Dual Immunofluorescence of γH2AX and 53BP1 in Human Peripheral Lymphocytes.

Double strand breaks (DSBs) are one of the most severe lesions that can occur in cell nuclei, and, if not repaired, they can lead to severe outcomes, including cancer. The cell is, therefore, provided with complex mechanisms to repair DSBs, and these pathways involve histone H2AX in its phosphorylated form at Ser-139 (namely γH2AX) and p53 binding protein 1 (53BP1). As both proteins can form foci at the sites of DSBs, identification of these markers is considered a suitable method to study both DSBs and their kinetics of repair. According to the molecular processes that lead to the formation of γH2AX and 53BP1 foci, it could be more useful to investigate their co-localization near the DSBs in order to set up an alternative approach that allows quantifying DSBs by the simultaneous detection of two DNA damage markers. Thus, this protocol aims to assess the genomic damage induced in human lymphocytes by the radiomimetic agent bleomycin through the presence of γH2AX and 53BP1 foci in a dual immunofluorescence. Using this methodology, we also delineated the variation in the number of γH2AX and 53BP1 foci over time, as a preliminary attempt to study the repair kinetics of bleomycin-induced DSBs.

Oxidative and DNA damage in obese patients undergoing bariatric surgery: A one-year follow-up study.

The pathogenesis of obesity and related comorbidities has long been associated with oxidative stress. The excess of adipose tissue contributes to the production of free radicals that sustain both a local and a systemic chronic inflammatory state, whereas its reduction can bring to an improvement in inflammation and oxidative stress. In our work, using the fluorescent lipid probe BODIPY® 581/591 C11 and the γH2AX foci assay, a well-known marker of DNA double strand breaks (DSB), we evaluated the extent of cell membrane oxidation and DNA damage in peripheral blood lymphocytes of normal weight (NW) controls and obese patients sampled before and after bariatric surgery. Compared to NW controls, we observed a marked increase in both the frequencies of oxidized cells or nuclei exhibiting phosphorylation of histone H2AX in preoperatory obese patients. After bariatric surgery, obese patients, resampled over one-year follow-up, improved oxidative damage and reduced the presence of DSB. In conclusion, the present study highlights the importance for obese patients undergoing bariatric surgery to also monitor these molecular markers during their postoperative follow-up.

High level of γH2AX phosphorylation in the cord-blood cells of large-for-gestational-age (LGA) newborns.

Newborns can experience adverse effects as a consequence of maternal or in utero exposure, altered growth of the fetus, or placental dysfunctions. Accurate characterization of gestational age allows monitoring of fetal growth, identification of deviations from the normal growth trajectory, and classification of babies as adapted, small, or large for gestational age (AGA, SGA, or LGA). The aim of this work was to evaluate nuclear and oxidative damage in umbilical cord-blood cells of newborns (sampled at birth), by applying the γH2AX assay and the fluorescent probe BODIPY581/591 C11, to detect DNA DSB and cell membrane oxidation, respectively. No statistically significant differences were observed in the proportion of oxidized cord-blood cells among the groups of newborns, although the LGA group showed the highest value. With regard to genome damage, elevated levels of γH2AX foci were detected in the cell nuclei from LGA newborns as compared to AGA or SGA babies, whose values did not differ from each other. Considering that the observed DNA damage, although still repairable, can represent a risk factor for obesity, metabolic diseases, or other pathologies, monitoring genome and cell integrity at birth can provide useful information for prevention of diseases later in life.

UCTD and SLE patients show increased levels of oxidative and DNA damage together with an altered kinetics of DSB repair.

