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A potential protective role of cognitive reserve proxies against frailty has been suggested in older adults. We explored the cross-sectional association between cognitive reserve indicators and frailty phenotype. Data were obtained from the UK Biobank. We included 31,975 dementia-free participants aged ≥ 60 years (50.7% females, 2.2% frail) who completed a web-based cognitive assessment (fluid intelligence, working memory, visuospatial attention and processing speed, and executive functioning). Frailty was defined according to the Fried's phenotype (unintentional weight loss, exhaustion, low physical activity, slowness, and weakness). Participants meeting three or more criteria were classified as frail. Cognitive performance was compared between nonfrail and frail groups, and regression models were employed to analyze the associations between cognitive reserve proxies (education, skill level of occupation, social support, and multiple deprivation index (MDI)) and the likelihood of frailty. Frail and nonfrail groups significantly differed on cognitive function, with frail individuals demonstrating poorer performance on all cognitive functions (all p < .05) except fluid intelligence. Regression analysis showed that, after adjusting for age and sex, a lower educational level (odds ratio (OR) .797, 95% confidence interval (CI) .673-.944, p = .009), having maintained occupations with low cognitive requirements (OR .790, 95% CI .668-.936, p = .006), having less social support (OR .755, 95% CI .631-.903, p = .002), and living in a region with a high rate of multiple deprivation (OR 1.025, 95% CI 1.019-1.031, p < .001), significantly increased the probability of experiencing frailty. Our findings support the relationship between declined cognitive functions and frailty emphasizing the importance of implementing public health measures to enhance cognitive reserve., (© 2024. The Author(s), under exclusive licence to American Aging Association.)

