N-hydroxyacetaminophen: a postulated toxic metabolite of acetaminophen.

The decomposition of N-hydroxyacetaminophen has been shown to occur via an initial first-order dehydration step to N-acetyl-p-benzoquinone imine with a rate constant at pH 7.6 of 8.66 x 10(-3) min-1 and a half-life of 80 min. This is followed by a complex reaction between the quinone imine and the N-hydroxy compound to ultimately yield p-nitrosophenol and acetaminophen. The glucuronide and sulfate conjugates of N-hydroxyacetaminophen have been observed as urinary metabolites of N-hydroxyacetaminophen. No N-hydroxylated metabolites were found among the metabolites of acetaminophen. These results have been interpreted to show that N-hydroxyacetaminophen is not a metabolite of acetaminophen. It is proposed that the hepatotoxicity and nephrotoxicity of acetaminophen are mediated by a direct oxidation of acetaminophen to the toxic reactive intermediate N-acetyl-p-benzoquinone imine by the cytochrome P450 dependent mixed-function oxidase system.

Studies on the mechanism of toxicity of acetaminophen. Synthesis and reactions of N-acetyl-2,6-dimethyl- and N-acetyl-3,5-dimethyl-p-benzoquinone imines.

N-Acetyl-2,6-dimethyl-p-benzoquinone imine and N-acetyl-3,5-dimethyl-p-benzoquinone imine were prepared from 2,6-dimethylacetaminophen and 3,5-dimethylacetaminophen by oxidation with lead tetraacetate. Reaction of N-acetyl-2,6-dimethyl-p-benzoquinone imine with hydrochloric acid gave 3'-chloro-2',6'-dimethyl-4'-hydroxyacetanilide, whereas ethanethiol, aniline, and ethanol gave tetrahedral adducts resulting from addition to the imine carbon. Water gave 2,6-dimethyl-p-benzoquinone. With N-acetyl-3,5-dimethyl-p-benzoquinone imine, water and aniline gave substitution on the imine carbon, yielding 2,6-dimethyl-p-benzoquinone and 3,5-dimethyl-N-phenyl-p-benzoquinone imine, respectively. Ethanethiol gave 3'.5'-dimethyl-2'-(ethylthio)-4'-hydroxyacetanilide. The toxicity of 2,6-dimethylacetaminophen and 3,5-dimethylacetaminophen was examined histologically in mice and rats. 3,5-Dimethylacetaminophen was slightly more nephrotoxic but showed a similar hepatotoxicity to acetaminophen. 2,6-Dimethylacetaminophen, like N-methylacetaminophen, showed very little tissue damage.

Clofibrate-induced in vitro hepatoprotection against acetaminophen is not due to altered glutathione homeostasis.

Prior induction of peroxisome proliferation protects mice against the in vivo hepatotoxicity of acetaminophen and various other bioactivation-dependent toxicants. The mechanisms underlying such chemoresistance are poorly understood, although they have been suggested to involve alterations in glutathione homeostasis. To clarify the role of glutathione in this phenomenon, we isolated hepatocytes from mice in which hepatic peroxisome proliferation had been induced with clofibrate. The cells were incubated with a range of acetaminophen concentrations and the extent of cell killing after up to 8 h was assessed by measuring lactate dehydrogenase leakage from the cells. Hepatocytes from clofibrate-pretreated mice were much less susceptible to acetaminophen than cells from vehicle-treated controls. However, the extent of glutathione depletion during exposure to acetaminophen was similar in both cell types, as were rates of excretion of the product of glutathione-mediated detoxication of acetaminophen's quinoneimine metabolite, 3-glutathionyl-acetaminophen. The glutathione-replenishing ability of clofibrate-pretreated cells after a brief exposure to diethyl maleate also resembled that of control cells. More importantly, prior depletion of glutathione by diethyl maleate did not abolish the resistance of clofibrate-pretreated cells to acetaminophen. Taken together, these findings indicate that although glutathione-dependent pathways may contribute to hepatoprotection during peroxisome proliferation, the resistance phenomenon is not due exclusively to this mechanism.

