Poisoning by members of the genus Cortinarius — a review

Mycol. Res. 94 (3): 289-298 (1990) Printed in Great Britain

289

Poisoning by members of the genus Corh'narius - a review*

D. MICHELOT U R A 401 CNRS, Laboratoire de Chimie, Mwe'um National &Histoire Naturelle, 63 rue Buffon, 75231-Paris Cedex 05, France

I. TEBBETT Department of Pharmacodynamics, University of Illinois at Chicago, Box 6998, Chicago, IL 60680, U.SA.

Poisoning by members of the genus Cortinarius - a review. Mycological Research

94 ( 3 ) :289-298

(1990)

Every year, Cortinariw mushrooms are responsible for severe poisonings all over Europe and they result in acute renal failure. The species concerned are mainly Cortinarius orellanus, C. speciosissimw and C. splendens, but other species in the genus may also prove to be toxic. The toxins in question are presumably orellanine and cortinarins; although their respective modes of action are still unknown, several assumptions have been made, justified by structural and biological similarities to chemical or pharmaceutical substances. This article reviews current knowledge of the r>isonings, the toxins incriminated and their mechanism of action. Key words: Orellanine, Cortinarins, Cortinarius, Nephrotoxicity, Poisonings. The genus Cortinarius Fr. represents a complex group of fungi which are found throughout Europe (Moser, 1983); the abundance of species, together with the fact that they contain some very potent toxins, has led to regular reports of serious intoxications - the toxicity of C . orellanw (Fr.) Fr., C. speciosissimus Kiihner & Romagn. and C. splendens Henry is well known and well documented. In addition, research in a number of disciplines has suggested that several other species within this genus are potentially dangerous. Cortinariw poisoning is characterized by a delayed renal insufficiency, and the nature of the chemical substances responsible has been partially elucidated. These are orellanine, which has a bipyridyl structure, and the cortinarins with a cyclopeptidic structure. The latter have been identified as being present, although detected in small amounts, in a large proportion of species within Cortinariw. The mechanism of action of these toxins is still hypothetical, being based on certain analogies drawn between the chemical structures of the toxins and poisonous substances of similar composition. Only a few review articles have outlined the main features of this unique type of intoxication (Schumacher & Hailand, 1983; Michelot & Tebbett, 1988).In view of the danger which this group of fungi presents to amateur mycologists, and in response to inquiries from toxicological services for further information, the present review article attempts to present all of the current knowledge concerning Corfinariw intoxications, the substances responsible and their mechanism of action. The present article is an updated and complementary restatement of a review already published by the same authors (Michelot & Tebbett, 1988).

HISTORY O F C O R T l N A R l U S INTOXICATION The first systematic census of cases of poisoning caused by Cortinariw was made in Poland Ltween 1953 and 1962 (Grzymala, 1957, 1964a, b). Twenty-three intoxications were reported which were attributed to Cortinariw. Taking into account the distinctive features of the dried samples collected during the poisonings and also the geographical location of the responsible species, Skirgiello & Nespiak (1957 )determined C . orellanw as the cause of poisoning. Ninety-six per cent of the people who had consumed the mushrooms were seriously poisoned and there were four fatalities. Cortinariw species soon took second place, behind Amanita phalloides (Vaill. : Fr.) Link, as being responsible for incidents of serious mushroomrelated intoxication. This earliest statement was very opportune, since it unambiguously proclaimed that this mushroom was not only 'non-edible' but also very toxic; it was highlighted throughout the mycological literature, and there were many more publications concerning this poisonous species (Pouchet, 1960). Subsequently reports of poisoning by C . orellanus emerged throughout Europe. These included three cases in Switzerland (Favre et al., 1976; Leski et al., 1976),two in the east of France (Marichal ef al., 1977), five in Germany (Farber & Feldmeier, 1977), and one collective poisoning in Brittany, France (Brousse et al., 1981). In France in 1987 many cases of poisoning were reported : one in the south (Perpignan, Delpech, 1987), two in Brittany (Brest, HenrC, 1987) and, most notably, twenty-six soldiers participating in a survival exercise in Morbihan in September were victims of a collective

