Antioxidative, anticancer and genotoxic properties of α-pinene on N2a neuroblastoma cells

Abstract

α-Pinene, an organic monoterpene, is found in essential oils of pine and coniferous trees. To date, although various biological activities of α-pinene have been demonstrated, its neurotoxicity has never been explored. Therefore in this study, we aimed to describe in vitro antiproliferative and/or cytotoxic properties by 3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test, genotoxic damage potentials by single cell gel electrophoresis, and oxidative effects by total antioxidant capacity (TAC) and total oxidative stress (TOS) analysis of α-pinene. Statistical analysis of MTT assay results indicated significant (p < 0.05) decreases of the cell proliferation rates in healthy neurons treated with α-pinene at only 400 mg/L, while significant decreases were observed in N2a cells at 100, 200 and 400 mg/L. On the other hand, the mean values of the total scores of cells showing DNA damage were not found significantly different from the control values on both cells. In addition, our results indicated that 10 and 25 mg/L of α-pinene treatment caused increases of TAC levels in primary rat neurons without any alterations of its level in N2a cells. However, α-pinene treatments at higher doses led to increases of TOS levels in both cell types. Overall our results suggest that α-pinene is of a limited therapeutic use as an anticancer agent.

This is a preview of subscription content, log in to check access.

Abbreviations

MTT:

3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

NB:

neuroblastoma

SCGE:

single cell gel electrophoresis

TAC:

total antioxidant capacity

TOS:

total oxidative stress

References

  1. Agullo G., Gamet-Payrastre L., Manenti S., Viala C., Remesy C., Chap H. & Payrastre B. 1997. Relationship between flavonoid structure and inhibition of phosphatidylinositol 3-kinase: a comparison with tyrosine kinase and protein kinase C inhibition. Biochem. Pharmacol. 53: 1649–1657.

    PubMed  Article  CAS  Google Scholar 

  2. Bae G.S., Park K.C., Choi S.B., Jo I.J., Choi M.O., Hong S.H., Song K., Song H.J. & Park S.J. 2012. Protective effects of α-pinene in mice with cerulein-induced acute pancreatitis. Life Sci. 91: 866–871.

    PubMed  Article  CAS  Google Scholar 

  3. Bayrak O., Seckiner I., Erturhan S., Aydin A. & Yagci F. 2012. Adult intrarenal neuroblastoma presenting as renal cell carcinoma. Can. Urol. Assoc. J. 6: E144–E146.

    PubMed  Article  Google Scholar 

  4. Bourgou S., Pichette A., Lavoie S., Marzouk B. & Legault J. 2012. Terpenoids isolated from Tunisian Nigella sativa L. essential oil with antioxidant activity and the ability to inhibit nitric oxide production. Flavour Fragr. J. 27: 69–74.

    Article  CAS  Google Scholar 

  5. Brodeur G.M. 2003. Neuroblastoma: biological insights into a clinical enigma. Nat. Rev. Cancer 3: 203–216.

    PubMed  Article  CAS  Google Scholar 

  6. Brusselmans K., Vrolix R., Verhoeven G. & Swinnen J.V. 2005. Induction of cancer cell apoptosis by flavonoids is associated with their ability to inhibit fatty acid synthase activity. J. Biol. Chem. 280: 5636–5645.

    PubMed  Article  CAS  Google Scholar 

  7. Cadet J.L. & Brannock C. 1998. Free radicals and the pathobiology of brain dopamine systems. Neurochem. Int. 32: 117–131.

    PubMed  Article  CAS  Google Scholar 

  8. Cai Y., Luo Q., Sun M. & Corke H. 2004. Antioxidant activity and phenolic compounds of 112 traditional chinese medicinal plants associated with anticancer. Life Sci. 74: 2157–2184.

    PubMed  Article  CAS  Google Scholar 

  9. Charles D.J. & Simon J.E. 1990. Comparison of extraction methods for the rapid determination of essential oil content and composition of basil. J. Amer. Soc. Hort. Sci. 115: 458–462.

    CAS  Google Scholar 

  10. Chen D., Daniel K.G., Chen M.S., Kuhn D.J., Landis-Piwowar K.R. & Dou Q.P. 2005. Dietary flavonoids as proteasome inhibitors and apoptosis inducers in human leukemia cells. Biochem. Pharmacol. 69: 1421–1432.

    PubMed  Article  CAS  Google Scholar 

  11. Chen Z.P., Schell J.B., Ho C.T. & Chen K.Y. 1998. Green tea epigallocatechin gallate shows a pronounced growth inhibitory effect on cancerous cells but not on their normal counterparts. Cancer Lett. 129: 173–179.

