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REVIEW

Poisonous Plants: Effects on Embryo and Fetal Development Kip E. Panter,* Kevin D. Welch, Dale R. Gardner, and Benedict T. Green Poisonous plant research in the United States began over 100 years ago as a result of livestock losses from toxic plants as settlers migrated westward with their flocks, herds, and families. Major losses were soon associated with poisonous plants, such as locoweeds, selenium accumulating plants, poison-hemlock, larkspurs, Veratrum, lupines, death camas, water hemlock, and others. Identification of plants associated with poisoning, chemistry of the plants, physiological effects, pathology, diagnosis, and prognosis, why animals eat the plants, and grazing management to mitigate losses became the overarching mission of the current Poisonous Plant Research Laboratory. Additionally, spin-off benefits resulting from the animal research have provided novel compounds, new techniques, and animal models to study human health conditions (biomedical research). The Poisonous Plant Research Laboratory has become an international leader of poisonous plant research as evidenced by the recent completion of the ninth International Symposium on Poisonous Plant Research held July 2013 in Hohhot, Inner Mongolia, China. In this article, we review plants that negatively impact embryo/fetal and neonatal growth and development, with emphasis on those plants that cause birth defects. Although this article focuses on the general aspects of selected groups of plants and their effects on the developing offspring, a companion paper in this volume reviews current understanding of the physiological, biochemical, and molecular mechanisms of toxicoses and teratogenesis. Birth Defects Research (Part C) 99:223–234, 2013. Published 2013 Wiley Periodicals, Inc. Key words: poisonous plants; alkaloids; birth defects; Veratrum; poisonhemlock; Nicotiana; lupine; reproduction; teratogenesis; embryo development

INTRODUCTION The impact of natural toxins from poisonous plants on the embryo, fetus, and neonate are dramatic, and the consequences economically important to the livestock industries and humanity throughout the world. The global significance of these toxicoses from poisonous plants lies in the ubiquitous nature of the plants and extensive examples of animals and humans being poisoned. The literature is replete with cases of human poisoning, either inadvertently or intentional. Thousands of species of plants in the world are known to

be hazardous to animals and many affect people either directly or indirectly. Reproductive success is dependent on many specifically timed biological events that must occur in a synchronized physiological sequence. The interference with one or more of these physiological events may result in birth of an abnormal, nonproductive offspring, total reproductive failure, or a more a subtle reduction in reproductive potential or compromised fetal/ neonatal growth and development. Table 1 provides a brief list of a few of the teratogenic plants discussed

in this article. Many factors must be considered when reproductive failure or birth defects occur (Wilson and Fraser, 1977). A few obvious factors include animal species and breed differences, stage of embryo, or fetal development when exposed to the teratogen, dose of the toxin or teratogen, and stage of plant growth when ingested. Nutritional status, range conditions, and environmental factors (climate, temperature, storms, season of the year, stress, etc.) all influence diet selection if toxic plants are present in pastures, ultimately impacting all phases of reproduction. To illustrate the global nature of the problem, there are over 350 known pyrrolizidine alkaloids (PAs) and over 6000 plant species that contain PAs. PAs act in various ways, depending on the structural characteristics of the alkaloids, the animal species implicated, and the dose and time frame of exposure. The PAs are generally hepatotoxins causing liver disease and secondary related health problems. These alkaloids are especially dangerous for the unborn fetus, neonate, and young, and cause venoocclusive disease, chronic or acute liver disease, and cancer. There are many examples of the dramatic effects that poisonous plants, such as Veratrum, Lupinus, poisonhemlock, tobaccos, locoweeds, and others, may have on the embryo or fetus. Although space limits the extent to which the subject may be

Kip E. Panter, Kevin D. Welch, Dale R. Gardner and Benedict T. Green USDA-Agricultural Research Service, Poisonous Plant Research Laboratory, 1150 East 1400 North, Logan, Utah 84341 *Correspondence to: Dr. Kip E. Panter, USDA-ARS-NPA Poisonous Plant Research Laboratory, 1150 East 1400 North, Logan, UT, 84341. E-mail: [email protected] View this article online at (wileyonlinelibrary.com). DOI: 10.1002/bdrc.21053

Published 2013. This article is a U.S. Government work and is in the public domain in the USA.

