JOURNAL OF MEDICINAL FOOD J Med Food 00 (0) 2015, 1–9 # Mary Ann Liebert, Inc., and Korean Society of Food Science and Nutrition DOI: 10.1089/jmf.2014.0130

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Hepatoprotective and Renoprotective Effects of Lavender (Lavandula stoechas L.) Essential Oils Against Malathion-Induced Oxidative Stress in Young Male Mice Slimen Selmi,1,2 Manel Jallouli,1 Najoua Gharbi,1 and Lamjed Marzouki 2 1

Laboratory of Metabolic and Endocrine Aggressions Physiology, Department of Life Sciences, Faculty of Sciences of Tunis, Tunis, Tunisia. 2 Laboratory of Nutrition and Animal Physiology, Higher Institute of Biotechnology of Beja, Beja, Tunisia ABSTRACT We aimed in the present study to investigate the hepato- and nephroprotective effects of Lavandula stoechas essential oils (LSEO) against malathion-induced oxidative stress in young male mice as well as the possible mechanism implicated in such protection. Animals were divided into eight groups of 12 each: Control, malathion (200 mg/kg b.w.); Various doses of LSEO (10, 30, and 50 mg/kg b.w.), malathion + various doses of LSEO. Malathion and LSEO were daily per orally (p.o.) administered by intragastric gavage during 30 days. We initially found that malathion treatment induced body weight gain decrease as well as a clear nephro- and hepatotoxicity as assessed by significant relative liver and kidney weight increase and related hemodynamic parameters deregulation. Malathion exposure of mice also induced a considerable perturbation of metabolic parameters. On the other hand, we showed that malathion administration was accompanied by an oxidative stress status assessed by an increase of malondialdehyde (MDA) and hydrogen peroxide (H2O2) levels as well as a depletion of sulfhydril group content (-SH) and antioxidant enzyme activities such as catalase (CAT) and glutathione peroxidase (GPx), total superoxide dismutase (SOD), Cu/Zn-SOD, Mn-SOD, and Fe-SOD in the kidney and liver. More importantly, LSEO treatment abolished all malathion-induced body gain loss, liver and kidney relative weight increase, hemodynamic and metabolic disorders, as well as hepatic and renal oxidative stress. In conclusion, our data suggest that LSEO exerted potential hepato- and nephroprotective effects against malathion-induced oxidative stress in mice. The beneficial effect of LSEO might be related, in part, to its antioxidant properties.

KEY WORDS:  essential oils  kidney  Lavandula stoechas  liver  malathion  mice  oxidative stress

and glutathione peroxidase (GPx). Excessive reactive oxygen species (ROS) accumulation in tissues leads to cellular injury initiated by impairment of vital macromolecules, such as DNA,10 protein,11 and lipid.12 The cellular antioxidant status determines the susceptibility to oxidative damage and is usually altered in response to oxidative stress. The cellular antioxidant action is reinforced by the presence of dietary antioxidants.13 Accordingly, interest has recently grown in the role and usage of natural antioxidants as strategy to prevent against oxidative damage in various health disorders, with oxidative stress as a factor in their pathophysiology.13 Natural antioxidants from fruits and vegetables are reported to provide substantial protection that slows down the process of oxidative damage caused by ROS.14 The beneficial effect of polyphenols is associated with a multitude of biological activities, including antioxidant and free radical-scavenging properties, antiplatelet aggregation, and inhibition of vascular smooth muscle cell proliferation. All these effects might interfere with atherosclerotic plaque development and stability. These observations might explain their cardiovascular protective properties.15 On the other hand, it is now

INTRODUCTION

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rganophosphate insecticides (OPs) are the major cause of morbidity and mortality, particularly among farmers in developing countries, including Tunisia.1 However, it is well known that OPs, for which suspicion of teratogenic, carcinogenic, and/or mutagenic effects in man exists, are still in use.2 Malathion has harmful effects on various organs systems, including the kidney,3 liver,3 lung,4 brain,5 and erythrocytes.6 This insecticide is metabolized in the liver by multiple specific cytochrome P450 enzymes through several reaction pathways.7 Indeed, malathion is known to inhibit the acetylcholinesterase activity in target tissues.8,9 The harmful effects of malathion are, in part, suspected to its ability to induce oxidative stress status characterized by depletion of endogenous antioxidant enzyme activities, such as superoxide dismutase (SOD), CAT, Manuscript received 18 August 2014. Revision accepted 11 February 2015. Address correspondence to: Slimen Selmi, PhD, Laboratory of Metabolic and Endocrine Aggressions Physiology, Department of Life Sciences, Faculty of Sciences of Tunis, Tunis 1062, Tunisia, E-mail: [email protected]

