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.     

Structural white matter abnormalities in patients with idiopathic dystonia

We investigated whether structural white matter abnormalities, in the form of disruption of axonal coherence and integrity as measured with diffusion tensor imaging (DTI), constitute an underlying pathological mechanism of idiopathic dystonia (ID), independent of genotype status. We studied seven subjects with ID: all had cervical dystonia as their main symptom (one patient also had spasmodic dysphonia and two patients had concurrent generalized dystonia, both DYT1‐negative). We compared DTI MR images of patients with 10 controls, evaluating differences in mean diffusivity (MD) and fractional anisotropy (FA). ID was associated with increased FA values in the thalamus and adjacent white matter, and in the white matter underlying the middle frontal gyrus. ID was also associated with increase in MD in adjacent white matter to the pallidum and putamen bilaterally, left caudate, and in subcortical hemispheric regions, including the postcentral gyrus. Abnormal FA and MD in patients with ID indicate that abnormal axonal coherence and integrity contribute to the pathophysiology of dystonia. These findings suggest that ID is not only a functional disorder, but also associated with structural brain changes. Impaired connectivity and disrupted flow of information may contribute to the impairment of motor planning and regulation in dystonia. © 2006 Movement Disorder Society     

Aktuelles zum Thema Latex-Allergie

Zusammenfassung Die Typ I-Allergien gegen Latex sind in den vergangenen Jahren zu einem zunehmenden berufsdermatologischen Problem geworden, zumal mindestens 10% der Angestellten im Gesundheitswesen betroffen sind. In der Dermatologischen Klinik der Universit?t Erlangen-Nürnberg stieg die Anzahl der j?hrlich diagnostizierten Patienten mit Latexallergien von 1989 bis 1995 auf das 12fache, wobei der Anteil der schweren, generalisierten Formen der Erkrankung von 10,7% (1989/1990) auf 44% (1994/1995) zunahm. Unter den m?glichen Ausl?sern der Latexallergie (wasserl?sliche Proteine mit Molekulargewichten von 2 bis 200 kD) sind mindestens 5 Hauptproteine mit bereits bekannter Prim?rstruktur zu berücksichtigen. Zus?tzlich gibt es Hinweise für Markerproteine, die in bestimmten Risikogruppen geh?uft zur Ausl?sung spezifischer IgE-Antik?rper führen (z.B. 46 kD-Protein in medizinischen Berufen, 14,6 kD- und 27 kD-Proteine bei Kindern mit Spina bifida). Das Vorkommen von Kreuzreaktionen zwischen Latex und unterschiedlichen Früchten (besonders Avocado, Kiwi, Banane, E?kastanie) bei 60 bis 70% der Latexallergiker ist bei der allergologischen Abkl?rung und Beratung dieser Patienten zu beachten. Wesentliche Aspekte der Prophylaxe umfassen die konsequente Umstellung medizinischer Einrichtungen auf ungepuderte Latexhandschuhe mit niedrigem Proteingehalt. Eine Zusammenstellung von OP- und Untersuchungshandschuhen, welche Angaben über die von uns ermittelten Proteinkonzentrationen (modifizierte Lowry-Methode und Hochdruck-Flüssigkeits-Chromatographie, HPLC) enth?lt, soll ein Leitfaden bei der Auswahl allergologisch geeigneter Handschuhe sein. Eingegangen am 10. August 1996 Angenommen am 21. August 1996     

Ca2+ dependence of the amphetamine, nomifensine, and Lu 19-005 effect on in vivo dopamine transmission

The present in vivo microdialysis study examined the role of vesicular- and carrier-mediated mechanisms underlying dopamine (DA) release, uptake and metabolism in halothane-anaesthetized rats. Omission of calcium (Ca2+) from the dialysis perfusing medium, thereby reducing the concentration of Ca2+ in the striatal microenvironment necessary for vesicular DA release, attenuated the elevation of DA normally induced by the potent DA uptake inhibitors, nomifensine and Lu 19-005. Consistent with the results of in vitro studies, amphetamine release DA in a Ca2+-independent manner. The release of DA induced by amphetamine could be effectively blocked by nomifensine and Lu 19-005, demonstrating that the in vivo movement of amines occurred via a transport carried-mediated mechanism. Additionally, the inhibition of DA metabolism produced by amphetamine could be reversed or blocked by prior or delayed treatment with DA uptake inhibitors. The results support a bidirectional in vivo capability of the amine transport carrier.     

