DNA damages promoted by the essential oil from leaves of Casearia sylvestris Sw . ( Salicaceae )

1 Programa de Pós-Graduação em Biotecnologia Vegetal, Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro (RJ), Brazil. 2 Instituto Brasileiro de Geografia e Estatística (IBGE), Diretoria de Geociências – Coordenadoria de Recursos Naturais – CREN, Rio de Janeiro (RJ), Brazil. 3 Departamento de Biofísica e Biometria, Universidade do Estado do Rio de Janeiro (UERJ), Rio de Janeiro (RJ), Brazil. 4 Departamento de Biologia, Universidade do Estado do Rio de Janeiro (UERJ), Rio de Janeiro (RJ), Brazil. 5 Instituto de Tecnologia em Fármacos, Departamento de Produtos Naturais, Fundação Oswaldo Cruz (Fiocruz), Rio de Janeiro (RJ), Brazil.


INTRODUCTION
Casearia sylvestris Sw. (Salicaceae) can be found throughout the Brazilian territory, including the State of Rio de Janeiro (Marquete and Vaz, 2007;Marquete and Mansano, 2013), and is popularly known as "guaçatonga".This species presents several medicinal properties, according to ethnobotanical surveys (Tomazi *Corresponding author.E-mail: flaviane.gp@gmail.com.Tel: 55 21 98189-1110.et al., 2014).It is one of the 71 plants of interest of the Brazilian Public Health System (Sistema Único de Saúde (SUS)), due to its wide use by the population to treat different conditions, such as herpes and tumors (Pereira et al., 2016;Ferreira et al., 2016).
Recent studies reported that the essential oils of Casearia genus are rich in sesquiterpenes, while monoterpenes are rare and arylpropanoids are absent (Esteves et al., 2005;Tininis et al., 2006;Sousa et al., 2007;Silva et al., 2008).In addition, the sesquiterpenes showed cytotoxic activity against different tumor cell lines (Silva et al., 2008;Bou et al., 2013) as well as genotoxic effects (Péres et al., 2009;Maistro et al., 2010;Ortiz et al., 2016).However, although this species is widely used by the Brazilian population, possible genotoxic effects promoted by the essential oil from leaves have not been investigated so far.

Essential oil extraction and analysis
Fresh leaves of C. sylvestris (1.5 kg) were collected in June 2014, chopped into to small pieces and led to hydrodistillation in a modified Clevenger-type apparatus for 2 h.Essential oil was directly separated from the aqueous phase yielding 1.2% (w/v), transferred to amber flasks and kept at low temperature (-20°C) until analysis.Essential oil was subjected to analysis by gas chromatography coupled to flame ionization detector (HP-Agilent 6890, GC-FID), and by gas chromatography coupled to mass spectrometry (HP Agilent GC 6890 -MS 5973), at the Analytical Platform of Institute of Pharmaceutical Technology, Farmanguinhos, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil.Briefly, the essential oil was diluted in dichloromethane (1.0 mg/ml) and analyzed by GC-MS to obtain the mass spectra and to perform chemical characterization.Concomitantly, another sample of essential oil (0.5 mg/ml) was analyzed by GC-FID for quantification of chemical constituents and to determine the retention indices (RI).The relative abundance of each essential oil component in the sample was quantified based in the individual component's relative peak area in the chromatogram.The substances in the essential oil were identified by comparing their mass spectra with database registration (WILEY7n) and by comparison of calculated Retention Indices (RI) with those from the literature (Adams, 2001;Pereira et al., 2016).RI were calculated from GC data of a homologous series of saturated aliphatic hydrocarbons within C8 to C20 (Sigma-Aldrich), performed using the same column and conditions used in the GC analysis for the essential oils, and with the equation proposed by Van dendool and Kratz (1963).

Evaluation of cell viability by the clonogenic assay
The A549 cell lineage was obtained from Microbiology Department Pereira et al. 819 from the Rio de Janeiro State University (Brazil).The cytotoxic potential of the essential oil was measured by the mitogenic capacity of treated cells using the clonogenic assay (Franken et al., 2006).

Comet assay
The procedure of comet assay was performed as described by Tice (2000), Cestari et al. (2004), and Miyaji et al. (2004) with modifications.Briefly, for each slide, 50 nuclei were randomly observed, always from the left to the right side, and total scoring (TS) was by obtained multiplying the number of cells in each class (nx) by the damage class , ranging from 0 to 150 (Dantas et al., 2002).Nuclei were visually classified according to the migration of fragments (tail size) to one of five classes as follows: class 0 (undamaged nuclei, absence of tail), class 1 (nuclei with short tails, smaller than their diameter), class 2 (tail length one to two times the diameter of the nuclei), class 3 (tails twice or the diameter of the nuclei), and class 4 (nuclei maximally damaged).

Statistical analysis
Statistical analysis was performed using the ANOVA and Tukey-Kramer multiple comparison tests by the statistical program InStat 3.01 version (GraphPad Software, San Diego, CA, USA).The significance level of p < 0.05 was adopted to compare data within the same experiment.

