CBMN-cyt Assay Research Articles

Preparation, characterization, and in vitro cytogenotoxic evaluation of a novel dimenhydrinate-β-cyclodextrin inclusion complex.

Dimenhydrinate (DMH), used to alleviate motion sickness symptoms such as nausea, vomiting, dizziness, and vertigo, encounters limitations in oral pharmaceutical formulations due to its poor water solubility and bitter taste. Our research hypothesized that inclusion complexation with β-cyclodextrin (β-CD) might address these drawbacks while ensuring that the newly formed complexes exhibit no cytotoxic or genotoxic effects on peripheral blood mononuclear cells (PBMCs). Inclusion complexes were prepared using the kneading method and the solvent evaporation method. The phase solubility analysis, attenuated total reflectance-fourier transform infrared spectroscopy (ATR-FTIR), and differential scanning calorimetry (DSC) were conducted to evaluate the complexation efficacy and stability constant of the new binary systems. The results demonstrated that both methods provided complete and efficient complexation. Cytogenotoxic analysis, including the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay, alkaline comet assay, and cytokinesis-block micronucleus cytome (CBMN-cyt) assay, was conducted to assess the cytogenotoxic potential of DMH-β-CD inclusion complexes, a topic previously unexamined. No cytotoxic or genotoxic effects were observed within the concentration range of 36.36 to 109.09 ng/mL. Cell viability of treated PBMCs exceeded 85% for all tested concentrations. No significant increases in DNA strand breaks were observed at any dose, and tail intensity of all complexes remained lower or up to 2.2% higher than the negative control. Parameters indicating genotoxic effects, as well as cytotoxic and cytostatic potential in the CBMN-cyt assay, did not significantly differ from untreated controls. These results suggest that inclusion complexation with β-CD might be a safe and promising solution to overcome the limitations of poor solubility and unpleasant taste of DMH, potentially providing opportunities for new and improved oral pharmaceutical dosage forms.

The increased chromosomal DNA damage in patients with Familial Mediterranean Fever

ABSTRACT Familial Mediterranean Fever (FMF) is an inherited autoinflammatory disease. In this study, we aimed to assess chromosomal DNA damage and cell proliferation by using cytokinesis-block micronucleus cytome (CBMN-cyt) assay in the peripheral blood lymphocytes of untreated FMF patients carrying M694V and R202Q mutations, which are the most common MEFV gene mutations in Turkish society. The study included 20 untreated FMF patients with M694V and R202Q mutations and 20 healthy individuals of similar age and sex as the control group. Micronucleus (MN), nucleoplasmic bridges (NPBs), and nuclear buds (NBUDs) were scored in the obtained bi-nucleated (BN) cells. Additionally, the nuclear division index (NDI) was calculated using the scores of mononuclear, binuclear, and multinuclear cells. We found that MN and NPBs frequencies in FMF patients were significantly higher than in controls, and number of metaphases was significantly lower (respectively, p < 0.05, p < 0.01, and p < 0.01). However, there was no significant difference in NBUDs frequencies and NDI values between FMF patients and controls (p > 0.05). Our study is the first to evaluate FMF patients’ lymphocytes using the CBMN-cyt assay, as no previous research has been found in this respect. Increased MN and NPB frequencies may be useful as biomarkers for chromosomal DNA damage, and may indicate a potential for elevated cancer risk in untreated FMF patients.

The effects of iodine 131 treatment on chromosomal and oxidative DNA damage in papillary thyroid carcinoma

