TOXICITA ULTRAJEMNÝCH ČÁSTIC V ŽIVOTNÍM PROSTŘEDÍ M. Machala Výzkumný ústav veterinárního lékařství, Brno machala@vri.cz
OBSAH PŘEDNÁŠKY 1. Co je nanotoxikologie? 2. Mechanismy toxicity PAHs a dalších chemických kontaminantů vázaných na vzduchové částice 3. Terénní studie: jsou nanočástice hlavními nosiči PAHs and jejich genotoxicity a dioxinové aktivity?
CO JE NANOTOXIKOLOGIE?
INTRODUCTION I. Nanotoxicology and human toxicology of engineered nanoparticles II. Airborne ultrafine and nanoparticles toxic effects of NPs as well as chemicals adsorbed on surface of particulate matter (PM), environmental health & human biomarkers of exposure III. Nanobiomaterials, nanomedicine, pharmacokinetics / particokinetics, adverse effects of biocompatible nanomaterials Nano-bioanalytical sciences (determination of size, surface, and other physico-chemical characteristics, chemical composition etc.) Modes of action of NPs and chemicals adsorbed on NPs Exposure scenarios and risk assessment of nanomaterials, nanosafety
Studies on effects of engineered and environmental nanoparticles - major biological models: in vitro models; ecotoxicology tests (bacteria Vibrio fischeri, Daphnia magna, Chlorella vulgaris, zebrafish etc.); experimental animal studies (rodents); human biomarkers (oxidative stress, inflammation and immunity parameters).
Studies on effects of engineered and environmental nanoparticles: major toxicological end-points DNA damage, oxidative stress; disruption of intercellular communication and cell adhesion, remodeling of membrane lipids and cytoskeleton and modulation of cell-surface mediated and intracellular signaling; modulation of gene expression; effects on cell populations (proliferation, cell death, differentiation).
Current limits of nanotoxicological studies: We have only limited understanding of fate, transport and toxicity of ENPs and airborne PM. The tools to study these interactions are being developed. Several potentially important toxic modes of action are currently not covered within nanotoxicology. Exposure protocols are based on classical toxicology paradigms and are often insufficient (NPs agregate, sorption, surface effects etc...).
HLAVNÍ MECHANISMY TOXICITY LÁTEK ADSORBOVANÝCH NA ČÁSTICE
HLAVNÍ MECHANISMY TOXICITY XENOBIOTIK Přímé mutageny / genotoxiny; metabolická aktivace promutagenů, adukty s proteiny a DNA, chromosomální aberace AhR-dependentní dioxinová aktivita Další receptor-dependentní mechanismy endokrinní disrupce (např. estrogenita) Oxidativní stres (tvorba ROS, peroxidace lipidů, oxidativní poškození DNA a proteinů), modulace signální transdukce pomocí ROS Neurotoxicita, imunotoxicita, poruchy metabolismu endogenních látek atd.
GENOTOXICITA METABOLICKÁ AKTIVACE POLYCYKLICKÝCH AROMATICKÝCH UHLOVODÍKŮ
GENOTOXICKÉ EFEKTY PAHs, POSTGENOTOXICKÉ SIGNÁLY, APOPTÓZA 1. Metabolická aktivace cytochromy P450 (CYP1A1, CYP1A2, CYP1B1) + alternativní dráha (AKR1C9, AKR1A1) Oxidativní stres Stabilní adukty DNA-PAH Oxidativní poškození DNA (oxidace bazí, apurinová místa)
GENOTOXICKÉ EFEKTY PAHs, POSTGENOTOXICKÉ SIGNÁLY, APOPTÓZA 2. DETEKCE POŠKOZENÍ DNA (stabilní adukty DNA s metabolity PAHs) Metoda 32P-postlabeling: expozice buněk, izolace DNA, digesce a značení 32P, TLC, detekce radioakt. fosforu DBalP, DBaeP, BgChry, BaP silně genotoxické PAHs; DBahA, BbF, Chry, BaA převážně negenotoxické efekty (aktivace AhR atd.)
