Protective potential of thymoquinone against peroxynitrite induced modifications in histone H2A: In vitro studies

doi:10.1016/j.ijbiomac.2018.01.157

International Journal of Biological Macromolecules

Volume 112, June 2018, Pages 169-174

https://doi.org/10.1016/j.ijbiomac.2018.01.157 Get rights and content

Abstract

Peroxynitrite (ONOO⁻) is a reactive oxidant involved in numerous pathological conditions. Thymoquinone (TQ) is an active constituent of Nigella sativa and is reported to have anti-disease activities, but its role on ONOO⁻ has never been investigated. This study was undertaken to investigate the role of TQ on ONOO⁻-induced damage of histone-H2A. Our novel data showed TQ significantly inhibited ONOO⁻-induced oxidative damage in histone-H2A. ONOO⁻ induces UV-hypochromicity of histone-H2A, whereas TQ reversed this effect to hyperchromicity. Tyrosine fluorescence was significantly reduced by ONOO⁻ and was significantly increased upon TQ treatment. TQ reduces ONOO⁻-induced hydrophobicity in histone-H2A and also reduces thermal stability of ONOO⁻-histone H2A complex. SDS-PAGE of native histone-H2A showed a single band, which disappeared when treated with ONOO⁻ alone. This changed was retained when protein samples were treated with TQ. Similar protective effects of TQ were found when protein carbonyl contents were estimated. In conclusion, this is the first study that shows the potential of TQ against ONOO⁻-induced damaged of histone-H2A. TQ inhibits oxidative modification of tyrosine, lysine, arginine, proline and threonine in histone-H2A. These results have importance for the development of novel therapeutic strategies for the treatment of disorders, where ONOO⁻ plays a role.

Introduction

Thymoquinone (TQ) is the most active and most abundant constituent of black seed oil (Nigella sativa) [1]. Several reports have shown that consumption of back seed has health benefits against many disorders including hypertension, headache, gastrointestinal problems, eczema, obesity, dysentery, bronchial asthma, etc. [2]. Studies undoubtedly suggested that major anti-disease activity of black seed is derived from TQ [[1], [2], [3]]. Now it is well documented that TQ plays an important roles in suppression of oxidative damage and also it ameliorates inflammation under several pathological conditions [[1], [2], [3], [4]]. Its anti-oxidant potential either directly or indirectly linked with its potential to alter “redox state” of biological systems and its inhibitory capacity against reactive oxygen species (ROS) through modulation of hepatic and extra hepatic antioxidant enzymatic system has been well defined [3,4]. Moreover, TQ is also known to suppress the cellular oxidative damage by modulating glutathione and other anti-oxidants [4]. Furthermore, TQ also provides kidney protection against toxicity induced by mercuric chloride, ifosfamide and cisplatin [3]. Not only have these, TQ also inhibits formation of lipid peroxide by reducing the malonaldehyde content in liver [3,4]. TQ also proved to be a powerful chemopreventive agent as it improves anti-oxidant enzymatic system and reduces the onset of hepatic lipid peroxides [[1], [2], [3], [4]]. Now it is well established that TQ has anti-cancer and anti-inflammatory effects as it slowdown proliferation of tumor cell via modulating of apoptotic/necrotic cell signaling events and to monitor angiogenesis [[1], [2], [3], [4]].

Peroxynitrite is a reactive oxygen and nitrogen species (RONS) with an anion formula ONOO⁻. It is highly unstable oxidizing/nitrating agent which is involved in a wide range of biomolecular damage in almost all biological systems [[5], [6], [7]]. In human, it is generated in response to endogenous/exogenous toxins, stress, ultraviolet light and many other stimuli under various pathological conditions [[5], [6], [7]]. It is now well known that most of the cytotoxic effects of nitric oxide are mainly due to peroxynitrite, which is more reactive and frequent to react with almost all biomolecules including proteins, lipids and nucleic acids [6]. Peroxynitrite not only causing oxidative biomolecular damage but also triggers number of cell signaling events which further inducing oxidative injury and committing cells to necrosis/apoptosis [[5], [6], [7]]. Importantly, peroxynitrite is also involved in the abnormal induction of many molecular pathways which directly associated with pathology of stroke, myocardial infarction, chronic heart failure, diabetes, inflammation, neurodegenerative disorders, cancer, etc. [[5], [6], [7]]. Not only have these, studies have also shown that peroxynitrite and others RONS are involved in the initiation and progression of various autoimmune responses [[8], [9], [10], [11]].

