Toxic Non-protein Amino Acid Research Articles

Effects of the Toxic Non-Protein Amino Acid β-Methylamino-L-Alanine (BMAA) on Intracellular Amino Acid Levels in Neuroblastoma Cells.

The cyanobacterial non-protein amino acid (AA) β-Methylamino-L-alanine (BMAA) is considered to be a neurotoxin. BMAA caused histopathological changes in brains and spinal cords of primates consistent with some of those seen in early motor neuron disease; however, supplementation with L-serine protected against some of those changes. We examined the impact of BMAA on AA concentrations in human neuroblastoma cells in vitro. Cells were treated with 1000 µM BMAA and intracellular free AA concentrations in treated and control cells were compared at six time-points over a 48 h culture period. BMAA had a profound effect on intracellular AA levels at specific time points but in most cases, AA homeostasis was re-established in the cell. The most heavily impacted amino acid was serine which was depleted in BMAA-treated cells from 9 h onwards. Correction of serine depletion could be a factor in the observation that supplementation with L-serine protects against BMAA toxicity in vitro and in vivo. AAs that could potentially be involved in protection against BMAA-induced oxidation such as histidine, tyrosine, and phenylalanine were depleted in cells at later time points.

Isolation and characterization of mimosine degrading enzyme from Arthrobacter sp. Ryudai-S1.

Leucaena leucocephala growing in the tropics and subtropics serves as potential forage for livestock because its foliage is rich in protein, fiber, and minerals. However, its use for livestock feed has been hindered by toxic nonprotein amino acid mimosine. Therefore, it is necessary to develop a method to reduce or eliminate mimosine from foliage. A previous study found that the fermentation of L. leucocephala foliage reduced the mimosine content and prompted the authors to isolate potent mimosine degrading microorganisms and characterize the mimosinase for the complete elimination of mimosine in the L. leucocephala foliage. The soil screening of the L. leucocephala tree surroundings led to the isolation of Arthrobacter sp. Ryudai-S1, which can degrade and assimilate mimosine as a nitrogen and carbon source. Mimosinase in this strain was found to be thermostable and showed strong activity. Docking model's inspection and the interaction energy calculation between mimosine-pyridoxal-5'-phosphate (PLP) complex and the active site of this enzyme identified 11 important amino acid residues that stabilized the binding. Of these amino acid residues, mutation experiment suggested that Tyr-263' and Phe-34 stabilizes the substrate binding and play a critical role in guiding the substrate to proper positions to accomplish high catalytic efficacy and selectivity. These observations suggest that Arthrobacter sp. Ryudai-S1 could be potentially useful for the development of L. leucocephala feed with reduced mimosine content.

SDS-PAGE Analyses and Electrophoretic Band Characterization of Three Cultivars of Grass Pea based on Seed Storage Proteins

Background: Grass pea (Lathyrus sativus L.) is valued for its high dietary protein content, high degree of adaptability under extreme conditions, disease resistance and low input requirement for its cultivation. This species is represented by several sub-species and important varieties which are not well distinguishable by their morphological characters. The objective of present study was to determine the cultivar differences based on seed storage protein by using SDS-PAGE and to obtain information useful for a breeding program. Methods: Gel electrophoresis was carried out in the discontinuous buffer system in a vertical electrophoresis apparatus and electrophoretic data were documented by using a gel documentation system. Seed protein sub-fractions (albumins, globulins, glutelins and prolamins) from seeds were extracted and quantified. ODAP (β-N-oxalyl-L-a,β-diaminopropionoc acid) an undesirable and toxic non-protein amino acid (responsible for ‘neuro-lathyrism’ disease) content was estimated. Result: The differences among cultivars were observed and clearly identifiable from their protein banding patterns in view of in number of bands, position of the bands and molecular weight of the bands etc. Quantity of total protein and sub-fractions of protein (albumins, globulin, glutelins and prolamins) of seed storage proteins also showed the variation among the three cultivars. ODAP content estimated in three grass pea cultivars was found very low ranging which is highly desirable for cultivation.

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.

