Combination of Biochemical, Molecular, and Synchrotron-Radiation-Based Techniques to Study the Effects of Silicon in Tomato (Solanum Lycopersicum L.).

The work focused on the analysis of two cultivars of tomato (Solanum lycopersicum L.), Aragon and Gladis, under two different treatments of silicon, Low, 2 L of 0.1 mM CaSiO3, and High, 0.5 mM CaSiO3, weekly, for 8 weeks, under stress-free conditions. We subsequently analyzed the morphology, chemical composition, and elemental distribution using synchrotron-based µ-XRF techniques, physiological, and molecular aspects of the response of the two cultivars. The scope of the study was to highlight any significant response of the plants to the Si treatments, in comparison with any response to Si of plants under stress. The results demonstrated that the response was mainly cultivar-dependent, also at the level of mitochondrial-dependent oxidative stress, and that it did not differ from the two conditions of treatments. With Si deposited mainly in the cell walls of the cells of fruits, leaves, and roots, the treatments did not elicit many significant changes from the point of view of the total elemental content, the physiological parameters that measured the oxidative stress, and the transcriptomic analyses focalized on genes related to the response to Si. We observed a priming effect of the treatment on the most responsive cultivar, Aragon, in respect to future stress, while in Gladis the Si treatment did not significantly change the measured parameters.

Target proteins reprogrammed by As and As + Si treatments in Solanum lycopersicum L. fruit.

BACKGROUND: Arsenic is an important contaminant of many arable soils worldwide, while silicon, one of the most abundant elements in the earth's crust, interacts with As in the context of plant metabolism. As toxicity results largely from its stimulation of reactive oxygen species, and it is believed that Si can mitigate this process through reduction of the level of oxidative stress. Experiments targeting the proteomic impact of exposure to As and Si have to date largely focused on analyses of root, shoot and seed of a range of mainly non-solanaceous species, thus it remains unclear whether oxidative stress is the most important manifestation of As toxicity in Solanum lycopersicum fruit which during ripening go through drastic physiological and molecular readjustments. The role of Si also needs to be re-evaluated. RESULTS: A comparison was drawn between the proteomic responses to As and As + Si treatments of the fruit of two tomato cultivars (cvs. Aragon and Gladis) known to contrast for their ability to take up these elements and to translocate them into fruits. Treatments were applied at the beginning of the red ripening stage, and the fruit proteomes were captured after a 14 day period of exposure. For each cultivar, a set of differentially abundant fruit proteins (from non-treated and treated plants) were isolated by 2DGE and identified using mass spectrometry. In the fruit of cv. Aragon, the As treatment reprogrammed proteins largely involved in transcription regulation (growth- regulating factor 9-like), and cell structure (actin-51), while in the cv. Gladis, the majority of differentially expressed proteins were associated with protein ubiquitination and proteolysis (E3 ubiquitin protein, and hormones (1-aminocyclopropane 1-carboxylase). CONCLUSIONS: The present experiments were intended to establish whether Si supplementation can be used to reverse the proteomic disturbance induced by the As treatment; this reprogram was only partial and more effective in the fruit of cv. Gladis than in that of cv. Aragon. Proteins responsible for the protection of the fruits' quality in the face of As-induced stress were identified. Moreover, supplementation with Si seemed to limit to a degree the accumulation of As in the tomato fruit of cv. Aragon.

Micronuclei and chromosome aberrations in subjects occupationally exposed to antineoplastic drugs: a multicentric approach.

