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Aim: Inflammatory algesia and pyresia are common pathological consequences of physiological defense. Phenacetin introduced as effective analgesic anti-pyretic agent, was proscribed from therapeutic use because of associated systemic toxicity. The aim of the study was to evaluate the potency of 1,2,3-triazole conjugation in reducing toxicity and increasing efficacy of the phenacetin nucleus., Main Methods: The amide bond implicated as the cause of phenacetin toxicity was bioisosterically replaced with 1,2,3-triazoles to yield a series of PhTCs(PhTC1, PhTC2 and PhTC3). The toxicology of the synthesized conjugates in reference to phenacetin was evaluated in accordance with OECD test guidelines 420, 425 and 407. For the purpose of evaluating anti-inflammatory potency carrageenan induced paw edema and croton oil induced ear edema models were evaluated. Anti-nociceptive efficacy was assessed using Eddy's hot plate and acetic acid induced writhing experimental models. For anti-pyretic efficacy, the conjugates were submitted to Brewer's yeast antipyretic assay., Key Findings: Toxicological examination of PhTCs in comparison to phenacetin revealed that, phenacetin treatment caused considerable nephrotoxicity and hepatotoxicity in experimental models PhTCs were devoid of such toxic manifestations. Results of pharmacological assays showed that the entire series of PhTCs possessed better anti-inflammatory, anti-nociceptive and anti-pyretic potential than phenacetin. Furthermore it was revealed that the pharmacological profile of PhTC1 with triazole substitution at para position of the phenol ring exhibited potency even better than that exhibited by the reference standards., Conclusion: Bioisosteric replacement of amide bond by 1,2,3-triazole in the phenacetin moiety yields conjugates with superior efficacy and diminished toxicity, thus opening neo avenues in treatment of inflammatory syndromes., (Copyright © 2019. Published by Elsevier Inc.)

Human cytochrome P450 (P450) 1A2 is involved in the oxidation of many important drugs and carcinogens. The prototype substrate phenacetin is oxidized to an acetol as well as the O-dealkylation product [Yun, C.-H., Miller, G. P., and Guengerich, F. P. (2000) Biochemistry 39, 11319-11329]. In an effort to improve rates of catalysis of P450 1A2 enzymes, we considered a set of p-alkoxyacylanilide analogues of phenacetin and found that variations in the O-alkyl and N-acyl substituents altered the rates of the two oxidation reactions and the ratio of acetol/phenol products. Moving one methylene group of phenacetin from the O-alkyl group to the N-acyl moiety increased rates of both oxidations approximately 5-fold and improved the coupling efficiency (oxidation products formed/NADPH consumed) from 6% to 38%. Noncompetitive kinetic deuterium isotope effects of 2-3 were measured for all O-dealkylation reactions examined with wild-type P450 1A2 and the E225I mutant, which has 6-fold higher activity. A trend of decreasing kinetic deuterium isotope effect for E225I > wild-type > mutant D320A was observed for O-demethylation of p-methoxyacetanilide, which follows the trend for k(cat). The set of O-dealkylation and acetol formation results for wild-type P450 1A2 and the E225I mutant with several of the protiated and deuterated substrates were fit to a model developed for the basic catalytic cycle and a set of microscopic rate constants in which the only variable was the rate of product formation (substrate oxygenation, including hydrogen abstraction). In this model, k(cat) is considerably less than any of the microscopic rate constants and is affected by several individual rate constants, including the rate of formation of the oxygenating species, the rate of substrate oxidation by the oxygenating species, and the rates of generation of reduced oxygen species (H(2)O(2), H(2)O). This analysis of the effects of the individual rate constants provides a framework for consideration of other P450 reactions and rate-limiting steps.

