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UNLABELLED: In the reperfusion era, a new paradigm of treating patients with endovascular treatment (EVT) and neuroprotective drugs is emerging as a promising therapeutic option for patients with acute ischemic stroke (AIS). In this context, ApTOLL, a Toll-like receptor 4 (TLR4) antagonist with proven neuroprotective effect in preclinical models of stroke and a very good pharmacokinetic and safety profile in healthy volunteers, is a promising first-in-class aptamer with the potential to address this huge unmet need. This protocol establishes the clinical trial procedures to conduct a Phase Ib/IIa clinical study (APRIL) to assess ApTOLL tolerability, safety, pharmacokinetics, and biological effect in patients with AIS who are eligible for EVT. This will be a multicenter, double-blind, randomized, placebo-controlled, Phase Ib/IIa clinical study to evaluate the administration of ApTOLL together with EVT in patients with AIS. The study population will be composed of men and non-pregnant women with confirmed AIS with a

Amide bond synthesis is ranked as the second most important challenge in key green chemistry research areas identified by the ACS Green Chemistry Institute. While developing more sustainable amide bond forming reactions has been in focus, significantly less attention has been given to human toxicity and environmental aspects of the underlying amine and acid substrates and their corresponding coupled products, a potentially important contribution to the overall sustainability of the amide-bond-forming reactions. Here, we explore biocatalytic amide bond formation from a safer-and-more-sustainable-by-design perspective in which commercially available amines and acids as well as their corresponding amide products were evaluated in silico based on potential human toxicity and environmental fate and exposure. This in silico filtering resulted in a panel of 188 amine and 54 acid building blocks that could be classified as safe, referred to herein as "safechems". To enable couplings of safechems, we generated a panel of robust and promiscuous ancestral ATP-dependent amide bond synthetases (ABS) using McbA from Marinactinospora thermotolerans SCSIO 00652 as a template. Ancestral ABS enzymes exhibited complementary specificities in the coupling of a representative safechem subset of 17 amines and 16 acids while showing an increased thermostability of up to 20 °C compared to the extant biocatalyst. Finally, the pool of safechems and their corresponding amides were evaluated by USEtox (the UNEP-SETAC toxicity model), analysing not only the intrinsic properties of the compounds but evaluating their complete impact pathway including fate, exposure and effects. The amides were in general predicted as more toxic compared to the starting acids and amines through non-additive effects, emphasising that focusing on the toxicity of the building blocks alone is not sufficient to strive towards low human and ecotoxicity impact. Pursuing a safer and more sustainable by design perspective in the implementation of safechems did not prevent us from generating an array of novel products with potentially potent applications as exemplified here by enzymatic synthesis of substructures that are part of drug candidates for e.g. cancer treatment. [ABSTRACT FROM AUTHOR]

BackgroundThe issue of endocrine disrupting chemicals (EDCs) is receiving wide attention from both the scientific and regulatory communities. Recent analyses of the EDC literature have been criticized for failing to use transparent and objective approaches to draw conclusions about the strength of evidence linking EDC exposures to adverse health or environmental outcomes. Systematic review methodologies are ideal for addressing this issue as they provide transparent and consistent approaches to study selection and evaluation. Objective methods are needed for integrating the multiple streams of evidence (epidemiology, wildlife, laboratory animal, in vitro, and in silico data) that are relevant in assessing EDCs.MethodsWe have developed a framework for the systematic review and integrated assessment (SYRINA) of EDC studies. The framework was designed for use with the International Program on Chemical Safety (IPCS) and World Health Organization (WHO) definition of an EDC, which requires appraisal of evidence regarding 1) association between exposure and an adverse effect, 2) association between exposure and endocrine disrupting activity, and 3) a plausible link between the adverse effect and the endocrine disrupting activity.ResultsBuilding from existing methodologies for evaluating and synthesizing evidence, the SYRINA framework includes seven steps: 1) Formulate the problem; 2) Develop the review protocol; 3) Identify relevant evidence; 4) Evaluate evidence from individual studies; 5) Summarize and evaluate each stream of evidence; 6) Integrate evidence across all streams; 7) Draw conclusions, make recommendations, and evaluate uncertainties. The proposed method is tailored to the IPCS/WHO definition of an EDC but offers flexibility for use in the context of other definitions of EDCs.ConclusionsWhen using the SYRINA framework, the overall objective is to provide the evidence base needed to support decision making, including any action to avoid/minimise potential adverse effects of exposures. This framework allows for the evaluation and synthesis of evidence from multiple evidence streams. Finally, a decision regarding regulatory action is not only dependent on the strength of evidence, but also the consequences of action/inaction, e.g. limited or weak evidence may be sufficient to justify action if consequences are serious or irreversible.

