Dose-rate effects in radiation biology and radiation protection.

Quantification of biological effects (cancer, other diseases, and cell damage) associated with exposure to ionising radiation has been a major issue for the International Commission on Radiological Protection (ICRP) since its foundation in 1928. While there is a wealth of information on the effects on human health for whole-body doses above approximately 100 mGy, the effects associated with doses below 100 mGy are still being investigated and debated intensively. The current radiological protection approach, proposed by ICRP for workers and the public, is largely based on risks obtained from high-dose and high-dose-rate studies, such as the Japanese Life Span Study on atomic bomb survivors. The risk coefficients obtained from these studies can be reduced by the dose and dose-rate effectiveness factor (DDREF) to account for the assumed lower effectiveness of low-dose and low-dose-rate exposures. The 2007 ICRP Recommendations continue to propose a value of 2 for DDREF, while other international organisations suggest either application of different values or abandonment of the factor. This paper summarises the current status of discussions, and highlights issues that are relevant to reassessing the magnitude and application of DDREF.

Mapping the subcellular localization of Fe3O4@TiO2 nanoparticles by X-ray Fluorescence Microscopy.

The targeted delivery of Fe3O4@TiO2 nanoparticles to cancer cells is an important step in their development as nanomedicines. We have synthesized nanoparticles that can bind the Epidermal Growth Factor Receptor, a cell surface protein that is overexpressed in many epithelial type cancers. In order to study the subcellular distribution of these nanoparticles, we have utilized the sub-micron resolution of X-ray Fluorescence Microscopy to map the locationof Fe3O4@TiO2 NPs and other trace metal elements within HeLa cervical cancer cells. Here we demonstrate how the higher resolution of the newly installed Bionanoprobe at the Advanced Photon Source at Argonne National Laboratory can greatly improve our ability to distinguish intracellular nanoparticles and their spatial relationship with subcellular compartments.

X-ray fluorescence microscopy for investigation of archival tissues.

Several recent efforts in the radiation biology community worldwide have amassed records and archival tissues from animals exposed to different radionuclides and external beam irradiation. In most cases, these samples come from lifelong studies on large animal populations conducted in national laboratories and equivalent institutions throughout Europe, North America, and Japan. While many of these tissues were used for histopathological analyses, much more information may still be obtained from these samples. A new technique suitable for imaging of these tissues is x-ray fluorescence microscopy (XFM). Following development of third generation synchrotrons, XFM has emerged as an ideal technique for the study of metal content, speciation, and localization in cells, tissues, and organs. Here the authors review some of the recent XFM literature pertinent to tissue sample studies and present examples of XFM data obtained from tissue sections of beagle dog samples, which show that the quality of archival tissues allows XFM investigation.

Proliferating cell nuclear antigen (PCNA): ringmaster of the genome.

Proliferating cell nuclear antigen (PCNA) protein is one of the central molecules responsible for decisions of life and death of the cell. The PCNA gene is induced by p53, while PCNA protein interacts with p53-controlled proteins Gadd45, MyD118, CR6 and, most importantly, p21, in the process of deciding cell fate. If PCNA protein is present in abundance in the cell in the absence of p53, DNA replication occurs. On the other hand, if PCNA protein levels are high in the cell in the presence of p53, DNA repair takes place. If PCNA is rendered non-functional or is absent or present in low quantities in the cell, apoptosis occurs. The evolution from prokaryotes to eukaryotes involved a change of function of PCNA from a 'simple' sliding clamp protein of the DNA polymerase complex to an executive molecule controlling critical cellular decision pathways. The evolution of multicellular organisms led to the development of multicellular processes such as differentiation, senescence and apoptosis. PCNA, already an essential molecule in the life of single cellular organisms, then became a protein critical for the survival of multicellular organisms.

Modulation of calmodulin by UV and X-rays in primary human endothelial cell cultures.

PURPOSE: Previous studies by the present authors and others have shown that the expression of many genes is modulated by radiation. The purpose of this study is to identify additional genes that are affected by UV and X-radiation. Identification of specific genes affected by radiation may allow the determination of pathways important in radiation responses as well as an examination of transcriptional elements that are involved in the process. MATERIALS AND METHODS: A modified differential display approach coupled with sequencing was used to identify genes that are modulated in response to UV and ionizing radiation, and Northern blot analysis was used to confirm specific gene modulation. RESULTS: Treatment of human primary umbilical vein endothelial cells with UV radiation resulted in the differential expression of several genes. Sequencing of the bands revealed that one of these was calmodulin. There was a 30% reduction in accumulation of calmodulin-specific mRNA 1 h post UV exposure, and a 50% decrease 3 h after treatment. X-rays also repressed accumulation of calmodulin mRNA. Radiation exposure of HeLa cells also resulted in a decrease in expression of this gene. CONCLUSIONS: UV and ionizing radiations cause a decrease in accumulation of calmodulin transcripts in the first 1-3 h following exposure. Repression of calmodium mRNA levels may be one mechanism of stress-induced intracellular Ca2+ modulation.

p53 gene deletions in paraffin-preserved lymphoid tumors from irradiated mice.

