in vitro interactions of sulfur-containing compounds with the hepatic mixed-function oxidase system...

I OYI(‘OL.OGY AND APPLIED PHARMACOLOGY 59, Ill- 124 ( 1981)

In Vitro Interactions of Sulfur-Containing Compounds with the Hepatic Mixed-Function Oxidase System in Mice: Effects on

Paracetamol’ Activation and Covalent Binding2

J. MICHAEI. TREDGER. HEATHER M. SMITH, MICHAEL DAVIS, AND ROGER WILLIAMS

I~I Vifrc, Interactions of Sulfur-Containing Compounds with the Hepatic Mixed-Function Oxidase System in Mice: Effects on Paracetamol Activation and Covalent Binding. TREDGER. .I. M.. SMITH. H. M.. Dsvrs. M., AND WILLIAMS, R. (1981). -Trj.t--ice/ Appl. Ph~rmtr~l. 59, 11 l- 124. The effects of five thiols ( N-acetylcysteine, cysteamine, cysteine. glutathione. 2-mercaptopropionylglycine) and methionine on various metabolic stages leading to the covalent binding of paracetamol to hepatic microsomes were examined using mouse liver preparations. All five thiols decreased paracetamol covalent binding at concentrations lo- to lOO-fold smaller than those needed to affect significantly the apparent dissociation equilibrium of substrates with oxidized cytochrome P-450. the flavoprotein- mediated reduction of the cytochrome or the oxygenation of cytochrome P-450 substrates. Thus, N-acetylcysteine. cysteamine. cysteine. glutathione, and 2-mercaptopropionylglycine decreased the covalent binding of [“Hlparacetamol-derived radioactivity by 50% at concentrations of 0.06, 0.01, 0.03, 0.13. and 0.11 mM, respectively. Only cysteamine and cysteine (5 mM) affected the spectral interactions of paracetamol (>l mM) with cytochrome P-450. Cysteamine also markedly decreased the affinity of cytochrome P-450 for ethylmorphine and aniline. Ri-Acetylcysteine, cysteamine, glutathione, and 2-mercaptopropionylglycine (I mM) decreased NADPH-cytochrome c reductase activity by <20’S%. This effect was augmented in the presence of paracetamol. N-Acetylcysteine. cysteamine, cysteine. glutathione. and 2-mercaptopropionylglycine inhibited the oxygenation of at least one of the mixed-function oxidase substrates acetanilide. benzo[ol]pyrene. biphenyl, and ethylmorphine up to 80’S, Methionine was ineffective in all the assays examined and the mechanism of its known effects in r,ircr must involve metabolites such as homocysteine, cysteine. and glutathione which are not produced by an in ~itru microsomal system. These data suggest that the effects of sulfur-containing compounds on the formation of a reactive paracetamol metabolite are of lesser importance than effects on its subsequent covalent binding, although they may influence the overall efficacy achieved,

The acute hepatotoxicity which has been observed clinically and experimentally following ingestion of large amounts of paracetamol occurs in association with the covalent binding of a chemically reactive metabolite to cellular components (Jollow rr

’ /V-Acetyl-4-aminophenol; acetaminophen. L Presented in part at The Annual General Meeting

of The Medical Research Society, London. December X-9, 1978 (Ch. Sri. 56, 13~. 1979).

~1.. 1973). The generation of this metabolite is thought to be increased after paracetamol overdose because alternative metabolic pathways are saturated (Andrews et ul., 1976; Davis et al., 1976) and it becomes covalently bound when cellular glutathione. with which it is normally conjugated, is depleted (Mitchell rt al.. 1973b; Davis et rrl., 1974; Moldeus, 1978). In laboratory animals, the amount of covalent binding

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112 TREDGER ET AL.

observed after administration of hepato- toxic amounts of paracetamol can be cor- related with the extent and severity of liver damage that ensues (Jollow et nl., 1973).

The involvement of the hepatic mixed- function oxidase system in the activation of paracetamol has been established from studies showing that pretreatment with enzyme-inducing agents potentiates binding and toxicity, whereas inhibitors of this enzyme system have the converse effects (Jollow et (II.. 1973; Potter rt (I/., 1974). Similarly, a decrease in the amount of paracetamol-induced covalent binding and hepatic necrosis has been demonstrated experimentally with several sulfur-containing compounds (Strubelt et al., 1974; McLean and Day, 1975: Piperno and Bers- senbruegge, 1976; Labadarios et trl., 1977) and some, including N-acetylcysteine, cysteamine, and methionine, have been used clinically with apparent benefit (Prescott et (II.. 1976, 1977). In the present study we have investigated the interactions between six sulfur-containing compounds (N-acetylcysteine, cysteamine, cysteine, glutathione. 9-mercaptopropionylglycine, and methionine) and the microsomal mixed-function oxidase system of mice to clarify the mechanisms by which such compounds may act. Various defined stages of the mixed-function oxidase reaction were examined. i.e.. the interaction of substrates with cytochrome P-450, the flavoprotein-mediated reduction of the cytochrome (measured as NADPH-cytochrome c reductase activity), the oxygenation ofP-450 substrates, and the kinetic parameters of paracetamol-associated covalent binding.

