Physicochemical Determinants of Beryllium Toxicity using in vitro and in vivo Models

1. Physicochemical Determinants of Beryllium Toxicity using in vitro and in vivo Models Gregory L. Finch, PhD Drug Safety Evaluation Pfizer Global Research & Development Groton, CT June 25/26, 2002 Beryllium Research Symposium, Bethesda MD Acknowledgement: Most of the research presented today was conducted at Inhalation Toxicology Research Institute [LRRI], Albuquerque, NM Be Research Symposium, GL Finch

2. CBD: An occupational health mystery Who? • current screening reveals many sensitized and diseased pts • only “susceptible” individuals appear to get CBD What? • granulomatous lesions with pronounced TH lymphocytic component, with pronounced Be-specific reactivity • a debilitating lung disease When? • a widely varying latency period following Be exposure; preceded by sensitization Where? • mostly occupational following exposure to various Be forms • no clear dose-response relationship has been defined Why? • an MHC-II restricted response • component of genetic susceptibility • importance of role of Be physicochemical form Be Research Symposium, GL Finch

3. Model of Be Interaction with Immune System From Newman, 1993 Be Research Symposium, GL Finch

4. Selected observations in role of physicochemical form in CBD • There are a wide variety of physicochemical forms encountered • Natural occuring mineral • Various “soluble”/”insoluble” forms in processing • Mostly insoluble forms delivered to end users • Early experience: • More soluble forms generally lead to acute Be disease • More insoluble forms generally lead to CBD • No exposure-dose-response apparent • Exposure-response relationships are now being revealed • CBD more likely following exposure to relatively insoluble forms • apparent excess risk for certain occupations/processes Be Research Symposium, GL Finch

5. How can in vitro/in vivo models help? • Understanding exposure-dose-response relationships • role of physicochemical form • acute, episodic, or chronic exposures • Linkage to health effects • Understanding pathogenesis of response • detailed characterization • manipulated and/or knock-in/out models • Seeking therapeutic intervention Be Research Symposium, GL Finch

6. Lovelace database on the properties and health effects of beryllium aerosols Be Research Symposium, GL Finch

7. Lovelace database on the properties and health effects of beryllium aerosols Be Research Symposium, GL Finch

8. Physicochemical properties and in vitro characteristics • Laboratory-produced preparations • BeO: produced with 7Be radiolabel and fired at 500 or 1000oC • Be metal: size-fractionated using an aerosol cyclone • “Field” preparations • Sawing/milling of alloys • Laser vaporization of Be metal Be Research Symposium, GL Finch

9. Be Metal Ni-Be Alloy Cu-Be Alloy Softer alloys yielded relatively more fine particles with identical machining processes Be Research Symposium, GL Finch

10. Beryllium Metal Particles Separated by an Aerosol Cyclone Similarities: particle morphology Differences: physical and aerodynamic size; specific surface area Be Research Symposium, GL Finch

11. Be Metal Particles Have an Oxide Surface Layer Initial dissolution behavior might be similar for Be and BeO Be Research Symposium, GL Finch

12. Solubility in an acidic environment Low fired BeO is more soluble than high fired or metal in an acidic environment Be Research Symposium, GL Finch

13. Solubility in a simulated lung extracellular fluid BeO is more soluble than Be in simulated lung fluid Be Research Symposium, GL Finch

14. Toxicity to Canine Alveolar Macrophages Toxicity increases with solubility of the Be material Be Research Symposium, GL Finch

15. In Vitro Toxicity based on: Mass Surface Area Normalization by “surface area dose” resulted in comparable toxicity Be Research Symposium, GL Finch

16. Large animal models of Be biokinetics and Be-induced toxicity – Studies in Dogs • Respirable preparations of 7BeO fired at either 500o or 1000oC were used • Dogs were exposed once by inhalation to achieve either low [17 g/kg] or high [50 g/kg] initial lung burdens [ILBs] • Dogs were sacrificed up through 365 days post-exposure • Biokinetic evaluation • Lung histopathology • A companion group was evaluated through 730 days post-exposure • Period lung lavage for cytology and lymphocyte simulation • Dogs were re-exposed at 2-yr then followed for an additional 210 days Be Research Symposium, GL Finch