Histones are highly conserved cationic proteins which bind DNA and remain confined in the nucleus [12]. There are five families of histones H1/H5, H2A, H2B, H3, and H4. Histone H2A is an important constituent of histone core that has tripartite union with central (H3-H4)2 flanked by two H2A-H2B dimmers, making histone H2A more prone to oxidative damage [13]. Recently, we have demonstrated that oxidative modifications of histone H2A lead to the generation of neoepitopes for the induction of autoantibodies in patients with systemic lupus erythematosus (SLE) [14]. More importantly, several studies have shown deleterious effects of peroxynitrite on histone H2A, causing extensive structural modifications, which directly or indirectly associated with the pathophysiology of several autoimmune disorders [15]. As we know studies revealed that TQ has the anti-oxidant activity [[1], [2], [3], [4]], but the effects of TQ on peroxynitrite-induced oxidative modification of histone H2A have never been investigated. Here, we have addressed the question for the first time of a possible inhibitory effect of TQ on peroxynitirite-induced damage of histone H2A. Our novel data may have importance for the development of novel therapeutic strategies for the treatment of disorders, where peroxynitrite plays a role.

Section snippets

Treatment of histone H2A by thymoquinone and peroxynitrite

Histone H2A (catalog # H9250, Sigma-Aldrich, St Louis, MO, USA) was treated simultaneously with TQ (catalog # 274666, Sigma-Aldrich) and peroxynitrite anion (ONOO⁻) in phosphate buffer saline (10 mM sodium phosphate buffer containing 150 mM NaCl, pH 7.4) as described previously [9,16] with some modifications. Briefly, histone H2A (1 mg) was treated with TQ (0.1–3.0 μM), and a mixture of SNP (0.5 mM; catalog # 13451, Sigma-Aldrich), pyrogallol (0.5 mM; catalog # P0381, Sigma-Aldrich) and DTPA

Results

Histone H2A was treated with peroxynitrite or TQ and the induced changes were analyzed by UV absorption spectroscopy. The UV absorption spectra of peroxynitrite histone H2A revealed 43.5% hypochromicity at 280 nm (Fig. 1A&B). Importantly, addition of TQ (0.1–3.0 μM) in the reaction mixture significantly decreased the peroxynitrite-induced hypochromicity in a dose-dependent manner (p < 0.05; Fig. 1C,D&E). The protective effect of TQ against peroxynitrite induced damage of histone H2A has been

Discussion

This is the first study that shows the protective effect of thymoquinone against peroxynitrite induced oxidative damage of histone H2A. It is now well documented that most of the pathogenicity induce by nitric oxide is due to the generation of peroxynitrite, which interacts with almost all biomolecules including protein, nucleic acids, lipids, etc. and causing oxidative structural damage which further cause induction of other free radical in chain reactions manner [[5], [6], [7],25]. These

Conclusions

This is the first study that shows the potential of thymoquinone against peroxynitrite induced oxidative damaged of histone H2A. Our novel data showed that TQ provides protection of tyrosine, lysine, arginine, proline, threonine of histone H2A against peroxynitrite. These results may have importance for the development of novel therapeutic strategies for the management of disorders, where peroxynitrite plays a role.

Disclosure statement

The authors declare no conflict of interest.

Acknowledgements

This work was funded by College of Medicine, Qassim University, KSA.

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