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C Mothersill

Publications and source records attributed to C Mothersill.

At least 37 records · Page 2Linked to original sources
PubMed2002

Early events in the apoptotic cascade initiated in cells treated with medium from the progeny of irradiated cells.

Genomic instability and the bystander effect have recently been linked experimentally. It has previously been shown that medium from irradiated cells can induce early events in the apoptotic cascade, such as mobilisation of intracellular calcium, loss of mitochondrial membrane potential and an increase in reactive oxygen species, in unhit cells. The aim of this study was to determine if medium from the progeny of irradiated cells could also initiate apoptosis in unhit cells. Human keratinocytes were irradiated (0.5 and 5 Gy) and medium was harvested up to the 7th passage post-irradiation and transferred to unirradiated keratinocytes. Intracellular calcium levels, mitochondrial membrane potential and the level of reactive oxygen species were all monitored for a period of 24 h following medium transfer. Rapid calcium fluxes (within 30 s), loss of mitochondrial membrane potential and increases in reactive oxygen species (from 6 h after medium transfer) were observed. There was no significant difference between medium generated by cells irradiated at the different doses. The data suggest that initiating events in the apoptotic cascade were induced in unhit cells by a signal produced by irradiated cells and that this signal can still be produced in the progeny of irradiated cells.

Apoptosis↗
PubMed2002

Bystander-induced apoptosis and premature differentiation in primary urothelial explants after charged particle microbeam irradiation.

The ureter primary explant technique was developed to study bystander effects under in vivo like conditions where stem and differentiated cells are present. Irradiation was performed with a 3He2+ charged particle microbeam available at the Gray Cancer Institute, with high (approximately 2 microns) precision. Tissue sections from porcine ureters were pre-irradiated with the microbeam at a single location with 10 3He2+ particles (5 MeV; LET 70 keV.micron-1). After irradiation, the tissue section was incubated for 7 days, thus allowing the explant outgrowth to form. Total cellular damage (total fraction of micronucleated and apoptotic cells) was measured according to morphological criteria. Apoptosis was also assessed using a 3'-OH DNA end-labelling technique. Premature differentiation was estimated using antibodies to uroplakin III, a specific marker of terminal urothelial differentiation. Results of our experiments demonstrated a significant bystander-induced differentiation and a less significant increase in apoptotic and micronucleated cells. A hypothesis based on the protective nature of the bystander effect is proposed.

Animals↗
PubMed2002

Initiation of apoptosis in cells exposed to medium from the progeny of irradiated cells: a possible mechanism for bystander-induced genomic instability?

Genomic instability and bystander effects have recently been linked experimentally both in vivo and in vitro. The aim of the present study was to determine if medium from irradiated cells several passages distant from the original exposure could initiate apoptosis in unirradiated cells. Human keratinocytes (from the HPV-G cell line) were irradiated with 0.5 Gy or 5 Gy gamma rays. Medium was harvested at each passage up to the 7th passage (approximately 35 population doublings) postirradiation and transferred to unirradiated keratinocytes. Intracellular calcium levels, mitochondrial membrane potential, and the level of reactive oxygen species were all monitored for 24 h after medium transfer. Rapid calcium fluxes (within 30 s), loss of mitochondrial membrane potential, and increases in reactive oxygen species (from 6 h after medium transfer) were observed in the recipient cells. There was no significant difference between medium conditioned by cells irradiated with 0.5 or 5 Gy. The effect of medium from progeny was the same as the initial effect reported previously and did not diminish with increasing passage number. The data suggest that initiating events in the cascade that leads to apoptosis are induced in unirradiated cells by a signal produced by irradiated cells and that this signal can still be produced by the progeny of irradiated cells for several generations.

Apoptosis↗
PubMed2001

Heavy metals of relevance to human health induce genomic instability.