GENOTOXICKÉ EFEKTY PAHs, POSTGENOTOXICKÉ SIGNÁLY, APOPTÓZA 3. POSTGENOTOXICKÉ SIGNÁLY (sensory poškození, regulátory DNA repairu a apoptózy) Roos, Kaina, 2006 Sensory genotoxicity, přenos post-genotoxických signálů a regulace: ATM, ATR, H2AX, ChK1, ChK2, p53
HLAVNÍ TYPY POŠKOZENÍ DNA ( apurinic sites a oxidačně modifikované báze) Oxidativní deaminace Depurinace (nestabilní modifikované nukleotidy)
Biomarker oxidativního poškození DNA (stanovení HPLC) PRODUKTY OXIDATIVNÍHO POŠKOZENÍ DNA
AhR ligand DIOXINOVÁ TOXICITA - AhR activation: hsp90 p23 XAP2 cytoplasm ARNT?? nucleus transcriptional coregulators (modified from Kewley et al., 2004) TNGCGTG Xenobiotic response genes
ENDOKRINNÍ DISRUPCE: AKTIVACE AhR A NUKLEÁRNÍCH RECEPTORŮ Aktivace AhR (indukce CYP1A1/1A2/1B1; modulace bun. cyklu, apoptózy, imunosuprese, syndromy dioxinové toxicity, vývojová toxicita, metabolismus steroidních hormonů) Xenoestrogeny a antiestrogeny (efekty na ERa, ERb a efekty na biosyntézu estrogenů) Xenoandrogeny / antiandrogeny (efekty na AR) Xenobiotika modulující thyroidní funkce efekty (efekty na transport a metabolismus thyroidů, modulace TR )
CHEMICKÁ KARCINOGENEZE
CHEMICKÁ KARCINOGENEZE 1. GENOTOXICKÉ POŠKOZENÍ - INICIACE 2. PŘEŽÍVANÍ / PROLIFERACE GENOTOXICKY POŠKOZENÝCH BUNĚK 3. TRANSFORMACE VE VÍCE AGRESÍVNÍ, METASTAZUJÍCÍ KLON(Y)
ZÁKLADNÍ MECHANISMY NÁDOROVÉ PROMOCE Negenotoxické efekty cizorodých látek Endogenní látky (cytokiny, růstové faktory, signál. transdukce) (Hannahan, Weinberg, 2000)
Základní stavební jednotkou jsou proteiny konexiny (connexin 32, 43 atd.), které tvoří hexamery (konexony); konexony sousedních buněk mohou tvořit společný kanál, kterým procházejí signální molekuly (camp, Ca 2+ atd.) INTERCELULÁRNÍ SPOJENÍ GJIC
INHIBICE GJIC (scrape-loading / dye transfer assay; prostup fluoreskující luciferové žluti monovrstvou buněk) kontrola expozice nádorovými promotery vede k inhibici GJIC
DISRUPCE ADHERENTNÍCH SPOJENÍ (ADHERENS JUNCTIONS)
DISRUPCE ADHERENTNÍCH SPOJENÍ (ADHERENS JUNCTIONS) signálování kateninu/cadherinu biosensor density kontrola bun. akumulace
ARE ULTRAFINE PARTICLES MAJOR CARRIERS OF c-pahs AND THEIR GENOTOXICITY AND DIOXIN- LIKE ACTIVITY?
Aim of the study: There is a wide range of organic and inorganic pollutants that become associated with NPs (including ultrafine/nanofraction of PM). There are two major goals to assess toxicity of NPs and to assess toxicity of chemicals adsorbed on the surface of NPs (toxicological impact of organic pollutants such as PAHs, PCBs, dioxins). Focus on the major toxic modes of action of PM genotoxicity and dioxin-like activity.
What is dioxin-like activity and how to determine it? 1) Aryl hydrocarbon receptor (AhR) is a key transcription factor of many developmental, metabolic and other processes. 2) Sustained AhR activation is associated with chemical carcinogenesis (including tumor progression), developmental perturbations and immunotoxicity. 3) Exposure to highly persistent TCDD and TCDD-like compounds as well as chronic exposure to PAHs leads to long-term activation of AhR and AhR-mediated toxicity.
