ToxSci Advance Access originally published online on July 13, 2006
Toxicological Sciences 2006 93(2):286-297; doi:10.1093/toxsci/kfl060
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Uranyl Nitrilotriacetate, a Stabilized Salt of Uranium, is Genotoxic in Nontransformed Human Colon Cells and in the Human Colon Adenoma Cell Line LT97
* Department of Nutritional Toxicology, Institute for Nutrition, Institute for Human Genetics and Anthropology, and Department of General and Visceral Surgery, Friedrich-Schiller-University Jena, 07743 Jena, Germany
1 To whom correspondence should be addressed at Department of Nutritional Toxicology, Institute for Nutrition, Friedrich-Schiller-University Jena, Dornburger Str. 25, 07743 Jena, Germany. Fax: +49 3641-949672. E-mail: b8pobe{at}uni-jena.de.
Received March 24, 2006; accepted June 28, 2006
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ABSTRACT |
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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 (75–2000µM, 72 h) and increased GSH (125–2000µM, 24 h). U-NTA was genotoxic (1000µM, 30 min) but did not inhibit the repair of DNA damage caused by hydrogen peroxide (H2O2), 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.
Key Words: colon cells; U-NTA; GSH; comet assay; TP53; chromosomal aberrations.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Previous extensive uranium mining in the "Wismut" region near Ronneburg (Thuringia, Germany) has enhanced environmental distribution of heavy metals and radionuclides. The compounds are considered to be hazardous based on what we know about their toxicity, but it is not known how environmental exposure to these compounds will affect human health. Several of the "Wismut"-related compounds probably are toxic through formation of free radical oxygen species (ROS) (Hei et al., 1998
), via irradiating mechanisms (radon) (Jostes, 1996), or by interacting directly with cellular macromolecules (e.g., inhibition of DNA repair) (Hartmann and Speit, 1996; Hartwig and Beyersmann 1989; Hartwig et al., 1996). The release of radionuclides and uranyl compounds into the environment during uranium mining is associated with the transfer of these compounds into the food chain (Fisenne et al., 1987; Thomas, 2000). The ingestion of contaminated soil or dust, moreover, increases the burden on the gastrointestinal system as well (Thomas, 2000).
Studies to determine the nutritional intake of radionuclides in Vietnam did not indicate an increased exposure to the noxious agents (Giang et al., 2001). The estimated uranium intake in these studies was 0.66 µg U238/day. This is lower than the estimated intake of 2.1 µg U/day at Ronneburg (Seeber, 1998). Kathren (2001) reported on the association between U238 inhalation and the risk of lung cancer. In contrast, there are hardly any investigations available on the importance of oral uranium intake.
The genotoxicity of uranium has not been investigated extensively in human cells, especially in cells of the colon, which is the major site of cancer related to nutrition. This is probably due to the lack of appropriate experimental methods. Since contamination of regional foods could increase the risk of gastrointestinal cancer, it is important that we assess the genotoxicity of "Wismut"-related compounds in human colon cells. We used normal primary colon cells, preneoplastic LT97 adenoma cells, and highly transformed tumor cells (HT29 clone 19A) to study the impacts of uranyl nitrilotriacetate (U-NTA). For this we determined a number of parameters related to the process of colorectal carcinogenesis, such as cell growth and DNA damage. The effects on DNA repair were studied as well since previous investigations had shown that heavy metals interfere with this important process of maintaining DNA integrity (Hartmann and Speit, 1996; Hartwig and Beyersmann, 1989; Hartwig et al., 1996, 2002). Furthermore, we used a new technique developed in our laboratory to determine specific damage in TP53, an important gene which is altered during colorectal carcinogenesis (Fearon and Vogelstein, 1990). For this we combined the techniques of single-cell gel electrophoresis (comet assay) and fluorescence in situ hybridization (FISH) (Schäferhenrich et al., 2003a). Last but not least, chromosomal aberrations in LT97 adenoma cells were detected with 24-color FISH. This is to our knowledge one of the first reports on the detection of cytogenetic lesions in human colon cells in vitro (Knoll et al., 2006a).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Compounds.
Uranyl acetate [UO2(OCOCH3)2·2H2O] from our laboratory stock (42.4 mg) was dissolved in 10 ml of double-distilled water (ddH2O). The solution was supplemented with the chelating agent disodium NTA (23.5 mg) (Sigma-Aldrich, Deisenhofen, Germany). The pH of the solution was adjusted to 7.4 by adding sodium hydrogen carbonate (NaHCO3). The final U-NTA concentration of this stock solution was 10mM.
