The monophenolic metabolites of the herbicide 2,6-dichlorobenzonitrile in animals as uncouplers of oxidative phosphorylation

1. Both monophenolic metabolites of 2,6-dichlorobenzonitrile (2,6-dichloro-3-hydroxybenzonitrile and its 4-hydroxy analogue) added to starved yeast cells incubated with a limited quantity of glucose cause a significant rise in oxygen consumption of the cells. 2. The same compounds induce adenosine-triphosphatase activity in isolated intact rat-liver mitochondria. 3. The possible role of the hydroxylation of 2,6-dichlorobenzonitrile in mammals in relation to hepatic injury is discussed.     

Chemical inhibition of cell wall formation and cytokinesis,but not of nuclear division,in protoplasts of Nicotiana tabacum L. cultivated in vitro

The effect of cytochalasin B, colchicine, coumarin and 2,6-dichlorobenzonitrile on cell wall formation and cellular division was studied by light and electron microscopy with tobacco mesophyll protoplasts cultivated in vitro. 2,6-dichlorobenzonitrile was found to be the most effective and reversible inhibitor of cell wall formation. The other inhibitors caused irreversible damage and/or inhibited mitosis. In protoplasts cultivated in the presence of 2,6-dichlorobenzonitrile the total inhibition of cell wall formation had no effect on nuclear division, but cytokinesis was totally inhibited so that multinucleate protoplasts were obtained.Abbreviations DB 2,6-dichlorobenzonitrile=dichlobenil - CB cytochalasin B     

The comparative metabolism of 2,6-dichlorothiobenzamide (prefix) and 2,6-dichlorobenzonitrile in the dog and rat

1. A single oral dose of either [(14)C]Prefix or 2,6-dichlorobenzo[(14)C]nitrile to rats is almost entirely eliminated in 4 days: 84.8-100.5% of (14)C from [(14)C]Prefix is excreted, 67.3-79.7% in the urine, and 85.8-97.2% of (14)C from 2,6-dichlorobenzo-[(14)C]nitrile is excreted, 72.3-80.7% in the urine. Only 0.37+/-0.03% of the dose of [(14)C]Prefix and 0.25+/-0.03% of the dose of 2,6-dichlorobenzo[(14)C]nitrile are present in the carcass plus viscera after removal of the gut. Rats do not show sex differences in the pattern of elimination of the respective metabolites of the two herbicides. The rates of elimination of (14)C from the two compounds in the 24hr. and 48hr. urines are not significantly different (P >0.05) from one another. 2. After oral administration to dogs, 85.9-106.1% of (14)C from [(14)C]Prefix is excreted, 66.6-80.9% in the urine, and 86.8-92.5% of (14)C from 2,6-dichlorobenzo[(14)C]nitrile is excreted, 60.0-70.1% in the urine. Dogs do not show sex differences in the pattern of eliminating the metabolites of either Prefix or 2,6-dichlorobenzonitrile. 3. Dogs and rats do not show species differences in the patterns of elimination of the two herbicides. 4. Prefix and 2,6-dichlorobenzonitrile are completely metabolized; unchanged Prefix and 2,6-dichlorobenzonitrile are absent from the urine and faeces, and from the carcasses when elimination is complete. In the hydrolysed urine of rats dosed with either [(14)C]Prefix or 2,6-dichlorobenzo[(14)C]nitrile, 2,6-dichloro-3-hydroxybenzonitrile accounts for approx. 42% of the (14)C, a further 10-11% is accounted for by 2,6-dichlorobenzamide, 2,6-dichlorobenzoic acid, 2,6-dichloro-3- and -4-hydroxybenzoic acid and 2,6-dichloro-4-hydroxybenzonitrile collectively, and 25-30% by six polar constituents, of which two are sulphur-containing amino acids. 5. In the unhydrolysed urines of rats dosed with either [(14)C]Prefix or 2,6-dichlorobenzo[(14)C]nitrile, there are present free 2,6-dichloro-3- and -4-hydroxybenzonitrile, their glucuronide conjugates, ester glucuronides of the principal aromatic acids that are present in the hydrolysed urines, and two sulphur-containing metabolites analogous to mercapturic acids or premercapturic acids. 6. Prefix is thus extensively transformed into 2,6-dichlorobenzonitrile: R.CS.NH(2)-->R.CN+H(2)S, where R=C(6)H(3)Cl(2). However, the competitive reaction: R.CS.NH(2)+H(2)O-->R.CO.NH(2)+H(2)S takes place to a very limited extent.     

