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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.
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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.
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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 analysed||Totald||Comet class||Scores|
| ||0||1||2||3|| |
|Peripheral blood (4-h sample)|
|Control||14.00 ± 4.43||86.00 ± 4.43||12.17 ± 4.62||1.33 ± 0.52||0.50 ± 0.84||16.33 ± 4.55|
|4-ME 500 mg kg–1||20.67 ± 1.63a||79.33 ± 1.63||20.00 ± 2.00a||0.67 ± 1.03||0.00 ± 0.00||21.33 ± 1.86|
|4-ME 1000 mg kg–1||19.00 ± 3.16||81.00 ± 3.16||18.83 ± 3.19||0.17 ± 0.41||0.00 ± 0.00||19.17 ± 3.19|
|4-ME 2000 mg kg–1||12.00 ± 3.58||88.00 ± 3.58||11.67 ± 3.93||0.33 ± 0.52||0.00 ± 0.00||12.33 ± 3.27|
|Doxorubicin 80 mg kg–1||32.50 ± 4.76c||67.50 ± 4.76c||28.00 ± 4.65c||3.17 ± 1.17c||1.33 ± 0.82a||38.33 ± 4.55c|
|Peripheral blood (24-h sample)|
|Control||12.33 ± 3.27||87.67 ± 3.27||11.50 ± 3.56||0.83 ± 0.75||0.00 ± 0.00||13.17 ± 3.13|
|4-ME 500 mg kg–1||20.50 ± 8.71||79.50 ± 8.71||20.00 ± 8.99||0.50 ± 0.55||0.00 ± 0.00||21.00 ± 8.46|
|4-ME 1000 mg kg–1||25.33 ± 5.47a||74.67 ± 5.47a||23.83 ± 4.62a||1.50 ± 1.38||0.00 ± 0.00||26.83 ± 6.49|
|4-ME 2000 mg kg–1||16.83 ± 3.13||83.17 ± 3.13||15.17 ± 3.60||1.50 ± 1.64||0.17 ± 0.41||18.67 ± 3.83|
|Doxorubicin 80 mg kg–1||83.67 ± 9.33c||16.33 ± 9.33c||76.00 ± 5.90c||6.50 ± 4.81c||1.17 ± 0.41c||92.50 ± 14.21c|
|Control||33.17 ± 4.36||66.83 ± 4.36||32.33 ± 4.08||0.83 ± 0.98||0.00 ± 0.00||34.00 ± 4.82|
|4-ME 500 mg kg–1||46.67 ± 9.16a||53.33 ± 9.16||45.67 ± 9.22||1.00 ± 1.10||0.00 ± 0.00||47.67 ± 9.22|
|4-ME 1000 mg kg–1||42.83 ± 7.70||57.17 ± 7.70||41.33 ± 8.07||1.50 ± 2.07||0.00 ± 0.00||44.33 ± 7.89|
|4-ME 2000 mg kg–1||32.00 ± 6.78||68.00 ± 6.78||32.00 ± 6.78||0.00 ± 0.00||0.00 ± 0.00||32.00 ± 6.78|
|Doxorubicin 80 mg kg–1||84.17 ± 6.62c||15.83 ± 6.62c||53.67 ± 11.00a||20.17 ± 6.74c||8.67 ± 11.13a||120.00 ± 26.37c|
|Control||21.67 ± 5.54||78.83 ± 5.54||21.33 ± 5.05||0.33 ± 0.82||0.00 ± 0.00||22.00 ± 6.10|
|4-ME 500 mg kg–1||24.67 ± 7.15||75.33 ± 7.15||24.67 ± 7.15||0.00 ± 0.00||0.00 ± 0.00||24.67 ± 7.15|
|4-ME 1000 mg kg–1||22.00 ± 3.69||78.00 ± 3.69||21.67 ± 3.33||0.33 ± 0.82||0.00 ± 0.00||22.33 ± 4.18|
|4-ME 2000 mg kg–1||29.67 ± 6.50||70.33 ± 6.50||28.83 ± 6.49||0.83 ± 0.75||0.00 ± 0.00||30.50 ± 6.60|
|Doxorubicin 80 mg kg–1||85.00 ± 6.29c||15.00 ± 6.29c||69.00 ± 10.84c||14.17 ± 10.19c||1.83 ± 1.47c||102.83 ± 16.42c|
|Control||54.00 ± 12.03||46.00 ± 12.03||52.83 ± 11.69||1.17 ± 1.17||0.00 ± 0.00||55.17 ± 12.48|
