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Keywords:

  • 4-methylesculetin;
  • coumarin derivative;
  • antigenotoxic effects;
  • micronucleus test;
  • comet assay

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

4-Methylesculetin (4-ME) is a synthetic derivative of coumarin that displays a potent reactive oxygen species (ROS) scavenger and metal chelating agent and therefore has been produced to help reduce the risk of human disease. The main objective of this study was to investigate the in vivo genotoxicity of 4-ME and initially to verify its potential antigenotoxicity on doxorubicin (DXR)-induced DNA damage. Different doses of 4-ME (500, 1000 and 2000 mg kg–1 body weight) were administered by gavage only or with a simultaneous intraperitoneal (i.p.) injection of DXR (80 mg kg–1). The following endpoints were analyzed: DNA damage in peripheral blood, liver, bone marrow, brain and testicle cells according to an alkaline (pH > 13) comet assay and micronucleus induction in bone marrow cells. Cytotoxicity was assessed by scoring polychromatic (PCE) and normochromatic (NCE) erythrocytes (PCE/NCE ratio). No differences were observed between the negative control and the groups treated with a 4-ME dose for any of the endpoints analyzed, indicating that it lacks genotoxic and cytotoxic effects. Moreover, 4-ME demonstrated protective effects against DXR-induced DNA damage at all tested doses and in all analyzed cell types, which ranged from 34.1% to 93.3% in the comet assay and 54.4% to 65.9% in the micronucleus test. Copyright © 2012 John Wiley & Sons, Ltd.


Introduction

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Coumarin is a naturally occurring secondary plant compound that taken together with its derivatives has a wide spectrum of biological activity (Rositca et al., 2002; Flasik et al., 2009), including antitumor (Nofal et al., 2000), anticoagulant (Smitha and Sanjeeva, 2004), anti-inflammatory (Garazd et al., 2007), antifungal (Satyanarayan et al., 2008), anti-HIV (Kotali et al., 2008) and antibacterial agents (Al-Amiery et al., 2012). Coumarins are widely used as pharmaceuticals and optical brighteners (Zabradnik, 1992), dispersing fluorescent and laser dyes (Heravi et al., 2008), and as additives to food, perfumes and cosmetics (Kennedy and Thornes, 1997).

Coumarin belongs to a group of herbs and spice components called ‘active principles’ that, although add to the flavoring effect, also have toxicological relevance. Coumarin is deemed to be hepatoxic and, administered in very high doses in animal experiments over long periods of time, has a carcinogenic effect (Cohen, 1979; EFSA, 2004).

Several previous studies have been conducted to evaluate the genotoxic potential of coumarin, with both positive and negative results reported. In the Ames test using Salmonella typhimurium strains TA98, TA1535, TA1537 and TA1538 with or without metabolic activation, the results were negative (NTP, 1993; Lake, 1999). However, a positive response was reported for strain TA100 with metabolic activation (Stolz and Scott, 1980; Norman and Wood, 1981; NTP, 1993). In Chinese hamster ovary (CHO) cells without the S9 fraction a positive response for inducing sister chromatid exchanges was observed. Chromosomal aberrations were increased in the presence of S9 but not in its absence (NTP, 1993). On the other hand, Sasaki et al. (1987) found no evidence of a sister chromatid exchange or chromosomal aberrations in this same cell line treated with coumarin.

In in vivo genotoxicity tests, no increases in micronuclei were observed in peripheral blood (NTP, 1993) or in bone marrow cells from male mice treated with coumarin (Morris and Ward, 1992). Moreover, this compound did not induce a sex-linked recessive lethal mutation in germ cells from Drosophila melanogaster adults and larvae (NTP, 1993). Api (2001) also observed that coumarin was negative in an in vivo mouse micronucleus assay.

Coumarins exhibits, among other properties, anti-oxidant activity (Farombi and Nwaokeafor, 2005). The structure of coumarins could bind a transition metal ion such as Fe(III), and thus inhibit hydroxyl radical and hydrogen peroxide formation produced by Fenton's reactions. Furthermore, their hydroxyl functions are potent H+ donors for free radical acceptors owing to electron delocalization across the molecule (Sharma et al., 2005). Thus, coumarin derivatives could be potent reactive oxygen species (ROS) scavengers and metal chelating agents. A large number of structurally novel coumarin derivatives have been synthesized to improve this antioxidant activity (Beillerot et al., 2008).

The major metabolic route of coumarin in the rat is by oxidation to form an unstable coumarin 3,4-epoxide intermediate (Born et al., 1997), which decomposes to o-hydroxyphenylacetaldehyde, which is the major metabolite of coumarin formed in rat liver microsomes (Lake et al., 1992). The major identified metabolite of coumarin in the rat in vivo is o-hydroxyphenylacetic acid (Lake, 1996). In the rat, single doses of coumarin produce hepatic necrosis (Lake, 1984), whereas chronic administration at high doses resulted in bile duct lesions and liver tumors (Lake et al., 1994). To our knowledge, there are no data in the literature about the biotransformation of the synthetic coumarin derivative 4-ME, and in spite of the therapeutic potential of the coumarin derivatives, studies on their possible genotoxic or antigenotoxic effects are lacking.

Given that data on the genotoxicity or protective effects of coumarin are partially conflicting and that a number of coumarin derivatives have been synthesized for potential use by humans, further studies must be performed to clarify the genotoxic/antigenotoxic potency of these compounds. The chemical structure of 4-ME is comprised of two hydroxyl groups on the benzene moiety which were more effective in scavenging radicals compared with other coumarin derivatives (Lin et al., 2008). In this context, the present study focuses on the in vivo analysis of the genotoxic potential of 4-methylesculetin (4-ME) for the first time, using a comet assay and a micronucleus test in different cells of mice, as well as an initial analysis of its antigenotoxic effects against oxidative DNA damage caused by doxorubicin, because this anthracycline antibiotic, during its metabolization, generates superoxide radicals which rapidly generate other ROS, such as hydroxyl radical and hydrogen peroxide.

