The herbicide 2,4-dichlorophenoxyacetic acid induces the generation of free-radicals and associated oxidative stress responses in yeast

The pro-oxidant action of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) is demonstrated in this study using Saccharomyces cerevisiae as a eukaryotic experimental model. Evidence is presented for the generation of hydroxyl-radicals, in yeast cells suddenly exposed to 2,4-D, detected by in vivo electron paramagnetic resonance (EPR) spectroscopy using 5,5'-dimethyl-1-pyrroline N-oxide and 5-tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide as spin-traps. The intensity of the EPR spectra was dependent on the concentration of herbicide tested and was consistently higher in a mutant (Deltasod1) devoid of the cytosolic CuZn-superoxide dismutase. A time-course-dependent variation of the level of free-radical adducts was registered upon sudden exposure of an yeast cell population to concentrations of 2,4-D that lead to an initial period of viability loss, before resumption of inhibited growth by the viable adapted population. The variation pattern of the level of hydroxyl-radical adducts correlated with the one determined for the activity of Sod1p, cytosolic catalase Ctt1p, and the dithiol glutaredoxins Grx1p and Grx2p.     

Expression of a bacterial gene in transgenic tobacco plants confers resistance to the herbicide 2,4-dichlorophenoxyacetic acid

Plants resistant to the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) were produced through the genetic engineering of a novel detoxification pathway into the cells of a species normally sensitive to 2,4-D. We cloned the gene for 2,4-D monooxygenase, the first enzyme in the plasmid-encoded 2,4-D degradative pathway of the bacterium Alcaligenes eutrophus, into a cauliflower mosaic virus 35S promoter expression vector and introduced it into tobacco plants by Agrobacterium-mediated transformation. Transgenic tobacco plants expressing the highest levels of the monooxygenase enzyme exhibited increased tolerance to 2,4-D in leaf disc and seed germination assays, and young plants survived spraying with levels of herbicide up to eight times the usual field application rate. The introduction of the gene for 2,4-D monooxygenase into broad-leaved crop plants, such as cotton, should eventually allow 2,4-D to be used as an inexpensive post-emergence herbicide on economically important dicot crops.     

Biological and molecular responses of Chironomus riparius (Diptera,Chironomidae) to herbicide 2,4-D (2,4-dichlorophenoxyacetic acid)

2,4-Dichlorophenoxyacetic acid (2,4-D) is an agricultural contaminant found in rural ground water. It remains to be determined whether neither 2,4-D poses environmental risks, nor is the mechanism of toxicity known at the molecular level. To evaluate the potential ecological risk of 2,4-D, we assessed the biological parameters including the survival rate, adult sex ratio of emerged adults, and mouthpart deformities in Chironomus riparius after long-term exposure to 2,4-D. The larvae were treated with 0.1, 1 or, 10 μg L^? 1 of 2,4-D for short- and long-term exposure periods. The sex ratio was changed in C. riparius exposed to only 10 μg L^? 1 of 2,4-D, whereas mouthpart deformities were observed as significantly higher in C. riparius exposed to 0.1 μg L^? 1 of 2,4-D. Survival rates were not significantly affected by 2,4-D. Furthermore, we evaluated the molecular and biochemical responses of biomarker genes such as gene expression of heat shock proteins (HSPs), ferritins and glutathione S-transferases (GSTs) in C. riparius exposed to 2,4-D for 24 h. The expressions of HSP70, HSP40, HSP90 and GST levels in C. riparius were significantly increased after exposure to a 10 μg L^? 1 concentration of 2,4-D, whereas ferritin heavy and light chain gene expressions were significantly increased at all concentrations of 2,4-D exposure. Finally, these results may provide an important contribution to our understanding of the toxicology of 2,4-D herbicide in C. riparius. Moreover, the 2,4-D-mediated gene expressions may be generated by 2,4-D is the causative effects on most probable cause of the observed alterations. These biological, molecular and morphological parameters and the measured parameters can be used to monitor 2,4-D toxicity in an aquatic environment.     

