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P Eyer

Publications and source records attributed to P Eyer.

At least 37 records · Page 2Linked to original sources
PubMed1998

Ring addition of the alpha-amino group of glutathione increases the reactivity of benzoquinone thioethers.

2-(Glutathion-S-yl)-1,4-benzoquinone was found to be remarkably unstable in phosphate buffer (pH 7.4) even in the absence of oxygen. Intramolecular addition of the alpha-amino group of the glutamate residue to the quinone ring yielded ultimately 2,3-(glutathion-N, S-yl)-1,4-benzoquinone and 2,6-(glutathion-N,S-yl)-1,4-benzoquinone in a 3:1 ratio along with 2-(glutathion-S-yl)-1,4-hydroquinone. Kinetic studies indicated that the cyclization reactions proceeded at a rate k1 of 0.093 min-1, while intermolecular reactions followed a second-order kinetics with a k2 of 94 M-1 min-1 (pH 7.4, 37 degreesC), resulting in multiple polymerization products. Both intramolecular amino adducts of 2-(glutathion-S-yl)-1,4-benzoquinone are prone to hydrolysis, leading to the insertion of an additional OH group in the ring. These S-substituted trihydroxybenzene derivatives are particularly susceptible to autoxidation. The model compound 6-(N-acetylcystein-S-yl)-2-hydroxy-1,4-hydroquinone was shown to form readily two atropoisomeric biphenyls upon autoxidation: 2,4'-bis(N-acetylcystein-S-yl)-2',3,3',4,6, 6'-hexahydroxybiphenyl, indicating C-C coupling, presumably via semiquinone radical intermediates. Thus, the sequence of glutathione S-addition, followed by oxidation, N-addition, oxidation, and hydrolysis, constitutes a novel and very effective activation pathway of quinones for eliciting oxidative stress. These data underline the fact that glutathione conjugates of autoxidizable aromatics are no obligatory stable end products of a detoxication reaction. The possible toxicological impacts of intra- and intermolecular addition reactions of quinoid thiol conjugates are discussed.

Acetylcysteine↗
PubMed1998

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.

Biotransformation↗
PubMed1998

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.

Alcohols↗
PubMed1998

Enzyme-based assay for quantification of paraoxon in blood of parathion poisoned patients.

1. Paraoxon concentration was estimated by means of inhibition kinetics observed with electric eel acetylcholinesterase (AChE) which was determined by a modified Ellman procedure. In human plasma, paraoxon was stabilized by inactivation of paraoxonase with EDTA and aluminon and by inhibition of butyrylcholinesterase with ethopropazine. Paraoxon (1-50 ng) was recovered at 86+/-1.7% (mean+/-s.e.m.) in ether extracts from 0.5 ml samples of spiked stabilized plasma. It could be stored without loss at - 20 degrees C for at least 1 month. 2. The enzyme-based assay was applied to follow the paraoxon plasma concentrations in three suicidal patients with severe parathion poisoning. In poisoning with excessive doses and initial paraoxon concentrations above 500 nM, therapeutic obidoxime concentrations of approximately 10 microM failed to essentially reactivate erythrocyte AChE in vivo, while reactivatability ex vivo was nearly complete. With the plasma concentrations of paraoxon dropping below 100 nM, however, reactivation by obidoxime became significant. Unexpectedly, paraoxon levels occasionally reincreased during treatment and resulted in re-inhibition of AChE, bearing some resemblance to the Intermediate Syndrome. 3. The paraoxon concentrations measured fitted satisfactorily the values calculated from the kinetic constants previously obtained for AChE inhibition and obidoxime-induced reactivation in vitro. This indicates that diethylphosphoryloxime formation during obidoxime-induced reactivation does not markedly contribute to the re-inhibition of AChE as observed in vitro.

Animals↗
PubMed1997

Reappraisal of indications and limitations of oxime therapy in organophosphate poisoning.