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established that hyperlipidemia represents a major risk factor for the premature development of atherosclerosis and its cardiovascular complications.16 A logical strategy to prevent against atherosclerosis and reduce the incidence of cardiovascular disease events is to target the hyperlipidemia and oxidative stress by diet and/or drug intervention. Lavender (Lavandula stoechas L.) is a medicinal plant largely used and included for centuries in the pharmacopoeia of several countries, including Tunisia. The phytochemical screening of this plant revealed that it is rich in bioactive molecules, such as phenolic compounds present a high scavenging capacity.17 Therefore, owing mainly to their antioxidant18,19 and anti-inflammatory20 properties, lavender extracts or its essential oils are known to exhibit antidiabetic,21 neuroprotective,22 antiallergic,23 antimicrobial,24 and anticancer25 activities. Recently, we have shown that L. stoechas essential oils (LSEO) protect against reproductive damage and oxidative stress in alloxan-induced diabetic rats.26 Hence, the present study aimed to investigate the hepatoand nephroprotective effects of LSEO against malathioninduced oxidative stress in young male mice as well as the possible mechanism implicated in such protection. MATERIALS AND METHODS Chemicals Malathion (96%), epinephrine, bovine catalase, 2-thiobarbituric acid (TBA), and butylated hydroxytoluene (BHT) were purchased from Sigma Chemicals Co. (Sigma-Aldrich GmbH, Steinheim, Germany). All other chemicals used were of analytical reagent grade. Plant collection L. stoechas aerial parts were collected in March 2010 from the area of Ain-Draham (North-West of Tunisia) and identified by the laboratory of taxonomy in the Faculty of Sciences of Tunis (FST)-Tunisia. The voucher specimens (No. L101) have been deposited with the herbarium of the Higher Institute of Biotechnology of Be´ja and also in the Department of Biological Sciences, Faculty of Science of Tunis, Tunisia. Plant extracts preparation The dried aerial parts were submitted to hydrodistillation for 3 h using a Clevenger-type apparatus. Briefly, the plant was immersed in water and heated to boiling, after which the essential oils were evaporated together with water vapour and finally collected in a condenser. The distillate was isolated and dried over anhydrous sodium sulfate. Animals and treatment Healthy male Swiss Albino mice (Mus musculus) (weighing 10 – 0.3 g, 3 weeks old, and housed six per cage) were purchased from SIPHAT (Tunis, Tunisia) and used in accordance with the Local Ethics Committee of Tunis Uni-

