The ascorbic acid paradox

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Biochemical and Biophysical Research Communications 400 (2010) 466–470

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The ascorbic acid paradox Michael Osiecki a, Parisa Ghanavi a, Kerry Atkinson b, Lars K. Nielsen c, Michael R. Doran a,⇑ a

Tissue Repair and Regeneration Program, Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Queensland 4059, Australia Adult Stem Cell Laboratory, Mater Medical Research Institute, Brisbane, Queensland 4101, Australia c Bioengineering Laboratory, Australian Institute for Bioengineering and Nanotechnology, University of Queensland, Brisbane, Queensland 4072, Australia b

a r t i c l e

i n f o

Article history: Received 12 August 2010 Available online 21 August 2010 Keywords: Pro-oxidant Antioxidant Tissue culture Differentiation

a b s t r a c t Ascorbic acid (AA) is a common culture medium and dietary supplement. While AA is most commonly known for its antioxidant properties, it is also known to function as a pro-oxidant under select conditions. However, the complexity and often unknown composition of biological culture systems makes prediction of AA behaviour in supplemented cultures challenging. The frequent observation of outcomes inconsistent with antioxidant behaviour suggests that AA may be playing a pro-oxidant role more often than appreciated. In this work we explored the intracellular and extracellular impact of AA supplementation on KG1a myeloid leukaemia cells over a 24-h culture period following the addition of the AA supplement. At 24 h we found that supplementation of AA up to 250 lM resulted in intracellular antioxidant behaviour. However, when these same cultures were evaluated at 2 or 4 h we observed pro-oxidant activity at the higher AA concentrations indicating that the outcome was very much time and dose dependent. In contrast, pro-oxidant activity was never observed in the extracellular medium. Paradoxically, and to our knowledge not previously reported, we observed that intracellular pro-oxidant activity and extracellular antioxidant activity could occur simultaneously. These results indicate that the precise activity of AA supplementation varies as a function of dose, time and cellular location. Further, these results demonstrate how in the absence of careful culture characterization the true impact of AA on cultures could be underappreciated. Ó 2010 Elsevier Inc. All rights reserved.

1. Introduction Ascorbic acid (AA) is well known for its antioxidant properties; however, a steady stream of papers [1–4] suggest the role of AA in cell culture is not fully appreciated. In this paper we briefly review related literature and use simple experiments to provide insight into both the counterintuitive and temporally dependent impact of AA in cell culture. 1.1. Reactive oxygen species in cell culture In vitro reactive oxygen species (ROS) formation is both a direct consequence of leakage from the mitochondria, electron transport chain and elevated spontaneous formation [5]. In mitochondria, oxygen is reduced to water one electron at a time; however, within some redox centres leakage of electrons to oxygen can result in the creation of superoxide anions (O 2 ) [6]. While the superoxide anion is not an especially strong oxidant, it can be transformed, by the Haber–Weiss reaction, to the much more potent hydroxyl radical ⇑ Corresponding author. Fax: +61 (0)7 3138 6030. E-mail addresses: [email protected] (P. Ghanavi), [email protected] (K. Atkinson), [email protected] (L.K. Nielsen), michael. [email protected], [email protected] (M.R. Doran). 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.08.052

[7]. This process is catalyzed by unbound transition metals (Cu2+ and Fe3+). Similarly elevated spontaneous ROS formation is an artefact thought to be the result of the high concentration of transition metals and oxygen in cell culture medium; this notion is supported by the fact that the addition of metal-chelating proteins significantly dampens spontaneous ROS production [8]. The trend towards the use of reduced protein or protein-free medium would therefore, in theory, exacerbate this artefact resulting in greater spontaneous ROS formation and therefore increase the ROS load on cultured cells. Cells are said to be in a state of oxidative stress when there is a serious imbalance between the production of ROS and antioxidant defences leading to potential cellular damage [9,10]. However, even in the absence of extreme conditions that physically damage cells, a subtle fluctuation in ROS may impact cultures by perturbing redox signalling pathways. 1.2. Redox signalling There is mounting evidence that ROS may function as second messengers in what is termed Redox signalling [11–13]. It is now thought that the cell redox state (often described as the balance of NAD+/NADH and NADP+/NADPH within the cell) can function to direct cell fate decisions rather than be a reflection of prior commitment to one [14,15]. This hypothesis is supported by the

