in vivo 23: 393-400 (2009)

Failure of Benzylpenicillin, N-Acetylcysteine and Silibinin to Reduce α-Amanitin Hepatotoxicity

JAN MAGDALAN1, ALINA OSTROWSKA1, ALEKSANDRA PIOTROWSKA2, ILONA IŻYKOWSKA2, MARCIN NOWAK3, ADAM SZELĄG1 and PIOTR DZIĘGIEL2,4

1Department of Pharmacology, 2Department of Histology and Embryology, Wrocław Medical University, Wrocław; 3Department of Pathological Anatomy, Pathophysiology, Microbiology and Forensic Veterinary Medicine, Wrocław University of Enviromental and Life Science, Wrocław; 4Department of Histology and Embryology, Poznań University of Medical Science, Poznań, Poland

Abstract. Background: Intoxications caused by amanitin- phalloides intoxications is principally a result of the containing represent an unresolved problem in acute liver failure following significant hepatocyte damage due to clinical toxicology. The objective of this study was a hepatocellular uptake of α-amanitin (α-AMA), the major comparative evaluation of benzylpenicillin (Bp), acetylcysteine (6-9). Severe intoxications caused by amanitin- (ACC) and silibinin (Sil) efficacy as antidotes in hepatocytes containing mushrooms represent an unresolved problem in intoxicated with α-amanitin (α-AMA). Materials and clinical toxicology since no specific and fully efficient antidote is Methods: All experiments were performed on cultured canine readily available. Several substances widely used in the past to hepatocytes. Cytotoxicity evaluation of cultured cells (MTT treat poisonings (steroids, cimetidine, thioctic acid) assay, extracellular lactate dehydrogenase activity) was are documented to be completely ineffective. Moreover, a performed at 12, 24 and 48 h of exposure to α-AMA and/or retrospective analysis revealed that the most often currently used antidotes. Results: Following 24 and 48 h exposure there was antidote – benzylpenicillin (Bp) shows poor clinical efficacy (1). a significant decline of hepatocyte viability and an increase of Overall results of this meta-analysis indicate that silibinin (Sil) lactate dehydrogenase activity in groups exposed to α-AMA or acetylcysteine (ACC) are found to be more effective in and in groups exposed simultaneously to α-AMA and therapy in humans. However, ACC, Sil and antidotes. Moreover, hepatocyte viability and lactate Bp were not effective in limiting hepatic injury after α-AMA dehydrogenase activity in all these groups were similar. poisoning in a murine model (10). Administration of studied antidotes without α-AMA, was not Since clinical course and symptoms of amanitin intoxication associated with any adverse effects in hepatocytes. Conclusion: in dogs are almost identical to those seen in humans, a concept All antidotes tested in this study against α-AMA were not of the treatment of this type of toxicity can be studied in a effective in canine hepatocyte cultures. canine hepatocyte model (11-14). The objective of this study was a comparative evaluation of ACC, Bp and Sil efficacy in Exposure to mushrooms containing is fairly common canine hepatocytes intoxicated with α-AMA. all over the world (1-6). Currently there are more than 30 known amatoxin-producing species belonging to the genera Amanita, Materials and Methods Galerina and Lepiota. The toadstool death cap () and its subspecies, () Chemicals and materials. Media and reagents used for hepatocyte and death angel () are responsible for nearly 95% isolation, including Hank’s balanced salt solution (HBSS), Leibovitz of all fatal mushroom poisonings (6). High mortality rate in (L-15) medium, EBSS (Earle’s balanced salt solution), Waymouth’s 752 medium, phosphate-buffered saline (PBS), ethylene glycolbis(aminoethylether)-tetraacetic acid (EGTA), glucose, gentamicin/amphotericin B solution, media supplements, fetal bovine Correspondence to: Jan Magdalan, Ph.D., Department of serum (FBS), collagenase type I and α-amanitin were purchased from Pharmacology, Wrocław Medical University, Mikulicza-Radeckiego Sigma Poland Chem. Corp. Collagen-coated plates (96- and 24-well) 2, PL 50-345 Wrocław, Poland. Tel/Fax: +48 717840094, e-mail: for hepatocyte culture were purchased from Becton Dickinson (USA). [email protected] MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] kit was from Sigma Poland Chem. Corp. Benzylpenicillin potasium salt Key Words: α-Amanitin, Antidotes, Amanita phalloides, intoxication, (cat. no. P7794), Silibinin (cat. no. S0417) and N-Acetyl-L-cysteine canine hepatocytes. (cat. no. A9165), were purchased from Sigma Poland Chem. Corp.

