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RNA as a new target for toxic and protective agents
Mutation Research 648 (2008) 15–22
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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
RNA as a new target for toxic and protective agents Carmela Fimognari a,∗ , Piero Sestili b , Monia Lenzi a , Anahi Bucchini c , Giorgio Cantelli-Forti a , Patrizia Hrelia a a
Dipartimento di Farmacologia, Alma Mater Studiorum – Università di Bologna, Via Irnerio 48, 40126 Bologna, Italy Istituto di Farmacologia e Farmacognosia, Università degli Studi di Urbino Carlo Bo, 61029 Urbino, Italy c Istituto di Botanica e Orto Botanico “P. Scaramella”, Università degli Studi di Urbino “Carlo Bo”, 61029 Urbino, Italy b
a r t i c l e
i n f o
Article history: Received 4 August 2008 Received in revised form 2 September 2008 Accepted 5 September 2008 Available online 18 September 2008 Keywords: RNA damage Human T-leukemia cells Reactive oxygen species Reactive nitrogen species Doxorubicin Pomegranate extract
a b s t r a c t In contrast to damage of genomic DNA and despite its potential to affect cell physiology, RNA damage is a poorly examined field in biomedical research. Potential triggers of RNA damage as well as its pathophysiological implications remain largely unknown. While less lethal than mutations in genome, such non-acutely lethal insults to cells have been recently associated with underlying mechanisms of several human chronic diseases. We investigated whether RNA damage could be related to the exposure of particular xenobiotics by testing the RNA-damaging activity of a series of chemicals with different mechanisms of action. Cultured human T-lymphoblastoid cells were treated with ethyl methanesulfonate (EMS), H2 O2 , doxorubicin, spermine, or S-nitroso-N-acetylpenicillamine (SNAP). Furthermore, we studied the potential protective activity of a pomegranate extract against RNA damage induced by different chemicals. Special attention has been paid to the protective mechanisms of the extract. The protective effect of pomegranate 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 (pre- and co-treatment) for understanding the mechanism of the inhibitory activity of pomegranate. We demonstrated that total RNA is susceptible to chemical attack. A degradation of total RNA could be accomplished with doxorubicin, H2 O2 , spermine and SNAP. However, EMS, a well-known DNA-damaging agent, was devoid of RNA-damaging properties, while spermine and SNAP, although lacking of DNA-damaging properties, were able to damage RNA. Pomegranate reduced the RNA-damaging effect of doxorubicin, H2 O2 , and spermine. Its inhibitory activity could be related with its ability to forms complexes with doxorubicin and H2 O2 , or interacts with the intracellular formation of reactive species mediating their toxicity. For spermine, an alteration of the rates of spermine absorption and uptake can also be involved. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Nucleic acids of all organisms are continuously damaged by extrinsic and intrinsic physical and chemical agents. 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. Nonetheless, DNA is not the only target for deleterious nucleic acids damaging agents. RNA may be more susceptible to damaging agents than DNA for different reasons. RNA is indeed mostly single-stranded and its bases are neither protected by hydrogen bonding nor located inside the double helix. Moreover, almost all of the cellular RNA have functional capacity for protein synthesis whether they are encoding proteins (mRNA) or involved
∗ Corresponding author. Tel.: +39 051 2095636; fax: +39 051 2095624. E-mail address: carmela.fi[email protected] (C. Fimognari). 0027-5107/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.mrfmmm.2008.09.003
in translation (rRNA and tRNA), whereas only 28% of the human genomic DNA is transcribed into RNA and only 5% of these transcribed sequences actually encode proteins [1]. However, it is now 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 [2–4]. Of particular interest, the complexity of an organism is poorly correlated with its number of protein-coding genes, but highly correlated with its number of ncRNAs [5]. Finally, it is usually considered that, in a cell, RNA is more abundant than DNA. In this view, it is highly probable that significant damage to RNA occurs when cells are exposed to nucleic acids damaging agents. Despite its potential to affect cell physiology, RNA damage is a poorly examined field in biomedical research. Potential triggers of RNA damage as well as its pathophysiological implications remain largely unknown. While less lethal than mutations in genome,
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such non-acutely lethal insults to cells have been recently associated with underlying mechanisms of several human diseases, especially chronic degeneration. A significant loss of RNA integrity has been demonstrated in advanced human atherosclerotic plaques as compared with nonatherosclerotic mammary arteries [6,7]. Furthermore, the increasing variety of ncRNAs being identified in the central nervous system suggests a strong connection between the biogenesis, dynamics of action, and combinational regulatory potential of ncRNAs and the complexity of the central nervous system [2,4]. Oxidative RNA damage has been described in several neurodegenerative diseases including Alzheimer disease, Parkinson disease, dementia with Lewy bodies, and prion diseases. Oxidative RNA damage is also a feature in vulnerable neurons at the earliest stages of these diseases, suggesting that RNA oxidation may actively contribute to the onset or to the development of disease [8–10]. Therefore, 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. The present study was designed to investigate whether RNA damage could be related to the exposure of particular chemical agents. We tested the RNA-damaging activity of a series of chemicals with different mechanisms of action. 