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- Analysis and evaluation of genotoxicity and carcinogenicity assessment in EU legislation to improve regulatory implementation of NAMs: A focus on in silico approachesPublication . Bossa, Cecília; Raitano, Giuseppa; Benfenati, Emilio; Alivernini, Silvia; Andreoli, Cristina; Aquilina, G.; Attias, L.; Dusinska, Maria; El Yamani, N.; Louro, Henriqueta; Marcon, Francesca; Rundèn-Pran, E.; Russo, Maria Teresa; Silva, Maria João; Battistelli, Chiara LauraGenotoxicity and carcinogenicity are key endpoints for the risk assessment of all types of substances. Research on alternatives to animal testing for these endpoints has been active for decades, leading to the development of short-term in vitro tests that are integrated into current testing strategies. Nevertheless, high relevance is still devoted to data from in vivo studies. In parallel, progress in the comprehension of mechanisms underpinning genotoxicity and genotoxic carcinogenicity processes, together with the analysis of the great wealth of experimental data produced, allowed the discovery of structural determinants utilized in quantitative and qualitative structure-activity relationships and enabling in silico predictions of these endpoints. Presented here is a case study part of the collective effort carried out within the European Partnership for the Assessment of Risks from Chemicals (PARC) to address the challenges associated with innovation in chemical risk assessment, including the phasing out of animal testing through the introduction of New Approach Methodologies (NAMs) [1,2]. The case study aims to analyze current practices of the regulatory evaluation of genotoxicity and carcinogenicity hazard in several EU frameworks, in order to highlight needs and challenges in the actual or potential use of NAMs as well as short-and long- term goals towards the overcoming of animal testing. Among other NAMs, we are focusing on the role of in silico approaches highlighting strategies to increase the regulatory application and acceptance of QSAR based approaches. To this aim, the OECD QSAR Assessment Framework [3,4] has been identified as a suitable tool for evaluating the models and their predictions and will be applied to selected case studies. Moreover, a list of human relevant carcinogens has been developed as reference chemicals to evaluate and possibly refine in silico methodologies supporting a human-centric paradigm shift in toxicology.
- BPA analogues affect intestinal barrier integrity and pro-inflammatory response in a coculture modelPublication . Pereira, Gonçalo; Barros, Patrícia; Matos, Paulo; Jordan, PeterBackground: Bisphenol (BP) A and its structural analogues are environmental pollutants with endocrine-disrupting properties and potential immune-modulating effects. Following legal restrictions on the use of BPA, structurally related compounds including BPAP, BPP, and BPS-MAE have been introduced as alternatives; however, their potential hazardous profiles remain largely unknown. Here we used a coculture cell model to investigate the effects of exposure to these BP analogues on intestinal barrier integrity, intestinal cell stress, and pro-inflammatory macrophage activation. Methods: As cellular model, Caco-2 cells were grown on filter membranes to a polarized epithelium-like layer, and cocultured with underlying basolateral THP-1 derived M0 macrophages. After apical exposure of the Caco-2 cells layer to BPs (0.1 -100 µM for 24 h), we determined transepithelial electrical resistance (TEER), polarized cell morphology by confocal microscopy, cellular stress markers (p-p38MAPK, p-JNK, p-eIF2α) by Western blot, and macrophage activation by IL-1β-transcript expression changes. In addition, M0-type macrophages were also directly incubated with BPA for comparison. Results: When M0 macrophages were directly exposed, BPA triggered IL-1β expression, an effect more evident after macrophage sensitization by the presence of interferon-γ. Apical exposure to BPA and BPS-MAE had little effect on TEER but induced some increase in epithelial stress markers, while BPAP and especially BPP clearly reduced TEER and polarized cell morphology, and showed a tendency to induce stress markers. In addition, apical cell exposure to BPP and BPS-MAE triggered clear expression of the pro-inflammatory marker IL-1β in sensitized M0 macrophages cocultured at the basolateral side, whereas BPAP and BPA were only effective at the highest concentration. Conclusion: Together, our data show that exposure of an intestinal epithelium-like layer to BPAP and BPS-MAE revealed adverse cellular effects similar to BPA, while BPP was clearly the most deleterious BP analogue. Pro-inflammatory macrophage activation was strongest after exposure to BPP followed by BPS-MAE.
