Catechol Metabolites Research Articles

Utilization of aromatic compounds by the Penicillium strain Bi 7/2

The Penicillium strain Bi 7/2 utilized phenol, catechol, resorcinol, hydroquinone, pyrogallol, hydroxyhydroquinone, phloroglucinol, m- and p-cresol, orcinol, 4-methylcatechol, 4-methoxyphenol, 4-aminophenol, benzyl alcohol, benzoic acid, 2-, 3- and 4-hydroxybenzoic acid, anthranilic acid, protocatechuic acid and gallic acid as sole sources of carbon and energy. The central metabolites catechol, protocatechuic acid and hydroxyquinone could be determined by HPLC with diode-array detection. Pathways for the degradation of aromatic substances were proposed.

Fast in vivo monitoring of electrically evoked dopamine release by differential pulse amperometry with untreated carbon fibre electrodes

Differential pulse amperometry has previously been used in combination with electrochemically treated carbon fibre electrodes. In order to improve the time resolution, this technique was combined here with untreated electrodes. Dopamine release was evoked in the nucleus accumbens of rats anaesthetized with urethane by electrical stimulation of the mesolimbic dopaminergic pathway. The differential oxidation current appearing at +200 mV was recorded every 1 s and was proportional to the dopamine concentration from 0.5 to 50 μM. At this voltage these untreated electrodes were not sensitive to the main catechol metabolite (DOPAC) and poorly sensitive to ascorbic acid. The electrically evoked increase in the oxidation current corresponded exclusively to dopamine. It was enhanced by nomifensine, amphetamine, haloperidol and pargyline and reduced by α-methyl- p-tyrosine (A-MPT). The results show that the evoked DA release was facilitated by increasing the stimulation frequency from 10 to 40 Hz. The method was sufficiently sensitive to detect dopamine release evoked by electrical stimulation at 10 Hz and its time resolution was 1 s.

Effects of handling or immobilization on plasma levels of 3,4-dihydroxyphenylalanine, catecholamines, and metabolites in rats.

In conscious animals, handling and immobilization increase plasma levels of the catecholamines norepinephrine (NE) and epinephrine (EPI). This study examined plasma concentrations of endogenous compounds related to catecholamine synthesis and metabolism during and after exposure to these stressors in conscious rats. Plasma levels of 3,4-dihydroxyphenylalanine (DOPA), NE, EPI, and dopamine (DA), the deaminated catechol metabolites 3,4-dihydroxyphenylglycol (DHPG), and 3,4-dihydroxyphenylacetic acid (DOPAC), and their O-methylated derivatives methoxyhydroxyphenylglycol (MHPG) and homovanillic acid (HVA) were measured using liquid chromatography with electrochemical detection at 1, 3, 5, 20, 60, and 120 min of immobilization. By 1 min of immobilization, plasma NE and EPI levels had already reached peak values, and plasma levels of DOPA, DHPG, DOPAC, and MHPG were increased significantly from baseline, whereas plasma DA and HVA levels were unchanged. During the remainder of the immobilization period, the increased levels of DOPA, NE, and EPI were maintained, whereas levels of the metabolites progressively increased. In animals immobilized briefly (5 min), elevated concentrations of the metabolites persisted after release from the restraint, whereas DOPA and catecholamine levels returned to baseline. Gentle handling for 1 min also significantly increased plasma levels of DOPA, NE, EPI, and the NE metabolites DHPG and MHPG, without increasing levels of DA or HVA. The results show that in conscious rats, immobilization or even gentle handling rapidly increases plasma levels of catecholamines, the catecholamine precursor DOPA, and metabolites of NE and DA, indicating rapid increases in the synthesis, release, reuptake, and metabolism of catecholamines.

The physiological role of estradiol 17-sulfate during pregnancy

To clarify the physiological role of estradiol 17-sulfate (ES) during pregnancy, experiments were conducted and the following results were obtained: (1) serum or urinary ES levels rose as a function of gestational age; (2) placental microsomes showed fairly high 2- and 4-hydroxylase activity for ES; and (3) the catechol products, 2- and 4-hydroxy-ES, had a strong inhibitory effect upon the in vitro production of lipid peroxides. These results suggest that ES actsas a precursor to the catechol metabolites which maintain normal gestation. This is coincident with the negative correlation of serum levels in ES and lipid peroxides observed in late pregnancy.

