Concomitant detection of CYP1A1 enzymatic activity and CYP1A1 protein in individual cells of a human urothelial cell line using a bilayer microfluidic device.

To understand molecular networking at the cellular level, analyses of processes and effects at the single-cell level are most appropriate. Usual biochemical or molecular biological analyses are based on integrated signals of numerous cells which differ, however, in their expression and activity profiles. Here we show that it is possible to determine different types of properties of individual cells by means of a specifically designed microfluidic device. As part of investigations to characterize the human urothelial cell line 5637 as a potential model system for studies of toxic and carcinogenic effects on urothelial cells, we use this cell line to assign cytochrome P450 activity, and expression of the enzymes involved, to individual cells. It is shown that the cell population is very heterogeneous with respect to the extent and kinetics of CYP1A1-dependent ethoxyresorufin O-deethylase (EROD). This is also true for the cells' CYP1A1 protein content. With some exceptions, the EROD activity largely coincides with the presence of CYP1A1 protein in the cells. The results obtained with the microfluidic device are promising and open up new perspectives with regard to multi-property determinations in individual cells and to studies focusing on the biochemical and molecular heterogeneity of cells.

Capillary electrophoresis-laser induced fluorescence analysis of endogenous damage in mitochondrial and genomic DNA.

Reactive oxygen molecules are formed in vivo as by-products of normal aerobic metabolism. All organisms dependent on oxygen are inevitably exposed to these species so that DNA damage can occur in both genomic and mitochondrial DNA (mtDNA). In order to determine endogenous DNA damage we have developed an analytical method that involves the isolation and hydrolysis of genomic DNA or mtDNA, the labeling of modified and unmodified nucleotides and micellar electrokinetic chromatography with laser-induced fluorescence detection. With this method we have found etheno-adenine, thymine glycol, uracil, hypoxanthine, and 5-methylcytosine. These were identified by the addition of internal standards to the genomic or mtDNA. There are a large number of other signals in the electropherograms of mtDNA that we have never found in genomic DNA analysis because they are at lower concentration in the genome. In the DNA of untreated patients with chronic lymphocytic leukemia (CLL), uracil and high levels of etheno-adenine were found, which can be explained by antioxidant enzyme alterations and oxidative stress in the CLL lymphocytes.