Human and canine exposure to potentially toxic mushrooms is relatively common. In 2007, the American Association of Poison Control Centers (AAPCC) reported a total of ~ 7700 calls related to mushroom exposure. The difficulty for the clinician is that rapid and proper identification of ingested mushrooms occurs infrequently.
Human and canine exposure to potentially toxic mushrooms is relatively common. In 2007, the American Association of Poison Control Centers (AAPCC) reported a total of ~ 7700 calls related to mushroom exposure. The difficulty for the clinician is that rapid and proper identification of ingested mushrooms occurs infrequently. For example, a specific mushroom was not identified in ~ 84% of the calls to the AAPCC. Fortunately, in the majority of human cases, adverse effects are uncommon. No human fatalities were reported in 2007 and there were only 35 cases in which a major adverse effect occurred. Presumably, this is also true for animal exposures, although good data are lacking. While most mushroom ingestions are benign, some mushrooms contain hepatotoxic cyclopeptides that, when ingested, cause life-threatening effects. Worldwide most human fatalities following mushroom ingestion are associated with those containing hepatotoxic cyclopeptides.
There are ten groups of toxins that have been identified in mushrooms: the aforementioned cyclopeptides, gyromitrin, muscarine, coprine, ibotenic acid and muscimol, psilocybin, general GI irritants, orellinine, allenic norleucine and myotoxins. This discussion will focus on the cyclopeptide hepatotoxins.
Hepatotoxic, cyclopeptide-containing species include the Amanita spp. (~ 9 species), Galerina spp. (~ 9 species) and Lepiota spp. (up to 24 species). In North America, Amanita spp., especially A. phalloides, (death cap or death angel) are most commonly implicated in causing significant disease in humans, although in Eastern Europe, Galerina sulpices is considered to be the species most often associated with mortality. Data specific to animals is lacking. However, based upon a series of documented cases in our laboratory, Amanita spp. (A. phalloides and A. ocreata) were the most commonly involved in intoxications. Both species are common in California and are associated with Quercus agrifolia or coast liveoak, but are found in other regions of the U.S. The distribution of A. ocreata in North America is provided in Figure 1.
Fig. 1: Approximate U.S. distribution of A. ocreata.
Hepatotoxic mushrooms contain three groups of cyclopeptides that vary in toxicity. These include the bicyclic octapeptide amatoxins and phallotoxins and the monocyclic heptapeptide virotoxins. Phallotoxins and verotoxins are not believed to have significant oral toxicity. Thus, the amatoxins are responsible for causing cellular damage. Amatoxins include α-, β-, γ-, and ε-amanitins, amanin, amanullin and proamanullin (see Figure 2 for α-amanitin structure). α- and β-amatoxin are present in approximately equal concentrations and account for over 90% of the amatoxin content of the mushroom.
Fig. 2: Î±-amantin, a bicyclic octapeptide.
α- and β-amantins are the most toxic amatoxins, with LD50s in mice of 0.1 to 0.75 mg/kg and 0.2 to 0.4 mg/kg b.w., respectively. An oral LD50 in dogs of methyl-γ-amanitin has been estimated to be 0.5 mg/kg body weight. Approximately 1.5 to 2.5 mg of total amanitin is present in 1 g of dry A. phalloides. Thus, a 20 g mushroom contains a potentially lethal dose (0.1 mg/kg or greater) for a human or a 10 kg dog. Interestingly, rats are resistant to the toxic effects of amanitins.
Toxicokinetics of Amanitins
The bioavailability of amanitins appears to vary with species with decreasing bioavailability reported for humans, dogs and mice and rabbits. Following systemic absorption, it is believed that hepatocytes take up α-amanitin via a sodium dependent, bile acid transporter or via an organic anion-transporting polypeptide.
α-amanitin has a low volume of distribution, no known plasma protein binding or liver metabolism and high renal clearance. After oral ingestion of A. phalloides in humans, α- and β-amanitins were detected in plasma for up to 36 hours and in urine for up to 72 hours post-exposure. In contrast, the half-life of amantins is short (25 to 50 minutes) in dogs given amanitins IV; they were detectable in plasma for only 4 to 6 hours. Amanitins have been detected in human liver and kidney tissue for up to 22 days post- exposure, with the highest concentrations detected in kidney tissue.
Mechanism of Action
Amanitins irreversibly bind with eukaryotic, DNA-dependent RNA polymerase II and inhibit elongation essential to transcription. This results in decreased mRNA formation and subsequent protein synthesis. Protein synthesis inhibition results in cell death; cells with a high metabolic rate such as intestinal crypt cells, hepatocytes and proximal convoluted tubule cells are particularly susceptible. Amanitins also alter hormones that regulate glucose, calcium and thyroid homeostasis, resulting in widespread endocrine abnormalities. Insulin and C-peptide concentrations are elevated before hepatic and renal dysfunction. This suggests direct toxicity to pancreatic β cells and the release of preformed hormone or induction of hormone synthesis. Hypoglycemia is a common finding in intoxicated individuals. Serum calcitonin concentrations can be elevated as well resulting in hypocalcemia.