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Hepatocyte isolation and culture. All experiments were performed Statistical analysis. Differences between values (MTT, LDH after approval by the Local Ethics Commission for Experiments activity) were analyzed by individual comparison with Mann- on Animals at the Institute of Immunology and Experimental Whitney U-test. Statistical analysis was carried out using the Therapy in Wroclaw (license no. 54/2007). The liver was Statistica 7.1 software (Stat Soft, Poland), and p<0.05 was obtained from a 5-year old beagle dog (male, weighing 16 kg). considered statistically significant. Briefly, after initial heparinization (200 IU/ kg, i.v. injection) the animal underwent a full midline incision under xylazine (2 Results mg/kg, i.m.) and ketamine (10 mg/kg, i.v.) general anesthesia. The liver was completely perfused through the portal vein and At 12 h of exposure hepatocyte viability (based on MTT then removed from the abdomen. Hepatocytes were isolated from assay) as well as expression of extracellular LDH activity in the left lateral lobe by a modified two-step perfusion as described all experimental groups were similar and no significant previously (13). The final cell suspension was centrifuged and the differences were observed compared to controls. Following medium aspirated. The viability and yield of isolated hepatocytes were estimated by trypan blue staining. The cells were 24 and 48 h exposure there was a significant decline of resuspended in L-15 plating medium supplemented with 10% hepatocyte viability and increase of LDH activity in all FBS, gentamicin and amphotericin B, and then dispensed into 24- experimental AMA groups of cultures (Figures 1-6). well collagen-coated plates. The cultures were incubated at 37˚C Additionally, viability of hepatocytes exposed simultaneously in a humidified atmosphere of 95% air with 5% CO2. After 4 h to α-AMA and studied antidotes declined, while the LDH of initial incubation, the plating medium was substituted with activity significantly increased as compared to the control defined culture medium (combination of EBSS and Waymouth’s group C. Hepatocyte viability and extracellular LDH activity 752/1, supplemented with 10% FBS). After the next 12 h incubation the medium was exchanged and primary hepatocyte in cell cultures exposed to antidotes were only stable and cultures were maintained for 48 h with 1 daily dose of α-AMA comparable to those of group C (Groups A, a; B, b; S, s; and/or antidotes (ACC, Bp, Sil) in the following final Figures 1-6). concentrations: Group C – control hepatocyte cultures received medium without α-AMA or antidotes; Group AMA: α-amanitin Discussion (10 μM); Group a: N-Acetyl-L-cysteine (0.5 mM); Group A: N- Acetyl-L-cysteine (1 mM); Group b: benzylpenicillin (0.5 mM); Conducting clinical evaluation of different medical Group B: benzylpenicillin (1 mM); Group s: silibinin (10 μM); Group S: silibinin (20 μM); Group AMA + a: α-amanitin (10 treatments of severe intoxications caused by amatoxin- μM) + acetylcysteine (0.5 mM); Group AMA + A: α-amanitin producing mushrooms is extremely difficult. A number of (10 μM) + acetylcysteine (1 mM); Group AMA + b: α-amanitin clinical factors need to be taken into account when (10 μM) + benzylpenicillin (0.5 mM); Group AMA + B: α- evaluating an effective treatment of mushroom poisoning, amanitin (10 μM) + benzylpenicillin (1 mM); Group AMA + s: including but not limited to age and gender of the patient, α-amanitin (10 μM) + silibinin (10 μM); Group AMA + S: α- health condition prior to intoxication, time from mushroom amanitin (10 μM) + silibinin (20 μM). ingestion to onset of therapy, and clinical condition at the According to Magdalan et al. (13) following 6-24 h exposure to 10 μM of α-AMA canine hepatocytes displayed a variety of time of therapy initiation. Moreover, using specific treatment morphological and functional changes (significant decrease of regimens including combined pharmacotherapy in one viability and significant increase of intracellular LDH). ACC patient significantly complicates such evaluation (1). concentrations corresponded to plasma levels obtained after the Therefore, primary hepatocyte cultures are a good model for recommended dosage during the treatment in paracetamol toxicity objective analysis of different antidotes without the necessity (15). Sil was used at concentrations corresponding to its of taking into consideration all aforementioned clinical therapeutic plasma levels (16, 17). Bp concentrations factors. In these studies, it was observed that decreased cell corresponded to plasma levels obtained after the dosage recommended in the therapy of the toadstool death cap poisoning, viability was correlated with LDH release measurements. i.e. 300.000 – 1.000.000 U/kg/day (6, 18). Cytotoxicity evaluation LDH is located almost entirely in the cytoplasm, and its of cultured cells was performed at 12, 24 and 48 h of exposure to leakage is a good marker of cell damage (20). Thus, it can α-AMA and/or antidotes. be concluded that decreased viability of hepatocytes treated with α-AMA was related to significant morphological Analytical methods. Retained functional integrity and viability derangements (disruption of cell membrane). of cultured hepatocytes was assessed using the MTT assay (19). The oldest antidote that has been widely used in Reduction of a yellow salt MTT by mitochondrial therapeutic treatment of intoxications caused by the toadstool dehydrogenases in viable cells to a purple formazan precipitate death cap is Bp (1). Initially, it was speculated that the major was determined by measuring the absorbance at 570 nm on a plate reader (Elx 800 Universal Microplate Reader, Bio-Tek mechanism of action of this antidote is related to expelling Instruments, USA). Lactate dehydrogenase (LDH) activity the amanitin from plasma albumin binding sites. However, it in hepatocyte culture supernatants was quantified spectro- has been found that such a mechanism is not relevant, since photometrically by using dimension analyzer (Panda, DADE amanitins do not form any bindings with plasma proteins Behring RxL Max, USA). (21, 22). Acording to Letschert et al. (23) Bp can inhibit

