Cytokinesis-block Micronucleus Cytome 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.

Cytotoxic and genotoxic profiles of the pyrethroid insecticide lambda-cyhalothrin and its microformulation Karate® in CHO-K1 cells.

Lambda-cyhalothrin (LCT) and its microformulation Karate® (25 % a.i.) were analysed for its genotoxicity and cytotoxicity on Chinese hamster ovary (CHO-K1) cells. Cytokinesis-block micronucleus cytome (CBMN-cyt) and alkaline single-cell gel electrophoresis (SCGE) bioassays were selected to test genotoxicity. Neutral red uptake (NRU), succinic dehydrogenase activity (MTT) and apoptogenic induction were employed for estimating cytotoxicity. Both compounds were analysed within a concentration range of 0.1-100µg/mL. Only LCT produced a significant augment in the frequency of micronuclei (MNs) when the cultures were exposed to highest concentrations of 10 and 100µg LCT/mL. A noticeable decrease in NDI was observed for cultures treated with LCT at 10 and 100µg/mL. Karate® induced the inhibition of both the proportion of viable cells and succinic dehydrogenase activity and triggered apoptosis 24h of exposition. Whilst an increased GDI in CHO-K1 cells was observed in the treatments with 1-100µg Karate®/mL, the GDI was not modified in the treatments employing LCT at equivalent doses. SCGE showed that Karate® was more prone to induce genotoxic effects than LCT. Only 50µg/mL of Karate® was able to increase apoptosis. Our results demonstrate the genomic instability and cytotoxic effects induced by this pyrethroid insecticide, confirming that LCT exposure can result in a severe drawback for the ecological equilibrium of the environment.

Genomic Instability and Cytotoxicity Evaluation of Two Communities Exposed to Pesticides in the Mexicali Valley by the L-CBMN Assay.

The continuous biomonitoring of a population directly or indirectly exposed to pesticides could be an additional tool for decision makers to improve their health conditions. In this work, we performed biomonitoring on two groups of people from the Mexicali Valley who were continuously exposed to pesticides using the cytokinesis-block micronucleus cytome assay (L-CBMN) to evaluate cytotoxic and genotoxic damage in human peripheral blood lymphocytes. The study groups comprised 14 indigenous Cucapah with non-vegetarian habits (NV group) from Ejido el Mayor (32.12594°, -115.27265°) and 21 lacto-ovo vegetarian (LOV) persons from the Seventh-day Adventist Church of Ejido Vicente Guerrero (32.3961°, -115.14023°). The L-CBMN assay determines the nuclear division index (NDI), apoptosis, necrosis, micronuclei (MNs), nuclear buds (NBUDs), and nucleoplasmic bridges (NPBs). Our results show that, regardless of diet or daily habits, both the studied groups presented with cytogenotoxic damage compared with non-exposed pesticide individuals, without modifications to the nuclear division index. In the rest of the evaluated biomarkers, the NV group exhibited greater cytotoxic and genotoxic damage than the LOV group. Nevertheless, individuals practicing a lacto-ovo vegetarian diet (LOV) showed lower damage than those with non-vegetarian habits (NV), suggesting a better antioxidant response that helps decrease the genotoxic damage due to the enhanced intake of folates and antioxidants from a plant-based diet.

Effect of Low Dietary Folate on Mouse Spermatogenesis and Spindle Assembly Checkpoint Dysfunction May Contribute to Folate Deficiency-Induced Chromosomal Instability in Cultured Mouse Spermatogonia.

