Protective effect of creatine against RNA damage

Mutation Research 670 (2009) 59–67

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Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis journal homepage: www.elsevier.com/locate/molmut Community address: www.elsevier.com/locate/mutres

Protective effect of creatine against RNA damage Carmela Fimognari a,∗ , Piero Sestili b , Monia Lenzi a , Giorgio Cantelli-Forti a , Patrizia Hrelia a a b

Dipartimento di Farmacologia, Alma Mater Studiorum - Università di Bologna, Via Irnerio 48, 40126 Bologna, Italy Dipartimento di Scienze Biomolecolari, Istituto di Ricerca sull’Attività Motoria, Università degli Studi di Urbino “Carlo Bo”, 61029 Urbino, Italy

a r t i c l e

i n f o

Article history: Received 25 May 2009 Received in revised form 9 July 2009 Accepted 14 July 2009 Available online 23 July 2009 Keywords: Creatine RNA damage Human T leukemia cells Reactive oxygen species Reactive nitrogen species Doxorubicin

a b s t r a c t It is well documented that damage to DNA could be very harmful for all cells and is the source of several consequences such as cancer development, apoptosis or genetic diseases. In contrast, RNA damage is a poorly examined field in biomedical research, despite its potential to affect cell physiology. For example, a significant loss of RNA integrity has been demonstrated in advanced human atherosclerotic plaques as compared with non-atherosclerotic mammary arteries, and oxidative RNA damage has been described in several neurodegenerative diseases including Alzheimer disease. In the present study, we investigated whether RNA damage could be related to the exposure of particular xenobiotics and then we studied the potential protective activity of creatine against RNA-damaging activity of a series of chemicals with different mechanisms of action [ethyl methanesulfonate (EMS), H2 O2 , doxorubicin, spermine NONOate, S-nitroso-N-acetylpenicillamine (SNAP)]. Since the protective effect against RNA damage can be mediated by different mechanisms, such as alterations of the rates of toxic agent absorption and uptake, trapping of electrophiles as well as free radicals, and protection of nucleophilic sites in RNA, we used two different treatment protocols (pre- and co-treatment) for understanding the mechanism of the inhibitory activity of creatine. We demonstrated that total RNA is susceptible to chemical attack by doxorubicin, H2 O2 , spermine and SNAP. Creatine significantly reduced the RNA-damaging activity of only two of the toxic tested agents (H2 O2 and doxorubicin), while it lacked activity in counterstaining the RNA damage induced by the NO donors spermine and SNAP. Its inhibitory activity could be at least partially dependent on its capacity to directly scavenge free radicals and/or to maintain phosphocreatine store and ATP regeneration. © 2009 Elsevier B.V. All rights reserved.

1. Introduction DNA damage in somatic cells plays a well-established role in cancer initiation. A similar mechanism has been postulated to be involved in the pathogenesis of other chronic degenerative diseases, such as smoke-related cardiomyopathies and atherosclerosis [1]. Nonetheless, DNA is not the only target for deleterious nucleic acids damaging agents. Although RNA could be subject to the same insults as DNA and other cellular macromolecules, damage to RNA has not been a major focus in toxicology studies. This is somewhat surprising since RNA is largely single-stranded and its bases not being protected by hydrogen bonding or binding proteins. This means RNA could theoretically be more susceptible to chemical insults than DNA [2]. It might also be noted that the relative abundance of RNA and its subcellular distribution in close proximity of mitochondria suggest an additional level of RNA vulnerability to reactive oxygen species (ROS) [3]. It is now evident that only a minority of genetic