Poisoning by Cortinarius species

290

intoxication. Cortinarius orellanus again seems to be the cause of this incident (Thomas, 1987). In addition to C. orellanw, it has been established that C. speciosissimus is equally toxic (Azkma, 1981). In 1974 four cases of poisoning by this species occurred in Finland (Hulmi ef al., 1974); further incidents were reported in Scotland (Short et al., 1980; Watling, 1982), in Sweden (Holmdahl et al., 1980; Holmdahl, Mulec & Ahlmen, 1984), in Norway (Fauchald & Westlie, 1982), in Italy (Busnach et al., 1982; Rovati ef al., 1984) and in Germany (Nolte et al., 1987). Poisonings by these members of Corfinarius have been reported all over Europe, and the poisonous capabilities of these two species are now unequivocally established. Intoxication by a further species, C . splendens, has been described. In 1979 it provoked a collective intoxication in Haute-Savoie, France (Finaz de Villaine, 1981; Gerault, 1981; Colon et al., 1981, 1982) and more recently a poisoning in Switzerland (Schliessbach ef al., 1983). Although in the first case the right determination of the C. splendens species has been much debated, the resulting clinical manifestations of both poisonings were similar to those produced by C. orellanu and C. speciosissimw, but the toxicity appeared to be lower. Two cases of renal tubular necrosis due to mushroom consumption have also been reported in the northern part of the United States; the available mushrooms were at the time inadequate for identification and the responsible species was assumed to be A. phalloides; but, in view of the symptomatology of both intoxications, the implication that mushrooms of the genus Corfinarius were the cause of poisoning seems more likely (Myler, Lee & Hopper, 1964). Although cases of intoxication by other species in the genus have not been formally reported, a large proportion are considered as suspect and their toxicity may come to light in the future. The identification of species responsible for a particular intoxication usually depends upon a determination

some time after the poisoning has taken place, often with poor-quality material. Conclusive identification of the species involved in the poisoning may thus prove difficult, and this is particularly true of members of the genus Cortinarius. Recent chemical analyses of substances present in Cortinariw species, together with animal experiments, have suggested that a number of other species of the genus are also toxic (H~iland, 1980; Flammer, 1982). The recently gained knowledge concerning Cortinariw poisoning, particularly the long latent period between onset of symptoms and investigation of the hmgi, suggests that Corfinariw species were responsible for several cases of renal insufficiency of unknown cause.

SYMPTOMS, CLINICAL CHARACTERISTICS A N D TREATMENT OF C O R T Z N A R Z U S POISONING Considering the clinical data published in the literature and related to the poisoning provoked by these three species (see above), it is already possible to sum up the common features of the Corfinarius syndrome (Fig. 1). Corfinarius intoxications are characterized by an exceptionally long latent period. This period of incubation greatly exceeds those usually observed with other types of mushroom poisoning. It should be emphasized that the latent period may be associated with repeated consumption of the fungi before the first symptoms become apparent. Toxic symptoms begin between two and twenty days after ingestion of the mushrooms. This has led to misdiagnosis of the poisoning, since the victim often does not associate his condition with a meal consumed several days earlier. It seems that the shorter the period of incubation, the greater the severity of the intoxication. Nausea, vomiting and diarrhoea accompany gastric upset and abdominal pain. These digestive problems disappear spontaneously, but after a brief delay an intense burning thirst

Fig. 1. Chronology and development of poisoning by Cortinan'w mushrooms. Variable latency period

