synergistic ameliorative effects of sesame oil and alpha- lipoic acid

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Synergistic ameliorative effects of sesame oil and alpha-

lipoic acid against subacute diazinon toxicity in rats: haematological, biochemical and antioxidant studies.

Journal: Canadian Journal of Physiology and Pharmacology

Manuscript ID cjpp-2015-0131.R1

Manuscript Type: Article

Date Submitted by the Author: 20-Jun-2015

Complete List of Authors: Abdel-Daim, Mohamed; Suez Canal University, Faculty of Veterinary

Medicine , Pharmacology Department Taha, Ramadan; Suez Canal University, Faculty of Veterinary Medicine, Clinical Pathology Ghazy, Emad; Kafrelsheikh University, Faculty of Veterinary Medicine, Clinical Pathology Department El-Sayed, Yasser; Damanhour University, Faculty of Veterinary Medicine, Veterinary Forensic Medicine and Toxicology

Keyword: diazinon, Sesame oil, Lipoic acid, antioxidant, oxidative stress

https://mc06.manuscriptcentral.com/cjpp-pubs

Canadian Journal of Physiology and Pharmacology

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Synergistic ameliorative effects of sesame oil and alpha-lipoic acid against 1

subacute diazinon toxicity in rats: haematological, biochemical and 2

antioxidant studies. 3

Mohamed M. Abdel Daim1*

, Ramadan Taha2, Emad W. Ghazy

3, Yasser S. El-Sayed

4 4

1 Pharmacology Department, Faculty of Veterinary Medicine, Suez Canal 5

University, Ismailia, 41522, Egypt. 6

2 Department of Clinical Pathology, Faculty of Veterinary Medicine, Suez Canal 7

University, Ismailia, 41522, Egypt. 8

3 Department of Clinical Pathology, Faculty of Veterinary Medicine, 9

Kafrelsheikh University, Kafr El-Sheikh 33516, Egypt. 10

4 Department of Veterinary Forensic Medicine and Toxicology, Faculty of 11

Veterinary Medicine, Damanhour University, Damanhour 22511, Egypt 12

13

Running title 14

Sesame oil and α-lipoic acid ameliorate diazinon toxicity 15

16

* Corresponding author: Mohamed M. Abdel Daim, Pharmacology Department, Faculty of 17

Veterinary Medicine, Suez Canal University, Ismailia, 41522, Egypt. 18

Tel/fax: +20-643-207-052; Mobile: +20-111-776-1570 19

E-mail: [email protected]; [email protected] 20

21

22

23

24

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Abstract 1

Diazinon (DZN) is a common organophosphorus insecticide extensively used for agriculture and 2

veterinary purposes. DZN toxicity is not limited to insects; it also induces harmful effects in 3

mammals and birds. Our experiment evaluated the protective and antioxidant potential of sesame 4

oil (SO) and/or alpha-lipoic acid (ALA) against DZN toxicity in male Wistar albino rats. DZN-5

treated animals exhibited macrocytic hypochromic anemia and significant increases in serum 6

biochemical parameters related to liver injury, including aspartate aminotransferase (AST), 7

alanine aminotransferase (ALT), alkaline phosphatase (ALP), γ-glutamyl transferase (γ-GT), 8

cholesterol, and triglycerides. They also had elevated levels of markers related to cardiac injury, 9

such as lactate dehydrogenase (LDH) and creatine phosphokinase (CPK), and increased 10

biomarkers of renal injury, urea and creatinine. DZN also increased hepatic, renal, and cardiac 11

lipid peroxidation and decreased antioxidant biomarker levels. SO and/or ALA supplementation 12

ameliorated the deleterious effects of DZN intoxication. Treatment improved hematology and 13

serum parameters, enhanced endogenous antioxidant status, and reduced lipid peroxidation. 14

Importantly, they exerted synergistic hepatoprotective, nephroprotective, and cardioprotective 15

effects. Our findings demonstrate that SO and/or ALA supplementation can alleviate the toxic 16

effects of DZN via their potent antioxidant and free radical-scavenging activities. 17

