Journal of Toxicology and Environmental Health, Part A Current Issues
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Molecular and structural changes induced by essential oils treatments in Vicia faba roots detected by genotoxicity testing Elena Sturchio, Priscilla Boccia, Miriam Zanellato, Claudia Meconi, Lucia Donnarumma, Giuseppe Mercurio & Mauro Mecozzi To cite this article: Elena Sturchio, Priscilla Boccia, Miriam Zanellato, Claudia Meconi, Lucia Donnarumma, Giuseppe Mercurio & Mauro Mecozzi (2016): Molecular and structural changes induced by essential oils treatments in Vicia faba roots detected by genotoxicity testing, Journal of Toxicology and Environmental Health, Part A, DOI: 10.1080/15287394.2015.1124059 To link to this article: http://dx.doi.org/10.1080/15287394.2015.1124059
Published online: 25 Feb 2016.
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Date: 26 February 2016, At: 15:13
JOURNAL OF TOXICOLOGY AND ENVIRONMENTAL HEALTH, PART A http://dx.doi.org/10.1080/15287394.2015.1124059
Molecular and structural changes induced by essential oils treatments in Vicia faba roots detected by genotoxicity testing Elena Sturchioa, Priscilla Bocciaa, Miriam Zanellatoa, Claudia Meconia, Lucia Donnarummab, Giuseppe Mercurioc, and Mauro Mecozzid
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a Italian Workers’ Compensation Authority (INAIL), Department of Technological Innovation and Safety of Plants, Product and Anthropic Settlements (DIT), Rome, Italy; bConsiglio per la Ricerca in Agricoltura e l’Analisi dell’Economia Agraria, Centro di Ricerca per la Patologia Vegetale (CREA-PAV), Rome, Italy; cItalian Red Cross–Army Corps–Central Laboratory, Rome, Italy; dLaboratory of Chemometrics and Environmental Applications (ISPRA), Rome, Italy
ABSTRACT
ARTICLE HISTORY
Over the last few years, there has been an increased interest in exploiting allelopathy in organic agriculture. The aim of this investigation was to examine the effects of essential oil mixtures in order to establish their allelopathic use in agriculture. Two mixtures of essential oils consisting respectively of tea tree oil (TTO) and clove plus rosemary (C + R) oils were tested. Phytotoxicity and genotoxicity tests on the root meristems of Vicia faba minor were performed. A phytotoxic influence was particularly relevant for C + R mixture, while genotoxicity tests revealed significant results with both C + R oil mixture and TTO. Phenotypic analysis on Vicia faba minor primary roots following C + R oil mixture treatment resulted in callose production, an early symptom attributed to lipid peroxidation. The approach described in this study, based on genotoxicity bioassays, might identify specific DNA damage induced by essential oil treatments. These tests may represent a powerful method to evaluate potential adverse effects of different mixtures of essential oils that might be useful in alternative agriculture. Future studies are focusing on the positive synergism of more complex mixtures of essential oils in order to reduce concentrations of potentially toxic components while at the same time maintaining efficacy in antimicrobial and antifungal management.
Received 4 March 2015 Accepted 20 November 2015
Currently, there is an increasing interest in using essential oils as alternative practice in agriculture due to their potential herbicide and fungicide properties (Tworkoski, 2002). Directive 2009/128/ EC, “Establishing a Framework for Community Action to Achieve the Sustainable Use of Pesticides,” proposed to reduce pesticide toxicity risk and impact on and environment and human health by development of alternative agricultural techniques through an integrated pest management program (European Commission, 2009). Within the alternative techniques in agriculture, essential oils play a fundamental role as substitutes for pesticides. In fact, essential oils were widely used in ancient times for medicinal and cosmetic applications, due to their antimicrobial, fungicidal and antiparasitical properties (Shelef, 1983; Mari et al., 2003). Essential oils are now
largely used in pharmaceutical, sanitary, cosmetic, and agricultural and food industries. Essential oils are mostly obtained by distillation from aromatic plants and are composed of a variety of volatile molecules, mainly terpenes. Although the mechanism of action remains poorly understood, Saad et al. (2013) reported that the activity of essential oils depends upon the structure and physicochemical properties of their components, which might affect various membrane molecular structures by modifying the calcium and potassium ion channels and receptors of the transport systems. It is well known that essential oil constituents act by multiple pathways and mechanisms involving cell cycle arrest, DNA repair modulation, and antiproliferative activity. Particularly relevant is the production of reactive oxygen species (ROS) that may lead to cell death
CONTACT Elena Sturchio [email protected] Italian Workers’ Compensation Authority (INAIL), Department of Technological Innovation and Safety of Plants, Product and Anthropic Settlements (DIT), Via Roberto Ferruzzi n. 38/40 - 00143 Roma, Italy. Color versions of one or more of the figures in the article can be found online atwww.tandfonline.com/uteh. © 2016 Taylor & Francis
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in cancer cells (Gautam et al., 2014) and inhibit growth of roots in plants (Ahuja et al., 2015). Previously, Sturchio et al. (2014) examined the use of tea tree oil (TTO) and combination of rosemary and clove essential oils (C + R) as potential fungicides in agriculture and showed that it was possible to obtain an alternative approach to management of powdery mildew diseases. The origin and properties of these oils are well recognized. Rosemary (Rosmarinus officinalis, Labiatae) originally comes from southern Europe. Rosemary leaves and oil are commonly used as spice and flavoring agents in food processing because of their desirable flavor and antimicrobial and antioxidant activity, due to phenolic presence of diterpenes, such as rosmarinic acid and carnosol (Maistro et al., 2010). Most health benefits and pharmacological effects of rosemary are the consequence of potent antioxidant activity attributed to the main chemical constituents, which include carnosol, carnosic acid, ursolic acid, rosmarinic acid, and caffeic acid. The antioxidant activity of rosemary constituents against lipid peroxidation and DNA damage induced by ROS was noted in rat liver mitochondria and microsomes at concentrations of 3–30 µM, demonstrating their ability to protect tissues and cells against oxidative stresses (Bradley, 2006). Clove oil is extracted from Eugenia caryophyllata plant and its main constituent is eugenol. Eugenol is an extraordinarily versatile molecule and has been included as an ingredient in cosmetics, and foods. Kamatou et al. (2012) demonstrated pharmacological properties, such as anaesthetic and analgesic effects, as well as antioxidant activity due to the presence of its phenolic groups. Further, Burt (2004) studied the antibiotic mechanism of eugenol and other flavoring agents, in which the cell wall becomes degraded, damaging the plasma membrane and membrane protein and exposing cellular content. The cytoplasm coagulates, producing the protonmotive force to move far away. Lu et al. (2008) investigated the in vitro antibacterial activity of eugenol by microdilution test and found that eugenol exerted antibacterial action against 12 common bacteria. The hydrophobic property of eugenol can separate lipids of cell membrane and
change its structure to increase cell membrane permeability. It can block the proton-motive force, electron stream, and active transport, and produce coagulation of cell content (Kong et al., 2014). Further, eugenol increases ROS generation leading to DNA fragmentation, a hallmark of apoptosis in treated colon cancer cells (Jaganathan et al., 2011). Tea tree oil is the essential oil obtained by steam distillation of the foliage and terminal branchlets of Melaleuca alternifolia, Melaleuca linariifolia, and Melaleuca dissitiflora, as well as other species of Melaleuca, provided that the oil obtained conforms to the requirements given in the International Standard (ISO 4730-2004). Tea tree oil from Melaleuca alternifolia contains various mono- and sesquiterpenes, as well as aromatic compounds. The monoterpenes terpinen-4-ol, αterpinene, γ-terpinene, 1,8-cineole, p-cymene, αterpineol, α-pinene, limonene, and sabinene account for 80–90% of the oil. The natural content of the individual terpenes in tea tree oil may vary considerably, depending upon (1) Melaleuca alternifolia population used, (2) climate, (3) leaf maceration, (4) age of the leaves, and (5) duration of distillation (Scientific Committee on Consumer Products [SCCP], n.d.). While these properties are increasingly well characterized, relatively limited data are available regarding the safety and toxicity associated with the use of the essential oil. TTO does not directly induce alterations of the cell wall but releases autolytic enzymes associated with the cell membrane, which may induce lysis and subsequent leakage of nucleic acids across damaged cytoplasmic membrane (Saad et al., 2013). The antimicrobial and antifungal activity of combinations of different essential oils as indication of their additive, synergistic, or antagonistic effects against individual microorganism tests was observed. Clove (Syzygium aromaticum L.) and rosemary (Rosmarinus officinalis L.) oils used in combination exhibited significant antimicrobial effects against all microorganisms tested (Fu et al., 2007). In this study the effects of essential oil were determined because their use in agriculture requires knowledge of their potential adverse influence on plants. In fact, essential oils might exert potential
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toxic effects by changing structural and molecular components by means of specific reactions and/or interactions. Thus, the roots of the probe organism Vicia faba were subjected to in vivo genotoxicity tests. Vicia faba is a common bioindicator plant employed due to its sensitivity to mutagenic effects, such as point mutations to full DNA damage induced by single or complex mixtures of chemicals. Further, plant bioassays may represent easy and inexpensive techniques and thus conducted under a wide range of environmental conditions (Patlolla, 2013). The genotoxicity tests enable detection of specific cell parameters related to DNA structures (Sturchio et al. 2012). By means of this approach, one can estimate stress factors initiated by pollutants and to assess specific molecular DNA damages, and oxidation and alterations of lipid structures, which affect membrane dynamics (Mecozzi et al., 2007; Pietroletti et al., 2010; Sturchio et al., 2012; Boccia et al., 2013).
