RESUMO
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.
Assuntos
Alternativas aos Testes com Animais , Neuropatia Óptica Tóxica , Bovinos , Animais , Testes de Toxicidade , Irritantes/toxicidade , Córnea/patologia , CabeloRESUMO
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).
Assuntos
Alternativas aos Testes com Animais , Bioensaio , Membrana Corioalantoide/irrigação sanguínea , Membrana Corioalantoide/efeitos dos fármacos , Córnea/efeitos dos fármacos , Irritantes/toxicidade , Neuropatia Óptica Tóxica , Testes de Toxicidade , Animais , Bovinos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Embrião de Galinha , Córnea/patologia , CoelhosRESUMO
Punicalagin is a phenolic compound extracted from Lafoensia pacari A. St.-Hil (Lythraceae) leaves. It has demonstrated interesting activity against pathogenic fungi, e.g., Cryptococcus gattii and Candida albicans, by inhibiting fungi growth in a minimum inhibitory concentration (MIC) at 4 µg/mL. However, the mechanisms behind its antifungal action are not well understood. In this study, certain parameters were investigated, by transmission electron microscopy, ergosterol synthesis inhibition, and flow cytometry analyses, to gain insight into the possible biological targets of punicalagin (4 or 16 µg/mL) against yeast cells. Data showed that, in contrast to untreated cells, punicalagin triggered severe ultrastructural changes in C. gattii and C. albicans, such as disorganization of cytoplasmic content and/or thickened cell walls. In addition, it caused a decrease in yeast plasma membrane ergosterol content in a concentration-dependent manner. However, it was unable to bring about significant fungal cell membrane rupture. On the other hand, punicalagin (16 µg/mL) significantly arrested C. albicans and C. gattii cells at the G0/G1 phase, with a consequent reduction in cells at the G2/M phase in both fungi isolates, and thereby prevented progression of the normal yeast cell cycle. However, these alterations showed no involvement of reactive oxygen species overproduction in C. albicans and C. gattii cells, although punicalagin triggered a significant loss of mitochondrial membrane potential in C. albicans. These findings suggest that punicalagin is a promising plant-derived compound for use in developing new antifungal therapies.
Assuntos
Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Cryptococcus gattii/efeitos dos fármacos , Ergosterol/metabolismo , Taninos Hidrolisáveis/farmacologia , Candida albicans/crescimento & desenvolvimento , Candida albicans/metabolismo , Candida albicans/ultraestrutura , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Cryptococcus gattii/crescimento & desenvolvimento , Cryptococcus gattii/metabolismo , Cryptococcus gattii/ultraestrutura , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Microscopia Eletrônica de TransmissãoRESUMO
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.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Piperidinas/farmacologia , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Pirazóis/farmacologia , Proteína Supressora de Tumor p53/deficiência , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Células 3T3 BALB , Humanos , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/enzimologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Camundongos , Mutação , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/genéticaRESUMO
In a regulatory context, skin sensitization hazard and risk evaluations of manufactured products and their ingredients (e.g. cosmetics) are mandatory in several regions. Great efforts have been made within the field of 21st Century Toxicology to provide non-animal testing approaches to assess the skin allergy potential of materials (e.g. chemicals, mixtures, nanomaterials, particles). Mechanistic understanding of skin sensitization process through the adverse outcome pathway (AOP) has promoted the development of in vitro methods, demonstrating accuracies superior to the traditional animal testing. These in vitro testing approaches are based on one of the four AOP key events (KE) of skin sensitization: formation of immunogenic hapten-protein complexes (KE-1 or the molecular initiating event, MIE), inflammatory keratinocyte responses (KE-2), dendritic cell activation (KE-3), and T-lymphocyte activation and proliferation (KE-4). This update provides an overview of the historically used in vivo methods as well as the current in chemico and in cell methods with and without OECD guideline designations to analyze the progress towards human-relevant in vitro test methods for safety assessment of the skin allergenicity potential of materials. Here our focus is to review 96 in vitro testing approaches directed to the KEs of the skin sensitization AOP.
