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Folic acid-mediated mitochondrial activation for protection against oxidative stress in human dental pulp stem cells derived from deciduous teeth
Biochemical and Biophysical Research Communications 508 (2019) 850e856
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Folic acid-mediated mitochondrial activation for protection against oxidative stress in human dental pulp stem cells derived from deciduous teeth Yu Zhang, Hiroki Kato*, Hiroshi Sato, Haruyoshi Yamaza, Yuta Hirofuji, Xu Han, Keiji Masuda**, Kazuaki Nonaka Section of Oral Medicine for Children, Division of Oral Health, Growth and Development, Faculty of Dental Science, Kyushu University, 3-1-1 Maidashi, Higashi-Ku, Fukuoka, 812-8582, Japan
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Article history: Received 7 November 2018 Accepted 27 November 2018 Available online 7 December 2018
Enzymatic antioxidant systems, mainly involving mitochondria, are critical for minimizing the harmful effects of reactive oxygen species, and these systems are enhanced by interactions with nonenzymatic antioxidant nutrients. Because fetal growth requires extensive mitochondrial respiration, pregnant women and fetuses are at high risk of exposure to excessive reactive oxygen species. The enhancement of the antioxidant system, e.g., by nutritional management, is therefore critical for both the mother and fetus. Folic acid supplementation prevents homocysteine accumulation and epigenetic dysregulation associated with one-carbon metabolism. However, few studies have examined the antioxidant effects of folic acid for healthy pregnancy outcomes. The purpose of this study was to elucidate the association between the antioxidant effect of folic acid and mitochondria in undifferentiated cells during fetal growth. Neural crest-derived dental pulp stem cells of human exfoliated deciduous teeth were used as a model of undifferentiated cells in the fetus. Pyocyanin induced excessive reactive oxygen species, resulting in a decrease in cell growth and migration accompanied by mitochondrial fragmentation and inactivation in dental pulp stem cells. This damage was significantly improved by folic acid, along with decreased mitochondrial reactive oxygen species, PGC-1a upregulation, DRP1 downregulation, mitochondrial elongation, and increased ATP production. Folic acid may protect undifferentiated cells from oxidative damage by targeting mitochondrial activation. These results provide evidence for a new benefit of folic acid in pregnant women and fetuses. © 2018 Elsevier Inc. All rights reserved.
Keywords: Oxidative stress Folic acid Mitochondria Stem cells from human exfoliated deciduous teeth
1. Introduction Reactive oxygen species (ROS) are inevitably produced by mitochondrial aerobic respiration [1]. While ROS at appropriate levels play important roles as signaling molecules, excessive ROS can have various deleterious effects on cells [2]. Antioxidant systems, including mitochondrial antioxidant enzymes and non-
Abbreviations: DRP1, dynamin related protein 1; FA, folic acid; PGC-1a, peroxisome proliferator-activated receptor gamma coactivator 1-alpha; PYO, pyocyanin; ROS, reactive oxygen species; SHED, stem cells from human exfoliated deciduous teeth. * Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (H. Kato), [email protected] (K. Masuda). https://doi.org/10.1016/j.bbrc.2018.11.169 0006-291X/© 2018 Elsevier Inc. All rights reserved.
enzymatic antioxidant nutrients, are therefore indispensable for cells [3]. During pregnancy in mammals, including humans, a large amount of ROS is generated due to enhanced mitochondrial respiration along with the growth of the fetus, uterus, and placenta [4]. The attenuation or exhaustion of antioxidant systems generates excessive ROS, which can have various adverse effects not only on the mother but also on the fetus [4]. Maternal nutritional management, including supplementation with antioxidant nutrients, is important for enhancing antioxidant systems in pregnant women throughout gestation [4]. Folic acid (FA) is a recommended supplement for healthy pregnancy outcomes [5]. FA contributes to epigenetic regulation via DNA and histone methylation as a result of one-carbon metabolism using methionine in cells [6,7]. Insufficient FA leads to the dysregulation of one-carbon metabolism, which may alter the expression profiles of epigenetically modified genes [8,9]. Furthermore, an FA
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deficiency disrupts the re-methylation of homocysteine to methionine in one-carbon metabolism, resulting in homocysteine accumulation, which is associated with various maternal and fetal abnormalities [5]. Thus, FA supplementation in pregnant women mainly contributes to maternal and fetal health by affecting onecarbon metabolism. In addition, previous studies have shown that FA exerts an antioxidant effect by functioning as a free radical scavenger [10]. However, few studies have focused on the antioxidant effect of FA during fetal development. Stem cells from human exfoliated deciduous teeth (SHED) are undifferentiated mesenchymal cells derived from the neural crest; they have multipotency, including neuron and bone differentiation potential [11]. Therefore, SHED are a potentially useful cellular model for analyzing the antioxidant effect of FA on undifferentiated embryonic cells. Pyocyanin (PYO) is produced by Pseudomonas aeruginosa and is commonly used as an experimental reagent to examine oxidative stress. PYO is reduced by accepting electrons from nicotinamide adenine dinucleotide phosphate (NADPH) under aerobic conditions in host cells, which reacts with O2 to produce a superoxide radical, generating ROS [12]. The purpose of this study was to elucidate the association between the antioxidant effect of FA and mitochondria in undifferentiated SHED derived from a healthy child with typical development. 2. Materials and methods 2.1. Isolation and preparation of SHED A human exfoliated deciduous tooth from a normal 7-year-old individual was provided by Pediatric Dentistry and Special Need Dentistry at Kyushu University Hospital in Japan. Written informed consent was obtained from the child's guardians. The isolation procedure was completed as previously described [13]. Briefly, the pulp tissue was subjected to enzymatic dissociation in 3 mg/mL collagenase I (Worthington Biochemical, Worthington, NJ, USA) and 4 mg/mL dispase II (Wako, Osaka, Japan) for 1 h and was then maintained at 37 C in a humidified 5% CO2 incubator in Eagle's Minimal Essential Medium, Alpha Modification (a-MEM; SigmaAldrich, MO, USA) containing 15% fetal bovine serum (SigmaAldrich), 100 mM L-ascorbic acid 2-phosphate (Wako), 2 mM L-
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glutamine (Life Technologies, Grand Island, NY, USA), 250 mg/mL Fungizone (Life Technologies), 100 U/mL penicillin (Life Technologies), and 100 mg/mL streptomycin (Life Technologies). Cells at not more than 10 passages were used for subsequent analyses. 2.2. Cell proliferation assay Cells (1 105) were seeded in 6-well plates. After 24 h, the cells were cultured in the presence or absence of 10e80 mM FA (Wako) for 6 h. Then, PYO (Sigma) was added to the wells at concentrations ranging from 0 to 100 mM. After 24 h, the cells were collected by trypsinization and stained with 0.4% trypan blue solution (Wako). The cells were counted using a hemocytometer. 2.3. Cell migration assay A Culture-Insert 2 Well (ibidi, Munich, Germany) was set into a 6-well plate (Corning, Inc., Corning, NY, USA). Cells (2.1 103) were seeded in the Culture-Insert 2 Well (ibidi). After 24 h, the CultureInsert 2 Well was removed and the cells were cultured in the presence or absence of 10e80 mM FA (Wako) for 6 h. Then, PYO (Sigma) was added to the assigned wells at a concentration of 100 mM, and the cells were cultured for an additional 48 h. Images of the wounded edges were obtained using an IX41 microscope (Olympus, Tokyo, Japan) equipped with a digital camera (NEX-5T; Sony, Tokyo, Japan) with an NY-1S adaptor (MeCan Imaging, Saitama, Japan). The images of wounded edges were collected 24 and 48 h after the addition of PYO and results were quantified using ImageJ [14]. 2.4. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) Cells (1 105) were seeded in a 6-well plate. After 24 h, the cells were cultured in the presence or absence of 80 mM FA (Wako) for 6 h. Then, PYO (Sigma) was added to the assigned wells at a concentration of 100 mM. After 8 h, total RNA extraction and RT-qPCR were performed as described previously [15]. The sequences of the primer sets used in this study were as follows: dynamin related protein 1 (DRP1), 50 -GCTCCAGGACGTCTTCAACA-30 (forward) and
Fig. 1. ROS production and growth inhibition by pyocyanin. (A, B) Cells were cultured in the presence or absence of the indicated concentration of pyocyanin (PYO). (A) Intracellular cell ROS production was measured. The relative levels were calculated, and the level in the absence of pyocyanin was set to 1. Means ± SEMs from three experiments are shown. *p < 0.05 (B) Cells were counted. Means ± SEMs from three experiments are shown. *p < 0.05, **p < 0.01.