Cell wall synthesis and salt (saline) sensitivity in cultured colt cherry (Prunus avium × pseudocerasus) protoplasts

In order to investigate the cellular basis of salt tolerance, Colt cherry (Prunus avium ×pseudocerasus) protoplasts from mesophyll tissues and root cell suspension cultures were cultured in the presence of NaCl, KCl or Na₂SO₄, at normalities of 25, 50, 100 or 200 mN for each salt and with or without 2,6-dichlorobenzonitrile, an inhibitor of cell wall synthesis. Results showed that the acquisition of salt tolerance was concomitant with the onset of cell wall regeneration, with protoplasts exhibiting a greater salt tolerance than cells.Abbreviations DBN 2,6-dichlorobenzonitrile - FDA fluorescein diacetate     

Controlled cell wall regeneration for efficient microinjections of Nicotiana tabacum var. Carlson protoplasts

Nicotiana tabacum var. Carlson protoplast culture conditions were modified to contain a cell wall inhibitor, 2,6-dichlorobenzonitrile, in order to delay cell wall regeneration and to allow efficient nuclear and cytoplasmic microinjections. Under modified conditions, the protoplast preparations appeared healthier as compared to the control protoplasts and showed no resistance at all during microinjection. Furthermore, the duration of protoplast microinjection was extended for up to 3–4 days. In order to set up nuclear microinjections, the nuclei of these protoplasts were stained either before or after immobilization without any adverse effect on their mitotic activity. Successful cytoplasmic microinjections were demonstrated by injecting Alfalfa mosaic virus (AMV) RNA, which resulted in viral infection of 14% of the injected protoplasts.Abbreviations AMV Alfalfa Mosaic virus - BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxy-acetic acid - DB 2,6-dichlorobenzonitrile - LR lissamine rhodamine - NAA 1-naphthalene-acetic acid     

2,6-Dichlorobenzonitrile and Boron Deficiency

Symptoms of 2,6-dichlorobenzonitrile poisoning in plants aredescribed and compared with the symptoms produced by phenylboronicacid and boron deficiency. The effects on the macroscopic andmicroscopic appearance of plants and on their ability to translocategrowth regulators are so alike that it is thought that both2,6-dichlorobenzonitrile and boron deficiency affect the samebasic process.     

Inhibition of conjugation of indole-3-acetic acid with amino acids by 2,6-dihydroxyacetophenone in Teucrium canadense

2,6-Dihydroxyacetophenone and five structurally related compounds were tested for their effects on metabolism of[2-¹⁴C]IAA in stem segments of 3-week-old American germander (Teucrium canadense). Pre-treatment of the plants with 2 mM 2,6-dihydroxyacetophenone for 12 hr significantly reduced the formation of two radioactive metabolites, which were tentatively identified as N-(indole-3-acetyl)-L-aspartic acid and N-(indole-3-acetyl)-L-glutamic acid. The chemical pre-treatment also decreased the level of a less polar metabolite chromatographically indistinguishable from oxindole-3-acetic acid, an oxidative product of IAA, and other unidentified metabolites of IAA. Concomitantly, the level of free [2-¹⁴C]IAA increased significantly in the treated tissue. 2,4-, 2,5- and 3,4-Dihydroxyacetophenones, as well as 3-bromo-2,6-dihydroxyacetophenone and 2-hydroxy-6-methoxyacetophenone, did not show a similar effect.     