∗ Corresponding author. Tel.: +39 051 2095636; fax: +39 051 2095624. E-mail address: carmela.fi[email protected] (C. Fimognari). 0027-5107/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.mrfmmm.2009.07.005

transcripts (2–3% in human) code for proteins. However, it is also evident that noncoding RNA (ncRNA), rather than being “junk,” not only has structural and catalytic functions but also plays a critical role in regulating the timing and rate of gene expression [4–6]. Of particular note, while DNA is constantly checked for mistakes and errors by repair enzymes, it is still unclear how and to what extent damaged RNA is removed [7]. Given these factors, it is not surprising that RNA damage has been hypothesized to be involved in the pathogenesis of different diseases. A significant loss of RNA integrity has been demonstrated in advanced human atherosclerotic plaques as compared with nonatherosclerotic mammary arteries [8,9]. Oxidative RNA damage has been described in several neurodegenerative diseases including Alzheimer disease, Parkinson disease, dementia with Lewy bodies, and prion diseases [10–12]. Additionally, RNA oxidation was demonstrated in muscle cells of patients with rimmed vacuole myopathy [13], and in smooth muscle of atherosclerotic plaques [8]. In aged human skeletal muscle, a recent study has also demonstrated increased RNA oxidation, possibly related to increased levels of non-heme iron [7]. RNA oxidation may play a particularly important role in skeletal muscles with aging, because it may affect the balance between protein degradation and synthesis which ulti-

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mately determines total muscle mass and therefore sarcopenia [14,15]. Further advances in studies on RNA damage and its surveillance may have a significant impact on the understanding of the pathophysiology of currently unresolved complex diseases. Creatine is a naturally occurring compound obtained in humans from endogenous production and consumption through the diet. It is used as an ergogenic aid to improve exercise performance and increase fat-free mass. Lately, creatine’s positive therapeutic benefits in various oxidative stress-associated diseases have been reported in literature and, more recently, creatine has also been shown to exert direct antioxidant effects and to protect circular and linear DNA from oxidative attacks [16,17]. The aim of the present study is to evaluate possible protective effects of creatine against RNA damage induced by different chemicals in an attempt to envisage its possible use in the prevention or amelioration of a wide range of human diseases where RNA damage may play an etiological role. We treated cultured human T-lymphoblastoid cells with ethyl methanesulfonate (EMS), an alkylating agent; H2 O2 , an oxidizing agent that also increases the levels of PtdIns(3,4,5)P3 and activates downstream signaling; doxorubicin, which acts as both an alkylating and an oxidizing agent; spermine and S-nitroso-N-acetylpenicillamine (SNAP), two donors of NO. As the protective effects of creatine can be mediated by a number of mechanisms, we treated the cells with creatine before and during treatment with the RNA-damaging agents. 2. Materials and methods 2.1. Chemicals The stock solution (50 mM) of SNAP was prepared by combining equal volumes of N-acetyl-d-penicillamine (19 mg/ml in 100% ethanol) and NaNO2 (7 mg/ml in RNasefree water). The mixture was acidified with 50 ␮l of hydrochloric acid (19%, v/v) per 1 ml of SNAP solution and incubated for at least 30 min at 4 ◦ C before use. The stock solution was prepared immediately before administration. Creatine (Sigma, St. Louis, MO) was dissolved in RPMI 1640 supplemented with 10% heat-inactivated bovine serum, 1% antibiotics (all obtained from Sigma) and used at the concentrations of 1, 3 and 10 mM. 2.2. Cell culture Jurkat T-leukemia cells were grown in suspension and propagated in RPMI 1640 supplemented with 10% heat-inactivated bovine serum, 1% antibiotics. To maintain exponential growth, the cultures were divided every third day by dilution to a concentration of 1 × 105 cells/ml. 2.3. Cell treatments Cells were treated with different concentrations of SNAP, spermine NONOate, EMS, or doxorubicin (all obtained from Sigma) for 24 h at 37 ◦ C. For H2 O2 , the protocol was slightly modified. The cultures were treated with different concentrations of H2 O2 in PBS for 6 h at 37 ◦ C. The range of concentrations of the potential RNA-damaging agents were selected considering the quantity of total RNA extracted per cell, as recently suggested [8,18,19]. In our experimental conditions, the highest concentration tested corresponded to 0.5 mM. For assessing the potential protective activity of creatine, two different treatment protocols were used: • Pre-treatment protocol: the cells were incubated for 24 h with creatine, then were washed and treated with the RNA toxic compounds for 24 h (6 h for H2 O2 ). • Co-treatment protocol: the cultures were incubated for 24 h with creatine and the RNA toxic compounds (6 h for H2 O2 ). 2.4. Cell viability Viability was determined immediately after treatments by using Guava EasyCyte Mini flow cytometry (Guava Technologies, Hayward, CA), according to the manufacturer’s recommendations. Briefly, cells were mixed with an adequate volume of Guava ViaCount Reagent (Guava Technologies) and allowed to stain for at least 5 min at room temperature. The Guava ViaCount Reagent provides absolute cell count and viability data based on the differential permeability of DNA-binding dyes and the analysis of forward scatter. The fluorescence of each dye is resolved operationally to

