Journal of Toxicology and Environmental Health, Part A, 78:109–118, 2015 Copyright © Taylor & Francis Group, LLC ISSN: 1528-7394 print / 1087-2620 online DOI: 10.1080/15287394.2014.943865
IN VITRO ASSESSMENT OF MUTAGENIC AND GENOTOXIC EFFECTS OF COUMARIN DERIVATIVES 6,7-DIHYDROXYCOUMARIN AND 4-METHYLESCULETIN Edson Luis Maistro1,2, Eduardo de Souza Marques1, Rafael Palhano Fedato1, Flora Tolentino2, Chayene de Andrade Cezário da Silva2, Marcela Stefanini Ferreira Tsuboy2, Flavia Aparecida Resende3, Eliana Aparecida Varanda3 1
Programa de Pós-Graduação em Biologia Geral e Aplicada, Universidade Estadual Paulista (UNESP), Instituto de Biociências, Botucatu, São Paulo, Brazil 2 Universidade Estadual Paulista–UNESP, Faculdade de Filosofia e Ciências, Departamento de Fonoaudiologia, Marília, SP, Brazil 3 Universidade Estadual Paulista–UNESP, Faculdade de Ciências Farmacêuticas, Departamento de Ciências Biológicas, Araraquara, SP, Brazil Coumarins are naturally occurring compounds, widely distributed throughout the plant kingdom (Plantae), and possess important pharmacological properties, including inhibition of oxidative stress. In this context, newly synthesized coumarin compounds are being produced due to their potent antioxidant activities. Therefore, the aim of the present study was to determine the in vitro cytotoxic, mutagenic, and genotoxic effects of 6,7-dihydroxycoumarin (6,7-HC) and 4-methylesculetin (4-ME) using the Salmonella/microsome test and in cultured human lymphocytes the comet assay and micronucleus test. The three coumarin derivatives concentrations evaluated in comet and MN assays were 2, 8, and 32 µg/mL, selected through a preliminary trypan blue-staining assay. In the Ames test, the 5 concentrations tested were 62.5, 125, 250, 500, and 750 µg/plate. Positive (methyl methane-sulfonate, MMS) and negative (dimethyl sulfoxide, DMSO) control groups were also included in the analysis. Our results showed that 4-ME induced greater cytotoxicity at high concentrations than 6,7-HC. In addition, both compounds were not mutagenic in the Ames test and not genotoxic or clastogenic/aneugenic in cultured human lymphocytes.
properties that include furanocoumarininduced photoxicity (Berenbaum, 1995), anticarcinogenesis/chemoprevention of cancer (Wattenberg et al., 1979; Cai et al., 1997; Tanaka et al., 1998; Kelly et al., 2000; Kleiner et al., 2002; Prince et al., 2006; Kim et al., 2009; Francisco et al., 2012), cancer cell cytotoxicity (Setzer et al., 2000), antimicrobial effects (Ngwendson et al., 2003), and modulation of cytochrome P450 3A4 (Guo and Yamazoe, 2004). The inhibition of oxidative stress is a contributing factor for the observed biological
Coumarins consist of a large class of phenolic substances detected in approximately 150 different plant species distributed around 30 different families, including Rutaceae, Apiaceae, Umbellifereae, and Moraceae (Murray, 1982), as well as Clusiaceae, Guttiferae, Caprifoliaceae, Oleraceae, and Nyctaginaceae (Venugopala et al., 2013). More than 1300 coumarins have been identified as secondary metabolites from plants, bacteria, and fungi (Iranshahi et al., 2009). Natural coumarins are of great interest due to their pharmacologic/toxicologic
Received 1 July 2014; accepted 8 July 2014. Address correspondence to Edson Luis Maistro, Universidade Estadual Paulista–UNESP, Faculdade de Filosofia e Ciências, Departamento de Fonoaudiologia. Av. Hygino Muzzi Filho, 737, Caixa Postal 181. Marília, SP, Brazil. 17525-900. E-mail: edson.maistro@ marilia.unesp.br 109
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effects of coumarins. Indeed, coumarins were reported to affect the formation and scavenging of reactive oxygen species (ROS) and influence free radical-mediated oxidative damage (Vianna et al., 2012). Based upon these antioxidant properties, there is a concerted effort to develop newly synthesized coumarin derivatives and determine the antioxidant behavior of these novel coumarins with different chemical substitutions. Hydroxycoumarins are newly synthesized compounds shown to possess effective antioxidant capacity (Beillerot et al., 2008; Vianna et al., 2012; Mitra et al., 2013). However, the use of some of the coumarins and their derivatives was found to produce adverse effects such as mild nausea, diarrhea, and hepatotoxicity (Lake and Grasso, 1996; Lake, 1999; Casley-Smith, 1999; Born et al., 2000). Bearing in mind the issue of side effects and considering that coumarin derivatives 6,7-dihydroxycoumarin (6,7-HC) (synonym esculetin) and 4-methylesculetin (4-ME) display a high antioxidant activity with potential use to humans, this study aimed to determine wheter these compounds exerted mutagenic and genotoxic effects using the following assays: DNA mutation in Salmonella typhimurium, and DNA damage and induction of micronuclei (MN) in human peripheral blood lymphocytes (PBL).
MATERIALS AND METHODS Chemical Compounds The 6,7-dihydroxycoumarin (6,7-HC) used in the tests was obtained from Aldrich (Cat.: 24,657-3, 98% purity), and the 4methylesculetin (4-ME) was obtained from Acrós Organics (CAS number 529-84-0, 99% purity) (Figure 1). Both were dissolved in 1% dimethyl sulfoxide (DMSO). Methyl methanesulfonate (MMS) (Aldrich, CAS number 66-273) was used as the positive control substance due to its potential for DNA damage established in the comet assay and micronucleus test. The other main chemicals were obtained from the following suppliers: normal melting
FIGURE 1. The chemical structures of coumarin, 6,7dihydroxycoumarin (esculetin), and 4-methylesculetin.
point (NMP) agarose (Invitrogen), low melting point (LMP) agarose (Invitrogen), sodium salt N-lauroyl sarcosine (Sigma), ethylenediamine tetraacetic acid (EDTA) (Merck), and Histopaque-1077 (Sigma-Aldrich, Co., St. Louis, MO). Salmonella/Microsome Assay 6,7-HC and 4-ME were evaluated in a bacterial mutation assay system using the Salmonella typhimurium tester strains TA98 (frameshift), TA100 (base-pair substitution), TA97a (frameshift), and TA102 (base-pair substitution) using preincubation methodology, with (+S9) and without (–S9) metabolization (Maron and Ames, 1983). Bacteria strains were kindly provided by Dr. B.N. Ames (Berkeley, CA). The strains were grown overnight from frozen cultures for 12–14 h in Oxoid nutrient broth no. 2. The metabolic activation mixture (S9 fraction), prepared from Sprague-Dawley
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mouse liver treated with the polychlorinated biphenyl mixture Aroclor 1254 (500 mg/kg), was purchased from Molecular Toxicology, Inc. (Boone, NC), and freshly prepared before each test. The metabolic activation system consisted of 4% S9 fraction, 1% 0.4 M MgCl2, 1% 1.65 M KCl, 0.5% 1 M D-glucose-6-phosphate disodium and 4% 0.1 M NADP, 50% 0.2 M phosphate buffer, and 39.5% sterile distilled water (Maron and Ames, 1983). For the mutagenic assessment, five different concentrations of 6,7HC and 4-ME (62.5, 125, 250, 500, and 750 µg/plate), diluted in DMSO, were tested. The concentrations of the samples were selected based upon preliminary toxicity tests that determined the highest non-toxic concentration and lowest toxic concentration. Samples were considered toxic when a thinning of the auxotrophic background (i.e., background lawn) was accompanied by a decrease in the number of histidine revertants (His+). The concentrations of test substances were added to 0.5 ml 0.2 M phosphate buffer or 0.5 ml 4% S9 mixture, with 0.1 ml bacterial culture, and then incubated at 37◦ C for 20–30 min. Two milliliters of surface agar was added, tubes were mixed, and the mixture was poured into a petri dish containing 20 ml minimal agar. The petri dishes were incubated at 37◦ C for 48 h and His+ revertant colonies were counted manually. The test was performed in triplicate. Data obtained were analyzed using the statistical software package Salanal 1.0 (U.S. Environmental Protection Agency, Monitorin Systems Laboratory, Las Vegas, NV, from the Research Triangle Institute, RTP, NC). Data (revertants/plate) were assessed by means of the analysis of variance (ANOVA) using the Bernstein et al. (1982) model followed by linear regression. The mutagenicity ratio (MR) was also determined for each concentration tested, where average number of revertants per plate with test compound was divided by average number of revertants per plate with negative (solvent) control. A test solution was considered mutagenic when a concentrationresponse relationship was detected and a twofold increase in number of mutants (MR ≥ 2) was observed in at least one concentration