- Co-Inheritance of alpha-thalassemia and sickle cell disease in a cohort of Angolan pediatric patientsPublication . Santos, Brígida; Delgadinho, Mariana; Ginete, Catarina; Germano, Isabel; Miranda, Armandina; Arez, Ana Paula; Faustino, Paula; Brito, MiguelIntrodution: Sickle cell disease (SCD) is a recessive hereditary disease and a major global health problem, affecting over 300.000 newborn infants each year, and it is estimated that 75% of these births occur in Sub-Saharan Africa. SCD is a monogenic disease; the clinical manifestations are very heterogeneous due to environmental and genetic factors; in particular, the co-inheritance of alpha-thalassemia and an innate ability to produce fetal haemoglobin are two major modifiers that substantially impact disease pathophysiology. Objectives: This study explores the association between alpha-thalassemia, fetal haemoglobin, haematological indices, and clinical adverse events in pediatric Angolan sickle cell disease pediatric patients. Methods: A total of 200 SCD children were selected, after guardian written informed consent. None of them was treated with hydroxyurea or transfusion in the last 3 months. A venous blood sample was collected from each participant in the context of the routine medical and used for hematological analyses, fetal hemoglobin quantification, and genotyping of 3.7 kb alpha-thalassemia deletion by GAP-PCR. Mean values, standard deviation, and frequency distributions were performed to estimate the hematological, clinical, and genetic data. Results: The frequency of the 3.7 kb alpha-thalassemia deletion in homozygosity was 12.5%, and in heterozygosity was 55.0%. An increase in alpha-thalassemia frequency was observed in children older than 5 years old (11.7% vs. 13.00%). Furthermore, 3.7 kb alpha-thalassemia deletion homozygotes had a significantly higher age of the first manifestation, lower number of blood transfusions by year, higher haemoglobin, lower mean corpuscular volume, mean corpuscular haemoglobin, and lower hemolytic rate observed by a lower number of reticulocytes count. There were no differences in fetal haemoglobin between the three genotypes. Moreover, the number of stroke events, osteomyelitis, splenomegaly, splenectomy, and hepatomegaly were lower when alpha-thalassemia was co-inherited. Conclusion: The effect of alpha-thalassemia deletion in SCD was analysed, and results reinforce that this trait influences the haematological and clinical aspects and produces a milder phenotype.
- Deciphering the genetic modifiers of sickle cell anemia in children: the role of CYB5R3 genePublication . Nascimento, Djanira; Lopes, Pedro; Kjollerstrom, Paula; Maia, Raquel; Faria, Teresa; Faustino, PaulaIntroduction: Sickle cell anemia (SCA) is an autosomal recessive disorder caused by the c.20A>T alteration in the beta-globin gene (HBB), leading the synthesis of abnormal hemoglobin S (HbS). Under hypoxic conditions, HbS polymerizes within red blood cells (RBCs), causing them to adopt the characteristic sickle shape. This results in a clinically heterogenous disease, characterized by chronic hemolytic anemia, vaso-occlusive crises, and multi-organ damage. The CYB5R3 gene encodes the enzyme NADH-cytochrome b5 reductase 3, which plays a crucial role in protecting hemoglobin (Hb) from oxidative damage to unfunctional methemoglobin. Patients with SCA may develop methemoglobinemia, particularly under conditions of oxidative stress. This study aimed to evaluate the potential modulatory effects of a CYB5R3 variant, along with other well-established genetic modifiers within the globin genes, on the phenotypic variability of SCA in pediatric age. Methodology: Eighty-one children with SCA were followed by pediatricians at two hospitals in the Greater Lisbon area, who characterized their clinical, hematological, and biochemical phenotypes. For this specific study, CYB5R3, HBG2, HBA1, and HBA2 genes were genotyped using PCR, Sanger sequencing, and Gap-PCR. Association analyses were performed using SPSS. Results and Discussion: Co-inheritance of α-thalassemia with SCA was observed in 43.2% of the children and proved to be beneficial, as it was associated with higher RBC counts, milder anemia, and a significant reduction in hemolysis biomarkers (bilirubin and reticulocyte counts). Similarly, elevated fetal Hb levels (HbF ≥10%) were also beneficial, leading to less severe hemolytic anemia. In the HBG2 gene, the rs7482144_T allele had a frequency of 15% and was associated with higher HbF, reduced hemolytic parameters, lower HbS level, milder anemia, and a lower frequency of clinical comorbidities, except for heart disease. In the CYB5R3 gene, the rs1800457_G allele showed a very high allelic frequency of 35% and appears to exert a deleterious effect because patients carrying this allele presented with more severe anemia, elevated hemolysis biomarkers, and a greater tendency toward hepatomegaly and cardiac comorbidities. This study contributes to understanding how genetic modifiers influence SCA severity, complication risk and eventually treatment response. Identifying these factors supports personalized medicine and may help uncover new therapeutic targets.
