Demonstrating the reliability of in vivo metabolomics based chemical grouping: towards best practice.

While grouping/read-across is widely used to fill data gaps, chemical registration dossiers are often rejected due to weak category justifications based on structural similarity only. Metabolomics provides a route to robust chemical categories via evidence of shared molecular effects across source and target substances. To gain international acceptance, this approach must demonstrate high reliability, and best-practice guidance is required. The MetAbolomics ring Trial for CHemical groupING (MATCHING), comprising six industrial, government and academic ring-trial partners, evaluated inter-laboratory reproducibility and worked towards best-practice. An independent team selected eight substances (WY-14643, 4-chloro-3-nitroaniline, 17α-methyl-testosterone, trenbolone, aniline, dichlorprop-p, 2-chloroaniline, fenofibrate); ring-trial partners were blinded to their identities and modes-of-action. Plasma samples were derived from 28-day rat tests (two doses per substance), aliquoted, and distributed to partners. Each partner applied their preferred liquid chromatography-mass spectrometry (LC-MS) metabolomics workflows to acquire, process, quality assess, statistically analyze and report their grouping results to the European Chemicals Agency, to ensure the blinding conditions of the ring trial. Five of six partners, whose metabolomics datasets passed quality control, correctly identified the grouping of eight test substances into three categories, for both male and female rats. Strikingly, this was achieved even though a range of metabolomics approaches were used. Through assessing intrastudy quality-control samples, the sixth partner observed high technical variation and was unable to group the substances. By comparing workflows, we conclude that some heterogeneity in metabolomics methods is not detrimental to consistent grouping, and that assessing data quality prior to grouping is essential. We recommend development of international guidance for quality-control acceptance criteria. This study demonstrates the reliability of metabolomics for chemical grouping and works towards best-practice.

Mode of action hypothesis testing in chemical safety assessments using metabolomics as supporting evidence: Phenobarbital and cyclobutrifluram metabolomics profile comparison.

In long term rodent studies administering Cyclobutrifluram (TYMIRIUM® Technology), a new agrochemical, there was a slight elevation of incidence of hepatocellular carcinomas in male CD-1 mice that was within the historical control range but appeared to be dose responsive. Cyclobutrifluram's ability to activate mouse constitutive androstane receptor (CAR) mediated gene transcription was confirmed in vitro, therefore a 28-day dietary toxicity study was conducted in vivo in male CD-1 mice to assess the CAR activation mode of action hypothesis of Cyclobutrifluram along with phenobarbital, a known CAR activator. In addition to other end points comprehensive (polar and lipidomic) hybrid metabolomics analyses were performed on terminal plasma and liver samples following 2-, 7- and 28-days dietary exposure to cyclobutrifluram and phenobarbital. The data generation and quality assessments were performed in line with the principles of the MEtabolomics standaRds Initiative in Toxicology (MERIT).First the full annotated feature set was used to compare the metabolomic changes induced by the administration of the two test substances using Shared and Unique Structures plots. This gave a comprehensive overview of the similarity of the two effect profiles showing good correlation and demonstrated that no other, alternative effect signatures were detected. Then the phenobarbital induced differentially abundant metabolites were selected, compared to the literature and their direction of change was assessed in cyclobutrifluram profiles, finding good agreement. Both approaches concluded that the metabolomics data supports the CAR activation hypothesis. Comparison of the metabolomic effect profiles can be a line of evidence in mode of action hypothesis testing in the chemical risk assessment process.

Solid-Phase Quasi-Intramolecular Redox Reaction of [Ag(NH3)2]MnO4: An Easy Way to Prepare Pure AgMnO2.

Two monoclinic polymorphs of [Ag(NH3)2]MnO4 containing a unique coordination mode of permanganate ions were prepared, and the high-temperature polymorph was used as a precursor to synthesize pure AgMnO2. The hydrogen bonds between the permanganate ions and the hydrogen atoms of ammonia were detected by IR spectroscopy and single-crystal X-ray diffraction. Under thermal decomposition, these hydrogen bonds induced a solid-phase quasi-intramolecular redox reaction between the [Ag(NH3)2]+ cation and MnO4- anion even before losing the ammonia ligand or permanganate oxygen atom. The polymorphs decomposed into finely dispersed elementary silver, amorphous MnOx compounds, and H2O, N2 and NO gases. Annealing the primary decomposition product at 573 K, the metallic silver reacted with the manganese oxides and resulted in the formation of amorphous silver manganese oxides, which started to crystallize only at 773 K and completely transformed into AgMnO2 at 873 K.

