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1.  The Effect of Lycopene Preexposure on UV-B-Irradiated Human Keratinocytes 
Lycopene has been reported as the antioxidant most quickly depleted in skin upon UV irradiation, and thus it might play a protective role. Our goal was to investigate the effects of preexposure to lycopene on UV-B-irradiated skin cells. Cells were exposed for 24 h to 10 M lycopene, and subsequently irradiated and left to recover for another 24 h period. Thereafter, several parameters were analyzed by FCM and RT-PCR: genotoxicity/clastogenicity by assessing the cell cycle distribution; apoptosis by performing the Annexin-V assay and analyzing gene expression of apoptosis biomarkers; and oxidative stress by ROS quantification. Lycopene did not significantly affect the profile of apoptotic, necrotic and viable cells in nonirradiated cells neither showed cytostatic effects. However, irradiated cells previously treated with lycopene showed an increase in both dead and viable subpopulations compared to nonexposed irradiated cells. In irradiated cells, lycopene preexposure resulted in overexpression of BAX gene compared to nonexposed irradiated cells. This was accompanied by a cell cycle delay at S-phase transition and consequent decrease of cells in G0/G1 phase. Thus, lycopene seems to play a corrective role in irradiated cells depending on the level of photodamage. Thus, our findings may have implications for the management of skin cancer.
PMCID: PMC4664803  PMID: 26664697
2.  The use of comet assay in plant toxicology: recent advances 
Frontiers in Genetics  2015;6:216.
The systematic study of genotoxicity in plants induced by contaminants and other stress agents has been hindered to date by the lack of reliable and robust biomarkers. The comet assay is a versatile and sensitive method for the evaluation of DNA damages and DNA repair capacity at single-cell level. Due to its simplicity and sensitivity, and the small number of cells required to obtain robust results, the use of plant comet assay has drastically increased in the last decade. For years its use was restricted to a few model species, e.g., Allium cepa, Nicotiana tabacum, Vicia faba, or Arabidopsis thaliana but this number largely increased in the last years. Plant comet assay has been used to study the genotoxic impact of radiation, chemicals including pesticides, phytocompounds, heavy metals, nanoparticles or contaminated complex matrices. Here we will review the most recent data on the use of this technique as a standard approach for studying the genotoxic effects of different stress conditions on plants. Also, we will discuss the integration of information provided by the comet assay with other DNA-damage indicators, and with cellular responses including oxidative stress, cell division or cell death. Finally, we will focus on putative relations between transcripts related with DNA damage pathways, DNA replication and repair, oxidative stress and cell cycle progression that have been identified in plant cells with comet assays demonstrating DNA damage.
PMCID: PMC4485349  PMID: 26175750
plant comet assay; genotoxicity; metal; phytocompounds; radiation; pollutants; nanoparticles; DNA damages biomarkers
3.  Clinical and Laboratory Studies of the Fate of Intranasal Allergen 
PLoS ONE  2015;10(5):e0127477.
The precise way in which allergen is handled by the nose is unknown. The objective of this study was to determine recovery of Der p 1 allergen following nasal administration and to determine whether Der p 1 can be detected in nasal biopsies after natural exposure and nasal challenge to allergen.
(1) 20 nonatopic non-rhinitics were challenged with Der p 1 and recovery was measured by ELISA in the nasal wash, nasal mucus and induced sputum up to 30 minutes. Particulate charcoal (<40 μm) served as control. (2) In 8 subjects (5 atopics), 30 to 60 minutes after challenge histological localisation of Der p 1 in the nasal mucosal epithelium, subepithelial mucous glands and lamina propria was performed. Co-localisation of Der p 1 with macrophages and IgE-positive cells was undertaken.
(1) Less than 25% of total allergen was retrievable after aqueous or particulate challenge, most from the nasal mucus during 1-5 min after the challenge. The median of carbon particles recovered was 9%. (2) Prechallenge Der p 1 staining was associated with the epithelium and subepithelial mucous glands. After challenge there was a trend for greater Der p 1 deposition in atopics, but both atopics and nonatopics showed increases in the number of Der p 1 stained cells and stained tissue compartments. In atopics, increased eosinophils, macrophages and IgE positive cells co-localized with Der p 1 staining.
