Increase In Chromosome Damage Research Articles

Influence of Asiatic acid on cell proliferation and DNA damage in vitro and in vivo systems.

Asiatic acid (AA) is a triterpene with promising pharmacological activity. In the present study, in vitro and in vivo assays were conducted to understand the effect of AA on cell proliferation and genomic instability. AA was cytotoxic to human tumor cell lines (M059J, HeLa, and MCF-7), with IC50 values ranging from 13.91 to 111.72 µM. In the case of M059J, AA exhibited selective cytotoxicity after 48 h of treatment (IC50 = 24 µM), decreasing the percentage of cells in the G0/G1 phase, increasing the percentage of cells in the S phase, and inducing apoptosis. A significant increase in chromosomal damage was observed in V79 cell cultures treated with AA (40 µM), revealing genotoxic activity. In contrast, low concentrations (5, 10, and 20 µM) of AA significantly reduced the frequencies of micronuclei induced by the mutagens doxorubicin (DXR), methyl methanesulfonate, and hydrogen peroxide. A reduction of DXR-induced intracellular free radicals was found in V79 cells treated with AA (10 µM). The antigenotoxic effect of AA (30 mg/kg) was also observed against DXR-induced chromosomal damage in Swiss mice. Significant reductions in p53 levels were verified in the liver tissue of these animals. Taken together, the data indicate that AA exerted antiproliferative activity in M059J tumor cells, which is probably related to the induction of DNA damage, leading to cell cycle arrest and apoptosis. Additionally, low concentrations of AA exhibited antigenotoxic effects and its antioxidant activity may be responsible, at least in part, for chemoprevention.

Preserved riparian forest protects endangered forest-specialists amphibian species against the genotoxic impact of sunlight and agrochemicals

Although agrochemicals and solar ultraviolet (UV) radiation are both environmental stressors currently associated with amphibian decline, little is known about their interactive effects on amphibian larvae. One potential hazardous consequence of this interaction could be the increase of chromosomal damage and induction of cell death, which culminate in tadpole mortality. In this study, the preservation level of the riparian forest of four different streams located at the Atlantic Forest in southern Brazil was evaluated through satellite imagery analysis. The incidence of solar UVB and UVA radiation were measured with a radiometer in the amphibian breeding sites, and the presence of agrochemicals in the water was quantified through liquid chromatography coupled in tandem to mass spectrometry. Tadpole population density of two endangered forest-specialist amphibian species (Boana curupi and Crossodactylus schmidti) was evaluated in each stream, and the chromosomal damage of the collected larvae was determined through cytogenetic analysis of blood samples. The results clearly indicate that the preservation of riparian forest efficiently blocks the incidence of UV radiation upon the water surface and reduce by 2.7-fold the entry of agrochemicals from adjacent agricultural crops. Tadpoles collected in streams with preserved and continuous riparian forest presented a significantly lower amount of micronuclei, as well as higher population density in comparison to tadpoles collected in streams located in converted agricultural landscape. Therefore, this study demonstrates that the preservation of the riparian forest has paramount importance in reducing the impact of environmental genotoxic agents into lotic breeding sites used by forest-specialist amphibian species.

86. Biological effects evaluation in peripheral blood lymphocytes from patients undergoing radium-223 (223RaCl2) therapy

Purpose The purpose of this study is to correlate the physical dose to non-target tissues (blood) to the radiation-induced chromosome damage in terms of the dicentrics and micronuclei (MN) in peripheral blood lymphocytes (PBL) after radium-223 (223RaCl2) therapy in patients with bone metastasis derived from prostate cancer [1] . Methods Four patients undergoing 223RaCl2 therapy for skeletal diseases have been enrolled in this prospective clinical study. The effective dose to blood per injected activity was calculated considering the alpha, beta and gamma emission of Ra-223 and considering a standard man weight of 70 kg. PBL cultures for dicentric assay and MN were performed before treatment (T0), 6 days (T1) and 30 days (T2) after the first cycle of treatment and after the end of the therapy (T3). Haematological toxicity parameters (blood cell count) have been monitored during therapy and analysed along with doses to blood. Results The administration of 223RaCl2 produces a high dose dependent increase of radiation-induced chromosome damage in terms of dicentrics and MN induction observed in the circulating lymphocytes. Surprisingly, the increase of chromosome damage observed between T1 and T2 is not due to an 223RaCl2 addition dose, suggesting that circulating lymphocytes were exposed to an extra dose by the emissions from the target areas. In addition, clinical monitoring during the treatment showed a progressive increasing fatigue, leucopenia and anemia with a partial recovery after some months after the end of therapy. Conclusions The cytogenetic data suggest a persistence of the radiation emission from the target tissue to non-target ones and seem to be correlated to the observed haematological toxicity, highlighting possible adverse effects related to this therapy. These data need further investigation in order to evaluate the potential side effects to normal, non-target tissue in patients treated with alpha emitters.