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(Resende et al., 2012). DMSO was used as negative (solvent) control. The mutagens used as positive controls in the tests without S9 mix were 4-nitro-o-phenylenediamine (NPD) (10 µg/plate) for TA98 and TA97a, sodium azide (SAZ) (1.25 µg/plate) for TA100, and mitomycin C (MMC) (0.5 µg/plate) for TA102. For the tests carried out in the presence of S9 mix, the positive control were 2-anthramine (2-AA) (1.25 µg/plate), used with TA98, TA97a, and TA100, and 2-aminofluorene (2-AF) (10 µg/plate), used with TA102. Cell Isolation and Cytotoxicity Assay In this study human peripheral blood lymphocytes (PBL) were used as the test material. Peripheral venous blood was collected from 3 healthy (2 males and 1 female) nonsmoking donors aged 18 to 27 yr old. Donors provided written informed consent at the time of donation for the use of their blood sample in this study. The Human Ethical Committee of the Universidade Estadual Paulista (UNESP), in Marília city, Brazil, approved the present study on August 8, 2012 (protocol 0014/2012). For the trypan blue-staining viability test, PBL were isolated using Histopaque1077 according to the protocol described by Panda et al. (2012). An aliquot of 2 × 105 cells was plated into 24-well plates in 2 ml RPMI culture medium per well at 37◦ C, under 95% air and 5% CO2 in a humidified incubator, and exposed to 6,7-HC and 4-ME at concentrations of 5, 10, 20, 40, 80, 160, 320, and 640 µg/ml for 24 h. Cellular viability testing for human PBL was performed according to Strober (1997). A freshly prepared solution of 10 µl trypan blue (0.05%) in distilled water was mixed with 10 µl cellular suspension for 20 min, spread onto a microscope slide, and covered with a coverslip. Nonviable cells appeared blue-stained. At least 100 cells per group were counted. Comet Assay An aliquot of 2 × 105 PBL was plated into 24-well plates in 2 ml RPMI culture medium per well at 37◦ C and incubated with tests
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compounds for 4 h at concentrations of 2, 8, or 32 µg/ml and MMS at 75 µM (positive control). The concentrations were selected due to the absence of toxicity in trypan blue exclusion test. Each experiment was performed three times (with separate blood from two donors) and in duplicate. The comet assay was performed according to Singh et al. (1988) and Tice et al. (2000). Cells were collected by centrifugation at 850 × g for 5 min, and the pellet was resuspended in 1 ml culture medium. A 20-µl aliquot of the cell suspension was mixed with 100 µl of 0.5% low-melting-point agarose at 37◦ C, and was immediately spread onto microscope slides precoated with 1.5% normal melting point agarose. Coverslips were added and the slides were allowed to gel at 4◦ C for 20 min. The coverslips were removed and slides were then immersed in cold, freshly prepared lysis buffer consisting of 89 ml stock solution (2.5 M NaCl, 100 mM EDTA, 10 nM Tris, pH 10) plus 1 ml Triton X-100 (Merck) and 10 ml DMSO. The slides were left for 1 h at 4◦ C, protected against light, then placed in the gel box, positioned at the anode end, and left for 20 min at 4◦ C, prior to electrophoresis, in a high-pH (>13) electrophoresis buffer (300 mM NaOH and 1 mM EDTA, pH 10) to allow DNA to unwind. Electrophoresis was performed in an ice bath (4◦ C) for 20 min at 25 V and 300 mA (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% ethyl alcohol for 10 min. The slides were dried and stored at least for an overnight before staining. For staining, slides were rinsed in distilled water, covered with 30 µl of 1× ethidium bromide staining solution, and covered with a coverslip. The material was evaluated immediately at 400× magnification using a fluorescence microscope (Olympus) with a 515–560 nm excitation filter and a 590-nm barrier filter. The extent and distribution of DNA damage indicated by the singl-cell gel electrophoresis (SCGE) assay was conducted by examining at least 100 randomly selected and nonoverlapping cells (50 cells per coded slide) per culture well in a blind analysis. These cells were
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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 one- to twofold greater than the diameter of the head; and class 3, tail length more than twice the diameter of the head. Comets with no heads with nearly the entire DNA in the tail or with a wide tail were excluded from the evaluation because these probably represented dead cells (Hartmann and Speit, 1997). The total score for 100 comets, which ranged from 0 (no damage) to 300 (severe damage), was obtained by the sum of multiplying the number of cells in each class by the damage class. Cytokinesis-Block Micronucleus Assay (CBMN) The cytokinesis-block micronucleus (CBMN) assay was performed according to the procedure described by Fenech (2000). Whole blood samples (0.4 ml) from 2 donors were added to 5 ml culture medium (RPMI 1640) supplemented with 10% fetal calf serum (GIBCO, Grand Island, NY). Phytohemagglutinin (PHA) was added to each culture flask at at 10 µl/ml, and PBL were incubated at 37◦ C, under 95% air and 5% CO2 in a humidified incubator for 72 h. Forty-four hours after starting PBL culture, cytocalasin-B (Sigma Chemical Co., St. Louis, MO) (6 µg/ml) was added to each culture. Four hours after cytocalasin-B addition, human PBL were exposed to different concentrations (2, 8, or 32 µg/ml) of 6,7-HC and 4-ME. The concentrations were selected based upon absence of toxicity in trypan blue exclusion test. The cells were harvest by centrifugation (5 min at 850 × g), and pellets were resuspended in a chilled hypotonic solution of 0.075 M KCl for 5 min. Subsequently, cells were washed once with 5 ml of cold methanol:acetic acid solution (3:1, v/v). The fixation procedure was applied three times. Formaldehyde (1%) was added to the last fixative to preserve the cytoplasm. The cell suspension was placed onto slides and stained in a solution of 5% Giemsa dye (Synth, Diadema, SP, Brazil) in phosphate buffer (pH
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6.8) for 5 min. In accordance to standard criteria (Fenech, 2000), micronuclei (MN) analysis was performed on coded slides by scoring 1000 binucleate lymphocytes for each subject. For the analysis, an optical microscope (Zeiss, Primo Star) at 100× magnification was used. As a measure of cytotoxicity, the nuclear division index (NDI) was calculated following the formula NDI = [M1 + 2(M2) + 3(M3) + 4(M4)]/N, where M1–M4 indicate the number of cells with 1–4 nuclei as assessed in 500 cells (N) (for each culture). As a positive control, MMS was used at final concentration of 150 µM. Statistical Analysis The results obtained in the comet and CBMN assays were subjected to analysis of variance (ANOVA) followed by Tukey’s test. GraphPad Prism software (version 5.02) was used to perform statistical analysis. In both tests, the results were considered statistically significant at p < .05.
RESULTS AND DISCUSSION Claxton et al. (1988) found that gene and chromosomal damage may be induced by exposure to carcinogenic chemicals, indicating that chemical agents produce marked alterations to cellular genetical material. Therefore, the aim of this study was to evaluate for the first time in vitro the genetic toxicity potential of coumarin derivatives 6,7-HC and 4-ME, as these compounds may possess significant beneficial usage as antioxidants in therapy. The use of in vitro studies provides important tools to enhance the understanding of hazardous effects by chemicals and for predicting these effects in humans (Broadhead and Combes, 2001). In the present study the Salmonella/ microsome assay was used to detect the potential of 6,7-HC and 4-ME to induce DNA mutations. This assay is commonly employed as an initial screen for genotoxic activity, in particular, point mutation-inducing activity