Keywords 18

diazinon; sesame oil; lipoic acid; antioxidant; oxidative stress 19

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1. Introduction 1

Organophosphorus insecticides are one of the largest groups of pesticides and are widely used 2

in agriculture for controlling insect pests on animals and crops. They constitute a major factor of 3

environmental pollution in different countries and are frequently detectable in soil, water, 4

vegetables, and air. The extensive use of organophosphates increases environmental pollution and 5

has potential health hazards including severe harmful effects on human and animals (Abdel-Daim 6

et al. 2014; Abdel-Daim and Halawa 2014). They are considered the most toxic class of 7

pesticides with regard to vertebrate animals. Numerous studies have shown that they induce 8

oxidative stress, which is known to be involved in the aging process and the pathogenesis of 9

several diseases including cancer, cataract, diabetes, stroke, coronary heart disease, Alzheimer’s 10

disease, and rheumatoid arthritis (Abdel-Daim 2014; Abdel-Daim and Ghazy 2015; Funasaka et 11

al. 2012; Lebda et al. 2012). Diazinon (DZN) is one of the most common organophosphorus 12

insecticides employed for agricultural, veterinary, and public health purposes. Humans and 13

animals can accidentally be exposed to DZN through ingestion and inhalation from the polluted 14

environment, in particular male individuals (Al-Attar 2015; Sarabia et al. 2009). The most 15

important characteristic of DZN toxicity is the irreversible acetylcholinesterase inhibition, and it 16

can cause animal death at high doses (Davies and Holub 1980). In addition, DZN is oxidized by 17

liver microsomal enzymes to produce hydroxydiazoxon, hydroxydiazinon, and diazoxon, which 18

are even more potent acetylcholinestrase inhibitors (WHO 1998). DZN intoxication also induces 19

erythrocyte lipid peroxidation (LPO) and causes significant changes in antioxidant enzyme 20

activities, suggesting that reactive oxygen species (ROS) may be involved in DZN toxicity 21

(Gultekin et al. 2000). These ROS induce oxidative damage resulting in hematologic, 22

hepatorenal, and cardiologic toxicity (Al-Attar and Abu Zeid 2013; Razavi et al. 2013). 23

Organisms have developed methods to defend against the harmful effects of free radicals. 24

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Superoxide dismutases (SOD), mitochondrial enzymes, and antioxidants are effective in 1

counteracting the harmful effects of free radicals. The primary mechanism by which the body 2

eliminates these radicals is through the donation of electrons between oxygen species and 3

antioxidants. ROS accumulation occurs when there is an imbalance between the formation and 4

removal of these species, leading to disturbances in cellular physiology and various pathological 5

conditions (Abdel-Daim et al. 2013; Al-Sayed and Abdel-Daim 2014; Al-Sayed et al. 2014; El-6

Sayed et al. 2015; Ibrahim and Abdel-Daim 2015). LPO increased in rat erythrocytes after DZN 7

administration (Sutcu et al. 2007), and DZN-induced oxidative stress decreased when various 8

antioxidants were administered (Ahmed et al. 2000; Cankayali et al. 2005; Gultekin et al. 2001). 9

Moreover, DZN exposure depletes antioxidant enzymes in rats (Shah and Iqbal 2010). 10

Sesame oil (SO) extracted from the seeds of Sesamum indicum is composed of different fatty 11

acids and non-fat antioxidants. The significant free radical-scavenging ability of SO may be due 12

to several antioxidants components (i.e. sesamolin, sesamin, tocopherol, and sesaminol) (Fukuda 13

1990), or the presence of phenolic compounds that inhibit the generation of ROS, and it has long 14

been used as a daily nutritional supplement to decrease LPO (Espin et al. 2000). Alpha-lipoic 15

acid (ALA) is a naturally occurring short-chain fatty acid necessary for mitochondrial enzyme 16

function (Abdou and Abdel-Daim 2014; Kim et al. 2004). It possesses antioxidant properties and 17

plays a crucial role in protecting cells and tissues against the deleterious effects of free radicals 18

and ROS. Considered as an ideal antioxidant found naturally in food, ALA appears to have 19

improved functional capacity when given as a supplement (Moini et al. 2002). ALA reportedly 20

scavenges free radicals by facilitating the regeneration of endogenous antioxidants in the body, 21

including vitamin C, vitamin E, and intracellular reduced glutathione (GSH) (Abdou and Abdel-22

Daim 2014; Wollin and Jones 2003). Therefore, the present study aimed to assess whether SO 23

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and/or ALA could attenuate the hepatotoxic, nephrotoxic, and cardiotoxic effects of subacute 1