Kinetics of piribedil and effects on dopamine metabolism: hepatic biotransformation is not a determinant of its dopaminergic action in rats

The importance of hepatic metabolism in relation to the central (dopaminergic) effects of piribedil (PD) is still not really known. Plasma and brain kinetics and the effects on striatal dopamine (DA) metabolism of the parent drug and its known metabolites were therefore evaluated in rats, a species widely used in neurochemical studies of this drug. PD injected intraperitoneally (IP, 15-60 mg/kg) and centrally (ICV, 100-200 micrograms/rat) lowered striatal 3,4-dihydroxyphenylacetic acid (DOPAC) and 3-methoxy-4-hydroxyphenylacetic acid (HVA) content and the intensity and time-course of the neurochemical effects were route- and dose-relatedly dependent on brain PD kinetics. The catechol (M1), p-hydroxylated (M2) and N-oxide (M3) metabolites of the drug were detectable only in trace amounts in rat brain and only at the highest IP dose tested; when administered ICV at doses equimolar to PD they caused no decrease in striatal DA metabolites, although striatal concentrations were higher than after IP PD, being comparable to or higher than those of the ICV parent drug. These data suggest that metabolites do not contribute to the dopaminergic effects of PD in rats.

3H]Monoamine releasing and uptake inhibition properties of 3,4-methylenedioxymethamphetaimne and p-chloroamphetamine analogues

The ability of several 3,4-methylenedioxymethamphetamine (MDMA) analogues to inhibit the uptake of [ 3H]serotonin (5-HT), dopamine (DA) and norepinephrine (NE) into synaptosomes was examined. In addition, the ability of the compounds to inhibit the uptake of [ 3H]-HT and DA into synaptosomes from rats pretreated with reserpine (5 mg/kg i.p., 16 h pretreatment) was compared to control experiments. All of the test compounds were found to be potent releasers of non-vesicular 5-HT (the reserpine IC 50 was significantly smaller than the control IC 50). The range of 5-HT inhibitory activity corresponds well to the small range of ED 50 values of the test compounds to substitute in drug discrimination experiments with animals trained to discriminate MDMA or S-(+)-N-methyl-1-(1,3-benzodioxol-5-yl)-2-aminobutane (S-MBDB) from saline. In contrast, there was a wide range of potency for the inhibition of NE and DA uptake. In addition, several of the analogues appeared to be pure uptake inhibitors of DA while others were found to be releasers of non-vesicular DA. Several of the compounds were very selective for 5-HT over DA or NE uptake inhibition, including 3-methoxy-4-methylamphetamine (MMA) and 5-methoxy-6-methyl-2-aminoindan (MMAI). A correlation was noted between the 5-HT neurotoxic potential of some of the test compounds and their relative ability to induce a release of non-vesicular DA. The potential catechol metabolites of the methylenedioxy-substituted compounds also showed potent monoamine releasing properties, suggesting that metabolism may play a role in the neurotoxic actions of some of these drugs. The present data support the hypothesis that drug-stimulated non-vesicular 5-HT release is primarily responsible for the discriminative cue of MDMA.

Catechol metabolites in the cerebrospinal fluid as possible markers in the early diagnosis of Parkinson's disease

This paper is a preliminary report of work aiming to elucidate the possible use of 5-S-cysteinyl metabolites of catechols in the cerebrospinal fluid as markers in the early diagnosis of Parkinson's disease (PD). The rationale for this approach is the hypothesis that this disorder is caused by a failure of antioxidative mechanisms to prevent the excessive autoxidation of dopamine and other catechols that yields highly reactive and cytotoxic semiquinones and quinones. 5-S-cysteinyl adducts of these quinones have been detected in human brains, analyzed postmortem, and appear to be formed at an increased rate in elderly individuals, who show an increased loss of dopaminergic neurons.

Inhibition of the metabolism of paracetamol by isoniazid.

1. The effect of isoniazid given daily for 7 days on paracetamol (acetaminophen) kinetics and metabolism was studied in 10 healthy volunteers. Paracetamol, 500 mg, was given before isoniazid, on day 7 of isoniazid administration, and 2 days after the last dose of isoniazid. 2. On day 7, isoniazid markedly inhibited the formation clearance of the glutathione and catechol metabolites by 69.7% and 62.2%, respectively. Total paracetamol clearance was lowered by 15.2%. There was no effect of isoniazid on the non-oxidative pathways of paracetamol elimination. 3. Two days after isoniazid was discontinued, paracetamol metabolism had returned to pre-isoniazid values.

