Characterization and applicability of a novel physiologically relevant 3D-tetraculture bronchial model for in vitro assessment of respiratory sensitization.

Chemical Respiratory Allergy (CRA) is triggered after exposure to Low Molecular Weight (LMW) sensitizers and manifests clinically as asthma and rhinitis. From a risk/toxicity assessment point of view, there are few methods, none of them validated, for evaluating the respiratory sensitization potential of chemicals once the in vivo-based models usually employed for inhalation toxicity addressment do not comprise allergenicity endpoints specifically. Based on that, we developed, characterized, and evaluated the applicability of a 3D-tetraculture airway model reconstructed with bronchial epithelial, fibroblasts, endothelial and monocytic cell lines. Moreover, we exposed the tissue to maleic anhydride (MA) aerosols to challenge the model and subsequently assessed inflammatory and functional aspects of the tissue. The reconstructed tissue presented phenotypic biomarkers compatible with human bronchial epithelium, and MA aerosol exposure triggered an increased IL-8 and IL-6 production, reactive oxygen species (ROS) formation, and apoptosis of epithelial cells. Besides, augmented IL-8 production by monocytic cells was also found, correlating with dendritic cell activation within the co-culture model after MA exposure. Our results demonstrated that the 3D-tetraculture bronchial model presents hallmarks related to human airways' structure and function. Additionally, exposure to a respiratory sensitizer induced inflammatory and functional alterations in the reconstructed tissue, rendering it a valuable tool for exploring the mechanistic framework of chemically induced respiratory sensitization.

Mitochondrial impairment related to the immunotoxicity of the herbicides clomazone, glyphosate and sulfentrazone in THP-1 cells.

Background: Pesticides are indispensable for the cultivation of crops, especially those of economic importance, such as soybeans. Data on the annual use of herbicides in crops show that they correspond to 50%, making it the most used in agriculture. Aim: Therefore, the aim of this study was to evaluate the toxicity of the three commercial herbicides (clomazone, glyphosate, and sulfentrazone) in THP-1 cells. Methods: Cells were incubated with 0-5,000 mg/L of the herbicides for 24 h at 37 °C for cytotoxicity evaluation. Additionally, a few toxicological pathways such as reactive species generation, mitochondrial impairment, and interleukin profile, which have been previously involved in the toxicity of pesticides, were also evaluated. Results: A potential immunotoxic effect of the herbicides on THP-1 cells was observed, especially glyphosate, as it is a powerful agent of cellular immunotoxicity. It was also possible to verify an increase in oxidative stress and IL-8 levels and mitochondrial dysfunction. Conclusion: All herbicides showed cytotoxic effects in THP-1 monocytes, which were related to mitochondrial impairment.

Biological effects triggered by chemical respiratory sensitizers on THP-1 monocytic cells.

Respiratory sensitization encompasses a group of diseases that manifest through airway hyperresponsiveness and airflow limitation. Although the concerns regarding human health, to date there are still no validated methods for preclinical assessment of this class of toxicants once the chemical respiratory allergy mechanistic framework is not fully understood. As Dendritic Cells (DCs) are the bridging elements between innate and adaptative immune responses, we preliminarily investigated the biological alterations triggered by seven different LMW respiratory allergens in the DC model THP-1. The results have shown that exposure to respiratory allergens promoted alterations in DCs maturation/activation status and triggered pro-inflammatory changes in these cells through increased expression for the CD86/HLA-DR/CD11c surface biomarkers and enhancement in IL-8 and IL-6 production by exposed THP-1 cells. Therefore, evidence was found to support the startpoint for chemical respiratory allergy pathogenesis elucidation, subsidizing the contribution of dendritic cells in such pathomechanisms.

Anxiolytic- and antidepressant-like effects of new phenylpiperazine derivative LQFM005 and its hydroxylated metabolite in mice.

