InhalT 90 Current Project Status 90-Day Nose-Only Inhalation Toxicity Study of Cerium Oxide Nanoparticles in Female Wistar Rats with subsequent Analysis of Gene Expression Profiles Sponsor s Study Monitor Federal Ministry of Education and Research Project Management Jülich Dr. Arndt Karschin Test Facility Fraunhofer Institute for Toxicology and Experimental Medicine Inhalation Toxicology Dr. Otto Creutzenberg Daniela Schwotzer Cooperation Leipzig University Institute of Medical Physics and Biophysics Dr. Irina Estrela-Lopis 19.05.2015 Daniela Schwotzer 1/17
Outline Objectives of the study (Geno-)toxicity of CeO 2 nanoparticles Study design + current status Exposure atmosphere Animal health in live data Bronchoalveolar lavage first results Gene expression analysis Processing of results Microscopy IBM & CRM pixabay.com pangea-intl.com nist.gov 2/17
Objectives of the study BASF: Chronic inhalation toxicity and carcinogenicity study with CeO 2 nanoparticles BMBF Satellite-Project: InhalT 90 Early indicators of (geno-)toxic and carcinogenic effects Investigation of adverse and adaptive effects of CeO 2 and BaSO 4 nanoparticles, following a 90-day nose-only inhalation in the rat Gene expression analyses as additional investigation Connection of observations to corresponding chronic inhalation toxicity and carcinogenicity study (BASF, Ludwigshafen, Germany) Foundation for the development of an in vitro screening system Generation of data concerning the health risks of nanomaterial for better risk assessment 3/17
(Geno-)toxicity of CeO 2 nanoparticles secondary genotoxicity (overload) Oxidation state Ce 3+ /Ce 4+ Oxidative stress Apoptosis Cell cycle arrest DNA repair CeO 2 NP Genotoxicity Inflammation Tumor formation Proliferation 4/17
(Geno-)toxicity of CeO 2 nanoparticles primary genotoxicity? Oxidation state Ce 3+ /Ce 4+ Oxidative stress Apoptosis Cell cycle arrest DNA repair CeO 2 NP Genotoxicity Inflammation Tumor formation Proliferation 5/17
Study design in vivo Standard 90-day inhalation toxicity study (OECD 413) in female Wistar Han rats Haematology and clinical chemistry Histopathology Immunhistochemistry Bronchoalveolar lavage Retention of test item Gene expression analysis in pneumocytes type II (kidney, liver tissue) as additional endpoint Localization and quantification of test items in cells/tissue (Leipzig University) Confocal Raman Microscopy (CRM) Ion Beam Microscopy (IBM) In vitro exposure of lung cell line to test items 6/17
Study design in vivo Standard 90-day inhalation toxicity study (OECD 413) in female Wistar Han rats Haematology and clinical chemistry Histopathology Immunhistochemistry Bronchoalveolar lavage Retention of test item Gene expression analysis in pneumocytes type II (kidney, liver tissue) as additional endpoint Localization and quantification of test items in cells/tissue (Leipzig University) Confocal Raman Microscopy (CRM) Ion Beam Microscopy (IBM) In vitro exposure of lung cell line to test items 6/17
Study design in vivo 90-day inhalation toxicity study (OECD 413) Test system Female Wistar rats [Crl:WI (Han)] Exposure Nose-only 6 h/day, 5 days/wk, 13 wks 13 wks recovery Dose group Test substance CeO 2 NM-212 BaSO 4 NM-220 Treatment Aerosolconc. (mg/m³) 1 Clean air - 2 CeO 2 very low 0.1 3 CeO 2 low 0.3 4 CeO 2 mid 1 5 CeO 2 high 3 6 BaSO 4 50 0 100 200 day 1 day 28 day 90+1 day 90+28 day 90+90 Haematology, Histology, Immunohistochemistry, Retention analytics, BAL, Gene expression (only at day 90+1) (only at day 1, 28, 90+1) 7/17
