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The comet assay is a versatile method to detect nuclear DNA damage in individual eukaryotic cells, from yeast to human. The types of damage detected encompass DNA strand breaks and alkali-labile sites (e.g., apurinic/apyrimidinic sites), alkylated and oxidized nucleobases, DNA-DNA crosslinks, UV-induced cyclobutane pyrimidine dimers and some chemically induced DNA adducts. Depending on the specimen type, there are important modifications to the comet assay protocol to avoid the formation of additional DNA damage during the processing of samples and to ensure sufficient sensitivity to detect differences in damage levels between sample groups. Various applications of the comet assay have been validated by research groups in academia, industry and regulatory agencies, and its strengths are highlighted by the adoption of the comet assay as an in vivo test for genotoxicity in animal organs by the Organisation for Economic Co-operation and Development. The present document includes a series of consensus protocols that describe the application of the comet assay to a wide variety of cell types, species and types of DNA damage, thereby demonstrating its versatility.The comet assay is commonly used to assess DNA damage. This collection of consensus protocols includes adaptations for a wide range of species and sample types, assay formats and detection of different types of DNA lesions.

[Abstract] Several studies have been conducted to address the potential adverse health risks attributed to exposure to nanoscale materials. While in vivo studies are fundamental for identifying the relationship between dose and occurrence of adverse effects, in vitro model systems provide important information regarding the mechanism(s) of action at the molecular level. With a special focus on exposure to inhaled (nano)particulate material toxicity assessment, this review provides an overview of the available human respiratory models and exposure systems for in vitro testing, advantages, limitations, and existing investigations using models of different complexity. A brief overview of the human respiratory system, pathway and fate of inhaled (nano)particles is also presented. The work was supported by the Fundação para a Ciência e a Tecnologia [POCI-01-0145-FEDER-029651, SFRH/BPD/122112/2016,PTDC/MED‐TOX/31162/2017, DL-57/INSA-06/2018,SFRH/BD/101060/2014,SFRH/BD/120646/2016,SIINN/0004/2014,UIDB/04750/2020, LA/P/0064/2020]; Spanish Ministry of Education, Culture and Sport [BEAGAL18/00142]; Spanish Ministry of Science and Innovation [PID2020-114908GA-I00] Portugal. Fundação para a Ciência e a Tecnologia; POCI-01-0145-FEDER-029651 Portugal. Fundação para a Ciência e a Tecnologia; SFRH/BPD/122112/2016 Portugal. Fundação para a Ciência e a Tecnologia; PTDC/MED‐TOX/31162/2017 Portugal. Fundação para a Ciência e a Tecnologia; DL-57/INSA-06/2018 Portugal. Fundação para a Ciência e a Tecnologia; SFRH/BD/101060/2014 Portugal. Fundação para a Ciência e a Tecnologia; SFRH/BD/120646/2016 Portugal. Fundação para a Ciência e a Tecnologia; SIINN/0004/2014 Portugal. Fundação para a Ciência e a Tecnologia; UIDB/04750/2020 Portugal. Fundação para a Ciência e a Tecnologia; LA/P/0064/2020

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 one 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 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) indicates 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.

[Abstract] Introduction: Immunosenescence and inflammaging have been implicated in the pathophysiology of frailty. Torquetenovirus (TTV), a single-stranded DNA anellovirus, the major component of the human blood virome, shows an increased replication rate with advancing age. An elevated TTV viremia has been associated with an impaired immune function and an increased risk of mortality in the older population. The objective of this study was to analyze the relation between TTV viremia, physical frailty, and cognitive impairment. Methods: TTV viremia was measured in 1,131 nonfrail, 45 physically frail, and 113 cognitively impaired older adults recruited in the MARK-AGE study (overall mean age 64.7 ± 5.9 years), and then the results were checked in two other independent cohorts from Spain and Portugal, including 126 frail, 252 prefrail, and 141 nonfrail individuals (overall mean age: 77.5 ± 8.3 years). Results: TTV viremia ≥4log was associated with physical frailty (OR: 4.69; 95% CI: 2.06-10.67, p < 0.0001) and cognitive impairment (OR: 3.49, 95% CI: 2.14-5.69, p < 0.0001) in the MARK-AGE population. The association between TTV DNA load and frailty status was confirmed in the Spanish cohort, while a slight association with cognitive impairment was observed (OR: 1.33; 95% CI: 1.000-1.773), only in the unadjusted model. No association between TTV load and frailty or cognitive impairment was found in the Portuguese sample, although a negative association between TTV viremia and MMSE score was observed in Spanish and Portuguese females. Conclusions: These findings demonstrate an association between TTV viremia and physical frailty, while the association with cognitive impairment was observed only in the younger population from the MARK-AGE study. Further research is necessary to clarify TTV's clinical relevance in the onset and progression of frailty and cognitive decline in older individuals. Ministerio de Ciencia e Innovación (España); PID2020-113788RB-I00 Xunta de Galicia; ED431B 2022/16 Xunta de Galicia; ED431F 2017/09