METHODS

Marrriu/s. Glucose 6-phosphate, glucose-6-phosphate dehydrogenase, N-acetylcysteine, cysteamine. cysteine, glutathione (reduced), methionine, homocysteine thiolactone, paracetamol, cytochrome c’, N-2. hydroxyethylpiperazine-N-2-ethanesulfonic acid (Hepes), and bovine serum albumin (fraction V) were obtained from Sigma. London, England: Aniline, bi-

phenyl, ethanolamine, and phenobarbital were pur- chased from British Drug Houses. Poole, England; benzo[cu]pyrene, P-naphthoflavone, and acetanilide from Aldrich Chemical Company Ltd, Gillingham. England: NADP+ from International Enzymes Ltd.. Windsor, England; and ethylmorphine hydrochloride from May and Baker, England. 2-Mercaptopropionyl- glycine was provided by Santen Pharmaceuticals Company Ltd. Milan, Italy. [“HlParacetamol (sp act. 5.74 $X/~mol) was prepared by Sterling-Winthrop Ltd., via4-[ring-“Hlaminophenol which was acetylated by acetic anhydride. The product was identical with authentic paracetamol using mass spectrometry, no- clear magnetic resonance spectrometry. ultraviolet spectrophotometry, and thin-layer chromatography. Its radiochemical purity was 99% as determined by thin-layer chromatography in chloroform/methanol (90/ 10. V/V).

A~imcrls. Adult KCH mice (30-38 g; 13 weeks of age) were from a colony bred at King’s College Hospital Medical School. They were housed four per cage and were fed Oxoid diet 4lB. modified (Oxoid Ltd., Basingstoke. England) and tap water rrd libirr,nr.

Ti.ss~r p~rpc~nition. Mice were killed between 8 and 9 4M by cervical dislocation and livers were excised into ice-cold KCI-Hepes (1.15% KC1 containing 10 mM Hepes. pH 7.6). At least four livers were pooled per sample. After homogenization. using six return passes of a motor-driven (2000 rpm) Teflon-glass Potter-Elvejhem homogenizer. a suspension of washed hepatic microsomes was prepared in KCI-Hepes as described elsewhere (Tredger @I ~1.. 1980). The protein content of this suspension was determined by the method of Lowry BC (I/. ( 1951). using standards of bovine serum albumin prepared in the appropriate dilution of KU-Hepes to negate the chromagenic effects of the buffer, and the microsomal suspensions were diluted as required prior to use in the enzyme assays.

Bioc~hemi~rtl drtrrmin~~tion.r The spectral interactions of ligands with washed microsomal suspensions were determined by the method of Schenkman ef trl. (1967). Where the induced spectral changes were determined in the presence of a sulfur-containing compound, the latter was added as a neutralized solution during the preparation of 6 ml of a diluted microsomal suspension containing 3 mg protein/ml 0. I M Hepes. pH 7.6, and before equal division of5 ml suspension between two cuvettes. Paracetamol was prepared as a 200 mM solution in warm water and stepwise additions (up to 100 ~1) were made to the

sample cuvette such that the final concentrations achieved were 0.24, 0.28, 0.40, 0.56. 1.0. 2.0. 4.0. and 8.0 mM. Equal volumes of water were added to the reference cuvette. The induced spectral changes were recorded using the front cell compartment of a Pye- Unicam SP 1800 dual-beam spectrophotometer and were measured as the total absorbance change (peak

SULFUR COMPOUNDS IN PARACETAMOL TOXICITY 113

to baseline plus peak to trough) after readjustment of the absorbance at 500 nm to a standard absorbance achieved in baseline scans.

NADPH-cytochrome L’ reductase was measured at 30°C using a Pye-Unicam SP 800 dual-beam spectrophotometer to monitor the reduction of cytochrome ( at 550 nm as described by Peters and Fouts ( 1970). The incubation system contained the following constituents, added in order: 250 Fmol Hepes, pH 8.0. 420 pmol KCI, 0.42 pmol KCN, 0.2 emol cytochrome c. protective agent to the concentration described elsewhere. 2.5 ,umol paracetamol (where appropriate), 100 pg microsomal protein, and water to 2.3 ml. The reaction was initiated by the addition to the sample cuvette of 0.2 ml of a NADPH-generating system containing 6.25 pmol glucose 6-phosphate, 6.25 pmol MgCl,, 1.25 pmol NADP+ and 2 units glucose-6-phosphate dehydrogenase. A molar absorption coefficient for reduced cytochrome c of 19.100 was used in the calculation of the results.

