RESUMO
The development of metabolically competent in vitro models is of utmost importance for predicting adverse drug reactions, thereby preventing attrition-related economical and clinical burdens. Using the antiretroviral drug nevirapine (NVP) as a model, this work aimed to validate rat hepatocyte 3D spheroid cultures as competent in vitro systems to assess drug metabolism and bioactivation. Hepatocyte spheroids were cultured for 12 days in a stirred tank system (3D cultures) and exposed to equimolar dosages of NVP and its two major Phase I metabolites, 12-OH-NVP and 2-OH-NVP. Phase I NVP metabolites were detected in the 3D cultures during the whole culture time in the same relative proportions reported in in vivo studies. Moreover, the modulation of SULT1A1 activity by NVP and 2-OH-NVP was observed for the first time, pointing their synergistic effect as a key factor in the formation of the toxic metabolite (12-sulfoxy-NVP). Covalent adducts formed by reactive NVP metabolites with N-acetyl-L-cysteine and bovine serum albumin were also detected by high-resolution mass spectrometry, providing new evidence on the relative role of the reactive NVP metabolites, 12-sulfoxy-NVP, and NVP quinone methide, in toxicity versus excretion pathways. In conclusion, these results demonstrate the validity of the 3D culture system to evaluate drug bioactivation, enabling the identification of potential biomarkers of bioactivation/toxicity, and providing new evidence to the mechanisms underlying NVP-induced toxic events. This model, integrated with the analytical strategies described herein, is of anticipated usefulness to the pharmaceutical industry, as an upstream methodology for flagging drug safety alerts in early stages of drug development.
Assuntos
Hepatócitos/efeitos dos fármacos , Nevirapina/farmacocinética , Esferoides Celulares/efeitos dos fármacos , Acetilcisteína/química , Acetilcisteína/metabolismo , Animais , Arilsulfotransferase/metabolismo , Biotransformação , Técnicas de Cultura de Células/métodos , Hepatócitos/metabolismo , Inativação Metabólica , Ratos , Reprodutibilidade dos TestesRESUMO
Sophorolipids (SLs) are glycolipid biosurfactants, produced as a mixture of several compounds by some nonpathogenic yeast. In the current study, separation of individual SLs from mixtures with further evaluation of their surface properties and biologic activity on MDA-MB-321 breast cancer cell line were investigated. SLs were biosynthesized by Starmerella bombicola in a culture media supplemented with borage oil. A reverse-phase flash chromatography method with an automated system coupled with a prepacked cartridge was used to separate and purify the main SLs. Compositional analysis of SLs was performed by high-performance liquid chromatography with electrospray ionization mass spectrometry and tandem mass spectrometry. The following diacetylated lactonic SLs were isolated and purified: C18:0, C18:1, C18:2, and C18:3. The critical micelle concentration (CMC) and surface tension at CMC (γCMC ) of the purified SLs showed an increase with the number of double bonds. High cytotoxic effect against MDA-MB-231 cells was observed with C18:0 and C18:1 lactonic SLs. The cytotoxic effects of C18:3 lactonic SL on cancerous cells were for the first time studied. This cytotoxic effect was considerably higher than the promoted by acidic SLs; however, it induced a lower effect than the previously mentioned SLs, C18:0 and C18:1. To our knowledge, for the first time, C18:1 lactonic SL, in selected concentrations, proved to be able to inhibit MDA-MB-231 cell migration without compromising cell viability and to increase intracellular reactive oxygen species.