Heavy metals used in medical prostheses or present in water supplies or tobacco can build up in tissues and blood and are well known to produce toxic effects. Normally, legislative controls on the levels of these substances are determined by reference to the acute toxicity data. This paper shows that cadmium and nickel can produce delayed effects in human cells in vitro, which are characteristic of genomic instability. The effects occur even at levels where no acute toxic effects can be demonstrated. Genomic instability can be demonstrated by persistent induction of cytogenetic abnormalities and delayed cell death in progeny of cells many generations after exposure. Formerly, this syndrome has only been definitively proven to occur following irradiation, but in these experiments cell populations exposed for only 1 or 24 hours were expanded over several months, involving eight passages, and the yield of chromosomal aberrations and cell loss due to lethal mutations did not decrease. The consequences of this genomic instability are not yet known but it is possible that many of the systemic symptoms associated with exposure to low concentrations of these metals could involve delayed expression of cellular damage. It is also clear that these effects cannot be predicted from acute toxicity data.

Cadmium↗
PubMed2001

Heat shock protein 70 levels in rainbow trout primary epidermal cultures in response to 2,4-dichloroaniline exposure: a novel in vitro aquatic toxicity marker.

The aim of this work was to investigate the use of the heat shock protein, HSP 70, as a sublethal measurement of ecotoxicity and to identify if the amount of HSP 70 synthesized is proportional to the chemical stress applied. This was achieved by quantifying the HSP 70 levels in primary cultured rainbow trout, Oncorhynchus mykiss (R.), skin epidermal cells in response to 2,4-dichloroaniline (2,4-DCA) exposure. The cellular stress response protects organisms from damage resulting from exposure to a wide variety of stressors including xenobiotics. The use of a HSP 70 polyclonal antibody on rainbow trout primary epidermal skin cultures exposed to 2,4-DCA was investigated as a possible biomarker for environmental stress using an immunocytochemical approach. The epidermis is highly susceptible, as it is the interface between the fish and its aquatic environment. In this study we have developed a simple in vitro system for aquatic-toxicity risk assessment. A method for the quantification of heat shock (stress) protein levels by immunocytochemistry is described. The antibody dilution range enabled the detection and quantification of only the inducible HSP 70 fraction. A 1:2000 dilution was decided upon. This assay was effective in detecting and quantifying the induced HSP 70. There was a direct toxicant concentration-dependent increase in the levels of the cellular stress protein in the primary epidermal cultures. Enhanced localization of HSP 70 in the nuclei of the epidermal cells was observed following exposure to 2,4-DCA. This work indicated the possibility of using heat shock protein induction and subsequent quantification as a sensitive system for aquatic toxicity risk assessment.

Aniline Compounds↗
PubMed2001

The further development of rainbow trout primary epithelial cell cultures as a diagnostic tool in ecotoxicology risk assessment.

The use of short-term cytotoxicity assays for the initial screening of chemicals not only aids in establishing priorities for the selection of chemicals that should be tested in vivo, but also decreases the time in which potential toxicants can be valued. Rainbow trout primary skin epithelial cell cultures are one such assay. Rainbow trout primary skin cell cultures contain two cell types, keratinocytes and goblet mucus cells. Two aquatic pollutants, copper and prochloraz were screened using this cell system. The influence of media composition on the effects of the aquatic pollutants was also studied by testing the chemicals in both serum-containing and serum-free medium and the morphological changes that occurred within the cell cultures recorded. The concentration of copper that causes a reduction of 90% in the residual of day 3 growth of the primary cell culture system was found to be approximately 10 fold more than that of prochloraz. Prochloraz was found to cause a greater reduction in growth area when added to the primary cell culture system in serum-free medium than in serum-containing medium. Copper, in contrast, was found to exert reduced toxicity when added to the test cultures in serum-free medium compared with addition in serum-containing medium. Prochloraz was found to kill the epithelial cells by a process of necrosis. Copper, was found to kill the epithelial cells by both necrosis and apoptosis in a ratio of 2:1. It was also observed that as the dose of both chemicals increased, the number of goblet cells contained in the cell cultures decreased. A PAS stain was carried out to determine if the goblet cells were exocytosing their contents onto the cell culture surface. It was found that as chemical exposure increased the number of cells expressing positivity for mucus also increased. The results of this study add further evidence to support that primary cell cultures are a very appropriate model for toxicity risk assessment.

