Exogenous factors can cause an imbalance in the redox state of biological systems, promoting the development of oxidative stress, especially reactive oxygen species (ROS). To monitor the intensity of ROS production in secondary keratinocytes (HaCaT) by diesel exhaust particles and thermoresponsive nanogels (tNG), electron paramagnetic resonance (EPR) spectroscopy after 1 and 24 h of incubation, respectively, was applied. Their cytotoxicity was analyzed by a cell viability assay (XTT). For tNG an increase in the cell viability and ROS production of 10% was visible after 24 h, whereas 1 h showed no effect. A ten times lower concentration of diesel exhaust particles exhibited no significant toxic effects on HaCaT cells for both incubation times, thus normal adult human keratinocytes (NHK) were additionally analyzed by XTT and EPR spectroscopy. Here, after 24 h a slight increase of 18% in metabolic activity was observed. However, this effect could not be explained by the ROS formation. A slight increase in the ROS production was only visible after 1 h of incubation time for HaCaT (9%) and NHK (14%).
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Valko, M., Leibfritz, D., Moncol, J., Cronin, M. T., Mazur, M., & Telser, J. (2007). Free radicals and antioxidants in normal physiological functions and human disease. The International Journal of Biochemistry & Cell Biology, 39, 44–84.
Bast, A., & Goris, R. J. (1989). Oxidative stress. Biochemistry and human disease. Pharmaceutisch weekblad. Scientific Edition, 11, 199–206.
Harman, D. (1956). Aging: A theory based on free radical and radiation chemistry. Journal of Gerontology, 11, 298–300.
Valko, M., Izakovic, M., Mazur, M., Rhodes, C. J., & Telser, J. (2004). Role of oxygen radicals in DNA damage and cancer incidence. Molecular and Cellular Biochemistry, 266, 37–56.
Ceriello, A. (2008). Possible role of oxidative stress in the pathogenesis of hypertension. Diabetes Care, 31(Suppl 2), S181–S184.
Cheeseman, K. H., & Slater, T. F. (1993). An introduction to free radical biochemistry. British Medical Bulletin, 49, 481–493.
Dikalov, S. I., & Harrison, D. G. (2014). Methods for detection of mitochondrial and cellular reactive oxygen species. Antioxidants & Redox Signaling, 20, 372–382.
Kalyanaraman, B., Darley-Usmar, V., Davies, K. J., Dennery, P. A., Forman, H. J., Grisham, M. B., et al. (2012). Measuring reactive oxygen and nitrogen species with fluorescent probes: Challenges and limitations. Free Radical Biology & Medicine, 52, 1–6.
Kundu, K., Knight, S. F., Willett, N., Lee, S., Taylor, W. R., & Murthy, N. (2009). Hydrocyanines: A class of fluorescent sensors that can image reactive oxygen species in cell culture, tissue, and in vivo. Angewandte Chemie, 48, 299–303.
Delaney, C. A., Green, I. C., Lowe, J. E., Cunningham, J. M., Butler, A. R., Renton, L., et al. (1997). Use of the comet assay to investigate possible interactions of nitric oxide and reactive oxygen species in the induction of DNA damage and inhibition of function in an insulin-secreting cell line. Mutation Research, 375, 137–146.
Fang, L., Neutzner, A., Turtschi, S., Flammer, J., & Mozaffarieh, M. (2015). Comet assay as an indirect measure of systemic oxidative stress. Journal of Visualized Experiments, 99, e52763.
Albrecht, S., Ahlberg, S., Beckers, I., Kockott, D., Lademann, J., Paul, V., et al. (2016). Effects on detection of radical formation in skin due to solar irradiation measured by EPR spectroscopy. Methods, 109, 44–54.
Ahlberg, S., Rancan, F., Epple, M., Loza, K., Hoppe, D., Lademann, J., et al. (2016). Comparison of different methods to study effects of silver nanoparticles on the pro- and antioxidant status of human keratinocytes and fibroblasts. Methods, 109, 55–63.
Lohan, S. B., Muller, R., Albrecht, S., Mink, K., Tscherch, K., Ismaeel, F., et al. (2016). Free radicals induced by sunlight in different spectral regions - in vivo versus ex vivo study. Experimental Dermatology, 25, 380–385.
