Objective
The principal risk from low doses of ionizing radiation is the induction of cancer. At present, determination of the risks of developing specific types of cancer are based on predictions made by methods which have not been validated at occupational radiation doses. At such low doses the use of animal models to study dose response relationships for cancer becomes prohibitively expensive. Cell transformation in vitro offers an alternative approach, however only one system, the C3H 10T1/2 assay, provides the relatively high precision required for measurements at low doses.
Even with this assay the sensitivity is low, consequently a collaborative effort involving several laboratories is necessary to measure effects at 10 mGy. A joint venture between AEA, Harwell; GSF, Frankfurt; Nuclear Electric, Berkeley and the Universities of Milan and Wurzburg has been established to carry out such a programme with the following objectives:
to standardize the C3H 10T1/2 cell transformation assay between European laboratories to ensure comparability of results;
to establish the shape of the doseAresponse relationships for survival and transformation of 10T1/2 cells exposed to a range of radiation qualities down to 10 mGy.
Comparison had been made between cell surface proteins from normal C3H/10T1/2 cells and those transformed by either cobalt-60 gamma rays or the chemical carcinogen 3-methylcholantherene (MCA). Membrance proteins from both normal and transformed cells were then analysed and compared by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). This led to the detection of consistent differences in membrane protein expression between the normal and transformed cells and in particular the identification of 4 proteins of special interest. These were of approximate molecular weight: 66, 49, 44 and 37 kD, the first of which showed greatest expression in the normal cells and was progressively lost in the transformed cells, whilst the reverse is true of the other 3.
Monoclonal antibodies have been produced against the 4 proteins of interest and commenced by isolating them from the SDS-PAGE gels by electroelution.
Monoclonal antibodies were then raised against a mixture of the 4 proteins using in vitro immunization techniques. By this approach 15 positive clones were identified, all of which were immunglobulin M (IgM) immunoglobulin isotype.
The fine specificity of these 15 monoclonal antibodies. Unfortunately, even by flow cytometry, only slight differences in antibody reactivity could be detected between normal and transformed cells, whilst the Western blotting indicated that the binding was nonspecific as the antibodies bound to multiple protein bands.
These combined results suggest that the antibodies react with epitopes common to a variety of cell surface proteins rather than to those which are transformation related, thus showing little difference in reactivity between the normal and transformed cells. Continuing work will investigate this problem and attempt to generate antibodies of the desired specificity.
With respect to the standardization of the C3H 10T1/2 transformation assay, cells were prepared for the standardization exercises for the 5 Gy intercomparison. The inverse dose rate effect using 2.2 MeV neutrons was studied and a direct comparison was made of the effect of protons and alpha-particles with the same linear energy transfer (LET).
The intercomparison experiments involved the detection of single transform cells using cell surface markers.
An intensive series of measurements involving the scoring of about 100 foci has shown the possibility of a small enhancement in the effectiveness of low dose rates of 2.2 MeV neutrons. The dose chosen for this comparison was 0.2 Gy given either acutely or over a period of 5 h. It was found that the transformation frequency at the low dose rate was 1.3 times higher than at the high dose rate (95% confidence limits +/-0.27).
In another series of collaborative experiments 10T1/2 cells were irradiated with either 0.5 1 or 2 Gy of protons and alpha-particles with LET's between 20 and 23 keV um{-1}. No significant difference in the transformation frequencies were seen for other endpoints.
In a comparative investigation the transformation rates of C3H 10T1/2 cells after exposure to chromium K alpha X-rays (5.4 keV) and cobalt-60-gamma rays have been determined. Soft X-rays are more effective than gamma for cell transformation. The relative biological effectiveness (RBE) value for soft X-rays versus gamma-rays was approximately 1.3 in the range of soft X-ray doses from 2 Gy to 5 Gy. There was no recognizable dependence of the RBE on dose, which contrasts the findings with alpha particles. The essential result of this soft X-ray study is that electrons of low energy and of ranges less than 1 um are more effective than fast electrons, not only for cell inactivation but also for cell transformation.
Induction of transformation incidence was examined in C3H 10T1/2 cells after exposure to single and fractionated doses of intermediate and high linear energy transfer (LET) particles, in order to investigate the possible LET dependence of an inversed dose rate effect for cell transformation.
Accelerated particles from the RARAF (Columbia University, New York) have been used to expose the cells to a single dose or 3 fractions with different time intervals between the fractions. Doses were chosen leading to surviving fractions of 0.6 to 0.7. The transformation frequency was influenced by extended exposure time for some but not all radiation qualities. Enhancement of transformation was evident for 40, 75, and 120 keV/um when the time between each fraction was greater than 15 min. As LET increases above 25 keV/um, the differences in transformation induction between acute and extended exposures increase to a maximum at 120 keV/um before disappearing at 200 keV/um. Further experiments with helium-4 ions (LET 150 keV/um) and with americium-alpha-particles (LET 147 keV/um) are underway to complete the data. The variation in enhancement with long intervals between fractionation appears to be consistent with a proposed mechanism in which a period of extra sensitivit y exists in the cell cycle (Rossi and Kellerer, 1986)
Research was carried out in order to evaluate parameters which affect the transformation frequency on C3H 10T1/2 cells and techniques were developed for the exposure to C3H 10T1/2 cells to characteristic ultrasoft X-rays.
Because of the extremely low range of CK characteristic X-rays special glass dishes with hostaphane foils were used. The glass dishes were made from glass cylinders and hostaphane was stretched across one side of the glass dish and glued to the dish. In order to avoid cells that may be shielded by an excess of glue or by attachment to the glass wall of the dish, a special device was constructed which produces first a ring of puremedium near the wall. Then 0.4 ml of cell suspension was distributed within this ring. After 5 h incubation, when cells are attached to the foil, 1.6 ml of medium was added and the incubation continued for another 48 h. The cell count at this time was about 1.2 E5 and cells were still in exponential growth phase when exposed to the ultrasoft X-rays.
