Antimutagenic and antitumor activities of a water-soluble fraction of soursop (syn Graviola, Annona muricata L.) fruit pulp.

Soursop (Annona muricata) is a tropical tree whose decoction derived from bark, root, seed, or leaf has been used for medicinal uses. In addition, the fruit itself is considered a food, and the juice is utilized to treat heart and liver diseases. The aim of this study was to determine the phenolic content. In addition, a water-soluble fraction of the soursop fruit pulp (WSSP) was examined for the following properties: antioxidant, mutagenic, and antimutagenicity. UV-visible spectrophotometry determined total phenolic content by the Folin-Ciocalteu method to be 11.22 ± 0.6 mg of gallic acid equivalent per gram dried extract, and free-radical scavenging activity by the 2,2'-diphenyl-1-picryl-hydrazyl (DPPH•) showed an EC50 of 1032 µg/ml. In the Salmonella/microsome assay, no marked mutagenicity was induced following WSSP treatment, and a chemopreventive capacity was observed in the antimutagenic assay. The cytotoxicity assays were carried out using the water-soluble tetrazolium salt and lactate dehydrogenase (LDH) assays demonstrated that WSSP induced significant cytotoxicity in MCF-7 and Caco-2 cells, indicating greater effectiveness of cytotoxic action by destroying cell membrane integrity. Data suggest that WSSP may exert beneficial effects as a DNA chemopreventive and antitumor agent.

Marketable 1,3-dimethylamylamine and caffeine-based thermogenic supplements: Regulatory genotoxicity assessment through in vitro and in silico approaches.

The consumption of dietary supplements to enhance physical performance has increased significantly in the last century, especially thermogenic pre-workout supplements. Nevertheless, this industry has faced criticism for inadequate safety measures surveillance in regulatory issues regarding their products. The aims of our study were to investigate two pre-workout supplements with respect to (1) mutagenicity utilizing Salmonella/microsome assay; (2) genotoxicity employing cytokinesis-block micronucleus (CBMN) assay protocols; and (3) hepatocytoxicity using WST cell proliferation, activities of lactate dehydrogenase (LDH) and alkaline phosphatase using human liver carcinoma (HepG2) and mouse fibroblast (F C3H) cells. Oxidative stress was determined through glutathione (GSH) measurement and in silico for predictions of pharmacokinetics and toxicity for the most abundant isolated substances present in these supplements. Both supplements induced mutagenicity in all examined bacterial strains, especially in the presence of exogenous metabolism. Further, tested supplements significantly elevated the formation of micronuclei (MN) as well as other cellular phenomena. Concentration- and time-dependent curves were observed for hepatotoxicity in both studied cell lines. In addition, both supplements decreased levels of intracellular and extracellular GSH. In silico predictions showed that the isolated individual compounds failed to induce the observed outcomes. Our findings provide contributions to the molecular mechanisms underlying two pre-workout supplement-induced toxicity and the need for surveillance.

In vitro and in vivo cytotoxicity assessment of glyphosate and imazethapyr-based herbicides and their association.

Resistance to glyphosate herbicide has initiated usage of combined application of herbicides as a weed control measure. Imazethapyr-based herbicides associated with glyphosate herbicide seem to be an alternative to suppress weed resistance. The aim of this study was to examine the adverse effects of Glyphosate Atanor 48® (ATN) and Imazethapyr Plus Nortox® (IMZT) formulations in both single forms and mixtures using HepG2 cells and zebrafish early-life stages models. Data demonstrated cytotoxicity due to exposure to ATN, IMZT for both models, as follows: (1) ATN (0.5 mg/L), IMZT (5 mg/L), and M3 (0.05 mg/L ATN + 5 mg/L IMZT) increased cytotoxicity by disturbing the mitochondrial activity of HepG2 cells 24 hr after exposure; (2) ATN and IMZT (5 mg/L), and M3 (0.05 mg/L ATN + 5 mg/L IMZT) also decreased the integrity of the membrane of HepG2 cells after 24 hr incubation; (3) only ATN and IMZT (5 mg/L) in their single forms diminished the mitochondrial potential of zebrafish; (4) ATN (single form) at 0.5 mg/L induced apoptosis in zebrafish larvae. In conclusion, these herbicides in their single forms appeared to produce greater cytotoxicity to HepG2 cells and zebrafish compared to the herbicide mixtures.