Due to their extensive use, the release of zinc oxide nanoparticles (ZnO NP) into the environment is increasing and may lead to unintended risk to both human health and ecosystems. Access of ZnO NP to the brain has been demonstrated, so their potential toxicity on the nervous system is a matter of particular concern. Although evaluation of ZnO NP toxicity has been reported in several previous studies, the specific effects on the nervous system are not completely understood and, particularly, effects on genetic material and on organism behaviour are poorly addressed. We evaluated the potential toxic effects of ZnO NP in vitro and in vivo, and the role of zinc ions (Zn 2+ ) in these effects. In vitro, the ability of ZnO NP to be internalized by A172 glial cells was verified, and the cytotoxic and genotoxic effects of ZnO NP or the released Zn 2+ ions were addressed by means of vital dye exclusion and comet assay, respectively. In vivo, behavioural alterations were evaluated in zebrafish embryos using a total locomotion assay. ZnO NP induced decreases in viability of A172 cells after 24 h of exposure and genetic damage after 3 and 24 h. The involvement of the Zn 2+ ions released from the NP in genotoxicity was confirmed. ZnO NP exposure also resulted in decreased locomotor activity of zebrafish embryos, with a clear role of released Zn 2+ ions in this effect. These findings support the toxic potential of ZnO NP showing, for the first time, genetic effects on glial cells and proving the intervention of Zn 2+ ions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The formamidopyrimidine DNA glycosylase (Fpg)-modified comet assay is widely used for the measurement of oxidatively generated damage to DNA. However, there has not been a recommended long-term positive control for this version of the comet assay. We have investigated potassium bromate as a positive control for the Fpg-modified comet assay because it generates many Fpg-sensitive sites with a little concurrent generation of DNA strand breaks. Eight laboratories used the same procedure for the treatment of monocytic THP-1 cells with potassium bromate (0, 0.5, 1.5, and 4.5 mM) and subsequent cryopreservation in a freezing medium consisting of 50% foetal bovine serum, 40% RPMI-1640 medium, and 10% dimethyl sulphoxide. The samples were analysed by the Fpg-modified comet assay three times over a 3-year period. All laboratories obtained a positive concentration-response relationship in cryopreserved samples (linear regression coefficients ranging from 0.79 to 0.99). However, there was a wide difference in the levels of Fpg-sensitive sites between the laboratory with the lowest (4.2% Tail DNA) and highest (74% Tail DNA) values in THP-1 cells after exposure to 4.5 mM KBrO3. In an attempt to assess sources of inter-laboratory variation in Fpg-sensitive sites, comet images from one experiment in each laboratory were forwarded to a central laboratory for visual scoring. There was high consistency between measurements of %Tail DNA values in each laboratory and the visual score of the same comets done in the central laboratory (r = 0.98, P < 0.001, linear regression). In conclusion, the results show that potassium bromate is a suitable positive comet assay control., (© The Author(s) 2023. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The comet assay is a simple and versatile method for measurement of DNA damage in eukaryotic cells. More specifically, the assay detects DNA migration from agarose gel-embedded nucleoids, which depends on assay conditions and the level of DNA damage. Certain steps in the comet assay procedure have substantial impact on the magnitude of DNA migration (e.g. electric potential and time of electrophoresis). Inter-laboratory variation in DNA migration levels occurs because there is no agreement on optimal assay conditions or suitable assay controls. The purpose of the hCOMET ring trial was to test potassium bromate (KBrO3) as a positive control for the formamidopyrimidine DNA glycosylase (Fpg)-modified comet assay. To this end, participating laboratories used semi-standardized protocols for cell culture (i.e. cell culture, KBrO3 exposure, and cryopreservation of cells) and comet assay procedures, whereas the data acquisition was not standardized (i.e. staining of comets and image analysis). Segregation of the total variation into partial standard deviation (SD) in % Tail DNA units indicates the importance of cell culture procedures (SD = 10.9), comet assay procedures (SD = 12.3), staining (SD = 7.9) and image analysis (SD = 0.5) on the overall inter-laboratory variation of DNA migration (SD = 18.2). Future studies should assess sources of variation in each of these steps. On the positive side, the hCOMET ring trial demonstrates that KBrO3 is a robust positive control for the Fpg-modified comet assay. In conclusion, the hCOMET ring trial has demonstrated a high reproducibility of detecting genotoxic effects by the comet assay, but inter-laboratory variation of DNA migration levels is a concern., (© The Author(s) 2023. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The comet assay is widely used in biomonitoring studies for the analysis of DNA damage in leukocytes and peripheral blood mononuclear cells. Rather than processing blood samples directly, it can be desirable to cryopreserve whole blood or isolated cells for later analysis by the comet assay. However, this creates concern about artificial accumulation of DNA damage during cryopreservation. In this study, 10 laboratories used standardized cryopreservation and thawing procedures of monocytic (THP-1) or lymphocytic (TK6) cells. Samples were cryopreserved in small aliquots in 50% foetal bovine serum, 40% cell culture medium, and 10% dimethyl sulphoxide. Subsequently, cryopreserved samples were analysed by the standard comet assay on three occasions over a 3-year period. Levels of DNA strand breaks in THP-1 cells were increased (four laboratories), unaltered (four laboratories), or decreased (two laboratories) by long-term storage. Pooled analysis indicates only a modest positive association between storage time and levels of DNA strand breaks in THP-1 cells (0.37% Tail DNA per year, 95% confidence interval: -0.05, 0.78). In contrast, DNA strand break levels were not increased by cryopreservation in TK6 cells. There was inter-laboratory variation in levels of DNA strand breaks in THP-1 cells (SD = 3.7% Tail DNA) and TK6 reference sample cells (SD = 9.4% Tail DNA), whereas the intra-laboratory residual variation was substantially smaller (i.e. SD = 0.4%-2.2% Tail DNA in laboratories with the smallest and largest variation). In conclusion, the study shows that accumulation of DNA strand breaks in cryopreserved mononuclear blood cell lines is not a matter of concern., (© The Author(s) 2023. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Measurement of DNA migration in the comet assay can be done by image analysis or visual scoring. The latter accounts for 20%-25% of the published comet assay results. Here we assess the intra- and inter-investigator variability in visual scoring of comets. We include three training sets of comet images, which can be used as reference for researchers who wish to use visual scoring of comets. Investigators in 11 different laboratories scored the comet images using a five-class scoring system. There is inter-investigator variation in the three training sets of comets (i.e. coefficient of variation (CV) = 9.7%, 19.8%, and 15.2% in training sets I-III, respectively). However, there is also a positive correlation of inter-investigator scoring in the three training sets (r = 0.60). Overall, 36% of the variation is attributed to inter-investigator variation and 64% stems from intra-investigator variation in scoring between comets (i.e. the comets in training sets I-III look slightly different and this gives rise to heterogeneity in scoring). Intra-investigator variation in scoring was also assessed by repeated analysis of the training sets by the same investigator. There was larger variation when the training sets were scored over a period of six months (CV = 5.9%-9.6%) as compared to 1 week (CV = 1.3%-6.1%). A subsequent study revealed a high inter-investigator variation when premade slides, prepared in a central laboratory, were stained and scored by investigators in different laboratories (CV = 105% and 18%-20% in premade slides with comets from unexposed and hydrogen peroxide-exposed cells, respectively). The results indicate that further standardization of visual scoring is desirable. Nevertheless, the analysis demonstrates that visual scoring is a reliable way of analysing DNA migration in comets., (© The Author(s) 2023. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Zinc oxide nanoparticles (ZnO NPs) are among the most widely used nanomaterials. They have multiple applications in cosmetics, textiles, paints, electronics and, recently, also in biomedicine. This extensive use of ZnO NPs notably increases the probability that both humans and wildlife are subjected to undesirable effects. Despite being among the most studied NPs from a toxicological point of view, much remains unknown about their ecotoxicological effects or how they may affect specific cell types, such as cells of the central nervous system. The main objective of this work was to investigate the effects of ZnO NPs on human glial cells and zebrafish embryo development and to explore the role of the released Zn 2+ ions in these effects. The effects on cell viability on human A172 glial cells were assessed with an MTT assay and morphological analysis. The potential acute and developmental toxicity was assessed employing zebrafish ( Danio rerio ) embryos. To determine the role of Zn 2+ ions in the in vitro and in vivo observed effects, we measured their release from ZnO NPs with flame atomic absorption spectrometry. Then, cells and zebrafish embryos were treated with a water-soluble salt (zinc sulfate) at concentrations that equal the number of Zn 2+ ions released by the tested concentrations of ZnO NPs. Exposure to ZnO NPs induced morphological alterations and a significant decrease in cell viability depending on the concentration and duration of treatment, even after removing the overestimation due to NP interference. Although there were no signs of acute toxicity in zebrafish embryos, a decrease in hatching was detected after exposure to the highest ZnO NP concentrations tested. The ability of ZnO NPs to release Zn 2+ ions into the medium in a concentration-dependent manner was confirmed. Zn 2+ ions did not seem entirely responsible for the effects observed in the glial cells, but they were likely responsible for the decrease in zebrafish hatching rate. The results obtained in this work contribute to the knowledge of the toxicological potential of ZnO NPs.

