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Abstract
Neolignans are secondary metabolites found in various groups of Angiosperms. They belong to a class of natural compounds with great diversity of chemical structures and pharmacological activities. These compounds are formed by linking two phenylpropanoid units. Several compounds that have ability to prevent genetic damage have been isolated from plants, and can be used to prevent or delay the development of tumor cells. Genetic toxicology evaluation is widely used in risk assessment of new drugs in preclinical screening tests. In this study, we evaluated the genotoxicity and cytotoxicity of the neolignan analogue 2-(4-nitrophenoxy)-1-phenylethanone (4NF) and its protective effect against DNA damage using the mouse bone marrow micronucleus test and the comet assay in mouse peripheral blood. Our results showed that this neolignan analogue had no genotoxic activity and was able to reduce induced damage both in mouse bone marrow and peripheral blood. Although the neolignan analogue 4NF was cytotoxic, it reduced cyclophosphamide-induced cytotoxicity. In conclusion, it showed no genotoxic action, but exhibited cytotoxic, antigenotoxic, and anticytotoxic activities.
Citation: Hanusch AL, Oliveira GRd, Sabóia-Morais SMTd, Machado RC, dos Anjos MM, Chen Chen L (2015) Genotoxicity and Cytotoxicity Evaluation of the Neolignan Analogue 2-(4-Nitrophenoxy)-1Phenylethanone and its Protective Effect Against DNA Damage. PLoS ONE 10(11): e0142284. https://doi.org/10.1371/journal.pone.0142284
Editor: David L. McCormick, IIT Research Institute, UNITED STATES
Received: June 15, 2015; Accepted: October 19, 2015; Published: November 10, 2015
Copyright: © 2015 Hanusch et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Data Availability: All relevant data are within the paper.
Funding: This study was supported by grants from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) to ALH, RCM. http://www.capes.gov.br/. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Neolignans are secondary metabolites found in various groups of Angiosperms. They belong to a class of natural compounds with great diversity of chemical structures and pharmacological activities. They are formed by connecting two phenylpropanoid units via a C-C bond different from β-β' [1].
The oxyneolignan group exhibits a great variety of biological properties such as anti-leishmanial, antioxidant, and antitumor activities [2–4]. Among these compounds, 8-O-4' type neolignans are known to exhibit antifungal, antioxidant, anti-inflammatory, antitumor, and antinitric oxide production activities [4–7]. Since the neolignan analogue 2-(4-nitrophenoxy)-1-phenylethanone (4NF) is a synthetic 8-O-4' type oxyneolignan, the investigation of its biological activities can provide information to help develop new drugs [8].
Many drugs present genotoxic risks and can cause changes in the genetic material of germ and/or somatic cells, accelerating carcinogenesis and the cell aging process [9]. In order to evaluate the genotoxic and carcinogenic risks for humans, the regulatory authorities of the European Union, Japan, and the United States recommend that genotoxic and carcinogenic evaluations are conducted before application for marketing approval of pharmaceuticals [10].
Important factors for lengthening human life span are cancer prevention and tumor control. It has been shown that many phytochemicals present potential to prevent DNA damage. The search for plants and phytochemicals that present antigenotoxic and cytotoxic activities is of great importance for the development of chemopreventive agents to be used in cancer therapies [11–14].
Several tests can be performed to assess the genotoxicity and cytotoxicity of phytochemicals. The mouse bone marrow micronucleus test has been extensively used in genotoxicity studies of chemicals. It is a simple and quick technique to evaluate the genotoxicity and cytotoxicity of compounds, originally developed using bone marrow cells of mice, but alternatively it can also use the erythrocytes of peripheral blood [15].
Another important test for genotoxicity evaluation is the comet assay or single cell gel electrophoresis (SCGE), which is a rapid and sensitive method to measure and detect double-strand breaks, single-strand breaks, incomplete excision repair sites, and alkali-labile sites in individualized eukaryotic cells. It is suitable to detect clastogenic compounds, but not aneugenic compounds. This test is used to evaluate the genotoxic effect of physical, chemical, or biological agents. It is based on the use of individual cells that have their membranes lysed and most of the proteins removed, allowing the movement of DNA during the electrophoretic run conducted in an alkaline buffer [16–18].
