Activity of Genes with Functions in Human Williams-Beuren Syndrome Is Impacted by Mobile Element Insertions in the Gray Wolf Genome.

In canines, transposon dynamics have been associated with a hyper-social behavioral syndrome, although the functional mechanism has yet to be described. We investigate the epigenetic and transcriptional consequences of these behavior-associated mobile element insertions (MEIs) in dogs and Yellowstone gray wolves. We posit that the transposons themselves may not be the causative feature; rather, their transcriptional regulation may exert the functional impact. We survey four outlier transposons associated with hyper-sociability, with the expectation that they are targeted for epigenetic silencing. We predict hyper-methylation of MEIs, suggestive that the epigenetic silencing of and not the MEIs themselves may be driving dysregulation of nearby genes. We found that transposon-derived sequences are significantly hyper-methylated, regardless of their copy number or species. Further, we have assessed transcriptome sequence data and found evidence that MEIs impact the expression levels of six genes (WBSCR17, LIMK1, GTF2I, WBSCR27, BAZ1B, and BCL7B), all of which have known roles in human Williams-Beuren syndrome due to changes in copy number, typically hemizygosity. Although further evidence is needed, our results suggest that a few insertions alter local expression at multiple genes, likely through a cis-regulatory mechanism that excludes proximal methylation

In vitro genotoxicity testing using the micronucleus assay in cell lines, human lymphocytes and 3D human skin models

The toxicological relevance of the micronucleus (MN) test is well defined: it is a multi-target genotoxic endpoint, assessing not only clastogenic and aneugenic events but also some epigenetic effects, which is simple to score, accurate, applicable in different cell types. In addition, it is predictive for cancer, amenable for automation and allows good extrapolation for potential limits of exposure or thresholds and it is easily measured in experimental both in vitro and in vivo systems. Implementation of in vitro micronucleus (IVMN) assays in the battery of tests for hazard and risk assessment of potential mutagens/carcinogens is therefore fully justified. Moreover, the final draft of an OECD guideline became recently available for this test. In this review, we discuss the prerequisites for an acceptable MN assay, including the cell as unit of observation, importance of cell membranes, the requirement of a mitotic or meiotic division and the assessment of cell division in the presence of the test substance. Furthermore, the importance of adequate design of protocols is highlighted and new developments, in particular the in vitro 3D human skin models, are discussed. Finally, we address future research perspectives including the possibility of a combined primary 3D human skin and primary human whole blood culture system, and the need for adaptation of the IVMN assays to assess the genotoxic potential of new materials, in particular nanomaterial

Seven benzimidazole pesticides combined at sub-threshold levels induce micronuclei in vitro

This article is made available through the Brunel Open Access Publishing Fund. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.Benzimidazoles act by disrupting microtubule polymerisation and are capable of inducing the formation of micronuclei. Considering the similarities in their mechanisms of action (inhibition of microtubule assembly by binding to the colchicine-binding site on tubulin monomers), combination effects according to the principles of concentration addition might occur. If so, it is to be expected that several benzimidazoles contribute to micronucleus formation even when each single one is present at or below threshold levels. This would have profound implications for risk assessment, but the idea has never been tested rigorously. To fill this gap, we analysed micronucleus frequencies for seven benzimidazoles, including the fungicide benomyl, its metabolite carbendazim, the anthelmintics albendazole, albendazole oxide, flubendazole, mebendazole and oxibendazole. Thiabendazole was also tested but was inactive. We used the cytochalasin-blocked micronucleus assay with CHO-K1 cells according to OECD guidelines, and employed an automated micronucleus scoring system based on image analysis to establish quantitative concentration–response relationships for the seven active benzimidazoles. Based on this information, we predicted additive combination effects for a mixture of the seven benzimidazoles by using the concepts of concentration addition and independent action. The observed effects of the mixture agreed very well with those predicted by concentration addition. Independent action underestimated the observed combined effects by a large margin. With a mixture that combined all benzimidazoles at their estimated threshold concentrations for micronucleus induction, micronucleus frequencies of ~15.5% were observed, correctly anticipated by concentration addition. On the basis of independent action, this mixture was expected to produce no effects. Our data provide convincing evidence that concentration addition is applicable to combinations of benzimidazoles that form micronuclei by disrupting microtubule polymerisation. They present a rationale for grouping these chemicals together for the purpose of cumulative risk assessment.United Kingdom Food Standards Agenc

A comparison of transgenic rodent mutation and in vivo comet assay responses for 91 chemicals.

