Sens-ocular model: Cell-based assay to evaluate eye stinging potential of chemicals and baby cosmetic formulations.

The TRPV1 receptor, which is known to contribute significantly to pain perception, has recently been identified as a useful tool for predicting eye stinging potential in cosmetics. In this study, HEK-293 cells with high TRPV1 expression were utilized to evaluate calcium influx related to receptor activation triggered by chemicals and cosmetic formulations. The cells were exposed to increasing concentrations of substances to cause or not some aggression to the eye, and TRPV1 activity was assessed by measuring intracellular FURA-2 AM fluorescence signal. To confirm TRPV1 channel activation, capsazepine, a capsaicin antagonist, was employed in addition to using capsaicin as a positive control. The study's results indicate that this novel model can identify compounds known to cause some aggression to the eye, such as stinging, considering a cut-off value of 60% of Ca2+ influx exposed to the lowest evaluated concentration (0.00032%). When applied to the cosmetic baby formulation, although the presented model exhibited higher sensitivity by classifying as stinging formulations that had previously undergone clinical testing and were deemed non-stinging, the assay could serve as a valuable in vitro tool for predicting human eye stinging sensation and can be used as a tier 1 in an integrated testing strategy.

Identification of toxicity-induced biomarkers in human non-immune airway cells exposed to respiratory sensitizers: A mechanistic approach.

Occupational asthma covers a group of work-related diseases whose clinical manifestations include airway hyperresponsiveness and airflow limitation. Although the chemical respiratory allergy (CRA) induced by Low Molecular Weight (LMW) sensitizers is a major concern, especially in terms of the regulatory framework, to date there are no methods available for preclinically addressing this toxicological outcome, as its mechanistic background is not fully understood at molecular or cellular levels. This paper proposes a mechanistic study applying New Approach Methodologies (NAM) of the pro-inflammatory and functional effects triggered by LMW respiratory allergens in different respiratory tract cell lines, including bronchial epithelial (BEAS-2B), lung fibroblast (MRC-5), and endothelial cells (EA.hy926), and an analysis of the capacity of such chemicals to interact with the mucin protein, to address certain toxicodynamic aspects of such compounds. The results showed that some of the sensitizers evaluated interact with mucin, the main protein mucus component, but the toxicant-mucin complex formation does not seem to be a common feature of different chemical classes of allergens. At a cellular level, sensitizers promoted an increase in IL-8, IL-6, and IL-1ß production in the evaluated cell types. It also impaired the MUC1 expression by bronchial cells and activated endothelial cells, thereby increasing the ICAM-I surface expression. Taken together, our results showed that these aforementioned cell types participate in the CRA Adverse Outcome Pathway and must be considered when developing preclinical testing strategies, particularly investigating danger signal production after exposure to LMW sensitizers in different tissue compartments.

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.

Brazilian National Network of Alternative Methods (RENAMA) 10th Anniversary: Meeting of the Associated Laboratories, May 2022.

The Brazilian National Network of Alternative Methods (RENAMA), which is linked to the Ministry of Science, Technology and Innovation, is currently comprised of 51 laboratories from CROs, academia, industry and government. RENAMA's aim is to develop and validate new approach methodologies (NAMs), as well as train researchers and disseminate information on their use - thus reducing Brazilian, and consequently Latin American, dependence on external technology. Moreover, it promotes the adoption of NAMs by educators and trained researchers, as well as the implementation of good laboratory practice (GLP) and the use of certified products. The RENAMA network started its activities in 2012, and was originally comprised of three central laboratories - the National Institute of Metrology, Quality and Technology (INMETRO); the National Institute of Quality Control in Health (INCQS); and the National Brazilian Biosciences Laboratory (LNBio) - and ten associated laboratories. In 2022, RENAMA celebrated its 10th anniversary, a milestone commemorated by the organisation of a meeting attended by different stakeholders, including the RENAMA-associated laboratories, academia, non-governmental organisations and industry. Ninety-six participants attended the meeting, held on 26 May 2022 in Balneário Camboriú, SC, Brazil, as part of the programme of the XXIII Brazilian Congress of Toxicology 2022. Significant moments of the RENAMA were remembered, and new goals and discussion themes were established. The lectures highlighted recent innovations in the toxicological sciences that have translated into the assessment of consumer product safety through the use of human-relevant NAMs instead of the use of existing animal-based approaches. The challenges and opportunities in accepting such practices for regulatory purposes were also presented and discussed.

