Nuclear Factor-Kappa-B Mediates the Advanced Glycation End Product-Induced Repression of Slc2a4 Gene Expression in 3T3-L1 Adipocytes.

Advanced glycated end products (AGEs) are cytotoxic compounds that are mainly increased in diabetes mellitus (DM), kidney failure, inflammation, and in response to the ingestion of AGE-rich diets. AGEs can also impair glycemic homeostasis by decreasing the expression of the Slc2a4 (solute carrier family 2 member 4) gene and its GLUT4 (solute carrier family 2, facilitated glucose transporter member 4) protein in muscle. However, the mechanisms underlying AGE's effect on adipocytes have not been demonstrated yet. This study investigated the effects of AGEs upon Slc2a4/GLUT4 expression in 3T3-L1 adipocytes, as well as the potential role of NFKB (nuclear factor NF-kappa-B) activity in the effects observed. Adipocytes were cultured in the presence of control albumin (CA) or advanced glycated albumin (GA) at concentrations of 0.4, 3.6, and 5.4 mg/mL for 24 h or 72 h. Slc2a4, Rela, and Nfkb1mRNAs were measured by RT-qPCR, GLUT4, IKKA/B, and p50/p65 NFKB subunits using Western blotting, and p50/p65 binding into the Slc2a4 promoter was analyzed by chromatin immunoprecipitation (ChIP) assay. GA at 0.4 mg/mL increased Slc2a4/GLUT4 expression after 24 h and 72 h (from 50% to 100%), but at 5.4 mg/mL, Slc2a4/GLUT4 expression decreased at 72 h (by 50%). Rela and Nfkb1 expression increased after 24 h at all concentrations, but this effect was not observed at 72 h. Furthermore, 5.4 mg/mL of GA increased the p50/p65 nuclear content and binding into Slc2a4 at 72 h. In summary, this study reveals AGE-induced and NFKB-mediated repression of Slc2a4/GLUT4 expression. This can compromise the adipocyte glucose utilization, contributing not only to the worsening of glycemic control in DM subjects but also the impairment of glycemic homeostasis in non-DM subjects under the high intake of AGE-rich foods.

Silybin restores glucose uptake after tumour necrosis factor-alpha and lipopolysaccharide stimulation in 3T3-L1 adipocytes.

Obesity is associated with a low-grade chronic inflammatory process characterized by higher circulating TNFα levels, thus contributing to insulin resistance. This study evaluated the effect of silybin, the main bioactive component of silymarin, which has anti-inflammatory properties, on TNFα levels and its impact on glucose uptake in the adipocyte cell line 3T3-L1 challenged with two different inflammatory stimuli, TNFα or lipopolysaccharide (LPS). Silybin's pre-treatment effect was evaluated in adipocytes pre-incubated with silybin (30 or 80 µM) before challenging with the inflammatory stimuli (TNFα or LPS). For the post-treatment effect, the adipocytes were first challenged with the inflammatory stimuli and then post-treated with silybin. After treatments, TNFα production, glucose uptake, and GLUT4 protein expression were determined. Both inflammatory stimuli increased TNFα secretion, diminished GLUT4 expression, and significantly decreased glucose uptake. Silybin 30 µM only reduced TNFα secretion after the LPS challenge. Silybin 80 µM as post-treatment or pre-treatment decreased TNFα levels, improving glucose uptake. However, glucose uptake enhancement induced by silybin did not depend on GLUT4 protein expression. These results show that silybin importantly reduced TNFα levels and upregulates glucose uptake, independently of GLUT4 protein expression.

Mineral matrix deposition of MC3T3-E1 pre-osteoblastic cells exposed to silicocarnotite and nagelschmidtite bioceramics: In vitro comparison to hydroxyapatite.