OBJECTIVES: Recently published works showed that occupational exposure to antineoplastic drugs (ANPD) is still frequent in hospital settings, despite significant safety policy improvements. The aim of this study was to assess the current level of occupational exposure to ANPD and any potentially associated cytogenetic damages in hospital nurses routinely handling ANPD. METHODS: Occupationally ANPD-exposed (n = 71) and ANPD-unexposed (n = 77; control) nurses were recruited on a voluntary basis from five hospitals in Northern and Central Italy. Evaluation of surface contamination and dermal exposure to ANPD was assessed by determining cyclophosphamide (CP) on selected surfaces (wipes) and on exposed nurses' clothes (pads). The concentration of unmetabolized CP­as a biomarker of internal dose­was measured in end-shift urine samples. Biomonitoring of genotoxic effects (i.e., biological effect monitoring) was conducted by analyzing micronuclei (MN) and chromosome aberrations (CA) in peripheral blood lymphocytes. Genetic polymorphisms for enzymes involved in metabolic detoxification (i.e., glutathione S-transferases) were analyzed as well. RESULTS: We observed a significant increase in MN frequency (5.30 ± 2.99 and 3.29 ± 1.97; mean values ± standard deviation; p < 0.0001) in exposed nurses versus controls, as well as in CA detection (3.30 ± 2.05 and 1.84 ± 1.67; p < 0.0001), exposed subjects versus controls. Our results provide evidence that, despite safety controlled conditions, ANPD handling still represents a considerable genotoxic risk for occupationally exposed personnel. CONCLUSIONS: Because both MN and CA have been described as being predictive of group-increased cancer risk, our findings point to a need for improving specific safety procedures in handling and administering ANPD.

Assessment of antioxidant and antiproliferative properties of spinach plants grown under low oxygen availability.

BACKGROUND: In the human diet, the consumption of fresh fruits and vegetables is important in maintaining good health and in preventing chronic diseases. It is known that plant-derived food is a powerful source of chemopreventive molecules, i.e. antioxidants, and spinach (Spinacia oleracea L., Chenopodiaceae) possesses a wide range of metabolites with such biological activity. Plant stress response could lead to the production of metabolites with high value for human health and this could be a tool to enhance the production of molecules with antioxidant activity in plants. RESULTS: Data reported in this paper confirm the antioxidant properties of spinach plants, and show a strong antiproliferative activity of leaf extract on HT-29 human cell line. Besides, the hypoxic stress seems to affect the pool of antioxidant molecules present in spinach leaves, as verified by means of HPLC-MS/MS analysis and the aluminium chloride and ABTS assays. CONCLUSION: Our findings represent a basis for improving the biological and pharmacological properties of spinach plants, including the use of different growth conditions to modulate the phytocomplex profile of spinach.

Multicentre study for the evaluation of mutagenic/carcinogenic risk in nurses exposed to antineoplastic drugs: assessment of DNA damage.

OBJECTIVES: People who handle antineoplastic drugs, many of which classified as human carcinogens by International Agency for Research on Cancer, are exposed to low doses in comparison with patients; however, the long duration of exposure could lead to health effects. The aim of this work was to evaluate DNA damage in white blood cells from 63 nurses who handle antineoplastic drugs in five Italian hospitals and 74 control participants, using different versions of the Comet assay. METHODS: Primary DNA damage was assessed by using the alkaline version of the assay on leucocytes, whereas to detect DNA oxidative damage and cryptic lesions specifically, the Comet/ENDO III assay and the Comet/araC assay were performed on leucocytes and lymphocytes, respectively. RESULTS: In the present study, no significant DNA damage was correlated with the work shift. The exposed population did not differ significantly from the reference group with respect to DNA primary and oxidative damage in leucocytes. Strikingly, in isolated lymphocytes treated with araC, lower data dispersion as well as a significantly lower mean value for the percentage of DNA in the comet tail was observed in exposed participants as compared with the control group (p<0.05), suggesting a potential chronic exposure to crosslinking antineoplastic drugs. CONCLUSIONS: Although stringent rules were adopted at national and international levels to prevent occupational exposure to antineoplastic drugs, data reported in this study support the idea that a more efficient survey on long-lasting exposures at very low concentrations is needed.

Evaluation of DNA damage induced by 2 polybrominated diphenyl ether flame retardants (BDE-47 and BDE-209) in SK-N-MC cells.

Polybrominated diphenyl ethers (PBDEs) are a class of flame retardants whose levels have increased in the environment and in human tissues in the past decades. Exposure to PBDEs has been associated with developmental neurotoxicity, endocrine dysfunction, and reproductive disorders. In spite of their widespread distribution and potential adverse health effects, only few studies have addressed the potential neurotoxicity of PBDEs. In the present study, we evaluated the cyto- and genotoxicity of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) and decabrominated diphenyl ether (BDE-209) in human neuroblastoma cells (SK-N-MC). The DNA damage was measured using the alkaline version of the Comet assay, while specific oxidative-generated DNA damage was evaluated by a modified version of the Comet assay with the repair enzyme formamidopyrimidine glycosylase (FPG). The results show that BDE-47 and BDE-209 (5-20 µmol/L) are able to induce DNA damage in human SK-N-MC cells. Pretreatment with the antioxidant melatonin significantly reduced the DNA damage induced by both congeners. The Comet assay carried out in the presence of FPG suggests that both congeners increase purine oxidation. In all cases, BDE-47 was more potent than BDE-209. The results indicate that 2 environmentally relevant PBDEs cause DNA damage which is primarily mediated by the induction of oxidative stress and may contribute to adverse health effects.