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. Highlights: Objective: to validate the use of salivary leucocytes in the challenge-comet assay; DNA damage of different nature was repaired over the evaluated time points; Results demonstrated that salivary leucocytes were as suitable as PBMC for this assay; Results were similar in fresh and after 5 months of cryopreservation; Recommendations are given depending on the type of DNA repair to be assessed. This work was funded by the Spanish Ministry of Science and Innovation: MCIN/AEI/10.13039/501100011033 (Grants PID2020- 113788RB-I00 and PID2020-114908 GA-I00), NanoBioBarriers project (PTDC/MED-TOX/31162/2017), Xunta de Galicia (ED431B 2022/16), co-financed by the Operational Program for Competitiveness and Internationalization (POCI) through European Regional Development Funds (FEDER/FNR), Spanish Ministry of Education, Culture and Sport [BEAGAL18/00142 to V.V.], and Spanish Ministry of Economy and Competitiveness, co-financed by the European Social Fund [RYC-2015- 18394 to L.L,-L,]. Funding info:eu-repo/semantics/publishedVersion

Recent studies exploring the relationship between DNA damage measured by the comet assay (single-cell gel electrophoresis) and cognitive function in both animal models and humans are reviewed and summarized. This manuscript provides an overview of studies exploring cognitive dysfunction related to DNA damage due to biological ageing process, cancer treatment, adverse environmental or occupational exposures, and prenatal genotoxic exposure. The review confirms the potential of comet assay to further explore the link between DNA damage, as indicative of genomic instability, and cognitive impairment in different research and clinical areas. Analysed studies support, in fact, the significant relationship between DNA damage and cognitive impairment, mainly affecting attention, working memory and executive functions. These cognitive domains are crucial to daily functioning and occupational performance, with important clinical implications. Although evidence support the relationship between DNA damage measured by the comet assay and cognitive function in different settings, further longitudinal research is needed to disentangle the temporal relationship between them over time, and to explore the potential of comet assay-detected DNA lesions to predict response to interventions. Highlights: Review on the link between DNA damage measured by the comet-assay and cognition; Ageing, cancer treatment, hazardous exposures and prenatal DNA damage are included; Studies support association affecting attention, memory and executive functions; Further longitudinal research will help to prevent and delay cognitive decline. This work was funded by the Spanish Ministry of Science and Innovation: MCIN/AEI/10.13039/501100011033 [Grant PID2020- 113788RB-I00], Xunta de Galicia (ED431B 2022/16, ED431F 2017/09), Ministry of Education, Culture and Sport [BEAGAL18/00142 to V.V.], and Ministry of Economy and Competitiveness, co-financed by the European Social Fund [RYC-2015–18394 to L.L-L]. Funding for open access charge: Universidade da Coruna/CISUG. info:eu-repo/semantics/publishedVersion

Peripheral blood leucocytes (PBL) have been traditionally used to investigate DNA damage by the comet assay in population studies, but validating alternative non-invasive samples would expand the application of this assay in human biomonitoring. The objectives of this study were (i) to test the validity of salivary leucocytes as a proper biomatrix for the comet assay, (ii) to evaluate the ability of this approach to detect different types of primary and oxidative DNA damage, and (iii) to determine whether frozen salivary leucocytes are still suitable for displaying those types of DNA damage. Fresh and frozen leucocytes isolated from saliva samples (six healthy non-smoking volunteers), were exposed to four genotoxic agents inducing different types of DNA damage, both primary (methyl methanesulfonate, actinomycin-D, ultraviolet radiation) and oxidative (potassium bromate), and standard or enzyme-modified comet assay was conducted. Results were compared with those obtained from PBL. Cells exposed to the four genotoxic agents showed dose-dependent increases of primary and oxidative DNA damage, demonstrating the suitability of all these samples to detect genetic damage from different origin. When comparing baseline levels of DNA damage, just a slight significant increase in primary DNA damage was observed in frozen salivary leucocytes regarding the other biomatrices, but similar results were obtained regarding sensitivity to DNA damage induction by all agents tested. This study demonstrates that salivary leucocytes can be employed in comet assay as an alternative or complement to blood samples. Frozen salivary leucocytes were proved to be a very convenient sample in large biomonitoring studies.

Part of the book series: Advances in Experimental Medicine and Biology (AEMB, volume 1357) Iron oxide nanoparticles (ION), with unique magnetic properties, have attracted huge scientific attention for a wide variety of uses, mostly in the biomedical field, due to their high biocompatibility, ability to cross biological membranes, appropriate surface architecture and easy conjugation with targeting ligands. Their current applications include diagnostic imaging, cell labelling, site-directed drug delivery and anticancer hyperthermia therapy. The ION surface may be modified by coating with different materials, aiming to stabilize the nanoparticles in different environments, to allow biomolecule binding favouring surface attachments with several molecules, and to prolong the recognition time by the immune system. Although the potential benefits of ION are considerable, and more and more ION are being manufactured to meet the demands of the rapidly proliferating field of nanomedicine, there is an urgent need to define their toxicological profile in order to avoid any potential health risks associated with their exposure and to reach optimal benefits of their use. The purpose of this chapter is to de-scribe the current knowledge on the ION toxicological features, addressing their structure and physicochemical characteristics, main exposure pathways and toxicokinetic aspects, interaction with cells, and their toxic effects, with special attention to those at the cellular and molecular level. The present work was supported by Xunta de Galicia (reference ED431B 2019/02), Ministerio de Ciencia e Innovación (PID2020-114908GA-I00), TOXI-NANO (SEM2021-A1), NeuroiNPact (SEM2021-B2), NanoBioBarriers (PTDC/MED-TOX/31162/2017) funded by Operational Program for Competitiveness and Internationalisation through European Regional Development Funds (FEDER/FNR) and through national funds by the Portuguese Foundation for Science and Technology (FCT), and NanoLegaTox (PTDC/SAU-PUB/29651/2017) project co-financed by COMPETE 2020, Portugal 2020 and European Union, through FEDER. Vanessa Valdiglesias was supported by Ministerio de Educación, Cultura y Deporte (reference BEAGAL18/00142), and Fátima Brandão by the Portuguese Foundation for Science and Technology (SFRH/BD/101060/2014). Authors would also like to acknowledge COST Action CA17140 “Cancer nanomedicine – from the bench to the bedside (NANO2CLINIC)”. info:eu-repo/semantics/publishedVersion