- Dissecting the DIS3L2 target-specificity of transcripts committed to nonsense-mediated decay in human cellsPublication . Garcia-Moreno, Juan F.; Carvalho, Miguel P.; Lacerda, Rafaela; Romão, LuísaNonsense-mediated mRNA decay (NMD) is a conserved surveillance mechanism that eliminates mRNAs harboring premature termination codons (PTCs) and regulates the expression of certain physiological transcripts. The 3’-to-5’ exoribonuclease DIS3L2 degrades different RNAs independently of the RNA exosome, following uridylation at the 3' end by the terminal uridylyl transferases TUT4 and TUT7. We and others have shown that DIS3L2 is involved in NMD in an uridylation-dependent manner, being its function in NMD target-specific (Kurosaki et al. 2018; da Costa et al. 2019). Now, we aim to characterize the mechanisms involved in DIS3L2/NMD-target specificity. We used our RNA-seq data already obtained and validated and compared the transcripts upregulated upon DIS3L2 knockdown (REF) with a validated NMD-target set (Colombo et al., 2017). We observed that about 7% of DIS3L2-sensitive transcripts overlap with known NMD-targets. Considering the different groups of transcripts, we then analyzed specific features that make some NMD-targets sensitive to DIS3L2 (so called DIS3L2/NMD-targets; group 1), versus the remaining NMD-targets (DIS3L2-resistant NMD-targets; group 2), the remaining DIS3L2-sensitive targets (group 3), or the remaining transcriptome (DIS3L2-resistant NMD-targets plus NMD-resistant transcriptome; group 4). We assessed the following genomic features: 5’ and 3’ untranslated region (UTR) lengths, 3’UTR GC-, AU-, G-, C-, A-, and U-contents, presence of 5’UTR upstream open reading frames (uORFs), and 3’UTR introns. Elevated G-, C-, and GC-contents in the 3’UTRs were the most consistent features distinguishing DIS3L2/NMD-targets from the group 4. Comparison between group 1 and 2, and 1 and 3 was not significant. To better characterize the importance of each transcript portion, we are also analyzing hybrid constructs combining regions of the DIS3L2/NMD-resistant human β-globin (HBB) gene and the DIS3L2/NMD-sensitive GADD45A gene expressed in DIS3L2 depleted cultured cells.