A database of 91 chemicals with published data from both transgenic rodent mutation (TGR) and rodent comet assays has been compiled. The objective was to compare the sensitivity of the two assays for detecting genotoxicity. Critical aspects of study design and results were tabulated for each dataset. There were fewer datasets from rats than mice, particularly for the TGR assay, and therefore, results from both species were combined for further analysis. TGR and comet responses were compared in liver and bone marrow (the most commonly studied tissues), and in stomach and colon evaluated either separately or in combination with other GI tract segments. Overall positive, negative, or equivocal test results were assessed for each chemical across the tissues examined in the TGR and comet assays using two approaches: 1) overall calls based on weight of evidence (WoE) and expert judgement, and 2) curation of the data based on a priori acceptability criteria prior to deriving final tissue specific calls. Since the database contains a high prevalence of positive results, overall agreement between the assays was determined using statistics adjusted for prevalence (using AC1 and PABAK). These coefficients showed fair or moderate to good agreement for liver and the GI tract (predominantly stomach and colon data) using WoE, reduced agreement for stomach and colon evaluated separately using data curation, and poor or no agreement for bone marrow using both the WoE and data curation approaches. Confidence in these results is higher for liver than for the other tissues, for which there were less data. Our analysis finds that comet and TGR generally identify the same compounds (mainly potent mutagens) as genotoxic in liver, stomach and colon, but not in bone marrow. However, the current database content precluded drawing assay concordance conclusions for weak mutagens and non-DNA reactive chemicals

Effect of oleic acid supplementation on prostaglandin production in maternal endometrial and fetal allantochorion cells isolated from late gestation ewes

Elevated circulating non-esterified fatty acids including oleic acid (OA) are associated with many pregnancy related complications. Prostaglandins (PGs) play crucial roles during parturition. We investigated the effect of OA supplementation on PG production using an in vitro model of ovine placenta

The Total Western Diet and Vancomycin Increase Inflammation Mediated Colorectal Cancer

Total Western Diet (TWD) fed mice have increased azoxymethane/dextran sodium sulfate (AOM/DSS) induced colorectal cancer (CRC). Using the AOM/DSS model, another group found that vancomycin (VM) reduced CRC via changes to the microbiome. This study was done to determine interactions between diet and VM induced changes to the microbiome on CRC. C57BL/6J mice were allotted into 4 treatments in a 2X2 factorial design: 1)AIN93G diet+no VM (AN, n=33); 2)AIN93G+VM in drinking water (2g/L, AVM, n=30); 3)TWD+no VM (TN, n=35); 4)TWD+VM (TVM, n=29). Food and water was provided ad libitum with intakes measured weekly. Fecal samples were collected for microbiome analysis. After 14 days, mice were injected with AOM (10 mg/kg) to induce CRC, followed by 1% DSS added to drinking water for 10 days to induce inflammation. Mice were evaluated for colitis disease activity (CAD). After recovery (24 days post AOM), 48 mice (n=12 per group) were sacrificed and colons were examined by histopathology. Remaining mice were kept on treatments for 9 additional weeks, sacrificed, and colons were examined for tumor burden and histopathology. TWD increased CAD compared to AIN93G during active colitis (P\u3c0.001); the same effect was found when assessing mucosal injury (P=0.01). TWD and VM increased tumor burden (P=0.02 for diet, P=0.002 for VM treatment) and multiplicity (P\u3c0.001 for diet and VM treatment); there were significant interactions between these factors for tumor burden (P=0.03) and multiplicity (P=0.04). The greatest difference for tumor burden was between TVM mice (49.7510.06mm3, meanSEM) and AN mice (12.343.67mm3, P\u3c0.01). Tumor multiplicity showed similar results. Beta diversity analysis of the microbiome demonstrated that VM determined whether samples clustered together. The TWD increased CRC; contrary to a prior report, we demonstrate VM increases CRC. There are significant interactions between these factors. These results suggest that diet and the gut microbiome modulate CRC

