Measuring DNA modifications with the comet assay: a compendium of protocols.

The comet assay is a versatile method to detect nuclear DNA damage in individual eukaryotic cells, from yeast to human. The types of damage detected encompass DNA strand breaks and alkali-labile sites (e.g., apurinic/apyrimidinic sites), alkylated and oxidized nucleobases, DNA-DNA crosslinks, UV-induced cyclobutane pyrimidine dimers and some chemically induced DNA adducts. Depending on the specimen type, there are important modifications to the comet assay protocol to avoid the formation of additional DNA damage during the processing of samples and to ensure sufficient sensitivity to detect differences in damage levels between sample groups. Various applications of the comet assay have been validated by research groups in academia, industry and regulatory agencies, and its strengths are highlighted by the adoption of the comet assay as an in vivo test for genotoxicity in animal organs by the Organisation for Economic Co-operation and Development. The present document includes a series of consensus protocols that describe the application of the comet assay to a wide variety of cell types, species and types of DNA damage, thereby demonstrating its versatility.

Biomarkers responses and polybrominated diphenyl ethers and their methoxylated analogs measured in Sparus aurata from the Lagoon of Bizerte, Tunisia.

This study aimed to the examination of the levels and effects of organobromine compounds (polybrominated diphenyl ethers: PBDEs and methoxylated brominated diphenyl ethers: MeO-PBDEs), in Sparus aurata native to the Lagoon of Bizerte. For that, different biomarkers of exposure (somatic indices, superoxide dismutase, and catalase activities) and effect (malondialdehyde level, histopathologic alterations, and DNA damage) as well as pollutant levels were measured in specimens collected from this impacted ecosystem and the Mediterranean Sea as a reference site. Bizerte Lagoon PBDE fish levels were higher than the Mediterranean Sea, whereas MeO-PBDEs were higher in the reference site. Fish from Bizerte Lagoon presented a higher hepatosomatic index, lower catalase and superoxide dismutase activity, higher level of malondialdehyde, and higher percentage of DNA tail in comparison to fish from the reference area. The histological study of the liver indicated substantial lesions in fish from the polluted site. The results showed strong positive correlations between the concentrations of the PBDE or MeO-PBDE and the MDA and DNA tail % levels and negative correlations for the activities of enzymes of SOD and CAT. Consequently, these findings could suggest a potential link between exposure to these pollutants and the observed biomarker responses in the Bizerte Lagoon seabream. Taken together, these results highlight the importance of biomarker selection and the selected sentinel fish species as useful tools for biomonitoring of aquatic pollution.

π-Donor/π-Acceptor Interactions for the Encapsulation of Neurotransmitters on Functionalized Polysilicon-Based Microparticles.

Bipyridinium salts, commonly known as viologens, are π-acceptor molecules that strongly interact with π-donor compounds, such as porphyrins or amino acids, leading their self-assembling. These properties have promoted us to functionalize polysilicon microparticles with bipyridinium salts for the encapsulation and release of π-donor compounds such as catecholamines and indolamines. In this work, the synthesis and characterization of four gemini-type amphiphilic bipyridinium salts (1·4PF6-4·4PF6), and their immobilization either non-covalently or covalently on polysilicon surfaces and microparticles have been achieved. More importantly, they act as hosts for the subsequent incorporation of π-donor neurotransmitters such as dopamine, serotonin, adrenaline or noradrenaline. Ultraviolet-visible absorption and fluorescence spectroscopies and high-performance liquid chromatography were used to detect the formation of the complex in solution. The immobilization of bipyridinium salts and neurotransmitter incorporation on polysilicon surfaces was corroborated by contact angle measurements. The reduction in the bipyridinium moiety and the subsequent release of the neurotransmitter was achieved using ascorbic acid, or Vitamin C, as a triggering agent. Quantification of neurotransmitter encapsulated and released from the microparticles was performed using high-performance liquid chromatography. The cytotoxicity and genotoxicity studies of the bipyridinium salt 1·4PF6, which was selected for the non-covalent functionalization of the microparticles, demonstrated its low toxicity in the mouse fibroblast cell line (3T3/NIH), the human liver carcinoma cell line (HepG2) and the human epithelial colorectal adenocarcinoma cell line (Caco-2).

A test battery for assessing the ecotoxic effects of textile dyes.