Determination of piribedil and its basic metabolites in plasma by high-performance liquid chromatography

A high-performance liquid chromatographic method for the determination of piribedil and its p-hydroxylated, catechol and N-oxide metabolites in plasma is described. After addition of an internal standard (buspirone), the plasma samples were subjected to a three-step extraction procedure. The final extracts were evaporated to dryness under nitrogen, and the residues were reconstituted in 100 microliters of mobile phase (0.01 M phosphate buffer-acetonitrile, 50:50, v/v) and chromatographed by acetonitrile gradient elution on a C18 reversed-phase column coupled to an ultraviolet detector set at 240 nm. The method was selective for piribedil and its metabolites, and sufficiently sensitive and precise for studies aimed at elucidating the role of the metabolites in the parent drug's pharmacological effects.

Metabolic fate of injected radiolabelled dopamine and 2-fluorodopamine in rats

In evaluating positron-emitting analogs of dopamine (DA) as possible imaging agents for visualizing tissue sympathetic innervation and function, the metabolic fate of systemically injected [ 3H]-DA or [ 14C]-DA was compared with that of [ 3H]-2-fluoroDA in plasma and in sympathetically innervated tissues (left ventricle, spleen and salivary glands) of rats. By 60 min after the injection of [ 3H]-DA or [ 3H]-2-fluoroDA, concentrations of [ 3H]-DA, [ 3H]-2-fluoroDA, [ 3H]-norepinephrine ([ 3H]-NE) and [ 3H]-2-fluoroNE in tissue exceeded concentrations in plasma by up to several thousand-fold. Whereas most of the radioactivity in tissue was in catechols, radioactivity in plasma was due to O-methylated metabolites of DA, including homovanillic acid (HVA) and of NE, including nonnetanephrine (NMN) and methoxyhydroxphenylglycol (MHPG). Estimated ratios of tissue: blood radioactivity at 60 min after injection of [ 3H]-2-fluoroDA were 4.10 for the heart, 1.91 for the spleen and 2.10 for the salivary glands. Thepatterns of levels of catechol metabolites and analogs of HVA in plasma and effects of blockade of neuronal uptake with desipramine suggested that [ 3H]-2-fluoroDA was not as efficiently removed by neuronal uptake and not as efficiently β-hydroxylated as the non-fluorinated compound. Concurrent administration of [ 3H]-DA and large amounts of non-radioactive 2-fluoroDA did not substantially alter the pattern of metabolites of [ 3H]-DA in plasma. After injection of [ 18F]-fluoroDA, visualization of sympathetic innervation of tissue should be feasible by positron emission tomography.

Role of Prostaglandin-H Synthase in Mediating Genotoxic and Carcinogenic Effects of Estrogens

Diethylstilbestrol (DES) has been found to be oxidized in Syrian hamster embryo (SHE) cells by prostaglandin-H synthase (PGH synthase). It is hypothesized that PGH synthase mediates adverse effects of DES and other carcinogenic estrogens such as induction of neoplastic transformation and genotoxicity. Interest in PGH synthase-catalyzed reactions focuses on two aspects: oxidation and metabolic activation of stilbene and steroid estrogens by PGH synthase, and modulation of prostaglandin biosynthesis via effects of these compounds on PGH synthase. Studies of the former aspect of PGH synthase-catalyzed in vitro metabolism have revealed that cooxidation of DES, DES analogues, and steroid estrogens gives rise to reactive intermediates; DES and DES analogues known to transform SHE cells are metabolized by PGH synthase in vitro; PGH synthase catalyzes both the formation and oxidation of catechol metabolites from steroid estrogens, and reactive intermediates from DES and from steroid estrogens are stable enough to bind both to the catalytic enzyme PGH synthase and to other proteins. The data support the contention that PGH synthase-catalyzed metabolic activation plays a role in the induction of neoplastic transformation by stilbene and steroid estrogens but is not conclusive evidence for a cause-effect relationship. More recently, two closely related DES indanyl analogues have been found to differ in their interaction with PGH synthase: indenestrol A is cooxidized and activated like DES, whereas indenestrol B inhibits the enzyme. They provide useful tools to test the above hypothesis from a new perspective.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of prostaglandin-H synthase in mediating genotoxic and carcinogenic effects of estrogens.