Geraniol and linalool anticandidal activity, genotoxic potential and embryotoxic effect on zebrafish.

Geraniol and linalool are major constituents of the essential oils of medicinal plants. Antifungal activity of geraniol and linalool were evaluated against five Candida species. The genotoxicity of these compounds was evaluated by the cytokinesis-block micronucleus test, and the embryotoxic assays use zebrafish model. Geraniol and linalool inhibited Candida growth, but geraniol was more effective. The geraniol at concentration of 800μg/ml and the linalool at concentration of 125μg/ml significantly increased chromosome damage. Geraniol was more toxic to zebrafish embryo than linalool: LC50 values were 31.3 and 193.3μg/ml, respectively. Geraniol and linalool have anticandidal activity, but they also exert genotoxic and embryotoxic effects at the highest tested concentrations.

OA168] Biological effects evaluation in peripheral blood lymphocytes from patients undergoing radium-223 chloride (223RaCl2) therapy

Purpose The purpose of this study is to correlate the physical dose to non-target tissues to the radiation-induced chromosome damage in terms of the dicentrics and micronuclei (MN) in peripheral blood lymphocytes (PBL) after radium-223 chloride (223RaCl2) therapy in patients with bone metastasis derived from prostate cancer [1] . Methods Four patients undergoing 223RaCl2 therapy for skeletal diseases have been enrolled in this prospective clinical study. The effective dose to non-target tissues per injected activity was calculated considering the alpha, beta and gamma emission of Ra-223 and considering a standard man weight of 70 kg. PBL cultures for dicentric assay and micronuclei induction were performed before treatment (T0), 6 days (T1) and 30 days (T2) after the first cycle of treatment. Haematological toxicity parameters (blood cell count) have been monitored during therapy and analysed along with doses to blood and non-target tissues. Results The administration of 223RaCl2 produces a high dose dependent increase of radiation-induced chromosome damage in terms of dicentrics and micronuclei observed in the circulating lymphocytes (non target-tissue). Surprisingly, this increase of chromosome damage observed is not due to an 223RaCl2 addition dose, suggesting that circulating lymphocytes were exposed to an extra dose by the emissions from the target organs. In addition, clinical monitoring during the treatment showed a progressive increasing fatigue, leucopenia and anemia with a partial recovery after some months after the end of therapy. Conclusions The cytogenetic data seem to suggest a persistence of the radiation emission from the target tissue to non-target ones and seem to be correlated to the observed haematological toxicity, highlighting possible adverse effects related to this therapy. These data need further investigation in order to evaluate the potential side effects to normal, non-target tissue in patients treated with alpha emitters.

Prevention of high-temperature-induced chromosome damage in mouse spermatozoa freeze-dried using Ca2+ chelator-containing buffer alkalinized with NaOH or KOH