Immunological tolerance is a critical feature of the immune system; its loss might lead to an abnormal response of lymphocytes causing autoimmune diseases. One of the most important groups belonging to autoimmune disorders is the connective tissue diseases (CTD). CTD are classified among systemic rheumatic diseases and include pathologies such as systemic lupus erythematosus (SLE), and undifferentiated CTD (UCTD). In this study, we evaluated oxidative and genome damage in peripheral blood lymphocytes from patients with SLE and UCTD, further classified on the basis of disease activity and the presence/absence of a serological profile. Oxidative damage was evaluated in cell membrane using the fluorescent fatty acid analogue BODIPY581/591 C11. The percentage of oxidised lymphocytes in both SLE and UCTD patients was higher than in the control group, and the oxidative stress correlated positively with both disease activity and autoantibody profile. The γH2AX focus assay was used to quantify the presence of spontaneous double strand breaks (DSBs), and to assess the abilities of DSBs repair system after T cells were treated with mitomycin C (MMC). Subjects with these autoimmune disorders showed a higher number of γH2AX foci than healthy controls, but no correlation with diseases activity and presence of serological profile was observed. In addition, patients displayed an altered response to MMC-induced DSBs, which led their peripheral cells to greatly increase apoptosis. Taken together our results confirmed an interplay among oxidative stress, DNA damage and impaired DNA repair, which are directly correlated to the aggressiveness and clinical progression of the diseases. We propose the evaluation of these molecular markers to better characterise SLE and UCTD, aiming to improve the treatment plan and the quality of the patients' life.

Role of oxidative stress, genome damage and DNA methylation as determinants of pathological conditions in the newborn: an overview from conception to early neonatal stage.

Increasing evidence suggests that early-life events can predispose the newborn to a variety of health issues in later life. In adverse pre- and perinatal conditions, oxidative stress appears to play an important role in the development of future pathological outcomes. From a molecular point of view, oxidative stress can result in genome damage and changes in DNA methylation that can in turn prime pathogenic mechanisms. Interestingly, both alterations have been related to a reciprocal regulation of oxidative stress. The aim of this review is to give a brief overview of the complex relationship linking oxidative stress to DNA damage and methylation and to go through the different sources of exposure that a neonate can encounter in utero or shortly after birth. In this context, the setup of methodologies to monitor the extent of oxidative stress, genomic damage and instability or the presence of altered methylation patterns contributes to the understanding on how the complex events occurring in early life can lead to either a healthy status or a pathological condition.

Assessing the cytotoxic/genotoxic activity and estrogenic/antiestrogenic potential of essential oils from seven aromatic plants.

Alternative therapies with new drugs are needed because the clinical efficacy of conventional chemotherapy is often reduced due to collateral effects. Many natural products of plant origin, including essential oils (EOs) have proved to be effective in prevention and therapy of several diseases such as bacterial infections, chronic diseases and cancer. In the present study, we investigated some biological activities of EOs extracted from seven plants: Rosmarinus officinalis, Salvia somalensis, Thymus vulgaris, Achillea millefolium, Helichrysum italicum, Pistacia lentiscus, Myrtus communis. In particular, we evaluated the cytotoxic and genotoxic activity using the cytochalasin B-blocked micronucleus assay (CBMN) in human peripheral lymphocytes, cytotoxicity in a human ovarian carcinoma cell line (A2780), and the estrogenic/antiestrogenic activity using a yeast strain expressing the human estrogen receptor alpha (ERα). Our results show that most EOs can have a strong cytotoxic and a slight/moderate genotoxic effect on human peripheral lymphocytes, and also a pronounced cytotoxic effect in A2780 cells. In addition, some EOs seem to have a marked antiestrogenic activity that could potentially perturb the estrogen-dependent tissues.

Increased level of DNA damage in some organs of obese Zucker rats by γ-H2AX analysis.

In a recent study, we showed that lymphocytes of obese Italian children/adolescents displayed levels of double strand breaks (DSB), assayed as serine 139-phosphorylated histone H2AX (γ-H2AX), about eightfold higher than normal weight controls, and that 30% of this damage-generated micronuclei. These findings suggested that obese children could be at increased risk of obesity-mediated cancer later in life. We therefore aimed to assess the level of γ-H2AX in a genetic animal model of obesity (Zucker rat) to identify a genotoxic/carcinogenic risk in some organs. The DSB marker was studied in 3- to 4-week-old rats and in 9- to 13-week-old rats. Paraffin-embedded sections of heart, thyroid, liver, pancreas, lung, kidney, esophagus, and gut from the fa-/fa- (obese) and the fa+/fa- (lean) control animals were processed for immunohistochemistry detection of γ-H2AX. Pancreas (0.0624 ± 0.0195), lung (0.1197 ± 0.0217), esophagus (0.1230 ± 0.0351), kidney (0.1546 ± 0.0149), and gut (0.1724 ± 0.0352) of 9- to 13-week-old obese rats showed a higher proportion of γ-H2AX-positive nuclei, than their lean counterparts (0.0092 ± 0.0033, 0.0416 ± 0.0185, 0.0368 ± 0.0088, 0.0686 ± 0.0318, and 0.0703 ± 0.0239, respectively). No difference was seen in the 3- to 4-week-old age group with regard to obesity, indicating that the DNA damage increased with older age of the rats. We hypothesize that the organs of the obese animals showing high levels of DSB could represent target tissues for the development of obesity-related cancers. Environ. Mol. Mutagen. 58:477-484, 2017. © 2017 Wiley Periodicals, Inc.