A study protocol for the evaluation of occupational mutagenic/carcinogenic risks in subjects exposed to antineoplastic drugs: a multicentric project.

BACKGROUND: Some industrial hygiene studies have assessed occupational exposure to antineoplastic drugs; other epidemiological investigations have detected various toxicological effects in exposure groups labeled with the job title. In no research has the same population been studied both environmentally and epidemiologically. The protocol of the epidemiological study presented here uses an integrated environmental and biological monitoring approach. The aim is to assess in hospital nurses preparing and/or administering therapy to cancer patients the current level of occupational exposure to antineoplastic drugs, DNA and chromosome damage as cancer predictive effects, and the association between the two. METHODS/DESIGN: About 80 healthy non-smoking female nurses, who job it is to prepare or handle antineoplastic drugs, and a reference group of about 80 healthy non-smoking female nurses not occupationally exposed to chemicals will be examined simultaneously in a cross-sectional study. All the workers will be recruited from five hospitals in northern and central Italy after their informed consent has been obtained.Evaluation of surface contamination and dermal exposure to antineoplastic drugs will be assessed by determining cyclophosphamide on selected surfaces (wipes) and on the exposed nurses' clothes (pads). The concentration of unmetabolized cyclophosphamide as a biomarker of internal dose will be measured in end-shift urine samples from exposed nurses. Biomarkers of effect and susceptibility will be assessed in exposed and unexposed nurses: urinary concentration of 8-hydroxy-2-deoxyguanosine; DNA damage detected using the single-cell microgel electrophoresis (comet) assay in peripheral white blood cells; micronuclei and chromosome aberrations in peripheral blood lymphocytes. Genetic polymorphisms for enzymes involved in metabolic detoxification (i.e. glutathione S-transferases) will also be analysed.Using standardized questionnaires, occupational exposure will be determined in exposed nurses only, whereas potential confounders (medicine consumption, lifestyle habits, diet and other non-occupational exposures) will be assessed in both groups of hospital workers.Statistical analysis will be performed to ascertain the association between occupational exposure to antineoplastic drugs and biomarkers of DNA and chromosome damage, after taking into account the effects of individual genetic susceptibility, and the presence of confounding exposures. DISCUSSION: The findings of the study will be useful in updating prevention procedures for handling antineoplastic drugs.

Biological Effect of Different Spinach Extracts in Comparison with the Individual Components of the Phytocomplex.

The Mediterranean-style diet is rich in fruit and vegetables and has a great impact on the prevention of major chronic diseases, such as cardiovascular diseases and cancer. In this work we investigated the ability of spinach extracts obtained by different extraction methods and of the single main components of the phytocomplex, alone or mixed, to modulate proliferation, antioxidant defense, and genotoxicity of HT29 human colorectal cells. Spinach extracts show dose-dependent activity, increasing the level of intracellular endogenous reactive oxygen species (ROS) when tested at higher doses. In the presence of oxidative stress, the activity is related to the oxidizing agent involved (H2O2 or menadione) and by the extraction method. The single components of the phytocomplex, alone or mixed, do not alter the intracellular endogenous level of ROS but again, in the presence of an oxidative insult, the modulation of antioxidant defense depends on the oxidizing agent used. The application of the phytocomplex extracts seem to be more effective than the application of the single phytocomplex components.

Cadmium sulfide quantum dots impact Arabidopsis thaliana physiology and morphology.

