Potential toxic effects produced by L-mimosine in the thyroid and reproductive systems. Evaluation in male rats.

Leucaena leucocephala is a worldwide plant used as forage; however, its use in animal production has been limited because of the presence of a toxic nonprotein amino acid, L-mimosine. L-mimosine exhibits negative effects not only in ruminants but also in monogastric animals; however, there is little information available on the effect of this amino acid in monogastric species. Thus, this study aimed to evaluate the general toxicity of L-mimosine in rats, as well as its effects on the endocrine and reproductive systems. L-mimosine was extracted from seeds of L. leucocephala that were administered orally by gavage to adult Wistar rats at different doses of 25, 40 and 60 mg/kg body weight/day for 28 days. The following parameters were evaluated: weight gain, feed intake, serum enzymes, histopathology (liver, kidney, thyroid, thymus, and spleen), serum hormones (testosterone, corticosterone, T3 and T4) and sexual behavior. No clinical signs of toxicity were observed in animals, but histopathology revealed consistent lesions in the thyroids. Additionally, rats exposed to L-mimosine presented low serum levels of testosterone, decreased mount numbers and increased mount intervals. Therefore, our study reinforces the assumption that L-mimosine has goitrogenic potential and causes impairment in male reproductive performance.

Systemic effects of Leucaena leucocephala ingestion on ringtailed lemurs (Lemur catta) at Berenty Reserve, Madagascar.

Leucaena (Leucaena leucocephala) is a leguminous tree that is nutritious forage for domestic livestock when ingested in limited amounts. Unfortunately, leucaena contains mimosine, a plant amino acid, that can be toxic when ingested at higher concentrations. Reported toxic effects include alopecia (fur loss), poor body condition, infertility, low birth weight, thyroid gland dysfunction, and organ toxicity. Originally native to Mexico and Central America, leucaena has been introduced throughout the tropics, including Berenty Reserve, Madagascar where it was planted as supplemental browse for livestock. In Berenty, a seasonal syndrome of alopecia in ringtailed lemurs (Lemur catta) is associated with eating leucaena. Although much is known about the toxic effects of leucaena and mimosine on domestic animals and humans, the systemic effects on wildlife had not been studied. In a comparison of lemurs that include leucaena in their diet and those that do not, we found that animals that ingest leucaena absorb mimosine but that ingestion does not affect body condition, cause kidney or liver toxicity, or affect the intestinal tract. Alopecia is due to mimosine's interference of the hair follicle cycle. Leucaena ingestion is associated with higher serum albumin, α-tocopherol, and thyroxine concentrations, suggesting that leucaena may provide some nutritional benefit and that lemurs can detoxify and convert mimosine to a thyroid stimulating metabolite. The primary conservation consequence of leucaena ingestion at Berenty may be increased infant mortality due to the infants' inability cling to their alopecic mothers. The widespread introduction of leucaena throughout the tropics and its rapid spread in secondary forest conditions mean that many other leaf-eating mammals may be including this tree in their diet. Thus, exposure to leucaena should be considered when wildlife health is being evaluated, and the potential effects on wildlife health should be considered when contemplating leucaena introduction into or near wildlife habitat.

Transgenic Leucaena leucocephala expressing the Rhizobium gene pydA encoding a meta-cleavage dioxygenase shows reduced mimosine content.

The use of the tree-legume Leucaena leucocephala (leucaena), which contains high levels of proteins in its foliage, is limited due to the presence of the toxic free amino acid mimosine. The goal of this research was to develop transgenic leucaena with reduced mimosine content. Two genes, pydA and pydB, encoding a meta-cleavage dioxygenase (EC 1.13.11.2) and a pyruvate hydrolase (EC 3.7.1.6), respectively, from the mimosine-degrading leucaena symbiont Rhizobium sp. strain TAL1145, were used to transform leucaena. These bacterial genes were sequence-optimized for expression in leucaena and cloned into the plant binary vector pCAMBIA3201 for Agrobacterium tumefaciens-mediated transformation. Using immature zygotic embryos as the start explant material, six pydA and three pydB transgenic lines were developed. The presence and expression of the bacterial genes in the transgenic lines were verified by PCR, reverse transcriptase PCR, and Southern analyses. HPLC analyses of the transgenic plants determined that the mimosine contents of the pydA-expressing lines were reduced up to 22.5% in comparison to the wild-type. No significant reduction in mimosine content was observed in the pydB-expressing lines. This is the first example of using a gene from a bacterial symbiont to reduce the toxicity of a tree-legume.

