Protoporphyrinogen Oxidase Inhibitors Research Articles

Cloning and expression of a Porphyromonas gingivalis gene for protoporphyrinogen oxidase by complementation of a hemG mutant of Escherichia coli.

Porphyromonas gingivalis, a bacterium implicated in periodontal pathogenesis, has a growth requirement for iron protoporphyrin IX. By complementation with a P. gingivalis 381 chromosomal DNA library, we were able to isolate a clone that enhanced the poor growth of a hemG mutant of Escherichia coli. The DNA sequence analysis of this clone revealed three open reading frames (ORFs). ORF3 encoded a protein of 466 amino acids with a calculated molecular weight of 51 695 Da. The deduced amino acid sequence of the ORF3 gene had significant similarity to sequences of protoporphyrinogen oxidase (PPO) from Myxococcus xanthus (30% identical residues). When the ORF3 gene was overexpressed in E. coli, the extract had much higher PPO activity than a control extract, and this activity was inhibited by acifluorfen, a specific inhibitor of PPO. Thus, ORF3 was named PgHemG. Furthermore, several porphyrin-related genes, including hemD, hemN and hemH, were identified in the data bases on the websites available on-line. We postulated that a porphyrin biosynthetic pathway to heme from preuroporphyrin may be conserved in P. gingivalis.

Potato (Solanum tuberosum) Variety and Weed Response to Sulfentrazone and Flumioxazin1

Field and greenhouse studies were conducted to evaluate sulfentrazone and flumioxazin as preemergence (PRE) herbicides for broadleaf weed control in potato. Sulfentrazone and flumioxazin were applied alone and in combination with s-metolachlor to determine the crop response and the weed spectrum controlled. These treatments were compared with metribuzin or rimsulfuron plus s-metolachlor treatments. Potato variety response to sulfentrazone and flumioxazin was evaluated in a separate field study. Sangre, Chipeta, Russet Norkotah, and Russet Nugget were treated with sulfentrazone from 0.14 to 0.28 kg/ha or flumioxazin from 0.035 to 0.07 kg/ha. Sulfentrazone and flumioxazin provided excellent broadleaf weed control at all the rates tested, whereas grass control increased as rate increased. Grass control improved when combined with s-metolachlor. Sulfentrazone and flumioxazin treatments were comparable with metribuzin and rimsulfuron treatments in weed control and total yield. Flumioxazin was safe when applied PRE to four selected varieties, whereas sulfentrazone produced initial phytotoxicity to Sangre and Chipeta at high rates but did not affect yields. Sulfentrazone increased the yield of U.S. No.1 potatoes compared with other treatments in the variety response study. Dose–response curves were used to generate the sulfentrazone, flumioxazin, and metribuzin herbicide rates required to reduce biomass by 50% (I50) for eight common weed species. Herbicides were applied PRE at several rates, and plant response was recorded. Log-logistic analysis was performed on bioassay data generated to estimate species sensitivity to each herbicide. Sulfentrazone reduced the biomass of hairy nightshade, black nightshade, redroot pigweed, kochia, common lambsquarters, and redstem filaree by more than 90% at 0.0175 kg/ha (the lowest rate evaluated), whereas flumioxazin had a similar effect on all broadleaf species except on kochia at 0.004 kg/ha (the lowest rate evaluated). Therefore, it was not possible to calculate I50 or even I80 values for most broadleaf species. Metribuzin I50 values could be calculated for most of the species tested. The metribuzin I50 value for hairy nightshade was 0.28 kg/ha, which was 16 and 70 times higher than the sulfentrazone and flumioxazin rates, respectively, that reduced hairy nightshade biomass by more than 90%. Sulfentrazone and flumioxazin appeared to be sufficiently safe when applied on potato and controlled several weed species common to potato production in the western United States.Nomenclature: Flumioxazin; sulfentrazone; black nightshade, Solanum nigrum L. #3 SOLNI; common lambsquarters, Chenopodium album L. # CHEAL; hairy nightshade, Solanum sarrachoides Sendtner # SOLSA; kochia, Kochia scoparia L. # KOCSC; redstem filaree, Erodium cicutarium L. # EROCI; redroot pigweed, Amaranthus retroflexus L. # AMARE; potato, Solanum tuberosum L. ‘Chipeta’, ‘Russet Norkotah’, ‘Russet Nugget’, ‘Sangre’.Additional index words: Arachis hypogaea, barnyardgrass, crop safety, diquat, ECHCG, Echinochloa crus-galli L., log-logistic analysis, MALNE, Malva neglecta Wallr., Panicum miliaceum L., PANMI, paraquat, Phaseolus lunatus, phytotoxicity, potato weed management, protoporphyrinogen oxidase inhibitors, Setaria viridis L., SETVI, weed dose–response, wild proso millet.Abbreviations: ALS, acetolactate synthase; I50, herbicide rate required to reduce biomass by 50%; OM, organic matter; POST, postemergence; PPO, protoporphyrinogen oxidase; PRE, preemergence; WAT, weeks after treatment.

