Christine Instanes

Cytotoxic and genotoxic effects of silver nanoparticles in testicular cells

Serious concerns have been expressed about potential risks of engineered nanoparticles. Regulatory health risk assessment of such particles has become mandatory for the safe use of nanomaterials in consumer products and medicines; including the potential effects on reproduction and fertility, are relevant for this risk evaluation. In this study, we examined effects of silver particles of nano- (20nm) and submicron- (200nm) size, and titanium dioxide nanoparticles (TiO(2)-NPs; 21nm), with emphasis on reproductive cellular- and genotoxicity. Ntera2 (NT2, human testicular embryonic carcinoma cell line), and primary testicular cells from C57BL6 mice of wild type (WT) and 8-oxoguanine DNA glycosylase knock-out (KO, mOgg1(-/-)) genotype were exposed to the particles. The latter mimics the repair status of human testicular cells vs oxidative damage and is thus a suitable model for human male reproductive toxicity studies. The results suggest that silver nano- and submicron-particles (AgNPs) are more cytotoxic and cytostatic compared to TiO(2)-NPs, causing apoptosis, necrosis and decreased proliferation in a concentration- and time-dependent manner. The 200nm AgNPs in particular appeared to cause a concentration-dependent increase in DNA-strand breaks in NT2 cells, whereas the latter response did not seem to occur with respect to oxidative purine base damage analysed with any of the particles tested.

Silver nanoparticles effects on epididymal sperm in rats.

The motivation of our study was to examine the acute effects of intravenously administered a single bolus dose of silver nanoparticles (AgNPs) on rat spermatogenesis and seminiferous tubules morphology. In the treated rats compared to the vehicle treated control animals, the experiments revealed a size-dependent (20nm and 200nm), dose-dependent (5 and 10mg/kg body mass) and time-dependent (24h, 7 and 28days) decrease the epididymal sperm count measured by histological methods. In parallel AgNPs injection increased the level of DNA damage in germ cells, as measured by alkaline comet assay. Histological examination of the testes showed change in the testes seminiferous tubule morphometry in 200nm Ag NPs treated rats. No change of body weight, adipose tissue distribution and the frequency of abnormal spermatozoa was observed. Twenty nanometers AgNP appeared to be more toxic than 200nm ones.

Chapter Five - Toxicity of Silver Nanomaterials in Higher Eukaryotes

Abstract The rapid expansion of nanotechnology promises to have significant benefits to society, yet there is increasing concern that exposure to nanoparticles (particles typically in vitro and in vivo uptake, biodistribution, and toxicity of AgNPs. Emphasis is placed on the systematization of data over animal and cell models, organs examined, doses applied, the type of particle administration, and the time of examination.

Genotoxicity and gene expression modulation of silver and titanium dioxide nanoparticles in mice.

Abstract Recently, we showed that silver nanoparticles (AgNPs) caused apoptosis, necrosis and DNA strand breaks in different cell models in vitro. These findings warranted analyses of their relevance in vivo. We investigated the genotoxic potential and gene expression profiles of silver particles of nano- (Ag20, 20 nm) and submicron- (Ag200, 200 nm) size and titanium dioxide nanoparticles (TiO2-NPs, 21 nm) in selected tissues from exposed male mice including the gonades. A single dose of 5 mg/kg bw nanoparticles was administered intravenously to male mice derived from C57BL6 (WT) and 8-oxoguanine DNA glycosylase knock-out (Ogg1−/− KO). Testis, lung and liver were harvested one and seven days post-exposure and analyzed for DNA strand breaks and oxidized purines employing the Comet assay with Formamidopyrimidine DNA glycosylase (Fpg) treatment, and sperm DNA fragmentation by the sperm chromatin structure assay (SCSA). Based on an initial screening of a panel of 21 genes, seven genes were selected and their expression levels were analyzed in all lung and testis tissues sampled from all animals (n = 6 mice/treatment group) using qPCR. AgNPs, in particular Ag200, caused significantly increased levels of DNA strand breaks and alkali labile sites in lung, seven days post-exposure. Fpg-sensitive lesions were significantly induced in both testis and lung. The transcript level of some key genes; Atm, Rad51, Sod1, Fos and Mmp3, were significantly induced compared to controls, particularly in lung samples from Ag200-exposed KO mice. We conclude that the Ag200 causes genotoxicity and distinct gene expression patterns in selected DNA damage response and repair related genes.

Gamma radiation at a human relevant low dose rate is genotoxic in mice

Even today, 70 years after Hiroshima and accidents like in Chernobyl and Fukushima, we still have limited knowledge about the health effects of low dose rate (LDR) radiation. Despite their human relevance after occupational and accidental exposure, only few animal studies on the genotoxic effects of chronic LDR radiation have been performed. Selenium (Se) is involved in oxidative stress defence, protecting DNA and other biomolecules from reactive oxygen species (ROS). It is hypothesised that Se deficiency, as it occurs in several parts of the world, may aggravate harmful effects of ROS-inducing stressors such as ionising radiation. We performed a study in the newly established LDR-facility Figaro on the combined effects of Se deprivation and LDR γ exposure in DNA repair knockout mice (Ogg1−/−) and control animals (Ogg1+/−). Genotoxic effects were seen after continuous radiation (1.4 mGy/h) for 45 days. Chromosomal damage (micronucleus), phenotypic mutations (Pig-a gene mutation of RBCCD24−) and DNA lesions (single strand breaks/alkali labile sites) were significantly increased in blood cells of irradiated animals, covering three types of genotoxic activity. This study demonstrates that chronic LDR γ radiation is genotoxic in an exposure scenario realistic for humans, supporting the hypothesis that even LDR γ radiation may induce cancer.

