Toxicogenetic and antioxidant activities of isopentyl ferulate

1 Postgraduate Program in Pharmaceutical Sciences, Federal University of Piauí, Teresina (Piaui)64.049-550, Brazil. 2 Laboratory of Toxicology and Genetics, Post-Graduate Program in Pharmaceutical Sciences, Federal University of Piauí, Teresina (Piauí)-64.009-550, Brazil. 3 Northeast Biotechnology Network (RENORBIO), Biotechnology, Federal University of Piauí, Teresina (Piauí)64.049550, Brazil. 4 Department of Pharmacy, Southern University Bangladesh, Mehedibag (Chittagong)-4000, Bangladesh.


INTRODUCTION
Identification of new molecules to safe and effective therapies has led to implementation of strategies to identify the most promising candidates during discovery phase and its safe advancement (Fielden and Kolaja, *Corresponding author.E-mail: rbiotufpi.br@gmail.com. Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License 2008; Bass et al., 2009).Approximately 25% of allopathic drugs are derived from herbal compounds currently, and many others synthetic analogues are developed in prototype compounds isolated from plant species (Bhat et al., 2013).Plants have a variety of compounds which have no obvious functions in growth and development, called secondary metabolites.Study of these metabolites led to a current focus on discovery of new drugs (Marques et al., 2013).Both the discovery of natural or synthetic compounds with pharmacological properties and their action mechanism have major challenges for drug discovery and development.However, toxicity information is required for both bioactive compounds from natural sources and their derivatives (Ping et al., 2013).
Application of toxicological research, before recommendation for development is a great promise for increasing success in selecting best compounds (Gross and Kramer, 2003;Bass et al., 2009).Results of these studies, in discovery phase, permit candidate exclusion before waste of time and typical resources of development.In addition, toxicology studies in discovery phase, serve to approach issues that may have arisen from study of another molecule (Bass et al., 2009).
Acute systemic toxicity studies are performed in many sectors including synthetic chemicals manufactured or used.The term ''acute toxicity'' is used to describe adverse effects of a substance that can result from a single or multiple exposures within a period of 24 h.Acute effects can be local (e.g.irritation of skin or eyes) and/or of systemic nature.Genetic toxicology studies already undertaken as a part of development of new drugs are considered by providing a better assessment of potential risks to human safety (Seidle et al., 2010).The genotoxic growth has been stimulated mainly through creation; evaluation and refinement of various techniques for detection of genetic material damage and repairs in a variety of cells and organisms (Mahadevan et al., 2011).A widely used method is comet assay which measures genotoxic potential even at the early stages of exposure (Widziewicz et al., 2012).
Otherwise, molecular oxygen (O 2 ) is relatively unreactive and harmless but may undergo partial reduction to form reactive oxygen species (ROS) rendering superoxide anion (O 2 •-) and hydrogen peroxide (H 2 O 2 ) which can still react to produce the highly reactive hydroxyl radical (OH • ) (Morano et al., 2012).The result of the imbalance between the antioxidant defense system and the formation of oxygen free radicals triggers oxidative stress (Farias et al., 2013).Increasing oxidative stress with age, for instance, may be partly due to a decline in endogenous cellular levels of antioxidants.Overall, the antioxidant defense seems to be in rough balance with the generation of ROS in vivo and there seems no great reserve of antioxidant defenses in mammals, perhaps because some oxygen species play roles of useful metabolites (Poljsak, 2011).
In recent years, there is a growing interest in the production and discovery of new molecules involved in antioxidant protection against ROS (Sá et al., 2013).
Plants that contain high levels of phenolic acids have potential as an important source of natural antioxidants and are used in many applications (Gonçalveset al., 2009;Farias et al., 2013;Tee-Ngam et al., 2013;Scherer and Godoy, 2014).Among other phenolic acids ubiquitous ferulic acid (FA) (3-4-hydroxy-methoxycinnamic acid) (Mori et al., 2013;Lin et al., 2013;Yang et al., 2013) is more readily absorbed in the body even than the ascorbic acid (Han et al., 2005;Tee-Ngam et al., 2013).It has also a wider range of therapeutic effects against various diseases such as cancer, diabetes, cardiovascular and neurodegenerative diseases (Srinivasan et al., 2007).Nowadays, FA has received a prominent attention in medicine for the prevention of atherosclerosis (Kwon et al., 2010) as well as cancers, especially those that occur in colon and rectum (Thakkar et al., 2015).The most beneficial human health effects of this phenolic compound can be attributed to its strong antioxidant activity (Monti et al., 2011).Due to the biochemical and molecular similarities to human cells; the yeast, Saccharomyces cerevisiae has proven to be a powerful model for understanding the eukaryotic cell response against damages caused by oxidative stress (Sá et al., 2013).
Thus, the aim of this study is to evaluate toxicity of a new synthetic derivative of FA called isopentanyl ferulate (IF) through determination of median lethal dose (LD 50 ), evaluation of biochemical and hematological parameters, observation of locomotor activity, genotoxic and antioxidative defense activities.

