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.

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.

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.

Loss of bile salt export pump aggravates lipopolysaccharide-induced liver injury in mice due to impaired hepatic endotoxin clearance.

BACKGROUND AND AIMS: Lipopolysaccharide (LPS) clearance is delayed in cholestatic liver diseases. While compromised clearance by Kupffer cells (KCs) is involved, the role of LPS uptake into hepatocytes and canalicular excretion remains unclear. APPROACH AND RESULTS: Wild-type (WT) and bile salt export pump (Bsep) knockout (KO) mice were challenged i.p. with LPS. Liver injury was assessed by serum biochemistry, histology, molecular inflammation markers, and immune cell infiltration. LPS concentrations were determined in liver tissue and bile. Subcellular kinetics of fluorescently labeled LPS was visualized by intravital two-photon microscopy, and the findings in Bsep KO mice were compared to common bile duct-ligated (BDL) and multidrug resistance protein 2 (Mdr2) KO mice. Changes in gut microbiota composition were evaluated by 16S ribosomal RNA gene amplicon sequencing analysis. Bsep KO mice developed more pronounced LPS-induced liver injury and inflammatory signaling, with subsequently enhanced production of proinflammatory cytokines and aggravated hepatic immune cell infiltration. After LPS administration, its concentrations were higher in liver but lower in bile of Bsep KO compared to WT mice. Intravital imaging of LPS showed a delayed clearance from sinusoidal blood with a basolateral uptake block into hepatocytes and reduced canalicular secretion. Moreover, LPS uptake into KCs was reduced. Similar findings with respect to hepatic LPS clearance were obtained in BDL and Mdr2 KO mice. Pretreatment with the microtubule inhibitor colchicine inhibited biliary excretion of LPS in WT mice, indicating that LPS clearance is microtubule-dependent. Microbiota analysis showed no change of the gut microbiome between WT and Bsep KO mice at baseline but major changes upon LPS challenge in WT mice. CONCLUSIONS: Absence of Bsep and cholestasis in general impair LPS clearance by a basolateral uptake block into hepatocytes and consequently less secretion into canaliculi. Impaired LPS removal aggravates hepatic inflammation in cholestasis.

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.

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.

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.

Interruption of bile acid uptake by hepatocytes after acetaminophen overdose ameliorates hepatotoxicity.

BACKGROUND & AIMS: Acetaminophen (APAP) overdose remains a frequent cause of acute liver failure, which is generally accompanied by increased levels of serum bile acids (BAs). However, the pathophysiological role of BAs remains elusive. Herein, we investigated the role of BAs in APAP-induced hepatotoxicity. METHODS: We performed intravital imaging to investigate BA transport in mice, quantified endogenous BA concentrations in the serum of mice and patients with APAP overdose, analyzed liver tissue and bile by mass spectrometry and MALDI-mass spectrometry imaging, assessed the integrity of the blood-bile barrier and the role of oxidative stress by immunostaining of tight junction proteins and intravital imaging of fluorescent markers, identified the intracellular cytotoxic concentrations of BAs, and performed interventions to block BA uptake from blood into hepatocytes. RESULTS: Prior to the onset of cell death, APAP overdose causes massive oxidative stress in the pericentral lobular zone, which coincided with a breach of the blood-bile barrier. Consequently, BAs leak from the bile canaliculi into the sinusoidal blood, which is then followed by their uptake into hepatocytes via the basolateral membrane, their secretion into canaliculi and repeated cycling. This, what we termed 'futile cycling' of BAs, led to increased intracellular BA concentrations that were high enough to cause hepatocyte death. Importantly, however, the interruption of BA re-uptake by pharmacological NTCP blockage using Myrcludex B and Oatp knockout strongly reduced APAP-induced hepatotoxicity. CONCLUSIONS: APAP overdose induces a breach of the blood-bile barrier which leads to futile BA cycling that causes hepatocyte death. Prevention of BA cycling may represent a therapeutic option after APAP intoxication. LAY SUMMARY: Only one drug, N-acetylcysteine, is approved for the treatment of acetaminophen overdose and it is only effective when given within ∼8 hours after ingestion. We identified a mechanism by which acetaminophen overdose causes an increase in bile acid concentrations (to above toxic thresholds) in hepatocytes. Blocking this mechanism prevented acetaminophen-induced hepatotoxicity in mice and evidence from patients suggests that this therapy may be effective for longer periods after ingestion compared to N-acetylcysteine.

