Characterization of water-soluble glucuronide and sulphate conjugates of aflatoxin B1. 2. Studies in primary cultures of rat hepatocytes

Fd Chem To~:tc Vol 23. No 9, pp 821-825. 1985

0278-6915 85 53 00+0 00 Pergamon Press Ltd

Printed m Great Bmaln

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. 2. S T U D I E S IN P R I M A R Y C U L T U R E S OF RAT H E P A T O C Y T E S * C I WE1 Food Science and Human Nutrition Department, University of Florida. Gaznesvllle. FL 32611 and D P H. HSmH Department of Environmental Toxicology. University of California. Davis, CA 95616, USA (Recewed 20 Juli' 1984)

Abstraet--Aflatoxln B~ and some of ~ts metabohtes were released from water-soluble aflatoxln conjugates isolated from rat primary hepatocyte cultures and hydrolysed by enzymes (fl-glucuromdase and sulphatase), by acid or by a combination of both treatments The presence of AFB~ in the hydrolysates was detected on TLC plates, or indicated indirectly by the Ames mutagenlclty assay The aflatoxm conjugates were not mutagenlc to Salmonella typhtmurlum strain TA98 m the presence of rat-liver S-9 mix However. following enzymatic hydrolysis, the chloroform extract of the hydroid,sate was h~ghly mutagenlc to the bacterm, Indicating the presence of mutagenlc AFB~ The conjugates AFB~-glucuromde and AFBt-sulphate are therefore produced from AFB~ in primary cultures of rat hepatocytes

INTRODUCTION

Primary cultures of adult rat hepatocytes have been developed as a model system for studying the metabohsm of foreign chemicals (Dougherty, Spilman, Green et al. 1980). By the use of a chemically defined medium containing amino acids, hormones and fatty acids, the cytochrome P-450 content of the primary hepatocyte cultures was maintained at levels close to those occurrmg m vwo (Decad, Hsieh & Byard, 1977, Dougherty et al 1980, Salocks, Hsleh & Byard, 1981). One advantage of using primary hepatocyte cultures m metabohsm studies ts that several experimental conditions can be compared using replicate dishes prepared from a single ammal Extensive research has been conducted recently on the use of rat-hepatocyte cultures to investigate the cytotoxlcity, metabolism and covalent binding of aflatoxm B~ (AFB~) to macromolecules (Decad et al 1977, Green, Rice, Hsieh & Byard, 1982, Loury, Hsteh & Byard, 1984, Salocks et al 1981) Hepatocyte cultures prepared from rats pretreated with butylated hydroxytoluene (BHT) or phenobarbttal were used to study the effects of these chemicals on the metabohsm of AFB~ Pretreatment of rats with BHT produced a protective effect against AFB~ cytotoxtcity and reduced the covalent binding of AFBt to cellular macromolecules in primary hepatocyte cultures (Salocks et al. 1981). Pretreatment of animals with phenobarbital also had a protectwe effect against the cytotoxlcity of AFB~ and reduced the covalent binding of AFB t to R N A and DNA, m additton It increased levels of mixed-function OXl*Florida Agricultural Experzment Station Journal Serzes No 5744 Abbreviations AF = aflatoxm, GSH = glutathmne, TLC = thin-layer chromatography

dases, glutathlone S-transferase activity and AFB~glutathlone (GSH) conjugate in liver-cell cultures (Loury et al. 1984) These results agree with previous reports that rats pretreated with phenobarbital were significantly less susceptible to the hepatoto,~lc effect of AFB~ m v w o (MgBo&le, Holscher & Neal, 1975), displayed reduced binding of AFB~ to hver nucleic acid (Garner, 1975) and were more resistant to the carcinogenic effect of AFB~ (McLean & Marshall, 1971, Swenson, L m , Miller & Mdler, 1977) Rathepatocyte cultures can thus provide a useful model for studying the tn vtvo interaction between phenobarbital and AFB~, or the mode of action of AFB~. Water-soluble AF conjugates were produced m hepatocyte cultures (Loury et al 1984) as well as in the urine samples of test animals, including the rat, mouse and monkey (Dalezlos & Wogan, 1972, Degan & Neumann, 1978, Raj & Lothkar, 1984, Wong, Wei, Rice & Hsleh, 1981) These include the AFB~-GSH conjugate (8,9-&hydro-8-S-glutathlonyl-9-hydroxyAFB~) and the glucoromde and sulphate conjugates of AF metabohtes such as AFM~ or AFP~ Since AFB~ was found to be excreted in urine as the glucuromde and sulphate conjugates (Wel, Marshall & Hsleh, 1985), it was antlc~pated that AFB~ could also be detected in the hydrolysates of a hepatocyte culture medium. This question was mveshgated in the studies reported here. EXPERIMENTAL

