SciCombinator

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Concept: Mutagen

271

Transmissible cancers are clonal lineages that spread through populations via contagious cancer cells. Although rare in nature, two facial tumor clones affect Tasmanian devils. Here we perform comparative genetic and functional characterization of these lineages. The two cancers have similar patterns of mutation and show no evidence of exposure to exogenous mutagens or viruses. Genes encoding PDGF receptors have copy number gains and are present on extrachromosomal double minutes. Drug screening indicates causative roles for receptor tyrosine kinases and sensitivity to inhibitors of DNA repair. Y chromosome loss from a male clone infecting a female host suggests immunoediting. These results imply that Tasmanian devils may have inherent susceptibility to transmissible cancers and present a suite of therapeutic compounds for use in conservation.

Concepts: DNA, Protein, Gene, Genetics, Cancer, Mutation, Signal transduction, Mutagen

171

Zinc-finger nucleases (ZFNs) and TAL effector nucleases (TALENs) have been shown to induce targeted mutations, but they have not been extensively tested in any animal model. Here, we describe a large-scale comparison of ZFN and TALEN mutagenicity in zebrafish. Using deep sequencing, we found that TALENs are significantly more likely to be mutagenic and induce an average of 10-fold more mutations than ZFNs. We observed a strong correlation between somatic and germ-line mutagenicity, and identified germ line mutations using ZFNs whose somatic mutations rates are well below the commonly used threshold of 1%. Guidelines that have previously been proposed to predict optimal ZFN and TALEN target sites did not predict mutagenicity in vivo. However, we observed a significant negative correlation between TALEN mutagenicity and the number of CpG repeats in TALEN target sites, suggesting that target site methylation may explain the poor mutagenicity of some TALENs in vivo. The higher mutation rates and ability to target essentially any sequence make TALENs the superior technology for targeted mutagenesis in zebrafish, and likely other animal models.

Concepts: DNA, Mutation, DNA repair, Model organism, Animal testing, Site-directed mutagenesis, Mutagenesis, Mutagen

155

Germline BRCA2 mutations are associated with poorer outcome prostate cancer (PCa) compared with sporadic tumours but this association remains to be characterized. In this study, we aim to assess if there is a signature set of copy number alterations (CNA) that could aid to the identification of BRCA2 mutated tumours and would assist us to understand their aggressive clinical behaviour.

Concepts: Immune system, Cancer, Mutation, Germline mutation, DNA repair, Prostate cancer, BRCA2, Mutagen

138

Benzene, a hazardous component of gasoline, is a genotoxic class I human carcinogen. This study evaluated the genotoxic effects of occupational exposure to benzene in gasoline stations. Genotoxicity of exposure to benzene was assessed in peripheral blood leucocytes of 62 gasoline station workers and compared with an equal numbers of matched controls using total genomic DNA fragmentation, micronucleus test and cell viability test. An ambient air samples were collected and analyzed for Monitoring of benzene, toluene, ethyl benzene and xylene (BTEX) in work environment and control areas. DNA fragmentation, micronucleus and dead cells percent were significantly higher in exposed workers than controls. Level of benzene, Toluene, Ethyl benzene and xylene in the work environment were higher than the control areas and the permissible limits. Gasoline station workers occupationally exposed to benzene are susceptible to genotoxic effects indicated by increased DNA fragmentation, higher frequency of micronucleus and decreased leukocytes viability.

Concepts: Benzene, Toluene, Mutagen

119

Exposure to thirdhand smoke (THS) is a newly described health risk. Evidence supports its widespread presence in indoor environments. However, its genotoxic potential, a critical aspect in risk assessment, is virtually untested. An important characteristic of THS is its ability to undergo chemical transformations during aging periods, as demonstrated in a recent study showing that sorbed nicotine reacts with the indoor pollutant nitrous acid (HONO) to form tobacco-specific nitrosamines (TSNAs) such as 4-(methylnitrosamino)-4-(3-pyridyl)butanal (NNA) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). The goal of this study was to assess the genotoxicity of THS in human cell lines using two in vitro assays. THS was generated in laboratory systems that simulated short (acute)- and long (chronic)-term exposures. Analysis by liquid chromatography-tandem mass spectrometry quantified TSNAs and common tobacco alkaloids in extracts of THS that had sorbed onto cellulose substrates. Exposure of human HepG2 cells to either acute or chronic THS for 24h resulted in significant increases in DNA strand breaks in the alkaline Comet assay. Cell cultures exposed to NNA alone showed significantly higher levels of DNA damage in the same assay. NNA is absent in freshly emitted secondhand smoke, but it is the main TSNA formed in THS when nicotine reacts with HONO long after smoking takes place. The long amplicon-quantitative PCR assay quantified significantly higher levels of oxidative DNA damage in hypoxanthine phosphoribosyltransferase 1 (HPRT) and polymerase β (POLB) genes of cultured human cells exposed to chronic THS for 24h compared with untreated cells, suggesting that THS exposure is related to increased oxidative stress and could be an important contributing factor in THS-mediated toxicity. The findings of this study demonstrate for the first time that exposure to THS is genotoxic in human cell lines.