Materials and Methods

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Chemicals

The 4-ME (Fig. 1) used in the tests was obtained from ACRÓS ORGANICS (ACRÓS ORGANICS, New Jersey, USA, CAS 529-84-0) and dissolved in sunflower oil. Doxorubicin (DXR) (Oncodox®; Meizler) was used as the positive control substance as a result of its potential for DNA damage recognizable in the comet and micronucleus assays. The other main chemicals were obtained from the following suppliers: normal melting point (NMP) agarose (Invitrogen), low-melting point (LMP) agarose (Invitrogen), sodium salt N-lauroyl sarcosine (Sigma) and ethylenediaminetetraacetic acid (EDTA) (Merck).

image

Figure 1. The chemical structure of 4-methylesculetin.

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Animals and dosing

Experiments were carried out on 10-week-old male mice of the strain albino Swiss, outbred (Mus musculus) weighing 25–30 g. The animals were acquired from the vivarium of the Universidade Estadual Paulista (UNESP), which is located in Botucatu, SP, Brazil, and kept in polyethylene cages in a climate-controlled environment (25 ± 4 °C, 55 ± 5% relative humidity) with a 12-h light/dark cycle (7.00 to 19.00 hours). Food (NUVILAB CR1, Nuvital) and tap water were available ad libitum. The mice were divided into eight experimental groups of six animals each. Considering the small size of the animals and the vehicle (sunflower oil), the 4-ME was administered in a single dose at 500, 1000 or 2000 mg kg–1 body weight. For this procedure, each animal was weighted individually and then the calculated dose was dissolved in 0.3 ml of the vehicle and administered by gavage. The dosage strengths were based on our acute toxicity studies in mice (> 2000 mg kg–1) and the limit dose recommended by OECD 420 for acute treatments in toxicology assays. Simultaneous treatment was carried out by administering the test substance and then DXR 1 h later. The negative control group received sunflower oil by gavage. The positive control group received an intraperitoneal (i.p.) injection of DXR 80 mg kg–1. The animals were sacrificed by cervical dislocation without anesthesia to avoid possible alterations in the DNA damage analysis. In accordance with Brazilian animal care regulations, the Animal Bioethics Committee of the Marília Medical School (Marília, SP, Brazil) approved the present study on 14 April 2010 (protocol number 085/10).

Comet assay

The comet assay (SCGE) was carried out according to Speit and Hartmann's method (1999), which is based on the original work of Singh et al. (1988) and included additional modifications, such as those introduced by Klaude et al. (1996). Peripheral blood samples from the tail vein were obtained from six mice of each group 4 and 24 h after treatment and before euthanasia. After the animals were sacrificed, liver, bone marrow, brain and testicle cell samples were collected and washed in saline solution in an ice bath. A small portion of each type (about 4 mm in diameter) was transferred to a Petri dish containing 4 ml of phosphate buffer solution (PBS, pH 7.4) and then gently homogenized with a small pair of tweezers and a syringe to remove any clumps of cells. An aliquot of 20 µl was removed from the supernatant of each cell type to determine cell viability. Cell counting was performed with a hemocytometer. Cell viability was determined by Trypan blue dye exclusion. The number of Trypan blue-negative cells, which was greater than 85% in all cases, was considered the number of viable cells. Another 20-µl aliquot of cells from each animal was mixed with 120 µl of 0.5% LMP agarose at 37 °C, and then quickly spread onto two microscope slides per animal that had been pre-coated with 1.5% NMP agarose. The slides were covered with coverslips and allowed to gel at 4 °C for 20 min. The coverslips were gently removed and the slides were then immersed in cold, freshly prepared lysing solution consisting of 89 ml of a stock solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, pH set to 10.0 with ~8 g solid NaOH, 890 ml of distilled water and 1% sodium lauryl sarcosine), plus 1 ml of Triton X-100 (Merck) and 10 ml of dimethyl sulfoxide (Merck). The slides, which were protected from light, were allowed to stand at 4 °C for 1 h and were then placed in the gel box, positioned at the anode end, and left in a high pH (> 13) electrophoresis buffer (300 mM NaOH, 1 mM EDTA that had been prepared from a stock solution of 10 N NaOH and 200 mM, pH 10.0, EDTA) at 4 °C for 20 min prior to electrophoresis to allow DNA unwinding. The electrophoresis run was carried out in an ice bath (4 °C) for 20 min at 300 mA and 25 V (0.722 V cm–1). The slides were then submerged in a neutralization buffer (0.4 M Tris-HCl, pH 7.5) for 15 min, dried at room temperature and fixed in 100% ethanol for 10 min. The slides were dried again and stored overnight or longer before staining. For the staining process, the slides were briefly rinsed in distilled water, covered with 30 µl of 1× ethidium bromide staining solution prepared from 10× stock (200 µg ml–1) and then covered with a coverslip. The material was evaluated immediately at 400× magnification with a fluorescence microscope (Olympus BX 50) with a 515–560 nm excitation filter and a 590 nm barrier filter. Only individual nucleoids were scored.

The extent and distribution of DNA damage indicated by the SCGE assay was evaluated by examining 100 randomly selected non-overlapping cells (50 cells per coded slide) per animal in a blind analysis (six mice per group). These cells were scored visually, according to tail size, into the following four classes: class 0, no tail; class 1, tail shorter than the diameter of the head (nucleus); class 2, tail length 1 to 2 times the diameter of the head; and class 3, tail length more than twice the diameter of the head. Comets with no heads, with nearly all of their DNA in the tail or with a very wide tail were excluded from the evaluation because they probably represented dead cells (Hartmann and Speit, 1997). The total score for 100 comets, which ranged from 0 (all undamaged) to 300 (all maximally damaged), was calculated according to the formula: score = (1 × n1) + (2 × n2) + (3 × n3), where n = the number of cells in each class analyzed.