In order to protect sperm chromosomes against degradation when they are being stored for relatively high temperatures, we investigated the optimal pH of the freeze-drying solution, EGTA/Tris-HCl buffered solution alkalinized by NaOH (Na-ETBS) or KOH (K-ETBS). Mouse spermatozoa suspended in Na-ETBS or K-ETBS were freeze-dried at pH 5.0–8.4 and stored at 4 °C or 50 °C for 3 days. Some freeze-dried samples were stored at 25 °C for 3 days or 1 month. After storage, samples injected into oocytes using intracytoplasmic sperm injection were assessed for chromosome damage in resulting zygotes. Irrespective of freeze-drying solutions and storage temperatures, almost all the zygotes (97–100%) produced using the spermatozoa freeze-dried at pH 5.0 had structural chromosome aberrations of sperm origin. When freeze-drying was conducted at pH 8.0–8.4 using Na-ETBS, the incidence of chromosome damage decreased to 14–17% in 4 °C storage and 24–26% in 50 °C storage. When freeze-dried in K-ETBS, the lowest level of chromosome damage occurred at pH levels of 7.7–8.4 at 4 °C storage (13–15%) and at pH 7.7–8.0 at 50 °C storage (16–23%). Spermatozoa freeze-dried in Na-ETBS at pH 8.2 and K-ETBS at pH 7.7 showed no significant increase in chromosome damage during 25 °C storage from 3 days to 1 month (11%–20% in Na-ETBS; 13%–18% in K-ETBS). Thus, use of the solutions optimized for short-term storage at high temperature (50 °C, 3 days) permits prolonged storage (1 month) of freeze-dried spermatozoa at room temperature.

Sensitivity and specificity prediction of the buccal micronucleus cytome assay in end-stage renal disease patients on dialysis: A case-control study

Patients with end-stage renal disease (ESRD) require hemodialysis. However, dialysis therapy may cause genomic damage due to increased oxidative stress. Non-invasive assessment of genotoxicity may be helpful for developing management strategies. We applied the buccal micronucleus cytome (BMCyt) assay to ESRD patients on dialysis. Patients (n=35, age 52±2 year) on dialysis therapy (20.9±0.8months) had low glomerular filtration rates (GFR=5.00±0.36ml/min/1.73m2); controls (n=21, age 51±2 year) were healthy adults with no known recent illnesses or exposures. Patients had significantly increased chromosome damage: clastogenic/aneugenic events (frequency of cells with MN), cellproliferation (basal cells), cytokinesis defects (binucleated cells), and celldeath (pyknotic cells); Repair Index was lower in the patient group. Receiver Operator Characteristic (ROC) curve analysis showed that cells with MN were the best predictor for discriminating between patients and controls. Other predictivebiomarkers were the frequencies of basal, binucleated,and pyknotic.

Hexavalent chromium induces chromosome instability in human urothelial cells

Numerous metals are well-known human bladder carcinogens. Despite the significant occupational and public health concern of metals and bladder cancer, the carcinogenic mechanisms remain largely unknown. Chromium, in particular, is a metal of concern as incidences of bladder cancer have been found elevated in chromate workers, and there is an increasing concern for patients with metal hip implants. However, the impact of hexavalent chromium (Cr(VI)) on bladder cells has not been studied. We compared chromate toxicity in two bladder cell lines; primary human urothelial cells and hTERT-immortalized human urothelial cells. Cr(VI) induced a concentration- and time-dependent increase in chromosome damage in both cell lines, with the hTERT-immortalized cells exhibiting more chromosome damage than the primary cells. Chronic exposure to Cr(VI) also induced a concentration-dependent increase in aneuploid metaphases in both cell lines which was not observed after a 24h exposure. Aneuploidy induction was higher in the hTERT-immortalized cells. When we correct for uptake, Cr(VI) induces a similar amount of chromosome damage and aneuploidy suggesting that the differences in Cr(VI) sensitivity between the two cells lines were due to differences in uptake. The increase in chromosome instability after chronic chromate treatment suggests this may be a mechanism for chromate-induced bladder cancer, specifically, and may be a mechanism for metal-induced bladder cancer, in general.

Genotoxicity of synthetic amorphous silica nanoparticles in rats following short-term exposure. Part 1: oral route.

