Differences in the Mechanisms of Toxicity of Nano and Ionic Ag in Bacteria

Transcription

1 Differences in the Mechanisms of Toxicity of Nano and Ionic Ag in Bacteria Professor Hilary Godwin, UCLA Copyright 2013 UC Center for Environmental Implications of Nanotechnology. The Regents of the University of California. All Rights Reserved. Contact to obtain permission to use copyrighted material. This material is based upon work supported by the National Science Foundation and the Environmental Protection Agency under Cooperative Agreement Number DBI and DBI Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation or the Environmental Protection Agency. This work has not been subjected to EPA review and no official endorsement should be inferred.

2 Core Questions: Are nanoparticles special in terms of their toxicology (compared to bulk or ionic analogs)? Does the one chemical-one pathway paradigm of toxicology make sense for most chemicals?

3 Silver (Ag) nanoparticles are an important test case because of bacterial relevance and regulatory implications: August 27, 2013: EPA Proposed Registration of Nanosilver Pesticide Product ( Nanosilva ) ( anosilver.html) Nowack, B.; Krug, H. F.; Height, M. 120 Years of Nanosilver History: Implications for Policy Makers. Environ. Sci. Technol. 2011, 45 (4), Costanza, J.; El Badawy, A. M.; Tolaymat, T. M. Comment on 120 Years of Nanosilver History: Implications for Policy Makers. Environ. Sci. Technol. 2011, 45 (17),

4 citrate-coated Ag NPs PVP-coated BPEI-coated Diameter (nm) Surface charge (mv) To address our core questions, we studied the magnitude and the mechanisms of toxicity of a series of Ag NPs with different sizes and surface coatings in bacteria (E. coli) Ivask et al, submitted to ACS Nano 4

5 (a): Particle sizes were also characterized in DI water and in bacterial media (b): Particle diameter, nm Ivask et al, submitted to ACS Nano 5

6 To address the question of whether Ag NPs are toxic simply because they generate, a bioluminescent reporter system for intracellular in E. coli was used regulatory gene promoter luciferase gene 6

7 To address the question of whether Ag NPs are toxic simply because they generate, a bioluminescent reporter system for intracellular in E. coli was used regulatory gene promoter luciferase gene RNAP Luciferase Response is triggered only by intracellular ions 7

8 Across the series, the magnitude of toxicity of the nanoparticles correlates with amount of delivered to cells (i.e., to the bioavailable Ag) Ivask et al, submitted to ACS Nano

9 The high toxicity and bioavailablity of positively charged Ag-BPEI appears to result from tight binding to cell membrane (a): control cells (b): cells+ag-cit 10 (c): cells+ag-pvp (d): cells + Ag-BPEI (e): Ag-cit 10 (f): Ag-PVP (g):ag-bpei Ivask et al, submitted to ACS Nano 9

10 HTS studies using ~4000 E. coli gene deletion strains were used to determine the mechanisms of toxicity for each Ag NP, for PS-NH 2 NP, and for Output: list of strains that are more sensitive than WT strain for each toxicant! Ivask et al, ES&T, 2011, 44(19), ; Ivask et al, submitted to ACS Nano

11 Gene Sensitivity Self organizing map analysis of HTS data reveals that each Ag NP has a unique mechanistic fingerprint Gene Clusters 17 clusters SOM 21-Sep-2012 Ivask et al, submitted to ACS Nano hscb rpod 95 ybgt ycib 90 ydfi 85 yfjf yfjg 80 mene cuer 75 tonb ycgh_2 70 yjjy yobh 65 cpxa dapf 60 kate reca 55 tolr 50 SOM 25-Sep-2012 SOM 25-Sep-2012 SOM 25-Sep clusters AgNO3 3 Ag (10 nm) Ag_10 Ag (20 Ag_20 nm) d SOM 25-Sep d SOM 25-Sep d SOM 25-Sep-2012 Only statistically significant correlations: Ag-cit(10 nm) and AgNO 3 Ag-BPEI and PS-NH 2 Ag_PVP Ag_PVP Ag_BPEI Ag_BPEI PS_NH2 PS_NH d d d

12 Results from cluster analysis of HTS data: number of confirmed hits per pathway for each toxicant GO term AgNO 3 Ag-cit 10 Ag-cit 20 Ag-PVP Ag-BPEI PS-NH 2 ATP binding; ATPase activity 12; ; ; ; ; ; --- Metal binding; iron binding 6; ; --- 4; - 3; ; ; 4 Antioxidant activity Protein transporter activity ; ; --- Flagellar motor activity Metal cluster or 4Fe-4S cluster binding Cell surface antigen activity (LPS) Quinone binding; oxidoreductase activity ; ; ; ; --- 7; 4 6; --- Protein kinase activity Ivask et al, submitted to ACS Nano

13 Results from cluster analysis of HTS data: gene ontology groups associated with categories of toxicants silver-specific nano-specific cationicspecific antioxidant activity Cell surface antigen activity (lipoplysaccharide) quinone binding; ubiquinone biosynthesis Ivask et al, submitted to ACS Nano

14 NSF: EF Conclusions: Although the magnitude of the toxicity of Ag NPs correlates with the amount of dissolved, the mechanism of toxicity is highly dependent on the size and coating/surface charge of the particles Each particle has its own fingerprint of pathways involved in bacterial response Positively charged Ag NP (Ag-BPEI) exhibits mechanisms of toxicity that are more similar to the cationic polystyrene particle (PS-NH 2 ) than to other Ag NPs

15 Acknowledgements -Angela Ivask -Chitrada Kaweeteerawat -Trish Holden (UCSB) -Allison Horst (UCSB) -Ivy Ji (UCLA) -Chong Hyun Chang (UCLA) -Donatello Telesca (UCLA) -David Boren (UCLA) -Cecile Low-Kam (UCLA) -Heidi Fischer (UCLA) -Yoram Cohen (UCLA) -Rong Liu (UCLA) -Amro ElBadawy (U Cinn) -Thabet Tolaymat (EPA) 15