The major role of glutathione in the metabolism and excretion of N,N-dimethyl-4-aminoazobenzene in the rat

Chem.-Biol. Interactions, 47 (1983) 307--323 307 Elsevier Scientific Publishers Ireland Ltd.

THE MAJOR ROLE OF GLUTATHIONE IN THE METABOLISM AND EXCRETION OF N,N-DIMETHYL-4-AMINOAZOBENZENE IN THE RAT

BRIAN COLES, SURJIT KAILA SINGH SRAI, H. BRYAN WAYNFORTH and BRIAN KETTERER

Courtauld Institute o f Biochemistry, Middlesex Hospital Medical School, London WIP 7PN (U.K.)

(Received March 4th, 1983) (Revision received July 8th, 1983) (Accepted July 19th, 1983)

SUMMARY

In the normal rat given a single dose of one mg N,N~limethyl-4-aminoazobenzene (DAB) via the hepatic portal vein the following biliary metabolites reached their maximal rates of excretion in the sequence: 4'-sulphonyloxy- DAB, N-(glutathion-S-methylene)-4-aminoazobenzene (GSCH2AB), 4'- sulphonyloxy-N-methyl-4-aminoazobenzene (4'-sulphonyloxy-MAB) 4'- sulphonyloxy-GSCH2AB and MAB-4'-/3-glucuronide.

The unusual and relatively unstable N-methylene glutathione conjugates were major metabolites accounting for up to 70% of the whole. It was shown that all the 4-aminoazobenzene (AB) and perhaps all of the 4'-sulphonyloxy- AB, which may be observed in bile, are artefacts due to decomposit ion of GSCH2AB and 4'-sulphonyloxy-GSCH2AB respectively and that biliary excretion of N-methyloxidised products of MAB and 4'-hydroxy-MAB is dependent on their conversion to the GSH conjugates, GSCH2AB and 4'- hydroxy-GSCH2AB respectively.

Sulphotransferase inhibition by pentachlorophenol caused a reduction in the excretion of all sulphate conjugates, but biliary excretion as a whole was not reduced significantly due to a compensatory increase in the excretion of l~AB-4'-~-glucuronide and the appearance of 4'-OH-GSCH2AB.

Glutathione (GSH) depletion by diethylmaleate caused a reduction in biliary metabolites of DAB by lowering the levels of GSH conjugates. This was because the amount of N-methyl oxidation of MAB and 4'-hydroxy- MAB were porportional to the amount of GSH present. The fall in N-methyl oxidation was not compensated for by an increase in 4'-hydroxylation and

Abbreviations: AB, 4-aminoazobenzene; DAB, N,N-dimethyl-4-aminoazobenzene; GSCH2AB , N-(glutathion-S-methylene)-4-aminoazobenzene; GSH, glutathione; 3-GSMAB, N-methyl-3-(glutathion-S-yl)-4-aminoazobenzene; HPLC, high pressure liquid chromatography; MAB, N-methyl-4-aminoazobenzene; TLC, thin-layer chromato~aphy.

0009-2797/83/$03.00 © 1983 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

308

was accompanied by a delay in the appearance of 4'-hydroxylated metabolites.

The administration of potential precursors of 4'-sulphonyloxy-GSCH2AB establishes the sequence of reactions resulting in its formation to be 4'- hydroxylation, N-methyloxidation, GSH conjugation and O-sulphation.

Key words: Biliary metabolites - -N,N-Dimethyl -4-aminoazobenzene- Glucuronide conjugates -- Glutathione conjugates -- Glutathione depletion, oxidative metabolism -- Sulphate conjuga tes - Sulphotransferase inhibition

INTRODUCTION

The study of the metabolism of the aminoazo-dye hepatocarcinogen, DAB, in the rat has a long history. Quite early on many of its metabolic transformations were understood, including N-demethylation 4'-hydroxylation azo-bond reduction and N-acetylation [1]. These are all detoxication products.

Sulphate and ~-glucuronide conjugates of 4'-hydroxylated metabolites have been identified in bile [2--10] and conjugates of the products resulting from azo-bond reduction in the urine [1,2,4]. More recently, strong evidence has been advanced that N-sulphonyloxy-N-methyl-4-aminoazobenzene (N-sulphonyloxy-MAB) is the toxic metabolite which is carcinogenic in DAB fed rats [11] and its detoxication product, the GSH conjugate 3-(glutathion~-yl)-N-methyl-4-aminoazobenzene (3-GSMAB) has been synthesized in vitro [12,13] and isolated from bile [12].

