FACTORS RELATED TO AFLATOXIN BIOSYNTHESIS

c. Fanelli': A.A. Fabbri'; G. Panfili' and S. Passi-'Diparumento di Btologta vegetale, Untversua di Roma 'La Sapienza: Roma, Italy,

2Istuuto San Galltcano (IFO), Roma, Italy

AbstractLipoper-oxi da t i on was pr-evi ous ly found by us to play a key role

In InducIng aflatoxIn bIosynthesIs. 'In vItro', the productIon ofaf'Latoxa ns IS strongly enhanced by the add i t i on of hydr-oper-oxi des ,epoxldes, UV-peroxldated sterols and halomethanes, WhICh InduceLipoper-oxi da t.ion WI t.hi n the f'ungi . 'In VIVO', on var zous OIly andstarchy seeds, we :found that the bIosynthesIs of a:flatoxlns wasd i rec t.Ly related 1:;0 the per-ox.ide number o:f the 011 content of theseeds. SInce It has been shown that llpoperoxldatlon IS a:f:fectedby the presence o:f an t i ox i dant.s and :free r-adi ca l scavengers In theenv i r-onrnerrt, t.h i s arivest i ga t.ion was undertaken to exarm.ne the e:f:fecto:f some common an t i ox i dant.s at d i f'f'er-ent concen tr-at i ons on af'Latox i.nproductIon Induced by the addItIon of carbon tetrachlorIde In culturesof AspergIllus parasltlcuS. Butylated hydroxy toluene (BHT), butylatedhydroxlanlsole (BHA), cysteamIne and sodIum thIosulfate (TIO) SIgnIfI-cantly InhIbIted the aflatoxIn output, but cysteIne, reduced glutathIo-ne, v i t.arm.n C and v i t.armn E greatly enhanced myco tox i n pr-oduc t i.onInduced by carbon tetrachlorIde. StudIes wIth mutant straIns ofAspergIllus parasl tlCUS and A. flavus demonstrated that the phenomenonof Ilpoperoxldatlon a:ffects not only the aflatoxIn bIosynthesIsbut also concerns theIr precursors. norsolorlnlc aCId, averufln,averantln, verSlcolorln A and sterlgmatocystln. In addItIon, thebIosynthesIs o:f these congeners IS InhIbIted by the addItIon ofBHA, BHT, TIO and cysteamIne to culture medIa.

IntroductIonPo i soru ng by t.ox.igen i c f'ungi o:f man and an i.maL are well known

from many countrIes over several centurIes. However, mycotoxIcosesreceIved lIttle attentIon untIl the early 1960s when a:flatoxlnswere d i acover-ed In England (the :famous Turkey X dasease ) and SIncethen have been detected on many Important cereal crops. Some aflatoxlnsshow a hIgh mutagenIc, carcInogenIc and teratogenIc actIvIty fora w i de r-ange o:f or-gani sms and cause hepa t i c car-cr.ncrna In mamma li a(Hea thco te and HIbbert, 1978). Due to t.hei r t.oxi c i ty the af'Lat.oxi ns

93

Proc 4th In! Work Con! Stored-Product Protection, Tel AVIV, Israel, Sept 1986rEds E Donahaye and S Navarro], pp 93-110

stimulated a search for other mould tox1ns and so the era of 'mycotox1ngold rush I began as Maggon et al , (1977) called the per-a od between1960 and 1975. Al though over 100 fungal t.oxi ns have been detectedand character1zed, aflatox1n rema1n the most 1mportant human andan1mal hazard, and the1r study dom1nates mycotox1n research. Countlessattempts have been made to cLar afy the d i f'f'er-errtfactors i.nvol.ved1n the b1osynthes1s of aflatox1ns as reported 1n many reV1ews (Maggonet al., 1977; Heathcote and H1bbert, 1978; Steyn et al., 1980; Bennettand Chr1stensen, 1983; Bet1na, 1984).

Many other r-evi ews on the pharmacology, d.is tr-i butn on , pr-oduct i onand econom1C 1mpact of aflatox1ns are also ava1lable (Goldblatt,1970; Ca eg ler' et al., 1971; Moss, 1972, 1977; AUStW1Ck, 1978). Mostof the works on aflatox1n b1osynthes1s have been cons1dered as dom1nantthemes 1n the research, such as the dynam1cs of carbohydrate metabol1sm,the enzyma t i c ac t i V1 t i es of the 'tr-i.car-boxyli c ac i.d cycle (TCA) andEmbden-Mayerhof (EM) pathway, the enzymes of polyket1de synthase,the role of metals (ma1nly zlnc), the role of nucleot1des.Many works have descr1bed that the or1g1n of aflato-X1n 8

1lS from acetyl CoA ch~ough pol1ket1de synthase(B1ollaz et al.,

1968,1970; HS1eh et al.,1975;St~yn et al. ,1975,1980,Pachler et al. ,1976),but the Ub1qU1ty of acetyl CoA , a key branch p01nt for catabol1c andanabol1c systems clouds a poss1ble clar1f1cat1on of spec1f1c factors re-lated to aflatox1n b1osynthes1s. For th1S reason many are the enV1ronmen-tal var1ables that can change the pool of acetyl CoA 1n fungal cells andthat have been reported 1n the enormous Ilter3ture descr1b1ng factorsWh1Ch affect aflatox1n b1osynthes1s.

