Genotoxicity Assessment of Birch-Bark Tar—A Most Versatile Prehistoric Adhesive
Author(s)
A. Baumgartner,
M. Sampol-Lopez,
E. Cemeli,
T. E. Schmid,
A. A. Evans,
R. E. Donahue,
D. Anderson*
Affiliation(s)
Division of Biomedical Sciences, University of Bradford, Bradford, UK. Department of Radiotherapy and Radiooncology, Klinikum Rechts der Isar, Technische Universit?t München, Munich, Germany. Division of Archaeological, Geographical and Environmental Sciences, University of Bradford, Bradford, UK.
Division of Biomedical Sciences, University of Bradford, Bradford, UK. Department of Radiotherapy and Radiooncology, Klinikum Rechts der Isar, Technische Universit?t München, Munich, Germany. Division of Archaeological, Geographical and Environmental Sciences, University of Bradford, Bradford, UK.
ABSTRACT
In the Mesolithic, birch-bark tar was commonly utilized across Europe and much of Asia as an adhesive to bind, seal and coat surfaces, but also quite frequently it was found to be chewed. The tar is known to contain biomarker triterpenoid compounds like betulin, crucial in preserving food and for medical applications. Aqueous, ethanolic and DMSO extracts were prepared from solid birch-bark tar and evaluated in vitro for the induction of DNA damage using Comet, micronucleus and sister-chromatid-exchange assays. Additionally, apoptosis induction was assessed. For the ethanolic extract, only the Comet assay showed a significant increase of DNA damage. All three extracts were able to significantly induce apoptosis. Thus, birch-bark tar seems capable of inducing genotoxic damage as well as apoptotic effects possibly originating from the triterpenoids’ antimicrobial properties. We examine why prehistoric tar is found with tooth marks, the beneficial effects of birch-bark tar, and evidence for increased genotoxic risk upon exposure.
In the Mesolithic, birch-bark tar was commonly utilized across Europe and much of Asia as an adhesive to bind, seal and coat surfaces, but also quite frequently it was found to be chewed. The tar is known to contain biomarker triterpenoid compounds like betulin, crucial in preserving food and for medical applications. Aqueous, ethanolic and DMSO extracts were prepared from solid birch-bark tar and evaluated in vitro for the induction of DNA damage using Comet, micronucleus and sister-chromatid-exchange assays. Additionally, apoptosis induction was assessed. For the ethanolic extract, only the Comet assay showed a significant increase of DNA damage. All three extracts were able to significantly induce apoptosis. Thus, birch-bark tar seems capable of inducing genotoxic damage as well as apoptotic effects possibly originating from the triterpenoids’ antimicrobial properties. We examine why prehistoric tar is found with tooth marks, the beneficial effects of birch-bark tar, and evidence for increased genotoxic risk upon exposure.
Cite this paper
Baumgartner, A. , Sampol-Lopez, M. , Cemeli, E. , Schmid, T. , Evans, A. , Donahue, R. & Anderson, D. (2012). Genotoxicity Assessment of Birch-Bark Tar—A Most Versatile Prehistoric Adhesive. Advances in Anthropology, 2, 49-56. doi: 10.4236/aa.2012.22006.
Baumgartner, A. , Sampol-Lopez, M. , Cemeli, E. , Schmid, T. , Evans, A. , Donahue, R. & Anderson, D. (2012). Genotoxicity Assessment of Birch-Bark Tar—A Most Versatile Prehistoric Adhesive. Advances in Anthropology, 2, 49-56. doi: 10.4236/aa.2012.22006.