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.

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.

Regenerative endodontic procedures for the treatment of necrotic mature teeth: A preliminary randomized clinical trial.

AIM: This preliminary randomized, prospective, controlled trial aimed to compare the clinical and radiographic outcomes of two regenerative endodontic procedures (REPs), revitalization and a platelet-rich fibrin (PRF)-based technique, in the treatment of mature permanent teeth with necrotic pulps. METHODOLOGY: The trial has been reported according to the Preferred Reporting Items for Randomized Trials in Endodontics 2020 guidelines. The study protocol was registered at the clinical trial registry (ClinicalTrials.gov) with identifier number NCT04158232. Twenty patients with mature necrotic anterior teeth with large periapical lesions were randomly allocated into two groups (n = 10): group I, treated with revitalization with the blood clot (BC) technique and group II, treated with a PRF-based technique. The follow-up was for 12 months. Periradicular healing was assessed using standardized radiographs taken at baseline, and at 6 and 12 months after treatment. An electric pulp tester was used to assess whether pulp sensibility had been regained during the follow-up period. Statistical analysis was conducted using Mann-Whitney test and Wilcoxon test for non-parametric data. For parametric data, repeated measures analysis of variance was used. The significance level was set at p ≤ .05. RESULTS: There was a significant increase in periradicular healing in both groups at 6 and 12 months, compared to that at baseline, with no significant difference between the studied groups after 12 months (p = .143). There was a significant difference between the tooth sensibility readings at baseline, 6-month and 12-month follow-up timepoints (p < .001). CONCLUSIONS: The findings of this preliminary trial indicate the potential for using REPs, such as revitalization or PRF-based techniques, as treatment options for mature teeth with necrotic pulps. A higher level of evidence obtained through adequately powered clinical trials and longer follow-up periods are required to conclusively validate the different outcomes of REPs.

Subcellular spatio-temporal intravital kinetics of aflatoxin B1 and ochratoxin A in liver and kidney.

Local accumulation of xenobiotics in human and animal tissues may cause adverse effects. Large differences in their concentrations may exist between individual cell types, often due to the expression of specific uptake and export carriers. Here we established a two-photon microscopy-based technique for spatio-temporal detection of the distribution of mycotoxins in intact kidneys and livers of anesthetized mice with subcellular resolution. The mycotoxins ochratoxin A (OTA, 10 mg/kg b.w.) and aflatoxin B1 (AFB1, 1.5 mg/kg b.w.), which both show blue auto-fluorescence, were analyzed after intravenous bolus injections. Within seconds after administration, OTA was filtered by glomeruli, and enriched in distal tubular epithelial cells (dTEC). A striking feature of AFB1 toxicokinetics was its very rapid uptake from sinusoidal blood into hepatocytes (t1/2 ~ 4 min) and excretion into bile canaliculi. Interestingly, AFB1 was enriched in the nuclei of hepatocytes with zonal differences in clearance. In the cytoplasm of pericentral hepatocytes, the half-life (t1/2~ 63 min) was much longer compared to periportal hepatocytes of the same lobules (t1/2 ~ 9 min). In addition, nuclear AFB1 from periportal hepatocytes cleared faster compared to the pericentral region. These local differences in AFB1 clearance may be due to the pericentral expression of cytochrome P450 enzymes that activate AFB1 to protein- and DNA-binding metabolites. In conclusion, the present study shows that large spatio-temporal concentration differences exist within the same tissues and its analysis may provide valuable additional information to conventional toxicokinetic studies.

Bile Microinfarcts in Cholestasis Are Initiated by Rupture of the Apical Hepatocyte Membrane and Cause Shunting of Bile to Sinusoidal Blood.

Bile duct ligation (BDL) is an experimental procedure that mimics obstructive cholestatic disease. One of the early consequences of BDL in rodents is the appearance of so-called bile infarcts that correspond to Charcot-Gombault necrosis in human cholestasis. The mechanisms causing bile infarcts and their pathophysiological relevance are unclear. Therefore, intravital two photon-based imaging of BDL mice was performed with fluorescent bile salts (BS) and non-BS organic anion analogues. Key findings were followed up by matrix-assisted laser desorption ionization imaging, clinical chemistry, immunostaining, and gene expression analyses. In the acute phase, 1-3 days after BDL, BS concentrations in bile increased and single-cell bile microinfarcts occurred in dispersed hepatocytes throughout the liver caused by the rupture of the apical hepatocyte membrane. This rupture occurred after loss of mitochondrial membrane potential, followed by entry of bile, cell death, and a "domino effect" of further death events of neighboring hepatocytes. Bile infarcts provided a trans-epithelial shunt between bile canaliculi and sinusoids by which bile constituents leaked into blood. In the chronic phase, ≥21 days after BDL, uptake of BS tracers at the sinusoidal hepatocyte membrane was reduced. This contributes to elevated concentrations of BS in blood and decreased concentrations in the biliary tract. Conclusion: Bile microinfarcts occur in the acute phase after BDL in a limited number of dispersed hepatocytes followed by larger infarcts involving neighboring hepatocytes, and they allow leakage of bile from the BS-overloaded biliary tract into blood, thereby protecting the liver from BS toxicity; in the chronic phase after BDL, reduced sinusoidal BS uptake is a dominant protective factor, and the kidney contributes to the elimination of BS until cholemic nephropathy sets in.

Spatio-temporal visualization of the distribution of acetaminophen as well as its metabolites and adducts in mouse livers by MALDI MSI.

Acetaminophen (APAP) is one of the most intensively studied compounds that causes hepatotoxicity in the pericentral region of the liver lobules. However, spatio-temporal information on the distribution of APAP, its metabolites and GSH adducts in the liver tissue is not yet available. Here, we addressed the question, whether APAP-GSH adducts and GSH depletion show a zonated pattern and whether the distribution of APAP and its glucuronide as well as sulfate conjugates in liver lobules are zonated. For this purpose, a matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) technique was established, where the MSI images were superimposed onto CYP2E1 immunostained tissue. A time-dependent analysis (5, 15, 30, 60, 120, 240, 480 min) after intraperitoneal administration of 300 mg/kg APAP and a dose-dependent analysis (56 up to 500 mg APAP/kg) at 30 min were performed. The results demonstrate that the MALDI MSI technique allows the assignment of compounds and their metabolites to specific lobular zones. APAP-GSH adducts and GSH depletion occurred predominantly in the CYP2E1-positive zone of the liver, although GSH also decreased in the periportal region. In contrast, the parent compound, APAP sulfate and APAP glucuronide did not show a zonated pattern and tissue concentrations showed a similar time course as the corresponding analyses were performed with blood from the portal and liver veins. In conclusion, the present study is in agreement with the concept that pericentral CYPs form NAPQI that in the same cell binds to and depletes GSH but a lower level of GSH adducts is also observed in the periportal region. The results also provide further evidence of the recently published concept of 'aggravated loss of clearance capacity' according to which also liver tissue that survives intoxication may transiently show decreased metabolic capacity.

Adverse outcome pathways: opportunities, limitations and open questions.

Adverse outcome pathways (AOPs) are a recent toxicological construct that connects, in a formalized, transparent and quality-controlled way, mechanistic information to apical endpoints for regulatory purposes. AOP links a molecular initiating event (MIE) to the adverse outcome (AO) via key events (KE), in a way specified by key event relationships (KER). Although this approach to formalize mechanistic toxicological information only started in 2010, over 200 AOPs have already been established. At this stage, new requirements arise, such as the need for harmonization and re-assessment, for continuous updating, as well as for alerting about pitfalls, misuses and limits of applicability. In this review, the history of the AOP concept and its most prominent strengths are discussed, including the advantages of a formalized approach, the systematic collection of weight of evidence, the linkage of mechanisms to apical end points, the examination of the plausibility of epidemiological data, the identification of critical knowledge gaps and the design of mechanistic test methods. To prepare the ground for a broadened and appropriate use of AOPs, some widespread misconceptions are explained. Moreover, potential weaknesses and shortcomings of the current AOP rule set are addressed (1) to facilitate the discussion on its further evolution and (2) to better define appropriate vs. less suitable application areas. Exemplary toxicological studies are presented to discuss the linearity assumptions of AOP, the management of event modifiers and compensatory mechanisms, and whether a separation of toxicodynamics from toxicokinetics including metabolism is possible in the framework of pathway plasticity. Suggestions on how to compromise between different needs of AOP stakeholders have been added. A clear definition of open questions and limitations is provided to encourage further progress in the field.