Elevated Micronucleus Frequency Research Articles

POST-SIEGE GENOTOXIC HAZARDS IN LAKE LANAO, PHILIPPINES BY MICRONUCLEUS ASSAY

Massive war pollutants brought by Marawi Siege in the Philippines have contaminated the city environs and the surrounding ancient Lake Lanao. Munition residues including heavy metals are reportedly genotoxic hence this study was conducted to determine the post-siege genotoxic hazards posed by the munition pollutants through micronucleus (MN) assay on the slow-moving edible gastropod snails Vivipara angularis Muller (locally known as “suso”) thriving in the lake. MN, a biomarker of genotoxicity was examined and counted in the hemocytes of exposed juvenile and adult snails. Results revealed that MN frequencies (no. of MN/1000 hemocytes) were significantly higher in snails sampled in Lake Lanao lakeshores than in the reference site Lake Dapao. Among adult samples, there was a decreasing trend of MN frequencies with increasing distance from lakeshore fronting warzone ‘ground zero’ where the battle was heaviest (site A) to sampling sites away from it: sites B, C, and D (Lake Dapao) which are 8.15 km, 24.41 km, and 34.45 km, respectively. Moreover, varied patterns of micronucleation were observed between age groups and sites, i.e., in all sites except C, adults had greater MN counts than juveniles which were significant in site A only. Unexpectedly juveniles displayed significantly high MN counts than adults in site C. The elevated MN frequencies in the snail hemocytes exposed to war pollution could be attributed to genotoxic munition residues eroded and washed into the lake water. Moreover, heavy metals which are common components of weaponries were also detected in the snail muscles, although at concentrations within safe levels but continued consumption may be cautioned to avoid biomagnification. Other genotoxins must be present in site C other than munition residues predisposing the juvenile snails. The results are baseline data on the MN frequencies in V. angularis exposed to war pollutants in Lake Lanao which need further investigation. Post-siege genotoxic hazards in the lake by MN assay cannot be ignored, thus local leaders must be informed of the results for possible intervention to protect the health and well-being of the community. 
 

Combined Approach: FFQ, DII, Anthropometric, Biochemical and DNA Damage Parameters in Obese with BMI ≥ 35 kg m-2.

Although obesity with its comorbidities is linked with higher cancer risk, the data on genome stability in the obese/severely obese are scarce. This is the first study with three DNA damage assessment assays (Fpg-modified and alkaline comet assays and micronucleus cytome assay) performed on a severely obese population (n = 53) where the results were compared with daily intake of food groups, nutrient intake, dietary inflammatory index (DII), and anthropometric and biochemical parameters usually measured in obese individuals. Results demonstrated the association between DNA damage levels and a decrease in cell proliferation with anthropometric measurements and the severity of obese status, together with elevated levels of urates, inorganic phosphates, chlorides, and hs troponin I levels. DII was connected with oxidative DNA damage, while BMI and basal metabolic rate (BMR) were associated with a decrease in cell proliferation and DNA damage creation. Measured daily BMR and calculated daily energy intake from the food frequency questionnaire (FFQ) demonstrated no significant difference (1792.80 vs. 1869.86 kcal day-1 mean values). Groups with higher DNA damage than expected (tail intensity in comet assay >9% and >12.4%, micronucleus frequency >13), consumed daily, weekly, and monthly more often some type of food groups, but differences did not show a clear influence on the elevated DNA damage levels. Combination of all three DNA damage assays demonstrated that some type of damage can start earlier in the obese individual lifespan, such as nuclear buds and nucleoplasmic bridges, then comes decrease in cell proliferation and then elevated micronucleus frequencies, and that primary DNA damage is not maybe crucial in the overweight, but in severely obese. Biochemically changed parameters pointed out that obesity can have an impact on changes in blood cell counts and division and also on genomic instability. Assays were able to demonstrate groups of sensitive individuals that should be further monitored for genomic instability and cancer prevention, especially when obesity is already connected with comorbidities, 13 different cancers, and a higher mortality risk with 7-10 disease-free years loss. In the future, both DNA damage and biochemical parameters should be combined with anthropometric ones for further obese monitoring, better insight into biological changes in the severely obese, and a more individual approach in therapy and treatment. Patients should also get a proper education about the foodstuff with pro- and anti-inflammatory effect.

Folic acid deficiency increases sensitivity to DNA damage by glucose and methylglyoxal.