versity for use and care of animals in conformity with the NIH recommendations. The animals were housed in standard cages (40 · 28 · 16 cm) under controlled conditions: 12-h light–12-h dark, 20–22C, food (BADR, Utique, Tunisia) and water are ad libitum. Mice were divided into eight groups of 12 animals each: control, malathion (200 mg/kg b.w.); various doses of LSEO (10, 30, and 50 mg/kg b.w.), malathion + various doses of LSEO. The animals were daily treated by gavage during 30 days with vehicle (control, NaCl 0.9%) or with various doses of LSEO (injection volume was 1 mL/kg b.w.). After 1 hour, mice also received malathion in corn oil (200 mg/kg b.w.) by gavage during 30 days. Group I: served as a negative control and received NaCl (0.9%) and corn oil by gavage during 30 days. Group II: served as a positive control and received NaCl (0.9%) and malathion (200 mg/kg b.w.) by gavage during 30 days. Groups III–V: received various doses of LSEO (10, 30, and 50 mg/kg b.w.) and corn oil during 30 days. Groups VI–VIII: received various doses of LSEO (10, 30, and 50 mg/kg b.w.) and malathion (200 mg/kg b.w.) during 30 days. Twenty-four hours after the last injection, animals were sacrificed and blood was collected in heparinized tubes. After centrifugation at 3000 g for 15 min, plasma was processed for biochemical parameter determinations. The liver and kidney were rapidly excised and homogenized in phosphate-buffered saline (PBS). After centrifugation at 10,000 g for 10 min at 4C, supernatants were used for biochemical determination. Body and reproductive organ weights Initial (weight at start point) and final (weight at end point) body weights were recorded. After decapitation, laparotomy was performed. The liver and kidney were stripped from fatty tissues and blood vessels, blotted, and their absolute weights were determined using precision balance Denver S-603 series. To normalize the data for statistical analysis, organ weights were expressed per 100 g body weight. Functional and metabolic parameters Assessment of liver function. Plasma albumin, bilirubin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), phosphatase alcaline (PAL), acid phosphatase (ACP), lactate dehydrogenase (LDH), and gamma-glutamyl transferase (c-GT) were measured using commercially available diagnostic kits (Biomaghreb, Ariana, Tunisia). Assessment of renal function. Plasma urea, creatinine, and uric acid analyses were performed using commercially available diagnostic kits (Biomaghreb). Metabolic parameters. Plasma total lipids (TL), total cholesterol (TC), low-density lipoprotein-cholesterol (LDL), high-density lipoprotein-cholesterol (HDL), and triglyceride (TG) concentrations were measured using commercially available diagnostic kits supplied by Randox Laboratories (Ardmore, Northern Ireland, United Kingdom).

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LSEO PROTECTION ON MALATHION-INDUCED INJURY Table 1. Sub-Chronic Effects of Lavandula stoechas Essential Oils and Malathion on Body Weight Gain and Relative Organ (Liver and Kidney) Weights in Young Male Mice Control

LSEO-10

LSEO-30

LSEO-50

Malathion

Malathion + LSEO-10

Malathion + LSEO-30

Malathion + LSEO-50

Body weight gain 270.29 – 1.99 278.01 – 5.16 274.92 – 3.62 281.46 – 5.44 185.37 – 5.01a 235.42 – 4.12b 248.68 – 2.37b 264.57 – 4.72b during 30 days (%) Relative liver weight 7.02 – 0.12 6.95 – 0.09 6.96 – 0.11 6.85 – 0.08 8.18 – 0.1a 7.74 – 0.09b 7.35 – 0.11b 7.12 – 0.12b (g/100 g b.w.) Relative kidney weight 1.04 – 0.07 1.01 – 0.06 1.03 – 0.04 1.02 – 0.06 1.42 – 0.03a 1.27 – 0.02b 1.16 – 0.03b 1.06 – 0.02b (g/100 g b.w.) Animals were treated during 30 days with LSEO (10, 30, and 50 mg/kg b.w., p.o.). One hour after essential oils administration, mice were daily exposed to malathion (200 mg/kg b.w., p.o.) or corn oil for the same period. Assays were carried out in triplicate (n = 12). a P < .05 compared to control group (NaCl + corn oil). b P < .05 compared to malathion group (NaCl + malathion). LSEO, Lavandula stoechas essential oils.

Lipid peroxidation Lipid peroxidation was detected by the determination of malondialdehyde (MDA) production by the method of Buege and Aust.27 Briefly, homogenates of the liver and kidney were centrifuged at 1000 g for 10 min at 4C to sediment cell debris. Supernatants were suspended in PBS, pH = 7.4, mixed with BHT-TCA solution (1& BHT dissolved in 0.2 g/mL TCA), centrifuged at 1000 g for 35 min, and finally, mixed with 0.5 N HCl and 120 mM TBA (thiobarbituric acid) in 26 mM Tris and heated in a water bath at 80C for 10 min. After cooling, the absorbance of the resulting chromophore was measured at 532 nm. MDA levels were determined by using an extinction coefficient for the MDA-TBA complex of 1.56 · 105 M - 1cm - 1. The results were expressed as nmol of malondialdehyde per milligram of protein (nmol MDA/mg protein). Thiol groups measurement Total concentration of thiol groups (-SH) was performed according to the Ellman’s method.28 Briefly, aliquots from

