Skip to main content

Effect of Trace Minerals and B Vitamins on the Proliferation/Cytotoxicity and Mineralization of a Gilthead Seabream Bone–Derived Cell Line

Abstract

Trace minerals and vitamins are known modulators of bone metabolism, and dietary optimization of these components may improve skeletal development and reduce the occurrence of skeleton deformities in farmed fish. As for larval stages, mineral and water-soluble vitamin nutrition requirements are lacking in research efforts and knowledge is scarce. An in vitro cell system developed from gilthead seabream vertebra and capable of mineralization was used to assess the effect of B vitamins (thiamin and pyridoxine) and trace minerals (copper, manganese, and zinc in a sulfated and chelated form) on cell proliferation and extracellular matrix (ECM) mineralization. Dependent on dose, inhibition of cellular proliferation and/or cytotoxic effects was observed for all nutrients tested and LD50 values were determined: copper, 67.4–69.5 ppm; manganese, 20.9–29.8 ppm; zinc, 37.1–42.8 ppm in sulfated and chelated form respectively; thiamin, 6273 ppm; pyridoxine, 14226 ppm. ECM mineralization was enhanced by mineral (dose and form dependent) and vitamin (dose dependent) supplementation, at non-toxic concentrations below the determined LD50s. This in vitro work confirmed the mineralogenic action of trace minerals and water-soluble vitamins and provided valuable insights for subsequent in vivo nutritional trials.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig 2
Fig. 3
Fig. 4
Fig. 5

References

  1. Boglione C, Gavaia P, Koumoundouros G et al (2013) Skeletal anomalies in reared European fish larvae and juveniles. Part 1: normal and anomalous skeletogenic processes. Rev Aquac 5:S99–S120. https://doi.org/10.1111/raq.12015

    Article  Google Scholar 

  2. Lall SP, Lewis-McCrea L (2007) Role of nutrients in skeletal metabolism and pathology in fish - an overview. Aquaculture 267:3–19. https://doi.org/10.1016/j.aquaculture.2007.02.053

    CAS  Article  Google Scholar 

  3. Boglione C, Gisbert E, Gavaia P et al (2013) Skeletal anomalies in reared European fish larvae and juveniles. Part 2: main typologies, occurrences and causative factors. Rev Aquac 5:S121–S167. https://doi.org/10.1111/raq.12016

    Article  Google Scholar 

  4. Lall SP (2002) The minerals. In: Hardy JE, Halver R (eds) Fish nutrition, 3rd edn. Academic Press, San Diego, pp 259–308

    Google Scholar 

  5. Watanabe T, Kiron V, Satoh S (1997) Trace minerals in fish nutrition. Aquaculture 151:185–207. https://doi.org/10.1016/S0044-8486(96)01503-7

    CAS  Article  Google Scholar 

  6. Dai Z, Koh W-P (2015) B-vitamins and bone health–a review of the current evidence. Nutrients 7:3322–3346. https://doi.org/10.3390/nu7053322

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  7. NRC (National Research Council) (2011) Nutrient requirements of fish and shrimp. The National Academies Press, Washington, DC

    Google Scholar 

  8. Dermience M, Lognay G, Mathieu F, Goyens P (2015) Effects of thirty elements on bone metabolism. J Trace Elem Med Biol 32:86–106. https://doi.org/10.1016/j.jtemb.2015.06.005

    CAS  Article  PubMed  Google Scholar 

  9. Halver JE (2002) The Vitamins. In: Hardy JE, Halver R (eds) Fish Nutrition, 3rd edn. Academic Press, San Diego, pp 61–141

    Google Scholar 

  10. Palacios C (2006) The role of nutrients in bone health, from A to Z. Crit Rev Food Sci Nutr 46:621–628. https://doi.org/10.1080/10408390500466174

    CAS  Article  PubMed  Google Scholar 

  11. Nguyen VT, Satoh S, Haga Y et al (2008) Effect of zinc and manganese supplementation in Artemia on growth and vertebral deformity in red sea bream (Pagrus major) larvae. Aquaculture 285:184–192. https://doi.org/10.1016/j.aquaculture.2008.08.030

    CAS  Article  Google Scholar 

  12. Terova G, Rimoldi S, Izquierdo M, Pirrone C, Ghrab W, Bernardini G (2018) Nano-delivery of trace minerals for marine fish larvae: influence on skeletal ossification, and the expression of genes involved in intestinal transport of minerals, osteoblast differentiation, and oxidative stress response. Fish Physiol Biochem 44:1375–1391. https://doi.org/10.1007/s10695-018-0528-7

    CAS  Article  PubMed  Google Scholar 

  13. Izquierdo MS, Ghrab W, Roo J et al (2016) Organic, inorganic and nanoparticles of Se, Zn and Mn in early weaning diets for gilthead seabream (Sparus aurata; Linnaeus, 1758). Aquac Res 48:1–16. https://doi.org/10.1111/are.13119

