5-Aminouracil and other inhibitors of DNA replication induce biphasic interphase-mitotic cells in apical root meristems of Allium cepa.

KEY MESSAGE: Induction of biphasic interphase-mitotic cells and PCC is connected with an increased level of metabolism in root meristem cells of Allium cepa. Previous experiments using primary roots of Allium cepa exposed to low concentrations of hydroxyurea have shown that long-term DNA replication stress (DRS) disrupts essential links of the S-M checkpoint mechanism, leading meristem cells either to premature chromosome condensation (PCC) or to a specific form of chromatin condensation, establishing biphasic organization of cell nuclei with both interphase and mitotic domains (IM cells). The present study supplements and extends these observations by describing general conditions under which both abnormal types of M-phase cells may occur. The analysis of root apical meristem (RAM) cell proliferation after prolonged mild DRS indicates that a broad spectrum of inhibitors is capable of generating PCC and IM organization of cell nuclei. These included: 5-aminouracil (5-AU, a thymine antagonist), characterized by the highest efficiency in creating cells with the IM phenotype, aphidicolin (APH), an inhibitor of DNA polymerase α, 5-fluorodeoxyuridine (FUdR), an inhibitor of thymidylate synthetase, methotrexate (MTX), a folic acid analog that inhibits purine and pyrimidine synthesis, and cytosine arabinoside (Ara-C), which inhibits DNA replication by forming cleavage complexes with topoisomerase I. As evidenced using fluorescence-based click chemistry assays, continuous treatment of onion RAM cells with 5-AU is associated with an accelerated dynamics of the DNA replication machinery and significantly enhanced levels of transcription and translation. Furthermore, DRS conditions bring about an intensified production of hydrogen peroxide (H2O2), depletion of reduced glutathione (GSH), and some increase in DNA fragmentation, associated with only a slight increase in apoptosis-like programmed cell death events.

Sediment drying-rewetting cycles enhance greenhouse gas emissions, nutrient and trace element release, and promote water cytogenotoxicity.

Increased periods of prolonged droughts followed by severe precipitation events are expected throughout South America due to climate change. Freshwater sediments are especially sensitive to these changing climate conditions. The increased oscillation of water levels in aquatic ecosystems causes enhanced cycles of sediment drying and rewetting. Here we experimentally evaluate the effects of induced drought followed by a rewetting event on the release of carbon dioxide (CO2), methane (CH4), nutrients (nitrogen and phosphorus), and trace elements (iron, manganese, and zinc) from the sediment of a tropical reservoir in southeastern Brazil. Furthermore, we used bulb onions (Allium cepa) to assess the potential cytogenotoxicity of the water overlying sediments after rewetting. We found peaks in CO2 and CH4 emissions when sediments first transitioned from wet to dry, with fluxes declining as sediments dried out. CO2 emissions peaked again upon rewetting, whereas CH4 emissions remained unaltered. Our experiment also revealed average increases by up to a factor of ~5000 in the release rates of nutrients and trace elements in water overlying sediments after rewetting. These increased release rates of potentially toxic compounds likely explain the lower replication of Allium cepa cells (up to 22% reduction) exposed to water overlying sediments after rewetting. Our findings suggest that increased events of drought followed by rewetting may lead to a range of changes in freshwater ecosystems, including nutrient enrichment, increased toxicity following resuspension of contaminants, and higher emission of greenhouse gases to the atmosphere.

Toxic, cytogenetic and antitumor evaluations of [6]-gingerol in non-clinical in vitro studies.

Gingerol - [6]-gingerol ((S)-5-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-3-decanone; [6]-G) - is a phenolic compound with several pharmacological properties. Herein, the aim of the study was to evaluate the toxicogenic effects of [6]-G on Artemia salina nauplii, Allium cepa, HL-60 cell line and Sarcoma 180 (S-180) ascitic fluid cells.For toxic and genotoxic analysis, it was used [6]-G concentrations of 5, 10, 20 and 40 µg mL-1. For cytotoxic evaluation using the MTT test (3- [4,5-dimethyl-thiazol-2-yl] -2,5-diphenyl tetrazolium bromide), serial [6]-G dilutions (1.56-100 µg mL-1) were performed, and S-180, HL-60 and peripheral blood mononuclear cells (PBMC) were treated for 72 h. The IC50 of [6]-G were 1.14, 5.73 and 11.18 µg mL-1 for HL-60, S-180 and PBMC, respectively, indicating a possible selectivity against tumor cell lines. At higher concentrations (>10 µg mL-1), toxicity and genotoxicity were observed in the A. cepa test, especially at 40 µg mL-1. Mechanisms indicating apoptosis, such as toxicity, cytotoxicity and nuclear abnormalities (bridges, fragments, delays, loose chromosomes and micronuclei) suggest that [6]-G has potential for antitumor pharmaceutical formulations.