This work presents the effect of the silicocarnotite (SC) and nagelschmidtite (Nagel) phases on in vitro osteogenesis. The known hydroxyapatite of biological origin (BHAp) was used as a standard of osteoconductive characteristics. The evaluation was carried out in conventional and osteogenic media for comparative purposes to assess the osteogenic ability of the bioceramics. First, the effect of the material on cell viability at 24 h, 7 and 14 days of incubation was evaluated. In addition, cell morphology and attachment on dense bioceramic surfaces were observed by fluorescence microscopy. Specifically, alkaline phosphatase (ALP) activity was evaluated as an osteogenic marker of the early stages of bone cell differentiation. Mineralized extracellular matrix was observed by calcium phosphate deposits and extracellular vesicle formation. Furthermore, cell phenotype determination was confirmed by scanning electron microscope. The results provided relevant information on the cell attachment, proliferation, and osteogenic differentiation processes after 7 and 14 days of incubation. Finally, it was demonstrated that SC and Nagel phases promote cell proliferation and differentiation, while the Nagel phase exhibited a superior osteoconductive behavior and could promote MC3T3-E1 cell differentiation to a higher extent than SC and BHAp, which was reflected in a higher number of deposits in a shorter period for both conventional and osteogenic media.

10­Gingerol, a novel ginger compound, exhibits antiadipogenic effects without compromising cell viability in 3T3­L1 cells.

Obesity is defined as excessive fat accumulation that can be detrimental to health and currently affects a large part of the global population. Obesity arises from excessive energy intake along with a sedentary lifestyle and leads to adipocytes with aggravated hypertrophy. Strategies have been designed to prevent and treat obesity. Nutrigenomics may serve a role in prevention of obesity using bioactive compounds present in certain foods with anti-obesogenic effects. Ginger (Zingiber officinale Roscoe) contains gingerols, key bioactive compounds that inhibit hypertrophy and hyperplasia of adipocytes. The present study aimed to evaluate the antiadipogenic activity of 10-gingerol (10-G) in the 3T3-L1 cell line. Three study groups were formed: Negative (3T3-L1 preadipocytes) and positive control (mature 3T3-L1 adipocytes) and 10-G (3T3-L1 preadipocytes stimulated with 10-G during adipogenic differentiation). Cell viability and lipid content were evaluated by MTT assay and Oil Red O staining, respectively. mRNA expression of CCAAT enhancer-binding protein α (C/ebpα), peroxisome proliferator-activated receptor γ (Pparγ), mechanistic target of rapamycin complex (Mtor), sterol regulatory element binding transcription factor 1 (Srebf1), acetyl-coenzyme A carboxylase (Acaca), fatty acid binding protein 4 (Fabp4), and 18S rRNA (Rn18s), was quantified by quantitative PCR. The protein expression of C/EPBα was analyzed by western blot. In the 10-G group, lipid content was decreased by 28.83% (P<0.0001) compared with the positive control; notably, cell viability was not affected (P=0.336). The mRNA expression in the 10-G group was higher for C/ebpα (P<0.001) and lower for Acaca (P<0.001), Fabp4 (P<0.001), Mtor (P<0.0001) and Srebf1 (P<0.0001) compared with the positive control group, while gene expression of Pparγ did not present significant changes. The presence of 10-G notably decreased C/EBPα protein levels in 3T3-L1 adipocytes. In summary, the antiadipogenic effect of 10-G during the differentiation of 3T3-L1 cells into adipocytes may be explained by mRNA downregulation of adipogenic transcriptional factors and lipid metabolism-associated genes.

Pharmacological and Chemical Analysis of Bauhinia divaricata L. Using an In Vitro Antiadipogenic Model.

Obesity is characterized by an excessive and abnormal accumulation of fat. According to the 2022 National Health and Nutrition Survey, in Mexico, the prevalence of overweight and obesity-diagnosed if one's body mass index (BMI) was ≥25 kg/m2-in adults was 75.2%. A strong association between the amount of visceral fat and diseases such as diabetes mellitus type II has been recognized. Species of the Bauhinia genus have lipid-lowering and antidiabetic properties. The aim of this work was to evaluate the lipolytic and antiadipogenic activity of Bauhinia divaricata L. in 3T3-L1 cells and to identify the major compounds in the bioactive treatments. The extraction of aerial parts allowed us to obtain hexanic (BdHex), ethyl acetate (BdEAc), and hydroalcoholic (BdHA) extracts. Lipid levels were measured in 3T3-L1 cells differentiated into adipocytes. Our evaluation of cell viability identified an IC50 > 1000 µg/mL in all the extracts, and our evaluation of the antiadipogenic activity indicated that there was a significant reduction (p < 0.001) in the accumulation of lipids with hydroalcoholic (60%) and ethyl acetate (75%) extracts of B. divaricate compared with metformin at 30 mM (65%). The major compounds identified in these extracts were as follows: triacetin (1), 2,3-dihydroxypropyl acetate (2), (3E)-2-methyl-4-(1,3,3-trimethyl-7-oxabicyclo[4.1.0]hept-2-yl)-3-buten-2-ol (3), 2,5-dihydroxyphenylacetic acid (4), (3R)-3-hydroxydodecanoic acid (5), kaempferol-3-O-rhamnoside (6), and quercetin 3-O-rhamnoside (7). Some of these naturally occurring compounds have been related to the anti-obesity effects of other medicinal plants; therefore, these compounds isolated from B. divaricata could be responsible for inhibiting the differentiation process from preadipocytes to mature adipocytes.