Diethylstilbestrol (DES) has been found to be oxidized in Syrian hamster embryo (SHE) cells by prostaglandin-H synthase (PGH synthase). It is hypothesized that PGH synthase mediates adverse effects of DES and other carcinogenic estrogens such as induction of neoplastic transformation and genotoxicity. Interest in PGH synthase-catalyzed reactions focuses on two aspects: oxidation and metabolic activation of stilbene and steroid estrogens by PGH synthase, and modulation of prostaglandin biosynthesis via effects of these compounds on PGH synthase. Studies of the former aspect of PGH synthase-catalyzed in vitro metabolism have revealed that cooxidation of DES, DES analogues, and steroid estrogens gives rise to reactive intermediates; DES and DES analogues known to transform SHE cells are metabolized by PGH synthase in vitro; PGH synthase catalyzes both the formation and oxidation of catechol metabolites from steroid estrogens, and reactive intermediates from DES and from steroid estrogens are stable enough to bind both to the catalytic enzyme PGH synthase and to other proteins. The data support the contention that PGH synthase-catalyzed metabolic activation plays a role in the induction of neoplastic transformation by stilbene and steroid estrogens but is not conclusive evidence for a cause-effect relationship. More recently, two closely related DES indanyl analogues have been found to differ in their interaction with PGH synthase: indenestrol A is cooxidized and activated like DES, whereas indenestrol B inhibits the enzyme. They provide useful tools to test the above hypothesis from a new perspective.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolism of methoxychlor by hepatic P-450 monooxygenases in rat and human. 1. Characterization of a novel catechol metabolite

Previous investigations demonstrated that the incubation of the chlorinated hydrocarbon pesticide methoxychlor [1,1,1-trichloro-2,2-bis(4-methoxyphenyl)ethane] with rat liver microsomes generates phenolic estrogenic metabolites. The current study shows that the incubation of liver microsomes from untreated and phenobarbital-treated rats and human donors, in the presence of NADPH, yields three phenolic metabolites. Identification of the metabolites was achieved by TLC, HPLC, GC/MS, and LC/MS and by hydrodynamic voltammetric analysis. These metabolites were identified as the mon- and didemethylated phenolic derivatives (mono-OH-M and bis-OH-M, respectively) and as a novel trihydroxy derivative (tris-OH-M). The tris-OH-M was demonstrated to be a catechol [1,1,1-trichloro-2-(4-hydroxyphenyl)-2-(3,4-dihydroxyphenyl)ethane]. Furthermore, the tris-OH-M becomes radiolabeled by [methyl-3H3]-S- adenosylmethionine (SAM) in a reaction catalyzed by catechol O-methyltransferase (COMT), indicating that tris-OH-M behaves like a catechol. Incubation of the monohydroxy metabolite with liver microsomes from phenobarbital-treated rats (PB microsomes) yields the dihydroxy and the trihydroxy metabolites. Furthermore, the time course of methoxychlor metabolism by PB microsomes demonstrated a rapid appearance and disappearance of the monohydroxy metabolite with the subsequent formation of the dihydroxy and trihydroxy metabolites. On the basis of these findings, it is proposed that the metabolic route of methoxychlor by mono-oxygenases involves sequential demethylations to the dihydroxy derivative and a subsequent ring hydroxylation.

Abnormal neurulation induced by 7-hydroxy-2-acetylaminofluorene and acetaminophen: Evidence for catechol metabolites as proximate dysmorphogens

