Ester Siniscalchi

Diet-related telomere shortening and chromosome stability.

Recent evidences have highlighted an influence of micronutrients in the maintenance of telomere length (TL). In order to explore whether diet-related telomere shortening had any physiological relevance and was accompanied by significant damage in the genome, in the present study, TL was assessed by terminal restriction fragment (TRF) analysis in peripheral blood lymphocytes of 56 healthy subjects for which detailed information on dietary habits was available and data were compared \with the incidence of nucleoplasmic bridges (NPBs), a marker of chromosomal instability related to telomere dysfunction visualised with the cytokinesis-blocked micronucleus assay. To increase the capability to detect even slight impairment of telomere function, the incidence of NPBs was also evaluated on cells exposed in vitro to ionising radiation. Care was taken to control for potential confounding factors that might influence TL, viz. age, hTERT genotype and smoking status. Data showed that higher consumption of vegetables was related with significantly higher mean TL (P = 0.013); in particular, the analysis of the association between micronutrients and mean TL highlighted a significant role of antioxidant intake, especially beta-carotene, on telomere maintenance (P = 0.004). However, the diet-related telomere shortening did not result in associated increased spontaneous or radiation-induced NPBs. The distribution of TRFs was also analysed and a slight prevalence of radiation-induced NPBs (P = 0.03) was observed in subjects with higher amount of very short TRFs (<2 kb). The relative incidence of very short TRFs was positively associate with ageing (P = 0.008) but unrelated to vegetables consumption and daily intake of micronutrients, suggesting that the degree of telomere erosion related with low dietary intake of antioxidants observed in this study was not so extensive to lead to chromosome instability.

Toxic and genotoxic effects of oral administration of furan in mouse liver

In this study, the effects induced in mouse liver by repeated oral exposure to furan were investigated. To this aim, the compound was given for 28 days by daily gavage to male B6C3F1 mice at 2, 4, 8 and 15 mg/kg body weight (b.w.)/day. Twenty-four hours after last administration, animals were sacrificed, liver was excised and the following parameters were evaluated: histological alterations, apoptosis, cell proliferation, polyploidy, overall DNA methylation, gene expression and DNA damage by the immunofluorescence detection of foci of phosphorylated histone H2AX (gamma-H2AX) and by alkaline comet assays, using both standard and modified protocols for the detection of DNA cross links. Liver DNA damage by comet assays was also evaluated in mice receiving furan as a single acute oral dose (15, 100 or 250 mg/kg b.w.). Microscopic analysis of liver sections indicated that repeated oral administration of furan was moderately toxic, producing mild histological alterations with necrotic figures, apoptosis and limited regenerative cell proliferation. The flow cytometric analysis of DNA content in single-cell suspensions of liver cells showed a statistically significant increase in polyploid (8N) cells at the highest dose. No treatment-related changes in overall DNA methylation, gamma-H2AX foci, DNA strand breaks and cross links were observed at the end of the 4-week exposure period. However, several genes involved in DNA damage response, beyond stress and liver toxicity, were over-expressed in mice treated with the highest furan dose (15 mg/kg b.w./day). Acute administration of furan induced evident liver toxicity at the highest dose (250 mg/kg b.w.), which was associated with a significant increase of DNA damage in the alkaline comet assay and with a distinct decrease in gamma-ray-induced DNA migration. Overall, the results obtained suggest that the contribution of genotoxicity to the mechanism of furan carcinogenicity in mouse liver should not be dismissed.

Novel Epigenetic Changes Unveiled by Monozygotic Twins Discordant for Smoking Habits

Exposure to cigarette smoking affects the epigenome and could increase the risk of developing diseases such as cancer and cardiovascular disorders. Changes in DNA methylation associated with smoking may help to identify molecular pathways that contribute to disease etiology. Previous studies are not completely concordant in the identification of differentially methylated regions in the DNA of smokers. We performed an epigenome-wide DNA methylation study in a group of monozygotic (MZ) twins discordant for smoking habits to determine the effect of smoking on DNA methylation. As MZ twins are considered genetically identical, this model allowed us to identify smoking-related DNA methylation changes independent from genetic components. We investigated the whole blood genome-wide DNA methylation profiles in 20 MZ twin pairs discordant for smoking habits by using the Illumina HumanMethylation450 BeadChip. We identified 22 CpG sites that were differentially methylated between smoker and non-smoker MZ twins by intra-pair analysis. We confirmed eight loci already described by other groups, located in AHRR, F2RL3, MYOG1 genes, at 2q37.1 and 6p21.33 regions, and also identified several new loci. Moreover, pathway analysis showed an enrichment of genes involved in GTPase regulatory activity. Our study confirmed the evidence of smoking-related DNA methylation changes, emphasizing that well-designed MZ twin models can aid the discovery of novel DNA methylation signals, even in a limited sample population.