Risk assessment of coffees of different qualities and degrees of roasting.

During the coffee beans roasting process, occurs the formation of polycyclic aromatic hydrocarbons, which are associated with the incidence of cancer in humans. This study aimed to evaluate the influence of coffee bean quality and roasting degree regarding mutagenicity, cytotoxicity and genotoxicity. Six samples of coffee drink made with roasted and ground Coffea arabica beans from different qualities and roast degrees were used after freeze-drying. Both commercial and special quality grains suffered light, medium and dark roasting. According to the Salmonella/microsome assay, the highest concentration of commercial grain sample (dark roast) significantly increased the number of revertants of the TA98 strain in the absence of metabolization. All the samples induced cytotoxicity to HepG2 cells. These effects can be ranked in the following order from most to least toxic: medium roast - special grain > light roast - special grain > dark roast - commercial grain > dark roast - special grain > light roast - commercial grain > medium roast - commercial grain. None of the samples induced genotoxicity in HepG2 cells. Our findings show that the harmful effects of coffee depend not only on the degree of roasting but also on the grain quality.

Immunocytochemistry Analysis of HepG2 Cell 3D Culture Encapsulated as Spheroids in Alginate Beads.

3D Cell culture is an alternative to animal use in many drug development and toxicity studies. The 3D cell culture can mimic and reproduce the original tissue microenvironment, morphology, and mechanical and physiological characteristics, to provide a more realistic and reliable response as compared to two-dimensional cultures. 3D cell culture encapsulated in alginate beads is a very simple and relatively inexpensive tool that is easy to handle and to maintain. The alginate beads function as a scaffold that imprisons cells and allows 3D cell growth, to generate spheroids that can have greater genic expression and cell-cell communication as a nano or microtissue. The HepG2 cell line is a human hepatocellular carcinoma cell derivative. HepG2 cells preserve several of the characteristics of hepatocytes and are therefore often used in toxicity studies. Here, we describe HepG2 cell encapsulation in alginate beads and analyze the resulting spheroids formed within the alginate beads by immunocytochemistry, by staining a certain structure with a specific antibody coupled with a fluorophore. This method preserves the beads and enables cell analysis by confocal microscopy.

Evaluation of the acute toxicity, phototoxicity and embryotoxicity of a residual aqueous fraction from extract of the Antarctic moss Sanionia uncinata.

BACKGROUND: Ultraviolet (UV) radiation is the main exogenous inductor of skin damage and so photoprotection is important to control skin disorders. The Antarctic moss Sanionia uncinata is an important source of antioxidants and the photoprotective activity of its organic extracts has been investigated. This study aimed to evaluate the potential photoprotection, cytotoxicity and embryotoxicity of residual aqueous fraction (AF) from the moss S. uncinata. METHODS: UV-visible spectrum and SPF (sun protection factor) were determined by spectrophotometry. Embryotoxicity potential was evaluated by Fish embryo-larval toxicity test using zebrafish (Danio rerio) as organism model. Cell death assays by water-soluble tetrazolium salt (WST-1) and lactate dehydrogenase (LDH) were investigated using HaCaT keratinocyte cell line cultured in monolayers and three dimensions (3D). Phototoxicity and association with UV-filters were performed by 3T3 neutral red uptake test. RESULTS: The AF showed sharp absorption bands in the UV region and less pronounced in the visible region. The SPF was low (2.5 ± 0.3), but the SPF values of benzophenone-3 and octyl-methoxycinnamate increased ~ 3 and 4 times more, respectively, in association with AF. The AF did not induce significant lethal and sublethal effects on zebrafish early-life stages. In monolayers, the HaCaT cell viability, evaluated by WST-1, was above 70% by ≤0.4 mg AF/mL after 48 and 72-h exposure, whereas ≤1 mg AF/mL after 24-h exposure. The LDH assay showed that the cell viability was above 70% by ≤0.4 mg AF/mL even after 72-h exposure, but ≤1 mg/mL after 24 and 48-h exposure. In 3D cell culture, an increased cell resistance to toxicity was observed, because cell viability of HaCaT cell by WST-1 and LDH was above ~ 90% when using ≤1 and 4 mg AF/mL, respectively. The AF demonstrated values of photo irritation factor < 2 and of photo effect < 0.1, even though in association with UV-filters. CONCLUSIONS: The residual AF absorbs UV-vis spectrum, increased SPF values of BP-3 and OMC and does not induce embryotoxicity to zebrafish early life-stage. The cell death assays allowed establishing non-toxic doses of AF and phototoxicity was not detected. AF of S. uncinata presents a good potential for skin photoprotection against UV-radiation.

