Characterization of water-soluble glucuronide and sulphate conjugates of aflatoxin B1. 1. Urinary excretion in monkey, rat and mouse

FdChem To~c Vol 23, No 9~ pp 809-819, 1985

0278-6915,'85 $300+000 Copyright ~" 1985 Pergamon Press Ltd

Printed xn Great Britain All rights reserved

C H A R A C T E R I Z A T I O N OF W A T E R - S O L U B L E G L U C U R O N I D E A N D S U L P H A T E C O N J U G A T E S OF A F L A T O X I N B 1. 1. U R I N A R Y E X C R E T I O N IN M O N K E Y , R A T AND MOUSE* C I. WE1 and M R MARSHALL Food Soence and Human Nutrition Department. University of Florida. Galnesvllle. FL 32611 and D P H HstEI4 Department of Environmental Toxicology, Umverslty of Cahforma, Davis, CA 95616. USA (Received 20 Juh' 1984)

Abstract--Water-soluble aflatoxm conjugates prepared from urine samples from rats. mice and rhesus monkeys dosed wah [~C]aflatoxln B~ (AFBt) ip or 1~ were hydrolysed by enzymes (fl-glucuron,dase and sulphatase), acid or a combination of both treatments Different amounts of AFB~ and its metabohtes were found in hydrolysates from different sources, indicating the presence of glucuronlde, sulphate and possibly mercapturate conjugates of aflatoxlns In addition to aflatoxms M~, P~. Q~ and B2a, AFB t was frequently identified in the products released from the hydrolysates These water-soluble aflatoxln conjugates were not mutagenlc to Salmonella t~phtrnurtum TA98 In the presence of rat-hver S-9 mix However. chloroform extracts of the hydrolysates from fl-glucuromdase and sulphatase treatment showed mutagenlc actlvlt~ m these bacteria m the presence of S-9 mix Although very low levels of AFB~ radioactivity were detected in the hydrolysates, the potent mutagemc actwlty of AFB~ contributed to the high numbers of revertant colonies AFP t was detected in urine samples from monkeys that were pretreated with phenobarbital before an Jv dose of AFB~ No mutagenlc activity was detected m the enzymatic hydrolysate of the sample from these monkeys The results thus mdlcate that AFB~ can form glucuronlde and:or sulphate conjugate(s) directly and be excreted m the urine

INTRODUCTION Aflatoxln B~ ( A F B 0 ~s a secondary fungal metabohte produced by Aspergdlus flat us and A. parasmcus It ~s a potent hepatotoxin and carcinogen affecting a wide variety of animal species (Newberne & Butler, 1969) The toxic, mutagenlc and carcmogemc effects of th~s mycotoxin are dependent on the metabohsm of the c o m p o u n d by liver mixed-function oxygenases ( M F O ) and other related enzymes (Campbell & Hayes, 1976). Metabolism of AFB~ includes epoxidation of the 8,9 double bond (kin, Kennan, Mdler & Miller, 1977, Swenson, Miller & Mdler, 1974) and hydroxylation on both furan and lactone rings (Hselh, Wong, W o n g et al. 1977), together with oxidative demethylation (Dalezios, Wogan & Weinreb, 1971), resulting m a variety of polar metabohtes These metabohtes are excreted as such or are conjugated as glucuromdes, sulphates or glutathione conjugates (Raj & Lothkar, 1984) before their excretlon m the bile and urine. Water-soluble conjugates of AFB~ are detoxlficat~on products Recently, Degan & N e u m a n n (1978) showed that the A F B ~ - G S H conjugate (8,9-&hydro-(S-glutathlonyl)-9-hydroxy-AFB0 *Florida Agricultural Experiment Station Journal Series No 5745 Abbrevlattons AF = aflatoxm, AFL = aflatoxlcol, GSH = glutathlone, HPLC = high-pressure llqmd chromatography, MFO=mlxed-funcuon ox~genases, PB = phenobarbital. TLC = thin-layer chromatograph)

is a major metabohte, not extractable with chloroform-ethyl acetate, in the bile of female Wlstar rats injected ip with AFB~ Gregory & Edds (1984) detected unidentified A F B t conjugate(s) in the livers of male turkey poults F o r m a t i o n of the watersoluble metabohte(s) was enhanced by dietary supplementatlon with selenium A similar conjugate was detected m the culture m e d m m of primary rat hepatocytes exposed to AFB~ (Loury, Hsieh & Byard, 1984) Pretreatment of rats with phenobarbital Increased the level of AFB~-GSH conjugate but reduced the total binding of AFB~ to D N A and R N A . Comparative studies (mctvo) in the rat and hamster indicated that inactivation ofAFB~ epoxide via glutathlone conjugation may play an important role m determining A F B L - D N A binding and hence AFB~ hepatocarcinogenesls in sensitive (rat) and resistant (hamster) speoes (Ra] & Lothkar, 1984). The hydroxylation and oxidative demethylatlon products of AFBt (AFM~, AFQ~ and A F P 0 have been shown to form glucuronlde and sulphate conjugates (Chipley, Mabee, Applegate & Dreyfuss, 1974, Dalezlos & Wogan, 1972, Dalezios et al 1971, Gregory, Goldsteln & Edds, 1983) Recently, Wei, Decad, W o n g et al (1978), Gregory et al (1983) and Gregory & Edds (1984) showed that direct conjugation of AFB~ with glucuronic acid and sulphate occurred both m vtvo and in hepatocyte cultures These conjugates can be hydrolysed by fl-glucuromdase and sulphatase or by acid to release AFB~ (Gregory et al. 1983, We1 et al 1978) Since