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, one of the few NO donors releasing authentic NO and reactive oxygen species (ROS); S-nitroso-Nacetylpenicillamine (SNAP), a donor of NO. Furthermore, we investigated the potential protective activity of a pomegranate extract against RNA damage induced by different chemicals. In the past two decades particular interest has been devoted to the identification of naturally occurring chemopreventive compounds from edible plants and fruits. Fruits and vegetables contain different compounds, such as vitamin C, vitamin E, carotenoids, polyphenols, isothiocyanates whose interactions with various biological systems contribute to the beneficial effect of this group of foods [11,12]. The last 7 years have seen the virtual explosion of interest in pomegranate as a medicinal and nutritional product showing to interfere with tumor and atherosclerotic lesion development, and able to counteract inflammation and oxidative stress [13–15]. As the protective effects of chemopreventive agents may be mediated by a number of mechanisms, we treated the cells with pomegranate extract before and during treatment with the RNAdamaging 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 RNase-free 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. Punica granatum fruits were collected in Urbino (458 m above sea level) in September 2006 and identified by D. Fraternale. A voucher specimen is deposited in the herbarium of the Botanical Garden of the University of Urbino, Italy. The extract was prepared from rinds which were manually removed, crashed in a mortar with Tris/HCl 50 mM pH 7.5 (1:2, w/v) and centrifuged at 2500 rpm for 10 min. All these steps were performed at ice bath temperature. The supernatant from the centrifugation step was recovered, microfiltered, aliquoted and immediately stored at −80 ◦ C. Total polyphenol content was determined by the Prussian Blue method [16]. 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 (all
obtained from Sigma, St. Louis, MO). To maintain exponential growth, the cultures were divided every third day by dilution to a concentration of 1 x 105 cells/ml. 2.3. Cell treatments Cells were treated with different concentrations of SNAP, H2 O2, 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 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 [6,17]. In our experimental conditions, the highest concentration tested corresponded to 0.5 mM. The extract of pomegranate was added to cultures at the concentration of 1% (v/v) and equilibrated for 20 min before addition of the toxic agents [18]. For assessing the potential protective activity of pomegranate extract, two different treatment protocols were used: - Pre-treatment protocol: the cells were incubated for 20 min with the extract, 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 20 min with the extract, and then treated with the RNA toxic compounds for 24 h (6 h for H2 O2 ). 2.4. Determination of the antioxidant capacity of pomegranate extract in cell-free systems The antioxidant capacity of pomegranate’s rinds extract was evaluated using two different methods: 2,2-diphenyl-p-picrylhydrazyl (DPPH) assay [19] and 5lipoxygenase inhibition assay [20]. The DPPH assay was conducted as follows: the extract (50 l) was added to 1.5 ml of a 100 M DPPH (in ethanol). The absorbance decrease at 517 nm after 10 min was recorded and the percent decrease (corrected for the control, without antioxidant agents added) was taken as an index of the antioxidant capacity. For lipoxygenase assay, 5-lipoxygenase (0.18 g/ml) was added to 1 ml of the reaction mixture (pre-equilibrated at 20 ◦ C for 20 min and containing 100 M linoleic acid, the sample or the same quantity of Tris/HCL as reference, and 50 mM sodium phosphate, pH 6.8) and the formation of hydroperoxides from linoleic acid was then observed spectrophotometrically at 235 nm at 20 ◦ C. 2.5. 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. 2.6. 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. 2.7. 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 [21]. 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,
C. Fimognari et al. / Mutation Research 648 (2008) 15–22 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, ten categories are defined from 1 (totally degraded RNA) to 10 (fully intact RNA) [22].
2.8. Statistical analysis All results, except those for polyphenols content and antioxidant activity of pomegranate’s rinds extract, are expressed as the mean ± S.D. of three experiments. The results for polyphenols content and antioxidant activity of pomegranate’s rinds extract are expressed as the mean ± S.E. of five determinations. 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.
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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% [23]. 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 contin-
Fig. 1. Effect of spermine, SNAP, doxorubicin, H2 O2 , EMS on viability of Jurkat cells. The viability was determined immediately after treatments, as detailed in Section 2. The data presented are averaged from three independent experiments with error bars denoting S.D. of the mean.
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ued 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 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 24S (I) as well as three 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), 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 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, 2 for SNAP, 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 the pomegranate extract. The extract exhibited a remarkable antioxidant capacity in acellular systems (Table 1), even higher than Trolox, an established antioxidant used as positive control. The high specific polyphenol content of the extract is likely to account for its antioxidant capacity [18]. For excluding an RNA-damaging activity of the extract, cultures were firstly treated with pomegranate extract only, which did not
Fig. 2. Electropherograms of RNA size distribution. The data are representative of three different experiments with similar results.