- Dysregulated gene expression in colorectal cancer upon exposure to bisphenol A alternatives - a new approachPublication . Lacerda, Rafaela; Ventura, Célia; Louro, Henriqueta; Silva, Maria João; Romão, LuísaBisphenol A (BPA) has been widely used in plastics and resins since the 1950s, making it a common part of everyday products like food containers and bottle linings. Alternative substances are increasingly replacing BPA, but they are raising health and environmental concerns. Some mimic BPA’s endocrine-disrupting effects, while others affect different biological pathways. Substitutions in bisphenols can alter their biological properties, including nuclear receptor activation. Some BPA alternatives, like BPS, BPF and BPZ, may also pose cancer risks by activating oestrogen receptors, potentially even more than BPA itself. They may also contribute to colorectal cancer (CRC). Research suggests that BPA and its substitutes can influence cancer progression by altering cellular pathways, promoting metastasis and affecting gene expression. One of the key steps in gene expression regulation is translation initiation, whose canonical pathway is globally impaired under stress conditions, like exposure to BPA alternatives. Thus, we will subject NCM460 (normal intestinal mucosa) and HCT116 (colorectal carcinoma) cells to BPS, BPF and BPZ exposure and identify the transcripts actively being translated in such conditions, using ribosome profiling. We will analyse data with the R package anota2seq and evaluate the positively identified targets (compared to total RNA sequencing) for the existence of alternative mechanisms of translation initiation regulating their expression. The accurate characterisation of such mechanisms will be crucial for designing antisense RNA oligomers (ASOs) for potential therapeutic approaches. We will evaluate the cytotoxic effects of BPS, BPF and BPZ in the presence or absence of the selected alternatively translated transcripts (functional or targeted with the designed ASOs). Cytotoxic effects will be assessed through in vitro assays, analysing metabolic activity, membrane integrity, and cell proliferation. Thus, our research explores protein synthesis dysregulation to reduce CRC risks from BPS, BPF and BPZ exposure — an emerging public health issue.
- Exposure to mycotoxins in the Portuguese adult populationPublication . Maris, Elias; Namorado, Sónia; Chen, A.; Pero-Gason, Roger; De Boevre, Marthe; De Saeger, Sarah; Silva, Maria João; Alvito, PaulaMycotoxins are toxic fungal metabolites commonly found in food, posing health risks such as immunosuppression, carcinogenicity, and endocrine disruption. Despite regulatory limits, chronic low-level exposure remains a concern. Understanding real-life exposure in populations is essential for effective risk assessment. This study aims to investigate mycotoxin exposure among young adults in Portugal, contributing to evidence-based public health interventions. This study leveraged data and biospecimens from the INSEF-ExpoQuim survey, a cross-sectional study nested within thPortuguese National Health Examination Survey (INSEF). Data was collected via REDCap-assisted telephone interviews, covering sociodemographic and exposure-relevant variables. A subset of 295 first morning urine samples was collected from adults aged 28–39 years between May 2019 and March 2020. Urine samples were analyzed by a newly optimized and validated LC-MS/MS method targeting 40 mycotoxins and/or their corresponding metabolites in urine. Urinary creatinine was measured using a validated colorimetric method to allow adjustment and standardization of mycotoxin concentrations, ensuring accurate exposure assessment and comparability. This methodological approach enabled a robust characterization of mycotoxin exposure in a representative Portuguese population cohort.The study included 58% females and 42% males. Most participants had medium to high education, and urbanization was nearly evenly split between towns/suburbs (36.9%) and rural areas (35.9%), with fewer living in cities (27.1%). The majority were employed, and sampling was primarily conducted in summer and autumn. The number of mycotoxin co-exposures in the Portuguese population ranged from 0 to 5, with two simultaneous exposures being most common (n = 160). Among the 40 mycotoxins analysed, deoxynivalenol and tenuazonic acid were most frequently detected, with frequency of detection of 85% and 96%, respectively. This study offers robust biomonitoring data on mycotoxin exposure in Portuguese young adults using a validated LC-MS/MS method. The high prevalence of deoxynivalenol and tenuazonic acid suggests low level dietary contamination. These findings support the need for continued monitoring and the integration ofhuman biomonitoring into national food safety strategies. Detailed sociodemographic analyses are planned to further clarify exposure patterns and enable targeted public health interventions.