Iron oxide nanoparticles (ION), with unique magnetic properties, have attracted huge scientific attention for a wide variety of uses, mostly in the biomedical field, due to their high biocompatibility, ability to cross biological membranes, appropriate surface architecture and easy conjugation with targeting ligands. Their current applications include diagnostic imaging, cell labelling, site-directed drug delivery and anticancer hyperthermia therapy. The ION surface may be modified by coating with different materials, aiming to stabilize the nanoparticles in different environments, to allow biomolecule binding favouring surface attachments with several molecules, and to prolong the recognition time by the immune system. Although the potential benefits of ION are considerable, and more and more ION are being manufactured to meet the demands of the rapidly proliferating field of nanomedicine, there is an urgent need to define their toxicological profile in order to avoid any potential health risks associated with their exposure and to reach optimal benefits of their use. The purpose of this chapter is to de-scribe the current knowledge on the ION toxicological features, addressing their structure and physicochemical characteristics, main exposure pathways and toxicokinetic aspects, interaction with cells, and their toxic effects, with special attention to those at the cellular and molecular level.

This study aimed to estimate chronic daily intake (CDI) and to predict the attributable lifetime cancer risk (LCR) and hazard index (HI) from concurrent exposure to four trihalomethanes (THMs; chloroform, bromodichloromethane, dibromochloromethane and bromoform), via multiple exposure routes (oral ingestion, dermal contact and inhalation), among 238 non-competitive attendees of 10 Portuguese public indoor swimming pools (SPs), using a probabilistic approach based on Monte Carlo simulations. Exposure parameters of study participants were collected via questionnaires and THMs levels in SPs water were determined according the respective normative standards. The CDI for total THMs calculated for male and female participants considering all routes was 7.52 and 8.97 mg/kg/day, respectively. SP attendees presented higher CDI through inhalation than via the other two exposure routes, and chloroform was the compound contributing the most to total THMs CDI. The risk analysis indicated that the total LCR and HI from the targeted THMs were higher than the negligible risk levels (1 × 10-6 and 1, respectively) in the scenarios examined (central tendency exposure and reasonable maximum exposure), and the health risk for females was slightly higher than for males. This study suggests that there are possible adverse health risks, thus, to protect pool attendees, adequate mitigation measures, and comprehensive regulatory guidelines on individual THMs concentrations are needed. Highlights: Chloroform was the main THM; inhalation route was found to be the most relevant; The carcinogenic risk exceeds U.S. EPA negligible level; The non-carcinogenic risk is several orders greater than the recommended value; Females are more susceptible to cancer and non-cancer risk from total THMs exposure. This work was supported by the Project NORTE-01-0145-FEDER 000010, Health, Comfort and Energy in the Built Environment (HEBE), co-financed by Programa Operacional Regional do Norte (NORTE2020), through the European Regional Development Fund and by the Foundation for Science and Technology – FCT (Portuguese Ministry of Science, Technology and Higher Education) under the project UIDB/04750/2020. Joana Madureira and Carla Costa are supported by FCT (SFRH/BPD/115112/2016 and SFRH/BPD/96196/2013 grants, respectively). Ri cardo Assunção was supported by FCT Individual CEEC 2018 Assistant Researcher Grant CEECIND/01570/2018. The authors are also grateful for the support from Xunta de Galicia [ED431B 2019/02], Ministerio de Educación, Cultura y Deporte [BEAGAL18/00142 to V.V.], Programa IACOBUS Estancias de Investigación 2021; and FCT/MCTES for the financial support to CESAM (UIDP/50017/2020+UIDB/50017/2020) through national funds. info:eu-repo/semantics/publishedVersion

The comet assay is a widely used test for the detection of DNA damage and repair activity. However, there are interlaboratory differences in reported levels of baseline and induced damage in the same experimental systems. These differences may be attributed to protocol differences, although it is difficult to identify the relevant conditions because detailed comet assay procedures are not always published. Here, we present a Consensus Statement for the Minimum Information for Reporting Comet Assay (MIRCA) providing recommendations for describing comet assay conditions and results. These recommendations differentiate between ‘desirable’ and ‘essential’ information: ‘essential’ information refers to the precise details that are necessary to assess the quality of the experimental work, whereas ‘desirable’ information relates to technical issues that might be encountered when repeating the experiments. Adherence to MIRCA recommendations should ensure that comet assay results can be easily interpreted and independently verified by other researchers., Here, members of the hCOMET COST Action program provide a consensus statement on the Minimum Information for Reporting Comet Assays (MIRCA).