Cells and culture conditions.
HT29 clone 19A is a permanent subclone derived from the carcinoma cell line HT29 treated with sodium butyrate (Augeron and Laboisse, 1984). The cells were grown in tissue culture flasks with Dulbecco modified Eaglemedium (Gibco BRL, Eggenstein, Germany) supplemented with 10% fetal calf serum (FCS) and 1% penicillin/streptomycin at 37°C in a humidified incubator (5% CO2/95% air). For this study we used passages 20–50.
The human colon adenoma cell line LT97 was established from a colon microadenoma of a patient with familial adenoma polyposis coli (Richter et al., 2002). LT97 was maintained in a culture medium containing MCDB 302 medium (Biochrom, Berlin, Germany) plus 20% of L15 Leibovitz medium, 2% FCS, and 1% penicillin/streptomycin. This medium was supplemented with 0.2nM triiodo-L-thyronine, 1 µg/ml hydrocortisone, 10 µg/ml insulin, 2 µg/ml transferrin, 5nM sodium selenite, and 30 ng/ml epidermal growth factor. Cells were grown in a humidified incubator under standardized culture conditions (5% CO2, 95% humidity, 37°C). Passages 17–33 were used for the experiments.
Primary human colon cells were isolated from surgical tissues (nontumor tissue) (Schäferhenrich et al., 2003b). The Ethical Committee of the Friedrich-Schiller-University Jena approved the study, and the tissues were made available from patients who had given their informed consent. The human colon epithelium was separated from the underlying layers of tissue by perfusion-supported mechanical disaggregation. The epithelium stripes were cut into small pieces and incubated with 2 mg/ml proteinase K (Sigma, Steinheim, Germany) and 1 mg/ml collagenase P (Boehringer, Mannheim, Germany) for 60–120 min in a shaking water bath at 37°C. After washing and centrifugation (5 min at 400 x g), the pellets were resuspended in phosphate-buffered saline (PBS: 8.0 g/l NaCl, 1.44 g/l Na2HPO4, 0.2 g/l KH2PO4, pH 7.3). Pellets containing erythrocytes were treated with erythrocyte lysis buffer (hypertonic ammonium chloride solution: 155µM NH4Cl, 5µM KCl, 0.06µM EDTA, pH 7.0), centrifuged, and resuspended in RPMI 1640 (Gibco BRL). Yield of the cells and their viability were determined using the trypan blue exclusion test. The average age of the donors was 54 ± 15 years (n = 3, male). For the fourth donor, the data are missing.
The investigations with long-time exposure (24–72 h; cell survival, glutathione [GSH] modulation, damage persistence) were done in the rapidly growing cancer cell line HT29 clone 19A to obtain just basic information on effective concentrations and basic biological activities. The comet assay was performed in all three cell types to characterize the genotoxic potential of U-NTA and the relative sensitivities of the cells in different stages of tumor development. The detection of gene-specific damage (Comet FISH) could only be performed with LT97 cells and primary colon cells since HT29 clone 19A cells have a polyploid karyotype, which prevents their usefulness in this type of assay (Schäferhenrich et al., 2003a). LT97 cells were also used to assess chromosomal aberrations because they are both capable of proliferating and have a diploid and relative stable karyotype.
Determination of cell survival.
This basal investigation was done in the fast-growing cancer cell line HT29 clone 19A to define nontoxic concentration ranges for the genotoxicity assays. Survival of HT29 clone 19A cells was determined in 96-well microtiter plates (Beyer-Sehlmeyer et al., 2003). Eight thousand cells per well were seeded, and after 24 h they were treated with 0–2000µM U-NTA dissolved in culture medium. Proliferation rates were determined after 24, 48, and 72 h of treatment. Quantification of DNA was achieved by first fixing and permeabilizing cells with methanol for 5 min, and then adding 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma-Aldrich) to stain the residual cellular DNA. DNA content was detected by fluorometric analysis at Ex/Em 360/465 nm, 30 min after application of DAPI in a Microplate Reader (Spectra Fluor Plus, Tecan, Austria; software: XFluor). Mean values of at least three independent experiments are shown in the figures. DNA content directly reflected the number of remaining cells.
Quantification of GSH.