Papillary thyroid carcinoma (PTC) is a common endocrine cancer with a good prognosis. Radioactive iodine is thought to be useful for individuals who have had a total or almost total thyroidectomy, but its effects are still controversial. The effects of radioactive iodine-131 (I-131) treatment on oxidative and chromosomal damage in PTC patients were examined in this study, which was carried out with 16 patients newly diagnosed with PTC and 20 healthy control subjects with similar age and gender. Blood samples were taken from patients with PTC at five sampling times (before total thyroidectomy, after total thyroidectomy, and seven days, six months, and one year after treatment) and from control subjects. The cytokinesis block micronucleus cytome (CBMN-cyt) assay parameters in peripheral blood lymphocytes of patients with PTC and controls were evaluated and plasma 8-hydroxydeoxyguanosine (8-OHdG) levels were measured. Furthermore, genome instability and oxidative DNA damage in peripheral blood lymphocytes and plasma of patients with PTC were evaluated before total thyroidectomy (n=16), after total thyroidectomy (before I-131 treatment) (n=16), seven days (n=10), six months (n=5), and one year after treatment (n=5). The numbers of CBMN-cyt assay parameters (micronucleus; MN and nucleoplasmic bridges; NPB) and 8-OHdG levels in patients with PTC were determined to be significantly higher than in those of the control subjects and these values significantly decreased after total thyroidectomy (before I-131 treatment). While the number of MN, apoptotic, and necrotic cells increased after I-131 treatment, it significantly decreased after six months and one year after treatment. The results achieved in this study suggest that I-131 treatment may pose a threat to cells and that radioactive iodine therapy should be avoided (if possible) for patients with PTC after total thyroidectomy.

Role of Oxidative Stress and DNA Damage on Preterm Birth Outcome.

Background: Preterm birth (PTB) poses a significant global health challenge and focused research is vital for improving maternal and neonatal health outcomes. The purpose of this study was to determine the effect of oxidative stress (OS) and DNA damage on PTB. Methods: There were two groups: (a) cases consisting of mothers with PTB (<37weeks of gestation, n = 100) and (b) controls consisting of mothers with term birth (>37weeks of gestation, n = 100). Women with vaginal infection, non-cephalic presentation, multiple gestations, fetal anomalies, Cesarean delivery, pregnancy with Mullerian anomalies, or preeclampsia were excluded from the study. OS analysis was conducted by measuring levels of superoxide dismutase (SOD), catalase (CAT), lipid peroxidation (LPO), and total protein and DNA damage were evaluated by CBMN-Cyt assay. Statistical analysis was performed using students' t-test and one-way ANOVA. Results: Low levels of antioxidants SOD and CAT (p < .0001), and total protein (p < .0001), besides high malondialdehyde (byproduct of LPO) (p < .0001) were observed in the PTB group. Moreover, high frequencies of micronuclei (p < .0001) and nucleoplasmic buds (p < .01) were detected in the PTB mothers compared to term birth mothers, while no significance was observed in the nucleoplasmic bridge frequencies. Conclusion: When the body's immune system and antioxidants fail to cope up with the generated OS, it can lead to PTB. Along with other body tests, OS markers and CBMN-Cyt tests have the potential to be used in diagnostics for early warning as well as monitoring and advising mothers for a better pregnancy outcome.

Unraveling gene expression and genetic instability in dental fluorosis: Investigating the impact of chronic fluoride exposure

Chronic fluoride exposure, even in small quantities, when continuously ingested by the human population, can lead to a significant public health concern known as fluorosis. Our understanding of the effects of fluoride on human health, as well as its potential to impact DNA, is limited. The present study aimed to assess genetic instability in 20 individuals diagnosed with dental fluorosis and 20 individuals without the condition from the state of Rio Grande do Sul, Brazil. The participants' dental fluorosis was evaluated using the Thylstrup-Fejerskov index (TF). To further evaluate genetic instability, several assays were conducted, including the alkaline and modified (+FPG) comet assay (using a visual score, VS), the buccal micronucleus (MN) cytome (BMCyt) assay, the cytokinesis-block MN (CBMN-Cyt) assay, and the measurement of telomere length (TL). In addition, the study utilized tools from Systems Biology to gain insights into the effects of fluoride exposure on humans, which aided in the selection and evaluation of mRNA expression levels of specific genes, namely PPA1 (inorganic pyrophosphatase 1), AQP5 (Aquaporin 5), and MT-ATP6 (Mitochondrially Encoded Adenosine Triphosphate Synthase Membrane Subunit 6). Furthermore, fluoride levels in the blood and urine were assessed using an ion-selective electrode, along with the evaluation of the inflammatory response in serum. The group with dental fluorosis exhibited 2.18 times higher MN frequencies specifically when assessed using the CBMN-Cyt assay, in comparison with individuals without fluorosis. Findings from the enzyme-modified comet assay indicated oxidative damage to purines in DNA. Furthermore, a decrease in TL was observed, along with elevated expression patterns of the PPA1 and AQP5 genes, and significant alterations in cytokine release. Significant correlations were identified between the TF and age, as well as the levels of necrotic cells. Additionally, noteworthy correlations were established between fluoride levels and the levels of MN, VS, and MT-ATP6. Although dental fluorosis results from fluoride exposure, our research highlights the potential influence of this condition on genomic instability and gene expression. Consequently, our findings stress the importance of continuously monitoring populations with a high incidence of dental fluorosis to enhance our comprehension of how genomic instability might correlate with the origins and consequences of health problems in these individuals.