- Impact of SHC-1 adaptor protein inhibitors on the plasma membrane abundance of the CFTR channel in epithelial cellsPublication . Barros, Patrícia; Pereira, Mariana F.L.; Tomilev, Alexey; Cortopassi, Gino; Jordan, Peter; Matos, PauloCFTR is a chloride and bicarbonate channel essential for ionic homeostasis in epithelial cells, and its plasma membrane (PM) abundance is often downregulated in chronic obstructive pulmonary disease (COPD) and colon cancer (CRC). CFTR endocytosis is promoted by Y512 phosphorylation by spleen tyrosine kinase, mediated by SHC-1, a phospho-tyrosine adaptor in MAPK signaling. Since the drug idebenone (IDE) disrupts SHC-1/phospho-insulin receptor interaction in hepatocytes, we tested its effects on CFTR internalization. In CFBE airway cells, IDE and a novel SHC-1 inhibitor (110#3) increased CFTR PM levels but also elevated PM levels of GLUT1 and E-cadherin, indicating a MAPK signaling-independent, non-specific action. Moreover, neither compound affected CFTR PM abundance or MAPK activity in 16HBE airway or Caco-2 intestinal cells. These findings underscore the context-dependent nature of SHC-1 signaling, suggesting IDE and related compounds may not universally disrupt SHC-1 interactions, or specifically block CFTR internalization in the airway or CRC cells.
- Loss of BCL6 transcriptional repressor impacts migration but not viability in MCF-7 breast cancer cellsPublication . Dyson-Jorge, João; Jordan, Peter; Barros, Patrícia; Matos, PauloBreast cancer (BC) incidence has risen over the past two decades, now being the most prevalent cancer worldwide and the second leading cause of cancer-related deaths. Despite advancements in BC treatment, challenges like acquired resistance, recurrence, and metastasis persist. BCL6, a transcriptional repressor, acts as an oncogene in BC, being downregulated in about half of primary tumors of all BC subtypes and associated with disease progression and poor patient prognosis. This underscores the need to better understand BCL6 role in BC development. Therefore, we used RNA interference to explore the impact of BCL6 depletion on the oncogenic progression of MCF-7 cells, a low-tumorigenic estrogen receptor-positive cell line. While BCL6 is known to regulate normal mammary cell proliferation and differentiation, its depletion did not affect MCF-7 cell proliferation or viability but significantly reduced their individual and collective migratory properties. RNA microarray analysis identified a set of genes upregulated following BCL6 depletion, including S100A7, previously reported to inhibit MCF-7 cell migration and invasion by reducing MMP9 secretion. However, our findings showed that S100A7 downregulation alone did not affect MCF-7 migration. Moreover, simultaneous depletion of BCL6 and S100A7 failed to restore MCF-7 cell migratory behavior.
- The potential function of alternative translation initiation of Argonaute 1 in cancerPublication . Vieira da Silva, Verónica; Lacerda, Rafaela; Romão, LuísaTranslation is one of the most regulated and energy-consuming cellular processes crucial for proper cell function. Translation is initiated by the canonical cap-dependent mechanism. However, under stress conditions, the initiation of canonical translation is inhibited, which allows the translation of specific proteins via alternative mechanisms. This project aims to understand the biological relevance of alternative protein synthesis mechanisms in Argonaute 1 (AGO1) expression. The AGO1 protein is involved in microRNA regulation, gene expression modulation and inhibition. AGO1 is also involved in the regulation of gene expression by RNA interference (RNAi), and its deregulation can lead to the activation of oncogenes or the suppression of tumor suppressor genes, contributing to the development and progression of cancer. Our work has shown that AGO1 mRNA can be translated through a cap-independent initiation mechanism. An upstream open reading frame (uORF) has also been identified in its 5’ untranslated region (5’UTR), which may play a role in the initiation of AGO1 translation. The results showed that the 5’UTR of human AGO1 supports a cap-independent mechanism of translation initiation, which is maintained under stress conditions. However, our analyses did not provide conclusive evidence for a regulatory role of the uORF in this initiation process. To this end, the 5’UTR of human AGO1 was cloned into a bicistronic vector containing Renilla (RLuc) and Firefly luciferase (FLuc), with FLuc positioned downstream of the 5’UTR. Luminometry assays will be used to evaluate the relative FLuc/RLuc translation efficiency under the control of the AGO1 5’UTR. The same approach will be used with monocistronistic reporter vectors, contaning only FLuc. This project aims to understand how these alternative mechanisms of mRNA translation initiation can influence AGO1 expression and help explain their potential roles in certain pathologies and cancer progression, such as colorectal cancer.