Radiolabeled herbicides

Through the further combined use of radiolabeled herbicides and additives in tracer and metabolic studies, it may be possible to develop new concepts regarding formulation of herbicides for differential absorption, translocation, and selective action, while at the same time minimizing chemical residues. 

Herbicides that inhibit photosynthetic electron flow prevent reduction of plastoquinone by the photosystem II acceptor complex. The properties of the photosystem II herbicide receptor proteins have been investigated by binding and displacement studies with radiolabeled herbicides. The herbicide receptor proteins have been identified with herbicide-derived photoaffinity labels. Herbicides, similar in their mode of action to 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) bind to a 34 kDa protein, whereas phenolic herbicides bind to the 43-51 kDa photosystem II reaction center proteins. At these receptor proteins, plastoquinone/herbicide interactions and plastoquinone binding sites have been studied, the latter by means of a plastoquinone-deriv-ed photoaffinity label. For the 34 kDa herbicide binding protein, whose amino acid sequence is known, herbicide and plastoquinone binding are discussed at the molecular level. 

In 1979, the concept of a photosystem II herbicide binding protein with different but overlapping binding sites for the various photosystem II herbicides was simultaneously established by Trebst and Draber ( 5) and Pfister and Arntzen (6). This idea of a herbicide receptor protein proved to be extremely fruitful because the techniques of receptor biochemistry were now applicable. Tischer and Strotmann (7) were the first investigators to study binding of radiolabeled herbicides in isolated thylakoids. 

As already stressed, photosystem II herbicides bind reversibly to their binding site. A1tough radiolabeled herbicides are available, it is impossible to identify the herbicide receptor protein without a chemical modification of the herbicide that allows for covalent... 

In conclusion, observations made in the last few years, especially the binding studies with radiolabeled herbicides, the photoaffinity labeling technique, and the advances of molecular biology have substantially added to our knowledge of the mechanism of action of photosynthetic herbicides. However, many questions also remain to be answered. 

The distinctly different behavior of the phenol-type herbicides following trypsin treatment suggests that different determinants within the PS II protein complex establish the "domains" that regulate the binding properties of these inhibitors. In spite of the fact that phenol-type herbicides will displace bound radiolabeled herbicides such as diuron, these inhibitors show noncompetitive inhibition (29, 30). At present, there are three lines of evidence which favor TH e involvement of two domains within the PS II complex that participate in creating the binding sites for these herbicides (a) isolated PS II particles can be selectively depleted of a polypeptide with parallel loss of atrazine sensitivity, but not dinoseb inhibition activity (33) (b) in resistant weed biotypes, chloroplast membranes that exhibit extreme triazine resistance have increased sensitivity to the phenol-type herbicides (13) and (c) experiments with azido (photoaffinity) derivatives of phenol and triazine herbicides result in the covalent labeling of different PS II polypeptides (, 31). 

Cellular Compartmentation of Herbicides. For detailed mechanism of action research, the Jja situ tissue distribution of DPE s should be known. One method to determine location is to treat with radiolabeled herbicide and, at some later time, fractionate the tissue into its various subcellular components... 

Herbicide binding — The amount of radiolabelled herbicide bound to membranes was followed at different illumination times and the activity expressed as per cent of initial value. 

The factors determining uptake by plants and subsequent movement in xylem and phloem have been discussed, and consideration should now be given as to how this information can be used to understand the detailed distribution patterns of compounds in plants. Such patterns are usually obtained by autoradiography of plants following application of radiolabeled herbicides, and it is important that the period of the experiment be sufficiently short that interpretation is not confused by extensive metabolism of the herbicide. 

Cl4-DBpD) may occur in trace amounts in the herbicide, 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) (I, 2). Radiolabeled preparations of this dioxin are needed to facilitate studies of its degradation chemistry, metabolism, and mode of action. 

It is a broad-spectrum herbicide active both pre- and post-emergent. Studies with radiolabelled compounds ( ) show it to be rapidly translocated particularly to the meristematic regions. Translocation to the underground storage organs of perennial weeds prevents regrowth of these weeds. Most herbaceous and woody plants are controlled in the field at 0.4-1.0 kg a.e./ha whereas most woody plants require 0.7-3.0 kg a.e./ha (10). 

Many inhibitors of photoinduced electron transport, including a large number of herbicides, have been shown to bind reversibly and competitively to the protein. The studies volve displacement of a radiolabeled inhibitor, such as [ C]-atrazine [2-chloro-4-(ethylamino)-6-(isopropylamino)- -trlazine], by an uy abeled inhibitor (29). Only marginal displacement of [ Cj-atrazine was observed by high concentrations of umbelliferone and naringenin. The other allelochemicals produced no measurable effects. 