    CAS  Article  Google Scholar 

  14. Gansner JM, Mendelsohn BA, Hultman KA, Johnson SL, Gitlin JD (2007) Essential role of lysyl oxidases in notochord development. Dev Biol 307:202–213. https://doi.org/10.1016/j.ydbio.2007.04.029

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  15. Apines-Amar MJS, Satoh S, Caipang CMA et al (2004) Amino acid-chelate: a better source of Zn, Mn and Cu for rainbow trout, Oncorhynchus mykiss. Aquaculture 240:345–358. https://doi.org/10.1016/j.aquaculture.2004.01.032

    CAS  Article  Google Scholar 

  16. Paripatananont T, Lovell RT (1997) Comparative net absorption of chelated and inorganic trace minerals in channel catfish Ictalurus punctatus diets. J World Aquacult Soc 28:62–67. https://doi.org/10.1111/j.1749-7345.1997.tb00962.x

    Article  Google Scholar 

  17. Katya K, Lee S, Bharadwaj AS et al (2016) Effects of inorganic and chelated trace mineral (Cu, Zn, Mn and Fe) premixes in marine rockfish, Sebastes schlegeli (Hilgendorf), fed diets containing phytic acid. Aquac Res 48:1–9. https://doi.org/10.1111/are.13236

    CAS  Article  Google Scholar 

  18. Pombinho AR, Laizé V, Molha DM, Marques SM, Cancela ML (2004) Development of two bone-derived cell lines from the marine teleost Sparus aurata; evidence for extracellular matrix mineralization and cell-type-specific expression of matrix Gla protein and osteocalcin. Cell Tissue Res 315:393–406. https://doi.org/10.1007/s00441-003-0830-1

    CAS  Article  PubMed  Google Scholar 

  19. Prabhu PAJ, Schrama JW, Mariojouls C et al (2014) Post-prandial changes in plasma mineral levels in rainbow trout fed a complete plant ingredient based diet and the effect of supplemental di-calcium phosphate. Aquaculture 430:1–10. https://doi.org/10.1016/j.aquaculture.2014.03.038

    CAS  Article  Google Scholar 

  20. Domínguez D, Rimoldi S, Robaina LE, Torrecillas S, Terova G, Zamorano MJ, Karalazos V, Hamre K, Izquierdo M (2017) Inorganic, organic, and encapsulated minerals in vegetable meal based diets for Sparus aurata (Linnaeus, 1758). PeerJ 5:e3710. https://doi.org/10.7717/peerj.3710

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  21. Viegas MN, Dias J, Cancela ML, Laizé V (2012) Polyunsaturated fatty acids regulate cell proliferation, extracellular matrix mineralization and gene expression in a gilthead seabream skeletal cell line. J Appl Ichthyol 28:427–432. https://doi.org/10.1111/j.1439-0426.2012.01994.x

    CAS  Article  Google Scholar 

  22. Tan F, Wang M, Wang W, Lu Y (2008) Comparative evaluation of the cytotoxicity sensitivity of six fish cell lines to four heavy metals in vitro. Toxicol in Vitro 22:164–170. https://doi.org/10.1016/j.tiv.2007.08.020

    CAS  Article  PubMed  Google Scholar 

  23. Clearwater SJ, Farag AM, Meyer JS (2002) Bioavailability and toxicity of dietborne copper and zinc to fish. Comp Biochem Physiol - C Toxicol Pharmacol 132:269–313. https://doi.org/10.1016/S1532-0456(02)00078-9

    Article  PubMed  Google Scholar 

  24. Domínguez D, Sarmiento P, Sehnine Z et al (2019) Effects of copper levels in diets high in plant ingredients on gilthead sea bream (Sparus aurata) fingerlings. Aquaculture 507:466–474. https://doi.org/10.1016/j.aquaculture.2019.04.044

    CAS  Article  Google Scholar 

  25. Arigony ALV, de Oliveira IM, Machado M et al (2013) The influence of micronutrients in cell culture: a reflection on viability and genomic stability. Biomed Res Int 2013:1–22

    Article  Google Scholar 

  26. Li S, Wang M, Chen X, Li SF, Li-Ling J, Xie HQ (2014) Inhibition of osteogenic differentiation of mesenchymal stem cells by copper supplementation. Cell Prolif 47:81–90. https://doi.org/10.1111/cpr.12083

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  27. Burghardt I, Lüthen F, Prinz C, Kreikemeyer B, Zietz C, Neumann HG, Rychly J (2015) A dual function of copper in designing regenerative implants. Biomaterials 44:36–44. https://doi.org/10.1016/j.biomaterials.2014.12.022