Plasmid as a measure of microbial degradation capacity for 2,4-dichlorophenoxyacetic acid

The purpose of this research was to pursuit the quantification of microbial degradation capacity for 2,4-dichlorophenoxyacetic acid (2,4-D) by detecting and quantifying a prominent 2,4-D degradation encoding plasmid. Batch reactor acclimation, de-acclimation, and re-acclimation tests were conducted during which periods the courses of 2,4-D dissipation and plasmid evolution were quantitatively measured. Pure cultures of bacterial strains were detected to give rise to a plasmid approximately the size of 90 kb after acclimation. The 90 kb plasmid content of Arthrobacter sp. increased when degradation occurred after acclimation, with a rate that corresponded closely to the degradation rate. During de-acclimation, plasmid content declined exponentially at a half-life of approximately 3.5 days. Re-acclimation saw a renewed induction of plasmid, but substrate consumption limited the rise of plasmid to a level much lower than after the first acclimation. This research recommends a method for measuring the microbial degradation capability for a xenobiotic.     

Introduction of 2,4-dichlorophenoxyacetic acid into soil with solvents and resulting implications for bioavailability to microorganisms

Slow equilibration of introduced chemicals through tortuous pore space limits uniform substrate distribution in soil biodegradation studies. The necessity of introducing poorly soluble xenobiotics via organic solvents, the volume of which is minimized to limit toxicity, likely also affects xenobiotic distribution. Our objective was to investigate relative effects of carrier solvent choice and volume on xenobiotic distribution, apparent solvent toxicity, and soil degradation of 2,4-dichlorophenoxy acetic acid. Incubations using four carrier solvents ranging in properties showed that the fraction of 2,4-D mineralized was a hyperbolic function of solvent volume used (0.02–10 μl g⁻¹), attributed to compensating effects of herbicide bioavailability and solvent toxicity. Substrate concentration influenced mineralization of herbicide introduced with organic carriers, but not water. Fraction of material readily desorbed increased when water was the carrier. Results suggest that solvent toxicity should be balanced with uniformity of substrate distribution when using organic carriers in soils. Substrate bioavailability is a ubiquitous issue in terrestrial microbiology research, thus limitations observed herein broadly apply to microbiology questions about introduced substances in soil. We advocate the development of tools to characterize variable conditions among soil compartments, estimates of substrate bioavailability, and linkage of this information to microbial data.     

Micronucleus frequency and proliferation in human lymphocytes after exposure to herbicide 2,4-dichlorophenoxyacetic acid in vitro and in vivo

Widespread use of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) and its association with non-Hodgkin’s lymphoma (NHL) and other cancers has raised public concern. Here, micronucleus (MN) formation has been used as a biomarker of genotoxicity, and replicative and mitotic indices (MIs) as biomarkers of cell cycle kinetics in human lymphocytes. Cells were cultured either as whole blood or isolated lymphocytes and treated with pure or commercial forms of 2,4-D at doses between 0.001 and 1 mM for 48 h. Exposure to 2,4-D produced a minimal increase in MN in whole blood and even smaller one in isolated lymphocyte cultures. This induction took place only at levels approaching cytotoxicity and was accompanied by a significant inhibition of replicative index (RI). At a low (0.005 mM) dose of commercial 2,4-D, a small, marginally significant increase in RI (12–15%) was found in two independent sets of experiments (P=0.052). Additionally, we found that lymphocyte RI was more affected by commercial 2,4-D containing 9.4% of the chemically pure 2,4-D, than with an equal concentration of the latter suggesting that other ingredients present in the commercial pesticide may be responsible or may enhance the effect of 2,4-D. Mitotic index, however, did not show any significant change with either commercial or pure 2,4-D. The lymphocytes of 12 male applicators exposed solely to 2,4-D during a 3-month period had a significantly higher RI than the same group prior to exposure and than a control group (P<0.01), in accordance with the in vitro finding of increased RI at low doses.     