An ultrastructural study of cell plate modifications induced by 2,6-dichlorobenzonitrile in onion root meristems

Summary The effect of 2,6-dichlorobenzonitrile on cytokinesis of meristematic cells of onion root during both treatment and recovery has been studied by electron microscopic techniques. 2,6-dichlorobenzonitrile interferes with cell plate formation in such a way that Golgi apparatus vesicles of treated cells appear to be different than controls and seem to coalesce as anomalous partial cell plates. During recovery, an apparently normal progression of cytokinesis is observed and abnormal portions of the cell plate are retained. Nuclear constrictions are observed frequently during recovery as a result of temporal alterations in cytokinesis. Our results show that 2,6-dichlorobenzo-nitrile induces anomalous and/or incomplete cell plates, which might be caused by an altered function of Golgi apparatus.     

Degradation and Mineralization of Nanomolar Concentrations of the Herbicide Dichlobenil and Its Persistent Metabolite 2,6-Dichlorobenzamide by Aminobacter spp. Isolated from Dichlobenil-Treated Soils

2,6-Dichlorobenzamide (BAM), a persistent metabolite from the herbicide 2,6-dichlorobenzonitrile (dichlobenil), is the pesticide residue most frequently detected in Danish groundwater. A BAM-mineralizing bacterial community was enriched from dichlobenil-treated soil sampled from the courtyard of a former plant nursery. A BAM-mineralizing bacterium (designated strain MSH1) was cultivated and identified by 16S rRNA gene sequencing and fatty acid analysis as being closely related to members of the genus Aminobacter, including the only cultured BAM degrader, Aminobacter sp. strain ASI1. Strain MSH1 mineralized 15 to 64% of the added [ring-U-¹⁴C]BAM to ¹⁴CO₂ with BAM at initial concentrations in the range of 7.9 nM to 263.1 μM provided as the sole carbon, nitrogen, and energy source. A quantitative enzyme-linked immunoassay analysis with antibodies against BAM revealed residue concentrations of 0.35 to 18.05 nM BAM following incubation for 10 days, corresponding to a BAM depletion of 95.6 to 99.9%. In contrast to the Aminobacter sp. strain ASI1, strain MSH1 also mineralized the herbicide itself along with several metabolites, including ortho-chlorobenzonitrile, ortho-chlorobenzoic acid, and benzonitrile, making it the first known dichlobenil-mineralizing bacterium. Aminobacter type strains not previously exposed to dichlobenil or BAM were capable of degrading nonchlorinated structural analogs. Combined, these results suggest that closely related Aminobacter strains may have a selective advantage in BAM-contaminated environments, since they are able to use this metabolite or structurally related compounds as a carbon and nitrogen source.     

Adaptation and growth of tomato cells on the herbicide 2,6-dichlorobenzonitrile leads to production of unique cell walls virtually lacking a cellulose-xyloglucan network

Suspension-cultured cells of tomato (Lycopersicon esculentum VF 36) have been adapted to growth on high concentrations of 2,6-dichlorobenzonitrile, an herbicide which inhibits cellulose biosynthesis. The mechanism of adaptation appears to rest largely on the ability of these cells to divide and expand in the virtual absence of a cellulose-xyloglucan network. Walls of adapted cells growing on 2,6-dichlorobenzonitrile also differ from nonadapted cells by having reduced levels of hydroxyproline in protein, both in bound and salt-elutable form, and in having a much higher proportion of homogalacturonan and rhamnogalacturonan-like polymers. Most of these latter polymers are apparently cross-linked in the wall via phenolic-ester and/or phenolic ether linkages, and these polymers appear to represent the major load-bearing network in these unusual cell walls. The surprising finding that plant cells can survive in the virtual absence of a major load-bearing network in their primary cell walls indicates that plants possess remarkable flexibility for tolerating changes in wall composition.     

Ethylene-induced lateral expansion in etiolated pea stems : kinetics, cell wall synthesis, and osmotic potential

Treatment of etiolated pea (Pisum sativum L.) internode tissue with ethylene gas inhibits elongation and induces lateral expansion. Precise kinetics of the induction of this altered mode of growth of excised internode segments were recorded using a double laser optical monitoring device. Inhibition of elongation and promotion of lateral expansion began after about 1 hour of treatment and achieved a maximum by 3 hours. Similar induction kinetics were observed after treating internodes with colchicine and 2,6-dichlorobenzonitrile, an inhibitor of cellulose synthesis. In sealed flask experiments, ethylene had no detectable effect on incorporation of label from [¹⁴C]glucose into any of the classical pectin, hemicellulose, or cellulose wall fractions. Ethylene inhibited fresh weight increase (total cell expansion) of both excised internode segments (in sealed flasks) and intact seedlings. Ethylene treatment resulted in an increase in cell sap osmolality in those tissues (intact and excised) which are inhibited by the gas. A model for ethylene-induced inhibition of elongation and induction of lateral expansion is presented.     