Non-animal approaches for photoallergenicity safety assessment: Needs and perspectives for the toxicology for the 21st century.

Certain chemicals and/or their byproducts are photoactivated by UV/VIS and trigger a dermal allergenic response, clinically recognized as photoallergic contact dermatitis (PACD). It is important to identify the chemicals which are potentially photoallergenic, not only for establishing the correct differential diagnosis between PACD and other photodermatoses, but also as causative agents which should be avoided as a preventative measure. Moreover, materials with photoallergenic properties need to be correctly identified to allow thorough safety assessments for their use in finished products (e.g. cosmetics). Development of methods for predicting photoallergenicity potential of chemicals has advanced at slow pace in recent years. To date, there are no validated methods for photosensitisation potential of chemicals for regulatory purposes, although it remains a required endpoint in some regions. The purpose of this review is to explore the mechanisms potentially involved in the photosensitisation process and discuss the methods available in the literature for identification of photosensitisers. The review also explores the possibilities of further research investment required to develop human-relevant new approach methodologies (NAMs) and next generation risk assessment (NGRA) approaches, considering the current perspectives and needs of the Toxicology for the 21st Century.

Effects of a mucoadhesive phytomedicine (Curcuma longa L. and Bidens pilosa L.) on radiotherapy-induced oral mucositis and quality of life of patients undergoing head and neck cancer treatment: randomized clinical trial.

PURPOSE: To assess the effect of a mucoadhesive herbal medicine containing curcuminoids and a glycerinated extract of Bidens pilosa L. (FITOPROT) in association with photobiomodulation (PBM) therapy and a Preventive Oral Care Program (POCP) compared to PBM and POCP in the treatment of radiotherapy (RT)-induced oral mucositis (ROM) and in the quality of life of these patients. METHODS: A double-blind clinical trial was performed with head and neck cancer patients undergoing RT or chemoradiotherapy. Participants were randomized into two groups: Group 1 (n=27): PBM and POCP; and Group 2 (n=25): PBM, POCP and FITOPROT. The PBM protocol was daily irradiation, 660 nm, 25mW, 0.25 J/point from the first until the last day of RT. The FITOPROT was used as mouthwash twice a day. ROM was evaluated based on the scales of the World Health Organization and National Cancer Institute. The quality of life was evaluated using the University of Washington Questionnaire, OHIP-14 and Patient-Reported Oral Mucositis Symptom Scale. The MMAS-8 questionnaire was used to evaluated the adherence to POCP and FITOPROT. Data were collected at baseline, 7th, 14th, 21st, and 30th RT sessions. RESULTS: No statistical differences were found between the groups for the ROM evaluation. Both groups experienced worsening of the quality of life during the RT. No statistically significant differences between groups were observed for any of the instruments evaluated. CONCLUSION: The results suggest that PBM associated with FITOPROT and POCP control the severity of ROM and stabilize the QoL of patients with head and neck cancer. TRIAL REGISTRATION: Brazilian Registry of Clinical Trials (ReBEC-RBR-9vddmr; UTN code: U1111-1193-2066), registered in August 8th, 2017.

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.

The Application of New Approach Methodologies in Respiratory Disease Research: Their Role in Improving Translational Medicine from Bench to Bedside.

The SARS-CoV-2 outbreak focused global attention on the shortcomings of the drug discovery process. It led to its acceleration in several areas, particularly in the processes associated with the development and approval of COVID-19 vaccines. This situation contrasts with the low approval rates of new drugs for respiratory system diseases (e.g. asthma, chronic obstructive pulmonary disease, cancer, tuberculosis), which are leading causes of morbidity and mortality worldwide. In this context, innovation in respiratory system drug discovery is surely needed, and it is most likely to succeed through the use of preclinical models that are cost-effective, high-throughput and generate predictive human-relevant outcomes. Here, we highlight several non-animal new approach methodologies (NAMs) and their applications in respiratory research. We describe their potential uses for efficacy and toxicity assessments, to optimise the drug development process and reduce the high failure rates in clinical trials.

Paclitaxel-Loaded Lipid-Coated Magnetic Nanoparticles for Dual Chemo-Magnetic Hyperthermia Therapy of Melanoma.