Essential oil chemical profile
According to GC-MS, GC-FID and Retention Indices (RI) analysis, it was possible to characterize 21 compounds, comprising 98.2% of the essential oil from leaves of C. sylvestris (Table 1).

Cytotoxic and genotoxic activities
The essential oil exhibited cytotoxic activity against A549 tumor cells, with EC 50 of 4.0 µg/ml, and a dose dependent pattern (r = -0.79,p = 0.03), as determined by linear regression test.It is interesting to note that no cytotoxic effect was detected on Vero cells (African green monkey kidney, maximum nontoxic concentration ≥ 250 µg/ml) (Table 2).The essential oil also presented suppressive effects on the colony-forming ability on A549 cells after the treatment.The results of the comet assay are shown in Table 3.All types of damages could be observed, especially classes 3 and 4 (p < 0.05), when compared with the control.

DISCUSSION
The comet assay was performed to evaluate potential genotoxic effects of essential oils, considering that it is a rapid and sensitive method to measure DNA damages (Tice et al., 2000;Almeida et al., 2012).Evaluation of cytotoxicity by clonogenic assay, and DNA damage of the essential oil have also been investigated.Considering that C. sylvestris is widely used in folk medicine, it is important to verify the toxic effects of its volatile fractions, especially considering that the National Sanitary Surveillance Agency (Agência Nacional de Vigilância Sanitária -ANVISA) recommends the assessment of possible mutagenicity of medicinal plants (Balbino and Dias, 2010).C. sylvestris essential oil is composed exclusively by sesquiterpenes and the main identified compounds were α-humulene (17.8%), spathulenol (11.8%), and α-copaene (8.5%) (Pereira et al., 2016).
For instance, sesquiterpenes were also identified as the major compounds in the essential oils from the leaves of C. sylvestris collected in the Brazilian states of São Paulo, Santa Catarina and Minas Gerais (Tininis et al., 2006;Silva et al., 2008;Esteves et al., 2008;Bou et al., 2013).However, the analyzed essential oil from the state of Rio de Janeiro showed α-humulene (α-caryophyllene) as the major compound.
There are few studies related to cytotoxic activity of essential oils from Salicaceae in tumor cell lines (Silva et al., 2008;Bou et al., 2013;Nikolic et al., 2014;Hayan et al., 2016).This data suggested that sesquiterpenes have cytotoxic activity, and indicated that further chemical and biological studies with other species from Casearia genus should be undertaken.
In recent years, the clonogenic assay is frequently used in cancer research laboratories to determine the survival and proliferation of tumor cells after experimental treatments (Hoffman, 1991;Tian et al., 2016).In these experiments, treatment with essential oil of C. sylvestris at 4.0 µg/ml reduced the colony-forming ability of A549 cells to 6% of the value obtained with the control.This effect could be due to interference of essential oil in the membrane integrity, DNA fragmentation or mitochondrial depolarization (Li et al., 2014;Ruan et al., 2015).
According to literature, the clonogenic assay is the method of choice to determine cell reproductive death after treatment with cytotoxic agents (Franken et al., 2006).The comet assay is widely used to evaluate the genotoxic potential in animal tissues, especially in studies on cancer therapy (Fairbairn et al., 1995;Dantas et al., 2002).According to other studies, the essential oils cause damages on tumor cell lines.For instance it has also been detected that the MCF-7 cell line is not able to repair the lesions on DNA, possibly due to the production of epoxides which induces DNA damage caused by essential oils, as well as by lipid peroxidation of essential oil rich in sesquiterpenes on V79 cells (Péres et al., 2009;Ortiz et al., 2016).There is evidence that DNA damages can be caused due to the epoxide formation by the induction of epoxide hydrolase 1 (EPHX1) responsible for the detoxification of exogenous chemicals (Ortiz et al., 2016).
In addition, this assay is extremely recommended when the toxicity of the agent tested is unknown, especially when there is no available data on cytotoxicity or genotoxicity (Tice et al., 2000).To the best of our Pereira et al. 821 knowledge, this is the first DNA damage study of the essential oil of C. sylvestris in a tumor cell line using two assays to detect potential harmful effects.

Conclusion
The essential oil from leaves of C. sylvestris exhibited potent DNA damage on A549 tumor cell line.
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Table 1 .
Chemical composition of the essential oil from fresh leaves of C. sylvestris.
RIcal = Retention Index values calculated;RIlit = Retention index values from literature data.

Table 2 .
Cytotoxicity activity of the essential oil from fresh leaves of C. sylvestris on A549 cell line.

Table 3 .
Comet assay scores for the essential oil of C. sylvestris on A549 tumor cell line.
Class 0: Undamaged nucleus, no tail; Class 1: nucleus with a short tail; Class 2: tail length is 1 and 2 times the diameter of the head; Class 3: tail is longer than the diameter of the nucleus; Class 4: nucleus totallydamaged; *p<0.05.