Kazimierz Wrobel

Universidad de Guanajuato, Ciudad Guanajuato, Guanajuato, Mexico

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Publications (91)245.7 Total impact

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    ABSTRACT: Rationale: A full understanding of the biological impact of nanomaterials demands analytical procedures suitable for the detection/quantification of epigenetic changes that occur in the exposed organisms. Here, the effect of CuO nanoparticles (NPs) on global methylation of nucleic acids in Lepidium sativum was evaluated by liquid chromatography/ion trap mass spectrometry. Enhanced selectivity toward cytosine-containing nucleosides was achieved by using their proton-bound dimers formed in positive electrospray ionization (ESI(+)) as precursor ions for multiple reaction monitoring (MRM) quantification based on one or two ion transitions. Methods: Plants were exposed to CuO NPs (0-1000 mg L(-1) ); nucleic acid extracts were washed with bathocuproine disulfate; nucleosides were separated on a Luna C18 column coupled via ESI(+) to an AmaZon SL mass spectrometer (Bruker Daltonics). Cytidine, 2´-deoxycytidine, 5-methylcytidine, 5-methyl-2´-deoxycytidine and 5-hydroxymethyl-2´-deoxycytidine were quantified by MRM based on MS(3) ([2M+H](+) /[M+H](+) /[M+H-132](+) or [M+H-116](+) ) and MS(2) ([2M+H](+) /[M+H](+) ). Results: Bathocuproine disulfate, added as Cu(I) complexing agent, allowed for elimination of [2M+Cu](+) adducts from the mass spectra. Poorer instrumental detection limits were obtained for MS(3) (20-120 fmol) as compared to MS(2) (9.0-41 fmol); however, two ion transitions helped to eliminate matrix effects in plant extracts. The procedure was tested by analyzing salmon sperm DNA (Sigma) and applied for the evaluation of DNA and RNA methylation in plants; in the absence of NPs, 13.03% and 0.92% methylated cytosines were found in DNA and RNA, respectively; for NPs concentration >50 mg L(-1) , DNA hypomethylation was observed with respect to unexposed plants. RNA methylation did not present significant changes upon plant exposure; 5-hydroxymethyl-2´-deoxycytidine was not detected in any sample. Conclusions: The MRM quantification proposed here of cytosine-containing nucleosides using their proton-bound homo-dimers as precursor ions proved its utility for the assessment of global methylation of DNA and RNA in plants under stress imposed by CuO NPs. Detection of copper adducts with cytosine-containing ions, and their elimination by washing extracts with Cu(I) chelator, calls for further investigation. Copyright © 2015 John Wiley & Sons, Ltd.
    No preview · Article · Jan 2016 · Rapid Communications in Mass Spectrometry
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    Full-text · Dataset · Dec 2015
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    ABSTRACT: The goal of this work was to establish a simple HPLC-ESI-ITMS/MS procedure, suitable for the determination of four common aliphatic polyamines in two different types of biological matrices. To this end, 1,6-diaminohexane was used as the internal standard (IS) and 4-fluoro-3-nitrobenzenotrifluoride (FNBT) as the derivatizing agent. Formation of fully derivatized compounds was confirmed by high resolution ESI-QTOFMS and MS/MS analysis. Reversed phase chromatographic separation was carried out by gradient elution with 0.1% (v/v) formic acid and methanol. In a positive ESI mode, the following pairs of precursor/quantifier ions were used for multiple reaction monitoring: 467.4/261.0 for PUT, 481.2/461.1 for CAD, 713.7/261.0 for SPD, 959.8/507.2 for SPM and 495.3/475.2 for IS. On-column instrumental detection limits of four polyamines were in the range 0.62-2.14fmol (0.039-0.215ng/ml). Versatility was demonstrated by analyzing plant extracts and human urine; prior to derivatization, all samples were cleaned-up by dichloromethane extraction. The evaluated signal suppression/enhancement was in the range 82.3-115.4% and the percentage recoveries obtained in the method of standard addition were in the range 83.7-114.4%. Statistically significant differences in polyamines concentrations were found in garden cress exposed to Cd(II) versus control seedlings (t-test, p<0.05); results obtained for urine from healthy volunteers and diabetic patients at different clinical conditions suggested possible utility of free polyamines as biomarkers of progressive diabetes. Copyright © 2015 Elsevier B.V. All rights reserved.