Micronucleus test

The assay was carried out according to standard protocols as recommended by Schmid (1975) and Krishna and Hayashi (2000). The same six male mice per group that were used in the comet assay were also used to this protocol. The bone marrow from one femur was flushed out using 3 ml of saline buffer (0.9% NaCl) and centrifuged for 7 min. The supernatant was discarded and smears were made on the slides. The slides were coded for a blind analysis, fixed with ethanol (70%) for 10 min and stained with Giemsa. For the micronucleated cell analysis, 2000 polychromatic erythrocytes (PCE) per animal were scored to determine the clastogenic/anticlastogenic property of 4-ME. To detect possible cytotoxic effects, a PCE/NCE (normochromatic erythrocytes) ratio of 200 erythrocytes per animal was calculated (Gollapudi and McFadden, 1995). The cells were blindly scored using a light microscope at 1000× magnification. The mean number of micronucleated polychromatic erythrocytes (MNPCE) in an individual mouse was used as the experimental unit, with variability (standard deviation) based on differences among animals within the same group.

Percentage reduction

The percentage that genotoxic agent-induced damage was reduced by 4-ME was calculated according to Waters et al. (1990) using the following formula:

  • display math

where A corresponds to the score or MNPCE mean observed in DXR treatment (positive control), B corresponds to the score or MNPCE mean observed in antigenotoxic treatment (extract plus DXR) and C corresponds to the score or MNPCE mean in controls.

Statistical analysis

After verifying normal distribution (KS normality test), the data obtained from the comet assay and micronucleus test were submitted to analysis of variance (anova) and the Tukey–Kramer multiple comparison test, using the GraphPad Instat® software (version 3.01). The results were considered statistically significant at P < 0.05.

Results

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The 4-ME comet assay genotoxicity results are shown in Table 1, where the results for the different doses of 4-ME and DXR positive controls were compared with negative controls (sunflower oil). The cell viability observed in the Trypan blue staining procedure was over 85% in all cases, which confirms the absence of cytotoxicity found by means of the PCE/NCE ratio in the MN test (Table 3). No deaths, morbidity or distinctive clinical signs were observed in the treated animals after 4-ME treatment. As expected, when positive controls were compared with negative controls, we found that DXR induced a significant increase (P < 0.001 or greater) in comet assay DNA migration for all cell types analyzed, indicating the validity of the species selected and the study design to detect genotoxic effects. No significant increase in comet assay DNA migration was found at any of the 4-ME doses or in any of the cells type analyzed. There was no statistical difference in DNA migration between the three tested doses of 4-ME in each cell type. In the few cells that presented DNA damage, it was predominantly minor (class 1) as was also observed in negative controls.

Table 1. DNA migration in the comet assay for assessing the genotoxicity of 4-methylesculetin (4-ME) in different cells of male Swiss mice in vivo [mean ± standard deviation (SD)]
Treatments and cells analysedTotaldComet classScores
 0123 
  1. a

    Significantly different from the negative control (P < 0.05).

  2. b

    Significantly different from the negative control (P < 0.01).

  3. c

    Significantly different from the negative control (P < 0.001).

  4. d

    Total number of damaged cells (class 1 + 2 + 3).

