Cristian Dan

Comparative Analysis of Cell Killing and Autosomal Mutation in Mouse Kidney Epithelium Exposed to 1 GeV/nucleon Iron Ions In Vitro or In Situ

Abstract Astronauts receive exposures to high-energy heavy ions from galactic cosmic radiation. Although high-energy heavy ions are mutagenic and carcinogenic, their mutagenic potency in epithelial cells, where most human cancers develop, is poorly understood. Mutations are a critical component of human cancer, and mutations involving autosomal loci predominate. This study addresses the cytotoxic and mutagenic effects of 1 GeV/nucleon iron ions in mouse kidney epithelium. Mutant fractions were measured for an endogenous autosomal locus (Aprt) that detects all types of mutagenic events contributing to human cancer. Results for kidneys irradiated in situ are compared with results for kidney cells from the same strain exposed in vitro. The results demonstrate dose-dependent cell killing in vitro and for cells explanted 3–4 months postirradiation in situ, but in situ exposures were less likely to result in cell death than in vitro exposures. Prolonged incubation in situ (8–9 months) further attenuated cell killing at lower doses. Iron ions were mutagenic to cells in vitro and for irradiated kidneys. No sparing was seen for mutant frequency with a long incubation period in situ. In addition, the degree of mutation induction (relative increase over background) was similar for cells exposed in vitro or in situ. We speculate that the latent effects of iron-ion exposure contribute to the maintenance of an elevated mutation burden in an epithelial tissue.

Comparison of Autosomal Mutations in Mouse Kidney Epithelial Cells Exposed to Iron Ions In Situ or in Culture

Abstract Exposure to accelerated iron ions represents a significant health risk in the deep space environment because it induces mutations that can cause cancer. A mutation assay was used to determine the full spectrum of autosomal mutations induced by exposure to 2 Gy of 1 GeV/nucleon iron ions in intact kidney epithelium, and the results were compared with mutations induced in cells of a kidney epithelial cell line exposed in vitro. A molecular analysis for loss of heterozygosity (LOH) for polymorphic loci on chromosome 8, which harbors Aprt, demonstrated iron-ion induction of mitotic recombination, interstitial deletion, and discontinuous LOH events. Iron-ion-induced deletions were detected more readily with the in vitro assay, whereas discontinuous LOH was detected more readily in the intact kidney. The specific induction of discontinuous LOH in vivo suggests that this mutation pattern may serve as an indicator of genomic instability. Interestingly, the frequency of small intragenic events increased as a function of time after exposure, suggesting non-targeted effects. In total, the results demonstrate that 1 GeV/nucleon iron ions can elicit a variety of autosomal mutations and that the cellular microenvironment and the sampling time after exposure can influence the distribution of these mutations in epithelial cell populations.

Comparative Analysis of Cell Killing and Autosomal Mutation in Mouse Kidney Epithelium Exposed to 1 GeV Protons In Vitro or In Vivo

Human exposure to high-energy protons occurs in space flight scenarios or, where necessary, during radiotherapy for cancer or benign conditions. However, few studies have assessed the mutagenic effectiveness of high-energy protons, which may contribute to cancer risk. Mutations cause cancer and most cancer-associated mutations occur at autosomal loci. This study addresses the cytotoxic and mutagenic effects of 1 GeV protons in mouse kidney epithelium. Mutant fractions were measured for an endogenous autosomal locus (Aprt) that detects all types of mutagenic events. Results for kidneys irradiated in vivo are compared with the results for kidney cells from the same strain exposed in vitro. The results demonstrate dose-dependent cell killing in vitro and for cells explanted 3–4 months postirradiation in vivo. Incubation in vivo for longer periods (8–9 months) further attenuates proton-induced cell killing. Protons are mutagenic to cells in vitro and for in vivo irradiated kidneys. The dose-response for Aprt mutation is curvilinear after in vitro or in vivo exposure, bending upward at the higher doses. While the absolute mutant fractions are higher in vivo, the fold-increase over background is similar for both in vitro and in situ exposures. Results are also presented for a limited study on the effect of dose fractionation on the induction of Aprt mutations in kidney epithelial cells. Dose-fractionation reduces the fraction of proton-induced Aprt mutants in vitro and in vivo and also results in less cell killing. Taken together, the mutation burden in the epithelium is slightly reduced by dose-fractionation. Autosomal mutations accumulated during clinical exposure to high-energy protons may contribute to the risk of treatment-associated neoplasms, thereby highlighting the need for rigorous treatment planning to reduce the dose to normal tissues. For low dose exposures that occur during most space flight scenarios, the mutagenic effects of protons appear to be modest.