Characterization of a novel cellulose synthesis inhibitor

The physiological effects of an experimental herbicide and cellulose synthesis inhibitor, N2-(1-ethyl-3-phenylpropyl)-6-(1-fluoro-1-methylethyl)-1,3,5-triazine-2,4-diamine, called AE F150944, are described. In the aminotriazine molecular class, AE F150944 is structurally distinct from other known cellulose synthesis inhibitors. It specifically inhibits crystalline cellulose synthesis in plants without affecting other processes that were tested. The effects of AE F150944 on dicotyledonous plants were tested on cultured mesophyll cells of Zinnia elegans L. cv. Envy, which can be selectively induced to expand via primary wall synthesis or to differentiate into tracheary elements via secondary wall synthesis. The IC₅₀ values during primary and secondary wall synthesis in Z. elegans were 3.91×10^–8 M and 3.67×10^–9 M, respectively. The IC₅₀ in suspension cultures of the monocot Sorghum halapense (L.) Pers., which were dividing and synthesizing primary walls, was 1.67×10^–10 M. At maximally inhibitory concentrations, 18–33% residual crystalline cellulose synthesis activity remained, with the most residual activity observed during primary wall synthesis in Z. elegans. Addition to Z. elegans cells of two other cellulose synthesis inhibitors, 1 M 2,6-dichlorobenzonitrile and isoxaben, along with AE F150944 did not eliminate the residual cellulose synthesis, indicating little synergy between the three inhibitors. In differentiating tracheary elements, AE F150944 inhibited the deposition of detectable cellulose into patterned secondary wall thickenings, which was correlated with delocalization of lignin as described previously for 2, 6-dichlorobenzonitrile. Freeze-fracture electron microscopy showed that the plasma membrane below the patterned thickenings of AE F150944-treated tracheary elements was depleted of cellulose-synthase-containing rosettes, which appeared to be inserted intact into the plasma membrane followed by their rapid disaggregation. AE F150944 also inhibited cellulose-dependent growth in the rosette-containing alga, Spirogyra pratensis, but it did not inhibit cellulose synthesis in Acetobacter xylinum or Dictyostelium discoideum, both of which synthesize cellulose via linear terminal complexes. Therefore, AE F150944 may inhibit crystalline cellulose synthesis by destabilizing plasma membrane rosettes.Abbreviations AE F150944 N2-(1-ethyl-3-phenylpropyl)-6-(1-fluoro-1-methylethyl)-1,3,5-triazine-2,4-diamine - CBI cellulose biosynthesis inhibiting - CGA CGA 325615, 1-cyclohexyl-5-(2,3,4,5,6-pentafluorophenoxy)-14,2,4,6-thiatriazin-3-amine - DCB 2,6-dichlorobenzonitrile - TE tracheary element     

Identification of a receptor protein in cotton fibers for the herbicide 2,6-dichlorobenzonitrile