PLGA nanoparticles optimized by Box-Behnken for efficient encapsulation of therapeutic Cymbopogon citratus essential oil.

This study aimed to optimize Cymbopogon citratus essential oil loaded into PLGA-nanoparticles by investigating the effect of processing variables (sonication time, ultrasound power, and essential oil/polymer ratio) on encapsulation efficiency and particle mean hydrodynamic diameter using Box-Behnken design. Nanoparticles were prepared by an emulsification/solvent diffusion method and physicochemically characterized by FTIR, DSC and TGA/DTA. Cytotoxicity was evaluated in human HaCat keratinocytes by WST-1 and LDH assays. The optimized formulation had a hydrodynamic mean diameter of 277 nm, a polydispersity index of 0.18, a Zeta potential of -16 mV and an encapsulation efficiency of 73%. Nanoparticle characterization showed that only citral was incorporated in nanocarriers, with some amount adsorbed on their surface, and highlighted the potential in increasing the oil thermal stability. The drug release profile demonstrated a biphasic pattern with a substantial sustained release depending on diffusion from the polymeric matrix. Toxicity effects on cell viability of pure essential oil at low concentrations were significantly eliminated when encapsulated. Results revealed the ability of PLGA-nanoparticles to improve essential oil physicochemical characteristics, by controlling release and reducing toxicity, suggesting their potential use in pharmaceutical preparations.

Toxicological evaluation of nail polish waste discarded in the environment.

Nail polish has been widely used around the world. However, the hazards of nail polishes discarded in the environment are still poorly investigated. Thus, the toxicogenetic effects of solubilized (SE) and leached (LE) extracts from nail polishes were investigated, simulating their disposal on water and landfill, respectively, and identifying their physicochemical properties and chemical constituents. Organic compounds and metals were detected in both extracts. SE and LE only induced mutagenic effects in TA98 Salmonella strain in the presence and absence of exogenous metabolic activation. Although both extracts did not significantly increase the frequency of micronucleated HepG2 cells, the cell viability was affected by 24-h exposure. No DNA damage was observed in gonad fish cells (RTG-2) exposed to both extracts; however, the highest SE and LE concentrations induced significant lethal and sublethal effects on zebrafish early-life stages during 96-h exposure. Based on our findings, it can be concluded that if nail polishes enter aquatic systems, it may cause negative impacts to the environment.

Differential toxicity of an organic PM2.5 extract to human lung cells cultured in three dimensions (3D) and monolayers.

