Ubiquinol decreases monocytic expression and DNA methylation of the pro-inflammatory chemokine ligand 2 gene in humans
© Fischer et al.; licensee BioMed Central Ltd. 2012
Received: 3 April 2012
Accepted: 20 September 2012
Published: 1 October 2012
Coenzyme Q10 is an essential cofactor in the respiratory chain and serves in its reduced form, ubiquinol, as a potent antioxidant. Studies in vitro and in vivo provide evidence that ubiquinol reduces inflammatory processes via gene expression. Here we investigate the putative link between expression and DNA methylation of ubiquinol sensitive genes in monocytes obtained from human volunteers supplemented with 150 mg/ day ubiquinol for 14 days.
Ubiquinol decreases the expression of the pro-inflammatory chemokine (C-X-C motif) ligand 2 gene (CXCL2) more than 10-fold. Bisulfite-/ MALDI-TOF-based analysis of regulatory regions of the CXCL2 gene identified six adjacent CpG islands which showed a 3.4-fold decrease of methylation status after ubiquinol supplementation. This effect seems to be rather gene specific, because ubiquinol reduced the expression of two other pro-inflammatory genes (PMAIP1, MMD) without changing the methylation pattern of the respective gene.
In conclusion, ubiquinol decreases monocytic expression and DNA methylation of the pro-inflammatory CXCL2 gene in humans. Current Controlled Trials ISRCTN26780329.
KeywordsCoenzyme Q10 Ubiquinol Gene expression DNA methylation Inflammation
Coenzyme Q10 (CoQ10) is a key component of the mitochondrial respiratory chain where it is mainly known for its role in oxidative phosphorylation. The reduced form of CoQ10, ubiquinol, serves as a potent antioxidant in mitochondria, lipid membranes and plasma lipoproteins [1, 2] as well as a regenerator of other lipid soluble antioxidants (e.g. vitamin E) [3, 4]. Several studies in vitro[5–7], in SAMP1 mice  and in humans  indicate that ubiquinol is involved in inflammatory processes and lipid metabolism via gene expression.
Gene expression as well as DNA methylation processes are affected by various dietary supplements and food nutrients [10–13]. Furthermore, DNA methylation is one of the epigenetic modifications that per se is able to determine the gene expression by regulating the chromatin organization [10, 14]. During methylation of mammalian DNA a methyl group is attached at the 5-position of the cytosine residue within the cytosine-guanine dinucleotides (CpG) resulting in the formation of 5-methylcytosine, which is designated as the fifth base of DNA . Although most genomic DNA in mammals is deficient in CpG sites, clusters of CpG dinucleotides (CpG islands) were described to be primarily located at promoter regions of genes . Here we investigated the effect of ubiquinol on the expression and methylation of CpG island promoter regions of pro-inflammatory genes in humans.
Materials and methods
Participants and study design
Sample characteristics of subjects and study design have been described lately . Briefly: fifty-three healthy male volunteers, 21–48 years of age, received 150 mg of the reduced form of CoQ10 (Q10H2, ubiquinol, KANEKA Corporation, Japan) daily in form of three capsules with each principal meal for 14 days. Fasting blood samples were taken before (T0) and after (T14) supplementation. The participants had an average Body Mass Index (BMI) of 24.1 ± 2.5 kg/m2, no history of gastrointestinal, hepatic, cardiovascular or renal diseases, a habit of non- or occasional smoking and maintenance of usual nutrition habits. The study was approved by the ethics committee of the Medical Faculty of Kiel University, Germany, and was conformed to Helsinki Declaration. All volunteers gave written informed consent.
Microarray-based gene expression analysis and qRT-PCR
Microarray experiment using the Affymetrix human genome U133 plus 2.0 GeneChip® were performed as described previously  with RNA samples from CD14-positive monocytes obtained from three volunteers before (T0) and after (T14) supplementation with ubiquinol. Based on microarray data, expression levels of selected genes including the CXCL2, MMD and PMAIP1 gene were verified by real-time qRT-PCR. Primer sequences for real-time qRT-PCR experiments were designed with Primer Express® Software 3.0 (Applied Biosystems, Darmstadt, Germany). Primer pairs were obtained from MWG Biotech AG (Ebersberg, Germany). cDNA synthesis with subsequent PCR amplification procedure has been described before .
