RESEARCH ARTICLE

Protective Effects of Essential Oils as Natural Antioxidants against Hepatotoxicity Induced by Cyclophosphamide in Mice Salah A. Sheweita1*, Lobna S. El-Hosseiny2, Munther A. Nashashibi1 1 Department of Biotechnology, Institute of Graduate Studies and Research, Alexandria University, Alexandria, Egypt, 2 Department of Environmental Studies, Institute of Graduate Studies and Research, Alexandria University, Alexandria, Egypt

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* [email protected]

Abstract OPEN ACCESS Citation: Sheweita SA, El-Hosseiny LS, Nashashibi MA (2016) Protective Effects of Essential Oils as Natural Antioxidants against Hepatotoxicity Induced by Cyclophosphamide in Mice. PLoS ONE 11(11): e0165667. doi:10.1371/journal. pone.0165667 Editor: Hans-Joachim Lehmler, University of Iowa, UNITED STATES Received: July 2, 2016 Accepted: October 14, 2016 Published: November 1, 2016 Copyright: © 2016 Sheweita et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: The author(s) received no specific funding for this work. Competing Interests: The authors have declared that no competing interests exist. Abbreviations: CP, Cyclophosphamide; CYP, Cytochrome P450; GST, glutathione S-transferase; GPx, glutathione peroxidase; GR, glutathione reductase; CAT, catalase; SOD, superoxide

Clinical application of cyclophosphamide (CP) as an anticancer drug is often limited due to its toxicity. CP is metabolized mainly in the liver by cytochrome P450 system into acrolein which is the proximate toxic metabolite. Many different natural antioxidants were found to alleviate the toxic effects of various toxic agents via different mechanisms. Therefore, the present study aimed at investigating the role of essential oils extracted from fennel, cumin and clove as natural antioxidants in the alleviation of hepatotoxicity induced by CP through assessment of hepatotoxicity biomarkers (AST, ALT, ALP), histopathology of liver tissues as well as other biochemical parameters involved in the metabolism of CP. The data of the present study showed that treatment of male mice with cyclophosphamide (2.5 mg/Kg BW) as repeated dose for 28 consecutive days was found to induce hepatotoxicity through the elevation in the activities of AST, ALT, and ALP. Combined administration of any of these oils with CP to mice partially normalized the altered hepatic biochemical markers caused by CP, whereas administration of fennel, clove or cumin essential oils alone couldn’t change liver function indices. Moreover, CP caused histological changes in livers of mice including swelling and dilation in sinusoidal space, inflammation in portal tract and hepatocytes, as well as, hyperplasia in Kuppfer cells. However, co-administration of any of the essential oils with CP alleviated to some extent the changes caused by CP but not as the normal liver. CP was also found to induce free radical levels (measured as thiobarbituric acid reactive substances) and inhibited the activities of superoxide dismutase, glutathione reductase, and catalase as well as activities and protein expressions of both glutathione S-transferase (GSTπ) and glutathione peroxidase. Essential oils restored changes in activities of antioxidant enzymes (SOD, CAT, GR, GST, and GPx) caused by CP to their normal levels compared to control group. In addition, treatment of mice with CP was found to induce the protein expression of CYP 3A4, 2B1/2, 2C6, 2C23. Moreover, the present study showed that essential oils reduced the expression of CYPs 2E1, 3A4 but could not restore the expression of CYP 2C6 and 2C23 compared to CP-treated mice. Interestingly, pretreatment of mice with essential oil of clove was found to restore activities of DMN-dI, AHH, and ECOD which were induced by CP to their normal control levels. It is concluded that EOs

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dismutase; TBARS, thiobarbituric acid reactive substances; ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, Alkaline phosphatase; GSH, glutathione; GSSG, oxidized gluathione; MDA, malondialdehyde; GC-MS, Gas chromatography-Mass spectroscopy (GC-MS); EO, Essential oil; DMN-dI, dimethylnitrosamine Ndemethylase I; ECOD, 7-ethoxycoumarin-Odeethylase; BSA, bovine serum albumin.

showed a marked hepatoprotective effect against hepatotoxicity induced by CP. In addition, co-administration of CP with any of these oils might be used as a new strategy for cancer treatment to alleviate the hepatotoxicity induced by CP.

