The mercapturic acid pathway metabolites of a glutathione conjugate of aflatoxin B1

139

Chem.-Biol. Interactions, 55 (1985) 139-155 Elsevier Scientific Publishers Ireland Ltd.

THE MERCAPTURIC ACID PATHWAY METABOLITES GLUTATHIONE CONJUGATE OF AFLATOXIN B1

ELIZABETH

J. MOSS*,

G.E. NEAL

OF A

and D.J. JUDAH

MRC Toxicology Unit, Medical Research Carshalton, Surrey, SM5 4EF (U.K.)

Council

Laboratories,

Woodmansterne

Road,

(Received January 30th, 1985) (Revision received May 27th, 1985) (Accepted June 17th, 1985)

SUMMARY

A glutathione conjugate of aflatoxin B1 (AFB,) which has previously been identified as 8,9-dihydro-8-(S-glutathionyl)-9-hydroxy aflatoxin B1 (AFB1-GSH) (E.J. Moss, D.J. Judah, M. Przybylski and G.E. Neal, Biochem. J., 210 (1983) 227-233) has been degraded in vitro to all of the intermediates of the mercapturic acid pathway (MAP) and the chromatographic and spectral characteristics of each of these compounds investigated. The cysteinylglycyl conjugate (AFB1-Cys.Gly) was prepared by incubating the AFB,-GSH conjugate with a rat hepatoma cell line rich in y-glutamyltranspeptidase (GGT). Incubations of the AFB,-Cys.Gly conjugate with dipeptidase produced a metabolite, which was purified and characterized by ‘H-NMR spectroscopy as 8,9dihydro-8-(S-cysteinyl)-9-hydroxy aflatoxin B1 (AFB,-Cys). The N-acetyl derivative of the AFB,-Cys conjugate resulted from the incubation of the AFB1-GSH conjugate in vitro with isolated rat kidney cells. Mass spectral data were consistent with the compound being 8,9-dihydro-8-(S-cysteinyl-(N-acetyl))-9-hydroxy aflatoxin B1 (AFB1-Nac.Cys). A chromatographically identical compound was obtained by the chemical acetylation of AFB1-Cys.

Key

words:

Aflatoxin

-

Glutathione

-

Mercapturic

acid -

Kidney

cells

*Present address: BIBRA, Woodmansterne Road, Carshalton, Surrey, U.K. Abbreviations: AFB,, aflatoxin B, ; AFB,-Cys, 8,9-dihydro-8-(S-cysteinyl)-9-hydroxy aflatoxin B,; AFB,-Cys.Gly, 8,9-dihydro-8-(S-cysteinylglycyl)-9-hydroxy aflatoxin B,; 8,9-dihydro-&(S-glutaAFB,-dhd, 8,9-dihydroxy-8,9-dihydro aflatoxin B,; AFB,-GSH, thionyl)-9-hydroxy afiatoxin B, ; AFB,-Nac.Cys, 8,9-dihydro-8-(S-cysteinyl)-(N-acetyl)9-hydroxy aflatoxin B,; FAB-MS, fast atom bombardment mass spectrometry; GGT, r-glutamyltranspeptidase (EC 2.3.2.2); GSH, reduced glutathione; HPLC, high performance liquid chromatography; MAP, mercapturic acid pathway; THF, tetrahydrofuran; ‘H-NMR, proton nuclear magnetic resonance. 0009-2797/85/$03.30 o 1985 Elsevier Scientific Publishers Printed and Published in Ireland

Ireland

Ltd.