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(Organization for Economic Cooperation and Development [OECD], 1997). Table 1 demonstrates the number of revertants per plate and mutagenicity ratio (MR) after treatment with each coumarin derivative, observed in S. typhimurium strains TA98, TA100, TA97a, and TA102, in the presence (+S9) and absence (−S9) of metabolic activation. Mutagenic activity was considered negative for all strains and all concentrations tested both in the absence and presence of metabolism, and enabled us to conclude that 6,7-HC and 4-ME exerted no direct or indirect mutagenic effects. Trypan blue exclusion test for cell viability is used to determine the number of viable cells present in a cell suspension. Data showed absence of toxicity for 6,7-HC at 5–320 µg/ml with cell viability higher than 84%, and at 5–40 µg/ml for 4-ME with cell viability higher than 76%. For the concentrations of 80–640 µg/ml of 4-ME, cell viability gradually decreased as the concentration increased. These results indicate that 4-ME produced more cytotoxicity at high concentrations than 6,7-HC. As highly damaged DNA may correspond to dead cells, the concentrations of test substance higher than 40 µg/ml were excluded from the comet study, since these may reflect possible cytotoxicity. The results obtained for genotoxic assessment of 6,7-HC and 4-ME in human PBL are shown in Table 2. None of the two tested compounds demonstrated significant changes in total number of damaged cells and scores at these tested concentrations compared to the negative control. As expected, an increase in total of damaged cells was observed only for the positive control MMS, confirming the sensitivity of the comet assay for detection of genotoxicity. The clastogenicity/aneugenicity potential of 6,7-HC and 4-ME in human PBL also was assessed in this study using the CBMN assay. Micronuclei (MN) are expressed in dividing cells that either contain chromosome breaks lacking centromeres (clastogenic effect) and/or whole chromosomes due to dysfunction of mitotic apparatus (aneugenic effect), and cytochalasin-B, which blocks the cytokinesis, enabling the identification of dividing cells by
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32 ± 5 39 ± 6(1.2) 47 ± 11(1.5) 37 ± 3(1.2) 26 ± 3(0.8) 27 ± 2(0.8) 797∗ ± 79b
32 ± 5 46 ± 7(1.5) 45 ± 8(1.4) 43 ± 5(1.4) 37 ± 6(1.2) 31 ± 5(1) 797∗ ± 79b
−S9
32 ± 4 28 ± 6(0.9) 25 ± 9(0.8) 32 ± 4(1) 20 ± 2(0.6) 19 ± 5(0.6) 2204∗ ± 255e
32 ± 4 33 ± 11(1) 30 ± 1(0.9) 32 ± 12(1) 38 ± 8(1.2) 28 ± 7(0.9) 2204∗ ± 255e
+S9
153 ± 6 157 ± 13(1) 163 ± 18(1.1) 141 ± 7(0.9) 145 ± 6(0.9) 141 ± 19(0.9) 1193∗ ± 39c
153 ± 6 156 ± 5(1) 178 ± 9(1.2) 168 ± 6(1.1) 150 ± 11(1) 142 ± 13(0.9) 1193∗ ± 39c
−S9
TA 100
136 ± 7 156 ± 5(1.1) 160 ± 5(1.2) 156 ± 12(1.2) 183 ± 6(1.4) 162 ± 20(1.2) 1229∗ ± 94e
136 ± 7 142 ± 10(1) 140 ± 6(1) 147 ± 28(1.1) 118 ± 13(0.9) 111 ± 2(0.8) 1229∗ ± 94e
+S9
355 ± 36 413 ± 20(1.2) 373 ± 9(1.1) 359 ± 28(1) 352 ± 21(1) 325 ± 18(0.9) 1192∗ ± 49d
355 ± 36 334 ± 19(0.9) 397 ± 24(1.1) 428 ± 16(1.2) 380 ± 35(1.1) 419 ± 53(1.2) 1192∗ ± 49d
−S9
TA 102
365 ± 43 401 ± 29(1.1) 393 ± 33(1.1) 376 ± 36(1) 372 ± 11(1) 336 ± 23(0.9) 1804∗ ± 43f
365 ± 43 455 ± 23(1.2) 400 ± 43(1.1) 412 ± 22(1.1) 369 ± 32(1) 321 ± 40(0.9) 1804∗ ± 43f
+S9
106 ± 23 113 ± 14(1.1) 129 ± 19(1.2) 128 ± 25(1.2) 109 ± 11(1) 112 ± 9(1.1) 716∗ ± 74b
106 ± 23 108 ± 9(1) 111 ± 16(1) 133 ± 27(1.3) 111 ± 14(1) 112 ± 26(1.1) 716∗ ± 74b
−S9
TA 97a
214 ± 15 263 ± 38(1.2) 252 ± 11(1.2) 256 ± 14(1.2) 222 ± 16(1) 203 ± 17(1) 2636∗ ± 132e
214 ± 15 249 ± 31(1.2) 241 ± 29(1.1) 232 ± 38(1.1) 220 ± 32(1) 220 ± 61(1) 2636∗ ± 132e
+S9
Note. Data are expressed as mean and standard deviation of the number of revertants/plate and mutagenic ratio (MR, in parentheses) in Salmonella typhimurium TA98, TA100, TA102, and TA97a strains treated with 6,7-dihydroxycoumarin (6,7-HC) and 4-methylesculetin (4-ME) at various concentrations, with (+S9) or without (–S9) metabolic activation. ∗ Significantly different at p < .05. a 0: Negative control: dimethyl sulfoxide (DMSO, 100 µl/plate). b Control+: 4-nitro-o-phenylenediamine (10 µg/ plate, TA98 and TA97a). C sodium azide(1.25 µg/plate, TA100). D mitomycin C (0.5 µg/ plate, TA102), in the absence of S9. e 2-Anthramine (1.25 µg/ plate, TA98, TA100, TA97a). f 2-Aminofluorene (10 µg/ plate, TA102), in the presence of S9.
6,7-HC 0a 62,5 125 250 500 750 C+ 4-ME 0a 62,5 125 250 500 750 C+
Treatments µg/plate
TA 98
Number of revertants (M ± SD)/plate and MR
TABLE 1. Mutagenic Activity Assessment
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TABLE 2. DNA Migration (Mean ± SD) in the Comet Assay for Assessing Genotoxicity of 6,7-Dihydroxycoumarin (6,7-HC) and 4Methylesculetin (4-ME) in Human Peripheral Blood Lymphocytes (4 h Treatment) Comet class Treatments
Totalc
0
Control 6,7-HC 2 µg/ml 6,7-HC 8 µg/ml 6,7-HC 32 µg/ml
22.83 ± 4.95 13.67 ± 1.75 15 ± 2.68 17 ± 5.42
77.17 ± 4.95 86.33 ± 1.75 85 ± 2.68 83 ± 5.42
18 ± 3.09 11 ± 2.28 13 ± 2.09 15 ± 3.96
4.83 ± 1.94 2.5 ± 1.04 1.83 ± 0.75 2 ± 1.91
0±0 0.16 ± 0.4 0.16 ± 0.4 0.00 ± 0.0
27.67 ± 6.86 16 ± 2 16.67 ± 3.38 19 ± 7.09
4-ME 2 µg/ml 4-ME 8 µg/ml 4-ME 32 µg/ml
17.5 ± 4.32 16.17 ± 5.03 14.67 ± 4.92
82.5 ± 4.32 83.83 ± 5.03 85.33 ± 4.92
13.67 ± 2.33 10.33 ± 3.72 12.5 ± 6.47
3.16 ± 2.31 4.66 ± 2.25 1.83 ± 1.83
0.66 ± 0.81 1.16 ± 0.75 0.33 ± 0.51
22 ± 7.4 23.17 ± 7.08 17.17 ± 4.16
MMS 75 µM (positive control)
99.67 ± 0.81a
12.83 ± 10.38b
82.33 ± 15.27a
277.2 ± 21.9a
1
0.33 ± 0.81a
2
4.5 ± 6.41
3
Scores
Note. SD, standard deviation. a Significantly different from the negative control (p < .05). b Significantly different from the negative control (p < .05). c Total number of damaged cells (class 1 + 2 + 3). TABLE 3. The Micronucleus Frequency and Nuclear Division Index in Human Lymphocytes Treated With 6,7-Dihydroxycoumarin (6,7HC) and 4-Methylesculetin (4-ME) Binucleated cells with MN (2000 cells scored)
Treatment Test substance
Períod (h)
Concentration (µg/ml)
N◦
%
NDI/1000 cells (mean ± SD)
Negative control MMS (Positive control) 6,7-HC
28 28 28 28 28 28 28 28
0 150a 2 8 32 2 8 32
2 52∗ 7 7 3 1 2 1
0.1 2.6∗ 0.35 0.35 0.15 0.05 0.1 0.05
2.04 ± 0.05 2.16 ± 0.11 1.99 ± 0.03 2.02 ± 0.01 2.02 ± 0.01 1.90 ± 0.07 1.97 ± 0.07 1.86 ± 0.07
4-ME
Note. MN, micronucleus, NDI, nuclear division index, SD, standard deviation. ∗ Significantly different from the negative control (p < .05). a Concentration, µM.
their binucleated appearance (Fenech, 2000). The MN data obtained demonstrated results similar to those observed using the comet assay (Table 3). CBMN test demonstrated no marked alterations in the number of micronucleated cells for the two tested substances at all tested concentrations. Similarly, the effects of coumarin derivatives on nuclear division did not display any significant change in cell division as measured by nuclear division index (NDI). No apparent previous reports evaluated the mutagenic or genotoxic effects of 6,7HC. In contrast, regarding 4-ME, there was
one in vivo assessment of genetic toxicity that reported absence of genotoxic effects in different mouse cells using comet assay and MN test (Fedato and Maistro, 2014), in agreement with our in vitro results. Fedato and Maistro (2014) also noted that 4-ME plays a role in preventing chemically induced DNA and chromosome damage induced by the antitumor agent doxorubicin, possibly by antioxidant mechanisms. Coumarin is the natural precursor of 4ME. Morris and Ward (1992) investigated the possibility that pretreatment with coumarin might inhibit the genotoxicity induced by
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benzo[a]pyrene (BaP) in ICR mice as evidenced by the bone-marrow MN test. Similar to findings in our in vitro studies with 4-ME, Morris and Ward (1992) demonstrated that coumarin treatment alone did not induce MN in either mouse gender. However, it is of interest that Morris and Ward (1992) also reported no inhibition in MN formation only in female mice pretreated with the same doses of coumarin used in males. Other in vivo studies showed that coumarin did not markedly influence MN frequencies in PBL of female and male B6C3F1 mice and in bone-marrow cells of Swiss mice (National Toxicology Program [NTP], 1993; Api, 2001). In conclusion, our initial screening of genetic toxicity potential of 6,7-HC and 4ME showed that both coumarin derivatives are not mutagenic in the Salmonella/microsome assay. In addition, no significant genotoxic and clastogenic/aneugenic effects were detected in human PBL in vitro by comet and CBMN assays. Due to its antioxidant potential and benefit for cancer prevention, further in vivo studies in eukaryotic cells are necessary to determine their safe use in humans. FUNDING This research was funded by the FAPESP, Fundação de Amparo à Pesquisa do Estado de São Paulo (grants 2010/07577-3 and 2012/17241-8), Brazil. REFERENCES Api, A. M. 2001. Lack of effect of coumarin on the formation of micronuclei in an in vivo mouse micronucleus assay. Food Chem. Toxicol. 39: 837–841. Baba, M., Jin, Y., Mizuno, A., Suzuki, H., Okada, Y., Takasuka, N., Tokuda, H., Nishino, H., and Okuyama, T. 2002. Studies on cancer chemoprevention by traditional folk medicines XXIV. Inhibitory effect of a coumarin derivative, 7isopentenyloxycoumarin, against tumor promotion. Biol. Pharm. Bull. 25: 244–246.