Cytotoxic allelochemicals induce ultrastructural modifications in Cassia tora L. and mitotic changes in Allium cepa L.: a weed versus weed allelopathy approach.

The stress induced by allelochemicals present in stem aqueous extract (SAE) of Nicotiana plumbaginifolia on alterations in growth, ultrastructure on Cassia tora L., and mitotic changes on Allium cepa L. were inspected. Application of SAE at different concentrations (0.5, 1, 2, and 4%) expressively reduced the growth of C. tora in terms of seedling length and dry biomass. Moreover, the ultrastructural variations induced in the epidermis of Cassia leaf (adaxial and abaxial surface) of 15-day-old saplings were analyzed through scanning electron microscopy (SEM). The variations noticed are rupturing and shrinking of cells along epidermis; damaged margins, extensively curled leaf apex along with the appearance of puff-like structures, grooves, and thread-like structures on the leaf surface. The epidermal cells of samples exposed to treatment no longer appear smooth relative to control, besides showing necrosis as well. Upon exposure to different concentrations of extract, A. cepa root tip cells showed aberrations in chromosome arrangement and disparity in the shape of the interphase and prophase nuclei along various phases of mitotic cycle as compared to control. The mitotic index (MI) showed a concentration-dependent decline in onion root tips exposed to SAE. The aberrations appearing frequently were formation of multinucleated cells, sticky metaphase and anaphase with bridges, sticky telophase, disturbed polarity, etc. The results also show the induction of elongated cells, giant cells, and cells with membrane damage by extract treatment. To our knowledge, this is the first gas chromatography-mass spectrometry (GC-MS) analysis of the methanolic extract of N. plumbaginifolia stem. Overall, 62 compounds were reported, covering 99.61% of the entire constituents, which can be considered responsible for the allelopathic suppression of C. tora. The chief component was 4-tert-butylcalix[4]arene with the highest composition of 19.89%, followed by palmitic acid (12.25%), palmitoleic acid (8.23%), precocene 2 (7.53%), isophytyl acetate (4.01%), and betastigmasterol (3.95%).

Phytosynthesis of gold and silver nanoparticles enhance in vitro antioxidant and mitostimulatory activity of Aconitum toxicum Reichenb. rhizomes alcoholic extracts.

Extracts obtained from different plant species proved to be a valuable tool in various biomedical applications. In the same time, the phytosynthesis of noble metal nanoparticles represents an already well-established route for obtaining nanoparticles with biological activity. The present paper studies the antioxidant activity and the cytogenetic effects of the alcoholic extracts from rhizomes of Aconitum toxicum Rchb., before and after the phytosynthesis of gold and silver nanoparticles, on the meristematic root cells of Allium cepa L., and on the general mitotic index and the progression rate through the mitotic phases, respectively, as well as on the genetic material organized in chromosomes. The extracts were characterized in terms of total polyphenolics content (1.49% and, respectively, 2.29%) and aconitine content (by HPLC - 4.891 mg/L and, respectively, 18.211 mg/L), while the phytosynthesis of metallic nanoparticles was monitored by UV-Vis spectrometry, X-ray diffraction, X-ray fluorescence and electron microscopy. Both the extracts and the obtained nanoparticles were evaluated for antioxidant potential (the antioxidant activity ranging between 78% and 84.32%) and cytogenetic effects. The obtained results prove the phytosynthesis of AgNPs and AuNPs with dimensions ranging from 9 nm to 15 nm for AuNPs, respectively from 53 nm to 67 nm for AgNPs. The extracts obtained from rhizomes of A. toxicum Rchb. induced mitotic stress, as well as a series of nuclear and mitotic aberrations. The biosynthesis of AgNPs and AuNPs intensified the antioxidant and mitostimulatory activity of the extracts.

Protective role of jaboticaba Plinia peruviana peel extract in copper-induced cytotoxicity in Allium cepa.

