ISCOMS - Crnković
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Oxidative stress response of Daphnia magna exposed to silver nanoparticles Tea Crnković Faculty of Pharmacy and Biochemistry University of Zagreb, Croatia
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Transcript of ISCOMS - Crnković
Oxidative stress response of Daphnia magna exposed to silver nanoparticles
Tea Crnković
Faculty of Pharmacy and Biochemistry
University of Zagreb, Croatia
Introduction
• Use of silver nanoparticles (Ag NPs)
Introduction
Lack of data
• The utility of biochemical approaches in environmental pollution monitoring
• Early warning indicators
• No published dana on oxidative stress response in Daphnia magna to either nano or ionic form of silver
Goal of this study
• A comprehensive toxicity assesment of silver nanoparticles (AgNPs) using a standardized test organism Daphnia magna
• Comparison of nano and ionic form of silver
Methods
1) Synthesis of Ag NPs and their purification
2) Characterisation and stability evaluation of Ag NPs
• Transmission electron microscopy
• Disperse light scatter method
3) Dissolution experiment
4) Acute toxicity test to D. magna
5) Determination of oxidative stress biomarkers
• Catalase
• Superoxide dismutase
• Reduced glutathione
• Reactive oxygen species production
Synthesis of citrate-capped Ag NPs and their purification
unpurified
purified
Characterisation and stability evaluation of Ag NPs
Medium
Peak I Peak II
ζ potential, mV
PdI
dH, nmMean
volume, %
dH, nmMean
volume, %
Ultrapurewater
18.2 ±10.1
85.3101.4 ±
48.312.4 - 19.6 ± 0.8 0.39
Standard culture
medium
46.2 ±23.8
49.3395.7 ±264.7
53.8 - 18.1 ± 0.4 0.58
Aggregation of Ag NPs
Ultrapure water Standard culture medium
Acute toxicity to Daphnia magna
• Daphnia magna neonates
• HRN EN ISO 6341:2013 protocol and OECD guidelines
• Immobilization and subsequent mortality depends on exposure concentration and time
• After 48 h
• LC50 for Ag NPs → 12.4 μg/L
• LC50 for Ag+ → 2.6 μg/L
Oxidative stress response
• Surviving neonates from the acute toxicity test
• 4 biochemical biomarkers:
• Catalase
• Superoxide dismutase
• Reduced glutathione
• Reactive oxygen species production
Reactive oxygen species production
• Fluorescent probes DCFH-DA and DHE
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μg/L Ag NPs μg/L Ag+
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DCFH-DA
Reactive oxygen species production
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lDHE
Catalase activity
0.0020.0040.0060.0080.00
100.00120.00140.00160.00180.00
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Level of reduced glutathione
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Superoxide dismutase activity
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Conclusion
• Ag NPs induced toxicity and a oxidative stress response in D. magna at 10-foldhigher concentrations than Ag+
• Biochemical results:
• Decreased reactive oxygen species level
• Increased reduced glutathione level and catalase activity
• No change in superoxide dismutase activity
Future perspective
• biochemical biomarkers as an early warning indicator of the population-level effect from sublethal concentration exposure
• chemical and biological processes that may modify Ag forms in real environmental matrices as well as different exposure pathways for silver to organisms should be analyzed and taken into account
Acknowledgements
• Co-authors:
• Lea Ulm, PhD
• Adela Krivohlavek, PhD
• Ivana Vinković Vrček, PhD (mentor)
• This research was supported by the Institute of Public Health “Dr. Andrija Štampar” and the Institute for Medical Research and Occupational Health, Analytical Toxicology and Mineral Metabolism Unit
References
• Bondarenko O, Juganson K, Ivask A, Kasemets K, Mortimer M, Kahru A. Toxicity of Ag, CuO and ZnO nanoparticles to selected environmentally relevant test organisms and mammalian cells in vitro: a critical review. Arch Toxicol. 2013; 87:1181-1200.
• Li H, Xia H, Wang D, Tao X. Simple synthesis of monodisperse, quasi-spherical, citrate-stabilized silver nanocrystals in water. Langmuir. 2013; 29:5074−5079.
• Held P. An Introduction to Reactive Oxygen Species -Measurement of ROS in Cells. Vermont: BioTek Instruments, Inc.; 2015.
• Ellman GL. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 1959; 82(1):70–77.
• Jemec A, Tišler T, Drobne D, Sepčić K, Jamnik P, Roš M. Biochemical biomarkers in chronically metal-stressed daphnids. Comp. Biochem. Physiol. part C. 2008; 147:61–68.
• Marklund SL, Marklund G. Involvement of the superoxide anion 574 radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur. J. Biochem. 1974; 47(3):469.
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