Role of albumin in the metabolism and excretion of ochratoxin A.

Ochratoxin A (OTA) is known to be strongly bound to serum albumin, but it remains unknown how albumin affects its metabolism and kinetics. To close this gap, we used a mouse model, where heterozygous albumin deletion reduces serum albumin to concentrations similar to hypoalbuminemic patients and completely eliminates albumin by a homozygous knockout. OTA and its potential metabolites (OTα, 4-OH-OTA, 7'-OH-OTA, OTHQ, OP-OTA, OTB-GSH, OTB-NAC, OTB) were time-dependently analyzed in plasma, bile, and urine by LC-MS/MS and were compared to previously published hepatotoxicity and nephrotoxicity data. Homozygous albumin deletion strongly accelerated plasma clearance as well as biliary and urinary excretion of the parent compound and its hydroxylation products. Decreasing albumin in mice by the heterozygous and even more by the homozygous knockout leads to an increase in the parent compound in urine which corresponded to increased nephrotoxicity. The role of albumin in OTA-induced hepatotoxicity is more complex, since heterozygous but not homozygous nor wild-type mice showed a strong biliary increase in the toxic open lactone OP-OTA. Correspondingly, OTA-induced hepatotoxicity was higher in heterozygous than in wild-type and homozygous animals. We present evidence that albumin-mediated retention of OTA in hepatocytes is required for formation of the toxic OP-OTA, while complete albumin elimination leads to rapid biliary clearance of OTA from hepatocytes with less formation of OP-OTA. In conclusion, albumin has a strong influence on metabolism and toxicity of OTA. In hypoalbuminemia, the parent OTA is associated with increased nephrotoxicity and the open lactone with increased hepatotoxicity.

Integrated data from intravital imaging and HPLC-MS/MS analysis reveal large interspecies differences in AFB1 metabolism in mice and rats.

Large interspecies differences between rats and mice concerning the hepatotoxicity and carcinogenicity of aflatoxin B1 (AFB1) are known, with mice being more resistant. However, a comprehensive interspecies comparison including subcellular liver tissue compartments has not yet been performed. In this study, we performed spatio-temporal intravital analysis of AFB1 kinetics in the livers of anesthetized mice and rats. This was supported by time-dependent analysis of the parent compound as well as metabolites and adducts in blood, urine, and bile of both species by HPLC-MS/MS. The integrated data from intravital imaging and HPLC-MS/MS analysis revealed major interspecies differences between rats and mice: (1) AFB1-associated fluorescence persisted much longer in the nuclei of rat than mouse hepatocytes; (2) in the sinusoidal blood, AFB1-associated fluorescence was rapidly cleared in mice, while a time-dependent increase was observed in rats in the first three hours after injection followed by a plateau that lasted until the end of the observation period of six hours; (3) this coincided with a far stronger increase of AFB1-lysine adducts in the blood of rats compared to mice; (4) the AFB1-guanine adduct was detected at much higher concentrations in bile and urine of rats than mice. In both species, the AFB1-glutathione conjugate was efficiently excreted via bile, where it reached concentrations at least three orders of magnitude higher compared to blood. In conclusion, major differences between mice and rats were observed, concerning the nuclear persistence, formation of AFB1-lysine adducts, and the AFB1-guanine adducts.

Acetaminophen overdose causes a breach of the blood-bile barrier in mice but not in rats.