394 Magdalan et al: Antidotes in α-Amanitin Hepatotoxicity

Figure 1. MTT activity in control (C) and experimental groups (AMA; AMA+A; AMA+a; A; a). The number of viable hepatocytes (one representative hepatocyte preparation) is proportional to the MTT reaction product, as determined by the optical density. Each point represents the mean ± SD, n=8, *p<0.05 vs. C (AMA – α-amanitin 10 μM; A - N-Acetyl-L-cysteine 1 mM; a - N-Acetyl-L-cysteine 0.5 mM).

Figure 2. MTT activity in control (C) and experimental groups (AMA; AMA+B; AMA+b; B; b). The number of viable hepatocytes (one representative hepatocyte preparation) is proportional to the MTT reaction product, as determined by the optical density. Each point represents the mean ± SD, n=8, *p<0.05 vs. C (AMA – α-amanitin 10 μM; B - benzylpenicillin 1 mM; b - benzylpenicillin 0.5 mM).

primary uptake of amanitin by hepatocytes. However, it is therapeutic treatment of the toadstool death cap intoxication unknown whether this inhibition process can result in (1). Currently, Bp is a widely used antidote (23, 24). alleviation of the cytotoxic effects on liver parenchymal However, it is always administered as a component of the cells. Reports published in the late 70s and early 80s combined pharmacotherapy, which makes the evaluation of indicated significant efficacy of Bp as an antidote in its real efficacy quite difficult. According to Letscher et al.

395 in vivo 23: 393-400 (2009)

Figure 3. MTT activity in control (C) and experimental groups (AMA; AMA+S; AMA+s; S; s). The number of viable hepatocytes (one representative hepatocyte preparation) is proportional to the MTT reaction product, as determined by the optical density. Each point represents the mean±SD, n=8, *p<0.05 vs. C (AMA – α-amanitin 10 μM; S - silibinin 20 μM; s - silibinin 10 μM).

Figure 4. Lactic dehydrogenase activity in culture medium of control (C) and in culture medium of experimental groups (AMA; AMA+A; AMA+a; A; a). Each point represents the mean ± SD, n=8, *p<0.05 vs. C. (AMA – α-amanitin 10 μM; A - N-Acetyl-L-cysteine 1 mM; a - N-Acetyl-L-cysteine 0.5 mM).

