Maternal exercise during pregnancy modulates genotoxicity caused by high fructose consumption in mice offspring.

Pregnancy is a period that is characterized by several metabolic and physiological changes and requires special attention, especially with regard to the relationship between feeding and foetal development. Therefore, the objective of this study was to evaluate whether the practice of voluntary physical exercise (VPE) in combination with chronic consumption of fructose (FRU) from the beginning of life and/or until the gestational period causes genotoxic changes in pregnant females and in their offspring. Seventy Swiss female mice received FRU in the hydration bottle and/or practiced VPE for 8 weeks (prepregnancy/pregnancy). After the lactation period, the offspring groups were separated by sex. It was observed that the consumption of FRU affected the food consumption, serum concentration of FRU, and glycemic profile in the mothers and that the VPE decreases these parameters. In addition, FRU was genotoxic in the mothers' peripheral tissues and VPE had a preventive effect on these parameters. The offspring showed changes in food consumption, serum FRU concentration, and body weight, in addition to an increase in the adiposity index in male offspring in the FRU (FRU) group and a decrease in the FRU + VPE group. FRU leads to hepatic steatosis in the offspring and VPE was able to decrease the area of steatosis. In addition, FRU led to genotoxicity in the offspring and VPE was able to modulate this effect, reducing damages. In conclusion, we observed that all interventions with VPE had nutritional, genetic, and biochemical benefits of the mother and her offspring.

Visual comet scoring revisited: a guide to scoring comet assay slides and obtaining reliable results.

Measurement of DNA migration in the comet assay can be done by image analysis or visual scoring. The latter accounts for 20%-25% of the published comet assay results. Here we assess the intra- and inter-investigator variability in visual scoring of comets. We include three training sets of comet images, which can be used as reference for researchers who wish to use visual scoring of comets. Investigators in 11 different laboratories scored the comet images using a five-class scoring system. There is inter-investigator variation in the three training sets of comets (i.e. coefficient of variation (CV) = 9.7%, 19.8%, and 15.2% in training sets I-III, respectively). However, there is also a positive correlation of inter-investigator scoring in the three training sets (r = 0.60). Overall, 36% of the variation is attributed to inter-investigator variation and 64% stems from intra-investigator variation in scoring between comets (i.e. the comets in training sets I-III look slightly different and this gives rise to heterogeneity in scoring). Intra-investigator variation in scoring was also assessed by repeated analysis of the training sets by the same investigator. There was larger variation when the training sets were scored over a period of six months (CV = 5.9%-9.6%) as compared to 1 week (CV = 1.3%-6.1%). A subsequent study revealed a high inter-investigator variation when premade slides, prepared in a central laboratory, were stained and scored by investigators in different laboratories (CV = 105% and 18%-20% in premade slides with comets from unexposed and hydrogen peroxide-exposed cells, respectively). The results indicate that further standardization of visual scoring is desirable. Nevertheless, the analysis demonstrates that visual scoring is a reliable way of analysing DNA migration in comets.

Inter-laboratory variation in measurement of DNA damage by the alkaline comet assay in the hCOMET ring trial.

The comet assay is a simple and versatile method for measurement of DNA damage in eukaryotic cells. More specifically, the assay detects DNA migration from agarose gel-embedded nucleoids, which depends on assay conditions and the level of DNA damage. Certain steps in the comet assay procedure have substantial impact on the magnitude of DNA migration (e.g. electric potential and time of electrophoresis). Inter-laboratory variation in DNA migration levels occurs because there is no agreement on optimal assay conditions or suitable assay controls. The purpose of the hCOMET ring trial was to test potassium bromate (KBrO3) as a positive control for the formamidopyrimidine DNA glycosylase (Fpg)-modified comet assay. To this end, participating laboratories used semi-standardized protocols for cell culture (i.e. cell culture, KBrO3 exposure, and cryopreservation of cells) and comet assay procedures, whereas the data acquisition was not standardized (i.e. staining of comets and image analysis). Segregation of the total variation into partial standard deviation (SD) in % Tail DNA units indicates the importance of cell culture procedures (SD = 10.9), comet assay procedures (SD = 12.3), staining (SD = 7.9) and image analysis (SD = 0.5) on the overall inter-laboratory variation of DNA migration (SD = 18.2). Future studies should assess sources of variation in each of these steps. On the positive side, the hCOMET ring trial demonstrates that KBrO3 is a robust positive control for the Fpg-modified comet assay. In conclusion, the hCOMET ring trial has demonstrated a high reproducibility of detecting genotoxic effects by the comet assay, but inter-laboratory variation of DNA migration levels is a concern.