Type 2 diabetes (T2D) is associated with elevated frequencies of micronuclei (MNi) and other DNA damage biomarkers. Interestingly, individuals with T2D are more likely to be deficient in micronutrients (folic acid, pyridoxal-phosphate, cobalamin) that play key roles in one-carbon metabolism and maintaining genomic integrity. Furthermore, it has recently been shown that deficiencies in these nutrients, in particular folic acid leaves cells susceptible to glucose-induced DNA damage. Therefore, we sought to investigate if the B lymphoblastoid WIL2-NS cell line cultured under folic acid-deficient conditions was more sensitive to DNA damage induced by glucose, or the reactive glycolytic byproduct methylglyoxal (MGO) and subsequent advanced glycation endproduct formation. Here, we show that only WIL2-NS cultured under folic acid-deficient conditions (23 nmol/l) experience an increase in MNi frequency when exposed to high concentrations of glucose (45 mmol/l) or MGO (100 µmol/l). Furthermore, we showed aminoguanidine, a well-validated MGO and free radical scavenger was able to prevent further MNi formation in folic acid-deficient cells exposed to high glucose, which may be due to a reduction in MGO-induced oxidative stress. Interestingly, we also observed an increase in MGO and other dicarbonyl stress biomarkers in folic acid-deficient cells, irrespective of glucose concentrations. Overall, our evidence shows that folic acid-deficient WIL2-NS cells are more susceptible to glucose and/or MGO-induced MNi formation. These results suggest that individuals with T2D experiencing hyperglycemia and folic acid deficiency may be at higher risk of chromosomal instability.

Micronucleus evaluation of exfoliated buccal epithelial cells from automobile spray painters – A preliminary study

Automobile spray painters in the Mechanic village at Coal Camp, Enugu State, Nigeria are exposed to genotoxic agents such as the automobile paints and spent engine lubricants which contaminate the soil of their working environment. The present study applied the micronucleus (MN) test on exfoliated oral mucosal cells as a means to assess the genotoxicity risk associated with occupational exposure to genotoxic agents. Two (2) groups of subjects which comprised 30 automobile spray painters and a control group of 30 apparently healthy unexposed volunteers were enrolled in the study. The study participants were all males. Moistened wooden spatulas were used to obtain buccal smears of the participating individuals, and the smears were stained. The presence of MN was assessed under light microscopy and a total of 1000 cells per individual were scored. The results obtained showed that statistically significant increase in MN frequency in buccal epithelial cells of automobile spray painters when compared with the control group (p<0.05). Elevated MN frequency was also observed withincreased age, smoking and alcohol consumption habits. MN frequency was significantly affected by the duration of working experience (years) of the spray painters whereas no difference was observed with number of workinghours/day. In conclusion, the present study has revealed that automobile spray painters in the Mechanic village of Coal Camp, Enugu State could be under risk of cytogenetic damage from exposure to genotoxic chemicals.Key words: Micronuclei frequency; Exfoliated buccal cells; Smoking; Alcohol consumption, Spray Painters; Genotoxicity;

Association Between Polymorphisms of Metabolic Enzyme Genes and Chromosomal Damage in Benzene-Exposed Workers in China.

To provide better understanding of genetic susceptibility for health risk among current benzene-exposed workers. Four hundred sixty one benzene-exposed workers and 88 matched controls were recruited, and their benzene exposure doses were monitored. Associations between genetic susceptibility for polymorphisms of metabolic enzymes CYP2E1 and NQO1, and expression of cytokinesis-block micronucleus (MN) were investigated. Mean MN frequency in the exposed workers was significantly higher than that in the control group (P < 0.01). Individuals with the NQO1 CC genotype showed significantly higher MN frequencies than those with the TT genotype (P < 0.05) in either single- or multiple-factor analyses. Age was an effect modifier for elevated MN frequency, while sex, smoking, and alcohol consumption had no relationship. Exposure to low dose of benzene among current workers can still cause health risk, especially among those with the NQO1 CC genotype.

