Reference cells and ploidy in the comet assay

In the comet assay single cells are analyzed with respect to their level of DNA damage. Discrimination of the individual cell or cell type based on DNA content, with concomitant scoring of the DNA damage, is useful since this may allow analysis of mixtures of cells. Different cells can then be characterized based on their ploidy, cell cycle stage, or genome size. We here describe two applications of such a cell type-specific comet assay: (i) Testicular cell suspensions, analyzed on the basis of their ploidy during spermatogenesis; and (ii) reference cells in the form of fish erythrocytes which can be included as internal standards to correct for inter-assay variations. With standard fluorochromes used in the comet assay, the total staining signal from each cell – whether damaged or undamaged – was found to be associated with the cell’s DNA content. Analysis of the fluorescence intensity of single cells is straightforward since these data are available in scoring systems based on image analysis. The analysis of testicular cell suspensions provides information on cell type specific composition, susceptibility to genotoxicants, and DNA repair. Internal reference cells, either untreated or carrying defined numbers of lesions induced by ionizing radiation, are useful for investigation of experimental factors that can cause variation in comet assay results, and for routine inclusion in experiments to facilitate standardization of methods, and comparison of comet assay data obtained in different experiments or in different laboratories. They can also be used – in combination with a reference curve – to quantify the DNA lesions induced by a certain treatment. Fish cells of a range of genome sizes, both greater and smaller than human, are suitable for this purpose, and they are inexpensive

Octyl Methoxycinnamate Modulates Gene Expression and Prevents Cyclobutane Pyrimidine Dimer Formation but not Oxidative DNA Damage in UV-Exposed Human Cell Lines

Octyl methoxycinnamate (OMC) is one of the most widely used sunscreen ingredients. To analyze biological effects of OMC, an in vitro approach was used implying ultraviolet (UV) exposure of two human cell lines, a primary skin fibroblast (GM00498) and a breast cancer (MCF-7) cell lines. End points include cell viability assessment, assay of cyclobutane pyrimidine dimers (CPDs) and oxidated DNA lesions using alkaline elution and lesion-specific enzymes, and gene expression analysis of a panel of 17 DNA damage–responsive genes. We observed that OMC provided protection against CPDs, and the degree of protection correlated with the OMC-mediated reduction in UV dose. No such protection was found with respect to oxidative DNA lesions. Upon UV exposure in the presence of OMC, the gene expression studies showed significant differential changes in some of the genes studied and the expression of p53 protein was also changed. For some genes, the change in expression seemed to be delayed in time by OMC. The experimental approach applied in this study, using a panel of 17 genes in an in vitro cellular system together with genotoxicity assays, may be useful in the initial screening of active ingredients in sunscreens

Molecular portrait of cisplatin induced response in human testis cancer cell lines based on gene expression profiles

<p>Abstract</p> <p>Background</p> <p>Testicular germ cell tumors (TGCTs) respond well to cisplatin-based chemotherapy and show a low incidence of acquired resistance compared to most somatic tumors. The reasons for these specific characteristics are not known in detail but seem to be multifactorial. We have studied gene expression profiles of testicular and colon cancer derived cell lines treated with cisplatin. The main goal of this study was to identify novel gene expression profiles with their functional categories and the biochemical pathways that are associated with TGCT cells' response to cisplatin.</p> <p>Results</p> <p>Genes that were differentially expressed between the TGCT cell lines vs the (somatic) HCT116 cell line, after cisplatin treatment, were identified using the significance analysis of microarrays (SAM) method. The response of TGCT cells was strikingly different from that of HCT116, and we identified 1794 genes that were differentially expressed. Functional classification of these genes showed that they participate in a variety of different and widely distributed functional categories and biochemical pathways. Database mining showed significant association of genes (n = 41) induced by cisplatin in our study, and genes previously reported to by expressed in differentiated TGCT cells. We identified 37 p53-responsive genes that were altered after cisplatin exposure. We also identified 40 target genes for two microRNAs, hsa-mir-372 and 373 that may interfere with p53 signaling in TGCTs. The tumor suppressor genes <it>NEO1 </it>and <it>LATS2</it>, and the estrogen receptor gene <it>ESR1</it>, all have binding sites for p53 and hsa-mir-372/373. <it>NEO1 </it>and <it>LATS2 </it>were down-regulated in TGCT cells following cisplatin exposure, while <it>ESR1 </it>was up-regulated in TGCT cells. Cisplatin-induced genes associated with terminal growth arrest through senescence were identified, indicating associations which were not previously described for TGCT cells.</p> <p>Conclusion</p> <p>By linking our gene expression data to publicly available databases and literature, we provide a global pattern of cisplatin induced cellular response that is specific for testicular cancer cell lines. We have identified cisplatin-responsive functional classes and pathways, such as the angiogenesis, Wnt, integrin, and cadherin signaling pathways. The identification of differentially expressed genes in this study may contribute to a better understanding of the unusual sensitivity of TGCT to some DNA-damaging agents.</p