Detection of <i>Synergistes jonesii</i> and genetic variants in ruminants from different geographical locations

Keynote paper presented at the International Leucaena Conference, 1‒3 November 2018, Brisbane, Queensland, Australia.Leucaena leucocephala is a nutritionally rich forage tree legume that contains a toxic non-protein amino acid, mimosine, from which other toxic compounds 3,4-dihydroxypyridone (3,4-DHP) and 2,3-DHP are formed in the rumen. The rumen bacterium Synergistes jonesii is able to degrade these DHP isomers into non-toxic end products. In this study we developed new PCR-based assays to improve the specificity and sensitivity of detection of S. jonesii in the rumen. Using these new assays in a survey of ruminants from different countries showed S. jonesii appeared to be ubiquitous rather than isolated geographically. The bacterium was present as a minor population (<106 cells/mL) in the rumen and was usually comprised of several genetic variants of the species. Although the indigenous nature of S. jonesii could imply animals are protected from toxicity, the relative abundance of the bacterium, potential variation in DHP degrading ability of genetic variants of the species, and amount of leucaena in the diet may determine the ability of the resident population in the rumen to protect the animal from toxicity.

Mimosine concentration in <i>Leucaena leucocephala</i> under various environmental conditions

Keynote paper presented at the International Leucaena Conference, 1‒3 November 2018, Brisbane, Queensland, Australia.Leucaena leucocephala (leucaena) is a multipurpose tropical tree-legume that is highly resistant to many biotic and abiotic stresses. Leucaena is used primarily as an animal fodder owing to its protein-rich foliage. However, leucaena foliage also contains mimosine, a toxic non-protein amino acid that can cause alopecia, goiter and other thyroid problems, infertility, and fetal death. Considering its toxicity and abundance in leucaena, it is important to quantify the mimosine concentrations in leucaena under different environmental conditions. Mimosine was extracted from various types of leucaena tissue exposed to a range of environmental conditions and then quantified by HPLC. The mimosine concentrations in leucaena treated with NaCl increased after 6 days of treatment and remained relatively high when treatment continued for 18 days. Interestingly, leucaena exposed to complete darkness for up to 5 days had a higher mimosine concentration than control plants exposed to normal light/dark photoperiods. On the other hand, drying leucaena leaflets or macerating them in an alkaline buffer significantly lowered their mimosine concentration. Mature leaflets that had fallen off the plant and dried out also contained significantly less mimosine than fresh leaflets. The results of this study indicate that mimosine concentrations in leucaena are affected by environmental conditions and this knowledge can assist in managing to prevent toxicity.

Non-protein amino acids in Australian acacia seed: Implications for food security and recommended processing methods to reduce djenkolic acid

Seed of Australian acacia species, Acacia colei, Acacia elecantha, Acacia torulosa, Acacia turmida and Acacia saligna, were analysed for the presence of toxic non-protein amino acids and the levels of essential amino acids. Amines were derivatised with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate before analysis using liquid chromatography electrospray ionisation triple quadrupole mass spectrometry (LC-ESI-QQQ-MS). Multiple reaction monitoring (MRM) with optimised transitions and collision energies for each analyte were employed. The known nephrotoxic compound djenkolic acid was found to be present at elevated levels in all species tested. The lowest levels were in A. colei (0.49% w/w) and the highest in A. saligna (1.85% w/w). Observed levels of djenkolic acid are comparable to measured and reported levels found in the djenkol bean. Subsequent testing of seed processing methods showed djenkolic acid levels can be significantly reduced by over 90% by dry roasting at 180°C rendering the seed safe for human consumption.

A Carbon-Nitrogen Lyase from Leucaena leucocephala Catalyzes the First Step of Mimosine Degradation

