Pronamide disrupts mitosis in a unique manner

Pronamide (3,5-dichloro(N-1,1-dimethyl-2-propynyl)benzamide) has traditionally been grouped with mitotic disrupters, such as colchicine or trifluralin, that inhibit polymerization of tubulin into microtubules. Many of the effects of pronamide (c-metaphases, polymorphic nuclei, isodiametric cells, abnormal xylem development, swollen root tips) are similar to these well-studied mitotic disrupters. However, immunofluorescence microscopy studies using anti-tubulin sera revealed that, unlike other mitotic disrupters, pronamide-treated cells have greatly shortened microtubules that are located only at the kinetochore region. These structures detected by immunofluorescence studies were determined by electron microscopy to be microtubules. Because these short kinetochore microtubules did not allow for mitosis to proceed normally, the nuclear envelope reforms around the chromosomes leading to polymorphic nuclei. Frequently, other cytoplasmic organelles normally excluded in the zone occupied by the chromosomes and spindle during mitosis were retained within the reformed nuclei. These data indicate that, although the net effect of pronamide is similar to those of colchicine and trifluralin, pronamide acts by a different mechanism.     

Absorption,translocation, and metabolism of ethalfluralin and trifluralin in Solanum spp

Seedlings of Solanum scabrum Mill. and Solanum ptycanthum Dun. were treated with [¹⁴C]ethalfluralin (N-ethyl-α,α,α-trifluoro-N-(methylallyl)-2,6-dinitro-p-toluidine) and [¹⁴C]trifluralin (α,α,α-trifluoro-2,6-dinitro-N,N-dipropyl-p-toluidine) supplied in nutrient solution to determine the basis for differences in response by these two species to these two herbicides. Plants of S. scabrum absorbed more [¹⁴C]ethalfluralin and [¹⁴C]trifluralin than plants of S. ptycanthum. During the first 24 h, S. scabrum seedlings, but not S. ptycanthum seedlings absorbed more [¹⁴C]ethalfluralin than did plants treated with [¹⁴C]trifluralin. More [¹⁴C]ethalfluralin than [¹⁴C]trifluralin was found in the shoots of plants of both species. Seventy-two hours after treatment with [¹⁴C]herbicides, the conversion to water-soluble metabolites was greater for [¹⁴C]ethalfluralin than for [¹⁴C]trifluralin. In the shoots of plants from both species an average of nearly 55% of the ¹⁴C recovered was found in the water-soluble fraction following [¹⁴C]ethalfluralin treatment whereas an average of only 40% was found in the water-soluble fraction following [¹⁴C]trifluralin treatment.     

Distribution and degradation of dinitroaniline herbicides in an aquatic ecosystem

The accumulation potential of six, structurally related, dinitroaniline herbicides was investigated in an aquatic ecosystem. The herbicides investigated were trifluralin (α,α,α-trifluoro-2,6-dinitro-N,N-dipropyl-p-toluidine), profluralin [N-(cyclopropylmethyl)-α,α,α-trifluoro-2,6-dinitro-N-propyl-p-toluidine], dinitramine [N³,N³-diethyl-2,4-dinitro-6-(trifluoromethyl)-m-phenylenediamine], chlornidine [N,N-bis(2-chloroethyl)-2,6-dinitro-p-toluidine], fluchloralin [N-(2-chloroethyl)-2,6-dinitro-N-propyl-4-(trifluoromethyl)aniline], and butralin [4-(1,1-dimethylethyl)-N-(1-methylpropyl)-2,6-dinitrobenzenamine]. The herbicide (0.1 mg) plus 1 μCi of ¹⁴C-labeled herbicide was adsorbed on 100 g of soil (1 ppm), added to individual aquariums, and flooded with 4 liters of water. Algae, snails, and daphnia were added, and ¹⁴C in water was monitored for 30 days. Fish were added on Day 30, and all components were harvested 3 days later. Bioaccumulation ratios (concentration in organism/concentration in water) for fish depended on the amount of their exposure to sunlight: Aquariums held in the dark had higher ratios for fish (235–755) than did those exposed to sunlight (32–83). Bioaccumulation ratios in the dark for fish based on ¹⁴C from bound soil residues of butralin and profluralin were 76 and 119, respectively. Direct repeated applications of profluralin (without soil) at 4-day intervals resulted in a rapid increase, then a decrease in bioaccumulation ratios for Gambusia, but a continuous increase for catfish.     

