Toxicology Letters 227 (2014) 139–149 Contents lists available at ScienceDirect Toxicology Letters j our na l ho me page: www.elsevier.com/locate/toxlet Basic Red 51, a permitted semi-permanent hair dye, is cytotoxic to human skin cells: Studies in monolayer and 3D skin model using human keratinocytes (HaCaT) Thalita B. Zanoni a,∗ , Manoela Tiago b , Fernanda Faião-Flores b , Silvia B. de Moraes Barros b , Aalt Bast c , Geja Hageman c , Danielle Palma de Oliveira a , Silvya S. Maria-Engler b a Department of Environmental Toxicology, School of Pharmaceutical Sciences, University of São Paulo, Ribeirao Preto, Brazil b Department of Clinical Chemistry & Toxicology, School of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil c Toxicology, Research Institute NUTRIM, Maastricht University, Maastricht, Netherlands h i g h l i g h t s • Basic Red 51 produced cytotoxic effects on HaCaT cells and 3D culture. • Oxidative stress is involved in Basic Red 51 toxicity. • Apoptosis and cell cycle were observed in human keratinocytes (HaCaT). • Tested doses are below the recommendation of consumers’ safety. a r t i c l e i n f o Article history: Received 7 October 2013 Received in revised form 10 March 2014 Accepted 12 March 2014 Available online 20 March 2014 Keywords: Temporary hair dye Basic Red 51 3D organotipic culture HaCaT a b s t r a c t The use of hair dyes is closely associated with the increase of cancer, inflammation and other skin disor- ders. The recognition that human skin is not an impermeable barrier indicates that there is the possibility of human systemic exposure. The carcinogenic potential of hair dye ingredients has attracted the atten- tion of toxicologists for many decades, mainly due to the fact that some ingredients belong to the large chemical family of aromatic amines. Herein, we investigated the cytotoxicity of Basic Red 51 (BR51) in immortalized human keratinocytes (HaCaT). BR51 is a temporary hair dye that belongs to the azo group (N N); the cleavage of this bond may result in the release of toxic aromatic amines. The half maximal effective concentration (EC 50 ) in HaCaT cells is 13 g/mL. BR51 induced a significant decrease on expres- sion of p21 in a dose dependent manner. p53 was not affected, whereas BR51 decreased procaspase 8 and cleaved procaspase 9. These results proved that caspase 3 is fully involved in BR51-induced apopto- sis. The dye was also able to stop this cell cycle on G2 in sub-toxic doses. Moreover, we reconstructed a 3D artificial epidermis using HaCaT cells; using this model, we observed that BR51 induced cell injury and cells were undergoing apoptosis, considering the fragmented nuclei. Subsequently, BR51 induced reactive oxygen species (ROS) leading to an increase on the levels of 8-oxo-dG. In conclusion, we provide strong evidence that consumer and/or professional exposure to BR51 poses risk to human health. © 2014 Elsevier Ireland Ltd. All rights reserved. 1. Introduction For many decades scientists have been investigating the car- cinogenic and mutagenic potential of hair dyes, since they are manufactured using aromatic amines. In the past, Ames et al. (1975) related 90% of ingredients used for hair dyes as muta- genic compounds. Several epidemiological studies regarding the ∗ Corresponding author. Tel.: +55 16 33019500; fax: +55 16 33016692. E-mail address: thalitaz@fcfrp.usp.br (T.B. Zanoni). association of cancer and hair dye exposure have been done, with variable results (Bolt and Golka, 2007; Czene et al., 2003; Golka et al., 2008; Nohynek et al., 2004). On the basis of mech- anistic studies, some ingredients used on permanent hair dyes such as p-phenylenediamine (PPD), toluene-2,5-diamine (PTD) and resorcinol were identified as potential skin sensitizers and induc- ers of contact allergy in animals and humans (Seo et al., 2012; Søsted et al., 2004; Goebel et al., 2012). Regarding toxicity of per- manent hair dyes, recently, other key ingredients used for hair dyes, like H 2 O 2 and monoethanolamine (MEA) were also con- sidered responsible for causing contact dermatitis and hair loss http://dx.doi.org/10.1016/j.toxlet.2014.03.007 0378-4274/© 2014 Elsevier Ireland Ltd. All rights reserved.