Genotoxic effects of two-generational selenium deficiency in mouse somatic and testicular cells

Many studies have investigated genotoxic effects of high Se diets but very few have addressed the genotoxicity of Se deprivation and its consequences in germ cells and none in somatic cells. To address these data gaps, C57BL/6 male mice were subjected to Se deprivation starting in the parental generation, i.e. before conception. Mice were given a diet of either low (0.01mg Se/kg diet) or normal (0.23mg Se/kg diet) Se content. Ogg1-deficient (Ogg1 (-/-) ) mice were used as a sensitive model towards oxidative stress due to their reduced capacity to repair oxidised purines. Ogg1 (-/-) mice also mimic the repair characteristics of human post-meiotic male germ cells which have a reduced ability to repair such lesions. The genotoxicity of Se deficiency was addressed by measuring DNA lesions with the alkaline single cell gel electrophoresis (+ Fpg to detect oxidised DNA lesions) in somatic cells (nucleated blood cells and lung cells) and male germ cells (testicular cells). Total Se concentration in liver and GPx activity in plasma and testicular cells were measured. Gene mutation was evaluated by an erythrocyte-based Pig-a assay. We found that Se deprivation of F1 from their conception and until early adulthood led to the induction of DNA lesions in testicular and lung cells expressed as significantly increased levels of DNA lesions, irrespective of the mouse genotype. In blood cells, Se levels did not appear to affect DNA lesions or mutant cell frequencies. The results suggest that the testis was the most sensitive tissue. Thus, genotoxicity induced by the low Se diet in the spermatozoal genome has potential implications for the offspring.

The Pig-a Gene Mutation Assay in Mice and Human Cells: A Review

This MiniReview describes the principle of mutation assays based on the endogenous Pig-a gene and summarizes results for two species of toxicological interest, mice and human beings. The work summarized here largely avoids rat-based studies, as are summarized elsewhere. The Pig-a gene mutation assay has emerged as a valuable tool for quantifying in vivo and in vitro mutational events. The Pig-a locus is located at the X-chromosome, giving the advantage that one inactivated allele can give rise to a mutated phenotype, detectable by multicolour flow cytometry. For in vivo studies, only minute blood volumes are required, making it easily incorporated into ongoing studies or experiments with limited biological materials. Low blood volumes also allow individuals to serve as their own controls, providing temporal information of the mutagenic process, and/or outcome of intervention. These characteristics make it a promising exposure marker. To date, the Pig-a gene mutation assay has been most commonly performed in rats, while reports regarding its usefulness in other species are accumulating. Besides its applicability to in vivo studies, it holds promise for genotoxicity testing using cultured cells, as shown in recent studies. In addition to safety assessment roles, it is becoming a valuable tool in basic research to identify mutagenic effects of different interventions or to understand implications of various gene defects by investigating modified mouse models or cell systems. Human blood-based assays are also being developed that may be able to identify genotoxic environmental exposures, treatment- and lifestyle-related factors or endogenous host factors that contribute to mutagenesis.

No adverse lung effects of 7- and 28-day inhalation exposure of rats to emissions from petrodiesel fuel containing 20% rapeseed methyl esters (B20) with and without particulate filter – the FuelHealth project

Abstract Increased use of biofuels raises concerns about health effects of new emissions. We analyzed relative lung health effects, on Fisher 344 rats, of diesel engine exhausts emissions (DEE) from a Euro 5-classified diesel engine running on petrodiesel fuel containing 20% rapeseed methyl esters (B20) with and without diesel particulate filter (DPF). One group of animals was exposed to DEE for 7 days (6 h/day), and another group for 28 days (6 h/day, 5 days/week), both with and without DPF. The animals (n = 7/treatment) were exposed in whole body exposure chambers. Animals breathing clean air were used as controls. Genotoxic effects of the lungs by the Comet assay, histological examination of lung tissue, bronchoalveolar lavage fluid (BALF) markers of pulmonary injury, and mRNA markers of inflammation and oxidative stress were analyzed. Our results showed that a minor number of genes related to inflammation were slightly differently expressed in the exposed animals compared to control. Histological analysis also revealed only minor effects on inflammatory tissue markers in the lungs, and this was supported by flow cytometry and ELISA analysis of cytokines in BALF. No exposure-related indications of genotoxicity were observed. Overall, exposure to DEE with or without DPF technology produced no adverse effects in the endpoints analyzed in the rat lung tissue or the BALF. Overall, exposure to DEE from a modern Euro 5 light vehicle engine run on B20 fuel with or without DPF technology produced no adverse effects in the endpoints analyzed in the rat lung tissue or the BALF.