P r e p a r a t i o n o f culture m e d i u m . Primary rathepatocyte cultures were prepared and provided by Dr J L Byard, Department of Environmental Toxicology, Umvers~ty of Cahfornm at Davis The hepatocytes were ~solated from 18(~210-g male Sprague-Dawley rats, obtained from Animal Re-

821

822

C 1 WEI and D P H HSIEH

source Service, University of Cahforma at Da',lS, and maintained dur, ng the study on commercml rodent laboratory cho~ The hepatoc.,,tes were cultured m a chemxcally defined medmm prexiousl,v shown to maintain the cytochrome P-450 content at lexels close to those found m t'no (Decad, Dougherty, Hs~eh & Byard, 1979, Decad et al 1977) Hepatoo'te cultwe.s The hepatocytes (15 × 10~ plate) were cultured on collagen-coated plastic culture d~shes (Panza, Yager, Goldfarb el al 1975) and incubated at 37 C in a CO, mr (5 95) mixture m a humidified incubator The culture medium was changed 21 hr after plating to remo,,e any unattached non-vmble cells and aas supplemented with 20 ml of fresh medmm containing [~4C]AFB~ (0 48 l, C1.6 Itg) m 20 itl propylene glycol lncubanon was continued for another 10hr and then the medmm from 15-20 plates was remo,,ed and pooled EAtractton attd h)'drolysts The pooled culture medmm was extracted with chloroform, run down an XAD-4 column, eluted and hydrolysed as shown m F~g 1 Hydrolysis was performed rather b) incubating the column eluate w~th an enzyme mixture (10,000 units //-glucuromdase:ml and 200 units sulphatase:ml) at 37 C for 48 hr or by incubating samples w~th an equal volume of 0 4 N-HCI at 90 C for 2 hr Details are reported m the preceding paper (Wel et al 1985)

R E S U L T S ~ND D I S C U S S I O N

The primary cultures of rat hepatocytes are capable of metabohzmg AFB~ to both chloroformextractable metabohtes and water-soluble AF conjugates As shown in Table 1, about 75°0 of the ra&oactwity was present m the aqueous phase and only about 22", was retained in the chloroformextractable phase, in which AFB~ was found to be the major constituent (nearly 14"0 of the total) Metabohtes AFM~, AFP~ and AFQ~. Menhfied by two-d~mensmnal TLC analysis, were also detected in the chloroform-extractable fraction, hut m smaller quantmes. Th~s result is m agreement w~th the findings of Decad et ul (1977) who found that AFB) was metabohzed to AFM) m rat-hepatocyte cultures The abfltty of hepatoc3,tes to metabohze AFB~ m t'ttro makes these cultures an appropriate model for studying AFB) metabolism Table I Dtstnbutmn of radloactlwt,, m fratAlons ol m e d m m lroln rat-hepatoc3te cultures treated vdth [~'~Claflatoxln B~ Fraction CHCI, extract of medtum* AFB~ AFQ~ AFP~ ,~FM~ Umdennfied Aqueous phase I Total reco~er3,:~

Rddloactl'vlt}* reco~ ered (",, ot total actl'~lD, in medium) 13 0 0 I 6 75

S5 22 12 36 7I 20

97 46

*Determined by scmttllahon counting tAflatoxm anal3,sls b~¢ t~'.o-dtmenstonal T L C and autotadtography *Radioactivity unaccounted for '.',as presumably bound to the mterphase generated during chloroform extractmn ol the culture medium Medtum from 15 20 plates v,a~ pooled

