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.     

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.     

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.     

Unusual clinical manifestations of disturbed CSF dynamics in hydrocephalic children

Objects: Clinical symptoms and signs of increased intracranial pressure (ICP) may be nonspecific and unreliable, or even entirely absent, in hydrocephalic infants and children. Even with a radiological examination, it is often difficult to distinguish between ”arrested hydrocephalus” and slowly progressive hydrocephalus requiring treatment. Methods: We present two cases with unusual and initially misleading clinical manifestations of increased ICP. In both cases, the disturbed cerebrospinal fluid (CSF) dynamics, i.e. raised ICP, were not recognised until demonstrated by a long-term ICP monitoring. In a 5-month-old boy with normal head circumference and normal psychomotor development, the sudden onset of episodes of torticollis and screaming were the only symptoms. No pathology underlying the developing hydrocephalus and the raised ICP could be established, but the boy’s condition improved after a shunt operation. In the other case, symptoms and signs consisted primarily in a slowly progressive dilatation of the facial veins with onset at the age of 2–3 years. As the girl also presented a congenital subvalvular aortic stenosis, the venous congestion was initially thought to reflect a vena cava superior syndrome. Further radiological examinations, however, revealed an extensive sinus thrombosis underlying the raised ICP. The girl underwent shunt insertion, and the venous congestion was reduced. Conclusions: The cases illustrate that though clinical and radiological findings may be very doubtful, or unusual for increased ICP, direct diagnostic long-term ICP monitoring should always be contemplated. Only ICP monitoring can reveal with certainty whether disturbed CSF dynamics are involved, requiring a CSF diverting operation to treat and stabilise the condition. Received: 6 December 1999     

A multidisciplinary memory clinic in a neurological setting: diagnostic evaluation of 400 consecutive patients

This report describes the operation of a multidisciplinary university hospital memory clinic in a neurological setting, and the diagnostic evaluations in 400 consecutive patients referred for cognitive symptoms and possible dementia during a period of 27 months (1 September 1995-31 December 1997). The mean age of the patients was 63.6 years (range 19-97). On clinical and neuropsychological examination, 46% of the patients fulfilled DSM IV criteria for dementia, 5% had selective amnesia, and 14% had other selective cognitive deficits. The remaining patients had either no significant cognitive deficits (31%) or were not evaluable (4%). A wide range of disorders from the fields of neurology, psychiatry, neurosurgery and internal medicine were identified as the underlying etiologies for the cognitive symptoms. Potentially reversible conditions were observed in 26% of the patients, not including the 11% in whom no specific underlying disease was identified. Concomitant conditions or risk factors with a potential influence on cognitive functions were identified in 61% of the patients. Diagnostic evaluation of patients with mild to moderate cognitive symptoms and possible dementia is an integrated multidisciplinary task, which should focus on the identification of non-progressive and potentially reversible etiologies, co-morbidity, selective cognitive deficits, and rare or atypical neurological conditions, as well as on the early identification of common progressive dementia disorders.