ACP Atmospheric Chemistry and Physics ACP 1680-7324 Copernicus GmbH Göttingen, Germany 10.5194/acp-12-9321-2012 On dithiothreitol (DTT) as a measure of oxidative potential for ambient particles: evidence for the importance of soluble \newline transition metals Charrier J. G. 1 2 Anastasio C. 1 2 Department of Land, Air and Water Resources, University of California, Davis, 1 Shields Ave. Davis, CA 95616, USA Agricultural and Environmental Chemistry Graduate Group, University of California, Davis, 1 Shields Ave. Davis, CA 95616, USA 15 10 2012 12 19 9321 9333 This is an open-access article ditributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. This article is available from http://www.atmos-chem-phys.net/12/9321/2012/acp-12-9321-2012.html The full text article is available as a PDF file from http://www.atmos-chem-phys.net/12/9321/2012/acp-12-9321-2012.pdf

The rate of consumption of dithiothreitol (DTT) is increasingly used to measure the oxidative potential of particulate matter (PM), which has been linked to the adverse health effects of PM. While several quinones are known to be very reactive in the DTT assay, it is unclear what other chemical species might contribute to the loss of DTT in PM extracts. To address this question, we quantify the rate of DTT loss from individual redox-active species that are common in ambient particulate matter. While most past research has indicated that the DTT assay is not sensitive to metals, our results show that seven out of the ten transition metals tested do oxidize DTT, as do three out of the five quinones tested. While metals are less efficient at oxidizing DTT compared to the most reactive quinones, concentrations of soluble transition metals in fine particulate matter are generally much higher than those of quinones. The net result is that metals appear to dominate the DTT response for typical ambient PM<sub>2.5</sub> samples. Based on particulate concentrations of quinones and soluble metals from the literature, and our measured DTT responses for these species, we estimate that for typical PM<sub>2.5</sub> samples approximately 80% of DTT loss is from transition metals (especially copper and manganese), while quinones account for approximately 20%. We find a similar result for DTT loss measured in a small set of PM<sub>2.5</sub> samples from the San Joaquin Valley of California. Because of the important contribution from metals, we also tested how the DTT assay is affected by EDTA, a chelator that is sometimes used in the assay. EDTA significantly suppresses the response from both metals and quinones; we therefore recommend that EDTA should not be included in the DTT assay.

 References Ayres, J. G., Borm, P., Cassee, F. R., Castranova, V., Donaldson, K., Ghio, A., Harrison, R. M., Hider, R., Kelly, F., Kooter, I. M., Marano, F., Maynard, R. L., Mudway, I., Nel, A., Sioutas, C., Smith, S., Baeza-Squiban, A., Cho, A., Duggan, S., and Froines, J.: Evaluating the toxicity of airborne particulate matter and nanoparticles by measuring oxidative stress potential - a workshop report and consensus statement, Inhal. Tox., 20, 75–99, 2008. Biorad: Chelex 100 and Chelex 20 chelating ion exchange resin instruction manual, http://www.bio-rad.com/webmaster/pdfs/9184_Chelex.PDF, 1991. Biswas, S., Verma, V., Schauer, J. J., Cassee, F. R., Cho, A. K., and Sioutas, C.: Oxidative Potential of Semi-Volatile and Non Volatile Particulate Matter (PM) from Heavy-Duty Vehicles Retrofitted with Emission Control Technologies, Env. Sci. Technol., 43, 3905–3912, 2009. Charrier, J. G. and Anastasio, C.: Impacts of antioxidants on hydroxyl radical production from individual and mixed transition metals in a surrogate lung fluid, Atmos. Environ., 45, 7555–7562, 2011. Cho, A. K., Di Stefano, E., You, Y., Rodriguez, C. E., Schmitz, D. A., Kumagai, Y., Miguel, A. H., Eiguren-Fernandez, A., Kobayashi, T., Avol, E., and Froines, J. R.: Determination of four quinones in diesel exhaust particles, SRM 1649a, an atmospheric PM$_2.5$, Aerosol Sci. Technol., 38, 68–81, 2004. Cho, A. K., Sioutas, C., Miguel, A. H., Kumagai, Y., Schmitz, D. A., Singh, M., Eiguren-Fernandez, A., and Froines, J. R.: Redox activity of airborne particulate matter at different sites in the Los Angeles Basin, Environ. Res., 99, 40–47, 2005. Chung, M. Y., Lazaro, R. A., Lim, D., Jackson, J., Lyon, J., Rendulic, D., and Hasson, A. S.: Aerosol-borne quinones and reactive oxygen species generation by