1. The hydroxamic acids N-hydroxyphenacetin and N-hydroxy-2-acetylaminofluorene were reduced to the corresponding amides, phenacetin and 2-acetylaminofluorene respectively by rabbit blood supplemented with both NAD(P)H and FAD. These reducing activities were found in erythrocytes but not in plasma, and were sensitive to inhibition by carbon monoxide and oxygen. When blood or erythrocytes were boiled, these activities were not abolished. 2. Haemoproteins such as haemoglobin and catalase exhibited the reductase activity in the presence of both NAD(P)H and FAD under anaerobic conditions. The activity was not abolished when the haemoproteins were boiled. 3. Haematin showed a significant reducing activity in the presence of these cofactors. The activity of haematin was also observed with the photochemically reduced form of FAD. 4. The reduction system in blood was composed of NAD(P)H, FAD and haemoglobin. Reduction appears to proceed in two steps, i.e. the reduction of FAD by NADH or NADPH, followed by the non-enzymatic reduction of the hydroxamic acids to the amides by reduced FAD, catalyzed by the haem group of haemoglobin in rabbit erythrocytes.

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.

Liver aldehyde oxidase (EC 1.2.3.1) was capable of reducing N-arylacetohydroxamic acids, N-hydroxy-2-acetyl-aminofluorene, N-hydroxy-4-acetylaminobiphenyl and N-hydroxyphenacetin, to the corresponding amides in the presence of an electron donor of the enzyme under anaerobic conditions. When supplemented with an electron donor of the enzyme, a significant reduction of N-hydroxy-2-acetylaminofluorene occurred, which was sensitive to an inhibitor of the enzyme. These observations were made with cytosolic fractions prepared from the livers of rabbits, guinea pigs, rats and mice.

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.

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.

1. The analgesic compound phenacetin and its O-alkyl homologues were metabolized by Cunninghamella elegans to yield the O-dealkylation product paracetamol (acetaminophen), and metabolites resulting from omega-1 hydroxylation and further oxidations. 2. Structural identification was based upon physical, spectral and chromatographic comparisons of isolated metabolites with synthetic standards generated by alkylation of paracetamol with the appropriate alkyl halide, epoxide, or alpha,beta-unsaturated ketone. 3. The rank order of O-dealkylation within the homologous series based upon either substrate disappearance or phenol formation was found to be ethyl greater than isopropyl greater than n-propyl greater than n-butyl greater than methyl.

Phenacetin was mutagenic in Salmonella typhimurium TA100 in plate assays when liver fractions from Aroclor-treated hamsters, but not rats, were used. Its known or putative metabolites were synthesized; of these, N-hydroxyphenacetin and N-acetoxyphenacetin were found to be mutagenic in liquid and plate assays, both requiring activation by liver fractions from Aroclor-treated hamsters. 2-Hydroxyphenacetin and 2-acetoxyphenacetin were nonmutagenic. N-Hydroxyphenetidine (the deacetylated metabolite of phenacetin) and p-nitrosophenetole were the only products that were found to be mutagenic per se when assayed under N2 in either Salmonella TA100 and TA100 NR (nitroreductase-deficient) strains. Phenacetin was administered to male BDVI rats and Syrian golden hamsters, and its urinary metabolites were deconjugated with beta-glucuronidase:arylsulfatase. After reactivation by hamsters liver fractions, mutagenicity was demonstrated in S. typhimurium TA100 with urine from phenacetin-treated hamsters, but not with that from rats. After treatment with deconjugating enzymes, N-hydroxyphenacetin was isolated from hamster urine by high-performance liquid chromatography and identified by mass spectral analysis. The data support the conclusions that (a) N-hydroxyphenacetin is a proximate mutagenic metabolite of phenacetin which, after N-deacylation, is responsible for the mutagenicity observed in vitro and in the urine of hamsters and (b) the higher yield of N-hydroxyphenacetin that is formed in the liver of hamsters as compared to rats explains the pronounced species-specific activation of phenacetin into bacterial mutagens.

Specific antibody producing human cell lines were established by preselecting antigen binding B-lymphocytes and subsequently transforming ("immortalizing") them with Epstein-Barr virus (EBV). NNP-binding B-cells were isolated from the blood of three donors with high anti-NNP titers. EBV-transformation led to polyclonal cell lines that had 15-18% NNP receptor positive cells. A similar fraction of the cells produced NNP-specific plaques in the Cunningham-Szenberg assay. Unconcentrated culture media agglutinated NNP-coupled erythrocytes in up to an 1/2,048 dilution and inactivated NNP-coupled T4 bacteriophage in up to an 1/10,000 dilution. EBV-transformed but not similarly NNP-preselected lines of the same donors were completely negative in the rosette, plaque and antibody secretion tests. Supernatants of the antibody producing lines gave no reaction with the non-coupled erythrocytes or with a number of other hapten coupled controls. Anti-NNP antibodies secreted by all three preselected lines were of the IgM kappa type, which was in contrast to the sera of the donors that contained both IgM and IgG antibodies, with both light chain types. Cloning of one NNP-antibody producing line yielded 9 antibody producers out of 30. The positive clones formed NNP rosettes and plaques with 31-86% of the cells and produced anti-NNP of class IgM, Kappa. The cell culture contained 5-16 micrograms IgM/ml cell culture.

