2,4-Dichlorophenoxyacetic acid causes chromatin and chromosome abnormalities in plant cells and mutation in cultured mammalian cells

The cytotoxic and mutagenic effects of the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) on shallot root tip cells and on V79 Chinese hamster fibroblast cells were examined and compared. In shallot root tips 2,4-D caused changes in mitotic activity, as well as changes in chromosome and chromatin structure, and also changes during the cell cycle. 2,4-D also showed mutagenic and cytotoxic effects on V79 cells in culture in concentrations higher than 10 micrograms/ml. The results in both systems (plant and mammalian cells) were in agreement showing mutagenic activity of 2,4-D in the concentration range higher than usually used in establishing plant tissue culture (greater than 5 micrograms/ml).     

Identification of 2,4-D-responsive proteins in embryogenic callus of Valencia sweet orange (<Emphasis Type="Italic">Citrus sinensis</Emphasis> Osbeck) following osmotic stress

The compound 2,4-Dicholorophenoxyacetic acid (2,4-D) is an important growth regulator which is used in the majority of embryogenic cell and tissue culture systems. However, 2,4-D also appears to have a negative effect on growth and development of plant tissues and organs cultured in vitro. For example, 2,4-D exerts inhibition on in vitro somatic embryo initiation and/or development of most citrus species. To understand the molecular mechanism by which 2,4-D inhibits somatic embryogenesis (SE), proteomic changes of Valencia sweet orange (Citrus sinensis) embryogenic callus induced by treatments with a high concentration of 2,4-D (6 mg l⁻¹) was investigated. Nine 2,4-D-responsive proteins were identified, of which eight were up-regulated and one was down-regulated. Interestingly, three of the eight up-regulated proteins were osmotic stress-associated, suggesting that 2,4-D induced osmotic stress in Valencia embryogenic callus. This speculation was supported by results from our physiological studies: 2,4-D treated callus cells exhibited increased cytoplasm concentration with a significant reduction in relative water content (RWC) and an obvious increase in levels of two osmolytes (proline and soluble sugar). Taken together, our results suggested that 2,4-D could inhibit somatic embryo initiation by, at least in part, inducing osmotic stress to citrus callus cells.     

Activated sludge treatment of a xenobiotic with or without a biogenic substrate during start-up and shocks

Lab-scale continuous flow activated sludge systems that were acclimated to 2,4-dichlorphenoxyacetic acid (2,4-D) under sole 2,4-D influent and without sludge wastage, were able to maintain successful 2,4-D treatment when both 2,4-D and a biogenic substrate were fed and the systems operated with finite mean cell residence times (theta(c)). When the systems were fed dual 2,4-D and biogenic substrates and operated with finite theta(c) from the start, treatment of 2,4-D fluctuated noticeably long after acclimation. At the reintroduction of 2,4-D after its absence from the influent for a period of time (2,4-D shock), the systems under both the sole and dual substrate conditions suffered similar treatment losses; the extent of treatment losses was related to the length of 2,4-D absence time. When shocked, systems with sole 2,4-D influent had a slight advantage over dual substrates by showing a faster recovery from shocks with the help of re-acclimation.     

Ames assays and unscheduled DNA synthesis assays on 2, 4-dichlorophenoxyacetic acid and its derivatives.

2,4-dichlorophenoxyacetic acid and several of its derivatives (collectively known as 2,4-D) are herbicides used to control a wide variety of broadleaf and woody plants. The genetic toxicity in vitro of 2,4-D and seven of its salts and esters were examined by employing gene mutation in bacteria (Ames test) and induction of DNA damage and repair in rat hepatocytes. In addition, an in vivo unscheduled DNA synthesis (UDS) assay was performed on 2,4-D. There were no indications of genotoxic potential for 2,4-D acid, or any of its derivatives, in these assays. These results are consistent with the reported lack of carcinogenic potential for 2,4-D in both mice and rats.     