S-phase progression stimulates both the mutagenic KU-independent pathway and mutagenic processing of KU-dependent intermediates, for nonhomologous end joining.

We used intrachromosomal substrates to directly monitor the effect of the cell cycle on the efficiency and the accuracy of nonhomologous end joining (NHEJ) in mammalian cells. We show that both KU and KU-independent (KU-alt) pathways are efficient when maintaining cells in G1/S, in G2/M or during dynamic progression through S phase. In addition, the accuracy of NHEJ is barely altered when the cells are blocked in G1/S or in G2/M. However, progression through S phase increases the frequency of deletions, which is a hallmark of the KU-alt pathway. Moreover, we show that the intermediates that are generated by the KU-dependent end joining of non-fully complementary ends, and which contain mismatches, nicks or gap intermediates, are less accurately processed when the cells progress through S phase. In conclusion, both KU and KU-alt processes are active throughout the cell cycle, but the repair is more error prone during S phase, both by increasing the mutagenic KU-alt pathway and decreasing the accuracy of the repair of the intermediates generated by the KU-dependent pathway.

Human hRad1 but not hRad9 protects hHus1 from ubiquitin-proteasomal degradation.

Three of the Rad family proteins, Rad9, Rad1, and Hus1, can interact with each other and form a heterotrimeric complex that is thought to play a role in the sensing step of the DNA integrity checkpoint pathways, but the nature of the Rad9-Rad1-Hus1 complex assembly remains enigmatic. Here, we demonstrate that the human hRad1 protein plays a significant role as molecular chaperone in the process of the hRad9-hRad1-hHus1 heterotrimeric complex formation. In contrast to hRad1, hHus1 is an unstable protein that is actively degraded via the ubiquitin-proteasome pathway. We show that treating cells with proteasome-specific inhibitors stabilizes hHus1 expression. Moreover, hRad1 can associate with hHus1 in the absence of hRad9 and protect hHus1 from ubiquitination and degradation in the cytoplasm. Importantly, genotoxic stress induces hRad1 expression and stabilizes the hHus1 protein. Taken together, these findings suggest a novel role of hRad1 as a potential intrinsic chaperone in the stabilization of hHus1 for the hRad9-hRad1-hHus1 checkpoint complex formation.

DNA breaks induction by mimosine.

Mouse erythroleukemic F4 N cells were treated with mimosine, etoposide, Fe(II)-EDTA, and Cu(II) in the presence of ascorbate. DNA was isolated and subjected to agarose gel electrophoresis and the size and distribution of the DNA fragments produced by the agents were compared. With increasing concentration of Cu(II) the production of DNA fragments was increased without decrease of the average length of the fragments, and their sizes were similar to those produced by etoposide as expected for cleavage of DNA at the nuclear matrix attachments sites. In contrast, mimosine and Fe(II) produced fragments of random size and with the progression of the reaction the average length of the fragments decreased. These results indicate that mimosine cuts DNA in a random fashion, regardless of its higher order chromatin organization. A conclusion is drawn that the DNA fragments obtained after mimosine treatment are a result of mimosine-assisted, Fe(II) dependent Fenton-like reactions randomly cutting chromosomal DNA.

Participation of kin17 protein in replication factories and in other DNA transactions mediated by high molecular weight nuclear complexes.