Derivatives of both N-hydroxy-2-acetylaminofluorene (N-OH-AAF) and N-hydroxyphenacetin (N-OH-P) were tested for their ability to cause DNA damage in Reuber (H4-II-E) cells using the alkaline elution technique. Reuber cells are devoid of N-OH-AAF deacylase, N,O-acyltransferase, and sulfotransferase activities. The hydroxamic acids themselves caused very little DNA damage, while N-hydroxy-2-aminofluorine (20 to 100 microM), N-hydroxyphenetidine (20 to 200 microM), and p-nitrosophenetole (10 to 100 microM) all caused dose-dependent damage. The dose-dependent DNA damage caused by N-acetoxy-2-acetylaminofluorene (5 to 25 microM) was completely inhibited by the deacylase inhibitor paraoxon (100 microM). In the presence of both partially purified rabbit liver cytosolic N,O-acyltransferase and guinea pig liver microsomal deacylase, N-OH-AAF was genotoxic. Neither paraoxon nor tRNA had any effect on the DNA damage induced by N-OH-AAF in the presence of N,O-acyltransferase, while paraoxon completely inhibited the damage when N-OH-AAF was incubated in the presence of guinea pig deacylase, and N-OH-P only caused slight DNA damage at higher concentrations of enzyme. In addition, partially purified guinea pig liver deacylase and N-OH-AAF (25 microM) caused 2600 revertants in the Salmonella test system, while only 380 revertants were seen with a 40-fold greater concentration of N-OH-P (1000 microM). The mutagenicity of both N-OH-AAF and N-OH-P was completely inhibited by paraoxon. Thus, it is clear that metabolites of N-OH-AAF formed outside the cell are capable of passing both the cellular and nuclear membranes to cause genotoxicity. Metabolic activation of N-OH-AAF by either the membrane-bound deacylase or the cytosolic N,O-acyltransferase caused genotoxicity via a deacetylation process. Metabolic activation of N-OH-P by guinea pig deacylase caused low levels of DNA damage, whereas activation by N,O-acyltransferase was not sufficient to cause genotoxicity.

Deacetylation of a homologous series of alkoxy acetanilides (p-methoxy-, p-ethoxy- (phenacetin), p-(n)-propoxy- and p-(n)-butoxyacetanilide) and three of the corresponding N-hydroxy derivatives was examined in microsomal fractions from the livers and kidneys of C57BL/6J mice. The rates of deacetylation of the phenacetin analogs to the corresponding amines were found to increase with increasing alkyl chain length. With the N-hydroxy derivatives, the apparent KM was found to decrease with increasing chain length, while the Vmax was relatively unaffected. Treatment of the microsomes with the esterase inhibitor bis-p-nitrophenylphosphate resulted in quite similar extents of inhibition of the deacetylation of the phenacetin analogs and their N-hydroxy derivatives.

N-hydroxyphenacetin, a phenacetin metabolite, was fed to rats as a 0.05-0.5% dietary supplement. After 9 months, tumours of the liver were found in 36 of 64 animals. One animal also developed a renal tumour. No tumours were found in control animals. The findings implicate phenacetin as a carcinogen and suggest that N-hydroxyphenacetin may be the metabolite responsible.

