The toxicology mechanism of endophytic fungus and swainsonine in locoweed.

Locoweed is a perennial herbaceous plant included in Astragalus spp. and Oxytropis spp. that contains the toxic indolizidine alkaloid swainsonine. The livestock that consume locoweed can suffer from a type of toxicity called locoism. There are aliphaticnitro compounds, selenium, selenium compounds, and alkaloids in locoweed. The toxic component in locoweed has been identified as swainsonine, an indolizidine alkaloid. Swainsonine inhibits lysosomal a-mannosidase and mannosidase II, resulting in altered oligosaccharide degradation and incomplete glycoprotein processing. Corresponding studies on endophytic fungi producing swainsonine have been isolated from a variety of locoweed, and these endophytic fungi and locoweed have a close relationship. Endophytic fungi can promote the growth of locoweed and increase swainsonine production. As a result, livestock that consume locoweed exhibit several symptoms, including dispirited behavior, staggering gait, chromatopsia, trembling, ataxia, and cellular vacuolar degeneration of most tissues by pathological observation. Locoism results in significant annual economic losses. Therefore, in this paper, we review the current research on locoweed, including that on locoweed species distribution in China, endophyte fungus in locoweed, the toxicology mechanism of locoweed, and the swainsonine effect on reproduction.

Pathogenesis and preventive treatment for animal disease due to locoweed poisoning.

Locoweeds are perennial herbaceous plants included in Astragalus spp. and Oxytropis spp. that contain the toxic indolizidine alkaloid swainsonine. The livestock that consume locoweed feeding can suffer from a type of toxicity called "locoism." There are aliphatic nitro compounds, selenium, selenium compounds and alkaloids in locoweed. The toxic component in locoweeds has been identified as swainsonine, an indolizidine alkaloid. Swainsonine inhibits lysosomal α-mannosidase and mannosidase II, resulting in altered oligosaccharide degradation and incomplete glycoprotein processing. As a result, livestock that consume locoweeds exhibit several symptoms, including dispirited behavior, staggering gait, chromatopsia, trembling, ataxia, and cellular vacuolar degeneration of most tissues by pathological observation. Locoism results in significant annual economic losses. Recently, locoweed populations have increased domestically in China and abroad, resulting in an increase in the incidence of poisoning. Therefore, in this paper, we review the current research on locoweed, including on species variation, pathogenesis, damage and poisoning prevention measures.

A simple and sensitive HPLC-UV method for the determination of swainsonine in rat plasma and its application in a pharmacokinetic study.

A simple, rapid, selective and sensitive HPLC-UV method was developed and validated for the determination of swainsonine (SWSN) in rat plasma. The analyte was extracted from rat plasma with methanol as the extraction solvent. The LC separation was performed on a Diamonsil® C18 (250×4.6 mm, 5 µm) analytical column with a mobile phase consisting of acetonitrile-potassium dihydrogen phosphate (25 mmol/l, pH=7.5) at a flow rate of 1.0 ml/min. There was a good linearity over the range of 10-500 ng/ml (r=0.9995) with a weighted (1/C2) least square method. The lower limit of quantification was proved to be 10 ng/ml. The accuracy was within 4.8% in terms of relative error and the intra- and inter-day precisions were less than 9.0% in terms of relative standard deviation. After validation, the method was successfully applied to characterize the pharmacokinetics of SWSN in rats.

Angiogenesis in the caprine caruncles in non-pregnant and pregnant normal and swainsonine-treated does.