Synthetic amorphous silica (SAS) in its nanosized form is now used in food applications although the potential risks for human health have not been evaluated. In this study, genotoxicity and oxidative DNA damage of two pyrogenic (NM-202 and 203) and two precipitated (NM-200 and -201) nanosized SAS were investigated in vivo in rats following oral exposure. Male Sprague Dawley rats were exposed to 5, 10, or 20 mg/kg b.w./day for three days by gavage. DNA strand breaks and oxidative DNA damage were investigated in seven tissues (blood, bone marrow from femur, liver, spleen, kidney, duodenum, and colon) with the alkaline and the (Fpg)-modified comet assays, respectively. Concomitantly, chromosomal damage was investigated in bone marrow and in colon with the micronucleus assay. Additionally, malondialdehyde (MDA), a lipid peroxidation marker, was measured in plasma. When required, a histopathological examination was also conducted. The results showed neither obvious DNA strand breaks nor oxidative damage with the comet assay, irrespective of the dose and the organ investigated. Similarly, no increases in chromosome damage in bone marrow or lipid peroxidation in plasma were detected. However, although the response was not dose-dependent, a weak increase in the percentage of micronucleated cells was observed in the colon of rats treated with the two pyrogenic SAS at the lowest dose (5 mg/kg b.w./day). Additional data are required to confirm this result, considering in particular, the role of agglomeration/aggregation of SAS NMs in their uptake by intestinal cells.

Arsenic is cytotoxic and genotoxic to primary human lung cells

Arsenic originates from both geochemical and numerous anthropogenic activities. Exposure of the general public to significant levels of arsenic is widespread. Arsenic is a well-documented human carcinogen. Long-term exposure to high levels of arsenic in drinking water has been linked to bladder, lung, kidney, liver, prostate, and skin cancers. Among them, lung cancer is of great public concern. However, little is known about how arsenic causes lung cancer and few studies have considered effects in normal human lung cells. The purpose of this study was to determine the cytotoxicity and genotoxicity of arsenic in human primary bronchial fibroblast and epithelial cells. Our data show that arsenic induces a concentration-dependent decrease in cell survival after short (24h) or long (120h) exposures. Arsenic induces concentration-dependent but not time-dependent increases in chromosome damage in fibroblasts. No chromosome damage is induced after either 24h or 120h arsenic exposure in epithelial cells. Using neutral comet assay and gamma-H2A.X foci forming assay, we found that 24h or 120h exposure to arsenic induces increases in DNA double strand breaks in both cell lines. These data indicate that arsenic is cytotoxic and genotoxic to human lung primary cells but lung fibroblasts are more sensitive to arsenic than epithelial cells. Further research is needed to understand the specific mechanisms involved in arsenic-induced genotoxicity in human lung cells.

Abstract B91: Combined ATM and ATR kinase inhibition selectively kills p53-mutated non-small cell lung cancer (NSCLC) cells.

Abstract Introduction: Cancer cells frequently exhibit defects in components of the DNA-damage response (DDR) and may as a consequence become highly dependent on remaining DDR pathways to survive DNA damage. Targeting such remaining DDR pathways to confer synthetic lethality has become a valuable new strategy for personalised cancer therapy, and an attractive target for this approach is the central DDR kinase Ataxia Telangiectasia and Rad3 related (ATR). Several small molecule inhibitors of ATR are now entering clinical development. However, the challenges remain to identify patient subgroups that might benefit from ATR inhibitors, and to integrate these agents with standard therapies. Two important components of the DDR, Ataxia Telangiectasia Mutated (ATM) and p53, are commonly mutated and inactivated in lung cancer. In this study, we therefore evaluate the potential of ATR kinase inhibition for the treatment of ATM- or p53-deficient human NSCLC, both as single agent and in combination with ionizing radiation (IR). Methods: We used proliferation and clonogenic survival assays to measure the sensitivity of a panel of NSCLC cell lines to the selective ATR kinase inhibitor VE-821. Inhibition of ATR mediated signalling was assessed by immunoblotting. Immunofluorescence and chromosome analysis were used to assess the induction of DNA damage following treatment of cells with either VE-821 alone, or in combination with the selective ATM inhibitor KU55933. FACS analysis was used to measure cell cycle phase distribution and DNA content. Results: NSCLC cells deficient in both ATM and p53 were highly sensitive to ATR inhibition (IC50 0.7 µM), but there was no clear association between sensitivity and mutation status of either ATM or p53 alone (IC50 3.9 - 8.9 µM). We observed similar relative sensitivities in clonogenic survival assays and in combination with IR. This increased cytotoxicity of ATR inhibition in a combined ATM- and p53-deficient background was associated with formation of giant micronucleated cells and evidence of mitotic catastrophe within 48 h of treatment. Furthermore, we found a marked increase in VE-821-induced chromosomal damage in cells deficient in ATM and p53, compared with cells deficient in either gene alone. We confirmed this dependency on p53 mutation status and ATM function for sensitivity to ATR inhibition by treating p53-mutated NSCLC cells with a combination of VE-821 and KU55933. In this setting, inhibition of ATM markedly sensitised tumour cells to VE-821 treatment, as shown by a reduction in clonogenic survival and an increase in chromosomal damage. Conclusion: Our data suggest that in NSCLC the functional status of both ATM and p53 determines the cellular response to ATR inhibition and that combined ATM and ATR kinase inhibition selectively kills p53 mutant cells. A combination of ATM and ATR inhibitors may therefore have a broad utility for the treatment of p53 mutated NSCLC. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B91. Citation Format: Anika M. Weber, Sivan M. Bokobza, Aoife M. Devery, Anderson J. Ryan. Combined ATM and ATR kinase inhibition selectively kills p53-mutated non-small cell lung cancer (NSCLC) cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B91.