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.

Epigenome-wide analysis of maternal exposure to green space during gestation and cord blood DNA methylation in the ENVIRONAGE cohort.

BACKGROUND: DNA methylation programming is sensitive to prenatal life environmental influences, but the impact of maternal exposure to green space on newborns DNA methylation has not been studied yet. METHODS: We conducted a meta-epigenome-wide association study (EWAS) of maternal exposure to green space during gestation with cord blood DNA methylation in two subsets of the ENVIRONAGE cohort (N = 538). Cord blood DNA methylation was measured by Illumina HumanMethylation 450K in one subset (N = 189) and EPICarray in another (N = 349). High (vegetation height>3 m (m)), low (vegetation height<3 m) and total (including both) high-resolution green space exposures during pregnancy were estimated within 100 m and 1000 m distance around maternal residence. In each subset, we sought cytosine-phosphate-guanine (CpG) sites via linear mixed models adjusted on newborns' sex, ethnicity, gestational age, season at delivery, sampling day, maternal parity, age, smoking, education, and estimated blood cell proportions. EWASs results were meta-analysed via fixed-effects meta-analyses. Differentially methylated regions (DMRs) were identified via ENmix-combp and DMRcate algorithms. Sensitivity analyses were additionally adjusted on PM2.5, distance to major roads, urbanicity and neighborhood income. In the 450K subset, cord blood expression of differentially methylated genes was measured by Agilent microarrays and associated with green space. RESULTS: 147 DMRs were identified, 85 of which were still significant upon adjustment for PM2.5, distance to major roads, urbanicity and neighborhood income, including HLA-DRB5, RPTOR, KCNQ1DN, A1BG-AS1, HTR2A, ZNF274, COL11A1 and PRSS36 DMRs. One CpG reached genome-wide significance, while 54 CpGs were suggestive significant (p-values<1e-05). Among them, a CpG, hypermethylated with 100 m buffer total green space, was annotated to PAQR9, whose expression decreased with 1000 m buffer low green space (p-value = 1.45e-05). CONCLUSIONS: Our results demonstrate that maternal exposure to green space during pregnancy is associated with cord blood DNA methylation, mainly at loci organized in regions, in genes playing important roles in neurological development (e.g., HTR2A).

Optimizing the Comet Assay-Based In Vitro DNA Repair Assay for Placental Tissue: A Pilot Study with Pre-Eclamptic Patients.

The comet assay-based in vitro DNA repair assay has become a common tool for quantifying base excision repair (BER) activity in human lymphocytes or cultured cells. Here, we optimized the protocol for studying BER in human placental tissue because the placenta is a non-invasive tissue for biomonitoring of early-life exposures, and it can be used to investigate molecular mechanisms associated with prenatal disorders. The optimal protein concentration of placental protein extracts for optimal damage recognition and incision was 2 mg protein/mL. The addition of aphidicolin did not lead to reduced non-specific incisions and was, therefore, not included in the optimized protocol. The interval between sample collection and analysis did not affect BER activity up to 70 min. Finally, this optimized protocol was tested on pre-eclamptic (PE) placental tissues (n = 11) and significantly lower BER activity in PE placentas compared to controls (n = 9) was observed. This was paralleled by a significant reduction in the expression of BER-related genes and increased DNA oxidation in PE placentas. Our study indicates that BER activity can be determined in placentas, and lower activity is present in PE compared with healthy. These findings should be followed up in prospective clinical investigations to examine BER's role in the advancement of PE.

Regenerative responses following DNA damage - ß-catenin mediates head regrowth in the planarian Schmidtea mediterranea.

Pluripotent stem cells hold great potential for regenerative medicine. Increased replication and division, such is the case during regeneration, concomitantly increases the risk of adverse outcomes through the acquisition of mutations. Seeking for driving mechanisms of such outcomes, we challenged a pluripotent stem cell system during the tightly controlled regeneration process in the planarian Schmidtea mediterranea Exposure to the genotoxic compound methyl methanesulfonate (MMS) revealed that despite a similar DNA-damaging effect along the anteroposterior axis of intact animals, responses differed between anterior and posterior fragments after amputation. Stem cell proliferation and differentiation proceeded successfully in the amputated heads, leading to regeneration of missing tissues. Stem cells in the amputated tails showed decreased proliferation and differentiation capacity. As a result, tails could not regenerate. Interference with the body-axis-associated component ß-catenin-1 increased regenerative success in tail fragments by stimulating proliferation at an early time point. Our results suggest that differences in the Wnt signalling gradient along the body axis modulate stem cell responses to MMS.