particulate matter extracts, Env. Sci. Technol., 40, 4880–4886, 2006. Connell, D. P., Winter, S. E., Conrad, V. B., Kim, M., and Crist, K. C.: The Steubenville Comprehensive Air Monitoring Program (SCAMP): Concentrations and solubilities of PM$_2.5$ trace elements and their implications for source apportionment and health research, J. Air Waste Manage. Assoc., 56, 1750–1766, 2006. Damodaran, S.: Estimation of disulfide bonds using 2-Nitro-5-thiosulfobenzoic acid – limitations, Anal. Biochem., 145, 200–204, 1985. Delhomme, O., Millet, M., and Herckes, P.: Determination of oxygenated polycyclic aromatic hydrocarbons in atmospheric aerosol samples by liquid chromatography-tandem mass spectrometry, Talanta, 74, 703–710, 2008. DiStefano, E., Eiguren-Fernandez, A., Delfino, R. J., Sioutas, C., Froines, J. R., and Cho, A. K.: Determination of metal-based hydroxyl radical generating capacity of ambient and diesel exhaust particles, Inhal. Tox., 21, 731–738, 2009. Eiguren-Fernandez, A., Shinyashiki, M., Schmitz, D. A., DiStefano, E., Hinds, W., Kumagai, Y., Cho, A. K., and Froines, J. R.: Redox and electrophilic properties of vapor- and particle-phase components of ambient aerosols, Environ. Res., 110, 207–212, 2010. Eyer, P., Worek, F., Kiderlen, D., Sinko, G., Stuglin, A., Simeon-Rudolf, V., and Reiner, E.: Molar absorption coefficients for the reduced Ellman reagent: Reassessment, Anal. Biochem., 312, 224–227, 2003. Geller, M. D., Ntziachristos, L., Mamakos, A., Samaras, Z., Schmitz, D. A., Froines, J. R., and Sioutas, C.: Physicochemical and redox characteristics of particulate matter (PM) emitted from gasoline and diesel passenger cars, Atmos. Environ., 40, 6988–7004, 2006. Ghio, A. J., Carraway, M. S., and Madden, M. C.: Composition of air pollution particles and oxidative stress in cells, tissues, and living systems, J. Toxicol. Environ. Health-Pt b-Crit. Rev., 15, 1–21, 2012. Held, K. D. and Biaglow, J. E.: Role of copper in the oxygen radical-mediated toxicity of the thiol-containing radioprotector dithiothreitol in mammalian-cells, Radiat. Res., 134, 375–382, 1993. Hu, S., Polidori, A., Arhami, M., Shafer, M. M., Schauer, J. J., Cho, A., and Sioutas, C.: Redox activity and chemical speciation of size fractioned PM in the communities of the Los Angeles-Long Beach harbor, Atmos. Chem. Phys., 8, 6439–6451, http://dx.doi.org/10.5194/acp-8-6439-2008doi:10.5194/acp-8-6439-2008, 2008. Kachur, A. V., Held, K. D., Koch, C. J., and Biaglow, J. E.: Mechanism of production of hydroxyl radicals in the copper-catalyzed oxidation of dithiothreitol, Radiat. Res., 147, 409–415, 1997. Krezel, A., Lesniak, W., Jezowska-Bojczuk, M., Mlynarz, P., Brasun, J., Kozlowski, H., and Bal, W.: Coordination of heavy metals by dithiothreitol, a commonly used thiol group protectant, J. Inorg. Biochem., 84, 77–88, 2001. Kumagai, Y., Koide, S., Taguchi, K., Endo, A., Nakai, Y., Yoshikawa, T., and Shimojo, N.: Oxidation of proximal protein sulfhydryls by phenanthraquinone, a component of diesel exhaust particles, Chem. Res. Tox., 15, 483–489, 2002. Leonard, S. S., Harris, G. K., and Shi, X. L.: Metal-induced oxidative stress and signal transduction, Free Radic. Biol. Med., 37, 1921–1942, 2004. Li, N., Sioutas, C., Cho, A., Schmitz, D., Misra, C., Sempf, J., Wang, M. Y., Oberley, T., Froines, J., and Nel, A.: Ultrafine particulate pollutants induce oxidative stress and mitochondrial damage, Environ. Health Perspect., 111, 455–460, 2003. Li, Q. F., Wyatt, A., and Kamens, R. M.: Oxidant generation and toxicity enhancement of aged-diesel exhaust, Atmos. Environ., 43, 1037–1042, 2009a. Li, Z., Porter, E. N., Sjodin, A., Needham, L. L., Lee, S., Russell, A. G., and Mulholland, J. A.: Characterization of PM$_(2.5)$-bound polycyclic aromatic hydrocarbons in Atlanta-Seasonal variations at urban, suburban, and rural ambient air monitoring sites, Atmos. Environ., 43, 4187–4193, 2009b. Lin, P. and Yu, J. Z.: Generation of reactive oxygen species mediated by humic-like substances in atmospheric aerosols, Env. Sci. Technol., 45, 10362–10368, 2011. Majestic, B. J., Schauer, J. J., and Shafer, M. M.: Application of synchrotron radiation for measurement of iron red-ox speciation in atmospherically processed aerosols, Atmos. Chem. Phys., 7, 2475–2487, http://dx.doi.org/10.5194/acp-7-2475-2007doi:10.5194/acp-7-2475-2007, 2007. McWhinney, R. D., Gao, S. S., Zhou, S. M., and Abbatt, J. P. D.: Evaluation of the effects of ozone oxidation