Deficiencies in DNA damage response and repair (DDRR) can cause serious pathological outcomes; therefore, having an ability to determine individual DDRR would enhance specificities in health risk assessment and in determining individual's response to cancer therapies. However, most methods for evaluating DDRR are not fully appropriate for population studies. The Challenge-Comet assay has gained acceptance for this purpose. The assay has traditionally used X-rays as challenge agent and isolated peripheral blood mononuclear cells (PBMC) as cell specimen. To enhance the usefulness of the assay, the objectives of this investigation were to use differently processed blood samples, to employ other challenge agents with different mechanisms of induction of DNA damage/repair, and to generate protocols for detecting different DDRR capacities. Fresh and frozen blood samples were challenged with bleomycin, methyl methanesulfonate (MMS) and ultraviolet light. Significant induction of damage after all treatments, and progressive and time-dependent DDRR were observed. No significant differences were obtained in the DDRR capacities of fresh or frozen whole blood samples as compared to PBMC, except that fresh blood samples showed higher MMS-induced DDRR capacity than PBMC. Results from this study show that the Challenge-Comet assay can be used as routine biomarker of DDRR capacity in human biomonitoring studies, and that whole blood is also a useful biomatrix for this assay. The collected data allow us to recommend different protocols for the Challenge-Comet assay which are useful for evaluating DDRR capacities in several key DNA repair pathways. Consequently, the usefulness of the Challenge-Comet assay can be greatly expanded.

A pesar de los avances considerables en cuestiones de igualdad de génerodesde la incorporación de las mujeres a la ciencia, las Reales Academias y organizaciones científicas similaressiguen manteniendo una infrarrepresentación femenina entre sus miembros. Estudiar la brecha de género en las Reales Academias y en premios o distinciones que otorgan es fundamental para influir en las políticas científicas, establecer prioridades, crear conciencia y corregir los desequilibrios de género. La Real Academia Gallega de Ciencias (RAGC) fundada en 1977 incorporó a las primeras mujeres científicas en 2014. A día de hoy la RAGC cuenta con una representación de Académicas inferior al 20%, la cual no se corresponde con la presencia de científicas en las universidades y centros de investigación de Galicia. Las Medallas de Investigación, de nominación indirecta, presentan un 80% de hombres galardonados, y otros premios científicos otorgados por la RAGC, como los Premios Ernesto Vieitez Cortizo o los Premios de Transferencia, presentan porcentajes desde el 53% hasta el 100% de hombres galardonados. De forma análoga, en lo que respecta al reconocimiento de la divulgación y periodismo científico, se observa una representación masculina en los galardonados desde el 42% al 83%. En ambos, hay una tendencia de aumento en la brecha de género, tanto en las candidaturas presentadas como en premios concedidos, a medida que se avanza en la carrera con la consiguiente repercusión económica y social del premio. En ello pueden influir factores como: la falta de diversidad en la composición de comités de selección, que puede favorecer la prevalencia de sesgos, conscientes o inconscientes, el lenguaje sexista o la invisibilidad de científicas, por ejemplo en el nombre de los premios, ya que apenas 3 llevan el nombre de científicas o en las 14 ediciones del Día de la Ciencia, de las cuales apenas 2 se han dedicado a mujeres científicas. Este informe concluye con una serie de recomendaciones que pretenden promover acciones positivas con el objetivo de permitir un mayor reconocimiento social de las contribuciones de las mujeres científicas, fomentar una imagen más inclusiva de la RAGC e incentivar la presentación de mujeres candidatas en los distintos premios, tanto de forma individual como por la comunidad científica e instituciones que representan.

In this work four vanadium complexes (compounds 1, 2, 3 and 4) and one molybdenum complex (compound 5) with hydrazone ligands derived from pyridoxal were synthesized and characterized. All compounds are mononuclear species, two of them (compounds 3 and 5) are dioxide complexes and the other three (compounds 1, 2 and 4) monoxide complexes. The vanadium atom of the compound 3 is five-coordinated and all the other compounds have a six coordinated environment polyhedron. The poses for the potential intercalation of the compounds 2 and 3 with DNA were obtained by using AutoDock software. Optimizations were also performed at PM6-D3H4 semi-empirical level whereas the study of the nature of the interaction was carried out by means of the Energy Decomposition Analysis and the Non-Covalent Interaction index by using in both cases Density Functional Theory computations. The cytotoxicity in lung cancer cells (A549 cell line) of all the compounds was also evaluated. After 24 h of treatment, vanadium complexes showed high values of IC50, between 419.93 ± 22.58 and 685.88 ± 46.55 μM. After 48 h, the results showed that the compound 3 had the lowest IC50 value, 65.32 ± 9.95 μM, and the compound 2 the highest value, 375.28 ± 32.09 μM. The molybdenum complex showed the lowest IC50 value at 48 h (11.22 ± 1.34 μM). The toxicity of the compounds 3, 4 and 5 was tested in vivo, using zebrafish model, and the molybdenum complex showed higher toxic effects than the studied vanadium complexes., This research was financially supported by Fundação para a Ciência e a Tecnologia (FCT) by means of the projects PTDC/QUI-QFI/29236/2017, UIDB/04046/2020, and UIDP/04046/2020. V.V. thanks Ministerio de Educación, Cultura y Deporte for a Beatriz Galindo Award [BEAGAL18/00142]. A.G. is thankful to ARAID – Fundación Agencia Aragonesa para la Investigación y el Desarrollo for current funding in the frame of ARAID researchers. We gratefully acknowledge Xunta de Galicia for the collaboration agreement “Development of research strategic actions UDC I+D+i 2021-2022: CICA-Disrupting Projects 2021SEM-A3 [NanoCool]”.