Acetaminophen (APAP) is known to cause a breach of the blood-bile barrier in mice that, via a mechanism called futile bile acid (BA) cycling, increases BA concentrations in hepatocytes above cytotoxic thresholds. Here, we compared this mechanism in mice and rats, because both species differ massively in their susceptibility to APAP and compared the results to available human data. Dose and time-dependent APAP experiments were performed in male C57BL6/N mice and Wistar rats. The time course of BA concentrations in liver tissue and in blood was analyzed by MALDI-MSI and LC-MS/MS. APAP and its derivatives were measured in the blood by LC-MS. APAP-induced liver damage was analyzed by histopathology, immunohistochemistry, and by clinical chemistry. In mice, a transient increase of BA in blood and in peri-central hepatocytes preceded hepatocyte death. The BA increase coincided with oxidative stress in liver tissue and a compromised morphology of bile canaliculi and immunohistochemically visualized tight junction proteins. Rats showed a reduced metabolic activation of APAP compared to mice. However, even at very high doses that caused cell death of hepatocytes, no increase of BA concentrations was observed neither in liver tissue nor in the blood. Correspondingly, no oxidative stress was detectable, and the morphology of bile canaliculi and tight junction proteins remained unaltered. In conclusion, different mechanisms cause cell death in rats and mice, whereby oxidative stress and a breach of the blood-bile barrier are seen only in mice. Since transient cholestasis also occurs in human patients with APAP overdose, mice are a clinically relevant species to study APAP hepatotoxicity but not rats.

A liver digital twin for in silico testing of cellular and inter-cellular mechanisms in regeneration after drug-induced damage.

This communication presents a mathematical mechanism-based model of the regenerating liver after drug-induced pericentral lobule damage resolving tissue microarchitecture. The consequence of alternative hypotheses about the interplay of different cell types on regeneration was simulated. Regeneration dynamics has been quantified by the size of the damage-induced dead cell area, the hepatocyte density and the spatial-temporal profile of the different cell types. We use deviations of observed trajectories from the simulated system to identify branching points, at which the systems behavior cannot be explained by the underlying set of hypotheses anymore. Our procedure reflects a successful strategy for generating a fully digital liver twin that, among others, permits to test perturbations from the molecular up to the tissue scale. The model simulations are complementing current knowledge on liver regeneration by identifying gaps in mechanistic relationships and guiding the system toward the most informative (lacking) parameters that can be experimentally addressed.

Role of Serine arginine protein kinase 1 and Minichromosome maintenance protein 2 in predicting epithelial ovarian cancer response to treatment and prognosis.

BACKGROUND: Serine-Arginine (SR) proteins are a conserved family of proteins involved in RNA splicing and are reported to be over-expressed in multiple cancers. The aim of the study is evaluation of the expression of Serine arginine protein kinase 1 (SRPK1) and Minichromosome maintenance protein 2 (MCM2) in epithelial ovarian cancer (EOC) and their correlation with clinicopathological features, response to therapy, progression-free survival (PFS), and cancer-specific survival (CSS). METHODS: This study was carried out on surgical specimens of 65 patients diagnosed with EOC which were submitted to immunohistochemical staining by SRPK1 and MCM2 antibodies. RESULTS: About 89.2% of cases showed SRPK1 expression and its high expression was significantly associated with type II tumors and advanced stage. All cases showed nuclear immunoreaction for MCM2 with high expression in 49.2% of cases. There was a significant relationship between high values of SRPK1 H-score and percentage of MCM2. Postmenopause, type II pathology, advanced stage, absence of complete response to the treatment, resistance to platinum-based chemotherapy, and surgery done by a general surgeon were the factors affecting PFS. Response to treatment and platinum sensitivity were the most independent factors affecting patients' PFS. The factors associated with shorter CSS were suboptimal debulking, advanced stage, absence of complete response to the treatment, platinum resistance, and high SRPK1. High SRPK1 expression and platinum sensitivity were the independent factors affecting patients' CSS. CONCLUSIONS: SRPK1 is an unfavorable biomarker in EOC patients because of its association with aggressive histologic type, advanced International Federation of Gynecology and Obstetrics (FIGO) stage, and worse survival. SRPK1 could promote the proliferation of EOC by up-regulation of MCM2.

The efficacy of various irrigation techniques on the removal of double antibiotic paste from simulated immature roots and the amount of apically extruded debris.