1 In vitro studies with human erythrocyte acetylcholinesterase (AChE) and the mouse diaphragm model were performed to unravel the various microscopic reaction parameters that contribute to the dynamic equilibrium of AChE inhibition, ageing and reactivation. These data may help to define more precisely the indications and limitations of oxime therapy in organophosphate (OP) poisoning. 2 Diethylphosphoryl-AChE resulting from intoxications with parathion, chlorpyrifos, chlorfenvinphos, diazinon and other OPs is characterized by slow spontaneous reactivation and low propensity for ageing. This kind of phosphorylated enzyme is particularly susceptible to reactivation by oximes. 3 None of the oximes tested (pralidoxime, obidoxime, HI 6 and HLö 7) can be regarded as a universally suitable reactivator. Obidoxime turned out to be the most potent and most efficacious oxime in reactivating AChE inhibited by various classes of OP insecticides and tabun. Obidoxime, however, was inferior to HI 6 against soman, sarin, cyclosarin and VX. Pralidoxime was generally less potent. 4 The kinetic data of reactivation established for diethylphosphoryl-AChE of human red cells indicate that the usually recommended dosage to attain a plasma concentration of 4 micrograms/ml does not permit exploitation of the full therapeutic potential of the oximes, in particular of pralidoxime. However, in suicidal mega-dose poisoning, oximes, even at optimal plasma concentrations, may be unable to cope with the fast re-inhibition of reactivated AChE in the first days following intoxication. 5 It is suggested that oximes be administered by continuous infusion following an initial bolus dose as long as reactivation can be expected and until permanent clinical improvement is achieved.

Acetylcholinesterase↗
PubMed1997

Cholinesterase status, pharmacokinetics and laboratory findings during obidoxime therapy in organophosphate poisoned patients.

1 The effectiveness of oxime therapy in organophosphate poisoning is still a matter of debate. It appears, however, that the often cited ineffectiveness of oximes may be due to inappropriate dosing. By virtue of in vitro findings and theoretical considerations we concluded in the preceding paper that oximes should preferably be administered by continuous infusion following an initial bolus dose for as long as reactivation of inhibited acetylcholinesterase (AChE) can be expected. This conclusion has called for a clinical trial to evaluate such oxime therapy on the basis of objective parameters. 2 Before transfer to the intensive care unit (ICU), 5 patients received primary care by an emergency physician. In the ICU, atropine sulphate was administered i.v. upon demand according to the endpoints: no bronchorrhoea, dry mucous membranes, no axillary sweating, heart rate of about 100/min. Obidoxime (Toxogonin) was given as an i.v. bolus (250 mg) followed by continuous infusion of 750 mg/24 h. 3 Intoxication and therapy were monitored by determining erythrocyte AChE (eryAChE) activity, reactivatability of the patient's eryAChE ex vivo, plasma cholinesterase activity, the presence of AChE inhibiting compounds, as well as the concentrations of obidoxime and atropine in plasma. 4 Obidoxime was effective in life-threatening parathion poisoning, in particular when the dose absorbed was comparably low. In mega-dose poisoning, net reactivation was not achieved until several days after ingestion, when the concentration of active poison in plasma had declined. Reactivatability in vivo lasted for a longer period than expected from in vitro experiments. 5 Obidoxime was quite ineffective in oxydemetonmethyl poisoning, when the time elapsed between ingestion and oxime therapy was longer than 1 day. When obidoxime was administered shortly after ingestion (1 h) reactivation was nearly complete. 6 Obidoxime levels of 10-20 microM were achieved by our regimen, and atropine could rapidly be reduced to approx. 20 microM, as attained by continuous infusion of 1 mg atropine sulphate/h. Maintenance of the desired plasma levels was not critical even when renal function deteriorated. 7 Signs of transiently impaired liver function were observed in patients who showed transient multiorgan failure. In the present stage of knowledge, we feel it advisable to keep the plasma concentration of obidoxime at 10-20 microM, although the full reactivating potential of obidoxime will not then be exploited. Still, the reactivation rate, with an apparent half-time of some 3 min, is twice that estimated for a tenfold higher pralidoxime concentration.

Acetylcholinesterase↗
PubMed1996

Pharmacokinetics of atropine in dogs after i.m. injection with newly developed dry/wet combination autoinjectors containing HI 6 or HLö 7.

