Genotoxicity Evaluation of the Soybean Isoflavone Genistein in Human Papillary Thyroid Cancer Cells. Study of Its Potential Use in Thyroid Cancer Therapy.

Genistein is one of the several known isoflavonic phytoestrogens found in a number of plants, with soybeans and soy products being the primary food source. The aim of the study is to evaluate if genistein is able to exert antineoplastic action in primary human papillary thyroid cancer (PTC) cells. Thyroid tissues were treated with genistein (1-10-50-100 µM). Cell viability, proliferation, DNA primary damage and chromosomal damage were evaluated. An antiproliferative effect was induced by the highest doses of genistein, and such an effect was synergistically enhanced by the cotreatment with the antineoplastic drug sorafenib. Comet assay did not show any genotoxic effect in terms of primary DNA damage at all the times (4 and 24 h) and tested doses. A reduction of hydrogen peroxide-induced DNA primary damage in primary thyrocytes from PTC cells pretreated with genistein was observed. Data suggest that genistein exerts antineoplastic action, does not induce genotoxic effects while reduces oxidative-induced DNA damage in primary thyrocytes from PTC cells, supporting its possible use in therapeutic intervention.

n-TiO2 and CdCl2 co-exposure to titanium dioxide nanoparticles and cadmium: Genomic, DNA and chromosomal damage evaluation in the marine fish European sea bass (Dicentrarchus labrax).

Due to the large production and growing use of titanium dioxide nanoparticles (n-TiO2), their release in the marine environment and their potential interaction with existing toxic contaminants represent a growing concern for biota. Different end-points of genotoxicity were investigated in the European sea bass Dicentrarchus labrax exposed to n-TiO2 (1mgL(-1)) either alone and combined with CdCl2 (0.1mgL(-1)) for 7 days. DNA primary damage (comet assay), apoptotic cells (diffusion assay), occurrence of micronuclei and nuclear abnormalities (cytome assay) were assessed in peripheral erythrocytes and genomic stability (random amplified polymorphism DNA-PCR, RAPD assay) in muscle tissue. Results showed that genome template stability was reduced after CdCl2 and n-TiO2 exposure. Exposure to n-TiO2 alone was responsible for chromosomal alteration but ineffective in terms of DNA damage; while the opposite was observed in CdCl2 exposed specimens. Co-exposure apparently prevents the chromosomal damage and leads to a partial recovery of the genome template stability.

Does the crystal habit modulate the genotoxic potential of silica particles? A cytogenetic evaluation in human and murine cell lines.

Crystalline silica inhaled from occupational sources has been classified by IARC as carcinogenic to humans; in contrast, for amorphous silica, epidemiological and experimental evidence remains insufficient. The genotoxicity of crystalline silica is still debated because of the inconsistency of experimental results ("variability of silica hazard"), often related to the features of the particle surfaces. We have assessed the role of crystal habit in the genotoxicity of silica powders. Pure quartz (crystalline) and vitreous silica (amorphous), sharing the same surface features, were used in an in vitro study with human pulmonary epithelial (A549) and murine macrophage (RAW264.7) cell lines, representative of occupational and environmental exposures. Genotoxicity was evaluated by the comet and micronucleus assays, and cytotoxicity by the trypan blue method. Cells were treated with silica powders for 4 and 24h. Quartz but not vitreous silica caused cell death and DNA damage in RAW264.7 cells. A549 cells were relatively resistant to both powders. Our results support the view that crystal habit per se plays a pivotal role in modulating the biological responses to silica particles.

Genomic and chromosomal damage in the marine mussel Mytilus galloprovincialis: Effects of the combined exposure to titanium dioxide nanoparticles and cadmium chloride.

Titanium dioxide nanoparticles (TiO2-NPs) continuously released into waters, may cause harmful effects to marine organisms and their potential interaction with conventional toxic contaminants represents a growing concern for biota. We investigated the genotoxic potential of nanosized titanium dioxide (n-TiO2) (100 µg L(-1)) alone and in combination with CdCl2 (100 µg L(-1)) in Mytilus galloprovincialis after 4 days of in vivo exposure. RAPD-PCR technique and Micronucleus test were used to study genotoxicity. The results showed genome template stability (GTS) being markedly reduced after single exposure to n-TiO2 and CdCl2. Otherwise, co-exposure resulted in a milder reduction of GTS. Exposure to n-TiO2 was responsible for a significant increase of micronucleated cell frequency in gill tissue, while no chromosomal damage was observed after CdCl2 exposure as well as after combined exposure to both substances.