OBJECTIVE: This study evaluated the effect of the XP-Endo Finisher (XPF), passive ultrasonic irrigation (PUI) and conventional irrigation using side-vented needle (SVN) on the amount of apically extruded debris and canal cleanliness following the removal of double antibiotic paste (DAP) from immature root canal models. MATERIAL AND METHODS: Forty-eight extracted mandibular premolars were drilled using peeso drills to simulate immature apices. The canals were filled with DAP and were randomly assigned into 3 groups according to the DAP removal method: XPF, PUI, and SVN (n = 16). The amount of extruded debris was assessed with an analytical balance then roots were split longitudinally and imaged using stereomicroscope to evaluate the residual medicament. Data were statistically analyzed using Kruskal-Wallis and Dunn's test. Spearman's correlation coefficient was used to determine significant correlation between extruded debris and the residual DAP scores. RESULTS: There was no significant difference between debris extrusion values for all groups (P value 0.237). For canal cleanliness, the amount of remaining DAP was significantly lower in the XPF and PUI compared to SVN (P value < 0.001). A non-significant positive (direct) correlation was found between the amounts of apically extruded debris and residual DAP scores (P value 0.087). CONCLUSION: XPF and PUI were associated with better canal cleanliness during removal of DAP, no difference could be found between the three irrigation techniques regarding the debris extrusion.

Inhibition of the renal apical sodium dependent bile acid transporter prevents cholemic nephropathy in mice with obstructive cholestasis.

BACKGROUND & AIMS: Cholemic nephropathy (CN) is a severe complication of cholestatic liver diseases for which there is no specific treatment. We revisited its pathophysiology with the aim of identifying novel therapeutic strategies. METHODS: Cholestasis was induced by bile duct ligation (BDL) in mice. Bile flux in kidneys and livers was visualized by intravital imaging, supported by MALDI mass spectrometry imaging and liquid chromatography-tandem mass spectrometry. The effect of AS0369, a systemically bioavailable apical sodium-dependent bile acid transporter (ASBT) inhibitor, was evaluated by intravital imaging, RNA-sequencing, histological, blood, and urine analyses. Translational relevance was assessed in kidney biopsies from patients with CN, mice with a humanized bile acid (BA) spectrum, and via analysis of serum BAs and KIM-1 (kidney injury molecule 1) in patients with liver disease and hyperbilirubinemia. RESULTS: Proximal tubular epithelial cells (TECs) reabsorbed and enriched BAs, leading to oxidative stress and death of proximal TECs, casts in distal tubules and collecting ducts, peritubular capillary leakiness, and glomerular cysts. Renal ASBT inhibition by AS0369 blocked BA uptake into TECs and prevented kidney injury up to 6 weeks after BDL. Similar results were obtained in mice with humanized BA composition. In patients with advanced liver disease, serum BAs were the main determinant of KIM-1 levels. ASBT expression in TECs was preserved in biopsies from patients with CN, further highlighting the translational potential of targeting ASBT to treat CN. CONCLUSIONS: BA enrichment in proximal TECs followed by oxidative stress and cell death is a key early event in CN. Inhibiting renal ASBT and consequently BA enrichment in TECs prevents CN and systemically decreases BA concentrations. IMPACT AND IMPLICATIONS: Cholemic nephropathy (CN) is a severe complication of cholestasis and an unmet clinical need. We demonstrate that CN is triggered by the renal accumulation of bile acids (BAs) that are considerably increased in the systemic blood. Specifically, the proximal tubular epithelial cells of the kidney take up BAs via the apical sodium-dependent bile acid transporter (ASBT). We developed a therapeutic compound that blocks ASBT in the kidneys, prevents BA overload in tubular epithelial cells, and almost completely abolished all disease hallmarks in a CN mouse model. Renal ASBT inhibition represents a potential therapeutic strategy for patients with CN.

Altering maternal calcium and phosphorus dietary intake induces persistent sex-specific changes in the dentition of the offspring.