Anthocyanin-enriched extract from Ribes nigrum inhibits triglyceride and cholesterol accumulation in adipocytes.

Aim: Obesity is a chronic pathology of epidemic proportions. Mature adipocytes from a 3T3-L1 cell line were used as in vitro obesity model to test different bioactive compounds. We aim to evaluate cassis (Ribes nigrum) extract antioxidant activity and its antiadipogenic effect on mature adipocytes. Results: We produced an extract by using enzyme that combines cellulase and pectinase; we obtained high yield of the bioactive compound anthocyanin. Extract showed high antioxidant capacity. We conducted in vitro assays by adding the extract to adipocytes culture medium. Extract reduced intracellular levels of triglyceride by 62% and cholesterol by 32%. Conclusion: Enzymatic extract's high antioxidant activity was likely attributable to its high concentration of anthocyanin. This extract inhibits lipid accumulation in adipocytes.

Obesity is a disease all over the world. By 2030, nearly 20% of adults are predicted to be obese. The consumption of processed foods is related to obesity in some countries such as Argentina. More natural food is needed. There are many different anti-obesity medicines but there is no good one to lose weight. We took extracts from cassis fruits and tested whether they could decrease fats like cholesterol within fat cells. We found that these extracts could successfully reduce the fat levels in the cells. Our results indicate that natural compounds like cassis fruit extract may be helpful in preventing future obesity epidemics.

A mix of ginger phenols exhibits anti­adipogenic and lipolytic effects in mature adipocytes derived from 3T3­L1 cells.

The prevalence of obesity has increased rapidly worldwide. Obesity is characterized by excessive adipose tissue in the body, which is related to hyperplasia and hypertrophy in adipocytes. Ginger (Zingiber officinale Roscoe) is a medicinal plant that possesses an anti-obesogenic effect mostly attributed to gingerols, the most abundant bioactive compounds in ginger. The anti-adipogenic and lipolytic effects of these phenols have been shown when investigated individually. Therefore, the present study aimed to evaluate the lipolytic and anti-adipogenic activity of a mix of the main ginger phenols 6-gingerol, 8-gingerol, 10-gingerol, 6-shogaol, 8-shogaol and 10-shogaol on the 3T3-L1 cell line. A total of four study groups were designed: Negative control (3T3-L1 preadipocytes); positive control (mature 3T3-L1 adipocytes); phenols-pre (3T3-L1 cells stimulated with the phenols mix during adipogenic differentiation); and phenols-post (mature 3T3-L1 adipocytes stimulated with the phenols mix). MTT viability cell assay and Oil Red O staining were performed. Glycerol concentration supernatants were determined using the VITROS 350 Chemistry System. Expression of mRNA was measured using qPCR. The treatment with a 2 µg/ml ginger phenol dose reduced the lipid content by 45.52±7.8 and 35.95±0.76% in the phenols-pre and -post group, respectively, compared with that in the positive control group. The phenols-post group presented a higher glycerol concentration in the supernatant compared with that in the positive control and the phenols-pre groups. The mRNA expression levels of CCAAT/enhancer-binding protein alpha, peroxisome proliferator activated receptor-γ, fatty acid-binding protein 4 and fatty acid synthase were higher in the phenols-pre and lower in the phenols-post groups, compared with those in the positive control group. To the best of our knowledge, the current study demonstrated for the first time the anti-adipogenic and lipolytic effects of a mix of the main bioactive compounds found in ginger, and it also established the basis to use this mix of phenols in in vivo studies and clinical trials.

