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C Mothersill

Publications and source records attributed to C Mothersill.

At least 19 recordsLinked to original sources

Communication of radiation-induced stress or bystander signals between fish in vivo.

We report data in this paper suggesting that fish irradiated to 0.5 Gy total body dose can release factors into the water that signal other unexposed fish and cause induction of bystander effects expressed as increased cell death in a reporter system. Radiation-induced bystander effects, resulting in the appearance of radiation damage or induction of typical radiation responses in unirradiated cells and tissues are now an established consequence of exposure to low doses of ionizing radiation, however little work has been done in vivo or in species other than humans or mice. In these experiments rainbow trout were irradiated and then paired with unirradiated fish for two hours. Additionally, unirradiated fish were placed in water which had previously been used to hold irradiated fish for 2 h. Sham-irradiated fish and absolute control fish were also examined all using blind protocols. Following a two h incubation period, at these various exposure regimes, the fish were killed by a blow to the head and dissected. Five organs were removed from each fish and tissue explants were cultured using an established technique. After 2 days, the culture medium was harvested and used in a reporter assay to determine whether a bystander effect had been induced. The explants were cultured on in Clonetics growth medium for a further 14 days then fixed for assay of radiation response proteins. The responses varied according to the cell type in the original explants, with the gill and fin showing the most pronounced response. The results suggest that communication signals leading to a typical radiation response can be passed between fish and seem to involve secretion of a chemical messenger into the water.

Animals↗

Effects of hTERT on metal ion-induced genomic instability.

There is currently a great interest in delayed chromosomal and other damaging effects of low-dose exposure to a variety of pollutants which appear collectively to act through induction of stress-response pathways related to oxidative stress and ageing. These have been studied mostly in the radiation field but evidence is accumulating that the mechanisms can also be triggered by chemicals, especially heavy metals. Humans are exposed to metals, including chromium (Cr) (VI) and vanadium (V) (V), from the environment, industry and surgical implants. Thus, the impact of low-dose stress responses may be larger than expected from individual toxicity projections. In this study, a short (24 h) exposure of human fibroblasts to low doses of Cr (VI) and V (V) caused both acute chromosome damage and genomic instability in the progeny of exposed cells for at least 30 days after exposure. Acutely, Cr (VI) caused chromatid breaks without aneuploidy while V (V) caused aneuploidy without chromatid breaks. The longer-term genomic instability was similar but depended on hTERT positivity. In telomerase-negative hTERT- cells, Cr (VI) and V (V) caused a long lasting and transmissible induction of dicentric chromosomes, nucleoplasmic bridges, micronuclei and aneuploidy. There was also a long term and transmissible reduction of clonogenic survival, with an increased beta-galactosidase staining and apoptosis. This instability was not present in telomerase-positive hTERT+ cells. In contrast, in hTERT+ cells the metals caused a persistent induction of tetraploidy, which was not noted in hTERT- cells. The growth and survival of both metal-exposed hTERT+ and hTERT- cells differed if they were cultured at subconfluent levels or plated out as colonies. Genomic instability is considered to be a driving force towards cancer. This study suggests that the type of genomic instability in human cells may depend critically on whether they are telomerase-positive or -negative and that their sensitivities to metals could depend on whether they are clustered or diffuse.

Apoptosis↗

The effect of melanin on the bystander effect in human keratinocytes.

The influence of melanin on radiation-induced bystander effects has been studied. Melanin is known to be a natural substance with proved radioprotective properties in different organisms and cell lines. It is non-toxic and is effective against acute and chronic irradiation. The lower the radiation dose, the higher the relative impact of melanin protection. In this study influence of melanin on human keratinocytes (HPV-G cells) has been studied using the colony-forming assay. We have shown that bystander donor medium from 0.5 Gy irradiated cells when transferred to unirradiated cells, caused almost the same effect as direct irradiation. Melanin increased the colony-forming ability of bystander recipient cells when it was added into culture medium before irradiation. The effect of melanin added after irradiation was to produce less protection in both the directly irradiated and bystander medium treated groups. The absorption spectrum of the filtered medium is identical to one of the intact culture medium showing that melanin was not present in filtered medium. Thus, it cannot protect recipient cells but reduces the amount of the bystander effect. It is concluded that melanin added before irradiation effectively decreased the radiation dose. The reduction of the impact of the bystander signal on recipient cells when melanin was added to the donor medium after harvest but before filtration, may mean that the bystander signal has a physical component as melanin can absorb all types of physical energy.