Beillerot, A., Domínguez, J. C. R., Kirsch, G., and Bagrel, D. 2008. Synthesis and protective effects of coumarin derivatives against oxidative stress induced by doxorubicin. Bioorg. Med. Chem. Lett. 18: 1102–1105. Berenbaum, M. 1995. Phototoxicity of plant secondary metabolites: Insect and mammalian perspectives. Arch. Insect Biochem. Physiol. 29: 119–134. Bernstein, L., Kaldor, J., Mccann, J., and Pike, M. C. 1982. An empirical approach to the statistical analysis of mutagenesis data from Salmonella test. Mutat. Res. 97: 267–281. Born, S. L., Hu, J. K., and Lehman-McKeeman, L. D. 2000. o-Hydroxyphenylacetaldehyde is a hepatotoxic metabolite of coumarin. Drug Metab. Dispos. 28: 218–223. Broadhead, C. L., and Combes, R. D. 2001. The current status for food additives toxicity testing and the potential for application of the three Rs. ATLA 29: 471–485. Cai, Y., Kleiner, H., Johnston, D., Dubowski, A., Bostic, S., Ivie, W., and DiGiovanni, J. 1997. Effect of naturally occurring coumarins on the formation of epidermal DNA adducts and skin tumors induced by benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene in SENCAR mice. Carcinogenesis 18: 1521–1527. Casley-Smith, J. R. 1999. Benzo-pyrones in the treatment of lymphoedema. Int. Angiol. 18: 31–41. Claxton, L. D., Stead, A. G., and Walsh, D. 1988. An analysis by chemical class Salmonella mutagenicity tests as precictors as animal carcinogenicity. Mutat. Res. 205: 1–4. Fedato, R. P., and Maistro, E. L. 2014. Absence of genotoxic effects of the coumarin derivative 4-methylesculetin in vivo and its potential chemovenction against doxorubicininduced DNA damage. J. Appl. Toxicol. 34: 33–39. Fenech, M. 2000. The in vitro micronucleus technique. Mutat. Res. 455: 81–95. Francisco, C. S., Rodrigues, L. R., Cerqueira, N. M., Oliveira-Campos, A. M., and Rodrigues, L. M. 2012. Synthesis of novel benzofurocoumarin analogues and their antiproliferative effect on human cancer cell lines. Eur. J. Med. Chem. 47: 370–376.
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Guo, L. Q., and Yamazoe, Y. 2004. Inhibition of cytochrome P450 by furanocoumarins in grapefruit juice and herbal medicines. Acta Pharmacol. Sin. 25: 129–136. Hartmann, A., and Speit, G. 1997. The contribution of cytotoxicity to DNA—Effects in the single cell gel test (comet assay). Toxicol. Lett. 90: 183–188. Iranshari, M., Askari, M., Sahebkar, A., and Hadjipavlou-Litina, D. 2009. Evaluation of antioxidant, anti-inflammatory and lipoxygenase inhibitory activities of the prenylated coumarin umbelliprenin. DARU J. Pharm. Sci. 17: 99–103. Kelly, V. P., Ellis, E. M., Manson, M. M., Chanas, S. A., Moffat, G. J., McLeod, R., Judah, D. J., Neal, G. E., and Hayes, J. D. 2000. Chemoprevention of aflotoxin B1 hepatocarcinogenesis by coumarin, a natural benzopyrone that is a potent inducer of aflatoxin B1-aldehyde reductase, the glutathione S-transferase A5 and P1 subunits, and NAD(P)H:quinone oxidoreductase in rat liver. Cancer Res. 60: 957–969. Kim, S. N., Kim, N. H., Park, Y. S., Kim, H., Lee, S., Wang, Q., and Kim, Y. K. 2009. 7-Diethylamino-3(2 -benzoxazolyl)coumarin is a novel microtubule inhibitor with antimitotic activity in multidrug resistant cancer cells. Biochem. Pharmacol. 77: 1773–1779. Kleiner, H. E., Vulimiri, S. V., Starost, M. F., Reed, M. J., and DiGiovanni, J. 2002. Oral administration of the citrus coumarin, isopimpinellin, blocks DNA adduct formation and skin tumor initiation by 7,12-dimethylbenz[a]anthracene in SENCAR mice. Carcinogenesis 23: 1667–1675. Lake, B. 1999. Coumarin metabolism, toxicity, and carcinogenicity: Relevance for human risk assessment. Food Chem. Toxicol. 37: 423–453 Lake, B. G., and Grasso, P. 1996. Comparison of the hepatotoxicity of coumarin in the rat, mouse, and Syrian hamster: A dose and time response study. Fundam. Appl. Pharmacol. 97: 311–323.
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Maron, D. M., and Ames, B. N. 1983. Revised methods for the Salmonella mutagenicity test. Mutat. Res. 113: 173–215. Mitra, I., Saha, A., and Roy, K. 2013. Predictive modeling of antioxidant coumarin derivatives using multiple approaches: Descriptor-based QSAR, 3D-pharmacophore mapping, and HQSAR. Sci. Pharm. 81: 57–80. Morris, D. L., and Ward, J. B. 1992. Coumarin inhibts micronuclei formation induced by benzo(a)pyrene in male but not female ICR mice. Environ. Mol. Mutagen. 19: 132–138. Murray, R. D. H., Mendez, J., and Brown, S. A., eds. 1982. The natural coumarins: Occurrence, chemistry and biochemistry, 97–111. New York, NY: John Wiley & Sons. National Toxicology Program. 1993. Toxicology and carcinogenic studies of coumarin (CAS No. 91-64-5) in F344/N mice and B6C3F1 mice (gavage studies). Technical report series no. 422. NIH Publication No. 92-31153. Bethesda, MD: U.S. Department of Health and Human Services. Ngwendson, J. N., Bedir, E., Efange, S. M., Okunji, C. O., Iwu, M. M., Schuster, B. G., and Khan, I. 2003. Constituents of Peucedanum zenkeri seeds and their antimicrobial effects. Pharmazie 58: 587–589. Organization for Economic Cooperation and Development. 1997. Guideline for testing of chemicals, Test guideline 471: Bacterial reverse mutation test. Paris, France: OECD. Panda, S. K., Kumar, S., Tupperwar, N. C., Vaidya, T., George, A., Rath, S., Bal, V., and Ravindran, B. 2012. Chitohexaose activates macrophages by alternate pathway through TLR4 and blocks endotoxemia. PLoS Pathol. 8: e1002717. Prince, M., Campbell, C. T., Robertson, T. A., Wells, A. J., and Kleiner, H. E. 2006. Naturally occurring coumarins inhibit 7,12dimethylbenz[a]anthracene DNA adduct formation in mouse mammary gland. Carcinogenesis 27: 1204–1213. Resende, F. A., Munari, C. C., Bentos, M. N. A., Tavares, D. C., Bastos, J. K., Silva-Filho,
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A. A., and Varanda, E. A. 2012. Comparative studies of the (anti) mutagenicity of Baccharis dracunculifolia and artepillin c by the bacterial reverse mutation test. Molecules 7: 2335–2350. Setzer, W. N., Setzer, M. C., Schmidt, J. M., Moriarity, D. M., Vogler, B., Reeb, S., Holmes, A. M., and Haber, W. A. 2000. Cytotoxic components from the bark of Stauranthus perforatus from Monteverde, Costa Rica. Planta Med. 66: 493–494. Singh, N. P., McCoy, M. T., Tice, R. R., and Schneider, E. L. 1988. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell Res. 175: 184–191. Strober, W. 1997. Trypan blue exclusion test of cell viability. Curr. Prot. Immunol. A.3B.1– A.3B.2. Tanaka, T., Kawabata, K., Kakumoto, M., Hara, A., Murakami, A., Kuki, W., Takahashi, Y., Yonei, H., Maeda, M., Ota, T., Odashima, S., Yamane, T., Koshimizu, K., and Ohigashi, H. 1998. Citrus auraptene exerts dosedependent chemopreventive activity in rat large bowel tumorigenesis: The inhibition
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correlates with suppression of cell proliferation and lipid peroxidation and with induction of phase II drug-metabolizing enzymes. Cancer Res. 58: 2550–2556. Tice, R. R., Agurell, E., Anderson, D., Burlinson, B., Hartmann, A., Kobayashi, H., Miyamae, Y., Rojas, E., Ryu, J. C., and Sasaki, Y. F. 2000. Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ. Mol. Mutagen. 35: 206–221. Venugopala, K. N., Rashmi, V., and Odhav, B. 2013. Review on natural coumarin lead compounds for their pharmacological activity. Biol. Med. Res. Int. 2013: 1–14. Vianna, D. R., Bulobs, G., Meirelles, G., Silva, B. V., Rocha, A., Lanznaster, M., Monserrat, J. M., Garcia, S. C., Poser, G. V., and EiflerLima, V. L. 2012. Evaluation of the antioxidant capacity of synthesized coumarins. Int. J. Mol. Sci. 13: 7260–7270. Wattenberg, L. W., Lam, L. K., and Fladmoe, A. V. 1979. Inhibition of chemical carcinogeninduced neoplasia by coumarins and alpha-angelicalactone. Cancer Res. 39: 1651–1654.
