Mutagenicity and genotoxicity assessment of the emerging mycotoxin Versicolorin A, an Aflatoxin B1 precursor.

Aflatoxin B1 (AFB1) is the most potent natural carcinogen among mycotoxins. Versicolorin A (VerA) is a precursor of AFB1 biosynthesis and is structurally related to the latter. Although VerA has already been identified as a genotoxin, data on the toxicity of VerA are still scarce, especially at low concentrations. The SOS/umu and miniaturised version of the Ames test in Salmonella Typhimurium strains used in the present study shows that VerA induces point mutations. This effect, like AFB1, depends primarily on metabolic activation of VerA. VerA also induced chromosomal damage in metabolically competent intestinal cells (IPEC-1) detected by the micronucleus assay. Furthermore, results from the standard and enzyme-modified comet assay confirmed the VerA-mediated DNA damage, and we observed that DNA repair pathways were activated upon exposure to VerA, as indicated by the phosphorylation and/or relocation of relevant DNA-repair biomarkers (γH2AX and 53BP1/FANCD2, respectively). In conclusion, VerA induces DNA damage without affecting cell viability at concentrations as low as 0.03 µM, highlighting the danger associated with VerA exposure and calling for more research on the carcinogenicity of this emerging food contaminant.

Brief Research Report: Anti-SARS-CoV-2 Immunity in Long Lasting Responders to Cancer Immunotherapy Through mRNA-Based COVID-19 Vaccination.

Cancer patients (CPs) have been identified as particularly vulnerable to SARS-CoV-2 infection, and therefore are a priority group for receiving COVID-19 vaccination. From the patients with advanced solid tumors, about 20% respond very efficiently to immunotherapy with anti-PD1/PD-L1 antibodies and achieve long lasting cancer responses. It is unclear whether an efficient cancer-specific immune response may also correlate with an efficient response upon COVID-19 vaccination. Here, we explored the antiviral immune response to the mRNA-based COVID-19 vaccine BNT162b2 in a group of 11 long-lasting cancer immunotherapy responders. We analysed the development of SARS-CoV-2-specific IgG serum antibodies, virus neutralizing capacities and T cell responses. Control groups included patients treated with adjuvant cancer immunotherapy (IMT, cohort B), CPs not treated with immunotherapy (no-IMT, cohort C) and healthy controls (cohort A). The median ELISA IgG titers significantly increased after the prime-boost COVID vaccine regimen in all cohorts (Cohort A: pre-vaccine = 900 (100-2700), 3 weeks (w) post-boost = 24300 (2700-72900); Cohort B: pre-vaccine = 300 (100-2700), 3 w post-boost = 8100 (300-72900); Cohort C: pre-vaccine = 500 (100-2700), 3 w post-boost = 24300 (300-72900)). However, at the 3 w post-prime time-point, only the healthy control group showed a statistically significant increase in antibody levels (Cohort A = 8100 (900-8100); Cohort B = 900 (300-8100); Cohort C = 900 (300-8100)) (P < 0.05). Strikingly, while all healthy controls generated high-level antibody responses after the complete prime-boost regimen (Cohort A = 15/15 (100%), not all CPs behaved alike [Cohort B= 12/14 (84'6%); Cohort C= 5/6 (83%)]. Their responses, including those of the long-lasting immunotherapy responders, were more variable (Cohort A: 3 w post-boost (median nAb titers = 95.32 (84.09-96.93), median Spike-specific IFN-γ response = 64 (24-150); Cohort B: 3 w post-boost (median nAb titers = 85.62 (8.22-97.19), median Spike-specific IFN-γ response (28 (1-372); Cohort C: 3 w post-boost (median nAb titers = 95.87 (11.8-97.3), median Spike-specific IFN-γ response = 67 (20-84)). Two long-lasting cancer responders did not respond properly to the prime-boost vaccination and did not generate S-specific IgGs, neutralizing antibodies or virus-specific T cells, although their cancer immune control persisted for years. Thus, although mRNA-based vaccines can induce both antibody and T cell responses in CPs, the immune response to COVID vaccination is independent of the capacity to develop an efficient anti-cancer immune response to anti PD-1/PD-L1 antibodies.