the liver and kidney were mixed with 100 lL of 10% SDS and 800 lL of 10 mM phosphate buffer (pH 8) and the absorbance was measured at 412 nm (A0). Then, 100 lL of DTNB were added and incubated at 37C during 60 min. After incubation, the absorbance of the sample was measured at 412 nm (A1). The concentration of thiol groups was calculated from A1 - A0 subtraction using a molar extinction coefficient of 13.6 · 103 M - 1cm - 1. Results were expressed as nmol of thiol groups per mg of protein. Antioxidant enzyme activity assays Liver and kidney SOD activities were determined by using a modified epinephrine assay of Misra and Fridovich.29 At alkaline pH, superoxide anion O2 - causes the autoxidation of epinephrine to adenochrome; while competing with this reaction, SOD decreased the adenochrome formation. One unit of SOD is defined as the amount of the extract that inhibits the rate of adenochrome formation by 50%. The enzyme extract was added in a 2 mL reaction mixture containing 10 lL of bovine catalase (0.4 U/lL), 20 lL of epinephrine (5 mg/mL), and 62.5 mM sodium carbonate/bicarbonate buffer pH 10.2.

Table 2. Subchronic Effects of Lavandula stoechas Essential Oils and Malathion on Plasma Levels of Albumin, Bilirubin, Aspartate Aminotransferase, Alanine Aminotransferase, Phosphatase Alcaline, Acid Phosphatase, Lactate Dehydrogenase, and Gamma-Glutamyl Transferase in Young Male Mice

Albumin (g/dL) Bilirubin (mg/dL) AST (U/L) ALT (U/L) ALP (U/L) ACP (U/L) LDH (U/L) c-GT (U/L)

Control

LSEO-10

LSEO-30

LSEO-50

Malathion

Malathion + LSEO-10

Malathion + LSEO-30

Malathion + LSEO-50

5.46 – 0.29 5.10 – 0.21 130.6 – 2.2 42.4 – 1.5 75.62 – 11.3 37.2 – 0.6 213 – 35.6 2.62 – 0.5

5.24 – 0.31 5.08 – 0.32 128.8 – 3.49 40.4 – 1.77 73.60 – 2.57 38.4 – 0.84 226.5 – 30.07 2.68 – 0.61

5.10 – 0.31 5.01 – 0.32 115.8 – 4.75 39.4 – 2.57 62.60 – 2.62 32.4 – 0.89 215.5 – 23.71 2.21 – 0.43

4.87 – 0.41 4.94 – 0.21 107.8 – 4.749 37.4 – 2.76 50.60 – 2.577 26.4 – 0.65 208.5 – 28.13 2.12 – 0.52

3.62 – 0.36a 6.89 – 0.31a 198.8 – 14.9a 59.2 – 2.63a 142.6 – 28.13a 46.4 – 2.25a 352.1 – 8.81a 4.64 – 0.27a

3.92 – 0.21 6.02 – 0.29 176.6 – 7.38 52.3 – 1.52b 96.8 – 3.91b 41 – 3.86 331.4 – 26 3.98 – 0.52

4.78 – 0.20b 5.76 – 0.29b 163.6 – 7.33b 48.6 – 1.23b 85.6 – 3.69b 39 – 2.26b 298.4 – 35b 3.63 – 0.27b

5.13 – 0.20b 5.26 – 0.29b 142.6 – 7.34b 45.6 – 1.36b 80.2 – 4.07b 36 – 3.29b 261.4 – 32.3b 2.82 – 0.41b

Animals were treated during 30 days with LSEO (10, 30, and 50 mg/kg b.w., p.o.). One hour after essential oils administration, mice were daily exposed to malathion (200 mg/kg b.w., p.o.) or corn oil for the same period. Assays were carried out in triplicate (n = 12). a P < .05 compared to control group (NaCl + corn oil). b P < .05 compared to malathion group (NaCl + malathion). AST, aspartate aminotransferase; ALT, alanine aminotransferase; PAL, phosphatase alcaline; ACP, acid phosphatase; LDH, lactate dehydrogenase; c-GT, gamma-glutamyl transferase.