The aim of this study was to evaluate the genotoxic and cytotoxic activities of the neolignan analogue 2-(4-nitrophenoxy)-1-phenylethanone (4NF) in mice and its protective effect against DNA damage.
Materials and Methods
Chemicals
4NF was obtained by the reaction between phenacyl bromide and p-nitrophenol in basic medium [19]. Under these conditions the product was obtained in 84% yield.
Cyclophosphamide was purchased from Hera Medicamentos (Belo Horizonte, MG, Brazil), fetal bovine serum from Laborclin (Campinas, SP, Brazil), and Giemsa from Doles (Goiânia, GO, Brazil). All the other reagents used were purchased from Vetec Química Fina Ltda (Duque de Caxias, RJ, Brazil) and Synth (Diadema, SP, Brazil).
Animal testing
This study was approved by the Ethics Committee on Animal Use of the Universidade Federal de Goiás (protocol no. 060/13). The experiments followed national and international standards of management and experimentation with animals [20,21].
Healthy, young, male adult outbred mice (Mus musculus, Swiss Webster), between 7 and 12 weeks old, weighing 30–40 g, obtained from the animal facilities of the same university, were randomly allocated to treatment groups. All animals were brought to the laboratory seven days before the experiments and housed in polyethylene cages (40 cm × 30 cm × 16 cm), lined with wood shavings, in groups of five animals, in air-conditioned rooms kept at 25 ± 2°C and 50 ± 10% relative humidity, with a 12-h light/dark natural cycle. Standard food pellets and water were provided ad libitum.
Treatments.
The mice were divided in groups of five animals each, treated intraperitoneally (i.p.) with 4NF at the doses of 50 mg/kg, 75 mg/kg, and 100 mg/kg dissolved in dimethylsulfoxide (DMSO). To evaluate the possible protective effect of the neolignan analogue against cyclophosphamide-induced damage, mice simultaneously received cyclophosphamide at the dose of 50 mg/kg i.p. The experiments were conducted for 24 h and 48 h. A positive control group (50 mg/kg cyclophosphamide i.p.) and two negative groups (10 μL/g DMSO and 10 μL/g saline) were also included.
Mouse bone marrow micronucleus test.
Experiments were performed according to von Ledebur and Schmid [22]. The animals were euthanized by cervical dislocation under sodium pentobarbital anesthesia and their bone marrow cells were flushed from both femurs in fetal calf serum. After centrifuging (300xg, 5 min), the bone marrow cells were smeared on glass slides. The slides were fixed in absolute methanol for 10 min and stained with buffered Giemsa solution at pH 6.8 for 10 min [15].
Slides were analyzed under light microscope (1000x) to detect possible changes and/or chromosomal losses in young red blood cells of bone marrow of mice submitted to the different treatments. The number of micronucleated polychromatic erythrocytes (MNPCE) in 2000 polychromatic erythrocytes (PCE) was assessed using two slides per animal, whereas cytotoxicity and anticytotoxicity were evaluated by calculating the ratio of PCE to normochromatic erythrocytes (NCE) [23].
Comet assay.
The comet assay was carried out under alkaline conditions according to the standard procedures described by Singh [24]. The same mice treated with 4NF in the micronucleus test were used in this assay.
Slides previously coated with normal melting point agarose (1.5%) received a homogenate of peripheral blood cells with low melting point agarose 0.75% at 37°C. The material was spread on the slides with coverslips and taken to a cold chamber. After gelation, the coverslips were carefully removed. The slides were immersed in lysis solution (1% triton X-100, 10% DMSO, 2.5 M NaCl, 100 mM Na2EDTA, and 10 mM Tris, pH 10.0) for 12–24 h, at 4°C. Subsequently, the slides were incubated with freshly made alkaline solution (300 mM NaOH, 1 mM EDTA, pH > 13) for 20 min, at 4°C for DNA unwinding. The electrophoresis was performed in the same buffer at 300 mA and 1 V/cm for 25 min at 4°C. After the electrophoresis, the slides were placed in a staining tray, covered with a neutralizing buffer (0.4 M Tris-HCl, pH 7.5). The slides were then stained with ethidium bromide and analyzed.