    CAS  Article  PubMed  Google Scholar 

  28. Rodríguez JP, Ríos S, González M (2002) Modulation of the proliferation and differentiation of human mesenchymal stem cells by copper. J Cell Biochem 85:92–100. https://doi.org/10.1002/jcb.10111

    CAS  Article  PubMed  Google Scholar 

  29. Hong H-H, Pischon N, Santana RB, Palamakumbura AH, Chase HB, Gantz D, Guo Y, Uzel MI, Ma D, Trackman PC (2004) A role for lysyl oxidase regulation in the control of normal collagen deposition in differentiating osteoblast cultures. J Cell Physiol 200:53–62. https://doi.org/10.1002/jcp.10476

    CAS  Article  PubMed  Google Scholar 

  30. Ashmead HD, Zunino H (1993) Factors which affect the intestinal absorbtion of minerals. In: Ashmead HD (ed) The roles of amino acid chelates in animal nutrition. Noyes Publications, New Jersey, pp 21–46

    Google Scholar 

  31. Lüthen F, Bulnheim U, Müller PD, Rychly J, Jesswein H, Nebe JG (2007) Influence of manganese ions on cellular behavior of human osteoblasts in vitro. Biomol Eng 24:531–536. https://doi.org/10.1016/j.bioeng.2007.08.003

    CAS  Article  PubMed  Google Scholar 

  32. Hallab NJ, Vermes C, Messina C, Roebuck KA, Glant TT, Jacobs JJ (2002) Concentration- and composition-dependent effects of metal ions on human MG-63 osteoblasts. J Biomed Mater Res 60:420–433. https://doi.org/10.1002/jbm.10106

    CAS  Article  PubMed  Google Scholar 

  33. Miola M, Vitale C, Maina G et al (2014) In vitro study of manganese-doped bioactive glasses for bone regeneration. Mater Sci Eng C 38:107–118. https://doi.org/10.1016/j.msec.2014.01.045

    CAS  Article  Google Scholar 

  34. Bracci B, Torricelli P, Panzavolta S, Boanini E, Giardino R, Bigi A (2009) Effect of Mg2+, Sr2+ , and Mn2+ on the chemico-physical and in vitro biological properties of calcium phosphate biomimetic coatings. J Inorg Biochem 103:1666–1674. https://doi.org/10.1016/j.jinorgbio.2009.09.009

    CAS  Article  PubMed  Google Scholar 

  35. Zafar N, Khan MA (2019) Growth, feed utilization, mineralization and antioxidant response of stinging catfish Heteropneustes fossilis fed diets with different levels of manganese. Aquaculture 509:120–128. https://doi.org/10.1016/j.aquaculture.2019.05.022

    CAS  Article  Google Scholar 

  36. Callaway DA, Jiang JX (2015) Reactive oxygen species and oxidative stress in osteoclastogenesis, skeletal aging and bone diseases. J Bone Miner Metab 33:359–370. https://doi.org/10.1007/s00774-015-0656-4

    CAS  Article  PubMed  Google Scholar 

  37. Arai M, Shibata Y, Pugdee K, Abiko Y, Ogata Y (2007) Effects of reactive oxygen species (ROS) on antioxidant system and osteoblastic differentiation in MC3T3-E1 cells. IUBMB Life 59:27–33. https://doi.org/10.1080/15216540601156188

    CAS  Article  PubMed  Google Scholar 

  38. Murphy CB, Martell AE (1957) Metal chelates of glycine and glycine peptides. J Biol Chem 226:37–50

    CAS  PubMed  Google Scholar 

  39. Nagata M, Lönnerdal B (2011) Role of zinc in cellular zinc trafficking and mineralization in a murine osteoblast-like cell line. J Nutr Biochem 22:172–178. https://doi.org/10.1016/j.jnutbio.2010.01.003

    CAS  Article  PubMed  Google Scholar 

  40. Yamaguchi M (1998) Role of zinc in bone formation and bone resorption. J Trace Elem Exp Med 11:119–135. https://doi.org/10.1002/(SICI)1520-670X(1998)11:2/3<119::AID-JTRA5>3.0.CO;2-3

  41. Sauer GR, Adkisson HD, Genge BR, Wuthier RE (1989) Regulatory effect of endogenous zinc and inhibitory action of toxic metal ions on calcium accumulation by matrix vesicles in vitro. Bone Miner 7:233–244. https://doi.org/10.1016/0169-6009(89)90080-9

    CAS  Article  PubMed  Google Scholar 

  42. Togari A, Arakawa S, Arai M, Matsumoto S (1993) Alteration of in vitro bone metabolism and tooth formation by zinc. Gen Pharmacol Vasc Syst 24:1133–1140. https://doi.org/10.1016/0306-3623(93)90360-A

    CAS  Article  Google Scholar 

  43. LeGeros RZ, Bleiwas C, Retino M, Rohanizadeh R, LeGeros J (1999) Zinc effect on the in vitro formation of calcium phosphates: Relevance to clinical inhibition of calculus formation. Am J Dent 12:65–71