Surugiu et al. [67] have introduced an Enzyme Immuno-Like Assays (EzILA) for the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). The label was a 2,4-D conjugate with the tobacco peroxidase (TOP) enzyme, which allows for both colorimetric and chemiluminescent detection. In this case, the polymer imprinted with 2,4-D was synthesized in the form of microspheres. In contrast, despite its higher binding capacity for radiolabeled 2,4-D, a conventional MIP prepared by bulk polymerization showed only weak binding of the 2,4-D-TOP tracer. 

Addition of colored, fluorescent (4), or radiolabeled tags (33) to the spray formulation are useful refinements. Biological indicators, particularly sensitive plants in the case of herbicides, are also employed in many experiments 0,4). 

Recently, Schloss et al (33) showed that IM and TP were able to quantitatively displace a radiolabelled SU herbicide from ALS, indicating competitive binding. Curiously, the SU ligand was also displaced by the quinone, Qo. It was proposed that SU, TP, and IM bind to ALS in a vestigial quinone binding site associated with the evolution of ALS from pyruvate oxidase. This enzyme is an FAD-protein that catalyzes the oxidation of pyruvate to acetate. 

Radiolabelled derivatives of the herbicide florasulam (N-(2,6-difluo-rophenyl)-5-methoxy-8-fluoro(l,2,4)-triazolo-[l,5-c]-pyrimidine-2-sulphon-amide) (VII) were exposed to natural sunlight in a sterile pH 5 buffer water and in a natural lake water collected from 20 to 30 cm below the surface [70]. The photo degradation was much faster in the natural water system, with a half-life of 3.3 days against 73 days in the buffered aqueous medium. Moreover, the photoproducts produced in the distilled and natural waters were found to be different. Direct photolysis led to the cleavage of the N - S bond with formation of the sulphonic acid derivative (Vila) after 10% of conversion (see Scheme 7). 

The first section of this book describes the application of LC/MS to the analysis of agricultural chemicals and their metabolites. Using LC/MS for residue analysis in agricultural chemistry has become routine in many laboratories. Many pesticides, such as the chlorophenoxy acid and sulfonyl urea herbicides or organophosphorus and methyl carbamate insecticides, are too polar or thermally labile for analysis via GC. The use of LC/MS for the identification of polar pesticide metabolites and conjugates, an area traditionally dominated by radiolabeled compounds, stands out as a particularly dramatic demonstration of the power of this technique. 

Metosulam (198) was C-radiolabeled in the 2-position or in the benzene ring by modified synthetic routes (97MI6). This way data on the pharmacokinetics in mammals could be provided (97MI4). Flumetsulam (197) was toxicologically examined (03MI5) and included in QSAR studies of herbicide toxicity (06BMC2779). 

Herbicide Binding Assays. Control and trypsin-treated chloroplast thylakoids were suspended in PSNM buffer. Buffer (1 ml volume) containing 50 Chi was incubated 3 min with l C-atrazine (specific activity 27.2 pCi/mg). Chloroplasts were pelleted and an aliquot of the supernatant was removed for determination of the amount of unbound atrazine. Details of this procedure are described elsewhere (6, 9). Radiolabeled atrazine was a gift of Dr. H. LeBaron, CTBA-GEIGY, N. Carolina. 

Figure 2. Effect of 1 (xM AFM on efflux of various radiolabeled substances from cucumber cotyledons ( ). At time zero, cotyledons were exposed to 600 fiE/m s (PAR) light and herbicide. A, Efflux of Cl. B, Efflux of C, Efflux of...

In an uptake experiment, over 90% of the radiolabeled pinoxaden was incorporated into the crops within 5 h when treatment solutions were applied in droplets to the adaxial leaf surface of two-leaved plants of barley, winter wheat or durum wheat. After 24 h, about 20% is translocated out of the treated leaf by basipetal movement below the treated area [87]. Cloquintocet does not affect the absorption or the movement of the herbicide within the crop. 

Organic phase MIA has also been applied to the herbicide atrazine, the groups of Stanker [35] and Mosbach [36] publishing similar assays simultaneously. In both cases, selectivity was demonstrated over related substituted triazines. The former assay performed in acetonitrile had a detection limit of 4.6 pM, the latter performed in toluene 250 nM. Other analytes of environmental interest for which conventional radiolabel MIAs have been developed include 2,4-dichlorophenoxyacetic acid [23] and 4-nitrophenol [48], although in the latter case the influence of interferents was not studied. 

For example, Dean-Raymond and Alexander (1976) have shown that radiolabeled NDMA incorporated into soil could be taken up by edible plants. A recent survey of dried waste sludge also indicates most sludges contain small amounts of VNA (Nbmma et al. 1982). If sludge is incorporated into soil uptake may be possible. It should be noted that no confirmed report of VNA in foods as a result of the use of pesticides or sludge in actual field use has appeared. On the contrary, Ross et al. (1978) analyzed soil, run off water and edible plant tissue after the application of a commercial herbicide containing NDPA and failed to detect the VNA in any sample. As pointed out by Oliver, (1981) it is difficult to draw conclusions about the VNA contamination of foods based on laboratory experiments. 

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