Correction to: Scientific Reports https://doi.org/10.1038/s41598-021-95976-7, published online 18 August 2021

[Abstract] 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 TiO2 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 TiO2 NP cellular uptake and MN induction as evaluated by flow cytometry (FCMN) were also assessed. TiO2 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 TiO2 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. This research was funded by Ministerio de Ciencia e Innovación (PID2020-114908GA-I00), Xunta de Galicia (ED431B 2019/02); NanoBioBarriers (PTDC/MED-TOX/31162/2017) funded by Operational Program for Competitiveness and Internationalisation through European Regional Development Funds (FEDER/FNR) and through national funds by the Portuguese Foundation for Science and Technology (FCT); NanoLegaTox (PTDC/SAU-PUB/29651/2017) project co-financed by COMPETE 2020, Portugal 2020 and European Union, through FEDER; Ministerio de Educación, Cultura y Deporte (BEAGAL18/00142, to V.V.); FCT (SFRH/BD/101060/2014, to F.B.); and CA17140 NANO2CLINIC COST Action Xunta de Galicia; ED431B 2019/02 Portugal. Fundação para a Ciência e a Tecnologia; PTDC/MED-TOX/31162/2017 Portugal. Fundação para a Ciência e a Tecnologia; PTDC/SAU-PUB/29651/2017 Portugal. Fundação para a Ciência e a Tecnologia; SFRH/BD/101060/2014

[Abstract] 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 (TiO₂), zinc oxide (ZnO), and cerium dioxide (CeO₂) 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. This research was funded by Xunta de Galicia [ED431B 2019/02]; Ministerio de Ciencia e Innovación (PID2020-114908GA-I00); Ministerio de Educación, Cultura y Deporte [BEAGAL18/00142, to V.V.]; CA17140 NANO2CLINIC COST Action; NanoBioBarriers (PTDC/MED-TOX/31162/2017) funded by Operational Program for Competitiveness and Internationalisation through European Regional Development Funds (FEDER/FNR) and through national funds by the Portuguese Foundation for Science and Technology (FCT). NanoLegaTox (POCI-01-0145-FEDER-029651) funded by Operational Program for Competitiveness and Internationalisation (COMPETE 2020) through FEDER, and through national funds by the Portuguese Foundation for Science and Technology (FCT). A.T. Reis funded by FCT Grant SFRH/BPD/122112/2016 Xunta de Galicia; ED431B 2019/02 European Cooperation in Science and Technology; CA17140 Portugal. Fundação para a Ciência e a Tecnologia; PTDC/MED-TOX/31162/2017 Portugal. Fundação para a Ciência e a Tecnologia; POCI-01-0145-FEDER-029651 Portugal. Fundação para a Ciência e a Tecnologia; SFRH/BPD/122112/2016

Background The aim of this study was to investigate serum neopterin levels in patients with acute pancreatitis (AP) and its predictive value for mortality in cases of severe acute pancreatitis (SAP). Methods Eighty-two patients with confirmed acute pancreatitis (AP) were included and divided into two groups, mild acute pancreatitis (MAP, n=51) and severe acute pancreatitis (SAP, n=31) according to severity of disease. From the SAP group, 8 cases went on to develop multiple organ dysfunction syndrome (MODS) and 6 subsequently died. Thirty healthy subjects from routine medical examination were included as the control group. The neopterin serum concentrations were determined and compared between groups. Results Serum neopterin concentrations from control, MAP, SAP, SAP_Non-MODS, MODS_survival and MODS_death groups were 6.85±2.42 (nmol/L), 0.91±4.83(nmol/L), 33.11±11.67(nmol/L), 30.39±9.97(nmol/L), 36.40±4.48(nmol/L) and 41.75±15.64(nmol/L) respectively, with statistical significant difference (p Conclusion: Serum neopterin levels in patients with acute pancreatitis were significantly elevated and correlated with the severity of disease. Neopterin may also be used a serological biomarker of mortality risk in patients with SAP.

Levels of DNA damage represent the dynamics between damage formation and removal. Therefore, to better interpret human biomonitoring studies with DNA damage endpoints, an individual's ability to recognize and properly remove DNA damage should be characterized. Relatively few studies have included DNA repair as a biomarker and therefore, assembling and analyzing a pooled database of studies with data on base excision repair (BER) was one of the goals of hCOME T (EU-COST CA15132). A group of approximately 1911 individuals, was gathered from 8 laboratories which r u n population studies with the comet-based in vitro DNA repair assay. BER incision activity data were normalized and subsequently correlated with various host factors. BER was found to be significantly higher in women. Although it is generally accepted that age is inversely related to DNA repair , no overal l effect of age was found, but se x differences were most pronounced in the oldest quartile (> 61 years). No effect of smoking or occupational exposures was found. A body mass index (BMI) above 25 kg/m(2) was related to higher levels of BER. However, when BMI exceeded 35 kg/m(2), repair incision activity was significantly lower. Finally, higher BER incision activity was related to lower levels of DNA damage detected by the comet assay in combination with formamidopyrimidine DNA glycosylase (Fpg), which is in line with the fact that oxidatively damaged DNA is repaired by BER. These data indicate that BER plays a role in modulating the steady-state level of DNA damage that is detected in molecular epidemiological studies and should therefore be considered as a parallel endpoint in future studies.

