Phytotoxicity of orellanine, a mushroom toxin

Taricrm, Vd. 2S, No . 3, pp . 730-334, 1987. Primed io t7ra~t Britain.

0041-OIOI/67 ß.00t .00 O 1917 Papmoa Jomo~b Ltd.

PHYTOTOXICITY OF ORELLANINE, A MUSHROOM TOXIN JEAN-MICHEL RICHARD, 1 PATRICK RAVANEL2 and DANIELLE CANTIN3 'Laboratoire de Chimie et Toxicologie, =Laboratoire de Pharmacognosie and'Laboratoire de Chimie Analytique, U .F .R. de Pharmacie, UniveraitE Scientifique, Technologique r< MEdiale de Grenoble, France (AmePted jor puäkation 26 AagrttiY 1986) J .-M . Rtctuan, P . Rwvwlvm. and D . Cerrrnv . Phytotoxidty of orellanine, a mushroom tOx1II . Tiaxicon 25, 330-334, 1987.-~Ordlanine, a toodc principle of Corrinarlut oneUmtur Fr., effidently inhibited the photosynthetic activity of duckweed, Larrrra minor L ., u a oonoentration of 0 .4 mM . A lower concentration (0 .06 mM) blocked the O= production in isolated spinadl sass A chloroplasts . However . no effect was observed on thyhrkaidr< (dace C dlbroPlatts), showing that orellanine does not interfere with the chloroplauic electron transfer chain and that ordlaaine . An does not ad like methylviologen (paraquat) in groea plants, as previously dectroch~iCal study of ordlanine and rdated compounds showed that oreitaaine tin neither be reduced by dxtroas derivod from H,O nor from NADPH, as ~ màhylviologen in plant and animal txW, reapectivdy .

was found (GRZYMALA, 1962; ANTKOWIAK and CiF.SSNER, 1975, 1985) to be responsible for the toxicity of several species of Coronaries mushrooms for the toxin animals and man (RICHARD et al., 1985; literature reviewed by SCHUMACHER and HOILAND, 1983). This compound was shown (ANTKOWIAK and CiESSNER, 1979) to be (2,2'-bipyridine~3,3',4,4'-tetrol-1,1'-dioxide, which has been synthesized (DE>:nKLOw and SCHULZ, 1985; TIECCb et al., 1986). We have confirmed that formula by establishing the complete crystal structure of a hydrogen bonded complex between onllanine and trifluoroacetic acid (Cohen-Added, C., Richard, J.-M. aced Guitel, J . C ., to be published). HOILAND (1983) showed that Coronaries speciosissimus Kiihn. 8c Romagn . aqueous extract is highly toxic for a green plant, the duckweed Lemna minor L. Taking into account the similar bipyridine structure of orellanine and of methylviologen (paraquat) or of diquat, two well-known herbicides, the author prrsumed that their mechanisms of phytotoxicity were the saint. In order to determine whether or not a crude raethanolic extract of Coronaries orellanes and pun orellanine itself have the same phytotoxic properties as methylviologen and to get more information on the toxicity mechanism of orellanine in plants, we have studied the effect of these products on duckweed cultures, on isolated spinach class A chloroplasts and on thylakoids~(class C chloroplasts) . We have also investigated the electrochemical behaviour of orellanine and compared it to that of methylviologen . Figure 1 shows that a crude methanolic extract of C. orellanes (3 mg/ml) and pure orellanine (0.4 mM) both strongly inhibit photosynthetic 02 production one day after ORELLANINE

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Fta. 1 . Et?PHCT of A METHANOUC eXrawcr of COrtinarlus Oi11lI/QAtQ AND OF PURE Overt 1 ~nnNe ON _rite rHOrosvrrntsnc wcrntrv of Ducfcwr~D cut nvw~D (a) trNDea uotrr oa (b) tN ttm nwafc - Control ; - "- methanolic extract (3 m~/mn ; -"- ordLnine (0.1 myml, 0 .4 mid (Kt .etN et al.,