Table 2 Dtsillbutlon ol radloactlVtt', in en/xlnatl~, h}drol.~sates ol at]alto\In conlugate~ extrat_ted trom rat-hepatoc'.te cultures treated ~ t h [taC]aftatoxln B, [-IdCtlOn* C H ( I ~ extract oi m e d m m Aqueous pha~e [ Colmnn elt]uent [ Column eluate Aqueou~ pha:,e l[ CHCI, extract II + APB~ ~. I-/vl L APQ~ &FP~ Umdenufied Total reco~erj,,~

Rddtoactl'~ 1I~~- recovered {",, Ol total actl~,ltV itl rlledlum} 22 30 31 2S I,~ ~¢) I IS 0 20 I 3,% (} 2"~ 3 70 78 b8

*See Fig 1 for scheme of procedures for preparing aflatoxm conjugates for hydrolysis, whtch was effected b~ m c u b a t m n wtth sulphatase 1200 umts ml) and fl-glucuromdase ( 10 000 umts ml) at 37 C tbr 48 hr t D e t e r m m e d by scmtfllatmn counting {Aflatoxm anabsts b.~ two-&mensaonal T L C and a u t o r a d m g r a p h y ~Radmacnvtt~ unaccounted for v.as presumably bound to the X A D - 4 resin and the mterphase generated during chloroform extractions Medium irom 15-20 plate~ v, as pooled

The dlstribuuon of radmacnvlty and the presence of AF metabol,tes m enzymat,c hydrolysates of rathepatocyte culture medmm, whmh had been extracted w,th chloroform and then run through the XAD-4 resin column (Fig l), are shown m Table 2 Some radioacuwt 5 remained m the column effluent and aqueous phase II, mdmatmg the presence of other aflatoxln conjugates, poss~bl2, the gIutathmne conjugates AFB~, AFM~, AFQt and AFP~ were released from the conjugated forms by enzyme hydro[ysts. AFB~ and AFQ~ m larger amounts than AFM~ and AFP~ In addmon to the two-dimens)onal T E e analysis and autora&ographs, the presence of mutagemc AFBL and AFMt m the enz~mahc hydrolysate was demonstrated b.~ the Ames assay m Salmonella t y p h m m r m m strata TA98 The hydrolysate with only 0 I l~g AFBt eqmvalents gave more than 700 revertant colomes (716+ 26:'plate for 3 determmauons) x~hereas the sample before enzymatic hydrolysis (0 3 I AFB~ eqmvalents) gave 123 +- 30 revertant colonies plate Th~s lov, mutagenlc activity m the sample pr,or to enzymatm hydrolysis was possibly due to the presence of a small quanttt 5 of AFB~ whmh was prevlousl.', shown by Wm et al (1985) to be released during column chromatography (XAD-4 resin) Further chloroform washings were performed to remove the AFs generated during column chromatograph.~ The d,stnbution of radioacuv~ty and the release of AFs from water-soluble conjugates after the chloroform washings, enzymatm treatment and then acM hydrolysis (Fig I) are shown m Table 3 ~ F B ~ , A F M ~ , a n d AFP~ were f o u n d m the chloroform extract of the column eluate immedmtel', aftra XAD-4 resin chromatography No AFB,, AFMt or AFP~ was released after enzymatic hydrolysis Hm~e,,er, AFB2, and a blue-fluorescent metaboI,te ~hich ~as more polar than AFB:, were released This metabohte had an R~ value lower than that of AFB2,, on the TLC plate The sample that had been subjected to enz~matic h)drolysts, chloroform wash-

Water-soluble conjugates of aflatoxm B~--2

Rot hepatocyte culture

823

medium

t

CHCI3 extraction ( x S )

]

F

CHCt 3 extract [ A ]

aqueous phase I

radioactivity measurement TLC analysm aut0rodl0graphy

XAD-4 resin coiumn

I

retamed lphose

column effluent I [13]

