Stimulatory effects of silibinin and silicristin from the milk thistle Silybum marianum on kidney cells.

The biochemical influence of flavonolignans from the milk thistle Silybum marianum has been tested on kidney cells of African green monkeys. Two nonmalignant cell lines were selected, with the focus of the work on the fibroblast-like Vero line. Proliferation rate, biosynthesis of protein and DNA, and the activity of the enzyme lactate dehydrogenase (as a measure of the cellular metabolic activity) were chosen as parameters for the effect of the flavonolignans. Silibinin and silicristin show remarkable stimulatory effects on these parameters, mainly in Vero cells; however, isosilibinin and silidianin proved to be inactive. In vitro experiments with kidney cells damaged by paracetamol, cisplatin, and vincristin demonstrated that administration of silibinin before or after the chemical-induced injury can lessen or avoid the nephrotoxic effects. The results warrant in vivo evaluations of the flavonolignan derivatives.

Additional pathways of S-conjugate formation during interaction of 4-nitrosophenetole with glutathione.

The rapid reactions of nitrosoarenes with cellular SH groups have proved to be main metabolic conversions during detoxication. Interactions of the phenacetin metabolite 4-nitrosophenetole with glutathione have been investigated in detail during the last years, revealing a complex pattern of products depending on the stoichiometry of the reactants and reaction conditions. Eight metabolites have been identified hitherto, and the present work extends this medley by six additional products. Three metastable sulfenamides, 4-ethoxy-2,N-bis(glutathion-S-yl)-aniline, N4-(glutathion-S-yl)-4-amino-4'-ethoxydiphenylamine, and N-(glutathion-S-yl)-4-aminophenol, as well as the N-sulfenylquinonimine N-(glutathion-S-yl)-1,4-benzoquinonimine were characterized by chemical reactivity, chromatographic behavior, UV/vis absorption, 1H NMR, and FAB-MS data. The structure of the sulfenamide 2,N4-bis(glutathion-S-yl)-4-amino-4'-ethoxydiphenylamine could not be proved unequivocally, but is strongly suggested due to the chemical reactivity, chromatographic behavior, and UV/vis absorption of the compound. Finally, traces of 4-aminophenol were detected. A reaction scheme is presented explaining the formation of all identified metabolites via a central sulfenamide cation. Molecular orbital calculations for this sulfenamide cation have been performed, corroborating the proposed reaction mechanisms on the basis of Klopman's generalized perturbation theory.

Formation of 4,4-dialkoxycyclohexa-2,5-dienone N-(thiol-S-yl)imine during reaction of 4-alkoxynitrosobenzenes with thiols in alcoholic solvents.

During the interaction of nitrosoarenes with glutathione in aqueous media, intermediate generation of a highly resonance-stabilized sulfenamide cation has been repeatedly suggested. Most intermediates and end products could be explained by reactions of this sulfenamide cation with different nucleophiles such as excess thiol, solvent water, and metabolically produced arylamine. The present paper presents evidence for adduct formation of the sulfenamide cation with solvent alcohol at neutral pH. Sulfenamide cations generated from 4-nitrosophenetole and 4-nitrosoanisole, respectively, are strongly suggested to form the metastable ketals 4-ethoxy-4-methoxycyclohexa-2,5-dienone N-(glutathion-S-yl)imine and 4,4-dimethoxycyclohexa-2,5-dienone N-(glutathion-S-yl)imine, respectively, during reaction with solvent methanol. Reaction of the two sulfenamide cations in ethanol yielded 4,4-diethoxycyclohexa-2, 5-dienone N-(glutathion-S-yl)imine and 4-ethoxy-4-methoxycyclohexa-2, 5-dienone N-(glutathion-S-yl)imine, respectively. Although the metastability of the ketals did not allow isolation of pure solid material, chromatographic and chemical behavior as well as tandem MS fragmentation substantiate a ketal structure of these intermediates. To confirm the proposed structure, new compounds, 2, 6-dimethyl-4-nitrosophenetole, 2,6-dimethyl-4-nitrophenetole, 2, 6-dimethyl-4-phenetidine, and N-(glutathion-S-yl)-N-hydroxy-4-aminoacetophenone, were synthesized and included in supportive experiments. In summary, the detection of ketals corroborates once more the occurrence of a sulfenamide cation which obviously not only reacts with soft nucleophiles such as GSH but, to a limited extent, also reacts with hard nucleophiles. The toxicological significance of this result is discussed.