140 INTRODUCTION

The potent hepatotoxin and hepatocarcinogen, AFBI, is believed to require metabolic activatioh by liver microsomal enzymes to exert its toxic/ harmful effect [l]. There is strong evidence that the reactive metabolite responsible for macromolecular binding is aflatoxin-8,9-epoxide [ 1,2] and the interaction of this reactive electrophile with nucleic acids and other cellular nucleophiles has been the subject of several investigations [3-71. The epoxide has not to date been isolated, but the hydrolysis product, 8,9-dihydroxy-8,9-dihydro aflatoxin Bi (AFB1-dhd) has been characterised and shown to be a major product of liver microsomal metabolism under suitable conditions [8,9]. Many reactive electrophiles are detoxified by conjugation with glutathione catalysed by glutathione transferase (EC 2.5.1.18) and are subsequently degraded by the MAP and then are eventually excreted as the mercapturates [lo]. A polar metabolite of AFB, originally reported to be formed in rat liver systems in vitro [ll] was subsequently characterised by ‘H-NMR and mass spectral studies and positively identified as a glutathione conjugate of AFB,-8,9-epoxide, the AFBi-GSH 1121. In view of the potential importance of this pathway in the metabolism and excretion of AFBi in exposed human populations, the present study was undertaken to characterise the MAP products of the metabolism of the AFBi-GSH conjugate, formed by various in vitro systems. MATERIALS

AND

METHODS

Chemicals AFBi was obtained from Makor Chemicals (Jerusalem, Israel) and [ 14C]AFBi (spec. radioact., 120 mCi mmol-‘) from Moravek Biochemicals (City of Industry, CA, U.S.A.). Compound AT125 (L-(aS,5S)-2-amino-2-(3chloro-4,5-dihydro-isoxazol-5-yl)acetic acid) was a gift generously provided by the Drug Synthesis and Chemistry Branch, Division of Cancer Treatment, National Cancer Institute (Bethesda, MD, U.S.A.) AFB,-GSH and [ 14C]AFB1-GSH were prepared from AFBi or [14C]AFB1 by the method described previously [12] using quail microsomes and mouse 100 000 X g supematant fraction. l-[14C]Acetyl CoA (53 mCi mmoll’) was obtained from Amersham International (U.K.). Cell and enzyme preparation and assays Dipeptidase was prepared from pig kidney, obtained fresh locally [ 131. A hepatoma cell line (JBl) rich in GGT on the outer membrane surface derived in this laboratory from an AFB1-induced liver tumour in a male Fischer F344 rat [14] was routinely cultured on plastic petri dishes (100 mm diam.) containing 9 cm3 of medium consisting of Williams Medium E (Flow Laboratories, Irvine, Scotland) supplemented with 5% foetal calf serum, 2 mM glutamine and 50 pg gentamycin/cm3.

141 Isolated cell suspensions were prepared from the kidneys of single rats (180-200 g body wt.) by collagenase perfusion (0.12% collagenase in Hanks buffered salt solution containing 4 mM CaCl,) by a modification of the method of Jones et al. [15]. Bovine albumin powder (Fraction V) (Armour Pharmaceutical Co. Ltd., Eastbourne, U.K.) was used in the first perfusion medium. Experiments in which the kidneys were incompletely blanched were not proceeded with. After perfusion with collagenase (2025 min) and trimming away the connective tissue, the kidneys were transferred to a measuring cylinder containing 50 cm3 Hanks Balanced Salt Solution without calcium or magnesium plus 3.5 cm3 of a 35% solution of bovine albumin in saline (Sigma (London) Chemical Co., U.K.). The kidneys were broken by gentle agitation with a glass rod and transferred to a round bottomed flask and rolled at room temperature for 5-10 min. The kidney tissue was disrupted further by sucking up and down in a widemouth 10 cm3 glass pipette. Tissue was sedimented by centrifugation at low speed followed by resuspension in approx. 20 cm3 Minimum Essential Medium with Earls salts minus phenol red (MEM). Tissue was then resedimented by centrifugation followed by resuspension in 10 cm3 of the MEM medium (total cells from 1 kidney/5 cm3 medium). Microscopic examination showed that the majority of the preparation consisted of short lengths of tubules. Addition of trypan blue demonstrated that the majority of the cells in the tubules excluded the dyestuff and on this criterion would be considered viable. An occasional cell in a tubule and a rather higher percentage of the small content of single cells became stained with trypan blue. Only preparations with overall viabihties in excess of 80% were proceeded with. Incubations were carried out in the MEM medium. Microsomal fractions were prepared from pig kidneys after homogenisation in 150 mM KCl. The microsomal fractions, after washing and resedimenting at 100 000 X g for 90 min, were stored at -70°C as suspensions in 150 mM KC1 (1 g fresh wt. tissue/l.6 cm3 suspension). Microsomal Nacetyl transferase activity was assayed using benzyl cysteine and l-[14C]acetyl CoA (0.64 pCi/assay) as substrates [ 161. N-Acetyl transferase activity using AFB,-Cys as substrate was assayed by HPLC. Protein was assayed by the method of Lowry et al. [17]. Incubation systems used to study the MAP metabolism of AFBl-GSH (a) Metabolism of AFBl-GSH to AFBI-Cys.Gly by GGT. AFB1-GSH or [14C]-AFBi-GSH (SA 0.7 mCi mmol-‘) was incubated with monolayer cultures of JBl cell line grown in 100 mm Petri dishes (approx. 10’ cells) from which the normal growth medium (Williams E) had been removed and replaced with 22 mM glycylglycine in phosphate-buffered saline (0.15 M NaCl/12 mM sodium phosphate buffer, pH 6.9). Substrate was added .to give a concentration of 5 PM in a final incubation volume of 10 cm3 and the cultures were incubated at 37°C in air/CO2 (19: 1). Portions (100 ~1) were removed at intervals and samples were prepared for HPLC and analyzed for disappearance of AFBi-GSH (see chromatographic procedures).