Der p 1 allergen is detected in nasal tissue independent of atopic status after natural exposure. After challenge the nose effectively retains allergen, which remains mucosally associated; in atopics there is greater Der p 1 deposition and inflammatory response than in nonatopics. These results support the hypothesis that nasal mucus and tissue act as a reservoir for the inhaled Der p 1 allergen leading to a persistent allergic inflammatory response in susceptible individuals.
PMCID: PMC4430540  PMID: 25969994
4.  Sulforaphane Induces Oxidative Stress and Death by p53-Independent Mechanism: Implication of Impaired Glutathione Recycling 
PLoS ONE  2014;9(3):e92980.
Sulforaphane (SFN) is a naturally-occurring isothiocyanate best known for its role as an indirect antioxidant. Notwithstanding, in different cancer cell lines, SFN may promote the accumulation of reactive oxygen species (ROS) and cause cell death e.g. by apoptosis. Osteosarcoma often becomes chemoresistant, and new molecular targets to prevent drug resistance are needed. Here, we aimed to determine the effect of SFN on ROS levels and to identify key biomarkers leading to ROS unbalance and apoptosis in the p53-null MG-63 osteosarcoma cell line. MG-63 cells were exposed to SFN for up to 48 h. At 10 μM concentration or higher, SFN decreased cell viability, increased the%early apoptotic cells and increased caspase 3 activity. At these higher doses, SFN increased ROS levels, which correlated with apoptotic endpoints and cell viability decline. In exposed cells, gene expression analysis revealed only partial induction of phase-2 detoxification genes. More importantly, SFN inhibited ROS-scavenging enzymes and impaired glutathione recycling, as evidenced by inhibition of glutathione reductase (GR) activity and combined inhibition of glutathione peroxidase (GPx) gene expression and enzyme activity. In conclusion, SFN induced oxidative stress and apoptosis via a p53-independent mechanism. GPx expression and activity were found associated with ROS accumulation in MG-63 cells and are potential biomarkers for the efficacy of ROS-inducing agents e.g. as co-adjuvant drugs in osteosarcoma.
PMCID: PMC3965485  PMID: 24667842
6.  The Redox State of Cytochrome C Modulates Resistance to Methotrexate in Human MCF7 Breast Cancer Cells 
PLoS ONE  2013;8(5):e63276.
Methotrexate is a chemotherapeutic agent used to treat a variety of cancers. However, the occurrence of resistance limits its effectiveness. Cytochrome c in its reduced state is less capable of triggering the apoptotic cascade. Thus, we set up to study the relationship among redox state of cytochrome c, apoptosis and the development of resistance to methotrexate in MCF7 human breast cancer cells.
Cell incubation with cytochrome c-reducing agents, such as tetramethylphenylenediamine, ascorbate or reduced glutathione, decreased the mortality and apoptosis triggered by methotrexate. Conversely, depletion of glutathione increased the apoptotic action of methotrexate, showing an involvement of cytochrome c redox state in methotrexate-induced apoptosis. Methotrexate-resistant MCF7 cells showed increased levels of endogenous reduced glutathione and a higher capability to reduce exogenous cytochrome c. Using functional genomics we detected the overexpression of GSTM1 and GSTM4 in methotrexate-resistant MCF7 breast cancer cells, and determined that methotrexate was susceptible of glutathionylation by GSTs. The inhibition of these GSTM isoforms caused an increase in methotrexate cytotoxicity in sensitive and resistant cells.
We conclude that overexpression of specific GSTMs, GSTM1 and GSTM4, together with increased endogenous reduced glutathione levels help to maintain a more reduced state of cytochrome c which, in turn, would decrease apoptosis, thus contributing to methotrexate resistance in human MCF7 breast cancer cells.
PMCID: PMC3652835  PMID: 23675469
7.  Can Physiological Endpoints Improve the Sensitivity of Assays with Plants in the Risk Assessment of Contaminated Soils? 
