The management of radiation injuries following a catastrophic event where large numbers of people may have been exposed to life-threatening doses of ionizing radiation will rely critically on the availability and use of suitable biodosimetry methods. In vivo electron paramagnetic resonance (EPR) tooth dosimetry has a number of valuable and unique characteristics and capabilities that may help enable effective triage. We have produced a prototype of a deployable EPR tooth dosimeter and tested it in several in vitro and in vivo studies to characterize the performance and utility at the state of the art. This report focuses on recent advances in the technology, which strengthen the evidence that in vivo EPR tooth dosimetry can provide practical, accurate, and rapid measurements in the context of its intended use to help triage victims in the event of an improvised nuclear device. These advances provide evidence that the signal is stable, accurate to within 0.5 Gy, and can be successfully carried out in vivo. The stability over time of the radiation-induced EPR signal from whole teeth was measured to confirm its long-term stability and better characterize signal behavior in the hours following irradiation. Dosimetry measurements were taken for five pairs of natural human upper central incisors mounted within a simple anatomic mouth model that demonstrates the ability to achieve 0.5 Gy standard error of inverse dose prediction. An assessment of the use of intact upper incisors for dose estimation and screening was performed with volunteer subjects who have not been exposed to significant levels of ionizing radiation and patients who have undergone total body irradiation as part of bone marrow transplant procedures. Based on these and previous evaluations of the performance and use of the in vivo tooth dosimetry system, it is concluded that this system could be a very valuable resource to aid in the management of a massive radiological event.

In order to meet the potential need for emergency large-scale retrospective radiation biodosimetry following an accident or attack, we have developed instrumentation and methodology for in vivo electron paramagnetic resonance spectroscopy to quantify concentrations of radiation-induced radicals within intact teeth. This technique has several very desirable characteristics for triage, including independence from confounding biologic factors, a non-invasive measurement procedure, the capability to make measurements at any time after the event, suitability for use by non-expert operators at the site of an event, and the ability to provide immediate estimates of individual doses. Throughout development there has been a particular focus on the need for a deployable system, including instrumental requirements for transport and field use, the need for high throughput, and use by minimally trained operators. Numerous measurements have been performed using this system in clinical and other non-laboratory settings, including in vivo measurements with unexposed populations as well as patients undergoing radiation therapies. The collection and analyses of sets of three serially-acquired spectra with independent placements of the resonator, in a data collection process lasting approximately five minutes, provides dose estimates with standard errors of prediction of approximately 1 Gy. As an example, measurements were performed on incisor teeth of subjects who had either received no irradiation or 2 Gy total body irradiation for prior bone marrow transplantation; this exercise provided a direct and challenging test of our capability to identify subjects who would be in need of acute medical care.

By using specific anti-ACAT-1 antibodies in immunodepletion studies, we previously found that ACAT-1, a 50-kDa protein, plays a major catalytic role in the adult human liver, adrenal glands, macrophages, and kidneys but not in the intestine. Acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity in the intestine may be largely derived from a different ACAT protein. To test this hypothesis, we produced specific polyclonal anti-ACAT-2 antibodies that quantitatively immunodepleted human ACAT-2, a 46-kDa protein expressed in Chinese hamster ovary cells. In hepatocyte-like HepG2 cells, ACAT-1 comprises 85-90% of the total ACAT activity, with the remainder attributed to ACAT-2. In adult intestines, most of the ACAT activity can be immunodepleted by anti-ACAT-2. ACAT-1 and ACAT-2 do not form heterooligomeric complexes. In differentiating intestinal enterocyte-like Caco-2 cells, ACAT-2 protein content increases by 5-10-fold in 6 days, whereas ACAT-1 protein content remains relatively constant. In the small intestine, ACAT-2 is concentrated at the apices of the villi, whereas ACAT-1 is uniformly distributed along the villus-crypt axis. In the human liver, ACAT-1 is present in both fetal and adult hepatocytes. In contrast, ACAT-2 is evident in fetal but not adult hepatocytes. Our results collectively suggest that in humans, ACAT-2 performs significant catalytic roles in the fetal liver and in intestinal enterocytes. Acyl-coenzyme A:cholesterol acyltransferase (ACAT) 1 is an integral membrane protein located in the endoplasmic reticulum. It catalyzes the formation of cholesteryl esters from long