The challenge-comet assay is a simple but effective approach that provides a quantitative and functional determination of DNA repair ability, and allows to monitor the kinetics of repair process. Peripheral blood mononuclear cells (PBMC) are the cells most frequently employed in human biomonitoring studies using the challenge-comet assay, but having a validated alternative of non-invasive biomatrix would be highly convenient for certain population groups and circumstances. The objective of this study was to validate the use of salivary leucocytes in the challenge-comet assay. Leucocytes were isolated from saliva samples and challenged (either in fresh or after cryopreservation) with three genotoxic agents acting by different action mechanisms: bleomycin, methyl methanesulfonate, and ultraviolet radiation. Comet assay was performed just after treatment and at other three additional time points, in order to study repair kinetics. The results obtained demonstrated that saliva leucocytes were as suitable as PBMC for assessing DNA damage of different nature that was efficiently repaired over the evaluated time points, even after 5 months of cryopreservation (after a 24 h stimulation with PHA). Furthermore, a new parameter to determine the efficacy of the repair process, independent of the initial amount of damage induced, is proposed, and recommendations to perform the challenge-comet assay with salivary leucocytes depending on the type of DNA repair to be assessed are suggested. Validation studies are needed to verify whether the method is reproducible and results reliable and comparable among laboratories and studies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Ageing is accompanied with a decline in several physiological systems. Frailty is an age-related syndrome correlated to the loss of homeostasis and increased vulnerability to stressors, which is associated with increase in the risk of disability, comorbidity, hospitalisation, and death in older adults. The aim of this study was to understand the relationship between frailty syndrome, immune activation, and oxidative stress. Serum concentrations of vitamins A and E were also evaluated, as well as inflammatory biomarkers (CRP and IL-6) and oxidative DNA levels. A group of Portuguese older adults (≥65 years old) was engaged in this study and classified according to Fried's frailty phenotype. Significant increases in the inflammatory mediators (CRP and IL-6), neopterin levels, kynurenine to tryptophan ratio (Kyn/Trp), and phenylalanine to tyrosine ratio (Phe/Tyr), and significant decreases in Trp and Tyr concentrations were observed in the presence of frailty. IL-6, neopterin, and Kyn/Trp showed potential as predictable biomarkers of frailty syndrome. Several clinical parameters such as nutrition, dependency scales, and polypharmacy were related to frailty and, consequently, may influence the associations observed. Results obtained show a progressive immune activation and production of pro-inflammatory molecules in the presence of frailty, agreeing with the inflammageing model. Future research should include different dimensions of frailty, including psychological, social, biological, and environmental factors.

The comet assay or single cell gel electrophoresis, is the most common method used to measure strand breaks and a variety of other DNA lesions in human populations. To estimate the risk of overall mortality, mortality by cause, and cancer incidence associated to DNA damage, a cohort of 2,403 healthy individuals (25,978 person-years) screened in 16 laboratories using the comet assay between 1996 and 2016 was followed-up. Kaplan-Meier analysis indicated a worse overall survival in the medium and high tertile of DNA damage (p < 0.001). The effect of DNA damage on survival was modelled according to Cox proportional hazard regression model. The adjusted hazard ratio (HR) was 1.42 (1.06-1.90) for overall mortality, and 1.94 (1.04-3.59) for diseases of the circulatory system in subjects with the highest tertile of DNA damage. The findings of this study provide epidemiological evidence encouraging the implementation of the comet assay in preventive strategies for non-communicable diseases., (© 2021. The Author(s).)

Standard toxicity tests might not be fully adequate for evaluating nanomaterials since their unique features are also responsible for unexpected interactions. The in vitro cytokinesis-block micronucleus (CBMN) test is recommended for genotoxicity testing, but cytochalasin-B (Cyt-B) may interfere with nanoparticles (NP), leading to inaccurate results. Our objective was to determine whether Cyt-B could interfere with MN induction by TiO 2 NP in human SH-SY5Y cells, as assessed by CBMN test. Cells were treated for 6 or 24 h, according to three treatment options: co-treatment with Cyt-B, post-treatment, and delayed co-treatment. Influence of Cyt-B on TiO 2 NP cellular uptake and MN induction as evaluated by flow cytometry (FCMN) were also assessed. TiO 2 NP were significantly internalized by cells, both in the absence and presence of Cyt-B, indicating that this chemical does not interfere with NP uptake. Dose-dependent increases in MN rates were observed in CBMN test after co-treatment. However, FCMN assay only showed a positive response when Cyt-B was added simultaneously with TiO 2 NP, suggesting that Cyt-B might alter CBMN assay results. No differences were observed in the comparisons between the treatment options assessed, suggesting they are not adequate alternatives to avoid Cyt-B interference in the specific conditions tested.

Metal oxide nanoparticles (NPs) have a wide variety of applications in many consumer products and biomedical practices. As a result, human exposure to these nanomaterials is highly frequent, becoming an issue of concern to public health. Recently, human salivary leucocytes have been proposed as an adequate non-invasive alternative to peripheral blood leucocytes to evaluate genotoxicity in vitro. The present study focused on proving the suitability of salivary leucocytes as a biomatrix in the comet assay for in vitro nanogenotoxicity studies, by testing some of the metal oxide NPs most frequently present in consumer products, namely, titanium dioxide (TiO 2 ), zinc oxide (ZnO), and cerium dioxide (CeO 2 ) NPs. Primary and oxidative DNA damage were evaluated by alkaline and hOGG1-modified comet assay, respectively. Any possible interference of the NPs with the methodological procedure or the hOGG1 activity was addressed before performing genotoxicity evaluation. Results obtained showed an increase of both primary and oxidative damage after NPs treatments. These data support the use of salivary leucocytes as a proper and sensitive biological sample for in vitro nanogenotoxicity studies, and contribute to increase the knowledge on the impact of metal oxide NPs on human health, reinforcing the need for a specific regulation of the nanomaterials use.