DZN toxicity in rats. 2

2. Materials and Methods 3

2.1. Chemicals and assay kits 4

Diazinon-60®

a commercial formulation containing 60% active ingredient, was purchased 5

from Drug Pharmaceuticals (Cairo, Egypt). SO was obtained from El-Captain Company for 6

extracting natural oils, herbs, and cosmetics (El-Obour City, Cairo, Egypt). ALA was obtained 7

from EVA Pharma (Cairo, Egypt). All kits were obtained from Biodiagnostics Co. (Cairo, 8

Egypt), except that to measure LDH, which was purchased from Randox Laboratories Ltd. 9

(Crumlin, UK), and CK, from Stanbio™ (CK-NAC [UV-Rate] kit; Boerne, TX, USA). All other 10

chemicals were of reagent grade and were commercially available from local scientific 11

distributors in Egypt. 12

2.2. Animals and treatments 13

Forty male Wistar rats, weighing 180 ± 20 g, were obtained from The Egyptian Organization 14

for Biological Products and Vaccines. The rats were kept in a ventilated animal house with a 15

controlled light-dark cycle (12 h light/dark) and constant temperature (25 ± 2°C). Food and water 16

were provided ad libitum. The Research Ethical Committee of the Faculty of Veterinary 17

Medicine, Suez Canal University, Ismailia, Egypt (approval no. 20149), approved the 18

experimental design and all animal handling protocols. All precautions were taken to avoid 19

animal stress. 20

Rats were allowed to acclimate for 2 weeks before the experimentation. They were randomly 21

divided into five different groups (n=8 each). The first group received normal saline and was 22

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used as a control. The second group received a daily dose of DZN (20 mg/kg body weight, 1

orally) (Hariri et al. 2010). The third group was given a daily dose of SO (5 ml/kg body weight, 2

orally) (Saleem et al. 2014). The fourth group received a daily dose of ALA (100 mg/kg body 3

weight, orally) (Abdou and Abdel-Daim 2014), and the fifth group received SO and ALA. Both 4

treatments were given an hour before DZN administration for four consecutive weeks. 5

At the end of experimental period (24 h after the last DZN dose), blood samples were 6

collected via the retro-orbital venous plexus under light ether anesthesia and were immediately 7

divided into aliquots with and without anticoagulant for hematological and serum biochemical 8

analysis, respectively. After blood collection, the rats were sacrificed by decapitation. The liver, 9

kidney and heart were rapidly excised from each animal, trimmed of connecting tissue, and 10

washed free of blood with 0.9% NaCl solution and distilled water to assess tissue oxidative status 11

and antioxidant indices. 12

2.3. Hematological examination 13

The aliquot contained EDTA (1 mg/ml) used for assessing red blood cells (RBCs), 14

hemoglobin (Hb), packed cell volume (PCV), mean corpuscular volume (MCV), mean 15

corpuscular hemoglobin concentration (MCHC), white blood cells (WBCs) and blood platelets 16

(Schalm 1986) using Orphee Mythic 22 CT Hematology Analyzer (Plan-les-Ouates, 17

Switzerland). 18

2.4. Serum biochemical parameters 19

The hepatic, renal and cardiac injury biomarkers, serum alanine aminotransferase (ALT), 20

aspartate aminotransferase (AST), alkaline phosphatase (ALP), LDH, gamma glutamyltransferase 21

(γGT) and CK enzyme activities, and cholesterol, creatinine, urea and uric acid contents were 22

measured spectrophotometrically according to manufacturers' instructions. 23

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2.5. Estimation of antioxidants and lipoperoxidation markers 1

The dissected tissues were washed with 50 mM sodium phosphate-buffered saline (100 mM 2

Na2HPO4/NaH2PO4, pH 7.4) in an ice-containing medium, with 0.1 mM EDTA to remove any 3

RBCs and clots. Then tissues were homogenized in 5–10 ml cold buffer per g tissue and were 4

centrifuged at 5000 rpm for 30 min. The resulting supernatant was transferred into an Eppendorf 5

tube and was preserved at -80°C into aliquots for the spectrophotometric estimation of tissue 6

LPO biomarker (MDA) (Mihara and Uchiyama 1978), SOD (Nishikimi et al. 1972), catalase 7

(CAT) (Aebi 1984), GSH (Beutler et al. 1963), glutathione peroxidase (GSH-Px) (Paglia and 8

Valentine 1967) and nitric oxide (NO) (Green et al. 1982). 9

2.6. Statistical analysis 10

All data are expressed as the mean ± S.E.M., and the levels of significance are cited at P ≤ 11