BACKGROUND: The maternal diet is essential to offspring development, but the specific effects on tooth morphology are still unknown. The aim of this study was to evaluate the effects of altering maternal calcium (Ca) and phosphorus (P) supplementation during gestation and lactation on offspring dentition. METHODS: Pregnant mice were fed an experimental diet containing a threefold increase in Ca and a threefold decrease in P compared to the standard mouse chow diet at embryonic Day 0.5 (E0.5). Offspring mice were maintained on standard or experimental diets from post-natal Day 0 to weaning, then fed control diets until 6 weeks of age. Six-week-old offspring heads were collected and scanned using micro-computed tomography. Dental morphometrics of offspring maxillary and mandibular first and third molars (n = 5-6 per diet/per sex) were determined. A two-way ANOVA test was employed to verify the existence of any significant differences between groups. The significance level was set at P < .05. RESULTS: A two-way ANOVA revealed a statistically significant interaction between the effects of diet and sex on the upper and lower dentition. Moreover, experimental diet-fed female offspring exhibited smaller molars with shorter mesiodistal width and larger pulp chambers relative to controls, while experimental diet-fed male offspring possessed larger molars with wider mesiodistal width and smaller pulp chambers. CONCLUSION: Our findings reveal that altering the maternal and offspring dietary Ca:P ratio during gestation, lactation and weaning led to significant, sex-specific changes in the offspring dentition. The differences in dentition appeared to be correlated with the sex-specific changes in the craniofacial skeleton.

Increased sinusoidal export of drug glucuronides is a compensative mechanism in liver cirrhosis of mice.

Rationale: Liver cirrhosis is known to affect drug pharmacokinetics, but the functional assessment of the underlying pathophysiological alterations in drug metabolism is difficult. Methods: Cirrhosis in mice was induced by repeated treatment with carbon tetrachloride for 12 months. A cocktail of six drugs was administered, and parent compounds as well as phase I and II metabolites were quantified in blood, bile, and urine in a time-dependent manner. Pharmacokinetics were modeled in relation to the altered expression of metabolizing enzymes. In discrepancy with computational predictions, a strong increase of glucuronides in blood was observed in cirrhotic mice compared to vehicle controls. Results: The deviation between experimental findings and computational simulations observed by analyzing different hypotheses could be explained by increased sinusoidal export and corresponded to increased expression of export carriers (Abcc3 and Abcc4). Formation of phase I metabolites and clearance of the parent compounds were surprisingly robust in cirrhosis, although the phase I enzymes critical for the metabolism of the administered drugs in healthy mice, Cyp1a2 and Cyp2c29, were downregulated in cirrhotic livers. RNA-sequencing revealed the upregulation of numerous other phase I metabolizing enzymes which may compensate for the lost CYP isoenzymes. Comparison of genome-wide data of cirrhotic mouse and human liver tissue revealed similar features of expression changes, including increased sinusoidal export and reduced uptake carriers. Conclusion: Liver cirrhosis leads to increased blood concentrations of glucuronides because of increased export from hepatocytes into the sinusoidal blood. Although individual metabolic pathways are massively altered in cirrhosis, the overall clearance of the parent compounds was relatively robust due to compensatory mechanisms.

Cognitive Functions, Neurotransmitter Alterations, and Hippocampal Microstructural Changes in Mice Caused by Feeding on Western Diet.

Metabolic Dysfunction Associated Steatotic Liver Disease (MASLD) is the most common chronic liver disease in Western countries. It is becoming increasingly evident that peripheral organ-centered inflammatory diseases, including liver diseases, are linked with brain dysfunctions. Therefore, this study aims to unravel the effect of MASLD on brain histology, cognitive functions, and neurotransmitters. For this purpose, mice fed for 48 weeks on standard (SD) or Western diet (WD) were evaluated by behavioral tests, followed by sacrifice and analysis of the liver-brain axis including histopathology, immunohistochemistry, and biochemical analyses. Histological analysis of the liver showed features of Metabolic Dysfunction-Associated Steatohepatitis (MASH) in the WD-fed mice including lipid droplet accumulation, inflammation, and fibrosis. This was accompanied by an elevation of transaminase and alkaline phosphatase activities, increase in inflammatory cytokine and bile acid concentrations, as well as altered amino acid concentrations in the blood. Interestingly, compromised blood capillary morphology coupled with astrogliosis and microgliosis were observed in brain hippocampus of the WD mice, indicating neuroinflammation or a disrupted neurovascular unit. Moreover, attention was impaired in WD-fed mice along with the observations of impaired motor activity and balance, enhanced anxiety, and stereotyped head-twitch response (HTR) behaviors. Analysis of neurotransmitters and modulators including dopamine, serotonin, GABA, glutamate, and acetylcholine showed region-specific dysregulation in the brain of the WD-fed mice. In conclusion, the induction of MASH in mice is accompanied by the alteration of cellular morphology and neurotransmitter expression in the brain, associated with compromised cognitive functions.