Several epidemiological studies have associated PM2.5 (particulate matter, aerodynamic diameter 2.5 µm) exposure with an increase in morbidity and mortality attributed to cardiopulmonary diseases. Based upon these observations and the growing effort to replace the use of animals in research, in vitro A549 cells cultured in three dimensions (3D), an alternative method to the use of animals, as well as monolayers were investigated to examine whether organic PM2.5 extract induced equivalent cytotoxic changes in vitro as compared to in vivo. PM2.5 was collected on Brazil Avenue, Rio de Janeiro, Brazil, from November 2010 to May 2011, except March, and analyzed for the ability to induce cytotoxicity in A549 cells using various established assays. Samples collected in all months significantly decreased viability of A549 cells using both types of cell death assays, and those collected in November showed lower cytotoxicity. It is worthwhile noting that for samples collected in all months except for April, PM2.5 induced greater toxicity in cells grown in monolayers than in 3D. Data demonstrated that cell behavior varied based upon type of culture system employed. Since the 3D cell culture mimics the architecture of in vivo tissue to a greater extent than monolayers, it is suggested that data from 3D studies resemble more closely human exposure conditions and thus may provide more reliable findings to be utilized in risk assessment following PM exposure than results obtained in traditional culture system.

Hepatotoxicity assessment of the azo dyes disperse orange 1 (DO1), disperse red 1 (DR1) and disperse red 13 (DR13) in HEPG2 cells.

During the dyeing process in baths approximately 10 to 15% of the dyes used are lost and reach industrial effluents, thus polluting the environment. Studies showed that some classes of dyes, mainly azo dyes and their by-products, exert adverse effects on humans and local biota, since the wastewater treatment systems and water treatment plants were found to be ineffective in removing the color and reducing toxicity of some dyes. In the present study, the toxicity of the azo dyes disperse orange 1 (DO1), disperse red 1 (DR1), and disperse red 13 (DR13) was evaluated in HepG2 cells grown in monolayers or in three dimensional (3D) culture. Hepatotoxicity of the dyes was measured using 3-(4,5-dimethylthiazol-2yl)2,5-diphenyltetrazolium (MTT) and cell counting kit 8 (CCK-8) assays after 24, 48, and 72 h of incubation of cells with 3 different concentrations of the azo dyes. The dye DO1 only reduced the mitochondrial activity in HepG2 cells grown in a monolayer after 72 h incubation, while the dye DR1 showed this deleterious effect in both monolayer and 3D culture. In contrast, dye DR13 decreased the mitochondrial activity after 24, 48, and 72 h of exposure in both monolayer and 3D culture. With respect to dehydrogenase activity, only the dye DR13 diminished the activity of this enzyme after 72 h of exposure in both monolayer and 3D culture. Our results clearly demonstrated that exposure to the studied dyes induced cytotoxicity in HepG2 cells.

An overview of biodiesel soil pollution: data based on cytotoxicity and genotoxicity assessments.

Biodiesel production has received considerable attention in the recent past as a nonpolluting fuel. However, this assertion has been based on its biodegradability and reduction in exhaust emissions. Assessments of water and soil biodiesel pollution are still limited. Spill simulation with biodiesel and their diesel blends in soils were carried out, aiming at analyzing their cytotoxic and genotoxic potentials. While the cytotoxicity observed may be related to diesel contaminants, the genotoxic and mutagenic effects can be ascribed to biodiesel pollutants. Thus, taking into account that our data stressed harmful effects on organisms exposed to biodiesel-polluted soils, the designation of this biofuel as an environmental-friendly fuel should be carefully reviewed to assure environmental quality.

Genotoxicity assessment of water soluble fractions of biodiesel and its diesel blends using the Salmonella assay and the in vitro MicroFlow® kit (Litron) assay.

The designation of biodiesel as an environmental-friendly alternative to diesel oil has improved its commercialization and use. However, most biodiesel environmental safety studies refer to air pollution and so far there have been very few literature data about its impacts upon other biotic systems, e.g. water, and exposed organisms. Spill simulations in water were carried out with neat diesel and biodiesel and their blends aiming at assessing their genotoxic potentials should there be contaminations of water systems. The water soluble fractions (WSF) from the spill simulations were submitted to solid phase extraction with C-18 cartridge and the extracts obtained were evaluated carrying out genotoxic and mutagenic bioassays [the Salmonella assay and the in vitro MicroFlow® kit (Litron) assay]. Mutagenic and genotoxic effects were observed, respectively, in the Salmonella/microsome preincubation assay and the in vitro MN test carried out with the biodiesel WSF. This interesting result may be related to the presence of pollutants in biodiesel derived from the raw material source used in its production chain. The data showed that care while using biodiesel should be taken to avoid harmful effects on living organisms in cases of water pollution.