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Table 1 Polyphenols content and antioxidant activity of pomegranate’s rinds extract. Polyphenolic content (mg/g DWa )
Rinds extract Troloxd a b c d
60.30 ± 0.62 –
Antioxidant activity DPPH assay IC50 (g/ml)b
Lipoxygenase assay IC50 (g/ml)c
11.3 ± 1.5 7.7 ± 0.9
89.7 ± 10.1 11.7 ± 0.13
DW: dry weight. Concentration of the extract (expressed as DW) or of Trolox promoting the reduction of 50% of the available DPPH. Concentration of the extract (expressed as DW) or of Trolox promoting a 50% decrease of linoleic acid peroxidation. The antioxidant activity of Trolox has been determined for comparative purpose.
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 three measures with error bars denoting S.D. of the mean.
modify the RIN value with respect to the untreated cells (9.9 vs. 9.8) (Fig. 4). The RNA-damaging activity of three of the toxic agents (spermine, doxorubicin, H2 O2 ) was reduced when the cells were also treated with pomegranate juice.
The highest protective activity of the extract was observed against the RNA-damaging effect of spermine using both the treatment protocols (pre- and co-treatment) (Fig. 5A). It is worth noting that the size distribution of the RNA fragments from sperminetreated cells is greatly affected by the addition of pomegranate extract (compare the electropherograms shown in Fig. 5B and C). In particular, the extract showed the greatest protective effect in the pre-treatment protocol, where the RNA damage induced by spermine was reduced by 61%. Although lower, the protective effect of the extract was evident also in the co-treatment protocol, where the damage by spermine was reduced by 39%. When the extract pre-loading was challenged against SNAP, we did not record any increase in the RIN value (Fig. 6). Surprisingly, in the co-treatment condition, an even greater RNA-damaging activity, with respect to SNAP alone, could be observed. Pomegranate extract produced an increase in the RIN value induced by doxorubicin (Fig. 7). However, the co-treatment protocol was the most effective, with an increase in the RIN value of 33%. In the pre-treatment protocol, the protective effect of the extract did not exceed 7%. The extract produced a significant decrease in the RNA damage induced by H2 O2 (Fig. 8). Co-treatment with pomegranate extract indeed increased the RIN value induced by H2 O2 by 55%. When used before the H2 O2 treatment, only a weak but not significant increase in the RIN value was detected. 4. Discussion
Fig. 4. RIN values calculated after cell treatment with pomegranate extract. The data presented are averaged from three measures with error bars denoting S.D. of the mean.
In contrast to damage of genomic DNA, the implications of RNA damage and the agents able to interact with RNA are not fully identified. Moreover, the RNA modifications are frequently overlooked, probably because they are much more difficult to detect. In the present study, we demonstrate that total RNA is susceptible to chemical attack. A degradation of total RNA could be accomplished with doxorubicin, H2 O2 , and the NO donors spermine and SNAP. It is not widely appreciated that many established DNAdamaging agents, such as doxorubicin and H2 O2 , also damage RNA. Given that there is at least as much RNA in a cell as DNA, it could be simply argued whenever DNA is damaged by such agents, RNA is surely damaged as well. However, a more complex scenario arises from our data. Indeed EMS, a well-known DNA-damaging agent [24], was devoid of RNA-damaging properties, while spermine and SNAP, although lacking of DNA-damaging properties [25,26], were able to damage RNA. Other than for its abundant cellular amount and its less protected structure, further characteristics of RNA can account for the different activity of spermine and SNAP on RNA vs. DNA. Whereas repair of DNA damage has been well documented in the literature, until recently it has been considered that damaged RNA may be only degraded rather than repaired. Degradation of RNA plays a central role in RNA metabolism, and damaged RNA can be removed through degradation by RNases, but selective degradation activity for oxi-
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Fig. 5. (A) RIN values calculated after cell treatment with spermine (0.5 mM) and pomegranate extract. The data presented are averaged from three measures with error bars denoting S.D. of the mean. (B) Electropherograms of RNA size distribution after treatment of cells with spermine. (C) Electropherograms of RNA size distribution after treatment of cells with spermine + pomegranate extract (pre-treatment protocol). The data are representative of three different experiments with similar results.
dized RNA has not been established for known RNases [27,28]. Oxidative stress induces cytoplasmic mRNA processing bodies (Pbodies), the site of active degradation of mRNA [29]. In contrast to mRNAs with rapid turnover, stable RNAs, consisting primarily of rRNAs and tRNAs and encompassing as much as 98% of total cellular RNA, may be protected against RNase action by tertiary structure, assembly into a ribonucleoprotein complex, or even blocking the RNA’s 3 terminus [27]. RNA repair has been recently suggested [30], but is yet to be fully characterized in vivo. However, this indicates that cells may have a greater investment in the protection of RNA than previously suspected.
The monofunctional alkylating agent EMS was devoid of RNAdamaging 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. On the whole, our results indicate that the global impact of chemicals on nucleic acids cannot be estimated by investigating exclusively the effects on DNA which, as it has been shown, are not predictive of the RNA-damaging potential, and vice versa. Considering the emerging role that RNA damage can play in several chronic diseases, it would be important to include the analysis of RNA damage in current testing strategies for better defining the toxicity profile of xenobiotics. Oxidized mRNAs indeed lead to loss of
Fig. 6. RIN values calculated after cell treatment with SNAP (0.5 mM) and pomegranate extract. The data presented are averaged from three measures with error bars denoting S.D. of the mean.