The Homo sapiens kin17 ((HSA)kin17) protein is a chromatin-associated protein conserved during evolution and overproduced in certain human tumor cell lines. For the first time, immunoelectron microscopy analysis of endogenous (HSA)kin17 protein revealed an ultrastructural co-localization of (HSA)kin17 and bromodeoxyuridine (BrdUrd) at sites of DNA replication after either short (15 min) or long (120 min) pulses of BrdUrd labeling. After hydroxyurea (HU) or L-mimosine (Mimo) block and withdrawal, we observed that (HSA)kin17 was recruited onto the chromatin during the re-entry and the progression in the S phase. These results are consistent with a major role of (HSA)kin17 protein in DNA replication factories. Other treatments hampering replication fork progression and/or inducing double-strand breaks also triggered an accumulation and a concentration of the chromatin-bound (HSA)kin17 protein into large intranuclear foci 24 h post-treatment. Moreover, HU- and Mimo-induced (HSA)kin17 foci were retained in the nucleus after detergent extraction, suggesting a strong association with nuclear structures. Gel filtration analyses of cellular extracts showed that endogenous (HSA)kin17 protein co-eluted with both replication proteins RPA32 and RPA70 in a fraction containing complexes of M(r) 600,000. Interestingly, HU-induced G(1)-S arrest triggered an increase in the molecular weight of complexes containing (HSA)kin17 protein. Hence, treatments interfering with either initiation and/or elongation of DNA replication also recruited chromatin-bound (HSA)kin17 protein. We hypothesize that in the presence of unrepaired DNA damage, (HSA)kin17 protein concentrated into high molecular weight complexes probably to create a bridge that contributes to the harmonization of DNA replication and repair.

Activation of the hypoxia-inducible factor-pathway and stimulation of angiogenesis by application of prolyl hydroxylase inhibitors.

Hypoxia-inducible transcription factors (HIF) mediate complex adaptations to reduced oxygen supply, including neoangiogenesis. Regulation of HIF occurs mainly through oxygen-dependent destruction of its alpha subunit. In the presence of oxygen, two HIFalpha prolyl residues undergo enzymatic hydroxylation, which is required for its proteasomal degradation. We therefore tested whether pharmacological activation of HIFalpha by hydroxylase inhibitors may provide a novel therapeutic strategy for the treatment of ischemic diseases. Three distinct prolyl 4-hydroxylase inhibitors-l-mimosine (L-Mim), ethyl 3,4-dihydroxybenzoate (3,4-DHB), and 6-chlor-3-hydroxychinolin-2-carbonic acid-N-carboxymethylamid (S956711)-demonstrated similar effects to hypoxia (0.5% O2) by inducing HIFalpha protein in human and rodent cells. L-Mim, S956711, and, less effectively, 3,4-DHB also induced HIF target genes in cultured cells, including glucose transporter 1 and vascular endothelial growth factor, as well as HIF-dependent reporter gene expression. Systemic administration of L-Mim and S956711 in rats led to HIFalpha induction in the kidney. In a sponge model for angiogenesis, repeated local injection of the inhibitors strongly increased invasion of highly vascularized tissue into the sponge centers. In conclusion, structurally distinct inhibitors of prolyl hydroxylation are capable of inducing HIFalpha and HIF target genes in vitro and in vivo and induce adaptive responses to hypoxia, including angiogenesis.

Radiation sensitization of mammalian cells by metal chelators.

