Research Symposium: Basic Mechanisms and Human Health
Session 3. Physico-Chemical Properties of Beryllium Metal and Alloys
Dosimetry of Beryllium in an Animal Model by Accelerator Mass Spectrometry
M.L. Chiarappa-Zucca1, R.C. Finkel1, J.E.
McAninch2, R.E. Martinelli1, and K.W. Turteltaub1
A method using accelerator mass spectrometry (AMS) was developed to measure low levels (femtomoles) of beryllium in biological samples. This method provides the sensitivity to investigate macromolecular complexes formed with beryllium at low exposure doses and will provide further understanding about the molecular targets involved in beryllium disease. Berylliosis is a debilitating, progressive and potentially fatal lung disease that develops in individuals exposed to beryllium. Proof of the method was tested by administering 0.05, 0.5 and 5.0 µg 9Be and 10Be by IP injection to 30 g male ICR mice. The mice were euthanized after 24 h and blood, femurs, feces, urine, kidneys, spleen, liver, thymus and lung were prepared for AMS analysis by acid digestion. Highest levels of Be were found in the liver and the spleen (6.0 and 2.0% of whole mouse dose, respectively) while the lowest levels were found in blood, lung and thymus. Beryllium levels were dose-dependent in the spleen and liver. The detection limit of Be in tissue by this method is approximately 2 amol and the analysis was linear over 2 orders of magnitude. Possible sample size effects for measuring Be by AMS showed that similar results were obtained when using samples that were between 3.0 and 150 mg of dosed liver tissue. Precision of 8 replicates of pooled liver tissue was 5% while the variability between 8 dosed livers was 10%. These results show that routine quantification of atto- to femtomole levels of Be in tissues is possible. This method should enable future studies to understand the molecular dosimetry and mechanisms of Be toxicity in biological studies.
This work performed under the auspices of the USDOE by LLNL (W- 7405-ENG-48) with support from the USDOE/OBER.
Identification of Non-Toxic Beryllium-Chelating Porphyrins and Method of Delivery to Mice in an Attempt to Reduce the Lung Beryllium (Be) Burden in Chronic Beryllium Disease (CBD)
Jim Klostergaard and George P. Sakalosky
We have proposed that Be entering the lungs is chelated by hemoglobin (Hb), some of which is fixed in place and constitutes a Be lung burden, and that such in-situ Be enhances the genetic and biological responses manifest as CBD. We have conducted in-vitro competitive binding experiments to evaluate (1) the interaction of Be with Hb to establish whether Be can replace the heme iron and thereby be chelated by the Hb; and, if this occurred, (2) the interaction of each of five different candidate porphyrin compounds with the Be-Hb complex to establish whether the Be could be chelated by and removed from the Be-Hb complex by one or all of these porphyrins. We observed that chelation of Be by Hb and subsequently from Hb by the porphyrin compounds does indeed occur. We have also examined parameters of toxicity of these porphyrins and intend to deliver via aerosols those identified as non-toxic into the lungs of Be-lung-burdened mice to determine whether Be can be chelated by these porphyrins and removed via urinary excretion. In our attempt to establish the parameters of toxicity associated with aerosol administration of these porphyrins, we (1) aerosolized aqueous solutions of the porphyrins using a constant-flow Pulmo-Mist pump and nebulizer system and (2) delivered these solutions into cages each housing five female 20+week-old C3H/HEJ mice over a period of twenty days. The group bodyweights of these mice were measured each morning prior to aerosol treatment. Regimens included multiple, constant dosing, and dose escalation. Mice in cages receiving multiple doses of 9mg of one of the select porphyrins began to lose weight (up to 14%) by the third day of treatment. When treatment was suspended after Day 5 to allow bodyweight normalization, and then resumed on Day 14, the weight loss was delayed compared to the first sequence, suggestive of some tolerance induction. Tolerance was also observed with the dose-escalation study, in that treatment with 18 mg on Day 6 resulted in a ~21% weight loss on Day 15; whereas, following weight normalization, treatment with 27 mg on Day 20 only resulted in ~12% weight loss. Treatments with two of the porphyrins generally reflected lower and delayed evidence of toxicity compared to that observed with one of the select porphyrins. Younger mice (~6 weeks old) were generally more tolerant than older mice, but the rank order of porphyrin toxicity was the same. Our results suggest that three of the select five beryllium-binding porphyrins may be safely delivered as aerosols to C3H/HEJ mice, to allow evaluation of reduction of a beryllium load in the lungs of mice as an intervention in development of CBD.