Microvascular corrosion casts of caruncles from non-pregnant and pregnant doe goats at 4, 7, 10, 13, 16, and 18 weeks were examined with scanning electron microscopy. The internal convex surface of the caruncles of non-pregnant does was covered with capillary meshes of regular diameter and form, without crypts. As pregnancy advanced the complexity of the vasculature increased: at 4 weeks the surface showed a pattern of ridges separated by troughs. At later stages, branches of radial arteries penetrated the periphery forming an extensive mesh of capillaries on the concave surface. Capillary diameters increased significantly during pregnancy, especially after 4 weeks, when large flattened sinusoids formed. These sinusoids had a great deal of surface area for potential contact with the fetal component. The caprine placenta is usually considered to have increased interhemal distance compared with endotheliochorial and hemochorial types: our results suggest that the very extensive development of sinusoids and crypts may compensate for any negative consequences of the placental architecture. Placental angiogenesis, which is physiologically normal, may serve as a general model of this process in other circumstances, such as tumor. The effect of swainsonine (active compound of locoweed and a potential anticancer drug) on vascular development showed no differences in sinusoidal diameters at 7 weeks, but a decrease in capillary density was noted. Swainsonine caused a great distortion to the vasculature at 18 weeks. The effects of this compound on the vascular development lend credibility to its potential as an anticancer agent.

Assessment of the perinatal effects of maternal ingestion of Ipomoea carnea in rats.

It is believed that Ipomoea carnea toxicosis induces abnormal embryogenesis in livestock. Studies on rats treated with I. carnea aqueous fraction (AF) during gestation, revealed litters with decreased body weight, but the characteristic vacuolar lesions promoted by swainsonine, its main toxic principle, were observed only in young rats on postnatal day (PND) 7. However, these alterations could have resulted as consequence of swainsonine placental passage and/or damage or even ingestion of the contaminated milk by pups. Thus, this perinatal work was performed to verify the transplacental passage of swainsonine and its excretion into milk employing the cross-fostering (CF) procedure as a tool of study. Females were treated with AF or vehicle during gestation and after birth pups were fostered between treated and untreated dams. Pup body weight gain (BWG) and histopathology to observe vacuolar degeneration were performed on PND 3 and 7. In addition, swainsonine detection was performed in amniotic fluid and milk from rats treated with the AF during gestation or lactation. BWG was significantly lower only in pups from mothers treated with the plant and fostered to other treated mothers (AF-AF group of pups). The histopathology revealed that pups from treated mothers fostered to untreated ones showed the characteristic vacuolar lesions; however, the lesions from the AF-AF pups were more severe in both periods evaluated. Amniotic fluid and milk analysis revealed the presence of swainsonine excretion into these fluid compartments. Thus, the results from CF and the chemical analysis allowed concluding that swainsonine passes the placental barrier and affects fetal development and milk excretion participates in I. carnea perinatal toxicosis.

Toxicokinetic profile of swainsonine following exposure to locoweed (Oxytropis sericea) in naïve and previously exposed sheep.

AIM: To determine the toxicokinetic profiles of swainsonine (SW) in sheep previously (subacute) and not previously (acute) exposed to locoweed. METHODS: Twenty-nine wethers were stratified by bodyweight (BW; 68.0 (SE 7.6) kg) and randomly assigned to one of six treatments. Treatments were: 0 (n=5), 0.4 (n=5), and 1.6 (n=5) mg SW/kg BW for Trial 1, and 0 (n=4), 0.2 (n=5), and 0.8 (n=5) mg SW/kg BW for Trial 2. Acute exposure in both trials included adaptation to blue grama (Bouteloua gracilis) hay for 14 days and no previous exposure to locoweed (i.e. SW), followed by administration of a single oral dose of SW prepared from an extract of locoweed, in the doses described above. Subacute exposure comprised ingestion of a blue grama and locoweed (428 microg SW/g locoweed) diet for 21 days in Trial 1 and 28 days in Trial 2, followed by removal from locoweed for 5 days, then an oral dose of SW, as above. Quantities of locoweed fed in the diet were adjusted to achieve the dose rates specified for each treatment. Blood samples were collected via jugular venepuncture twice daily for 3 days prior to initial exposure to SW and then every 7 days for the duration of the trials, to monitor serum alkaline phosphatase (Alk-P) and aspartate aminotransferase (AST) activities. For intensive sampling periods, SW was administered immediately following blood sampling at 0 h, and blood samples were collected at hourly intervals from 0-12 h, 3-h intervals from 15-24 h, 6-h intervals from 30-48 h, and 12-h intervals from 60-168 h. Concentrations of SW in serum and locoweed extract were determined using the alpha-mannosidase inhibition assay (detection limit=25 ng/ml). Rates of absorption and elimination of SW from serum were calculated for each animal, using exponential curve fits of the concentration of SW in serum concentration vs time plots. RESULTS: In both trials, SW was detected in serum in all animals exposed to locoweed. Elevated (p<0.05) serum Alk-P and AST activities indicated that subclinical SW intoxication was induced during the subacute exposure phase. Calculated rates of elimination were faster (p<0.001) for the 1.6 vs 0.4 (Trial 1) and 0.8 vs 0.2 (Trial 2) mg SW/kg BW doses. Rates of elimination indicated that, in both trials, SW was removed from serum faster (p<0.06) following acute exposure than subacute exposure. Higher exposure rates to SW resulted in higher concentrations of SW in serum within a trial. CONCLUSIONS: Multiple compartments were involved in the kinetics of SW, and dose and previous exposure altered the toxicokinetics of SW. CLININCAL RELEVANCE: Should the true elimination half-life prove to be as high or higher than the 95 h demonstrated for the treatment using 0.4 mg SW/kg BW in Trial 1, then withdrawal periods for clearing SW from sheep should be >40 days (assuming 10 half-lives to clear the compound).