Animals
Swiss albino mice (Mus musculus) of either sex (2 months old, 25-30 g) were collated from the Center for Pharmaceutical Technology (CPT) of the Federal University of Piauí, Brazil.Animals were acclimated at 25 ± 2°C and kept in acrylic cages of 30×30 cm 2 with light/dark cycle of alternating 12 h with standard chow like Purina and water ad libitum.Experiments were developed in accordance with guidelines for care and use of laboratory animals of Department of Health and Human Services of United States of America (USA).The project was previously approved by Ethics Committee on Animal Experimentation of Federal University of Piauí under the protocol number #030/13.

Chemicals and reagents
Isopentyl ferulate (IF) and all necessary reagents and chemicals were purchased from Sigma Chem.Co., St. Louis, MO, USA.

Preparation of isopentyl ferulate (IF) for investigations
IF in a dose range of 1000 to 3000 mg/kg was emulsified in 0.05% Tween 80 dissolved in saline (0.9% NaCl solution) and tested for LD50, serum biochemical and hematological parameters, locomotor and genotoxic activities whereas, doses of 0.10 to 0.30 g/ml was used for S. cerevisiae test.

Acute toxicity study in M. musculus
In this study, animals were divided into three groups.Each group was divided into four subgroups of five in each (according to sex and administration route).After fasting for 12 h, different doses of IF (1000, 2000 and 3000 mg/kg) were given orally via orogastric tube while (1000, 1500 and 2000 mg/kg) via intraperitoneal (i.p.) route.Thereafter, animals were placed in plastic cages with food and water ad libitum and general behavior was monitored at 0.5, 1, 2, 3, 4 and 24 h which then extended to 48 h and followed by a total of 14 days.Behavioral parameters were observed according to hippocratic test described by Malone (1977).Signs of toxicity or death were monitored.For LD50 determination, the number of deaths in each group was expressed as a percentage of total number of animals that received IF.Determination of LD50 was made by semi-logarithmic interpolation being put on ordinate axis values corresponding to probabilistic percentage of deaths and the abscissa, administrated doses of product.
For biochemical analysis, blood centrifuged at 3500 rpm for 10 min was subjected to the determination of glucose, urea, creatinine, aminotransferase aspartate (AST), aminotransferase alanine (ALT), total cholesterol, triglycerides, alkaline phosphatase (ALP) and uric acid.The tests were performed in Labmax 240 automatic machine with Labtest ® commercial systems.In hematological assessment, number of erythrocytes (RBC), leukocytes (WBC) and platelets, hemoglobin, hematocrit and RBC indices such as mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC) were determined immediately after collection of blood by using an automatic analyzer cell hematologic (Advia 120/Hematology Siemens).Differential counts for WBC were performed in colored extensions with May-Grünwald-Giemsa.Each assay was performed with 100 cells (Malone, 1977;Al-Habori et al., 2002).

Locomotor activity
This experiment was accomplished with the same experimental groups used in acute toxicity test and treated according to the experimental protocols.Motor activity of animals was verified by an open acrylic field (OFT) (transparent walls and black floor, 30 × 30 Taimo et al. 647 × 15 cm) divided into nine equal quadrants (Archer, 1973).On day seven treated animals, one by one, were placed in center of OFT apparatus where number of intersections with four legs (spontaneous locomotor activity, SLA), number of self-cleaning behavior (grooming) and number of stand up without lean against the wall (rearing), were observed for 5 min.After each animal exposure, OFT apparatus was cleaned with 70% ethanol before introducing new mice, in order to avoid the possible influences of the odor left by the previous (Frussa- Filho and Palermo-Neto, 1990).

