Annamaria Buschini

Effects of temperature on baseline and genotoxicant-induced DNA damage in haemocytes of Dreissena polymorpha

The potential application of the Comet assay for monitoring genotoxicity in the freshwater mussel Dreissena polymorpha was explored and a preliminary investigation was undertaken of the baseline levels of DNA damage in mussel haemocytes of animals kept at different temperatures. In addition, in vitro cell sensitivity against genotoxicants was assessed in relation to increasing temperatures. The mussels were kept at four different constant temperatures (4, 18, 28 and 37 degrees C) for 15 h. The haemocytes withdrawn were treated in vitro with melphalan, as a model genotoxic compound, or sodium hypochlorite, a common water disinfectant capable of producing mutagenic/carcinogenic by-products, at the established temperatures for 1h. The data obtained in vivo, in cells directly withdrawn from the mussels showed a significant (P<0.001, Students t test) inter-individual variability, probably due to genetic and epigenetic factors and an increasing amount of DNA damage at increasing temperature. Mussel haemocytes showed a clear dose-response effect after in vitro melphalan treatment. Hypochlorite treatment also significantly increased DNA migration: the damage was temperature dependent, with a similar increase at 4 and 28 degrees C and a minimum level at 18 degrees C. This study demonstrates the potential application of the Comet assay to haemocytes of D. polymorpha. However, these findings suggest that temperature could alter both DNA damage baseline levels in untreated animals and cell sensitivity towards environmental pollutants in in vitro conditions. Therefore, more information is needed about seasonal variations and the natural background levels of DNA damage in mussels living in the wild, before they are used for the monitoring of genotoxic effects in aquatic environments.

Urban airborne particulate: genotoxicity evaluation of different size fractions by mutagenesis tests on microorganisms and comet assay.

The genotoxic effects of different size fractions of airborne particulate (Total, PM10 and PM25), extracted with acetone or toluene, were evaluated by: the Ames plate test (TA98 and TA100 strains, w/o S9), gene conversion and reversion (w/o endogenous metabolic activation) in the Saccharomyces cerevisiae D7 strain, and the comet assay on human leukocytes. The data on human leukocytes confirm the sensitivity of the comet assay and its applicability to assess genotoxicity in environmental samples. The PM2.5 fraction of airborne particulate generally shows the highest concentration of DNA-damaging compounds. Genotoxic response, in all the test systems applied, is highly dependent on extraction solvent used. Acetone seems to extract compounds with more similar genotoxic responses in the three test systems used than toluene extracts. Toluene appears to extract air pollutants genotoxic on yeast and leukocytes but is mainly cytotoxic on Salmonella.

Synthesis, characterization and deepening in the comprehension of the biological action mechanisms of a new nickel complex with antiproliferative activity.

Thiosemicarbazones are versatile organic compounds that present considerable pharmaceutical interest because of a wide range of properties. In our laboratory we synthesised some new metal-complexes with thiosemicarbazones derived from natural aldehydes which showed peculiar biological activities. In particular, a nickel complex [Ni(S-tcitr)(2)] (S-tcitr=S-citronellalthiosemicarbazonate) was observed to induce an antiproliferative effect on U937, a human histiocytic lymphoma cell line, at low concentrations (IC(50)=14.4microM). Therefore, we decided to study the interactions of this molecule with various cellular components and to characterise the induced apoptotic pathway. Results showed that [Ni(S-tcitr)(2)] causes programmed cell death via down-regulation of Bcl-2, alteration of mitochondrial membrane potential and caspase-3 activity, regardless of p53 function. The metal complex is not active on G(0) cells (i.e. fresh leukocytes) but is able to induce perturbation of the cell cycle on stimulated lymphocytes and U937 cells, in which a G(2)/M block was detected. It reaches the nucleus where it induces, at low concentrations (2.5-5.0microM), DNA damage, which could be partially ascribed to oxidative stress. [Ni(S-tcitr)(2)] is moreover able to strongly reduce the telomerase activity. Although the biological target of this metal complex is still unknown, the reported data suggest that [Ni(S-tcitr)(2)] could be a good model for the synthesis of new metal thiosemicarbazones with specific biological activity.

Evaluation of the migration of mutagens/carcinogens from PET bottles into mineral water by Tradescantia/micronuclei test, Comet assay on leukocytes and GC/MS.