However, some authors have reported a relat1onsh1p betweenIlP1d metabol1sm and aflatox1n b1osynthes1s even 1f the subjectlS st i Ll debated. Detroy and Hessel t i ne (1969) reported an a nver-serelatlonsh1p between fatty aCld and aflatox1n blosynthesls; Shlhand Marth (1974) reported that the seLec t ave rnh i b i t i on of t.er-nu.nalresp1rat1on of Asperg1llus paras1t1cuS Speare by sod1um aZ1de enhancedboth af'Latoxi n and IlP1d b i osyrrthee i s . Also, 1t 1S rnt.er-est i ng tonote that all the exper-arnent.s have been performed under d i f'f'er-entexper1mental cond1t1ons and th1S can expla1n the d1sagreements ofmany results.

In many cases af'Latoxa n pr-oduct i on appeared not to be alwaysrelated to fungal growth (Bu'Lock, 1967, 1975, Turner, 1971). Int.hi s connec t i on , both the pr i mar-y and the secondary me tabo I i sm off'ungi have been taken rnt.o account even rf generally most myco t.oxi nsare cons1dered secondary metabolltes Slnce they are produced whenthe exponent1al growth phase (or trophophase) stops and the 1d1ophase

94

starts. However, the whole problem, WhlCh BulLock (1967) deflned'metabollc grld' lS extremely complex and It would be dlfflcultto synthetlze all the structural and blochemlcal aspects lnvolvedIn the dlfferent blosynthetlc pathways of mycotoxlns.

In the followlng report we present malnly the stlmulatlng factorsof the blosynthesls of aflatoxlns, thelr congeners and thelr lnhlbltlon.

Materlals and methods

Asperglllus parasltlcuS (NRRL 2999), A. flavus Llnk. ex Fr.(ATCC 22548) and other r so lat.ed In our Department from wheat seedswere used as toxlgenlc stralns (106 conldla from 15 days old culturesgrown on Czapek Dox agar medlum were used as lnoculum).

'In vltro' experlments

The seeds used In the exper-imen t.s were: wheat (Tr-i.tlcum vulgareHost. cv.Manltoba), malze (Zea mays L. cv.Decalb 365), sunflowerHellanthus annuus L. cv.Unlflor 70), peanuts (Arachls hypogaea L.cv. Bombay). CuI 't i var cond i tn ons , t i rne and temperature of rncuba t.i.on,mOlsture of seeds and aflatoxln analysls were reported prevlously(Fabbrl et al., 1980). In some experlments, sunflower seeds of dlffere-nt ages and di f'f'er-ent per-oxi de number In t.hei r- o i I.s were anocul.at.edwlth toxlgenlc stralns of Asperglllus.

'In vltro' experlments

In all exper i merrt.s the Li qu i d culture med i.a used were CzapekDox broth plus 5 mg/l ZnS04 . 7 H20 and 1 mg/l Na2Mo04 . 2 H20. The ll-qUld culture medla, lnoculated wlth aflatoxlgenlc stralns, weresupplemented wlth the followlng compounds:

1. o i Ls extracted from d i f'f'er-ent seeds (.a • e. sunflower, peanutsand malze) (Fanelll et al., 1981 a).

2. synthetlc free fatty aClds (olelc aCld), trlglycerldes (trlole-In) under experlmental condltlons (Fanelll et al., 1981b) •

3. ce r-ul e n i. n, dl-, tetra- and hexahydr-ocer-ulen i n (Fane ILa etal , 1983 a).

4. synthetlc epoxldes such as 9,10and 9,10.12,13 dlepoxymethylstearateb) •

epoxymethylstearate(Fanelll et al.

and1983

95

5. synthet1c hydroperox1des obta1ned by the act10n of soybean 11-pox1genase on Ilnole1c aC1d, UV perox1dated sterols and UV pe-rox1da~ed free fatty aC1ds (Fabbr1 et al., 1983).

6. carbon tetrachlor1de (CCI ) alone or 1n assoc1at1on w1th other4drugs such as SKF 525 A and phenobarb1tal (Pass1 et al., 1984;Fanell1 et al., 1984).

7. d1fferent halomethanes alone or 1n assoc1at1on w1th phenobarb1-tal (Pass1 et al., 1985).

8. d1fferent organ1c solvents alone or 1n assoc1at1on w1th pheno-barb1tal (Fanell1 et al., 1985 a).

9. common ant1ox1dants and free rad1cal scavengers alone or 1n as-soc1at1on w1th CCI (Fanell1 et al., 1985 b).

4

The fungal growth was measured as mycel1um dry we1ght as prev10uslydescr1bed (Fanell1 et al., 1980).

Preparat10n of m1tochondr1a and m1crosomes.

Mycel1a were homogen1zed 1n hyposmot1c med1um (10 mM- Tr1s/EDTA).The cellular fract10ns were separated by d1fferent1al centr1fugat1on(Pass1 et al., 1985)' the heavy fract10n (Wh1Ch conta1ns nucle1, plasmamembrane and some non d1srupted cells) was pelletted at 800 g for 15m1n, the m1tochondr1al fract10n at 7000 g for 20 m1n and the m1crosomalfractlon at 100 000 g for 60 m1n.

Analysls of m1tochondr1a and mlcrosomal llplds.

Measured al1quots of m1crosomes and m1tochondrla were extractedw1th CHCI /MEOH (2'1 v/v). After evaporat1on of the solvents under

3vacuum, the extracted IlP1ds were fract10nated by thln layer chromato-graphy (TLC) (Pass1 et al., 1981) to separate the IlP1d fract1ons. The11Pld spots correspondlng to phosphol1P1ds (PL), free fatty aC1ds (FFA),tr1glycerldes (TG) and sterol esters (SE) were scraped off separatelyfrom the plate and the scrap1ngs extracted three t1mes w1th perox1de-free dlethylether. The FFA fractlon was d1rectly methylated w1th d1azo-methane; PL, TG and SE were separately transester1f1ed by reflux1ng for3 h wlth 2% v/v concentrated sulphur1c aCld 1n red1st1llated methanol.A measured al1quot of FA methylester m1xture of each fract10n was used

96

for gas lIqUId chromatography (GLC) on a fused sIlIca gel, 30 m, 0.32 mmInternal dIam., Supercowax 10 caplilary column (Supelco) to 5890 A Hew-lett-Packard gas chromatograph equIpped wlth a hydrogen flame lonlzatlondetector.

