DNA methylation of genes regulating appetite and prediction of weight loss after bariatric surgery in obese individuals.

PURPOSE: Epigenetic traits are influenced by clinical variables; interaction between DNA methylation (DNAmeth) and bariatric surgery-induced weight loss has been scarcely explored. We investigated whether DNAmeth of genes encoding for molecules/hormones regulating appetite, food intake or obesity could predict successful weight outcome following Roux-en-Y gastric bypass (RYGB). METHODS: Forty-five obese individuals with no known comorbidities were stratified accordingly to weight decrease one-year after RYGB (excess weight loss, EWL ≥ 50%: good responders, GR; EWL < 50%: worse responders, WR). DNAmeth of leptin (LEP), ghrelin (GHRL), ghrelin receptor (GHSR) and insulin-growth factor-2 (IGF2) was assessed before intervention. Single nucleotide polymorphisms of genes affecting DNAmeth, DNMT3A and DNMT3B, were also determined. RESULTS: At baseline, type 2 diabetes was diagnosed by OGTT in 13 patients. Post-operatively, GR (n = 23) and WR (n = 22) achieved an EWL of 67.7 ± 9.6 vs 38.2 ± 9.0%, respectively. Baseline DNAmeth did not differ between GR and WR for any tested genes, even when the analysis was restricted to subjects with no diabetes. A relationship between GHRL and LEP methylation profiles emerged (r = 0.47, p = 0.001). Searching for correlation between DNAmeth of the studied genes with demographic characteristics and baseline biochemical parameters of the studied population, we observed a correlation between IGF2 methylation and folate (r = 0.44, p = 0.003). Rs11683424 for DNMT3A and rs2424913 for DNMT3B did not correlate with DNAmeth of the studied genes. CONCLUSIONS: In severely obese subjects, the degree of DNAmeth of some genes affecting obesity and related conditions does not work as predictor of successful response to RYGB.

Diagnostic approach to mitochondrial disorders: the need for a reliable biomarker.

Mitochondrial diseases (MD) are disorders caused by impairment of the mitochondrial electron transport chain (ETC). Phenotypes are polymorphous and may range from pure myopathy to multisystemic disorders. The genetic defect can be located on mitochondrial or nuclear DNA. The ETC is needed for oxidative phosphorylation (which provides the cell with the most efficient energetic outcome in terms of ATP production), and consists of five multimeric protein complexes located in the inner mitochondrial membrane. The ETC also requires cytochrome c and a small electron carrier, coenzyme Q10. One of the pathogenic mechanisms of ETC disorders is excessive accumulation of reactive oxygen species (ROS). Mitochondrial dysfunction and oxidative stress appear to have a strong impact also on the pathogenesis of neurodegenerative diseases. At present, diagnosis of MD requires a complex approach: measurement of serum lactate, exercise testing, electromyography, magnetic resonance spectroscopy, muscle histology and enzymology, and genetic analysis. Biomarkers are molecules associated with biological processes or regulatory mechanisms. A reliable biomarker for the screening or diagnosis of MD is still needed. In this paper we review the diagnostic approach to MD, from serum lactate to other blood and urinary markers, from muscular biopsy to imaging studies, and we highlight some potentially interesting perspectives in this field.

Genotoxicity induced by arsenic compounds in peripheral human lymphocytes analysed by cytokinesis-block micronucleus assay.

This work focuses on the analysis of genotoxic effects on human peripheral lymphocytes exposed in vitro to different arsenic (As) compounds by means of the cytokinesis-block micronucleus assay. The study was carried out by challenging peripheral human lymphocytes with six As compounds in trivalent or pentavalent forms such as arsenite (As(III)) and arsenate (As(V)) and organoarsenic species such as monomethylarsonous acid (MMAs(III)), monomethylarsonic acid (MMAs(V)), dimethylarsinic acid (DMAs(V)) and trymethylarsine oxide (TMAO(V)). For As(III) and As(V) at concentrations of 4 and 32 microM, respectively, an increase of micronuclei (MN) frequency was found. MMAs(III) and MMAs(V) induced a statistically significant increase of MN frequency at the dose of 2 and 500 microM, respectively. For DMAs(V), no significant increase of MN was observed, although a decrease of the nuclear division index (NDI) was evident, indicating a cytotoxic effect. The genotoxic mechanism of action of MMAs(III) was further evaluated by means of fluorescence in situ hybridization analysis. Due to a higher percentage of centromere-positive MN, MMAs(III) showed a clear aneuploidogenic property. Finally, for TMAO(V) no genotoxicity was observed up to 1 mM. These results show how speciation is important in determining the genotoxic and cytotoxic effects of As compounds in human peripheral lymphocytes and support the emerging hypothesis that the induction of aneuploidy could be a mechanism by which As exerts its carcinogenic properties.

