N-hydroxy-N-arylacetamides--IV. Differences in the mechanism of haemoglobin oxidation in vitro between N-hydroxy-N-arylacetamides and arylhydroxylamines.

In solutions of purified human haemoglobin N-hydroxy-4-chloroacetanilide (N-hydroxy-4ClAA] was one of the most active compounds and N-hydroxy-acetanilide (N-hydroxy-AA) was the least active compound among the six N-hydroxy-N-arylacetamides tested for ferrihaemoglobin (HbFe3+)-forming activity. Co-oxidation of haemoglobin by N-hydroxy-4-chloroacetanilide was compared with that of N-hydroxy-4-chloroaniline(N-hydroxy-4ClA) and found to differ in the kinetics of HbFe2+-oxidation, in the catalytic activity of the two compounds, in the activation energy, and in the product pattern, indicating that the mechanism by which N-hydroxy-N-arylacetamides oxidize oxyhaemoglobin in vitro is different from that of arylhydroxylamines. Attempts have failed to detect by EPR spectroscopy acetyl 4-chlorophenyl nitroxide radical, the postulated catalytically-active oxidation product of N-hydroxy-4-chloroacetanilide.

N-hydroxy-N-arylacetamides. III: Mechanism of haemoglobin oxidation by N-hydroxy-4-chloroacetanilide in erythrocytes in vitro.

N-Hydroxy-4-chloroacetanilide(N-hydroxy-4C1AA) was the most active, and N-hydroxy-2-acetylaminofluorene(N-hydroxy-2AAF) the least active compound among six N-hydroxy-N-arylacetamides, in forming ferrihaemoglobin(HbFe3+) in bovine erythrocytes in the presence of 11 mM glucose. N-Hydroxy-4C1AA oxidized 25 equiv. of HbFe2+, both in the presence and absence of glucose or lactate. Therefore, its catalytic properties did not depend on metabolic regeneration by the NADPH- or NADH-dependent erythrocyte reductases. In contrast, N-hydroxy-4-chloroaniline(N-hydroxy-4C1A) oxidized 760 equiv. of HbFe2+ in the presence of glucose, but only 81 equiv. of HbFe2+ in the presence of lactate. These results indicate that the catalytic activity depended on the metabolic regeneration from 4-chloronitrosobenzene(4-C1NOB) by NADPH-dependent erythrocyte reductases. A relationship was established between HbFe3+ concn. and the concn. of N-hydroxy-4C1A and 4-C1NOB(determined together), 4-chloroacetanilide(4-C1AA) and 4-chloroaniline(4-C1A), indicating co-oxidation of N-hydroxy-4C1AA and oxyhaemoglobin in erythrocytes and partial reduction of the newly formed 4-C1NOB to 4-C1A. In rat blood in vitro incubated with N-hydroxy-4C1AA, 4-C1NOB concn. increased with increasing HbFe3+ concn., indicating that 4-C1NOB was formed by co-oxidation of oxyhaemoglobin and N-hydroxy-4C1AA, and not by enzymic N-deacetylation.

N-hydroxy-N-arylacetamides. II: Molecular aspects of ferrihaemoglobin formation by N-hydroxy-N-arylacetamides and arylhydroxylamines in the rat.

Ferrihaemoglobin (HbFe3+) formation in rats after i.p. injection of 6 N-hydroxy-N-arylacetamides has shown that N-hydroxy-4-chloroacetanilide(N-hydroxy-4ClAA) was the most active and N-hydroxy-2-acetylaminofluorene(N-hydroxy-2AAF) the least active compound tested. As N-hydroxy-N-arylacetamides were thought to produce HbFe3+ only after enzymic N-deacetylation, the corresponding arylhydroxylamines were also tested for HbFe3+-forming activity and were found to be more active, N-hydroxy-4-chloroaniline(N-hydroxy-4ClA) being one of the most active and N-hydroxy-2-aminofluorene(N-hydroxy-2AF) the least active compound tested. N-Hydroxy-4-chloroacetanilide given i.p. to rats more rapidly invaded the blood and produced larger amounts of ferrihaemoglobin than did N-hydroxy-2-acetylaminofluorene, due to differences in their availability in plasma. Injection of 50 mg/kg of N-hydroxy-4-chloroacetanilide gave similar concn of HbFe3+ and 4-chloronitrosobenzene(4-CINOB) as injections of 8 mg/kg of N-hydroxy-4-chloroaniline, indicating that the arylhydroxylamine, after N-deacetylation, was the active molecule in vivo. The concn of 4-chloronitrosobenzene declined faster than HbFe3+ concn. 4-Chloronitrosobenzene therefore is a further example of a 'hit-and-run' chemical. Inhibition by the microsomal carboxylesterase inhibitor, bis(4-nitrophenyl)phosphate(BNPP), indicated that ferrihaemoglobin formation by 4-chloroacetanilide, but not by N-hydroxy-4-chloroacetanilide, depends on the enzymic activity of hepatic microsomal carboxylesterases.

