DNA damage in storage cells of anhydrobiotic tardigrades

In order to recover without any apparent damage, tardigrades have evolved effective adaptations to preserve the integrity of cells and tissues in the anhydrobiotic state. Despite those adaptations and the fact that the process of biological ageing comes to a stop during anhydrobiosis, the time animals can persist in this state is limited; after exceedingly long anhydrobiotic periods tardigrades fail to recover. Using the single cell gel electrophoresis (comet assay) technique to study the effect of anhydrobiosis on the integrity of deoxyribonucleic acid, we showed that the DNA in storage cells of the tardigrade Milnesium tardigradum was well protected during transition from the active into the anhydrobiotic state. Specimens of M. tardigradum that had been desiccated for two days had only accumulated minor DNA damage (2.09 ± 1.98% DNA in tail, compared to 0.44 ± 0.74% DNA in tail for the negative control with active, hydrated animals). Yet the longer the anhydrobiotic phase lasted, the more damage was inflicted on the DNA. After six weeks in anhydrobiosis, 13.63 ± 6.41% of DNA was found in the comet tail. After ten months, 23.66 ± 7.56% of DNA was detected in the comet tail. The cause for this deterioration is unknown, but oxidative processes mediated by reactive oxygen species are a possible explanation.     

Polymorphisms in XPC and XPD genes modulate DNA damage in pesticide-exposed agricultural workers of Punjab,North-West India

Molecular Biology Reports - The genetic susceptibility of individuals to the genotoxic effect of pesticides may be modulated by variations in genes involved in nucleotide excision repair (NER)...     

Simplified method for the collection, storage, and comet assay analysis of DNA damage in whole blood

Single-cell gel electrophoresis (comet assay) is one of the most common methods used to measure oxidatively damaged DNA in peripheral blood mononuclear cells (PBMC), as a biomarker of oxidative stress in vivo. However, storage, extraction, and assay workup of blood samples are associated with a risk of artifactual formation of damage. Previous reports using this approach to study DNA damage in PBMC have, for the most part, required the isolation of PBMC before immediate analysis or freezing in cryopreservative. This is very time-consuming and a significant drain on human resources. Here, we report the successful storage of whole blood in ~ 250 μl volumes, at − 80 °C, without cryopreservative, for up to 1 month without artifactual formation of DNA damage. Furthermore, this blood is amenable for direct use in both the alkaline and the enzyme-modified comet assay, without the need for prior isolation of PBMC. In contrast, storage of larger volumes (e.g., 5 ml) of whole blood leads to an increase in damage with longer term storage even at − 80 °C, unless a cryopreservative is present. Our “small volume” approach may be suitable for archived blood samples, facilitating analysis of biobanks when prior isolation of PBMC has not been performed.     

Nuclear anomalies in the buccal cells of calcite factory workers

The micronucleus (MN) assay on exfoliated buccal cells is a useful and minimally invasive method for monitoring genetic damage in humans. To determine the genotoxic effects of calcite dust that forms during processing, MN assay was carried out in exfoliated buccal cells of 50 (25 smokers and 25 non-smokers) calcite factory workers and 50 (25 smokers and 25 non-smokers) age- and sex-matched control subjects. Frequencies of nuclear abnormalities (NA) other than micronuclei, such as binucleates, karyorrhexis, karyolysis and 'broken eggs', were also evaluated. Micronuclei and the other aforementioned anomalies were analysed by two way analysis of covariance. The linear correlations between the types of micronucleus and nuclear abnormalities were determined by Spearman's Rho. There was a positive correlation between micronuclei and other types of nuclear abnormalities in accordance with the Spearman's Rho test. Results showed statistically significant difference between calcite fabric workers and control groups. MN and NA frequencies in calcite fabric workers were significantly higher than those in control groups (p < 0.05). The results of this study indicate that calcite fabric workers are under risk of significant cytogenetic damage.     

