In vitro evaluation of the cytotoxicity and eye irritation potential of preservatives widely used in cosmetics

Abstract The consumption of cosmetics has been increasing every year and is expected to reach $675 billion by 2020 at an estimated growth rate of 6.4% per year. Exposure to skin irritants is the major cause of non-immunological inflammation of the skin. Therefore, the safety evaluation of cosmetic preservatives should be increased. Thus, the present work aimed to evaluate the cytotoxicity as the viability endpoint and the eye irritation potential of preservatives used in cosmetics. Cytotoxicity assays were performed using MTT and NRU in human keratinocytes (HaCaT), human dermal fibroblasts, adult (HDFa), and human hepatoma cells (HepG2). The eye irritation potential was evaluated using the Hen's Egg Test-chorioallantoic membrane (HET-CAM). The evaluated preservatives were methylparaben (MP), propylparaben (PP), phenoxyethanol (PE), and a mixture of methylchloroisothiazolinone and methylisothiazolinone (CMI/MI). All preservatives showed cytotoxic potential within the permitted concentrations for use in cosmetic products. In the HET-CAM test, PE and CMI/MI, MP, and PP were classified as severe, moderate, and poor irritants, respectively. Our results indicate that proper safety evaluations are required to ensure the beneficial properties of preservatives on cosmetic products without exceeding exposure levels that would result in adverse health effects for consumers.

Role of cytochrome P450 1A2 and N-acetyltransferase 2 in 2, 6-dimethylaniline induced genotoxicity

Abstract The purpose of the current work was to assess a possible role of cytochrome P450 1A2 (CYP1A2) and N-acetyltransferase 2 (NAT2) in the metabolic activation of 2,6-dimethylaniline (2,6-DMA) and also clarify the function of DNA repair in affecting the ultimate mutagenic potency. Two cell lines, nucleotide excision repair (NER)-deficient 5P3NAT2 and proficient 5P3NAT2R9 both expressing CYP1A2 and NAT2, were treated with 2,6-DMA for 48 h or its metabolites for 1 h. Cell survival determined by trypan blue exclusion and MTT assays, and 8-azaadenine-resistant mutants at the adenine phosphoribosyltransferase (aprt) gene locus were evaluated. 5P3NAT2 and 5P3NAT2R9 cells treated with 2,6-DMA and its metabolites showed a dose-dependent increase in cytotoxicity and mutant fraction; N-OH-2,6-DMA and 2,6-DMAP in serum-free α-minimal essential medium (MEM) are more potent than 2,6-DMA in complete MEM. 5P3NAT2 cells was more sensitive to the cytotoxic and mutagenic action than 5P3NAT2R9 cells. H2DCFH-DA assay showed dose-dependent ROS production under 2,6- DMAP treatment. These findings indicate that the genotoxic effects of 2,6-DMA are mediated by CYP1A2 activation via N-hydroxylation and the subsequent esterification by the phase II conjugation enzyme NAT2, and through the generation of ROS by hydroxylamine and/or aminophenol metabolites. NER status is also an important contributor

Análise proteômica de células BEAS-2B expostas a benzo[α]pireno e nicotinamida ribosídeo / Proteomic Analysis of BEAS-2B cells exposed to benzo[α]pyrene and nicotinamide riboside