allow the quantitative assessment of both viable and non-viable cells present in a suspension. 2.5. Extraction of RNA After cell treatment, RNA was isolated with an Agilent Total RNA isolation Mini Kit (Agilent Technologies, Palo Alto, CA), according to the manufacturer’s recommendations. Briefly, 350–400 ␮l of lysis solution were added to cell pellet and the cell homogenate was centrifuged through a mini-prefiltration column. The flowthrough was mixed with an equal volume of 70% ethanol, incubated for 5 min at room temperature and centrifuged through a mini-isolation column. The flow-through was discarded and the RNA-loaded column was transferred into an RNase-free final collection tube. Then, the purified RNA was eluted by addition of 10–15 ␮l of nuclease-free water. 2.6. Analysis of RNA damage RNA analysis was performed by microfluidic capillary electrophoresis with the Agilent 2100 bioanalyzer. The bioanalyzer is an automated bio-analytical device using microfluidics technology that provides electrophoretic separations in an automated and reproducible manner [20]. Tiny amounts of RNA samples are separated in the channels of the microfabricated chips according to their molecular weight and subsequently detected via laser-induced fluorescence detection. The result is visualized as an electropherogram where the amount of measured fluorescence correlates with the amount of RNA of a given size. A software algorithm then allows the calculation of an RNA Integrity Number (RIN). The RIN algorithm is based on a selection of informative features from the electropherograms. For this purpose, each electropherogram is divided into the following nine adjacent segments covering the entire electropherogram: a pre-region, a marker-region, a 5S-region, a fast-region, an 18S-region, an inter-region, a 28S-region, a precursor-region and a post-region. In addition, several global features are extracted, i.e., features that span several segments. Among these, the average and maximum height, areas and their ratios, total RNA ratio and the 28S area ratio are the most important features. The gradual degradation of rRNA is reflected by a continuous shift towards shorter fragment sizes. For classification of RNA integrity, 10 categories are defined from 1 (totally degraded RNA) to 10 (fully intact RNA) [21]. 2.7. Statistical analysis All results are expressed as the mean ± S.D. of at least two independent experiments. Differences among treatments were evaluated by ANOVA, followed by Bonferroni t-test, using GraphPad InStat version 3.00 for Windows 95 (GraphPad Software, San Diego, CA, USA). P < 0.05 was considered significant.

3. Results Jurkat were treated with different concentrations of SNAP, H2 O2 , spermine NONOate, EMS, or doxorubicin. Cell viability is shown in Fig. 1. A general approach in performing genotoxicity test such as micronucleus is to avoid the testing of doses that decrease viability, compared to the concurrent control cultures, by more than 60% [22]. Spermine, SNAP, H2 O2 and EMS did not modify the viability up to the highest concentrations tested and within the considered time interval. The analysis of RNA-damaging activity was therefore performed at the concentration 0.5 mM for spermine, SNAP, H2 O2 and EMS. However, the viability of doxorubicin-treated cells showed a gradual dose-dependent decrease (Fig. 1), starting at 0.01 mM, where the viability decreased by about 55%. Cell viability continued decreasing up to the concentration 0.5 mM, where doxorubicin reduced the viability by more than 94% (Fig. 1). Since the only concentration exhibited less than 60% toxicity was the lowest tested, the analysis of RNA-damaging activity was therefore conducted at the concentration of 0.01 mM. In the first part of the study, we analyzed the RNA-damaging activity of spermine, SNAP, doxorubicin, H2 O2 and EMS. To this aim, cells were treated with spermine, SNAP, doxorubicin or EMS for 24 h or with H2 O2 for 6 h. The RNA-damaging effect of the different compounds was assessed by RIN measurement. As reported in Fig. 2, a prototype electropherogram of total RNA containing a marker peak at about 24 s (I) as well as 3 prominent peaks corresponding to small RNAs (peak II), 18S (peak III), and 28S (peak IV) rRNA was obtained for untreated cells. In treated cells, aside from three prominent peaks (small RNAs, 18S and 28S rRNA),