Chromosomal stability of second polar bodies in mouse embryos

Incorporation of a second polar body (PB2) into one of the blastomeres has been considered as a causal mechanism underlying diploid/triploid mixoploidy in humans. Using a mouse model, we examined whether PB2s can participate in the formation of mixoploidy. Uptake of BrdU was examined to determine DNA synthesis in PB2s up to 28 h after fertilization. PB2s from embryos at 4-6 (1-cell), 24 (2-cell), 48 (4-cell), and 72 h (morula) were fused with MII oocytes to induce premature chromosome condensation. Caspase and TUNEL assays were used to detect apoptotic PB2s at 24, 48, and 72 h. PB2s were fused with one of the blastomeres of the 2-cell embryos to produce mixoploid embryos. DNA synthesis in the PB2s continued until 22 h after fertilization. At 4-6 h, nearly all of the PB2s showed G1-type chromosomes and there was no significant increase in chromosome damage. At 24, 48, and 72 h, S-type chromatin predominated. Few PB2s showed apoptotic response until 72 h. Regardless of the fusion with the PB2, more than 90 % of the embryos developed to 4-cell stage, and over 80 % of the resultant 4-cell embryos had daughter blastomeres with a morphologically normal nucleus. Some of the daughter blastomeres displayed triploidy. The PB2 is viable for at least 72 h after fertilization, with slow progression through the cell cycle. Once the PB2 has been incorporated into a blastomere, the cell cycle of the PB2 might be synchronized with that of the host resulting in diploid/triploid mixoploidy.

Phenolic antioxidant 2,6-di-tert-butyl-p-cresol (vitamin E synthetic analogue) does not inhibit 1,1’-dimetyl-4,4’-bipyridium dichloride (paraquat)-induced structural chromosomal damage in cultured leukocytes of the dark-spotted-frog Pelophylax (Rana) nigr

Pro-oxidative effect of phenolic antioxidant (vitamin E) in combination with the initiators on human low-density lipoprotein is known. Recently I reported that oxidative stress induced by vitamin E in combination with the herbicide paraquat enhances structural chromosomal damage in cultured anuran leukocytes. In the present study, the phenolic antioxidant vitamin E-synthetic-analogue 2,6-di-tert-butyl-p-cresol (BHT) in combination with paraquat was found to enhance structural chromosomal damage in cultured Pelophylax (Rana) nigromaculatus leukocytes more than paraquat only and paraquat plus nicotinamido adenine dinucleotido phosphate served as positive control, although BHT only had no effect on induction of structural chromosomal damage. Paraquat plus BHT-enhanced structural chromosomal damage was inhibited by combination of the superoxide dismutase mimic Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin and the hydrogen peroxide scavenger catalase. In test based on reduction of paraquat cation, BHT was found to reduce paraquat cation chemically to paraquat monocation radical. These results suggest that BHT functions in chemically donating electron to paraquat and thereby induces an acute accumulation of reactive oxygen species, resulting in increase in chromosomal damage.