    Full-text · Article · Aug 2015 · Journal of chromatography. B, Analytical technologies in the biomedical and life sciences
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    ABSTRACT: Over the past few decades, reduction of hexavalent chromium (Cr(VI)) has been studied in many physicochemical contexts. In this research, we reveal the mechanism underlying the favorable effect of Mn(II) observed during Cr(VI) reduction by oxalic acid using liquid chromatography with spectrophotometric diode array detector (HPLC-DAD), nitrogen microwave plasma atomic emission spectrometry (HPLC-MP-AES), and high resolution mass spectrometry (ESI-QTOFMS). Both reaction mixtures contained potassium dichromate (0.67mM Cr(VI)) and oxalic acid (13.3mM), pH 3, one reaction mixture contained manganese sulfate (0.33mM Mn(II)). In the absence of Mn(II) only trace amounts of reaction intermediates were generated, most likely in the following pathways: (1) Cr(VI)→Cr(IV) and (2) Cr(VI)+Cr(IV)→2Cr(V). In the presence of Mn(II), the active reducing species appeared to be Mn(II) bis-oxalato complex (J); the proposed reaction mechanism involves a one-electron transfer from J to any chromium compound containing CrO bond, which is reduced to CrOH, and the generation of Mn(III) bis-oxalato complex (K). Conversion of K to J was observed, confirming the catalytic role of Mn(II). Since no additional acidification was required, the results obtained in this study may be helpful in designing a new, environmentally friendly strategy for the remediation of environments contaminated with Cr(VI). Copyright © 2015. Published by Elsevier B.V.
    Full-text · Article · Jul 2015 · Journal of hazardous materials
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    ABSTRACT: Phosphate (Pi) availability is a significant limiting factor for plant growth and productivity in both natural and agricultural systems. To cope with such limiting conditions, plants have evolved a myriad of developmental and biochemical strategies to enhance the efficiency of Pi acquisition and assimilation to avoid nutrient starvation. In the past decade, these responses have been studied in detail at the level of gene expression; however, the possible epigenetic components modulating plant Pi starvation responses have not been thoroughly investigated. Here, we report that an extensive remodeling of global DNA methylation occurs in Arabidopsis plants exposed to low Pi availability, and in many instances, this effect is related to changes in gene expression. Modifications in methylation patterns within genic regions were often associated with transcriptional activation or repression, revealing the important role of dynamic methylation changes in modulating the expression of genes in response to Pi starvation. Moreover, Arabidopsis mutants affected in DNA methylation showed that changes in DNA methylation patterns are required for the accurate regulation of a number of Pi-starvation–responsive genes and that DNA methylation is necessary to establish proper morphological and physiological phosphate starvation responses.