To cope with the rapid onset of the life-threatening cholinergic crisis after poisoning with organophosphorus compounds, atropine-oxime preparations should be available in autoinjectors allowing i.m. administration also in the absence of a physician. Such a scenario is conceivable in the battlefield, when nerve agents are disseminated, and can no longer be excluded in civilian areas, as demonstrated most recently in Tokyo. In addition, autoinjectors may be of value in agriculture when medical care is remote. The use of second generation oximes with broad antidotal spectrum, e.g., HI 6 (1-(((4-(aminocarbonyl)pyridinio)methoxy)methyl)-2-((hydr oxyimino)methyl) pyridinium dichloride monohydrate; CAS 34433-31-3) and HLö 7 (1-(((4-(aminocarbonyl)pyridinio)methoxy)methyl) 2,4-bis((hydroxyimino)methyl) pyridinium dimethanesulfonate; CAS 145613-73-6) is only possible in dry/wet autoinjectors because their stability is limited in concentrated solution. To detect a possible delay in atropine absorption by the two oximes, the pharmacokinetics of atropine after "autoinjection" in beagle dogs were determined. Commercially available autoinjectors from two manufacturers [STI International Ltd (BJ) and Astra Tech (AT)] were filled with atropine sulfate, either alone (2 mg) or in combination with HI 6 (500 mg) and HLö 7 (200 mg), respectively, and injected according to a complete cross-over design. Atropine concentration was determined as l-hyoscyamine equivalents in a radioreceptor assay (RRA). In the range of 0.1-6.9 ng/ml, atropine sulfate displaced [N-methyl-3H]-scopolamine methyl chloride ([3H]NMS) competitively from rat cerebral cortex membranes. At 200 pmol/l [3H]NMS, IC50 was 1.4 +/- 0.1 x 10(-9) M atropine (CV = 8.1%). The intra-assay deviation was about 6%; day-to-day deviation in determination of 1 nM (0.695 ng/ml) atropine was 2.6% (CV = 5.2%). AT autoinjectors containing HI 6 delivered only 1.81 mg atropine sulfate while 2.14 mg was released by the other injectors. According to the manufacturer, the reduced delivery was caused by a defective Teflon-coated O-ring as detected later on in the batch used. To allow comparison of the bioavailability of atropine from various autoinjectors, the AUCs were normalized to a constant dose. The atropine absorption half-time (7 min) was not affected either by the autoinjector type or by the combination with oximes. The other pharmacokinetic data likewise did not reveal any differences between the groups. Maximal plasma concentration was 33 ng ml-1, elimination half-life 52 min, Vapp 3.2 l kg-1 and Clpl 44 ml min-1 kg-1. The relatively high clearance of l-hyoscyamine is discussed.

Animals↗
PubMed1995

Oxidation versus addition reactions of glutathione during the interactions with quinoid thioethers of 4-(dimethylamino)phenol.

4-(Dimethylamino)phenol (DMAP) is a potent cyanide antidote which forms many equivalents of ferrihemoglobin in vivo and in vitro. During this process formation of phenoxyl radicals was observed which are reduced by ferrohemoglobin, thereby sustaining a catalytic cycle of ferrihemoglobin formation, or which disproportionate to give the quinone imine of DMAP. In the presence of thiols, e.g., glutathione (GSH), formation of 4-(dimethylamino)-2-(glutathion-S-yl)phenol (2-GS-DMAP), 4-(dimethylamino)-2,6-bis(glutathion-S-yl)phenol (2,6-bis-GS-DMAP), and 4-(dimethylamino)-2,3,6-tris(glutathion-S-yl)phenol (2,3,6-tris-GS-DMAP) was observed. While the trisubstituted glutathione conjugate is a stable end product, 2-GS-DMAP and 2,6-bis-GS-DMAP were still reactive and produced ferrihemoglobin. It is concluded that formation of polysubstituted DMAP thioethers is a result of sequential oxidation/addition reactions with quinoid intermediates. Formation of glutathione disulfide (GSSG) was minimal during the interaction of oxidized DMAP or 2-GS-DMAP with glutathione but became significant when oxidized 2,6-bis-GS-DMAP reacted with GSH. Thus it is conceivable that the bulky glutathione substituents in 2,6-bis-GS-DMAP render the addition of a third GSH molecule to the quinone imine derivative more difficult, and other reactions may get a chance. The reaction mechanism of GSSG formation has not been fully resolved, but a radical pathway mechanism involving thiyl radicals is proposed. Oxidation and addition reactions were also observed in the absence of oxygen when ferrihemoglobin served as oxidant. In the presence of oxygen, however, GSSG formation was increased, Partly due to hydrogen peroxide formation, partly due to an additional trapping reaction of the glutathione disulfide radical anion.(ABSTRACT TRUNCATED AT 250 WORDS)

Aminophenols↗
PubMed1995

Reactivity of glutathione adducts of 4-(dimethylamino)phenol. Formation of a highly reactive cyclization product.

During ferrihemoglobin formation, 4-(dimethylamino)phenol (DMAP), a potent cyanide antidote, forms a quinoid compound that is prone to sequential oxidation/addition reactions. In human red cells and hemoglobin solutions fortified with glutathione, a transient adduct has been isolated and identified as 4-(dimethylamino)-2-(glutathion-S-yl)phenol (2-GS-DMAP). This compound still formed ferrihemoglobin but differed from parent DMAP in that the reaction rate was roughly proportional to the oxygen concentration and exhibited a lag phase, pointing to a reactive autoxidation product. The compound was isolated and tentatively identified as an intramolecular cyclization product of 2-GS-DMAP. Formation of this product includes three reaction steps: (1) formation of a quinoid intermediate, (2) addition of the alpha-amino nitrogen atom of the glutamate residue to the aromatic ring, and (3) autoxidation of the cyclization product to give a highly reactive o-quinone imine. The isolated compound existed in two isomeric states (1H-NMR) which upon reduction could be separated by HPLC. The isolated reduced isomers mutually converted into each other. A model compound which was synthesized to mimic the most important structural features, 4-(dimethylamino)-6-[S-(2'-hydroxyethyl)-thio]-N-(2"-phenylethyl)-1,2- quinone imine, had a very similar visible spectum and exhibited an even higher ferrihemoglobin activity than the cyclization product. A similar phenomenon of intramolecular cyclization of a thioether of DMAP had been observed earlier: DMAP covalently bound to the SH groups of the beta-chains in hemoglobin formed a cross-link with the C-terminal histidine residue in the presence of oxygen but not in its absence.(ABSTRACT TRUNCATED AT 250 WORDS)

Aminophenols↗
PubMed1995

Neuropsychopathological changes by organophosphorus compounds--a review.

1. Available literature dealing with neuropsychopathological changes after exposure to organophosphate insecticides is reviewed. 2. Subacute neurological sequelae following acute organophosphate intoxication include the 'intermediate syndrome', probably a myopathy elicited by excess acetylcholine, and the 'organophosphate-induced delayed neuropathy' (OPIDN), which is caused by particularly neurotoxic organophosphates that inhibit neuropathy target esterase. 3. Long-term toxic effects affecting behaviour as well as mental and visual functions are occasionally observed after exposure to high doses of organophosphates with repeated acute, clinically significant intoxications. 4. The available data do not indicate that asymptomatic exposure to organophosphates is connected with an increasing risk of delayed or permanent neuropsychopathological effects.

Cholinesterase Inhibitors↗
PubMed1994

Pharmacokinetics of the oximes HI 6 and HLö 7 in dogs after i.m. injection with newly developed dry/wet autoinjectors.

