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Thylakoid dismantling of damaged unfunctional chloroplasts modulates the Cab and RbcS gene expression in wheat leaves.

Thylakoid membrane dismantling and Lhcb and RbcS nuclear gene expression have been analysed in leaves of wheat plants grown in high fluence rate light and deprived of photoprotective carotenoids by treatments with the two bleaching herbicides, either norflurazon or amitrole. The Lhcb transcript was not detectable in cells of norflurazon-supplied leaves, having chloroplasts totally devoid of both inner membranes and pigments. In contrast, a substantial amount of Lhcb mRNA could be found in cells of amitrole-treated leaves, whose severely damaged organelles still contained few strikingly altered and photosynthetically unfunctional thylakoids, as well as chlorophyll traces. A possible relationship between chlorophyll synthesis and Lhcb expression, with the transcript level depending on the rate of pigment production in photodamaged chloroplasts is discussed. Also the RbcS expression was linked to the chloroplast membrane photodamage. However, a detectable level of transcript was still produced in norflurazon-treated cells, despite complete thylakoid demolition. Thus, the wheat cell behaviour had to be placed between that of species, such as maize, in which the RbcS expression is broken off in these conditions, and that of species, such as pea, in which it is slightly lowered. Interestingly, the dramatically photodamaged chloroplasts still maintained the ability to synthesize proteins and this allowed SSU and LSU Rubisco subunits to be found in the organelles of both norflurazon- and amitrole-treated plants.

Amitrole↗

Genotoxicity testing of five herbicides in the Drosophila wing spot test.

Four triazine herbicides: amitrole, metribuzin, prometryn and terbutryn, and the bipyridal compound diquat dibromide have been evaluated for genotoxicity in the wing somatic mutation and recombination test of Drosophila melanogaster, following standard procedures. Third-instar larvae trans-heterozygous for the third chromosome recessive markers multiple wing hairs (mwh) and flare-3 (flr(3)) were chronically fed with different concentrations of the test compounds. Feeding ended with pupation of the surviving larvae. Genetic changes induced in somatic cells of the wing's imaginal discs lead to the formation of mutant clones on the wing blade. Point mutation, chromosome breakage and mitotic recombination produce single spots; while twin spots are produced only by mitotic recombination. Exposure to 0.5 mM and 1 mM of amitrole clearly increased the frequency of small single, large single and total spots. Terbutryn, at the concentration of 5 mM, induced a slight increase in the frequency of small single and total spots, but this result could be false positive. The other three herbicides tested did not show any genotoxic effect. When heterozygous larvae for mwh and the multiple inverted TM3 balancer chromosomes were treated, significant increases in the frequency of mutant spots were only detected for amitrole. The observed spot frequencies were lower than those found in mwh/flr(3)50%) of the total spot induction was due to mitotic recombination.

Amitrole↗

Dietary subacute toxicity of ethylene thiourea in the laboratory rat.

Ethylene thiourea (ETU) was fed to groups of rats at 0, 1, 5, 125 or 625 ppm for up to 90 days. Other groups of rats received either propylthiouracil (PTU; 125 ppm) or amitrole (50 ppm) in their diets as positive controls. Only those rats which received ETU at 125 or 625 ppm and those ingesting PTU or amitrole demonstrated a measurable toxic response. This toxicity was reflected as an alteration in thyroid function and a significant change in thyroid morphology. Ingestion of 625 ppm ETU or 125 ppm PTU resulted in very substantial decrease in serum triiodothyronine (T-3) and thyroxine (T-4). Marked increases in serum thyroid stimulating hormone (TSH) levels were found in the 625 and 125 ppm ETU rats, the 125 PTU rats, and the rats receiving amitrole, each time this hormone was measured. Rats which ingested 625 ppm ETU also exhibited a decrease in iodide uptake by the thyroid. While a statistically significant increase in serum T-4 and degree of thyroid hyperplasia was observed for rats ingesting 25 ppm ETU for 60 days, normal thyroid hormone levels and thyroid morphology was found in rats on 25 ppm ETU for either 30 or 90 days. Based on diochemical and microscopic changes examined, the no-effect level for dietary ETU in this 90-day study is considered to be 25 ppm.