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TABLE 1. Poisonous Plants that Cause Birth Defects Plant Veratrum californicum (skunk cabbage, false hellebore) Veratrum eschscholtizi Veratrum album Oxytropis, Astragalus Lupinus spp L. caudatus L. sericeus L. nootkatensis L. laxiflorus L. sulphureus L. formosus L. arbustus L. argenteus Nicotiana tabacum, N. glauca

Toxicant Steroidal alkaloids, cyclopamine, jervine, cycloposine Unknown, possibly same as above Same as above Swainsonine, swainsonine N-oxide Quinolizidine alkaloid Anagyrine

Piperidine alkaloid Ammodendrine Anabasine

Conium maculatum (Poison-hemlock)

Coniine and c-coniceine

Prunus serotina (wild black cherry)

Cyanogenic compounds suspected

Datura stramonium (jimsonweed)

Unknown, possibly alkaloids

Sorghum vulgare, S. Sudanese Lathyrus spp L. cicera L. odoratus Mimosa and Leucaena

Cyanogenic compounds suspected Lathyrogens

Mimosine?

discussed, this chapter will review some of the most dramatic examples of poisonous plant-induced effects on the developing embryo and fetus. This chapter is not intended to be all inclusive, and the readers are encouraged to seek other literature sources for specific interests on the subject.

VERATRUM CALIFORNICUM A discussion of the Veratrum spp. and the birth defects associated with ingestion of this plant in early gestation represent one of the most bizarre and dramatic examples of the impact that natural toxins can have on a biological

Effects Cyclopia, cleft palate, limb defects, tracheal stenosis and embryonic death Cyclopia

Cattle, goats, sheep

Cyclopia Bowed limbs, embryo or fetal death Cleft palate, contracture- type skeletal defects

Llamas and alpacas Sheep, cattle, horses: most stages of pregnancy Cattle

Cleft palate, contracture-type skeletal defects Cleft palate, contracture-type skeletal defects Cleft palate, contracture-type skeletal defects Cleft palate, contracture-type skeletal defects Cleft palate, contracture-type skeletal defects Contracture-type skeletal defects Skeletal defects

Cattle, sheep, goats

Skeletal defects and craniofacial malformations

Cattle, sheep, goats

system. The early events leading up to the discovery that Veratrum was responsible for severe and truly unbelievable craniofacial birth defects is historically interesting (James, 1999). Early research began in the late 1950s with the discovery that Veratrum caused the “monkey-faced lamb” syndrome in sheep (Binns et al., 1965; Fig. 1). This plant caused large losses to the sheep industry on rangelands in southeastern and central Idaho in multiple years. Field and subsequent laboratory research followed by management recommendations have essentially eliminated the problem for livestock producers. Once the plant

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Species affected

Horses

Pigs, cattle, sheep, goats

Pigs, cattle, sheep, goats

Pigs

Pigs

Horses Cattle, sheep

was identified, the time of insult determined, and the steroidal alkaloids elucidated, the solution to the problem was quickly resolved. However, and quite importantly, the teratogenic steroidal alkaloids, which were isolated and characterized at the USDA-ARS Poisonous Plant Research Laboratory, were discovered to impact a multitude of hedgehog pathway-controlled biological mechanisms. This discovery has lead to chemical synthesis of molecular probes, elucidation of critical gene pathways of disease, and biomedical treatments. Over the last 30 years, these steroidal alkaloids and their optimized derivatives have had an

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Figure 1. Monkey-faced lamb associated with maternal ingestion of Veratrum californicum on day 14 of gestation. Published with permission of USDA Poisonous Plant Research Laboratory.

Figure 2. Line drawing of Veratrum californicum. Note the large lily-like leaves and long inflorescence.

impressive impact on biomedical research, development of pharmaceuticals, and treatment of difficult diseases such as cancer.

Veratrum belongs to the Liliaceae (Lily) family (Fig. 2), and comprises at least five species in North America. Veratrum californi-

cum grows primarily in the high mountain ranges of the western US (Knight and Walter, 2001; Burrows and Tyrl, 2013). Veratrum viride is the most widespread species and grows in the northwestern US north through western Canada into Alaska, and is also widespread in the northeastern US. V. insolitum grows in a relatively small region of northwestern California and southwestern Oregon; V. parviflorum grows in the central southeastern states; and V. woodii grows from Ohio to Missouri, Oklahoma, and Arkansas. Two other species have been reported to cause poisoning in other countries, V. japonicum in Korea and V. album in Europe. Common names include western false hellebore, hellebore, skunk cabbage, corn lily, Indian poke, wolfsbane, and so forth. Caution should be used when referring to common names as they may be used interchangeably within these genera but also in unrelated genera, that is, hellebore is also used for the genus Helleborus in the buttercup family. Most Veratrum spp. are found in similar habitats of moist, open alpine meadows or open woodlands, marshes, along waterways, in swamps or bogs, and along lake edges in high mountain ranges (Burrows and Tyrl, 2013). Most species grow at higher elevations. All species are similar with coarse, erect plants about 1–2.5 m tall, with short perennial rootstalks. The leaves are smooth, alternate, parallel veined, broadly oval to lanceolate, up to 30-cm long, 15-cm wide, in three ranks, and sheathed at the base. The inflorescence is a panicle of flowers, the lower ones often staminate and the upper ones perfect. The flowers of V. viride are distinctly green and the fruit is three-chambered with several seeds. Toxicity of V. californicum has been studied extensively because of large outbreaks of craniofacial birth defects in sheep during the mid 1900s. On some operations, 25% of pregnant ewes that grazed on infested pastures in the mountains of central Idaho gave birth to