Animals↗
PubMed2001

Oxidative stress in cells exposed to low levels of ionizing radiation.

The ability of medium from gamma-irradiated cells to induce early events in the apoptotic cascade, such as the mobilization of intracellular calcium, loss of mitochondrial membrane potential and increased levels of reactive oxygen species, in unirradiated cells was investigated. Medium from irradiated human keratinocytes was harvested and transferred to unirradiated keratinocytes. Intracellular calcium levels, mitochondrial membrane potential and the level of reactive oxygen species were all monitored for a period of 24 h following medium transfer. Rapid calcium fluxes (within 30 s), loss of mitochondrial membrane potential and increases in reactive oxygen species (from 6 h after medium transfer) were observed. There was no significant difference between the effects of medium generated by cells irradiated at 0.5 Gy or 5 Gy. The data suggest that a signal that leads to apoptosis is released from cells undergoing radiation-induced oxidative stress.

Apoptosis↗
PubMed2001

Individual variation in the production of a 'bystander signal' following irradiation of primary cultures of normal human urothelium.

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, we have developed an assay where fragments of human tissue can be irradiated ex vivo and the media harvested and added to unirradiated, allogenic explants or to a clonogenic cell line which has a well characterized and stable response to the bystander signal. The variation in production of the signal from patient to patient can thus be assessed using molecular and cellular endpoints. A study using tissue from over 100 patients and from mouse strains with well characterized responses to low level radiation exposure shows that there is variation in the effect of the signal produced by irradiated tissue from different patients. Gender, smoking status and the existence of a bladder malignancy influence the expression of the signal by normal urothelium. 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.

Animals↗
PubMed2001

Radiation-induced bystander effects: past history and future directions.

There has been a recent upsurge of interest in the phenomenon now known as radiation-induced bystander effects. This is largely due to the increased awareness of the contribution of indirect and delayed effects, such as genomic instability, to cellular outcomes after low-dose exposures. It is also due to the availability of tools such as the microbeam and advanced cell culture systems and to the ability to study end points such as gene or protein expression at low doses which were previously difficult to study. This review looks at the history of bystander effects in the earlier literature, in which the clastogenic effect of plasma from irradiated patients was well known. The effect was known to persist for several years and to cause transgenerational effects, making it similar to what we now call genomic instability. The review then examines the current data and controversies which are now beginning to resolve the questions concerning the mechanisms underlying the induction and transmission of both bystander effects and genomic instability. Finally, the possible impact of data concerning radiation-induced bystander effects on radiotherapy and radiation protection is discussed.

Animals↗
PubMed2001

Effect of low doses of ionizing radiation on cells cultured from the hematopoietic tissue of the Dublin Bay prawn, Nephrops norvegicus.

Explant cultures from the hematopoietic tissue of the Dublin Bay prawn, Nephrops norvegicus, were exposed to low doses of (60)Co gamma radiation. Cells growing from the explants were examined 7 days after irradiation using light and transmission electron microscopy and were also tested for their ability to produce signals indicative of a bystander effect. The exposed cultures displayed pronounced damage and were orders of magnitude more sensitive than the data in the literature would suggest for arthropod cells. The cultures were also more sensitive than mammalian cells that were exposed to similar doses. Cellular abnormalities included damage to cytoplasmic organelles, particularly the cytoskeleton. Abnormal mitochondria were also prominent. At low doses (0.5 Gy), nuclear damage was not apparent in the cultures, but there was evidence of a dose-dependent increase in apoptosis. The irradiated cultures released a factor into the medium that was capable of inducing apoptosis and cell death in unirradiated fish and human cells. This bystander effect was of a similar magnitude to that reported for mammalian cell systems. It is suggested that these crustaceans may be highly sensitive to radiation, unlike terrestrial arthropods and certain other invertebrates, which are generally considered to be radioresistant.