Classically, amanitin intoxication is characterized by four phases. Phase 1 is a latency period of approximately 8 to 12 hours, duration during which the person or animal appears normal. Phase 2 is characterized by severe GI signs including nausea, emesis, bloody diarrhea and severe abdominal pain. Phase 3 involves a period of apparent clinical improvement of several hours to several days. Phase 4, beginning approximately 36 to 80 hours post-exposure, is characterized by multi-organ failure including fulminant liver and acute renal failure, coma and death. Coagulopathy and encephalopathy are frequent complications of liver failure.
Interestingly, there is a relative paucity of detailed reports describing gross and histopathologic lesions associated with amanitin intoxication. As mentioned, fulminant liver and acute proximal tubular necrosis are classically described. In one confirmed case of amanitin intoxication in a dog, the liver was pale tan to yellow and slightly swollen grossly. The lungs were wet and exuded clear fluid from cut surfaces; numerous petechiae were noted in the lungs as well. Microscopically, there was panlobular, uniform coagulative necrosis of hepatocytes extending from the central veins to portal areas. Hepatic plates were collapsed and sinusoids were obliterated. The majority of hepatocytes contained multiple, small cytoplasmic vacuolations with a smaller number containing dark, eosinophilic, finely granular cytoplasm and pyknotic or karyorrhetic nuclei. Lung alveolar septa were expanded by eosinophilic, proteinaceous fluid; multiple foci of alveolar hemorrhage were noted. No renal lesions were described.
A number of analytical procedures have been developed to detect amanitins. ELISA assays hold promise for rapid detection of the toxins, although such tests are not widely available in clinical settings. In veterinary medicine, the confirmation of amanitin intoxication has historically been difficult in the absence of a history of ingestion of a mushroom and subsequent positive identification of the ingested mushroom as a species containing amanitin. A rapid LC-MS/MS/MS method was developed by our laboratory for the detection of α- and β-amanitin in serum, urine, liver and kidneys. The availability of a specific and sensitive analytical test has resulted in our ability to confirm amanitin intoxication in a number of animal cases (see below).
Amanitin can be detected in urine before the onset of clinical signs. Therefore, urine is the preferred specimen for antemortem testing. Unfortunately, urine amantin concentrations do not correlate with the severity of clinical signs and are not prognostic of case outcome. Kidney tissue is a preferred postmortem sample, since amanitins are found at higher concentrations and persist for longer periods in kidneys compared to liver. Interestingly, in one case involving the deaths of a bitch and her 3 week old puppy, amanitin was detected in a deparaffinized kidney tissue sample. Thus, retrospective assessment of exposure, in the absence of urine or fresh tissue samples, is possible.
No antidote is available for the treatment of amanitin-intoxication and, unfortunately, no specific therapeutic protocol has proven to be universally effective. The mortality rate in exposed people ranges from 20 to 40%. Early decontamination procedures such as induction of emesis or administration of activated charcoal (AC) are recommended if exposure is highly suspected and the patient presents within 1 to 2 hours of exposure. More delayed administration of AC might interrupt enterohepatic recycling of amanitins and therefore increase their clearance. In humans, specific therapies have included silymarin complex and silibinin (from Mediterranean milk thistle seeds), β-lactam antibiotics (primarily penicillin G) and N-acetylcysteine (NAC) along with general supportive measures. Silymarin and silibinin (one of the three bioactive flavonolignan isomers in silymarin) are believed to reduce amanitin uptake by hepatocytes. They also have free radical scavenging ability, inhibit cyclooxygenase and 5-lipooxygenase pathways of arachindonic acid metabolism, and activate of DNA-dependent RNA polymerase I. Clinical evidence supports the use of silymarin and silibinin in treating amanitin intoxication. β-lactam antibiotics are also believed to reduce amanitin uptake by hepatocytes, although their clinical efficacy is questionable.3 NAC, which is beneficial in preventing liver damage following acetaminophen overdose, appears to be as efficacious as silymarin or silibinin in reducing human mortalities. Liver transplantation is an option in human medicine.
Summary of CAHFS' Cases
A search of the CAHFS' database since the development of the amanitin assay in 2005 for cases positive for amanitin was conducted. A total of 32 cases were identified. All cases except three involved dogs; one case involved a human and two cases involved cats. Twenty of the 32 cases originated in California, two cases originated in Mississippi, and one case each originated in Virginia, Kentucky, Massacheusetts, Georgia and Ontario, Canada. Cases originating from California were primarily from central coastal and Sierra foothill counties where toxic Amanita spp. are commonly found. The majority of cases occurred between May and June, although fall and winter months were also represented. The largest percentage of affected dogs was less than one year of age (ranging from 3 weeks to 13 years). In many cases there was no known mushroom ingestion. In those cases in which a mushroom was known to have been ingested, clinical signs occurred as soon as 12 hours post-ingestion. The most common presenting signs were non-specific and included acute onset of lethargy, emesis and diarrhea. Consistent clinical pathologic changes included high ALT values (ranging from 542 to 20,213 U/L, hypoglycemia (as low as 19 mg/dl) and prolonged prothrombin and partial thromboplastin times. Fifteen of 21 dogs for which information was available died or were euthanized; six dogs recovered. The most consistent postmortem lesion in those dogs for which a necropsy was performed was panlobular hepatic necrosis, although significant gastrointestinal lesions were noted in a number of cases as well.
Acknowledgements: Drs. Birgit Puschner, Asheesh Tiwary and Motoko Mukai were toxicologists of record for many of the CAHFS' cases and deserve recognition for their contribution to the above summary.
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