1. 08 February 2006(RASS: Moss presentation.)1 Dosimetry can change mechanistic models: The challenge of scrutinizing the source before gathering and analyzing the data Owen R. Moss, Ph.D.

2. 08 February 2006(RASS: Moss presentation.)2 Particulate Matter Health Effects Dosimetry of pulmonary hypersensitivity Macrophage uptake of nanoparticles Biomarkers of long- term response PGD 2 -N-methyl-2-picolinyl ester

3. 08 February 2006(RASS: Moss presentation.)3 Nanoparticle Toxicology Warheit (2005) –“Toxicity depends on surface characteristics, particularly surface area and free radical generation by interaction of particles with cells”

4. 08 February 2006(RASS: Moss presentation.)4 When nanoparticles get in the way: Impact of projected area on in vivo and in vitro macrophage function. Moss, O. R. and Wong, V. A. (2006) Inhalation Toxicology (in review January 2006)

5. 08 February 2006(RASS: Moss presentation.)5 Frontiers in the “application” of nanoparticle dosimetry Application in experimental design to determine mechanisms of action of inhaled nanoparticles. Two examples: –In vivo example from the toxicology literature. –In vitro example from confocal microscopy.

6. 08 February 2006(RASS: Moss presentation.)6 Oberdorster et al. 1994 Oberderster et al. (1994) Correlation between Particle Size, in Vivo Particle Persistence, and Lung Injury, Environmental Health Perspectives Vol. (102), Supplement 5, 1-11 … “A correlation between particle surface area and [impairment of macrophage function] was observed.” Was that chemical interaction or physical obstruction? Was that particle surface area or particle projected surface?

7. 08 February 2006(RASS: Moss presentation.)7 A story of 4 spheres 12,400 nm diameter macrophage 3,000 nm diameter PSL particles 250 nm diameter TiO 2 particles 20 nm diameter TiO 2 particles.

8. 08 February 2006(RASS: Moss presentation.)8 Coverage 250 nm TiO 2 10,000. 20 nm TiO 2 1,600,000

9. 08 February 2006(RASS: Moss presentation.)9 Oberdorster 1994 Experimental Design (12 week TiO 2 exposure; 29 week clearance) EXPOSURE**CLEARANCE 250 nm diameter TiO 2 particles* PSL t (1/2) PSL t (1/2) PSL t (1/2) EXPOSURE**CLEARANCE 20 nm diameter TiO 2 particles* PSL t (1/2) PSL t (1/2) PSL t (1/2) * Mass deposition of 250 nm and 20 nm diameter particles the same. ** Target no-overload: Alveolar space TiO 2 particle volume < 6% of macrophage volume. 1000 to 1300 to 11E+6 to 14E+5 to 1

10. 08 February 2006(RASS: Moss presentation.)10 Macrophage toxicity and “surface area” 250 nm TiO 2 20 nm TiO 2 (PSL clearance half-time for controls = 66 d)

11. 08 February 2006(RASS: Moss presentation.)11 Impact of masking on macrophage mediated clearance. 250 nm TiO 2 20 nm TiO 2 (PSL clearance half-time for controls = 66 d)

12. 08 February 2006(RASS: Moss presentation.)12 Impact of masking on macrophage mediated clearance. 250 nm TiO 2 20 nm TiO 2 (PSL clearance half-time for controls = 66 d)

13. 08 February 2006(RASS: Moss presentation.)13 In vitro tests 2x10 13 fluorescent 26 nm diameter PSL beads per ml 0.2 ml injected 300,000 cells 1.3x10 7 fluorescent particles per cell time-lapse photography on confocal scope resolution: 300x increase in concentration.

14. 08 February 2006(RASS: Moss presentation.)14 Confocal images

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18. 08 February 2006(RASS: Moss presentation.)18 60 seconds

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20. 08 February 2006(RASS: Moss presentation.)20 160 seconds

21. 08 February 2006(RASS: Moss presentation.)21 240 seconds

22. 08 February 2006(RASS: Moss presentation.)22 300 seconds

23. 08 February 2006(RASS: Moss presentation.)23 Number of Beads per Cell

24. 08 February 2006(RASS: Moss presentation.)24 Nanometer particle uptake Cell 4 - Minutes - 26 nm PSL 1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9

25. 08 February 2006(RASS: Moss presentation.)25 Dose metrics Impairment of macrophage function can be directly related to the potential for TiO 2 particles to mask the surface of the macrophage. Nanoparticle deposition modeling is needed in resolving chemical and physical impact on cell and organ function.

