DNA strand break levels in cryopreserved mononuclear blood cell lines measured by the alkaline comet assay: results from the hCOMET ring trial.

The comet assay is widely used in biomonitoring studies for the analysis of DNA damage in leukocytes and peripheral blood mononuclear cells. Rather than processing blood samples directly, it can be desirable to cryopreserve whole blood or isolated cells for later analysis by the comet assay. However, this creates concern about artificial accumulation of DNA damage during cryopreservation. In this study, 10 laboratories used standardized cryopreservation and thawing procedures of monocytic (THP-1) or lymphocytic (TK6) cells. Samples were cryopreserved in small aliquots in 50% foetal bovine serum, 40% cell culture medium, and 10% dimethyl sulphoxide. Subsequently, cryopreserved samples were analysed by the standard comet assay on three occasions over a 3-year period. Levels of DNA strand breaks in THP-1 cells were increased (four laboratories), unaltered (four laboratories), or decreased (two laboratories) by long-term storage. Pooled analysis indicates only a modest positive association between storage time and levels of DNA strand breaks in THP-1 cells (0.37% Tail DNA per year, 95% confidence interval: -0.05, 0.78). In contrast, DNA strand break levels were not increased by cryopreservation in TK6 cells. There was inter-laboratory variation in levels of DNA strand breaks in THP-1 cells (SD = 3.7% Tail DNA) and TK6 reference sample cells (SD = 9.4% Tail DNA), whereas the intra-laboratory residual variation was substantially smaller (i.e. SD = 0.4%-2.2% Tail DNA in laboratories with the smallest and largest variation). In conclusion, the study shows that accumulation of DNA strand breaks in cryopreserved mononuclear blood cell lines is not a matter of concern.

Long-term cryopreservation of potassium bromate positive assay controls for measurement of oxidatively damaged DNA by the Fpg-modified comet assay: results from the hCOMET ring trial.

The formamidopyrimidine DNA glycosylase (Fpg)-modified comet assay is widely used for the measurement of oxidatively generated damage to DNA. However, there has not been a recommended long-term positive control for this version of the comet assay. We have investigated potassium bromate as a positive control for the Fpg-modified comet assay because it generates many Fpg-sensitive sites with a little concurrent generation of DNA strand breaks. Eight laboratories used the same procedure for the treatment of monocytic THP-1 cells with potassium bromate (0, 0.5, 1.5, and 4.5 mM) and subsequent cryopreservation in a freezing medium consisting of 50% foetal bovine serum, 40% RPMI-1640 medium, and 10% dimethyl sulphoxide. The samples were analysed by the Fpg-modified comet assay three times over a 3-year period. All laboratories obtained a positive concentration-response relationship in cryopreserved samples (linear regression coefficients ranging from 0.79 to 0.99). However, there was a wide difference in the levels of Fpg-sensitive sites between the laboratory with the lowest (4.2% Tail DNA) and highest (74% Tail DNA) values in THP-1 cells after exposure to 4.5 mM KBrO3. In an attempt to assess sources of inter-laboratory variation in Fpg-sensitive sites, comet images from one experiment in each laboratory were forwarded to a central laboratory for visual scoring. There was high consistency between measurements of %Tail DNA values in each laboratory and the visual score of the same comets done in the central laboratory (r = 0.98, P < 0.001, linear regression). In conclusion, the results show that potassium bromate is a suitable positive comet assay control.

Role of Chemical Reduction and Formulation of Graphene Oxide on Its Cytotoxicity towards Human Epithelial Bronchial Cells.

Graphene-based materials may pose a potential risk for human health due to occupational exposure, mainly by inhalation. This study was carried out on bronchial epithelial 16HBE14o- cells to evaluate the role of chemical reduction and formulation of graphene oxide (GO) on its cytotoxic potential. To this end, the effects of GO were compared to its chemically reduced form (rGO) and its stable water dispersion (wdGO), by means of cell viability reduction, reactive oxygen species (ROS) generation, pro-inflammatory mediators release and genotoxicity. These materials induced a concentration-dependent cell viability reduction with the following potency rank: rGO > GO >> wdGO. After 24 h exposure, rGO reduced cell viability with an EC50 of 4.8 µg/mL (eight-fold lower than that of GO) and was the most potent material in inducing ROS generation, in contrast to wdGO. Cytokines release and genotoxicity (DNA damage and micronucleus induction) appeared low for all the materials, with wdGO showing the lowest effect, especially for the former. These results suggest a key role for GO reduction in increasing GO cytotoxic potential, probably due to material structure alterations resulting from the reduction process. In contrast, GO formulated in a stable dispersion seems to be the lowest cytotoxic material, presumably due to its lower cellular internalization and damaging capacity.

