Acta Medica Mediterranea, 2015, 31: 1181

WHAT IS THE RESTORING ROLE OF CHRYSIN IN ALPHA AMANITIN TOXICITY?

SINAN BAKIRCI1, RECEP BAYRAM 2, ERTUGRUL KAYA 3, KURSAT OGUZ YAYKASLI 4, ISMAIL YILMAZ5 1Department of Anatomy, Faculty of Medicine, Duzce University, Duzce - 2Department of Medical Pharmacology, Faculty of Medicine, Abant Izzet Baysal University, Bolu - 3Department of Medical Pharmacology, Faculty of Medicine, Duzce University, Duzce - 4Department of Medical Biology, Faculty of Medicine, Kahramanmaraş Sutcu Imam University, Kahramanmaraş. - 5Department of Medical Pharmacology, Izmir Bozyaka Training and Research Hospital, Izmir, Turkey

ABSTRACT

Aim: To explore whether chrysin has any protective effect against the deadly effect of alpha amanitin on hepatocytes that is one of the major toxins in the structure of phalloides, which is considered the most important mushroom responsible for fatal mushroom poisonings. Materials and methods: For this study, alpha amanitin was purified from A phalloides mushroom using the preparative HPLC (high performance liquid chromatography) method as described in the literature. Four hours after administering alpha ama- nitin in a concentration of 10 µg/mL on the cells in a hepatocyte cell line (C3A), silibinin and chrysin were administered in various concentrations. The MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] test was used to determine cell viability. Results: The alpha amanitin was obtained in high purity from the mushrooms and was found to decrease cell viability down to a level of 63% after 48 hours due to its toxic effect on the liver cells in the cell culture. In the groups given silibinin and chrysin, both substances were seen to reduce the toxicity caused by alpha amanitin. Conclusion: Chrysin may play a role in decreasing the tissue damage caused by toxins and lowering the mortality rates in fatal mushroom poisonings associated with alpha amanitin.

Key words: Chrysin, Alpha amanitin, C3A human hepatocyte cell line, Mushroom poisoning, Protection.

Received March 16, 2015; Accepted May 02, 2015

Introduction The toxins that are found in largest amounts in a mushroom are alpha amanitin with a neutral char- Mushroom poisonings, especially those occur- acter and beta amanitin with an acidic character. ring due to constitute a serious Amanitins are present in a bicyclic form because the health problem worldwide as they result in liver sulfur atom in the cysteine contained in it forms a necrosis and death. Amanita type poisonous mush- bond with the indoleamine group of tryptophane and rooms have , and virotoxins this aspect is important in toxicity(1). Amanitins are in their structures. A phalloides, as one of these stable toxins that are considerably resistant to heat species, is a mushroom known with its greenish cap and chemicals(2). They can cause deaths in very small and white gills. The toxins that are responsible for doses (lethal dose=0.1 mg/kg) and a single mush- poisonings are amatoxins in the cyclopeptide struc- room weighing around 50 grams has this lethal ture and these amatoxins are divided into various amount(3). subgroups such as alpha amanitin, beta amanitin and After having been absorbed by the gastrointesti- gamma amanitin. nal system, the first organ amanitins come in contact 1182 Sinan Bakirci, Recep Bayram et Al with is the liver. Proteins such as OATP1B3 and then they were pulverized and left in 100 g/l L of dis- OATP2B1 contained in liver cells facilitate the pas- tilled water for 24 hours at room temperature to sage of toxins from sinusoids into hepatocytes. obtain a mushroom extract. The extract was cen- Amanitins cause liver necrosis stopping protein syn- trifuged at 5000 rpm for 10 minutes. The supernatant thesis by binding to eukaryotic RNA polymerase II. was removed and the rest of the liquid was vaporized About 60% of the toxins that come to the liver are on a heater. The remaining dry extract was diluted eliminated with bile, but are reabsorbed by the diges- with 10 mL of HPLC mobile phase and taken to the tive system and the enterohepatic circulation formed purification process. The preparative HPLC