Hepatotoxicity of phenacetin and paracetamol in the Gunn rat.

Both phenacetin and paracetamol produce acute centrilobular liver necrosis in the homozygous Gunn rat. Paracetamol is more hepatotoxic than phenacetin, and both are more hepatotoxic to the homozygous Gunn rat than to the heterozygous Gunn rat or to the albino rat. These findings have relevance to the role of the compounds in the clinical syndromes of paracetamol induced liver necrosis and analgesic nephropathy.

Neoplasia in the rat induced by N-hydroxyphenacetin, a metabolite of phenacetin.

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 nephrotoxicity of p-aminophenol. I. The effect on microsomal cytochromes, glutathione and covalent binding in kidney and liver.

p-Aminophenol administration lowered the microsomal cytochrome P-450 and b5 content and decreased the activity of NADPH cytochrome c reductase in kidney, but not in liver. Kidney GSH was depleted to 29% of the control value at 2 h, and only partly restored (50% of control) at 24 h. Liver GSH was transiently decreased, the lowest levels (77% of control) occurring at 30 min. The maximum level of covalently bound radioactivity was at two hours when 16.8% of the total radioactivity in kidney, 1.5% in liver and 3.6% in plasma was protein bound. At this time 81% of the total radioactivity in kidney and 95% of that in the liver was present in the soluble fraction.

The nephrotoxicity of p-aminophenol. II. The effect of metabolic inhibitors and inducers.

Inducers and inhibitors of the microsomal mixed function oxidase system have no consistent effect upon the nephrotoxicity of p-aminophenol, or on binding of the compound in vivo to cell protein. p-[ring-3H]Aminophenol was bound in vitro to kidney microsomal protein and to a lesser extent to liver. The binding was enhanced by preincubation of the p-aminophenol in air and inhibited by ascorbate, GSH, N2 and NADPH. These findings indicate that in contrast to paracetamol hepatoxicity which is dependent upon the mixed function oxidase system, that nephrotoxicity of p-aminophenol is dependent upon oxidation to a toxic metabolite by some other pathway. A similar metabolite may be responsible for the nephrotoxic action of phenacetin.

The metabolism and toxicity of paracetamol in Sprague-Dawley and Wistar rats.

Urinary paracetamol metabolites from Sprague-Dawley and Wistar rats were analysed by reversed-phase HPLC. Variations in the metabolic profile were observed as a function of dose, age, sex, species and route of administration. In addition the effect of 3-methylcholanthrene as an inducer of cytochrome P450 mixed function oxidase on paracetamol metabolism was also studied. Increased oxidative metabolism which lead to the formation of 3-thiomethylparacetamol conjugates along with paracetamol mercapturic acid could be correlated with increased susceptibility to hepatic damage. Furthermore it appears that the length of time taken for excretion and the level of free drug excreted may be involved in the aetiology of chronic renal damage.

Complete separation of urinary metabolites of paracetamol and substituted paracetamols by reversed-phase ion-pair high-performance liquid chromatography.

A reversed-phase high-performance liquid chromatographic procedure has been developed for the separation of thirteen urinary metabolites of the analgesic drug paracetamol. The method involved the use of radially compressed columns packed with octadecylsilica with a particle diameter of 5 micron. Metabolites were chromatographed by linear gradient elution using an ion-pair solvent system composed of tetrabutylammonium hydroxide and Tris buffered to pH 5.0 with phosphoric acid, and acetonitrile as the organic solvent. Analyses can be performed at the rate of three per hour. This method enables the direct identification of sulphate and glucuronide conjugates of 3-thiomethylparacetamol and 3-thiomethylparacetamol sulphoxide which have only previously been detected following enzyme hydrolysis of urine samples. The application of this fully optimised separation to the study of the metabolism of substituted paracetamols is also discussed.

Port Pirie lead abatement Program, 1992.

