Biochemistry of hemlock (Conium maculatum L.) alkaloids and their acute and chronic toxicity in livestock. A review

PERGAMON

Toxicon 37 (1999) 841±865

Biochemistry of hemlock (Conium maculatum L.) alkaloids and their acute and chronic toxicity in livestock. A review T.A. LoÂpez a, *, M.S. Cid b, c, M.L. Bianchini d a

Laboratorio de ToxicologõÂa Veterinaria, EstacioÂn Experimental Agropecuaria Balcarce (INTA), C.C. 276, 7620 Balcarce, Buenos Aires, Argentina b Grupo de Interacciones Planta-Animal, Facultad de Ciencias Agrarias, Universidad Nacional de Mar del Plata, C.C. 276, 7620 Balcarce, Buenos Aires, Argentina c Consejo Nacional de Investigaciones Cientõ®cas y TeÂcnicas, Rivadavia 1917, 1033 Buenos Aires, Argentina d CaÂtedra de Fisico-QuõÂmica, Universidad Nacional de Mar del Plata, C.C. 276, 7620 Balcarce, Buenos Aires, Argentina

Received 15 July 1998; accepted 18 August 1998

Abstract The literature on Conium maculatum biochemistry and toxicology, dispersed in a large number of scienti®c publications, has been put together in this review. C. maculatum is a weed known almost worldwide by its toxicity to many domestic animals and to human beings. It is an Umbelliferae, characterized by long, hollow stems, reaching up to 2 m height at maturity, producing a large amount of lush foliage during its vegetative growth. Its ¯owers are white, grouped in umbels formed by numerous umbellules. It produces a large number of seeds that allow the plant to form thick stands in modi®ed soils, sometimes encroaching on cultivated ®elds, to the extent of impeding the growth of any other vegetation inside the C. maculatum area of growth. Eight piperidinic alkaloids have been identi®ed in this species. Two of them, g-coniceine and coniine are generally the most abundant and they account for most of the plant acute and chronic toxicity. These alkaloids are synthesized by the plant from eight acetate units from the metabolic pool, forming a polyketoacid which cyclises through an aminotransferase and forms g-coniceine as the parent alkaloid via reduction by a NADPH-dependent reductase. The acute toxicity is observed when animals ingest C. maculatum vegetative and ¯owering plants and seeds. In a short time the alkaloids produce a neuromuscular blockage conducive to death when the respiratory muscles are a€ected. The chronic toxicity a€ects only

* Corresponding author. 0041-0101/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 1 - 0 1 0 1 ( 9 8 ) 0 0 2 0 4 - 9

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pregnant animals. When they are poisoned by C. maculatum during the fetuses organ formation period, the o€spring is born with malformations, mainly palatoschisis and multiple congenital contractures (MCC; frequently described as arthrogryposis). Acute toxicity, if not lethal, may resolve in the spontaneous recovery of the a€ected animals provided further exposure to C. maculatum is avoided. It has been observed that poisoned animals tend to return to feed on this plant. Chronic toxicity is irreversible and although MCC can be surgically corrected in some cases, most of the malformed animals are lost. Since no speci®c antidote is available, prevention is the only way to deal with the production loses caused by this weed. Control with herbicides and grazing with less susceptible animals (such as sheep) have been suggested. C. maculatum alkaloids can be transferred to milk and to fowl muscle tissue through which the former can reach the human food chain. The losses produced by C. maculatum chronic toxicity may be largely underestimated, at least in some regions, because of the diculty in associate malformations in o€spring with the much earlier maternal poisoning. # 1999 Elsevier Science Ltd. All rights reserved.

1. Introduction Conium maculatum is an Umbelliferae native to Europe and western Asia. It has been introduced in America and Oceania as an ornamental plant (Holm et al., 1997). In Europe (hemlock; cigueÈ) it is known for its acute toxicity in horses and cattle (Derivaux and LieÂgeois, 1962; Jean-Blain and Grisvard, 1973). There are references of its teratogenicity in the United Kingdom (Dyson and Wrathall, 1977; Hannam, 1985). In Australia (carrot fern) it has been found naturalized in all the states (Everist, 1981), particularly in the South; however, it seems not to be of signi®cance in terms of toxicity to livestock (Seawright, 1982). It is widely distributed in New Zealand (hemlock), where cases of the acute toxicity of hemlock in livestock and in human beings have been reported (Connor, 1977). In North America (poison hemlock) it is frequently found in the United States and southern Canada (Kingsbury, 1964). C. maculatum is also found in nearly all South American countries (Holm et al., 1997). It is considered as poisonous in Brazil (cicuta) but seemingly without particular signi®cance as a problem in livestock production (Tokarnia et al., 1992). In Argentina (cicuta) it is well known as a toxic plant, there exist records of both acute and teratogenic toxicity (Ragonese and Milano, 1984; LoÂpez et al., 1991). It is widespread throughout this country (Marzocca et al., 1993), particularly in the large region known as Pampa, where it coincides with areas of high livestock production. Since the literature on C. maculatum biology, biochemistry and toxicology is dispersed in a large number of papers in di€erent scienti®c magazines and books, it was worthwhile bringing them together in a single article. In this review we will consider di€erent aspects of this plant: botanic description, habitat, growth habits, toxic components and their biosynthetic pathways, metabolism in animals, acute

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and chronic toxicity and the mechanisms of both. The high toxicity of this plant will be emphasized since it seems to have been largely underestimated, at least in Argentina, in terms of the high risk of livestock losses that it represents1.