Cell Communication↗

Increased radiosensitivity in cells of two human cell lines treated with bystander medium from irradiated repair-deficient cells.

Radiation-induced bystander factors have been shown to be more toxic if they are from medium harvested from irradiated repair-deficient cells. The aim of this study was to test the hypothesis that the radiosensitivity of repair-proficient cells can be increased by exposing them to medium-borne factors harvested from sensitive cells and vice versa. Cells from a mismatch repair (MMR)-deficient cell line (Raji 10) with a sensitive response to radiation or the wild-type parent cell line were irradiated to 0.5 Gy gamma rays and then monitored for growth rate in their own medium or in the alternative conditioned medium. In other experiments, cells or conditioned medium were added to reporter cells (HPV-G, which are relatively sensitive keratinocytes, or highly radioresistant HT29 cells). The subsequent responses of the two cell lines to a 0.5-Gy dose of (60)Co gamma rays were measured. The results show that prior exposure of resistant cells to medium from irradiated sensitive cells reduced the clonogenic survival of the subsequently irradiated resistant cells. The reverse is also true. Measurement of the apoptosis index and BCL2 expression confirmed that the harvested medium was capable of modulating apoptosis after irradiation. This may have important applications in tumor therapy and also in the understanding of mechanisms involved in induction of adaptive responses.

Apoptosis↗

The involvement of calcium and MAP kinase signaling pathways in the production of radiation-induced bystander effects.

Much evidence now exists regarding radiation-induced bystander effects, but the mechanisms involved in the transduction of the signal are still unclear. The mitogen-activated protein kinase (MAPK) pathways have been linked to growth factor-mediated regulation of cellular events such as proliferation, senescence, differentiation and apoptosis. Activation of multiple MAPK pathways such as the ERK, JNK and p38 pathways have been shown to occur after exposure of cells to radiation and a variety of other toxic stresses. Previous studies have shown oxidative stress and calcium signaling to be important in radiation-induced bystander effects. The aim of the present study was to investigate MAPK signaling pathways in bystander cells exposed to irradiated cell conditioned medium (ICCM) and the role of oxidative metabolism and calcium signaling in the induction of bystander responses. Human keratinocytes (HPV-G cell line) were irradiated (0.005-5 Gy) using a cobalt-60 teletherapy unit. The medium was harvested 1 h postirradiation and transferred to recipient HPV-G cells. Phosphorylated forms of p38, JNK and ERK were studied by immunofluorescence 30 min-24 h after exposure to ICCM. Inhibitors of the ERK pathway (PD98059 and U0126), the JNK pathway (SP600125), and the p38 pathway (SB203580) were used to investigate whether bystander-induced cell death could be blocked. Cells were also incubated with ICCM in the presence of superoxide dismutase, catalase, EGTA, verapamil, nifedipine and thapsigargin to investigate whether bystander effects could be inhibited because of the known effects on calcium homeostasis. Activated forms of JNK and ERK proteins were observed after exposure to ICCM. Inhibition of the ERK pathway appeared to increase bystander-induced apoptosis, while inhibition of the JNK pathway appeared to decrease apoptosis. In addition, reactive oxygen species, such as superoxide and hydrogen peroxide, and calcium signaling were found to be important modulators of bystander responses. Further investigations of these signaling pathways may aid in the identification of novel therapeutic targets.

Bystander Effect↗

A test battery approach for the ecotoxicological evaluation of estuarine sediments.