Materials and methods Sample preparation
The essential oils consisted of TTO and C + R oil mixture. The composition of tested essential oil mixtures was previously analyzed by chromatographic techniques (Sturchio et al., 2014). Oils were emulsified with 0.05% Tween 20 (Reuveni et al., 1996; Terzi et al., 2007) before their application. Treatments were performed for each product with doses indicated on the respective labels or in literature (Galletti et al., 2008; Copping, 2009; Vardi and Reuveni, 2009) and as already described by preliminary investigation previously (Sturchio et al., 2014). The tested concentrations of essential oils represent a range where there is no marked effect on growth inhibition. TTO was added at 0.3, 0.6, or 0.9 ml/L concentrations and C + R oils in the following concentrations: C 0.2 ml/L + R 0.4 ml/L, C 0.4 ml/L + R 0.8 ml/L, and C 0.8 ml/L + R 1.6 ml/L (Table 1). The experimental protocol consisted of phytotoxicity and genotoxicity tests (comet assay and micronuclei [MN] analysis) on primary roots of Vicia faba grown in quartz sand soil treated with the different concentrations of essential oils in aluminum basins. Each basin, containing 250 g quartz sandy soil and 25 Vicia faba seeds, was treated
Table 1. Treatment Mutagenicity Tests.
Programs
Treatment Tween 20: Control test Tween 20 + tea tree oil Tween 20 + tea tree oil Tween 20 + tea tree oil Tween 20 + clove oil + rosemary oil Tween 20 + clove oil + rosemary oil Tween 20 + clove oil + rosemary oil
and
Doses
Applied
3
to
Dose, mL/L 0.3 0.6 0.9 C 0.2 + R 0.4 C 0.4 + R 0.8 C 0.8 + R 1.6
Note. Oils were emulsified with 0.05% Tween 20 before their application.
with 50 ml of each emulsion of essential oils. Basins wetted only with Tween 20 were used as negative controls. Three basins were set up for each treatment. To verify the effects of Tween 20 used as vehicle to dissolve the oils, Vicia faba seeds were allowed also to grow in absence of Tween 20 but wetted only with H2O. Each aluminum basin was sealed with Parafilm and incubated in a climatic chamber (21 ± 1ºC, 60% relative humidity) for 5 d to enable germination. All experiments were repeated three times. TTO and the mixtures of C + R oils were tested in two separate trials. The primary roots of seedlings were measured for phytotoxicity and removed for genotoxicity tests. Phytotoxicity testing
After 5 d of incubation, exposed Vicia faba seedlings were collected and total germination percentage (Gt) was calculated as Gt = (n/N × 100), where n is the total number of germinated seeds at the end of the experiment of the given oil treatment and N is the total number of seeds used for germination test. Phytotoxicity was determined by measuring primary root length. The mean of primary root length (L) of each sample was compared with controls, providing an indication of oil effects. Germination Index (GI), evaluated as [(Gt sample × L sample)/(Gt Tween 20 × L Tween 20)] × 100, is a maturity test based on seed germination and initial plant growth where Gt = (n/N × 100) is the Gt percentage, and L is the average length of the primary root of the n germinated seeds. GI reflects the phytotoxicity of the essential oil, where low toxicity affects root growth, while high toxicity affects germination (Selim et al., 2012).
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Genotoxicity testing
Statistical analysis
Comet assay Single-cell gel electrophoresis (SCGE) is a cytogenetic test used to assess in a single cell genetic damage induced by mutagenic agents. The test is a simple, sensitive, and rapid shortterm genotoxicity method for measuring under alkaline conditions, pH ≥ 13.5, all DNA structural damages, including double-strand/singlestrand (ds/ss) breaks, alkali-labile sites such abasic sites, and oxidized bases (Koppen and Verschaeve, 1996; Menke et al., 2000). The comet test was performed under an alkaline unwinding/alkaline electrophoresis (A/A) using a Comet assay kit (Trevigen, Gaithersburg, MD) according to the manufacturer’s protocol. Tail length (TL) has been used as a parameter of DNA damage. Each experiment was repeated three times (in three biological replicates).
Phytoxicity and genotoxicity experimental data sets were subjected to one-way analysis of variance (ANOVA) followed by Dunnett’s test for comparing the difference of mean and standard error at the .05 level for statistical significance versus control using the statistical software package SPSS (Chicago, IL).
Micronucleus test Micronuclei (MN) are Feulgen-positive corpuscles localized within the cell wall in the cytoplasmic area surrounding the main nucleus. MN are formed by chromosomes or chromosome fragments that are not incorporated into daughter nuclei at the time of cell division (Ma, 1982). Micronuclei test evaluates the frequency of micronucleated cells in root meristems of Vicia faba, analyzing 15,000 cells (15 root tips, 1,000 cells for tip). Frequency of cell division was also analyzed. The experiment was performed as described by Sturchio et al. (2011) and repeated three times (in three biological replicates).
Results Phytotoxicity and genotoxicity testing
Vicia faba seeds were allowed to grow in the presence and in absence of Tween 20 used as vehicle to dissolve the oil, in order to determine any potential effects of Tween 20. Since no significant differences were observed between seeds grown with and without Tween 20 (data not shown), the experiments described hereafter report only results obtained in the presence of Tween 20 as control. A primarily phenotypic analysis of exposed Vicia faba primary roots presented callose production only at higher concentration of C + R oil treatment, as an early symptom attributed to lipid peroxidation as reported by Yamamoto et al. (2001) (Figure 1). A phytotoxic effect was observed in both trials (Figures 2A and 2B). It was more marked for the C + R mixture, exhibiting a linear dose dependence with concomitant decrease in GI and of the Gt% (Figures 3A and 4A). The GI and Gt parameters displayed no significant effects with TTO oil treatment (Figure 3B and Figure 4 B). Comet assay showed DNA damage at the intermediate concentration of C + R oil mixture (C 0.4 ml/L + R 0.8 ml/L) and at the intermediate and higher concentrations tested of TTO (0.6 ml/L and 0.9 ml/L) (Figures 5A and 5B). The MN test per-
Figure 1. Phenotypic analysis of Vicia faba roots. Sample treated with clove oil 0.8 ml/L + Rosemary oil 1.6 ml/L. Meristems showed a callose production after the treatment as an early symptom caused by lipid peroxidation.