. Ductwad, Lanna ntlnor L ., was c:uhivated at room temperature (2 j fresh weight üt a 20 ml Petrl dish) oo a modified wlution of Johmon (ErsrmN, 1972). If required, catltura were continuously illumi~ted by a thwrescent tube ~ivin~ an irradiana of 2000 hur at the surface of the Petri dishes . Aliquou of 200 m~ of fra6 ductweed (about 20 plants) in 2 ml of culture medium were used for follwvin; O, evaltrtion . Each point is the mean of three detaminadom made ao two identical cultures. Standard error was beLnv 0.3 unit : of meaturemeat. Chlorophyll coaantratlom were daa~mined aocordina to the apecuop6otometrk method of ARNON (1949) . O~ release west followed poLuo~raphicaüy at 25° C using a Clam-type datrode >Haan purchased from Iiaasatah Ltd (Hardwict IndumW Baste, Kinfi't Lynn, U .K.) . The light source wa: a 130 W Xenon Limp tivlna an irradiana of 40001ux at the atrfs~e of the reaction vessel . treatment in duckweed growing under light . With pun orellanine a progressive loss of this inhibition was shown (Fig. la), probably due to photodestruction of this toxin, giving the non-toxic product onlline (Richard, J .-M ., unpublished results ; ANrxowlwx and C3BSSrrBIt, 198 . During the experiment the chlorophyll a and b and the carotenoid contents of duckweed decreased to 60-SO% of the initial values, as a result of photosynthesis inhibition, which enhances siaglet oxygen emission from PS II (photoaystem In and reduces the quenching of it by carotaroids (FBrn'KB, 1982). In duckweed treatment in the dark, either by orellanine or by crude methanolic extract, photosyatheaia was also inhibited (Fig . lb) . The products studied were therefor investigated for their inhibitory effect on isolated spinach class A chloroplasts . These chloroplasts were prepared using the method of

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Fia. 2. Hr~acz of oaei .urm~ oN senvecx cEn.oao~iwsrs: (a) oN O, avo~.tmoH err ~sou~u cuss ~ c~n.os~oruars; (b) ~m.t~craaar~r~rBa u+ Txrueoms . Number on traces refs to g relare or oonaimption (mmole/hr/mt dilorophyll) . Chl. = spinach dan A ; T7~y .=thylakoids (dan C chloroplasts) . Substrates: NH,C3 S mM uncoupler): mahylvioloten(MV) 0.1 mM (ebctroa acceptor from protein 7~ : 3-(3,4~iChlorophmyl}l,l'~imethyhma (DCMIn 0.01 mM (inhibitor of elatron tramfa betwan coaozyme Q and p~Lstoqulnooas) : (DPIP) 0.1 mM (ebdron donorto cytochro®e n; asoorbate 0.5 mM (ebdron donor to DP1P). Radien medium : 330 mM mannitol, SO mM mo~rphoänopropane snlfonic said (MOPS)-NaOH (pH 7.6), 2 mM BDTA, S mM NaHCO 0.1 mM YH,PO. and diloroplasts (0.07-0.08 mt of ddaphyll in a finalvolume of 1 ~. The radian medh~m was aaaed with nitroten before chloroplast addition and eq~librated with the axyten ebcuode at 23°C. Càloroplasp were prepared (Nn>v+rent and &+:seer, 1977) and purI~ed (Pace d aL, 1979) as previously daxibed. Tbylakaids (dan C diloropLsts) wen obtained from dan A diloroplasts by osmotic shock (Mwct~m, d d., 1986). Back acperiment was reported six lima whh at bast two different preparations of chloroplasb. The varlabWty of the g evohttionrate was3~ . Far the prooedura for folbwint Oa evohrtion and determidnt pipnmt oonoentratioos sa Pit 1 .