809

810

(

I WEI et al

these enz}mes are also wldel} d~strlbuted in animal tissues, such as the hver, k~dne~, spleen and mammary gland, and even m intestinal juice and the bile (Wakabayshk 1970), the sulphate and glucuronide conjugates of AFB~ may present health hazards This report investigates the occurrence of AFB~ conjugates in inonkey, mouse and rat urine, while a subsequent paper (Win & Hsmh. 1985) describes AFBt conjugates in primary hepatocjte cultures EXPERIMENTAL 4ntmali Male rhesus monkeys (Macaca mulatta), weighing (~8 kg, were purchased from and housed at the Cahforma Primate Research Center, University of Califorma at Daxls The animals were fed Purina Monkey Chow-15 (Ralston Purina Co., Inc, St Lores, MO) supplemented with fresh fruit, throughout the experiment Tap-water was provided ad hb The monkeys were housed individually in stainlesssteel Labcare squeeze cages (Research Eqmpment C o , Byran, TX) They were placed m restraining chairs during the administration of AFB~ Male Sprague-Dawle3 rats (240 260 g) and Swiss-Webster mine (40 50 g) were acquired from Animal Resource Serxlces, Um~ersity of Cahforma at Da,,lS, and were maintained on commercial rodent laboratory' chow during the study To collect urine and faeces samples, Stanford metabolism cages were used (Stanford Glassblowtng Laborator,es, Palo Alto, CA) Alhttoxm compound* AFB, was purchased from Calblochem (San Diego, CA) Ring-labelled [~aC]AFB~ was isolated from cultures of A parastttcus ATCC 15517 supplemented with [1-'4C]sodIum acetate according to Hsleh & Mateles (1971) Purification of AFB~ consisted of repeated TLC on silica gel (Brinkman Instruments, Inc, Westbur',, NY), followed by HPLC as described by Hsleh. Fltzell. Miller & Smber (1976) The pur|ty of the non-radmactl~e AFB~ was checked by TLC and HPLC, while the purity of the radioactive ~.FB~ was confirmed by autoradlography of thin-layer chromatograms and by HPLC AFM~ was obtamed from cultures of A lhwus N R R L 3251 cultured on rice AFB~. was prepared by treating AFB~ with 0.5 ~-HCI at 37 C for 2hr IPohland, Cushmac & Andrellos. 1968) Aflatoxlcol (AFL) was prepared by incubating AFB~ m i m o with the hver cytosol reductase system of rainbow trout (Salhab & Edwards, 1977) Purification of these aflatoxln metabohtes ~as achle,~ed by repeated TLC on silica gel Standards of AFB~ metabohtes, including AFL, AFP~. AFQt. AFM~ and AFB,,, x~ere available from prewous m t tt~o studies described by Hs~eh. Dalezlos. Krleger et al (1974) and Salhab & Hsleh (1975) Phenobarbital pretreatment oJ rhesus monkey.~ Sodium phenobarbital (PB J T Baker Chemical C o , Philhpsburg, N J) was dissolved in physiological sahne (400 mg,'ml) and sterlhzed by Mfllipore filtratmn (0 45/xm) Monkeys were given PB intramuscularly in a dose of 15 mg kg body ~mght twice daily for 7 days Th~s dose regimen stgmficantly reduces hepatic MFO m rhesus monkeys (Hucker. Stauffer & White, 1972) Preparation and admmt vtt atton o/[t4C],4 FB : R~nglabelled [t4C]AFB~ ~as &luted ~ t h non-radmactlve

AFB~ to :field specific acnvltles of 0 5, 0 6 and 1 5 Cl,mol for monkey, rat and mouse treatments. respectively For both ix and ~p administration, each test ammal recmved a dose of [~4C]AFB~ approvmately equivalent to 10"o of the LD~,,, assuming LD,,, values to be 3, 10 and 15 mg/kg body ~mght for the monke), rat and mouse, respectively Each quantity of [~4C]AFB, was taken from a common source. transferred in chloroform to half-dram vials, dried under nitrogen and redlssolved in physiological sahne-dlmethylsulphoxlde (3 I.~.v) so that each monkey, rat and mouse received I 5.0 3 and 0 05 ml of solution, respectl~e[v The rhesus monkeys ~ere given 0 1-0 3 mg [~4C]AFB~,:kg either lp or by a rapid infusion into the right saphenous vein Rats and mice ~ere administered [~4C]AFB~ into the tall veto using a cannula apparatus This consisted of a 27-gauge needle connected to a 10-pm capillary tube, which was joined to a short length of polyethylene (PE 100) tubing The cannula terminated in a blunt 18-gauge needle, and all joints were sealed with a cyanoacrylate adhesive (Permabond 102, Pearl Chemical C o . N Miami Beach, FL) During AFB~ administration, both rat~ and mice were placed m a restrainer consisting of ,l pol)vlnyl chloride canmster eqmpped ~lth a screen outlet for the tad Urine ~ollectton After f~ or lp adinlnlstratlon of [~4C]AFBL, the test anunals were housed m glass or stainless-steel metabolism cages for collection of urine For 4 days after dosing, inonkey urine free of faecal contamination ~as collected dady in Erlenmeyer flasks kept m dr> me Mouse and rat urine was collected In a slmdar manner for 2 day's, using graduated cyhnders All samples ~ere collected under subdued hghtlng and stored frozen until anab sed Ptepwatton oJ ~later-~oluhle COnlugate~ lot acid m e n o m a t t c hydrolysis A flow chart describing the procedure for preparing the hydrolysed samples is given m Fig 1 After removal of pamculate matter through cheese cloth, the urine samples were extracted five times w~th equal volumes of chloroform The chloroform extracts were pooled, evaporated to dr.,hess and redissol~ed m a small quanmy of chloroform Ahquots of these samples were used to measure radloacmqty and to determine the percentage dlsmbutlon of extractable metabohtes in urine sample.~ For deterinlnlng the radloacmlty m the res,dual aqueous phase, the samples were concentrated b3 recycling (fi~e rimes) through an Amberhte XAD-4 resin column (Rhom & Haas, Phdadelphm, P&} Retained AF conjugates ~ere eluted with acetone and methanol, taken to dryness under ~acuum and redlssolved m 0 2 M-sodmm acetate acetic acfd buffer (pH 5 0) These were then subjected to ia&oactlvaty measurement, the Ames assay fm mutagenlclty, enzymatic or acid hydrolysis, ol another chloroform washing Enzymatic hydrolysis was carrmd out b.~ incubating the dlssoked conjugates ~lth a mixture of sulphatase (type V. 200 units ml) and fl-glucuronldase (type V-A. 10.000 unlts'ml), both fiom Sigma Chemical Co (St Louis, MO). for 48 hr m a shakmg water-bath at 37 C The aqueous solutions were acidified w~th HCI to 0 2 ,,. transferred to

