Toxicity and Metabolism of Pyrrolizidine Alkaloids P. R. Cheeke J ANIM SCI 1988, 66:2343-2350.

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T O X I C I T Y A N D M E T A B O L I S M OF P Y R R O L I Z I D I N E
P. R. Cheeke 2 Oregon State University, Corvallis 97331
ABSTRACT

ALKALOIDS 1

Pyrrolizidine alkaloids (PA) are found mainly in plants of three families: Boraginaceae, Compositae and Leguminosae. In North America, PA poisoning of livestock is caused primarily by consumption of Senecio and Crotalaria spp. The PA of Senecio spp. cause irreversible hepatic damage; toxicity signs are a consequence of impaired liver function. Crotalaria intoxication leads to pulmonary damage as a primary effect; hepatic effects are less prominent. Large species differences exist in susceptibility to PA toxicosis. Small herbivores such as sheep, goats, rabbits, guinea pigs and other herbivorous laboratory animals are highly resistant to PA toxicity, associated with a low rate of hepatic production of reactive metabolites (pyrroles) and (or) a high rate of activity of detoxifying enzymes. Diester PA common to Heliotropium and Ecbium spp. are metabolized in the ovine tureen to 1-methyl metabolites, whereas the macrocyclic ester PA of Senecio spp. are not. Exposure to PA results in high concentrations of liver Cu, reduced liver Zn, and abnormal Fe metabolism with hematopoesis markedly impaired. Pyrrolizidine alkaloid toxicity alters vitamin A metabolism in rats, depressing plasma and liver levels of vitamin A. Synthetic antioxidants in the diet confer protective activity in laboratory animals (e.g., rats, mice) against PA toxicoses. The PA and their metabolites are secreted in the milk of lactating animals, but this probably does not represent a significant human health hazard. (Key Words: Pyrrolizidine Alkaloids, Senecio, Poisonous Plants, Hepatotoxins.)

I ntroduction

Alkaloids are compounds that contain N, usually in a heterocyclic ring, and are generally basic (alkali-like = alkaloid). They usually are bitter in taste, physiologically or pharmacologically active, and they function in the chemical defenses of plants against herbivory (Cheeke and Shull, 1985). Pyrrolizidine alkaloids (PA) constitute a large group of alkaloids containing the pyrrolizidine nucleus. Many of the PA are hepatotoxins, causing irreversible liver damage, and several PA are carcinogens. A comprehensive recent review on the structure, analysis, physical and chemical properties, metabolism, toxicity and carcinogenicity of PA

1Technical Paper No. 8343, Oregon Agric. Exp. Sta. Presented as an invited paper in a symposium on Poisonous Plants and Animal Toxicoses at the 79th Annu. Mtg. of the Am. Soc. of Anita. Sci., July 31, 1987, at Utah State Univ., Logan. a Dept. of Anita. Sci. Received October 2, 1987. Accepted March 14, 1988.

is available (Mattocks, 1986) and should be consulted for in-depth treatment of the subject. Plants containing PA are widespread throughout the world and responsible for extensive poisoning of humans and livestock. Most PA-conraining plants are found in three plant families, the Boraginaceae, Compositae and Leguminosae. Some of the more important toxic plants of these families are shown in Table 1. Ecbium plantagineum and HeIiotropium europaeum are the two most important plants in the family Boraginaceae that cause livestock toxicoses. These grow extensively in Australia and are responsible for poisoning of sheep (Seaman, 1985), cattle (Harper et al., 1985), horses (Giesecke, 1986), swine (Jones et al., 1981) and poultry (Pass et al., 1979). An interesting aspect o f the toxicity of these PA-containing plants, to be discussed in more detail later, is that chronic Cu poisoning, especially in sheep, is often the principal sign of toxicity. An outbreak of PA toxicity in humans in Afghanistan in 1974 was caused by contamination of wheat with Heliotropium seeds (Mohabbat et al., 1976). Over 1,600 people J. Anim. Sci. 66:2343-2350