Type of InJectIon, spiltiess; carrler gas, HelIum; flow 2 ml/mln;operatIng temp. Injector, 250°C; 300°C; InItIal oven temperature, 50°Cfor 0.5 mIn, gradIent to 160°C at 20°C/mIn and then to 250°C at 2°C/mIn.

Assay of mIcrosomal enzymes.

A measured allquot of mlcrosomes was adjusted to a concentratIonof approxImately 2 mg proteIn per ml In 0.1 M-TrIs-HCl buffer, pH 7.4.MIcrosomal proteIn was determIned by a method of Lowry et al., (1951),uSlng bOVIne serum albumlne as standard. Cytochrome P-450 was assayedby the method of Omura and Sato (1964). NADPH cytochrome C reductase~assayed by the method of Mazel (1971). Amlnopyrlne demethylase was assay-ed by measurIng the amount of formaldehyde produced In the presence ofNADPH generatIng system (Werrlngloer, 1978).

Results and dIScussIon

Our studIes started some years ago from the observatIon that 'lnVIVO' aflatoxIn output was normally much hlgher In OIly than In st~seeds contamlned by A. flavus and A. parasltlcuS (Fabbrl et al.,1980).In order to clarIfy If the aflatoxIn bIosyntheSIS could be related toIIPlds present In olly seeds , we supplemented llqUld culture medIa ~culated WIth A. flavus and A. parasltlcuS wlth olls extracted from dlffe-rent seeds (l.e. sunflower, peanuts, maIze) (Fanelll et al., 1981 a). Asynthetlc medIum was chosen for a better understandlng of what could re-present selectIve factors leadIng to an Increased aflatoxln productIon.Flg.1 shows the fungal growth and aflatoxln productIon after 15 days ofIncubatlon under these experImental condltlons. It IS eVldent that allthe olls added show a defInIte stImulatIng effect only on fungal growthWhlCh was enhanced by the addltlon to the medlum of a well known deter-gent, Trlton X 100. In fact, the added detergent Increases solubllltyof OIls and so faCllltates ItS uptake. It IS also eVIdent that aflatoXlnproductIon IS not slgniflcantly affected by the presence of dlfferentOIls. The results were conflrmed by the addltlon of synthetlc free fattyaCIds (oleIC aCId), trIglycerldes (trloleln) under the same experImentalcondltlons (Fanelll et al., 1981 b).

The above results dld not agree wlth those of the authors who sho-wed a relatIonshIp between lIpId metabolIsm and aflatOXIn biosyntheSIS.

97

Fig. 1 EFFECT OF OILS OF SUNFLOWER I MAIZE AND PEANUTS SEEDS

ON GROWTH AND AFLATOXIN PRODUCTION OF A.FbAyUS AFTER

115 DAYS OF INCUBATION

2000

DRY WEIGHT"'1l/SOml..

1500

AFLATOXINnll150ml

1:·:-:-31000

500

SyntheticmedIum(am)

Czapek

+TritonX 100

+Sunfl_r

011 +Triton

.'00

+Pe.nut

011+T~il88

+Sunflower

011

+~.nut

011

+Mei.e

011

.M.lzeoil +

Tii1~8

Therefore for a better understanding of the problem of the relationshipbetween lipid metabolism and aflatoxin production we used some well-known antibiotic substances (cerulenin and tetrahydrocerulenin) that areinhibitors of the fatty acid synthetase. Cerulenin and tetrahydroceru-lenin stimulated significantly aflatoxin biosynthesis by about twentyto twenty five times as compared with controls (Fanelli et al., 1983 a).Such an effect was stricly related to the epoxide ring present in thecerulenin and tetrahydrocerulenin molecules. In fact the stimulatingeffect disappears following the transformation of epoxide of ceruleninand tetrahydrocerulenin to the corresponding dioles di- and hexahydroce-rulenin. The importance of the epoxide ring in the stimulation of afla-toxin biosynthesis was confirmed by adding synthetic epoxide such as9,10-epoxymethylstearate and 9,10:12,13 diepoxymethylstearate to cultu-res of A. flavus and A. parasiticus. The output of aflatoxins increasedover one hundred times, and proved to be affected by the process of li-poperoxidation in general (Fanelli et al., 1983 b).

Lipid epoxides may be formed in limited amounts during the lipidperoxidation of unsaturated lipids which primarily produces lipoperoxi-des (lipid peroxy radical, hydroperoxides) and subsequently many otherbreckdown products responsible for rancidity in naturally occurring

98

fats (Flg.2).

Flg.2 L1POPEROXIDATION OF LINOLEIC ACID(C182 •.69-12)

13 12 11 10 9ALKYL RADICAL -CH=CH-CH=CH -~H-CH2

ISOMER t +029

-CH-I0-0·

PEROXV RADICAL +xy ~9

HYDROPEROXIDE - Y - + x.O-OH~

18 14 13 12 11 10 9 8 1CH3(C~kCH:fCH =CH-c;H- CH=CH-CH:rICH2)6 - COOH

/ '\ ALKYL RADICAL

13 12 11 10 9-CH-CH=CH-CH=CH-

. I +02

~ 13-CH-I0-0·

/' '\"XH PEROXV RADICAL

13-C- +x.

