T RAINING

Pyrrolizidine alkaloids are toxic secondary metabolites of a wide variety of plants found in various environments throughout the world, from the colder temperate climates to sparsely vegetated hot dry regions. Plants containing pyrrolizidine alkaloids have been responsible for numerous outbreaks of poisoning of livestock on several continents and continue to cause serious economic damage. Although most of the respective pasture-contaminating plants are unpalatable to grazing animals, they may be foraged in times of food shortage or ingested via contaminated silage. In recent years, pyrrolizidine alkaloids have been identified as causing human deaths in less developed countries as a result of contamination of cereal crops and harvested seed, and they have been suspected of causing illness following intentional ingestion as vegetables and in the form of herbal remedies. Several authors have described specific aspects of pyrrolizidine chemistry and toxicity or have given overviews: some of the more comprehensive reviews have been presented by Bull et al. (1968), Peterson and Culvenor (1983), Mattocks (1986), World Health Organisation (1988), Rizk (1991b), Stegelmeier et al.

(1999), and Fu et al. (2001).

The pyrrolizidine structure is based upon two fused five-membered rings that share a bridgehead nitrogen atom, forming a tertiary alkaloid. The nitrogen atom is very often present as the oxide. In nature the rings are most frequently substituted with a hydroxymethylene group at position C-1 and a simple hydroxyl group at position C-7, forming a structure known as a necine base. The bases most commonly encountered are heliotridine, retronecine, supinidine, and otonecine; the structures of which are shown in Fig. 2.1.

N O OH H

C7

Heliotridine

N O OH H

C7

Retronecine

N OH H

Supinidine

O OH H

N O

CH₃ Otonecine Fig. 2.1 The major bases of the pyrrolizidine alkaloids.

N

The necine base alcohols are normally esterified with any of a series of characteristic (necic) acids to form pyrrolizidine alkaloids. The esterification may be in the form of C-1 mono-esters, open-chain diesters or, more frequently, a macrocyclic diester. The substituting acids are mostly highly branched chains of five to ten carbon atoms substituted with methyl, methylene, hydroxyl and/or keto groups, producing several relatively complex alkaloids.

Summaries of the structures of many of the pyrrolizidine alkaloids, especially those most commonly associated with human toxicity, have been assembled by Rizk (1991b), Hartmann and Witte (1995), and Roeder (1995). Over 350 pyrrolizidine alkaloids have so far been identified and characterized, not including the N-oxide forms. Examples of some important representatives are shown in Fig. 2.2.

2.3 OCCURRENCE

Pyrrolizidine alkaloids are found mainly in the families Compositae (Asteraceae), Borag-inacea, and Leguminosae, but also in Apocyanacae, Ranunculacae, and Scrophulariacae.

The genera Senecio, Eupatorium, Symphytum, Cynoglossum, Heliotropium, and Crotalaria contain the species most frequently associated with human illness. These genera are widely distributed throughout different climates and their pyrrolizidine alkaloid-containing species could comprise as much as 3% of the world’s flowering plants (Smith and Culvenor, 1981).

Comprehensive lists of plants containing unsaturated pyrrolizidine alkaloids have been pub-lished (Smith and Culvenor, 1981; Mattocks, 1986; Rizk, 1991b; Hartmann and Witte, 1995).

Each species contains an unusually characteristic range of pyrrolizidine alkaloids and a spe-cific ratio of free base to N-oxide (Molyneux and James, 1990). Some species may contain essentially only a single pyrrolizidine alkaloid, notably riddelliine in Senecio riddelli, but most contain over five.

Alkaloid content varies between species, plant organ, site, and season, and can be up to several percent of the plant’s dry weight. In general, levels are considerably higher in roots than in leaves and they are higher in buds, inflorescences and young leaves than in older leaves, and lower still in stems. In some species, notably Crotalaria, high levels (up to 5%

dry weight) are often found in the seed (Johnson and Molyneux, 1984; Johnson et al., 1985), presenting a serious threat to health in cases of contamination of grain intended for human consumption. Several necine bases lack the 1,2-unsaturation and their alkaloids are relatively nontoxic. These saturated pyrrolizidine alkaloids may be found associated with their toxic counterparts in particular species, but they are much less widespread.

