Environmental Science and Pollution Research

, Volume 24, Issue 17, pp 15187–15195 | Cite as

Effect of cadmium on cytosine hydroxymethylation in gastropod hepatopancreas

  • Dragos NicaEmail author
  • Cristina Popescu
  • George Draghici
  • Ionela Privistirescu
  • Maria Suciu
  • Reinhard Stöger
Short Research and Discussion Article


5-Hydroxymethylcytosine (5hmC) is an important, yet poorly understood epigenetic DNA modification, especially in invertebrates. Aberrant genome-wide 5hmC levels have been associated with cadmium (Cd) exposure in humans, but such information is lacking for invertebrate bioindicators. Here, we aimed to determine whether this epigenetic mark is present in DNA of the hepatopancreas of the land snail Cantareus aspersus and is responsive to Cd exposure. Adult snails were reared under laboratory conditions and exposed to graded amounts of dietary cadmium for 14 days. Weight gain was used as a sublethal endpoint, whereas survival as a lethal endpoint. Our results are the first to provide evidence for the presence of 5hmC in DNA of terrestrial mollusks; 5hmC levels are generally low with the measured values falling below 0.03%. This is also the first study to investigate the interplay of Cd with DNA hydroxymethylation levels in a non-human animal study system. Cadmium retention in the hepatopancreas of C. aspersus increased from a dietary Cd dose of 1 milligram per kilogram dry weight (mg/kg d. wt). For the same treatment, we identified the only significant elevation in percentage of samples with detectable 5hmC levels despite the lack of significant mortalities and changes in weight gain among treatment groups. These findings indicate that 5hmC is an epigenetic mark that may be responsive to Cd exposure, thereby opening a new aspect to invertebrate environmental epigenetics.


Cadmium 5-Hydroxymethylcytosine Land snails Hepatopancreas Dietary exposure 



The present work was supported by a grant of the Romanian National Authority for Scientific Research and Innovation, CNCS-UEFISCDI, Research Council, project number PN-II-RU-TE-2014-4-0776 (awarded to DVN). We are very grateful for the anonymous reviewers’ constructive comments and recommendations, which have helped us greatly increase the quality of the present article. In addition, the authors wish to thank Prof. Iosif Gergen (BUASVMT) for the help and guidance in performing chemical analyses.


  1. ATSDR (2008) Toxicological profile for cadmium. US Department of Health and Human Services. Public Health Service, AtlantaGoogle Scholar
  2. Baurand PE, Pedrini-Martha V, De Vaufleury A, Niederwanger M et al (2015) Differential expression of metallothionein isoforms in terrestrial snail embryos reflects early life stage adaptation to metal stress. PLoS One 10:e0116004. doi: 10.1371/journal.pone.0116004 CrossRefGoogle Scholar
  3. Bergman Y, Cedar H (2013) DNA methylation dynamics in health and disease. Nat Struct Mol Biol 20:274–281. doi: 10.1038/nsmb.2518 CrossRefGoogle Scholar
  4. Branco MR, Ficz G, Reik W (2011) Uncovering the role of 5-hydroxymethylcytosine in the epigenome. Nat Rev Genet 13:7–13. doi: 10.1038/nrg3080 CrossRefGoogle Scholar
  5. Cingolani P, Cao X, Khetani RS, Chen CC, Coon M et al (2013) Intronic non-CG DNA hydroxymethylation and alternative mRNA splicing in honey bees. BMC Genomics 14:666. doi: 10.1186/1471-2164-14-666 CrossRefGoogle Scholar
  6. Coeurdassier M, Gomot DVA, Lovy C, Badot PM (2002) Is the cadmium uptake from soil important in bioaccumulation and toxic effects for snails? Ecotox Environ Saf 53(3):425–431. doi: 10.1016/S0147-6513(02)00004-0 CrossRefGoogle Scholar