3-GS-MAB is a minor component of the total biliary metabolites (Srai et al., unpublished) and some recent papers have assumed that this product is the only one in which GSH conjugation is involved [7]. However, we have shown that N-methylene-GSH conjugates, namely GSCH2AB [9] and 4'- sulphonyloxy-GSCH2AB [10], are excreted in abundance, but have been overlooked in the past because of their relative instability.

In the present study we have traced the sequence of excretion of all the major biliary metabolites arising from a single intra-hepatic portal dose of DAB. The effects of GSH depletion and sulphotransferase inhibition have been compared. In addition, the fate of possible precursors of the unusual, doubly-conjugated, 4'-su!phonyloxy-GSCH2AB has enabled the mechanism of formation of this conjugate to be determined.

MATERIALS AND METHODS

Materials DAB was purchased from BDH Ltd., (Poole, Dorset, U.K.) and purified

before use. [Prime ring-14C] DAB (7.16 mCi/mmol) was purchased from New England Nuclear Corp. (Boston, MA, U.S.A.). 4'-sulphonyloxy-DAB and 4'-sulphonyloxy-AB were synthesized from the corresponding phenols

309

[14,15] and purified by silica thin-layer chromatography (TLC) as prev- ously described [10]. GSCH2AB was synthesized [9] as described previously and 4'-OH-GSCH2AB and its 4'-/3-glucuronide were synthesized using the method for 4'-sulphonyloxy-GSCH2AB [10], replacing 4'-sulphonyloxy-AB with 4r-hydroxy-AB or its glucuronide as appropriate. 1+C-Labelled DAB metabolites were isolated by cellulose TLC (E. Merck AG No 5716, Darmstadt, F.R.G.) from the bile of rats injected with [14C]DAB as described elsewhere [9] and further purified by HPLC (see below). Larger quantities of unlabelled MAB conjugates were obtained similarly. The identity of metabolites was confirmed by u.v. and visible spectroscopy, mass spectrometry, NMR spectroscopy, the action of ~-glucuronidase or arylsulphatase as appropriate [2--5] and by comparison with authentic materials. Bovine serum albumin and propylene glycol were purchased from Sigma Chemicals Co. (St. Louis, MO, U.S.A.) and pentachlorophenol from Fluka AG (CH-947, Buchs, Switzerland). Other chemicals were of analytical purity, purchased from BDH Ltd., (Eastleigh, Hants, U.K.).

Methods HPLC was carried out as described elsewhere [10] on a 6.4 × 250 mm

/~Bondapak C-18 column (Waters Associates, Milford, MA, U.S.A.)using the solvent programme: 10% methanol in water to 100% methanol, linear gradient, during 15 min, flow rate 2 ml min -1, monitored at 405 nm and connected to a Hewlett Packard (Avondale, PA, U.S.A.) 3390 A plotting integrator; the integrator was calibrated for read out in nmol by the use of [14C]DAB metabolites of proven identity. The retention times for this HPLC system are given in Table I.

Collection and analysis o f biliary metabolites Experiments were performed with male Wistar rats (200 g) inbred at the

Courtauld Institute of Biochemistry. One mg of the appropriate aminoazo-dye, suspended in 1.0 ml of 1%

bovine serum albumin in 0.9% saline, was injected into the hepatic portal vein and bile collected as previously described [9,16]. Fractions were collected each 5 min for 2 h, kept at -10°C protected from light, and stored, prior to analysis, at -30°C. After measurement of its volume, each fraction was diluted to 1.0 ml with water and 20 pl of this solution analysed by HPLC. Additional bile samples, from identically treated animals, were examined immediately after collection.

When rats were to be treated with diethylmaleate, 200 ~1 of diethylo maleate was given, i.p., 1 h before administration of DAB. When rats were to be treated with sulphotransferase inhibitor, 2.0 mg of pentachlorophenol in 200 pl of propylene glycol was given i.p. 30 min prior to the administration of aminoazo-dye; in these experiments, rats which received propylene glycol alone were used as controls.