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CHEEKE

TABLE 1. IMPORTANT POISONOUS PLANTS CONTAINING PYRROLIZIDINE ALKALOIDS Botanical name (Genus, species) Family Boraginaceae Amsinckia intermedia Borago officinalis Cynoglossum officinale Ecbium lycopsis (plantagineum )
Ecbium vulgate Heliotropium europaeurn Sympbytum officinale Family Compositae Senecio alpinus S. brasiliensis S. cineraria S. glabellus S. integerrimus S. jacobaea S. Iongilobus S. riddellii S. vulgaris Family Leguminosae Crotalaria retusa C. spectabilis

Common name (s)

tatweed borage hound's tongue echium, Paterson's curse, Salvation Jane echium heliotrope comfrey

Dusty Miller tansy ragwort common groundsel erotalaria erotalaria, rattle pod

were poisoned, with many fatalities. Another important member of the Boraginaceae is comfrey, a plant commonly grown as a forage and a medicinal herb. Comfrey contains at least eight hepatotoxic PA (Culvenor et al., 1980), some of which are known to be carcinogenic. Hirono et al. (1978) demonstrated that the leaves and roots of comfrey are carcinogenic to rats. Recently, PA-toxicity in humans has been attributed to consumption of comfrey tea (Ridker et al., 1985). The widespread use of comfrey in so-caUed "health food" products indicates extensive human exposure to hepatotoxic and carcinogenic PA, warranting full assessment of the hazards and risks associated with the consumption of this plant. Another herb, borage, also contains PA (Larson et al., 1984). In North America, the main Boraginaceae species of interest in livestock poisoning is Cynoglossum officinale (Knight et al., 1984). A number of toxic Senecio spp. (Table 1) are important poisonous plants. In North America, S. jacobaea (tansy ragwort) is of greatest concern, being widespread in the temperate west coastal areas and eastern Canada. S. vulgaris (common groundsel) also is important in some areas. S. jacobaea occurs in temperate areas worldwide and is significant in Australia, New Zealand, South America, Africa, western Europe and North America. In South

America, S. brasiliensis is a problem in Brazil and Argentina (Tokarnia and Dobereiner, 1984). Many Senecio species are important in Africa, with over 250 (many of which contain PA) in South Africa alone (Vahrmeijer, 1981). Two Crotalaria species in the family Leguminosae are toxic (Table 1). In contrast to the other plants mentioned, for which concern is mainly with the toxicity of the foilage, the seeds of crotalaria are the primary cause of poisoning. Crotalaria is a common weed of grain fields; Crotalaria seed contamination of grain and grain screenings has resulted in livestock and poultry poisonings in the U.S. (Burguera et aI., 1983) and Australia (Hooper, 1978). Crotalaria seed contamination of millet grain caused epidemics of human mortality in India in the early 1970s (Mattocks, 1986). Chemical Structure o f Pyrrolizidine Alkaloids. Most of the hepatoxic PA are esters of the necine bases retronecine and heliotridine, which are diastereomers with opposite configurations at C7. Their toxicity is influenced by structure. For hepatotoxicity, there must be a 1,2-double bond and a branch in an esterified side chain. Cyclic diesters are the most toxic, noncyclic diesters are of intermediate toxicity, and monoesters are the least toxic. Representative structures are shown in Figure 1. Further information on structural relationships to

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PYRROLIZlDINE ALKALOID TOXICITY AND METABOLISM

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?"

(a)

Retrorsine

"F, .CH=
CH _~;_ ~I~ t ~ 9 ,,.- ~ "~ M CH~ " -O H 0 "C ~ I CI.~-O-C-C-CH-CH 2 I I 3

CT.)

.o oc..

(b)

Lasiocarpine

.,cc.