0.05. GraphPad Prism statistical package version 5.0 for Windows (GraphPad Software Inc., La 12

Jolla, CA). was used for all data analysis. Differences in values were analyzed by One-Way 13

analysis of variance, followed by Tukey's multiple range tests. 14

3. Results 15

3.1. Hematological findings 16

The effects of DZN intoxication and the preventive effects of SO and/or ALA on the 17

hematological parameters are shown in Table 1. We observed significant (P ≤ 0.05) decreases in 18

blood parameters (RBCs, HB, PCV and platelets) in DZN-intoxicated rats compared to the 19

control group (73.4%, 69.6%, 84.5% and 41.3%, respectively). Contrary to these results, a 20

significant (P ≤ 0.05) increase in WBCs was observed (160.4%). 21

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SO significantly (P ≤ 0.05) reversed the changes in hematological parameters (RBCs, Hb, 1

PCV, and platelets) compared to the DZN-treated group (121.2%, 126.2%, 108.2%, and 150.5%, 2

respectively), and the WBCs count was also significantly (P ≤ 0.05) decreased (67.6% of the 3

DZN count). Similarly, ALA significantly (P ≤ 0.05) increased RBCs (127%), Hb (137.1%), 4

PCV (109.5%), and platelets (189.6%), and significantly (P ≤ 0.05) decreased WBCs (66.5%) 5

compared to the DZN-treated group. At the same time, simultaneous supplementation of SO and 6

ALA significantly (P ≤ 0.05) restored the reductions in blood parameters (RBCs, HB, PCV and 7

platelets) (135.1%, 143.8%, 115.7% and 235%, respectively) and significantly (P ≤ 0.05) 8

decreased WBCs (63.1%) in comparison with DZN-treated rats. 9

3.2. Tissues injury biomarkers 10

The deleterious effects of DZN intoxication and the preventive effects of SO and/or ALA on 11

serum biochemical measurements are shown in Table 2. Significant (P ≤ 0.05) increases in serum 12

liver function marker enzymes (AST, 227.5%; ALT, 325.9%; ALP, 254.2%; and γGT, 194.7%) 13

were recorded in DZN-treated rats compared to the control group. Similarly, we observed 14

significant (P ≤ 0.05) increases in serum cholesterol and triglycerides (177% and 196.1%, 15

respectively) and cardiac muscle enzyme markers (LDH, 201.1%; CPK, 171%). Serum renal 16

products (urea, creatinine, and uric acid) were also significantly (P ≤ 0.05) increased by about 17

315.2%, 536.4%, and 304.1%, respectively, compared with control rats. 18

Treatment with SO and/or ALA ameliorated most of the negative effects of DZN on the 19

studied serum parameters. The results indicate that SO and/or ALA effectively reduced DZN-20

induced hepatorenal toxicity. Serum hepatic enzymes were significantly (P ≤ 0.05) decreased in 21

the DZN-SO group (AST, 59%; ALT, 54.9%; ALP, 53.2%; and γGT, 68.1%). Moreover, serum 22

cholesterol and triglycerides were significantly (P ≤ 0.05) reduced by 80.5% and 73.3%, 23

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respectively. SO treatment also caused significant (P ≤ 0.05) declines in serum muscle enzymes 1

(LDH, 80.7%; CPK, 74.9%) and renal function markers (urea, 64%; creatinine, 58.6%; uric acid, 2

55.7%) compared to the DZN-treated group. 3

Regarding to DZN-ALA group, hepatic serum biomarker enzymes were significantly (P ≤ 4

0.05) decreased (AST, 52.7%; ALT, 46%; ALP, 49.8%; and γGT, 62.3%). Serum cholesterol and 5

triglycerides were also significantly (P ≤ 0.05) reduced (76.4% and 60.9%, respectively). ALA 6

supplementation induced a significant (P≤0.05) declines in serum muscle enzymes (LDH, 73.7%; 7

CPK, 68.7%) and serum renal injury markers (urea, 58%; creatinine, 48.6%; and uric acid, 8

54.3%) compared with DZN-treated rats. Co-administration of SO and ALA significantly (P ≤ 9

0.05) reduced hepatic serum biomarker enzymes (AST, 45.4%; ALT, 32%; ALP, 41%; and γGT, 10

54.9%) and serum cholesterol (52.8%) and triglycerides (53.9%). We also observed significant (P 11