Impact of radiofrequency radiation on DNA damage and antioxidants in peripheral blood lymphocytes of humans residing in the vicinity of mobile phone base stations

ABSTRACTRadiofrequency radiations (RFRs) emitted by mobile phone base stations have raised concerns on its adverse impact on humans residing in the vicinity of mobile phone base stations. Therefore, the present study was envisaged to evaluate the effect of RFR on the DNA damage and antioxidant status in cultured human peripheral blood lymphocytes (HPBLs) of individuals residing in the vicinity of mobile phone base stations and comparing it with healthy controls. The study groups matched for various demographic data including age, gender, dietary pattern, smoking habit, alcohol consumption, duration of mobile phone use and average daily mobile phone use. The RF power density of the exposed individuals was significantly higher (p < 0.0001) when compared to the control group. The HPBLs were cultured and the DNA damage was assessed by cytokinesis blocked micronucleus (MN) assay in the binucleate lymphocytes. The analyses of data from the exposed group (n = 40), residing within a perimeter of 80 m of mobile base stations, showed significantly (p < 0.0001) higher frequency of micronuclei when compared to the control group, residing 300 m away from the mobile base station/s. The analysis of various antioxidants in the plasma of exposed individuals revealed a significant attrition in glutathione (GSH) concentration (p < 0.01), activities of catalase (CAT) (p < 0.001) and superoxide dismutase (SOD) (p < 0.001) and rise in lipid peroxidation (LOO) when compared to controls. Multiple linear regression analyses revealed a significant association among reduced GSH concentration (p < 0.05), CAT (p < 0.001) and SOD (p < 0.001) activities and elevated MN frequency (p < 0.001) and LOO (p < 0.001) with increasing RF power density.

Association of elevated frequency of micronuclei in peripheral blood lymphocytes of type 2 diabetes patients with nephropathy complications.

The increasing incidence of type 2 diabetes mellitus globally has increased the incidence of diabetes-associated complications such as nephropathy. DNA damage induced by oxidative stress might be one of the important mechanisms in the pathogenesis of diabetic complications. Two hundred Iranian individuals with the conditions of type 2 diabetes, diabetic nephropathy and nephropathy patients with no sign of diabetes and normal unaffected sex- and age-matched controls (50 in each group) were enrolled in the study. The background and the net levels of micronucleus (MN) formation as well as other cellular damages induced after in vitro treatment with 25 µg/ml of bleomycin (BLM) were evaluated using cytokinesis block MNs cytome assay (CBMN cyt) in peripheral blood lymphocytes. The background and net BLM-induced levels of MNs were significantly higher in all patient groups compared with the control (P < 0.01, P < 0.001, respectively). The frequency of MNs was significantly higher in those patients with prior incidence of nephropathy than those without. A positive association was observed between basal and net MN frequency among study groups and also between net genetic damages and serum creatinine value and duration of diabetes. The rate of basal and net apoptosis was significantly higher in patients with hyperglycemia. Our results indicate that increased genomic instability expressed as MNs is associated with nephropathy in all pathological stages. Therefore, implementation of MN assay in clinical level may potentially enhance the quality of management of patients with diabetes and its complications such as nephropathy.

Elevated micronucleus frequency in patients with type 2 diabetes, dyslipidemia and periodontitis.

The over-production of reactive oxygen species (ROS) can cause oxidative damage to a large number of molecules, including DNA, and has been associated with the pathogenesis of several disorders, such as diabetes mellitus (DM), dyslipidemia and periodontitis (PD). We hypothesise that the presence of these diseases could proportionally increase the DNA damage. The aim of this study was to assess the micronucleus frequency (MNF), as a biomarker for DNA damage, in individuals with type 2 DM, dyslipidemia and PD. One hundred and fifty patients were divided into five groups based upon diabetic, dyslipidemic and periodontal status (Group 1 - poor controlled DM with dyslipidemia and PD; Group 2 - well-controlled DM with dyslipidemia and PD; Group 3 - without DM with dyslipidemia and PD; Group 4 - without DM, without dyslipidemia and with PD; and Group 5 - without DM, dyslipidemia and PD). Blood analyses were carried out for fasting plasma glucose, HbA1c and lipid profile. Periodontal examinations were performed, and venous blood was collected and processed for micronucleus (MN) assay. The frequency of micronuclei was evaluated by cell culture cytokinesis-block MN assay. The general characteristics of each group were described by the mean and standard deviation and the data were submitted to the Mann-Whitney, Kruskal-Wallis, Multiple Logistic Regression and Spearman tests. The Groups 1, 2 and 3 were similarly dyslipidemic presenting increased levels of total cholesterol, low density lipoprotein cholesterol and triglycerides. Periodontal tissue destruction and local inflammation were significantly more severe in diabetics, particularly in Group 1. Frequency of bi-nucleated cells with MN and MNF, as well as nucleoplasmic bridges, were significantly higher for poor controlled diabetics with dyslipidemia and PD in comparison with those systemically healthy, even after adjusting for age, and considering Bonferroni's correction. Elevated frequency of micronuclei was found in patients affected by type 2 diabetes, dyslipidemia and PD. This result suggests that these three pathologies occurring simultaneously promote an additional role to produce DNA impairment. In addition, the micronuclei assay was useful as a biomarker for DNA damage in individuals with chronic degenerative diseases.