.o,
(c)
Hellotrine

0 C~ HOCH3

Figure 1. Representative examples of toxic pyrrolizidine alkaloids: (1) cyclic diesters, e.g., retrorsine; (b) noncyclic diesters, e.g., lasiocarpine; (c) monoesters, e.g., heliotrine.

toxicity can be obtained from the book by Mattocks (1986). Some non-hepatotoxic PA, lacking the 1,2-double bond, occur in tall rescue and have been implicated in a number of pathologies associated with tall rescue toxicity, including fescue foot, summer fescue toxicosis and fat necrosis. Examples include loline, N-acetyl loline and N-formyl loline. Their presence in fescue is a result of infection with an endophytic fungus (Epicbloe typbina); they may be phytoalexins produced in response to the fungal infection (Lyons et al., 1986).

whereby the side chain(s) are cleaved from the necine nucleus, is a detoxification reaction, because neither the necine nor necic acid moities produced are toxic. Although hydrolysis of synthetic PA has been well studied (Mattocks, 1986) there is little evidence that this is an important pathway for plant PA. Dehydrogenation of PA to yield pyrrole derivatives is the classic pathway of PA metabolism (Mattocks, 1986). Pyrroles are very reactive and are strong alkylating agents; they cross-link strands of DNA and thus impair cell division and protein synthesis. The signs of toxicity are a reflection of these effects. The first lesions are swollen hepatocytes followed by frank enlargement of the hepatocytes and their nuclei, giving the classic histologic signs of megalocytosis and karyomegaly of cells of the centrilobular region. Karyomegaly is the result of the antimiotic effect, whereas megalocytosis is due to an interaction between antimitotic effects and the stimulus for regeneration. Other lesions include progressive fibrosis, bile duct proliferation, veno-occlusion and loss of hepatic metabolic functions 9because of impaired protein synthesis. Levels of serum proteins are depressed because of impaired hepatic protein synthesis (Miranda et al., 1980), leading to ascites and edema (Cheeke and Garman, 1974). The liver becomes small, hard and fibrotic. Liver function tests, such as dye clearance, show marked impairment (Garrett et al., 1984; Cheeke et al., 1985). Secondary photosensitization may be evident. Death occurs from liver failure. Segall et al. (1985) have provided evidence of additional important metabolites. These workers suggested that trans-4-hydroxy-2-hexenal (t-4HH) may be the ultimate metabolite. This is a reactive aldehyde that causes lipid peroxidation. The involvement of t-4HH could explain the protective effects of antioxidants against PA toxicosis.

Pyrrolizidine Alkaloid Metabolisrm--Signs of Toxicosis. Pyrrolizidine alkaloid toxicity results

in irreversible liver damage. Ingested PA are absorbed and metabolized in the parenchymal cells (hepatocytes) of the liver, especially the cells of Zone 3 (centrilobular region) that have the highest activity of drug-metabolizing enzymes. There are several pathways of PA metabolism, discussed in detail by Mattocks (1986). These include hydrolysis, dehydration, N-oxidation and minor pathways such as hydroxylation and epoxidation. Hydrolysis,

Species Differences in Pyrrolizidine Alkaloid Toxicity and Metabolism. A fascinating aspect

of PA toxicity is the great variation in susceptibility among animal species. For cattle (Cheeke et al., 1985) and horses (Garrett et al., 1984), intakes (DM basis) of tansy ragwort of only about 5% of BW are a lethal dose, whereas for sheep (Swick et al., 1983a,b; White et al., 1984) and goats (Goeger et al., 1982a), cumulative intakes of several hundred percent of body weight are required. Among laboratory animals, rats and mice are highly susceptible, whereas a

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CHEEKE TABLE 2. COMPARATIVE RATES OF IN VITRO PYRROLE PRODUCTION (NMOL 9MIN -1 o MG LIVER -1 (WET WT) FROMECH1UM PLANTAGINEUM PYRROLIZIDINE ALKALOID BY RAT AND SHEEP LIVER MICROSOMAL PREPARATIONS a Alkaloid Mixed echium PA .27 (.29 to .24) .05 (.05 to .04)