b One moderate consumption Recovery

Chronic renal insufficiency: repeated dialysis, renal transplantation

Lumbar, abdominal pain, nausea, vomiting, diarrhoea,

Acute renal failure

Multiple or abundant consumption

Extrarenal epuration

I

I

I

I

2 days

2 0 days

// //

) Death I

I

3 months

6 months

Time

D. Michelot and I. Tebbett (Dehmlow & Schulz, 1985, 1987; Tiecco, 1986; Tiecco et al., 1984, 1986, 1987; Hasseberg & Gerlach, 1988). Antkowiak & Gessner have also isolated orellanine from C. speciosissimus and were the first to mention that this compound undergoes chemical reduction and decomposition by heat or light to the orellinine (2) and finally to a non-toxic compound, the orelline (3) (Antkowiak & Gessner, 1985). This formula, first-proposed by Antkowiak & Gessner (1975), has been confirmed afterwards by various authors and recently by the crystal structure of an orellanine-trifluoroacetic complex (CohenAddad ef al., 1987). Four years later, Kumsteiner & Moser (1981) also isolated a lethal toxin from C . orellanus which was unstable after having been illuminated for a long period. This toxin showed close similarities in absorption spectroscopy (u.v.) and (i.r.) to the orellanine described by Antkowiak & Gessner. This compound, like orellanine, also formed a non-toxic breakdown product. Nevertheless, there were some properties, such as thermal stability and solubility, which differed from those previously reported by Antkowiak & Gessner. Holmdahl et al. (1987) have recently isolated orellanine from C. speciosissirnus and confirmed its nephrotoxicity by a study in rats. KellerDilitz, Moser & Ammirati (1985) have also detected orellanine together with other fluorescent components in C. speciosissimus, C. orelhnoides Henry and C. rainierensis Smith & Stuntz. Furthermore, Andary et al. (1986) have only found orellanine in the following species: C. orellanus, C. orellanoides, C. speciosissimus, C. rainierensis, C. brunneofulvus Fr., C. fluorescens Horak, C. henrici Reum. which are all members of the section Orelhni (subgenus Leprocybe Mos.). Nevertheless, C. fulvaureus Henry, which is still a member of the same group, does not contain this compound. The authors assert that orellanine is not present in 28 Corfinarius species of the subgenera Cortinarius Fr., Leprocybe Mos. (section Orellani excepted) and Phlegmacium (Fr.) Fr. nor in 1 3 Dermocybe species (Rapior & Andary, 1988; Rapior, Andary & Privat, 1988).This statement, mainly suggesting a chemotaxonomic marker for the Orellani section, is very interesting in other respects; as a matter of fact, species within these latter orellanine-free groups have also been reported responsible for orellanw-like intoxications, in clinical cases (C. splendens, Colon ef al., 1981; Finaz de Villaine, 1981, 1982; Gerault, 1981; Schliessbach et al., 1981), or during in vivo experiments (C. cinnamomeus (L.:Fr.) Fr., C. THE TOXINS, THE SUSPICIOUS FUNGI malicorius Fr., C. phoeniceus R. Maire, C. sanguineus (Wulfen: SPECIES Fr.) Fr., Viallier ef al., 1968; C. gentilis (Fr.) Fr., Mottonen, In 1962, Grzymala isolated a substance from C. orellanus Nieminen & Heikkila, 1975 ; Holmdahl et al., 1980; C. limonius which he called orellanine (Grzymala, 1962).Experiments with (Fr.:Fr.) Fr., Holmdahl et al., 1980). These novel and additional this toxin in animals produced the same toxic effects as the data imply that other toxins different from orellanine are mushrooms themselves. Testa (1970, 1982) suggested that involved in Cortinarius nephrotoxicity. The yield of orellanine in these species is usually reported orellanine was a mixture of four major compounds, namely as being approximately 2% of the dried mushroom; the grzymaline, benzonine a and b, and cortinarine. The first pioneer step towards realizing a chemical content is slightly lower in C. speciosissimus (Prast et al., 1988). identification of the toxins was accomplished by Antkowiak & Orellanine has also been detected chromatographically in the Gessner (1975). A product was isolated (Antkowiak & mycelium of C. orellanw, but in very small amounts (Rapior, Gessner, 1979) and the structure of this molecule was Andary & Mousain, 1987). In fact, distinct fluorescent elucidated as 3,3',4,4'-tetrahydroxy-2,2'-bipyridine-l,lf-diox- components, previously referred to, have been extracted from ide (I) which was confirmed by the same authors by chemical C. speciosissimus. They were found to be present in most synthesis (Antkowiak & Gessner, 1984). This synthesis has species of Cortinariw, and structural analysis of these subsequently been improved and extended to other analogues compounds indicated that they have a cyclopeptide structure