Nucleoid images were captured with an epifluorescence Leica DM 2000 Citogen microscope (Leica Microsystems, Wetzlar, Germany), equipped with a Jenoptik ProgRes® MF camera (Optronics, Goleta, CA, USA), driven by Lucia CytogeneticsTM version 2.5 software (Laboratory Imaging Ltd, Prague, Czech Republic). Cell images were analyzed using the Comet Assay Software Project (CASP) version 1.2.3beta2 [24–26]. The calibration parameters used were: head center threshold = 0.95, thresholds comet = 0:05, head threshold = 0:05, tail threshold = 0.1, and profile 1. DNA damage was evaluated assessing 100 nucleoids per animal using the following parameters: percentage of DNA in the tail, tail moment, and Olive tail moment.
Statistical analysis
The experiments were performed in triplicate, and the values obtained are expressed as mean ± standard deviation. The GraphPad Prism software 5 was used for the statistical analyses. Prior to the hypothesis test, tests of normality and homoscedasticity were applied to data. Homoscedastic data were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett’s post-hoc multiple-comparison test. Heteroscedastic data were analyzed using Kruskal-Wallis test followed by post-hoc Dunn's test. The results were considered significantly different when p < 0.05.
Results
Mouse bone marrow micronucleus test
The results of the bone marrow micronucleus test in mice treated with 4NF did not show significant increase in MNPCE frequency at 24 h (p > 0.05) and 48 h (p > 0.05) of exposure (Table 1). Cytotoxicity was calculated by the ratio of PCE/NCE. Only the dose of 100 mg/kg was significantly different from the negative control group at 24 h of exposure (p < 0.05), demonstrating the cytotoxic action of the neolignan analogue 4NF at this dose.
The evaluation of antigenotoxicity using the micronucleus test revealed that 4NF at the doses of 75 mg/kg and 100 mg/kg reduced micronucleus frequency induced by cyclophosphamide at 24 h (p < 0.05). At the dose of 100 mg/kg, this neolignan analogue also decreased MNPCE frequency by 52% at 48 h (p < 0.05) (Table 2). In the evaluation of anticytotoxicity the ratio of PCE/NCE increased at 24 h at the dose of 100 mg/kg (p < 0.05) and at 48 h at the doses of 50 mg/kg and 100 mg/kg (p < 0.05), demonstrating a protective action against the cytotoxicity induced by cyclophosphamide.
Comet assay
The comet assay, applied to evaluate the levels of primary DNA damage in peripheral blood leukocytes of mice treated with 4NF, showed no significant difference between the doses tested and the negative control group for the parameters percentage of DNA in the tail, tail moment, and Olive tail moment at 24 h and 48 h (Fig 1). Therefore, this neolignan analogue exhibited no genotoxic effects at all doses tested.
SAL, saline (negative control); DMSO, dimethylsulfoxide (negative control); CP, cyclophosphamide (50 mg/kg) (positive control). * p < 0.05, ** p < 0.01 compared to the negative control.
Antigenotoxicity evaluation showed decrease in the values of all parameters assessed using the comet assay at the doses of 75 mg/kg and 100 mg/kg at 24 h of exposure (p < 0.05). However, at 48 h the values were close to zero for all treatments, including the positive control with cyclophosphamide, which limited the measurement for this time of exposure (Fig 2).
SAL, saline (negative control); DMSO, dimethylsulfoxide (negative control); CP, cyclophosphamide (50 mg/kg) (positive control). * p < 0.05, ** p < 0.01, *** p < 0.001 compared to the positive control.
Discussion
Genotoxicity tests are of fundamental importance for drug development, since genotoxic compounds can cause mutation and chromosomal damage events that are essential to initiate carcinogenesis [27]. The pharmacological and toxicological activities of the neolignan analogue 4NF have not been studied yet. In our study, this compound showed no significant genotoxic activity in mice at the doses tested assessed by the micronucleus test in bone marrow as well as by the comet assay in peripheral blood leukocytes.
Studies with neolignans extracted from plants also showed no genotoxic effects induced by these compounds. The possible genotoxic effects of a plant extract containing 94% of magnolol neolignan and 1.5% of honokiol neolignan were investigated. The authors concluded that the standardized extract showed no mutagenic activity using the Ames mutagenicity test and did not induce micronucleus formation in immature erythrocytes of Swiss albino mice [28].
