Diphenyl Ether Herbicides Research Articles

Effects of a diphenyl-ether herbicide, oxyfluorfen, on human BFU-E/CFU-E development and haemoglobin synthesis

The diphenyl-ether herbicides exert their phytotoxic activity by preventing chlorophyll formation in plants as a result of inhibition of protoporphyrinogen oxidase. This enzyme is the last step of the common pathway for chlorophyll and haem biosynthesis. The aim of this work is to determine whether herbicide inhibitors of plant protoporphyrinogen oxidase could act on the human protoporphyrinogen oxidase involved in haemoglobin synthesis and cause heamatologic diseases. Human erythroblastic progenitors (BFU-E/CFU-E: Burst Forming Unit-Erythroid and Colony Forming Unit-Erythroid) were exposed to oxyfluorfen, a diphenyl-ether herbicide in the presence of erythropoietin, and the haematoxicity evaluated in vitro by scoring the development of BFU-E/CFU-E colonies after 7 and 14 days of culture. The toxic effect on differentiation has been evaluated using four criteria: morphology, total protein, total porphyrin, and haemoglobin content. The study of BFU-E/CFU-E proliferation and differentiation showed a cytotoxic effect of oxyfluorfen only at very high concentrations. In contrast, haemoglobin synthesis can be inhibited by concentration of oxyfluorfen (10(-4) M) that have no adverse effect on cellular proliferation.

Cytotoxicity of organochlorine pesticides and lipid peroxidation in isolated rat hepatocytes.

The cytotoxicity and lipid peroxidation of pesticides containing a halogen group were examined in isolated rat hepatocytes. We examined 9 pesticides of 3 different representative chemical families, chlorinated aromatic fungicides (pentachlorophenol (PCP), pentachloronitrobenzene (PCNB), chlorothalonil, fthalide), polyhaloalkylated thio fungicides (dichlofluanid, captan) and diphenyl ether herbicide (2,4-dichlorophenyl 4-nitrophenyl ether (NIP), 4-nitrophenyl2,4,6-trichlorophenyl ether (CNP), chlomethoxynil). The contents of the hydroperoxides in phospholipid, phosphatidylcholine hydroperoxide (PCOOH) and phosphatidylethanolamine hydroperoxide (PEOOH) were determined by the HPLC-chemiluminescence (CL-HPLC) method, which is sensitive and specific for lipid hydroperoxide. Chlorothalonil, dichlofluanid and captan were the most potent cytotoxicants evaluated by lactate dehydrogenase (LDH) leakage. PCP, NIP and CNP exhibited intermediate cytotoxicity. PCNB, fthalide and chlomethoxynil showed low cytotoxicity. The cellular phospholipid hydroperoxide (PCOOH and PEOOH) levels were remarkably increased by chlorothalonil (PCOOH, 23 times and PEOOH, 7 times), dichlofluanid (PCOOH, 523 times and PEOOH, 22 times) and captan (PCOOH, 518 times and PEOOH, 16 times) as compared with the control group. The PCOOH content was slightly increased by PCP (4.8 times) and NIP (6.3 times), whereas the other 4 pesticides did not change the phospholipid hydroperoxide level. Severe cytotoxicity was observed with a remarkable increase of phospholipid hydroperoxide by chlorothalonil, dichlofluanid and captan.