    PubMed  Article  CAS  Google Scholar 

  12. Clarke M.F. 2004. At the root of brain cancer. Nature 432: 281–282.

    PubMed  Article  CAS  Google Scholar 

  13. Constantinou A., Mehta R., Runyan C., Rao K., Vaughan A. & Moon R. 1995. Flavonoids as DNA topoisomerase antagonists and poisons: structure-activity relationships. J. Nat. Prod. 58: 217–225.

    PubMed  Article  CAS  Google Scholar 

  14. Daikhin Y. & Yudkoff M. 2000. Compartmentation of brain glutamate metabolism in neurons and glia. J. Nutr. 130: 1026–1031.

    Google Scholar 

  15. Das G.P., Shaik A.P. & Jamil K. 2006. Cytotoxicity and genotoxicity induced by the pesticide profenofos on cultured human peripheral blood lymphocytes. Drug Chem. Toxicol. 29: 313–322.

    Article  CAS  Google Scholar 

  16. Dorman H.J.D., Figueiredo A.C., Barroso J.G. & Deans S.G. 2000. In vitro evaluation of antioxidant activity of essential oils and their components. Flavour. Fragr. J. 15: 12–16.

    Article  CAS  Google Scholar 

  17. Erel O. 2004. A novel automated method to measure total antioxidant response against potent free radical reactions. Clin. Biochem. 37: 112–119.

    PubMed  Article  CAS  Google Scholar 

  18. Erel O. 2005. A new automated colorimetric method for measuring total oxidant status. Clin. Biochem. 38: 1103–1111.

    PubMed  Article  CAS  Google Scholar 

  19. Gminski R., Tang T. & Mersch-Sundermann V. 2010. Cytotoxicity and genotoxicity in human lung epithelial A549 cells caused by airborne volatile organic compounds emitted from pine wood and oriented strand boards. Toxicol. Lett. 196: 33–41.

    PubMed  Article  CAS  Google Scholar 

  20. Gomes-Carneiro M.R., Viana M.E., Felzenszwalb I. & Paumgartten F.J. 2005. Evaluation of β-myrcene, α-terpinene and (+)- and (−)-α-pinene in the Salmonella/microsome assay. Food Chem. Toxicol. 43: 247–252.

    PubMed  Article  CAS  Google Scholar 

  21. Halliwell B. 2006. Oxidative stress and neurodegeneration: where are we now? J. Neurochem. 97: 1634–1658.

    PubMed  Article  CAS  Google Scholar 

  22. Heaton P.R., Ransley R. & Charlton C.J. 2002. Application of single-cell gel electrophoresis (comet) assay for assessing levels of DNA damage in canine and feline leukocytes. J. Nutr. 132: 1598S–1603S.

    PubMed  CAS  Google Scholar 

  23. Heck J.E., Ritz B., Hung R.J., Hashibe M. & Boffetta P. 2009. The epidemiology of neuroblastoma: a review. Paediatr. Perinat. Epidemiol. 23: 125–143.

    PubMed  Article  Google Scholar 

  24. Him A., Ozbek H., Turel I. & Oner A.C. 2008. Antinociceptive activity of α-pinene and fenchone. Pharmacol. Online 3: 363–369.

    Google Scholar 

  25. Ho C.L. & Su Y.C. 2012. Composition, antioxidant and antimicrobial activities of the leaf essential oil of Machilus japonica from Taiwan. Nat. Prod. Commun. 7: 109–112.

    PubMed  CAS  Google Scholar 

  26. Kazi A., Wang Z., Kumar N., Falsetti S.C., Chan T.H. & Dou Q.P. 2004. Structure activity relationships of synthetic analogs of (−)-epigallocatechin-3-gallate as proteasome inhibitors. Anticancer Res. 24: 943–954.

    PubMed  CAS  Google Scholar 

  27. Kizilian N., Wilkins R.C. & Reinhardt P. 1999. Silver stained comet assay for detection of apoptosis. Biotechniques 27: 926–930.

    PubMed  CAS  Google Scholar 

  28. Kusano C. & Ferrari B. 2008. Total antioxidant capacity: a biomarker in biomedical and nutritional studies. J. Cell Mol. Biol. 7: 1–15.

    CAS  Google Scholar 

  29. Lepley D.M., Li B., Birt D.F. & Pelling J.C. 1996. The chemopreventive flavonoid apigenin induces G2/M arrest in keratinocytes. Carcinogenesis 17: 2367–2375.

    PubMed  Article  CAS  Google Scholar 

  30. Lima C.F., Carvalho F., Fernandes E., Bastos M.L., Santos-Gomes P.C., Fernandes-Ferreira M. & Pereira-Wilson C. 2004. Evaluation of toxic/protective effects of the essential oil of Salvia officinalis on freshly isolated rat hepatocytes. Toxicol. In Vitro 18: 457–465.