Mutagenic joining of enzymatically induced DNA double-strand breaks, accompanied by persistent unrepaired DNA damage and a secretory protein phenotype, in HZE-exposed human cells

High charge and energy (HZE) particles are a component of galactic cosmic rays. They cause complex damage to DNA and other cellular components, leading to both direct and indirect biological effects. Here, we investigate the hypothesis that one of these indirect effects is to compromise the accuracy of the DNA repair machinery, reducing the ability to cope with subsequent genotoxic insults. To this end, we used a new human reporter cell line with single-copy integrated fluorescent reporter cassettes that allow measurement of the frequency of mutagenic repair. Introduction of the rare cutting I-SceI nuclease stimulates both translocations (joining of two I-SceI sites on different chromosomes), and deletions (joining of two I-SceI sites after deletion of an intervening fragment) [ 1]. These can be measured simultaneously in the same cell population using different color reporter genes. To test the effect of HZE exposure on the frequency of translocations and deletions in this assay, cells were exposed to 600 MeV/u 56Fe ions or 1000 MeV/u 48Ti ions at doses of 0.3 or 1.0 Gy. They were allowed to recover and challenged with I-SceI at 1, 7, 14, 21 and 28 days post-irradiation. Results showed that HZE particle irradiation significantly increased the frequency of I-SceI translocations and deletions above baseline levels. There was an increase in translocations by up to threefold, seen in both 56Fe- and 48Ti-treated populations. There was also a more modest, but significant increase in I-SceI-mediated deletions seen in a population that received the higher dose (1.0 Gy) of 56Fe particles. The increased frequency of I-SceI-induced translocations and deletions persisted for 2–3 weeks with 56Fe (but not with 48Ti). The increased frequency of translocations and deletions was not observed in populations treated with low-LET radiation at doses up 3 Gy. Thus, the phenomenon, which we term the ‘mutagenic repair phenotype’ depends on both dose and radiation quality. The mutagenic repair phenotype was associated with an elevated frequency of micronuclei and excess DNA repair foci, suggesting that persistent genomic stress might be one causative factor [ 2].To further explore the mechanism underlying the mutagenic DNA repair phenotype, we performed genome-wide expression profiling on cells that were harvested 7 days post-56Fe ion exposure. Results showed significant alterations in 234 genes. Many of the most highly induced genes encode secreted proteins that have previously been implicated in cellular senescence or pro-inflammatory processes. These findings suggest that a paracrine mechanism (induction of a set of senescence or inflammation-related proteins) may also contribute to the mutagenic repair phenotype.Altered regulation of cellular double-strand break repair, with an increased reliance on an error-prone pathway, is a novel mechanism whereby an indirect effect of HZE exposure may amplify cancer risk.

Elevated frequencies of micronuclei in pregnant women with type 1 diabetes mellitus and in their newborns

Pregestational diabetes mellitus (type 1 and type 2) affects about 1% of the obstetric population. In diabetes, persistent hyperglycemia can be a source of DNA damage via overproduction of reactive oxygen species (ROS). Using the cytokinesis-block micronucleus (CBMN) test, we measured the frequencies of micronuclei (MN) per 1000 binucleated (BN) cells in pregnant women (mothers) with type 1 diabetes mellitus (T1DM) and in their newborns. Peripheral blood lymphocytes were collected from 17 pregnant women with T1DM and cord-blood lymphocytes from their 17 newborns. The control group included 40 pregnant women (mothers) without diabetes mellitus (DM) and their 40 newborns. In the group of pregnant women with T1DM, the mean number of MN per 1000 BN cells was 2.35 (±1.07), significantly (p<0.001) higher than in the control group of pregnant women (0.86±0.90). The frequency value in the group of newborns of T1DM mothers was 1.42 (±0.60), significantly (p<0.05) higher than in the corresponding control group (0.67±0.79). The value in the group of mothers with T1DM was significantly (p<0.05) higher than in their newborns. Comparing mothers without DM with their newborns, no significant frequency differences were observed. No significant correlations were observed between MN frequencies in mothers with T1DM and either the frequencies in their newborns, the duration of diabetes, or HbA1C levels. Our results indicate that T1DM is accompanied by increased frequencies of MN in pregnant women and their newborns.