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Primary root lenght (mm) 40 35 30
*
25
*
*
20 15 10
Tea tree oil 0.9 mL/L
B)
Tea tree oil 0.6 mL/L
A)
Tea tree oil 0.3 mL/L
0
Tween
5
formed with C + R oil mixture revealed an elevated MN frequency at the intermediate concentration (C 0.4 ml/L + R 0.8 ml/L) and a decreased cell division frequency at the higher concentration (C 0.8 ml/L + R 1.6 ml/L) (Figure 6A and 7A). No marked effects on MN and frequency of cell divisions were observed in TTO trials (Figure 6B and 7B). Discussion Although essential oils have been demonstrated to possess antimicrobial properties (Shelef, 1983), antimycotic activity (Azzouz and Bullerman, 1982; Akgül and Kivanç, 1988; Mari et al., 2003), and antiviral activity (Bishop, 1995) and to be effective against a broad spectrum of plant pathogenic fungi (Vardi and Reuveni, 2009) their mechanisms of action remain to be determined. As potential lipophiles, essential oils pass through the
cytoplasmic membrane and disrupt the structure of their different layers of polysaccharides, fatty acids, and phospholipids, and permeabilize them. Cytotoxicity includes this type of membrane damage. Damage to cell wall and membrane leads to leakage of intracellular macromolecules and lysis (Bakkali et al., 2008). The results obtained performing a phenotypic analysis on samples of Vicia faba-treated roots exposed at higher concentration of C + R oils yielded callose structures probably due to lipid peroxidation as reported by Yamamoto et al. (2001) (Figure 1). Lipid peroxidation is one of the most important expressions of oxidative stress induced by ROS. ROS readily react with unsaturated lipids and produce polar lipid hydroperoxides that produce increased membrane fluidity by disturbing hydrophobic phospholipids (Dix and Aikens, 1993; Van Ginkel and Sevanian, 1994; Khan et al., 2011).
Germination Index (%)
Germination Index (%)
120 140
100
120
80
*
100
60
80
*
40 20
Tea tree oil 0.9 mL/L
B)
Tea tree oil 0.6 mL/L
0 Tea tree oil 0.3 mL/L
Clove oil 0.4 mL/L + Rosemary oil 0.8 mL/L
Clove oil 0.2 mL/L + Rosemary oil 0.4 mL/L
A)
Clove oil 0.8 mL/L + Rosemary oil 1.6 mL/L
*
20
0
60
Tween
40
Tween
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Figure 2. (A) Phytotoxicity tests performed on Vicia faba roots treated with mixture of clove oil and rosemary oil. (B) Phytotoxicity tests performed on Vicia faba roots treated with tea tree oil. Asterisk indicates significant differences compared to Tween 20 by ANOVA with Dunnett test at p < .05.
Figure 3. (A) Germination index calculated on Vicia faba roots treated with mixture of clove oil and rosemary oil. (B) Germination index calculated on Vicia faba roots treated with tea tree oil. Asterisk indicates significant differences compared to Tween 20 by ANOVA with Dunnett test at p < .05.
E. STURCHIO ET AL.
Total Germination percentage (%)
Total Germination percentage (%)
100
80
90
70
*
80 70
60
60
50
50 40
*
30 20
Tea tree oil 0.9 mL/L
B)
Tea tree oil 0.6 mL/L
0 Tween
Clove oil 0.8 mL/L + Rosemary oil 1.6 mL/L
Clove oil 0.4 mL/L + Rosemary oil 0.8 mL/L
Tween
10 Clove oil 0.2 mL/L + Rosemary oil 0.4 mL/L
A)
30 20
10 0
40
Tea tree oil 0.3 mL/L
6
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Figure 4. (A) Total germination percentage (Gt) calculated on Vicia faba roots treated with mixture of clove oil and rosemary oil. (B) Total germination percentage (Gt) calculated on Vicia faba roots treated with tea tree oil. Asterisk indicates significant differences compared to Tween 20 by ANOVA with Dunnett test at p < .05.
Lipid peroxidation has been also related to MN induction. At the intermediate concentration of essential oils (C + R oils) a significant increase of MN frequency was noted (Figure 6A). Conversely, a significant reduction in frequency of cell division was evident at the higher concentration where it was not possible to detect micronucleated cells (Figure 7A). ROS overproduction coupled with deficiency of antioxidant defense mechanisms may be an important factor contributing to elevated MN frequency. Rosa et al. (2003) found that there was a relationship between the MN formation and lipid peroxidation under several stress conditions. These results agree with the phytotoxicity test and germination rate, which also showed the significant effect of C + R oils at the higher concentration together with a dose dependent trend (Figure 2A and Figure 4A).
Several studies reported that essential oils, by penetrating through the cell wall and cytoplasmic membrane, disrupt and permeabilize them, especially damaging mitochondrial membranes. The mitochondria, by changes in electron flow through the electron transport chain, produce free radicals, which oxidize and damage lipids, proteins, and DNA (Sakihama et al., 2002; Jimenez Del Rio and Velez-Pardo, 2004; Azmi et al., 2006). Further, eugenol, the major constituent in clove essential oil, showed its antimicrobial activity by its ability to permeabilize the cell membrane and interact with proteins. Hemaiswarya and Doble (2009) demonstrated that eugenol action on membranes occurs mainly by a nonspecific permeabilization, as evidenced by increased transport of potassium and ATP out of the cells. Finally, TTO seems to affect cell
Figure 5. (A) Comet assay performed on Vicia faba roots treated with mixture of clove oil and rosemary oil. (B) Comet assay performed on Vicia faba roots treated with tea tree oil. Tail Length is used as parameter to measure DNA damage. Asterisk indicates significant differences compared to Tween 20 by ANOVA with Dunnett test at p < .05.
JOURNAL OF TOXICOLOGY AND ENVIRONMENTAL HEALTH, PART A
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membrane in many microorganisms (Saad et al., 2013). In our study, DNA damage was also confirmed by the Comet test. The intermediate concentration of both treatments showed significant results and may represent the useful concentration to perform genotoxicity tests because at the higher concentration the synergistic effect of the combination of different essential oils may lead to cell death (Figures 5A and 5B). The possible mechanism of essential oil toxicity is either to react with DNA or, by the capability to generate ROS, to induce DNA damage (Gupta et al., 2010). During normal metabolism of the cell, ROS are generated in low amounts and regulate various biological processes such as signal transduction pathways. At high and/or sustained levels, ROS severely damage DNA, protein, and lipids (Lau
et al., 2008), according to the scheme in Figure 8. TTO seems to exhibit a repairable DNA damage as evidenced by inhibition of root growth and the Comet test. However, the C + R oils mixture displayed a genotoxic effect (MN induction), together with a dose-dependent phytotoxicity. It is therefore conceivable that the combination of essential oils may determine a synergistic effect. It is important to understand the effect of the combination of different oils, even if used at lower concentrations, compared to the treatment with a single oil (Tisserand and Young, 2013). These assumptions are important in light of increasing interests in the use of essential oil combinations to improve their natural antimicrobial and antifungal activities (Sturchio et al., 2014). It is important to evaluate the concentration of some active molecules of essential oils as well as
Frequency of cell divisions 3,5 3 2,5
*
2 1,5 1 0,5
A)
Tea tree oil 0.9 mL/L
Tea tree oil 0.6 mL/L
Tea tree oil 0.3 mL/L
0
Tween
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Figure 6. (A) Micronuclei test (MN) performed on Vicia faba roots treated with mixture of clove oil and rosemary oil. (B) The frequency of micronuclei is expressed as a percentage. Asterisk indicates significant differences compared to Tween 20 by ANOVA with Dunnett test at p < .05.
