RESUMO
In the present study, pyroligneous acid, also known as wood vinegar, has been employed as reducing and stabilizing agent in the synthesis of silver nanoparticles (AgNPs) anchored on nanocellulose (NC). The idea is to confer the latter bactericidal properties for its typical uses such as in cosmetics and food-packing. It has been demonstrated that AgNPs can be directly produced onto NC in one-pot fashion while dramatically enhancing the kinetics of AgNPs synthesis (2 h for reaction completion) in comparison to the NC-less counterpart (10 days for reaction completion). Furthermore, NC allowed for a narrower size distribution of AgNPs. NC-supported and non-supported AgNPs had sizes of 5.1 ± 1.6 nm and 16.7 ± 4.62 nm, respectively. Immortalized human keratinocytes (HaCat) cells were then employed as model to evaluate the cytotoxicity of the AgNPs-NC compound. The latter was found not to impact cell proliferation at any formulation, while decreasing the viability by only 6.8% after 72 h. This study contributes to the development of more environmentally benign routes to produce nanomaterials and to the understanding of their impact on cells.
Assuntos
Sobrevivência Celular , Celulose , Células HaCaT , Nanopartículas Metálicas , Prata , Humanos , Prata/química , Nanopartículas Metálicas/química , Celulose/química , Celulose/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Queratinócitos/efeitos dos fármacos , Queratinócitos/citologia , Tamanho da Partícula , Proliferação de Células/efeitos dos fármacos , Ácido Acético/química , Ácido Acético/farmacologiaRESUMO
Diisopentyl phthalate (DiPeP) is primarily used as a plasticizer or additive within the production of polyvinyl chloride (PVC), and has many additional industrial applications. Its metabolites were recently found in urinary samples of pregnant women; thus, this substance is of concern as relates to human exposure. Depending upon the nature of the alcohol used in its synthesis, DiPeP may exist either as a mixture consisting of several branched positional isomers, or as a single defined structure. This article investigates the skin sensitization potential and immunomodulatory effects of DiPeP CAS No. 84777-06-0, which is currently marketed and classified as a UVCB substance, by in silico and in vitro methods. Our findings showed an immunomodulatory effect for DiPeP in LPS-induced THP-1 activation assay (increased CD54 expression). In silico predictions using QSAR TOOLBOX 4.5, ToxTree, and VEGA did not identify DiPeP, in the form of a discrete compound, as a skin sensitizer. The keratinocyte activation (Key Event 2 (KE2) of the adverse outcome pathway (AOP) for skin sensitization) was evaluated by two different test methods (HaCaT assay and RHE assay), and results were discordant. While the HaCaT assay showed that DiPeP can activate keratinocytes (increased levels of IL-6, IL-8, IL-1α, and ILA gene expression), in the RHE assay, DiPeP slightly increased IL-6 release. Although inconclusive for KE2, the role of DiPeP in KE3 (dendritic cell activation) was demonstrated by the increased levels of CD54 and IL-8 and TNF-α in THP-1 cells (THP-1 activation assay). Altogether, findings were inconclusive regarding the skin sensitization potential of the UVCB DiPeP-disagreeing with the results of DiPeP in the form of discrete compound (skin sensitizer by the LLNA assay). Additional studies are needed to elucidate the differences between DiPeP isomer forms, and to better understand the applicability domains of non-animal methods in identifying skin sensitization hazards of UVCB substances.
Assuntos
Simulação por Computador , Queratinócitos , Ácidos Ftálicos , Humanos , Queratinócitos/efeitos dos fármacos , Ácidos Ftálicos/toxicidade , Células HaCaT , Pele/efeitos dos fármacos , Pele/imunologia , Pele/metabolismo , Relação Quantitativa Estrutura-Atividade , Plastificantes/toxicidade , Células THP-1 , Molécula 1 de Adesão Intercelular/metabolismo , Molécula 1 de Adesão Intercelular/genética , Linhagem CelularRESUMO
The aim of this study was twofold: (1) evaluate the effect of benzo[a]pyrene (BaP) on expression levels of AQP3 and Notch1 genes in HaCaT cells exposed "in vitro" and (2) investigate the possible biological role of assessed genes by bioinformatics methods. Cells were exposed to increasing concentrations of BaP (0.0-4.0 µM) for 1-4 days. After treatments, cell viability and expression levels of AhR, CYP1A1, AQP3, and Notch1 genes were evaluated. The possible biological role of assessed genes was evaluated using bioinformatics tools. Low cytotoxicity in HaCaT cells dosed with BaP was detected. A significant overexpression (p < .05) of CYP1A1, AQP3, and Notch1 was found in exposed HaCaT cells. The gene expression upregulation was dependent on AhR activation. The bioinformatics analysis showed that these genes were enriched in related cancer signaling pathways. The findings suggest that AQP3 and Notch1 are upregulated by AhR activation in HaCaT cells exposed to BaP.