    PubMed  Article  CAS  Google Scholar 

  31. Linares D., Fontanille P. & Larroche C. 2009. Exploration of α-pinene degradation pathway of Pseudomonas rhodesiae CIP 107491. Application to novalic acid production in a bioreactor. Food Res. Int. 42: 461–469.

    Article  CAS  Google Scholar 

  32. Loza-Tavera H. 1999. Monoterpenes in essential oils. Biosynthesis and properties. Adv. Exp. Med. Biol. 464: 49–62.

    PubMed  Article  CAS  Google Scholar 

  33. Maidment S.L. & Pilkington GJ. 2001. Brain cancers. Encyclopaedia of Life Sciences Nature Publishing Group/Macmillan (Invited Reference Work for Electronic Publication).

    Google Scholar 

  34. Maris J.M. & Matthay K.K. 1999. Molecular biology of neuroblastoma. J. Clin. Oncol. 17: 2264–2279.

    PubMed  CAS  Google Scholar 

  35. Matsuo A.L., Figueiredo C.R., Arruda D.C., Pereira F.V., Scutti J.A.B., Massaoka M.H., Travassos L.R., Sartorelli P. & Lago J.H.G. 2011. α-Pinene isolated from Schinus terebinthifolius Raddi (Anacardiaceae) induces apoptosis and confers antimetastatic protection in a melanoma model. Biochem. Biophys. Res. Commun. 411: 449–454.

    PubMed  Article  CAS  Google Scholar 

  36. Mecocci P., Mac Garvey U. & Beal M.F. 1994. Oxidative damage to mitochondrial DNA is increased in Alzheimer’s disease. Ann. Neurol. 36: 747–751.

    PubMed  Article  CAS  Google Scholar 

  37. Montes M., Veiga M.C. & Kennes C. 2010. Two-liquid-phase mesophilic and thermophilic biotrickling filters for the biodegradation of α-pinene. Bioresour. Technol. 101: 9493–9499.

    PubMed  Article  CAS  Google Scholar 

  38. Mueller W.P., Coppenrath E. & Pfluger T. 2013. Nuclear medicine and multimodality imaging of pediatric neuroblastoma. Pediatr. Radiol. 43: 418–427.

    PubMed  Article  Google Scholar 

  39. Nijholt W.W. & McMullen L.H. 1980. Pine oil prevents mountain pine beetle attack on living lodgepole pine trees. Bimonthly Research Notes 36: 1–2.

    Google Scholar 

  40. Okumura N., Yoshida H., Nishimura Y., Kitagishi Y. & Matsuda S. 2012. Terpinolene, a component of herbal sage, downregulates AKT1 expressionin K562 cells. Oncol. Lett. 3: 321–324.

    PubMed  CAS  Google Scholar 

  41. Ozkan A., Erdogan A., Sokmen M., Tugrulay S. & Unal O. 2010. Antitumoral and antioxidant effect of essential oils and in vitro antioxidant properties of essential oils and aqueous extracts from Salvia pisidica. Biologia 65: 990–996.

    Article  CAS  Google Scholar 

  42. Özen T. & Kinalioğlu K. 2008. Determination of antioxidant activity of various extracts of Parmelia saxatilis. Biologia 63: 211–216.

    Article  Google Scholar 

  43. Plaumann B., Fritsche M., Rimpler H., Brandner G. & Hess RD. 1996. Flavonoids activate wild-type P53. Oncogene 13: 1605–1614.

    PubMed  CAS  Google Scholar 

  44. Rene E.R., Lopez M.E., Veiga M.C. & Kennes C. 2010. Steadyand transient-state operation of a two-stage bioreactor for the treatment of a gaseous mixture of hydrogen sulphide, methanol and α-pinene. J. Chem. Technol. Biotechnol. 85: 336–348.

    Article  CAS  Google Scholar 

  45. Rupar-Gadd K., Bagherpour M.B., Holmstedt G., Welander U. & Sanati M. 2006. Solid phase micro extraction fibers, calibration for use in biofilter applications. Biochem. Eng. J. 31: 107–112.

    Article  CAS  Google Scholar 

  46. Saleha Banu B., Dana Devi K. & Mahboob M. 2000. In vivo genotoxic effect of zinc sulfate in mouse peripheral blood leukocytes using comet assay. Drug Chem. Toxicol. 24: 63–73.

