Aflatoxin M1 in Brazilian goat milk and health risk assessment.

Contamination of goat milk with aflatoxin M1 (AFM1) is a public health concern. This study investigated filamentous fungi in goat feed and quantified AFM1 in milk samples (n = 108) from goat fed forage and concentrate. Based on the detected AFM1 concentration, risk assessment analyses were performed concerning the Estimated Daily Intake (EDI) for one-year-old children and adults. Filamentous fungi were found in goat feed samples in a range of 3.1 ± 1.9 to 4.2 ± 0.2 log CFU/g. Five genera were identified, to cite Aspergillus, Penicillium, Fusarium, Rhizopus and Acremonium. Aspergillus species comprised A. flavus, A. niger, and A. ochraceus. All goat milk samples were contaminated with AFM1 (5.60-48.20 ng/L; mean 21.90 ± 10.28 ng/L) in amounts below the limits imposed by regulatory agencies. However, EDI values for AFM1 through goat milk estimated for one-year-old children were above the Tolerable Daily Intake. The calculated Hazard Index for one-year-old children indicated potential risk of liver cancer due to goat milk consumption. The Margin of Exposure values to AFM1 in one-year-old children and adults consuming goat milk as the unique milk source indicated increased health risk. Therefore, contamination of goat milk with AFM1 should be considered a high priority for Brazil's risk management actions.

Multi-element composition, physicochemical and pollen attributes of honeys from the Paraguaçu River (Bahia, Brazil) by inductively coupled plasma-optical emission spectrometry (ICP OES).

Honey is a food of nutritional, medicinal and commercial importance. The physicochemical characteristics, pollen spectrum and mineral composition of eighteen honey samples obtained from regions (Cachoeira, Coqueiros, Maragojipe and Santiago do Iguape) near the Paraguaçu River, Bahia, Brazil were evaluated. Botanical families Asteraceae, Leguminosae, Malvaceae, Myrtaceae and Palmae were most frequently found. Five samples had water contents above the maximum limit established by the Brazilian legislation (> 20%). The mineral composition was determined by ICP OES, after microwave digestion. Ca, K, Mg and Na were measured (mg Kg-1) in the range from: 18.85 to 79.61; 366.74 to 1214.98; 12.46 to 44.59 and 11.56 to 85.39, respectively. Cu, Fe, Mn and Zn had variable concentration ranges, between 0.05 and 6.13 mg Kg-1. Al, Ba, Cd, Co, Cr, Ni, Pb, Se and V showed values below the LOD. Principal component analysis (PCA) and hierarchical cluster analysis (HCA) demonstrated that there are no similarities of mineral composition among honey samples.

Bioactivity and Molecular Docking Studies of Derivatives from Cinnamic and Benzoic Acids.

Over the last decade, there has been a dramatic increase in the prevalence and gravity of systemic fungal diseases. This study aimed therefore at evaluating the antifungal potential of ester derivatives of benzoic and cinnamic acids from three Candida species. The compounds were prepared via Fischer esterification, and the antifungal assay was performed by the microdilution method in 96-well microplates for determining the minimal inhibitory concentrations (MICs). The findings of the antifungal tests revealed that the analogue compound methyl ferulate, methyl o-coumarate, and methyl biphenyl-3-carboxylate displayed an interesting antifungal activity against all Candida strains tested, with MIC values of 31.25-62.5, 62.5-125, and 62.5 µg/ml, respectively. A preliminary Structure-Activity Relationship study of benzoic and cinnamic acid derivatives has led to the recognition of some important structural requirements for antifungal activity. The results of molecular docking indicate that the presence of the enoate moiety along with hydroxyl and one methoxy substitution in the phenyl ring has a positive effect on the bioactivity of compound 7 against Candida albicans. These observations further support the hypothesis that the antifungal activity of compound 7 could be due to its binding to multiple targets, specifically to QR, TS, and ST-PK. Additional experiments are required in the future to test this hypothesis and to propose novel compounds with improved antifungal activity.

Antifungal activity of naphthoquinoidal compounds in vitro against fluconazole-resistant strains of different Candida species: a special emphasis on mechanisms of action on Candida tropicalis.

In recent decades, the incidence of candidemia in tertiary hospitals worldwide has substantially increased. These infections are a major cause of morbidity and mortality; in addition, they prolong hospital stays and raise the costs associated with treatment. Studies have reported a significant increase in infections by non-albicans Candida species, especially C. tropicalis. The number of antifungal drugs on the market is small in comparison to the number of antibacterial agents available. The limited number of treatment options, coupled with the increasing frequency of cross-resistance, makes it necessary to develop new therapeutic strategies. The objective of this study was to evaluate and compare the antifungal activities of three semisynthetic naphthofuranquinone molecules against fluconazole-resistant Candida spp. strains. These results allowed to us to evaluate the antifungal effects of three naphthofuranquinones on fluconazole-resistant C. tropicalis. The toxicity of these compounds was manifested as increased intracellular ROS, which resulted in membrane damage and changes in cell size/granularity, mitochondrial membrane depolarization, and DNA damage (including oxidation and strand breakage). In conclusion, the tested naphthofuranquinones (compounds 1-3) exhibited in vitro cytotoxicity against fluconazole-resistant Candida spp. strains.