Responsável por milhões de óbitos anuais e um grande custo para a saúde pública, o câncer é a segunda maior causa de mortes no mundo. Dentre seus diversos tipos, o câncer de pulmão, além da alta incidência, é um dos mais letais. A exposição a substâncias tóxicas provenientes da combustão de matéria orgânica, assim como o consumo de cigarro, são os principais responsáveis pela alta incidência de câncer de pulmão. Dentre estas substâncias, está o benzo[α]pireno (B[α]P), um carcinógeno completo, ou seja, capaz de iniciar e promover o processo de carcinogênese. Resultados anteriores obtidos pelo grupo demonstraram que células BEAS-2B expostas a 1 µM de B[α]P apresentaram alterações das concentrações de metabólitos intracelulares, indução de estresse redox e hipermetilação do DNA. A exposição a 1 µM de nicotinamida ribosídeo (NR), um dos precursores de NAD+, foi capaz de proteger as células BEAS-2B contra a transformação induzida por B[α]P, além de impedir totalmente que células não expostas a B[α]P formassem colônias em soft-agar. A utilização da proteômica neste trabalho permitiu verificar a abundância das proteínas nos quatro diferentes grupos de exposição: Controle, B[α]P, B[α]P + NR e NR. Após 120 h de exposição as células foram coletadas, as proteínas extraídas e preparadas para análise. Foram descobertas 3024 proteínas posteriormente analisadas com o objetivo de elucidar vias possivelmente envolvidas na proteção contra o processo de transfomação maligna. Os grupos NR e Controle demonstram ser mais parecidos em relação ao seu conteúdo, enquanto os grupos B[α]P e B[α]P + NR foram mais semelhantes entre si. A análise de proteínas exclusivas revelou menos processos relacionados ao reparo de DNA no grupo tratado apenas com B[α]P quando comparado com B[α]P + NR. A análise estatística do total de proteínas utilizando o teste ANOVA (p < 0,05, N = 5) revelou 564 proteínas diferencialmente expressas entre os grupos. A clusterização nos permitiu observar a diferença na abundância de proteínas entre os quatro tratamentos. As proteínas estão envolvidas em funções como a regulação do metabolismo, resposta a estresse, transdução de sinal, regulação de expressão gênica e morte celular. Um dos clusters (cluster 1), contendo 59 proteínas, revelou poucos processos na análise de enriquecimento, mas as proteínas contidas nele apresentam funções como controle da divisão celular, apoptose e proteção ao estresse redox. Nele podemos observar que, no geral, o tratamento com B[α]P aumentou a abundância de algumas proteínas, o que foi revertido no grupo B[α]P + NR. O tratamento apenas com NR diminuiu a abundância das proteínas contidas nesse cluster. Outro cluster (cluster 4) apresentou 51 proteínas de abundância diminuída durante a exposição ao B[α]P, o que se reverteu no grupo B[α]P + NR. As proteínas desse cluster estão envolvidas em etapas importantes da via glicolítica, de crescimento, adesão, migração e invasão celular. Apesar de ser descrito que a exposição a NR pode aumentar a eficiência do reparo de DNA, os resultados apresentados nesse trabalho indicam que o efeito protetor pode estar relacionado com a modulação do ciclo celular ou alterações na adesão celular

Responsible for millions of annual deaths and a great health expense, cancer is the second leading cause of death in the world. Among its many types, lung cancer, besides its high incidence, is also one of the most lethal. Exposure to toxic substances resulting from the combustion of organic matter, as well as cigarette consumption, are the mainly responsible for the high incidence of lung cancer. One of these substances is benzo[α]pyrene (B[α]P), a complete carcinogen, able to initiate and promote the carcinogenesis process. Results obtained previously demonstrated that BEAS-2B cells exposed to 1 µM BaP presented alterations in the levels of intracellular metabolites, induction of oxidative stress, and hypermethylation of DNA. The exposure to 1 µM nicotinamide riboside (NR), one of the precursors of NAD+, was able to protect BEAS-2B cells against the transformation induced by B[α]P, moreover, it also totally prevented the colonies formation on soft agar in cells not exposed to B[α]P. The use of proteomics allowed us to verify the abundance of proteins in the four different exposure groups: Control, B[α]P, B[α]P + NR e NR. After 120h of exposure, the cells were collected followed by the extraction of the proteins. A total of 3024 proteins were identified and analyzed aiming to elucidate possible pathways involved in the protective effect against the malignant transformation induced by B[α]P. The NR and Control groups showed to be more similar, while B[α]P and B[α]P + NR were more similar. The analysis of exclusive proteins revealed fewer processes related to DNA repair in B[α]P when compared with B[α]P + NR. The statistical analysis of the total proteins using the ANOVA test (p <0.5, N = 5) revealed 564 proteins differentially expressed between the groups. The heatmap showed the difference in protein abundance between the four treatments. Proteins are involved in functionssuch asthe regulation of metabolism, stress response, signal transduction, regulation of gene expression, and cell death. One of the clusters (cluster 1), containing 59 proteins, revealed a few processes in the enrichment analysis, but the proteins contained in it have functions such as control of cell division, apoptosis, and protection from redox stress. It is possible to observe, in general, treatment with B[α]P increased the abundance of some proteins, which was partially reversed in group B[α]P + NR. On the other hand, the NR treatment decreased the abundance of proteins contained in this cluster. Another cluster (cluster 4) showed 51 proteins of decreased abundance during exposure to B [α] P, which was partially reversed in group B[α]P + NR. The proteins in this cluster are involved in important stages of the glycolytic pathway, also in growth, adhesion, migration, and cell invasion. Although it has been described that exposure to NR can increase the efficiency of DNA repair, the results presented in this work indicate that the protective effect may be related to the modulation of the cell cycle or cell adehsion modifications