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50 -TAGCACTGAGCTCTTTCCGC-30 (reverse); peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1a), 50 -GGCAGAAGGCAATTGAAGAG-30 (forward) and 50 TCAAAACGGTCCCTCAGTTC-30 (reverse); 18S rRNA, 50 -CGGCTACCACATCCAAGGAA-30 (forward) and 50 -GCTGGAATTACCGCGGCT-30 (reverse). The relative expression levels of the target genes were analyzed using the comparative threshold cycle method by normalization against the levels of 18S rRNA.
2.5. Detection of intracellular ROS A total of 1 105 cells was seeded in 6-well plates (Corning). After 24 h, the cells were cultured in the presence or absence of 80 mM FA (Wako) for 6 h. Then, PYO (Sigma) was added to the assigned wells at a concentration of 100 mM. After 24 h, the cells
were incubated with 5 mM Cell ROX Green (Life Technologies) for 30 min. The cells were then treated with Tryple Express (Life Technologies) for detachment from the culture plate. The fluorescence signal was measured using a FACSVerse instrument (BD Biosciences, San Jose, CA, USA).
2.6. Detection of mitochondrial ROS Cells (1 105) were seeded in a 6-well plate (Corning). After 24 h, the cells were cultured in the presence or absence of 80 mM FA (Wako) for 6 h. Then, PYO (Sigma) was added to the assigned wells at a concentration of 100 mM. After 8 h, the cells were incubated with 5 mM MitoSOX Red (Life Technologies) for 10 min and treated with Tryple Express (Life Technologies) for detachment from the culture plate. The fluorescence signal was measured using a
Fig. 2. Recovery of the PYO-induced inhibition of cell growth and migration by FA. Cells were cultured in the presence or absence of the indicated concentrations of pyocyanin (PYO) and folic acid (FA). (A) Cells were counted. Means ± SEMs from four experiments are shown. **p < 0.01, ***p < 0.001. n.s., not significant. (B, C) Cell migration was measured. (B) Images of wounded edges. Left panels show original images and other panels show grayscale images converted using ImageJ. Scale bar ¼ 250 mm. (C) Images of wounded edges were quantitated using ImageJ. Values for the wounded edges at time 0 were set to 100%. Means ± SEMs from three experiments are shown. n.s., not significant. ###p < 0.001 compared with PYO‒ FA‒. *p < 0.05 compared with PYO þ FAþ. ***p < 0.001 compared with PYO þ FAþ.
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FACSVerse instrument (BD Biosciences). 2.7. Measurement of intracellular ATP levels Cells (1 105) were seeded in a 6-well plate. After 24 h, the cells were cultured in the presence or absence of 80 mM FA (Wako) for 6 h. Then, PYO (Sigma) was added to the assigned wells at a concentration of 100 mM. After 24 h, the cells were harvested in icecold PBS. The CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, WI, USA) was used to measure intracellular ATP levels. The ATP levels were normalized to the protein content of the samples. 2.8. Immunocytochemistry Cells (1 105) were seeded on a cover glass in a 6-well plate. After 24 h, the cells were cultured in the presence or absence of 80 mM FA (Wako) for 6 h. Then, PYO (Sigma) was added to the assigned wells at a concentration of 100 mM. After 8 h, the cells were fixed with 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4) for 10 min at 25 C and then permeabilized with 0.1% Triton X-100 in PBS for 5 min. The cells were blocked with 2% bovine serum albumin (BSA; Wako Pure Chemical Industries) in PBS for 20 min and then incubated with an anti-Tom20 antibody (1:200, #sc-11415; Santa Cruz Biotechnology, Santa Cruz, CA, USA). After 90 min, the cells were incubated with Alexa Fluor-conjugated secondary antibody (Life Technologies) in the dark for 60 min. After staining with antibodies, the nuclei were stained with 1 mg/mL 40 ,6diamidino-2-phenylindole dihydrochloride (DAPI; Dojindo, Kumamoto, Japan). The cells were then mounted using ProLong Diamond (Life Technologies). Fluorescence images were captured using an Axio Imager M2 (Zeiss, Oberkochen, Germany) equipped with the ApoTome2 (Zeiss). 2.9. Statistical analysis Statistical analyses were performed by Student's t-tests using JMP version 13 (SAS Institute, Raleigh, NC, USA). A value of p < 0.05 was considered statistically significant.