Chromosomal instability in peripheral blood lymphocytes of patients with reproductive failure assessed by micronucleus assay

We investigated chromosomal instability in peripheral blood lymphocytes (PBL) of patients with reproductive failure in respect to age, smoking habits, gender, miscarriages, and semen parameters. The study involved 36 individual cases of reproductive failure (18 men and 18 women) attended at the Clinical Centre of Kragujevac, Serbia, and 30 healthy subjects (15 men and 15 women). Micronuclei (MN) frequency was estimated in PBL using the cytokinesis-block micronucleus (CBMN) assay. The baseline MN frequencies were significantly higher (p=0.031; p<0.001) in male [(9.22 ± 4.70) MN per 1000 BN cells] and female patients [(13.50 ± 2.5) MN per 1000 BN cells] than in male and female healthy controls [(6.27 ± 2.66) MN per 1000 BN cells; (6.80 ± 2.98) MN per 1000 BN cells]. The mean baseline MN frequency did not significantly differ between miscarriage groups and between patients with and without normal values of semen parameters. The correlations between poor sperm concentration (<20x106 mL-1), rapid progressive motility (<25 %), normal morphology (<30 %), and MN frequencies were negative, but not statistically significant. We found that only gender significantly influenced the MN rates in analysed patients. There were no significant differences between age groups and between smokers and non-smokers in patients and control samples. We conclude that the increase in baseline MN frequency in PBL of patients with reproductive failure corresponds to the increase in chromosomal damage, which occurs as a result of complex events that cause reproductive disorders.

Zoledronic acid and radiation: toxicity, synergy or radiosensitization?

Zoledronic acid (Z) is a bisphosphonate used in hypercalcaemia-related cancer, in complications for bone metastasis and in postmenopausal osteoporosis and it has been related to osteoradionecrosis, especially when associated with radiation to the head and neck structures. To determine the radiosensitization capacity of zoledronic acid in the combined treatment with ionizing radiation (IR) by evaluating its genotoxic and cytotoxic capacities in non-tumoral cells. The genotoxic effect of Z was studied by means of the micronucleus test in cytokinesis-blocked cells of human lymphocytes irradiated before and after a 2Gy irradiation, while the cytotoxic effect was studied by a cell viability test in the PNT2 cell line before and after exposure to different X-ray doses (0-20Gy) in four groups (Z alone, radiation alone, Z+IR and IR+Z). A dose-dependent and time-dependent cytotoxic effect of Z and IR on PNT2 cells in vitro (p>0.001) was demonstrated. With the concentrations recommended for humans, the combined treatment had a more pronounced effect than individual treatments (p<0.001). The effect was synergic (CI<1), increasing the Z enhancement ratio (2.6) and sensitization factor (56%); the effect of Z was always greater after IR exposure. In the genotoxic effect, only the administration of Z after irradiation (IR+Z) increased chromosome damage (p<0.001) and the sensibilization factor (35.7%). High concentrations of Z are toxic, but the concentrations recommended for clinical practice in humans give it the characteristics of a radiosensitization agent, whose effect is even greater when administered after IR.

Genotoxic effects of banding procedure with different orthodontic cements on human oral mucosa cells