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Fig. 1. Effect of spermine, SNAP, doxorubicin, H2 O2 , and EMS on viability of Jurkat cells. The viability was determined immediately after treatments, as detailed in Section 2. The data presented are averaged from two independent experiments with error bars denoting S.D. of the mean.

an electropherogram of the size distribution of cellular RNAs shows a broad range of molecular weights with much weaker signals. With increasing RNA damage, heights of 18S and 28S peaks gradually decrease, additional peak signals appear in a molecular weight range between small RNAs and the 18S peak, and the baseline signal increases. A decrease of the heights of 18S and 28S peaks was well evident in cells treated with SNAP (Fig. 2). An increase in the baseline signal was particularly marked in spermine-, doxorubicinand H2 O2 -treated cells, along with faint signals from cellular RNAs with a broad range of molecular weights especially in H2 O2 -treated cells (Fig. 2). Most of the tested agents were therefore able to damage RNA. However, the values of RIN indicated a different RNA-damaging potency. The RIN values were about 3.8 for spermine, 1.6 ± 0.1 for SNAP, and 5 for doxorubicin and H2 O2 . All these RIN values were significantly lower than the RIN value (9.8) recorded in untreated cells (Fig. 3). It is interesting to note that EMS did not induce any decrease in the RIN value with respect to the control cultures (Fig. 3). The next series of experiments focused on the protective activity of creatine.

For excluding an RNA-damaging activity of creatine, cultures were firstly treated with creatine only, which did not modify the RIN value with respect to the untreated cells (9.8 ± 0.0 vs. 9.9 ± 0.1) (Fig. 4). Creatine significantly reduced the RNA-damaging activity of only two of the toxic agents tested (H2 O2 and doxorubicin). When the creatine preloading was challenged against spermine, we recorded a slight but not significant increase in the RIN value at the highest concentration of creatine tested (7.3 ± 0.4 vs. 6.4 ± 0.1) (Fig. 5A). In the co-treatment condition, the RIN value registered in cultures treated with creatine plus spermine was similar to that observed in cultures treated with only spermine (Fig. 5B). When creatine was tested against the strong RNA-damaging activity of SNAP, the pre- and co-treatment protocols resulted in no protective activity. The highest RIN value in the presence of creatine was similar to that observed in cultures treated with SNAP alone (2.1 ± 0.1 vs. 2.2 ± 0.1) (Fig. 6). Creatine produced a weak but significant decrease in the RNA damage induced by H2 O2 at all the concentrations tested. Cotreatment with creatine 10 mM indeed increased the RIN value induced by H2 O2 by 15%. Similar values were observed in cultures

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Fig. 2. Electropherograms of RNA size distribution. The data are representative of two different experiments with similar results.