Comet assay in peripheral blood and bone marrow
Comet assay was performed with oral administration: G1 (negative control, saline 10 ml/kg), G2 (IF 3000 mg/kg), G3 (positive control; methylmethanesulfonate, MMS (40 µM)); and intraperitoneal administration: G1 (negative control, saline 10 ml/kg), G2 (IF 1500 mg/kg), G3 (positive control, MMS).Each group was comprised of three mice for a length of treatment for 14 days.This test is accomplished in alkaline conditions, pH>13, according to Tice et al. (2000), modified by Da Silva (2000).Biological material was obtained by capillary puncture of tail and bone marrow (extracted from femur of animal).After collecting 5 µl of blood, it was transferred and mixed with 95 µl of 0.75% lowmelting point agarose (LMPA) which was then subsequently spread on previously prepared normal melting point agarose (1%) (NMPA) coated slides and covered with adequate cover slips.After drying and removing the cover slips, slides were placed in electrophoretic buffer for 20 min followed by electrophoresis at 300 mA for 15 min, then neutralization of slides was done with neutral buffer for 5 min (three times).Slides were then washed with distilled water (DW) two times and dried in room temperature over night.Those were submitted to fixing solution for 10 min, washed and dried again similarly.Those were followed by silver nitrate staining and three times washing with DW covering with stop solution (acetic acid) for 5 min, dried again and made ready for photomicrography at 100X magnification.Hundred cells were considered for the determination of frequency and damage index.

Evaluation of antioxidant activity S. cerevisiae (in vivo)
This test was performed according to the aerobic metabolism pathway described by Fragoso et al. (2008).In brief, previously sub-cultured strains were linearly swabbed to the sterile YEPD media (0.5% yeast extract, 2% peptone, 2% dextrose and 2% bacteriological agar).Then 0.01 ml of test sample/controls (specified concentrations) were applied on sterile paper disks and were treated accordingly such as in pretreatment and posttreatment, IF with specified concentrations and hydrogen peroxide (H2O2 as stressor), then subjected to incubation at 35 ± 1°C for 3 h whichever then followed by the addition of H2O2 and IF, respectively.To the co-treatment group, IF plus H2O2 (40 mM) were added immediately.The negative control (sterilized by filtration) (0.9% NaCl) and H2O2 were added subsequently.Treatments were done immediately after swabbing the organisms in petridishes.Dishes were then inverted (180°C), transferred to an incubator maintaining temperature of 35 ± 1°C for 72 h, followed by the measurement of zones of inhibition in millimeters (mm) with a range from 0 mm (full growth) to 40 mm (no growth); these values being the size of the petridishes procured.All the treatments were performed in duplicate.

Statistical analyses
Values are expressed as mean ± standard error of mean (SEM).

Acute toxicity study in M. musculus
Toxicity symptoms for all tested doses were checked by both oral and intraperitoneal administration of IF after single dose (Tables 1 and 2).Common manifestations to all groups were decreased in the general activities.
Mortality to the animals was evidenced after intraperitoneal administration of IF (Tables 2 and 3).LD 50 calculated at i.p. dose of 1494.54 mg/kg (Table 3).However, there was no mortality observed in case of oral doses.Hematological parameters were also found to be unaltered, with only the exception of increased platelets by IF at 3000 mg/kg oral administration in comparison to control (Table 5).

Effects of IF on locomotor activity in mice
There were no significant behavioral changes (number of intersections, grooming and rearing) by IF treated groups when compared to control

Genotoxic effect in peripheral blood and bone marrow cells of mice
Comet assay performed in blood and bone marrow cells of mice suggests that IF at doses 1500 mg/kg (i.p.) and 3000 mg/kg (p.o.) caused increase in DNA damage when compared to the negative control (Figures 1 and 2).

Antioxidative defense of IF in S. cerevisiae cells
Antioxidant activity test in S. cerevisiae proficient and deficient (single/double) strains delineates that IF at doses 0.15 and 0.20 mg/ml produced better antioxidative defense in pre-, post-and cotreatments in comparison to the stressor H 2 O 2 (Figure 3, 4 and 5).