Effects of Protoporphyrinogen Oxidase Inhibitors on Soybean (Glycine max L.) Response, Sclerotinia sclerotiorum Disease Development, and Phytoalexin Production by Soybean1

Sclerotinia stem rot is an important soybean disease. An increase in phytoalexin production with herbicide treatments may reduce the incidence of this disease in soybean. Research was conducted to determine soybean response, Sclerotinia sclerotiorum lesion development, and phytoalexin production in glyphosate-resistant and -susceptible soybean cultivars treated with protoporphyrinogen oxidase–inhibiting herbicides. Necrosis of soybean leaves 7 d after postemergence application of oxyfluorfen at 17.5 g ai/ha, carfentrazone at 1.8 g ai/ha, sulfentrazone at 9.0 g ai/ha, fomesafen at 280 g ai/ha, acifluorfen at 425 g ai/ha, flumiclorac at 30 g ai/ha, CGA-248757 at 4 g ai/ha, and oxadiazon at 280 g ai/ha was equal to or less than lactofen at 70 g ai/ha. In a detached leaf bioassay, S. sclerotiorum lesion diameter was reduced by oxyfluorfen, carfentrazone, sulfentrazone, lactofen, fomesafen, flumiclorac, and oxadiazon compared with the untreated control. Furthermore, lesion diameter on untreated leaves of soybean treated with oxyfluorfen, carfentrazone, sulfentrazone, lactofen, fomesafen, acifluorfen, flumiclorac, CGA-248757, and oxadiazon was reduced compared with the untreated control. Lactofen and sulfentrazone increased leaf phytoalexin production similarly, but neither herbicide affected stem phytoalexin production compared with the untreated control. Glyphosate-resistant and near-isogenic–susceptible cultivars responded similarly when inoculated with S. sclerotiorum in the detached leaf bioassay. Glyphosate-resistant S20-B9 and P93B01 produced more phytoalexins than glyphosate-susceptible S 19-90 and P9281. Herbicide treatments may increase phytoalexin production in leaves of treated plants, but levels in the stem do not explain protection from Sclerotinia stem rot.Nomenclature: Acifluorfen; carfentrazone; CGA-248757, [[2-chloro-4-fluoro-5-[(tetrahydro-3-oxo-1H,3H-[1,3,4]thiadiazolo[3,4-a]pyridazin-1-yliden)amino]phenyl]thio]acetate (proposed common name, fluthiacet-methyl); flumiclorac; fomesafen; lactofen; oxadiazon; oxyfluorfen; sulfentrazone; soybean, Glycine max (L.) Merr. ‘Novartis S 19-90’ (‘S 19-90’), ‘Novartis S20-B9’ (‘S20-B9’), ‘Great Lakes 2415’ (‘GL2415’), ‘Great Lakes 2600’ (‘GL2600’), ‘Pioneer 9281’ (‘P9281’), ‘Pioneer 93B01’ (‘P93B01’); Sclerotinia stem rot, Sclerotinia sclerotiorum (Lib.) de Bary.Additional index words: Aryl triazinone, cultivar, cyclic imide, diphenyl ether, herbicide resistant, isolines, oxadiazole, phytotoxicity, postemergence, protoporphyrinogen inhibitor, Sclerotinia stem rot.Abbreviations: DAT, days after treatment; NIS, nonionic surfactant; GR, glyphosate resistant; protox, protoporphyrinogen oxidase; TLC, thin-layer chromatography; UAN, 28% urea ammonium nitrate.

