María Sánchez-Flores

γH2AX Assay as DNA Damage Biomarker for Human Population Studies: Defining Experimental Conditions

H2AX histone phosphorylation represents an early event in the cellular response against DNA double-strand breaks (DSBs), and plays a central role in sensing and repairing DNA damage. Therefore, the analysis of H2AX phosphorylated (γH2AX) may be possibly used as biomarker of genotoxicity and genomic instability with a number of applications in human epidemiology. However, the lack of an experimental standard leads to a wide heterogeneity in the results obtained and their interpretation, affecting the reliability of the assay. To address the most critical issues limiting the use of the γH2AX assay in human population studies, a flow cytometry analysis was performed to establish differences in γH2AX levels between fresh or cryopreserved peripheral blood lymphocytes, and to assess the influence of phytohemagglutinin (PHA) stimulation. To this purpose, cells were treated with 4 known genotoxic chemicals with different mechanisms of DSB induction, ie, bleomycin, methyl methanesulfonate, camptothecin, and actinomycin. According to our results, both unstimulated and stimulated fresh lymphocytes can be efficiently employed to evaluate γH2AX levels, but the sensitivity of the assay is depending upon the kind of damage observed. On the other hand, cryopreserved lymphocytes require PHA stimulation since unstimulated cells showed too high basal damage. Consequently, the protocol conditions will depend on the expected mechanism of production of DSB and the characteristics of the study design (sample collection and storage conditions, type of epidemiological study). Further studies are required to standardize the protocol of γH2AX assay to be employed as biomarker of genotoxicity or genomic instability in human population studies.

Lymphocyte Subsets in a Population of Nonfrail Elderly Individuals.

Age-related frailty is characterized by increased vulnerability to stress due to decline in homeostatic reserve, which results in increased risk of adverse health outcomes including disability, hospitalization, and death. The relationship between frailty and immunological system alterations is well established. Thus, analysis of immunological changes, such as alterations in lymphocyte subsets, during senescence may provide useful markers for frailty and associated pathologies. Since reference ranges currently used for lymphocyte subsets do not specifically differentiate the elderly group, the aim of this study was to (1) establish reference ranges in nonfrail elderly individuals and (2) assess the evolution of these parameters with age. Further, the influence of other physiological and lifestyle factors was also evaluated. The study was performed on 144 elderly individuals (aged 65–95) from Galicia (in northwestern Spain). Percentages of lymphocyte subpopulations (CD3+ T lymphocytes, CD4+ T-helper lymphocytes, CD8+ T-cytotoxic lymphocytes, CD19+ B lymphocytes, and CD56+16+ natural killer cells) were analyzed in peripheral blood by flow cytometry, and reference ranges were calculated. The individual status as nonfrail or prefrail did not markedly affect the immunological parameters, but an apparent influence of age was obtained for %CD3+, %CD4+, and %CD19+ cells, all of which fell with increasing age. Women showed higher levels of %CD19+ lymphocytes. No significant influence of smoking habits, physical activity, or drinking alcohol or caffeine beverages was observed. The results obtained may serve as a basis to establish comparisons between frail and nonfrail elderly individuals, in order to determine the usefulness of lymphocyte subsets as immunological biomarkers of frailty.

Oxidative stress, genomic features and DNA repair in frail elderly: A systematic review

Frailty is an emerging geriatric syndrome characterized by higher vulnerability to stressors, with an increased risk of adverse health outcomes such as mortality, morbidity, disability, hospitalization, and institutionalization. Although it is generally recognized to have a biological basis, no particular biological trait has been consistently associated to frailty status so far. In this work, epidemiological studies evaluating association of frailty status with alterations at cellular level - namely oxidative stress, genomic instability and DNA damage and repair biomarkers -were revised and compared. A total of 25 studies fulfilled inclusion/exclusion criteria and, consequently, were included in the review. Variations of oxidative stress biomarkers were often associated to frailty status in older people. On the contrary, genomic instability seems not to be linked to frailty. The only study which addressed the possible relationship between DNA repair modulations and frailty status also failed in finding association. Despite the large number of cellular alterations known to be associated with frailty, studies on this issue are still very scarce and limited to some of the possible cellular targets. The established link between DNA repair, genomic instability, and age and age-related disorders, encourage deeper investigations on this line.

Frailty Syndrome and Genomic Instability in Older Adults: Suitability of the Cytome Micronucleus Assay As a Diagnostic Tool

Frailty, a condition involving increased risk of disability and mortality in older adults, has emerged as a reliable way to predict the effect of aging. Genomic instability may help to anticipate recognition of frail individuals and improving frailty outcomes. Our objective was to evaluate the potential of the micronucleus frequency, evaluated in lymphocytes and buccal cells, to anticipate frailty identification and improve diagnosis reliability. Our results, from a group of older adults over 65, showed that frail individuals had significantly higher frequencies of micronucleus in lymphocytes (19.16 ± 0.66 vs. 13.07 ± 0.78, p < .001) and of binucleated buccal cells (82.65 ± 3.42 vs. 37.16 ± 2.85, p < .001) and lower frequencies of pyknotic and condensed chromatin buccal cells, than nonfrail subjects. When cognitive status was considered, similar results were obtained. Moreover, the presence of frailty and cognitive impairment were independently related to increases in frequencies of lymphocyte micronucleus and binucleated buccal cells. Our results encourage the use of micronucleus frequency in lymphocytes as a complement to clinical parameters in frailty identification. However, these results have to be further evaluated in prefrail patients, to better understand the connection between genomic instability and frailty and to establish these parameters as actual biomarkers of frailty in clinical practice.