Animals↗
PubMed2000

Development and characterization of primary cell cultures from the hematopoietic tissues of the Dublin Bay prawn, Nephrops norvegicus.

Improved maintenance in vitro of the hematopoietic tissue of the Dublin Bay prawn Nephrops norvegicus (L.) resulted by using 10% (v/v) 2x Leibovitz's medium prepared in seawater (salinity = 25 per thousand), and supplemented with 10% (v/v) heat inactivated fetal bovine serum plus 5% (v/v) Nephrops serum or 5% (v/v) Nephrops muscle extract, and 0.06 g/l of L-proline and 1 g/l of glucose. Pronase at 100 microg/ml improved tissue dissociation and subsequent spreading of hematopoietic cell cultures. The addition of epithelial growth factor (EGF), based fibroblast growth factor (bFGF) or insulin growth factor 1 (IGF-I) did not enhance cell growth. Cell culture contained several types of maturing hemocytes, in the size range of 6-24 microm diameter. Acid phosphatase, alpha-naphthyl butyrate esterase, alpha-naphthyl acetate esterase, naphthyl AS-D chloroacetate esterase activity and phenoloxidase activity was demonstrated, but not so alkaline phosphatase or peroxidase. Small PAS (= Periodic acid Schiff) positive granules, unsaturated lipids and phospholipids were observed. Cultures remained functional for over two weeks. Mitosis was noticed occasionally; however, cell proliferation was not recorded by use of nuclear proliferation markers.

Acid Phosphatase↗
PubMed2000

Involvement of energy metabolism in the production of 'bystander effects' by radiation.

These experiments were done to determine if interference with energy metabolism and REDOX biochemistry during low LET radiation exposure would alter the ability of medium harvested from the irradiated cells to induce a bystander effect in unirradiated cells. Human keratinocyte cells and CHO-K1 mutant cell lines were irradiated using cobalt 60. Clonogenic assays were used to determine the reproductive death of the cells exposed to direct irradiation or medium from irradiated cells. The persistence in progeny was also examined. Use of apoptosis inhibitors or medium from the LDH or G6PD null cell lines, reduced or prevented the bystander effect. Transfection with G6PD recovered the effect. Treatment with anti-oxidant substances, L-lactate and L-deprenyl prevented bystander factor associated cell kill. The lactate analogue, oxamate, was less effective. Data from experiments where media harvested from the different cell lines was exchanged suggest that signal production and cellular response may involve different mechanisms. The effects on exposed cells were transmitted to progeny which also showed excessive levels of cell death for several generations. The results suggest that energy/REDOX metabolism may be involved in the expression of a radiation induced bystander response. Given the aberrant energy metabolism in tumour cells, this may have implications for dose escalation in radiotherapy.

Animals↗
PubMed2000

Production of a signal by irradiated cells which leads to a response in unirradiated cells characteristic of initiation of apoptosis.

This study investigated the ability of medium from irradiated cells to induce early events in the apoptotic cascade, such as mobilization of intracellular calcium, loss of mitochondrial membrane potential and increase in reactive oxygen species, in cells which were never exposed to radiation. Medium from irradiated human keratinocytes was harvested and transferred to unirradiated keratinocytes. Endpoints characteristic of the initiation of apoptosis were monitored for a period of 24 h following medium transfer. Clonogenic survival was also measured. Rapid calcium fluxes (within 30 s), loss of mitochondrial membrane potential, increases in reactive oxygen species (from 6 h after medium transfer), an increase in the number of apoptotic cells (48 hours after medium transfer) and a marked reduction in clonogenic survival (after 9 days) were observed. There was no significant difference between medium generated by cells irradiated at 0.5 Gy or 5 Gy. The data suggest that initiating events in the apoptotic cascade were induced in unexposed cells by a signal produced by irradiated cells.

Apoptosis↗
PubMed2000

Dose- and time-response relationships for lethal mutations and chromosomal instability induced by ionizing radiation in an immortalized human keratinocyte cell line.