2. Botanic description, habitat and growth C. maculatum is an annual or biennial herb (perennial in favorable conditions (Panter and Keeler, 1988)); the stems are glabrous, 0.6 to 2 m tall, erect and hollow except at the nodes, striated, rami®ed, ®stulous, with numerous purple spots. Leaves are petiolated, pinnaticompound2, folioles pinnatisected, lower alternate, higher usually opposite. During the ®rst year of growth C. maculatum reaches 45 cm height forming dense stands around the parent plants. During the second year the new plants grow from rosettes, with larger leaves which are dark green, bisected, triangular, glabrous and pinnatisected 4 to 5 times (Panter and Keeler, 1988). Flowers are white, grouped in umbels, composed of 10 to 20 umbellules. It produces a large number of green fruits, 2 to 3 mm long and 2 to 2.3 mm wide, grayish at maturity and formed by two indehiscent mericarps (Cabrera, 1965; Ragonese and Milano, 1984; Marzocca et al., 1993). The seeds are non-dormant (Baskin and Baskin, 1998). Because of the large seed production, it may dominate small areas with a high density of plants and encroach on alfalfa ®elds, grass pastures and meadows (Panter et al., 1992a,b). C. maculatum has a white solid taproot, similar to that of parsnip (Pastinaca sativa L.). C. maculatum grows in modi®ed soils, along fences, roadsides and ditches, around windmills, abandoned constructions and under and around woods, under which it can vegetate during the winter. It is frequently found as a weed in pastures and crops, which makes possible the presence of the plant in hays and silages and its seeds as impurities in grains. In temperate climates, C. maculatum initiates its vegetative growth in midwinter, when the growth rates of other forages is low and can represent most of the available biomass. Under these conditions the fast formation of dense stands makes voluntary or involuntary ingestion almost inevitable for herbivores. Furthermore, during this season its most toxic component in our region, gconiceine, predominates (de la Torre et al., unpublished results). It may remain in vegetative state throughout the winter, when most of the other forages are scarce (Panter et al., 1992a,b). 1 C. maculatum can pose some degree of danger to the plant production since it has been found to be a natural reservoir of the carrot thin leaf virus, celery mosaic virus and alfalfa mosaic virus (Howell and Mink, 1981). 2 C. maculatum leaves have sometimes been described as pinnaticompound, although they have been characterized as simple, 2 to 4 times or 4 to 5 times pinnatisected (Panter and Keeler, 1988; S. Alonso, personal communication). This description takes into account the number of lead blade divisions and the depth of the segments in each division (Petetin and Molinari, 1977).

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Fig. 1. The known alkaloids from Conium maculatum.

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3. Alkaloids of C. maculatum There are 8 known piperidinic alkaloids produced by C. maculatum (Panter et al., 1988a) (Fig. 1)3. Two of them, g-coniceine (I) and coniine (II) are usually found in largest amounts (Panter et al., 1988a,b,c) and both combined account for most of the biological activity of C. maculatum, II being about 8 times more toxic than I (The Merck Index, 1996). We have found a large variability in both the qualitative and quantitative results of the alkaloid analysis carried out by di€erent researchers, in di€erent stages of growth of di€erent organs of the plant (Tables 1 and 2). There are other parameters in¯uencing the alkaloid concentration in C. maculatum: rain (increase of g-coniceine concentration in fruits and ¯owers in rainy weather) and temperature (Fairbairn and Challen, 1959; Leete and Adityachaudhury, 1967); there seem to be even diurnal changes in the plant alkaloid content (Panter et al., 1983). The amount of alkaloids found in the fruits during sunny seasons is twice the amount found in cloudy seasons (Fairbairn and Challen, 1959; Fairbairn and Suwal, 1961). No information was found related to similar changes in other parts of the plant. In fruits the alkaloids are accumulated in two cellular layers contiguous to the endocarp (Fairbairn and Challen, 1959). Their concentrations increase as fruits ripen; this increase depends also on the solar exposure and the soil moisture available. The alkaloid concentration in fruits reaches a maximum while ripening, but lowers near maturity, green fruits containing larger amounts of alkaloids than mature fruits and seeds. Coniine prevails in a sunny dry summer; coniine and coniceine are produced in similar amounts in a cloudy, rainy summer (Fairbairn and Challen, 1959). We have found a marked increase in the alkaloid content of C. maculatum when it grows in nitrogen fertilized soils (de la Torre et al., unpublished results).

4. Biosynthesis of C. maculatum alkaloids For a long time secondary plant metabolites were considered as waste substances, accumulating during the plant life. Actually, they have a dynamic interaction with the primary metabolism and are involved in turnover and catabolic processes, fully participating in the whole scheme of the plant metabolism (Barz and Koster, 1981). C. maculatum alkaloids are no exception. Their concentrations can undergo frequent changes during the plant growth. It is remarkable that seeds present a high alkaloid content, while during the early stages of growth a much lower alkaloid content is found in the foliage (de la Torre et al., unpublished results). Rapid changes in the alkaloid concentration are 3

Coniine was the ®rst synthesized alkaloid (Clarke and Clarke, 1963; Ladenburg, 1886).

± ND ± ± 0.29 0.86 0.75 ± 0.36 ± 0.23 0.01 ± 0.15 ± ± ± ± ± 0.18 ± ± ± ±

± 0.026 ± ± ND 0.06 0.2 ± 0.42 ± ND 0.42 ± ND ± ± ± ± ± 0.01 ± ± ± ±

Coniine (%DM) ± 0.074 ± ± 0.03b 0.02a 0.09a ± 0.25 ± ± 0.85a ± 0.02a ± ± ± ± ± ± ± ± ± ±

Other (%DM)

Dashes indicate that the corresponding values were not mentioned. ND: not detected. %DM: percentage in dry matter. a N-methylconiine. b Conhydrine.