DNA damage and repair activity are often assessed in blood samples from humans in different types of molecular epidemiology studies. However, it is not always feasible to analyse the samples on the day of collection without any type of storage. For instance, certain studies use repeated sampling of cells from the same subject or samples from different subjects collected at different time-points, and it is desirable to analyse all these samples in the same comet assay experiment. In addition, flawless comet assay analyses on frozen samples open up the possibility of using this technique on biobank material. In this article we discuss the use of cryopreserved peripheral blood mononuclear cells (PBMCs), buffy coat (BC) and whole blood (WB) for analysis of DNA damage and repair using the comet assay. The published literature and the authors' experiences indicate that various types of blood samples can be cryopreserved with only a minor effect on the basal level of DNA damage. There is evidence to suggest that WB and PBMCs can be cryopreserved for several years without much effect on the level of DNA damage. However, care should be taken when cryopreserving WB and BCs. It is possible to use either fresh or frozen samples of blood cells, but results from fresh and frozen cells should not be used in the same dataset. The article outlines detailed protocols for the cryopreservation of PBMCs, BCs and WB samples., (© The Author(s) 2021. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society.All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Frailty is a geriatric syndrome defined as a status of extreme vulnerability to stressors, leading to a higher risk of negative health-related outcomes. "Inflammaging", an age-related state of low-grade chronic inflammation, is characterized by an increased concentration of pro-inflammatory cytokines and acute phase proteins. Inflammaging has been postulated as an underlying mechanism of frailty, and several studies tested the relationship between frailty and concentration of inflammatory mediators. The aim of this systematic review and meta-analysis was to test whether inflammatory mediators are overproduced in frail older adults. Among the 758 articles identified in the literature search, 50 were included in the systematic review, and 39 in the three meta-analyses, i.e., C-reactive protein (CRP), interleukin 6 (IL6), and tumor necrosis factor α. To reduce heterogeneity, meta-analyses were restricted to studies identifying frailty by the Fried et al. [1] [J. Gerontol. A. Biol. Sci. Med. Sci. 56, M146-56] phenotypic criteria. Quantitative analyses measuring the association between frailty and biomarker concentrations showed significant differences when frail subjects were compared to non-frail and pre-frail subjects for CRP and IL6. This work established strong association between inflammatory biomarkers and frailty, confirming the role of age-related chronic inflammation in frailty development.

The comet assay is a popular assay in biomonitoring studies. DNA strand breaks (or unspecific DNA lesions) are measured using the standard comet assay. Oxidative stress-generated DNA lesions can be measured by employing DNA repair enzymes to recognise oxidatively damaged DNA. Unfortunately, there has been a tendency to fail to report results from assay controls (or maybe even not to employ assay controls). We believe this might have been due to uncertainty as to what really constitutes a positive control. It should go without saying that a biomonitoring study cannot have a positive control group as it is unethical to expose healthy humans to DNA damaging (and thus potentially carcinogenic) agents. However, it is possible to include assay controls in the analysis (here meant as a cryopreserved sample of cells i.e. included in each experiment as a reference sample). In the present report we tested potassium bromate (KBrO3) as a positive comet assay control for the formamidopyrimidine DNA glycosylase (Fpg)-modified comet assay. Ten laboratories used the same procedure for treatment of monocytic THP-1 cells with KBrO3 (0.5, 1.5 and 4.5 mM for 1 h at 37°C) and subsequent cryopreservation. Results from one laboratory were excluded in the statistical analysis because of technical issues in the Fpg-modified comet assay. All other laboratories found a concentration-response relationship in cryopreserved samples (regression coefficients from 0.80 to 0.98), although with different slopes ranging from 1.25 to 11.9 Fpg-sensitive sites (%DNA in tail) per 1 mM KBrO3. Our results demonstrate that KBrO3 is a suitable positive comet assay control., (© The Author(s) 2020. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Serum vitamin D deficiency is widespread among older adults and is a potential modifiable risk factor for frailty. Moreover, frailty has been suggested as an intermediate step in the association between low levels of vitamin D and mortality. Hence, we conducted a systematic review of the literature and meta-analysis to test the possible association of low concentrations of serum 25-hydroxyvitamin D (25(OH)D), a marker of vitamin D status, with frailty in later life. We reviewed cross-sectional or longitudinal studies evaluating populations of older adults and identifying frailty by a currently validated scale. Meta-analyses were restricted to cross-sectional data from studies using Fried's phenotype to identify frailty. Twenty-six studies were considered in the qualitative synthesis, and thirteen studies were included in the meta-analyses. Quantitative analyses showed significant differences in the comparisons of frail (standardized mean difference (SMD)-1.31, 95% confidence interval (CI) (-2.47, -0.15), p = 0.0271) and pre-frail (SMD-0.79, 95% CI (-1.58, -0.003), p = 0.0491) subjects vs. non-frail subjects. Sensitivity analyses reduced heterogeneity, resulting in a smaller but still highly significant between-groups difference. Results obtained indicate that lower 25(OH)D levels are significantly associated with increasing frailty severity. Future challenges include interventional studies testing the possible benefits of vitamin D supplementation in older adults to prevent/palliate frailty and its associated outcomes.

Titanium dioxide nanoparticles (TiO 2 NPs) have a wide variety of applications in many consumer products, including as food additives, increasing the concern about the possible hazards that TiO 2 NPs may pose to human health. Although most previous studies have focused on the respiratory system, ingestion must also be considered as an important exposure route. Furthermore, after inhalation or ingestion, TiO 2 NPs can reach several organs, such as the liver, brain or lungs. Taking this into consideration, the present study focuses on the uptake and potential genotoxicity (micronuclei induction) of TiO 2 NPs on four human cell lines of diverse origin: lung cells (A549), liver cells (HepG2), glial cells (A172) and neurons (SH-SY5Y), using flow cytometry methods. Results showed a concentration-, time- and cell-type- dependent increase in TiO 2 NPs uptake but no significant induction of micronuclei in any of the tested conditions. Data obtained reinforce the importance of cell model and testing protocols choice for toxicity assessment. However, some questions remain to be answered, namely on the role of cell culture media components on the agglomeration state and mitigation of TiO 2 NPs toxic effects., Competing Interests: The authors declare no conflicts of interest.