|4-ME 500 mg kg–1||36.67 ± 4.89||63.33 ± 4.89||34.33 ± 5.75||2.33 ± 1.63||0.00 ± 0.00||39.00 ± 4.47|
|4-ME 1000 mg kg–1||40.17 ± 8.23||59.83 ± 8.23||38.17 ± 9.66||2.00 ± 2.10||0.00 ± 0.00||42.17 ± 7.14|
|4-ME 2000 mg kg–1||40.50 ± 15.93||59.50 ± 15.93||39.83 ± 16.49||0.67 ± 1.03||0.00 ± 0.00||41.17 ± 15.43|
|Doxorubicin 80 mg kg–1||82.83 ± 5.98c||17.17 ± 5.98c||57.67 ± 7.76c||22.00 ± 9.98c||3.17 ± 3.06c||111.17 ± 19.14c|
|Control||27.00 ± 6.93||73.00 ± 6.93||24.50 ± 6.63||2.33 ± 2.42||0.17 ± 0.41||29.67 ± 8.43|
|4-ME 500 mg kg–1||27.17 ± 2.04||72.83 ± 2.04||27.17 ± 2.04||0.00 ± 0.00||0.00 ± 0.00||27.17 ± 2.04|
|4-ME 1000 mg kg–1||24.83 ± 2.93||75.17 ± 2.93||23.67 ± 2.66||1.17 ± 1.17||0.00 ± 0.00||26.00 ± 3.58|
|4-ME 2000 mg kg–1||26.50 ± 8.09||73.50 ± 8.09||22.67 ± 8.64||3.50 ± 2.07||0.33 ± 0.52||30.67 ± 8.26|
|Doxorubicin 80 mg kg–1||65.50 ± 5.13c||34.50 ± 5.13c||55.83 ± 10.57c||8.33 ± 4.27c||1.33 ± 2.34||76.50 ± 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 analyzed||Totald||Comet class||Scores||Reduction % (*)|
|Peripheral blood (4-h sample)|
|4-ME 500 mg kg–1 + DXR 80 mg kg–1||23.33 ± 2.73b||76.67 ± 2.73b||23.00 ± 2.61||0.33 ± 0.52c||0.00 ± 0.00c||23.67 ± 2.94c||66.7|
|4-ME 1000 mg kg–1 + DXR 80 mg kg–1||22.33 ± 5.32b||77.67 ± 5.32c||22.17 ± 5.27||0.17 ± 0.41c||0.00 ± 0.00c||22.50 ± 5.39c||72.0|
|4-ME 2000 mg kg–1 + DXR 80 mg kg–1||30.67 ± 3.27||69.33 ± 3.27||30.50 ± 3.21||0.17 ± 0.41c||0.00 ± 0.00c||30.83 ± 3.37a||34.1|
|Doxorubicin (DXR) 80 mg kg–1||32.50 ± 4.76||67.50 ± 4.76||28.00 ± 4.65||3.17 ± 1.17||1.33 ± 0.82||38.33 ± 4.55||--|
|Peripheral blood (24-h sample)|
|4-ME 500 mg kg–1+ DXR 80 mg kg–1||22.17 ± 6.65c||77.83 ± 6.65c||21.00 ± 6.90c||1.33 ± 1.21b||0.00 ± 0.00c||23.67 ± 6.86c||86.8|
|4-ME 1000 mg kg–1 + DXR 80 mg kg–1||22.00 ± 3.46c||78.00 ± 3.46c||21.50 ± 3.78c||0.50 ± 0.55c||0.00 ± 0.00c||22.50 ± 3.21c||82.8|
|4-ME 2000 mg kg–1 + DXR 80 mg kg–1||33.83 ± 10.19c||66.17 ± 10.19c||33.67 ± 9.87c||0.50 ± 0.84c||0.00 ± 0.00c||34.67 ± 10.71c||72.9|
|Doxorubicin (DXR) 80 mg kg–1||83.67 ± 9.33||16.33 ± 9.33||76.00 ± 5.90||6.50 ± 4.81||1.17 ± 0.41||92.50 ± 14.21||--|
|4-ME 500 mg kg–1+ DXR 80 mg kg–1||67.33 ± 6.80||32.67 ± 6.80||60.17 ± 11.67||6.67 ± 4.72c||0.33 ± 0.82a||74.50 ± 3.89c||52.9|
|4-ME 1000 mg kg–1 + DXR 80 mg kg–1||70.50 ± 6.28||29.50 ± 6.28||59.33 ± 11.15||10.50 ± 9.09a||0.67 ± 0.82a||82.33 ± 12.56b||43.8|
|4-ME 2000 mg kg–1 + DXR 80 mg kg–1||50.50 ± 19.21c||49.50 ± 19.21c||48.83 ± 17.06||1.67 ± 2.25c||0.00 ± 0.00a||52.17 ± 21.39c||78.9|