    Full-text · Article · Jun 2015 · Proceedings of the National Academy of Sciences
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    ABSTRACT: Somatic embryogenesis is a powerful biotechnological tool for the mass production of economically important cultivars. Due to the cellular totipotency of plants, somatic cells under appropriate conditions are able to develop a complete functional embryo. During the induction of somatic embryogenesis, there are different factors involved in the success or failure of the somatic embryogenesis response. Among these factors, the origin of the explant, the culture medium and the in vitro environmental conditions have been the most studied. However, the secretion of molecules into the media has not been fully addressed. We found that the somatic embryogenesis of Coffea canephora , a highly direct embryogenic species, is disrupted by the metabolites secreted from C . arabica , a poorly direct embryogenic species. These metabolites also affect DNA methylation. Our results show that the abundance of two major phenolic compounds, caffeine and ch
    Full-text · Article · Jun 2015 · PLoS ONE
  • Katarzyna Wrobel · Kazimierz Wrobel
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    ABSTRACT: Ensuring the efficient uptake of micronutrients and protection against toxicity of non-essential elements are the two important issues in trace element research for human nutrition. Since the uptake, in vivo utilization, specific biological functions or toxic effects of a given element are subject to its actual physicochemical form, food speciation analysis has received much attention over the past few decades. Different analytical schemes have been developed, depending on elements/species of interest, their physicochemical properties, total concentrations, chemical composition of the sample and the specific goal of the analysis. In this chapter, the methodological aspects of food speciation analysis are presented, including the description of different fractionation schemes, non-chromatographic approaches and hyphenated techniques. Studies evaluating the effect of food elaboration and gastrointestinal ingestion have also been considered. The role of atomic spectrometry techniques as the primary detection tools in analysis of known species/forms is highlighted and the applications of structure-characterizing techniques for species confirmation/identification in innovating speciation studies are also mentioned. Representative examples from the recent literature are reviewed in order to present the trends in sample pretreatment, species separation and quatification. New challenges due to the expanding knowledge of trace elements in human nutrition, the production of selenium-fortified prducts, the use of transgenic food and the presence of metal/metalloid-based nanomaterials have been featured. Finally, an outlook on the approaches to quantification and quality control is briefly presented.
    No preview · Article · Apr 2015
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    ABSTRACT: Melatonin, a hormoneknown for its effects onfree radical scavenging and antioxidant activity, can reduce lead toxicity in vivo and in vitro.We examined the effects ofmelatonin on lead bio-distribution. Rats were intraperitoneally injected with lead acetate (10, 15 or 20mg/kg/day) with or without melatonin (10mg/kg/day) daily for 10 days. In rats intoxicated with the highest lead doses, those treated with melatoninhad lower leadlevelsin blood and higher levels in urine and feces than those treated with lead alone, suggesting that melatonin increaseslead excretion. To explore the mechanism underlying this effect, we firstassessed whether lead/melatonin complexes were formed directly. Electronic density functional (DFT) calculations showed that a lead/melatonin complex is energetically feasible; however, UV spectroscopy and NMRanalysis showed noevidence of such complexes. Next, we examined the liver mRNA levels of metallothioneins (MT) 1 and 2. Melatonin cotreatment increased the MT2 mRNA expression in theliver ofrats that received the highest doses of lead. The potential effectsof MTs on the tissue distribution and excretion of lead are not well understood. This is the first report to suggest that melatonindirectlyaffectslead levels in organisms exposed to subacute lead intoxication. Copyright © 2015. Published by Elsevier Ireland Ltd.
    Full-text · Article · Jan 2015 · Toxicology Letters
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    ABSTRACT: The Cr(VI) reducing capability of growing cells of the environmental A. tubingensis Ed8 strain is remarkably efficient compared to reference strains A. niger FGSC322 and A. tubingensis NRRL593. Extracellular glucose oxidase (GOX) activity levels were clearly higher in colonies developed in solid medium and in concentrated extracts of the spent medium of liquid cultures of the Ed8 strain in comparison with the reference strains. In addition, concentrated extracts of the spent medium of A. tubingensis Ed8, but not those of the reference strains, exhibited the ability to reduce Cr(VI). In line with this observation, it was found that A. niger purified GOX is capable of mediating the conversion of Cr(VI) to Cr(III) in a reaction dependent on the presence of glucose that is stimulated by organic acids. Furthermore, it was found that a decrease in Cr(VI) may occur in the absence of the GOX enzyme, as long as the reaction products gluconolactone and hydrogen peroxide are present; this conversion of Cr(VI) is stimulated by organic acids in a reaction that generates hydroxyl radicals, which may involve the formation of an intermediate peroxichromate(V) complex. These findings indicated that fungal glucose oxidase acts an indirect chromate reductase through the formation of Cr(VI) reducing molecules, which interact cooperatively with other fungal metabolites in the biotransformation of Cr(VI). Copyright © 2014 Elsevier Ltd. All rights reserved.
