Mutation Research 758 (2013) 6–11 Contents lists available at ScienceDirect Mutation Research/Genetic Toxicology and Environmental Mutagenesis j o ur nal hom epa g e: www.elsevier.com/locate/gentox Co mm u nit y add ress: www.elsevier.com/locate/mutres Effect of blueberries (BB) on micronuclei induced by N-methyl-N ′ -nitro-N-nitrosoguanidine (MNNG) and 7,12-dimethylbenz(a)anthracene (DMBA) in mammalian cells, assessed in in vitro and in vivo assays Gaetano Pepe 1 , Maria Rosaria Grossi 1 , Andrea Berni 1 , Silvia Filippi, Rathina Kumar Shanmugakani, Cristiano Papeschi, Pasquale Mosesso, Adayapalam T. Natarajan, Fabrizio Palitti ∗ Department of Ecological and Biological Sciences, Università degli Studi della Tuscia, Largo dell’Università, snc, I-01100 Viterbo, Italy a r t i c l e i n f o Article history: Received 18 October 2012 Received in revised form 16 June 2013 Accepted 8 July 2013 Available online 20 September 2013 Keywords: Blueberries Micronucleus assay MNNG DMBA HepG2 Mice bone marrow a b s t r a c t The protective effect of blueberry (BB) on the clastogenic effects of MNNG and DMBA was evaluated with the induced micronucleus (MN) frequency as a biomarker, both in vitro and in vivo. Human hep- atoma HepG2 cells, which contain most of the metabolic activating enzymes was used for the in vitro test. MN frequencies were determined in binucleated cells generated by blocking cytokinesis by use of cytochalasin-B. The MN frequency in vivo was determined in polychromatic erythrocytes (PCEs) from the bone marrow of treated mice. BB by itself was not toxic both in vivo and in vitro. There was no evidence of a potential physico-chemical interaction between BB and the test carcinogens in vitro. Pre-treatment with BB reduced the MN frequency induced by MNNG. But, simultaneous treatment and post-treatment with BB did not affect the frequency of MNNG-induced MN. BB did not affect the frequency of DMBA- induced MN in vitro under any test condition. Under in vivo conditions, BB reduced the frequencies of MNNG- and DMBA-induced MN in PCEs, but in the case of the protective effect of BB against DMBA a dramatic reduction in the percentage of PCEs was observed, suggesting increased cytotoxicity. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Several studies have demonstrated that a diet rich in fruits and vegetables exerts protective effects against infectious diseases and age-related diseases such as neural degeneration, diabetes, and cancer [1–3]. One such dietary agent with a beneficial impact is the blueberry and its constituent phytochemicals. Blueberry is a species of the genus Vaccinium belonging to the family Ericaceae. Blueberries are sold fresh or processed as individually quick frozen (IQF) fruit, juice or dried or infused berries, which in turn may be used in a variety of consumer products such as jellies, jams, pies, muffins, snack foods and cereals. Blueberries have been shown to reduce the risk of artherosclerosis in apolipoprotein-E deficient mice, which are highly prone to oxidative stress and antioxidant deficiencies in situations of high blood cholesterol [4]. Blueberry extract was found to be effective in inhibiting proliferation of HL60 human leukemia and HCT116 human colon carcinoma cells ∗ Corresponding author. Tel.: +39 0761357206; fax: +39 0761357242. E-mail address: palitti@unitus.it (F. Palitti). 1 These authors contributed equally to this work. in vitro. Blueberry extracts and anthocyanins purified from these extracts have been shown to induce apoptosis in HL60 cells, sug- gesting a role of growth inhibitory and apoptosis-inducing effects of blueberry in cancer prevention [5]. Wild blueberry was found to decrease H 2 O 2 -induced DNA damage in Sprague-Dawley (SD) rats as evaluated in lymphocytes by means of the comet assay [6]. Extracts of berries of the Vaccinium species inhibit the induction of ornithine decarboxylase activity by the tumor promoter phorbol- 12-myristate-13-acetate (TPA) [7]. Blueberry extracts have been shown to inhibit the growth of HT29 cells in vitro even at a con- centration of 10 mg/ml whereas other berries like black currant, strawberry, raspberry and lingonberry show similar inhibitory effects only at higher concentrations (30, 40 and 60 mg/ml). Low bush “wild” blueberries (Vaccinium angustifolium) possess a high antioxidant potential due to their high concentration of polyphe- nolic anthocyanins [8–12]. This anthocyanin content gives the blueberry a high Trolox-equivalent antioxidant capacity (TEAC) of 27.60 M/g of fresh weight [13], however, these important mech- anisms of protection are strictly dependent on the bio-availability of the compounds. There is no significant difference between the transport and absorption efficiency of anthocyanins in blueberry extracts. It is well known that anthocyanins are rapidly absorbed, 1383-5718/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.mrgentox.2013.07.012