Comparative potency approach based on H2AX assay for estimating the genotoxicity of polycyclic aromatic hydrocarbons

Polycyclic Aromatic Hydrocarbons (PAHs) constitute a family of over one hundred compounds and can generally be found in complex mixtures. PAHs metabolites cause DNA damage which can lead to the development of carcinogenesis. Toxicity assessment of PAH complex mixtures is currently expressed in terms of toxic equivalents, based on Toxicity Equivalent Factors (TEFs). However, the definition of new TEFs for a large number of PAH could overcome some limitations of the current method and improve cancer risk assessment. The current investigation aimed at deriving the relative potency factors of PAHs, based on their genotoxic effect measured in vitro and analyzed with mathematical models. For this purpose, we used a new genotoxic assay (γH2AX) with two human cell lines (HepG2 and LS-174T) to analyze the genotoxic properties of 13 selected PAHs at low doses after 24 h treatment. The dose-response for genotoxic effects was modeled with a Hill model; equivalency between PAHs at low dose was assessed by applying constraints to the model parameters. In the two cell lines tested, we observed a clear dose-response for genotoxic effects for 11 tested compounds. LS-174T was on average ten times more sensitive than HepG2 towards PAHs regarding genotoxicity. We developed new TEFs, which we named Genotoxic Equivalent Factor (GEF). Calculated GEF for the tested PAHs were generally higher than the TEF usually used. Our study proposed a new in vitro based method for the establishment of relevant TEFs for PAHs to improve cancer risk assessment.     

Assessment in vitro of cytogenetic and genotoxic effects of propolis on human lymphocytes

We evaluated the genetic damage by ethanolic extract of propolis (EEP) induced to human lymphocytes which were exposed to increasing concentrations (0-2000μgml(-1)). The results indicated that EEP reduced significantly the mitotic index (MI) and proliferation index (PI) when high concentrations of EEP were used. Sister chromatid exchange (SCE) rates indicated that EEP could have genotoxic effects at high concentrations. Exposure of the cells to the amount of ethanol used as solvent did not alter either the MI and cell proliferation kinetics (CPK), or the rate of SCE. The results showed: (a) statistical increase in the percentage the cells with CAs and in the frequency of SCE at the highest concentrations, (b) a decrease in MI and in the CPK values was observed, (c) no effect was noticed in negative controls. In conclusion, it can be assumed that high concentrations of EEP have a cyto and genotoxic effect, in vitro, for human peripheral lymphocytes.     

Reactive oxygen species mediated DNA damage in human lung alveolar epithelial (A549) cells from exposure to non-cytotoxic MFI-type zeolite nanoparticles

Increasing utilization of engineered nanoparticles in the field of electronics and biomedical applications demands an assessment of risk associated with deliberate or accidental exposure. Metal based nanoparticles are potentially most important of all the nanoparticles in terms of health risks. Microporous alumino-silicates and pure silicates named as zeolites and zeo-type materials with variety of structures, chemical compositions, particle sizes and morphologies have a significant number of industrial uses such as in catalysis, sorption and ion-exchange processes. In particular, the nanosized particles due to their unique properties are used in hybrid organic-inorganic materials for photography, photonics, electronics, labeling, imaging, and sensing. The aim of the current study is to investigate pure silica MFI-type zeolites nanoparticles with sizes of 50 nm and 100 nm (samples MFI-50 and MFI-100) under suspended conditions and their toxicological effects on human lung alveolar (A549) cells under in vitro conditions.     

Silver nanoparticles: evaluation of DNA damage, toxicity and functional impairment in human mesenchymal stem cells

Silver nanoparticles (Ag-NPs) are the most frequent commercialized nanomaterial currently. Due to a distinct lack of information on hazardous properties of Ag-NPs in human cells, a study was conducted to evaluate Ag-NP induced DNA damage, cell death and functional impairment in human mesenchymal stem cells (hMSCs).Initially, Ag-NPs and their cellular distribution were characterized by transmission electron microscopy (TEM). Ag-NPs were exposed to hMSCs for 1, 3 and 24 h. Cytotoxicity was measured by the trypan blue exclusion test and the fluorescein-diacetate test, DNA damage was evaluated by the comet assay and chromosomal aberration test. Cytokine release of IL-6, IL-8 and VEGF was observed using the ELISA technique. Additionally, hMSC migration capability was tested in a transwell system.TEM revealed a Ag-NP distribution to cytoplasm and nucleus. Cytotoxic effects were seen at concentrations of 10 μg/ml for all test exposure periods. Both, comet assay and chromosomal aberration test showed DNA damage after 1, 3, and 24 h at 0.1 μg/ml. A significant increase of IL-6, IL-8 and VEGF release indicates hMSC activation. Migration ability was not impaired at subtoxic concentrations.In conclusion, we demonstrated cyto- and genotoxic potential of Ag-NPs in hMSCs at significantly higher concentrations as compared to antimicrobial effective levels.     

Genotoxicity of carbon nanofibers: are they potentially more or less dangerous than carbon nanotubes or asbestos?