References
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Nature, 251, 156-158. doi:10.1038/251156a0 [26] Pool-Zobel, B. L., Klein, R. G., Liegibel, U. M., Kuchenmeister, F., Weber, S., & Schmezer, P. (1992). Systemic genotoxic effects of tobacco-related nitrosamines following oral and inhalational administration to Sprague-Dawley rats. Clinical Investigator, 70, 299-306. doi:10.1007/BF00184666 [27] Regert, M. (2004). Investigating the history of prehistoric glues by gas chromatography—Mass spectrometry. Journal of Separation Science, 27, 244-254. doi:10.1002/jssc.200301608 [28] Regert, M., & Rolando, C. (1996). Archéologie des résidus organiques. De la chimie analytique à l'archéologie: Un état de la question. Techne, 3, 118-128. [29] Saleem, M., Murtaza, I., Witkowsky, O., Kohl, A. M., & Maddodi, N. (2009). Lupeol triterpene, a novel diet-based microtubule targeting agent: Disrupts survivin/cFLIP activation in prostate cancer cells. Biochemical and Biophysical Research Communications, 388, 576- 582. doi:10.1016/j.bbrc.2009.08.060 [30] Sauter, F., Jordis, U., Graf, A., Werther, W., & Varmuza , K. (2000). Studies in organic archaeometry I: Identification of the prehistoric adhesive used by the “Tyrolean Iceman” to fix his weapons. Archive for Organic Checmistry, 1, 735-747. [31] Schlegel, R. A., & Williamson, P. (2001). Phosphatidylserine, a death knell. Cell Death & Differentiation, 8, 551-563. doi:10.1038/sj.cdd.4400817 [32] Sieroszewski, W., Stepanov, S. A., Zhornitskaia, M. I. A., & Mukhamediarov, S. F. (1993). Iakuty: Opyt etnograficheskogo issledovaniia. Moskva: Assotsiatsiia “rossiiskaia polit entsiklopediia”. [33] Stern, B., Clelland, S. J., Nordby, C. C., & Urem-Kotsu, D. (2006). Bulk stable light isotopic rations in archaeological birch bark tars. Applied Geochemistry, 21, 1668-1673. doi:10.1016/j.apgeochem.2006.07.004 [34] Tice, R. R., Agurell, E., Anderson, D., Burlinson, B., Hartmann, A., Kobayashi, H., Miyamae, Y., Rojas, E., Ryu, J. C., & Sasaki, Y. F. (2000). Single cell gel/comet assay: Guidelines for in vitro and in vivo genetic toxicology testing. Environmental and Molecular Muta- genesis, 35, 206-221. doi:10.1002/(SICI)1098-2280(2000)35:3<206::AID-EM8>3.0.CO;2-J [35] Urem-Kotsou, D., Stern, B., Heron, C., & Kotsakis, K. (2002). Birch- bark tar at Neolithic Makriyalos, Greece. Antiquity, 76, 962-967. [36] Vermes, I., Haanen, C., Steffens-Nakken, H., & Reutelingsperger, C. (1995). A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. Journal of Immunological Methods, 184, 39-51. doi:10.1016/0022-1759(95)00072-I [37] Yogeeswari, P., & Sriram, D. (2005). Betulinic acid and its derivatives: A review on their biological properties. Current Medicinal Chemistry, 12, 657-666. doi:10.2174/0929867053202214
[1] Abdel-Rahman, W. M. (2008). Genomic instability and carcinogenesis: An update. Current Genomics, 9, 535-541. doi:10.2174/138920208786847926 [2] Alakurtti, S., Makela, T., Koskimies, S., & Yli-Kauhaluoma, J. (2006). Pharmacological properties of the ubiquitous natural product betulin. European Journal of Pharmaceutical Sciences, 29, 1-13. doi:10.1016/j.ejps.2006.04.006 [3] Anderson, D., Yu, T. W., Dobrzynska, M. M., Ribas, G., & Marcos, R. (1997). Effects in the comet assay of storage conditions on human blood. Teratogenesis, Carcinogenesis, and Mutagenesis, 17, 115-125. doi:10.1002/(SICI)1520-6866(1997)17:3<115::AID-TCM3>3.0.CO;2-K [4] Anderson, D., Yu, T. W., & McGregor, D. B. (1998). Comet assay responses as indicators of carcinogen exposure. Mutagenesis, 13, 539-555. doi:10.1093/mutage/13.6.539 [5] Aveling, E. (1997). Chew, chew that ancient chewing gum. British Archaeology, 21, 1-2. [6] Aveling, E. M., & Heron, C. (1999). Chewing tar in the early Holocene: an archaeological and ethnographic evaluation. Antiquity, 73, 579- 584. [7] Charters, S., Evershed, R. P., Goad, L. J., Heron, C., & Blinkhorn, P. (1993). Identification of an adhesive used to repair a Roman jar. Archaeometry, 35, 91-101. doi:10.1111/j.1475-4754.1993.tb01025.x [8] Dominguez-Carmona, D. B., Escalante-Erosa, F., Garcia-Sosa, K., Ruiz-Pinell, G., Gutierrez-Yapu, D., Chan-Bacab, M. J., Gimenez- Turba, A., & Pena-Rodriguez, L. M. (2010). Antiprotozoal activity of betulinic acid derivatives. Phytomedicine, 17, 379-382. doi:10.1016/j.phymed.2009.08.002 [9] Drag, M., Surowiak, P., Drag-Zalesinska, M., Dietel, M., Lage, H., & Oleksyszyn, J. (2009). Comparision of the cytotoxic effects of birch bark extract, betulin and betulinic acid towards human gastric carcinoma and pancreatic carcinoma drug-sensitive and drug-resistant cell lines. Molecules, 14, 1639-1651. doi:10.3390/molecules14041639 [10] Dudd, S. N., & Evershed, R. P. (1999). Unusual triterpenoid fatty acyl ester components of archaeological birch bark tars. Tetrahedron Letters, 40, 359-362. doi:10.1016/S0040-4039(98)02311-9 [11] Eiznhamer, D. A., & Xu, Z. Q. (2004). Betulinic acid: A promising anticancer candidate. IDrugs, 7, 359-373. [12] Fenech, M. (2007). Cytokinesis-block micronucleus cytome assay. Nature Protocols, 2, 1084-1104. doi:10.1038/nprot.2007.77 [13] Fenech, M., Chang, W. P., Kirsch-Volders, M., Holland, N., Bonassi, S., Zeiger, E., & Uman, H. (2003). HUMN project: Detailed description of the scoring criteria for the cytokinesis-block micronucleus assay using isolated human lymphocyte cultures. Mutation Research, 534, 65-75. [14] Grünberg, J. M. (2002). Middle Palaeolithic birch-bark pitch. Antiquity, 76, 15-16. [15] Hartmann, A., & Speit, G. (1997). The contribution of cytotoxicity to DNA-effects in the single cell gel test (comet assay). Toxicology Letters, 90, 183-188. doi:10.1016/S0378-4274(96)03847-7 [16] Hayek, E. W. H., Jordis, U., Moche, W., & Sauter, F. (1989). A bicentennial of betulin. Phytochemistry, 28, 2229-2242. doi:10.1016/S0031-9422(00)97961-5 [17] Henderson, L., Wolfreys, A., Fedyk, J., Bourner, C., & Windebank, S. (1998). The ability of the Comet assay to discriminate between genotoxins and cytotoxins. Mutagenesis, 13, 89-94. doi:10.1093/mutage/13.1.89 [18] Itkonen, T. I. (1984). Suomen lappalaiset vuoteen 1945. Porvoo: S?derstr?m. [19] Koller, J., Baumer, U., & Mania, D. (2001). High-tech in the middle Palaeolithic Neandertal-manufactured pitch identified. European Journal of Archaeology, 4, 385-397. doi:10.1177/146195710100400315 [20] Krasutsky, P. A. (2006). Birch bark research and development. Natural Product Reports, 23, 919-942. doi:10.1039/b606816b [21] Krause, A. W., Carley, W. W., & Webb, W. W. (1984). Fluorescent erythrosin B is preferable to trypan blue as a vital exclusion dye for mammalian cells in monolayer culture. Journal of Histochemistry & Cytochemistry, 32, 1084-1090. doi:10.1177/32.10.6090533 [22] Kumaravel, T. S., & Jha, A. N. (2006). Reliable Comet assay measurements for detecting DNA damage induced by ionising radiation and chemicals. Mutation Research, 605, 