Imaging of shifted stimulated echoes and multiple spin echoes

It is shown that a repetitive pulse sequence consisting of two 90° pulses and gradients in a 1:2 ratio around the second 90° pulse generates interscan shifted stimulated echoes (SSTEs) and intrascan multiple spin echoes (MSEs). Separation of these two types of signals is accomplished using specific gradient crusher schemes. The intensity of the SSTEs is an order of magnitude larger than that of the MSEs and determines the signal contrast if both effects are selected simultaneously. The SSTE sequence generates improved contrast between gray and white matter, even at high field, which is explained in terms of increased inverse T₁-weighting for the interscan echo. The MSE image has low signal to noise and no detectable contrast. The effect of interscan diffusion weighting is also discussed.     

Temporal relationship of hepatocellular dysfunction and ischemia in sepsis

To determine whether hepatic dysfunction in sepsis results from hypoperfusion or direct cellular injury, Sprague-Dawley rats underwent either cecal ligation and puncture or sham operation. After either two or six hours, effective hepatic blood flow was measured using the galactose clearance method. Hepatocytes were isolated and intracellular sodium and potassium and glucose production were measured. Hepatic blood flow in septic rats decreased as early as two hours after sepsis when compared with sham-operated rats (3.8 +/- 1.4 vs 8.7 +/- 3.1 mL/min/100 g body weight). Intracellular sodium and potassium levels and glucose production in septic rats were not significantly different when compared with controls at two hours. After six hours, hepatic blood flow remained depressed and intracellular sodium level was increased compared with sham-operated rats (41.7 +/- 10.4 vs 31.4 +/- 5.9 mmol/L [41.7 +/- 10.4 vs 31.4 +/- 5.9 mEq/L]) and potassium decreased compared with controls (90.7 +/- 7.9 vs 111.5 +/- 6.7 mmol/L [90.7 +/- 7.9 vs 111.5 +/- 6.7 mEq/L]). Glucose production was decreased in septic rats after six hours when compared with controls (4.7 +/- 1.5 vs 15.4 +/- 6.4 mumol/g hepatocytes). These data suggest that hepatic blood flow is decreased before alterations in intracellular sodium and potassium as well as glucose production.     

A comparison of the relative risk of vessel injury with conical versus pyramidal laparoscopic trocars in a rabbit model

OBJECTIVE: Our purpose was to compare the relative risk of vessel injury after use of a 5 mm conical-tipped trocar, a 5 mm pyramidal-tipped trocar, and a 10 mm pyramidal-tipped trocar in a rabbit vessel model.STUDY DESIGN: Plastic templates were placed in front of and behind 108 mesenteric vessels in 11 anesthetized New Zealand White rabbits. Laparoscopic trocars were inserted through the templates and mesentery. The incidence of vessel injury was determined at distances from the vessels ranging from 0 to 5 mm.RESULTS: The 5 mm conical trocar resulted in a vessel injury rate of 88% at 0 mm from the vessel but 0% at 1 or 2 mm. The 5 mm pyramidal trocar resulted in 100%, 88%, and 62% injury rates of 0, 1, and 2 mm from the vessels, respectively. The 10 mm pyramidal trocar resulted in a 100% injury rate at 0, 1, 2, or 3 mm from the vessels and 80% and 40% at 4 mm and 5mm, respectively.CONCLUSION: The relative risk of vessel injury is significantly increased by the use of pyramidal-tipped trocars when compared with conical-tipped trocars, especially if larger diameter trocars are used.     