Water-soluble conjugates of aflatoxln Bt--I

811

Urine sample

I

CHC1 extract=on (x5) 3 I

I

I

aqueous phase I

CHC13 extract [A]

XAD-4 resin column

radioaetw~ty measurement

1

i i

i

column effluent [B]

retained phase

f

I

rad0oaetw~ty measurement

eluted w=th acetone and methanol

f

column eluate t evaporation, dissolved m 0 2 M-sodlurn acetate buffer (pH 5 0) '

Ames mutagen assay

second CHC13 washing, (x5) CHIC13

Enzymatic or acid hydrolysis 1

extract~ll [C]

Ames mutagen assay

rad=oactw=ty

measurement

CHC13 extractmn (x5)

I

I

CHC13 extract II

aquet°us phase III

TLC analys=s, autoradmgraphy

enzymat=c hydrolysis

f

I

aqueous phase I I

CHC1 extract=on (x5)

~ radioactivity measurement I'

i

aqueous

TLC analysis, autoradmgraphy

CHC1

phase IV

[ r0,oct,v,t

3

extract IV

measurement

acid hydrolysis

TLC analysis) autoradJography

f CHC1 extractmon(x5)

a j

I

CHC1 extract V TLC analysis) autoradmgraphy

'

I

aqueous phase V radloactwlty measurement

Fig 1 Procedures for the preparation of water-soluble aflato,~m conjugates prior to enzymatic and acid hydrolysis and Ames mutagemclty assays [A], [B] and [C] are fracttons slmdarly =denufied m Table 4

and sealed m a container and heated m an oven for 2 hr at 9 O C Chloroform extracuon, ra&oacuvtty measurements and T L C analysis w~th autora&ography were used to identify chloroformextractable aflatoxm metabohtes released from conjugates For the other half of the experiment (right half of Fig 1), A F conjugates, present m 0.2M-sodium acetate-aceuc acid buffer (pH 5 0), were subjected to enzymatic hydrolysis as before. After the enzymatic hydrolysis, a chloroform extraction was followed by acid hydrolysis of the aqueous phase to determine the nature of the A F conjugates.

TLC analysts o f AF metabohtes and autoradtography. The hydrolysed ml,~tures were extracted with chloroform, concentrated and analysed for A F

metabohtes using one- or two-&mensmnal T L C Chloroform extracts were spiked w~th cold authenuc A F metabohtes and kept overmght at room temperature before autora&ography Th~s allowed better vls~bd]ty of minute quanutles of the metabohtes For one-dimensional T L C analys~s, the plates were developed m chloroform-acetone-~sopropanol (85 15 2 5, by vol ) and two-dimensional T L C analysis was developed first m the above solvent system and then m toluene-ethyl acetate-formic acid (6 3 1, by vol ) These solvent s)stems provided satisfactory separation of the metabohtes, which fluoresced under UV hght Ethyl ether was sometimes used for one&mens~onal T L C Identification of metabohtes was carried out by comparing their R e values with cochromatographed standards Metabohtes present on

C I WEt et al

812

AFM1, AFL, AFB2a dtssolved m 1 ml methanol

+ 100 ml d=stflled water

l

X A D - 4 resin column

I

I'

I

column effluent

retained phase

CHC13 extractLon (x5)

eluted with methanol and acetone

t

f

aqueous phase

column eluate evaporation, dissolved m 0.2 ml methanol

I

t

CHC1

3 extract evaporated to dryness, dissolved m

+ 15 ml 0.2 M-sodium

CHC13

acetate buffer (pH 5.0)

1

TIC analysis

enzymatm hydrolysis CHC13 extractton (xS)

t aqueous phase

I

CHC13;xtract T

evaporated to dryness, dissolved in CHC1 3 TLC analys,s Fig 2 Processing of pure samples of aflatoxlcol (AFL), and aflatoxms 1M=and B:, (AFM t and AFB:~) for enzymaUc h~drolysls

TLC plates were also confirmed by autoradlography A sheet of X-ray film (Kodak Medtcal No-Screen, Eastman Kodak C o , Rochester, NY) was pressed against the chromatogram w~th a glass plate, then wrapped m alumimum foil, and finally stored for 1 month to allow exposure After development and fixation, the dark spots on the film were compared w~th the fluorescent spots on the TLC plates Sihca gel containing the metabohtes was scraped from the chromatoplates and transferred into vmls containing 0 1 ml dlstxlled water Thxs reded m releasing silica-bound aftatoxm metabohtes Scintillation counting was performed after addmon of 13 ml Handlfluor (New England Nuclear Boston, MA) to each vml. Measurement oJ radtoaetwtty Radioactivity of all samples was measured by scmtxllataon counting xn 13ml Handifluor using a Tn-Carb LNmd Scmtdlataon Spectrometer (Model 2425, Packard Instrument Co., Downers Grove, ILl w~th automatic exter-

nal standardization [~4C]Toluene INew England Nuclear) was used as an internal standard Enzymatw hydroh'sts o/AFL. AFM~ and AFBa, In order to verify AFL, AFM~ or AFB,_, as the possible precursors of AFB~, these compounds (335 #g AFL, 200pg AFM~ and 500 l~g AFB,,) were subJected to the treatment procedures outhned m Fig. 2 The metabohtes were dtssolved m 1 ml methanol, diluted with 100ml water and then passed three ames through an XAD-4 resin column The compounds retained m the column were eluted w~th acetone and methanol, evaporated to dryness and d~ssolved m 0 2 ml methanol and 15 m[ 0 2 M-sodmm acetate aceUc acid buffer (pH 5 0) This soluuon was then subJected to enzymauc hydrolysis as before Chloroform extracts of the hydrolysates were analysed using both one- and two-dlmensmnal TLC analys~s as prewously described Ames mutagemcttv testing The Ames Salmonella,'mlcrosome assay (Ames, McCann &

