Agents and Actions vol. 9/2 (1979) Birkh/iuser Verlag, Basel

221

On the Nephrotoxicity of a-Amanitin and the Antagonistic Effects of Silymarin in Rats by G. VOGEL1), R. BRAATZ l) and U. MENGS 2)

Dr Madaus u. Co., Pharmakologische Abtlg.~), Abtlg. f/ir Pathologie und Toxikologie2), Ostmerheimer Str. 198, D-5000 K61n 9 l, Federal Republic of Germany

Abstract In human beings, poisoning by the deathcup mush-

room eauses renal lesions in addition tO extremely severe liver damage. It is known from animal experiments that silymarin, a polyhydroxyphenylehromanone, is capable of counteracting this ct-amanitin-induced liver damage. The purpose of the present work was to ascertain whether renal damage could be indueed in rats by giving cr-amanitin, and whether silymadn would have any effeet on sueh renal damage. The fact that ct-amanitin produces pathological changes in the kidneys and that these lesions can be almost completely prevented by pretreatlng rats with silymarin has now been amply demonstrated by biochemieai and hlsto- logical teehniques alike.

Introduction The effects of deathcup mushroom poison-

ing in man include pulmonary and cerebral oedema [1, 21 and pathological changes in the kidneys [1-91 in addit ion to liver damage. The intense toxicity of the green deathcup mush- room, Arnanita phalloides, is mainly due to two cyclic oligopeptides, phalloidin and c~-amanitin. Both toxins are freely absorbed from the human gastro-intestinal tract. Phalloidin has specific hepatotoxic effects, in that it reacts with the hepatocyte membranes and des t roys them. The result is an outflow of intracellular K § which is osmotical ly balanced by an inflow of N a § and H20 [10]. ct-Amanitin binds to the nucleus, where, even in low concentrat ions, it inhibits R N A - p o l y m e r a s e II [1 1-16]. This interferes with the synthesis of nucleic acids and, hence, with protein synthesis [17]. o:-Amanitin is not liver specific, but appears to interact mainly with cells having high protein synthesis rates.

Clinically, the symptoms and signs of death- cup mushroom poisoning begin between 8 and 24 hours after ingestion of the toadstools . The first phase lasts approximate ly two days and is

character ized by nausea and vomiting leading to dehydrat ion. After a latent period o f roughly one day, this is followed by the onset of hepatic and renal tubular necrosis. Dea th is usually due to hepatic coma [18, 19]. Numerous studies in experimental animals have shown that silymarin is capable of neutralizing the toxic action of ct- amanit in on the liver. Given prophylact ical ly , it will prevent the toxic effects; given thera- peutically, it will reduce them.

The purpose of the present s tudy was to ascertain whether ct-amanitin can induce renal damage in rats, and whether such lesions can be counteracted by si lymarin [20].

After it had been shown that renal damage caused by et-amanitin could in fact be counter- acted by silymarin, the work was continued to find out whether si lymarin, likewise, had a beneficial influence on the toxic effects of cr- amanit in on the blood and on hair growth.

Materials and methods The animals used for this work were male Wistar rats

(Mus rattus AG, Brunnthal) weighing 200 to 300 g. In order to produce renal damage the rats were given 5 mg/ml • kg ct-amanitin in aqueous solution intraperitoneally; this dosage is in the region of the LDmi n.

For prophylactic testing, silymarin was administered as the sodium hemisuccinate in a dose of 100 mg/5 ml • kg, intravenously, 60 or 20 minutes before the toxic dose of ct- amanitin. To test the curative efficacy of silymarin it was given intravenously in a dose of 100 mg/5 ml x kg, 5, 10 and 20 minutes after administration of the toxin or in doses of 300 mg/10 ml x kg, 5 and 10 minutes afterwards. The development of the renal damage was followed by bio- chemical tests on urine and plasma and by histological examination of frozen sections. Fifty-five hours after injec- tion of the toxin, some of the rats (unanaesthetized) were placed in diuresis funnels for 16 hours. Creatinine and urea were measured in the collected urine and the plasma.