The Relationship between Telomere Length and Nucleoplasmic Bridges and Severity of Disease in Prostate Cancer Patients

Telomeres are repetitive nucleotide (TTAGGG) sequences that stabilize the chromosome ends and play an important role in the prevention of cancer initiation and progression. Nucleoplasmic bridges (NPBs) are formed when chromatids remain joined together during mitotic anaphase either due to mis-repair of DNA breaks or due to chromatid end fusion as a result of telomere loss or telomere dysfunction. We tested the hypotheses that (i) telomere length (TL) is shorter in prostate cancer (PC) patients relative to healthy age-matched individuals, (ii) TL differs in different stages of PC and (iii) shorter TL is significantly correlated with NPBs formation in PC cases. TL was measured in whole blood by well-established quantitative PCR method and the frequency of NPBs was measured in lymphocytes using cytokinesis-block micronucleus cytome (CBMNcyt) assay. Our results indicate that TL is shorter and NPBs are increased in PC patients relative to age-matched healthy controls. Furthermore, TL was significantly shorter (p = 0.03) in patients with a Gleason score more than 7 and there was also a significant trend of decreasing TL across all three stages (p trend = 0.01; Gleason score &lt;7, 7 and &gt;7). Furthermore, TL was significantly inversely correlated with NPB frequency in PC patients (r = −0.316; p = 0.001) but not in controls (r = 0.163; p = 0.06) and their relationships became stronger with higher Gleason scores. More studies are required that can confirm our observations and explore mechanistic differences in the role of telomeres in NPB formation in PC cases relative to non-cancer cases.

Assessment of genotoxic instability markers in peripheral blood lymphocytes of breast cancer patients: a case control study

Changes in genetic constitution of an individual leads to uncontrollable cell growth and tumour formation. The acquisition of genomic instability predisposes cells to accumulate stable genome mutations causing carcinogenesis. The cytokinesis-block micronucleus cytome assay (CBMN), a well-established marker assay for chromosomal mutagen sensitivity, was applied in this study enrolling breast cancer patients and age and sex-matched controls. This work aimed to assess the predictive value of the frequency of genotoxic markers in peripheral blood lymphocytes for the risk/susceptibility of breast cancer. Samples from a hundred untreated breast cancer patients and age and sex matched controls were enrolled in the study from Government Medical College, Alappuzha. The genomic instability was assessed using cytokinesis block micronucleus assay where cytome events were marked. The results showed a significant increase in the frequency of micronucleus, nucleoplasmic bridge, and buds in the binucleated cells of breast cancer patients compared to the control samples. The variability was assessed by CBMN Cyt assay. The frequency of Micronuclei and Nucleoplasmic buds was significantly higher in the patient groups than in the controls (p < 0.0001). In Breast cancer patients, the median (IQR) range of MNi was 12(6), the Nucleoplasmic bridge 3(3) and the Nuclear buds were 2(1) and, in the controls, it was 6(5), 1(2) and 1(1) respectively. A larger difference in the frequency of genetic markers in cancer patients over control cases support a significant role of these markers in the population screening of individuals at high risk of cancer. Communicated by Ramaswamy H. Sarma

Effects of folate on telomere length and chromosome stability of human fibroblasts and melanoma cells in vitro: a comparison of folic acid and 5-methyltetrahydrofolate.