Lymphocyte DNA damage in bus manufacturing workers

To study the effect of occupational exposure, smoking, and drinking on lymphocyte DNA damage in bus manufacturing workers, 346 employees (106 women and 240 men) from six job categories (welders, mechanics, painters, and assembling, auxiliary and managerial workers) in a bus manufacturing factory in Guangzhou were included. Significant differences of tail moment among the six job categories were found (P=0.003) with adjustment for age and gender. Smoking increased tail moment significantly (3.14 (2.89-3.40) versus 2.79 microm (2.63-2.97), P=0.023). Analysis of covariance showed that occupational exposure (P=0.001) and smoking (P=0.019) had significant effect on tail moment after adjusting for all factors, whereas age and gender had no effect on DNA damage. Stratified analysis showed that painters (P=0.002), auxiliary workers (P=0.011), and mechanics (P=0.044) had larger tail moments than managerial workers after adjusting for age, gender, smoking, and drinking.     

Micronucleus formation and DNA damage in buccal epithelial cells of Indian street boys addicted to gasp 'Golden glue'

Genotoxicity of glue sniffing/huffing and tobacco use has been examined in 302 street boys (median age 13 years) and 50 age-matched control school boys who were neither tobacco nor glue users. All the street boys were tobacco users. In addition, 155 were addicted to gasp an industrial adhesive popularly known as 'Golden glue'. Micronucleus (MN) frequency was determined as a measure of chromosomal breakage in exfoliated buccal epithelial cells (BECs) and DNA double strand breaks were quantitatively assessed by counting γ-H2AX foci using immunofluorescence microscopy. Micronucleated cell frequencies (MCFs) in BEC of glue non-addicted (only tobacco) and addicted (tobacco plus glue) street boys were 1.87 ± 1.06‰ and 4.04 ± 2.55‰ respectively, which were significantly higher than that of control (0.32 ± 0.11‰, p<0.0001). Similarly, the numbers γ-H2AX foci in nuclei of BEC were 2.3- and 5.2-times more than control in glue non-addicted and addicted street boys respectively (p<0.0001). Spearman's rank correlation revealed a strong positive association between years of glue addiction with MCFs and γ-H2AX foci numbers, and the association between glue addiction and chromosomal and DNA damage remained positive and significant after controlling income, spending on addiction and loss of appetite as potential confounders in multivariate logistic regression analysis. Thus, addiction to tobacco among the street children in India is associated with chromosomal and DNA damage in BECs and the severity of these changes is significantly increased by the habit of sniffing/huffing of industrial glue.     

Genotyping accuracy for whole-genome amplification of DNA from buccal epithelial cells.

We compared the accuracy of genotyping for DNA extracted from lymphocytes to that of DNA amplified from buccal epithelial cells. Amplification was via a rolling circle/phi29 DNA polymerase commercial kit. Paired buccal and lymphocyte DNA samples were available from 30 individuals. All samples were genotyped for 12 SNPs, 5 microsatellites and 2 VNTRs. The accuracy of genotyping (no-call proportions, reproducibility, and concordance) was similar for DNA from lymphocytes in comparison to amplified DNA from buccal samples. If used with caution, these data suggest that rolling-circle whole-genome amplification can be used to increase the DNA mass available for large-scale genotyping projects based on DNA from buccal cells.     

DNA breakage detection-fluorescence in situ hybridization in buccal cells

DNA breakage detection-fluorescence in situ hybridization (DBD-FISH) is a recently developed technique that allows cell-by-cell detection and quantification of DNA breakage in the whole genome or within specific DNA sequences. The present investigation was conducted to adapt the methodology of DBD-FISH to the visualization and evaluation of DNA damage in buccal epithelial cells. DBD-FISH revealed that DNA damage increased significantly according to H₂O₂ concentration (r²=0.91). In conclusion, the DBD-FISH technique is easy to apply in buccal cells and provides prompt results that are easy to interpret. Future studies are needed to investigate the potential applicability of a buccal cell DBD-FISH model to human biomonitoring and nutritional work.Key words: DNA damage, buccal cell, DNA breakage detection/fluorescence in situ hybridization.     

Evaluation of DNA damage in agricultural workers exposed to pesticides using single cell gel electrophoresis (comet) assay

BACKGROUND:

Pesticides are used in agriculture to protect crops, but they pose a potential risk to farmers and environment. The aim of the present study is to investigate the relation between the occupational exposure to various pesticides and the presence of DNA damage.