Formaldehyde (FA) is a high-volume production chemical manufactured worldwide to which many people are exposed to both environmentally and occupationally. FA was recently reclassified as a human carcinogen. Several epidemiological studies have revealed an increased risk of cancer development among workers exposed to FA. Although FA genotoxicity was confirmed in a variety of experimental systems, data from human studies are conflicting. The aim of the present study was to evaluate the occupational exposure to FA in a multistage approach relating the exposure with different biomarkers (dose and effect) and individual susceptibility. Air monitoring was performed to estimate the level of exposure to FA during shift work. Eighty-five workers from hospital anatomy-pathology laboratories exposed to FA and 87 controls were tested for cytogenetic alterations in lymphocytes (micronucleus, MN; sister-chromatid exchange, SCE) and T-cell receptor (TCR) mutation assay. The frequency of MN in exfoliated buccal cells, a first contact tissue was also assessed. Percentages of different lymphocyte subpopulations were selected as immunotoxicity biomarkers. The level of formic acid in urine was investigated as a potential biomarker of internal dose. The effects of polymorphic genes of xenobiotic metabolising enzymes and DNA repair enzymes on the endpoints studied were determined. The mean level of FA exposure was 0.38 ± 0.03 ppm. MN (in lymphocytes and buccal cells) and SCE were significantly increased in FA-exposed workers compared to controls. MN frequency positively correlated with FA levels of exposure and duration. Significant alterations in the percentage of T cytotoxic lymphocytes, NK cells and B lymphocytes were found between groups. Polymorphisms in CYP2E1, GSTP1 and FANCA genes were associated with increased genetic damage in FA-exposed subjects. The obtained information may provide new important data to be used by health and safety care programs and by governmental agencies responsible for setting the acceptable levels for occupational exposure to FA., (Copyright © 2019. Published by Elsevier Inc.)

Iron oxide nanoparticles (ION) have great potential for an increasing number of medical and biological applications, particularly those focused on nervous system. Although ION seem to be biocompatible and present low toxicity, it is imperative to unveil the potential risk for the nervous system associated to their exposure, especially because current data on ION effects on human nervous cells are scarce. Thus, in the present study potential toxicity associated with silica-coated ION (S-ION) exposure was evaluated on human A172 glioblastoma cells. To this aim, a complete toxicological screening testing several exposure times (3 and 24 h), nanoparticle concentrations (5-100 μg/ml), and culture media (complete and serum-free) was performed to firstly assess S-ION effects at different levels, including cytotoxicity - lactate dehydrogenase assay, analysis of cell cycle and cell death production - and genotoxicity - H2AX phosphorylation assessment, comet assay, micronucleus test and DNA repair competence assay. Results obtained showed that S-ION exhibit certain cytotoxicity, especially in serum-free medium, related to cell cycle disruption and cell death induction. However, scarce genotoxic effects and no alteration of the DNA repair process were observed. Results obtained in this work contribute to increase the knowledge on the impact of ION on the human nervous system cells., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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.

Background: Numerous 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., Methods: GiBiEx 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., Results: No 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., Conclusions: None 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 Registration: ClinicalTrials.gov Identifier: NCT03004508 , December 20, 2016. Trial retrospectively registered.

Cell cultures from neuronal and glial origin have proven to be powerful tools for elucidating cellular and molecular mechanisms of nervous system development and physiology, and as neurotoxicity models to evaluate in vitro the possible effects of chemicals. But cellular heterogeneity of nervous system is considerable and these cells have been shown to respond diversely to neurotoxic insults, leading to disparate results from different studies. To shed more light on suitability of cellular models of nervous origin for neurotoxicity screening, the objective of this study was to compare the sensitivity to genetic damage induction of two nervous cell lines. To this aim, neurons (SH-SY5Y) and glial (A172) cells were treated with differently-acting genotoxic agents (bleomycin, actinomycin-D, methyl methanesulfonate, mitomycin C, and griseofulvin). After discarding cytotoxicity, genotoxicity was evaluated by a battery of assays encompassing detection of different genetic lesions. Results obtained showed that glial cells are generally more resistant to genotoxic damage induced by clastogenic agents, but more sensitive to aneugenic effects. These results highlight the need of proper design of in vitro neurotoxicology studies, especially for neurogenotoxicity screening, emphasizing the importance of employing more than one nervous cell type for testing the potential toxicity of a particular exposure., (Copyright © 2017. Published by Elsevier Ltd.)

Our aim was to estimate the prevalence of cognitive impairment in rural and urban elderly populations and to examine the relationship between lifetime occupation and general cognitive performance. A cross-sectional study was carried out covering a representative sample ( n = 749) of adults aged ≥65 years. Two categories were created to define the degree of urbanization using a criterion of geographical contiguity in combination with a minimum population threshold: densely populated (urban) areas and intermediate-thinly populated (rural) areas. Occupational histories were ranked by skill level requirements according to the Spanish National Classification of Occupations. Prevalence estimates of cognitive impairment were measured with the Mini-Mental State Examination. Results show that rural residence was not significantly associated with higher risk of cognitive impairment. A protective effect of cognitive demands at work against age-related cognitive decline was observed. However, this effect was not independent of confounder factors, such as age and education. A low overall prevalence of cognitive impairment was observed (6.5%), compared with previous estimates, possibly due to the sample selection in senior centers. Occupation during active life is not an isolated protective factor against cognitive impairment, and it is closely related to educational level. In future geriatric programs, description of both factors should be taken into consideration in screening older adults at increased risk of cognitive impairment and dementia.