The rapid onset of cholinergic crisis after intoxication with highly toxic organophosphorus compounds calls for pre-clinical administration of effective antidotes as early as possible. For this purpose, i.m. administration of the antidotes by autoinjectors is desired to allow early treatment also in the absence of a physician. Besides atropine, oximes with broad antidotal spectrum are considered valuable adjuncts that should be included in antidotal mixtures. To circumvent the problem of limited stability of the new-generation oximes, dry/wet autoinjectors were developed in which the unstable solid is dissolved by a diluent in an adjacent chamber upon activation of the device. In this study the tolerance, bioavailability and pharmacokinetics of 500 mg HI 6 [1-(((4-(aminocarbonyl) pyridinio)methoxy) methyl)-2-((hydroxyimino)methyl) pyridinium dichloride monohydrate] or 200 mg HLö 7 [1-(((4-(aminocarbonyl) pyridinio)methoxy)methyl)-2,4- bis((hydroxyimino)methyl)pyridinium dimethanesulfonate] in combination with 2 mg atropine sulfate versus atropine alone, delivered by two dry/wet autoinjector types, were investigated in eight male beagle dogs (16 kg) in a complete cross-over design. The dogs tolerated the six injections with 3-week intervals without any symptoms of discomfort. Nonetheless, CPK activity increased, peaking at 6 h after injection. In contrast to atropine which merely led to a marginal increase, HI 6 plus atropine increased the baseline CPK activity about 10-fold, and HLö 7 plus atropine about 20-fold, regardless of the injector type. The HI 6 autoinjectors from Astra Tech were from an irregular production batch which did not deliver the declared HI 6 dose. The HLö 7 autoinjectors from Astra Tech and both Binaject autoinjectors from STI functioned well: the bioavailability was complete with tmax values of about 25 min as observed after conventional i.m. injection. The absorption half-time was about 8 min, elimination t1/2 about 50 min, and Vapp 0.26 l/kg. The urinary recovery of unchanged oximes was 70-80%, the renal clearance being the same as for inulin. Unexpectedly, hematocrit and hemoglobin content of blood decreased by about 15% within 2 h and reached pre-treatment values after 6-24 h. This decrease was observed with all three drug treatments and could not be accounted for by blood loss (< 4%), thus pointing to an atropine effect. In conclusion, the newly developed dry/wet autoinjectors appear suitable for the administration of atropine and an oxime stored in solid form.

Animals↗
PubMed1994

Reactions of oxidatively activated arylamines with thiols: reaction mechanisms and biologic implications. An overview.

Aromatic amines belong to a group of compounds that exert their toxic effects usually after oxidative biotransformation, primarily in the liver. In addition, aromatic amines also undergo extrahepatic activation to yield free arylaminyl radicals. The reactive intermediates are potential promutagens and procarcinogens, and responsible for target tissue toxicity. Since thiols react with these intermediates at high rates, it is of interest to know the underlying reaction mechanisms and the toxicologic implications. Phenoxyl radicals from aminophenols and aminyl radicals from phenylenediamines quickly disproportionate to quinone imines and quinone diimines. Depending on the structure, Michael addition or reduction reactions with thiols may prevail. Products of sequential oxidation/addition reactions (e.g., S-conjugates of aminophenols) are occasionally more toxic than the parent compounds because of their higher autoxidizability and their accumulation in the kidney. Even after covalent binding of quinone imines to protein SH groups, the resulting thioethers are able to autoxidize. The quinoid thioethers can then cross-link the protein by addition to neighboring nucleophiles. The reactions of nitrosoarenes with thiols yield a so-called "semimercaptal" from which various branching reactions detach, depending on substituents. Compounds with strong pi-donors, like 4-nitrosophenetol, give a resonance-stabilized N-(thiol-S-yl)-arylamine cation that may lead to bicyclic products, thioethers, and DNA adducts. Examples of toxicologic implications of the interactions of nitroso compounds with thiols are given for nitrosoimidazoles, heterocyclic nitroso compounds from protein pyrolysates, and nitrosoarenes. These data indicate that interactions of activated arylamines with thiols may not be regarded exclusively as detoxication reactions.

Amines↗
PubMed1994

Structural requirements for the ferrihemoglobin-forming activity of glutathione S-conjugates of 4-dimethylaminophenol.