The cell cycle effects, alteration in radiation response, and inherent cytotoxicity of the metal chelators mimosine, desferrioxamine (DFO), N,N'-bis(o-hydroxybenzyl)-ethylenediamine-N,N'-diacetic acid (HBED), and deferiprone (L1) were studied in exponentially growing Chinese hamster V79 cells. Incubation of cells with 200-1000 microM mimosine for 12 h reduced clonogenic survival to 50-60%, while incubation for 24 h reduced survival further to 0.5%. Mimosine treatment resulted in cell cycle blocks at the G(1)/S-phase border and in S phase. Pulse labeling with 5-bromodeoxyuridine indicated that the S-phase cells ceased to actively replicate DNA after only 2 h of mimosine treatment and were unable to replicate DNA for extended periods. Treatment of V79 cells with 600 microM mimosine for 12 h resulted in radiosensitization, yielding a sensitizer enhancement ratio (SER) of 2.7 +/- 0.3 at the 10% survival level. To study the kinetics of the sensitization, V79 cells were incubated with mimosine for various times up to 12 h and irradiated with a single 10-Gy dose of X rays. It was found that the radiosensitization increased continually up to 8 h (from a 3- to a 100-fold difference in survival) and then reached a plateau after 8 h. Mimosine also equally radiosensitized human lung cancer cells having either a normal or mutated TP53 gene, suggesting a TP53-independent mechanism. To test whether iron binding by mimosine was responsible for the observed radiosensitization, additional experiments were performed using the iron chelators DFO, HBED and L1. V79 cells treated with 500 microM of these agents for 8 h followed by various doses of X rays gave SERs similar to that for mimosine (2.0-2.7). These studies indicate that metal chelators are potent radiosensitizers in V79 and human cells. Importantly, when the DFO was preloaded together with Fe(3+) [Fe(III)-DFO], the radiosensitizing effect was lost. These preliminary findings warrant further studies for the possible application of metal chelators as radiation sensitizers in radiation oncology.

Osteopathy in broiler chicks fed toxic mimosine in Leucaena leucocephala.

Further studies of mimosine toxicity in broiler chicks were done to clarify a possibility of osteopathy. The mineral content and density of femur and the strength, ductility, and toughness for the index of mechanical properties significantly decreased in the 1% mimosine group, compared with those in the control and restricted groups. The stiffness had a decreasing tendency in the 1% mimosine group. Consequently, it was concluded that chicks fed ad libitum a 1% mimosine diet for 12 days developed osteopathy. The bone mineral density and the strength of the restricted group were lower than those of the control group, and those of the 1% mimosine group were still lower than those of the restricted group. Contents of pyridinoline and deoxypyridinoline in the excrement were significantly higher in the restricted group than those in the control group, but the contents in the 1% mimosine group were significantly lowest among the groups. Osteopathy in chicks fed mimosine, therefore, seemed to be done by loss of appetite and changing to a low turnover of bone caused by mimosine.

Nutritional and economic benefits of Leucaena and Gliricidia as feed supplements for small ruminants in humid West Africa.

Considering leguminous trees Leucaena and Gliricidia as good sources of quality food, on-station and on-farm studies were conducted in the humid zone of West Africa to establish animal responses to levels, times and forms of browse supplementation, to develop alternative feeding strategies for utilising limited feed supply and to assess the economic benefits of feed supplements as against the use of tree foliage as mulch for crop production. Results indicate that at any level of supplement, sheep grew twice as fast as goats. The main benefits of supplementation came through increased growth and survival. Form and level of supplementation had significant effect on intake. Economic analyses showed that crop response to mulching was the principal competing determinant of whether the use of tree foliage as feed supplement was economic.

Effects of mimosine administered to a perfused area of skin in Angora goats.