Peripheral blood (4-h sample)
Control14.00 ± 4.4386.00 ± 4.4312.17 ± 4.621.33 ± 0.520.50 ± 0.8416.33 ± 4.55
4-ME 500 mg kg–120.67 ± 1.63a79.33 ± 1.6320.00 ± 2.00a0.67 ± 1.030.00 ± 0.0021.33 ± 1.86
4-ME 1000 mg kg–119.00 ± 3.1681.00 ± 3.1618.83 ± 3.190.17 ± 0.410.00 ± 0.0019.17 ± 3.19
4-ME 2000 mg kg–112.00 ± 3.5888.00 ± 3.5811.67 ± 3.930.33 ± 0.520.00 ± 0.0012.33 ± 3.27
Doxorubicin 80 mg kg–132.50 ± 4.76c67.50 ± 4.76c28.00 ± 4.65c3.17 ± 1.17c1.33 ± 0.82a38.33 ± 4.55c
Peripheral blood (24-h sample)
Control12.33 ± 3.2787.67 ± 3.2711.50 ± 3.560.83 ± 0.750.00 ± 0.0013.17 ± 3.13
4-ME 500 mg kg–120.50 ± 8.7179.50 ± 8.7120.00 ± 8.990.50 ± 0.550.00 ± 0.0021.00 ± 8.46
4-ME 1000 mg kg–125.33 ± 5.47a74.67 ± 5.47a23.83 ± 4.62a1.50 ± 1.380.00 ± 0.0026.83 ± 6.49
4-ME 2000 mg kg–116.83 ± 3.1383.17 ± 3.1315.17 ± 3.601.50 ± 1.640.17 ± 0.4118.67 ± 3.83
Doxorubicin 80 mg kg–183.67 ± 9.33c16.33 ± 9.33c76.00 ± 5.90c6.50 ± 4.81c1.17 ± 0.41c92.50 ± 14.21c
Liver
Control33.17 ± 4.3666.83 ± 4.3632.33 ± 4.080.83 ± 0.980.00 ± 0.0034.00 ± 4.82
4-ME 500 mg kg–146.67 ± 9.16a53.33 ± 9.1645.67 ± 9.221.00 ± 1.100.00 ± 0.0047.67 ± 9.22
4-ME 1000 mg kg–142.83 ± 7.7057.17 ± 7.7041.33 ± 8.071.50 ± 2.070.00 ± 0.0044.33 ± 7.89
4-ME 2000 mg kg–132.00 ± 6.7868.00 ± 6.7832.00 ± 6.780.00 ± 0.000.00 ± 0.0032.00 ± 6.78
Doxorubicin 80 mg kg–184.17 ± 6.62c15.83 ± 6.62c53.67 ± 11.00a20.17 ± 6.74c8.67 ± 11.13a120.00 ± 26.37c
Bone marrow
Control21.67 ± 5.5478.83 ± 5.5421.33 ± 5.050.33 ± 0.820.00 ± 0.0022.00 ± 6.10
4-ME 500 mg kg–124.67 ± 7.1575.33 ± 7.1524.67 ± 7.150.00 ± 0.000.00 ± 0.0024.67 ± 7.15
4-ME 1000 mg kg–122.00 ± 3.6978.00 ± 3.6921.67 ± 3.330.33 ± 0.820.00 ± 0.0022.33 ± 4.18
4-ME 2000 mg kg–129.67 ± 6.5070.33 ± 6.5028.83 ± 6.490.83 ± 0.750.00 ± 0.0030.50 ± 6.60
Doxorubicin 80 mg kg–185.00 ± 6.29c15.00 ± 6.29c69.00 ± 10.84c14.17 ± 10.19c1.83 ± 1.47c102.83 ± 16.42c
Brain
Control54.00 ± 12.0346.00 ± 12.0352.83 ± 11.691.17 ± 1.170.00 ± 0.0055.17 ± 12.48
4-ME 500 mg kg–136.67 ± 4.8963.33 ± 4.8934.33 ± 5.752.33 ± 1.630.00 ± 0.0039.00 ± 4.47
4-ME 1000 mg kg–140.17 ± 8.2359.83 ± 8.2338.17 ± 9.662.00 ± 2.100.00 ± 0.0042.17 ± 7.14
4-ME 2000 mg kg–140.50 ± 15.9359.50 ± 15.9339.83 ± 16.490.67 ± 1.030.00 ± 0.0041.17 ± 15.43
Doxorubicin 80 mg kg–182.83 ± 5.98c17.17 ± 5.98c57.67 ± 7.76c22.00 ± 9.98c3.17 ± 3.06c111.17 ± 19.14c
Testicle
Control27.00 ± 6.9373.00 ± 6.9324.50 ± 6.632.33 ± 2.420.17 ± 0.4129.67 ± 8.43
4-ME 500 mg kg–127.17 ± 2.0472.83 ± 2.0427.17 ± 2.040.00 ± 0.000.00 ± 0.0027.17 ± 2.04
4-ME 1000 mg kg–124.83 ± 2.9375.17 ± 2.9323.67 ± 2.661.17 ± 1.170.00 ± 0.0026.00 ± 3.58
4-ME 2000 mg kg–126.50 ± 8.0973.50 ± 8.0922.67 ± 8.643.50 ± 2.070.33 ± 0.5230.67 ± 8.26
Doxorubicin 80 mg kg–165.50 ± 5.13c34.50 ± 5.13c55.83 ± 10.57c8.33 ± 4.27c1.33 ± 2.3476.50 ± 6.57c

Table 2 shows the extent of DNA damage in different cells simultaneously exposed to 4-ME and DXR. There was a significant reduction in the extent of DNA damage for all three tested doses and in all analyzed cell types compared with the group treated with DXR alone. DNA damage reduction was high for all doses and cell types, ranging from 93.3% in the bone marrow cells of animals treated with 1000 mg kg–1 of 4-ME to 34.1% in the peripheral blood cells of animals treated with 2000 mg kg–1. The results showed that 4-ME clearly protected against the DNA-damaging agent DXR.

Table 2. DNA migration in the comet assay for assessing the antigenotoxicity of 4-methylesculetin (4-ME) in different cells of male Swiss mice in vivo (mean ± standard deviation (SD)]
Treatments and cells analyzedTotaldComet classScoresReduction % (*)
 0123
  1. a

    Significantly different from doxorubicin (DXR) (P < 0.05).

  2. b

    Significantly different from DXR (P < 0.01).

  3. c

    Significantly different from DXR (P < 0.001).

  4. d

    The total number of damaged cells (class 1 + 2 + 3).

  5. (*) How much 4-ME decreased the genotoxicity of the DXR, according to Waters et al. (1990).