Folate, as the initial substrate in one-carbon metabolism, is involved in the synthesis of important substances such as DNA, RNA, and protein. Folate deficiency (FD) is associated with male subfertility and impaired spermatogenesis, yet the underlying mechanisms are poorly understood. In the present study, we established an animal model of FD to investigate the effect of FD on spermatogenesis. GC-1 spermatogonia were used as a model to investigate the effect of FD on proliferation, viability, and chromosomal instability (CIN). Furthermore, we explored the expression of core genes and proteins of spindle assembly checkpoint (SAC), a signaling cascade ensuring accurate chromosome segregation and preventing CIN during mitosis. Cells were maintained in medium containing 0, 20, 200, or 2000 nM folate for 14 days. CIN was measured by using a cytokinesis-blocked micronucleus cytome assay. We found that sperm counts decreased significantly (p < 0.001) and the rate of sperm with defects in the head increased significantly (p < 0.05) in FD diet mice. We also found, relative to the folate-sufficient conditions (2000 nM), cells cultured with 0, 20, or 200 nM folate exhibited delayed growth and increased apoptosis in an inverse dose-dependent manner. FD (0, 20, or 200 nM) significantly induced CIN (p < 0.001, p < 0.001, and p < 0.05, respectively). Moreover, FD significantly and inverse dose dependently increased the mRNA and protein expression of several key SAC-related genes. The results indicate that FD impairs SAC activity, which contributes to mitotic aberrations and CIN. These findings establish a novel association between FD and SAC dysfunction. Thus, FD-impaired spermatogenesis may be partly due to genomic instability and proliferation inhibition of spermatogonia.

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.

Reduction of oral pathogens and oxidative damage in the CAL 27 cell line by Rosmarinus officinalis L. and Taraxacum officinale Web. extracts

Ethnopharmacological relevanceDandelion (Taraxacum officinale Web.) and rosemary (Rosmarinus officinalis L.) are treasured botanicals with a long usage history in traditional herbal practices worldwide. Dandelion was used to treat kidney, spleen, and liver disease, as well as cardiovascular disease, diabetes, and bacterial infections, whereas rosemary was used to treat pain, spasms, and to improve blood circulation. Aim of the studyThe aim of this study was to determine the influence of rosemary and dandelion leaves aqueous extracts on the human tongue epithelial carcinoma cell line (CAL 27) at the level of interaction between oral microbiota and tongue epithelial cells, genomic damage, and H2O2 - induced oxidative damage protection. Materials and methodsThe polyphenolic composition of the extracts was determined by spectrophotometric and HPLC analyses. After extract treatment, cytotoxic impact and ROS generation in CAL 27 cells were measured using the MTT assay and the 2',7'-dichlorofluorescein-diacetate (DCFH-DA) assay, respectively. Microdilutions were applied to investigate the antimicrobial and adhesive properties against representatives of the oral microbiota. The single-cell gel electrophoresis (comet assay) and cytokinesis-blocked micronucleus cytome assay (CBMN cyt) were used to detect induced genomic damages. ResultsBoth extracts increased the adhesion of the lactic acid bacteria L. plantarum but decreased the adhesion of the bacterial pathogens S. enterica serovar Typhimurium LT21 and E. coli K-12 MG1655 adhesion onto CAL 27 cells. 1 h treatment with 5x concentrated dandelion extract and 1x, 2.5x, and 5x of rosemary extract caused an increase in comet tail intensity. CBMN cyt results demonstrated a significant increase in micronucleus formation even at concentrations several times lower than the usual bioactive compound concentrations found in a cup of beverage, with higher concentrations also inducing cell apoptosis and necrosis. Rosemary extract showed a protective effect against H2O2 - induced oxidative damage by decreasing the apoptotic cell number, probably preventing mutations leading to tumor aggressiveness, invasion, and metastasis. ConclusionsBoth tested extracts demonstrated their usefulness in maintaining good oral bacteria balance and their protective capability as powerful antitumor agents by causing a protective apoptotic effect in tumor cell line already at the dosage of an average daily cup.

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

Toxicogenic effects of the mushroom Ganoderma lucidum on human liver and kidney tumor cells and peripheral blood lymphocytes.

Ganoderma lucidum (Curtis) P. Karst., a bioactive mushroom with medicinal properties, is known to exert immunomodulatory, anti-inflammatory, hypocholesterolemic, hypoglycemic, and hepatoprotective effects. In this study, the effects of the G. lucidum fruiting body dry extract (GLE) on human liver (HepG2/C3A) and kidney (786-O) tumor cells and peripheral blood lymphocytes were evaluated. MTT-based cytotoxicity, trypan blue-based cell viability, comet, and cytokinesis-block micronucleus cytome assays were performed, and the production of reactive oxygen species was evaluated in vitro. GLE was toxic to the tumor cells, decreasing their viability by increasing their production of reactive oxygen species and inducing damage to their DNA. By contrast, only high concentrations of GLE were toxic to lymphocytes and decreased their viability, whereas low concentrations increased lymphocyte viability. Moreover, primary DNA damage was induced by GLE only at the highest concentration tested. G. lucidum shows potential antitumor effects against cancerous kidney and liver cells, exhibiting cytotoxic and genotoxic activity at low concentrations, whereas the same effects in lymphocytes are mediated only at high concentrations. This mushroom has the potential to be biotechnologically developed into a therapeutic agent for diseases, such as cancer.