Mitochondrial dysfunction and delayed hepatotoxicity: another lesson from troglitazone.

AIMS/HYPOTHESIS: Troglitazone was approved for treatment of type 2 diabetes mellitus, but by 2000 it had been removed from all world markets due to severe drug-induced liver injury. Even today, we still do not know how many patients sustained a long-term liver injury. No system is in place to acquire that knowledge. Regarding toxicity mechanisms, controversy persists as to which ones are class effects of thiazolidinediones (TZDs) and which are unique to troglitazone. This study aims to provide long-term outcome data and new insights on mechanisms of troglitazone-induced liver injury. METHODS: This case series reports the liver injuries sustained by eleven type 2 diabetic patients treated with troglitazone between 1997 and 2000. Exhaustive review of medical records was performed for all patients. Long-term outcomes were available for all the non-fatal cases. A comprehensive literature review was also performed. RESULTS: Long-term liver injury progressing to cirrhosis was identified in seven patients. All eleven cases had liver injury patterns consistent with troglitazone toxicity. Analysis of these cases and of the experimental troglitazone toxicity data points to mitochondrial toxicity as a central factor. The general clinical patterns of mitochondrial hepatotoxic events are reviewed, as are the implications for other members of the TZD family. CONCLUSIONS/INTERPRETATION: This analysis enables the liver injury induced by troglitazone to be better understood. In future cases of delayed drug-induced liver injury that progresses after discontinuation, the possibility of mitochondrial toxicity should be considered. When appropriate, this can then be evaluated experimentally. Such proactive investigation may anticipate clinical risk before a large-scale therapeutic misadventure occurs. Drug-induced liver injury due to mitochondrial hepatotoxins may be less unpredictable than has previously been surmised.

A potential endogenous ligand for the aryl hydrocarbon receptor has potent agonist activity in vitro and in vivo.

The aryl hydrocarbon receptor (AhR) is best known as a mediator of toxicity of a diverse family of xenobiotic chemicals such as dioxins and PCBs. However, many naturally occurring compounds also activate AhR. One such compound, 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE), was isolated from tissue and found to be potent in preliminary tests [J. Song, M. Clagett-Dame, R.E. Peterson, M.E. Hahn, W.M. Westler, R.R. Sicinski, H.F. DeLuca, Proc. Natl. Acad. Sci. USA 99 (2002) 14694-14699]. We have synthesized ITE and [(3)H]ITE and further evaluated its AhR activity in several in vitro and in vivo assays in comparison with the toxic ligand, TCDD. AhR in Hepa1c1c7 cell cytosol bound [(3)H]ITE with high affinity and the AhR.ITE complex formed in vitro bound dioxin response element (DRE) oligonucleotide as potently as TCDD.AhR. In cells treated with ITE, nuclear translocation of AhR, and induction of CYP1A1 protein and of a DRE-dependent luciferase reporter gene were observed. ITE administered to pregnant DRE-LacZ transgenic mice activated fetal AhR, observed as X-gal staining in the same sites as in TCDD-treated mice. However, unlike TCDD, ITE did not induce cleft palate or hydronephrosis. TCDD but not ITE induced thymic atrophy in young adult mice, but both ITE and TCDD caused similar loss of cells and alterations of cell profiles in cultured fetal thymi. These data demonstrate that ITE is a potent AhR agonist in cell extracts, cultured cells, and intact animals, but does not cause the toxicity associated with the more stable xenobiotic ligand, TCDD.

Identification of potential aryl hydrocarbon receptor antagonists in green tea.

Previous investigations have implicated green tea to exert chemopreventive effects in animal models of chemical carcinogenesis, including polycyclic aryl hydrocarbon-induced cancers. In an effort to understand the compound(s) responsible for this protection, the effects of green tea extracts (GTE) and individual green tea catechins on aryl hydrocarbon receptor (AhR) gene induction were determined. Green tea (GT) was organically extracted and subsequently fractionated by column chromatography. The chemical composition of each fraction was determined by NMR. Several fractions inhibited tetrachlorodibenzo-p-dioxin-induced transcription of a dioxin responsive element-dependent luciferase reporter in stably transfected mouse hepatoma cells in a concentration-dependent manner. To determine the GT component(s) responsible for the observed effects, individual catechins were tested in the luciferase reporter system at concentrations found within the active fractions. Of the catechins tested, epigallocatechingallate (EGCG) and epigallocatechin (EGC) were the most potent antagonists, with IC(50) values of 60 and 100 microM, respectively. Re-creation of the active fractions using commercially available catechins further confirmed the identification of EGCG and EGC as the active AhR antagonists in green tea. These data suggest that EGCG and EGC are capable of altering AhR transcription and are responsible for most, if not all, of the AhR antagonist activity of GTE.

