# Presentation: 515536

## 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**

**Notes:**
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

## 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 **

**Notes:**
-fate
-distribution 
In animals, cells, humans

## 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**

##                          What is AMS?

**                         ****What is AMS?**

## 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 

**     **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 < t****1/2 ****> 10****7 ****yrs) radioisotopes**

**Notes:**
Sensitivity depends on isotope measured

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

## 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**

- 14C BioAMS

- Tandem; multiple isotopes including 10Be

**Notes:**
Small machine funded in part by DOE and other part by NIH
10,000 sq ft building

## Routine sensitivity is ~0.02 fg
Experiments require fCi total activities
10Be has a long half life (1.6 My) and therefore can be measured in samples from months to years
10Be in low dose experiments can typically be handled as non-radioactive and non-hazardous  

**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**

##                 Sample Preparation Steps

**                ****Sample Preparation Steps**

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

**Notes:**
Briefly:
Nitric acid, hydrofluoric acid,with  hydrogen peroxide and ammonium persulfate
Sample taken to dryness and re-suspended in water
Be is precipitated with NaOH and centrifuged
Added to the disposable crucible
Oxidized to BeO at 800 degrees centrigrade
Mixed with Niobium powder and packed

##            AMS Measurements of Standards 

**           ****AMS Measurements of Standards **

## Each data point represents the mean ± SD of three independently prepared standards 
Instrument precision is 1-3%

**Instrument precision is 1-3%**

**AMS measurement of standards is linear in the range of interest with good precision   **

**Notes:**
Define MDL: mean of replicate control tissue samples plus 3 SD
Standards counted by LSC and compared to NIST standards
High precision; 
Standards are accurate and the dynamic range is 3 orders of magnitude for our specific application 

##               Proof-of-Concept Experiment

**              ****Proof-of-Concept Experiment**

## 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

**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**

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

**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 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)

**Notes:**
We measured nanogram quantities of  Be in all the tissues 
Graph shows Be content (y axis) and Dose administered on the x axis
One 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 Be
Be measured in  tissues is dose-dependent between 0.05 and 5 μg range given
Extrapolation
Environmental exposures-for comparison purposes 
More experiments underway to test this

## 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**

**Notes:**
Study the fate and distribution of Be in animals 
We can also trace Be and any associated proteins inside or outside a cell
Correlation and endpoints:  such as the relationship to genetic variants thought to be involved with CBD and animal models as they develop

## Funding

**Office of Biological and Environmental Research**
**U.S. Department of Energy**

**Notes:**
Thank you and  I will be happy to take any questions