26. 08 February 2006(RASS: Moss presentation.)26 The Toxicology of Numbers The Avalanche Scenario implies that –snowflakes are toxic because avalanches are lethal The toxicology of nanoparticles includes: –the impact of individual nanoparticles –the impact of the composite

27. 08 February 2006(RASS: Moss presentation.)27 Dosimetry Counts: Molecular hypersensitivity may not drive pulmonary hyperresponsiveness (1) CIIT Centers for Health Research; (2) University of California, Irvine Moss, O. R. (1) and Oldham, M. J. (2) (2006) J. Aerosol Med (in second review February 2006)

28. 08 February 2006(RASS: Moss presentation.)28 Reanalysis of Previous Research DeLorme, M.P. and O.R. Moss. 2002. Pulmonary function assessment by whole-body plethysmography in restrained versus unrestrained mice. J. Pharmacol. Toxicol. Meth. 47:1–10. Oldham, M.J. and R.F. Phalen, 2002. Dosimetry implications of upper tracheobronchial airway anatomy in two mouse varieties. Anat. Rec. 268:59–65. Oldham, M.J., R.F. Phalen, G.M. Schum, and D.S. Daniels. 1994. Predicted nasal and tracheobronchial particle deposition efficiencies for the mouse. Ann. Occup. Hyg. 38 (Supp. 1):135–141.

29. 08 February 2006(RASS: Moss presentation.)29 Airway response Bronchoconstrictive agonist –Murine model Most Responsive AJ > BALB/c > CD-1 > B6C3F1 Least Responsive

30. 08 February 2006(RASS: Moss presentation.)30 Airway Response Measurement Change in airway resistance –Based on pulmonary function values Change in enhanced pause (Penh) reflects change in resistance –DeLorme and Moss (2002) J Pharm Tox. Methods 47:1-10.

31. 08 February 2006(RASS: Moss presentation.)31 Chamber Airway Response Generator Methacholine Solution (mg/ml) C1C2…CnC1C2…Cn

32. 08 February 2006(RASS: Moss presentation.)32 Whole Body Plethysmograph

33. 08 February 2006(RASS: Moss presentation.)33 Enhanced Pause Penh = ( T e /T r – 1)( PEP/PIP ) START of breath END of breath PIP TeTe 36% of area TrTr PEP TiTi inhalation time exhalation

34. 08 February 2006(RASS: Moss presentation.)34 PC200R (BALB/c) BALB/c PC200R = 19.6 mg/ml

35. 08 February 2006(RASS: Moss presentation.)35 Methacholine for 200% increase in resistance 12x

36. 08 February 2006(RASS: Moss presentation.)36 Airway Diameters Oldham and Phalen, 2002, Anatomical Record 268:59-65

37. 08 February 2006(RASS: Moss presentation.)37 Particle deposition at PC200R

38. 08 February 2006(RASS: Moss presentation.)38 Different aerosols 02.5 5 10204080160320 Methacholine Solution Concentrations (mg/ml)

39. 08 February 2006(RASS: Moss presentation.)39 Size Distribution at PC200R

40. 08 February 2006(RASS: Moss presentation.)40 P”D”200R 3.6x

41. 08 February 2006(RASS: Moss presentation.)41 3.6x difference in hypersensitivity Airway resistance from nasal tissue –response time Close enough –possible but dosimetry seems incomplete Molecular biology component –genomic component may be morphometry

42. 08 February 2006(RASS: Moss presentation.)42 Smooth Muscle Constriction DEP  ( dFdF –) =-K  L0L0 Change in Circumference d0d0 d0d0 d0d0 dFdF

43. 08 February 2006(RASS: Moss presentation.)43 Smooth Muscle Constriction DEP  ( dFdF –) =-K  L0L0 Change in Circumference d0d0 d0d0 d0d0 dFdF

44. 08 February 2006(RASS: Moss presentation.)44 ∆ in Resistance to Flow DEP RFRF = 1 1- 1 K  4 Change in Resistance R0R0 L0L0 (d 0 ) 2

45. 08 February 2006(RASS: Moss presentation.)45 Comparing equal resistance ∆ = = If then Equal Resistance Change R a0 R b0 R aF R bF DEP a La0La0 (d a0 ) 2 DEP b Lb0Lb0 (d b0 ) 2

46. 08 February 2006(RASS: Moss presentation.)46 Representative airway generation By Volume By Sensitivity

47. 08 February 2006(RASS: Moss presentation.)47 Airway Volumes

48. 08 February 2006(RASS: Moss presentation.)48 Airway Generation Sensitivity Sensitivity as a multiple of the sensitivity of generation 1, the trachea.

49. 08 February 2006(RASS: Moss presentation.)49 Airway Generation Sensitivity Sensitivity as a multiple of the sensitivity of generation 1, the trachea.

50. 08 February 2006(RASS: Moss presentation.)50 Airway Generation Sensitivity Sensitivity as a multiple of the sensitivity of generation 1, the trachea.

51. 08 February 2006(RASS: Moss presentation.)51 Physiological dose for 200% ∆R

52. 08 February 2006(RASS: Moss presentation.)52 Microdosimetry Time delay in nasal response Constant Smooth Muscle Response Deposition models

53. 08 February 2006(RASS: Moss presentation.)53 Pharyngeal Aspiration aerosol deposition

54. 08 February 2006(RASS: Moss presentation.)54 Changing Mechanistic Models Diameter-squared has three meanings: –chemical reactivity; –physical obstruction; –or both. Dose Counts –pulmonary morphometry can impact response