M. Osiecki et al. / Biochemical and Biophysical Research Communications 400 (2010) 466–470

observation that purposeful modification of the cell redox state through supplementation of the medium with pro-oxidants or antioxidants can, in itself, direct cell fate decisions [15,16]. Thus, if purposeful modification of the culture ROS environment can direct cell fate, then it would be logical to assume that even subtle manipulations of environmental and consequently the internal cell redox could also drastically alter culture outcomes. 1.3. Ascorbic acid In the body redox balance is, in part, maintained by enzymes that metabolize ROS and small molecules, such as ascorbic acid (AA), that function to quench ROS [17]. In vitro, and potentially in vivo, the antioxidant potential of AA hangs in a tenuous balance with other cellular and medium constituents such as free metal ions. There are now many reports of AA functioning as a pro-oxidant in vitro [18–20], or demonstrating behaviour inconsistent with that of an antioxidant [1,2]. While the literature is not united in its explanation of this phenomenon, it is generally accepted that the oxidation of ascorbic acid in vitro is mediated by the reduction of Fe3+ to Fe2+ (or similarly with other transition metals), which then via the Haber–Weiss reaction [7,21,22] generates the potent hydroxyl radical and pro-oxidant activity. If such metals are necessary for the observed pro-oxidant activity of AA, then it is assumed that this process will be mitigated in vivo by the presence of proteins such as ferritin, transferrin and ceruloplasmin that chelate such metals [23]. Again, this theory is supported by the general observation that pro-oxidant activity is dampened in cell cultures by the addition of serum which is rich in metal-chelating proteins [8]. As AA is commonly found in our diet, and is frequently used as a dietary supplement, much investigation has already been pursued to expose potential in vivo pro-oxidant activity. Most [24– 28], but not all [24,29], in vivo studies report outcomes suggesting that AA behaves as an antioxidant. Interestingly, pro-oxidant activity is often regarded as an artefact of the analysis methods [21,30]. Furthermore, in potentially genuine cases where trauma has occurred, the pro-oxidant activity is thought to be the result of the release of cellular contents including transition metal ions [21,30]. However, even in the event of trauma the story may be more complex. Recent work presented by Niethammer et al. demonstrates that a tissue-scale gradient of hydrogen peroxide is generated by cells in response to a wound [31]. Thus the observed pro-oxidant activity may be the result of purposeful signalling mechanisms rather than simply the spillage of intracellular metal ions. The complexity of the redox hierarchy, lack of clarity on mechanism, and the probable variability between cell types make predicting precise environmental outcomes difficult. In cell culture this challenge has been previously noted and variable pro-oxidant activity has been attributed to culture medium selection [19]. Nearly a decade ago, Clement et al. made the following statement – ‘‘. . . it is tempting to wonder if some of the alleged antioxidant effects of ascorbic acid in cells in vitro could be attributed to low-level H2O2 production in the medium. . .” [19]. In order to provide further clarity on this issue we explored the dose and temporal impact of AA medium supplementation on KG1a (human myeloid leukaemia cells [32]) cultured in RPMI medium. In addition we independently evaluated changes in both intra and extracellular ROS. 2. Materials and methods KG1a cells were maintained in Roswell Park Memorial Institute (RPMI) medium (Gibco, Carlsbad CA) containing 10% foetal bovine serum (FBS) (Lonza, Basel, Switzerland) and 100 U/ml penicillin,