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Figure 3. X-ray computed tomography scan of a monkey-faced lamb showing the synopthalmia.

malformed lambs (Binns et al., 1965; James, 1999). The gross malformations ranged from a series of lethal craniofacial defects, including synophthalmia (congenital cylcops), to less severe deformities of the upper and lower jaws, cleft palate, tracheal stenosis, and limb abnormalities (Binns et al., 1965; Welch et al., 2009; Fig. 3). The Basque shepherds called the grossly deformed cyclops lambs “chatto” which translates as “monkey-faced” lamb. Although many years of research at the USDA-ARS Poisonous Plant Research Laboratory and subsequent management recommendations essentially eliminated livestock losses from Veratrum, new frontiers in biomedical research and disease treatment were discovered. Based on these research findings and the steroidal alkaloids isolated and characterized at the Poisonous Plant Research Laboratory, new molecular probes and research tools to study the hedgehog gene signaling pathway emerged (Gaffield and Keeler, 1996; James et al., 2004; Scales and Sauvage, 2009). Over 50 complex steroidal alkaloids have been identified from the Veratrum spp. (Keeler, 1978; Brown and Keeler, 1978; Keeler, 1984; Keeler et al., 1993; Gaffield and Keeler, 1996). Five classes of

steroidal alkaloids have been characterized based on structure and function: veratrines, cevanines, jervanines, solanidines, and cholestanes. The veratrines and cevanines are of considerable interest in toxicology, as they are neurological toxins and hypotensive agents that bind to sodium channels, delaying closure, and causing cardiotoxic and respiratory effects. The cevanine alkaloids are also found in Zygadenus spp., members of the lily family. The jervanines are most significant in the teratogenic effects, the most notable of which were named cyclopamine and jervine (Keeler, 1978; Fig. 4). Both are potent inducers of the congenital cyclopia “monkey-faced lamb” syndrome. This cyclopic defect is induced in the sheep embryo during the blastocyst stage of development when the pregnant mother ingests the plant during the 14th day of gestation (range 13–15 days; Welch et al., 2009, 2012). Limb defects and tracheal stenosis occur when maternal ingestion includes days 28–33 of gestation (Keeler et al., 1985; Keeler and Stuart, 1987; Keeler, 1990). The solanidine alkaloids found in potatoes and other plants of the nightshade family are structurally similar to those in Veratrum, and

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are toxic and some teratogenic. The cholestanes and other similar alkaloids found in these plants have been used as hypotensive drugs, but are structurally less likely to induce the birth defects. Structure activity relationship is the key for potency to produce the craniofacial birth defects (Keeler et al., 1993; Gaffield and Keeler, 1994). This structure activity relationship impacts the mechanism of action, that being inhibition of the hedgehog signaling gene pathway (Gaffield and Keeler, 1996). This hedgehog gene pathway and the subsequent down-stream regulation of gene expression have now been implicated in numerous human diseases, such as cancer, birth defects, and so forth. The teratogen, cyclopamine, has become a significant biomedical tool in the study of the very complex hedgehog pathway, and cyclopamine derivatives such as IPI-926 have shown great promise in treatment of certain cancers and other hedgehog driven diseases (Scales and Sauvage, 2009; Olive et al., 2009).

LUPINES, POISONHEMLOCK, AND NICOTIANA GLAUCA Lupines, poison-hemlock (Conium maculatum), and Nicotiana spp. (N. tabacum, N. glauca) will be discussed together because the congenital malformations (flexure-type skeletal contractures and cleft palates) and the mechanism of action are the same (Panter et al., 1990, 1999a). The lupines are by far the most economically important of the three genera to livestock producers. The teratogenic effects of lupines are primarily a problem in cattle, whereas poison-hemlock is reported to cause field cases of skeletal defects in cattle, horses, and pigs, and poisoning in a multitude of animal species, including avian species and humans. Nicotiana tabacum has caused outbreaks of malformations in piglets born to pasture-grazed pregnant sows. Although N. glauca has caused death losses in livestock, it has not

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been reported as a cause of malformations in livestock in field cases. It is a rich source of the teratogenic alkaloid anabasine, which

is an important chemical tool in our laboratory for basic and biomedical research using a congenital goat cleft palate model

(Weinzweig et al., 1999, 2008). Lupines contain quinolizidine and a few piperidine alkaloids; poisonhemlock and N. glauca contain piperidine alkaloids; and N. tabacum contains pyridine and piperidine alkaloids. Piperidine and quinolizidine alkaloids are widely distributed in nature, and most possess a certain level of toxicity (Fig. 5). Some alkaloids are teratogenic, depending on structural characteristics which are now substantially understood.