Fig. 7. RIN values calculated after cell treatment with doxorubicin (0.01 mM) and pomegranate extract. The data presented are averaged from three measures with error bars denoting S.D. of the mean.
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Fig. 8. RIN values calculated after cell treatment with H2 O2 (0.5 mM) and pomegranate extract. The data presented are averaged from three measures with error bars denoting S.D. of the mean.
normal protein level and protein function and potentially produce defective proteins leading to protein aggregation, a common feature of neurodegenerative diseases [31]. Recently it has been shown that the translation of damaged mRNA in rabbit reticulocyte lysate and human HEK293 cells causes the accumulation of short polypeptides, which is a result of premature termination of the translation process of the damaged mRNA and/or the proteolytic degradation of the modified protein containing the translation errors due to the damaged mRNA [32]. Additionally, it has also been shown that the oxidative damage to E. coli 16S rRNA results in the formation of short complementary DNA (cDNA) [33]. Also the biological consequences of ribosomal damage have been investigated in vitro and showed a significant reduction of protein synthesis [34]. Previous studies demonstrate that the brains of subjects with Alzheimer disease and mild cognitive impairment present ribosomal dysfunction associated with RNA damage [35,36]. Isolated polyribosome complexes from Alzheimer disease and mild cognitive impairment brains show decreased rate of protein synthesis without alteration in the polyribosome content. Decreased rRNA and tRNA levels are accompanied by the ribosomal dysfunction [35]. These findings indicate that RNA damage has detrimental effects on cellular function whether the damaged RNA species are coding for proteins (mRNA) or performing translation (rRNA and tRNA). In the second part of our study, we investigated the potential protective activity of pomegranate extract against the RNAdamaging properties of spermine, SNAP, doxorubicin and H2 O2 . Special attention has been paid to the protective mechanisms of the extract. The protective effect of pomegranate 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 pomegranate. Pomegranate reduced the RNA-damaging effect of doxorubicin, H2 O2 , and spermine. For doxorubicin and H2 O2 the extract was effective only in the co-treatment protocol. The inhibitory activity of the extract in the co-treatment suggests that pomegranate forms complexes with doxorubicin and H2 O2 , or interacts with the intracellular formation of species mediating their toxicity, namely ROS [37,38]. This latter is the most likely hypothesis. Both H2 O2 and doxorubicin are indeed known to produce ROS capable of
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damaging DNA. Similarly, RNA might be attacked and damaged by these radicals. Thus pomegranate protective activity against H2 O2 - and doxorubicin-induced RNA damage may depend on its well-known capacity to directly scavenge ROS and/or to chelate intracellular iron ions, which participate in the production of the radicals generated by both the agents. Notably, pomegranate extract contains high amounts of punicalagin and other polyphenols acting either as radical-scavengers or as iron chelators. As to doxorubicin it is unlikely that its intercalating capacity (i.e. the major mechanism responsible for anthracycline-induced DNA damage) is involved in the formation of RNA lesions, since RNA is mostly present as single-stranded chains which cannot be intercalated. For spermine the protective activity of pomegranate was evident under all two treatment conditions. When the extract is present in the culture medium before treatment with spermine (as in the pre-treatment), an extra-cellular mode of protection could alter the rates of spermine absorption and uptake, and/or preventively increase cellular defenses against radical nitrogen species produced by the subsequent treatment of cultures with spermine. However, the extract was not active against the RNA-damaging activity of SNAP, which also represents a donor of radical nitrogen species. Pomegranate is a rich source of potent polyphenolic, flavonoid antioxidants (anthocyanins), which have been shown to possess anti-atherogenic properties [39] related to the protection of NO against oxidative destruction, increase of the production of NO by vascular endothelial cells, and enhancement of its biological actions. The different activity of pomegranate against the RNAdamaging activity of spermine and SNAP could lie in the oxidation reaction to which spermine is subjected at cellular level, which causes the formation of ROS [40]. It is possible to speculate that pomegranate reduces the RNA-damaging properties of spermine by exclusively counteracting the reactive oxygen but not nitrogen species. Further experiments with other donors of RNS are necessary for exploring this hypothesis. In conclusion, the present data unequivocally show that cellular RNA is a sensitive target for different xenobiotics. This may present serious ramifications for cellular biochemical processes, particularly involving de novo protein synthesis, thus contributing to cytotoxic events. The observation may also further support efforts to establish the assay of RNA integrity in studying the toxicological profile of a xenobiotic. There is a clear need for further quantitative and qualitative validations in this area. 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 FIRB Piattaforme/Reti 2006. References [1] D. Baltimore, Our genome unveiled, Nature 409 (2001) 814–816. [2] 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. [3] F.F. Costa, Non-coding RNAs: new players in eukaryotic biology, Gene 357 (2005) 83–94. [4] M.F. Mehler, J.S. Mattick, Non-coding RNAs in the nervous system, J. Physiol. 575 (2006) 333–341. [5] R.J. Taft, M. Pheasant, J.S. Mattick, The relationship between non-protein-coding DNA and eukaryotic complexity, Bioessays 29 (2007) 288–299. [6] 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.