Effect of Electrospun PLGA/Collagen Scaffolds on Cell Adhesion, Viability, and Collagen Release: Potential Applications in Tissue Engineering.

The development of scaffolding obtained by electrospinning is widely used in tissue engineering due to porous and fibrous structures that can mimic the extracellular matrix. In this study, poly (lactic-co-glycolic acid) (PLGA)/collagen fibers were fabricated by electrospinning method and then evaluated in the cell adhesion and viability of human cervical carcinoma HeLa and NIH-3T3 fibroblast for potential application in tissue regeneration. Additionally, collagen release was assessed in NIH-3T3 fibroblasts. The fibrillar morphology of PLGA/collagen fibers was verified by scanning electron microscopy. The fiber diameter decreased in the fibers (PLGA/collagen) up to 0.6 µm. FT-IR spectroscopy and thermal analysis confirmed that both the electrospinning process and the blend with PLGA give structural stability to collagen. Incorporating collagen in the PLGA matrix promotes an increase in the material's rigidity, showing an increase in the elastic modulus (38%) and tensile strength (70%) compared to pure PLGA. PLGA and PLGA/collagen fibers were found to provide a suitable environment for the adhesion and growth of HeLa and NIH-3T3 cell lines as well as stimulate collagen release. We conclude that these scaffolds could be very effective as biocompatible materials for extracellular matrix regeneration, suggesting their potential applications in tissue bioengineering.

Aloe vera and copaiba oleoresin-loaded chitosan films for wound dressings: microbial permeation, cytotoxicity, and in vivo proof of concept.

Chitosan films are commonly used for wound dressing, provided that this polymer has healing, mucoadhesiveness and antimicrobial properties. These properties can be further reinforced by the combination of chitosan with polysaccharides and glycoproteins present in aloe vera, together with copaiba oleoresin's pharmacological activity attributed to sesquiterpenes. In this work, we developed chitosan films containing either aloe vera, copaiba oil or both, by casting technique, and evaluated their microbial permeation, antimicrobial activity, cytotoxicity, and in vivo healing potential in female adult rats. None of the developed chitosan films promoted microbial permeation, while the cytotoxicity in Balb/c 3 T3 clone A31 cell line revealed no toxicity of films produced with 2 % of chitosan and up to 1 % of aloe vera and copaiba oleoresin. Films obtained with either 0.5 % chitosan or 0.5 % copaiba oleoresin induced cell proliferation which anticipate their potential for closure of wound and for the healing process. The in vivo results confirmed that tested films (0.5 % copaiba-loaded chitosan film and 0.5 % aloe vera-loaded chitosan film) were superior to a commercial dressing film. For all tested groups, a fully formed epithelium was seen, while neoformation of vessels seemed to be greater in formulations-treated groups than those treated with the control. Our work confirms the added value of combining chitosan with aloe vera and copaiba oil in the healing process of wounds.

Mesoporous silica nanoparticles adsorb aflatoxin B1 and reduce mycotoxin-induced cell damage.

The present study examined the effects of mesoporous silica nanoparticles (MSNs) on its adsorption capacity of aflatoxin B1 (AFB1). Moreover, the study evaluated the toxicity of MSNs with AFB1 using NIH3T3 cells and hemolysis test. The obtained MSNs were spherical, irregular-like in shape, having a mean size of 39.97 ± 7.85 nm and a BET surface area of 1195 m2/g. At 0.1 mg mL-1 concentration of MSN, the AFB1 adsorption capacity was 30%, which reached 70% when the MSN concentration increased to 2.0 mg mL-1. Our findings showed that AFB1 was adsorbed (∼67%) in the first few minutes on being in contact with MSNs, reaching an adsorption capacity of ∼70% after 15 min. Thereafter, the adsorption capacity remained constant in solution, demonstrating that the MSNs adsorbed toxins even beyond overnight. MSN treatment (0.5-2.0 mg mL-1) using NIH3T3 cells did not result in any reduction in cell viability. In addition, MSN treatment completely reversed the cytotoxic effect of AFB1 at all concentrations. Hemolysis test also revealed no hemolysis in MSNs evaluated alone and in those combined with AFB1. To the best of our knowledge, this study is the first to demonstrate that MSN can reduce cell toxicity produced by AFB1 due to its potential to adsorb mycotoxins.

