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Development of new molecular procedures for the detection of genetic alterations in man

Objective

To identify, understand and characterize the main processes involved in the bioavailability of pollutants in soils and sediments, to develop reliable risk assessment methods and to design innovative practices for biological remediation taking into account bioavailability aspect.

The Restriction Site Mutation (RSM) system is a procedure for detecting mutations of a deoxyribonucleic acid (DNA) sequence at any unselected locus. The mutations are identified as alterations of the DNA sequence at any chosen site which is normally the target for digestion by a restriction enzyme. The mutation will change the DNA sequence so that it can no longer be digested by the restriction enzyme. It is intended to develop the technique such that mutations can be detected with a sensitivity of 1 in 10{7} using either cultured human cells after mutagen treatment, experimental animals, or exposed human populations.

The sensitivity and specificity of the procedure has been assessed in bacteria and in mammalian cells using reconstruction experiments. Mutant DNA could be detected at a frequency of about 1E-7 in bacteria and 2E-7 in mammalian cells. Using the molecular assay, it is able to show that much more mutant DNA is produced in Xeroderma pigmentosum (XP) than in normal cells following UV irradiation. No cellular selection was applied. The mutant DNA has been sequenced and contains a high proportion of double and tandem base mutations. At this stage using the RSM system, it is able to detect mutations produced at relatively high frequency in cultured cells by chemicals and UV radiation. The principal problem encountered is the appearance of false positives, ie samples of DNA from untreated cells which produce mutant DNA bands.
The research investigations into risk assessment and reclamation of abandoned waste disposal sites and contaminated industrial sites elicite two majorr bottlenecks. The first one deals with the processes governing the biological availability (bioavailability) of pollutants in the environment and especially in soil, sediment, subsoil and groundwater systems. The second one deals with the lack of suitable pilot scale soil (or sediment) reactors to identify, understand and characterize the former processes.
Concerning the bioavailability of pollutants in soil and sediments, the main processes influence both the acuteness of induded toxicity effect on organisms and the kinetics of biological degradation and thus the safety and efficiency of remedial practices. They include physico-chemical processes related to pollutants availability but also processes related to organism availability : microbial mobility limitations, physiological and ecological limiting factors. The expected achievement for this first main task is the development of suitable concepts and associated numerical models which will be able to simulate the main processes governing the bioavailability of pollutants in soils or sediments. These tools will be designed, improved and validated using pilot soil reactors either with a scale-up or a scale-down approach. Practical tools for risk assessment will be checked and validated on real scale contaminated systems. This application and validation stage will give to the developed practices and risk assessment methods a precompetitive character and will allow to test rapid non invasive techniques for characterizing the bioavailability of real scale contaminations.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

MRC Cell Mutation Unit
Address
University Of Sussex Falmer
BN1 9RR Brighton
United Kingdom

Participants (1)

UNIVERSIDAD DE CORDOBA
Spain
Address
S/n,avenida San Alberto Maquo S/n
14071 Cordoba