Lupine Toxicity

Figure 4. Steroidal alkaloid teratogens cyclopamine and jervine from Veratrum.

Stockmen long ago recognized the toxicity of lupines, especially in late summer and fall when the pods and seeds are present. Thousands of sheep died in the late 1800s and early 1900s, when sheep producers harvested lupine hay for winter feed. Not understanding the danger of the alkaloids

Figure 5. Quinolizidine and piperidine alkaloid teratogens found in lupines, poison–hemlock, and Nicotiana spp. Birth Defects Research (Part C) 99:223–234 (2013)

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Figure 6. Lupine-induced crooked calf syndrome (CCS).

in the seed pods and late summerharvested lupine hay resulted in very toxic forage, which killed many sheep during the winter months. The principle clinical signs were muscular weakness, ataxia, and death from respiratory failure (Panter et al., 1999a). Clinical signs appear as early as 15–30 min after ingestion of lupine, and death may result within hours to a few days, depending on the alkaloid content and concentration in the plant and how fast the animal consumes it. Generally, if death does not occur within this time frame, the animal recovers completely. Because of their foraging behavior and preference for forbs, sheep are more likely to die from acute lupine poisoning than cattle. However, in the mid 1900s, a second syndrome associated with lupine emerged in cattle and was referred to as “crooked calf syndrome” (CCS; Fig. 6). This problem has been much more difficult to manage, because so many cattle ranges in the west are infested with lupines that cause the CCS. This continues to be a major economic problem today for cattle ranchers

throughout the western US, and especially in the northwest. Quinolizidine alkaloids are the primary group of toxins in lupines, but a few species also contain the piperidine alkaloid ammodendrine and its chemical derivatives. More than 150 quinolizidine alkaloids have been structurally identified from the Leguminosae family, including Lupinus, Laburnum, Cytisus, Thermopsis, and Sophora (Schmeller et al., 1994; Wink et al., 1995). Quinolizidine alkaloids occur naturally as N-oxides as well as free bases, but very little research has been done on the toxicity or teratogenicity of the N-oxides. However, the N-oxides of PAs are reduced to the corresponding free bases in the rumen, and it seems likely that quinolizidine alkaloids could undergo a similar conversion (R. J. Molyneux, personal communication). All of these alkaloids are toxic; however, the degree of toxicity is dependent on chemical structural characteristics. Interestingly, only the quinolizidine alkaloid anagyrine is teratogenic in cattle and causes CCS (Keeler, 1976).

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Piperidine and quinolizidine alkaloid content as well as individual proportions vary in plants, depending on environmental conditions, season of the year, and stage of plant growth (Wink and Carey, 1994). Typically, alkaloid content is highest during early growth stages, decreases through the flower stage, and increases in the seed and pod stage. This knowledge has been used in management strategies to reduce losses to cattle producers. Alkaloid profiles vary considerably within and between lupine species (Wink and Carey, 1994). Season, environment, and location influence alkaloid profile and concentration in a given species of lupine. Site differences in alkaloid levels have been described and are substantial (Carey and Wink, 1994). Total alkaloid content decreases as elevation increases, and was shown to be six times higher in plants at 2700 m versus plants collected at 3500 m. This phenomenon persists even when seedlings from the highest and lowest elevations were grown under identical greenhouse conditions, suggesting genetic differences as plants adapt to elevation changes. Generally, alkaloid content is highest in young plants and in mature seeds. For many lupines, the time and degree of seeding varies from year to year. Most direct death losses have occurred under conditions in which animals consume large amounts of pods or toxic plants in a brief period. This is especially true in sheep, and happens when livestock are driven through an area of heavy lupine growth, unloaded into such an area, trailed through an area where the grass is covered by snow exposing lupine only, or when animals are forced to eat the plants due to overgrazing (Panter et al., 1999a). Most poisonings occur in the late summer or early fall because seed pods are present and lupine remains green after other forage has matured or dried. Most calf losses occur because of teratogenic effects resulting from their mothers grazing lupine plants or pods during susceptible stages of pregnancy. Lupine-induced “crooked calf syndrome” results when pregnant

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cattle graze lupines containing either the quinolizidine alkaloid anagyrine or piperidine alkaloids of which ammodendrine, N-methyl ammodendrine, and N-acetyl hystrine are the most likely teratogens (Panter et al., 1999a). Subsequently, calves may be born with multiple skeletal contracture malformations and/or cleft palate, depending on the stage of pregnancy when the dam grazes the lupines.

