experimental sub-jects, and are willing to share the original data and materials if sorequested. The experimental animals were treated according toGuidelines for Animal Study, No. 282–12/2002 (Ethics Committeeof the Military Medical Academy, Belgrade, Serbia and Montenegro).

Disclosure statements

Results presented in the manuscript have not been published orsubmitted for publication elsewhere.

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Funding sources

This work was supported by Grants from the Ministry of Educa-tion and Science, Republic of Serbia (Project No. III41018) and bythe Ministry of Defense of the Republic of Serbia (Project No.MMA/06-10/B.3).

Acknowledgements

We are grateful to Krishnakumar Balasubramanian, Ph.D. (Uni-versity of Pittsburgh, USA); Ms. Marija Tvrtkovic (Graduate studentat the Mc Gill University, Montreal, CA); and to Mrs. Ana Djuric,PhD student, Ms. Slavica Eric, PhD, Associate Professor and Mr. Vla-dimir Savic, PhD, Professor (Faculty of Pharmacy, University of Bel-grade, Serbia) for their assistance. We are particularly grateful toour reviewers for contributively criticisms and helpful suggestions.This work was supported by Grants from the Ministry of Educationand Science, Republic of Serbia (Project No. III41018) and by theMinistry of Defense of the Republic of Serbia (Project No. MMA/06-10/B.3).

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