Water-soluble conjugates of aflatoxln B~--I

813

Table 1 D~strtbutmn of radtoact~v~ty m fractions of urine samples from rhesus monkeys treated v.lth [~4C]aflatoxm B~ and in h.,,drol.,,sates of the urmary aflatoxm conjugates Rad~oacm~ty* recovered (°o of total urmar3 actwtt)) W~thout PB Route of AFB~ administration Method of hydrol3 s~s+ CHCI~ extract of urine Aqueous phase I Column effluent Column eluate Aqueous phase It CHCI extract II AFB) AFMI AFP~ AFB:, Umdentlhed Recovery]l

With PB

lp

IV

I ~-

Enz~

a,c~d,~

Enz +

A.c,d~

Enz~

15 00

15 00

28 00

28 00

21 20

31 37

31 37

26 57

2{) 57

44 05

3 30

4 03

10 67

24 49

21 73

0 20 16 45 I 37

0 27 -I 00 22 63 4 77 79 07

0 2I 0 35 2 56

0 08 0 28 -0 30 2 [7 81 89

6 40 74 09

2 95 71 31

0 44 l 95 89 37

PB ~ Phenobarbital pretreatment *Determined by sclntdlatmn counting +By m c u b a n o n wtth sulphatase (200 umts ml) and fl-glucuromdase I10,000 umts m b at 37 C for 48 hr IEnz) or 0 4 ~ t h s - H C I I I I. v v) at 90 C for 2hr, prmr to the second C H C L extracnon (lZ~g I) ~Aflatoxln analys~s b.~ one-dtmensmnal T L C §Aflatoxm analys~s by two-&mensmnal T L C and a u t o r a d m g r a p h 3 IIRadtoactlvtt3 anaccounted for v, as presumably bound to the X ~ D - 4 resin and the mterphase generated d u r m g C H C L extracuons Each sample contained urine from three or four animals

YamasakL 1975) mo&fied for aflatoxms (Wong & Hsaeh, 1976) was used m the presence of rat-hver S-9 mix to determine the mutagemc activity of the watersoluble conjugates before and after enzymanc hydrolys~s S. typhlmurtum strata TA98 was prowded by Dr B N Ames, Bmchem~stry Department, Umvers~ty of Cahfornm at Berkeley The protocol comphed with the workshop recommendation on th~s assay (de Serres & Shelby, 1979) regarding basra techmques, such as strata maintenance, genotypm characteristic checks, S-9 preparatmns and background lawn exammarion for toxm~ty RESULTS

The dlstrlbutmn of radmact~vlty m monkey-urine samples and the identity of the types of AF released from the AF conJugates after enzymatic or acid hydrolyms are shown m Table 1 In monkeys dosed iv with [~4C]AFBt, PB pretreatment reduced the level of radmact~v~ty m the chloroform extract of the urine samples The level was even lower m the chloroform extract of urine samples from monkeys gaven [~4C]AFB~ lp Radmactlvlty was stdl detected m the column effluents obtained after recychng the urine samples through the XAD-4 column, the sample from the PB-treated monkeys given AFB~ iv showed the hlghest actw~ty followed by the sample from ~p-treated animals and then by that from the ammals treated only wzth AFB~ w AFB~ was found m the enzymatm and acid hydrolysates of samples after ~v and ~p treatment w~thout PB, but only AFP~ was identified m those from PB-treated animals. More AFB~ was released from the ~p-treatment acld-hydrolysed sample than from the ~v-treatment ac~d-hydrolysed sample m terms of the percentage of total urine, but similar amounts were released from the ~p- and w-treatment samples by enzymanc hydrolysis Urine samples collected from ammals not gwen PB pretreatment were found

to contain AFM~ and AFP~ after chloroform extraction of the enzymatic hydrolysates, more AFM~ being released from the lp than from the iv sample No AFM~ was found m the acid hydrolysate of the lp sample AFB,. was only identified in samples subjected to aod hydrolysis, more being found m the lp than m the iv sample The sample from PB-treated animals given AFB~ iv showed the highest percentage recovery of radmact~vtty, whde the enzymehydrolysed other iv sample (no PB treatment) had the lowest The m~ssmg radmact~wty was retained b~ the XAD-4 resin or lost in the mterphase formed during chloroform washing The dlstnbutmn of ra&oactlvlty m urme samples of rat and mouse is shown m Table 2 A greater Table 2 Distribution o) radmactl~.lt} m fracttons o! urine samples from rats and trace treated ,atth [HC]aflatoxm BE and m hydrolysates* of the urinary, aflatoxm conjugates Radmactlvlt3,t reco,.ered C,. ol total urmar', acu;lt', Fractmn

Rat++

Mouse++

CHCla extract of urine Aqueous phase I Column effluent I Column eluate Aqueous phase II CHCI~ extract II AFB~ AFM~ AFQI AFPf Polar Umdent~fied Reco'~ery~

35 70

If) 10

24 07

"~5 I I

9 16

26 41

17 0 26

-3 40 2 61 3 42

0

--

0 0 4 73

35 20 06 97

12 65 93 70

*Enzymatic h3.drol3.sts by mcubatlon '*lth sulphatase 1,200 unus ml) and ,8-glucuromdase 110,000 umt'~ m h at ~'7 C for 48 hr, prior to the second CHCI, extraction *Determined by scmtdlatlon counting +Aflatoxm analysis by one-dimensional T L C .}Radloactl;ity unaccounted for v, as presumably bound to the XAD4 resin and the mterphase generated during CHCI~ extractions Each urine sample contained urme from bet~,een sl~. and ten rats