Upregulation of mNEIL3 in Ogg1-null cells is a potential backup mechanism for 8-oxoG repair.

Reactive oxygen species formation and resultant oxidative damage to DNA are ubiquitous events in cells, the homeostasis of which can be dysregulated in a range of pathological conditions. Base excision repair (BER) is the primary repair mechanism for oxidative genomic DNA damage. One prevalent oxidised base modification, 8-oxoguanine (8-oxoG), is recognised by 8-oxoguanine glycosylase-1 (OGG1) initiating removal and repair via BER. Surprisingly, Ogg1 null mouse embryonic fibroblasts (mOgg1-/- MEFs) do not accumulate 8-oxoG in the genome to the extent expected. This suggests that there are backup repair mechanisms capable of repairing 8-oxoG in the absence of OGG1. In the current study, we identified components of NER (Ercc1, Ercc4, Ercc5), BER (Lig1, Tdg, Nthl1, Mpg, Mgmt, NEIL3), MMR (Mlh1, Msh2, Msh6) and DSB (Brip1, Rad51d, Prkdc) pathways that are transcriptionally elevated in mOgg1-/- MEFs. Interestingly, all three nucleotide excision repair genes identified: Ercc1 (2.5 ± 0.2-fold), Ercc4 (1.5 ± 0.1-fold) and Ercc5 (1.7 ± 0.2-fold) have incision activity. There was also a significant functional increase in NER activity (42.0 ± 7.9%) compared to WT MEFs. We also observed upregulation of both Neil3 mRNA (37.9 ± 1.6-fold) and protein in mOgg1-/- MEFs. This was associated with a 3.4 ± 0.4-fold increase in NEIL3 substrate sites in genomic DNA of cells treated with BSO, consistent with the ability of NEIL3 to remove 8-oxoG oxidation products from genomic DNA. In conclusion, we suggest that in Ogg1-null cells, upregulation of multiple DNA repair proteins including incision components of the NER pathway and Neil3 are important compensatory responses to prevent the accumulation of genomic 8-oxoG.

Collection and storage of human white blood cells for analysis of DNA damage and repair activity using the comet assay in molecular epidemiology studies.

DNA damage and repair activity are often assessed in blood samples from humans in different types of molecular epidemiology studies. However, it is not always feasible to analyse the samples on the day of collection without any type of storage. For instance, certain studies use repeated sampling of cells from the same subject or samples from different subjects collected at different time-points, and it is desirable to analyse all these samples in the same comet assay experiment. In addition, flawless comet assay analyses on frozen samples open up the possibility of using this technique on biobank material. In this article we discuss the use of cryopreserved peripheral blood mononuclear cells (PBMCs), buffy coat (BC) and whole blood (WB) for analysis of DNA damage and repair using the comet assay. The published literature and the authors' experiences indicate that various types of blood samples can be cryopreserved with only a minor effect on the basal level of DNA damage. There is evidence to suggest that WB and PBMCs can be cryopreserved for several years without much effect on the level of DNA damage. However, care should be taken when cryopreserving WB and BCs. It is possible to use either fresh or frozen samples of blood cells, but results from fresh and frozen cells should not be used in the same dataset. The article outlines detailed protocols for the cryopreservation of PBMCs, BCs and WB samples.

Potassium bromate as positive assay control for the Fpg-modified comet assay.

The comet assay is a popular assay in biomonitoring studies. DNA strand breaks (or unspecific DNA lesions) are measured using the standard comet assay. Oxidative stress-generated DNA lesions can be measured by employing DNA repair enzymes to recognise oxidatively damaged DNA. Unfortunately, there has been a tendency to fail to report results from assay controls (or maybe even not to employ assay controls). We believe this might have been due to uncertainty as to what really constitutes a positive control. It should go without saying that a biomonitoring study cannot have a positive control group as it is unethical to expose healthy humans to DNA damaging (and thus potentially carcinogenic) agents. However, it is possible to include assay controls in the analysis (here meant as a cryopreserved sample of cells i.e. included in each experiment as a reference sample). In the present report we tested potassium bromate (KBrO3) as a positive comet assay control for the formamidopyrimidine DNA glycosylase (Fpg)-modified comet assay. Ten laboratories used the same procedure for treatment of monocytic THP-1 cells with KBrO3 (0.5, 1.5 and 4.5 mM for 1 h at 37°C) and subsequent cryopreservation. Results from one laboratory were excluded in the statistical analysis because of technical issues in the Fpg-modified comet assay. All other laboratories found a concentration-response relationship in cryopreserved samples (regression coefficients from 0.80 to 0.98), although with different slopes ranging from 1.25 to 11.9 Fpg-sensitive sites (%DNA in tail) per 1 mM KBrO3. Our results demonstrate that KBrO3 is a suitable positive comet assay control.

