6-Hydroxydopamine and lipopolysaccharides induced DNA damage in astrocytes: Involvement of nitric oxide and mitochondria

https://doi.org/10.1016/j.mrgentox.2014.12.007Get rights and content

Highlights

  • 6-OHDA/LPS caused increased expression of GFAP and DNA damage in astrocytes.

  • 6-OHDA/LPS treatment caused increased expression of iNOS and nitrite level.

  • iNOS inhibitor, aminoguanidine attenuates the DNA damage in astrocytes.

  • 6-OHDA/LPS caused mitochondrial impairment and apoptosis in astrocytes.

  • Nitric oxide has emerged as key player in DNA damage in astrocytes.

Abstract

The present study was conducted to investigate the effect of the neurotoxins 6-hydroxydopamine and lipopolysaccharide on astrocytes. Rat astrocyte C6 cells were treated with different concentration of 6-hydroxydopamine (6-OHDA)/lipopolysaccharides (LPS) for 24 h. Both neurotoxins significantly decreased the viability of astrocytes, augmented the expression of inducible nitric oxide synthase (iNOS) and the astrocyte marker – glial fibrillar acidic protein. A significantly decreased mitochondrial dehydrogenase activity, mitochondrial membrane potential, augmented reactive oxygen species (ROS) level, caspase-3 mRNA level, chromatin condensation and DNA damage was observed in 6-OHDA/LPS treated astroglial cells. 6-OHDA/LPS treatment also caused the significantly increased expression of iNOS and nitrite level. Findings showed that 6-OHDA/LPS treatment caused mitochondrial dysfunction mediated death of astrocytes, which significantly involve the nitric oxide. Since we have observed significantly increased level of iNOS along with mitochondrial impairment and apoptotic cell death in astrocytes, therefore to validate the role of iNOS, the cells were co-treated with iNOS inhibitor aminoguanidine (AG, 100 μM). Co-treatment of AG significantly attenuated the 6-OHDA/LPS induced cell death, mitochondrial activity, augmented ROS level, chromatin condensation and DNA damage. GFAP and caspase-3 expression were also inhibited with co-treatment of AG, although the extent of inhibition was different in both experimental sets. In conclusion, the findings showed that iNOS mediated increased level of nitric oxide acts as a key regulatory molecule in 6-OHDA/LPS induced mitochondrial dysfunction, DNA damage and apoptotic death of astrocytes.

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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