Research Article Effect of Annatto on Micronuclei Induction by Direct and Indirect Mutagens in HepG2 Cells Gustavo Rafael Mazzaron Barcelos, 1 * Jos e Pedro Friedmann Angeli, 3 Juliana Mara Serpeloni, 1 Bruno Alves Rocha, 2 M ario S ergio Mantovani, 3 and Lus^ ania Maria Greggi Antunes 1 1 Departamento de Ana ´lises Clı´nicas, Bromatolo ´gicas e Toxicolo ´gicas de Faculdade de Cieˆncias Farmaceˆuticas de Ribeira˜o Preto, Universidade de Sa˜o Paulo Av. do Cafe´, s/n., Campus Universita ´rio da USP, Ribeira˜o Preto, SP, 14040-903, Ribeira˜o Preto, Sa˜o Paulo, Brasil 2 Departamento de Cieˆncias Farmaceˆuticas, Faculdade de Cieˆncias Farmaceˆuticas de Ribeira˜o Preto, Universidade de Sa˜o Paulo Av. do Cafe´, s/n., Campus Universita ´rio da USP, Ribeira˜o Preto, SP, 14040-903, Ribeira˜o Preto, Sa˜o Paulo, Brasil 3 Departamento de Biologia Geral, Centro de Cieˆncias Biolo ´gicas, Universidade Estadual de Londrina, Rodovia Celso Garcia Cid km 380, 86051-990, Londrina, Parana ´, Brasil Annatto (AN), a natural food colorant rich in caro- tenoids, has been reported as being an effective antioxidant, but little is known about its potential chemopreventive properties. In this study, we eval- uated the ability of AN to protect human hepatoma cells (HepG2) from micronucleus (MN) induction against three different mutagens: benzo(a)pyrene (B(a)P), doxorubicin (DXR), and methyl methanesul- fonate (MMS). In an attempt to clarify the possible mechanism of antimutagenicity of AN, three proto- cols of treatment were applied (pretreatment; simul- taneous treatment, and post-treatment with AN fol- lowing treatment with the mutagens). Also, cells exposed only to AN were assayed for cytotoxicity and mutagenicity. A dosage up to 10 lg/ml of AN was devoid of mutagenic activity. Protective effects were seen on micronuclei induced by B(a)P and DXR using pre and simultaneous treatment, but AN had no significant effect on MN induction by MMS in any of the protocols. Our results also show that exposure of cells to concentrations of AN higher than 10 lg/ml decreased cell viability. Taken together, our findings indicate that AN presents antimutagenic activity in vitro, but its pro- tective effect is dependent on the mutagen and on type of treatment suggesting its potential use as a chemopreventive agent. Environ. Mol. Mutagen. 50:808–814, 2009. V V C 2009 Wiley-Liss, Inc. Key words: annatto; bixin; micronuclei; HepG2; antimutagenicity INTRODUCTION Dietary chemoprevention has emerged as a cost-effec- tive approach to control most prevalent chronic diseases, including cancer. Epidemiological studies have provided evidence that high consumption of fruits and vegetables effectively decrease the risk for cardiovascular disease and cancer by improving the antioxidant capacity [Bhuva- neswari and Nagini, 2005]. These epidemiological studies also suggest that over two-thirds of cancers might be pre- vented through lifestyle modification [Ferguson, 1999]. Dietary imbalance, especially the lack of sufficient amounts of dietary fruits and vegetables, is one of the major influences on risk of cancer [Ames and Gold, 1997]. The beneficial health effects from the consumption *Correspondence to: Gustavo Rafael Mazzaron Barcelos, Departamento de Ana ´lises Clı ´nicas, Bromatolo ´gicas e Toxicolo ´gicas de Faculdade de Cie ˆncias Farmace ˆuticas de Ribeira ˜o Preto, Universidade de Sa ˜o Paulo Av. do Cafe ´, s/n., Campus Universita ´rio da USP, Ribeira ˜o Preto, SP, 14040-903, Ribeira ˜o Preto, Sa ˜o Paulo, Brasil. E-mail: barcelos@fcfrp.usp.br Grant sponsors: Conselho Nacional de Desenvolvimento Cientı ´fico e Tecnolo ´gico (CNPq, Brazil), Fundac ¸a ˜o de Amparo a ` Pesquisa do estado de Sa ˜o Paulo (FAPESP). Received 1 April 2008; provisionally accepted 13 March 2009; and in final form 17 March 2009 DOI 10.1002/em.20494 Published online 28 April 2009 in Wiley InterScience (www. interscience.wiley.com). V V C 2009 Wiley-Liss, Inc. Environmental and Molecular Mutagenesis 50:808^814 (2009)