(4-Methoxyphenyl)(3,4,5-trimethoxyphenyl)methanone inhibits tubulin polymerization, induces G2/M arrest, and triggers apoptosis in human leukemia HL-60 cells.

(4-Methoxyphenyl)(3,4,5-trimethoxyphenyl)methanone (PHT) is a known cytotoxic compound belonging to the phenstatin family. However, the exact mechanism of action of PHT-induced cell death remains to be determined. The aim of this study was to investigate the mechanisms underlying PHT-induced cytotoxicity. We found that PHT displayed potent cytotoxicity in different tumor cell lines, showing IC50 values in the nanomolar range. Cell cycle arrest in G2/M phase along with the augmented metaphase cells was found. Cells treated with PHT also showed typical hallmarks of apoptosis such as cell shrinkage, chromatin condensation, phosphatidylserine exposure, increase of the caspase 3/7 and 8 activation, loss of mitochondrial membrane potential, and internucleosomal DNA fragmentation without affecting membrane integrity. Studies conducted with isolated tubulin and docking models confirmed that PHT binds to the colchicine site and interferes in the polymerization of microtubules. These results demonstrated that PHT inhibits tubulin polymerization, arrests cancer cells in G2/M phase of the cell cycle, and induces their apoptosis, exhibiting promising anticancer therapeutic potential.

Genetic toxicology evaluation of essential oil of Alpinia zerumbet and its chemoprotective effects against H(2)O(2)-induced DNA damage in cultured human leukocytes.

Essential oil (EO) of Alpinia zerumbet leaves, at non-toxic concentrations (50-300 µg/mL), did not induce genotoxicity in human leukocytes. However, at the highest concentration (500 µg/mL) tested caused a reduction in cell proliferation and viability, and an increase in DNA damage. Moreover, in vivo experiments showed that EO (400 mg/kg) did not exert mutagenicity on peripheral blood cells and bone marrow in mice. In DPPH test, EO showed scavenging effects against DPPH radicals, and other free radicals (determination of intracellular GSH and lipid peroxidation assays). Furthermore, EO was able to reduce the intracellular levels of ROS, and prevented leukocytes DNA against oxidative damage. The ability of EO to reduce H(2)O(2) toxicity was observed only when cells were treated with EO during and after exposure to H(2)O(2). With the co- and post-treatment procedures, EO decreased the frequency of apoptotic and micronucleated leukocytes as well DNA strand breaks. However, a synergistic effect was observed in cultures exposed to 500 µg/mL EO. In conclusion, EO at concentrations up to 300 µg/mL or doses up to 400mg/kg are not mutagenic in leukocytes and in mice, but do have antioxidative and protective effects against the cytotoxicity and clastogenesis induced by H(2)O(2).

Assessment of genotoxic effects of (4-methoxyphenyl)(3,4,5-trimethoxyphenyl)methanone in human lymphocytes.

(4-Methoxyphenyl)(3,4,5-trimethoxyphenyl)methanone (PHT) belongs to the phenstatin family. This compound has been studied due to its potent cytotoxicity and ability to inhibit tubulin assembly. The present study aimed to evaluate the mutagenic potential of PHT in human lymphocytes. PHT displayed cytotoxicity in human lymphocytes with an IC50 value of 5.68 µM, and therefore, concentrations of 0.25, 0.5, 1.0, 2.0, and 4.0 µM were used for all protocols. The alkaline comet assay and chromosome aberration (CA) analysis were performed in different phases of the cell cycle (G1, G1/S, transition, and G2), to evaluate the DNA-damaging and clastogenic effects of PHT, respectively. CA analysis was carried out in the presence or absence of colchicine to evaluate the action of PHT in the mitotic phase. PHT was cytotoxic and significantly reduced the mitotic index with drug exposure in all phases of cell cycle. Interestingly, it induced an increase in mitotic index in experimental protocols without colchicine, corroborating its action as an antitubulin agent. It also induced DNA damage and was clastogenic with drug exposure in all phases of the cell cycle, in the presence or absence of colchicine. In conclusion, PHT induces DNA damage and exerts clastogenic effects in human lymphocytes.

Preclinical genotoxicology of nor-ß-lapachone in human cultured lymphocytes and Chinese hamster lung fibroblasts.