The current treatments available for anxiety and depression are only palliative. Full remission has remained elusive, characterizing unmet medical needs. In the scope of an academic drug discovery program, we describe here the design, synthesis, in vitro metabolism prediction and pharmacological characterization of a new piperazine compound, 1-(4-methoxyphenyl)-4-((1-phenyl-1H-pyrazol-4-yl)methyl)piperazine (LQFM005), and of its main putative metabolite, 4-(4-((4-(4-methoxyphenyl)piperazin-1-yl)methyl)- 1H-pyrazol-1-yl)phenol (LQFM235). The production of the metabolite was initially performed by in vitro biotransformation of LQFM005 using Aspergillus candidus and then by chemical synthesis. Oral administration of either 12 or 24 µmol/kg LQFM005 to mice did not affect spontaneous locomotor activity but increased the time spent in the center of the open field. Both LQFM005 and LQFM235 (24 µmol/kg) increased the time spent by the mice in the open arms of the elevated plus maze (EPM), a good indication of anxiolytic-like effect, and decreased the immobility time in the forced swimming test (FST), suggesting an antidepressant-like effect. The previous administration of WAY-100635 (a 5-HT1A antagonist) abolished the effects of LQFM005 in both EPM and FST. Binding experiments showed that LQFM005 and its metabolite bind to the 5-HT1A receptor with a moderate affinity (Ki around 5-9 µM). The two compounds are relatively safe, as indicated by cytotoxic assessment using the 3T3 fibroblast cell line and estimated LD50 around 600 mg/kg. In conclusion, oral administration of the newly synthesized phenylpiperazines produced anxiolytic- and antidepressant-like effects in behavioral tests, putatively in part through the activation of 5-HT1A receptors.

Safety and efficacy of a mucoadhesive phytomedication containing curcuminoids and Bidens pilosa L. extract in the prevention and treatment of radiochemotherapy-induced oral mucositis: Triple-blind, randomized, placebo-controlled, clinical trial.

BACKGROUND: Oral mucositis (OM) is the significant complication of radio/chemotherapy treatment. This study evaluated the safety and efficacy of a mucoadhesive phytomedication containing curcuminoids and Bidens pilosa L. (FITOPROT) in the prevention/treatment of OM. METHODS: Sixty-two patients were randomized into the group's intervention and placebo. Adverse effect assessment, OM grading, pain, and saliva collection were carried at the 1st, 15th, 21st, and final of radiotherapy (RT). Inflammatory salivary mediators were measured. RESULTS: FITOPROT decreased the severity of OM from the 15th to the final RT, while the placebo showed an increase in the severity (p < 0.05). Intervention group had a lower number of patients with ulcerated OM at the final RT (p < 0.05). Phytomedication prevented increases of IL-8 levels and reduced the salivary nitrite during RT. CONCLUSIONS: FITOPROT does not promote adverse effects, it appears to be effective at reducing the severity of OM, and it controls the concentration of pro-inflammatory mediators.

Strategy Combining Nonanimal Methods for Ocular Toxicity Evaluation.

Historically, the ocular toxicity of manufactured consumer materials has been evaluated using the rabbit in vivo Draize rabbit eye test. The animal data obtained were used by the United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS) to define the classification and labelling (C&L) for eye damage/irritation endpoint. However, the Draize test, a method which was never formally validated, has been widely criticized because of its technical limitations. In addition, ethical and economic issues and advances in scientific knowledge, and political and public pressures have made animal experimentation unsustainable. This scenario has consequently led to the development of nonanimal testing and protocols/approaches with considerable predictive value and relevance for humans. It is widely accepted that one single nonanimal method cannot cover all the criteria of damage/inflammation assessed by regulatory adopted in vivo animal testing. Thus, integrated testing strategies (ITS) have been proposed, including a tiered testing approach combining different nonanimal testing with different endpoints, which have been used for regulatory purposes, on a case-by-case basis and within integrated approaches to testing and assessment (IATA), to identify materials according to their ability to trigger eye damage. In particular, the top-down and bottom-up approaches have been recommended for the C&L of materials, which cause serious eye damage or eye irritation, respectively. This chapter describes detailed protocols for eye irritation testing based on cells (Short Time Exposure-STE, OECD No. 491/2017), a vascularized membrane (the Hen's Egg Test-Chorioallantoic Membrane-HET-CAM) and corneal tissue (Bovine Corneal Opacity and Permeability-BCOP, OECD No. 437/2017), which can be applied using top-down or bottom-up approaches. In addition, it suggests making a corneal histomorphometric evaluation as an additional parameter in the BCOP method to differentiate materials that cause serious eye tissue damage (UN GHS Cat. 1) from materials that have reversible eye irritation effects (UN GHS Cat. 2).

Assessing Agricultural Toxicity in Brazil: Advances and Opportunities in the 21st Century.