Expression of cytokeratins 7 and 20 in carcinomas of the gastrointestinal tract.

AIMS: The differential expression of cytokeratin (CK) 7 and 20 by carcinomas may help in determining the primary site of a metastatic tumour. The aim of this study was to extend the published data on CK7 and CK20 expression in epithelial neoplasms of the gastrointestinal tract by considering the degree of differentiation and including some unusual neoplasms. METHODS AND RESULTS: Cases referred to the Armed Forces Institute of Pathology were studied prospectively for immunohistochemical expression of CK7 and CK20. Lesions from 105 patients were analysed. Adenocarcinomas of the upper gastrointestinal tract were positive for both CK7 and CK20 in 78% of cases; only poorly differentiated lesions were CK7-. Well-differentiated and moderately differentiated adenocarcinomas of the large intestine, including appendix, were CK7-/CK20+ in the great majority of cases, as were goblet cell carcinoids, but half of the poorly differentiated adenocarcinomas exhibited aberrant expression, as did most of the mixed goblet cell carcinoid/adenocarcinomas. All five high-grade neuroendocrine carcinomas were negative for both CK7 and CK20. CONCLUSIONS: Not only the site but also the grade and histological type of a gastrointestinal carcinoma should be considered when assessing cytokeratin phenotype.

Synthesis and biological evaluation of prostaglandin-F alkylphosphinic acid derivatives as bone anabolic agents for the treatment of osteoporosis.

A series of novel C(1) alkylphosphinic acid analogues of the prostaglandin-F family have been evaluated at the eight human prostaglandin receptors for potential use in the treatment of osteoporosis. Using molecular modeling as a tool for structure-based drug design, we have discovered that the phosphinic acid moiety (P(O)(OH)R) behaves as an isostere for the C(1) carboxylic acid in the human prostaglandin FP binding assay in vitro and possesses enhanced hFP receptor selectivity when compared to the parent carboxylic acid. When evaluated in vivo, the methyl phosphinic acid analogue (4b) produced a bone anabolic response in rats, returning bone mineral volume (BMV) [corrected], to intact levels in the distal femur in the ovariectomized rat (OVX) model. These results suggest that prostaglandins of this class may be useful agents in the treatment of diseases associated with bone loss.

First total synthesis of (+/-)-stemonamide and (+/-)-isostemonamide.

[structure: see text] The total synthesis of the tetracyclic alkaloids stemonamide (1) and isostemonamide (2) is presented. The key step is the reaction between a silyloxyfuran and an N-acyliminium ion. The second quaternary center is created by an intramolecular aldol spirocyclization. After 1,4-addition of an appropriate side chain, the methyl and double bond are installed by Mannich reaction. The seven-membered ring is closed by intramolecular nucleophilic displacement.

Quinolone signaling in the cell-to-cell communication system of Pseudomonas aeruginosa.

Numerous species of bacteria use an elegant regulatory mechanism known as quorum sensing to control the expression of specific genes in a cell-density dependent manner. In Gram-negative bacteria, quorum sensing systems function through a cell-to-cell signal molecule (autoinducer) that consists of a homoserine lactone with a fatty acid side chain. Such is the case in the opportunistic human pathogen Pseudomonas aeruginosa, which contains two quorum sensing systems (las and rhl) that operate via the autoinducers, N-(3-oxododecanoyl)-L-homoserine lactone and N-butyryl-L-homoserine lactone. The study of these signal molecules has shown that they bind to and activate transcriptional activator proteins that specifically induce numerous P. aeruginosa virulence genes. We report here that P. aeruginosa produces another signal molecule, 2-heptyl-3-hydroxy-4-quinolone, which has been designated as the Pseudomonas quinolone signal. It was found that this unique cell-to-cell signal controlled the expression of lasB, which encodes for the major virulence factor, LasB elastase. We also show that the synthesis and bioactivity of Pseudomonas quinolone signal were mediated by the P. aeruginosa las and rhl quorum sensing systems, respectively. The demonstration that 2-heptyl-3-hydroxy-4-quinolone can function as an intercellular signal sheds light on the role of secondary metabolites and shows that P. aeruginosa cell-to-cell signaling is not restricted to acyl-homoserine lactones.

Cryptic Mucor infection leading to massive cerebral infarction at initiation of antileukemic chemotherapy.