Cytotoxicity evaluation of haloperidol, clozapine and a new molecule with antipsychotic potential, PT-31, in NIH-3T3 cells

Abstract Schizophrenia is an illness that affects 26 million people worldwide. However, conventional antipsychotics present side effects and toxicity, highlighting the need for new antipsychotics. We aimed to evaluate the cytotoxicity of haloperidol (HAL), clozapine (CLO), and a new molecule with antipsychotic potential, PT-31, in NIH-3T3 cells. The neutral red uptake assay and the MTT assay were performed to evaluate cell viability and mitochondrial activity, morphological changes were assessed, and intracellular reactive oxygen species (ROS) detection was performed. HAL and CLO (0.1 µM) showed a decrease in cell viability in the neutral red uptake assay and in the MTT assay. In addition, cell detachment, content decrease, rounding and cell death were also observed at 0.1 µM for both antipsychotics. An increase in ROS was observed for HAL (0.001, 0.01 and 1 µM) and CLO (0.01 and 1 µM). PT-31 did not alter cell viability in any of the assays, although it increased ROS at 0.01 and 1 µM. HAL and CLO present cytotoxicity at 0.1 µM, possibly through apoptosis and necrosis. In contrast, PT-31 does not present cytotoxicity to NIH-3T3 cells. Further studies must be performed for a better understanding of these mechanisms and the potential risk of conventional antipsychotics

Bioactive Compounds and Adipocyte Browning Phenomenon.

Overweight and obesity have become worldwide health issues in most countries. Current strategies aimed to prevent or reduce overweight and obesity have mainly focused on the genes and molecular mechanisms that give the functional characteristics to different types of adipose tissue. The Browning phenomenon in adipocytes consists of phenotypic and metabolic changes within white adipose tissue (WAT) activated by thermogenic mechanisms similar to that occurring in brown adipose tissue (BAT); this phenomenon has assumed great relevance due to its therapeutic potential against overweight and obesity. In addition, the study of inflammation in the development of overweight and obesity has also been included as a relevant factor, such as the pro-inflammatory mechanisms promoted by M1-type macrophages in adipose tissue. Studies carried out in this area are mainly performed by using the 3T3-L1 pre-adipocyte cell line, testing different bioactive compound sources such as plants and foods; nevertheless, it is necessary to standardize protocols used in vitro as well to properly scale them to animal models and clinical tests in order to have a better understanding of the mechanisms involved in overweight and obesity.

Causative Agents of American Tegumentary Leishmaniasis Are Able to Infect 3T3-L1 Adipocytes In Vitro.

Although macrophages have long been considered key players in the course of Leishmania infections, other non-professional phagocytes have lately been shown to maintain low levels of the parasite in safe intracellular niches. Recently, it was demonstrated that the adipose tissue is capable of harboring Old World L. (L.) infantum in mice. However, there is no evidence of experimental adipocyte infection with New World Leishmania species so far. In addition, it was not known whether adipocytes would be permissive for formation of the unique, large and communal parasitophorous vacuoles that are typical of L. (L.) amazonensis in macrophages. Here we evaluated the ability of L. (L.) amazonensis and L. (V.) braziliensis promastigotes and amastigotes to infect 3T3-L1 fibroblast-derived adipocytes (3T3-Ad) using light and transmission electron microscopy. Our results indicate that amastigotes and promastigotes of both species were capable of infecting and surviving inside pre- and fully differentiated 3T3-Ad for up to 144 h. Importantly, L. (L.) amazonensis amastigotes resided in large communal parasitophorous vacuoles in pre-adipocytes, which appeared to be compressed between large lipid droplets in mature adipocytes. In parallel, individual L. (V.) braziliensis amastigotes were detected in single vacuoles 144 h post-infection. We conclude that 3T3-Ad may constitute an environment that supports low loads of viable parasites perhaps contributing to parasite maintenance, since amastigotes of both species recovered from these cells differentiated into replicative promastigotes. Our findings shed light on the potential of a new host cell model that can be relevant to the persistence of New World Leishmania species.

