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Application of adverse outcome pathways (AOP) and integration of quantitative in vitro to in vivo extrapolation (QIVIVE) may support the paradigm shift in toxicity testing to move from apical endpoints in test animals to more mechanism-based in vitro assays. Here, we developed an AOP of proximal tubule injury linking a molecular initiating event (MIE) to a cascade of key events (KEs) leading to lysosomal overload and ultimately to cell death. This AOP was used as a case study to adopt the AOP concept for systemic toxicity testing and risk assessment based on in vitro data. In this AOP, nephrotoxicity is thought to result from receptor-mediated endocytosis (MIE) of the chemical stressor, disturbance of lysosomal function (KE1), and lysosomal disruption (KE2) associated with release of reactive oxygen species and cytotoxic lysosomal enzymes that induce cell death (KE3). Based on this mechanistic framework, in vitro readouts reflecting each KE were identified. Utilizing polymyxin antibiotics as chemical stressors for this AOP, the dose-response for each in vitro endpoint was recorded in proximal tubule cells from rat (NRK-52E) and human (RPTEC/TERT1) in order to (1) experimentally support the sequence of key events (KEs), to (2) establish quantitative relationships between KEs as a basis for prediction of downstream KEs based on in vitro data reflecting early KEs and to (3) derive suitable in vitro points of departure for human risk assessment. Time-resolved analysis was used to support the temporal sequence of events within this AOP. Quantitative response-response relationships between KEs established from in vitro data on polymyxin B were successfully used to predict in vitro toxicity of other polymyxin derivatives. Finally, a physiologically based kinetic (PBK) model was utilized to transform in vitro effect concentrations to a human equivalent dose for polymyxin B. The predicted in vivo effective doses were in the range of therapeutic doses known to be associated with a risk for nephrotoxicity. Taken together, these data provide proof-of-concept for the feasibility of in vitro based risk assessment through integration of mechanistic endpoints and reverse toxicokinetic modelling.
Proteolytic cleavage of the extracellular domain affects signaling of parathyroid hormone 1 receptor
(2022)
Parathyroid hormone 1 receptor (PTH1R) is a member of the class B family of G protein-coupled receptors, which are characterized by a large extracellular domain required for ligand binding. We have previously shown that the extracellular domain of PTH1R is subject to metalloproteinase cleavage in vivo that is regulated by ligand-induced receptor trafficking and leads to impaired stability of PTH1R. In this work, we localize the cleavage site in the first loop of the extracellular domain using amino-terminal protein sequencing of purified receptor and by mutagenesis studies. We further show, that a receptor mutant not susceptible to proteolytic cleavage exhibits reduced signaling to G\(_s\) and increased activation of G\(_q\) compared to wild-type PTH1R. These findings indicate that the extracellular domain modulates PTH1R signaling specificity, and that its cleavage affects receptor signaling.
Adipositas ist eine Erkrankung, die durch ein erhöhtes Krebsrisiko neben zahlreichen anderen Komorbiditäten mit weitreichenden Folgen für die Gesundheit adipöser Patient*innen einhergeht. In der Pathogenese der adipositas-assoziierten Krebsarten sind dabei ein erhöhter oxidativer Stress sowie die damit einhergehende Schädigung der DNS maßgeblich beteiligt. Im Umkehrschluss wurde in der vorliegenden Arbeit der Einfluss eines durch bariatrische Chirurgie induzierten Gewichtsverlusts auf den oxidativen Stress und DNS-Schaden in adipösen Patient*innen anhand von Blutproben präoperativ sowie 6 und 12 Monate postoperativ untersucht. In einer Subpopulation der Patient*innen konnte eine tendenzielle Verringerung des DNS-Schadens anhand des Comet-Assays in peripheren Lymphozyten beobachtet werden. Im Hinblick auf den oxidativen Stress wurde im Plasma die Eisenreduktionsfähigkeit als Maß für die antioxidative Kapazität sowie Malondialdehyd als Surrogatmarker für das Ausmaß an Lipidperoxidation bestimmt. Weiterhin wurde in Erythrozyten das Gesamtglutathion und das oxidierte Glutathion bestimmt. Die oxidativen Stressparameter zeigten insgesamt nach einer initialen Zunahme im oxidativen Stress 6 Monate postoperativ eine rückläufige Tendenz im oxidativen Stress am Studienende. Somit geben die Beobachtungen dieser Arbeit Anlass zur Hoffnung, dass adipöse Patient*innen durch einen bariatrisch induzierten Gewichtsverlust von einer Verringerung des Krebsrisikos profitieren könnten.
