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Journal of Trace Elements in Medicine and Biology 28 (2014) 89– 93

Contents lists available at ScienceDirect

Journal of Trace Elements in Medicine and Biology

j ourna l h omepage: www.elsev ier .de / j temb

oxicology

ntioxidant effect of selenium on lipid peroxidation, hyperlipidemiand biochemical parameters in rats exposed to diazinon

atma M. El-Demerdasha,∗, Hoda M. Nasrb

Department of Environmental Studies, Institute of Graduate Studies and Research, Alexandria University, Alexandria, EgyptDepartment of Pest Control and Environmental Protection, Faculty of Agriculture, Damanhour University, Damanhour, Egypt

r t i c l e i n f o

rticle history:eceived 16 June 2013ccepted 4 October 2013

eywords:iazinoneleniumnzymesxidative stressipids profile

a b s t r a c t

Diazinon (DZN) is one of the most organophosphate insecticides that widely used in agriculture andindustry. Selenium is generally recognized to be a trace mineral of great importance for human health,protecting the cells from the harmful effects of free radicals. Therefore, the present study was carried outto investigate the alterations in biochemical parameters, free radicals and enzyme activities induced bydiazinon in male rat serum, and the role of selenium in alleviating the negative effects of DZN. Animalswere divided into four groups of seven rats each; the first group was used as control. Groups 2, 3 and 4 weretreated with selenium (Se; 200 �g/kg BW), diazinon (DZN; 10 mg/kg BW) and diazinon plus selenium,respectively. Rats were orally administered their respective doses daily for 30 days. Results obtainedshowed that DZN significantly induced thiobarbituric acid reactive substances (TBARS) and decreasedthe activities of glutathione S-transferase (GST), superoxide dismutase (SOD), catalase (CAT), glutathioneperoxidase (GPx) and glutathione reductase (GR) and the levels of reduced glutathione (GSH) in rat sera.Aminotransferases (AST, ALT), phosphatases (AlP, AcP) and lactate dehydrogenase (LDH) activities weresignificantly increased while acetylcholinesterase (AChE) activity was decreased due to DZN adminis-

tration. Also, DZN treatment caused significant perturbations in lipids profile and serum biochemicalparameters. On the other hand, Se alone significantly decreased the levels of TBARS, total lipids, choles-terol, urea and creatinine, while increased the activities of antioxidant enzymes and glutathione content,total protein (TP) and albumin. In addition, Se in combination with DZN partially or totally alleviatedits toxic effects on the studied parameters. In conclusion, Se has beneficial effects and could be able toantagonize DZN toxicity.

ntroduction

Pesticides are ubiquitous in the environment and have signif-cant economic, environmental and public health impact. Theirsage helps to improve human nutrition through greater availabil-

ty, longer storage life and lower costs of food. Organophosphateompounds (Ops) are occasionally used indiscriminately in largemounts causing environmental pollution and therefore, are aause of concern [1,2]. Residual amounts of OP compounds haveeen detected in the soil, water bodies, vegetables, grains and other

oods products [3]. Diazinon (diethoxy-[(2-isopropyl-6-methyl--pyrimidinyl) oxy]-thioxophosphorane) is an organophosphorusompound with anticholinesterase mode of action. It is used

∗ Corresponding author at: Department of Environmental Studies, Institute ofraduate Studies and Research, Alexandria University, 163 Horreya Avenue, P.O.ox 832, Alexandria 21526, Egypt. Tel.: +20 34 29 50 07; fax: +20 34 24 14 85.

E-mail addresses: eldemerdashf@yahoo.com (F.M. El-Demerdash),ohm hm@yahoo.com (H.M. Nasr).

946-672X/$ – see front matter © 2013 Elsevier GmbH. All rights reserved.ttp://dx.doi.org/10.1016/j.jtemb.2013.10.001

© 2013 Elsevier GmbH. All rights reserved.

extensively to control flies, lice, insect pests of ornamental plantsand food crops, as well as nematodes and soil insects in lawnsand croplands [4]. In addition, oxidant and antioxidant system[1], bio-element levels [2], immune system [5], hematologicaland biochemical parameters [1] could be affected by OP toxicity.Furthermore, OP insecticides induced toxic effects that probablyoccur through the generation of reactive oxygen species (ROS),causing damage to various membranous components of the cell[6]. For this reason, treatment with antioxidants and free radicalscavengers can decrease the oxidative stress and LPO related toOP-induced toxicity.