≤ 0.05) decreases in serum muscle enzymes (LDH, 53.5%; CPK, 63.1%) and serum renal injury 12

markers (urea, 35.5%; creatinine, 22.1%; and uric acid, 37.5%). Notably, the reductions were 13

greater than when each of them was used alone. 14

3.3. Oxidative stress and antioxidant biomarkers 15

Liver. The deleterious effects of DZN and preventive effects of SO and/or ALA on hepatic LPO 16

and antioxidant parameters are shown in Figure 1. Significant (P ≤ 0.05) increases in hepatic 17

MDA and NO content were observed (180.4% and 146.3%, respectively). Liver GSH content, 18

and GSH-Px, SOD, and CAT activities were significantly (P ≤ 0.05) decreased (69.9%, 49.7%, 19

41.2% and 36%, respectively) compared with the control group. 20

In the DZN-SO group, liver MDA and NO levels were decreased (66% and 74.1%, 21

respectively), and liver GSH content, and GSH-Px, SOD, and CAT activities were increased 22

(131.1%, 146.5%, 182.8%, and 229.6%, respectively). Regarding the DZN-ALA group, liver 23

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MDA and NO levels were significantly (P ≤ 0.05) decreased (56.7% and 72.2%, respectively), 1

whereas liver GSH content, and GSH-Px, SOD, and CAT activities were significantly (P ≤ 0.05) 2

increased (115.6%, 123.1%, 152.5% and 209.6%, respectively) in comparison with DZN-3

intoxicated rats. Furthermore, co-administration of SO and ALA significantly (P ≤ 0.05) reduced 4

liver MDA and NO levels (56.7% and 65.8%, respectively) and increased GSH, GSH-Px, SOD, 5

and CAT more than each of them used alone (140.9%,192%, 214.4% and 258.1%, respectively) 6

compared with the DZN-intoxicated rats (Figure 1). 7

Heart. The effects of DZN treatment with or without SO and/or ALA on cardiac LPO and 8

antioxidant parameters are shown in Figure 2. A significant (P ≤ 0.05) increase in cardiac MDA 9

and NO content (180.4% and 169.4%, respectively) was observed compared with the control 10

group, but cardiac antioxidants were significantly (P ≤ 0.05) decreased (GSH, 67%; GSH-Px, 11

62.6%; SOD, 41.2%; and CAT, 36%, respectively). 12

Regarding the DZN-SO group, cardiac MDA and NO levels were reduced (56.7% and 13

68.7%, respectively), whereas antioxidants were elevated (GSH, 120.7%; GSH-Px, 129.2%; 14

SOD, 152.5%; and CAT, 209.6%) compared to the DZN-treated group. In the DZN-ALA group, 15

cardiac MDA and NO levels were significantly (P ≤ 0.05) reduced to 66% and 65.4%, 16

respectively. Cardiac antioxidant levels were significantly (P ≤ 0.05) increased (GSH, 136.9%; 17

GSH-Px, 146.6%; SOD, 182.8%; and CAT, 229.6%) in comparison with DZN-treated rats. 18

Similarly, co-administration of SO and ALA significantly (P ≤ 0.05) decreased cardiac tissue 19

MDA and NO levels (56.9% and 60.4%, respectively) and increased antioxidant levels more than 20

each of them used alone (GSH, 147.2%; GSH-Px, 157.6%; SOD, 214.4%; and CAT, 258.1%) 21

compared with the DZN-treated rats (Figure 2). 22

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Kidney. The effects of DZN-intoxication and the preventive effects of SO and/or ALA on renal 1

LPO and antioxidant parameters are shown in Figure 3. A significant (P ≤ 0.05) increase in renal 2

MDA and NO content (207.6% and 167.4%, respectively) was observed compared with the 3

control group. Renal antioxidants were significantly (P≤0.05) decreased (GSH, 63.6%; GSH-Px, 4

50.7%; SOD, 36.2%; and CAT, 35.1%). 5

Concerning the DZN-SO group, renal MDA and NO levels were reduced (76.5% and 6

73.8%), but renal antioxidants were elevated (GSH, 126.6%; GSH-Px, 138.9%; SOD, 205.1%; 7

and CAT, 216%) compared to DZN-treated group. In DZN-ALA rats, renal MDA and NO levels 8

were significantly (P ≤ 0.05) decreased (65.4% and 65.2%, respectively), while renal antioxidant 9

levels were significantly (P≤0.05) increased (GSH, 148.1%; GSH-Px, 148.5.1%; SOD, 217.8%; 10

and CAT, 225.2%) in comparison with DZN-treated rats. Furthermore, combination of SO and 11