Toxicity of 40 Herbicides to the Green Alga Chlorella vulgaris

The effects on the green alga Chlorella vulgaris of 40 herbicides in 19 different chemical structure classes and with 11 dissimilar modes of action were studied through 96-h acute toxicity tests. Experimental results indicated that the average acute toxicity of acetolactate synthase (ALS)-inhibiting herbicides to C. vulgaris was close to those of the acetyl-CoA carboxylase (ACCase)-inhibiting herbicides and the lipid synthesis-inhibiting protoporphyrinogen oxidase (Protox) inhibitor. Their acute toxicities were higher than those of the microtubule process inhibitor, mitotic process inhibitor, the glutamine synthase inhibitors, and 5-enolpyruvyl-shikimate-3-phosphate synthase (EPSP synthase) inhibitors. The acute toxicities of auxin herbicides to C. vulgaris were the lowest among all herbicides tested and that of the photosynthesis-inhibiting herbicides was the highest.

Gene targeting in Arabidopsis.

Precise modification by gene targeting (GT) provides an important tool for studies of gene function in vivo. Although routine with many organisms, only isolated examples of GT events have been reported for flowering plants. These were at low frequencies precluding reliable estimation of targeting efficiency and evaluation of GT mechanisms. Here we present an unambiguous and straightforward system for detection of GT events in Arabidopsis using an endogenous nuclear gene encoding protoporphyrinogen oxidase (PPO), involved in chlorophyll and heme syntheses. Inhibition of PPO by the herbicide Butafenacil results in rapid plant death. However, the combination of two particular mutations renders PPO highly resistant to Butafenacil. We exploited this feature for selection of GT events by introducing the mutations into the PPO gene by homologous recombination. We have estimated the basal GT frequency to be 2.4 x 10(-3). Approximately one-third of events were true GT (TGT) leading to the anticipated modification of the chromosomal PPO copy. The remaining events could be classified as ectopic GT (EGT) arising by modification of vector DNA by the chromosomal template and its random integration into the Arabidopsis genome. Thus the TGT frequency in our experimental setup is 0.72 x 10(-3). In view of the high efficiency of Arabidopsis transformation, GT experiments of a reasonable size followed by a PCR screen for GT events should also allow for modification of non-selectable targets. Moreover, the system presented here should contribute significantly to future improvement of GT technology in plants.

Resistance of a soybean cell line to oxyfluorfen by overproduction of mitochondrial protoporphyrinogen oxidase.

The diphenyl ether herbicide oxyfluorfen (2-chloro-4-trifluoromethylphenyl 3-ethoxy-4-nitrophenyl ether) inhibits protoporphyrinogen oxidase (Protox) which catalyzes the oxidation of protoporphyrinogen IX (Protogen) to protoporphyrin IX (Proto IX), the last step of the common pathway to chlorophyll and haeme biosynthesis. We have selected an oxyfluorfen-resistant soybean cell line by stepwise selection methods, and the resistance mechanism has been investigated. No growth inhibition was observed in resistant cells at a concentration of 10(-7) M oxyfluorfen, a concentration at which normal cells did not survive. While the degree of inhibition of total extractable Protox by oxyfluorfen was the same in both cell types, the enzyme activity in the mitochondrial fraction from non-treated resistant cells was about nine-fold higher than that from normal cells. Northern analysis of mitochondrial Protox revealed that the concentration of mitochondrial Protox mRNA was much higher in resistant cells than that in normal cells. There were no differences in the absorption and metabolic breakdown of oxyfluorfen. The growth of resistant cells was also insensitive to oxadiazon [5-tert-butyl-3-(2,4-dichloro-5-isopropoxyphenyl)-1,3,4-oxadiazol-2-(3H)- one], the other chemical class of Protox inhibitor. Therefore, the resistance of the selected soybean cell line to oxyfluorfen is probably mainly due to the overproduction of mitochondrial Protox.