B)
Figure 7. (A) Frequency of cell divisions calculated on Vicia faba roots treated with mixture of clove oil and rosemary oil. (B) Frequency of cell divisions calculated on Vicia faba roots treated with tea tree oil. Asterisk indicates significant differences compared to Tween 20 by ANOVA with Dunnett test at p < .05.
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Figure 8. Scheme of reactive oxygen species (ROS) activity, which, according to a second messenger, induce oxidative damage to lipids, proteins, and DNA in plants.
their biological activity and to identify the range of concentration to attain minimal toxicity. Data suggest that the antibacterial activity of high concentrations of TTO showed a similar mode of action involving the human cell membrane and bacterial cell wall (Söderberg et al., 1996), while lower concentrations of TTO were lethal to bacteria and yet not toxic to human fibroblasts (Loughlin et al., 2008). Further, eugenol and similar compounds rapidly neutralize free radicals, but are themselves converted to phenoxyl radicals. These radicals and their secondary metabolites appear to be responsible for cytotoxicity. In conclusion, data suggest that it is difficult to predict the interactions between chemical constituents of essential oils in a mixture, because their combinations lead to different effects, such as additive, synergistic, or antagonist (Tisserand and Young, 2013). In light of this assumption, it is important to evaluate more comprehensively the biological activity of essential oils and to identify the range of concentration producing greater efficacy for antimicrobial and antifungal activities, at the same time minimizing potential toxicity. The preliminary results showed that synergistic effect of essential oil components in the mixture of the three oils exerted
a positive synergism, considering the decrease of the genotoxic effect compared to the mixture of two oils (Sturchio et al., 2015). In conclusion, genotoxicity analysis may represent an effective method to understand essential oil-induced toxicity and assess specific DNA damage. Future studies need to focus on ROS quantification or a modified comet assay with specific enzymes that recognize the oxidized bases in order to elucidate the essential oil mechanism of actions.
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components and commercial antifungal agents. J. Food Protect. 45: 1298–1301. Bakkali, F., Averbeck, S., Averbeck, D., and Idaomar, M. 2008. Biological effects of essential oils—A review. Food Chem Toxicol 46: 446–475. Bishop, C. D. 1995. Antiviral activity of the essential oil of Melaleuca alternifolia (Maiden and Betche) Cheel (tea tree) against tobacco mosaico virus. J. Essent. Oil Res. 7: 641–644. Boccia, P., Meconi, C., Mecozzi, M., and Sturchio, E. 2013. Molecular modifications induced by inorganic arsenic in Vicia faba investigated by FTIR, FTNIR spectroscopy and genotoxicity testing. J. Toxicol. Environ. Health A 76: 281– 290. Bradley, P. 2006. British herbal compendium. Volume 2: A handbook of scientific information of widely used plant drugs. Bournemouth: British Herbal Medicine Association. Burt, S. 2004. Essential oils: Their antibacterial properties and potential applications in foods-a review. Int. J. Food Microbiol. 94: 223–253. Copping, L. G., ed. 2009. The manual of biocontrol agents, 4th ed. Farnham: BCPC. Dix, T. A., and Aikens, J. 1993. Mechanisms and biological relevance of lipid peroxidation initiation. Chem. Res. Toxicol. 6: 2–18. European Commission. 2009. Directive 2009/128/EC of the European Parliament and of the Council of 21 October 2009 establishing a framework for Community action to achieve the sustainable use of pesticides, 24-11-2009. 2009. Off. J. Eur. Union L 309:71. Fu, Y., Zu, Y., Chen, L., Shi, X., Wang, Z., Sun, S., and Efferth, T. 2007. Antimicrobial activity of clove and rosemary essential oils alone and in combination. Phytother. Res. 21: 989–994. Galletti B., Collina, M., Sedda, G., Portilli, I., and Brunelli, A. 2008. Verifiche di laboratorio, serra e campo, dell’attività di estratti vegetali contro fitopatogeni fungini. Proc. Gior. Fitopatol. 2: 525–532. Gautam, N., Mantha, A. K. and Mittal, S. 2014. Essential oils and their constituents as anticancer agents: A mechanistic view. BioMed. Res. Int. article 154106. http://dx.doi.org/10. 1155/2014/154106. Gupta, S. C., Mishra, M., Sharma, A., Deepak Balaji, T. G. R., Kumar, R., Mishra, R. K., and Chowdhuri, D. K. 2010. Chlorpyrifos induces apoptosis and DNA damage in Drosophila through generation of reactive oxygen species. Ecotoxicol. Environ. Safety 73: 1415–1423. Hemaiswarya, S., and Doble, M. 2009. Synergistic interaction of eugenol with antibiotics against gram-negative bacteria. Phytomedicine 16: 997–1005. Jaganathan, S.K., Mazumdar, A., Mondhe, D., and Mandal, M. 2011. Apoptotic effect of eugenol in human colon cancer cell lines. Cell Biol. Int. 35: 607–615. Jimenez Del Rio, M., and Velez-Pardo, C. 2004. Transition metal-induced apoptosis in lymphocytes via hydroxyl radical generation, mitochondria dysfunction, and caspase-3 activation: An in vitro model for neurodegeneration. Arch. Med. Res. 35: 185–193.
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Kamatou, G. P., Vermaak, I., and Viljoen, A. M. 2012. Eugenol—From the remote Maluku Islands to the international market place: A review of a remarkable and versatile molecule. Molecules 17: 6953–6981. Khan, A., Ahmad, A., Akhtar, F., Yousuf, S., Xess, I., Khan, L. A., and Manzoor, N. 2011. Induction of oxidative stress as a possible mechanism of the antifungal action of three phenylpropanoids. FEMS Yeast Res 11: 114–122. Kong, X., Liu, X., Li, J., and Yang, Y. 2014. Advances in pharmacological research of eugenol. Curr. Opin. Complement. Altern. Med. 1: 8–11. Koppen, G., and Verschaeve, L. 1996. The alkaline comet test on plant cell: A new genotoxicity test for DNA strand breaks in Vicia faba root cells. Mutat. Res. 360: 193–200. Lau, A. T., Wang, Y., and Chiu, J.-F. 2008. Reactive oxygen species: Current knowledge and applications in cancer research and therapeutic. J. Cell. Biochem. 104: 657–667. Loughlin, R., Gilmore, B. F., McCarron, P. A., and Tunney, M. M. 2007. Comparison of the cidal activity of tea tree oil and terpinen-4-ol against clinical bacterial skin isolates and human fibroblast cells. Lett. Appl. Microbiol. 46: 428–433 Lu, S. M., Chen, Z. L., Chen, J. X., et al. 2008. A study on in vitro bacteriostatic action of eugenol. Shi Pin Ke Xue; 29: 122–124. Ma, T. H. 1982. Tradescantia cytogenetic tests root-tip mitosis, pollen mitosis, pollen mother-cell meiosis. Mutat. Res. 99: 293–302. Maistro, E. L., Mota, S. F., Lima, E. B., Bernardes, B. M., and Goulart, F. C. 2010. Genotoxicity and mutagenicity of Rosmarinus officinalis (Labiatae) essential oil in mammalian cells in vivo. Genet. Mol. Res. 9: 2113–2122. Mari, M., Bertolini P., and Pratella, G. C. 2003. Non-conventional methods for the control of post-harvest pear diseases. J. Appl. Microbiol. 94: 761–766. Mecozzi, M., Pietroletti, M., and Di Mento, R. 2007. Application of FTIR spectroscopy in ecotoxicological studies supported by multivariate analysis and 2D correlation spectroscopy. Vib. Spectrosc. 44: 228–235. Menke, M., Angelis, K. J., and Schubert, I. 2000. Detection of specific DNA lesions by a combination of comet assay and fish in plants. Environ. Mol. Mutagen. 35: 132–138. Patlolla, A. K. 2013. Environmental toxicity monitoring of nanomaterials using Vicia faba GENE-TOX Assay. J. Nanomed. Nanotechol. 4: e129. Perromat, A., Mewlin, A., and Déléris, G. 2001. Pharmacologic application of Fourier transform infrared spectroscopy: The in vivo toxic effect of carrageenan. Appl. Spectrosc. 55: 1165–1171. Pietroletti, M., Capobianchi, A., Ragosta, E., and Mecozzi, M. 2010. Preliminary evaluation of hydrocarbon removal power of Caulerpa racemosa in seawater by means of infrared and visible spectroscopic measurements. Spectrochim. Acta A 77: 673–679. Reuveni, M., Agapov, V., and Reuveni, R. 1996. Controlling powdery mildew caused by Sphaerotheca fuliginea in cucumber by foliar sprays of phosphate and potassium salts. Crop Protect. 15: 49–53.