2.3.1 Formation and function

The biosynthetic routes of some of the major pyrrolizidine alkaloids have been studied in detail (Hartmann and Ober, 2000). In the biosynthesis of the base retronecine, depending on plant species, eitherL-ornithine orL-arginine or both are combined to form two molecules of putrescine, and the biosynthesis proceeds via homospermidine.

The necic acids are synthesized from the α-amino acidsL-valine,L-leucine,L-isoleucine, andL-threonine by a route compiled by Roeder (1995). These reactions occur in the roots of the plant where the primary product in most species studied is the N-oxide (Hartmann and Toppel, 1987; van Dam et al., 1994). The oxide is much more water-soluble than the base and can be transported within the plant. The mechanism and purpose of pyrrolizidine alkaloid transport are not yet well understood, but the alkaloids are specifically channeled via the phloem to the younger leaves and flowering parts of the plant, where they accumu-late. The purpose of this is possibly to provide important tissues with a deterrent towards herbivores. Many grazing animals avoid eating plants that contain pyrrolizidine alkaloids, although sheep and goats have some tolerance. Some insects, particularly certain moths and butterflies, accumulate the toxins, which they can then use as a defense against predators or as intermediates in the production of substituted carbonyl pyrrolizidines that act as pheromones.

Concise and informative reviews have been published describing the varied functions and uses of pyrrolizidine alkaloids by insects (Boppr´e, 1990; Hartmann and Witte, 1995).

2.4 EXPOSURE

The two most significant sources of exposure of humans to pyrrolizidine alkaloids are the acci-dental contamination of foodstuffs and the intentional ingestion of plants containing the alka-loids in the form of culinary vegetables or herbal medicines. The incidence of pyrrolizidine poisoning of humans has probably been underestimated owing to the lack of association between plants and disease, poor recognition of chronic effects, and the time lag between in-gestion and the appearance of symptoms in subacute poisoning (Roitman, 1983). Many plants that contain pyrrolizidine alkaloids are deliberately consumed as food or herbal remedies in all parts of the world and reports of toxins in materials are increasing in number (Mattocks, 1986; Hirono, 1993; Roeder, 1995, 2000; Bertram et al., 2001; Fu et al., 2002b).

2.4.1 Contamination of foods

Direct accidental contamination of grain with seed from pyrrolizidine alkaloid-containing plants occasionally leads to a major incident. The plants usually responsible are Heliotropium lasiocarpum, H. popovii and H. europaeum which grow well with wheat (Prakash et al., 1999).

In 1976 in Afghanistan over 20% of a population of 7200 villagers who had consumed wheat contaminated with the seed of Heliotropium showed signs of liver disease (Mohabbat et al., 1976). The wheat had been consumed over a period of 2 years with an estimated minimum intake of 1.5 g of the alkaloid in the form of the N-oxide. In the previous year, contamination of local grain heavily contaminated with seeds of Crotalaria species was responsible for the death of 28 patients from 67 people affected in four villages in India (Tandon et al., 1976).

A more recent outbreak (1992–1993) was precipitated by a famine and a delay in the wheat harvest which allowed growth of Heliotropium within the wheat crop. It has been documented in some detail (Chauvin et al., 1994).

The use of plants containing pyrrolizidine alkaloids as foods is limited mostly to Japan where Petasites, Tussilago, Symphytum, and Farfugium are consumed (Hirono, 1993); how-ever, there is a paucity of information on the exposure to the alkaloids from these sources.

Contamination of foods on a smaller scale occurs where there is an intermediate agent between the plant source and the foodstuff. Examples of this are the transfer of pyrrolizidine alkaloids from plants into milk by herbivores, into eggs via chickens, and into honey by bees.