The production of carbon nanofibers and nanotubes (CNF/CNT) and their composite products is increasing globally. CNF are generating great interest in industrial sectors such as energy production and electronics, where alternative materials may have limited performance or are produced at a much higher cost. However, despite the increasing industrial use of carbon nanofibers, information on their potential adverse health effects is limited. In the current study, we examine the cytotoxic and genotoxic potential of carbon-based nanofibers (Pyrograf®-III) and compare this material with the effects of asbestos fibers (crocidolite) or single-walled carbon nanotubes (SWCNT). The genotoxic effects in the lung fibroblast (V79) cell line were examined using two complementary assays: the comet assay and micronucleus (MN) test. In addition, we utilized fluorescence in situ hybridization to detect the chromatin pan-centromeric signals within the MN indicating their origin by aneugenic (chromosomal malsegregation) or clastogenic (chromosome breakage) mechanisms. Cytotoxicity tests revealed a concentration- and time-dependent loss of V79 cell viability after exposure to all tested materials in the following sequence: asbestos > CNF > SWCNT. Additionally, cellular uptake and generation of oxygen radicals was seen in the murine RAW264.7 macrophages following exposure to CNF or asbestos but not after administration of SWCNT. DNA damage and MN induction were found after exposure to all tested materials with the strongest effect seen for CNF. Finally, we demonstrated that CNF induced predominately centromere-positive MN in primary human small airway epithelial cells (SAEC) indicating aneugenic events. Further investigations are warranted to elucidate the possible mechanisms involved in CNF-induced genotoxicity.     

Evaluation of toxic effects of a diet containing fish contaminated with methylmercury in rats mimicking the exposure in the Amazon riverside population

This study was designed to evaluate the effects of a diet rich in fish contaminated with MeHg, mimicking the typical diet of the Amazon riverside population, in rats. Animals were randomly assigned to one of three groups with eight rats in each group: Group I—control, received commercial ration; Group II—received a diet rich in uncontaminated fish; Group III—received a diet rich in fish contaminated with MeHg. Treatment time was 12 weeks. Oxidative stress markers were evaluated, as well as the effects of this diet on DNA stability, systolic blood pressure (SBP), nitric oxide (NO) levels and histological damage in different tissues. There was a significant increase in SBP values in rats fed with MeHg-contaminated fish diet after the 10th week of the treatment. As far as oxidative stress biomarkers are concerned, no differences were observed in reduced glutathione and protein carbonyl levels, glutathione peroxidase, catalase, superoxide dismutase or δ-aminolevulinate dehydratase activities between the groups of animals receiving contaminated and uncontaminated fish diets. On the other hand, malondialdehyde levels increased significantly in rats fed with contaminated fish. NO levels were similar in all groups. DNA migration showed augmented in rats exposed to contaminated fish and histopathological analyses showed weak but significant leukocyte infiltration. Thus, we conclude that the MeHg-contaminated fish diet induced a slight lipid peroxidation and genotoxicity. However, these effects seem to be much less pronounced than when rats are exposed to aqueous solution containing CH₃HgCl.Our findings support the contention that the chemical form of MeHg in fish or fish nutrients such as polyunsaturated fatty acids, Se or vitamin E could minimize the toxic effects of MeHg exposure in fish-eating communities.     

Mutagenic impact on fish of runoff events in agricultural areas in south-west France

When heavy rainfall follows herbicide application, the intense surface runoff causes stream water contamination. Aquatic organisms are then briefly exposed to a complex mixture of contaminants. The aim of the present study is to investigate the genotoxic impact of such events on fish. A model fish, the Crucian carp (Carassius carassius) was exposed in controlled conditions, for 4 days, to water sampled daily in the Save River (France). The watershed of this stream is representative of agricultural areas in south-west France. Three hydrological conditions were compared: basal flow, winter flood, and spring flood. Chemical analysis of the water samples confirmed the higher contamination of the spring flood water, mainly explained by a peak of metolachlor. Genotoxicity was evaluated by micronucleus (MN) test and comet assay in peripheral erythrocytes. A significant increase in DNA breakdowns compared to controls was detected by the comet assay for all conditions. Exposure to spring flood water resulted in the highest damage induction. Moreover, induced chromosomal damage was only detected in this condition. In addition, fish were exposed, for 4 days, to an experimental mixture of 5 herbicides representative of the spring flood water contamination. Fish exhibited moderate DNA damage induction and no significant chromosomal damage. The mutagenicity induced by field-collected water is then suspected to be the result of numerous interactions between contaminants themselves and environmental factors, stressing the use of realistic exposure conditions. The results revealed a mutagenic impact of water contamination during the spring flood, emphasizing the need to consider these transient events in water quality monitoring programs.     