co-treated with creatine 1 and 3 mM (13% and 11%, respectively) (Fig. 7B). When used before the H2 O2 treatment, creatine did not affect the RNA-damaging activity of H2 O2 (Fig. 7A). The highest protective activity of creatine was observed against the RNA-damaging effect of doxorubicin using both the treatment protocols (pre- and co-treatment) (Fig. 8A and B). It is worth noting that the size distribution of the RNA fragments from doxorubicintreated cells is greatly affected by the addition of creatine (compare the electropherograms shown in Fig. 8C and D). In particular, in the pre-treatment protocol, creatine increased the RIN value by 48%. However, the co-treatment protocol was the most effective, with an increase in the RIN value of 52%. 4. Discussion The present study unequivocally shows that cellular RNA is a sensitive target for different xenobiotics. A degradation of total RNA

could be accomplished with doxorubicin, H2 O2 , and the NO donors spermine and SNAP. The monofunctional alkylating agent EMS was devoid of RNA-damaging properties. This indicates that the formation of ROS and/or reactive nitrogen species (RNS) seems to be essential for the reaction of a xenobiotic with RNA. Moreover, our data suggest that creatine possesses an interesting protective activity against different RNA-damaging agents. Special attention has been paid to the protective mechanisms of creatine. The protective effect of creatine can be mediated by alterations of the rates of toxic agent absorption and uptake, by trapping of electrophiles as well as free radicals, and protection of nucleophilic sites in RNA. We used two different treatment protocols in an effort to understand the mechanism of the inhibitory activity of creatine. However, it is worth noting that creatine accumulates in the cell during pre-treatment, then it would be partially present also during the subsequent treatment with xenobiotics. Accordingly, similar effects of creatine, with the

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Fig. 3. RIN values calculated after cell treatment with spermine (0.5 mM), SNAP (0.5 mM), doxorubicin (0.01 mM), H2 O2 (0.5 mM) and EMS (0.5 mM), respectively. The data presented are averaged from two measures with error bars denoting S.D. of the mean.

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Fig. 4. RIN values calculated after cell treatment with creatine. The data presented are averaged from two measures with error bars denoting S.D. of the mean.

Fig. 5. RIN values calculated after cell treatment with spermine (0.5 mM) and creatine: (A) pre-treatment protocol and (B) co-treatment protocol. The data presented are averaged from two measures with error bars denoting S.D. of the mean.

Fig. 6. RIN values calculated after cell treatment with SNAP (0.5 mM) and creatine: (A) pre-treatment protocol and (B) co-treatment protocol. The data presented are averaged from two measures with error bars denoting S.D. of the mean.

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Fig. 7. RIN values calculated after cell treatment with H2 O2 (0.5 mM) and creatine: (A) pre-treatment protocol and (B) co-treatment protocol. The data presented are averaged from two measures with error bars denoting S.D. of the mean.

exception for H2 O2 , were observed in the pre- and co-treatment protocol. Creatine is the most popular supplement proposed as an ergogenic aid. It is distributed throughout the body with 95% found in skeletal muscle and the remaining 5% in the brain, liver, kidney, and testes [23]. Creatine taken up by cells from the blood

through a specific Na+ - and Cl− -dependent transporter is mostly converted into its phosphorylated form creatine phosphate by creatine kinase using ATP as phosphate donor. Creatine kinase, catalyzing the reversible transphosphorylation between ATP and phosphocreatine, is able to stock the “high energy” of ATP in the form of phosphocreatine and, vice versa, to use phosphocreatine to

Fig. 8. RIN values calculated after cell treatment with doxorubicin (0.01 mM) and creatine: (A) pre-treatment protocol and (B) co-treatment protocol. The data presented are averaged from two measures with error bars denoting S.D. of the mean. (C) Electropherograms of RNA size distribution after treatment of cells with doxorubicin. (D) Electropherograms of RNA size distribution after treatment of cells with doxorubicin + creatine 3 mM (pre-treatment protocol). The data are representative of two different experiments with similar results.