Vinpocetine protects against chloroquine-induced cardiotoxicity by mitigating oxidative stress.

Chloroquine (CQ) and hydroxychloroquine (HCQ) are classical antimalarial drugs, and recently have been used for other applications including coronavirus disease 2019 (COVID-19). Although they are considered safe, cardiomyopathy may associate CQ and HCQ applications particularly at overdoses. The goal of the present study was to evaluate the potential protective effect of the nootropic agent vinpocetine against CQ and HCQ adverse effects with a specific focus on the heart. For this purpose, a mouse model of CQ (0.5 up to 2.5 g/kg)/HCQ (1 up to 2 g/kg) toxicity was used, and the effect of vinpocetine was evaluated by survival, biochemical, as well as histopathological analyses. Survival analysis revealed that CQ and HCQ caused dose-dependent lethality, which was prevented by co-treatment with vinpocetine (100 mg/kg, oral or intraperitoneal). To gain deeper understanding, a dose of 1 g/kg CQ-which did not cause death within the first 24 h after administration-was applied with and without vinpocetine administration (100 mg/kg, intraperitoneal). The CQ vehicle group showed marked cardiotoxicity as evidenced by significant alterations of blood biomarkers including troponione-1, creatine phosphokinase (CPK), creatine kinase-myocardial band (CK-MB), ferritin, and potassium levels. This was confirmed at the tissue level by massive alteration of the heart tissue morphology and coincided with massive oxidative stress. Interestingly, co-administration of vinpocetine strongly ameliorated CQ-induced alterations and restored the antioxidant-defense system of the heart. These data suggest that vinpocetine could be used as an adjuvant therapy together with CQ/HCQ applications.

Effect of continuous chelation on the dentinal tubule penetration of a calcium silicate-based root canal sealer: a confocal laser microscopy study.

BACKGROUND: This study aimed to evaluate the effect of various irrigation protocols on the penetration depth of a calcium silicate-based sealer into dentinal tubules using confocal laser scanning microscopy (CLSM). METHODS: Twenty single-rooted mandibular premolars were endodontically prepared and divided into the following two groups according to the irrigation protocol used (n = 10): Group I: NaOCl + EDTA and Group II: continuous chelation (NaOCl/Dual Rinse). Obturation was performed with the warm vertical compaction technique using TotalFill HiFlow bioceramic sealer mixed with a fluorophore dye. Samples were observed using CLSM at 10× to measure the percentage of sealer penetration and its maximum depth into the dentinal tubules. Data were analysed using one-way ANOVA followed by Tukey's post-hoc test. The significance level was set at p < 0.05 within all tests. RESULTS: Comparing the overall results of all sections tested, no statistically significant differences existed between the groups regarding the percentage of sealer penetration (p = 0.612) and maximum depth of penetration (p > 0.05). CONCLUSIONS: With both types of irrigation used, dentinal tubule penetration was higher in the coronal section than in the apical section. Continuous chelation using NaOCl/Dual Rinse HEDP performed better in the coronal segments, while irrigation using NaOCl + EDTA promoted a higher percentage of sealer penetration in the apical segment.

Proficiency of some synthetic alginate derivatives for sequestration of Iodine-131 from radioactive liquid waste.