The mechanism-based inactivation (suicide inactivation) by N-hydroxyphenacetin (NHP) of N-arylhydroxamic acid N,O-acyltransferase (AHAT) and p-aminobenzoic acid N-acetyltransferase (PABA NAT) activities of a partially purified hamster liver preparation was investigated. The inactivation of both enzyme activities was irreversible, but a partial protection of PABA NAT could be achieved by inclusion of the nucleophile cysteine in the incubation mixture; cysteine did not reduce the extent of inactivation of AHAT by NHP. Hepatic AHAT and PABA NAT activities were separated by affinity chromatography, and the resolved enzyme activities were subjected to incubation in the presence of NHP, N-hydroxy-2-acetamidofluorene (N-OH-AAF), and N-hydroxy-4-acetamidobiphenyl (N-OH-AABP); AHAT, but not PABA NAT, was inactivated by NHP, N-OH-AAF and N-OH-AABP. Incubation of hamster heptic PABA NAT with radiolabeled N-OH-AAF resulted in the formation of only 15% as much fluorenylamine-tRNA adduct as was formed when N-OH-AAF was bioactivated with hamster hepatic AHAT. Hamster intestinal AHAT and PABA NAT activities also were resolved by affinity chromatography; the intestinal AHAT fractions were much more effective than the PABA NAT fractions in bioactivating N-OH-AAF. These results demonstrate that hamster liver and intestine contain at least two arylamine transacetylating activities, one of which is much more effective than the other in the bioactivation of toxic and carcinogenic N-arylhydroxamic acids.

This work describes a method for the quantitative determination of the labile, toxic N-hydroxy metabolite of phenacetin in urine. A thin-layer chromatography step was used for the preliminary purification of extracts, and the specificity of the assay was based on the monitoring of specific metastable decompositions in a forward geometry double-focussing mass spectrometer, in a manner analogous to conventional tandem mass spectrometry. This precluded the need for a gas chromatographic separation, thus minimizing thermal decomposition which can occur with these compounds, as well as enabling very rapid analyses.

The N-methyl analogs of p-hydroxyacetanilide (acetaminophen) and p-ethoxyacetanilide (phenacetin) were prepared and tested for toxicity. N-Methylacetaminophen was found to cause no hepatic necrosis in mice, rats, or hamsters in doses that caused massive hepatic necrosis in the same animals when acetaminophen was administered. Neither acetaminophen nor its N-methylated analog caused methemoglobinemia at these dose levels. Fischer rats that were administered large doses of acetaminophen (900 mg/kg s.c.) sustained necrosis in the proximal renal tubules, whereas N-methylacetaminophen caused no renal injury at higher dose levels (1800 mg/kg s.c.). N-Methylphenacetin caused no observable hepatic necrosis in 3-methylcholanthrene (3-MC) pretreated hamsters at dose levels higher than those in which phenacetin caused hepatic necrosis. Also, in contrast to phenacetin, N-methylphenacetin did not cause extensive methemoglobinemia in mice, rats, or hamsters.

Deacylation has been proposed as a mechanism of activation of arylhydroxamic acids. In the present studies solubilized preparations from guinea pig liver microsomes, a source of high deacylase activity, were subjected to gel filtration on Sephacryl S-200. A single peak (peak I) of activity was found when column fractions were assayed colorimetrically for deacylation of N-hydroxy-2-acetylaminofluorene (N-OH-AAF). Corresponding to this peak were the following activities: binding of [3H-ring]-N-hydroxy-phenacetin (N-OH-P) to tRNA and deacylation of N-OH-P and N-OH-AAF, measured by the formation of nitrosophenetole (N = O-P) and nitrosofluorene (N = O-F), respectively. The binding of [3H-ring]-N-OH-AAF to tRNA was catalyzed by peak I, but to a greater extent by a second peak (II). The binding of both N-OH-P and N-OH-AAF to tRNA was inhibited by paraoxon, an esterase inhibitor. H.p.l.c. analysis revealed that for peak I, the major ether-extractable metabolites of N-OH-P and N-OH-AAF were the corresponding nitroso derivatives. In the presence of peak II, little metabolism to organic-extractable metabolites occurred. These data indicate that more than one mechanism is involved in the activation of N-OH-P and N-OH-AAF in this system, and that the difference in the activation of these arylhydroxamic acids cannot be explained by differences in the formation of deacylated metabolites.