Cytological studies of dinitroaniline-resistant Eleusine

Ultrastructural studies of primary roots (goosegrass) from dinitroaniline-resistant (R) and susceptible (S) biotypes of Eleusine indica (L). Gaertnr. establish a possible cytological basis for trifluralin resistance. Although the S biotype has a normal ultrastructure when grown in water, exposure to trifluralin solutions (between 10⁻⁸ and 10⁻⁵M) for 24 h results in a swelling of the root tip, typical of herbicides that affect microtubule production. The loss of spindle microtubules in the S biotype results in a mitosis arrested at prometaphase and the loss of cortical microtubules results in the formation of isodiametric cells in the zone of elongation. Nuclear membranes reform around the chromosomes in arrested prometaphase, producing abnormal, polymorphic nuclei. The mitotic index is increased in the S biotype after trifluralin treatment because many of the cells are arrested in prometaphase. The root tips of R biotypes are not swollen by even 10⁻⁵M trifluralin treatment. Trifluralin does not markedly affect cell division in the R biotype nor are the mitotic irregularities noted in the S biotype after treatment. However, even when the R biotype is not exposed to trifluralin, the microtubules are less abundant than in the S biotype and frequently cell walls are oriented abnormally or are incompletely formed. The level of resistance exhibited by the R biotype, the apparent difference in microtubule number and function between the two biotypes, and the lack of effect on the microtubules at high trifluralin concentrations indicate a site-of-action mutation.     

The biochemical mechanism of action of the dinitroaniline herbicide oryzalin

Dinitroaniline herbicides such as oryzalin (3,5-dinitro-N⁴,N⁴-dipropylsulfanilamide) disrupt mitosis in the meristematic cells of seedling plants by inhibiting the formation of microtubules. For further understanding of the biochemical mechanism of action of oryzalin, laboratory analyses with isolated plant tubulin must be employed. Plant tubulin from flagella of the alga Chlamydomonas was isolated and purified. This tubulin was incubated with [¹⁴C]oryzalin, and free oryzalin was separated from oryzalin bound to plant tubulin by miniature DEAE-cellulose chromatography. Scatchard analysis predicts a molar ratio of oryzalin bound to plant tubulin of 1.0 ± 0.1 when oryzalin is incubated with plant tubulin for 30 min at pH 6.9 and 25°C. The association constant for the oryzalin-tubulin complex is 2.08 ± 0.08 × 10⁵M^?1 at 25°C. The thermodynamic values for the formation of the oryzalin-tubulin complex at 25°C are ΔG^o = ?7.25 ± 0.02 kcal mol^?1, ΔH^o = 6.5 ± 0.2 kcal mol^?1, and ΔS^o = 46 ± 2 cal mol^?1 deg^?1 (mean ± standard error). Oryzalin has little or no affinity for intact microtubules, previously denatured plant tubulin, actin, bovine serum albumin, calmodulin, ferredoxin, trypsin, or urease, indicating oryzalin is specific for the biologically active conformation of plant tubulin. Oryzalin binds to plant tubulin to form a complex that may be incapable of polymerizing into microtubules.     

Trifluralin effects on tobacco callus tissue: Mitosis and selected metabolic effects

Inhibition of growth of pith callus of tobacco (Nicotiana tabacum, var. S-73) by the herbicide trifluralin (α,α,α - trifluoro - 2,6 - dinitro - N,N- dipropyl - p - toluidine) was previously observed. Inhibition of cell division in callus tissue of varying age by this herbicide was investigated using the Feulgen reagent and light microscopy. Upon staining and counting the number of cells in each phase of mitosis, a decrease in the number of cells in metaphase, anaphase, and telophase in the treated tissues was found. In addition to this reduction, arrested metaphases and multinucleated cells were observed. Similar results were observed with 10^?4M colchicine. The effects of trifluralin on incorporation of ¹⁴C-precursors into callus RNA, DNA, and protein were also investigated. Apparent RNA, DNA, and protein synthesis in callus were inhibited by trifluralin (5 × 10^?6M) treatment. The inhibition, however, was not expressed until 5–7 days after initiating treatment. Colchicine also affected apparent RNA, DNA, and protein synthesis; however, these effects were different than those observed with trifluralin. Incorporation of ¹⁴C-amino acids into protein was most severely inhibited by colchicine.     