Appearance and disappearance of swainsonine in serum and milk of lactating ruminants with nursing young following a single dose exposure to swainsonine (locoweed; Oxytropis sericea).

A series of experiments were conducted to investigate the elimination of swainsonine in the milk of lactating ruminants following a single dose oral exposure to swainsonine (locoweed; Oxytropis sericea) and to assess subsequent subclinical effects on the mothers and their nursing young. In a preliminary experiment, lactating ewes were gavaged with locoweed providing 0.8 mg swainsonine/kg BW (n = 4; BW = 75.8 +/- 3.6 kg; lactation = d 45) and lactating cows were offered up to 2.0 mg swainsonine/kg BW free choice (n = 16; BW = 389.6 +/- 20.9 kg; lactation = d 90). Serum and milk were collected at h 0 (before treatment), 3, 6, 12, and 24 for ewes, and h 0 (before treatment), 6, 12, 18, and 24 for cows. Swainsonine was highest (P < 0.05) by h 6 in the serum and milk of ewes. Consumption of at least 0.61 mg swainsonine/kg BW induced consistent (> 0.025 microg/mL) appearance of swainsonine in cow serum and milk. In response to the results obtained in the preliminary experiment, a subsequent experiment utilizing lactating ewes (n = 13; BW = 74.8 +/- 6.4 kg; lactation = d 30) and cows (n = 13; BW = 460.8 +/- 51.9 kg; lactation = d 90) was conducted. Each lactating ruminant was gavaged with a locoweed extract to provide 0 (control), 0.2, or 0.8 mg swainsonine/kg BW and individually penned with her nursing young. Serum and milk from the mothers and serum from the nursing young were collected at h 0 (before treatment), 3, 6, 9, 12, 24 and 48 (an additional sample was obtained at h 72 for ewes and lambs). Serum and milk swainsonine was higher (P < 0.05) in the 0.8 mg treated groups and maximal (P < 0.05) concentrations occurred from h 3 to 6 for ewes and h 6 to 12 h for cows (P < 0.05). Rises in alkaline phosphatase activity indicated subclinical toxicity in the treated ewes (P < 0.05). Following a single dose oral exposure to 0.2 and 0.8 mg swainsonine/kg BW provided by a locoweed extract, swainsonine was detected in the serum and milk of lactating ewes and cows, and rises in serum alkaline phosphatase activity were observed in the ewes. Neither swainsonine nor changes in alkaline phosphatase activity was detected in the serum of the lambs and calves nursing the ewes and cows dosed with swainsonine.

Tissue swainsonine clearance in sheep chronically poisoned with locoweed (Oxytropis sericea).

