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.

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.

MALDI-HRMS imaging and HPLC-HRESI-MSn characterisation of kaurane diterpenes in the fruits of Xylopia aethiopica (Dunal) A. Rich (Annonaceae).

INTRODUCTION: Kaurane diterpenes, notably xylopic acid, have demonstrated important biological activities including analgesia, anti-oxidant, antimicrobial and cytotoxicity. The fruits of Xylopia aethiopica have been reported to be a rich source of kaurane diterpenes. OBJECTIVE: An analytical approach for detailed imaging and characterisation of selected kaurane diterpenes was developed using matrix-assisted laser desorption/ionisation high-resolution mass spectrometry (MALDI-HRMS) imaging techniques and high-performance liquid chromatography-high resolution electrospray ionisation-tandem mass spectrometry (HPLC-HRESI-MSn ) studies, respectively. METHODS: The images of the compounds were constructed based on selected ions from their HRESI-MS spectra. The matrix employed comprised a solution of α-cyano-4-hydroxycinnamic acid (HCCA) in acetonitrile-water with trifluoroacetic acid (TFA). HPLC-HRESI-MSn measurements were conducted on an LTQ-Orbitrap spectrometer equipped with a heated electrospray ionisation (HESI)-II source. RESULTS: The analytical strategy adopted showed the spatial distribution of the compounds in the fruits of X. aethiopica based on the dominant ions at m/z 301.2163 [M + H - HOCOCH3 ]+ and m/z 399.1932 [M + K]+ for xylopic acid, m/z 317.2111 [M + H]+ and m/z 355.1670 [M + K]+ for 15-oxo-ent-kaur-16-en-19-oic acid and m/z 303.2319 [M + H]+ for ent-kaur-16-en-19-oic acid. The fragmentation patterns of the compounds were proposed based on the HRESI-MSn measurements. CONCLUSIONS: The study revealed the spatial variability, differential behaviours and specificity of the selected kaurane diterpenes in the fruit, seed and pericarp. The compounds under study were predominantly restricted to the pericarp of the fruit with trace amounts in the seed.

Chemometric evaluation of hypericin and related phytochemicals in 17 in vitro cultured Hypericum species, hairy root cultures and hairy root-derived transgenic plants.

OBJECTIVES: The objective of this study was to ascertain the presence and correlations among eight important secondary metabolites viz. hypericin, pseudohypericin, emodin, hyperforin, rutin, hyperoside, quercetin and quercitrin in different organs of 17 in vitro cultured Hypericum species, along with H. tomentosum and H. tetrapterum hairy root cultures, and hairy root-derived transgenic plants of H. tomentosum. METHODS: Samples were extracted and analysed by LC-MS. The LC-MS data were subjected to chemometric evaluations for metabolite profiling and correlating the phytochemical compositions in different samples. KEY FINDINGS: Hypericin, pseudohypericin and their proposed precursor emodin were detected in various levels in the leaves of eight Hypericum species. The highest content of hypericins and emodin was found in H. tetrapterum, which contains the studied secondary metabolites in all plant organs. A significant positive correlation between hypericins and emodin was observed both by principal component analysis (PCA) and multidimensional scaling (MDS), indicating the role of emodin as a possible precursor in the biosynthetic pathway of hypericins. Flavonoids were found in all tested plant organs except roots of H. pulchrum. The hairy roots lacked hypericin, pseudohypericin, emodin, hyperforin and rutin. However, the hairy root-derived transgenic plants showed a significant increase in flavonoids. CONCLUSIONS: This study broadens knowledge about the phytochemical composition of selected in vitro cultured Hypericum species, compared to that of hairy root cultures and hairy root-derived transgenic plants.

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.

Localization and Organization of Scopolamine Biosynthesis in Duboisia myoporoides R. Br.

Tropane alkaloids (TAs), especially hyoscyamine and scopolamine, are important precursors for anticholinergic and antispasmodic drugs. Hyoscyamine and scopolamine are currently obtained at commercial scale from hybrid crosses of Duboisia myoporoides × Duboisia leichhardtii plants. In this study, we present a global investigation of the localization and organization of TA biosynthesis in a Duboisia myoporoides R. Br. wild-type line. The tissue-specific spatial distribution of TAs within D. myoporoides is presented, including quantification of the TAs littorine, 6-hydroxy hyoscyamine, hyoscyamine, scopolamine and, additionally, hyoscyamine aldehyde as well as scopolamine glucoside. Scopolamine (14.77 ± 5.03 mg g-1), and to a lesser extent hyoscyamine (3.01 ± 1.54 mg g-1) as well as 6-hydroxy hyoscyamine (4.35 ± 1.18 mg g-1), are accumulated in leaves during plant development, with the highest concentration of total TAs detected in 6-month-old plants. Littorine, an early precursor in TA biosynthesis, was present only in the roots (0.46 ± 0.07 mg g-1). During development, the spatial distribution of all investigated alkaloids changed due to secondary growth in the roots. Transcripts of pmt, tr-I and cyp80f1 genes, involved in early stages of TA biosynthesis, were found to be most abundant in the roots. In contrast, the transcript encoding hyoscyamine 6ß-hydroxylase (h6h) was highest in the leaves of 3-month-old plants. This investigation presents the spatial distribution of biochemical components as well as gene expression profiles of genetic factors known to participate in TA biosynthesis in D. myoporoides. The results of this investigation may aid in future breeding or genetic enhancement strategies aimed at increasing the yields of TAs in these medicinally valuable plant species.