Concepts: DNA, Gene, Cell, Chromosome, Smoking, Tobacco, Cell culture, Mutagen

114

Tobacco smoking increases the risk of at least 17 classes of human cancer. We analyzed somatic mutations and DNA methylation in 5243 cancers of types for which tobacco smoking confers an elevated risk. Smoking is associated with increased mutation burdens of multiple distinct mutational signatures, which contribute to different extents in different cancers. One of these signatures, mainly found in cancers derived from tissues directly exposed to tobacco smoke, is attributable to misreplication of DNA damage caused by tobacco carcinogens. Others likely reflect indirect activation of DNA editing by APOBEC cytidine deaminases and of an endogenous clocklike mutational process. Smoking is associated with limited differences in methylation. The results are consistent with the proposition that smoking increases cancer risk by increasing the somatic mutation load, although direct evidence for this mechanism is lacking in some smoking-related cancer types.

Concepts: DNA, Cancer, Ionizing radiation, Mutation, Ultraviolet, Tobacco, Tobacco smoking, Mutagen

93

Ionizing radiation is a potent carcinogen, inducing cancer through DNA damage. The signatures of mutations arising in human tissues following in vivo exposure to ionizing radiation have not been documented. Here, we searched for signatures of ionizing radiation in 12 radiation-associated second malignancies of different tumour types. Two signatures of somatic mutation characterize ionizing radiation exposure irrespective of tumour type. Compared with 319 radiation-naive tumours, radiation-associated tumours carry a median extra 201 deletions genome-wide, sized 1-100 base pairs often with microhomology at the junction. Unlike deletions of radiation-naive tumours, these show no variation in density across the genome or correlation with sequence context, replication timing or chromatin structure. Furthermore, we observe a significant increase in balanced inversions in radiation-associated tumours. Both small deletions and inversions generate driver mutations. Thus, ionizing radiation generates distinctive mutational signatures that explain its carcinogenic potential.

Concepts: DNA, Cancer, Ionizing radiation, Mutation, Ultraviolet, DNA repair, DNA replication, Mutagen

91

Haematopoietic stem cells renew blood. Accumulation of DNA damage in these cells promotes their decline, while misrepair of this damage initiates malignancies. Here we describe the features and mutational landscape of DNA damage caused by acetaldehyde, an endogenous and alcohol-derived metabolite. This damage results in DNA double-stranded breaks that, despite stimulating recombination repair, also cause chromosome rearrangements. We combined transplantation of single haematopoietic stem cells with whole-genome sequencing to show that this damage occurs in stem cells, leading to deletions and rearrangements that are indicative of microhomology-mediated end-joining repair. Moreover, deletion of p53 completely rescues the survival of aldehyde-stressed and mutated haematopoietic stem cells, but does not change the pattern or the intensity of genome instability within individual stem cells. These findings characterize the mutation of the stem-cell genome by an alcohol-derived and endogenous source of DNA damage. Furthermore, we identify how the choice of DNA-repair pathway and a stringent p53 response limit the transmission of aldehyde-induced mutations in stem cells.

Concepts: DNA, Gene, Genetics, Mutation, Bacteria, Chromosome, DNA repair, Mutagen

88

A current threat to the marine ecosystem is the high level of solar ultraviolet radiation (UV). Large whales have recently been shown to suffer sun-induced skin damage from continuous UV exposure. Genotoxic consequences of such exposure remain unknown for these long-lived marine species, as does their capacity to counteract UV-induced insults. We show that UV exposure induces mitochondrial DNA damage in the skin of seasonally sympatric fin, sperm, and blue whales and that this damage accumulates with age. However, counteractive molecular mechanisms are markedly different between species. For example, sperm whales, a species that remains for long periods at the sea surface, activate genotoxic stress pathways in response to UV exposure whereas the paler blue whale relies on increased pigmentation as the season progresses. Our study also shows that whales can modulate their responses to fluctuating levels of UV, and that different evolutionary constraints may have shaped their response strategies.

Concepts: DNA, Mutation, Ultraviolet, Sun, Sunlight, Titanium dioxide, Mutagen, Humpback whale

75

We describe a single-color digital PCR assay that detects and quantifies cancer mutations directly from circulating DNA collected from the plasma of cancer patients. This approach relies on a double-stranded DNA intercalator dye and paired allele-specific DNA primer sets to determine an absolute count of both the mutation and wild-type-bearing DNA molecules present in the sample. The cell-free DNA assay uses an input of 1 ng of nonamplified DNA, approximately 300 genome equivalents, and has a molecular limit of detection of three mutation DNA genome-equivalent molecules per assay reaction. When using more genome equivalents as input, we demonstrated a sensitivity of 0.10% for detecting the BRAF V600E and KRAS G12D mutations. We developed several mutation assays specific to the cancer driver mutations of patients' tumors and detected these same mutations directly from the nonamplified, circulating cell-free DNA. This rapid and high-performance digital PCR assay can be configured to detect specific cancer mutations unique to an individual cancer, making it a potentially valuable method for patient-specific longitudinal monitoring.

Concepts: DNA, Genetics, Cancer, Mutation, Polymerase chain reaction, DNA replication, Gene duplication, Mutagen