Peripheral blood (4-h sample)
4-ME 500 mg kg–1 + DXR 80 mg kg–123.33 ± 2.73b76.67 ± 2.73b23.00 ± 2.610.33 ± 0.52c0.00 ± 0.00c23.67 ± 2.94c66.7
4-ME 1000 mg kg–1 + DXR 80 mg kg–122.33 ± 5.32b77.67 ± 5.32c22.17 ± 5.270.17 ± 0.41c0.00 ± 0.00c22.50 ± 5.39c72.0
4-ME 2000 mg kg–1 + DXR 80 mg kg–130.67 ± 3.2769.33 ± 3.2730.50 ± 3.210.17 ± 0.41c0.00 ± 0.00c30.83 ± 3.37a34.1
Doxorubicin (DXR) 80 mg kg–132.50 ± 4.7667.50 ± 4.7628.00 ± 4.653.17 ± 1.171.33 ± 0.8238.33 ± 4.55--
Peripheral blood (24-h sample)
4-ME 500 mg kg–1+ DXR 80 mg kg–122.17 ± 6.65c77.83 ± 6.65c21.00 ± 6.90c1.33 ± 1.21b0.00 ± 0.00c23.67 ± 6.86c86.8
4-ME 1000 mg kg–1 + DXR 80 mg kg–122.00 ± 3.46c78.00 ± 3.46c21.50 ± 3.78c0.50 ± 0.55c0.00 ± 0.00c22.50 ± 3.21c82.8
4-ME 2000 mg kg–1 + DXR 80 mg kg–133.83 ± 10.19c66.17 ± 10.19c33.67 ± 9.87c0.50 ± 0.84c0.00 ± 0.00c34.67 ± 10.71c72.9
Doxorubicin (DXR) 80 mg kg–183.67 ± 9.3316.33 ± 9.3376.00 ± 5.906.50 ± 4.811.17 ± 0.4192.50 ± 14.21--
Liver
4-ME 500 mg kg–1+ DXR 80 mg kg–167.33 ± 6.8032.67 ± 6.8060.17 ± 11.676.67 ± 4.72c0.33 ± 0.82a74.50 ± 3.89c52.9
4-ME 1000 mg kg–1 + DXR 80 mg kg–170.50 ± 6.2829.50 ± 6.2859.33 ± 11.1510.50 ± 9.09a0.67 ± 0.82a82.33 ± 12.56b43.8
4-ME 2000 mg kg–1 + DXR 80 mg kg–150.50 ± 19.21c49.50 ± 19.21c48.83 ± 17.061.67 ± 2.25c0.00 ± 0.00a52.17 ± 21.39c78.9
Doxorubicin (DXR) 80 mg kg–184.17 ± 6.6215.83 ± 6.6253.67 ± 11.0020.17 ± 6.748.67 ± 11.13120.00 ± 26.37--
Bone marrow
4-ME 500 mg kg–1+ DXR 80 mg kg–132.83 ± 6.08c67.17 ± 6.08c30.33 ± 6.50c2.50 ± 2.07c0.00 ± 0.00c35.33 ± 6.35c84.6
4-ME 1000 mg kg–1 + DXR 80 mg kg–127.00 ± 6.42c73.00 ± 6.42c25.67 ± 7.09c1.33 ± 1.75c0.00 ± 0.00c28.33 ± 6.19c93.3
4-ME 2000 mg kg–1 + DXR 80 mg kg–147.67 ± 19.20c52.33 ± 19.20c45.67 ± 20.10b2.00 ± 1.79c0.00 ± 0.00c49.67 ± 18.44c66.6
Doxorubicin (DXR) 80 mg kg–185.00 ± 6.2915.00 ± 6.2969.00 ± 10.8414.17 ± 10.191.83 ± 1.47102.83 ± 16.42--
Brain
4-ME 500 mg kg–1+ DXR 80 mg kg–161.17 ± 4.71a38.83 ± 4.71a58.67 ± 4.682.50 ± 0.84c0.00 ± 0.00c63.67 ± 4.89c84.8
4-ME 1000 mg kg–1 + DXR 80 mg kg–159.17 ± 13.38b40.83 ± 13.38b51.00 ± 9.728.17 ± 5.81c0.00 ± 0.00c67.33 ± 18.20c78.3
4-ME 2000 mg kg–1 + DXR 80 mg kg–162.67 ± 13.22a37.33 ± 13.22a59.67 ± 12.823.00 ± 1.26c0.00 ± 0.00c65.67 ± 13.72c81.3
Doxorubicin (DXR) 80 mg kg–182.83 ± 5.9817.17 ± 5.9857.67 ± 7.7622.00 ± 9.983.17 ± 3.06111.17 ± 19.14--
Testicle
4-ME 500 mg kg–1+ DXR 80 mg kg–147.83 ± 4.6252.17 ± 4.62c44.83 ± 6.183.00 ± 1.79b0.00 ± 0.0050.83 ± 3.31c54.8
4-ME 1000 mg kg–1 + DXR 80 mg kg–152.00 ± 4.2048.00 ± 4.20b48.67 ± 5.793.33 ± 1.63b0.00 ± 0.0055.33 ± 2.66c45.2
4-ME 2000 mg kg–1 + DXR 80 mg kg–129.67 ± 3.88c70.33 ± 3.88c26.17 ± 3.76c3.17 ± 0.75b0.33 ± 0.5233.50 ± 4.32c91.8
Doxorubicin (DXR) 80 mg kg–165.50 ± 5.1334.50 ± 5.1355.83 ± 10.578.33 ± 4.271.33 ± 2.3476.50 ± 6.57--

The results of experiments designed to assess the clastogenicity of 4-ME in the bone marrow cells of mice are shown in Table 3. As expected, the positive mutagen DXR induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes (MNPCE). However, the clastogenicity test revealed no increase in the mean number of MNPCE at any tested dose. 4-ME did not cause a statistically significant decrease in PCE/NCE ratios. These data indicate that 4-ME had no clastogenic or cytotoxic effects on the bone marrow cells of mice.

Table 3. The number of micronucleated polychromatic erythrocytes (MNPCE) observed in the bone marrow cells of male Swiss mice (M1-6) treated with 4-methylesculetin (4-ME) and respective controls. Two thousand cells were analyzed
TreatmentsNumber of MNPCE per AnimalMNPCE (Mean ± SD)PCE/NCE (Mean ± SD)Reduction (%) (*)
 M1M2M3M4M5M6
  1. a

    Significantly different from doxorubicin (DXR) (P < 0.001).

  2. b

    Significantly different from the negative control (P < 0.001).

  3. (*) How much 4-ME decreased the clastogenicity of the DXR, according to Waters et al. (1990).