Cellular responses in the cyprinid Leuciscus cephalus from a contaminated freshwater ecosystem.

The response of wild chubs (Leuciscus cephalus) to chemical pollution was assessed in a metal contaminated river (Cecina River, Italy) through a wide battery of biomarkers which included: Comet assay detecting DNA strand breaks; diffusion assay for apoptosis induction; micronucleus test assessing chromosomal alterations; ethoxyresorufin O-deethylase (EROD) activity for the induction of cytochrome P 4501A; acetylcholinesterase (AChE) activity responsive to pesticide exposure; vitellogenin gene expression in males revealing estrogenic effects. Bioaccumulation of mercury, chromium and polycyclic aromatic hydrocarbons (PAHs) was also determined. Levels of mercury and PAHs were higher in tissues of chubs sampled from the most downstream station, reflecting an anthropogenic pollution of industrial origin. Otherwise, accumulation of Cr was quite similar in fish along the entire course of Cecina River confirming a natural origin due to local geochemical features. Biomarker responses revealed a significant increase of apoptotic cells, DNA stand breaks and micronucleus frequency in chubs from the more impacted sites. A slight EROD induction and AChE inhibition were only seen at the most downstream station demonstrating a limited impact due to PAHs and pesticides. On the other hand, the induction of vitellogenin gene in male chubs was measured in all the sites, suggesting a diffuse estrogenic effect. This study confirmed the utility of large batteries of biomarkers in biomonitoring studies and the suitability of wild chub as bioindicator organism for river basins.

Effects of algal extracts (Polysiphonia fucoides) on rainbow trout (Oncorhynchus mykiss): a biomarker approach.

The genotoxicity of algal extracts (Polysiphonia fucoides) was investigated in erythrocytes of rainbow trout (Oncorhynchus mykiss). Trout were exposed to 0.5% of the algal extract for 7 days. Comet assay (alkaline and neutral versions) and Micronucleus test were used to assess DNA damage, and Diffusion Assay to detect apoptotic cells. EROD activities and oxidative stress parameters in rainbow trout liver were also measured. A significant induction of DNA single strand breaks comparable to the ones induced by the in vivo exposure to 20 mg/kg B[a]P was observed at the end of the treatment, while increases of double strand breaks and apoptotic cells were not observed. The absence of activation of antioxidant responses seems to underline a mechanism of action of the genotoxic algal extract which does not involve oxidative stress.

Cellular biomarkers for monitoring estuarine environments: transplanted versus native mussels.

In developed countries, estuarine environments are often subjected to chemical pollution, whose biological impact is profitably evaluated by the use of multi-biomarker approaches on sentinel species. In this paper, we investigate genotoxicity and lysosomal alterations in the Mediterranean mussel (Mytilus galloprovincialis), from the estuary of the River Cecina (Tuscany, Italy), selected as "pilot basin" within the Water Frame Directive (2000/60 European Community). Both native and 1 month transplanted mussels were used in order to compare these two approaches in terms of sensitiveness of specific biomarker responses. Genotoxic effects were evaluated as strand breaks, by single cell gel electrophoresis (or Comet assay), and as chromosomal alterations, by the micronucleus test in gill cells. Lysosomal alterations were assessed by the neutral red retention time (in haemocytes), lipofuscin accumulation and ultrastructure (in digestive cells). Heavy metal bioaccumulation was also analysed. Mussels from the River Cecina showed a general alteration of all the biomarkers investigated, accompanied by an elevation of tissue metal levels. However, some differences in specific responses occurred between transplanted and native mussels. Early biomarkers, such as those based on DNA and lysosomal membrane integrity, were induced at similar degree in native and transplanted mussels; while alterations resulting from cumulative events, as the increase of micronuclei frequency were much more elevated in native specimens (23.1+/-7.6) than in transplanted (9.3+/-4.7) and reference ones (5.8+/-5.2). Similarly, the comparison between lipofuscin accumulation and mean lysosomal diameter in impacted and control sites, gave significant differences exclusively with transplanted mussels. These results suggest that the parallel use of caged and native mussels in environmental biomonitoring can improve the characterization of the study area.

Induction of DNA strand breakage and apoptosis in the eel Anguilla anguilla.