Direct additions to culture media of either acetaminophen (APAP) or 7-hydroxy-2-acetylaminofluorene (7-OH-AAF) resulted in abnormal closure of the anterior neuropores of cultured rat embryos in the absence of an exogenous bioactivation system. Concentrations required to produce a 50% incidence of the defect were approximately 500 and 250 μ m for APAP and 7-OH-AAF, respectively. Losses of viability were not evident at these concentrations but 7-OH-AAF elicited a somewhat greater effect on growth parameters and generalized embryotoxicity. Transplacental induction with 3-methylcholanthrene (MC) of P450IA1 in subsequently cultured rat embryos did not detectably alter the capacity of APAP or 7-OH-AAF to effect embryotoxicity or neuropore closure. However, additions to the culture medium of exogenous hepatic bioactivating systems (S9) from MC-induced vs phenobarbital (PB)-induced adult rats produced profoundly different effects on neuropore closure. Coincubation with S9 from MC-induced rats reduced the incidence of 7-OH-AAF-elicited abnormal neuropores from 45 to 19%, whereas coincubation with S9 from PB-induced rats increased the incidence to 77%. Coincubation with MC-induced S9 produced no statistically significant effect on APAP-elicited neuropore abnormalities but, with PB-induced S9, resulted in a significant increase from 60 to 86%. Additions of 3-OH-APAP (0.1 – 0.2 m m) but not N-acetyl- p-benzoquinoneimine (NAPQI, 0.1 – 0.5 m m) to the culture medium elicited the typical neuropore abnormality. Experiments with APAP and 7-OH-AAF as substrates demonstrated that embryonic enzymes catalyzed their conversion to the corresponding catechols. Considered together, the results provided evidence that embryonic conversion of APAP or 7-OH-AAF to the corresponding catechol metabolites may be instrumental in effecting the abnormal anterior neuropore closure observed after exposure of embryos to the respective parent compounds.

Influences of estradiol and of catechol and non-catechol estrogens on the output of prostaglandins in uteri from spayed rats

The effects of 17-beta estradiol and of some catechol and non-catecholestrogens on the synthesis and output of prostaglandins (PGs) E and F by uteri from ovariectomized rats, were explored. Uteri from castrated animals released twice as much PGE than PGF. When uterine tissue was obtained from spayed rats injected prior to sacrifice with a low dose of 17-beta estradiol (0.5 ± 1.0 μg, on two consecutive days), the output of PGE diminished significantly. With a higher dose of the hormone (0.5 + 50.0 μg) the depressive influence on the synthesis and release of PGE was even more marked, whereas the output of PGF rose significantly. Low or high doses of estrone or of estriol failed to affect the release of either one of the PGs determined. On the other hand, 20H-estradiol at a low dose had no action but at a higher one inhibited the release of PGE without influencing PGF. Neither low nor high doses of 2-0H estriol or of 2-0H estrone affected the synthesis and release of uterine PGs. It was also observed that all the compounds tested evoked a significant uterotrophic action. It appears plausible that some catechol metabolites of 17-beta estradiol, but not other catechol-estrogens, could be involved in the mechanism of action of 17-beta estradiol modulating the production of PGs by the rat uterus.

Studies on the metabolism of conjugated drugs. III. 3-Deoxyestradiol 17-sulfate. (3). The metabolism by rat liver microsomes

When 17 beta-hydroxyestra-1,3,5(10)-triene 17-sulfate was incubated with rat liver microsomes, the hydroxylations occurred predominantly at the position 2, followed by the 3. The hydroxylation at 4 was negligible. With proceeding the incubation time, two catechol metabolites hydroxylated at the positions 2,3 and 3,4 were produced. These catechols were confirmed as the second products derived from the above phenolic metabolites. Some kinetic parameters obtained by these hydroxylations showed that all the hydroxylases concerned are different enzymes.

Dissimilation of aromatic substances by fungi

Summary Chaetomium cupreum, Drechslera oryzae and Fusarium oxysporum f. sp. vasinfectum utilized phenol, resorcinol, phloroglucinol, pyrogallol, benzoic acid, m -hydroxybenzoic acid, p -hydroxybenzoic acid, phloroglucinolcarboxylic acid, phenylacetic acid, m -hydroxyphenylacetic acid as sole carbon source. The central metabolites catechol, protocatechuic acid, hydroxyquinol, gentisic acid and homogentisic acid were isolated by replacement culture. The fungi elaborated catechol 1,2-dioxygenase, protocatechuate 3,4-dioxygenase, hydroxyquinol 1,2-dioxygenase, gentisate oxygenase and homogentisate oxygenase during growth on aromatic substances. Pathways for the degradation of aromatic substances were proposed.

Semi-quinone formation from the catechol and ortho-quinone metabolites of the antitumor agent VP-16-213.