100 lg/ml streptomycin (1% p/s, Gibco). Twenty-four hours prior to initiating experiments, cultures were diluted to 105 cells/ml to generate consistency in log phase expansion and metabolic behaviour. 2.1. Cell culture Cell density was again adjusted to 105 cells/ml at the onset of each experiment. Medium was supplemented with 250 ng/ml of dihydrorhodamine 123 (DHR123, Sigma–Aldrich, St. Louis, MO), a ROS scavenger that fluoresces when oxidized [33]. Medium was titrated with L-ascorbic acid (Sigma–Aldrich) such that the working concentration in the cultures ranged from 10 to 250 lM. At least four replicate 200 ll cultures for each condition were maintained in 96-well plates (Nunc, Rochester, NY). Cultures were incubated in a 5% CO2 atmosphere at 37 °C for 2, 4 or 24 h. Cultures maintained for 2 or 4 h were evaluated with or without FBS supplementation. Twenty-four hour serum containing cultures were performed where DHR123 was added to cultures either at time zero or after completion of 22 h of the total 24-h culture period. 2.2. Intracellular ROS analysis Immediately following the designed culture period, cells were analysed by flow cytometry (FC-500, Beckman Coulter, Brea, CA) to assess DHR123 relative fluorescence (read using FL2 channel). For graphical purposes, raw fluorescence measurements were normalized against cultures containing DHR123, but without AA supplementation. 2.3. Extracellular ROS analysis Parallel cultures, and medium cocktails void of cells, were maintained in black tissue culture plates (Nunc, Rochester, NY) for evaluation of bulk culture or medium fluorescence at the same time points. Fluorescence was evaluated using a Polar Star Optima reader (BMG labtech, Offenburg, Germany) at 504 excitation and 530 emission. 2.4. Statistics Unless otherwise stated, SPSS 17.0 (SPSS Inc., Chicago, IL) was used for one-way analysis of variance (ANOVA) with Tukey post hoc tests to assess statistical significance, which was defined as p < 0.05. In all cases error bars represent one standard deviation. All conditions are replicated n = 4 times, and all experiments were repeated at least three times. Flow cytometry samples were 100 ll of the 200 ll total culture volume. Unless otherwise stated, all fluorescent measurements were normalized against a zero ascorbic acid control. 3. Results and discussion KG1a cultures were supplemented with L-ascorbic acid (AA) where the final concentration ranged from 10 to 250 lM. This concentration range represents concentrations commonly utilized in mesenchymal induction medium [3,34], shown to influence embryonic stem cell differentiation [2,35], and which enhance the reprogramming of somatic cells [1]. The relative intracellular and extracellular ROS formation was gauged by measuring the increase in fluorescence associated with oxidized DHR123. DHR123 can be oxidized to various extents by an assortment of ROS, including peroxide and hydroxyl radicals [36,37]. While the reduced form of DHR123 passes freely through the cell membrane, the oxidized form is trapped within the cell, thus providing a


M. Osiecki et al. / Biochemical and Biophysical Research Communications 400 (2010) 466–470

relative indication of the cumulative intracellular ROS formation [37]. Fig. 1A shows results from 24-h cultures where ROS probe DHR123 was added to cultures at time zero. An increased relative fluorescence reflects an increase in intracellular ROS. The results in Fig. 1A suggest that there is no net antioxidant or pro-oxidant activity associated with AA medium supplementation over the 24-h culture period. However, results shown in Fig. 1B, where DHR123 was added to cultures in the final 2 h of the 24 h culture period, indicate that supplementation of cultures with AA does result in net antioxidant behaviour. Further, the observed trend appears to indicate that increased AA supplementation correlates with increased antioxidant activity. This trend is consistent with the popular assumption that AA functions as an antioxidant. In contrast to the results shown in Fig. 1B, we observed that if the same cultures were assessed at 2 h then pro-oxidant activity rather than antioxidant activity dominated. Fig. 2A shows that in cultures supplemented with serum that intracellular pro-oxidant activity was statistically significant for concentrations of AA greater than 200 lM. Interestingly, when we fit a curve to this data the p-values for the coefficients suggest, with 99% confidence, that there is a parabolic relationship with antioxidant activity at lower concentrations of AA, before pro-oxidant activity is established at higher AA concentrations. Fig. 2B shows that in serum-free cultures that pro-oxidant activity onset occurs at a lower AA concentration and that this activity is also greater in magnitude than that observed in serum-supplemented cultures. This result is consistent with previous reports that the chelation of free metal ions in culture medium by serum proteins at least partially mitigates AA driven ROS production [8]. However, it is likely that this relationship is more complex. We observed that the base-line ROS formation in

Fig. 1. In (A) there was no net intracellular pro-oxidant or antioxidant activity, where DHR123 was added to cultures at time zero of the 24 h culture. In contrast (B) shows that when DHR123 was added at 22 h of the 24 h culture, there was net intracellular antioxidant behaviour.

Fig. 2. In (A) intracellular pro-oxidant activity of ascorbic acid at 2 h in serumsupplemented medium is shown. The titration follows a statistically significant (p < 0.01) parabolic shape indicating that lower concentrations of AA resulted in true antioxidant behaviour, before net pro-oxidant behaviour dominated. In (B) the onset of intracellular pro-oxidant behaviour occurred at a lower concentrations of AA and that the magnitude of this behaviour was greater in serum-free cultures is shown.