The ability of benzo[a]pyrene, Aroclor 1254, 2-3-7-8-tetrachlorodibenzo-p-dioxin and beta-naphthoflavone to induce DNA strand breaks (SB) and apoptosis in erythrocytes of the European eel (Anguilla anguilla) was investigated following by in vivo exposure. DNA damage was evaluated by the Comet assay, while the diffusion assay was used to investigate the induction of apoptosis 7 days after a single intraperitoneal administration. 2-3-7-8-Tetrachlorodibenzo-p-dioxin induced the highest genotoxic effect, followed by benzo[a]pyrene, while the other two substances had limited effects. A significant induction of apoptosis was observed at the highest doses after exposure to benzo[a]pyrene, when DNA damage was also elevated. The occurrence of apoptotic cells after exposure to Aroclor, 2-3-7-8-tetrachlorodibenzo-p-dioxin and beta-naphthoflavone was quite variable and did not show clear dose-related responses. The role of oxidative stress in mediating DNA damage was also discussed.

Vitellin polypeptide pathways in late insect yolk sacs.

A panel of monoclonal antibodies was raised against late yolk sacs of the stick insect Carausius morosus and tested by immunoblotting to establish the extent vitellin polypeptides are processed proteolytically during embryonic development. Cryosections of late yolk sacs were also examined by confocal laser microscopy to determine how vitellin cleavage products become spatially distributed amongst yolk granules during the same developmental period. Distinct labelling patterns were obtained on yolk granules depending on: (1) the nature of the proteolytic processing; (2) the origin of vitellin cleavage products; and ultimately (3) their molecular sizes. Monoclonal antibodies raised against vitellin cleavage products resulting from proteolytic processing appeared to label: (1) the entire volume of many yolk granules; (2) their limiting membrane; or (3) a number of small vesicles interposed between larger yolk granules. On the other hand, monoclonal antibodies against vitellin cleavage products that remain invariant throughout development appeared to label either the serosa membrane or the cytosolic space comprised between adjacent yolk granules. Data are interpreted as indicating that vitellin cleavage products may leak out from the yolk granules, gain access to the cytosolic space of the vitellophages and eventually percolate through the serosa membrane enclosing the yolk sac.

Genotoxic hazard of pollutants in cetaceans: DNA damage and repair evaluated in the bottlenose dolphin (Tursiops truncatus) by the Comet Assay.

Single cell gel electrophoresis (or Comet Assay) was used for evaluation of the in vitro genotoxicity of hydrogen peroxide (used as a positive control), polychlorinated biphenyls (PCBs) (Aroclor 1254) and methyl mercury chloride, in isolated bottlenose dolphin leukocytes. Results showed that hydrogen peroxide and methyl mercury induced DNA strand breakage in a dose-dependent manner, while PCBs did not induce a clear dose-effect response at the low doses investigated. Efficiency in repairing DNA breakage induced by methyl mercury was also evaluated. Findings demonstrated that dolphin cells are characterized by higher efficiency in DNA repair when compared to human leukocytes. The observed resistance to methyl mercury toxicity in dolphins was hypothesized to be a defence strategy developed to combat high dietary exposure and compensate for limited capacity to excrete persistent pollutants.

DNA integrity and total oxyradical scavenging capacity in the Mediterranean mussel, Mytilus galloprovincialis: a field study in a highly eutrophicated coastal lagoon.

In Mediterranean coastal lagoons, the combination of human impact and wide variability of natural environmental factors can lead to upsets in ecosystem homeostasis resulting in biodiversity decline. Oxidative damage has been causally linked to various kinds of environmental stress, both natural and artificial, the result being impairment of cellular functions. DNA damage and the efficiency of antioxidant defences in Mytilus galloprovincialis from the highly eutrophicated Orbetello Lagoon (Tuscany, Italy) were investigated, respectively by the single cell gel electrophoresis (or Comet test) and the total oxyradical scavenging capacity assay. Results showed significantly higher levels of DNA damage in mussels collected from the inner parts of the lagoon compared to specimens from more external sites. Specimens with the lower genetic integrity also exhibited a reduced efficiency in neutralizing three potent cellular oxidizing species, namely peroxyl radicals (ROO*), hydroxyl radicals (*OH) and peroxynitrite (HOONO), suggesting the involvement of reactive oxygen species in mediating the genetic damage. The analyzed biological parameters also showed a seasonal variability with a minimum of both DNA integrity and antioxidant scavenging efficiency during the warm months and an opposite trend in winter. The potential of analyzed techniques is discussed for the assessment of both anthropogenic and natural disturbance.