Topical noninvasive retinal drug delivery of a tyrosine kinase inhibitor: 3% cediranib maleate cyclodextrin nanoparticle eye drops in the rabbit eye.

PURPOSE: Tyrosine kinase inhibitors inhibit VEGF receptors. If delivered to the retina, they might inhibit oedema and neovascularization such as in age-related macular degeneration and diabetic retinopathy. The aim of this study was to formulate cediranib maleate, a potent VEGF inhibitor, as γ-cyclodextrin nanoparticle eye drops and measure the retinal delivery and overall ocular pharmacokinetics after a single-dose administration in rabbits. METHODS: A novel formulation technology with 3% cediranib maleate as γ-cyclodextrin micro-suspension was prepared by autoclaving method. Suitable stabilizers were tested for heat-stable eye drops. The ophthalmic formulation was topically applied to one eye in rabbits. The pharmacokinetics in ocular tissues, tear film and blood samples were studied at 1, 3 and 6 hr after administration. RESULTS: γ-cyclodextrin formed complex with cediranib maleate. The formation of γ-cyclodextrin nanoparticles occurred in concentrated complexing media. Combined stabilizers prevented the degradation of drug during the autoclaving process. Three hours after administration of the eye drops, treated eyes showed cediranib levels of 737 ± 460 nM (mean ± SD) in the retina and 10 ± 6 nM in the vitreous humour. CONCLUSIONS: Cediranib maleate in γ-cyclodextrin nanoparticles were stable to heat in presence of stabilizers. The drug as eye drops reached the retina in concentrations that are more than 100 times higher than the 0.4 nM IC50 value reported for the VEGF type-II receptor and thus, presumably, above therapeutic level. These results suggest that γ-cyclodextrin-based cediranib maleate eye drops deliver effective drug concentrations to the retina in rabbits after a single-dose administration.

Versicolorin A enhances the genotoxicity of aflatoxin B1 in human liver cells by inducing the transactivation of the Ah-receptor.

Aflatoxins are a group of mycotoxins that have major adverse effects on human health. Aflatoxin B1 (AFB1) is the most important aflatoxin and a potent carcinogen once converted into a DNA-reactive form by cytochrome P450 enzymes (CYP450). AFB1 biosynthesis involves the formation of Versicolorin A (VerA) which shares structural similarities with AFB1 and can be found in contaminated commodities, often co-occurring with AFB1. This study investigated and compared the toxicity of VerA and AFB1, alone or in combination, in HepG2 human liver cells. Our results show that both toxins have similar cytotoxic effects and are genotoxic although, unlike AFB1, the main genotoxic mechanism of VerA does not involve the formation of DNA double-strand breaks. Additionally, we show that VerA activates the aryl hydrocarbon receptor (AhR) and significantly induce the expression of the CYP450-1A1 (CYP1A1) while AFB1 did not induce AhR-dependent CYP1A1 activation. Combination of VerA with AFB1 resulted in enhanced genotoxic effects, suggesting that AhR-activation by VerA influences AFB1 genotoxicity by promoting its bioactivation by CYP450s to a highly DNA-reactive metabolite. Our results emphasize the need for expanding the toxicological knowledge regarding mycotoxin biosynthetic precursors to identify those who may pose, directly or indirectly, a threat to human health.

Proteome changes induced by a short, non-cytotoxic exposure to the mycoestrogen zearalenone in the pig intestine.