The tree legume Leucaena leucocephala contains a large amount of a toxic nonprotein aromatic amino acid, mimosine, and also an enzyme, mimosinase, for mimosine degradation. In this study, we isolated a 1,520-bp complementary DNA (cDNA) for mimosinase from L. leucocephala and characterized the encoded enzyme for mimosine-degrading activity. The deduced amino acid sequence of the coding region of the cDNA was predicted to have a chloroplast transit peptide. The nucleotide sequence, excluding the sequence for the chloroplast transit peptide, was codon optimized and expressed in Escherichia coli. The purified recombinant enzyme was used in mimosine degradation assays, and the chromatogram of the major product was found to be identical to that of 3-hydroxy-4-pyridone (3H4P), which was further verified by electrospray ionization-tandem mass spectrometry. The enzyme activity requires pyridoxal 5'-phosphate but not α-keto acid; therefore, the enzyme is not an aminotransferase. In addition to 3H4P, we also identified pyruvate and ammonia as other degradation products. The dependence of the enzyme on pyridoxal 5'-phosphate and the production of 3H4P with the release of ammonia indicate that it is a carbon-nitrogen lyase. It was found to be highly efficient and specific in catalyzing mimosine degradation, with apparent Km and Vmax values of 1.16×10(-4) m and 5.05×10(-5) mol s(-1) mg(-1), respectively. The presence of other aromatic amino acids, including l-tyrosine, l-phenylalanine, and l-tryptophan, in the reaction did not show any competitive inhibition. The isolation of the mimosinase cDNA and the biochemical characterization of the recombinant enzyme will be useful in developing transgenic L. leucocephala with reduced mimosine content in the future.

MidD-encoded ‘rhizomimosinase’ from Rhizobium sp. strain TAL1145 is a C–N lyase that catabolizes L-mimosine into 3-hydroxy-4-pyridone, pyruvate and ammonia

Rhizobium sp. strain TAL1145 catabolizes mimosine, which is a toxic non-protein amino acid present in Leucaena leucocephala (leucaena). The objective of this investigation was to study the biochemical and catalytic properties of the enzyme encoded by midD, one of the TAL1145 genes involved in mimosine degradation. The midD-encoded enzyme, MidD, was expressed in Escherichia coli, purified and used for biochemical and catalytic studies using mimosine as the substrate. The reaction products in the enzyme assay were analyzed by HPLC and mass spectrometry. MidD has a molecular mass of ~45 kDa and its catalytic activity was found to be optimal at 37 °C and pH 8.5. The major product formed in the reaction had the same retention time as that of synthetic 3-hydroxy-4-pyridone (3H4P). It was confirmed to be 3H4P by MS/MS analysis of the HPLC-purified product. The K m, V max and K cat of MidD were 1.27 × 10(-4) mol, 4.96 × 10(-5) mol s(-1) mg(-1), and 2,256.05 s(-1), respectively. Although MidD has sequence similarities with aminotransferases, it is not an aminotransferase because it does not require a keto acid as the co-substrate in the degradation reaction. It is a pyridoxal-5'-phosphate (PLP)-dependent enzyme and the addition of 50 μM hydroxylamine completely inhibited the reaction. However, the supplementation of the reaction with 0.1 μM PLP restored the catalytic activity of MidD in the reaction containing 50 μM hydroxylamine. The catalytic activity of MidD was found to be specific to mimosine, and the presence of its structural analogs including L-tyrosine, L-tryptophan and L-phenylalanine did not show any competitive inhibition. In addition to 3H4P, we also identified pyruvate and ammonia as other degradation products in equimolar quantities of the substrate used. The degradation of mimosine into a ring compound, 3H4P with the release of ammonia indicates that MidD of Rhizobium sp. strain TAL1145 is a C-N lyase.

Development of leucaena mimosine-degrading bacteria in the rumen of sheep in Myanmar

Myanmar has an agricultural base, and about 70% of people reside in rural areas. They depend for survival on agriculture and small-scale crop production, with ruminant livestock consuming fibrous agricultural residues. For optimal ruminant production, concentrates are needed as supplements to these residues. As concentrates are expensive, researchers are testing alternative protein sources like legumes, including foliage from leguminous trees such as leucaena (Leucaena leucocephala). Leucaena is the most widely used leguminous tree as a ruminant feed because it is rich in protein (~ 22%) and contains easily digestible fiber (23% neutral detergent fiber, 16.6% acid detergent fiber; Ni Ni Maw 2004). Khin Htay Myint (2005) noted that 25% of leucaena in the ration tended to increase nitrogen retention without decreasing dry matter and organic matter digestibilities. However, leucaena leaves contain a toxic non-protein amino acid, called mimosine. Research workers have endeavored to reduce mimosine toxicity in animals fed leucaena in Myanmar (Aung Aung 2007; Wink Phyo Thu 2010) and one avenue of research was the development of mimosine-degrading bacteria in the rumen of sheep fed leucaena. In this paper we describe an experiment tracing the development of mimosine-degrading bacteria in the rumen of sheep.