Introduction For more than 50 years cyclophosphamide (CP) has been widely used to treat various forms of cancers, including lymphoma, breast cancer and leukemia [1]. However, clinical application of CP is often restricted due to its deleterious side effects [2], especially hepatotoxicity [3]. The toxic effects of CP are mainly due to the generation of two major metabolites namely phosphoramide mustard which is the antineoplastic moiety and acrolein metabolite which is the most toxic agent. These metabolites are generated by cytochrome P450 isozymes including CYP 3A4, 2B6, 2C9, and 2C19 [4]. Acrolein is a highly reactive α, β- unsaturated aldehyde, and was identified as the initiator of lipid peroxidation. This reactivity is the main reason of the cytotoxicity in all cells exposed to acrolein [5] which limits using CP in clinical practice. CPinduced oxidative stress through the generation of free radicals leading to biochemical and physiological disturbances in animal models [6]. Protection of cells from the lethal effects of toxic compounds was observed due to the presence of abundant amounts of glutathione which is an important determinant of cellular sensitivity to various drugs and other toxic compounds [7,8]. Depletion of GSH levels in the cells could promote tumor development in different animal species [9]. Supporting this suggestion, depletion of GSH level and inhibition of GST activity were found to reduce the covalent binding of the ultimate metabolites of both aflatoxin B1 and benzo[a]pyrene, with DNA [10], and decreasing of hepatocarcinogenesis caused by these compounds was correlated with low the level of DNA adducts [10]. Several studies suggested that dietary antioxidants supplementation can reduce the development of adverse effects associated with anticancer drugs including CP [11,12]. It has been found that some plants contain a wide variety of antioxidant phytochemicals or bioactive molecules which can neutralize free radicals and decreased the oxidative stress that plays a critical role in the incidence of adverse toxic effects associated with chemotherapies [13]. Fennel (Foeniculum vulgare Miller) is a medicinal and aromatic plant with a diverse pharmacological spectrum and of considerable importance in food industries. Previous studies have shown that various extracts of fennel possess a range of pharmacological actions ranging from antispasmodic and anxiolytic to cytoprotective and anti-inflammatory effects due to the presence of terpenes and terpenoids [14]. Cumin (Cuminum Cyminum L.) is belonging to the family of Apiaceous. Cumin seeds are used in folklore therapy [15,16]. Clove (Syzygiumaromaticum L.) is one of the most valuable spices that have been used for centuries as a food preservative and for many medicinal purposes including antimicrobial, anti-inflammatory, antioxidant and anticancer activities [17]. Phenolic compounds (eugenol, eugenol acetate, and gallic acid) which possess pharmaceutical, cosmetic, food and agricultural applications are the main constituent of clove oil. Recently, the protective effects of Foeniculum vulgare (fennel) essential oil (FEO) and Syzygium cumini extract against genotoxicity and oxidative stress induced by CP was investigated [18,19]. It has been shown that CP produced a significant increase in the average percentage of aberrant metaphases and chromosomal aberrations, excluding gap and micronuclei formation in polychromatic erythrocytes, produced cytotoxicity in mouse bone marrow cells [18,19]. CP

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also markedly inhibited the activities of SOD, CAT, and GSH and increased MDA content. Pretreatments with FEO and Syzygium cumini extract significantly inhibited the frequencies of aberrant metaphases, chromosomal abreactions, micronuclei formation, and cytotoxicity in mouse bone marrow cells induced by CP and antagonized the reduction of CP-induced SOD, CAT, and GSH activities and inhibited increased MDA content in the liver of mice [18,19]. In addition, FEO and Syzygium cumini extract inhibited genotoxicity and oxidative stress induced by CP (Tripathi et al., 2013a&b). In another study, the lyophilized ethanolic leaf of Eugenia dysenterica exhibited protection against CP induced genotoxic and cytotoxic actions at all doses tested [20]. CP-induced hepatotoxicity is critical issues during the clinical application of CP. Oxidative stress, caused by the active metabolites of CP, exhibited deleterious effects on hepatocellular membranes and other tissues. Therefore, finding a strategy to reduce the oxidative stress might be a key, at least in part, to alleviate the CP-induced hepatotoxicity. To the best of our knowledge, no previous study investigating the protective effects of essential oils of cumin and/or clove either alone or in combination with CP has been conducted. However, the influence of Foeniculum vulgare (fennel) essential oil either alone or in combination with CP was tested in the livers of mice on the activities of antioxidant enzymes but not on the protein expression of CYP450 [19]. Therefore, the present study aimed at investigating the extracted essential oils from fennel, clove and cumin and their roles in alleviating the toxic effects exerted by CP administration to mice through determination of activities and protein expressions of cytochrome P450 isozymes. In addition, the present study showed the influence of essential oils either alone or in combination with CP on levels of free radicals and activities and protein expressions of antioxidant enzymes including glutathione peroxidase and glutathione S-transferase after treatment of mice.

