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Projeto de investigação
Partnership for the Assessment of Risks from Chemicals
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A regulatory perspective on the applicability of NAMs in genotoxicity and carcinogenicity assessment in EU: current practices and future directions
Publication . Bossa, Cecilia; Alivernini, Silvia; Andreoli, Cristina; Aquilina, Gabriele; Attias, Leonello; Benfenati, Emilio; Dusinska, Maria; El Yamani, Naouale; Louro, Henriqueta; Marcon, Francesca; Raitano, Giuseppa; Rundén-Pran, Elise; Russo, Maria Teresa; Silva, Maria João; Battistelli, Chiara Laura
New Approach Methodologies (NAMs) are gaining significant momentum globally to reduce animal testing and enhance the efficiency and human relevance of chemical safety assessment. Even with substantial EU commitment from regulatory agencies and the academic community, the full regulatory adoption of NAMs remains a distant prospect. This challenge is further complicated by the fact that the academic world, oriented toward NAMs development, and regulatory agencies, focused on practical application, frequently operate in separate spheres. Addressing this disconnect, the present paper, developed within the European Partnership for the Assessment of Risks from Chemicals (PARC), provides a clear overview of both the available non-animal tests and current evaluation practices for genotoxic and carcinogenic hazard assessment, while simultaneously highlighting existing regulatory needs, gaps, and challenges toward greater human health protection and the replacement of animal testing through NAMs adoption. The analysis reveals a complex landscape: while the EU is deeply committed to developing and adopting NAMs, as outlined in its Chemical Strategy for Sustainability and supported by initiatives like PARC, prescriptive regulations such as Classification, Labelling and Packaging (CLP) and Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) still heavily mandate in vivo animal data for hazard classification, particularly for germ cell mutagenicity and carcinogenicity. This reliance creates a "too-short-blanket-problem," where efforts to reduce animal testing may impact human health protection because of the current in vivo-based classification criteria. In contrast, sectors such as cosmetics and certain European Food Safety Authority (EFSA)-regulated products demonstrate greater flexibility toward progressive integration of NAMs. While the deep mechanistic understanding of genotoxicity and carcinogenicity has significantly advanced the integration of alternatives to animal tests into regulatory chemical hazard assessment, their broader and full implementation faces considerable challenges due to both scientific complexities (i.e., the development and validation of fit-for-purpose NAMs) and existing legislative provisions.
Dysregulated gene expression in colorectal cancer upon exposure to bisphenol A alternatives - a new approach
Publication . Lacerda, Rafaela; Ventura, Célia; Louro, Henriqueta; Silva, Maria João; Romão, Luísa
Bisphenol 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.
BPA analogues affect intestinal barrier integrity and pro-inflammatory response in a coculture model
Publication . Pereira, Gonçalo; Barros, Patrícia; Matos, Paulo; Jordan, Peter
Background: 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.
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Entidade financiadora
European Commission
Programa de financiamento
HORIZON Programme Cofund Actions
Número da atribuição
101057014