Effect of Tiagabine and Topiramate on Porphyrin Metabolism in an in vivo Model of Porphyria

Abstract: Administration of many antiepileptic drugs to patients with porphyria can precipitate an acute porphyric crisis. Information on the porphyrogenic activity of new antiepileptic drugs is still limited. In the presented study, the effects of tiagabine and topiramate on porphyrin metabolism were evaluated in an in vivo model of porphyria. Administration of the protoporphyrinogen oxidase inhibitor oxadiazon (12.5 mg/kg/day) for four days to male Wistar rats caused a partial block of porphyrin biosynthesis, thus mimicking the condition of quiescent variegate porphyria. Administration of phenobarbital (75 mg/kg/day) to oxadiazon‐pretreated rats increased liver porphyrin content, liver porphobilinogen content (means 480 nmol/g, control less than 20 nmol/g) and urinary excretion of porphobilinogen (means 1000 μmol/l, control less than 20 μmol/l). Tiagabine (75 mg/kg/day) and topiramate (75 mg/kg/day) increased liver porphobilinogen content (means 33 and 53 nmol/g respectively) and urinary porphobilinogen concentration (240 and 490 μmol/l respectively). Similar results were obtained in oxadiazon‐treated BALB/c mice. In untreated rats, tiagabine and topiramate caused a moderate increase of hepatic pentoxyresorufin‐O‐dealkylase activity (approximately 100 and 200 pmol/min./mg respectively, controls 15 pmol/min./mg). These data demonstrate that administration of tiagabine or topiramate to oxadiazon‐treated animals can provoke a condition resembling an acute porphyric attack and suggest that administration of these drugs to patients with suspected porphyria should be avoided. However, 5‐day administration of both tiagabine and topiramate (75 mg/kg) is considerably less porphyrogenic than phenobarbital administered at the same dose.

Effect of tiagabine and topiramate on porphyrin metabolism in an in vivo model of porphyria.

Administration of many antiepileptic drugs to patients with porphyria can precipitate an acute porphyric crisis. Information on the porphyrogenic activity of new antiepileptic drugs is still limited. In the presented study, the effects of tiagabine and topiramate on porphyrin metabolism were evaluated in an in vivo model of porphyria. Administration of the protoporphyrinogen oxidase inhibitor oxadiazon (12.5 mg/kg/day) for four days to male Wistar rats caused a partial block of porphyrin biosynthesis, thus mimicking the condition of quiescent variegate porphyria. Administration of phenobarbital (75 mg/kg/day) to oxadiazon-pretreated rats increased liver porphyrin content, liver porphobilinogen content (means 480 nmol/g, control less than 20 nmol/g) and urinary excretion of porphobilinogen (means 1,000 micromol/l, control less than 20 micromol/l). Tiagabine (75 mg/kg/day) and topiramate (75 mg/kg/day) increased liver porphobilinogen content (means 33 and 53 nmol/g respectively) and urinary porphobilinogen concentration (240 and 490 micromol/l respectively). Similar results were obtained in oxadiazon-treated BALB/c mice. In untreated rats, tiagabine and topiramate caused a moderate increase of hepatic pentoxyresorufin-O-dealkylase activity (approximately 100 and 200 pmol/min./mg respectively, controls 15 pmol/min./mg). These data demonstrate that administration of tiagabine or topiramate to oxadiazon-treated animals can provoke a condition resembling an acute porphyric attack and suggest that administration of these drugs to patients with suspected porphyria should be avoided. However, 5-day administration of both tiagabine and topiramate (75 mg/kg) is considerably less porphyrogenic than phenobarbital administered at the same dose.