The herbicide 2,6-dichlorobenzonitrile (DCB) is an effective and apparently specific inhibitor of cellulose synthesis in higher plants. We have synthesized a photoreactive analog of DCB (2,6-dichlorophenylazide [DCPA]) for use as an affinity-labeling probe to identify the DCB receptor in plants. This analog retains herbicide activity and inhibits cellulose synthesis in cotton fibers and tobacco cells in a manner similar to DCB. When cotton fiber extracts are incubated with [³H]DCPA and exposed to ultraviolet light, an 18 kilodalton polypeptide is specifically labeled. About 90% of this polypeptide is found in the 100,000g supernatant, the remainder being membrane-associated. Gel filtration and nondenaturing polyacrylamide gel electrophoresis of this polypeptide indicate that it is an acidic protein which has a similar size in its native or denatured state. The amount of 18 kilodalton polypeptide detectable by [³H]DCPA-labeling increases substantially at the onset of secondary wall cellulose synthesis in the fibers. A similar polypeptide, but of lower molecular weight (12,000), has been detected upon labeling of extracts from tomato or from the cellulosic alga Chara corallina. The specificity of labeling of the 18 kilodalton cotton fiber polypeptide, coupled with its pattern of developmental regulation, implicate a role for this protein in cellulose biosynthesis. Being, at most, only loosely associated with membranes, it is unlikely to be the catalytic polypeptide of the cellulose synthase, and we suggest instead that the DCB receptor may function as a regulatory protein for β-glucan synthesis in plants.     

Dioxygenative cleavage of C-methylated hydroquinones and 2,6-dichlorohydroquinone by Pseudomonas sp. HH35

The dioxygenolytic catabolism of five C-methylated hydroquinones and 2,6-dichlorohydroquinone in Pseudomonas sp. strain HH35 was elucidated. This organism, which is known to catabolise 2,6-dimethylhydroquinone by 1,2-cleavage, accumulated metabolites from 2-methyl-, 2,3-dimethyl-, 2,5-dimethyl-, 2,3,5-trimethyl- and 2,3,5,6-tetramethylhydroquinone which we isolated and characterised by mass spectrometry and ¹H NMR and UV spectroscopy. The identification of these metabolites defined the impact of methyl groups present in the hydroquinone and showed how each substitution pattern determined the site of the initial enzymic attack. With the exception of the 2,3,5,6-tetramethylhydroquinone, all C-methylated hydroquinones were catabolised by an initial dioxygenolytic cleavage occurring adjacent (1,2- or 3,4-cleavage) to a hydroxy group. In addition, our results indicated that the 2,6-dichlorohydroquinone is catabolised in a similar way by this strain.     

Animal-specific membrane components visualised on the surface of animal/plant heterokaryons

Summary Immunofluorescence was used to demonstrate the presence and distribution of animalspecific membrane components within the plasma membranes ofXenopus/carrot heterokaryons. The inhibitor of cellulose synthesis, 2,6-dichlorobenzonitrile, was used to impair cell wall regeneration so that the plasma membranes of cultured heterokaryons would remain accessible to antibodies.Xenopus-Specific surface components were observed in heterokaryons after 14 days of culture.     

3,7-Dichloroquinolinecarboxylic Acid Inhibits Cell-Wall Biosynthesis in Maize Roots

The mode of action of the herbicide 3,7-dichloroquinolinecar-boxylic acid (quinclorac) was examined by measuring incorporation of [14C]glucose, [14C]acetate, [3H]thymidine, and [3H]uridine into maize (Zea mays) root cell walls, fatty acids, DNA, and RNA, respectively. Among the precursors examined, 10 [mu]M quinclorac inhibited [14C]glucose incorporation into the cell wall within 3 h. Fatty acid and DNA biosynthesis were subsequently inhibited, whereas RNA biosynthesis was unaffected. In contrast to the cellulose synthesis inhibitor 2,6-dichlorobenzonitrile, quinclorac strongly inhibited cellulose and a hemicellulose fraction presumed to be glucuronoarabinoxylan. However, the synthesis of (1->3),(1->4)-[beta]-D-glucans was only slightly inhibited. The degree of inhibition was time- and dose-dependent. By 4 h after treatment, the concentration that inhibited [14C]glucose incorporation into the cell wall, cellulose, and the sensitive hemicellulose fraction by 50% was about 15, 5, and 20 [mu]M, respectively. Concomitant with an inhibition of [14C]glucose incorporation into the cell wall, quinclorac treatment led to a marked accumulation of radioactivity in the cytosol. The increased radioactivity was found mostly in glucose and fructose. However, total levels of glucose, fructose, and uridine diphosphate-glucose were not changed greatly by quinclorac. These data suggest that quinclorac acts primarily as a cell-wall biosynthesis inhibitor in a susceptible grass by a mechanism that is different from that of 2,6-dichlorobenzonitrile.     

