In Vitro Cell.Dev.Biol.—Animal DOI 10.1007/s11626-014-9760-3
Single-walled carbon nanotubes induce cytotoxicity and DNA damage via reactive oxygen species in human hepatocarcinoma cells Saud Alarifi & Daoud Ali & Ankit Verma & Fahad N. Almajhdi & Ahmed A. Al-Qahtani
Received: 12 December 2013 / Accepted: 7 April 2014 / Editor: T. Okamoto # The Society for In Vitro Biology 2014
Abstract Carbon nanotubes (CNTs) are gradually used in various areas including drug delivery, nanomedicine, biosensors, and electronics. The current study aimed to explore the DNA damage and cytotoxicity due to single-walled carbon nanotubes (SWCNTs) on human hepatocarcinoma cells (HepG2). Cellular proliferative assay showed the SWCNTs to exhibit a significant cell death in a dose- and timedependent manner. However, SWCNTs induced significant intracellular reactive oxygen species (ROS) production and elevated lipid peroxidation, catalase, and superoxide dismutase in the HepG2 cells. SWCNTs also induced significant decrease in GSH and increase caspase-3 activity in HepG2 cells. DNA fragmentation analysis using the alkaline singlecell gel electrophoresis showed that the SWCNTs cause genotoxicity in a dose- and time-dependent manner. Therefore, the study points towards the capability of the SWCNTs to induce oxidative stress resulting cytotoxicity
Daoud Ali and Saud Alarifi are contributed equally to this work. S. Alarifi : D. Ali (*) Department of Zoology, College of Science, King Saud University, Box 2454, 11451 Riyadh, Saudi Arabia e-mail: [email protected] D. Ali e-mail: [email protected] A. Verma Ram Manohar Lohiya Institute of Medical Sciences, Lucknow, UP, India F. N. Almajhdi Department of Botany and Microbiology, College of Science, King Saud University, Riyadh, Saudi Arabia A. A. Al-Qahtani Department of Infection and Immunity, Research Center, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia
and genomic instability. This study warrants more careful assessment of SWCNTs before their industrial applications. Keywords SWCNTs . Human hepatocarcinoma cells . Oxidative stress . MTT assay . DNA damage
Introduction Carbon nanotubes (CNTs) are currently used in sunscreens, paints, cosmetics, electronics, and industrial lubricants. The low density and small size of carbon nanotubes make respiratory exposures likely, with the highest exposures expected to occur occupationally, either during production or their incorporation into several products. Single-walled carbon nanotubes (SWCNTs) produce reactive oxygen species (ROS) and oxidative stress in human keratinocyte cells (Shvedova et al. 2003). SWCNTs reveal the greater toxicity as compared with multiwalled carbon nanotubes and C60 fullerenes (Jia et al. 2005). The different types of nanoparticle have been shown to disrupt phagocytosis in mammalian macrophages including carbon nanotubes and aluminum nanoparticles (Jia et al. 2005). Despite the existing studies on the toxicity of SWCNTs, the underlying mechanism leading to toxicity remains unclear. Moreover, none of the studies, so far, have explored the adverse effects of SWCNTs in the human liver which is primary the organ of metabolism. The workers involved in the synthesis of SWCNTs may be exposed by unintentional hand to mouth transfer of nanomaterials. Yamago et al. (1995) reported that the major percentage of radiolabelled functionalized fullerenes were detected in the liver after administrated intravenously to rats. Their potential toxicological impacts are still a matter of investigation, and our actual knowledge on the effects of engineered nanosized contaminants on biological systems remains incomplete (Singh et al. 2009; Skocaj et al.
ALARIFI ET AL.
2011). Exposure of rats/mice to SWCNTs by inhalation or intratracheal instillation caused inflammation, fibrosis, granuloma formation, and immunosuppression (Mitchell et al. 2007). Some in vitro studies have demonstrated that both multiwalled carbon nanotubes and SWCNTs can induce oxidative stress, inflammatory cytokines, cytotoxic effects, apoptosis, and altered protein expression in various types of cell (Barillet et al. 2010). Free oxygen radical generation and oxidative stress elicit a wide variety of cellular events including DNA damage and apoptosis (Ostrovsky et al. 2009). The genotoxic potential of nanomaterial is of particular concern since the changes of the genetic material have potential for cell death, tissue malfunction, cancer development, and reproductive adverse effects. Oxidative stress has been implicated as an explanation behind the nanoparticles toxicity (Nel et al. 2006). Recent studies reported nanoparticle-induced oxidative stress as determined by increasing membrane lipid peroxidation, reactive oxygen species, and decreasing intracellular glutathione reduced (Wang et al. 2011). We evaluated the oxidative stress biomarkers including GSH as an antioxidant; ROS generation as a collective marker of superoxide anion (O2−), hydroxyl radi c a l ( H O ) , a nd h y d r o g en p e r o x i d e ( H 2 O 2 ) ; a n d malondialdehyde (MDA) as an end product of membrane LPO in response to SWCNTs exposure. Therefore, the current study was designed to assess the cellular toxicity and genotoxic potential of SWCNTs in human hepatocarcinoma cells as well as to understand its possible mechanism.