Assuntos
Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Glicolipídeos/biossíntese , Glicolipídeos/farmacologia , Saccharomycetales/fisiologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Cromatografia Líquida/métodos , Meios de Cultura/química , Feminino , Humanos , Óleos de Plantas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem , Ácido gama-Linolênico/farmacologiaRESUMO
Microtiter plates were used as minireactors to study Starmerella bombicola growth and sophorolipid (SL) production. Compositional analysis of SL mixtures by liquid chromatography with electrospray ionization tandem mass spectrometry showed similar results on SLs produced using the laboratory scale (shake flask) and the microscale (24-well microtiter plates (MTP)) approach. MTP suitability on SL production was proven, being this approach, especially advantageous on SL screening. Several hydrophilic carbon sources, hydrophobic co-substrates and nitrogen sources were supplied to culture media, and their influence on SL production was evaluated. The selection of specific hydrophobic co-substrate and nitrogen sources influenced the ratio acidic/lactonic SLs. In fact, it was observed that the production of acidic C18:1 diacetylated hydroxy fatty acid SLs was favoured when culture media was supplied with avocado, argan, sweet almond and jojoba oil or when NaNO(3) was supplied instead of urea. This last case was observed after 144 h of cultivation. A new SL, lactonic C18:3 hydroxy fatty acid diacetylated SL, was detected when borage and onagra oils were used individually as co-substrates. Overall results indicated the potential of the selective production of different and new sophorolipids by Starmerella bombicola based on the selection of carbon and nitrogen sources to culture media.
Assuntos
Ascomicetos/crescimento & desenvolvimento , Ascomicetos/metabolismo , Meios de Cultura/química , Glicolipídeos/metabolismo , Carbono/metabolismo , Cromatografia Líquida , Ensaios de Triagem em Larga Escala/métodos , Nitrogênio/metabolismo , Óleos de Plantas/metabolismo , Espectrometria de Massas em TandemRESUMO
Rhodotorula bogoriensis is known as the producer of longer chain acidic sophorolipids (SLs) with a unique hydroxylation position where the sophorose unit is linked to the 13-hydroxydocosanoic acid. The influence of initial inoculum concentration, hydrophilic and hydrophobic carbon, and nitrogen sources on R. bogoriensis growth and SL production was evaluated to obtain a selective SL production. Experiments took place in microtiter plates, used as minireactors, after the verification of its suitability compared with shake flasks. The common structure of SLs is the 13-[2'-O-ß-d-glucopyranosyl-ß-d-glucopyranosyloxy]-docosanoic acid SL. The analysis of the fermentation media using high-performance liquid chromatography with evaporative light scattering detector showed the production of four main SLs, respectively, in the following forms: (i) deacetylated (peak A) (C22:0 SL), (ii) 6â³monoacetylated (peak B) (C22:0-6â³Ac SL), (iii) 6'monoacetylated (peak C) (C22:0-6'Ac SL), and (iv) 6',6â³ diacetylated (peak D) (C22:0-6',6â³Ac SL). The identification of compounds in SL mixtures was performed by liquid chromatography with electrospray ionization mass spectrometry analysis, and no differences were observed. Besides the four compounds detected using high-performance liquid chromatography with evaporative light scattering detector chromatograms, three other SLs was identified, corresponding to mono- and diacetylated C24:0 hydroxy fatty acid SLs. To our knowledge, this work presents for the first time the production and identification of C24:0 SLs. A longer hydrophobic tail on SLs had an important role in the improvement of surface active properties. The selection of a specific time for fermentation end and the use of different carbon (e.g. glucose, fructose, mannose, lactose, galactose, xylose) and nitrogen (e.g. peptone, (NH(4))(2)SO(4) and NaNO(3)) sources led to a selective production of de-, mono-, and diacetylated SLs by R. bogoriensis.