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Rosa, E. V., Valgas, C., Souza-Sierra, M. M., Correa, A. X., and Radetski, C. M. 2003. Biomass growth, micronucleus induction, and antioxidant stress enzyme responses in Vicia faba exposed to cadmium in solution. Environ. Toxicol. Chem. 22: 645–649. Saad, N. Y., Muller, C. D., and Lobstein, A. 2013. Major bioactivities and mechanism of action of essential oils and their components. Flavour Fragr. J. 28: 269–279. Sakihama, Y., Cohen, M. F., Grace, S. C., and Yamasaki, H. 2002. Plant phenolic antioxidant and prooxidant activities: Phenolics-induced oxidative damage mediated by metals in plants. Toxicology 177: 67–80. Scientific Committee on Consumer Products. n.d. SCCP opinion on tea tree oil. SCCP/1155/08. http://ec.europa.eu/ health/ph_risk/risk_en.html. Selim, S. M., Zayed Mona, S., and Atta Houssam, M. 2012. Evaluation of phytotoxicity of compost during composting process. Nat. Sci. 10: 69–77. Shelef, L. A. 1983. Antimicrobial effects of spice. J. Food. Safety 6: 29–44. Söderberg, T. A., Johansson, A., and Gref, R. 1996. Toxic effects of some conifer resin acids and tea tree oil on human epithelial and fibroblast cells. Toxicology 107: 99–109. Sturchio, E., Boccia, P., Meconi, C., Zanellato, M., Marconi, S., Beni, C., Aromolo, R., Ciampa, A., and Valentini, M. 2011. Methods to investigate arsenic contamination on soil–plant system. Chem. Ecol. 27: 1–12. Sturchio, E., Donnarumma, L., Annesi, T., Milano, F., Casorri, L., Masciarelli, E., Zanellato, M., Meconi, C., and Boccia, P. 2014. Essential oils: An alternative approach to management of powdery mildew diseases. Phytopathol. Mediter. 53: 385–395.
Sturchio, E., Mecozzi, M., Boccia, P., Meconi, C., Zanellato, M., and Donnarumma, L. 2015. Genotoxic effects of different mixtures of essential oils. Proceedings of the Fifth International Conference on Environmental Management, Engineering, Planning and Economics (CEMEPE 2015) and SECOTOX Conference, June 14-18, 2015, Mykonos Island, Greece. Sturchio, E., Napolitano, P., Beni, C., and Mecozzi, M. 2012. Evaluation of arsenic effects in Vicia faba by FTIR and FTNIR spectroscopy. Global NEST J. 14: 86–92. Terzi, V., Morcia, C., Faccioli, P., Valè, G., Sacconi, G., and Malnati, M. 2007. In vitro antifungal activity of the tea tree (Melaleuca alternifolia) essential oil and its major components against plant pathogens. Lett. Appl. Microbiol. 44: 613–618. Tisserand, R., and Young, R. 2013. Essential oil safety: A guide for health care professionals. Edinburgh: Elsevier Health Sciences. Tworkoski, T. 2002. Herbicide effects of essential oils. Weed Sci. 50: 425–431. Van Ginkel, G., and Sevanian, A. 1994. Lipid peroxidation induced membrane structural alterations. Methods Enzymol. 233: 273–288. Vardi, Y., and Reuveni, M. 2009. Antifungal activity of a new broad spectrum biocide in the controlling of plant diseases. Phytopathology 99: 5134. Yamamoto, Y., Kobayashi, Y., and Matsumoto, H. 2001. Lipid peroxidation is an early symptom triggered by aluminum, but not the primary cause of elongation inhibition in pea roots. Plant Physiol. 125: 199–208.
ISSN: 1528-7394 (Print) 1087-2620 (Online) Journal homepage: http://www.tandfonline.com/loi/uteh20
Molecular and structural changes induced by essential oils treatments in Vicia faba roots detected by genotoxicity testing Elena Sturchio, Priscilla Boccia, Miriam Zanellato, Claudia Meconi, Lucia Donnarumma, Giuseppe Mercurio & Mauro Mecozzi To cite this article: Elena Sturchio, Priscilla Boccia, Miriam Zanellato, Claudia Meconi, Lucia Donnarumma, Giuseppe Mercurio & Mauro Mecozzi (2016): Molecular and structural changes induced by essential oils treatments in Vicia faba roots detected by genotoxicity testing, Journal of Toxicology and Environmental Health, Part A, DOI: 10.1080/15287394.2015.1124059 To link to this article: http://dx.doi.org/10.1080/15287394.2015.1124059
Published online: 25 Feb 2016.
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Date: 26 February 2016, At: 15:13
JOURNAL OF TOXICOLOGY AND ENVIRONMENTAL HEALTH, PART A http://dx.doi.org/10.1080/15287394.2015.1124059
Molecular and structural changes induced by essential oils treatments in Vicia faba roots detected by genotoxicity testing Elena Sturchioa, Priscilla Bocciaa, Miriam Zanellatoa, Claudia Meconia, Lucia Donnarummab, Giuseppe Mercurioc, and Mauro Mecozzid
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a Italian Workers’ Compensation Authority (INAIL), Department of Technological Innovation and Safety of Plants, Product and Anthropic Settlements (DIT), Rome, Italy; bConsiglio per la Ricerca in Agricoltura e l’Analisi dell’Economia Agraria, Centro di Ricerca per la Patologia Vegetale (CREA-PAV), Rome, Italy; cItalian Red Cross–Army Corps–Central Laboratory, Rome, Italy; dLaboratory of Chemometrics and Environmental Applications (ISPRA), Rome, Italy
ABSTRACT
ARTICLE HISTORY
Over the last few years, there has been an increased interest in exploiting allelopathy in organic agriculture. The aim of this investigation was to examine the effects of essential oil mixtures in order to establish their allelopathic use in agriculture. Two mixtures of essential oils consisting respectively of tea tree oil (TTO) and clove plus rosemary (C + R) oils were tested. Phytotoxicity and genotoxicity tests on the root meristems of Vicia faba minor were performed. A phytotoxic influence was particularly relevant for C + R mixture, while genotoxicity tests revealed significant results with both C + R oil mixture and TTO. Phenotypic analysis on Vicia faba minor primary roots following C + R oil mixture treatment resulted in callose production, an early symptom attributed to lipid peroxidation. The approach described in this study, based on genotoxicity bioassays, might identify specific DNA damage induced by essential oil treatments. These tests may represent a powerful method to evaluate potential adverse effects of different mixtures of essential oils that might be useful in alternative agriculture. Future studies are focusing on the positive synergism of more complex mixtures of essential oils in order to reduce concentrations of potentially toxic components while at the same time maintaining efficacy in antimicrobial and antifungal management.