    Article  Google Scholar 

  47. Saverini M., Catanzaro I., Sciandrello G., Avellone G., Indelicato S., Marci G. & Palmisano L. 2012. Genotoxicity of Citrus wastewater in prokaryotic and eukaryotic cells and efficiency of heterogeneous photocatalysis by TiO2. J. Photochem. Photobiol. B 108: 8–15.

    PubMed  Article  CAS  Google Scholar 

  48. Shon M.Y., Choi S.D., Kahng G.G., Nam S.H. & Sung N.J. 2004. Antimutagenic, antioxidant and free radical scavenging activity of ethyl acetate extracts from white, yellow and red onions. Food Chem. Toxicol. 42: 659–666.

    PubMed  Article  CAS  Google Scholar 

  49. Simonsen J.L. 1957. The terpenes (2nd Edition), Cambridge University Press, Cambridge, 105-191 pp.

    Google Scholar 

  50. Singh D.K. & Lippman S.M. 1998. Cancer chemoprevention part 1: retinoids and carotenoids and other classic antioxidants. Oncology 12: 1643–1660.

    PubMed  CAS  Google Scholar 

  51. Singh H.P., Mittal S., Kaur S., Batish D.R. & Kohli R.K. 2009. Characterization and antioxidant activity of essential oils from fresh and decaying leaves of Eucalyptus tereticornis. J. Agric. Food Chem. 57: 6962–6966.

    PubMed  Article  CAS  Google Scholar 

  52. Singh N.P., McCoy M.T. & Tice R.R. 1998. A simple technique for quantitation of low level of DNA damage in individual cells. Exp. Cell Res. 17: 184–191.

    Google Scholar 

  53. Tice R.R., Agurell E. & Anderson D. 2000. Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ. Mol. Mutagen. 35: 206–221.

    PubMed  Article  CAS  Google Scholar 

  54. Turkez H. 2011. The role of ascorbic acid on titanium dioxideinduced genetic damage assessed by the comet assay and cytogenetic tests. Exp. Toxicol. Pathol. 63: 453–457.

    PubMed  Article  CAS  Google Scholar 

  55. Turkez H. & Aydin E. 2012. Anti-genotoxic role of eicosapentaenoic acid against imazalil-induced DNA damage in vitro. Toxicol. Ind. Health (in press); DOI:10.1177/0748233711433943.

    Google Scholar 

  56. Turkez H. & Geyikoglu F. 2010. Boric acid: a potential chemoprotective agent against aflatoxin B(1) toxicity in human blood. Cytotechnology 62: 157–165.

    PubMed  Article  CAS  Google Scholar 

  57. Turkez H., Geyikoglu F., Dirican E. & Tatar A. 2012. In vitro studies on chemoprotective effect of borax against aflatoxin B1-induced genetic damage in human lymphocytes. Cytotechnology 64: 607–612.

    PubMed  Article  CAS  Google Scholar 

  58. Turner S.D., Tinwell H., Piegorsch W., Schmezer P. & Ashby J. 2001. The male rat carcinogens limonene and sodium saccharin are not mutagenic to male big blue rats. Mutagenesis 16: 329–332.

    PubMed  Article  CAS  Google Scholar 

  59. Wang W., Li N., Luo M., Zu Y. & Efferth T. 2012. Antibacterial activity and anticancer activity of Rosmarinus officinalis L. essential oil compared to that of its main components. Molecules 17: 2704–2713.

    CAS  Google Scholar 

  60. Wang W., Wu N., Zu Y.G. & Fu Y.J. 2008. Antioxidative activity of Rosmarinus officinalis L. essential oil compared to its main components. Food Chem. 108: 1019–1022.

    CAS  Google Scholar 

  61. Wei A. & Shibamoto T. 2007. Antioxidant activities and volatile constituents of various essential oils. J. Agric. Food Chem. 55: 1737–1742.

    PubMed  Article  CAS  Google Scholar 

  62. Wu C.S., Chen Y.J., Chen J.J., Shieh J.J., Huang C.H., Lin P.S., Chang G.C., Chang J.T. & Lin C.C. 2012. Terpinen-4-ol induces apoptosis in human nonsmall cell lung cancer in vitro and in vivo. Evid. Based Complement. Alternat. Med., Article ID: 818261.

    Google Scholar 

Download references

Author information

Affiliations

Authors

Corresponding author

Correspondence to Elanur Aydin.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Aydin, E., Türkez, H. & Geyikoğlu, F. Antioxidative, anticancer and genotoxic properties of α-pinene on N2a neuroblastoma cells. Biologia 68, 1004–1009 (2013). https://doi.org/10.2478/s11756-013-0230-2

Download citation

Key words

  • alpha-pinene
  • comet assay
  • MTT assay
  • neurotoxicity
  • N2a neuroblastoma cell line
  • oxidative status