Experiments were performed to measure deletions in the p53 gene in paraffin-embedded tissues (tumors and control) derived from mice exposed to gamma-rays or neutrons up to 28 years ago. Deletions in exons 1, 3, 4, 5, 6, 7 and 9 were monitored by PCR and Southern blotting techniques. The results of these experiments demonstrated p53 deletions in only 1/6 spontaneous tumors but in 5/6 gamma-ray-induced and 5/6 neutron-induced tumors. Exons deleted in tumors from gamma-ray exposed mice were similar to those deleted in tumors from neutron-exposed mice. They document differences in spectra of p53 deletions in comparing spontaneous radiation-induced tumors.

Identification of genes regulated by UV/salicylic acid.

PURPOSE: Previous work from the authors' group and others has demonstrated that some of the effects of UV irradiation on gene expression are modulated in response to the addition of salicylic acid to irradiated cells. The presumed effector molecule responsible for this modulation is NF-kappaB. In the experiments described here, differential-display RT-PCR was used to identify those cDNAs that are differentially modulated by UV radiation with and without the addition of salicylic acid. MATERIALS AND METHODS: Differential-display RT-PCR was used to identify differentially expressed genes. RESULTS: Eight such cDNAs are presented: lactate dehydrogenase (LDH-beta), nuclear encoded mitochondrial NADH ubiquinone reductase 24 kDa (NDUFV2), elongation initiation factor 4B (eIF4B), nuclear dots protein SP100, nuclear encoded mitochondrial ATPase inhibitor (IF1), a cDNA similar to a subunit of yeast CCAAT transcription factor HAP5, and two expressed sequence tags (AA187906 and AA513156). CONCLUSIONS: Sequences of four of these genes contained NF-kappaB DNA binding sites of the type that may attract transrepressor p55/p55 NF-kappaB homodimers. Down-regulation of these genes upon UV irradiation may contribute to increased cell survival via suppression of p53 independent apoptosis.

Detection of codon 12 point mutations of the K-ras gene from mouse lung adenocarcinoma by 'enriched' PCR.

PURPOSE: Recent studies have shown that chemical carcinogens induce a high frequency of point mutations in the K-ras oncogene from mouse lung tumours at codons 12, 13 and 61. These experiments were performed to identify K-ras mutations in tissues from control and radiation-exposed mice. MATERIALS AND METHODS: By modifying the technique of the 'enriched' polymerase chain reaction (PCR), it was possible to detect point mutations at codon 12 of the K-ras oncogene from 25-year-old paraffin-embedded normal lungs and lung adenocarcinomas from mice exposed to radiation. Together, a total of 120 lung tissues were screened for point mutations at codon 12 of the K-ras oncogene in this study. RESULTS: A significant increase in K-ras codon 12 point mutations was observed in the normal lungs from mice exposed to 24 once-weekly neutron irradiations (100%), compared with normal lungs from mice with sham-irradiation (50%) (p<0.05). Lung adenocarcinomas from mice receiving 24 once-weekly neutron irradiations also had a significantly higher frequency of K-ras codon 12 point mutations (100%) than the lung adenocarcinomas of mice receiving 24 or 60 once-weekly gamma-ray irradiations (50%), but the higher frequency was not significantly different from that in spontaneous lung adenocarcinomas from mice (75%; p > 0.05). The validity of the technique was confirmed by sequencing two of the mutants. In doing so, a K-ras 13(Asp) point mutation was observed. CONCLUSIONS: The data suggest that high-linear energy transfer (LET) neutron radiation was more effective than low-LET gamma-rays in inducing K-ras point mutations at codon 12 in the lungs of B6CF1 mice.

Rb and p53 gene deletions in lung adenocarcinomas from irradiated and control mice.