The catechol metabolite of the antitumor agent VP-16-213 and the ortho-quinone of VP-16-213--a secondary metabolite formed from the catechol--easily undergo auto-oxidation into a free radical at pH greater than or equal to 7.4. By elevation of the pH from 7.4 to 10, an increase in the production of the free radical was observed, which was accompanied by the formation of products with higher hydrophylicity than the catechol and ortho-quinone, as found by HPLC-analysis. The hyperfine structure of the free radical indicates that it is the semi-quinone radical of VP-16-213. At pH 12.5 a secondary radical is formed from the catechol and the ortho-quinone of VP-16-213 besides the semi-quinone radical. One-electron oxidation of the catechol with horseradish peroxidase/hydrogen peroxide resulted in the formation of the same radical as observed under alkaline conditions and subsequent oxidation to the ortho-quinone. If the ortho-quinone was incubated with NADPH cytochrome P-450 reductase, a free radical was detected by spin-trapping with POBN, but not without spin-trapping. Studies on inactivation of phi X174 DNA by the system ortho-quinone of VP-16-213/NADPH cytochrome P-450 reductase suggest that the semi-quinone radical may play a role in the process of inactivation of DNA.

Cytochrome P-450 isozyme selectivity in the oxidation of acetaminophen.

Highly purified isozymes of cytochrome P-450 catalyzed the formation of 3-glutathion-S-ylacetaminophen (GS-APAP) and 3-hydroxyacetaminophen (3-OH-APAP) from acetaminophen (APAP). A major isozyme from untreated male rats (P-450UT-A) catalyzed the formation of ca. 2.0 nmol/nmol of P-450/10 min of 3-OH-APAP and approximately 7.2 nmol of GS-APAP/nmol of P-450/10 min. Antibodies specific for cytochrome P-450UT-A caused a decrease in the amounts of both metabolites produced in microsomal incubations. In contrast to these results, two other constitutive P-450 isozymes from rat liver, cytochrome P-450UT-F and the female specific isozyme P-450UT-I, produced less of both oxidative metabolites. Moreover, they produced significantly more of the catechol metabolite than the glutathione conjugate. These results are in accord with the observation that male rats are more susceptible to acetaminophen hepatotoxicity than female rats. Isozymes induced by phenobarbital also produced more of the catechol than the glutathione conjugate. Conversely, the major isozyme induced by beta-naphthoflavone, cytochrome P-450 beta NF-B, produced a significantly greater amount of GS-APAP than 3-OH-APAP. When comparison was made to a major phenobarbital inducible form (cytochrome P-450PB-B) a definite isozyme specificity for the formation of the two metabolites was seen. The catechol was formed at rates of 2.21 and 0.53 nmol/nmol of P-450/10 min by cytochromes P-450PB-B and P-450 beta NF-B, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Dose-dependent pharmacokinetics of acetaminophen: evidence of glutathione depletion in humans.

The time course of excretion of acetaminophen and its metabolites in urine was determined in eight healthy adults (seven men and one woman) who ingested 1 gm of the drug and collected timed urine samples for 24 hours. The mean time of peak excretion rate was 1.3 to 3.7 hours for acetaminophen, its glucuronide, sulfate, cysteine, mercapturate, and methoxy metabolites but 13.5 hours for methylthioacetaminophen. The mean half-life of acetaminophen was 3.1 hours and the mean half-life of the metabolites other than methylthioacetaminophen ranged from 4.1 to 5.7 hours. The half-life of methylthiometabolite could not be determined because of its very late peak time. In a second study the effect of dose on the clearance of acetaminophen was determined in nine healthy adult subjects (eight men and one woman) who received doses of 0.5 and 3 gm acetaminophen on separate occasions, separated by 4 to 10 days. The renal clearance of acetaminophen and the formation clearances of the sulfate, glutathione, and catechol metabolites were lower (by 38%, 41%, 35%, and 46%, respectively) at the higher dose. The renal clearance of acetaminophen sulfate and glucuronide conjugates were not different between doses. In a third study (10 men), 10 gm N-acetylcysteine was found to increase the formation clearance of the sulfate conjugate by 27% and that of the glutathione conjugate by 10%. The data suggest that the hepatic supply of reduced glutathione and 3'-phosphoadenosine 5'-phosphosulfate begins to be depleted over the range of 0.5 to 3 gm acetaminophen and that the depletion is overcome by the administration of N-acetylcysteine.