PLoS ONE  2013;8(4):e59748.
Site-specific risk assessment of contaminated areas indicates prior areas for intervention, and provides helpful information for risk managers. This study was conducted in the Ervedosa mine area (Bragança, Portugal), where both underground and open pit exploration of tin and arsenic minerals were performed for about one century (1857 – 1969). We aimed at obtaining ecotoxicological information with terrestrial and aquatic plant species to integrate in the risk assessment of this mine area. Further we also intended to evaluate if the assessment of other parameters, in standard assays with terrestrial plants, can improve the identification of phytotoxic soils. For this purpose, soil samples were collected on 16 sampling sites distributed along four transects, defined within the mine area, and in one reference site. General soil physical and chemical parameters, total and extractable metal contents were analyzed. Assays were performed for soil elutriates and for the whole soil matrix following standard guidelines for growth inhibition assay with Lemna minor and emergence and seedling growth assay with Zea mays. At the end of the Z. mays assay, relative water content, membrane permeability, leaf area, content of photosynthetic pigments (chlorophylls and carotenoids), malondialdehyde levels, proline content, and chlorophyll fluorescence (Fv/Fm and ΦPSII) parameters were evaluated. In general, the soils near the exploration area revealed high levels of Al, Mn, Fe and Cu. Almost all the soils from transepts C, D and F presented total concentrations of arsenic well above soils screening benchmark values available. Elutriates of several soils from sampling sites near the exploration and ore treatment areas were toxic to L. minor, suggesting that the retention function of these soils was seriously compromised. In Z. mays assay, plant performance parameters (other than those recommended by standard protocols), allowed the identification of more phytotoxic soils. The results suggest that these parameters could improve the sensitivity of the standard assays.
PMCID: PMC3615127  PMID: 23565165
8.  Is the Interplay between Epigenetic Markers Related to the Acclimation of Cork Oak Plants to High Temperatures? 
PLoS ONE  2013;8(1):e53543.
Trees necessarily experience changes in temperature, requiring efficient short-term strategies that become crucial in environmental change adaptability. DNA methylation and histone posttranslational modifications have been shown to play a key role in both epigenetic control and plant functional status under stress by controlling the functional state of chromatin and gene expression. Cork oak (Quercus suber L.) is a key stone of the Mediterranean region, growing at temperatures of 45°C. This species was subjected to a cumulative temperature increase from 25°C to 55°C under laboratory conditions in order to test the hypothesis that epigenetic code is related to heat stress tolerance. Electrolyte leakage increased after 35°C, but all plants survived to 55°C. DNA methylation and acetylated histone H3 (AcH3) levels were monitored by HPCE (high performance capillary electrophoresis), MS-RAPD (methylation-sensitive random-amplified polymorphic DNA) and Protein Gel Blot analysis and the spatial distribution of the modifications was assessed using a confocal microscope. DNA methylation analysed by HPCE revealed an increase at 55°C, while MS-RAPD results pointed to dynamic methylation-demethylation patterns over stress. Protein Gel Blot showed the abundance index of AcH3 decreasing from 25°C to 45°C. The immunohistochemical detection of 5-mC (5-methyl-2′-deoxycytidine) and AcH3 came upon the previous results. These results indicate that epigenetic mechanisms such as DNA methylation and histone H3 acetylation have opposite and particular dynamics that can be crucial for the stepwise establishment of this species into such high stress (55°C), allowing its acclimation and survival. This is the first report that assesses epigenetic regulation in order to investigate heat tolerance in forest trees.
PMCID: PMC3543447  PMID: 23326451
9.  Cytogenetic characterization and genome size of the medicinal plant Catharanthus roseus (L.) G. Don 
AoB Plants  2012;2012:pls002.
The genome size and organization of the important medicinal plant Catharanthus roseus is shown to correspond to 1C = 0.76 pg (~738 Mbps) and 2n = 16 chromosomes. The data provide a sound basis for future studies including cytogenetic mapping, genomics and breeding.
