Pyrrolizidine alkaloids and seneciosis in farm animals. part 1: occurrence, chemistry and toxicology

Pyrrolizidine alkaloids belong to a class of phytotoxins which are present in more than 6000 plant species. The disease seneciosis in farm animals represents the severe poisoning by pyrrolizidine alkaloids from plants of the genus Senecio. This form of poisoning has been known since the end of the 19th century in Germany, the USA, Canada and New Zealand, and is mainly caused by Senecio jacobaea and related Senecio spp. in farm animals, including poultry. Animal poisoning by pyrrolizidine alkaloids is of worldwide importance. In Germany poisoning of horses and cattle by Senecio jacobaea, which was earlier named Schweinsberg disease, is of renewed relevance for veterinary medicine. The disease occurs almost entirely as a consequence of chronic poisoning and in general ends fatally. The ultimate cause is the formation of toxic metabolites of pyrrolizidine alkaloids in the liver, and their covalent binding to nucleic acids and proteins leading to liver cirrhosis. Because many pyrrolizidine alkaloids possess mutagenic, and a few also carcinogenic properties, European and international authorities are concerned about possible residue levels in food of animal origin. The review addresses in its first part several aspects, being the occurrence, the chemistry, and the toxicology of pyrrolizidine alkaloids as well as animal intoxications by poisonous plants. In the second part (46) clinical characteristics of animal seneciosis, the therapeutic interventions, the significant species differences and a critical assessment of so-called nontoxic amounts of Senecio plants in animal fodder with reference to cumulative lethal toxin doses are presented.     

The toxicity of Senecio inaequidens DC

This study was designed to confirm the toxicity of a plant implicated in an outbreak of poisoning of stock in Frankfort, Free State Province, South Africa. Cows died acutely after being introduced into a camp, where an abundant, green shrublet was noted to be heavily grazed. This plant was subsequently identified as Senecio inaequidens DC. (Asteraceae) by the South African National Biodiversity Institute (SANBI). Extraction and chemical analyses for pyrrolizidine alkaloids (PAs) in Senecio inaequidens revealed the presence of 4 different compounds, namely retrorsine and senecionine (known to be hepatotoxic) and 2 unidentified compounds. The average total PA (free base plus N-oxide) concentration in plant parts of S. inaequidens collected at Frankfort during the outbreak was 0.81%, compared with the total alkaloid content in the dried, milled S. inaequidens plant material, collected 7 weeks after the outbreak, of only 0.18%. Male Sprague-Dawley rats (n = 4), aged 8-9 weeks, were dosed per os. Each rat received a different dose of the crude Senecio inaequidens extract, ranging from 0.049 mg/g body weight (b.w.) to 0.25 mg/g b.w. No clinical signs were observed in the rat receiving the lowest dose. Rats receiving higher doses showed depression, an unsteady gait, pilo-erection and jaundice, which was particularly noticeable in the ears. Clinical chemistry evaluation revealed an increase in the activities of ALP (except Rat 4), AST and GGT in all animals. Total serum bilirubin, creatinine and urea concentrations were also elevated. All rats had low serum globulin concentrations with an A/G ratio above 1.2. Post mortem examination of the rats revealed marked hepatic lesions. Histopathologically, these changes were characterised by necrosis (variable in extent) of the centrilobular and midzonal hepatocytes (but sparing the portal hepatocytes), with extensive haemorrhage and congestion. Proliferation of the bile ducts, fibrosis and oedema were also present. Ultrastructural changes in affected rats were characterised by margination of chromatin, the presence of numerous autolysosomes in necrotic hepatocytes, intramitochondrial woolly inclusions and changes in the endoplasmic reticulum. A sheep, also dosed with the crude extract, failed to exhibit clinical signs, clinical chemistry aberrations or macroscopic lesions; however, examination of the liver of this sheep revealed histopathological and ultrastructural changes similar, though milder, to those displayed by the rats. Pyrrolizidine alkaloids were extracted from the liver and kidneys of the rats and the sheep. In the case of the sheep, retrorsine was also detected in the lungs, urine and bile.     