Isolation and characterization of dolichol-linked oligosaccharides from Haloferax volcanii.

Biosynthesis of procaryotic glycoproteins has been studied in some detail only in the extreme halophile Halobacterium halobium. To extend these studies for a moderate halophile, dolichol phosphate-linked oligosaccharides were isolated and characterized from Haloferax volcanii. Mannosyl (beta 1-->4)galactosyl phosphodolichol could be characterized as a main component by GC/MS permethylation analysis, mass spectrometry and 1H-NMR-spectroscopy. Furthermore, two additional components, present in lower quantities, were partially characterized and identified as a tetrasaccharyl phosphodolichol containing mannose, galactose, and rhamnose in the ratio of 2:1:1 as well as another dihexosyl phosphodolichol. Both the latter compounds contain an additional charged group. All these oligosaccharides were found to be linked to a dolichol consisting of 11 or 12 isoprene units including a saturated omega-terminal isoprene residue.

Biotransformation of the fungal toxin fomannoxin by conifer cell cultures.

Fomannoxin [(+/-)-5-Formyl-2-isopropenyl-2,3-dihydrobenzofurane] is a phytotoxic secondary metabolite, which is produced by the forest pathogenic basidiomycete Heterobasidion annosum during the infection process. Fomannoxin shows growth-inhibiting effects on callus and suspension cultures of conifer cells. By investigating the interaction of the phytotoxin with Pinus sylvestris cells a detoxification of fomannoxin was detected, presumably as a defense reaction of the plant cells. Undifferentiated green cell lines of Pinus sylvestris were used as target cells. To provide rac-fomannoxin as a substrate a simple method for the chemical synthesis was developed. It was found that the aromatic aldehyde group is reduced by the plant cells producing the non-toxic fomannoxin alcohol which was isolated and identified by spectroscopy. After longer incubation times, also fomannoxin acid-beta-glucoside could be isolated as another detoxification metabolite. For comparison this glucoside was also synthesized.

Influence of the Gloeophyllum metabolite oosponol and some synthetic analogues on protein and RNA synthesis in target cells.

Oosponol [4-(hydroxyacetyl)-8-hydroxy-1H-2-benzopyran-1-one], a toxic metabolite of the basidiomycete Gloeophyllum abietinum, and some synthetic analogues were studied to clarify the molecular basis of their fungicidal, bactericidal, and phytotoxic effects. The antibiotic activity was due to a strong inhibition of RNA and protein synthesis in target cells.

Chemical reactivity of the toxin oosponol from Gloeophyllum abietinum with nucleophilic agents.

It is shown that contrary to most isocoumarins the antibiotic compound oosponol from the fungus Gloeophyllum abietinum reacts instantaneously with nucleophilic agents like thiol compounds. The reason for this high reactivity is due to the vinylogous acid anhydride character of the compound which is produced by a single oxidation step of the non toxic biological precursor oospoglycol. One must assume that reactions of oosponol with HS-groups of polypeptides form the basis for its toxicity. The ring-opening reactions were studied in detail with the synthetic analogue 4-acetyl-isocoumarin. The structures of some reaction products have been analysed mainly with H-1 and C-13 NMR spectroscopy.

Secondary fungal metabolites and their biological activities, V. Investigations concerning the induction of the biosynthesis of toxic secondary metabolites in basidiomycetes.

In cultures of the basidiomycetes Heterobasidion annosum, Gloeophyllum abietinum or Armillaria ostoyae, the biosynthesis of some of their toxic secondary metabolites is enhanced up to 400-fold when they grow in the presence of an antagonist. This stimulation is induced before any cell contact occurs. The "inducing signals" are not macromolecules, polypeptides or constituents of the cell membranes, but the same toxins which are synthesized already in monocultures in very low concentrations. After excretion and diffusion into the media, the fungi thus recognize the proximity of the antagonist in dualcultures. As a consequence, the enormous stimulation of toxin synthesis takes place. The increasing toxin concentrations in the fungal dual cultures finally cause the formation of mycel-free growth inhibition zones between the fungi, or sometimes one of the contrahents is killed. This stimulation of toxin synthesis is achieved by an enhanced de novo synthesis of enzymes of the corresponding secondary pathways.