1 I column etuote

etute with acetone

radlooctlvlt y measurement

evaporatmn~ d~ssolved

and

methanol

~n 0 2 M sodium acetate Duffer (pH 5 0 ) I

~Ames

mutagen assay

second CHCt3Jwashmg [x 5 ] t

i

enzymatic hydrolysis

......... l

CHCl~-extroct I1| [C]

aqueous phase III

I

rod IoacT~ v}ty measurement

CHCI 3 extrachon (x 5) . . . . . . . . [ ~ "1 CHCl 3 extract II aqueous phase ]1

TLC anal ysJs, Outorod,o(~rophy

acid hydrolysis

L

enzymatmc h ydrolys~s I

CHCI 3 e x t r a c t i o n

L

r a d ~ o a c h w t y measurement TLC anal ys~s, autorad~og rophy

t CHCI,~ ext ract IV

i

aqueous phase IV

1

acid hydrolysis

TLC analysis,

I

.....

CHCI 3 extract V

~

outoradtography

CHCI~, extrochon (x 5) i

aqueous phase VI

CHCI

extract VI

rodtoact iv i t y

measurement

I

aqueous phase V

radloactlvlt y measurement TLC onalyspst autorodlography Fig

!

(xS~

CHCI 3 e x t r a c t i o n ( K 5 )

Procedures f o r the p r e p a r a t i o n o f water-soluble a l t a t o x m conjugates f o r e n z y m a t i c and a o d

hydrolysis and for the Ames mutagemclty assay

824

C

1 WEt a n d D

Table 3 Dlstrlbutlou oI radloactl,.lt) In afldtoxln conjugateb m unh_~drol_',sed attd hydrobsed fractmns of medmm from rathepatoc~te cultures ntcubated w~th [HC]aflatoxm B~ Fraction* CHCL extract ol medmm [~.] Aqueous phase I Column effluent I [B] Column eluate

Rddloachvu.~ i" recovered 1",, of total dCllVIt% in medium} 22 30 3l 28

After second CHCL washmg (a)+. a,queou., phase III 13 89 CHCI, extract lit,~ &F B~ ', 95 M-NI~ 0 I1 ~FQ, AFP~ 0 77 Reco~ ery'] 72 30 After (a) and enz)matic h~drolys~s (b).+

[a] + [81 CHCI~ extract Ill [(] ~queou> phase Ill a~queous pha>c I~, CHCL extract l\'~ AFK

53 58 8 58 S 34

~FNL

AFP~ ~XFB:~ Polar metabohte '~ Reco'. erv I1

0 08 II 0S 70 66

¢,fter (a), (b) and acid hydrol)sts~ [A] + [B] + [el 62 16 ( H C I , extra~.t IV 2 85 ~queous pha>e ['~ ~queous phase V 64 CHCI, extract ~,,~ AFB~ 02 AFQ~ 0 04 at[- p~ 0 27 AFB:, 0 22 Recover.,,] I 72 14 *See Fig 1 t\')r scheme of procedures for preparing water-soluble AF conjugates tor enz3manc and acid h3drolys~b [A], [B] and [(2] are fractions smaflarl3 ~dennfied m Fig 1 fDetermnted b~ sclnnllatton counting +CHCI, washing by extracuon of sample> five nines ~lth an equal tolume of CHCI~ cnz3manc hydrol3sl> b 3 incubation wtth sulphata>e (2(10 umts nail and fl-glucuromdase 110 000 units roll at 3; C Ior 48 hr, acid h3drolysts b 3 mcubatmn ~lth an equal volume ot II4,,.-HCI at 91) C for 2 h r ~Aflatoxm analxst> b3 t~o-dtmensmnal TLC and autoradtograph~ ~(Rad~oactt~a 3 unaccounted for was presumabb bound to the X~kD-4 ream and the mterphase generated durmg CHCI. extrdctlons ¶ Blue-fluorescent polar metabohte ~ u h an R~ xalue lo~er than that of ~FB2~ Medmm lrom 15 20 plate> ~as pooled

P H

HSIEH

Table 4 Radloucuxtty In CHCl:v,a,,hed and acM-hydrolssed medmm from hepatoc3te cultures incubated ~lth ['lC]aflatoxnl B I RddlOdCtl'~It} l" reco',cred C',, of total dCtlX.'ltV in medium)