Received 4 March 2015 Accepted 20 November 2015
Currently, there is an increasing interest in using essential oils as alternative practice in agriculture due to their potential herbicide and fungicide properties (Tworkoski, 2002). Directive 2009/128/ EC, “Establishing a Framework for Community Action to Achieve the Sustainable Use of Pesticides,” proposed to reduce pesticide toxicity risk and impact on and environment and human health by development of alternative agricultural techniques through an integrated pest management program (European Commission, 2009). Within the alternative techniques in agriculture, essential oils play a fundamental role as substitutes for pesticides. In fact, essential oils were widely used in ancient times for medicinal and cosmetic applications, due to their antimicrobial, fungicidal and antiparasitical properties (Shelef, 1983; Mari et al., 2003). Essential oils are now
largely used in pharmaceutical, sanitary, cosmetic, and agricultural and food industries. Essential oils are mostly obtained by distillation from aromatic plants and are composed of a variety of volatile molecules, mainly terpenes. Although the mechanism of action remains poorly understood, Saad et al. (2013) reported that the activity of essential oils depends upon the structure and physicochemical properties of their components, which might affect various membrane molecular structures by modifying the calcium and potassium ion channels and receptors of the transport systems. It is well known that essential oil constituents act by multiple pathways and mechanisms involving cell cycle arrest, DNA repair modulation, and antiproliferative activity. Particularly relevant is the production of reactive oxygen species (ROS) that may lead to cell death
CONTACT Elena Sturchio [email protected] Italian Workers’ Compensation Authority (INAIL), Department of Technological Innovation and Safety of Plants, Product and Anthropic Settlements (DIT), Via Roberto Ferruzzi n. 38/40 - 00143 Roma, Italy. Color versions of one or more of the figures in the article can be found online atwww.tandfonline.com/uteh. © 2016 Taylor & Francis
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in cancer cells (Gautam et al., 2014) and inhibit growth of roots in plants (Ahuja et al., 2015). Previously, Sturchio et al. (2014) examined the use of tea tree oil (TTO) and combination of rosemary and clove essential oils (C + R) as potential fungicides in agriculture and showed that it was possible to obtain an alternative approach to management of powdery mildew diseases. The origin and properties of these oils are well recognized. Rosemary (Rosmarinus officinalis, Labiatae) originally comes from southern Europe. Rosemary leaves and oil are commonly used as spice and flavoring agents in food processing because of their desirable flavor and antimicrobial and antioxidant activity, due to phenolic presence of diterpenes, such as rosmarinic acid and carnosol (Maistro et al., 2010). Most health benefits and pharmacological effects of rosemary are the consequence of potent antioxidant activity attributed to the main chemical constituents, which include carnosol, carnosic acid, ursolic acid, rosmarinic acid, and caffeic acid. The antioxidant activity of rosemary constituents against lipid peroxidation and DNA damage induced by ROS was noted in rat liver mitochondria and microsomes at concentrations of 3–30 µM, demonstrating their ability to protect tissues and cells against oxidative stresses (Bradley, 2006). Clove oil is extracted from Eugenia caryophyllata plant and its main constituent is eugenol. Eugenol is an extraordinarily versatile molecule and has been included as an ingredient in cosmetics, and foods. Kamatou et al. (2012) demonstrated pharmacological properties, such as anaesthetic and analgesic effects, as well as antioxidant activity due to the presence of its phenolic groups. Further, Burt (2004) studied the antibiotic mechanism of eugenol and other flavoring agents, in which the cell wall becomes degraded, damaging the plasma membrane and membrane protein and exposing cellular content. The cytoplasm coagulates, producing the protonmotive force to move far away. Lu et al. (2008) investigated the in vitro antibacterial activity of eugenol by microdilution test and found that eugenol exerted antibacterial action against 12 common bacteria. The hydrophobic property of eugenol can separate lipids of cell membrane and
change its structure to increase cell membrane permeability. It can block the proton-motive force, electron stream, and active transport, and produce coagulation of cell content (Kong et al., 2014). Further, eugenol increases ROS generation leading to DNA fragmentation, a hallmark of apoptosis in treated colon cancer cells (Jaganathan et al., 2011). Tea tree oil is the essential oil obtained by steam distillation of the foliage and terminal branchlets of Melaleuca alternifolia, Melaleuca linariifolia, and Melaleuca dissitiflora, as well as other species of Melaleuca, provided that the oil obtained conforms to the requirements given in the International Standard (ISO 4730-2004). Tea tree oil from Melaleuca alternifolia contains various mono- and sesquiterpenes, as well as aromatic compounds. The monoterpenes terpinen-4-ol, αterpinene, γ-terpinene, 1,8-cineole, p-cymene, αterpineol, α-pinene, limonene, and sabinene account for 80–90% of the oil. The natural content of the individual terpenes in tea tree oil may vary considerably, depending upon (1) Melaleuca alternifolia population used, (2) climate, (3) leaf maceration, (4) age of the leaves, and (5) duration of distillation (Scientific Committee on Consumer Products [SCCP], n.d.). While these properties are increasingly well characterized, relatively limited data are available regarding the safety and toxicity associated with the use of the essential oil. TTO does not directly induce alterations of the cell wall but releases autolytic enzymes associated with the cell membrane, which may induce lysis and subsequent leakage of nucleic acids across damaged cytoplasmic membrane (Saad et al., 2013). The antimicrobial and antifungal activity of combinations of different essential oils as indication of their additive, synergistic, or antagonistic effects against individual microorganism tests was observed. Clove (Syzygium aromaticum L.) and rosemary (Rosmarinus officinalis L.) oils used in combination exhibited significant antimicrobial effects against all microorganisms tested (Fu et al., 2007). In this study the effects of essential oil were determined because their use in agriculture requires knowledge of their potential adverse influence on plants. In fact, essential oils might exert potential
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JOURNAL OF TOXICOLOGY AND ENVIRONMENTAL HEALTH, PART A
toxic effects by changing structural and molecular components by means of specific reactions and/or interactions. Thus, the roots of the probe organism Vicia faba were subjected to in vivo genotoxicity tests. Vicia faba is a common bioindicator plant employed due to its sensitivity to mutagenic effects, such as point mutations to full DNA damage induced by single or complex mixtures of chemicals. Further, plant bioassays may represent easy and inexpensive techniques and thus conducted under a wide range of environmental conditions (Patlolla, 2013). The genotoxicity tests enable detection of specific cell parameters related to DNA structures (Sturchio et al. 2012). By means of this approach, one can estimate stress factors initiated by pollutants and to assess specific molecular DNA damages, and oxidation and alterations of lipid structures, which affect membrane dynamics (Mecozzi et al., 2007; Pietroletti et al., 2010; Sturchio et al., 2012; Boccia et al., 2013).