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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
RNA as a new target for toxic and protective agents Carmela Fimognari a,∗ , Piero Sestili b , Monia Lenzi a , Anahi Bucchini c , Giorgio Cantelli-Forti a , Patrizia Hrelia a a
Dipartimento di Farmacologia, Alma Mater Studiorum – Università di Bologna, Via Irnerio 48, 40126 Bologna, Italy Istituto di Farmacologia e Farmacognosia, Università degli Studi di Urbino Carlo Bo, 61029 Urbino, Italy c Istituto di Botanica e Orto Botanico “P. Scaramella”, Università degli Studi di Urbino “Carlo Bo”, 61029 Urbino, Italy b
a r t i c l e
i n f o
Article history: Received 4 August 2008 Received in revised form 2 September 2008 Accepted 5 September 2008 Available online 18 September 2008 Keywords: RNA damage Human T-leukemia cells Reactive oxygen species Reactive nitrogen species Doxorubicin Pomegranate extract
a b s t r a c t In contrast to damage of genomic DNA and despite its potential to affect cell physiology, RNA damage is a poorly examined field in biomedical research. Potential triggers of RNA damage as well as its pathophysiological implications remain largely unknown. While less lethal than mutations in genome, such non-acutely lethal insults to cells have been recently associated with underlying mechanisms of several human chronic diseases. We investigated whether RNA damage could be related to the exposure of particular xenobiotics by testing the RNA-damaging activity of a series of chemicals with different mechanisms of action. Cultured human T-lymphoblastoid cells were treated with ethyl methanesulfonate (EMS), H2 O2 , doxorubicin, spermine, or S-nitroso-N-acetylpenicillamine (SNAP). Furthermore, we studied the potential protective activity of a pomegranate extract against RNA damage induced by different chemicals. Special attention has been paid to the protective mechanisms of the extract. The protective effect of pomegranate 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 (pre- and co-treatment) for understanding the mechanism of the inhibitory activity of pomegranate. We demonstrated that total RNA is susceptible to chemical attack. A degradation of total RNA could be accomplished with doxorubicin, H2 O2 , spermine and SNAP. However, EMS, a well-known DNA-damaging agent, was devoid of RNA-damaging properties, while spermine and SNAP, although lacking of DNA-damaging properties, were able to damage RNA. Pomegranate reduced the RNA-damaging effect of doxorubicin, H2 O2 , and spermine. Its inhibitory activity could be related with its ability to forms complexes with doxorubicin and H2 O2 , or interacts with the intracellular formation of reactive species mediating their toxicity. For spermine, an alteration of the rates of spermine absorption and uptake can also be involved. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Nucleic acids of all organisms are continuously damaged by extrinsic and intrinsic physical and chemical agents. 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. Nonetheless, DNA is not the only target for deleterious nucleic acids damaging agents. RNA may be more susceptible to damaging agents than DNA for different reasons. RNA is indeed mostly single-stranded and its bases are neither protected by hydrogen bonding nor located inside the double helix. Moreover, almost all of the cellular RNA have functional capacity for protein synthesis whether they are encoding proteins (mRNA) or involved
∗ Corresponding author. Tel.: +39 051 2095636; fax: +39 051 2095624. E-mail address: carmela.fi[email protected] (C. Fimognari). 0027-5107/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.mrfmmm.2008.09.003
in translation (rRNA and tRNA), whereas only 28% of the human genomic DNA is transcribed into RNA and only 5% of these transcribed sequences actually encode proteins [1]. However, it is now 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 [2–4]. Of particular interest, the complexity of an organism is poorly correlated with its number of protein-coding genes, but highly correlated with its number of ncRNAs [5]. Finally, it is usually considered that, in a cell, RNA is more abundant than DNA. In this view, it is highly probable that significant damage to RNA occurs when cells are exposed to nucleic acids damaging agents. Despite its potential to affect cell physiology, RNA damage is a poorly examined field in biomedical research. Potential triggers of RNA damage as well as its pathophysiological implications remain largely unknown. While less lethal than mutations in genome,
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such non-acutely lethal insults to cells have been recently associated with underlying mechanisms of several human diseases, especially chronic degeneration. A significant loss of RNA integrity has been demonstrated in advanced human atherosclerotic plaques as compared with nonatherosclerotic mammary arteries [6,7]. Furthermore, the increasing variety of ncRNAs being identified in the central nervous system suggests a strong connection between the biogenesis, dynamics of action, and combinational regulatory potential of ncRNAs and the complexity of the central nervous system [2,4]. Oxidative RNA damage has been described in several neurodegenerative diseases including Alzheimer disease, Parkinson disease, dementia with Lewy bodies, and prion diseases. Oxidative RNA damage is also a feature in vulnerable neurons at the earliest stages of these diseases, suggesting that RNA oxidation may actively contribute to the onset or to the development of disease [8–10]. Therefore, 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. The present study was designed to investigate whether RNA damage could be related to the exposure of particular chemical agents. We tested the RNA-damaging activity of a series of chemicals with different mechanisms of action. 