Adverse outcome pathways for grouping of nanomaterials

Transcription

1 Adverse outcome pathways for grouping of nanomaterials Sabina Halappanavar, PhD Research Scientist, Genomics and Nanotoxicology Laboratory, HC Adjunct Professor, Department of Biology, University of Ottawa Environmental Health Science and Research Bureau, Health Canada, Ottawa, Canada

2 Adverse outcome pathways (AOPs) Conceptual constructs that portray existing knowledge concerning the linkages between a direct molecular/initiating event and an adverse outcome at a biological level of organization relevant to risk assessment (Ankley et al 2010, Environ. Toxicol. Chem., 29(3): ). Molecular (Initiating Event) Key (Intermediate) Events Adverse Outcome MIE KE1 KE2 KE3 AO AOP Knowledge Base; OECD's Series on Adverse Outcome Pathways; OECD AOP users' handbook. (Villeneuve et al 2014, Toxicological Sciences, 142(2), )

3 SAR AOPs for categorising chemicals Chemicals that activate the same AOP are grouped together Example: Exposure to a group of (Source chemicals) chemicals will induce the same AO A new chemical (Target chemical) that is structurally similar lacks data An AOP is available, SAR is established for the MIE. Both source and target chemicals induce SAR, thus assumed to induce the same AO Source 1-3 Target 1-2 Source 1-3 Source 1-3 Source 1-3 OECD(2017), Guidance document for the use of adverse outcome pathways in developing integrated approaches to testing and assessment (IATA), Series on Testing & Assessment No. 260, Environment, Health and Safety, Environment Directorate, OECD.

4 Nikota, J., Banville, A., Goodwin, L. R., Wu, D., Williams, A., Yauk, C. L., Halappanavar, S. (2017). Stat-6 signaling pathway and not Interleukin-1 mediates multi-walled carbon nanotube-induced lung fibrosis in mice: insights from an adverse outcome pathway framework. Particle and Fibre Toxicology, 14, 37. A putative/qualitative AOP for lung fibrosis AOP 173: Substance interaction with the resident cell membrane components leading to lung fibrosis - OECD EAGMS internal review completed Sabina Halappanavar, Monita Sharma, Hakan Wallin, Ulla Vogel, Kristie Sullivan, Amy J. Clippinger Halappanavar et al., manuscript in preparation Gene/protein expression, Frustrated phagocytosis, Trigger perturbed homeostasis Gene/protein expression, Acute responses BALF cellularity Biopersistence, tissue injury - tipping point BALF cellularity ( temporal), bioperistence Adaptive response/active disease phase Immunohistochemistryfibroblast markers Disease manifestation Histopathology collagen stain, alveolar thickness Substance interaction with the resident cell membrane components. Increased, proinflammatory and profibrotic mediators Increased, recruitment of proinflammatory cells Loss of alveolar capillary membrane integrity T-helper type 2 cells, Activation Fibroblast/ myofibroblast proliferation ECM deposition Lung Fibrosis Labib, S., Williams, A., Yauk, C. L., Nikota, J. K., Wallin, H., Vogel, U., & Halappanavar, S. (2016). Nano-risk Science: application of toxicogenomics in an adverse outcome pathway framework for risk assessment of multi-walled carbon nanotubes. Particle and Fibre Toxicology, 13, 15.

5 AOP for categorisation and read-across Gene/protein expression, Frustrated phagocytosis Substance interaction with the resident cell membrane components. High aspect ratio materials (structural alert) MWCNTs, SAR not available potentially fibrogenic to lungs upon inhalation Frustrated Experimentally phagocytosis assessed(mie?) Experimental data available Short Target MWCNTs Source MWCNTs Producer CNT length (±SEM) CNT length CNT diameter CNT diameter nm 1. NM-401 IO-LE- 4048±366 nm(±sem) 67 (24-138) nm 1. NRCWE NRCWE-043 TECNanomaterials Nanocyl (NC-7000) Cheap (CP-0006-SG) Tubes, Brattleboro, VT 847±102 nm (±3471) (±6.88) 2. Mitsui-7 Mitsui/Hadoga 5700±490 nm 74 (29-173) 3. NRCWE-044 Cheap Tubes, Brattleboro, VT 1330 (±2454) 32.55(±14.44) 4. NRCWE-045 Cheap Tubes, Brattleboro, VT 1553(±2954) 28.07(±13.85) 5. NRCWE-046 Cheap Tubes, Brattleboro, VT 717.2(±1214) 17.22(±5.77) 6. NRCWE-047 Cheap Tubes, Brattleboro, VT 532.5(±591.9) 12.96(±4.44) 7. NRCWE-048 Cheap Tubes, Brattleboro, VT 1604(±5609) 15.08(±4.69) 8. NRCWE-049 Cheap Tubes, Brattleboro, VT 731.1(±1473) 13.85(±6.09) Gene/protein expression, Increased, proinflammatory and profibrotic mediators BALF cellularity Increased, recruitment of proinflammatory cells BALF cellularity ( temporal), bioperistence Loss of alveolar capillary membrane integrity T-helper type 2 cells, Activation Histopathology Immunohistochemistryfibroblast markers collagen stain, alveolar thickness Fibroblast/ myofibroblast proliferation Excess ECM deposition/ Lung fibrosis Source 1-2 Source 1-2 Source 1-2 Source 1-2 Source 1-2 Target 1-8 Target 1-8 Target 2-8 ~ Thin Jackson et al, EMM, 56:183^203 (2015); Poulsen et al, Nanotoxicology, 2016; 10(9):