Phytotoxic Response and Yield of Soybean (Glycine max) Varieties Treated with Sulfentrazone or Flumioxazin1

Flumioxazin and sulfentrazone were compared for phytotoxicity on 15 soybean varieties in a greenhouse study and four varieties in the field. In the greenhouse, injury from sulfentrazone was greater overall than with flumioxazin, 10% compared with 1%, respectively, when averaged across varieties at the labeled rates. The varieties P9552, P91B01, P9362, and P9305 were more sensitive to sulfentrazone than the 11 other varieties on the basis of visible injury and plant height reductions. Sulfentrazone at 224 g ai/ha (1× rate) reduced plant height 23 to 53% and caused 18 to 38% visible injury on the four sensitive varieties. Visible injury symptoms consisted of stunting, crinkling of leaves, and chlorotic spots. Varieties P9305, P9306, P9352, and P9362 were compared in a field study with flumioxazin at 105, 210, and 420 g ai/ha or sulfentrazone at 224, 448, and 896 g ai/ha, corresponding to 1, 2, and 4× labeled rates. An untreated control of each variety was included for comparison. Contrary to the results of the greenhouse studies, injury from flumioxazin was similar to that from sulfentrazone in the field experiments. In 1998, emergence counts were reduced 19 to 52% with flumioxazin and 27 to 73% with sulfentrazone at the 1× rate. Visible injury and stand count reductions resulting from both herbicides were also apparent in that year. Emergence count reductions, stand count reductions, and visible injury resulting from the herbicides were less severe in 1999. At the 1× rate of each herbicide, yields were not affect in either year, except P9305 treated with sulfentrazone in 1999. At the 4× rate, P9305 was most sensitive to sulfentrazone, as indicated by a 53 and 23% reduction yield in 1998 and 1999, respectively. Avoidance of sensitive varieties will reduce the potential for injury and yield reduction with sulfentrazone, and maybe flumioxazin.Nomenclature: Flumioxazin, 2-[7-fluoro-3,4-dihydro-3-oxo-4-(2-propynl)-2H-1,4 benzoxazin-6-yl[-4,5,6,7-tetrahydro-1H-isoindole-1,3(2H)-dione; sulfentrazone, N-[2,4-dichloro-5-[4-(difluoromethyl)-4,5-dihydro-3-methyl-5-oxo-1H-1,2,4-triazol-1-yl]phenyl] methanesulfonamide; soybean, Glycine max (L.).Additional index words: Protoporphyrinogen oxidase inhibitors, differential herbicide tolerance.Abbreviations: DAE, days after emergence; DAP, days after planting; DAT, days after treatment; PPO, protoporphyrinogen oxidase; PRE, preemergence.

A key role for the mitochondrial benzodiazepine receptor in cellular photosensitisation with δ-aminolaevulinic acid

The aim of this study was to determine the part played by the mitochondrial benzodiazepine receptor in cellular photosensitisation with the protoporphyrin IX precursor, δ-aminolaevulinic acid. Evaluation of the δ-aminolaevulinic acid-concentration dependence and kinetics of fluorescent protoporphyrin IX accumulation in monolayers of rat AR4 2J pancreatoma cells established a basis for assessing pharmacological modulation of the biosynthetic pathways for protoporphyrin IX production and photocytotoxicity. Iron chelation enhanced the accumulation of photo-active protoporphyrin IX whereas 1-(2-chlorophenyl)- N-methyl- N-(1-methylpropyl)-3-isoquinolinecarboxamide (PK11195), dipyridamole, or 7-(dimethylcarbamoloxy)-6-phenylpyrrolo-[2,1- d]benzothiazepine (DPB), competitive ligands of the mitochondrial benzodiazepine receptor, all diminished protoporphyrin IX accumulation, as did acifluorfen, a mitochondrial protoporphyrinogen oxidase inhibitor. In addition to protoporphyrin IX (Em max: 630 nm), δ-aminolaevulinic acid-treated cells also generated a fluorophore of Em max 580 nm; this compound was identified as Zn-protoporphyrin IX. Mitochondrial benzodiazepine receptor ligands increased the formation of the zinc porphyrin whilst decreasing that of protoporphyrin IX. The involvement of the mitochondrial benzodiazepine receptor in the translocation of porphyrins and the formation of Zn-protoporphyrin IX have wide implications for the use of δ-aminolaevulinic acid in photodynamic therapy.