PURPOSE: To investigate the relationship between two well-established delayed effects of ionizing radiation, experiments were conducted to determine the induction and expression of lethal mutations (delayed reproductive death) and chromosomal instability with respect to dose and time in a human immortalized keratinocyte cell line. METHODS: HPV-G cells were gamma- or alpha-irradiated and maintained in culture for up to 72 population doublings. At intervals, measurements were made of cloning efficiency and the cells examined for apoptosis and cytogenetic aberrations. RESULTS: The descendants of cells surviving 1 or 3 Gy gamma-irradiation, but not 0.5 Gy gamma-irradiation, exhibited a reduced colony-forming efficiency. The reduction persisted at a constant rate of 15-20% clonogenic cell loss per population doubling for up to 72 population doublings. Apoptosis was demonstrated in all colonies in the 1 and 3 Gy groups at 30 and 72 population doublings post-irradiation but not in the 0.5 Gy group. A significant persistent reduction in colony-forming ability (approximately 80%) was demonstrated in the progeny of cells irradiated with 0.5 Gy alpha-particles. After 30 population doublings, the proportion of chromosomally aberrant cells was significantly greater than control values for all doses of both high- and low-LET radiations. The major cytogenetic aberrations (chromatid breaks, chromosome fragments and minutes) were consistent with the transmission of chromosomal instability. The expression of instability declined between 30 and 72 population doublings in the 0.5 Gy and 3 Gy gamma-irradiation groups, but persisted up to 72 population doublings in the 1 Gy group. The expression of chromosomal instability was greater in the descendants of alpha-irradiated cells and showed little evidence of reduction with time. CONCLUSIONS: Unstable aberrations characteristic of radiation-induced chromosomal instability may commonly result in apoptosis and account for a component of the delayed reproductive death/lethal mutation phenotype in HPV-G cells. However, the absence of lethal mutations in the descendants of 0.5 Gy gamma-irradiated cells indicates a low-LET threshold effect for this particular endpoint. Overall, and particularly at low doses, there is no direct correlation between the two endpoints, indicating the absence of a simple relationship between these manifestations of radiation-induced genomic instability.

Alpha Particles↗
PubMed2000

Relative contribution of bystander and targeted cell killing to the low-dose region of the radiation dose-response curve.

Human keratinocytes show a bystander effect when exposed to low doses of low-LET radiation. In this paper, data are presented showing a method of correcting the overall survival curve to enable analysis of the relative contributions of the bystander effect and the effect attributable to direct interaction of the radiation with the target cell. The technique used is to obtain a standard clonogenic survival curve using the assay of Puck and Marcus and, with a different set of flasks containing cloning densities of unirradiated cells, to assay the cell killing caused by medium harvested from 2 x 10(5) cells irradiated with the same doses. The data show that for this human epithelial cell line, doses of 0.01-0. 5 Gy show clonogenic death by the bystander effect only, if maximum potential bystander killing is assumed. The magnitude of the effect is relatively constant, and it appears to saturate at doses in the range of 0.03-0.05 Gy. After doses greater than 0.5 Gy, the curves for clonogenic death are the result of a dose-dependent non-bystander effect and a dose-independent bystander effect. If these particular dose-response effects occur in epithelial cells in vivo, they may have important consequences for therapy and studies of low-dose risk.

Apoptosis↗
PubMed1999

Antigenic characterization of Nephrops norvegicus (L.) hepatopancreas cells.

During this work structural, differentiation and proliferation antigenic markers developed for mammals were applied in paraffin sections of Nephrops norvegicus (L.) hepatopancreas. The purpose was to establish standards for the characterization of invertebrate cells in vitro. Antibody concentration was optimized for quantification of cell proliferation. There are no antibodies specific for crustaceans on the market. An avidin-biotin immunoperoxidase method was used to visualize cell antigen expression. The immunocytochemical results indicate that the epithelium in the Nephrops hepatopancreas digestive tubules does express cytokeratins and proliferating cell nuclear antigen. The results of this work indicate that some mammalian antibodies cross-react with crustacean epitopes. This may facilitate cell characterization of cell types cultured in vitro.

Animals↗