The current effort in environmental remediation is aimed at removing iodine-131 radionuclide from radioactive liquid waste produced by an Egyptian nuclear power plant using some synthesised alginate derivatives. Two different copolymers, namely sodium alginate poly (acrylic acid) (P1) and sodium alginate poly (acrylic acid-methacrylic acid) (P2), are prepared using gamma radiation. The ability of these polymers to remove 131I radionuclide as sorbents has been investigated. The synthesised polymers exhibit excellent adsorption performance for 131I ions, and the adsorption equilibrium requires only 30 min, which reveals that the sorption process is kinetically faster than most of the other materials reported previously. The removal percents for 131I radionuclide at a pH of 3.0 at room temperature on P1 and P2 are 77.7% and 84.2%, respectively. The sorption capacities of the two polymers demonstrate that P2 > P1, with capacities of 67.9 and 58.5 mg/g, respectively. Four linear kinetic models are investigated: pseudo-first order, pseudo-second order, Elovich, and Weber-Morris models. Regarding their calculated parameters, these models indicate that the adsorption process of I-ions on both P1 and P2 is controlled by chemisorption. Four equilibrium isotherm models (Redlich-Peterson, Langmuir, Freundlich, and Harkin-Jura) are investigated, revealing that the adsorption process is a monolayer and multilayer process on a heterogeneous surface.

In vitro to in vivo acetaminophen hepatotoxicity extrapolation using classical schemes, pharmacodynamic models and a multiscale spatial-temporal liver twin.

In vitro to in vivo extrapolation represents a critical challenge in toxicology. In this paper we explore extrapolation strategies for acetaminophen (APAP) based on mechanistic models, comparing classical (CL) homogeneous compartment pharmacodynamic (PD) models and a spatial-temporal (ST), multiscale digital twin model resolving liver microarchitecture at cellular resolution. The models integrate consensus detoxification reactions in each individual hepatocyte. We study the consequences of the two model types on the extrapolation and show in which cases these models perform better than the classical extrapolation strategy that is based either on the maximal drug concentration (Cmax) or the area under the pharmacokinetic curve (AUC) of the drug blood concentration. We find that an CL-model based on a well-mixed blood compartment is sufficient to correctly predict the in vivo toxicity from in vitro data. However, the ST-model that integrates more experimental information requires a change of at least one parameter to obtain the same prediction, indicating that spatial compartmentalization may indeed be an important factor.

Comparative histological analysis of the skin for forensic investigation of some animal species.

Macroscopical and histological analysis of the skin is fundamental in both human and veterinary forensic investigations. However, databases of differential skin histology of various animal species are rare. The aim of the present study is to identify species-specific differential histological features of the skin that could be used in forensic investigations including animal identification. For this purpose, skin specimens were collected from the neck region of various farm animals including buffalo, cow, camel, sheep, goat, dog, and donkey, and were processed for histological analysis. Our comparative analysis revealed specific histological features in the skin that could differentiate between the studied animal species. The epidermis layer of the skin was very thick in buffalo, intermediate in cow, sheep, goat, dog, and donkey, but very thin in camel. The papillomatous epidermis was very frequent in buffalo, but very rare in cow. In the dermis layer of the skin, four structures were located which showed differential features between the studied animal species: the papillary layer, which was thick in buffalo, camel, sheep, dog, and donkey but thin in cow and goat. The sweat glands, which were few in buffalo, cow, goat, and dog, but numerous and deeply located in the dermis of sheep; they were individually located in all studied animals except in camel and donkey they were arranged in clusters. The hair follicles were characteristic for the skin of sheep as they were present at two different levels in the dermis with simple and compound hair follicles. The sebaceous glands were large and multi-lobular in buffalo, but small and uni-lobular in cow and camel. The hypodermis layer of the skin was very thick in sheep and goat in contrast to all other analyzed animals. In conclusion, the present study provides comprehensive information on the differential histological features of the skin of seven different domestic animal species that could be used as a key in forensic investigations.

Inhibition of cytochrome P450 enhances the nephro- and hepatotoxicity of ochratoxin A.