    Full-text · Article · Jan 2015 · Chemosphere
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    ABSTRACT: Selenium biofortified yeast is the most common dietary Se supplement in human nutrition and in farm animals. Therefore, the production and routine quality control of commercial products are highly demanded. In this work, a simple and cost-effective procedure is proposed for the determination of SeMet and Se(iv) in hydrolyzed yeast, consisting of ion-pair reversed phase separation, post-column hydride generation and Se quantification by atomic emission spectrometry with microwave plasma sustained by nitrogen (HPLC-HG-MP-AES). Freeze-dried biomass was hydrolyzed with methanesulfonic acid; chromatographic separation was performed with a mobile phase containing 0.08% v/v heptafluorobutyric and methanol (92-8) at a flow rate of 1 mL min-1; the column effluent was on-line mixed with an alkaline solution of potassium persulfate (K2S2O8 6% m/v, NaOH 3% m/v), passed through a reaction coil submerged in a water bath at 60 °C, and then 10 M hydrochloric acid was added prior to hydride generation in the MP-AES multimode sample introduction system (NaBH4 2% m/v, NaOH 0.3% m/v). The total chromatographic run was accomplished in 5 min and the evaluated on-column quantification limits were 59 ng Se mL-1 for Se(iv) and 0.52 μg mL-1 for SeMet. The procedure was tested using standardized Seleno Excell® high selenium yeast and then applied for the analysis of Saccharomyces cerevisiae biofortified under different fermentation and exposure conditions. The procedure was capable of detecting differences in selenium concentration among cultures and the results were consistent with those obtained while coupling HPLC separation directly to ICP-MS detection.
    Full-text · Article · Jan 2015 · Journal of Analytical Atomic Spectrometry
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    ABSTRACT: Three α-ketoaldehydes, potentially present in high fructose agave syrups (HFASs) as intermediates of the Maillard reaction, were determined. A previously reported HPLC-FLD procedure based on pre-column derivatisation with 4-methoxy-o-phenylenediamine was adopted, yielding the method quantification limits 0.11mg/kg, 0.10mg/kg, 0.09mg/kg for glyoxal, methylglyoxal (MGo) and diacetyl, respectively. The obtained results revealed high concentrations of methylglyoxal in HFASs (average 102±91mg/kg, range 15.6-315mg/kg) as compared to commercial Mexican bee honeys or corn syrups. Hydrogen peroxide was generated in all HFASs upon dilution, yet to less extent than in bee honeys. HFASs presented bacteriostatic activity against Bacillus subtilis and Escherichia coli; catalase addition had minimum effect on the assay results in syrups with elevated MGo. Principal component analysis revealed direct association between growth inhibition and MGo. It is concluded that elevated concentration of MGo in HFASs is at least in part responsible for their non-peroxide bacteriostatic activity.
    Full-text · Article · Dec 2014 · Food Chemistry
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    ABSTRACT: A straightforward synthetic protocol to directly incorporate stabilized 1,3-dicarbonyl C nucleophiles to the meso position of BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) is reported. Soft nucleophiles generated by deprotonation of 1,3-dicarbonyl derivatives smoothly displace the 8-methylthio group from 8-(methylthio)BODIPY analogues in the presence of CuI thiophenecarboxylate in stoichiometric amounts at room temperature. Seven highly fluorescent new derivatives are prepared with varying yields (20–92 %) in short reaction times (5–30 min). The excellent photophysical properties of the new dyes allow focusing on applications never analyzed before for BODIPYs substituted with stabilized C nucleophiles such as pH sensors and lasers in liquid and solid state, highlighting the relevance of the synthetic protocol described in the present work. The attainment of these dyes, with strong UV absorption and highly efficient and stable laser emission in the green spectral region, concerns to one of the greatest challenges in the ongoing development of advanced photonic materials with relevant applications. In fact, organic dyes with emission in the green are the only ones that allow, by frequency-doubling processes, the generation of tunable ultraviolet (250-350 nm) radiation, with ultra-short pulses.