The textile dyeing industry is one of the main sectors contributing to environmental pollution, due to the generation of large amounts of wastewater loaded with dyes (ca. 2-50% of the initial amount of dyes used in the dye baths is lost), causing severe impacts on human health and the environment. In this context, an ecotoxicity testing battery was used to assess the acute toxicity and genotoxicity of the textile dyes Direct Black 38 (DB38; azo dye) and Reactive Blue 15 (RB15; copper phthalocyanine dye) on different trophic levels. Thus these dyes were tested using the following assays: Filter paper contact test with earthworms (Eisenia foetida); seed germination and root elongation toxicity test (Cucumis sativus, Lactuca sativa and Lycopersicon esculentum); acute immobilization test (Daphnia magna and Artemia salina); and the Comet assay with the rainbow trout gonad-2 cell fish line (RTG-2) and D. magna. Neither phytotoxicity nor significant effects on the survival of E. foetida were observed after exposure to DB38 and RB15. Both dyes were classified as relatively non-toxic to D. magna (LC50 > 100 mg/L), but DB38 was moderately toxic to A. salina with a LC50 of 20.7 mg/L. DB38 and RB15 induced significant effects on the DNA of D. magna but only DB38 caused direct (alkaline comet assay) and oxidative (hOGG1-modified alkaline comet assay) damage to RTG-2 cells in hormetic responses. Therefore, the present results emphasize that a test battery approach of bioassays representing multiple trophic levels is fundamental in predicting the toxicity of textile dyes, aside from providing the information required to define their safe levels for living organisms in the environment.

Gemini pyridinium amphiphiles for the synthesis and stabilization of gold nanoparticles for drug delivery.

HYPOTHESIS: Gemini pyridinium-based amphiphiles can play a triple role as: gold nanoparticles (AuNPs) synthesis facilitator, particle stabilizer and anion recognition centre. The so formed nanoparticles should be able to bind and release anionic drugs. EXPERIMENTS: We describe (a) Synthesis, by a phase transfer method, of both new organic media and water soluble AuNPs using gemini-type surfactants based on bis-pyridinium salts as ligands, acting as transfer agents into organic media and also as nanoparticle stabilizers, (b) Examination of their stability in solution, (c) Chemical and physical characterization of the nanoparticles, (d) Toxicity data concerning both the bis-pyridinium ligands and the bis-pyridinium coated nanoparticles, and (e) Study of their ability for delivering anionic pharmaceuticals such as ibuprofen and piroxicam. FINDINGS: Pyridinium gemini-type surfactants show the ability to play multiple roles such as transfer agent and stabilizer, as well as ionophores: They are responsible for the preparation, stability, and delivery properties of these AuNPs, which gold core is stabilized by the anions present in the bis-pyridinium salts. The tetrahydropyridine resulting from the reduction of the bis-pyridinium salt is capable of reduce gold, due to its spontaneous oxidation to the corresponding pyridinium salt, leading to the formation of stable AuNPs.

Textile dyes induce toxicity on zebrafish early life stages.

Textile manufacturing is one of the most polluting industrial sectors because of the release of potentially toxic compounds, such as synthetic dyes, into the environment. Depending on the class of the dyes, their loss in wastewaters can range from 2% to 50% of the original dye concentration. Consequently, uncontrolled use of such dyes can negatively affect human health and the ecological balance. The present study assessed the toxicity of the textile dyes Direct Black 38 (DB38), Reactive Blue 15 (RB15), Reactive Orange 16 (RO16), and Vat Green 3 (VG3) using zebrafish (Danio rerio) embryos for 144 h postfertilization (hpf). At the tested conditions, none of the dyes caused significant mortality. The highest RO16 dose significantly delayed or inhibited the ability of zebrafish embryos to hatch from the chorion after 96 hpf. From 120 hpf to 144 hpf, all the dyes impaired the gas bladder inflation of zebrafish larvae, DB38 also induced curved tail, and VG3 led to yolk sac edema in zebrafish larvae. Based on these data, DB38, RB15, RO16, and VG3 can induce malformations during embryonic and larval development of zebrafish. Therefore, it is essential to remove these compounds from wastewater or reduce their concentrations to safe levels before discharging textile industry effluents into the aquatic environment.