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SELMI ET AL. Table 3. Subchronic Effects of Lavandula stoechas Essential Oils and Malathion on Plasma Levels of Urea, Creatinine, and Uric Acid in Young Male Mice Control

LSEO-10

LSEO-30

LSEO-50

Malathion + LSEO-10

Malathion

Malathion + LSEO-30

Malathion + LSEO-50

9.21 – 0.23b 7.89 – 0.41b 7.36 – 0.33b Urea (mM) 7.06 – 0.4 7.23 – 0.4 6.82 – 0.36 5.83 – 0.53 9.72 – 0.32a a b Creatinine (lM) 102.83 – 5.36 110.29 – 6.43 102.62 – 3.23 92.17 – 3.86 142 – 6.59 132 – 4.36 122.72 – 3.59 119.3 – 4.63b Uric acid (lM) 288.64 – 14.87 289.86 – 12.43 302.5 – 9.38 346.64 – 14.29 143.17 – 11.42a 176.56 – 13.21 207.64 – 11.66b 274.64 – 17.48b Animals were treated during 30 days with LSEO (10, 30, and 50 mg/kg b.w., p.o.). One hour after essential oils administration, mice were daily exposed to malathion (200 mg/kg b.w., p.o.) or corn oil for the same period. Assays were carried out in triplicate (n = 12). a P < .05 compared to control group (NaCl + corn oil). b P < .05 compared to malathion group (NaCl + malathion).

Changes in absorbance were recorded at 480 nm. Characterization of SOD isoforms was performed using KCN (2 mM), which inhibits Cu/Zn-SOD or H2O2 (5 mM) and affecting both Cu/Zn-SOD and Fe-SOD, whereas Mn-SOD was insensitive to both inhibitors.30 Liver and kidney CAT activities were assayed by measuring the initial rate of H2O2 disappearance at 240 nm.31 The reaction mixture contained 33 mM H2O2 in 50 mM phosphate buffer pH 7.0, and the CAT activity was calculated using the extinction coefficient of 40 mM - 1cm - 1 for H2O2. The GPx activity was measured by the procedure of Flohe´ and Gu¨nzler.32 Briefly, 1 mL of reaction mixture containing 0.2 mL of sample supernatant (liver or kidney), 0.2 mL of phosphate buffer 0.1 M pH 7.4, 0.2 mL of GSH (4 mM), and 0.4 mL of H2O2 (5 mM) was incubated at 37C for 1 min and the reaction stopped by addition of 0.5 mL TCA (0.05 g/ mL). After centrifugation at 1500 g for 5 min, an aliquot (0.2 mL) from the supernatant was mixed with 0.5 mL of phosphate buffer 0.1 M pH 7.4 and 0.5 mL DTNB (10 mM) and absorbance recorded at 412 nm. The GPx activity was expressed as nmol of GSH consumed/min/mg protein. H2O2 determination The liver and kidney H2O2 levels were performed according to Dingeon et al.33 Briefly, the hydrogen peroxide reacts with p-hydroxybenzoic acid and 4-aminoantipyrine in

the presence of peroxidase leading to the formation of quinoneimine, which has a pink color detected at 505 nm. Protein determination The protein concentration was determined according to the Bradford method.34 Serum albumin was used as a standard. Statistical analysis The data were analyzed by one-way analysis of variance (ANOVA) and expressed as mean – standard error of the mean (SEM). The data are representative of 12 independent experiments. All statistical tests were two-tailed, and a P value < 0.05 was considered significant. RESULTS Mortality and macroscopic symptoms of toxicity No mortality was noted until the end of the experiment in control and treated groups. Concerning the liver and kidney, no macroscopic alterations were found. However, cholinergic signs of adverse toxicological effects were observed such as sluggishness, muscular tremors, irregular movements, and abdominal tremble in the malathion-treated group. LSEO administration reversed all adverse cholinergic effects induced by malathion intoxication.