For the GSH measurement, HT29 clone 19A cells were seeded in 96-well microtiter plates at 0.4 x 106 cells per well. After preincubating the cells for 24 h, U-NTA (0–2000µM) was added for another 24 h. Cell morphology was judged and the cells were harvested. Cell numbers and viability of the cells were determined using the trypan blue exclusion test. The cell suspensions were centrifuged (855 x g, 10 min, 4°C). GSH was measured in the cytosols. Therefore, the pellets were resuspended in 5% metaphosphoric acid (MPA, 2.5 g in 50 ml) and disrupted by ultrasound for 1 min. Fluorescence, which is proportional to the GSH content, was quantified at a wavelength of 400 nm in a microtiter plate fluorescence photometer against MPA.
Determination of genetic damage.
The alkaline version of the comet assay was performed according to the guidelines published by Tice et al. (2000). This method allows the detection of compound-induced DNA damage, including DNA single-strand breaks (SSBs), alkali-labile sites, DNA-DNA/DNA-protein cross-links, and SSB resulting from incomplete excision repair (Tice et al., 2000). LT97 cells grown in 25-cm2 tissue flasks were washed with PBS containing EDTA (5mM) and subsequently removed from the surface of the flasks with trypsin/versene (10% [vol/vol], Invitrogen, Karlsruhe, Germany) at 37°C for 2–5 min. The cells were then taken up in RPMI to yield suspension cultures. HT29 clone 19A cells were processed in the same manner. Primary human colon cells were isolated as described above. Cell numbers and viabilities were determined with the trypan blue exclusion test, and the cell number was adjusted to 2 x 106 cells/ml. Aliquots of these cell suspensions were centrifuged (380 x g, 5 min), and the cell pellets were incubated with 0–1000µM U-NTA at 37°C for 30 min on a thermomixer. We used Fe-NTA as positive or reference control in the comet assay investigations of this study. NTA treatment alone was not performed since our previous studies had not shown an effect of NTA using comet assay and Comet FISH (Knöbel et al., 2006). The suspensions were then centrifuged (380 x g, 5 min), and the cell pellets were washed in PBS. Twenty microliters of the cell suspensions was stored on ice for determination of cell numbers and viability. The remaining cell suspensions were centrifuged (380 x g, 5 min), and the pellets were mixed with agarose and distributed onto microscopic slides. After the agarose solidified, cells on slides were lysed for at least 60 min (pH 10). For DNA unwinding (pH > 13), the slides were placed in an electrophoresis chamber. After 20 min the electrophoresis was carried out at 1.25 V/cm and at 300 mA for another 20 min. The slides were neutralized by washing three times, each for 5 min, and afterward they were stained with SYBR-Green (1 µl/ml, 30 µl per slide). All steps of the comet assay were conducted under red light.
The images were quantified by microscopic evaluation using the image analysis system of Kinetic Imaging (Liverpool, UK). The percentage of fluorescence in the tail (tail intensity) was measured for 50 cells per slide. The means of three replicate slides were used to calculate the means of at least three independently reproduced experiments.
Persistence of DNA damage (DNA repair).
To investigate the influence of a low uranium dose on the persistence of induced DNA damage, HT29 clone 19A cells were incubated for 24 h with 10µM U-NTA or RPMI and then were harvested as described above. After centrifugation, the cell pellets were resuspended in PBS or RPMI containing 75µM hydrogen peroxide (H2O2), 200µM 4-hydroxy-2-nonenal (HNE), or 25µM 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]-pyridine (NOH-PhIP) for 5 min at 4°C (H2O2) or for 30 min at 37°C (HNE and NOH-PhIP). One quarter of the cells were applied to the slides, and the remaining cells were incubated at 37°C to allow DNA repair processes to begin (postincubation period). These cells were distributed onto slides after 30, 60, and 120 min of postincubation. All slides were then incubated in lysis solution for at least 60 min. The H2O2-damaged DNA was also digested with endonuclease III (Endo III) for 45 min to determine oxidized pyrimidine bases along with other endonuclease-specific lesions. Alternatively, the slides were treated with formamidopyrimidine-glycosylase (Fpg) for 30 min to determine oxidized purine bases, among other Fpg-specific lesions (Collins et al., 1995). Subsequently, all steps of the comet assay were carried out as described above.
Determination of gene-specific damage in TP53 (Comet FISH).