Poison-Hemlock (Conium maculatum) Poison-hemlock (Conium maculatum) has interesting historic significance as the “hemlock tea” used for execution in ancient Greece and the decoction used to put the philosopher Socrates to death (Daugherty, 1995). Toxicoses in livestock frequently occur and field reports of teratogenic effects in cattle and pigs have been reported (Edmonds et al., 1972; Panter et al., 1985a,b; Panter et al., 1999a). Poison-hemlock contains at least five piperidine alkaloids, all of which are believed to contribute to the toxicity. Coniine and c-coniceine predominate and are believed to also contribute to the teratogenic effects. The teratogenic effects are the same as those induced in cattle by lupines, that is, cleft palate and multiple congenital skeletal contractures. Clinical signs of poisoning are similar to those described for lupine, although onset may be more rapid. The early clinical signs are stimulation progressing to depression, resulting in relaxation, recumbency, and eventually death from respiratory paralysis if the dose is high enough. Poisoning has been reported in cattle, pigs, goats, elk, wild geese and quail, domestic turkeys and chickens, and people (Copithorne, 1937; MacDonald, 1937; Edmonds et al., 1972; Jessup et al., 1986; Frank and Reed, 1990; Frank et al., 1995). Coniine, c-coniceine, and Nmethyl coniine are the principal alkaloids in Conium, with relative concentration depending on the stage of plant growth. All are toxic and teratogenic, and structural

characteristics impart potency (cconiceine > coniine > N-methyl coniine) (Leete and Olson, 1972; Keeler and Balls, 1978; Lee et al., 2008, 2013; Green et al., 2013). More detailed research on molecular bioactivity of these toxins is reported in a separate paper by Green et al. (2013) in this volume.

Nicotiana spp. Nicotiana tabacum has been responsible for large outbreaks of skeletal malformations and cleft palate in newborn pigs in the late 1960s in Kentucky (Menges et al., 1970). Research determined that nicotine was not the teratogen, but a simple piperidine called anabasine found in the tobacco stalks was the cause. Although Nicotiana glauca has not been associated with field cases of teratogenesis in livestock, there have been reported cases of toxicoses (Plumlee et al., 1993). Subsequent experiments determined that birth defects could be induced in pigs, cattle, sheep, and goats, and these birth defects mimicked those caused by lupine in cattle (Keeler et al., 1981, 1984; Panter and Keeler, 1992). N. glauca is a rich source of anabasine and has been used extensively in our research to elucidate mechanism, characterize biochemical, and molecular activity of this class of teratogens, and for biomedical research. Because of its chemical stability and repeatability, N. glauca was used to develop a cleft palate goat model to research novel methods of treatment for cleft palate in children. For further information, review Weinzweig et al. (1999, 2008).

Teratogenicity of Lupines, Poison-Hemlock and Nicotiana glauca The teratogenic mechanism of action of Lupinus, Conium, and Nicotiana spp. is the same, but bioactivity and potency of the individual alkaloids between and within these plants varies, depending on structural characteristics. Detailed information on the biochemical and molecular activity at

nicotinic acetylcholine receptors of the individual piperidine alkaloids is reviewed by Green et al. (2013) in this volume. Briefly, it was determined using ultrasound imaging that the piperidine alkaloid teratogens inhibit fetal movement during the critical stages of pregnancy, resulting in the skeletal contracture defects and cleft palate. The periods of gestation when the fetus is susceptible to these plant teratogens have been defined in cattle, sheep, goats, and swine (Table 2; Shupe et al., 1967; Panter et al., 1985a,b, 1997, 1999a; Panter and Keeler 1992). The severity and type of the malformations are dependent on alkaloid dosage ingested, the stage of pregnancy when the plants are eaten, and the length of time ingestion takes place. In swine, cleft palate only occurred when Conium was fed during days 30– 41 of gestation (Panter et al., 1985b). Skeletal defects, predominantly the forelimbs, spine, and neck without cleft palate, were induced when pregnant sows were fed Conium during gestation days 40–53 (Panter et al., 1985a). When feeding of Conium included days 50–63, rear limbs were affected also. When the feeding period for Conium included days 30–60, all combinations of the defects described occurred. In sheep and goats, the teratogenic insult period is similar to pigs, and includes days 30–60 (Keeler and Crowe, 1984; Panter et al., 1990). In goats, a narrow period for cleft palate induction was defined to include days 35–41 (Panter and Keeler, 1992). The cleft palate induction period in cattle was defined from 40–50 days (Panter et al., 1998). For CCS in general, the critical gestational period for cattle producers is 40–100 days, and any variation of birth defects may be expected depending on the amount of alkaloid ingested and the exact time frame when ingestion occurred. Even though research at the Poisonous Plant Research Laboratory has been limited to the three genera aforementioned, there are