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replenish cellular ATP pools [24]. Intramuscular and cerebral stores of creatine, as well as phosphocreatine, increase with oral creatinesupplementation. The increase of these stores can offer therapeutic benefits by preventing ATP depletion, stimulating protein synthesis or reducing protein degradation, and stabilizing biological membranes [25]. Moreover, other authors have demonstrated that creatine supplementation is also beneficial in the prevention or treatment of some oxidative stress-associated diseases [26–29]. In these diseases RNA represents an important target for oxidative damage. Indeed, RNA damage has recently been reported as being an etiological factor in oxidative stress-related disorders including cardiovascular diseases and neurodegenerative disorders, and the normal aging process [8–12]. Under our experimental conditions, the RNA damage induced by H2 O2 was reduced in the co-treatment protocol; using the pretreatment protocol, the RNA-damaging activity of H2 O2 was not affected by creatine. H2 O2 exerts its toxic activity through the induction of oxidative damage to RNA. Lawler et al. reported that creatine is capable of directly quenching aqueous radical and reactive species ions in vitro [30]. A more recent study showed that creatine exerts direct antioxidant activity via a scavenging mechanism rather than through iron-chelation [16]. This mechanism would have no effect when creatine is administered before H2 O2 . Moreover, a recent study reported that creatine preloading did not augment the activity of catalase or glutathione peroxidase [16], and this can explain its lack of activity in the pre-treatment protocol. In this study creatine was capable to afford protection also when administered prior to the oxidative insult, while in the present one pre-incubation seems to lack any protective effect. This apparent contradiction might largely depend on the different treatment conditions used in the two studies. Indeed a far longer exposure time to a high H2 O2 concentration (6 h with 500 ␮M vs. 1 h with 100–300 ␮M H2 O2 in Ref. [16]) were used throughout the present study. Such a longer exposure time, selected to induce a significant RNA damage, is likely to yield a level of ROS which overwhelms the scavenging potential of the intracellular free-creatine stores accumulated over the 24 h preloading. Thus, to obtain an effective antioxidant activity here, creatine needs to be present in the treatment milieu where it would either scavenge H2 O2 extracellularly or contribute to maintain high intracellular free-creatine levels over the 6 h oxidative challenge. Finally, the protective effects of creatine are not likely to depend on the induction of catalase or glutathione peroxidase activity which has repeatedly been shown to be unaffected by creatine loading in various cell lines [16,17,31]. The behavior of creatine against the RNA-damaging activity of doxorubicin seems more complex. Under our experimental conditions, the RNA damage induced by doxorubicin was significantly reduced in both the pre- and co-treatment protocols. Doxorubicin is an anthracycline antibiotic, useful in the treatment of diverse malignancies, which exerts cytotoxic effects by different mechanisms. The molecular mechanisms of anthracycline toxicity are still far from being clear. Anthracyclines impair mitochondrial functions, such as respiratory rate, and generation of high-energy phosphates. Numerous mechanisms for inactivation of the mitochondrial respiratory chain by anthracyclines have been proposed, such as generation of free radicals, interaction with mitochondrial DNA, disruption of gene expression, alteration of calcium exchange, lipid peroxidation inducing disturbance of mitochondrial membranes, and apoptosis [32–35]. Several lines of evidence suggest the creatine kinase/ phosphocreatine system to be important targets of anthracycline toxicity. Decreased creatine kinase activity after treatment with doxorubicin alone or in combination with horseradish peroxidase was observed with rat heart and cardiomyocyte cultures [36], heart homogenates, or purified cytosolic creatine kinase