For the Comet FISH experiments, we used LT97 adenoma cells and primary colon cells. Direct Texas Red–labeled TP53 probe was a mixture of directly labeled probes that were specific for genomic sequences, which included the TP53 locus (microdissection derived probe spanning 17pter-p12) (Liehr et al., 2002). FISH experiments were performed according to Schäferhenrich et al. (2003a) but were adapted to the hybridization conditions needed for the Texas Red–labeled TP53 probes used here. The dehydrated slides from the comet assay were rehydrated in ddH2O for 10 min. Afterward, the target DNA was denatured with 0.5M NaOH for 30 min and then neutralized in 0.01M PBS for 1 min. The slides were dehydrated through an ethanol series (70, 80, and 95%, each for 5 min) and dried at room temperature. Thirty microliters of Hybrisol VI (Oncor, Gaithersburg, UK) was dropped on each slide gel and spread with a plastic coverslip. The TP53 probe (5 µl for each slide) was diluted with Hybrisol VI (7 µl for each slide) and denatured at 75°C for 5 min, 4°C for 2 min, and 37°C for 30 min. This hybridization mixture (12 µl per slide) was added to the slides, covered with a plastic coverslip (24 x 24 mm), and incubated in hybridization chambers for 72 h at 37°C. The coverslips were then removed, and the slides were washed for 5 min in 2x saline sodium citrate (0.3M NaCl, 0.03M Na citrate, pH 7.2) without formamide at 65°C. This step was followed by a 5-min washing in 1x phosphate-buffered detergent (Oncor) at room temperature. SYBR-Green (1 µl/ml, 30 µl per slide) was used to counterstain the Texas Red–labeled probes. Negative controls were included for each concentration on the same slide using Hybrisol VI without the DNA probe (24 x 24-mm coverslip).
For evaluating Comet FISH experiments, the comets were categorized into four classes representing different degrees of DNA damage ranging from nondamaged to severely damaged images (classes 1–4) (Wollowski et al., 1999). Also, the total numbers of TP53 signals per cell were scored. The expected number of spots in a normal interphase nucleus is two. The hybridization efficiency was calculated on the basis of the number of cells without signals, with only one signal, and with two and more signals. Moreover, cells with broken fluorescent signals were counted as an indication of breaks which occur directly in the gene. For the further Comet FISH evaluation, only cells with two fluorescent spots were used and the localization of the TP53 signals in the comet head or comet tail was recorded. The parameter of TP53 migration was based on the percentage of damaged cells (belonging to comet classes 2–4), in which we could find a migration of at least one TP53 signal into the comet tail. One hundred to 120 images per slide were measured during the Comet FISH evaluation.
Fluorescence microscopy was performed using a ZEISS Axiovert M100 (Carl Zeiss Jena GmbH, Jena, Germany) equipped with filters to detect Texas Red (red, ZEISS filter No. 15) and SYBR-Green (green, ZEISS filter No. 09). Images were captured using a MicroMAX Digital CCD camera (BFI OPTILAS GmbH, Puchheim, Germany). Meta View Imaging Software (Visitron Systems GmbH, Puchheim, Germany) was used to capture and process the images.
Determination of chromosomal aberrations (24-color FISH).
The protocol was based on the guidelines of the OECD (1997). To detect U-NTA–induced chromosomal aberrations, subconfluent grown LT97 cells of the same passage were treated with medium, U-NTA (0.5, 1, and 2mM), the solvent control NTA, or the positive control compound ethylmethanesulfonate (EMS, 1mM) for 6 h without gas exchange. Cell culture medium served as a negative control. LT97 cells were grown under standard conditions. After the medium was changed, the culture was further incubated for 46 h to allow the minimum one reproduction cycle.
Thereafter, metaphase chromosomes were prepared according to standard protocols for 24-color FISH (Claussen et al., 2002). Further steps were performed as described by Schäferhenrich et al. (2003a). In short, prepared slides for cytogenetic analyses were pretreated to perform multicolor FISH (M-FISH) hybridization according to previously described techniques (Senger et al., 1998). After incubating the samples for 48 h (37°C) with the probe mixture, they were subsequently washed and the detection was carried out. Metaphase images were captured using a fluorescence microscope (Axioplan 2, Zeiss, Germany) with a PCO VC45 CCD camera (PCO, Kehl, Germany) and suitable filter combinations (DAPI/fluorescein isothiocyanate/Spectrum Orange/Texas Red/Cyanine 5/Diethylamino-coumarine). Evaluation was done with the ISIS3-software (MetaSystems, Altlussheim, Germany). Using this method, each of the 24 different chromosomes (22 autosomes, X, Y) was labeled with a specific combination of fluorochromes that allowed an unambiguous identification of the origin of the chromosomal material (Speicher et al., 1996). Fifty metaphases were analyzed per concentration.
