Access Type

Open Access Dissertation

Date of Award

January 2014

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Biological Sciences

First Advisor

James D. Tucker

Abstract

Chapter 2:

Micronuclei have been used extensively in studies as an easily-evaluated indicator of DNA damage but little is known about their association with other types of damage such as nucleoplasmic bridges and nuclear buds. Radiation-induced clastogenic events were evaluated via the cytokinesis-block micronucleus assay in two normal human lymphoblastoid cell lines exposed to neutrons or gamma radiation. Micronuclei, nucleoplasmic bridges and nuclear buds were enumerated by recording the coincident presence of these endpoints within individual cells, and the associations among these three endpoints were evaluated for all treatment conditions. The common odds ratios for micronuclei and nucleoplasmic bridges were found to be significantly larger than unity, indicating that the presence of one or more micronuclei in a cell imposes a significant risk for having one or more nucleoplasmic bridges in that same cell, and vice versa. The strength of this association did not change significantly with radiation dose. Common odds ratios for association between micronuclei and buds, and between bridges and buds were also found to be significantly higher than unity. However, associations between micronuclei and buds could not be calculated for some treatments due to heterogeneity in the odds ratios, and hence may depend on radiation dose. This study provides evidence for how paired analyses among genetic endpoints in the cytokinesis-block micronucleus assay can provide information concerning abnormalities of cell division and possibly about structural chromosomal rearrangements induced by radiation.

Chapter 3:

Bystander effects have been observed repeatedly in mammalian cells following photon and alpha particle irradiation. However, few studies have been performed to investigate bystander effects arising from neutron irradiation. Here we asked whether neutrons also induce a bystander effect in two normal human lymphoblastoid cell lines. These cells were exposed to fast neutrons produced by targeting a near-monoenergetic 50.5 MeV proton beam at a Be target (17 MeV average neutron energy), and irradiated-cell conditioned media (ICCM) was transferred to unirradiated cells. The cytokinesis-block micronucleus assay was used to quantify genetic damage in radiation-naïve cells exposed to ICCM from cultures that received 0 (control), 0.5, 1, 1.5, 2, 3 or 4 Gy neutrons. Cells grown in ICCM from irradiated cells showed no significant increase in the frequencies of micronuclei or nucleoplasmic bridges compared to cells grown in ICCM from sham irradiated cells for either cell line. However, the neutron beam has a photon dose-contamination of 5%, which may modulate a neutron-induced bystander effect. To determine whether these low doses of contaminating photons can induce a bystander effect, cells were irradiated with cobalt-60 at doses equivalent to the percent contamination for each neutron dose. No significant increase in the frequencies of micronuclei or bridges was observed at these doses of photons for either cell line when cultured in ICCM. As expected, high doses of photons induced a clear bystander effect in both cell lines for micronuclei and bridges (p > 0.0001). These data indicate that neutrons do not induce a bystander effect in these cells. Finally, neutrons had a relative biological effectiveness of 2.0 ± 0.13 for micronuclei and 5.8 ± 2.9 for bridges compared to cobalt-60. These results may be relevant to radiation therapy with fast neutrons and for regulatory agencies setting standards for neutron radiation protection and safety.

Chapter 4:

The shape of the ionizing radiation response curve at very low doses has been the subject of considerable debate. Linear-no-threshold (LNT) models are widely used to estimate risks associated with low dose exposures. However, the low-dose hyper-radiosensitivity (HRS) phenomenon, in which cells are especially sensitive at low doses but then show increased radioresistance at higher doses, provides evidence of nonlinearity in the low dose region. HRS is more prominent in the G2 phase of the cell cycle than the G0/G1 or S phases. Here I provide the first cytogenetic evidence of low-dose hyper-radiosensitivity in human peripheral blood lymphocytes using structural chromosomal aberrations. Human peripheral blood lymphocytes from two normal healthy female donors were acutely exposed to cobalt-60 gamma rays in either G0 or G2 using closely-spaced doses ranging from 0-1.5 Gy. Structural chromosomal aberrations were enumerated and the slopes of the regression lines at low doses (0-0.4 Gy) were compared with doses of 0.5 Gy and above. HRS was clearly evident in both donors for cells irradiated in G2. No HRS was observed in cells irradiated in G0. The radiation effect per unit dose was 2.5-3.5 fold higher for doses ≤0.4 Gy than >0.5 Gy. These data provide the first cytogenetic evidence for the existence of HRS in human cells irradiated in G2 and suggest that LNT models may not always be optimal for making radiation risk assessments at low doses.

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