Amitrole Toxicity Research Articles

First structural evidence for the mode of diffusion of aromatic ligands and ligand-induced closure of the hydrophobic channel in heme peroxidases

The mode of binding of aromatic ligands in the substrate binding site on the distal heme side in heme peroxidases is well understood. However, the mode of diffusion through the extended hydrophobic channel and the regulatory role of the channel are not yet clear. To provide answers to these questions, the crystal structure of the complex of lactoperoxidase and 3-amino-1,2,4-triazole (amitrole) has been determined, which revealed the presence of two ligand molecules, one in the substrate binding site and the second in the hydrophobic channel. The binding of ligand in the channel induced a remarkable conformational change in the side chain of Phe254, which flips from its original distant position to interact with the trapped ligand in the hydrophobic channel. As a result, the channel is completely blocked so that no ligand can diffuse through it to the substrate binding site. Another amitrole molecule is bound to lactoperoxidase in the substrate binding site by replacing three water molecules, including the crucial iron-bound water molecule, W1. In this arrangement, the amino nitrogen atom of amitrole occupies the position of W1 and interacts directly with ferric iron. As a consequence, it prevents the binding of H2O2 to heme iron. Thus, the interactions of amitrole with lactoperoxidase obstruct both the passage of ligands through the hydrophobic channel as well as the binding of H2O2. This explains the amitrole toxicity. From binding studies, the dissociation constant (Kd) for amitrole with lactoperoxidase was found to be approximately 5.5x10(-7) M, indicating high affinity.

Primary Site of Action of Amitrole in Arabidopsis thaliana Involves Inhibition of Root Elongation but Not of Histidine or Pigment Biosynthesis

Interference with histidine metabolism, inhibition of pigment biosynthesis, or both have been the principal candidates for the primary site of action of 3-amino 1,2,4-triazole (amitrole). Arabidopsis thaliana is sensitive to 1,2,4-triazole-3-alanine, a feedback inhibitor of histidine biosynthesis, and this effect is reversed by histidine. The combination of triazolealanine and histidine, however, does not reverse the herbicidal effect of amitrole. This indicates that amitrole toxicity is not caused by histidine starvation, nor is it caused by the accumulation of a toxic intermediate of the histidine pathway. Amitrole inhibits root elongation at lower concentrations than it causes pigment bleaching in the leaves. In contrast, fluridone, a known inhibitor of the carotenoid biosynthetic pathway does not block root elongation. Fluridone also inhibits carotenoid accumulation in etiolated seedlings in the dark, but amitrole does not. Last, gabaculine and acifluorfen, but not amitrole, prevent chlorophyll accumulation in greening etiolated seedlings of Arabidopsis. These experiments cast doubt on pigment biosynthesis as the primary site of action of amitrole.

Mutagenicity and toxicity of amitrole. I. Drosophila tests

Amitrole was highly toxic at early larval stages of Drosophila (LD 50 is 40 ppm in medium). Toxicity of amitrole was also revealed by prolongation of development time even at 10 ppm. However, no mutagenic effects of amitrole were observed either in the sex chromosome non-disjunction test (females reared on medium containing amitrole at 10 ppm) or in the sex-linked recessive lethal test (males reared on medium containing amitrole at 10 ppm).

Mutagenicity and toxicity of amitrole. III. Microbial tests

Amitrole (3-amino-1,2,4-triazole) inhibits bacterial growth both in Escherichia coli and Salmonella typhimurium at a concentration of 0.5% in minimal medium. Repression of growth already occurs at a concentration of 0.1% of amitrole in this medium. In complete medium the bacteria tolerate concentrations of amitrole as high as 1.7–2.4% before growth ceases. Mutagenicity was tested by differential growth comparisons on E. coli strains W 3110 thy pol A1, defective in DNA polymerase I, and its revertant pol A +. Known mutagens (MMS, NTG, mitomycin C) were used as positive controls. Analogous negative results were also obtained in a revertant test when several trp mutant strains of Salmonella were used.

Mutagenicity and toxicity of amitrole. II. Human lymphocyte culture tests

Effects of amitrole (3-amino-1,2,4-triazole) on human leucocytes in culture were investigated. Amitrole interfered with lymphoblast transformation and inhibited cell growth in concentrations of 0.2% w/v and higher. Selected metaphases were examined for the presence of chromosome and chromatid aberrations. No clastogenic effects were observed.

Beeinflussung der Amitroltoxizität durch Riboflavin in Haferkeimpflanzen

The following parameters were determined to characterize the amitrole toxicity in oat seedlings: growth, wet and dry weight, content of pigments, and activity of catalase. Riboflavin reduces the inhibitory actions of amitrole only in plants, which have been treated with these substances in the light. In the dark riboflavin had no effect on amitrole toxicity. Riboflavin- 14 C is not translocated from the roots to the leaves, and this behaviour is not changed by amitrole. Riboflavin does not affect the uptake and the translocation of amitrole- 14 C. We suggest that in oat the effect of riboflavin in the light is exclusively due to the well-known riboflavin-mediated photodecomposition of amitrole but not to physiological processes within the plants. Furthermore we conclude from experiments with amitrole-[5- 14 C], which was supplied and metabolized in the dark, that the site of action of amitrole must be connected with light-independent reactions which are essential for subsequent light-dependent processes.