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, one of the few NO donors releasing authentic NO and reactive oxygen species (ROS); S-nitroso-Nacetylpenicillamine (SNAP), a donor of NO. Furthermore, we investigated the potential protective activity of a pomegranate extract against RNA damage induced by different chemicals. In the past two decades particular interest has been devoted to the identification of naturally occurring chemopreventive compounds from edible plants and fruits. Fruits and vegetables contain different compounds, such as vitamin C, vitamin E, carotenoids, polyphenols, isothiocyanates whose interactions with various biological systems contribute to the beneficial effect of this group of foods [11,12]. The last 7 years have seen the virtual explosion of interest in pomegranate as a medicinal and nutritional product showing to interfere with tumor and atherosclerotic lesion development, and able to counteract inflammation and oxidative stress [13–15]. As the protective effects of chemopreventive agents may be mediated by a number of mechanisms, we treated the cells with pomegranate extract before and during treatment with the RNAdamaging 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 RNase-free 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. Punica granatum fruits were collected in Urbino (458 m above sea level) in September 2006 and identified by D. Fraternale. A voucher specimen is deposited in the herbarium of the Botanical Garden of the University of Urbino, Italy. The extract was prepared from rinds which were manually removed, crashed in a mortar with Tris/HCl 50 mM pH 7.5 (1:2, w/v) and centrifuged at 2500 rpm for 10 min. All these steps were performed at ice bath temperature. The supernatant from the centrifugation step was recovered, microfiltered, aliquoted and immediately stored at −80 ◦ C. Total polyphenol content was determined by the Prussian Blue method [16]. 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 (all
obtained from Sigma, St. Louis, MO). To maintain exponential growth, the cultures were divided every third day by dilution to a concentration of 1 x 105 cells/ml. 2.3. Cell treatments Cells were treated with different concentrations of SNAP, H2 O2, 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 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 [6,17]. In our experimental conditions, the highest concentration tested corresponded to 0.5 mM. The extract of pomegranate was added to cultures at the concentration of 1% (v/v) and equilibrated for 20 min before addition of the toxic agents [18]. For assessing the potential protective activity of pomegranate extract, two different treatment protocols were used: - Pre-treatment protocol: the cells were incubated for 20 min with the extract, 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 20 min with the extract, and then treated with the RNA toxic compounds for 24 h (6 h for H2 O2 ). 2.4. Determination of the antioxidant capacity of pomegranate extract in cell-free systems The antioxidant capacity of pomegranate’s rinds extract was evaluated using two different methods: 2,2-diphenyl-p-picrylhydrazyl (DPPH) assay [19] and 5lipoxygenase inhibition assay [20]. The DPPH assay was conducted as follows: the extract (50 l) was added to 1.5 ml of a 100 M DPPH (in ethanol). The absorbance decrease at 517 nm after 10 min was recorded and the percent decrease (corrected for the control, without antioxidant agents added) was taken as an index of the antioxidant capacity. For lipoxygenase assay, 5-lipoxygenase (0.18 g/ml) was added to 1 ml of the reaction mixture (pre-equilibrated at 20 ◦ C for 20 min and containing 100 M linoleic acid, the sample or the same quantity of Tris/HCL as reference, and 50 mM sodium phosphate, pH 6.8) and the formation of hydroperoxides from linoleic acid was then observed spectrophotometrically at 235 nm at 20 ◦ C. 2.5. 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. 2.6. 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. 2.7. 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 [21]. 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,
C. Fimognari et al. / Mutation Research 648 (2008) 15–22 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, ten categories are defined from 1 (totally degraded RNA) to 10 (fully intact RNA) [22].
2.8. Statistical analysis All results, except those for polyphenols content and antioxidant activity of pomegranate’s rinds extract, are expressed as the mean ± S.D. of three experiments. The results for polyphenols content and antioxidant activity of pomegranate’s rinds extract are expressed as the mean ± S.E. of five determinations. 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.
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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% [23]. 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 contin-
Fig. 1. Effect of spermine, SNAP, doxorubicin, H2 O2 , EMS on viability of Jurkat cells. The viability was determined immediately after treatments, as detailed in Section 2. The data presented are averaged from three independent experiments with error bars denoting S.D. of the mean.
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ued 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 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 24S (I) as well as three 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), 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 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, 2 for SNAP, 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 the pomegranate extract. The extract exhibited a remarkable antioxidant capacity in acellular systems (Table 1), even higher than Trolox, an established antioxidant used as positive control. The high specific polyphenol content of the extract is likely to account for its antioxidant capacity [18]. For excluding an RNA-damaging activity of the extract, cultures were firstly treated with pomegranate extract only, which did not
Fig. 2. Electropherograms of RNA size distribution. The data are representative of three different experiments with similar results.
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Table 1 Polyphenols content and antioxidant activity of pomegranate’s rinds extract. Polyphenolic content (mg/g DWa )
Rinds extract Troloxd a b c d
60.30 ± 0.62 –
Antioxidant activity DPPH assay IC50 (g/ml)b
Lipoxygenase assay IC50 (g/ml)c
11.3 ± 1.5 7.7 ± 0.9
89.7 ± 10.1 11.7 ± 0.13
DW: dry weight. Concentration of the extract (expressed as DW) or of Trolox promoting the reduction of 50% of the available DPPH. Concentration of the extract (expressed as DW) or of Trolox promoting a 50% decrease of linoleic acid peroxidation. The antioxidant activity of Trolox has been determined for comparative purpose.
