Gunnar Brunborg

Paternal lifestyle as a potential source of germline mutations transmitted to offspring

Paternal exposure to high levels of radioactivity causes heritable germline minisatellite mutations. However, the effect of more general paternal exposures, such as cigarette smoking, on germline mutations remains unexplored. We analyzed two of the most commonly used minisatellite loci (CEB1 and B6.7) to identify germline mutations in blood samples of complete mother‐father‐child triads from the Norwegian Mother and Child Cohort Study (MoBa). The presence of mutations was subsequently related to general lifestyle factors, including paternal smoking before the partner became pregnant. Paternally derived mutations at the B6.7 locus (mutation frequency 0.07) were not affected by lifestyle. In contrast, high gross yearly income as a general measure of a healthy lifestyle coincided with low‐mutation frequencies at the CEB1 locus (P=0.047). Income was inversely related to smoking behavior, and paternally derived CEB1 mutations were dose dependently increased when the father smoked in the 6 mo before pregnancy, 0.21 vs. 0.05 in smoking and nonsmoking fathers, respectively (P=0.061). These results suggest that paternal lifestyle can affect the chance of heritable mutations in unstable repetitive DNA sequences. To our knowledge, this is the first study reporting an effect of lifestyle on germline minisatellite mutation frequencies in a human population with moderate paternal exposures.—Linschooten, J. O., Verhofstad, N., Gutzkow, K., Olsen, A.‐K., Yauk, C., Oligschläger, Y., Brunborg, G., van Schooten, F. J., Godschalk, R. W. L. Paternal lifestyle as a potential source of germline mutations transmitted to offspring. FASEB J. 27, 2873‐2879 (2013). www.fasebj.org

The comet assay: topical issues

The comet assay is a versatile and sensitive method for measuring single- and double-strand breaks in DNA. The mechanism of formation of comets (under neutral or alkaline conditions) is best understood by analogy with nucleoids, in which relaxation of DNA supercoiling in a structural loop of DNA by a single DNA break releases that loop to extend into a halo-or, in the case of the comet assay, to be pulled towards the anode under the electrophoretic field. A consideration of the simple physics underlying electrophoresis leads to a better understanding of the assay. The sensitivity of the assay is only fully appreciated when it is calibrated: between one hundred and several thousand breaks per cell can be determined. By including lesion-specific enzymes in the assay, its range and sensitivity are greatly increased, but it is important to bear in mind that their specificity is not absolute. Different approaches to quantitation of the comet assay are discussed. Arguments are presented against trying to apply the comet assay to the study of apoptosis. Finally, some of the advantages and disadvantages of using the comet assay on lymphocyte samples collected in human studies are rehearsed.

Differentiation of Human Embryonal Carcinomas In vitro and In vivo Reveals Expression Profiles Relevant to Normal Development

Embryonal carcinoma is a histologic subgroup of testicular germ cell tumors (TGCTs), and its cells may follow differentiation lineages in a manner similar to early embryogenesis. To acquire new knowledge about the transcriptional programs operating in this tumor development model, we used 22k oligo DNA microarrays to analyze normal and neoplastic tissue samples from human testis. Additionally, retinoic acid-induced in vitro differentiation was studied in relevant cell lines. We identified genes characterizing each of the known histologic subtypes, adding up to a total set of 687 differentially expressed genes. Among these, there was a significant overrepresentation of gene categories, such as genomic imprinting and gene transcripts associated to embryonic stem cells. Selection for genes highly expressed in the undifferentiated embryonal carcinomas resulted in the identification of 58 genes, including pluripotency markers, such as the homeobox genes NANOG and POU5F1 (OCT3/4), as well as GAL, DPPA4, and NALP7. Interestingly, abundant expression of several of the pluripotency genes was also detected in precursor lesions and seminomas. By use of tissue microarrays containing 510 clinical testicular samples, GAL and POU5F1 were up-regulated in TGCT also at the protein level and hence validated as diagnostic markers for undifferentiated tumor cells. The present study shows the unique gene expression profiles of each histologic subtype of TGCT from which we have identified deregulated components in selected processes operating in normal development, such as WNT signaling and DNA methylation.