Micronucleus frequency and proliferation in human lymphocytes after exposure to herbicide 2,4-dichlorophenoxyacetic acid in vitro and in vivo

Widespread use of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) and its association with non-Hodgkin's lymphoma (NHL) and other cancers has raised public concern. Here, micronucleus (MN) formation has been used as a biomarker of genotoxicity, and replicative and mitotic indices (MIs) as biomarkers of cell cycle kinetics in human lymphocytes. Cells were cultured either as whole blood or isolated lymphocytes and treated with pure or commercial forms of 2,4-D at doses between 0.001 and 1 mM for 48 h. Exposure to 2,4-D produced a minimal increase in MN in whole blood and even smaller one in isolated lymphocyte cultures. This induction took place only at levels approaching cytotoxicity and was accompanied by a significant inhibition of replicative index (RI). At a low (0.005 mM) dose of commercial 2,4-D, a small, marginally significant increase in RI (12-15%) was found in two independent sets of experiments (P=0.052). Additionally, we found that lymphocyte RI was more affected by commercial 2,4-D containing 9.4% of the chemically pure 2,4-D, than with an equal concentration of the latter suggesting that other ingredients present in the commercial pesticide may be responsible or may enhance the effect of 2,4-D. Mitotic index, however, did not show any significant change with either commercial or pure 2,4-D. The lymphocytes of 12 male applicators exposed solely to 2,4-D during a 3-month period had a significantly higher RI than the same group prior to exposure and than a control group (P<0.01), in accordance with the in vitro finding of increased RI at low doses.     

Activation and significance of vacuolar H+-ATPase in Saccharomyces cerevisiae adaptation and resistance to the herbicide 2,4-dichlorophenoxyacetic acid

The stimulation of the activity of the H(+)-ATPase present in the vacuolar membrane (V-ATPase) of Saccharomyces cerevisiae is here described in response to a moderate stress induced by 2,4-dichlorophenoxyacetic acid (2,4-D). This in vivo activation (up to 5-fold) took place essentially during the adaptation period, preceding cell division under herbicide stress, in coordination with a marked activation of plasma membrane H(+)-ATPase (PM-ATPase) (up to 30-fold) and the decrease of intracellular and vacuolar pH values, suggesting that activation may be triggered by acidification. Single deletion of VMA1 and genes encoding other V-ATPase subunits led to a more extended period of adaptation and to slower growth under 2,4-D stress. Results suggest that a functional V-ATPase is required to counteract, more rapidly and efficiently, the dissipation of the physiological H(+)-gradient across vacuolar membrane registered during 2,4-D adaptation.     

Cotton plants transformed with a bacterial degradation gene are protected from accidental spray drift damage by the herbicide 2,4-dichlorophenoxyacetic acid

The agronomic performance of broad leaved crop plants such as cotton would be greatly improved if genetically-engineered resistance to broadleaf herbicides could both protect the plants from accidental spray drift damage and allow the suppression of problem broadleaf weeds by chemical means. Followingin vitro modification and the addition of plant expression signals, the gene for 2,4-D monooxygenase, a bacterial enzyme that degrades the broadleaf herbicide 2,4-dichlorophenoxyacetic acid (2,4-D), was introduced into cotton plants byAgrobacterium-mediated transformation. First generation homozygous progeny of regenerated transgenic cotton plants carrying this gene exhibited up to a 50–100 fold increase in tolerance to 2,4-D compared with untransformed controls, and glasshouse trials suggest that the genetically-engineered plants would be completely protected from spray drift of 2,4-D, at least up to the normal field application rates commonly used on neighbouring cereal crops.     

Differential response of young and adult leaves to herbicide 2,4-dichlorophenoxyacetic acid in pea plants: role of reactive oxygen species

In this work the differential response of adult and young leaves from pea (Pisum sativum L.) plants to the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) (23 mm) applied by foliar spraying was investigated. The concentration of 2,4-D (23 mm) and the time of treatment (72 h) were previously optimized in order to visualize its toxic effects on pea plants. Under these conditions, the herbicide induced severe disturbances in mesophyll cells structure and proliferation of vascular tissue in young leaves and increased acyl-CoA oxidase (ACX), xanthine oxidase (XOD) and lipoxygenase (LOX) activities in young leaves, and only ACX and LOX in adult leaves. This situation produced reactive oxygen species (ROS) over-accumulation favoured by the absence of significant changes in the enzymatic antioxidants, giving rise to oxidative damages to proteins and membrane lipids. An increase of ethylene took place in both young and adult leaves and the induction of genes encoding the stress proteins, PRP4A and HSP 71,2, was observed mainly in young leaves. These results suggest that ROS overproduction is a key factor in the effect of high concentrations of 2,4-D, and ROS can trigger a differential response in young and adult leaves, either epinasty development in young leaves or senescence processes in adult tissues.     