MATERIALS AND METHODS:

Blood samples of 210 exposed workers (after a day of intense spraying) and 50 control subjects belonging to various districts of Punjab (India) were evaluated using Comet assay. Sixty workers who showed DNA damage were selected for follow up at 5-6 months after the first sampling during a low or null spraying period.

RESULTS:

Significant differences were found in DNA damage between freshly exposed workers and controls and freshly exposed and followed up cases. There was significant increase in the comet parameters viz. mean comet tail length and frequency of cells showing migration in exposed workers as compared to controls (72.22 ± 20.76 vs. 46.92 ± 8.17, P<0.001; 31.79 vs. 5.77, P<0.001). In the second samples, followed up cases showed significant decrease in frequency of damaged cells as compared to freshly exposed workers of first sampling (P<0.05). The confounding factors such as variable duration of pesticide exposure, age, smoking, drinking and dietary habits etc which were expected to modulate the damage, were instead found to have no significant effect on DNA fragmentation.

CONCLUSION:

The evidence of a genetic hazard related to exposure resulting from the intensive use of pesticides stresses the need for educational programs for agricultural workers to reduce the use of chemicals in agriculture.     

Effects of Bacillus anthracis hydrophobicity and induction of host cell death on sample collection from environmental surfaces

The objective of this study is to determine whether DNA signature recovery of Bacillus anthracis strains from different environmental substrates correlates with pathogen cell surface hydrophobicity and induction of host cell death. We compared recovery of DNA signatures from a panel of B. anthracis strains collected from two environmental substrates, non-porous surfaces and soil, using real-time qPCR. We further assessed both cell surface hydrophobicity of the B. anthracis strains by contact angle measurements and host cell viability in response to B. anthracis infection in a mouse macrophage cell model system. Our studies demonstrated correlation between reduced B. anthracis sample recovery from environmental substrates and increased cell surface hydrophobicity. Surprisingly, the most hydrophilic strain, K4596, which exhibited the highest level of recovery from the environmental surfaces, induced the highest level of host cell cytotoxicity compared to more hydrophobic B. anthracis strains in the panel. Our results suggest that cell surface hydrophobicity may play a leading role in mediating pathogen adherence to environmental surfaces. These findings can contribute to the optimization of pathogen detection efforts by understanding how bacterial parameters such as hydrophobicity and induction of host cell death affect bacterial adherence to environmental surfaces.     

Optimized pH method for DNA elution from buccal cells collected in Whatman FTA cards

DNA is the most accessible biologic material for obtaining information from the human genome because of its molecular stability and its presence in every nucleated cell. Currently, single nucleotide polymorphism genotyping and DNA methylation are the main DNA-based approaches to deriving genomic and epigenomic disease biomarkers. Upon the discontinuation of the Schleicher & Schuell IsoCode product (Dassel, Germany), which was a treated paper system to elute DNA from several biologic sources for polymerase chain reaction (PCR) analysis, a high-yielding DNA elution method was imperative. We describe here an improved procedure of the not fully validated Whatman pH-based elution protocol. Our DNA elution procedure from buccal cells collected in Whatman FTA cards (Whatman Inc., Florham Park, NJ) yielded approximately 4 microg of DNA from a 6-mm FTA card punch and was successfully applied for HLA-DQB1 genotyping. The genotypes showed complete concordance with data obtained from blood of the same subjects. The achieved high DNA yield from buccal cells suggests a potential cost-effective tool for genomic and epigenomic disease biomarkers development.     

Evaluation of oxidative DNA damage promoted by storage in sperm from sex-reversed rainbow trout