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.

Clinical significance of sperm DNA damage in assisted reproduction outcome

BACKGROUND Sperm DNA damage shows great promise as a biomarker of infertility. The study aim is to determine the usefulness of DNA fragmentation (DF), including modified bases (MB), to predict assisted reproduction treatment (ART) outcomes. METHODS DF in 360 couples (230 IVF and 130 ICSI) was measured by the alkaline Comet assay in semen and in sperm following density gradient centrifugation (DGC) and compared with fertilization rate (FR), embryo cumulative scores (ECS(1)) for the total number of embryos/treatment, embryos transferred (ECS(2)), clinical pregnancy (CP) and spontaneous pregnancy loss. MB were also measured using formamidopyrimidine DNA glycosylase to convert them into strand breaks. RESULTS In IVF, FR and ECS decreased as DF increased in both semen and DGC sperm, and couples who failed to achieve a CP had higher DF than successful couples (+12.2% semen, P = 0.004; +9.9% DGC sperm, P = 0.010). When MB were added to existing strand breaks, total DF was markedly higher (+17.1% semen, P = 0.009 and +13.8% DGC sperm, P = 0.045). DF was not associated with FR, ECS or CP in either semen or DGC sperm following ISCI. In contrast, by including MB, there was significantly more DNA damage (+16.8% semen, P = 0.008 and +15.5% DGC sperm, P = 0.024) in the group who did not achieve CP. CONCLUSIONS DF can predict ART outcome for IVF. Converting MB into further DNA strand breaks increased the test sensitivity, giving negative correlations between DF and CP for ICSI as well as IVF.

Pulmonary exposure to carbon black by inhalation or instillation in pregnant mice: Effects on liver DNA strand breaks in dams and offspring

Abstract Effects of maternal pulmonary exposure to carbon black (Printex 90) on gestation, lactation and DNA strand breaks were evaluated. Time-mated C57BL/6BomTac mice were exposed by inhalation to 42 mg/m3 Printex 90 for 1 h/day on gestation days (GD) 8–18, or by four intratracheal instillations on GD 7, 10, 15 and 18, with total doses of 11, 54 and 268 μg/animal. Dams were monitored until weaning and some offspring until adolescence. Inflammation was assessed in maternal bronchoalveolar lavage (BAL) 3–5 days after exposure, and at weaning. Levels of DNA strand breaks were assessed in maternal BAL cells and liver, and in offspring liver. Persistent lung inflammation was observed in exposed mothers. Inhalation exposure induced more DNA strand breaks in the liver of mothers and their offspring, whereas intratracheal instillation did not. Neither inhalation nor instillation affected gestation and lactation. Maternal inhalation exposure to Printex 90-induced liver DNA damage in the mothers and the in utero exposed offspring.

Nanotitanium dioxide toxicity in mouse lung is reduced in sanding dust from paint

BackgroundLittle is known of how the toxicity of nanoparticles is affected by the incorporation in complex matrices. We compared the toxic effects of the titanium dioxide nanoparticle UV-Titan L181 (NanoTiO2), pure or embedded in a paint matrix. We also compared the effects of the same paint with and without NanoTiO2.MethodsMice received a single intratracheal instillation of 18, 54 and 162 μg of NanoTiO2 or 54, 162 and 486 μg of the sanding dust from paint with and without NanoTiO2. DNA damage in broncheoalveolar lavage cells and liver, lung inflammation and liver histology were evaluated 1, 3 and 28 days after intratracheal instillation. Printex 90 was included as positive control.ResultsThere was no additive effect of adding NanoTiO2 to paints: Therefore the toxicity of NanoTiO2 was reduced by inclusion into a paint matrix. NanoTiO2 induced inflammation in mice with severity similar to Printex 90. The inflammatory response of NanoTiO2 and Printex 90 correlated with the instilled surface area. None of the materials, except of Printex 90, induced DNA damage in lung lining fluid cells. The highest dose of NanoTiO2 caused DNA damage in hepatic tissue 1 day after intratracheal instillation. Exposure of mice to the dust from paints with and without TiO2 was not associated with hepatic histopathological changes. Exposure to NanoTiO2 or to Printex 90 caused slight histopathological changes in the liver in some of the mice at different time points.ConclusionsPulmonary inflammation and DNA damage and hepatic histopathology were not changed in mice instilled with sanding dust from NanoTiO2 paint compared to paint without NanoTiO2. However, pure NanoTiO2 caused greater inflammation than NanoTiO2 embedded in the paint matrix.