Background and aims
Catharanthus roseus is a highly valuable medicinal plant producing several terpenoid indole alkaloids (TIAs) with pharmaceutical applications, including the anticancer agents vinblastine and vincristine. Due to the interest in its TIAs, C. roseus is one of the most extensively studied medicinal plants and has become a model species for the study of plant secondary metabolism. However, very little is known about the cytogenetics and genome size of this species, in spite of their importance for breeding programmes, TIA genetics and emerging genomic research. Therefore, the present paper provides a karyotype description and fluorescence in situ hybridization (FISH) data for C. roseus, as well as a rigorous characterization of its genome size.
The organization of C. roseus chromosomes was characterized using several DNA/chromatin staining techniques and FISH of rDNA. Genome size was investigated by flow cytometry using an optimized methodology.
Principal results
The C. roseus full chromosome complement of 2n = 16 includes two metacentric, four subtelocentric and two telocentric chromosome pairs, with the presence of a single nucleolus organizer region in chromosome 6. An easy and reliable flow cytometry protocol for nuclear genome analysis of C. roseus was optimized, and the C-value of this species was estimated to be 1C = 0.76 pg, corresponding to 738 Mbp.
The organization and size of the C. roseus genome were characterized, providing an important basis for future studies of this important medicinal species, including further cytogenetic mapping, genomics, TIA genetics and breeding programmes.
PMCID: PMC3292738  PMID: 22479673
10.  Two New Nuclear Isolation Buffers for Plant DNA Flow Cytometry: A Test with 37 Species 
Annals of Botany  2007;100(4):875-888.
Background and Aims
After the initial boom in the application of flow cytometry in plant sciences in the late 1980s and early 1990s, which was accompanied by development of many nuclear isolation buffers, only a few efforts were made to develop new buffer formulas. In this work, recent data on the performance of nuclear isolation buffers are utilized in order to develop new buffers, general purpose buffer (GPB) and woody plant buffer (WPB), for plant DNA flow cytometry.
GPB and WPB were used to prepare samples for flow cytometric analysis of nuclear DNA content in a set of 37 plant species that included herbaceous and woody taxa with leaf tissues differing in structure and chemical composition. The following parameters of isolated nuclei were assessed: forward and side light scatter, propidium iodide fluorescence, coefficient of variation of DNA peaks, quantity of debris background, and the number of particles released from sample tissue. The nuclear genome size of 30 selected species was also estimated using the buffer that performed better for a given species.
Key Results
In unproblematic species, the use of both buffers resulted in high quality samples. The analysis of samples obtained with GPB usually resulted in histograms of DNA content with higher or similar resolution than those prepared with the WPB. In more recalcitrant tissues, such as those from woody plants, WPB performed better and GPB failed to provide acceptable results in some cases. Improved resolution of DNA content histograms in comparison with previously published buffers was achieved in most of the species analysed.
WPB is a reliable buffer which is also suitable for the analysis of problematic tissues/species. Although GPB failed with some plant species, it provided high-quality DNA histograms in species from which nuclear suspensions are easy to prepare. The results indicate that even with a broad range of species, either GPB or WPB is suitable for preparation of high-quality suspensions of intact nuclei suitable for DNA flow cytometry.
PMCID: PMC2749623  PMID: 17684025
Cytosolic compounds; flow cytometry; general purpose buffer; genome size; lysis buffers; nuclear DNA content; nuclear DNA staining; propidium iodide; woody plant buffer
11.  Distribution of Flower Morphs, Ploidy Level and Sexual Reproduction of the Invasive Weed Oxalis pes-caprae in the Western Area of the Mediterranean Region 
Annals of Botany  2007;99(3):507-517.
Background and Aims
Oxalis pes-caprae is a widespread invasive weed in regions with a Mediterranean climate. In its native habitat (southern Africa) this species has been reported as heterostylous with trimorphic flowers and a self- and morph-incompatible reproductive system. In most of the areas invaded, only a pentaploid short-styled morphotype that reproduces mainly asexually by bulbils is reported, but this has only been confirmed empirically. This study aims to analyse the floral morph proportions in a wide distribution area, test the sexual female success, and explain the causes of low sexual reproduction of this species in the western area of the Mediterranean Basin.