  4. SD, standard deviation from the mean; PCE/NCE, polychromatic/normochromatic erythrocytes.

Control (sunflower oil)1221211.50 ± 0.55a1.32 ± 0.12 
4-ME (500 mg kg–1)1211421.83 ± 0.83a1.10 ± 0.04a, b 
4-ME (1000 mg kg–1)2343232.83 ± 0.82a1.27 ± 0.10 
4-ME (2000 mg kg–1)2412222.17 ± 0.98a1.22 ± 0.08 
4-ME (500 mg kg–1) + DXR (80 mg kg–1)7578656.33 ± 1.21a, b1.35 ± 0.0865.9
4-ME (1000 mg kg–1) + DXR (80 mg kg–1)7675676.33 ± 0.82a, b1.20 ± 0.0865.9
4-ME (2000 mg kg–1) + DXR (80 mg kg–1)7796897.67 ± 1.21a, b1.29 ± 0.0654.4
DXR (80 mg kg–1)16111315211815.67 ± 3.56b1.36 ± 0.09 

The frequencies of MNPCEs in bone marrow cells of Swiss mice treated with different doses of 4-ME plus DXR are also presented in Table 3. The oral administration of different doses of 4-ME concomitantly with an injection of DXR led to a significant reduction in the frequency of MNPCEs compared with animals treated only with DXR. This reduction ranged from 65.9% to 54.4% in the micronucleus test. A gradual increase in the 4-ME concentration did not result in a proportional decrease in DXR-induced clastogenicity, indicating the lack of a dose-response relationship.

Discussion

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The alkaline comet assay (pH > 13.0) detects primary DNA damage such as single and double-strand breaks, alkali-labile sites (apurinic/apyrimidinic sites) or errors in the sugar molecule (Collins et al., 2008). This assay is widely used in in vivo experiments to test the genotoxicity of new chemicals (Brendler-Schwaab et al., 2005; Collins et al., 2008). The damage is thus detected before the action of the DNA repair system, which repairs it in an even shorter time than is necessary to fix a mutation. Furthermore, the DNA lesion can be measured in the absence of mitotic activity. On the other hand, for a better evaluation of the genotoxic potential of any compound, the concomitant use of a widely-accepted complementary test system such as the micronucleus test is necessary (Krishna and Hayashi, 2000). In this test, the cells have already gone through at least one cellular cycle and the repair system has already been activated.

Hematopoietic mouse cells are generally used to test the genotoxicity of compounds investigated in in vivo assays with the micronucleus test (Krishna and Hayashi, 2000). This means that little information is available about the test substance's genotoxicity in other organs. Additionally, organ-specific genotoxicity closely parallels cell-specific carcinogenicity (Tsuda et al., 2000). In an effort to improve this situation, the present study analyzed the genotoxic effects of 4-ME in the cells of five different organs, including somatic metabolizing and non-metabolizing cells, and germ cells. Cell viability in each of the analyzed cell types was over 90% (data not shown). The negative control group showed different natural endogenous genotoxicity thresholds; the lowest genotoxicity scores were observed in peripheral blood leukocytes and the highest were in the brain cells. On the other hand, the use of the damage-inducing agent DXR led to a clear increase (P < 0.001) in genotoxicity in each type of cell, indicating the validity of both the species selected and the study design for detecting genotoxic effects.

The genotoxicity data from the comet assay showed that 4-ME, in the doses administered via gavage in mice, did not promote a significant increase in damage scores compared with negative controls for any of the cell types evaluated. Similarly, 4-ME did not promote a significant increase in MNPCE frequency. These results indicate that 4-ME had neither genotoxic nor clastogenic effects on the different cells analyzed. No other study has investigated the genotoxic potential of 4-ME. On the other hand, other in vivo genotoxicity tests evaluating coumarin, the natural precursor of 4-ME, have demonstrated that it does not increase micronuclei frequencies in either bone marrow cells from ICR mice (Morris and Ward, 1992) or the peripheral blood of male or female B6C3F1 mice (NTP, 1993). The same was observed in the bone marrow cells of Swiss mice (Api, 2001).

DXR is a potent chemotherapeutic agent, often used in the treatment of solid tumors, especially for breast and ovarian cancer, but its therapeutic use is limited by its cardiotoxicity. DXR-induced cardiomyopathy includes the formation of oxygen-free radicals (Takemura and Fujiwara, 2007). The semiquinone form, produced by the reduction of DXR by several endogenous enzymes, generates superoxide radicals that rapidly generate other reactive oxygen species, such as hydroxyl and hydrogen peroxide radicals, which can induce apoptosis in cardiomyocytes (Menna et al., 2007). Thus, it is important to test strategies for decreasing the amount of ROS without affecting therapeutic efficiency in order to reduce the cardiac toxicity of this agent.

In the present study's antigenotoxicity tests, 4-ME appeared as an important inhibitor of DXR-induced DNA damage. The comet assay revealed that 4-ME reduced damage up to 93.3% in the bone marrow cell samples of mice treated with 1000 mg kg–1 4-ME simultaneously with 80 mg kg–1 DXR. The micronucleus test in bone marrow cells demonstrated that 4-ME caused a reduction in MNPCE frequency of up to 65.9% in doses of 500 and 1000 mg kg–1.