Methylation analysis of genomic regions of CXCL2, MMD and PMAIP1 gene
The presence of CpG islands within the CXCL2, MMD and PMAIP1 genes was predicted using the European Molecular Biology Open Software Suite CpGplot, respectively. Quantitative methylation analysis was performed on the MassARRAY® system (Sequenom, Hamburg, Germany) at BioGlobe (Hamburg, Germany) applying the MassCLEAVETM (hMC) biochemistry after bisulfit treatment of DNA samples and MALDI-TOF mass spectrometry for analyte detection. All reactions were performed according to the standard protocols recommended by the supplier. Genomic DNA was extracted from human monocytes obtained from five volunteers (H1/H1_1 to H5/H5_1) before (T0, H1-H5) and after (T14, H1_1-H5_5) using the DNeasy Tissue Kit (Quiagen). Analysis was carried out from both, forward and reverse strand.
The protocol starts with a bisulfit treatment of provided genomic DNA sample, which converts native cytosine (“C”) nucleotides into uracil (“U”), whereas 5-methyl-protected cytosine residues remain as “C”. The resulting artificial sequence variation is conserved during PCR amplification using methylation independent primers. One primer for each PCR is tagged with T7 RNA polymerase promoter sequence facilitating the transformation of double stranded PCR product into single stranded RNA together with a second level of amplification. The in vitro transcription product is “U-specific” cleaved with RNase A. The generated fragments represent unique portions of the amplified region of interest and are displayed based on their molecular weight in the mass spectrum, which is acquired after sample conditioning with a MassARRAY® Analyzer Compact. Automated data analysis was performed with EpiTyper Software.
Results and discussion
Ubiquinol supplementation reduces the expression of low and high abundant mRNA steady-state levels of pro-inflammatory genes in human monocytes
Normalized steady-state mRNA expression levels (AU) of the CXCL2, PMAIP1 and MMD gene in monocytes of human volunteers before (T 0 ) and after (T 14 ) supplementation with ubiquinol
(T0 vs. T14)
1271 ± 35
578 ± 106
147 ± 30
85 ± 19
1150 ± 865
93 ± 30
Ubiquinol supplementation reduces the methylation status of six adjacent CpG islands within the promoter of the CXCL2 gene
Position, length and number of CpG islands of amplicons covering the analysed genomic regions of the human CXCL2, PMAIP1 and MMD gene
left primer (+Tag)
right primer (+Tag)
The effect of ubiquinol on DNA methylation seems to be rather gene specific and might depend on the extent of ubiquinol induced alteration of gene expression
Our study provides a first hint towards a modifying effect of ubiquinol on DNA methylation in humans. This effect is in line with another human study demonstrating that a supplementation with a mixture of CoQ10, niacin and riboflavin reduces DNA methylation of the tumor suppressor gene RASSF1A in breast cancer patients undergoing tamoxifen therapy . Moreover, literature indicates that global methylation patterns are affected by several other dietary supplements and micronutrients [27–32]. The effect of ubiquinol on DNA methylation seems be rather gene specific because we identified two genes (PMAIP1, MMD) which are regulated by ubiquinol without changing DNA methylation. A recent SAMP-mice study from our group  found that ubiquinol alters hepatic expression of PPARα target genes without influencing DNA methylation in the respective gene promoters.
The effect of ubiquinol on DNA methylation might be linked to the extent of ubiquinol dependent alteration of gene expression. In the case of the CXCL2 gene, ubiquinol reduces its expression more than 10-fold accompanied by a reduced methylation status within certain CpG islands. This finding seems to be contradictory to common models of gene expression, because increased DNA methylation of a gene leads to reduced expression levels , whereas demethylation correlates with the transcription of the gene . On the other hand there is evidence, especially in cancer cells, that DNA methylation status does not correlate with gene expression [36, 37]. However, the mechanism regarding effects of ubiquinol on DNA methylation and expression remains unclear and has to be studied in the future. The reduced expression of pro-inflammatory genes under ubiquinol supplementation supports recent findings from our lab [5, 6, 8, 9, 38–41] and other groups [42, 43] suggesting that CoQ10 displays anti-inflammatory properties. As a summary, we found in a human intervention study that ubiquinol decreases expression and DNA methylation of the pro-inflammatory CXCL2 gene in monocytes. Further studies will be necessary to investigate the mechanistic link between ubiquinol dependent gene expression, DNA methylation and inflammation.