The comet assay in human biomonitoring: cryopreservation of whole blood and comparison with isolated mononuclear cells.

The comet assay is often applied in human biomonitoring. Most of the time the assay is performed with isolated peripheral blood mononuclear cells (PBMC). However, using whole blood instead of isolated cells reduces processing time, and only 20 µl is sufficient for analysis. In this study, a cryopreservation protocol for human whole blood for application in the comet assay was optimised by removing excess plasma before adding freezing medium. Cryopreservation of whole blood samples (n = 30) did not increase the detected level of strand breaks and formamidopyrimidine DNA glycosylase (FPG)-sensitive sites. Although there was no significant correlation with breaks measured in fresh whole blood, strand breaks detected in frozen whole blood were significantly correlated with breaks measured in frozen PBMC (Pearson correlation r = 0.54, P < 0.01). This correlation was however not observed for FPG-sensitive sites. Since we do not yet know the full extent to which cryopreservation might influence the blood cell population, care should be taken to ensure a similar cell type and storage conditions for all samples in one study.

RELIC: a novel dye-bias correction method for Illumina Methylation BeadChip.

BACKGROUND: The Illumina Infinium HumanMethylation450 BeadChip and its successor, Infinium MethylationEPIC BeadChip, have been extensively utilized in epigenome-wide association studies. Both arrays use two fluorescent dyes (Cy3-green/Cy5-red) to measure methylation level at CpG sites. However, performance difference between dyes can result in biased estimates of methylation levels. RESULTS: Here we describe a novel method, called REgression on Logarithm of Internal Control probes (RELIC) to correct for dye bias on whole array by utilizing the intensity values of paired internal control probes that monitor the two color channels. We evaluate the method in several datasets against other widely used dye-bias correction methods. Results on data quality improvement showed that RELIC correction statistically significantly outperforms alternative dye-bias correction methods. We incorporated the method into the R package ENmix, which is freely available from the Bioconductor website ( https://www.bioconductor.org/packages/release/bioc/html/ENmix.html ). CONCLUSIONS: RELIC is an efficient and robust method to correct for dye-bias in Illumina Methylation BeadChip data. It outperforms other alternative methods and conveniently implemented in R package ENmix to facilitate DNA methylation studies.

The next three decades of the comet assay: a report of the 11th International Comet Assay Workshop.

The International Comet Assay Workshops are a series of scientific conferences dealing with practical and theoretical aspects of the Comet Assay (single-cell gel electrophoresis)-a simple method for detecting DNA strand breaks. The first paper describing such an assay was published over 30 years ago in 1984 by Swedish researchers O. Ostling and K. J. Johanson. Appropriately, the theme for the 2015 meeting was looking to the future: 'The Next 3 Decades of the Comet Assay'. The programme included 25 oral and 43 poster presentations depicting the latest advances in technical developments as well as applications of the comet assay in genotoxicity testing (in vitro and in vivo) and biomonitoring of both humans and the environment. Open discussion sessions based on questions from the participants allowed exchange of practical details on current comet assay protocols. This report summarises technical issues of high importance which were discussed during the sessions. We provide information on ways to improve the assay performance, by testing for cytotoxicity, by using reference samples to reduce or allow for inter-experimental variation, and by standardising quantification of the damage, including replicates and scoring enough comets to ensure statistical validity. After 30 years of experimentation with the comet assay, we are in a position to control the important experimental parameters and make the comet assay a truly reliable method with a wealth of possible applications.

Causes of genome instability: the effect of low dose chemical exposures in modern society.