Nor-ß-lapachone has shown several biological properties. Regarding cytotoxic activity against cancer cell lines, it has been recognized as an important prototype. However, quinonoid drugs present a major challenge because of their toxicity. In this study, we evaluated the cytotoxicity and genetic toxicity of nor-ß-lapachone in human lymphocytes and HL-60 leukemia cells and murine V79 fibroblasts, to shed some light on its selectivity toward cancer cells. As measured by MTT test, exposure of V79 cells to nor-ß-lapachone resulted in a weak cytotoxicity (IC(50) = 13.41 µM), and at a concentration up to 21.9 µM, no cytotoxic effect was observed in lymphocytes, while in HL-60 cells, nor-ß-lapachone elicited significantly greater cytotoxicity (IC(50) = 1.89 µM). Cultures coexposed to GSH-OEt showed an increased viability, which may indicate a neutralization of ROS generated by quinonoid treatment. In fact, only the highest concentrations of nor-ß-lapachone (10 or 20 µM) caused an increase in oxidative stress in nontumor levels cells as measured by TBARS and nitrite/nitrate detection. This was accompanied by an alteration in intracellular thiol content. However, NAC pre-exposure restored the redox equilibrium of the cells and the concentration of thiol levels to control values. Nor-ß-lapachone at 2.5 and 5 µM failed to induce DNA damage in nontumor cells, but at the highest concentrations tested, it induced single and double DNA strand breaks and increased the frequency of chromosomal aberrations. Interestingly, these damages were prevented by NAC pretreatment or exacerbated by prior exposure to the GSH-depleting agent 1-bromoheptane. In electrochemical experiments, nor-ß-lapachone at the same concentrations as those used in genotoxic tests did not damage DNA directly, but at the highest concentration tested (200 µM), it caused a very weak DNA interaction. Corroborating electrochemical data, oxidative modifications of DNA bases were observed, as checked by DNA repair enzymes EndoIII and FPG, which reinforced the indirect actions caused by nor-ß-lapachone through ROS generation and not via DNA intercalation. The DNA repair capacities were higher for nontumor cells than for leukemia cells, which may be related to the selective cytoxicity of nor-ß-lapachone toward cancer cells. Our data suggest that ROS play an important role in nor-ß-lapachone toxicity and that its DNA-damaging effect occurs only at concentrations several times higher than that needed for its antiproliferative effect on cancer cells.

In vitro cytotoxic activity of Brazilian Middle West plant extracts

Cytotoxic activity of eight plant extracts, native from the Mid-West of Brazil comprising Cerrado, Pantanal and semideciduous forest, was evaluated for MDA-MB-435, SF-295, and HCT-8 cancer cell strains. A single 100 µg.mL-1 dose of each extract was employed with 72 h of incubation for all tests. Doxorubicin (1 µg.mL-1) was used as the positive control and the MTT method was used to detect the activity. Cytotoxicity of distinct polarities was observed in thirty extracts (46 percent), from different parts of the following species: Tabebuia heptaphylla (Vell.) Toledo, Bignoniaceae, Tapirira guianensis Aubl., Anacardiaceae, Myracrodruon urundeuva Allemão, Anacardiaceae, Schinus terebinthifolius Raddi, Anacardiaceae, Gomphrena elegans Mart., Amaranthaceae, Attalea phalerata Mart. ex Spreng., Arecaceae, Eugenia uniflora L., Myrtaceae, and Annona dioica A. St.-Hil., Annonaceae. Extracts of at least two tested cell strains were considered to be highly active since their inhibition rate was over 75 percent.

In vitro and in vivo antiproliferative activity of Calotropis procera stem extracts.

The cytotoxic potential of stem organic extracts from Calotropis procera (Asclepiadaceae) was firstly evaluated against cancer cell lines by MTT assay. Subsequently, samples considered cytotoxic were tested for antimitotic activity on sea urchin egg development and for in vivo antiproliferative activity in mice bearing Sarcoma 180 tumor. Among the five extracts (hexane, dichloromethane, ethyl acetate, acetone and methanol), ethyl acetate and acetone extracts displayed higher cytotoxic potential against tumor cells, with IC50 ranging from 0.8 to 4.4 microg/mL, while methanolic extract was weakly cytotoxic. Cytotoxic extracts also exhibited cell division inhibition capacity by antimitotic assay, revealing IC50 values lower than 5 microg/mL. In the in vivo antitumor assessments, ethyl acetate- and acetone-treated animals showed tumor growth inhibition ratios of 64.3 and 53.1%, respectively, with reversible toxic effects on liver and kidneys. Further studies are in progress in order to identify C. procera cytotoxic compound(s) and to understand the mechanism of action responsible for this tumor-decreasing potential.