Genotoxic and cytotoxic effects of thiomersal and paracetamol combination

Background: Thiomersal is used as a preservative of some vaccines or as a trace from the pathogen inactivation process in vaccine production. Prophylactic use of paracetamol upon vaccination is still common, even though paracetamol decreases immune response on some vaccines. Considering the cytotoxic and genotoxic potential of thiomersal and paracetamol and possible interaction in vivo, in vitro study was performed. Methods: The genotoxic and cytotoxic effects of thiomersal and paracetamol combination were examined on human lymphocyte cultures by using two methods: analysis of chromosomal aberrations and cytokinesis-block micronucleus cytome assay. Blood samples of three healthy donors were analyzed with the following concentrations of tested substances: thiomersal 1 µg/ml and 0.5 µg/ml, paracetamol 20 µg/ml, thiomersal 0.5 µg/ml with paracetamol 20 µg/ml and thiomersal 1.0 µg/ml with paracetamol 20 µg/ml. Results: The analysis of structural chromosomal aberrations was significantly increased in all treated cultures. In cells treated with the combination of thiomersal 1 µg/ml and 20 µg/ml of paracetamol, the number of aberrations was significantly decreased. Cytokinesis-block micronucleus cytome assay analyses showed significantly increased micronucleus frequency in lymphocytes cultivated with thiomersal 1 µg/ml compared to lymphocytes cultures exposed to thiomersal 0.5 µg/ml. Conclusions: Induction of structural chromosome aberrations and micronucleus is shown as a sign of genotoxicity for the examined concentrations of thiomersal and paracetamol. The suppressing effect of paracetamol on thiomersal genotoxicity in lymphocytes culture treated with thiomersal was shown to be indicative of further examination of paracetamol use in the prevention of genotoxicity.

Investigation Of Genotoxic Effect Of Octyl Gallate Used As An Antioxidant Food Additive In In Vitro Test Systems.

Several antioxidant food additives are added to oils, soups, sauces, chewing gum, potato chips etc. One of them is octyl gallate. The purpose of this study was to evaluate the potential genotoxicity of octyl gallate in human lymphocytes, using in vitro chromosomal abnormalities (CA), sister chromatid exchange (SCE), cytokinesis block micronucleus cytome (CBMN-Cyt), micronucleus-FISH (MN-FISH), and comet tests. Different concentrations (0.50, 0.25, 0.125, 0.063, and 0.031 μg/mL) of octyl gallate were used. A negative (distilled water), a positive (0.20 μg/mL Mitomycin-C), and a solvent control (8.77 μL/mL ethanol) were also applied for each treatment. Octyl gallate did not cause changes in chromosomal abnormalities, micronucleus, nuclear bud (NBUD) and nucleoplasmic bridge (NPB) frequency. Similarly, there was no significant difference in DNA damage (comet assay), percentage of centromere positive and negative cells (MN-FISH test) compared to the solvent control. Moreover, octyl gallate did not affect replication and nuclear division index. On the other hand, it significantly increased the SCE/cell ratio in three highest concentrations compared to solvent control at 24h treatment. Similarly, at 48h treatment, the frequency of SCE raised significantly compared to solvent controls at all the concentrations (except 0.031 μg/mL). An important reduce was detected in mitotic index values in highest concentration at 24h treatment and almost all concentrations (except 0.031 and 0.063 µg / mL) at 48h treatment. The results obtained suggest that octyl gallate has no an important genotoxicological action on human peripheral lymphocytes at the concentrations applied in this study.

