Development of a Rapid Cell-free Method for Cytotoxicity Assessment of Vapor Phase of Cigarette Smoke

Cahours, X; Blanchet, M; Dubois, M; Haond, C

doi:10.2478/cttr-2013-0893

Abstract

Currently, several in vitro tests are widely used to measure toxicological properties of mainstream smoke (Neutral Red Uptake Assay, Micronucleus assay, Ames Test). These tests are necessary to assess cytotoxicity, genotoxicity, and mutagenicity, but are time consuming. This is essentially due to the preparation and the handling of cells. It is difficult to use these in vitro tests as screening method for product testing and development. For a better assessment of the cytotoxicity of the vapor phase, a rapid cell-free method has been developed. This paper describes a capillary electrophoresis cell-free method, based on the depletion of an anti-oxidant L-gamma-glutamyl-L-cysteinylglycine (GSH), applied to an aliquot of vapor phase phosphate buffered saline (PBS)-trapped cigarette smoke (as recommended for in vitro testing). The correlation between this method and the survival/viability test (Neutral Red cytotoxicity) is excellent (coefficient of correlation (r = 0.99)).

References

1. Health Canada: Bacterial Reverse Mutation Assay for Mainstream Tobacco Smoke; Official method T-501; (2004) Version 1. 1, pp. 1-19.
Search in Google Scholar
2. Health Canada: Neutral Red Uptake Assay for Mainstream Tobacco Smoke; Official method T-502; (2004) Version 1. 1, pp. 1-18.
Search in Google Scholar
3. Health Canada: In vitro Micronucleus Assay for Mainstream Tobacco Smoke; Official method T-503; (2004) Version 1. 1, pp. 1-17.
Search in Google Scholar
4. Leuchtenberger, C, R. Leuchtenberger, and I. Zbinden: Gas vapor phase constituents and SH reactivity of cigarette smoke influence lung cultures; Nature 247 (1973) 565-567.
Search in Google Scholar
5. Scherer, G, H. W. Hagedorn, G Gilch, and D. Janket: Mechanistically-based tests for toxicity of tobacco smoke; CORESTA Meeting, Smoke Science/Product Technology Groups, Freiburg, Germany, 2003.
Search in Google Scholar
6. Guengerich, F. P. and D. C. Liebler: Enzymatic activation of chemicals to toxic metabolites; Crit. Rev. Toxicol. 14 (1985) 259-307.
Search in Google Scholar
7. Meister, A. and M. E. Anderson: Glutathion; Annu. Rev. Biochem. 52 (1983) 711-760.10.1146/annurev.bi.52.070183.0034316137189
Search in Google Scholar
8. Church, D. F. and WA. Pryor: Free-radical chemistry of cigarette smoke and its toxicological implications; Environ Health Perspect. 64 (1985)111-126.
Search in Google Scholar
9. Pryor, W. A.: Biological effects of cigarette smoke, wood smoke, and the smoke from plastics: the use of electron spin resonance; Free Radic. Biol. Med. 13 (1992) 659–76.
Search in Google Scholar
10. Counts, M. E., F. S. Hsu, S. W. Laffoon, R. W. Dwyer, and R. H. Cox: Mainstream smoke constituent yields and predicting relationships from a worldwide market sample of cigarette brands: ISO smoking conditions; Annu. Rev. Biochem. 39 (2004) 111–34.
Search in Google Scholar
11. Meacher, D. M. and D. B. Menzel: Glutathione depletion lung cells by low-molecular-weight aldehydes; Cell Biol. Toxicol. 15 (1999) 163–171.
Search in Google Scholar
12. Reddy, S., E. I. Finkelstein, P. S. Y. Wong, A. Phung, C. E. Cross, and A. Van der Vliet: Identification of glutathione modifications by cigarette smoke; Free Radic. Biol. Med. 33 (2002) 1490–1498.
Search in Google Scholar
13. International Organization for Standardization: ISO 3308: Routine analytical cigarette-smoking machine -Definitions and standard conditions, 2000.
Search in Google Scholar
14. Bombick, D. W. P. H. Ayres, and D. J. T. Doolittle: Cytotoxicity assessment of whole smoke and vapor phase of mainstream and sidestream cigarette smoke from three Kentucky reference cigarettes; Toxicol. Mech. Methods 7 (1997) 177–190.
Search in Google Scholar
15. Bombick D. W., B. R. Bombick, P. H. Ayres, K. Putnam, J. Avalos, M. F. Borgerding, and D. J. T. Doolittle: Evaluation of the genotoxic and cytotoxic potential of mainstream whole smoke and smoke condensate from a cigarette containing a novel carbon filter; Fundam. Appl. Toxicol. 39 (1997) 11–7.
Search in Google Scholar
16. Putnam, K. P., D. W. Bombick, and D. J. T. Doolittle: Evaluation of eight in-vitro assays for assessing the cytotoxicity of cigarette smoke condensate; Toxicol. In Vitro 16 (2002) 599–607.
Search in Google Scholar
17. Owens, I. and R. V. Belcher: A colorimetric micro-method for the determination of glutathione; Biochem. J. 94 (1965) 705–710.
Search in Google Scholar
18. Yoshida, T.: Determination of reduced and oxidized glutathione in erythrocytes by high-performance liquid chromatography with ultraviolet absorbance detection; J. Chromatogr. B 678 (1996) 157–164.
Search in Google Scholar
19. Lenton, K. J., H. Therriault, and J. R. Wagner: Analysis of glutathione disulfide in whole cells and mitochondria by postcolumn derivatisation high-performance liquid chromatography with ortho-phtalaldehyde; Anal Biochem. 274 (1999) 125–30.
Search in Google Scholar
20. Havel, K., K. Pritts, and T. Wielgos: Quantification of oxidized and reduced glutathione in plasma by micellar electrokinetic capillary electrophoresis; J. Chromatogr A 853 (1999) 215–23.
Search in Google Scholar
21. Carru, C., A. Zinellu, S. Sotgia, G. Marongiu, M. G. Farina, M. F. Usai, G. M. Pes, B. Tadolini, and L. Deina: Optimization of the principal parameters for the ultrarapid electrophoretic separation of reduced and oxydised glutathione by capillary electrophoresis; J. Chromatogr A. 1017 (2003) 233–238.
Search in Google Scholar
22. Newman, D.: The distribution of range in samples from a normal population, expressed in terms of an independent estimate of standard deviation; Biometrika 31 (1939) 20–30.
Search in Google Scholar

Development of a Rapid Cell-free Method for Cytotoxicity Assessment of Vapor Phase of Cigarette Smoke

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