Author's personal copy Mutation Research 695 (2010) 81–86 Contents lists available at ScienceDirect Mutation Research/Genetic Toxicology and Environmental Mutagenesis journal homepage: www.elsevier.com/locate/gentox Community address: www.elsevier.com/locate/mutres Aneugenic effects of benzoxazinones in cultured human cells Elena Arroyo a , Nuria Chinchilla a , José M.G. Molinillo a , Francisco A. Macias a , Antonio Astola b , Manuela Ortiz b , Manuel M. Valdivia b,∗ a Grupo de Alelopatía, Departamento de Química Orgánica, Facultad de Ciencias, 11510 Puerto Real, Cádiz, Spain b Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias, 11510 Puerto Real, Cádiz, Spain article info Article history: Received 27 January 2009 Received in revised form 31 July 2009 Accepted 10 September 2009 Available online 16 December 2009 Keywords: Benzoxazinones Micronucleus Aneuploidy HeLa cells abstract Benzoxazinones (BAs) are natural products that are present in Gramineae and represent part of the plant defence system against pests. In recent years, sprouts of maize, wheat and rye have been used for the production of dietary supplements. We have investigated the potential genotoxic activities of a diverse range of synthetic derivatives of the most abundant natural BA, namely DIBOA (2,4-dihydroxy- 1,4-benzoxazin-3-one), proposed for use as a potential herbicide. We have tested 18 synthetic BAs for potential effects in cultured HeLa cells. We found significantly higher micronucleus (MN) induction over the background level, with the solvent DMSO used as an internal control. Concentration-dependent effects were found between 1 nM and 20 nM for all the synthetic compounds studied. Immunostaining with an anticentromere antibody showed that >80% of MN induced gave a centromere-positive signal. Similarly, fluorescence in situ hybridization (FISH) analysis with alphoid centromere probes showed a positive hybridization signal, indicating that all compounds analyzed are aneugenic. Chemical modification of the N in the heterocyclic aromatic amine served us to suggest a relationship between the structure and the aneugenic effects of the compounds analyzed. Our findings indicate that benzoxazinoids could be potential genotoxins for human cells. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Benzoxazinones (BAs) were identified in the early 1960s as secondary metabolites of the grasses that function as natural pes- ticides and exhibit allelophatic properties [1,2]. BAs are abundant in sprouts of Gramineae, including major agricultural crops such as wheat, maize and rye, and they can be present in concentrations as high as 1–10 nmol/mg wet weight [3]. BAs are part of the plant defence system against pests including insects, bacteria and fungi [4,5]. These compounds also play a role in the allochemical system [6,7]. Several studies on the acute toxic effects of BAs in insects and bacteria have been published [8]. Observations indicate that BAs may be activated through N-acetylation in a similar way to aromatic and heterocyclic aromatic amines [9]. Research on the chemistry and bioactivity of benzoxazinoids yielded a wide vari- ety of natural and synthetic derivatives with different substituent combinations in the aromatic ring. A complete structure–activity relationship (SAR) study with benzoxazinoids and related com- pounds, including synthetic analogues, was presented previously and was directed toward the search for natural herbicide mod- els based on allelochemical structures [10–13]. Accordingly, there ∗ Corresponding author. Tel.: +34 956016388. E-mail address: manuel.valdivia@uca.es (M.M. Valdivia). are precedents concerning the use of benzoxazinoid derivatives as active ingredients in commercial herbicides [14,15]. In recent years the consumption of wheat and other grami- neous sprouts has increased markedly and sprout extracts are also used for the production of food supplements and functional foods [16]. Recently, a study on the genotoxic effects in human cultured cells of the two most abundant natural BAs, namely DIMBOA (2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one) and DIBOA (2,4-dihydroxy-1,4-benzoxazin-3-one) was presented [17]. In the present study we investigated the genotoxic activity of 18 synthetic derivatives of benzoxazinoids analyzed by use of immunofluorescence (IF) in cultured HeLa cells. 2. Material and methods 2.1. Chemicals and compounds The chemical structures of the synthetic benzoxazinones related to DIMBOA used in this study are shown in Table 1; these materials were prepared as described in previous reports [11–13]. Analysis by HPLC showed a purity of >98% for all com- pounds tested. In addition, a lactam compound derived from the chemical structure of BAs was assayed. Dimethyl sulfoxide (DMSO) was obtained from Sigma (St. Louis, MO, USA) and bisbenzimidine H33342 fluorochrome (Hoechst) was obtained from Calbiochem. Dulbecco’s minimal essential medium (DMEM), trypsin, antibiotics, fungizone and the labelling kit for FISH were purchased from Invitrogen. Avidin- FITC and Vectashield antifade were obtained from Vector laboratories (Burlingame, USA). 1383-5718/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2009.12.006