Assuntos
Benzo(a)pireno , Citocromo P-450 CYP1A1 , Humanos , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Benzo(a)pireno/metabolismo , Células HaCaT , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Regulação para CimaRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Leishmaniasis are widely distributed among tropical and subtropical countries, and remains a crucial health issue in Amazonia. Indigenous groups across Amazonia have developed abundant knowledge about medicinal plants related to this pathology. AIM OF THE STUDY: We intent to explore the weight of different pharmacological activities driving taxa selection for medicinal use in Amazonian communities. Our hypothesis is that specific activity against Leishmania parasites is only one factor along other (anti-inflammatory, wound healing, immunomodulating, antimicrobial) activities. MATERIALS AND METHODS: The twelve most widespread plant species used against leishmaniasis in Amazonia, according to their cultural and biogeographical importance determined through a wide bibliographical survey (475 use reports), were selected for this study. Plant extracts were prepared to mimic their traditional preparations. Antiparasitic activity was evaluated against promastigotes of reference and clinical New-World strains of Leishmania (L. guyanensis, L. braziliensis and L. amazonensis) and L. amazonensis intracellular amastigotes. We concurrently assessed the extracts immunomodulatory properties on PHA-stimulated human PBMCs and RAW264.7 cells, and on L. guyanensis antigens-stimulated PBMCs obtained from Leishmania-infected patients, as well as antifungal activity and wound healing properties (human keratinocyte migration assay) of the selected extracts. The cytotoxicity of the extracts against various cell lines (HFF1, THP-1, HepG2, PBMCs, RAW264.7 and HaCaT cells) was also considered. The biological activity pattern of the extracts was represented through PCA analysis, and a correlation matrix was calculated. RESULTS: Spondias mombin L. bark and Anacardium occidentale L. stem and leaves extracts displayed high anti-promatigotes activity, with IC50 ≤ 32 µg/mL against L. guyanensis promastigotes for S. mombin and IC50 of 67 and 47 µg/mL against L. braziliensis and L. guyanensis promastigotes, respectively, for A. occidentale. In addition to the antiparasitic effect, antifungal activity measured against C. albicans and T. rubrum (MIC in the 16-64 µg/mL range) was observed. However, in the case of Leishmania amastigotes, the most active species were Bixa orellana L. (seeds), Chelonantus alatus (Aubl.) Pulle (leaves), Jacaranda copaia (Aubl.) D. Don. (leaves) and Plantago major L. (leaves) with IC50 < 20 µg/mL and infection rates of 14-25% compared to the control. Concerning immunomodulatory activity, P. major and B. orellana were highlighted as the most potent species for the wider range of cytokines in all tested conditions despite overall contrasting results depending on the model. Most of the species led to moderate to low cytotoxic extracts except for C. alatus, which exhibited strong cytotoxic activity in almost all models. None of the tested extracts displayed wound healing properties. CONCLUSIONS: We highlighted pharmacologically active extracts either on the parasite or on associated pathophysiological aspects, thus supporting the hypothesis that antiparasitic activities are not the only biological factor useful for antileishmanial evaluation. This result should however be supplemented by in vivo studies, and attracts once again the attention on the importance of the choice of biological models for an ethnophamacologically consistent study. Moreover, plant cultural importance, ecological status and availability were discussed in relation with biological results, thus contributing to link ethnobotany, medical anthropology and biology.