Materials and methods Sample preparation
The essential oils consisted of TTO and C + R oil mixture. The composition of tested essential oil mixtures was previously analyzed by chromatographic techniques (Sturchio et al., 2014). Oils were emulsified with 0.05% Tween 20 (Reuveni et al., 1996; Terzi et al., 2007) before their application. Treatments were performed for each product with doses indicated on the respective labels or in literature (Galletti et al., 2008; Copping, 2009; Vardi and Reuveni, 2009) and as already described by preliminary investigation previously (Sturchio et al., 2014). The tested concentrations of essential oils represent a range where there is no marked effect on growth inhibition. TTO was added at 0.3, 0.6, or 0.9 ml/L concentrations and C + R oils in the following concentrations: C 0.2 ml/L + R 0.4 ml/L, C 0.4 ml/L + R 0.8 ml/L, and C 0.8 ml/L + R 1.6 ml/L (Table 1). The experimental protocol consisted of phytotoxicity and genotoxicity tests (comet assay and micronuclei [MN] analysis) on primary roots of Vicia faba grown in quartz sand soil treated with the different concentrations of essential oils in aluminum basins. Each basin, containing 250 g quartz sandy soil and 25 Vicia faba seeds, was treated
Table 1. Treatment Mutagenicity Tests.
Programs
Treatment Tween 20: Control test Tween 20 + tea tree oil Tween 20 + tea tree oil Tween 20 + tea tree oil Tween 20 + clove oil + rosemary oil Tween 20 + clove oil + rosemary oil Tween 20 + clove oil + rosemary oil
and
Doses
Applied
3
to
Dose, mL/L 0.3 0.6 0.9 C 0.2 + R 0.4 C 0.4 + R 0.8 C 0.8 + R 1.6
Note. Oils were emulsified with 0.05% Tween 20 before their application.
with 50 ml of each emulsion of essential oils. Basins wetted only with Tween 20 were used as negative controls. Three basins were set up for each treatment. To verify the effects of Tween 20 used as vehicle to dissolve the oils, Vicia faba seeds were allowed also to grow in absence of Tween 20 but wetted only with H2O. Each aluminum basin was sealed with Parafilm and incubated in a climatic chamber (21 ± 1ºC, 60% relative humidity) for 5 d to enable germination. All experiments were repeated three times. TTO and the mixtures of C + R oils were tested in two separate trials. The primary roots of seedlings were measured for phytotoxicity and removed for genotoxicity tests. Phytotoxicity testing
After 5 d of incubation, exposed Vicia faba seedlings were collected and total germination percentage (Gt) was calculated as Gt = (n/N × 100), where n is the total number of germinated seeds at the end of the experiment of the given oil treatment and N is the total number of seeds used for germination test. Phytotoxicity was determined by measuring primary root length. The mean of primary root length (L) of each sample was compared with controls, providing an indication of oil effects. Germination Index (GI), evaluated as [(Gt sample × L sample)/(Gt Tween 20 × L Tween 20)] × 100, is a maturity test based on seed germination and initial plant growth where Gt = (n/N × 100) is the Gt percentage, and L is the average length of the primary root of the n germinated seeds. GI reflects the phytotoxicity of the essential oil, where low toxicity affects root growth, while high toxicity affects germination (Selim et al., 2012).
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Genotoxicity testing
Statistical analysis
Comet assay Single-cell gel electrophoresis (SCGE) is a cytogenetic test used to assess in a single cell genetic damage induced by mutagenic agents. The test is a simple, sensitive, and rapid shortterm genotoxicity method for measuring under alkaline conditions, pH ≥ 13.5, all DNA structural damages, including double-strand/singlestrand (ds/ss) breaks, alkali-labile sites such abasic sites, and oxidized bases (Koppen and Verschaeve, 1996; Menke et al., 2000). The comet test was performed under an alkaline unwinding/alkaline electrophoresis (A/A) using a Comet assay kit (Trevigen, Gaithersburg, MD) according to the manufacturer’s protocol. Tail length (TL) has been used as a parameter of DNA damage. Each experiment was repeated three times (in three biological replicates).
Phytoxicity and genotoxicity experimental data sets were subjected to one-way analysis of variance (ANOVA) followed by Dunnett’s test for comparing the difference of mean and standard error at the .05 level for statistical significance versus control using the statistical software package SPSS (Chicago, IL).
Micronucleus test Micronuclei (MN) are Feulgen-positive corpuscles localized within the cell wall in the cytoplasmic area surrounding the main nucleus. MN are formed by chromosomes or chromosome fragments that are not incorporated into daughter nuclei at the time of cell division (Ma, 1982). Micronuclei test evaluates the frequency of micronucleated cells in root meristems of Vicia faba, analyzing 15,000 cells (15 root tips, 1,000 cells for tip). Frequency of cell division was also analyzed. The experiment was performed as described by Sturchio et al. (2011) and repeated three times (in three biological replicates).
Results Phytotoxicity and genotoxicity testing
Vicia faba seeds were allowed to grow in the presence and in absence of Tween 20 used as vehicle to dissolve the oil, in order to determine any potential effects of Tween 20. Since no significant differences were observed between seeds grown with and without Tween 20 (data not shown), the experiments described hereafter report only results obtained in the presence of Tween 20 as control. A primarily phenotypic analysis of exposed Vicia faba primary roots presented callose production only at higher concentration of C + R oil treatment, as an early symptom attributed to lipid peroxidation as reported by Yamamoto et al. (2001) (Figure 1). A phytotoxic effect was observed in both trials (Figures 2A and 2B). It was more marked for the C + R mixture, exhibiting a linear dose dependence with concomitant decrease in GI and of the Gt% (Figures 3A and 4A). The GI and Gt parameters displayed no significant effects with TTO oil treatment (Figure 3B and Figure 4 B). Comet assay showed DNA damage at the intermediate concentration of C + R oil mixture (C 0.4 ml/L + R 0.8 ml/L) and at the intermediate and higher concentrations tested of TTO (0.6 ml/L and 0.9 ml/L) (Figures 5A and 5B). The MN test per-
Figure 1. Phenotypic analysis of Vicia faba roots. Sample treated with clove oil 0.8 ml/L + Rosemary oil 1.6 ml/L. Meristems showed a callose production after the treatment as an early symptom caused by lipid peroxidation.
JOURNAL OF TOXICOLOGY AND ENVIRONMENTAL HEALTH, PART A
5
Primary root lenght (mm) 40 35 30
*
25
*
*
20 15 10
Tea tree oil 0.9 mL/L
B)
Tea tree oil 0.6 mL/L
A)
Tea tree oil 0.3 mL/L
0
Tween
5
formed with C + R oil mixture revealed an elevated MN frequency at the intermediate concentration (C 0.4 ml/L + R 0.8 ml/L) and a decreased cell division frequency at the higher concentration (C 0.8 ml/L + R 1.6 ml/L) (Figure 6A and 7A). No marked effects on MN and frequency of cell divisions were observed in TTO trials (Figure 6B and 7B). Discussion Although essential oils have been demonstrated to possess antimicrobial properties (Shelef, 1983), antimycotic activity (Azzouz and Bullerman, 1982; Akgül and Kivanç, 1988; Mari et al., 2003), and antiviral activity (Bishop, 1995) and to be effective against a broad spectrum of plant pathogenic fungi (Vardi and Reuveni, 2009) their mechanisms of action remain to be determined. As potential lipophiles, essential oils pass through the
cytoplasmic membrane and disrupt the structure of their different layers of polysaccharides, fatty acids, and phospholipids, and permeabilize them. Cytotoxicity includes this type of membrane damage. Damage to cell wall and membrane leads to leakage of intracellular macromolecules and lysis (Bakkali et al., 2008). The results obtained performing a phenotypic analysis on samples of Vicia faba-treated roots exposed at higher concentration of C + R oils yielded callose structures probably due to lipid peroxidation as reported by Yamamoto et al. (2001) (Figure 1). Lipid peroxidation is one of the most important expressions of oxidative stress induced by ROS. ROS readily react with unsaturated lipids and produce polar lipid hydroperoxides that produce increased membrane fluidity by disturbing hydrophobic phospholipids (Dix and Aikens, 1993; Van Ginkel and Sevanian, 1994; Khan et al., 2011).