N-Hydroxy-N-arylacetamides. I. Toxicity of certain polycyclic and monocyclic N-hydroxy-N-arylacetamides in rats.

Of the two carcinogenic N-hydroxy-N-arylacetamides tested, N-hydroxy-4-acetylaminobiphenyl was as active as the monocyclic analogs in the oxidation of hemoglobin, whereas N-hydroxy-2-acetylaminofluorene produced less ferrihemoglobin after IP injection into female and male rats. Monocyclic N-hydroxy-N-arylacetamides, such as N-hydroxy-4-chloroacetanilide or N-hydroxyphenacetin, were more toxic than the parent N-arylacetamides, LD50 in mice being 190 mg/kg for N-hydroxy-4-chloroacetanilide vs 755 mg/kg for 4-chloroacetanilide, and 702 mg/kg for N-hydroxyphenacetin versus 1,220 mg/kg for phenacetin. The higher acute toxicities are probably due, at least in part, to the production of more ferrihemoglobin by the N-hydroxy-N-arylacetamides. Chronic toxicity of N-hydroxy-4-chloroacetanilide was tested on 10 male and 10 female Sprague Dawley rats after IP or SC injection of 20 mg (0.11 mmol)/kg twice weekly for 16 weeks into two groups of 10 animals each (five males, five females, total dose: 3.5 mmol/kg). The experiment, which was terminated after 2 years, did not yield any hint that N-hydroxy-4-chloroacetanilide was carcinogenic in the rat. Subchronic toxicity of N-hydroxyphenacetin was tested in two experiments on male and female Sprague Dawley rats after IP or SC injection of 50 or 100 mg (0.26 or 0.51 mmol)/kg. In the first experiment, two groups of 15 rats each (seven males, eight females) were injected either IP or SC with 50 and 100 mg/kg twice weekly for 29 weeks, and in the second experiment groups of 10 males and 10 females were injected SC with 100 mg/kg twice daily on 5 days a week for 12 weeks. The experiments, which were terminated after 29 weeks and 12 weeks treatment, respectively, did not provide evidence for chronic interstitial nephritis or tumor growth in the kidney. N-Hydroxy-N-arylacetamides were found to be inferior to the corresponding arylhydroxylamines in their ferrihemoglobin-forming capabilities in female rats. Large differences in activity of the arylhydroxylamines and no close relation to the number of rings was observed, N-hydroxy-2-acetylaminofluorene being the least active and N-hydroxy-4-acetylaminobiphenyl being as active as the monocyclic compounds, and exceeding all in the duration of its activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Peroxidase activity of oxyhaemoglobin in vitro.

In bovine erythrocyte suspensions incubated with 16 mM aniline, 4-phenetidine, 4-chloro- or 3,4-dichloroaniline for three hours at 37 degrees C, HbFe3+ concentrations of 10, 35, 77 and 93%, respectively, were found. N- and C-oxygenation products of aniline, 4-chloro-, and 3,4-dichloroaniline were formed, which can explain the oxidation of HbFe3+, indicative of peroxygenase activity of oxyhaemoglobin. The same N- and C-oxygenated derivatives of 4-chloro- and 3,4-dichloroaniline were also formed by hepatic microsomes, although at a 25- to 5000-fold higher rate. HbFe3+ was formed more readily on incubation of either bovine erythrocytes or purified human Hb with various N-arylacetohydroxamic acids. The metabolites of N-(4-chlorophenyl)-N-hydroxyacetamide are the same as the products of chemical oxidation of NOH-4ClAA by PbO2 or KMnO4, indicating the peroxidase activity of oxyhaemoglobin.