Species Rats Sheep

Number 6 7

Lasiocarpine 1.00 (1.07 to .93) .13 (.15 to .11)

Heliotrine .15 (.17 to .13) .04 (.04 to .03)

aFrom Peterson and Jago, 1984.

number of small herbivores, such as rabbits (Pierson et al., 1977), guinea pigs (Swick et al., 1982b), gerbils and hamsters (Cheeke and Pierson-Goeger, 1983) are highly resistant. Chickens and turkeys are susceptible to PA toxicosis (Cheeke and Pierson-Goeger, 1983), but Japanese quail can tolerate a tansy ragwort cumulative intake of several thousand percent of b o d y weight (Buckmaster et al., 1977). These species differences in resistance and susceptibility appear to be primarily a consequence of differences in hepatic PA metabolism. Using hepatic microsomes prepared from several species, Shull et al. (1976) found that the rate of in vitro pyrrole production correlated with susceptibility to PA toxicosis. Susceptible species such as rats, cattle and horses had a high rate of pyrrole production, whereas PA-resistant animals such as sheep and Japanese quail had low pyrrole production rates. Peterson and Jago (1984) observed similar results comparing sheep and rats. Rats fed a diet containing echium developed classical signs of PA toxicosis, but sheep fed the same material for an extensive period showed little or no pathology. The rate of in vitro pyrrole production from the echium PA was much higher with rat than with sheep tissue (Table 2). In some cases, as for example with rabbits, the in vitro pyrrole production rate is high (Shull et al., 1976) even though the animals are resistant to PA toxicosis (Pierson et al., 1977). A tenable explanation is that in such cases high activity of detoxifying enzymes exists. For example, Swick et al. (1983b) suggested that part of the resistance of sheep to PA toxicity may be because of their high activity of enzymes such as glutathione-S-transferase and epoxide hydrolase, which are involved in PA excretion. A complete understanding of the mechanisms

of resistance will require information on the specific pathways of PA metabolism, which may vary among species.

Ruminal Metabolism of Pyrrolizidine Alkaloids. Although hepatic metabolism probably

explains the majority of species differences in the susceptibility to PA, ruminal metabolism of PA may be relevant. Monoester PA found in echium and heliotrope is metabolized in the sheep rumen. Lanigan (1970) demonstrated in vitro PA metabolism in sheep rumen fluid, resulting in the conversion of open ester PA to 1-methyl derivaties (Figure 2). Subsequent work resulted in the isolation of a rumen

OH :

CHocHs . ~ I CHz-O-C-C-CH-CH3
.0.

Heliotrine ~n)e o

OH

I - methylene derivative

l-methyl derivative
Figure 2. Metabolism of heliotrine in the sheep l u men.

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PYRROLIZIDINE A L K A L O I D TOXICITY AND METABOLISM T A B L E 3. METABOLIC CONVERSION OF ECHIUM P Y R R O L I Z I D I N E A L K A L O I D S INTO 1-METHYLENE DERIVATIVES IN OVINE R U M E N F L U I D IN VITROa, b Alkaloid

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Group
Before echium feedingC Echium diet Control After 12 wk echium feeding Echium diet Control

Uplandicine

Echimidine

Echiumine

Heliotrine

Lasiocarpine
4 0 89 0

21 10 100 0

30 4 100 0

33 0 100 0

14 6 100 0

aFrom Culvenor et al. (1984). bvalues are % of PA metabolized in 24 h.

