Francesca Mussi

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.

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.

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.

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.

An intrinsically fluorescent glycoligand for direct imaging of ligand trafficking in artificial and living cell systems

Glycoligands, sugar-based molecules able to complex metal cations, constitute a new class of molecules with great potential for biological and biochemical applications. To analyze their behaviour in a biological environment, we have synthesized an intrinsically fluorescent glycoligand and analyzed its trafficking in both living (U937 human cancer cells) and artificial (giant unilamellar vesicles) cell systems. We have found that this ligand has moderate cytotoxicity accompanied by specific accumulation in both living and reconstituted membranes, which it can cross to reach inner compartments.

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.

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.

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.

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.

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.