  7. Dallinger R, Rainbow PS (1993) Ecotoxicology of metals in invertebrates. Lewis Publishers, Boca RatonGoogle Scholar
  8. Dallinger R, Berger B, Hunziker PE, Birchler N, Hauer CR, Kägi JH (1993) Purification and primary structure of snail metallothionein. Similarity of the N-terminal sequence with histones H4 and H2A. Eur J Biochem 216(3):739–746CrossRefGoogle Scholar
  9. Dallinger R, Berger B, Triebskorn-Kohler R, Kohler H (2001) Soil biology and ecotoxicology. In: Barker GM (ed) The biology of terrestrial molluscs. CABI Publishing, Wallingford, pp 489–525CrossRefGoogle Scholar
  10. Dallinger R, Lagg B, Egg M, Schipflinger R, Chabicovsky M (2004) Cd accumulation and Cd-metallothionein as a biomarker in Cepaea hortensis (Helicidae, Pulmonata) from laboratory exposure and metal-polluted habitats. Ecotoxicology 13(8):757–772. doi: 10.1007/s10646-003-4474-4 CrossRefGoogle Scholar
  11. Dao T, Cheng RYS, Revelo MP, Mitzner W, Tang WY (2014) Hydroxymethylation as a novel environmental biosensor. Environ Health Rep 1(1):1–10. doi: 10.1007/s40572-013-0005-5 CrossRefGoogle Scholar
  12. Erdmann RM, Souza AL, Clish CB, Gehring M (2015) 5-Hydroxymethylcytosine is not present in appreciable quantities in Arabidopsis DNA. G3: Genes Genomes Genetics 5(1):1–8. doi: 10.1534/g3.114.014670 CrossRefGoogle Scholar
  13. European Comission (2006) Regulation No. 1881/2006 of 19 December 2006 setting maximum levels for certain contaminants in foodstuffs. Official Journal of the European Union L Series 364:5–24Google Scholar
  14. Feliciello I, Parazajder J, Akrap I, Ugarkovic D (2013) First evidence of DNA methylation in insect Tribolium castaneum. Environmental regulation of DNA methylation within heterochromatin. Epigenetics 8(5):534–541. doi: 10.4161/epi.24507 CrossRefGoogle Scholar
  15. Fneich S, Dheilly N, Adema C, Rognon A, Reichelt M et al (2013) 5-Methyl-cytosine and 5-hydroxy-methyl-cytosine in the genome of Biomphalaria glabrata, a snail intermediate host of Schistosoma mansoni. Parasite Vector 6:1. doi: 10.1186/1756-3305-6-167 CrossRefGoogle Scholar
  16. Garcia A, Perea JM, Mayoral A, Acero R, Martos J et al (2006) Laboratory rearing conditions for improved growth of juvenile Helix aspersa Müller snails. Lab Anim 40(3):309–316. doi: 10.1258/002367706777611505 CrossRefGoogle Scholar
  17. Gimbert F, de Vaufleury A, Douay F, Scheifler R, Coeurdassier M, Badot PM (2006) Modelling chronic exposure to contaminated soil. A toxicokinetic approach with terrestrial snail Helix aspersa. Environ Int 32:866–875. doi: 10.1016/j.envint.2006.05.006 CrossRefGoogle Scholar
  18. Gimbert F, Mench M, Coeurdassier C, Badot PM, de Vaufleury A (2008) Kinetic and dynamic aspects of soil-plant-snail transfer of cadmium in the field. Environ Pollut 152:736–745. doi: 10.1016/j.envpol.2007.06.044 CrossRefGoogle Scholar
  19. Globisch D, Munzel M, Muller M, Michalakis S, Wagner M et al (2010) Tissue distribution of 5-hydroxymethylcytosine and search for active demethylation intermediates. PLoS One 5:e15367. doi: 10.1371/journal.pone.0015367 CrossRefGoogle Scholar
  20. Gomot A (1997) Dose-dependent effects of cadmium on the growth of snails in toxicity bioassays. Arch Environ Contam Toxicol 33(2):209–216CrossRefGoogle Scholar
  21. Hanna CW, Bloom MS, Robinson WP, Kim D, Parsons PJ et al (2012) DNA methylation changes in whole blood is associated with exposure to the environmental contaminants, mercury, lead, cadmium and bisphenol A, in women undergoing ovarian stimulation for IVF. Hum Reprod 27:1401–1410. doi: 10.1093/humrep/des038 CrossRefGoogle Scholar
  22. Head JA, Dolinoy DC, Basu N (2012) Epigenetics for ecotoxicologists. Environ Toxicol Chem 31:221–227. doi: 10.1002/etc.1707 CrossRefGoogle Scholar