Amitrole↗

Cab gene expression in bleached leaves of carotenoid-deficient maize.

The chloroplast photo-oxidation and the expression of the Cab gene Lhcb1, encoding the Lhcb1 light-harvesting chlorophyll a/b-protein of PS II, have been studied in leaf cells of maize treated with the two bleaching herbicides norflurazon and amitrole and of the two carotenoid-free mutants vp9 and vp2 grown under high photodamaging light. Both herbicides and mutations caused severe photo-oxidation of organelles. However, the plastids of norflurazon-treated and vp2 leaves were totally devoid of thylakoids and did not contain any chlorophyll, while the organelles of amitrole-treated and vp9 leaves still had a few altered and photosynthetically unfunctional membranes and very small quantities of chlorophylls. Despite the dramatic photodamage undergone by the plastids over several days, the cells of amitrole-treated and vp9 leaves maintained a certain expression of the Lhcb1 gene which, on the contrary, was completely blocked in the cells of norflurazon-treated and vp2 leaves. The experimental results, obtained by integrating biochemical and molecular analyses with ultrastructural observations, show that the maintainance of Cab gene expression does not strictly depend on intact and functional chloroplasts. The transcription of these genes, still maintained in cells with greatly photo-oxidized organelles, seems to be inversely related to the degree of thylakoid demolition, which can affect the last steps of chlorophyll biosynthesis.

Journal Article↗

Selection of Glyphosate-Tolerant Tobacco Calli and the Expression of this Tolerance in Regenerated Plants.

From nonmutagenized haploid suspensions of Nicotiana tabacum L. cv Wisconsin 38 cells, 51 cell lines capable of growth in the presence of 1 millimolar glyphosate (N-phosphonomethyl glycine) were initially isolated at a frequency of 2.3 x 10(-8). Eighteen cell lines retained tolerance when grown on selective medium for 3 years. Tolerance persisted for at least 14 months in six cell lines cultured in the absence of glyphosate. Some plants regenerated from four glyphosate-tolerant cell lines were tolerant. Glyphosate-tolerant tissue was isolated from some sensitive as well as some tolerant regenerated plants. Six of the tolerant cell lines were also tolerant to the herbicide amitrole (3-amino-1,2,4-triazole). Five cell lines selected for amitrole tolerance were glyphosate tolerant. Some plants regenerated from three of these five cell lines were glyphosate tolerant and glyphosate-tolerant tissue was obtained from several of these regenerated plants. Amitrole uptake in suspension cultures of several variants was assessed in terms of influx rate constants. This parameter was not sufficiently different indicating that altered membrane properties could not account for the herbicide tolerance.

Journal Article↗

Role of catalase and oxidative stress in hepatic peroxisome proliferator-induced morphological transformation of Syrian hamster embryo cells.

Several hepatic peroxisome proliferators (HHPs) such as di(2-ethylhexyl)phthalate (DEHP), mono(2-ethylhexyl)-phthalate, clofibrate and tiadenol, induce morphological transformation of Syrian hamster embryo (SHE) cells in vitro. According to one hypothesis, the hepatocarcinogenic effect of HPPs is caused by an oxidative stress due to increased H2O2-production from the strongly induced peroxisomal beta-oxidation of fatty acids. Thus, increased transformation frequencies by HPPs should be obtained when catalase was inhibited by 3-amino-1,2,4-triazole (amitrole). However, co-exposure to HPPs and amitrole did not enhance the transformation frequencies for any of the HPPs. The sensitivity of SHE cells for oxidative agents was studied by using menadione and H2O2. Menadione only induced transformation at a toxic concentration, while H2O2 induced transformation at non-toxic concentrations. To study the generation of oxidative radicals in SHE cells, electron spin resonance was employed. No oxidative radical formation was detected in tiadenol- or DEHP-exposed SHE cells. When menadione or H2O2 were added during the measurements, oxidative radicals were found. A transmission electron microscopic study showed a small number of peroxisomes, and did not reveal any increase in the number of peroxisomes in clofibrate-treated SHE cells.