The microsomal activities of ethylmorphine Ri- demethylase, acetanilide 4-hydroxylase. biphenyl 4- hydroxylase. and benzo[ oclpyrene hydroxylase were determined using slight modifications of the assays described by Bend CI rtl. ( 1972), Krisch and Staudinger (1961 1, Creaven c’t cl/. (1965) and Wattenberg CI II/. (19621, respectively. Each incubation contained 200 gmol Hepes (pH 7.6 for ethylmorphine, pH 8.1 for biphenyl. pH 7.8 for acetanilide, and pH 7.4 for benzolalpyrene). 5 Kmol glucose h-phosphate. 10 pmol MgCI,. 1 pmol NADP’, I unit gluose-6-phosphate dehydrogenase, sulfur-containing compound to the concentration required. microsomal protein (3 mg for ethylmorphine. biphenyl, and acetanilide. 50 pg for benrot alpyrene). and water to a 2 ml total volume. In addition, IO rmol semicarbazide was included in the ethylmorphine assay and 2 mg bovine serum albumin in the benzo[ crlpyrene assay. After 5 min preincubation. reactions were started by the addition of substrate ( 18gmol ethylmorphine, 16pmol acetanilide. 9.6 pmol biphenyl in 2.59 Tween 80, and 0.1 pmol henzo[a]pyrene in 50 ~1 acetone). and proceeded for IO min for benzo[a]pyrcne and 15 min for the other substrates. Standards of formaldehyde,4-hydroxybi- phenyl. and paracetamol were carried through the assays for ethylmorphine. biphenyl. and acetanilide oxygenations. respectively. Benzo[a]pyrene hydroxy-

lase activity was determined by measuring the Auores- cence of the phenolic products (394 nm excitation: 514 nm emission) and was expressed in relative nuorescence (RF) units, where the fluorescence of a

solution containing 3 pg quinine sulfate/ml 0.1 M H,SO, is 167 RF units (excitation. 350 nm: emis-

sion, 450 nm). The irreversible binding of [“H]paracetamol-derived

material to microsomal macromolecules was determined using a modified version of the method of

Potter er (I/. (1973). To each incubation was added, in order, 80 pmol Hepes, pH 7.7, sulfur-containing com-

pound (aqueous solution, pH 6.5-7.5) and paracetamol to the concentration described elsewhere. 5 x IO” dpm [“Hlparacetamol, 3 mg microsomal protein. and water to 1.5 ml. After a 5-min preincubation the reaction was initiated by addition of 0.5 ml of a NADPH- generating system comprising 120 /*mol Hepes. pH 7.7. 5 pmol glucose 6-phosphate. 5 pmol MgCl,, 1 pmol NADP+, and 1 unit glucose-6-phosphate dehydrogenase, and continued for 20 min before termination. In those incubations in which the amount of mixed- function oxidase-independent binding was being measured, the glucose-&phosphate dehydrogenase was omitted. The amount of irreversibly bound [“HI- paracetamol-derived material was determined as described elsewhere (Tredger cut (II.. 1980) and expressed as nanomoles of paracetamol equivalents bound per milligram of microsomal protein per minute.

Dtrfrl htrr~dli!tg. Double reciprocal plots of induced spectral change versus paracetamol concentration were made according to Lineweaver and Burk (1934) and linear regression analysis was performed using the method of least squares. The constants derived from these plots are presented i I SE of estimate (Moroney. 1974). Values of K,,, and V,,~,, for paracetamol covalent binding were derived for each microsomal pool in the presence and absence of a sulfur- containing compound from plots of binding vet-sub paracetamol concentration fitted by maximum likeli- hood using computer analysis (Wilkinson, 1961). Dif- ferences between the two sets of data were evaluated using Student’s paired t test. I,,, value\ were cal- culated by linear regression analysis of a plot of the amount of covalent binding versus log inhibitor concentration, and are presented -+ I SE of estimate.

RESULTS

The addition of paracetamol to an oxidized microsomal suspension produced an absorbance difference spectrum character- ized by a peak around 424 nm and a trough around 390 nm (Table 1). Using a double reciprocal plot, the spectral change achieved on addition of increasing paracetamol concentrations followed a linear relationship with concentration above and below a dis-

114 TREDGER ET AL.

TABLE 1

CHARACTERISTICS OF THE SPECTRAL CHANGES INDUCED IN MOUSE MICROSOMES USING PARACETA- MOL. VARIOUS SULFUR-CONTAINING COMPOUNDS. ANILINE,ETHYLMORPHINE.AND ETHANOL

Absorbance (nm)

Maximum Minimum

Paracetamol 424 390 N-Acetylcysteine 428 408 Cysteamine 426 390 Cysteine 421 397 Glutathione 414 2-Mercaptopropionylglycine 416 398 Methionine 422 390 Aniline 422 388 Ethylmorphine 386 418 Ethanol 416 389

No/r. The positions of maximal and minimal absorbance wel-e determined using 5 mM concentrations of the ligands as described under Methods. No obvious absorbance maximum was observed using glutathione.

continuity in the plot around 1 mM paracetamol concentrations (Fig. 1). The two dissociation constants (K,) derived from these linear parts of the curve were of the order of 1 mM (high affinity) and 3.0 mM (low affinity) although some variability in the values was observed when measurements were made on different occasions during the several months over which the studies were performed. The corresponding values of the theoretical maximal spectral change (A,,,) were around 0.04 and 0.08 A, respectively.