  23. HHS, US Department of Health and Human Services (2001) Guidance for industry, bioanalytical method validation.
  24. Hill PW, Amouroux R, Hajkova P (2014) DNA demethylation, Tet proteins and 5-hydroxymethylcytosine in epigenetic reprogramming: an emerging complex story. Genomics 104(5):324–333. doi: 10.1016/j.ygeno.2014.08.012 CrossRefGoogle Scholar
  25. Hispard F, Schuler D, de Vaufleury A, Scheifler R, Badot PM, Dallinger R (2008) Metal distribution and metallothionein induction after cadmium exposure in the terrestrial snail Helix aspersa (Gastropoda, Pulmonata). Environ Toxicol Chem 27(7):1533–1542. doi: 10.1897/07-232.1 CrossRefGoogle Scholar
  26. Höckner M, Stefanon K, De Vaufleury A, Monteiro F, Pérez-Rafael S et al (2011) Physiological relevance and contribution to metal balance of specific and non-specific metallothionein isoforms in the garden snail, Cantareus aspersus. Biometals 24(6):1079–1092. doi: 10.1007/s10534-011-9466-x CrossRefGoogle Scholar
  27. Hödl E, Felder E, Chabicovsky M, Dallinger R (2010) Cadmium stress stimulates tissue turnover in Helix pomatia: increasing cell proliferation from metal tolerance to exhaustion in molluscanmidgut gland. Cell Tissue Res 341(1):159–171. doi: 10.1007/s00441-010-0980-x CrossRefGoogle Scholar
  28. Hood RD (2016) Developmental and reproductive toxicology: a practical approach. CRC Press, Boca RatonGoogle Scholar
  29. Hossain MB, Vahter M, Concha G, Broberg K (2012) Low-level environmental cadmium exposure is associated with DNA hypomethylation in Argentinean women. Environ Health Perspect 120:879–884. doi: 10.1289/ehp.1104600 CrossRefGoogle Scholar
  30. Itziou A, Dimitriadis VK (2011) Introduction of the land snail Eobania vermiculata as a bioindicator organism of terrestrial pollution using a battery of biomarkers. Sci Total Environ 409(6):1181–1192. doi: 10.1016/j.scitotenv.2010.12.009 CrossRefGoogle Scholar
  31. Itziou A, Kaloyianni M, Dimitriadis V (2011) In vivo and in vitro effects of metals in reactive oxygen species production, protein carbonylation, and DNA damage in land snails Eobania vermiculata. Arch Environ Contam Toxicol 60:697–707. doi: 10.1007/s00244-010-9583-5 CrossRefGoogle Scholar
  32. Jiang G, Xu L, Song S, Zhu C, Wu Q, Zhang L, Wu L (2008) Effects of long-term low-dose cadmium exposure on genomic DNA methylation in human embryo lung fibroblast cells. Toxicology 244:49–55. doi: 10.1016/j.tox.2007.10.028 CrossRefGoogle Scholar
  33. Jones PA (2012) Functions of DNA methylation: islands, start sites, gene bodies and beyond. Nat RevGenet 13:484–492. doi: 10.1038/nrg3230 CrossRefGoogle Scholar
  34. Kerney MP, Cameron RAD (1979) A field guide to the land snails of Britain and northwestern Europe. William Collins Sons and Co., LondonGoogle Scholar
  35. Kucharski R, Maleszka J, Foret S, Maleszka R (2008) Nutritional control of reproductive status in honeybees via DNA methylation. Science 319:1827–1830. doi: 10.1126/science.1153069 CrossRefGoogle Scholar
  36. Laskowski R, Hopkin SP (1996) Effect of Zn, Cu, Pb, and Cd on fitness in snails (Helix aspersa). Ecotoxicol Environ Saf 34:59–69. doi: 10.1006/eesa.1996.0045 CrossRefGoogle Scholar
  37. Li W, Liu M (2011) Distribution of 5-hydroxymethylcytosine in different human tissues. J Nucleic Acids 2011:870726. doi: 10.4061/2011/870726 CrossRefGoogle Scholar
  38. Lian S, He Y, Li X, Zhao B, Hou R et al (2015) Changes in global DNA methylation intensity and DNMT1 transcription during the aging process of scallop Chlamysfarreri. J OceanU China 14:685–690. doi: 10.1007/s11802-015-2507-2 CrossRefGoogle Scholar
  39. Liu J, Hesson LB, Ward RL (2013) Liquid chromatography tandem mass spectrometry for the measurement of global DNA methylation and hydroxymethylation. J Proteomics Bioinform S2:005. doi: 10.4172/jpb.S2-005 CrossRefGoogle Scholar
  40. Lyko F, Foret S, Kucharski R, Wolf S, Falckenhayn C, Maleszka R (2010) The honey bee epigenomes: differential methylation of brain DNA in queens and workers. PLoS Biol 8(11):e1000506. doi: 10.1371/journal.pbio.1000506 CrossRefGoogle Scholar