Characterization and screening of bacteria from rhizosphere of maize grown in Indonesian and Pakistani soils

Thirty rhizobacteria isolated from maize grown in Pakistani and Indonesian soils were evaluated for their morphological characteristics, nitrogen fixation, P-solubilization, indole acetic acid (IAA) and siderophores production. Nitrogenase activity was detected in nineteen isolates ranging from 21.8-3624 n moles C2H4 produced/h/mg protein. Most of the isolates produced IAA, ten were capable of siderophore production while four were P-solubilizers. Ultrastructural studies of Pseudomonas sp. F14 indicated characteristic rhizospheric colonization within 48 h that was observed to change considerably with the passage of time from few bacteria to micro colonies. Random amplified polymorphic DNA (RAPD) analysis of 30 bacterial strains using 30 oligonucleotide primers resulted in considerable level of genetic diversity, with genetic distance ranging from 2-16%. Indonesian isolates were found to be more diverse as compared to Pakistani isolates. The characterization and screening of rhizobacteria of maize rhizosphere has helped in selection of isolates F7, LS-1, 3.1.1.C, F2, F3 and F13 as superior strains for use as bioinoculant. Moreover isolate F14 identified, as Pseudomonas fulgida by partial 16S rRNA sequence analysis is a novel strain regarding its tremendous potentials for inoculum production to enhance the yield of maize.     

Isolation and phenogenetics of a novel circadian rhythm mutant in zebrafish

Widespread use of zebrafish (Danio rerio) in genetic analysis of embryonic development has led to rapid advances in the technology required to generate, map and clone mutated genes. To identify genes involved in the generation and regulation of vertebrate circadian rhythmicity, we screened for dominant mutations that affect the circadian periodicity of larval zebrafish locomotor behavior. In a screen of 6,500 genomes, we recovered 8 homozygous viable, semi-dominant mutants, and describe one of them here. The circadian period of the lager and lime (lag(dg2)) mutant is shortened by 0.7 h in heterozygotes,and 1.3 h in homozygotes. This mutation also shortens the period of the melatonin production rhythm measured from cultured pineal glands, indicating that the mutant gene product affects circadian rhythmicity at the tissue level, as well as at the behavioral level. This mutation also alters the sensitivity of pineal circadian period to temperature, but does not affect phase shifting responses to light. Linkage mapping with microsatellite markers indicates that the lag mutation is on chromosome 7. A zebrafish homolog of period1(per1) is the only known clock gene homolog that maps near the lag locus. However, all sequence variants found in per1 cDNA from lag(dg2) mutants are also present in wild type lines, and we were unable to detect any defect in per1 mRNA splicing, so this mutation may identify a novel clock gene.     

Use of subtractive hybridization to identify a diagnostic probe for a cystic fibrosis epidemic strain of Pseudomonas aeruginosa

A multiresistant strain of Pseudomonas aeruginosa is widespread among cystic fibrosis (CF) patients attending clinics in Liverpool, United Kingdom. Suppression subtractive hybridization was used to identify sequences present in the Liverpool CF epidemic strain but absent from strain PAO1. Using dot blot and PCR amplification assays, the prevalence of such sequences among a panel of CF isolates was determined. Several sequences were found only in the Liverpool epidemic strain. Some sequences were present in the Liverpool epidemic strain and in a minority of other isolates, including sequences with homology to genes implicated in O6 serotype and siderophore production. The Liverpool epidemic strain and 81% of nonepidemic isolates contained a sequence identified as part of the PAGI-1 genomic island. Other strains implicated in epidemic spread, which were from Manchester, United Kingdom, and Melbourne, Australia, were also screened. None of the sequences identified was present in the Manchester strain. However, one of two Melbourne strains contained some of the sequences found in the Liverpool epidemic strain. All isolates implicated in epidemic spread and 76% of sporadic isolates contained the exoS gene. A sequence present in all isolates of the Liverpool epidemic strain was used to develop a diagnostic PCR test for identification of the strain from colonies or directly from sputum samples.