develops together with dryness of the mouth, a sensation of cold, anorexia, muscle fatigue and headaches. An acute renal insufficiency develops progressively, resulting in oliguria and anuria. Examination of the urine shows albuminuria and haematuria. The blood balance shows an increase in urea and creatinine levels. Histopathological examination of renal tissue shows, in most cases, a tubulo-interstitial nephritis, with necrosis of the renal tubules and leucocyte infiltration of the parenchyma. If these lesions become serious, and in the absence of relevant treatment, the renal insufficiency becomes chronic (up to 5 0 % of the cases reported by different authors) and requires repeated dialysis or a transplant in order to restore renal function. In addition to these renal manifestations, neurological problems may also develop. These include drowsiness, loss of consciousness, convulsions and muscle tremors of the face. A mild cytolytic hepatitis may also eventually become apparent. Death, as a result of renal insufficiency, usually occurs in severe cases two to three months after ingestion of the fungi. Treatment of Cortinarius poisoning must be primarily orientated towards the elimination of the toxin from the blood circulation. This is achieved by haemoperfusion and haemodialysis using the appropriate membranes, the approach being the same even if ingestion of the mushrooms was several days earlier (Myler et al., 1964; Heath ef al., 1980; Busnach et al., 1982; Schumacher & Hoiland, 1983; Holmdahl ef al., 1984; Rovati et al., 1984; Thomas, 1987). With serious intoxications, the appropriate treatment is the same as that advocated for acute renal insufficiency (Larcan, Lamarche & Lambert, 1979; Gamier, 1983). We must emphasize that forced diuresis should be avoided. As a matter of fact, instead of being a solution to the anuric crisis, forced passage through the kidneys could rather hasten and amplify the nephrotoxic process, since it possibly accentuates the accumulation of the toxins in the kidney (see above). A peculiarity of Cortinarius intoxication is the secondary nature of hepatotoxicity. This feature allows for the distinction between this type of poisoning and other types of intoxications by fungi such as A. phalloides (Wieland, 1986; Michelot & Labia, 1988) and Gyromitra esculenta (Pers.) Fr. (Michelot, 1989).

Poisoning by Corfinariw species Fig. 2. Chemical structures of orellanine, orellinine and orelline.

(Caddy ef al., 1982). Three principal components of C. speciosissimw, all having similar structures, have been isolated and identified. These compounds were referred to as cortinarins A 4, B 5 and C 6, the first two being nephrotoxic in mice (Tebbett ef al., 1983; Tebbett & Caddy, 1983, 1984a; Tebbett, 1984, 1986). Sixty different species of Corfinariw were also examined by thin-layer chromatography in order to determine the presence and the quantity of cortinarins. Among these, cortinarin A has been detected, in various concentrations, in all of the species examined with the exception of C. violacew (L.:Fr.) Fr., which is one of the few species of Cortinariw still considered to be edible. Three species were found to contain cortinarin B, namely C. orellanw, C. orellanoides and C. speciosissimw, which also contain large quantities of cortinarin A and are considered to be the most toxic species within the genus. Cortinarin C, which is non-toxic, was found in all 60 species examined. Concentrations of cortinarin A ranged - from 0.47% of the dry weight (C. speciosissimw) to 0.0004% (C. croceifoliw (Peck) Mos.). Concentrations of cortinarin B were 0.60% (C. speciosissimw), 0.52% (C. orellanw) and 0.47% (C. orellanoides). These values suggest that the toxicity of these

species is proportional to the sum of cortinarins A and B in the fungus (Tebbett & Caddy, 1984 b). In the light of these observations it seems that the toxicity of the Corfinariw species is due to substances which are highly resistant to heat, freezing and drying. At present this remarkable nephrotoxicity cannot be attributed specifically and exclusively to one molecular type, but rather to a combination of the compounds already described present in different amounts in the mushroom, and perhaps others still unknown. These substances, of whatever chemical structure, have to be henceforth added to the list of well-known nephrotoxins (Ringoir, Schoots & Vanholder, 1988; Gibson, 1986; Walker & Duggin, 1988).

IN V Z V O A N D IN V Z T R O STUDIES O N TOXICITY OF C O R T Z N A R Z U S SPECIES The determination of the toxicity of different species of Corfinariw to living organisms has been the object of a number of experimental studies. Grzymala (1964 b, c) was one of the first toxicologists to verify experimentally the toxicity

Fig. 3. Chemical structures of cortinarins A, B and C.