    CAS  PubMed  Google Scholar 

  44. Hamre K, Yúfera M, Rønnestad I et al (2013) Fish larval nutrition and feed formulation: knowledge gaps and bottlenecks for advances in larval rearing. Rev Aquac 5:S26–S58. https://doi.org/10.1111/j.1753-5131.2012.01086.x

    Article  Google Scholar 

  45. Haley TJ, Flesher AM (1946) A toxicity study of thiamine hydrochloride. Science 104:567–568

    CAS  Article  Google Scholar 

  46. Waagbø R (2010) Water-soluble vitamins in fish ontogeny. Aquac Res 41:733–744. https://doi.org/10.1111/j.1365-2109.2009.02223.x

    CAS  Article  Google Scholar 

  47. Alvarez OM, Gilbreath RL (1982) Thiamine influence on collagen during the granulation of skin wounds. J Surg Res 32:24–31. https://doi.org/10.1016/0022-4804(82)90180-9

    CAS  Article  PubMed  Google Scholar 

  48. Mccormick RJ (1989) The influence of nutrition on collagen metabolism and stability. Reciprocal Meat Conf Proc 42:137–148

    Google Scholar 

  49. Vrolijk MF, Opperhuizen A, Jansen EHJM et al (2017) The vitamin B6 paradox: supplementation with high concentrations of pyridoxine leads to decreased vitamin B6 function. Toxicol in Vitro 44:206–212. https://doi.org/10.1016/j.tiv.2017.07.009

    CAS  Article  PubMed  Google Scholar 

  50. Molina A, Oka T, Muñoz SM et al (1997) Vitamin B6 suppresses growth and expression of albumin gene in a human hepatoma cell line HepG2. Nutr Cancer 28:206–211. https://doi.org/10.1080/01635589709514576

    CAS  Article  PubMed  Google Scholar 

  51. DiSorbo DM, Litwack G (1981) Vitamin B6 kills hepatoma cells in culture. Nutr Cancer 3:216–222. https://doi.org/10.1080/01635588109513725

    Article  Google Scholar 

  52. Herrmann M, Umanskaya N, Wildemann B et al (2007) Accumulation of homocysteine by decreasing concentrations of folate, vitamin B12 and B6 does not influence the activity of human osteoblasts in vitro. Clin Chim Acta 384:129–134. https://doi.org/10.1016/j.cca.2007.06.016

    CAS  Article  PubMed  Google Scholar 

  53. Murray JC, Levene CI (1977) Evidence for the role of vitamin B-6 as a cofactor of lysyl oxidase. Biochem J 167:463–467. https://doi.org/10.1042/bj1670463

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  54. Fedde KN, Lane CC, Whyte MP (1988) Alkaline phosphatase is an ectoenzyme that acts on micromolar concentrations of natural substrates at physiologic pH in human osteosarcoma (SAOS-2) cells. Arch Biochem Biophys 264:400–409. https://doi.org/10.1016/0003-9861(88)90305-0

    CAS  Article  PubMed  Google Scholar 

  55. Dodds RA, Catterall A, Bitensky L, Chayen J (1986) Abnormalities in fracture healing induced by vitamin B6-deficiency in rats. Bone 7:489–495. https://doi.org/10.1016/8756-3282(86)90008-6

    CAS  Article  PubMed  Google Scholar 

  56. Rafael MS, Marques CL, Parameswaran V et al (2010) Fish bone-derived cell lines: an alternative in vitro cell system to study bone biology. J Appl Ichthyol 26:230–234. https://doi.org/10.1111/j.1439-0426.2010.01411.x

    Article  Google Scholar 

Download references

Funding

This research has been carried out with the financial support of the LARVAMIX project (grant no. 17925) supported by Portugal and the European Union through FEDER, COMPETE 2020, and CRESC Algarve 2020, in the framework of Portugal 2020. MV received financial support by FCT/MCTES (Portugal) through a doctoral fellowship (grant PDE/BDE/113672/2015). VL acknowledges the financial support of the Portuguese Foundation for Science and Technology (FCT) through project UID/Multi/04326/2019 (CCMAR). 

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Michael N. Viegas.

Ethics declarations

Conflict of Interest

The authors declare that they have no conflicts of interest.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Viegas, M.N., Laizé, V., Salgado, M.A. et al. Effect of Trace Minerals and B Vitamins on the Proliferation/Cytotoxicity and Mineralization of a Gilthead Seabream Bone–Derived Cell Line. Biol Trace Elem Res 196, 629–638 (2020). https://doi.org/10.1007/s12011-019-01939-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12011-019-01939-5

Keywords

  • Trace minerals
  • Vitamins
  • Cell culture
  • Mineralization
  • Gilthead seabream