Analogues of 2,3-dihydrobenzo(b)furan-5-ol were profiled.

The preparation of water-soluble diaryl tellurides, alkyl aryl tellurides and dialkyl tellurides that carry sulfopropyl groups is examined. Results indicate that they possess peroxide decomposing and antioxidative capacity.

The role of endothelial cells in regulating the flow of metabolic precursors of intracellular AA and GSH (glutathione) in the human umbilical vein smooth muscle cells (HUVSMCs) and the mechanisms influencing GSH and ascorbate levels in human vascular walls were described. The findings showed that the intercellular antioxidative capacity of HUVSMCs was enhanced by the uptake of extracellular precursors of GSH synthesis and AA. The data may aid in the development of applications for endogenous water-soluble antioxidants in treating human diseases.

The 1H nuclear magnetic resonance method is employed to study the glutathione peroxidase-like property of diaryl ditellurides. The classical coupled reductase assay is also used to study the aforementioned activity. The mechanistic study reveals that diaryl ditellurides are immediately transformed into tellurosulfides in the presence of hydrogen peroxide and thiol. On the other hand, the thiol, in the presence of thiol/hydrogen peroxide, is swiftly converted to the corresponding disulfide. These conversions have led to the formulation of a mechanism for the thiol peroxidase reaction of ditellurides.

Reactive Oxygen Species in Biological Systems An Interdisciplinary Approach Daniel L. Gilbert and Carol A. Colton, Eds. Kluwer/Plenum, New York, 1999. 733 pp $165, 97.50 £. ISBN 0-306-4576-3 Free Radicals [...]

SEURAT-1 is a European public-private research consortium that is working towards animal-free testing of chemical compounds and the highest level of consumer protection. A research strategy was formulated based on the guiding principle to adopt a toxicological mode-of-action framework to describe how any substance may adversely affect human health. The proof of the initiative will be in demonstrating the applicability of the concepts on which SEURAT-1 is built on three levels: (i) Theoretical prototypes for adverse outcome pathways are formulated based on knowledge already available in the scientific literature on investigating the toxicological modes-of-action leading to adverse outcomes (addressing mainly liver toxicity); (ii) adverse outcome pathway descriptions are used as a guide for the formulation of case studies to further elucidate the theoretical model and to develop integrated testing strategies for the prediction of certain toxicological effects (i.e., those related to the adverse outcome pathway descriptions); (iii) further case studies target the application of knowledge gained within SEURAT-1 in the context of safety assessment. The ultimate goal would be to perform ab initio predictions based on a complete understanding of toxicological mechanisms. In the near-term, it is more realistic that data from innovative testing methods will support read-across arguments. Both scenarios are addressed with case studies for improved safety assessment. A conceptual framework for a rational integrated assessment strategy emerged from designing the case studies and is discussed in the context of international developments focusing on alternative approaches for evaluating chemicals using the new 21st century tools for toxicity testing. [ABSTRACT FROM AUTHOR]

The principles of the 3Rs, Replacement, Reduction and Refinement, are being increasingly incorporated into legislations, guidelines and practice of animal experiments in order to safeguard animal welfare. In the present study we have studied the systematic application of 3R principles to toxicological research in the pharmaceutical industry, with particular focus on achieving reductions in animal numbers used in regulatory and investigatory in vivo studies. The work also details major factors influencing these reductions including the conception of ideas, cross-departmental working and acceptance into the work process. Data from 36 reduction projects were collected retrospectively from work between 2006 and 2010. Substantial reduction in animal use was achieved by different strategies, including improved study design, method development and project coordination. Major animal savings were shown in both regulatory and investigative safety studies. If a similar (i.e. 53%) reduction had been achieved simultaneously within the twelve largest pharmaceutical companies, the equivalent reduction world-wide would be about 150,000 rats annually. The results point at the importance of a strong 3R culture, with scientific engagement, collaboration and a responsive management being vital components. A strong commitment in leadership for the 3R is recommended to be translated into cross-department and inter-profession involvement in projects for innovation, validation and implementation. Synergies between all the three Rs are observed and conclude that in silico-, in vitro- and in vivo-methods all hold the potential for applying the reduction R and should be consequently coordinated at a strategic level. [ABSTRACT FROM AUTHOR]