Micronutrients are dietary minerals required by the humanbody in a very small quantity. They probably interact with xenobi-otics at several sites like, during absorption and excretion, transportof metals in the body, binding to target proteins, metabolism andsequestration of toxic metals, and oxidative stress [7]. Besides this,

they may also serve as required prosthetic groups in active sites oras co-enzymes for indispensable metalloenzymes. Several studiesshowed that antioxidant nutrients protect cells against deleteriouseffects of environmental agents [2,8]. Selenium (SE) has received

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0 F.M. El-Demerdash, H.M. Nasr / Journal of Trace

onsiderable attention as an essential micronutrient for both ani-al and human beings [8]. It has been detected that Se functions

n the active site of glutathione peroxidase. It is important in manyiochemical and physiological processes including the biosynthesisf coenzyme Q, regulation of ion fluxes across membranes, main-enance of the integrity of keratins, and stimulation of antibodyynthesis [7]. The protective effects of Se seem to be primarily asso-iated with its presence in the seleno-enzymes, which are knowno protect DNA and other cellular components from oxidative dam-ge [9]. Therefore, the present study was undertaken to evaluatehe ameliorating effect of Se on antioxidant status and biochem-cal changes against diazinon-induced toxicity in serum of maleats.

aterials and methods

hemicals

DZN (purity; 99%) was purchased from Wako Pure Chemicalndustries Ltd. (Osaka, Japan). Sodium selenite (Na2SeO3) used inhis study was purchased from Aldrich Chemical Company (Mil-aukee, USA). All other reagents used were of analytical reagent

rade.

nimals and care

Twenty-eight male Sprague–Dawely rats (weighing 150–170 g)ere obtained from the animal house of the Faculty of Medicine,lexandria University, Alexandria, Egypt. The local committeepproved the design of the experiments, and the protocol conformso the guidelines of the National Institutes of Health (NIH). Animalsere caged in groups of seven and given food and water ad libi-

um. The animal room was maintained at 21–24 ◦C and 40–60%elative humidity with 12-h light–dark cycles, the light cycle coin-iding with the day light hours. After 2 weeks of acclimation, theroups were assigned at random to one of the following treatments:roup 1 served as control that was given corn oil, while groups 2nd 3 were treated with Se (200 �g/kg BW) and diazinon (10 mg/kgW), respectively. Group 4 received diazinon (10 mg/kg BW) pluse (200 �g/kg BW). The diazinon dose used in the present experi-ent was selected according to the previous study of Ogutcu et al.

10]. Animals were treated daily with the tested compounds by oralavages for 30 days.

erum samples and parameters measured

Blood samples were taken by cardiac puncture and allowedo stand for 30 min at room temperature to clot before beingentrifuged at 3000 × g for 15 min. Serum was obtained by cen-rifugation and stored at −60 ◦C. Serum samples were aliquoted inppendorf tubes to use each one for one time. Serum glutathione-transferase (GST; EC 2.5.1.18) activity was determined accord-ng to Habig et al. using para-nitrobenzylchloride as a substrate11]. Superoxide dismutase (SOD; EC 1.15.1.1) was assayed accord-ng to Misra and Fridovich [12]. The assay procedure involves thenhibition of epinephrine auto-oxidation in an alkaline mediumpH 10.2) to adrenochrome, which is markedly inhibited by theresence of SOD. Catalase (CAT; EC 1.11.1.6) activity was measuredpectrophotometrically at 240 nm by calculating the rate of degra-ation of H2O2, the substrate of the enzyme [13]. The Se-dependentlutathione peroxidase (GPx; EC 1.11.1.9) and glutathione reduc-

ase (GR; EC 1.6.4.2) activities were measured by the methodescribed by Hafeman et al. [14]. Glutathione content was mea-ured in serum after reaction with 5,5′-dithiobis-(2-nitrobenzoiccid) using the method of Ellman [15]. Thiobarbituric acid-reactive

ents in Medicine and Biology 28 (2014) 89– 93

substances (TBARS) were measured using the method of Ohkawaet al. [16].