Assuntos
Antiprotozoários/farmacologia , Leishmania/efeitos dos fármacos , Extratos Vegetais/farmacologia , Plantas Medicinais/química , Animais , Antiprotozoários/isolamento & purificação , Brasil , Células HaCaT , Células Hep G2 , Humanos , Leishmaniose/tratamento farmacológico , Leishmaniose/parasitologia , Leucócitos Mononucleares/parasitologia , Medicina Tradicional , Camundongos , Células RAW 264.7 , Células THP-1RESUMO
The objective of this study was to determine the chemical composition as well as antioxidant, antibacterial, and cytotoxic properties of the essential oil of Mentha piperita L. (peppermint). Fifteen chemical constituents were identified in the essential oil, for a total of 99.99% of the compounds. The essential oil exhibited antimicrobial activity against two Gram-positive bacteria Staphylococcus aureus and Listeria monocytogenes. The minimum inhibitory concentration (MIC) of essential oil of Mentha piperita L. for Staphylococcus aureus and Listeria monocytogenes was 1.84 µg/ml, whereas the minimum bactericidal concentration (MBC) values were 3.7 and 7.43 µg/ml, respectively. The oil displayed potent antioxidant activity inhibiting up to approximately73% of 2,2'-azinothiobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radicals. In the cytotoxicity assay, the highest essential oil concentration (100 µg/ml) resulted in viability of approximately 90% human epidermal keratinocyte (HaCaT) cells. With respect to antitumor activity in C6 rat glioma cells, there was significant reduction in cell viability: 56-74% in 24 hr, and 71-77% in 48 hr. Data suggest that in presence of the essential oil of Mentha piperita L. antioxidant, antibacterial, antitumor and non-cytotoxic properties were noted.
Assuntos
Antibacterianos/farmacologia , Antioxidantes/farmacologia , Óleos de Plantas/farmacologia , Células HaCaT , Humanos , Listeria monocytogenes/efeitos dos fármacos , Mentha piperita/química , Mentha piperita/toxicidade , Testes de Sensibilidade Microbiana , Óleos Voláteis/farmacologia , Óleos Voláteis/toxicidade , Extratos Vegetais/farmacologia , Óleos de Plantas/química , Óleos de Plantas/toxicidade , Medição de Risco , Staphylococcus aureus/efeitos dos fármacosRESUMO
The solar ultraviolet (UV) radiation that reaches the Earth is composed of 95% of UVA (320 to 400 nm) and 5% of UVB (280 to 320 nm) radiation. UVB is carcinogenic, generating potentially mutagenic DNA lesions. The solar UVA radiation also causes DNA damage, but this fact does not fully account for its biological impact. UVA is absorbed by non-DNA cellular chromophores, generating reactive oxygen species such as singlet oxygen. Knowing the proteome mediates stress responses in cells, here we investigated the cellular effects of a non-cytotoxic dose of UVA radiation, equivalent to about 20 minutes of midday sun exposure, on the proteome of human keratinocytes. Using a combination of mass spectrometry-based proteomics, bioinformatics, and conventional biochemical assays, we analyzed two aspects of UVA-induced stress: spatial remodeling of the proteome in subcellular compartments 30 minutes after stress and long-term changes in protein levels and secretion (24 hours and 7 days postirradiation). In the first part of this thesis, we quantified and assigned subcellular localization for over 3000 proteins, of which about 600 potentially redistribute upon UVA exposure. Protein redistributions were accompanied by redox modulations, mitochondrial fragmentation and DNA damage. In the second part of the work, our results showed that primary human keratinocytes enter senescence upon exposure to a single dose of UVA, mounting antioxidant and inflammatory responses. Cells under UVA-induced senescence further elicit paracrine responses in neighboring premalignant HaCaT epithelial cells via inflammatory mediators. Altogether, these results reiterate the role of UVA radiation as a potent metabolic stressor in the skin
A radiação ultravioleta (UV) solar que atinge a superfície terrestre é composta por 95% de radiação UVA (320 a 400 nm) e 5% de radiação UVB (280 a 320 nm). A radiação UVB é carcinogênica e gera lesões potencialmente mutagênicas no DNA. A radiação UVA solar também gera danos no DNA, mas a genotoxicidade dessa radiação não explica inteiramente o seu impacto biológico. Atualmente, sabe-se que a radiação UVA é absorvida por cromóforos celulares, gerando espécies reativas de oxigênio, como o oxigênio singlete. Sabendo que o proteoma é um mediador de respostas ao estresse celular, nós investigamos os efeitos celulares de uma dose não-citotóxica de radiação UVA, equivalente a cerca de 20 minutos de exposição ao sol, no proteoma de queratinócitos humanos. Utilizando espectrometria de massas, bioinformática e ensaios bioquímicos