The mycotoxin ochratoxin A (OTA) is a contaminant in food that causes nephrotoxicity and to a minor degree hepatotoxicity. Recently, we observed that OTA induces liver damage preferentially to the cytochrome P450 (CYP)-expressing pericentral lobular zone, similar to hepatotoxic substances known to be metabolically toxified by CYP, such as acetaminophen or carbon tetrachloride. To investigate whether CYP influences OTA toxicity, we used a single dose of OTA (7.5 mg/kg; intravenous) with and without pre-treatment with the pan CYP-inhibitor 1-aminobenzotriazole (ABT) 2 h before OTA administration. Blood, urine, as well as liver and kidney tissue samples were collected 24 h after OTA administration for biochemical and histopathological analyses. Inhibition of CYPs by ABT strongly increased the nephro- and hepatotoxicity of OTA. The urinary kidney damage biomarkers kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL) were increased > 126-fold and > 20-fold, respectively, in mice treated with ABT and OTA compared to those receiving OTA alone. The blood biomarkers of liver damage, alanine transaminase (ALT) and aspartate transaminase (AST) both increased > 21- and 30-fold, respectively, when OTA was administered to ABT pre-treated mice compared to the effect of OTA alone. Histological analysis of the liver revealed a pericentral lobular damage induced by OTA despite CYP-inhibition by ABT. Administration of ABT alone caused no hepato- or nephrotoxicity. Overall, the results presented are compatible with a scenario where CYPs mediate the detoxification of OTA, yet the mechanisms responsible for the pericental liver damage pattern still remain to be elucidated.

Spatial modeling reveals nuclear phosphorylation and subcellular shuttling of YAP upon drug-induced liver injury.

The Hippo signaling pathway controls cell proliferation and tissue regeneration via its transcriptional effectors yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ). The canonical pathway topology is characterized by sequential phosphorylation of kinases in the cytoplasm that defines the subcellular localization of YAP and TAZ. However, the molecular mechanisms controlling the nuclear/cytoplasmic shuttling dynamics of both factors under physiological and tissue-damaging conditions are poorly understood. By implementing experimental in vitro data, partial differential equation modeling, as well as automated image analysis, we demonstrate that nuclear phosphorylation contributes to differences between YAP and TAZ localization in the nucleus and cytoplasm. Treatment of hepatocyte-derived cells with hepatotoxic acetaminophen (APAP) induces a biphasic protein phosphorylation eventually leading to nuclear protein enrichment of YAP but not TAZ. APAP-dependent regulation of nuclear/cytoplasmic YAP shuttling is not an unspecific cellular response but relies on the sequential induction of reactive oxygen species (ROS), RAC-alpha serine/threonine-protein kinase (AKT, synonym: protein kinase B), as well as elevated nuclear interaction between YAP and AKT. Mouse experiments confirm this sequence of events illustrated by the expression of ROS-, AKT-, and YAP-specific gene signatures upon APAP administration. In summary, our data illustrate the importance of nuclear processes in the regulation of Hippo pathway activity. YAP and TAZ exhibit different shuttling dynamics, which explains distinct cellular responses of both factors under physiological and tissue-damaging conditions.

Colchicine overdose impairs the capacity of Kupffer cells to clear foreign particles and endotoxins.