Degradation of isomeric monochlorobenzoates and 2,4-dichlorophenoxyacetic acid by a constructed Pseudomonas sp.

Summary A 4-chlorobenzoate-degrading Pseudomonas sp. US1 was mated with a strain of Escherichia coli JMP 397 (harbouring the plasmid pJP4). An ex-conjugant designated Pseudomonas sp. US1 ex that could utilize all the isomeric monochlorobenzoates and 2,4-dichlorophenoxyacetate was obtained. The ex-conjugant released stoichiometric amounts of chloride when grown on these chloroaromatics as sole sources of carbon and energy.     

Biosurveillance for invasive insect pest species using an environmental DNA metabarcoding approach and a high salt trap collection fluid

1. With the increase in global trade and warming patterns, the movement, introduction, and establishment of non‐native insect species has increased. A rapid and effective early detection biosurveillance program to identify species of concern is needed to reduce future impacts and costs associated with introduced non‐native species. One of the challenges facing insect surveillance trapping methods is the sheer volume of individual specimens in the collections. Although molecular identification methods are improving, they currently have limitations (e.g., destructive processing of specimens) and a protocol addressing these limitations can support regulatory applications that need morphological evidence to corroborate molecular data.
2. The novel protocol presented here uses a metabarcoding approach to amplify environmental DNA from a saturated salt solution trap fluid, which retains trap specimens for downstream morphological identifications. The use of a saturated salt solution to preserve specimens in traps addresses issues with the high evaporation rate of ethanol in traps, and public safety concerns with other fluid preservation options with unattended traps in public settings.
3. Using a metabarcoding approach, a 407‐nucleotide segment of the cytochrome c oxidase subunit 1 (COI) animal barcode region was successfully amplified from Lindgren funnel trap collection fluids. These traps were placed in forested areas to survey for wood‐boring beetles of regulatory concern. Our results displayed successful amplification of target taxa, including the molecular identification of the Japanese Beetle Popillia japonica, a species regulated in Canada. A second species, Anisandrus maiche, recently introduced to North America, was identified in every trap. The genus Lymantria, which contains numerous species of concern to North American woodlands, was also detected. Also, there were six other species identified of interest due to their potential impacts on native and crop flora and fauna.
4. Our results show how this protocol can be used as an efficient method for the surveillance of insects using a trap with a saturated salt solution and eDNA metabarcoding to detect species of regulatory concern.

     

The effect of 2,4-dichlorophenoxyacetic acid upon the metabolism and composition of soybean hypocotyl mitochondria

Summary Dark-grown, 3-day-old soybean seedlings were sprayed with 1 mM 2,4-dichlorophenoxyacetic acid 24 hours before harvest. Mitochondria from 2,4-D-treated lower hypocotyls were found to be larger and showed greater incorporation in vivo, of amino acids into protein and phosphate into phospholipids and RNA, than mitochondria from untreated tissue. Mitochondria isolated from 2,4-D-treated hypocotyls showed an enhanced energy-dependent incorporation of amino acids into protein, although the incorporation of nucleoside triphosphates into the RNA of isolated mitochondria was not affected. No effect of 2,4-D, applied in vitro, was noted, and no enhancement of mitochondrial respiratory efficiency followed auxin treatment. A method of isolating mitochondria with a very low level of bacterial contamination is reported.     

Studies on the growth of rice tissue under submerged condition II. The effect of light and 2,4-dichlorophenoxyacetic acid on elongation of coleoptile and mesocotyl rice seedlings

Plant Systematics and Evolution -     

Comparison of a gene probe with classical methods for detecting 2,4-dichlorophenoxyacetic acid (2,4-D)-biodegrading bacteria in natural waters