The aminoazo-dye conjugates N-sulphonyloxy-DAB, GSCH2AB and 4'- OH-GSCH2AB were dosed at 0.5 mg, 0.1 mg and 0.3 mg/animal, respectively.

310

RESULTS

Separation of metabolites Biliary metabolites of DAB separate on cellulose TLC using n-butanol/

water/pyridine/acetic acid into four main bands (I--IV, Table I and Refs. 9 and 10), corresponding to sulphate (I), glucuronide (II) and glutathione (III, IV) conjugates. HPLC of these bands shows each to be a mixture of conjugates. Thus the sulphate conjugates separate into three peaks, corresponding to 4'-sulphonyloxy-DAB, -MAB and -AB [2--4] ; the glucuronide conjugates into 4'-glucuronides of DAB, -MAB and -AB [2--4] band III into GSCH2AB, its decomposition product AB [9] and minor components including 3-GSMAB [9,12,13] and band IV into 4'-sulphonyloxy-GSCH2AB, its decomposition product 4'-sulphonyloxy-AB [10] and some minor components, which include the glutathione conjugates 4'-hydroxy-GSCH~AB and its glucuronide (see below). These last two conjugates do not separate in the HPLC system used here, they are shown to be present by their decomposition products 4'-hydroxy-AB and the 4'-~-glucuronide of AB. Retention time data for these metabolites and their decomposition products are given in Table I.

TABLE I

CHROMATOGRAPHIC DATA OF SOME COMPOUNDS

DAB METABOLITES AND RELATED

Compound Rt a Rf b

G c GSCH2AB 10.1 0.63 Band III E 4'-Hydroxy-GSCH2AB d 8.8 0.60 A 4'-Sulphonyloxy-GSCH~AB e 4.4 0.46 Band IV

B 4'-Sulphonyloxy-AB 7.5 F 4'-Sulphonyloxy-MAB 9.1 H 4'-Sulphonyloxy-DAB 10.4

0.86--0.94 g Band I

D AB-4'-~-glucuronide 8.4 C MAB-4'-~-glucuronide 8.0 0.70--0.78 g Band II

DAB-4'-~-glucuronide 9.6

K AB 14.2 I 4'-Hydroxy-AB 12.7

4'-Hydroxy-MAB f 13.8 4'-Hydroxy-DAB f 14.8

1.0 g

aHPLC retention time in min; see Methods for HPLC details. bR -values in TLC, using as solvent n-BuOH/acetic acid/pyridine/water (15 :3 : 10:12). ? CLetters refer to peaks of Fig. 1. dDoes not separate from its 4'-~-glucuronide under the conditions used here. eThe R t is very sensitive to column conditions and varies from 3--6 rain. fNot observed as a metabolite in this study. gCompounds within the bands are incompletely resolved.

311

HPLC analysis of small quantitites of unfractionated bile also results in good separation of ]2AB metabolites (Fig. 1). ]2irect HPLC has the advantage that the decomposit ion of unstable glutathione conjugates which occurs on TLC is obviated and a good separation of metabolites is possible in a single chromatographic analysis.

Quantitative analyses o f biliary metabolites o f DA B in the normal rat The proport ions and absolute amounts of biliary metabolites of I~AB,

excreted by the normal rat during 2-h period following intravenous injection of the aminoazo-dye are given in Table II.

GSH conjugates form approx. 60% of the biliary conjugates and consist of approximately equal quantities of GSCH2AB and 4'-sulphonyloxy- GSCH2AB. Sulphate conjugates form the bulk of the remaining biliary metabolites and glucuronide conjugates are a relatively minor component . 4'-OH-DAB-/3-glucuronide was not detected in these experiments.

AB and 4'-sulphonyloxy-AB are very low in HPLC analyses of fresh bile. They are approx. 1% of G SCF2AB and 3% of 4'-sulphonyloxy GSCH2AB levels respectively and higher in stored bile~ but still correlated with the respective GSCH2 AB and 4'-sulphonyloxy-GSCH2AB levels.