ALA significantly (P≤0.05) reduced renal tissue MDA and NO levels (49.1% and 62.1%, 12

respectively) and increased antioxidant levels (GSH, 155.1%; GSH-Px, 190.3%, SOD, 248%; 13

and CAT, 277.3%) to a greater degree than when used separately compared with DZN-treated 14

rats (Figure 3). 15

4. Discussion 16

The present study investigated the effect of SO and/or ALA supplementation on the toxic 17

effects of DZN organophosphorus insecticide on hematological, biochemical and antioxidant 18

biomarkers. The results of the present study revealed a significant reduction in the erythrogram 19

parameters (RBCs, Hb, PCV, MCV and MCHC) in the DZN-intoxicated group. Reductions in 20

RBCs may be due to either direct destruction of erythrocytes by the insecticide or indirect 21

destruction through its adverse effects on the bone marrow (Rajini et al. 1987). It is likely that 22

macrocytic hypochromic anemia developed during the period of exposure due to red cell 23

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destruction or hemorrhagic anemia, both of which stimulate reticulocyte production. Coles 1

(1986) Reported that low PCV in anemia stimulates early reticulocyte release from bone marrow 2

(Rajini et al. 1987; Yassa et al. 2011). The increment in MCV and reduction in MCHC after DZN 3

treatment suggest either hemorrhagic or hemolytic anemia. Others have attributed decreases in 4

Hb concentration, PCV values, and RBCs counts to the ability of pesticides to interfere with Hb 5

biosynthesis, thus shorting circulating erythrocyte lifespan (Patil et al. 2003), as well as their 6

effects on erythropoietic tissue and erythropoietin hormone production (Kalender et al. 2006). 7

The toxic effects of DZN on hematologic parameters may be due to enhanced ROS production 8

and decreased antioxidant levels. Because ROS are highly reactive and can oxidize cellular 9

macromolecules (i.e. phospholipids, DNA, and proteins), they can exert different biologic effects 10

such as increased membrane rigidity, osmotic fragility, decreased cellular deformability, reduced 11

erythrocyte survival, membrane fluidity, and fine structural damage to RBC membranes, 12

ultimately resulting in hemolytic anemia (Hogg 1998; Kaplowitz and Tsukamoto 1996). The 13

increment in total leukocytic count (TLC) observed in the DZN-treated rats could indicate 14

activation of the immune system in response to tissue inflammation and necrosis induced by the 15

pesticide (Kalender et al. 2006; Mohamed et al. 2007). Increased numbers of WBCs despite 16

decreases in all other erythrogram and thrombocyte counts could indicate that the bone marrow is 17

not completely depressed. While DZN depressed erythropoiesis and thrombopoiesis, we found 18

that it enhanced granulopoiesis. 19

Similarly, the stimulatory effect of DZN on liver enzymes was likely due to the production of 20

ROS, which enhanced LPO and the production of toxic aldehydes such as MDA. The exhaustion 21

of antioxidant defense enzymes ultimately led to hepatocellular injury and necrosis and the 22

release of intracellular enzymes including ALT, AST, γGT, ALP, and LDH as well as cholesterol 23

and triglycerides. Increases in these biomarkers are evidence of active liver dysfunction. These 24

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elevations could be attributed to direct damage to hepatocytes caused by the insecticide, which 1

was confirmed by measuring antioxidant levels in liver tissue. At the same time, the nephrotoxic 2

and cardiotoxic effects of subacute DZN toxicity may be also due to oxidative stress caused by 3

free radical production and the subsequent depletion of intracellular antioxidant enzymes, 4

ultimately elevating serum levels of creatinine and urea. 5

DZN treatment mediates oxidative stress as evidenced by elevated tissues MDA and NO 6

levels and depleted enzymatic and non-enzymatic antioxidant defenses, including CAT, SOD, 7

GSH-Px and GSH. ROS are highly reactive and can oxidize cellular macromolecules such as 8

lipids, causing LPO and other destructive processes. Furthermore, they induce gene expression 9

alterations by activating the transcription factor nuclear factor-κB or altering mitochondrial 10

permeability, with lethal consequences (Abdel-Daim 2014; Abdel-Daim and Halawa 2014; El-11