142 Control incubations were carried out in which 1 mM AT125, a specific inhibitor of GGT [18], was included. Samples of AFB1-Cys.Gly. to be used in further incubations were isolated by HPLC (see Chromatographic Procedures). Metabolites were quantified by reference to 14C-labelling. (b) The metabolism of AFB,-Cys.Gly to AFB,-Cys using pig kidney dipeptidase. AFB1-Cys.Gly (15 pg, 30 nmol) was incubated at 37°C with

dipeptidase (250 ~1) in 0.1 M Tris-HCl, pH 8, 10 mM MgC12) in a final volume of 2 cm3. Portions (100 ~1) were removed at intervals and samples were prepared for HPLC and analyzed for disappearance of AFB,-Cys.Gly. (c) The metabolism

of AFB1-GSH

to AFBl-Nac.Cys

using rat kidney

cells. AFBI-GSH (9.6 nmol in analytical and 50 nmol in preparative experiments) was incubated with rat kidney cell preparations. The AFB,-GSH substrate was dissolved in 600 ~1 of MEM and added to 5 cm3 MEM without phenol red containing the total cell product from 1 rat kidney in a 100 cm3 round-bottomed flask. Flasks were incubated at 37°C in a swirling incubation apparatus under a stream of OZ/COZ (19: 1, v/v). Aliquots (50 ~1) were taken at intervals and analysed by HPLC after addition of methanol followed by centrifugation and filtration. Samples were analysed for the disappearance of AFB1-GSH, and the appearance of new metabolites. In preparative experiments the incubations were carried out as above and continued for 16 hrs. The flasks were then removed from the incubation apparatus and the contents freeze dried. Samples were reconstituted in water/methanol as described under Chromatographic Procedures. (d) Incubation system used in an unsuccessful attempt to form AFB1Nac.Cys. Experiments were carried out in which AFBI-Cys (9.6 nmol)

was incubated with pig kidney microsomal fractions in the presence of 0.1 mM [14C]acetyl-CoA. The microsomal fractions had been found to be active in N-acetylating benzyl cysteine as a substrate in preliminary experiments. Samples were analysed by HPLC. Chromatographic procedures (a) Analysis of metabolites by HPLC. Samples

were prepared in methanol/ water (1: 1, v/v) and clarified by centrifuging at 1500 X g for 45 min at -15°C. All samples (except those analyzed by solvent systems 4 and 5, see below) were maintained at pH 3-4 which improved the profiles of the peaks eluted from the HPLC columns. Gradient HPLC was carried out using either 2 M6000 A pumps coupled to a 660 gradient programmer (Waters Associates, Northwich, Cheshire, U.K.) or 2 Constametric III pumps (LDC/Milton Roy, Stone, Staffs, U.K.) controlled by an Apple II+ computer (Apple UI Ltd., Hemel Hempstead, Herts, U.K.). Solvent mixing was improved by passing the eluting solvent through a 50 mm X 4.6 mm column packed with 40 pm diam. glass beads. UV monitoring at 365 nm was carried out using a Spectromonitor III detector (LDC/Milton Roy) and fluorescence monitoring (excitation 370 nm, emission 418 nm long pass) using either a model 420 (Waters Associates) or a Fluoromonitor III (LDC/ Milton Roy) fluorescence detector. All separations were performed at

