Nrf2 Protects the Lung Against Inflammation Induced by Titanium Dioxide Nanoparticles: A Positive Regulator Role of Nrf2 on Cytokine Release Norma L. Delgado-Buenrostro,1 Estefany I. Medina-Reyes,1 Isabel Lastres-Becker,2 nica Freyre-Fonseca,1,3 Zhaoxia Ji,4 Rogelio Herna ndez-Pando,5 Brenda Marquina,5 Vero 6 2 Pedraza-Chaverri, Sandra Espada, Antonio Cuadrado,2 Yolanda I. Chirino1 Jose 1
xico, Unidad de Biomedicina, Facultad de Estudios Superiores Iztacala, UNAM, Estado de Me xico 54059, Me 2
dicas “Alberto Sols” Departamento de Bioquımica e Instituto de Investigaciones Biome noma de Madrid Consejo Superior de Investigaciones Cientıficas (CSIC), Universidad Auto ~ (UAM), Madrid, Espana 3
gicas, Instituto Polite cnico Nacional, Distrito Federal, CP Escuela Nacional de Ciencias Biolo xico 11340, Me
4
California NanoSystems Institute, University of California, Los Angeles, California 90095
5
Department of Pathology, Experimental Pathology Section, National Institute of Medical Science and Nutrition, Salvador Zubiran, Mexico City, Mexico
6
Departamento de Biologıa, Facultad de Quımica, Laboratorio 209, Edificio F, UNAM, Distrito xico Federal, 04510, Me
Received 22 November 2013; revised 8 January 2014; accepted 9 January 2014 ABSTRACT: Titanium dioxide nanoparticles (TiO2 NPs) have been classified as possibly carcinogenic to humans and they are an important nanomaterial widely used in pharmaceutical and paint industries. Inhalation is one of the most important routes of exposure in occupational settings. Several experimental models have shown that oxidative stress and inflammation are key mediators of cell damage. In this regard, Nrf2 modulates cytoprotection against oxidative stress and inflammation, however, its role in inflammation induced by TiO2 NPs exposure has been less investigated. The aim of this work was to investigate the role of Nrf2 in the cytokines produced after 4 weeks of TiO2 NPs exposure (5 mg/kg/2 days/week) using wild-type and Nrf2 knockout C57bl6 mice. Results showed that Nrf2 protects against
Additional Supporting Information may be found in the online version of this article. Correspondence to: Y. I. Chirino; e-mail: [email protected] Contract grant sponsor: CONACyT. Contract grant numbers: 166727 and 129838 Contract grant sponsor: DGAPA PAPIIT. Contract grant numbers: IB201112 and IN210713 Contract grant sponsor: Spanish Ministry of Economy and Competitiveness.
Contract grant number: SAF2010-17822 Contract grant sponsor: FES-Iztacala. Contract grant number: Project 28 Contract grant sponsor: CONACyT and Escuela Nacional de Ciencias Biol ogicas at Instituto Politecnico Nacional (Doctorado en Ciencias en Alimentos) (to V.F.-F.). Contract grant number: 202805 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/tox.21957
C 2014 Wiley Periodicals, Inc. V
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DELGADO-BUENROSTRO ET AL.
