http://informahealthcare.com/phb ISSN 1388-0209 print/ISSN 1744-5116 online Editor-in-Chief: John M. Pezzuto Pharm Biol, 2015; 53(1): 92–97 ! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2014.910674
ORIGINAL ARTICLE
Genoprotective effects of Origanum vulgare ethanolic extract against cyclophosphamide-induced genotoxicity in mouse bone marrow cells
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Emran Habibi1,2, Mohammad Shokrzadeh1,3, Amirhossein Ahmadi1, Aroona Chabra4, Farshad Naghshvar5, and Razieh Keshavarz-Maleki3 1
Pharmaceutical Sciences Research Center, Faculty of Pharmacy, 2Department of Pharmacognosy, Faculty of Pharmacy, 3Department of Toxicology and Pharmacology, Faculty of Pharmacy, 4Student Research Committee, Faculty of Pharmacy, and 5Department of Pathology, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran Abstract
Keywords
Context: Cyclophosphamide (CP), an alkylating chemotherapeutic agent, can bind DNA, causing chromosome breaks, micronucleus (Mn) formation, and cell death. Because Origanum vulgare L. (Lamiaceae) has antioxidative properties, it might protect against DNA damage. Objective: The genoprotective effect of O. vulgare ethanolic extract against CP-induced genotoxicity in mouse bone marrow cells was evaluated using a Mn assay. Materials and methods: Mice were pre-treated with aerial parts of O. vulgare ethanolic extract at different doses (50, 100, 200, or 400 mg/kg) for 7 d. One hour after the last administration of O. vulgare, animals were injected with CP at 200 mg/kg. After 24 h, the bone marrow cells of both femurs were flushed and the frequency of MnPCEs was evaluated to measure the chromosomal damages. In addition, the number of PCEs per 1000 NCEs in each animal was recorded to evaluate the bone-marrow suppression; mitotic activity was calculated as [PCE/(PCE + NCE)] 100 to assess the cell division. Results: At 400 mg/kg, O. vulgare displayed its maximum protective effect, reduced the number of MnPCEs from 10.52 ± 1.07 for CP group to 2.17 ± 0.26 and completely normalized the mitotic activity (p50.001). Origanum vulgare also led to significant proliferation and hypercellularity of immature myeloid elements after the mice were treated with CP, mitigating the bone marrow suppression. Discussion and conclusion: Origanum vulgare ethanolic extract exerts a potent genoprotective effect against CP-induced genotoxicity in mice bone marrow, which might be possibly due to the scavenging of free radicals during oxidative stress conditions.
Antioxidant activity, bone marrow suppression, DNA damage, micronucleus assay
Introduction Cyclophosphamide [N,N-bis(2-chloroethyl)tetrahydro-2H1,3,2-oxazaphosphorin-2-amine-2-oxide, CP] is an oxazophosphorine derivative of nitrogen mustard and is commonly used during cancer chemotherapy. The tumor cell-killing activity of CP is primarily attributed to DNA alkylation. This drug also exhibits significant immunosuppressive activity and is used to treat autoimmune diseases as well as for renal and bone marrow transplantations (Dollery, 1999). The chronic carcinogenic side-effects of alkylating agents create concern during their use in combination drug therapies. Numerous studies have demonstrated the mutagenic effects of CP in both humans and animals (De Ridder et al., 1998; Ember et al., 1995). The chemically reactive metabolic products of CP induce cytotoxicity by alkylating DNA and protein as well as Correspondence: Amirhossein Ahmadi, 18 kilometer of Farah Abad Road, Pharmaceutical Sciences Research Center, Faculty of Pharmacy, Mazandaran University of Medical Sciences, P. Box: 48175-861, Sari, Iran. Tel/Fax: +98 151 3543084. E-mail: Amirhossein_pharma@ yahoo.com
History Received 6 September 2013 Revised 28 March 2014 Accepted 28 March 2014 Published online 2 2 2
by cross-linking DNA (Hales, 1982). The antineoplastic and toxic effects of CP, such as apoptosis, necrosis, and oncosis, are linked with two active metabolites: acrolein and phosphoramide (Kern & Kehrer, 2002). CP produces carbonium ions that react with the electron-rich sites on nucleic acids and proteins, triggering gene mutations, DNA-strand breaks, chromosome aberrations (CA), micronuclei induction, and the production of ROS. The high reactivity of free radicals causes cellular damage using various mechanisms (Bendich, 1990). The most deleterious effects of free radicals lead to DNA damage that causes cancer (Richter et al., 1988). The genotoxic effects of CP have been evaluated in in vivo and in vitro studies (Hartmann et al., 1995). Recently, the search for compounds from plants capable of minimizing the toxicity chemotherapy-induced without compromising antineoplastic activity has received great interest (Pratheeshkumar & Kuttan, 2010). Epidemiological studies have shown the intake of phenolic foods is associated with protection from various diseases (Morton et al., 2000). These phenolic compounds display tremendous antioxidant and chemoprotective properties in vivo (Zhao et al., 2004).
