June 25/26, 2002Be Research Symposium, GL Finch1 Physicochemical Determinants of Beryllium Toxicity using in vitro and in vivo Models Gregory L. Finch,

June 25/26, 2002 Be Research Symposium, GL Finch 1

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

Gregory L. Finch, PhD

Drug Safety EvaluationPfizer 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


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

sensitizationWhere?

– 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


Model of Be Interaction with Immune System

From Newman, 1993


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


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


BeO 500oC

BeO 1000oC

Be metal size fractions

Be metal ultrafine

BeO ultrafine

Be Soluble salts

Be alloys

Be Work- Place

Physicochem Properties

X X X X X X X X

In vitro cell culture X X X X X X

Biokinetics Dogs/inhl Rodents/inhl

X

X

X

X (it)

Cancer Rats/inhl Mice/inhl

X X

Granulomatous LD Dogs/inhl Monkeys/inst Rats/inhl Mice/inhl

X X

X

X X X

X(it)

Lovelace database on the properties and health effects of beryllium aerosols


BeO 500oC

BeO 1000oC

Be metal size fractions

Be metal ultrafine

BeO ultrafine

Be Soluble salts

Be alloys

Be Work- Place

Physicochem Properties

X X X X X X X X

In vitro cell culture X X X X X X

Biokinetics Dogs/inhl Rodents/inhl

X

X

X

X (it)

Cancer Rats/inhl Mice/inhl

X X

Granulomatous LD Dogs/inhl Monkeys/inst Rats/inhl Mice/inhl

X X

X

X X X

X(it)

Lovelace database on the properties and health effects of beryllium aerosols


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 Metal Ni-Be Alloy Cu-Be Alloy

Softer alloys yielded relatively more fine particles with identical machining processes


Beryllium Metal Particles Separated by an Aerosol Cyclone

Similarities: particle morphology

Differences: physical and aerodynamic size; specific surface area


Be Metal Particles Have an Oxide Surface Layer

Initial dissolution behaviormight be similar for

Be and BeO


Solubility in an acidic environment

Low fired BeO is more soluble than high fired or metal in an acidic environment


Solubility in a simulated lung extracellular fluid

BeO is more soluble than Be in simulated lung fluid


Toxicity to Canine Alveolar Macrophages

Toxicity increases with solubility of the Be material


In Vitro Toxicity based on:Mass Surface Area

Normalization by “surface area dose” resulted in comparable toxicity


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


BeO Clearance/Translocation in Dogs

Lower-fired BeO cleared more rapidly from lung, and persisted at higher levels in extrapulmonary compartments

LUNGS


Lung lesion in BeO-exposed dogs


Interstitial granuloma in BeO-exposed dogs


Relative severity of pulmonary lesions in BeO-exposed dogs


Influence of BeO Temperature Historyon the Influx of Neutrophils


Lymphocyte numbers and SIs in Dogs


Lymphocyte SIs following re-exposure to BeOGrouped by first exposure to high or low ILBs of 500 or 1000oBeO


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


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-induced lesions in monkey


Be-induced lesion in monkey


Lymphocytes recovered by lavage


Lymphocyte proliferation


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


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


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


Acknowledgements

LRRI Principal Investigators:Greg FinchMark HooverPat Haley

LRRI ScientistsEd BarrBill BechtoldDave BiceFletcher HahnCharles HobbsTom MarchBruce MuggenburgKris NikulaBill GriffithJanet BensonSteve Belinsky

Technical Support Staff:Lee BlairDee EsparzaAnna HolmesApplied Toxicology GroupExposure Operations GroupAnimal Care UnitNecropsy/Histology LabLung Cancer Program

Collaborations:Bill Carlton, DVM, PurdueAlan Rebar, DVM, Purdue

Funding from the US Department of Energy

Documents

June 25/26, 2002Be Research Symposium, GL Finch1 Physicochemical Determinants of Beryllium Toxicity using in vitro and in vivo Models Gregory L. Finch,