Fifty-five populations of O. pes-caprae were sampled in the Iberian Peninsula and Morocco to evaluate the floral morph ratio and individual fruit set. In plants from a dimorphic population, hand-pollination experiments were performed to evaluate the effect of the pollen source on pollen tube growth through the style. The ploidy level and genome size of individuals of each floral morph were analysed using flow cytometry.
Key Results
From the populations studied 89·1 % were monomorphic, with most of them containing the short-styled (SS) floral morph, and 10·9 % were dimorphic containing long-styled (LS) and SS morphs. In some of these, isoplethy was verified but no fruit production was observed in any population. A sterile form was also recorded in several populations. Hand-pollination experiments revealed that pollen grains germinated over recipient stigmas. In intermorph crossings, pollen tubes were able to develop and fruit initiation was observed in some cases, while in intramorph pollinations, pollen tube development was sporadic and no fruit initiation was observed. All individuals within each floral form presented the same DNA ploidy level: SS plants were pentaploid and LS and the sterile form were tetraploid.
The low or null sexual reproduction success of this species in the area of invasion studied seems related with the high frequency of monomorphic populations, the unequal proportion of floral morphs in dimorphic populations and the presence of different ploidy levels between SS and LS morphs. The discovery of the occurrence of an LS floral morph and a sterile form, whose invading capacity in these areas is as yet unknown, will be valuable information for management programmes.
PMCID: PMC2802954  PMID: 17218342
Flow cytometry; genome size; heterostyly; invasive plant; Oxalis pes-caprae; ploidy level; reproductive biology; weed
12.  Comparison of Four Nuclear Isolation Buffers for Plant DNA Flow Cytometry 
Annals of Botany  2006;98(3):679-689.
• Background and Aims DNA flow cytometry requires preparation of suspensions of intact nuclei, which are stained using a DNA-specific fluorochrome prior to analysis. Various buffer formulas were developed to preserve nuclear integrity, protect DNA from degradation and facilitate its stoichiometric staining. Although nuclear isolation buffers differ considerably in chemical composition, no systematic comparison of their performance has been made until now. This knowledge is required to select the appropriate buffer for a given species and tissue.
• Methods Four common lysis buffers (Galbraith's, LB01, Otto's and Tris.MgCl2) were used to prepare samples from leaf tissues of seven plant species (Sedum burrito, Oxalis pes-caprae, Lycopersicon esculentum, Celtis australis, Pisum sativum, Festuca rothmaleri and Vicia faba). The species were selected to cover a wide range of genome sizes (1·30–26·90 pg per 2C DNA) and a variety of leaf tissue types. The following parameters were assessed: forward (FS) and side (SS) light scatters, fluorescence of propidium iodide-stained nuclei, coefficient of variation of DNA peaks, presence of debris background and the number of nuclei released from sample tissue. The experiments were performed independently by two operators and repeated on three different days.
• Key Results Clear differences among buffers were observed. With the exception of O. pes-caprae, any buffer provided acceptable results for all species. LB01 and Otto's were generally the best buffers, with Otto's buffer providing better results in species with low DNA content. Galbraith's buffer led to satisfactory results and Tris.MgCl2 was generally the worst, although it yielded the best histograms in C. australis. A combined analysis of FS and SS provided a ‘fingerprint’ for each buffer. The variation between days was more significant than the variation between operators.
• Conclusions Each lysis buffer tested responded to a specific problem differently and none of the buffers worked best with all species. These results expand our knowledge on nuclear isolation buffers and will facilitate selection of the most appropriate buffer depending on species, tissue type and the presence of cytosolic compounds interfering with DNA staining.