The literature cites protective effects of coumarin and its derivatives such as 4-ME. Beillerot et al. (2008) studied a wide variety of coumarin derivatives in cancer cells (MCF7) and observed that 4-ME had an antioxidant effect on DXR-induced oxidative damage without affecting the cytotoxic efficacy of this anthracycline. They also pointed out the importance of evaluating these effects in healthy cells. Lin et al. (2008) evaluated the antioxidant activity of coumarin compounds, including 4-ME, in neuroblastoma cells, and observed that these drugs had high antioxidant activity, inhibiting xanthine oxidase and reducing DPPH radicals (1,1-diphenyl-2-picrylhydrazyl hydrate). These and other free radicals such as ROS can interact directly with DNA molecules, resulting in genotoxic damage to cells (Ribeiro et al., 2005). We suggest, therefore, that the antigenotoxic and anticlastogenic activities of 4-ME observed in the present study may have been as a result of the antioxidant activity in this compound that, by diminishing the formation of free radicals, impedes oxidative genetic damage. The hypothesis outlined in this study is supported by the studies of Hiramoto et al. (1996), who reported that esculetin also scavenges hydroxyl radicals, and Beillerot et al. (2008), who reported that 4-ME was able to reduce the oxidative stress induced by DXR in the cancerous MCF7 cells without affecting its therapeutic efficacy. However, further studies are necessary to test if the chemoprevenction of 4-ME against DXR is exclusively by antioxidant effects, once that DXR also cause mutagenicity acting as a topoisomerase II inhibitor and is able to intercalate between base pairs of the DNA affecting DNA replication and RNA synthesis (Ferguson and Pearson, 1996), and also to verify if the antigenotoxic effects of the 4-ME may result in a lesser efficacy of the DXR against the target tumors.

In conclusion, the results obtained in this study showed that 4-ME administered by gavage was not genotoxic in mice peripheral blood leukocytes, liver, bone marrow, brain or testicle cells according to the comet assay or in bone marrow cells according to the micronucleus test. In addition, 4-ME was also observed to play a role in preventing chemically induced DNA and chromosome damage (breakage and loss) in vivo by the antitumor agent DXR. Further in vivo and in vitro studies are necessary to better characterize the protective role of this compound and to explore its therapeutic relevance as antioxidant and as an adjuvant in DXR treatments for humans.

Acknowledgments

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

This study was supported by the Brazilian agency FAPESP - Fundação de Amparo à Pesquisa do Estado de São Paulo (2010/07577-3). We would like to thank Patrícia C. Martins Mello for her technical assistance.