The impact of aromatic amines on the environment: risks and damages.

Aromatic amines are a group of chemicals whose ubiquitous presence in the environment is a result of the multitude of sources from which they originate. These compounds are widely used as raw materials or at intermediate stages in the manufacturing of industrial chemicals such as pesticides, medicines, dyestuffs, polymers, surfactants, cosmetics and corrosion inhibitors, especially in dyestuff factories. As with most chemical carcinogens, aromatic amines need to be metabolized into reactive electrophiles in order to exert their carcinogenic effects. This activation typically involves N-oxidation of arylamines to yield N-hydroxyarylamines. Since these amines are potential carcinogenic agents and are discharged into the atmosphere, water and soil, they constitute an important class of environmental pollutants of enormous concern due to the potential for human exposure.

The azo dye Disperse Orange 1 induces DNA damage and cytotoxic effects but does not cause ecotoxic effects in Daphnia similis and Vibrio fischeri.

Azo dyes constitute the largest group of colorants used in industry and can pass through municipal waste water plants nearly unchanged due to their resistance to aerobic treatment, which potentially exposes humans and local biota to adverse effects. Unfortunately, little is known about their environmental fate. Under anaerobic conditions, some azo dyes are cleaved by microorganisms forming potentially carcinogenic aromatic amines. In the present study, the azo dye Disperse Orange 1, widely used in textile dyeing, was tested using the comet, Salmonella/microsome mutagenicity, cell viability, Daphnia similis and Microtox(®) assays. The human hepatoma cell line (HepG2) was used in the comet assay and for cell viability. In the mutagenicity assay, Salmonella typhimurium strains with different levels of nitroreductase and o-acetyltransferase were used. The dye showed genotoxic effects with respect to HepG2 cells at concentrations of 0.2, 0.4, 1.0, 2.0 and 4.0µg/mL. In the mutagenicity assay, greater responses were obtained with the strains TA98 and YG1041, suggesting that this compound mainly induces frameshift mutations. Moreover, the mutagenicity was greatly enhanced with the strains overproducing nitroreductase and o-acetyltransferase, showing the importance of these enzymes in the mutagenicity of this dye. In addition, the compound induced apoptosis after 72h in contact with the HepG2 cells. No toxic effects were observed for either D. similis or Vibrio fischeri.

Elderly and drugs: risks and necessity of rational use

In recent decades, the world has undergone a demographic transformation with a rapid growth of the elderly population, resulting in an increased demand for funds to maintain their health and drug consumption. Pharmacokinetic and pharmacodynamic changes occurring in the elderly can interfere directly in the adverse effects of drugs and increase the risk of intoxication. In addition, there are external factors interfering with the pharmacotherapy of the elderly, such as inappropriate use and the lack of access to information. Many therapeutic classes of drugs should be used with caution or avoided in the elderly population, such as anti-inflammatory and some anti-hypertensive drugs, diuretics and digitalis. If not managed carefully, these medicines can affect the safety and quality of life in the elderly. Thus, the aim of this review was to identify drugs that should be used with caution in elderly patients in order to avoid intoxication and/or adverse drug events.

Durante as últimas décadas, o mundo passou por uma transformação demográfica, com um rápido crescimento da população idosa e, portanto, tanto a demanda para a manutenção da saúde deste grupo populacional, quanto o consumo de medicamentos estão aumentando. Ainda, as mudanças farmacocinéticas e farmacodinâmicas que ocorrem em idosos podem interferir diretamente nos efeitos adversos dos medicamentos e aumentar o risco de intoxicação. Além disso, há fatores externos que interferem na farmacoterapia dos idosos, tais como o uso inadequado e a falta de acesso à informação. Existem várias classes terapêuticas de medicamentos que devem ser utilizados com cautela ou evitados na população idosa, tais como antiinflamatórios, alguns anti-hipertensivos, diuréticos, digitálicos entre outros. Estes medicamentos, se não forem utilizados com cuidado, podem afetar a qualidade de vida e a segurança desta população. Assim, este trabalho visa identificar medicamentos que devem ser utilizados com cuidado em pacientes idosos para evitar intoxicações e/ou eventos adversos aos medicamentos.