Mrakic-Sposta, S., Gussoni, M., Montorsi, M., Porcelli, S., & Vezzoli, A. (2014). A quantitative method to monitor reactive oxygen species production by electron paramagnetic resonance in physiological and pathological conditions. Oxidative Medicine and Cellular Longevity, 2014, 306179.
Lohan, S. B., Bauersachs, S., Ahlberg, S., Baisaeng, N., Keck, C. M., Muller, R. H., et al. (2015). Ultra-small lipid nanoparticles promote the penetration of coenzyme Q10 in skin cells and counteract oxidative stress. European Journal of Pharmaceutics and Biopharmaceutics, 89, 201–207.
Fuchs, J., Groth, N., Herrling, T., & Zimmer, G. (1997). Electron paramagnetic resonance studies on nitroxide radical 2,2,5,5-tetramethyl-4-piperidin-1-oxyl (TEMPO) redox reactions in human skin. Free Radical Biology & Medicine, 22, 967–976.
Lauer, A. C., Groth, N., Haag, S. F., Darvin, M. E., Lademann, J., & Meinke, M. C. (2013). Radical scavenging capacity in human skin before and after vitamin C uptake: An in vivo feasibility study using electron paramagnetic resonance spectroscopy. The Journal of Investigative Dermatology, 133, 1102–1104.
Venugopal, J. R., & Ramakrishna, S. (2016). Nanotechnology: 21st century revolution in restorative healthcare. Nanomedicine, 11, 1511–1513.
Wilczewska, A. Z., Niemirowicz, K., Markiewicz, K. H., & Car, H. (2012). Nanoparticles as drug delivery systems. Pharmacological Reports, 64, 1020–1037.
Moller, P., Jacobsen, N. R., Folkmann, J. K., Danielsen, P. H., Mikkelsen, L., Hemmingsen, J. G., et al. (2010). Role of oxidative damage in toxicity of particulates. Free Radical Research, 44, 1–46.
Lai, P., Lechtman, E., Mashouf, S., Pignol, J. P., & Reilly, R. M. (2016). Depot system for controlled release of gold nanoparticles with precise intratumoral placement by permanent brachytherapy seed implantation (PSI) techniques. International Journal of Pharmaceutics, 515, 729–739.
Ishihara, T., Kaneko, K., Ishihara, T., & Mizushima, T. (2014). Development of biodegradable nanoparticles for liver-specific ribavirin delivery. Journal of Pharmaceutical Sciences, 103, 4005–4011.
Sahle, F. F., Giulbudagian, M., Bergueiro, J., Lademann, J., & Calderon, M. (2017). Dendritic polyglycerol and N-isopropylacrylamide based thermoresponsive nanogels as smart carriers for controlled delivery of drugs through the hair follicle. Nanoscale, 9, 172–182.
Rancan, F., Giulbudagian, M., Jurisch, J., Blume-Peytavi, U., Calderon, M., & Vogt, A. (2016). Drug delivery across intact and disrupted skin barrier: Identification of cell populations interacting with penetrated thermoresponsive nanogels. European Journal of Pharmaceutics and Biopharmaceutics, 116, 4–11.
Giulbudagian, M., Rancan, F., Klossek, A., Yamamoto, K., Jurisch, J., Neto, V. C., et al. (2016). Correlation between the chemical composition of thermoresponsive nanogels and their interaction with the skin barrier. Journal of Controlled Release , 243, 323–332.
Cuggino, J. C., Alvarez, I. C. I., Strumia, M. C., Welker, P., Licha, K., Steinhilber, D., et al. (2011). Thermosensitive nanogels based on dendritic polyglycerol and N-isopropylacrylamide for biomedical applications. Soft Matter, 7, 11259–11266.
Giulbudagian, M., Asadian-Birjand, M., Steinhilber, D., Achazi, K., Molina, M., & Calderon, M. (2014). Fabrication of thermoresponsive nanogels by thermo-nanoprecipitation and in situ encapsulation of bioactives. Polymer Chemistry, 5, 6909–6913.