143 ambient temperature. In all systems used, 100% final solvent conditions were maintained for 2 min on completion of the gradient. The following chromatographic systems were developed having the particular usages mentioned: System 1. Elution was achieved by a linear gradient methanol/water (15% (solvent A) to 40% (solvent B)) in a constant 8% acetonitrile, 0.02% phosphoric acid, over 9 min, with a flow rate of 1.25 cm3 min-‘. A laboratory packed Magnusphere 5 ODS (Magnus Scientific Instrumentation, Aylesbury, Bucks, U.K.) column (100 mm X 4.6 mm) was used coupled to a guard column (60 mm X 2.1 mm) packed with Co Pell ODS (Whatman LabSales Ltd., Maidstone, Kent, U.K.). This system gave good general separations of the MAP metabolites of AFB,. System 2. All details as for system 1 except that the column used was a cartridge (100 mm X 3 mm) packed with 8 pm CP sphere-C,, (Chrompack (U.K.) Ltd., London, U.K.) and a guard column was not employed. This system, using a commercially-obtained column in place of a laboratory packed one, also gave good general resolution of the MAP metabolites of AFB, (see Table I). System 3. Elution was achieved by a linear tetrahydrofuran/water gradient (4% to 9% over 10 min) in a constant 0.01% phosphoric acid with a flow rate of 1.6 cm3 min-’ . The column details were as in system 1. This system gave improved resolution of AFB1-GSH and AFBI-Cys (see Fig. 4 and Table I). System 4. As in system 1 except that 0.1% formic acid was substituted for phosphoric acid in Solvent A and acid was omitted from Solvent B and the gradient time was reduced to 6 min (see Fig. 6). This solvent system incorporating a volatile acid was used in the purification of samples of AFB1-Nac.Cys for ‘H-NMR and FAB-MS. System 5. All details as for system 4. except elution achieved by a linear gradient of acetonitrile/water (10-40s over 6 min) without acidification. This solvent system was also used in the further purification of samples of AFB,-NacCys for ‘H-NMR and FAB-MS. System 6. Methanol/water gradient as in System 1, but omitting acetonitrile and replacing phosphoric acid with 0.01% formic acid. This system was used in the purification of samples of AFBi-Cys for ‘H-NMR analyses and AFB,-Cys.Gly to be used in further incubations. (b) Preparation of samples for NMR and FAB-MS analysis. Samples of AFB,-Cys resulting from the incubation of AFB1-Cys.Gly with kidney dipeptidase to be used for ‘H-NMR were purified by ion-exchange chromatography (DE-52 DEAE-Cellulose, Whatman Ltd., U.K.) and gel filtration chromatography (Sephadex G-15 bed volume 195 cm3 followed by Sephadex LH-20) by modification of the procedures of Degen and Neumann [ 111. The product was further purified by HPLC using solvent System 6. Samples of AFB1-Nac.Cys resulting from the incubation of AFBI-GSH (approx. 50 nmol/incubation flask) with rat kidney cells were purified by resuspending the freeze dried residues of the incubations in water followed