Placental transfer of perfluorinated compounds is selective--a Norwegian Mother and Child sub-cohort study.

Perfluorinated compounds (PFCs) comprise a large group of man-made fluorinated chemicals used in a number of consumer products and industrial applications. PFCs have shown to be persistent, bio-accumulative and widespread in the environment. Animal studies have demonstrated hepatotoxicity, immunotoxicity, developmental toxicity as well as hormonal effects. We investigated prenatal exposure to several PFCs and detected up to seven different PFCs in 123 paired samples of human maternal and cord blood, from a subcohort of the Norwegian Mother and Child Cohort Study (MoBa). The maternal and foetal levels were significantly correlated for all PFCs tested with median PFC concentrations in cord blood ranging between 30 and 79% of the maternal concentrations, demonstrating placental passage. The composition of the different PFCs varied between cord and maternal blood, with a higher proportion of shorter chained PFCs together with a higher amount of the branched isomers of perfluorooctane sulfonate (PFOS) in cord blood. Additionally, the sulfonate group seems to impede transfer efficiency. This indicates a selective placental passage of the different PFCs and hence a specific foetal exposure.

Genotoxic effects of the drinking water mutagen 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX) in mammalian cells in vitro and in rats in vivo

The potent bacterial mutagen 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]- furanone) (MX), which is formed during chlorination of drinking water and accounts for about one third of the Ames mutagenicity of tap water, has been studied with respect to its genotoxicity in vitro and in vivo. Treatment with 30-300 microM MX (1 h) induced DNA damage in a concentration-dependent manner in suspensions of rat hepatocytes, as measured by an automated alkaline elution system. The effect was similar in hepatocytes from PCB-induced and uninduced rats. DNA damage was induced in V79 Chinese hamster cells and in isolated rat testicular cells, at the same concentration level as in hepatocytes. Pretreating testicular cells with diethylmaleate, which depletes 85% of cellular glutathione, had no significant effect on the DNA damage induced by MX. The treatment conditions used in the alkaline elution experiments were not cytotoxic to any of the cell types used, as determined by trypan blue exclusion. V79 cells exposed to 2-5 microM MX (2 h) showed an increased frequency of sister-chromatid exchanges (SCE) whereas no significant effect on HGPRT mutation induction was observed. Higher concentrations (greater than 10 microM, 2 h) apparently blocked cell division. The data indicate that MX can react directly with DNA or that MX is metabolized to an ultimate mutagen via some enzyme which is common in mammalian cells. The in vivo experiments showed no evidence of genotoxicity after intraperitoneal (18 mg/kg, 1 h) or oral (18, 63 or 125 mg/kg, 1 h) administration of MX, as measured by alkaline elution, in any of the following organs: the pyloric part of the stomach, the duodenum, colon ascendens, liver, kidney, lung, bone marrow, urinary bladder and the testes. In conclusion, MX is a direct-acting genotoxicant in vitro but no in vivo genotoxicity was detected.

Towards a more reliable comet assay: Optimising agarose concentration, unwinding time and electrophoresis conditions

The comet assay is now the method of choice for measuring most kinds of DNA damage in cells. However, due to the lack of a standardised protocol inter-laboratory comparisons are of limited value. The aim of this paper is to demonstrate how small changes in comet-assay variables may significantly affect the results. We examined the effect of varying agarose concentrations, alkaline unwinding time, electrophoresis time, voltage and current, by use of two cell types, viz. human peripheral blood lymphocytes and the lymphoblastoid cell line TK-6. All these variables have marked effects on assay performance and, therefore, on the determination of DNA damage. Here we identify factors of particular importance.