Impact of physico-chemical properties on the toxicological potential of reduced graphene oxide in human bronchial epithelial cells.

The increasing use of graphene-based materials (GBM) requires their safety evaluation, especially in occupational settings. The same physico-chemical (PC) properties that confer GBM extraordinary functionalities may affect the potential toxic response. Most toxicity assessments mainly focus on graphene oxide and rarely investigate GBMs varying only by one property. As a novelty, the present study assessed the in vitro cytotoxicity and genotoxicity of six reduced graphene oxides (rGOs) with different PC properties in the human bronchial epithelial 16HBE14o - cell line. Of the six materials, rGO1-rGO4 only differed in the carbon-to-oxygen (C/O) content, whereas rGO5 and rGO6 were characterized by different lateral size and number of layers, respectively, but similar C/O content compared with rGO1. The materials were characterized by transmission electron microscopy, X-ray photoelectron spectroscopy, laser diffraction and dynamic light scattering, and Brunauer-Emmett-Teller analysis. Cytotoxicity (Luminescent Cell Viability and WST-8 assays), the induction of reactive oxygen species (ROS; 2',7'-dichlorofluorescin diacetate-based assay), the production of cytokines (enzyme-linked immunosorbent assays) and genotoxicity (comet and micronucleus assays) were evaluated. Furthermore, the internalization of the materials in the cells was confirmed by laser confocal microscopy. No relationships were found between the C/O ratio or the lateral size and any of the rGO-induced biological effects. However, rGO of higher oxygen content showed higher cytotoxic and early ROS-inducing potential, whereas genotoxic effects were observed with the rGO of the lowest density of oxygen groups. On the other hand, a higher number of layers seems to be associated with a decreased potential for inducing cytotoxicity and ROS production.

In Vivo Genotoxicity and Toxicity Assessment of Sterigmatocystin Individually and in Mixture with Aflatoxin B1.

Mycotoxins are natural food and feed contaminants produced by several molds. The primary mode of exposure in humans and animals is through mixtures. Aflatoxin B1 (AFB1) and sterigmatocystin (STER) are structurally related mycotoxins that share the same biosynthetic route. Few in vivo genotoxicity assays have been performed with STER. In the present genotoxicity study, Wistar rats were dosed orally with STER (20 mg/kg b.w.), AFB1 (0.25 mg/kg b.w.) or a mixture of both in an integrated micronucleus (bone marrow) and comet study (liver and kidney). STER was dosed at the highest feasible dose in corn oil. No increase in the percentage of micronuclei in bone marrow was observed at any condition. Slight DNA damage was detected in the livers of animals treated with AFB1 or the mixture (DNA strand breaks and Fpg (Formamidopyrimidine DNA glycosylase)-sensitive sites, respectively). Plasma, liver, and kidney samples were analyzed with LC-MS/MS demonstrating exposure to both mycotoxins. General toxicity parameters (organs absolute weight, biochemistry, and histopathology) were not altered either individually or in the mixture. The overall absence of individual genotoxicity did not allow us to set any type of interaction in the mixture. However, a possible toxicokinetic interaction was observed.

Genotoxicity of Graphene-Based Materials.