Amitrole↗

[Metabolism of 3-amino-1,2,4-triazole in rats].

3-Amino-1,2,4-triazole[5-(14)C] was administered orally to rats as a single dose of 50 mg/kg body weight. Excretion in urine and feces was followed during a period of 3 days. Within the first 24 hrs the main part of the radioactivity was found in the urine as unchanged amitrole. 3-Amino-5-mercapto-1,2,4-triazole and 3-amino-1,2,4-triazolyl-(5)-mercapturic acid were isolated from urine and identified by comparison with synthetic compounds. The total amount of these metabolites in the urine was about 6% of the dose. The metabolic pathways of amitrole and the possible relations between biotransformation and toxicity are discussed.

Acetylcysteine↗

Genetic toxicity of six carcinogens and six non-carcinogens in the Drosophila wing spot test.

Six rodent carcinogens, 5 of which are also human carcinogens, and 6 compounds recognized as non-carcinogens were tested for their genotoxic activity in the Drosophila melanogaster wing spot test. 72-h-old larvae trans-heterozygous for the recessive wing cell markers 'multiple wing hairs' (mwh) and 'flare' (flr3) were fed various concentrations of the test compounds for a period of 48 h. With amitrole and 4-aminobiphenyl, larvae of the same age were also given an acute treatment of 6 h with higher concentrations, and, in addition, 48-h-old larvae were fed for a longer period of 72 h. Repeats of all experiments document the good reproducibility of the results in the wing spot test. Amitrole and 4-aminobiphenyl were genotoxic after both 48-h and 72-h treatments, but their activity could not be detected following acute exposure of only 6 h. Chlorambucil and melphalan were clearly genotoxic. The carcinogens sodium arsenite and sodium arsenate, however, which are highly toxic to Drosophila, could only be tested at low exposure levels and were negative under these treatment conditions. The 6 non-carcinogens (ascorbic acid, 2-aminobiphenyl, mannitol, piperonyl butoxide, stannous chloride and titanium dioxide) were all definitely non-genotoxic in the Drosophila wing spot test. The data for the non-carcinogens demonstrate that non-genotoxic compounds can be identified in the wing spot test with a reasonable experimental effort.

Aminobiphenyl Compounds↗

Adsorption of pesticides on resins.

The objective of this work was to assess the capability of organic hydrophobic polymeric resins Amberlite XAD-4 and XAD-7 to remove the pesticides alachlor and amitrole from water. The pesticides adsorption on the two different adsorbents was measured by batch equilibrium technique and isotherm types and parameters were estimated. Two theoretical models were applied based on a Freundlich and a Langmuir isotherms. The effect of pesticides chemical composition and structure as well as the nature of solid surface on the efficiency of adsorption was evaluated. The influence of pH also was studied. In low pH solutions adsorption of amitrole was higher upon the nonionic aliphatic acrylic ester copolymer XAD-7 in comparison to the nonionic, crosslinked macroreticular copolymer of styrene divinylbenzene XAD-4. In neutral and intermediate pH solutions the polar acrylic ester copolymer XAD-7 was more effective to the retention of alachlor. The acrylic ester copolymer showed at pH 3 the lower effectiveness in alachlor removal from water. The data of the adsorption isotherms of pesticides upon the examined polymeric resins seemed to conform to both the Freundlich and the Langmuir isotherm models.

Acetamides↗

The radioimmunological measurement of thyroglobulin secretion in vitro under the influence of some herbicides.

A radioimmunoassay (RIA) technique of human thyroglobulin (Tgl) was developed using 125I-Tgl and a rabbit anti-Tgl serum at the initial dilution of 1:100,000. The sensitivity of the technique was of 2.5 ng/ml. The RIA of Tgl was applied as a screening test for the in vitro secretory activity of human thyroid cells under the influence of two herbicides of the heterocyclic group (Amitrol and Atrazin). Three levels of concentration of Amitrol and Atrazin were administered to the tissue culture tubes containing in the first series of experiments perinodular tissue (Typ), and in the second one nodular tissue (TyN). The thyroid gland was surgically obtained from a patient with the diagnosis of polynodular goiter. Only the high levels (10 mg and 1.0 mg/tube, respectively) of herbicide concentration inhibited the secretion and release of Tgl, the nodular tissue being more resistant to the toxic. However, parallel studies including the dosage of the active hormones (T3, T4) are necessary in order to appreciate the value of Tgl determination as an indicator of hormogenesis in this type of experiments.