Materials and Methods Chemicals Cyclophosphamide was obtained from Baxter Oncology, Germany. Nicotinamide adenine dinucleotide phosphate (NADP) sodium salt, nicotinamide adenine dinucleotide phosphate reduced (NADPH) sodium salt, cumene hydroperoxide, 1-chloro-2,4-dinitrobenzene (CDNB) and all other chemicals were purchased from Sigma Chemical Co., UK. Anti-cytochrome P450 3A4, 2B1/2, 2C6, 2C23, 2E1 and NADPH-cytochrome c reductase were purchased from ABCAM, UK. Cytochrome P450 Western blotting detection kits were purchased from Amersham, UK. Diagnostic kits for aspartate aminotransferase, alanine amino transferase, and alkaline phosphatase were obtained Spectrum Diagnostics, Hannover, Germany. Dimethylsulfoxide (DMSO) pharmaceutical grade (C2H6OS, purity 99%) was obtained from Gaylord Chemical Co., LA, USA. All other agents and solvents were of analytical grade.

Essential oil extraction and characterization by GC-MS Dried seeds of fennel, cumin and flower buds of clove authenticated by Prof. Salama El Darier, Professor of Plant Ecology, Botany and Microbiology Department, Faculty of Science, Alexandria University were used for the extraction of essential oils. 100 gm of the respective dried seeds or flower buds were subjected to hydrodistillation for 4 hr using Clevenger type apparatus. The distillate oil was collected, dried over anhydrous sodium sulfate and stored in dark sealed vials at 4°C prior to characterization of their chemical composition. Compositional analysis of the extracted essential oils was assessed using Thermo scientific GC-MS version 5 system. Qualitative identification of the oil constituents was carried out on the basis of their retention indices and matching their mass spectral fragmentation patterns

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with NIST library data base [21]. Quantitative analysis of the oil components expressed as relative percentage of peak area, was estimated using peak area normalization measurements.

Animal treatments Sixty-four Swiss male albino mice, weighing 25±2 g were obtained from the animal house of the Faculty of Medicine, Alexandria University. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the College of Medicine, Alexandria University. The protocol was approved by the committee of postgraduate studies & research on the Ethics of Animal Experiments of the University of Alexandria. All surgery was performed under diethyl ether anesthesia, and all efforts were made to minimize suffering. Mice were housed in a stainless steel wire bottom cages placed in a wellventilated animal house, maintained for one week for acclimatization period on food and water ad libitum, and subjected to the natural photoperiod of 12 hours light:dark cycle. The animals were assigned into 8 groups comprising 8 mice/cage in each group and fed the same diet throughout the experimental period (28 days). Group I served as the control and received 0.1 ml of DMSO orally. Group II (CP) was orally administered with the aqueous solution of CP at a dose of 2.5 mg/Kg BW. Groups III, IV and V were orally treated with the essential oils of fennel, cumin and clove dissolved in DMSO at doses of 1/50 their LD50 corresponding to 0.12 ml/ Kg BW, 0.10 ml/Kg BW and 0.106 ml/Kg BW respectively. Groups VI, VII, and VII were administered orally by a combination of CP with fennel, cumin or clove essential oils at the same doses scheduled in groups II, III, IV and V. At the end of the experimental period, mice were starved overnight and then sacrificed. Blood and liver samples were collected. Serum was obtained by centrifugation of coagulated blood at 3000 xg for 5 min. Livers were washed with cold phosphate buffer saline and the S9 fraction was prepared by homogenizing the livers in 33% W/V of 0.1 M phosphate buffered solution (pH 7.4) and centrifuging at 10000 xg for 20 min. Liver microsomal fractions were prepared by centrifugation of the S9 fraction supernatant at 105,000 xg for 1 hour at 4°C, then the microsomal pellets were suspended in 0.1 M phosphate buffer (pH 7.4) and stored in aliquots at -80°C [22].