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 TiO2 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 TiO2 NP cellular uptake and MN induction as evaluated by flow cytometry (FCMN) were also assessed. TiO2 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 TiO2 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.

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.

[Abstract] The alkaline comet assay, or single cell gel electrophoresis, is one of the most popular methods for assessing DNA damage in human population. One of the open issues concerning this assay is the identification of those factors that can explain the large inter-individual and inter-laboratory variation. International collaborative initiatives such as the hCOMET project - a COST Action launched in 2016 - represent a valuable tool to meet this challenge. The aims of hCOMET were to establish reference values for the level of DNA damage in humans, to investigate the effect of host factors, lifestyle and exposure to genotoxic agents, and to compare different sources of assay variability. A database of 19,320 subjects was generated, pooling data from 105 studies run by 44 laboratories in 26 countries between 1999 and 2019. A mixed random effect log-linear model, in parallel with a classic meta-analysis, was applied to take into account the extensive heterogeneity of data, due to descriptor, specimen and protocol variability. As a result of this analysis interquartile intervals of DNA strand breaks (which includes alkali-labile sites) were reported for tail intensity, tail length, and tail moment (comet assay descriptors). A small variation by age was reported in some datasets, suggesting higher DNA damage in oldest age-classes, while no effect could be shown for sex or smoking habit, although the lack of data on heavy smokers has still to be considered. Finally, highly significant differences in DNA damage were found for most exposures investigated in specific studies. In conclusion, these data, which confirm that DNA damage measured by the comet assay is an excellent biomarker of exposure in several conditions, may contribute to improving the quality of study design and to the standardization of results of the comet assay in human populations. This article is based upon work from COST Action hCOMET CA15132, supported by COST (European Cooperation in Science and Technology www.cost.eu) - STSM fellowships for Mirta Milić (IMROH, EU 19); IMROH, Zagreb, Croatia, Institute for Medical Research and Occupational Health (IMROH), Zagreb, Croatia, and the Ministry of Science, Education and Sports of the Republic of Croatia (Grant No. 022-0222148-2125) (EU4); Cancer Plan for PestiBG; Grant number: no ENV201401(EU 8, EU9); Italian Ministry of Education, University and Research PRIN 2005, prot. 2005058197 and Cariplo Foundation (Milan, Italy), Rif. Pratica 2007-5810 and Rif. Pratica 2010.2303 (EU 18); Associazione Italiana per la Ricerca sul Cancro (AIRC) (IG 2015/17564). (EU19); European Union Integrated Projects New Generis, 6th Framework Programme, Priority 5: Food Quality and Safety; Newborns and Genotoxic Exposure Risks, FOOD-CT-2005-016320 (EU22); ACT project No. 036APy/09 and No. 005DBB/12 (EU 24); FCT-SFRH/BPD/96196/2013, SFRH/BPD/100948/2014, Portugal (EU 26); MZ 2012/8-UKBA-8; VEGA 1/0703/13, APVV 15-0063 (EU30); Xunta de Galicia (XUGA 10605B98; INCITE08PXIB106155PR; ED481B2016/190-0; Grants ED431B2019/02), Spain (EU 32); Grant 01 173034, Ministry of Education, Science and Technological Development of the Republic of Serbia (EU 42); The Centre for Industrial and Technological Development within National Strategic Consortia for Techical Research (Industrial Research diets and food with specific characteristics for elderly, SENIFOOD); University of Navarra LE/97; Physiopathology of Obesity and Nutrition (CIBER Obn); Carlos III Health Research Institute (CB12/03/30002); Ministerio de Economia y Compatitividad (‘Ramón y Cajal’ Programme, RYC-2013-14370) of the Spanish Government for personal support (EU 45); the Ministry of Education, Youth and Sports of the Czech Republic project Healthy Aging in Industrial Environment HAIE (CZ.02.1.01/0.0/0.0/16_019/0000798) which is co-financed by the European Union (European Structural and Investment funds; Operation Programme Research, Development and Education); MYES LO 1508 (EU 46); MICRODIAB Study; ClinicalTrials.org (#NCT02231736) (EU 52); The study was funded by the Italian Ministry for Education, University and Scientific Research (MIUR) - Research No. 2005-062547 (EU14, EU53); Projects financed from Serbian Ministry of Education, Science and Technological Development #11146002, #175035, #173034 (EU 54); Mehr foundation organisation, UK (EU 55); MCTI/CNPQ No. 01/2016-Universal; FAPESC No. 09/2015; MEC/MCTI/CAPES/CNPQ/FAPS/ No. 09/2014, Brazil (CSA 6); the National Nuclear Energy Agency of Indonesia (Badan Tenaga Nuklir Nasional) with contract number 080.01.06 3447.001 001.052.A (AS4); Slovak Grant Agency (APVT-21 013202, APVT-21- 017704); Ministry of Health, Slovak Republic (2005/43-SZU-21, 2006/07- SZU-02 MZ SR, 2005/42-SZU-20