The purpose of this study was to evaluate the overall sensitivity and applicability of a number of bioassays representing multiple trophic levels, for the preliminary ecotoxicological screening (Tier I) of estuarine sediments. Chemical analyses were conducted on sediments from all sampling sites to assist in interpreting results. As sediment is an inherently complex, heterogeneous geological matrix, the toxicity associated with different exposure routes (solid, porewater and elutriate phases) was also assessed. A stimulatory response was detected following exposure of some sediment phases to both the Microtox and algal bioassays. Of the bioassays and endpoints employed in this study, the algal test was the most responsive to both elutriates and porewaters. Salinity controls, which corresponded to the salinity of the neat porewater samples, were found to have significant effects on the growth of the algae. To our knowledge, this is the first report of the inclusion of a salinity control in algal toxicity tests, the results of which emphasise the importance of incorporating appropriate controls in experimental design. While differential responses were observed, the site characterised as the most polluted on the basis of chemical analysis was consistently ranked the most toxic with all test species and all test phases. In terms of identifying appropriate Tier I screening tests for sediments, this study demonstrated both the Microtox and algal bioassays to be more sensitive than the bacterial enzyme assays and the invertebrate lethality assay employing Artemia salina. The findings of this study highlight that salinity effects and geophysical properties need to be taken into account when interpreting the results of the bioassays.

Aliivibrio fischeri↗

Assessing the potential of fish cell lines as tools for the cytotoxicity testing of estuarine sediment aqueous elutriates.

In the present study, we assess the potential of fish cell lines (CHSE, EPC and RTG-2) to be used as screening tools for the ecotoxicological assessment of estuarine sediments. The processing of sediment to a form suitable for in vitro exposure is an inherent problem when using cell cultures. The approach employed in this study was to prepare aqueous elutriate extracts from whole sediments, which were subsequently used to reconstitute powdered media. This procedure allowed the exposure of cell cultures to concentrations of up to and including 100% of the original aqueous sample. Cytotoxicity was assessed using multiple endpoint measurements. Cell viability was quantified using the neutral red and alamar blue colorimetric assays, which specifically assess lysosomal and mitochondrial function, respectively. In addition, the total protein content of the cells was measured using the coomassie blue assay. Initial tests were conducted to ensure that any resultant cytotoxicity was due to sample contaminants and not osmotic stress. In addition, elutriate samples were spiked with a model toxicant to verify the ability of the cell lines to detect and respond to bioavailable contaminants. Chemical analyses were conducted on sediments from all sampling sites to assist in interpreting any observed cytotoxicity. A differential response was observed for the cytotoxicity assays following exposure treatments, which emphasises the importance of employing multiple endpoints for the determination of toxicity. Of the three cell lines utilised in this study, RTG-2 cells were the most suitable for the testing of estuarine aqueous elutriate samples on the basis of tolerance to osmolality effects. Slight toxicity was observed following exposure to the aqueous elutriates tested in this study using RTG-2 cells and the alamar blue assay. In order to fully evaluate the overall sensitivity of this cell line, further research is warranted using an extensive range of test sites incorporating more polluted sediments.

Animal Testing Alternatives↗

Elevated G2 chromosomal radiosensitivity in Irish breast cancer patients: a comparison with other studies.

Previous studies have shown that a significant proportion of breast cancer patients exhibit elevated G2 chromosomal radiosensitivity in contrast to controls (approximately 40%). In this study, the G2 assay was applied to a small number of Irish breast cancer patients who were recorded as sporadic cases and they were compared with a control group to compare and contrast with the previous documented studies. Lymphocyte cultures were set up on whole blood samples and stimulated with phytohaemagglutinin. The cultures were irradiated 74 h later with 0.5 Gy gamma-radiation and cells were arrested in metaphase by treating the cultures with colcemid. The chromosomes were harvested and the aberrations scored per 100 metaphases to assign a G2 score. The assay was first carried out on four donor controls to estimate intra-individual variation and then ten controls for inter-individual variation to measure assay reproducibility. The G2 assay was then applied to 27 breast cancer patients. Good intrinsic assay reproducibility was observed in the coefficient of variation (CV) data in three out of four controls. Intra-individual variation was similar in three out of four of the donors (4.6 - 5.1%) with one donor showing a higher CV compared with the others (22.9%). Inter-individual variation was calculated at 30.5% for all controls. No significant difference was observed between intra- and inter-individual variation using the variance ratio F-test. A G2 radiosensitivity cut-off of 110 aberrations/100 metaphases was calculated from the controls, and from this 70.4% of breast cancer patients and 7.7% of controls were calculated as G2 radiosensitive. This proportion of G2-sensitive breast cancer patients is the highest recorded in studies to date. It is thought that the G2 radiosensitivity assay is a biomarker of breast cancer predisposition genes of low penetrance, suggesting the presence of these genes in the Irish breast cancer patients used in this study who were recorded as sporadic cases. A larger number of Irish patients would be required to consolidate these findings and be representative of the Irish breast cancer population.