Analysis of the cytotoxic and genotoxic effects in a population chronically exposed to coal mining residues

During coal mining activities, many compounds are released into the environment that can negatively impact human health. Particulate matter, polycyclic aromatic hydrocarbons (PAHs), metals, and oxides are part of the complex mixture that can affect nearby populations. Therefore, we designed this study to evaluate the potential cytotoxic and genotoxic effects in individuals chronically exposed to coal residues from peripheral blood lymphocytes and buccal cells. We recruited 150 individuals who lived more than 20 years in La Loma-Colombia and 120 control individuals from the city of Barranquilla without a history of exposure to coal mining. In the cytokinesis-block micronucleus cytome (CBMN-Cyt) assay, significant differences in the frequency of micronucleus (MN), nucleoplasmic bridge (NPB), nuclear bud (NBUD), and apoptotic cells (APOP) were observed between the two groups. In the buccal micronucleus cytome (BM-Cyt) assay, a significant formation of NBUD, karyorrhexis (KRX), karyolysis (KRL), condensed chromatin (CC), and binucleated (BN) cells was observed in the exposed group. Considering the characteristics of the study group, a significant correlation for CBMN-Cyt was found between NBUD and vitamin consumption, between MN or APOP and meat consumption, and between MN and age. Moreover, a significant correlation for BM-Cyt was found between KRL and vitamin consumption or age, and BN versus alcohol consumption. Using Raman spectroscopy, a significant increase in the concentration of DNA/RNA bases, creatinine, polysaccharides, and fatty acids was detected in the urine of individuals exposed to coal mining compared to the control group. These results contribute to the discussion on the effects of coal mining on nearby populations and the development of diseases due to chronic exposure to these residues.

The influence of vitamin D supplementation and strength training on health biomarkers and chromosomal damage in community-dwelling older adults

Older adults lack of proper physical activity which is often accompanied by vitamin D deficiency. Those factors are known to contribute to health issues in the later years of life. The main goal of this intervention study was to investigate the effect of different vitamin D supplementation strategies for 4 weeks solely or combined with a 10-week strength training program on chromosomal stability in peripheral blood mononuclear cells in community-dwelling older people. One hundred women and men (65–85 years) received either vitamin D3 daily (800 IU), a monthly dose (50.000 IU) or placebo for 17 weeks. All groups received 400 mg calcium daily. The fitness status of the study participants was measured using the 30- second chair stand test, the handgrip strength test and the 6-min walk test. The cytokinesis block micronucleus cytome (CBMN) assay was applied to analyze chromosomal anomalies, including cytotoxic and genotoxic parameters. Changes in antioxidant markers were measured in plasma.Walking distance and chair stand performance improved significantly. Increased levels of the parameters of the CBMN assay were detected for all intervention groups at study end. At baseline micronuclei (MNi) frequency correlated significantly with BMI in both sexes (females: r = 0.369, p = 0.034; males: r = 0.265, p = 0.035), but not with vitamin D serum levels. In females, body fat (r = 0.372, p < 0.001) and functional parameter using the 30-s chair stand test (r = 0.311, p = 0.002) correlated significantly with MNi frequency. Interestingly, not vitamin D supplementation but 10 weeks of resistance training increased MNi frequency indicating elevated chromosomal instability and also adverse effects on antioxidant markers including glutathione and FRAP were detected in the group of community-dwelling older adults.

Effects of Curcumin and Soy Isoflavones on Genomic Instability of Human Colon Cells NCM460 and SW620.

Curcumin (CUR) and soy isoflavones (SIs) are two plant-based polyphenols that have attracted much attention, because of their extensive anticancer and health maintenance effects. However, the relevant molecular mechanisms are still uncertain. Genomic instability (GIN) refers to a combination of gene abnormal amplification, sequence deletion, ectopic, and other types of gene damage in cells, and it is one of the main factors causing cells to lose normal physiological functions. Therefore, we used the cytokinesis-block micronucleus cytome (CBMN-Cyt) assay as the main research method to analyze the effects of CUR and SIs on the GIN of human normal colon cells NCM460 and colon cancer cells SW620. Results show that CUR (12.5 μM) could reduce the apoptosis of NCM460 and maintain its genomic stability while inhibiting the proliferation of SW620 and promoting its apoptosis. There was no difference in the promoting effect of GIN between SW620 and NCM460 using SIs (3.125-50 μM). When the two polyphenols (v/v = 1/1, 1.5625-6.25 μM) were mixed, they could promote the proliferation and GIN of the NCM460 and SW620 cells, but we did not find that combining the two produced a better effect on the cells. In conclusion, CUR has more prominent health and anticancer effects, and it may become a dietary recommendation for daily health maintenance and a potential adjuvant drug for cancer treatment.