Phosphorylation of H2AX histone (γH2AX) represents an early event in the DNA damage response against double-strand breaks (DSB); hence, its measurement provides a surrogate biomarker of DSB. Recently, we reported initial steps in the standardization of γH2AX assay in peripheral blood leukocytes (PBL), addressing the possibility of using cryopreserved samples, and the need of phytohaemagglutinin (PHA) stimulation prior analysis (Toxicol Sci 2015, 144:406-13). Validating the use of whole blood samples as cell specimen for this assay would be particularly useful for human population studies. Hence, in the current study we determined for the first time the feasibility of whole blood samples, both fresh and frozen, to be used in the γH2AX assay, evaluated by flow cytometry, and the convenience of PHA stimulation. Freshly collected and cryopreserved whole blood samples were treated with bleomycin (BLM), actinomycin-D (Act-D) and mitomycin C (MMC); half of the samples were previously incubated with PHA. Results were compared with those from PBL. Negative responses in MMC treatments were probably due to the quiescence of unstimulated cells, or to the short treatment time in PHA stimulated cells. Fresh whole blood samples exhibited a more intense response to BLM and Act-D treatments in stimulated cells, probably due to DSB indirectly produced from other less relevant types of DNA damage. Results obtained in frozen whole blood samples indicate that PHA stimulation is not advisable. In conclusion, this study demonstrates that whole blood samples can be used to assess DSB-related genotoxicity by the flow cytometry γH2AX assay. This work was supported by Xunta de Galicia [ED431B 2019/02], Ministerio de Educación, Cultura y Deporte [BEAGAL18/00142 to V.V], and Deputación Provincial de A Coruña [to M.S.-F. and N.F.-B.].

Genome instability denotes an increased tendency to alterations in the genome during cell life cycle, driven by a large variety of endogenous and exogenous insults. Ageing is characterized by the presence of damage to various cellular constituents, but genome alterations, randomly accumulating with age in different tissues, constitute the key target in this process, and are believed to be the main factor of ageing. Age-related failure of DNA repair pathways allows DNA lesions to occur more frequently, and their accumulation over time contributes to the age-associated decrease in genome integrity in somatic cells. The micronucleus (MN) test is one of the most widely used assays to evaluate genomic instability in different surrogate tissues. A large number of studies has consistently shown a progressive increase in MN frequency with age, starting from very young age groups onwards. Therefore, MN frequency is a suitable biomarker of genomic instability in ageing. Frailty is a multidimensional geriatric syndrome of unsuccessful ageing, characterized by decreased biological reserves and increased vulnerability to external stressors, involving a higher risk of negative health outcomes. Although there is a well-founded belief that genome instability is involved in the frailty syndrome, only two studies investigated the relationship between MN frequency and frailty, not allowing to draw a definite conclusion on the utility of this biomarker for frailty detection. The use of MN and other genomic biomarkers in the detection and follow-up of patients affected by or at risk of frailty has the potential to accumulate evidence on the clinical impact of this approach in the identification and control of frailty in older people. This research was funded by Xunta de Galicia [grant number ED431B 2019/02], Ministerio de Educación, Cultura y Deporte [grant number BEAGAL18/00142 to V.V.], and Fundação para a Ciência e a Tecnologia [grant number SFRH/BPD/100948/2014 to S.C.].

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. This research was funded by Xunta de Galicia [ED431B 2019/02]; Ministerio de Educación, Cultura y Deporte [BEAGAL18/00142 to V.V, PRX19/00353 to B.L.]; and Deputación Provincial de A Coruña [to D.M.-P. and M.S.-F.]. info:eu-repo/semantics/publishedVersion

The main objective of the present work was to assess the cellular uptake and potential genotoxicity (micronuclei induction) of TiO2 NPs on four diverse human cell lines. unta de Galicia (ED431B 2019/02), Ministerio de Educación, Cultura y Deporte (BEAGAL18/00142 to V.), Deputación Provincial de A Coruña (to N.F.-B.), and FCT (subsidized by national fund of MCTES, SFRH/BD/101060/2014 [to F.B.]). Authors would also like to acknowledge COST Action CA15132 “The comet assay as a human biomonitoring tool (hCOMET)” N/A

Deficiencies in DNA damage response and repair (DDRR) can cause serious pathological outcomes; therefore, having an ability to determine individual DDRR would enhance specificities in health risk assessment and in determining individual's response to cancer therapies. However, most methods for evaluating DDRR are not fully appropriate for population studies. The Challenge-Comet assay has gained acceptance for this purpose. The assay has traditionally used X-rays as challenge agent and isolated peripheral blood mononuclear cells (PBMC) as cell specimen. To enhance the usefulness of the assay, the objectives of this investigation were to use differently processed blood samples, to employ other challenge agents with different mechanisms of induction of DNA damage/repair, and to generate protocols for detecting different DDRR capacities. Fresh and frozen blood samples were challenged with bleomycin, methyl methanesulfonate (MMS) and ultraviolet light. Significant induction of damage after all treatments, and progressive and time-dependent DDRR were observed. No significant differences were obtained in the DDRR capacities of fresh or frozen whole blood samples as compared to PBMC, except that fresh blood samples showed higher MMS-induced DDRR capacity than PBMC. Results from this study show that the Challenge-Comet assay can be used as routine biomarker of DDRR capacity in human biomonitoring studies, and that whole blood is also a useful biomatrix for this assay. The collected data allow us to recommend different protocols for the Challenge-Comet assay which are useful for evaluating DDRR capacities in several key DNA repair pathways. Consequently, the usefulness of the Challenge-Comet assay can be greatly expanded.