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TABLE 2. Susceptible Periods of Gestation for Alkaloid-Induced Cleft Palate and Multiple Skeletal Contractures (MCC) in Cattle, Sheep, Goats, and Swine Days gestation Defect Cleft palate MCCa

Cattle

Sheep

Goats

Swine

40–50 40–70 40–100

35–41 30–60

35–41 30–60

30–40 40–53 50–63 30–60

a MCC–multiple congenital contractures–include arthrogryposis, scoliosis, kyphosis, and torticollis. Rib cage anomalies and asymmetry of the head also occur.

many other plant species that contain piperidine and quinolizidine alkaloids structurally similar to what we would expect to be both toxic and teratogenic. These include species in the following genera: Genista, Prosopis, Lobelia, Cytisus, Sophora, Pinus, Punica, Duboisia, Sedum, Withania, Carica, Hydrangea, Dichroa, Cassia, Ammondendron, Liparia, Colidium, and others (Keeler and Crowe, 1984). Many plant species or varieties from these genera may be included in animal and human diets; however, toxicity and teratogenicity are a matter of dose, rate of ingestion, and alkaloid concentration and composition in the plant.

LOCOWEEDS (ASTRAGALUS AND OXYTROPIS SPP.) Locoweeds, species of the Oxytropis and Astragalus genera containing the indolizidine alkaloid toxin swainsonine (Molyneux and James, 1982; Molyneux et al., 1991), reduce reproductive performance in livestock (Panter et al., 1999b). Most aspects of reproduction are affected, including mating behavior, libido and spermatogenesis in males, estrus behavior and conception in females, fetal growth and development, reproductive maturity at puberty, and neonatal/ maternal behavior. Although extensive research has been done to characterize and describe the histological changes, we have just begun to understand the magni-

tude of the physiological problems, the mechanism of action of reproductive dysfunction, and management strategies needed to prevent losses. Once animals begin to graze locoweed, nearly immediate measurable increases in serum swainsonine with concomitant decreases in a-mannosidase occur (Stegelmeier et al., 1995a,b). Although these measurable changes are diagnostic for locoweed ingestion, the rapid clearance of swainsonine from serum (t1=2  20 hr) and accompanying rapid recovery of amannosidase (t1=2  65 hr) limits serum analysis of these parameters as a reliable test for past or longterm locoweed exposure (Stegelmeier et al., 1995a). Currently, diagnosis of locoweed poisoning relies on history of locoweed ingestion, behavioral changes, loss of condition, and in terminal cases, histological evidence of neurovisceral vacuolation. Histological lesions induced by locoweed and pure swainsonine (James et al., 1991; Panter et al., 1999b) have been compared and are the same. Lesions appear to develop in a threshold-like fashion over time (Van Kampen and James, 1969, Van Kampen, 1970; James et al., 1970a, b; Stegelmeier et al., 1999a, 1999b), and animal tissues (such as liver and kidney) that accumulate high swainsonine concentrations develop lesions more rapidly than other organ systems (Stegelmeier et al., 1998). Even

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though a-mannosidase activity in serum recovers quickly, tissue repair and return to normal organ function occur much more slowly (Stegelmeier et al., 1999a,b), which is especially true for reproductive function. Some neurological lesions do not resolve and longterm behavioral changes appear to be permanent. Locoweeds affect almost every aspect of reproduction in the female, such as estrus behavior, estrous cycle length, ovarian function, conception, embryonic and fetal viability, embryo and fetal growth and development, and maternal/infant behavior and bonding (Hartley and James, 1975; Panter et al., 1987, 1999b; Pfister et al., 1993, 2006; Panter and Stegelmeier, 2000). Locoweed fed to cattle and sheep at various times and dosages temporarily altered ovarian function, increased estrous cycle length, altered breeding behavior, reduced conception rates, and caused birth defects in offspring (Panter et al., 1999c; Fig. 7). Experimental feeding trials in sheep with locoweeds (A. mollissimus, A. lentiginosus, and O. sericea) altered the estrous cycle, delayed and shortened estrus behavior, decreased conception rates, and reduced the number and quality of viable embryos. Although only a few abnormal morula-stage embryos were collected from ewes after 30 days of feeding locoweed, in vitro data using bovine oocytes demonstrated that swainsonine added to culture media at different concentrations (up to 6.4 mg/mL) did not directly interfere with bovine oocyte maturation, in vitro fertilization (IVF), or preimplantation embryo growth and development (Wang et al., 1999). Furthermore, pregnancy rates were not different from controls when these swainsoninecultured (in vitro maturation/in vitro fertilization/in vitro culture) bovine embryos were transferred to recipient heifers. This research suggests that the negative effects locoweed has on early embryo viability and development in vivo are not from direct effects of swainsonine on the oocyte or the preimplantation