and BARBER (19Th and purified following the method of PRICE et al. (1979) . It is known that intact isolated chloroplaste product oxygen in light when supplemented with NaHC03 (S mIVn and phosphate (0.1 mM I{H2P0~. Figure 2a shows that in isolated class A chloroplasts pun orellanine (0.06 mIVI) fully inhibits O= evolution. Class A chloroplasts broken by osmotic shock (MACHEREL et al., 1986) become thylakoids (class C chloroplasts), which an able, under light, to transfer electrons from H2O to an appropriate electron acceptor, such as Fe(CN)s'- or methylviologen . Figure 2b shows that in light O.1mM orellanine is unable to accept electrons from the thylakoid electron transfer chain (PS II + PS n . It is known that when methylviologen is added an intense electron transfer appears, which leads to H202 formation and therefore to oxygen consumption (AsHTnly and CRAFTS, 1973). Comparing this 02 consumption to that of class C chloroplasts without orellanine in the presence of methylviologen, we found that NAKATANI

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the intensity of the electron transfer remained the same. These results suggest that 0.1 mM onllanine cannot inhibit electron transfer from H2O to methylviologen through PS II and PS I. After addition of 0.01 mM 3-(3,4-dichlorophenyl}l,l'-dimethylurea (DCML>) this electron transfer was fully inhibited under light, even in the presence of dichlorophenolindophenol (DP1P) . Orellanine (0.1 mM) is therefore unable either to inject electrons directly to the transfer chain at the level of cytochrome for to reduce (as does ascorbate) dichlorophenolindophenol, which is sufficiently lipophilic to transfer electrons to PS I in light. In order to confirm these results, an electrochemical study of orellanine and related compounds was undertaken (Canon, D., Richard, J.-M ., Alary, J. and Serve, D., unpublished results) . Pyridine-N-oxide, 2,2'-bipyridine-N-oride and N,N'-dioxide were reduced in buffered aqueous solutions at the dropping mercury electrode. We thought that orellanine could also be reduced under such conditions . Polarograms wen obtained in Britton - Robinson buffer at different pH values . Direct current polarography and differential-pulse polarography were usod with a saturaied calomel electrode (S.C.E.) as reference at 2510 .1°C. In an acidic medium orellanine exhibited a single direct current and differential-pulse polarographic reduction wave which was strongly dependent on concentration and pH. This wave was attributed to the reduction of the fully protonated N-oxide function . Orellanine, like most bipyridines, developed important adsorption phenomena at the dropping mercury electrode, which made this study more complicated. In the BrittonRobinson system the half-wave potentials (E,n) or peak potentials (E~ moved cathodically with increasing pH. In the pH region above 5.3 the peak was partially or totally overlapped by the supporting electrolyte discharge current. Cyclic voltammetry showed that the reduction mechanism is irreversible . Therefor, no stable radical could be observed in aqueous buffered solutions. The electrochemical behaviour of methylviologen under the same operating conditions is totally different. This paraquaternary salt is more easily electroreducible . The polarogram exhibits two reduction waves. The first wave is due to a reversible one electron process (literature reviewod by Btxn and Kt1fW, 1981). The toxic cation radical produced (Ha~~rt~ and SMITH, 1977 ; ASHTOty sad CRAFTS, 1973) is very stable under an inert atmosphere . We used the same experimental conditions as for orellanine (BrittonRobinson buffer at pH 2.1) in order to determine the potential of the fn-st peak of methylviologen . At that pH the first peak had an E, value of -0.47 t 0.01 V/S.C .E (Volt vs. saturated calomel electrode) with 2.10'' M methylviologen. For 10'6 M orellaaine the EP obtained was -1 .11 t 0.01 V/S.C.E. Therefore, the difference between the Eo of methylviologen and orellanine is as large as 0.64 V, From the foregoing observations it appears that the electrochemical reduction behaviours of onllanine and of methyviologen are quite different. Orellanine can neither be easily reduced nor can it form a stable radical. Neither the electrons derived from H2 O nor those derived from NADPH [Ep = -0.90 V, pH independent, calculated from E,n given by CUNNINoHAM and UNDERWOOI) (19fî6)] can educe orellaniae, as is the case for methylviologen is plant and animal cells, re.4poctivdy . Hence, the mode of action of orellanine is clearly different from that of methylviologen, contrary to the hypothesis of HOLLAND (1983) . Furthermore, orellanine is more difficult to oxidize than is ascorbate . Orellaaine (10-` M) gave a relatively well developed wave at a platinum rotating disk electrode. The Ep value determined by differential-pulse polarography in phosphate buffer, pH 6.2, was