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by FA supplementation together with PYO, 75% of the open wound area was closed. A quantitative analysis showed a significant difference between treatments at 48 h (Fig. 2C). These results suggested that the inhibition of SHED growth and migration by PYO-mediated oxidative stress is improved by the antioxidant effect of FA. 3.3. Effect of FA on PYO-mediated oxidative stress The antioxidant effect of FA on extensive ROS generation by PYO was evaluated (Fig. 3). Mitochondrial ROS levels were significantly higher in the presence of 100 mM PYO alone than in the control cultures. A significant reduction of mitochondrial ROS levels was observed by treatment with 80 mM FA and 100 mM PYO compared to PYO alone, although mitochondrial ROS levels did not recover to the level observed in the control. These results suggested that FA influences PYO-mediated oxidative stress by the attenuation of excessive mitochondrial ROS in SHED. 3.4. Enhanced mitochondrial activation with elongation and biogenesis in SHED by FA The antioxidant system of cells depends on mitochondrial activity, which is enhanced by mitochondrial elongation and biogenesis. The association of mitochondrial morphology and biogenesis with the antioxidant effect of FA was examined. While mitochondrial fragmentation was observed in the presence of PYO alone, it was diminished by supplementation with FA (Fig. 4A and B). Consistent with this observation, the mRNA expression of DRP1, an essential factor for mitochondrial fragmentation, was upregulated by treatment with PYO alone and downregulated by supplementation with FA (Fig. 4C). Furthermore, the mRNA expression of PGC-1a, a master regulator of mitochondrial biogenesis, was downregulated by treatment with PYO alone and upregulated by supplementation with FA (Fig. 4D). Intracellular ATP levels decreased in the presence of PYO alone and increased by supplementation with FA (Fig. 4E). These results suggested that the antioxidant effect of FA on PYO-
3. Results 3.1. Intracellular ROS production and growth inhibition by PYO PYO elevated intracellular ROS levels in SHED in a dosedependent manner; ROS levels were highest for 50 mM PYO (Fig. 1A). PYO suppressed the growth of SHED in a dose-dependent manner and showed the maximal inhibitory effect at 100 mM (Fig. 1B and Supplemental fig.). These results suggested that there is an association between PYO-mediated oxidative stress and cellular growth inhibition in SHED. 3.2. Recovery of the PYO-induced inhibition of growth and migration by FA The effect of FA on the PYO-induced inhibition of SHED growth was examined. In the presence of 100 mM PYO, which showed the maximal inhibitory effect on SHED growth, FA supplementation improved cell growth in a dose-dependent manner and showed the maximal effect at 80 mM (Fig. 2A). In the absence of PYO, 80 mM FA did not alter cell growth. Furthermore, differences in the effects of PYO and FA on cell migration were observed (Fig. 2B and C). In control cultures without PYO and FA, the open wound area was completely closed after 48 h by SHED migration. In the presence of PYO alone, only 50% of the open wound area was closed. However,
Fig. 3. Reduction of PYO-induced mitochondrial ROS by folic acid. Cells were cultured in the presence or absence of 100 mМ pyocyanine (PYO) and 80 mM folic acid (FA) and mitochondrial ROS levels were measured. The relative levels were calculated, and the level in the absence of pyocyanin and folic acid was set to 1. Means ± SEMs from four experiments are shown. *p < 0.05.
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Fig. 4. Recovery of the pyocyanin-induced fragmentation of mitochondria and reduction in mitochondrial activity by folic acid. (A) Cells were cultured in the presence or absence of pyocyanin and folic acid, stained with an antibody against Tom20 (red), a mitochondrial marker, and counterstained with DAPI (blue). Lower panels show a detailed view of the boxed region in the upper panels. Yellow arrows indicate elongated mitochondria. White arrows indicate fragmented mitochondria. Scale bar ¼ 25 mm (upper panel), 5 mm (lower panel). (B) Cells with fragmented mitochondria were counted. Percentages of the total cell number were calculated. Data represent mean ± SEMs from the analysis of 30 cells from 5 immunostained images. *p < 0.05, **P < 0.01. (C, D) mRNA expression of DRP1 (C) and PGC-1a (D) were measured by RT-qPCR. The relative levels were calculated, and the level in the absence of pyocyanin and folic acid was set to 1. Data represent the means ± SEMs from four experiments. *p < 0.05, **p < 0.01, ***p < 0.001. (E) Intracellular ATP levels were measured by an ATP luminescence assay. The relative levels were calculated, and the level in the absence of pyocyanin and folic acid was set to 1. Data represent means ± SEMs from four experiments. *p < 0.05, ***p < 0.001.
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mediated oxidative stress was associated with enhanced mitochondrial activity to diminish excessive ROS by promoting mitochondrial elongation and biogenesis. 4. Discussion In this study, PYO-induced ROS production inhibited SHED growth and migration, with mitochondrial inactivation and fragmentation, and these changes were alleviated by FA via the upregulation of PGC-1a and downregulation of DRP1. These results suggested that FA targeted mitochondria to activate antioxidant systems, thereby protecting undifferentiated cells from oxidative stress. SHED are human dental pulp stem cells derived from the cranial neural crest [11]. In the third week after fertilization, the neural fold and groove are formed from the ectodermal germ layer, followed by neural tube formation [16]. During this stage, a neural crest is formed at the tip of the neural fold, from which neural crest cells dissociate and invade the mesoderm to form various organs [17]. The cranial neural crest cells migrate to branchial arches that will form facial tissues, including the bone, muscle, fat, dentin, pulp, and trigeminal nerve system [18]. Considering that SHED can differentiate into neurons, osteoblasts, adipocytes, odontoblasts, or myoblasts in vitro, they may at least partially maintain the features of fetal cranial neural crest cells [11,19]. A recent study has shown that FA has an effect on fetal neural tube defects using human induced pluripotent stem cells as a model of undifferentiated cells [20]. Embryonic stem cells (ESCs) have also been proposed as a model to examine FA-mediated one-carbon metabolism and epigenetic regulation, but the use of human ESCs has ethical limitations [21]. Despite previous studies, the antioxidant effect of FA is unresolved. SHED are a promising human cellular model to elucidate the mechanism underlying the effects of FA on oxidative stress at the fetal stage. In this study, the inhibition of cellular functions by PYO-induced oxidative stress in SHED was improved by FA, with decreased levels of mitochondrial ROS. Since previous studies of cell-free systems have shown that FA is a free radical scavenger, it is possible that excessive free radicals induced by PYO was attenuated by this reaction in SHED [10]. In contrast, mitochondria have a major role in ROS regulation by superoxide dismutase 2, a mitochondrial antioxidant enzyme [22]. Excessive ROS damages various cellular components, including mitochondrial enzymes, leading to the inactivation of the mitochondrial antioxidant system. While mitochondrial inactivation has been associated with mitochondrial fragmentation in various kind of damaged cells, acceleration of mitochondrial fusion and