Antioxidant Properties of Amanita Phalloides Mushroom Toxins

Original Contribution ANTIOXIDANT PROPERTIES OF AMANITA PHALLOIDES MUSHROOM TOXINS

A. Zheleva*, V. Gadjeva, S. Popova

Thracian University, Medical Faculty, Department Chemistry and Biochemistry

SUMMARY Alpha amanitin and phalloidin are powerful hepatotoxins that belong to the main classes of toxins namely, amatoxins and phallotoxins, isolated from the deadly poisonous mushroom Amanita phalloides. It is well known that disruptions of antioxidant enzyme defense that include superoxide dismutase (SOD), catalase, glutathione peroxidase lead to generation of reactive oxygen species, followed by increase in the products of lipid peroxidation. Our previous studies showed that alpha amanitin and phalloidin could inhibit catalase activity. These generated the interest to study the in vitro behaviour of both toxins on SOD activity using spectrophotometry. Our result showed some slight increase of SOD after incubation of the enzyme and toxins. Superoxide anion scavenging activity (SSA) of alpha amanitin and phalloidin was also studied. It was established that both toxins, like the enzyme, exhibit SSA activity. It was obvious that at our experimental conditions alpha amanitin and phalloidin behaved as antioxidants.

Key words: Alpha amanitin, phalloidin, superoxide anion scavenging activity.

INTRODUCTION primary toxic effects of phalloidin poisoning (7). Moreover, Ca2+ release from Amatoxins and phallotoxins are powerful mitochondria induced by prooxidants has hepatotoxins. Amatoxins - alpha and beta been discussed in details (8, 9). amanitins, and phallotoxins - phalloidin are bridged oligopeptides (Figure 1, Figure 2) HO H found in a few Amanita mushroom species, H C CH2OH mainly Amanita phalloides (1, 2)* H C C Amatoxins elicit toxicity by inhibiting 3 CH CO NH CH CO NH CH2 the RNA polymerase II of the eukaryotic NH cells (3,4). They cause dramatic toxic CH2 CO consequences mainly within the liver (5) and CO NH CH3 kidney (6). However, the different types of O NH 6 OH CH S amatoxins (alpha-, beta-, etc amanitins) do CH CH H N CH C2H5 not show any straight correlation between in HO 2 CO vivo LD and inhibitory constants (Ki) CO 50 CH NH CO CH NH CO CH2 NH obtained from in vitro studies (5). CH CO R Hepatotoxic effects of the phallotoxins 2 including phalloidin, have been attributed to their specific binding to F-actin, thus strongly stabilizing the structure of the assembled Figure 1. Chemical structure of alpha amanitin (R filaments (2). Later studies showed their = NH2) ability to deplete the mitochondrial Ca2+ pool after treatment of intact hepatocytes by phalloidin. This finding was related to the

* Correspondence to: A. Zheleva, Trakia University, Medical Faculty, Department of Chemistry and Biochemistry, 11 Armeiska Str., 6000 Stara Zagora, Bulgaria; Tel: 042 28 19 227; E-mail: [email protected] 28 Trakia Journal of Sciences, Vol. 2, No. 3, 2004 ANNIVERSARY ISSUE Zheleva A.. et al. H H CH2OH µM), 62.4 µl (0.032 µM)], or phalloidin [100 5 6 7 H3C C CO NH CH CO NH C CH2 C CH3 µl (0.057 µM)], 850 µl, 820 µl or 780 µl PBS

CH2 OH buffer and 400 µl xanthine oxidase (0.25 NH CO mg/ml PBS) were added to a final volume of S N 2 ml. Activity of control standard SOD after CO H NH incubation at the same experimental CH CH2 3 conditions was also studied. Tubes were 1C stirred, stoppered and incubated at 37oC. 4 N CO C H H HO D D Absorbance of the standard incubated NH CO CH NH CO enzyme was taken as 100 % activity. After 20 CH min incubation, samples were put on ice and HO CH3 absorbance at 560 nm was measured. Control Figure 2. Chemical structure of phalloidin sample did not, of course, contain either toxin or enzyme. Their quantities were replaced by Our previous studies have demonstrated that equivalent amounts of PBS buffer. LPO process took place in the livers of alpha- Absorbance at 560 nm of the controls amanitin poisoned mice (10). It is known that presented the quantity of the formed .- in vivo increased levels of LPO products superoxide anion (O2 ) taken as 100 %. might be due to increased levels of reactive oxygen species (ROS) as a result of damage RESULTS AND DISCUSSION to the antioxidant enzyme defense system comprising essentially of superoxide Results from SOD activity of the incubated dismutase, gluthatione peroxidase and mixtures of corresponding amatoxin catalase. Using in vitro studies we have concentration or phalloidin plus enzyme demonstrated that alpha amanitin, beta compared to that of the SOD standard amanitin and phalliodin could inhibit catalase showed some increase in SOD activity. At a (CAT) activity to a considerable degree (11). concentration of 0.016 µM of the alpha Based on the above, it became necessary to amanitin, strength of SOD activity of the study the in vitro effect of alpha amanitin and mixture was 107.99 ± 5.83 %. At a phalloidin on superoxide dismutase (SOD) concentration of 0.032 µM, strength of SOD activity, as well. activity of the mixture was 103.44 ± 13.59 %. At a concentration of 0.057 µM of the MATERIALS AND METHODS phalloidin, strength of SOD activity of the mixture was 105.26 ± 6.53 %. These results Alpha amanitin, beta amanitin, phalloidin, demonstrated slight activating effect of the enzyme SOD and all other reagents were toxins towards the enzyme. To check whether purchased from Sigma Chemical Co (St. that effect was due to superoxide anion Louis, USA). scavenging activity (SSA) of the toxins we All visible and UV spectrophoto- studied absorbance of samples containing metrical studies were performed on a nonincubated toxin (0.016 µM and 0.032 µM Pharmacia LKB Ultrospec spectrophotometer for alpha amanitin and 0.057 M for (Sweden). µ phalloidin) alone in comparison with that of