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+O.SO t 0.02 V/S .C.E. One can compare this value to the one given by LINDQuIST and (1973) for ascorbic acid in acetate buffer, pH S.S (+0.217 V/S.C .E.). Orellanine is thus not an electron donor to dichlorophenolindophenol, in contrast to ascorbate, and the toxin is not oxidized by the chloroplast electron transfer chain. Thus orellanine cannot interfere with the electron transfer chain in chloroplasts . Since this should also be the case in mitochondria, we checked the effect of a crude methanolic extract and of pure orellanine on electron transfer in mitochondria . No significant inhibition was shown and there was no uncoupling of oxidative phosphorylation. Orellanine is a photosynthesis inhibitor which affects a target located inside the class A chloroplast but not in the thylakoid. This could be the photophosphorylation process, the Calvin cycle or the envelope transporters . FARROHA

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Ast rox, F. M. and Caws, A. S. (1973) Bipyridyliuma. In : Mode ojAction of Herbicides, p. 183 . New York : Whey and Sons . Barn, C. L. and Kutnv, A. T. (1981) Electrochemistry of the viologene. Chern. Soc. Rev. 10, 49 . Gt~r~t~uaa~, A. J. and Urmmewoon, A. L. (1966) Electrochemical reduction of triphoaphopyridine nucleotide . Arehs Biodrem. Blophys. 117, 88 . Det~aa.ow, E. V. and Scxutz, H:J. (1985) Synthesis of orellanine, the lethal poison of a toadstool. Tetrsdudron Lets . 26, 4903 . EPSretrt, E. (1972) Mihesal Nutrition ojPlants. Principles and Piaspectives, p. 39 (LN.C ., Ed .). New York : Whey and Sons . Ft?Dree, C. (1982) Biochemistry and Physiology ojXerbicide Action, p. 68 . Halin: Springs Vorlag . GazYMwu, S. (1962) L'isolertxnt de l'orellanine, poison du Cortinarlrrs oreAanus Fries, a l'hude de sea effete anatomo-pathologiquea. Bull. Soc. myrnl. Fr. 78, 394. Hexrtl, D. and SMmt, P. (1977) The pathology of the lung in paraquat poisoning. In: BiochemicalMechanisms ojAsr~aquat Taxictty, p. 39 (Atrroa, A. P., Ed .) . New York Academic Press. Hettwrm, K. (1983) Extracts of Cortlnarius spalasissimus affecting the photosynthetic apparatus of Lemma minor. Trpns. Br. mycol. Soc. gl, 633. Kt ew, G., Rtct~nn, J.-M. and S~'rae, M. (1986) Effect of a mushroom toxin, orellanine, on the cellular slime mold Dktyastdium dfscoidnrm and the bacterium Encherfchla colt Mkrobiol. Lett., Fedn eur. microblol Socs 33, 19. LtNnQU~sr, J. and Fwattow~, S. M. (1975) Application of diffsential-pulse polarography to sassy of vitamins. Analyst 109, 377. MACNSast., D., Ttssur, M., Nunrr, F., RAVANEL, P., BeaaoN, M. and Crc~otv, J. P. (1986) Inhibitory action of as isopropyl carbanylate series on mitosis, respiration and photosynthesis . Physlol. Ve