IN VITRO ASSESSMENT OF MUTAGENIC AND GENOTOXIC EFFECTS OF COUMARIN DERIVATIVES 6,7-DIHYDROXYCOUMARIN AND 4-METHYLESCULETIN Edson Luis Maistro1,2, Eduardo de Souza Marques1, Rafael Palhano Fedato1, Flora Tolentino2, Chayene de Andrade Cezário da Silva2, Marcela Stefanini Ferreira Tsuboy2, Flavia Aparecida Resende3, Eliana Aparecida Varanda3 1
Programa de Pós-Graduação em Biologia Geral e Aplicada, Universidade Estadual Paulista (UNESP), Instituto de Biociências, Botucatu, São Paulo, Brazil 2 Universidade Estadual Paulista–UNESP, Faculdade de Filosofia e Ciências, Departamento de Fonoaudiologia, Marília, SP, Brazil 3 Universidade Estadual Paulista–UNESP, Faculdade de Ciências Farmacêuticas, Departamento de Ciências Biológicas, Araraquara, SP, Brazil Coumarins are naturally occurring compounds, widely distributed throughout the plant kingdom (Plantae), and possess important pharmacological properties, including inhibition of oxidative stress. In this context, newly synthesized coumarin compounds are being produced due to their potent antioxidant activities. Therefore, the aim of the present study was to determine the in vitro cytotoxic, mutagenic, and genotoxic effects of 6,7-dihydroxycoumarin (6,7-HC) and 4-methylesculetin (4-ME) using the Salmonella/microsome test and in cultured human lymphocytes the comet assay and micronucleus test. The three coumarin derivatives concentrations evaluated in comet and MN assays were 2, 8, and 32 µg/mL, selected through a preliminary trypan blue-staining assay. In the Ames test, the 5 concentrations tested were 62.5, 125, 250, 500, and 750 µg/plate. Positive (methyl methane-sulfonate, MMS) and negative (dimethyl sulfoxide, DMSO) control groups were also included in the analysis. Our results showed that 4-ME induced greater cytotoxicity at high concentrations than 6,7-HC. In addition, both compounds were not mutagenic in the Ames test and not genotoxic or clastogenic/aneugenic in cultured human lymphocytes.
properties that include furanocoumarininduced photoxicity (Berenbaum, 1995), anticarcinogenesis/chemoprevention of cancer (Wattenberg et al., 1979; Cai et al., 1997; Tanaka et al., 1998; Kelly et al., 2000; Kleiner et al., 2002; Prince et al., 2006; Kim et al., 2009; Francisco et al., 2012), cancer cell cytotoxicity (Setzer et al., 2000), antimicrobial effects (Ngwendson et al., 2003), and modulation of cytochrome P450 3A4 (Guo and Yamazoe, 2004). The inhibition of oxidative stress is a contributing factor for the observed biological
Coumarins consist of a large class of phenolic substances detected in approximately 150 different plant species distributed around 30 different families, including Rutaceae, Apiaceae, Umbellifereae, and Moraceae (Murray, 1982), as well as Clusiaceae, Guttiferae, Caprifoliaceae, Oleraceae, and Nyctaginaceae (Venugopala et al., 2013). More than 1300 coumarins have been identified as secondary metabolites from plants, bacteria, and fungi (Iranshahi et al., 2009). Natural coumarins are of great interest due to their pharmacologic/toxicologic
Received 1 July 2014; accepted 8 July 2014. Address correspondence to Edson Luis Maistro, Universidade Estadual Paulista–UNESP, Faculdade de Filosofia e Ciências, Departamento de Fonoaudiologia. Av. Hygino Muzzi Filho, 737, Caixa Postal 181. Marília, SP, Brazil. 17525-900. E-mail: edson.maistro@ marilia.unesp.br 109
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effects of coumarins. Indeed, coumarins were reported to affect the formation and scavenging of reactive oxygen species (ROS) and influence free radical-mediated oxidative damage (Vianna et al., 2012). Based upon these antioxidant properties, there is a concerted effort to develop newly synthesized coumarin derivatives and determine the antioxidant behavior of these novel coumarins with different chemical substitutions. Hydroxycoumarins are newly synthesized compounds shown to possess effective antioxidant capacity (Beillerot et al., 2008; Vianna et al., 2012; Mitra et al., 2013). However, the use of some of the coumarins and their derivatives was found to produce adverse effects such as mild nausea, diarrhea, and hepatotoxicity (Lake and Grasso, 1996; Lake, 1999; Casley-Smith, 1999; Born et al., 2000). Bearing in mind the issue of side effects and considering that coumarin derivatives 6,7-dihydroxycoumarin (6,7-HC) (synonym esculetin) and 4-methylesculetin (4-ME) display a high antioxidant activity with potential use to humans, this study aimed to determine wheter these compounds exerted mutagenic and genotoxic effects using the following assays: DNA mutation in Salmonella typhimurium, and DNA damage and induction of micronuclei (MN) in human peripheral blood lymphocytes (PBL).
MATERIALS AND METHODS Chemical Compounds The 6,7-dihydroxycoumarin (6,7-HC) used in the tests was obtained from Aldrich (Cat.: 24,657-3, 98% purity), and the 4methylesculetin (4-ME) was obtained from Acrós Organics (CAS number 529-84-0, 99% purity) (Figure 1). Both were dissolved in 1% dimethyl sulfoxide (DMSO). Methyl methanesulfonate (MMS) (Aldrich, CAS number 66-273) was used as the positive control substance due to its potential for DNA damage established in the comet assay and micronucleus test. The other main chemicals were obtained from the following suppliers: normal melting
FIGURE 1. The chemical structures of coumarin, 6,7dihydroxycoumarin (esculetin), and 4-methylesculetin.
point (NMP) agarose (Invitrogen), low melting point (LMP) agarose (Invitrogen), sodium salt N-lauroyl sarcosine (Sigma), ethylenediamine tetraacetic acid (EDTA) (Merck), and Histopaque-1077 (Sigma-Aldrich, Co., St. Louis, MO). Salmonella/Microsome Assay 6,7-HC and 4-ME were evaluated in a bacterial mutation assay system using the Salmonella typhimurium tester strains TA98 (frameshift), TA100 (base-pair substitution), TA97a (frameshift), and TA102 (base-pair substitution) using preincubation methodology, with (+S9) and without (–S9) metabolization (Maron and Ames, 1983). Bacteria strains were kindly provided by Dr. B.N. Ames (Berkeley, CA). The strains were grown overnight from frozen cultures for 12–14 h in Oxoid nutrient broth no. 2. The metabolic activation mixture (S9 fraction), prepared from Sprague-Dawley
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mouse liver treated with the polychlorinated biphenyl mixture Aroclor 1254 (500 mg/kg), was purchased from Molecular Toxicology, Inc. (Boone, NC), and freshly prepared before each test. The metabolic activation system consisted of 4% S9 fraction, 1% 0.4 M MgCl2, 1% 1.65 M KCl, 0.5% 1 M D-glucose-6-phosphate disodium and 4% 0.1 M NADP, 50% 0.2 M phosphate buffer, and 39.5% sterile distilled water (Maron and Ames, 1983). For the mutagenic assessment, five different concentrations of 6,7HC and 4-ME (62.5, 125, 250, 500, and 750 µg/plate), diluted in DMSO, were tested. The concentrations of the samples were selected based upon preliminary toxicity tests that determined the highest non-toxic concentration and lowest toxic concentration. Samples were considered toxic when a thinning of the auxotrophic background (i.e., background lawn) was accompanied by a decrease in the number of histidine revertants (His+). The concentrations of test substances were added to 0.5 ml 0.2 M phosphate buffer or 0.5 ml 4% S9 mixture, with 0.1 ml bacterial culture, and then incubated at 37◦ C for 20–30 min. Two milliliters of surface agar was added, tubes were mixed, and the mixture was poured into a petri dish containing 20 ml minimal agar. The petri dishes were incubated at 37◦ C for 48 h and His+ revertant colonies were counted manually. The test was performed in triplicate. Data obtained were analyzed using the statistical software package Salanal 1.0 (U.S. Environmental Protection Agency, Monitorin Systems Laboratory, Las Vegas, NV, from the Research Triangle Institute, RTP, NC). Data (revertants/plate) were assessed by means of the analysis of variance (ANOVA) using the Bernstein et al. (1982) model followed by linear regression. The mutagenicity ratio (MR) was also determined for each concentration tested, where average number of revertants per plate with test compound was divided by average number of revertants per plate with negative (solvent) control. A test solution was considered mutagenic when a concentrationresponse relationship was detected and a twofold increase in number of mutants (MR ≥ 2) was observed in at least one concentration