~ 6-0H HYDROPEROXIDE

ALKYL RADICALISOMER

PRO PAGATION

!ALKOXY RADICALSCYCLIC PEROXIDES

AND BYPRODUCTS

Synthetlc hydroperoXldes obtalned by the actlon of soybean IlPO-xlgenase on Ilnolelc aCld, UV peroxldated sterols and UV peroxldatedunsaturated free fatty aClds paralleled the effect of epoxldes (Fabbrlet al., 1983) (Flg.3).

Followlng these results 'In vltro' we started to lnvestlgate therole of Ilpoperoxldatlon 'In V1VO' by lncubatlng sunflower seeds ofdlfferent ages and dlfferent peroxlde numbers of thelr olls wlth ~.fla-vus and A. parasltlcuS . Sunflower seeds were chosen because the trlgly-cerldes In thelr 011 contaln a hlgh percentage of polyunsaturated fattyaClds and especlally Ilnolelc aCld. It lS eVldent that the hlgher theperoxlde number of the 011 content of the seeds, the hlgher the aflato-Xln productlon by toxlgenlc fungl (Passl et al., 1984) (Flg.4).

The problem of Ilpoperoxldatlon 'In V1VO' lS extremely compllca~,and in addition to the degree of unsaturatlon of fatty aClds (polyunsa-turated are more easlly and rapldly peroxldable than monounsaturated)many other physlcochemlcal factors ln the envlronment such as radlatlon,'humidlty, temperature, the presence of varlOUS metal salts, antloxldants

99

Fig.3

1000

900

E 800

oIt)

';;;'-700

(J)

Z

X 5000l-et...J 400LLet

300

200

GO~-~..Z

)(

0 2~C...III.C

Effect of UV peroxidated sitosterols, UV peroxidated linoleicacid, synthetic hydroperoxides on aflatoxin production in liquidcultures inoculated with A.parasiticus.

... , '

'00

SM.UV PEROXIDATED C,8:2SM.UV PEROXIDATED SITOSTEROLSM. C,8:2 HYDROPEROX.(O.5mg/ml)SM +C,8:2 HYDROPERox.c025mg/m/)SM .C,8:2(O.2mg/ml)CONTROl(SM)

8 10 12

DAYS OF INCUBATION

15T

Fig.4 AFLATOXIN PRODUCED ON SUNFLOWER SEEDS OF DIFFERENTAGES INOCULATED WITH ASPERGILLUS PARASITICUS

(DAYS OF INCUBATION :30 )

PEROXIDE NUMBER 11-0_.8_1_'_~O_E_A_~_.2__ 1_.6-+1_'2_.8_Y_E_3_:_R_S_4_._8 9.8 11.1 17.0 I3 YEARS

100

and so on, may act on the phenomenon of Ilpoperox1dat1on. The confl1c-t1ng results reported 1n the Ilterature on the amount of aflatox1ns 1nagr1cultural crops may be expla1ned on th1S bas1s.

Follow1ng the above results show1ng the st1mulat1ng effect of theexogenous IJpOperOx1des added to the cultures of tox1gen1c fung1, we1nvest1gated the possib1llty of enhanclng the aflatoxln output by sub-stances capable of 1nduc1ng Ilpoperoxldat1on of the 1nternal membraneof fung1, that lS endogenous Ilpoperox1dat1on.

The presence of components of polysubstrate monoxygenase act1v1tyhas been establ1shed, and 1n partlcular cytochrome P-450 and NADPH P-450 reductase 1n m1crosomes of A. paras1t1cuS (Bhatnagar et al., 1982).As 1t has been postulated, on good eV1dence, that the bas1s of tOX1C1tyof some halomethanes and 1n part1cular carbon tetrachlor1de, Wh1Ch 1San extens1vely stud1ed hepatotox1n, lS perox1dat1on of IlP1ds of theendoplasm1c retlculum of Ilver cells by hlghly react1ve tr1chloromethylradlcals CCI . formed by the lnteractlon of carbon tetrachlor1de w1th

3the cytochrome P-450 system, we stud1ed the effect of CCI on aflatoxlnn 4product1on 1n cultures or A. paras1t1cuS and A. flavus. The presence

of CCI 1n cultures of A. paras1t1cuS and A. flavus h1ghly st1mulated4aflatox1n output. Th1S effect m1ght be due to the perox1dat1on of IlP1dsof endoplasm1c ret1culum of Asperg1llus by h1ghly react1ve CCI . rad1GllS

3formed by the 1nteract1on of halomethane w1th the NADPH cytochrome P-450system of the fungus Wh1Ch produce pr1mary and secondary d1sturbances(Fanell1 et al., 1984). Th1S hypothes1s lS supported by the results ob-ta1ned by add1ng to cultures, together wlth CCI , other drugs such asd1ethylam1noethyld1phen1l-propylacetate (SKF 5~5 A) and phenobarbltal(Pass1 et al., 1984)'F1g.5).

Phenobarb1tal 1ncreases endoplasm1c ret1culum synthes1s of prote1nand phosphol1P1ds (Orren1us and Ernster, 1964) and the components of~NADPH cytochrome P-450 system are all 1nduced to st1mulate the metabol1~of CCI and other drugs. Our results are 1n line w1th th1S: 1n fact the

4add1tlon to cultures conta1n1ng 0.8% CCI of phenobarb1tal enhanced the4effect of eCl by about 1.7 times on the 14th day of 1ncubat1on.In thlS gonnect1on also the addltlon of dlfferent halomechanes alo-

ne or 1n assoc1at1on w1th phenobarb1tal have been tested 1n A. paras1t1-cus cultures. F1g.6 shows shows the growth (dry we1ght of mycel1um) andaflatox1n product1on of A. paras1t1cuS grown for 14 days at 30°C on syn-thet1c med1um supplemented w1th d1fferent halomethanes 1n the presenceand absence of phenobarb1tal.