This study was conducted on mouse lung adenocarcinoma tissues that were treated with formalin and embedded in paraffin 25 years ago to investigate the large gene deletions of Rb and p53 in B6CF1 male mice. A total of 80 lung tissue samples from irradiated mice and 40 lung samples from nonirradiated controls were selected randomly and examined in the Rb portion of this study. The results showed a significantly (P < 0.05) higher percentage of Rb deletions in lung adenocarcinomas from mice exposed to 60 once-weekly gamma-ray doses than those from mice receiving 24 once-weekly gamma-ray doses at low doses and low dose rates; however, the percentage was not significantly different (P > 0.05) from that for spontaneous lung adenocarcinomas or lung adenocarcinomas from mice exposed to single-dose gamma irradiation at a similar total dose. Rb fragments 3 (71%) and 5 (67%), the parts of the gene that encoded the pocket binding region of Rb protein to adenovirus E1A and SV40 T-antigen, were the most frequently deleted fragments. Analysis of p53 gene deletion was carried out on normal lungs and lung adenocarcinomas that were initially found to bear Rb deletions. Exons 1, 4, 5, 6 and 9 were chosen to be analyzed. The data showed that 30 (97%) of 31 normal lungs and lung adenocarcinomas had p53 deletions. Exons 4 (83%) and 5 (90%) were the most frequently deleted among tested exons. Mice exposed to neutrons 60 times on a once-weekly schedule had a higher percentage of complete p53 deletions (5/8; 63%) than those exposed to gamma rays 60 times on a once-weekly schedule (2/8; 25%). We conclude that p53 deletions may be one of the major mutational events in the tumorigenesis of lung adenocarcinomas in the irradiated B6CI, mice.

The effects of vinblastine on the expression of human immunodeficiency virus type 1 long terminal repeat.

Previous work by our group has demonstrated induction of the HIV-LTR following exposure of cells to various DNA-damaging agents such as ultraviolet (UV) light, cisplatin, and doxorubicin. The current experiments were designed to determine the relative effects of the anti-mitotic drug vinblastine on expression of the HIV-LTR. Using human cervical carcinoma (HeLa) cells stably transfected with the chloramphenicol acetyl transferase (CAT) reporter transcriptionally driven by the HIV-LTR promoter, we demonstrated a 9-10-fold induction at 48-72 h following vinblastine treatment. Previous experiments had demonstrated repression of cisplatin or doxorubicin-mediated HIV induction by treatment with salicylic acid. The vinblastine induction also was repressed by salicylic acid treatment, but not by treatment with indomethacin, suggesting a role for the NF kappa B pathway in the inductive response. When UV exposure was coupled to the vinblastine treatment, there was no additive or synergistic effect evident, suggesting similar paths of induction between the two agents. Northern blots demonstrated that these agents were operating at the level of transcription and that salicylic acid inhibited vinblastine-mediated induction of HIV-LTR-CAT mRNA only if administered at the same time as vinblastine; addition of salicylic acid 2 h later had no effect on transcript accumulation. All combinations of treatments with vinblastine and/or salicylic acid markedly reduced cell survival.

HIV expression is induced in dying cells.

Using HeLa cells stably transfected with an HIV-LTR-CAT construct, we demonstrated a peak in CAT induction that occurs in viable (but not necessarily cell-division-competent) cells 24 h following exposure to some cell-killing agents. gamma rays were the only cell-killing agent which did not induce HIV transcription; this can be attributed to the fact that gamma-ray-induced apoptotic death requires functional p53, which is not present in HeLa cells. For all other agents, HIV-LTR induction was dose-dependent and correlated with the amount of cell killing that occurred in the culture. Doses which caused over 99% cell killing induced HIV-LTR transcription maximally, demonstrating that cells that will go on to die by 14 days are the cells expressing HIV-LTR-CAT.

Effect of passage number on cellular response to DNA-damaging agents: cell survival and gene expression.

The effect of different passage numbers on plating efficiency, doubling time, cell growth, and radiation sensitivity was assessed in Syrian hamster embryo (SHE) cells. Changes in gene expression after UV or gamma-ray irradiation at different passage numbers were also examined. The SHE cells were maintained in culture medium for up to 64 passages. Cells were exposed to 60Co gamma rays or 254-nm UV radiation. Differential display of cDNAs and Northern blots were used for the study of gene expression. With increasing passage number, SHE cells demonstrated decreased doubling time, increased plating efficiency, and a decreased yield in the number of cells per plate. Between passages 41 and 48 a 'crisis' period was evident during which time cell growth in high serum (20%) was no longer optimal, and serum concentrations were reduced (to 10%) to maintain cell growth. Sensitivity to ionizing radiation was no different between early- and intermediate-passage cells. However, after UV exposure at low passages (passage 3), confluent cells were more sensitive to the killing effects of UV than were log-phase cells. At intermediate passages (passages 43, 48), confluent cells were slightly more radioresistant than were log-phase cells. By passage 64, however, both confluent and log-phase cells showed similar patterns of UV sensitivity. Expression of gamma-actin, PCNA, and p53 transcripts did not change following UV exposure. p53 mRNA was induced following gamma-ray exposure of the intermediate (passage 45) epithelial cells. The observed differences in radiation sensitivity associated with increasing passage number may be influenced by radiation-induced gene expression. We are conducting experiments to identify these genes.

Mechanisms of radiation-induced gene responses.