Surface Area of Respirable Beryllium Metal, Oxide, and Copper Alloy Aerosols and Implications for Assessment of Exposure Risk of Chronic Beryllium Disease
A.B. Stefaniak1,2, M.D. Hoover3,9, R.M. Dickerson4,
E.J. Peterson5, G.A. Day6,7, P.N. Breysse2,
M.S. Kent8, and R.C. Scripsick1
The continued prevalence of chronic beryllium disease (CBD) suggests the current occupational exposure limit of 2 µg beryllium per cubic meter of air does not adequately protect workers from CBD. An understanding of the role of particle surface area, including its influence on beryllium particle dissolution kinetics, could allow for development of an exposure metric based on bioavailability of dissolved beryllium. Therefore, we examined the morphology and measured the particle surface area of aerodynamically size-separated powders and process-sampled particles of beryllium metal, beryllium oxide, and copper-beryllium alloy. The beryllium metal powder consisted of compact particles, while the beryllium oxide powder and particles were clusters of smaller primary particles. Large relative differences in SSA were observed as a function of particle size for the beryllium metal powder, from 4.0± 0.01 m2/g (for the particle size fraction >6 µm) to 20.8± 0.44 m2/g (for the particle size fraction <=0.4 µm). In contrast, little relative difference in SSA (<25%) was observed as a function of particle size for the beryllium oxide powder and particles collected from the screening operation. The SSA of beryllium metal powder decreases with increasing particle size, as expected for compact particles, and the SSA of the beryllium oxide powders and particles remains constant as a function of particle size, which might be expected for clustered particles. These associations illustrate how process-related factors can influence the morphology and SSA of beryllium materials. To avoid errors in predicting bioavailability of beryllium and the associated risks for CBD, the mechanisms of particle formation should be understood and the SSA of beryllium particles should be measured directly.
Examining Beryllium Chemistry with Modern Analytical Techniques
M. Sutton, S.R. Burastero, C. Mundy, and J. Quong
There are increasing numbers of cases of occupational Chronic Beryllium Disease (CBD) being reported and there is currently no cure for CBD. Be body burden has been identified in many CBD patients and may play a role in CBD pathogenesis. Chelation therapy has been suggested to provide a potential therapeutic treatment for the reduction of a beryllium body burden.
Currently available thermodynamic data has been used to assess the solubility, speciation and chelation of beryllium particles in a number of biological matrices. In cases where thermodynamic data is not available, laboratory experiments such as potentiometric titrations have been used to measure formation constants for potential chelators to add to the model database. In addition, multi-scale quantum molecular dynamics has been used to investigate the interaction of chelators with beryllium on the quantum level, and in the process, predicting free energy data that can be compared with previously reported constants and those determined in the laboratory. Modern imaging techniques to identify small quantities of Be in tissues will be discussed.
A large number of possible chelators have been assessed and a ranking system developed based upon the effectiveness of the chelator in dissolving beryllium particles and competition for metal chelation with other chemical systems in the body. This has led to the selection and development of three candidates for selective and effective chelators for further study — sulfo-naphthoic acids, diphosphonic acids and disulfonic acids.
The research currently being undertaken at LLNL brings together a number of scientific capabilities to study the physical-, analytical- and biological- chemistry of beryllium particles in both the body and the workplace environment.
Original Announcements: Agenda - Symposium Information - Speakers - Symposia - Hotel Accommodations - Registration Form - Abstract Submission Form - Poster (pdf, 1365 KB)