Statistical evaluation.
Each experiment was independently reproduced three to four times, as is specified in the legends to the figures. There were two to three replicates for each exposure level within each experiment. The GraphPad Prism software Version 4.0 (GraphPad Software, Inc., San Diego, CA) was used for establishing significance levels. Statistics were carried out on the mean values of the data, and these are also presented (mean and standard deviation) in the figures and tables. As is specified in the figure legends, one-way analysis of variance (ANOVA) with Dunnett posttest or two-way ANOVA with Bonferroni posttest was used whenever appropriate.
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RESULTS |
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Cell Survival
U-NTA was analyzed for its effects on cell growth (or rather cell survival) to determine cytotoxic dose, in relation to treatment time and concentration. These studies were performed with the most highly proliferating cells, namely, with the HT29 clone 19A tumor cell line. Figure 1 shows that at 24- and 48-h treatments with U-NTA, there was a significant growth arrest only at the highest tested U-NTA concentration, which was 2000µM (p < 0.01). After 72 h incubation, however, U-NTA was cytotoxic in a wide concentration range. A significant decrease in cell number was observed from 75 to 2000µM U-NTA (p < 0.01, p < 0.001). The 50% effective concentration (EC50) at 72 h was reached already at 215.2 ± 19.2µM U-NTA.
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GSH Content in the Cells
Reduced GSH catalyzes the detoxification of electrophilic compounds, such as reactive oxygen species and free radicals. Moreover, intracellular GSH levels are directly related to the cell density and thus to growth properties. Therefore, we also investigated the influence of a uranium incubation on the GSH level in HT29 clone 19A cells. HT29 clone 19A cells were treated for 24 h with U-NTA. Figure 2 shows that there was a significant increase of the GSH content in the U-NTA–treated cells in comparison with control cells (a, vs. medium 125–2000µM U-NTA; b, vs. NTA 500–2000µM U-NTA).
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Effects on DNA Damage
The key aim of this work was to determine whether U-NTA is genotoxic in human colon cells. For this we first analyzed DNA strand breaks at concentrations which did not affect cell growth (30 min, 0–1000µM). We also did not observe cytotoxic effects of our substances at these tested concentrations when using other methods to determine viability. Thus, with the trypan blue exclusion assay, the viability was between 70 and 100%. Table 1 shows these data as well as the impact of U-NTA on induction of strand breaks in HT29 clone 19A cells, LT97 cells, and primary colon cells after 30 min of treatment. At these doses, U-NTA was genotoxic in the different cell types. These effects were significant in HT29 clone 19A cells at 1000µM U-NTA (p < 0.01), in LT97 cells at 500µM (p < 0.05) and at 1000µM (p < 0.01), and in primary colon cells at 1000µM U-NTA (p < 0.05). HT29 clone 19A cells reacted differently than the other two cell types and were significantly less sensitive.
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Persistence of DNA Damage (DNA Repair)
The effects of U-NTA on repair of damage by putative colon carcinogens were investigated at 10µM for 30, 60, and 120 min. These were conditions that had been found to be nongenotoxic and were also previously reported to be effective for other metals. We did not observe an influence of 10µM U-NTA (24 h, 37°C) on the repair of damage induced by our genotoxic agents. For instance, H2O2-induced damage (75µM) was removed after 30 to 120 min postincubation (p < 0.01), without additional effects of U-NTA (Table 2). In addition to SSBs, H2O2 induces oxidized bases. H2O2-induced oxidized pyrimidine bases plus strand breaks were also repaired significantly (p < 0.01) after 30 min, but U-NTA again did not have an additional effect on the persistence of this type of damage (Table 3).
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NOH-PhiP–induced DNA damage was significantly repaired after 60 min postincubation (p < 0.01). Preincubation with the nongenotoxic U-NTA concentration of 10µM, however, did not affect damage persistence (Table 2).
Finally, HNE-induced DNA damage was also repaired, but without statistical significance. Again, there was no detectable influence of U-NTA on the persistence of induced DNA damage (Table 2).