Objective: Malnutrition is a common and relevant syndrome in elderly people due to its influence on quality of life. The main aim of the present study was to identify health determinants of malnutrition or risk of malnutrition., Design: Cross-sectional study collecting information on sociodemographic and health factors (co-morbidity, cognitive or affective problems, prescription medication use, frailty status, self-rated health) as determinants of nutritional status, assessed by the short form of the Mini Nutritional Assessment., Setting: Forty-three senior centres from Galicia (north-western Spain) participated to recruit participants., Subjects: A representative community-dwelling sample of 749 elderly people aged ≥65 years., Results: Of the total participants, 14·3 % were malnourished/at risk of malnutrition. Presence of overweight or obesity, depressive symptoms, polypharmacy (use of five or more prescription medications), presence of pre-frailty or frailty status and poor self-rated health showed the strongest relationship to malnutrition/risk of malnutrition. This model predicted 86·0 % of the cases correctly. The best determinants for women were polypharmacy and poor self-rated health, reaching 82·8 % of cases of malnourishment/risk of malnutrition predicted correctly. In men, the main determinants were overweight or obesity, depressive symptomatology and polypharmacy, with 89·8 % of cases of malnourishmen/risk of malnutrition predicted correctly., Conclusions: Screening for nutritional status and its determinant factors should be included as part of comprehensive assessments to ensure an early screening of malnutrition and to propose possible intervention strategies that would be important for both elderly people and the health-care system.

Okadaic acid (OA) and dinophysistoxins (DTXs) are the main toxins responsible for diarrhetic shellfish poisoning (DSP) intoxications during harmful algal blooms (HABs). Although the genotoxic and cytotoxic responses to OA have been evaluated in vitro, the in vivo effects of these toxins have not yet been fully explored. The present work fills this gap by evaluating the in vivo effects of the exposure to the DSP-toxin-producing dinoflagellate Prorocentrum lima during the simulation of an early HAB episode in the mussel Mytilus galloprovincialis. The obtained results revealed that in vivo exposure to this toxic microalgae induced early genotoxicity in hemocytes, as a consequence of oxidative DNA damage. In addition, the DNA damage observed in gill cells seems to be mainly influenced by exposure time and P. lima concentration, similarly to the case of the oxidative damage found in hemocytes exposed in vitro to OA. In both cell types, the absence of DNA damage at low toxin concentrations is consistent with the notion suggesting that this level of toxicity does not disturb the antioxidant balance. Lastly, in vivo exposure to growing P. lima cell densities increased apoptosis but not necrosis, probably due to the presence of a high number of protein apoptosis inhibitors in molluscs. Overall, this work sheds light into the in vivo genotoxic and cytotoxic effects of P. lima. In doing so, it also demonstrates for the first time the potential of the modified (OGG1) comet assay for assessing oxidative DNA damage caused by marine toxins in marine invertebrates.

[This corrects the article DOI: 10.1039/c5tx00206k.].

Iron oxide nanoparticles (ION) have been widely used in biomedical applications, for both diagnosis and therapy, due to their unique magnetic properties. They are intensively explored in neuromedicine mostly because of their ability to cross the blood brain barrier. Hence, their potential harmful effects on neuronal cells need to be carefully assessed. The objective of this study was to evaluate the toxicity of silica-coated ION (S-ION) (10-200 μg ml -1 ) on human neuronal SHSY5Y cells. Alterations in the cell cycle, cell death by apoptosis or necrosis, and membrane integrity were assessed as cytotoxicity parameters. Genotoxicity was determined by a γH2AX assay, a micronucleus (MN) test, and a comet assay. Complementarily, possible effects on DNA damage repair were also analysed by means of a DNA repair competence assay. All analyses were performed in complete and serum-free cell culture media. Iron ion release from the nanoparticles was notable only in complete medium. Despite being effectively internalized by the neuronal cells, S-ION presented in general low cytotoxicity; positive results were only obtained in some assays at the highest concentrations and/or the longest exposure time tested (24 h). Genotoxicity evaluations in serum-free medium were negative for all conditions assayed; in complete medium, dose and time-dependent increase in DNA damage not related to the production of double strand breaks or chromosome loss (according to the results of the γH2AX assay and MN test), was obtained. The presence of serum slightly influenced the behaviour of S-ION; further studies to investigate the formation of a protein corona and its role in nanoparticle toxicity are necessary.

Background: Telomere shortening in peripheral blood mononuclear cells (PBMCs) has been associated with biological age and several chronic degenerative diseases. However, the relationship between telomere length and sarcopenia, a hallmark of the aging process, is unknown. The aim of the present study was therefore to determine whether PBMC telomeres obtained from sarcopenic older persons were shorter relative to non-sarcopenic peers. We further explored if PBMC telomere length was associated with frailty, a major clinical correlate of sarcopenia., Methods: Analyses were conducted in 142 persons aged ≥65 years referred to a geriatric outpatient clinic (University Hospital). The presence of sarcopenia was established according to the European Working Group on Sarcopenia in Older People criteria, with bioelectrical impedance analysis used for muscle mass estimation. The frailty status was determined by both the Fried's criteria (physical frailty, PF) and a modified Rockwood's frailty index (FI). Telomere length was measured in PBMCs by quantitative real-time polymerase chain reaction according to the telomere/single-copy gene ratio (T/S) method., Results: Among 142 outpatients (mean age 75.0 ± 6.5 years, 59.2% women), sarcopenia was diagnosed in 23 individuals (19.3%). The PF phenotype was detected in 74 participants (52.1%). The average FI score was 0.46 ± 0.17. PBMC telomeres were shorter in sarcopenic subjects (T/S = 0.21; 95% CI: 0.18-0.24) relative to non-sarcopenic individuals (T/S = 0.26; 95% CI: 0.24-0.28; p = 0.01), independent of age, gender, smoking habit, or comorbidity. No significant associations were determined between telomere length and either PF or the FI., Conclusion: PBMC telomere length, expressed as T/S values, is shorter in older outpatients with sarcopenia. The cross-sectional assessment of PBMC telomere length is not sufficient at capturing the complex, multidimensional syndrome of frailty.