Microbial dealkylation of trifluralin in pure culture

Mixed populations of soil microorganisms were enriched in the presence of trifluralin (α,α,α-trifluoro-2,6-dinitro-N,N-dipropyl-p-toluidine) and 180 pure strains were subsequently isolated. About a third were able to liberate 1.5–6% ¹⁴CO₂ from 0.15 mM [propyl-1-¹⁴C]trifluralin after growing for 21 days on a complex medium. One strain, identified as a Candida sp., showed a ¹⁴CO₂ evolution of 11%. The amount of liberated ¹⁴CO₂ could not be enhanced by adding small concentrations (<3%) of solvents to the culture, by varying the concentration of trifluralin, or by varying the composition of the complex medium. The Candida sp. was unable to cleave the aromatic ring of trifluralin or to use trifluralin as a sole source of carbon or nitrogen. Only traces (< 1%) of dealkylated trifluralin were accumulated in the culture.     

Microbial metabolism of dinitramine

The microbial metabolism of N³,N³-diethyl 2,4-dinitro-6-trifluoromethyl-m-phenylenediamine (dinitramine), N-sec-butyl-4-tert-butyl-2,6-dinitroaniline (A-820), N-n-propyl-N-cyclopropylmethyl-4-trifluoromethyl-2,6-dinitroaniline (CGA-10832), and α,α,α-trifluoro-2,6-dinitro-N,N-dipropyl-p-toluidine (trifluralin) by Aspergillus fumigatus Fres., Fusarium oxysporum Schlecht and Paecilomyces sp. was investigated. The dinitrodiamine and dinitroanilines were most readily metabolized by Paecilomyces sp. The metabolism of dinitramine was examined in detail, and four metabolites were isolated: N³-ethyl 2,4-dinitro-6-trifluoromethyl-m-phenylenediamine; 2,4-dinitro-6-tri-fluoromethyl-m-phenylenediamine; 6-amino-1-ethyl-2-methyl-7-nitro-5-trifluoromethylbenzimidazole and 6-amino-2-methyl-7-nitro-5-trifluoromethylbenzimidazole. The presence of a dinitramine-degrading enzyme system in A. fumigatus was demonstrated. The enzyme dealkylates dinitramine and requires NADPH and Fe²⁺ as cofactors.     

Metabolism of trifluralin,profluralin, and fluchloralin by rat liver microsomes

Three structurally related [¹⁴C]dinitroaniline herbicides, trifluralin, profluralin, and fluchloralin, were extensively metabolized in vitro by both normal and phenobarbital-induced rat liver microsomes. Identification of the metabolites in the ethyl acetate extracts indicated that aliphatic hydroxylation, N-dealkylation, reduction of a nitro group, and cyclization were the predominant metabolic routes for these herbicides in vitro. Of particular interest was the formation of a benzimidazole metabolite.     

Behavior of 14C oryzalin in soil and plants

Radiochemical studies of field soil treated with ¹⁴C oryzalin (3,5-dinitro-N⁴,N⁴-dipropylsulfanilamide) indicated that the compound was readily degradable. One year after soil treatment with oryzalin, 45% of the original radioactivity had dissipated, 25% was extractable, and 30% was “soil bound”. The extractable fraction contained oryzalin and several degradation products, some of which were isolated and identified. No single degradation product accounted for more than 3% of the applied oryzalin. The “soil-bound” radioactivity was extractable with hot alkali. No significant radioactive residues were detectable in either seed or forage of soybean and wheat plants. No specific metabolites of oryzalin were identified in soybean plants. Trace amounts of radioactivity found in plant tissue appeared to be associated with the various plant constituents.     