In vitro and in vivo antiproliferative activity of Calotropis procera stem extracts

The cytotoxic potential of stem organic extracts from Calotropis procera (Asclepiadaceae) was firstly evaluated against cancer cell lines by MTT assay. Subsequently, samples considered cytotoxic were tested for antimitotic activity on sea urchin egg development and for in vivo antiproliferative activity in mice bearing Sarcoma 180 tumor. Among the five extracts (hexane, dichloromethane, ethyl acetate, acetone and methanol), ethyl acetate and acetone extracts displayed higher cytotoxic potential against tumor cells, with IC50 ranging from 0.8 to 4.4 μg/mL, while methanolic extract was weakly cytotoxic. Cytotoxic extracts also exhibited cell division inhibition capacity by antimitotic assay, revealing IC50 values lower than 5 μg/mL. In the in vivo antitumor assessments, ethyl acetate- and acetone-treated animals showed tumor growth inhibition ratios of 64.3 and 53.1 percent, respectively, with reversible toxic effects on liver and kidneys. Further studies are in progress in order to identify C. procera cytotoxic compound(s) and to understand the mechanism of action responsible for this tumor-decreasing potential.

O potencial citotóxico de extratos orgânicos do caule de Calotropis procera (Asclepiadaceae) foi primeiramente avaliado frente a linhagens de células tumorais através do ensaio de MTT. Aquelas amostras consideradas citotóxicas foram sub-sequentemente testadas para atividade antimitótica sobre o desenvolvimento de ovos de ouriço-do-mar e para atividade antiproliferativa in vivo em camundongos transplantados com tumor Sarcoma 180. Dentre os cinco extratos estudados (hexano, diclorometano, acetato de etila, acetona e metanol), os extratos acetato de etila e acetona mostraram maior potencial citotóxico contra células tumorais, com CI50 variando de 0,8 to 4,4 μg/mL, enquanto o extrato metanólico revelou ser fracamente citotóxico. s extratos citotóxicos também exibiram capacidade de inibição da divisão celular com valores de CI50 menores que 5 μg/mL. Nas avaliações antitumorais in vivo, os animais tratados com os extratos acetato de etila e acetona mostraram taxas de inibição do crescimento tumoral de 64,3 e 53,1 por cento, respectivamente, com efeitos tóxicos reversíveis sobre o fígado e os rins.

Kauren-19-oic acid induces DNA damage followed by apoptosis in human leukemia cells.

This study evaluated the potential cytotoxicity of the natural diterpenoids kauren-19-oic acid (KA), 14-hydroxy-kaurane (1) and xylopic acid (2), and semi-synthetic derivatives of KA (3-5) towards human cancer cell lines (K562, HL60, MDA-MB435 and SF295) and lymphocytes. Mouse erythrocytes were used to verify a possible hemolytic activity Cytotoxicity mechanisms were investigated in HL60 cells. KA showed a moderate antiproliferative effect in MTT assay towards all cancer cells (IC(50), 9.1-14.3 microg ml(-1)). However, KA appeared not selective to cancer cells, since it also inhibited the lymphocytes proliferation (IC(50), 12.6 microg ml(-1)). Unlike KA, compounds 1-5 displayed no cytotoxicity and were also free from antiproliferative and hemolytic effects, suggesting that the exocyclic double bond (C16) unit may be the active pharmacophore of KA cytotoxicity. KA-treated HL60 cells displayed decreased proliferation (5-bromo-2';-deoxyuridine incorporation assay) and topoisomerase I activity (DNA relaxation assay). These assays revealed that KA primarily intercalates with DNA and not with topoisomerase I. Fluorescence microscopy using AO/EB (acridine orange/ethidium bromide) staining indicated that KA can induce both apoptosis and necrosis in HL-60 cell cultures, which corroborate the findings with MTT. From these findings, we conclude that KA, although demonstrating cytotoxic potential, may have a limited or poor therapeutic potential due to lack of selectivity to tumor cells. Further studies on the structure modification of KA and the mechanism of the new derivatives are currently in progress.

Estudo fitoquimico de Auxemma glazioviana Taub

Phytochemical investigation of heartwood of Auxemma glazioviana (Boraginaceae) has led to the isolation of three benzoquinones, one hydroquinone identified as: oncocalyxone A (1), oncocalyxone C (2), glazioviana A (3), glaziovianol (4) besides allantoin (5) á-bisabolol (6) and á-cadinol (7). Their structures were determined based on spectral analysis, including 2D NMR experiments. Essential oil obtained from heartwood by hydrodistillation was analyzed by GC/MS. The major constituents were identified as á-bisabolol and á-cadinol. The oil was active against larvae of dengue (Aedes aegypti ) with DL50 2.98 ppm.