Jaboticaba Plinia peruviana (Poir.) Govaerts is a Brazilian berry that presents high levels of polyphenols, which may play a key role in preventing cytotoxic and genotoxic effects of harmful agents. Although copper is an essential micronutrient that plays an important role in organisms, high copper concentrations may trigger toxicity to animals and plants. Here, we investigated whether Plinia peruviana hydroalcoholic extract prevents copper-induced cytotoxicity in Allium cepa root cells. Five different anthocyanins and phenolic compounds were identified in Plinia peruviana extract. Importantly, the exposure to 1.53 mg/L copper for 24 h impaired mitotic index, as well as increased mitosis disturbances and triggered DNA damage. Pre-incubation with Plinia peruviana extract (0.25 g/L and 0.75 g/L) for 3 h prevented copper-induced changes in the mitotic index and reduced the number of abnormal cells. In conclusion, we suggest that Plinia peruviana peel extract has protective effects against cellular and genetic disturbances induced by copper.

Cytogenetic and genotoxic effects of Rosmaniric Acid on Allium cepa L. root meristem cells.

Rosmarinic acid (RA) is a natural polyphenol carboxylic acid, an ester of caffeic acid with 3,4-dihydroxyphenyllactic acid, found in many species. Current study was aimed to investigate the mitotic division, chromosomal and genotoxic effects of RA on Allium cepa root meristematic cells. In Allium root growth inhibition test, EC50 value was found as 100 ppm. Three concentrations (50, 100, and 200 ppm) of RA under different exposure periods (24, 48, 72 and 96 h) were employed to onion tuber roots. Distilled water and methyl methane sulfonate (MMS, 10 ppm) were used as a negative and positive control, respectively. 100 (except 24 h) and 200 ppm of RA significantly decreased mitotic index (MI). There was an increase of total chromosomal aberrations (CAs) at 50 ppm and simultaneous decrease of CAs at 200 ppm concentrations (p < 0.05). A significant increase in DNA damage was also observed at 200 ppm by Comet assay. Quantitative analysis of RA in A. cepa root meristem cells was also done by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Further investigations are required to explore the molecular mechanism involved in the cytotoxicity and genotoxicity of RA on plants.

Toxic, cytotoxic and genotoxic potential of synthetics food flavorings / Potencial tóxico, citotóxico y genotóxico de saborizantes sintéticos de alimentos

Food flavorings in general are few studied for the toxicological aspect. This condition justifies toxicity, cytotoxicity and genotoxicity assessments of the substances. In the present study, the toxicity of banana, cherry and hazelnut flavorings was evaluated in meristematic cells of roots of Allium cepa, in pure form (as marketed) and in the concentrations of 12.5; 25 and 50%, after 24 and 48 hours of exposure. Toxic potential of these food additives was also evaluated against Artemia salina nauplii at concentrations of 0.78; 1.56; 3.12; 6.25; 12.5; 25 and 50%, after 24 hours of exposure. The three additives, in all treatments and times of analysis considered, caused significant inhibition of cell division in A. cepa, however did not cause cellular alterations to the evaluated meristems. These food flavorings also caused significant mortality to micro crustaceans with LC50<100 μg/mL. From this, under the conditions of mentioned analyzes, cherry, banana and hazelnut flavorings induced significant toxicity and cytotoxicity to the bioassays used.

En general, los aspectos toxicológicos de los saborizantes de los alimentos son poco estudiados. Esta condición justifica las evaluaciones de toxicidad, citotoxicidad y genotoxicidad de estas sustancias. En el presente estudio, se evaluó la toxicidad de los aromas de plátano, cereza y avellana en células meristemáticas de raíces de Allium cepa, en forma pura (según comercializa) y en concentraciones de 12.5; 25 y 50%, después de 24 y 48 horas de exposición. El potencial tóxico de estos aditivos alimentarios también se evaluó frente a nauplios de Artemia salina a concentraciones de 0,78; 1,56; 3.12; 6.25; 12.5; 25 y 50%, después de 24 horas de exposición. Los tres aditivos, en todos los tratamientos y tiempos de análisis considerados, causaron inhibición significativa de la división celular en A. cepa, sin embargo, no causaron alteraciones celulares a los meristemos evaluados. Estos saborizantes alimentarios también causaron una mortalidad significativa a microcrustáceos con LC50 <100 μg/ mL. A partir de esto, bajo las condiciones de los análisis descriptos, los aromatizantes de cereza, plátano y avellana indujeron toxicidad significativa y citotoxicidad para los bioensayos utilizados.

Mitodepressive, antioxidant, antifungal and anti-inflammatory effects of wild-growing Romanian native Arctium lappa L. (Asteraceae) and Veronica persica Poiret (Plantaginaceae).