7-16. [23] Modugno, F., Ribechini, E., & Colombini, M. P. (2006). Chemical study of triterpenoid resinous materials in archaeological findings by means of direct exposure electron ionisation mass spectrometry and gas chromatography/mass spectrometry. Rapid Communications in Mass Spectrometry, 20, 1787-1800. doi:10.1002/rcm.2507 [24] Muceniece, R., Saleniece, K., Rumaks, J., Krigere, L., Dzirkale, Z., Mezhapuke, R., Zharkova, O., & Klusa, V. (2008). Betulin binds to gamma-aminobutyric acid receptors and exerts anticonvulsant action in mice. Pharmacology Biochemistry & Behavior, 90, 712-716. doi:10.1016/j.pbb.2008.05.015 [25] Perry, P., & Wolff, S. (1974). New Giemsa method for the differential staining of sister chromatids. Nature, 251, 156-158. doi:10.1038/251156a0 [26] Pool-Zobel, B. L., Klein, R. G., Liegibel, U. M., Kuchenmeister, F., Weber, S., & Schmezer, P. (1992). Systemic genotoxic effects of tobacco-related nitrosamines following oral and inhalational administration to Sprague-Dawley rats. Clinical Investigator, 70, 299-306. doi:10.1007/BF00184666 [27] Regert, M. (2004). Investigating the history of prehistoric glues by gas chromatography—Mass spectrometry. Journal of Separation Science, 27, 244-254. doi:10.1002/jssc.200301608 [28] Regert, M., & Rolando, C. (1996). Archéologie des résidus organiques. De la chimie analytique à l'archéologie: Un état de la question. Techne, 3, 118-128. [29] Saleem, M., Murtaza, I., Witkowsky, O., Kohl, A. M., & Maddodi, N. (2009). Lupeol triterpene, a novel diet-based microtubule targeting agent: Disrupts survivin/cFLIP activation in prostate cancer cells. Biochemical and Biophysical Research Communications, 388, 576- 582. doi:10.1016/j.bbrc.2009.08.060 [30] Sauter, F., Jordis, U., Graf, A., Werther, W., & Varmuza , K. (2000). Studies in organic archaeometry I: Identification of the prehistoric adhesive used by the “Tyrolean Iceman” to fix his weapons. Archive for Organic Checmistry, 1, 735-747. [31] Schlegel, R. A., & Williamson, P. (2001). Phosphatidylserine, a death knell. Cell Death & Differentiation, 8, 551-563. doi:10.1038/sj.cdd.4400817 [32] Sieroszewski, W., Stepanov, S. A., Zhornitskaia, M. I. A., & Mukhamediarov, S. F. (1993). Iakuty: Opyt etnograficheskogo issledovaniia. Moskva: Assotsiatsiia “rossiiskaia polit entsiklopediia”. [33] Stern, B., Clelland, S. J., Nordby, C. C., & Urem-Kotsu, D. (2006). Bulk stable light isotopic rations in archaeological birch bark tars. Applied Geochemistry, 21, 1668-1673. doi:10.1016/j.apgeochem.2006.07.004 [34] Tice, R. R., Agurell, E., Anderson, D., Burlinson, B., Hartmann, A., Kobayashi, H., Miyamae, Y., Rojas, E., Ryu, J. C., & Sasaki, Y. F. (2000). Single cell gel/comet assay: Guidelines for in vitro and in vivo genetic toxicology testing. Environmental and Molecular Muta- genesis, 35, 206-221. doi:10.1002/(SICI)1098-2280(2000)35:3<206::AID-EM8>3.0.CO;2-J [35] Urem-Kotsou, D., Stern, B., Heron, C., & Kotsakis, K. (2002). Birch- bark tar at Neolithic Makriyalos, Greece. Antiquity, 76, 962-967. [36] Vermes, I., Haanen, C., Steffens-Nakken, H., & Reutelingsperger, C. (1995). A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. Journal of Immunological Methods, 184, 39-51. doi:10.1016/0022-1759(95)00072-I [37] Yogeeswari, P., & Sriram, D. (2005). Betulinic acid and its derivatives: A review on their biological properties. Current Medicinal Chemistry, 12, 657-666. doi:10.2174/0929867053202214