Secondary fungal metabolites and their biological activities, IV. Synthesis of compounds with structural similarities to the toxic metabolites of the pathogenic fungus Heterobasidion annosum and investigation of their antibiotic activities.

A series of synthetic analogues of fomannoxin and fomajorin S, the toxic metabolites of the basidiomycete Heterobasidion annosum, was prepared in order to investigate which partial structures of the molecules are responsible for their biological activities. Of the compounds synthesized the five indane-2-acid derivatives 5-acetylindane-2-carboxylic acid (2), (+/-)5-formylindane-2-carboxylic acid (3), (+/-)indane-2,5-dicarboxylic acid (4), (+/-)formyl-2-methylindane-2-carboxylic acid (7) and 5-acetyl-indane-2,2-dicarboxylic acid (10) and 4-(2'-ethylbutyloxy)benzaldehyde (18) have not yet been described. Different biological test systems were employed to investigate the antibiotic activities of the synthesized analogues. The experimental results suggest that in the case of fomajorin S, the structural element essential for its toxicity is the 2-H- or 2-methylindane 2-acid. From the results obtained with the synthetic analogues of fomannoxin, it is now evident that the toxicity of this fungal metabolite can be attributed to an aromatic ring with a carbonyl or carboxyl substituent combined with a branched lipophilic side chain in p-substitution.

Secondary fungal metabolites and their biological activities, III. Further metabolites from dual cultures of the antagonistic basidiomycetes Heterobasidion annosum and Gloeophyllum abietinum.

In addition to a preceding publication (Sonnenbichler, J., Bliestle, I.M., Peipp, H. & Holdenrieder, O. (1989) Biol. Chem. Hoppe-Seyler 370, 1295-1303), six further metabolites formed in dual cultures of the antagonistic fungi Heterobasidion annosum and Gloeophyllum abietinum are described; three of them represent unknown structures. The producing organisms have been identified. The metabolites fommanoxin from H. annosum and oosponol from G. abietinum show that the toxins can be detoxified by metabolization by the antagonistic fungus. The newly-described compounds have been tested for their fungicidal properties.

[Side reactions in peptide synthesis. V. O-sulfonation of serine and threonine during removal of pmc- and mtr-protecting groups from arginine residues in fmoc-solid phase synthesis]. / Nebenreaktionen bei Peptidsynthesen, V. O-Sulfonierung von Serin und Threonin während der Abspaltung der Pmc- und Mtr-Schutzgruppen von Argininresten bei Fmoc-Festphasen-Synthesen.

A novel side reaction in Fmoc-solid-phase synthesis, which occurs during removal of protecting groups and detachment from the resin, was elucidated by investigations on model peptides: During the cleavage of Pmc- or Mtr-protecting groups from arginine residues by trifluoroacetic acid in peptides with O-tert-butyl-protected aliphatic hydroxyamino acids, peptides containing O3-sulfo-serine and O3-sulfo-threonine are formed as side-products in high yields, if suitable scavengers are absent. Subsequent to their isolation and purification, the structures of these peptide sulfuric acid mono-esters could unequivocally be proven by chemical and spectroscopic (MS, NMR, IR) methods.

Secondary fungal metabolites and their biological activities, II. Occurrence of antibiotic compounds in cultures of Armillaria ostoyae growing in the presence of an antagonistic fungus or host plant cells.

We found that in the presence of host plant cells or some antagonistic fungi, the highly forest-pathogenic basidiomycete Armillaria ostoyae is strongly stimulated to produce a series of toxic secondary metabolites which are capable of inhibiting the growth of the antagonist or of killing the plant cells still before cell contact. The chemical structures of the metabolites have been identified, of which two of them are new compounds. The time dependence and sites of synthesis in the mycelium have been determined in order to lay the foundation for future studies concerning the induction mechanism for the synthesis of the toxins.

Di-myo-inositol-1,1'-phosphate: a new inositol phosphate isolated from Pyrococcus woesei.

A new inositol derivative could be isolated from the Archaeum Pyrococcus woesei and identified as di-myo-inositol-1,1'-phosphate by 1H, 31P NMR spectroscopy, mass spectrometry and thin layer chromatography. In P. woesei, this inositol phosphate represents the dominant counterion of K+ which ranges from 500 to 600 mM. The role of the potassium salt of di-myo-inositol-1,1'-phosphate as thermostabilizer is discussed.