Toxicological evaluation of ammonium 4,8-dioxa-3H-perfluorononanoate,a new emulsifier to replace ammonium perfluorooctanoate in fluoropolymer manufacturing

Ammonium 4,8-dioxa-3H-perfluorononanoate (ADONA) was developed to replace ammonium perfluorooctanoate (APFO) as an emulsifier in the manufacture of fluoropolymers. The toxicity of ADONA was evaluated in acute and repeat-dose studies of up to 90-days duration, and in eye and skin irritation, dermal sensitization, genotoxicity, and developmental toxicity studies. ADONA was also evaluated as a peroxisome proliferator-activated receptor alpha (PPARα) agonist in rats. ADONA was moderately toxic orally and practically non-toxic dermally in acute studies in rats. It was a mild skin irritant and a moderate to severe eye irritant in rabbits. It was a weak dermal sensitizer in local lymph node assays in mice. ADONA was not genotoxic based on the weight of evidence from five assays. It was not developmentally toxic in rats except at maternally toxic doses. ADONA was a possible PPARα agonist in male rats. The liver was the primary target organ in male rats and the kidney was the primary target organ in female rats. NOAELs in 28- and 90-day oral studies in rats were 10 mg/kg/day for males and 100 mg/kg/day for females. These findings demonstrate that the toxicity profile for ADONA is acceptable for its intended use and is superior to that of APFO.     

Antioxidant and antigenotoxic properties of compounds isolated from Marrubium deserti de Noé

In our continual course toward the valorization of traditionally used endemic flora through the analysis of its chemobiodiversity, the phytochemical analysis of aerial parts of Marrubium deserti de Noé was undertaken. Dichloromethane and methanol extracts led to the isolation of terpenoid derivatives among which two were new labdane diterpenes named marrulibacetal A and desertine, respectively. Six of them were known compounds (a mixture of the isomers cyllenin A and 15-epi-cyllenin A, marrubiin, marrulactone, marrulibacetal and β-stigmasterol) and seven known phenolic compounds were also isolated: apigenin and several 7-O-substituted derivatives (apigenin-7-O-β-neohesperidoside, apigenin-7-O-glucoside, terniflorin and apigenin-7-O-glucuronide) together with two phenylethanoid glucosides (acteoside and forsythoside B). The structures and relative configurations of the new compounds were elucidated by MS and a series of 1D and 2D NMR analyses. Some pure compounds have been evaluated for their antioxidant activities through different methods: DPPH and ABTS assays as well as CUPRAC assay. Genotoxic and antigenotoxic activities of extracts and pure compounds were also evaluated in vitro on Escherichia coli PQ37 cells by the SOS Chromotest. Some of the isolated compounds like phenylethanoid derivatives showed stronger antioxidant capacity than trolox and were also able to significantly inhibit β-galactosidase induction caused by the mutagen agent nitrofurantoin.     

Ellagic acid, a natural polyphenol protects rat peripheral blood lymphocytes against nicotine-induced cellular and DNA damage in vitro: with the comparison of N-acetylcysteine

The present work is aimed at evaluating the protective effect of ellagic acid (EA), a natural polyphenolic compound that is widely distributed in fruits and nuts against nicotine-induced toxicity in rat peripheral blood lymphocytes. The effect of EA against nicotine toxicity was compared with N-acetylcysteine (NAC), a well-known antioxidant. Lymphocytes were exposed to nicotine at the doses of 0.125, 0.25, 0.5, 1, 2, 3 and 4 mM for 1h in culture media. Thiobarbituric acid reactive substances (TBARS), a lipid peroxidative marker and reduced glutathione (GSH), as indicative of endogenous antioxidant status were analyzed to fix the optimum dose. The lowest concentration eliciting significant damage was 1 mM nicotine and maximum damage was observed with 3 mM concentration, as evidenced by increased levels of TBARS and decreased levels of GSH. Hence, the test concentration was fixed at 3 mM nicotine. To establish most effective protective support we used five different concentrations of EA (10, 50, 100, 150 and 300 microM) against 3 mM nicotine. A dose-dependent inhibitory effect was observed with all doses of EA. Maximum protection was observed at the dose of 100 microM EA. So, 100 microM dose was used for further studies. We have tested five different concentrations of NAC-0.25, 0.5, 1, 2 and 4 mM to elucidate the optimum protective dose against nicotine toxicity. One millimolar NAC showed a significant protection against nicotine toxicity. Protective effect of EA against nicotine toxicity was elucidated by analyzing the lipid peroxidative index, viz., TBARS, hydroperoxides (HP) and endogenous antioxidant status, viz., superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), reduced glutathione (GSH), Vitamins A, E and C. DNA damage and repair were assessed by using alkaline single-cell microgel electrophoresis (Comet assay) and micronucleus assay. There was a significant increase in the levels of lipid peroxidative index, severity in DNA damage and micronuclei number in nicotine-treated group, which was positively modulated by EA treatment. Antioxidant status was significantly depleted in nicotine-treated group, which was effectively restored by EA treatment. The protection of EA against nicotine toxicity was equally effective to that of NAC. EA and NAC treatment alone did not produce any damage to the normal lymphocytes at their effective doses. These findings suggest the potential use and benefit of EA as a modifier of nicotine-induced genotoxicity.