cells cultured in serum-free medium was nearly twofold greater than seen in cells maintained in serum-supplemented medium (fold increase = 1.92 ± 0.09, n = 4, average ± standard deviation). Upregulation of redox signalling and apoptotic processes are known to be triggered in serum-starved cells [38], and this process is believed to be mediated by both mitochondria and NOX ROS generation [39]. Thus, from our analysis it is not possible to be certain to what extent purposeful redox signalling driven by serum starvation or less encumbered Haber–Weiss reactions is the cause of the greater intracellular ROS content observed in serum-free medium. Intracellular ROS levels were further elevated in AA containing cultures at 4 h (Fig. 3A and B). At 4 h, pro-oxidant behaviour was statistically significant down to concentration of 100 lM in both serum containing and serum-free medium. The fold-increase saturated in serum-free conditions above 150 lM AA and the level at 250 lM AA was no longer greater than in serum containing conditions at 250 lM AA. It is possible that the saturation reflects a technical limitation of the assay rather than a true saturation. It is also critical to recognize that the transport of reduced and oxidized AA into the cell occurs via two different transport mechanisms and that it is possible that saturation of these mechanisms may play a role in the temporal observations made [40]. To summarize, the critical insight provided by Figs. 1–3 is that if the intracellular redox state of AA supplemented cultures are evaluated at 22–24 h, then net antioxidant behaviour is observed, while if the same cultures are evaluated at 2–4 h then intracellular pro-oxidant activity is observed. This temporal relationship may

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Fig. 3. In (A) intracellular pro-oxidant activity at AA concentrations greater than 100 lM in serum-supplemented medium is shown. In (B) intracellular pro-oxidant activity is prevalent at AA concentrations greater than 100 lM in serum-free medium is shown.

Fig. 4. (A) Reveals that, with the exception of medium containing cells and serum, the addition of 200 lM AA always results in an antioxidant effect (p < 0.05, n = 4, Student’s t-test). (B) Reveals that at 24 h the addition of higher concentrations of AA results in increased antioxidant activity in cell-free medium.

suggest that studies assessing intracellular redox at a single time point and reporting antioxidant behaviour may have simply over-looked a period of pro-oxidant activity. This possibility is enhanced by the very nature by which we maintain cells in culture. For example, the daily exchange and analysis of culture medium in human embryonic stem cell cultures might very well result in brief periods of pro-oxidant activity being over-looked. Such activity might explain DNA demethylation events observed in response to AA supplementation [4]. Extracellular conditions for each intracellular time point were replicated, with the addition of a cell-free control to elucidate the role that cells have on the final bulk culture outcome. However, even in medium containing cells the measurements made using a fluorescence plate reader are assumed to be representative of the extracellular environment as at 105 cells/ml (assuming an average cell diameter of 10 lm) the cells comprise only a 0.05% fraction of the total culture volume. Fig. 4A provides a summary plot of the extracellular results obtained over the first 2–4 h of culture, while Fig. 4B summarizes results at 24 h. The most critical observation is that the addition of AA to the medium never resulted in pro-oxidant behaviour in the extracellular environment. This data is shown in more detail in Fig. S1 (Supplementary data). While the addition of AA to medium containing cells and serum did not result in a measurable antioxidant effect, the ROS levels in these cultures was as low or lower than any other test condition measured at this same time point. Thus, it may be that AA is behaving similarly in these cultures, but that cells functioning normally in serum-supplemented cultures mask the effect of AA supplementation. Work from our laboratory (results not shown), as well as other published work [41], demonstrates that haematopoietic cells do reduce the ROS content

of the extracellular environment, likely through the release of antioxidant enzymes such as catalase. Thus, we propose that AA does continue to function extracellularly as an antioxidant, at these concentrations, even in medium containing both cells and serum and that this effect is masked by the antioxidant environmental conditioning provided by cells cultured in serum-supplemented medium. At 24 h (Fig. 4B) we did not evaluate cells cultured in the absence of serum, as the cells (KG1a) undergo apoptosis when serum-starved and the oxidative mechanism of apoptosis, and the fact that cells would not be behaving normally, would confound results [38]. Only in the absence of cells is there measurable antioxidant activity as a result of AA supplementation. Again, the raw values of the cell containing medium are equal to the lowest ROS values in the AA supplemented cell-free medium suggesting that the cells are modifying the environment and masking the effects of AA supplementation. Perhaps the most interesting observation is realized if we contrast results shown in Figs. 2 and 3 with Fig. 4A which reveals that it is possible for intracellular pro-oxidant activity and extracellular antioxidant activity to occur simultaneously in AA supplemented cultures. One possible explanation for this phenomenon might be the accumulation, and thus concentration of iron in cultured cells relative to the bulk medium [42]. This counterintuitive result indicates that not only do temporal considerations need to be made when assessing the impact of AA supplementation on intracellular ROS, but equally critical will be the selection of either an intracellular or extracellular marker to indicate the relevant oxidative environment. For example, inconsistencies found in previous in vivo studies could be related to this same phenomenon. Previous in vivo studies in which extracellular markers were assessed


