Browsing by Author "Pires, Joana"
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- Comparative Analysis of the Toxicity Profile of Eleven Consumer-Relevant Nanomaterials in Human Intestinal and Placental Barrier CellsPublication . Pires, Joana; Moreira, Luciana; Teixeira, João Paulo; Fraga, SóniaBackground: The growing number of items incorporating nanomaterials (NM) has prompted considerable concerns about human health and safety [1]. Metal nanoparticles, inorganic non-metallic, and carbon-based NM are among the types with the highest market volume [2]. Objective: The purpose of this study was to determine the effect of chemical composition [Ag, Au, TiO2, SiO2, and graphene oxide (nano_GO)], primary size (10, 30 and 60 nm AgNP and AuNP), crystal structure (TiO2NP rutile/anatase and anatase), and surface coating (citrate and PEGylated AuNP) on potential toxicity to human intestinal (Caco-2) and placental (BeWo b30) epithelial cells. Methods: Changes in cell morphology, metabolic activity, plasma membrane integrity, intracellular ROS and ATP levels, and DNA integrity were assessed to investigate their potential toxicity at 24 h after exposure. Results: In both barrier models, the toxicity profile was similar, however placentalwere more sensitive than intestinal epithelial cells. Overall, NM may be ranked for cytotoxicity as AgNP > nano_GO > AuNP ~ TiO2NP ~ SiO2NP, with the effects becoming more evident at greater concentrations. The influence of size was more pronounced for AgNP than for AuNP, with the smaller nanoparticles producing higher cytotoxic effects. The cytotoxicity of AuNP was prevented by PEG capping. AgNP and nano_GO exposure markedly raised the levels of ROS, indicating that oxidative stress may play a role in their cytotoxicity. Except for 10 nm AuNP, every NM tested markedly increased intracellular ATP levels. One interesting finding was that a higher cytotoxic potential did not necessarily equate to a higher genotoxic potential, since only AgNP (classified as positive) and anatase TiO2NP (classified as equivocal) caused DNA damage. Conclusions: Our findings alert to the potential risks associated with human barriers exposure to NM, where the physicochemical properties are important determinants of their toxicity. Additional research is needed for a deeper understanding of NM impact on human barriers.
- Cumulus cell DNA damage linked to fertilization success in females with an ovulatory dysfunction phenotypePublication . Rodrigues, Bárbara; Sousa, Vanessa; Esteves, Filipa; Vale-Fernandes, Emídio; Costa, Solange; Sousa, Daniela; Brandão, Raquel; Leal, Carla; Pires, Joana; Gaivão, Isabel; Teixeira, João Paulo; Nogueira, António J.A.; Jorge, PaulaIntracytoplasmic sperm injection (ICSI) is a widely used technique in fertility centers. ICSI success depends on both nuclear and cytoplasmic oocyte maturation. Cumulus cells, which surround the oocytes, play a pivotal role in oocyte competence. However, the significance of DNA damage in cumulus cells as a marker of fertilization success remains largely unexplored. This study aims to investigate the relationship between DNA damage in cumulus cells of females undergoing ICSI, and oocyte competence, with a focus on in vitro fertilization (IVF) outcomes. We employed the alkaline comet assay to assess DNA damage levels (%TDNA) in cumulus cells and whole blood from 22 potentially fertile females and 35 infertile females, including 20 with an ovulatory disfunction phenotype. Our results revealed significant differences between the levels of %TDNA in cumulus cells and blood. Females with an ovulatory dysfunction phenotype exhibited higher levels of %TDNA in cumulus cells compared to potentially fertile females. Additionally, within the group of females with ovulatory dysfunction, a significant correlation was observed between %TDNA levels and the number of oocytes with two pronuclei. Our findings suggest that blood does not accurately reflect DNA damage in cumulus cells, which was correlated with the fertilization success in females with ovulatory dysfunction. High levels of %TDNA in cumulus cells were associated with a higher likelihood of successful fertilization. Moreover, our results imply that low levels of %TDNA may be linked to oocytes that fail to complete maturation and, consequently, do not fertilize (oocytes with zero pronuclei). Further research with larger cohorts is necessary to validate these findings and to explore potential applications in female fertility. However, our study provides evidence that DNA damage in cumulus cells may serve as a valuable biomarker for predicting fertilization success and oocyte competence.