The addition of 5 mM concentrations of each of the various sulfur-containing compounds to oxidized microsomal suspensions produced difference spectra with the characteristics shown in Table 1. The spectral changes induced by aniline (type II), ethylmorphine (type I1 and ethanol (reverse type I) are included for comparative pur- poses. In agreement with published data (Castro et trl., 1972) cysteamine induced a type II spectral change. Type II spectral changes were also obtained using N-acetylcysteine, cysteine, and methionine. In con-

trast , 2-mercaptopropionylglycine induced a reverse type I spectral change, while glutathione induced an atypical spectrum.

When the same sulfur-containing compounds were included in microsomal suspensions during the determination of the spectrally apparent interactions of paracetamol, modifications to the double reciprocal plot obtained were noted. These effects are shown in Table 2 as changes in the values of the high- and low-affinity K, and A,,;,,. N-Acetylcysteine and methionine had no significant effect on the high-affinity spectral constants, i.e., those derived using paracetamol concentrations below 1 mM. Cys- teine significantly decreased the high-affinity K, (from 1.5 to 0.9 mM), while both glutathione and 2-mercaptopropionylglycine increased K, (Table 2). Using paracetamol concentrations greater than 1 mM to derive the low-affinity K,, N-acetylcysteine, glutathione, 2-mercaptopropionylglycine, and methionine had no effect, whereas cysteamine and cysteine increased K, approxi- mately twofold.

In addition, significant changes in the A,,,;,, derived for the high-affinity interactions were noted with cysteamine (50% reduction), cysteine (50% reduction), and 7- mercaptopropionylglycine (95cJ increase).

In view of their marked effects on paracetamol-binding spectra, the effects of cysteine and cysteamine on the spectrally apparent binding of the model substrates ethylmorphine and aniline were also examined. Cysteamine (5 mM), but not cysteine, markedly decreased the magnitude of the interactions of both aniline and ethylmorphine with mouse hepatic microsomes and this was reflected by changes in both the apparent K, and A,,, (Table 3).

It~tercictiot~s of SlrI~t4~-ContrritlitlK Con- politlds ,Ilith NADPH-Cvtr,chrottlc (’ Reducttrse ,4cti\litJ

When the assay mixture for determination of NADPH-cytochrome c reductase activity


I /

[PARACETAMOL] c m ~1.‘)

FIG. I. Typical double reciprocal plot of paracetamol concentration vs the magnitude of the spectral change induced. The spectral interactions of paracetamol and oxidized microsomal suspensions were determined as described under Methods. The spectrally apparent constants appropriate to the high- or low-affinity interactions are given by K>’ and A,,,’ or K,’ and A,,,‘, respectively. and were derived from three separate determinations made at each paracetamol concentration.

was constituted as described under Methods, it was noted that 10 mM concentrations of N-acetylcysteine. cysteamine, glutathione, and 2-mercaptopropionylglycine and 1 and IO rnbl concentrations of cysteine mediated an NADPH-independent autoreduction of cytochrome (‘. Even if these compounds were added to both sample and reference cuvettes simultaneously. the rates ofautore- duction of cytochrome c induced were so extensive that the resulting depletion of cytochrome c prevented an accurate quantitation of its enzymically mediated reduction. Consequently, only the results obtained using concentrations of the inhibitors which caused minimal autoreduction are presented in Fig. 2. When added to the reaction mixture at concentrations of 1 mM,

:V-acetylcysteine, glutathione, and 2-mercaptopropionylglycine had no significant effect on NADPH-cytochrome c reductase activity, and the same was true for 0.1 mM cysteine and 10 mM methionine. However, under the same conditions 1 mM cysteamine

decreased NADPH-cytochrome c reductase activity by 21% (Fig. 2).

When NADPH-cytochrome c reductase activities were studied in the presence of paracetamol, a slight increase in the basal rate was observed (195 k 3 versus 186 2 3 nmol cytochrome c reducedlmglmin: mean 2 SE, II = 24; p < 0.05, Student’s paired f test). Relative to this increased activity. 1 mM N-acetylcysteine, cysteamine, glutathione, and 2-mercaptopropionylglycine significantly decreased reductase activity by 23, 27, 17, and 19%, respectively (Fig. 2). Cysteine (0.1 mM) and methionine (10 mM) had no significant effect (Fig. 2), but 0.1 mM cysteamine caused a 7% decrease in activity (p < 0.05 by Student’s paired t test).

Effects of Sulfirr-Containirlg Cotr~pounds on Miued-Function Osidase Activities

Since some paracetamol-derived material becomes covalently bound to microsomal

116 TREDGER ET AL.

TABLE 2

EFFECTS OF SULFUR-CONTAINING COMPOUNDS ON THE SPECTRALLY DERIVED CONSTANTS

FOR PARACETAMOL BINDING TO MOUSE HEPATIC MICROSOMES

Compound and constant

High-affinity interaction Low-affinity interaction

Control Test Control Test

N-Acetylcysteine K, 0.90 2 0.05 0.96 t 0.04 2.7 t 0.4 2.4 2 0.4 &XiX 40 f 2 41 -i- 2 79 -c 8 76 + 8