A comparison of 2,4-dichlorophenoxyacetic Acid metabolism in cultured soybean cells and in embryogenic carrot cells

The removal or reduction in concentration of auxin is often a successful method for obtaining morphogenesis in cell cultures of higher plants, such as carrot, but not for soybean. For this reason, the metabolism of one auxin, 2,4-dichlorophenoxyacetic acid (2,4-D), was compared in both carrot and soybean cells. Whereas soybean cells conjugated a high percentage of their 2,4-D to amino acids, carrot cells contained primarily free 2,4-D. Moreover, after long-term exposure to 2,4-D, carrot cells released much more 2,4-D upon transfer to 2,4-D-free (embryogenic) medium than did soybean cells. It appears that the retention of 2,4-D by soybean cells might interfere with subsequent morphogenesis. Because no impairment of 2,4-D efflux was found with short-term exposure to radiolabeled 2,4-D, it was concluded that 2,4-D retention in soybean cells might be due to a time-dependent, metabolic process. The conjugation of 2,4-D to amino acids was shown to be one such time-dependent process. Additionally, the release of 2,4-D from the cells was shown to be due primarily to a loss of free 2,4-D and not 2,4-D-amino acid conjugates. It seems that the greater retention of 2,4-D by soybean cells upon transfer to 2,4-D-free medium is due to greater formation of 2,4-D-amino acid conjugates.     

Two stage cultures for the production of berberine in cell suspension cultures of Thalictrum rugosum.

Two stage culture systems were examined in cell suspensions of Thalictrum rugosum which produce berberine in a growth associated manner. The utilization of indole 3-acetic acid (IAA) as a growth regulator, in place of 2,4-dichlorophenoxyacetic acid (2,4-D), at various growth phases was investigated. Although the enhancing effect was clear when it was used separately, in combination with 2,4-D effect of IAA was suppressed and medium change from 2,4-D to IAA was detrimental to product formation. The decrease of berberine production in this two stage culture may be due to the loss of conditioning factors associated with medium replacement.     

Herbicide 2,4-dichlorophenoxyacetic acid (2,4-D)-induced cytogenetic damage in human lymphocytes in vitro in presence of erythrocytes

The genotoxic effects of 2,4-D and its commercial derivative 2,4-D DMA were studied by measuring sister chromatid exchange (SCE), cell-cycle progression and mitotic index in human whole blood (WBC) and plasma leukocyte cultures (PLC). Concentrations of 10, 25, 50 and 100 microg herbicide/ml were used during 72 h. In WBC, a significant increase in SCE frequency was observed within the 10-50 microg 2,4-D/ml and 25-100 microg 2,4-D DMA/ml dose range. Contrarily, in PLC, none of the concentrations employed affected the SCEs frequency. A significant delay in cell proliferation was observed in WBC after treatments with 25 and 50 microg 2,4-D/ml and 50 and 100 microg 2,4-D DMA/ml. In PLC, only 100.0 microg 2,4-D/ml altered cell-cycle progression. For both chemicals, a progressive dose-related inhibition of mitotic activity was observed. The results demonstrated that the presence of erythrocytes in the culture system modulated the DNA and cellular damage inflicted by 2,4-D and 2,4-D DMA into human lymphocytes in vitro as well as both 2,4-D and 2,4-D DMA were more potent genotoxic agents in the presence of human red cells.     

Chemotaxis of Ralstonia eutropha JMP134(pJP4) to the herbicide 2,4-dichlorophenoxyacetate

Ralstonia eutropha JMP134(pJP4) and several other species of motile bacteria can degrade the herbicide 2,4-dichlorophenoxyacetate (2,4-D), but it was not known if bacteria could sense and swim towards 2,4-D by the process of chemotaxis. Wild-type R. eutropha cells were chemotactically attracted to 2,4-D in swarm plate assays and qualitative capillary assays. The chemotactic response was induced by growth with 2,4-D and depended on the presence of the catabolic plasmid pJP4, which harbors the tfd genes for 2,4-D degradation. The tfd cluster also encodes a permease for 2,4-D named TfdK. A tfdK mutant was not chemotactic to 2,4-D, even though it grew at wild-type rates on 2,4-D.     