Effects on TP53 (Comet FISH)
This set of experiments addresses the possibility that U-NTA specifically damages genes, which are known to be altered during the carcinogenesis process. For Comet FISH experiments, we used LT97 and primary colon cells. Damage induced in TP53 was evaluated by first grouping the images into comet classes. We determined the total number of signals per slide and per cell. A normal metaphase or interphase nucleus has two spots. The initial evaluation was done to exclude cells without and with only one signal and to determine the hybridization efficiency. This was found to be 86.8 ± 2.2% for primary cells and 83.4 ± 1.2% for LT97 cells. Only very few comets had three signals, which would indicate an actual break of the TP53 gene. For further Comet FISH evaluation, only cells with two spots were used.
Figure 3 shows the frequency of comet classes 2–4 (damaged cells) resulting from the treatment of primary colon cells and LT97 with U-NTA (30 min, 37°C). It is apparent that the total score of damaged cells increased with increasing concentrations of the test compound. A significant shift was observed primarily in the direction of the stronger damaged cells. Thus, U-NTA significantly caused DNA damage, in comparison to the medium control, already at 100µM U-NTA in primary human colon cells and in LT97 adenoma cells. The differences in the response between primary colon cells and LT97 adenoma cells were not significant.
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Figure 4 shows the proportion of damaged cells with the TP53 signals in the comet tail. We observed a concentration-dependent TP53 migration into the comet tail, which was significant compared to medium control at 500 and 1000µM for primary colon cells and at 1000µM for LT97 cells. Significant differences between responses of the two cell types were not apparent.
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Effects on Chromosome-Specific Damage (24-color FISH)
Figure 5 exemplifies some cytogenetic alterations in representative 24-color-FISH karyograms of each data point. The depicted alterations in the U-NTA–treated cells are compound related and have not been observed in the medium control and during previous routine analysis of LT97 karyotype (Schäferhenrich et al., 2003a
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Figure 6 describes the range of observed chromosomal abnormalities in LT97 adenoma cells, which were translocations, deletions, and isochromosomes. It is apparent that EMS mainly caused translocations, whereas uranium caused more deletions. The proportion of translocations, however, increased with increasing U-NTA concentrations as well. The number of aberrant metaphases is shown in Figure 7. The total score of aberrant metaphases increased with increasing concentrations of U-NTA. Interestingly, when regarding only chromosomes 5, 12, and 17, which harbor the tumor-related target genes APC (adenomatous polyposis coli), KRAS (Kirsten rat sarcoma), and TP53, 2mM U-NTA caused a relatively higher proportion of damage than was expected for EMS. The data clearly point to a cytogenetic potential of U-NTA in human colon adenoma cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Natural uranium is weakly radioactive and potentially chemotoxic (Burkart, 1991
). Target organs for uranium toxicity are the kidneys, as has also been shown for cadmium, lead, and mercury (Meinrath et al., 2003). Drinking water and food are the primary sources of natural uranium (Fisenne et al., 1987; UNSCEAR, 2000). The daily intake is estimated to be 1.5 µg from water and 1–2 µg from diet (ATSDR, 1999). The Agency for Toxic Substances and Disease Research sets the tolerable daily intake (TDI) level for uranium to 1 µg/kg/day (ATSDR, 1999). Jacob et al. (1997) suggests a TDI of 0.7 µg/kg/day, whereas the World Health Organization (WHO, 1998) recommends a TDI of less than 0.6 µg/kg/day. In recent years, several groups have published articles related to the toxicological assessment of uranium and other radionuclides (Cardis and Richardson, 2002; Priest, 2001). Some of this work was related to studies of exposure to depleted uranium (DU) used in the production of artillery (Durakovic, 2001; Giannardi and Domininci, 2003; McDiarmid et al., 2000). The radiological toxicity of uranium results from its alpha-, beta-, and gamma-emissions (Meinrath et al., 2003). Because of the long half-life of U-NTA and the relatively short incubation times in our experiments (0.5–72 h), the results of our studies probably only reflect the chemical toxicity of the compound.