Assuntos
Alternativas aos Testes com Animais , Haptenos/toxicidade , Animais , Dermatite Alérgica de Contato/etiologia , Humanos , Técnicas In Vitro , Medição de RiscoRESUMO
In Brazil, efforts towards the regulatory acceptance and implementation of innovative methods to replace experimental animal use in various fields began to gather force in 2008, with the approval of Law No. 11,794/2008 (the Arouca Law). This law represented a milestone, as it created the National Council for the Control of Animal Experimentation (CONCEA) to deal with the ethical and legal issues related to the use of laboratory animals. In 2014, CONCEA put together a framework for expanding the implementation of non-animal methodologies for use in research and education. It also promoted the regulatory acceptance in Brazil of 24 test guidelines, including 15 in vitro approaches. It should be emphasised that, in Brazilian legislation, replacement is generally based on the toxicological endpoint and not on the category of product, as tends to be the case in other countries (e.g. cosmetics in the European Union). The resolution-dependent deadlines for the obligatory replacement of in vivo methods with the CONCEA-approved tests are 2019 and 2021. Brazil has advanced considerably towards the replacement of animal experimentation, and in certain aspects, this has been in a highly progressive manner. However, there is still a lot of work to be done, especially considering the current political scenario with reduced investment in research, development and innovation. The chronology of significant events following the approval of the Arouca Law, which have contributed to the promotion of the Three Rs alternatives in Brazil, will be examined.
Assuntos
Experimentação Animal , Alternativas aos Testes com Animais , Alternativas aos Testes com Animais/tendências , Animais , Animais de Laboratório , Brasil , Projetos de PesquisaRESUMO
BACKGROUND: Allergic contact dermatitis caused by henna-based hair-colouring products has been associated with adulteration of henna with p-phenylenediamine (PPD). OBJECTIVES: To develop a testing approach based on in vitro techniques that address key events within the skin sensitization adverse outcome pathway in order to evaluate the allergenic potential of hair-colouring products. METHODS: The following in vitro assays were used to test the sensitizing capacity of hair dye ingredients: the micro-direct peptide reactivity assay (mDPRA); the HaCaT keratinocyte-associated interleukin (IL)-18 assay; the U937 cell line activation test (U-SENS)/IL-8 levels; the blood monocyte-derived dendritic cell test; and genomic allergen rapid detection (GARD skin). Those techniques with better human concordance were selected to evaluate the allergenic potential of 10 hair-colouring products. RESULTS: In contrast to the information on the label, chromatographic analyses identified PPD in all products. The main henna biomarker, lawsone, was not detected in one of the 10 products. Among the techniques evaluated by testing hair dye ingredients, the mDPRA, the IL-18 assay, GARD skin and the U-SENS correlated better with human classification (concordances of 91.7%-100%) and were superior to the animal testing (concordance of 78.5%). Thus, these assays were used to evaluate hair-colouring products, which were classified as skin sensitizers by the use of different two-of-three approaches. CONCLUSIONS: Our findings highlight the toxicological consequences of, and risks associated with, the undisclosed use of PPD in henna-based "natural" "real-life" products.
Assuntos
Tinturas para Cabelo/efeitos adversos , Naftoquinonas/efeitos adversos , Fenilenodiaminas/efeitos adversos , Antígeno B7-2/metabolismo , Bioensaio/métodos , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Células Dendríticas/metabolismo , Dermatite Alérgica de Contato/etiologia , Tinturas para Cabelo/química , Humanos , Técnicas In Vitro , Interleucina-18/metabolismo , Interleucina-8/metabolismo , Queratinócitos/metabolismo , Naftoquinonas/análise , Fenilenodiaminas/análiseRESUMO
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 24â¯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 24â¯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.