DISCUSSION
After administration of all doses of IF via oral and intraperitoneal routes, toxicity manifestations in animals were attributed (Table 1 and 2).Decrease of general activities also appeared in all groups.Besides this symptom, others such as decrease in corneal reflex and piloerection were seen at lowest doses, while tremors and hypnosis were observed with the increased doses.Nevertheless, no animals died with oral IF doses.According to the Globally Harmonized System (2011), substances can be allocated to one of five categories of toxicity based on acute oral toxicity, where IF is occupied in category 5 with relatively low acute toxicity (> 2000 to ≤ 5000 mg/kg).Median lethal dose (LD 50 ) was first introduced in 1927 by Trevan for testing substances in human use.However, in 1970s, the test which aims to find lethal dose of a unique substance that kills half of animals in a test group, had become generally accepted as a basis for comparison and classification for toxicity of chemicals and gradually has become a popular and essential test for various regulatory entities including new drugs/other products (Botham, 2004;Seidle et al., 2010).Maximum mortality of test animals was observed with i.p.IF at 2000 mg/kg.In this study, using IF LD 50 of 1494.54 mg/kg (i.p.) is however lower than the LD 50 of FA (>2370 mg/kg; p.o.) in mice.Tolerable daily intake (TDI) in humans can be established from boundary data of LD 50 in mice.Assuming that standard uncertainty factor representing that variation between species is 10, TDI at 14.20 g of FA was calculated to 60 kg man (Mori et al., 2013).
Blood is an important index of physiological and pathological condition in humans and animals and parameters measured usually are total RBC counts and its indexes, hemoglobin, hematocrit, total WBC counts and its differential counts, platelet count and biochemical parameters, such as liver and kidney function tests (Oduola et al., 2007;Adeneye et al., 2010).Normal range of these parameters can be changed by ingestion of some toxic substances (Adedapo et al., 2004;Adeneye et al., 2010).
In the present study, the results showed no significant difference between groups treated with IF and vehicle (control) after 24 h in male mice.Although, there was no significant differences to the tested biochemical parameters but IF at 1500  4).
Otherwise, an almost unaltered hematological parameters were observed compared to control, with an exception to increasing platelets at 3000 mg/kg oral IF dose (Table 5).
However, an unchanged serum ALT and AST indicate no deleterious effects of IF on liver functions in association with biliary tract as it caused no alteration to the levels of ALP (Adeneye et al., 2010), suggesting an indication of oral safety of IF on liver function.There is a change in urea, which is an important biomarker for renal function, unchanged levels of glucose, uric acid cholesterol as well as triglycerides were observed, which may be connected to its low/free metabolic disorders.In addition, insignificant alteration of hematological parameters suggests no detrimental effects of IF in hematopoietic and leukopoietic systems, especially by i.p. administration.
OFT test evaluated with three parameters such as number of intersections with four legs, exploratory animal activity and number of rearing and grooming mainly changed by the sedation or fear (anxiety) effects caused by anxiolytic or anxiogenic agents (Marques et al.,2013).No significant changes in these parameters compared to control were attributed to the lack of interference in psychomotor activity by IF (Table 6).Toxigenomic and statistical methods have been employed to measure carcinogenicity for acute and subchronic doses involving identification of compounds having carcinogenic or non-carcinogenic properties.DNA damage, activating of proliferation and signaling in survival are characteristics of genotoxic carcinogens; however carcinogens are not genotoxic and normally may cause oxidative stress thus affecting cell cycle and regenerative processes.
Multi-factorial pathways associated with gene expression profile are internal mechanisms for prediction of carcinogens (Guyton et al., 2009).Major sources and types of DNA damage as well as repair mechanisms are associated with aging, can lead to cell dysfunction.DNA damage (genotoxicity) and mutations (mutagenicity) accumulate with age in tissues, suggesting that high level of DNA damage can accelerate decline in physiological functions and proceed to cancer.High DNA damage can induce signaling pathways such as apoptosis, which results in cellular system depletion and accelerates aging (Freitas and Magalhães, 2011).Comet assay is based on the principle of quantification of denatured DNA fragments that migrate out from the nucleus during electrophoresis.While separation by an applied electric field, the remaining DNA at the site of of cell irrigated, partially anchored to residual nucleus structures, migrates towards anode with appropriate  speed to its fragmentation.Migration speed to DNA in agarose is directly proportional to the degree of damage.Image obtained in this procedure looks like a "comet" with distinct head and tail consisting of relaxed loops and fragments of damaged DNA (Widziewicz et al., 2009).Amount of DNA on heads and tails of comets provides an estimate of frequency of filament breaks.To some lesions types, modified DNA can be directly reverted to original sequence (Spivak et al., 2009), these DNA repair mechanisms allow estimating a possible adaptation to low concentrations of genotoxic element (Widziewicz et al., 2009).

Sodwt
In our test, IF showed genotoxicity in peripheral blood and bone marrow cells of mice that was evidenced by the increase in frequency and index of damage at two tested doses with different administration routes (Figures 1 and  2).Chemicals with positive results in standard genotoxicity tests are generally known to induce cancer via genotoxic and/or mutagenic mode of action that is an indication of human risks (Dearfield and Moore, 2005;Cimino, 2006).However there are numerous protective strategies to prevent deleterious effects of DNA oxidation.Bases excision repair is the most important strategy in repairing oxidative damage induced by ROS, but proteins involved in nucleotide excision repair, that are mutated in human syndromes, can be involved in repair.Identification of human diseases related to mutations in repair genes to oxidative damage shows that these defects can increase incidence of neurological diseases as well as cancer (D'errico et al., 2008).