Locoweed poisoning is seen throughout the world and annually costs the livestock industry millions of dollars. Swainsonine inhibits lysosomal alpha-mannosidase and Golgi mannosidase II. Poisoned animals are lethargic, anorexic, emaciated, and have neurologic signs that range from subtle apprehension to seizures. Swainsonine is water-soluble, rapidly absorbed, and likely to be widely distributed in the tissues of poisoned animals. The purpose of this study was to quantify swainsonine in tissues of locoweed-poisoned sheep and determine the rate of swainsonine clearance from animal tissues. Twenty-four crossbred wethers were gavaged with ground Oxytropis sericea to obtain swainsonine doses of 1 mg swainsonine x kg(-1) BW x d(-1) for 30 d. After dosing, the sheep were killed on d 0, 1, 2, 3, 4, 6, 14, 30, 60, and 160. Animal weights and feed consumption were monitored. Serum was collected during dosing and withdrawal periods, and tissues were collected at necropsy. Serum swainsonine concentrations were determined using an alpha-mannosidase inhibition assay. Swainsonine concentrations in skeletal muscle, heart, brain, and serum were similar at approximately 250 ng/g. Clearance from these tissues was also similar, with half-lives (T(1/2)) of less than 20 h. Swainsonine at more than 2,000 ng/g, was detected in the liver, spleen, kidney, and pancreas. Clearance from liver, kidney, and pancreas was about T(1/2) 60 h. These findings imply that poisoned sheep have significant tissue swainsonine concentrations and animals exposed to locoweed should be withheld from slaughter for at least 25 d (10 T(1/2)) to ensure that the locoweed toxin has cleared from animal tissues and products.

Phase IB clinical trial of the oligosaccharide processing inhibitor swainsonine in patients with advanced malignancies.

The indolizidine alkaloid swainsonine, a potent inhibitor of Golgi alpha-mannosidase II, has been shown to reduce tumor cell metastasis, enhance cellular immune responses, and reduce solid tumor growth in mice. In our previous Phase I study, swainsonine administered by 5-day continuous infusion inhibited L-phytohemagglutinin-reactive N-linked oligosaccharide expression on peripheral blood lymphocytes. Significant toxicities included edema and elevated serum aspartate aminotransferase (AST). One patient with head and neck cancer had objective (>50%) tumor remission. Two patients showed symptomatic improvement. The objectives of this Phase IB trial were to examine the pharmacokinetics, toxicities, and biochemical effects of bi-weekly oral swainsonine at escalating dose levels (50-600 microgram/kg) in 16 patients with advanced malignancies and 2 HIV-positive patients unsuitable for conventional therapy. Eastern Cooperative Oncology Group performance status was 20% of patients included increase in serum AST (all patients), fatigue (n = 9), anorexia (n = 6), dyspnea (n = 6), and abdominal pain (n = 4). Inhibition of Golgi alpha-mannosidase II occurred in a dose-dependent manner. Examination of immunological parameters revealed a transient decrease in CD25(+) peripheral blood lymphocytes and, in seven of eight patients, an increase in CD4(+):CD8(+) ratios at 2 weeks. Serum drug levels peaked 3-4 h following a single oral dose in most patients and were proportional to dose at levels >/=150 microgram/kg. We conclude that oral swainsonine is tolerated by chronic intermittent administration at doses up to 150 microgram/kg/day. Adverse events considered drug related were similar to those observed in the infusional study but with fatigue and neurological effects also noted. Investigations of alternative dosing schedules with low starting doses are suggested for further clinical testing.

Interaction of swainsonine with lymphoid and highly perfused tissues: a pharmacokinetics explanation for sustained immunomodulation.