Alendronate inhibits triglyceride accumulation and oxidative stress in adipocytes and the inflammatory response of macrophages which are associated with adipose tissue dysfunction.

Alendronate, a bisphosphonate used to prevent osteoporosis, stimulates osteogenesis but impairs adipogenesis. Different clinical trials suggest that the incidence of diabetes may be lower in patients treated with alendronate. Taking into account the importance of adipocytes and macrophages of adipose tissue in insulin resistance and type 2 diabetes, it is necessary to evaluate the effect of alendronate in both cell types. In this paper, we investigated the effect of alendronate on the differentiation to adipocytes of 3T3-L1 fibroblasts, the cell line most used to study adipogenesis, and also its effect on lipid content and oxidative stress in mature adipocytes as well as on the inflammatory response of macrophages. We found that alendronate inhibits differentiation of 3T3-L1 fibroblasts to adipocytes in keeping with reports in other cell lines. On the other hand, treatment of 3T3-L1 adipocytes with alendronate was able to decrease triglyceride content and to prevent H2O2-induced lipid peroxidation which was evaluated as an indicator of oxidative stress. In addition, it was found that activation of RAW 264.7 macrophages to a pro-inflammatory M1 type is inhibited by this bisphosphonate. These results suggest that alendronate may contribute to prevent adipocyte excessive enlargement and the induction of oxidative stress in 3T3-L1 adipocytes as well as the activation of macrophages to a pro-inflammatory M1 type, which are events associated with adipose tissue dysfunction and insulin resistance. In this study, we unraveled the underlying mechanisms of events that were previously observed in clinical trials.

Fabrication of bioactive novel scaffold by solid-state reaction sintering for bone regeneration.

In this work we present a new type of scaffold obtained by solid-state reaction, simultaneous sintering of a mixture of precursor oxides, carbonates, and organic substances, the latter used for pore generation. Having variable local composition, exhibits excellent overall physicochemical and bioactivity response. Open porosity is about 50%-60% and its permeability 10-11  m2 . X-ray diffraction exhibits the presence of a sodium-calcium silicate and sodium-calcium phosphate crystalline phases. Additionally, by mechanical compression tests the range of failure stress obtained for the scaffolds was 0.3-1.1 MPa. The bioactivity and dissolution rate of the scaffolds were evaluated by in vitro tests. After 1 week soaking in simulated body fluid, the formation of a continuous hydroxyapatite layer, which does not differentiate local compositions, was observed. Our results from cell culture tests clearly indicate that during hydroxyapatite layer formation, scaffolds do not liberate any cytotoxic substances. Moreover, cells seeded in the hydroxyapatite-covered scaffolds grew better than the control.

Biocompatibility analysis of high molecular weight chitosan obtained from Pleoticus muelleri shrimps. Evaluation in prokaryotic and eukaryotic cells.

The search for the exploitation and recycling of biomaterials is increasing for reducing the use of non-renewable resources and minimizing environmental pollution caused by synthetic materials. In this context, Chitosan (CS) being a naturally occurring biopolymer becomes relevant. The aim of the present work was to explore the effects of High Molecular Weight CS (H-CS) from Argentinean shrimp's wastes in prokaryotic and eukaryotic in vitro cell cultures. Ultrastructure of H-CS was analysed by SEM and TEM. In vitro studies were performed in prokaryotic (Lactobacillus casei BL23) and eukaryotic (Caco-2, ARPE-19, EA.hy926 and 3T3-L1) culture cells. High performance microscopic techniques were applied to examine culture cells. No changes in morphology were found in any of the cell types. In addition, fluorescent-dyed H-CS revealed that eukaryotic cells could internalize it optimally. Viability was maintained and proliferation rate even increased for Caco-2, ARPE-19 and 3T3-L1 cells under H-CS treatment. Besides, viability was neither altered in L. casei nor in EA.hy926 cells after H-CS exposure. In conclusion, H-CS could be a suitable biopolymer to be exploited for biomedical or food industry applications.

Effect of lead on proliferation, oxidative stress and genotoxic damage of 3T3-L1 fibroblasts.