Table 4. Subchronic Effects of Lavandula stoechas Essential Oils and Malathion on Plasma Levels of Total Lipids, Total Cholesterol, Low-Density Lipoprotein-Cholesterol, High-Density Lipoprotein-Cholesterol, and Triglyceride in Young Male Mice Control

LSEO-10

LSEO-30

LSEO-50

Malathion

Malathion + LSEO-10

Malathion + LSEO-30

TL (mg/dL) 278.9 – 7.00 257.23 – 7.41 256.82 – 0.36 231.31 – 10.4 375.4 – 23.45a 352.21 – 0.23 315.89 – 0.41b TC (mg/dL) 80.6 – 7.48 76.29 – 6.40 69.62 – 3.23 63 – 3.51 109.2 – 8.97a 98 – 4.36 84.72 – 3.59b TG (mg/dL) 98.6 – 2.6 93.86 – 2.43 82.5 – 3.38 76.4 – 3.49 124.3 – 3.67a 110.56 – 13. 21b 106.64 – 6.56b HDL (mg/dL) 50.44 – 1.195 58.38 – 3.34 59.43 – 3.64 64.57 – 2.69 31.75 – 3.04a 43.27 – 2.86b 45.18 – 3.63b LDL (mg/dL) 47.98 – 1.13 42.71 – 2.84 39.56 – 2.43 37.18 – 2.30 63.53 – 2.40a 53. 92 – 1.38b 51.09 – 1.99

Malathion + LSEO-50 306.4 – 2.6b 79.8 – 09.52b 101.2 – 7.76b 45.82 – 4.12b 49.63 – 2.91b

Animals were treated during 30 days with LSEO (10, 30, and 50 mg/kg b.w., p.o.). One hour after essential oils administration, mice were daily exposed to malathion (200 mg/kg b.w., p.o.) or corn oil for the same period. Assays were carried out in triplicate (n = 12). a P < .05 compared to control group (NaCl + corn oil). b P < .05 compared to malathion group (NaCl + malathion). TL, total lipids; TC, total cholesterol; LDL, low-density lipoprotein-cholesterol; HDL, high-density lipoprotein-cholesterol; TG, triglyceride.

LSEO PROTECTION ON MALATHION-INDUCED INJURY

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Body and relative organ weights Data from Table 1 showed that malathion-treated mice showed a significant decrease of body weight gain. Malathion intoxication also increased the relative weights of the liver and kidney by 19% and 27%, respectively. LSEO administration significantly and dose dependently protected against malathion-induced body weight decrease as well as liver and kidney relative weights increase. Effects of malathion and LSEO on liver and kidney functions To test the in vivo effect of LSEO on malathion-induced liver injury, mice were treated with various doses of LSEO and malathion during 30 days. As shown in Table 2, malathion-induced hepatotoxicity was shown by a decrease of plasma albumin content as well as a significant increase of plasma bilirubin, AST, ALT, ALP, ACP, LDH, and c-GT levels. Interestingly, when animals were coadministrated with LSEO, the levels of these parameters were practically restored in a dose-dependent manner. We also studied in the present work, the effect of LSEO on malathion-induced renal dysfunction (Table 3). Malathion per se increased plasma creatinine and urea levels when the uric acid content decreased significantly. However, the LSEO treatment significantly and dose dependently protects against malathioninduced renal hemodynamic parameters deregulation. Effects of malathion and LSEO on lipid metabolic parameters Table 4 showed that malathion exposure induced a considerable metabolic disorder, as assessed by a significant increase of TL, TG, TC, and LDL plasma contents as well as a decreased plasma HDL level. Importantly, we showed that LSEO supplementation significantly protects against the disturbance of lipid metabolic parameters induced by malathion intoxication. Effects of malathion and LSEO on lipid peroxidation and thiol groups content We further looked at the effect of malathion and essential oils on oxidative stress conditions. We initially showed that oxidative stress induced by malathion treatment is accompanied by a significant increase of liver and kidney MDA, hydrogen peroxide, as well as a decrease of thiol groups content (Fig. 1). Treatment with LSEO protected against lipoperoxidation, H2O2 increase, as well as (-SH) groups decrease induced by malathion intoxication. Effects of malathion and LSEO on antioxidant enzyme activities We also studied the hepatic and renal antioxidant enzyme activities (Figs. 2 and 3). As expected, malathion per se significantly decreased CAT, GPx, total SOD, Cu/Zn-SOD, and Mn-SOD activities. The subchronic administration of LSEO protected against the depletion of hepatic and renal