Phe

-Val -Om

NH--CH-€0

I

Leu

Phe

NH---CH-40 -Val

I

-Om

Leu

LY~

S

I CH2 Gly

-Thr

I

OC-CH-NH

- IsoLeu

4 Cortinarin A, toxic

R=OCH3

5 Cortinarin B, toxic

R=OH

Gly

-Thr 6 Cortinarin C, non-toxic

Ala

-IsoLeu

D. Michelot and I. Tebbett of C. orellanus in the cat, guinea pig or mouse by different modes of administration (oral, subcutaneous or peritoneal injection). Similar toxicity and histopathological changes were observed in these animals as were seen in humans after serious intoxication (Grzymala, 1964 b, c; Prast & Pfaller, 1988). The kidney is always the target organ, lesions becoming apparent in the tubular epithelium. The effects of single sublethal doses of C. speciosissimus on rat kidney were studied by transmission electron microscopy. Epithelial cells and proximal tubules are primarily affected and no changes were seen in the glomeruli. Ultrastructural changes in the renal cortex developed after two days and necrosis of proximal tubular cells was prominent after five days; on the other hand, regeneration started at ten days and seemed to be achieved after two months (Lahtipera, Naukkarinen & Collan, 1986). Subsequent necrosis results in renal insufficiency, the seriousness of this being determined by the size of the dose. It has been noted that there is no difference in the toxicity of fresh, dried or cooled mushrooms. An LD,, dose in the present kind of intoxication, does not express events other than death, such as acute and chronic renal failure. However, many experiments have been performed in order to estimate it. Nevertheless, the average lethal dose (LD,,) in rats of crude orellanine has been determined and is of the order of 5 mg/kg body weight (Grzymala, 1964 b). This value (equivalent to 2 g/kg of dried mushroom, Viallier, Oddoux & Casanova, 1965; 2.20 g/kg of dried C. orellanus and 3.12 g/kg of C. speciosissimus, Prast ef al., 1988) has been mentioned in an early estimation of Viallier and coworkers (1968); they also showed that in the rat and guinea pig C. speciosissimus and C. orellanoides produced toxic effects similar to C. orellanus. The human lethal dose early estimated as 100-200 g of fresh mushroom per kg of body weight is in accordance with the data concerning the higher sensitivity of humans published afterwards (Grzymala, 1957). Other species of Cortinarius (C. cinnamomeus, C. phoeniceus and C. sanguineus) also produced toxic effects and the eventual death of the animal, but after a much longer latent period. Values of the LD,, of orellanine have been determined in mice - by intraperitoneal injection as 12.5 mg/kg (Richard, Louis & Cantin, 1988) and per os as 90 mg/kg (Richard et al., 1988) or 33 mg/kg (Prast ef al., 1988). This differs from the calculated figure of 5 g/kg determined by comparison with structurally similar compounds. As a result of this difference between calculated and actual LD,,, the authors call into question the true chemical nature of the toxic entity and of the mechanism of action (Richard, Taillandier & Benoit-Guyod, 1985); actually, pharmacological data may prove that orellanine itself is not the true toxin, but rather a precursor. The resistance of animals to Cortinarius poisoning is very variable. The same dose of C. speciosissimus, when given to a group of rats, induced severe renal lesions in some subjects whilst others showed no kidney damage whatsoever (Mottonen et al., 1975). Studies by the same authors indicated that, in the rat, the first signs of Cortinarius intoxication are manifested in the form of interstitial infiltrates two days after ingestion of the mushroom. Inflammation of the kidney occurs after four days, and finally necrosis of the tubules in the renal cortex becomes apparent (Nieminen et al., 1975). Andraud