Leucaena toxicity: a new perspective on the most widely used forage tree legume

The tree legume Leucaena leucocephala (leucaena) is a high quality ruminant feed, vitally important for livestock production in the tropics, despite the presence of mimosine in the leaves. This toxic non-protein amino acid has the potential to limit productivity and adversely affect the health of animals. In the 1980s, the ruminal bacterium Synergistes jonesii was discovered and subsequently distributed in Australia as an oral inoculum to overcome these toxic effects. However, in recent times, a number of factors, including: surveys of the status of toxicity worldwide; improved understanding of the chemistry and mode of action of the toxins; new techniques for molecular sequencing; and concerns about the efficacy of the in vitro inoculum; have cast doubt on some past understanding of leucaena toxicity and provide new insights into the geographical spread of S. jonesii . There is also confusion and ignorance regarding the occurrence and significance of toxicity in many countries worldwide. Ongoing research into the taxonomy and ecology of the Synergistetes phylum, improved methods of inoculation, and improved management solutions, along with aware-ness-raising extension activities, are vital for the future success of leucaena feeding systems.

Application of near-infrared reflectance spectroscopy for the estimation of protein and L-canavanine contents in seeds of one-flowered vetch (Vicia articulata Hornem.)

In this work it has been evaluated the effectiveness of near-infrared reflectance spectroscopy (NIRS) for the estimation of two constituents affecting the nutritional quality of the seeds of one-flowered vetch (Vicia articulata Hornem.): protein and L-canavanine, a toxic non-protein amino acid. The NIRS calibrations showed good statistics with coefficients of multiple determination (R2) of 0.97 for total protein and 0.95 for L-canavanine. The developed equations were further used to estimate the contents of protein and L-canavanine of a set of unknown samples (external validation). The equation for total protein was able to predict with an accuracy similar to that of the reference method showing a correlation (r2) between the reference and predicted values of 0.95. In the case of L-canavanine, r2 was only 0.72 and the equation was only effective to discriminate samples into groups of low, medium and high contents.

Bad Taste Protects Fruit Flies from Eating a Toxic Amino Acid in Plants

Bad Taste Protects Fruit Flies from Eating a Toxic Amino Acid in Plants

Amino acid discrimination by arginyl‐tRNA synthetases as revealed by an examination of natural specificity variants

L-canavanine occurs as a toxic non-protein amino acid in more than 1500 leguminous plants. One mechanism of its toxicity is its incorporation into proteins, replacing L-arginine and giving rise to functionally aberrant polypeptides. A comparison between the recombinant arginyl-tRNA synthetases from a canavanine producer (jack bean) and from a related non-producer (soybean) provided an opportunity to study the mechanism that has evolved to discriminate successfully between the proteinogenic amino acid and its non-protein analogue. In contrast to the enzyme from jack bean, the soybean enzyme effectively produced canavanyl-tRNA(Arg) when using RNA transcribed from the jack bean tRNA(ACG) gene. The corresponding k(cat)/K(M) values gave a discrimination factor of 485 for the jack bean enzyme. The arginyl-tRNA synthetase does not possess hydrolytic post-transfer editing activity. In a heterologous system containing either native Escherichia coli tRNA(Arg) or the modification-lacking E. coli transcript RNA, efficient discrimination between L-arginine and L-canavanine by both plant enzymes (but not by the E. coli arginyl-tRNA synthetase) occurred. Thus, interaction of structural features of the tRNA with the enzyme plays a significant role in determining the accuracy of tRNA arginylation. Of the potential amino acid substrates tested, apart from L-canavanine, only L-thioarginine was active in aminoacylation. As it is an equally good substrate for the arginyl-tRNA synthetase from both plants, it is concluded that the higher discriminatory power of the jack bean enzyme towards L-canavanine does not necessarily provide increased protection against analogues in general, but appears to have evolved specifically to avoid auto-toxicity.