Use of Amplification Fragment Length Polymorphism to Genotype Pseudomonas stutzeri Strains Following Exposure to Ultraviolet Light A.

Changes in ultraviolet light radiation can act as a selective force on the genetic and physiological traits of a microbial community. Two strains of the common soil bacterium Pseudomonas stutzeri, isolated from aquifer cores and from human spinal fluid were exposed to ultraviolet light. Amplification length polymorphism analysis (AFLP) was used to genotype this bacterial species and evaluate the effect of UVA-exposure on genomic DNA extracted from 18 survival colonies of the two strains compared to unexposed controls. AFLP showed a high discriminatory power, confirming the existence of different genotypes within the species and presence of DNA polymorphisms in UVA-exposed colonies.

Different repair kinetic of DSBs induced by mitomycin C in peripheral lymphocytes of obese and normal weight adolescents.

In 2013, 42 million children under the age of 5 years were overweight or obese. In the context of obesity, we recently showed that (1) peripheral lymphocytes of obese children/adolescents had an 8-fold increase in double strand breaks (DSBs), expressed as g-H2AX foci, than normal weight adolescents, and (2) 30% of the damage was retained into chromosome mutations. Thus, we investigated DSBs repair efficiency in a group of obese adolescents assessing the kinetic of H2AX phosphorylation in mitomycin C (MMC)-treated lymphocytes harvested 2 h- or 4 h-post mutagen treatment. According to our previous studies, these harvesting times represent the peak of DSBs induction and the time in which an appreciable DSBs reduction was observed. In addition, we evaluated the expression of the high mobility group box-1 protein (HMGB1), a chromatin remodelling protein involved in DSBs repair and obesity. Compared to normal weight adolescents, obese subjects 1) showed higher levels of g-H2AX foci at either 2 h- (0.239±0.041 vs. 0.473±0.048, P=0.0016) or 4 h- (0.150±0.026 vs. 0.255±0.030, P=0.0198) post mutagen treatment, and 2) have repaired a greater amount of the initial lesions (0.088±0.033 vs. 0.218±0.045, P=0.0408). Concordantly, 1) HMGB1 levels of obese individuals increased and decreased at 2h- or 4 h-post mutagen treatment, respectively, and 2) the opposite occurred for the normal weight adolescents where the protein was down-expressed at 2h and over-expressed at 4h. In conclusion, lymphocytes of obese and normal weight adolescents showed a distinct temporal kinetic of repairing MMC-induced DSBs, together with a different expression of HMGB1. The finding that obesity may modulate the repair of DNA damage induced in lymphocytes by genotoxic agents should be confirmed by further experiments.

Vincristine-induced bystander effect in human lymphocytes.