- Littarru GP, Tiano L: Bioenergetic and antioxidant properties of coenzyme Q10: recent developments. Mol Biotechnol. 2007, 37 (1): 31-37. 10.1007/s12033-007-0052-y.PubMedView ArticleGoogle Scholar
- Littarru GP, Tiano L: Clinical aspects of coenzyme Q10: an update. Nutrition. 2010, 26 (3): 250-254. 10.1016/j.nut.2009.08.008.PubMedView ArticleGoogle Scholar
- Mukai K, Itoh S, Morimoto H: Stopped-flow kinetic study of vitamin E regeneration reaction with biological hydroquinones (reduced forms of ubiquinone, vitamin K, and tocopherolquinone) in solution. J Biol Chem. 1992, 267 (31): 22277-22281.PubMedGoogle Scholar
- Crane FL, Navas P: The diversity of coenzyme Q function. Mol Aspects Med. 1997, 18 (Suppl): S1-S6.PubMedView ArticleGoogle Scholar
- Schmelzer C, Doring F: Identification of LPS-inducible genes downregulated by ubiquinone in human THP-1 monocytes. Biofactors. 2010, 36 (3): 222-228. 10.1002/biof.93.PubMedView ArticleGoogle Scholar
- Schmelzer C, Kohl C, Rimbach G, Doring F: The reduced form of coenzyme Q10 decreases the expression of lipopolysaccharide-sensitive genes in human THP-1 cells. J Med Food. 2011, 14 (4): 391-397. 10.1089/jmf.2010.0080.PubMedView ArticleGoogle Scholar
- Groneberg DA, Kindermann B, Althammer M, Klapper M, Vormann J, Littarru GP, Doring F: Coenzyme Q10 affects expression of genes involved in cell signalling, metabolism and transport in human CaCo-2 cells. Int J Biochem Cell Biol. 2005, 37 (6): 1208-1218. 10.1016/j.biocel.2004.11.017.PubMedView ArticleGoogle Scholar
- Schmelzer C, Kubo H, Mori M, Sawashita J, Kitano M, Hosoe K, Boomgaarden I, Doring F, Higuchi K: Supplementation with the reduced form of Coenzyme Q10 decelerates phenotypic characteristics of senescence and induces a peroxisome proliferator-activated receptor-alpha gene expression signature in SAMP1 mice. Mol Nutr Food Res. 2010, 54 (6): 805-815.PubMedView ArticleGoogle Scholar
- Schmelzer C, Niklowitz P, Okun JG, Haas D, Menke T, Doring F: Ubiquinol-induced gene expression signatures are translated into altered parameters of erythropoiesis and reduced low density lipoprotein cholesterol levels in humans. IUBMB Life. 2011, 63 (1): 42-48. 10.1002/iub.413.PubMedView ArticleGoogle Scholar
- Vucetic Z, Kimmel J, Totoki K, Hollenbeck E, Reyes TM: Maternal high-fat diet alters methylation and gene expression of dopamine and opioid-related genes. Endocrinology. 2010, 151 (10): 4756-4764. 10.1210/en.2010-0505.PubMedPubMed CentralView ArticleGoogle Scholar
- Yubero-Serrano EM, Gonzalez-Guardia L, Rangel-Zuniga O, Delgado-Lista J, Gutierrez-Mariscal FM, Perez-Martinez P, Delgado-Casado N, Cruz-Teno C, Tinahones FJ, Villalba JM: Mediterranean diet supplemented with coenzyme Q10 modifies the expression of proinflammatory and endoplasmic reticulum stress-related genes in elderly men and women. J Gerontol A: Biol Sci Med Sci. 2012, 67 (1): 3-10.View ArticleGoogle Scholar
- van den Donk M, van Engeland M, Pellis L, Witteman BJ, Kok FJ, Keijer J, Kampman E: Dietary folate intake in combination with MTHFR C677T genotype and promoter methylation of tumor suppressor and DNA repair genes in sporadic colorectal adenomas. Cancer epidemiology, biomarkers & prevention: a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology. 2007, 16 (2): 327-333. 10.1158/1055-9965.EPI-06-0810.View ArticleGoogle Scholar
- van Engeland M, Weijenberg MP, Roemen GM, Brink M, de Bruine AP, Goldbohm RA, van den Brandt PA, Baylin SB, de Goeij AF, Herman JG: Effects of dietary folate and alcohol intake on promoter methylation in sporadic colorectal cancer: the Netherlands cohort study on diet and cancer. Cancer Res. 2003, 63 (12): 3133-3137.PubMedGoogle Scholar
