Effects of Multivitamins and Known Teratogens on Chick Cardiomyocytes Micromass Culture Assay

Memon, Samreen; Pratten, Margaret

doi:10.22038/ijbms.2013.1681

Effects of Multivitamins and Known Teratogens on Chick Cardiomyocytes Micromass Culture Assay

Document Type : Original Article

Authors

Samreen Memon ¹
Margaret Pratten ²

¹ Department of Anatomy Liaquat University of Medical Sciences, Pakistan

² School of Biomedical Sciences, University of Nottingham, UK

10.22038/ijbms.2013.1681

Abstract

Objective(s):
This study aimed to find out whether the chick cardiomyocyte micromass (MM) system could be employed to predict the teratogenecity of common environmental factors. Different multivitamins and over the counter drugs were used in this study.

Materials and Methods:
White Leghorn 5-day-old embryo hearts were dissected and trypsinized to produce a cardiomyocyte cell suspension in Dulbecco's Modified Eagle's Medium. The cultures were incubated at 370C in 5% CO2 in air, and observations were made at 24, 48 and 144 hr, for the detection of cell beating. Cellular viability was assessed using the resazurin assay and cell protein content was assessed by the kenacid blue assay. It was observed that while not affecting total cell number folic acid, vitamin C, sodium fluoride and ginseng did not significantly reduced cell activity and beating. However cadmium chloride significantly reduced the beating, cell viability and cell protein content in micromass cultures.
Results:
The results demonstrate the potential of the chick cardiomyocyte MM culture assay to identify teratogens/embryotoxins that alter morphology and function, which may result in either teratogenic outcome or cytotoxicity.
Conclusion:
This could form part of a screen for developmental toxicity related to cardiac function

Keywords

Chick cardiomyocyte Environmental teratogens Micromass culture Multivitamins

References

to fluoride-induced oxidative stress damage and mitochondrial transmembrane potential loss. Toxicol Appl Pharmacol. 2008;230:352-357.

39. Verma RJ, Sherlin DMG. Vitamin C ameliorates fluoride-induced embryotoxicity in pregnant rats. Human Exp Toxicol. 2001;20:619-623.

40. Heindel JJ, Bates HK, Price CJ, Marr MC, Myers CB, Schwetz BA. Developmental Toxicity Evaluation of Sodium Fluoride Administered to Rats and Rabbits in Drinking Water. Toxicol Sci. 1996;30:162-177.

41. Fu Y, Ji LL. Chronic ginseng consumption attenuates ageassociated oxidative stress in rats. J Nutr. 2003;133:3603-3609.

42. Kitts D, Hu C. Efficacy and safety of ginseng. Public Health Nutr. 2000; 3:473-485.

43. Liu P, Xu Y, Yin H, Wang J, Chen K, Li Y. Developmental toxicity research of ginsenoside Rb1 using a whole mouse embryo culture Model. Birth Defects Res B Dev Reprod Toxicol. 2005;74:207-209.

44. Liu P, Xu YJ, Yin HJ, Zhang ZF, Wang J, Chen K, et al. Effects of ginsenoside Rb1 on mouse embryonic development in vitro. Wei Sheng Yan Jiu. 2005;34:175-177.

45. Liu P, Yin H, Xu Y, Zhang Z, Chen K, Yong L. Effects of ginsenoside Rg1 on postimplantation rat and mouse embryos cultured in vitro. Toxicol in Vitro 2006;20: 234-238.

46. Poindexter BJ, Allison AW, Bick RJ, Dasgupta A. Ginseng: Cardiotonic in adult rat cardiomyocytes, cardiotoxic in neonatal rat cardiomyocytes. Life Sci. 2006;79:2337-2344.

1. Bailey J, Knight A, Balcombe J. The future of teratology research is in vitro. Biogenic Amin. 2005;19:97-145.

2. Brown NA, Spielmann H, Bechter R, Flint OP, Freeman S, Jelinek RJ, et al. Screening chemicals for reproductive toxicity: the current alternatives. Altern Lab Anim. 1995;23:868-882.

3. Bournias-Vardiabasis N, Teplitz RL. Use of Drosophilia embryo cell cultures as an in vitro teratogen assay. Teratog Carcinog Mutagen. 1982;2:333-341.

4. Hartung T, Bremer S, Casati S, Coecke S, Corvi R, Fortaner S, et al. ECVAM’s response to the changing political environment for alternatives: consequences of the European union chemicals and cosmetics policies. Altern Lab Anim. 2003;31:473-481.

5. Genschow E, Scholz G, Brown N, Piersma A, Brady M, Clemann N, et al. Development of prediction models for three in vivo embryotoxicity tests in an ECVAM validation study. In vitr Mol Toxicol. 2000;13:51-66.

6. Genschow E, Spielmann H, Scholz G, Seller A, Brown N, Piersma A, et al. The ECVAM international validation study on in vitro embryotoxicity tests: results of the definitive phase and evaluation of prediction models. Altern Lab Anim. 2002;30:151-176.

7. Brown NA. Selection of test chemicals for the ECVAM international validation on in vitro embryotoxicity tests. Altern Lab Anim. 2002;30:177-198.

8. Spielmann H, Genschow E, Scholz G, Brown NA, Piersma AH, Brady M, et al. Preliminary results of the ECVAM validation study on three in vitro embryotoxicity tests. Altern Lab Anim. 2001;29:301-303.

9. Flint OP, Orton TC. An in vitro assay for teratogens with cultures of rat embryo midbrain and limb bud cells. Toxicol Appl Pharmacol. 1984;76:383-395.

10. Parsons JF, Rockley J, Richold M. In vitro micromass teratogen test: Interpretation of results from a blind trial of 25 compounds using three separate criteria. Toxicol in Vitro. 1990;4:609-611.

