1. Dosimetry of Beryllium in an Animal Model by Accelerator Mass Spectrometry (AMS) Marina Chiarappa-Zucca R.C. Finkel J.E. McAninch R.E. Martinelli K.W. Turteltaub Lawrence Livermore National Laboratory University of California

2. Our aim is to develop a method to quantify attomole (10 -18 ) amounts of Be in biological samples This method should enable the identification of molecular targets of Be at very low doses and provide detailed characterization of Be dosimetry Demonstration of this capability will facilitate research collaborations on the cellular and molecular mechanisms of CBD

3. Outline Introduction – What is accelerator mass spectrometry (AMS)? Experimental Approach – Sample preparation steps for extracting Be for AMS – AMS measurement of Be standards – Data from experiments showing Be distribution in mouse tissues Conclusions

4. What is AMS? What is AMS?

5. AMS counts nuclei directly rather then measuring radioactive decay This results in 3-9 orders of magnitude more sensitivity relative to scintillation counting Allows analysis of attomole quantities in small samples (µg-mg) with low activity levels (nCi-fCi) LLNL has 15 years of experience with AMS and has pioneered the bioscience applications AMS provides high sensitivity measurements of long-lived (10 10 7 yrs) radioisotopes

6. Accelerators at Lawrence Livermore National Laboratory 64 samples (plus standards and blanks) measured as a set >100 unknowns can be quantified in 24 hr of accelerator time 14 C BioAMS Tandem; multiple isotopes including 10 Be

7. 10 Be has significant advantages compared to other Be isotopes Routine sensitivity is ~0.02 fg Experiments require fCi total activities 10 Be has a long half life (1.6 My) and therefore can be measured in samples from months to years 10 Be in low dose experiments can typically be handled as non-radioactive and non-hazardous AMS can be used to measure 10 Be

8. Sample Preparation Steps Sample Preparation Steps

9. There are multiple steps for extracting Be from samples for AMS measurement AMS Ion Source Packing into AMS target Oxidizing to BeO Disposable Quartz Crucibles PrecipitationAcid digestion

10. AMS Measurements of Standards AMS Measurements of Standards

11. Each data point represents the mean ± SD of three independently prepared standards Instrument precision is 1-3% AMS measurement of standards is linear in the range of interest with good precision 1.0E+05 1.0E+07 1.0E+09 1.00E+051.00E+071.00E+09 Expected 10 Be atoms Method Detection Limit 10 9 10 7 10 5 Measured 10 Be atoms 10 7 10 9

12. Proof-of-Concept Experiment Proof-of-Concept Experiment

13. 30 g male ICR mice Intraperitoneal injection of 0.05, 0.5, and 5.0 µg Be (~2-200 µg/kg body weight) Three mice used for each dose 24 hr exposure Liver, spleen, kidneys, lung, blood, and femurs prepared for AMS analysis Proof of concept experiments show Be distribution in mouse tissues

14. Be measured in tissues is dose-dependent within dose range studied 10 5 10 -3 10 0.001 0.1 10 Be (ng/g wet tissue) Total Dose (µg Be) method detection limit * ** Spleen and blood data extrapolated to determine dose limits with current MDL ~ 200 pg (.007 µg/kg bw) * ~ 0.01 µg (0.3 µg/kg bw) ** These doses are below environmental Be exposures (e.g. for humans 0.9  0.5 µg/kg bw)

15. Conclusions AMS provides high sensitivity 10 Be measurements in biological samples Our future direction is to study mechanisms of Be disease – Explore molecular dosimetry – Identify molecular (e.g. protein) targets that are responsible or involved in CBD – Evaluate the correlation between these endpoints and susceptibility to CBD We are ready now to collaborate with other researchers that have specific applications for this capability

16. Funding Office of Biological and Environmental Research U.S. Department of Energy