Genoprotective effects of O. vulgare
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DOI: 10.3109/13880209.2014.910674
Natural products have exerted protective effects against genotoxicity induced by CP in mouse bone marrow cells when these compounds were administered before CP treatment. Antioxidant activity might explain the chemoprotective effects of these natural products (Ahmadi et al., 2008; Hosseinimehr et al., 2010). Origanum vulgare L. (Lamiaceae), also known as oregano, is a flavoring herb widely used around the world. Origanum vulgare is an aromatic plant with a wide distribution throughout Asia, particularly in Iran. It is used to cure respiratory diseases, hypoglycemic disease, and leukemia (Sheibani et al., 2010). Because of its antioxidant properties, oregano could become a helpful agent for the treatment of cancer, heart disease, and high blood pressure (Pirigharnaei et al., 2011). The results of various studies indicated that oregano’s antioxidant effects might be a result of the dominant constituents, including carvacrol and thymol, present in its essential oil (Lagouri et al., 1993). Origanum’s high antioxidant activity seems to arise from the phenolic –OH groups present in the plant’s flavonoid and phenolic compounds, which donate hydrogen atoms to the peroxy radicals produced during the first step of lipid oxidation (Roofchaee et al., 2011). Because it has been used extensively as an additive agent and is known to be safe, this study was undertaken to assess the effects of O. vulgare ethanolic extract against the genotoxicity induced by CP in mouse bone marrow cells using the Mn assay. Histological examination of the bone marrow was also used for to observe the possible mitigating effects of the O. vulgare ethanolic extract against the myelosuppressive effect of CP and bone marrow suppression.
Materials and methods Preparation of extracts Dried aerial parts of O. vulgare powder were purchased from the Giah Essence Phyto-Pharmaceutical Co., Golestan Province, Iran. Three hundred gram samples of the dried plant powder were extracted with 3000 ml of ethanol (75%) for 72 h. After evaporating the solvent under vacuum at a temperature below 50 C, 35 g of dried powder was obtained. Animals Male Naval Medical Research Institute (NMRI) mice weighing 22 ± 3 g were obtained from the Pasteur Institute of Iran (Amol, Iran). The mice were housed in the university animal facility and were maintained under a controlled 12 h light/dark cycle at 23 ± 1 C. The animals were acclimated for 1 week before the study and were provided with standard food pellets and water ad libitum. All of the animals were cared for according to the ‘‘Care and Use of Laboratory Animals’’ at the Mazandaran University of Medical Sciences. The protocol for the study was approved by the Research Committee of Mazandaran University of Medical Sciences in Sari, Iran. Experimental treatment For the Mn assay, the animals were divided into seven groups (n ¼ 5 for each group): the negative control group,
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administered distilled water (10 ml/kg b.w.) via intraperitoneal (i.p.) injection for 7 d; the positive control group, administered a single toxic CP dose (200 mg/kg b.w., i.p.) in distilled water (10 ml/kg b.w.) at seventh days of experiment; OV (O. vulgare) 50, 100, 200, and 400 plus CP groups, administered increasing doses of O. vulgare ethanolic extract (50, 100, 200, or 400 mg/kg b.w., i.p., respectively) in distilled water (10 ml/kg b.w.) each day for 7 consecutive days, which was followed by a single i.p. CP dose 1 h after the last O. vulgare ethanolic extract administration; and OV 400 group, administered only O. vulgare ethanolic extract (400 mg/kg b.w. by i.p. injection) in distilled water (10 ml/kg b.w.) once per day for 7 d. Mn assay The Mn test was performed as previously described (Ahmadi et al., 2008; Hosseinimehr et al., 2010). The bone marrow Mn test is a well-known in vivo assay used to assess genotoxicity and DNA damage in animals, such as mice and rats. The number of MnPCEs is increased in rodent bone marrow cells exposed to hazardous chemical and chromosome-damaging agents. A Mn is round and is 1/20th–1/5th the size of that in an erythrocyte. The ratio of PCE to NCE in bone marrow preparations is useful for estimating any perturbations in hematopoiesis caused by the treatment in exposed animals (Gollapudi & McFadden, 1995; Schmid, 1975). The femurs from the animals were used to estimate the Mn frequency and mitotic activity. Mice were sacrificed by cervical dislocation 24 h after CP injection. The bone marrow of both femurs was removed as a fine suspension in a centrifuge tube with FCS. The cells were dispersed by gentle pipetting and collected by centrifuge at 1500 rpm for 10 min. The cell pellet was resuspended in a drop of FCS, and smears were prepared. The slides were coded to avoid any observed bias. After 48 h of air drying, the smears were stained with May-Grunwald/Giemsa. For each experimental point, five mice were used, and 5000 PCEs were scored blindly and microscopically to determine the percentage of MnPCE. In addition, the number of PCEs per 1000 NCEs in each animal was recorded to evaluate the bone-marrow suppression; mitotic activity was calculated as [PCE/(PCE + NCE)] 100 to assess the cell division. Histology of bone marrow For the histological examination of the myeloid hyperplasia in the bone marrow, mice were pretreated with 200 and 400 mg/kg of O. vulgare ethanolic extract solutions for 7 d before CP (200 mg/kg b.w.) was administered 1 h after the last injection of O. vulgare ethanolic extract. Both femurs were removed from the mice; the mice were killed by cervical dislocation 24 h after CP administration. The femurs were immersed in 10% formalin; the bones were decalcified before being processed using a microtome to generate micron scale slides. Routine H & E staining was performed on 6 mm paraffin sections, and the slides were evaluated under a light microscope. The slides were photographed using a camera microscope (Olympus, Orinpasu Kabushiki-gaisha, Japan) at 400 magnification.
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Statistical analysis The data are presented as the mean ± standard deviation (SD). ANOVA test with post hoc Tukey test were used for the comparison of Mn data. A p value below 0.05 was considered statistically significant.