Assuntos
Alternativas aos Testes com Animais/métodos , Alternativas aos Testes com Animais/normas , Irritantes/toxicidade , Queratinócitos/efeitos dos fármacos , Testes de Toxicidade Aguda/métodos , Testes de Toxicidade Aguda/normas , Bioensaio/normas , Linhagem Celular , Citocinas/análise , Ensaio de Imunoadsorção Enzimática , Humanos , Inflamação/induzido quimicamente , Concentração Inibidora 50 , Queratinócitos/química , Modelos BiológicosRESUMO
AIMS: This study evaluated parameters of toxicity and antiproliferative effects of (+)-N(1)-4-fluorobenzaldehyde-N(4)-{1-methyl-1-[(1R)-4-methylcyclohexene-3-il]-ethyl}-thiossemicarbazone (4-FTSC) in PC-3 adenocarcinoma prostate cells. MAIN METHODS: Cytotoxicity of 4-FTSC in PC-3 cells was evaluated using MTT assay. Morphology examination of PC-3 cells treated with 4-FTSC was also performed as well as the cell death mechanisms induced were investigated using flow cytometry. Parameters of toxicity of 4-FTSC was conducted by the investigation of its potential myelotoxicity and lymphotoxicity, hemolytic activity and acute oral toxicity profile. KEY FINDINGS: 4-FTSC showed promising cytotoxic effects against PC-3 cells (IC50â¯=â¯18.46⯵M). It also triggered apoptotic morphological changes, phosphatidylserine externalization and a significant increase of DNA fragmentation in PC-3 cells. Moreover, 4-FTSC did not show changes in the PC-3 cell cycle with levels of p21, p27, NFĸB and cyclin D1 similar to those found in both control and treated cells. 4-FTSC also promoted an increase of p53 levels associated with mitochondrial impairment through loss of ∆Ψm and ROS overproduction. 4-FTSC-induced cell death mechanism in PC-3 cells involved activation of caspase-3/-7 through apoptosis intrinsic pathway via caspase-9. Regarding toxicological profile, 4-FTSC showed in vitro lymphotoxicity, although with low cytotoxicity for bone marrow progenitors and no hemolytic potential. Moreover, it was classified as GHS category 5 (LD50â¯>â¯2000-5000â¯mg/Kg), suggesting it has low acute oral systemic toxicity. SIGNIFICANCE: 4-FTSC seems to be a promising candidate to be used as a clinical tool in prostate cancer treatment. Further studies are required to better clarify its toxicopharmacological effects found in this compound.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Benzaldeídos/química , Cicloexenos/farmacologia , Neoplasias da Próstata/patologia , Tiossemicarbazonas/farmacologia , Animais , Antineoplásicos/química , Células 3T3 BALB , Benzaldeídos/farmacologia , Ciclo Celular , Proliferação de Células/efeitos dos fármacos , Cicloexenos/química , Humanos , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Tiossemicarbazonas/química , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Piperazine is a promising scaffold for drug development due to its broad spectrum of biological activities. Based on this, the new piperazine-containing compound LQFM018 (2) [ethyl 4-((1-(4-chlorophenyl)-1H-pyrazol-4-yl)methyl)piperazine-1-carboxylate] was synthetized and some biological activities investigated. In this work, we described its ability to bind aminergic receptors, antiproliferative effects as well as the LQFM018 (2)-triggered cell death mechanisms, in K562 leukemic cells, by flow cytometric analyses. Furthermore, acute oral systemic toxicity and potential myelotoxicity assessments of LQFM018 (2) were carried out. LQFM018 (2) was originally obtained by molecular simplification from LASSBio579 (1), an analogue compound of clozapine, with 33% of global yield. Binding profile assay to aminergic receptors showed that LQFM018 (2) has affinity for the dopamine D4 receptor (Kiâ¯=â¯0.26⯵M). Moreover, it showed cytotoxicity in K562 cells, in a concentration and time-dependent manner; IC50 values obtained were 399, 242 and 119⯵M for trypan blue assay and 427, 259 and 50⯵M for MTT method at 24, 48 or 72â¯h, respectively. This compound (427⯵M) also promoted increase in LDH release and cell cycle