Telomere length (TL), which is maintained by human telomerase reverse transcriptase (hTERT; component of telomerase) and/or TRF1/TRF2 (core components of shelterin) via different mechanisms, is essential for chromosomal stability and cell survival. Folates comprise a group of essential B9 vitamin that involve in DNA synthesis and methylation. This study aimed to evaluate the effects of folic acid (FA) and 5-methyltetrahydrofolate (5-MeTHF) on TL, chromosome stability, and cell survival of telomerase-negative BJ and telomerase-positive A375 cells in vitro. BJ and A375 cells were cultured in modified medium with FA or 5-MeTHF (22.6 or 2260 nM) for 28 days. TL and mRNA expression were determined by RT-qPCR. Chromosome instability (CIN) and cell death were measured by CBMN-Cyt assay. Results showed that abnormal TL elongation was observed in FA and 5-MeTHF deficient BJ cells. The TL of A375 cells showed no obvious alterations under the FA-deficient condition but was significantly elongated under the 5-MeTHF-deficient condition. In both BJ and A375 cells, FA and 5-MeTHF deficiency caused decreased TRF1, TRF2, and hTERT expression, increased CIN and cell death; while a high concentration of 5-MeTHF induced elongated TL, elevated CIN, increased TRF1 and TRF2 expression and decreased hTERT expression, when compared with the FA counterpart. These findings concluded that folate deficiency induced TL instability in both telomerase-negative and -positive cells, and FA was more efficient in maintaining TL and chromosome stability compared with 5-MeTHF.

Effects of folate on telomere length and chromosome stability of human fibroblasts and melanoma cells in vitro: A comparison of folic acid and 5-methyltetrahydrofolate.

Telomere length (TL), which is maintained by hTERT (component of telomerase) and/or TRF1/TRF2 (core components of shelterin) via different mechanisms, is essential for chromosomal stability and cell survival. Folates comprise a group of essential B9 vitamin that involve in DNA synthesis and methylation. This study aimed to evaluate the effects of folic acid (FA) and 5-methyltetrahydrofolate (5-MeTHF) on TL, chromosome stability, and cell survival of telomerase-negative BJ and telomerase-positive A375 cells in vitro. BJ and A375 cells were cultured in modified medium with FA or 5-MeTHF (22.6 or 2260nM) for 28 days. TL and mRNA expression were determined by RT-qPCR. Chromosome instability (CIN) and cell death were measured by CBMN-Cyt assay.Results showed that abnormal TL elongation was observed in FA and 5-MeTHF deficient BJ cells. The TL of A375 cells showed no obvious alterations under the FA deficient condition but was significantly elongated under the 5-MeTHF deficient condition. In both BJ and A375 cells, FA and 5-MeTHF deficiency caused decreased TRF1, TRF2, and hTERT expression, increased CIN and cell death; while a high concentration of 5-MeTHF induced elongated TL, elevated CIN, increased TRF1 and TRF2 expression and decreased hTERT expression, when compared with the FA counterpart. These findings concluded that folate deficiency induced TL instability in both telomerase-negative and positive cells, and FA was more efficient in maintaining TL and chromosome stability compared with 5-MeTHF.