Graphene-based materials (GBMs) are a broad family of novel carbon-based nanomaterials with many nanotechnology applications. The increasing market of GBMs raises concerns on their possible impact on human health. Here, we review the existing literature on the genotoxic potential of GBMs over the last ten years. A total of 50 articles including in vitro, in vivo, in silico, and human biomonitoring studies were selected. Graphene oxides were the most analyzed materials, followed by reduced graphene oxides. Most of the evaluations were performed in vitro using the comet assay (detecting DNA damage). The micronucleus assay (detecting chromosome damage) was the most used validated assay, whereas only two publications reported results on mammalian gene mutations. The same material was rarely assessed with more than one assay. Despite inhalation being the main exposure route in occupational settings, only one in vivo study used intratracheal instillation, and another one reported human biomonitoring data. Based on the studies, some GBMs have the potential to induce genetic damage, although the type of damage depends on the material. The broad variability of GBMs, cellular systems and methods used in the studies precludes the identification of physico-chemical properties that could drive the genotoxicity response to GBMs.

In Vitro Genotoxicity Evaluation of an Antiseptic Formulation Containing Kaolin and Silver Nanoparticles.

Worldwide antimicrobial resistance is partly caused by the overuse of antibiotics as growth promoters. Based on the known bactericidal effect of silver, a new material containing silver in a clay base was developed to be used as feed additive. An in vitro genotoxicity evaluation of this silver-kaolin clay formulation was conducted, which included the mouse lymphoma assay in L5178Y TK+/- cells and the micronucleus test in TK6 cells, following the principles of the OECD guidelines 490 and 487, respectively. As a complement, the standard and Fpg-modified comet assays for the evaluation of strand breaks, alkali labile sites and oxidative DNA damage were also performed in TK6 cells. The formulation was tested without metabolic activation after an exposure of 3 h and 24 h; its corresponding release in medium, after the continuous agitation of the silver-kaolin for 24 h was also evaluated. Under the conditions tested, the test compound did not produce gene mutations, chromosomal aberrations or DNA damage (i.e., strand breaks, alkali labile sites or oxidized bases). Considering the results obtained in the present study, the formulation seems to be a promising material to be used as antimicrobial in animal feed.

Genotoxicity and Toxicity Assessment of a Formulation Containing Silver Nanoparticles and Kaolin: An In Vivo Integrative Approach.

A new material composed of a kaolin base with silver nanoparticles (AgNPs) attached to its surface was developed, as an alternative to antibiotics used as supplements in animal feed. As part of its safety assessment, an in vivo geno-toxicological evaluation of this material was conducted in rats. First, a preliminary dose finding study was carried out to decide the doses to be tested in the main study: 50, 300 and 2000 mg/kg b.w. For the main study, a combined strategy composed of the MN test (TG 474) and the comet assay (TG 489), integrated in a repeated dose 28-day oral toxicity study (TG 407), was performed. A No Observed Adverse Effect Level (NOAEL) of 2000 mg of the silver-kaolin formulation/kg b.w. by oral route, for 28 days, was determined. The silver-kaolin formulation did not induce micronuclei in bone marrow, or DNA strand breaks (SBs) or alkali labile sites (ALS) in liver, spleen, kidney or duodenum at any dose. The modified Fpg comet assay did not reveal oxidized bases in the same tissues at the dose of 2000 mg/kg b.w. Silver was quantified by ICP-MS in all the target organs, confirming the negative results obtained under these conditions.

Genotoxicity of Silver Nanoparticles.

Silver nanoparticles (AgNPs) are widely used in diverse sectors such as medicine, food, cosmetics, household items, textiles and electronics. Given the extent of human exposure to AgNPs, information about the toxicological effects of such products is required to ensure their safety. For this reason, we performed a bibliographic review of the genotoxicity studies carried out with AgNPs over the last six years. A total of 43 articles that used well-established standard assays (i.e., in vitro mouse lymphoma assays, in vitro micronucleus tests, in vitro comet assays, in vivo micronucleus tests, in vivo chromosome aberration tests and in vivo comet assays), were selected. The results showed that AgNPs produce genotoxic effects at all DNA damage levels evaluated, in both in vitro and in vivo assays. However, a higher proportion of positive results was obtained in the in vitro studies. Some authors observed that coating and size had an effect on both in vitro and in vivo results. None of the studies included a complete battery of assays, as recommended by ICH and EFSA guidelines, and few of the authors followed OECD guidelines when performing assays. A complete genotoxicological characterization of AgNPs is required for decision-making.