SENECIO LAUTUS TOXICITY IN CATTLE

Mortalities and a poor growth syndrome in cattle in the Hunter River valley of New South Wales were associated with pyrrolizidine alkaloid toxicity caused by Senecio lautus (common fireweed). A clinical problem of poor growth rate on a large property, where S. lautus was very common, was associated with consistent evidence of some degree of hepatic pyrrolizidine alkaloid lesions in young growing cattle and cows. S. lautus subsp maritimus from this property caused the death of 2 experimentally fed calves within 77 days and depressed growth rate in a third calf. Pyrrolizidine alkaloidosis was evident histopathologically in the liver, biliary tree and gall bladder and other organs, especially kidney, of these calves. Histopathology of liver biopsy samples was the most sensitive monitoring technique of those used to indicate exposure to pyrrolizidine alkaloids in this investigation.     

Evaluation of amino acids, B vitamins and butylated hydroxyanisole as protective agents against pyrrolizidine alkaloid toxicity in rats

Supplementation of the diets of rats with branched chain amino acids (BCAA: leucine, isoleucine, valine) did not alter their susceptibility to chronic poisoning by tansy ragwort (Senecio jacobaea), which contains hepatotoxic pyrrolizidine alkaloids (PA). Phenobarbital in the diet, which alters liver microsomal enzyme activity, also did not alter susceptibility to PA poisoning. A combination of butylated hydroxyanisole (BHA), cysteine and BCAA did increase (P less than .05) survival time of rats fed tansy ragwort. Dietary BHA and cysteine increased the survival time of rats injected with the PA monocrotaline, with evidence that addition of vitamin B12 and folic acid improved the effectiveness of this treatment. In a chronic feeding trial with tansy ragwort, a combination of BHA and cysteine increased (P less than .05) the survival times of rats, showing protective activity against PA poisoning. A mixture of B-complex vitamins, or vitamin B12-folic acid, was not effective in improving the response.     

青霉酸毒性研究进展

青霉酸是圆弧青霉代谢产物的主要成分。现已证明,青霉酸对各种动物具有毒性,主要引起心脏、肝脏和肾脏等器官的损伤,并具有潜在致癌性。青霉酸在饲料中的含量很高,而且污染极其广泛。因此,研究青霉酸的毒性、中毒机理及与其他毒素的联合作用,对保证饲料卫生和动物健康具有很重要的意义,该文就青霉酸的器官毒性、细胞毒性、分子毒性及联合毒性的研究进行综述。     

Oxidative stress in the erythrocytes of cattle intoxicated with Senecio sp

BACKGROUND: Intoxication caused by Senecio sp is characterized by irreversible damage to liver cells and may be associated with oxidative stress. OBJECTIVES: The aim of this study was to evaluate the effects of intoxication by Senecio sp on lipoperoxidation, antioxidant defenses, and the osmotic resistance of erythrocytes in cattle. METHODS: Blood samples from 30 intoxicated animals (group 1) and 30 healthy animals (group 2) were analyzed. The diagnosis of poisoning by Senecio sp was based on histopathologic lesions verified through hepatic biopsy. The following biochemical parameters of oxidative stress in the erythrocytes were determined: thiobarbituric acid-reactive substances (TBARS), copper-zinc superoxide dismutase (CuZnSOD) activity, and nonprotein sulfhydryl (NPSH) groups. Erythrocyte osmotic fragility also was evaluated. RESULTS: TBARS concentration and CuZnSOD activity were significantly (P <.001) higher in group 1 when compared with group 2. The concentration of erythrocyte NPSH groups was significantly (P <.03) lower in group 1 when compared with group 2. Osmotic fragility was more pronounced in the erythrocytes of group 1 when compared with group 2 (P < .001). CONCLUSIONS: The results of this study indicate that poisoning by Senecio sp causes an increase in lipoperoxidation, oxidation of NPSH groups, and consequently, oxidative stress in bovine erythrocytes that may contribute to hemolysis. These findings may contribute to a better understanding of the mechanisms involved in cell damage in animals intoxicated by Senecio sp.     

Pyrrolizidine alkaloid poisoning of yaks (Bos grunniens) and confirmation by recovery of pyrrolic metabolites from formalin-fixed liver tissue.