Melanoma is the most aggressive and metastasis-prone form of skin cancer. Conventional therapies include chemotherapeutic agents, either as small molecules or carried by FDA-approved nanostructures. However, systemic toxicity and side effects still remain as major drawbacks. With the advancement of nanomedicine, new delivery strategies emerge at a regular pace, aiming to overcome these challenges. Stimulus-responsive drug delivery systems might considerably reduce systemic toxicity and side-effects by limiting drug release to the affected area. Herein, we report the development of paclitaxel-loaded lipid-coated manganese ferrite magnetic nanoparticles (PTX-LMNP) as magnetosomes synthetic analogs, envisaging the combined chemo-magnetic hyperthermia treatment of melanoma. PTX-LMNP physicochemical properties were verified, including their shape, size, crystallinity, FTIR spectrum, magnetization profile, and temperature profile under magnetic hyperthermia (MHT). Their diffusion in porcine ear skin (a model for human skin) was investigated after intradermal administration via fluorescence microscopy. Cumulative PTX release kinetics under different temperatures, either preceded or not by MHT, were assessed. Intrinsic cytotoxicity against B16F10 cells was determined via neutral red uptake assay after 48 h of incubation (long-term assay), as well as B16F10 cells viability after 1 h of incubation (short-term assay), followed by MHT. PTX-LMNP-mediated MHT triggers PTX release, allowing its thermal-modulated local delivery to diseased sites, within short timeframes. Moreover, half-maximal PTX inhibitory concentration (IC50) could be significantly reduced relatively to free PTX (142,500×) and Taxol® (340×). Therefore, the dual chemo-MHT therapy mediated by intratumorally injected PTX-LMNP stands out as a promising alternative to efficiently deliver PTX to melanoma cells, consequently reducing systemic side effects commonly associated with conventional chemotherapies.

Challenges and opportunities for overcoming dog use in agrochemical evaluation and registration.

Progress in developing new tools, assays, and approaches to assess human hazard and health risk provides an opportunity to re-evaluate the necessity of dog studies for the safety evaluation of agrochemicals. A workshop was held where partic­ipants discussed the strengths and limitations of past use of dogs for pesticide evaluations and registrations. Opportunities were identified to support alternative approaches to answer human safety questions without performing the required 90-day dog study. Development of a decision tree for determining when the dog study might not be necessary to inform pesticide safety and risk assessment was proposed. Such a process will require global regulatory authority participation to lead to its acceptance. The identification of unique effects in dogs that are not identified in rodents will need further evaluation and determination of their relevance to humans. The establishment of in vitro and in silico approaches that can provide critical data on relative species sensitivity and human relevance will be an important tool to advance the decision process. Promising novel tools including in vitro comparative metabolism studies, in silico models, and high-throughput assays able to identify metabolites and mechanisms of action leading to development of adverse outcome pathways will need further development. To replace or eliminate the 90-day dog study, a collaborative, multidisciplinary, international effort that transcends organi­zations and regulatory agencies will be needed in order to develop guidance on when the study would not be necessary for human safety and risk assessment.

The Potential of Vouacapanes from Pterodon emarginatus Vogel Against COVID-19 Cytokine Storm.

Purpose: The emergence of the COVID-19 pandemic has led to the search for potential therapeutic responses for various aspects of this disease. Fruits of Pterodon emarginatus Vogel (Fabaceae), sucupira, have been used in Brazilian traditional medicine because of their anti-inflammatory properties, which have been proven in vivo, in vitro, and in silico. Therefore, the aim of this work is to evaluate P. emarginatus oleoresin and isolated diterpenes by in vitro anti-inflammatory models. Methods: In this study, the mechanisms underlying the anti-inflammatory activity of P. emarginatus oleoresin and vouacapanes 6α,19ß-diacetoxy-7ß,14ß-dihydroxyvouacapan (V1), 6α-acetoxy-7ß,14ß-dihydroxyvouacapan (V2), and methyl 6α-acetoxy-7ß-hydroxyvouacapan-17ß-oate (V3) were investigated in HaCaT cells. Results: Oleoresin, V2, and V3 inhibited phospholipase A2 (30.78%, 24.96%, and 77.64%, respectively). Both vouacapanes also inhibited the expression of COX-2 (28.3% and 33.17%, respectively). The production of interleukin 6 (IL-6) was inhibited by oleoresin by 35.47%. However, oleoresin did not interfere with Nrf-2 expression or IL-8 production. Conclusion: The results support the ethnomedicinal use of P. emarginatus oleoresin as an anti-inflammatory herbal medicine, and also highlight P. emarginatus oleoresin and isolated vouacapanes as an attractive therapeutic approach for COVID-19 through the reduction or chronological control of the inflammatory mediators IL-6, cyclooxygenase-2 (COX-2), phospholipase A2, and INF-y (indirectly) during the SARS-CoV-2 infection process.