Analysis of the Frequency of Kidney Toxicity in Preclinical Safety Studies using the eTOX Database
(2022)
This research aimed to obtain reliable data on the frequency of different
types of renal toxicity findings in 28-day oral gavage studies in Wistar rats, their
consistency across species and study duration, as well as the correlation between histopathological endpoints and routinely used clinical chemistry parameters indicative of kidney injury. Analysis of renal histopathological findings was
carried out through extraction of information from the IMI eTOX database.
Spontaneous renal histopathological findings in 28-day oral gavage studies in control Wistar rats and beagle dogs confirmed tubular basophilia and renal
dilation as the most frequent incidental findings in controls, whereas necrosis
and glomerulosclerosis were not identified at all or only rarely as a background
lesion.
Histopathological evidence of necrosis and glomerulosclerosis was associated with changes in clinical chemistry parameters in 28-day oral gavage
Wistar rat studies. Necrosis was frequently accompanied by a statistically significant rise in serum creatinine and serum urea, whereas serum albumin was
frequently found to decrease statistically significantly in treatment groups in
which necrosis was recorded. In contrast to necrosis, glomerulosclerosis was
not associated with statistically significant changes in serum creatinine and urea
in any of the 28-day oral gavage Wistar rat treatment groups, but appears to be
best reflected by a pattern of statistically significantly lowered serum albumin
and serum protein together with a statistically significant increase in serum cholesterol. As might have been expected based on the high background incidences
of tubular basophilia and dilation, no consistent changes in any of the clinical
chemistry parameters were evident in animals in which renal lesions were confined to renal tubular basophilia or dilation. In summary, the routinely provided
clinical chemistry parameters are rather insensitive - novel kidney biomarkers
such as Cystatin C, β-trace protein and Kidney injury molecule 1 should further
be evaluated and integrated into routine preclinical and clinical practice. However, evaluation of clinical chemistry data was limited by the lack of individual
animal data. Even though an extensive amount of preclinical studies is accessible
through the eTOX database, comparison of consistency across time was limited
by the limited number of shorter- and longer term studies conducted with the
compounds identified as causing renal histopathological changes within a 28-
day study in rats. A high consistency across time for both treatment-related tubular basophilia and treatment-related dilation cannot be confirmed for either of
the two effects as these two findings were both induced only rarely in studies
over a different treatment-duration other than 28 days after administration of the
compounds which provoked the respective effect in a 28-day study. For the
finding of necrosis consistency across time was low with the exception of
“AZ_GGA_200002321”, in which renal papillary necrosis was identified consistently throughout different treatment durations (2, 4, 26, 104 weeks). No shorter and longer-term studies were available for the compounds identified as causing
glomerulosclerosis within a 28-day study in rats.
No consistent findings of the selected histopathological endpoints were
identified in any of the corresponding 28-day oral gavage beagle dog studies
after treatment with the identical compounds, which caused the respective effect after 28-day treatment in rats. However, in the overwhelming majority of
cases, beagle dogs were administered lower doses in these studies in comparison to the corresponding 28-day Wistar rat studies.
Searching the eTOX database yielded no 28-day oral gavage studies in
Wistar and Wistar Han rats in which accumulation of hyaline droplets, tubular
atrophy or hyperplasia was recorded. Only one 28-day oral gavage Wistar rat
study was identified with the histopathological result of neutrophilic inflammation. Consequently, evaluation of these four renal findings in relation to clinical
chemistry parameters and consistency across time and species cannot be
made.
In summary, this work contributes knowledge through mining and evaluating the eTOX database on a variety of specific renal endpoints that frequently
occur after administration of trial substances in 28-day oral gavage studies in
Wistar rats in the field of preclinical toxicity with specific focus on their frequency relation to background findings, as well as consistency across time and species. Targeted statistical evaluation of in vivo data within joint research ventures
such as the eTOX project, presents an enormous opportunity for an innovative
future way of aiding preclinical research towards a more efficient research in the
preclinical stage of drug development. This could be achieved through the augmentation of methodological strategies and possibly novel software tools in order to predict in vivo toxicology of new molecular entities by means of information that is already available before early stages of the drug development
pipeline begin.