Intestinal epithelial homeostasis is regulated by a complex network of signaling pathways. Among them is estrogen signaling, important for the proliferation and differentiation of epithelial cells, immune signaling and metabolism. The mycotoxin zearalenone (ZEN) is an estrogen disruptor naturally found in food and feed. The exposure of the intestine to ZEN has toxic effects including alteration of the immune status and is possibly implicated in carcinogenesis, but the molecular mechanisms linked with these effects are not clear. Our objective was to explore the proteome changes induced by a short, non-cytotoxic exposure to ZEN in the intestine using pig jejunal explants. Our results indicated that ZEN promotes little proteome changes, but significantly related with an induction of ERα signaling and a consequent disruption of highly interrelated signaling cascades, such as NF-κB, ERK1/2, CDX2 and HIF1α. The toxicity of ZEN leads also to an altered immune status characterized by the activation of the chemokine CXCR4/SDF-1 axis and an accumulation of MHC-I proteins. Our results connect the estrogen disrupting activity of ZEN with its intestinal toxic effect, associating the exposure to ZEN with cell-signaling disorders similar to those involved in the onset and progression of diseases such as cancer and chronic inflammatory disorders. SIGNIFICANCE: The proteomics results presented in our study indicate that the endocrine disruptor activity of ZEN is able to regulate a cascade of highly inter-connected signaling events essential for the small intestinal crypt-villus cycle and immune status. These molecular mechanisms are also implicated in the onset and progress of intestinal immune disorders and cancer indicating that exposure to ZEN could play an important role in intestinal pathogenesis.

Intrinsic alterations in peripheral neutrophils from cystic fibrosis newborn piglets.

BACKGROUND: The hallmark of the cystic fibrosis (CF) lung disease is a neutrophil dominated lung environment that is associated to chronic lung tissue destruction and ultimately the patient's death. It is unclear whether the exacerbated neutrophil response is primary related to a defective CFTR or rather secondary to chronic bacterial colonization and inflammation. Here, we hypothesized that CF peripheral blood neutrophils present intrinsic alteration at birth before the start of an inflammatory process. METHODS: Peripheral blood neutrophils were isolated from newborn CFTR+/+ and CFTR-/- piglets. Neutrophils immunophenotype was evaluated by flow cytometry. Lipidomic and proteomic profile were characterized by liquid chromatography/tandem mass spectrometry (LC-MS/MS), intact cell matrix-assisted laser desorption/ionization mass spectrometry (ICM-MS) followed by top-down high-resolution mass spectrometry (HRMS), respectively. The ability of CF neutrophils to kill pseudomonas aeruginosa was also evaluated. RESULTS: Polyunsaturated fatty acid metabolites analysis did not show any difference between CFTR+/+ and CFTR-/- neutrophils. On the other hand, a predictive mathematical model based on the ICM-MS proteomic profile was able to discriminate between both genotypes. Top-down proteomic analysis identified 19 m/z differentially abundant masses that corresponded mainly to proteins related to the antimicrobial response and the generation of reactive oxygen species (ROS). However, no alteration in the ability of CFTR-/- neutrophils to kill pseudomonas aeruginosa in vitro was observed. CONCLUSIONS: ICM-MS demonstrated that CFTR-/- neutrophils present intrinsic alterations already at birth, before the presence of any infection or inflammation.

Acute Exposure to Zearalenone Disturbs Intestinal Homeostasis by Modulating the Wnt/ß-Catenin Signaling Pathway.

The mycotoxin zearalenone (ZEN), which frequently contaminates cereal-based human food and animal feed, is known to have an estrogenic effect. The biological response associated with exposure to ZEN has rarely been reported in organs other than the reproductive system. In the intestine, several studies suggested that ZEN might stimulate molecular changes related to the activation of early carcinogenesis, but the molecular mechanisms behind these events are not yet known. In this study, we investigated gene expression and changes in protein abundance induced by acute exposure to ZEN in the jejunum of castrated male pigs using an explant model. Our results indicate that ZEN induces the accumulation of ER but not ER, modulates Wnt/ß-catenin and TGF- signaling pathways, and induces molecular changes linked with energy sensing and the antimicrobial activity without inducing inflammation. Our results confirm that the intestine is a target for ZEN, inducing changes that promote cellular proliferation and could contribute to the onset of intestinal pathologies.

