Dosimetry of Beryllium in an Animal Model by Accelerator Mass Spectrometry (AMS)Marina Chiarappa-Zucca R.C. Finkel J.E. McAninch R.E. MartinelliK.W. Turteltaub

Lawrence Livermore National Laboratory University of California

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

Outline

IntroductionWhat 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

What is AMS?

AMS provides high sensitivity measurements of long-lived (10 < t1/2 > 107 yrs) radioisotopesAMS counts nuclei directly rather then measuring radioactive decay This results in 3-9 orders of magnitude more sensitivity relative to scintillation countingAllows analysis of attomole quantities in small samples (g-mg) with low activity levels (nCi-fCi) LLNL has 15 years of experience with AMS and has pioneered the bioscience applications

Accelerators at Lawrence Livermore National Laboratory64 samples (plus standards and blanks) measured as a set>100 unknowns can be quantified in 24 hr of accelerator time14C BioAMSTandem; multiple isotopes including 10Be

AMS can be used to measure 10BeRoutine sensitivity is ~0.02 fgExperiments require fCi total activities10Be has a long half life (1.6 My) and therefore can be measured in samples from months to years10Be in low dose experiments can typically be handled as non-radioactive and non-hazardous

10Be has significant advantages compared to other Be isotopes

Sample Preparation Steps

There are multiple steps for extracting Be from samples for AMS measurement

AMS Measurements of Standards

AMS measurement of standards is linear in the range of interest with good precision Each data point represents the mean SD of three independently prepared standards Instrument precision is 1-3%

Proof-of-Concept Experiment

Proof of concept experiments show Be distribution in mouse tissues30 g male ICR miceIntraperitoneal injection of 0.05, 0.5, and 5.0 g Be (~2-200 g/kg body weight) Three mice used for each dose 24 hr exposureLiver, spleen, kidneys, lung, blood, and femurs prepared for AMS analysis

Be measured in tissues is dose-dependent within dose range studied

*** Spleen and blood data extrapolated to determine dose limits with current MDL

~ 200 pg (.007 g/kg bw) * ~ 0.01 g (0.3 g/kg bw) **These doses are below environmental Be exposures (e.g. for humans 0.9 0.5 g/kg bw)

Chart2

3.77170546847.89765865221.73669062170.04109199880.19340648591.0477366845

2.14569816247.20763742892.31701663850.07941291280.12377329820.6756918431

10.20822284011.63011791591.13326149690.04391236630.12824886081.1235906625

20.704418004779.448638924346.34682765532.44942345472.68703490244.9599909109

4.112684842764.029046314914.89823984880.618891377231.7987111452.7446725152

6.0022592893121.289148120519.88711496581.71246215432.49922261834.5067601522

62.0671950111145.813078192953.200645243925.557979398323.0775803248

155.25094355341387.099290524252.653355661546.578035996359.5358522573

159.39358618795515.905083781463.260774886841.8791965453

Femur

Spleen

Liver

Blood

Lung

Kidney

Sheet1

9Be/tissue

microgrammicrog/g

DoseLivererror (ng)Spleenerror (ng)Femurerror (ng)Blooderror (ng)Lungerror (ng)Kidneyerror (ng)

0.051.7370.0187.8980.1633.7720.0720.0410.0040.1930.0101.0480.024

0.052.3170.0267.2080.1122.1460.0700.0790.0030.1240.0080.6760.012

0.051.1330.0131.6300.04110.2081.5160.0440.0050.1280.0101.1240.034

0.546.3470.41979.4492.35520.7040.1662.4490.0292.6870.0884.9600.053

0.514.8980.49264.0291.5454.1130.0480.6190.01331.7990.3392.7450.031

0.519.8870.223121.2891.9876.0020.1541.7120.0242.4990.0354.5070.048

51145.81311.20162.0671.1313.2010.03825.5580.17723.0781.022

51387.09915.293155.2515.7972.6530.03146.5780.45559.5361.659

5159.39411.58415.9050.18063.2610.43741.8791.585

microgram

DoseLiverlogSpleenFemurBloodbloodLungKidney

0.051.7370.2407.8980.8973.7720.041-1.3860.1931.048

0.052.3170.3657.2080.8582.1460.079-1.1000.1240.676

0.051.1330.0541.6300.21210.2080.044-1.3570.1281.124

0.546.3471.66679.4491.90020.7042.4490.3892.6874.960

0.514.8981.17364.0291.8064.1130.619-0.20831.7992.745

0.519.8871.299121.2892.0846.0021.7120.2342.4994.507

51145.8133.05962.0673.2010.50525.55823.078

51387.0993.142155.2512.6530.42446.57859.536

5159.39415.9051.20263.26141.879

Sheet1

Femur

Spleen

Liver

Blood

Lung

Kidney

Sheet2

Sheet3

ConclusionsAMS provides high sensitivity 10Be measurements in biological samplesOur future direction is to study mechanisms of Be diseaseExplore molecular dosimetryIdentify molecular (e.g. protein) targets that are responsible or involved in CBDEvaluate 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

Funding

Office of Biological and Environmental ResearchU.S. Department of Energy

Our group has been doing research focused on understanding the effects of very low levels of toxicants and drugs on animal and human health. We study the molecular mechanisms involved and how they might relate to disease. One of the tools we have been using for the past 15 years is AMS. We thought our contribution to understanding CBD was to develop an AMS method that could measure low levels of Be-fate-distribution In animals, cells, humans

Sensitivity depends on isotope measured

Method developed for tritium and dual label tracing of 3H and 14C

Small machine funded in part by DOE and other part by NIH10,000 sq ft buildingBriefly:Nitric acid, hydrofluoric acid,with hydrogen peroxide and ammonium persulfateSample taken to dryness and re-suspended in waterBe is precipitated with NaOH and centrifugedAdded to the disposable crucibleOxidized to BeO at 800 degrees centrigradeMixed with Niobium powder and packedDefine MDL: mean of replicate control tissue samples plus 3 SDStandards counted by LSC and compared to NIST standardsHigh precision; Standards are accurate and the dynamic range is 3 orders of magnitude for our specific application We measured nanogram quantities of Be in all the tissues Graph shows Be content (y axis) and Dose administered on the x axisOne group had a higher Be content then the other. Femurs, Spleen, and Liver (shown in yellow) Be content in Blood, lung, and kidney (shown in blue) had about 10X lower BeBe measured in tissues is dose-dependent between 0.05 and 5 g range givenExtrapolationEnvironmental exposures-for comparison purposes More experiments underway to test thisStudy the fate and distribution of Be in animals We can also trace Be and any associated proteins inside or outside a cellCorrelation and endpoints: such as the relationship to genetic variants thought to be involved with CBD and animal models as they developThank you and I will be happy to take any questions