elongation has been shown to improve mitochondrial activity and cellular function [23‒26]. It is therefore likely that the FA-mediated Drp1 downregulation observed in SHED may have improved the mitochondrial antioxidant activity through modulation of mitochondrial morphology. PGC-1a has been shown to regulate several molecules involved in mitochondrial fusion and fission, including DRP1 [27]. In SHED, FA-mediated PGC-1a upregulation may have been involved in the mitochondrial fusion and activation through Drp1 downregulation in the downstream pathway. The effect of FA on mitochondrial elongation and activation may be a novel mechanism for the protection of undifferentiated cells from oxidative damage. There are several limitations of this research. First, the regulatory mechanism by which FA alters the expression of PGC-1a and DRP1 for mitochondrial activation remains to be elucidated. The epigenetic status of these genes may be altered by FA-mediated one-carbon metabolism in SHED. Further studies are required to elucidate the expression profile or epigenetic modulation of the genes in the pathway downstream of FA leading to mitochondrial
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activation. Second, the antioxidant effect of FA and underlying mechanism during cell differentiation are unresolved. An experimental system that differentiates SHEDs into neurons or osteoblasts would be useful for addressing this issue. Third, the relevance of FA doses recommended for pregnant women and FA concentrations effective for inducing antioxidant stress in fetal undifferentiated cells remains unclear. It may be necessary to examine the in vivo administration of FA in pregnant animals. In conclusion, FA may target and activate the mitochondrial antioxidant system to protect undifferentiated cells from oxidative damage. These results provide new evidence to support the effectiveness of FA in pregnant women to protect the fetus from oxidative damage. Conflicts of interest The authors have no conflicts of interest to declare. Acknowledgments We thank all the members of the Department of Pediatric & Special Needs Dentistry at Kyushu University Hospital for valuable suggestions, technical support, and materials. We appreciate the technical assistance provided by the Research Support Center at the Research Center for Human Disease Modeling, Kyushu University Graduate School of Medical Sciences. This work was supported by the Japan Society for the Promotion of Science (KAKENHI grant numbers JP25670877, and JP16K15839), Japan. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.11.169. References [1] V. Adam-Vizi, C. Chinopoulos, Bioenergetics and the formation of mitochondrial reactive oxygen species, Trends Pharmacol. Sci. 27 (2006) 639e645. [2] W. Droge, Free radicals in the physiological control of cell function, Physiol. Rev. 82 (2002) 47e95. [3] A.Y. Andreyev, Y.E. Kushnareva, A.A. Starkov, Mitochondrial metabolism of reactive oxygen species, Biochemistry (Mosc.) 70 (2005) 200e214. [4] K.H. Al-Gubory, P.A. Fowler, C. Garrel, The roles of cellular reactive oxygen species, oxidative stress and antioxidants in pregnancy outcomes, Int. J. Biochem. Cell Biol. 42 (2010) 1634e1650. [5] K. Fekete, C. Berti, I. Cetin, M. Hermoso, B.V. Koletzko, T. Decsi, Perinatal folate supply: relevance in health outcome parameters, Matern. Child Nutr. Suppl 2 (2010) 23e38. [6] J.T. Fox, P.J. Stover, Folate-mediated one-carbon metabolism, Vitam. Horm. 79 (2008) 1e44. [7] S.J. Mentch, J.W. Locasale, One-carbon metabolism and epigenetics: understanding the specificity, Ann. N. Y. Acad. Sci. 1363 (2016) 91e98. rez, E. S [8] N. Alata Jimenez, S.A. Torres Pe anchez-V asquez, J.I. Fernandino, P.H. Strobl-Mazzulla, Folate deficiency prevents neural crest fate by disturbing the epigenetic Sox 2 repression on the dorsal neural tube, Dev. Biol. S0012e1606 (2018) 30595eX, https://doi.org/10.1016/j.ydbio.2018.08.001. [9] A. Caffrey, R.E. Irwin, H. McNulty, J.J. Strain, D.J. Lees-Murdock, B.A. McNulty, M. Ward, C.P. Walsh, K. Pentieva, Gene-specific DNA methylation in newborns in response to folic acid supplementation during the second and third trimesters of pregnancy: epigenetic analysis from a randomized controlled trial, Am. J. Clin. Nutr. 107 (2018) 566e575. [10] R. Joshi, S. Adhikari, B.S. Patro, S. Chattopadhyay, T. Mukherjee, Free radical scavenging behavior of folic acid: evidence for possible antioxidant activity, Free Radic. Biol. Med. 30 (2001) 1390e1399. [11] M. Miura, S. Gronthos, M. Zhao, B. Lu, L.W. Fisher, P.G. Robey, S. Shi, SHED: stem cells from human exfoliated deciduous teeth, Proc. Natl. Acad. Sci. U. S. A 100 (2003) 5807e5812. [12] B. Rada, T.L. Leto, Pyocyanin effects on respiratory epithelium: relevance in Pseudomonas aeruginosa airway infections, Trends Microbiol. 21 (2013) 73e81. [13] H. Kato, T.T.M. Pham, H. Yamaza, K. Masuda, Y. Hirofuji, X. Han, H. Sato, T. Taguchi, K. Nonaka, Mitochondria regulate the differentiation of stem cells from human exfoliated deciduous teeth, Cell Struct. Funct. 116 (2017) 105e116, https://doi.org/10.1247/csf.17012.
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[14] C.A. Schneider, W.S. Rasband, K.W. Eliceiri, NIH Image to ImageJ: 25 years of image analysis, Nat. Methods 9 (2012) 671e675. [15] H. Kato, X. Han, H. Yamaza, K. Masuda, Y. Hirofuji, H. Sato, T.T.M. Pham, T. Taguchi, K. Nonaka, Direct effects of mitochondrial dysfunction on poor bone health in Leigh syndrome, Biochem. Biophys. Res. Commun. 493 (2017) 207e212. [16] N.D. Greene, A.J. Copp, Neural tube defects, Annu. Rev. Neurosci. 37 (2014) 221e242. [17] R. Mayor, E. Theveneau, The neural crest, Development 11 (2013) 2247e2251. [18] P.M. Kulesa, C.M. Bailey, J.C. Kasemeier-Kulesa, R. McLennan, Cranial neural crest migration: new rules for an old road, Dev. Biol. 344 (2010) 543e554. [19] M. La Noce, L. Mele, V. Tirino, F. Paino, A. De Rosa, P. Naddeo, P. Papagerakis, G. Papaccio, V. Desiderio, Neural crest stem cell population in craniomaxillofacial development and tissue repair, Eur. Cell. Mater. 28 (2014) 348e357. [20] V. Sahakyan, R. Duelen, W.L. Tam, S.J. Roberts, H. Grosemans, P. Berckmans, G. Ceccarelli, G. Pelizzo, V. Broccoli, J. Deprest, F.P. Luyten, C.M. Verfaillie, M. Sampaolesi, Folic acid exposure rescues spina bifida aperta phenotypes in human induced pluripotent stem cell model, Sci. Rep. 8 (2018) 2942. [21] S. Kasulanati, V. Venkatesan, Understanding pluripotency under folic acid deficiency using embryonic stem cells as an in vitro model, Med. Hypotheses
111 (2018) 24e26. [22] R.A. Weisiger, I. Fridovich, Mitochondrial superoxide simutase. Site of synthesis and intramitochondrial localization, J. Biol. Chem. 248 (1973) 4793e4796. [23] T. Wai, T. Langer, Mitochondrial dynamics and metabolic regulation, Trends Endocrinol. Metabol. 27 (2016) 105e117. [24] C. Hu, Y. Huang, L. Li, Drp1-Dependent mitochondrial fission plays critical roles in physiological and pathological progresses in mammals, Int. J. Mol. Sci. 18 (2017) 144. [25] L.C. Gomes, L. Scorrano, Mitochondrial elongation during autophagy: a stereotypical response to survive in difficult times, Autophagy 7 (2011) 1251e1253. [26] A.A. Rosdah, J.K. Holien, L.M. Delbridge, G.J. Dusting, S.Y. Lim, Mitochondrial fission - a drug target for cytoprotection or cytodestruction? Pharmacol. Res. Perspect. 4 (2016), e00235. [27] K. Peng, L. Yang, J. Wang, F. Ye, G. Dan, Y. Zhao, Y. Cai, Z. Cui, L. Ao, J. Liu, Z. Zou, Y. Sai, J. Cao, The interaction of mitochondrial biogenesis and fission/fusion mediated by PGC-1a regulates rotenone-induced dopaminergic neurotoxicity, Mol. Neurobiol. 54 (2017) 3783e3797.