Sky1 is the only member of the SR (Serine-Arginine) protein kinase family in Saccharomyces cerevisiae. When yeast cells are treated with the anti-cancer drug cisplatin, Sky1 kinase activity is necessary to produce the cytotoxic effect. In this study, proteome changes in response to this drug and/or SKY1 deletion have been evaluated in order to understand the role of Sky1 in the response of yeast cells to cisplatin. Results reveal differential expression of proteins previously related to the oxidative stress response, DNA damage, apoptosis and mitophagy. With these precedents, the role of Sky1 in apoptosis, necrosis and mitophagy has been evaluated by flow-cytometry, fluorescence microscopy, biosensors and fluorescence techniques. After cisplatin treatment, an apoptotic-like process diminishes in the ∆sky1 strain in comparison to the wild-type. The treatment does not affect mitophagy in the wild-type strain, while an increase is observed in the ∆sky1 strain. The increased resistance to cisplatin observed in the ∆sky1 strain may be attributable to a decrease of apoptosis and an increase of mitophagy.

Okadaic acid (OA) is one of the most frequent and worldwide distributed marine toxins. It is easily accumulated by shellfish, mainly bivalve mollusks and fish, and, subsequently, can be consumed by humans causing alimentary intoxications. OA is the main representative diarrheic shellfish poisoning (DSP) toxin and its ingestion induces gastrointestinal symptoms, although it is not considered lethal. At the molecular level, OA is a specific inhibitor of several types of serine/threonine protein phosphatases and a tumor promoter in animal carcinogenesis experiments. In the last few decades, the potential toxic effects of OA, beyond its role as a DSP toxin, have been investigated in a number of studies. Alterations in DNA and cellular components, as well as effects on immune and nervous system, and even on embryonic development, have been increasingly reported. In this manuscript, results from all these studies are compiled and reviewed to clarify the role of this toxin not only as a DSP inductor but also as cause of alterations at the cellular and molecular levels, and to highlight the relevance of biomonitoring its effects on human health. Despite further investigations are required to elucidate OA mechanisms of action, toxicokinetics, and harmful effects, there are enough evidences illustrating its toxicity, not related to DSP induction, and, consequently, supporting a revision of the current regulation on OA levels in food.

In November 2002 the oil tanker Prestige spilled 63,000tonnes of heavy oil off the northwest coast of Spain, impacting more than 1000km of coastline. A general concern led to a huge mobilization of human and technical resources, and more than 300,000 people participated in cleanup activities, which lasted up to 10months. Some endocrine and immunological alterations were reported in Prestige oil exposed subjects for several months. Therefore, the objective of this study was to evaluate if these alterations are still present seven years after the exposure. Fifty-four individuals exposed for at least 2months were compared to 50 matched referents. Prolactin and cortisol plasma concentrations, percentages of lymphocyte subsets (CD3(+), CD4(+), CD8(+), CD19(+), and CD56(+)16(+)), plasma levels of circulating cytokines (interleukin (IL) 2, IL4, IL6, IL10, tumour necrosis factor α, and interferon γ), and serum concentrations of neopterin, tryptophan and kynurenine were determined in peripheral blood samples. Results showed significant differences in exposed individuals vs. referents only in cortisol (increase), kynurenine and %CD16(+)56(+) lymphocytes (both decrease). Time of exposure to the oil or using protective clothes did not influence the results, but effect of using protective mask was observed on neopterin, %CD8(+), CD4(+)/CD8(+) ratio and IL4. Surveillance of the exposed individuals for early detection of possible health problems related to the endocrine or immunological systems is recommended., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Harmful Algal Blooms (HABs) constitute one of the most important sources of contamination in the oceans, producing high concentrations of potentially harmful biotoxins that are accumulated across the food chains. One such biotoxin, Okadaic Acid (OA), is produced by marine dinoflagellates and subsequently accumulated within the tissues of filtering marine organisms feeding on HABs, rapidly spreading to their predators in the food chain and eventually reaching human consumers causing Diarrhetic Shellfish Poisoning (DSP) syndrome. While numerous studies have thoroughly evaluated the effects of OA in mammals, the attention drawn to marine organisms in this regard has been scarce, even though they constitute primary targets for this biotoxin. With this in mind, the present work aimed to provide a timely and comprehensive insight into the current literature on the effect of OA in marine invertebrates, along with the strategies developed by these organisms to respond to its toxic effect together with the most important methods and techniques used for OA detection and evaluation.