Dose rate dependent reduction in chromatin accessibility at transcriptional start sites long time after exposure to gamma radiation

Ionizing radiation (IR) impact cellular and molecular processes that require chromatin remodelling relevant for cellular integrity. However, the cellular implications of ionizing radiation (IR) delivered per time unit (dose rate) are still debated. This study investigates whether the dose rate is relevant for inflicting changes to the epigenome, represented by chromatin accessibility, or whether it is the total dose that is decisive. CBA/CaOlaHsd mice were whole-body exposed to either chronic low dose rate (2.5 mGy/h for 54 d) or the higher dose rates (10 mGy/h for 14 d and 100 mGy/h for 30 h) of gamma radiation (60Co, total dose: 3 Gy). Chromatin accessibility was analysed in liver tissue samples using Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-Seq), both one day after and over three months post-radiation (>100 d). The results show that the dose rate contributes to radiation-induced epigenomic changes in the liver at both sampling timepoints. Interestingly, chronic low dose rate exposure to a high total dose (3 Gy) did not inflict long-term changes to the epigenome. In contrast to the acute high dose rate given to the same total dose, reduced accessibility at transcriptional start sites (TSS) was identified in genes relevant for the DNA damage response and transcriptional activity. Our findings link dose rate to essential biological mechanisms that could be relevant for understanding long-term changes after ionizing radiation exposure. However, future studies are needed to comprehend the biological consequence of these findings.publishedVersio

Risk assessment of grilled and barbecued food

When grilling, more harmful substances can be formed than when frying in a pan. For most people, there is a low risk associated with eating grilled food. Grilling food at a high temperature and/or on a campfire, often, and eating a lot of it may damage one's health. Distance to the heat source, how early food is placed onto the grill, and the type of fuel used can affect the formation of harmful substances. It is known that heat treatment such as grilling and frying can give rise to unwanted toxic compounds in the food, so-called process-induced contaminants. Grilling is a common way of preparing food in Norway, and the grilling season has become longer. The food selection has become ever wider and sales of different types of grills are increasing. Many factors may thus have changed since VKM's previous assessment of health risks from the consumption of grilled food, which was published in 2007. To be able to give current and relevant advice to consumers and others who sell or offer grilled food, the Norwegian Food Safety Authority has asked VKM for updated knowledge about the formation of process-induced contaminants in different food products by different grilling methods, and an assessment of what risk this may pose. Main findings: Harmful substances can be formed at high cooking temperatures. When grilling, the temperature is higher and less controllable than when frying in a pan. There is good evidence that two groups of genotoxic and carcinogenic substances, heterocyclic amines (HAA) and polycyclic aromatic hydrocarbons (PAH), are formed in higher concentrations in grilled food than in fried food. VKM has assessed PAH, for which there are good studies of occurrence in barbecued food. PAHs are formed when fat burns after dripping from the food onto the heat source. PAHs can also be released from coal, briquettes and wood. The smoke with PAH settles on the food. The occurrence of PAH in grilled food varies greatly and depends on how the food is grilled. The concentration of PAH is highest in very well-done meat with a high fat content, such as pork ribs and hamburgers. The highest concentration of PAH was found in sausage grilled on a campfire. This is caused by unburnt fat or soot/smoke from the fire that stick to the sausage. By avoiding fat dripping directly onto the heat source, preventing smoke from coming into contact with the food, and not overcooking the food, the amount of PAH in grilled food can be reduced. When using charcoal, the PAH emission is higher immediately after lighting, and the amount of PAH in the food can be reduced by waiting sufficiently long before start grilling. It is not the number of times you grill that is a critical factor, but how. A "worst-case" scenario shows that the annual consumption of more than 15-25 meals that have been grilled in a way that produces a high PAH incidence may provide too low margin of exposure* for the risk to be low. If, on the other hand, you prefer lean barbecue food that is not overcooked, then you can eat it more than 100 times a year according to our calculations and still have a high enough margin of exposure. * Margin of exposure is the ratio between the lowest dose (reference point) that causes increased cancer in experimental animals and the calculated intake. Exposure margin below 10,000 is considered a public health concern. Norsk: Ved grilling kan det dannes flere helseskadelige stoffer enn ved steking i panne. For de fleste er det lav risiko knyttet til å spise grillmat.Risk assessment of grilled and barbecued foodpublishedVersionacceptedVersionacceptedVersio

Assessing the carcinogenic potential of low-dose exposures to chemical mixtures in the environment: the challenge ahead.