293

et al. (1965) reported that subcutaneous injections of extracts of C. orellanus diminished the amount of sulphydrylic groups in the hepatic tissue; the authors considered this decrease as proportional to the doses of toxic agent, and also to the importance of the lesions. Although the data are not comprehensive, this observation might reinforce an active implication of the liver in the metabolic course of the intoxication. The fact that some animals appeared to be resistant to Cortinarius poisoning, irrespective of dose, was explained by . to thirty genetic variability (Nieminen & Pyy, 1 9 7 6 ~ )Twenty per cent of the animals showed total resistance to the toxins. In the others, the seriousness of the renal lesions observed was related to the size of the dose (Nieminen, 1976). Nieminen also reported that there were sex-linked differences in the susceptibility of rats to Cortinarius poisoning (Nieminen & Pyy, 1976 b). Females appear to be more resistant than males to the toxins. These observations were confirmed by Finaz de Villaine (1981). Recently, of two patients who had ingested equivalent quantities of C. orellanus, a mild renal failure was reported with the female, although the male presented severe renal failure (Hen;, 1987). Furthermore, a sexual difference in individual sensitivity has been later reported in mice during experimental intoxications by Amanifa virosa (Fr.) Bertillon; in this latter case, males appear to be more resistant than females (Nieminen et al., 1977). In order to try to understand the mechanism of Cortinarius toxicity more fully and eventually to propose a course of treatment, Nieminen and co-workers studied the effects of concomitant administration of C. speciosissimus together with furosemide, phenobarbitone, phenylbutazone and cyclophosphamide in the rat (Nieminen, 1976). The administration of the diuretic furosemide before the ingestion of the mushroom potentiabed the renal necrosis without changing the degree of inflammation (Nieminen, Pyy & Hirsimaki, 1976). It was concluded that the toxin must reach the kidney in high concentration several hours after ingestion, but the appearance of visible histological necrosis requires a minimum delay period of two days. Pretreatment with phenobarbitone, which augments hepatic metabolism, also intensifies the renal lesions in the cortical zone, but not the inflammation. The authors concluded that it is highly probable that toxicity is due to a product of hepatic metabolism and not to the actual compounds present in the fungi. Nieminen also suggested the existence of two different types of toxins with distinct biological properties, one with a rapid action, the other acting more slowly. Phenylbutazone did not seem to have any effect on the nephrotoxicity of C. speciosissimus, whilst cyclophosphamide, an immunodepressant, administered at the same time as the mushroom prevented renal inflammation, and the only lesions observed were situated in the collecting ducts and the outer medullary zone (Nieminen et al., 1976). It was deduced from these observations that these were the initial sites of action of the toxin. These observations are in good agreement with the current theoretical concepts related to the biochemical mechanisms of nephrotoxicity. Similarly, Holmdahl and co-workers (1980) have shown that C. speciosissimus, C. gentilis and C. orellanus are nephrotoxic in mice. In addition C. limoniw (Fr.:Fr.) Fr., a species up till then

Poisoning by Cortinariw species considered to be non-toxic, was also found to cause renal damage in mice. The LD,, for the mouse was determined as being about 2 g/kg of dried C. orellanus. Such estimates can, however, only be approximations in view of the variable concentrations of the toxin in different mushrooms and also the differences in the susceptibility of individual animals to the toxins. Gerault (1981) has demonstrated the toxicity of C. splendens in the rat and confirmed the observations of Nieminen that some animals are totally resistant to Cortinarius mushroom poisoning. This result may be compared to the resistance of humans; in fact of the French soldiers who consumed the mushrooms, a few never presented any symptom of poisoning (Thomas, 1987). Some experiments dealing with the toxic properties of cortinarins A and B have been carried out. Single i.p. administration (5 mg) induced after four days the death of mice showing severe kidney damage (Tebbett & Caddy, 1984). When injecting the sulphoxide of cortinarin A or B, the latwcy period was significantly shortened (Tebbett, unpubl.). A comparative study between C. orellanus, C. splendens and C. vitellinw Mos., performed by Gerault (1981) and interpreted by Finaz de Villaine (1981), showed that all three fungi caused nephrotoxicity and emphasized the potential toxicity of a high proportion of Cortinariw species. Furthermore, the action of the toxins of C. orellanus has been studied at the cellular level. The bipyridine orellanine has a molecular structure similar to diquat or paraquat. The effects of analogous substances (2,2'- and 4,4'-bipyridines) have been studied on pig kidney epithelial cells. The morphology of the cells tested was similar to kidney cells of rats poisoned In vivo with C. orellanus. In addition, these synthetic chemicals caused a comparable change in enzymic activity (alkaline phosphatase and y-glutamyl transpeptidase) in the cell cultures to that observed with Cortinariw mushrooms (Gstraunthaler & Prast, 1983 ; Prast & Pfaller, 1988). Similar reactions were observed when purified orellanine was incubated with kidney cells. The absence of damage to the cellular membrane seems to confirm the intracellular mode of action of orellanine (Heufler, Felmayer & Prast, 1984, 1987). Additionally, orellanine has been demonstrated to be toxic to unicellular organisms. This natural compound inhibits the growth of the amoeboid phase of Dicfyosfelium discoideum Raper and of the bacterium Escherichia coli (Klein, Richard & Satre, 1986). No significant effect on phagocytosis or pinocytosis was observed, although Ahlmen ef al. (1983) reported a strong inhibition of the pinocytosis in Amoeba proteus when using the crude extract of C. speciosissimw.