Ozone Photodissociation In The Singlet Channel At 226 Nm

Ozone photodissociation plays an important role in atmospheric chemistry and has been the focus of many experimental and theoretical studies. In the Hartley band (200-300 nm) there are two spin-allowed dissociation channels, one forming excited state, singlet products (O2(a 1$\Delta$g) + O(1D)), and one forming ground state, triplet products (O2(X 3$\Sigma$g-) + O(3P)). The singlet channel is the primary dissociation channel in the Hartley band, and numerous studies have characterized the dissociation at longer wavelengths within the Hartley band. There has been considerable interest in the triplet channel dissociation at 226 nm following the observation of low velocity O(3P) fragments, but the singlet channel dissociation dynamics at 226 nm has not been previously reported. We report the rotational state distribution and vector correlations of the O2(a 1$\Delta$g, v=0) fragments arising from the 226 nm photodissociation of jet-cooled O3. Consistent with previously reported trends, the rotational distribution is shifted to higher rotational states with decreasing wavelength. We observe highly suppressed odd rotational state populations due to a strong $\Lambda$-doublet propensity. The measured rotational distribution is in agreement with classical trajectory calculations for the v=0 products, although the distribution is narrower than predicted. The spatial anisotropy follows the previously observed trend of decreasing $\beta$ with increasing photon energy with $\beta$ = 0.72 $\pm$ 0.14 for v=0, j=38. As expected for a triatomic molecule, the v-j correlation is consistent with v perpendicular to j, but the measured correlation is non-limiting due to rotational and translational depolarization. The j-dependent linewidth of the O2(a 1$\Delta$g) REMPI spectrum is also discussed in connection with the lifetime of the resonant O2(d 1$\Pi$g) state and predissociation via the II 1$\Pi$g valence state

REMPI AND IMAGING STUDIES OF SINGLET O₂ FOLLOWING SPIN-FORBIDDEN PHOTODISSOCIATION OF OZONE

Ozone photodissociation in the Hartley (200-300 nm) and Huggins (310-370 nm) bands has been the focus of numerous studies due to its critical role in atmospheric chemistry. While Hartley band dissociation occurs primarily through two spin-allowed dissociation channels, three additional spin-forbidden channels have previously been observed following dissociation in the Huggins band. We report 1-D and 2-D REMPI spectra and velocity-mapped ion images of O₂(a¹∆g) and O₂(b¹Σ⁺) fragments following spin-forbidden dissociation in the Huggins band. We have previously observed a preference for the formation of even rotational states of O₂(a¹∆g) arising from Hartley band dissociation due to a Λ-doublet propensity. In the Huggins band, however, odd rotational states are enhanced in the REMPI spectrum, which we attribute to greater coupling between the initial excited state of O₃ and ³A′′ states producing odd rotational states of O₂(a¹∆g), than between the initial excited state and ³A′ states producing even rotational states. Ion image angular distributions of the O₂(a¹∆g) fragment in odd and even rotational states showed significant differences following Hartley band dissociation, supporting the Λ-doublet propensity model, but are indistinguishable following Huggins band dissociations. This supports a preference for the A′ Λ-doublet and even rotational states following O₃ transitions from the B state to ³A′ states and a preference for the A′′ Λ-doublet and odd rotational states following O₃ transitions from the B state to the ³A′′ states, but indicates that in the Huggins band, the A′′ Λ-doublet does not originate from a warmer distribution of parent molecules as seen in the Hartley band. 2D-REMPI allows simultaneous measurements of the rotational distributions for v=0-2 of the b¹Σ⁺ state as well as v=0 of the a¹∆g state. The relative signal in v=0-2 of the b¹Σ⁺ can provide information about the vibrational distribution, and rotational state distributions of each vibrational state can be fit individually. Spectra indicate a broad rotational distribution of the O₂(a¹∆g) fragment and a narrow distribution of the O₂(b¹Σ⁺) fragment. While determination of the O₂(a¹∆g) rotational distribution is limited due to the highly perturbed resonant state accessed in the REMPI scheme, a broad distribution is additionally supported by the multimodality of the radial distributions in the ion images