Marked aneuploidy and loss of multiple chromosomes are common in autosomal mutants isolated from normal mouse kidney epithelium

Marked aneuploidy and loss of multiple chromosomes are hallmarks of cancer, but whether these events are only present in malignant cells is not known. In prior work, we showed that approximately half of spontaneous autosomal mutants isolated directly from normal kidney epithelium arose from loss of a marker chromosome 8 containing the wild type Aprt gene. Chromosome loss was detected by loss of heterozygosity (LOH) for all chromosome 8 polymorphic loci examined. To determine whether loss of chromosome 8 reflected a larger mitotic event, LOH was examined for polymorphic loci on 11 nonselected chromosomes in Aprt mutants that lost the selected chromosome 8 homologue. LOH events were detected for one or more nonselected chromosomes in 38% of these mutants. The additional LOH events also reflected apparent chromosome loss based on the molecular analysis. Metaphase spreads from mutants that lost chromosome 8 were markedly aneuploid, and chromosome painting revealed reduced levels for any chromosome shown to be lost with the LOH analysis. In contrast, LOH on nonselected chromosomes was infrequent in Aprt mutants exhibiting intragenic events or mitotic recombination for chromosome 8, and marked aneuploidy was absent. These observations suggest that the mechanism leading to chromosome loss in somatic mammalian cells is often not a simple nondisjunction event and instead could result from a single catastrophic event. They also suggest that cells with characteristics of malignancy are present in normal appearing tissue.

Autosomal mutations in mouse kidney epithelial cells exposed to high-energy protons in vivo or in culture.

Proton exposure induces mutations and cancer, which are presumably linked. Because protons are abundant in the space environment and significant uncertainties exist for the effects of space travel on human health, the purpose of this study was to identify the types of mutations induced by exposure of mammalian cells to 4–5 Gy of 1 GeV protons. We used an assay that selects for mutations affecting the chromosome 8-encoded Aprt locus in mouse kidney cells and selected mutants after proton exposure both in vivo and in cell culture. A loss of heterozygosity (LOH) assay for DNA preparations from the in vivo-derived kidney mutants revealed that protons readily induced large mutational events. Fluorescent in situ hybridization painting for chromosome 8 showed that >70% of proton-induced LOH patterns resembling mitotic recombination were in fact the result of nonreciprocal chromosome translocations, thereby demonstrating an important role for DNA double-strand breaks in proton mutagenesis. Large interstitial deletions, which also require the formation and resolution of double-strand breaks, were significantly induced in the cell culture environment (14% of all mutants), but to a lesser extend in vivo (2% of all mutants) suggesting that the resolution of proton-induced double-strand breaks can differ between the intact tissue and cell culture microenvironments. In total, the results demonstrate that double-strand break formation is a primary determinant for proton mutagenesis in epithelial cell types and suggest that resultant LOH for significant genomic regions play a critical role in proton-induced cancers.

Accelerated 48Ti Ions Induce Autosomal Mutations in Mouse Kidney Epithelium at Low Dose and Fluence

Exposure to high-energy charged particles (HZE ions) at low fluence could significantly affect astronaut health after prolonged missions in deep space by inducing mutations and related cancers. We tested the hypothesis that the mutagenic effects of HZE ions could be detected at low fluence in a mouse model that detects autosomal mutations in vivo. Aprt heterozygous mice were exposed to 0.2, 0.4 and 1.4 Gy of densely ionizing 48Ti ions (1 GeV/amu, LET = 107 keV/μm). We observed a dose-dependent increase in the Aprt mutant fraction in kidney epithelium at the two lowest doses (an average of 1 or 2 particles/cell nucleus) that plateaued at the highest dose (7 particles/cell nucleus). Mutant cells were expanded to determine mutation spectra and translocations affecting chromosome 8, which encodes Aprt. A PCR-based analysis for loss of heterozygosity (LOH) events on chromosome 8 demonstrated a significant shift in the mutational spectrum from Ti ion exposure, even at low fluence, by revealing “radiation signature” mutations in mutant cells from exposed mice. Likewise, a cytogenetic assay for nonreciprocal chromosome 8 translocations showed an effect of exposure. A genome-wide LOH assay for events affecting nonselected chromosomes also showed an effect of exposure even for the lowest dose tested. Considered in their entirety, these results show that accelerated 48Ti ions induce large mutations affecting one or more chromosomes at low dose and fluence.