IMPROVEMENT OF ERYTHROBLASTIC PROGENITOR CULTURE FOR TOXICOLOGICAL INVESTIGATIONS

The rapid rate of blood cell renewal makes the hematopoietic system a susceptible target for xenobiotic toxicity, and xenobiotics could interfere with cell proliferation and differentiation. Hematotoxic molecules can affect one or more hematopoietic lineages, leading to blood disorders (neutropenia, agranulocytosis, thrombopenia, anemia, and pancytopenia). Erythropoiesis is the part of hematopoiesis responsible for red blood cell production by proliferation and differentiation of specific erythroblastic progenitors, the BFU-E/ CFU-E (Burst and Colony-Forming Unit-Erythroid). Some drugs and chemicals (antituberculosis drugs, chloramphenicol, penicillamines, ethanol, and lead) are able to cause dysfunction in heme biosynthesis and are responsible for hematological diseases such as porphyrias. The aim of this work was to obtain a useful tool to predict the toxic effect of xenobiotics in the proliferation or erythroblastic progenitors and on their differentiation. Optimal growth conditions for erythroblastic progenitor culture were investigated to define a cell culture model for toxicological investigations that can perform a morphological study of cells as well as an evaluation of cell abilities to synthetize porphyrins and hemoglobin in vitro. Biochemical dosages by spectrophotometry and chromatography have been adapted to this clonogenic assay on cells obtained after BFU-E/ CFU-E proliferation and differentiation. The effects of two pesticides (lindane \[organochlorine insecticide] and oxyfluorfen \[diphenyl-ether herbicide]) and two mycotoxins (T-2 toxin \[trichothecene mycotoxin] and fumonisin B1) were studied on BFU-E/ CFU-E development to validate this extension of clonogenic assay to toxicology.

Protoporphyrinogen Destruction by Plant Extracts and Correlation with Tolerance to Protoporphyrinogen Oxidase-Inhibiting Herbicides

Herbicidal damage by photobleaching diphenylether herbicides is the indirect result of inhibition of an enzyme in chlorophyll biosynthesis. The substrate of the inhibited enzyme, protoporphyrinogen, accumulates and is subsequently converted to protoporphyrin, a potent photoactive compound which causes light-dependent membrane damage. In the present study, we report characteristics of a factor in the soluble fraction of leaves which can decompose protoporphyrinogen to nonporphyrin products. This process may be important in protecting plants from herbicide damage, since it would interfere with accumulation of the phototoxic porphyrin, protoporphyrin. We found that this protoporphyrinogen destruction is associated with the protein fraction of the soluble leaf homogenate, suggesting its enzymatic nature. Protoporphyrinogen destruction is stable to mild heat, but is eliminated by boiling. Protoporphyrinogen destruction is present in the soluble leaf homogenate but is not localized within the stromal fraction of the chloroplast. The reductants dithiothreitol and β-mercaptoethanol, but not glutathione, inhibit protoporphyrinogen destruction at high concentrations. In contrast, ascorbic acid markedly inhibits destruction even at low concentrations, suggesting a role for cellular ascorbic acid in protecting protoporphyrinogen from destruction, thereby enhancing herbicide action. Protoporphyrinogen destruction was least active in young cucumber leaves, a plant highly susceptible to herbicides. Higher levels of protoporphyrinogen destruction were found in leaves of broadleaf mustard and radish, two plants exhibiting herbicide tolerance. For cucumber, the extent of destruction increased with the age of the plant. These findings suggest a correlation between increased protoporphyrinogen destruction and herbicide tolerance in some plant species.

Protoporphyrinogen Oxidase of Myxococcus xanthus: EXPRESSION, PURIFICATION, AND CHARACTERIZATION OF THE CLONED ENZYME

Protoporphyrinogen oxidase (EC 1.3.3.4) catalyzes the six electron oxidation of protoporphyrinogen IX to protoporphyrin IX. The enzyme from the bacterium Myxococcus xanthus has been cloned, expressed, purified, and characterized. The protein has been expressed in Escherichia coli using a Tac promoter-driven expression plasmid and purified to apparent homogeneity in a rapid procedure that yields approximately 10 mg of purified protein per liter of culture. Based upon the deduced amino acid sequence the molecular weight of a single subunit is 49,387. Gel permeation chromatography in the presence of 0.2% n-octyl-beta-D-glucopyranoside yields a molecular weight of approximately 100,000 while SDS gel electrophoresis shows a single band at 50,000. The native enzyme is, thus, a homodimer. The purified protein contains a non-covalently bound FAD but no detectable redox active metal. The M. xanthus enzyme utilizes protoporphyrinogen IX, but not coproporphyrinogen III, as substrate and produces 3 mol of H2O2/mol of protoporphyrin. The apparent Km and kcat for protoporphyrinogen in assays under atmospheric concentrations of oxygen are 1.6 microM and 5.2 min-1, respectively. The diphenyl ether herbicide acifluorfen at 1 microM strongly inhibits the enzyme's activity.

Etiological Studies on the Gallbladder Cancer Clustered in the Niigata Plains.