A 74-year-old man with newly diagnosed acute myelogenous leukemia unexpectedly suffered a massive cerebral infarct on day 2 of induction chemotherapy. Clinically, the hemorrhagic infarct was thought to be due to leukostasis and thrombocytopenia. Necropsy, however, revealed that Zygomycetes-type hyphae had infiltrated cerebral vessels in and near the infarct. The fungal infection was clinically silent otherwise, although fungal elements were also identified in the lung at autopsy. This case illustrates how closely fungal infection may resemble a leukemia-associated cerebrovascular accident.

Flavone antagonists bind competitively with 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) to the aryl hydrocarbon receptor but inhibit nuclear uptake and transformation.

Previous analyses suggested that potent aryl hydrocarbon receptor (AhR) antagonists were planar, with a lateral electron-rich center. To further define structural requirements and mechanism for antagonism, ten additional flavone derivatives were synthesized. Based on their ability to 1) compete with 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) for binding to the AhR; 2) inhibit TCDD-elicited binding of AhR to dioxin-responsive elements (DRE) in vitro; and 3) inhibit TCDD-induced transcription of DRE-dependent luciferase in stably transfected hepatoma cells, the most potent flavones contained a 3'-methoxy group and a 4'-substituent having one or more terminal atoms of high electron density (-N3, -NO2, or -NCS). Furthermore, these had low agonist activity as assessed by their inability to elicit AhR. DRE binding or to induce luciferase. Compounds containing bulkier 3' or 4'-substituents, or a 3'-OH group were less potent antagonists, and some were partial agonists. In rat liver cytosol, 3'-methoxy-4'-azido- and 3'-methoxy-4'-nitroflavones bound competitively (with TCDD) to the AhR, indicating that they bind to the TCDD-binding site. When hepatoma cells were exposed to these flavones, AhR complexes were primarily immunoprecipitable from the cytosol and contained 90 kDa heat shock protein. In contrast, AhR in TCDD-treated cells was primarily immunoprecipitated from nuclear extracts and was associated with Arnt but not 90 kDa heat shock protein. Immunocytofluorescence analysis in intact cells further indicated that the potent antagonist inhibited nuclear uptake of AhR and blocked TCDD-dependent down-regulation of AhR. Together, these data indicate that the most potent antagonists bind the AhR with high affinity but cannot initiate receptor transformation and nuclear localization.

Thietanium ion formation from the food mutagen 2-chloro-4-(methylthio)butanoic acid.

2-Chloro-4-(methylthio)butanoic acid (1) is a direct-acting mutagen and suspected gastric carcinogen isolated from fish preserved with salt and nitrite. The reactive intermediates formed from acid 1 that may be associated with its mutagenicity have not been identified. A candidate reactive intermediate is proposed in this work. 1-Methyl-2-thietaniumcarboxylic acid may result from internal displacement of chloride by neighboring-group participation of sulfide sulfur in the solvolysis of acid 1. Evidence for the formation of 1-methyl-2-thietaniumcarboxylic acid was derived from experiments in which 4-chlorophenol and aniline were included to react with electrophilic intermediates formed from acid 1. Incubation of acid 1 in the absence of the aniline or 4-chlorophenol resulted in the formation of 2,4-bis(methylthio)butanoic acid. Incubation of acid 1 with 4-chlorophenol or aniline gave adducts that were identified by 1H NMR spectroscopy and GC/MS as 2-(4-chlorophenoxy)-4-(methylthio)butanoic acid and 4-(4-chlorophenoxy)-2-(methylthio)butanoic acid or 2-anilino-4-(methylthio)butanoic acid and 4-anilino-2-(methylthio)butanoic acid, respectively. The structures of these adducts indicate the intermediate formation of 1-methyl-2-thietaniumcarboxylic acid as a reactive intermediate derived from acid 1 that may be associated with its observed mutagenicity.

[3H]benzylpempidine, a new radioligand for probing a putative channel site on nicotinic cholinergic receptors.

A study was undertaken to assess the receptor binding characteristics of [3H]4-benzylpempidine to an allosteric site on calf brain membranes associated with nicotinic cholinergic receptors and to compare the binding affinity of novel arylpempidine analogs with their ability to antagonize the behavioral effects of nicotine in mice. Scatchard analysis of the binding yielded a K(d) of 20 nM and a B(max) of 330 fmols/mg membrane protein. [3H]4-benzylpempidine appears to be a more satisfactory ligand than [3H]mecamylamine, since it possessed a 50-fold greater affinity and its binding was far less sensitive to inorganic ions and Tris. Among the arylpempidine analogs 4-m-chlorobenzylidenepempidine and 4-benzylidenepempidine had the lowest K(i) values (1.4 nM and 5.0 nM, respectively) and were the most potent in antagonizing nicotine-induced seizures in mice. Although the K(i) values for pempidine and mecamylamine were 1-2 orders of magnitude greater than any of the arylpempidines, the dose required to antagonize nicotine-induced seizures in mice was comparable to the arylpempidines. One explanation for this apparent discrepancy in the correlation of binding affinity and nicotine antagonism is the lower brain penetration of arylpempidines compared to mecamylamine, following their systemic administration to mice.