Elevated micronucleus frequencies in patients with pleural plaque secondary to environmental exposure to asbestos

We examined genotoxic damage and frequency of micronuclei in people living in the Bekilli and Suller districts of Denizli city who had been diagnosed with pleural plaques as a result of environmental exposure to asbestos. Peripheral blood samples were obtained from 30 volunteer patients 59-86 years old who did not smoke or consume alcohol and who were diagnosed with calcified pleural plaques. We also examined 30 healthy controls with similar features, who also lived in downtown Denizli. Micronucleus frequencies, nuclear division index, and mitotic index were determined. Micronucleus frequency, nuclear division index, and mitotic index were significantly higher in patients who had been exposed to asbestos than in the controls. We conclude that asbestos had a genotoxic effect, resulting in an increase in micronucleus frequency and other changes in patients diagnosed with pleural plaques secondary to asbestos exposure.

The suppression of hematopoiesis function in Balb/c mice induced by prolonged exposure of microcystin-LR

Microcystins (MCs) cause normocytic anemia in patients in a hemodialysis unit in Caruaru, Brazil in 1996, but the underlying mechanisms are still unclear. In the present study, Balb/c mice were intraperitoneally injected with microcystin-LR (MC-LR) at the doses of 0.5, 2 and 8μg/kg body weight (bw) every 48h for 30d. After the prolonged exposure of MC-LR, significant decreases of red blood cell count (RBC), hemoglobin (Hb) and hematocrit (Ht) were observed in 2 and 8μg/kg bw groups, but erythrocyte mean corpuscular volume (MCV) showed no significant changes. Significantly elevated micronucleus frequency was observed in bone marrow cells (BMCs) in all MC-LR treatments. The proliferation of BMCs significantly declined in both 2 and 8μg/kg bw groups. Serum levels of some hematopoietic growth factors significantly changed in 8μg/kg bw group, mainly including granulocyte-macrophage (GM-CSF), erythropoietin (EPO), interleukin-3 (IL-3) and TNF-α. The transcriptional levels of these 4 genes in BMCs were also significantly changed in 8μg/kg bw group. MC-LR exposure significantly increased the apoptosis rates in all MC-LR treatments. The present study indicates prolonged exposure of MC-LR induces normocytic anemia, and the disturbed hematopoietic growth factors and BMCs apoptosis are responsible for this normocytic anemia.

Dioxin Induces Genomic Instability in Mouse Embryonic Fibroblasts

Ionizing radiation and certain other exposures have been shown to induce genomic instability (GI), i.e., delayed genetic damage observed many cell generations later in the progeny of the exposed cells. The aim of this study was to investigate induction of GI by a nongenotoxic carcinogen, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Mouse embryonic fibroblasts (C3H10T1/2) were exposed to 1, 10 or 100 nM TCDD for 2 days. Micronuclei (MN) and expression of selected cancer-related genes were assayed both immediately and at a delayed point in time (8 days). For comparison, similar experiments were done with cadmium, a known genotoxic agent. TCDD treatment induced an elevated frequency of MN at 8 days, but not directly after the exposure. TCDD-induced alterations in gene expression were also mostly delayed, with more changes observed at 8 days than at 2 days. Exposure to cadmium produced an opposite pattern of responses, with pronounced effects immediately after exposure but no increase in MN and few gene expression changes at 8 days. Although all responses to TCDD alone were delayed, menadione-induced DNA damage (measured by the Comet assay), was found to be increased directly after a 2-day TCDD exposure, indicating that the stability of the genome was compromised already at this time point. The results suggested a flat dose-response relationship consistent with dose-response data reported for radiation-induced GI. These findings indicate that TCDD, although not directly genotoxic, induces GI, which is associated with impaired DNA damage response.

Tempol suppresses micronuclei formation in astrocytes of newborn rats exposed to 50-Hz, 10-mT electromagnetic fields under bleomycin administration

