Replicative senescence of human dermal fibroblasts affects structural and functional aspects of the Golgi apparatus

It is well recognized that the world population is ageing rapidly. Therefore, it is important to understand ageing processes at the cellular and molecular levels to predict the onset of age‐related diseases and prevent them. Recent research has focused on the identification of ageing biomarkers, including those associated with the properties of the Golgi apparatus. In this context, Golgi‐mediated glycosylation of proteins has been well characterized. Additionally, other studies show that the secretion of many compounds, including pro‐inflammatory cytokines and extracellular matrix–degrading enzymes, is modified during ageing, resulting in physical and functional skin degradation. Since the Golgi apparatus is a central organelle of the secretory pathway, we investigated its structural organization in senescent primary human dermal fibroblasts using confocal and electron microscopy. In addition, we monitored the expression of Golgi‐related genes in the same cells. Our data showed a marked alteration in the Golgi morphology during replicative senescence. In contrast to its small and compact structure in non‐senescent cells, the Golgi apparatus exhibited a large and expanded morphology in senescent fibroblasts. Our data also demonstrated that the expression of many genes related to Golgi structural integrity and function was significantly modified in senescent cells, suggesting a relationship between Golgi apparatus function and ageing.     

Application of the adverse outcome pathway framework to genotoxic modes of action

In May 2017, the Health and Environmental Sciences Institute's Genetic Toxicology Technical Committee hosted a workshop to discuss whether mode of action (MOA) investigation is enhanced through the application of the adverse outcome pathway (AOP) framework. As AOPs are a relatively new approach in genetic toxicology, this report describes how AOPs could be harnessed to advance MOA analysis of genotoxicity pathways using five example case studies. Each of these genetic toxicology AOPs proposed for further development includes the relevant molecular initiating events, key events, and adverse outcomes (AOs), identification and/or further development of the appropriate assays to link an agent to these events, and discussion regarding the biological plausibility of the proposed AOP. A key difference between these proposed genetic toxicology AOPs versus traditional AOPs is that the AO is a genetic toxicology endpoint of potential significance in risk characterization, in contrast to an adverse state of an organism or a population. The first two detailed case studies describe provisional AOPs for aurora kinase inhibition and tubulin binding, leading to the common AO of aneuploidy. The remaining three case studies highlight provisional AOPs that lead to chromosome breakage or mutation via indirect DNA interaction (inhibition of topoisomerase II, production of cellular reactive oxygen species, and inhibition of DNA synthesis). These case studies serve as starting points for genotoxicity AOPs that could ultimately be published and utilized by the broader toxicology community and illustrate the practical considerations and evidence required to formalize such AOPs so that they may be applied to genetic toxicity evaluation schemes. Environ. Mol. Mutagen. 61:114–134, 2020. © 2019 Wiley Periodicals, Inc.     

Comparison of the peripheral blood micronucleus test using flow cytometry in rat and mouse exposed to aneugens after single-dose applications

Detection of clastogenic compounds in the peripheral blood micronucleus test (MNT) in rats is a well-established methodology. However, the results obtained on the induction of micronuclei by aneugens in rat peripheral blood are controversial. Our aim was a comparative evaluation of the peripheral blood flow cytometry MNT in Wistar Han rat and CD1 mouse exposed to three aneugens (vinblastine, vincristine and colchicine) after single-dose applications. In addition, the same compounds were tested in the rat bone marrow MNT. The treatment with vinblastine (0.25, 0.5, 1, mg/kg), vincristine (0.025, 0.05, 0.1 mg/kg) or colchicine (0.7, 1, 1.3 mg/kg) induced no statistically significant increase in MN-PCEs (micronucleated polychromatic erythrocytes or reticulocytes) in rat peripheral blood. In rat bone marrow, a clear statistically significant increase in MN-PCE was found with vincristine and vinblastine. However, colchicine showed a clear increase in MN-PCE frequency without reaching statistically significant level only at 1 mg/kg. The positive effect in the bone marrow MNT shows that the target organ was exposed to the appropriate concentration levels of the respective aneugens. In mouse, the peripheral blood flow cytometry analysis after the treatment with vinblastine, vincristine and colchicine showed clear statistically significant increase in MN-PCE with all three compounds. The experiments with splenectomized rats treated with vincristine and colchicine were performed and statistically significant increases in MN-PCE were found with 0.05, 0.1, 0.15 mg/kg of vincristine and 0.7 and 1 mg/kg of colchicine. Our results demonstrate that micronucleated cells induced by aneugens are removed from rat peripheral blood by the spleen due to the large size of micronuclei. Based on our data, it is concluded that the flow cytometry peripheral blood MNT after single-dose applications is an appropriate test system for evaluating the genotoxic effects of aneugens in mice. However, in rats peripheral blood MNT aneugen detection might require multiple-dose applications to overwhelm the spleen effect.     