814

C I WE} et al

Table 3 Mutagemclt~ assays (m Salmonelh* t~phtmurmm TA98) of h.~drolysed and unh~,drobsed aflatoxm conjugate~ extracted from the urine o f m o n k e y s treated v, tth aflato~.m B~ Treatment

Sample ~FB~ eqm~alenl+ h~,dro[3 sis* (,u g. plate)

AFB~ Ip + 4FB~ t~ + AFB~ ~ -tPB m~

+

[-r 70 16 20 t) 60 t) 32 2 76 2 84

Re~ertants (no plate){ v 4 + 13 253 + 3S 26 ~- 4 359 _+ 60 23 t- 8 [6 ± 2

-kFB, = ~flatoxm B~ PB - Phenobarbital *Where used I + J h.~ incubating samples ~ t h sulphatase (200 umts mll and fl-glucuromdase I I0 000 units ml) at ~7 ( for 48 hr "I'a~FB~ eqm~alents were calculated by c o m p a r i n g the rad~oa~.tt~tty of the C H C I , extracts of the h~,drobsates (or corresponding unhydrolysed sample} ~ I h the specific acnvit~ ot the [~aC]AFB~ admlmstered :~Means___ S D for tnphcate plates calculated after subtraction of background numbers of revertant~

proportion of radloactlvlD was found m the chloroform extract of rat urine than In that of mouse urine. However, the column effluent of the mouse sample contained the higher proportmn of ra&oactwlty AFB~ was detected m rat-urine samples after enzymatic hydrolysis but not m mouse samples AFMt and AFPt were detected m both rat- and mouse-urine samples, but the greater quantity was found m the mouse samples AFQ~ was only detected in mouse urine. Unlike the monkey' samples, enzymatmally hydrolysed urine samples from the rat and the mouse did not contain AFB,_~. To check the completeness of enzymatic hydrolysxs, chloroform extracts of the mouse and rat urinary' enzymatic hydrolysates were further subjected to acid hydrolysis The aflatoxln metabohtes released were extracted with chloroform and analysed by two-dimensional TLC and autoradtography Only AFBe~ was detected m rat urine and the amount accounted for 14 9°0 of the chloroform extract or 1 05°o of the total actlwty in the urine (data not shown) The Ames Salmonella:mlcrosome assa~, was used to determine the changes m mutagemc activity' of AF conjugates found m the urine of monkeys after enzymatic hydrolysis Except for samples from monkeys pretreated wuth PB and given AFB~ w, higher numbers of revertant colonies resulted from exposure to the enzymanc hydrolysates than to conjugated forms (Table 3) Since more AFB~ was detected in the anne or animals given AFB~ iv than m that of the animals given AFB~ lp (Table I), more revertant colonies xn the sample from animals treated iv were to be expected No mutagemc acm, tty was found in the enzymatic hydrolysate of the urine of monkeys pretreated with PB and dosed iv with AFBt This agrees w~th the results m Table I, which showed that only the non-mutagemc AFPt was detected m th~s sample Shght mutagemc actlwty was detected in the urine sample from lp-treated monkeys prior to enzymanc hydrolysis (Table 3) This result suggested that some mutagemc AF metabohtes, such as AFB~, AFM~ or AFL could be released during the XAD-4 column recycling process or during the purification of conjugates TLC analysis of the chloroform washings of

these samples prior to enzymatic hydrolysis re~ealed the presence of AF metabohtes (data not shown) An additional chlorofolm ~xashmg step was included In all subsequent experiments to retno~e anx releabed metabohtes during the XAD-4 cohmm lecychng process or other purification steps (Fig l, right-hand section of flo~x chart) The dlstilbUtlOn of radioactivity m mine samples that were washed with chloroform and hjdrolysed bx enzymes and then b', acid (Fig I) is presented m Table 4, all the tabulated data being from ix-treated ammals AFB~ was Rmnd in chlorofolm extract III of the rat-urine sample Metabohtes AFM~, AFQ~ and AFP~ in different combinations x~ele also found in these chloroform extracts ot the samples More AFM~ and AFP~ t~ere found in the mouse than in the rat sample Enzymatic hydrolysis of chloroform-~xashed ~.onjugate samples (Table 4) caused the release of AF metabohtes A,FB~ was only' detected in the chloroform extract 1V of the urine from inonkeys not pretreated x~lth PB However, AFPj ~as detected only in the other three samples and AFM~ only in the other monkey and the mouse samples More AFP~ was again found in the lnouse- than ul the rat-urine extract After acid hydrobsls, AFBt was again only' found in the sample from monkeys not pretreated with PB (Table 41 AFBza was found m both the rat and mouse urine samples AFP~ was only found in the sample from PB-treated monkeys The detection of AFBt before and after different treatment procedures (Tables 1, 2 & 4) was of particular interest TLC analysis of aqueous phase 1 showed that ~FB~ was completely remoxed by initial chloroform extraction of urine samples (data not sho~n) Therefore, one possible explanation for the pretence of AFBt in treated samples ~xas that AFB~ was converted from &FL, AFM~ or AFB2, dullng the handling procedures To xerll~ thlb possibility, sampies of pure AFL, AFMI and A F B , x~ele dlsbol~ed in water and subjected to the same treatment plocedures (Fig 2) The chloroform extracts from the XAD-4 column effluents and the enzytnattc hydrolysates xxere analysed by' TLC As shown in Fig 3a, only AFM~ and AFB,, samples from the XAD-4 column effluents re~ealed any' faint AFB~ spot For those subjected to enzymatic hydrolysis, samples of AFL and AFM, shorted a faint AFBj spot, ~hlle salnple AFB:, demonstrated a distinct AFB~ spot The A F B z hydrolysate also contained a blue fluorescent spot right below AFB~, which was buspected of being AFL The enz.~matlc h.',drolysates of AI-L and AFB:, samples ~xele also chromatographed ~xlth ethyl ether This solvent system was found to be appropriate (Wong & Hsleh, 1978 & 1980} for AFL analysis on TLC plates (Fig 3by The &FL hydrolybate revealed AFL, i t S l s o m e r a n d a f a l n t s p o t o f A F B ~ T h e A F B , , salnple sho~ed a faint AFB) spot, but no & F L spot The spot In Fig 3a suspected of being AFL was, thelefore, not AFL Tyro-dimensional TLC analysis of samples *kFM~ and AFB,~ after enzymatic hydrolysis (Fig 4) also showed the presence of AFB~ spots No AFL ~as found m the AFB:, sample, again confirming that