New generation of protox-inhibiting herbicides

Benzfendizone belongs to the 2-[(4-heterocyclic-phenoxymethyl)-phenoxy]alkanoate class of herbicides which are potent inhibitors of the plant enzyme protoporphyrinogen oxidase (Protox). Benzfendizone, when applied postemergence, provides control of a number of key grass and broadleaf weeds in orchards and no-till crop situations. Benzfendizone can also be used as a potato desiccant and cotton defoliant. Crops such as soyabean and corn showed no injury when planted 2–3 days following postemergence application of benzfendizone.

Light-dependent herbicides: an overview

Activities of a surprisingly large number of herbicide families are directly or indirectly influenced by light. Herbicide mechanisms that are dependent on light or are enhanced by light fall into several different categories. These herbicides inhibit electron flow in photosystem II in the photosynthetic light reaction (e.g., triazines, phenyl ureas, and uracils), capture electrons in photosystem I in the photosynthetic light reaction (e.g., bipyridiniums such as paraquat), inhibit glutamine synthetase in the nitrogen assimilation pathway (e.g., glufosinate), inhibit protoporphyrinogen oxidase during chlorophyll biosynthesis (e.g., diphenyl ethers [DPEs] and N-phenyl heterocycles), directly inhibit carotenoid biosynthesis by inhibiting one of the desaturase enzymes (e.g., norflurazon and fluridone), or indirectly inhibit carotenoid biosynthesis by inhibiting a quinone cofactor involved in the desaturase reaction (e.g., triketones and isoxazoles). Surprisingly, in all instances the involvement of light in herbicide action is not a direct interaction of light and herbicide at the primary site of action. In addition, the events that cause tissue damage (necrosis) are not associated with the primary target and are always due to membrane damage caused by lipid peroxidation of polyunsaturated fatty acids.

Mitotic Index and Cell Proliferation Kinetics as Additional Variables for Assessment of Genotoxic Effect of The Herbicide Modown

·ivikova K. , J . Dianovsk : Mitotic Index and Cell Proliferation Kinetics as Additional Variables for Assessment of Genotoxic Effect of The Herbicide Modown. Acta Vet. Brno 2000, 69: 45–50. The in vitro effect of the herbicide Modown (with active component bifenox) was tested for the ability to influence cell proliferation of PHA-stimulated bovine peripheral lymphocytes. Mitotic (MI) and proliferation (PI) indices were determined as an alternative for the screening of the cytostatic activity. The herbicide Modown exerted a clear effect on the inhibition of MI and PI over a concentration-tested range of 25 μg/ml to 1000 μg/ml. An expressive proliferation delays was found after the treatment with herbicide at a dose of 250 μg/ml (P < 0.001), while the higher doses of 500 and 1000 μg/ml had caused nearly complete mitotic inhibition in each donor (P < 0.05 and P < 0.001, respectively). A correlation between the PI and MI inhibition refers rather to cytostatic than cytotoxic effects of the herbicide. The results support the possibility of immunosuppression by herbicide exposure. Herbicide Modown, bovine peripheral lymphocytes, cell proliferation, cytostatic effect Nitrodiphenylether herbicides are persistent and lipophilic environmental contaminants may contribute to the impairment of animal health and production. The presence of pesticide residues has been demonstrated in raw bovine milk and in different domestic animal tissues (Lioi et al. 1998). All nitrodiphenylether herbicides exert their phytotoxic activity in chlorophyll of plants by the reduction itself to radical species, which initiate destructive reactions in membrane lipids, leading to the cell leakage (Corbet t et al. 1984). Members, as acifluorfen and oxyfluorfen, e.g. are extremely potent inhibitors of protoporphyrinogen oxidase, a membrane–bound enzyme involved in the heme and chlorophyll biosynthesis pathways (Camadro et al. 1995). The later was studied for the induction of haematological diseases in human erythroid progenitor cells but a cytotoxic effect had been seen only at very high concentrations (Rio et al. 1997). Bifenox is a selective herbicide used in control of annual broad-leaved weeds and some grasses, e. g. in cereals, maize, soya beans, rice and other crops. J inno et al. (1999) described the cytotoxic and porphyrogenic effects of bifenox in rat hepatocytes. They found the maximum porphyrin accumulation at 0.25 mM of the herbicide and an inhibion of protoporphyrogen oxidase, resulting in the accumulation of protoporphyrin IX. Francis et al. (1999) evaluated maternal and developmental toxicity of ten diphenylether herbicides (including bifenox) in mice. They found a correlation among the position of chlorine substituents and the potential for inducing prenatal and postnatal syndroms. But no prenatal and postnatal embryotoxicity was shown. ACTA VET. BRNO 2000, 69: 45–50 Address for correspondence: RNDr. Katarina ·ivikova, CSc. Department of Veterinary Genetics, University of Veterinary Medicine Komenskeho 73, 041 81 Ko‰ice, Slovak Republic Phone: +421 95 63 321 11, (ext. 482) Fax: +421 95 63 236 66 E-mail: sivikova@uvm.sk http://www.vfu.cz/acta-vet/actavet.htm