Oxidative and chromosomal DNA damage in patients with type I Gaucher disease and carriers

Background and aimsGaucher disease (GD) is caused by a genetic deficiency of the beta-glucocerebrosidase enzyme which results in the accumulation of glucosylceramide in macrophages. This accumulation may induce oxidative stress, resulting in DNA damage in patients with GD. The aim of this study was to assess plasma 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels and cytokinesis-block micronucleus cytome (CBMN-cyt) assay parameters in the peripheral blood lymphocytes of patients with GD and carriers, evaluate the possible associations of these values with GD, and determine whether they can be used as potential biomarkers in GD. MethodsThis study included 20 patients with type 1 GD, six carriers, and 27 age- and sex-matched healthy controls. CBMN-cyt assay parameters in peripheral blood lymphocytes of the patients with GD, carriers, and controls were evaluated and 8-OHdG levels in their plasma samples were measured. ResultsCBMN-cyt assay parameters in patients with GD and carriers were not significantly different when compared with controls (p > 0.05). However, plasma 8-OHdG levels were found to be higher in both patients with GD and carriers than in control subjects (p < 0.01). ConclusionsOxidative DNA damage may be a useful prognostic tool, whereas the CBMN-cyt assay cannot be used as a predictive biomarker of GD.

Genetic Instability among Hitnü People Living in Colombian Crude-Oil Exploitation Areas.

Oil exploitation, drilling, transportation, and processing in refineries produces a complex mixture of chemical compounds, including polycyclic aromatic hydrocarbons (PAHs), which may affect the health of populations living in the zone of influence of mining activities (PZOI). Thus, to better understand the effects of oil exploitation activities on cytogenetic endpoint frequency, we conducted a biomonitoring study in the Hitnü indigenous populations from eastern Colombia by using the cytokinesis micronucleus cytome assay (CBMN-cyt). PAH exposure was also measured by determine urine 1-hydroxypyrene (1-OHP) using HPLC. We also evaluated the relationship between DNA damage and 1-OHP levels in the oil exploitation area, as well as the modulating effects of community health factors, such as Chagas infection; nutritional status; and consumption of traditional hallucinogens, tobacco, and wine from traditional palms. The frequencies of the CBMN-cyt assay parameters were comparable between PZOI and Hitnü populations outside the zone of influence of mining activities (POZOI); however, a non-significant incremental trend among individuals from the PZOI for most of the DNA damage parameters was also observed. In agreement with these observations, levels of 1-OHP were also identified as a risk factor for increased MN frequency (PR = 1.20) compared to POZOI (PR = 0.7). Proximity to oil exploitation areas also constituted a risk factor for elevated frequencies of nucleoplasmic bridges (NPBs) and APOP-type cell death. Our results suggest that genetic instability and its potential effects among Hitnü individuals from PZOI and POZOI could be modulated by the combination of multiple factors, including the levels of 1-OHP in urine, malnutrition, and some traditional consumption practices.

In vitro analysis of thimerosal genotoxicity and cytotoxicity effect

Background: Thimerosal is an organomercury compound with high mercury content. Thimerosal is located in the market as an effective bacteriostatic in a series of pharmaceutical products, ophthalmic and nasal products, immunoglobulin preparations, and as a preservative in vaccines. Aims and Objectives: Since it is a compound with living content, this study aimed to examine the genotoxic and cytotoxic potential of thimerosal in human lymphocytes culture. Materials and Methods: We used chromosome aberration analysis and cytokinesis-block micronucleus cytome (CBMN-Cyt) assay to test its genotoxic and cytotoxic potential in human lymphocyte culture. Results: Results showed that the frequency of structural chromosome aberrations and CBMN-cyt assay was significantly increased in treated cultures (1 μg/ml and 0.5 μg/ml) compared to the negative control. Conclusion: Obtained results and statistical analysis show that thimerosal is genotoxic and cytotoxic in human lymphocytes in tested concentrations.