Assuntos
Carbono/metabolismo , Ácidos Graxos/química , Glucanos/química , Glicolipídeos/biossíntese , Nitrogênio/metabolismo , Rhodotorula/metabolismo , Cromatografia Líquida de Alta Pressão , Meios de Cultura , Ácidos Graxos/metabolismo , Fermentação , Glucanos/metabolismo , Glicolipídeos/química , Glicolipídeos/isolamento & purificação , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Conjugated linoleic acid (CLA) has been reported as having body fat lowering properties and the ability to modulate the inflammatory system in several models. In the present study, the effects of CLA added to saturated fat diets, from vegetable and animal origins, on the serum adipokine profile of obese Zucker rats were assessed. In addition, the fatty acid composition of epididymal and retroperitoneal adipose tissues was determined and a principal component analysis (PCA) was used to assess possible relationships between fatty acids and serum metabolites. Atherogenic diets (2 % cholesterol) were formulated with palm oil and ovine fat and supplemented or not with 1 % of a mixture (1:1) of cis-9, trans-11 and trans-10, cis-12-CLA isomers. CLA-fed animals exhibited lower daily feed intake, final body and liver weights, and hepatic lipids content. Total and LDL-cholesterol levels were increased in CLA-supplemented groups. CLA also promoted higher adiponectin and lower plasminogen activator inhibitor-1 (PAI-1) serum concentrations. In contrast to palm oil diets, ovine fat increased insulin resistance and serum levels of leptin, TNF-alpha and IL-1beta. Epididymal and retroperitoneal adipose tissues had similar deposition of individual fatty acids. The PCA analysis showed that the trans-10, cis-12-CLA isomer was highly associated with adiponectin and PAI-1 levels. Summing up, CLA added to vegetable saturated enriched diets, relative to those from animal origin, seems to improve the serum profile of adipokines and inflammatory markers in obese Zucker rats due to a more favourable fatty acid composition.
Assuntos
Adipocinas/sangue , Tecido Adiposo/química , Gorduras na Dieta/administração & dosagem , Ácidos Graxos/análise , Ácidos Linoleicos Conjugados/administração & dosagem , Obesidade/metabolismo , Animais , Composição Corporal , Colesterol/sangue , Gorduras Insaturadas na Dieta/administração & dosagem , Epididimo , Gordura Intra-Abdominal/química , Lipídeos/análise , Fígado/anatomia & histologia , Fígado/química , Masculino , Obesidade/sangue , Tamanho do Órgão , Óleo de Palmeira , Óleos de Plantas/administração & dosagem , Inibidor 1 de Ativador de Plasminogênio/sangue , Ratos , Ratos Zucker , OvinosRESUMO
OBJECTIVE: To evaluate the effect of postpolymerization treatment based on ethanol-aqueous solutions on the residual monomer (RM) content, flexural strength, microhardness, and cytotoxicity of hard chairside reline resins (Kooliner, Ufi Gel Hard). METHODS: After polymerization, specimens were immersed in water, 20%, 50%, or 70% ethanol solutions at 23°C or 55°C for 10 minutes. Controls were left untreated. HPLC was used for the determination of RM content. Specimens were submitted to Vickers microhardness and 3-point loading flexural strength tests. Cytotoxicity of resin eluates was determined on human fibroblasts by assessing cellular mitochondrial function and lactate dehydrogenase release. RESULTS: Higher concentrations of ethanol promoted lower RM content at 55°C in both materials. The mechanical properties were maintained after 50% and 20% ethanol treatments in Kooliner and Ufi Gel Hard, respectively. Specimens submitted to those treatments showed significant reduction on cytotoxicity compared to immersion in hot water, the treatment of choice in the recent literature. SIGNIFICANCE: Immersion of relined dentures in specific ethanol solutions at 55°C for 10 minutes can be considered an effective postpolymerization treatment contributing to increase materials biocompatibility. The proposed protocol is expeditious and easy to achieve with simple equipment in a dental office.
Assuntos
Resinas Acrílicas/síntese química , Resinas Acrílicas/farmacologia , Reembasamento de Dentadura/métodos , Etanol/química , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Materiais Biocompatíveis/síntese química , Materiais Biocompatíveis/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Etanol/farmacologia , Teste de Materiais , Polímeros/síntese química , Polímeros/farmacologia , Resinas Sintéticas/síntese química , Resinas Sintéticas/farmacologiaRESUMO
High-performance liquid chromatography (HPLC) with evaporative light scattering detection (ELSD) and HPLC with electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) methods were implemented and optimized to separate and identify sophorolipids (SLs) produced by Rhodotorula bogoriensis and Starmerella bombicola. SLs are carbohydrate-based amphiphilic biosurfactants with increased interest in pharmaceutical and environmental areas. Rhodotorula bogoriensis and Starmerella bombicola are mainly producers of respectively C22, and C16 and C18 SLs. Mass fragmentation patterns of SLs produced by both yeasts were investigated by HPLC-ESI-MS/MS in the positive mode for [M+Na]+. Based on the established fragmentation pattern, SLs produced by both yeasts were identified and characterized. A correlation between HPLC-ELSD and HPLC- ESI-MS/MS methods was established and made possible the identification of SLs by the HPLC-ELSD technique. TLC is a common tool for the analysis of SLs mixtures. In this work, TLC scrapped bands were analysed by HPLC-ELSD and HPLC-MS allowing the correlation between R(F) values and the identification of sophorolipids by this technique. Identification of monoacetylated and diacetylated C24:0 hydroxy fatty acids sophorolipids produced by Rhodotorula bogoriensis was for the first time accomplished with this study. Although present in lower quantity these longer chain SLs can assume special importance regarding their biological activity and surface active properties.