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Folic acid-mediated mitochondrial activation for protection against oxidative stress in human dental pulp stem cells derived from deciduous teeth Yu Zhang, Hiroki Kato*, Hiroshi Sato, Haruyoshi Yamaza, Yuta Hirofuji, Xu Han, Keiji Masuda**, Kazuaki Nonaka Section of Oral Medicine for Children, Division of Oral Health, Growth and Development, Faculty of Dental Science, Kyushu University, 3-1-1 Maidashi, Higashi-Ku, Fukuoka, 812-8582, Japan
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a b s t r a c t
Article history: Received 7 November 2018 Accepted 27 November 2018 Available online 7 December 2018
Enzymatic antioxidant systems, mainly involving mitochondria, are critical for minimizing the harmful effects of reactive oxygen species, and these systems are enhanced by interactions with nonenzymatic antioxidant nutrients. Because fetal growth requires extensive mitochondrial respiration, pregnant women and fetuses are at high risk of exposure to excessive reactive oxygen species. The enhancement of the antioxidant system, e.g., by nutritional management, is therefore critical for both the mother and fetus. Folic acid supplementation prevents homocysteine accumulation and epigenetic dysregulation associated with one-carbon metabolism. However, few studies have examined the antioxidant effects of folic acid for healthy pregnancy outcomes. The purpose of this study was to elucidate the association between the antioxidant effect of folic acid and mitochondria in undifferentiated cells during fetal growth. Neural crest-derived dental pulp stem cells of human exfoliated deciduous teeth were used as a model of undifferentiated cells in the fetus. Pyocyanin induced excessive reactive oxygen species, resulting in a decrease in cell growth and migration accompanied by mitochondrial fragmentation and inactivation in dental pulp stem cells. This damage was significantly improved by folic acid, along with decreased mitochondrial reactive oxygen species, PGC-1a upregulation, DRP1 downregulation, mitochondrial elongation, and increased ATP production. Folic acid may protect undifferentiated cells from oxidative damage by targeting mitochondrial activation. These results provide evidence for a new benefit of folic acid in pregnant women and fetuses. © 2018 Elsevier Inc. All rights reserved.
Keywords: Oxidative stress Folic acid Mitochondria Stem cells from human exfoliated deciduous teeth
1. Introduction Reactive oxygen species (ROS) are inevitably produced by mitochondrial aerobic respiration [1]. While ROS at appropriate levels play important roles as signaling molecules, excessive ROS can have various deleterious effects on cells [2]. Antioxidant systems, including mitochondrial antioxidant enzymes and non-
Abbreviations: DRP1, dynamin related protein 1; FA, folic acid; PGC-1a, peroxisome proliferator-activated receptor gamma coactivator 1-alpha; PYO, pyocyanin; ROS, reactive oxygen species; SHED, stem cells from human exfoliated deciduous teeth. * Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (H. Kato), [email protected] (K. Masuda). https://doi.org/10.1016/j.bbrc.2018.11.169 0006-291X/© 2018 Elsevier Inc. All rights reserved.
enzymatic antioxidant nutrients, are therefore indispensable for cells [3]. During pregnancy in mammals, including humans, a large amount of ROS is generated due to enhanced mitochondrial respiration along with the growth of the fetus, uterus, and placenta [4]. The attenuation or exhaustion of antioxidant systems generates excessive ROS, which can have various adverse effects not only on the mother but also on the fetus [4]. Maternal nutritional management, including supplementation with antioxidant nutrients, is important for enhancing antioxidant systems in pregnant women throughout gestation [4]. Folic acid (FA) is a recommended supplement for healthy pregnancy outcomes [5]. FA contributes to epigenetic regulation via DNA and histone methylation as a result of one-carbon metabolism using methionine in cells [6,7]. Insufficient FA leads to the dysregulation of one-carbon metabolism, which may alter the expression profiles of epigenetically modified genes [8,9]. Furthermore, an FA
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deficiency disrupts the re-methylation of homocysteine to methionine in one-carbon metabolism, resulting in homocysteine accumulation, which is associated with various maternal and fetal abnormalities [5]. Thus, FA supplementation in pregnant women mainly contributes to maternal and fetal health by affecting onecarbon metabolism. In addition, previous studies have shown that FA exerts an antioxidant effect by functioning as a free radical scavenger [10]. However, few studies have focused on the antioxidant effect of FA during fetal development. Stem cells from human exfoliated deciduous teeth (SHED) are undifferentiated mesenchymal cells derived from the neural crest; they have multipotency, including neuron and bone differentiation potential [11]. Therefore, SHED are a potentially useful cellular model for analyzing the antioxidant effect of FA on undifferentiated embryonic cells. Pyocyanin (PYO) is produced by Pseudomonas aeruginosa and is commonly used as an experimental reagent to examine oxidative stress. PYO is reduced by accepting electrons from nicotinamide adenine dinucleotide phosphate (NADPH) under aerobic conditions in host cells, which reacts with O2 to produce a superoxide radical, generating ROS [12]. The purpose of this study was to elucidate the association between the antioxidant effect of FA and mitochondria in undifferentiated SHED derived from a healthy child with typical development. 2. Materials and methods 2.1. Isolation and preparation of SHED A human exfoliated deciduous tooth from a normal 7-year-old individual was provided by Pediatric Dentistry and Special Need Dentistry at Kyushu University Hospital in Japan. Written informed consent was obtained from the child's guardians. The isolation procedure was completed as previously described [13]. Briefly, the pulp tissue was subjected to enzymatic dissociation in 3 mg/mL collagenase I (Worthington Biochemical, Worthington, NJ, USA) and 4 mg/mL dispase II (Wako, Osaka, Japan) for 1 h and was then maintained at 37 C in a humidified 5% CO2 incubator in Eagle's Minimal Essential Medium, Alpha Modification (a-MEM; SigmaAldrich, MO, USA) containing 15% fetal bovine serum (SigmaAldrich), 100 mM L-ascorbic acid 2-phosphate (Wako), 2 mM L-
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glutamine (Life Technologies, Grand Island, NY, USA), 250 mg/mL Fungizone (Life Technologies), 100 U/mL penicillin (Life Technologies), and 100 mg/mL streptomycin (Life Technologies). Cells at not more than 10 passages were used for subsequent analyses. 2.2. Cell proliferation assay Cells (1 105) were seeded in 6-well plates. After 24 h, the cells were cultured in the presence or absence of 10e80 mM FA (Wako) for 6 h. Then, PYO (Sigma) was added to the wells at concentrations ranging from 0 to 100 mM. After 24 h, the cells were collected by trypsinization and stained with 0.4% trypan blue solution (Wako). The cells were counted using a hemocytometer. 2.3. Cell migration assay A Culture-Insert 2 Well (ibidi, Munich, Germany) was set into a 6-well plate (Corning, Inc., Corning, NY, USA). Cells (2.1 103) were seeded in the Culture-Insert 2 Well (ibidi). After 24 h, the CultureInsert 2 Well was removed and the cells were cultured in the presence or absence of 10e80 mM FA (Wako) for 6 h. Then, PYO (Sigma) was added to the assigned wells at a concentration of 100 mM, and the cells were cultured for an additional 48 h. Images of the wounded edges were obtained using an IX41 microscope (Olympus, Tokyo, Japan) equipped with a digital camera (NEX-5T; Sony, Tokyo, Japan) with an NY-1S adaptor (MeCan Imaging, Saitama, Japan). The images of wounded edges were collected 24 and 48 h after the addition of PYO and results were quantified using ImageJ [14]. 2.4. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) Cells (1 105) were seeded in a 6-well plate. After 24 h, the cells were cultured in the presence or absence of 80 mM FA (Wako) for 6 h. Then, PYO (Sigma) was added to the assigned wells at a concentration of 100 mM. After 8 h, total RNA extraction and RT-qPCR were performed as described previously [15]. The sequences of the primer sets used in this study were as follows: dynamin related protein 1 (DRP1), 50 -GCTCCAGGACGTCTTCAACA-30 (forward) and
Fig. 1. ROS production and growth inhibition by pyocyanin. (A, B) Cells were cultured in the presence or absence of the indicated concentration of pyocyanin (PYO). (A) Intracellular cell ROS production was measured. The relative levels were calculated, and the level in the absence of pyocyanin was set to 1. Means ± SEMs from three experiments are shown. *p < 0.05 (B) Cells were counted. Means ± SEMs from three experiments are shown. *p < 0.05, **p < 0.01.
