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)

Nephrotoxicity of aminophenols: effects of 4-dimethylaminophenol on isolated rat kidney tubules.

In isolated rat kidney tubules DMAP was found to inhibit the gluconeogenesis from lactate, pyruvate, or dihydroxyacetone. The ratio DMAP/protein rather than the calculated concentration of DMAP determined the strength of the effect, 20--25 nmoles DMAP/mg protein inhibiting the rate of gluconeogenesis by about 50%. The inhibition was not reversible. Phenacetin, 4-aminophenol and 4-acetamidophenol were much less effective than DMAP in inhibiting gluconeogenesis in isolated rat kidney tubules. DMAP 14C-labeled in the ring was quickly bound to proteins in kidney tubules. A portion of DMAP which did not exceed about 4 nmoles/mg protein, was bound in compounds soluble in perchloric acid. From this portion tris-GS-DMAP was isolated. DMAP diminished the glutathione content of isolated rat kidney tubules. Reduced glutathione added before DMAP prevented the inhibition of gluconeogenesis and diminished the binding of DMAP to proteins. The binding of DMAP required oxygen and was inhibited by carbon monoxide or cyanide. Several enzymes from isolated kidney tubules were found to be inhibited by DMAP doses which inhibited gluconeogenesis. Large DMAP doses also diminished the sums of ATP + ADP + AMP as well as NAD + NADH and NADP + NADPH. This effect corresponded to an increase in nucleotide degradation products and to increased activity of extracellular LDH. The results indicate that the inhibition of gluconeogenesis by DMAP is not due to a specific effect on one enzyme or on membranes but to unspecific reactions with many substances.

Effects of 4-dimethylaminophenol and Co2EDTA on circulation, respiration, and blood homeostasis in dogs.

The effects of intravenously injected 4-dimethylaminophenol and Co2EDTA on peripheral circulation, respiration, acid-base balance, and several other physiological and biochemical parameters were studied on dogs. DMAP increased the respiratory minute volume and mean arterial pressure, diminished the lactate-to-pyruvate ratio, and induced an increase in arterial oxygen pressure caused by liberation of oxygen from oxyhemoglobin during the formation of ferrihemoglobin. A study in vitro of the fate of the oxygen during the reaction between DMAP and oxyhemoglobin showed that only 30--40% of the oxygen released by the formation of ferrihemoglobin appeared in the gas phase. Co2EDTA caused circulatory depression, hyperventilation, and metabolic acidosis resulting in a decrease in base-excess and pH. The concentrations of lactate, pyruvate, potassium, and urea nitrogen and the hemoglobin content were increased by Co2EDTA. The side effects of Co2EDTA in therapeutic doses were more serious than those of DMAP. Thus the latter is superior in the therapy of cyanide poisoning, all the more since it detoxifies more cyanide.

Absorption in rats, dogs, pigs, and humans of nicotinic acid after oral administration of phosphatidyl inositol pentanicotinate hydrochloride (PIN).

Bound nicotinic acid in feces after oral administration of phosphatidyl inositol pentanicotinate (PIN) was determined by chromatographical isolation from acid hydrolysate and UV absorbance of the eluted nicotinic acid. With all species tested, the absorption of nicotinic acid after administration of PIN was found to be incomplete, proportions from 5 to 25% of the amount of nicotinic acid administered with PIN being recovered from feces. Humans absorbed about 75% of the nicotinic acid administered with 700 mg PIN, i.e. 230 mg. It is concluded that the presence of bound nicotinic acid in feces is due to slow absorption of the PIN or slow hydrolysis of the nicotinic acid ester. Rabbit liver homogenate, human blood plasma, and human duodenal juice were found to liberate nicotinic acid from PIN.

Pharmacokinetics of cyanide in poisoning of dogs, and the effect of 4-dimethylaminophenol or thiosulfate.

Cyanide in blood, plasma, and urine of dogs after administration of K14CN was determined with the isotope dilution technique. The addition of large amounts of inactive KCN as soon as possible to a sample to be analyzed inhibited the decrease of the original cyanide concentration. After administration of several lethal doses of cyanide into the stomach or by slow intravenous infusion a concentration of about 40 micron cyanide in plasma was found at the moment of respiratory arrest. Since 60% of the cyanide in plasma was bound to proteins the concentration of free cyanide which stopped respiration was about 16 micron. Quick formation of ferrihemoglobin by i.v. injection of 4-dimethylaminophenol after plasma cyanide had risen to or above 40 micron decreased the cyanide concentration in plasma and restored respiration, while cyanide was accumulated in red cells by formation of ferrihemoglobin cyanide. Equilibrium constants calculated for the reaction between ferrihemoglobin and cyanide in vivo indicated that the reaction approached equilibrium in a few minutes. Up to 60% of the radioactive cyanide absorbed was found as non-cyanide radioactivity in the urine.

