Accepted Manuscript Title: Investigation on cellular interactions of astrocytes with Zinc oxide nanoparticles using rat C6 cell lines Author: S. Sruthi P.V. Mohanan PII: DOI: Reference:
S0927-7765(15)00354-9 http://dx.doi.org/doi:10.1016/j.colsurfb.2015.05.041 COLSUB 7114
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
22-1-2015 20-5-2015 22-5-2015
Please cite this article as: S. Sruthi, P.V. Mohanan, Investigation on cellular interactions of astrocytes with Zinc oxide nanoparticles using rat C6 cell lines, Colloids and Surfaces B: Biointerfaces (2015), http://dx.doi.org/10.1016/j.colsurfb.2015.05.041 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Investigation on cellular interactions of astrocytes with Zinc oxide nanoparticles using rat C6 cell lines Sruthi S, Mohanan P.V.*
ip t
Toxicology Division, Biomedical Technology Wing Sree Chitra Tirunal Institute for Medical Sciences and Technology,
* Corresponding Author: Dr. PV. Mohanan
an
Email: [email protected] or [email protected]
us
cr
Thiruvananthapuram 695 012, Kerala, India
M
Abstract
Zinc oxide nanoparticles (ZnO NPs) are widely used in cosmetic industries and have also found important applications in electrical and chemical industries. It is well documented that inhaled ZnO NPs can reach the brain
d
through the olfactory neuronal pathway and can interfere with the brain zinc homeostasis. Most of the studies focus
te
on the toxicity of ZnO NPs on neuronal cells and microglia. Not much work is available on the bio interaction of
Ac ce p
ZnO NPs with astrocytes, the major cells involved in brain homeostasis. Therefore, this study focuses on the interaction of ZnO NPs with rat C6 glial cells. The results of this study reveal that the nanoparticles are taken up by the astrocytes and induce a time and dose dependent toxicological response which is indicated by the nanoparticle uptake studies and cell viability assays. Also the results of DCFH-DA (2’7’-dichlorofluorescien diacetate) assay show that the ZnO NPs induce strong oxidative stress in cells at 3 and 6h. However, at 24h the reactive oxygen species (ROS) detected in the nanoparticles treated groups were same as that of control. The mode of cell death induced by ZnO NPs was apoptosis as revealed by the nuclear condensation studies, live dead assay using Acridine orange/ ethidium bromide and apoptosis detection kit. This study, which explores the interaction of ZnO NPs with astrocytes, concludes that the persistence of particle can continue to have a damaging effect on the astrocytes. And hence the time of exposure and particle clearance by the system should be evaluated more thoroughly to prevent the health hazards due to these particles. Key words: ZnO NPs, astrocytes, neurotoxicity, particle uptake, oxidative stress, apoptosis
1 Page 1 of 31
Introduction The era of nanotechnology that we live in has influenced our lives in a broad manner. Nanomaterials are used in a wide variety of products ranging from sophisticated machines and sensors to cosmetics and daily care products. In
ip t
cosmetic industry ZnO NPs are used as a sunscreen agent. They are used in some toothpaste and wound dressing because of their antibacterial properties and are being investigated as an antimicrobial agent in a variety of
cr
applications [1-3]. Research is undergoing world wide for the exploitation of ZnO NPs as a tool for biomedical application and therapeutic intervention owing to their high stability, inherent photoluminescence and wide band-
us
gap semiconductor properties. Many studies project ZnO NPs as potential agents for various biomedical applications like bio-sensing [4], cancer therapy, [5,6] gene targeting [7,8] and drug delivery [9,10]. Also, there are studies
an
stating the use of ZnO NPs in cell imaging [11], in diabetes treatment as dietary modulators, as cholesterol
M
biosensors [12] and as anti-inflammatory agents [13].