Fracuon* CHCI, extract ot medmm Aqueous phase l Column effluent l Column eluate CHCI, extract 111 Aqueous phase Ill{ Aqueous phase V[ CHCI~ extract Vlb

22 30 ~1 28 8 58 9 91 0 25 2 46

~FB:,

Umdentlfied Reco~ er)]]

74 "78

*See Fig I for scheme of procedures for preparing gater-soluble AF conjugate> fDetermmed b,, ~cmtlllatlon courJtlng {Aqueous pha,,e Ill ,*as hydrolysed by mt.ubatlon v, lth an equal ~olume of 0 4 ~ - H C I at 90 C for 2b~ ~Aflatoxm anal.,,sls b,. t~o-dmlen~mnal TLC and autoradmgraphy IlRadtoactl',lt'y unaccounted for wa~ presumabb bound to the XAD-4 resin and the mterpha>e generated during CHCL extractions Medlutn from 15 2(I plates ~a', pooled

The potential enol-keto tautomertsm m the cyclopentenone rmg could account for the aNht.,, of AFB~ to form glucuromde and sulphate conjugates (Gregory et al 1983, Wet et al 1985) The hydrox) metabohtes, such as AFM~, AFQ~, AFP~ and AFB_,, ~ere also released from watersoluble conjugates and were thus able to form glucuronlde and sulphate conjugates dtrect b Similar conjugates ha~e been reported prevmusly tn monkeyurme samples (Dalezms, Wogan & Wemreb, 19111 and m rat hepatocyte culture medmm (Decad et al 1977. Loury et al 1984) Because some radtoacttvtt} was still detected m the culture medium preparations after actd or enzymatic hydrolysis, the presence of AFB~-GSH conjugate was exMent This conjugate has been detected b} Loury et ul 11984) m rat primary hepatoc) te culture and has been shown to be the detoxlficatmn product of AFB~ 4 cl, nou ledgement,--Thls r e s e a r c h ~ as s u p p o r t e d m p a r t b y f u n d s frona R e g i o n a l Projects S-175 a n d W - 1 2 2 a n d the F l o r i d a AgricuLtural E x p e r i m e n t S t a t m n T h e a u t h o r s are g r a t e f u l to D r J k B y a r d l b r his t e c h n i c a l a s s i s t a n c e a n d helpful d i s c u s s i o n s REFERENCES

lng and then actd hydrolysis contamed AFB~, AFQ~, AFP~ and &FB2, When the aqueous phase III preparation was acid h}drolysed d~rectly, on b AFBz~ was recovered Thts AFB,. mtght have originated from AFBt (Table 4/ Water-soluble glucurontde and sulphate conjugates of AFB~. which had prewously been shown to be present m the urme samples of rats, mine and monk%s (Wet et ul 1985) and m the hver and muscle of turke~ poults dosed with AFB~ (Gregoty, Goldstem & Edds. 1983). mere also produced m the primary hepatoc)te culture tnedtum These conJugates were not mutagemc to S tvphtmu~tum TA98 m the presence of rat-h,,er S-9 preparaUon Enzymatic hydro[ysts. hinderer, released AFB~ and greatly increased the mutagenlc acttvtt), because &FB~ is htghly mutagenlc to thts tester strum (Wong & Hsmh, 1976)

D a l e z l o s J &; W o g a n G N ~1972) M e t a b o h s m o f a f l a t o x l n B I m rhesus m o n k e y s Cancel Res 32. 2297 D a l e z l o s J , W o g a n G N & W e m r e b S M 11971) A f l a t o x m P~ A new a f l a t o x m m e t a b o [ t t e tn i n o n k e , , s Scten(e. N Y 171, 584 Decad G M, Doughert) K K. Hsteh D P H & B y a r d J L 11979) M e t a b o h s m o f a f l a t o x m B~ m c u l t u r e d m o u s e h e p a t o c ) t e s C o m p a r i s o n v, tth rat a n d effects o f c.,,clohexme o x i d e a n d d l e t h ) l m a l e a t e To,~u appl Pharma¢ 50. 429 D e c a d G M , H s t e h D P H & B y a r d J L (19771 M a i n t e n a n c e o f c ) t o c h r o m e P-450 a n d m e t a b o l i s m ol a f l a t o x m B, m p r l m a r x h e p a t o c y t e c u l t u r e s Blochml bloph_t'~ Res Commun 78. 279 Degan G H & Neumann H G (lq78)The major metabo h t e o f a f l a t o x m B r in the rat ts a g l u t a t h m n e c o n j u g a t e Chemu o-Blol lnteracuon~ 22, 239 Doughert5 K K. Spflman S D. Green C E. Steward A R & B ' , a r d J L Ilq8(B P r i m a r y c u l t m e s o f a d u l t