Halomethanes, because of the1r h1gh speclf1c grav1ty and Ilm1tedwater solub1l1ty, settled at the bottom of the flask, after the1r add1-t10n to cultures. However, after 12 days of 1ncubat1on they had d1sap-

101

Fig. 5

0.3E0~ClgCIIZ

0.2)(0l-e(

..JI&.e(

0.1

0.4

E0~Cl 0.3!.CIIz)(

0I-

0.2e(

..Ju,e(

0.1

Effect of carbon tetrachloride(CCl ) alone or in association4with SKF 525 A and phenobarbital on aflatoxin production in

liquis cultures inoculated with A. parasiticus.

• __ • SM +0.8't.CC'4 +2mg/ml Ph.noberbital

...- -_ ....SM+0.8't.cCI4+20Jl9/ml SKF 525 A~-•........• SM+0.8't.CCI4 ......--

.-_.SM+0.8't.CCI4+4.,gfml SKF525 A • __ ... - _ ... -

o--oSM (Synth.tic m.dium) ......... - -,,,I" .." _ .........•....

,,' .../ ...., ...-

.~~5·····/.~~.: .....r···· -i 1h ,

14

DAYS OF INCUBATION

Fig. 6 HALOMETHANES(HAL) AND AFLATOXIN PRODUCTION

~t:;3

~

HAL + Phenobarbital

HAL.'.'.''.'.'

11~1'.''.'::::::4.*•..'..'..'..'..'..'..'..:::........

102

peared from the liquid cultures with the exception of 0.2% and 0.1%tetraiodomethane (CI ) and carbon tetrabromide (CBr ) and 0.2% iodoform4 4(CHI ). Trichlorobromomethane (BrCCl ) (0.1%), bromoform (CHBr ) (0.2%),carb6ntetrachloride (CCI )(0.2%) and3carbon tetrabromide (CBr ; (0.02%)

4 4in decreasing order highly stimulated the aflatoxin production, the di-alomethanes were less effective.

The primary physicochemical factor in the cleavage of the halocarl:x:>nto a free radical is the potential for activation to a free radical spe-cies which increases with both increasing halogenation and decreasingelectronegativity of the halogen atoms. Therefore CBr is more likely to

4be activated than CCI to a free radical because bromine is less elec~4negative than chlorine, and CCI is more easily activated to a free radi-

4cal than chloroform (CHCl ) because of the greater number of halogen abr3ms. Then as expected, the addition of phenobarbital to cultures contai-

ning halomethanes significantly enhanced the effect of the halomethanesin inducing aflatoxin production (Passi et al., 1985).

Moreovel:', the addition of different organic solvents to culturesof A. parasiticus and A. flavus have shown a stimulating effect on afla-toxin biosynthesis which is further enhanced by the presence in culturesof phenobarbital. On the analogy with the halomethanes we suggest thatthe production of aflatoxins depends on lipoperoxidation of unsaturatedlipids of endoplasmic reticulum of Aspergillus due to formation of freeradical species derived from microsomal activation of solvents (Fig.7)(Fanelli et al., 1985 a).

Fig· 7 ORGANIC SOLVENTS (O.SJ AND AFLATOXIN PRODUCTION

E~o.-C)

!.~o.)(

oI-

~o.3IL

C

0.2

0.1

I o.s.

I O.S. plus Phenobarbital

Control Benune Acetone Diann Ciclo- Ethyl- Eth ... ol Hexane M.thanolh."ane acatate

103

In the case of A.paras1t1cuS treated w1th halomethanes, we have produced1nd1rect eV1dence of the format1on of free rad1cal 1ntermed1ates (a) thedeterm1nat1on of a structure-act1v1ty relatlonsh1p w1th1n the dlfferenthalomethanes, between propens1ty for act1vat1on to free rad1cals and afla-tox1n product1on (Pass1 et al., 1985), (b) drugs WhlCh act by 1nh1b1t1ng(SKF 525 A) or act1vat1ng (phenobarbltal) the cytochrome P-450 system,respectlvely 1nh1b1t or act1vate the effect of CCI 1n cultures of A.fla-

4vus and A.paras1t1cuS (Pass1 et al., 1984). Attempts to conf1rm the presen-ce of, for exemple, tr1chloromethyl rad1cals (CCI .) by d1rect eV1dence3uS1ng electron sp1n resonance measurements In the presence of sp1n trappers,were unsuccessful. However, It must be p01nted out that even 1n the Ilverof an1mals treated w1th CCI there are controvers1al results demonstrat1ng4the presence of CCI " and that there lS large d1fference between mammals

3and fung1 1n the rate of metabol1sm of CCI . The rate of metabol1sm of CCI4 4lS very rap1d In mammals and 1n contrast, lS slow 1n cultures of A. paras1-

t1CUS and A. flavus.Other 1nd1rect eV1dence was found for the role of Ilpoperox1datlon In

the 1nduct1on of the b1osynthes1s of aflatox1ns by carbon tetrachlor1de 1ncultures'

1. 1n 15-days-old cultures of Asperg1llus supplemented w1th CC14 thephosphol1P1d fract10ns of m1crosomes and partly of m1tochondr1a,showed a large decrease 1n the unsaturated/saturated rat10 1ndl-cat1ng that Ilpoperox1dat1on proceeds more slowly, and at lowerextent 1n m1tochondr1a than 1n m1crosomes (Pass1 et al.,1986)(Table 1).