Geographical distribution of standardized mortality ratio (SMR) for biliary tract cancer (BTC) showed a characteristic clustering pattern; high in the northeastern regions and low in the southwestern regions of Japan. Among the 47 prefectures (corresponding to counties in the U. S.) with high SMRs, Niigata Prefecture has been the highest in both sexes for the last two decades. It was found that the cities, towns and villages in Niigata where the mortalities from BTC were high were correspondent with rice producing areas.In addition, it was revealed that the sources of tap water in the cities with high SMRs in Niigata were commonly big rivers, whereas in those with low SMRs they were either reservoirs located in the mountains, underground water or small river originating from the mountains. Based on these findings, the contamination of tap water by agricultural chemicals form paddy fields was suspected as a cause of the high mortality from BTC. Among several chemicals examined, diphenylether herbicide, chlornitrofen (CNP) and its derivative (CNP-amino) were detected high in tap water in the cities with higher SMRs and they seem to be related to the occurrence of BTC, particularly of female gallbladder cancer.

Synthesis and QSAR of Herbicidal 3-Pyrazolyl α,α,α-Trifluorotolyl Ethers

Pyrazole nitrophenyl ethers (PPEs) were recently identified as a novel class of chemistry exerting herbicidal effects by inhibition of protoporphyrinogen IX oxidase. This area of chemistry has been extended to include herbicidal pyrazolyl fluorotolyl ethers. In these compounds, a trifluoromethyl group substitutes for the 4‘-nitro group found in the original herbicides and in “classical” nitrodiphenyl ether hebicides. Fluoroanisole pyrazole ethers, in which a trifluoromethoxy group replaces the nitro group of diphenyl ether herbicides, are also described. The shift from 4‘-nitro to 4‘-trifluoromethyl substitution, which is conservative in terms of electrostatics, produced a novel class of herbicide with substantial pre-emergent activity on narrowleaf weed species. Quantitative structure/activity relationships obtained with respect to substitution on the pyrazole ring and at the 3‘-position of the fluorotolyl moiety can be summarized effectively by comparative molecular field analysis. In general, 5-methanesulfonyl fluorotoluidide ethers were found to be most active. Keywords: Herbicide; protoporphyrinogen oxidase; structure/activity; QSAR; pyrazole phenyl ether; comparative molecular field analysis; CoMFA

Mutagenicity and Hepatotoxicity of Diphenyl Ether Herbicides (Proceedings of the 21st Symposium on Toxicology and Environmental Health)

Mutagenicity and Hepatotoxicity of Diphenyl Ether Herbicides (Proceedings of the 21st Symposium on Toxicology and Environmental Health)

Cloning, Sequence, and Expression of Mouse Protoporphyrinogen Oxidase

Protoporphyrinogen oxidase (EC 1.3.3.4) is the penultimate enzyme in the heme biosynthetic pathway, catalyzing the six-electron oxidation of protoporphyrinogen to protoporphyrin. A dominantly inherited genetic deficiency in this enzyme results in the disease variegate porphyria. We now report the cloning, sequence, and expression of mouse protoporphyrinogen oxidase. The cDNA for mouse protoporphyrinogen oxidase was obtained by complementation ofEscherichia coliSASX38, a protoporphyrinogen oxidase-deficient strain, with a mouse erythroleukemia (MEL) cell expression library. The sequence of this cDNA along with 5′ untranslated sequence obtained by 5′ rapid amplification of cDNA ends of MEL cell mRNA is 1814 bp in length and contains an open reading frame of 1431 bp. This encodes a protein of 477 amino acid residues with a calculated molecular weight of 50,870. The protein as expressed inE. coliis sensitive to inhibition by the diphenyl ether herbicide acifluorfen. Northern blot analyses of RNA from uninduced and induced MEL cells as well as mouse hepatoma cells all show two major mRNA species of 1.8 and 3.6 kb.

Generation of Resistance to the Diphenyl Ether Herbicide Acifluorfen by MEL Cells

The diphenyl ether herbicide acifluorfen has been shown to act by inhibition of the terminal enzyme of the protoporphyrin biosynthetic pathway, protoporphyrinogen oxidase (E.C. 1.3.3.4) (PPO), in plant and animal cells. In the present study we show that long term maintenance of murine erythroleukemia (MEL) cells in acifluorfen, which is normally toxic to these cells at 5 μM concentration, results in cells that grow at a near normal rate in 100 μM acifluorfen. Acifluorfen resistant cells do not have increased levels of PPO activity, nor does the PPO made by these cells have increased resistance to acifluorfenin, but these cells accumulate porphyrin and have elevated levels of heme. Data is presented that suggests the resistance of these MEL cells to acifluorfen may be attributable to induction of a cytochrome P450(s).