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.

Hexacyclopeptides secreted by an endophytic fungus Fusarium solani N06 act as crosstalk molecules in Narcissus tazetta.

The basis of chemical crosstalk in plants and associated endophytes lies in certain so-called communication molecules that are responsible for plant-microbe and microbe-microbe interactions. Consequently, elucidating the factors that affect the nature, distribution, and amount of these molecules and how they impact the interaction among endophytes and associated organisms is essential to understand the true potential of endophytes. In the present study, we report the discovery of nine hexacyclopeptides from an endophytic fungus, Fusarium solani, isolated from the bulb of Narcissus tazetta, and their selective accumulation by an endophytic bacterium, Achromobacter xylosoxidans isolated from the same tissue. We used matrix-assisted laser desorption ionization imaging high-resolution mass spectrometry (MALDI-imaging-HRMS) to firstly identify and visualize the spatial distribution of the hexacyclopeptides produced by endophytic F. solani. After culture condition optimization, their sequence was identified to be cyclo((Hyp or Dhp)-Xle-Xle-(Ala or Val)-Thr-Xle) (Dhp: dehydroproline) by the characteristic a, b, or y ions using liquid chromatography tandem mass spectrometry (LC-HRMS(n)). These hexacyclopeptides were confirmed to be fungal biosynthetic products by deuterium labeling experiments. Finally, in order to understand the plausible ecological relevance of one or more of the discovered hexacyclopeptides within the contexts of microbial "neighbor communication," we devised a dual-culture setup to visualize using MALDI-imaging-HRMS how the hexacyclopeptides released by the endophytic fungus are accumulated by another endophytic bacterium, A. xylosoxidans, isolated from the same bulb tissue. This work exemplifies the relevance of cyclopeptides in endophyte-endophyte interspecies neighbor communication occurring in nature. Such communication strategies are evolved by coexisting endophytes to survive and function in their distinct ecological niches.

Spatial chemo-profiling of hypericin and related phytochemicals in Hypericum species using MALDI-HRMS imaging.

Advanced analytical imaging techniques, including matrix-assisted laser desorption/ionization high-resolution mass spectrometry (MALDI-HRMS) imaging, can be used to visualize the distribution, localization, and dynamics of target compounds and their precursors with limited sample preparation. Herein we report an application of MALDI-HRMS imaging to map, in high spatial resolution, the accumulation of the medicinally important naphthodianthrone hypericin, its structural analogues and proposed precursors, and other crucial phytochemical constituents in the leaves of two hypericin-containing species, Hypericum perforatum and Hypericum olympicum. We also investigated Hypericum patulum, which does not contain hypericin or its protoforms. We focused on both the secretory (dark glands, translucent glands, secretory canals, laminar glands, and ventral glands) and the surrounding non-secretory tissues to clarify the site of biosynthesis and localization of hypericin, its possible precursors, and patterns of localization of other related compounds concomitant to the presence or absence of hypericin. Hypericin, pseudohypericin, and protohypericin accumulate in the dark glands. However, the precursor emodin not only accumulates in the dark glands but is also present outside the glands in both hypericin-containing species. In hypericin-lacking H. patulum, however, emodin typically accumulates only in the glands, thereby providing evidence that hypericin is possibly biosynthesized outside the dark glands and thereafter stored in them. The distribution and localization of related compounds were also evaluated and are discussed concomitant to the occurrence of hypericin. Our study provides the basis for further detailed investigation of hypericin biosynthesis by gene discovery and expression studies.

Quorum quenching is an antivirulence strategy employed by endophytic bacteria.

Bacteria predominantly use quorum sensing to regulate a plethora of physiological activities such as cell-cell crosstalk, mutualism, virulence, competence, biofilm formation, and antibiotic resistance. In this study, we investigated how certain potent endophytic bacteria harbored in Cannabis sativa L. plants use quorum quenching as an antivirulence strategy to disrupt the cell-to-cell quorum sensing signals in the biosensor strain, Chromobacterium violaceum. We used a combination of high-performance liquid chromatography high-resolution mass spectrometry (HPLC-ESI-HRMS(n)) and matrix-assisted laser desorption ionization imaging high-resolution mass spectrometry (MALDI-imaging-HRMS) to first quantify and visualize the spatial distribution of the quorum sensing molecules in the biosensor strain, C. violaceum. We then showed, both quantitatively and visually in high spatial resolution, how selected endophytic bacteria of C. sativa can selectively and differentially quench the quorum sensing molecules of C. violaceum. This study provides fundamental insights into the antivirulence strategies used by endophytes in order to survive in their ecological niches. Such defense mechanisms are evolved in order to thwart the plethora of pathogens invading associated host plants in a manner that prevents the pathogens from developing resistance against the plant/endophyte bioactive secondary metabolites. This work also provides evidence towards utilizing endophytes as tools for biological control of bacterial phytopathogens. In continuation, such insights would even afford new concepts and strategies in the future for combating drug resistant bacteria by quorum-inhibiting clinical therapies.