Study design in vivo 90-day inhalation toxicity study (OECD 413) Test system Female Wistar rats [Crl:WI (Han)] Exposure Nose-only 6 h/day, 5 days/wk, 13 wks 13 wks recovery Dose group Test substance CeO 2 NM-212 BaSO 4 NM-220 Treatment Aerosolconc. (mg/m³) 1 Clean air - 2 CeO 2 very low 0.1 3 CeO 2 low 0.3 4 CeO 2 mid 1 5 CeO 2 high 3 6 BaSO 4 50 0 100 200 day 1 day 28 day 90+1 day 90+28 day 90+90 Haematology, Histology, Immunohistochemistry, Retention analytics, BAL, Gene expression (only at day 90+1) (only at day 1, 28, 90+1) 7/17
aerosol conc. [mg/m³] aerosol concentration [mg/m³] Exposure atmosphere up to day 70 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 80 60 40 20 0 0 10 20 30 40 50 60 exposure day 0 10 20 30 40 50 60 exposure day Mean [mg/m³] SD [mg/m³] 0.12 0.04 0.33 0.10 1.07 0.12 3.05 0.28 49.41 4.34 MMAD [µm] GSD 0.71 3.59 0.63 3.83 0.68 4.23 0.79 3.50 2.95 2.43 0.1 mg/m³ CeO2 0.3 mg/m³ CeO2 1.0 mg/m³ CeO2 3.0 mg/m³ CeO2 50 mg/m³ BaSO4 8/17
food consumption [g/cage] water consumption [g/cage] bodyweight [g] Animal health in live data up to day 70 300 200 100 0-20 0 20 40 60 80 time [days] clean air 0.1 mg/m³ CeO2 0.3 mg/m³ CeO2 1.0 mg/m³ CeO2 3.0 mg/m³ CeO2 50 mg/m³ BaSO4 20 35 15 30 25 10 5 * * * 20 15 10 5 * ** 0 0 4 8 12 time [weeks] 0 0 4 8 12 time [weeks] Statistics: ANOVA with Dunnett post-hoc, * p < 0.05, ** p < 0.01 vs. clean air control 9/17
Endpoints in vivo 90-day inhalation toxicity study (OECD 413) + gene expression analysis 0 100 200 day 1 day 28 day 90+1 day 90+28 day 90+90 Histology Immunohisto- Retention Bronchoalveolar Gene chemistry analytics lavage expression Left lung lobe, trachea, larynx, pharynx, nasal cavities, LALN Preservation of all organs Left lung lobe Genotoxicity (γ- H2AX, 8-OHdG) Proliferation (Ki- 67) Apoptosis (Caspase-3) Right lung lobes Preservation of blood, liver, spleen, kidney, bone Solubility in artificial body fluids Total cell count Differential cell count LDH, β- glucuronidase, total protein Cytokines Pneumocytes type II Genotoxicity Inflammation Oxidative stress Apoptosis Lung cancer Preservation of liver, kidney 10/17
Endpoints in vivo 90-day inhalation toxicity study (OECD 413) + gene expression analysis 0 100 200 day 1 day 28 day 90+1 day 90+28 day 90+90 Histology Immunohisto- Retention Bronchoalveolar Gene chemistry analytics lavage expression Left lung lobe, trachea, larynx, pharynx, nasal cavities, LALN Preservation of all organs Left lung lobe Genotoxicity (γ- H2AX, 8-OHdG) Proliferation (Ki- 67) Apoptosis (Caspase-3) Right lung lobes Preservation of blood, liver, spleen, kidney, bone Solubility in artificial body fluids Total cell count Differential cell count LDH, β- glucuronidase, total protein Cytokines Pneumocytes type II Genotoxicity Inflammation Oxidative stress Apoptosis Lung cancer Preservation of liver, kidney 10/17
Bronchoalveolar lavage first results day 1 & day 28 day 1 day 28 100.0 TP 100.0 TP PMN 10.0 LDH PMN 10.0 LDH 1.0 1.0 0.1 0.1 LYMPH GL LYMPH GL MPH Cell count MPH Cell count 0.1 mg/m³ CeO2 0.3 mg/m³ CeO2 1 mg/m³ CeO2 3 mg/m³ CeO2 50 mg/m³ BaSO4 Treatment groups presented as fold change of clean air control 11/17
LDH [% of control] GL [% of control] PMN [%] TP [% of control] Bronchoalveolar lavage first results day 1 & day 28 10 200 ** 8 6 4 *** 150 100 2 50 0 day 1 day 28 0 day 1 day 28 250 200 ** 250 200 * 150 150 100 100 50 50 0 day 1 day 28 0 day 1 day 28 clean air 0.1 mg/m³ CeO2 0.3 mg/m³ CeO2 1 mg/m³ CeO2 3 mg/m³ CeO2 50 mg/m³ BaSO4 Statistics: ANOVA with Dunnett post-hoc, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. clean air control 12/17