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 three measures with error bars denoting S.D. of the mean.
modify the RIN value with respect to the untreated cells (9.9 vs. 9.8) (Fig. 4). The RNA-damaging activity of three of the toxic agents (spermine, doxorubicin, H2 O2 ) was reduced when the cells were also treated with pomegranate juice.
The highest protective activity of the extract was observed against the RNA-damaging effect of spermine using both the treatment protocols (pre- and co-treatment) (Fig. 5A). It is worth noting that the size distribution of the RNA fragments from sperminetreated cells is greatly affected by the addition of pomegranate extract (compare the electropherograms shown in Fig. 5B and C). In particular, the extract showed the greatest protective effect in the pre-treatment protocol, where the RNA damage induced by spermine was reduced by 61%. Although lower, the protective effect of the extract was evident also in the co-treatment protocol, where the damage by spermine was reduced by 39%. When the extract pre-loading was challenged against SNAP, we did not record any increase in the RIN value (Fig. 6). Surprisingly, in the co-treatment condition, an even greater RNA-damaging activity, with respect to SNAP alone, could be observed. Pomegranate extract produced an increase in the RIN value induced by doxorubicin (Fig. 7). However, the co-treatment protocol was the most effective, with an increase in the RIN value of 33%. In the pre-treatment protocol, the protective effect of the extract did not exceed 7%. The extract produced a significant decrease in the RNA damage induced by H2 O2 (Fig. 8). Co-treatment with pomegranate extract indeed increased the RIN value induced by H2 O2 by 55%. When used before the H2 O2 treatment, only a weak but not significant increase in the RIN value was detected. 4. Discussion
Fig. 4. RIN values calculated after cell treatment with pomegranate extract. The data presented are averaged from three measures with error bars denoting S.D. of the mean.
In contrast to damage of genomic DNA, the implications of RNA damage and the agents able to interact with RNA are not fully identified. Moreover, the RNA modifications are frequently overlooked, probably because they are much more difficult to detect. In the present study, we demonstrate that total RNA is susceptible to chemical attack. A degradation of total RNA could be accomplished with doxorubicin, H2 O2 , and the NO donors spermine and SNAP. It is not widely appreciated that many established DNAdamaging agents, such as doxorubicin and H2 O2 , also damage RNA. Given that there is at least as much RNA in a cell as DNA, it could be simply argued whenever DNA is damaged by such agents, RNA is surely damaged as well. However, a more complex scenario arises from our data. Indeed EMS, a well-known DNA-damaging agent [24], was devoid of RNA-damaging properties, while spermine and SNAP, although lacking of DNA-damaging properties [25,26], were able to damage RNA. Other than for its abundant cellular amount and its less protected structure, further characteristics of RNA can account for the different activity of spermine and SNAP on RNA vs. DNA. Whereas repair of DNA damage has been well documented in the literature, until recently it has been considered that damaged RNA may be only degraded rather than repaired. Degradation of RNA plays a central role in RNA metabolism, and damaged RNA can be removed through degradation by RNases, but selective degradation activity for oxi-
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Fig. 5. (A) RIN values calculated after cell treatment with spermine (0.5 mM) and pomegranate extract. The data presented are averaged from three measures with error bars denoting S.D. of the mean. (B) Electropherograms of RNA size distribution after treatment of cells with spermine. (C) Electropherograms of RNA size distribution after treatment of cells with spermine + pomegranate extract (pre-treatment protocol). The data are representative of three different experiments with similar results.
dized RNA has not been established for known RNases [27,28]. Oxidative stress induces cytoplasmic mRNA processing bodies (Pbodies), the site of active degradation of mRNA [29]. In contrast to mRNAs with rapid turnover, stable RNAs, consisting primarily of rRNAs and tRNAs and encompassing as much as 98% of total cellular RNA, may be protected against RNase action by tertiary structure, assembly into a ribonucleoprotein complex, or even blocking the RNA’s 3 terminus [27]. RNA repair has been recently suggested [30], but is yet to be fully characterized in vivo. However, this indicates that cells may have a greater investment in the protection of RNA than previously suspected.
The monofunctional alkylating agent EMS was devoid of RNAdamaging 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. On the whole, our results indicate that the global impact of chemicals on nucleic acids cannot be estimated by investigating exclusively the effects on DNA which, as it has been shown, are not predictive of the RNA-damaging potential, and vice versa. Considering the emerging role that RNA damage can play in several chronic diseases, it would be important to include the analysis of RNA damage in current testing strategies for better defining the toxicity profile of xenobiotics. Oxidized mRNAs indeed lead to loss of
Fig. 6. RIN values calculated after cell treatment with SNAP (0.5 mM) and pomegranate extract. The data presented are averaged from three measures with error bars denoting S.D. of the mean.