The present study aims to evaluate the potential uses of hydroalcoholic extracts obtained from Romanian native wild-growing plants. The hydroalcoholic extracts were obtained from the burdock roots and respectively the aerial parts of birdeye speedwell. The extracts were characterised by HPLC (quantifying 13 compounds in the V. persica extract, 6 compounds in the A. lappa extract and confirming the presence of arctiin and arctigenin in the burdock extract). The antioxidant potential of the crude extracts was evaluated using two methods: the DPPH assay (79.91% for speedwell extract, 76.23% for burdock extract) and the phosphomolybdate method (296.5 mg/g ascorbic acid equivalents for burdock, 324.4 mg/g for speedwell). The crude extracts were found to be active against both fungal lines used (Aspergillus niger and Penicillium hirsutum), inhibition zones - 17.1 mm and 13.1 mm against P. hirsutum, respectively ca. 22 mm for both extracts against A. niger. The cytogenetic effects (assessed using the Allium cepa assay) revealed a series of chromosomal aberrations and nuclear aberrations induced in the meristematic root cells. The anti-inflammatory effect, estimated in two inflammation experimental models, showed a significant effect, especially for the speedwell extract. The results recommend the evaluated extracts as promising sources of biologically-active compounds.

Marinobufagin, a molecule from poisonous frogs, causes biochemical, morphological and cell cycle changes in human neoplasms and vegetal cells.

Skin toad secretion present physiologically active molecules to protect them against microorganisms, predators and infections. This work detailed the antiproliferative action of marinobufagin on tumor and normal lines, investigate its mechanism on HL-60 leukemia cells and its toxic effects on Allium cepa meristematic cells. Initially, cytotoxic action was assessed by colorimetric assays. Next, HL-60 cells were analyzed by morphological and flow cytometry techniques and growing A. cepa roots were examined after 72 h exposure. Marinobufagin presented high antiproliferative action against all human tumor lines [IC50 values ranging from 0.15 (leukemia) to 7.35 (larynx) µM] and it failed against human erythrocytes and murine lines. Human normal peripheral blood mononuclear cells (PBMC) were up to 72.5-fold less sensitive [IC50: 10.88 µM] to marinobufagin than HL-60 line, but DNA strand breaks were no detected. Leukemia treaded cells exhibited cell viability reduction, DNA fragmentation, phosphatidylserine externalization, binucleation, nuclear condensation and cytoplasmic vacuoles. Marinobufagin also reduced the growth of A. cepa roots (EC50: 7.5 µM) and mitotic index, caused cell cycle arrest and chromosomal alterations (micronuclei, delays and C-metaphases) in meristematic cells. So, to find out partially targeted natural molecules on human leukemia cells, like marinobufagin, is an amazing and stimulating way to continue the battle against cancer.

Expression of the MYB transcription factor gene BplMYB46 affects abiotic stress tolerance and secondary cell wall deposition in Betula platyphylla.

Plant MYB transcription factors control diverse biological processes, such as differentiation, development and abiotic stress responses. In this study, we characterized BplMYB46, an MYB gene from Betula platyphylla (birch) that is involved in both abiotic stress tolerance and secondary wall biosynthesis. BplMYB46 can act as a transcriptional activator in yeast and tobacco. We generated transgenic birch plants with overexpressing or silencing of BplMYB46 and subjected them to gain- or loss-of-function analysis. The results suggest that BplMYB46 improves salt and osmotic tolerance by affecting the expression of genes including SOD, POD and P5CS to increase both reactive oxygen species scavenging and proline levels. In addition, BplMYB46 appears to be involved in controlling stomatal aperture to reduce water loss. Overexpression of BplMYB46 increases lignin deposition, secondary cell wall thickness and the expression of genes in secondary cell wall formation. Further analysis indicated that BplMYB46 binds to MYBCORE and AC-box motifs and may directly activate the expression of genes involved in abiotic stress responses and secondary cell wall biosynthesis whose promoters contain these motifs. The transgenic BplMYB46-overexpressing birch plants, which have improved salt and osmotic stress tolerance, higher lignin and cellulose content and lower hemicellulose content than the control, have potential applications in the forestry industry.

Characterization of RsMYB28 and RsMYB29 transcription factor genes in radish (Raphanus sativus L.).