Titanium dioxide (TiO2) are among most frequently used nanoparticles (NPs). They are present in a variety of consumer products, including food industry in which they are employed as an additive. The potential toxic effects of these NPs on mammal cells have been extensively studied. However, studies regarding neurotoxicity and specific effects on neuronal systems are very scarce and, to our knowledge, no studies on human neuronal cells have been reported so far. Therefore, the main objective of this work was to investigate the effects of two types of TiO₂ NPs, with different crystalline structure, on human SHSY5Y neuronal cells. After NPs characterization, a battery of assays was performed to evaluate the viability, cytotoxicity, genotoxicity and oxidative damage in TiO₂ NP-exposed SHSY5Y cells. Results obtained showed that the behaviour of both types of NPs resulted quite comparable. They did not reduce the viability of neuronal cells but were effectively internalized by the cells and induced dose-dependent cell cycle alterations, apoptosis by intrinsic pathway, and genotoxicity not related with double strand break production. Furthermore, all these effects were not associated with oxidative damage production and, consequently, further investigations on the specific mechanisms underlying the effects observed in this study are required., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Okadaic acid (OA) is one of the most common and highly distributed marine toxins. It can be accumulated in several molluscs and other marine organisms and cause acute gastrointestinal symptoms after oral consumption by humans, called diarrheic shellfish poisoning. However other toxic effects beyond these gastrointestinal symptoms were also reported. Thus, OA was found to induce important chromosomal abnormalities and other genetic injuries that can lead to severe pathologies, including cancer. Furthermore, the relationship between OA and carcinogenic processes has been previously demonstrated in in vivo studies with rodents, and also suggested in human epidemiological studies. In this context, further research is required to better understand the underlying mechanisms of OA-related tumourigenesis. In a previous study, we identified 247 genes differentially expressed in SHSY5Y neuroblastoma cells exposed to 100nM OA at different times (3, 24 and 48h) by means of suppression subtractive hybridization. These genes were involved in relevant cell functions such as signal transduction, cell cycle, metabolism, and transcription and translation processes. However, due to the high potential percentage of false positives that may be obtained by this approach, results from SSH are recommended to be analyzed by an independent method. In the present study, we selected ten genes related to cancer initiation or progression, directly or indirectly, for further quantitative PCR analysis (ANAPC13, PTTG1, CALM2, CLU, HN1, MALAT1, MAPRE2, MLLT11, SGA-81M and TAX1BP1). Results obtained showed important alterations in the expression patterns of all the genes evaluated at one or more treatment times, providing, for the first time, a possible explanation at the molecular level of the potential relationship between the consumption of OA-contaminated shellfish and the incidence of different cancers in humans. Nevertheless, given the complexity of this process, more exhaustive studies are required before drawing any final conclusion., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Zinc oxide nanoparticles (ZnO NPs) are one of the most abundantly used nanomaterials in consumer products and biomedical applications. As a result, human exposure to these NPs is highly frequent and they have become an issue of concern to public health. Although toxicity of ZnO NPs has been extensively studied and they have been shown to affect many different cell types and animal systems, there is a significant lack of toxicological data for ZnO NPs on the nervous system, especially for human neuronal cells and tissues. In this study, the cytotoxic and genotoxic effects of ZnO NPs on human SHSY5Y neuronal cells were investigated under different exposure conditions. Results obtained by flow cytometry showed that ZnO NPs do not enter the neuronal cells, but their presence in the medium induced cytotoxicity, including viability decrease, apoptosis and cell cycle alterations, and genotoxicity, including micronuclei production, H2AX phosphorylation and DNA damage, both primary and oxidative, on human neuronal cells in a dose- and time-dependent manner. Free Zn(2+) ions released from the ZnO NPs were not responsible for the viability decrease, but their role on other types of cell damage cannot be ruled out. The results obtained in this work contribute to increase the knowledge on the genotoxic and cytotoxic potential of ZnO NPs in general, and specifically on human neuronal cells, but further investigations are required to understand the action mechanism underlying the cytotoxic and genotoxic effects observed., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

There are substantial inter-laboratory variations in the levels of DNA damage measured by the comet assay. The aim of this study was to investigate whether adherence to a standard comet assay protocol would reduce inter-laboratory variation in reported values of DNA damage. Fourteen laboratories determined the baseline level of DNA strand breaks (SBs)/alkaline labile sites and formamidopyrimidine DNA glycosylase (FPG)-sensitive sites in coded samples of mononuclear blood cells (MNBCs) from healthy volunteers. There were technical problems in seven laboratories in adopting the standard protocol, which were not related to the level of experience. Therefore, the inter-laboratory variation in DNA damage was only analysed using the results from laboratories that had obtained complete data with the standard comet assay protocol. This analysis showed that the differences between reported levels of DNA SBs/alkaline labile sites in MNBCs were not reduced by applying the standard assay protocol as compared with the laboratory's own protocol. There was large inter-laboratory variation in FPG-sensitive sites by the laboratory-specific protocol and the variation was reduced when the samples were analysed by the standard protocol. The SBs and FPG-sensitive sites were measured in the same experiment, indicating that the large spread in the latter lesions was the main reason for the reduced inter-laboratory variation. However, it remains worrying that half of the participating laboratories obtained poor results using the standard procedure. This study indicates that future comet assay validation trials should take steps to evaluate the implementation of standard procedures in participating laboratories.

Background: Okadaic acid (OA), a toxin produced by several dinoflagellate species is responsible for frequent food poisonings associated to shellfish consumption. Although several studies have documented the OA effects on different processes such as cell transformation, apoptosis, DNA repair or embryogenesis, the molecular mechanistic basis for these and other effects is not completely understood and the number of controversial data on OA is increasing in the literature., Results: In this study, we used suppression subtractive hybridization in SHSY5Y cells to identify genes that are differentially expressed after OA exposure for different times (3, 24 and 48 h). A total of 247 subtracted clones which shared high homology with known genes were isolated. Among these, 5 specific genes associated with cytoskeleton and neurotransmission processes (NEFM, TUBB, SEPT7, SYT4 and NPY) were selected to confirm their expression levels by real-time PCR. Significant down-regulation of these genes was obtained at the short term (3 and 24 h OA exposure), excepting for NEFM, but their expression was similar to the controls at 48 h., Conclusions: From all the obtained genes, 114 genes were up-regulated and 133 were down-regulated. Based on the NCBI GenBank and Gene Ontology databases, most of these genes are involved in relevant cell functions such as metabolism, transport, translation, signal transduction and cell cycle. After quantitative PCR analysis, the observed underexpression of the selected genes could underlie the previously reported OA-induced cytoskeleton disruption, neurotransmission alterations and in vivo neurotoxic effects. The basal expression levels obtained at 48 h suggested that surviving cells were able to recover from OA-caused gene expression alterations.