- Silahtaroglu A, Stenvang J: MicroRNAs, epigenetics and disease. Essays Biochem. 2010, 48 (1): 165-185. 10.1042/bse0480165.PubMedView ArticleGoogle Scholar
- Delaval K, Feil R: Epigenetic regulation of mammalian genomic imprinting. Curr Opin Genet Dev. 2004, 14 (2): 188-195. 10.1016/j.gde.2004.01.005.PubMedView ArticleGoogle Scholar
- Takai D, Jones PA: Comprehensive analysis of CpG islands in human chromosomes 21 and 22. Proc Natl Acad Sci USA. 2002, 99 (6): 3740-3745. 10.1073/pnas.052410099.PubMedPubMed CentralView ArticleGoogle Scholar
- Bender AT, Beavo JA: PDE1B2 regulates cGMP and a subset of the phenotypic characteristics acquired upon macrophage differentiation from a monocyte. Proc Natl Acad Sci USA. 2006, 103 (2): 460-465. 10.1073/pnas.0509972102.PubMedPubMed CentralView ArticleGoogle Scholar
- Bourdonnay E, Morzadec C, Sparfel L, Galibert MD, Jouneau S, Martin-Chouly C, Fardel O, Vernhet L: Global effects of inorganic arsenic on gene expression profile in human macrophages. Mol Immunol. 2009, 46 (4): 649-656. 10.1016/j.molimm.2008.08.268.PubMedView ArticleGoogle Scholar
- Liang F, Seyrantepe V, Landry K, Ahmad R, Ahmad A, Stamatos NM, Pshezhetsky AV: Monocyte differentiation up-regulates the expression of the lysosomal sialidase, Neu1, and triggers its targeting to the plasma membrane via major histocompatibility complex class II-positive compartments. J Biol Chem. 2006, 281 (37): 27526-27538. 10.1074/jbc.M605633200.PubMedView ArticleGoogle Scholar
- Liu Q, Zheng J, Yin DD, Xiang J, He F, Wang YC, Liang L, Qin HY, Liu L, Liang YM, Han H: Monocyte to macrophage differentiation-associated (MMD) positively regulates ERK and Akt activation and TNF-alpha and NO production in macrophages. Mol Biol Rep. 2012, 39 (5): 5643-5650. 10.1007/s11033-011-1370-5.PubMedView ArticleGoogle Scholar
- Yu J, Zhang L: The transcriptional targets of p53 in apoptosis control. Biochem Biophys Res Commun. 2005, 331 (3): 851-858. 10.1016/j.bbrc.2005.03.189.PubMedView ArticleGoogle Scholar
- Kim HY, Kim HS: Upregulation of MIP-2 (CXCL2) expression by 15-deoxy-Delta(12,14)-prostaglandin J(2) in mouse peritoneal macrophages. Immunol Cell Biol. 2007, 85 (1): 60-67. 10.1038/sj.icb.7100001.PubMedView ArticleGoogle Scholar
- Lkhagvaa B, Tani K, Sato K, Toyoda Y, Suzuka C, Sone S: Bestatin, an inhibitor for aminopeptidases, modulates the production of cytokines and chemokines by activated monocytes and macrophages. Cytokine. 2008, 44 (3): 386-391. 10.1016/j.cyto.2008.10.011.PubMedView ArticleGoogle Scholar
- Rehli M, Krause SW, Schwarzfischer L, Kreutz M, Andreesen R: Molecular cloning of a novel macrophage maturation-associated transcript encoding a protein with several potential transmembrane domains. Biochem Biophys Res Commun. 1995, 217 (2): 661-667. 10.1006/bbrc.1995.2825.PubMedView ArticleGoogle Scholar
- Shen L, Kondo Y, Guo Y, Zhang J, Zhang L, Ahmed S, Shu J, Chen X, Waterland RA, Issa JP: Genome-wide profiling of DNA methylation reveals a class of normally methylated CpG island promoters. PLoS Genet. 2007, 3 (10): 2023-2036.PubMedView ArticleGoogle Scholar
- Premkumar VG, Yuvaraj S, Shanthi P, Sachdanandam P: Co-enzyme Q10, riboflavin and niacin supplementation on alteration of DNA repair enzyme and DNA methylation in breast cancer patients undergoing tamoxifen therapy. Br J Nutr. 2008, 100 (6): 1179-1182. 10.1017/S0007114508968276.PubMedView ArticleGoogle Scholar
- Brunaud L, Alberto JM, Ayav A, Gerard P, Namour F, Antunes L, Braun M, Bronowicki JP, Bresler L, Gueant JL: Effects of vitamin B12 and folate deficiencies on DNA methylation and carcinogenesis in rat liver. Clin Chem Lab Med: CCLM / FESCC. 2003, 41 (8): 1012-1019.View ArticleGoogle Scholar