Short-term storage and cryopreservation of sperm are two common procedures in aquaculture, used for routine practices in artificial insemination reproduction and gene banking, respectively. Nevertheless, both procedures cause injuries affecting sperm motility, viability, cell structure and DNA stability, which diminish reproductive success. DNA modification is considered extremely important, especially when sperm storage is carried out with gene banking purposes. DNA damage caused by sperm storage is not well characterized and previous studies have reported simple and double strand breaks that have been attributed to oxidative events promoted by the generation of free radicals during storage.The objective of this study was to reveal DNA fragmentation and to explore the presence of oxidized bases that could be produced by oxidative events during short-term storage and cryopreservation in sex-reversed rainbow trout (Oncorhynchus mykiss) spermatozoa. Sperm from six males was analyzed separately. Different aliquots of the samples were stored 2 h (fresh) or 5 days at 4 °C or were cryopreserved. Then spermatozoa were analyzed using the Comet assay, as well as combining this method with digestion with two endonucleases from Escherichia coli (Endonuclease III, that cut in oxidized cytosines, and FPG, cutting in oxidized guanosines). Both storage procedures yielded DNA fragmentation, but only short-term storage oxidative events were clearly detected, showing that oxidative processes affect guanosines rather than cytosines. Cryopreservation increases DNA fragmentation but the presence of oxidized bases was not noticed, suggesting that mechanisms other than oxidative stress could be involved in DNA fragmentation promoted by freezing.     

Effects of blood collection conditions on ovarian cancer serum markers

Background

Evaluating diagnostic and early detection biomarkers requires comparing serum protein concentrations among biosamples ascertained from subjects with and without cancer. Efforts are generally made to standardize blood processing and storage conditions for cases and controls, but blood sample collection conditions cannot be completely controlled. For example, blood samples from cases are often obtained from persons aware of their diagnoses, and collected after fasting or in surgery, whereas blood samples from some controls may be obtained in different conditions, such as a clinic visit. By measuring the effects of differences in collection conditions on three different markers, we investigated the potential of these effects to bias validation studies.

Methodology and Principle Findings

We analyzed serum concentrations of three previously studied putative ovarian cancer serum biomarkers–CA 125, Prolactin and MIF–in healthy women, women with ovarian cancer undergoing gynecologic surgery, women undergoing surgery for benign ovary pathology, and women undergoing surgery with pathologically normal ovaries. For women undergoing surgery, a blood sample was collected either in the clinic 1 to 39 days prior to surgery, or on the day of surgery after anesthesia was administered but prior to the surgical procedure, or both. We found that one marker, prolactin, was dramatically affected by collection conditions, while CA 125 and MIF were unaffected. Prolactin levels were not different between case and control groups after accounting for the conditions of sample collection, suggesting that sample ascertainment could explain some or all of the previously reported results about its potential as a biomarker for ovarian cancer.

Conclusions

Biomarker validation studies should use standardized collection conditions, use multiple control groups, and/or collect samples from cases prior to influence of diagnosis whenever feasible to detect and correct for potential biases associated with sample collection.     

Effects of sample handling and storage on quantitative lipid analysis in human serum

There is sparse information about specific storage and handling protocols that minimize analytical error and variability in samples evaluated by targeted metabolomics. Variance components that affect quantitative lipid analysis in a set of human serum samples were determined. The effects of freeze-thaw, extraction state, storage temperature, and freeze-thaw prior to density-based lipoprotein fractionation were quantified. The quantification of high abundance metabolites, representing the biologically relevant lipid species in humans, was highly repeatable (with coefficients of variation as low as 0.01 and 0.02) and largely unaffected by 1–3 freeze-thaw cycles (with 0–8% of metabolites affected in each lipid class). Extraction state had effects on total lipid class amounts, including decreased diacylglycerol and increased phosphatidylethanolamine in thawed compared with frozen samples. The effects of storage temperature over 1 week were minimal, with 0–4% of metabolites affected by storage at 4°C, −20°C, or −80°C in most lipid classes, and 19% of metabolites in diacylglycerol affected by storage at −20°C. Freezing prior to lipoprotein fractionation by density ultracentrifugation decreased HDL free cholesterol by 37% and VLDL free fatty acid by 36%, and increased LDL cholesterol ester by 35% compared with fresh samples. These findings suggest that density-based fractionation should preferably be undertaken in fresh serum samples because up to 37% variability in HDL and LDL cholesterol could result from a single freeze-thaw cycle. Conversely, quantitative lipid analysis within unfractionated serum is minimally affected even with repeated freeze-thaw cycles.     