Monitoring gene expression in mixed microbial communities by using DNA microarrays

A DNA microarray to monitor the expression of bacterial metabolic genes within mixed microbial communities was designed and tested. Total RNA was extracted from pure and mixed cultures containing the 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacterium Ralstonia eutropha JMP134, and the inducing agent 2,4-D. Induction of the 2,4-D catabolic genes present in this organism was readily detected 4, 7, and 24 h after the addition of 2,4-D. This strain was diluted into a constructed mixed microbial community derived from a laboratory scale sequencing batch reactor. Induction of two of five 2,4-D catabolic genes (tfdA and tfdC) from populations of JMP134 as low as 10(5) cells/ml was clearly detected against a background of 10(8) cells/ml. Induction of two others (tfdB and tfdE) was detected from populations of 10(6) cells/ml in the same background; however, the last gene, tfdF, showed no significant induction due to high variability. In another experiment, the induction of resin acid degradative genes was statistically detectable in sludge-fed pulp mill effluent exposed to dehydroabietic acid in batch experiments. We conclude that microarrays will be useful tools for the detection of bacterial gene expression in wastewaters and other complex systems.     

Degradation of 2,4-dichlorophenoxyacetic acid by mixed cultures of bacteria

Summary We explored the feasibility of using mixed cultures for herbicide degradation, with the ultimate aim of application for effluent treatment. The present study reports on mixed cultures which were developed to grow aerobically with 2,4-dichlorophenoxyacetic acid (2,4-D) as the sole carbon substrate. Degradation of 2,4-D was verified by HPLC and UV-spectroscopic analysis of the residual 2,4-D concentration in the test cultures. Cultures that were initially developed with 2,4-D also grew readily with glucose, but the degradation of 2,4-D was effectively prevented under mixed substrate conditions. Mamor intermediates or metabolites resulting from 2,4-D degradation were not detected with the HPLC methodology except 2,4-dichlorophenol which appeared to accumulate transiently in the growth medium.     

Genotoxic effect of 2,4-dichlorophenoxy acetic acid and its metabolite 2,4-dichlorophenol in mouse.

The cytogenetic effect of 2,4-dichlorophenoxy acetic acid (2,4-D) and its metabolite 2,4-dichlorophenol (2,4-DCP) was studied in bone-marrow, germ cells and sperm head abnormalities in the treated mice. Swiss mice were treated orally by gavage with 2,4-D at 1.7, 3.3 and 33 mg kg(-1)BW (1/200, 1/100 and 1/10 of LD(50)). 2,4-DCP was intraperitoneally (i.p.) injected at 36, 72 and 180 mg kg(-1)BW (1/10, 1/5, 1/2 of LD(50)). A significant increase in the percentage of chromosome aberrations in bone-marrow and spermatocyte cells was observed after oral administration of 2,4-D at 3.3 mg kg(-1)BW for three and five consecutive days. This percentage increased and reached 10.8+/-0.87 (P<0.01) in bone-marrow and 9.8+/-0.45 (P<0.01) in spermatocyte cells after oral administration of 2,4-D at 33 mg kg(-1)BW for 24 h. This percentage was, however, lower than that induced in bone-marrow and spermatocyte cells by mitomycin C (positive control). 2,4-D induced a dose-dependent increase in the percentage of sperm head abnormalities. The genotoxic effect of 2,4-DCP is weaker than that of 2,4-D, as indicated by the lower percentage of the induced chromosome aberrations (in bone-marrow and spermatocyte cells) and sperm head abnormalities. Only the highest tested concentration of 2,4-DCP (180 mg kg(-1)BW, 1/2 LD(50)) induced a significant percentage of chromosome aberrations and sperm head abnormalities after i.p. injection. The obtained results indicate that 2,4-D is genotoxic in mice in vivo under the conditions tested. Hence, more care should be given to the application of 2,4-D on edible crops since repeated uses may underlie a health hazard.     

Colonisation of seedling roots by 2,4-D degrading bacteria: A plant-microbial model

The development of model plant-microbial associations between Gram negative soil microbes capable of degrading phenoxyacetate herbicides, such as 2,4-D and 2,4-D methyl ester, and the crops canola and wheat was described. Both an Acinetobacter baumannii pJP4 transconjugant and Alcaligenes eutrophus JMP 134 colonised non-parasitically on the roots of sterilised seedlings in a hydroponic system. Laser scanning confocal microscopy has shown that colonisation occurred both on the root surface and deeper inside the mucilage layer or inside some surface root cells. When 2,4-D was added to the hydroponic medium supporting the growth of those seedlings colonised by 2,4-D degrading bacteria, the gas chromatographic analysis showed a rapid decrease in the concentration of this herbicide. These bacteria colonising the root system were shown to be responsible for the degradation of 2,4-D. Plants inoculated with the 2,4-D degrading microbes were subsequently found to be less susceptible to damage by the herbicide in such hydroponic systems.     