Glucosinolates (GSLs) are important secondary metabolites in Brassicaceae plants. Previous studies have mainly focused on GSL contents, types, and biosynthesis-related genes, but the molecular characterization patterns of GSL biosynthesis-related transcription factors remain largely unexplored in radish (Raphanus sativus L.). To isolate transcription factor genes regulating the GSL biosynthesis, genomic DNA and cDNA sequences of RsMYB28 and RsMYB29 genes were isolated in radish. Two R2R3-MYB domains were identified in the deduced amino acid sequences. Subcellular localization and yeast-one hybrid assays indicated that both the RsMYB28 and RsMYB29 genes were located in the nucleus and possessed transactivation activity. Reverse transcription quantitative analysis showed that the RsMYB28 and RsMYB29 genes were expressed in seeds, leaves, stems, and roots at the seedling, taproot thickening, and mature stages. Both genes were highly expressed during the seedling and taproot thickening stages. The expression level of RsMYB28 was found to be up-regulated following wounding, glucose, and abscisic acid treatments, whereas RsMYB29 was up-regulated following wounding and methyl jasmonate treatments. These results provide insights into the biological function and characterization of the RsMYB28 and RsMYB29 genes, and facilitate further dissection of the molecular regulatory mechanism underlying the GSL biosynthesis in radish.

Uso do bioensaio com Allium cepa L. e análises físico-químicas e microbiológicas para avaliação da qualidade do Rio da Ilha, RS, Brasil / Allium cepa L. bioassay and physicochemical and microbiological analysis to evaluate the water quality of the Ilha River, RS, Brazil

O estudo avalia a toxicidade, citotoxicidade, genotoxicidade e análises físico-químicas e microbiológicas de amostras de águas coletadas em dois pontos (nascente e foz) do Rio da Ilha - um dos principais afluentes do Rio dos Sinos, RS, Brasil - em dois períodos: inverno (2014) e verão (2015), através do bioensaio com Allium cepa que fornece esses dados através da mensuração das raízes dos bulbos, índice mitótico e presença de aberrações cromossômicas. Os resultados demonstraram níveis de citotoxicidade principalmente na foz do rio, e alguns parâmetros (DBO5, fósforo, alumínio, chumbo, ferro, níquel e coliformes termotolerantes) acima da legislação estabelecida, mesmo a região sofrendo pouco impacto de origem antrópica.

This study evaluates the toxicity, cytotoxicity, genotoxicity and physicochemical and microbiological analysis of water samples collected at sites (source and mouth) of the Ilha River -one of the main tributaries of the Sinos River, RS, Brazil - in winter (2014) and summer (2015), by Allium cepa bioassay which provided the data by measuring the roots of the bulbs, mitotic index and presence of chromosomal aberrations. The results show levels of cytotoxicity especially at the mouth of the river, and some parameters (DBO5, phosphorus, aluminum, lead, iron, nickel and fecal coliforms) above the limits established by the Brazilian legislation, despite the localization of the region in an area under minor anthropic impact.

Antimitotic and antimutagenic action of the Hymenaea stigonocarpa bark on dividing cells / Ação antimitótica e antimutagênica do ritidoma de Hymenaea stigonocarpa Mart ex Hayne na divisão celular

Abstract The objective of this study was to evaluate the action of Hymenaea stigonocarpa bark hydroalcoholic extract against a mutagenic compound using A. cepa meristematic root cells as a test system. The treatment groups were: Negative Control (NC) – distilled water; Positive Control (PC) – paracetamol at a concentration of 0.008 mg/mL, Jatoba Control (JC) – aqueous fraction jatobá-do-cerrado at 0.5 or 1.0 or 1.5 mg/mL, and Simultaneous Treatment (ST) - jatobá-do-cerrado aqueous fraction at a concentration of 0.5 or 1.0 or 1.5 mg/mL associated with paracetamol solution at a concentration of 0.008 mg/mL. All groups were analyzed at 24 and 48 h. Five onion bulbs (five replications) were used for each treatment group. The root tips were fixed in Carnoy and slides prepared by the crush technique. Cells were analyzed throughout the cell cycle, totaling 5,000 for each treatment group at each exposure time. Mitotic indices were subjected to statistical analysis using the chi-square test (p<0.05). From the results it was found that the ST group, at the three concentrations, significantly potentiated the antiproliferative effect of the test system cells when compared to PC, NC and TJ at the three concentrations. Furthermore, the three ST concentrations significantly reduced the number of cell aberrations when compared to the number of aberrant cells obtained for the PC, demonstrating antimutagenic action on the A. cepa test system cells.