The Port Pirie Lead Decontamination Program commenced in 1984 with a ten year mandate. The abatement programme involves identification of children with elevated blood lead levels, house decontamination, soil treatment, general City greening, family education and support and community education. Since 1984 the smelter has also implemented substantial new environmental controls.Blood lead and air monitoring programmes as well as some investigations of recontamination are in place. The blood lead monitoring programme has shown a significant decrease in the mean blood lead levels of the children, with the magnitude of the reduction being greatest in areas remote from the smelter.The results of the air monitoring programme suggest that there has been little change in the general air lead levels in the City over the period of the abatement programme. Analysis of the data suggests that re-entrainment of lead from the contaminated areas within the City is only a small contributor to air-borne lead levels compared with that from the smelter and its environs.Sources and pathways of lead for absorption by the children in Port Pirie are discussed.

Reversed-phase chromatography of urinary metabolites of paracetamol using ion suppression and ion pairing.

High-performance liquid chromatography (HPLC) has proven particularly useful for the study of paracetamol metabolism. Two alternative methods were developed using reversed-phase C18 columns. A rapid ion suppression technique was used for the analysis of free paracetamol, paracetamol mercapturic acid and cysteine conjugate in urine samples obtained from isolated perfused rat kidney preparations, which has conveniently demonstrated the oxidative metabolic capacity of the kidney towards paracetamol. A somewhat longer, but higher resolution, ion-pair HPLC procedure was developed for the analysis of paracetamol metabolites in urine samples from experimental animals. The ion-pairing solvent was composed of tetrabutylammonium hydroxide, Tris and EDTA buffered to pH 7.2 with phosphoric acid. Gradient programming was further used to enhance resolution. Using this system two new metabolites, the sulphate and glucuronide conjugates of 3-thiomethyl-paracetamol were detected and routinely determined along with other known paracetamol metabolites, viz. free paracetamol, paracetamol sulphate, glucuronide, mercapturic acid, and cysteine conjugates, 3-methoxyparacetamol glucuronide and sulphate, p-aminophenol and its O-glucuronide and O-sulphate conjugates. Phenolic O-substituted glucuronide and sulphate conjugates of N-hydroxyparacetamol were also separated.

Improved high-performance liquid chromatographic separation of urinary paracetamol metabolites using radially compressed columns.

Methods have been adapted for the high-performance liquid chromatographic (HPLC) analysis of urinary paracetamol metabolites on radial compression columns. Enhanced resolution and decreased analysis time were two major advances. Various modifications to existing methods were made to counter the effect of the different C18 surface. Thus in ion suppression HPLC the addition of triethylamine at pH 3.0 (phosphate buffer) was necessary to block residual hydroxyl sites, while in ion-pair HPLC a higher tetrabutyl-ammonium hydroxide concentration of 0.01 M at pH 5.0 was used to enhance selectivity. The methods were successfully applied to the study of the metabolism of paracetamol, its glutathione conjugate and 3-thiomethylparacetamol in Sprague-Dawley rats. 3-Thiomethyl-paracetamol sulphoxide and its glucuronide and sulphate conjugates were shown to be metabolites of both 3-thiomethylparacetamol and paracetamol. 3-Thiomethylparacetamol sulphate was unresolved from the sulphates of paracetamol and 3-methoxyparacetamol in ion-pair HPLC. This raises a previously unrecognised problem in which the peak normally attributed to paracetamol sulphate contains metabolites arising from an oxidative metabolic pathway. Elevated levels of 3-methoxyparacetamol conjugates were found in human overdose urine and to some extent in analgesic nephropathy.

Metabolism of paracetamol by the isolated perfused kidney of the homozygous Gunn rat.

1. Isolated kidneys from homozygous Gunn rats were perfused with paracetamol in concentrations lower and higher than Km for paracetamol oxidation in the albino rat kidney. 2. Glucuronylation of paracetamol was not detected at either concentration. 3. An increase in oxidative metabolism at the higher concentration, similar to that seen with the Sprague-Dawley rat kidney, did not occur with kidneys from homozygous Gunn rats. 4. This finding does not support the hypothesis that the enhanced nephrotoxicity of paracetamol observed in the homozygous Gunn rat in vivo is due to increased intrarenal formation of reactive metabolites.