Original ContributionPyridoxamine protects intestinal epithelium from ionizing radiation-induced apoptosis
Introduction
Exposure to ionizing radiation (IR) can produce severe health impairments owing to injury and failure of susceptible organs. The detrimental effects of IR on biological tissues are, for the most part, mediated via increased production of reactive oxygen species (ROS) and reactive carbonyl species (RCS). Hydroxyl radical produced during radiolysis of water can trigger the oxidation of lipids, amino acids, and saccharides leading to formation of various secondary free radicals and reactive carbonyl compounds as shown in Fig. 1 [1], [2], [3]. These toxic products chemically modify DNA, proteins, and lipids, causing cellular damage.
Because the intestinal epithelium is one of the fastest proliferating tissues in the body, the gastrointestinal (GI) tract is injured during intended or accidental radiation exposure [4], [5], [6], [7]. In the GI tract, epithelial cells of the small intestinal crypts are the most susceptible to radiation-induced apoptosis [8]. Whereas only occasional apoptotic cells can be observed in the intestinal crypts of healthy mice and humans, as low as 1 Gy of radiation induces a dramatic increase in apoptosis in mouse small intestinal crypt within 3 to 6 h after exposure, predominantly in the stem cell region [9].
Radioprotectors are essential in safeguarding the normal tissue during intended radiation exposure. Various radioprotective strategies have been explored, including thiol compounds that can scavenge free radicals and modulate the DNA repair process; or growth factors and cytokines that function through receptor-mediated mechanisms and can modify cellular responses to radiation [10], [11], [12]. At present, thiol compounds are the most effective class of radioprotectors; however, they have significant shortcomings, including relatively high toxicity and unfavorable routes of administration, which negatively affect their application and efficacy [13]. Therefore, there is a need for safer and even more effective radioprotective treatments.
Pyridoxamine (PM) is one of the natural forms of vitamin B6 and an intermediate in transamination reactions catalyzed by vitamin B6-dependent enzymes. PM can also inhibit pathogenic oxidative reactions (reviewed in [14]) and is a prospective pharmacological agent for treatment of chronic conditions involving oxidative and carbonyl stress such as diabetic complications [15], [16], [17]. Clinical trials have also demonstrated that PM is safe at the effective pharmacological dose [18], [19]. Investigation of its mechanism of action showed that PM can scavenge RCS and ROS derived from the oxidation of sugars and lipids [20], [21], [22], [23], [24], [25], [26], [27].
Because ROS and RCS are important mediators of IR-induced damage in biological systems (Fig. 1), we reasoned that PM might be protective against the detrimental effects of radiation. We demonstrate here that PM treatment inhibits IR-induced GI epithelial apoptosis in cell culture and in an animal model. PM was more effective at protecting from radiation-induced apoptosis compared to amifostine (Ethyol), a synthetic thiol compound and the only FDA-approved radioprotector. PM, therefore, has potential as an effective and safe radioprotective agent.
Section snippets
Chemicals
Bovine serum albumin, pyridoxamine hydrochloride, xanthine, and glycolaldehyde were purchased from Sigma–Aldrich. Xanthine oxidase was from Roche; and amifostine was from Medimmune Oncology (Gaithersburg, MD, USA).
Generation and determination of hydroxyl radical
Hydroxyl radical was generated using the xanthine:xanthine oxidase:Fe3+system [1]. Quantification of hydroxyl radical was performed using the salicylate hydroxylation method [28]. Briefly, xanthine oxidase (16 mU) was added to 150 mM potassium phosphate buffer, pH 7, supplemented with
Pyridoxamine inhibits both accumulation of hydroxyl radical and carbonyl-induced protein oxidation
Significant amounts of hydroxyl radical were generated in the xanthine/xanthine oxidase system (Fig. 2A). PM inhibited detectable hydroxyl radical in a concentration-dependent manner (Fig. 2A), consistent with an earlier report of the PM inhibitory effect toward hydroxyl radical in the Fenton reaction [14]. In a similar concentration-dependent fashion, PM inhibited the oxidative carboxylation of albumin lysine residues induced by reactive carbonyl glycolaldehyde (Fig. 2B). These data reflect
Discussion
Radioprotective treatments that have been proposed over the past several decades include thiol compounds that can scavenge free radicals, growth factors and cytokines that function through receptor-mediated mechanisms to modify cellular responses to radiation, and natural antioxidants and extracts [10], [11], [12], [33]. Thiol compounds are the most effective and the longest studied radioprotectors. The synthetic thiol amifostine (Ethyol) is the only FDA-approved radioprotective treatment
Acknowledgments
This work has been supported by Grants DK66415 (P.V.), DK65138 (B.H.), and CA89674 (D.H.) from the National Institutes of Health and by research grants from the Pardee Foundation (E.Y.) and Whitmer Family Foundation (D.H.).
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