The most-probable-number enumeration of dichlobenil and 2,6-dichlorobenzamide (BAM) degrading microbes in Finnish aquifers

In groundwater subsurface deposits and a topsoil from five aquifers having 2,6-dichlorobenzamide (BAM) in water, we determined the most-probable-number (MPN) of 2,6-dichlorobenzonitrile (dichlobenil) and metabolite BAM degrading microorganisms. Dichlobenil and BAM were combined nitrogen sources in the MPN tubes, which were scored positive at concentrations <75% after 1 month incubation. Aerobic and anaerobic microbes degrading dichlobenil and BAM were common in samples in low numbers of 3.6–210 MPN g dw⁻¹. Additional degradation occurred in high MPN dilutions of some samples, the microbial numbers being 0.11–120 × 10⁵ MPN g dw⁻¹. The strains were isolated from low and high dilutions of one deposit, and degradation in pure cultures was confirmed by HPLC. According to the 16S rDNA sequencing, strains were from genera Zoogloea, Pseudomonas, Xanthomonas, Rhodococcus, Nocardioides, Sphingomonas, and Ralstonia. Dichlobenil (45.5 ± 18.3%) and BAM (37.6 ± 14%) degradation was low in the MPN tubes. Despite of microbial BAM degradation activity in subsurface deposits, BAM was measured from groundwater.     

Distribution of Microsomal Epoxide Hydrolase and Glutathione S-transferase in the Rat Olfactory Mucosa: Relevance to Distribution of Lesions Caused by Systemically-administered Olfactory Toxicants

This study represents part an of ongoing effort to understandthe mechanism underlying the distribution of the olfactory mucosallesion resulting from the systemic administration of compoundssuch as 2,6-dichlorobenzonitrile (dichlobenil) and ß,ß'-iminodipropionitrile(IDPN). Immunohistochemistry was performed to localize the microsomalform of epoxide hydrolase (mEH) and glutathione S-transferase(GST) isozymes     

Transformation of the herbicide 2,6-dichlorobenzonitrile to the persistent metabolite 2,6-dichlorobenzamide (BAM) by soil bacteria known to harbour nitrile hydratase or nitrilase

In soil the herbicide 2,6-dichlorobenzonitrile (dichlobenil) is degraded to the persistent metabolite 2,6-dichlorobenzamide (BAM) which has been detected in 19% of samples taken from Danish groundwater. We tested if common soil bacteria harbouring nitrile-degrading enzymes, nitrile hydratases or nitrilases, were able to degrade dichlobenil in vitro. We showed that several strains degraded dichlobenil stoichiometrically to BAM in 1.5–6.0 days; formation of the amide intermediate thus showed nitrile hydratase rather than nitrilase activity, which would result in formation of 2,6-dichlorobenzoic acid. The non-halogenated␣analogue benzonitrile was also degraded, but here the benzamide intermediate accumulated only transiently showing nitrile hydratase followed by amidase activity. We conclude that a potential for dichlobenil degradation to BAM is found commonly in soil bacteria, whereas further degradation of the BAM intermediate could not be demonstrated.     

Oxidation of carbon tetrachloride,bromotrichloromethane, and carbon tetrabromide by rat liver microsomes to electrophilic halogens

In order to determine whether CCl₄, CBrCl₃, CBr₄ or CHCl₃ undergo oxidative metabolism to electrophilic halogens by liver microsomes, they were incubated with liver microsomes from phenobartital pretreated rats in the presence of NADPH and 2,6-dimethylphenol. The analysis of the reaction mixtures by capillary gas chromatography mass spectrometry revealed that 4-chloro-2,6-dimethylphenol was a metabolite of CCl₄ and CBrCl₃ whereas 4-bromo-2,6-dimethylphenol was a metabolite of CBr₄. The formation of the metabolites was significantly decreased when the reactions were conducted with heat denatured microsomes, in the absence of NADPH or under an atmosphere of N₂. These results indicate that the chlorines of CBrCl₃ and CCl₄ and the bromines of CBr₄ are oxidatively metabolized by rat liver microsomes to electrophilic and potentially toxic metabolites.