What is dioxin-like activity and how to determine it? TCDD and Related Compounds + AhR Src HSP90 1 ARNT HSP90 HSP90 2 P Nuclear Factors AhR ARNT HSP90 Src Activated DRE-Luc P Increased Protein Phosphorylation Modulation of Gene Expression Light Membrane Proteins P Cytosolic Proteins Luciferase Adapted from Blankenship (1994)
Materials and Methods Air sampling in 4 sites by HiVol cascade impactor (ultrafine aerosol collected on PTFE coated Glass Micro Fiber Absolute filters) Extraction by DCM and HPLC/FD analysis of c-pahs In vitro acellular assay with DNA adduct analysis by P 32 -postlabeling and TLC Determination of AhR-dependent reporter gene expression ( dioxin-like activity ) in H4IIE.GudLuc cells exposed to DCM extracts of subfractions (DR-CALUX assay)
Diesel engine PM agregates (carbonaceous nano-pm aggregates) (Murr, Garza, 2009)
Example of formation of PM (soot formation from PAHs and other compounds) PAHs, dioxins,... adsorbed on PM surface
Distribution of c-pahs in extractable organic matter prepared from various size fractions of ambient-air PM (ng/mg PM)
Distribution of c-pahs in extractable organic matter prepared from various size fractions of ambient-air PM (ng/m 3 )
DNA formation after exposure to isolated subfractions of ambient PM (acellular genotoxicity assay: a sample + calf thymus DNA +/- S9) d ae :1 10 m 0.5 1 m 0.17 0.5 m <0.17 m +S9 -S9
DNA formation after exposure to isolated subfractions of ambient PM (acellular genotoxicity assay: a sample +calf thymus DNA + S9; expressed as DNA adducts/mg PM)
Normalized data from acellular genotoxicity assay (+S9 metabolic activation, DNA adducts/m 3 )
1-10 0.5-1 0.17-0.5 < 0.17 1-10 0.5-1 0.17-0.5 < 0.17 1-10 0.5-1 0.17-0.5 < 0.17 1-10 0.5-1 0.17-0.5 < 0.17 pg TCDD/m3 pg TCDD/m3 Dioxin-like activity of PM fractions (expressed as TCDD equivalents per m 3 ) 80 TEQ - DR-Calux 70 60 50 40 30 20 80 60 40 Strip mine Highway City centre Background station 10 0 20 0 1-10 0.5-1 0.17-0.5 < 0.17 Strip mine Highway City centre Background station
Chemická analýza velikostních frakcí (pouze PAHs, vzorek Praha 2010) ng/m 3 Compound Name 1-10 µm 0.5-1.0 µm 0.17-0.5 µm <0.17 µm 1 Phenanthrene 0.26 1.53 0.55 0.85 2 Anthracene 0.04 0.14 0.05 0.06 3 Fluoranthene 0.61 1.60 0.54 0.61 4 Pyrene 0.46 1.21 0.39 0.40 5 Benz[a ]anthracene 0.24 0.37 0.13 0.12 6 Chrysene 0.41 0.56 0.18 0.14 7 Benzo[b ]fluoranthene 0.35 0.48 0.14 0.11 8 Benzo[k ]fluoranthene 0.16 0.23 0.08 0.05 9 Benzo[a ]pyrene 0.25 0.45 0.15 0.10 10 Dibenz[a,h ]anthtacene 0.02 0.03 0.01 0.01 11 Benzo[g,h,i ]perylene 0.56 0.61 0.22 0.15 12 Indeno[1,2,3-cd ]pyrene 0.27 0.29 0.13 0.09 1 4H-Cyclopenta[def]phenanthrene 0.05 0.17 0.06 0.07 2 Benzo[c]phenanthrene 0.06 0.13 0.02 0.03 3 Triphenylene 0.08 0.09 0.03 0.02 4 Cyclopenta[cd]pyrene 0.14 0.36 0.11 0.06 5 Benzo[a]fluoranthene 0.08 0.11 0.00 0.00 6 Benzo[j]fluoranthene 0.20 0.32 0.10 0.08 7 Benzo[e]pyrene 0.17 0.22 0.07 0.05 8 Perylene 0.04 0.08 0.03 0.02 11 Dibenz[a,c]anthracene 0.02 0.03 0.01 0.01 12 Dibenz[a,j]anthracene 0.02 0.04 0.02 0.01 14 Coronene 0.10 0.10 0.04 0.02 22 Dibenzo[b,k ]fluoranthene 0.02 0.03 0.01 0.01 24 Naphtho[2,1-a ]pyrene 0.03 0.06 0.02 0.01 26 Naphtho[2,3-e ]pyrene 0.02 0.03 0.01 0.01 27 Naphtho[1,2-b ]fluoranthene 0.04 0.07 0.02 0.01 28 Naphtho[1,2-k ]fluoranthene 0.04 0.06 0.02 0.02 31 Benzo[a ]coronene 0.02 0.04 0.01 0.01 Sum of PAHs 4.78 9.45 3.17 3.15 30 25 20 15 10 5 0 30 25 20 15 10 5 0 Relativní koncentrace PAU s MW 252 [%] 30 1-10 µm 0.5-1.0 µm 25 20 15 10 5 0 BaF BbF BkF BjF BaP BeP Per BaF BbF BkF BjF BaP BeP Per 30 0.17-0.5 µm <0.17 µm 25 20 15 10 5 0 BaF BbF BkF BjF BaP BeP Per BaF BbF BkF BjF BaP BeP Per