  41. Moroz LL, Kohn AB (2013) Single-neuron transcriptome and methylome sequencing for epigenomic analysis of aging. Methods Mol Biol 1048:323–352. doi: 10.1007/978-1-62703-556-9_21 CrossRefGoogle Scholar
  42. Moroz LL, Kocot KM, Citarella MR, Dosung S, Norekian TP et al (2014) The ctenophore genome and the evolutionary origins of neural systems. Nature 510:110–114. doi: 10.1038/nature13400 CrossRefGoogle Scholar
  43. Murata A, Baba Y, Ishimoto T, Miyake K, Kosumi K et al (2015) TET family proteins and 5-hydroxymethylcytosine in esophageal squamous cell carcinoma. Oncotarget 6(27):23372–23382. doi: 10.18632/oncotarget.4281 CrossRefGoogle Scholar
  44. MWFEP (Ministry of Waters, Forests and Environmental Protection, Romania) (2002) Ordinul nr. 756/1997 al Ministerului Apelor, Padurilor şi Protecţiei Mediului pentru aprobarea Reglementării privind evaluarea poluarii mediului modificat de Ordinul nr. 1144/2002 al Ministerului Apelor si Protectiei Mediului. Accessed 10.01.2017
  45. Nica DV, Filimon MN, Bordean DM, Harmanescu M, Draghici GA et al (2015) Impact of soil cadmium on land snails: a two-stage exposure approach under semi-field conditions using bioaccumulative and conchological end-points of exposure. PLoS One 10:e0116397. doi: 10.1371/journal.pone.0116397 CrossRefGoogle Scholar
  46. Nica DV, Popescu C, Draghici GA, Andrica FM, Privistirescu IA, Stöger R (2016) Cadmium hepatotoxicity: an epigenetic gastropod-based approach. SETAC/iEOS Joint Focused Topic, Environmental and (Eco)toxicological Omics and Epigenetics: Science, Technology and Regulatory Applications ISSN 2310–3191Google Scholar
  47. Notten M, Oosthoek A, Rozema J, Aerts R (2006) The landsnail Cepaeanemoralis regulates internal Cd levels when fed on Cd-enriched stinging nettle (Urticadioica) leaves at low, field-relevant concentrations. Environ Pollut 139:296–305. doi: 10.1016/j.envpol.2005.05.007 CrossRefGoogle Scholar
  48. Oehlmann J, Schulte-Oehlmann U (2003) Molluscs as bioindicators. In: Markert BA, Breure AM, Zechmeister HG (eds) Trace metals and other contaminants in the environment, vol. 6. Elsevier, Oxford, pp 577–635. doi: 10.1016/S0927-5215(03)80147-9 CrossRefGoogle Scholar
  49. Palacios O, Pagani A, Perez-Rafael S, Egg M, Hockner M et al (2011) Shaping mechanisms of metal specificity in a family of metazoan metallothioneins: evolutionary differentiation of mollusc metallothioneins. BMC Biol 9(1):1. doi: 10.1186/1741-7007-9-4 CrossRefGoogle Scholar
  50. Pells S, Koutsouraki E, Morfopoulou S, Valencia-Cadavid S, Tomlinson SR et al (2015) Novel human embryonic stem cell regulators identified by conserved and distinct CpG Island methylation state. PLoS One 10(7):e0131102. doi: 10.1371/journal.pone.0131102 CrossRefGoogle Scholar
  51. Pierron F, Baillon L, Sow M, Gotreau S, Gonzalez P (2013) Effect of low-dose cadmium exposure on DNA methylation in the endangered European eel. Environ Sci Technol 48:797–803. doi: 10.1021/es4048347 CrossRefGoogle Scholar
  52. Pirola CJ, Scian R, Gianotti TF, Dopazo H, Rohr C et al (2015) Epigenetic modifications in the biology of nonalcoholic fatty liver disease: the role of DNA hydroxymethylation and TET proteins. Medicine 94(36):e1480. doi: 10.1097/MD.0000000000001480 CrossRefGoogle Scholar
  53. Rabitsch WB (1996) Metal accumulation in terrestrial pulmonates at a lead/zinc smelter site in Arnoldstein, Austria. Bull Environ Contam Toxicol 56:734–741CrossRefGoogle Scholar
  54. Rasmussen EM, Vågbø CB, Münch D, Krokan HE, Klungland A et al (2016) DNA base modifications in honey bee and fruit fly genomes suggest an active demethylation machinery with species- and tissue-specific turnover rates. Biochem Biophys Rep 6:9–15. doi: 10.1016/j.bbrep.2016.02.011 CrossRefGoogle Scholar