Serum alanine aminotransferase (ALT; EC 2.6.1.2) and aspar-tate aminotransferase (AST; EC 2.6.1.1) activities were assayed bythe method of Reitman and Frankel [17]. Alkaline phosphatase(AlP; EC 3.1.3.1) activity was measured at 405 nm by the for-mation of paranitrophenol from para-nitrophenylphosphate as asubstrate using the method of Principato et al. [18]. For assayingacid phosphatase (AcP; EC 3.1.3.2) activity, the method of Mosswas used [19]. Lactate dehydrogenase (LDH; EC 1.1.1.27) wasdetermined by the method of Cabaud and Wroblewski [20]. Acetyl-cholinesterase (AChE; EC 3.1.1.7) activity was estimated in serumusing acetylthiocholine iodide as a substrate according to themethod of Ellman et al. [21]. Serum total cholesterol (TC), trigly-cerides (TG), total lipids (TL), high-density lipoprotein-cholesterol(HDL-C), low-density lipoprotein-cholesterol (LDL-C) and very-lowdensity lipoprotein-cholesterol (VLDL-C) were assayed using com-mercial reagent kits. Total protein (TP) was determined using themethod of Lowry et al. [22]. Folin and Ciocalteus phenol reagentwas used to develop the blue color that was measured spec-trophotometrically at 750 nm. Bovine serum albumin was used as astandard. Albumin concentrations were determined by the methodof Doumas et al. [23]. Globulin concentrations were determinedby difference (TP-albumin). Glucose level was measured accord-ing to Hyvarinen and Nikkila [24] and concentrations of urea andcreatinine were determined by the methods of Patton and Crouchand Henry et al., respectively [25,26]. Total bilirubin was measuredusing the method of Walters and Gerade [27].

Statistical analysis

Data were analyzed as a completely randomized design accord-ing to Steel and Torrie [28]. Statistical significance of the differencein values of control and treated animals was calculated by (F) testwith 5% significance level (P < 0.05). Data of the present study werestatistically analyzed by using Duncan’s Multiple Range Test, SAS[29].

Results

Lipid peroxidation and glutathione content

As shown in Table 1, a significant (P < 0.05) increase in TBARSconcentration, the indicator of LPO, after the administration of DZNto rats as compared to control while rats treated with DZN + Seshowed a significant decrease in TBARS concentration as com-pared to DZN-treated groups. On the other hand, DZN treated ratsshowed a significant decrease in glutathione content (GSH). Nev-ertheless, Se co-treatment to DZN significantly raised GSH contentwhen compared to DZN-treated group. Treatment with Se alonecaused significant decrease in serum TBARS, while GSH content wassignificantly increased.

Antioxidant enzymes

Data concerning serum antioxidant enzyme activities (SOD, CAT,GPx, GR and GST) are presented in Table 1. A significant (P < 0.05)reduction in the antioxidant enzymes activity was observed in DZNtreated rats as compared to control. However, rats treated with DZN

and supplemented with Se, antioxidant enzymes activity showeda significant recovery as compared to DZN treated group. On theother hand, treatment with Se alone caused significant (P < 0.05)increase in some of the antioxidant enzyme activities in rat serum.

F.M. El-Demerdash, H.M. Nasr / Journal of Trace Elements in Medicine and Biology 28 (2014) 89– 93 91

Table 1Lipid peroxidation and antioxidant indices in serum of male rats treated with selenium, diazinon and/or their combination.

Parameters Control Se DZN DZN + Se

TBARS (nmol/mL) 2.69 ± 0.079c 2.18 ± 0.037d 3.65 ± 0.081a 3.14 ± 0.053b

GSH (�mol/mL) 19.30 ± 0.59b 22.43 ± 0.78a 13.00 ± 0.44d 16.29 ± 0.52c

SOD (U/mL) 1.12 ± 0.037b 1.31 ± 0.026ab 0.72 ± 0.016d 0.91 ± 0.017c

CAT (U/mL) 45.17 ± 1.15b 52.57 ± 1.32a 31.00 ± 1.15d 38.14 ± 0.91c

GST (�mol/h) 1.09 ± 0.031b 1.21 ± 0.025a 0.72 ± 0.026d 0.89 ± 0.026c

GPx (U/mL) 24.56 ± 0.751b 29.29 ± 0.680a 17.43 ± 0.481d 21.57 ± 0.571c

GR (U/mL) 15.22 ± 0.432b 17.11 ± 0.635a 10.66 ± 0.293d 12.86 ± 0.340c

Values are expressed as means ± standard errors; n = 7 for each treatment group.Mean values within a row not sharing a common superscript letter (a, b, c, d) were significantly different (P < 0.05).T superop