Bystander effect is a known radiobiological effect, widely described using ionizing radiations and which, more recently, has also been related to chemical mutagens. In this study, we aimed to assess whether or not a bystander response can be induced in cultured human peripheral lymphocytes by vincristine, a chemotherapeutic mutagen acting as spindle poison, and by mitomycin-C, an alkylating agent already known to induce this response in human lymphoblastoid cells. Designing a modified ad hoc protocol for the cytokinesis blocked micronucleus (MN) assay, we detected the presence of a dose-dependent bystander response in untreated cultures receiving the conditioned medium (CM) from mitomycin-C (MMC) or vincristine (VCR) treated cultures. In the case of MMC, MN frequencies, expressed as micronucleated binucleates, were: 13.5±1.41 at 6µM, 22±2.12 at 12µM or 28.25±5.13 at 15µM vs. a control value of 4.75±1.59. MN levels for VCR, expressed as micronucleated mononucleates were: 2.75±0.88 at 0.0µM, 27.25±2.30 at 0.4µM, 46.25±1.94 at 0.8µM, 98.25±7.25 at 1.6µM. To verify that no mutagen residual was transferred to recipient cultures together with the CM, we evaluated MN levels in cultures receiving the medium immediately after three washings following the chemical treatment (unconditioned medium). We further confirmed these results using a cell-mixing approach where untreated lymphocytes were co-cultured with donor cells treated with an effect-inducing dose of MMC or VCR. A distinct production pattern of both reactive oxygen species and soluble mediator proteins by treated cells may account for the differences observed in the manifestation of the bystander effect induced by VCR. In fact, we observed an increased level of ROS, IL-32 and TGF-ß in the CM from VCR treated cultures, not present in MMC treated cultures.

Genomic instability and cellular stress in organ biopsies and peripheral blood lymphocytes from patients with colorectal cancer and predisposing pathologies.

Inflammatory bowel disease (IBD) and polyps, are common colorectal pathologies in western society and are risk factors for development of colorectal cancer (CRC). Genomic instability is a cancer hallmark and is connected to changes in chromosomal structure, often caused by double strand break formation (DSB), and aneuploidy. Cellular stress, may contribute to genomic instability. In colorectal biopsies and peripheral blood lymphocytes of patients with IBD, polyps and CRC, we evaluated 1) genomic instability using the γH2AX assay as marker of DSB and micronuclei in mononuclear lymphocytes kept under cytodieresis inhibition, and 2) cellular stress through expression and cellular localization of glutathione-S-transferase omega 1 (GSTO1). Colon biopsies showed γH2AX increase starting from polyps, while lymphocytes already from IBD. Micronuclei frequency began to rise in lymphocytes of subjects with polyps, suggesting a systemic genomic instability condition. Colorectal tissues lost GSTO1 expression but increased nuclear localization with pathology progression. Lymphocytes did not change GSTO1 expression and localization until CRC formation, where enzyme expression was increased. We propose that the growing genomic instability found in our patients is connected with the alteration of cellular environment. Evaluation of genomic damage and cellular stress in colorectal pathologies may facilitate prevention and management of CRC.

Transcriptional alterations of ET-1 axis and DNA damage in lung tissue of a rat obesity model.

Obesity has been implicated in the development of many cancers. This can lead to genome damage, especially in the form of double-strand break, the presence of which is now easily detected through nuclear phosphorylation of histone H2AX (γ-H2AX) focus assay. Recently, the endothelin (ET) axis has also been shown to have a role in the growth and progression of several tumors, including lung cancer. The aim of this study was to evaluate the ET-1 system transcriptional alterations and γ-H2AX in lung tissue of Zucker rats subdivided into obese (O, n=22) and controls (CO, n=18) rats: under either fasting conditions (CO(fc)-O(fc)) or acute hyperglycemia (CO(AH)-O(AH)). Significantly higher prepro-ET-1 (p=0.05) and ET-converting enzyme (ECE)-2 mRNA expression was observed in O with respect to CO. A significant positive association was observed between prepro-ET-1 and ET-A in the whole rat population (p=0.009) or in the obese group alone (p=0.007). The levels of γ-H2AX in O and in O(AH) rats were significantly higher (p=0.019) than in the corresponding CO and CO(AH) rats (p=0.038). The study shows an inappropriate secretion of ET-1 in O animals with a parallel DNA damage in their lungs, providing novel mechanisms by which ET receptor antagonist may exert organ protection.

In vitro cultures of Bituminaria bituminosa: pterocarpan, furanocoumarin and isoflavone production and cytotoxic activity evaluation.