Time course o f excretion o f DAB metabolites in the normal rat The time courses for excretion expressed as the average rate during a

5-min collection period against time, are shown in Fig. 2. The figure shows that the excretion of DAB metabolites is sequential. 4 '-Sulphonyloxy-DAB is excreted first, and, at much the same time, GSCH2AB, these are then

B K

A E

I I 0 1 2 3 4 5 6 7 8 9 10 lll 112 1~] 14 15

rain

Fig. 1. HPLC separat ion of biliary metabol i tes of DAB. Bile was obtained f rom the normal rat 30 rain after in t raper i toneal inject ion of 1 mg DAB. For ident i f icat ion of metabol i tes see Table I. The peak D (AB-4'-~-glucuronide) is no t detectable at this t ime poin t and under the exper imenta l condi t ions (see Table II and Fig. 2). The peak (L) at 12.3 min is due to bile pigments.

312

TABLE II

BILIARY METABOLITES OF DAB IN THE NORMAL, GSH-DEPLETED AND SULPHOTRANSFERASE INHIBITED RAT

Percentage of total biliary metabolites 2 h after administration of aminoazo-dye (absolute amounts in brackets expressed in nmol). Sulphate conjugates were analysed separately in a single experiment only. Figures are the range for 3 experiments.

Metabolite Control + Diethyl- + Pentachloro- maleate phenol

GSCH:AB 24.6--35.6 8.7--14.9 39.5--40.3 (210--221) (24--90) (157--234)

4~-Sulphonyloxy - 20.6--30.0 5.6--14.5 8.6--10.0 GSCH~AB (150--180) (23--40) (39--50)

4'-Hydroxy-GSCH 2 AB 2.1--4.6 Nil 8.6--9.7 and its/3-glucuronide (13--36) (50--88)

Total GSH 47.3--70.2 4.3--29.4 56.7--60.0 conjugates (373--437 ) (47--130) (246--372)

4'-Sulphonyloxy-AB

4'-Sulphonyloxy-MAB

4'-Sulphonyloxy-DAB

Total sulphate conjugates

6.9--10.2 5.5(9) 3.9--8.7 (43--81) (23--35)

10.6--15.4 29.8(49) 3.5--5.7 (66--135) (20--23)

11.4--19.4 23.4(38) 5.0--6.1 (71--171) (24--29)

28.9--45.0 47.5--64.2 12.4--20.5 (180--387) (78--384) (67--87)

AB 4'-~-glucuronide 0.1--0.2 Nil 0.5--1.0 (2--3) (7--8)

MAB 4'-~-glucuronide 4.7--6.8 16.1--25.0 20.5--28.5 (34--60) (17--50) (82--165)

Total glucuronide 4.8--7.0 16.1--25.0 21.0--29.5 conjugates (36--63) (17--50) (89--173)

% of dose excreted 16--22 6--15 10--15 in 2 h (681--882) (193--598) (398--580)

fo l lowed by 4 ' - su lphonyloxy-MAB and 4 ' - su lphonyloxy-GSCH2AB. 4'- Su lphonyloxy-AB is exc re ted at the same t ime as 4 ' - su lphonyloxy-GSCH2AB. ~-Glucuronide conjugates are the last to be excre ted . The absolute pos i t ion of the ma x im a observed in these t ime courses varies slightly f rom one expe r imen t to another , bu t the order n o t e d above is always sustained. The average maximal rates of exc re t ion occur at the fol lowing times: 4 ' -sulphony- l o xy DAB, 22 min; GSCH2AB, 23 min; 4 ' - su lphonyloxy-MAB, 30 min; 4 ' - su lphonyloxy-GSCH~AB, 52 min; 4 ' - su lphonyloxy-AB, 52 min; MAB-4'- ~-glucuronide, 65 min.

The effect of diethylmaleate treatment The dose o f d ie thy lmalea te used here lowers the levels of hepat ic GSH

to app rox ima te ly one f i f th o f tha t in normal animals and this lower level is

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maintained throughout the experiment [17,18] . GSH conjugate levels are reduced some 4-5~ fold in quanti ty while levels of sulphate and 3-glucuronide conjugates are similar to those found in the normal rat (Table II). Concern- ing decomposi t ion products of the GSH conjugates, 4'-sulphonyloxy-AB is very low and AB is not detected.

Although GSCH2AB excretion is low, the point at which the maximal rate of excretion is achieved is essentially unchanged (Fig. 2I~). However, excretion of sulphated metabolites is much delayed (Fig. 2E).