Adult↗

Genotoxicity of field-collected inter-tidal sediments from Cork Harbor, Ireland, to juvenile turbot (Scophthalmus maximus L.) as measured by the Comet assay.

The alkaline single cell gel electrophoresis (SCGE) or Comet assay was employed to test the potential of surficial sediment collected from Cork Harbor, Ireland, to induce DNA damage in turbot (Scophthalmus maximus L.) in a laboratory exposure experiment. Turbot were exposed for 21 days to field-collected sediment from Cork Harbor and from a relatively clean reference site at Ballymacoda and sampled at 0, 7, 14, and 21 days. As a positive control for the sediment exposure experiment, a subsample of the turbot was exposed to cadmium chloride-spiked seawater. DNA damage analysis was performed on epidermal, gill, spleen, liver, and whole blood cell preparations. Liver, gill, and blood were the most sensitive tissues while a lower level of damage was detected in the epidermis and spleen. The blood was determined to be a suitable predictor of DNA damage in the whole organism. Chemical analysis of the sediment indicated that polycyclic aromatic hydrocarbons formed the bulk of the contaminants, with the harbor sites having almost double the levels of those from the reference site. The data indicated that turbot exposed to sediments from Cork Harbor elicited a significant increase in DNA damage in comparison with those exposed to sediment from the reference site and that exposure to the contaminated sediments caused a multi-organ genotoxic response. Results from the study indicate a relationship between the presence of genotoxicants in sediment and DNA damage. This finding was encouraging with regard to the potential use of the Comet assay as part of a marine biomonitoring strategy.

Animals↗

Implications of seasonal priming and reproductive activity on the interpretation of Comet assay data derived from the clam, Tapes semidecussatus Reeves 1864, exposed to contaminated sediments.

We explore the use of the clam Tapes semidecussatus Reeves 1864 as an indicator for the presence of potentially genotoxic substances in estuarine sediments. The limitations associated with the interpretation of Comet assay data (expressed as % DNA in tail) in terms of clam reproductive state, size (age) and thermal exposure history following laboratory acclimation are discussed. Hatchery-reared clams, subjected to ambient temperature fluctuations during growth, were exposed in vivo under laboratory conditions for three weeks to sediment samples collected from a polluted site and a "clean" reference site. The DNA damage observed in haemocytes, gill and digestive gland cells was significantly higher in animals exposed to contaminated sediment compared to those exposed to sediment from the reference site. The extent of DNA damage recorded was not correlated with size (age). Spawning was not observed during the experiment. Nevertheless, clams with well-developed gonads showed a statistically higher degree of DNA damage in gill and digestive gland cells- but not haemocytes, demonstrating an increased sensitivity to potential genotoxic compounds, possibly caused by impaired DNA repair capacity due to reproductive activity. Furthermore, the degree of DNA damage in clams exposed to contaminated sediments was higher in autumn and winter compared to spring and summer, suggesting an effect of seasonal priming.

Analysis of Variance↗

An assessment of the pollutant status of surficial sediment in Cork Harbour in the South East of Ireland with particular reference to polycyclic aromatic hydrocarbons.

Surface sediment from three polluted sites within Cork Harbour, Ireland, and from a relatively clean reference site were collected and analysed for polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), organochlorine pesticides (OCPs), brominated flame retardants (BFRs), organotins (OTs), and heavy metals. PAHs were determined to be the most abundant class of contaminant. Concentrations of the sum (Sigma) of the 21 PAHs measured from the Harbour sites (2877.70 ng g(-1), 1000.7 ng g(-1) and 924.40 ng g(-1) dry weight respectively) were significantly higher than that of the sediment from the reference site (528.30 ng g(-1) dry weight). An inner harbour site, Douglas being the more contaminated of the three harbour sites. A similar pattern was observed with the other contaminants however, these compounds, with the exception of the heavy metals, all tended to be detected at concentrations on or below detection limits.

Environmental Monitoring↗

In vitro cytotoxicity testing of three zinc metal salts using established fish cell lines.