Protective Effects of Arbutus unedo L. Honey in the Alleviation of Irinotecan-Induced Cytogenetic Damage in Human Lymphocytes-An In Vitro Study.

Strawberry tree (Arbutus unedo L.) honey (STH) has been used since ancient times as a folk medicine remedy, especially in certain Mediterranean countries. This honey, rich in phenolic content, is well recognized for its antioxidant, anti-inflammatory, and antimicrobial activities, and is used for the treatment of skin lesions as well as gastrointestinal and respiratory disorders. This study investigated whether STH alleviates genome damage in human peripheral blood lymphocytes produced by the cytotoxic drug irinotecan. The phenolic profile of STH was previously estimated by ultra-high-performance liquid chromatography coupled to a linear ion trap-Orbitrap hybrid mass spectrometer. The effects of STH were evaluated at three concentrations (1×, 5×, and 10×), based on the daily consumption of the honey by an adult person. After 2 h of in vitro exposure, standard lymphocyte cultures for the analysis of chromosome aberrations and the cytokinesis-block micronucleus cytome assay were established. Our results demonstrate that STH offered remarkable geno- and cytoprotection when administered with irinotecan. These findings are relevant for drawing preliminary conclusions regarding the in vitro safety of the tested honey. However, further studies are needed with the application of more complex experimental models.

Association of MTHFR C677T polymorphism with risk of preterm birth in Indian mothers: a case–control study

BackgroundSince MTHFR is the key enzyme in folate metabolism, its reduction can lead to hyperhomocysteinemia, which can have a negative impact on pregnancy outcome. Moreover, MTHFR polymorphism has also been linked with oxidative stress and genotoxicity. Identifying its ethnicity-specific association can help to reduce the incidence of preterm birth (PTB). Material and methods: Age-matched preterm birth mothers (< 37 weeks) and full-term mothers (> 37 weeks) were carefully selected for the study. The polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) method was adopted to analyse MTHFR C677T polymorphism. Oxidative stress (OS) analysis was performed by measuring the levels of antioxidants (superoxide dismutase (SOD) and catalase (CAT)) and OS damage markers (lipid peroxidation (LPO), total protein). Genotoxicity was confirmed by the cytokinesis-block micronucleus cytome (CBMN-Cyt) assay. The statistical analysis was performed by Student’s t test, chi-square test, and one-way ANOVA. The relevant risk of premature delivery was represented by odds ratios (ORs) with 95% confidence intervals (95% CIs).ResultsThe MTHFR polymorphism showed statistical significance for PTB outcome with CT and TT genotype frequencies at p < 0.01 and p < 0.05, respectively, between cases and controls. Within the PTB categories (extreme-, very-, moderate-PTB), TT genotype showed statistical significance at (p < 0.05), while CT genotype remained insignificant. Also, statistically high oxidative stress and DNA damage were observed in cases compared to controls for all genotypes. Furthermore, the T allele of the MTHFR gene was found to be linked with significantly increased OS and DNA damage on comparison within the groups.ConclusionsThis study confirms the MTHFR C677T polymorphism, oxidative stress, and genotoxicity biomarkers are associated with the PTB outcome. Analysis of these biomarkers during pregnancy can be of clinical significance.

Tatlandırıcı Olan Xylitol'ün In Vitro Genotoksik Etkilerinin İncelenmesi

Xylitol (XYL) is a sweetener used as a food additive in the food industry. In the present study, four different genotoxicity assays (chromosomal aberration=CA, sister chromatid exchange=SCE, cytokinesis-block micronucleus cytome=CBMN-Cyt, and comet assays) were conducted to assess the potential genotoxicity of XYL in human lymphocytes. Four concentrations (125, 250, 500, and 1000 μg mL-1) of XYL were applied to lymphocytes obtained from three healthy young donors. The frequency of CA was not significantly affected by 24-h administration of XYL (except 1000 μg mL-1 for the number of CAs). 48 h treatment of XYL increased the frequency of CAs and abnormal cells. However, this increase was significant at only two highest concentrations. XYL significantly increased SCE/cell rate at the two highest concentrations at both treatment times (24 and 48h). Similarly, the frequency of MN, Nuclear buds (NBUDs), and Nucleoplasmic bridges (NPBs) significantly increased by XYL only at the high concentrations. It raised the comet parameters at the two highest concentrations. These observations showed that XYL, only at high concentrations, may have a genotoxic effect on human lymphocytes in vitro. For this reason, it can be concluded that its use at low concentrations may not cause DNA or chromosomal damage.