- Cumulus cells damage can help to indirectly predict oocyte quality in infertile females undergoing ICSIPublication . Rodrigues, Bárbara; Sousa, Vanessa; Esteves, Filipa; Pires, Joana; Sousa, Daniela; Brandão, Raquel; Leal, Carla; Santos, Rosário; Vale-Fernandes, Emídio; Nogueira, António; Costa, Solange; Jorge, PaulaIntroduction: Intracytoplasmic sperm injection (ICSI) is currently used in clinical practice for couples with fertility issues. Some studies have shown an association between male reproductive ability and sperm DNA damage levels, assessed by comet assay. However, little is known regarding this endpoint and female fertility, mostly due to tissue accessibility. To overcome this, we used cumulus cells (CC) to analyze DNA damage in search of correlation with clinical parameters evaluated in the context of infertility. Methodology: DNA damage was assessed via comet assay, in two different tissues, blood and CC, from females undergoing ICSI: 22 potentially fertile and 35 infertile. DNA damage levels (%TDNA) were compared between the two groups (fertile vs infertile), and correlated, within each group, with hormone levels, stimulation days, number of cumulus-oocyte complexes (COCs) retrieved, and oocytes injected. All analysis were performed using SigmaPlot version 14.0 (Systat Software®Inc., Chicago, IL, United States). Results: No significant differences were found in %TDNA levels between the 2 groups. However, the DNA damage observed in CC was notably increased in the infertile females when compared to the potentially fertile, although it did not reach statistical significance. Interestingly, %TDNA in CC was significantly correlated with the number of oocytes injected, in both groups. This finding was not observed in the blood. Moreover, the difference between number of COCs retrieved and oocytes injected was significantly higher in the infertile females group and showed a correlation with the damage observed in CC. Discussion: Our results established a correlation between DNA damage in CC and oocyte quality. CC support and nurture oocytes during development, but DNA damage in CC can predict a reduced oocyte quality and availability for injection. This finding underscores the importance of CC in oocyte development and emphasizes the need to consider tissue-specific effects in DNA damage studies, particularly those related to fertility and reproductive health. Nevertheless, further studies are needed to confirm our results.
- From forests to health impacts: integrating human biomonitoring on wildfire researchPublication . Esteves, Filipa; Madureira, Joana; Pires, Joana; Teixeira, João Paulo; Costa, SolangeAbout the impact of Wildfires, that can cause significant impacts on the environment, economy, and people.
- How can exposure to engineered nanomaterials influence our epigenetic code? A review of the mechanisms and molecular targetsPublication . Moreira, Luciana; Costa, Carla; Pires, Joana; Teixeira, João Paulo; Fraga, SóniaEvidence suggests that engineered nanomaterials (ENM) can induce epigenetic modifications. In this review, we provide an overview of the epigenetic modulation of gene expression induced by ENM used in a variety of applications: titanium dioxide (TiO2), silver (Ag), gold (Au), silica (SiO2) nanoparticles and carbon-based nanomaterials (CNM). Exposure to these ENM can trigger alterations in cell patterns of DNA methylation, posttranscriptional histone modifications and expression of non-coding RNA. Such effects are dependent on ENM dose and physicochemical properties including size, shape and surface chemistry, as well as on the cell/organism sensitivity. The genes affected are mostly involved in the regulation of the epigenetic machinery itself, as well as in apoptosis, cell cycle, DNA repair and inflammation related pathways, whose long-term alterations might lead to the onset or progression of certain pathologies. In addition, some DNA methylation patterns may be retained as a form of epigenetic memory. Prenatal exposure to ENM may impair the normal development of the offspring by transplacental effects and/or putative transmission of epimutations in imprinting genes. Thus, understanding the impact of ENM on the epigenome is of paramount importance and epigenetic evaluation must be considered when assessing the risk of ENM to human health.
- Microbiological and chemical quality of ice used to preserve fish in Lisbon marketplacesPublication . Teixeira, Pedro; Brandão, João; Silva, Susana; Babič, Monika Novak; Gunde-Cimerman, Nina; Pires, Joana; Costa, Sílvia; Valério, ElisabeteIce is widely used to preserve fish on markets, playing a major role in the food industry. If manufactured, stored or distributed in inadequate sanitary conditions, it can represent a considerable health risk for both consumers and professionals. The present study characterizes ice used in marketplaces, on microbiological and chemical parameters. The aim was to assess potential risks on occupational exposure and consumer safety and therewith plan orientation guidelines. Sampling took place in 18 marketplaces in Lisbon, with ice samples collected at three different stages— production (n = 29), storage (n = 30), and product-contact (n = 29). No significant contamination was found in production and storage stages, demonstrating that ice production and storage procedures are adequate regarding sanitary conditions. With exception for Salmonella, significantly higher concentrations of microbial parameters were identified in product-contact stages, specifically Total Coliforms (548 MPN/100 mL), Escherichia coli (1 MPN/100 mL), Enterococci (29 MPN/100 mL), Staphylococcus aureus (271 CFU/100 mL), HPC at 5 , 22 , and 37 (>301 CFU/mL). Our study also shows that the majority of samples from ice production and storage comply with national regulations regarding drinking water quality. Yet, occupational health and consumer risks may still exist at the final product-contact stage, as significant contamination was detected.. Practical applications The present study can be used as a model for ice quality monitoring in fish preservation, serving as a tool for quality control and fast contamination detection during the several process stages. This monitoring model is an important contribution for assuring the safety of the preserved products, as well as, occupational health improvement and consumer risk protection.