convencionais, nós analisamos dois aspectos do estresse induzido por radiação UVA: o remodelamento espacial do proteoma 30 minutos depois do estresse e alterações nos níveis e na secreção de proteínas no longo prazo (24 horas e 7 dias depois da irradiação). Na primeira parte desta tese, nós quantificamos e atribuímos classificações de localização subcelular a mais de 3000 proteínas. Dentre essas proteínas, 600 tem potencialmente a sua distribuição subcelular alterada em resposta à radiação. As redistribuições subcelulares são acompanhadas de modulações redox, fragmentação mitocondrial e danos no DNA. Na segunda parte da tese, os nossos resultados mostraram que queratinócitos humanos primários entram em senescência sob exposição a uma única dose de radiação UVA, montando respostas antioxidantes e pró-inflamatórias. Células sob senescência induzida por UVA, por sua vez, desencadeiam respostas parácrinas em queratinócitos pré-tumorais (células HaCaT) por meio de mediadores inflamatórios. Em conjunto, esses resultados reiteram o papel da radiação UVA como um potente estressor metabólico em células da pele
Assuntos
Pele , Raios Ultravioleta/efeitos adversos , Queratinócitos/química , Proteômica/classificação , Doses de Radiação , Espectrometria de Massas/métodos , DNA , Células Epiteliais/classificação , Genotoxicidade/efeitos adversos , Células HaCaT/classificação , Antioxidantes/efeitos adversosRESUMO
Once weak ultraviolet ray-B (UVB) irradiates the skin cells, the generation of reactive nitrogen species (RNS), but not reactive oxygen species (ROS), is stimulated for the mislocalization of claudin-1 (CLDN1), an essential protein for forming tight junctions (TJs). Since our skin is constantly exposed to sunlight throughout our lives, an effective protection strategy is needed to maintain the skin barrier against weak UVB. In the present study, we investigated whether an ethanol extract of Brazilian green propolis (EBGP) and flavonoids had a protective effect against weak UVB irradiation-induced barrier dysfunction in human keratinocyte-derived HaCaT cells. A pretreatment with EBGP suppressed TJ permeability, RNS production, and the nitration level of CLDN1 in the weak UVB-exposed cells. Among the propolis components, apigenin and apigenin-like flavonoids have potent protective effects against NO production and the mislocalization of CLDN1 induced by UVB. The analyses between structures and biological function revealed that the chemically and structurally characteristic flavonoids with a hydroxyl group at the 4' position on the B-ring might contribute to its protective effect on barrier dysfunction caused by weak UVB irradiation. In conclusion, EBGP and its component apigenin protect HaCaT cells from weak UVB irradiation-induced TJ barrier dysfunction mediated by suppressing NO production.
Assuntos
Apigenina/farmacologia , Claudina-1/metabolismo , Óxido Nítrico/metabolismo , Própole/farmacologia , Substâncias Protetoras/farmacologia , Brasil , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Etanol/química , Células HaCaT , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Extratos Vegetais/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismo , Raios UltravioletaRESUMO
Cervical cancer is the fourth most common cause of cancer in women worldwide in terms of both incidence and mortality. Persistent infection with high-risk types of human papillomavirus (HPV), namely 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68, constitute a necessary cause for the development of cervical cancer. Viral oncoproteins E6 and E7 play central roles in the carcinogenic process by virtue of their interactions with cell master proteins such as p53, retinoblastoma (Rb), mammalian target of rapamycin (mTOR), and c-MYC. For the synthesis of E6 and E7, HPVs use a bicistronic messenger RNA (mRNA) that has been studied in cultured cells. Here, we report that in cervical tumors, HPV-18, -39, and -45 transcribe E6/E7 mRNAs with extremely short 5' untranslated regions (UTRs) or even lacking a 5' UTR (i.e., zero to three nucleotides long) to express E6. We show that the translation of HPV-18 E6 cistron is regulated by the motif ACCaugGCGCG(C/A)UUU surrounding the AUG start codon, which we term Translation Initiation of Leaderless mRNAs (TILM). This motif is conserved in all HPV types of the phylogenetically coherent group forming genus alpha, species 7, which infect mucosal epithelia. We further show that the translation of HPV-18 E6 largely relies on the cap structure and eIF4E and eIF4AI, two key translation initiation factors linking translation and cancer but does not involve scanning. Our results support the notion that E6 forms the center of the positive oncogenic feedback loop node involving eIF4E, the mTOR cascade, and p53.
