Exposure to space radiation can harm an individual by mutating genetic material, DNA, which exists in the body's cells. These changes may lead to increased health risks in long-term manned space missions. Measuring mutations in tissues in animals have been limited due to technical difficulties. However, by using specialized breeds of mice, where pieces of "target" DNA was engineered into the normal DNA sequence of every cell in the animal, Dr. Polly Chang is able to measure mutations in any tissue in mice that are exposed to space-like radiation doses. In the current research, Dr. Chang is determining the cellular effects and comparing mutation frequencies in the "target" gene in brain and spleen tissues at both short and long times after radiation exposure. She will also test whether the cytokine interleukin-1, a molecule that is naturally present in the body, will reduce the long-term consequences to radiation exposure.
Polly Chang, Ph.D.
Evaluations of risks involving alterations in the genome using whole animal systems are essential to missions in space. The lacZ transgenic-mouse model is the only system available to date for the assessment of alterations in the genome in every tissue of the animal. In this model system, every cell of the animal contains multiple copies of an integrated but inert target transgene.
Radiation-induced mutations can be measured and specific genetic alterations characterized using established protocols. Genetic alterations in tissues that are of high priority in NASAs Strategic Program Plans but are not accessible using conventional techniques, e.g., the central nervous system, can be evaluated using this model system. In addition to measuring short (one week) and long-term (up to 16 weeks after treatment) mutagenicity in the reporter transgene, concomitant evaluation of the clastogenic potential of particle radiation can also be done using the same experimental animals. Some of these evaluations include examining radiation responses in the hematopoietic system by enumerating micronuclei (MN) in peripheral blood, evaluating chromosomal damage in either circulating or bone-marrow lymphocytes by using fluorescence in situ hybridization (FISH) techniques, and induced gene expressions in tissues by using RT-PCR.
Specifically, our research aims for this project included the use of lacZ transgenic mice to characterize the dose- and time-dependent radiation-induced responses in lacZ transgenic mice after high-LET iron-particle beams generated at Brookhaven National Laboratory and low-LET proton irradiation at the Loma Linda University Medical Accelerator. We measured the initial effects and long-term residual consequences of radiation exposure in tissues that are of high priority to NSBRI and NASA, namely the brain (CNS), and compared these responses to another tissue such as the spleen that is known to be a highly proliferative tissue with stem cell populations. We hypothesized that the lacZ mutation frequency (MF) in individual tissues would increase as a function of dose for each tissue, that this response is LET-dependent but the level of induction of MF is dependent on the specific tissues analyzed.
Micronuclei (MN) in peripheral blood have been used extensively as a biomarker to evaluate radiation toxicity in the human population. We aimed to examine radiation responses in the hematopoietic system (immature polychromatic reticulocytes (RETs) and mature normochromatic erythrocytes (NCEs)) and lymphatic system in the same experimental animals. We expected that the level of genetic damage as well as the kinetics of removal of aberrant cells and the spectrum of chromosome aberrations was dose-dependent.
Variations in genetic background have been shown to impact an individuals sensitivity to radiation exposure. The tumor suppressor p53 gene function has been shown to be radiation-responsive and very important in the regulation of cell growth, proliferation, differentiation and apoptotic signaling pathways in many tissues. We cross-bred the C57 lacZ transgenic mice that are wild type for Tp53(Tp53+/+) with Tp53 nullizygous (Tp53 -/-) mice to establish breeding colonies of transgenic mice possessing the lacZ transgene and are either hemizygous or nullizygous for Tp53. These animals will be used to assess tissue-specific Tp53-dependent (or -independent) molecular and genetic mechanisms in radiation-induced damage resulting from exposure to particle beams in the energy range corresponding to space radiation.
Specifically, animals with different Tp53 genetic backgrounds will be exposed to a range of doses of either iron particles or proton radiation, and tissue-specific radiation responses using the same endpoints as mentioned in the previous section will be evaluated. Results from these studies will reveal the impact of variation of genetic background to an individuals sensitivity to radiation exposure of different LETs.
Polly Chang, Ph.D.
The space atmosphere is comprised of particle radiations in a wide spectrum of energies and charges, and radiation exposure poses a serious health hazard to humans in long-term, manned space explorations. We are using the plasmid-based lacZ transgenic mouse-mutation model system to examine the acute and long-term tissue-specific mutagenic responses of CNS and rapidly renewing organ systems after exposure to protons and HZE particles.
We have cross-bred the lacZ animals with Tp53 knock-out animals in order to examine the impact of Tp53 genetic backgrounds on radiation responses. Transgenic mouse-mutation model systems are accepted by regulatory agencies for toxicology studies. In addition to the aforementioned goals, we also aim to develop this mutation model system to be used to examine the effectiveness of targeted countermeasures such as shielding materials, known radioprotective pharmaceutical or cytokines in the reduction of tissue-specific mutation frequencies or genetic damage in vivo.
Analysis of genetic damage in different tissues in lacZ/Tp53 transgenics will allow us to test the efficacy of each countermeasure in the context of protection by reducing radiation damage and examine the impact of Tp53-dependent apoptosis in radiation protection. The results of this ground-based research project will provide information to characterize differences in tissue-specific mutagenic and clastogenic effects in vivo after particle radiation with different LETs, the acute and long-term consequences in tissue such as the brain, spleen and hematopoietic system, and the effectiveness of available countermeasures in mitigating the effects of radiation exposure in a variety of tissues in vivo.