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.

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.

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.

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.