Contamination of milk and its effects on suckling animals has been reviewed by Panter and James (1990), most studies not showing serious effects on the offspring. The proportion of pyrrolizidine alkaloids passed into milk from goats fed Senecio jacobaea (ragwort) was determined to be about 0.1% of that ingested (Deinzer et al., 1982); however, there is evidence that water-soluble metabolites (Eastman et al., 1982) and pyrrolizidine N-oxides (Candrian et al., 1991) are present. With the pooling of milk samples being widespread practice, any risk to health is probably confined to the consumption of milk from individual animals and particularly species such as goats, which are comparatively willing to eat pyrrolizidine-containing plants (Molyneux and James, 1990).

In a report of contamination in hens’ eggs, levels of a number of pyrrolizidines in eggs laid by hens accidentally poisoned with seeds of Heliotropum and other pyrrolizidine-containing plants reached about 40 μg per egg (Edgar and Smith, 2000).

Relatively high concentrations (up to 4 mg/kg) of pyrrolizidines have been measured in honey produced from S. jacobaea (Deinzer and Thompson, 1977; Crews et al., 1997) and Echium plantagineum (Culvenor et al., 1981). Pioneering studies into this problem have been carried out in Australia at the Commonwealth Scientific and Industrial Research Organisa-tion (CSIRO) where the occurrence of a range pyrrolizidine alkaloids from Heliotropium and Echium have recently been confirmed in Australian honeys at levels of about 2 mg/kg (Beales et al., 2004; Betteridge et al., 2005). In cases where honey is contaminated, the plant respon-sible is usually the predominant nectar source, during its flowering season, in the locality of the hives (Fig. 2.3). There is therefore considerable potential for the localized contamination of honey. Transfer has been associated with the nectar but pollen, which is actively collected by bees and is a constituent of honey, has also been shown to be a major site of pyrrolizidine alkaloid storage in the plant (Boppr´e et al., 2005). Honeys produced in study hives situated adjacent to sites in the UK where Senecio jacobaea flowered were found to contain Senecio pollen, and alkaloids at concentrations ranging from 0.01 to 0.06 mg/kg (Crews et al., 1997).

Human exposure to pyrrolizidine alkaloids from contaminated honey is considered to be a problem and has been reviewed in detail (Edgar et al., 2002). A wide range of plants containing pyrrolizidine alkaloids make a significant contribution to honey production

Fig. 2.3 Wild plants of Echium vulgare in New Zealand. A rich monofloral source of nectar and pyrrolizidine alkaloids. Photo courtesy of Barrie Wills. For a color version of this figure, please see Plate 1 of the color plate section that falls between pages 224 and 225. (Reprinted with permission from Bet-teridge et al. in Journal of Agricultural and Food Chemistry, 53, 1894–1902, Figure 4. Copyright 2005 with permission from the American Chemical Society.)

world-wide. Europeans eat an average of about 1 g of honey per capita per day but some consumers, including infants in the UK (Ministry of Agriculture, Fisheries and Food, 1995), and in Australia (Edgar et al., 2002), can consume far more than this.

Another potential dietary source of pyrrolizidine alkaloids is oil obtained from borage seed, Borago officinalis, which is popular in Europe on account of its high content of the beneficial gamma-linolenic acid. However, the pyrrolizidine content of this plant is very low (Larson et al., 1984; L¨uthy et al., 1984) and that of its oil even lower (Wretensjo and Karlberg, 2003). The European borage should not be confused with the various and more toxic colloquially named borages derived from Echium species found in New Zealand.

Numerous reports of poisonings directly related to herbal teas and similar products have been published (Weston et al., 1981; Kumana et al., 1985; Margalith et al., 1985; Ridker et al., 1985; Culvenor et al., 1986; Ridker and McDermott, 1989). The practice in Jamaica of brewing teas from uncultivated plants for medicinal purposes (bush teas) has been asso-ciated with epidemics of pyrrolizidine poisoning (Bras et al., 1954). However, government campaigns there have led to a reduction in the frequency of these incidents.