Nanostructured lipid carriers as a novel tool to deliver sclareol: physicochemical characterisation and evaluation in human cancer cell lines

Sclareol (SC) is arousing great interest due to its cytostatic and cytotoxic activities in several cancer cell lines. However, its hydrophobicity is a limiting factor for its in vivo administration. One way to solve this problem is through nanoencapsulation. Therefore, solid lipid nanoparticles (SLN-SC) and nanostructured lipid carriers (NLC-SC) loaded with SC were produced and compared regarding their physicochemical properties. NLC-SC showed better SC encapsulation than SLN-SC and was chosen to be compared with free SC in human cancer cell lines (MDA-MB-231 and HCT-116). Free SC had slightly higher cytotoxicity than NLC-SC and produced subdiploid DNA content in both cell lines. On the other hand, NLC-SC led to subdiploid content in MDA-MB-231 cells and G2/M checkpoint arrest in HCT-116 cells. These findings suggest that SC encapsulation in NLC is a way to allow the in vivo administration of SC and might alter its biological properties

Lipid-rich fraction of the sclerotium of Tiger Milk Mushroom Lignosus rhinocerotis (Agaricomycetes) attenuates LPS-induced inflammation in BV2 cells via Nrf2 pathway

Lignosus rhinocerotis (tiger milk mushroom) is widely used by the indigenous people of Malaysia as a traditional remedy. The present study was carried out in order to evaluate the antioxidant, cytotoxic and anti-neuroinflammatory activities of L. rhinocerotis extract on brain microglial cells (BV2). The antioxidant activity was evaluated by 2,2-diphenyl-1-picryhydrazyl (DPPH•), 2,2'-azinobis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS•+) scavenging assays, and ferric reducing antioxidant power (FRAP). The FRAP, DPPH and ABTS•+ scavenging capacities of the TE3 fraction were 420.77 mg FE/g, 58.01%, and 7%, respectively. The cytotoxic activity was determined by MTS assay. The in vitro model of anti-neuroinflammatory property was evaluated by measuring the production of nitric oxide (NO) in lipopolysaccharide (LPS)-induced BV2 cells. The TE3 fraction showed a significant NO reduction at 1 to 100 µg/mL. The TE3 fraction down-regulated inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX2) genes while it upregulated heme oxygenase (HO-1) and NADPH quinone acceptor oxidoreductase-1 (NQO-1) genes. The nuclear factor (erythroid-derived 2)-like 2 (Nrf2) transcription was also activated. The chemical component of the active fraction (TE3) was identified by gas chromatography-mass spectrometry (GCMS). Overall, the BV2 in vitro model anti-neuroinflammatory activity of L. rhinocerotis may be caused by the lipid constituents identified in the fraction

Microscopía electrónica de la interacción de membranas plasmáticas con la matriz extracelular de fibrinoide placentario / Electron microscopy of the plasma membranes interaction with the extracellular matrix of placentary fibrinoid

Estudiar la interacción de la matriz extracelular con membranas plasmáticas de células fetales y maternas con microscopía electrónica de trasmisión y microscopía electrónica de barrido complementada por histoquímica ultraestructural usando azul alcián. Imágenes simultáneas de microfotografías con microscopía electrónica de trasmisión y microscopía electrónica de barrido son correlacionadas en sectores de membranas con relación a la matriz extracelular teñida con azul alcián. Centro de Investigación y Análisis Docente Asistencial del Núcleo Aragua. Facultad de Ciencias de la Salud. Universidad de Carabobo. Maracay-Venezuela. Microfilamentos conectan matriz hipoplasmática con matriz extracelular por intermedio de la membrana. Matriz extracelular de aspecto gránulo-filamentoso; se notó en dos tonalidades contrastadas, granulos electrón densos de células deciduales o trofoblásticas se observan en la periferia celular. Fibrina, colágena degenerada, fibrinoide y gránulos de diversos tamaños conforman una red compleja en el espacio extracelular. Fibrinoide tipo matriz fue reactivo al azul alcián y conforma parte del espacio extracelular observado simultáneamente con imágenes de microscopía electónica de trasmisión y microscopía electrónica de barrido a baja resolución, donde reacciones fisiológicas son de notable importancia clínica