Analysis of structural requirements for Ah receptor antagonist activity: ellipticines, flavones, and related compounds.

A number of studies have examined the structure-activity relationships for the agonist activity of Ah receptor (AhR) ligands. Fewer studies have considered the structural basis for potential antagonist properties. Certain ellipticine derivatives have been reported to bind to the AhR and inhibit the ability of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to transform the AhR to a form that recognizes a dioxin-responsive enhancer element (DRE) upstream of the cytochrome P4501A1 gene. In the present study, over 30 ellipticine derivatives and structurally related compounds were examined for their ability to bind to the AhR, activate it to a DRE-binding form, induce the luciferase gene under control of a DRE-containing enhancer, and block activation of the AhR by TCDD. The ability of several ellipticine derivatives to inhibit TCDD-elicited DRE binding and TCDD-induced luciferase activity was inversely related to their ability to alone stimulate these responses. The most potent antagonist activity was related to good AhR binding characteristics in terms of conforming to previously predicted 14 x 12 x 5 A van der Waals dimensions and the presence of an electron-rich ring nitrogen at or near a relatively unsubstituted X-axis terminal position. Based on these data, a number of flavone derivatives were synthesized and tested for their relative agonist/antagonist activity. These additional data were consistent with the hypothesis that an electron-rich center near or along a lateral position of the van der Waals binding cavity is a characteristic that enhances AhR antagonist activity.

Functional analysis of the Pseudomonas aeruginosa autoinducer PAI.

A series of structural analogs of the Pseudomonas aeruginosa autoinducer [PAI, N-3-oxo-dodecanoyl homoserine lactone] were obtained and tested for their ability to act as autoinducers in stimulating the expression of the gene for elastase (lasB) by measuring beta-galactosidase production from a lasB-lacZ gene fusion in the presence of the transcriptional activator LasR. The data suggest that the length of the acyl side chain of the autoinducer molecule is the most critical factor for activity. Replacement of the ring O by S in the homoserine lactone moiety can be tolerated. Tritium-labelled PAI ([3H]PAI) was synthesized and used to demonstrate the association of [3H]PAI with cells overexpressing LasR. The PAI analogs were also tested for their ability to compete with [3H]PAI for binding of LasR. Results from the competition assays suggest that once again the length of the acyl side chain appears to be crucial for antagonist activity. The presence of the 3-oxo moiety also plays a significant role in binding since analogs which lacked this moiety were much less effective in blocking binding of [3H]PAI. All analogs demonstrating competition with PAI in binding to LasR also exhibited the ability to activate lasB expression, suggesting that they are functional analogs of PAI.

Bilateral acetylsulfapyridine nephrolithiasis associated with chronic sulfasalazine therapy.

We report on formation of bilateral renal calculi secondary to sulfasalazine therapy for juvenile rheumatoid arthritis. The condition was successfully treated with extracorporeal shock wave lithotripsy. Analysis of the fragments with thin layer chromatography and nuclear magnetic resonance revealed acetylsulfapyridine, a metabolite of sulfasalazine.

5-Isothiocyanonicotine: a high-affinity irreversible ligand for brain nicotinic receptors.

A newly synthesized affinity ligand, (R,S)-5-isothiocyanonicotine (ISCN-N) was found to inhibit irreversibly the binding of [3H]methylcarbamylcholine (a specific nicotinic receptor ligand) to brain membranes. Plots of percent inhibition versus ligand concentration yielded an IC50 of 7 x 10(-8) M for SCN-N and Ki values of 6 x 10(-9) and 2 x 10(-9) M for (R,S)-5-aminonicotine and (S)-nicotine, respectively. The IC50 value for irreversible inhibition of [3H]methylcarbamylcholine by SCN-N was 2 x 10(-7) M. The affinity ligand irreversibly inhibited brain nicotinic receptors in vivo in a dose-dependent manner, the inhibition being 49% at a dose of 20 mumol/kg. Behavioral studies in mice revealed that SCN-N had less than one-fifth the potency of nicotine in producing muscle weakness and seizures, whereas 5-aminonicotine was without significant behavioral effects at doses up to 20 mumol/kg.