Cysteamine KS 1.48 2 0.07 1.38 2 0.14 3.8 lr 0.4 7.6 i 1.1" A,,, 46 2 4 31 t 2” 87 2 8 772 1'

Cysteine K, 1.48 2 0.07 0.93 2 0.12” 3.8 k 0.4 7.0 + 0.8, A nt,,s 46 t 4 23 t 3" 87 t- 8 99 i 11

Glutathione K, 1.03 k 0.11 1.79 f 0.15” 4.0 2 0.3 3.2 t 0.3 A ma* 43 k 5 60 + 5 106 f 5 94 k 6

2-Mercapto- propionylglycine

K, 1.48 f 0.07 4.66 f 0.27" 3.8 iz 0.4 3.6 -c 0.2 A mar 46 2 4 90 t 5" 87 + 8 76 -c 5

Methionine KS 1.63 -+ 0.15 1.34 f 0.09 2.3 + 0.3 2.6 t 0.3 &XiS 53 i 5 43 2 7 65 + 6 68 i I

Note. The spectral interactions were determined as described under Methods using 5 mM concentrations of the sulfur compounds and paracetamol concentrations of 0.24 to 1.0 mM for the high-affinity interactions and 1.0 to 8.0 IIIM for the low-affinity interactions. K, values are presented as mM concentrations: A,,,;,, values are absorbance units x 103. Each result is the mean obtained from three separate experiments and is presented 2 1 SE of estimate (Methods).

’ Values so denoted differed from the appropriate control by more than twice the sum of the standard errors of estimate. Since 95% of all values lie within two standard errors of estimate, such differences were considered significantly different (p < 0.05).

macromolecules following its mixed-function taining compounds inhibited the mixed- oxidation, quantitation of its total metab- function oxygenation of at least one of the olism by measurement of the products of substrates used (Fig. 3). Benzo[a]pyrene the oxygenation is not technically feasible. hydroxylase activity was decreased by Measurement of paracetamol disappear- cysteamine (61% inhibition), acetanilide ance may also be unreliable if the break- hydroxylase activity by cysteine and cyste- down of paracetamol-thiol adducts to the amine (31 and 64% inhibition, respectively), parent compound occurs. We have there- and biphenyl hydroxylase activity by cyste- fore used the model substrates acetanilide, ine, cysteamine, and 2-mercaptopropionyl- benzo[a]pyrene, biphenyl. and ethylmor- glycine (23, 75, and 31% inhibition, re- phine to study the effects of sulfur-con- spectively). Ethylmorphine N-demethylase taining compounds on mixed-function oxy- activity seemed most sensitive to the in- genation. With the exception of methionine, hibiting effects of thiols as demethylation 10 mM concentrations of all the sulfur-con- was inhibited by 8, 15, 27. 74, and 82%


by 10 mM concentrations of N-acetylcysteine, glutathione, 2-mercaptopropionylgly- tine, cysteine, and cysteamine, respectively. Concentrations of 1 mM cysteine and cysteamine also decreased ethylmorphine demethylase activity (by 40 and 30%. respectively).

lntcmctions of Sulfkr-Contuining Corn- pounds ir+th the Irreversible Microsortrci~ Binding of [“H]Parncetamol-Derided Rrr- dioclctivity

Values for K,,, and V,,, derived from plots of covalent binding versus paracetamol concentration in the absence and presence of 0.1 and 1.0 mM concentrations of six sulfur-containing compounds are shown in Table 4. Neither 0.1 nor 1 .O mM methionine significantly affected the covalent binding of [“Hlparacetamol-derived radioactivity. The remaining sulfhydryl-group-containing compounds (thiols) produced changes which were both quantitatively and qualitatively different depending on the compound used, and its concentration. Thus, all the thiols decreased (by 65-95%) the apparent V,,, when present at 1 mM concentrations, and at 0.1 mM concentrations of the same compounds all but 2-mercaptopropionylglycine were also effective. N-Acetylcysteine and glutathione had no effect on K,,, at any of the concentrations used, while increases in K,,, were observed using 0.1 mM cysteamine, 0. I mM cysteine, and 0.1 and 1.0 mM 2- mercaptopropionylglycine.

The relative efficacies of the different thiols were evaluated by determining their I,,, concentrations, i.e., the concentration of thiol required to inhibit the covalent binding found in control incubations by 50%. When determined in incubations containing 1 mM paracetamol and thiols at four or more of a range of six concentrations (0.01, 0.025, 0.04, 0.1, 0.2, and 1.0 mM), the I,, concentration of methionine massively exceeded 1 mM since even at this concentration methionine inhibited covalent

TABLE 3

EFFECTS OF 5 mM CYSTEAMINE AND CYSTEINE ON

THE SPECTRALLY APPARENT ASSOCIATION OF ANILINE

AND ETHYLMORPHINE WITH MOUSE HEPATK MICRO- SOMAL SUSPENSIONS

Constants derived in the presence of

Ligand Water Cysteamine Cysteine

Ethyl- morphine

x, 0.40 f 0.16 7.2 5 0.b” 0.43 2 0.09 A rrM\ 0.03 t 0.01 0.04 t 0.01 0.03 + 0.01

Aniline K;, 0.70 k 0.04 3.1 i 0.3” 0.70 4 0.03 Ama, 0.13 2 0.01 0.07 r 0.01” 0.12 -+ 0.01

Nore. K, values are rnM concentrations: A,,, values are absorbance units r 103. Each result is the mean 2 SE of three separate determinations made using 0.35. 0.70. 1.4, 3.5. and 7.0 rnM concentrations of aniline, and 0.2. 0.5. 1.0, and 2.0 rnM concentrations of ethylmorphine.