FIG. 1. Subchronic effects of Lavandula stoechas essential oils (LSEO) and malathion on malondialdehyde (MDA) (A) and -SH group (B) levels in the liver and kidney of young male mice. Animals were treated during 30 days with LSEO (10, 30, and 50 mg/kg b.w., p.o.). One hour after essential oils administration, mice were daily exposed to malathion (200 mg/kg b.w., p.o.) or corn oil for the same period. Assays were carried out in triplicate (n = 12). aP < .05 compared to control group (NaCl + corn oil) and bP < 0.05 compared to malathion group (NaCl + malathion).

antioxidant enzyme activities induced by malathion intoxication in a dose-dependent manner. ROEO alone had no antioxidant enzyme activities. Effects of malathion and LSEO on antioxidant enzyme activities The effects of malathion and LSE on hydrogen peroxide level are reported in Figure 4. We showed that malathion intoxication significantly increased the liver and kidney H2O2 levels. LSEO administration significantly protected against malathion-induced hydrogen peroxide increase. DISCUSSION In the present work, we studied the protective role of LSEO against malathion-induced injury in the liver and kidney of young male mice as well as the implication of oxidative stress in such protection.

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FIG. 2. Subchronic effects of LSEO and malathion on CAT (A) and glutathione peroxidase (GPx) (B) activities in the liver and kidney of young male mice. Animals were treated during 30 days with LSEO (10, 30, and 50 mg/kg b.w., p.o.). One hour after essential oils administration, mice were daily exposed to malathion (200 mg/kg b.w., p.o.) or corn oil for the same period. Assays were carried out in triplicate (n = 12). aP < .05 compared to control group (NaCl + corn oil) and bP < .05 compared to malathion group (NaCl + malathion).

We initially revealed that malathion exposure (200 mg/kg b.w.) of young male mice during 30 induced a significant decrease of body weight as well as an increase of liver and kidney relative weights. The increased relative weights of liver and kidney were also observed with several cases of OP poisoning, in mice, such as chlorfenvinfos, fenthion, and dimethoate.35 This loss of corporal mass has consequently numerous toxic effects in male mice, such as their feeding ability, and therefore, their reproductive performances.36 LSEO coadministration protects in a dose-dependent manner against malathion-induced body and relative organ weights deregulation. We also showed in the present work that malathion treatment resulted in clear hepatotoxicity as evidenced by a decrease of plasma albumin content as well

as a significant increase of plasma bilirubin, AST, ALT, ALP, ACP, LDH, and c-GT levels, used as indexes of liver injury. On the other hand, malathion administration also induced renal dysfunction as assessed by an increase of plasma creatinine and urea levels when uric acid content decreased significantly. However, LSEO treatment significantly and dose dependently protects against malathioninduced liver injury and renal hemodynamic parameters deregulation. The high level of ALT and AST in the plasma compartment is a central indication of the degree of damage to the liver caused by OP administration.37,38 We suggest that LSEO prevent against Kupffer cell activation by decreasing the formation of inflammatory and fibrogenic mediators. The same mechanism was, in part, proposed by

LSEO PROTECTION ON MALATHION-INDUCED INJURY

FIG. 3. Subchronic effects of LSEO and malathion on total-SOD, Cu/Zn-SOD, Mn-SOD, and Fe-SOD activities in the liver (A) and kidney (B) of young male mice. Animals were treated during 30 days with LSEO (10, 30, and 50 mg/kg b.w., p.o.). One hour after essential oils administration, mice were daily exposed to malathion (200 mg/kg b.w., p.o.) or corn oil for the same period. Assays were carried out in triplicate (n = 12). aP < .05 compared to control group (NaCl + corn oil) and bP < .05 compared to malathion group (NaCl + malathion).