The differential mechanisms of CdS QDs (Quantum Dots) and Cd ion toxicity to Arabidopsis thaliana (L.) Heynh were investigated. Plants were exposed to 40 and 60 mg L-1 for CdS QDs and 76.9 and 115.2 mg L-1 CdSO4·7H2O and toxicity was evaluated at 5, 20, 35 (T5, T20, T35) days after exposure. Oxidative stress upon exposure was evaluated by biochemical essays targeting non-enzymatic oxidative stress physiological parameters, including respiration efficiency, total chlorophylls, carotenoids, ABTS and DPPH radicals reduction, total phenolics, GSH redox state, lipid peroxidation. Total Cd in plants was measured with AAS. Root and leaf morphology and element content were assessed in vivo utilizing low-vacuum Environmental Scanning Electron Microscopy (ESEM) with X-ray microanalysis (EDX). This integrated approach allowed identification of unique nanoscale CdS QDs toxicity to the plants that was distinct from CdSO4 exposure. The analyses highlighted that CdS QDs and Cd ions effects are modulated by the developmental stage of the plant, starting from T20 till T35 the plant development was modulated by the treatments, in particular CdS QDs induced early flowering. Both treatments induced Fe accumulation in roots, but at different intensities, while CdS QDs was associated with Mn increase into plant leaf. CdSO4 elicited higher levels of oxidative stress compared with QDs, especially the former treatment caused more intense respiration damages and reduction in chlorophyll and carotenoids than the latter. The two types of treatments impact differently on root and leaf morphology.

Oxygen Availability during Growth Modulates the Phytochemical Profile and the Chemo-Protective Properties of Spinach Juice.

Fruits and vegetables are a good source of potentially biologically active compounds. Their regular consumption in the human diet can help reduce the risk of developing chronic diseases such as cardiovascular diseases and cancer. Plants produce additional chemical substances when subject to abiotic stress or infected by microorganisms. The phytochemical profile of spinach leaves (Spinaciaoleracea L.), which is a vegetable with widely recognized health-promoting activity, has been affected by applying root hypoxic and re-oxygenation stress during plant growth. Leaf juice at different sampling times has been subject to liquid chromatography mass spectrometry (LC-MSn) analysis and tested on the human colorectal adenocarcinoma cell line HT29 by using the Comet assay. The cells were previously treated with H2O2 to simulate the presence of an oxidative stress (as in colon cancer condition) and the leaf juice application resulted in a significant antioxidant and protective in vitro effect. The duration of the hypoxic/re-oxygenation stress imposed on the plant reflects the antioxidant leaf juice content. After hypoxic stress (24 hours) and reoxygenation (2 hours), we show a decrease (50%) of the relative abundance of the principal identified antioxidant molecules but a higher antioxidant activity of the spinach juice on HT29 cells (20%). Data shows a complex relation between plant growing conditions and the modulation of secondary metabolites content in leaf juice that results in different chemo-protective activities in colon cancer cells.

Structural and Functional Features of Chars From Different Biomasses as Potential Plant Amendments.

Biochars result from the pyrolysis of biomass waste of plant and animal origin. The interest in these materials stems from their potential for improving soil quality due to increased microporosity, carbon pool, water retention, and their active capacity for metal adsorption from soil and irrigation water. Applications in agriculture have been studied under different conditions, but the overall results are still unclear. Char structure, which varies widely according to the pyrolysis process and the nature of feedstock, is thought to be a major factor in the interaction of chars with soil and their metal ion adsorption/chelation properties. Furthermore, biochar nutrients and their elemental content can modify soil fertility. Therefore, the use of biochars in agricultural settings should be examined carefully by conducting experimental trials. Three key problems encountered in the use of biochar involve (i) optimizing pyrolysis for biomass conversion into energy and biochar, (ii) physicochemically characterizing biochar, and (iii) identifying the best possible conditions for biochar use in soil improvement. To investigate these issues, two types of wood pellets, plus digestate and poultry litter, were separately converted into biochar using different technologies: pyrolysis/pyrogasification or catalytic (thermo)reforming. The following physicochemical features for the different biochar batches were measured: pH, conductivity, bulk density, humidity and ash content, particle size, total organic substances, and trace element concentrations. Fine porous structure analysis and total elemental analysis were performed using environmental scanning electron microscopy along with energy-dispersive X-ray spectrometry (EDX). Phytotoxicity tests were performed for each biochar. Finally, we were able to (i) differentiate the biochars according to their physicochemical properties, microstructure, elemental contents, and original raw biomass; (ii) correlate the whole biochar features with their respective optimal concentrations when used as plant fertilizers or soil improvers; and (iii) show that biochars from animal origin were phytotoxic at lower concentrations than those from plant feedstock.