  55. Riviere G, Wu GC, Fellous A, Goux D, Sourdaine P, Favrel P (2013) DNA methylation is crucial for the early development in the oyster C. gigas. Mar Biotechnol 15:739–753. doi: 10.1007/s10126-013-9523-2 CrossRefGoogle Scholar
  56. Russell LK, DeHaven JI, Botts RP (1981) Toxic effects of cadmium on the garden snail (Helix aspersa). Bull Environ Contam Toxicol 26:634–640. doi: 10.1007/BF01622148 CrossRefGoogle Scholar
  57. Sanders A, Smeester L, Rojas D, De Bussycher T, Wu M et al (2014) Cadmium exposure and the epigenome: exposure-associated patterns of DNA methylation in leukocytes from mother-baby pairs. Epigenetics 9:212–221. doi: 10.4161/epi.26798 CrossRefGoogle Scholar
  58. Spurgeon DJ, Rowland P, Ainsworth G, Rothery P, Long S, Black HI (2008) Geographical and pedological drivers of distribution and risks to soil fauna of seven metals (Cd, Cu, Cr, Ni, Pb, V and Zn) in British soils. Environ Pollut 153(2):273–283CrossRefGoogle Scholar
  59. Strepetkaite D, Alzbutas G, Astromskas E, Lagunavicius A, Sabaliauskaite R et al (2016) Analysis of DNA methylation and hydroxymethylation in the genome of crustacean Daphnia pulex. Genes 7(1):1. doi: 10.3390/genes7010001 CrossRefGoogle Scholar
  60. Takiguchi M, Achanzar WE, Qu W, Li G, Waalkes MP (2003) Effects of cadmium on DNA-(cytosine-5) methyltransferase activity and DNA methylation status during cadmium-induced cellular transformation. Exp Cell Res 286:355–365. doi: 10.1016/S0014-4827(03)00062-4 CrossRefGoogle Scholar
  61. Tellez-Plaza M, Wan-yee T, Shang Y, Umans JG, Francesconi KA et al (2014) Association of global DNA methylation and global DNA hydroxymethylation with metals and other exposures in human blood DNA samples. Environ Health Perspect 122(9):946–954. doi: 10.1289/ehp.1306674 CrossRefGoogle Scholar
  62. Wang B, Li Y, Shao C, Tan Y, Cai L (2012) Cadmium and its epigenetic effects. Curr Med Chem 19:2611–2620. doi: 10.2174/092986712800492913 CrossRefGoogle Scholar
  63. Wang H, He L, Song J, Cui W, Zhang Y et al (2016) Cadmium-induced genomic instability in Arabidopsis: molecular toxicological biomarkers for early diagnosis of cadmium stress. Chemosphere 150:258–265. doi: 10.1016/j.chemosphere.2016.02.042 CrossRefGoogle Scholar
  64. Wattanaphansak S, Asawakarn T, Gebhart CJ, Deen J (2008) Development and validation of an enzyme-linked immunosorbent assay for the diagnosis of porcine proliferative enteropathy. J Vet Diagn Investig 20(2):170–177. doi: 10.1177/104063870802000205 CrossRefGoogle Scholar
  65. Wojciechowski M, Rafalski D, Kucharski R, Misztal K, Maleszka J et al (2014) Insights into DNA hydroxymethylation in the honeybee from in-depth analyses of TET dioxygenase. Open Biol 4(8) doi:  10.1098/rsob.140110 CrossRefGoogle Scholar
  66. Zhang C, Liang Y, Lei L, Zhu G, Chen X et al (2013) Hypermethylations of RASAL1 and KLOTHO is associated with renal dysfunction in a Chinese population environmentally exposed to cadmium. Toxicol Appl Pharmacol 271:78–85. doi: 10.1016/j.taap.2013.04.025 CrossRefGoogle Scholar
  67. Zhang J, Mu X, Xu W, Martin FL, Alamdar A et al (2014) Exposure to arsenic via drinking water induces 5-hydroxymethylcytosine alteration in rat. Sci Total Environ 498:618–625. doi: 10.1016/j.scitotenv.2014.08.009 CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  • Dragos Nica
    • 1
    • 2
    Email author
  • Cristina Popescu
    • 2
    • 3
  • George Draghici
    • 2
  • Ionela Privistirescu
    • 4
  • Maria Suciu
    • 1
    • 5
  • Reinhard Stöger
    • 6
  1. 1.Faculty of Pharmacy“Victor Babes” University of Medicine and PharmacyTimisoaraRomania
  2. 2.Institute of Life Sciences“Vasile Goldis” Western University of AradAradRomania
  3. 3.Faculty of Pharmacy“Vasile Goldis” Western University of AradAradRomania
  4. 4.Faculty of Medicine“Victor Babes” University of Medicine and PharmacyTimisoaraRomania
  5. 5.“Babes-Bolyai” UniversityCluj-NapocaRomania
  6. 6.School of BiosciencesUniversity of NottinghamLeicestershireUK

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