Carrot microtubules are dinitroaniline resistant. I. Cytological and cross-resistance studies

Treatment of both tolerant and susceptible species with dinitroaniline herbicides results in root swelling typical of mitotic disrupters, however, no root swelling was observed in carrots treated with saturated solutions of all the dinitroaniline herbicides tested, with the exception of oryzalin treatments at ≥ 10^?5 M. These levels of treatment are 100–1000 times the levels that caused root-tip swelling, even in tolerant plants. This properly classifies carrot as dinitroaniline-resistant rather than merely tolerant. Cross-resistance was noted to the structurally related mitotic disrupter, hexanitrodiphenylamine, and to the structurally unrelated herbicide, amiprophos-methyl. No resistance was noted to a number of other mitotic-disrupting herbicides. Both immunofluorescence and electron microscopy indicate that the microtubules of carrot roots were unaffected by dinitroaniline treatment. All organized microtubule configurations (cortical, pre-prophase bands, spindle, and phragmoplast) were observed in the treated material.     

Enhanced Mineralization of [14C]Atrazine in Kochia scoparia Rhizospheric Soil from a Pesticide-Contaminated Site

Mineralization of atrazine (6-chloro-N²-ethyl-N⁴-isopropyl-1,3,5- triazine-2,4-diamine) in soil treated with a mixture of atrazine and metolachlor (2-chloro-6′-ethyl-N-(2-methoxy-1-methylethyl)acet-o-toluidide at concentrations typical of point-source contamination (50 μg g⁻¹ each) was significantly greater (P<0·001) in rhizospheric soil from Kochia scoparia (L.) Roth., a herbicide-resistant plant, than in non-vegetated and control soils. Soils were collected from an agrochemical dealership contaminated with several herbicides, including atra-zine, metolachlor, trifluralin (α,α,α-trifluoro-2,6-dinitro-N,N-dipropyl-p-toluidine and pendimethalin (N-(1-ethylpropyl)-2,6-dinitro-3,4-xylidene), at concentrations well exceeding the field application rates. Mineralization rates of ring-labeled atrazine in both rhizospheric and non-vegetated soils were quite high (>47% of the initial ¹⁴C applied after 36 days) compared to literature values. These results suggest that plants such as Kochia might be managed at pesticide-contaminated sites to help facilitate microbial degradation of wastes such as atrazine in soil.     

Interactions between soil-applied herbicides in the roots of some legume species

The effects on plant growth of applying trifluralin or nitralin combination with simazine, atrazine, prometryne and linuron to the upper 5-cm root region of vetch (Vicia sativa L.), pea (Pisum sativum L.) and soybean (Glycine max) were investigated. Foliar injury due to herbicides of the second group was markedly reduced in each species by simultaneous treatment with trifluralin or nitralin both of which inhibited lateral root growth without affecting aerial plant growth or tap root extension growth. This inhibition of lateral root growth in roots treated with trifluralin or nitralin was associated with reduced uptake and subsequent transport to the foliage of ¹⁴C-labelled simazine in vetch and pea and ¹⁴C-labelled atrazine in soybean. This probably accounted for the reduction in simazine and atrazine phytotoxicity. In the presence of trifluralin or nitralin comparatively higher amounts of radioactivity were retained in the roots of pea and soybean and this reduced the amount of ¹⁴C available for transport to the foliage. This was not evident in vetch.     

Growth regulator inhibition of tobacco callus growth

The activity of two groups of growth regulators, substituted dinitroanilines and nitrophenylhydrazines, were evaluated in a tobacco (Nicotiana tabacum L. “X-73”) callus tissue bioassay. Molar concentrations required to inhibit fresh weight gain by 50% (I₅₀) was determined by using linear regression analysis on data obtained by testing a range of five concentrations of each chemical. All chemicals tested were inhibitory to callus tissue grown in the dark. Cell division seemed to be the primary activity inhibited. The most active of the dinitroaniline series was α,α,α-trifluoro-2,6-dinitro-N-ethyl-N-2′,6′-dichlorobenzyl-p-toluidine (I) (I₅₀ = 1.5 × 10^?10M). I and two other N-(o-halobenzyl) dinitroanilines were more active than α,α,α-trifluoro-2,6-dinitro-N-ethyl-N-2′-chloro-6′-fluorobenzyl-p-toluidine (IV), which is being developed commercially for suppression of axillary buds in tobacco. The two most active nitrophenylhydrazines tested were 1,1-dimethyl-2-(2′,6′-dinitro-3′-n-propylamino-α,α,α-trifluoro-p-tolyl)hydrazine (XVIII) and 3′,5′-dinitro-p-(2,2-diethylhydrazino)-N-methoxy-N-methylbenzamide (XIX) (I₅₀ values of 7.9 × 10^?9 and 9.3 × 10^?9M, respectively). Factors such as electronic distribution, steric hindrance, and lipid solubility were considered to influence the biological activity of the compounds tested.     