Results
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Bone marrow Mn assay The effect of increasing doses of O. vulgare ethanolic extract on the MnPCE frequency in bone marrow cells 24 h after CP injection is shown in Figure 1. The frequency of Mn significantly increased in the mice treated with CP compared with the control group (p50.001). In the mice pretreated with three doses of O. vulgare ethanolic extract, the MnPCE frequency induced by CP was significantly decreased compared with those treated with only CP (p50.01–p50.001). The MnPCE frequency was lower in the O. vulgare ethanolic extract + CP groups by factors of 2.33, 4.28, and 4.85 for the 100, 200, and 400 mg/kg doses, respectively, compared with the CP treated group. The data showed that the O. vulgare ethanolic extract suppress the activity of CP on the clastogenic effects. Moreover, the mitotic activity (%PCE) in CP-treated Figure 1. Frequency of MnPCE in the bone marrow cells of mice treated with the O. vulgare ethanolic extract and/or CP (200 mg/kg). OV, Origanum vulgare; CP, cyclophosphamide; MnPCE, micronucleated polychromatic erythrocyte. (a) p50.001; CP versus control. (b) p50.001; (OV 200 mg + CP) and (OV 400 mg + CP) versus CP-treated group. (c) p50.01; (OV 100 mg + CP) versus CP-treated group. (d) p40.05; (OV 400 mg) versus control. Values are presented as mean ± SD for each group of five mice. The data were analyzed using ANOVA test with the post hoc Tukey test.
Figure 2. Frequency of mitotic activity (%PCE) in the bone marrow cells of mice treated with the O. vulgare ethanolic extract and/or CP (200 mg/kg). OV, Origanum vulgare; CP, cyclophosphamide; PCE, polychromatic erythrocyte; NCE, normochromatic erythrocyte. (a) p50.001; CP versus control. (b) p50.001; (OV 200 mg + CP) and (OV 400 mg + CP) versus CP-treated group. (c) p50.01; (OV 100 mg + CP) versus CP-treated group. (d) p40.05; (OV 400 mg) versus control. Values are presented as mean ± SD for each group of 5 mice. The data were analyzed using ANOVA test with the post hoc Tukey test.
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mice showed a pronounced cytotoxic effect on bone marrow proliferation, and this behavior was significantly decreased in the mouse bone marrow after the CP treatment (p50.001). Treating the mice with O. vulgare ethanolic extract arrested the CP-induced decline in mitotic activity (%PCE) (Figure 2). Origanum vulgare ethanolic extract treatment at 200, or 400 mg/kg completely prevented the cytotoxicity induced by CP in the mouse bone marrow, increasing mitotic activity (%PCE) by increasing the bone marrow proliferation (p50.001). Origanum vulgare ethanolic extract alone did not cause any genotoxicity in the mice bone marrow cells at high dose of 400 mg/kg (p40.05) (Figure 2). Histology of bone marrow Figure 3 presents myeloid hypoplasia and hemorrhage in a representative femur 24 h after CP administration (200 mg/ kg). Figure 4 presents myeloid hyperplasia and hemorrhage reduction induced by administering the O. vulgare ethanolic for 7 consecutive days) before CP treatment. The histological examinations of the bone marrow showed that administering CP induced myelosuppressive effects. Administering 200 or 400 mg/kg of O. vulgare ethanolic extract led to marked proliferation and hypercellularity in the immature myeloid
DOI: 10.3109/13880209.2014.910674
elements after CP treatment, in addition to mitigating bone marrow suppression and hemorrhage reduction. The relative increase in the proportion of myeloid to erythroid precursors after O. vulgare ethanolic extract administration is notable in figures.
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Discussion The active metabolites of CP are phosphoramide and acrolein; these compounds slow the growth of cancer cells because they interfere with cellular DNA. Hydroxy–cyclophosphamide and deschloroethyl–cyclophosphamide are the two intermediate compounds that lead to the formation of the agent responsible the mutagenic effect of CP, phosphoramide. Phosphoramide induces cross-linking and strand lesions in DNA (Hengstler et al., 1997). We previously reported that CP induced DNA damage and genotoxicity in mouse bone marrow cells (Ahmadi et al., 2008; Shokrzadeh et al., 2014). DNA damage is critical for the initiation and subsequent promotion of carcinogenesis induced by different genotoxicants, as indicated by the experimental evidence. Errors in DNA molecules cause chromosomal aberrations. Mn is wellcharacterized biomarkers for structural and numerical chromosomal damage. The Mn in young erythrocytes arises
Figure 3. Myeloid hypoplasia and hemorrhage in a femur 24 h after 200 mg/kg CP administration. CP induced myelosuppressive effects (hematoxylin and eosin-stained paraffin sections; H&E 400).
