Student Abstracts: Medical & Health Sciences at BNL

Evaluation of the in vivo and ex vivo binding of novel CB1 cannabinoid receptor radiotracers. ASHLEY MILLER (University of Connecticut, Storrs, CT 06269) DR. JOHN GATLEY (Brookhaven National Laboratory, Upton, NY 11973) .
The primary active ingredient of marijuana, 9-tetrahydrocannabinol, exerts its psychoactive effects by binding to cannabinoid CB1 receptors. These receptors are found throughout the brain with high concentrations in the hippocampus and cerebellum. The current study was conducted to evaluate the binding of a newly developed putative cannabinoid antagonist, AM630, and a classical cannabinoid 8-tetrahydrocannabinaol as potential PET and/or SPECT imaging agents for brain CB1 receptors. For both of these ligands in vivo and ex vivo studies in mice were conducted. AM630 showed good overall brain uptake (as measure by %IA/g) and a moderately rapid clearance from the brain with a half-clearance time of approximately 30 minutes. However, AM630 did not show selective binding to CB1 cannabinoid receptors. Ex vivo autoradiography supported the lack of selective binding seen in the in vivo study. Similar to AM630, 8-tetrahydrocanibol also failed to show selective binding to CB1 receptor rich brain areas. The 8-tetrahydrocanibol showed moderate overall brain uptake and relatively slow brain clearance as compared to AM630. Further studies were done with AM2233, a cannabinoid ligand with a similar structure as AM630. These studies were done to develop an ex vivo binding assay to quantify the displacement of [131I]AM2233 binding by other ligands in Swiss-Webster and CB1 receptor knockout mice. By developing this assay we hoped to determine the identity of an unknown binding site for AM2233 present in the hippocampus of CB1 knockout mice.

Lack of Potentiation of Boron Neutron Capture by Gadolinium Neutron Capture . NINA NAMI (Binghamton University, Binghamton, NY 13902) LOUIS A. PEÑA, PH.D. (Brookhaven National Laboratory, Upton, NY 11973) .
DNA damage is central to research in many fields, especially cancer research and toxicology. In this experiment we used normal endothelial cells (HAEC) and a tumor cell line (9L GS) to compare the atomic neutron capture reactions by boron-10, gadolinium-157, and by the combination of both. Cell death/DNA damage was measured by clonogenic survival assays and with single cell gel electrophoresis, also known as the comet assay. The clonogenic assay measures the cell's ability to divide and form colonies after exposure to irradiation. Whereas in the comet assay, electrophoresis causes broken DNA to move from the nucleus towards the anode forming an image resembling the tail of a comet, with the greater the extent of damage, the greater the tail. Our results indicate that the gadolinium-157 containing compound, Gd-DTPA, does not potentiate in the clonogenic assay or in the comet assay. The presence of Gd-DTPA in combination with the boron-10 containing, BPA, attenuated the biological effect of BPA in both HAEC and 9L cell types.

The Effect of Endogenous Serotonin on the in vivo binding of Radiotracers of the 5-HT Receptors in Mice. ADENIKE OLAODE (Monroe comunity College, Rochester, NY 14621) ANDREW GIFFORD (Brookhaven National Laboratory, Upton, NY 11973) .
Serotonin is a group of chemical messenger, which is also known as neurotransmitters. Different radiotracers examined in previous studies showed insensitivity to changes in endogenous Serotonin. The importance of this study is to reveal the relationship between radiotracer used in PET and endogenous Serotonin level in brain. Numerous studies have suggested that some neurotransmitters (i.e., dopamine) are able to have competition on certain radiotracers binding on the receptors. This phenomenon is a critical issue in PET that uses those radiotracers on the study of brain function. In order to more fully understand the role of neurotransmitters on radiotracer binding in vivo, the present project was designed to investigate the changes of binding of [3H]WAY 100635 and [3H]NMS in mice brains that had the depletion of Serotonin by 5,7-dihydroxytryptamine (5,7-DHT, i.c.v. 5mg/kg) and p-chlorophenylalanine (PCPA, i.p. 150mg/kg twice/day for 4 days). Our results indicated that the Serotonin level has decreased approximately more than 50% and 80% by 5, 7-DHT alone and 5, 7-DHT with PCPA respectively in both front cortex and hippocampus of mice brain. However, there were no significant changes of radiotracers binding in mice that had these decreases of Serotonin in brain. Thus, our results suggest that the depletion of Serotonin in brain has no significant effect on in vivo binding of radiotracers of both 5-HT1A and 5-HT2A receptors in mice.

Developing a Ribonuclease Protection Assay to Evaluate Peptide Nucleic Acids for use in Antisense Research. JORDAN PLIESKATT (The George Washington University, Washington, DC 20052) DR. ANDREW GIFFORD (Brookhaven National Laboratory, Upton, NY 11973) .
In an effort to continue to expand the ability to treat and detect different diseases, researchers have turned to antisense technology. Traditional drugs bind to the targeted protein and block its action. Antisense agents differ from traditional drugs by stopping the protein from ever being translated by binding to the transcribed mRNA. Such technology can be used both in therapeutic applications to stop destructive proteins and also in gene specific imaging. Peptide nucleic acids (PNA) are ideal antisense probes due to their high cellular uptake and resistance to cellular nucleases. In this study, mRNA for the glial fibrillary acidic protein was used as the antisense target because of its high content in glial cells and because the mRNA expression can be readily regulated both in vivo and in vitro. Various 15mer PNA GFAP antisense probes were created including 131I labeled versions, which were tested in their ability to bind to complimentary GFAP mRNA. A ribonuclease protection assay, employing radiolabeled peptide nucleic acids rather than conventional radiolabeled cDNA probes, was developed to test, isolate and visualize these hybridized PNA/RNA duplexes on a native polyacrylamide gel. In conclusion, this study confirmed that peptide nucleic acids can hybridize to mRNA and protect it from RNase digestion in vitro.

Microbeam Radiation Therapy Cancer Research. ALLISON SAWCHUK (University of Michigan, Ann Arbor, MI 48109) AVRAHAM DILMANIAN (Brookhaven National Laboratory, Upton, NY 11973) .
High-grade malignant gliomas currently represent 60% of all primary brain tumors, at an incidence of over 8000 cases per year. However, these highly malignant tumors of the delicate central nervous system are difficult to treat, and alarmingly, very few viable treatment modalities are currently available. X-ray radiotherapy, XRT, has been the leading treatment method, used in adjunction to chemotherapy and surgery. However, XRT offers little, if any hope for these highly malignant tumors of the central nervous system, such as a glioblastoma multiforme. Conventional x-ray therapy is a potentially palliative and incomplete treatment prescribed for these highly malignant cancers. It often causes more harm than good in its destruction of both mutagenic cancer tissues and the normal brain tissues. Therefore, the novel technique referred to as microbeam radiation therapy, MRT, provides medical researchers with a fresh perspective on these difficult cancers. Current research on rats and mice suggests that this treatment preferentially destroys malignant gliomas while leaving the healthy tissues relatively unharmed. This phenomenon may support the "endothelial replacement" hypothesis, a possible explanation of the biological mechanism motivating this effective tumor ablation. Microbeam radiation therapy, and its possible foundation, the "endothelial replacement" hypothesis, provides new hope in effective cancer research. Furthermore, this innovative radiotherapy modality, supported by the "endothelial replacement" hypothesis might provide the medical community with a viable treatment regimen to treat these highly malignant brain tumors.