6 AOP for toxicity categorisation Benchmark Dose (BMD)-response analysis Most sensitive apical endpoint measured in vivo Apical endpoint BMD pro-inflammatory cell influx in lung fluid 24hr Most sensitive pathways with the lowest BMD in vivo NCEA, USEPA (USEPA 2010b). Transcriptomics -pathway BMD 24hr (MIE)

7 AOP for toxicity categorisation Dose-response analysis Halappanavar et al. in review BMD neutrophil influx µg/mouse Mitsui-7 NM-401 NRCWE-026 NRCWE-043 NRCWE-045 NRCWE-044 NRCWE-046 NRCWE-048 NRCWE-047 NRCWE-049 Transcriptional/pathway BMD (Square Rooted) Reflective of the MIE substance interaction with the membrane components MWCNTs Lowest pathway BMD (µg/mouse)

8 AOP-guided categorisation or prioritisation (further testing) High aspect ratio Length, Diameter, Rigidity Long and thick Rigid Short and thin Rigid Short and thin Tangled Toxicity Tox category High High Source MWCNTs NM-401 Mitsui-7 Tox category Target MWCNTs NRCWE-043 NRCWE-044 NRCWE-045 NRCWE-046 NRCWE-047 NRCWE-048 NRCWE-049 Tox category Target MWCNTs NRCWE-026

9 AOP-aligned in vitro assays reflective of in vivo responses Substance interaction with the resident cell membrane components. Increased, proinflammatory and profibrotic mediators Increased, recruitment of proinflammatory cells Loss of alveolar capillary membrane integrity T-helper type 2 cells, Activation Fibroblast/ myofibroblast proliferation ECM deposition Lysosomal uptake DAMP release Receptor signaling Phys-chem ROS synthesis Pro-inflammatory cytokines Fibrogenic factors (Arg-1, IL1-β, TGFβ1, OPN) Inflammosome activation Th2 response genes/proteins Fibroproliferation assays, α-sma Sircol collagen assay Hydroxyproline assay Collagen genes I, III/proteins Tiered - Combinations - Defined approaches - Cell models Cell types - Cell viability/cytotoxicity assays - Colony forming/proliferation assay - Loss of gap junctions - Transepithelial electrical resistance multi-cell type cultures - Persistent ROS synthesis - Lysosomal destabilisation - Vacuolisation - Imbalanced proteases/antiprotease - TNF α, IL-1 β Halappanavar et al., in preparation 9

10 Halappanavar et al., in preparation Positive Positive AOP-aligned in vitro assays reflective of in vivo fibrogenic responses in lungs Decision tree IATA QSAR structural/chemical alerts Lysosomal uptake (M/IE) Frustrated phagocytosis (M/IE) ROS synthesis (M/IE) Negative Increased pro-inflammatory and pro-fibrogenic factors (KE2) Cytotoxicity (KE3) Vacuolisation/lysosomal destablisation (KE3) Negative Low or no toxicity Sircol collagen assay (KE6) Yes/No Potency ranking

11 Exposure Phys-chem characteristics Dose-response IATA Predictive modelling Risk assessment Conclusions Prioritisation, categorisation Read across Mechanistic insights, IATA triggers, Weight of evidence Improved in vivo testing strategies, refine test guidelines Mechanisms-based in vitro testing strategies, alternative testing methods Improved QSAR models Qualitative, semi-quantitative Quantitative For further reading, refer to Validation of Alternative Methods for Toxicity Testing, Chantra Eskes, Maurice Whelan (eds). Advances in Experimental Medicine and Biology 856, Springer. 11

12 Acknowledgements Genomics and Nanotoxicology Laboratory, Health Canada Dongmei Wu Luna Rahman, PhD Jake Nikota, PhD Andrew Williams - Biostatistician National Research Center for the Working Environment Ulla Vogel Hakan Wallin Funding Chemicals Management Plan Nano Genomics Research and Development Initiative