system in this way plays an important role in increasing the was used for purification. As a mobile phase, effect of the toxin(4-7). methanol + acetonitrile + ammonium acetate (50 The toxin passes into the systemic circulation mM, pH 5.5) (10+10+80; v/v/v) was used in the sys- and is absorbed by the tubulus cells after having tem at a 6 mL/min flow rate. A preparative column been eliminated from glomeruli in the kidneys and of 10x250 mm C18 (particle diameter 5 µm) this results in a tubular damage in the kidneys. A spe- (Agilent USA) was used. The extract obtained was cific antidote against toxins could not be found for purified using the preparative HPLC method twice mushroom poisonings. Therapeutic agents such as consecutively. The alpha amanitin fractions were col- penicillin, silibinin, prednisolone and N acetylcys- lected according to the involvement time of its stan- teine are being tried for treatment(8-11). The substances dard. Alpha amanitin was calculated by proportion- frequently preferred for treatment in clinical practice ing its toxic peak area to the total peak area in the are silibinin and penicillin and both of these hinder analytic HPLC system. The ultraviolet spectrum and the transportation of toxins to liver cells by competit- involvement time of the alpha amanitin obtained ing effect. In the cases of poisoning where treatment were compared to the ultraviolet spectrum and is started late or all treatment efforts remain inade- involvement time of the alpha amanitin standard. quate, an irreversible liver necrosis develops within a period of 7 days and liver transplantation becomes Cell culture the last remedy for survival. However, this treatment The C3A type hepatocellular cancer cells that approach usually cannot be practiced since an appro- were used in the study were kept alive in priate donor cannot be found readily and the time of DMEM/F12 (Dulbecco’s modified eagle medium: mortality is short(12). nutrient mixture F-12) containing 10% FCS (fetal Chrysin is a flavonoid in the form of “5.7- calf serum) and 1% antibiotic in a humid environ- Dihydroxy-2-phenyl-4H-chromen-4-one” contained ment with 5% CO2 at 37oC. The hepatocytes were in some passion flowers (Passiflora caerulea), oyster proliferated by dissociating them with trypsin-EDTA mushroom (Pleurotus ostreatus), which is an edible (ethylenediaminetetraacetic acid) twice a week. The mushroom, chamomile, honeycomb and a flowering medium on the cells was replenished every third day. plant Oroxylum indicum commonly known as mid- After collecting the cells with increased reproduction night horror. It has antioxidant(13), anti-carcinogenic(14- function, they were incubated in 96 well-plates in 16), anti-inflammatory(17, 18), anti-atherogenic(19), anti- equal amounts to form a concentration of estrogenic(20), anxiolytic(21), anti-aging(22), and anti-dia- 30x10^3/100 microliters. We waited about a day to betogenic properties. Chrysin has also been shown to allow the incubated cells to adhere. A control group, have protective effect on hepatocyte damages caused an alpha amanitin group where 10 μg/ml of alpha by D-galactosamine and carbon tetrachloride amanitin was given and three silibinin and chrysin (CCl4)(24, 25). groups where various doses (1, 10, 100 µg/mL) of Our study aims at investigating whether chrysin each were administered 4 hours after giving alpha with its broad-spectrum effects has any protective amanitin were established for the study. The groups effect on liver cells against alpha amanitin that caus- were tested with MTT for viability 48 hours after the es lethal mushroom poisonings. procedure. To this end, about 10% MTT solution was added on each well-plate. To fully dissolve the for- Material and methods mazan product that emerged 4 hours later, 100 microliters of DMSO (dimethyl sulfoxide) was Extraction of alpha amanitin added to the well-plates and for dissolution, they The A. phalloides mushrooms were dried under were kept in a drying oven at 37oC in a damp atmos- hot airflow between 50° and 60oC for 24 hours and phere for a night. What is the restoring role of chrysin in alpha amanitin toxicity? 1183