Susceptibility to chromosome malsegregation in lymphocytes of women who had a Down syndrome child in young age.

Recent findings seem to converge towards a unified hypothesis trying to relate Down's syndrome (DS), trisomy 21 and Alzheimer's disease (AD). The majority of DS individuals develop neuropathological characteristics of AD by the age of 40. Previous cytogenetic studies performed by us showed an increased frequency of aneuploidy in peripheral lymphocytes and fibroblasts of AD patients and a preferential occurrence of chromosome 21 in malsegregation events. An increased frequency of AD among young mothers of individuals with DS (MDS) is reported. This study investigates the cytogenetic characteristics and the predisposition to chromosome malsegregation of peripheral blood lymphocytes in a group of women (n = 35) who had a Down syndrome child in young age (<35 years) and in a control group (n = 30). We applied the micronucleus assay and the dual-color FISH in order to assess the susceptibility to malsegregation events. The results indicate a higher frequency of binucleated micronucleated cells in MDS in respect to the control group (16.1+/-9.1 per thousand versus 8.7+/-5.4 per thousand). Moreover, our data reveal that peripheral lymphocytes of MDS are more prone to chromosome non-disjunction with both chromosomes, 13 and 21, equally involved.

Oxidative DNA damage in peripheral leukocytes of mild cognitive impairment and AD patients.

It is well established that oxidative stress plays a key role in the degenerative neuronal death and progression of Alzheimer's disease (AD), although it is not clear if it is the primary triggering event in the pathogenesis of this disorder. Mild cognitive impairment (MCI) is a clinical condition between normal aging and AD, characterized by a memory deficit without loss of general cognitive and functional abilities. We performed this study by a comet assay analysis to evaluate the level of primary and oxidative DNA damage in two groups of MCI and AD patients, compared to healthy controls. Data showed a significantly higher level of primary DNA damage in leukocytes of AD and also of MCI patients compared to control individuals (average: 2.09+/-0.79 and 2.47+/-1.01, respectively for AD and MCI, versus 1.04+/-0.31 in controls). Moreover, the amount of oxidised DNA bases (both purines and pyrimidines) was significatively higher in the two groups of patients (AD and MCI) compared to controls. Our results give a further indication that oxidative stress, at least at the DNA level, is an earlier event in the pathogenesis of AD.

Searching for the role and the most suitable biomarkers of oxidative stress in Alzheimer's disease and in other neurodegenerative diseases.

The contribution of oxidative stress to neurodegeneration is not peculiar of a specific neurodegenerative disease, oxidative stress has been found implicated in a number of neurodegenerative disorders among which Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS). Even increasing are studies dealing with the search for peripheral biomarkers of oxidative stress in biological fluids or even in peripheral tissues themselves such as fibroblasts or blood cells. The application of the modified version of the comet assay for the detection of oxidised purines and pyrimidines in peripheral blood leukocytes results particularly useful if the study requires repeated blood drawn from the same individual, for instance if a clinical trial is performed with a preventive therapy. Likely damage occurs to every category of biological macromolecules and we consider, in the context of neurodegenerative diseases, particularly critical the proteic level. The identification of subjects at risk to develop AD or with pre-pathogenic conditions, the possibility to use "a battery of assays" for the detection of oxidative damage at peripheral level, together with recent advances in brain imaging, will allow to better address studies aimed not only to therapeutic purposes but also mainly to primary prevention.

Chromosome and oxidative damage biomarkers in lymphocytes of Parkinson's disease patients.

As cancer development usually results from exposure to several environmental risk factors in interaction with the genetic susceptibility of the host, it could be of interest to investigate if neurodegeneration, as occurs in Parkinson's disease (PD) patients can be attributed at least partially, to environmental risk factors. There is growing evidence that oxidative stress could play a significant role as a risk factor in the aetiology and pathogenesis of neurodegenerative diseases, emphasising the need for new individual and human-based approaches. The aim of our research is to explore the relation between chromosome instability and oxidative stress biomarkers in Parkinson's disease using a variety of strategies. We determined peripheral markers for oxidative damage in PD by testing for spontaneous and induced chromosomal damage, DNA strand breaks, oxidised pyrimidines and altered purines both in peripheral blood and cultured lymphocytes. We also measured glutathione S-transferase activity in the plasma of patients and controls. Compared to healthy controls, PD patients show higher frequencies of micronuclei (17.2 +/- 4.8 vs. 9.0 +/- 3.4, p < 0.001) and a significant increase in the levels of single strand breaks (SSB). Significant differences were also obtained in the distribution of oxidised purine bases between the two groups. Preliminary data obtained by fluorescence in situ hybridization analysis showed that the percentage of centromere negative micronuclei is higher than that of centromere positive micronuclei. Glutathione S-transferase activity in plasma from PD patients and controls was also measured and the enzymatic activity in PD patients was lower than in healthy controls.