Distribución diferencial de células cebadas en el cuello uterino de cerdas con desarrollo folicular y cuerpo lúteo ováricos / Differential distribution of mast cells in cervix uteri of sows with folicular development and ovaric corpus luteum

Diversos hallazgos experimentales sugieren la participación de las hormonas esteroides ováricas sobre la regulación de la población de células cebadas (CC) en órganos del aparato reproductor femenino. Con el propósito de conocer la distribución de la población de células cebadas en tres porciones anatómicas del cuello uterino (CU) de cerdas con diferentes características morfológicas ováricas, se estudiaron los CU de 20 cerdas púberes clínicamente sanas, 10 con desarrollo folicular y las restantes con cuerpo lúteo funcional. Se tomaron fragmentos de 2 cm2 de los segmentos craneal, medio y caudal del CU y se procesaron mediante la técnica de inclusión en parafina. Los cortes fueron teñidos con azul de tolouidina y posteriormente se contaron las CC presentes por mm2. Hubo diferencia estadística significativa (P< 0.05) entre los tres segmentos anatómicos estudiados, en las dos fases del ciclo estral, resultando una mayor cantidad de CC en el CU de cerdas con desarrollo folicular respecto de las cerdas con cuerpo lúteo. En cada fase se encontró diferencia significativa entre el segmento craneal con respecto a los dos segmentos anatómicos restantes. Estos resultados sugieren que la dinámica funcional de las CC del CU de la cerda varía dependiendo de su porción anatómica y de la condición morfofuncional ovárica que predomina en determinado momento del ciclo estral

Fibrotecoma (Fibroma)de ovario / Fecofibrona (fibrona) of ovary

Se describe un caso de fibrotecoma de ovàrio en una mujer de 48 años, con la presencia de tumoraciòn pèlvica dolorosa y ascitis. Microscopicamente muestra la presencia de cèlulas fusiformes con disposiciòn entrecruzada sin mitosis anormales entremezcladas con nidos de cèlulas tecales. Tumor que se origina en el estroma ovàrico raras veces en un fibroma de, consistencia dura, al corte con aspecto arremolinado blanquesino, con tonos amarillentos, benigno

Resistencia a la insulina, hiperinsulinemia e hipertensión: existe una relación patogénica? / Insulin resistance, hyperinsulinemia and hypertension: existent a patogenic relation?

La relación existente entre insulino-resistencia, hiperinsulinemia e hipertensión es bien conocida, pero la correlación entre estas no prueba que se trata de una interrelación causal. Ha sido postulado que la insulino-resistencia y la hiperinsulinemia compensadora, contribuyen a la patogénesis de la hipertensión, posiblemente estimulando el sistema nervioso simpático, promoviendose la reabsorción de sodio, modulando el transporte de cationes y/o estimulando la hipertrofia del músculo liso vascular. Es nuestro propósito en esta revisión examinar críticamente la hipótesis de la insulinoresistencia-hiperinsulinemia-hipertensión

Antitumoral activity of duodenum and colon extracts of rat and duodenum of mouse pn INHRR-984 carcinoma cells

In former papers we have repoted the existence of a duodenl factor that prevents the developement of Walker's carcinoma when is mixed whith Walker's tumor cells before their inoculation in outbred Sprague- Dawley rats by i.p.,s.c.ori.m.injection. We have also reported, that the duodenal extract (DE) obatined inmediatly after parturition has a stronger inhibitory effect than the duodenal extract from vigin female rats or that of male rats. We have also described some characteristics of the action of the H² Blockers and tyhe sodium-bicarbonate effect.In this paper we are reporting the action of duodenum and colon extracts of rat and duodenum of mouse extract on the transplantable mouse mamary carcinoma (INHRR-984 Ca) originated spontaneouly in outbred N:NIH (S) mouse described elsewhere