222 On the Nephrotoxicity of a-Amanitin and the Antagonistic Effects of Silymarin in Rats

Sixty-four hours after the toxic dose of cc-amanitin, other rats were anaesthetized with 40-50 mg/kg pento- barbital sodium i.p. and both ureters were catheterized. Creatinine and PAH were injected into the jugular vein, each in a dose of 20 mg/kg. Twenty minutes later the rats were given infusions of creatinine, at a rate of 3 mg/kg x min and PAH, at 0.2 mg/kg x min, the infusion volume being 0.2 ml/min x rat. The infusion fluid was mannitol-Ringer solution of the following composition:

NaC1 140 mmol/1 KCI 2.7 mmol/1 CaC12 1.8 mmol/l NaHCO 3 12.0 mmol/1 mannitol 50 g/l

One hour after starting the infusion, urine was collected for a five minute period and at the same time a blood sample was taken from one carotid artery.

These samples were 'used to measure the clearances and the quantities of creatinine, urea, PAH and Na + secreted or reabsorbed in relation to the amounts filtered through the glomeruli.

Measurements of glucose, protein, osmolality, GPT, GOT and bilirubin were made in urine and/or serum.

To obtain renal tissue for histological examination, some of the rats were killed 60 hours after the toxic dose of ct-amanitin and the kidneys were fixed in 10% formalin. Frozen and paraffin sections were stained with haema- toxylin-eosin or scharlach red. The PAS reaction was also carried out on paraffin sections.

Results 1. B iochemica l f indings

The in toxica t ion o f the rats wi th cr-amanitin did not p roduce any charac te r i s t i c changes in

urine v o l u m e or compos i t i on as m e a s u r e d be- tween 55 and 72 hours af terwards . H o w e v e r , the p l a s m a concen t ra t ions o f creat in ine and urea rose to u raemic values. P re t r ea tmen t o f the ra ts with 100 mg s i l ymar in /kg a lmos t comple te ly preven- ted these abnormal i t i es (Tables 1 and 2).

W h e n s i lymar in was given cura t ive ly , the t ime-effect re la t ionship o f its ac t ion against renal d a m a g e was very similar to that of its an tago- nistic effect agains t cr-amanit in-induced l iver damage . W h e n the s i lymarin was given 10 minutes after the cr-amanitin it gave good, but no t 100%, pro tec t ion . W h e n the de lay was leng- thened to 2 0 - 3 0 minutes the effect o f s i lymar in was no longer demons t rab le . W h e n s i lymar in was given cura t ively , in doses o f f rom 100 to 300 m g / k g body weight , there was no evidence o f any dose-effect relat ionship.

Stat is t ical analysis o f the c lea rance tests on anaes the t ized rats gave the fol lowing pic ture : Po i son ing with cr-amanitin caused a p r o n o u n c e d fall in G F R and in the absolu te and f rac t iona l re- absorp t ion o f HEO and N a § toge ther with a reduc t ion in P A H and u rea c learances and a rise in p l a sma urea concent ra t ion . These abnor- mali t ies were s ignif icantly r educed by p rophy- lact ic admin is t ra t ion of s i l y m a r i n (Table 1). D i sc r iminan t analysis d e m o n s t r a t e d the clear dif- fe rence be tween the two exper imenta l g roups (A = tr-amanitin, B = s i lymar in + tr-amanitin) as

Figure 1 Extensive necrotic lesions in the renal tubules of the cortex 60 h after a-amanitin (5 mg/kg i.p.) intoxication. Magnification: 160 x.

On the Nephrotoxicity of 0c-Amanitin and the Antagonistic Effects of Silymarin in Rats 223

Figure 2 Single cell necrosis in significant less marked renal damage after prophylactic intravenous injection of silymarin ( 1 O0 mg/kg). Magnification: 160x.

assessed by the indices G F R (I)3), urea clearance (3) and P A H clearance (4) (but not Na + clearance (2)) and as assessed by the relative re- absorption of H20 (5) and Na + (6), but not by that o f urea (7) or P A H secretion (8).