Agriculture in the 21st century faces multiple challenges to produce food for the growing population using ethical/sustainable and efficient methods safely for humans and the environment. Brazil today is a world leader in terms of production of food of plant origin, both for human consumption and animal feed. Agriculture and livestock raising are critical economic activities in maintaining a positive balance in its economy. As a consequence, the registration and use of pesticides in Brazil have grown at an accelerated rate. This work shows the current situation in Brazil in terms of the prevailing laws about the registration of pesticides, with a focus on the toxicological aspects related to human health. The regulatory aspects of registration of pesticides in Brazil, the mandatory testing for evaluating pesticide toxicity, adoption of the Globally Harmonized System of Classification and Labeling of Chemicals, and recent progress toward nonanimal methods to toxicity evaluation were explored in this work. In this field, Brazil has advanced and there are opportunities and challenges. There is still much to be done and investments to be made so that Brazil can definitively consolidate its conduct within the context of a Modern Regulatory Toxicology, which has entered the 21st century.

Innovative strategy based on mechanisms to substitute animal testing for ocular toxicity assessment of agrochemical formulations market in Brazil.

Considering the successful employment of alternative methods for eye toxicity assessment of products for regulatory purposes, and the recent advances in Brazilian legislative scenario, which adopted the UN GHS classification system for agrochemical formulations toxicity assessment, there is an emerging demand for strategies that allow the evaluation of such products. Based on this, the present study aimed to address the applicability of a mechanistic-based defined approach for eye toxicity assessment of agrochemical formulations. It was investigated the opacity/permeability, depth and location of corneal injury in bovine cornea, and vascular events in chorioallantoic membrane induced for different Brazilian agrochemicals using a Sequential Testing Strategy (STS). Cytotoxicity induced by the agrochemical formulations was evaluated by Short Time exposure (STE) (OECD TG 491) assay (step 1), corneal injury was investigated by standard Bovine Corneal Opacity and Permeability (BCOP) (OECD TG 437) followed by histopathological evaluation (step 2), and Hen Chorionic-allantoic Membrane test (HET-CAM) was used to evaluate vascular injury (step 3). The results demonstrated that the proposed defined approach enabled a classification corresponding UN GHS classification of agrochemical formulations while minimizing the use of live animals. Therefore, this approach may be useful for categorization of agrochemicals in Brazil according to the new regulatory scenario.

Small-molecule MDM2 inhibitor LQFM030-induced apoptosis in p53-null K562 chronic myeloid leukemia cells.

Our group designed and synthesized the N-phenyl-piperazine LQFM030 [1-(4-((1-(4-chlorophenyl)-1H-pyrazol-4-yl)methyl) piperazin-1-yl) ethanone], a small molecule derived from molecular simplification of the Nutlin-1, an inhibitor of the human homologue of murine double minute 2 (MDM2) protein that is expressed in several types of cancer. To better investigate the effects of LQFM030 regarding the p53 mutation status, this study investigated the antiproliferative activity of LQFM030 against the p53-null K562 leukemia cells as well as the cell death pathways involved. In addition, the effects of LQFM030 on the levels of the p53/MDM2 complex were also carried out using 3T3 cells as a p53 wild-type model. Our data suggest that LQFM030 triggered apoptosis in K562 cells via different mechanisms including cell cycle arrest, caspase activation, reduction of mitochondrial activity, decrease in MDM2 expression, and transcriptional modulation of MDMX, p73, MYC, and NF-ĸB. Additionally, it promoted effects in p53/MDM2 binding in p53 wild-type 3T3 cells. Therefore, LQFM030 has antiproliferative effects in cancer cells by a p53 mutation status-independent manner with different signaling pathways. These findings open new perspectives to the treatment of leukemic cells considering the resistance development associated with cancer treatment with conventional cytotoxic drugs.

Corneal histomorphometric analysis: The depth of damage induced in the bovine cornea correlates with the severity of the ocular toxicity.