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(Resende et al., 2012). DMSO was used as negative (solvent) control. The mutagens used as positive controls in the tests without S9 mix were 4-nitro-o-phenylenediamine (NPD) (10 µg/plate) for TA98 and TA97a, sodium azide (SAZ) (1.25 µg/plate) for TA100, and mitomycin C (MMC) (0.5 µg/plate) for TA102. For the tests carried out in the presence of S9 mix, the positive control were 2-anthramine (2-AA) (1.25 µg/plate), used with TA98, TA97a, and TA100, and 2-aminofluorene (2-AF) (10 µg/plate), used with TA102. Cell Isolation and Cytotoxicity Assay In this study human peripheral blood lymphocytes (PBL) were used as the test material. Peripheral venous blood was collected from 3 healthy (2 males and 1 female) nonsmoking donors aged 18 to 27 yr old. Donors provided written informed consent at the time of donation for the use of their blood sample in this study. The Human Ethical Committee of the Universidade Estadual Paulista (UNESP), in Marília city, Brazil, approved the present study on August 8, 2012 (protocol 0014/2012). For the trypan blue-staining viability test, PBL were isolated using Histopaque1077 according to the protocol described by Panda et al. (2012). An aliquot of 2 × 105 cells was plated into 24-well plates in 2 ml RPMI culture medium per well at 37◦ C, under 95% air and 5% CO2 in a humidified incubator, and exposed to 6,7-HC and 4-ME at concentrations of 5, 10, 20, 40, 80, 160, 320, and 640 µg/ml for 24 h. Cellular viability testing for human PBL was performed according to Strober (1997). A freshly prepared solution of 10 µl trypan blue (0.05%) in distilled water was mixed with 10 µl cellular suspension for 20 min, spread onto a microscope slide, and covered with a coverslip. Nonviable cells appeared blue-stained. At least 100 cells per group were counted. Comet Assay An aliquot of 2 × 105 PBL was plated into 24-well plates in 2 ml RPMI culture medium per well at 37◦ C and incubated with tests
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compounds for 4 h at concentrations of 2, 8, or 32 µg/ml and MMS at 75 µM (positive control). The concentrations were selected due to the absence of toxicity in trypan blue exclusion test. Each experiment was performed three times (with separate blood from two donors) and in duplicate. The comet assay was performed according to Singh et al. (1988) and Tice et al. (2000). Cells were collected by centrifugation at 850 × g for 5 min, and the pellet was resuspended in 1 ml culture medium. A 20-µl aliquot of the cell suspension was mixed with 100 µl of 0.5% low-melting-point agarose at 37◦ C, and was immediately spread onto microscope slides precoated with 1.5% normal melting point agarose. Coverslips were added and the slides were allowed to gel at 4◦ C for 20 min. The coverslips were removed and slides were then immersed in cold, freshly prepared lysis buffer consisting of 89 ml stock solution (2.5 M NaCl, 100 mM EDTA, 10 nM Tris, pH 10) plus 1 ml Triton X-100 (Merck) and 10 ml DMSO. The slides were left for 1 h at 4◦ C, protected against light, then placed in the gel box, positioned at the anode end, and left for 20 min at 4◦ C, prior to electrophoresis, in a high-pH (>13) electrophoresis buffer (300 mM NaOH and 1 mM EDTA, pH 10) to allow DNA to unwind. Electrophoresis was performed in an ice bath (4◦ C) for 20 min at 25 V and 300 mA (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% ethyl alcohol for 10 min. The slides were dried and stored at least for an overnight before staining. For staining, slides were rinsed in distilled water, covered with 30 µl of 1× ethidium bromide staining solution, and covered with a coverslip. The material was evaluated immediately at 400× magnification using a fluorescence microscope (Olympus) with a 515–560 nm excitation filter and a 590-nm barrier filter. The extent and distribution of DNA damage indicated by the singl-cell gel electrophoresis (SCGE) assay was conducted by examining at least 100 randomly selected and nonoverlapping cells (50 cells per coded slide) per culture well in a blind analysis. These cells were
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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 one- to twofold greater than the diameter of the head; and class 3, tail length more than twice the diameter of the head. Comets with no heads with nearly the entire DNA in the tail or with a wide tail were excluded from the evaluation because these probably represented dead cells (Hartmann and Speit, 1997). The total score for 100 comets, which ranged from 0 (no damage) to 300 (severe damage), was obtained by the sum of multiplying the number of cells in each class by the damage class. Cytokinesis-Block Micronucleus Assay (CBMN) The cytokinesis-block micronucleus (CBMN) assay was performed according to the procedure described by Fenech (2000). Whole blood samples (0.4 ml) from 2 donors were added to 5 ml culture medium (RPMI 1640) supplemented with 10% fetal calf serum (GIBCO, Grand Island, NY). Phytohemagglutinin (PHA) was added to each culture flask at at 10 µl/ml, and PBL were incubated at 37◦ C, under 95% air and 5% CO2 in a humidified incubator for 72 h. Forty-four hours after starting PBL culture, cytocalasin-B (Sigma Chemical Co., St. Louis, MO) (6 µg/ml) was added to each culture. Four hours after cytocalasin-B addition, human PBL were exposed to different concentrations (2, 8, or 32 µg/ml) of 6,7-HC and 4-ME. The concentrations were selected based upon absence of toxicity in trypan blue exclusion test. The cells were harvest by centrifugation (5 min at 850 × g), and pellets were resuspended in a chilled hypotonic solution of 0.075 M KCl for 5 min. Subsequently, cells were washed once with 5 ml of cold methanol:acetic acid solution (3:1, v/v). The fixation procedure was applied three times. Formaldehyde (1%) was added to the last fixative to preserve the cytoplasm. The cell suspension was placed onto slides and stained in a solution of 5% Giemsa dye (Synth, Diadema, SP, Brazil) in phosphate buffer (pH
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6.8) for 5 min. In accordance to standard criteria (Fenech, 2000), micronuclei (MN) analysis was performed on coded slides by scoring 1000 binucleate lymphocytes for each subject. For the analysis, an optical microscope (Zeiss, Primo Star) at 100× magnification was used. As a measure of cytotoxicity, the nuclear division index (NDI) was calculated following the formula NDI = [M1 + 2(M2) + 3(M3) + 4(M4)]/N, where M1–M4 indicate the number of cells with 1–4 nuclei as assessed in 500 cells (N) (for each culture). As a positive control, MMS was used at final concentration of 150 µM. Statistical Analysis The results obtained in the comet and CBMN assays were subjected to analysis of variance (ANOVA) followed by Tukey’s test. GraphPad Prism software (version 5.02) was used to perform statistical analysis. In both tests, the results were considered statistically significant at p < .05.
RESULTS AND DISCUSSION Claxton et al. (1988) found that gene and chromosomal damage may be induced by exposure to carcinogenic chemicals, indicating that chemical agents produce marked alterations to cellular genetical material. Therefore, the aim of this study was to evaluate for the first time in vitro the genetic toxicity potential of coumarin derivatives 6,7-HC and 4-ME, as these compounds may possess significant beneficial usage as antioxidants in therapy. The use of in vitro studies provides important tools to enhance the understanding of hazardous effects by chemicals and for predicting these effects in humans (Broadhead and Combes, 2001). In the present study the Salmonella/ microsome assay was used to detect the potential of 6,7-HC and 4-ME to induce DNA mutations. This assay is commonly employed as an initial screen for genotoxic activity, in particular, point mutation-inducing activity