The linear discriminant function for the ct- amanitin group A is hence as follows:

YA = --15"08Xl + 17"97X3 + 0"156X4 + 1.66X 5 -- 0.387X 7 -- 59.05

and for the silymarin pretreated group B:

YB = --6.7xl + 8.21x3 -- 0.104x4 + 2.14x 5 + 0.201x 7 -- 84.41

When silymarin was given 10 minutes after the toxic dose of cc-amanitin, it had a significant antagonistic effect against the resulting renal damage (Table 2). In these experiments there was even a difference in the amounts of urea re- absorbed, so that the discriminant functions are as follows:

C for the c~-amanitin group:

Yc = 29.14x2 + 3.33x3 - 1-18x4 - 0.603xs + 0.902x 6 + 0.170x 7 - 18.39

and D for the ct-amanitin + silymarin group:

YD = 17"28X2 -- 3"9X3 + l ' l l x 4 + 0-456Xs + 0.0327X6 -- 0.0599X 7 -- 29.83

9) The numerals are the indices for the relevant parameters and are reproduced in the discriminant functions.

2. Histopathologic findings in the kidneys Sixty hours after intraperitoneal adminis-

tration of c~-amanitin, 5 mg/kg, the rats ' kidneys showed severe degenerative changes in the renal tubules of the cortex. These took the form of cloudy swelling and vacuolar degeneration of the cytoplasm with distended, chromatin-poor nuclei, progressing, in some instances, to extensive necrotic lesions involving groups of cells (Fig. 1). Nuclear pyknosis and single cell necrosis were found here and there in the epithelial cells of Henle's loops and the collecting tubules in the medulla. The lumina of the tubules contained massive amounts o f necrotic material. The glomeruli showed no lesions visible by the light microscope.

Prophylactic intravenous injections of sily- marin, 100 mg/kg, gave a definite quantitative reduction in the renal lesions just described. The degree of damage was significantly less marked, especially in regard to the necrotic lesions in the renal tubules. Single cell necrosis was still evident (Fig. 2), but necrotic lesions involving cell groups were now very infrequent.

3. Other findings, not involving the kidneys When taking blood samples it was noted that

the blood from rats poisoned with ct-amanitin seemed more viscous than that from normal animals. The average haematocrit of the poisoned rats was 62.0 + 2.51. When silymarin was given

224

Table I

On the Nephrotoxicity of a-Amanitin and the Antagonistic Effects of Silymarin in Rats

Urine Creatinine Urea GFR Na + volume plasma conc. plasma conc. (ml/min x kg) clearance (ml/min x kg) (rag/100 ml) (mg/100 ml) (ml/min x kg)

A cr-Amanitin 2 0.784 35.4 252.65 2.73 0.672 n = 10 • 0.296 5.8 94.30 1.40 0.25

B Silymarin (prophylactic) + a-amanitin J? 0.884 25.3 51.78 8.82 0.737 n = 10 • 0.268 5.0 17.40 2.33 0.31

Table 2

Urine Creatinine Urea GFR NA § volume plasma conc. plasma conc. (ml/min x kg) clearance (ml/min x kg) (mg/100 ml) (mg/100 ml) (ml/min x kg)

A a-Amanitin 2 0.815 n=10 • 0.221

B cr-Amanitin + silymarin (curative) 2 0.874 n = 10 • 0.180

45.05 330.1 1.54 0.738 3.02 102.6 0.67 0.266

28.47 210.9 5.28 0.775 6.09 104.3 2.80 0.233

prophylact ical ly , the average haematocri t , measured 65 hours after the toxic dose, was 47.8 + 1.55. This difference has a significance o f p < 0.1%.

Measurements of the relative protein com- posit ion of the p lasma and its total protein content showed no differences between the two test groups.