arrest in G2/M phase. Furthermore, it triggered necrotic morphologies in K562 cells associated with intense cell membrane rupture as confirmed by Annexin V/propidium iodide double-staining. LQFM018 (2) also triggered mitochondrial disturb through loss of ΔΨm associated with increase of ROS production. No significant accumulation of cytosolic cytochrome c was verified in treated cells. Furthermore, it was verified an increase of expression of TNF-R1 and mRNA levels of CYLD with no involviment in caspase-3 and -8 activation and NF-κB in K562 cells. LQFM018 (2) showed in vitro myelotoxicity potential, but it was orally well tolerated and classified as UN GHS category 5 (LD50â¯>â¯2000-5000â¯mg/Kg). Thus, LQFM018 (2) seems to have a non-selective action considering hematopoietic cells. In conclusion, it is suggested LQFM018 (2) promotes cell death in K562 cells via necroptotic signaling, probably with involvement of dopamine D4 receptor. These findings open new perspectives in cancer therapy by use of necroptosis inducing agents as a strategy of reverse cancer cell chemoresistance.
Assuntos
Apoptose/efeitos dos fármacos , Piperazinas/farmacologia , Receptores de Dopamina D4/metabolismo , Testes de Toxicidade , Células 3T3 , Administração Oral , Animais , Ligação Competitiva/efeitos dos fármacos , Caspase 3/genética , Caspase 3/metabolismo , Caspase 8/genética , Caspase 8/metabolismo , Ciclo Celular/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Citocromos c/metabolismo , Enzima Desubiquitinante CYLD/metabolismo , Exocitose/efeitos dos fármacos , Feminino , Humanos , Células K562 , Cinética , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , NF-kappa B/metabolismo , Necrose , Fosfatidilserinas/metabolismo , Piperazina , Piperazinas/síntese química , Piperazinas/química , Ligação Proteica/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismoRESUMO
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.
Assuntos
Ligases/metabolismo , Ligases/farmacologia , Estomatite/prevenção & controle , Anticarcinógenos/farmacologia , Antioxidantes/farmacologia , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Curcuma/metabolismo , Curcuma/fisiologia , Citocinas/metabolismo , Flavonoides/farmacologia , Fluoruracila/efeitos adversos , Fluoruracila/farmacologia , Humanos , Interleucina-1beta/farmacologia , Interleucina-6/farmacologia , Queratinócitos/metabolismo , Ligases/uso terapêutico , NF-kappa B/metabolismo , Substâncias Protetoras/farmacologia , Estomatite/tratamento farmacológico , Estomatite/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
AIMS: This study reports the biological properties of LQFM030 in vivo, a molecular simplification of the compound nutlin-1. MAIN METHODS: Ehrlich ascites tumor (EAT)-bearing mice were treated intraperitoneally with LQFM030 (50, 75 or 150mg/kg) for 10days to determine changes in ascites tumor volume, body weight, cytotoxicity and angiogenesis. Moreover, flow cytometric expression of p53 and p21 proteins and caspase-3/7, -8 and -9 activation were investigated in EAT cells from mice treated. Acute oral systemic toxicity potential of LQFM030 in mice was also investigated using an alternative method. KEY FINDINGS: Treatment of EAT-bearing mice with LQFM030 resulted in a marked decline in tumor cell proliferation and the vascular endothelial growth factor (VEGF) levels along with enhanced survival of the mice. Apoptotic tumor cell death was detected through p53 and p21 modulation and increase of caspase-3/7, -8 and -9 activity. LQFM030 also showed orally well tolerated, being classified in the UN GHS category 5 (LD50>2000-5000mg/Kg). SIGNIFICANCE: LQFM030 seems to be a promising antitumor candidate for combinatory therapy with typical cytotoxic compounds, reducing the toxicity burden while allowing a superior anticancer activity. Moreover, these data also open new perspectives for LQFM030 as an antiangiogenic agent for treatment of diseases involving VEGF overexpression.
