Cytotoxic effects of cadmium in mammary epithelial cells: Protective role of the macrocycle [15]pyN 5 Sandrina Gonçalves a,1 , Ana Sofia Fernandes a,b,1 , Nuno G. Oliveira a,⇑ , Joana Marques a , Judite Costa a , M. Fátima Cabral a , Joana Miranda a , Madalena Cipriano a , Patrícia S. Guerreiro a , Matilde Castro a a Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL), Faculty of Pharmacy, University of Lisbon, Av. Prof. Gama Pinto, 1649-003 Lisbon, Portugal b C BIOS, Universidade Lusófona, Campo Grande 376, 1749-024 Lisbon, Portugal article info Article history: Received 26 December 2011 Accepted 3 April 2012 Available online 16 April 2012 Keywords: Cadmium Breast Cytotoxicity Chelation therapy Macrocycle abstract Human exposure to cadmium (Cd) occurs via different routes, including diet. The increasing amount of data linking Cd with different cellular effects in the mammary gland justifies additional toxicological assessments using human mammary epithelial cells. This work aimed therefore to assess the cytotoxic effects of Cd in MCF10A cells and to characterize the cytoprotective role of the macrocycle [15]pyN 5 in the form of calcium salt. Cadmium chloride revealed to be cytotoxic to MCF10A cells, decreasing cell via- bility and proliferation in a concentration-dependent manner. Comparable dose–response curves and IC50 values (57–63 lM, 24 h treatment) were obtained using the MTT reduction, crystal violet and BrdU assays. In terms of reactive oxygen species formation, only a slight increase in superoxide radical anion was observed at very high Cd concentrations (P100 lM). Chelation should thus constitute the primary strategy to mitigate the cytotoxic effects induced by Cd in mammary cells. In this context, [15]pyN 5 which presents appropriate chemical and thermodynamic features was studied as a Cd chelator. This macrocycle (25 and 50 lM) significantly reduced or even abolished Cd-induced cytotoxicity. Protective effects were observed in terms of cell viability, cell proliferation and morphological alterations, being the protection mostly attributed to a chelating-based mechanism. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction Cadmium is one of the most toxic metals possessing several industrial applications including the manufacture of Ni-Cd batter- ies, pigments, coatings, and plastics stabilizer (NTP, 2011). This highly persistent and bio-accumulating element is also present in the tobacco smoke. Foodstuffs are also a very important source of Cd, being considered the main source of Cd exposure for the non-smoking general population (EFSA, 2009). The average cad- mium intake from food generally varies between 8 and 25 lg per day (Jarup and Akesson, 2009). Cd is present in a wide variety of foods and high levels of Cd can be found in offal products such as liver and kidney, species of shellfish, like oysters and other bi- valve mollusks, crabs and cephalopods (Olsson et al., 2002; EFSA, 2009; Jarup and Akesson, 2009). Cereals such as rice and wheat, green leafy vegetables, potato and root vegetables may also con- tain significant amounts of Cd (Olsson et al., 2002). Cd has been an issue of concern for the scientific community for many years (Nordberg, 2009), inducing several acute and chronic deleterious effects, including cancer (IARC, 1993). The Agency for Toxic Substances and Disease Registry (ATSDR, 2008) ranked cadmium in the 7th position in the Priority List of Hazardous Sub- stances highlighting the importance of cadmium for public health. Cd has been found in considerable amounts in breast tissue of women from different regions (Antila et al., 1996; Strumylaite et al., 2011). This metal has been quantified in human milk usually in small amounts (Honda et al., 2003; Kippler et al., 2009; Orun et al., 2011), being these levels lower than the ones found in breast tissue, suggesting a possible bioaccumulation of Cd in the mam- mary gland (Antila et al., 1996; Petersson and Oskarsson, 2000). This increased Cd accumulation in breast tissue has also been ob- served during lactation in rodents pointing out that the mammary gland may act as a protective barrier, similarly to the placenta (Petersson and Oskarsson, 2000). In rodent models, Cd induced involution-like histological alterations in the mammary gland, which were associated with the disturbance of the regulation of cellular calcium in the lactating mammary epithelium (Ohrvik et al., 2006, 2011). Cd binds to the human estrogen receptor (ER) and different studies suggested that Cd is an endocrine disruptor, acting as a possible metalloestrogen (Garcia-Morales et al., 1994; Johnson et al., 2003). In addition, epidemiological studies have suggested 0278-6915/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fct.2012.04.006 ⇑ Corresponding author. Tel.: +351 217946400. E-mail address: ngoliveira@ff.ul.pt (N.G. Oliveira). 1 Both authors equally contributed Food and Chemical Toxicology 50 (2012) 2180–2187 Contents lists available at SciVerse ScienceDirect Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox