Anwendung der hochaufgelösten bildgebenden MALDI-MS zur Aufklärung der räumlichen Verteilung des Acetaminophens und seiner Biotransformationsprodukte sowie von Taurocholaten in der Leber von Mäusen in Folge einer Acetaminophen Intoxikation

Überdosierungen an APAP führen zu hepatotoxischen Auswirkungen in der Leber von Säugetieren. Die Hepatotoxizität ist u.a. eine Folge der metabolischen Aktivierung des Acetaminophens durch CYP. Dabei treten pericentrale Schadensmuster in den Leberläppchen auf. Es fehlten bisher Informationen über die räumlich-zeitliche Verteilung vom APAP und seinen Biotransformationsprodukten in der Leber bis zur Veröffentlichung von Teilen dieser vorliegenden Arbeit. Um die Fragestellung zu beantworten, wurde eine Methode basierend auf der leistungsstarken HR-MALDI-MSI Technik entwickelt. Dadurch konnte die Verteilung vom APAP, seinen Hauptmetaboliten APAP-GLC und APAP-SUL und seinem Addukt APAP-GSH in der Leber von C57BL/6 Mäusen, denen APAP intraperitoneal verabreicht wurde und deren Leber sowohl zeit- als auch dosisabhängig beprobt wurden, detektiert werden. Zuvor wurden intensive Anstrengungen unternommen, um das toxische Intermediat NAPQI im Lebergewebe durch MALDI-MSI zu erfassen. Ziel war es, Informationen über seinen Bildungs- und Wirkungsort im Lebergewebe zu gewinnen. Hervorzuheben ist dabei ein Versuchsansatz bei dem H2S zur Derivatisierung von NAPQI eingesetzt wurde. Diese innovative Methode könnte zudem das Portfolio an Derivatisierungsmöglichkeiten für MALDI-MSI Experimente erweitern. Allerdings konnte das NAPQI weder direkt noch über seine artifiziellen Derivate im Lebergewebe detektiert werden, was vermutlich auf einen Mangel an nicht-abreagiertem NAPQI im Lebergewebe zurückzuführen ist. Die durch Gewebehistologie unterstützte Superpositionierung von Ionendichtebildern unter Einbeziehung der Eigenschaften verschiedener Imaging Software ermöglichte es, präferentielle Verteilungen der Analyten bezüglich der metabolischen Zonen zu erfassen. Während für APAP, APAP-SUL und APAP-GLC keine zonale Verteilung festgestellt werden konnte, konnte eine präferentielle Bildung des APAP-GSHs sowie ein schwerpunktmäßiger Abbau des GSHs in der pericentralen Zone verortet werden. Der Abbau an GSH war aber nicht ausschließlich auf die pericentralen Zonen beschränkt, sondern konnte auch für die periportalen Zonen demonstriert werden. 120 min nach der APAP Injektion konnte ein Influx an APAP-GSH und an Taurocholaten von den Gallengefäßen in die pericentrale Zone und von dort ins Blut beobachtet werden. Dieser massive Eintrag von Gallensäuren beweist, dass sich ein „sekundäres“ Toxizitätsevent ereignet, welches vormals durch die Aktivierung des APAPs in der Leber gestartet wurde.Acetaminophen induces hepatotoxicity in the liver of mammal organisms, after the uptake of an overdose. It is well reported, that CYP mediated activation of APAP is thereby causing a pericentral damage pattern in the liver lobules. However, no direct spatio-temporal information about the distribution pattern of APAP and its biotransformation products in the liver were available until the publication of parts of this work. Herein, the development of an analytical method based on of the sophisticated HR-MALDI-MSI technique is reported. The aim was to detect the distribution of APAP, its main metabolites APAP-GLC and APAP-SUL and adduct APAPGSH, known to be formed during detoxification of the toxic intermediate metabolite NAPQI by GSH, in the liver of C57BL/6 mice, which were treated intraperitoneally with APAP and whose livers were collected in a time-and dose-dependent manner. Previously, intensive efforts were put in the analytical investigation of the toxic intermediate NAPQI from liver tissue by MALDIMSI since its detection would have revealed valuable information about its sites of formation and impact. For this purpose, an innovative derivatization approach with H2S gas was applied, which in general, could also enrich the portfolio of derivatization methodologies used for MALDIMSI experiments. However, the detection of NAPQI and its on-tissue derivatization attempts failed, presumably due to the lack of unreacted NAPQI in the liver. Histology-directed superposition of ion density images in consideration of special features of different imaging software made it possible to determine the differences in the distribution pattern of the analytes. While no specific zonation could be detected for APAP, APAP-SUL and APAP-GLC, a preferential formation of APAP-GSH and depletion of GSH in the pericentral zones of the lobule were revealed. However, the depletion of GSH was not restricted to pericentral zones but was also demonstrated for the periportal areas. 120 min after APAP injection a series of drastic events happened: APAP-GSH as well as taurocholates entered from the bile vessels into the pericentral zone and from there leaked into the systemic blood circulation. The massive leakage of bile acids into the hepatocytes is providing the evidence of a “secondary” toxicity event initiated by the activation of APAP in the liver

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

Background & aimsAcetaminophen (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.MethodsWe 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.ResultsPrior 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.ConclusionsAPAP 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 summaryOnly 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

Occurrence and Distribution of Phytochemicals in the Leaves of 17 In vitro Cultured Hypericum spp. Adapted to Outdoor Conditions

A plethora of plants belonging to the genus Hypericum have been investigated so far owing to the biological efficacies of pharmacologically important secondary metabolites produced by several Hypericum species. However, there is currently a dearth of information about the localization (accumulation) of these compounds in the plants in situ. In particular, the biosynthetic and ecological consequence of acclimatization of in vitro cultured Hypericum spp. to outdoor conditions is not fully known. Herein, we report an application of matrix-assisted laser desorption/ionization high-resolution mass spectrometry (MALDI-HRMS) to reveal the distribution of major naphthodianthrones hypericin, pseudohypericin, protohypericin and their proposed precursor emodin as well as emodin anthrone, along with the phloroglucinol derivative hyperforin, the flavonoids quercetin, quercitrin, rutin and hyperoside (and/or isoquercitrin), and chlorogenic acid in Hypericum leaves. Plants encompassing seventeen Hypericum species classified into eleven sections, which were first cultured in vitro and later acclimatized to outdoor conditions, were studied. We focused both on the secretory (dark and translucent glands, other types of glands, and glandular-like structures) as well as the non-secretory leaf tissues. We comparatively analyzed and interpreted the occurrence and accumulation of our target compounds in different leaf tissues of the seventeen species to get an intra-sectional as well as inter-sectional perspective. The naphthodianthrones, along with emodin, were present in all species containing the dark glands. In selected species, hypericin and pseudohypericin accumulated not only in the dark glands, but also in translucent glands and non-secretory leaf tissues. Although hyperforin was localized mainly in translucent glands, it was present sporadically in the dark glands in selected species. The flavonoids quercetin, quercitrin and hyperoside (and/or isoquercitrin) were distributed throughout the leaves. Rutin was present only within sections Hypericum, Adenosepalum, Ascyreia and Psorophytum. Our study provides insights into the prospects and challenges of using in vitro cultured Hypericum plants, further adapted to field conditions, for commercial purposes

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

International audienceIn 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 homogeneous compartment pharmaco-dynamic (PD) models and a 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 pharmaco-kinetic 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

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

International audienceBile 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 f luorescent bile salts (BS) and non-BS organic anion analogues. Key f indings 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. (Hepatology2019;69:666-683