Activation and detoxication of aminophenols. II. Synthesis and structural elucidation of various thiol addition products of 1,4-benzoquinoneimine and N-acetyl-1,4-benzoquinoneimine.

1. Nine thioethers of 4-aminophenol with beta-hydroxyethylmercaptan, ranging from mono- to tetra-substituted thioadducts, were prepared from synthetic 1,4-benzoquinoneimine and characterized by 1H-n.m.r. and u.v. spectroscopy. For each compound, extinction coefficients and pKa values of the amino group were determined. 2. Five thioethers of 4-aminophenol with glutathione (GSH) were prepared and characterized by 1H-n.m.r. and u.v. spectroscopy with their respective extinction coefficients and pKa values. Two further thioadducts were tentatively assigned by their u.v. spectroscopic properties. 3. Reaction products of 1,4-[U-14C]benzoquinoneimine and GSH were studied, indicating formation of 4-amino-2-(glutathione-S-yl)phenol, 4-amino-2,3,6-tris(glutathione-S-yl)phenol as the main products. Formation of glutathione disulphide (GSSG) was not detected. In contrast, N-acetyl-1,4-[U-14C]benzoquinoneimine was partly reduced by GSH and formed only the 2-substituted thioadduct. 4. Investigation of the product orientation in the reductive addition of GSH to 2-(glutathione-S-yl)-1,4-benzoquinoneimine and 3-(glutathione-S-yl)-1,4-benzoquinoneimine, respectively, showed that the 3-substituted derivative formed mainly the 3,5-di-substituted thioadduct, whereas the 2-substituted compound formed mainly the 2,3,6-tri-substituted thioadduct. 5. Formation of thioadducts which autoxidize markedly faster than the parent aminophenol indicates that thioether formation is not an obligatory detoxication process.

Activation and detoxication of aminophenols. III. Synthesis and structural elucidation of various glutathione addition products to 1,4-benzoquinone.

1. Four thioethers of 1,4-hydroquinone with glutathione (GSH) were prepared from 1,4-benzoquinone and characterized by 1H-n.m.r. and partly by 13C-n.m.r. spectroscopy. The structures of three additional thioethers were tentatively assigned by u.v. spectroscopy. 2. The corresponding thioethers of 1,4-benzoquinone with GSH were obtained by oxidation of the corresponding 1,4-hydroquinone thioadducts with PbO2 or potassium ferricyanide. 3. Relative redox potentials of the hydroquinone/benzoquinone thioethers were estimated by determination of their redox equilibria with benzoquinone/hydroquinone. The redox potential of the mono-substituted derivative was 30 mV lower, and that of the di-substituted derivatives 70 mV lower, than that of the unsubstituted couple, thus explaining the readiness of sequential oxidation and addition reactions of the produced thioethers. 4. By use of 1,4-[U-14C]benzoquinone the reaction products with GSH were quantified to elucidate the product orientation. As observed with 1,4-benzoquinoneimine and its thioethers, formation of GSSG was not detected at physiological pH. 5. The high susceptibility of particular thioethers of 1,4-hydroquinone towards (aut)oxidation characterizes these products as reactive intermediates rather than as definitive detoxication products.

Secondary fungal metabolites and their biological activities, I. Isolation of antibiotic compounds from cultures of Heterobasidion annosum synthesized in the presence of antagonistic fungi or host plant cells.

Heterobasidion annosum (Syn. Fomes annosus), one of the most pathogenic basidiomycetes in conifer forests, produces a series of new metabolites specifically in the presence of antagonistic fungi or some plant cells. These "ecological metabolites" have been isolated and chemically characterized. The time course of production was measured with regard to their biosynthetic pathway. In bio-assays with other fungi, E. coli and cell cultures of Picea abies and Nicotiana tabacum, it could be shown that some of the compounds have antibiotic and growth-inhibiting properties.

Pharmacokinetics and biotransformation of diethylene glycol and ethylene glycol in the rat.