Demerdash et al. 2013; Kaplowitz and Tsukamoto 1996). Chronic DZN administration induced 12

significant oxidative damage in DNA in liver and kidney tissue and simultaneously reduced 13

plasma GSH, CAT, and TAC levels (Tsitsimpikou et al. 2013). Furthermore, subacute DZN 14

toxicity significantly increased serum levels of tumor necrosis factor-a, uric acid, AST, ALT, and 15

LDH (Hariri et al. 2010). These results point toward the major role of ROS in DZN-mediated 16

injury and toxicity. 17

The present data also demonstrate that SO alone or in combination with ALA can counteract 18

the DZN-induced reductions in total RBCs, PCV, Hb concentration, and platelet count. This may 19

be due to the free radical-scavenging properties and the antioxidant activities of both SO and 20

ALA that protect against LPO of the erythrocyte membrane. In addition, these compounds 21

inhibited cellular ROS generation and maintained the mitochondrial membrane potential during 22

oxidative stress (Wollin and Jones 2003). Supplementation of SO and/or ALA reduced serum 23

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hepatic, renal, and cardiac injury biomarkers. They also reduced LPO and restored the 1

antioxidant enzyme levels in these organs. The antioxidant and protective effects of SO are 2

attributable to its non-fat antioxidants including tocopherol, sesamin, sesamolin, and sesamol 3

(Fukuda 1990). Its free radical-scavenging capacity could also be due to the presence of phenolic 4

compounds, which inhibit the generation of reactive oxygen free radicals. Collectively, our 5

findings support the use of SO as a daily nutritional supplement for lowering oxidative stress. 6

ALA also acts also by regenerating endogenous antioxidants (Wollin and Jones 2003). The 7

abilities of SO and ALA to exert this protective effect is positively correlated with their abilities 8

to suppress NO overproduction, maintain cellular antioxidant defense mechanisms and LPO. 9

Therefore, SO and/or ALA might play a role in reducing the deleterious effects of DZN, likely 10

via their powerful antioxidant properties. This hypothesis is supported by our observations of 11

lowered MDA and NO levels and improved GSH levels, and SOD, CAT and GSH-Px activities 12

in the liver, kidney and heart. Indeed, a considerable body of evidence indicates that SO and ALA 13

protect against oxidative stress in vital organs via antioxidant properties (Abdel-Zaher et al. 2008; 14

Amudha et al. 2006; Dulundu et al. 2007; Hsu and Liu 2004; Kaur and Saini 2000). 15

5. Conclusion 16

In summary, the protective effect of SO and/or ALA against oxidative stress induced by DZN 17

toxicity in rats could be mediated either directly by scavenging ROS, inhibiting LPO and 18

suppressing NO overproduction or indirectly through the enhancement of antioxidant enzymes 19

depleted. We found that SO and ALA synergistically exerted protective effects and consequently 20

can be used in combination to minimize and prevent the toxic effects of DZN via their free 21

radical-scavenging and potent antioxidant activities. 22

23

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Conflict of interest statement 1

The authors declare that there are no conflicts of interest. 2

3

Acknowledgement 4

This research work received no fund from any organization. 5

6

References 7

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10.1371/journal.pone.0072991. 17

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Tables 1

Table 1: Hematological parameters in control and treated groups. 2

Groups RBCs (×106/µl) Hb (g/dl) Pl (×10

3/µl) PCV (%) WBCs (×10

3/µl)

Control 7.27±0.17a

14.4±0.24a

724±37.2a

45.1±.63a

10.39±025a

DZN 5.33±0.14c 10.0±0.31

c 299.2±14

d 38.1±0.79

c 16.66±0.51

b

DZN-SO 6.47±0.16b 12.6±0.32

bc 450.2±14.2

c 41.2±.64

b 11.27±.46

a

DZN-LA 6.78±0.16ab

13.7±0.29ab

567.2±14b 41.7±.79b

11.08±.50

a

DZN-SO-LA 7.21±0.27a 14.1±0.25

a 703.2±37

a 44.1±.81

ab 10.51±.26

a

The data are presented as means ± S.E. (n = 8). Diazinon (DZN), sesame oil (SO), alpha-lipoic 3

acid (ALA), hemoglobin (Hb), red blood corpuscles (RBCs), platelets (Pl), packed cell volume 4

