Protective effect of kavain in meristematic cells of Allium cepa L.

: Kavain is one of the main kavalactones of Piper methysticum (Piperaceae) with anxiolytic, analgesic, and antioxidant activities. Therefore, the aim of the study was to evaluate the cytotoxic, mutagenic, and antimutagenic potential of kavain in Allium cepa cells. Roots of A. cepa were transferred to the negative (2% acetone) and positive (10 µg/mL of Methylmethanesulfonate, MMS) controls and to the concentrations of kavain (32, 64 and 128 µg/mL) for 48 h. A total of 5,000 meristematic cells were analyzed under an optical microscope to determine the mitotic index, mean number of chromosomal alterations and percentage of damage reduction. Data were analyzed by Kruskal-Wallis test ( p <0.05). All concentrations of kavain were not cytotoxic and did not show significant chromosomal changes when compared to 2% acetone. Kavain showed a cytoprotective effect in the pre (128 μg/mL) and in the post-treatment (32 and 64 μg/ mL) and reduced damage against the mutagenic action of MMS in all concentrations of the pre and simultaneous and at the highest of post (128 μg/mL). Kavain promoted a significant reduction in micronuclei, nuclear buds and chromosomal losses in relation to MMS. The observed data indicate the importance of kavain for the inhibition of damage and chemoprevention.


INTRODUCTION
Medicinal plants have antifungal, antimicrobial, insecticidal, and antiseptic activities (Hosseinzadeh et al. 2015), and are used in many different countries and cultures for the treatment of urinary tract infections, epilepsy and diabetes, playing a key role in research on herbal medicines and the development of new drugs (Asadbeigi et al. 2014, Dias et al. 2014, Aragão et al. 2015).In addition, studies show that approximately 80% of the world population uses medicines of plant origin (Delfan et al. 2014), however they are still used empirically by the population and can cause toxic effects (Bae et al. 2015).
Piper methysticum G. Forster is a perennial shrub of the family Piperaceae known as Kava, kava-kava or awa (Einbonda et al. 2017).Originating from the Pacific Ocean islands, the infusion prepared from dry roots and rhizomes is traditionally used by island communities in religious rituals to induce a relaxed psychological state (Singh & Singh 2002, Lebot & Legendre 2016).In Europe, it is marketed without prescription, as an alternative to benzodiazepines to treat anxiety and insomnia (Chua et al. 2016).
Kava has in its chemical composition several constituents; the main ones are called kavapyrones or kavalactones (Ketola et al. 2015) present mainly in the rhizome (Singh & Singh 2002).In total, 18 kavalactones have been identified, with an emphasis on kavain, yangonin, desmethoxyyangonin, dihydrokavain, methysticin and dihydromethysticin that have greater pharmacological importance (Kuchta et al. 2017); and kavain is present in greater amount in kava extracts (Chua et al. 2016).
Kavain and other kavalactones alone or combined with the extract of kava have the capacity to inhibit several isoforms of cytochrome P450 (CYP450), being: CYP1A2, CYP2C9; CYP2C19; CYP2D6, CYP3A4; CYP4A9 and CYP4A9/11 (Mathews et al. 2005).This property is the source of numerous interactions, mainly pharmacokinetic, with other drugs, as it decreases their metabolism by inhibiting enzymes of the CYP450 complex, which can induce toxicity (Zou et al. 2004, Mathews et al. 2002).However, evidence of pharmacokinetic and/or pharmacodynamic interactions remains unsustainable and only a few investigations have been carried out on the potential of kava and kavalactone preparations to interact with specific drugs.
Studies on the medicinal potential of kavain and its interaction with DNA and spindle fibers are important for safety in its administration.The Allium cepa L. (onion) test system has often been used to monitor the toxicogenetic activity of isolated compounds (Luz et al. 2012, Liman et al. 2019, Shetty et al. 2017, De Souza et al. 2017).The test system for chromosomal changes in A. cepa is widely cited in the literature as a bioindicator for the evaluation of cytotoxicity, genotoxicity and protective effect of chemical compounds, as it has rapid cell multiplication, large and few chromosomes, which allows better analysis of structural and numerical alterations (Bonciu et al. 2018, Leme & Marin-Morales 2009).The A. cepa bioassay stands out for being an excellent bioindicator of mutagenic compounds, has low cost, reliability and agreement with other test organisms, helping studies to prevent damage to human health (Oliveira et al. 2013, Firbas & Amon 2014, Kumar et al. 2015, Liman et al. 2015).According to Rank & Nielsen (1997) and Fedel-Miyasato et al. (2014), a comparison of the mutagenicity assays in A. cepa with tests in rodents shows a agreement of approximately 80%, and therefore allows the confirmation of the mutagenic and antimutagenic mode of action.Besides that, it has a good correlation with cytotoxicity and genotoxicity tests in vitro or in vivo (Eren & Özata 2014, Sá et al. 2019).
A. cepa is also used to assess the reduction of DNA damage (Mauro et al. 2014).A chemoprotection can be desmutagenic or bio-antimutagenic.Desmutagenic substances neutralize mutagenic agents either directly or through their derivatives to inactivate them chemically in the extra-or intracellular medium (Fedel-Miyasato et al. 2014, Felicidade et al. 2014).Bio-antimutagenic agents stimulate repair and replication of DNA and act at the cellular level by increasing reliability of replication, error-free repair and inhibiting repair systems that are subject to error (Oliveira et al. 2006).
With this in mind, the present study aimed to evaluate the mutagenic and antimutagenic potential of this molecule in meristematic cells of A. cepa.