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the carpal or tarsal joints; curvature and rigidity of most joints; over extension or contracture of the pasterns; lateral rotation of the forelimbs; scoliosis; kyphosis; torticollis; and brachygnathia. Some of the contracture malformations may resolve spontaneously but many are permanent. Neonate behavior is usually compromised reducing nursing ability, maternal/ infant bonding, and survival. Although research and field observations have demonstrated that locoweed affects almost every aspect of reproduction in livestock, several questions still remain unanswered. How long and how much locoweed can livestock eat before reproduction declines? What functions are affected first? When are reproductive effects irreversible? What are the modes of action? How long does it take for reproductive function to return to normal once locoweed ingestion stops? Answers to these fundamental questions will aid in management decisions to improve reproductive performance and allow better utilization of locoweed-infested ranges. Figure 7. “Windswept” lamb produced by maternal ingestion of locoweed. Variations of skeletal defects may be seen and the mechanism of action is unknown.

embryo. Therefore, the oocyte/preimplantation embryo effects are apparently secondary and result from effects on other facets of reproduction, such as the maternal pituitary/hypothalamic axis where glycoprotein gonadotropins are produced and released, at the ovarian/ utero interface, or the utero/placental interaction once pregnancy recognition is established. Although gross and microscopic lesions in the dam may begin to resolve quickly after locoweed ingestion ceases, fetal effects may be prolonged as a result of abnormal placentation (Hafez et al., 2007) and severe enough to result in fetal/embryo death and resorption or abortion, birth of small weak offspring, or reduced maternal/infant bonding and impaired nursing ability of the neonate (Panter et al., 1987, 1992; Pfister et al., 2006). Locoweed ingestion during gestation days 100–130 disrupted normal maternal infant

bonding compared to control ewelamb pairs (Pfister et al., 2006). Lambs from mothers ingesting locoweed failed to suckle within 2 h after birth, were slower to stand, and were less vigorous than control lambs. Maternal ingestion of locoweed also disrupted the learning ability of neonatal lambs. Swainsonine is also excreted in the milk and can result in further intoxication of nursing offspring or exacerbate intoxication when offspring begin to graze locoweeds and continue to nurse their locoweed-grazing mothers (James and Hartley, 1977). Locoweed ingestion causes skeletal birth defects and behavioral changes in offspring of cattle, sheep, and horses (James, 1977). These birth defects or abortion may be expected when pregnant dams ingest locoweed during most stages of pregnancy. The congenital malformations may include one or more of the following: flexure of

OTHER TERATOGENIC PLANT SPECIES Table 1 lists other poisonous plants with suspected or known teratogenic effects. Although experimental substantiation is not complete with many of these plants, research is ongoing.

Cyanogenic Plants Although the implication of malformations induced by plants containing cyanogenic glycosides lacks experimental substantiation, reports of skeletal defects in pigs, calves, and foals associated with maternal ingestion of cyanogenic plants species is cause for further investigation. Limb contractures in calves and foals have been reported with a known history of maternal ingestion of sudan or sorghum (Van Kampen, 1970; Seaman et al., 1981). Other signs of toxicoses and pathology in mares, such as posterior ataxia, cystitis, and myelomalacia, indicate that a potential

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teratogenic effect may exist. Similar contracture malformations in pigs have been implicated with consumption of wild black cherries by pregnant sows (Selby et al., 1971). Confirmation of a specific cyanogenic glycoside or group of glycosides is lacking, although fetal anoxia from HCN hydrogen cyanide (prussic acid) has been theorized in livestock.

Nightshades The nightshade family of plants is large and comprises over 2300 species worldwide. Although most contain toxic alkaloids, only a few genera, including Datura, Solanum, and Nicotiana, have been associated with birth defects in United States. Spirosolane alkaloids found widely in the Solanum genus are structurally related to the teratogenic Veratrum alkaloids and contribute to the list of toxins with known teratogenic activity. Although solasodine was teratogenic in a hamster model, neither tomatidine nor the form of solasodine which lacks a nitrogen atom (diosgenin) showed any teratogenic activity. The solanidane alkaloids in potatoes and other plants are less closely related to the Veratrum alkaloids but research studies demonstrated that the alkaloid glycosides a-solanine and a-chaconine and the aglycone epimers solanidine and demissidine were teratogenic in a hamster model, although at a reduced level of activity (Keeler et al., 1993). Jimson weed (Datura stramonium) was reported to be the cause of an outbreak of skeletal deformities in newborn pigs in Kansas in 1971 (Leipold et al., 1973). Experimental feeding trials in Hampshire sows that included gestation days 28–90 failed to demonstrate the birth defects (Keeler, 1981). Research evidence and suggested mechanisms of action are inconclusive and further studies are needed.