Similar results were obtained studying the lignan daurinol. The compound showed no genotoxic effect on human peripheral blood leukocytes using an in vitro test [29]. The lignan cubebin showed no genotoxic action in a study employing the mouse bone marrow micronucleus test [30].
The antitumor activity of neolignans due to the presence of cytotoxic action has also been tested [4]. Cytotoxicity of natural compounds has received great attention during recent years due to their important role in the prevention and treatment of diseases, as well as in the development of promising chemotherapeutic candidates. In the last 20 years, over 60% of the new drugs for the treatment of cancer are of natural origin [4]. The neolignan analogue 4NF showed cytotoxicity due to the decrease in PCE/NCE ratio in the bone marrow of mice at the dose of 100 mg/kg (p < 0.05).
Studies have found lignoids with cytotoxic activity specific for some tumor cell lines, such as neolignan licarin A and the lignans galbacin, sesamin, machilin A and G. These lignoids were effective in inhibiting proliferation of tumor cell lines A549 (human lung carcinoma), MCF-7 (human breast adenocarcinoma), and HCT-15 (human colon adenocarcinoma) by inhibition of phospholipase Cγ1, which is an important factor for tumor cell proliferation. Lignan sesamin proved to be cytotoxic to tumor cell lines CCRF-CEM and CEM/ADR5000 (human T-leukemia) [31,32].
Antigenotoxic and antimutagenic compounds have chemopreventive properties against genotoxins. In the present study, the neolignan analogue 4NF was able to significantly reduce cyclophosphamide-induced cytotoxic and genotoxic damages assessed by the mouse bone marrow micronucleus test and the comet assay. The neolignan grandidisin also reduced MNPCE frequency in mice treated with cyclophosphamide, a known genotoxic agent [33].
The neolignan magnolol has shown to be antimutagenic as assessed by the Ames mutagenicity test using Salmonella typhimurium strains TA98 and TA100 [34]. The same compound also showed antigenotoxic activity using the mouse bone marrow micronucleus test. Additionally, magnolol inhibited oxidative damage induced by X-ray due to an increase in the activity of catalase, superoxide dismutase, glutationa-transferase, and uridine diphosphate-glucuronosyl transferase, enzymes that act in the detoxification process [35].
The lignin hinokinin is able to reduce the chromosomal damage caused by methyl methane sulfonate (MMS) acting as a desmutagenic substance [36]. Hinokinin showed no genotoxic activity, but proved to be antigenotoxic in an in vivo trial with Wistar rats previously treated with doxorubicin [37].
The lignan cubebin was able to inhibit the genotoxicity induced by doxorubicin due to its antioxidant activity [33]. This action has been identified by the determination method using 2,2-diphenyl-1-picrylhydrazyl (DPPH), and this compound was considered a desmutagenic agent [38].
Conclusion
The neolignan analogue 4NF did not exhibit genotoxic effect at the tested doses. This compound was able to reduce cytotoxic and genotoxic effects of cyclophosphamide. The possible use of the neolignan analogue 4NF as a chemopreventive and/or therapeutic agent still requires further investigation using different mutagenicity/genotoxicity tests, long-term tests in animals, and research in molecular biology to carry out comprehensive studies of gene expression.
Acknowledgments
This study was supported by grants from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).
Author Contributions
Conceived and designed the experiments: ALH LCC. Performed the experiments: ALH LCC GRO SMTSM RCM MMA. Analyzed the data: ALH LCC. Contributed reagents/materials/analysis tools: LCC GRO SMTSM MMA. Wrote the paper: ALH LCC SMTSM.
References
- 1. Kumar BS, Singh A, Kumar A, Singh J, Hasanain M, Singh A, et al. Synthesis of neolignans as microtubule stabilisers. Bioorg Med Chem. 2014;22: 1342–1354. pmid:24457094.
- 2. Barata LES, Santos LS, Ferri PH, Phillipson JD, Paine A, Croft SL. Anti-leishmania activity of neolignans from Virola species and synthetic analogues. Phytochemistry. 2000;55: 589–595.
- 3. Kónya K, Varga Zs, Antus S. Anti-oxidant properties of 8.O.4' neolignans. Phytomedicine. 2001;8: 454–459. pmid:11824520.