Biochemical analyses Activities of biomarker enzymes for hepatocellular damage, including alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) were determined in serum of different groups according to the well-established spectrophotometric methods according to the manuals supplied with the commercial kits obtained from Spectrum Diagnostics, Hannover, Germany. A substrate concentration of 4 mM dimethylnitrosamine, which represents the saturation level for dimethylnitrosamine N-demethylase I (DMN-dI) activity, was used [23,24]. Also, the microsomal 7-ethoxycoumarin-O-deethylase (ECOD) activity was measured as described by Greenle and Poland, 1978 [25] and the intensity of 7-hydroxy-coumarin was measured fluorometrically at excitation and emission wavelengths of 338 and 458 nm respectively. NADPHcytochrome c reductase activity in hepatic microsomal fraction was assayed by measuring the reduction of oxidized cytochrome cat 550nm using an extinction coefficient of 21 mM-1 Cm-1 [26]. The activity of GPx was determined in the S9 fraction of cell homogenates according to the method of Chiu et al., 1976 [27]. The principle of determination is based on the decrease in absorbance of NADPH at 340 nm. The specific activity was expressed in terms of the amount of enzyme that oxidizes 1 μmol of NADPH per min. Glutathione reductase activity in hepatic

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S9 fraction was monitored by the oxidation of NADPH in the presence of GSSG according to the method of David and Richard, 1983 [28]. Glutathione-S-transferase activity was assayed according to the method of Habig et al., 1974 [29], which is based on the measurement of 1-chloro-2,4-dinitrobenzene (CDNB) conjugate formed at 340 nm. A molar extinction coefficient of 9.6 mM-1 Cm-1 was used for the calculation of GST activity. One μmole of CDNB conjugate/mg protein/min is expressed as a unit/mg protein. Catalase activity was determined in the hepatic S9 fraction according to the method of Beers and Sizer, 1952 [30], which is based on decreasing in absorbance of H2O2 solution decomposed by the catalase enzyme (assay kit was obtained from Randox Laboratory, UK). The quantity of H2O2 was measured at 240 nm and catalase activity was expressed as U/mg protein. Superoxide dismutase (SOD) was assayed according to the method of Misra and Fridovich, 1972 [31]. SOD quantitation was dependent on the generation of superoxide radicals by xanthine and xanthine oxidase, which react with nitro blue tetrazolium to form the formazin dye. SOD activity was measured at 560 nm and expressed as U/mg protein. The protein content was measured in both S9 fraction and microsomal pellets according to the method of Lowry et al., 1950 [32] using bovine serum albumin as standard. Quantitative measurement of lipid peroxidation was performed in cell homogenates according to the methods of Tappel and Zalkin, 1959 [33], which is based on the formation of thiobarbituric acid reactive substances (TBARS) and expressed as the extent of malondialdehyde (MDA) production. TBARS was expressed as μmole MDA per gram tissue. Glutathione level was measured in the S9 fractions according to the method of Mitchell et al., 1973 [34], after protein precipitation with sulfosalicylic acid. After centrifugation at 3000 xg for 20 min, one ml of clear supernatant was added to bis-(3-carboxy-4-nitrophenyl)-disulphide for color development. The developed color solution was spectrophotometrically at 412 nm. Glutathione levels were expressed as μmole GSH per gram tissue.

Western Immunoblotting From each pooled group (10 mice each), forty microgram of the S9 and the microsomal fractions were mixed with the sample application buffer (SAB), then boiled for 3 minutes and loaded on a 10% SDS-polyacrylamide gel. After electrophoresis, proteins were transferred to nitrocellulose membranes using a semidry trans blotter. Each membrane was blocked in 0.5% BSA for 2 hours with gentle shaking at room temperature prior to antibody incubations. Primary antibodies of GSTπ, glutathione peroxidase, NADPH-cytochrome c reductase, CYP2E1, CYP3A4, CYP2B1/2, CYP2C23 and CYP2C6 in 0.5% bovine serum albumin were used in the present study. The secondary antibody (anti-mouse conjugated-peroxidase) was used after incubation with the membrane for a minimum of 30 minutes at room temperature. Signals of immune-reactive proteins were detected using X-ray films according to the manufacturer's instructions [35]. The band intensity was measured using quantity one software program.

Histological examination of the liver Formalin fixed paraffin embedded hepatic tissues were sectioned and stained routinely with hematoxylin and eosin (H&E). Hepatic damage in H&E-stained sections was examined from a digital image captured at 200x magnification [36].

Statistical Analysis Data presented in the study were shown as mean ± standard error. Mean of different treatment groups were tested for significance using one-way analysis of variance (ANOVA) and were

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compared using posthoc Duncan's Multiple Range Test (DMRT). Difference were considered significant at P

Protective Effects of Essential Oils as Natural Antioxidants against Hepatotoxicity Induced by Cyclophosphamide in Mice.

Clinical application of cyclophosphamide (CP) as an anticancer drug is often limited due to its toxicity. CP is metabolized mainly in the liver by cyt...
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