- Fischer A, Gaedicke S, Frank J, Doring F, Rimbach G: Dietary vitamin E deficiency does not affect global and specific DNA methylation patterns in rat liver. Br J Nutr. 2010, 104 (7): 935-940. 10.1017/S0007114510001649.PubMedView ArticleGoogle Scholar
- Ly A, Hoyt L, Crowell J, Kim YI: Folate and DNA Methylation. Antioxid Redox Signal. 2012Google Scholar
- Uekawa A, Katsushima K, Ogata A, Kawata T, Maeda N, Kobayashi K, Maekawa A, Tadokoro T, Yamamoto Y: Change of epigenetic control of cystathionine beta-synthase gene expression through dietary vitamin B12 is not recovered by methionine supplementation. J Nutrigenet Nutrigenomics. 2009, 2 (1): 29-36. 10.1159/000165374.PubMedView ArticleGoogle Scholar
- Waterland RA: Assessing the effects of high methionine intake on DNA methylation. J Nutr. 2006, 136 (6 Suppl): 1706S-1710S.PubMedGoogle Scholar
- Wolff GL, Kodell RL, Moore SR, Cooney CA: Maternal epigenetics and methyl supplements affect agouti gene expression in Avy/a mice. FASEB J: Off Publ Fed Am Soc Exp Biol. 1998, 12 (11): 949-957.Google Scholar
- Schmelzer C, Okun JG, Haas D, Higuchi K, Sawashita J, Mori M, Doring F: The reduced form of coenzyme Q10 mediates distinct effects on cholesterol metabolism at the transcriptional and metabolite level in SAMP1 mice. IUBMB Life. 2010, 62 (11): 812-818. 10.1002/iub.388.PubMedView ArticleGoogle Scholar
- Ehrlich M: Expression of various genes is controlled by DNA methylation during mammalian development. J Cell Biochem. 2003, 88 (5): 899-910. 10.1002/jcb.10464.PubMedView ArticleGoogle Scholar
- De Smet C, Lurquin C, Lethe B, Martelange V, Boon T: DNA methylation is the primary silencing mechanism for a set of germ line- and tumor-specific genes with a CpG-rich promoter. Mol Cell Biol. 1999, 19 (11): 7327-7335.PubMedPubMed CentralView ArticleGoogle Scholar
- Gama-Sosa MA, Slagel VA, Trewyn RW, Oxenhandler R, Kuo KC, Gehrke CW, Ehrlich M: The 5-methylcytosine content of DNA from human tumors. Nucleic Acids Res. 1983, 11 (19): 6883-6894. 10.1093/nar/11.19.6883.PubMedPubMed CentralView ArticleGoogle Scholar
- Baylin SB, Herman JG, Graff JR, Vertino PM, Issa JP: Alterations in DNA methylation: a fundamental aspect of neoplasia. Adv Cancer Res. 1998, 72: 141-196.PubMedView ArticleGoogle Scholar
- Schmelzer C, Kitano M, Rimbach G, Niklowitz P, Menke T, Hosoe K, Doring F: Effects of ubiquinol-10 on microRNA-146a expression in vitro and in vivo. Mediators Inflamm. 2009, 2009: 415437-PubMedPubMed CentralView ArticleGoogle Scholar
- Schmelzer C, Lindner I, Rimbach G, Niklowitz P, Menke T, Doring F: Functions of coenzyme Q10 in inflammation and gene expression. Biofactors. 2008, 32 (1–4): 179-183.PubMedView ArticleGoogle Scholar
- Schmelzer C, Lindner I, Vock C, Fujii K, Doring F: Functional connections and pathways of coenzyme Q10-inducible genes: an in-silico study. IUBMB Life. 2007, 59 (10): 628-633. 10.1080/15216540701545991.PubMedView ArticleGoogle Scholar
- Schmelzer C, Lorenz G, Lindner I, Rimbach G, Niklowitz P, Menke T, Doring F: Effects of Coenzyme Q10 on TNF-alpha secretion in human and murine monocytic cell lines. Biofactors. 2007, 31 (1): 35-41. 10.1002/biof.5520310104.PubMedView ArticleGoogle Scholar
- Dominguez PM, Ardavin C: Differentiation and function of mouse monocyte-derived dendritic cells in steady state and inflammation. Immunol Rev. 2010, 234 (1): 90-104. 10.1111/j.0105-2896.2009.00876.x.PubMedView ArticleGoogle Scholar
- Sohet FM, Neyrinck AM, Pachikian BD, de Backer FC, Bindels LB, Niklowitz P, Menke T, Cani PD, Delzenne NM: Coenzyme Q10 supplementation lowers hepatic oxidative stress and inflammation associated with diet-induced obesity in mice. Biochem Pharmacol. 2009, 78 (11): 1391-1400. 10.1016/j.bcp.2009.07.008.PubMedView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.