Germination Index (%)
Germination Index (%)
120 140
100
120
80
*
100
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*
40 20
Tea tree oil 0.9 mL/L
B)
Tea tree oil 0.6 mL/L
0 Tea tree oil 0.3 mL/L
Clove oil 0.4 mL/L + Rosemary oil 0.8 mL/L
Clove oil 0.2 mL/L + Rosemary oil 0.4 mL/L
A)
Clove oil 0.8 mL/L + Rosemary oil 1.6 mL/L
*
20
0
60
Tween
40
Tween
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Figure 2. (A) Phytotoxicity tests performed on Vicia faba roots treated with mixture of clove oil and rosemary oil. (B) Phytotoxicity tests performed on Vicia faba roots treated with tea tree oil. Asterisk indicates significant differences compared to Tween 20 by ANOVA with Dunnett test at p < .05.
Figure 3. (A) Germination index calculated on Vicia faba roots treated with mixture of clove oil and rosemary oil. (B) Germination index calculated on Vicia faba roots treated with tea tree oil. Asterisk indicates significant differences compared to Tween 20 by ANOVA with Dunnett test at p < .05.
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Total Germination percentage (%)
Total Germination percentage (%)
100
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80 70
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50 40
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30 20
Tea tree oil 0.9 mL/L
B)
Tea tree oil 0.6 mL/L
0 Tween
Clove oil 0.8 mL/L + Rosemary oil 1.6 mL/L
Clove oil 0.4 mL/L + Rosemary oil 0.8 mL/L
Tween
10 Clove oil 0.2 mL/L + Rosemary oil 0.4 mL/L
A)
30 20
10 0
40
Tea tree oil 0.3 mL/L
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Figure 4. (A) Total germination percentage (Gt) calculated on Vicia faba roots treated with mixture of clove oil and rosemary oil. (B) Total germination percentage (Gt) calculated on Vicia faba roots treated with tea tree oil. Asterisk indicates significant differences compared to Tween 20 by ANOVA with Dunnett test at p < .05.
Lipid peroxidation has been also related to MN induction. At the intermediate concentration of essential oils (C + R oils) a significant increase of MN frequency was noted (Figure 6A). Conversely, a significant reduction in frequency of cell division was evident at the higher concentration where it was not possible to detect micronucleated cells (Figure 7A). ROS overproduction coupled with deficiency of antioxidant defense mechanisms may be an important factor contributing to elevated MN frequency. Rosa et al. (2003) found that there was a relationship between the MN formation and lipid peroxidation under several stress conditions. These results agree with the phytotoxicity test and germination rate, which also showed the significant effect of C + R oils at the higher concentration together with a dose dependent trend (Figure 2A and Figure 4A).
Several studies reported that essential oils, by penetrating through the cell wall and cytoplasmic membrane, disrupt and permeabilize them, especially damaging mitochondrial membranes. The mitochondria, by changes in electron flow through the electron transport chain, produce free radicals, which oxidize and damage lipids, proteins, and DNA (Sakihama et al., 2002; Jimenez Del Rio and Velez-Pardo, 2004; Azmi et al., 2006). Further, eugenol, the major constituent in clove essential oil, showed its antimicrobial activity by its ability to permeabilize the cell membrane and interact with proteins. Hemaiswarya and Doble (2009) demonstrated that eugenol action on membranes occurs mainly by a nonspecific permeabilization, as evidenced by increased transport of potassium and ATP out of the cells. Finally, TTO seems to affect cell
Figure 5. (A) Comet assay performed on Vicia faba roots treated with mixture of clove oil and rosemary oil. (B) Comet assay performed on Vicia faba roots treated with tea tree oil. Tail Length is used as parameter to measure DNA damage. Asterisk indicates significant differences compared to Tween 20 by ANOVA with Dunnett test at p < .05.
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membrane in many microorganisms (Saad et al., 2013). In our study, DNA damage was also confirmed by the Comet test. The intermediate concentration of both treatments showed significant results and may represent the useful concentration to perform genotoxicity tests because at the higher concentration the synergistic effect of the combination of different essential oils may lead to cell death (Figures 5A and 5B). The possible mechanism of essential oil toxicity is either to react with DNA or, by the capability to generate ROS, to induce DNA damage (Gupta et al., 2010). During normal metabolism of the cell, ROS are generated in low amounts and regulate various biological processes such as signal transduction pathways. At high and/or sustained levels, ROS severely damage DNA, protein, and lipids (Lau
et al., 2008), according to the scheme in Figure 8. TTO seems to exhibit a repairable DNA damage as evidenced by inhibition of root growth and the Comet test. However, the C + R oils mixture displayed a genotoxic effect (MN induction), together with a dose-dependent phytotoxicity. It is therefore conceivable that the combination of essential oils may determine a synergistic effect. It is important to understand the effect of the combination of different oils, even if used at lower concentrations, compared to the treatment with a single oil (Tisserand and Young, 2013). These assumptions are important in light of increasing interests in the use of essential oil combinations to improve their natural antimicrobial and antifungal activities (Sturchio et al., 2014). It is important to evaluate the concentration of some active molecules of essential oils as well as
Frequency of cell divisions 3,5 3 2,5
*
2 1,5 1 0,5
A)
Tea tree oil 0.9 mL/L
Tea tree oil 0.6 mL/L
Tea tree oil 0.3 mL/L
0
Tween
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Figure 6. (A) Micronuclei test (MN) performed on Vicia faba roots treated with mixture of clove oil and rosemary oil. (B) The frequency of micronuclei is expressed as a percentage. Asterisk indicates significant differences compared to Tween 20 by ANOVA with Dunnett test at p < .05.
B)
Figure 7. (A) Frequency of cell divisions calculated on Vicia faba roots treated with mixture of clove oil and rosemary oil. (B) Frequency of cell divisions calculated on Vicia faba roots treated with tea tree oil. Asterisk indicates significant differences compared to Tween 20 by ANOVA with Dunnett test at p < .05.
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Figure 8. Scheme of reactive oxygen species (ROS) activity, which, according to a second messenger, induce oxidative damage to lipids, proteins, and DNA in plants.
their biological activity and to identify the range of concentration to attain minimal toxicity. Data suggest that the antibacterial activity of high concentrations of TTO showed a similar mode of action involving the human cell membrane and bacterial cell wall (Söderberg et al., 1996), while lower concentrations of TTO were lethal to bacteria and yet not toxic to human fibroblasts (Loughlin et al., 2008). Further, eugenol and similar compounds rapidly neutralize free radicals, but are themselves converted to phenoxyl radicals. These radicals and their secondary metabolites appear to be responsible for cytotoxicity. In conclusion, data suggest that it is difficult to predict the interactions between chemical constituents of essential oils in a mixture, because their combinations lead to different effects, such as additive, synergistic, or antagonist (Tisserand and Young, 2013). In light of this assumption, it is important to evaluate more comprehensively the biological activity of essential oils and to identify the range of concentration producing greater efficacy for antimicrobial and antifungal activities, at the same time minimizing potential toxicity. The preliminary results showed that synergistic effect of essential oil components in the mixture of the three oils exerted
a positive synergism, considering the decrease of the genotoxic effect compared to the mixture of two oils (Sturchio et al., 2015). In conclusion, genotoxicity analysis may represent an effective method to understand essential oil-induced toxicity and assess specific DNA damage. Future studies need to focus on ROS quantification or a modified comet assay with specific enzymes that recognize the oxidized bases in order to elucidate the essential oil mechanism of actions.
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