Food safety problems are a major hindrance to achieving food security, trade, and healthy living in Africa. Fungi and their secondary metabolites, known as mycotoxins, represent an important concern in this regard. Attempts such as agricultural, storage, and processing practices, and creation of awareness to tackle the menace of fungi and mycotoxins have yielded measurable outcomes especially in developed countries, where there are comprehensive mycotoxin legislations and enforcement schemes. Conversely, most African countries do not have mycotoxin regulatory limits and even when available, are only applied for international trade. Factors such as food insecurity, public ignorance, climate change, poor infrastructure, poor research funding, incorrect prioritization of resources, and nonchalant attitudes that exist among governmental organisations and other stakeholders further complicate the situation. In the present review, we discuss the status of mycotoxin regulation in Africa, with emphasis on the impact of weak mycotoxin legislations and enforcement on African trade, agriculture, and health. Furthermore, we discuss the factors limiting the establishment and control of mycotoxins in the region.
Vibrational spectroscopy can detect characteristic biomolecular signatures and thus has the potential to support diagnostics. Fabry disease (FD) is a lipid disorder disease that leads to accumulations of globotriaosylceramide in different organs, including the heart, which is particularly critical for the patient’s prognosis. Effective treatment options are available if initiated at early disease stages, but many patients are late- or under-diagnosed. Since Coherent anti-Stokes Raman (CARS) imaging has a high sensitivity for lipid/protein shifts, we applied CARS as a diagnostic tool to assess cardiac FD manifestation in an FD mouse model. CARS measurements combined with multivariate data analysis, including image preprocessing followed by image clustering and data-driven modeling, allowed for differentiation between FD and control groups. Indeed, CARS identified shifts of lipid/protein content between the two groups in cardiac tissue visually and by subsequent automated bioinformatic discrimination with a mean sensitivity of 90–96%. Of note, this genotype differentiation was successful at a very early time point during disease development when only kidneys are visibly affected by globotriaosylceramide depositions. Altogether, the sensitivity of CARS combined with multivariate analysis allows reliable diagnostic support of early FD organ manifestation and may thus improve diagnosis, prognosis, and possibly therapeutic monitoring of FD.
The potential of human-induced pluripotent stem cells (hiPSCs) to be differentiated into cardiomyocytes (CMs) mimicking adult CMs functional morphology, marker genes and signaling characteristics has been investigated since over a decade. The evolution of the membrane localization of CM-specific G protein-coupled receptors throughout differentiation has received, however, only limited attention to date. We employ here advanced fluorescent spectroscopy, namely linescan Fluorescence Correlation Spectroscopy (FCS), to observe how the plasma membrane abundance of the β\(_1\)- and β\(_2\)-adrenergic receptors (β\(_{1/2}\)-ARs), labelled using a bright and photostable fluorescent antagonist, evolves during the long-term monolayer culture of hiPSC-derived CMs. We compare it to the kinetics of observed mRNA levels in wildtype (WT) hiPSCs and in two CRISPR/Cas9 knock-in clones. We conduct these observations against the backdrop of our recent report of cell-to-cell expression variability, as well as of the subcellular localization heterogeneity of β-ARs in adult CMs.
Diabetes, a chronic group of medical disorders characterized byhyperglycemia, has become a global pandemic. Some hormones may influence the course and outcome of diabetes, especially if they potentiate the formation of reactive oxygen species (ROS). There is a close relationship between thyroid disorders and diabetes. The main objective of this investigation was to find out whether peripheral blood mononuclear cells (PBMCs) are more prone to DNA damage by triiodothyronine (T\(_3\)) (0.1, 1 and 10 μM) at various stages of progression through diabetes (obese, prediabetics, and type 2 diabetes mellitus—T2DM persons). In addition, some biochemical parameters of oxidative stress (catalase-CAT, thiobarbituric acid reactive substances—TBARS) and lactate dehydrogenase (LDH) were evaluated. PBMCs from prediabetic and diabetic patients exhibited increased sensitivity for T\(_3\) regarding elevated level of DNA damage, inhibition of catalase, and increase of TBARS and LDH. PBMCs from obese patients reacted in the same manner, except for DNA damage. The results of this study should contribute to a better understanding of the role of thyroid hormones in the progression of T2DM.