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(Organization for Economic Cooperation and Development [OECD], 1997). Table 1 demonstrates the number of revertants per plate and mutagenicity ratio (MR) after treatment with each coumarin derivative, observed in S. typhimurium strains TA98, TA100, TA97a, and TA102, in the presence (+S9) and absence (−S9) of metabolic activation. Mutagenic activity was considered negative for all strains and all concentrations tested both in the absence and presence of metabolism, and enabled us to conclude that 6,7-HC and 4-ME exerted no direct or indirect mutagenic effects. Trypan blue exclusion test for cell viability is used to determine the number of viable cells present in a cell suspension. Data showed absence of toxicity for 6,7-HC at 5–320 µg/ml with cell viability higher than 84%, and at 5–40 µg/ml for 4-ME with cell viability higher than 76%. For the concentrations of 80–640 µg/ml of 4-ME, cell viability gradually decreased as the concentration increased. These results indicate that 4-ME produced more cytotoxicity at high concentrations than 6,7-HC. As highly damaged DNA may correspond to dead cells, the concentrations of test substance higher than 40 µg/ml were excluded from the comet study, since these may reflect possible cytotoxicity. The results obtained for genotoxic assessment of 6,7-HC and 4-ME in human PBL are shown in Table 2. None of the two tested compounds demonstrated significant changes in total number of damaged cells and scores at these tested concentrations compared to the negative control. As expected, an increase in total of damaged cells was observed only for the positive control MMS, confirming the sensitivity of the comet assay for detection of genotoxicity. The clastogenicity/aneugenicity potential of 6,7-HC and 4-ME in human PBL also was assessed in this study using the CBMN assay. Micronuclei (MN) are expressed in dividing cells that either contain chromosome breaks lacking centromeres (clastogenic effect) and/or whole chromosomes due to dysfunction of mitotic apparatus (aneugenic effect), and cytochalasin-B, which blocks the cytokinesis, enabling the identification of dividing cells by
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32 ± 5 39 ± 6(1.2) 47 ± 11(1.5) 37 ± 3(1.2) 26 ± 3(0.8) 27 ± 2(0.8) 797∗ ± 79b
32 ± 5 46 ± 7(1.5) 45 ± 8(1.4) 43 ± 5(1.4) 37 ± 6(1.2) 31 ± 5(1) 797∗ ± 79b
−S9
32 ± 4 28 ± 6(0.9) 25 ± 9(0.8) 32 ± 4(1) 20 ± 2(0.6) 19 ± 5(0.6) 2204∗ ± 255e
32 ± 4 33 ± 11(1) 30 ± 1(0.9) 32 ± 12(1) 38 ± 8(1.2) 28 ± 7(0.9) 2204∗ ± 255e
+S9
153 ± 6 157 ± 13(1) 163 ± 18(1.1) 141 ± 7(0.9) 145 ± 6(0.9) 141 ± 19(0.9) 1193∗ ± 39c
153 ± 6 156 ± 5(1) 178 ± 9(1.2) 168 ± 6(1.1) 150 ± 11(1) 142 ± 13(0.9) 1193∗ ± 39c
−S9
TA 100
136 ± 7 156 ± 5(1.1) 160 ± 5(1.2) 156 ± 12(1.2) 183 ± 6(1.4) 162 ± 20(1.2) 1229∗ ± 94e
136 ± 7 142 ± 10(1) 140 ± 6(1) 147 ± 28(1.1) 118 ± 13(0.9) 111 ± 2(0.8) 1229∗ ± 94e
+S9
355 ± 36 413 ± 20(1.2) 373 ± 9(1.1) 359 ± 28(1) 352 ± 21(1) 325 ± 18(0.9) 1192∗ ± 49d
355 ± 36 334 ± 19(0.9) 397 ± 24(1.1) 428 ± 16(1.2) 380 ± 35(1.1) 419 ± 53(1.2) 1192∗ ± 49d
−S9
TA 102
365 ± 43 401 ± 29(1.1) 393 ± 33(1.1) 376 ± 36(1) 372 ± 11(1) 336 ± 23(0.9) 1804∗ ± 43f
365 ± 43 455 ± 23(1.2) 400 ± 43(1.1) 412 ± 22(1.1) 369 ± 32(1) 321 ± 40(0.9) 1804∗ ± 43f
+S9
106 ± 23 113 ± 14(1.1) 129 ± 19(1.2) 128 ± 25(1.2) 109 ± 11(1) 112 ± 9(1.1) 716∗ ± 74b
106 ± 23 108 ± 9(1) 111 ± 16(1) 133 ± 27(1.3) 111 ± 14(1) 112 ± 26(1.1) 716∗ ± 74b
−S9
TA 97a
214 ± 15 263 ± 38(1.2) 252 ± 11(1.2) 256 ± 14(1.2) 222 ± 16(1) 203 ± 17(1) 2636∗ ± 132e
214 ± 15 249 ± 31(1.2) 241 ± 29(1.1) 232 ± 38(1.1) 220 ± 32(1) 220 ± 61(1) 2636∗ ± 132e
+S9
Note. Data are expressed as mean and standard deviation of the number of revertants/plate and mutagenic ratio (MR, in parentheses) in Salmonella typhimurium TA98, TA100, TA102, and TA97a strains treated with 6,7-dihydroxycoumarin (6,7-HC) and 4-methylesculetin (4-ME) at various concentrations, with (+S9) or without (–S9) metabolic activation. ∗ Significantly different at p < .05. a 0: Negative control: dimethyl sulfoxide (DMSO, 100 µl/plate). b Control+: 4-nitro-o-phenylenediamine (10 µg/ plate, TA98 and TA97a). C sodium azide(1.25 µg/plate, TA100). D mitomycin C (0.5 µg/ plate, TA102), in the absence of S9. e 2-Anthramine (1.25 µg/ plate, TA98, TA100, TA97a). f 2-Aminofluorene (10 µg/ plate, TA102), in the presence of S9.
6,7-HC 0a 62,5 125 250 500 750 C+ 4-ME 0a 62,5 125 250 500 750 C+
Treatments µg/plate
TA 98
Number of revertants (M ± SD)/plate and MR
TABLE 1. Mutagenic Activity Assessment
GENOTOXIC EFFECTS OF COUMARIN DERIVATIVES
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TABLE 2. DNA Migration (Mean ± SD) in the Comet Assay for Assessing Genotoxicity of 6,7-Dihydroxycoumarin (6,7-HC) and 4Methylesculetin (4-ME) in Human Peripheral Blood Lymphocytes (4 h Treatment) Comet class Treatments
Totalc
0
Control 6,7-HC 2 µg/ml 6,7-HC 8 µg/ml 6,7-HC 32 µg/ml
22.83 ± 4.95 13.67 ± 1.75 15 ± 2.68 17 ± 5.42
77.17 ± 4.95 86.33 ± 1.75 85 ± 2.68 83 ± 5.42
18 ± 3.09 11 ± 2.28 13 ± 2.09 15 ± 3.96
4.83 ± 1.94 2.5 ± 1.04 1.83 ± 0.75 2 ± 1.91
0±0 0.16 ± 0.4 0.16 ± 0.4 0.00 ± 0.0
27.67 ± 6.86 16 ± 2 16.67 ± 3.38 19 ± 7.09
4-ME 2 µg/ml 4-ME 8 µg/ml 4-ME 32 µg/ml
17.5 ± 4.32 16.17 ± 5.03 14.67 ± 4.92
82.5 ± 4.32 83.83 ± 5.03 85.33 ± 4.92
13.67 ± 2.33 10.33 ± 3.72 12.5 ± 6.47
3.16 ± 2.31 4.66 ± 2.25 1.83 ± 1.83
0.66 ± 0.81 1.16 ± 0.75 0.33 ± 0.51
22 ± 7.4 23.17 ± 7.08 17.17 ± 4.16
MMS 75 µM (positive control)
99.67 ± 0.81a
12.83 ± 10.38b
82.33 ± 15.27a
277.2 ± 21.9a
1
0.33 ± 0.81a
2
4.5 ± 6.41
3
Scores
Note. SD, standard deviation. a Significantly different from the negative control (p < .05). b Significantly different from the negative control (p < .05). c Total number of damaged cells (class 1 + 2 + 3). TABLE 3. The Micronucleus Frequency and Nuclear Division Index in Human Lymphocytes Treated With 6,7-Dihydroxycoumarin (6,7HC) and 4-Methylesculetin (4-ME) Binucleated cells with MN (2000 cells scored)
Treatment Test substance
Períod (h)
Concentration (µg/ml)
N◦
%
NDI/1000 cells (mean ± SD)
Negative control MMS (Positive control) 6,7-HC
28 28 28 28 28 28 28 28
0 150a 2 8 32 2 8 32
2 52∗ 7 7 3 1 2 1
0.1 2.6∗ 0.35 0.35 0.15 0.05 0.1 0.05
2.04 ± 0.05 2.16 ± 0.11 1.99 ± 0.03 2.02 ± 0.01 2.02 ± 0.01 1.90 ± 0.07 1.97 ± 0.07 1.86 ± 0.07
4-ME
Note. MN, micronucleus, NDI, nuclear division index, SD, standard deviation. ∗ Significantly different from the negative control (p < .05). a Concentration, µM.
their binucleated appearance (Fenech, 2000). The MN data obtained demonstrated results similar to those observed using the comet assay (Table 3). CBMN test demonstrated no marked alterations in the number of micronucleated cells for the two tested substances at all tested concentrations. Similarly, the effects of coumarin derivatives on nuclear division did not display any significant change in cell division as measured by nuclear division index (NDI). No apparent previous reports evaluated the mutagenic or genotoxic effects of 6,7HC. In contrast, regarding 4-ME, there was
one in vivo assessment of genetic toxicity that reported absence of genotoxic effects in different mouse cells using comet assay and MN test (Fedato and Maistro, 2014), in agreement with our in vitro results. Fedato and Maistro (2014) also noted that 4-ME plays a role in preventing chemically induced DNA and chromosome damage induced by the antitumor agent doxorubicin, possibly by antioxidant mechanisms. Coumarin is the natural precursor of 4ME. Morris and Ward (1992) investigated the possibility that pretreatment with coumarin might inhibit the genotoxicity induced by
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benzo[a]pyrene (BaP) in ICR mice as evidenced by the bone-marrow MN test. Similar to findings in our in vitro studies with 4-ME, Morris and Ward (1992) demonstrated that coumarin treatment alone did not induce MN in either mouse gender. However, it is of interest that Morris and Ward (1992) also reported no inhibition in MN formation only in female mice pretreated with the same doses of coumarin used in males. Other in vivo studies showed that coumarin did not markedly influence MN frequencies in PBL of female and male B6C3F1 mice and in bone-marrow cells of Swiss mice (National Toxicology Program [NTP], 1993; Api, 2001). In conclusion, our initial screening of genetic toxicity potential of 6,7-HC and 4ME showed that both coumarin derivatives are not mutagenic in the Salmonella/microsome assay. In addition, no significant genotoxic and clastogenic/aneugenic effects were detected in human PBL in vitro by comet and CBMN assays. Due to its antioxidant potential and benefit for cancer prevention, further in vivo studies in eukaryotic cells are necessary to determine their safe use in humans. FUNDING This research was funded by the FAPESP, Fundação de Amparo à Pesquisa do Estado de São Paulo (grants 2010/07577-3 and 2012/17241-8), Brazil. REFERENCES Api, A. M. 2001. Lack of effect of coumarin on the formation of micronuclei in an in vivo mouse micronucleus assay. Food Chem. Toxicol. 39: 837–841. Baba, M., Jin, Y., Mizuno, A., Suzuki, H., Okada, Y., Takasuka, N., Tokuda, H., Nishino, H., and Okuyama, T. 2002. Studies on cancer chemoprevention by traditional folk medicines XXIV. Inhibitory effect of a coumarin derivative, 7isopentenyloxycoumarin, against tumor promotion. Biol. Pharm. Bull. 25: 244–246.