Clinical and genomic safety of treatment with Ginkgo biloba L. leaf extract (IDN 5933/Ginkgoselect®Plus) in elderly: A randomised placebo-controlled clinical trial [GiBiEx]

BackgroundNumerous health benefits have been attributed to the Ginkgo biloba leaf extract (GBLE), one of the most extensively used phytopharmaceutical drugs worldwide. Recently, concerns of the safety of the extract have been raised after a report from US National Toxicology Program (NTP) claimed high doses of GBLE increased liver and thyroid cancer incidence in mice and rats. A safety study has been designed to assess, in a population of elderly residents in nursing homes, clinical and genomic risks associated to GBLE treatment.MethodsGiBiEx is a multicentre randomized clinical trial, placebo controlled, double blinded, which compared subjects randomized to twice-daily doses of either 120-mg of IDN 5933 (also known as Ginkgoselect®Plus) or to placebo for a 6-months period. IDN 5933 is extracted from dried leaves and contains 24.3% flavone glycosides and 6.1% of terpene lactones (2.9% bilobalide, 1.38% ginkgolide A, 0.66% ginkgolide B, 1.12% ginkgolide C) as determined by HPLC. The study was completed by 47 subjects, 20 in the placebo group and 27 in the treatment group. Clinical (adverse clinical effect and liver injury) and genomic (micronucleus frequency, comet assay, c-myc, p53, and ctnnb1 expression profile in lymphocytes) endpoints were assessed at the start and at the end of the study.ResultsNo adverse clinical effects or increase of liver injury markers were reported in the treatment group. The frequency of micronuclei [Mean Ratio (MR) = 1.01, 95% Confidence Intervals (95% CI) 0.86–1.18), and DNA breaks (comet assay) (MR = 0.91; 95% CI 0.58–1.43), did not differ in the two study groups. No significant difference was found in the expression profile of the three genes investigated.ConclusionsNone of the markers investigated revealed a higher risk in the treatment group, supporting the safety of IDN 5933 at doses prescribed and for duration of six months.Trial registrationClinicalTrials.gov Identifier: NCT03004508, December 20, 2016. Trial retrospectively registered.

Exploring Genetic Outcomes as Frailty Biomarkers

Frailty has emerged as a reliable measure of the aging process. Because the early detection of frailty is crucial to prevent or even revert it, the use of biomarkers would allow an earlier and more objective identification of frail individuals. To improve the understanding of the biological features associated with frailty as well as to explore different biomarkers for its early identification, several genetic outcomes-mutagenicity, different types of genetic damage, and cellular repair capacity-were analyzed in a population of older adults classified into frail, prefrail, and nonfrail. Besides, influence of clinical parameters-nutritional status and cognitive status-was evaluated. No association of mutation rate or primary DNA damage with frailty was observed. However, DNA repair capacity showed a nonsignificant tendency to decrease with frailty, and persistent levels of phosphorylated H2AX, as indicative of DNA breakage, increased progressively with frailty severity. These results support the possible use of H2AX phosphorylation to provide information regarding frailty severity. Further investigation is necessary to determine the consistency of the current findings in different populations and larger sample sizes, to eventually standardize biomarkers to be used in clinics, and to fully understand the influence of cognitive impairment.

Frailty in Older Adults Is Associated With Plasma Concentrations of Inflammatory Mediators but Not With Lymphocyte Subpopulations

Frailty denotes a multidimensional syndrome that gives rise to vulnerability to stressors and leads to an increase of the age-related decline of different physiological systems and cognitive abilities. Aging-related alterations of the immune system may compromise its competence culminating in a chronic low-grade inflammation state. Thus, it has been proposed that frailty is associated with alterations in the concentration of pro-inflammatory molecules and in different lymphocyte subpopulations. To provide further support to the validity of that hypothesis, we conducted a cross-sectional study in a population of Spanish older adults (N = 259, aged 65 and over) classified according to their frailty status. Biomarkers analyzed included percentages of several lymphocyte subsets and several inflammation mediators, namely concentrations of interleukin 6 (IL6), C-reactive protein (CRP), tumor necrosis factor α (TNFα), and 75 kDa soluble TNFα receptor II (sTNF-RII). Reference ranges for the inflammation mediators were established for the first time in robust older adults. A significant increase in the CD4+/CD8+ ratio and a significant decrease in the % CD19+ cells were observed in the frail group. Progressive increases with frailty severity were obtained in all inflammatory mediator concentrations, especially notable for IL6 and sTNF-RII. Area under the receiver-operating characteristic curve obtained for sTNF-RII (0.90, 95% CI 0.85–0.94, P < 0.001) indicates a high accuracy in the predictive value of this biomarker for frailty. Although results from the current study revealed limited strength associations between frailty and the lymphocyte subsets assessed, data obtained for the inflammatory mediators provide further support to involvement of inflammaging in frailty status in older adults.

Silica-coated iron oxide nanoparticles do not induce DNA double strand breaks or aneugenicity in SHSY5Y neuronal cells

This work was supported by Xunta de Galicia (EM 2012/079), the project NanoToxClass (ERA ERASIINN/001/2013), and by TD1204 MODENA COST Action.