The 96 well-plates were tested by an elisa read- Discussion er at 570 nm wavelength. Percentage absorbance val- ues were calculated based on mean values of the In mushroom poisoning cases involving A control groups. The tests were repeated 3 times in all phalloides, a high level of mortality is observed groups. because there is no known antidote of amanitins and this rate is much higher in children as com- Statistical analysis pared to adults. The substance first comes to mind The avarage absorbance values of control group as an antidote against the toxin is silibinin. It can be were accepted as 100%. The average absorbance val- used alone or in combination with penicillin G or N ues of other samples were calculated by comparing acetylcysteine. In particular, the combined use of to control groups. Data are given as the mean value some antitoxic substances have different mecha- ±SEM (standart error means). The statistical analysis nisms of action may increase the hepatoprotective between the viability values was assessed using the effect. SPSS program and between dual groups using the In mushroom poisonings, the soonest the diag- Mann Whitney U Test. The level of statistical signifi- nosis is made and the earliest the treatment is start- cance has been set at p< 0.05. ed, the least the liver damage is and thus the lowest the mortality rate is. While the mortality is 40% Results with early treatment, it goes up to 75% in the case of a delay(26-28). According to the MTT test, cell viability went The first of silibinin’s action mechanisms is down to an average of 63% at hour 48 following the that silibinin competitively races with amanitins for administration of alpha amanitin in concentrations of OATP1B3, a subunit of the anion transport system 10 µg/mL. In the groups where silibinin was given in in the plasma membrane, which is used by aman- concentrations of 1, 10 and 100 µg/mL 4 hours after itins to enter hepatocytes. Its other action mecha- alpha amanitin administration, cell viability rates nism is that it increases protein synthesis by bind- appeared to be 66%, 63% and 56% respectively at ing to a specific region in RNA polymerase II, hour 48 and in the groups where chrysin was given in enabling regeneration of the liver(10). concentrations of 1, 10 and 100 µg/mL 4 hours after Magdalan et al. argued that silibinin, benzyl alpha amanitin administration, the cell viability rates penicillin and acetylcysteine had a considerably were 66%, 69% and 67% respectively (Figure 1). The strong protective effect against cell damage caused concentrations of 10 µg/mL for chrysin and 1 µg/mL by alpha amanitin in human hepatocyte cultures and for silibinin had the best efficacy on cell viability and this protective effect emerging independent of the when both of these were compared to the alpha aman- dose of these three antidotes was due to the high itin group, the difference in between was statistically efficacy of these substances(10). They showed in a significant (p<0.05). When the efficacies of the most similar study that these antidotes were not effective efficient doses of the silibinin and chrysin groups were in dog hepatocytes that had been exposed to alpha compared, there was again a significant difference amanitin(11). We used a human hepatocyte cell line (p<0.05). in our study and found that silibinin had a protec- tive effect on hepatocytes, a result similar to that of Magdalan et al. Additionally, we also showed in our study that chrysin, a plant-based flavonoid with a fairly broad spectrum, had also protective effect on liver cells at rates similar to those of silibinin. Silibinin is also a considerably strong anti- inflammatory substance that prevents leukocyte migration and a free radical scavenger. Amanitins not only inhibit protein synthesis by binding to the Figure 1: The ratio of cell viability after silibinin and chrysin RBPI subunit of RNA polymerase II, they also treatments. The cell line stimulated by alpha amanitin at cause an increase in free radicals in hepatocytes by 10μg/ml concentration, and was treated with silibinin or chry- inhibiting the catalase enzyme activity in the sin 4 hours after toxin administration. After incubating 48 (29) hours, the MTT test performed to evaluate the cell viability. liver . Magdalan et al. after reducing the catalase (*:p<0.05 chrysin or silibinin groups against control group. #: activity in hepatocytes (from 52 to 32 on the aver- p<0.05 chrysin group against silibinin group). 1184 Sinan Bakirci, Recep Bayram et Al age) by giving alpha amanitin through extracorpo- mg/dl to 237.8 mg/dl) in a period of 7 days in some real perfusion, investigated the separate effects of groups of rats that had been made diabetic with six substances, namely penicillin, rifampicin, silib- streptazocin(23). It was found in the same study that inin, acetylcysteine, beta estradiol, and taurocholate giving alkyl derivates of chrysin (5.7-dialkoxy- sodium, on the catalase activity. They demonstrated chrysin and 5-hydroxy-7-alcooxychrysin) in doses that from the six groups, penicillin was not effec- as high as 500 mg/kg did not result in any changes tive (mean 32) in the catalase activity that had been in general characteristics or eating habits of ani- decreased by alpha amanitin; rifampicin, beta estra- mals(23). This shows that chrysin may be used in diol and taurocholate (average 43, 41 and 41 very high doses when needed. respectively) produced a significant increase, but It is known that the sulfur atom in the mole- could not come close to the catalase activity level in cule structure of alpha amanitin plays an important the control group; silibinin and acetylcysteine role. In fact, the dethio-amanitin molecule that brought the catalase activity close to normal values comes into being when the sulphoxide part, which (average 48 and 48)(29). Silibinin and acetylcysteine forms a bond with the indole structure, is removed also have a free radical scavenger activity; they from the molecule has been shown to be nontoxic. inhibit lipid peroxidation, protect membrane struc- It is possible that chrysin gains an antitoxic poten- tures and play a protective role against the proox- tial when the isoflavonoid residue contained in the idative effects of alpha amanitin(29). Penicillin G chrysin molecule structurally mimics the sulphox- does not improve catalase activity decreased due to ide-indole part in the amanitin molecule but without toxins. Therefore, silibinin and acetylcysteine give showing any toxicity. Penicillin with a beta lactam better results than penicillin G in clinical applica- structure and silibinin with an isoflavonoid struc- tions(29). ture are also used in treating amanitin-based mush- Pushpavalli et al. showed in a study that in D- room poisonings. It is possible that the beta lactam galactosamine induced hepatotoxicity, chrysin and isoflavonoid structures of these molecules also caused an increase in the levels of the enzymes that act by mimicking the same part. work as free radical scavengers (superoxide dismu- tase, catalase and glutation peroxidase) and restored Conclusion the increase in the liver enzyme levels that was associated with the toxicity caused by D-galac- Our results showed that chrysin decreased as tosamine. In that study, the effects of chrysin and much as silibinin the toxic effect made by alpha sylimarin (silibinin used in mushroom poisoning is amanitin, one of the poisonous mushroom toxins, the most active component of sylimarin) were com- on human hepatocytes. After further animal experi- pared and chrysin was shown to be more effective ments with chrysin and studies on its action mecha- than sylimarin(25). Similarly, chrysin was seen to nism have been completed, chrysin will become a restore the increased glutamic oxaloacetic transmi- candidate to be used as a new healing agent in nase and glutamic pyruvate transaminase enzyme mushroom poisonings by itself or in combination levels and decreased catalase, superoxide, dismu- with another therapeutic drug like penicillin that tase and glutation peroxidase levels, that were asso- has a different action mechanism. ciated with the tissue damage occurred in the livers of rats exposed to CCl4(24). We found in our study the effects of silibinin and chrysin against the toxic- ity caused by alpha amanitin in human hepatocyte cultures were close to each other, the effect of References chrysin being even better. The amount of absorbed toxins through diges- 1) Vetter J. Toxins of Amanita Phalloides. Toxicon 1998; tion is important in in mushroom poisonings. 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