on redox-cycling activity of two-stroke engine exhaust particles, Env. Sci. Technol., 45, 2131–2136, 2011. Netto, L. E. S. and Stadtman, E. R.: The iron-catalyzed oxidation of dithiothreitol is a biphasic process: Hydrogen peroxide is involved in the initiation of a free radical chain of reactions, Arc. Biochem. Biophys., 333, 233–242, 1996. Ntziachristos, L., Froines, J. R., Cho, A. K., and Sioutas, C.: Relationship between redox activity and chemical speciation of size-fractionated particulate matter, Part. Fibre Toxicol., 4, 2007. Park, J. H., Gopishetty, S., Szewczuk, L. M., Troxel, A. B., Harvey, R. G., and Penning, T. M.: Formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGuo) by PAH o-quinones: Involvement of reactive oxygen species and copper(II)/copper(I) redox cycling, Chem. Res. Tox., 18, 1026–1037, 2005. Rattanavaraha, W., Rosen, E., Zhang, H. F., Li, Q. F., Pantong, K., and Kamens, R. M.: The reactive oxidant potential of different types of aged atmospheric particles: An outdoor chamber study, Atmos. Environ., 45, 3848–3855, 2011. Sauvain, J. J., Deslarzes, S., and Riediker, M.: Nanoparticle reactivity toward dithiothreitol, Nanotoxicology, 2, 121–129, 2008. Shen, H. and Anastasio, C.: Formation of hydroxyl radical from San Joaquin Valley particles extracted in a cell-free surrogate lung fluid, Atmos. Chem. Phys., 11, 9671–9682, http://dx.doi.org/10.5194/acp-11-9671-2011doi:10.5194/acp-11-9671-2011, 2011. Shen, H. and Anastasio, C.: A comparison of hydroxyl radical and hydrogen peroxide generation in ambient particle extracts and laboratory metal solutions, Atmos. Environ., 46, 665–668, 2012. Shen, H., Barakat, A. I., and Anastasio, C.: Generation of hydrogen peroxide from San Joaquin Valley particles in a cell-free solution, Atmos. Chem. Phys., 11, 753–765, http://dx.doi.org/10.5194/acp-11-753-2011doi:10.5194/acp-11-753-2011, 2011. Shima, H., Koike, E., Shinohara, R., and Kobayashi, T.: Oxidative ability and toxicity of n-hexane insoluble fraction of diesel exhaust particles, Toxicol. Sci., 91, 218–226, 2006. Shinyashiki, M., Eiguren-Fernandez, A., Schmitz, D. A., Di Stefano, E., Li, N., Linak, W. P., Cho, S. H., Froines, J. R., and Cho, A. K.: Electrophilic and redox properties of diesel exhaust particles, Environ. Res., 109, 239–244, 2009. Verma, V., Ning, Z., Cho, A. K., Schauer, J. J., Shafer, M. M., and Sioutas, C.: Redox activity of urban quasi-ultrafine particles from primary and secondary sources, Atmos. Environ., 43, 6360–6368, 2009a. Verma, V., Polidori, A., Schauer, J. J., Shafer, M. M., Cassee, F. R., and Sioutas, C.: Physicochemical and toxicological profiles of particulate matter in Los Angeles during the October 2007 Southern California wildfires, Environ. Sci. Technol., 43, 954–960, 2009b. Verma, V., Pakbin, P., Cheung, K. L., Cho, A. K., Schauer, J. J., Shafer, M. M., Kleinman, M. T., and Sioutas, C.: Physicochemical and oxidative characteristics of semi-volatile components of quasi-ultrafine particles in an urban atmosphere, Atmos. Environ., 45, 1025–1033, 2011. Vidrio, E., Phuah, C. H., Dillner, A. M., and Anastasio, C.: Generation of hydroxyl radicals from ambient fine particles in a surrogate lung fluid solution, Environ. Sci. Technol., 43, 922–927, 2009. Walgraeve, C., Demeestere, K., Dewulf, J., Zimmermann, R., and Van Langenhove, H.: Oxygenated polycyclic aromatic hydrocarbons in atmospheric particulate matter: Molecular characterization and occurrence, Atmos. Environ., 44, 1831–1846, 2010. Wang, Y., Arellanes, C., Curtis, D. B., and Paulson, S. E.: Probing the source of hydrogen peroxide associated with coarse mode aerosol particles in Southern California, Env. Sci. Technol., 44, 4070–4075, 2010. Ward, T. J., Hamilton, R. F., and Smith, G. C.: The Missoula Valley semivolatile and volatile organic compound study: Seasonal average concentrations, J. Air Waste Manage. Assoc., 55, 1007–1013, 2005. Wilson, D. W., Aung, H. H., Lame, M. W., Plummer, L., Pinkerton, K. E., Ham, W., Kleeman, M., Norris, J. W., and Tablin, F.: Exposure of mice to concentrated ambient particulate matter results in platelet and systemic cytokine activation, Inhal. Toxicol., 22, 267–276, 2010. Xiao, Z. G., Brose, J., Schimo, S., Ackland, S. M., La Fontaine, S., and Wedd, A. G.: Unification of the Copper(I) binding affinities of the cetallo-chaperones Atx1, Atox1, and related proteins detection probes and affinity standards, J. Biol. Chem., 286, 11047–11055, 2011.