The effect o f pentachlorophenol treatment Intraperitoneal injection of the sulphotransferase inhibitor, pentachloro-

phenol [19,20] causes the amounts of all sulphated metabolites to be reduced to approximately one third of that in the normal rat while the amounts of MAB and AB glucuronides is correspondingly increased some three-fold (Table II). The absolute amount of GSCH2AB (which is largely excreted within 1.5 h, see Fig. 2A) is similar in pentachlorophenol-treated and control animals.

Under these conditions of partial sulphotransferase inhibition an additional peak of R T 9.2 min becomes evident. This was found to contain both 4'-hydroxy-GSCH2AB and its 4'-3-glucuronide. The compounds showed the same lability, on standing or on acid treatment, as the well characterised GSH conjugates GSCH2AB [9] and 4'-sulphonyloxy-GSCH2AB [ 10] and were identified by their decomposi t ion products and by comparison with syn- thetic materials. The increase in importance of these GSH conjugates on sulphotransferase inhibition maintains the high level of C SH conjugates in the bile.

Metabolism and excretion of individual metabolites The proportions and absolute amounts of biliary metabolites which result

from a single i.v. injection of four selected DAB metaboli te in the normal and pentachlorophenol-treated rat are given in Table III.

(1) 4'-OH-DAB. Biliary metabolites of 4'-OH-PAB account for a greater proport ion of the dose than those of DAB. However, GSH conjugates again form some 60% of the biliary metabolites and the 4'-sulphates of -EAB, -I~AB and -AB prove to be of no more relative importance than in the case of DAB, despite the presence of preformed hydroxyl group. The time courses of excretion of biliary metabolites common to 4'-CH-BAB and l~AB (Fig. 3) are also similar.

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material which is presumably unchanged together with conjugates of 4'-GH- AB, apparently derived from deconjugated GSCH2AB. Neither 4'-CH- GSCH2AB nor 4'-sulphonyloxy-GSCH2AB were detected as metabolites of GSCH:AB. It is not known whether deconjugation of GSCH2AB in vivo occurs spontaneously or as the result of the action of an enzyme such as 7-glutamyl transpeptidase which has been shown to cause decomposition to AB in vitro [9].

(3) 4'-OH-GSCH2AB. 4'-CH-GSCH2AB is excreted as rapid pulses of 4'-GH-GSCH2AB and then 4'-sulphonyloxy-GSCH2AB (Fig. 5) followed by sulphate and glucuronide conjugates of 4'-OH-AB. Sulphotransferase inhibition affects excretion in much the same way as it does 4'-OH-DAB metabolism.

(4) 4'-sulphonyloxy-DAB. The pattern of excretion of biliary metabolites of 4'-sulphonyloxy-DAB (Fig. 6) shows that although a portion of the 4'-sulphonyloxy-DAB appears to be excreted unchanged (cf. Figs. 6B and 6D

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Fig. 5. Biliary excret ion o f 4 ' - sulphonyloxy-GSCH2AB as the result o f i.v. injection of 4' -hydroxy-GSCH2AB. (The means o f 2 experiments) .

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319

with Figs. 3B and 3D) some clearly undergoes deconjugation to 4'-OH- DAB. This fraction undergoes all the reactions for 4'-OH-DAB described above including G SH conjugation and sulphation and consequently is sensitive to sulphotransferase inhibition.

DISCUSSION

Role of GSH in biliary excretion of DAB metabolites Our results clearly indicate that, contrary to earlier assumptions [6--8],

GSH conjugates form the major biliary metabolites of I~AB in the rat. The GSH conjugates concerned are GSCH2AB [9] and 4'-sulphonyloxy-GSCH2AB [10]; together they account for up to 70% of the total biliary metabolites. Previous authors incubated bile with enzyme preparations intended to hydrolyse sulphates and glucuronides, a procedure which we now know also deconjugates the above GSH conjugates which are relatively unstable. In previous work AB appeared to be an important component [3--8]; this is contrary to expectation, since biliary metabolites are usually conjugated or, if not conjugated, already amphipathic, like indocyanine green [21]. The presence of AB in bile in this earlier work would appear to be due to the decomposi t ion of GSCH2AB [9]. Likewise most, if not all, of the 4'-sulphonyloxy-AB observed in earlier work is probably a decomposit ion product of 4'-sulphonyloxy-GSCH2AB [ 10]. The excretion of AB and 4 '-sulphonyloxy AB are simultaneous with that of GSCH2AB and 4'-sulphonyloxy-GSCH2AB, respectively (Fig. 7 ).