Effects of polar substituents on the biological activity of thiosemicarbazone metal complexes.

In this paper, citronellal, vanillin and pyridoxal thiosemicarbazones were modified with polar substituents, namely ethylmorpholine and glucose, to increase their polarity and compare the effects of these moieties on their biological activity. Altogether, nine ligands were synthesized and for each of them also their copper(II) and nickel(II) complexes were prepared and used for the biological tests. Eventually, assays on proliferation inhibition were conducted using leukemic cell line U937, already used as a model for previous citronellal thiosemicarbazone tests. Biological tests were also performed on solid tumor cell line HT29. From the first screenings, two of the metal complexes showed remarkable interesting properties, and, therefore, were also tested for histosensitivity.

Abiotic Stress Response to As and As+Si, Composite Reprogramming of Fruit Metabolites in Tomato Cultivars.

The toxic element arsenic interacts with the beneficial element silicon at many levels of the plant metabolism. The ability of the tomato plant to take up and translocate As into its fruit has risen concerns that it could facilitate the entry of this element into the human food chain above the admitted level. Here, the fruit of two contrasting tomato cultivars, Aragon and Gladis, were evaluated following exposures of either 48 h or 14 days to As-contaminated irrigation water, with or without supplementary Si. The focus was on selected biochemical stress response indicators to dissect metabolic fruit reprogramming induced by As and Si. A multivariate statistical approach was utilized to establish the relationship between tissue As and Si concentrations and selected biochemical aspects of the stress response mechanisms to identify a set of relevant stress response descriptors. This resulted in the recognition of strong cultivar and temporal effects on metabolic and biochemical stress parameters following the treatments. In this paper the metabolic changes in H2O2 content, lipid peroxidation, lycopene and carotenoids content, ascorbate and GSH redox state, total phenolics, ABTS and DPPH radicals inhibition were in favor of an oxidative stress. The significance of some of these parameters as reliable arsenic exposition biomarkers is discussed in the context of the limited knowledge on the As-induced stress response mechanisms at the level of the ripening fruit which presents a distinctive molecular background dissimilar from roots and shoots.

In vitro evaluation of the activity of thiosemicarbazone derivatives against mycotoxigenic fungi affecting cereals.

With a steadily increasing world population, a more efficient system of food production is of paramount importance. One of the major causes of food spoilage is the presence of fungal pathogens and the production and accumulation of mycotoxins. In the present work we report a study on the activity of a series of functionalized thiosemicarbazones (namely cuminaldehyde, trans-cinnamaldehyde, quinoline-2-carboxyaldehyde, 5-fluoroisatin thiosemicarbazone and 5-fluoroisatin N(4)-methylthiosemicarbazone), as antifungal and anti-mycotoxin agents, against the two major genera of cereal mycotoxigenic fungi, i.e. Fusarium and Aspergillus. These thiosemicarbazones display different patterns of efficacy on fungal growth and on mycotoxin accumulation depending on the fungal species. Some of the molecules display a greater effect on mycotoxin synthesis than on fungal growth.

Unravelling mechanisms behind the biological activity of bis(S-citronellalthiosemicarbazonato)nickel(II).

Bis(S-citronellalthiosemicarbazonato)nickel(II), [Ni(tcitr)2], is a compound that inhibits proliferation of tumour line U937 by inducing a G2/M block and leading the cancer cells to apoptosis. This nickel derivative shows no activity on non proliferating healthy cells. In this paper we report our studies on the action mechanisms of [Ni(tcitr)2]. Apoptosis in U937 cells exposed to [Ni(tcitr)2] takes place through activation of caspase-9, and therefore through an intrinsic triggering mechanism. Given the DNA damage observed in the Comet assay, the mutagenic activity of the metal complex was tested, including with the Ames test, micronuclei and DNA damage recovery, but neither mutagenicity nor recovery were detected. Nickel-complex-DNA interactions were analyzed by direct action of the compound on plasmidic and linear DNA by UV-vis and CD spectroscopy, gel electrophoresis and Atomic Force Microscopy. These experiments reveal that [Ni(tcitr)2] does not cause DNA breaks and does not intercalate, but significantly alters the DNA conformation creating knot-like structures and hairpins.