Amitrole↗

Effect of pesticides on blue-green algae and nitrogen-fixation.

The effects of the pesticides, amitrol, a derivative of amitrol (viz. 3,5-diamino-1,2,4-triazole), diquat, paraquat, linuron, MCPA, malathion, and monuron, were studied on the nitrogen-fixing algae, Anabaena cylindrica, Aulosira sp., Calothrix elenkenii. Chlorogloeae frischii, Cylindrospermum muscicola, Nostoc sp. from Collema tenax, Nostoc muscorum Tolypothrix tenuis, and Westiellopsis sp. In general, two types of response were discernible; an initial period of depression succeeded by an increased activity and an initial period of depression followed by a distinct decrease on prolonged incubation. The results indicate that some pesticidal compounds can severely limit the nitrogen-fixing capacities of blue-green algae, thereby affecting the overall nitrogen economy of soils in general.

Culture Media↗

Performance of 181 chemicals in a Drosophila assay predominantly monitoring interchromosomal mitotic recombination.

An evaluation is presented of the effects of 181 chemicals in the (white/white+) (w/w+) eye mosaic assay, an in vivo short-term test measuring genetic damage in somatic cells of Drosophila after treatment of larvae. The genetic principle of this system is loss of heterozygosity for the wild-type reporter gene w+, an event predominantly resulting from homologous interchromosomal mitotic recombination between the two X chromosomes of female genotypes. The w/w+ eye mosaic test detects a broad spectrum of DNA modifications, since all distinct classes of genotoxins are monitored. Non-DNA-reactive chemicals are in principle not detected by this system. Occasional positive responses obtained for chemicals such as amitrole, ethionine and hexachloeroethane are probably not related to the mechanism responsible for their tumorigenicity. The principle outcome of this analysis is the necessity for classification of responses into three categories. (i) Positive, '++'. The 92 chemicals (Tables II and III) falling into this category were clearly recombinagenic in the assay, meaning that dose-response relations were obtained (or could have been established as was evident from the strong responses obtained at one or two exposure doses). Among the 92 chemicals were 49 promutagens including volatile chemicals such as vinyl bromide and vinyl chloride. (ii) Marginally positive, '+w'. The definition of a weakly positive response is the absence of a dose-response relationship due to the fact that a weak but reproducible effect, in most cases no more than a doubling of the spontaneous clone frequency, is inherently related to toxicity. The 40 chemicals (Tables IV and V) belonging to this category mainly represented four distinct types. (a) Procarcinogens, such as 2-acetylaminofluorene, dibenz[a,h]anthracene, p-dimethylaminoazobenzene, 2-naphthylamine and safrole, for which metabolic conversion was the apparent problem in the assay. (b) Electrophilic chemicals of high nucleophilic selectivity: acrolein, acrylamide, acrylonitrile, epichlorohydrin, chloroethylisocyanate, 1,2-epoxybutane, N-methyl-n-vinylacetamide, methyl vinylketone, 2-methyl-2-vinyloxirane and methyl vinylsulfone. These chemicals have a relatively low DNA reactivity. Chloroethylisocyanate was active only in the absence of excision repair, suggesting that efficient DNA repair is the cause for the weak genotoxic effectiveness of these genotoxins. (c) Spindle poisons (Table V) were active at rather low but toxic exposure levels. Irregularities in the structure of ommatidia were seen at dose levels producing no more than 2- to 3-fold increases in clone frequencies. (d) The fourth group consisted of chemicals generally regarded as non-genotoxic carcinogens: amitrole, ethionine, ethylurea, tetrachloroethylene and thiourea. Their weak responses were always accompanied by signs of toxicity.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

Regulation of tryptophan biosynthetic enzymes in Neurospora crassa.