Assuntos
Ascomicetos/química , Cromatografia Líquida de Alta Pressão/métodos , Lipídeos/química , Rhodotorula/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodos , Ascomicetos/metabolismo , Metabolismo dos Lipídeos , Rhodotorula/metabolismoRESUMO
The effect of dietary conjugated linoleic acid (CLA) supplementation in combination with fat from vegetable versus animal origin on the fatty acid deposition, including that of individual 18:1 and 18:2 (conjugated and non-conjugated) isomers, in the liver and muscle of obese rats was investigated. For this purpose, 32 male Zucker rats were randomly assigned to one of four diets containing palm oil or ovine fat, supplemented or not with 1% of 1:1 cis(c)9,trans(t)11 and t10,c12 CLA isomers mixture. Total fatty acid content decreased in the liver and muscle of CLA-fed rats. In the liver, CLA increased saturated fatty acids (SFA) in 11.9% and decreased monounsaturated fatty acids (MUFA) in 6.5%. n-3 Polyunsaturated fatty acids (PUFA) relative proportions were increased in 30.6% by CLA when supplemented to the ovine fat diet. In the muscle, CLA did not affect SFA but decreased MUFA and PUFA percentages. The estimation of Δ9-indices 16 and 18 suggested that CLA inhibited the stearoyl-CoA desaturase activity in the liver (a decrease of 13-38%), in particular when supplemented to the ovine fat diet. Concerning CLA supplementation, the t10,c12 isomer percentage was 60-80% higher in the muscle than in the liver. It is of relevance that rats fed ovine fat, containing bio-formed CLA, had more c9,t11 CLA isomer deposited in both tissues than rats fed palm oil plus synthetic CLA. These results highlight the importance to further clarify the biological effects of consuming foods naturally enriched in CLA, alternatively to CLA dietary supplementation.
Assuntos
Gorduras na Dieta/farmacologia , Ácidos Linoleicos Conjugados/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Músculos/efeitos dos fármacos , Óleos de Plantas/farmacologia , Tecido Adiposo/efeitos dos fármacos , Animais , Composição Corporal/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Gorduras na Dieta/metabolismo , Relação Dose-Resposta a Droga , Ácidos Graxos Monoinsaturados/metabolismo , Ácidos Graxos Insaturados/biossíntese , Ácidos Linoleicos Conjugados/metabolismo , Fígado/metabolismo , Masculino , Músculos/metabolismo , Obesidade/metabolismo , Obesidade/fisiopatologia , Óleo de Palmeira , Óleos de Plantas/metabolismo , Ratos , Ratos Zucker , Carneiro Doméstico , Estearoil-CoA Dessaturase/metabolismoRESUMO
The fatty acid composition of phospholipids plays a key role in the structural and functional properties of cellular membrane. In this study, it was hypothesized that conjugated linoleic acid (CLA) isomer supplementation changes the unsaturation degree of the fatty acids of neutral lipids (NLs) but not those of polar lipids (PLs). Thus, the main goal was to determine the pattern of fatty acid incorporation into hepatic PL and NL fractions. Wistar male rats were fed cis(c)9,trans(t)11 and t10,c12 CLA isomers, separately or as a mixture. Whereas the t10,c12 isomer incorporation in the PL fraction was similar when supplemented either individually or as a mixture, the c9,t11 isomer reached the highest values of incorporation when combined with t10,c12. In the PL fraction, the linoleic acid did not change; but the arachidonic acid decreased, especially in the rats given the mixture. Also in this fraction, the t10,c12 isomer, either separately or as a mixture, decreased the amounts of n-6 long-chain (LC) polyunsaturated fatty acids (PUFA) and increased those of the n-3 LC PUFA relative to the control. In the NL fraction, linoleic acid incorporation followed the diet composition, whereas the arachidonic acid was similar among treatments. Facing CLA isomer supplementation, the present study suggests that fatty acid incorporation into phospholipids, through the balance between n-6 and n-3 LC PUFA, is dependent upon maintaining the unsaturation degree of cellular membrane.