Seeds

Shoots (vegetative growth)

Ripe fruits

Flowers Green fruits

Dry stems Leaves (vegetative growth)

Roots

Coniceine (%DM) <0.01 0.5 0.009 1.49 0.32 0.94 1.04 1.62 1.03 3 ± 1.07 0.217 0.17 0.075 0.023 0.029 0.02 0.761 ± 0.75 0.92 0.217 0.019

Total alkaloids

Hull, England Chelsea, England Buenos Aires, Argentina Hull, England Illinois, USA Hull, England Utah, USA Illinois, USA Illinois, USA California, USA California, USA California, USA Utah, USA Illinois, USA Illinois, USA Indiana, USA

Hull, England Hull, England Hull, England

Hull, England California, USA Utah, USA

Source of C. maculatum

Table 1 Individual and total alkaloid contents of Conium maculatum of diverse origin as found by di€erent authors

Cromwell (1956) Jessup et al. (1986) Keeler (1974) Madaus and Schindler (1938) Cromwell (1956) (1st year of growth) Cromwell (1956) (2nd year of growth) Cromwell (1956) Cromwell (1956) Cromwell (1956) Fairbairn and Challen (1959) de la Torre et al. (1998) (pers. comm.) Cromwell (1956) Panter et al. (1985) Cromwell (1956) Keeler (1974) Panter et al. (1985) Panter et al. (1985) Jessup et al. (1986) Jessup et al. (1986) Galey et al. (1992) Keeler (1974) Panter et al. (1985) Panter et al. (1985) Frank and Reed (1987)

Refs.

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T.A. LoÂpez et al. / Toxicon 37 (1999) 841±865 Table 2 Non-quantitative comparisons of alkaloid contents in di€erent plant organs of Conium maculatum

Fully formed fruit, still green Half-ripe fruits Green fruits Stems Stems

Organs

Refs.

>young leaves>roots >ripe fruits>>roots >ripe fruits >leaves>fruits>roots >leaves>>fruits>roots

Forsyth (1968) Connor (1977) Kingsbury (1964) Edmonds et al. (1972) Steyermark (1962)

observed in di€erent circumstances: wet or dry weather, di€erent seasons and degree of herbivory. In early experiments related to changes of the concentrations of the two major alkaloids found in C. maculatum (coniine and g-coniceine, the latter in much lower concentrations), it was noticed that coniine reached a maximum of about 6 mg/fruit at 4 to 5 weeks of growth depending on the year of collection, while gconiceine simultaneously, although not stoichiometrically, decreased to a minimum. Diurnal, large changes, were also noticed in the concentrations of both alkaloids, the increase in one corresponding to a decrease in the other (Fairbairn and Suwal, 1961). According to these authors minor quantities of other alkaloids were noticed: when coniine was at its maximum, N-methylconiine co-occurred, while conhydrine occurred in small amounts when coniceine predominated, but in this case N-methylconiine was absent. It was proposed that the piperidine ring of C. maculatum alkaloids is produced by cyclization of lysine (Robinson, 1917). However, attempts to demonstrate that lysine is a precursor of the piperidine ring in coniine by feeding D,L-lysine-2-14 C to the stems of C. maculatum plants, failed in producing labeled coniine (Leete, 1964). Uniformly labeled L-lysine-14 C administered to hemlock plants, a€orded radioactive coniine (Schiedt and HoÈss, 1958), but no degradative studies were produced by these authors to prove that labeling was present in the piperidine ring. Instead, feeding sodium acetate-1-14 C to two-year-old C. maculatum plants indicated that alkaloids obtained from the plant (coniine, g-coniceine and conhydrine), presented labeling. Chemical degradation of the obtained coniine showed that activity was mostly and evenly distributed in even numbered C atoms, which allowed to speculate that hemlock alkaloids are derived from an eight C aliphatic polyketo chain produced by the linear attachment of four acetate units (Leete, 1963, 1964) probably through the condensation of 4 molecules of acetyl-CoA (Leete et al., 1975). The results of Schiedt and HoÈss (1958) may be due to metabolisation of lysine-14 C to radioactive acetate units, then incorporated to coniine (Leete, 1964). Feeding g-coniceine-1 0 -14 C to C. maculatum plants showed that all the activity was transferred to the C-1 0 of the propyl side chains of coniine and pseudoconhydrine indicating that g-coniceine can be the precursor of the former. It is interesting to note that similar feeding experiments conducted in a greenhouse and outdoors, produced conhydrine and pseudo-conhydrine, respectively, suggesting

Fig. 2. Biosynthetic pathways of the alkaloids of Conium maculatum.

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that changes in the environment can induce changes in the alkaloid composition of C. maculatum (Leete and Adityachaudhury, 1967). Later experimental work provided evidence that the 8 carbon polyketoacid intermediate in the synthesis of g-coniceine is derived from octanoic acid since this acid was shown to be readily incorporated into coniine (Leete, 1970). Further work indicated that 5-keto-octanoic acid and 5-keto-octanal were produced during the biosynthesis of g-coniceine (Leete, 1970; Leete and Olson, 1970). A transaminase (L-alanine: 5-keto-octanal aminotransferase) was obtained from C. maculatum. This transaminase catalyzes the reaction between 5-keto-octanal with L-alanine as the amino group donor to form the piperidinic ring and the propyl side chain (Roberts, 1971, 1977). Another C. maculatum alkaloid, Nmethylconiine, was shown to be produced by another enzyme from the plant: a coniine methyltransferase which acts as a transmethylator utilizing S-adenosyl-Lmethionine as a methyl group donor (Roberts, 1974a,b). The production of coniine is catalyzed by a NADPH-dependent g-coniceine reductase (Roberts, 1975) in a reversible reaction, since earlier experiments showed a fast interconversion between both substances, even in an hourly basis (Fairbairn and Suwal, 1961). Glutamate-oxalacetate aminotransferase (GOT) and GPT, possible aliphatic aminotransferases in the amination of 5-keto-octanal instead of L-alanine:5-ketooctanal aminotransferase, were excluded from that function by further work which separated the activities of the former from that of the latter, in turn shown to be composed of two isozymes, although no individual function was described (Roberts, 1978). g-Coniceine has also been proposed as the source of pseudo-conhydrine. Tautomerization of the double bond between N and C2 of the piperidinic ring could produce the shift of the double bond to N and C6, yielding an allylic C5 which in turn could be oxidized to produce a hydroxy C5 (Leete and Adityachaudhury, 1967). It has been proposed that conhydrine is produced by oxidation of g-coniceine to conhydrinone and its subsequent reduction to conhydrine (Roberts, 1975) (Fig. 2). Further information, particularly related to the stereochemistry of these reactions and of the substances involved, can be found in Waller and Dermer (1981).