Lead (Pb) is an environmental and industrial contaminant that still represents a public health problem. In this paper, we investigated the effect of Pb on proliferation, lipid peroxidation and the number of micronucleated cells in exponentially growing 3T3-L1 fibroblasts, a cell line previously used to evaluate different environmental contaminants. We found that Pb (10 µM or higher) was able to inhibit proliferation of exponentially growing cells after 24-h treatment, which was evaluated by the MTT assay and cell counting in Neubauer chamber, but cell survival was not affected according to the trypan blue exclusion assay. On the other hand, Pb was able to increase lipid peroxidation and the number of micronucleated cells, which are indicative of oxidative stress and genotoxic damage respectively. We also found that removal of Pb after 24-h treatment allowed cells to recover proliferation. Our results indicate that Pb was able to induce oxidative stress and genotoxicity in this cell line under standardized conditions, which supports the involvement of Pb in similar effects observed in human exposed to this heavy metal. In addition, Pb inhibits proliferation of exponentially growing fibroblasts but cells resume proliferation after removal of this metal, which suggests that it is important to move away Pb-exposed individuals from the source of contamination.

Efecto citotóxico y genotóxico in vitro del extracto crudo y etanólico del rizoma de Curcuma longa L / In vitro cytotoxic and genotoxic effect of the crude and ethanolic extract from the rhizome of Curcuma longa L

RESUMEN Objetivos: Determinar el efecto citotóxico y genotóxico in vitro del extracto crudo y etanólico del rizoma de Curcuma longa L. Materiales y métodos: El efecto citotóxico fue evaluado utilizando líneas celulares DU-145, HT-29, 3T3 BALB/c. Se hallaron los porcentajes de crecimiento en 48 horas y se determinó la concentración inhibitoria 50 (CI50). El efecto genotóxico en el ADN genómico humano se determinó mediante el método Tomasevich. Resultados: El extracto crudo produjo una CI50 de 12,98 ± 0,21 μg/mL para la línea celular tumoral HT-29, que es inferior a DU-145 con una CI50 de 36,77 ± 9,12 μg/mL; el extracto etanólico presentó una CI50 de 13,24 ± 0,77 y 20,54 ± 2,58 µg/mL para ambas líneas celulares, respectivamente; el compuesto estándar curcumina presentó una CI50 de 3,96 ± 0,60 y 13,94 ± 2,79 μg/mL, respectivamente. El extracto crudo a concentraciones de 50 y 100 mg/mL fragmentó entre el 40% a 95% de ADN genómico humano; mientras que, a 200 mg/mL, la fragmentación fue mayor al 95%. El extracto etanólico a todas las concentraciones no fragmentó el ADN. La curcumina a 200 mg/mL fragmentó menos del 5% de ADN genómico humano. Conclusiones: Los extractos crudo y etanólico de Curcuma longa L. demuestran efecto citotóxico in vitro diferencial para la línea celular tumoral humana DU-145 y HT29 semejante al compuesto estándar curcumina. El extracto crudo de Curcuma longa L. presenta una potente actividad genotóxica in vitro frente al ADN genómico humano, esta actividad está ausente en el extracto etanólico.

ABSTRACT Objectives: To determine the in vitro cytotoxic and genotoxic effect of the crude and ethanolic extract from the Curcuma longa L. rhizome. Materials and methods: The cytotoxic effect was evaluated using DU-145, HT-29, 3T3 BALB/c cell lines. The growth percentages in 48 hours; and the half maximal inhibitory concentration (IC50) were determined. The genotoxic effect on human genomic DNA was determined using the Tomasevich method. Results: Crude extract produced an IC50 of 12.98 ± 0.21 μg/mL for the HT-29 tumor cell line, which is lower than the value obtained for DU-145, with an IC50 of 36.77 ± 9.12 μg/mL. The ethanolic extract presented an IC50 of 13.24 ± 0.77 and 20.54 ± 2.58 μg/mL for both cell lines, respectively; the curcumin standard compound presented an IC50 of 3.96 ± 0.60 and 13.94 ± 2.79 μg/mL, respectively. Crude extract concentrations of 50 and 100 mg/mL fragmented between 40% to 95% of human genomic DNA; while at 200 mg/mL, fragmentation was greater than 95%. The ethanolic extract at all concentrations did not fragment the DNA. Curcumin at 200 mg/mL fragmented less than 5% of human genomic DNA. Conclusions: The crude and ethanolic extracts of Curcuma longa L. demonstrate different in vitro cytotoxic effects for the human tumor cell lines DU-145 and HT-29; similar to the standard curcumin compound. The crude extract of Curcuma longa L. shows a potent genotoxic in vitro activity against human genomic DNA; this type of effect is not produced by the ethanolic extract.