|Doxorubicin (DXR) 80 mg kg–1||84.17 ± 6.62||15.83 ± 6.62||53.67 ± 11.00||20.17 ± 6.74||8.67 ± 11.13||120.00 ± 26.37||--|
|4-ME 500 mg kg–1+ DXR 80 mg kg–1||32.83 ± 6.08c||67.17 ± 6.08c||30.33 ± 6.50c||2.50 ± 2.07c||0.00 ± 0.00c||35.33 ± 6.35c||84.6|
|4-ME 1000 mg kg–1 + DXR 80 mg kg–1||27.00 ± 6.42c||73.00 ± 6.42c||25.67 ± 7.09c||1.33 ± 1.75c||0.00 ± 0.00c||28.33 ± 6.19c||93.3|
|4-ME 2000 mg kg–1 + DXR 80 mg kg–1||47.67 ± 19.20c||52.33 ± 19.20c||45.67 ± 20.10b||2.00 ± 1.79c||0.00 ± 0.00c||49.67 ± 18.44c||66.6|
|Doxorubicin (DXR) 80 mg kg–1||85.00 ± 6.29||15.00 ± 6.29||69.00 ± 10.84||14.17 ± 10.19||1.83 ± 1.47||102.83 ± 16.42||--|
|4-ME 500 mg kg–1+ DXR 80 mg kg–1||61.17 ± 4.71a||38.83 ± 4.71a||58.67 ± 4.68||2.50 ± 0.84c||0.00 ± 0.00c||63.67 ± 4.89c||84.8|
|4-ME 1000 mg kg–1 + DXR 80 mg kg–1||59.17 ± 13.38b||40.83 ± 13.38b||51.00 ± 9.72||8.17 ± 5.81c||0.00 ± 0.00c||67.33 ± 18.20c||78.3|
|4-ME 2000 mg kg–1 + DXR 80 mg kg–1||62.67 ± 13.22a||37.33 ± 13.22a||59.67 ± 12.82||3.00 ± 1.26c||0.00 ± 0.00c||65.67 ± 13.72c||81.3|
|Doxorubicin (DXR) 80 mg kg–1||82.83 ± 5.98||17.17 ± 5.98||57.67 ± 7.76||22.00 ± 9.98||3.17 ± 3.06||111.17 ± 19.14||--|
|4-ME 500 mg kg–1+ DXR 80 mg kg–1||47.83 ± 4.62||52.17 ± 4.62c||44.83 ± 6.18||3.00 ± 1.79b||0.00 ± 0.00||50.83 ± 3.31c||54.8|
|4-ME 1000 mg kg–1 + DXR 80 mg kg–1||52.00 ± 4.20||48.00 ± 4.20b||48.67 ± 5.79||3.33 ± 1.63b||0.00 ± 0.00||55.33 ± 2.66c||45.2|
|4-ME 2000 mg kg–1 + DXR 80 mg kg–1||29.67 ± 3.88c||70.33 ± 3.88c||26.17 ± 3.76c||3.17 ± 0.75b||0.33 ± 0.52||33.50 ± 4.32c||91.8|
|Doxorubicin (DXR) 80 mg kg–1||65.50 ± 5.13||34.50 ± 5.13||55.83 ± 10.57||8.33 ± 4.27||1.33 ± 2.34||76.50 ± 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
|Treatments||Number of MNPCE per Animal||MNPCE (Mean ± SD)||PCE/NCE (Mean ± SD)||Reduction (%) (*)|
|Control (sunflower oil)||1||2||2||1||2||1||1.50 ± 0.55a||1.32 ± 0.12|| |
|4-ME (500 mg kg–1)||1||2||1||1||4||2||1.83 ± 0.83a||1.10 ± 0.04a, b|| |
|4-ME (1000 mg kg–1)||2||3||4||3||2||3||2.83 ± 0.82a||1.27 ± 0.10|| |
|4-ME (2000 mg kg–1)||2||4||1||2||2||2||2.17 ± 0.98a||1.22 ± 0.08|| |
|4-ME (500 mg kg–1) + DXR (80 mg kg–1)||7||5||7||8||6||5||6.33 ± 1.21a, b||1.35 ± 0.08||65.9|
|4-ME (1000 mg kg–1) + DXR (80 mg kg–1)||7||6||7||5||6||7||6.33 ± 0.82a, b||1.20 ± 0.08||65.9|
|4-ME (2000 mg kg–1) + DXR (80 mg kg–1)||7||7||9||6||8||9||7.67 ± 1.21a, b||1.29 ± 0.06||54.4|
|DXR (80 mg kg–1)||16||11||13||15||21||18||15.67 ± 3.56b||1.36 ± 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.
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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.