ZnO NPs in bulk formulation is approved as non-toxic by FDA. Nevertheless, with advancement in nanotechnology, these bulk particles are being replaced by nanoformulations. In many cases nanoparticles are incorporated into
d
various products without proper investigation on their biocompatibility and cytotoxicity. A growing number of
te
evidences suggest that the particle in nanoformulations differs from their parental particle in their physical and
Ac ce p
chemical properties [14, 15]. Increased surface area when compared to the bulk particle of same mass makes them highly reactive. Hence the particle in its nano form can interact with the macromolecules like DNA and proteins of the biological system in many unexpected ways and can accumulate at unanticipated sites [16]. With increase in use of ZnO NPs, the chances of both occupational and consumer exposure to these nanoparticles are increasing and this has raised public concern regarding the adverse health effects. A number of studies are available demonstrating the toxicological effect of ZnO NPs on a wide variety of organisms, some of which include macroalgae [17] and [18], bacteria [19], yeast [20], protozoa [21], zebrafish [22] and mice [23]. In vitro studies on human and mouse cancerous and normal cell lines reports that the response of different cell lines varies with the molecular and genetic composition of the cells and particle size [24-26]. Despite the extensive studies conducted on ZnO NPs, the exact mechanism of its toxicity is not yet clear. Some studies attribute them to the ability of ZnO NPs to generate reactive oxygen species [27-31] whereas others to the particle dissolution into free Zn2+ ions [32-33].
2 Page 2 of 31
Bio-distribution studies do not provide evidence for ZnO NPs reaching brain. However, along with the finding that other nanoparticles like carbonaceous and MnO2 particle reaches brain following nasal exposure [34-37], researchers has demonstrated the entry of ZnO NPs into the brain through olfactory neuronal pathway [38-39]. Also, there are studies suggesting disruption of mineral homeostasis by accumulated ZnO NPs in the brain [40]. In vitro
ip t
studies focus mainly on the interaction with neuronal cells [41-42]. However, the effect of ZnO NPs on the other major cells of the brain, including astrocytes, microglia and oligodentrocytes needs to be explored. Astrocytes being
cr
the principle cells involved in brain homeostasis have a key role in balancing the zinc ion concentration in the brain
us
and in maintaining blood brain barrier [43] and hence demands the need for understanding the biology of ZnO nanoparticle astrocyte interaction. C6 glial cells, a cell line of astrocyte origin forms an ideal invitro cell model for
an
ellucidating ZnO NP interaction with astrocytes. Hence the present study investigates the bio-interaction of astrocytes with ZnO NPs using rat C6 cell lines.
M
Materials and Methods Chemicals
d
Phosphate buffered saline (Ca2+, Mg2+ free; PBS), Dulbecco’s modified eagles medium F12 (DMEM), minimal
te
essential medium (MEM), fetal bovine serum (FBS), trypsin EDTA, GlutaMAX™, antibiotic and antimycotic
Ac ce p
solution (10,000 units/mL of penicillin, 10,000 µg/mL of streptomycin and 25 µg/mL of Fungizone® Antimycotic) were purchased from Gibco (Grand Island NY, USA). Sterile plastics for tissue culture were obtained from Corning (Corning, NY, USA). Sodium pyruvate, sodium bicarbonate, Ethidium bromide, 3-(4,5-Dimethylthiazol-2-yl)-2,5Diphenyltetrazolium Bromide (MTT), Neutral red dye and trypan blue were purchased from Sigma Chemicals Co. Ltd. (St. Louis, MO USA). 2’7’-dichlorofluorescien diacetate (DCFH-DA) and Rhodamine Phalloidin were purchased from Molecular probes, Invitrogen (Carlsbad, CA, USA). Giemsa stain, acridine orange (AO), 4, 6Diamidino-2-Phenylindole (DAPI) were purchased from Hi-media Pvt. Ltd. (Mumbai, India). All other chemical were obtained from Sigma Chemicals Co. Ltd. (St. Louis, MO USA).