The utilisation of fish cell lines has proven to be a valuable, rapid and cost-effective tool in the ecotoxicological assessment of chemicals and environmental samples. The main objective of this study was to investigate the value of multiple endpoint measurements in evaluating the cytotoxicity of three divalent zinc salts in three established fish cell lines (EPC, CHSE and RTG-2) and the potential for their employment as effective screening tools for zinc contaminated environmental samples. A significant stimulatory effect was detected with the neutral red assay in EPC and RTG-2 cells exposed to the lower doses of some zinc compounds. Significant (p < or = 0.01) lactate dehydrogenase release was detectable only with the highest exposure concentration of ZnCl2. Toxicity ranking based on IC50 values calculated from the neutral red and coomassie blue assay data found that in general, ZnC2 was the most cytotoxic metal compound to the cell lines employed. Differential cell sensitivities were observed to be dependant on the particular compound tested and the endpoint employed. It was found that the use of light microscopy in the identification of cell morphological changes was a valuable adjunct in verifying the results of colorimetric tests. In conclusion, careful consideration should be given to study design and statistics applied and use of a battery style approach is recommended for toxicological screening studies.

Animals↗

Ionizing radiation induces a stress response in primary cultures of rainbow trout skin.

Fish skin is very vulnerable to damage from physical and chemical pollutants because it is in direct contact with the aquatic environment. In this study, the effect of gamma radiation on primary cultures of rainbow trout skin was investigated. Primary cultures containing two cell types, epidermal cells and goblet mucous cells, were exposed to doses ranging from 0.5-15 Gy 60Co gamma radiation. Expression of PCNA, c-myc and BCL2 was investigated as well as growth and levels of apoptosis and necrosis. Morphological and functional changes were also studied. The irradiated cultures showed evidence of a dose-dependent increase in necrosis and enhanced proliferation as well as morphological damage. In addition, mucous cell area was found to decrease significantly after irradiation. The study shows the value of these primary cultures as in vitro models for studying radiation effects. They provide an effective alternative to whole-animal exposures for radiation risk assessment.

Animals↗

Relationship between clonogenic radiosensitivity, radiation-induced apoptosis and DNA damage/repair in human colon cancer cells.

The intrinsic radiation sensitivity of normal and tumour tissue is a major determinant of the outcome of radiotherapy. There is currently no established test that can be used routinely to measure the radiosensitivity of the cells in an individual patient's cancer in a manner that can inform treatment planning. The purpose of this study was to evaluate, in four human colorectal adenocarcinoma cell lines, two possible end points as surrogate markers of radiation response--apoptosis and induction of DNA single-strand breaks--and to compare the results with those of a conventional clonogenic assay. Cell lines (SW707 SW480, SW48 and HT29) known to differ in radiosensitivity were exposed to single doses of X-rays ranging from 0.5 to 5 Gy and cell survival was measured using the clonogenic assay. Apoptosis was determined on the basis of morphology under fluorescent microscopy and DNA damage/repair was measured, as tail moment, using an adaptation of the alkaline comet assay. The relationship between surviving fraction at 2 Gy (SF2) and the percentage of apoptotic cells 24 h after the same dose was complex, but apoptosis accurately predicted the order of radiosensitivities as measured by SF2. Initial damage measured after 2 Gy using the alkaline comet assay gave a close correlation with SF2 (r2=0.95), whereas there was no correlation between initial DNA damage repair rate and SF2.

Adenocarcinoma↗

Monoamine oxidase inhibitors l-deprenyl and clorgyline protect nonmalignant human cells from ionising radiation and chemotherapy toxicity.