The effect of mimosine on a perfused area of skin tissue was studied using an isolated perfusion technique. Four mature Angora wethers (body weight 35 (SE 2.3) kg) were cannulated bilaterally with indwelling silicone catheters in the superficial branches of the deep circumflex iliac artery and vein. Mimosine (40 mg/kg metabolic weight (W)0.75) per d) was infused intra-arterially into one iliac artery of each goat for 3 d and saline was infused in the contralateral (control) iliac artery. Iliac venous blood samples were taken from both sides along with arterial samples from the carotid artery. Mimosine infusion elevated plasma mimosine in the carotid artery (52.6 (SEM 19.21) mumol/l) and iliac vein on the saline-treated side to 54.1 (SEM 16.31) mumol/l and in the iliac vein on the mimosine-treated side to 191.3 (SEM 19.14) mumol/l (P < 0.01). Mimosine decreased feed intake (2.3 v. 0.6 kg/d, SEM 0.29; P < 0.001) and water consumption (5.2 v. 1.3 litres/d, SEM 0.67; P < 0.001). Mimosine did not cause defleecing in the area of infusion and was cleared from the bloodstream within 12 h of cessation of infusion. The following effects were also observed during mimosine infusion: decrease in plasma amino acids to half pre-infusion values (methionine 22.7 v. 13.1 mumol/l, SEM 1.41; lysine 95.9 v. 37.4 mumol/l, SEM 4.28; P < 0.001); decreases in plasma triiodothyronine (1495 v. 695 ng/l, SEM 43.1; P < 0.001), thyroxine (61.5 v. 19.5 micrograms/l, SEM 1.8; P < 0.001) and insulin (28.7 v. 17.3 microIU/ml, SEM 1.89; P < 0.01) concentrations; increase in plasma cortisol (14 v. 62 micrograms/l, SEM 0.35; P < 0.001) concentration; decreases in levels of plasma Zn and Mg (0.97 v. 0.49 mg/l, SEM 0.063; P < 0.001 and 21.4 v. 14.6 mg/l, SEM 1.74; P < 0.001 respectively). All reported variables returned to their normal values 24 h after cessation of mimosine infusion except feed intake which was affected for a longer period. Mohair length and diameter were not affected by mimosine infusion. The toxicity of mimosine may be due to the drastic depletion of Zn and Mg in the blood as mimosine possesses very strong chelating properties and is excreted in the urine as a chelate.

Mimosine is a potent clastogen in primary and transformed hamster fibroblasts but not in primary or transformed human lymphocytes.

The cytogenetic effects of mimosine, a naturally occurring plant amino acid known to arrest cell-cycle progression at the G1-S border in cultured cells, have been studied. It was found that mimosine inhibits the cell-cycle progression in a dose-dependent manner in primary and transformed Chinese hamster fibroblasts as well as primary lymphocytes and transformed lymphoblastoid cells of human origin. In the Chinese hamster fibroblast cells, the first division metaphases analysed were found to be highly damaged or pulverized. The damaged cells which could pass through the next cell division, showed very high frequencies of sister chromatid exchanges (SCEs) compared with untreated second division cells. No such cytogenetic alterations could be detected in the human cells. The absence of clastogenic effect in cells of lymphoid origin appears to be related to the known capacity of these cells to undergo apoptosis, thereby efficiently eliminating cells with high frequencies of chromosomal aberrations. Our study demonstrates the clastogenic potency of mimosine and suggests the need for a careful interpretation of the results while using mimosine for cellular or molecular studies pertaining to cell cycle events.

Mimosine-induced cell death and related chromatin changes.

The non-protein amino acid mimosine has recently been shown to induce an unusual mode of cell death that differs from necrosis and apoptosis, the two fundamental schemes of cell death. The drug affects primarily the cell nucleus and induces first condensation of the chromatin into a regular network of fibrils and then gradual decondensation. The present study was performed to evaluate effective mimosine concentrations, time dependency of toxicity, and mimosine-related alterations of the chromatin, DNA and histones. To this end, primarily cultured carp hepatocytes were exposed to a wide range of mimosine (10(-1) to 10(-4) M) up to 12 h and investigated by means of light microscopy, electron microscopy, and histochemistry for DNA and histones. With 10(-1) M mimosine severe cytopathological transformations were obtained already after 3 h whereas 10(-2) M was cytotoxic only after 12 h. Lower concentrations were ineffective within the experimental period. Cytopathology started with condensation of the chromatin into a homogeneous network of ca. 25 nm wide fibrils and segregation of the nucleolus. In parallel, the nuclei were depleted from histones leaving the pattern of DNA fluorescence largely unchanged. The following period of chromatin decondensation was characterized by removal of electron-dense components from the condensed chromatin fibrils and gradual loss of the DNA. The segregated nucleolus and also the nuclear pores remained well preserved until final cell lysis.

Blood metabolite and regulatory hormone concentrations and response to metabolic challenges during the infusion of mimosine and 2,3-dihydroxypyridine in alpine goats.