Discuss ion These results - though they are at var iance

with those of FLUME et al. [21] - showed that intraperi toneal injections of a-amanit in can induce renal damage in rats. Physiologically, the lesion is character ized by serious depression of glomerular filtration rate, though, histologically, glomerular changes are hardly perceptible. Microscopic examinat ion suggests that urine is dammed back by the accumulat ion of necrotic material in the tubules, with a consequent rise in intra tubular counterpressure and, hence, a fall in effective filtration pressure. I t must, therefore, be concluded that, in rats, there is some reabsorp- tion of cr-amanitin in the renal tubules. The discrepancy between the results of FLUME et al. and those described in this communicat ion is

undoubtedly due to the fact that previous investigations were continued for only 24 hours after the toxic dose and that the dose of the poison used in the present work was higher by a factor of 10.

In contras t to the clinical picture of death- cut ingestion in human beings, there were no signs of gastroenteritis in rats given a-amanit in by the intraperi toneal route. This means that the rise in haematocr i t value, also found in man [4], must be solely due to restriction of water reabsorpt ion in the kidneys consequent on the renal damage. However, it is not certain whether the water loss, caused in this way, is large enough to support the hypothesis that the a-amanit in- induced renal lesions are nothing more than the secondary consequences of hypotension and hypovolaemia [31.

The renal damage induced by a-amanit in can be counteracted by silymarin, a phenomenon analogous to that previously demonst ra ted in a- amanit in-induced liver damage [20].

In molecular terms, the cause of the cell degeneration which follows c~-amanitin poisoning is to be found in inhibition of the enzyme polymerase II, situated in the cell nucleus. Inhibition of this enzyme leads to b reakdown of

On the Nephrotoxicity of a-Amanitin and the Antagonistic Effects of Silymarin in Rats 225

Table 1 (continued)

Urea PAH Relative Relative Relative Secretion clearance clearance reabsorption reabsorption reabsorption of PAH (ml/min x kg) (ml/min x kg) of H20 of Na + (% of of urea (/2g/rain x kg)

(% of GFR) filtered Na +) (% of GFR)

1.76 9.65 68.3 71.6 34.78 108.0 0.83 5.65 9.7 11.1 9.90 58.1

5.62 23.85 89.6 91.5 36.72 113.1 1.76 6.33 3.3 3.3 9.90 42.0

Table 2 (continued)

Urea PAH Relative Relative Relative Secretion clearance clearance reabsorption reabsorption reabsorption of PAH (ml/min x kg) (ml/min x kg) of H20 ofNa + (% of of urea (gg/min x kg)

(% of GFR) filtered Na +) (% of GFR)

1.505 5.61 41.45 46.31 16.394 98.08 0.628 2.71 17.85 20.29 14.211 41.29

3.066 15.30 79.76 81.19 39.71 97.91 1.383 4.83 8.08 9.55 9.79 21.82

the synthesis of messenger-RNA, and, as a result, the translation and, hence, further protein bio- synthesis become impossible [11-17]. This sug- gests that all cells having a high protein turnover should be particularly susceptible towards cr- amanitin. Administration of sublethal doses of c~- amanifin to rats was followed within seven days by massive hair loss. However, no other patho- logical phenomena were observed.

Although silymarin is a reliable antagonist of ~-amanifin-induced renal damage and hepatic damage, it failed to prevent this hair loss and seemed even to accentuate it. This fact appears to exclude any direct molecular interaction between a-amanitin and silymarin as the cause of the pharmacological activity of the latter, although certain results obtained by Sonnenbichler with isolated polymerase II seemed to point towards direct interaction (personal communication).

An alternative hypothesis is that silymarin and cc-amanitin compete for the same receptors on the cell membrane. One fact in favour of this hypothesis is that the effectiveness of silymarin in animals poisoned with ct-amanitin falls steeply as the time interval between administration of the

toxin and the antidote lengthens. This would imply that hepatocytes and renal tubule cells must possess identical receptor structures. The fact that silymarin does not counteract the a-amanitin- induced hair loss might be explained by its pharmacokinetic properties. Silymarin is excreted principally via the liver and to a lesser extent via the kidneys, so that in these organs the drug will reach higher concentrations than it does in the vicinity of the hair follicle cells [22, 23].

Acknowledgment This paper was presented in part on the 18th Spring

Meeting of the Deutsche Pharmakologische Gesellschaft, March 16-18, 1977, Mainz.

Received 5 September 1978.

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