Materials and Methods Chemicals and reagents. Single-walled carbon nanotube (SWCNTs) (product no. 750522 and AD
Single-walled carbon nanotubes induce cytotoxicity and DNA damage via reactive oxygen species in human hepatocarcinoma cells Saud Alarifi & Daoud Ali & Ankit Verma & Fahad N. Almajhdi & Ahmed A. Al-Qahtani
Received: 12 December 2013 / Accepted: 7 April 2014 / Editor: T. Okamoto # The Society for In Vitro Biology 2014
Abstract Carbon nanotubes (CNTs) are gradually used in various areas including drug delivery, nanomedicine, biosensors, and electronics. The current study aimed to explore the DNA damage and cytotoxicity due to single-walled carbon nanotubes (SWCNTs) on human hepatocarcinoma cells (HepG2). Cellular proliferative assay showed the SWCNTs to exhibit a significant cell death in a dose- and timedependent manner. However, SWCNTs induced significant intracellular reactive oxygen species (ROS) production and elevated lipid peroxidation, catalase, and superoxide dismutase in the HepG2 cells. SWCNTs also induced significant decrease in GSH and increase caspase-3 activity in HepG2 cells. DNA fragmentation analysis using the alkaline singlecell gel electrophoresis showed that the SWCNTs cause genotoxicity in a dose- and time-dependent manner. Therefore, the study points towards the capability of the SWCNTs to induce oxidative stress resulting cytotoxicity
Daoud Ali and Saud Alarifi are contributed equally to this work. S. Alarifi : D. Ali (*) Department of Zoology, College of Science, King Saud University, Box 2454, 11451 Riyadh, Saudi Arabia e-mail: [email protected] D. Ali e-mail: [email protected] A. Verma Ram Manohar Lohiya Institute of Medical Sciences, Lucknow, UP, India F. N. Almajhdi Department of Botany and Microbiology, College of Science, King Saud University, Riyadh, Saudi Arabia A. A. Al-Qahtani Department of Infection and Immunity, Research Center, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia
and genomic instability. This study warrants more careful assessment of SWCNTs before their industrial applications. Keywords SWCNTs . Human hepatocarcinoma cells . Oxidative stress . MTT assay . DNA damage
Introduction Carbon nanotubes (CNTs) are currently used in sunscreens, paints, cosmetics, electronics, and industrial lubricants. The low density and small size of carbon nanotubes make respiratory exposures likely, with the highest exposures expected to occur occupationally, either during production or their incorporation into several products. Single-walled carbon nanotubes (SWCNTs) produce reactive oxygen species (ROS) and oxidative stress in human keratinocyte cells (Shvedova et al. 2003). SWCNTs reveal the greater toxicity as compared with multiwalled carbon nanotubes and C60 fullerenes (Jia et al. 2005). The different types of nanoparticle have been shown to disrupt phagocytosis in mammalian macrophages including carbon nanotubes and aluminum nanoparticles (Jia et al. 2005). Despite the existing studies on the toxicity of SWCNTs, the underlying mechanism leading to toxicity remains unclear. Moreover, none of the studies, so far, have explored the adverse effects of SWCNTs in the human liver which is primary the organ of metabolism. The workers involved in the synthesis of SWCNTs may be exposed by unintentional hand to mouth transfer of nanomaterials. Yamago et al. (1995) reported that the major percentage of radiolabelled functionalized fullerenes were detected in the liver after administrated intravenously to rats. Their potential toxicological impacts are still a matter of investigation, and our actual knowledge on the effects of engineered nanosized contaminants on biological systems remains incomplete (Singh et al. 2009; Skocaj et al.
ALARIFI ET AL.
2011). Exposure of rats/mice to SWCNTs by inhalation or intratracheal instillation caused inflammation, fibrosis, granuloma formation, and immunosuppression (Mitchell et al. 2007). Some in vitro studies have demonstrated that both multiwalled carbon nanotubes and SWCNTs can induce oxidative stress, inflammatory cytokines, cytotoxic effects, apoptosis, and altered protein expression in various types of cell (Barillet et al. 2010). Free oxygen radical generation and oxidative stress elicit a wide variety of cellular events including DNA damage and apoptosis (Ostrovsky et al. 2009). The genotoxic potential of nanomaterial is of particular concern since the changes of the genetic material have potential for cell death, tissue malfunction, cancer development, and reproductive adverse effects. Oxidative stress has been implicated as an explanation behind the nanoparticles toxicity (Nel et al. 2006). Recent studies reported nanoparticle-induced oxidative stress as determined by increasing membrane lipid peroxidation, reactive oxygen species, and decreasing intracellular glutathione reduced (Wang et al. 2011). We evaluated the oxidative stress biomarkers including GSH as an antioxidant; ROS generation as a collective marker of superoxide anion (O2−), hydroxyl radi c a l ( H O ) , a nd h y d r o g en p e r o x i d e ( H 2 O 2 ) ; a n d malondialdehyde (MDA) as an end product of membrane LPO in response to SWCNTs exposure. Therefore, the current study was designed to assess the cellular toxicity and genotoxic potential of SWCNTs in human hepatocarcinoma cells as well as to understand its possible mechanism.
Materials and Methods Chemicals and reagents. Single-walled carbon nanotube (SWCNTs) (product no. 750522 and AD