Particle synthesis and characterization of ZnO NPs In house synthesized ZnO NPs (wet precipitation method) were used for entire study. The Detailed characterization of the ZnO NPs was reported by our own group (Syama et al. 2014) [44]. In brief the particles were synthesized
3 Page 3 of 31
using zinc nitrate and sodium hydroxide. The synthesized particles were characterized by transmission electron microscopy (TEM), X-ray diffractometer (XRD). Purity of the ZnO NPs was assessed by Fourier transform infrared spectroscopy (FTIR). The size of the ZnO NPs used for the study was
S0927-7765(15)00354-9 http://dx.doi.org/doi:10.1016/j.colsurfb.2015.05.041 COLSUB 7114
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
22-1-2015 20-5-2015 22-5-2015
Please cite this article as: S. Sruthi, P.V. Mohanan, Investigation on cellular interactions of astrocytes with Zinc oxide nanoparticles using rat C6 cell lines, Colloids and Surfaces B: Biointerfaces (2015), http://dx.doi.org/10.1016/j.colsurfb.2015.05.041 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Investigation on cellular interactions of astrocytes with Zinc oxide nanoparticles using rat C6 cell lines Sruthi S, Mohanan P.V.*
ip t
Toxicology Division, Biomedical Technology Wing Sree Chitra Tirunal Institute for Medical Sciences and Technology,
* Corresponding Author: Dr. PV. Mohanan
an
Email: [email protected] or [email protected]
us
cr
Thiruvananthapuram 695 012, Kerala, India
M
Abstract
Zinc oxide nanoparticles (ZnO NPs) are widely used in cosmetic industries and have also found important applications in electrical and chemical industries. It is well documented that inhaled ZnO NPs can reach the brain
d
through the olfactory neuronal pathway and can interfere with the brain zinc homeostasis. Most of the studies focus
te
on the toxicity of ZnO NPs on neuronal cells and microglia. Not much work is available on the bio interaction of
Ac ce p
ZnO NPs with astrocytes, the major cells involved in brain homeostasis. Therefore, this study focuses on the interaction of ZnO NPs with rat C6 glial cells. The results of this study reveal that the nanoparticles are taken up by the astrocytes and induce a time and dose dependent toxicological response which is indicated by the nanoparticle uptake studies and cell viability assays. Also the results of DCFH-DA (2’7’-dichlorofluorescien diacetate) assay show that the ZnO NPs induce strong oxidative stress in cells at 3 and 6h. However, at 24h the reactive oxygen species (ROS) detected in the nanoparticles treated groups were same as that of control. The mode of cell death induced by ZnO NPs was apoptosis as revealed by the nuclear condensation studies, live dead assay using Acridine orange/ ethidium bromide and apoptosis detection kit. This study, which explores the interaction of ZnO NPs with astrocytes, concludes that the persistence of particle can continue to have a damaging effect on the astrocytes. And hence the time of exposure and particle clearance by the system should be evaluated more thoroughly to prevent the health hazards due to these particles. Key words: ZnO NPs, astrocytes, neurotoxicity, particle uptake, oxidative stress, apoptosis
1 Page 1 of 31
Introduction The era of nanotechnology that we live in has influenced our lives in a broad manner. Nanomaterials are used in a wide variety of products ranging from sophisticated machines and sensors to cosmetics and daily care products. In
ip t
cosmetic industry ZnO NPs are used as a sunscreen agent. They are used in some toothpaste and wound dressing because of their antibacterial properties and are being investigated as an antimicrobial agent in a variety of
cr
applications [1-3]. Research is undergoing world wide for the exploitation of ZnO NPs as a tool for biomedical application and therapeutic intervention owing to their high stability, inherent photoluminescence and wide band-
us
gap semiconductor properties. Many studies project ZnO NPs as potential agents for various biomedical applications like bio-sensing [4], cancer therapy, [5,6] gene targeting [7,8] and drug delivery [9,10]. Also, there are studies
an
stating the use of ZnO NPs in cell imaging [11], in diabetes treatment as dietary modulators, as cholesterol
M
biosensors [12] and as anti-inflammatory agents [13].