144

by methanol (final ratio 1: 1, v/v) and centrifuged (60 min/1500 X g) at -15°C. Successive samples of 100-150 ~1 were purified by HPLC using solvent system 4. Acid was omitted because preliminary experiments had indicated a considerable loss of AFB,-Nac.Cys when samples were prepared following chromatography at the pH normally employed. Peaks containing the presumed AFB,-Nac.Cys were combined and freeze dried. The freeze dried material was reconstituted in water/methanol (1: 1, v/v) and rechromatographed in solvent System 5. Fractions containing the presumed AFBl metabolites were collected, concentrated by freeze drying before finally drying down in 5 cm’ micro reaction vials for ‘H-NMR and in the case of AFB1-Nac.Cys for FAB-MS studies. (c) TLC analyses. Compounds were analysed using normal and reversedphase silica TLC plates. (1) Silica gel G plates (10 X 25 cm, 0.25 mm thick, Analtech Uniplates, supplied by Anachem, Luton, Beds, U.K.) or Silica 60 plates (aluminium foil, 0.2 mm thick, Merck, supplied by BDH, Poole, U.K.) were developed in n-butanollglacial acetic acid/water (2 : 1: 1, by vol.). (2) HETLC-RPS (reversed-phase) silica plates (2.5 X 10 cm, Analtech Uniplates, as above) were developed in methanol/acetonitrile/orthophosphoric acid/water (15: 8:0.02: 77, by vol., solvent A in System 1, see previous section). In all cases compounds were located by viewing under long wave UV light. of all metabolites were estimated indi(d) UV, NMR and MS. The E,, vidually by reference to 14C-labelling. These assays were based on pooled HPLC peak fractions obtained following repetitive injections of products of relevant incubations. Background radioactivities were routinely subtracted. The 360 mHz ‘H-NMR spectra were obtained with approx. 0.05 pmol of AFBI-Cys or 0.02 pmol of AFB1-Nac.Cys dissolved in 99.98% ‘Hz0 (40 ~1) by using a Bruker model WH 360 spectrometer operating in the Fourier-transform mode and equipped with a 2.5 mm microprobe. FAB-MS was performed with a VG Analytical 7070E medium resolution mass spectrometer using xenon as bombarding gas at 8 KV energy. The sample was applied in a glycerol matrix. Chemical

acetylation

of AFB1-Cys

AFB,-Cys (25 pg) was dissolved in 0.5 cm3 of a 50% saturated solution of sodium acetate (pH 7.8) containing 2.5 ~1 acetic anhydride in a mini reaction vial. After 30 min stirring in ice reaction was complete. Reaction solution was diluted to 5 ml with water, and passed through a Cl8 ‘Sep-pak’ cartridge (Waters Associates). After washing with 20 cm3 water, product was eluted with 10 cm3 methanol. RESULTS

AND

DISCUSSION

(i) The conversion

AFB,-GSH

of AFB,-GSH

was converted

to AFB,-Cys.Gly

to AFB,-Cys.Gly

using GGT positive

by the transfer

cells

of the y-

145 glutamyl moiety to glycylglycine, catalyzed by GGT present on the cell surface of a ccl! ‘%e. The cell line (JBl) was derived from carcinogeninduced rat hepatoma cells, and has been shown to have high levels of GGT on the external cell surface [14,19]. Incubations of AFB,-GSH or [14C]AFB,-GSH with these cells in the presence of glycylglycine resulted in the disappearance of the conjugate as determined by HPLC analysis (Fig. 1). This disappearance of AFBi-GSH correlated with the appearance of a new radioactive, fluorescent and UV absorbing material which had a longer retention time on reverse phase HPLC (Table I). The kinetics of the conve 4sion of AFBi-GSH to the new aflatoxin metabolite and its inhibition by the specific GGT inhibitor, AT125 are illustrated in Fig. 1. This evidence would suggest that the new metabolite had arisen from the transfer of the y-glutamyl moiety from the glutathione conjugate to glycylglycine in the incubation medium. The new metabolite was tentatively identified as the cysteinyl glycyl conjugate of AFBi (AFBi-Cys.Gly) (Fig. 2). The initial rate of loss of ATB,-GSH was estimated to be 1.66 nmol/h/106 cells. (ii)

The conversion

of AFB1-Cys.Gly

to AFB,-Cys

using kidney

dipeptidase

Incubations of the presumed AFBi-Cys.Gly with kidney dipeptidase preparations resulted in the disappearance of AFB,-Cys.Gly and the appearance of a new aflatoxin metabolite with a shorter retention time on reverse phase HPLC (Table I). The kinetics of its formation are illustrated in Fig. 3. The new metabolite was tentatively identified as AFB,-Cys (Fig. 2). The rate of metabolism of AFB,-Cys.Gly by the dipeptidase preparation was estimated from two determinations to be 20-26 nmol/mg protein/h. An elution system (solvent System 3) was developed to give improved resolution of AFB,-GSH, AFB,-Cys.Gly and AFB,-Cys. The separation of these three metabolites of [ 14C]AFB1 is illustrated in Fig. 4.

