In the present work, application of the previously established reversed-phase liquid chromatography procedure based on fluorescent labeling of cytosine and methylcytosine moieties with 2-bromoacetophenone (HPLC-FLD) is presented for simultaneous evaluation of global DNA and total RNA methylation at cytosine carbon 5. The need for such analysis was comprehended from the recent advances in the field of epigenetics that highlight the importance of non-coding RNAs in DNA methylation and suggest that RNA methylation might play a similar role in the modulation of genetic information, as previously demonstrated for DNA. In order to adopt HPLC-FLD procedure for DNA and RNA methylation analysis in a single biomass extract, two extraction procedures with different selectivity toward nucleic acids were examined, and a simplified calibration was designed allowing for evaluation of methylation percentage based on the ratio of chromatographic peak areas: cytidine/5-methylcytidine for RNA and 2'-deoxycytidine/5-methyl-2'-deoxycytidine for DNA. As a proof of concept, global DNA and total RNA methylation were determined in Lepidium sativum hydroponically grown in the presence of different Cd(II) or Se(IV) concentrations, expecting that plant exposure to abiotic stress might affect not only global DNA but also total RNA methylation. The results obtained showed the increase of DNA methylation in the treated plants up to concentration levels 2 mg L(-1) Cd and 1 mg L(-1) Se in the growth medium. For higher stressors' concentration, global DNA methylation tended to decrease. Most importantly, an inverse correlation was found between DNA and RNA methylation levels (r = -0.6788, p = 0.031), calling for further studies of this particular modification of nucleic acids in epigenetic context.

In eukaryotes, actual DNA methylation patterns provide biologically important information, for which both, genome-wide and locus-specific methylation at cytosine residues have been extensively studied. The original contribution of this work relies on the selective derivatization of cytosine moieties with 2-bromoacetophenone for the determination of global DNA methylation by reversed phase high performance liquid chromatography with spectrofluorimetric detection. The important features of the proposed procedure are as follows: (1) no need for the elimination of RNA, (2) detection limits for cytidine, 2'-deoxycytidine, 5-methylcytidine, and 5-methyl-2'-deoxycytidine in the range of 14.4-22.7 fmol, (3) feasibility for the detection of 0.06% of methylation in a low amount of DNA (80 ng), (4) potential viability for the evaluation of RNA methylation, and (5) relative simplicity in terms of analytical instrumentation and personnel training. The results obtained in the analysis of salmon testes DNA and nucleic acids from plant, human blood, and earthworms demonstrate the utility of the proposed procedure in biological studies and, in particular, for evaluation of the potential effect of environmental factors on actual DNA methylation in different types of living organisms.

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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 À1 ; the column effluent was on-line mixed with an alkaline solution of potassium persulfate (K 2 S 2 O 8 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 (NaBH 4 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 À1 for Se(IV) and 0.52 mg mL À1 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.

In this work, the effect of cadmium (0-5.0 mg L(-1) as cadmium chloride, Cd(II)) and selenium (0-2.0 mg L(-1) as sodium selenite, Se(IV)) was studied in Lepidium sativum with specific focus on glyoxal (GO) and methylglyoxal (MGO) and on the cellular distribution of both elements under different exposure conditions. The concentrations of two reactive α-ketoaldehydes present as natural metabolites and as by-products of lipid peroxidation, were increased in plants treated with Cd(II), providng complementary experimental evidence on element phytotoxicity in garden cress, in terms of oxidative damage. Even though for higher than 1.0 mg L(-1) Se in medium similar adverse effect was found, under simultaneous exposure to both elements the changes in GO and MGO concentrations were clearly attenuated as compared to a single stressor treatment. This effect was accompanied by lower uptake of the two elements, significant decrease of their relative distribution in the fraction containing polar compounds and their increase in fraction corresponding to insoluble cell fragments/components, suggesting that the direct in vivo interaction between two element forms might be involved in the favorable effects of simultaneous treatment with Cd(II) + Se(IV). The fluorescence spectra obtained for biomass extracts corresponding to different exposure conditions suggested possible in vivo formation of CdSe quantum dots; however further studies are needed for ultimate identification and characterization of such nanoparticulate species.