A rare form of human ACAT1 mRNA, containing the optional long 5 -untranslated region, is produced as a 4.3-kelonucleotide chimeric mRNA through a novel interchromosomal trans-splicing of two discontinuous RNAs transcribed from chromosomes 1 and 7 (Li, B. L., Li, X. L., Duan, Z. J., Lee, O., Lin, S., Ma, Z. M., Chang, C. C., Yang, X. Y., Park, J. P., Mohandas, T. K., Noll, W., Chan, L., and Chang, T. Y. (1999) J. Biol. Chem. 274, 11060 -11071). To investigate its function, we express the chimeric ACAT1 mRNA in Chinese hamster ovary cells and show that it can produce a larger ACAT1 protein, with an apparent molecular mass of 56 kDa on SDS-PAGE, in addition to the normal, 50-kDa ACAT1 protein, which is produced from the ACAT1 mRNAs without the optional long 5 -untranslated repeat. To produce the 56-kDa ACAT1, acat1 sequences located at both chromosomes 7 and 1 are required. The 56-kDa ACAT1 can be recognized by specific antibodies prepared against the predicted additional amino acid sequence located upstream of the N-terminal of the ACAT1 ORF . The translation initiation codon for the 56-kDa protein is GGC, which encodes for glycine, as deduced by mutation analysis and mass spectrometry. Similar to the 50-kDa protein, when expressed alone, the 56-kDa ACAT1 is located in the endoplasmic reticulum and is enzymatically active. The 56-kDa ACAT1 is present in native human cells, including human monocyte-derived macrophages. Our current results show that the function of the chimeric ACAT1 mRNA is to increase the ACAT enzyme diversity by producing a novel isoenzyme. To our knowledge, our result provides the first mammalian example that a trans-spliced mRNA produces a functional protein.Acyl-coenzyme A:cholesterol acyltransferase (ACAT) 1 is an intracellular enzyme that plays important roles in lipid metabolism. It catalyzes the formation of cholesteryl esters, using long-chain fatty acyl coenzyme A and cholesterol as the two substrates. In mammals, two ACAT genes have been identified (reviewed in Refs. 1-4). The first ACAT gene, acat1, was identified by isolating a human cDNA (ACAT cDNA K1) that functionally complements a Chinese hamster ovary cell mutant (clone AC29) lacking endogenous ACAT activity (5). The second ACAT gene, acat2, was identified by homology cloning, based on the nucleotide sequence of ACAT1 cDNA. The ACAT1 and ACAT2 proteins share extensive sequence homology at their C-terminal halves but not at their N-terminals. Both enzymes are integral membrane proteins. Human ACAT1 (hACAT1) contains seven transmembranes (6), whereas hACAT2 contains only two detectable transmembranes (7). A conserved histidine (His-460 in hACAT1 and His-432 in hACAT2), located within a long stretch of hydrophobic residues, may serve as an active site for ACAT catalysis (7,8). Human ACAT1 message and protein are present in many tissues and various cell types examined, including adrenal, kidney, hepatocytes, Kupffer cells, intestinal enterocytes, fibroblasts, macrophages, and neurons in the brain (5, 9 -12). In contrast, abundant ACA...

As a result of terrorism, accident, or war, populations potentially can be exposed to doses of ionizing radiation that could cause direct clinical effects within days or weeks. There is a critical need to determine the magnitude of the exposure to individuals so that those with significant risk have appropriate procedures initiated immediately, while those without a significant probability of acute effects can be reassured and removed from the need for further consideration in the medical/emergency system. In many of the plausible scenarios there is an urgent need to make the determination very soon after the event and while the subject is still present. In vivo EPR measurements of radiation-induced changes in the enamel of teeth is a method, perhaps the only such method, which can differentiate among doses sufficiently for classifying individuals into categories for treatment with sufficient accuracy to facilitate decisions on medical treatment. In its current state, the in vivo EPR dosimeter can provide estimates of absorbed dose with an error approximately +/- 50 cGy over the range of interest for acute biological effects of radiation, assuming repeated measurements of the tooth in the mouth of the subject. The time required for acquisition, the lower limit, and the precision are expected to improve, with improvements in the resonator and the algorithm for acquiring and calculating the dose. The magnet system that is currently used, while potentially deployable, is somewhat large and heavy, requiring that it be mounted on a small truck or trailer. Several smaller magnets, including an intraoral magnet are under development, which would extend the ease of use of this technique.