C Twelve weeks after a previous period o f Ecbiura feeding.

microbe, Peptococcus beliotrinreducans, that accomplished this conversion (Lanigan, 1976). Culvenor et al. (1983) observed that echium alkaloids were rapidly metabolized to 1-methylene derivaties in sheep rumen fluid (Table 3); the conversion was greater in inocula obtained from sheep that had been fed echium for 12 wk, suggesting an adaptation process. The Australian workers (Lanigan, 1970, 1976; Culvenor et al., 1984) suggest that the metabolism of PA in the sheep rumen may explain the resistance of sheep to PA toxicity. Unfortunately, comparative studies with cattle and sheep have not been conducted to determine if PA indeed are detoxified in the sheep but not bovine rumen. Detoxification of the closed ester PA of Senecio spp. does not seem to occur in the rumen. Shull et al. (1976) found that in vitro incubation of tansy ragwort did not alter its toxicity to rats. This finding was confirmed by Swick et al. (1983a), who further noted that 1-methyl derivatives of Senecio PA could not be detected in sheep rumen fluid.They suggested that because of steric hindrance the closed ester PA are less likely to be hydrolyzed and methylated than are the open ester PA.

Interactions of Pyrrolizidine Alkaloids with Mineral and Vitamin Metabolism. In Australia,

sheep consuming echium and helitrope often develop chronic Cu toxicosis (Bull et al., 1956; St. George-Granbauer and Rac, 1962). Exposure to PA apparently increases the avidity of liver cells for Cu; liver Cu concentrations increase to high levels, followed by the hemolytic crisis of Cu toxicosis. Mild hepatic damage seems to be a predisposing factor. The PA-

induced chronic Cu toxicity may be analagous to secondary photosensitization; both conditions are a consequence of impaired liver function. Culvenor et al. (1984) reported that Merino sheep fed echium for a prolonged period did not develop liver pathology, and liver Cu levels were not elevated, indicating that liver damage may be a prerequisite factor. However, Swick et al. (1983b) and White et al. (1984) observed normal liver Cu in lambs that died of hepatoxic effects of tansy ragwort consumption. Therefore, the conditions that lead to secondary Cu toxicosis in PA-exposed sheep are not entirely clear. In rats, Swick et al. (1982c) found that dietary Cu concentration had a marked effect on PA-induced liver Cu accumulation. Pyrrolizidine alkaloid toxicosis impairs or saturates normal subcellular Cu excretory mechanisms and may involve a lysosomal defect (Swick et al., 1982b). Besides causing elevations in liver Cu, PA toxicosis also results in reduced liver Zn (Swick et al., 1982a,c). Pronounced effects on Fe metabolism also are noted. Swick et al. (1982c) observed that in chronic PA toxicosis in rats, erythropoesis is almost totally impaired, anemia develops, and the spleen is greatly enlarged. Spleen enlargement is a result of storage of Fe that cannot be utilized because of impaired erythroycte formation (Swick et al., 1982c), which, in turn, is likely a result of impaired hepatic protein synthesis. Moghaddam and Cheeke (unpublished data) examined the effect of PA toxicosis in rats on vitamin A metabolism. Rats fed tansy ragwort had markedly lower (40 to 50%) liver and plasma vitamin A levels than control afiimals.