inflammation and oxidative damage induced by TiO2 NPs exposure, however, Nrf2 is a positive mediator in the expression of IFN-g, TNF-a, and TGF-b in bronchial epithelium and alveolar space after 4 weeks of exposure. These results suggest that Nrf2 has a central role in up-regulation of cytokines released during inflammation induced by TiO2 NPs and those cytokines are needed to cope with histological alterations in C 2014 Wiley Periodicals, Inc. Environ Toxicol 00: 000–000, 2014. lung tissue. V Keywords: titanium dioxide nanoparticles; Nrf2; lung inflammation
INTRODUCTION Occupational exposure, mostly by inhalation to titanium dioxide nanoparticles (TiO2 NPs) occurs during their manufacturing and during their industrial use in pigments for paints, varnishes, enamels, lacquers, and paper coatings to impart whiteness, opacity, and brightness (Hext et al., 2005). The potential hazard of these NPs, which is part of the engineered nanomaterials (ENM), has led to its classification as "possibly carcinogenic to humans in Group 2B” by the International Agency for Research in Cancer (IARC) based on the fact that there is sufficient evidence of carcinogenicity in experimental animals but inadequate evidence for human carcinogenicity (Baan, 2007), and also is classified as “potential occupational carcinogen” by the National Institute for Occupational Safety and Health (NIOSH). Effects of long-term exposure of TiO2 NPs have been less explored than short-term outcome, which has been associated with acute inflammation (Grassian et al., 2007; Bonner et al., 2013). In this regard, inflammatory response has been characterized by an increase of alveolar macrophages, neutrophils and lymphocytes in bronchoalveolar lavage after TiO2 NPs inhalation in experimental animals (Grassian et al., 2007). Inflammation induced by TiO2 NPs can be resolved after 7 days of a single oropharyngeal aspiration in mice (Bonner et al., 2013). However, short-term exposure should not be underestimated as intranasal TiO2 NPs exposure not only induces inflammation but also inhibits lung development in neonatal mice (Ambalavanan et al., 2013). Inflammation has been assessed by the recovered cells from bronchoalveolar lavage fluid composed by macrophages, lymphocytes and eosinophils at short-term (Bonner et al., 2013) but also unremitting inflammation has been observed at long-term exposure (Hwang et al., 2010; Cui et al., 2011; Winter et al., 2011; Sun et al., 2012a). In this regard, longterm exposure is associated with up-regulation of heme oxygenase 1 (HO-1) expression, decrease of glutathione content and increase of reactive oxygen species (ROS) of 90-dayTiO2 NPs exposed mice (Hwang et al., 2010; Cui et al., 2011; Winter et al., 2011; Sun et al., 2012a). Those alterations can be modulated by the transcription factor nuclear factor erythroid-derived 2 (Nrf2), which is considered a master regulator of redox homeostasis and play a central role in the antioxidant and anti-inflammatory defense (Iizuka et al., 2005). Indeed, administration of 10 mg/kg TiO2 NPs caused an increase in Nrf2 expression in lung mice (Sun et al., 2012a). However, the relationship between Nrf2 expression
Environmental Toxicology DOI 10.1002/tox
and the cytokine profile produced by TiO2 NPs in the lung has not been described, especially under sub-chronic conditions. Thus, the aim of this study was to determine the role of Nrf2 in the cytokine pattern of TNF-a, IFN-g, and TGFb, induced by TiO2 NPs exposure in lung mice.
METHODS TiO2 NPs Characterization TiO2 NPs (99.7%,
xico, Unidad de Biomedicina, Facultad de Estudios Superiores Iztacala, UNAM, Estado de Me xico 54059, Me 2
dicas “Alberto Sols” Departamento de Bioquımica e Instituto de Investigaciones Biome noma de Madrid Consejo Superior de Investigaciones Cientıficas (CSIC), Universidad Auto ~ (UAM), Madrid, Espana 3
gicas, Instituto Polite cnico Nacional, Distrito Federal, CP Escuela Nacional de Ciencias Biolo xico 11340, Me
4
California NanoSystems Institute, University of California, Los Angeles, California 90095
5
Department of Pathology, Experimental Pathology Section, National Institute of Medical Science and Nutrition, Salvador Zubiran, Mexico City, Mexico
6
Departamento de Biologıa, Facultad de Quımica, Laboratorio 209, Edificio F, UNAM, Distrito xico Federal, 04510, Me
Received 22 November 2013; revised 8 January 2014; accepted 9 January 2014 ABSTRACT: Titanium dioxide nanoparticles (TiO2 NPs) have been classified as possibly carcinogenic to humans and they are an important nanomaterial widely used in pharmaceutical and paint industries. Inhalation is one of the most important routes of exposure in occupational settings. Several experimental models have shown that oxidative stress and inflammation are key mediators of cell damage. In this regard, Nrf2 modulates cytoprotection against oxidative stress and inflammation, however, its role in inflammation induced by TiO2 NPs exposure has been less investigated. The aim of this work was to investigate the role of Nrf2 in the cytokines produced after 4 weeks of TiO2 NPs exposure (5 mg/kg/2 days/week) using wild-type and Nrf2 knockout C57bl6 mice. Results showed that Nrf2 protects against
Additional Supporting Information may be found in the online version of this article. Correspondence to: Y. I. Chirino; e-mail: [email protected] Contract grant sponsor: CONACyT. Contract grant numbers: 166727 and 129838 Contract grant sponsor: DGAPA PAPIIT. Contract grant numbers: IB201112 and IN210713 Contract grant sponsor: Spanish Ministry of Economy and Competitiveness.