Role of Fas and granule exocytosis pathways in tumor-infiltrating T lymphocyte-induced apoptosis of autologous human lung-carcinoma cells

We have isolated a cytotoxic T lymphocyte (CTL) clone, Heu161, that reacts specifically with the human autologous lung carcinoma cell line IGR-Heu. We first demonstrated that IGR-Heu lacked Fas-receptor expression and was resistant to CD95-induced apoptosis. To further elucidate the role of Fas in tumor immune surveillance, we have stably transfected IGR-Heu with a Fas-expression vector and isolated CD95-sensitive and -resistant clones. Our data indicated that the resistance of 2 selected Fas-transfected clones to CD95-mediated lysis correlated with down-regulation of caspase-8 or its lack of cleavage and subsequent activation. All Fas transfectants, either sensitive or resistant to anti-Fas agonistic antibody, were as efficiently lysed by the CTL clone as the parental cell line. In addition, neither anti-Fas-blocking antibody nor Fas-Fc molecule inhibited T-cell lysis of Fas-sensitive tumor clone. This cytotoxicity was extracellular Ca(2+)-dependent and abolished in the presence of EGTA, indicating that it was mainly granzyme-mediated. Interestingly, although the caspase inhibitor z-VAD-fmk had no effect on tumor-cell lysis, it efficiently blocked target DNA damage triggered by autologous CTLs via the granule exocytosis pathway, indicating that the latter event was caspase-dependent. The present results suggest that lung carcinoma-specific CTLs use mainly a granule exocytosis-dependent pathway to lyse autologous target cells and that these effectors are able to circumvent alteration of the Fas-triggered intracellular signalling pathway via activation of a caspase-independent cytoplasmic death mechanism.     

Flow cytometric analysis of micronuclei in mammalian cell cultures: past, present and future

The relative simplicity of the in vitro micronucleus (MNvit) endpoint has made it amenable to several automated scoring approaches. Flow cytometry is one such scoring platform that has been successfully employed. This review describes the origins of the MNvit assay, as well as the evolution and properties of flow cytometry-based scoring systems. While the current state-of-the-art methods acquire micronucleus (MN) frequency data very efficiently, it is becoming clear that they also endow the assay with high information content. For instance, simultaneous with MN frequency determinations, several additional endpoints are acquired that provide insights into cytotoxicity, cell cycle perturbations and, in the event of MN induction, information about genotoxic mode of action. This review concludes with a discussion regarding data gaps and also recommendations for additional work that is needed to more fully realise the potential of flow cytometric MNvit scoring.     

Fistules artérioveineuse post-traumatiques des membres : expérience de 26 cas

Introduction

Posttraumatic arteriovenous fistulas are not a rare event in military facilities during periods of armed conflicts, but are seldom seen in the civilian health care system.

Patients and method

We report our approach to the management of 26 cases of posttraumatic limb arteriovenous fistulas colligated from 1996 to 2006.

Results

The main cause of our posttraumatic arteriovenous fistulas is the penetrating traumatism. Arteriography was the mostly used imaging exploration. The treatment was surgical for 24 patients. One case of thrombosis was reported.

Conclusion

Our series of posttraumatic fistulas is characterized by a particularly high rate of penetrating trauma, in which case intervention is the rule. Our study shows that, in our experience, surgical treatment is still a safe and efficient therapeutic attitude.     

Indications for a threshold of chemically-induced aneuploidy in vitro in human lymphocytes

The possible existence of a threshold for compounds inducing chromosomal loss was investigated for four known aneugens (colchicine, COL; carben-dazim, MBC; mebendazole, MEB; nocodazole, NOC) and two clastogens (methyl methanesulfo-nate, MMS; mitomycin C, MMC) using the micronu-cleus (MN) test in human lymphocytes. The presence of a whole chromosome in the MN was studied by fluorescent in situ hybridization (FISH) using a synthetic pancentromeric oligonucleotide probe. FISH was applied on two different MN preparations: cytokinesis-blocked MN (MNCB) assay, and MN sorted by flow cytometry. At subtoxic concentrations analyzed by MNCB and FISH, COL, MEB, MBC, and NOC induced a concentration-dependent increase in centromere-positive MN (MNCen+), MMC seemed to induce an increase in both types of MN (MNCen- and MNCen+), while MMS induced only MNCen-. On the sorted micronuclei (in a wide range of low to subtoxic concentrations), the concentration-effect profile for MNCen+, with the four aneugens tested, showed a statistically nonsignificant increase over a range of concentrations, followed by a second range of high concentrations with a statistically significant increase. To analyze the existence of a threshold, a piecewise linear regression was applied to the data. The first concentration that showed a statistically significant increase in MNCen+ was chosen as a breakpoint (0.037 μM for COL, 2.62 μM for MBC, 0.27 μM for MEB, and 0.066 μM for NOC). The statistical correlation between observed and predicted values showed a high correlation (r = 0.99), indicating a clear threshold for aneuploidy induction. However, for MMS the concentration-effect profile for MNCen+ showed a continuous concentration-dependent decrease with no threshold. With the two cytotoxicity assays used (Bio-Rad and MTT), no significant reduction was detected either in the protein content or in mitochondrial succinate dehydrogenase activity with all chemicals tested for MN induction. Therefore, our data suggest that the observed thresholds were not due to indirect toxic effects but to real aneugenic effects. © 1995 Wiley-Liss, Inc.