While identifying genes differentially expressed in cells exposed to ultraviolet radiation, we identified a transcript with a 25-nucleotide region that is highly conserved among a variety of species, including Bacillus circulans, pumpkin, yeast, Drosophila, mouse, and man. In the 5' untranslated region of a gene, the sequence is predominantly in a +/+ orientation with respect to the coding DNA strand; while in the coding region and the 3' untranslated region, the sequence is most frequently in a -/+ orientation. The element is found in many different genes that have diverse functions. Gel mobility shift assays demonstrated the presence of a protein in HeLa cell extracts that binds to the sense and antisense single-stranded consensus oligomers, as well as to double-stranded oligonucleotide. When double-stranded oligomer was used, the size shift demonstrated an additional protein-oligomer complex larger than the one bound to either sense or antisense single-stranded consensus oligomers alone. This element may bind to protein(s) that maintain DNA in a single-stranded orientation for transcription, or be important in the transcription-coupled DNA repair process.

Regulation of thymus PCNA expression is altered in radiation-sensitive wasted mice.

Mice bearing the autosomal recessive mutation 'wasted' (wst/wst) express a disease syndrome characterized by neurologic dysfunction, immunodeficiency, and increased sensitivity to the killing effects of ionizing radiation relative to normal littermates (wst/-) and to parental control mice (BCF1, BALB/c, and C57BL/6). Many of these abnormalities, evident as early as 21 days of age, have been localized to thymic tissues and T-lymphocyte populations. Comparison of two-dimensional gel electrophoresis patterns of proteins from wst/wst and control mouse thymus revealed that an acidic protein with a molecular mass of approximately 30 kDa was consistently expressed at lower levels in wasted mice than in controls. Microsequencing of this protein revealed a sequence of 19 N-terminal amino acids identical to the sequence of murine proliferating cell nuclear antigen (PCNA). Northern blot analyses of PCNA expression in thymus and spleen demonstrated lower accumulation of PCNA-specific transcripts in wasted mice compared with that in controls. Because PCNA expression is associated with cell cycle progression, the percentages of thymic and splenic cells in each stage of the cell cycle were examined; there were no differences in the cell stage distribution of lymphocytes freshly isolated from wasted mice compared with littermate or parental controls. After activation with concanavalin A, however, splenocytes from wst/wst mice showed a lower percentage of cells in S phase compared with that in controls. Southern blots with PCNA probes showed that the PCNA loci from the wasted mice and their normal littermates have the same restriction maps. While differences in polymerase chain reaction (PCR) priming were obtained, these could be attributed to strain-specific differences in mouse PCNA pseudogenes. These results suggest the presence of an alteration in the pathway leading to PCNA expression in radiation-sensitive tissues of wasted mice.

The effects of 5-fluorouracil and doxorubicin on expression of human immunodeficiency virus type 1 long terminal repeat.

Previous work by many groups has documented induction of the human immunodeficiency virus (HIV) long terminal repeat (LTR) following exposure of cells to ultraviolet light and other DNA damaging agents. Our experiments set out to determine the relative activation or repression of the HIV-LTR in response to two classes of chemotherapeutic agents: Doxorubicin is a DNA damage-inducing agent, and 5-fluorouracil has an antimetabolic mode of action. Using HeLa cells stably transfected with a construct in which HIV-LTR drives expression of the chloramphenicol acetyl transferase reporter gene, we demonstrated an up to ten-fold induction following doxorubicin treatment at 24 h post-treatment. This induction was repressed by treatment with salicylic acid, suggesting a role for prostaglandin/cyclo-oxygenase pathways and/or NF-kappa B in the inductive response. Induction by 5-fluorouracil, in contrast, was more modest (two-fold at most) though it was consistently elevated over controls.

The effects of cisplatin and methotrexate on the expression of human immunodeficiency virus type 1 long terminal repeat.

Previous work by many groups has documented the induction of HIV-LTR (human immunodeficiency virus-long terminal repeat) following exposure of cells or whole animals to ultraviolet (UV) light and other DNA damaging agents. In these experiments we set out to determine whether exposure to the cancer chemotherapeutic agents methotrexate and cisplatin had any effect on the expression of the HIV-LTR. Using HeLa cells stably transfected with a construct in which HIV-LTR drives the expression of the reporter gene chloramphenicol acetyl transferase (CAT), we demonstrated induction of HIV-LTR 24-48 h following exposure to 50 microM cisplatin. When UV exposure (10 Jm-2) was coupled with cisplatin (50 microM) treatment (which also causes DNA damage), HIV-LTR induction was additive relative to either treatment alone. Methotrexate, which depletes the medium of tetrahydrofolate and does not induce DNA damage, induced HIV-LTR at later (6-7 days) time points than cisplatin or UV treatments. When methotrexate (128 microM) and UV (10 Jm-2) treatments were combined, the agents were synergistic with regard to HIV induction. For both drugs, though, induction was not due to generalized transcriptional activation since both cisplatin and methotrexate induced a repression of total transcription as measured in nuclear run-on assays.