Bituminaria bituminosa L. is known for producing several compounds with considerable pharmaceutical interest, such as phenylpropanoids, furanocoumarins and pterocarpans. In vitro cultures of seedlings, shoots, and callus have been produced to obtain plant materials useful for the production of these metabolites. The secondary metabolite profile was evaluated by HPLC-DAD. The extracts of all the in vitro material contained the flavonoid daidzein, while plicatin B, erybraedin C and bitucarpin A were found only in the extracts of the in vitro shoots and in wild shoots. The furanocoumarins angelicin and psoralen were found in in vivo and in vitro plants, but in the callus were not detectable. The extracts were also tested for cytotoxic activity in HeLa cell culture; the highest level of cytotoxicity was found in in vitro shoot extracts.

High levels of γ-H2AX foci and cell membrane oxidation in adolescents with type 1 diabetes.

Oxidative stress caused by an excess of free radicals is implicated in the pathogenesis and development of type 1 diabetes mellitus (T1DM) and, in turn, it can lead to genome damage, especially in the form of DNA double-strand break (DSB). The DNA DSB is a potentially carcinogenic lesion for human cells. Thus, we aimed to evaluate whether the level of oxidative stress was increased in peripheral blood lymphocytes of a group of affected adolescents. In 35 T1DM adolescents and 19 healthy controls we assessed: (1) spontaneous and H2O2-induced oxidation of cell membrane using a fluorescence lipid probe; (2) spontaneous and LPS-induced expression of iNOS protein and indirect NO determination via cytofluorimetric analysis of O2(-); (3) immunofluorescent detection of the basal level of histone H2AX phosphorylation (γ-H2AX foci), a well-validated marker of DNA DSB. In T1DM, the frequencies of oxidized cells, both spontaneous and H2O2-induced (47.13±0.02) were significantly higher than in controls (35.90±0.03). Patients showed, in general, both a reduced iNOS expression and production of NO. Furthermore, the level of spontaneous nuclear damage, quantified as γ-H2AX foci, was markedly increased in T1DM adolescents (6.15±1.08% of γ-H2AX(+) cells; 8.72±2.14 γ-H2AXF/n; 9.26±2.37 γ-H2AXF/np), especially in females. In the present study, we confirmed the role that oxidative stress plays in the disease damaging lipids of cell membrane and, most importantly, causing genomic damage in circulating white blood cells of affected adolescents. This also indicates that oxidative stress can affect several tissues in the body. However, although the observed DNA damage is a clear indication that the proper DNA repair mechanisms are activated, the risk for young T1DM subjects of developing not only cardiovascular complications but also some type of cancer cannot be ruled out. In this view, females, probably due to hormonal imbalance typical of adolescence, might represent a more susceptible population.

Kinetics of nuclear phosphorylation (γ-H2AX) in human lymphocytes treated in vitro with UVB, bleomycin and mitomycin C.

After double-strand break induction, formation of γ-H2AX foci due to phosphorylation at Ser-139 of histone H2AX represents an early event of the DNA damage response (DDR). γ-H2AX foci are then rapidly dephosphorylated as signal for the subsequent recruitment of effector proteins. The induction and disappearance of the foci can be, therefore, used to monitor the functioning of the DDR machinery in a cell population exposed to genotoxic stress. Here, we investigated the time-course of γ-H2AX in unstimulated or cultured peripheral lymphocytes in vitro treated with UVB, bleomycin and mitomycin C (MMC). Once the mutagen exposure was performed, cells were harvested at different interval times from 0.5 to 5h. The results show that (i) in 20-h stimulated peripheral lymphocytes, UVB irradiation caused extensive and dose-dependent increases in nuclear phosphorylation, and disappearance of γ-H2AX foci progressed, proportionally to the UV fluence, with increasing the harvesting time; (ii) UVB-exposed G0 cells cultured for 20-h post-irradiation displayed low amounts of DNA phosphorylation, depicting a time-course in which the maximum effect was reached at 0.5h and dephosphorylation started after 1h; (iii) treatment of unstimulated lymphocytes with bleomycin sulphate induced an increase in nuclear phosphorylation of several folds higher than that of untreated cells, depicting kinetics comparable to those observed for UVB-exposed G1 cells; (iv) in stimulated cells, MMC caused a severe and dose-dependent high degree of H2AX phosphorylation together with a very slower kinetic of dephosphorylation with respect to the other experimental treatments. This study confirms the feasibility of the γ-H2AX focus assay as a genotoxic end-point and supports the view that the proposed type of analysis should be introduced in biomonitoring studies of human populations. This could also represent a feasible and useful tool in the screening and diagnosis of precancerous states or very early stages of other diseases.