Water-soluble conjugates of aftatoxm B~--I

815

Table 4 Distribution of radloactl',lt} In extracts of urine froirl monkeys, rats and mice treated ~v v.~th [~C]aflatoxln B~ and m the hydrol)~ates of the aflatoxm conjugates present after the second chloroform ~ashmg Radloachvltyt recovered (°,, of total urmar) actlvlt,,) Monke3 Urine fraction*

No PB:~

CHCI, e,~tract of urine [A] Aqueous phase l Column effluent I [B1 Column eluate Aqueous phase II1 CHCI~ extract III [C]

PB-pretreated~

Rat,~

Mouse~

28 00

21 20

35 70

10 10

26 57

44 05

24 O7

35 I [

I I 44

40 22

After second CHCI~ washing (a)l[ I 1 63 30 39 6 47

~FB~ AFM~

AFQt AFP~ Reco,.ery~

ND

-

001

ND ND ND 72 67

0 06 -0 11 95 81

010 021 71 53

331 89 73

099

After (a) and enz,,mane h)drol)sis (b)ll

[A] + [B] CHCL extract Ill [C] Aqueous phase Ill ~queous phase IV CHCI~ extract IV AFB~ AFM t AFP~ AFB2. Reco'~er~~

54 57 6 47

65 25 2 39

59 77 2 16

45 21 14 57

9 89

11 21

8 60

21 4O

0 0I ---

0 27 1 13

0 09

115 1 67

70 94

80 25

70 62

84 00

After (a,) (b) and acid hydrolysisll

[A] -I- [B] + [C] CHCI, extract IV Aqueous phase IV ~queous phase V CHCI~ extract V "~FB~ AFQ~ AFP~ ~FB2,

61 04 I 7~

67 64 3 86

61 93 I 09

59 78 7 48

9 33

9 05

6 2l

13 80

024 69 47

82 27

001

Reeover:,~

--

0 10

72 12

80 65

I 21

PB = PhenobarNtal AF = Aflatoxm *See Fig 1 for scheme of procedures for preparing water-soluble AF conjugates and for enz)matlc and acid h)drol)sls [A] [B] and [C] are fractions similarly identified m Fig I ~'Determmed by sclntlllatlon counting ~Aflatoxms analysed b) one-dlmensmnal TLC ~Aflatoxms analysed by two-dlmensmnal TLC and autoradmgraph) [ICHCI~ washing by extraction of samples fives times with an equal volume of CHCI~ enz~manc h)drolysls by mcubahon ~lth sulphatase (200 urals roll and fl-glucuromdase (I0 000 umts mh at 37 C for 48 hr aczd hydrolysis b~ meubanon x~lth an equal volume of 0 4 N-HCI at 90 C for 2hr ~Radloactl~lt_v unaccounted for v.as presumably bound to the XAD-4 resin and the mterphase generated during CHCI~ extractton~

AFB2. w a s n o t c o n v e r t e d to A F L d u r i n g e n z y m a t i c hydrolysis

DISCUSSION

T h e s u s c e p t l b d l t y o f an a n i m a l species to the toxic a n d c a r c i n o g e n i c effects o f AFB~ is a s s o c m t e d with the m e t a b o h s m o f th~s c o m p o u n d ( H s l e h et al 1977. P a t t e r s o n , 1973, S a l h a b & E d w a r d s , 1977) A c o m p a r a u v e m e t a b o h c s t u d y c o n d u c t e d by W o n g & H s l e h (1980) s h o w e d that a species that is relanvely resistant to the a c u t e toxicity o f A F B t, s u c h as the mouse, had a lower volume of AFBt dlstrtbutlon, a l o w e r e q u l h b r m m t r a n s f e r rate c o n s t a n t , l o w e r levels o f total a f l a t o x m s m hver a n d p l a s m a , a n d a s h o r t e r p l a s m a b m l o g l c a l half-hfe o f AFB~ T h i s species w a s also m o r e active in c o n v e r t i n g A F B I to AFP~ a n d w a t e r - s o l u b l e m e t a b o l l t e s Raj & L o t h k a r (1984) s h o w e d t h a t a b o u t 10-15°o o f AFB~ m ' e c t e d lp ~ a s F C T 2~9 C

excreted in the urine o f the h a m s t e r , w h i c h ~s m o r e resistant to AFB~ toxicity t h a n is the rat. T o t a l e x c r e n o n o f AFB~ w a s 50°0 h i g h e r m the h a m s t e r t h a n m the rat A l t h o u g h tt was n o t o u r objective in this s t u d y to c o r r e l a t e a n i m a l s u s c e p t l b t h t y to AFB~ with its m vwo m e t a b o l i s m , the results s h o w e d that a h i g h e r perc e n t a g e o f r a d l o a c h v l t y was detected m the c h l o r o f o r m - e x t r a c t a b l e fraction o f rat t h a n o f m o u s e urine (Table 2). A h~gher p e r c e n t a g e o f rad,oactlVlt) was also detected m the c h l o r o f o r m - e x t r a c t a b l e fracn o n o f m o n k e y urine after l~ t h a n after lp a d m i n i s t r a t i o n o f A F B t, the level m m o n k e y urine f o l l o w i n g PB p r e t r e a t m e n t a n d iv l n j e c h o n o f AFB~ was b e t w e e n the t w o T h e s e results agreed with the findings o f Hsleh et al (1977) a n d W o n g , Wel, Rice & Hs~eh (1981) that the m o r e resistant a m m a l species s h o w e d the g r e a t e r liver e n z y m e activity in c o n v e r t i n g AFB~ to the less toxic AFQ~ a n d . o r w a t e r - s o l u b l e metabohtes