17. BeO Clearance/Translocation in Dogs LUNGS Lower-fired BeO cleared more rapidly from lung, and persisted at higher levels in extrapulmonary compartments Be Research Symposium, GL Finch

18. Lung lesion in BeO-exposed dogs Be Research Symposium, GL Finch

19. Interstitial granuloma in BeO-exposed dogs Be Research Symposium, GL Finch

20. Relative severity of pulmonary lesions in BeO-exposed dogs Be Research Symposium, GL Finch

21. Influence of BeO Temperature History on the Influx of Neutrophils Be Research Symposium, GL Finch

22. Lymphocyte numbers and SIs in Dogs Be Research Symposium, GL Finch

23. Lymphocyte SIs following re-exposure to BeOGrouped by first exposure to high or low ILBs of 500 or 1000oBeO Be Research Symposium, GL Finch

24. Summary of results in dogs • Compared to high fired BeO, low-fired BeO: • cleared from lungs more rapidly • produced more marked inflammatory response • Increased numbers of lymphocytes • Increased lymphocyte stimulation indices • Responses peaked several months after exposure • Previous exposure history did not influence responses to a 2nd exposure to low-fired BeO, and the effects were not cumulative Be Research Symposium, GL Finch

25. Comparative toxicity of Be metal vs. BeO in monkeys • Study used low-fired BeO [500oC] and size fractionated Be metal • Animals were exposed by bronchoscopic, intrabronchiolar instillation • Regimen 1: • Graded doses of BeO [saline, 2.5, 12.5, 37.5 g] or Be metal [saline, 1, 50, 150 g] into different lung lobes • Doses based on estimated dissolution over 80 dpe • Histological evaluation of granulomas guided dose selection for regimen 2 • Regimen 2: • Single doses of 12.5 g BeO or 50 g Be metal • Lavages through 120 dpe and sacrifice at 180 dpe Be Research Symposium, GL Finch

26. Be-induced lesions in monkey Be Research Symposium, GL Finch

27. Be-induced lesion in monkey Be Research Symposium, GL Finch

28. Lymphocytes recovered by lavage Be Research Symposium, GL Finch

29. Lymphocyte proliferation Be Research Symposium, GL Finch

30. Large animal studies - summary • Clear differences between BeO [and temperature history] and Be metal demonstrated • Biologically: • Granulomatous lesions were produced • Lymphocytes were increased in number • Increased lymphocyte proliferation demonstrated • However: • Biological responses were not progressive • Additional efforts were devoted to murine studies using Be metal Be Research Symposium, GL Finch

31. Summary of importance of physicochemical form • It is important for “relatively insoluble” particles • The amount of surface presented appears to control dissolution and toxicity • Form and preparation influences disposition, biokinetics, and in vivo toxicity • Exposure-dose-response need not be rejected • Just need to look in the right place • Compare “equivalent” exposures • Account for host factors, genetic susceptibility Be Research Symposium, GL Finch

32. Conclusion – a hypothesis • A hypothesis: there is a critical balance in lung between solubility and retained or newly deposited dose • Solubility/stimulus: needed to release Be++ to produce antigenic stimulus and induction of sensitization • Retention/re-challenge: needed to provide long term challenge depot of Be++ once sensitization is achieved • Both form/solubility and chronicity of exposure undoubtably work in concert – with host factors - to drive CBD Be Research Symposium, GL Finch

33. LRRI Principal Investigators: Greg Finch Mark Hoover Pat Haley LRRI Scientists Ed Barr Bill Bechtold Dave Bice Fletcher Hahn Charles Hobbs Tom March Bruce Muggenburg Kris Nikula Bill Griffith Janet Benson Steve Belinsky Technical Support Staff: Lee Blair Dee Esparza Anna Holmes Applied Toxicology Group Exposure Operations Group Animal Care Unit Necropsy/Histology Lab Lung Cancer Program Collaborations: Bill Carlton, DVM, Purdue Alan Rebar, DVM, Purdue Funding from the US Department of Energy Acknowledgements Be Research Symposium, GL Finch