The Bovine Corneal Opacity and Permeability (BCOP) assay is an alternative method used to ocular toxicity potential assessment of chemicals and mixtures. The standard BCOP test provides information about permeability and opacity, however, corneal histopathological analysis has been recommended as an additional parameter to better categorize eye irritants. Moreover, such analysis associated with depth of substance-induced corneal injury analysis may provide additional scientific measurement for the refinement of BCOP test. The aim of this study was to measure the depth of injury into the bovine cornea induced by eye irritants and associate it with the damage severity. For this purpose, BCOP assay was performed for 12 substances from different Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS) categories and, additionally, corneal sections of 5 µm thickness were obtained and analyzed by fluorescence microscopy. The results showed that the fluorescein permeation depth was directly proportional to the substances irritation degree. Severe irritants promoted highest rates of permeation followed by moderate and mild irritants, while non-irritants showed similar permeation indexes to the negative control. The refinement of BCOP by the depth of injury analysis through epithelial permeation of fluorescein can be considered a useful quantitative parameter to better categorize eye irritants.

Increased Nose-to-Brain Delivery of Melatonin Mediated by Polycaprolactone Nanoparticles for the Treatment of Glioblastoma.

PURPOSE: Intranasal administration has been extensively applied to deliver drugs to the brain. In spite of its unfavorable biopharmaceutic properties, melatonin (MLT) has demonstrated anticancer effects against glioblastoma. This study describes the nose-to-brain delivery of MLT-loaded polycaprolactone nanoparticles (MLT-NP) for the treatment of glioblastoma. METHODS: MLT-NP were prepared by nanoprecipitation. Following intranasal administration in rats, brain targeting of the formulation was demonstrated by fluorescence tomography. Brain and plasma pharmacokinetic profiles were analyzed. Cytotoxicity against U87MG glioblastoma cells and MRC-5 non-tumor cells was evaluated. RESULTS: MLT-NP increased the drug apparent water solubility ~35 fold. The formulation demonstrated strong activity against U87MG cells, resulting in IC50 ~2500 fold lower than that of the free drug. No cytotoxic effect was observed against non-tumor cells. Fluorescence tomography images evidenced the direct translocation of nanoparticles from nasal cavity to the brain. Intranasal administration of MLT-NP resulted in higher AUCbrain and drug targeting index compared to the free drug by either intranasal or oral route. CONCLUSIONS: Nanoencapsulation of MLT was crucial for the selective antitumoral activity against U87MG. In vivo evaluation confirmed nose-to-brain delivery of MLT mediated by nanoparticles, highlighting the formulation as a suitable approach to improve glioblastoma therapy.

Mechanistic-based non-animal assessment of eye toxicity: Inflammatory profile of human keratinocytes cells after exposure to eye damage/irritant agents.

Eye toxicity is a mandatory parameter in human risk and safety evaluation for products including chemicals, pesticides, medicines and cosmetics. Historically, this endpoint has been evaluated using the Draize rabbit eye test, an in vivo model that was never formally validated. Due to advances in scientific knowledge, economic and ethical issues, non-animal methods based on mechanisms of toxicity are being developed and validated for increasing the capability of these models to predict eye toxicity. In this study, the Cytometric Bead Array (CBA) and ELISA assays were used to evaluate the inflammatory cytokine profile produced by HaCaT human keratinocytes after exposure to chemicals with different UN GHS eye toxicity classifications, aiming to stablish a correlation between inflammatory endpoints and eye toxicity (damage/irritation) potential. As a first step, cytotoxic profile of the chemicals, including 3 non-irritants and 10 eye toxicants (GHS Category 1, 2A and 2B), was evaluated after 24 h exposure using MTT assay and Inhibitory Concentration of 20% of cell viability (IC20) was calculated for each chemical. Then, the cells were exposed to these chemicals at IC20 for 24 h and supernatants and cell lysates were analyzed by CBA assay for quantification of the following cytokines: IL-6, IL-8, IL-10, IL-1ß, TNF and IL-12p70. Regarding cytotoxicity evaluation, chemicals showed different cytotoxicity profiles and data demonstrated no correlation with their UN GHS classification. Among the cytokines evaluated, IL-1ß production has changed after exposure and such alterations were confirmed by quantification employing ELISA method. The higher intracellular levels of IL-1ß were found in GHS Category 1 chemicals, followed by Category 2A and 2B, while non irritants did not induce such increase. Thus, these findings show that IL-1ß measurement, using HaCaT model, can be a considerable biomarker to identify chemicals according to their potential in promote eye toxicity, differentiating damage from irritation potential.

Chemopreventive effects of FITOPROT against 5-fluorouracil-induced toxicity in HaCaT cells.