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50 -TAGCACTGAGCTCTTTCCGC-30 (reverse); peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1a), 50 -GGCAGAAGGCAATTGAAGAG-30 (forward) and 50 TCAAAACGGTCCCTCAGTTC-30 (reverse); 18S rRNA, 50 -CGGCTACCACATCCAAGGAA-30 (forward) and 50 -GCTGGAATTACCGCGGCT-30 (reverse). The relative expression levels of the target genes were analyzed using the comparative threshold cycle method by normalization against the levels of 18S rRNA.
2.5. Detection of intracellular ROS A total of 1 105 cells was seeded in 6-well plates (Corning). After 24 h, the cells were cultured in the presence or absence of 80 mM FA (Wako) for 6 h. Then, PYO (Sigma) was added to the assigned wells at a concentration of 100 mM. After 24 h, the cells
were incubated with 5 mM Cell ROX Green (Life Technologies) for 30 min. The cells were then treated with Tryple Express (Life Technologies) for detachment from the culture plate. The fluorescence signal was measured using a FACSVerse instrument (BD Biosciences, San Jose, CA, USA).
2.6. Detection of mitochondrial ROS Cells (1 105) were seeded in a 6-well plate (Corning). After 24 h, the cells were cultured in the presence or absence of 80 mM FA (Wako) for 6 h. Then, PYO (Sigma) was added to the assigned wells at a concentration of 100 mM. After 8 h, the cells were incubated with 5 mM MitoSOX Red (Life Technologies) for 10 min and treated with Tryple Express (Life Technologies) for detachment from the culture plate. The fluorescence signal was measured using a
Fig. 2. Recovery of the PYO-induced inhibition of cell growth and migration by FA. Cells were cultured in the presence or absence of the indicated concentrations of pyocyanin (PYO) and folic acid (FA). (A) Cells were counted. Means ± SEMs from four experiments are shown. **p < 0.01, ***p < 0.001. n.s., not significant. (B, C) Cell migration was measured. (B) Images of wounded edges. Left panels show original images and other panels show grayscale images converted using ImageJ. Scale bar ¼ 250 mm. (C) Images of wounded edges were quantitated using ImageJ. Values for the wounded edges at time 0 were set to 100%. Means ± SEMs from three experiments are shown. n.s., not significant. ###p < 0.001 compared with PYO‒ FA‒. *p < 0.05 compared with PYO þ FAþ. ***p < 0.001 compared with PYO þ FAþ.
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FACSVerse instrument (BD Biosciences). 2.7. Measurement of intracellular ATP levels Cells (1 105) were seeded in a 6-well plate. After 24 h, the cells were cultured in the presence or absence of 80 mM FA (Wako) for 6 h. Then, PYO (Sigma) was added to the assigned wells at a concentration of 100 mM. After 24 h, the cells were harvested in icecold PBS. The CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, WI, USA) was used to measure intracellular ATP levels. The ATP levels were normalized to the protein content of the samples. 2.8. Immunocytochemistry Cells (1 105) were seeded on a cover glass in a 6-well plate. After 24 h, the cells were cultured in the presence or absence of 80 mM FA (Wako) for 6 h. Then, PYO (Sigma) was added to the assigned wells at a concentration of 100 mM. After 8 h, the cells were fixed with 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4) for 10 min at 25 C and then permeabilized with 0.1% Triton X-100 in PBS for 5 min. The cells were blocked with 2% bovine serum albumin (BSA; Wako Pure Chemical Industries) in PBS for 20 min and then incubated with an anti-Tom20 antibody (1:200, #sc-11415; Santa Cruz Biotechnology, Santa Cruz, CA, USA). After 90 min, the cells were incubated with Alexa Fluor-conjugated secondary antibody (Life Technologies) in the dark for 60 min. After staining with antibodies, the nuclei were stained with 1 mg/mL 40 ,6diamidino-2-phenylindole dihydrochloride (DAPI; Dojindo, Kumamoto, Japan). The cells were then mounted using ProLong Diamond (Life Technologies). Fluorescence images were captured using an Axio Imager M2 (Zeiss, Oberkochen, Germany) equipped with the ApoTome2 (Zeiss). 2.9. Statistical analysis Statistical analyses were performed by Student's t-tests using JMP version 13 (SAS Institute, Raleigh, NC, USA). A value of p < 0.05 was considered statistically significant.