(23) Bp shows little efficacy as an antidote in intoxications damage effects in cultures exposed simultaneously to α- with amatoxin-containing mushrooms in humans. These AMA and different concentrations of Bp were comparable observations are in agreement with the results of the present to those treated with α-AMA alone. study indicating that there is no protective effect of Bp on ACC is approved to reduce the extent of intoxication after dog hepatocytes exposed to α-AMA. Moreover, hepatocyte acetaminophen overdose. Administration of ACC is beneficial

396 Magdalan et al: Antidotes in α-Amanitin Hepatotoxicity

Figure 5. Lactic dehydrogenase activity in culture medium of control (C) and in culture medium of experimental groups (AMA; AMA+P; AMA+p; P; p). Each point represents the mean±SD, n=8, *p<0.05 vs. C. (AMA – α-amanitin 10 μM; B - benzylpenicillin 1 mM; b - benzylpenicillin 0.5 mM).

Figure 6. Lactic dehydrogenase activity in culture medium of control (C) and in culture medium of experimental groups (AMA; AMA+S; AMA+s; S; s). Each point represents the mean±SD, n=8, *p<0.05 vs. C. (AMA – α-amanitin 10 μM; S - silibinin 20 μM; s - silibinin 10 μM).

in preventing hepatic injury through stimulation of marked decreases in the intracellular glutathione content (26). glutathione synthesis, enhancing transformation of toxic It seems likely that ACC is also efficient as a free radical acetaminophen metabolite to non-toxic mercaptide conjugate scavenger, and according to some reports free radical (25). Introduction of ACC dosing in mushroom poisoning formation might contribute to the severe amatoxin was based on reports describing that incubation of rat hepatotoxicity (27, 28). Nevertheless, there is no hepatocytes with the extracts of Amanita virosa can cause experimental data revealing that ACC is an efficient antidote