5. Structure±activity relationship of C. maculatum alkaloids The molecular structure determines the teratogenicity (chronic toxicity) of C. maculatum alkaloids (no information has been found relating the acute toxicity and the structure of these alkaloids). The side chain must be a propyl group or larger. For instance, 2-ethylpiperidine has been shown to be non-teratogenic. Keeler and Dell Balls (1978), have experimented in ewes the teratogenicity of several piperidinic substances with di€erent substituents and di€erent degrees of

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Fig. 3. Teratogenic alkaloids from Conium maculatum and some non-teratogenic homologous substances.

nuclear unsaturation. Their results are shown in Fig. 3. Partial unsaturation seems to increase toxicity since coniceine is more toxic than coniine, while 2propylpyridine is non-teratogenic, i.e. aromatization of the ring suppresses toxicity. This structure±activity relationship is supported by the ®nding that swainsonine (IV) (Fig. 4), an alkaloid of Astragalus spp causing locoism, is also teratogenic (James et al., 1967; James, 1972; Molyneux et al., 1985). It is considered as an indolizidinic alkaloid, but it can also be understood as a piperidinic alkaloid where the piperidine ring has a three C atoms cyclic

Fig. 4. Swainsonine (IV, V), a teratogenic alkaloid from Oxytropys and Astragalus spp., showing its resemblance with teratogenic piperidinic alkaloids from Conium maculatum.

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substituent. This seems to be a general situation since, as will be discussed later, the same teratogenic properties are found in similar substances produced by several plants of di€erent genera.

6. Acute toxicity There are reports on acute toxicity in domestic animals, caused by the ingestion of fresh C. maculatum. Monogastric animals and ruminants show similar symptoms, but di€erent susceptibilities. There are no indications of macro or microscopic lesions related to poisoning with this plant. The a€ected species are: cattle (Penny, 1953); sheep (Panter et al., 1988a,b,c); goats (Capithorne, 1937); horses (MacDonald, 1937); elk (Jessup et al., 1986); pigs (Buckingham, 1936; Edmonds et al., 1972) and poultry by seed ingestion: range turkeys (Frank and Reed, 1987) and quail (Kennedy and Grivetti, 1980). Alkaloid extracts of C. maculatum have been found to be toxic for chickens and chicks (Bowman and Sanghvi, 1963) and coniine for chickens, quails and turkeys (Frank and Reed, 1990). Acute lethal doses of fresh C. maculatum have been reported by di€erent authors (Table 3). The data show that the acute toxicity of fresh C. maculatum is higher in sows, lower in cows and moderate to low in ewes (Table 4). Further comments on these data will be presented in Section 9. There is information concerning the acute toxicity of pure coniine, g-coniceine and N-methylconiine. The oral LD50's in mice are 12 mg/kg for coniceine, 100 mg/kg for coniine and 204.5 mg/kg for N-methylconiine (Bowman and Sanghvi, 1963; The Merck Index, 1996). The single oral doses of pure coniine inducing acute toxic e€ects in some domestic animals are indicated in Table 5. There are cases of acute poisoning of domestic animals which ingested C. maculatum foliage despite a good forage availability (Panter and Keeler, 1988). Preference for or apparent willingness to ingest more hemlock (even after su€ering from intoxication) has also been recorded in di€erent animal species: cows (Penny, 1953; Panter et al., 1992a,b), pigs (Panter et al., 1983; Holm et al., 1997), goats (Capithorne, 1937), elk (Jessup et al., 1986) and chickens (Frank and Reed, 1990). Table 3 Acute lethal oral doses of fresh Conium maculatum in some domestic animals Animal species

Lethal dose (g/kg LW)

Alkaloid content (%)

g-Coniceine content (%)

g-Coniceine dosis (g/kg LW)

Refs.

Sows Cows Sheep

8.0 5.3 10.0

0.023 0.40 0.40

98 98 98

1.8 21 39

Panter et al. (1983, 1985) Keeler and Dell Balls (1978) Panter et al. (1988b)

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T.A. LoÂpez et al. / Toxicon 37 (1999) 841±865 Table 4 Degree of toxicity of fresh Conium maculatum in di€erent animal species Toxicity

Decreasing susceptibility

Acute Chronic

cattle>sheep = goats>pigs cows>sows>sheep

Human beings have been poisoned accidentally by hemlock after mistaking it for other Umbelliferae: the root for parsnip, the leaves for parsley and the seeds for anise (James et al., 1980; Hulbert and Oehme, 1981). There are records of acute, sometimes lethal, poisoning in human beings, particularly children, who used to blow through the green, hollow stems in the manner of pea-shooters, ¯utes or whistles (Everist, 1981). It has been argued whether the meat of birds which eat C. maculatum seeds during migratory ¯ights becomes poisonous to human beings as is the case of quail migrating to Europe from Northern Africa and Western Asia (Forsyth, 1968; Frank and Reed, 1990). It has been found that the ingestion of C. maculatum by dairy cattle reduces milk production, transmitting to it unpleasant taste and smell (Forsyth, 1968; Marzocca et al., 1993). The velocity of the onset of toxicity symptoms after single oral doses of coniine varies depending on the animal species. Signs started after 30±40 min in mares and in 1.5±2 h in cows and sheep (Keeler and Dell Balls, 1978), possibly because of a faster absorption in monogastric than in ruminant animals. In pigs, symptoms started after 15 min of the oral dose of the fresh plant or seeds (Panter et al., 1985). Sheep dosed orally with 6 g of fresh C. maculatum with high gconiceine content (98% of total alkaloids) showed symptoms after 10 min. Higher doses produced death after 5 days of administration (Panter et al., 1988b). In birds, symptoms started between 5 and 60 min of the oral administration of coniine, independently of dose (Frank and Reed, 1990). Table 5 Acute e€ects of single oral doses of coniine in di€erent domestic animals Species