MECHANISM OF TOXICITY

Whilst the mechanism of action of the Corfinarius toxins is still unknown, a number of hypotheses have been proposed. In the current literature, there is no close chemical model, structurally and functionally related to orellanine, e.g. pyridine N-oxides, which also induces nephrotoxicity ;however, a few hypotheses attempt to explain this action. One of the first presented analogies between the structures and biological activities of orellanine and the herbicides diquat

and paraquat. Diquat and paraquat have bipyridyl structures which are toxic to mammals, and their toxicity is primarily associated with pulmonary lesions. Symptoms of acute renal failure associated with toxic lesions in other organs, provoked by paraquat and diquat, have also been observed (Szepietowski & Adamiec, 1987). It is generally thought that the toxicity of these compounds involves redox reactions. Both in vivo and in vitro paraquat is reduced to an intermediary species PQ+ by the action of NADPH (Nicatinamide-Adenine Dinucleotide Phosphate Hydrogenat&. This species instantaneously produces PQ2+ ions in the presence of oxygen with the simultaneous generation of H 2 0 2 , which can produce free peroxy or hydroperoxy radicals; these very toxic compounds cause peroxidation of lipid membranes. Another consequence of this transport of electrons is the total transformation of NADPH to NADP. The level of NADPH consequently falls below the level at which chemical and biochemical functions cannot operate. A combination of these two effects results in the death of the cell (Smith, 1985, 1987). By comparison with the cytotoxic mechanism of diquat and paraquat, Schumacher & Hailand (1983) proposed a mechanism for the toxicity of orellanine implicating oxidation/reduction chain reactions, with the ultimate formation of free radicals and the lowering of NADPH concentration. This hypothesis was illustrated by reference to the destructive action of extracts of C. speciosissimw on the chloroplasts of the water lentil, Lemna minor L., widely used for testing herbicides. In contrast, extracts of C. gentilis, C. limoniw, C. cinnamomeus and C. a m i l l a f w (Fr.) Fr. were without effect (Hailand, 1983). It was suggested that orellanine is directly implicated in this action, being absent or in low concentrations in all of the species tested apart from C. speciosissimus. It has been shown that the electrochemical activity of orellanine is totally different from that of diquat and paraquat. Richard et al. (1988) showed that orellanine was unable either to be easily reduced in vivo or to form a reactive radical species. In effect the electrons issued from water or from NADPH were unable to reduce orellanine in animal cells as suggested by Hailand's hypothesis (Richard, Ravanel & Cantin, 1987; Cantin ef al., 1988; Richard et al., 1988). Unexpectedly, purified orellanine, kept from light, and administered orally in low doses of 50 mg/kg, is reported as ineffectivein mice (Andary ef al., 1986). The same dose of this purified compound, after being subjected to daylight, caused the death of the animal. Considering the non-toxic nature of both orellanine before being subjected to light and orelline the product of total photodecomposition of orellanine - the authors suggested that toxicity was due to a light-induced product. They postulate that this compound would be a new isoxazolinium which may be formed in vivo capable of bonding covalently with a number of proteins in the organism. Nevertheless, the hypothesis of an intermediary toxin (supposedly photoactivated, but more probably a liver metabolite) may g o some way to explaining the findings of Richard et al. (1985). who showed that the observed toxicity of orellanine does not correspond to the theoretical concentration. At present it is unreasonable to assert a molecular biological mode of action for orellanine or its metabolites, and the

D. Michelot and I. Tebbett

295

Fig. 4. Metabolic pathways of Cortinariw toxins.

T

Cortinarius toxin (orellanine, cortinarine . . .)

>

Gastrointestinal tract Pathway a

Pathway h

Systemic circulation

-

X

'Stable' metabolite

Liver

+ X

-

+ )Y

Reactive metabolite

1

Detoxification

I

C

1 Covalent binding or peroxidative reaction

+

Damage to kidney

mechanisms are as yet undefined; nevertheless, considering the molecular formula of this toxin and its functional requirements for exhibiting its nephrotoxic potency, i.e. one N-oxide group and one hydroxyl in position 3 or 3', we assume that the toxic events involve complexation reactions and/or oxido-reduction steps (perhaps generating in situ highly cytotoxic superoxide species). Cortinarins A and B are cyclopeptides bridged by a tryptathionine group. Certain analogies have been drawn between the pharmacological action of the hormone vasopressin and the symptoms of cortinarin intoxication. There are also some similarities in the structure-activity relationships of both compounds : both the cortinarins and vasopressin have a cyclic structure (Tebbett, 1984). Opening of the disulphide bridge renders vasopressin inactive, and opening of the thioether bridge transforms cortinarins A and B into the nontoxic cortinarin C. Vasopressin acts on the distal tubule and collecting ducts of the kidney, causing water retention. It stimulates the gastrointestinal tract causing nausea, cramps and diarrhoea. Vasoconstriction, hypertension and pallor are