- 4. Huang XX, Zhou CC, Li LZ, Li FF, Lou LL, Li DM, et al. The cytotoxicity of 8-O-4′ neolignans from the seeds of Crataegus pinnatifida. Bioorg Med Chem Lett. 2013;23: 5599–5604. pmid:23999046.
- 5. Pinheiro AAC, Borges RS, Santos LS, Alves CN. A QSAR study of 8.O.4′-neolignans with antifungal activity. J Mol Struct: Theochem. 2004;672: 215–219.
- 6. Lee WS, Baek YI, Kim JR, Cho KH, Sok DE, Jeong TS. Antioxidant activities of a new lignan and a neolignan from Saururus chinensis. Bioorg Med Chem Lett. 2004;14: 5623–5628. pmid:15482936.
- 7. Cao GY, Xu W, Yang XW, Gonzalez FJ, Li F. New neolignans from the seeds of Myristica fragrans that inhibit nitric oxide production. Food Chem. 2015;173: 231–237. pmid:25466017.
- 8. Souza VA, Nakamura CV, Corrêa AG. Atividade antichagásica de lignanas e neolignanas. Rev Virtual Quim. 2012;4: 197–207.
- 9. Kahl VFS, Reyes JM, Sarmento MS, Silva J. Mitigation by vitamin C of the genotoxic effects of nicotine in mice, assessed by the comet assay and micronucleus induction. Mutat Res/Genet Toxicol Environ Mutagen. 2012;744: 140–144. pmid:22331007.
- 10. Brambilla G, Martelli A. Genotoxicity and carcinogenicity studies of analgesics, anti-inflammatory drugs and antipyretics. Pharmacol Res. 2009;60: 1–17. pmid:19427580.
- 11. Rhee CH, Park HD. Three glycoproteins with antimutagenic activity identified in Lactobacillus plantarum KLAB21. Appl Environ Microbiol. 2001;67: 3445–3449. PMCID: PMC93041. pmid:11472917
- 12. Antunes LMG, Araújo MCP. Mutagenicidade e antimutagenicidade dos principais corantes para alimentos. Rev Nutr. 2000;13: 81–88.
- 13. Cordell JL. A guide to developing clinical pathways. Med Lab Obs. 1995;27: 35–39. pmid:10141919.
- 14. Lourenço JA, Pitangui CP, Jordão AA, Vannucchi H, Cecchi AO. Ausência de mutagenicidade e antimutagenicidade do extrato obtido das flores do ipê roxo [Tabebuia impetiginosa (Mart. ex DC.) Standl.]. Rev Bras Plantas Med. 2010;12: 414–420.
- 15. Heddle JA. A rapid in vitro test for chromosomal damage. Mutat Res. 1973;18: 187–190. pmid:4351282.
- 16. Singh NP, McCoy MT, Tice RR, Schneider EL. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res. 1988;175: 184–191. pmid:3345800.
- 17. Tice RR, Agurell E, Anderson D, Burlinson B, Hartmann A, Kobayashi H, et al. Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol Mutagen. 2000;35: 206–221. pmid:10737956.
- 18. Collins AR, Oscoz AA, Brunborg G, Gaivão I, Giovannelli L, Kruszewski M, et al. The comet assay: topical issues. Mutagenesis. 2008; 23: 143–151. pmid:18283046.
- 19. Aveniente M, Pinto EF, Santos LS, Rossi-Bergmann B, Barata LE. Structure-activity relationship of antileishmanials neolignan analogues. Bioorg Med Chem. 2007; 15: 7337–7343.
- 20. Schnaider TB, Souza C. Aspectos éticos da experimentação animal. Rev Bras Anestesiol. 2003; 53: 278–285.
- 21.
Olfert ED, Cross BM, McWilliam AA. Guide to the care and use of experimental animals. v. 1. Ottawa: Canadian Council on Animal Care; 1993.
- 22. von Ledebur M, Schmid W. The micronucleus test methodological aspects. Mutat Res/Fundam Mol Mech Mutagen. 1973;19: 109–117. pmid:4792278.
- 23. Schmid W. The micronucleus test. Mutat Res. 1975;31: 9–15. pmid:48190.