Water-soluble conjugates of aflatoxm B~--2 mouse and rat hepatocytes for studying the m e t a b o h s m of forexgn chemicals Btochem Pharmae 29, 2117 Garner R C (1975) Reduction m binding of[JSC]aflatoxln B I to rat hver macromolecules by phenobarbltone pretreatment Btochem Pharmac 24, 1553 Green C E , Rice D W , Hsleh D P H & Byard J L (1982) The comparatwe m e t a b o h s m and toxic potenc) of aflatoxm B t and aflatoxm Mt m primary cultures of adult-rat hepatocytes Fd Chem Toxtc 20, 53 Gregory J F I I I , Goldstem S L & Edds G T (1983) Metabohte distribution and rate of residue clearance m turkeys fed a diet containing aflatoxm B~ Fd Chem To_xw 21, 463 Loury D J , Hsleh D P H & Byard J L (1984) The effect of phenobarbital pretreatment on the metabolism. covalent binding, and cytotoxlc~ty of aflatoxm B t m primary cultures of rat hepatocytes J To.xtcol envtr Hlth 13, 145 McLean A E M & M a r s h a l l A (1971) Reduced carcmogemc effects o f a f l a t o x m m rats gwen phenobarbltone Br J e:~p Path 52, 322 MgBodfle M U K , Holscher M & Neal R A (1975) A possible protectwe role for reduced glutath~one m aflatoxm B~ toxicity Effect of pretreatment of rats w~th phenobarbital and 3-methylcholanthrene on aflatoxm toxicity Toxic appl Pharmac 34, 128 P a n z a M W , Yager J D . Goldfarb S , Jr, Gurr J A , Yanagl S , G r o s s m a n S H . Becket J E . B a r b e r T A

825

& Potter V R (1975) Biochemical, autoradlographlc, and electron mlcroscop~c studies of adult rat hver parenchymal cells m the primary culture In Gene Expresston and Carcinogenesis m Cultured Lines p 137 Academic Press, New York Raj H G & Lothkar P D (1984) Urinary excretion of th~ol conjugates of aflatoxm B~ m rats and hamsters Cancer Lett 22, 125 Salocks C B , Hsleh D P H & Byard J L (1981) Butylated hydroxytoluene pretreatment protects against cytotox~c~ty and reduced covalent binding of aflatoxm B~ m primary hepatocyte cultures Toxtc appl Pharmac 59, 33 [ Swenson D H , L m J - K . Miller E C & Miller J A (1977) Aflatoxm B~-2,3-o~lde as a probable intermediate m the covalent binding of aflatoxm B~ and B2 to rat hver D N A and ribosomal R N A tn elt;O Cancer Res 37, 172 Wel C I, Marshall M R & Hsleh D P H. (1985) Characterization of water-soluble glucuromde and sulphate conjugates of aflatoxm B~ I U n n a r y excretion in monke), rat and mouse Fd Chem Toxw 23, 809 Wong J J & Hsleh D P H (1976) Mutagemc~ty o f aflatoxms related to their m e t a b o h s m and carcmogemc potentml Proc natn Acad Scz U S A 73, 2241 W o n g Z A , W e ~ C l , R l c e D W & H s l e h D P H (1981) Effects of phenobarbital pretreatment on the m e t a b o h s m and tOXlcokmetlcs of aflatoxm B t m the rhesus monkey To~:w appl Pharmac 60, 387