Time lhrl

Fig. 1. The conversion of AFB,-GSH (0) to AFB,-Cys. Gly (m) using JBl cell line. In the and in the presence’(o-- -0) of AT125. (HPLC assays performed absence (0 -0) using solvent System 1, see Materials and Methods).

I AND CHROMATOGRAPHIC

PROPERTIES

OF AFB,-GSH,

AFB,-CYS.

2.27

8.6

12.0 0.3

6.9 0.3

0.41 0.32 0.50

20 000 365

AFB,-Cys

Gly

in methanol. in butanobacetic acid/water (2 : 1: 1, by vol.) in methanol/acetonitrile/water/phosphoric acid (15 : 8 : 77 : 0.02, by vol.).

1.97

Gel filtration Sephadex G15 velvo

*Determined bDeveloped ‘Developed

6.7

10.2 1.1

HPLC -System 2 k’ Peak height ratio (UV/fluor) 3

5.4 1.0

HPLC - System I k’ Peak height ratio (UV/fluor)

HPLC -System k’

0.30 0.36 0.65

TLC (Rfvalues)

20 000 365

Chromatographic Silica gel 60b Silica gel G HETLC-RPS=

UV max (1 mol-’ cm-l)” max (acid and alkali)

AFB,-GSH

n.d., not determined; k’, elution time of compound of interest - elution time of unretained pounds. For details of the HPLC solvent systems see Materials and Methods.

SOME SPECTRAL

TABLE AFB,-CYS

3.43

5.9

8.8 0.8

5.0 0.7

0.42 0.34 0.48

20 000 365

AFB, Cys

compounds/elution

GLY,

CYS

n.d.

8.0

11.5 1.2

5.5 1.1

0.52 0.68 0.70

20 000 365

--

AFB,-Nat

time of unretained

and AFB,-NAC.

Cys

com-

147

00 AFB ,

Macromolecular bindinq

4-

-AFtI,-dhd

AFB

- 8.9

epxide Clulathione

COOH

-S -tranrlerase

HO

CO. NHCH2.

COOH AFE 1 GSH

qfycylqfycine &

qlutamyltranrpeptidase

b -qlutamylqlycylqlyclne

HO

CO.NH.CI~~.COOH

ME, Kidney

acetyl

-Cys. Gly.

dwetiv&se

CoA N -aceiyltransferase

CoA q

Fig. 2. Proposed metabolic pathway for formation of AFB,-GSH conjugate from AFB, and subsequent further metabolism of the conjugate via the mercapturic acid pathway.

Fig. 3. The conversion of AFB,-Cys. Gly (0) to AFB,-Cys (m) using kidney (HPLC assays performed using solvent System 1, see Materials and Methods).

I

dipeptidase.

2

1

3

3

A..L r 2

1

.-2 P .k E' Ii -2

2

200 100 0

1

3

,J!LL. 4 8

Retention

time

12

16

( min 1

Fig. 4. HPLC analysis of [“CJAFB,-Cys Gly (3). (Solvent System 3, see Materials tion in arbitrary units. Background subtracted

(l), [“CIAFB,-GSH (2) and [“C]AFB,-Cys. and Methods). Fluorescence and UV absorpfrom radioactive data.

149 (iii) The conversion of AFB,-GSH to AFB,-Nac.Cys using a rat kidney cell preparation When AFB1-GSH (approx. 9.6 nmol) was incubated with rat kidney cell preparations, rapid metabolism to AFB,-Cys.Gly followed by conversion to AFBI-Cys took place. There was subsequently a progressive conversion to a new metabolite. The kinetics of the conversion are illustrated in Fig. 5 and the HPLC properties of the new metabolite, the presumed AFB1-Nat. Cys in Table I. The reason for the lag in the production of the AFB1-Nat. Cys is not known and is being further investigated. When the incubations were repeated on a preparative scale and allowed to proceed for 16 h, there was an almost complete loss of AFB1-Cys, and the presumed AFB,-Nac.Cys was the predominant product. There was however evidence for the presence of another aflatoxin metabolite (Fig. 6). The identity of this further metabolite is at present under investigation. Some loss of metabolites was also evident which was only partially accounted for by the production of the unknown metabolite. This is also being further investigated. The presumed AFB,-Nac.Cys was further purified to allow chemical characterisation. (iv) Attempts to metabolise AFB,-Cys to form AFB,-Nac.Cys Incubations utihsing pig kidney microsome preparations, which catalysed the N-acetylation of approx. 4 nmol of the model substrate benzyl cysteine/h under the incubation conditions used, were not effective in catalysing the N-acetylation of AFB,-Cys as determined by the HPLC assay. The reasons for the failure of the system is at present under investigation.

