Effect of different hemodialysis regimens on genomic damage in end-stage renal failure.

Patients with end-stage renal disease display enhanced genomic damage that may have pathophysiologic relevance for cancer development and cardiovascular complications. We investigated to what extent the genomic damage in peripheral blood lymphocytes can be modulated #1: by initiation of standard hemodialysis (SHD) in formerly conservatively treated end-stage renal disease patients, #2: by a switch from SHD to hemodiafiltration, and #3: daily dialysis (DHD). Genomic damage was evaluated by the micronuclei (MN) frequency test and the comet assay (CA). In a prospective study we found that initiation of SHD did not induce significant changes of genomic damage in peripheral blood lymphocytes whereas the change to hemodiafiltration improved the percentage of DNA in the tail as measured by CA without modulating the MN frequency. In a cross-sectional investigation the degree of genomic damage as evaluated by MN frequency was significantly lower in a patient group treated by DHD as compared with a group treated by SHD. In the DHD patients there also was a significant decrease of the plasma concentrations of urea and the advanced glycation end products imidazolone A, carboxymethyllysine, and of advanced glycation end product-associated fluorescence.

Time-dependent effects of sodium arsenite on DNA breakage and apoptosis observed in the comet assay.

To assess genotoxic effects of sodium arsenite (NaAsO2) the single-cell gel electrophoresis (comet assay) had been conducted in various studies indicating genotoxicity. However, DNA fragmentation due to NaAsO2-induced apoptosis may constitute a bias in the interpretation of the results. Apoptotic cells can show typically large and diffuse comets, which are usually excluded during genotoxicity analysis. It is controversial whether there is a time-window in which the apoptotic process generates comets that would falsely be interpreted to be the result of genotoxic DNA damage. Therefore, we evaluated frequency histograms for single-cell measures of tail DNA (% DNA in comet tail) in 30-min intervals after incubation of mouse lymphoma L5178Y cells with sodium arsenite (NaAsO2). In parallel, we evaluated apoptosis by measuring annexin V-positive cells with flow cytometry, and visualized apoptotic cells on slides by Hoechst bisbenzimide 33258 staining. The first observed effect at 30 min after treatment was an increase in annexin V-positive cells. At about 60 min the number of cells with moderate DNA migration increased in the comet-assay analysis. After 90 min, an increase in the number of cells with high levels of DNA migration was observed, which resulted in a bimodal distribution of cells with moderate and high levels of DNA migration. Hoechst-stained apoptotic cells could only be observed at later times (> or = 120 min). This means that the treatment would have been considered to be genotoxic if analysed at 120 min even if the cells with high levels of DNA migration would have been excluded. The occurrence of annexin V-positive cells preceded the appearance of cells with moderate levels of DNA migration. We hypothesize that these cells were early apoptotic cells and not indicative of genotoxic damage. We conclude that DNA-damaging effects of NaAsO2 cannot adequately be interpreted if the comet assay is not accompanied by separate analysis of early endpoints for induction of apoptosis.