Rhamnose-coated superparamagnetic iron-oxide nanoparticles: an evaluation of their in vitro cytotoxicity, genotoxicity and carcinogenicity.

Tumor recurrence after the incomplete removal of a tumor mass inside brain tissue is the main reason that scientists are working to identify new strategies in brain oncologic therapy. In particular, in the treatment of the most malignant astrocytic tumor glioblastoma, the use of magnetic nanoparticles seems to be one of the most promising keys in overcoming this problem, namely by means of magnetic fluid hyperthermia (MFH) treatment. However, the major unknown issue related to the use of nanoparticles is their toxicological behavior when they are in contact with biological tissues. In the present study, we investigated the interaction of glioblastoma and other tumor cell lines with superparamagnetic iron-oxide nanoparticles covalently coated with a rhamnose derivative, using proper cytotoxic assays. In the present study, we focused our attention on different strategies of toxicity evaluation comparing different cytotoxicological approaches in order to identify the biological damages induced by the nanoparticles. The data show an intensive internalization process of rhamnose-coated iron oxide nanoparticles by the cells, suggesting that rhamnose moiety is a promising biocompatible coating in favoring cells' uptake. With regards to cytotoxicity, a 35% cell death at a maximum concentration, mainly as a result of mitochondrial damages, was found. This cytotoxic behavior, along with the high uptake ability, could facilitate the use of these rhamnose-coated iron-oxide nanoparticles for future MFH therapeutic treatments.

The Comet Assay and its applications in the field of ecotoxicology: a mature tool that continues to expand its perspectives.

Since Singh and colleagues, in 1988, launched to the scientific community the alkaline Single Cell Gel Electrophoresis (SCGE) protocol, or Comet Assay, its uses and applications has been increasing. The thematic areas of its current employment in the evaluation of genetic toxicity are vast, either in vitro or in vivo, both in the laboratory and in the environment, terrestrial or aquatic. It has been applied to a wide range of experimental models: bacteria, fungi, cells culture, arthropods, fishes, amphibians, reptiles, mammals, and humans. This document is intended to be a comprehensive review of what has been published to date on the field of ecotoxicology, aiming at the following main aspects: (i) to show the most relevant experimental models used as bioindicators both in the laboratory and in the field. Fishes are clearly the most adopted group, reflecting their popularity as bioindicator models, as well as a primary concern over the aquatic environment health. Amphibians are among the most sensitive organisms to environmental changes, mainly due to an early aquatic-dependent development stage and a highly permeable skin. Moreover, in the terrestrial approach, earthworms, plants or mammalians are excellent organisms to be used as experimental models for genotoxic evaluation of pollutants, complex mix of pollutants and chemicals, in both laboratory and natural environment. (ii) To review the development and modifications of the protocols used and the cell types (or tissues) used. The most recent developments concern the adoption of the enzyme linked assay (digestion with lesion-specific repair endonucleases) and prediction of the ability to repair of oxidative DNA damage, which is becoming a widespread approach, albeit challenging. For practical/technical reasons, blood is the most common choice but tissues/cells like gills, sperm cells, early larval stages, coelomocytes, liver or kidney have been also used. (iii) To highlight correlations with other biomarkers. (iv) To build a constructive criticism and summarize the needs for protocol improvements for future test applications within the field of ecotoxicology. The Comet Assay is still developing and its potential is yet underexploited in experimental models, mesocosmos or natural ecosystems.

Oxidative stress, genotoxicity and histopathology biomarker responses in Mugil cephalus and Dicentrarchus labrax gill exposed to persistent pollutants. A field study in the Bizerte Lagoon: Tunisia.

The use of biomarkers has become an important tool for modern environmental assessment as they can help to predict pollutants involved in the monitoring program. Despite the importance of fish gill in several functions (gaseous exchange, osmotic and ionic regulation, acid-base balance and nitrogenous waste) its use in coastal water biomonitoring focusing on protection and damage is scarce. This field study investigates biochemical (catalase, superoxide dismutase, lipid peroxidation), molecular (DNA integrity) and morphological (histology) parameters in gill of mullet (Mugil cephalus) and sea bass (Dicentrarchus labrax) and originating from Bizerte lagoon (a coastal lagoon impacted by different anthropogenic activities) and from the Mediterranean Sea (a reference site). Remarkable alterations in the activities of oxidative stress enzymes and DNA integrity in the tissue of the two studied fish species were detected in Bizerte Lagoon. The study of histopathological alterations of gills in both two fish species from Bizerte Lagoon suggest thickening of primary lamellae, cellular hyperplasia, aneurism, curving, shortening and fusion of secondary lamellae. The adopted approach, considering simultaneously protection responses and damaging effects, revealed its usefulness on the pollution assessment.