Effects of spindle poisons in peripheral human lymphocytes by the in vitro cytokinesis-block micronucleus assay.

The search for micronuclei (MN) in binucleated cells is not always the best choice to recognize microtubule-perturbing agents, as they give rise to (micronucleated) mononucleated cells, mainly via mitotic slippage. We therefore treated peripheral lymphocytes with vincristine (VCR), nocodazole (NOC) and colcemid (COL): (i) to quantify the formation of MN in mononucleated cells and the occurrence of abnormal mitoses (c-anaphases, endoreduplicated or tetraploid metaphases); (ii) to investigate the role of cytokinesis inhibition in determining or modulating the cytogenetic effects induced by the spindle poisons (we used either cytochalasin B (cyt B) or latrunculin A, a cytokinesis inhibitor that acts differently as compared with cyt B); (iii) to assess the ploidy of cells bearing MN by fluorescence in situ hybridisation (FISH) analysis; and (iv) to evaluate the levels of the mitotic arrest deficient (MAD2) protein, that blocks the cell at the metaphase-anaphase transition, by immunoblotting. We observed the induction of numerous abnormal mitoses and tetraploid interphase nuclei, as well as of MN in mononucleated cells, a high percentage of which had a diploid complement. We also found that the effects were generally not dose but chemical dependent, where NOC was proven to be more effective than COL and VCR in inducing overall MN formation and, specifically, diploid micronucleated lymphocytes. Aneugens damaged cells to a greater extent in the presence of cytokinesis inhibitors rather than in their absence. MAD2 protein was expressed in controls to an extent reflecting the amount of lymphocytes which were initially in the G2/M transition phase. The same trend was seen in aneugen-treated cells where MAD2 levels decreased with increasing spindle poison concentration. Here, we demonstrate that micronucleated mononucleated cells and aberrant mitoses can be considered useful markers of exposure to aneugens-like spindle poisons causing preferentially, but not exclusively, mitotic slippage. Assessment of MAD2 levels can be used to confirm the cell-damaging activity of the compounds.

Non-thermal effects of 2.45 GHz microwaves on spindle assembly, mitotic cells and viability of Chinese hamster V-79 cells.

The production of mitotic spindle disturbances and activation of the apoptosis pathway in V79 Chinese hamster cells by continuous 2.45 GHz microwaves exposure were studied, in order to investigate possible non-thermal cell damage. We demonstrated that microwave (MW) exposure at the water resonance frequency was able to induce alteration of the mitotic apparatus and apoptosis as a function of the applied power densities (5 and 10mW/cm(2)), together with a moderate reduction in the rate of cell division. After an exposure time of 15 min the proportion of aberrant spindles and of apoptotic cells was significantly increased, while the mitotic index decreased as well, as compared to the untreated V79 cells. Additionally, in order to understand if the observed effects were due to RF exposure per se or to a thermal effect, V79 cells were also treated in thermostatic bath mimicking the same temperature increase recorded during microwave emission. The effect of temperature on the correct assembly of mitotic spindles was negligible up to 41°C, while apoptosis was induced only when the medium temperature achieved 40°C, thus exceeding the maximum value registered during MW exposure. We hypothesise that short-time MW exposures at the water resonance frequency cause, in V79 cells, reversible alterations of the mitotic spindle, this representing, in turn, a pro-apoptotic signal for the cell line.

Cytotoxicity and genotoxicity studies of two free-radical generators (AAPH and SIN-1) in human microvascular endothelial cells (HMEC-1) and human peripheral lymphocytes.