Dose (mg/kg)

Degree of symptoms

Cowsa Maresa Ewesa Quailsb Chicksb Turkey chicksb

3.3 15.3 44.0 25.0 50.0 100.0

severe severe moderate severe severe severe

Keeler et al., 1980; Frank and Reed, 1990. a Adult animals (maximum non lethal doses). b Three to six-week-old animals (lethal to some individuals).

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The signs of acute poisoning with C. maculatum are similar in the di€erent animal species. Cattle, sheep and swine present muscular weakness, incoordination, trembling, midriasis, support on the metacarpophalangic joints, excessive salivation, cyanotic membranes and cold limbs. These signs are followed by initial stimulus of the central nervous system, then by depression, fast and shallow respiration, turning to slow and laborious, dilated pupils, frequent micturition and defecation, coma and death caused by respiratory paralysis (Panter et al., 1985, 1988a,b; Galey et al., 1992). Poisoned ruminants eruct frequently. Cattle and swine may become temporarily blind due to the closing of the nictitating membrane. They also present `moussy' odour in breath and urine (Panter et al., 1988a,b; Galey et al., 1992). In birds, the following signs have been noticed: excitation followed by depression, hypermetria, seizures, opistostonous and ¯accid paralysis (Frank and Reed, 1990). With non-toxic doses a sedative or depressive e€ect of the central nervous system, producing deep sleep, is noticed. These anesthetic e€ects were observed in several piperidinic substances by Hunt and Fosbinder (1940). Animals surviving acute poisoning usually recover without sequels, but abortions may result (Agriculture Research Service, ARS, 1968; Forsyth, 1968). The necropsy ®ndings are consistent with those expected from a respiratory arrest: dark and dense blood, dark, congested liver; the right side of the heart is found full of blood while the left is empty; the lungs are congestive and dark colored, showing clear bands where the ribs have been in contact with the lungs (Forsyth, 1968; Everist, 1981). There are no antidotes. The treatment is symptomatic. The use of stimulants and large volumes of water has been suggested for the treatment of poisoned livestock (ARS, 1968; Forsyth, 1968). In human beings the administration of alcoholic beverages, tea, co€ee has been suggested and also the induction of vomit with a tablespoon of salt dissolved in warm water, repeating the treatment until the vomit is clear, keeping the victim laying down, resting, covered and under medical control (ARS, 1968; Forsyth, 1968).

7. Chronic teratogenic toxicity Genetic arthrogryposis, associated with palatoschisis, scoliosis and torticollis, has been described (Nawroth et al., 1980). However, in the early studies of C. maculatum toxicity, it was shown that the breeding of 11 cows with arthrogryposis with a bull with palatoschisis, produced the birth of 11 healthy calves, which supported the current belief that the many malformations observed in livestock in the western states of USA (including Alaska) and in southern Canada, were not of hereditary but of toxic origin (Shupe et al., 1967b; Leipold et al., 1970). In a particular case it was observed that malformed calves were born to cows feeding on range, while calves born to cows feeding on pastures (mostly crested wheatgrass) of the same area, were healthy. It was then suggested that plants might be involved in the production of malformed o€spring. Lupinus spp.,

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abundant in the area, were suspected and subsequently experimentally shown to be teratogenic (Shupe et al., 1967a). It seems that this ®nding was the precursor of the identi®cation of C. maculatum teratogenicity. It had been stated that C. maculatum would prove to be teratogenic since it contains piperidinic alkaloids similar to those found in tobacco stalks, already known to be teratogenic (Keeler, 1972). In 1972, a case of malformation in piglets born to sows that ingested C. maculatum during their pregnancies, was one of the ®rst indications of the relationship between ingestion of this weed during pregnancy and the occurrence of malformation in the o€spring (Edmonds et al., 1972). C. maculatum related malformations have been described in calves, piglets and lambs: at birth, they present arthrogryposis4, scoliosis, torticollis, palatoschisis and excessive ¯exure of the carpal joints. These lesions were experimentally reproduced in calves after maternal forced administration of the fresh plant in non-lethal, daily toxic doses, during the 55±75 days of pregnancy (Keeler, 1974, 1975; Keeler and Dell Balls, 1978; Keeler et al., 1980). Toxic amounts of fresh hemlock administered to dairy cows during 42±70 days of pregnancy, produced palatoschisis in calves (other authors had not noticed this type of malformation in cattle produced by C. maculatum (Panter et al., 1985)) while the same administration during 55±70 days produced arthrogryposis (Galey et al., 1992). The arthrogrypotic lesions in calves worsen as the animals grow up. The ¯exure of the front limbs becomes more severe, the a€ected animals cannot support their own weights, and are forced to `walk' on the carpal joints (Keeler and Dell Balls, 1978). Sows gave birth to piglets with variable teratogenic lesions according to di€erent authors. Palatoschisis alone was produced after forced toxic, non-lethal, maternal administration of fresh plant during 30±45 days of gestation (Panter et al., 1985). If the same administration was given during 43±61 days of pregnancy, piglets were born with limb malformations, despite the fact that limbs start being formed during 17 and 18 days of gestation, which seemed to indicate that damages were produced in soft tissues formed thereafter, since no bone malformations were observed (Edmonds et al., 1972). Administration to sows of fresh plants or seeds during 43±53 days of pregnancy produced skeletal terata in the piglets (Panter et al., 1985). Other authors have cited both limb and skeletal malformations in piglets (Dyson and Wrathall, 1977). It has also been found that maternal feeding of C. maculatum or its seeds to sows during 42±70 days of pregnancy resulted in piglets with palatoschisis, while administration during days 43±53 produced excessive ¯exure of the carpal joints and arthrogryposis and 4 Arthrogryposis has been questioned as an accurate name for the malformation and it has been proposed to be replaced by multiple congenital contracture (MCC) (Swinyard and Bleck, 1985). These authors found that MCC is caused by loss of muscle mass with imbalance of muscle power at the joints which provokes a collagenic response consisting of partial replacement of muscle volume and collagenous thickening of the joint capsules, the latter leading to joint ®xation.