DNA-damaging agent
The MMS was used to induce DNA damage in meristematic cells of A. cepa.MMS (10 μg/ mL) is an alkylating agent with direct activity, inducing disturbances such as DNA breaks, bridges and chromosome loss, which are also expressed as micronuclei (Bianchi et al. 2016, Couto et al. 2019).

A. cepa test
One hundred seeds of A. cepa were germinated at the Genetics Laboratory of FACIME at room temperature in Petri dishes containing filter paper moistened with distilled water.The seeds with roots approximately 2 cm long were subjected to different treatments to evaluate mutagenicity and antimutagenicity according to Couto et al. (2019) andPereira et al. (2020).
To assess mutagenicity, 30 seeds were transferred to the negative control (NC = acetone 2%), solvent (distilled water), positive control I (PC I = 10 μg/mL of MMS solubilized in distilled water), positive control II (PC II = 10 μg/mL of MMS solubilized in acetone 2%) and 32, 64 and 128 μg/mL concentrations of Kavain in separate dishes for each control and concentration.The Kavain concentrations used in this study were pre-determined based on the non-mutagenic effect in D. melanogaster (Silva et al. 2021).
For the pretreatment group, the seeds were transferred to 32, 64 and 128 μg/mL concentrations of Kavain for 24 h, then to MMS solution for additional 24 h.For the simultaneous treatment, the seeds were transferred to ultrapure water for 24 h, then transferred to the 32, 64 and 128 μg/ mL concentrations of Kavain and MMS solution at the same time for an additional 24 h.For the post-treatment group, the seeds were grown in MMS for 24 h and germinated for an additional 24 h in 32, 64 and 128 μg/mL of Kavain.
After mutagenic and antimutagenic treatments, the root tips were fixed in 3:1 methanol:acetic acid and stored at -20 ºC until slide preparation.The root tips were washed in distilled water three times for 5 min each, then hydrolyzed at 60 ºC for 10 min in 1 N HCl.After hydrolysis, the root tips were washed again in distilled water, transferred to amber glass bottles containing Schiff's reagent and kept there for 2 h in the dark.The root tips then were washed until the reagent was removed, transferred onto slides, squashed with one drop of 2% acetic carmine and mounted with Entellan® (107960; Merck Millipore) (Almeida et al. 2015).
The mitotic index (MI) indicates cytotoxicity and chromosome alterations, which reflects mutagenicity.To determine the MI, the number of cells in different phases of mitosis was divided by the total number of cells.For chromosome alterations, the number of alterations was divided by the total number of cells.We scored 5,000 meristematic cells on ten slides/treatment using light microscopy at 400 x magnification (Olympus CX 21, Zhejiang, China).Chromosome alterations included those resulting from aneugenic activity, e.g., C-metaphases, metaphases with chromosome adherence, lost chromosomes, multipolar anaphases, binucleate cells and polyploid metaphases, or clastogenic effects, e.g., chromosome fragments in metaphase or anaphase and chromosome bridges.MN may arise from either aneugenic or clastogenic effects (Anacleto et al. 2017).
Antimutagenic activity was assessed the percentage of damage reduction (%DR).The %DR was calculated for each treatment using the formula: %DR = [(a -b)/(a -c)] x 100 where a = number of damaged cells in the PC, b = number of damaged cells in each treatment, c = number of damaged cells in the NC (Waters et al. 1990).

Statistical analysis
Data were evaluated using the nonparametric test of Kruskal-Wallis followed by the post hoc test of Student-Newman-Keuls using the program, BioEstat 5.3 (Ayres et al. 2007).Values for p ≤ 0.05 were considered statistically significant.