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[37]. Anthracycline treatment reduced transcriptional rate of cytosolic creatine kinase and adenylate translocator, as well as phosphocreatine levels [33,38]. Doxorubicin, exhibiting high affinity to cardiolipin, was shown to diminish binding of creatine kinase to this phospholipid [39] and, after complexation with iron, to decrease enzymatic activity of creatine kinase in isolated mitochondria [40]. Involvement of creatine kinase in anthracycline toxicity is also suggested by the fact that creatine kinase is a prime target of damage by peroxynitrite and oxygen radicals [41,42], which are known to be generated by anthracyclines [38,43]. Damage at low and moderate anthracycline concentrations (≤100 ␮M) was caused mainly by oxidation of sulfhydryl groups, as indicated by protective and reversal effects of ␤-mercaptoethanol, whereas the protective effect of superoxide dismutase at higher drug concentrations (>100 ␮M) points to additional damage by ROS (e.g., superoxide anion) [44]. Creatine kinase is known to be particularly sensitive to oxidative and radical injury [42,41] as well as inactivation by thiol-specific reagents [45]. Anthracyclines, because of their quinone structure and intrinsic electrophilicity, are potent redox-active agents [46,47]. Thus, anthracyclines would oxidize creatine kinase thiols (direct effect) and generate ROS that contribute to further damage (indirect effect). The proposed mechanism for creatine protection against RNA damage induced by doxorubicin could be attributed to a buildup of phosphocreatine stores, which increase the efficiency of ATP regeneration. However, while ATP and chronic supplementation of creatine increase cell survival and improve biochemical parameters [48,49], their role in RNA damage modulation is not defined. We can therefore not exclude that the protective effect of creatine against RNA damage induced by doxorubicin was independent from ATP regeneration. An inversion of the treatment protocol (cells treated with doxorubicin, washed and then treated with creatine) can clarify this aspect. The radical scavenging mechanism of creatine and its activity in the pre-treatment protocol also point to the role of freecreatine as an antioxidant and suggest that the effect of creatine on RNA damage induced by doxorubicin might represent an important mechanism contributing to its cytoprotective activity in cells subjected to oxidative stress. In this case, the protective activity of the creatine pre-incubation protocol, which was inactive against H2 O2 -induced RNA damage, might depend on the fact that the intracellular free-creatine stores accumulated over the 24 h preloading are sufficient to scavenge the lower level of ROS generated by doxorubicin treatment. Creatine did not affect the RNA damage induced by the NO donors spermine and SNAP. It has been demonstrated that the S-nitrosation of creatine kinase active-site thiol groups inhibits enzyme activity [50]. Two S-nitrosothiol groups were formed in the creatine kinase dimer after nitrosation of rabbit skeletal muscle creatine kinase in solution. Creatine kinase inactivation due to S-nitrosation was time- and concentration-dependent, and was rapidly reversible with the sulfhydryl dithiothreitol [50]. Wolosker et al. [51] demonstrated that S-nitrosoglutathione could also inactivate creatine kinase by a nitric oxide dependent mechanism. Considering the key role of creatine kinase in cellular energetics, it is possible that spermine and SNAP, as NO donors, affect creatine kinase activity and make it cells virtually unable to form phosphocreatine. This blocks the creatine kinase/phosphocreatine circuit and impairs cellular energy generation. An additional aspect worth considering is that spermine and SNAP could generate radicals that are not creatine-sensitive. Other experiments are necessary for supporting this hypothesis. In conclusion, the present study shows that cellular RNA is a sensitive target for different xenobiotics. Moreover, our data suggest creatine possesses an interesting protective activity against different RNA-damaging agents, at least partially dependent on its