Fig. 7. RIN values calculated after cell treatment with doxorubicin (0.01 mM) and pomegranate extract. The data presented are averaged from three measures with error bars denoting S.D. of the mean.
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Fig. 8. RIN values calculated after cell treatment with H2 O2 (0.5 mM) and pomegranate extract. The data presented are averaged from three measures with error bars denoting S.D. of the mean.
normal protein level and protein function and potentially produce defective proteins leading to protein aggregation, a common feature of neurodegenerative diseases [31]. Recently it has been shown that the translation of damaged mRNA in rabbit reticulocyte lysate and human HEK293 cells causes the accumulation of short polypeptides, which is a result of premature termination of the translation process of the damaged mRNA and/or the proteolytic degradation of the modified protein containing the translation errors due to the damaged mRNA [32]. Additionally, it has also been shown that the oxidative damage to E. coli 16S rRNA results in the formation of short complementary DNA (cDNA) [33]. Also the biological consequences of ribosomal damage have been investigated in vitro and showed a significant reduction of protein synthesis [34]. Previous studies demonstrate that the brains of subjects with Alzheimer disease and mild cognitive impairment present ribosomal dysfunction associated with RNA damage [35,36]. Isolated polyribosome complexes from Alzheimer disease and mild cognitive impairment brains show decreased rate of protein synthesis without alteration in the polyribosome content. Decreased rRNA and tRNA levels are accompanied by the ribosomal dysfunction [35]. These findings indicate that RNA damage has detrimental effects on cellular function whether the damaged RNA species are coding for proteins (mRNA) or performing translation (rRNA and tRNA). In the second part of our study, we investigated the potential protective activity of pomegranate extract against the RNAdamaging properties of spermine, SNAP, doxorubicin and H2 O2 . Special attention has been paid to the protective mechanisms of the extract. The protective effect of pomegranate 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 pomegranate. Pomegranate reduced the RNA-damaging effect of doxorubicin, H2 O2 , and spermine. For doxorubicin and H2 O2 the extract was effective only in the co-treatment protocol. The inhibitory activity of the extract in the co-treatment suggests that pomegranate forms complexes with doxorubicin and H2 O2 , or interacts with the intracellular formation of species mediating their toxicity, namely ROS [37,38]. This latter is the most likely hypothesis. Both H2 O2 and doxorubicin are indeed known to produce ROS capable of
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damaging DNA. Similarly, RNA might be attacked and damaged by these radicals. Thus pomegranate protective activity against H2 O2 - and doxorubicin-induced RNA damage may depend on its well-known capacity to directly scavenge ROS and/or to chelate intracellular iron ions, which participate in the production of the radicals generated by both the agents. Notably, pomegranate extract contains high amounts of punicalagin and other polyphenols acting either as radical-scavengers or as iron chelators. As to doxorubicin it is unlikely that its intercalating capacity (i.e. the major mechanism responsible for anthracycline-induced DNA damage) is involved in the formation of RNA lesions, since RNA is mostly present as single-stranded chains which cannot be intercalated. For spermine the protective activity of pomegranate was evident under all two treatment conditions. When the extract is present in the culture medium before treatment with spermine (as in the pre-treatment), an extra-cellular mode of protection could alter the rates of spermine absorption and uptake, and/or preventively increase cellular defenses against radical nitrogen species produced by the subsequent treatment of cultures with spermine. However, the extract was not active against the RNA-damaging activity of SNAP, which also represents a donor of radical nitrogen species. Pomegranate is a rich source of potent polyphenolic, flavonoid antioxidants (anthocyanins), which have been shown to possess anti-atherogenic properties [39] related to the protection of NO against oxidative destruction, increase of the production of NO by vascular endothelial cells, and enhancement of its biological actions. The different activity of pomegranate against the RNAdamaging activity of spermine and SNAP could lie in the oxidation reaction to which spermine is subjected at cellular level, which causes the formation of ROS [40]. It is possible to speculate that pomegranate reduces the RNA-damaging properties of spermine by exclusively counteracting the reactive oxygen but not nitrogen species. Further experiments with other donors of RNS are necessary for exploring this hypothesis. In conclusion, the present data unequivocally show that cellular RNA is a sensitive target for different xenobiotics. This may present serious ramifications for cellular biochemical processes, particularly involving de novo protein synthesis, thus contributing to cytotoxic events. The observation may also further support efforts to establish the assay of RNA integrity in studying the toxicological profile of a xenobiotic. There is a clear need for further quantitative and qualitative validations in this area. 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 FIRB Piattaforme/Reti 2006. References [1] D. Baltimore, Our genome unveiled, Nature 409 (2001) 814–816. [2] 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. [3] F.F. Costa, Non-coding RNAs: new players in eukaryotic biology, Gene 357 (2005) 83–94. [4] M.F. Mehler, J.S. Mattick, Non-coding RNAs in the nervous system, J. Physiol. 575 (2006) 333–341. [5] R.J. Taft, M. Pheasant, J.S. Mattick, The relationship between non-protein-coding DNA and eukaryotic complexity, Bioessays 29 (2007) 288–299. [6] 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.
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