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mainly from chromosomal fragments that are not incorporated into the daughter nuclei during the cell division of the erythropoietic blast cells (Salamone & Heddle, 1983). The decreased PCE:NCE ratio induces the bone marrow cytotoxicity (Al-Majed et al., 2006). CP treatment is cytotoxic and is highly clastogenic in nature. In the present study, CP induced Mn formation because it decreased the PCE:NCE ratio; O. vulgare ethanolic extract effectively restored this PCE:NCE ratio back to normal levels. CP also helps to generate DNA strand breaks through the interaction of acrolein with DNA, causing DNA disintegration or smearing due to necrosis and MnPCEs formation. Prophylactic treatments of O. vulgare ethanolic extract significantly and dose dependently restore these hallmarks of genotoxicity to normal levels. Although the exact mechanism of the chemoprotective effect is unknown, free-radical scavenging is apparently responsible for the inhibitory effects of herbal extract and natural compounds on the clastogenic activity induced by genotoxic agents (Hosseinimehr et al., 2010). We recently showed that melatonin, a natural pineal secretory product, has a potent chemoprotective effect against the genotoxicity induced by diazinon in human blood lymphocyte cells, and this protective effect might be the results of free radicalscavenging properties (Karimian et al., 2013). Natural compounds, including flavonoids and phenolic compounds, may scavenge the free radicals, such as hydroxyl radicals, generated by chemical hazardous agents. Increased intracellular levels of reactive oxygen species are frequently referred to as oxidative stress; this state represents a potentially toxic insult that interacts with macromolecules, inducing DNA damage (Hosseinimehr et al., 2011). Another recent study also provided evidence that Citrullus colocynthis (L.) Schrad. (Cucurbitaceae) fruits extract pretreatment attenuates CPinduced oxidative stress and the subsequent DNA damage in mice. It had a potent anticlastogenic effect against the genotoxicity induced by CP in mice bone marrow cells, which might be due to the scavenging of free radicals and inhibition of lipid proxidation of the plant (Shokrzadeh et al., 2013). We also showed that O. vulgare ethanolic extract with high amount of flavonoids and phenolic compounds protects human lymphocytes against genotoxicity induced by internal irradiation. The results suggested that O. vulgare ethanolic
Figure 4. Myeloid hyperplasia and hemorrhage reduction induced by administering the O. vulgare extract (200 mg/kg; right and 400 mg/kg; left for 7 consecutive days) before CP treatment. Origanum vulgare led to marked proliferation and hypercellularity of immature myeloid elements after the mice were treated with CP, the bone marrow suppression was mitigated and the hemorrhage was reduced. The relative increase in the proportion of myeloid to erythroid precursors after O. vulgare administration is notable (hematoxylin and eosin-stained paraffin sections; H&E 400).
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extract acts effectively as a free-radical scavenger and provides concentration-dependent radioprotection and protective effect against DNA damage (Arami et al., 2013). Origanum vulgare L. is the only species of the Origanum genus that grows in the wild of Iran (Zargari, 1997). Several studies have shown that O. vulgare have a high amount of flavonoids and phenolic compounds. The major aqueous constituents of oregano are rosmarinic acid, eriocitrin, luteolin-7-O-glucoside, apigenin-7-O-glucoside, origanol A and B, and ursolic acid (Sheibani et al., 2010). Rosmarinic acid and origanol A and B, which are the most abundant components of the aqueous extract of oregano, have antioxidative activities (Kulisic et al., 2007; Matsuura et al., 2003). Previous studies have reported that the essential oil of O. vulgare has antioxidant capacity, which has been linked to components such as thymol, carvacrol, d-terpinene, and p-cymene (Halici et al., 2005; Odabasoglu et al., 2004; Russo et al., 2002). In addition, there are many reports showing that components of the aqueous oregano extract, such as ursolic acid and rosmarinic acid, exert potent antioxidant activities by scavenging free radicals (Di Sotto et al., 2010; Lambert et al., 2001). Several studies have shown that thymol and carvacrol have chemoprotective effects against the toxicity and genotoxicity induced by both chemical agents and ionizing radiation (Archana et al., 2009; Horvathova et al., 2007; Slamenova´ et al., 2007; Vicun˜a et al., 2010). The essential oil from O. onites as well as carvacrol and thymol showed a concentration-dependent protective (antioxidant) effect against the cytotoxic effects and membrane damage induced by H2O2 on HepG2 cells ¨ zkan, 2011). In another study, the incubation of Hep G2 and (O Caco-2 cells in the presence of numerous different concentrations of carvacrol or thymol protected the cells from the DNA strand breaks induced by hydrogen peroxide (Slamenova´ et al., 2007). Aydin et al. (2005) also observed that thymol and carvacrol significantly reduced oxidative damage in human lymphocytes. Carvacrol and thymol displayed dose-dependent antiproliferative effects on human uterine carcinoma cells (Mastelic´ et al., 2008). Origanum vulgare may act as a potent chemoprotective agent due to its prevalent flavonoids and phenolic compounds. These compounds are rich in OH groups and can scavenge the free radicals induced by CP. Therefore, we evaluated the chemoprotective effects of the O. vulgare ethanolic extract against the genotoxicity induced by CP in the mouse bone marrow. The O. vulgare ethanolic extract exhibited dose-dependent protective effects, reduced the frequency of MnPCE induced by CP and increased the proliferation of bone marrow cellularity that was affected by CP. Administering 200 and 400 mg/kg of O. vulgare ethanolic extract to mice before CP injection effectively reduced the frequency of MnPCE by approximately 4.28- and 4.85-fold, respectively. The O. vulgare ethanolic extract treatment also increased the PCE/ PCE + NCE ratio, which declined in mice treated with CP. Origanum vulgare ethanolic extract pre-treatment might reduce the CP-induced oxidative stress, suggested that the protection provided by O. vulgare ethanolic extract might occur by modulating the cellular antioxidant levels and reducing the oxidative stress markers.
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In conclusion, our study confirms that O. vulgare ethanolic extract displays potent protective effects against the genotoxicity and Mn formation induced by CP in mouse bone marrow cells. Histological examinations of the bone marrow also revealed that the O. vulgare ethanolic extract mitigates both the myelosuppressive effect and the bone marrow suppression caused by CP. The DNA protective effect of O. vulgare could be explained by its ability to scavenge the ROS and quench the free radicals that induce DNA strand breaks. Therefore, O. vulgare ethanolic extract might help defend the body against the side effects, particularly DNA damage, induced by hazardous chemical agents.