” p -Z 0.05 as in Table 1.

binding by only 2% (Table 5). However, the remaining protective agents showed I,, concentrations in the range 9 to 150 pM

and their efficacy was in the order cysteamine > cysteine > N-acetylcysteine > 2- mercaptopropionylglycine = glutathione.

To establish the relative importance of chemical substituents in the molecules used, the effects of homocysteine and ethanolamine on covalent binding were also determined (Table 5). Homocysteine, the S- demethylated derivative of methionine, con- tains a free terminal sulfhydryl group and showed an Is, concentration of 42 PM when tested under the above conditions at concentrations of 0.01, 0.025, 0.05, 0.1, and 0.2 mM. In contrast 10 mM ethanolamine (the hydroxyl group containing analogue of the sulfhydryl compound cysteamine) inhibited binding by only 29%, while the same concentration of cysteamine produced a 96% inhibition.

DISCUSSION

In a preliminary study (Tredger et al., 1980) we reported that thiols demonstrated

118 TREDGERETAL

[PROTECTIVE AGENT]

N-AcCYS

IrnM

CYS

O.lmM

CYSNH2

IrnM

GSH

IrnM

2-MPG

ImM

METH

lOiT?M

FIG. 2. Effect of sulfur-containing compounds on NADPH-cytochrome c reductase activity. Reductase activities are presented as percentage mean decreases in activity (*SE. n = 4) relative to the appropriate control which contained no N-acetylcysteine (N-AcCYS), cysteine (CYS), cysteamine (CYSNH,), glutathione (GSH), 2-mercaptopropionylglycine (2-MPG), and methionine (METH). Negative values indicate increased activities. Unshaded histograms show comparisons made in the presence of sulfur-compound alone, while cross-hatched histograms show the same comparisons made in the presence of 1 mM paracetamol. The appropriate control (no sulfur compound) activities were (mean t SE, ~1 = 24): -, no paracetamol, 187 + 3: paracetamol added, 195 t 3 (nmol cytochrome r reducedimg microsomal proteinimin). Asterisks denote values which are significantly different from the appropriate control (p i 0.05: Student’s paired I test).

a lo-fold range in their efficacy toward decreasing the covalent binding of paracetamol as determined by their Ij0 concentrations. While these differences could be explained by the relative nucleophilicity of the thiols for the reactive paracetamol metabolite, it seemed likely that interactions at earlier stages in the generation of this metabolite might also be involved. The results of the present study show that several thiols can effect the apparent dissociation equilibrium of substrate with oxidized cytochrome P-450, the rate of NADPH-dependent reduction mediated by

microsomal flavoprotein and the mixed- function oxygenation of cytochrome P-450 substrates. However, the effects of thiols at these early stages in the mixed-function oxidation of paracetamol are only mediated at concentrations which far exceed those which substantially decrease covalent binding.

Of the spectrally apparent constants derived from the type II spectral change induced by paracetamol, only the values pertaining to the low-affinity interaction are comparable with those reported by Aikawa rt ~1. (1977). However, the second dissociation constant derived by Aikawa and


*

* I I

II.

-

CON N-AC TVS

CYS C’s NH> GSH ‘-

MPG METH

FIG. 3. Effect of 10 mM concentrations of sulfur-containing compounds on the oxygenation of acetanilide, benzo[or]pyrene. biphenyl, and ethylmorphine. All results are expressed as the mean -t SE of four separate determinations and are presented as the amount of product formedimg microsomal proteinimin. (Methods). Asterisks denote values which are significantly different (p < 0.05: Student’s paired t test) from the appropriate control (CON) containing no protective agents (abbreviations as in Fig. 2).

his colleagues fell outside the range of paracetamol concentrations used for its determination, and this, as well as other differences in experimental conditions. may explain the inconsistency observed. Using methods more similar to our own, Lake has obtained results more comparable with those presently reported.3

The ability of sulfur-containing compounds to induce binding spectra may ex-

:I B. G. Lake, personal communication

plain their capacity to affect the spectrally apparent interactions of paracetamol with oxidized microsomal suspensions. At lower paracetamol concentrations (< 1 mM) cysteamine, cysteine, glutathione, and 2-mercaptopropionylglycine affected paracetamol binding, but only cysteamine and cysteine significantly decreased the affinity of the P-450-paracetamol interactions (expressed as increased K, values) at concentrations which did not massively exceed those of the substrate. Cysteamine, but not cysteine,