Assuntos
Proteínas de Ligação a DNA/genética , Fator de Iniciação 4A em Eucariotos/genética , Fator de Iniciação 4E em Eucariotos/genética , Papillomavirus Humano 18/genética , Proteínas Oncogênicas Virais/genética , RNA Mensageiro/genética , Regiões 5' não Traduzidas/genética , Linhagem Celular Tumoral , Códon de Iniciação/genética , Proteínas de Ligação a DNA/biossíntese , Feminino , Regulação Viral da Expressão Gênica/genética , Células HEK293 , Células HaCaT , Células HeLa , Papillomavirus Humano 18/metabolismo , Humanos , Proteínas Oncogênicas Virais/biossíntese , Iniciação Traducional da Cadeia Peptídica/genética , RNA Viral/genética , Serina-Treonina Quinases TOR/genética , Proteína Supressora de Tumor p53/genética , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/virologiaRESUMO
Industrial application of lycopene is limited due to its chemical instability and low bioavailability. This study proposes the development of fucan-coated acetylated cashew gum nanoparticles (NFGa) and acetylated cashew gum nanoparticles (NGa) for incorporation of the lycopene-rich extract from red guava (LEG). Size, polydispersity, zeta potential, nanoparticles concentration, encapsulation efficiency, transmission electron microscopy (TEM) and atomic force microscopy (AFM) were used to characterize nanoparticles. The antioxidant activity was determinated and cell viability was evaluated in the human breast cancer cells (MCF-7) and human keratinocytes (HaCaT) by MTT assay. The toxic effect was evaluated by hemolysis test and by Galleria mellonella model. NFGa showed higher stability than NGa, having a size of 162.10 ± 3.21 nm, polydispersity of 0.348 ± 0.019, zeta potential -30.70 ± 0.53 mV, concentration of 6.4 × 109 nanoparticles/mL and 60% LEG encapsulation. Microscopic analysis revealed a spherical and smooth shape of NFGa. NFGa showed antioxidant capacity by ABTS method and ORAC assay. The NFGa presented significant cytotoxicity against MCF-7 from the lowest concentration tested (6.25-200 µg/mL) and did not affect the cell viability of the HaCaT. NFGa showed non-toxic effect in the in vitro and in vivo models. Therefore, NFGa may have a promising application in LEG stabilization for antioxidant and antitumor purposes.
Assuntos
Anacardium/química , Antineoplásicos/administração & dosagem , Antioxidantes/administração & dosagem , Licopeno/administração & dosagem , Nanopartículas/química , Gomas Vegetais/química , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Antioxidantes/química , Antioxidantes/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Células HaCaT , Humanos , Licopeno/química , Licopeno/farmacologia , Células MCF-7 , Polissacarídeos/química , Psidium/química , OvinosRESUMO
Marine-derived fungi proved to be a rich source of biologically active compounds. The genus Penicillium has been extensively studied regarding their secondary metabolites and biological applications. However, the photoprotective effects of these metabolites remain underexplored. Herein, the photoprotective potential of Penicillium echinulatum, an Antarctic alga-associated fungus, was assessed by UV absorption, photostability study, and protection from UVA-induced ROS generation assay on human immortalized keratinocytes (HaCaT) and reconstructed human skin (RHS). The photosafety was evaluated by the photoreactivity (OECD TG 495) and phototoxicity assays, performed by 3T3 neutral red uptake (3T3 NRU PT, OECD TG 432) and by the RHS model. Through a bio-guided purification approach, four known alkaloids, (-)-cyclopenin (1), dehydrocyclopeptine (2), viridicatin (3), and viridicatol (4), were isolated. Compounds 3 and 4 presented absorption in UVB and UVA-II regions and were considered photostable after UVA irradiation. Despite compounds 3 and 4 showed phototoxic potential in 3T3 NRU PT, no phototoxicity was observed in the RHS model (reduction of cell viability < 30%), which indicates their very low acute photoirritation and high photosafety potential in humans. Viridicatin was considered weakly photoreactive, while viridicatol showed no photoreactivity; both compounds inhibited UVA-induced ROS generation in HaCaT cells, although viridicatol was not able to protect the RHS model against UVA-induced ROS production. Thus, the results highlighted the photoprotective and antioxidant potential of metabolites produced by P. echinulatum which can be considered a new class of molecules for photoprotection, since their photosafety and non-cytotoxicity were predicted using recommended in vitro methods for topical use.