A number of epidemiological studies have suggested that exposure to environmental and occupational electromagnetic fields (EMFs) contribute to the induction of brain tumors. The aim of this study was to investigate the mutagenetic effects of co-exposure to 50-Hz, 10-mT EMFs and bleomycin (BLM) using an ex vivo newborn rat astrocyte micronucleus assay. We also investigated whether the mutagenetic effects of EMFs were related to active oxygen species by using 4-hydroxy-2,2,6,6-tetramethyl piperidine-1-oxyl (tempol), a superoxide radical scavenger. Three-day-old male Sprague-Dawley rats were co-exposed to 50-Hz EMFs and BLM (5 or 10mg/kg body weight (BW)) in each group (n=6; total 6 group), and were co-exposed to 50-Hz EMFs and 10mg/kg BW BLM with administration of 200μmol/kg BW tempol in each group (total 4 group). Brain cells were dissociated into single cells, cultured for 96h, incubated with an antibody against glial fibrillary acidic protein, and stained with acridine orange. The frequency of micronucleated astrocytes was determined using a fluorescence microscope. The frequency of micronucleated astrocytes in the 10mg/kg BW bleomycin plus EMF exposure group (Mean±SD: 19.8±5.2‰) was 1.6 times higher than that in the 10mg/kg BW bleomycin plus sham-exposure group (Mean±SD: 12.7±3.3‰) (p<0.05). Analysis of the frequency of micronuclei in astrocytes after co-exposure to EMF and bleomycin for 72h and administration of tempol revealed that, in the EMF exposure group, the frequency of micronuclei in rats administered with 10mg/kg BW bleomycin and treated with tempol (Mean±SD: 11.2±1.9‰) was 40% of that in rats administered with the same dose of bleomycin and physiological saline (Mean±SD: 28.0±15.0‰) (p<0.01). Results of the current study suggested that the mechanism responsible for the elevated frequency of micronuclei in astrocytes of rats co-exposed to BLM and EMFs is related to active oxygen species.

Zinc deficiency or excess within the physiological range increases genome instability and cytotoxicity, respectively, in human oral keratinocyte cells

Zinc (Zn) is an essential component of Zn-finger proteins and acts as a cofactor for enzymes required for cellular metabolism and in the maintenance of DNA integrity. The study investigated the genotoxic and cytotoxic effects of Zn deficiency or excess in a primary human oral keratinocyte cell line and determined the optimal concentration of two Zn compounds (Zn Sulphate (ZnSO(4)) and Zn Carnosine (ZnC)) to minimise DNA damage. Zn-deficient medium (0μM) was produced using Chelex treatment, and the two Zn compounds ZnSO(4) and ZnC were tested at concentrations of 0.0, 0.4, 4.0, 16.0, 32.0 and 100.0μM. Cell viability was decreased in Zn-depleted cells (0 μM) as well as at 32μM and 100μM for both Zn compounds (P<0.0001) as measured via the MTT assay. DNA strand breaks, as measured by the comet assay, were found to be increased in Zn-depleted cells compared with the other treatment groups (P<0.05). The Cytokinesis Block Micronucleus Cytome assay showed a significant increase in the frequency of both apoptotic and necrotic cells under Zn-deficient conditions (P<0.05). Furthermore, elevated frequencies of micronuclei (MNi), nucleoplasmic bridges (NPBs) and nuclear buds (NBuds) were observed at 0 and 0.4μM Zn, whereas these biomarkers were minimised for both Zn compounds at 4 and 16μM Zn (P<0.05), suggesting these concentrations are optimal to maintain genome stability. Expression of PARP, p53 and OGG1 measured by western blotting was increased in Zn-depleted cells indicating that DNA repair mechanisms are activated. These results suggest that maintaining Zn concentrations within the range of 4-16μM is essential for DNA damage prevention in cultured human oral keratinocytes.

The effect of zinc sulphate and zinc carnosine on genome stability and cytotoxicity in the WIL2-NS human lymphoblastoid cell line

Zinc (Zn) is an essential cofactor required by numerous enzymes that are essential for cell metabolism and the maintenance of DNA integrity. We investigated the effect of Zn deficiency or excess on genomic instability events and determined the optimal concentration of two Zn compounds that minimize DNA-damage events. The effects of Zn sulphate (ZnSO 4) and Zn carnosine (ZnC) on cell proliferation were investigated in the WIL2-NS human lymphoblastoid cell line. DNA damage was determined by the use of both the comet assay and the cytokinesis-block micronucleus cytome (CBMN-Cyt) assay. Zn-deficient medium (0 μM) was produced using Chelex treatment, and the two Zn compounds (i.e. ZnSO 4 and ZnC) were tested at concentrations of 0.0, 0.4, 4.0, 16.0, 32.0 and 100.0 μM. Results from an MTT assay showed that cell growth and viability were decreased in Zn-depleted cells (0 μM) as well as at 32 μM and 100 μM for both Zn compounds ( P < 0.0001). DNA strand-breaks, as measured by the comet assay, were found to be increased in Zn-depleted cells compared with the other treatment groups ( P < 0.05). The CBMN-Cyt assay showed a significant increase in the frequency of both apoptotic and necrotic cells under Zn-deficient conditions ( P < 0.0001). Elevated frequencies of micronuclei (MNi), nucleoplasmic bridges (NPBs) and nuclear buds (NBuds) were induced in Zn-depleted cells ( P < 0.0001), whereas genome damage was reduced in supplemented cultures for both Zn compounds at 4 μM and 16 μM, possibly suggesting that these concentrations may be optimal for genome stability. The potential protective effect of ZnSO 4 and ZnC was also investigated following exposure to 1.0 Gy γ-radiation. Culture in medium containing these compounds at 4–32 μM prior to irradiation displayed significantly reduced frequencies of MNi, NPBs and NBuds compared with cells maintained in 0 μM medium ( P < 0.0001). Expression of γ-H2AX and 8-oxoguanine glycosylase measured by western blotting was increased in Zn-depleted cells. These results suggest that Zn plays important role in genomic stability and that the optimal Zn concentration-range for prevention of DNA damage and cytotoxicity in vitro lies between 4 and 16 μM.