Severe losses of yaks (Bos grunniens) were investigated in the most eastern region of Bhutan. The most serious disease was a fatal chronic skin disease with emaciation and anaemia. Post mortem examinations revealed major lesions in the liver, consisting of hepatic megalocytosis, bile duct proliferation, fibrosis and remodelling of the hepatic structure. There was also renal megalocytosis, ascites and mild to moderate icterus. Pyrrolizidine alkaloid poisoning was diagnosed and confirmed by demonstrating sulphur-bound pyrrolic metabolites of the alkaloids in preserved liver tissue. Skin lesions with hyaline parakeratosis were an important feature. Similar lesions were found in the mucosa of the oral cavity. No records could be found of such skin lesions in any other species or disease. Pyrrolizidine alkaloid poisoning seriously affects the livelihood of the local population which depends almost entirely on the yak. Various control measures are discussed. The key to be found is the identification of the toxic plants.     

Phytic acid-induced metabolic changes in the rat

Phytic acid (PA) is present in considerable amounts in the seeds of many plant species consumed by animals. Little is known about its influence on metabolic processes. In the performed experiment the effect of rats feeding a standard laboratory diet (Murigran) with increasing supplements of PA on some hormones and biochemical parameters was tested. Male Wistar rats were divided into five groups of eight animals each. In the first group (control) rats were fed a diet without any supplement whereas the groups II-V received food with added PA: 0.1, 0.2, 0.3 and 1%. After 20 days animals were decapitated, blood serum, liver and tight muscles were sampled. In rats fed a diet enriched in PA the concentration of thyroid hormones was diminished. Simultaneously, T(3)/T(4) ratio was slightly reduced. These changes were accompanied by a rise in blood glucose level and an augmentation in liver and muscle glycogen stores and were found in spite of unchanged blood insulin. Consumption of food with increased amounts of PA resulted in a substantial reduction of liver triglyceride content, but serum triglycerides were not affected. In rats from groups II and III serum free-fatty acids concentration was reduced. However, in animals receiving highest PA supplement this effect was not observed. Serum calcium and magnesium were not affected by PA. Serum iron was significantly reduced, but only in rats on the highest supplement of the tested compound. Results obtained in this experiment clearly indicate that the PA is able to induce hormonal and metabolic changes in animals. These changes seem to result not only from reduced bioavailability of minerals but also from interactions of PA with nutrients and enzymes in the liver.     

峨眉千里光对小鼠急性毒性及蓄积毒性研究

评价峨眉千里光毒性作用,采用机率单位法（Bliss法）和固定剂量蓄积系数法考察了峨眉千里光叶和全草水提物对小鼠经口急性毒性及蓄积毒性。急性毒性结果显示,不同剂量的峨眉千里光叶和全草水提物均能导致小鼠发生中毒和死亡,引起小鼠肺和肝脏损伤;叶水提物LD50为62.28 g/kg,95%可信限为52.68-73.65 g/kg;全草水提物LD50为81.65 g/kg,95%可信限为70.15-94.53 g/kg。蓄积毒性结果显示,峨眉千里光叶和全草的蓄积系数K〉5。结果表明,峨眉千里光叶和全草水提物对小鼠有急性毒性作用,叶的毒性比全草强,肺和肝脏为毒性靶器官;两种水提物均有轻度蓄积毒性。     

Effect of alpha-interferon and alpha-tocopherol in reversing hepatic cirrhosis in rats

The aim of this study was to assess the effects of alpha-interferon and alpha-tocopherol (vitamin E), or a combination of both, in reversing hepatic fibrosis following the induction of cirrhosis using thioacetamide by histological and biochemical analysis. Fifty male Wistar rats were used in this study. The animals were divided equally into five groups. Animals in group I were used as controls. The remaining animals (groups II-V) were provided with 0.5 g/L of thioacetamide in order to induce liver cirrhosis. Group II animals were used as the cirrhotic control. Animals of groups III, IV and V were given alpha-interferon, alpha-tocopherol and interferon together with alpha-tocopherol, respectively, for 30 days. After 30 days the animals were killed and following gross morphological examination of the liver, the hepatic tissues were processed for histological analysis and the serum was used for liver function tests. Morphological analysis showed a decrease in the number of nodules on the surface of the liver in both interferon- as well as vitamin E-treated cirrhotic rats. Histopathological analysis showed that the abnormalities of the cirrhotic liver were partially reversed and liver function tests showed an overall improvement following treatment of animals of groups III, IV and V. Combination therapy using both interferon and alpha-tocopherol did not have any substantial effect on the rats compared with that when they were given separately. These findings suggest that alpha-interferon and alpha-tocopherol may have therapeutic value in reversing liver cirrhosis.     