    No preview · Article · Dec 2014 · Chemistry - A European Journal
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    ABSTRACT: Experimental evidence indicates that diabetic patients and individuals with impaired copper homeostasis could be at risk of molybdenum toxicity. A self-administered food frequency questionnaire revealed that in central Mexico, diabetic patients with severe complications tend to consume beans more often than individuals with less advanced disease. Four varieties of Phaseolus vulgaris were comparatively evaluated as the dietary sources of two elements; the results showed molybdenum concentration decreasing in the order peruvian > pinto > mayflower > black, whereas for copper, the order was peruvian > pinto ∼ black > mayflower. The two elements were determined in pre-soaking water, cooked legumes, and broth obtained in cooking procedure; an in vitro gut model was also applied to assess potentially bioavailable fraction of both elements in cooked beans. The results indicated that the black variety would be the healthiest bean choice for diabetic patients and individuals susceptible to Mo toxicity. Relatively low total molybdenum was found in this variety (2.9 ± 1.4 versus 4.3-10.9 μg g(-1) in other types), element availability was also low (15 % in supernatant from enzymolysis, 24.9 % in combined broth + supernatant fractions), and the molar ratio of Cu/Mo was the highest among four types (41, versus Cu/Mo <10 in peruvian, pinto, or mayflower). Considering peruvian and pinto beans, broth elimination would help to lower molybdenum intake with marginal effect on Cu/Mo molar ratio. This recommendation would be especially important for peruvian variety, which provided 1090, 803, and 197 μg day(-1) of molybdenum in raw grains, broth + supernatant, and supernatant, respectively (based on 100-g portion), exceeding the recommended daily allowance of 45 μg day(-1).
    Full-text · Article · Nov 2014 · Biological Trace Element Research
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    ABSTRACT: The ability of human serum albumin to capture unbound copper under different clinical conditions is an important variable potentially affecting homeostasis of this element. Here, we propose a simple procedure based on size-exclusion chromatography with on-line UV and nitrogen microwave-plasma atomic-emission spectrometry (MP-AES) for quantitative evaluation of Cu(II) binding to HSA upon its glycation in vitro. The Cu-to-protein molar ratio for non-glycated albumin was 0.98 ± 0.09; for HSA modified with glyoxal (GO), methylglyoxal (MGO), oxoacetic acid (GA), and glucose (Glc), the ratios were 1.30 ± 0.22, 0.72 ± 0.14, 0.50 ± 0.06, and 0.95 ± 0.12, respectively. The results were confirmed by using ICP-MS as an alternative detection system. A reduced ability of glycated protein to coordinate Cu(II) was associated with alteration of the N-terminal metal-binding site during incubation with MGO and GA. In contrast, glycation with GO seemed to generate new binding sites as a result of tertiary structural changes in HSA. Capillary reversed-phase liquid chromatography with electrospray-ionization quadrupole-time-of-flight tandem mass spectrometry enabled detection and identification of Cu(II) coordinated to the N-terminal metal-binding site (Cu(II)–DAHK) in all tryptic digests analyzed. This is the first report confirming Cu(II)–DAHK species in HSA by means of high-resolution tandem mass spectrometry, and the first report on the use of MP-AES in combination with chromatographic separation. Graphical Abstract General scheme designed to study Cu(II) binding to glycated versus non-glycated albumin and MPAESinstrumentation used
    Full-text · Article · Nov 2014 · Analytical and Bioanalytical Chemistry
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    ABSTRACT: In this work, the sorption of pentachlorophenol (PCP) by non-viable biomass of Rhizopus oryzae ENHE was evaluated. The kinetics and isotherm studies were performed at pH 5.0, 6.0, and 8.0. The point of zero charge of the biomass was determined; this value allowed us to explain the changes of pH during sorption studies. The analyzed experimental kinetic data revealed that Ho's model adjusted better to the experimental data than Lagergren's model. PCP sorption was fast; an equilibrium sorption time was reached within 30 min, regardless of pH. PCP sorption at pH 5.0 and 6.0 was better described by the Freundlich isotherm than by the Langmuir isotherm. In contrast, at pH 8.0, the Langmuir isotherm describes better the PCP sorption. Sorption data showed that at pH 5.0 and 6.0, the sorption capacity of PCP was higher than at pH 8.0. Sorption of PCP by the fungal biomass occurred spontaneously; it was endothermic and due to physical sorption. Finally, FT-IR analysis of the dried biomass indicated that amino and hydroxyl groups were involved in the sorption of PCP. This work is one of the few reporting the effect of pH and temperature on the sorption of PCP by microbial biomass from a filamentous fungus belonging to the genus Rhizopus.