No elevated genomic damage in children and adolescents with attention deficit/hyperactivity disorder after methylphenidate therapy

Background and objective Attention-deficit/hyperactivity disorder (ADHD) is the most frequent psychiatric disorder in children and adolescents and is often treated with methylphenidate (MPH), resulting in MPH exposure in more than 1% of all children in many countries. A 2005 report on cytogenetic effects in peripheral lymphocytes from 12 ADHD children treated for 3 months with MPH raised questions about its genetic toxicity and potential carcinogenicity. In 2007, we described no elevated micronucleus frequency in 21 children after 3 months of MPH-treatment; since the difference between the two studies could not be explained we now enlarged the overall sample size, and added a healthy control group, a new chronically treated group and positive control slides. Furthermore, micronuclei were analyzed in a second tissue, buccal mucosa. Study participants A healthy control group (23 individuals), a chronically MPH-treated (>12 months) group (21 patients), and a drug naïve group of ADHD-affected children (26 patients), which was analyzed again after 3 months (17 patients) and 6 months (11 patients), provided samples for analysis of micronucleated lymphocytes. With inclusion of 14 previously obtained blood samples, an overall group size of 31 patients was reached for the comparison of the 3 months observation time with before for micronucleated lymphocytes. For buccal mucosa cells, an additional inclusion of 10 more chronically treated patients (no lymphocytes donated) yielded sample numbers of 22 (healthy), 17 (chronically treated), 23 (ADHD drug naïve), 14 (3 months) and 11 (6 months). Results No significant alteration in genomic damage as seen as micronucleus frequency in peripheral lypmphocytes or buccal mucosa cells was detected after MPH treatment. Conclusions No indication for genomic damage induced by MPH was obtained in this study, as in our previous study. Together with our previous study, our overall number of MPH-treated patients is now 68 (30 chronically treated, 38 prospectively followed), plus 23 healthy controls. Ongoing studies in the USA, as well as continuation of recently published epidemiological cancer incidence analysis should provide additional reassurance for MPH-treated ADHD patients.

Interlaboratory evaluation of a flow cytometric, high content in vitro micronucleus assay