Colchicine is an anti-inflammatory drug with a narrow therapeutic index. Its binding to tubulin prevents microtubule polymerization; however, little is known about how depolymerization of microtubules interferes with the phagocytosis function of Kupffer cells (KC). Here, we applied functional intravital imaging techniques to investigate the influence of microtubule disruption by colchicine on KC morphology, as well as its capacity to clear foreign particles and bacterial lipopolysaccharide (LPS) in anesthetized mice. Intravital imaging of KC in healthy mice showed the typical elongated morphology, localization at the luminal side of the sinusoidal endothelial cells, and moving cell protrusions. In contrast, at colchicine doses of 1 mg/kg and higher (intraperitoneal), KC appeared roundish with strongly reduced protrusions and motility. To study the functional consequences of these alterations, we analyzed the capacity of KC to phagocytose fluorescent nanospheres (100 nm-size) and LPS. After tail vein injection, the nanospheres formed aggregates of up to ~ 5 µm moving along the sinusoidal bloodstream. In controls, the nanosphere aggregates were rapidly captured by the Kupffer cell protrusions, followed by an internalization process that lasted up to 10 min. Similar capture events and internalization processes were observed after the administration of fluorescently labeled LPS. In contrast, capture and internalization of both nanospheres and LPS by KC were strongly reduced in colchicine-treated mice. Reduced phagocytosis of LPS was accompanied by aggravated production of inflammatory cytokines. Since 0.4 mg/kg colchicine in mice has been reported to be bio-equivalent to human therapeutic doses, the here-observed adverse effects on KC occurred at doses only slightly above those used clinically, and may be critical for patients with endotoxemia due to a leaky gut-blood barrier.

Hypoalbuminemia affects the spatio-temporal tissue distribution of ochratoxin A in liver and kidneys: consequences for organ toxicity.

Hypoalbuminemia (HA) is frequently observed in systemic inflammatory diseases and in liver disease. However, the influence of HA on the pharmacokinetics and toxicity of compounds with high plasma albumin binding remained insufficiently studied. The 'lack-of-delivery-concept' postulates that HA leads to less carrier mediated uptake of albumin bound substances into hepatocytes and to less glomerular filtration; in contrast, the 'concept-of-higher-free-fraction' argues that increased concentrations of non-albumin bound compounds facilitate hepatocellular uptake and enhance glomerular filtration. To address this question, we performed intravital imaging on livers and kidneys of anesthetized mice to quantify the spatio-temporal tissue distribution of the mycotoxin ochratoxin A (OTA) based on its auto-fluorescence in albumin knockout and wild-type mice. HA strongly enhanced the uptake of OTA from the sinusoidal blood into hepatocytes, followed by faster secretion into bile canaliculi. These toxicokinetic changes were associated with increased hepatotoxicity in heterozygous albumin knockout mice for which serum albumin was reduced to a similar extent as in patients with severe hypoalbuminemia. HA also led to a shorter half-life of OTA in renal capillaries, increased glomerular filtration, and to enhanced uptake of OTA into tubular epithelial cells. In conclusion, the results favor the 'concept-of-higher-free-fraction' in HA; accordingly, HA causes an increased tissue uptake of compounds with high albumin binding and increased organ toxicity. It should be studied if this concept can be generalized to all compounds with high plasma albumin binding that are substrates of hepatocyte and renal tubular epithelial cell carriers.

Influence of Optimum Torque Reverse Motion on Dentinal Cracks after Root Canal Preparation with Two Nickel-Titanium Rotary Systems.

Objectives: This study was aimed to evaluate the cause-effect relationship between canal preparation with ProTaper Next (PTN) and ProTaper Gold (PTG) using optimum torque reverse (OTR) motion or continuous rotation and dentinal crack formation. Materials and Methods: Fifty distobuccal roots of human maxillary first molars were divided into five groups; Group I: PTG Full rotation, Group II: PTG in OTR, Group III: PTN Full rotation, Group IV: PTN in OTR, Group V: unprepared (control group). After mechanical preparation, the distobuccal roots were sectioned horizontally at 3, 6, and 9 mm from the apex. Images were captured using a stereomicroscope at 25X to determine the presence or absence of dentinal cracks. Friedman test was used to compare between root sections followed by Wilcoxon signed-rank test for pairwise comparison. Kruskal-Wallis test was used to compare between tested rotary systems followed by pairwise comparison with Dunn Bonferroni correction (α = 0.05). Results: Crack development was significantly higher in PTG using OTR motion 36.7% followed by PTN using OTR 33.3%, while the control group showed no cracks. PTG and PTN with full rotation showed crack development with 23.3% and 13.3%, respectively. Conclusions: The type of motion kinematics used during mechanical preparation have an impact on dentinal crack formation. Nickel-titanium instruments with larger taper tend to induce more cracks.