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indicate no pro-oxidant activity [28], while similar studies in which intracellular markers were evaluated pro-oxidant activity was observed [29]. These results parallel our own finding, suggesting that perhaps in vivo two different mechanisms are simultaneously at play. 4. Conclusion Recently there have been reports of AA influencing culture outcomes in a manner that could not be attributed to antioxidant activity [1–4]. In this paper we show that intracellular doses of AA up to 250 lM appear to drive intracellular antioxidant behaviour at 24 h. However, if these same cells are assessed at 2 or 4 h time points then we observed pro-oxidant activity at higher AA concentrations. Paradoxically, at these early time points, it is possible to observe extracellular antioxidant activity and intracellular pro-oxidant activity simultaneously. This result has not, to our knowledge, been previously reported and may give insight to a number of previously unexplained results. Acknowledgments The Authors would like to thank Inner Wheel Australia and The Queensland University of Technology’s Vice Chancellor’s fellowship program for funding this work. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2010.08.052. References [1] M.A. Esteban, T. Wang, B. Qin, J. Yang, D. Qin, J. Cai, W. Li, Z. Weng, J. Chen, S. Ni, K. Chen, Y. Li, X. Liu, J. Xu, S. Zhang, F. Li, W. He, K. Labuda, Y. Song, A. Peterbauer, S. Wolbank, H. Redl, M. Zhong, D. Cai, L. Zeng, D. Pei, Vitamin C enhances the generation of mouse and human induced pluripotent stem cells, Cell Stem Cell 6 (2010) 71–79. [2] T. Takahashi, B. Lord, P.C. Schulze, R.M. Fryer, S.S. Sarang, S.R. Gullans, R.T. Lee, Ascorbic acid enhances differentiation of embryonic stem cells into cardiac myocytes, Circulation 107 (2003) 1912–1916. [3] K.M. Choi, Y.K. Seo, H.H. Yoon, K.Y. Song, S.Y. Kwon, H.S. Lee, J.K. Park, Effect of ascorbic acid on bone marrow-derived mesenchymal stem cell proliferation and differentiation, J. Biosci. Bioeng. 105 (2008) 586–594. [4] T.L. Chung, R.M. Brena, G. Kolle, S.M. Grimmond, B.P. Berman, P.W. Laird, M.F. Pera, E.J. Wolvetang, Vitamin C promotes widespread yet specific DNA demethylation of the epigenome in human embryonic stem cells, Stem Cells (2010). [5] A. Grzelak, B. Rychlik, G. Bartosz, Reactive oxygen species are formed in cell culture media, Acta Biochim. Pol. 47 (2000) 1197–1198. [6] J.F. Turrens, Mitochondrial formation of reactive oxygen species, J. Physiol. 552 (2003) 335–344. [7] J.R. Bucher, M. Tien, L.A. Morehouse, S.D. Aust, Redox cycling and lipid peroxidation: the central role of iron chelates, Fundam. Appl. Toxicol. 3 (1983) 222–226. [8] K. Satoh, Y. Ida, S. Kimura, K. Taguchi, M. Numaguchi, K. Gomi, M. Kochi, H. Sakagami, Chelating effect of human serum proteins on metal-catalyzed ascorbate radical generation, Anticancer Res. 17 (1997) 4377–4380. [9] B. Halliwell, M. Whiteman, Measuring reactive species and oxidative damage in vivo and in cell culture: how should you do it and what do the results mean?, Br J. Pharmacol. 142 (2004) 231–255. [10] H. Sies, Oxidative stress: oxidants and antioxidants, Exp. Physiol. 82 (1997) 291–295. [11] C. Piccoli, A. D’Aprile, M. Ripoli, R. Scrima, L. Lecce, D. Boffoli, A. Tabilio, N. Capitanio, Bone-marrow derived hematopoietic stem/progenitor cells express multiple isoforms of NADPH oxidase and produce constitutively reactive oxygen species, Biochem. Biophys. Res. Commun. 353 (2007) 965–972. [12] L.S. Haneline, Redox regulation of stem and progenitor cells, Antioxid. Redox Signal. 10 (2008) 1849–1852. [13] M. Ushio-Fukai, N. Urao, Novel role of NADPH oxidase in angiogenesis and stem/progenitor cell function, Antioxid. Redox Signal. 11 (2009) 2517– 2533.

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