Genome instability is a prerequisite for the development of cancer. It occurs when genome maintenance systems fail to safeguard the genome's integrity, whether as a consequence of inherited defects or induced via exposure to environmental agents (chemicals, biological agents and radiation). Thus, genome instability can be defined as an enhanced tendency for the genome to acquire mutations; ranging from changes to the nucleotide sequence to chromosomal gain, rearrangements or loss. This review raises the hypothesis that in addition to known human carcinogens, exposure to low dose of other chemicals present in our modern society could contribute to carcinogenesis by indirectly affecting genome stability. The selected chemicals with their mechanisms of action proposed to indirectly contribute to genome instability are: heavy metals (DNA repair, epigenetic modification, DNA damage signaling, telomere length), acrylamide (DNA repair, chromosome segregation), bisphenol A (epigenetic modification, DNA damage signaling, mitochondrial function, chromosome segregation), benomyl (chromosome segregation), quinones (epigenetic modification) and nano-sized particles (epigenetic pathways, mitochondrial function, chromosome segregation, telomere length). The purpose of this review is to describe the crucial aspects of genome instability, to outline the ways in which environmental chemicals can affect this cancer hallmark and to identify candidate chemicals for further study. The overall aim is to make scientists aware of the increasing need to unravel the underlying mechanisms via which chemicals at low doses can induce genome instability and thus promote carcinogenesis.

Maternal folate depletion and high-fat feeding from weaning affects DNA methylation and DNA repair in brain of adult offspring.

The mechanisms through which environmental and dietary factors modulate DNA repair are still unclear but may include dysregulation of gene expression due to altered epigenetic markings. In a mouse model, we investigated the effect of maternal folate depletion during pregnancy and lactation, and high-fat feeding from weaning, on base excision repair (BER) and DNA methylation and expression of selected BER-related genes in the brain of adult offspring. While folate depletion did not affect BER activity of the mothers, BER increased in the offspring at weaning (P=0.052). In the long term, as observed in 6-mo-old offspring, the double insult, i.e., maternal low-folate supply and high-fat feeding from weaning, decreased BER activity significantly in the cortex, cerebellum, hippocampus, and subcortical regions (P≤0.017). This fall in BER activity was associated with small changes in methylation or expression of BER-related genes. Maternal folate depletion led to slightly increased oxidative DNA damage levels in subcortical regions of adult offspring, which may increase sensitivity to oxidative stress and predispose to neurological disorders. In summary, our data suggest that low-folate supply during early life may leave an epigenetic mark that can predispose the offspring to further dietary insults, causing adverse effects during adult life.

Further development of CometChip technology to measure DNA damage in vitro and in vivo: Comparison with the 2 gels/slide format of the standard and enzyme-modified comet assay.

DNA damage plays a pivotal role in carcinogenesis and other diseases. The comet assay has been used for more than three decades to measure DNA damages. The 1-2 gels/slide format is the most used version of the assay. In 2010, a high throughput 96 macrowell format with a spatially encoded array of microwells patterned in agarose was developed, called the CometChip. The commercial version (CometChip®) has been used for the in vitro standard version of the comet assay (following the manufacturer's protocol), although it has not been compared directly with the 2 gels/slide format. The aim of this work is to developed new protocols to allow use of DNA repair enzymes as well as the analysis of in vivo frozen tissue samples in the CometChip®, to increase the throughput, and to compare its performance with the classic 2 gels/slide format. We adapted the manufacturer's protocol to allow the use of snap frozen tissue samples, using male Wistar rats orally dosed with methyl methanesulfonate (MMS, 200 mg/kg b.w.), and to detect altered nucleobases using DNA repair enzymes, with TK6 cells treated with potassium bromate (KBrO3, 0-4 mM, 3 h) and formamidopyrimidine DNA glycosylase (Fpg) as the enzyme. Regarding the standard version of the comet, we performed thee comparison of the 2 gel/slide and CometChip® format (using the the manufacturer's protocol), using TK6 cells with MMS (100-800 µM, 1 h) and hydrogen peroxide (H2O2, 7.7-122.5 µM, 5 min) as testing compounds. In all cases the CometChip® was performed along with the 2 gels/slide format. Results obtained were comparable and the CometChip® is a good alternative to the 2 gels/slide format when a higher throughput is required.

Tissue differences in BER-related incision activity and non-specific nuclease activity as measured by the comet assay.

DNA repair mechanisms are important for genome stability and to prevent accumulation of DNA damage, which contributes to cellular ageing and cancer development. Study of these physiological processes requires robust and practical assays to quantify DNA repair capacity. The in vitro comet-based assay is a simple, yet reliable, assay for measurement of DNA repair and has been modified recently to quantify DNA incision activity in mouse brain and liver. In this study, we applied this assay to assess DNA incision activity in other mouse tissues, i.e. lung and colon, and found that high, non-specific nuclease activity was a problem when measuring DNA incision activity, especially in the colon. We tested the utility of multiple optimisation steps including addition of aphidicolin, ATP and polyAT and used multiple wash steps, which resulted in modest improvements in performance of the assay. Washing the tissues before protein extraction and decreasing the protein concentration in the assay were the most effective steps in reducing non-specific nuclease activity. Using the comet-based assay with these further modifications, we found that base excision repair incision activity changed with age differently in each tissue. This study shows that non-specific nuclease activity in the comet-based assay for DNA repair is more pronounced in some tissues than others so care should be taken to optimise the protocol when applying the assay to a new tissue. Our data suggest the importance of using control cells (noRo cells incubated with extract) in the assay to assess for non-specific nuclease activity. In conclusion, the comet-based DNA repair assay can be easily adapted to study a range of mammalian tissues.