The frequency of CC to TT tandem mutations in mismatch repair-deficient cells is increased in a cytosine run

Mononucleotide runs are hot spots for frameshift mutations in mismatch repair (MMR)-deficient cells. However, a role for mononucleotide runs in the formation of base pair substitutions has not been tested. Previously, we demonstrated that ultraviolet radiation C (UVC)- or reactive oxygen species-induced CC to TT tandem mutations are markedly enhanced in MMR-deficient cells. The target for the mutational analysis was two cytosines in a run of five cytosines (5C) within mouse Aprt. Because mutation from C to T for either or both of the two critical cytosines created a codon yielding a functional Aprt protein, this assay allowed both single and tandem substitutions to be quantified and the relative ratios compared. To determine if the cytosine run increased the frequency of single and/or tandem base pair substitutions, alternative constructs were created in which the cytosine run was disrupted by flanking the target cytosines with either thymines (2Cpyr) or adenines (2Cpur). Disruption of the cytosine run dramatically decreased the frequency of UVC-induced tandem mutations in the 2Cpyr and 2Cpur constructs, as compared with the 5C construct. Moreover, CC to TT tandem mutations occurred spontaneously or were induced by oxidative stress only within the 5C construct. These results demonstrate that CC to TT tandem mutations in MMR-deficient cells form more readily in a homocytosine run than in a sequence limited to two cytosines.

Simulated space radiation-induced mutants in the mouse kidney display widespread genomic change

Exposure to a small number of high-energy heavy charged particles (HZE ions), as found in the deep space environment, could significantly affect astronaut health following prolonged periods of space travel if these ions induce mutations and related cancers. In this study, we used an in vivo mutagenesis assay to define the mutagenic effects of accelerated 56Fe ions (1 GeV/amu, 151 keV/μm) in the mouse kidney epithelium exposed to doses ranging from 0.25 to 2.0 Gy. These doses represent fluences ranging from 1 to 8 particle traversals per cell nucleus. The Aprt locus, located on chromosome 8, was used to select induced and spontaneous mutants. To fully define the mutagenic effects, we used multiple endpoints including mutant frequencies, mutation spectrum for chromosome 8, translocations involving chromosome 8, and mutations affecting non-selected chromosomes. The results demonstrate mutagenic effects that often affect multiple chromosomes for all Fe ion doses tested. For comparison with the most abundant sparsely ionizing particle found in space, we also examined the mutagenic effects of high-energy protons (1 GeV, 0.24 keV/μm) at 0.5 and 1.0 Gy. Similar doses of protons were not as mutagenic as Fe ions for many assays, though genomic effects were detected in Aprt mutants at these doses. Considered as a whole, the data demonstrate that Fe ions are highly mutagenic at the low doses and fluences of relevance to human spaceflight, and that cells with considerable genomic mutations are readily induced by these exposures and persist in the kidney epithelium. The level of genomic change produced by low fluence exposure to heavy ions is reminiscent of the extensive rearrangements seen in tumor genomes suggesting a potential initiation step in radiation carcinogenesis.

Autosomal Mutants of Proton-Exposed Kidney Cells Display Frequent Loss of Heterozygosity on Nonselected Chromosomes