Assuntos
Inibidores da Angiogênese/farmacologia , Carcinoma de Ehrlich/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Piperidinas/farmacologia , Pirazóis/farmacologia , Fator A de Crescimento do Endotélio Vascular/biossíntese , Inibidores da Angiogênese/toxicidade , Animais , Apoptose/efeitos dos fármacos , Carcinoma de Ehrlich/patologia , Caspases/biossíntese , Feminino , Injeções Intraperitoneais , Masculino , Camundongos , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/patologia , Proteína Oncogênica p21(ras)/biossíntese , Proteína Oncogênica p21(ras)/genética , Piperidinas/toxicidade , Pirazóis/toxicidade , Proteína Supressora de Tumor p53/biossíntese , Proteína Supressora de Tumor p53/genéticaRESUMO
Co-encapsulation of anticancer drugs paclitaxel and imatinib in nanocarriers is a promising strategy to optimize cancer treatment. Aiming to combine the cytotoxic and antiangiogenic properties of the drugs, a liposome formulation targeted to folate receptor co-encapsulating paclitaxel and imatinib was designed in this work. An efficient method was optimized for the synthesis of the lipid anchor DSPE-PEG(2000)-folic acid (FA). The structure of the obtained product was confirmed by RMN, FT-IR, and ESI-MS techniques. A new analytical method was developed and validated for simultaneous quantification of the drugs by liquid chromatography. Liposomes, composed of phosphatidylcholine, cholesterol, and DSPE-mPEG(2000), were prepared by extrusion. Their surface was modified by post-insertion of DSPE-PEG(2000)-FA. Reaction yield for DSPE-PEG(2000)-FA synthesis was 87%. Liposomes had a mean diameter of 122.85 ± 1.48 nm and polydispersity index of 0.19 ± 0.01. Lyophilized formulations remained stable for 60 days in terms of size and drug loading. FA-targeted liposomes had a higher effect on MCF7 cell viability reduction (p < 0.05) when compared with non-targeted liposomes and free paclitaxel. On PC-3 cells, viability reduction was greater (p < 0.01) when cells were exposed to targeted vesicles co-encapsulating both drugs, compared with the non-targeted formulation. VEGF gene expression was reduced in MCF7 and PC-3 cells (p < 0.0001), with targeted vesicles exhibiting better performance than non-targeted liposomes. Our results demonstrate that multifunctional liposomes associating molecular targeting and multidrug co-encapsulation are an interesting strategy to achieve enhanced internalization and accumulation of drugs in targeted cells, combining multiple antitumor strategies.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Receptores de Folato com Âncoras de GPI , Mesilato de Imatinib/administração & dosagem , Paclitaxel/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Ácido Fólico/química , Humanos , Mesilato de Imatinib/farmacologia , Lipossomos , Células MCF-7 , Paclitaxel/farmacologia , Fosfatidiletanolaminas/química , Polietilenoglicóis/química , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Scientific evidences have highlighted 5-(1-(3-fluorophenyl)-1H-pyrazol-4-yl)-2H-tetrazole (LQFM021) as a promising anti-inflammatory, analgesic and antinociceptive agent due to its effects on peripheral opioid receptors associated with activation of the nitric oxide/cGMP/KATP pathway. Despite these important pharmacological findings, toxicity data of LQFM021 are scarce. Thus, this study investigated the in vitro genotoxicity of LQFM021 through cytokinesis-block micronucleus assay (OECD Nº 487/2014). Moreover, zebrafish model was used to assess the embryotoxicity potential of LQFM021 using fish embryo toxicity test (OECD Nº 236/2013) with extended exposure to evaluate subchronic larval development. In vivo subchronic toxicity of LQFM021 in rats (OECD Nº 407/2008) was also conducted. This compound at the lower concentrations tested (3.1 and 31 µg/mL) did not promote changes in micronuclei frequency in HepG2 cells. However, in the higher concentrations of LQFM021 (310 and 620 µg/mL) triggered a significant increase of micronucleated HepG2 cells, showing an alert signal of potential genotoxicity. Regarding the oral treatment of rats with LQFM021 (62.5, 125 or 250 mg/kg) for 28 days, the main findings showed that LQFM021 promoted renal and liver changes in a dose-dependent manner, being irreversible damage for kidneys while liver tissue showed a recovery after 14 days post treatment. Regarding embryotoxicity, although the lower concentrations used did not show toxicity, the concentration of LQFM021 (39.8 and 100 mg/L) promoted malformations in zebrafish embryo-larvae stage, in especial cardiac tissue changes. In conclusion, anti-inflammatory compound LQFM021 seems to have some limiting factors as a new therapeutic option to be used orally and in high repeated doses, related to those found in the non-steroidal anti-inflammatory drugs (NSAIDs).