Lifestyle factors are responsible for a considerable portion of cancer incidence worldwide, but credible estimates from the World Health Organization and the International Agency for Research on Cancer (IARC) suggest that the fraction of cancers attributable to toxic environmental exposures is between 7% and 19%. To explore the hypothesis that low-dose exposures to mixtures of chemicals in the environment may be combining to contribute to environmental carcinogenesis, we reviewed 11 hallmark phenotypes of cancer, multiple priority target sites for disruption in each area and prototypical chemical disruptors for all targets, this included dose-response characterizations, evidence of low-dose effects and cross-hallmark effects for all targets and chemicals. In total, 85 examples of chemicals were reviewed for actions on key pathways/mechanisms related to carcinogenesis. Only 15% (13/85) were found to have evidence of a dose-response threshold, whereas 59% (50/85) exerted low-dose effects. No dose-response information was found for the remaining 26% (22/85). Our analysis suggests that the cumulative effects of individual (non-carcinogenic) chemicals acting on different pathways, and a variety of related systems, organs, tissues and cells could plausibly conspire to produce carcinogenic synergies. Additional basic research on carcinogenesis and research focused on low-dose effects of chemical mixtures needs to be rigorously pursued before the merits of this hypothesis can be further advanced. However, the structure of the World Health Organization International Programme on Chemical Safety 'Mode of Action' framework should be revisited as it has inherent weaknesses that are not fully aligned with our current understanding of cancer biology

Genetic association study of QT interval highlights role for calcium signaling pathways in myocardial repolarization.

The QT interval, an electrocardiographic measure reflecting myocardial repolarization, is a heritable trait. QT prolongation is a risk factor for ventricular arrhythmias and sudden cardiac death (SCD) and could indicate the presence of the potentially lethal mendelian long-QT syndrome (LQTS). Using a genome-wide association and replication study in up to 100,000 individuals, we identified 35 common variant loci associated with QT interval that collectively explain ∼8-10% of QT-interval variation and highlight the importance of calcium regulation in myocardial repolarization. Rare variant analysis of 6 new QT interval-associated loci in 298 unrelated probands with LQTS identified coding variants not found in controls but of uncertain causality and therefore requiring validation. Several newly identified loci encode proteins that physically interact with other recognized repolarization proteins. Our integration of common variant association, expression and orthogonal protein-protein interaction screens provides new insights into cardiac electrophysiology and identifies new candidate genes for ventricular arrhythmias, LQTS and SCD

Assessing the carcinogenic potential of low-dose exposures to chemical mixtures in the environment: the challenge ahead

Summary Low-dose exposures to common environmental chemicals that are deemed safe individually may be combining to instigate carcinogenesis, thereby contributing to the incidence of cancer. This risk may be overlooked by current regulatory practices and needs to be vigorously investigated

Assessing the Carcinogenic Potential of Low-Dose Exposures to Chemical Mixtures in the Environment: The Challenge Ahead

Lifestyle factors are responsible for a considerable portion of cancer incidence worldwide, but credible estimates from the World Health Organization and the International Agency for Research on Cancer (IARC) suggest that the fraction of cancers attributable to toxic environmental exposures is between 7% and 19%. To explore the hypothesis that low-dose exposures to mixtures of chemicals in the environment may be combining to contribute to environmental carcinogenesis, we reviewed 11 hallmark phenotypes of cancer, multiple priority target sites for disruption in each area and prototypical chemical disruptors for all targets, this included dose-response characterizations, evidence of low-dose effects and cross-hallmark effects for all targets and chemicals. In total, 85 examples of chemicals were reviewed for actions on key pathways/mechanisms related to carcinogenesis. Only 15% (13/85) were found to have evidence of a dose-response threshold, whereas 59% (50/85) exerted low-dose effects. No dose-response information was found for the remaining 26% (22/85). Our analysis suggests that the cumulative effects of individual (non-carcinogenic) chemicals acting on different pathways, and a variety of related systems, organs, tissues and cells could plausibly conspire to produce carcinogenic synergies. Additional basic research on carcinogenesis and research focused on low-dose effects of chemical mixtures needs to be rigorously pursued before the merits of this hypothesis can be further advanced. However, the structure of the World Health Organization International Programme on Chemical Safety \u27Mode of Action\u27 framework should be revisited as it has inherent weaknesses that are not fully aligned with our current understanding of cancer biology