the control samples and that of nonincubated Enzyme assay SOD. Results obtained (see Figure 3) Determination of SOD activity was based on showed that at a concentration of 0.016 µM the generation of superoxide anion radical alpha amanitin exhibited 3.50 ± 1.47 % SSA .- O2 in a system containing activity, while at a concentration of 0.032 .- hypoxanthine/xanthine oxidase in which O2 µM this activity was 15.07 ± 3.97 %. reduced nitroblue tetrazolium to formazan; Phalloidin exhibited 12.49 ± 2.36 % SSA the absorbance of the latter was measured at activity; at the same time nonincubated SOD

560 nm. SOD activity was determined exhibited 42.95 ± 3.94 % SSA activity. As is according to Sun et al., (12) with slight seen SSA activity of the amatoxin depended modifications. Briefly, in glass tubes were on its concentration. Based on the results added consecutively the following reagents: obtained, activating abilities of alpha 200 µl hypoxanthine (0.075mg/ml of PBS), amanitin and phalloidin towards SOD could 20 µl EDTA (0.085 mM), 400 µl NBT be explained by their SSA activity at our (0.05mg/ml of PBS), and then incubated for 3 experimental conditions. Lack of the additive h at 37oC. Mixtures of SOD (100 µl, 0.0011 activating effect might be explained by mg/ml) plus alpha amanitin [31.2 µl (0.016 possible formation of the complex between

Trakia Journal of Sciences, Vol. 2, No. 3, 2004 29 ANNIVERSARY ISSUE Zheleva A.. et al. the toxins and enzyme during the incubation 2. Wieland, Th., Faulsthich, H., Peptide which hampered active toxin groups toxins from Amanita. In: Keeler R, (probably phenolic group in 6’- Tu A, (eds), Handbook of Natural hydroxyindole moiety and/or sulphoxide Toxins, Marcel Dekker, New York, from the bridge for alpha amanitin and Vol. 1, pp. 585-634, 1983. sulphur from the bridge structure for 3. Fiume, L.and Stirpe, F., Decreased RNA phalloidin, see , ) to exhibit Figure 1 Figure 2 content in mouse liver nuclei after their SSA activity completely. intoxication with alpha amanitin.

Biochim Biophys Acta, 123: 643-647, Figure 3. SSA activity at concentrations of alpha 1966. amanitin ( 1 - 0.016 µM; 2 - 0.032 µM), 4. Seifart, T., Sekeris, C.E., Alpha- phalloidin (3 - 0.057 µM) and SOD (4 - 0.0011 mg / ml) amanitin, a specific inhibitor of transcription by mammalian RNA CONCLUSION polymerase. Z Naturforsch, B 34: 1538- 1542, 1969. Our recent in vitro studies had demonstrated 5. Wieland, Th., Peptides of poisonous Amanita mushrooms. In: Rich A, (ed), SOD Spinger Series in Molecular Biology, 45 Springer –Verlag, New York, 1986. 40 6. Beaudreuil, S., Sharobeem, R., Maitre, 35 F., Karsenti, D., Grezard, O., Pierre, D., 30 Biopsy proven Amanita Phalloides renal 25 alpha toxicity. Presse Medicale, 27: 1434, 20 ama phalloidin 1998. 15 7. Faulstich, H. and Munter, K. New 10 aspects of phalloidin poisoning, Klin 5 Wochenschr, 64: 66-70, 1986. 0 2+ 1 2 3 4 8. Richer, Ch., Frei, B., Ca Release from mitochondria induced by prooxidants, that both toxins inhibit CAT activity. Our Free Rad Biol & Med,.4: 365-375, 1988. present studies have shown that alpha 9. Novgorodov, S.A., Gudz, T.I., amanitin and phalloidin possessed SSA Permeability transition pore of the inner activity. It could be suggested that alpha mitochondrial membrane can operate in amanitin and phalloidin exhibited prooxidant two open states with different or antioxidant properties at our experimental selectivities. J Bioener Biomembr, 28(2): conditions. That means they could be 139-146, 1996. involved in free radical reactions. It seems 10. Zhelev, Zh., Zheleva, A., Benov, L., very likely that they penetrate liver cell Halacheva, K., Michelot, D., Mode of membranes and enter the hepatocytes where action of amatoxins - 1. Effect of Alpha they constitute reactive free radicals. This amanitin on lipid peroxidation, a . - might stimulate creation of ROS ( O2 H2O2, preliminary study. In: Tan N H, Oo S L, .OH) and as a result induce chain reactions in Thambyrajan V, Azila N, (eds), the membrane polyunsaturated fatty acids. Advances in venom and toxin research, Moreover, if both toxins could inhibit in vivo Kuala Lumpur, Malaysia, pp. 247-253, some of the antioxidant enzymes, such as 1994. CAT, they could additionally lead to increase 11. Antoaneta Zheleva, Momchil Zhelev, in the levels of ROS and LPO products and Vesselina Gadzheva, Amanita phalloides contribute to their severe hepatotoxicity. mushroom toxins inhibit in vitro catalase activity. Medical - biological sciences, REFERENCES prophylactic medicine and social health. 1. Faulsthich, H., The Amatoxins. In: Hahn Vol 1, 75-79, 2002 FE (ed), Progress in Molecular and 12. Sun, Y., Oberley, L.W., Li,Y., A simple Subcellular Biology Springer, Berlin, method for clinical assay of superoxide Vol. 7, p 88, 1980. dismutase. Clin Nephrol 53: S9-17, 1988.

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