AIMS: This study evaluated the mechanisms involved in the chemopreventive effects of a mucoadhesive formulation (FITOPROT), containing curcuminoids from Curcuma longa L. (Zingiberaceae) and Bidens pilosa L. (Asteraceae) extract, against 5-FU-induced cellular toxicity using an in vitro oral mucositis model. MAIN METHODS: Effects of FITOPROT on 5-FU-induced cytotoxicity in HaCaT and SSC-4 cells were evaluated by MTT assay. For mechanistic analyses, HaCaT cells were first pretreated with FITOPROT (0.005%) for 24h followed by treatment with FITOPROT and simultaneously exposed to 5-FU (10µg/mL) for additional 24h. KEY FINDINGS: FITOPROT was able to protect HaCaT cells from 5-FU-triggered cell damage. Moreover, the FITOPROT+5-FU association showed higher cytotoxic effects on SSC-4 cancer cells. Flow cytometry and/or fluorescence microscopy analysis showed FITOPROT was able to significantly reduce ROS generation and prevent mitochondrial changes in HaCaT cells. In addition, it avoided the release of cytochrome c from mitochondria to the cytoplasm in cells exposed to 5-FU, and restored their proliferative activity via Ki-67 expression. Furthermore, FITOPROT regulated 5-FU-induced oxidative stress via Nrf2 involvement. HaCaT cells pretreated/treated with FITOPROT also showed normal expression of TNF-R1 and NF-κB inflammatory proteins and decreased levels of pro-inflammatory cytokines (TNF, IL-1ß, IL-6 and IL-8). Moreover, a high-resolution liquid chromatography-mass spectrometry analysis showed the presence of flavonoids rutin, glucoronylated quercetin and dimethylquercetin rutenoside in FITOPROT. SIGNIFICANCE: It was showed that FITOPROT, an antioxidant phytochemicals-rich mucoadhesive formulation, exerts chemopreventive effects against 5-FU-triggered toxicity through antioxidant and anti-inflammatory mechanisms and restoration of proliferative capacity in HaCaT cells.

A novel chalcone derivative, LQFM064, induces breast cancer cells death via p53, p21, KIT and PDGFRA.

This study shows the design, synthesis and antitumoral potential evaluation of a novel chalcone-like compound, (E)-3- (3, 5-di-ter-butyl-4-hydroxyphenyl)-1- (4-hydroxy-3-methoxyphenyl) prop-2-en-1-one [LQFM064) (4)], against human breast adenocarcinoma MCF7 cells. Some toxicological parameters were also investigated. LQFM064) (4) exhibited cytotoxic activity against MCF7 cells (IC50=21µM), in a concentration dependent-manner, and triggered significant changes in cell morphology and biochemical/molecular parameters, which are suggestive of an apoptosis inductor. LQFM064) (4) (21µM) induced cell cycle arrest at G0/G1 phase with increased p53 and p21 expressions. It was also shown that the compound (4) did not interfere directly in p53/MDM2 complexation of MCF7 cells. In these cells, externalization of phosphatidylserine, cytochrome c release, increased expression of caspases-7, -8 and -9, reduced mitochondrial membrane potential and ROS overgeneration were also detected following LQFM064 (4) treatment. Further analysis revealed the activation of both apoptotic pathways via modulation of the proteins involved in the extrinsic and intrinsic pathways with an increase in TNF-R1, Fas-L and Bax levels and a reduction in Bcl-2 expression. Furthermore, KIT proto-oncogene receptor tyrosine kinase, insulin-like growth factor (IGF1) and platelet-derived growth factor receptor A (PDGFRA) were downregulated, while glutathione S-transferase P1 (GSTP1) and interferon regulatory factor 5 (IRF5) expressions were increased by LQFM064 (4)-triggered cytotoxic effects in MCF7 cells. Moreover, it can be inferred that compound (4) has a moderate acute oral systemic toxicity hazard, since its estimated LD50 was 452.50mg/kg, which classifies it as UN GHS Category 4 (300mg/kg>LD50<2000mg/kg). Furthermore, LQFM064 (4) showed a reduced potential myelotoxicity (IC50=150µM for mouse bone marrow hematopoietic progenitors). In conclusion, LQFM064 (4) was capable of inducing breast cancer cells death via different cytotoxic pathways. Thus, it is a promising alternative for the treatment of neoplasias, especially in terms of the drug resistance development.