397 in vivo 23: 393-400 (2009) for amanitin toxicity (29). However, a retrospective analysis References suggests that this formulation can be used for therapeutic treatment of amanitin intoxication. Unfortunately, in most of 1 Enjalbert A, Rapior S, Nouguier-Soule J, Guillon S, Amoroux N all analyzed cases ACC was used as one of the components in and Cabot C: Treatment of Amatoxin Poisoning: 20-Year a combined poly-chemotherapy (1), hence the actual Retrospective Analysis. J Toxicol Clin Toxicol 40: 715-757, 2002. evaluation of its benefit was impossible. Since there are no 2 Adukauskiene D, Dockiene I, Naginiene R, Kevalaitis E, reports on standard dosage regimen in mushroom poisoning, Pundzius J and Kupcinskas L: Acute liver failure in Lithuania. ACC was tested in concentrations corresponding to its plasma Medicina 44: 536-540, 2008. levels after the recommended dosage during the treatment in 3 Erguven M, Yilmaz O, Deveci M, Aksu N, Dursun F, Pelit M acetaminophen toxicity. In this study, the lack of protective and Cebeci N: Mushroom poisoning. Indian J Pediatr 74: 847- effect of ACC against α-AMA in cultured canine hepatocytes 852, 2007. was demonstrated. However, administration of ACC, like 4 Joshi A, Awale P, Shrestha A and Lee M: Acute mushroom poisoning: a report of 41 cases. JNMA J Nepal Med. Assoc 46: other antidotes tested in this study, was not associated with 7-12, 2007. any adverse effects in the tested hepatocytes. 5 Kotwica M and Czerczak S: Acute poisonings registered since The flavonolignan sylimarin and one of its structural 1970: trends and characteristics. Analysis of the files collected components, Sil are substances with documented hepato- in the National Poison Centre, Lódz, Poland. Int J Occup Med protective properties (30). The data in the literature indicate Environ Health 20: 38-43, 2007. that Sil acts as a scavenger of free radicals, and regulates high 6 Schneider SM: Mushrooms. In: Clinical Toxicology. Ford MD, anti-inflammatory effect in vivo by inhibition of leukocyte Delaney KA, Ling LJ, Erickson WB (eds.). Philadelphia, London New York, St. Louis, Sydney, Toronto, Saunders migration into the inflamed site (31, 32). The inhibition of Company, pp. 899-909, 2001. amanitin uptake into hepatocytes exposed to Sil has also been 7 Jaeger A, Jehl F, Flesch F, Sauder P and Kopferschmitt J: documented (23, 33). Vogel et al. (34) reported specific Kinetics of amatoxins in human poisoning: therapeutic protection of Sil against Amanita phalloides intoxication in implications. J Toxicol Clin Toxicol 31: 63-80, 1993. dogs. However, clinical observations of the Sil efficacy are 8 Krenova M, Pelclova D and Navratil T: Survey of Amanita ambiguous, mostly due to the fact that this substance is more phalloides poisoning: clinical findings and follow-up evaluation. usually used in combination with other antidotes, like Bp and Hum Exp Toxicol 26: 955-961, 2007. ACC (1). According to Ganzert et al. (35) lower death and 9 Yildiz BD, Abbasoglu O, Saglam A and Sökmensüer C: Urgent liver transplantation for Amanita phalloides poisoning. Pediatr transplantation rates were observed in patients treated with Sil Transplant 12: 105-108, 2008. than in patients treated with Sil combined with Bp. 10 Tong TC, Hernandez M, Richardson WH 3rd, Betten DP, Presumably, different efficacy of Sil depends upon the route of Favata M, Riffenburgh RH, Clark RF and Tanen DA: administration as well as a type of used formulation (sylimarin, Comparative treatment of alpha-amanitin poisoning with N- or purified Sil). Thus, considering low bioavailability of acetylcysteine, benzylpenicillin, cimetidine, thioctic acid, and sylimarin, Sil plasma levels are significantly lower after oral silybin in a murine model. Ann Emerg Med 50: 282-288, dosing of sylimarin (parent compound) than after intravenous 2007. 11 Faulstich H and Fauser U: The course of amanita intoxication in administration of purified Sil (16, 17). In this study no beagle dogs. In: Amanita toxins and poisoning. Faulstich H, protective effect of Sil on cultured canine hepatocytes exposed Kemmerell B and Wieland TH (eds.). New York, Gerhard to α-AMA was demonstrated. This can be caused by Witzstrock, pp. 115-123, 1980. application of too small concentrations of the antidote, 12 Liggett AD and Weiss R: Liver necrosis caused by mushroom corresponding rather with the levels usually obtained in patients poisoning in dogs. J Vet Diagn Invest 1: 267-269, 1989. after oral dosing of Sil in the form of Liverman capsules (16). 13 Magdalan J, Ostrowska A, Podhorska-Okołów M, Piotrowska A, All antidotes (Bp, ACC and Sil) tested in this study Iżykowska I, Nowak M, Dolińska-Krajewska B, Zabel M, Szeląg A and Dzięgiel P: Early morphological and functional alterations against toxic α-AMA were not effective in canine hepatocyte in canine hepatocytes due to α-amanitin, a major toxin of cultures. However, considering possible interspecies Amanita phalloides. Arch Toxicol (in press). differences it seems reasonable to independently reproduce 14 Puschner B, Rose HH and Filigenzi MS: Diagnosis of Amanita all experiments using primary human hepatocyte cultures. Toxicosis in a Dog with Acute Hepatic Necrosis. J Vet Diagn Evaluation of a real efficacy of all those antidotes may Invest 19: 312-317, 2007. contribute to improved therapy in amanitin-containing 15 Prescott LF, Donovan JW, Jarvie DR and Proudfoot AT: The mushroom intoxication. disposition and kinetics of intravenous N-acetylcysteine in patients with paracetamol overdosage. Eur J Clin Pharmacol 37: 501-506, 1989. Acknowledgements 16 Kim YC, Kim EJ, Lee ED, Kim JH, Jang SW, Kim YG, Kwon JW, Kim WB and Lee MG: Comparative bioavailability of This work is a part of a project granted by Polish Ministry of silibinin in healthy male volunteers. Int J Clin Pharmacol Ther Science and Higher Education (grant no. N 401 2809 33). 41: 593-596, 2003.

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