Molecular determinants of Bim(BH3) peptide binding to pro-survival proteins.

Proteins of the Bcl-2 family regulate apoptosis through the formation of heterodimers between antiapoptotic or pro-survival proteins and proapoptotic or pro-death proteins. Overexpression of antiapoptotic proteins not only contributes to the progression of many cancers, but also confers resistance to the chemo- and radiotherapeutic treatments. It has been demonstrated that peptides containing the BH3 domain of proapoptotic Bcl-2 family members are able to bind and inhibit antiapoptotic proteins. For this reason, the design of small molecules mimicking the BH3 domain of proapoptotic proteins has emerged as a promising therapeutic strategy for cancer treatment during the last years. However, BH3 domains exhibit different affinities for binding to antiapoptotic proteins; whereas Bim(BH3) and Puma(BH3) are able to bind all antiapoptotic proteins, others like Bad(BH3) and Bmf(BH3) show preference for some proteins over others. Consequently, the ability of a BH3-mimetic to kill tumor cells will depend on the BH3 peptide used as template and thus will have a selective or pan-inhibition effect. Recently, it has been suggested that this last approach could be interesting. Therefore, the present work is aimed to elucidate how the nonselective peptide Bim(BH3) is able to bind to all of the Bcl-2 family antiapoptotic proteins. To unravel the molecular determinants of this pan-inhibition, we used the MM-PB/GBSA approaches to calculate the binding free energy of the different complexes studied and to determine which residues of the peptide have the largest contribution to complex formation. Results obtained in the present work show that the binding of Bim(BH3) to pro-survival proteins is mainly hydrophobic and that specific interactions are fully distributed along the peptide sequence.

Rational design of new class of BH3-mimetics as inhibitors of the Bcl-xL protein.

The Bcl-2 family of proteins plays an important role in the intrinsic pathway of cell apoptosis. Overexpression of pro-survival members of this family of proteins is often associated with the development of many types of cancer and confers resistance against conventional therapeutic treatments. Accordingly, antagonism of its protective function has emerged as an encouraging anticancer strategy. In the present work, we use a pharmacophore for describing interaction between the BH3 domain of different pro-apoptotic members and the pro-survival protein Bcl-x(L) in order to identify new lead compounds. In the strategy followed in the present work, the pharmacophore was derived from molecular dynamics studies of different Bcl-x(L)/BH3 complexes. This pharmacophore was later used as query for 3D database screening. Hits obtained from the search were computationally assessed, and a subset proposed for in vitro testing. Two of the 15 compounds assayed were found able to disrupt the Bcl-x(L)/Bak(BH3) complex with IC(50) values in the lower micromolar range. Finally, docking studies were performed to explore the binding mode of these compounds to Bcl-x(L) for further modifications.

[Follow-up and monitoring of hypertension. Is this performed the same in the autochthonous and immigrant populations?]. / Seguimiento y control de la hipertensión arterial. Se efectúa igual en la población autóctona y en la inmigrante?

OBJECTIVES: To evaluate the degree of control of blood pressure (BP) in the autochthonous and immigrant populations and to find the variables linked to good control. DESIGN: Cross-sectional, observational study. SETTING: Urban primary care team, Spain. PARTICIPANTS: All patients with hypertension seen between 1/1/2000 and 1/7/2005 and whose origin was known: 1.063 patients in all, 931 autochthonous and 132 immigrant ones. MAIN MEASUREMENTS: The main variable was hypertension control the last time BP was taken (BP

An update of the mechanisms of resistance to EGFR-tyrosine kinase inhibitors in breast cancer: Gefitinib (Iressa) -induced changes in the expression and nucleo-cytoplasmic trafficking of HER-ligands (Review).