Declaration of interest The authors declare no conflict of interest. This study was supported by a grant from the Student Research Committee, Mazandaran University of Medical Sciences, Sari, Iran.
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ORIGINAL ARTICLE
Genoprotective effects of Origanum vulgare ethanolic extract against cyclophosphamide-induced genotoxicity in mouse bone marrow cells
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Emran Habibi1,2, Mohammad Shokrzadeh1,3, Amirhossein Ahmadi1, Aroona Chabra4, Farshad Naghshvar5, and Razieh Keshavarz-Maleki3 1
Pharmaceutical Sciences Research Center, Faculty of Pharmacy, 2Department of Pharmacognosy, Faculty of Pharmacy, 3Department of Toxicology and Pharmacology, Faculty of Pharmacy, 4Student Research Committee, Faculty of Pharmacy, and 5Department of Pathology, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran Abstract
Keywords
Context: Cyclophosphamide (CP), an alkylating chemotherapeutic agent, can bind DNA, causing chromosome breaks, micronucleus (Mn) formation, and cell death. Because Origanum vulgare L. (Lamiaceae) has antioxidative properties, it might protect against DNA damage. Objective: The genoprotective effect of O. vulgare ethanolic extract against CP-induced genotoxicity in mouse bone marrow cells was evaluated using a Mn assay. Materials and methods: Mice were pre-treated with aerial parts of O. vulgare ethanolic extract at different doses (50, 100, 200, or 400 mg/kg) for 7 d. One hour after the last administration of O. vulgare, animals were injected with CP at 200 mg/kg. After 24 h, the bone marrow cells of both femurs were flushed and the frequency of MnPCEs was evaluated to measure the chromosomal damages. In addition, the number of PCEs per 1000 NCEs in each animal was recorded to evaluate the bone-marrow suppression; mitotic activity was calculated as [PCE/(PCE + NCE)] 100 to assess the cell division. Results: At 400 mg/kg, O. vulgare displayed its maximum protective effect, reduced the number of MnPCEs from 10.52 ± 1.07 for CP group to 2.17 ± 0.26 and completely normalized the mitotic activity (p50.001). Origanum vulgare also led to significant proliferation and hypercellularity of immature myeloid elements after the mice were treated with CP, mitigating the bone marrow suppression. Discussion and conclusion: Origanum vulgare ethanolic extract exerts a potent genoprotective effect against CP-induced genotoxicity in mice bone marrow, which might be possibly due to the scavenging of free radicals during oxidative stress conditions.
Antioxidant activity, bone marrow suppression, DNA damage, micronucleus assay
Introduction Cyclophosphamide [N,N-bis(2-chloroethyl)tetrahydro-2H1,3,2-oxazaphosphorin-2-amine-2-oxide, CP] is an oxazophosphorine derivative of nitrogen mustard and is commonly used during cancer chemotherapy. The tumor cell-killing activity of CP is primarily attributed to DNA alkylation. This drug also exhibits significant immunosuppressive activity and is used to treat autoimmune diseases as well as for renal and bone marrow transplantations (Dollery, 1999). The chronic carcinogenic side-effects of alkylating agents create concern during their use in combination drug therapies. Numerous studies have demonstrated the mutagenic effects of CP in both humans and animals (De Ridder et al., 1998; Ember et al., 1995). The chemically reactive metabolic products of CP induce cytotoxicity by alkylating DNA and protein as well as Correspondence: Amirhossein Ahmadi, 18 kilometer of Farah Abad Road, Pharmaceutical Sciences Research Center, Faculty of Pharmacy, Mazandaran University of Medical Sciences, P. Box: 48175-861, Sari, Iran. Tel/Fax: +98 151 3543084. E-mail: Amirhossein_pharma@ yahoo.com
History Received 6 September 2013 Revised 28 March 2014 Accepted 28 March 2014 Published online 2 2 2
by cross-linking DNA (Hales, 1982). The antineoplastic and toxic effects of CP, such as apoptosis, necrosis, and oncosis, are linked with two active metabolites: acrolein and phosphoramide (Kern & Kehrer, 2002). CP produces carbonium ions that react with the electron-rich sites on nucleic acids and proteins, triggering gene mutations, DNA-strand breaks, chromosome aberrations (CA), micronuclei induction, and the production of ROS. The high reactivity of free radicals causes cellular damage using various mechanisms (Bendich, 1990). The most deleterious effects of free radicals lead to DNA damage that causes cancer (Richter et al., 1988). The genotoxic effects of CP have been evaluated in in vivo and in vitro studies (Hartmann et al., 1995). Recently, the search for compounds from plants capable of minimizing the toxicity chemotherapy-induced without compromising antineoplastic activity has received great interest (Pratheeshkumar & Kuttan, 2010). Epidemiological studies have shown the intake of phenolic foods is associated with protection from various diseases (Morton et al., 2000). These phenolic compounds display tremendous antioxidant and chemoprotective properties in vivo (Zhao et al., 2004).