The kinetics of inhibition of metastasis by the immunomodulator swainsonine (SW) is effective 1 to 3 days after administration. It is likely that SW's prolonged antimetastatic effect is due to its mitogenic property (spleenocytes isolated from animals treated with SW for 42-72 hours stimulated DNA synthesis that remained elevated for up to 3 days after removal of the drug from the drinking water). An analysis of SW in lymphoid (spleen and thymus) and highly perfused tissues was undertaken to determine if SW's sustained antimetastatic effect could be correlated to its retention. C57BL/6 mice received [3H]SW in drinking water for 24-72 hours and thereafter, received SW-free drinking for 24, 48, and 72 hours. Lymphoid and highly perfused tissues were analyzed for [3H]SW. At 24, 48, and 72 hours, spleen SW levels are, respectively, at least 2.33, 2.25, and 2.00 times greater than the perfused tissue; and thymus are, respectively, 1.44, 1.50, and 1.77 as great as the perfused tissue (kidney) with the highest SW level. These studies suggest that SW is predominantly retained for at least 72 hours, in lymphoid tissue. The targeting and retention of SW for lymphoid tissue days after removal of SW from animal drinking water is consistent with a) the immunomodulatory/mitogenic property and b) the sustained antimetastatic effect attributed to SW.

Tissue and serum swainsonine concentrations in sheep ingesting Astragalus lentiginosus (locoweed).

Locoweed intoxication or locoism results when animals continuously graze certain plants of the general Astragalus or Oxytropis. The locoweed toxin, swainsonine, is water soluble and is rapidly absorbed and eliminated. The purpose of this study was to determine the distribution of swainsonine in tissues of sheep eating locoweed and to determine if the tissue swainsonine concentrations change with continued locoweed ingestion. Fifteen cross-breed whethers were divided into 3 groups of 5 each and fed alfalfa pellets (Group 1) or alfalfa pellets with 10% Astragalus lentiginosus for 13 d (Group 2) or for 21 d (Group 3). After the feeding periods, the animals were slaughtered and tissues were collected, frozen and later analyzed for swainsonine using an in vitro, alpha-mannosidase inhibition assay. Significant alpha-mannosidase inhibitory activity (interpreted as ng/ml of swainsonine) was detected in whole blood, skeletal muscle, brain, kidney, liver, thyroid and urine. The swainsonine concentrations in tissues were significantly correlated with daily swainsonine intake (r = 0.58 to 0.96). With the exception of kidney, longer exposure did not result in significant increases in the swainsonine concentrations in blood, muscle, brain, liver or thyroid. Liver had the highest swainsonine concentrations with 3049 +/- 1952 and 3947 +/- 457 ng/ml (mean +/- SD) in Groups 2 and 3 respectively. Swainsonine concentrations varied widely within the groups suggesting individual animal variability in swainsonine absorption, metabolism and excretion. These findings suggest that swainsonine is present in tissues of animals eating locoweed and that in most tissues the amount was directly correlated to the swainsonine dose ingested, but not to the length of exposure.

Serum swainsonine concentration and alpha-mannosidase activity in cattle and sheep ingesting Oxytropis sericea and Astragalus lentiginosus (locoweeds).

Serum alpha-mannosidase activity and swainsonine concentration were determined in 4 cattle and 15 sheep (3 groups of 5 each) that were administered ground locoweed (Oxytropis sericea or Astragalus lentiginosus) containing swainsonine at dosages of approximately 0.8 mg/kg of body weight/d (cows, 30 days each) and 0, 1.0, and 1.5 mg/kg/d (sheep, 11 days each). The cattle developed mild clinical signs of locoism, including signs of depression, lethargy, and slight intention tremors. Clinical signs of toxicosis were not observed in the sheep. Within 24 hours of initial treatment, serum alpha-mannosidase activity in cows and sheep, measured by the release of 4-methylumbelliferone from an artificial substrate, was markedly decreased to 28 and 40 mumol of 4-methylumbelliferone/L, respectively. Mean serum alpha-mannosidase activity of control cows and sheep was 400 +/- 94 and 422 +/- 42 (mean +/- SD), respectively. In the treated animals, decreased serum alpha-mannosidase activities returned to normal or higher activities within 6 days after treatment was discontinued. Using a jack bean alpha-mannosidase assay, increased swainsonine activity (153, 209, and 381 ng/ml, respectively) was detected in the serum of cattle and of sheep in the high- and low-dose groups within 24 hours after treatment with locoweed. Swainsonine concentration remained high, with mean concentrations of 204, 432, and 395 ng/ml (cows and 2 sheep groups, respectively) during the treatment period. After treatment, swainsonine was rapidly cleared, with estimated serum half-life of 16.4, 17.6, and 20.3 hours (cows, and high- and low-dose sheep groups, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Carbonoyloxy analogs of the anti-metastatic drug swainsonine. Activation in tumor cells by esterases.