l-Deprenyl (R-(-)-deprenyl, selegiline) is an inhibitor of monoamine oxidase-B (MAO-B) that is known to protect nerve cells from a variety of chemical and physical insults. As apoptosis is a common mechanism of radiation-induced cell death, the effect of l-deprenyl on the survival of cultured cells and tissue explants was studied following exposure to gamma radiation. The results obtained were compared with the effects of the less-selective MAO-B inhibitor pargyline and the MAO-A inhibitor clorgyline. l-Deprenyl at a concentration of 10(-9) M protected the nontumorigenic cell line (HaCaT) and normal human urothelial explants from the effects of cobalt-60 gamma radiation, but did not protect tumorigenic human cell lines HaCaT-ras, HPV-transfected human keratinocytes (HPV-G cells), or PC3. Human bladder carcinoma explants were not protected. Clorgyline showed a smaller protective effect of normal cells, whereas pargyline had no effect. Radiation-induced delayed effects (genomic instability measured as delayed cell death) were prevented in normal cells by l-deprenyl but, interestingly, deprenyl appeared to increase the amount of delayed death in the tumorigenic cell lines. Studies using l-deprenyl prior to the exposure of nonmalignant cells to cisplatin showed that cell death due to this agent was also reduced. Treatment of cultures of nontumorigenic cells with l-deprenyl or clorgyline significantly increased the levels of the protein Bcl-2 following irradiation, but there was no such effect on the already-elevated levels of this protein in the tumour samples. Since the Bcl-2 has been shown to be an inhibitor of apoptosis or programmed cell death, this would imply that the protective effects of l-deprenyl and clorgyline involve activation of antiapoptotic pathways within the normal cell. This hypothesis is supported by data showing reduced levels of apoptosis in HaCAT cells and in normal bladder explant cultures following treatment with l-deprenyl.

Animals↗

A proliferation-dependent bystander effect in primary porcine and human urothelial explants in response to targeted irradiation.

The aim of this study was to test whether radiation-induced bystander effects are involved in the response of multicellular systems to targeted irradiation. A primary explant technique was used that reconstructed the in vivo microarchitecture of normal urothelium with proliferating and differentiated cells present. Sections of human and porcine ureter were cultured as explants and irradiated on day 7 when the urothelial outgrowth formed a halo around the tissue fragment. The Gray Cancer Institute charge particle microbeam facility allowed the irradiation of individual cells within the explant outgrowth with a predetermined exact number of (3)He(2+) ions (which have very similar biological effectiveness to alpha-particles). A total of 10 individual cell nuclei were irradiated with 10 (3)He(2+) ions either on the periphery, where proliferating cells are located, or at the centre of the explant outgrowth, which consisted of terminally differentiated cells. Samples were fixed 3 days after irradiation, stained and scored. The fraction of apoptotic and micronucleated cells was measured and a significant bystander-induced damage was observed. Approximately 2000-6000 cells could be damaged by the irradiation of a few cells initially, suggesting a cascade mechanism of cell damage induction. However, the fraction of micronucleated and apoptotic cells did not exceed 1-2% of the total number of the cells within the explant outgrowth. It is concluded that the bystander-induced damage depends on the proliferation status of the cells and can be observed in an in vitro explant model.

Animals↗

Characterisation of a bystander effect induced in human tissue explant cultures by low let radiation.

The existence of a bystander effect following both alpha and gamma irradiation of many cell lines is not now in dispute. The significance of this effect for cancer risk assessment and radiotherapy treatment planning requires demonstration of its relevance in vivo. The problem in demonstrating the existence of the effect in vivo is that other systemic effects may mask or confound the effect being investigated and it is practically impossible to attribute an effect in a particular cell to a signal produced in another irradiated cell. To approach this problem, an assay has been developed where fragments of human tissue can be irradiated ex vivo and the media harvested and added to unirradiated, clonogenic cells which have a well characterised and stable response to the bystander signal. The variation in the production of a signal from patient to patient can thus be assessed. The results of a study using tissue from over 100 patients attending Beaumont and St Vincent's Hospitals in Dublin for investigation of urological disorders including follow-up after treatment for transitional cell carcinoma (TCC) and resection of suspect prostatic lesions, are now available. Blood samples from the prostate group were also obtained. The results show that there is variation in the effect of the signal produced by irradiated tissue from different patients. This holds for bladder, prostate and blood. Gender, smoking status and the existence of a malignancy influence the expression of the signal by normal tissue. Male gender, smoking and a pre-existing malignancy all reduce the amount or effect of the signal produced into medium when the tissue is exposed. The effects of exposure to medium containing the signal are transmitted to distant progeny of the exposed cell population. The results may be important not only for understanding radiation risk mechanisms for protection but also for radiotherapy treatment planning where they may open new avenues for development of drugs for combined therapy.

Alpha Particles↗