Departamento de Genética Humana
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- Deliverable D5.4 -1st closing data gaps report WP5 – Task 5.1. Partnership for the Assessment of Risks from ChemicalsPublication . Partnership for the Assessment of Risks from ChemicalsThis deliverable summarizes the preliminary results of the PARC WP5 Task 5.1, aiming to close key data gaps for substances of emerging concern. Focus is placed on two major substance groups: natural toxins and bisphenol A (BPA) alternatives, addressing both human and environmental health. For human health, prioritized mycotoxins (enniatins including beauvericin and Alternaria toxins) were tested across genotoxicity, endocrine disruption, developmental neurotoxicity, and immunotoxicity endpoints, employing both OECD test guidelines and new approach ethodologies (NAMs). In a parallel project, hazard testing of BPA alternatives examined metabolism, bioactivation, and toxicological profiles using as well OECD test guidelines and in vitro systems aligned with regulatory relevance. On the environmental side, aquatic organisms such as Daphnia magna, Lymnaea stagnalis, and Chlorella vulgaris were used to assess the ecotoxicity of selected natural toxins and BPA alternatives under standard OECD and ISO guidelines. Both single and mixture exposures were tested, revealing species- and compound specific effects and emphasizing the relevance of temperature and combined stressors in ecological risk assessment. Altogether, this first data gap report contributes to strengthening risk assessment capabilities across the EU by supporting better prioritization and regulation of under-studied substances. The data are being shared and discussed with regulatory agencies (EFSA, ECHA, EEA), and further testing is ongoing to complete hazard characterizations.
- Generation of Cellular Models for Fabry Disease: Unlocking the Potential of iPSCs and Gene EditingPublication . Duarte, Ana Joana; Moreira, Luciana; Ribeiro, Diogo; Alves, Sandra; Gaspar, Paulo; Bragança, José; Amaral, OlgaIntroduction: Fabry Disease (FD) is a lysosomal storage disorder caused by mutations in the GLA gene, resulting in a defective α-GAL A enzyme. This deficiency leads to the accumulation of Gb3 and lyso-Gb3 within lysosomes, resulting in a multisystem disease. Through reprogramming, we obtained induced pluripotent stem cells (iPSCs) derived from fibroblasts of a patient with FD2 and from a wild-type (WT) control. We used CRISPR/Cas9 to correct the c.860G>A mutation present in the patient’s cells, as well as to generate a WT GLA knockout (KO). The resulting cells were then differentiated into cardiomyocytes, a cell type affected by this disease. Methods: We reprogrammed the fibroblasts into iPSCs using episomal vectors or Sendai virus. For gene editing, single-guide RNAs (sgRNAs) and Cas9 were nucleofected, and the editing was confirmed by Sanger sequencing. Following colony selection, isogenic cell lines were established. The FD iPSCs, the corrected FD iPSCs, and the WT iPSCs were then differentiated into iPSC-derived cardiomyocytes (iPSC-CMs). Results: Seven new cell models were generated. Functional studies of the FD iPSCs showed the maintenance of the molecular and biochemical characteristics and a normal karyotype. The KO cell line recapitulated the biological features observed in FD patient cells, with reduced GLA expression, lower α-Galactosidase A (α-Gal A) activity (1.5 nmol/h/mg protein), and Gb3 accumulation. The corrected cell line was generated with 75.8% efficiency and 69.6% on-target efficacy. Enzyme activity increased to 579 nmol/h/mg protein (vs. 0.78 nmol/h/mg protein in FD iPSCs), accompanied by a marked reduction in Gb3 levels. We successfully generated iPSC-CM lines, which were validated by qRT-PCR and immunofluorescence. Discussion: Cell modelling is essential for studying the pathophysiology of disease mechanisms. By retaining the characteristics of the original cells, iPSCs are a valuable biological resource for generating specific differentiated cell types affected by the disease, which would otherwise be difficult to access. This study also explored the therapeutic potential of gene editing as a promising approach to altering the course of rare diseases.