Assuntos
Gorduras Insaturadas na Dieta/metabolismo , Ácidos Linoleicos Conjugados/administração & dosagem , Ácidos Linoleicos Conjugados/metabolismo , Fígado/metabolismo , Animais , Ácido Araquidônico/metabolismo , Dieta , Suplementos Nutricionais , Isomerismo , Ácido Linoleico/metabolismo , Masculino , Fosfolipídeos/metabolismo , Ratos , Ratos WistarRESUMO
We hypothesized that reducing weight properties of conjugated linoleic acid (CLA) are due to adipocyte apoptosis and that CLA differentially modulates the apoptotic responses in hepatic lipotoxicity from rats fed saturated fat diets. Obese Zucker rats were fed atherogenic diets (2%w/w of cholesterol) formulated with high (15%w/w) saturated fat, from vegetable or animal origin, supplemented or not with 1% of a mixture (1:1) of cis-9, trans-11 and trans-10, cis-12 CLA isomers for 14 weeks. CLA induced no changes on retroperitoneal fat depot weight, which was in line with similar levels of apoptosis. Interestingly, CLA had a contrasting effect on cell death in the liver according to the dietary fat. CLA increased hepatocyte apoptosis, associated with upregulation of Fas protein in rats fed palm oil, compared to rats receiving palm oil alone. However, rats fed ovine fat alone displayed the highest levels of hepatic cell death, which were decreased in rats fed ovine fat plus CLA. This reducing effect of CLA was related to positively restoring endoplasmic reticulum (ER) ATF-6α, BiP and CHOP protein levels and increasing phosphorylated c-Jun NH(2)-terminal kinase (JNK) and c-Jun, thus suggesting an adaptive response of cell survival. These findings reinforce the role of CLA as regulator of apoptosis in the liver. Moreover, the dietary fat composition is a key factor in activation of apoptosis.
Assuntos
Apoptose/efeitos dos fármacos , Gorduras na Dieta/administração & dosagem , Ácidos Linoleicos Conjugados/administração & dosagem , Fígado/efeitos dos fármacos , Obesidade/metabolismo , Óleos de Plantas/administração & dosagem , Animais , Caspase 3/metabolismo , Gorduras na Dieta/farmacologia , Gorduras Insaturadas na Dieta/administração & dosagem , Gorduras Insaturadas na Dieta/farmacologia , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Isomerismo , Ácidos Linoleicos Conjugados/química , Ácidos Linoleicos Conjugados/farmacologia , Fígado/citologia , Fígado/metabolismo , Masculino , Obesidade/induzido quimicamente , Óleo de Palmeira , Óleos de Plantas/farmacologia , Ratos , Ratos Zucker , OvinosRESUMO
OBJECTIVES: The development of different types of materials with application in dentistry is an area of intense growth and research, due to its importance in oral health. Among the different materials there are the acrylic based resins that have been extensively used either in restorations or in dentures. The objective of this manuscript was to review the acrylic based resins biodegradation phenomena. Specific attention was given to the causes and consequences of materials degradation under the oral environment. DATA AND SOURCES: Information from scientific full papers, reviews or abstracts published from 1963 to date were included in the review. Published material was searched in dental literature using general and specialist databases, like the PubMED database. STUDY SELECTION: Published studies regarding the description of biodegradation mechanisms, in vitro and in vivo release experiments and cell based studies conducted on acrylic based resins or their components were evaluated. Studies related to the effect of biodegradation on the physical and mechanical properties of the materials were also analyzed. CONCLUSIONS: Different factors such as saliva characteristics, chewing or thermal and chemical dietary changes may be responsible for the biodegradation of acrylic based resins. Release of potential toxic compounds from the material and change on their physical and mechanical properties are the major consequences of biodegradation. Increasing concern arises from potential toxic effects of biodegradation products under clinical application thus justifying an intensive research in this area.