Assuntos
Anti-Inflamatórios/toxicidade , Mutagênicos/toxicidade , Pirazóis/toxicidade , Tetrazóis/toxicidade , Animais , Embrião não Mamífero/efeitos dos fármacos , Feminino , Células Hep G2 , Humanos , Testes de Mutagenicidade , Ratos Wistar , Peixe-ZebraRESUMO
Dual 5-LOX/COX inhibitors are potential new dual drugs to treat inflammatory conditions. This research aimed to design, synthesis and to evaluate the anti-inflammatory and antinociceptive effects of the new compound, which is derived from nimesulide and darbufelone lead compounds. The new dual inhibitor 5-LOX/COX has the possible advantage of gastrointestinal safety. A voltammetric experiment was conducted to observe the drug's antioxidative effect. A formalin test, a hot plate test and carrageenan-induced mechanical hyperalgesia were employed to evaluate the analgesic nature of LQFM-091. To evaluate anti-inflammatory activity, we measured edema, leukocyte count, myeloperoxidase activity and cytokines levels in carrageenan-induced inflammation tests. We elucidated the underlying mechanisms by assessing the interaction the with COXs and LOX enzymes by colorimetric screening assay and molecular docking. The lethal dose (LD50) was estimated using 3T3 Neutral Red Uptake assay. Our results indicate that the LQFM-091 prototype is a powerful antioxidant, as well as able to inhibit COX-1, COX-2 and LOX activities. LQFM091 was classified in GHS category 4 (300Assuntos
Inibidores de Ciclo-Oxigenase/uso terapêutico
, Inibidores de Lipoxigenase/uso terapêutico
, Fenóis/uso terapêutico
, Células 3T3
, Animais
, Carragenina
, Sobrevivência Celular/efeitos dos fármacos
, Inibidores de Ciclo-Oxigenase/farmacologia
, Citocinas/imunologia
, Edema/induzido quimicamente
, Edema/tratamento farmacológico
, Feminino
, Temperatura Alta
, Hiperalgesia/induzido quimicamente
, Hiperalgesia/tratamento farmacológico
, Contagem de Leucócitos
, Lipoxigenase/metabolismo
, Inibidores de Lipoxigenase/farmacologia
, Camundongos
, Simulação de Acoplamento Molecular
, Medição da Dor
, Peroxidase/imunologia
, Fenóis/farmacologia
, Estimulação Física
, Pleurisia/induzido quimicamente
, Pleurisia/tratamento farmacológico
, Pleurisia/imunologia
, Prostaglandina-Endoperóxido Sintases/metabolismo
, Úlcera Gástrica/induzido quimicamente
, Úlcera Gástrica/tratamento farmacológico
, Sulfonamidas
RESUMO
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.