The metabolism of phenacetin to reactive intermediates in humans was estimated from the excretion of thio adducts in urine. N-Hydroxyphenacetin, a precursor of reactive metabolites, was also quantified. Following an oral dose of phenacetin (10 mg/kg) to humans, these metabolites in 24 h urine were: paracetamol-3-cysteine, 4.4% dose; paracetamol-3-mercapturate, 3.9%; 3-thiomethylparacetamol, 0.4%; N-hydroxyphenacetin, 0.5%. Rats showed a considerable increase in N-hydroxyphenacetin excretion after chronic dosing with phenacetin at high dosage (500 mg/kg) for one month. chronic dosing with a low dose (50 mg/kg) did not increase N-hydroxyphenacetin excretion, but a marked increase occurred on concomitant administration of aspirin and caffeine.

1. A comparative study of the metabolism of [acetyl-14C]phenacetin, [acetyl-14C]methacetin, [acetyl-14C]paracetamol and [acetyl=14C]acetanilide in the rat is reported. 2. The extent of N-deacetylation, evidenced by the measurement of respired 14CO2, varied, being greatest with acetanilide (25-31%) and least with paracetamol (6%). 3. The major urinary metabolites in each case were N-acetyl-p-aminophenyl sulphate and N-acetyl-p-aminophenyl glucuronide; the relative proportions varied with the sex of the animals and as a result of extended dosage. 4. The metabolism of [ethyl-14C]phenacetin and [ethyl-14C]phenetidine was investigated and the extent of O-dealkylation determined by measurement of respired 14CO2. 5. The metabolic pathways of some related glycolanilides and oxanilic acids included N-deacylation, and in the glycolanilides, oxidation of the glycollic group.

Addition of p-[3H]nitrosophenetole to protein incubations resulted in quantitative binding of radiolabel to protein. Preincubation of protein with N-methylmaleimide to derivatize sulfhydryl groups followed by addition of p-[3H]nitrosophenetole resulted in a 90% decrease in covalent binding. Treatment of the p-[3H]nitrosophenetole protein-bound residue, after extensive solvent washing, with HCl decreased binding by 72% and the corresponding amine, p-phenetidine was detected in the aqueous phase. Inasmuch as incubation of the bound residue with reduced glutathione did not alter protein binding appreciably the involvement of a sulfinanilide S-oxide is suggested. Incubation of p-[3H]phenetidine with microsomal incubation mixtures resulted in NADPH-dependent covalent binding to protein. Treatment of the p-[3H]phenetidine protein-bound residue after extensive solvent washing with HCl decreased binding by 64%. Administration of either p-[3H]phenetidine (500 mg/kg i.p.) or [14C]phenacetin (500 mg/kg i.p.) to mice resulted in covalent binding of radiolabel to protein of liver, lung, kidney, small intestine and blood. Incubation of solvent-washed protein with acid resulted in an approximately 35% decrease in protein binding in lung and blood of both treatment groups.

The mutagenicity of N-hydroxy-2-acetylaminofluorene and N-hydroxyphenacetin and their respective deacetylated metabolites, N-hydroxy-2-aminofluorene and 2-nitrosofluorene, and N-hydroxyphenetidine and rho-nitrosophenetole was determined in nitroreductase deficient Salmonella tester strains TA 98FR and TA100FR. The mutagenicity of N-hydroxy-2-acetylaminofluorene medicated by either rat liver microsomes or rat liver 105 000 g supernatant fractions was no different in either TA98 (nitroreductase proficient) or TA98FR (nitroreductase deficient). Similarly the mutagenicity of N-hydroxyphenacetin mediated by hamster liver microsomes was not affected by either the presence or absence of nitroreductase activity in TA100. N-Hydroxy-2-aminofluorene and 2-nitrosofluorene were equipotent direct acting mutagens in both TA98 and TA98FR, as were both N-hydroxyphenetidine and rho-nitrosophenetole in TA100 and TA 100FR. Ascorbate (5 mM) and NADPH (1 mM) had no significant effect on the mutagenicity of either N-hydroxy-2-acetylaminofluorene, N-hydroxy-2-aminofluorene, or 2-nitrosofluorene in TA98 or TA98FR whereas ascorbate and NADPH markedly inhibited the mutagenicity of both N-hydroxyphenetidine and rho-nitrosophenetole in both TA100 and TA100FR. Ascorbate appears to be inhibiting the mutagenicity of N-hydroxyphenetidine and rho-nitrosophenetole as a result of the nonenzymatic chemical reduction of these compounds to non-mutagenic derivatives.