11. Tsuchiya T, Bürgin H, Tsuchiya M, Winternitz P, Kistler A. Embryolethality of new herbicides is not detected by the micromass teratogen tests 1991;65:145-149.

12. Wiger R, Strottum A, Brunborg G. Estimating chemical developmental hazard in chicken embryo limb bud micromass system. Pharmacol Toxicol. 1988;62:32-37.

13. L'Huillier N, Pratten MK, Clothier RH. The relative embryotoxicity of 1,3-dichloro-2-propanol on

primary chick embryonic cells. Toxicol in Vitro. 2002;16:433-42.

14. Meyer MP, Swann K, Burnstock G, Clarke JDW. The extracellular ATP receptor, cP2Y1, inhibits cartilage formation in micromass cultures of chick limb mesenchyme. Dev Dyn. 2001;222:494-505.

15. Hurst HS, Clothier RH, Pratten M. An evaluation of a novel chick cardiomyocyte micromass culture assay with two teratogens/embryotoxins associated with heart defects. Altern Lab Anim. 2007;35:505-514.

16. Ahir B, Pratten MK. Association of anxiolytic drugs diazepam and lorazepam, and the antiepileptic valproate, with heart defects-effects on cardiomyocytes in micromass (MM) and embryonic stem cell culture. Reprod Toxicol. 2011;31:66-74.

17. Memon S, Pratten MK. Developmental toxicity of ethanol in chick heart in ovo and in micromass culture can be prevented by addition of vitamin C and folic acid. Reprod Toxicol. 2009;28:262-269.

18. Memon S, Pratten MK. Teratogenic effects of diabetic conditions in chick heart in ovo and micromass culture may be prevented by addition of vitamin C and folic acid. Reprod Toxicol. 2013;35:117-124.

19. Atterwill CK, Johnston H, Thomas SM. Models for the in vitro assessment of neurotoxicity in the nervous system in relation to xenobiotics and neurotrophic factor-mediated events. Neurotoxicology. 1992;13:39-53.

20. Tang LS, Wlodarczyk BJ, Santillano DR, Miranda RC, Finnel RH. Developmental cosequences of abnormal folate transport during murine heart morphogenesis. Birth Defects Res A Clin Mol Teratol. 2004;70:449-458.

21. Torrens C, Brawley L, Anthony FW, Dance CS, Dunn R, Jackson AA, et al. Folate supplementation during pregnancy improves offspring cardiovascular dysfunction induced by protein restriction. Hypertention. 2006;47:982-987.

22. Jaffe GM. Vitamin C. In: Machlin L, ed Handbook of vitamins. New York: Marcel Dekker Inc; 1984. p. 199-244.

23. Gilani SH, Alibhai Y. Teratogenicity of metals to chick embryos. J Toxicol Environ Health. 1990;30:23 - 31.

24. Thompson J, Hipwell E, Loo HV, Bannigan J. Effects of cadmium on cell death and cell proliferation in chick embryos. Reprod Toxicol. 2005;20:539-548.

25. Verma RJ, Guna Sherlin DM. Sodium fluoride-induced hypoproteinemia and hypoglycemia in parental and F1-generation rats and amelioration by vitamins. Food Cheml Toxicol. 2002;40:1781-1788.

26. Gibson PS, Powrie R, Star J. Herbal and alternative medicine use during pregnancy: a cross-sectional survey. Obstet Gynecol. 2001;97:S44-S5.

27. Clothier R, Starzec G, Pradel L, Baxter V, Jones M, Cox H, et al. The prediction of human skin responses by using the combined in vitro fluorescence leakage/Alamar blue (resazurin) assay. Altern Lab Anim. 2002;30:493–504.

28. Knox P, Uphill PF, Fry JR, Benford J, Balls M. The FRAME multicentre project on in vitro cytotoxicology. Food Chem Toxicol. 1986;24:457-463.

29. Brown NA, Spielmann H, Bechter R, Flint OP, Freeman S, Jelinek RJ, et al. Screening chemicals for

reproductive toxicity: the current alternatives. Altern Lab Anim. 1995;23:868-882.

30. Zhang Z, Xu Y, Li L, Han J, Zheng L, Liu P, et al. Prevention of retinoic acid-induced early craniofacial abnormalities by folinic acid and expression of endothelin-1/dHAND in the branchial arches in mouse. Br J Nutr. 2006;96:418-425.

31. Botto LD, Correa A. Decreasing the burden of congenital heart anomalies: an epidemiologic evaluation of risk factors and survival. Progress in Pediatr Cardiol. 2003;18:111-121.

32. Huhta JC, Hernandez-Robles JA. Homocysteine, Folate, and Congenital Heart Defects. Fetal & Pediatr Pathol. 2005;24:71-79.

33. Botto LD, Mulinare J, Erickson JD. Do mutivitamin or folic acid supplements reduce the risk for congenital heart defects? Am J Medl Genet A. 2003;121A:95-101.

34. Chan PK, Cheng SH. Cadmium-induced ectopic apoptosis in zebrafish embryos Arch Toxicol. 2003;77:69-79.

35. Yano CL, Marcondes MCCG. Cadmium chloride-induced oxidative stress in skeletal muscle cells in vitro. Free Radicl Bio Med. 2005;39:1378-1384.

36. Chow HES, Cheng SH. Cadmium Affects Muscle Type Development and Axon Growth in Zebrafish Embryonic Somitogenesis. Toxicol Sci. 2003;73:149-159.

37. Chan PK, Cheng SH. Cadmium-induced ectopic apoptosis in zebrafish embryos. Journal Arch Toxicol 2003;77:69-79.

38. Izquierdo-Vega JA, Sánchez-Gutiérrez M, Del Razo LM. Decreased in vitro fertility in male rats exposed