Gene promoter methylation and DNA repair capacity in monozygotic twins with discordant smoking habits

The influence of DNA repair capacity, plasma nutrients and tobacco smoke exposure on DNA methylation was investigated in blood cells of twenty-one couples of monozygotic twins with discordant smoking habits. All study subjects had previously been characterized for mutagen sensitivity with challenge assays with ionizing radiation in peripheral blood lymphocytes. Plasma levels of folic acid, vitamin B12 and homocysteine were also available from a previous investigation. In this work DNA methylation in the promoter region of a panel of ten genes involved in cell cycle control, differentiation, apoptosis and DNA repair (p16, FHIT, RAR, CDH1, DAPK1, hTERT, RASSF1A, MGMT, BRCA1 and PALB2) was assessed in the same batches of cells isolated for previous studies, using the methylation-sensitive high-resolution melting technique. Fairly similar profiles of gene promoter methylation were observed within co-twins compared to unrelated subjects (p= 1.23 × 10(-7)), with no significant difference related to smoking habits (p = 0.23). In a regression analysis the methylation index of study subjects, used as synthetic descriptor of overall promoter methylation, displayed a significant inverse correlation with radiation-induced micronuclei (p = 0.021) and plasma folic acid level (p = 0.007) both in smokers and in non-smokers. The observed association between repair of radiation-induced DNA damage and promoter methylation suggests the involvement of the DNA repair machinery in DNA modification. Data also highlight the possible modulating effect of folate deficiency on DNA methylation and the strong influence of familiarity on the individual epigenetic profile.

Evaluation of radiation-induced chromosome instability in subjects with a family history of gastric cancer

Gastric cancer (GC) shows a familiar predisposition which is largely unexplained. In this study the hypothesis that radiation sensitivity is implicated in the familiar predisposition to GC was investigated by means of the cytokinesis-block micronucleus assay. Data indicate that a family history of GC is not associated with any of the biomarkers investigated and does not interact with the demographic variables considered. When study subjects were dichotomized around the median age, a significant prevalence of micronuclei was observed in older subjects. Age and both spontaneous and radiation-induced micronuclei were linearly correlated. The effect of age was not modified by gender or smoking habits.

DNA damage response in monozygotic twins discordant for smoking habits

Previous studies in twins indicate that non-shared environment, beyond genetic factors, contributes substantially to individual variation in mutagen sensitivity; however, the role of specific causative factors (e.g. tobacco smoke, diet) was not elucidated. In this investigation, a population of 22 couples of monozygotic twins with discordant smoking habits was selected with the aim of evaluating the influence of tobacco smoke on individual response to DNA damage. The study design virtually eliminated the contribution of genetic heterogeneity to the intra-pair variation in DNA damage response, and thus any difference in the end-points investigated could directly be attributed to the non-shared environment experienced by co-twins, which included as main factor cigarette smoke exposure. Peripheral lymphocytes of study subjects were challenged ex vivo with γ-rays, and the induction, processing, fixation of DNA damage evaluated through multiple approaches. Folate status of study subjects was considered significant covariate since it is affected by smoking habits and can influence radiosensitivity. Similar responses were elicited by γ-rays in co-twins for all the end-points analysed, despite their discordant smoking habits. Folate status did not modify DNA damage response, even though a combined effect of smoking habits, low-plasma folic acid level, and ionising radiation was observed on apoptosis. A possible modulation of DNA damage response by duration and intensity of tobacco smoke exposure was suggested by Comet assay and micronucleus data, but the effect was quantitatively limited. Overall, the results obtained indicate that differences in smoking habits do not contribute to a large extent to inter-individual variability in the response to radiation-induced DNA damage observed in healthy human populations.

Telomerase activity, telomere length and hTERT DNA methylation in peripheral blood mononuclear cells from monozygotic twins with discordant smoking habits

Increased telomerase expression has been implicated in the pathogenesis of lung cancer and, since the primary cause of lung cancer is smoking, an association between telomerase reactivation and tobacco smoke has been proposed. In this work an investigation has been performed to assess the relationship between tobacco smoke exposure and telomerase activity (TA) in peripheral blood mononuclear cells of healthy smokers. The methylation status of the catalytic subunit of telomerase hTERT was concurrently investigated to assess the possible association between epigenetic modifications of hTERT and TA. Besides, the association between smoke and telomere length (TL) has been evaluated. Healthy monozygotic twins with discordant smoking habits were selected as study population to minimize inter‐individual differences because of demographic characteristics and genetic heterogeneity. Statistically significant higher values of TA and TL were observed in smokers compared to nonsmoker co‐twins. The multivariate analysis of data showed, besides smoking habits (P = 0.02), an influence of gender (P = 0.006) and BMI (P = 0.001) on TA and a borderline effect of gender (P = 0.05) on TL. DNA methylation analysis, focused on 100 CpG sites mapping in hTERT, highlighted nine CpG sites differentially methylated in smokers. When co‐twins were contrasted, selecting as variables the intra‐twin difference in TA and hTERT DNA methylation, a statistically significant inverse correlation (P = 0.003) was observed between TA and DNA methylation at the cg05521538 site. In conclusion, these results indicate an association of tobacco smoke with TA and TL and suggest a possible association between smoke‐induced epigenetic effects and TA in healthy smokers. Environ. Mol. Mutagen. 58:551–559, 2017.