Effect of the Peroxisome Proliferating Diphenyl Ether Herbicide Fomesafen on Liver Porphyrin Biosynthesis and Neoplastic Transformation -- Synergism with Iron Pretreatment

Effect of the Peroxisome Proliferating Diphenyl Ether Herbicide Fomesafen on Liver Porphyrin Biosynthesis and Neoplastic Transformation -- Synergism with Iron Pretreatment

Synthesis of Novel Diphenyl Ether Herbicides

The benzoxazine derivatives are a new chemical family of diphenyl ether herbicides, which exhibit a strong peroxidizing herbicidal activity on mono- and dicotyledonous species in preemergence and postemergence tests. Twenty derivatives were synthesized, and their herbicidal activity was determined to examine structure-activity relationships. Among the compounds investigated, it was found that the fluorine atom introduction into the trifluoromethylbenzene moiety together with an oxazine ring instead of a nitro group led to the most active herbicide.

Herbicidal Activity of UCC-C4243 and Acifluorfen is Due to Inhibition of Protoporphyrinogen Oxidase

Laboratory and greenhouse studies were conducted to determine the mode of action of soil- and foliar-applied UCC-C4243. Experiments demonstrated that UCC-C4243 required light for phytotoxicity, phytotoxic symptoms were similar to inhibitors of porphyrin synthesis such as acifluorfen, and UCC-C4243 potently inhibited protoporphyrinogen oxidase. Germination and emergence of field pennycress and lentil in the dark were not affected by soil-incorporated UCC-C4243 at rates more than 10 times greater than like treatments that killed all plants in the light. Soil-incorporated UCC-C4243 required light for activity and killed seedlings within 1 d after emergence; sublethal doses caused desiccation, veinal necrosis, and leaf deformation. Field pennycress and lentil were susceptible to soil-incorporated UCC-C4243 and acifluorfen in the light, but were 5 to 93 times less sensitive to the herbicides in the dark. Wheat was not affected by either herbicide in the light or dark. Injury symptoms from UCC-C4243 applied POST to redroot pigweed were similar to symptoms from diphenyl ether and bipyridinium herbicides: rapid, light-dependent chlorophyll bleaching, desiccation, and necrosis. UCC-C4243, acifluorfen-methyl, and acifluorfen acid caused light- and concentration-dependent chlorophyll bleaching and electrolyte leakage from cucumber leaf disks (I50= 1.0, 1.8, and 4.3 μM, respectively). An inhibitor of the porphyrin synthesis pathway, 4,6-dioxoheptanoic acid, almost completely inhibited herbicide-induced electrolyte leakage. δ-Aminolevulinic acid, a tetrapyrrole precursor and stimulator of the porphyrin synthesis pathway, caused synergistic effects with each herbicide. Protoporphyrinogen oxidase from barley etioplast preparations was inhibited 50% by 40 nM UCC-C4243. Barley leaf sections treated with 100 μM UCC-C4243 accumulated protoporphyrin IXin vivoto levels > 75 times non-treated controls. These data indicate the light-requiring herbicide activity of UCC-C4243, like acifluorfen, is due to inhibition of protoporphyrinogen oxidase.

Synthesis and Herbicidal Activity of Cyperin

Cyperin is a phytotoxic diphenyl ether natural product. Total synthesis of cyperin has been achieved and its herbicidal activity evaluated. The synthesis is highlighted by an Ullmann coupling to construct the diphenyl ether moiety contained within cyperin. It was found that cyperin inhibited root growth of Cyperus rotundus grown on agar; however, root growth of Cyperus grown in soil was unaffected. Cyperin also inhibited growth of Arabidopsis thaliana end Agrostis palustris grown in agar. The mode of action of cyperin is different from that of commercial diphenyl ether herbicides that inhibit protoporphyrinogen oxidase.