Abstract: Cells phenotypically adapt to alterations in their intra- and extracellular environment via organised alterations to gene and protein expression. Many chemical and physical stimuli are known to drive such responses, including the induction of oxidative stress and heat shock. Increasing use of mobile telephony in our society, has brought focus on the potential for radio frequency (microwave) electromagnetic radiation to elicit biological stress responses, in association with potentially detrimental effects of this to human health. Here we review evidence suggesting altered gene and protein expression in response to such emissions, with particular focus on heat shock proteins. Non-thermal induction of heat shock proteins has been claimed by a number of investigations in in vitro cellular systems, and appears pleiotrophic for many other regulatory events. However, many of these studies are flawed by inconsistencies in exposure models, cell types used and the independent reproducibility of the findings. Further, the paucity of evidence from in vivo experimentation is largely contradictory. Therefore, the validity of these effects in human health risk assessment remain unsubstantiated. Where possible, suggestions for further experimental clarification have been provided. [Copyright &y& Elsevier]

The fjord-region PAH dibenzo[a,l]pyrene (DBP) is considerably more carcinogenic than the bay-region benzo[a]pyrene (BP). This fact can be ascribed to differences in DNA binding efficiency of their ultimate carcinogenic diol epoxide (DEs) intermediates, differences in structural features of the DNA adducts, and differences in DNA adduct recognition and the subsequent lesion removal by nucleotide excision repair (NER). In order to further substantiate previous findings in cell-free human systems (1), cultured cells (2), and in rodents (3) we have studied DNA adduct formation of anti-DE of DBP [(±)-anti-DBPDE], in A549 human epithelial lung carcinoma cells and monitored the levels of adducts as a function of time. A high-performance liquid chromatography (HPLC) procedure that allows monitoring of all cis- and trans-nucleoside adducts of dA and dG was used. Circular dichroism and UV-spectroscopy have been employed to gain information on adduct structural features. Incubation of cells with 0.1 μM (±)-anti-DBPDE resulted in rapid formation of adducts, followed by a decline. After 6 h of incubation about 20% of the adducts remained. Repeating the experiment with 0.01 μM (±)-anti-DBPDE resulted in a correspondingly lower adduct level initially, but in this case a larger proportion (35%) of the adducts remained after 6 h of incubation. Notably, at this time point, similar amounts of adducts are observed with both (±)-anti-DBPDE concentrations studied. Independent of diol epoxide concentration and incubation time, the dA/dG adducts were constant in number (∼⃒2.8). However, within the different adduct categories, the adduct distribution seemed to be time dependent. Although the data are preliminary, a selection with regard to adduct removal seems to have taken place. In comparative experiments with (+)-anti-BPDE, no significant difference in rates of adduct removal was evident. Preliminary results from global gene expression analysis, with focus on genes involved in DNA maintenance and cell cycle checkpoints, demonstrate interesting differences in the stress response elicited in the cells following exposure to the distorted and flexible nonplanar DBPDE or the rigid and planar BPDE molecule. As expected some major common induction events were clearly related to the activation of p53-dependent cell cycle checkpoint. However, distinct clusters of gene expression alterations were detected which mark one or other treatment specifically, indicating a high degree of subtlety in the stress response to closely related DNA adduct forming species. [ABSTRACT FROM AUTHOR]