The CK photon beam was vertical and photons entered through the bottom of the glass dish, through the hostaphane foil. The dishes were placed in a holder with the hostaphane foil facing the photon beam. Dosimetry was performed using a special ionization chamber. The dose measurements corresponded to the entrance dose, ie the dose in cells just behind the 1.5 um hostaphane foil. The dose rate amounts to about 5 Gy min{-1}.
For quantitation of cell transformation it was necessary to measure cell survival as a function of dose. C3H 10T1/2 cells attached to the hostphane foil are very flat. The mean thickness of the cell nucleus amounted to about 2 um so that the average dose to the cell nucleus relative to the entrance dose was found to be 0.55. Survivial curves were drawn of C3H 10T1/2 cells after exposure to cobalt-60 gamma rays (reference radiation) and CK photons. The relative biological effectiveness (RBE) values of the CK photons relative to cobalt-60 gamma rays for va rious survival levels of C3H 10T1/2 cells were calculated. The RBE values were dose dependant, mainly due to the less pronounced shoulder in the survival curve obtained after CK photon exposure.
Transformation and inactivation frequencies induced in 10T1/2 cells exposed to 4.3 MeV alpha particles were determined in the range interval between 0.2 and 300 cGy. Survival data are well fitted by an exponential function of the dose with a mean lethal dose value of 0.61+/-0.02 Gy. Transformation frequencies per surviving cell versus dose were calculated. The initial cell density was kept to between 2 and 4 cells/cm{2}. The transformation curve shows a complex behaviour. For less than 0.02 Gy, data are well described by a staight line with an average slope of (8.5+/-2) E-3 Gy{-1}. In this dose region there is a negligible probability for a cell or its nucleus to be hit by 2 independent radiation tracks. Therefore a linear relationship between transformation frequency and dose was expected.
In the interval between 0.02 and 0.2 Gy there is an apparent constancy. This could be explained either by assuming an inducible repair process (in this dose interval the contribution of multiple traversals of the cells becomes significant), or, as due to the phenomena that occur at low doses and tend towards saturation. Such phenomena could be an alteration with its own probability of leading to transformation, or a period in the cell cycle during which the cells are especially sensitive. The analysis of the curve in the light of these models is in progress in the laboratory. Experiments on dose fractionation effect are underway. A total dose of 0.21 Gy was delivered either as a single fraction or as 3 equal fractions at time invervals of 1.5 h between the doses. In each experiment single and fractionated dose were delivered in parallel and with the same experimental conditions. Preliminary results show that transformation frequencies after fractionated exposure are higher than after single exposure by a factor of about 1.3.
Initial discussions revealed several differences in the assay procedures used by the participating laboratories:
Comparative evaluation of scoring transformants.
As a first step towards harmonizing the assay procedures each laboratory contributed a selection of culture vessels containing transformed foci from previous experiments. The vessels were then circulated around the laboratories and each laboratory independently recorded the number of foci they considered to be transformed. A comparison between the scores revealed some differences but overall there was good agreement. On the basis of roundtable discussions the criteria for defining a transformed focus was further refined and the criteria for identifying transformants standardized between the participating laboratories.
Intercomparison of experimental protocols.
A comparison of the standard procedures used by the different labs showed some minor differences but it is not clear whether these affected the final transformation frequency. At this stage, no attempt has been made to derive and impose a consensus standard procedure for the assay, However, certain additional steps have been taken to harmonize the technical procedures used by the different laboratories. In particular, the plating density of viable cells at the beginning of a transformation experiment has, initially, been set to 3 to 5 cellcm{A2}, and a common method of staining the monolayer at the end of the experiment has been adopted.
Multicentre measurement of transformation frequency.
In order to establish whether other differences in technique influence the outcome of a transformation experiment 10T1/2 cells irradiated at one laboratory will be shipped to the other participants who will then perform a transformation assay according to their normal procedures. Preliminary studies on the optimum method of shipping the cells, by the University of Wurzburg and Berkeley, revealed that they travelled best at 0 C in an ice-water mixture. Using this procedure, recovery was high and the plating efficiency was unaffected by 48 hours in transit. Freezing in liquid nitrogen followed by shipping on dry ice was unsatisfactory; recovery was poor and the plating efficiency was reduced by approximately 50%, irradiated and unirradiated cells being affected to the same extent.
Future objectives:
Having established the feasibility of shipping irradiated cells between the participants with no loss of viability 10T1/2 cells will be irradiated with 5 Grays at one centre and shipped to each participant along with appropriate controls. Each participant will initiate a transformation assay from the cells in parallel, with a transformation assay using their own cells as an internal control. The results will be reviewed and, if necessary, the experimental procedures revised to achieve greater uniformity. If the results are in reasonable agreement (ie no significant difference between the labs so the results can be pooled), the procedure will be repeated for a dose of 1 Gy and subsequently in steps to lower doses, the final target being 10 mGy.
Fields of science (EuroSciVoc)
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques. See: The European Science Vocabulary.
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques. See: The European Science Vocabulary.
- natural sciences chemical sciences inorganic chemistry transition metals
- natural sciences biological sciences biochemistry biomolecules proteins
- natural sciences physical sciences nuclear physics
- natural sciences chemical sciences electrochemistry electrophoresis
- natural sciences physical sciences theoretical physics particle physics photons
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OX11 0RA Didcot - Oxfordshire
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