Original Contributions
Creatine supplementation affords cytoprotection in oxidatively injured cultured mammalian cells via direct antioxidant activity

https://doi.org/10.1016/j.freeradbiomed.2005.10.035Get rights and content

Abstract

A growing body of evidence suggests that creatine (Cr) might exert protective effects in a variety of pathologies where oxidative stress plays a concausal etiologic role; furthermore, it has been recently reported that Cr displays direct antioxidant activity in a cell-free setting. However, at present, no research has been specifically aimed to directly test the antioxidant potential of Cr on oxidatively injured cultured cells. Here, the effects of Cr were studied using cultured human promonocytic (U937) and endothelial (HUVEC) cells, and murine myoblasts (C2C12) exposed to H2O2, tert-butylhydroperoxide (tB-OOH) and, in the case of U937 cells, peroxynitrite. Cr (0.1–10 mM) attenuated the cytotoxic effects caused by the oxidants in all the cell lines; under our conditions, cytoprotection was invariably associated with elevation of the intracellular fraction of Cr but it seemed to be unrelated to the levels of Cr phosphate (CrP); Cr did not affect the activity of catalase (CAT) and glutathione peroxidase (GpX), but it prevented H2O2- or tB-OOH-induced consumption of the nonprotein sulfhydryl (NPSH) pool in U937 and HUVEC cells; mass spectrometry experiments showed that a 136 MW molecule, which is likely to represent an oxidation by-product of Cr, formed in reaction buffers containing Cr and H2O2 as well as in cellular extracts from H2O2- or tB-OOH- treated Cr-preloaded U937 cells; finally, Cr cytoprotection appeared to be unrelated to chelation of Fe2+.

In conclusion, it is suggested that Cr exerts a mild, although significant, antioxidant activity in living cells, via a mechanism depending on direct scavenging of reactive oxygen (in particular hydroxyl radical) and nitrogen species.

Introduction

Creatine (Cr) monohydrate is a popular supplement in the sports industry, reputed to maintain high-energy phosphates during exercise; although normal levels of Cr can be obtained through an omnivorous diet and/or by endogenous synthesis in the liver, kidney, and pancreas, dietary consumption of supplements containing Cr is on the rise. The wide use of Cr as an ergogenic aid among athletes finds its rationale in that 90% of total Cr in the body is stored in skeletal muscles where it represents, along with ATP, the fundamental energy pool. Cr taken up by cells from the blood through a specific Na+- and Cl dependent transporter is mostly converted into its phosphorylated form Cr phosphate (CrP) by Cr kinase (CK) using ATP as phosphate donor. Under acute demand conditions, such as physical exercise, CrP serves as an energy buffer, since it tranfers back to ADP phosphoryl groups, quickly restoring adequate ATP levels. However, it should be pointed out that the measurable effect Cr has on ATP regeneration happens within 10 s-or even less-from initiation of the exercise [1] and after that, ATP generation depends primarily on glycolysis, glycogenolysis, and oxidative phosphorylation.

It is well documented that high-dose oral Cr supplementation further elevates intracellular energy pools of muscle tissue, as well as serum Cr levels in vivo [2]: indeed administration of very high Cr doses (up to 20 g daily) dose dependently augments plasma Cr up to 2 mM as well as intracellular Cr and CrP levels [3], [4]. Although the widespread use of Cr supplementation is mostly driven by anecdotal reports, consistent data in the scientific and exercise literature indicate that high-dose oral Cr produces some performance benefits in healthy young males [3], [5], [6], [7], and also in men [8] and women [3].

Apart from its use as an ergogenic aid, Cr research is garnering more attention since it has been suggested that Cr supplementation may be beneficial in the prevention and/or treatment of a number of muscular, neurological, and cardiovascular diseases such as gyrate atrophy [9], [10], [11], McArdle disease [12], Duchenne dystrophy [13], [14], myastenia gravis [15], amyotrophic lateral sclerosis [16], Parkinson's disease [17], ischemic brain [18] and heart [19] injury, dilated cardiomyopathy [20], and congestive heart disease [21]. The putative benefits of Cr in these disorders have been generally attributed to the Cr-induced buffering of cellular ATP levels, whose fall would lead to the accumulation of intracellular Ca2+, stimulation of formation of reactive oxygen species (ROS), and tissue oxidative damage [22]. Indeed, most of these pathologies recognize multiple aetiological factors among which the detrimental role of oxidative stress has been stably recognized [23], [24]. Interestingly, Lawler et al. [25] have recently reported that Cr, in a cell-free system, displays antioxidant scavenging properties against various oxidative agents using 2,2′-azino-bis-3-ethylbenzothiazolamine-6-sulfonic acid quenching as a marker: they hypothesized that this effect, in vivo, might contribute to the prevention of muscular fatigue. Along the same line, it would be attractive to speculate that the beneficial effects of Cr supplementation in the aforementioned pathologies derive, beyond from its contribution to cellular energetics, also from its antioxidant activity. Then it would be important to know whether Cr acts as an antioxidant in oxidatively injured living cells. The purpose of this study was to directly test Cr antioxidant activity in cultured mammalian cells exposed to an array of oxidative agents and to garner more information on the mechanism(s) responsible for Cr-afforded cytoprotection in oxidatively injured cells.

Section snippets

Reagents

H2O2, tert-butylhydroperoxide (tB-OOH), Cr, 3-amino-1,2,4-triazole (ATZ), buthionine sulfoximine (BSO), β-guanidinopropionic acid (GPA), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT), xylenol orange, reagents of analytical grade, and chemicals for light and electron microscopy were purchased from Sigma Chemical Co. (St. Louis, MO). Cell culture media, sera, and antibiotics were from Cambrex Corporate (East Rutherford, NJ).

Cell culture and treatment conditions

Cells were grown at 37°C in an atmosphere of 95%

Effect of Cr on the survival of oxidatively injured cells

The aim of this work was to assess the actual antioxidant activity of Cr in living cells exposed to an array of oxidative stressing agents. For this purpose the effect of Cr on the cytotoxic response of U937 promonocytic cells exposed to three different oxidative agents, namely H2O2 (Fig. 1A), tB-OOH (Fig. 1B), and peroxynitrite (Fig. 1C), was investigated. For U937 human promonocytic cells, the toxicity paradigm involved 2 h preincubation with or without Cr, followed by treatments with H2O2 or

Discussion

Our results demonstrate that Cr exerts antioxidant activity in cultured mammalian cells exposed to various oxidative agents. To the best of our knowledge, this is the first study that identifies the potential of Cr to directly counteract oxidative stress in living cells. Our data strengthen and extend the observation of Lawler et al. [25], who found that very high concentrations of Cr (20–60 mM) quenched free radicals in a cell-free in vitro setting. In the present study we show that Cr

Acknowledgments

This research was supported by COFIN 2004 (P.S., E.F.) and by CIPE 36/2002 E 17/2003-DGR 618 e 1245 2003 (P.S.).

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