References

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  • Api AM. 2001. Lack of effect of coumarin on the formation of micronuclei in an in vivo mouse micronucleus assay. Food Chem. Toxicol. 39: 837841.
  • Al-Amiery AA, Al-Bayati R, Saour K, Radi M. 2012. Cytotoxicity, antioxidant and antimicrobial activities of novel 2-quinolone derivatives derived from coumarins. Res. Chem. Interm. 38:559569.
  • Beillerot A, Domínguez JCR, Kirsch G, Bagrel D. 2008. Synthesis and protective effects of coumarin derivatives against oxidative stress induced by doxorubicin. Bioor. Med. Chem. Let. 18:11021105.
  • Born SL, Rodriguez PA, Eddy CL, Lehman-McKeeman LD. 1997. Synthesis and reactivity of coumarin 3,4-epoxide. Drug Metab. Dispos. 25: 13181323.
  • Brendler-Schwaab S, Hartmann A, Pfuhler S, Speit G. 2005. The in vivo comet assay: use and status in genotoxicity testing. Mutagenesis 20: 245254.
  • Cohen AJ. 1979. Critical review of the toxicology of coumarin with special reference to interspecies differences in metabolism and hepatotoxic response and their significance to man. Food Chem. Toxicol. 17: 277289.
  • Collins AR, Oscoz AA, Brunborg G, Gaivão I, Giovanelli L, Kruszewski M, Smith CC, Stetina R. 2008. The comet assay: topical issues. Mutagenesis 23: 143151.
  • EFSA (European Food Safety Authority). 2004. Opinion of the scientificpanel on food additives, flavourings, processing aids and materials in contact with food (AFC) on a request from the commission related to coumarin, adopted on 6 october 2004. EFSA J. 104: 136.
  • Farombi EO, Nwaokeafor IA. 2005. Anti-oxidant mechanisms of kolaviron: studies on serum lipoprotein oxidation, metal chelation and oxidative membrane damage in rats. Clin. Exp. Pharmacol. Physiol. 32: 667674.
  • Ferguson LR, Pearson AE. 1996. The clinical use of mutagenic anticancer drugs. Mutat. Res. 355: 112.
  • Flasik R, Stankovicová H, Gáplovsky A, Donovalová J, 2009. Synthesis and study of novel coumarin derivatives potentially utilizable as memory media. Molecules 14: 48384848.
  • Garazd MM, Muzychka OV, Voyk AI, Nagorichna IV, Ogorodniichuk AS. 2007. Modified coumarins. 27. Synthesis and antioxidant activity of 3-substituted 5,7-dihydroxy-4-methylcoumarins. Chem. Nat. Comp. 43: 1923.
  • Gollapudi BB, McFadden LG. 1995. Sample size for the estimation of polychromatic to normochromatic erithrocyte ratio in the bone marrow micronucleus test. Mutat. Res. 347: 9799.
  • Hartmann A, Speit G. 1997. The contribution of cytotoxicity to DNA-effects in the single cell gel test (comet assay). Toxicol. Lett. 90: 183188.
  • Heravi M, Sadjadi S, Oskooie H, Shoar R, Bamoharram F. 2008. The synthesis of coumarin-3-carboxylic acids and 3-acetyl-coumarin derivatives using heteropolyacids as heterogeneus and recyclable catalysts. Catal. Commun. 9: 470474.
  • Hiramoto K, Ojima N, Sako K, Kikugawa K. 1996. Effect of plant phenolics on the formation of the spin-adduct of hydroxyl radical and the DNA strand breaking by hydroxyl radical. Biol. Pharm. Bull. 19: 558563.
  • Kennedy RO, Thornes RD. 1997. Coumarins: Biology, Applications and Mode of Action. John Wiley and Sons: Chichester.
  • Klaude M, Eriksson S, Nygren J, Ahnstrom G. 1996. The comet assay: mechanisms and technical considerations. Mutat. Res. 363: 8996.
  • Kotali A, Lafazanis I, Harris P. 2008. Synthesis of 6,7-diacylcoumarins via the transformation of a hydroxyl into a carbonyl group. Synth. Commun. 38: 39964006.
  • Krishna G, Hayashi M. 2000. In vivo rodent micronucleus assay: protocol, conduct and data interpretation. Mutat. Res. 455: 155166.
  • Lake BG. 1984. Investigations in to the mechanism of coumarin-induced hepatotoxicity in the rat. Arch. Toxicol. 7: 1629.
  • Lake BG. 1999. Coumarin metabolism, toxicity and carcinogenicity: relevance for human risk assessment. Food Chem. Toxicol. 37: 423453.
  • Lake BG, Osborne DJ, Walters DG, Price RJ. 1992. Identification of o-hydroxyphenylacetaldehyde as a major metabolite of coumarin in rat hepatic microsomes. Food Chem. Toxicol. 30: 99104.
  • Lake BG, Evans JG, Lewis DFV, Price RJ. 1994. Comparison of the hepatic effects of coumarin, 3,4-dimethylcoumarin, dihydrocoumarin and 6-methylcoumarin in the rat. Food Chem. Toxicol. 32: 743751.
  • Lake BG. 1996. Coumarin: species differences in metabolism. Proceedings from the Seventh International Information Exchange. pp. 3344. Reserach Institute for Fragrance Materials, Inc.: New Jersey.
  • Lin HC, Tsai SH, Chen CS, Chang YC, Lee CM, Lai ZY, Lin CM. 2008. Structure-activity relationship of coumarin derivatives on santhine oxidase-inhibiting and free radical-scavenging activities. Biochem. Pharmacol. 75: 14161425.
  • Menna P, Minotti G, Salvatorelli E. 2007. In vitro modeling of the structure-activity determinants of anthracycline cardiotoxicity. Cell Biol. Toxicol. 23: 4962.
  • Morris DL, Ward JB. 1992. Coumarin inhibits micronuclei formation induced by benzo(a)pyrene in male but not female ICR mice. Environ. Mol. Mutag. 19: 132138.
  • Nofal ZM, El-Zahar M, Abd-El-Karim S. 2000. Novel coumarin derivatives with expectedbiological activity. Molecules 5: 99113.
  • Norman RL, Wood AW. 1981. Assessment of the mutagenic potential of coumarin in histidine-dependent strains of Salmonella typhimurium. Proc. Am. Assoc. Cancer Res. 22, Abs 433.
  • NTP (National Toxicology Program). 1993. Toxicology and carcinogenic studies of coumarin (CAS No. 91-64-5) in F344/N rats and B6C3F1 mice (Gavage studies). Technical Report Series No. 422. NIH Publication No. 92-31153, US Department of Health and Human services, Bethesda, MD.
  • Ribeiro SMR, Queiroz JH, Pelúzo MCG, Costa NMB, Matta SLP, Queiroz MELR. 2005. The formation and the effects of the reactive oxygen species in biological media. Biosci. J. 21: 133149.
  • Rositca DN, Vayssilov GN, Rodios N, Bojilova A. 2002. Regio- and stereoselective [2 + 2] photodimerization of 3-substituted 2-Alkoxy-2-oxo-2H-1,2-benzoxaphosphorines. Molecules 7: 420432.
  • Sasaki YF, Imanishi H, Ohta T, Shirasu Y. 1987. Effect of antimutagenic flavorings on SCEs induced by chemical mutagens in cultured Chinese hamster cells. Mutat. Res. 189: 313318.
  • Satyanarayan VS, Sreevani P, Sivakumar A. 2008. Synthesis and antimicrobial activity of new Schiff bases containing coumarin moiety and their spectral characterization. Arkivoc 17: 221233.
  • Sharma SD, Rajor HK, Chopra S, Sharma RK. 2005. Studies on structure activity relationship of some dihydroxy-4-methylcoumarin antioxidants based on their interaction with Fe(III) and ADP. Biometals 18: 143154.
  • Schmid W. 1975. The micronucleus test. Mutat. Res. 31: 915.
  • Singh NP, McCoy MT, Tice RR, Schneider EL. 1988. A simple technique for quantitation of low levels of DNA damage in individual cells. Exper. Cell Res. 175: 184191.
  • Smitha G, Sanjeeva R. 2004. ZrCl4-catalyzed Pechmann reaction: Synthesis of coumarins under solvent-free conditions. Synth. Commun. 34: 39974003.
  • Speit G, Hartmann A. 1999. The comet assay (single-cell gel test). In DNA Repair Protocols: Eukaryotic Systems, Henderson DS (ed.). Methods in Molecular Biology, Vol. 113. Humana Press: Totowa, NJ; 203-212.
  • Stolz DR, Scott PM. 1980. Mutagenicity of coumarin and related compounds for Salmonella typhimurium. Can. J. Genet. Cytol. 22: 679.
  • Takemura G, Fujuwara H. 2007. Doxorubicin-induced cardiomyopathy from the cardiotoxic mechanisms to management. Prog. Cardiovasc. Dis. 49: 330352.
  • Tsuda S, Matsusaka N, Madarame H, Miyamae Y, Ishida K, Satoh M, Sekihashi K, Sasaki YF. 2000. The alkaline single cell electrophoresis assay with eight mouse organs: results with 22 mono-functional alkylating agents (including 9 dialkyl N-nitrosoamines) and 10 DNA crosslinkers. Mutat. Res. 467: 8398.
  • Waters MD, Brady AL, Stack HF, Brockmann HE. 1990. Antimutagenicity profiles for some model compounds. Mutat. Res. 238: 5785.
  • Zabradnik M. 1992. The Production and Application of Fluorescent Brightening Agents. John Wiley and Sons: New York, NY, USA.