Dilated cardiomyopathy (DCM) is a common cause of heart failure (HF) and is of familial origin in 20–40% of cases. Genetic testing by next-generation sequencing (NGS) has yielded a definite diagnosis in many cases; however, some remain elusive. In this study, we used a combination of NGS, human-induced pluripotent-stem-cell-derived cardiomyocytes (iPSC-CMs) and nanopore long-read sequencing to identify the causal variant in a multi-generational pedigree of DCM. A four-generation family with familial DCM was investigated. Next-generation sequencing (NGS) was performed on 22 family members. Skin biopsies from two affected family members were used to generate iPSCs, which were then differentiated into iPSC-CMs. Short-read RNA sequencing was used for the evaluation of the target gene expression, and long-read RNA nanopore sequencing was used to evaluate the relevance of the splice variants. The pedigree suggested a highly penetrant, autosomal dominant mode of inheritance. The phenotype of the family was suggestive of laminopathy, but previous genetic testing using both Sanger and panel sequencing only yielded conflicting evidence for LMNA p.R644C (rs142000963), which was not fully segregated. By re-sequencing four additional affected family members, further non-coding LMNA variants could be detected: rs149339264, rs199686967, rs201379016, and rs794728589. To explore the roles of these variants, iPSC-CMs were generated. RNA sequencing showed the LMNA expression levels to be significantly lower in the iPSC-CMs of the LMNA variant carriers. We demonstrated a dysregulated sarcomeric structure and altered calcium homeostasis in the iPSC-CMs of the LMNA variant carriers. Using targeted nanopore long-read sequencing, we revealed the biological significance of the variant c.356+1G>A, which generates a novel 5′ splice site in exon 1 of the cardiac isomer of LMNA, causing a nonsense mRNA product with almost complete RNA decay and haploinsufficiency. Using novel molecular analysis and nanopore technology, we demonstrated the pathogenesis of the rs794728589 (c.356+1G>A) splice variant in LMNA. This study highlights the importance of precise diagnostics in the clinical management and workup of cardiomyopathies.
Recent analyses conducted by German official food control reported detection of the aromatic amides N-(2,4-dimethylphenyl)acetamide (NDPA), N-acetoacetyl-m-xylidine (NAAX) and 3-hydroxy-2-naphthanilide (Naphthol AS) in cold water extracts from certain food contact materials made from paper or cardboard, including paper straws, paper napkins, and cupcake liners. Because aromatic amides may be cleaved to potentially genotoxic primary amines upon oral intake, these findings raise concern that transfer of NDPA, NAAX and Naphthol AS from food contact materials into food may present a risk to human health. The aim of the present work was to assess the stability of NDPA, NAAX and Naphthol AS and potential cleavage to 2,4-dimethylaniline (2,4-DMA) and aniline during simulated passage through the gastrointestinal tract using static in vitro digestion models. Using the digestion model established by the National Institute for Public Health and the Environment (RIVM, Bilthoven, NL) and a protocol recommended by the European Food Safety Authority, potential hydrolysis of the aromatic amides to the respective aromatic amines was assessed by LC–MS/MS following incubation of the aromatic amides with digestive fluid simulants. Time-dependent hydrolysis of NDPA and NAAX resulting in formation of the primary aromatic amine 2,4-DMA was consistently observed in both models. The highest rate of cleavage of NDPA and NAAX was recorded following 4 h incubation with 0.07 M HCl as gastric-juice simulant, and amounted to 0.21% and 0.053%, respectively. Incubation of Naphthol AS with digestive fluid simulants did not give rise to an increase in the concentration of aniline above the background that resulted from the presence of aniline as an impurity of the test compound. Considering the lack of evidence for aniline formation from Naphthol AS and the extremely low rate of hydrolysis of the amide bonds of NDPA and NAAX during simulated passage through the gastrointestinal tract that gives rise to only very minor amounts of the potentially mutagenic and/or carcinogenic aromatic amine 2,4-DMA, risk assessment based on assumption of 100% cleavage to the primary aromatic amines would appear to overestimate health risks related to the presence of aromatic amides in food contact materials.