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CHEEKE meat, milk and eggs produced by animals exposed to dietary PA. Because the PA are metabolized in the liver, this is the major organ in which PA residues would be expected. The liver, and probably the carcass, of an animal with chronic PA hepatotoxicosis would be condemned because of the gross lesions. The level of PA or pyrrole bound to liver tissue decreases rapidly after PA administration (Mattocks, 1986) to undetectable levels. The tissue damage, but not the PA content, is cumulative. Therefore, in m y judgement,. PA residues in meat are unlikely to represent a hazard to human health. Alkaloids are excreted in the milk of cows and goats fed a PA source. Goeger et al. (1982a,b) fed a diet containing 25% tansy ragwort to dairy goats. A concentration of .33 to .81 ppm Senecio PA was detected in milk (Deinzer et al., 1982). Rats fed the milk for 180 d, with a calculated PA intake of .96 mg/rat, had swollen hepatocytes of centrilobular distribution, and biliary hyperplasia indicative of mild PA toxicosis (Goeger et al., 1982b). The results indicate that PA are transferred in goats' milk and may produce hepatotoxic effects in animals fed the milk. However, the hazard to human health, with the possible exception of instances in which a family goat might be grazed on tansy ragwort, probably is very slight. Johnson (1976) also demonstrated the secretion of PA in milk of cows fed a high dose of tansy ragwort, but concluded the health hazard was slight. Luthy et al. (1983) administered labeled Senecio alkaloid to lactating rats and found that only .08% of the radioactivity was excreted in the milk, and 70 to 80% of this was in the form of water-soluble metabolites. Similarly, Eastman et al. (1982) found that only a very small (.04%) amount of a dose of labeled Senecio PA was recovered in the milk of lactating mice; 84% of the radioactivity was associated with water-soluble metabolites.

Because of the reduced bile excretion in PA toxicosis, a possible mechanism for this effect would be impaired vitamin A absorption because of lack of bile excretion. Low plasma vitamin A could reflect impaired hepatic synthesis of retinol-binding protein. Additionally, we noted a susceptibility to in vitro hemolysis of erythrocytes of PA-fed rats, suggesting a possible vitamin E deficiency, which also could result from impaired bile excretion. This could be particularly important in the pathogenesis of PA toxicosis, because Segall et al..(1985) have reported that the ultimate metabolites of PA metabolism cause lipid peroxidation at the subcellular level. It is obvious that PA toxicosis causes pronounced alterations in mineral and vitamin metabolism, reflecting the importance of normal liver function in the metabolism of many nutrients.

Dietary Protective Agents Against Pyrrolizidine Alkaloid Toxicosis. Because many animals
are resistant to PA toxicosis, the possibility exists that the metabolism of susceptible species might be modified by dietary means to increase their resistance. Both liver and rumen metabolic processes can be altered by diet. Various inducers and inhibitors of liver drug metabolizing enzymes are known; mQdification of PA metabolism to minimize pyrrole production and maximize conjugation and excretion certainly is possible theoretically. Partial .success in protecting laboratory animals from PA toxicosis has been achieved. Buckmaster et al. (1976) found that feeding 1% cysteine to rats had a pronounced protective effect against tansy ragwort toxicity, increasing the survival time from 88 to 187 d, with an average tansy ragwort intake of 54.7 and 161.4 g per rat, respectively. Cysteine could be acting as a glutathione precursor, increasing glutathione conjugation of PA. Synthetic antioxidants such as ethoxyquin and butylated hydroxyanisole (BHA) increase glutathione-S-transferase activity, which catalyzes glutathione conjugation. In a series of studies with rats and mice (Miranda et al., 1981a,b; Garrett and Cheeke, 1984), synthetic antioxidants were shown to be protective against PA activity. However, in tests with ponies (Garrett et al., 1984) and beef cattle (Cheeke et al., 1985), a dietary additive containing sulfur amino acids and synthetic antioxidants was not effective in protecting against tansy ragwort toxicity.

Conclusions

Pyrrolizidine Alkaloids in Animal Products.

Little work has been reported on PA residues in

Pyrrolizidine alkaloids are of significance in both human and animal health. There are marked differences among animal species in the susceptibility to PA toxicosis, reflecting variations in hepatic and ruminal PA metabolism. A full understanding of these mechanisms should increase the likelihood of successful development of dietary additives to protect susceptible livestock (cattle and horses) from PA toxicity. The PA have pronounced effects on mineral

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PYRROLIZIDINE ALKALOID TOXICITY AND METABOLISM a n d vitamin m e t a b o l i s m , w i t h practical signific a n c e in d e v e l o p m e n t o f s e c o n d a r y Cu t o x i c o s i s a n d p o s s i b l y in h y p o v i t a m i n o s i s A and E.

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