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administration during 51±61 days produced only moderate ¯exure of the fetlock and carpal joints (Panter et al., 1983). The gavage of goats with fresh hemlock or its seeds at toxic levels, during 30± 40 days of gestation produced the birth of goat kids with severe teratogenic lesions: arthrogryposis, scoliosis, lordosis, palatoschysis, rib cage depressions and wedging of vertebrae (Panter et al., 1990). There seems to be some degree of resistance of sheep and mares to the teratogenic e€ects of C. maculatum. Sheep fed fresh hemlock in toxic levels during 30±60 days of gestation gave birth to lambs with slight carpal ¯exure and scoliosis which disappeared spontaneously in 8 weeks (Panter et al., 1988b). The same lesions and outcome were observed in lambs whose dams were dosed orally with coniine at toxic levels during 25±35 days of gestation (Keeler et al., 1980). Lambs were born healthy to ewes dosed coniine during 12±30, 30±50 and 35±65 days of gestation (Keeler et al., 1980). Pregnant mares administered coniine to produce severe toxicity during 45±75 days of pregnancy (with fetal development similar to bovine fetuses that su€er teratogenic e€ects), produced normal foals, which may indicate a resistance of equine to the teratogenic e€ects of this plant (only two mares were used in the experiment; two others died of overdosing before parturition) (Keeler et al., 1980). We have found no references of malformations in foals, produced by maternal ingestion of C. maculatum or its teratogenic alkaloids. Experimental evidence of teratogenic e€ects of C. maculatum has been demonstrated only for coniine and for the whole plant and seeds. g-Coniceine is considered teratogenic because fresh C. maculatum containing alkaloids of which 98% were g-coniceine, was found to be teratogenic in cattle (Keeler and Dell Balls, 1978). The same material produced numerous piglets with palatoschysis (Panter et al., 1985). It seems that the other piperidinic alkaloids of C. maculatum have little or no teratogenicity since C. maculatum containing 0% coniine, 19.8% coniceine and 80.2% of the other alkaloids, produced toxicity but not malformations in pigs (Panter et al., 1985)5. Table 6 summarizes part of the described teratogenic ®ndings. Drying of fresh hemlock under the sun during 7 days produces an important loss of biological activity. Administering this dried material to three pregnant cows produced slight signs of toxicity in one of them and no malformations in the calves, while the same material administered fresh caused arthrogryposis in three of three calves. The fresh material contained 0.4% alkaloids, while the dried material contained 0.03% (Keeler and Dell Balls, 1978). 5

The lack of teratogenic e€ects of this plant material indicates a very low or nil teratogenic activity of the alkaloids besides g-coniceine and coniine. N-methylconiine (the other mostly cited alkaloid in C. maculatum) has not been tested for its capacity of teratogenicity, although this substance has the required structural characteristics for the production of malformations.

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Table 6 Periods of gestation during which fetuses of di€erent animal species are susceptible to the teratogenic e€ects of Conium maculatum Species

Administration period of gestation (days)

Lesions

Refs.

Cattle (283)a Swine (115)a

55±75 42±70 30±45 43±53 51±61

Keeler (1978a,b) Galey et al. (1992) Panter et al. (1985) Panter et al. (1985) Panter et al. (1983)

Goats (150)a

30±60

Sheep (147)a

30±60

arthrogryposis palatoschisis palatoschisis arthrogryposis moderate ¯exure of fetlock and carpal joints arthrogryposis, palatoschisis and other arthrogryposis

a

Panter et al. (1990) Galey et al. (1992)

Total days of gestation.

It has been stated that the chronic toxicity of C. maculatum a€ects more cows and sows than ewes (Panter and Keeler, 1988), apparently causing no e€ects on mares (Keeler et al., 1980). Arthrogrypotic lesions have been surgically corrected in 25±34 calves (Verschooten et al., 1969).

8. Mechanisms of toxicity 8.1. Acute toxicity It has been proposed that the acute toxic activity of coniine, g-coniceine and Nmethylconiine consists in the blockaging of the spinal re¯exes through the action on the medulla: they produce an initial stimulus followed by the depression of the autonomic gangliae. High doses produce a stimulus of the skeletal muscles and a subsequent neuromuscular blockage through the action on nicotinic receptors. Death occurs when the phrenic nerve is a€ected and respiratory muscles become paralyzed (Bowman and Sanghvi, 1963; Panter et al., 1988a; Galey et al., 1992). Experimental poisoning of cattle with vegetative C. maculatum containing 0.01% coniine and 0.18% coniceine indicated that at least part of the alkaloids are eliminated through urine as coniine. It is not known whether coniceine is reduced in the rumen (probably not in the liver since this organ tends to produce oxidative processes) or is destroyed in the liver (Galey et al., 1992). There is a di€erence of about 10 times between the susceptibility of cattle and that of sheep to the acute toxicity of coniceine and of coniine. This di€erence seems not to be attributable to a structural modi®cation of this substance in the liver or in the rumen, or to a di€erent degree of intestinal absorption, since