Table 1. Unsaturated/saturated fatty aCld ratlo of SE, TG,FFA and PL fractlons of IlPldextracts of mltochondrla(Mlt) and mlcrosomes(Mlc) of A paras,t,cuS after 10 and15 days of lncubatlon at 30°C Unsaturate FA C +C ,saturaced FA C +C16 0+C18 0 Cyst. cysteamlne 18 1 18 2 14 0

Control CCI CC14+cystL1Pld fractlons Days Mlt M1C Mlt 4 M1C Mlt M1C

Sterol ester(SE) 10 4 2 10 3 3 9 9 4 4 3 10 6

15 4 2 9 1 4.6 7 5 4 7 10.5

Trlglycerldes(TG) 10 2 1 3.0 2.0 2 8 2 7 2 8

15 2 4 3 0 2 2 1.5 2 7 2 9

Free fatty aClds(FFA) 10 5 1 3 2 4.8 3 0 4 9 3 4

15 6.2 4.5 6 0 3.1 6 7 4 9

Phosphollplds(PL) 10 57.2 215.4 50.2 164.3 75 5 236 9

15 51.2 186.7 18 1 3.5 70 4 204 0

104

2. as axpected, peroxldatlve decomposltlon of structural Ilpldsled to a loss of enzymatlc activltles, l.e.cytochrome P-450,NADPH cytochrome C reductase and Amlnopyrlne N-demethylase(Table 2). In fact It lS well known that the normal structuresand functlon of the endoplasmlc retlculum depends on the lnte-grlty of structural llplds, malnly phosphollplds (Passl et al.,1986) .

Table ~ Effect of CC14 1n presence and 1n absence of cysteam1ne on m1crosomeal parametersof A paras1t1cuS at 10 and 15 days of growth at 30.C n d not detected

Parameters Days Control CCI CCl 4+Cyst4

Cytochrome P450(n mol/mg prote1n) 10 0.31+0.1 0.06+0.1 0 36+0 1715 0.27+0.13 n.d 0 40+0 14

NADPH cyt C reductase 10 40 1 +7 9 16.0 +2 5 51 5 +17 0(n mol cyt C reduced/m1n/mg prot ) 15 33 2 +10.3 3 1 +2 5 105 2 +26 3

Am an opy r an e N-<lemethylase 10 4 5 +1 6 2.6 +1 9 4 2 +1 4(n mol HCHO formed/10 m1n/mg prot ) 15 3.8 +1 9 n ,d. 6 3 +2 2

3. If the Ilpoperoxldatlon hypothesls lS well founded, commonantloxldants and free radlcal scavengers such as cysteamlne,butylated hydroxyanlsole (BHA), butylated hydroxy toluene (BHT),cystelne, vltamln C and vltamln E should lnhlblt the stlmulac~ng effect on aflatoxln output lnduced by CCI4.

As Flg. 8 shows, the addltlon of CCI to cultures of A. parasltlcuS4together wlth the most common antloxldants and free radlcal scavengers

gave surprlslng results. Cystelne, vltamln C and vltamln E not only dldnot lnhlblt aflatoxln blosynthesls lnduced by CCI but further lncreased

4aflatoxln productlon slgnlflcantly as compared wlth cultures supplemen-ted only wlth CCI . Thls phenomenon mlght be ascrlbed to the formatlon

4In cultures of more or less reactlve speCles, l.e. thyll radlcals, ascor-bll radlcals, a-tocopherol free radlcals, whose effect were synerglC Wlththat of CC13· . In addltlon, cystelne and vltamln C stlmula~ed aflatoxlnblosynthesls even wlthout CCl (Fanelll et al., 1985 b).

4 --Conversely, cysteamlne, BHA and BHT lnhlblted aflatoxln productlon

lnduced by the halomethane. The cysteamlnyl radlcal(R·), whlch lS produ-ced durlng scavengers actlon, dld not show a prooxldant effect on CCl .

3 'probably because It preferenclally underwent a reactlon In comblnatlonwlth another cysteamlnyl radlcal ('R·) (R· + 'R·=R'R) formlng unreaC'lve

105

DRY VEIGHT

'SOIl1.AfLATOXINS

fZZZJ

Fig. 8 EFFECT OF DIFFERENT SCAVENGERS

0lDITIIIJI. CCI .. OCI ... CCI ... CCI • CCI .. . 1:C1.. . 1:1:1.. ,..

(0.!41 Cll1w-l ... Cll1 .... l... RIIA HilT VI L.C VI L.IC

(o.!.SI (o.;>sl (o.o;>Sl (0.1J?S1 (o.ISI (o.ISI

cystamine. A similar mechanism might explain the inhibitory action of BHAand BHT . The phenoxyradical (Ph'), stable because of sterical hinderancedue to the electron donor terbutyl groups in the ortho position, has apreferential bent for reacting with other phenoxy radical ('Ph') or pero-xy radical (ROO') with production of unreactive compounds. The lower in-hibiting effect of BHT as compared to BHA could explained by its higherinsolubility in liquid cultures.

The phenomenon of lipoperoxidation is not restricted to the inducti-on of aflatoxins but concerns all the stable known aflatoxin congeners(norsolorinic acid, averantin, averufin, versicolorin A, and sterigmato-cystin) (Figs. 9 & 10). In fact, as the figures show, cultures of diffe-rent mutant strains of A. parasiticus and A. flavus added with CC14 orlipoperoxides or epoxides highly enhanced the congeners production con-firming the effect described for the aflatoxins.

In conclusion, we suggest that lipoperoxides from any sources (syn-thetic lipoperoxides, naturally occurring lipoperoxides in aged or storedseeds, endogenous lipoperoxides formed by the interaction of xenobioticdrugs interacting with the mixed function oxidase system of A. parasiti-cus, and probably many of their reactive bypr-oducts) playa key role. in

106

Z2.0ii:

:>a:w><t

ol(

z- 1.,Ji-- ECo010.J_Oat~Ern_a:w> 1.0

Z

i=zol(a:Il.t

o.a><

Eo..,.......Cl

50

zi= c.im<00~~~ ~ 25

~SQ::mlila:1-0m z

..~.CONTROL(SM)

Fig.9 lIPOPEROXIDATION AND PRODUCTION OF AVERANTIN _,AVERUFIN~ ANDVERSICOLORIN AImJ BY MUTANT STRAINS

II...:::~~~Ij~j ';;11.:.: .:.