Lathyrus and Vicia Certain members of the Lathyrus and Vicia genera contain com-

pounds called osteolathyrogens which cause congenital skeletal defects in offspring when their pregnant mothers eat the plant. The malformations are characterized as contracture or flexure of the pastern and carpal joints, lateral rotation of the forelimbs, scoliosis, kyphosis, torticollis, and front limb abductions (Keeler and James, 1971). The extent to which these two genera contribute to congenital malformations in livestock grazing on native ranges in the US is unknown, but believed to be minimal. Feeding the osteolathyrogen aminoacetonitrile for as few as 10 days anytime during gestation days 20–129 in sheep produced birth defects (Keeler and James, 1971). The natural lathyrogen b-aminopropionitrile is believed to be the putative teratogen.

Mimosa and Leucaena Leucaena and Mimosa are tropical plants of high protein content used for forages in tropical areas including the US Virgin Islands, Hawaii, South America, and so forth. Mimosine is known to be toxic and is believed to possess teratogenic activity; other derivatives of mimosine are also believed to contribute to the teratogenic effects of the plants. In South American countries, Mimosa tenuiflora and M. ophthalmocentra are responsible for various skeletal and craniofacial malformations, including flexure of the forelimbs, cleft palate, brachygnathia, agnathia, cranioschisis, xyphosis, harelip, and soft tissue anomalies, including segmental stenosis of the colon and meningocele. In livestock, the first trimester is believed to be the most susceptible gestational period (Riet-Correa et al., 2006; Pessoa et al., 2011).

CONCLUSIONS AND IMPLICATIONS Birth defects in livestock are economically important and significantly affect the livelihood of producers, whereas birth defects in humans are of economic importance to society and emotionally

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devastating for families. Similar types of birth defects occur in children, and although relatively few of these would be specifically caused by plant or plant product ingestion during pregnancy, there may be genetic, environmental, or nutritional factors that contribute. The plant-induced birth defects in animals and the ensuing research provide important knowledge, investigative techniques, chemical tools or probes, and animal models to study these defects in people. The research on teratogenic effects of poisonous plants and the subsequent understanding of the structure activity relationship between the chemistry and the molecular events serve as a template for biomedical research and discovery of novel techniques and new pharmaceuticals or neutraceuticals to treat human and animal diseases. In animals, reproductive success is the single most important economic multiplier for livestock producers in the US. The recognition that poisonous plants may have a major impact on reproductive performance is relatively new and not fully realized. Compared to carcass and growth traits, reproductive performance is considered to be 5 and 10 times more significant, respectively. It must be kept in mind that if reproduction fails, the other values have little relevance. Reproductive performance not only relates to an animal’s ability to produce offspring, but to produce it at a proper time interval and provide proper neonatal care and nutrition. Effects on spermatogenesis, oogenesis, libido, fertilization, placentation, embryo/fetal survival, development (birth defects) and growth, postpartum intervals, and neonatal survival and development are all factors affected by poisonous plants. The following basic concepts have been recommended to livestock producers and will reduce risk of poisonous plant losses. In limited situations, these ideas are adaptable for basic understanding of the dangers of some plants to humans also. 1. Recognize the plants on your range or in your yard and seek

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2.

3.

4.

5. 6. 7.

8.

9.

appropriate help from extension, university, or federal agencies for identification. Know the potential hazards of these plants and understand conditions in which poisoning may occur. Avoid introducing naive animals into unfamiliar pastures or ranges where poisonous plants grow. Do not introduce animals into poisonous plant infested ranges before adequate quality feed is available. Many toxic plants emerge before grasses are sufficient for grazing. Do not discard grass, shrubs, or tree clippings where livestock have access. For example, yew clippings are a common cause of poisoning in cattle and horses. Provide free choice access to fresh water and trace mineral salt. Do not overstock or overgraze pastures. Avoid bedding, lambing/calving, watering, salting, or unloading hungry animals near poisonous plant populations. Avoid excess stress to affected animals, especially when animals may be showing clinical effects. Control poisonous plants if economically feasible, either through hand grubbing, mechanical clipping, or herbicide treatment.

ACKNOWLEDGMENT The authors thank Ms. Terrie Wierenga of the USDA Poisonous Plant Research Laboratory for technical assistance and for editorial and formatting oversight of the manuscript. No conflicts of interest exist for the authors.

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