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intramuscular injection (IM) of coniine maintains the di€erent toxicity in both species: an IM dose of 240 mg/kg is required to kill a sheep, while a dose of only 16 mg/kg kills a cow. This indicates that the acute toxic e€ect of coniine is due to its direct action on receptors (Keeler et al., 1980). 8.2. Chronic toxicity The fetal mandibular movements (particularly, the downward movement of the lower jaw) and tongue retraction are required for palatal shelves to become closer, allowing a normal closure of the upper jaw. The absence of these movements can produce palatoschysis, since the tongue occupies the place where the palatal shelves must fuse (Humphrey, 1969). A group of simple derivatives of piperidine has anesthetic spinal activities, which in some of the derivatives resulted more intense than those produced by cocaine, procaine and piperocaine (g-2 methylpipediridilpropyl benzoate) (Hunt and Fosbinder, 1940). It is possible that the known anesthetic e€ect of the alkaloids of C. maculatum inhibits the mentioned fetal movements. In consequence, the tongue obstructs the place where the shelves fuse together. Furthermore, the closure of the palatal shelves also depends on the separation of the fetal inferior jaw from the breast bone, which in turn depends on the fetal movements. If these movements are impaired, the lower jaw compresses the tongue against the upper part of the oral cavity, which impedes the mutual approaching of both palatal shelves (Poswillo, 1966; Walker, 1969). There is a mechanism of control of the amniotic liquid volume, at least in human beings (Bourne, 1962). If the volume of the amniotic liquid increases, it exerts more pressure on the fetus. If the volume decreases, the placenta may compress it. In both cases there is an impairment in the fetal movements leading to palatoschysis. It is possible that the C. maculatum piperidinic alkaloids may act upon the mechanism that regulates the amniotic liquid, adding to the production of malformations. Finally, lack of fetal movement, whatever its origin, can also cause limb malformations (Poswillo, 1966). Seeds and fresh C. maculatum administered orally to goats twice daily during 30±60 days of gestation produced malformations in the o€spring (palatoschisis and arthrogryposis). Ultrasonic control of fetal movement was performed during 12 h at 45, 51, 55 and 60 days of pregnancy. C. maculatum seeds produced almost total fetal immobility, while the fresh plant produced the same e€ect, but lasting only 5 h. Goat kids born to dams dosed with seeds showed palatoschisis and arthrogryposis, while those born to dams fed with the fresh plant, showed minor ¯exures of forelimbs, which receded a few weeks after birth (Panter et al., 1992a,b). This lack of fetal movement caused by C. maculatum had been also observed in pigs (Panter et al., 1985) and in sheep (Panter et al., 1988c). In the latter, only slight front limb abnormalities were found, which receded in about 8 weeks, this being consistent with the already mentioned lower chronic toxicity produced by C. maculatum in sheep.

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9. Discussion and conclusions In Argentina few precautions seem to be taken by farmers, as regards the access of livestock to C. maculatum, despite its high densities, the large areas invaded and its early growth during the year (it starts to vegetate in July (midwinter) in the southeast of Buenos Aires province). This allows hemlock to grow when the growth rates of grasses and most perennial pastures is low and to become an important source of dense, lush forage. In protected sites such as forests it may vegetate throughout the year. In general, ®eld workers know of the toxic nature of C. maculatum, but its e€ects are considered to be transient and the risk of death after acute poisoning, the teratogenic e€ects, abortions (Forsyth, 1968) and the production losses such as decreased meat and milk production (Marzocca et al., 1993; Holm et al., 1997) are disregarded. An assessment of the economic loss in a farm were 45 pigs and 24 pregnant dairy cows (and their 26 to be born calves) were lost to hemlock, produced an estimate of US$50,000, not including accessory losses such as milk disposal, additional veterinary costs, reposition costs and delay in the return to normal activity (Panter and Keeler, 1988). There is no information concerning possible losses in poultry production caused by the ingestion of C. maculatum seeds, such as low weight gains, decreased egg production and teratogenicity in chicks. In our experience these seeds can be found as contaminants in grains and seeds used in poultry feeding; however, we did not ®nd references concerning teratogenic e€ects produced by ingestion of C. maculatum seeds in these species. It has been shown that anabasine, an alkaloid of Nicotiana tabacum (Fig. 5), causes malformations in chicks (Panter et al., 1992a,b). This alkaloid has a piperidinic structure satisfying the requirements for teratogenicity (Keeler, 1974; Keeler, 1979). For these reasons it is possible that C. maculatum alkaloids can be teratogenic to poultry because of its high content of g-coniceine and coniine.

Fig. 5. Alkaloids from Nicotiana tabacum. Nicotine has been shown to be non-teratogenic, while anabasine is. Probably IX and X are also teratogenic since they are in agreement with the required structural conditions for teratogenicity.

