Optimizaciones metodológicas del ensayo del cometa y su aplicación en biomonitorización humana

Student thesis: Doctoral thesis

Abstract

The DNA integrity is a fundamental aspect for human health and for the suitable functions of our organism. However, the genetic material is susceptible to be damaged by many agents and processes. Although the majority of damage induced in our genetic material is repaired efficiently, a part of this damage escapes from this process and becomes one of the substrates for the potential development of a carcinogenic process. During the last years new methodologies have been developed to assess the DNA damage by performing techniques which allow to measure single strand breaks (SSB) and double strand breaks (DSB) directly. The comet assay has become one of the most popular approaches to evaluate this type of damage. Although this technique has been proposed as an appropriate tool, many aspects must be still reconsidered. For this reason, the aim of this Thesis was to contribute to the optimization of the comet assay as a biomarker of DNA damage and its application in radiology, by means of the construction of a calibration curve and, also, biomonitoring of two cohorts composed by mothers and newborns recruited from two hospitals of the Barcelona province. The methodologies of the comet assay, which are applied in the different laboratories, are variable but, with no doubt, the alkaline version is the most used. This technique is more sensitive to detect lower levels of damage at pH>13. For this reason, we have chosen it as the standard version to be used in our study. We have developed numerous experiments with modifications which have optimized the assay in three aspects: the type of support utilized, which allows the manipulation of many samples in the same experiment; the use of repair enzymes in order to detect the oxidative damage; and the cryopreservation to keep many samples which can be properly evaluated in the assay. As regards the applications, the construction of a calibration curve was an important contribution because, with its use we have been able to establish the basal genetic damage of the population, in terms of breaks /109 Da DNA. Moreover, it has been proposed that the individual radiosensitivity can be measured with the comet assay. This supportsthat this technique is a satisfactory indicator of individual genomic instability. Finally, in the area of human biomonitoring, we have found a strong correlation for both the DNA basal damage and oxidative damage between mothers and children; but we couldn't clearly establish a significant influence of any of the confusion factors that has been considered in this study. This evidence has revealed related problems with the recent use of comet assay in biomonitoring studies, and has resulted in the necessity to develop specific studies in order to determine these factors, which can influence significantly on the levels of DNA damage in human populations.
Date of Award30 Nov 2009
Original languageUndefined/Unknown
SupervisorAmadeu Creus Capdevila (Director) & Ricardo Marcos Dauder (Director)

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