Assuntos
Antineoplásicos/farmacologia , Chalconas/farmacologia , Células 3T3 , Animais , Apoptose/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Caspases/metabolismo , Ciclo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Chalconas/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Humanos , Células MCF-7 , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
This study evaluated the applicability of a modified Direct Peptide Reactivity Assay (DPRA) (OECD N° 442C, 2015) through the 10-fold reduction of reaction volume (micro-DPRA, mDPRA) for skin sensitization evaluation of six commercial glyphosate-containing formulations. In addition, another modification of DPRA was proposed by adding a UVA (5J/cm2) irradiation step, namely photo-mDPRA, to better characterize (photo)sensitizer materials. The phototoxicity profile of pesticides was also evaluated using the 3T3 Neutral Red Uptake Phototoxicity Test (3T3-NRU-PT) (OECD N° 432, 2004). The mDPRA could represent an environmentally acceptable test approach, since it reduces costs and organic waste. Peptide depletion was greater in photo-mDPRA and changed the reactivity class of each test material, in comparison to mDPRA. Thus, the association of mDPRA with photo-mDPRA was better for correctly characterizing human (photo)sensitizer substances and pesticides. In general, cysteine depletion was greater than that of lysine for all materials tested in both mDPRA and photo-mDPRA. Furthermore, while 3T3-NRU-PT is unable to predict (photo)sensitizers, it was capable of correctly identifying the phototoxic potential of the tested agrochemical formulations. In conclusion, mDPRA plus photo-mDPRA and 3T3-NRU-PT seem to be preliminary non-animal test batteries for skin (photo)sensitization/phototoxicity assessment of chemicals, agrochemical formulations and their ingredients.
Assuntos
Dermatite Fototóxica/patologia , Glicina/análogos & derivados , Herbicidas/toxicidade , Transtornos de Fotossensibilidade/induzido quimicamente , Pele/efeitos dos fármacos , Alternativas aos Testes com Animais , Animais , Células 3T3 BALB , Cisteína/metabolismo , Composição de Medicamentos , Glicina/toxicidade , Humanos , Lisina/metabolismo , Camundongos , Vermelho Neutro , Transtornos de Fotossensibilidade/patologia , Pele/patologia , Raios Ultravioleta , GlifosatoRESUMO
In the context of developing a new natural product-based cosmetic, the in vitro efficacy and safety evaluations of a complex botanical mixture based on Eugenia dysenterica leaf hydroalcoholic extract (EDE) (2.5-1000µg/mL) were carried out. Chromatographic analysis demonstrated the presence of the tannin (ellagic acid) and flavonoids (quercetin and gallic acid) which characterize the EDE as a polyphenol-rich mixture. Using HFF-1 fibroblasts, it was shown that EDE promoted cell regeneration after UVA exposure. It also led to the inhibition of the collagenase, elastase and tyrosinase enzymes, which are involved in skin-related disorders. In terms of toxicological evaluation, the EDE was classified as non-phototoxic through the 3T3 Neutral Red Uptake Phototoxicity Test (OECD N° 432, 2004) and non-eye irritant by Bovine Corneal Opacity and Permeability (OECD N° 437, 2013) assay, in conjunction with corneal histomorphometric analysis. Furthermore, the EDE has no skin sensitization potential as demonstrated by a two-out-of-three prediction model [protein-binding/haptenization (OECD N° 442C, 2015), keratinocyte and dendritic cell activations]. In addition, it was shown that the EDE seems to be non-genotoxic through the cytokinesis-block micronucleus assay (OECD N° 487, 2014) using HepG2 cells. When considered together, these findings support the use of EDE botanical mixture in cosmetic/pharmaceutical products.