~-·C·C""'14""+"""--Lin~ic ~EPor;;tea"'ra""'t-e----Cysteamine hydroperoxide

(0.5%) (0.25%)(o.S%)

Fig. lOll POPEROXIDATION AND PRODUCTION OF NORSOLORINIC AC .•AND STERIGMATOCYSTIN m BY MUTANT STRAINS

75

CONTROL(SM)

.............~:::rJ.·.

J.~~~~.:.:.:

CCI ..(O.S")

~

III

iii:::x~..

CCIJ,OB") 9.10EPOXY· 9.10EPOXY- C18'2+ STEARATE STE~RATE HYDRoPEROXIDE

CYSTEAMINE (O.5mg/ml) CYSTEAMINE (0.25 mg/mll(0.5%) (0.5 %)

107

aflatoxln productlon.However, It must be pOlnted out that although the above studles can

provlde valuable data as to WhlCh scavengers may be effectlve 'In vltro' ,In terms of reactlvlty wlth CCl . and llpoperoxldes, the only real testwlll come wlth the demonstratlo~ of satlsfactory scavenglng 'In V1VO'.

AcknowledgementsThlS study was supported by European Communlty Grant n.TSD.A.111.I(S).

References

AUStW1Ck P.K.C.(1978) AflatoxlcoS1S In poul+ry.MycotoxlC fungl,mycotoxlnf~mycotoxycoses. An encyclopedlc handbooj~. Vol.2 Marcel Decker, Inc.New York and Basel. pp.279-301.

Bennett J.W. and Chrlstensen S.B.(l983) New perspectlves on aflatoxln blO-synthesls. Adv.Appl.Mlcroblol. 29,53-92.

Betlna V.(1984) Mycotoxlns,Productlon,Isolatlon,Separatlon and Purlflcatl-on Elsevler Sc.Comp.Amsterdam.

Bhatnagar R.K., Ahmad S., Kohil K.K., MukerJlK.G.and Venka t.asubr-amam an T.A.(1982) Inductlon of polysubstrate monoxygenase and aflatoxln produc-tlon by phenobarbltone In Asperglllus parasltlcuS. Blochem.Bloph.Res.Comm. 104,1287-1292.

Blollaz M., Buchl G. and Mllne G.(1968) Blosynthesls of aflatoxlns.Theblogenesls of blsfuranolds In the genus Asperglllus. Journ.Am.Chem.Soc. 90,5017-5020.

Blollaz M., Buchl G. and Mllne G.(1970) The blosynthesls of aflatoxlns.Journ.Am.Chem.Soc. 92,1035-1043.

Bu'Lock J.0.(1967) The regulatlon of secondary blosynthesls. Essay In blO-synthesls and mlcroblal development. John Wlley & Sons.lnc.New York.

Bu'Lock J.D.(1975) Secondary metabollsm In fungl and ltS relatlonshlp togrowth and development. The fllamentous fungl. Vol.1 Industrlal Mycolo-gy. pp.33-58 Edward Arnold Press, London Halsted Press, New York.

Clegler A.,Kadls S. and AJl S.J.(1971) Fungal toxlns. Mlcroblal toxlns' acomprehenslve treatlse. Vol.6 Academlc Press Inc.New York.

Oetroy R.W. and Hesseltlne C.W.(1969) Net synthesls of14C-labeled IlPldsand aflatoxlns In restlng cells of Asperglllus ~SltlcUS. Oev.lnd.Mlcroblol. 10,127-133.

Fabbrl A.A., Fanelll C. and Seraflnl M.(1980) Aflatoxln productlon oncereals, 011 seeds and some organlc fractlons extracted from sunflo-wer . Accad.Naz.Sc.(del XL) 98,219-228.

Fabbrl A.A.,Fanelll C.,Panflll G.,Passl S. and Fasella P. (1983).Llpoperoxl-datlon and aflatoxln blosynthesls by Asperglllus parasiticus and A.

108

flavus. Journ.Gen.Mlcroblol. 129, 3447-3453.Fanelll C., Fabbrl A.A. and PaSSl S.(1980) Growth requlrements an llpld

metabollsm of Asperglilus flavus. Trans.Br.mycol.Soc. 75,371-375.Fanelll C., Fabbrl A.A., Seraflnl M. and Flnottl E.(1981 a) Fattorl con-

neSSl con la slntesl delle aflatosslne 'In vltro' e 'In VlVO'. Lacontamlnazlone da mlcotosslne dl allmentl,manglml e foraggl.pp.113-120 A.l.A.C. e S.l.M.A. Ed.

Fanelll C., Fabbrl A.A. and PaSSl S. (1981 b) Aflatoxln productlon byAsperglilus flavus durlng lncubatlon wlth llpld sources In culturemedla. Trans.Br.mycol.Soc. 77,416-419.

Fanelll C., Fabbrl A.A., Flnottl E., Panflll G., and PaSSl S.(1983 a)Cerulenln and tetrahydrocerulenln. stlmulatlng factors of aflatoxlnblosynthesls. Trans.Br.mycol.Soc. 81,201-204.

Fanelll C., Fabbrl A.A., Flnottl E. and PaSSl S.(1983 b) Stlmulatlon ofaflatoxln blosynthesls by llpophlilc epoxldes. Journ.Gen.Mlcroblol.129, 1721-1723.

