Mutation Research/Genetic Toxicology and Environmental Mutagenesis
6-Hydroxydopamine and lipopolysaccharides induced DNA damage in astrocytes: Involvement of nitric oxide and mitochondria
Introduction
The neurotoxic effects of the nitric oxide (NO) are widespread [1], [2] as reported in stroke, brain trauma, brain infections, brain aging and various neurodegenerative diseases [3], [4]. It is a well-known inhibitor of the mitochondrial electron transport system [5], causes depleted level of mitochondrial adenosine triphosphate due to the loss of mitochondrial respiration and disrupts membrane potential across the mitochondrial inner membrane [6]. NO mediated mitochondrial membrane collapse is associated with mitochondrial swelling, disruption of the outer mitochondrial membrane, release of proapoptotic factors and consequent apoptotic death [7], [8]. Direct links between impaired mitochondrial electron transport and apoptosis have also been reported [9].
In response to neuronal damage the astrocytes can enter a state called reactive astrogliosis in brain which is characterized by hypertrophy and increased expression of glial fibrillary acidic protein (GFAP). The astrocytes constitute the major population of non-neuronal cells, play an important role in neuronal survival and transmission and actively contribute in mediating the physiologic and pathologic effects on neurons [10], [11]. Initially activated astrocytes exert neuroprotective effects but their over activation lead to both beneficial and detrimental effects on surrounding neurons [12], [13]. Their role in disease initiation and progression has also been reported [13]. The neuroprotective and neurodegenerative roles of astrocytes depend largely on the molecules that they release into and take up from the extracellular space. Astrocytes release neurotrophic factors along with various neurotoxic factors like proinflammatory cytokines and increased ROS generation [13], [14], [15], [16], [17]. Evidence of astrogliosis in animal models and human post mortem studies of neurological disorders has been reported and showed their association in neuronal death [18], [19], [20], [21].
6-OHDA and LPS are neurotoxic to dopaminergic neurons and have been used to induce experimental models of Parkinsonism. 6-OHDA causes increased oxidative and nitrosative stress in dopaminergic neurons of substantia nigra [22], [23] while LPS causes damage to these neurons via inflammatory reactions mediated from neurons themselves and glial cells [24], [25]. 6-OHDA/LPS injection in rat brain caused significantly increased expression of inducible nitric oxide synthase and mitochondria mediated apoptotic death of neurons within 72 h [26], [27]. However, the specific effect of neurotoxin on astrocyte viability is not well studied. Though under in vivo conditions the specific effects of neurotoxin on single population of astrocytes is not possible therefore, the present study was performed in astrocytes cell culture to investigate the effect of neurotoxins on astrocyte viability and involvement of NO in neurotoxins induced mitochondrial dysfunction and death of astrocytes.
Section snippets
Chemicals used
Agarose, BSA, calcium chloride (CaCl2), copper sulphate (CuSO4·5H2O), dichlorofluorescein diacetate (DCF-DA), DMEM culture medium (GIBCO BRL, NY), DMSO (Dimethyl sulfoxide), ethidium bromide (EtBR), EDTA, fetal bovine serum (GIBCO BRL, NY), FK-12 culture medium (GIBCO BRL, NY), glucose, HEPES, magnesium chloride (MgCl2), MTT (3-(4,5-dimethylthiazolyl 2,5-diphenyltetrazolium bromide)), Disodium hydrogen phosphate (Na2HPO4), NADH, paraformaldehyde, penicillin and streptomycin antibiotics (GIBCO
Astrocytes viability and morphological alteration
Neurotoxins 6-OHDA/LPS treatment to cells caused significant altered cellular morphology as showed in Fig. 1a. Enzyme activity of lactate dehydrogenase was estimated in culture medium of control (untreated) and toxin treated cells. It is cytosolic enzyme and would be release in culture medium due to lysis of cells. Significant (p < 0.01, Fig. 1b) increase in LDH activity was found in culture medium of 6-OHDA/LPS treated cells in concentration dependant manner.
The LDH activity in culture medium of
Discussion
The present study demonstrated that 6-OHDA/LPS treatment exhibited significant DNA damage and apoptotic death of C6 cells in which NO emerged as key player. 6-OHDA/LPS treatment caused significant lysis of cells as reflected by release of cytosolic enzyme lactate dehydrogenase (LDH) in culture medium. Expression of nNOS was not observed in control or treated cells however iNOS expression was significantly augmented in neurotoxins treated cells in comparison to control cells. LPS induced iNOS
Acknowledgements
Authors wish to thank Dr. A. K. Balapure, Head, Tissue and Cell Culture Unit, CDRI and his team for providing cells for experimentation. Poonam Goswami wishes to thanks Indian Council of Medical Research, India for the senior research fellowship.
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