Resumo O objetivo do presente trabalho foi avaliar a ação do extrato hidroalcólico do ritidoma de Hymenaea stigonocarpa frente a um composto mutagênico, utilizando como sistema teste as células meristemáticas de raízes de A. cepa. Os grupos tratamentos avaliados foram: Controle Negativo (CN) – água destilada; Controle Positivo (CP) – paracetamol na concentração de 0,008 mg/mL, Controle Jatobá (CJ) – fração aquosa de jatobá-do-cerrado na concentração de 0,5 ou 1,0 ou 1,5 mg/mL, e Tratamento Simultâneo (TS) – fração aquosa de jatobá-do-cerrado na concentração de 0,5 ou 1,0 ou 1,5 mg/mL associada a solução de paracetamol na concentração de 0,008 mg/mL. Todos os grupos foram analisados nos tempos de 24 e 48 h. Para cada grupo tratamento cinco bulbos de cebolas (cinco repetições) foram utilizados. As radículas foram fixadas em Carnoy e as lâminas preparadas pela técnica de esmagamento. Analisaram-se células em todo ciclo celular, totalizando 5.000 para cada grupo tratamento em cada tempo de exposição. Os índices mitóticos obtidos foram submetidos à análise estatística do Qui-quadrado (p<0,05). A partir dos resultados verificou-se que o grupo TS, nas três concentrações, potencializou o efeito antiproliferativo significativo as células do sistema teste quando comparado ao CP, CN e TJ nas três concentrações. Ainda, o TS nas três concentrações reduziu de forma significativa o número de aberrações celulares quando comparado com o número de células aberrantes obtidas para o CP, demonstrando ação antimutagênica as células do sistema teste A. cepa.

Cytotoxicity of Cheese and Cheddar Cheese food flavorings on Allim cepa L root meristems / Citotoxicidade de aromatizantes alimentares de Queijo e Queijo Cheddar sob meristemas de raízes de Allim cepa L

Abstract Despite their great importance for the food industry, flavorings, in general, raise a number of questions regarding their cytotoxicity, mutagenicity and carcinogenicity, since, in the literature, there are few studies found evaluating the toxicity on the systemic and cellular level, of these chemical compounds. The root meristems of Allium cepa (onion) are widely used for the assessment of toxicity of chemical compounds of interest. Thus, this study aimed to evaluate, in A. cepa meristematic cells, individually and in combination at the cellular level, the toxicity of synthetic Cheese and Cheddar Cheese food flavorings, identical to the natural, at doses of 1.0 and 2.0 mL, at exposure times of 24 and 48 hours. In combination we used 0.5 mL of Cheese flavor associated with 0.5 mL of Cheddar flavor; and 1.0 mL of Cheese flavor associated with 1.0 mL of Cheddar flavor, at exposure times of 24 and 48 hours. For these evaluations, we used groups of five onion bulbs, which were first embedded in distilled water and then transferred to their respective doses. The root tips were collected and fixed in acetic acid (3:1) for 24 hours. The slides were prepared by crushing and were stained with 2% acetic orcein. Cells were analyzed throughout the cell cycle, totaling 5,000 for each control and exposure time. The mitotic indices calculated and cellular aberrations observed were subjected to statistical analysis using the chi-square test (p <0.05). No chromosomal abnormalities nor those of mitotic spindle were observed for the treatments performed. The results, both individually and in combination, showed that the flavorings under study significantly reduced the cell division rate of the test system cells used. Therefore, under the conditions studied, the two flavorings were cytotoxic.

Resumo Apesar da grande importância para a indústria alimentícia, os aromatizantes, em geral, suscitam uma série de dúvidas quanto a sua citotoxicidade, mutagenicidade e carcinogenicidade, visto que, na literatura, poucos são os trabalhos encontrados avaliando a toxicidade, em nível sistêmico e celular, destes compostos químicos. Os meristemas de raízes de Allium cepa (cebola) são muito utilizados para a avaliação da toxicidade de compostos químicos de interesse. Desta forma, este trabalho teve por objetivo avaliar em células meristemáticas de A. cepa, de forma individual, a toxicidade em nível celular de aromatizantes alimentares sintéticos, idênticos aos naturais, de sabores Queijo e Queijo Cheddar, nas doses de 1,0 e 2,0 mL, nos tempos de exposição de 24 e 48 horas; e de forma associada, onde se utilizou 0,5 mL do aromatizante sabor Queijo associado a 0,5 mL do aromatizante sabor Queijo Cheddar; e 1,0 mL do aromatizante sabor Queijo associado a 1,0 mL do aromatizante sabor Queijo Cheddar, nos tempos de exposição de 24 e 48 horas. Para estas avaliações utilizou-se grupos de cinco bulbos de cebolas, que primeiramente foram enraizados em água destilada, e em seguida transferidos para as suas respectivas doses. As radículas foram coletadas e fixadas em ácido acético (3:1) por 24 horas. As lâminas foram preparadas pela técnica de esmagamento e coradas com orceína acética a 2%. Analisaram-se células em todo ciclo celular, totalizando 5.000 para cada controle e tempo de exposição. Os índices mitóticos calculados e as aberrações celulares observadas foram submetidos à análise estatística do Qui-quadrado (p<0,05). Não foram observadas alterações cromossômicas e anomalias de fuso mitótico para nenhum dos tratamentos realizados. Os resultados obtidos, tanto individualmente como de forma associada, mostraram que os aromatizantes em estudos reduziram de forma significativa os índices de divisões celulares das células do sistema teste utilizado. Portanto, nas condições analisadas, os dois aromatizantes foram citotóxicos.