Fanelll C., Fabbrl A.A., Flnottl E., Fasella P. and PaSSl S.(1984) Freeradlcals and aflatoxln blosynthesls. Experlentla, 40,191-193.

Fanelll C., Fabbrl A.A., Plerettl S., Panflll G. and PaSSl S.(1985 a)Effect of organlc solvents on aflatoxln productlon In culturesof Asperglilus parasltlcuS. Trans.Br.mycol.Soc. 84,591-593.

Fanelll C., Fabbrl A.A., Plerettl S., Flnottl E. and PaSSl S.(1985 b)Effect of dlfferent antloxldants and free radlcal scavengers onaflatoxln productlon. Mycot.Res. 1,65-69.

Goldblatt L.A.(1970) Chemlstry and control of aflatoxlns. Pure Appl.Chem. 21,331-353.

Gupta S.K., Maggon K.K. and Venkltasubramanlan T.A.(1977) Effect of Zlncon TCA cycle lntermedlates and enzymes In relatlon to aflatoXln blO-synthesls. Journ.Gen.Mlcroblol. 99,43-48.

Heathcote J.C. and Hlbbert J.R.(1978) Aflatoxlns.chemlcal and blologlcaspects. Elsevler, Amsterdam.

Hs i eh D.P.H., Se i ber- J.N., ReeceC.A., Fi.tze lI D.L.,YangS.L., Delez i os G.N.,La Mar D.L., Budd D.L. and Motell E. (1975) 13C nuclear magnetlc resonan-

ce spectra of aflatoxln B1 derlved from acetate. Tetrahedron 31,661-663.

Lowry O.H., Rosenbrough N.J., Farr A.L. and Randall R.J. (1951) Protelnmeasurements wlth the Folln phenol reagent. Journ.Blol.Chem.193,265-275.

Maggon K.K., Gupta S.K. and Venkltasubramanlan T.A. (1977) Blosyntheslsof aflatoxlns. Bact.Rev. 41, 822-855.

Mazel P. (1971) General prlnclples and procedures for drug metabollsmIn vltro. Fundamental of Drug Metabollsm and Dlsposltlon. pp. 527-545. Wllilams and Wllllams Baltlmore.

109

Moss M.O. (1972) Aflatox1ns and other mycotox1ns. Phytochem1cal ecologypp. 125-144. Academ1c Press, New York.

Moss M.O. (1977) Asperg1llus mycotox1ns. Genet1cs and PhYS10logy of As-perg1llus. pp. 499-524. Academ1c Press, New York.

Omura J. and Sato R. (1964) The carbon monox1de b1nd1ng p1gment of llverm1crosomes. I. iV1dence for 1tS hemoprote1n. Journ.B1ol.Chem. 293,2370-2378.

Orren1us S. and Ernster L. (1964) Phenobarb1tal-1nduced synthes1s of theoX1dat1ve demethylat1ng enzymes of rat llver m1crosomes. B1ochem.B1oph.Res.Comm. 16, 60-64.

Pachler K.C.R. , Steyn P.S., Vleggaar R., Wessels P.L. and Scott D.B.(1976) Carbon-13C-nuclear magnet1c resonance ass1gnments and b10-synthes1s of aflatox1n Band ster1gmatocyst1n. Journ.Chem.Soc.Per.

1Trans. 1976: 1182-1189.Pass1 S., Rothsh1ld -Boros M.C., Fasella P., Nazzaro-Porro M. and Wh1te-house D. (1981) An appl1cat1on of h1gh performance llqU1d chromatography

to analys1s of llP1ds 1n archeolog1cal samples. Journ.L1p1d Res.22, 778-784.

Pass1 S., Nazzaro-Porro M., Fanell1 C., Fabbr1 A.A. and Fasella P. (1984)Role of llpoperox1dat1on 1n aflatox1n product1on. Appl.M1crob1ol.Blothechn. 19, 186-190.

PaSS1 S., Fanell1 C., Fabbr1 A.A.,FlDott1 E., Panf1l1 G. and Nazzaro-PorroM. (1985) Effect of halomethanes on aflatox1n 1nduct1on 1n cultures of

Asperg1llus paras1t1cuS. Journ.Gen.M1crob1ol. 131, 687-691.PaSS1 S., Nazzaro-Porro M., P1cardo M., F1nott1 E., Fabbr1 A.A. andFanell1 C. (1986) M1crosomal and m1tochondr1al 1nvolvement 1n the pro-

duct10n of aflatox1ns 1nduced by carbon tetrachlor1de and hydrope-rox1de 1n cultures of Asperg1llus paras1t1cuS. Trans.Br.mycol.Soc.87(3), 451-456.

Sh1h C.N. and Marth E.H. (1974) Aflatox1n format1on, llP1d synthes1s andglucose metabol1sm by Asperg1llus paras1t1cuS dur1ng 1ncubat1on w1thand w1thout agltat1on. B1ochem.B1oph.Acta 338, 286-296.

Steyn P.S., Vleggaar R. and Wessels P.L. and Scott D.B. (1975) B1osyntes1sof aflatox1n B from 2_13C and 1_13C acetate. Journ.Chem.Soc.Chem.

1Commun. 1975, 193-195.Steyn P.S., Vleggaar R. and Wessels P.L. (1980) The B1osynthes1s of Afla-

tox1ns and 1tS congeners. The B1osynthes1s of Mycotox1ns . A study1n secondary metabol1sm. pp.105-155. Academ1c Press, New York.

Turner W.B. (1971) Fungal metabol1tes. Academ1c Press, New York.Werr1ngloer J. (1978) Assay of formaldehyde generated dur1ng m1crosomal OX1-

dat10n reactlon. Methods 1n Enzymology 52, 297-302.

110