Bio-monitoring the covalent binding of nitrosoarenes to the SH groups of human hemoglobin has been proposed as a reliable approach to get an integral parameter for exposure control and possibly risk assessment of persons exposed to aromatic amines and nitro compounds. Availability of nitrosoarenes to bind to the cysteine residues is greatly influenced by the competition of hemoglobin iron with nitrosoarenes. In contrast to earlier reports, we found that nitrosobenzene has a 14 fold higher affinity for "stripped" human hemoglobin than oxygen. The binding mode is similar to gaseous ligands and exhibits the same free energy of cooperation and sensitivity to heterotropic effectors like inositol hexaphosphate. To elucidate the electronic influence of para substituents, 4-chloronitrosobenzene, 4-nitrosotoluene and 4-nitrosophenetole were tested. A linear free energy relationship was found for all equilibrium parameters with a reaction constant rho = 3, when using Hammett sigma p constants. Similarly, the apparent second order rate constants for binding of para-substituted nitrosobenzenes to the cysteine residues (Cys beta 93) in hemoglobin followed the Hammett relationship with lg k-lg k0 = 1.7 X sigma p (r2 = 0.99). In case of 4-chloronitrosobenzene covalent binding proceeded biphasically and a "semimercaptal"-like intermediate was observed. The affinities for hemoglobin iron and for the SH groups were highest with 4-chloronitrosobenzene and lowest with 4-nitrosophenetole. All nitrosobenzenes were capable to produce ferrihemoglobin. In the absence of oxygen, 4-chloronitrosobenzene hemoglobin decayed with formation of ferrihemoglobin. Presumably the nitroxide radical anion is formed as an intermediate which comproportionates into the azoxy derivative. It is assumed that the efficiency of the microscopic compartmentation of nitrosoarenes by binding to hemoglobin iron has important impacts on the toxicokinetics of these compounds.

N-Hydroxy-2-acetylaminofluorene has been found to be an effective inhibitor of aldehyde oxidase. At concentrations of 1 X 10(-6) M and 1 X 10(-5) M, 38% and 88% inhibition was observed on the oxidase activity towards N1-methylnicotinamide. The inhibition was of noncompetitive type and had a Ki value of 4.4 X 10(-6) M. In contrast, little inhibition of the enzyme was observed with 2-aminofluorene, 2-acetylaminofluorene and acetohydroxamic acid even at a concentration of 1 X 10(-4) M.

N-Hydroxyphenacetin has been found in the urine of rats dosed with phenacetin, extending previous reports that phenacetin is N-hydroxylated by liver microsomes in vitro. After an oral dose of phenacetin (500 mg/kg) urine was collected for 24 hr, conjugates hydrolyzed with extract of Helix pomatia, and the metabolites extracted with dichloromethane and treated with diazomethane. Methylation of N-hydroxyphenacetin produced a stable derivative, N-methoxyphenacetin, which was separated from most other metabolites by thin layer chromatography. Identification of N-methoxyphenacetin was by combined gas chromatography-mass spectrometry and comparison with the synthetic reference compound. Quantification by gas chromatography with flame-ionization detection showed that 0.023% of the dose phenacetin was recovered from urine as N-hydroxyphenacetin. It is probable that this value considerably underestimates the extent of phenacetin N-hydroxylation in vivo, inasmuch as N-hydroxyphenacetin is known to be rapidly degraded in biological systems.

(1) Hemoglobin-free single-pass perfusion of isolated rat livers was carried out with various concentrations of 4-nitrosophenetol (NOPt). (2) NOPt, up to 2 mumol/min/g liver wet wt., was reduced by the liver with formation of N-hydroxy-4-phenetidine (NHOHPt), 4-phenetidine (NH2Pt), phenacetin and polar metabolites. (3) Three per cent of NOPt was irreversibly bound to liver proteins after a load of 20 mumol/g liver wet wt. After 30 min perfusion, 0.2 mumol of liver glutathione was lost by 1 mumol NOPt infused. (4) Bile flow and glutathione release by the bile decreased rapidly during NOPt perfusion. (5) Glutathione release of the livers into venous effluent was diminished by NOPt and was stimulated only slightly by t-butylhydroperoxide (BOOH). Because BOOH reduction and glutathione peroxidase were not altered and intracellular glutathione disulfide (GSSG) levels were elevated, inhibition of the GSSG excretory mechanism is assumed.