Fluorosilicic acid and cotinine, separately and in combination, induce genotoxicity and telomeric reduction in human osteoblast cell line MG63

Skeletal fluorosis is a severe case in which bone deformations and bone tissue weakening occur due to excessive fluorine deposition. Recently, data on smoking have been published that smoke constituents can indirectly influence bone mass and interfere in the metabolism of fluorides in humans. Thus, the present in vitro study aimed to assess the genetic instability in human osteoblast MG63 cells exposed to fluorosilicic acid (FA) and cotinine (COT), separately and in combination, in concentrations found in human plasma. For this, cell cytotoxicity was performed by MTT assay; DNA damage was performed by alkaline comet assay (CA), modified by repair endonucleases (+FPG); micronuclei test (MN) using CBMN-Cyt assay; and telomere length (TL) by qPCR in MG63 cells. No cytotoxicity was observed for all concentrations tested in this study. Alkaline CA results showed a significant increase in DNA damage at all FA concentrations (0.03125–0.300 mg/L), in the two highest concentrations of COT (125 and 250 ng/mL), and the highest concentration of FA+COT (0.300 mg/L+250 ng/mL). Alkaline CA+FPG test was used to detect oxidized nucleobases, which occurred at the two highest concentrations of FA, COT, and FA+COT. Micronuclei test showed an increase in the frequency of MN at all concentrations of FA (0.075–0.300 mg/L) except in the lowest concentration (0.03125 mg/L), in the two highest concentrations of COT (125 and 250 ng/mL), and all concentrations of FA+COT. There was no significant difference in nuclear division index, binucleated cells, nucleoplasmic bridge, and nuclear bud. A TL reduction was observed in cells treated with the highest concentrations of FA alone (0.300 mg/L) and FA+COT (0.300 mg/L+250 ng/mL). Finally, our study showed that FA and COT (mainly alone) at concentrations found in human plasma induced oxidative damage and genetic instability in human osteoblast cells.

Evidence of chromosomal damage in scleroderma

Evidence of chromosomal damage in scleroderma

Genotoxicity of advanced glycation end products in vitro is influenced by their preparation temperature, purification and cell exposure time.

Advanced glycation end products (AGEs) are formed via non-enzymatic reactions between amino groups of proteins and the carbonyl groups of reducing sugars. Previous studies have shown that highly glycated albumin prepared using a glucose-bovine serum albumin (Glu-BSA) model system incubated at 60°C for 6 weeks induces genotoxicity in WIL2-NS cells at 9 days of exposure measured by the cytokinesis-block micronucleus cytome (CBMNcyt) assay. However, this AGE model system is not physiologically relevant as normal body temperature is 37°C and the degree of glycation may exceed the extent of albumin modification in vivo. We hypothesised that the incubation temperature and purification method used in these studies may cause changes to the chemical profile of the glycated albumin and may influence the extent of genotoxicity observed at 3, 6 and 9 days of exposure. We prepared AGEs generated using Glu-BSA model systems incubated at 60°C or 37°C purified using trichloroacetic acid (TCA) precipitation or ultrafiltration (UF) and compared their chemical profile (glycation, oxidation, and aggregation) and genotoxicity in WIL2-NS cells using the CBMNcyt assay after 3, 6 and 9 days of exposure. The number of micronuclei (MNi) was significantly higher for cells treated with Glu-BSA incubated at 60°C and purified via TCA (12 ± 1 MNi/1000 binucleated cells) compared to Glu-BSA incubated at 37°C and purified using UF (6 ± 1 MNi/1000 binucleated cells) after 9 days (P < 0.0001). The increase in genotoxicity observed could be explained by a higher level of protein glycation, oxidation, and aggregation of the Glu-BSA model system incubated at 60°C relative to 37°C. This study highlighted that the incubation temperature, purification method and cell exposure time are important variables to consider when generating AGEs in vitro and will enable future studies to better reflect in vivo situations of albumin glycation.

Homogentisic acid, a main phenolic constituent of strawberry tree honey, protects human peripheral blood lymphocytes against irinotecan-induced cytogenetic damage in vitro