- Impact of nanocelluloses on genome-wide DNA methylation pattern of human pulmonary and intestinal cellsPublication . Ventura, Célia; Vital, Nádia; Valente, Ana; Vieira, Luís; Louro, Henriqueta; Silva, Maria JoãoObjective: Nanocellulose is an innovative nanomaterial with interesting physicochemical properties for several industrial and biomedical applications and its safety for human health must be ensured. This study aimed to identify DNA methylation changes in human pulmonary and intestinal cells after exposure to two fibrillar celluloses with different physicochemical properties, both derived from Eucaliptus globulus. Their cellular effects were investigated in silico by functional pathway and gene ontology (GO) analysis. Methods: We applied Reduced Representation Bisulfite Sequencing to analyze the methylation differences in DNA CpG-rich regions from human bronchial (BEAS-2B) and intestinal (Caco-2) cells exposed for 24h to 14.3 µg/mL of cellulose nanofibrils (CNF) or microfibrils (CMF) versus non-exposed ones. A bioinformatics pipeline was implemented for identifying differentially methylated genes (DMGs), functional pathways, and GO associations. Results: CNF and CMF exposure resulted in 11 and 14 DMGs, respectively, in BEAS-2B cells, 6 being common to both nanocelluloses. In Caco-2 cells, 36 and 31 DMGs were identified, sharing 12 DMGs. No DMGs were shared between these cell lines. Hypomethylation predominated in BEAS-2B cells, and hypermethylation in Caco-2 cells. In BEAS-2B cells, both nanocelluloses affected similar pathways and GO terms (e.g., regulation of DNA replication, damage repair and senescence, telomere maintenance, and D-glucose transport). In Caco-2 cells, both CNF and CMF enriched, for instance, signal transduction, glycosylation, and cytoskeletal dynamics. Each nanocellulose type also affected other different pathways and terms. Conclusions: Nanocellulose may have a wide impact on the metabolism and survival of pulmonary and intestinal cells through several regulatory pathways, which depend on nanocellulose physicochemical properties. Cell type also influences the outcome, suggesting tissue-specific effects. These findings highlight the relevance of DNA methylation in nanotoxicology, providing insights into underlying molecular mechanisms of action. Keywords: gene ontology; nanomaterial; pathway analysis; RRBS
- Interlaboratory Validation of the Cell Transformation Assay (CTA) for Carcinogenic Assessment of BPA AlternativesPublication . El Yamani, N.; Aimonen, K.; Dusinska, M.; Guichard, Y.; Honza, T.; Louro, H.; Pereira, M.J.; Rundén-Pran, E.; SenGupta, T.; Tavares, A.M.; Silva, M.J.Bisphenol A (BPA) has long been used in various plastic products, resins and coatings, making human exposure to this chemical inevitable. Due to its harmful health effects, including endocrine disruption, and immunotoxicity, BPA has been increasingly replaced by several alternative compounds. However, there are still significant gaps in research regarding the safety of these BPA alternatives, particularly concerning their potential carcinogenicity. One of the in vitro assays to assess carcinogenic potential of chemicals is the Bhas-42 cell transformation assay (CTA). The assay can detect both genotoxic and non-genotoxic carcinogens It is valuable in identifying potential cancer risks before widespread exposure occurs, contributing to the development of safer chemicals and products, as well as better regulatory standards while adhering to the 3R concept. The EU-Partnership for the Assessment of Risks from Chemicals (PARC) project is addressing these research gaps to enhance the risk assessment of BPA alternatives. BPA and some alternatives, including BPZ, BPE, BPAP, BPA-MAE, BPP, and TCBPA, were selected for evaluation of their carcinogenic potential using the in vitro 2-stage Bhas-42 CTA. A key objective of the project is to validate the CTA as a reliable in vitro method for assessing carcinogenicity. To ensure consistency and accuracy across participating labs, an interlaboratory comparison was initiated and a standardized SOP was developed, including concentration ranges for controls and BPA analogues, in alignment with OECD guidance document. The first results from the protocol harmonization, using the selected controls, were consistent across all participating labs. BPA and its analogues are being tested, and the results are under evaluation. The data generated will contribute to the overall weight of evidence on the hazards posed by these chemicals and, when combined with findings from other endpoints, will provide a solid basis for refining their regulation.