Pre-clinical safety of topically administered sunitinib-loaded lipid and polymeric nanocarriers targeting corneal neovascularization.

Three different types of sunitinib-loaded (SUN-loaded) nanocarriers were compared, aiming at the topical treatment of corneal neovascularization (CNV): polymeric nanospheres (NS), liposomes (LIP), and solid lipid nanoparticles (SLN). Three out of eleven formulations prepared for an optimization study - the best SUN-loaded nanocarrier of each assessed type (NS, LIP, and SLN) - were selected, based on their size, polydispersity index (PdI), drug load (DL), and encapsulation efficiency (EE). These three optimal formulations were further characterized by nanoparticle tracking analysis (NTA), electron paramagnetic resonance (EPR) spectroscopy, and zeta potential. In vitro SUN release profiles were obtained for the optimal formulations, along with ex vivo corneal permeability/retention studies, and ocular tolerance assays, namely: the bovine corneal opacity and permeability (BCOP) assay, the HET-CAM test (hen's egg test - chorioallantoic membrane), and hemolytic potential (HP) assay. None of the optimal formulations exhibited toxicity or potential for ocular irritation. SLN showed higher surface fluidity, drug release more suitable for topical ocular applications, besides greater SUN corneal retention. Our results suggest that SLN are the best CNV-targeting SUN-loaded nanocarriers for clinical translation when compared to their NS and LIP analogues.

Development of a novel ex vivo model for chemical ocular toxicity assessment and its applicability for hair straightening products.

This study developed an air-liquid interface (ALI) corneal model using explants bovine eyes for ocular toxicity assessment of ten chemicals and seven hair straightening mixtures. It was successfully maintained physiologically viable and normal for six days. Both eye damage (GHS cat. 1) and irritating (GHS cat. 2) chemicals induced corneal injury in our model. However, cat. 2 irritants triggered moderate damage when compared to cat. 1 agents, which induced a marked cytotoxicity profile. The mixtures were also able to trigger viability reduction associated with histopathological changes in the corneal tissues, especially when the exposure was via aerosol particles. Thus, the chemical exposure microenvironment simulation seemed to provide more reliable toxicological data. Moreover, mixture-induced corneal damage correlated with increased ROS levels, suggesting a close correlation between tissue death and oxidative stress. Besides mixtures showing the potential to induce moderate/mild ocular toxicity, we could verify that the corneal tissue damage showed reversibility due to the recovery from the injury after exposure to some of the mixtures. Hence, our ex vivo corneal model seems to be a simple and cost-effective approach for future studies related to further investigating the reversibility of damage in the cornea triggered by chemicals and their mixtures.

The Perceptions of Students and Lecturers on the Use of Animals in Biomedical Science Undergraduate Education in Brazil.

The use of animals in research and education is a controversial topic that has raised extensive debates. Undergraduate students (n = 404) and lecturers (n = 62) from biomedical science schools at the Federal University of Goiás (UFG) in the municipality of Goiânia, Jataí and Catalão, Goiás, Brazil, were asked about their knowledge and opinions on bioethics, the use and importance of animals in education, the replacement of animal use with non-animal alternatives, and the current legislation of the National Council for the Control of Animal Experimentation (CONCEA) that bans animal use in some practical classes within technical and higher education (i.e. Resolution No. 53/2021). Most students and lecturers agreed not only that animal use can contribute to education, but also that it is important to replace this animal use with innovative non-animal alternatives where appropriate. The lecturers emphasised that the replacement of animal models will be possible only with the provision of appropriate training to improve the skills of educators in their use, as well as ensuring reliable access to suitable facilities and materials.

Folate-Targeted PEGylated Magnetoliposomes for Hyperthermia-Mediated Controlled Release of Doxorubicin.