- Multiparametric In Vitro Toxicity Assessment of Consumer-relevant Nanomaterials in a Human Placental ModelPublication . Pires, Joana; Moreira, Luciana; Teixeira, João Paulo; Fraga, SóniaThe increasing number of consumer products containing nanomaterials (NM) has raised serious concerns regarding their environmental and human safety. Among the categories of NM of large market volume are metal nanoparticles (M-NP), inorganic non-metallic and carbon-based NM [1]. So far, little is known about the impact of NM exposure on placental development and function. Thus, our study aimed at evaluating the in vitro toxicity of NM of consumer relevance in human trophoblastic BeWo clone b30 epithelial cells, a widely used human placental model. BeWo b30 cells were exposed for 24 h to varied concentrations (0.8-48 µg/cm2) of NM of different chemical composition [Au, Ag, TiO2, SiO2 and graphene oxide (nano_GO)], primary size (10, 30 and 60 nm Au- and AgNP), capping (citrate- and PEGylated AuNP) and crystal structure (TiO2 NP rutile and anatase forms). In vitro cytotoxicity was assessed by determining changes in cell morphology (optical microscopy), metabolic activity (AlamarBlue assay), plasma membrane integrity (LDH release) and intracellular reactive oxygen species (ROS; DCFH-DA assay) and ATP levels (luminescent assay). In vitro genotoxicity (DNA strand breaks and oxidative damage) was assessed by the comet assay. No evident changes in cell morphology were observed after exposure to any tested NM. Overall, NM can be ranked for cytotoxicity as AgNP > nano_GO > AuNP ~TiO2 NP ~ SiO2 NP, being the effects more visible at higher concentrations. Regarding M-NP, the influence of the size in the cytotoxic-induced effects was more evident for AgNP than for AuNP, with the smaller NP causing more cytotoxicity in BeWo b30 cells. PEG capping was an effective protection, preventing the cytotoxic effects that were visible in cells exposed to the tested citrate-capped AuNP. No significant differences between rutile-anatase and anatase TiO2 NP-induced cytotoxicity were observed. Exposure to AgNP and nano_GO significantly increased ROS levels of the exposed cells suggesting that oxidative stress is a possible mechanism underlying their cytotoxicity in BeWo b30 cells. All tested NM significantly increased intracellular ATP levels compared to control cells, except for 10 nm AuNP. No significant changes in both DNA strand-breaks levels and DNA oxidative damage were detected for all tested NM. Our findings highlight the potential hazard associated with human placental exposure to NM, where the physicochemical properties are important determinants of their toxicity. Additional research is needed for a deeper understanding of NM impact on human placental barrier, to unravel their mechanisms of action and the properties responsible for NM-mediated toxicity, and thus support regulatory decisions that protect consumers and ultimately assist in the development of safer NM.
- Occupational exposure to wildland firefighting and its effects on systemic DNA damagePublication . Esteves, Filipa; Madureira, Joana; Costa, Carla; Pires, Joana; Barros, Bela; Alves, Sara; Vaz, Josiana; Oliveira, Marta; Slezakova, Klara; Fernandes, Adília; Pereira, Maria do Carmo; Morais, Simone; Valdiglesias, Vanessa; Bonassi, Stefano; Teixeira, João Paulo; Costa, SolangeBackground: Portugal is among the European Union countries more devastated by forest fires. Wildland firefighters are at the forefront of this battle, facing exposure to a wide range of harmful pollutants. Epidemiological studies have highlighted a potential link between occupational firefighting exposure and several diseases, including cancer. To date, very few studies have explored the biological mechanisms associated with such exposure. The present longitudinal study aims to assess changes in early effect biomarkers following wildland firefighters’ occupational exposure to a real wildfire event. Methods: Paired blood samples from 59 healthy Portuguese wildland firefighters were collected at two different time points: before wildfire season and after a fire event during wildfire season. Sociodemographic variables (e.g., age, sex) and work-related factors (e.g., years of service) were assessed via a self-reported questionnaire. Levels of early effect biomarkers, such as primary DNA damage and oxidative DNA damage (oxidised purines) were assessed via comet assay. DNA double-strand breaks (DSBs) were evaluated by phosphorylated H2AX (γH2AX). Moreover, hydroxylated polycyclic aromatic hydrocarbon metabolites (OHPAHs) and metal(loid)s were quantified in urine samples. The influence of urinary OHPAHs, urinary metal(loid)s, and other exposure-related factors (e.g., firefighting duration) on changes (Δ) in early effect biomarkers (post-vs. baseline levels) was investigated. Results: Firefighting activities led to a significant increase in both primary DNA damage and oxidative DNA damage by 22 % (95 % CI: 1.11–1.35; p < 0.05) and 23 % (95 % CI: 1.04–1.45; p < 0.05), respectively. Results from linear regression revealed that per each unit increase of urinary 2-hydroxyfluorene (2-OHFlu) (μmol/mol creatinine), the risk of ⧍ oxidative DNA damage increased by 20 % [FR: 1.20 (1.09–1.32); p < 0.01]. Additionally, each unit increase in urinary cesium (Cs) (μg/L) resulted in a significant 4 % increase in Δ primary DNA damage [FR: 1.04 (1.01–1.06); p < 0.05] and a 3 % increase in Δ oxidative DNA damage [FR: 1.03 (1.01–1.05); p < 0.05]. Post-exposure levels of γH2AX were significantly correlated with urinary 2-OHFlu levels assessed after firefighting (r = 0.30; p < 0.05). Furthermore, exposure duration and reported breathing difficulties during firefighting were significantly associated with increased levels of primary DNA damage. Conclusion: Results obtained provide insights into the potential human health effects of wildland firefighting occupational exposure at the genetic and molecular levels, offering new and important mechanistic data. These findings are crucial for implementing health and safety measures, recommendations, and best practices to mitigate occupational risks and protect the health of wildland firefighters.