Genoprotective effects of O. vulgare
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DOI: 10.3109/13880209.2014.910674
Natural products have exerted protective effects against genotoxicity induced by CP in mouse bone marrow cells when these compounds were administered before CP treatment. Antioxidant activity might explain the chemoprotective effects of these natural products (Ahmadi et al., 2008; Hosseinimehr et al., 2010). Origanum vulgare L. (Lamiaceae), also known as oregano, is a flavoring herb widely used around the world. Origanum vulgare is an aromatic plant with a wide distribution throughout Asia, particularly in Iran. It is used to cure respiratory diseases, hypoglycemic disease, and leukemia (Sheibani et al., 2010). Because of its antioxidant properties, oregano could become a helpful agent for the treatment of cancer, heart disease, and high blood pressure (Pirigharnaei et al., 2011). The results of various studies indicated that oregano’s antioxidant effects might be a result of the dominant constituents, including carvacrol and thymol, present in its essential oil (Lagouri et al., 1993). Origanum’s high antioxidant activity seems to arise from the phenolic –OH groups present in the plant’s flavonoid and phenolic compounds, which donate hydrogen atoms to the peroxy radicals produced during the first step of lipid oxidation (Roofchaee et al., 2011). Because it has been used extensively as an additive agent and is known to be safe, this study was undertaken to assess the effects of O. vulgare ethanolic extract against the genotoxicity induced by CP in mouse bone marrow cells using the Mn assay. Histological examination of the bone marrow was also used for to observe the possible mitigating effects of the O. vulgare ethanolic extract against the myelosuppressive effect of CP and bone marrow suppression.
Materials and methods Preparation of extracts Dried aerial parts of O. vulgare powder were purchased from the Giah Essence Phyto-Pharmaceutical Co., Golestan Province, Iran. Three hundred gram samples of the dried plant powder were extracted with 3000 ml of ethanol (75%) for 72 h. After evaporating the solvent under vacuum at a temperature below 50 C, 35 g of dried powder was obtained. Animals Male Naval Medical Research Institute (NMRI) mice weighing 22 ± 3 g were obtained from the Pasteur Institute of Iran (Amol, Iran). The mice were housed in the university animal facility and were maintained under a controlled 12 h light/dark cycle at 23 ± 1 C. The animals were acclimated for 1 week before the study and were provided with standard food pellets and water ad libitum. All of the animals were cared for according to the ‘‘Care and Use of Laboratory Animals’’ at the Mazandaran University of Medical Sciences. The protocol for the study was approved by the Research Committee of Mazandaran University of Medical Sciences in Sari, Iran. Experimental treatment For the Mn assay, the animals were divided into seven groups (n ¼ 5 for each group): the negative control group,
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administered distilled water (10 ml/kg b.w.) via intraperitoneal (i.p.) injection for 7 d; the positive control group, administered a single toxic CP dose (200 mg/kg b.w., i.p.) in distilled water (10 ml/kg b.w.) at seventh days of experiment; OV (O. vulgare) 50, 100, 200, and 400 plus CP groups, administered increasing doses of O. vulgare ethanolic extract (50, 100, 200, or 400 mg/kg b.w., i.p., respectively) in distilled water (10 ml/kg b.w.) each day for 7 consecutive days, which was followed by a single i.p. CP dose 1 h after the last O. vulgare ethanolic extract administration; and OV 400 group, administered only O. vulgare ethanolic extract (400 mg/kg b.w. by i.p. injection) in distilled water (10 ml/kg b.w.) once per day for 7 d. Mn assay The Mn test was performed as previously described (Ahmadi et al., 2008; Hosseinimehr et al., 2010). The bone marrow Mn test is a well-known in vivo assay used to assess genotoxicity and DNA damage in animals, such as mice and rats. The number of MnPCEs is increased in rodent bone marrow cells exposed to hazardous chemical and chromosome-damaging agents. A Mn is round and is 1/20th–1/5th the size of that in an erythrocyte. The ratio of PCE to NCE in bone marrow preparations is useful for estimating any perturbations in hematopoiesis caused by the treatment in exposed animals (Gollapudi & McFadden, 1995; Schmid, 1975). The femurs from the animals were used to estimate the Mn frequency and mitotic activity. Mice were sacrificed by cervical dislocation 24 h after CP injection. The bone marrow of both femurs was removed as a fine suspension in a centrifuge tube with FCS. The cells were dispersed by gentle pipetting and collected by centrifuge at 1500 rpm for 10 min. The cell pellet was resuspended in a drop of FCS, and smears were prepared. The slides were coded to avoid any observed bias. After 48 h of air drying, the smears were stained with May-Grunwald/Giemsa. For each experimental point, five mice were used, and 5000 PCEs were scored blindly and microscopically to determine the percentage of MnPCE. In addition, the number of PCEs per 1000 NCEs in each animal was recorded to evaluate the bone-marrow suppression; mitotic activity was calculated as [PCE/(PCE + NCE)] 100 to assess the cell division. Histology of bone marrow For the histological examination of the myeloid hyperplasia in the bone marrow, mice were pretreated with 200 and 400 mg/kg of O. vulgare ethanolic extract solutions for 7 d before CP (200 mg/kg b.w.) was administered 1 h after the last injection of O. vulgare ethanolic extract. Both femurs were removed from the mice; the mice were killed by cervical dislocation 24 h after CP administration. The femurs were immersed in 10% formalin; the bones were decalcified before being processed using a microtome to generate micron scale slides. Routine H & E staining was performed on 6 mm paraffin sections, and the slides were evaluated under a light microscope. The slides were photographed using a camera microscope (Olympus, Orinpasu Kabushiki-gaisha, Japan) at 400 magnification.
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Statistical analysis The data are presented as the mean ± standard deviation (SD). ANOVA test with post hoc Tukey test were used for the comparison of Mn data. A p value below 0.05 was considered statistically significant.
