The C3H 10T1/2 cell transformation system has been widely used for a number of years to measure the malignant transformation by radiation and by chemicals. The radiosensitivity of this system is, however, rather low and experiments to determine the transformation frequency at low doses require a large number of samples in order to obtain significantly meaningful results. The exact details of the experimental protocol used can also significantly affect the measured transformation frequency. Evidence of this can be deduced from the scientific literature.
The data from collaborative experiments are based on a large number of transformed foci (759 in total), far in excess of the numbers reported in any previous publication involving the C3H 10T1/2 cell transformation system. As such the results have a firm statistical basis. Moreover, experimental conditions which were found most likely to affect transformation frequencies were identified and well controlled.
In summary the specific objectives have been achieved by this collaborative project:
(i) The standard protocol on cell transformation in vitro using the C3H 10T1/2 mouse fibroblast system as recommended by the IARC working group (Kakunaga and Yamasaki, IARC Scientific Publication No. 67, 207-219, 1985) has been extended and further standardised by suitable inter-comparison experiments. A catalogue of transformed and non-transformed foci depicting several different morphologies has been produced. Additional recommendations that should be taken into account are:
- seeding density of cultures should be ~2 viable cells per square centimetre;
- cultures should be incubated for a fixed period of four weeks after confluence has been reached;
- regular consensus scoring is required;
- type II and type III (after Reznikoff et. al.) should be regarded as positively transformed clones, but with some modification to the scoring criteria;
- type X foci should be scored as positively transformed.
(ii) Inter-comparison experiments and the results of pooling data from the participating laboratories has determined the dose response relationships for cell survival and cell transformation to a high precision.
In-vitr cell transformation studies have become an important alternative for the study of the carcinogenic potential of different types of ionising radiation and other cancer-causing agents. They do not provide direct quantitative risk estimates, but produce important information about dose-effect relationships and their dependence on dose-rate, radiation quality and on the combined effects With other agents. However, only one cell transformation assay, the C3H 10Tl/2mouse fibroblast system, provides the high precision needed for Work at l0W absorbed doses. This sytem is routinely used in a number of laboratories in Europe and the USA.
The proposed project Will be a collaborative study of dose-response relationships for cell transformation In-vitro using the C3H 10Tl/2mouse fibroblast system at as low a dose as can be achieved. A realistic minimum dose at which to assess transformation frequency with adequate precision and achieve meaningful comparisons between participating laboratories during the next two years is 0.1 gy. Ultimately it is expected that, by pooling data from each individual laboratory, it will be possible to extend the determination of dose-response relationships down to a dose of 0.01 Gy. other objectives of this proposal are the preparation of a standard manual for cell transformation assays and the introduction of epithelial cell systems and assays that have increased relevance to human risk estimation. The advantages of standardisation and inter comparison are that they will allow data from different laboratories to be interpreted uniformly. The resultant information for low doses will enable current models of radiation risk to be improved which can lead toincreased efficiency and financial savings in radiological protection as the uncertainties in risk estimation at low doses will be reduced.
In addition, subgroups in varying combinations of the participants will continue more specialised investigations on: (i) dose-rate effects with densely ionising radiations, (ii) role of neighbouring entities of high ionisation density using ultrasoft X-rays and Auger-emitters, (iii) transformation frequency and the cell cycle, (iv) alternative indicators of transformation and (v) alternative transformation assays.
Funding SchemeCSC - Cost-sharing contracts
OX11 0RA Didcot
00060 Santa Maria Di Galeria Roma