C I WEI et al

816

Solvent front

2nd

Plate (o) STD ~

AFB,

i,

~,/

~-qI-~ > ~-q -n

AFL yellow-green

~ AFQ~

0 AFB'

~ AAFM~ FB~o

I

I

I

I

AFL AFM, AFB2~ STD .i

'XAD-4effluent

I

I

I

#~blue-green (DE2/ Oyellow-green

i

i

Enzymat)chydrolysate

AFM~ Solvenf front

AFB~I

~

0 blue- green

Origin

AFL AFM, AFB~o

yellow-green

AFM~ sQmple

AFL I~

(~ ,AFQ

AFM~ ~

AFB2o

STD

P l a t e (b)

o

49

2nd

o

~ ~

I

AFL

I

O

O

I

I

I

~

AFL

yellow-green

0 r~gm

AFL AFB~o AFB~ AFL ' ' STD STD STD Enzymat)c hydrolysote

F~g 30ne-d~mens~onal thin-layer chromatograph) of the chloroform extracts of XAD-4 column effluents or enzymanc hydrolysates of AFM~. AFL and AFB,. preparations as indicated m Ftg 2 The solvent system used for the development of plate (a) was chloroform-acetone~sopropanol (85 15 2 5. by vol ) and for plate (b) was ethyl ether The dotted areas md~cate faint spots The route of a d m m B t r a u o n of AFB~ affected the profile of A F conjugates (Table 1) More A F M j was released from the enzymauc hydrolysate of urine from the 1p-dosed than from the w-dosed monkeys, indicating the possible mvoNement of intestinal ep~thehal cells m A F metabohsm. Following actd hydrolysls of the monkey urines, more AFB2. was released after lp than after w admmstratlon, suggesting that A F B . , could come d~rectly from AFB~ as a result of acid treatment Pretreatment of the monkey w~th PB increased its reststance to aflatoxlcos~s (Wong et al 1981), and only the non-toxic AFP~ was found m the enzymattc hydrolysate of urine from the PB-treated monkeys AFB~ was found m the other four chloroform extracts of enzymattc or acid hydrolysates (Table 1) The presence of AFB~ was indicated by the h~gh level of mutagomc achvtty demonstrated m the Ames Salmonella,"m~crosome assay (Table 3) Th~s assay has been w~dely used to detect small quantities of a h~ghly mutagemc component such as AFB~ m a complex m~xture (McCann & Ames, 1977) The enzymahc hydrolysate of urine from PB-treated monkeys revealed no mutagentc actw~ty, supporting the T L C detecuon of only AFP~, which has no mutagemc acttwty m the Ames assay (Wong & Hs~eh, 1976)

#

{)AFQ,

oOOc~FBz°

(~ AFBzo

~AFM~ O

AFB2o sample

STD

F~g 4 Two-dimensional thin-layer chromatography of the chloroform extracts of enzymatic hydrolysates of aflatoxms M~ and B~ preparations (Fig 2) The solvent systems used ~ere chloroform-acetone-tsopropanol (85 15 2 5. by vol) followed by toluene~ethyl acetate-lbrm~c acid (6 31 by vol ) AFB, was also found m the enzymauc hydrolysate of rat urine but not of mouse urine These samples were not tested for mutagemc acuwty Other A F metabohtes were detected m these hydrolysates. More AFM~. AFQ~ and AFPt were found m the mouse hydrolysate than m the corresponding rat sample (Table 2) There are three possLble explanahons for the surprising presence of AFB, m the enzymauc or acid hydrolysates of the monkey and rat urines. The first ~s the possxble retenuon of AFB~ by the X A D - 4 resin column and its later eluuon into the column eluate because of incomplete mmal chloroform washing of the urine samples However, if the m m a l chloroform washing was complete, A F L . AFM~ or AFB_,. released during X A D - 4 column eluuon could have been converted to AFB~ Thirdly, through enol-keto t a u t o m e n s m (Gregory et al 1983) AFB~ can form conjugates directly and can be hydrolysed by acid or enzymatic treatment to release AFB~ T L C analysis of the aqueous phase I sample re-

ILoLo

0

ConlugaT~on

1

LJ-.o~o~.,

0

ROH///

AFB1

0 0 H z O ~

~s~

o

Conjugation

,,RNHz

O'~

0

H

ConjugaTion

Ftg 5 Possible reactions of aflatoxm B t (AFB I) wJlh amines (RNH2), thlols (RSH), alcohols (ROH) and H~O to form conjugates