The effects of N-hydroxyphenacetin on DNA function and structure were investigated to elucidate the involvement of phenacetin in analgesic nephropathy and transitional cell carcinoma. N-Hydroxyphenacetin or a metabolite inhibited synthesis of DNA, RNA and protein; DNA inhibition was greater at higher pH. No single-strand breaks were detectable in DNA after N-hydroxyphenacetin treatment and no appreciable effect on cell viability was observed at concentrations up to 5 mM. N-Hydroxyphenacetin-induced alteration to chromatin structure was detected using nucleoid sedimentation analysis. Direct binding to plasmid DNA was not observed. These observations are consistent with a role for phenacetin metabolites in renal disease.

Phenacetin, a constituent of several analgesic and antipyretic formulations has been made responsible for a variety of toxic and carcinogenic actions. 4-Nitrosophenetol, the N-oxydation product of intermediate 4-phenetidine, forms methemoglobin and binds covalently to sulfhydryl groups of proteins and glutathione. In the reaction of 4-nitrosophenetol with glutathione and other thiols an intermediate so-called "semimercaptal" is formed from which N-(thiol-S-yl)-4-phenetidine S-oxide, N-(thiol-S-yl)-4-phenetidine and 4-phenetidine derive. Besides thiol adducts, a yellow compound is formed which was isolated as a pure crystalline product (elemental analysis) and identified by FAB-MS, EI-MS, 13C-, 1H-NMR, and UV-VIS spectroscopy as 4-ethoxy-4'-nitrosodiphenylamine. This nitrosoarene is formed by an unknown mechanism from 4-nitrosophenetol and 4-phenetidine under liberation of ethanol. In human erythrocytes this compound is easily reduced to 4-amino-4'-ethoxydiphenylamine (FAB-MS, EI-MS, 13C-NMR). During the reaction of 4-nitrosophenetol with red cells only traces of 4-ethoxy-4'-nitrosodiphenylamine were formed, whereas up to 10% appeared as the reduction product 4-amino-4'-ethoxydiphenylamine. This latter compound is unstable in red cells and is metabolized further to unidentified products.

Two mutagenic metabolites of phenacetin, p-nitrosophenetol and N-hydroxy-p-phenetidine, were tested in S. typhimurium strains TA100, its nitroreductase-deficient derivative TA100NR, and O-acetylase-deficient strains TA100 Tn5-1,8-DNP1011 and -DNP1012 in the presence or absence of an exogenous metabolic activation system. The results indicate that bacterial nitroreductase(s) and O-acetylase(s), shown to be involved in the conversion of certain nitroarenes, are not required for the intrabacterial activation of the two phenacetin metabolites to bacterial mutagens. In view of the low reactivity of nitrosoarenes towards nucleophiles at neutrality, the mechanism by which they exert such a high mutagenic effect in S. typhimurium strains remains to be clarified, but is discussed.

N-Acetyl-p-benzoquinone imine (4) can be detected by UV spectrophotometry and HPLC during the hydrolysis of N-(pivaloyloxy)phenacetin (3c). This material serves as a model for the sulfate and glucuronide conjugates (3a and 3b) of N-hydroxyphenacetin (2). Direct detection of 4 during the hydrolysis of 3a and 3b is precluded by the extreme instability of 3a and the very slow hydrolysis of 3b. Our data show that greater than 90% of the hydrolysis of 3c proceeds through the intermediate 4 at all pH values in the range 1.0-8.0. All our results indicate that 4 is produced through a nitrenium ion mechanism. Many of the differences in the hydrolysis reactions of 3b and 3c can be explained in terms of a mechanism involving tight and solvent-separated nitrenium ion pairs. Other differences may be due to a homolysis pathway, which is more important for the material with the poorer leaving group (3b).