Effects of various classes of herbicides on four species of algae

Chlorella pyrenoidosa, Chlorococcum sp., Lyngbya sp., and Anabaena variabilis were cultured in Bold's basal medium. They were treated with 0.1, 1.0, and 10 μM concentrations of 2-chloro-2′, 6′-diethyl-N-(methoxymethyl)acetanilide (alachlor), 2-chloro-4-(ethylamino)-6-(tert-butyl-amino)-s-triazine (terbuthylazine), 2-sec-butyl-4,6-dinitrophenol (dinoseb), 1,1-dimethyl-3-(α,α,α-trifluoro-2,6-dinitro-N-propyl-p-toluidine) (profluralin), 2, 4-bis(isopropylamino)-6-(methylthio)-s-triazine (prometryne), and (2,4-dichlorophenoxy)acetic acid (2,4-D). Growth of all algal species tested was markedly reduced by the triazines. Alachlor, dinoseb, and fluometuron inhibited growth of some algae at higher concentrations while 2,4-D and profluralin did not inhibit growth at the concentrations tested. Photosynthesis was greatly inhibited by the triazines, even at the 0.1 μM concentration. Fluometuron was very toxic to the blue-green algae but had less effect on the green algae tested. Lyngbya was most susceptible to photosynthesis reduction by the herbicides. The concentrations of herbicides tested had little effect on respiration of the algae species. It appears that effects on algal growth were due primarily to inhibition of photosynthesis rather than to other metabolic processes.     

Herbicides and fungicides inhibit Ca2+ uptake by plant mitochondria: A possible mechanism of action

The herbicides trifluralin, oryzalin, ioxynil, dichlofopmethyl, and dinoseb, and the fungicides chloraniformethan and dichlofluanid inhibited energy-dependent Ca²⁺ uptake in plant mitochondria at concentrations where they did not inhibit ADP phosphorylation and did not interfere with tubulin polymerization. We suggest as a common mechanism of action for these pesticides the inhibition of the regulation of cytoplasmic free Ca²⁺ concentration which may in turn lead to many physiological malfunctions.     

The herbicide flamprop-M-methyl has a new antimicrotubule mechanism of action

BACKGROUND: The herbicidal mode of action of flamprop‐M‐methyl [methyl N‐benzoyl‐N‐(3‐chloro‐4‐fluorophenyl)‐D ‐alaninate] was investigated. RESULTS: For initial characterization, a series of bioassays was used, which indicated a mode of action similar to that of mitotic disrupter herbicides. Cytochemical fluorescence studies, which included monoclonal antibodies against polymerized tubulin, were applied to elucidate effects on mitosis and microtubule assembly in maize roots. When seedlings were root treated with 50 µM of flamprop‐M‐methyl, cell division activity in meristematic root tip cells ceased within 4 h. The compound severely disturbed the orientation of spindle and phragmoblast microtubules, leading to defective spindle and phragmoblast structures. Cortical microtubules were only slightly affected. In late anaphase and early telophase cells, phragmoblast microtubules were disorganized in multiple arrays that hampered regular cell plate deposition in cytokinesis. Microtubules of the spindle apparatus were found attached to chromosomal kinetochores, but did not show regular organization associated with a zone of microtubule‐organizing centres at the opposite ends of the cell. On account of this loss of spindle organization, chromosomes remained in a condensed state of prometaphase or metaphase. Unlike known microtubule disrupter herbicides, flamprop‐M‐methyl and its biologically active metabolite flamprop did not inhibit soybean tubulin polymerization to microtubules in vitro at 50 µM . In contrast, soybean plants responded sensitively to the compounds. CONCLUSION: The results indicate that flamprop‐M‐methyl is a mitotic disrupter herbicide with a new antimicrotubule mechanism of action that affects orientation of spindle and phragmoblast microtubules, possibly by minus‐end microtubule disassembly. Copyright © 2008 Society of Chemical Industry     