Sodwt
Advanced technological and scientific studies have compared remarkable story of mutation, chromosomes breaks and biological consequences of these events with hazard identification and risk estimation of cancer.Driven by regulatory concerns, the main reason for conducting in vitro and in vivo genotoxicity tests has been a try to predict if molecules are likely to be carcinogenic agents or not (Mahadevan et al., 2011).
Mutagenicity is attributed to permanent induction of DNA damage that can be a result of hereditary changes.Antimutagenic agents are able to modify effects of mutagens.These agents can be natural or synthetic compounds.Compounds with antioxidant activity can inhibit mutagens activation.These aspects are important for cancer prevention and therapy.Mutations can alter a single gene, block genes and lose chromosomes.The mutations alter sequence of gene, are most frequent alterations in DNA and can be divided into base pair substitutions, deletions and insertions (Słoczyńska et al., 2014).
In antioxidant test, IF showed protective effects when compared to the H 2 O 2 treated group by intracellular mechanisms in the proficient strain (Sod wt) and cytoplasmic (Sod1Δ) and mitochondrial superoxide dismutase (Sod2Δ), liver catalase (Cat1Δ) and double mutants (Sod1ΔSod2Δ, Sod1ΔCat1Δ).Recent studies have shown interest in natural compounds with antioxidant properties to minimize the levels of oxidative stress as a strategy for prevention of various diseases (Farias et al., 2013).The IF showed protective (Figure 3), antioxidant (Figure 4) and repair (Figure 5) capacities in all the test strains.According to Sherer and Godoy (2014) FA has been proven for its promising antioxidant capacity.We should also emphasize that, the phenylpropanoids exhibit antioxidant activities, which are considered important for prevention of several diseases, including cancer.To minimize the harmful effects of ROS, aerobic organisms have evolved both enzymatic and non-enzymatic antioxidant defenses.The non-enzymatic defenses include intrinsic antioxidant compounds such as vitamins C and E. The enzymatic defenses such as superoxide dismutase (SODs), catalase (CAT) and peroxidases, protect directly by cleaning of O 2 •-and H 2 O 2 by converting these to less reactive species (Scandalios, 2005;Sá et al., 2013).The SODs (Sod1ΔSod2Δ) deal with the first product of univalent reduction of O 2 , catalyzing the dismutation of O 2 to H 2 O 2 (Scandalios, 2005;Abreu and Cabelli, 2010), which must then be destroyed by CAT, which reduces H 2 O 2 to 2H 2 O. Thus, SOD and CAT serve together as front line to the antioxidant defenses (Scandalios, 2005).
The antioxidant capacity of IF was observed in all strains, which suggests that the protective effect (Figure 3) may increase the activity of Sod and Cat, demonstrating the role of this compound in cellular antioxidant defense.The strain of S. cerevisiae wild type (Sod wt) showed a higher level of survival in co-treatment (Figure 4) and post-treatment (Figure 5), whereas strains deficient in antioxidant defenses were more sensitive to H 2 O 2 , which shows the importance of CAT and O 2 •-in cellular protection against oxidative stress.
Among the SODs, Cu/Zn-SOD is conceived as the first line defense against the toxicity of ROS and is found in many eukaryotic organelles.The function of the Mn-SOD located in the mitochondria seems to be restricted to protecting cells against radical products of respiration and/or other processes within the mitochondria.Notably, the yeast strains are highly damaged when mutations are present in Sod1Δ (Sá et al., 2013).

CONCLUSION
Our study indicates isopentyl ferulate with low toxicity in oral doses and insignificant changes in biochemical and hematological parameters, genotoxic and antioxidant with damage repair capacity, thus IF may be a new drug candidate with pharmaceutical importance.However, to our knowledge, these tests are performed for the first time.So before developing more studies, it is highly appreciated for further investigation of IF's safety profile.

Table 1 .
Effects of IF in mice after acute oral (p.o.) administration.

Table 2 .
Effects of IF in mice after acute intraperitoneal (i.p.) administration.

Table 4 .
Effects of IF on biochemical parameters in male mice after 24 h of oral (p.o.) and intraperitoneal (i.p.) administration.

Table 5 .
Effects of IF on hematological parameters in male mice after 24 h of oral (p.o.) and intraperitoneal (i.p.) administration.
the IF treated groups compared to control group (p<0.05).IF with i.p. 1500 and 2000 mg/kg only decreased the urea parameter significantly (P<0.05) as compared to control treated group while ALT by 1000 and 2000 mg/kg and uric acid by 2000 mg/kg with orally administered IF.

Table 6 .
Effects of IF (p.o./i.p.) in locomotor activity performed by open field test.