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BARS, thiobarbituric acid reactive substances; GSH, reduced glutathione; SOD,

eroxidase; GR, glutathione reductase.

valuation of aminotransferases, phosphatases and lactateehydrogenase

For the determination of liver damage induced by DZN andhe protective effect of Se, the activities of some hepatic enzymesere used as hepatotoxic biomarkers. Following DZN administra-

ion, ALT, AST, ALP, AcP and LDH activities increased significantlyP < 0.05) as compared to control (Table 2) indicating the occurrencef hepatic injury. Furthermore, DZN treatment caused significantP < 0.05) decrease in the activity of serum AChE as compared to theontrol. Nevertheless all enzymes activities showed a significantecovery in Se + DZN treated group as compared to DZN-treatedne. Treatment with Se alone did not cause any significant changen all measured enzyme activities.

hanges in lipids profile

In diazinon treated rats, a significant increase in total lipids, totalholesterol, TG, LDL-C and VLDL-C levels (P < 0.05) were observedhile the level of HDL-C was decreased. On one hand, in diazi-on + Se-treated group, a significant decrease in total lipids, totalholesterol, TG, LDL-C and VLDL-C levels, and significant increasen HDL-C as compared with diazinon group. On the other hand,reatment with Se alone did not cause any significant change inipid profile of rat serum (Table 3).

erum biochemical parameters

Data presented in Table 4 showed that treatment with daizinonaused significant increase (P < 0.05) in serum urea, creatinine, uriccid, total bilirubin and glucose, while the levels of TP and albu-in were significantly decreased as compared with control. On

he other hand, globulin insignificantly changed. Rats treated withe alone showed significant alterations in some of the measuredarameters. The presence of Se with DZN maintained the levels ofost parameters closer to the normal values.

iscussion

Organophosphate insecticides have been shown to interfereith membrane dependent processes, including nerve conduc-

ance and plasma membrane and organelle enzyme activities.xidative damage primarily occurs through production of reactivexygen species (ROS) and can damage lipids, proteins and DNA.herefore, oxidative damage may contribute to loss of enzymaticctivity and structural integrity of enzymes and activate inflamma-

ory processes [30,31]. The increase of TBARS and decreased GSHbserved in serum following DZN exposure was probably ascribedo the excessive production of ROS, which could be related withepatocyte enzyme leakage. The present results are in agreement

xide dismutase; CAT, catalase; GST, glutathione S-transferase; GPx, glutathione

with the finding of Ogutcu et al. [10] who demonstrated a signifi-cant increase in LPO level of rat heart tissue by DZN treatment.

GSH redox cycles serve as a crucial component of cellularantioxidant defenses and are essential for the tissues to protectthemselves against the ROS damage. They participate in the elim-ination of ROS, acting both as a non-enzymatic oxygen radicalscavenger and as a substrate for various enzymes such as GPx [32].Several researches have demonstrated that a shortage of sulfhydrylgroups brings about the cells/tissues at risk of oxidative damage[33,34]. GSH could be synthesized de novo, being this synthesisregulated by oxidants, antioxidants, and growth factors [35] orregenerated from GSSG by a reaction catalyzed by GR [36]. GPx is animportant selenocysteine-containing enzyme for cellular antiox-idant defense [37]. The observed reduction of GSH content andantioxidant enzyme activities could be attributable to the directeffect of DZN on the enzyme and/or due to depletion of the enzymesubstrates themselves. Furthermore, it may be due to enhancedfree radical production as evidenced by increased LPO. SOD andCAT are considered to be a primary defense that prevents biologicalmacromolecules from oxidative damage. It is known that xenobi-otics can induce superoxide radical production and if additionallySOD is inhibited the amount of oxygen radicals formed in cell canreach dangerous levels. In addition, superoxide radical is a potentinhibitor of CAT [38].