Contract grant number: SAF2010-17822 Contract grant sponsor: FES-Iztacala. Contract grant number: Project 28 Contract grant sponsor: CONACyT and Escuela Nacional de Ciencias Biol ogicas at Instituto Politecnico Nacional (Doctorado en Ciencias en Alimentos) (to V.F.-F.). Contract grant number: 202805 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/tox.21957
C 2014 Wiley Periodicals, Inc. V
1
2
DELGADO-BUENROSTRO ET AL.
inflammation and oxidative damage induced by TiO2 NPs exposure, however, Nrf2 is a positive mediator in the expression of IFN-g, TNF-a, and TGF-b in bronchial epithelium and alveolar space after 4 weeks of exposure. These results suggest that Nrf2 has a central role in up-regulation of cytokines released during inflammation induced by TiO2 NPs and those cytokines are needed to cope with histological alterations in C 2014 Wiley Periodicals, Inc. Environ Toxicol 00: 000–000, 2014. lung tissue. V Keywords: titanium dioxide nanoparticles; Nrf2; lung inflammation
INTRODUCTION Occupational exposure, mostly by inhalation to titanium dioxide nanoparticles (TiO2 NPs) occurs during their manufacturing and during their industrial use in pigments for paints, varnishes, enamels, lacquers, and paper coatings to impart whiteness, opacity, and brightness (Hext et al., 2005). The potential hazard of these NPs, which is part of the engineered nanomaterials (ENM), has led to its classification as "possibly carcinogenic to humans in Group 2B” by the International Agency for Research in Cancer (IARC) based on the fact that there is sufficient evidence of carcinogenicity in experimental animals but inadequate evidence for human carcinogenicity (Baan, 2007), and also is classified as “potential occupational carcinogen” by the National Institute for Occupational Safety and Health (NIOSH). Effects of long-term exposure of TiO2 NPs have been less explored than short-term outcome, which has been associated with acute inflammation (Grassian et al., 2007; Bonner et al., 2013). In this regard, inflammatory response has been characterized by an increase of alveolar macrophages, neutrophils and lymphocytes in bronchoalveolar lavage after TiO2 NPs inhalation in experimental animals (Grassian et al., 2007). Inflammation induced by TiO2 NPs can be resolved after 7 days of a single oropharyngeal aspiration in mice (Bonner et al., 2013). However, short-term exposure should not be underestimated as intranasal TiO2 NPs exposure not only induces inflammation but also inhibits lung development in neonatal mice (Ambalavanan et al., 2013). Inflammation has been assessed by the recovered cells from bronchoalveolar lavage fluid composed by macrophages, lymphocytes and eosinophils at short-term (Bonner et al., 2013) but also unremitting inflammation has been observed at long-term exposure (Hwang et al., 2010; Cui et al., 2011; Winter et al., 2011; Sun et al., 2012a). In this regard, longterm exposure is associated with up-regulation of heme oxygenase 1 (HO-1) expression, decrease of glutathione content and increase of reactive oxygen species (ROS) of 90-dayTiO2 NPs exposed mice (Hwang et al., 2010; Cui et al., 2011; Winter et al., 2011; Sun et al., 2012a). Those alterations can be modulated by the transcription factor nuclear factor erythroid-derived 2 (Nrf2), which is considered a master regulator of redox homeostasis and play a central role in the antioxidant and anti-inflammatory defense (Iizuka et al., 2005). Indeed, administration of 10 mg/kg TiO2 NPs caused an increase in Nrf2 expression in lung mice (Sun et al., 2012a). However, the relationship between Nrf2 expression
Environmental Toxicology DOI 10.1002/tox
and the cytokine profile produced by TiO2 NPs in the lung has not been described, especially under sub-chronic conditions. Thus, the aim of this study was to determine the role of Nrf2 in the cytokine pattern of TNF-a, IFN-g, and TGFb, induced by TiO2 NPs exposure in lung mice.
METHODS TiO2 NPs Characterization TiO2 NPs (99.7%,