To assess the genotoxic and cytotoxic effects of banding procedure with 5 different orthodontic cements on human oral buccal epithelium cells. Materials and methods: Fifty healthy volunteers (mean age: 14.54 ± 2.37 years) were randomly divided into 5 groups of 10. Preformed stainless steel molar bands (3M Unitek) were cemented to the upper and lower first molar teeth with 5 different orthodontic cements (Durelon, GC Fuji Ortho Band LC Paste Pak, Meron, Ultra Band Lok, and 3M Unitek Multi Cure) in each group. Genotoxic effects of the banding procedure for a 1-month period were evaluated using the micronucleus test (MNT). To monitor cytotoxic effects, binucleated cells (BNs), karyolysis (KL), and karyorrhexis (KR) were also evaluated in this setting. Results: Analysis of micronuclei in buccal epithelial cells revealed a significant increase in chromosomal damage in all groups (P < 0.01). Significant differences were found in the number of BNs in the groups receiving treatment with Meron, Ultra Band Lok, Durelon, and 3M Multi Cure (P < 0.01). Banding with GC Fuji and Durelon significantly elevated KL frequencies (P < 0.05). Durelon had a significant effect on KR (P < 0.01). Conclusion: Band cementation with conventional glass ionomer cement (Meron) showed the least genotoxic effects. The highest cytotoxic effects on human oral buccal epithelium cells were found in the polycarboxylate cement (Durelon) group.

Evaluating chromosomal damage in workers exposed to hexavalent chromium and the modulating role of polymorphisms of DNA repair genes

Welders have been chronically exposed to hexavalent chromium with potential consequences on chromosomal integrity. Our study is focused on the extent of any such chromosomal aberrations with respect to chromium levels in the blood of welders as well as on the tentative modulating role of polymorphisms in DNA repair genes XPD Lys751Gln, XPG Asn114His, XPC Lys939Gln, hOGG1 Ser326Cys and XRCC1 Arg399Gln on chromosomal damage. The study was conducted on 144 individuals consisting of 73 welders exposed to chromium for 10.2 ± 1.67 years and 71 control individuals without known exposures. Chromosomal aberrations, their chromatid-type and chromosome-type aberrations were detected by conventional cytogenetic analysis. XPD, XPG, XPC, hOGG1 and XRCC1 gene polymorphisms were assayed for by Taqman SNP genotyping assay ("Assay-by-Demand") using Real-Time allelic discrimination on AB 7500 equipment. Chromium concentration in the blood was determined by atomic absorption spectrophotometry. The level of chromium in the blood of welders ranged between 0.032 and 0.182 μmol l(-1) and was significantly higher than that in controls (0.07 ± 0.04 μmol l(-1) vs. 0.03 ± 0.007 μmol l(-1)). Parameters of chromosomal damage were similar in both the exposed and the control individuals (1.89% vs. 1.70% for total chromosomal aberrations, 0.97% vs. 0.88% for chromosome-type and 0.92% vs. 0.80% for chromatid-type, respectively). Chromatid-type of aberrations positively correlated with the level of chromium in the blood (r = 0.28; P = 0.02). Significantly higher total chromosomal aberrations were detected in individuals with homozygous variant polymorphism in XRCC1 Arg399Gln gene as compared to those with heterozygous and homozygous wild-type genotypes (2.20, 1.89 and 1.48%, respectively; P = 0.01). A similar tendency was found for chromatid-type aberrations (1.30% for homozygous variant genotype bearers, 0.94% for those with heterozygous genotype and 0.75% for carriers of homozygous wild-type genotype, respectively; P = 0.04). Although no apparent increase in chromosomal damage was recorded in chromium-exposed welders in comparison with controls, genetic make-up in DNA repair genes may increase susceptibility toward adverse effect of chromium.

Comparison of the Micronucleus and Chromosome Aberration Techniques for the Documentationof Cytogenetic Damage in Radiochemotherapy-Treated Patients with Rectal Cancer

The goal of the interdisciplinary Clinical Research Unit KFO179 (Biological Basis of Individual Tumor Response in Patients with Rectal Cancer) is to develop an individual Response and Toxicity Score for patients with locally advanced rectal cancer treated with neoadjuvant radiochemotherapy. The aim of the present study was to find a reliable and sensitive method with easy scoring criteria and high numbers of cell counts in a short period of time in order to analyze DNA damage in peripheral blood lymphocytes. Thus, the cytokinesis-block micronucleus (CBMN) assay and the chromosome aberration technique (CAT) were tested. Peripheral blood lymphocytes obtained from 22 patients with rectal cancer before (0 Gy), during (21.6 Gy), and after (50.4 Gy) radiochemotherapy were stimulated in vitro by phytohemagglutinin (PHA); the cultures were then processed for the CBMN assay and the CAT to compare the two methods. A significant increase of chromosomal damage was observed in the course of radiochemotherapy parallel to increasing radiation doses, but independent of the chemotherapy applied. The equivalence of both methods was shown by Westlake's equivalence test. The results show that the CBMN assay and the CAT are equivalent. For further investigations, we prefer the CBMN assay, because it is simpler through easy scoring criteria, allows high numbers of cell counts in less time, is reliable, sensitive, and has higher statistical power. In the future, we plan to integrate cytogenetic damage during radiochemotherapy into the planned Response and Toxicity Score within our interdisciplinary Clinical Research Unit.