Evaluation of extracts from Coccoloba mollis using the Salmonella/microsome system and in vivo tests.

The common everyday use of medicinal plants is an ancient, and still very widespread practice, whereby the need for studies on their possible toxicity and mutagenic properties. The species Coccoloba mollis has been much used in phytotherapy, mainly in cases involving loss of memory and stress. In order to investigate its genotoxic and mutagenic potential, ethanolic extracts from the leaves and roots underwent Salmonella/microsome assaying (TA98 and TA100 strains, with and without exogenous metabolism - S9), besides comet and micronucleus tests in vivo.There was no significant increase in the number of revertants/plate of Salmonella strains in any of the analyzed root-extract concentrations, although the extract itself was extremely toxic to the Salmonella TA98 strain in the tests carried out with S9 (doses varying from 0.005 to 0.5 µg/plate). On the other hand, the leaf-extract induced mutations in the TA98 strain in the absence of S9 in the highest concentration evaluated, although at very low mutagenic potency (0.004 rev/ µg). Furthermore, there was no statistically significant increase in the number of comets and micronuclei, in treatments involving Swiss mice. It was obvious that extracts of Coccoloba mollis, under the described experimental conditions, are not mutagenic.

Evaluation of extracts from Coccoloba mollis using the Salmonella/microsome system and in vivo tests

The common everyday use of medicinal plants is an ancient, and still very widespread practice, whereby the need for studies on their possible toxicity and mutagenic properties. The species Coccoloba mollis has been much used in phytotherapy, mainly in cases involving loss of memory and stress. In order to investigate its genotoxic and mutagenic potential, ethanolic extracts from the leaves and roots underwent Salmonella/microsome assaying (TA98 and TA100 strains, with and without exogenous metabolism - S9), besides comet and micronucleus tests in vivo.There was no significant increase in the number of revertants/plate of Salmonella strains in any of the analyzed root-extract concentrations, although the extract itself was extremely toxic to the Salmonella TA98 strain in the tests carried out with S9 (doses varying from 0.005 to 0.5 µg/plate). On the other hand, the leaf-extract induced mutations in the TA98 strain in the absence of S9 in the highest concentration evaluated, although at very low mutagenic potency (0.004 rev/µg). Furthermore, there was no statistically significant increase in the number of comets and micronuclei, in treatments involving Swiss mice. It was obvious that extracts of Coccoloba mollis, under the described experimental conditions, are not mutagenic.

The azo dyes Disperse Red 1 and Disperse Orange 1 increase the micronuclei frequencies in human lymphocytes and in HepG2 cells.

The use of azo dyes by different industries can cause direct and/or indirect effects on human and environmental health due to the discharge of industrial effluents that contain these toxic compounds. Several studies have demonstrated the genotoxic effects of various azo dyes, but information on the DNA damage caused by Disperse Red 1 and Disperse Orange 1 is unavailable, although these dyes are used in dyeing processes in many countries. The aim of the present study was to evaluate the mutagenic activity of Disperse Red 1 and Disperse Orange 1 using the micronucleus (MN) assay in human lymphocytes and in HepG2 cells. In the lymphocyte assay, it was found that the number of MN induced by the lowest concentration of each dye (0.2 microg/mL) was similar to that of the negative control. At the other concentrations, a dose response MN formation was observed up to 1.0 microg/mL. At higher dose levels, the number of MN decreased. For the HepG2 cells the results were similar. With both dyes a dose dependent increase in the frequency of MN was detected. However for the HepG2, the threshold for this increase was 2.0 microg/mL, while at higher doses a reduction in the MN number was observed. The proliferation index was also calculated in order to evaluate acute toxicity during the test. No differences were detected between the different concentrations tested and the negative control.