Biotransformation and some effects of 2-dimethylamino-4-(N-methylanilino)-phenol in dogs.

2-Dimethylamino-4-(N-methylanilino)-phenol (MP), an active metabolite of N,N-dimethylaniline-N-oxide in the autocatalytic formation of ferrihemoglobin, reacted quickly in dogs after intravenous injection. A dose of 14C-labeled MP which oxidized 40% of the hemoglobin disappeared from the blood in 20 min. During this period of time MP transferred catalytically electrons from ferrohemoglobin to oxygen, reacted with sulfotransferases to form the sulfuric acid ester, and was covalently bound in blood and other tissues. In the urine, in addition to the sulfuric acid ester of MP (25%), methylamine, dimethylamine, and N-methylaniline were found. Their amount indicated that most of the MP not esterified with sulfuric acid had lost a nitrogen by hydrolysis of the quinonimine. The metabolites which were covalently bound in blood and other tissues disappeared slowly, traces of radioactivity being found in blood and urine 7 days after i.v. injection of MP, 15 mg/kg. The formation of methylamines as well as N-methylaniline from MP in vivo and in blood in vitro proves that the oxidation product of MP, a purple dye, is a resonance hybrid of the two structures 2-dimethylamino-N-methyl-N-phenyl-1,4-benzoquinone-4-imonium and 4-(N-methylanilino)-N,N-dimethyl-1,2-benzoquinone-2-imonium. In addition to ferrihemoglobin MP produced numerous Heinz bodies in red cells and caused hemolytic anemia. After lethal doses necroses in the kidney tubules were found.

The effect of tris(2-chloroethyl)amine on human hemoglobin.

The reaction of tris(2-chloroethyl)amine (TCEA) with purified hemoglobin and its effect on properties of hemoglobin was studied using 14C-labeled TCEA. Hemoglobin remained soluble after binding as much as 4 TCEA per heme. In concentrations which did not denature hemoglobin TCEA reacted only with a small proportion of the free SH groups; blockade of the SH groups with PMB did not noticeably affect the binding of TCEA to hemoglobin. Hydrolysis by trypsin or chymotrypsin of hemoglobin which had reacted with TCEA yielded radioactive peptides besides not radioactive peptides and radioactive compounds not reacting with ninhydrin. The reaction with TCEA caused a change in electrophoretic mobility of hemoglobin and prevented its complete disintegration by PMB into subunits. After reaction with TCEA the affinity of hemoglobin for oxygen was strongly increased and the heme-heme interaction strongly diminished. The Bohr effect and the effect of 2,3-diphosphoglycerate on oxygen affinity remained unchanged. The effect of TCEA on the properties of hemoglobin points to specificity in its reaction with functional groups of hemoglobin.

Biotransformation of 4-dimethylaminophenol: reaction with glutathione, and some properties of the reaction products.

4-Dimethylaminophenol (DMAP) forms ferrihemoglobin by catalytic transfer of electrons from ferrohemoglobin to oxygen. In solutions of purified human hemoglobin, quick binding of oxidized DMAP to the globin moiety of hemoglobin terminates this reaction. Reduced glutathione in high concentrations, as in the red cell, substantially diminished binding of oxidized DMAP to hemoglobin by formation of S,S,S-(2-dimethylamino-5-hydroxy-1,3,4-phenylene)-tris-glutathione (tris-(GS)-DMAP), which does not form ferrihemoglobin. In the presence of reduced glutathione, DMAP disappeared more rapidly from hemoglobin solutions than in its absence. The formation of tris(GS)-DMAP in red cells was found to be of importance for the termination of catalytic ferrihemoglobin formation by DMAP in vivo. With low concentrations of GSH, DMAP in hemoglobin solutions formed another conjugate, (GS)-DMAP, S,S(2-dimethylamino-5-hydroxy-1,3-phenylene)-bis-glutathione. Similar to DMAP, bis(GS)-DMAP catalyzed the formation of ferrihemoglobin. As the oxidized bis(GS)-DMAP was bound to hemoglobin more slowly and to a lesser extent, it produced more ferrihemoglobin than DMAP. In contrast to the reactions of DMAP with hemoglobin, hydrogen peroxide and superoxide radicals are involved in the ferrihemoglobin formation by bis(GS)-DMAP. The radicals accelerate the oxidation of bis(GS)-DMAP and thereby the ferrihemoglobin formation.