Homogentisic acid (HGA) is the most abundant phenolic compound in strawberry tree (Arbutus unedo L.) honey and an intermediate in the metabolism of phenylalanine and tyrosine. Since HGA exerts its dual nature (pro-oxidant and antioxidant), which depends on the concentration and cell type, the aim of study was to determine whether HGA possess cytoprotective effects and could counteract the cyto- and genotoxic effects of the antineoplastic drug irinotecan (IRI). Tested concentrations corresponded to HGA content in average daily dose of strawberry tree honey as well as five- and ten-fold higher concentrations. Cyto- and genoprotective effects were tested on human peripheral blood lymphocytes using chromosomal aberrations assay and cytokinesis-block micronucleus cytome assay. HGA, even at concentrations 10-fold higher than the one present in the daily amount of consumed strawberry tree honey, posed a non-significant cytotoxic threat to lymphocytes, had a negligible potential for causing cytogenetic damage in treated cells, and did not significantly impair their proliferation. Results of the chromosomal aberration assay and CBMN Cyt assay also showed that HGA efficiently counteracted the detrimental cytogenetic effects of IRI in vitro. The finding on cyto- and genoprotective effects of HGA merits further research in order to better explain the safety profile of this compound and to assess its potency for the development of novel nutraceutical products.

Methylglyoxal induces chromosomal instability and mitotic dysfunction in lymphocytes.

Type 2 diabetes is associated with elevated levels of DNA damage, in particular micronuclei (MNi) which are formed by acentric chromosome fragments caused by double-stranded DNA breaks (DSBs), or whole chromosomes which fail to segregate during mitosis. We investigated if methylglyoxal (MGO), a reactive dicarbonyl known to be elevated in type 2 diabetes is capable of increasing chromosomal instability and DNA damage as measured by the cytokinesis block micronucleus cytome (CBMNcyt) assay in B-lymphoblastoid WIL2-NS cells and primary peripheral blood lymphocytes (PBL). We also investigated the level of various dicarbonyl stress biomarkers, including extracellular and intracellular MGO, protein and MGO modifications of DNA. WIL2-NS cells exposed to either MGO or a glyoxalase 1 inhibitor showed increases in MNi and nuclear buds, which were associated with an increase in intracellular MGO. DNA damage in the form of MNi and nucleoplasmic bridges were observed in primary PBL exposed to 10 µM MGO, suggesting low concentrations of MGO may be genotoxic. Furthermore, we showed, using fluorescent in situ hybridisation, that the majority of MNi caused by MGO in WIL2-NS cells were caused by whole chromosome loss events, rather than DSBs. Our data suggest that MGO, a reactive metabolite elevated in type 2 diabetes and other pathologies, can affect genomic integrity by impairing chromosome segregation during mitosis.

Exposure to hypomethylating 5-aza-2′-deoxycytidine (decitabine) causes rapid, severe DNA damage, telomere elongation and mitotic dysfunction in human WIL2-NS cells

Background5-aza-2′-deoxycytidine (5azadC, decitabine) is a DNA hypomethylating agent used in the treatment of myelodysplastic syndromes. Due to cytotoxic side effects dose optimization is essential. The aim of this study was to define and quantify the effects of 5azadC on biomarkers of chromosomal stability, and telomere length, in human lymphoblastoid cell line, WIL2-NS, at clinically relevant dosages. MethodsHuman WIL2-NS cells were maintained in complete medium containing 0, 0.2 or 1.0 μM 5azadC for four days, and analysed daily for telomere length (flow cytometry), chromosomal stability (cytokinesis-block micronucleus cytome (CBMN-cyt) assay), and global methylation (%5me-C). ResultsDNA methylation decreased significantly in 1.0 μM 5azadC, relative to control (p < 0.0001). Exposure to 1.0 μM 5azadC resulted in 1.7-fold increase in telomere length (p < 0.0001), in parallel with rapid increase in biomarkers of DNA damage; (micronuclei (MN, 6-fold increase), nucleoplasmic bridges (NPB, a 12-fold increase), and nuclear buds (NBud, a 13-fold increase) (all p < 0.0001). Fused nuclei (FUS), indicative of mitotic dysfunction, showed a 5- and 13-fold increase in the 0.2 μM and 1.0 μM conditions, respectively (p = 0.001) after 4 days. ConclusionsThese data show that (i) clinically relevant concentrations of 5azadC are highly genotoxic; (ii) hypomethylation was associated with increased TL and DNA damage; and (iii) longer TL was associated with chromosomal instability. These findings suggest that lower doses of 5azdC may be effective as a hypomethylating agent, while potentially reducing DNA damage and risk for secondary disease.