Intrinsic resistance to the epidermal growth factor receptor (EGFR; HER1) tyrosine kinase inhibitor (TKI) gefitinib, and more generally to EGFR TKIs, is a common phenomenon in breast cancer. The availability of molecular criteria for predicting sensitivity to EGFR-TKIs is, therefore, the most relevant issue for their correct use and for planning future research. Though it appears that in non-small-cell lung cancer (NSCLC) response to gefitinib is directly related to the occurrence of specific mutations in the EGFR TK domain, breast cancer patients cannot be selected for treatment with gefitinib on the same basis as such EGFR mutations have been reported neither in primary breast carcinomas nor in several breast cancer cell lines. Alternatively, there is a general agreement on the hypothesis that the occurrence of molecular alterations that activate transduction pathways downstream of EGFR (i.e., MEK1/MEK2 right curved arrow ERK1/2 MAPK and PI-3'K right curved arrow AKT growth/survival signaling cascades) significantly affect the response to EGFR TKIs in breast carcinomas. However, there are no studies so far addressing a role of EGF-related ligands as intrinsic breast cancer cell modulators of EGFR TKI efficacy. We recently monitored gene expression profiles and sub-cellular localization of HER-1/-2/-3/-4 related ligands (i.e., EGF, amphiregulin, transforming growth factor-alpha, beta-cellulin, epiregulin and neuregulins) prior to and after gefitinib treatment in a panel of human breast cancer cell lines. First, gefitinib-induced changes in the endogenous levels of EGF-related ligands correlated with the natural degree of breast cancer cell sensitivity to gefitinib. While breast cancer cells intrinsically resistant to gefitinib (IC50 > or =15 microM) markedly up-regulated (up to 600 times) the expression of genes codifying for HER-specific ligands, a significant down-regulation (up to 10(6) times) of HER ligand gene transcription was found in breast cancer cells intrinsically sensitive to gefitinib (IC50 < or =1 microM). Second, loss of HER1 function differentially regulated the nuclear trafficking of HER-related ligands. While gefitinib treatment induced an active import and nuclear accumulation of the HER ligand NRG in intrinsically gefitinib-resistant breast cancer cells, an active export and nuclear loss of NRG was observed in intrinsically gefitinib-sensitive breast cancer cells. In summary, through in vitro and pharmacodynamic studies we have learned that, besides mutations in the HER1 gene, oncogenic changes downstream of HER1 are the key players regulating gefitinib efficacy in breast cancer cells. It now appears that pharmacological inhibition of HER1 function also leads to striking changes in both the gene expression and the nucleo-cytoplasmic trafficking of HER-specific ligands, and that this response correlates with the intrinsic degree of breast cancer sensitivity to the EGFR TKI gefitinib. The relevance of this previously unrecognized intracrine feedback to gefitinib warrants further studies as cancer cells could bypass the antiproliferative effects of HER1-targeted therapeutics without a need for the overexpression and/or activation of other HER family members and/or the activation of HER-driven downstream signaling cascades.

Mechanism of action of potato carboxypeptidase inhibitor (PCI) as an EGF blocker.

The epidermal growth factor receptor (EGFR) signal transduction pathway plays a prominent role in the development of carcinomas, and is an interesting target for antitumoral therapy. We have previously described how potato carboxypeptidase inhibitor (PCI), a 39-amino acid protease inhibitor with a T-Knot motif, binds to EGFR receptor and inhibits the activation of receptor protein tyrosine kinase. In this paper it is shown that PCI interferes with EGFR activation through inhibition of receptor dimerization and receptor transphosphorylation induced by epidermal growth factor (EGF) and by transforming growth factor alpha (TGF-alpha). Moreover, PCI blocks the formation and activation of ErbB1/ErbB-2 heterodimers that have a prominent role in carcinoma development. As a result of these effects, PCI interferes in the EGFR signal transduction pathway by reversing the effects of EGF on the growth of two tumoral cell lines, A431 and MDA-MB-453, and promotes EGFR down-regulation. These results show that PCI acts as an EGF/TGF-alpha antagonist, which suggests its therapeutic potential in the treatment of carcinomas.