Isolation and functional characterization of a cold responsive phosphatidylinositol transfer-associated protein, ZmSEC14p, from maize (Zea may L.).

KEY MESSAGE: A Sec14-like protein, ZmSEC14p , from maize was structurally analyzed and functionally tested. Overexpression of ZmSEC14p in transgenic Arabidopsis conferred tolerance to cold stress. Sec14-like proteins are involved in essential biological processes, such as phospholipid metabolism, signal transduction, membrane trafficking, and stress response. Here, we reported a phosphatidylinositol transfer-associated protein, ZmSEC14p (accession no. KT932998), isolated from a cold-tolerant maize inbred line using the cDNA-AFLP approach and RACE-PCR method. Full-length cDNA that consisted of a single open reading frame (ORF) encoded a putative polypeptide of 295 amino acids. The ZmSEC14p protein was mainly localized in the nucleus, and its transcript was induced by cold, salt stresses, and abscisic acid (ABA) treatment in maize leaves and roots. Overexpression of ZmSEC14p in transgenic Arabidopsis conferred tolerance to cold stress. This tolerance was primarily displayed by the increased germination rate, root length, plant survival rate, accumulation of proline, activities of antioxidant enzymes, and the reduction of oxidative damage by reactive oxygen species (ROS). ZmSEC14p overexpression regulated the expression of phosphoinositide-specific phospholipase C, which cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) and generates second messengers (inositol 1,4,5-trisphosphate and 1,2-diacylglycerol) in the phosphoinositide signal transduction pathways. Moreover, up-regulation of some stress-responsive genes such as CBF3, COR6.6, and RD29B in transgenic plants under cold stress could be a possible mechanism for enhancing cold tolerance. Taken together, this study strongly suggests that ZmSEC14p plays an important role in plant tolerance to cold stress.

Cytotoxicity of Cheese and Cheddar Cheese food flavorings on Allim cepa L root meristems.

Despite their great importance for the food industry, flavorings, in general, raise a number of questions regarding their cytotoxicity, mutagenicity and carcinogenicity, since, in the literature, there are few studies found evaluating the toxicity on the systemic and cellular level, of these chemical compounds. The root meristems of Allium cepa (onion) are widely used for the assessment of toxicity of chemical compounds of interest. Thus, this study aimed to evaluate, in A. cepa meristematic cells, individually and in combination at the cellular level, the toxicity of synthetic Cheese and Cheddar Cheese food flavorings, identical to the natural, at doses of 1.0 and 2.0 mL, at exposure times of 24 and 48 hours. In combination we used 0.5 mL of Cheese flavor associated with 0.5 mL of Cheddar flavor; and 1.0 mL of Cheese flavor associated with 1.0 mL of Cheddar flavor, at exposure times of 24 and 48 hours. For these evaluations, we used groups of five onion bulbs, which were first embedded in distilled water and then transferred to their respective doses. The root tips were collected and fixed in acetic acid (3:1) for 24 hours. The slides were prepared by crushing and were stained with 2% acetic orcein. Cells were analyzed throughout the cell cycle, totaling 5,000 for each control and exposure time. The mitotic indices calculated and cellular aberrations observed were subjected to statistical analysis using the chi-square test (p <0.05). No chromosomal abnormalities nor those of mitotic spindle were observed for the treatments performed. The results, both individually and in combination, showed that the flavorings under study significantly reduced the cell division rate of the test system cells used. Therefore, under the conditions studied, the two flavorings were cytotoxic.

Laser dissection sampling modes for direct mass spectral analysis.