Selective determination of mimosine and its dihydroxypyridinyl derivative in plant systems

Our observations on the growth stimulatory nature of mimosine, (beta-(3-hydroxy-4-pyridon-1-yl)-L-alanine), the toxic non-protein plant amino acid, in some model experimental systems, warranted sensitive and selective routine estimations. For the determination of both mimosine and DHP, an indirect spectrophotometric method was developed based on their individual reaction with known excess of DZSAM and by estimating the remaining DZSAM with N-(1-naphthyl)ethylene-diamine (NEDA). The resultant decrease in the secondary coupled product was measured at 540 nm. On equimolar basis, DHP had 40% of the reactivity of mimosine while interference from other relevant compounds was 15-35%. The determination of mimosine and DHP in tissue samples under different physiological conditions was effected after paper chromatographic separation of mimosine and DHP with distinctly differing Rf, from other compounds. The indirect method is superior in terms of absolute selectivity, sensitivity and ease of applicability with linear decreases in absorbance, proportional to increasing concentrations of mimosine from 0.1 to 0.75 microM or DHP from 0.2 to 1.5 microM and with recoveries of 99.2 to 100.5%.

Iron-mediated H2O2 Production as a Mechanism for Cell Type-specific Inhibition of Tumor Necrosis Factor α-Induced but Not Interleukin-1β-induced IκB Kinase Complex/Nuclear Factor-κB Activation

Coordinated and specific regulation of tumor necrosis factor (TNF) and interleukin (IL)-1 signaling pathways and how and whether they are modified by different agents are key events for proper immune responses. The IkappaB kinase complex (IKK)/NF-kappaB and JNK/AP-1 pathways are central mediators of TNF and IL-1 during inflammatory responses. Here we show that l-mimosine, a toxic non-protein amino acid that has been shown to reduce serum TNFalpha levels and affect inflammatory responses, specifically inhibits TNF-induced IKK but not JNK in a cell type-specific manner. l-Mimosine did not affect IKK and NF-kappaB activation by IL-1beta. l-Mimosine caused cell cycle arrest at G(1)-S phase, but inhibition of IKK was found to be independent of cell cycle arrest. Treatment of cells with l-mimosine resulted in production of H(2)O(2). Addition of FeSO(4) restored IKK activation by TNFalpha as did ectopic expression of catalase or pretreatment of cells with N-aceltyl-l-cysteine, indicating a role for intracellular H(2)O(2) as a mediator of inhibition. Cleavage and degradation of TNF pathway components TNFR1, RIP, and Hsp90 were observed in l-mimosine and H(2)O(2) treated cells indicating a putative mechanism for selective inhibition of TNF but not IL-1beta-induced IKK activation.

Cassava cyanogens and free amino acids in raw and cooked leaves

Cassava leaves ( Manihot esculenta Crantz) constitute the main daily source of protein as supplement to the major staple food, the processed cassava roots in remote rural areas of Africa. Konzo, an upper motoneurone disease with permanent spastic paralysis of both legs, has been reported among populations consuming this unbalanced diet. In commercial pounded cassava leaves residual cyanogens and the presence of inherent potentially toxic non-protein amino acids were analysed to check their safety. The initial total cyanogens before cooking ranged from 35.9±0.4 to 107.5±0.8 mg HCN (hydrogen cyanide) equivalent kg −1 dry weight. After cooking, the residual cyanogens were significantly reduced ( P<0.05) ranging from 0.30±0.04 to 1.9±0.2 mg HCN equivalent kg −1 dry weight, and were below the recommended FAO/WHO safe limit set at 10 mg HCN equivalent kg −1 dry weight. The total free amino acids and trigonelline (N-methyl-nicotinic acid) detected varied from 10.8 g kg −1 dry weight to 38.2 g kg −1 dry weight in the raw and from 7.4 g kg −1 dry weight to 25.6 g kg −1 dry weight in the cooked cassava leaves. The non-protein amino acids γ-amino butyric acid (GABA) and α-amino butyric acid (α-ABA) were detected. No known potentially toxic non-protein amino acid was found. In konzo-affected areas, cassava leaves with inadequate preparation and cooking can be a non-negligible source of dietary exposure to cyanogens apart from the cassava roots that are suggested to be involved in the aetiology of konzo.