In the present study, rats treated with DZN for 30 days showedincreases in ALP, ALT, AST, AcP and LDH activities. These enzymesare secreted into the blood after liver and kidney injury, resultingin an increase of their activities in serum samples [39]. Lipid per-oxidation is known to disturb the integrity of cellular membranes,leading to the leakage of cytoplasmic enzymes [40]. Therefore, theincreased activities of these enzymes in serum observed in thepresent study could be due to the necrosis of liver, kidney and lung[41]. AChE activity is used as a standard biomarker of organophos-phate pesticide toxicity. The inhibition in serum cholinesteraseactivity probably referred to the effect of DZN on the synaptic trans-mission, altering acetylcholine release or metabolism and alteringmuscle contraction [6].

Lipids are thought to be among the most sensitive biologicalmolecules in terms of ROS susceptibility. In particular, unsaturatedfatty acids, which are located in cellular membranes, tissuesand blood, are prone to ROS attack. Previous studies reportedperturbations in lipids profile in serum of experimental animalsand workers exposed to organophosphates insecticides includingDZN [42]. The observed increase in the level of serum cholesterolmay be due to an increased cholesterol synthesis in the liver orit may be a sign of liver damage that can be attributed to theeffect of pesticides on the permeability of liver cell membrane.

Moreover, the increase in serum total cholesterol level may beattributed to the blockage of liver bile ducts causing reductionor cessation of its secretion to the duodenum [1]. The elevationin serum triglycerides has been attributed to an inhibition of the

92 F.M. El-Demerdash, H.M. Nasr / Journal of Trace Elements in Medicine and Biology 28 (2014) 89– 93

Table 2Enzyme activities in plasma of male rats treated with selenium, diazinon and/or their combination.

Parameters Control Se DZN DZN + Se

AST (IU/L) 106 ± 2.92c 98 ± 1.83cd 135 ± 3.32a 121 ± 2.33b

ALT (IU/L) 54.0 ± 1.65d 49.1 ± 1.14cd 68.5 ± 1.28a 60.1 ± 1.5b

AlP (IU/L) 124 ± 2.26c 114 ± 1.59cd 154 ± 1.46a 140 ± 1.79b

AcP (IU/L) 10.04 ± 0.36c 9.10 ± 0.31cd 12.98 ± 0.34a 11.51 ± 0.25b

LDH (IU/L) 338 ± 7.04c 302 ± 4.50cd 439 ± 9.06a 383 ± 10.04b

AChEA 3.60 ± 0.11b 3.94 ± 0.09ab 2.55 ± 0.08d 3.03 ± 0.07c

Values are expressed as means ± standard errors; n = 7 for each treatment group.Mean values within a row not sharing a common superscript letter (a, b, c, d) were significantly different (P < 0.05).AST, aspartate aminotransferase; ALT, alanine aminotransferase; AlP, alkaline phosphatase; AcP, acid phosphatase; LDH, lactate dehydrogenase; AChE, acetylcholinesterase.

A AChE activity: �mole substrate hydrolyzed/min.

Table 3Lipids profile of male rats treated with selenium, diazinon and/or their combination.

Parameters (mg/dl) Control Se DZN DZN + Se

TL 452 ± 8.50c 394 ± 12.30cd 552 ± 8.63a 508 ± 8.73b

TC 142 ± 2.86c 130 ± 2.59cd 177 ± 3.62a 158 ± 4.56b

TG 109 ± 1.74c 97 ± 1.13cd 139 ± 0.86a 119 ± 2.87b

HDL-C 46.38 ± 1.27b 51.14 ± 0.88ab 34.29 ± 0.81d 41.71 ± 0.57c

LDL-C 44.23 ± 1.39c 38.54 ± 0.86cd 55.96 ± 1.44a 50.23 ± 1.25b

VLDL-C 15.22 ± 0.43c 16.34 ± 0.67cb 20.67 ± 0.69a 17.96 ± 0.50b

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alues are expressed as means ± SE; n = 7 rats for each treatment group.ean values within a row not sharing a common superscript letter (a, b, c, d) were

L, total lipids; TC, total Cholesterol; TG, triglycerides; HDL-C, high density lipoprot

ipase enzyme activity of both the hepatic triglycerides and plasmaipoproteins [43]. HDL-C is mainly synthesized in the liver andntestinal cells. It plays an important role in cholesterol efflux fromissues and carries it back to the liver for removal as bile acids [44].t has been established that the decreased serum HDL-C levels arentiatherogenic [45], whereas the reduced levels are associatedith an increased risk for coronary artery disease [46].