[Abstrac] Titanium dioxide nanoparticles (TiO2 NPs) have a wide variety of applications in many consumer products, including as food additives, increasing the concern about the possible hazards that TiO2 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, TiO2 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 TiO2 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 TiO2 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 TiO2 NPs toxic effects. The present work was supported by Xunta de Galicia (reference ED431B 2019/02), NanoBioBarriers (PTDC/MED-TOX/31162/2017) funded by Operational Program for Competitiveness and Internationalisation through European Regional Development Funds (FEDER/FNR) and through national funds by the Portuguese Foundation for Science and Technology (FCT) and NanoLegaTox (PTDC/SAU-PUB/29651/2017) project co-financed by COMPETE 2020, Portugal 2020 and European Union, through FEDER. F.B. are M.J.B. recipients of FCT fellowships (SFRH/BD/101060/2014 and SFRH/BD/12046/2016); V.V. is supported by a Beatriz Galindo Research Fellowship (reference BEAGAL18/00142); and NF-B by a Deputación Provincial da Coruña fellowship (2019000011550) Xunta de Galicia; ED431B 2019/02 Portugal. Fundação para a Ciência e a Tecnologia; PTDC/MED-TOX/31162/2017 Portugal. Fundação para a Ciência e a Tecnologia; PTDC/SAU-PUB/29651/2017 Portugal. Fundação para a Ciência e a Tecnologia; SFRH/BD/101060/2014 Portugal. Fundação para a Ciência e a Tecnologia; SFRH/BD/12046/2016 Deputación Provincial da Coruña; 2019000011550

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. Research funded by Xunta de Galicia (ED431B 2016/013) and by EU COST Action IS1402. V. Valdiglesias was supported by a Xunta de Galicia postdoctoral fellowship (reference ED481B 2016/190-0). D. Marcos-Pérez and M. Sánchez-Flores were supported by INDITEX-UDC fellowships. The work of S. Bonassi was supported by grants funded by the Associazione Italiana per la Ricerca sul Cancro (AIRC).

Frailty is an age-related syndrome expected to increase over the next decades. This syndrome has been identified to be the most common condition leading to disability, institutionalisation and death in the elderly. The aim of this pilot study is to investigate a possible link between frailty status, biomarkers and environmental exposures. A group of 71 older adults (≥65 years old) was engaged in this study. The study population was classified as 45.1% robust, 45.1% pre-frail and 9.8% frail. A significant higher prevalence of second-hand smokers was found in the pre-frail group when compared to robust. Furthermore, a higher prevalence of robust individuals was found among those consuming home-produced vegetables and water from well/springs. Significant differences were found between data collected in a lifetime exposure questionnaire (LTEQ) and the levels of genotoxicity endpoints and the mercury levels analysed regarding some exposure-related parameters, namely, smoking habits, intake of home-produced vegetables and the use of pesticides in agriculture. Understanding if the way we live(d) or worked can impact the way we age are important questions to be explored. Data obtained in this pilot study encourage further studies on this matter, exploring the role of exposures history and its impact on health. Highlights: Frailty is an age-related syndrome expected to increase over the next years; A prevalence of second-hand smokers was found in the pre-frail group; Home-produced vegetables consumers had lower %TDNA levels; Exposure-related factors should be included when studying disease aetiology; The way we live(d) or worked may have an impact on the way we age. Armanda Teixeira-Gomes and Solange Costa are supported by Fundação para a Ciência e Tecnologia (FCT-MCTES), and by the European Social Fund, through Programa Operacional Capital Humano (POCH), under the grants SFRH/BD/121802/2016 and SFRH/BPD/ 100948/2014, respectively; Vanessa Valdiglesias is supported by Beatriz Galindo Research FellowshipBEAGAL18/00142. The authors also acknowledge the contribution of the COST Action CA15132 “The comet assay as a human biomonitoring tool (hCOMET)” to this study info:eu-repo/semantics/publishedVersion

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. Highlights: Micronucleus frequency (MN) positively correlated with formaldehyde levels of exposure and exposure duration; Strong association of MN levels between lymphocytes and buccal cells; Significant alterations in the percentage of T cytotoxic lymphocytes, NK cells and B lymphocytes; Polymorphisms on CYP2E1, GSTP1 and FANCA genes were associated with the effect endpoints levels. Solange Costa, Carla Costa, Joana Madureira and Armanda Teixeira- Gomes are supported by Portuguese national funds, through Fundação para a Ciência e Tecnologia (FCT-MCTES), and by the European Social Fund, through Programa Operacional Capital Humano (POCH), respectively under the grants SFRH/BPD/100948/2014, SFRH/BPD/ 96196/2013, SFRH/BPD/115112/2016 and SFRH/BD/121802/2016. Vanessa Valdiglesias was supported by Xunta de Galicia (Spain) postdoctoral fellowship (reference ED481B 2016/190-0). info:eu-repo/semantics/publishedVersion