    No preview · Article · May 2014 · Water Air and Soil Pollution
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    ABSTRACT: Tequila is elaborated from Agave tequilana Weber blue variety and it is commercialized at different stages of aging. Chemical composition of this product has often been addressed; however, data on phenolic compounds are scarce. In this work, a high-performance liquid chromatography–electrospray ionization-ion trap mass spectrometry (HPLC–ESI-ITMS) procedure has been established for the determination of 34 small phenolic compounds. The combination of suitable separation conditions with extraction of chromatograms at individual m/z values has enabled for total analysis run of 17 min (11 min separation plus 6 min column cleaning/equilibration) with the detection limits in the range 1.28–75.0 μg l−1 (0.07–6.1 pmol on-column). Commercial tequilas analyzed included 6 white, 12 rested, and 4 aged. The following acids were found and quantified: gallic, procatechuic, 4-hydroxybenzoic, vanillic, syringic, homovanillic, 3-hydroxybenzoic, ferulic, salicylic, and benzoic. The white tequilas contained fewer compounds and lower total phenolics concentrations (range 36–408 μg l−1) as compared to the rested and aged liquors (515–4,296 and 2,048–3,249 μg l−1, respectively). In the latter products, syringic, vanillic, procatechuic, and gallic acids were the most abundant, which indicates that maturation in wooden barrels is the main source of small phenolics in tequila. On the other part, homovanillic acid was found in all tequila types (medians for white, rested, and aged products 82, 153, and 162 μg l−1, respectively), suggesting that some phenolics may originate from the raw material or might be formed during liquor elaboration.
    Full-text · Article · Apr 2014 · Food Analytical Methods
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    ABSTRACT: Bioanalytical relevance of glyoxal (Go) and methylglyoxal (MGo) arises from their role as biomarkers of glycation processes and oxidative stress. The third compound of interest in this work is diacetyl (DMGo), a component of different food products and alcoholic beverages and one of the small α-ketoaldehydes previously reported in urine. The original idea for the determination of the above compounds by reversed phase high-performance liquid chromatography (HPLC) with fluorimetric detection was to use 4-methoxy-o-phenylenediamine (4MPD) as a derivatizing reagent and diethylglyoxal (DEGo) as internal standard. Acetonitrile was added to urine for matrix precipitation, and derivatization reaction was carried out in the diluted supernatant at neutral pH (40 °C, 4 h); after acidification, salt-induced phase separation enabled recovery of the obtained quinoxalines in the acetonitrile layer. The separation was achieved within 12 min using a C18 Kinetex column and gradient elution. The calibration detection limits for Go, MGo, and DMGo were 0.46, 0.39, and 0.28 μg/L, respectively. Within-day precision for real-world samples did not exceed 6%. Several urine samples from healthy volunteers, diabetic subjects, and juvenile swimmers were analyzed. The sensitivity of the procedure proposed here enabled detection of differences between analyte concentrations in urine from patients at different clinical or exposure-related conditions.