Einfluß von Amitrol auf die Zusammensetzung der freien Aminosäuren in Haferkeimpflanzen

Oat seeds were germinated 72 h in the dark and thereafter cultivated in solutions with or without 10 − 3 M amitrole 72 h in the dark and 36 h in the light (3000 lx). Plants treated in this way become chlorotic (content of chlorophyll: 25% of the control) without reduction of weight. The chlorophyll suppression is connected with increased concentrations of amino acids and free ammonia. Slight differences in the degree of enhancement between the various amino acids are not specific for definite metabolic groups. In contrast to results with microorganisms, the biosynthesis of histidine is not inhibited by amitrole; an alternative pathway of histidine biosynthesis may exist in higher plants. The serine-glycine-pool is not depleted, that means that the reaction of amitrole with serine is not responsible for the amitrole chlorosis in oat plants. Some of the visible symptoms of amitrole toxicity may be due to the action of accumulated ammonia.

Der Einfluß von 3-Amino-1,2,4-triazol (Amitrol) auf die löslichen Nukleotide und weitere Phosphorverbindungen in Haferkeimpflanzen

Oat seedlings were exposed for 3 or 7 days to light at 3000 lx in a medium with or without 10− 3 M amitrole. The amitrole-treated plants contained slightly more acid-soluble nucleotides than the non-treated ones. Purine nucleotides were accumulated as much as pyrimidine nucleotides. The content of UDPG was extremely high, after 7 d 204% per g wet weight. The concentration of ATP was increased, this suggests oxidative phosphorylation not affected. There was no detectable depletion of any nucleotide. The increase of nucleotides is not connected with a decrease of RNA and DNA. The amount of soluble phosphorus compounds not adsorbed to Norit A, was insignificantly increased; the content of phospholipids was unchanged. The treated plants were chlorotic, and their weight was reduced. These symptoms of amitrole toxicity cannot be explained by an interaction of amitrole with the synthesis of purines or nucleic acids. We also describe a simple device for generating variable concentration gradients and give equations for their evaluation.

The toxicity of amitrole in the rat

A single oral dose of 4080 mg/kg and a single dermal dose of 2500 mg/kg of amitrole, the highest doses given, were not lethal to adult rats. Dietary levels of 0, 1000, 500, 100 and 25 ppm amitrole were fed to groups of 10 male and 10 or 12 female rats. The dietary levels of 1000 and 500 ppm were fed for 107–110 days, the dietary levels of 100 and 25 ppm for 240–247 days to the F 0 generation and for 141 days to the F 1b generation. At the dietary levels of 1000 and 500 ppm the pups that were born were smaller and had atropic thymuses and spleens indicative of runt disease. Most of them had died with-in a week after weaning. Reproduction was not affected at the lower dietary levels. Hyperplasia of the thyroid was seen in all rats fed dietary levels of 100 ppm or greater. In the 25 ppm group 4 10 F 0 and 4 10 F 1b females and 6 10 F 1b males, but none of the F 0 males developed hyperplasia of the thyroid.

The herbicide 3-amino-1,2,4-triazole (Amitrole) as teratogen

The teratogenic activity of the herbicide 3-amino-1,2,4-triazole (Amitrole) was tested by injecting aqueous solutions into the yolk sac of chicken eggs at 0, 24, 48, and 96 hours of incubation. The toxicity of Amitrole was relatively high at 0 hours, moderate at 24 and 48 hours, and very high at 96 hours. It showed clear dosage dependence. The principal malformations produced by Amitrole concern the beak. Abnormalities of the tibia shaft occurred as a much less common defect. With a dosage of 20 mg/egg at 0, 24, and 48 hours of incubation Amitrole led to a relatively low incidence of malformations, but this was followed by a threshold to high teratogenicity at 96 hours. Treatment with 40 mg/egg of the teratogen, on the other hand, produced at all stages between 0 and 96 hours a similarly high incidence of beak defects. A series of compounds tested as supplement to Amitrole was of no avail in reducing teratogenicity. A comparison of dimethyl sulfoxide and water as solvents of Amitrole showed that in the former the compound is much less detrimental to chick development than in the latter.

Regulation of histidine biosynthesis in cultured plant cells: Evidence from studies on amitrole toxicity

Early exponential phase cells of Paul's Scarlet rose in suspension culture respond to the herbicide amitrole (3-amino-1,2,4-triazole) by accumulating the histidine precursor imidazoleglycerol phosphate and its derivative, imidazoleglycerol. This accumulation is inhibited by l-histidine, which provides evidence for the operation of endproduct inhibition in vivo.

