In this work the capillary electrophoresis method developed by Schmitz for the determination of the genome-wide DNA methylation level was used and further optimised.
The surplus of the fluorescence marker BODIPY FL EDA left after derivatisation leads with the method used to fluorophoric decomposition products that produce interfering signals in the electropherogram and after some hours' storage in the autosampler prevent the analysis of a sample. To increase the sample throughput of the method, after the derivatisation the surplus of the fluorescence marker was precipitated with sodium tetraphenyl borate, dissolved in dichloromethane and separated by centrifugation from the aqueous phase, which contains the phosphoramidate to be analyzed. The sample throughput was thereby increased from 75 to 250 analyses per week .
The method was to be used i. a. to examine whether the regulation of the methylation level is suitable as a genotoxicity test. With in vivo experiments, a significant change of the methylation level in the small intestine and the lung of rats could be ascertained as early as 48 h after a single application of 3-nitrobenzanthrone. In vitro experiments then carried out with V79 cells (which express cytochrome P450 enzyme) with four cancerogens showed, because of surprisingly low DNA-adduct formation, no significant change in the methylation level. In order to understand these unexpected results better, in collaboration with several European working groups, knock-out mice, in which the cytochrome P450 was inactivated, were treated with benzo[α]pyrene (B[α]P) and their metabolism examined. To this end an HPLC method with fluorescence detection was developed, by means of which the clearance of B[α]P from mouse blood could be determined. In cooperation with the other working groups it was found that, as a result of the absence of the cytochrome P450 activity, increased DNA-adduct formation took place . This is contrary to the popular opinion that the increased level of DNA adducts results from the formation of PAH metabolites by cytochrome P450.
With the new sample preparation, the DNA-methylation level in non-mammals, such as the marbled crayfish, could also be investigated for the first time. On account of the size of these animals, only 1-μg DNA samples could be used. The methylation level was found to correlate with the size and weight of the animals .
Further, the first experiments were carried out which might indicate a connection between oxidative stress and the methylation level. For this, DNA samples of Israeli combat divers who dive with hyperbaric oxygen were compared with those of non-diving elite soldiers. However, no significant differences could be ascertained among the samples to be analysed. Perhaps there is no correlation between oxidative stress and the methylation level, but it is also possible that the duration of the oxidative stress (five months) was not long enough for a definitive statement to be made.
To determine whether the capillary electrophoresis method for determining the genome-wide methylation level could be used to analyse tissue from tumour banks, as well, this study examined whether the customary fixation of tissue with formalin has an influence on the determinable methylation level. It turned out that the methylation level of frozen kidney tissue was about 20% lower than in kidney tissue fixed with formalin. A follow-up study was therefore designed to look at whether the fixation time had a significant influence on the methylation level. For this, bladder tissue from five patients was fixed for 0, 6, 12, 18, 24, 30, 36, 42 and 48 h in formalin, and in each case the DNA methylation level was analysed. The methylation level varied greatly in both directions. This was probably due to the inhomogeneous samples, because it was afterwards discovered that the bladder tissue in all cases was inflamed and in two samples a tumour was even present. Even if the results of the study indicate the problems with tissue fixation, the relevance of the methylation level in various illnesses nevertheless becomes clear.