120 TREDGER ET AL.

TABLE 4

KINETIC CONSTANTS FOR THE IRREVERSIBLE IN VJTRO BINDING OF [“HJPARACETAMOL-DERIVED MATERIAL

IN THE PRESENCE OF SEVERAL SULFUR-CONTAINING COMPOUNDS

Compounds and K, (mM) ~‘nlax (nmol = boundimgimin) concentration

(rnM) Control Test Control Test

N-Acetylcysteine 1.0 0.87 e 0.04 1.41 2 0.27 4.0 z 0.1 0.84 2 0.14” 0.1 1.19 2 0.24 1.15 2 0.12 5.3 t 0.5 2.4 2 0.1”

Cysteamine 1.0 h h b h

0.1 0.80 2 0.06 3.91 k 0.42” 1.8 F 0.1 0.24 -e 0.04”

Cysteine I .o 0.85 f 0.06 0.91 t 0.16 4.5 2 0.2 0.25 2 0.05” 0.1 1.03 t 0.02 3.32 f 0.26” 5.4 2 0.3 2.2 2 0.06”

Glutathione 1.0 0.68 2 0.05 0.82 2 0.10 4.6 2 0.2 0.92 -t 0.06” 0.1 0.67 2 0.12 0.74 -t 0.02 3.4 t 0.3 2.1 2 0.04”

2-Mercapto- propionylglycine

1.0 0.70 2 0.05 2.61 f 0.39” 1.7 ? 0.1 0.58 zt 0.04” 0.1 0.63 -c 0.03 2.34 + 0.34” 1.8 -t 0.1 1.9 c 0.3

Methionine 1.0 0.83 rt_ 0.06 0.85 2 0.08 3.5 2 0.1 3.6 t 0.1 0.1 0.73 z 0.04 0.73 2 0.03 2.9 k 0.4 2.8 ) 0.2

Note. Values of K,,, and V,,, were obtained as described under Methods and represent the mean 2 SE of three sets of data derived using paracetamol concentrations of 0.04. 0.06, 0.10, 0.13. 0.25. 0.50. 1.0. and 2.0 mM in the assay.

” Significantly different from the corresponding control (p < 0.05; Student’s paired I test). ” Data using I.0 mM cysteamine are not shown since inhibition was too great to permit calculation of

the kinetic constants.

also affected the attachment of ethylmorphine or aniline to cytochrome P-450, despite the higher affinity associated with such interactions relative to paracetamol (as judged by K, values). Consequently, despite the difficulties in equating spectral interactions with metabolism (Mailman et al.. 1974), it would seem that both cysteamine and cysteine may affect the interactions of paracetamol with cytochrome P-450. but that cysteamine may be more effective.

Of the compounds used, cysteamine, again, was the most effective inhibitor of NADPH-cytochrome c reductase and this supports reports of its inhibiting effects of NADPH-cytochrome c reductase in mice

(Mull et al., 1977) and on P-450 reductase activity in rats (Castro et ul., 1972). Para- cetamol increased the inhibition of reductase activity noted with several thiols, including cysteamine, and this may reflect the variable capacity of NADPH to reduce different P-450-substrate complexes (Gigon et ul., 1969).

Sulfur-containing compounds inhibited the oxygenation of several model cytochrome P-450 substrates by variable amounts depending on the thiol and substrate used. These differences did not appear to be related to the dissimilar catalytic properties of the various types of cytochrome P-450 present in liver preparations since we have


TABLE 5

QUANTITATIVE COMPARISON OF THE EFFECTS OF

NI:CLEOPHILIC COMPOUNDS ON THE IRREVERSIBLE

BINDING OF [3H]P~~~~~~~~~~-D~~~~~~ RADIO-

ACTIVITY TO MOUSE MKROSOMES

Additions (concentrations)

Binding (nmol =/mg I 5u proteinimin) (FM)

None I.61 k 0.06 -

.!v-Acetylcysteine t 1 mMl 0.26 + 0.02 57 + 6 Cysteamine (1 mM) 0.05 IT 0.01 9-+ I Cysteine (I mM) 0.09 2 0.02 31 t4 Glutathione (I mMl 0.36 -t 0.01 126 f 13 ?-Mercaptopropionyl-

glycine (1 mMl 0.37 t 0.03 112 2 II Methionine (1 mMl 1.58 2 0.08 BlOOO

Cysteamine t 10 mM) 0.06 t 0.01 9kl Homocysteine t 10 mMl 0.08 It 0.03 42 ? 5 Ethanolamine (IO mM) 1.15 2 0.19 > 10000

Notr. Covalent binding and I,,, values were determined as described under Methods. Each value is the mean t SE of at least three determinations. All additions except methionine significantly reduced covalent binding relative to control values (P < 0.05: Student’s paired f test).

shown that cysteamine and cysteine inhibit microsomal enzyme activities to the same extent in control mice as in those pretreated with phenobarbital, P-naphthoflavone, and pregnenolone 16cY-carbonitrile.” Nonetheless, the inhibition of acetanilide hydroxylation by cysteamine and cysteine may reflect similar effects of these two thiols on paracetamol oxygenation because of the similar properties of oxidations involving N- acetylarylamines (Guenthner and Nebert, 1978).-’ This conclusion is supported by the recent findings of Buckpitt et al. (1979) who showed that the rate of disappearance of paracetamol from microsomal incubations was decreased by cysteamine and cysteine.