Membrane Potential of Cultured Carrot Cells in Relation to the Synthesis of Anthocyanin and Embryogenesis

Membrane potentials of cultured carrot cells in culture mediumwere about –40 mV and did not change with addition ofsalts of addition (or depletion) of 2,4-dichlorophenoxyaceticacid (2,4-D). When the measurement was performed in the testmedium (containing low concentration of salts), the values werewidely distributed (from –60 to –110mV) and changedlargely with external concentration of K⁺ but not Mg²⁺ nor Ca²⁺.When the cells were fractionated by Ficoll density gradientcentrifugation, the membrane potential of the cells of higherdensity (> 14% Ficoll) was about –150 mV in the testmedium and did not change during embryogenesis with depletionof 2,4-D. The membrane potential of cells of lower density (bandingbetween 6– 10% Ficoll) was less negative (– 60 to– 110 mV) in the test medium. When these cells were transferredand cultured in medium containing zeatin but lacking 2,4-D,the membrane potential was shifted negatively by about 15 mVprior to anthocyanin synthesis. When 2,4-D was added to anthocyanin-synthesizingcells in the medium containing zeatin, a transient hyperpolarizationand subsequent depolarization of the membrane were observedprior to the inhibition of anthocyanin synthesis. (Received October 22, 1987; Accepted April 20, 1988)     

Electrical patterns of tobacco cells in media containing indole-3-acetic acid or 2,4-dichlorophenoxyacetic acid

A simple, inexpensive, and stable drive-unit for a vibrating probe is described. It was used to measure transcellular electrical currents and their stability in cells from suspension cultures of Nicotiana tabacum L. var. virginica. The cells were highly variable in size, morphology and current-pattern. The magnitude and pattern of the currents depended on the age of the culture, the morphology of the cells and the auxin in the culture medium. Currents in small cell clusters were weakest during the lag-phase of growth and strongest when the cultures were actively growing. The shape of the cells was related to the electrical pattern surrounding them, electrically polar cells tending to be elongated. The proportion of polar cells depended on the auxin composition of the culture medium. About 75% of the cells from suspensions grown in the presence of indole-3-acetic acid (IAA) were electrically polar. These cells normally divided at right angles to their electrical axes to form filaments. Only around 20% of the cells grown in medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) were electrically polar, the remainder had randomly oriented currents and divided in random directions to form irregular clusters rather than filaments. The electrical patterns of cells in 2,4-D were much less stable than those of cells in IAA. When currents were measured repeatedly at fixed locations on cells, those in 2,4-D were about twice as likely to disappear, arise de novo, or change direction as those in IAA. When cells were transferred from 2,4-D to IAA media, the percentage of polar cells increased from 25 to 40 within 1 d, but when they were transferred from IAA to 2,4-D, this percentage decreased from 48 to 26. It is suggested that one of the reasons that 2,4-D suppresses organogenesis in tobacco cultures (and possibly why it also functions as a herbicide) is that it reduces the stability of transcellular currents and disrupts the electrical patterns of cells so that they become less capable of organized polar growth.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid The authors are indebted to the Agricultural and Food Research Council of the UK for their financial support and to the Royal Society for the provision of the vibrating probe. We would also like to thank Dr. A. Lagoa for his help in culturing the cells.     

Chemotaxis of Ralstonia eutropha JMP134(pJP4) to the Herbicide 2,4-Dichlorophenoxyacetate

Ralstonia eutropha JMP134(pJP4) and several other species of motile bacteria can degrade the herbicide 2,4-dichlorophenoxyacetate (2,4-D), but it was not known if bacteria could sense and swim towards 2,4-D by the process of chemotaxis. Wild-type R. eutropha cells were chemotactically attracted to 2,4-D in swarm plate assays and qualitative capillary assays. The chemotactic response was induced by growth with 2,4-D and depended on the presence of the catabolic plasmid pJP4, which harbors the tfd genes for 2,4-D degradation. The tfd cluster also encodes a permease for 2,4-D named TfdK. A tfdK mutant was not chemotactic to 2,4-D, even though it grew at wild-type rates on 2,4-D.     