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by FA supplementation together with PYO, 75% of the open wound area was closed. A quantitative analysis showed a significant difference between treatments at 48 h (Fig. 2C). These results suggested that the inhibition of SHED growth and migration by PYO-mediated oxidative stress is improved by the antioxidant effect of FA. 3.3. Effect of FA on PYO-mediated oxidative stress The antioxidant effect of FA on extensive ROS generation by PYO was evaluated (Fig. 3). Mitochondrial ROS levels were significantly higher in the presence of 100 mM PYO alone than in the control cultures. A significant reduction of mitochondrial ROS levels was observed by treatment with 80 mM FA and 100 mM PYO compared to PYO alone, although mitochondrial ROS levels did not recover to the level observed in the control. These results suggested that FA influences PYO-mediated oxidative stress by the attenuation of excessive mitochondrial ROS in SHED. 3.4. Enhanced mitochondrial activation with elongation and biogenesis in SHED by FA The antioxidant system of cells depends on mitochondrial activity, which is enhanced by mitochondrial elongation and biogenesis. The association of mitochondrial morphology and biogenesis with the antioxidant effect of FA was examined. While mitochondrial fragmentation was observed in the presence of PYO alone, it was diminished by supplementation with FA (Fig. 4A and B). Consistent with this observation, the mRNA expression of DRP1, an essential factor for mitochondrial fragmentation, was upregulated by treatment with PYO alone and downregulated by supplementation with FA (Fig. 4C). Furthermore, the mRNA expression of PGC-1a, a master regulator of mitochondrial biogenesis, was downregulated by treatment with PYO alone and upregulated by supplementation with FA (Fig. 4D). Intracellular ATP levels decreased in the presence of PYO alone and increased by supplementation with FA (Fig. 4E). These results suggested that the antioxidant effect of FA on PYO-
3. Results 3.1. Intracellular ROS production and growth inhibition by PYO PYO elevated intracellular ROS levels in SHED in a dosedependent manner; ROS levels were highest for 50 mM PYO (Fig. 1A). PYO suppressed the growth of SHED in a dose-dependent manner and showed the maximal inhibitory effect at 100 mM (Fig. 1B and Supplemental fig.). These results suggested that there is an association between PYO-mediated oxidative stress and cellular growth inhibition in SHED. 3.2. Recovery of the PYO-induced inhibition of growth and migration by FA The effect of FA on the PYO-induced inhibition of SHED growth was examined. In the presence of 100 mM PYO, which showed the maximal inhibitory effect on SHED growth, FA supplementation improved cell growth in a dose-dependent manner and showed the maximal effect at 80 mM (Fig. 2A). In the absence of PYO, 80 mM FA did not alter cell growth. Furthermore, differences in the effects of PYO and FA on cell migration were observed (Fig. 2B and C). In control cultures without PYO and FA, the open wound area was completely closed after 48 h by SHED migration. In the presence of PYO alone, only 50% of the open wound area was closed. However,
Fig. 3. Reduction of PYO-induced mitochondrial ROS by folic acid. Cells were cultured in the presence or absence of 100 mМ pyocyanine (PYO) and 80 mM folic acid (FA) and mitochondrial ROS levels were measured. The relative levels were calculated, and the level in the absence of pyocyanin and folic acid was set to 1. Means ± SEMs from four experiments are shown. *p < 0.05.
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Fig. 4. Recovery of the pyocyanin-induced fragmentation of mitochondria and reduction in mitochondrial activity by folic acid. (A) Cells were cultured in the presence or absence of pyocyanin and folic acid, stained with an antibody against Tom20 (red), a mitochondrial marker, and counterstained with DAPI (blue). Lower panels show a detailed view of the boxed region in the upper panels. Yellow arrows indicate elongated mitochondria. White arrows indicate fragmented mitochondria. Scale bar ¼ 25 mm (upper panel), 5 mm (lower panel). (B) Cells with fragmented mitochondria were counted. Percentages of the total cell number were calculated. Data represent mean ± SEMs from the analysis of 30 cells from 5 immunostained images. *p < 0.05, **P < 0.01. (C, D) mRNA expression of DRP1 (C) and PGC-1a (D) were measured by RT-qPCR. The relative levels were calculated, and the level in the absence of pyocyanin and folic acid was set to 1. Data represent the means ± SEMs from four experiments. *p < 0.05, **p < 0.01, ***p < 0.001. (E) Intracellular ATP levels were measured by an ATP luminescence assay. The relative levels were calculated, and the level in the absence of pyocyanin and folic acid was set to 1. Data represent means ± SEMs from four experiments. *p < 0.05, ***p < 0.001.
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mediated oxidative stress was associated with enhanced mitochondrial activity to diminish excessive ROS by promoting mitochondrial elongation and biogenesis. 4. Discussion In this study, PYO-induced ROS production inhibited SHED growth and migration, with mitochondrial inactivation and fragmentation, and these changes were alleviated by FA via the upregulation of PGC-1a and downregulation of DRP1. These results suggested that FA targeted mitochondria to activate antioxidant systems, thereby protecting undifferentiated cells from oxidative stress. SHED are human dental pulp stem cells derived from the cranial neural crest [11]. In the third week after fertilization, the neural fold and groove are formed from the ectodermal germ layer, followed by neural tube formation [16]. During this stage, a neural crest is formed at the tip of the neural fold, from which neural crest cells dissociate and invade the mesoderm to form various organs [17]. The cranial neural crest cells migrate to branchial arches that will form facial tissues, including the bone, muscle, fat, dentin, pulp, and trigeminal nerve system [18]. Considering that SHED can differentiate into neurons, osteoblasts, adipocytes, odontoblasts, or myoblasts in vitro, they may at least partially maintain the features of fetal cranial neural crest cells [11,19]. A recent study has shown that FA has an effect on fetal neural tube defects using human induced pluripotent stem cells as a model of undifferentiated cells [20]. Embryonic stem cells (ESCs) have also been proposed as a model to examine FA-mediated one-carbon metabolism and epigenetic regulation, but the use of human ESCs has ethical limitations [21]. Despite previous studies, the antioxidant effect of FA is unresolved. SHED are a promising human cellular model to elucidate the mechanism underlying the effects of FA on oxidative stress at the fetal stage. In this study, the inhibition of cellular functions by PYO-induced oxidative stress in SHED was improved by FA, with decreased levels of mitochondrial ROS. Since previous studies of cell-free systems have shown that FA is a free radical scavenger, it is possible that excessive free radicals induced by PYO was attenuated by this reaction in SHED [10]. In contrast, mitochondria have a major role in ROS regulation by superoxide dismutase 2, a mitochondrial antioxidant enzyme [22]. Excessive ROS damages various cellular components, including mitochondrial enzymes, leading to the inactivation of the mitochondrial antioxidant system. While mitochondrial inactivation has been associated with mitochondrial fragmentation in various kind of damaged cells, acceleration of mitochondrial fusion and elongation has been shown to improve mitochondrial activity and cellular function [23‒26]. It is therefore likely that the FA-mediated Drp1 downregulation observed in SHED may have improved the mitochondrial antioxidant activity through modulation of mitochondrial morphology. PGC-1a has been shown to regulate several molecules involved in mitochondrial fusion and fission, including DRP1 [27]. In SHED, FA-mediated PGC-1a upregulation may have been involved in the mitochondrial fusion and activation through Drp1 downregulation in the downstream pathway. The effect of FA on mitochondrial elongation and activation may be a novel mechanism for the protection of undifferentiated cells from oxidative damage. There are several limitations of this research. First, the regulatory mechanism by which FA alters the expression of PGC-1a and DRP1 for mitochondrial activation remains to be elucidated. The epigenetic status of these genes may be altered by FA-mediated one-carbon metabolism in SHED. Further studies are required to elucidate the expression profile or epigenetic modulation of the genes in the pathway downstream of FA leading to mitochondrial