The mechanism of formation of GSCH2AB has been studied in vitro, by the incubation of MAB with a microsomal system in the presence or absence of GSH and an inhibitor of N-methyloxidation [9]. It was shown that GSCH2AB results from the reaction of GSH with a reactive N-methyl oxidized derivative of MAB such as an N-methylol or an N-methimine. This reaction is non-enzymic with the result that the rate of formation of GSCH2AB is in direct proport ion to GSH concentration [9] . The mechanism of formation of 4'-OH-GSCH2AB from 4'-OH-MAB is assumed to be similar. The present work shows that the above mechanism (established from in vitro experiments) also operates in vivo. The depletion of liver cytosolic levels of GSH by diethyl maleate from approx. 10 to 2 mM causes a fall in absolute values for total GSH conjugates excreted over 2 h from a mean of 405 nmol to a mean of 88 nmol, i.e. a drop in hepatic GSH concentration to approx. 20% results in a similar drop in GSH conjugate formation. If electrophilic metabolites of DAB were substrates for GSH transferases it is likely that not only would the rate of conjugation be greater but also, since the Kin-value for GSH with regard to most GSH transferases is 0.2 mM [22], substantial GSH depletion could occur without affecting the rate of GSH conjugation.

Pathways of DAB metabolism Assuming that neither conjugation nor biliary excretion are rate limiting,

the sequential excretion of biliary metabolites of DAB, reflects rates of

320

30

c 20

=e ;o

o A

I I I l I [ I i I L I I 29 40 50 80 100 120

Minu tes

¢ GSCH2AB ~ AB

20

$.. E 10

E 0

fl 20 40 60 80 l O0 120 Minu tes

o 4Lsulphonyloxy .GSCI~AB ^ 4t.sulphonyloxy .AB

Fig. 7. Biliary excretion of metabolites of DAB: (A) GSCH2AB and AB; (B) 4'-sulphonyloxy-GSCH~AB and 4'-sulphonyloxy-AB. (Data from a single experiment to show the coincidence maximal rates of excretion of (A) GSCH:AB and AB and (B) of 4'-sulphonyloxy-GSCH~AB and 4'-sulphonyloxy-AB).

oxidation by mixed function oxygenation. For example the excretion of 4'-sulphonyloxy-DAB and GSCH~AB are almost simultaneous i.e. with DAB as substrate the two N-methyloxidations occur within the same time period as the 4'-hydroxylation. On the other hand 4'-sulphonyloxy-lVAB is excreted later than 4'-sulphonyloxy-DAB or GSCH2AB and 4'-sulphonyloxy-GSCH2AB later than 4'-sulphonyloxy-MAB. Apparently the introduction of the 4'- hydroxyl group reduces the rate of the two N-methyloxidations so that they become readily separated in time.

Steps in DAB metabolism have also been examined by Levine et al. [23] using microsomal oxidation in vitro. As GSH was not included in the incubation mixtures, N-methyloxidation of DAB and MAB gave the desmethyl compounds only. Even so ip. agreement with our findings DAB and MAB were found to be more rapidly N-methyl oxidized than 4'-OH-I)AB and 4'-OH-MAB, respectively.

A comparison of the effects o f GSH depletion and sulphotransferase inhibition

GSH depletion decreases the excretion of GSH conjugates but does not

321

result in a compensatory increase in either sulphate or glucuronide conjugates: rather GSH depletion causes a delay in the excretion of sulphate conjugates. The result is a marked reduction in total biliary excretion of DAB metabolites during the period of observation. It would seem that aminoazo-dye may be less rapidly hydroxylated in the absence of GSH, but the mechanism of this effect is not known.

In contrast, sulphotransferase inhibition reduces sulphate conjugation but there tends to be a compensatory increase in glucuronide conjugates and little effect on GSH conjugates. It is interesting to note that 4'-OH-GSCH2AB as well as its glucuronide are excreted in place of 4'-sulphonyloxy-GSCH2AB. Although the total excretion of BAB metabolites is reduced somewhat by sulphotransferase inhibition, it would seem (Fig. 3) that much of this de- crease is due to the slow excretion of glucuronide conjugates.