Detection of mutagenicity of diphenyl ether herbicides in Salmonella typhimurium YG1026 and YG1021

Three kinds of diphenyl ether herbicides, 4-nitrophenyl 2,4,6-trichlorophenyl ether (CNP, chlornitrofen), 2,4-dichlorophenyl 3-methoxy-4-nitrophenyl ether (chlomethoxynil) and 2,4-dichlorophenyl 3-methoxycarbonyl-4-nitrophenyl ether (bifenox), were tested for mutagenicity in Salmonella typhimurium YG1026 and YG1021, which have high nitroreductase activity, and also in S. typhimurium TA100 and TA98. CNP and chlomethoxynil showed mutagenicity in S. typhimurium YG1026, without S9 mix, inducing 50 and 304 revertants per μg. These mutagenicities were suppressed by the addition of S9 mix. CNP and chlomethoxynil were also mutagenic to YG1021 with and without S9 mix, and their mutagenicities were lower than those to YG1026. On the other hand, bifenox was mutagenic to YG1026 only with S9 mix, inducing 3.0 revertants per μg. These three herbicides showed no mutagenicity in S. typhimurium TA100 and TA98 either with or without S9 mix.

Porphyrin Stability in Plant Supernatant Fractions: Implications for the Action of Porphyrinogenic Herbicides

Photobleaching diphenyl ether herbicides act by inhibiting the chloroplast and mitochondrial enzyme which converts protoporphyrinogen to protoporphyrin, a step in the porphyrin synthesis pathway. This results in accumulation of the phototoxic compound protoporphyrin and subsequent light-dependent membrane damage. Recent findings suggest that protoporphyrin accumulation is a result of protoporphyrinogen accumulation in the herbicide-inhibited chloroplast followed by export into the cytoplasm and subsequent oxidation to the phototoxic protoporphyrin by herbicide-resistant mechanisms associated with extraorganellar plant membranes. The present study indicates that accumulated protoporphyrinogen can be decomposed to nonporphyrin (and therefore nonphototoxic) products by factors in the soluble fraction of barley and mustard leaves. In contrast, protoporphyrin is quite stable in these fractions. The rate of protoporphyrinogen destruction is dependent upon the amount of soluble leaf fraction present and the time of incubation. Protoporphyrinogen destruction is suppressed in the presence of the reducing agent dithiothreitol (5 m M) and by ascorbic acid (0.5 m M). Boiling of the soluble extract also suppresses protoporphyrinogen destruction. Uroporphyrinogen I is not destroyed by soluble leaf extracts. These findings suggest that the protoporphyrinogen which accumulates outside of the chloroplast upon herbicide treatment may decompose to nonporphyrin (and therefore nontoxic) products if it is not rapidly oxidized to protoporphyrin by herbicide-resistant mechanisms in extraorganellar membranes. Protoporphyrinogen destruction may be a mechanism for providing protection from the toxic affects of protoporphyrin accumulation. Some evidence for this was provided by our observation that older mustard leaves are more active in protoporphyrinogen destruction than younger leaves. Other studies have shown that older weeds are more herbicide tolerant than younger weeds.

Synthesis and properties of a photoaffinity labeling reagent for protoporphyrinogen oxidases, the target enzymes of diphenyl ether herbicides

A diazoketone 3 has been synthesized in two steps from acifluorfen 1, a diphenyl ether herbicide. Like the parent compound 1, the diazoketone 3 is toxic to plant cells and inhibits protoporphyrinogen oxidase, the molecular target of diphenyl ether herbicides. On photolysis of 3 in methanol, the generated carbene mainly undergoes the Wolff rearrangement to a ketene which further adds methanol, but many other products are observed. A tritiated derivative of 3 has been prepared which is suitable for photoaffinity labeling experiments.