ZnO NPs in bulk formulation is approved as non-toxic by FDA. Nevertheless, with advancement in nanotechnology, these bulk particles are being replaced by nanoformulations. In many cases nanoparticles are incorporated into
d
various products without proper investigation on their biocompatibility and cytotoxicity. A growing number of
te
evidences suggest that the particle in nanoformulations differs from their parental particle in their physical and
Ac ce p
chemical properties [14, 15]. Increased surface area when compared to the bulk particle of same mass makes them highly reactive. Hence the particle in its nano form can interact with the macromolecules like DNA and proteins of the biological system in many unexpected ways and can accumulate at unanticipated sites [16]. With increase in use of ZnO NPs, the chances of both occupational and consumer exposure to these nanoparticles are increasing and this has raised public concern regarding the adverse health effects. A number of studies are available demonstrating the toxicological effect of ZnO NPs on a wide variety of organisms, some of which include macroalgae [17] and [18], bacteria [19], yeast [20], protozoa [21], zebrafish [22] and mice [23]. In vitro studies on human and mouse cancerous and normal cell lines reports that the response of different cell lines varies with the molecular and genetic composition of the cells and particle size [24-26]. Despite the extensive studies conducted on ZnO NPs, the exact mechanism of its toxicity is not yet clear. Some studies attribute them to the ability of ZnO NPs to generate reactive oxygen species [27-31] whereas others to the particle dissolution into free Zn2+ ions [32-33].
2 Page 2 of 31
Bio-distribution studies do not provide evidence for ZnO NPs reaching brain. However, along with the finding that other nanoparticles like carbonaceous and MnO2 particle reaches brain following nasal exposure [34-37], researchers has demonstrated the entry of ZnO NPs into the brain through olfactory neuronal pathway [38-39]. Also, there are studies suggesting disruption of mineral homeostasis by accumulated ZnO NPs in the brain [40]. In vitro
ip t
studies focus mainly on the interaction with neuronal cells [41-42]. However, the effect of ZnO NPs on the other major cells of the brain, including astrocytes, microglia and oligodentrocytes needs to be explored. Astrocytes being
cr
the principle cells involved in brain homeostasis have a key role in balancing the zinc ion concentration in the brain
us
and in maintaining blood brain barrier [43] and hence demands the need for understanding the biology of ZnO nanoparticle astrocyte interaction. C6 glial cells, a cell line of astrocyte origin forms an ideal invitro cell model for
an
ellucidating ZnO NP interaction with astrocytes. Hence the present study investigates the bio-interaction of astrocytes with ZnO NPs using rat C6 cell lines.
M
Materials and Methods Chemicals
d
Phosphate buffered saline (Ca2+, Mg2+ free; PBS), Dulbecco’s modified eagles medium F12 (DMEM), minimal
te
essential medium (MEM), fetal bovine serum (FBS), trypsin EDTA, GlutaMAX™, antibiotic and antimycotic
Ac ce p
solution (10,000 units/mL of penicillin, 10,000 µg/mL of streptomycin and 25 µg/mL of Fungizone® Antimycotic) were purchased from Gibco (Grand Island NY, USA). Sterile plastics for tissue culture were obtained from Corning (Corning, NY, USA). Sodium pyruvate, sodium bicarbonate, Ethidium bromide, 3-(4,5-Dimethylthiazol-2-yl)-2,5Diphenyltetrazolium Bromide (MTT), Neutral red dye and trypan blue were purchased from Sigma Chemicals Co. Ltd. (St. Louis, MO USA). 2’7’-dichlorofluorescien diacetate (DCFH-DA) and Rhodamine Phalloidin were purchased from Molecular probes, Invitrogen (Carlsbad, CA, USA). Giemsa stain, acridine orange (AO), 4, 6Diamidino-2-Phenylindole (DAPI) were purchased from Hi-media Pvt. Ltd. (Mumbai, India). All other chemical were obtained from Sigma Chemicals Co. Ltd. (St. Louis, MO USA).
Particle synthesis and characterization of ZnO NPs In house synthesized ZnO NPs (wet precipitation method) were used for entire study. The Detailed characterization of the ZnO NPs was reported by our own group (Syama et al. 2014) [44]. In brief the particles were synthesized
3 Page 3 of 31
using zinc nitrate and sodium hydroxide. The synthesized particles were characterized by transmission electron microscopy (TEM), X-ray diffractometer (XRD). Purity of the ZnO NPs was assessed by Fourier transform infrared spectroscopy (FTIR). The size of the ZnO NPs used for the study was