Sixteen Alpine wethers (average BW 35 +/- 2 kg) were used to evaluate the effect of continuous 48-h intravenous infusions of saline (CON), mimosine (MIM; 200 mg.kg.75.d-1), 2-hydroxy-3(1H)-pyridine (2,3-DHP; 200 mg.kg.75.d-1, or MIM+2,3-DHP (100 mg of MIM plus 100 mg of 2,3-DHP-kg.75.d-1) on hepatic function and selected blood metabolite and circulating hormone concentrations. Neither MIM nor 2,3-DHP affected plasma ammonia N, glucose, cortisol, or insulin concentrations over time (P > .10). Jugular plasma total protein concentration was greater in the MIM group (P < .07). Plasma triiodothyronine (P < .01) and thyroxine (P < .08) concentrations were higher in the goats receiving the MIM, 2,3-DHP, and MIM+2,3-DHP infusions than in the goats receiving the CON infusion. Plasma urea N concentration was decreased by MIM (P < .10) and MIM+2,3-DHP (P < .03) compared with the CON infusion. A Propionate Load Test was conducted at 24 to 28 h into the infusion to assess the toxins' effects on the liver's ability to increase circulating glucose concentrations in the presence of elevated propionate levels. The results indicated that neither 2,3-DHP nor MIM reduced the liver's ability to respond to a bolus dose of propionate (P > .10). Following a Urea Load Test, circulating ammonia N and glucose concentrations in the MIM, 2,3-DHP, and MIM+2,3-DHP treatments had lower peak values than that in the CON treatment (P < .01).(ABSTRACT TRUNCATED AT 250 WORDS)

Successful transfer of DHP-degrading bacteria from Hawaiian goats to Australian ruminants to overcome the toxicity of Leucaena.

Cattle and goats in Australia lack the ability to totally degrade 3-hydroxy-4(1H)-pyridone, also known as 3,4-dihydroxy pyridine (3,4 DHP), the ruminal metabolite of mimosine, a toxic aminoacid present in the leguminous shrub Leucaena leucocephala. Ruminants in Hawaii have this capacity due to the presence of micro-organisms able to rapidly degrade the DHP. A mixed bacterial population capable of rapidly degrading DHP in vitro was isolated from a goat on the island of Maui. Cultures were grown anaerobically, without added sugars, in Medium 98-5 containing DHP. Cultures at a dilution of 10(-12) from the original rumen fluid were introduced into Townsville and further sub-cultured and multiplied in vitro in strict isolation at the Oonoonba Veterinary Laboratory, Townsville. Infusion of the culture into a goat and a steer fed a 100% leucaena diet resulted in cessation of DHP excretion in the urine. After 60 days the serum thyroxine levels and thyroid size were normal and there were no clinical signs of disease. The ability of the rumen fluid to degrade DHP in vitro showed that the bacteria had become established in the rumen. In the absence of any disease in the animals, clearance has been given for the wider use of these cultures in areas where leucaena is grown. The limited evidence suggests that the leucaena toxicity problem can be solved by the use of these introduced bacteria.

Comparative toxicities of mimosine and some chemically related compounds to mouse bone marrow cells in liquid culture.

Mimosine, a plant amino acid which is toxic in mammals, was shown to be a potent inhibitor of incorporation of [3H]thymidine in mouse bone marrow cells in liquid culture (is greater than 70% inhibition at a concentration of 2 x 10(-4) M). To determine the parts of the molecule responsible for the inhibitory mechanism the effects of 13 chemically related compounds were examined in this system. The structural features necessary for inhibitory activity of the 4(1H)-pyridones were (1) the 3-hydroxyl-4-oxo function of the pyridone ring together with (2) an alpha-alanine or a 2-aminoethyl side chain. Compounds based on several other hydroxy heterocyclic functions were either weakly active or inactive. 3-Hydroxy-4(1H)-pyridone, the goitrogen to which mimosine is converted in ruminants, was only slightly inhibitory. These results are compared with published information on the effects of some of these compounds on other types of mammalian cells in vitro. The mouse bone marrow system in which inhibition of incorporation of [3H]thymidine is used as an index of cytotoxicity was shown to be sensitive and reproducible, and could be useful for structure-activity investigations of other cytotoxic compounds.