1Or

TIME

(hr.)

Fig. 5. The conversion of AFB,-GSH (0) to AFB,-Nat. Cys (*) with intermediate formation of AFB,-Cys. Gly (=) and AFB,-Cys (A) using rat kidney cells. (HPLC assays performed using solvent System 1, see Materials and Methods).

A

a-

6 C

I:

A

aC

6a

4-

2-

0’ Retention Time Fig. 6. Formation of longed incubation of metabolite AFB,dhd System 4, see Materials

( nlin~ AFB,-Nac.Cys (A) and unidentified metabolites 0) by the ProAFB,-GSH with rat kidney cells. The highly fluorescent AFB, (C) is included as an internal standard (HPLC analysis using &vent and Methods).

(v) Characterisation of the metabolites

of AFB1-GSH ‘H-NMR data were obtained using purified AFB,-Cys and interpretation of the 360 MHz spectrum obtained (Fig. 7) was assisted by homonudear decoupling experiments and by comparison with the 360 MHz ‘H-NMR spectrum of AFBI-GSH [ 121. Two doublets of doublets at 6 3.11 and 3.36 integrated for one proton each and both collapsed to a doublet when irradiation at the position of one proton triplet at S4.33 was carried out. This information, together with the similar pattern obtained from the fl-cysteine protons of AFB1GSH identified these protons as those of cysteine. The triplet at 64.33

151

8

7

6

5

4

3

2

1

PPnl

Fig. 7. ‘H-NMR For experimental

spectrum of AFB,-Cys details see text.

conjugate

and proposed

structure

of conjugate.

was assigned to the a-proton. The splitting pattern of the fl-cysteine protons appears to be characteristic of attachment of cysteine via the sulphur atom. The aromatic proton and the aromatic O-methyl protons of aflatoxin were readily identified by comparison with literature values as the one proton singlet at 6.63 and the three proton singlet at 64.01. A singlet at 6 5.58 was assigned to the proton (h) at the C-8 position adjacent both to a sulphur and oxygen atom. The proton at C-9 would be expected to appear between 4.5 and 5 ppm from previous data for AFBi-GSH and other literature reports for AFB1-metabolites [20] but the spectrum obtained for AFB,-Cys had a very large signal in this area due to residual protons in *H,O. The signal for the proton at C-9 was not observed. Two doublets at 64.21 and 6.79 have previously been observed in ‘HNMR spectra for AFBi-metabolites [4] and were assigned to the two protons between the furan rings. The signal from the higher field frequency was assigned to the C-9a proton and the lower field signal to the proton adjacent to the two oxygen atoms, O-6 and O-7. Identical coupling constants and decoupling experiments confirmed the cis configuration of these two protons. The signals for the protons of the cyclopentenone ring of aflatoxin were identified as two multiplets at 6 2.67 and 3.45. Irradiation at 63.45 caused a sharpening of the signal at 62.67 and the two proton multiplet at 6 3.45 collapsed to a doublet (J = 6 Hz) on irradiation at S 2.67. Previous workers have assigned the lower field signal to the C-3 protons [4]. Integration for the proton signal at 2.67 ppm suggested slightly more than two protons (2.3-2.5 protons) which may be due to some impurity resonating at the same frequency. The integration for the signal at 3.45 ppm was satisfactory for two protons. This is an important finding in view of a problem that arose in interpreting the AFB,-GSH spectrum described in a previous paper [ 121. The data for AFBi-GSH suggested that one of the pro-