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It is not known how dangerous C. maculatum can be as an impurity of forages in silages. The required air-tightness an compaction may preclude the evaporation of its alkaloids, resulting in a poisonous feed. There are no references related to the possibility of passage of hemlock alkaloids to milk. However, the foul smell and taste of the milk from animals feeding in paddocks where hemlock grows, is a possible indicator that in fact they are incorporated to milk, which in turn would imply the risk of poisoning in lactating o€spring and in humans. It has been found that coniine orally administered to turkeys in single doses, persists in muscle tissue and in the liver (80±120 and 100±200 ppm, respectively) 7 days after administration (Frank and Reed, 1990). For these reasons, both, meat and milk may be vehicles of human exposure to the toxic alkaloids. The presence of alkaloid residues in muscle and liver tissues of birds might support the hypothesis that the `coturnism', an acute poisoning of human beings due to ingestion of quail (Coturnix coturnix coturnix), is caused by those animals after feeding on C. maculatum seeds before or during their migratory ¯ights. Coturnism is known since biblical times and even today controversy persists on the etiology of the disease. There are some interesting scienti®c works with opposing conclusions. One author found evidence that quail reaching Europe from Northern Africa had fed on C. maculatum seeds and attributed to this the deaths occurring in human beings after eating these animals (Sergent, 1948). This fact was later supported by Derivaux and LieÂgeois (1962). Finally, other authors fed quail with C. maculatum seeds, ®nding that these animals su€ered acute poisoning that can result in death. For this reason it was claimed that, under these conditions, the animals could not perform their migratory ¯ights (Kennedy and Grivetti, 1980). A comparative analysis of acute and chronic toxicity of fresh C. maculatum among di€erent animal species is shown in Table 4. In places highly invaded by this weed, it has been recommended to reduce the risk of poisoning of livestock by an initial grazing with sheep, incorporating later other species. However, preliminary ®ndings of the content of g-coniceine seem to indicate that the g-coniceine concentration increases in the regrowth after hand defoliation of the plants (de la Torre et al., unpublished results). This response to defoliation is con®rmed by observations that some plant species increase their synthesis of deterrent antihervibory substances following intensive foraging (Bryant, 1981). The proposed handling of paddocks containing abundant C. maculatum plants would be valid if the access of cattle and swine follows sheep defoliation and the former are utilized in large numbers to both avoid excessive regrowth of hemlock and to decrease the amount of C. maculatum eaten by each animal. Plants synthesize poisonous substances which were considered as waste products like urea and uric acid in animals (Robinson, 1980). In fact, those plant substances are metabolites acting as antihervibory defenses as a response to the selective pressure of insect grazing. It has been proposed that their toxicity to large mammals may be accidental since the defenses evolved before these animals were present (Molyneux and Ralphs, 1992). The plant production of these

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substances is favored by natural selection when their cost of synthesis is lower than the bene®t obtained from their protection (Coley et al., 1985). For this reason, plant species growing in environments without resource restrictions, produce defensive substances such as alkaloids and phenolic and cyanogenic glycosides, which are present in low concentrations, have high toxicity, have a low cost of synthesis and present great mobility (qualitative defensive substances (Feeny, 1975)). C. maculatum is a weed of rapid growth, colonizing places recently altered; its chemical defenses (alkaloids) belong to the qualitative type (Feeny, 1975) and it could be expected to be rapidly synthesized after defoliation, increasing their concentration in the regrowth. Our preliminary results tend to con®rm this behaviour, ®nding more alkaloid concentration in the regrowth (15 days after arti®cial defoliation) than in the plants that were not defoliated. In particular, g-coniceine synthesis is increased, while coniine remains at constant concentration (de la Torre et al., unpublished results). These results may indicate that in its defensive response C. maculatum synthesizes only its most toxic alkaloid and that during that time it does not synthesize substances of lower toxicity such as coniine of which the former is the precursor. The main problem related to the diagnosis of the chronic poisoning of livestock with C. maculatum is the diculty to associate the malformed o€spring with the much earlier maternal poisoning (Keeler, 1978a,b). This serious situation is further aggravated by the fact that, except for cases of moderate arthrogryposis which can be surgically solved, the rest of the possible toxic malformations are irreversible and the a€ected animals are lost. In these conditions, prevention of the chronic poisoning seems mandatory and consists in either avoiding the access of pregnant animals to heavily infested paddocks, removing these animals from further ingestion of C. maculatum as soon as maternal signs of poisoning are noticed (particularly during their susceptible lapse of gestation) or to control the plant, which is sensitive to common herbicides such as 2,4-D and MCPA, spraying the plants before ¯owering (Panter and Keeler, 1988; Marzocca et al., 1993). The improvement of pastures decreases the number of available hemlock plants, which has also been suggested as a way to avoid livestock poisoning (Keeler, 1978a,b). In di€erent occasions we have been asked how much of the fresh plant must be eaten by an animal to become acutely or chronically poisoned. According to Table 1, the hemlock alkaloid content is highly variable, even at the same growth stage, depending on the source of the plant and other factors. The animal species is also involved in the degree of susceptibility. For these reasons, there cannot be an accurate or even orientative answer to that question. Breeding of cattle in our area and in most of the Pampanean region is performed during October, November and December. For this reason, the period of fetal susceptibility (up to 75 days of pregnancy) coincides with the time of the year of large hemlock availability (vegetative, preblooming and blooming stages) and high g-coniceine content. These concurrent factors (high C. maculatum o€er and maximum fetal sensitivity), allow us to suspect that the local cases of C. maculatum-produced teratogenesis may be more frequent than the modest prevalence recorded. The full recovery of acutely poisoned animals in a rather

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short time, undoubtedly contributes to minimize the risks associated with this plant. Furthermore, we consider quite probable that the teratogenic e€ects of C. maculatum are largely unknown to many farmers and professionals devoted to animal production. We have mentioned the possibility of C. maculatum alkaloids reaching the human food chain. It is of concern that many other plant genera (some of them used in human foodstu€s) contain piperidinic alkaloids satisfying the structural requirements for teratogenicity: Ammodendron, Carica (papaya), Cassia, Collidium, Dichroa, Duboisia, Gemista, Hydrangea, Liparia, Lupinus (lupine), Prosopis (mesquite), Punica (pomegranate), Sedum and Withania (Keeler and Ward Crowe, 1985; Keeler, 1988). This implies that C. maculatum and other piperidinic alkaloid producing plants represent not only a risk for the animal production, but also for human health.

10. Suggested readers Dr. K.E. Panter: USDA, ARS, Poisonous Plants Research Laboratory, 1150 East 1400 North, Logan, UT 84321, USA. Dr. A.A. Frank: Animal Disease Diagnostic Laboratory, Purdue University, School of Veterinary Medicine, West Lafayette, IN 47907, USA. Professor Dr. G.G. Habermehl: EichhoÈrnchensteg 18, 30657 Hannover, Germany.

Acknowledgements We are grateful to Marina LoÂpez Casoli for her valuable suggestions on the writing of this paper.

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