DNA-repair measurements by use of the modified comet assay: an inter-laboratory comparison within the European Comet Assay Validation Group (ECVAG).

The measurement of DNA-repair activity by extracts from cells or tissues by means of the single-cell gel electrophoresis (comet) assay has a high potential to become widely used in biomonitoring studies. We assessed the inter-laboratory variation in reported values of DNA-repair activity on substrate cells that had been incubated with Ro19-8022 plus light to generate oxidatively damaged DNA. Eight laboratories assessed the DNA-repair activity of three cell lines (i.e. one epithelial and two fibroblast cell lines), starting with cell pellets or with cell extracts provided by the coordinating laboratory. There was a large inter-laboratory variation, as evidenced by the range in the mean level of repair incisions between the laboratory with the lowest (0.002incisions/10(6)bp) and highest (0.988incisions/10(6)bp) incision activity. Nevertheless, six out of eight laboratories reported the same cell line as having the highest level of DNA-repair activity. The two laboratories that reported discordant results (with another cell line having the highest level of DNA-repair activity) were those that reported to have little experience with the modified comet assay to assess DNA repair. The laboratories were also less consistent in ordering the repair activity of the other two cell lines, probably because the DNA-repair activity by extracts from these cell lines were very similar (on average approximately 60-65% of the cell line with the highest repair capacity). A significant correlation was observed between the repair activity found in the provided and the self-made cell extracts (r=0.71, P<0.001), which indicates that the predominant source for inter-laboratory variation is derived from the incubation of the extract with substrate cells embedded in the gel. Overall, we conclude that the incubation step of cell extracts with the substrate cells can be identified as a major source of inter-laboratory variation in the modified comet assay for base-excision repair.

Analysis of DNA methylation at birth and in childhood reveals changes associated with season of birth and latitude.

BACKGROUND: Seasonal variations in environmental exposures at birth or during gestation are associated with numerous adult traits and health outcomes later in life. Whether DNA methylation (DNAm) plays a role in the molecular mechanisms underlying the associations between birth season and lifelong phenotypes remains unclear. METHODS: We carried out epigenome-wide meta-analyses within the Pregnancy And Childhood Epigenetic Consortium to identify associations of DNAm with birth season, both at differentially methylated probes (DMPs) and regions (DMRs). Associations were examined at two time points: at birth (21 cohorts, N = 9358) and in children aged 1-11 years (12 cohorts, N = 3610). We conducted meta-analyses to assess the impact of latitude on birth season-specific associations at both time points. RESULTS: We identified associations between birth season and DNAm (False Discovery Rate-adjusted p values < 0.05) at two CpGs at birth (winter-born) and four in the childhood (summer-born) analyses when compared to children born in autumn. Furthermore, we identified twenty-six differentially methylated regions (DMR) at birth (winter-born: 8, spring-born: 15, summer-born: 3) and thirty-two in childhood (winter-born: 12, spring and summer: 10 each) meta-analyses with few overlapping DMRs between the birth seasons or the two time points. The DMRs were associated with genes of known functions in tumorigenesis, psychiatric/neurological disorders, inflammation, or immunity, amongst others. Latitude-stratified meta-analyses [higher (≥ 50°N), lower (< 50°N, northern hemisphere only)] revealed differences in associations between birth season and DNAm by birth latitude. DMR analysis implicated genes with previously reported links to schizophrenia (LAX1), skin disorders (PSORS1C, LTB4R), and airway inflammation including asthma (LTB4R), present only at birth in the higher latitudes (≥ 50°N). CONCLUSIONS: In this large epigenome-wide meta-analysis study, we provide evidence for (i) associations between DNAm and season of birth that are unique for the seasons of the year (temporal effect) and (ii) latitude-dependent variations in the seasonal associations (spatial effect). DNAm could play a role in the molecular mechanisms underlying the effect of birth season on adult health outcomes.