Presents a study on the analytical developments in the assay of intra- and extracellular glutathione (GSH) homeostasis. Identification of novel protein substrates for S-glutathionylation during oxidative stress and constitutive metabolism; Glutathione and GSH-protein compartmentalisations and population distributions in intact cells; Determination of GSH redox states in the extracellular space of intact human tissues.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has unmasked mankind's vulnerability to biological threats. Although higher age is a major risk factor for disease severity in COVID-19, several predisposing risk factors for mortality are related to low cardiorespiratory and metabolic fitness, including obesity, cardiovascular disease, diabetes, and hypertension. Reaching physical activity (PA) guideline goals contribute to protect against numerous immune and inflammatory disorders, in addition to multi-morbidities and mortality. Elevated levels of cardiorespiratory fitness, being non-obese, and regular PA improves immunological function, mitigating sustained low-grade systemic inflammation and age-related deterioration of the immune system, or immunosenescence. Regular PA and being non-obese also improve the antibody response to vaccination. In this review, we highlight potential physiological, cellular, and molecular mechanisms that are affected by regular PA, increase the host antiviral defense, and may determine the course and outcome of COVID-19. Not only are the immune system and regular PA in relation to COVID-19 discussed, but also the cardiovascular, respiratory, renal, and hormonal systems, as well as skeletal muscle, epigenetics, and mitochondrial function. [ABSTRACT FROM AUTHOR]

Apoptotic regulatory mechanisms in skeletal muscle have not been revealed. This is despite indications that remnant apoptotic events are detected following exercise, muscle injury and the progression of dystrophinopathies. The recent elicitation of a cytochrome c-mediated induction of caspases has led to speculation regarding a cytochrome c mechanism in muscle. We demonstrate that cytosols from skeletal muscle biopsies from healthy human volunteers lack the ability to activate type-II caspases by a cytochrome c-mediated pathway despite the confirmed presence of both procaspase-3 and -9. This was not due to the presence of an endogenous inhibitor, as the muscle cytosols enhanced caspase activity when added to a control cytosol, subsequently activated by cytochrome c and dATP. In addition, we demonstrate that muscle cytosols lackthe apoptosis protease activator protein-1 (APAF-1), both at the protein and mRNA levels. These data indicate that human skeletal muscle cells will be refractory to mitochondrial-mediated events leading to apoptosis and thus can escape a major pro-apoptotic regulatory mechanism. This may reflect an evolutionary adaptation of cell survival in the presence of the profusion of mitochondria required for energy generation in motility. [ABSTRACT FROM AUTHOR]

In this comunication we report on the effects of serial passage of human umbilical vein endothelial cells on the levels of GSH and activities of selenium-dependent GSH peroxidase (SeGSHpx) and GSSG reductase (GSSGred) in confluent monolayers of human umbilical vein endothelial cells derived from 4 individual donors. The mean levels of GSH in confluent monolayers of individual human umbilical vein endothelial cells batches fell from an average of 35 nmol/mg protein to 16 nmol/mg protein from primary culture to the third passage, with little interindividual variation noted. Simultaneously, the mean activities of SeGSHpx and GSSGred remained unaltered during serial culture, again with little interindividual variation noted. However, serial passage of the cells from different individuals did not affect the sensitivity of the cells to sublethal injury induced by H2O2 (10 or 100 μM), as judged by the stimulated release of (3H)-deoxyglucose. When these GSH-dependent parameters were assayed during the coarse of the growth of an individual passage of cells, GSH levels declined following trypsinisation, but the activities of SeGSHpx and GSSGred remained essentially unaltered, when the data were standardised to cell number. On the other hand, standardisation of these data to cell protein revealed decreases in all three parameters following trypsinisation, indicating the introduction of artefacts into the data and the unsuitability of this standardisation procedure. For the first four hours following plating, all of the GSH-dependent parameters remained essentially unaltered, but began increasing thereafter. Thus, GSH levels climbed to a peak prior to the onset of cell division and the activity of GSSGred rose to a peak by 24 hr, whilst the activity of SeGSHpx rose steadily during the first 72 hr of culture. When these low density cell cultures were tested for their sublethal response to H2O2 (3, 10 and 100 μM), a dose-dependent increase in the release of (3H)-deoxyglucose was noted, which remained constant during the first 72 hr of culture. Due to the relative lack of interindividual variations in the GSH parameters tested and in the sensitivity to H2O2, these data suggest that confluent cultures of pooled cells of any passage up to the third may be used for model studies of the sublethal effects of H2O2 in these human cells. The data also suggest that the sensitivity of confluent human umbilical vein endothelial cells derived from different individuals to the sublethal effects of H2O2 is not predictable from their individual GSH-dependent potential to detoxicate the peroxide. Additionally, fluctuations in GSH-dependent potential to detoxicate H2O2 may be related to the onset of growth in sub-confluent cultures of human umbilical vein endothelial cells, but appear, once again, not to be related to the sensitivity of the cells to sublethal effects of H2O2. [ABSTRACT FROM AUTHOR]