PMCID: PMC2803574  PMID: 16820407
Angiosperms; flow cytometry; genome size; lysis buffers; nuclear DNA content; nuclear isolation buffers; propidium iodide; stoichiometric error
13.  Flow Cytometric and Microscopic Analysis of the Effect of Tannic Acid on Plant Nuclei and Estimation of DNA Content 
Annals of Botany  2006;98(3):515-527.
• Background and Aims Flow cytometry (FCM) is extensively used to estimate DNA ploidy and genome size in plants. In order to determine nuclear DNA content, nuclei in suspension are stained by a DNA-specific fluorochrome and fluorescence emission is quantified. Recent studies have shown that cytosolic compounds may interfere with binding of fluorochromes to DNA, leading to flawed data. Tannic acid, a common phenolic compound, may be responsible for some of the stoichiometric errors, especially in woody plants. In this study, the effect of tannic acid on estimation of nuclear DNA content was evaluated in Pisum sativum and Zea mays, which were chosen as model species.
• Methods Nuclear suspensions were prepared from P. sativum leaf tissue using four different lysis buffers (Galbraith's, LB01, Otto's and Tris.MgCl2). The suspensions were treated with tannic acid (TA) at 13 different initial concentrations ranging from 0·25 to 3·50 mg mL−1. After propidium iodide (PI) staining, samples were analysed using FCM. In addition to the measurement of nuclei fluorescence, light scatter properties were assessed. Subsequently, a single TA concentration was chosen for each buffer and the effect of incubation time was assessed. Similar analyses were performed on liquid suspensions of P. sativum and Z. mays nuclei that were isolated, treated and analysed simultaneously. FCM analyses were accompanied by microscopic observations of nuclei suspensions.
• Key Results TA affected PI fluorescence and light scatter properties of plant nuclei, regardless of the isolation buffer used. The least pronounced effects of TA were observed in Tris.MgCl2 buffer. Samples obtained using Galbraith's and LB01 buffers were the most affected by this compound. A newly described ‘tannic acid effect’ occurred immediately after the addition of the compound. With the exception of Otto's buffer, nuclei of P. sativum and Z. mays were affected differently, with pea nuclei exhibiting a greater decrease in fluorescence intensity.
• Conclusions A negative effect of a secondary metabolite, TA, on estimation of nuclear DNA content is described and recommendations for minimizing the effect of cytosolic compounds are presented. Alteration in light scattering properties of isolated nuclei can be used as an indicator of the presence of TA, which may cause stoichiometric errors in nuclei staining using a DNA intercalator, PI.
PMCID: PMC2803573  PMID: 16820406
Cytosolic compounds; dye accessibility; genome size; flow cytometry; nuclear DNA content; Pisum sativum; propidium iodide; tannic acid; Zea mays
14.  SP-A binds alpha1-antitrypsin in vitro and reduces the association rate constant for neutrophil elastase 
Respiratory Research  2005;6(1):146.
α1-antitrypsin and surfactant protein-A (SP-A) are major lung defense proteins. With the hypothesis that SP-A could bind α1-antitrypsin, we designed a series of in vitro experiments aimed at investigating the nature and consequences of such an interaction.
Methods and results
At an α1-antitrypsin:SP-A molar ratio of 1:1, the interaction resulted in a calcium-dependent decrease of 84.6% in the association rate constant of α1-antitrypsin for neutrophil elastase. The findings were similar when SP-A was coupled with the Z variant of α1-antitrypsin. The carbohydrate recognition domain of SP-A appeared to be a major determinant of the interaction, by recognizing α1-antitrypsin carbohydrate chains. However, binding of SP-A carbohydrate chains to the α1-antitrypsin amino acid backbone and interaction between carbohydrates of both proteins are also possible. Gel filtration chromatography and turnover per inactivation experiments indicated that one part of SP-A binds several molar parts of α1-antitrypsin.
We conclude that the binding of SP-A to α1-antitrypsin results in a decrease of the inhibition of neutrophil elastase. This interaction could have potential implications in the physiologic regulation of α1-antitrypsin activity, in the pathogenesis of pulmonary emphysema, and in the defense against infectious agents.
PMCID: PMC1343571  PMID: 16351724

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