RATIONALE: Laser microdissection coupled directly with mass spectrometry provides the capability of on-line analysis of substrates with high spatial resolution, high collection efficiency, and freedom on shape and size of the sampling area. Establishing the merits and capabilities of the different sampling modes that the system provides is necessary in order to select the best sampling mode for characterizing analytically challenging samples. METHODS: The capabilities of laser ablation spot sampling, laser ablation raster sampling, and laser 'cut and drop' sampling modes of a hybrid optical microscopy/laser ablation liquid vortex capture electrospray ionization mass spectrometry system were compared for the analysis of single cells and tissue. RESULTS: Single Chlamydomonas reinhardtii cells were monitored for their monogalactosyldiacylglycerol (MGDG) and diacylglyceryltrimethylhomo-Ser (DGTS) lipid content using the laser spot sampling mode, which was capable of ablating individual cells (~4-15 µm) even when agglomerated together. Turbid Allium Cepa cells (~150 µm) having unique shapes difficult to precisely measure using the other sampling modes could be ablated in their entirety using laser raster sampling. Intact microdissections of specific regions of a cocaine-dosed mouse brain tissue were compared using laser 'cut and drop' sampling. Since in laser 'cut and drop' sampling whole and otherwise unmodified sections are captured into the probe, 100% collection efficiencies were achieved. Laser ablation spot sampling has the highest spatial resolution of any sampling mode, while laser ablation raster sampling has the highest sampling area adaptability of the sampling modes. CONCLUSIONS: Laser ablation spot sampling has the highest spatial resolution of any sampling mode, useful in this case for the analysis of single cells. Laser ablation raster sampling was best for sampling regions with unique shapes that are difficult to measure using other sampling modes. Laser 'cut and drop' sampling can be used for cases where the highest sensitivity is needed, for example, monitoring drugs present in trace amounts in tissue.

Pre-apoptotic activity of aqueous extracts of Cynanchum sarcomedium Meve & Liede on cells of Allium cepa and human erythrocytes.

Cynanchum sarcomedium Meve & Liede is a member of Apocynaceae, seen in dry and rocky areas. The present study highlights the cytotoxic potential of C. sarcomedium mediated by apoptosis on cells of Allium cepa and human red blood cells (RBCs). Cytogenetic changes in A. cepa and in situ visualization of cell death were revealed through acetocarmine and Evans blue staining techniques. Quantitative estimation of cell death was carried out at 600 nm in a spectrophotometer. Membrane characteristics of RBC in response to the treatment were evaluated by May-Grünwald-Giemsa staining and scanning electron microscopy (SEM). Cell membrane damage is a major factor for assessing apoptosis which is observed in the present study (90.91 %). Cell shrinkage, cytoplasmic fragmentation, condensed chromatin and presence of apoptotic bodies were the common cytological changes in A. cepa associated with apoptosis. Blebs in RBC evidenced by SEM revealed the membrane damage potential of the plant. Results obtained hereby suggest that the plant is an effective source to be used in toxicological studies and anti-cancer therapy.

Induction of mitotic and chromosomal abnormalities on Allium cepa cells by pesticides imidacloprid and sulfentrazone and the mixture of them.

To evaluate the cytotoxic and genotoxic effects of low concentrations of pesticides in non-target organisms, seeds of Allium cepa were exposed for 24 h to the imidacloprid insecticide, sulfentrazone herbicide and to the mixture of them, followed by recovery periods of 48 and 72 h. Imidacloprid results indicated an indirect genotoxic effect by inducing different types of chromosome aberration (CA), mainly bridges and chromosomal adherences. Cells with micronucleus (MN) were not significant in the analyzed meristems. Moreover, the 72-h recovery tests indicated that the two lower concentrations of the insecticide (0.036 and 0.36 g L(-1)) had their genotoxic effects minimized after discontinuation of treatment, differently to the observed for the field concentration (3.6 g L(-1)). Sulfentrazone herbicide at field concentration (6 g L(-1)) caused cytotoxic effects by inducing nuclear fragmentation and inhibition of cell division. The other concentrations (0.06, 0.6 and 1.2 g L(-1)) indicated genotoxic effects for this herbicide. The concentration of 0.06 g L(-1) induced persistent effects that could be visualized both by the induction of CA in the recovery times as by the presence of MN in meristematic and F1 cells. The induction of MN by this lowest concentration was associated with the great amount of breakage, losses and chromosomal bridges. The mixture of pesticides induced genotoxic and cytotoxic effects, by reducing the MI of the cells. The chromosomal damage induced by the mixture of pesticides was not persistent to the cells, since such damage was minimized 72 h after the interruption of the exposure.