Quantification of major isoflavonoids and l-canavanine in several organs of kudzu vine ( Pueraria montana) and in starch samples derived from kudzu roots

Isoflavonoids (daidzein, genistein, daidzin, genistin and puerarin) were extracted and analyzed quantitatively by high pressure liquid chromatography from different organs of kudzu vine ( Pueraria montana) and starch samples derived from kudzu roots. Aerial parts of kudzu plants (young shoots, leaf blades and leaf petioles) contained relatively low levels of all isoflavonoids examined, whereas seeds and seedlings were intermediate in isoflavonoid levels and roots consistently had the highest levels, particularly puerarin and the glucosyl conjugates of genistein and daidzein, namely, genistin and daidzin. Interestingly, commercially available kudzu root starch from Japan does not contain the isoflavonoids of interest, whereas homemade kudzu root starch contains all studied isoflavonoids in various amounts, and especially, high levels of puerarin. Shoots of light-grown kudzu seedlings, when compared with shoots of dark-grown seedlings had higher levels of all isoflavonoids with the exception of daidzin. In contrast, for seedling roots, such differences were not greatly different between light-grown and dark-grown plants. Light-grown intact kudzu seedlings had significantly higher levels of soluble proteins than dark-grown seedlings. Mass spectrometer analyzes of all kudzu samples for the toxic non-protein amino acid, l-canavanine, indicated it to be absent using this method of detection and level of sensitivity.

L-Canavanine: a higher plant insecticidal allelochemical.

L-Canavanine, L-2-amino-4-(guanidinooxy)butyric acid, is a potentially toxic nonprotein amino acid of certain leguminous plants. Many species are prolific canavanine producers; they divert enormous nitrogen resource to the storage of this single natural product. Canavanine, a highly effective protective allelochemical, provides a formidable chemical barrier to predation and disease. The accumulated experimental evidence leaves little doubt that the key element in the ability of canavanine to function as an effective protective allelochemical is its subtle structural mimicry of arginine which makes it an effective substrate for amino acid activation and aminoacylation, and its marked diminution in basicity relative to arginine which mediates the production of structural aberrant, dysfunctional canavanyl proteins. The biological burdens of canavanyl protein formation by canavanine-treated Manduca sexta larvae were carried throughout their remaining life cycle. Protein-based sequestration of canavanine prevented turnover and clearance of the free amino acid, and undoubtedly contributed significantly to the antimetabolic character of this protective allelochemical.

The effect of nonprotein amino acid administration on the lysozyme activity of the tobacco hornworm, Manduca sexta [Sphingidae

Abstract A group of seven naturally-occurring, potentially toxic nonprotein amino acid antimetabolites, namely l -azetidine-2-carboxylic acid, l -homoarginine, l -canavanine, 5-hydroxy- l -lysine, l -albizzine, seleno- l -methionine, or 2-aminoethyl- l -cysteine were administered to terminal instar larvae of the tobacco hornworm, Manduca sexta [Sphingidae]. These potentially toxic secondary metabolites were provided concurrently with bacterial cell wall fragments that induce de novo -synthesis of lysozyme. Analysis of hemolymph lysozyme activity revealed that some of these compounds, such as 5-hydroxylysine, homoarginine, and albizzine, had no discernible effect on the activity of the induced lysozyme. On the other hand, providing canavanine, azetidine-2-carboxylic acid, selenomethionine, or 2-aminoethylcysteine, at the time of bacterial challenge, resulted in a significant loss of lysozyme activity. Concurrent administration of canavanine and 2-aminoethylcysteine markedly intensified their individual capacity to inhibit enzyme activity. Selenomethionine had a dramatic sparing effect on canavanine-mediated disruption of lysozyme activity. The individual deleterious effect of azetidine-2-carboxylic acid and canavanine were not exacerbated when these two toxicants were co-administered.