also associated with large doses of vasopressin. Following clinical observations of victims of Cortinarius poisoning and experimental intoxication of animals, it has been reported that the cortinarins act on the distal tubule and the collecting ducts, resulting in oliguria and anuria. Cortinarius poisoning also causes intestinal upset with nausea, vomiting and diarrhoea. It provokes hypertension and a sensation of cold. It would seem that cortinarins A and B are metabolized into the same toxin compound, a sulphoxide of cortinarin B, lately detected in the kidneys. This derivative, and also orellanine, have been detected in the plasma and urine of a patient 12 days after the ingestion of C. orellanus (Michelot & Tebbett, in prep.). Perhaps the cortinarin intermediate activated by the liver attacks the same target receptors as vasopressin through the bioactivated sulphoxide function (Tebbett, 1984, 1986; Flynn & Ash, 1983; Ash et al., 1984; Wieland, 1984; Michelot, Mattioni & Labia, 1985; Michelot & Labia, 1988). This hypothesis is still in agreement with the observations of Nieminen, who has suggested that the toxin is a metabolite. It would also explain two elements of the intoxication. First, the latent period, between ingestion of the mushroom and onset of toxic symptoms, may be due to the active metabolite being slowly produced and released by the hepatic system, and only when sufficient concentrations of this toxin have reached the kidney do symptoms become apparent. Secondly, the genetic and sex-linked variation in sensitivity of animals to the toxins may be due to differences in the hepatic or renal cytochrome Pa,,-dependent mixed-function oxidase systems, which would be responsible for the 0-demethylation and Soxidation reactions required for the conversion of cortinarins A and B to the active metabolite (Takata et al., 1983; Kalow, 1987). The mechanisms by which chemicals induce renal damage are several and complex, since a combination of biochemical and physiological events may participate in the nephrotoxicity (Hook & Smith, 1985);however, taking into account all of the correlated data which concern this intoxication, we can speculate as to the metabolic pathway which may mediate chemically induced renal damage produced by the ingestion of toxic Cortinarius mushrooms. We postulate the uptake of the pro-toxins by the liver from the blood; their biotransformation could occur through the gastrointestinal mucosa (pathway a), as has already been demonstrated in a few cases (Back & Rogers, 1987), but most probably in the liver (pathway b), yielding a metabolite X stable enough to enter and remain in the systemic circulation during the latency period. Considering the unique ability of the kidney to accumulate certain chemicals, the concentrations of the reactive and toxic metabolites would become sufficient to produce specific organ damage. This compound itself may be toxic to the kidney or following further (intrarenal?) metabolism to afford the entity Y (Fig. 4). If detoxification reactions do not take place, the active metabolite close to its target, would contribute to the toxic reactions underlying cellular damage. This commonly involves covalent binding (arylation), but more likely peroxidative attack of any cellular entity to generate cellular dysfunction, eventually resulting in the destruction of the kidney. Further data will shortly give answers to biochemical and physiological questions.

Poisoning b y Cortinariw species

CONCLUSION T h e part played b y each of the different toxins found in C. orellanus, C . speciosissimus and even C . splendens is still not clearly established. However, their similar actions a t the renal level allow us t o suggest that Cortinarius intoxication may be due t o a number of different compounds rather than one specific toxin. Progress in the elucidation of the molecular properties of the toxins o r the putative bioactivators and receptors and of the metabolites directly involved in t h e nephrotoxic process should therefore contribute to the understanding of the renal toxicity of foreign chemicals. Numerous other species of t h e genus Cortinariw, especially within the section Orellani, which have been shown t o contain one o r more of these compounds, are potentially very dangerous, even if intoxications b y these species have not yet been reported. It is anticipated that the mode of action of Cortinarius toxins will be identified in the near future, leading t o t h e specific determination of species within the genus which are toxic. Until such time, however, and in spite of the opinion of a few amateur mycologists, it is imperative that the consumption of all Corfinurius species b e avoided if mass poisonings, such as the one which recently occurred in France, are to be prevented.

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