Critical factors to be considered when testing nanomaterials for genotoxicity with the comet assay.

The comet assay is widely used to test the genotoxicity of engineered nanomaterials (ENMs) but outcomes may vary when results from different laboratories, or even within one laboratory, are compared. We address some basic methodological considerations, such as the importance of carrying out physico-chemical characterisation of the ENMs in test-medium, performing uptake and cytotoxicity tests, and testing several genotoxicity-related endpoints. In this commentary, we discuss the different ways in which concentration of ENMs can be expressed, and stress the need to include appropriate controls and reference standards to monitor variation and avoid interference. Treatment conditions, including cell number, cell culture plate format and volume of treatment medium on the plate are crucial factors that may impact on results and thus should be kept constant within the study.

In vivo inflammatory effects of ceria nanoparticles on CD-1 mouse: evaluation by hematological, histological, and TEM analysis.

The attention on CeO2-NPs environmental and in vivo effects is due to their presence in diesel exhaust and in diesel filters that release a more water-soluble form of ceria NPs, as well as to their use for medical applications. In this work, acute and subacute in vivo toxicity assays demonstrate no lethal effect of these NPs. Anyhow, performing in vivo evaluations on CD-1 mouse systems, we demonstrate that it is even not correct to assert that ceria NPs are harmless for living systems as they can induce status of inflammation, revealed by hematological-chemical-clinical assays as well as histological and TEM microscope observations. TEM analysis showed the presence of NPs in alveolar macrophages. Histological evaluation demonstrated the NPs presence in lungs tissues and this can be explained by assuming their ability to go into the blood stream and lately into the organs (generating inflammation).

An integrated approach for detecting embryotoxicity and developmental toxicity of environmental contaminants using in vitro alternative methods.

The main available alternatives for testing embryotoxicity are cellular tests with stem cells and in vitro-ex vivo tests with embryos. In cellular tests, the most developed alternative is the embryonic stem cell test, while the most developed tests involving embryos are the zebrafish and whole embryo culture test. They are technically more complex than cellular tests, but offer the advantage of determining the expectable phenotypic alteration caused by the exposure. Many efforts are currently being made, basically through proteomic and genomic approaches, in order to obtain improvements in predictivity of these tests. Development is a very complex process, and it is highly unlikely that a single alternative test can yield satisfactory performance with all types of chemicals. We propose a step-wise approach where model complexity, and consequently technical skills and economical costs, gradually increase if needed. The first level would be run short cellular assays to detect effects in early differentiation stages. The second level would involve longer cellular embryotoxicity tests to search embryotoxicants that have an effect on late differentiation stages. The third stage would consider tests with embryos because they allow the determination of hazards based on molecular and morphological alterations, and not only on differentiating cells.

Lysine-based surfactants in nanovesicle formulations: the role of cationic charge position and hydrophobicity in in vitro cytotoxicity and intracellular delivery.

Understanding nanomaterial interactions within cells is of increasing importance for assessing their toxicity and cellular transport. Here, the authors developed nanovesicles containing bioactive cationic lysine-based amphiphiles and assessed whether these cationic compounds increase the likelihood of intracellular delivery and modulate toxicity. Different cytotoxic responses were found among the formulations, depending on surfactant, cell line and endpoint assayed. The induction of mitochondrial dysfunction, oxidative stress and apoptosis were the general mechanisms underlying cytotoxicity. Fluorescence microscopy analysis demonstrated that nanovesicles were internalised by HeLa cells and evidenced that their ability to release endocytosed materials into cell cytoplasm depends on the structural parameters of amphiphiles. The cationic charge position and hydrophobicity of surfactants determine the nanovesicle interactions within the cell and, thus, the resulting toxicity and intracellular behaviour after cell uptake of the nanomaterial. The insights into some toxicity mechanisms of these new nanomaterials contribute in reducing the uncertainty surrounding their potential health hazards.