RESULTS AND DISCUSSION
Recent studies have focused on the identification of phytochemicals/isolated compounds with beneficial effects, and on the elucidation of mechanisms that are related to protective action in the cell (Qian et al. 2016, Sharma et al. 2012, Zhang et al. 2016).This reinforces the concern of researchers in the search and development of new drugs that are more efficient against cancer, more effective in protecting and repairing DNA and preventing the formation of tumors (Słoczyńska et al. 2014).Therefore, considering the medicinal importance of kavain and the need for more toxicogenetic information, the present study aimed to investigate the cytotoxic, mutagenic and antimutagenic effect of this molecule on the meristematic cells of A. cepa.
The results of the present study showed that all kavain concentrations (32, 64 and 128 μg/mL) were not cytotoxic, as there was no significant reduction in the mitotic index (MI) of A. cepa cells in relation to the negative control (2% acetone) (Table I).Thus, kavain allowed the progression of the cell cycle of A. cepa, which reinforces its non-interference in DNA synthesis and/or in the inhibition of the G1/S and G2/M checkpoints in cells of A. cepa, as proposed by Mauro et al. (2014) with the inulin isolate.The non-cytotoxic effect has also been found in previous studies on kavain in human liver hepatocellular carcinoma cells (HepG2) using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide], lactate dehydrogenase (LDH) and ethidium bromide (EB) (Tang et al. 2011) assays in hippocampus cells (Mulholland & Prendergast 2002), in astrocytoma and bone cells of rats, using the LDH and MTS assays (Wruck et al. 2008, Guo et al. 2018).In these studies, the concentrations were lower (ranging from 0.23 to 23 μg/mL), however even at higher concentrations (IC 50 = 166 ± 14 μg/mL), kavain also showed low toxicity to Hepa1c1c7 liver cells (Shaik et al. 2009).
All kavain concentrations were not mutagenic (total mean chromosomal changes) to A. cepa cells, as the mean values were not significantly different from the negative control (NC) (Table I).
In addition, all chromosomal changes assessed individually were also not significant, except for micronuclei (MN) with lower mean values than NC (Table II) (Figure 1), showing that kavain did not interfere with the chromatin condensation processes, polymerization of mitotic spindle Data are means ± SD.NC, negative control (acetone 2%); MMS, methyl methanesulfonate (10 μg/mL, positive control); pretreatment, 24 h Kavain + 24 h MMS; simultaneous treatment, 24 h distilled water + 24 h combined Kavain and MMS; posttreatment, 24 h MMS + 24 h Kavain.* Significant difference for the Kavain compared to NC. + Significant difference for pre, simultaneous and post-treatment compared to MMS.Significant by Kruskal-Wallis test with a posteriori Student-Newman-Keuls test ( + p < 0.05; **/++ p < 0.01).Data are for 5,000 cells/treatment.The acetone 2% was used as a negative control, but how the results were statistical identical to solvent (distilled water), the data using water were omitted.The positive control I (PC I = 10 μg/mL of MMS solubilized in distilled water) and positive control II (PC II = 10 μg/mL of MMS solubilized in acetone 2%) also were statistical identical, the data using PC I were omitted.
64 μg/mL) compared to MMS (Table I).While in the other concentrations of the pre-(32 and 64 μg/mL), the simultaneous (32, 64 and 128 μg/ mL) and the post-treatment (128 μg/mL), even though no significant difference was detected in relation to MMS, there was an increase in MI compared to MMS, except for the intermediate concentration of the simultaneous, contributing to a "trend" of the cytoprotective effect.Thus, the cytoprotective effect indicated the possible interaction of kavain in a direct and/or indirect way with MMS, decreasing and/or neutralizing its cytotoxic action.
Alkylating agents, such as MMS, reduced glutathione-S-transferase (GST) in mammalian cells, cause oxidative stress (Liu et al. 1996).MMS likely reduced GST in the meristematic cells of A. cepa, which normally contain high levels of GST (Hossain et al. 2007).Loss of GST decreases the antioxidant defense of cells, which results in accumulation of reactive oxygen species (ROS).ROS may increase the risk of DNA damage, including cell division with unrepaired or misrepaired damage, which cause mutations (Kehrer & Klotz 2015).In addition, ROS may be associated with decreased MI in meristematic cells of A. cepa (Bianchi et al. 2016), because they cause oxidation of lipids, alterations in membrane fluidity and DNA damage.The reduced MI in response to DNA damage mainly during the G1 and G2 phases occurs to allow the cells to repair damage before replicating their DNA and starting mitosis (Feng et al. 2010).Kavain may have neutralized the free radicals resulting from the action of MMS, since the isolate has antioxidant activity (Singh et al. 2018).