Vascular endothelial cells, smooth muscle cells, macrophages and other cell types in the arterial wall may develop oxidative/nitrosative damage by generation of reactive oxygen/nitrogen species, which could alter endothelial cell function. These changes could play a key role in acute inflammatory processes, atherosclerosis and neurodegenerative pathogenesis. A human microvascular endothelial cell line (HMEC-1) and human peripheral lymphocytes were employed to investigate the cytotoxic and genotoxic effects induced by reactive peroxyl radicals and peroxynitrite generated from 2,2'-azo-bis-(2-amidinopropane)-dihydrochloride (AAPH) and 3-morpholinosydnonimine (SIN-1), respectively. The peroxides generated by AAPH were cytotoxic but not genotoxic in HMEC-1 cells and in peripheral lymphocytes (in separate culture and in whole blood). SIN-1 showed progressive cytotoxicity to HMEC-1 at doses of 10-75µM. In the same range of concentrations a significant increase in apoptotic cells and micronuclei was observed. DNA flow-cytometric analysis indicated that 100 and 200µM SIN-1 significantly increased the proportion of cells in G(2) phase compared with the control. SIN-1 decomposition products, NO and superoxide anion or peroxynitrite, induced greater cytotoxicity in lymphocyte cultures (separately and in whole blood) supplemented with HEPES - the organic buffer that is widely used to maintain stable physiological pH in cell cultures -, due to H(2)O(2) production, than in cultures without HEPES. In contrast, increased genotoxicity was observed in both lymphocyte cultures in the absence of HEPES due to the reduced cytotoxicity. In the cell systems employed in this study the genotoxic effect appears closely dependent on the nature of radical species generated by SIN-1.

Nuclear damage in peripheral lymphocytes of obese and overweight Italian children as evaluated by the gamma-H2AX focus assay and micronucleus test.

Childhood obesity, often characterized by a chronic low-grade inflammation, has been associated with an increased risk of developing some types of cancer later in life. Nuclear γ-H2AX foci represent the first detectable response of cells to DNA tumorigenesis lesions, such as the double-strand breaks (DSBs). An excess of micronucleated peripheral lymphocytes was found in subjects with cancer or inflammation-based diseases. We set out to investigate the expression of genome damage, from DNA lesions to chromosome mutations (micronuclei), in overweight and obese children. Using the γ-H2AX focus assay and micronucleus (MN) test, we analyzed peripheral lymphocytes from 119 Italian children classified as normal weight (n=38), overweight (n=20), or obese (n=61). Cultures treated with bleomycin (BLM) were also set up for each child in both assays to check functioning of the apparatus that ensures DNA integrity. We measured serum TNF-α, IL-6, and C-reactive protein (CRP) as markers of inflammation. Overweight and obese children had significantly higher levels of H2AX phosphorylation (0.0191±0.0039 and 0.0274±0.0029 γ-H2AXF/n) and increased MN frequencies (2.30±0.25 and 2.45±0.22‰) than normal-weight children (0.0034±0.0006 γ-H2AXF/n, and 0.92±0.12‰ MN), while all subjects responded to BLM induction, irrespective of their weight status. The fold increase of spontaneous MN frequencies in overweight and obese subjects was 2.5 and 2.7, respectively, well below the corresponding increase in the γ-H2AX foci (5.6- and 8.0-fold, respectively). IL-6 and CRP mean values were significantly higher in obese and overweight children than in controls. Here, we demonstrated that peripheral cells of overweight and obese children showed increased levels of DSBs, which were not completely repaired as part of them has been converted into micronuclei. Characterization of childhood obesity inflammation could be implemented using molecular markers of genome damage.

Antiestrogenic and antigenotoxic activity of bee pollen from Cystus incanus and Salix alba as evaluated by the yeast estrogen screen and the micronucleus assay in human lymphocytes.

The estrogenic/antiestrogenic activity and the genotoxicity/antigenotoxicity of bee pollen from Salix alba L. and Cystus incanus L. and its derivative extracts in yeast and human cells was investigated. All samples showed a marked inhibitory effect on the activity of the natural estrogen 17 beta-estradiol (higher than 90% for extracts 2) and failed to cause estrogenic activity and chromosome damage. At least one preparation from each species showed a marked antigenotoxic effect against the action of the anticancer drugs mytomicin C, bleomycin, and vincristine. Bee pollens from C. incanus and S. alba were found to be neither genotoxic nor estrogenic as well as effective estrogen inhibitors, and able to reduce the chromosome damage induced by the three cancer drugs used, thus supporting their use as a safe food supplement and future chemoprotective/chemopreventive agents.