Substituent effects on the biological properties of Zn-salophen complexes.

The synthesis, characterization, and luminescent properties of a series of 5,5'-X-substituted salophen ligands, X = OCH3, Br, and NO2, and the corresponding Zn(II) complexes are reported here. Their biological activity has been analyzed and related to the different Lewis acid character of the complexes. In vitro studies (AFM and absorption and emission titrations) show that the strongest interaction with free plasmid DNA is observed for 5,5'-dinitro-substituted Zinc-salophen complex 3b. Semiempirical theoretical calculations together with redox potential measurements suggest that this might be interpreted as a direct consequence of this compound having the hardest Lewis acid character. Cellular uptake and cytotoxicity studies undertaken with these metal complexes show that they enter the cells but are not cytotoxic.

Cytotoxicity and genotoxicity of ceria nanoparticles on different cell lines in vitro.

Owing to their radical scavenging and UV-filtering properties, ceria nanoparticles (CeO(2)-NPs) are currently used for various applications, including as catalysts in diesel particulate filters. Because of their ability to filter UV light, CeO(2)-NPs have garnered significant interest in the medical field and, consequently, are poised for use in various applications. The aim of this work was to investigate the effects of short-term (24 h) and long-term (10 days) CeO(2)-NP exposure to A549, CaCo2 and HepG2 cell lines. Cytotoxicity assays tested CeO(2)-NPs over a concentration range of 0.5 µg/mL to 5000 µg/mL, whereas genotoxicity assays tested CeO(2)-NPs over a concentration range of 0.5 µg/mL to 5000 µg/mL. In vitro assays showed almost no short-term exposure toxicity on any of the tested cell lines. Conversely, long-term CeO(2)-NP exposure proved toxic for all tested cell lines. NP genotoxicity was detectable even at 24-h exposure. HepG2 was the most sensitive cell line overall; however, the A549 line was most sensitive to the lowest concentration tested. Moreover, the results confirmed the ceria nanoparticles' capacity to protect cells when they are exposed to well-known oxidants such as H(2)O(2). A Comet assay was performed in the presence of both H(2)O(2) and CeO(2)-NPs. When hydrogen peroxide was maintained at 25 µM, NPs at 0.5 µg/mL, 50 µg/mL, and 500 µg/mL protected the cells from oxidative damage. Thus, the NPs prevented H(2)O(2)-induced genotoxic damage.

In vitro safety toxicology data for evaluation of gold nanoparticles-chronic cytotoxicity, genotoxicity and uptake.

Safety and toxic effects of nanoparticles are still largely unexplored due to the multiple aspects that influence their behaviour toward biological systems. Here, we focus the attention on 12 nm spherical gold nanoparticle coated or not with hyaluronic acid compared to its precursor counterpart salt. Results ranging from the effects of a 10-days exposure in an in vitro model with BALB/c 3T3 fibroblast cells show how 12 nm spherical gold nanoparticles are internalized from 3T3 cells by endo-lysosomal pathway by an indirect measurement technique; and how gold nanoparticles, though not being a severe cytotoxicant, induce DNA damage probably through an indirect mechanism due to oxidative stress. While coating them with hyaluronic acid reduces gold nanoparticles cytotoxicity and slows their cell internalization. These results will be of great interest to medicine, since they indicate that gold nanoparticles (with or without coating) are suitable for therapeutic applications due to their tunable cell uptake and low toxicity.

Oxidative stress, genotoxicity and histopathology biomarker responses in mullet (Mugil cephalus) and sea bass (Dicentrarchus labrax) liver from Bizerte Lagoon (Tunisia).

The aim of the study was to evaluate the impact of environmental contaminants on oxidative stress, genotoxic and histopathologic biomarkers in liver of mullet (Mugil cephalus) and sea bass (Dicentrarchus labrax) collected from a polluted coastal lagoon (Bizerte Lagoon) in comparison to a reference site (the Mediterranean Sea). Antioxidant enzyme activities were lower in fish from the polluted site compared with fish from the reference site, suggesting deficiency of the antioxidant system to compensate for oxidative stress. DNA damage was higher in both fish species from the contaminated site indicating genotoxic effects. The liver histopathological analysis revealed alterations in fish from Bizerte Lagoon. Hepatocytes from both fish species featured extensive lipid-type vacuolation and membrane disruption. Results suggest that the selected biomarkers in both fish species are useful for the assessment of pollution impacts in coastal environments influenced by multiple pollution sources.