One important mechanism of chemical toxicity could be the generation of oxidative stress. Uranium has been shown to catalytically oxidize ascorbate to dehydroascorbate at low pH (0.99–2.00) in the presence of dioxygen (Taqui Khan and Martell, 1969). Hamilton et al. (1997) could show that uranyl nitrate produces hydroxyl radicals in the presence of H2O2 at pH < 4. In in vitro experiments with plasmid DNA, Yazzie et al. (2003) showed that uranyl-acetate induced SSBs after reaction with ascorbate. Their data suggest that uranium may be directly genotoxic and may, like chromium, react with DNA by more than one pathway. Lund et al. (1998) showed an association of ROS with toxicity related to crypt cell proliferation in the large intestine of rats. Finally, another response to oxidative stress could be an enhanced cellular level of the tripeptide GSH, which is essential for detoxification. Metals can inhibit antioxidative enzymes and decrease intracellular GSH (Quig, 1998). Hg and Cd have high affinities for GSH, so they lead to a GSH depletion in the cells. In contrast, Seymen et al. (1997) showed that iron supplementation increases GSH, GSH-Px, and superoxide dismutase (SOD) levels in erythrocytes. This finding corresponds to our results with Fe-NTA (Knöbel et al., 2006). In this study, U-NTA also significantly increased intracellular GSH.
Next to cytotoxicity, ROS are also genotoxic, and uranium has also been shown to have DNA-damaging effects. Reactions of H2O2 with U(IV) may generate the DNA-damaging hydroxyl radical (Taqui Khan and Martell, 1969). Yazzie et al. (2003) reported genotoxic effects of U(VI) in plasmid DNA after reduction with ascorbate, but the authors could not confirm the Fenton-type chemistry. Oxidative damage was recently found in DU-treated calf thymus DNA in the presence of H2O2 and ascorbate (Miller et al., 2002). Another possible mechanism of uranium genotoxicity could be the direct interaction of uranyl cation with the DNA, as has been suggested previously (Franklin, 2001).
HNE and H2O2 are genotoxic products of oxidative stress (Abrahamse et al., 1999; Knoll et al., 2006a). In a recent study we have been able to show that both compounds are genotoxic in human colon cells and that their genotoxicity can be reduced by GSH/glutathione-S-transferases (Pool-Zobel et al., 1996). To study more interactions, we therefore assessed how low doses of U-NTA affect the genotoxicity of these compounds and the repair of the DNA damage that they cause. It had, for instance, been shown that very low doses of Fe-NTA clearly increased the DNA-damaging effects of HNE, indicating synergistic potentials of the combination HNE + Fe-NTA (Knöbel et al., 2006). Here, using the same approach with uranium, we did not see similar effects. We also did not observe that U-NTA affected the DNA repair of H2O2-mediated damage or of HNE- and NOH-PhiP–induced DNA damage. In contrast, similar types of interactions have been detected with other carcinogenic metal compounds, such as nickel, cadmium, arsenic, and cobalt. Nickel, cadmium, cobalt, and arsenic interfere with the nucleotide and base excision repair at low, nongenotoxic concentrations (Hartwig and Schwerdtle, 2002). As a possible mechanism of repair inhibition, it was suggested that Ni(II) replaced other divalent ions in repair enzymes, which are essential for enzyme activity (Hartwig and Beyersmann, 1989). Here, we saw no indication that U-NTA modulates the DNA repair of damage caused by different genotoxins, and thus U-NTA probably does not inhibit repair by this mechanism.
Putative carcinogens initiate colon cancer, presumably by first damaging the APC gene. This step is followed by alterations of KRAS and SMAD4 (mothers against decapenta-plegic homolog 4) (Potter, 1999). These genetic alterations may cause cells to proliferate more rapidly and thus form aberrant crypts, polyps, and microadenomas. Mutations in the tumor suppressor gene TP53 are the final genetic changes in colon carcinogenesis and they are responsible for converting adenoma cells into invasive carcinoma cells. In order to study whether U-NTA damages human colon cells in tumor-relevant genes, such as in TP53, we stained comet images of the comet assay slides using FISH technique. U-NTA caused a dose-dependent migration of TP53 into the comet tail in both LT97 and primary colon cells. Treatment of cells with high U-NTA concentrations was associated with a detectable migration of TP53 in about 40–50% of the damaged cells. Thus, we can conclude that the tumor suppressor gene TP53 is sensitive toward U-NTA at high concentrations, especially in primary human colon cells. Comparable results were obtained by Schäferhenrich et al. (2003a) with HNE- and H2O2-mediated genotoxic damage in LT97 cells. They concluded that these compounds were more effective in damaging TP53 than total genomic DNA.