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activation. Second, the antioxidant effect of FA and underlying mechanism during cell differentiation are unresolved. An experimental system that differentiates SHEDs into neurons or osteoblasts would be useful for addressing this issue. Third, the relevance of FA doses recommended for pregnant women and FA concentrations effective for inducing antioxidant stress in fetal undifferentiated cells remains unclear. It may be necessary to examine the in vivo administration of FA in pregnant animals. In conclusion, FA may target and activate the mitochondrial antioxidant system to protect undifferentiated cells from oxidative damage. These results provide new evidence to support the effectiveness of FA in pregnant women to protect the fetus from oxidative damage. Conflicts of interest The authors have no conflicts of interest to declare. Acknowledgments We thank all the members of the Department of Pediatric & Special Needs Dentistry at Kyushu University Hospital for valuable suggestions, technical support, and materials. We appreciate the technical assistance provided by the Research Support Center at the Research Center for Human Disease Modeling, Kyushu University Graduate School of Medical Sciences. This work was supported by the Japan Society for the Promotion of Science (KAKENHI grant numbers JP25670877, and JP16K15839), Japan. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.11.169. References [1] V. Adam-Vizi, C. Chinopoulos, Bioenergetics and the formation of mitochondrial reactive oxygen species, Trends Pharmacol. Sci. 27 (2006) 639e645. [2] W. Droge, Free radicals in the physiological control of cell function, Physiol. Rev. 82 (2002) 47e95. [3] A.Y. Andreyev, Y.E. Kushnareva, A.A. Starkov, Mitochondrial metabolism of reactive oxygen species, Biochemistry (Mosc.) 70 (2005) 200e214. [4] K.H. Al-Gubory, P.A. Fowler, C. Garrel, The roles of cellular reactive oxygen species, oxidative stress and antioxidants in pregnancy outcomes, Int. J. Biochem. Cell Biol. 42 (2010) 1634e1650. [5] K. Fekete, C. Berti, I. Cetin, M. Hermoso, B.V. Koletzko, T. Decsi, Perinatal folate supply: relevance in health outcome parameters, Matern. Child Nutr. Suppl 2 (2010) 23e38. [6] J.T. Fox, P.J. Stover, Folate-mediated one-carbon metabolism, Vitam. Horm. 79 (2008) 1e44. [7] S.J. Mentch, J.W. Locasale, One-carbon metabolism and epigenetics: understanding the specificity, Ann. N. Y. Acad. Sci. 1363 (2016) 91e98. rez, E. S [8] N. Alata Jimenez, S.A. Torres Pe anchez-V asquez, J.I. Fernandino, P.H. Strobl-Mazzulla, Folate deficiency prevents neural crest fate by disturbing the epigenetic Sox 2 repression on the dorsal neural tube, Dev. Biol. S0012e1606 (2018) 30595eX, https://doi.org/10.1016/j.ydbio.2018.08.001. [9] A. Caffrey, R.E. Irwin, H. McNulty, J.J. Strain, D.J. Lees-Murdock, B.A. McNulty, M. Ward, C.P. Walsh, K. Pentieva, Gene-specific DNA methylation in newborns in response to folic acid supplementation during the second and third trimesters of pregnancy: epigenetic analysis from a randomized controlled trial, Am. J. Clin. Nutr. 107 (2018) 566e575. [10] R. Joshi, S. Adhikari, B.S. Patro, S. Chattopadhyay, T. Mukherjee, Free radical scavenging behavior of folic acid: evidence for possible antioxidant activity, Free Radic. Biol. Med. 30 (2001) 1390e1399. [11] M. Miura, S. Gronthos, M. Zhao, B. Lu, L.W. Fisher, P.G. Robey, S. Shi, SHED: stem cells from human exfoliated deciduous teeth, Proc. Natl. Acad. Sci. U. S. A 100 (2003) 5807e5812. [12] B. Rada, T.L. Leto, Pyocyanin effects on respiratory epithelium: relevance in Pseudomonas aeruginosa airway infections, Trends Microbiol. 21 (2013) 73e81. [13] H. Kato, T.T.M. Pham, H. Yamaza, K. Masuda, Y. Hirofuji, X. Han, H. Sato, T. Taguchi, K. Nonaka, Mitochondria regulate the differentiation of stem cells from human exfoliated deciduous teeth, Cell Struct. Funct. 116 (2017) 105e116, https://doi.org/10.1247/csf.17012.
856
Y. Zhang et al. / Biochemical and Biophysical Research Communications 508 (2019) 850e856
[14] C.A. Schneider, W.S. Rasband, K.W. Eliceiri, NIH Image to ImageJ: 25 years of image analysis, Nat. Methods 9 (2012) 671e675. [15] H. Kato, X. Han, H. Yamaza, K. Masuda, Y. Hirofuji, H. Sato, T.T.M. Pham, T. Taguchi, K. Nonaka, Direct effects of mitochondrial dysfunction on poor bone health in Leigh syndrome, Biochem. Biophys. Res. Commun. 493 (2017) 207e212. [16] N.D. Greene, A.J. Copp, Neural tube defects, Annu. Rev. Neurosci. 37 (2014) 221e242. [17] R. Mayor, E. Theveneau, The neural crest, Development 11 (2013) 2247e2251. [18] P.M. Kulesa, C.M. Bailey, J.C. Kasemeier-Kulesa, R. McLennan, Cranial neural crest migration: new rules for an old road, Dev. Biol. 344 (2010) 543e554. [19] M. La Noce, L. Mele, V. Tirino, F. Paino, A. De Rosa, P. Naddeo, P. Papagerakis, G. Papaccio, V. Desiderio, Neural crest stem cell population in craniomaxillofacial development and tissue repair, Eur. Cell. Mater. 28 (2014) 348e357. [20] V. Sahakyan, R. Duelen, W.L. Tam, S.J. Roberts, H. Grosemans, P. Berckmans, G. Ceccarelli, G. Pelizzo, V. Broccoli, J. Deprest, F.P. Luyten, C.M. Verfaillie, M. Sampaolesi, Folic acid exposure rescues spina bifida aperta phenotypes in human induced pluripotent stem cell model, Sci. Rep. 8 (2018) 2942. [21] S. Kasulanati, V. Venkatesan, Understanding pluripotency under folic acid deficiency using embryonic stem cells as an in vitro model, Med. Hypotheses
111 (2018) 24e26. [22] R.A. Weisiger, I. Fridovich, Mitochondrial superoxide simutase. Site of synthesis and intramitochondrial localization, J. Biol. Chem. 248 (1973) 4793e4796. [23] T. Wai, T. Langer, Mitochondrial dynamics and metabolic regulation, Trends Endocrinol. Metabol. 27 (2016) 105e117. [24] C. Hu, Y. Huang, L. Li, Drp1-Dependent mitochondrial fission plays critical roles in physiological and pathological progresses in mammals, Int. J. Mol. Sci. 18 (2017) 144. [25] L.C. Gomes, L. Scorrano, Mitochondrial elongation during autophagy: a stereotypical response to survive in difficult times, Autophagy 7 (2011) 1251e1253. [26] A.A. Rosdah, J.K. Holien, L.M. Delbridge, G.J. Dusting, S.Y. Lim, Mitochondrial fission - a drug target for cytoprotection or cytodestruction? Pharmacol. Res. Perspect. 4 (2016), e00235. [27] K. Peng, L. Yang, J. Wang, F. Ye, G. Dan, Y. Zhao, Y. Cai, Z. Cui, L. Ao, J. Liu, Z. Zou, Y. Sai, J. Cao, The interaction of mitochondrial biogenesis and fission/fusion mediated by PGC-1a regulates rotenone-induced dopaminergic neurotoxicity, Mol. Neurobiol. 54 (2017) 3783e3797.