Toxicity studies with the blue-green alga Oscillatoria agardhii from two eutrophic Norwegian lakes

Extracts of the blue-green alga Oscillatoria agardhii were tested for acute toxicity on laboratory mice and rats. Material originating from lake Gjersjøen proved to be toxic to the animals, samples from the nearby lake Årungen did not. Clinical symptoms culminated in the development of a fatal shock due to decrease in circulating blood volume. Pathological examination revealed heavy pooling of blood in the liver and severe damage to the organ. Blood analyses also indicated liver damage. Effects were the same with extracts from a laboratory clone culture as from a natural water bloom, but the toxin content was higher in the bloom material. Toxicity was not affected by heat, acid or alkali treatment.     

A mechanistic insight into 3,4-methylenedioxymethamphetamine ("ecstasy")-mediated hepatotoxicity

3,4-Methylenedioxymethamphetamine (MDMA, "ecstasy") is a popular drug of abuse among young people with stimulant and hallucinogenic properties. The drug is generally thought to be safe among consumers due to its low-mortality rates. However, MDMA-adverse effects can occur and the risks are not clearly associated to a specific pattern since the consumption quantity seems not to be correlated with the initiation and severity of the injury. MDMA-mediated adverse health effects have been widely studied and can be evoked by multiple factors such as hyperthermia, polydrug abuse (drug-drug interactions), the altered release of neurotransmitters, impairment of mitochondrial function and apoptosis, metabolism and immune responses. Another adverse effect often associated with MDMA is liver toxicity, yet the mechanism of MDMA-induced liver toxicity is not completely understood. A critical starting point appears to be the hepatic metabolism of MDMA by phase I and II enzymes, leading to reactive metabolites. Elucidating the mechanism of hepatic injury mediated by MDMA is of high toxicological and clinical relevance. In this review, an overview of the literature and the latest findings with respect to the mechanism of MDMA-mediated liver toxicity is described.     

Zoonoses transmitted by mouse and rat maintained as laboratory or pet animals

Large numbers of mice (Mus spp.) and rats (Rattus spp.) are maintained for scientific reasons and as pet animals in Germany. While laboratory animals are monitored for pathogenic agents, the hygienic status of pet animals is usually completely unknown. Despite great efforts, zoonotic infections were reported even in laboratory settings, e.g. with Hantavirus (Seoul virus), Streptobacillus moniliformis, and Trichphyton mentagrophytes. However, in current reports, zoonotic infections were transmitted by mice and rats maintained as pet animals. This includes infections by Lymphocytic choriomeningitis virus, Leptospira interrogans, Streptobacillus moniliformis, Salmonella enterica, Trichophyton mentagrophytes, and Ornithonyssus bacoti. Furthermore, entero-hepatic Helicobacter spp. of rats and mice are currently discussed to be involved in the etiology of hepatobiliary diseases. Pasteurella spp. of mice and rats do not present a risk for human disease comparable to those species that are transmitted by dogs or cats and might induce serious disease after bites. Altogether, this article lists potential zoonotic agents that were detected in mice and rats and are present in Germany, as well as agents that were reportedly transmitted by mice and rats maintained as laboratory or pet rodents.     

Milk transfer of pyrrolizidine alkoloids in cattle.

A study was conducted to determine if pyrrolizidine alkaloids from Senecio jacobaea (tansy ragwort) are passed into milk of lactating cows. Four cows were given dried tansy ragwort via rumen cannula at a dosage of 10 g/kg/day for 2 weeks. Chemical assay of pyrrolizidine alkaloid content in the dried plants averaged 0.16% by weight. By use of thin-layer chromatography, 5 alkaloids were isolated from the plant materials. The only alkaloid isolated from the milk was jacoline. Condition of the cows and nursing calves was monitored via measurement of blood leukocyte count, and serum protein, albumin, and globulin content. Sorbitol dehydrogenase values and liver biopsy specimens were used to assess liver function. In the cows, marked changes were observed in blood leukocyte count, sorbitol dehydrogenase values, and liver biopsy findings. No changes of any type were observed in the calves. Pyrrolizidine alkaloid content of the milk ranged from 9.4 to 16.7 mug/100 ml of milk.     