Kavain promoted the protective effect in all concentrations of the pre-(81.49to 94.76%) and simultaneous (85.53 to 100.53%) and in the highest concentration of the post-treatment (88.33%) against mutagenic action of MMS (Table I).In the pre-treatment (demutagenic action), the isolated compound may have directly interacted with MMS in the intracellular fibers, chromosomal breaks and/or mitotic segregation of A. cepa cells (Bianchi et al. 2016, Pereira et al. 2020).Similar results were reported by Silva et al. (2021), who evidenced the non-mutagenic and/or recombinogenic effect (32, 64 and 128 μg/mL) in D. melanogaster.The antioxidant potential of kavain (Wruck et al. 2008, Sing et al. 2018) probably prevented the damage to the genetic material, and thus contributed to the reduction of chromosomal changes in the present study.
As the percentage of damage reduction (%DR) in the present study was higher for simultaneous treatment, the mechanism of action would be both demutagenic and bioantimutagenic.However, the protective action of the pre-was greater than the post-treatment, which shows that the major mechanism of action of kavain was demutagenic.The protective effect of kavain has also been observed in mice in pre-treatment against the toxicity of 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP), which is a neurotoxin that causes symptoms of Parkinson's disease by destroying dopaminergic neurons in rat nerve cells (Schmidt & Ferger 2001).Wruck et al. (2008) observed the neuroprotective effect of kavain in the pre-treatment against the accumulation of β-amyloid plaques, which can block the signaling between nerve cells in the synapses.
Silva et al. ( 2021) administered only kavain (32, 64 and 128 μg/mL) simultaneously with Doxorubicin (DXR) in drosophilas and showed the protective effect only at the concentration of 32 μg/mL (75.94%) and 64 μg/mL (53.66%) at ST (standard) and HB (High bioactivation) crossings, respectively.This result demonstrates the sensitivity of the A. cepa test system, more able to detect antimutagenic events, as predicted by Leme & Marin-Morales (2009).On the other hand, the higher efficiency of the reduction in micronuclei (MN), nuclear buds (NB) and chromosomal losses (CL) in all concentrations, except for NB at 64 μg/mL in the pre-, in relation to MMS (Table II).This result reinforces that kavain when interacting with MMS, probably prevented the aneugenic and/ or clastogenic action of MMS in provoking the mentioned chromosomal alterations.A similar result for NB and CL was also found in all posttreatment concentrations.However, only at the highest concentration (128 μg/mL), there was a significant reduction in MN, which contributed to the highest %RD.
The results of this study demonstrated that kavain did not interfere with the progression of the cell cycle (mitotic index) and did not result in significant chromosomal changes caused by aneugenic and/or clastogenic mechanisms, indicating the absence of cytotoxicity and mutagenicity in A. cepa.In addition, kavain demonstrated an important chemopreventive activity, which is indirectly related to prevention and/or treatment of diseases, such as cancer.However, further studies are required to elucidate the biochemical mechanisms of interaction between kavain and the agent that induces DNA damage.
A. cepa test compared to drosophila can be explained by the metabolism of the organisms.The cytochrome P450 enzyme is responsible for 50% of the metabolism of therapeutic agents, and the comparison of the presence of this enzyme complex leads to the conclusion that plants have a lower concentration compared to mammals and insects (Rocha et al. 2016, Leme & Marin-Morales 2009).Thus, kavain, by inhibiting several cytochrome P450 isoforms (Mathews et al. 2002, Zou et al. 2004) may have decreased DXR metabolism and resulted in the least protective effect observed by Silva et al. (2021).
MMS was used in the present study as an inducer of DNA damage in the A. cepa assay.There are two main mechanisms by which this compound can act.The first is its known capacity for alkylation and methylation, which can cause breaks in the double strand of DNA and inhibit the replication fork (Chatterjee & Walker 2017).The second is its induction of high levels of oxidative stress, which can lead to apoptosis, cell death and DNA damage (Jiang et al. 2016).Studies demonstrate the ability to deplete Glutathione-S-transferase (Liu et al. 1996) and Glutathione (Siddique et al. 2019) of MMS, which impairs cellular antioxidant defenses and leads to the accumulation of free radicals generated as byproducts from normal cell function (Raza 2011).Probably, kavain acted by neutralizing the action of MMS by the two mechanisms mentioned, once the direct mutagenic action of MMS was reduced in the protocols, mainly in the pre-and simultaneous and at the highest concentration of post.In addition, kavain may also have acted by the second mechanism mentioned, in which the isolated molecule would have neutralized the free radicals resulting from the action of MMS, since the isolate has antioxidant activity (Singh et al. 2018).
The protective effect of kavain in the preand simultaneous is related to the significant Table II.Chromosomal alterations in meristematic cells of A. cepa.

Table I .
Mitotic index, total chromosomal alterations and percentage of damage reduction (%DR) in meristematic cells of A. cepa.
PROTECTIVE EFFECT OF KAVAIN IN Allium cepa L. ERASMO P. DO VALE JUNIOR et al.PROTECTIVE EFFECT OF KAVAIN IN Allium cepa L.