Throughout our study we noted that the amount of covalent binding achieved was variable but generally greater than that obtained in earlier investigations (Potter et al.,

’ J. M. Tredger, H. M. Smith. M. Davis, and R. Wil- liams. unpublished observations.

1973: Davis et al., 1974) or by ourselves using C57B16 mice.4 We feel that these higher values may result primarily from the use of KCH mice in our studies, since mice of this strain appear to metabolize paracetamol considerably faster than other strains. This is borne out by the high susceptibility of KCH mice to the toxic effects of paracetamol, as judged both by relative mortality rates and the extent of hepatic necrosis compared with those found in other strains following administration of identical amounts of the drug (cf. Labadarios et al., 1976; Mitchell et al., 1973a). None- theless, the variations in basal activities we have experienced during the course of our experiments do not affect the overall effect of the compounds we have tested on different occasions. Consequently, the mode of action and relative efficacy of the thiols tested should apply irrespective of the basal activities achieved in their absence.

In quantitative terms, the efficacy of all the sulfhydryl-group-containing compounds in decreasing the covalent binding of paracetamol-derived radioactivity was considerably greater than would be predicted from their effects on the prior metabolic stages examined. Moreover, using N-acetylcysteine and glutathione, the results of our studies of the kinetic parameters of covalent binding suggest that interactions at stages during the formation of the reactive metabolite con- tribute little toward the efficacy of these compounds, since they affected covalent binding only by decreasing V,,, values. This result, involving changes in the rate of product formation alone, is consistent with a noncompetitive type of inhibition such as would be expected when an inhibitor exerts minimal effects on the association between an enzyme and its substrate. However, with cysteamine, cysteine, and 2-mercaptopropionylglycine, additional changes in the values of the apparent K,ri as well as V,,, were observed, suggestive of effects on both the rate of product formation and the interaction of enzyme and substrate. The latter effect may reflect the ability of cysteamine,

122 TREDGER ET AL.

cysteine, and 2-mercaptopropionylglycine to influence various stages in the mixed- function oxidation of paracetamol, as al- ready discussed.

Current evidence suggests that deactiva- tion of the reactive paracetamol metabolite may be achieved by the formation of adducts with sulfhydryl compounds (Buckpitt et al.. 1977, 1979; Moldeus, 1978). Because several of these adducts may be prepared from oxidized paracetamol by chemical means (Gemborys et al., 1980),5 it is possible that this interaction may not be entirely enzymically mediated (Rollins and Buckpitt. 1979), although the presence of cytoplasmic glutathione S-transferases may particularly facilitate the conjugation of paracetamol with glutathione in V~\YI, since the transferases have a high specific requirement for this thiol cosubstrate (Habig et al., 1974). Nonetheless, the demonstration that endo- plasmic reticulum also possesses glutathione S-transferase activity and that this activity may be augmented in the presence of sulfhydryl-group donors (Morganstern et cl/., 1979) may be relevant to the formation of paracetamol-thiol adducts in viva.

In c!i\~, sulfhydryl compounds are trans- formed to metabolites which may not be produced in an in t,itro microsomal system, but which may decrease paracetamol toxicity. Since methionine had no effect at any of the stages we examined, its activity in rhvo must be achieved after transformation to one or more of its known metabolites which include cysteine, glutathione, and sulfate anions (White et al., 1968; Reed and Orrenius, 1979). Each of these compounds is first derived from homocysteine, the proximal demethylated metabolite of methionine (White et rrl., 1968). Our results have shown homocysteine to be a potent inhibitor of covalent binding in vitro and methionine may exert its effects in r,i~a via this metabolite. Furthermore, the differential efficacies of methionine and homocysteine emphasize the importance of an intact thiol

3 R. S. Andrews. personal communication.

group in reducing covalent binding-an observation endorsed by the substantially different effects on covalent binding we observed using identical concentrations of the sulfhydryl compound cysteamine and its hydroxyl-group-containing analog ethanolamine.

Finally, because of the dissimilar means by which different thiols may achieve their overall efficacy in vitro, there appears to be some foundation for considering that com- binations of protective agents may be more effective than a single agent in the clinical situation. Moreover, those compounds such as cysteine, N-acetylcysteine, and methionine which supplement the cellular supplies of glutathione (Thor et (I/., 1979) may have the additional advantage of restoring the levels of this endogenous electrophile whose importance in the maintenance of cellular homeostasis is becoming increas- ingly apparent (Sies, 1978).

ACKNOWLEDGMENTS

We gratefully acknowledge the assistance of Dr. M. Crowder, Department of Mathematics. University of Surrey, for his analyses of kinetic data, and the Departments of Immunology and Chemical Pathology, King’s College Hospital, for use of their equipment. We also thank Sterling Winthrop Ltd. for provision of [“Hjparacetamol. and Santen Pharmaceuticals for 2-mercaptopropionylglycine and other assistance.

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