The rise in blood glucose may indicate disrupted carbohy-rate metabolism due to enhanced breakdown of liver glycogen,ossibly mediated by an increase in adrenocorticotrophic andlucagon hormones and/or reduced insulin activity [47]. Thelevation in serum urea and creatinine levels in DZN-treatedats is considered as a significant marker of renal dysfunc-ion and it may be related to metabolic disturbances in liverunction, as urea is the end-product of protein catabolism. Fur-hermore, xenobiotics intensify the acid-secretory function ofidney and change the transport of sodium [48]. The increasen serum total bilirubin may result from decreased liver uptake,onjugation or increased bilirubin production from hemoly-is [8]. The decrease in the levels of protein in DZN treatedats might be due to changes in protein synthesis and/oretabolism.

Selenium is an essential dietary trace element, which has the

bility to counteract free radicals and protect the structure andunction of proteins, DNA and chromosomes against the injuryf oxidation [49]. Its biological function is expressed through

able 4erum biochemistry in male rats treated with selenium, diazinon and/or their combinatio

Parameters (mg/dl) Control Se

TP 7.65 ± 0.184b 8.65 ±

Albumin 5.54 ± 0.169b 6.32 ±

Globulin 2.52 ± 0.077a 2.73 ±

Urea 37.74 ± 0.628c 30.63 ±

Creatinine 0.64 ± 0.017d 0.55 ±

Uric acid 2.77 ± 0.050c 2.44 ±

Total bilirubin 0.63 ± 0.015c 0.57 ±

Glucose 105 ± 2.65c 99 ±

alues are expressed as means ± standard errors; n = 7 for each treatment group.ean values within a row not sharing a common superscript letter (a, b, c, d) were signifi

P, total protein.

cantly different (P < 0.05).DL-C, low density lipoprotein; VLDL-C, very low density lipoprotein.

biologically active compounds including GPx and GST [50]. Accu-mulating evidence suggests that many selenoproteins, whichcontain selenium in the form of amino acid selenocysteine, haveimportant enzymatic functions associated with antioxidant activ-ity. Furthermore, Se is important in sulphur amino acid metabolismthat protects animals against several diseases associated with lowintakes of Se. In this way, the sulphur amino acids methionineand cystine can spare Se through their antioxidant role [51]. Thepresent results showed that treatment with Se alone caused asignificant decrease in the levels of TBARS, and an increase inSOD, CAT, GPx, GR and GST activity and GSH content in rat serumas compared to control. On the other hand, changes in TBARSlevels, antioxidant enzymes activity, lipids profile and biochemicalindices observed in the group administered Se plus DZN arerelated to the decrease in the level of free radicals due to seleniumsupplementation. The present results are in good accordance withthose obtained by El-Demerdash who found that Se maintainedthe levels of antioxidants [52], membrane-bound enzymes andthe activities of antioxidant enzymes near normal levels, thusemphasizing its effect as antioxidant.

In conclusion, our results showed that DZN treatment inducedLPO and generation of free radicals in rat sera. Additionally, the

antioxidant defense system, lipids profile and biochemical indiceswere significantly affected by DZN toxicity. Moreover, Se adminis-tration showed a significant protective effect against DZN-inducedtoxicity due to its antioxidant effect.

n.

DZN DZN + Se

0.220a 5.47 ± 0.175d 6.52 ± 0.147c

0.187a 4.15 ± 0.0.98d 4.97 ± 0.089c

0.091a 2.33 ± 0.034a 2.53 ± 0.058a

0.781d 47.16 ± 1.366a 42.74 ± 1.097b

0.011c 0.79 ± 0.018a 0.73 ± 0.019ab

0.081cd 3.33 ± 0.062a 3.09 ± 0.074b

0.017c 0.80 ± 0.019a 0.71 ± 0.023b

1.33c 135 ± 2.32a 116 ± 2.50b

cantly different (P < 0.05).

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onflict of interest statement

There is no conflict of interest.

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