Mechanism of the autocatalytic formation of ferrihemoglobin by N,N-dimethylaniline-N-oxide. Structure and ferrihemoglobin forming activity of the purple dye.

The structure of the leuco compound of the purple dye which is formed in mixtrues of N,N-dimethylaniline-N-oxide (DANO) and ferrihemoglobin or ferricytochrome c was elucidated. IR, NMR, mass spectroscopy, and synthesis by oxidation of mixtures of N-methylaniline and 2-dimethylaminophenol showed that the leuco compound is produced by condensation of these two compounds. But only X-ray analysis proved the structure: 2-dimethylamino-4-(N-methylanilino)-phenol. The purple dye was produced from the leuco compound by withdrawal of two electrons and may be considered as resonance hybrid of the p-quinonimine and the o-quinonimine. When DANO was incubated with ferrihemoglobin or ferricytochrome c the oxygen of DANO was used for the production of the dye by oxidation of N-methylaniline and 2-dimethylaminophenol. The amount of N,N-dimethylaniline found in the incubation mixtures corresponded with the amount of purple dye produced. In the absence of molecular oxygen from incubation mixtures of DANO with cytochrome c the purple dye was formed at the same rate as under air. In blood in vitro the purple dye catalytically transferred electrons from ferrohemoglobin to molecuar oxygen. Its ferrihemoglobin-forming activity was lower than that of 4-dimethylaminophenol but higher than that of 2-dimethylaminophenol. The chemical mechanism of the autocatalytic formation of ferrihemoglobin by DANO is described.

The fate of phenylhydroxylamine in human red cells.

Phenylhydroxylamine added to human red cells under aerobic conditions and in the presence of glucose was partly reduced to aniline. About half the hydroxylamine was recovered as amine after a 2-hr incubation. The aniline, after acetylation, was identified as acetanilide by melting point, Rf-value in TCL as well as UV, IR, and NMR spectroscopy. The fate of the remaining phenylhydroxylamine was followed by use of 14C-labeled phenylhydroxylamine. About 30% of the total radioactivity was bound to hemoglobin or other proteins and about 20% was found in highly polar low-molecular substances which were insoluble in organic solvents. The elucidation of the sites at which phenylhydroxylamine was bound to hemoglobin was complicated by the lability of the bonds. When purified human hemoglobin had reacted with radioactive phenylhydroxylamine, large proportions of the radioactivity bound to hemoglobin were removed by treatment with acid or with PMB for separation of alpha- and beta-chains. The radioactive compound liberated from hemoglobin by acid was found to be aniline. After reaction with phenylhydroxylamine the number of SH groups titrable with PMB was found to be diminished. Pretreatment of hemoglobin with N-ethylmaleimide or PMB decreased the amount of phenylhydroxylamine bound to hemoglobin but did not fully prevent the reaction. Tryptic digestion of hemoglobin after reaction with radioactive phenylhydroxylamine yielded tryptic peptides with lower specific activity than that of hemoglobin. Chymotryptic digestion of the tryptic core yielded a core with specific activity much higher than that of hemoglobin. Fingerprinting of the tryptic or chymotryptic hydrolyzates showed the presence of peptides with high and other ones with low or no radioactivity and of radioactive compounds which did not react with ninhydrin. In the covalent binding of phenylhydroxylamine to globin the SH group beta93 plays an important role, but other yet unknown sites are also reactive.

Ferrihemoglobin and kidney lesions in rats produced by 4-aminophenol or 4-dimethylaminophenol.

4-Dimethylaminophenol hydrochloride (DMAP), 20mg/kg i.v., was found to oxidize in rats as much as 50% of the hemoglobin to ferrihemoglobin but did not cause kidney lesions. 4-Aminophenol hydrochloride, 400 mg/kg i.v., oxidized only 25% of the hemoglobin and produced large tubular necroses. In highly toxic doses only, e.g., twice the LD50, DMAP also produced tubular necroses.