N-Hydroxyphenacetin (100 mg/kg) injected i.p. into rats rapidly appeared in the blood and disappeared with a t1/2 of 14 min; phenacetin and 4-acetamidophenol were major metabolites in blood. Ferrihaemoglobin was formed, but 4-nitrosophenetole was not detected in blood. N-Hydroxyphenacetin injected i.p. into rats was excreted in the urine unchanged (partly conjugated 2.1% of the dose, 2% was excreted as phenacetin, 19% as 4-acetamidophenol) and 1.8% as 2-hydroxyphenacetin. In addition, small amounts of 3-hydroxyphenacetin (0.4%) and traces of N-[4-(2-hydroxyethoxy)phenyl]acetamide (beta-HAP) (0.05%) were found. Time-course kinetics have shown that N-hydroxyphenacetin is metabolized in vitro to phenacetin, 2- and 3-hydroxyphenacetin, and 4-acetamidophenol by microsomal and cytosolic preparations of rat and rabbit liver. However, after the initial reaction, the formation of phenacetin and 2- and 3-hydroxyphenacetin did not continue with time, indicating that these products were not formed enzymically. N-Hydroxyphenacetin incubated with rat erythrocytes formed ferrihaemoglobin; the relationship between ferrihaemoglobin, phenacetin and 4-nitrosophenetole concn indicated that N-hydroxyphenacetin was oxidized by oxyhaemoglobin to acetyl 4-ethoxyphenyl nitroxide, which yielded phenacetin and 4-nitrosophenetole spontaneously.

The direct-acting mutagens, N-hydroxy-p-phenetidine and p-nitrosophenetole, are known to be metabolites of the analgesic phenacetin and may be responsible for its carcinogenic activity. In this study, the potential detoxification of these metabolites by glutathione was examined. Glutathione reacted rapidly with p-nitrosophenetole, which was quantitatively converted to a single product as determined by high-pressure liquid chromatography. The analysis of the product by fast atom bombardment mass spectrometry and 500-MHz 1H-NMR spectroscopy established its structure as N-(glutathion-S-yl)-p-phenetidine. The same glutathione conjugate was also formed when N-hydroxy-p-phenetidine was incubated with glutathione. However, since conjugate formation from N-hydroxy-p-phenetidine occurred slowly and was decreased in the presence of an argon atmosphere as well as by higher levels of glutathione, it was concluded that the conjugate resulted from oxidation of the N-hydroxy arylamine to the nitrosoarene, which subsequently reacted with glutathione. N-(Glutathion-S-yl)-p-phenetidine was semistable in water (half-life, 6-7 hr) and very unstable in the presence of nucleophiles such as 10 mM glutathione (half-life, 7 min), quantitatively decomposing to p-phenetidine. The conjugate was also very unstable in acidic buffers (half-life, 17 min, pH 5). Radiolabeled N-hydroxy-p-phenetidine, but not p-nitrosophenetole, was shown to bind covalently to calf thymus DNA in vitro, and 4 times more binding was detected at pH 5 than at pH 7. Glutathione did not significantly decrease binding of the N-hydroxy derivative at either pH, nor did purified ring-radiolabeled N-(glutathion-S-yl)-p-phenetidine significantly bind to DNA at either pH. Thus, we hypothesize that an important detoxification pathway for phenacetin in vivo could involve the facile oxidation of N-hydroxy-p-phenetidine to p-nitrosophenetole, which then reacts rapidly with glutathione to form an excretable conjugate.

N-Hydroxyphenacetin was activated to a mutagen in the Salmonella-Ames test by rabbit liver acyltransferase, rat liver cytosol, and rat liver microsomes. N-[ring]3H]-Hydroxyphenacetin was bound to transfer RNA when activated by acyltransferase from rabbit or rat liver or rat liver microsomes. The acyltransferase-catalyzed binding was not inhibited by paraoxon, a deacetylase inhibitor. The use of N-hydroxyphenacetin radioactively labeled in the acetyl group, as well as the ring, indicated that deacetylation was involved in the microsome-catalyzed binding reaction. In addition, the microsome-catalyzed binding was inhibited 90% by paraoxon. p-Nitrosophenetole, a deacetylated derivative of N-hydroxyphenacetin, was synthesized and bound to transfer RNA without enzymatic activation. Activation of N-hydroxyphenacetin by sulfate conjugation was also found to lead to binding to transfer RNA. The data implicated acyl transfer, deacetylation, and sulfate conjugation as possible routes for the activation of N-hydroxyphenacetin.