4-Dimethylaminophenol (DMAP) is a suitable cyanide antidote that rapidly forms ferrihemoglobin by catalytic transfer of electrons from ferrohemoglobin to oxygen. Deleterious methemoglobinemia, because of the catalytic cycling, is prevented by side reactions of oxidized DMAP with thiols, particularly with glutathione (GSH). In human red cells, both in vitro and in vivo, the formation of a transient bis-glutathione and a stable tris-glutathione adduct was observed. To investigate the reactivity of GSH adducts of DMAP, we synthesized various thioethers by oxidizing DMAP with PbO2 in 0.1 M sulfuric acid followed by reaction with GSH. The following compounds were isolated and characterized by 1H-NMR spectroscopy and determination of the pK values: 4-dimethylamino-2-(glutathione-S-yl)-phenol (2-GS-DMAP), 4-dimethylamino-3-(glutathione-S-yl)-phenol (3-GS-DMAP), 4-dimethylamino-2,5-bis(glutathione-S-yl)-phenol (2,5-bis GS-DMAP), 4-dimethylamino-2,6-bis(glutathione-S-yl)-phenol (2,6-bis GS-DMAP), and 4-dimethylamino-2,3,6-tris(glutathione-S-yl)-phenol (2,3,6-tris GS-DMAP). Ferrihemoglobin-forming activity was investigated with oxyhemoglobin, alkylated with N-ethylmaleimide (Hb-NES) to prevent binding of oxidized compounds to the protein SH groups. DMAP, 2,6-bis-GS-DMAP, and 2-GS-DMAP (0.1 mM each) completely oxidized Hb-NES (0.6 mM) in a decreasing order of activity (pH 7.4, 37 degrees C, air); the other derivatives were quite inactive. The same thioether reactivity was observed during autoxidation. Ferrihemoglobin formation by the reactive thioethers was greatly enhanced when the oxygen tension was increased from 2 to 100%. In contrast, variation of the oxygen tension had only marginal effects on the activity of DMAP.(ABSTRACT TRUNCATED AT 250 WORDS)

Aminophenols↗
PubMed1994

Additional pathways of S-conjugate formation during the interaction of thiols with nitrosoarenes bearing pi-donating substituents.

During the well-established reaction pathways of nitrosoarenes interacting with thiols, a reactive N-(thiol-S-yl)-arylamine cation was implicated in the so-called rearrangement reaction which transforms the semimercaptal to the sulfinamide. In the case of nitrosoarenes with pi-donating substituents, this cationic transition state includes resonance structures bearing the positive charge in 2 and 4 position, thereby facilitating the attack of nucleophiles to the aromatic ring. Investigating the reaction products of 4-nitrosophenetol and reduced glutathione in chemical systems and human red cells, some glutatione S-conjugates were detected other than the already known sulfenamide and sulfinamide. Three of them were separated by HPLC and identified by mass spectroscopy, 1H-NMR, and UV-visible spectroscopy, by determination of pKa values and chemical behavior. The hitherto unknown conjugates are 4-ethoxy-2-(glutathione-S-yl)-aniline, N-(4-ethoxyphenyl)-N'-(glutathione-S-yl)-benzoquinonediimine, and 4-amino-4'-ethoxy-2-(glutathione-S-yl)-diphenylamine. In preliminary experiments, some of these conjugates were shown to be highly active in producing ferrihemoglobin. Considerations on the formation pathways of these metabolites lend further support to the electrophilic N-(glutathione-S-yl)arylamine cation as a reactive intermediate that may be implicated in nitrosoarene toxicity.

Amino Acid Sequence↗
PubMed1994

Collagen gel immobilization: a useful cell culture technique for long-term metabolic studies on human hepatocytes.