Influence of folic acid, vitamin B2 and B6 supplementation on feed intake, body and organs weight, and liver fatty acids composition in rats subjected to severe protein deprivation

Growing rats fed for 3 months a low-protein (LP) diet (4.5% of energy from protein), possessed about 29% lower body weight than animals consuming adequate-protein diet (20% energy from protein). The LP diet feeding caused an increase in daily feed intake followed by a decrease in feed conversion efficiency. The enrichment of LP diet with folic acid, vitamin B2 and B6 (3 times above the level applied in the control diet) did not have any impact on rats BW and supplementation with these vitamins minimize the effect of LP diet on feed intake. The use of examined vitamins had a tendency to diminish an increase in feed conversion ratio caused by the LP nutrition. This effect was significant when all vitamins were added together. Rats fed the LP diet had higher relative weights of lungs, heart, liver and testis. Vitamins enriching the LP diet were observed to decrease a relative weight of lungs (folic acid, vitamin B6 and vitamin mixture), and liver (vitamin B6 and vitamin mixture). A tendency of increasing relative testis weight was also revealed in rats given the LP diet enriched with vitamins. The lower content of hepatic polyunsaturated fatty acids (FA) and a tendency for monounsaturated FA content to be higher were found in rats fed the LP diet. The LP diet enrichment with folic acid caused that these changes were more pronounced and statistically significant. Enrichment of LP diet with vitamins tested may cause a partial reverse of changes observed in the hepatic FA composition.     

Toxicity of Riddell's groundsel (Senecio riddellii) to cattle

The toxicity of Riddell's groundsel (Senecio riddellii) fed to calves in gelatin capsules, by gavage, or mixed in their hay ration was determined. Dosages were varied according to the pyrrolizidine alkaloid (PA) content of the plant, and the calves' responses were measured by clinical signs, serum enzyme changes, survival time, and histopathologic changes. Calves fed S riddellii to provide 10 mg of PA/kg of body weight/day in capsules or by gavage for 20 consecutive days did not develop clinical signs of seneciosis and did not have meaningful serum enzyme changes. However, feedings of the plant that provided 15 to 20 mg of PA/kg/day or more (gavaged or fed in capsules for the same time period) resulted in high mortality. The Senecio plant mixed in calves' hay ration was eaten slowly and reluctantly and was tolerated at dosages greater than 20 mg/kg/day, emphasizing that the toxicity was dependent on the rate at which the dosage was consumed and that mortality was not necessarily dependent on the cumulative dosage. Because of its high PA content, S riddellii presents a great hazard to cattle managed under conditions where they can consume quantities of the plant in short periods of time.     

Effects of dietary copper and molybdenum on tansy ragwort (Senecio jacobaea) toxicity in sheep

Dried tansy ragwort (Senecio jacobaea), which contains pyrrolizidine alkaloids (PA), was fed to sheep to determine whether PA consumption influenced liver Cu concentrations. Nine Polypay wether lambs were allotted to 3 groups according to the diets: control diet + 50 micrograms of Cu/g of feed (control + Cu); tansy ragwort (TR) diet + 50 micrograms of Cu/g of feed (TR + Cu); and TR diet + 50 micrograms of Cu/g of feed and 10 micrograms of Mo/g of feed (TR + Cu + Mo). Liver Cu concentrations were sharply increased (13-fold) after 8 weeks of feeding the diets, and then decreased slightly. There were no significant differences in concentrations of liver Cu among treatment groups. All lambs in the group fed TR + Cu + Mo died by the end of 23 weeks, with the usual signs of PA poisoning, including liver necrosis, hepatic megalocytosis, and biliary hyperplasia. The TR intake was about 2.7 to 3.0 kg/kg of their initial body weight. All lambs in the group fed the control + Cu diet survived. The results indicated that sheep fed TR do not preferentially accumulate liver Cu. There was an indication that dietary Mo increased susceptibility to TR intoxication.     

