Susana Pastor

Chronic inhalation exposure to nanoplastics, specifically polyethylene terephthalate (PET) nanoplastics (PET-NPLs) is an emerging health concern, yet the long-term consequences for genomic stability and DNA damage response (DDR) capacity in bronchial epithelial cells remain poorly characterized. For this study, human bronchial epithelial BEAS-2B cells were continuously exposed to PET-NPLs for over 20 weeks, after which elevated basal DNA genotoxic damage was observed, as assessed by the alkaline comet assay. In addition, a broad transcriptional suppression of the DDR, with 27 of 84 profiled genes involved in DDR showing reduced expression relative to passage-matched control was observed. The suppressed genes span ATM/ATR checkpoint signaling, homologous recombination (HR), base excision repair (BER), nucleotide excision repair (NER), and apoptotic pathways. To determine whether chronic PET-NPL exposure altered susceptibility to acute genotoxic challenge in a damage-type-specific manner, cells were treated with methyl methanesulfonate (MMS), ultraviolet-C (UV-C) radiation, or bleomycin. While MMS and UV-C induced comparable levels of DNA damage in control and PET-exposed cells, bleomycin produced significantly greater damage in PET-exposed cells, indicating selective sensitization to doble-strand breaks (DSB)-type and oxidative genotoxic insults. Transcriptional profiling during bleomycin challenge identified 18 DDR genes with relatively higher expression in PET-exposed cells compared to passage-matched controls, encompassing HR, BER, ATM/ATR signaling, the Fanconi anemia pathway, and apoptosis. Furthermore, PET-exposed cells retained significantly higher residual DNA damage after 3 h of bleomycin challenge, indicating a persistent early repair deficit. Together, these findings suggest that chronic PET-NPL exposure specifically compromises the bronchial epithelial DDR, with potential implications for long-term genomic stability in respiratory epithelia subjected to nanoplastic inhalation.

Bisphenol (BP) compounds are widely present in the environment, primarily due to their use as plastic additives. These substances involve health risks, particularly as endocrine disruptors. While the general population is chronically exposed, patients with end-stage chronic kidney disease undergoing hemodialysis (HD-CKD) represent a particularly vulnerable group. This is due to both impaired renal clearance of circulating BPs and potential contamination from plastic-containing dialyzers used in extracorporeal blood circulation. In this longitudinal study, from the 35 HD-CKD patients initially selected, 25 changed their conventional dialyzers to BPA-free dialyzers for 6 months. Blood serum samples were collected, at baseline and after the intervention, to quantify levels of five BP analogues: Bisphenol A (BPA), Bisphenol AF (BPAF), Bisphenol F (BPF), Bisphenol B (BPB), and Bisphenol S (BPS). Genotoxicity was assessed using the comet assay and the micronucleus test on peripheral white blood cells. Among the analyzed BPs, only BPAF showed a statistically significant reduction when using BPA-free dialyzers. In terms of genotoxicity, a significant decrease was observed only in primary DNA damage (mainly DNA strand breaks), with no notable changes in chromosomal damage. This is the first study to detect multiple BP analogues in HD-CKD patients, beyond BPA, and to associate human exposure to BPs with DNA damage biomarkers. The observed reduction in DNA damage in parallel with decreased BPAF levels highlights the importance of monitoring and minimizing BP exposure of this high-risk population.

One important environmental/health challenge is to determine, in a feasible way, the potential carcinogenic risk associated with environmental agents/exposures. Since a significant proportion of tumors have an environmental origin, detecting the potential carcinogenic risk of environmental agents is mandatory, as regulated by national and international agencies. The challenge mainly implies finding a way of how to overcome the inefficiencies of long-term trials with rodents when thousands of agents/exposures need to be tested. To such an end, the use of in vitro cell transformation assays (CTAs) was proposed, but the existing prevalidated CTAs do not cover the complexity associated with carcinogenesis processes and present serious limitations. To overcome such limitations, we propose to use a battery of assays covering most of the hallmarks of the carcinogenesis process. For the first time, we grouped such assays as early, intermediate, or advanced biomarkers which allow for the identification of the cells in the initiation, promotion or aggressive stages of tumorigenesis. Our proposal, as a novelty, points out that using a battery containing assays from all three groups can identify if a certain agent/exposure can pose a carcinogenic risk; furthermore, it can gather mechanistic insights into the mode of the action of a specific carcinogen. This structured battery could be very useful for any type of in vitro study, containing human cell lines aiming to detect the potential carcinogenic risks of environmental agents/exposures. In fact, here, we include examples in which these approaches were successfully applied. Finally, we provide a series of advantages that, we believe, contribute to the suitability of our proposed approach for the evaluation of exposure-induced carcinogenic effects and for the development of an alternative strategy for conducting an exposure risk assessment.