1. Primary cultures of human hepatocytes have already been employed in various applications for the study of xenobiotic metabolism. Most of these approaches were performed either on freshly isolated cells or on short-term primary cultures. Standard culture techniques do not maintain functional stability of P450 enzymes for > 1 week in vitro. 2. The aim of this study was to demonstrate the beneficial effect of an easy to apply, extracellular matrix configuration on the long-term performance of cultured human liver cells. Light microscopical examination of the cultures indicated that the cells remained viable over 1 month. As revealed by electron microscopy, hepatocytes exhibited bile canaliculi and desmosomes and were rich in mitochondria and endoplasmatic reticulum, indicating metabolic activity. 3. An early culture phase (3 days after isolation) could be described with decreasing DNA content of the cultures, peak values of alanine-amino-transferase (ALAT), and increasing albumin synthesis. After this adaptive period stable levels for DNA content and albumin synthesis were noted; ALAT returned to low values. 4. Functional activity was monitored by measurements of P450 1A1-dependent O-demethylation of p-nitroanisole to p-nitrophenol, which appeared to be constant over 3 weeks and weakly inducible by 1 mM phenobarbital. Another set-up examined conjugation of acetaminophen at subtoxic concentrations: acetaminophen was metabolized to its glucuronide and sulphate; 3-(glutathione-S-yl)-acetaminophen was not detected. Almost identical metabolism was found, comparing day 3 with 16 of culture. 5. We concluded that collagen gel immobilization not only provides mechanical support to cultured hepatocytes, but also supports long-term differentiated function of the cells for metabolic studies.

Acetaminophen↗
PubMed1993

Effects of the phenacetin metabolite 4-nitrosophenetol on glycolysis and pentose phosphate pathway in human red cells.

Human erythrocytes exposed to 4-nitrosophenetol showed marked alterations of their endogenous metabolism. Rapid ferrihemoglobin formation mediated by the NADPH-dependent enzymic cycling of the nitrosoarene ("Kiese cycle") and extensive GSSG production caused an immediate drain of G-6-P into the pentose phosphate pathway at maximal flow. Despite a 2.4-fold increase in glucose phosphorylation rate and a branching ratio of 97:3 between pentose phosphate pathway and Embden-Meyerhof pathway, the G-6-P supply was obviously insufficient to meet the immense NADPH demand. Thus, a significant recycling of pentose phosphate pathway-derived F-6-P was observed in the order of 65%. Comparison of NADPH regeneration and ferrihemoglobin formation indicates the "Kiese cycle" to be a minor mechanism in ferrihemoglobin production in the case of high 4-nitrosophenetol concentrations. Most probably, reactive intermediates of 4-nitrosophenetol other than N-hydroxy-4-phenetidine, i.e. bicyclic arylamines and glutathione S-conjugates are formed which produce ferrihemoglobin without involvement of NADPH. The experiments have shown that red cells are remarkable robust to tackle the massive oxidative stress as elicited by 4-nitrosophenetol. The immediate metabolic response of the pentose phosphate pathway allows rapid regeneration of reduced glutathione. Thereby, SH-containing enzymes are effectively protected and/or regenerated and hemolysis is kept minimal. Hence, red cells are favourably suited for clearing the blood from N-oxygenated arylamines before they can reach more sensitive target organs.

Adenosine Triphosphate↗
PubMed1993

Effects of the phenacetin metabolite 4-nitrosophenetol on the glutathione status and the transport of glutathione S-conjugates in human red cells.

The extent of ferrihemoglobin formation in human erythrocytes by 4-nitrosophenetol and its metabolisation rate strongly depended on the availability of cellular GSH. Ferrihemoglobin formation rate was increased by inhibition of the red cell glutathione reductase, and 4-nitrosophenetol disappeared more slowly. When red cells were completely depleted from SH groups, ferrihemoglobin formation was retarded, despite 4-nitrosophenetol was hardly metabolized. In turn, the glutathione status of human red cells was strongly affected by 4-nitrosophenetol. GSSG, which was produced in large amounts, was reduced, as long as the reducing system was intact. The decreased total glutathione content, however, did not recover completely, indicating formation of stable glutathione S-conjugates. The active export of the stable model glutathione thioether S-(2,4-dinitrophenyl)glutathione was strongly inhibited by 4-nitrosophenetol. A Lineweaver-Burk plot of the transport data suggested a competitive inhibition mechanism, presumably caused by glutathione adducts. The results indicate that the strong pi-donor substituent in 4-nitrosophenetol enables metabolic reactions with glutathione, producing biological effects hitherto not observed with nitrosobenzene. Bicyclic arylamines and glutathione S-conjugates may cause ferrihemoglobin formation that is not brought about by the diaphorase reaction. The latter may be responsible for transport inhibition of GSSG and other glutathione S-conjugates.

Biological Transport↗