Menadione and diquat cause toxicity in isolated hepatocytes. The toxicities of both menadione and diquat are primarily due to redox cycling and consequent oxidative stress. Menadione toxicity, however, has another component as the compound also possesses alkylating and oxidating properties allowing it to interact directly with cellular nucleophiles. Sulfite afforded considerable protection of isolated rat hepatocytes against the toxicity of menadione. This protective effect of sulfite may have several components. Sulfite effectively competed with glutathione (GSH) for conjugation with menadione, sparing intracellular GSH which may continue to detoxify reactive oxygen species formed through menadione redox cycling. The menadione sulfite conjugate undergoes much slower redox cycling than both menadione and the menadione glutathione conjugate. Sulfite also showed some degree of protection of hepatocytes from the toxicity of diquat. Diquat is a 'pure' redox cycling agent and the protective effect of sulfite may involve the liberation of GSH from GSSG by sulfitolysis. This would again bolster intracellular GSH levels allowing further GSH-dependent detoxification of reactive oxygen species through cellular GSH peroxidases. In conclusion, our data illustrate the potential of inorganic sulfite to support the intracellular detoxification function of GSH, both against reactive electrophilic metabolites and against agents undergoing redox cycling. [ABSTRACT FROM AUTHOR]

Benzo[α]pyrene 7,8-dihydrodiol 9,10-epoxide is an established carcinogen, known to covalently bind to DNA, in articular to the exocyclic aminogroup of dG, and thereby cause conformational changes to the double helix. AP-1 is a well-studied transcription factor that specifically binds to the DNA sequence 5'-d(TGAGTCA). The effects of more or less randomly distributed BPDE adducts on DNA have been studied in different contexts, as well as the effects of different stimuli on transcription factor binding affinity and expression, but so far no investigation has been made concerning the effect of specific modification of a transcription factor binding site. In this study we have specifically modified the binding site of the transcription factor AP-1 with the (+)-anti- or (−)-syn-enantiomers of BPDE, and have studied how this affects the binding of the Fos-Jun proteins. Both (−)-syn- and (+)-anti-BPDE, giving rise to a cis- and a trans-adduct, respectively, have been used and, in both cases, the binding of AP-1 like proteins from HeLa cell nuclear extracts to the modified binding site decreased by approximately 50% as compared to controls. There was no apparent difference in response between the different diastereomers, so it seems that the binding geometry of the adduct (either intercalated or pointing towards the 5'-end in the minor groove, respectively) is of less importance. An interesting feature was the apparent yield of three differently shifted bands using the modified binding site. This can be due to conformational changes of the complex and/or the presence of less specific complexes as an effect of the adduct. Recombinant, truncated Fos-Jun proteins completely failed to bind to modified binding sites when performing the same experiments as detailed above and their binding to unmodified oligonucleotide was 50% less than for native proteins from the nuclear extract Supershift assays, using antibodies specific for c-Fos and c-Jun proteins, and competition experiments with various unlabelled oligonucleotides, were performed in order to check the specificity of binding in the observed bands. The results using the oligonucleotide containing the unmodified binding sequence and HeLa cell nuclear extract were fully consistent with binding of c-Fos and c-Jun, whereas the binding to oligonucleotides containing BPDE-modified binding sequences was not This implies involvement of other proteins in this event. [ABSTRACT FROM PUBLISHER]