Pyrrolizidine alkaloids and seneciosis in farm animals. Part 2: clinical signs, species-specific sensitivity, food residues, feed contamination, limit values

At the forefront of pyrrolizidine alkaloid (PA) poisoning is the chronic ingestion of contaminated hay, which causes liver damage resulting in an ongoing fatal liver cirrhosis or in the veno-occlusive disease in liver or lung, respectively. The symptomatology of PA-poisoning is not identical for all animal species, and also includes central nervous symptoms. In affected horses significantly elevated levels of hepatogenic serum enzymes and an increase of the retention time for bromosulfophthalein indicates the fatal outcome of the intoxication. Chronic seneciosis of horses is incurable. Rabbits, Japanese quails, and guinea pigs are regarded as poison-resistant species. Sheep and in particular goats are insensitive unless extremely high amounts of plants which exceed the animal's body weight by several-fold are ingested. In contrast, pigs, cattle, and horses as well as chicken and likewise man are very sensitive to poisonings by PA-containing plants. In sensitive animal species a very small amount of contaminated dry hay is needed to exceed the daily dose of 1μg/kg body weight PA which is taken as harmless for man by health authorities. Therefore, all feed with visible pieces of Senecio jacobaea plants are not acceptable as animal fodder and should be destroyed.     

Natural plant toxicants in milk: a review

Elimination of plant toxicants via milk by lactating animals is considered a minor route of excretion; however, it may be important when the health of the neonate or food safety in humans is considered. Among plant toxicants excreted in milk is tremetol or tremetone, the toxin in white snakeroot (Eupatorium rugosum) and rayless goldenrod (Haplopappus heterophyllus). These plants have been responsible for intoxication of cows and their suckling calves and for many human poisonings. Other plant toxins excreted through the milk that pose a toxicity hazard include pyrrolizidine alkaloids in Senecio, Crotalaria, Heliotropium, Echium, Amsinckia, Symphytum (comfrey), Cynoglossum (hounds tongue) and Festuca (tall fescue); piperidine alkaloids in Conium, tobacco and others; quinolizidine alkaloids in Lupinus; sesquiterpene lactones of bitterweed and rubber weed; and glucosinolates in Amoracia (horseradish), Brassica (cabbage, broccoli, etc.), Limnanthes (meadowfoam), Nasturtium (watercress), Raphanus (radish) and Thlaspi (stinkweed). Many plants such as Astragalus, Oonopsis, Stanleya, Xylorrhiza, Aster, Atriplex, Sideranthus and Machaeranthera accumulate selenium and may cause intoxication when grazed. Selenium is found in the milk at concentrations relative to the amounts ingested by the lactating animal. Excretion of selenium via the milk is important in the deficiency state, but when in excess it may cause toxicity to offspring.     

Effect of dietary conjugated linoleic acid on vitamin A status of lactating rats and their offspring

The absorption and metabolism of vitamin A is linked with that of lipids. It is known that conjugated linoleic acid (CLA) affects the lipid metabolism in growing and lactating animals. In the present study, the hypothesis was investigated that dietary CLA influences vitamin A status of lactating rats and their pups during the suckling period. For this purpose, Wistar Han rats were fed either a control diet (control group, n = 14) or a diet containing 0.87% of cis‐9, trans‐11 and trans‐10, cis‐12 (1:1) CLA (CLA group, n = 14) during pregnancy and lactation. Vitamin A concentrations in various body tissues were determined 14 days after delivery in dams and 1, 7 and 14 days after birth in pups, and expression of selected genes involved in metabolism of retinoids was determined in dams. Vitamin A concentrations in liver, plasma and muscle were similar in control and CLA‐fed dams. Expression of genes involved in retinoid transport, storage and degradation in liver and adipose tissue in dams was also not different between control and CLA‐fed dams. Vitamin A concentrations in milk curd, sampled at d 1, 7 and 14 of lactation were not different between control and CLA‐fed dams. Vitamin A concentrations in liver, lung and adipose tissue were also not different in pups from control dams and pups from CLA‐fed dams. In conclusion, we show for the first time that dietary CLA has little effect on vitamin A concentrations and vitamin A metabolism in lactating rat dams and, moreover, does not influence tissue vitamin A concentrations in their newborn and suckling pups.