1. 14C-Diethylene glycol (DEG), administered orally to rats at 1, 5, and 10 ml/kg, gave elimination half-lives of 6, 6, and 10 h, respectively, from urinary excretion data. Half-logarithmic plots of urinary 14C excretion rates versus time indicated zero-order elimination for the first 9 and 18 h after oral doses of 5 and 10 ml of 14C-DEG/kg, respectively. 14C-DEG urinary elimination kinetics changed into first-order 6, 9, and 18 h after oral doses of 1, 5, and 10 ml/kg, with a half-life of 3 h. 2. After oral doses of 3 and 5 ml ethylene glycol (EG)/kg, half-lives of 4.5 and 4.1 h were estimated from cumulative urinary excretion data for non-metabolized EG. A half-life of 2 h was determined from half-logarithmic plots of urinary excretion rates of non-metabolized EG after the same oral doses of EG. 3. The urinary concentrations of non-metabolized DEG and its metabolite, 2-hydroxyethoxyacetic acid (2-HEAA), determined by high-resolution n.m.r. spectroscopy in the urine of rats doses with DEG were 61-68% and 16-31% dose, respectively. 4. Urinary concentrations of non-metabolized EG and its metabolite, glycolic acid (GA), determined by n.m.r., gave 62-67% for non-metabolized EG and 28.7% for GA following oral doses of EG. 5. Oxidation of DEG and EG in rats was accompanied by a change of urinary pH, reflecting metabolic acidosis. 6. Comparison of the KM for DEG oxidation in vitro by ADH with that of ethanol oxidation, showed a 680-fold difference in substrate affinity. DEG inhibited ethanol oxidation non-competitively, the Ki being 0.44 M.

Studies on the stability and decomposition of the Hagedorn-oxime HLö 7 in aqueous solution.

HLö 7, (pyridinium, 1-[[[4-(aminoarbonyl)pyridinio]methoxy]methyl] -2,4-bis- [(hydroxyimino)methyl] diiodide) has been shown to be efficacious in soman poisoning of mice even in the absence of atropine. To assess possible risks involved in the administration of HLö 7 its degradation products were analyzed at pH 2.5 and pH 7.4, respectively. At pH 2.5, where HLö 7 in aqueous solution was assumed to possess maximal stability, the predicted shelf life (10% decomposition) was about 8 years for 10 mM solutions at 8 degrees C. The apparent energy of activation was 117 kJ/mol. At pH 2.5, attack on the aminal-acetal bond predominated with formation of pyridine-2,4-dialdoxime, 2-cyanopyridine-4-aldoxime, isonicotinamide, and formaldehyde. At pH 7.4, primary attack on the 2-aldoxime group resulted in formation of an intermediate 2-cyano-4-aldoxime derivative which mainly decomposed into cyanide and the corresponding 2-pyridinone, 1-[[[4-(aminocarbonyl)-pyridinio]methoxy]methyl]-4- [(hydroxyimino)methyl] diiodide. In addition, liberated cyanide reacted with the intermediate 2-cyano-4-aldoxime derivative with formation of 2-pyridinone, 1-[[[4-(aminocarbonyl)-pyridinio]-methoxy]methyl]-6-cyano-4- [(hydroxyimino)methyl] diiodide. This cyanide sequestering pathway became significant only at high concentrations (10 mM) of HLö 7, and was marginal at 1 mM HLö 7.

Wedelolactone and coumestan derivatives as new antihepatotoxic and antiphlogistic principles.

For the study of structure-activity relationships, the antihepatotoxic wedelolactone (7-methoxy-5,11,12-trihydroxy-coumestan) and 6 coumestan derivatives were synthesized by the application of a modified method of Wanzlich. An evaluation of the biological characteristics of the synthetic compounds and acuminatin from Musa acuminata showed that most of the wedelolactone derivatives significantly protected primary cultured liver cells from the toxicity of CCl4, galactosamine (Galc), and phalloidin, and strongly inhibited the activity of 5-lipoxygenase in porcine leukocytes. The hepatocyte protective activity was dependent on the C-7 substitution with pharmacological efficacy decreasing in the following order: EtO greater than MeO greater than OH greater than CH3(CH2)9. In addition, a free OH at C-5 of the wedelolactone molecule was shown to be important in protecting hepatocytes from CCl4 and Galc damage. Similar observation regarding the effect of C-7 substitution in wedelolactone was obtained in the 5-lipoxygenase test. In general, an increase in the lipophilicity in ring A increased the inhibition of 5-lipoxygenase activity. The synthetic wedelolactone was also found to have stimulatory effect on the RNA synthesis in isolated nuclei from hepatocytes.