- Portuguese wildland firefighters: assessing exposure and cytogenetic effects in non-fire settings during the pre-fire seasonPublication . Pires, Joana; Esteves, Filipa; Slezakova, Klara; Madureira, Joana; Vaz, Josiana; Fernandes, Adília; Pereira, Maria do Carmo; Morais, Simone; Teixeira, João Paulo; Costa, SolangeIARC has recently listed occupational exposure as a firefighter carcinogenic to humans. However, firefighter is among the least studied occupations. The goal of the present study was to assess the level of cytogenetic damage in buccal mucosa (first-contact exposure tissue) of Portuguese firefighters during the pre- wildland fire season, as well as, to evaluate firefighters ́ exposure to particulate matter (PM) on fire stations as a baseline for occupational exposure characterization. A group of 172 Portuguese wildland firefighters were enrolled in the study, relevant information was obtained through a questionnaire. The frequency of micronucleus and other endpoints were evaluated via buccal micronucleus cytome assay (BMCyt) along with the influence of potential risk factors (e.g., smoking habits). Estimated inhalation doses of PM10 and PM2.5 (indoor/outdoor) were estimated for a group of 80 firefighters. No significant associations were found between estimated inhaled doses and BMCyt endpoints. Some lifestyle/behavioural variables were shown to significantly decrease the levels of cytogenetic endpoints, whereas others were found to increase them. Firefighters of the Permanent Intervention Team also shown to have significantly higher levels of BMCyt endpoints compared to other firefighters. Fire seasons are getting longer and dangerous, and this trend will continue in a progressive warming world. Implementing effective mitigation strategies for firefighters’ occupational exposure is an urgent public health issue.
- Protecting Wildland Firefighters’ Health: saving the lives of those who fight to save usPublication . Esteves, Filipa; Madureira, Joana; Pires, Joana; Teixeira, João Paulo; Costa, SolangeClimate change and global warming have contributed to the rise of forest fires across the globe. The proximity of firefighters to wildfires exposes them to a complex mixture of pollutants. In June 2022, occupational exposure as a firefighter was classified as “carcinogenic to humans” by the International Agency for Research on Cancer. However, up to date, very few studies have been focused on the relationship between wildland firefighting occupational exposure and health outcomes. Bio4Fox study aims to characterize wildland firefighters’ exposure in a pre- and during a wildfire season to identify a set of appropriate (bio)markers for the surveillance of wildland firefighters’ health. We aim to enroll around 200 northern Portuguese wildland firefighters. Here we present some preliminary data characterizing the exposure of these firefighters in the fire stations before the wildfire season. Around 172 northern Portuguese wildland firefighters (141 males and 31 females; mean age of 37.5± 10.9) recruited before the wildfire season of 2021 were enrolled in this analysis. Information on sociodemographic data, lifestyle and occupational exposure was obtained via a comprehensive questionnaire. Genetic instability was assessed in buccal cells through the Buccal Mucosa Cytome (BMCyt) assay. Lower frequencies of micronuclei and pycnotic cells (cell death biomarker) were observed among firefighters taking vitamin supplements and consuming vegetables daily, respectively (p<0.05). Being part of Permanent Intervention Teams (full-time firefighters) or part of the Command team, contributed to higher frequencies of cells with condensed chromatin (cell death marker) and karyolitic cells, respectively (p<0.05). Our findings will furnish a better characterization of Portuguese wildland firefighters in a pre- wildfire season. We expect to contribute to the implementation of health and safety measures highly needed in this sector.