Werner protein protects nonproliferating cells from oxidative DNA damage

Werner syndrome, caused by mutations of the WRN gene, mimics many changes of normal aging. Although roles for WRN protein in DNA replication, recombination, and telomere maintenance have been suggested, the pathology of rapidly dividing cells is not a feature of Werner syndrome. To identify cellular events that are specifically vulnerable to WRN deficiency, we used RNA interference (RNAi) to knockdown WRN or BLM (the RecQ helicase mutated in Bloom syndrome) expression in primary human fibroblasts. Withdrawal of WRN or BLM produced accelerated cellular senescence phenotype and DNA damage response in normal fibroblasts, as evidenced by induction of gammaH2AX and 53BP1 nuclear foci. After WRN depletion, the induction of these foci was seen most prominently in nondividing cells. Growth in physiological (3%) oxygen or in the presence of an antioxidant prevented the development of the DNA damage foci in WRN-depleted cells, whereas acute oxidative stress led to inefficient repair of the lesions. Furthermore, WRN RNAi-induced DNA damage was suppressed by overexpression of the telomere-binding protein TRF2. These conditions, however, did not prevent the DNA damage response in BLM-ablated cells, suggesting a distinct role for WRN in DNA homeostasis in vivo. Thus, manifestations of Werner syndrome may reflect an impaired ability of slowly dividing cells to limit oxidative DNA damage.     

Effects of simulated microgravity on metabolic activities related to DNA damage and repair in lymphoblastoid cells

We adopted a simple experimental framework to follow the dependence of structural aberrations and the modifications in selected metabolic processes correlated with the exposure of cells to microgravity. Alterations to the cellular metabolism induced by exposure to microgravity are evidentiated in the modification of PARP activity (strongly dependent to the presence of DNA damages and to the altered gene expression), in the modification of the repair ability and in the cell's energy homeostasis (NAD and ATP). Cells are exposed continuously to microgravity in a Random Positioning Machine (RPM) in complete medium for 48 hours. At the end of this period a part of these cells are immediately analysed for the parameters reported above and the remaining were furtherly incubated in standard laboratory conditions to document eventual defects during the phases of the recovery process. A part of cells, just after exposure to microgravity, were also subjected to treatment with a strong damaging agent, KBrO3, and these cells were subsequently analyzed. This final treatment was meant to amplify the eventual deficiencies experienced by microgravity-exposed cells in the DNA repair process also in dependence with the alterated metabolic conditions resulting after the exposure to microgravity.     

Effects of blood storage on rheology and damage in low-stress shear flow

Data are presented on the rheological and hemolytic behavior of whole human blood as it ages while stored at 4 degrees C (as in blood banking practice) up to 26 days. The viscometric properties of steady shear viscosity eta and oscillatory (complex) viscosity eta * = eta' - i eta" reported over ranges of shear rate gamma and radian frequency omega of 33 less than gamma less than 4130 s-1 and 1.5 less than omega less than 48 s -1; data on autologous plasma are given for reference. The Cox-Merz relation, eta (gamma) = [eta *(omega)] omega = gamma, is found to be a good approximation, with eta greater than or equal to [eta *], over the range studied. Release of hemoglobin (Hgb) and lactate dehydrogenase (LDH) into the plasma during shearing is tracked as a function of time for 30 min, and its sensitivity to gamma magnitude is measured. Bloods from four different donors are studied, with primary attention given to one (SSR). For all bloods, the release of both Hgb and LDH increases with storage age, but differences in such aging characteristics between different bloods can be substantial (even when rheological properties are identical). A post-shear incubation at 4 degrees C for one day shows no enhancement of plasma Hgb and LDH levels beyond those expected from normal aging after the shearing experience, demonstrating the absence of significant delayed-action effects as a consequence of shearing trauma.     

Paraquat-induced DNA damage in mammalian cells

We have examined the possibility that paraquat (PQ) may exert its toxicity by inducing DNA damage. Mouse lymphoblasts in culture exhibited inhibition of colony forming ability and DNA single strand breaks following a 2 hour exposure to PQ. These phenomenon are dose dependent and increase when a rat liver S9 fraction is included in the incubation mixture. The presence of superoxide dismutase and catalase did not prevent the effects of PQ. Our data indicate that DNA should be considered as a possibile macromolecular target for the lethal effects of paraquat.