Expression of DNA repair and apoptosis genes in mitochondrial mutant and normal cells following exposure to ionizing radiation

In double-strand DNA damage repair, nonhomologous end joining (NHEJ) is more error-prone than homologous recombination repair (HRR), indicating that the relative prevalence of NHEJ may lead to more incorrect repair and thus to increases in chromosome damage. If DNA damage is extensive and cells are unable to repair that damage they typically undergo apoptosis. The mechanism(s) by which cells decide to switch from DNA repair to apoptosis is unknown. Since DNA repair and apoptosis are both energy-demanding processes, the answer may involve ATP utilization. We used human mitochondrial mutant cell lines obtained from people with phenotypic manifestations of compromised ATP generation. We hypothesized that these cells may not have adequate capacity for dealing with the additional demands for ATP required for repairing DNA damage after genotoxic exposure, perhaps making the cells more prone to undergo apoptosis instead of initiating repair. This study describes changes in the expression of genes involved in NHEJ or HRR, as well as genes involved in apoptosis, in one normal and two mitochondrial mutant human cell lines following ionizing radiation exposure. Compared to normal cells, both mutant cell lines showed reduced expression of genes involved in NHEJ and HRR. Analysis of expression changes in genes involved in apoptosis revealed marked increases in expression in the mutants compared to normal cells. These results indicate that following ionizing radiation exposure, mitochondrial mutant cells have decreased levels of mRNA expression of DNA repair genes and increased expression levels of genes involved in apoptosis compared to normal cells. This study provides information that might be useful in characterizing energy dependent processes following exposure to stress or genotoxic agents.

Increased chromosome damage in systemic sclerosis skin fibroblasts

Objective: To evaluate chromosome damage, by means of micronucleus frequency, in dermal fibroblasts from affected and non-affected skin from systemic sclerosis (SSc) patients and from controls.Methods: Primary fibroblast cultures were obtained by biopsy from affected and non-affected skin from SSc patients. Control fibroblasts were derived from skin remnants from plastic surgery in healthy adults. The number of micronuclei-bearing cells per 1000 binucleated cells (MN+ cells/1000 BN) was determined in cultures with and without clastogenic stimulus (bleomycin 3 μg/mL).Results: Primary cultures from 10 SSc patients (affected and non-affected skin) and nine controls were analysed by two blinded examiners. In the absence of bleomycin, the frequency of MN+ cells was higher in cultures from affected (14.01 ± 11.96 MN+ cells/1000 BN; p = 0.004) and non-affected (15.41 ± 13.58 MN+ cells/1000 BN; p = 0.005) skin from SSc patients as compared to fibroblasts from healthy controls (4.74 ± 3.30 MN+ cells/1000 BN). In bleomycin-treated cultures, the frequency of MN+ cells was higher in SSc affected (38.03 ± 26.14 MN+ cells/1000 BN; p = 0.041) and non-affected skin (38.47 ± 17.88 MN+ cells/1000 BN; p = 0.034) as compared to healthy control fibroblasts (20.54 ± 13.09 MN+ cells/1000 BN). There was no difference in the frequency of MN+ cells in cultures from affected and non-affected skin of SSc patients.Conclusions: This is the first demonstration that dermal fibroblasts from SSc patients present an increased frequency of spontaneous and clastogen-induced micronuclei. Increased clastogenesis seems to be a widespread phenomenon in SSc because fibroblasts from clinically affected and non-affected skin presented the equivalent increased micronuclei counts.