A colorimetric bioassay for quantitation of both basal and insulin-induced glucose consumption in 3T3-L1 adipose cells.

INTRODUCTION: The quantitation of glucose consumption in animal cell cultures is mainly based on the use of radiolabeled or fluorescent analogues, resulting in expensive and tedious procedures, requiring special equipment and, sometimes, with potential health and environmental risks. OBJECTIVES: The objective of this work was to evaluate the application of a blood plasma colorimetric assay to quantify glucose consumption in in vitro cultures of adipose cells. METHODS: We worked with 3T3-L1 adipose cells differentiated by 7-8 days, which were exposed to different initial glucose concentrations (5.5, 2.8 and 1.4 mM) for variable times, either in the absence or the presence of 100 nM insulin. Using a commercial colorimetric glucose assay, extracellular glucose was determined, and glucose uptake was calculated as the difference between the initial and final glucose concentration. RESULTS: The colorimetric assay allowed us to quantify glucose uptake in our cell model, observing a linear response over time (r 2 ≥0.9303) to the different glucose concentrations, both in the basal and insulin-induced condition. The insulin-stimulated glucose consumption was higher than basal consumption at all glucose concentrations evaluated, but significant differences were observed at 120-, 360- and 480-min in glucose 5.5 mM (p ≤ 0.01, n = 5), and 240 min in glucose 1.4 mM (p ≤ 0.01, n = 5). A V max of 4.1 and 5.9 nmol/ml/min (basal and insulin-induced, respectively) and a K m of 1.1 mM (same in basal vs insulin-stimulated) were calculated. The bioassay was also useful in a pharmacological context: in glucose 1.4 mM, glucose consumption showed an effect that depended on insulin concentration, with a calculated EC50 of 18.4 ± 1.1 nM. CONCLUSIONS: A simple and low-cost bioassay is proposed to quantify glucose consumption in 3T3-L1 adipose cells.

Docetaxel-loaded solid lipid nanoparticles prevent tumor growth and lung metastasis of 4T1 murine mammary carcinoma cells.

BACKGROUND: Metastasis causes the most breast cancer-related deaths in women. Here, we investigated the antitumor effect of solid lipid nanoparticles (SLN-DTX) when used in the treatment of metastatic breast tumors using 4T1-bearing BALB/c mice. RESULTS: Solid lipid nanoparticles (SLNs) were produced using the high-energy method. Compritol 888 ATO was selected as the lipid matrix, and Pluronic F127 and Span 80 as the surfactants to stabilize nanoparticle dispersion. The particles had high stability for at least 120 days. The SLNs' dispersion size was 128 nm, their polydispersity index (PDI) was 0.2, and they showed a negative zeta potential. SLNs had high docetaxel (DTX) entrapment efficiency (86%), 2% of drug loading and showed a controlled drug-release profile. The half-maximal inhibitory concentration (IC50) of SLN-DTX against 4T1 cells was more than 100 times lower than that of free DTX after 24 h treatment. In the cellular uptake test, SLN-DTX was taken into the cells significantly more than free DTX. The accumulation in the G2-M phase was significantly higher in cells treated with SLN-DTX (73.7%) than in cells treated with free DTX (23.0%), which induced subsequent apoptosis. TEM analysis revealed that SLN-DTX internalization is mediated by endocytosis, and fluorescence microscopy showed DTX induced microtubule damage. In vivo studies showed that SLN-DTX compared to free docetaxel exhibited higher antitumor efficacy by reducing tumor volume (p < 0.0001) and also prevented spontaneous lung metastasis in 4T1 tumor-bearing mice. Histological studies of lungs confirmed that treatment with SLN-DTX was able to prevent tumor. IL-6 serum levels, ki-67 and BCL-2 expression were analyzed and showed a remarkably strong reduction when used in a combined treatment. CONCLUSIONS: These results indicate that DTX-loaded SLNs may be a promising carrier to treat breast cancer and in metastasis prevention.