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capacity to directly scavenge free radicals and/or to maintain phosphocreatine store and ATP regeneration. Apart from its use as an ergogenic aid, creatine research is garnering more attention. Recent findings have indicated that creatine supplementation has a therapeutic role in several diseases characterized by atrophic conditions, weakness, and metabolic disturbances (i.e., in the muscle, bone, lung, and brain). Accordingly, there has been an evidence indicating that creatine supplementation is capable of attenuating the degenerative state in some muscle disorders (i.e., Duchenne and inflammatory myopathies), central nervous diseases (i.e., Parkinson’s, Huntington’s, and Alzheimer’s), and bone and metabolic disturbances (i.e., osteoporosis and type II diabetes) [52–63]. The putative benefits of creatine in these disorders have been generally attributed to the creatine-induced buffering of cellular ATP levels, whose fall would lead to the accumulation of intracellular Ca2+ , stimulation of formation of ROS, and tissue oxidative damage [25]. Most of these pathologies recognize multiple aetiological factors among which damage to RNA, as recently demonstrated [7–12]. Our results indicate that the beneficial effects of creatine supplementation in the aforementioned pathologies derive, beyond from its contribution to cellular energetics, also from its ability to protect a critical target, such as RNA. Conflict of interest statement We declare that there are no conflicts of interest. Acknowledgments This research was supported by Fondazione Cassa di Risparmio in Bologna (Italy) and Ministero dell’Istruzione dell’Università e della Ricerca (PRIN 2007). References [1] S. De Flora, A. Izzotti, F. D’Agostini, R.M. Balansky, D. Noonan, A. Albini, Multiple points of intervention in the prevention of cancer and other mutation-related diseases, Mutat. Res. 480–481 (2001) 9–22. [2] R.J. Castellani, A. Nunomura, R.K. Rolston, P.I. Moreira, A. Takeda, G. Perry, M.A. Smith, Sublethal RNA oxidation as a mechanism for neurodegenerative disease, Int. J. Mol. Sci. 9 (2008) 789–806. [3] A. Nunomura, G. Perry, M.A. Pappolla, R. Wade, K. Hirai, S. Chiba, M.A. Smith, RNA oxidation is a prominent feature of vulnerable neurons in Alzheimer’s disease, J. Neurosci. 19 (1999) 1959–1964. [4] X. Cao, G. Yeo, A.R. Muotri, T. Kuwabara, F.H. Gage, Noncoding RNAs in the mammalian central nervous system, Annu. Rev. Neurosci. 29 (2006) 77–103. [5] F.F. Costa, Non-coding RNAs: new players in eukaryotic biology, Gene 357 (2005) 83–94. [6] M.F. Mehler, J.S. Mattick, Non-coding RNAs in the nervous system, J. Physiol. 575 (2006) 333–341. [7] T. Hofer, E. Marzetti, J. Xu, A.Y. Seo, S. Gulec, M.D. Knutson, C. Leeuwenburgh, E.E. Dupont-Versteegden, Increased iron content and RNA oxidative damage in skeletal muscle with aging and disuse atrophy, Exp. Gerontol. 43 (2008) 563–570. [8] W. Martinet, G.R.Y. De Meyer, A.G. Herman, M.M. Kockx, Reactive oxygen species induce RNA damage in human atherosclerosis, Eur. J. Clin. Invest. 34 (2004) 323–327. [9] W. Martinet, G.R.Y. De Meyer, A.G. Herman, M.M. Kockx, RNA damage in human atherosclerosis, RNA Biol. 2 (2005) 4–7. [10] A. Nunomura, K. Honda, A. Takeda, K. Hirai, X. Zhu, M.A. Smith, G. Perry, Oxidative damage to RNA in neurodegenerative diseases, J. Biomed. Biotechnol. 2006 (2006) 1–6. [11] A. Nunomura, R.J. Castellani, X. Zhu, P.I. Moreira, G. Perry, M.A. Smith, Involvement of oxidative stress in Alzheimer disease, J. Neuropathol. Exp. Neurol. 65 (2006) 631–641. [12] A. Nunomura, P.I. Moreira, A. Takeda, M.A. Smith, G. Perry, Oxidative RNA damage and neurodegeneration, Curr. Med. Chem. 14 (2007) 2968–2975. [13] M. Tateyama, A. Takeda, Y. Onodera, M. Matsuzaki, T. Hasegawa, A. Nunomura, K. Hirai, G. Perry, M.A. Smith, Y. Itoyama, Oxidative stress and predominant Abeta42(43) deposition in myopathies with rimmed vacuoles, Acta Neuropathol. (Berl.) 105 (2003) 581–585. [14] K. Honda, M.A. Smith, X. Zhu, D. Baus, W.C. Merrick, A.M. Tartakoff, T. Hattier, P.L. Harris, S.L. Siedlak, H. Fujioka, Q. Liu, P.I. Moreira, F.P. Miller, A. Nunomura, S. Shimohama, G. Perry, Ribosomal RNA in Alzheimer disease is oxidized by bound redox-active iron, J. Biol. Chem. 280 (2005) 20978–20986.

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