The formation of enzymatic activities involved in the biosynthesis of tryptophan in Neurospora crassa was examined under various conditions in several strains. With growth-limiting tryptophan, the formation of four enzymatic activities, anthranilic acid synthetase (AAS), anthranilate-5-phosphoribosylpyrophosphate phosphoribosyl transferase (PRAT), indoleglycerol phosphate synthetase (InGPS), and tryptophan synthetase (TS) did not occur coordinately. AAS and TS activities began to increase immediately, whereas PRAT and InGPS activities began to increase only after 6 to 12 hr of incubation. In the presence of amitrole (3-amino-1,2,4-triazole), the formation of TS activity in a wild-type strain was more greatly enhanced than were AAS and InGPS activities. With a tr-3 mutant, which ordinarily exhibits an elevated TS activity, amitrole did not produce an increase in TS activity greater than that observed on limiting tryptophan. With tr-3 mutants, the increased levels of TS activity could be correlated with the accumulation of indoleglycerol in the medium; prior genetic blocks which prevented or reduced the synthesis of indoleglycerol also reduced the formation of TS activity. The addition of indoleglycerol to cultures of a double mutant (tr-1, tr-3) which could not synthesize indoleglycerol markedly stimulated the production of TS activity but not PRAT activity; the production of TS activity reached the same level with limiting or with excess tryptophan. A model explaining these and other related observations on enzyme formation in N. crassa is proposed.

Cell-Free System↗

Goitre and wasting induced in hamsters by hexachlorobenzene.

Male Syrian hamsters were treated with hexachlorobenzene (HCB) in their diet at levels of 100 ppm for 28 weeks, 200 ppm for 18 and 28 weeks, and 500 ppm for 6 weeks. All treatments caused at least a 2.5- to 3-fold increase in thyroid size, mainly by enlargement of some follicles. Serum thyroxine (T4) levels were unchanged, whereas levels of triiodothyronine (T3) eventually became depressed by greater than 60%. Uptake of 131I into thyroids was induced approximately 3-fold when estimated after feeding HCB (500 ppm) for 3 or 6 weeks. Hamsters also lost weight by depletion of adipose tissue, leading to 50% mortality in longer experiments. Results were distinct from the effects of the known antithyroid agent 3-aminotriazole or amitrole (200 ppm for 28 weeks), which did not affect survival and although causing thyroid enlargement depressed serum T4 and significantly elevated T3. The effects of HCB in hamsters were also different from those in rats (500 ppm HCB for 6 weeks) in which there was only a small increase in thyroid size (1.3-fold), serum levels of T3 were only slightly depressed but T4 levels were reduced by 74%. These studies are discussed with reference to the effects of other polyhalogenated aromatic chemicals on the thyroid, serum thyroid hormone levels and lethality.

Adipose Tissue↗

Evaluation of the isobologram method for the assessment of mixtures of chemicals. Combination effect studies with pesticides in algal biotests.

The aim of this paper is an evaluation of isobolograms, a method proposed for the assessment of combined effects of chemicals. In order to examine potentials and shortcomings of this approach for ecotoxicological purposes, algal biotests with selected pesticidal compounds were performed. Additivity, as defined by the model, is demonstrated for the combination of atrazine and metribuzin for different combination ratios, response levels, and parameters. Subadditivity is shown for amitrole and glufosinate-ammonium. The results and inherent biometrical features are discussed in terms of criteria considered suitable for comparative evaluation of biometrical models for the assessment of mixtures of chemicals.

Aminobutyrates↗

Effects of environmental synthetic chemicals on thyroid function.