Assuntos
Cosméticos/química , Cosméticos/toxicidade , Fármacos Dermatológicos/química , Fármacos Dermatológicos/toxicidade , Eugenia/química , Eugenia/toxicidade , Animais , Bovinos , Células Cultivadas , Misturas Complexas , Qualidade de Produtos para o Consumidor , Córnea/efeitos dos fármacos , Células Dendríticas/efeitos dos fármacos , Dermatite Fototóxica , Humanos , Interleucina-18/metabolismo , Irritantes/toxicidade , Queratinócitos/efeitos dos fármacos , Camundongos , Testes para Micronúcleos , Extratos Vegetais/química , Extratos Vegetais/toxicidade , Folhas de Planta/químicaRESUMO
A new molecule, LQFM048, originally designed through molecular hybridization using green chemistry approach, is in development as a photoprotective agent. Eye irritation, skin toxicity and genotoxicity evaluations are mandatory for predicting health risks. In this context, the purpose of this study was to investigate the eye irritation potential of LQFM048 by combining Short Time Exposure (STE), Bovine Corneal Opacity and Permeability (BCOP) associated with corneal histomorphometry and Hen's Egg Test-Chorioallantoic Membrane (HET-CAM). Additionally, skin toxicity was evaluated by interleukin-18 production in the HaCaT keratinocyte, Local Lymph Node Assay (LLNA:BrdU-ELISA) method, 3T3 Neutral red uptake (NRU) assay and in vivo phototoxicity test. Genotoxic potential of LQFM048 was also analyzed by cytokinesis-block micronucleus assay (MNvit test-cytoB) in HepG2 cells. Our results showed that LQFM048 did not induce eye irritation and it was classified as UN GHS No Category for both STE and BCOP assays and non-irritating for HET-CAM test. LQFM048 showed non-potential skin sensitization with stimulation index (SI=0.7) in the LLNA:BrdU-ELISA method. Corroborating in vivo tests, it did not promote significant cytotoxicity in HaCaT cells and it showed similar levels of IL-18 when compared to control. Furthermore, LQFM048 induced non-phototoxic potential with photo-irritation factor (PIF) and mean photo effect (MPE) of 1 and -0.138, respectively, for 3T3 cells. Similarly, it was not phototoxic for in vivo testing with or without exposure to UVA, showing SI values of 1 and 1.2, respectively. The micronucleus test showed that LQFM048 was not genotoxic, under the conditions tested.In conclusion, LQFM048, a heterocyclic compound obtained through an environmentally acceptable simple synthetic route, seems to be safe for human use, especially for the development of a new sunscreen product, since it is neither an eye irritant, nor a contact allergen, nor mutagenic and nor phototoxic.
Assuntos
Córnea/efeitos dos fármacos , Irritantes/toxicidade , Pele/efeitos dos fármacos , Protetores Solares/toxicidade , Células 3T3 , Animais , Bovinos , Linhagem Celular Transformada , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Sobrevivência Celular/efeitos da radiação , Galinhas , Córnea/fisiologia , Córnea/efeitos da radiação , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Feminino , Células Hep G2 , Humanos , Irritantes/administração & dosagem , Camundongos , Camundongos Endogâmicos BALB C , Testes de Mutagenicidade/métodos , Distribuição Aleatória , Pele/metabolismo , Pele/efeitos da radiação , Raios Ultravioleta/efeitos adversosRESUMO
Jabuticaba is an exotic fruit native to Brazil that has been arousing medicinal interest. Using chemical (HPLC-PDA, resonance mass spectra, and NMR), electroanalytical (differential pulse voltammetry, radical scavenging assay), and pharmacological (in vivo and in vitro) approaches, we have identified its bioactive compounds and hypotensive effects on hypertensive rats. The hydroalcoholic extract of jabuticaba (HEJ) presents a great quantity of phenolic compounds, and several molecules with hydroxyl groups present high efficiency as an antioxidant. The treatment with HEJ (100 and 300 mg/kg/day, for four weeks) presented hypotensive effects on L-NAME-induced hypertensive rats, possibly improving the nitric oxide bioavailability because of its high antioxidant potential. Furthermore, renal and cardiac hypertrophies were also attenuated after the HEJ treatment. Moreover, the vascular responses to contractile and dilating agonists were improved with the HEJ treatment, which is also able to induce nitric oxide production in endothelial cells.