Colony hybridizations with a gene probe for enumeration of 2,4-dichlorophenoxy-acetic acid (2,4-D)-degrading bacteria were compared with classical enrichment and radiolabel most-probable-number (MPN) assay methods. Two natural water samples (rivers) and raw sewage were tested by each method. UV scans of enrichment cultures revealed 2,4-D degradation with raw sewage occurred in 4–11 days, 4–>22 days with Mary's River water, and 5–>22 days with Willamette River water. [¹⁴C]-2,4-D MPN analysis, measuring release of¹⁴CO₂, yielded estimates of bacteria per milliliter able to degrade 2,4-D. Raw sewage estimates were 1.4 × 10⁵ 2,4-D degraders/ml, Mary's River >1.6 × 10⁵/ml, and Willamette River water 1.6 × 10⁴/ml. Activities noted by UV scan enrichment data supported these results.Autoradiograms of colony blots were also used to estimate numbers of 2,4-D-degrading bacteria. These estimates were also supported by the UV scan data from enrichment cultures. Raw sewage gave counts between 5 × 10⁴ and 2.9 × 10⁵ 2,4-D-degrading bacteria/ml, which correlates well with the estimates obtained by¹⁴C-MPN analyses. River waters, both much lower in total bacterial counts and organic carbon than raw sewage, yielded fewer 2,4-D-degrading bacteria than estimated by¹⁴C-MPN. Media composition and cometabolism may account for discrepancies in estimates for 2,4-D-degrading bacteria observed when colony blot and¹⁴C-MPN analyses were compared.Replica plating made it possible to test for 2,4-D biodegradation from colonies reactive in autoradiograms. Five of 12 (42%) colonies reacting in the colony hybridization exhibited biodegradation activities. Nonreactive colonies failed to degrade 2,4-D.     

The regeneration potential of wheat shoot meristems in the presence and absence of 2,4-dichlorophenoxyacetic acid

Summary Tissue cultures capable of plant regeneration were successfully initiated from extremely immature shoot meristems of 21 randomly selected genotypes of wheat on nutrient media containing 2,4-dichlorophenoxyacetic acid (2,4-D). By means of scanning electron microscopy it was demonstrated that cultures consisted of teratomatous primordia, which were kept in a proliferating budding state by the 2,4-D. These are characteristic of cereal tissue cultures. Release of the primordia and outgrowth of normal shoots and roots occurred when the cultures were no longer exposed to 2,4-D. Shoot primordia which were clearly identifiable were always associated with root primordia in a quasi-bipolar fashion. Sometimes regions assumed the shape of zygotic embryos, but the transition from apparently normal embryos with scutellum to abnormal configurations with shoot and root regions was gradual. The differences between genotypes in shoot regeneration potential was minimal compared to cultures derived from explants which were taken from regions temporally and spatially more distant from the shoot apex. It is concluded that the ability to give rise to cultures capable of shoot regeneration was lost within a fraction of a millimeter distance from the apical meristem in many genotypes. The proliferating tissues were subcultured at regular intervals over a period of one year and the regeneration potential was monitored. Areas capable of shoot regeneration tended to deteriorate more or less rapidly and were overgrown by root-type tissue in a number of genotypes. The results are discussed in the context of the frequently observed, but largely unexplained, variability in the regeneration potential of cereal tissue cultures.     

In vivo and in vitro binding of 2,4-dichlorophenoxyacetic acid to a rat liver mitochondrial protein

2,4-dichlorophenoxyacetic acid (2,4-D) is a hormonal herbicide widely used in the world because of its efficacy in the control of broadleaf and woody plants. In this study we have demonstrated in vivo covalent binding of the phenoxyherbicide 2,4-D to a single protein of 52 kD (from rat liver mitochondrial preparation) detected through immunoblotting studies with the specific antiserum for 2,4-D. The direct involvement of 2,4-D in the formation of the adduct has also been demonstrated in vitro, using liver mitochondrial preparations exposed to 14C-UL-2,4-D. Radiolabeled protein separated by SDS-PAGE and afterwards electroeluted showed a single labeled protein of 52 kD. When mitochondria exposed to radiolabeled xenobiotic were devoid of their outer membrane, the specific activity observed suggest that protein involved in covalent interaction belongs to the inner mitochondrial membrane. We propose that covalent binding of the phenoxyherbicide 2,4-D to a very specific single protein of 52 kD observed in vitro and in vivo may be related to known alterations of the mitochondrial function.