Evidence is mounting supporting a role for oxidative stress in the mechanism of tumour promotion in response to agents such as 12--tetradecanoylphorbol-13-acetate (TPA). In this paper we demonstrate that glutathione peroxidase-mimetic xenobiotics, ebselen, ebselen-glutathione, a-(phenyl-selenenyl) acetophenone and bis-(4-aminophenyl) telluride (at concentrations between 10 μM and 50 μM) all demonstrate protective effects on TPA-induced downregulation of gap-junctional intercellular communication (GJIC) between WB-F344 rat liver epithelial cells. These effects were, in each case, diminished if the cells were depleted of their intracellular glutathione, and potentiated if glutathione was supplemented into the incubations. Additionally, bis-(4-aminophenyl) selenide and several -substituted analogues, possessing potent antioxidant activity but being devoid of GSH peroxidase-mimetic activity, demonstrated remedial activity against TPA-induced downregulation of GJIC. Structure-activity relationships between these molecules showed a strong correlation to the oxidation potential of the selenium atom in the compound as the bis-(4-nitrophenyl)- and bis-(4-cyanophenyl)- derivatives, which possess poor antioxidant capacity and a half-wave redox potential well above +1.0 V, did not affect TPA-induced effects on GJIC. Examination of the mechanism of action of these redox-active compounds demonstrated correlations between their abilities to (i) prevent TPA-induced downregulation of GJIC, (ii) abolish the accumulation of intracellular oxidants and (iii) prevent the hyper-phosphorylation and internalization of connexin 43 in the cells. The active compounds were also able to prevent the rapid, TPA-induced translocation of protein kinase C to the particulate fraction of the cells, without affecting phorbol ester binding. These data support a synergistic role for oxidants. and other TPA-dependent responses within the cell in mediating the downregulation of GJIC. Such oxidative metabolism may play a role in the control of translocation of protein kinase C from the cytosol to membranes in response to TPA within these cells. Despite the nature of the test system studied, the data also clarify the molecular basis for a potential anti-tumour promotive effect of antioxidants, based on established redox chemistries of several series of structurally-related molecules. [ABSTRACT FROM PUBLISHER]

Sodium nitrite was shown to enhance the metabolism of -7,8-dihydroxy-7,8-dihydrobenzo[]pyrene (BP-7,8-diol) to 7/8,9,10- and 7,10/8,9-tetrahydroxy-7,8,9,10-tetrahydro-benzo[]pyrene (tetraols) in phorbol myristate acetate (PMA- stimulated polymorphonuclear leukocytes (PMNs).The production of these tetraols implicates the intermediate formation of the corresponding -7,8-dihydroxy-9,10-epoxy-7,8-9,10-tetrahydrobenzo[]pyrene (and-BPDE). A 2- to 3-fold increase in the tetraol yield was observed in the presence of nitrite in excess of 1 mM. Sodium azide, an inhibitor of myeloperoxidase and catalase, reduced the nitrite-stimulated metabolism of BP-7,8-diol in PMA-activated leukocytes. Diphenylene iodonlum sulphate, a NADPH-oxidase inhibitor, lowered the production of tetraols in PMA-stimulated leukocytes both in the absence and presence of nitrite. Additionally, nitrite markedly enhanced the covalent binding of metabolites derived from [H](−)-BP-7,8-diol to leukocyte proteins as well as to DNA present extracellularly. The nitrite-stimulated covalent binding to both proteins and DNA was inhibited by the presence of sodium azide. The mechanism underlying the effect of nitrite on the metabolism of BP-7,8-diol to reactive intermediates in PMA-activated human polymorphonuclear leukocytes is not known. However, the results are compatible with a peroxidase-dependent mechanism although other possible pathways may contribute to the enhanced rate of metabolism. [ABSTRACT FROM PUBLISHER]

Exposure of human umbilical vein endothelial cells to oxidants such as hydrogen peroxide, tertbutyl hydroperoxide and diamide has been shown to induce oxidant-specific S-thiolation of cellular proteins. In this study one of the main S-thiolated proteins in hydrogen-peroxide-treated cells was identified as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase. Additionally, we have shown that the post-translational modification of the cysteinyl thiols of glyceraldehyde-3-phosphate dehydrogenase accompanies an inhibition of the enzyme and that both events are simultaneously and rapidly reversed upon the removal of the oxidative stimulus. [ABSTRACT FROM AUTHOR]