Characterization of the genotoxic profile of antineoplastic drugs using the cytokinesis-block micronucleus cytome assay

Since antineoplastic agents are frequently used in cancer therapy and able to affect the patient’s DNA, it is important to know the genotoxic consequences on non-cancerous tissue. Therefore, we aimed to characterize the genotoxic profile of antineoplastic drugs belonging to different classes, using the cytokinesis-block micronucleus cytome assay in a human monocytic cell line (THP-1). All tested antineoplastic agents resulted in increased micronucleus formation. Exposure to anthracyclines led to an increased number of vacuolated cells and cell death, while for mitotic spindle inhibitors, (different stages of) cell death and an increased nuclear bud formation was observed. Alkylating agents induce a high proportion of vacuolated cells and increased nuclear bud formation. No striking differences of nuclear division index or nucleoplasmic bridge formation were observed between exposed and non-exposed cells. The here presented class-specific aberrations may facilitate interpretation of genotoxic aberrations when evaluating clinical samples from patients treated with these antineoplastic agents.

Increased oxidative and chromosomal DNA damage in patients with ankylosing spondylitis: its role in pathogenesis.

Increased DNA damage has been suggested to contribute to the pathogenesis of chronic inflammatory diseases, but controlled studies are lacking in ankylosing spondylitis (AS). Therefore, we assessed oxidative stress, oxidative DNA damage, chromosomal DNA damage, cell proliferation and cell death in the peripheral blood lymphocytes of patients with AS as well as the possible role of DNA damage in the development of the disease. In total, 25 newly diagnosed AS patients who had not received anti-inflammatory agents and 25 healthy controls were recruited. Oxidative DNA damage was assessed by plasma 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels, and chromosomal DNA damage was assessed by the cytokinesis-block micronucleus cytome (CBMN-cyt) method. Compared to controls, the micronucleus (MN) frequencies, nucleoplasmic bridge (NPB) frequencies, nuclear bud (NBUD) frequencies, apoptotic cell frequencies, necrotic cell frequencies and plasma 8-OHdG levels were significantly higher in patients with AS (p < 0.001, p < 0.05, p < 0.01, p < 0.001, p < 0.001, and p < 0.001, respectively), and the metaphase cell numbers, binucleated (BN) cell frequencies and nuclear division index (NDI) values were significantly lower in patients with AS (p < 0.01, p < 0.001 and p < 0.001, respectively). Thus, the present findings suggested that oxidative stress, oxidative DNA damage, and chromosomal DNA damage may be involved in the pathogenesis of AS similar to other chronic inflammatory diseases. In addition, the increased plasma 8-OHdG levels, MN frequencies, NPB frequencies and NBUD frequencies in AS patients may reflect an increased cancer risk.

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.

PARADOXICAL DUAL ROLES OF SOME CYTOKINES INTERPRETED BY CYTOGENETICS INVESTIGATIONS IN IRRADIATED HUMAN BLOOD CULTURES.

Four different cytokines (IL1-β, IL-6, IL-10 and TNF-α) and the cytokinesis-block micronucleus (CBMN) cytome assay investigations were evaluated in six human blood samples. They were divided into the control (nonirradiated) and five gamma-irradiated groups which were exposed to five different doses (0.5, 1, 2, 4 and 8 Gy). Blood groups were cultured in triplets for 72h following 1h of irradiation. Immunological and cytogenetics were investigated parallelly at different irradiation doses to understand the connection between them. Our aim is anchoring the active proliferation action of cytokines by presence of binucleated cells and resting immune system by mononuclear cell. Also, cell death by increasing necrotic cell count and TNF-α concentration. When compared with the control group, 0.5, 1, 2 and 4Gy irradiation groups recorded a gradual increase in the cytokines levels, an increase in the total micronucleated cells (binucleated and mononucleated cells), an increase in necrotic and apoptotic cells counts. While 8Gy irradiation leads to depletion in TNF-α concentration, although the number of necrotic cells was high.