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Zhong et al.39 to explain the protective effect of polyphenols extracted from Camellia sinensis against cholestasisinduced liver fibrosis in rats. Our results also showed that malathion exposure induced an increase in serum HDL as well as a decrease of TL, TC, TG, and LDL levels when compared to the control group. LSEO coadministration protected against all malathioninduced metabolic lipid parameters deregulation. HDL may hasten the removal of cholesterol from the peripheral tissue to the liver for catabolism and excretion. Also, the high level of HDL may compete with LDL receptor sites on arterial smooth muscle cells and thus partially inhibit the uptake and degradation of LDL. Also, HDL could protect LDL against oxidation in vivo, because the lipids in HDL are preferentially oxidized before those in LDL.40 Malathion-induced hepato- and nephrotoxicity was also assessed by an increase in lipoperoxidation, a decrease in SH- group levels, as well as depletion of antioxidant enzyme activities such as CAT, GPx, total SOD, Cu/Zn-SOD, MnSOD, and Fe-SOD. Malathion-induced tissue oxidative stress was widely documented in many organ systems such as the liver,3 kidney,3 heart,41 brain,9 and erythrocytes.9 However, OP exposure can lead to oxidative stress through the generation of ROS42 produced by cellular metabolic activities leading to the reduction of the cellular antioxidant defence capacity.6 Hydroxyl radical (OH) reacts with all biological substances and the most susceptible ones are polyunsaturated fatty acids. Reaction with these cell membrane constituents leads to lipid peroxidation.43 Increased LPO impairs membrane function by decreasing membrane fluidity and changing the activity of membrane-bound enzymes and receptors.43 More importantly, LSEO treatment protected against malathion-induced hepatic and renal oxidative stress. These data fully corroborated all previously reported in vivo and in vitro antioxidant and anti-inflammatory properties of

FIG. 4. Subchronic effects of LSEO and malathion on hydrogen peroxide level in the liver and kidney of young male mice. Animals were treated during 30 days with LSEO (10, 30, and 50 mg/kg b.w., p.o.). One hour after essential oils administration, mice were daily exposed to malathion (200 mg/kg b.w., p.o.) or corn oil for the same period. Assays were carried out in triplicate (n = 12). aP < .05 compared to control group (NaCl + corn oil) and bP < .05 compared to malathion group (NaCl + malathion).

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L. stoechas.21 Malathion-induced oxidative stress as well as liver and kidney dysfunctions have been shown to be attenuated by N-acetylcysteine,44 vitamin C, and vitamin E.45 However, to the best our knowledge, our report is the first one to deal with the LSEO protective effect on malathion-induced oxidative stress in the liver and kidney of young mice. The chemical composition of LSEO using the GC-MS technique has been done in our previous report, which allowed the identification of 22 compounds.21 The principal compounds detected are as follows: d-Fenchone (29.28%), apinene (23.18%), Camphor (15.97%), Camphene (7.83%), Eucapur (3.29%), Limonene (2.71%), Linalool (2.01%), Endobornyl Acetate (1.03%). The essential oils also contained smaller percentages of Tricyclene, Cymene, DeltaCadinene, Selina-3,7(11)-diene. These molecules are the primal source of the antioxidant ability of this plant, by scavenging free radicals as hydroxyl radical (OH), which is the major cause of lipid peroxidation.46 More importantly, our data showed also that LSEO treatment abolished malathion-induced increase in H2O2 levels in the liver and kidney. However, we can now suggest that LSEO exert beneficial effects by scavenging H2O2 leading to antioxidant properties, as recently described by Souli et al.47 Further experiments are needed to assess the effect of LSEO on labile iron levels, known for its implication in the H2O2mediated oxidative injury through its participation in the Fenton pathway.48 CONCLUSION Our data clearly demonstrate that LSEO exert protective effects against malathion-induced injury in the rat liver and kidney owing, in part, to their antioxidant properties.

ACKNOWLEDGMENT The financial support of the Tunisian Ministry of Higher Education and Scientific Research is gratefully acknowledged.

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AUTHOR DISCLOSURE STATEMENT No competing financial interests exist.

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Hepatoprotective and Renoprotective Effects of Lavender (Lavandula stoechas L.) Essential Oils Against Malathion-Induced Oxidative Stress in Young Male Mice.

We aimed in the present study to investigate the hepato- and nephroprotective effects of Lavandula stoechas essential oils (LSEO) against malathion-in...
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