Apo-neocarzinostatin: a protein carrier for Cu(II) glycocomplexes and Cu(II) into U937 and HT29 cell lines.

In the field of pharmaceuticals there is an increasing need for new delivery systems to overcome the issues of solubility, penetration, toxicity and drug resistance. One of the possible strategies is to use biocarriers such as proteins to encourage the cell-penetration of drugs. In this paper, the use of the apo-protein neocarzinostatin (apo-NCS) as a carrier-protein for two Cu(II) glycocomplexes, previously characterized, and Cu(II) ions was investigated. Its interaction with the metallic compounds was analyzed using microcalorimetry. The dissociation constants were shown to be in the micromolar range. The Cu(II) glycocomplexes, in absence of apo-NCS, were found to be cytotoxic in the U937 and HT29 cell lines whereas the corresponding glycoligands showed no toxicity. The leukemic cell line (U937) seems to be more sensitive to glycocomplexes than the colon cancer cell line (HT29). Interestingly, apo-NCS was shown to increase systematically the antiproliferative activity by a factor of 2 and 3 for Cu(II) glycocomplexes and Cu(II) respectively. The antiproliferative activity detected was not related to proteasome inhibition. This result stresses the importance of new molecular tools for the delivery of Cu(II) to tumor cells using non-covalent association with carriers proteins.

Copper(II) thiosemicarbazonate molecular modifications modulate apoptotic and oxidative effects on U937 cell line.

To improve the solubility in aqueous media of bis(citronellalthiosemicarbazonato)copper(II) [Cu(S-tcitr)(2)], a compound that is effective in inhibiting cell growth of U937 cell line, the ligand was modified adding an ethylmorpholine group. [Cu(S-tcitr)(2)] and [Cu(Etmorph-S-tcitr)(2)] cytotoxic effects are compared using as a model U937 cells. [Cu(Etmorph-S-tcitr)(2)] results more effective in cell growth inhibition (IC(50:) 2.3 vs 14.8 µM). Apoptosis in [Cu(Etmorph-S-tcitr)(2)] treated cells was apparent after 8h, with increased caspase activities, and these effects were not observed for [Cu(S-tcitr)(2)]. During the exposure to [Cu(Etmorph-S-tcitr)(2)], ROS (reactive oxygen species) and TBARS (Thiobarbituric acid reactive substances) significantly increased, coupled with reduced glutathione (GSH) levels and significant activation of superoxide dismutase (SOD). These intracellular scavengers seem to limit the early ROS and TBARS increases in U937 cells exposed to [Cu(S-tcitr)(2)]. Both complexes interacted in vitro with naked DNA: UV-visible and CD titration reveal that they can induce DNA structure modifications in a distinct way. Furthermore, the complexes induced DNA damage on U937 cells at concentrations higher than IC(50). The mechanisms of action and the effects of these two complexes are remarkably different even though they have the same coordination geometry around copper(II) and differ only for the presence of the ethylmorpholine group.

Genotoxicity revaluation of three commercial nitroheterocyclic drugs: nifurtimox, benznidazole, and metronidazole.

Nitroheterocyclic compounds are widely used as therapeutic agents against a variety of protozoan and bacterial infections. However, the literature on these compounds, suspected of being carcinogens, is widely controversial. In this study, cytotoxic and genotoxic potential of three drugs, Nifurtimox (NFX), Benznidazole (BNZ), and Metronidazole (MTZ) was re-evaluated by different assays. Only NFX reduces survival rate in actively proliferating cells. The compounds are more active for base-pair substitution than frameshift induction in Salmonella; NFX and BNZ are more mutagenic than MTZ; they are widely dependent from nitroreduction whereas microsomal fraction S9 weakly affects the mutagenic potential. Comet assay detects BNZ- and NFX-induced DNA damage at doses in the range of therapeutically treated patient plasma concentration; BNZ seems to mainly act through ROS generation whereas a dose-dependent mechanism of DNA damaging is suggested for NFX. The lack of effects on mammalian cells for MTZ is confirmed also in MN assay whereas MN induction is observed for NFX and BNZ. The effects of MTZ, that shows comparatively low reduction potential, seem to be strictly dependent on anaerobic/hypoxic conditions. Both NFX and BNZ may not only lead to cellular damage of the infective agent but also interact with the DNA of mammalian cells.