Endpoints in vivo standard 90-day inhalation toxicity study + gene expression analysis 0 100 200 day 1 day 28 day 90+1 day 90+28 day 90+90 Histology Immunohisto- Retention Bronchoalveolar Gene chemistry analytics lavage expression Left lung lobe, trachea, larynx, pharynx, nasal cavities, LALN Preservation of all organs Left lung lobe Genotoxicity (γ- H2AX, 8-OHdG) Proliferation (Ki- 67) Apoptosis (Caspase-3) Right lung lobes Preservation of blood, liver, spleen, kidney, bone Solubility in artificial body fluids Total cell count Differential cell count LDH, β- glucuronidase, total protein Cytokines Pneumocytes type II Genotoxicity Inflammation Oxidative stress Apoptosis Lung cancer Preservation of liver, kidney 13/17
Endpoints in vivo standard 90-day inhalation toxicity study + gene expression analysis 0 100 200 day 1 day 28 day 90+1 day 90+28 day 90+90 Histology Immunohisto- Retention Bronchoalveolar Gene chemistry analytics lavage expression Left lung lobe, trachea, larynx, pharynx, nasal cavities, LALN Preservation of all organs Left lung lobe Genotoxicity (γ- H2AX, 8-OHdG) Proliferation (Ki- 67) Apoptosis (Caspase-3) Right lung lobes Preservation of blood, liver, spleen, kidney, bone Solubility in artificial body fluids Total cell count Differential cell count LDH, β- glucuronidase, total protein Cytokines Pneumocytes type II Genotoxicity Inflammation Oxidative stress Apoptosis Lung cancer Preservation of liver, kidney 13/17
Gene expression analysis RT² Profiler PCR Array (SABioscience/Qiagen) Pathway related set of 84 genes Inflammatory cytokines and receptors Oxidative stress DNA Repair Apoptosis Lung Cancer 1. Isolation of pneumocytes type II from lung 2. RNA Isolation 3. cdna Synthesis 4. RTqPCR with PCR Array 5. Data analysis selection of marker genes Additionally: analysis of liver and kidney tissue sabiosciences.com 14/17
Gene expression analysis RT² Profiler PCR Array (SABioscience/Qiagen) Pathway related set of 84 genes Inflammatory cytokines and receptors Oxidative stress DNA Repair Apoptosis Lung Cancer 1. Isolation of pneumocytes type II from lung 2. RNA Isolation 3. cdna Synthesis 4. RTqPCR with PCR Array 5. Data analysis selection of marker of marker genes genes Additionally: analysis of liver and kidney tissue sabiosciences.com 14/17
Processing of results selection of marker genes comparison In vivo In vitro Histology Retention analytics Immunohistochemistry Bronchoalveolar lavage Air liquid- exposure A549 cells Same test substances as in vivo P.R.I.T ALI Culture- and Exposure-System Gene expression analysis of selected marker genes 15/17
Processing of results selection of marker genes comparison In vivo In vitro Histology Retention analytics Immunohistochemistry Bronchoalveolar lavage Air liquid- exposure A549 cells Same test substances as in vivo In vivo/in vitro Microscopy of cells and tissue P.R.I.T ALI Culture- and Exposure-System Gene expression analysis of selected marker genes 15/17
Ion Beam Microscopy H + Y RBS PIXE RBS - Rutherford Backscattering PIXE - Particle induced X ray emission µpixe X Cell scanning of fixed cell with ion beam gather RBS and PIXE spectra information about elemental composition and visualisation determine information about impact of CeO 2 NP internalisation in cell 16/17
Confocal Raman Microspectroscopy label free technique in living cells Information about functional groups and cell compartments information about localisation, distribution, co-localisation and internalisation of CeO 2 NP Identification of spectral Biomarkers Lamellar Body Nucleus Cytoplasma Cerium NP 17/17
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