Assuntos
Resinas Acrílicas/química , Resinas Acrílicas/metabolismo , Resinas Acrílicas/toxicidade , Animais , Biotransformação , Análise do Estresse Dentário , Humanos , Hidrólise , Metilmetacrilatos/químicaRESUMO
Previous reports have demonstrated that conjugated linoleic acid (CLA) acts on body fat accumulation in a variety of animal models. The aim of the present study was to investigate the effect of cis (c)-9,trans (t)-11 and t10,c12 CLA isomers on the number and size of adipocytes from the inguinal and retroperitoneal fats in Wistar male rats. A 5.1% palm oil-based diet was supplemented with CLA isomers as follows: 0.6% of c9,t11, 0.6% of t10,c12, 1.3% of c9,t11 and t10,c12 isomers in mixture, and a control nonsupplemented group for comparative purposes. Fat tissues were prepared on microscope slides for histologic examination using an image-analysis software to count the number of adipocytes and measure cell sizes. The results showed that CLA isomers did not affect (P > .05) either final body and fat depot weights or serum lipids (with the exception of triacylglycerols) and adipocytokines (leptin and adiponectin). Animals fed the c9,t11 CLA isomer diet showed larger adipocytes when compared to other groups. Independently of the CLA dietary treatment, retroperitoneal fat showed larger adipocytes (3319 microm(2)) and therefore a smaller number of adipocytes per unit of area, compared to inguinal fat (3055 microm(2)). Taken together, the data suggest that a palm oil-based diet supplemented with the c9,t11 CLA isomer in Wistar rats, in contrast to the t10,c12 isomer and the mixture of both isomers, increases adipocyte dimensions in inguinal and retroperitoneal fat depots, while having a minor effect in serum lipids and adipocytokines.
Assuntos
Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Ácido Linoleico/administração & dosagem , Adiponectina/sangue , Animais , Contagem de Células , Tamanho Celular/efeitos dos fármacos , Dieta , Suplementos Nutricionais , Virilha , Leptina/sangue , Lipídeos/sangue , Masculino , Ratos , Ratos Wistar , Espaço RetroperitonealRESUMO
The present study provides a detailed overview of the contents of conjugated linoleic acid (CLA) isomers in the most consumed Portuguese CLA-rich foods (milk, butter, yoghurt, cheese, beef and lamb meat), by using silver ion-HPLC. In addition, the contribution of these ruminant-derived foods to the daily intake of CLA isomers was estimated based on Portuguese consumption habits. The total CLA concentration in milk and dairy products ranged from 4.00 mg/g fat in yoghurt to 7.22 mg/g fat in butter, and, regarding meats, from 4.45 mg/g fat in intensively produced beef to 11.29 mg/g fat in lamb meat. The predominant CLA isomers identified in these products were cis-9, trans-11 (59.89-79.21 %) and trans-7, cis-9 (8.04-20.20 %). The average estimated total CLA intake for the Portuguese population was 73.70 mg/d. Milk and cheese are probably the two products with the highest contribution to the final CLA intake, as a result of their high fat content and consumption values. The results also suggested that cis-9, trans-11 and trans-7, cis-9 are the isomers most represented, with, respectively, 76.10 and 12.56 % of the total CLA intake. Being the first detailed report on the contents of total and individual CLA isomers in Portuguese commercial ruminant-derived foods, we further discuss the implication of the results for diet characteristics and human health.