High-energy protons found in the space environment can induce mutations and cancer, which are inextricably linked. We hypothesized that some mutants isolated from proton-exposed kidneys arose through a genome-wide incident that causes loss of heterozygosity (LOH)-generating mutations on multiple chromosomes (termed here genomic LOH). To test this hypothesis, we examined 11 pairs of nonselected chromosomes for LOH events in mutant cells isolated from the kidneys of mice exposed to 4 or 5 Gy of 1 GeV protons. The mutant kidney cells were selected for loss of expression of the chromosome 8-encoded Aprt gene. Genomic LOH events were also assessed in Aprt mutants isolated from isogenic cultured kidney epithelial cells exposed to 5 Gy of protons in vitro. Control groups were spontaneous Aprt mutants and clones isolated without selection from the proton-exposed kidneys or cultures. The in vivo results showed significant increases in genomic LOH events in the Aprt mutants from proton-exposed kidneys when compared with spontaneous Aprt mutants and when compared with nonmutant (i.e., nonselected) clones from the proton-exposed kidneys. A bias for LOH events affecting chromosome 14 was observed in the proton-induced Aprt mutants, though LOH for this chromosome did not confer increased radiation resistance. Genomic LOH events were observed in Aprt mutants isolated from proton-exposed cultured kidney cells; however the incidence was fivefold lower than in Aprt mutants isolated from exposed intact kidneys, suggesting a more permissive environment in the intact organ and/or the evolution of kidney clones prior to their isolation from the tissue. We conclude that proton exposure creates a subset of viable cells with LOH events on multiple chromosomes, that these cells form and persist in vivo, and that they can be isolated from an intact tissue by selection for a mutation on a single chromosome.

Genotoxicity of charged particles of importance in space flight using murine kidney epithelial cells

Ionizing radiation presents significant challenges for human space flight including an increased cancer risk. High-energy heavy ions in the galactic cosmic radiation can produce qualitative and quantitative differences in biological effects when compared with sparsely ionizing radiations. Mutations are induced by charged particle exposure and are integral to the formation and/or progression of human cancers. Most cancer-associated mutations occur on autosomal chromosomes, and most solid cancers occur in epithelial tissues. Here, a combined in vitro/in vivo approach was used to evaluate cell killing and the induction of mutations at a model autosomal locus, Aprt, in mouse kidney epithelium. For in vitro exposures, Aprt heterozygous kidney cells (clones 1a, 4a or 6a) were used from C57BL/6×DBA/2 mice. Additional experiments were performed using whole body irradiation of mice with the same genotype. Both males and females were irradiated in approximately equal numbers. Irradiations were performed at the NASA Space Radiation Laboratories at Brookhaven National Laboratory. For in vitro studies, cells from primary kidney clones were irradiated and seeded at limiting dilution immediately post-irradiation to determine the toxicity of the treatment. The irradiated kidney cells were also seeded in mutation assays within 1 week post-irradiation to determine the Aprt mutant fraction at the earliest time post-exposure. This work was complemented by studies wherein mice were exposed to the same ions with kidneys harvested several months post-irradiation to determine the residual toxicity and the Aprt mutant fraction. Our previous studies focused on sparsely ionizing 1 GeV protons (LET = 0.24 keV/µm) and densely ionizing 1 GeV/amu Fe ions (LET = 151 keV/µm). Our most recent studies have included work with Si ions (240 MeV/amu for in vitro studies, LET = 78 keV/µm; 263 MeV/amu initial energy for in vivo studies to achieve 78 keV/µm near the midline of the animal) and O ions (250 MeV/amu in vitro studies only, LET = 25 keV/µm). Toxicity for the cultured kidney cells in vitro follows this pattern: Fe > Si > O > protons when the results are expressed per unit dose. D0 values were 92 cGy for Fe ions, 103 cGy for Si ions, 192 cGy for O ions and 340 cGy for protons. With regard to the induction of Aprt mutations, Fe ions were more mutagenic than protons. Si ions were also quite mutagenic with evidence for a linear dose–response for Aprt mutations in kidney cells exposed in vitro or in kidneys harvested from mice irradiated several months earlier. These results are consistent with the linear dose–response data obtained previously for Aprt mutation induction following Fe ion exposure in vitro or in vivo, but the results for Si ions differ from the curvilinear dose–response data we recently published following similar exposures to energetic protons [ 1, 2]. Our most recent studies examined the molecular characteristics of Si ion-induced Aprt mutants following in vitro exposure. A dose of 160 cGy was used to collect 58 Aprt kidney cell mutants. Mutational events were classified as follows based on PCR-based analyses of polymorphic markers along mouse chromosome 8: intragenic events, apparent mitotic recombination, interstitial deletions of Aprt only, multilocus deletions, discontinuous loss of heterozygosity or whole chromosome loss. The results for this group of mutants will be compared against our previous studies on Aprt mutants arising after exposure to sparsely ionizing 1 GeV protons or densely ionizing 1 GeV/amu Fe ions. Additional studies are ongoing to define mutational spectra following Si ion exposure to kidney epithelium in vivo. Clinical Trial Registration number: not applicable.