The presence of nanomaterials (NMs) in the environment may represent a serious risk to human health, especially in a scenario of chronic exposure. To evaluate the potential relationship between NM-induced epigenetic alterations and carcinogenesis, the present study analyzed a panel of 33 miRNAs related to the cell transformation process in BEAS-2B cells transformed by TiO₂NP and long-term MWCNT exposure. Our battery revealed a large impact on miRNA expression profiling in cells exposed to both NMs. From this analysis, a small set of five miRNAs (miR-23a, miR-25, miR-96, miR-210, and miR-502) were identified as informative biomarkers of the transforming effects induced by NM exposures. The usefulness of this reduced miRNA battery was further validated in other previously generated transformed cell systems by long-term exposure to other NMs (CoNP, ZnONP, MSiNP, and CeO₂NP). Interestingly, the five selected miRNAs were consistently overexpressed in all cell lines and NMs tested. These results confirm the suitability of the proposed set of mRNAs to identify the potential transforming ability of NMs. Particular attention should be paid to the epigenome and especially to miRNAs for hazard assessment of NMs, as wells as for the study of the underlying mechanisms of action.

The increasing presence of micro- and nanoplastics (MNPLs) in the environment, and their consequent accumulation in trophic niches, could pose a potential health threat to humans, especially due to their chronic ingestion. In vitro studies using human cells are considered pertinent approaches to determine potential health risks to humans. Nevertheless, most of such studies have been conducted using short exposure times and high concentrations. Since human exposure to MNPLs is supposed to be chronic, there is a lack of information regarding the potential in vitro MNPLs effects under chronic exposure conditions. To this aim, we assessed the accumulation and potential outcomes of polystyrene nanoparticles (PSNPs), as a model of MNPLs, in undifferentiated Caco-2 cells (as models of cell target in ingestion exposures) under a relevant long-term exposure scenario, consisting of eight weeks of exposure to sub-toxic PSNPs concentrations. In such exposure conditions, culture-media was changed every 2–3 days to maintain constant exposure. The different analyzed endpoints were cytotoxicity, dysregulation of stress-related genes, genotoxicity, oxidative DNA damage, and intracellular ROS levels. These are endpoints that showed to be sensitive enough in different studies. The obtained results attest that PSNPs accumulate in the cells through time, inducing changes at the ultrastructural and molecular levels. Nevertheless, minor changes in the different evaluated genotoxicity-related biomarkers were observed. This would indicate that no DNA damage or oxidative stress is observed in the human intestinal Caco-2 cells after long-term exposure to PSNPs. This is the first study dealing with the long-term effects of PSNPs on human cultured cells.

Chronic kidney disease (CKD) patients have many affected physiological pathways. Variations in the genes regulating these pathways might affect the incidence and predisposition to this disease. A total of 722 Spanish adults, including 548 patients and 174 controls, were genotyped to better understand the effects of genetic risk loci on the susceptibility to CKD. We analyzed 38 single nucleotide polymorphisms (SNPs) in candidate genes associated with the inflammatory response (interleukins IL-1A, IL-4, IL-6, IL-10, TNF-α, ICAM-1), fibrogenesis (TGFB1), homocysteine synthesis (MTHFR), DNA repair (OGG1, MUTYH, XRCC1, ERCC2, ERCC4), renin-angiotensin-aldosterone system (CYP11B2, AGT), phase-II metabolism (GSTP1, GSTO1, GSTO2), antioxidant capacity (SOD1, SOD2, CAT, GPX1, GPX3, GPX4), and some other genes previously reported to be associated with CKD (GLO1, SLC7A9, SHROOM3, UMOD, VEGFA, MGP, KL). The results showed associations of GPX1, GSTO1, GSTO2, UMOD, and MGP with CKD. Additionally, associations with CKD related pathologies, such as hypertension (GPX4, CYP11B2, ERCC4), cardiovascular disease, diabetes and cancer predisposition (ERCC2) were also observed. Different genes showed association with biochemical parameters characteristic for CKD, such as creatinine (GPX1, GSTO1, GSTO2, KL, MGP), glomerular filtration rate (GPX1, GSTO1, KL, ICAM-1, MGP), hemoglobin (ERCC2, SHROOM3), resistance index erythropoietin (SOD2, VEGFA, MTHFR, KL), albumin (SOD1, GSTO2, ERCC2, SOD2), phosphorus (IL-4, ERCC4 SOD1, GPX4, GPX1), parathyroid hormone (IL-1A, IL-6, SHROOM3, UMOD, ICAM-1), C-reactive protein (SOD2, TGFB1,GSTP1, XRCC1), and ferritin (SOD2, GSTP1, SLC7A9, GPX4). To our knowledge, this is the second comprehensive study carried out in Spanish patients linking genetic polymorphisms and CKD.