This study monitored the release of mutagenic/carcinogenic compounds into mineral water (natural and carbonated) from polyethylene terephthalate (PET) bottles, using a plant mutagenicity test which reveals micronuclei formation in Tradescantia pollen cells (Trad/MCN test), a DNA damage assay (Comet assay) on human leukocytes and gas chromatography/mass spectrometry (GC/MS) for the characterisation of migrants. The water samples were collected at a bottling plant and stored in PET bottles for a period ranging from 1 to 12 months. Every month some samples were randomly collected and lyophilised, the residual powders were extracted with organic solvents and then analysed by GC/MS and tested for DNA damage in human leukocytes, or reconstituted with distilled water to obtain concentrates for the exposure of Tradescantia inflorescences. Micronuclei increase in pollen was found only in natural mineral water stored for 2 months. DNA-damaging activity was found in many of the natural and carbonated water samples. Spring water was negative in the plant micronuclei test and the Comet assay, whereas distributed spring water showed DNA-damaging effects, suggesting a possible introduction of genotoxins through the distribution pipelines. GC/MS analysis showed the presence in mineral water of di(2-ethylhexyl)phthalate, a nongenotoxic hepatocarcinogenic plasticizer, after 9 months of storage in PET bottles.

Bleomycin genotoxicity and amifostine (WR-2721) cell protection in normal leukocytes vs. K562 tumoral cells

Recent advances in chemotherapy have focused on the benefit of high dose regimens, increasing the dose intensity of conventional chemotherapy. However, unacceptable cytotoxicity and genotoxicity on normal cells often impairs the proper management of patients. Phosphoaminothiol WR-1065, the active metabolite of amifostine, appears to protect normal cells and tissues against cytotoxic exposure to radiation or chemotherapeutic agents. Nevertheless, there is disagreement in findings on amifostine protection against bleomycin-induced severe side effects which have suggested that amifostine effectiveness against bleomycin-induced genotoxicity in normal leukocytes and tumour line cells K562 be studied. DNA damage was detected by single cell gel electrophoresis (or Comet) assay, a technique able to detect DNA strand breaks, alkali-labile sites and incomplete excision repair events in individual cells and which appears to be an ideal tool for assessing variability in response of different cell types in vitro. WR-2721 appears to selectively protect healthy leukocytes but not K562 tumoral cells. On the other hand, data on the inter- and intra-individual sensitivity to bleomycin and amifostine suggest that individual metabolic/genetic differences and other factors relating to lifestyle may be responsible for response variability. Application of the Comet assay in appropriate clinical settings to test the sensitivity of patients when undergoing chemotherapy appears possible.

Synthesis, structural characterization and antiproliferative and toxic bio-activities of copper(II) and nickel(II) citronellal N4-ethylmorpholine thiosemicarbazonates

This paper reports the syntheses and characterization of ethylmorpholine substituted citronellal thiosemicarbazone copper(II) and nickel(II) metal complexes. The compounds were characterized through elemental analyses and spectroscopic (IR, UV-Vis, NMR, MS) methods. The X-ray analysis of the two complexes shows that both Ni and Cu derivatives present a square planar coordination, where the coordinating homologous donor atoms bind in trans to each other. The compounds were tested for their biological activity after determination of their octanol-saline partition coefficients, followed by their radical scavenging properties. Eventually the complexes were tested for their proliferation inhibition on human histiocytic lymphoma U937 cell line. The GI(50) values resulted to be 2.3microM for the copper derivative and 12.3microM for the nickel derivative.

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.

Drinking water quality: An in vitro approach for the assessment of cytotoxic and genotoxic load in water sampled along distribution system

An in vitro approach was performed to assess the quality of drinking water collected at two treatment/distribution networks located near the source (Plant #1) and the mouth of River Po (Plant #2). The water was sampled at different points of each distribution network, before (raw water) and after the chlorine dioxide disinfection, and in two points of the pipeline system to evaluate the influence of the distribution system on the amount and quality of the disinfection by-product. Cytotoxicity and genotoxicity of water extracts were evaluated in human peripheral lymphocytes and Hep-G2 cells by the use of the micronucleus (MN) test and Comet assay. Raw water samples of both plants induced cytotoxic effects, but not the increases of MN frequency in Hep-G2 cells and in human lymphocytes. Increases of DNA damage in human leukocytes was detected by Comet assay for raw water of Plant #2 at concentration > or = 0.25 Leq/mL. The disinfection process generally has reduced the toxicity of water samples, even if potential direct DNA-damaging compounds have been detectable in drinking water samples. The proposal approach, if currently used together with chemical analysis, can contribute to improve the monitoring drinking water.

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.

Evaluation of adhesion properties and antibacterial activities of the infant gut commensal Bifidobacterium bifidum PRL2010.

Bifidobacteria are extensively exploited by the food industry as health-promoting microorganisms. However, very little is known about the molecular mechanisms responsible for these beneficial activities, or the molecular players that sustain their ability to colonize and persist within the human gut. Here, we have investigated the enteric adaptation features of the gut commensal Bifidobacterium bifidum PRL2010, originally isolated from infant feces. This strain was able to survive under gastrointestinal challenges, while it was shown to adhere to human epithelial intestinal cell monolayers (Caco 2 and HT-29), thereby inhibiting adhesion of pathogenic bacteria such as Escherichia coli and Cronobacter sakazakii.