Con|ugahon

0

"-.4

O~

I

O

8

t~

o

818

c

I WEI r, trl

vealed no AFB, spot Therefore. the uutlnl chloroform uashlng was complete and the first powble explanation IS not plausible The presence of hydrouyl groups IS required for the formation of glucuromdes and sulphate conlug‘ltes (WIlllams. 1967) Metabohtes IIke AFQ,. AFP,. 4FL and AFM, are capable of formtng conluguteb .4FP,. the 0-demethylatlon product of AFB,, was m,unl~ excreted m cotyugated form into the urine of male rhesus monkeys (Dalezlos & Wognn. 1972) .4ppro\lmately 50”,, of AFP, was the glucuromde conJugate. while IO”, was present as the sulphate conjugate These AF metabohtes could be released from plucurontde or sulphnte coriJugates during the elutlon process from the X.4D-4 column and then be converted to AFB, during enzymatic hydrolysis To tect this posslblhty. pure AFB,,. AFM, or AFL uas subjected to slmilnr preparation (Fig 2) and analksed b> TLC Although very small amounts of AFB,,. .4FL and AFM, were found to be conberted to 4FG, after enzymatic hydrolpsls. this could not account for the quantltles of AFB, detected Therefore. AFB, appears to be capable of formmg glucuromde and sulphatr conlugates by Itself, powbly through a rearrangement of Its structure In the presence of water, alcohols. thlols or ammes. the keto-group of the cyclopentenone IS converted to J C-OH group. uhlch can then form glucuromde and sulphate conlugates (Fig 5) Further evidence that AFB, can form glucut omde and sulphate coryugates IS shown III Table 1 .4FB, uas found m chloroform extract III of the rat urine and m chloroform extracts IV from enzjmatlc h)drolys~s and V from enzqmatlc and acid hydrolqsls of urine from IL-treated monkeys Other AF metabohtes m various comblnatlons uere detected In samples from other treatments The release of AF after ncld hydrolysis of the samples pretreated wth enzymes may Indicate the presence of mercnpturate coyugates of AF The AFB,-GSH conjugate ma! account fat the radloactl\lty of the aqueous phase after the samples had been treated wth both enzhme5 and ‘lcld hydrolysis and chloroform washmp (Table 3) Degnn & Neumann (1978) reported th‘it AFB,&SH conlugate IS the major aflatoxm metabohte found III the bile of female Wlstal rats dosed up with 4FB, It nccounted for about lo”,, of the admlmstered dose R~J & Lothkar (1984) reported that AFB,-GSH coryugate and AFB,+ystemylglyme uere prewnt In both rat and hamster urme samples They belleked that InactIvatIon of AFB,-epoklde \~a GSH conjugatlon might play an Important role In drtermlmng AFB,-DNA binding and hence 4FB, hcpitocarcinogenesis in animal species Direct formation of AFB, conlugate \\a~ also reported In a rat hepatocyte culture medium (Wei t’t al 1978) and m tissue samples of turkey poults fed a diet contalmng 4FB, (Gregory er (II 1983) Because of the potency of AFB, m causmg tow effects. the release of AFB, from water-soluble 4FB, conjugates by enzymes (/j-glucuromdase and sulphatase) \ecreted from mtestmal eplthehal cells or the lntestlnal mlcroflora (Schelme. 1975) may account for effects seen In organ? other than the hver The hldney has been shown to be the target organ of 4FB, 111 the guinea-pig (Madhavan & Rao. 1967). hamster

(Herrold. 1969) and rat (Butler &I LiJmsky, 1971. Newberne. Rogers & Wogan. 1968) particularly when the anmals \\ere fed a ION hpotroplc diet The colon of the rat IS another site of atlatoxln carclnogenesls \Lhen the rat IS deprlked of \ltamm A (Newberne & Rogers. 1972) The presence of AF metdbohtes m the chloroformwashing (chloroform extract II) of the coqugatecontalnmg fractions after XAD-4 resin column chromatogr~ph~ (Table -I) suggests that some of the bound AF compounds here Iele,wd when passed through the XAD-4 rehln These metabohtes wet-r proh‘tbly non-covalentI> bound to protelm or other components and mere not tour conJugntes Thus, when XPID-4 rem I:, ubed to Isolate conjugates ot drugs 01 towants from bIologIcal samples. a chloroform wnshlng of the elu;ltes IS recommended to temo\e released metabohte5 before the eluates ‘ire wblected to enrqmatlc or xtd hydrolyslz

REFERENCES

Chlplq J R Mahce hl 5. L\pplsgtc K L & Drckt‘uss hl S ( 1974) Further chdrrwtew,uwn 01 tissue dlstrlhutwn dnd met,thollsm of [“C]~~tl~~tovn 9, III chlckenc -f/v/ Ifrc rohrr,/ 28, IO?7

Water-soluble conjugates of aflato,~ln B~--I Hsleh D P H , Wong Z A , Wong J J . Michas C & Ruebner B H (1977) Comparative metabolism of aflatoxlns In Mlcoto~ms ltl Human and 4ntmal Health Edited by J V Rodrlcks. C W Hesseltme & M A Mehlman p 37 Pathoto,q Park Forest South. IL H u c k e r H B Stauffer S C & W h i t e s E (1972) Effect of halofenate on binding of various drugs to h u m a n plasma proteins and on the plasma half-life of antlpyrlne in monkeys J pha~m S~I 61. 1490 LtnJ-K,KennanK A Mtller E C & M d l e r J A (1977) Reduced nlCotlnamlde adenine dznucleotlde phosphatedependent formation of 2,3-dlh)dro-2 3-dlhydro,~yaflatoxln from aflatoxln B~ by hepatic mlcrosomes Cancel Re~ 38, 2424 Lour~, D J . Hsleh D P H & Byard J L (1984) The effect of phenobarbital pretreatment on the metabolism. covalent binding, and cytotoxlclty of aflatoxtn B~ m primary cultures of rat hepatocytes J ToAtcol enrlr Hlth 13, 145 M c C a n n J & Ames B N (1977) The Salmonella mlcrosome mutagenlclty test Predictive value for ammal carcmogemctty In Origins o f Human Cancer Book C Human Risk Assessment p 1431 Cold S p n n g Harbor LaboratoD. New York M a d h a ~ a n T V & Rao K S (19671 Tubular eplthehal reflux in the kidney in aflatoxm poisoning J Path Bact 93, 329 Newberne P M & Butler W H (1969) Acute and chronic effects of aflatoxln on the liver of domestic and laboratory animals A review Cancer Res 29, 236 Newberne P M & Rogers A E (1972) Vitamin A. hver and colon carcinoma m rats fed low levels of aflatoxln (Abstract) To.~w appl Pharma~ 22, 280 Nev, berne P M , Rogers A E & Wogan G N (19681 Hepatorenal lesions in rats fed a low hpotrope diet and exposed to aflatoxln J .Vtttr 94, 331 Patterson D S P (1973~ Metabolism as a factor in determining the tO~.lC action of aflatoxms in different species Fd Co,met ToAwol 11, 287 Pohland A E , C u s h m a c M E & Andrellos P J (1968) Aflatoxln Bj hemlacetal J 4s~ off analyt Chem 51,907 Raj H G & Lothkar P D I1984) Urmar,, excretion of

819

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