Embryotoxicity of cobalt ferrite and gold nanoparticles: a first in vitro approach.

Nanoparticles (NPs) are emerging as promising biomedical tools thanks to their peculiar characteristics. Our purpose was to investigate the embryotoxicity of cobalt ferrite and gold NPs through the Embryonic Stem Cell Test (EST). The EST is an in vitro standard assay, which permits to classify substances as strongly, weakly or non-embryotoxic. Due to the particular physical-chemical nature of nanoparticles, we introduced a modification to the standard protocol exposing the Embryonic Stem Cells (ES-D3) to nanoparticles only during the first 5 days of the assay. Moreover, we proposed a method to discriminate and compare the embryotoxicity of the substances within the weakly embryotoxic range. Our ID(50) results permit to classify cobalt ferrite nanoparticles coated with gold and silanes as non-embryotoxic. The remaining nanoparticles have been classified as weakly embryotoxic in this decreasing order: gold salt (HAuCl(4).3H(2)O)>cobalt ferrite salt (CoFe(2)O(4))>cobalt ferrite nanoparticles coated with silanes (Si-CoFe)>gold nanoparticles coated with hyaluronic acid (HA-Au).

Chronic exposure to MDMA (ecstasy) increases DNA damage in sperm and alters testes histopathology in male rats.

3,4-Methylenedioxymethamphetamine (MDMA or "ecstasy") is consumed mainly by young population. For this reason, it is especially relevant to take into consideration the effects on the reproductive system. The influence of MDMA on the fertility and reproduction of the male rat was assessed in this study. MDMA was administered subcutaneously at 0 mg/kg (control), 0.5 mg/kg, 5 mg/kg and 10 mg/kg to SD male rats once a day, 3 consecutive days a week during 12 weeks, simulating human weekend associated consumption. Hormonal, haematological, biochemical, histological, genotoxicological and testicular and sperm parameters were evaluated in half of the rats. The remaining animals were mated with untreated sexually receptive females to evaluate the mating and pregnancy rates. A significantly higher incidence of DNA damage in Comet Test in sperm, tubular degeneration and interstitial oedema in testes was found. At all doses tested, sperm motility, morphology, mating and pregnancy rates, and number of implantation sites were not affected. This study fills the existing gap of knowledge about the chronic effects of MDMA in reproductive function using a realistic experimental design. Taking into account the higher sensitivity of human males, some concerns about the effects on the reproductive health still remain.

Use of transduction proteins to target trabecular meshwork cells: outflow modulation by profilin I.

PURPOSE: Fusion proteins containing a protein transduction domain (PTD4) are able to cross biological membranes. We tested the applicability of the protein transduction method for study of the aqueous humor trabecular outflow pathway by targeting the actin cytoskeleton, which is known to be involved in outflow facility regulation. METHODS: Expression vectors useful for generating fusion proteins with the PTD4 domain and the actin-binding protein Profilin I were constructed. The transductional and functional properties of these proteins were tested in bovine trabecular meshwork cells in culture. The effects of PTD4-Profilin I on outflow facility were evaluated in perfused bovine anterior segments. PTD4-beta-galactosidase was used to visually check correct delivery of fusion proteins to trabecular meshwork cells. RESULTS: The fusion proteins generated were characterized by western blot. Immunocytochemistry experiments showed intracellular staining for PTD4-Profilin I in trabecular meshwork cells in culture. The fusion protein was found in the cytoplasm associated with actin filaments and in the leading edge of the cellular membrane. In contrast, control Profilin I, without the PTD4 domain, was unable to cross the cell membrane. In perfused anterior segments, 2 microM PTD4-Profilin I increased trabecular outflow facility in a reversible manner, while Profilin I had no significant effect. Anterior segments perfused with PTD4-beta-galactosidase showed positive staining in the trabecular meshwork tissue. CONCLUSIONS: Protein transduction technology is a valuable tool for targeting trabecular meshwork tissue, not only for performing physiological studies, but also as a potential drug-delivery method. Profilin I action on the actin cytoskeleton further reinforces the importance of this structure in outflow facility regulation.