Sister chromatid exchange and micronucleus frequency in human lymphocytes of 1,650 subjects in an Italian population: I. Contribution of methodological factors.

The influence of several methodological factors on mean values of sister chromatid exchanges (SCEs) and micronuclei (MN) in peripheral lymphocytes of 1,650 subjects was analyzed. Donors belonged to a general healthy population living in Pisa and in two nearby small cities: Cascina and Navacchio (Ca-Na). Blood samples were collected over a period of 29 months and processed in three different laboratories of the some institute. Slides were analyzed by several scorers. Our data showed that lymphocyte proliferation indexes (PIs) and baseline mean values of SCEs were affected mainly by sampling period. This factor accounted for a percentage ranging from roughly 10% (Pisa) to 20% (Ca-Na) of total SCE variance and from roughly 10% (Pisa) to 13% (Ca-Na) of total PIs variance. A marginal effect was attributable to the different laboratories involved (maximum 3% for SCEs and 7% for PIs). The sampling period variable included many sources of variability such as culture media batches, fetal calf serum, PHA, BrdUrd, and seasonality. MN counts revealed a more marked dependence on processing laboratories. This factor accounted for a percentage of roughly 10% (Pisa and Ca-Na) of total variance, while the sampling period was marginally effective (about 1-4% of total variability). Because laboratories were equipped and supplied with the same materials and consumables and technicians were rotated constantly, the only variable ascertained was represented by the three different models of CO2 incubators used for lymphocyte culturing. When "month" and "incubator" variables were considered jointly, experimental variability accounted for 15-20% of total variance, both for PIs and mean values SCEs and MN. The variability due to slide scoring was reduced by assigning each slide to five different scorers and matching low with high scorers in each group. Present data show that when the study is performed under these controlled conditions, about 20% of total interdonor variability can be explained by experimental or seasonal factors.

Monitoring human exposure to urban air pollutants.

A multidisciplinary study on a general population exposed to vehicle exhaust was undertaken in Pisa in 1991. Environmental factors such as air pollution and those associated with lifestyle were studied. Meanwhile, biological and medical indicators of health condition were investigated. Chromosomal aberrations, sister chromatid exchanges (SCEs), and micronuclei in lymphocytes were included for the assessment of the genotoxic risk. Because of the large number (3800) of subjects being investigated, standardization of protocols was compulsory. The results on data reproducibility are reported. To assess the reliability of the protocol on a large scale, the population of Porto Tolle, a village located in northeast Italy, was studied and compared to a subset of the Pisa population. Preliminary results showed that probable differences between the two populations and individuals were present in terms of SCE frequencies. The study was potentially able to detect the effects of several factors such as age, smoking, genetics, and environment. The in vitro treatment of lymphocytes with diepoxybutane confirmed the presence of more responsive individuals and permitted us to investigate the genetic predisposition to genetic damage. The possible influence of environmental factors was studied by correlation analyses with external exposure to air pollutants as well as with several lifestyle factors.

C-mitosis and numerical chromosome aberration analyses in human lymphocytes: 10 known or suspected spindle poisons.

As a part of a coordinated EEC project to validate suitable assays for chemically induced genomic mutations, numerical chromosomal aberrations and spindle effects were studied in human lymphocyte cultures exposed to cadmium chloride, chloral hydrate, colchicine, diazepam, econazole, hydroquinone, pyrimethamine, thiabendazole, thimerosal and vinblastine. Chromosome number analysis was carried out after treatment for 48 and 72 h; spindle effects, i.e., increases in the mitotic indices and c-mitoses, were analyzed in cultures treated 5 h before fixation. Dose-related numerical chromosomal aberrations are induced by colchicine and vinblastine, the only chemicals that also induce c-mitotic effects in a wide range of doses. Hyperdiploidy is induced by chloral hydrate, cadmium chloride and thimerosal without dose-effect relationship; chloral hydrate and thimerosal affect spindle functions while only a weak spindle effect is produced by cadmium chloride. Tetraploid and/or endoreduplicated cells are induced without dose-effect relationship by hydroquinone, thiabendazole and thimerosal, all of them able to produce c-mitotic effects. Diazepam and econazole induce only hypodiploidy; pyrimethamine does not induce numerical chromosomal aberrations.