(PCV), white blood corpuscles (WBCs). Different superscript letters within a column indicate 5

significantly different mean values (p ≤ 0.05). 6

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Table 2: Hematological parameters in control and treated groups. 1

Groups Control DZN DZN-SO DZN-LA DZN-SO-LA

AST (U/L) 65.9±2.9b

150±11.8a 88.5±3.6

b 79±3.69

b 68±3.6

b

ALT (U/L) 28.2±1.16c

91.9±2.9a 50.5±2.04

b 42.3±1.8

b 29.4±1.7

c

ALP (U/L) 68.9±3.14c

175.2±10.3a 93.15±1.9

b 87.2±2.14

bc 73.2±1.4

bc

γGT (U/L)) 2.9±0.2b

5.6±0.38a 3.8±0.22

b 3.5±0.22

b 3.1±0.25

b

LDH (U/L) 209.3±10.8c

420.9±13.7a 339±11.2

b 310.3±7.7

b 225.2±12.7

c

CPK (U/L) 199.4±10.8c

340.9±13.7a 255±9.5

b 234.2±7.2

b 215.2±12.7

bc

Cholesterol

(mg/dL) 75.54±4.2

c 134.0±4.2

a 107.8±3.72

b 102.4±3.1

b 70.8±2.7

c

Triglyceride

(mg/dL) 103.2±4.4

c 202.4±9.2

a 148.3±2.9

bb 123.3±4.4

c 109.2±4.6

c

Urea (mg/dL) 21.7±1.1c 68.4±4.2

a 43.8±1.9

b 39.7±1.9

b 24.6±2.02

c

Creatinine (mg/dL) 0.63±0.06c

3.4±0.30a 1.98±0.14

b 1.6±0.12

b 0.75±0.09

b

Uric acid (mg/dL) 23.8±1.8c

72.3±3.44a 40.28±1.86

b 39.3±1.8

b 27.1±1.2

c

2

Data are expressed as means ± S.E. (n = 8). Diazinon (DZN), sesame oil (SO), lipoic acid (ALA), 3

aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), 4

gamma glutamyl transferase (γGT), lactate dehydrogenase (LDH), creatine phosphokinase 5

(CPK), creatinine (Cr). Different superscript letters within a column indicate significantly 6

different mean values (p ≤ 0.05). 7

8

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Figures legends 1

Figure 1. Hepatic lipid peroxidation and antioxidant parameters in control and treated groups; 2

(A) MDA: malondialdehyde, (B) NO: nitric oxide, (C) GSH: reduced glutathione, (D) 3

GSH-Px; glutathione peroxidase, (E) SOD: superoxide dismuatse and (F) CAT: 4

catalase. Different letters indicate significantly different mean values (p ≤ 0.05). 5

Figure 2. Renal lipid peroxidation and antioxidant parameters in control and treated groups; (A) 6

MDA: malondialdehyde, (B) NO: nitric oxide, (C) GSH: reduced glutathione, (D) 7



Figure 3. Cardiac lipid peroxidation and antioxidant parameters in control and treated groups; 10

(A) MDA: malondialdehyde, (B) NO: nitric oxide, (C) GSH: reduced glutathione, (D) 11



14

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Hepatic lipid peroxidation and antioxidant parameters in control and treated groups; (A) MDA: malondialdehyde, (B) NO: nitric oxide, (C) GSH: reduced glutathione, (D) GSH-Px; glutathione peroxidase, (E) SOD: superoxide dismuatse and (F) CAT: catalase. Different letters indicate significantly different mean

values (p ≤ 0.05). 333x359mm (150 x 150 DPI)

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Renal lipid peroxidation and antioxidant parameters in control and treated groups; (A) MDA: malondialdehyde, (B) NO: nitric oxide, (C) GSH: reduced glutathione, (D) GSH-Px; glutathione peroxidase, (E) SOD: superoxide dismuatse and (F) CAT: catalase. Different letters indicate significantly different mean

values (p ≤ 0.05). 335x337mm (150 x 150 DPI)

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Cardiac lipid peroxidation and antioxidant parameters in control and treated groups; (A) MDA: malondialdehyde, (B) NO: nitric oxide, (C) GSH: reduced glutathione, (D) GSH-Px; glutathione peroxidase, (E) SOD: superoxide dismuatse and (F) CAT: catalase. Different letters indicate significantly different mean

values (p ≤ 0.05). 336x336mm (150 x 150 DPI)

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synergistic ameliorative effects of sesame oil and alpha- lipoic acid

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