Asadian-Birjand, M., Bergueiro, J., Rancan, F., Cuggino, J. C., Mutihac, R. C., Achazi, K., et al. (2015). Engineering thermoresponsive polyether-based nanogels for temperature dependent skin penetration. Polymer Chemistry, 6, 5827–5831.
Rancan, F., Asadian-Birjand, M., Dogan, S., Graf, C., Cuellar, L., Lommatzsch, S., et al. (2016). Effects of thermoresponsivity and softness on skin penetration and cellular uptake of polyglycerol-based nanogels. Journal of Controlled Release, 228, 159–169.
Ahlberg, S., Meinke, M. C., Werner, L., Epple, M., Diendorf, J., Blume-Peytavi, U., et al. (2014). Comparison of silver nanoparticles stored under air or argon with respect to the induction of intracellular free radicals and toxic effects toward keratinocytes. European Journal of Pharmaceutics and Biopharmaceutics, 88, 651–657.
Kuhn, D. M., Balkis, M., Chandra, J., Mukherjee, P. K., & Ghannoum, M. A. (2003). Uses and limitations of the XTT assay in studies of Candida growth and metabolism. Journal of Clinical Microbiology, 41, 506–508.
Riss, T. L., Moravec, R. A., Niles, A. L., Duellman, S., Benink, H. A., Worzella, T. J., et al. (2004). Cell viability assays. In G. S. Sittampalam, N. P. Coussens, H. Nelson, M. Arkin, D. Auld, C. Austin, et al. (Eds.) Assay guidance manual. The National Center for Advancing Translational Sciences (NCATS), Bethesda, MD.
Saitoh, Y., Ohta, H., & Hyodo, S. (2016). Protective effects of polyvinylpyrrolidone-wrapped fullerene against intermittent ultraviolet-A irradiation-induced cell injury in HaCaT cells. Journal of Photochemistry and Photobiology B, 163, 22–29.
Gerecke, C., Edlich, A., Giulbudagian, M., Schumacher, F., Zhang, N., Said, A., et al. (2017). Biocompatibility and characterization of polyglycerol-based thermoresponsive nanogels designed as novel drug delivery systems and their intracellular fate in keratinocytes. Nanotoxicology, Under review.
Zhang, Y., Chan, H. F., & Leong, K. W. (2013). Advanced materials and processing for drug delivery: the past and the future. Advanced Drug Delivery Reviews, 65, 104–120.
Dockery, D. W. (2009). Health effects of particulate air pollution. Annals of Epidemiology, 19, 257–263.
Kuhlbusch, T. A., John, A. C., & Quass, U. (2009). Sources and source contributions to fine particles. Biomarkers, 14(Suppl 1), 23–28.
Ema, M., Naya, M., Horimoto, M., & Kato, H. (2013). Developmental toxicity of diesel exhaust: A review of studies in experimental animals. Reproductive Toxicology, 42, 1–17.
Fiorito, S., Mastrofrancesco, A., Cardinali, G., Rosato, E., Salsano, F., Sheng Su, D. et al. (2011). Effects of carbonaceous nanoparticles from low-emission and older diesel engines on human skin cells. Carbon, 49, 1–9.
Takenaka, S., Takenaka-Dornhöfer, H., & Muhle, H. (1986). Alveolar distribution of fly ash and of titanium dioxide after long-term inhalation by Wistar rats. Journal of Aerosol Science, 17, 361–364.
Droge, W. (2002). Free radicals in the physiological control of cell function. Physiological Reviews, 82, 47–95.
Choi, M., & Lee, C. (2015). Immortalization of primary Keratinocytes and its application to skin research. Biomolecules & Therapeutics, 23, 391–399.
The authors acknowledge the support of the Deutsche Forschungsgemeinschaft (DFG)/ German Research Foundation via SFB 1112, Projects A04, B01.
Conflict of Interest
The authors declare that they have no competing interests.
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Lohan, S.B., Ahlberg, S., Mensch, A. et al. EPR Technology as Sensitive Method for Oxidative Stress Detection in Primary and Secondary Keratinocytes Induced by Two Selected Nanoparticles. Cell Biochem Biophys 75, 359–367 (2017). https://doi.org/10.1007/s12013-017-0823-4
- Oxidative stress
- Electron paramagnetic resonance spectroscopy
- Cell viability