2.4.2 Pyrrolizidines in herbal preparations

Increasing interest in “alternative” therapies and herbal medicines in Europe and the USA has led to preparations of pyrrolizidine alkaloid-containing plants being made widely

Fig. 2.4 Herbal products sold in the UK derived from pyrrolizidine-containing plants.

available commercially and publicized for their health-giving properties. Comfrey (Sym-phytum officinale), in particular, has long been a popular herb in Europe and the USA.

Preparations of comfrey in the form of dried leaves, dried root, and root powder tablets and capsules, often mixed with other herbs, have been sold with active promotion of the plant’s supposed healing and digestive properties. Some examples of herbal products sold in the UK which are derived from plants likely to contain pyrrolizidine alkaloids are shown in Fig. 2.4.

They include tinctures and flowers from coltsfoot (Tussilago) and teas, leaf, and root material from comfrey, with the latter bearing a suitable health warning.

Herbal preparations of Symphytum, Tussilago, Borago, and Eupatorium, in the form of leaf, root powders, tablets and root extract tinctures, sold in the UK in 1994 were surveyed for pyrrolizidine alkaloid content (Ministry of Agriculture, Fisheries and Food, 1994). Com-frey (Symphytum) tablets contained up to 5000 mg/kg and root powders up to 8300 mg/kg of pyrrolizidine alkaloids, giving estimated potential intakes in excess of 35 mg/day. Com-frey and borage (Borago) leaf preparations intended for consumption as teas contained less than 100 mg/kg total pyrrolizidine alkaloids. About 50% of the total acetyllycopsamine and symphytine but only about 5% of the lycopsamine were extracted into the water on brew-ing comfrey leaf teas, possibly due to bindbrew-ing of the more polar lycopsamine to the plant tissue.

A survey of comfrey leaf and root products sold in the USA in 1989 showed them to contain up to 1200 mg/kg of pyrrolizidine alkaloids (Betz et al., 1994). Teas prepared from comfrey root and leaf showed preferential extraction of acetyllycopsamine and acetylintermedine

over lycopsamine and intermedine. Later studies confirmed that pyrrolizidine alkaloids were present in many comfrey preparations sold in the USA (Altamirano et al., 2005). In one case where the N-oxides were reduced prior to determination the level of symphytine measured increased from 0.1 to 1 mg/L (Oberlies et al., 2004).

More recently, political changes in China have made aspects of Chinese culture more accessible to the West and this has raised the popularity of traditional Chinese remedies.

This activity has been followed by studies of the composition of such medicines which has revealed that of the wide range of herbal plants used in China, about 50 have so far been identified as containing toxic pyrrolizidine alkaloids (Zhao et al., 1989; Roeder, 2000; Fu et al., 2002b).

The misidentification of plants is an additional risk to consumers of vegetables and herbs.

This is particularly likely to occur where the intended plant is closely related to a more toxic species. For example, Sperl et al. (1995) reported a poisoning case in which Adenostyles had been gathered and consumed in place of the less toxic Tussilago. Similar and additional problems of herbal products have been described by Huxtable (1990a).

2.5 REGULATIONS

Regulations and recommendations have been introduced in some countries in attempts to limit human exposure. Regulations introduced in Germany by the Federal Health Bureau (Germany Federal Health Bureau, 1992) limit the tolerable pyrrolizidines in herbal medicines to levels providing less than 1 μg per day orally based on an assessment of genotoxic carcinogenicity, reduced to 0.1 μg per day when used for over 6 weeks. The regulations are notable for the fact that the limits can easily be exceeded by consumption of contaminated foods such as eggs and honey, nevertheless similar limits are likely to be adopted across Europe (Roeder, 2000). Food Standards Australia New Zealand (FSANZ) has set a provisional exposure level of 1 μg/kg body weight per day and advised heavy consumers not to eat honey from Echium plantagineum every day (FSANZ, 2004). Herbal preparations containing comfrey root were removed voluntarily from the market in the UK (Ministry of Agriculture, Fisheries and Food, 1994) and in the USA (FDA).