Ultrastructural effects of glyphosate on Glycine max seedlings

Roots and leaves of glyphosate-treated and untreated control soybean [Glycine max (L.) Merr. cv. Hill] were examined by scanning and transmission electron microscopy to elucidate some of the events involved in glyphosate phytotoxicity. Study of the gross morphology of the roots by scanning electron microscopy revealed a swollen, “club” morphology at the root tip similar, but not identical, to that noted after treatment with herbicides such as trifluralin that disrupt mitosis. The swelling occurs much more slowly with glyphosate treatment than with the classical mitosis disrupters that react directly with tubulin. Nuclei in the glyphosate-treated root cells showed symptoms of microtubule loss; cells with heavily lobed nuclei or micronuclei and cells with arrested stages of division were frequently observed. Immunoblot quantitation of tubulin from control and glyphosate-treated roots revealed the presence of less tubulin protein in the glyphosate-treated roots. Some amelioration of the microtubule stability during fixation was provided by addition of the Ca²⁺ chelator EGTA. Chloroplasts of glyphosate-treated leaves were distinctly abnormal, with lamellae arranged in spiral-shaped grana stacks. Starch and stroma lamellae were also much less abundant in the glyphosate-treated plants.     

Herbicidal cyanoacrylates with antimicrotubule mechanism of action

The herbicidal mode of action of the new synthetic cyanoacrylates ethyl (2Z)-3-amino-2-cyano-4-ethylhex-2-enoate (CA1) and its isopropyl ester derivative CA2 was investigated. For initial characterization, a series of bioassays was used indicating a mode of action similar to that of mitotic disrupter herbicides such as the dinitroaniline pendimethalin. Cytochemical fluorescence studies including monoclonal antibodies against polymerized and depolymerized tubulin and a cellulose-binding domain of a bacterial cellulase conjugated to a fluorescent dye were applied to elucidate effects on cell division processes including mitosis and microtubule and cell wall formation in maize roots. When seedlings were root treated with 10 microM of CA1 or CA2, cell division activity in meristematic root tip cells decreased within 4 h. The chromosomes proceeded to a condensed state of prometaphase, but were unable to progress further in the mitotic cycle. The compounds caused a complete loss of microtubular structures, including preprophase, spindle, phragmoplast and cortical microtubules. Concomitantly, in the cytoplasm, an increase in labelling of free tubulin was observed. This suggests that the herbicides disrupt polymerization and microtubule stability, whereas tubulin synthesis or degradation appeared not to be affected. In addition, cellulose labelling in cell walls of root tip cells was not influenced. The effects of CA1 and CA2 were comparable with those caused by pendimethalin. In transgenic Arabidopsis plants expressing a green fluorescent protein-microtubule-associated protein4 fusion protein, labelled arrays of cortical microtubules in living epidermal cells of hypocotyls collapsed within 160 min after exposure to 10 microM CA1 or pendimethalin. Moreover, a dinitroaniline-resistant biotype of goosegrass (Eleusine indica (L) Gaertn) with a point mutation in alpha-tubulin showed cross-resistance against CA1 and CA2. The results strongly indicate that the cyanoacrylates are a new chemical class of herbicide which possess the same antimicrotubule mechanism of action as dinitroanilines, probably including interaction with the same binding site in alpha-tubulin.     

Adsorption and bioactivity of di-allate,tri-allate and trifluralin*

Studies were conducled to cstmiate the adsorption parameter k and the bioactivity (in terms of G R₅₀) of di-allate [S-2,3-dichloroallyl N, N-di-isopropyl (thiocar bamate)], iri-allate [S-2,3,3-trichloroallyl N, N -di-isopropyl (thiocar bamate], and [trifiuralin (2,6-dinitro N, N-dlpropyl-4-trifluoromelhylaniline) in a number of Saskatchewan soil. The k values ratiged from 5 for di-allate adsorption m Asquilh loamy sand to 315 for trifluralin adsorption on Melfort loam and were closely related to the soil organic matter content. The relative degree of adsorption was irifluralin > tri-allate >di-allaie. For each herbicide, the G R₅₀ values were positively correlated wich organic matter conienl atid wilh k. It was suggested that these nonionic herbicides may be amenable to a predictive approach for field application rates in different soils. Among herbicides for any one soil, however, there was not the same relationship between G R₅₀andk. since the G R₅₀ was least for trifluralin and there was no significant difference between di-allate and tri-allate.