Uranyl nitrilotriacetate, a stabilized salt of uranium, is genotoxic in nontransformed human colon cells and in the human colon adenoma cell line LT97.

Previous uranium mining in the "Wismut" region in Germany enhanced environmental distribution of heavy metals and radionuclides. Carryover effects may now lead to contamination of locally produced foods. Compounds of "Wismut" origin are probably genotoxic via their irradiating components (radon) or by interacting directly with cellular macromolecules. To assess possible hazards, we investigated the genotoxic effects of uranyl nitrilotriacetate (U-NTA) in human colon tumor cells (HT29 clone 19A), adenoma cells (LT97), and nontransformed primary colon cells. These are target cells of oral exposure to environmentally contaminated foods and represent different cellular stages during colorectal carcinogenesis. Colon cells were incubated with U-NTA. Cell survival, cytotoxicity, cellular glutathione (GSH) levels, genotoxicity, and DNA repair capacity (comet assay), as well as gene- and chromosome-specific damage combination of comet assay and fluorescence in situ hybridization [FISH], 24-color FISH) were determined. U-NTA inhibited growth of HT29 clone 19A cells (75-2000 microM, 72 h) and increased GSH (125-2000 microM, 24 h). U-NTA was genotoxic (1000 microM, 30 min) but did not inhibit the repair of DNA damage caused by hydrogen peroxide (H(2)O(2)), 4-hydroxynonenal, and 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]-pyridine. U-NTA was also genotoxic in LT97 cells and primary colon cells, where it additionally increased migration of TP53 into the comet tail. In LT97 cells, 0.5-2mM U-NTA increased chromosomal aberrations in chromosomes 5, 12, and 17, which harbor the tumor-related genes APC, KRAS, and TP53. It may be concluded that uranium compounds could increase alimentary genotoxic exposure in humans if they reach the food chain in sufficient amounts.

New revival of an old biomarker: characterisation of buccal cells and determination of genetic damage in the isolated fraction of viable leucocytes.

Buccal cells serve as targets to assess oral exposures. We have refined isolation methods to characterise yield, viabilities, types of cells and baseline levels of genetic damage. Buccal cells were isolated from mouthwashes of 27 volunteers. They were characterised microscopically and different methods (using antibody-labelled magnetic beads, filtration and gradient centrifugation) were compared to separate epithelial cells from leucocytes. Viability of cells, DNA damage, and activity of glutathione S-transferase (GST) were measured with dye exclusion, microgelelectrophoresis, and biochemically. Mouthwashes contained approximately equal amounts of epithelial cells and leucocytes with detectable GST-activities. Repetitive determinations with mouthwashes from four individuals yielded per sample (3.5+/-1.4)x10(6) epithelial cells and (4.7+/-3.9)x10(6) leucocytes with viabilities of 8 and 94%, respectively. Epithelial cells could not be isolated using antibody-labelled beads, but cell separation with the leukocyte-specific antibody CD45 succeeded, yielding 37% leucocytes with a purity of 95% and viability of 65%. Filtering the mouthwash through a 10 microm filter yielded 57% leucocytes, with 86% purity and 94% viability. When using density gradient centrifugation as the separation method, the recovery of leucocytes was low (22%), but good results were scored for purity (95%) and cell viability (88%). This method was used to isolate leucocytes, which were then subjected to a micro-scale comet assay-modification. It was found that buccal leucocytes obtained from smokers had more DNA damage than cells from non-smokers. In conclusion, suspensions of buccal cells consist in approximately equal parts of epithelial cells and leucocytes. Only leucocytes are sufficiently viable for measuring parameters of cytotoxicity and genotoxicity or for studying modulation of gene expression. The cells are useful targets of non-invasive biomarkers, which could be incorporated as tools in many types of intervention studies.