The importance of GSH in N-methyloxidation of MAB It appears from this work that GSH is fundamental to the detoxicati(~,:

of MAB. Whereas N-methyloxidat ion of DAB gives the desmethyl compound MAB, N-methyloxidat ion of MAB gives an N-methylol or methimine which requires to be conjugated with GSH if the N-methyloxidat ion of MAB is to proceed at a rapid rate in vivo. GSH depletion does not result in an increase in the desmethyl product of MAB, AB, in vivo, indeed AB levels fall with GSH depletion.

The importance of duration of bile collection Because of the sequential excretion of biliary metabolites of E AB, the

time-period of collection has a pronounced effect on the relative contribu- tions of individual metabolites to the whole. A short collection period, gives a false impression of the importance of GSCH2AB and 4'-sulphonyloxy-DAB [6--8]. The 2 h collection period used in the present work has enabled a number of biliary metabolites to be determined. Even so the contr ibution of glucuronides is low in this time scale. If a larger dose is given, e.g. 50 mg i.p. and the time of collection is longer, e.g. 24 h, the relative proport ion of glucuronide fraction becomes greater [9] and metabolites include 4'- OH-DAB-~-glucuronide (Coles et al., unpublished).

The mechanism of formation of 4'-sulphonyloxy-GSCH2AB 4'-Sulphonyloxy-GSCH2AB, which is shown to be such an important

metaboli te in the present work, appears to be an uncommon though perhaps not unique [24] type of double conjugate. From information provided in this paper it is now possible to propose a mechanism for its formation. For example, whereas 4'-OH-DAB and 4'-OH-GSCH2AB are precursors, GSCH2AB is not. This implies a reaction pathway in which 4'-hydroxylation is the first step, N-methyloxidat ion the second, GSH conjugation the third and 4'-O-sulphation the final step. Sulphotransferase inhibition which results in the excretion of 4'-OH-GSCH2AB and its glucuronide provide confirma- tion that 4'-O-sulphation is the final step.

322

Although the results presented here provided what might be regarded as a new view of aminoazo-dye detoxication, it should be noted that it was presaged by the work of Mueller and Miller [25], who observed that the addition of GSH to an in vitro system for the N-demethylation of 3-methyl- DAB produced a water soluble labile dye which was suggested to be a GSH conjugate.

ACKNOWLEDGEMENT

We thank the Cancer Research Campaign, of which BK is Ralph Bateman Fellow, for their generous support.

REFERENCES

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3 M. Ishidate, Z. Tamura and K. Samejima, Metabolism of 4-dimethylaminoazobenzene and related compounds. III. Metabolites of 4-dimethylaminoazobenzene in rat bile and influence of DAB feeding on their amounts, Chem. Pharm. Bull., 11 (1963) 1014.

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7 W.G. Levine and T.T. Finkelstein, A role of liver glutathione in the hepatobiliary fate of N,N-dimethyl-4-aminoazobenzene, J. Pharmacol. Exp. Ther., 208 (1979) 399.

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9 B. Ketterer, S.K.S. Srai, B. Waynforth, D.L. Tullis, F.E. Evans and F.F. Kadlubar, Formation of N-(glutathion-S-methylene)-4-aminoazobenzene following metabolic activation of the N-methyl group of the carcinogen, N-methyl-4-aminoazobenzene, Chem.-Biol. Interact., 38 (1982) 287.

10 B. Coles, S.K.S. Srai, B. Ketterer, B. Waynforth and F.F. Kadlubar, Identification of 4'-sulphonyloxy-N-(glutathion-S-methylene)-4-aminoazobenzene, a compound conjugated with both sulphate and glutathione, which is a major biliary metabolite of N, N-dimethyl-4-aminoazobenzene, Chem.-Biol. Interact., 43 (1983) 123.

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12 B. Ketterer, F. Kadlubar, T. Flammang, T. Carne and G. Enderby, Glutathione

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14 J.T. Hewitt and W. Thomas, The colour and constitution of azo compounds, Part HI, J. Chem. Soc., 95, 1282.

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Documents

The major role of glutathione in the metabolism and excretion of N,N-dimethyl-4-aminoazobenzene in the rat