- 24. Srinivasan M, Kalpana KB, Devipriya N, Menon VP. Protective effect of lycopene on whole body irradiation induced liver damage of Swiss albino mice: pathological evaluation. Biomed Prev Nutr. 2014;4: 87–94.
- 25. Yang Q, Shi L, Huang K, Xu W. Protective effect of N-acetylcysteine against DNA damage and S-phase arrest induced by ochratoxin A in human embryonic kidney cells (HEK-293). Food Chem Toxicol. 2014;70: 40–47. pmid:24799199.
- 26. Yamamoto ML, Chapman AM, Schiestl RH. Effects of side-stream tobacco smoke and smoke extract on glutathione- and oxidative DNA damage repair-deficient mice and blood cells. Mutat Res/Fundam Mol Mech Mutagen. 2013;749: 58–65. pmid:23748015.
- 27. Jena GB, Kaul CL, Ramarao P. Genotoxicity testing, a regulatory requirement for drug discovery and development: impact of ICH guidelines. Indian J Pharmacol. 2002;34: 86–99.
- 28. Li N, Song Y, Zhang W, Wang W, Chen J, Wong AW, et al. Evaluation of the in vitro and in vivo genotoxicity of magnolia bark extract. Regul Toxicol Pharmacol. 2007;49: 154–159. pmid:17692444.
- 29. Kang K, Oh SH, Yun JH, Jho EH, Kang JH, Batsuren D, et al. A novel topoisomerase inhibitor, daurinol, suppresses growth of HCT116 cells with low hematological toxicity compared to etoposide. Neoplasia. 2011;13: 1043–1057. pmid:22131880.
- 30. Rezende AAA, Munari CC, Oliveira PF, Ferreira NH, Tavares DC, Silva MLA, et al. A comparative study of the modulatory effects of (−)-cubebin on the mutagenicity/recombinogenicity induced by different chemical agents. Food Chem Toxicol. 2013;55: 645–652. pmid:23402860.
- 31. Lee JS, Kim J, Yu YU, Kim YC. Inhibition of phospholipase Cγ1 and cancer cell proliferation by lignans and flavans from Machilus thunbergii. Arch Pharm Res. 2004;27: 1043–1047. pmid:15554262.
- 32. Saeed M, Khalid H, Sugimoto Y, Efferth T. The lignan, (−)-sesamin reveals cytotoxicity toward cancer cells: pharmacogenomic determination of genes associated with sensitivity or resistance. Phytomedicine. 2014;21: 689–696. pmid:24556122.
- 33. Oliveira LM Júnior, Guerra MT, Vieira MS, Valadares MC. Investigação do potencial mutagênico da grandisina. Rev Eletrôn Farm. 2007;4: 82–84.
- 34. Saito J, Sakai Y, Nagase H. In vitro anti-mutagenic effect of magnolol against direct and indirect mutagens. Mutat Res. 2006;609: 68–73. pmid:16884943.
- 35. Saito J, Shibuya K, Nagase H. Anti-clastogenic effect of magnolol on benzo(a)pyrene-induced clastogenicity in mice. Food Chem Toxicol. 2008;46: 694–700. pmid:17967502.
- 36. Resende FA, Tomazella IM, Barbosa LC, Ponce M, Furtado RA, Pereira AC, et al. Effect of the dibenzylbutyrolactone lignan(−)-hinokinin on doxorubicin and methyl methanesulfonate clastogenicity in V79 Chinese hamster lung fibroblasts. Mutat Res/Genet Toxicol Environ Mutagen. 2010;700: 62–66. pmid:20452459.
- 37. Medola JF, Cintra VP, Silva EPP, Royo VA, Silva R, Saraiva J, et al. (−)-Hinokinin causes antigenotoxicity but not genotoxicity in peripheral blood of Wistar rats. Food Chem Toxicol. 2007;45: 638–642. pmid:17141387.
- 38. Sinigaglia M, Lehmann M, Baumgardt P, Amaral VS, Dihl RR, Reguly ML, et al. Vanillin as a modulator agent in SMART test: inhibition in the steps that precede N-methyl-N-nitrosourea-, N-ethyl-N-nitrosourea-, ethylmethanesulphonate- and bleomycin-genotoxicity. Mutat Res/Genet Toxicol Environ Mutagen. 2006;607: 225–230. pmid:16777474.