Chromosomal and oxidative DNA damage in non-functioning pituitary adenomas.

Clinically non-functioning pituitary adenomas (NFPA) are common tumours of the pituitary gland and are mainly considered as benign. The primary aim of this study was to research the effects of NFPA on genome instability in patients with non-functioning pituitary adenoma by using the cytokinesis-block micronucleus cytome (CBMN-cyt) assay and 8-hydroxy- 2'-deoxyguanosine (8-OHdG) assay. The second objective of this study was to assess whether there is a relationship between age, pituitary adenoma diameters, 8-OHDG levels, CBMN site assay parameters, and tumour aggressiveness. The study was performed on 30 patients who had been diagnosed with NFPA and were admitted to the Department of Endocrinology and Metabolism, and 20 healthy subjects of similar age and sex. Micronucleus (MN), nucleoplasmic bridges (NPBs), nuclear bud (NBUD) frequencies, and apoptotic and necrotic cell frequencies in patients with NFPA were found to be significantly higher than in control subjects, and plasma 8-OHdG levels in patients with NFPA were statistically significantly lower than control subjects in this study. It is believed that this is the first study to evaluate the aggressiveness of tumour with chromosome/oxidative DNA damage in patients with NFPA. However, further studies are needed in order to understand the cause of NFPA aggression and to evaluate these patients in terms of risk of cancer.

Prognostic value of the micronucleus assay for clinical endpoints in neoadjuvant radiochemotherapy for rectal cancer

BackgroundThe question whether lymphocyte radiosensitivity is representative of patients’ response to radiotherapy (RT) remains unsolved. We analyzed lymphocyte cytogenetic damage in patients who were homogeneously treated with preoperative radiochemotherapy (RCT) for rectal cancer within clinical trials. We tested for interindividual variation and consistent radiosensitivity after in-vivo and in-vitro irradiation, analyzed the effect of patients’ and RCT characteristics on cytogenetic damage, and tested for correlations with patients’ outcome in terms of tumor response, survival and treatment-related toxicity.MethodsThe cytokinesis-block micronucleus cytome (CBMNcyt) assay was performed on the peripheral blood lymphocytes (PBLCs) of 134 patients obtained before, during, at the end of RCT, and during the 2-year follow-up. A subset of PBLCs obtained before RCT was irradiated in-vitro with 3 Gy. RCT included 50.4 Gy of pelvic RT with 5-fluorouracil (5-FU) alone (n = 78) or 5-FU plus oxaliplatin (n = 56). The analyzed variables included patients’ age, gender, RT characteristics (planning target volume size [PTV size], RT technique), and chemotherapy characteristics (5-FU plasma levels, addition of oxaliplatin). Outcome was analyzed as tumor regression, patient survival, and acute and late toxicity.ResultsCytogenetic damage increased significantly with the radiation dose and varied substantially between individuals. Women were more sensitive than men; no significant age-dependent differences were observed. There was a significant correlation between the cytogenetic damage after in-vitro irradiation and in-vivo RCT. We found a significant effect of the PTV size on the yields of cytogenetic damage after RCT, while the RT technique had no effect. Neither the addition of oxaliplatin nor the 5-FU levels influenced cytogenetic damage. We found no correlation between patient outcome and the cytogenetic damage.ConclusionsWe found consistent cytogenetic damage in lymphocytes after in-vivo RCT and in-vitro irradiation. Gender was confirmed as a well-known, and the PTV size was identified as a less well-known influencing variable on lymphocyte cytogenetic damage after partial-body irradiation. A consistent level of cytogenetic damage after in-vivo and in-vitro irradiation may indicate the importance of genetic factors for individual radiosensitivity. However, we found no evidence that in-vivo or in-vitro irradiation-induced cytogenetic damage is an adequate biomarker for the response to RCT in rectal cancer patients.