    Full-text · Article · Mar 2014 · Analytical Biochemistry
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    ABSTRACT: The purpose of this study was to investigate the effects of cadmium [Cd(II) as cadmium chloride], selenium [Se(IV) as sodium selenite] and their mixtures on phenolic compounds (PCs) and antioxidant activity (AOx) in Lepidium sativum. The biomass fractions corresponding to free (F1), soluble glycoside-bound (F2) and cell wall ester-bound phenolics (F3) were obtained for each treatment and PCs were screened by gas chromatography (GC-FID); F1 and F2 fractions were also analyzed by liquid chromatography with UV and fluorimetric detection. The treated plants presented different profiles of PCs as compared to controls, specifically in F1 and F2 fractions; the plant response was element-, and concentration-dependent. The cultures challenged with Cd(II) up to 5 mg L−1, presented higher AOx with respect to controls, and this increase was associated with glycoside-bound PCs, whereas for Se(IV) the increase of AOx was less marked and associated with free PCs. Under simultaneous exposure to Cd(II) + Se(IV) (0.5–2.0 mg L−1 each), the AOx values were relatively constant and lower than those found in the presence of Se(IV) or Cd(II) alone, providing further evidence of the protective role of Se(IV) against stress imposed by Cd(II) in this plant species. The evaluation of AOx of individual PCs and the results of principal component analysis enabled to attain several relationships among exposure conditions, antioxidant activity, free- and glycoside-bound phenolic compounds.
    Full-text · Article · Jan 2014 · Acta Physiologiae Plantarum
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    ABSTRACT: Arsenic release from the abandoned mines and its fate in a local stream were studied. Physicochemical parameters, metals/metalloids and arsenic species were determined. One of the mine drainages was found as a point source of contamination with 309 μg L(-1) of dissolved arsenic; this concentration declined rapidly to 10.5 μg L(-1) about 2 km downstream. Data analysis confirmed that oxidation of As(III) released from the primary sulfide minerals was favored by the increase of pH and oxidation reduction potential; the results obtained in multivariate approach indicated that self-purification of water was due to association of As(V) with secondary solid phase containing Fe, Mn, Ca.
    No preview · Article · Sep 2013 · Bulletin of Environmental Contamination and Toxicology
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    ABSTRACT: Plant cells have the capacity to generate a new plant without egg fertilization by a process known as somatic embryogenesis (SE), in which differentiated somatic cells can form somatic embryos able to generate a functional plant. Although there have been advances in understanding the genetic basis of SE, the epigenetic mechanism that regulates this process is still unknown. Here, we show that the embryogenic development of Coffea canephora proceeds through a crosstalk between DNA methylation and histone modifications during the earliest embryogenic stages of SE. We found that low levels of DNA methylation, histone H3 lysine 9 dimethylation (H3K9me2) and H3K27me3 change according to embryo development. Moreover, the expression of LEAFY COTYLEDON1 (LEC1) and BABY BOOM1 (BBM1) are only observed after SE induction, whereas WUSCHEL-RELATED HOMEOBOX4 (WOX4) decreases its expression during embryo maturation. Using a pharmacological approach, it was found that 5-Azacytidine strongly inhibits the embryogenic response by decreasing both DNA methylation and gene expression of LEC1 and BBM1. Therefore, in order to know whether these genes were epigenetically regulated, we used Chromatin Immunoprecipitation (ChIP) assays. It was found that WOX4 is regulated by the repressive mark H3K9me2, while LEC1 and BBM1 are epigenetically regulated by H3K27me3. We conclude that epigenetic regulation plays an important role during somatic embryogenic development, and a molecular mechanism for SE is proposed.
    Full-text · Article · Aug 2013 · PLoS ONE