Degradation of 2,4-dichlorophenoxyacetic acid by Pseudomonas cepacia DBO1(pRO101) in a dual-substrate chemostat.

To determine the effect of a secondary carbon source on biodegradation of a chloroaromatic compound, Pseudomonas cepacia DBO1(pRO101) was grown in continuous cultures on basal salts media containing various mixtures of 2,4-dichlorophenoxyacetic acid (2,4-D) and succinate. Both succinate and 2,4-D were metabolized over the entire range of dilution rates and compositions analyzed (0.05 to 0.6 h-1). 2,4-Dichlorophenol (DCP), the only intermediate detected, accumulated to significant amounts (10 to 21 mg/liter) in the chemostat only when the dilution rate was 0.4 h-1 or greater. At these concentrations, DCP reduced the apparent growth rate of P. cepacia DBO1(pRO101) in batch cultures by 15 to 35% over the apparent growth rate on succinate alone. Succinate fed to the chemostat increased the cell density as well as the percentage of 2,4-D that was consumed at each dilution rate. When the amount of succinate in the feed exceeded the amount of 2,4-D, the specific rates of 2,4-D degradation in the chemostat or by washed cells were significantly lower than the specific rates for cells grown on 2,4-D alone, suggesting repression by succinate. However, when the amount of 2,4-D in the feed exceeded the amount of succinate, the specific rates of 2,4-D degradation remained at values equivalent to or higher than the specific rate for cells grown on 2,4-D alone. DCP accumulated significantly in the washed-cell assay, suggesting that the level of DCP hydroxylase is rate limiting.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of 2,4-dichlorophenoxyacetic acid on the activity of o-methyltransferase in carrot cell culture

The activity of caffeic acid-O-methyltransferase (OMT) in carrot cells was greatly affected by the amount of 2,4-dichlorophenoxyacetic acid (2,4-D) supplemented to the culture medium. The OMT fraction was purified by (NH₄)₂SO₄ followed by ultrafiltration and gel filtration or DEAE-Sephadex chromatography after cells were cultured in the medium containing [2-¹⁴C]-2,4-D. Thus, this purified fraction revealed high OMT activity and was still radioactive. The OMT activity was about eight-fold higher (or more) in cells cultured at 0.05 ppm 2,4-D than in those at 1.0 ppm 2,4-D. The ratio of radioactivity to OMT activity was about four-fold higher in cells cultured at 1.0 ppm 2,4-D than those at 0.05 ppm 2,4-D. On the other hand, the OMT fraction was separated into two radioactive protein fractions by DEAE-Sephadex chromatography. The radioactive fractions became Et₂O-soluble after HCl hydrolysis, but not after salt-urea treatment. From these results, it was concluded that 2,4-D is covalently bound to proteins in the OMT fraction. Such 2,4-D protein conjugates may play a role in the regulation of OMT activity.     

Auxin transport at cellular level: new insights supported by mathematical modelling

The molecular basis of cellular auxin transport is still not fully understood. Although a number of carriers have been identified and proved to be involved in auxin transport, their regulation and possible activity of as yet unknown transporters remain unclear. Nevertheless, using single-cell-based systems it is possible to track the course of auxin accumulation inside cells and to specify and quantify some auxin transport parameters. The synthetic auxins 2,4-dichlorophenoxyacetic acid (2,4-D) and naphthalene-1-acetic acid (NAA) are generally considered to be suitable tools for auxin transport studies because they are transported specifically via either auxin influx or efflux carriers, respectively. Our results indicate that NAA can be metabolized rapidly in tobacco BY-2 cells. The predominant metabolite has been identified as NAA glucosyl ester and it is shown that all NAA metabolites were retained inside the cells. This implies that the transport efficiency of auxin efflux transporters is higher than previously assumed. By contrast, the metabolism of 2,4-D remained fairly weak. Moreover, using data on the accumulation of 2,4-D measured in the presence of auxin transport inhibitors, it is shown that 2,4-D is also transported by efflux carriers. These results suggest that 2,4-D is a promising tool for determining both auxin influx and efflux activities. Based on the accumulation data, a mathematical model of 2,4-D transport at a single-cell level is proposed. Optimization of the model provides estimates of crucial transport parameters and, together with its validation by successfully predicting the course of 2,4-D accumulation, it confirms the consistency of the present concept of cellular auxin transport.