Results
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Bone marrow Mn assay The effect of increasing doses of O. vulgare ethanolic extract on the MnPCE frequency in bone marrow cells 24 h after CP injection is shown in Figure 1. The frequency of Mn significantly increased in the mice treated with CP compared with the control group (p50.001). In the mice pretreated with three doses of O. vulgare ethanolic extract, the MnPCE frequency induced by CP was significantly decreased compared with those treated with only CP (p50.01–p50.001). The MnPCE frequency was lower in the O. vulgare ethanolic extract + CP groups by factors of 2.33, 4.28, and 4.85 for the 100, 200, and 400 mg/kg doses, respectively, compared with the CP treated group. The data showed that the O. vulgare ethanolic extract suppress the activity of CP on the clastogenic effects. Moreover, the mitotic activity (%PCE) in CP-treated Figure 1. Frequency of MnPCE in the bone marrow cells of mice treated with the O. vulgare ethanolic extract and/or CP (200 mg/kg). OV, Origanum vulgare; CP, cyclophosphamide; MnPCE, micronucleated polychromatic erythrocyte. (a) p50.001; CP versus control. (b) p50.001; (OV 200 mg + CP) and (OV 400 mg + CP) versus CP-treated group. (c) p50.01; (OV 100 mg + CP) versus CP-treated group. (d) p40.05; (OV 400 mg) versus control. Values are presented as mean ± SD for each group of five mice. The data were analyzed using ANOVA test with the post hoc Tukey test.
Figure 2. Frequency of mitotic activity (%PCE) in the bone marrow cells of mice treated with the O. vulgare ethanolic extract and/or CP (200 mg/kg). OV, Origanum vulgare; CP, cyclophosphamide; PCE, polychromatic erythrocyte; NCE, normochromatic erythrocyte. (a) p50.001; CP versus control. (b) p50.001; (OV 200 mg + CP) and (OV 400 mg + CP) versus CP-treated group. (c) p50.01; (OV 100 mg + CP) versus CP-treated group. (d) p40.05; (OV 400 mg) versus control. Values are presented as mean ± SD for each group of 5 mice. The data were analyzed using ANOVA test with the post hoc Tukey test.
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mice showed a pronounced cytotoxic effect on bone marrow proliferation, and this behavior was significantly decreased in the mouse bone marrow after the CP treatment (p50.001). Treating the mice with O. vulgare ethanolic extract arrested the CP-induced decline in mitotic activity (%PCE) (Figure 2). Origanum vulgare ethanolic extract treatment at 200, or 400 mg/kg completely prevented the cytotoxicity induced by CP in the mouse bone marrow, increasing mitotic activity (%PCE) by increasing the bone marrow proliferation (p50.001). Origanum vulgare ethanolic extract alone did not cause any genotoxicity in the mice bone marrow cells at high dose of 400 mg/kg (p40.05) (Figure 2). Histology of bone marrow Figure 3 presents myeloid hypoplasia and hemorrhage in a representative femur 24 h after CP administration (200 mg/ kg). Figure 4 presents myeloid hyperplasia and hemorrhage reduction induced by administering the O. vulgare ethanolic for 7 consecutive days) before CP treatment. The histological examinations of the bone marrow showed that administering CP induced myelosuppressive effects. Administering 200 or 400 mg/kg of O. vulgare ethanolic extract led to marked proliferation and hypercellularity in the immature myeloid
DOI: 10.3109/13880209.2014.910674
elements after CP treatment, in addition to mitigating bone marrow suppression and hemorrhage reduction. The relative increase in the proportion of myeloid to erythroid precursors after O. vulgare ethanolic extract administration is notable in figures.
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Discussion The active metabolites of CP are phosphoramide and acrolein; these compounds slow the growth of cancer cells because they interfere with cellular DNA. Hydroxy–cyclophosphamide and deschloroethyl–cyclophosphamide are the two intermediate compounds that lead to the formation of the agent responsible the mutagenic effect of CP, phosphoramide. Phosphoramide induces cross-linking and strand lesions in DNA (Hengstler et al., 1997). We previously reported that CP induced DNA damage and genotoxicity in mouse bone marrow cells (Ahmadi et al., 2008; Shokrzadeh et al., 2014). DNA damage is critical for the initiation and subsequent promotion of carcinogenesis induced by different genotoxicants, as indicated by the experimental evidence. Errors in DNA molecules cause chromosomal aberrations. Mn is wellcharacterized biomarkers for structural and numerical chromosomal damage. The Mn in young erythrocytes arises
Figure 3. Myeloid hypoplasia and hemorrhage in a femur 24 h after 200 mg/kg CP administration. CP induced myelosuppressive effects (hematoxylin and eosin-stained paraffin sections; H&E 400).