Využití chemických dat pro výpočet relativních toxických potencí Karcinogenita: CEQ [ng BaP eq./m 3 ] =RF (RPF) x konc. [ng/m 3 ] RPF (Relativní faktory karcinogenní potence): US EPA (2010): Development of a relative potency factor (RPF) approach for polycyclic aromatic hydrocarbon (PAH) mixtures Mutagenita: MEQ [ng BaP eq./m 3 ] = RMF x konc. [ng/m 3 ] RMFs (Relativní mutagenní faktory): Durant et al., 1996; Durant et al., 1999. Dioxinová aktivita: IEQ [pg TCDD eq./m 3 ] = IEF x konc. [pg/m 3 ] IEF (indukční ekvivalenční faktory): Machala et al., 2001; Švihálková et al., 2007; Marvanová et al., 2008 a další články (užito testu DR-CALUX).
Toxické efekty vypočtené z koncentrací toxikantů Karcinogenní potence 2.0 CEQs [ng BaP eq. /m3] 4.0 Mutagenní potence MEQs [ng BaP eq. /m3] Dioxinová potence 1.0 IEQs [pg TCDD/m3] 3.0 1.0 2.0 0.5 1.0 0.0 1-10 µm 0.5-1.0 µm 0.17-0.5 µm <0.17 µm 0.0 1-10 µm 0.5-1.0 µm 0.17-0.5 µm <0.17 µm 0.0 1-10 µm 0.5-1.0 µm 0.17-0.5 µm <0.17 µm
1-10 0.5-1 0.17-0.5 <0.17 1-10 0.5-1 0.17-0.5 <0.17 1-10 0.5-1 0.17-0.5 <0.17 1-10 0.5-1 0.17-0.5 <0.17 Dioxin-like activity of c-pahs (calculated from concentration data and induction equivalency factors of individual PAHs) 2,0 1,8 1,6 1,4 1,2 1,0 0,8 0,6 0,4 0,2 0,0 1,4 1,2 1,0 0,8 0,6 0,4 0,2 IEQ Strip of c-pah mine calculated from chemical data 1-10 0.5-1 0.17-0.5 <0.17 0,0 Strip mine Highway BaA City Chrycenter BbFBackground BkF station BaP DBahA IPY
CONCLUSIONS The upper accumulation fractions (0.5-1 um) of ambient-air aerosols are major carriers of PAHs and exhibit the highest production of DNA adducts (when normalized per m 3 ). The fractions of ultrafine particles (1-170 nm) showed generally lower concentrations of PAHs and lower efficiency to form DNA adducts. Similarly, the most potent induction of AhRdependent gene expression ( dioxin-like activity, assessed in DR-CALUX assay) was found for the upper accumulation fraction; ultrafine fractions elicit the lowest activity. Nevertheless, chemical contaminants associated with nanoparticles contribute significantly to the overall toxicity of airborne particulate matter. Toxicity of nanoparticles themselves?
FUTURE EXPERIMENTS: Toxicity of ultrafine and nanoparticles themselves vs. toxicity of coarse, upper accumulation and lower accumulation particles in lung epithelial and bronchial cells. Studies on effects of NPs and adsorbed chemical contaminants associated with carcinogenic and tumor promoting processes. Risk assessment of environmental NPs.
Acknowledgements K. Pěnčíková, M. Ciganek, J, Neča, J. Turánek (Dept. Chemistry and Toxicology, Veterinary Research Institute, Brno, Czech Republic) J. Vondráček (Department of Cytokinetics, Institute of Biophysics, ASCR, Brno) A. Milcová, J. Schmuczerová, R. Šrám, J. Topinka (Laboratory of Genetic Toxicology, IEM, Prague) Supported by the Centre of Excelence CENATOX (Czech Science Foundation, project No. P503/12/G147)