Influence of Oils on the Toxicity of Amitrole to Quackgrass

In field trials, 2 gpa of oil enhanced the herbicidal activity of 3-amino-1,2,4-triazole (amitrole) and a mixture of amitrole + ammonium thiocyanate (hereinafter referred to as amitrole-T) on quackgrass (Agropyron repens(L.) Beauv.). None of the oils consistently increased the toxicity of other foliarly-applied herbicides. A paraffinic mineral oil enhanced the activity of amitrole on quackgrass to a greater degree than NH4SCN. The inclusion of a paraffinic mineral oil enhanced both the amount and the rate of14C-amitrole and amitrole-T translocated from the treated leaf to the shoots and roots, this being more pronounced with amitrole (twelvefold) than with amitrole-T (twofold).

Reversal by Histidine of the Inhibition of Prototheca Growth due to 3-Amino-1,2,4-triazole

3-AMINO-l,2,4-triazole (amitrole) is used as a systemic herbicide. It also inhibits the growth of a number of micro-organisms including several algae. Organic acids have long been known to inhibit the growth of algae by combining with iron1. Aaronson2 concluded that amitrole was also working in this way as the inhibition of Ochromonas danica could be abolished by the supply of extra iron to cultures in the presence of the inhibitor. Later Wolf3, examining Chlorella pyrenoidosa, and Boney4 certain marine red algae, reported that the inhibition by amitrole could be reversed by adenine. In this the algae are similar to Escherichia coli5. Histidine is effective in releasing the inhibition of Torula cremoris5 and of Saccharomyces cerevisiae6. Since the standard medium used by Aaronson contained histidine7, an investigation of the reversal of amitrole toxicity towards Prototheca zopfii was carried out. This colourless alga is believed to be closely related to Chlorella and it will grow in a mineral medium supplemented with glucose and thiamine8. Aaronson and Scher7 showed that there was no significant difference in the concentration of amitrole required to achieve inhibition of Euglena and Ochromonas growth in the presence of an organic substrate in the dark as compared with the light although chlorophyll synthesis was inhibited.

Riboflavin Nullification of Inhibitory Actions of 3-Amino-1,2,4-Triazole on Seedling Growth

The physiological mechanislmis involved in the toxic actions of 3-amino-1,2,4-triazole (amitrole) have been actively investigated since the chemical was first introduced as a potential herbicide in 1954 (2). Much of the research has been guided by hypotheses involving one of three major metabolic processes: A, porphyrin synthesis, B, cation functions, and more recently C, the metabolism of purines and their derivatives. The extensive literature associated with the first two categories will not be considered herein. Amitrole interferences in some of the biochemiiical processes of the third category now appear to be the actions of nmajor physiological importance in the response of plants to the herbicide. Much of the pertinent research deals with attempts to find physiological compounds capable of reducing the toxicity of amitrole to various organisms. The most effective conmpounds are purines (23, 24) or histidine (13, 23) for unicellular plants anid riboflavin or riboflavin derivatives (22, 24) for higher plants. The investigations reported herein Nwere initiated as a result of my observations that the riboflavin effect could not be denmonstrated with barley seedlings by tecliiques that had been used successfully with other herbicidemetabolite combinations (9, 14, 18). The objectives of this study were: A, to develop techniques by whiclh the riboflavin nullification of amitrole toxicity could be observed with higher plants, and to compare adenine and histidine with riboflavin by these techniques; BR, to consider the possibilitv that the riboflavin effect was the result of non-physiological processes: C, to compare the effectiveness of riboflavin in species of higher plants that differ in their response to amitrole, and D, to determine if certain other metabolites of theoretical interest might give additional reductionis in amitrole toxicity.

Dalapon, amitrole, and weed oil compared for effectiveness in control of bermudagrass in California citrus orchards

Dalapon, amitrole, and weed oil, used alone, as mixtures, or in combination, were compared for effectiveness in the control of bermudagrass. Split applications of dalapon, properly timed, were more effective than single applications with the same total amount of herbicide. Amitrole, and to a lesser extent dalapon, was more effective when applied late in the growing season. Variation in spray volume, the presence or absence of surfactant, or the inclusion of small amounts of kerosene had no effect on the toxicity of amitrole. Mixtures of dalapon and amitrole were less toxic than was dalapon applied alone at the same rate. Split applications of amitrole were less effective than single applications containing the same total amount of herbicide. Repeat spraying with weed oil at three-week intervals was highly effective and gave control equal to the more frequent oil spraying. In a screening program, atratone and prometone at rates of 10 pounds per acre controlled bermudagrass. Ipazine, trietazine, simetone, simazine, CIPC, EPTC, and maleic hydrazide were not highly toxic to bermudagrass. Sodium 2,3-dichloroisobutyrate was only an eighth as effective as dalapon. This paper reports research on the evaluation of several chemicals as herbicides for the control of bermudagrass. No recommendations for or against the use of these chemicals are intended or implied. Federal and state laws regulate the use of agricultural chemicals, and persons wishing to use chemicals mentioned in this publication should consult local agricultural regulatory officials.