Swainsonine (SW), a plant alkaloid and inhibitor of alpha-mannosidases, has been shown to inhibit N-linked oligosaccharide processing and to block tumor cell metastasis in mice. In this study, a series of SW analogs were chemically synthesized and compared for inhibition of complex-type N-linked oligosaccharide processing in cultured MDAY-D2 tumor cells, for inhibition of alpha-mannosidases in vitro, and for stimulation of bone marrow proliferation in vivo. Carbonoyloxy substitutions at the 2 and 8 carbons of SW reduced inhibitor activity by 2-3 orders of magnitude for Jack Bean and MDAY-D2 tumor cell lysosomal alpha-mannosidases in vitro. However, 2-p-nitrobenzoyloxy-, 2-octanoyloxy- and 2-butanoyloxy-derivatives of SW retained full activity as inhibitors of Golgi oligosaccharide processing in viable MDAY-D2 tumor cells. Inhibition of oligosaccharide processing was reduced by the esterase inhibitor diethyl p-nitrophenyl phosphate, suggesting that although 2-p-nitrobenzoyloxy-SW, 2-octanoyloxy-SW and 2-butanoyloxy-SW are relatively poor inhibitors of alpha-mannosidases in vitro, the compounds enter cells at a rate comparable to that of SW, and are converted to SW by cellular esterases. The more lipophilic esters, 2-benzoyloxy-SW, 2-toluoyloxy-SW, 8-palmitoyloxy-SW and 8-myristinoyloxy-SW, showed IC50 values at least 10 times higher for inhibition of Golgi oligosaccharide processing, probably due to less efficient entry of the compounds into tumor cells. The anti-metastatic activities of SW and two analogs were tested and shown to correlate with the IC50 values for inhibition of Golgi oligosaccharide processing in cultured tumor cells. In vivo, SW and the analogs were administered intraperitoneally to mice and found to have comparable activities as stimulators of bone marrow cell proliferation. Carbonoyloxy substitutions at the 2- or 8-position of SW with other chemical groups may lead to new drugs with improved pharmacokinetics and anti-cancer activity.

A preliminary pharmacokinetic evaluation of the antimetastatic immunomodulator swainsonine: clinical and toxic implications.

The pharmacokinetics of swainsonine (SW) was investigated in mice after intravenous administration of 3 micrograms/ml. The time course of SW blood levels followed a three-compartment open pharmacokinetic model which consisted of biphasic distribution, and a rapid elimination phase (terminal half-life, 31.6 min). After completion of the distribution, SW was widely distributed to the extravascular space (Vss, 22ml; Vd, 33ml). Free fractions of this substance were indistinguishable from unity, indicating little or no protein binding. The rate-limiting step in the elimination of SW from the body appears to be the slow return from the deep compartment into the central one. Accordingly, SW blood levels may be low and yet significant amounts of this agent may be present in different body organs and tissues. A comparison of SW tissue levels indicates that the highest amounts appeared in the bladder, kidney, and thymus, (3.8 0.5, and 2.2 nmoles/g wet wt) with the lowest levels consistently appearing in the brain (< 0.1 nmoles/g wet wt). Hence, this study suggest that: 1) SW has high affinity for the thymus, which is in part consistent with its previously published immunomodulatory action; 2) SW should be infused for at least 2 1/2 hrs for its concentration to approach a plateau (this is based on the short half-life of SW and its time to steady state); and 3) CNS toxicity may be dose-limiting and not be present at SW levels preventing metastasis.