Doxorubicin (DOX) is a chemotherapeutic agent commonly used for the treatment of solid tumors. However, the cardiotoxicity associated with its prolonged use prevents further adherence and therapeutic efficacy. By encapsulating DOX within a PEGylated liposome, Doxil® considerably decreased DOX cardiotoxicity. By using thermally sensitive lysolipids in its bilayer composition, ThermoDox® implemented a heat-induced controlled release of DOX. However, both ThermoDox® and Doxil® rely on their passive retention in tumors, depending on their half-lives in blood. Moreover, ThermoDox® ordinarily depend on invasive radiofrequency-generating metallic probes for local heating. In this study, we prepare, characterize, and evaluate the antitumoral capabilities of DOX-loaded folate-targeted PEGylated magnetoliposomes (DFPML). Unlike ThermoDox®, DOX delivery via DFPML is mediated by the heat released through dynamic hysteresis losses from magnetothermal converting systems composed by MnFe2O4 nanoparticles (NPs) under AC magnetic field excitation-a non-invasive technique designated magnetic hyperthermia (MHT). Moreover, DFPML dismisses the use of thermally sensitive lysolipids, allowing the use of simpler and cheaper alternative lipids. MnFe2O4 NPs and DFPML are fully characterized in terms of their size, morphology, polydispersion, magnetic, and magnetothermal properties. About 50% of the DOX load is released from DFPML after 30 min under MHT conditions. Being folate-targeted, in vitro DFPML antitumoral activity is higher (IC50 ≈ 1 µg/ml) for folate receptor-overexpressing B16F10 murine melanoma cells, compared to MCF7 human breast adenocarcinoma cells (IC50 ≈ 4 µg/ml). Taken together, our results indicate that DFPML are strong candidates for folate-targeted anticancer therapies based on DOX controlled release.

Cytotoxic activity in basal and tumoral cell lines of the C0K3N3 protein from the snake venom Crotalus durissus collilineatus, variety crotamine negative.

Snake venoms are natural sources of bioactive substances with therapeutic potential. In this work, we evaluated the cytotoxicity of the Crotalus durissus collilineatus, negative crotamine variety and the isolated fraction C0K3N3 in BALB C/3T3 and K562 cell lines. The results indicate that the C0K3N3 protein is more cytotoxic against the K562 tumor cell line than in the 3T3 baseline.

In vitro and in vivo cytotoxicity assessment of glyphosate and imazethapyr-based herbicides and their association.

Resistance to glyphosate herbicide has initiated usage of combined application of herbicides as a weed control measure. Imazethapyr-based herbicides associated with glyphosate herbicide seem to be an alternative to suppress weed resistance. The aim of this study was to examine the adverse effects of Glyphosate Atanor 48® (ATN) and Imazethapyr Plus Nortox® (IMZT) formulations in both single forms and mixtures using HepG2 cells and zebrafish early-life stages models. Data demonstrated cytotoxicity due to exposure to ATN, IMZT for both models, as follows: (1) ATN (0.5 mg/L), IMZT (5 mg/L), and M3 (0.05 mg/L ATN + 5 mg/L IMZT) increased cytotoxicity by disturbing the mitochondrial activity of HepG2 cells 24 hr after exposure; (2) ATN and IMZT (5 mg/L), and M3 (0.05 mg/L ATN + 5 mg/L IMZT) also decreased the integrity of the membrane of HepG2 cells after 24 hr incubation; (3) only ATN and IMZT (5 mg/L) in their single forms diminished the mitochondrial potential of zebrafish; (4) ATN (single form) at 0.5 mg/L induced apoptosis in zebrafish larvae. In conclusion, these herbicides in their single forms appeared to produce greater cytotoxicity to HepG2 cells and zebrafish compared to the herbicide mixtures.

Ex vivo pulmonary assay applied for screening of toxicity potential of chemicals.

Acute inhalation toxicity testing for chemical classification and labeling has been performed using animal models, however, these models have limited predictibility of the toxicity on the respiratory system. Thus, non-animal models have been emerging as alternatives for preclinical assessment of respiratory toxicity of chemicals, comprising in chemico, in vitro, ex vivo, and in silico approaches. In this study, we characterized and evaluated the applicability of a new ex vivo bovine bronchial model for addressing key aspects of pulmonary toxicity. Standardized bronchial fragments were cultured at an air-liquid interface for seven days showing cell viability, morphology, and function during the ex vivo time of cultivation. Different exposure ways, liquid or aerosol exposure, were also studied using paraformaldehyde (PFA) as a positive control. In a concentration-dependent manner, a decrease in tissue viability was observed for aerosols instead of direct liquid exposure upon tissue surface. Moreover, PFA exposure allowed the addressment of several damage biomarkers, including epithelium thickness, mitochondrial activity, ROS production, and caspase-3 activation. Besides, the bronquial tissue was exposed to chemicals from different UN GHS inhalation toxicity categories and presented a concentration-dependent response for most of the evaluated materials. The proposed airway ex vivo model represents a low-cost and reproducible tool applicable for pulmonary toxicity assessment of chemicals.

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.