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mainly from chromosomal fragments that are not incorporated into the daughter nuclei during the cell division of the erythropoietic blast cells (Salamone & Heddle, 1983). The decreased PCE:NCE ratio induces the bone marrow cytotoxicity (Al-Majed et al., 2006). CP treatment is cytotoxic and is highly clastogenic in nature. In the present study, CP induced Mn formation because it decreased the PCE:NCE ratio; O. vulgare ethanolic extract effectively restored this PCE:NCE ratio back to normal levels. CP also helps to generate DNA strand breaks through the interaction of acrolein with DNA, causing DNA disintegration or smearing due to necrosis and MnPCEs formation. Prophylactic treatments of O. vulgare ethanolic extract significantly and dose dependently restore these hallmarks of genotoxicity to normal levels. Although the exact mechanism of the chemoprotective effect is unknown, free-radical scavenging is apparently responsible for the inhibitory effects of herbal extract and natural compounds on the clastogenic activity induced by genotoxic agents (Hosseinimehr et al., 2010). We recently showed that melatonin, a natural pineal secretory product, has a potent chemoprotective effect against the genotoxicity induced by diazinon in human blood lymphocyte cells, and this protective effect might be the results of free radicalscavenging properties (Karimian et al., 2013). Natural compounds, including flavonoids and phenolic compounds, may scavenge the free radicals, such as hydroxyl radicals, generated by chemical hazardous agents. Increased intracellular levels of reactive oxygen species are frequently referred to as oxidative stress; this state represents a potentially toxic insult that interacts with macromolecules, inducing DNA damage (Hosseinimehr et al., 2011). Another recent study also provided evidence that Citrullus colocynthis (L.) Schrad. (Cucurbitaceae) fruits extract pretreatment attenuates CPinduced oxidative stress and the subsequent DNA damage in mice. It had a potent anticlastogenic effect against the genotoxicity induced by CP in mice bone marrow cells, which might be due to the scavenging of free radicals and inhibition of lipid proxidation of the plant (Shokrzadeh et al., 2013). We also showed that O. vulgare ethanolic extract with high amount of flavonoids and phenolic compounds protects human lymphocytes against genotoxicity induced by internal irradiation. The results suggested that O. vulgare ethanolic
Figure 4. Myeloid hyperplasia and hemorrhage reduction induced by administering the O. vulgare extract (200 mg/kg; right and 400 mg/kg; left for 7 consecutive days) before CP treatment. Origanum vulgare led to marked proliferation and hypercellularity of immature myeloid elements after the mice were treated with CP, the bone marrow suppression was mitigated and the hemorrhage was reduced. The relative increase in the proportion of myeloid to erythroid precursors after O. vulgare administration is notable (hematoxylin and eosin-stained paraffin sections; H&E 400).
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extract acts effectively as a free-radical scavenger and provides concentration-dependent radioprotection and protective effect against DNA damage (Arami et al., 2013). Origanum vulgare L. is the only species of the Origanum genus that grows in the wild of Iran (Zargari, 1997). Several studies have shown that O. vulgare have a high amount of flavonoids and phenolic compounds. The major aqueous constituents of oregano are rosmarinic acid, eriocitrin, luteolin-7-O-glucoside, apigenin-7-O-glucoside, origanol A and B, and ursolic acid (Sheibani et al., 2010). Rosmarinic acid and origanol A and B, which are the most abundant components of the aqueous extract of oregano, have antioxidative activities (Kulisic et al., 2007; Matsuura et al., 2003). Previous studies have reported that the essential oil of O. vulgare has antioxidant capacity, which has been linked to components such as thymol, carvacrol, d-terpinene, and p-cymene (Halici et al., 2005; Odabasoglu et al., 2004; Russo et al., 2002). In addition, there are many reports showing that components of the aqueous oregano extract, such as ursolic acid and rosmarinic acid, exert potent antioxidant activities by scavenging free radicals (Di Sotto et al., 2010; Lambert et al., 2001). Several studies have shown that thymol and carvacrol have chemoprotective effects against the toxicity and genotoxicity induced by both chemical agents and ionizing radiation (Archana et al., 2009; Horvathova et al., 2007; Slamenova´ et al., 2007; Vicun˜a et al., 2010). The essential oil from O. onites as well as carvacrol and thymol showed a concentration-dependent protective (antioxidant) effect against the cytotoxic effects and membrane damage induced by H2O2 on HepG2 cells ¨ zkan, 2011). In another study, the incubation of Hep G2 and (O Caco-2 cells in the presence of numerous different concentrations of carvacrol or thymol protected the cells from the DNA strand breaks induced by hydrogen peroxide (Slamenova´ et al., 2007). Aydin et al. (2005) also observed that thymol and carvacrol significantly reduced oxidative damage in human lymphocytes. Carvacrol and thymol displayed dose-dependent antiproliferative effects on human uterine carcinoma cells (Mastelic´ et al., 2008). Origanum vulgare may act as a potent chemoprotective agent due to its prevalent flavonoids and phenolic compounds. These compounds are rich in OH groups and can scavenge the free radicals induced by CP. Therefore, we evaluated the chemoprotective effects of the O. vulgare ethanolic extract against the genotoxicity induced by CP in the mouse bone marrow. The O. vulgare ethanolic extract exhibited dose-dependent protective effects, reduced the frequency of MnPCE induced by CP and increased the proliferation of bone marrow cellularity that was affected by CP. Administering 200 and 400 mg/kg of O. vulgare ethanolic extract to mice before CP injection effectively reduced the frequency of MnPCE by approximately 4.28- and 4.85-fold, respectively. The O. vulgare ethanolic extract treatment also increased the PCE/ PCE + NCE ratio, which declined in mice treated with CP. Origanum vulgare ethanolic extract pre-treatment might reduce the CP-induced oxidative stress, suggested that the protection provided by O. vulgare ethanolic extract might occur by modulating the cellular antioxidant levels and reducing the oxidative stress markers.
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In conclusion, our study confirms that O. vulgare ethanolic extract displays potent protective effects against the genotoxicity and Mn formation induced by CP in mouse bone marrow cells. Histological examinations of the bone marrow also revealed that the O. vulgare ethanolic extract mitigates both the myelosuppressive effect and the bone marrow suppression caused by CP. The DNA protective effect of O. vulgare could be explained by its ability to scavenge the ROS and quench the free radicals that induce DNA strand breaks. Therefore, O. vulgare ethanolic extract might help defend the body against the side effects, particularly DNA damage, induced by hazardous chemical agents.
Declaration of interest The authors declare no conflict of interest. This study was supported by a grant from the Student Research Committee, Mazandaran University of Medical Sciences, Sari, Iran.
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