An international, multi-lab trial was conducted to evaluate a flow cytometry-based method for scoring micronuclei in mouse lymphoma L5178Y cells [S.L. Avlasevich, S.M. Bryce, S.E. Cairns, S.D. Dertinger, In vitro micronucleus scoring by flow cytometry: differential staining of micronuclei versus apoptotic and necrotic chromatin enhances assay reliability, Environ. Mol. Mutagen. 47 (2006) 56–66]. A reference laboratory investigated the potential of six chemicals to induce micronuclei—the genotoxicants mitomycin C (MMC), etoposide (ETOPO), and vinblastine (VB), and the non-genotoxicants sucrose (SUC), staurosporine (STS), and dexamethasone (DEX). The latter two non-genotoxicants were selected as extreme challenges to the assay because of their potent apoptogenic activity. Three collaborating laboratories were supplied with prototype In Vitro MicroFlow™ kits, and each was assigned one genotoxicant and one non-genotoxicant. Cells were treated continuously for 24 h over a range of concentrations up to 5 mg/ml, or overtly cytotoxic concentrations. Micronuclei were scored via standard microscopy and flow cytometry. In addition to enumerating micronucleus frequencies, a cytotoxicity measurement that is simultaneously acquired with the flow cytometric micronucleus scoring procedure was evaluated (Flow-NBR). With this method, latex particles served as counting beads, and facilitated relative survival measurements that exclude the presence of dead/dying cells. For comparison purposes, additional cytotoxicity endpoints were measured, including several that are based on cell number, and others that reflect compromised membrane integrity, including dye permeability and/or phospholipid distribution. Key findings for this set of compounds include the following: (1) significant discrepancies in top concentration selection were found when cytotoxicity measurements were based on different methods, with the Flow-NBR approach tending to be the most sensitive, (2) both microscopy- and flow cytometry-based scoring methods detected concentration-dependent micronucleus formation for the three genotoxic agents studied, with good agreement between the reference laboratory and the collaborating laboratories, and (3) whereas flow cytometric analyses showed no significant increases for the non-genotoxicants when top concentration selection was based on Flow-NBR, significantly elevated micronucleus frequencies were observed for concentrations that were chosen based on less-sensitive cytotoxicity assays. Collectively, these results indicate that rapid assessment of genotoxicity can be accomplished with a relatively simple flow cytometric technique, and that the scoring system is transferable across laboratories. Furthermore, a concurrent assessment of cytotoxicity, Flow-NBR, may help reduce the occurrence of irrelevant positive results, as it may represent a more appropriate means for choosing top concentration levels. Finally, the data presented herein reinforce concerns about the manner in which cytotoxicity limits are described in guidance documents, since these recommendations tend to cite fixed cut-off values without reference to methodology.

Micronuclei in diabetes: Folate supplementation diminishes micronuclei in diabetic patients but not in an animal model

Diabetes mellitus (DM) is associated with a high risk of health complications, mainly due to excessive free radical (FRs) production that could result in an increased frequency of micronuclei. The consumption of antioxidants, like folic acid (FA), may mitigate the effects of the FRs. In the present study, micronucleated polychromatic erythrocyte (MNPCE) frequencies were determined in blood sampled weekly from the tails of pregnant female Wistar rats and pregnant Wistar rats with experimental diabetes that were given unsupplemented diets and diets supplemented with FA. At birth, the pups were sampled to analyze micronucleated erythrocyte (MNE) and MNPCE frequencies. Moreover micronucleated cells (MNCs) were evaluated in buccal mucosa samples taken from 81 healthy adult subjects, 48 patients with DM, and 30 DM patients who were sampled before and after FA treatment. Increases in MNPCE frequencies were significant only at the first sampling ( P < 0.01 and P < 0.03) in pregnant rats with experimental diabetes. In addition, the pups from the diabetic group and from diabetic group treated with FA had higher frequencies of MNEs ( P < 0.03 and P < 0.001, respectively) and MNPCEs ( P < 0.009 and P < 0.05, respectively) than the controls. No differences were found in diabetic rats and newborn rats born to diabetic mothers treated with FA compared with untreated animals. Patients with DM had a higher frequency of MNCs compared with healthy subjects ( P < 0.001). Also FA reduced the frequency of MNCs in DM patients ( P < 0.001). The results of this study indicate that diabetes results in elevated frequencies of micronuclei, and that, at least in humans, FA can protect against the elevation.

Influence of Antimonite, Selenite, and Mercury on the Toxicity of Arsenite in Primary Rat Hepatocytes

The long-term toxicity of arsenic (As) as a result of exposure to contaminated drinking water might be modified by coinciding exposures to elements like selenium, antimony, or mercury. In this study the influence of tetravalent selenite, trivalent antimonite, and divalent mercury was investigated in vitro using cultured primary rat hepatocytes. The cell vitality was assessed in the 3-[4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide] (MTT), assay with concurrent exposures of the cells to up to 50 microM sodium arsenite(III) and a potential modifier [50 microM sodium(IV) selenite, 10 microM antimony(III) chloride, 25 microM mercuric(II) chloride], which indicated an additive increase in the combined cytotoxicity. Sodium arsenite was tested for genotoxicity in the micronucleus test in a concentration range of 0.25 up to 7.5 microM. In this range, the MTT conversion was at least 80%, indicating high cell viability. Adose-dependent induction of micronuclei was observed. The lowest concentration causing a significantly elevated frequency of micronuclei was 1 microM As (p < 0.05). A significant influence (i.e., reduction of the combined genotoxicity as a result of the presence of a potential modifier) was only observed for 10 and 25 microM antimony chloride (p < 0.05, Fisher's exact test). The metabolic methylation of arsenite was not affected by concurrent incubation with any of the potential modifiers.