Synthetic chemicals are released into the environment by design (pesticides) or as a result of industrial activity. It is well known that natural environmental chemicals can cause goiter or thyroid imbalance. However, the effects of synthetic chemicals on thyroid function have received little attention, and there is much controversy over their potential clinical impact, because few studies have been conducted in humans. This article reviews the literature on possible thyroid disruption in wildlife, humans, and experimental animals and focuses on the most studied chemicals: the pesticides DDT, amitrole, and the thiocarbamate family, including ethylenethiourea, and the industrial chemicals polyhalogenated hydrocarbons, phenol derivatives, and phthalates. Wildlife observations in polluted areas clearly demonstrate a significant incidence of goiter and/or thyroid imbalance in several species. Experimental evidence in rodents, fish, and primates confirms the potentiality for thyroid disruption of several chemicals and illustrates the mechanisms involved. In adult humans, however, exposure to background levels of chemicals does not seem to have a significant negative effect on thyroid function, while exposure at higher levels, occupational or accidental, may produce mild thyroid changes. The impact of transgenerational, background exposure in utero on fetal neurodevelopment and later childhood cognitive function is now under scrutiny. There are several studies linking a lack of optimal neurological function in infants and children with high background levels of exposure to polychlorinated biphenyls (PCBs), dioxins, and/or co-contaminants, but it is unclear if the effects are caused by thyroid disruption in utero or direct neurotoxicity.

Amitrole↗

Lung pH and pulmonary absorption of nonvolatile drugs in the rat.

The effect of pH on pulmonary absorption of nonvolatile drugs was investigated in the rat. Krebs-Ringer phosphate solutions (pH 6.2 and 7.4), Krebs-Ringer pyrophosphate solution (pH 8.4), or an unbuffered salt solution containing a drug were administered through tight-fitting tracheal cannulas to anesthetized animals. After 3 min, the lungs were removed and assayed for the amount of drug that remained. Weak acids and a weak base were absorbed most rapidly at pH values at which the drugs were least ionized. For example, with the base procainamide, 36% of the dose was absorbed at pH 6.2 and 76% at pH 8.4. With the acid sulfisoxazole, 71% was absorbed at pH 6.2 and 55% at pH 8.4. Similarly, with p-aminosalicylic acid, 77% was absorbed at pH 6.2 and 40% at pH 8.4. In contrast to these results, compounds such as urea and amitrole, which remain completely nonionized over the pH range studied, showed no change in absorption rate when the pH was varied. The two weak acids and the weak base were absorbed from an unbuffered solution as though the pH at the site of drug absorption was between 6.2 and 7.4. The absorption rate for each weak electrolyte from unbuffered solution, when compared graphically with the respective absorption rates from buffered solutions, indicated that the pH at the site of drug absorption is about 6.6.

Absorption↗

Binding of organic compounds to rat liver and lung.

The binding of various radioisotopically labeled organic compounds to rat liver and lung was investigated in vitro. Pieces of rat lung and slices of rat liver were incubated at 37 degrees C under a nitrogen atmosphere in a modified Krebs-Ringer phosphate solution (pH 7.4) CONTAININg the compound to be studied. Of the neutral compounds investigated, digitoxin, digoxin and dexamethasone were highly bound to both liver and lung tissue, whereas the degree of binding of amitrole, erythritol, and ouabain was 20% or less. The weak acids which were bound to the greatest extent in both liver and lung were phenobarbital, pentobarbital, and diphenylhydantoin. Barbital was poorly bound, and there was no evidence for the binding of 5,5-dimethyloxazolidine-2,4-dione or p-aminohippuric acid in either tissue. Binding of the cardiac glycosides and the barbiturates directly paralleled their lipid solubilities. The degree of binding of neutral compounds and weak acids to lung and liver tissue did not vary greatly with concentration, even though broad concentration ranges were studied. This was also true of the weak base morphine. On the other hand, the binding to liver and lung of the organic bases nicotine, pilocarpine, d-amphetamine, lidocaine, erythromycin, and chloroquine, did vary with concentration. The quaternary ammonium compound decamethonium was bound only to liver, and this binding also varied with concentration. Two additional quaternary ammonium compounds, tetraethylammonium and N1-methylnicotinamide, were not significantly bound to either tissue. Comparisons on the basis of equal content of solids revealed that the binding of diverse organic compounds in liver is greater than or equal to that in lung.

Amitrole↗