152 tons at C-3 was absent. This raised the possibility that deuterium exchange might have occurred, and that one proton in particular, was favoured to exchange because of the spatial proximity of the amine of the glutamyl moiety in AFBi-GSH. In AFB,-Cys however, the cysteine moiety is too short to fold over and bring the cysteinyl amine adjacent to the C-3 proton. Therefore, if the proposed mechanism is correct, deuterium exchange would not be expected in the AFB,-Cys. The satisfactory integration for two protons in the AFBi-Cys spectrum would confirm that no loss of a proton has occurred, and therefore supports the proposed mechanism for loss of a proton in the AFBiGSH spectrum. As the AFBI-Cys was prepared from AFBi-GSH, the satisfactory integration also confirms that the loss of a proton in the AFBI-GSH spectrum was not due to a permanent substitution at C-3. (‘H-NMR data is summarised in Table II). ‘H-NMR data was also obtained on the proposed AFB,-Nac.Cys (22 nmol) after purification by HPLC as described in the methods section. Homonuclear decoupling experiments and comparison with the ‘H-NMR data from other aflatoxin conjugates assisted interpretation and the data was consistent with an aflatoxin derived material. There was evidence for impurities in the sample, particularly from the signals obtained between 1 ppm and 4 ppm making accurate interpretation of the spectrum difficult. A singlet at 2.01 ppm integrated for 3 protons when compared with the single aromatic proton of the aflatoxin group at 6.66 ppm. This 3 proton signal would be consistent with the presence of a CH,-CO-N-R group in the sample. TABLE ‘H-NMR

II DATA

FOR PROPOSED

AFB,-CYS

CONJUGATE

The ‘H-NMR data for AFB,-Cys. refer to the 360 MHz spectrum in ‘H,O/‘HCI. Chemical shifts are given in p.p.m. from an external standard sodium 3-trimethylsilyl[2,2,3,3,-‘H, ]propionate. Proton

Chemical shift (6, p3.m.)

Relative no. of protons?

Multiplicity

Coupling

ab (1)

3.11 2.67

12-3

dmofd

3.36

1

d of d

J = J= J= J=

ii

3.45 4.01

23

Sm

H

4.33 4.21

1

td

Not visible 5.58 6.63 6.79

1 1 1

-

it i j

(2)

‘Relative

no. of protons

estimated

to the nearest proton

7.5 Hz 14Hz 6Hz 14Hz

JJ== 5.2 6Hz Hz

S

sl from the integration.

J = 5.2 Hz

153

90 80 70 bo -

50

53

40 30 20 -

275

33 P

i

,-

I II

‘0

50

100

Fig. 8. FAB-Mass

150

200

Spectrum

250

300

350

400

450

500

550

of the AFB,-Nac.Cys.

The negative ion spectrum obtained when the proposed AFB,-Nac.Cys conjugate was subjected to FAB mass spectrometry is shown in (Fig. 8). The M-l ion at m/e 490 is consistent with a molecular weight for the compound of 491 g mol-’ which would agree with the structure of AFB,Nac.Cys. The major ions in this spectrum, m/e 91 and 183, are related to the glycerol used as solvent. (vi) Analysis of chemically acetylated AFB,-Cys The material, isolated by absorption onto a ‘Sep-Pak’ cartridge following the chemical acetylation of AFB,-Cys, was subjected to HPLC analyses using the solvent systems described above. In all separations the predominant UV absorbing, fluorescent product (>90% of the total UV absorbing/fluorescing material) was found to elute at a similar retention time to that of the enzymically-formed presumed AFB,-Nac.Cys. Identity was further indicated by co-chromatography of the two compounds in ‘spiking’ analyses. (vii) Possible relevance of results The present study has positively identified the individual MAP metabolite of the AFB1-GSH conjugate. The whole pathway can be catalysed by rat kidney cells. It is possible that identification and quantification of AFBimercapturate could be of value in monitoring exposure of human populations to the toxin. This possibility is supported by the results of preliminary experiments in which we have positively identified AFB,-mercapturate in urine samples collected from marmoset monkeys following the administration of AFB1. These studies are being continued.

154 ACKNOWLEDGEMENTS

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