Our LT97 adenoma cells were derived from preneoplastic stages of tumorgenesis and were thus interesting target cells to study clastogenic effects of U-NTA (Knoll et al., 2006a). EMS was used as the positive control compound. It is an alkylating agent which induces apurinic and apyrimidine sites (AP sites) and forms SSBs in the next S-phase. These SSBs are converted into double-strand breaks in the second S-phase (Kaufmann and Paules, 1996). We incubated our cells for 6 h, after which a medium change was performed. The culture was further incubated for 46 h to allow the minimum one reproduction cycle. Since the cells are not all in the same phase of the cycle when the incubation starts, the cells pass different numbers of S-phases. In our study we could also demonstrated that EMS induce translocations or chromosome deletions (Knoll et al., 2006b). Here, uranyl acetate was shown to be clastogenic in LT97 cells, where it also caused deletions and translocations. The proportion of tumor-related chromosomes in the total score of damaged chromosomes was about 30% (2mM U-NTA). These results support the hypothesis that chromosomal rearrangements might be causally involved in early stages of carcinogenesis. Lin et al. (1993) found increased frequencies of micronuclei, sister-chromatid exchanges, and chromosomal aberrations in CHO cells after incubation with uranyl nitrate (10–300µM). Opposed to this, McDiarmid et al. (2000) did not observe chromosomal alterations in peripheral blood lymphocytes of Gulf War veterans, who were exposed to DU. The same was true of uranium-mining workers experiencing higher and longer exposures to uranium-associated compounds, who did not show an increased incidence of chromosomal aberrations in the white blood cells (Lloyd et al., 2002). In contrast, a group of workers from the "Wismut" mines exhibited a significantly increased incidence of micronuclei in their lung macrophages (Popp et al., 2000). These differences could possibly be due to varying types and the extents of exposures or to differences in the target cells that were examined.
We were able to detect significant genotoxic and gene-specific effects at higher concentrations (at least 100µM) that normally do not occur in our environment. There are, however, geographical regions with high concentrations of uranium and other radionuclides in the groundwater resulting from uranium mining or processing or from special events like reactor accidents or use of atomic bombs. This is associated with a higher transfer of these compounds into the water cycle and food chain (Fisenne et al., 1987; Priest, 2001). The burden of the gastrointestinal system by consuming contaminated soil, dust, food, or drinking water has been shown to increase (Thomas, 2000). Kurttio et al. (2002) measured 131 µg U/l drinking water in South Finland, which exceeded the WHO (2004) guideline level of 15 µg/l. The authors postulated that an increase in the daily intake of uranium from drinking water by 1 µg was associated with an increase of 0.21 ng U/mmol creatinine in urine. They detected a mean urine concentration of 424 µg U/l. This concentration (424 µg U/l = 1.8µM) is only about 50-fold lower than the concentration, which was effective in our study. Considering the longer term exposure durations in humans as compared to the treatment periods in our cell culture experiments, the respective doses may be even more similar to each other. A number of other investigations have also reported on detectable urinary uranium concentrations after environmental uranium exposure, which, however, did not reach the levels reported by Kurttio et al. (2002; Hooper et al., 1999; Orloff et al., 2004). In any case, the available data do suggest that the contamination of regional foods in the human food chain could increase genotoxic exposure and thus the risk of gastrointestinal diseases for exposed populations (Küppers and Schmidt, 1994).
In summary, U-NTA was cytotoxic in HT29 clone 19A tumor cells. U-NTA was also genotoxic in nontransformed human colon cells and in a cell line derived from a human colon adenoma. Health effects from environmental uranium exposures have been well documented in the past, but the potential genotoxicity to the gastrointestinal tract via dietary exposure was not previously considered. The results of our study, therefore, add support to the assumption that this contaminant may increase alimentary genotoxicity in humans if it reaches the food chain in sufficient amounts. It is therefore necessary, in the future, that these types of exposures be included in environmental studies.
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NOTES |
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The authors certify that all research involving human subjects was done under full compliance with all government policies and the Helsinki Declaration.
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ACKNOWLEDGMENTS |
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This work has been carried out with financial support by Deutsche Forschungsgemeinchaft project number PO 284/6-1 and PO 284/6-2. Furthermore, we are thankful to Dr. William M. Pool for editorial assistance and to Prof. B. Marian, Institute of Cancer Research, University of Vienna, Austria for the generous gift of LT97 adenoma cells.
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