Beillerot, A., Domínguez, J. C. R., Kirsch, G., and Bagrel, D. 2008. Synthesis and protective effects of coumarin derivatives against oxidative stress induced by doxorubicin. Bioorg. Med. Chem. Lett. 18: 1102–1105. Berenbaum, M. 1995. Phototoxicity of plant secondary metabolites: Insect and mammalian perspectives. Arch. Insect Biochem. Physiol. 29: 119–134. Bernstein, L., Kaldor, J., Mccann, J., and Pike, M. C. 1982. An empirical approach to the statistical analysis of mutagenesis data from Salmonella test. Mutat. Res. 97: 267–281. Born, S. L., Hu, J. K., and Lehman-McKeeman, L. D. 2000. o-Hydroxyphenylacetaldehyde is a hepatotoxic metabolite of coumarin. Drug Metab. Dispos. 28: 218–223. Broadhead, C. L., and Combes, R. D. 2001. The current status for food additives toxicity testing and the potential for application of the three Rs. ATLA 29: 471–485. Cai, Y., Kleiner, H., Johnston, D., Dubowski, A., Bostic, S., Ivie, W., and DiGiovanni, J. 1997. Effect of naturally occurring coumarins on the formation of epidermal DNA adducts and skin tumors induced by benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene in SENCAR mice. Carcinogenesis 18: 1521–1527. Casley-Smith, J. R. 1999. Benzo-pyrones in the treatment of lymphoedema. Int. Angiol. 18: 31–41. Claxton, L. D., Stead, A. G., and Walsh, D. 1988. An analysis by chemical class Salmonella mutagenicity tests as precictors as animal carcinogenicity. Mutat. Res. 205: 1–4. Fedato, R. P., and Maistro, E. L. 2014. Absence of genotoxic effects of the coumarin derivative 4-methylesculetin in vivo and its potential chemovenction against doxorubicininduced DNA damage. J. Appl. Toxicol. 34: 33–39. Fenech, M. 2000. The in vitro micronucleus technique. Mutat. Res. 455: 81–95. Francisco, C. S., Rodrigues, L. R., Cerqueira, N. M., Oliveira-Campos, A. M., and Rodrigues, L. M. 2012. Synthesis of novel benzofurocoumarin analogues and their antiproliferative effect on human cancer cell lines. Eur. J. Med. Chem. 47: 370–376.
GENOTOXIC EFFECTS OF COUMARIN DERIVATIVES
Guo, L. Q., and Yamazoe, Y. 2004. Inhibition of cytochrome P450 by furanocoumarins in grapefruit juice and herbal medicines. Acta Pharmacol. Sin. 25: 129–136. Hartmann, A., and Speit, G. 1997. The contribution of cytotoxicity to DNA—Effects in the single cell gel test (comet assay). Toxicol. Lett. 90: 183–188. Iranshari, M., Askari, M., Sahebkar, A., and Hadjipavlou-Litina, D. 2009. Evaluation of antioxidant, anti-inflammatory and lipoxygenase inhibitory activities of the prenylated coumarin umbelliprenin. DARU J. Pharm. Sci. 17: 99–103. Kelly, V. P., Ellis, E. M., Manson, M. M., Chanas, S. A., Moffat, G. J., McLeod, R., Judah, D. J., Neal, G. E., and Hayes, J. D. 2000. Chemoprevention of aflotoxin B1 hepatocarcinogenesis by coumarin, a natural benzopyrone that is a potent inducer of aflatoxin B1-aldehyde reductase, the glutathione S-transferase A5 and P1 subunits, and NAD(P)H:quinone oxidoreductase in rat liver. Cancer Res. 60: 957–969. Kim, S. N., Kim, N. H., Park, Y. S., Kim, H., Lee, S., Wang, Q., and Kim, Y. K. 2009. 7-Diethylamino-3(2 -benzoxazolyl)coumarin is a novel microtubule inhibitor with antimitotic activity in multidrug resistant cancer cells. Biochem. Pharmacol. 77: 1773–1779. Kleiner, H. E., Vulimiri, S. V., Starost, M. F., Reed, M. J., and DiGiovanni, J. 2002. Oral administration of the citrus coumarin, isopimpinellin, blocks DNA adduct formation and skin tumor initiation by 7,12-dimethylbenz[a]anthracene in SENCAR mice. Carcinogenesis 23: 1667–1675. Lake, B. 1999. Coumarin metabolism, toxicity, and carcinogenicity: Relevance for human risk assessment. Food Chem. Toxicol. 37: 423–453 Lake, B. G., and Grasso, P. 1996. Comparison of the hepatotoxicity of coumarin in the rat, mouse, and Syrian hamster: A dose and time response study. Fundam. Appl. Pharmacol. 97: 311–323.
117
Maron, D. M., and Ames, B. N. 1983. Revised methods for the Salmonella mutagenicity test. Mutat. Res. 113: 173–215. Mitra, I., Saha, A., and Roy, K. 2013. Predictive modeling of antioxidant coumarin derivatives using multiple approaches: Descriptor-based QSAR, 3D-pharmacophore mapping, and HQSAR. Sci. Pharm. 81: 57–80. Morris, D. L., and Ward, J. B. 1992. Coumarin inhibts micronuclei formation induced by benzo(a)pyrene in male but not female ICR mice. Environ. Mol. Mutagen. 19: 132–138. Murray, R. D. H., Mendez, J., and Brown, S. A., eds. 1982. The natural coumarins: Occurrence, chemistry and biochemistry, 97–111. New York, NY: John Wiley & Sons. National Toxicology Program. 1993. Toxicology and carcinogenic studies of coumarin (CAS No. 91-64-5) in F344/N mice and B6C3F1 mice (gavage studies). Technical report series no. 422. NIH Publication No. 92-31153. Bethesda, MD: U.S. Department of Health and Human Services. Ngwendson, J. N., Bedir, E., Efange, S. M., Okunji, C. O., Iwu, M. M., Schuster, B. G., and Khan, I. 2003. Constituents of Peucedanum zenkeri seeds and their antimicrobial effects. Pharmazie 58: 587–589. Organization for Economic Cooperation and Development. 1997. Guideline for testing of chemicals, Test guideline 471: Bacterial reverse mutation test. Paris, France: OECD. Panda, S. K., Kumar, S., Tupperwar, N. C., Vaidya, T., George, A., Rath, S., Bal, V., and Ravindran, B. 2012. Chitohexaose activates macrophages by alternate pathway through TLR4 and blocks endotoxemia. PLoS Pathol. 8: e1002717. Prince, M., Campbell, C. T., Robertson, T. A., Wells, A. J., and Kleiner, H. E. 2006. Naturally occurring coumarins inhibit 7,12dimethylbenz[a]anthracene DNA adduct formation in mouse mammary gland. Carcinogenesis 27: 1204–1213. Resende, F. A., Munari, C. C., Bentos, M. N. A., Tavares, D. C., Bastos, J. K., Silva-Filho,
118
A. A., and Varanda, E. A. 2012. Comparative studies of the (anti) mutagenicity of Baccharis dracunculifolia and artepillin c by the bacterial reverse mutation test. Molecules 7: 2335–2350. Setzer, W. N., Setzer, M. C., Schmidt, J. M., Moriarity, D. M., Vogler, B., Reeb, S., Holmes, A. M., and Haber, W. A. 2000. Cytotoxic components from the bark of Stauranthus perforatus from Monteverde, Costa Rica. Planta Med. 66: 493–494. Singh, N. P., McCoy, M. T., Tice, R. R., and Schneider, E. L. 1988. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell Res. 175: 184–191. Strober, W. 1997. Trypan blue exclusion test of cell viability. Curr. Prot. Immunol. A.3B.1– A.3B.2. Tanaka, T., Kawabata, K., Kakumoto, M., Hara, A., Murakami, A., Kuki, W., Takahashi, Y., Yonei, H., Maeda, M., Ota, T., Odashima, S., Yamane, T., Koshimizu, K., and Ohigashi, H. 1998. Citrus auraptene exerts dosedependent chemopreventive activity in rat large bowel tumorigenesis: The inhibition
E. L. MAISTRO ET AL.
correlates with suppression of cell proliferation and lipid peroxidation and with induction of phase II drug-metabolizing enzymes. Cancer Res. 58: 2550–2556. Tice, R. R., Agurell, E., Anderson, D., Burlinson, B., Hartmann, A., Kobayashi, H., Miyamae, Y., Rojas, E., Ryu, J. C., and Sasaki, Y. F. 2000. Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ. Mol. Mutagen. 35: 206–221. Venugopala, K. N., Rashmi, V., and Odhav, B. 2013. Review on natural coumarin lead compounds for their pharmacological activity. Biol. Med. Res. Int. 2013: 1–14. Vianna, D. R., Bulobs, G., Meirelles, G., Silva, B. V., Rocha, A., Lanznaster, M., Monserrat, J. M., Garcia, S. C., Poser, G. V., and EiflerLima, V. L. 2012. Evaluation of the antioxidant capacity of synthesized coumarins. Int. J. Mol. Sci. 13: 7260–7270. Wattenberg, L. W., Lam, L. K., and Fladmoe, A. V. 1979. Inhibition of chemical carcinogeninduced neoplasia by coumarins and alpha-angelicalactone. Cancer Res. 39: 1651–1654.