Changes in the concentrations of chlornitrofen (CNP) and CNP-amino in river and faucet water in Niigata, Japan

It has been reported that the standardized mortality ratios (SMRs) of biliary tract cancer (BTC) (female gallbladder cancer (GBC) in particular) are high in the Niigata plain and low in other parts of Niigata Prefecture. Based on epidemiological studies, the presence of environmental water pollution by agricultural chemicals is likely to be associated with the occurrence of GBC, and one of the most suspected of these is a diphenylether herbicide, chlornitrofen (CNP). The purpose of the present study was to investigate whether or not there was a difference in concentrations of CNP and its reduction product (CNP-amino) in river and faucet water between Niigata and Joetsu Cities, which are located in the Niigata plain and another part of Niigata, respectively. By employing a new method for CNP and CNP-amino extraction, the detection of CNP and CNP-amino was undertaken by using a gas-chromatograph with an electron-capture detector. The maximum levels of CNP concentrations were 871 and 554 ng/l in river and faucet water, respectively on May 6, 1992 in Niigata City. The concentration of CNP in faucet water was well correlated with that in river water. In Joetsu City, the maximum concentration in river water was 183 ng/l on May 6 and had decreased rapidly towards the background level on July 29, 1992. As to the concentrations of CNP in faucet water in Joetsu City, they fluctuated between the levels of 2 and 9 ng/l. In the case of CNP-amino concentrations in faucet water, a clear-cut difference was evident between the two cities; 384 ng/l in Niigata and under the quantity limit in Joetsu.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of a cloned protoporphyrinogen oxidase.

The previously cloned hem Y gene of Bacillus subtilis (Hansson, M., and Hederstedt, L. (1992) J. Bacteriol. 174, 8081-8093) has been expressed in Escherichia coli. The expressed protein has been shown to be the penultimate enzyme of the heme biosynthetic pathway, protoporphyrinogen oxidase (EC 1.3.3.4) and, thus, the gene designation should be hem G. This represents the first report of the expression of a cloned protoporphyrinogen oxidase from any source. The enzyme is present in the soluble cytoplasmic fraction and is, thus, unlike all previously reported eukaryotic or prokaryotic protoporphyrinogen oxidases, which are membrane-bound. It utilizes molecular oxygen as a terminal electron acceptor, and protoporphyrinogen IX, mesoporphyrinogen IX, and coproporphyrinogen III serve as substrates. The diphenyl ether herbicide acifluorfen, which is a strong inhibitor of the eukaryotic enzyme, is only weakly inhibitory. The enzyme has a predicted molecular weight of 51,200, which corresponds well with molecular weight determination via high performance liquid chromatography and SDS-polyacrylamide gel electrophoresis. In addition the enzyme contains a putative dinucleotide binding region at the amino terminus, which is consistent with the previously demonstrated presence of a flavin moiety in the characterized mammalian enzymes.

Superoxide Dismutase Enhances Tolerance of Freezing Stress in Transgenic Alfalfa (Medicago sativa L.)

Activated oxygen or oxygen free radicals have been implicated in a number of physiological disorders in plants including freezing injury. Superoxide dismutase (SOD) catalyzes the dismutation of superoxide into O2 and H2O2 and thereby reduces the titer of activated oxygen molecules in the cell. To further examine the relationship between oxidative and freezing stresses, the expression of SOD was modified in transgenic alfalfa (Medicago sativa L.). The Mn-SOD cDNA from Nicotiana plumbaginifolia under the control of the cauliflower mosaic virus 35S promoter was introduced into alfalfa using Agrobacterium tumefaciens-mediated transformation. Two plasmid vectors, pMitSOD and pChlSOD, contained a chimeric Mn-SOD construct with a transit peptide for targeting to the mitochondria or one for targeting to the chloroplast, respectively. The putatively transgenic plants were selected for resistance to kanamycin and screened for neomycin phosphotransferase activity and the presence of an additional Mn-SOD isozyme. Detailed analysis of a set of four selected transformants indicated that some had enhanced SOD activity, increased tolerance to the diphenyl ether herbicide, acifluorfen, and increased regrowth after freezing stress. The F1 progeny of one line, RA3-ChlSOD-30, were analyzed by SOD isozyme activity, by polymerase chain reaction for the Mn-SOD gene, and by polymerase chain reaction for the neo gene. RA3-ChlSOD-30 had three sites of insertion of pChlSOD, but only one gave a functional Mn-SOD isozyme; the other two were apparently partial insertions. The progeny with a functional Mn-SOD transgene had more rapid regrowth following freezing stress than those progeny lacking the functional Mn-SOD transgene, suggesting that Mn-SOD serves a protective role by minimizing oxygen free radical production after freezing stress.