Positional Isomers of Aryloxyphenyl Cyanurates Have Different Sites of Action

Site of action of aryloxyphenyl cyanurate analogues was found to vary significantly depending upon the placement of cyanurate moiety. Plants treated with the analog having the cyanuric acid in the para-position showed chlorosis and took 2–3 days before the first necrotic symptoms appeared. Additional tests indicated that this compound is a strong photosystem II inhibitor. In contrast, plants treated with the analog having cyanuric acid in the meta-position exhibited necrosis within a few hours after treatment. Further tests revealed that this compound is a potent inhibitor of protoporphyrinogen oxidase. Therefore, depending upon the placement of cyanuric acid, the site of action of these herbicidally active compounds changes substantially. These results also show that aryloxyphenyl cyanurates are new classes of PSII and PROTOX inhibitors.

Differential Susceptibilities of Wheat and Barley to Diphenyl Ether Herbicide Oxyfluorfen

Wheat is known to be relatively tolerant to diphenyl ether herbicides. Growth and physiological responses of wheat to diphenyl ether herbicide oxyfluorfen were examined in comparison with those of oxyfluorfensusceptible barley. Compared to barley, wheat was significantly less susceptible to the herbicide with preemergence and postemergence treatments. The differential susceptibilities of wheat and barley to the herbicide were more apparent with postemergence than with preemergence treatment. The effects of the herbicide on causing cellular leakage, chlorophyll loss, and lipid peroxidation were much lower in wheat than in barley leaves. Protoporphyrin IX accumulated in both wheat and barley leaves treated with oxyfluorfen, in a concentration-dependent manner. However, the magnitude of the protoporphyrin IX accumulation was much lower in wheat than in barley leaves treated with oxyfluorfen at a concentration of higher than 0.33 μM. Protoporphyrinogen oxidase from wheat leaves was found to be less susceptible to oxyfluorfen than that from barley leaves. The I50 concentrations of oxyfluorfen on protoporphyrinogen oxidase activity were approximately 0.2 and 0.06 μM for wheat and barley etioplasts, respectively. Whereas the inhibition of protoporphyrinogen oxidase in barley etioplasts increased with increasing concentration of oxyfluorfen, protoporphyrinogen oxidase in wheat etioplasts was not further inhibited beyond 0.33 μM oxyfluorfen concentrations. In addition, both plant species similarly responded to superoxide anion-generating paraquat and to singlet oxygen-generating rose bengal, implying that the relative tolerance of wheat to oxyfluorfen is not due to protective mechanism against active oxygen species. Our results suggest that the differential susceptibilities of wheat and barley to oxyfluorfen are at least partly due to the differential inhibition of protoporphyrinogen oxidase by the herbicide, and thereby wheat is relatively tolerant to the herbicide.

Structural replacements for the benzoxazinone protox inhibitors

Substituted benzoxazinones are a class of highly active inhibitors of protoporphyrinogen oxidase (protox) which are effective at controlling grass and broadleaf weeds at low rates. As part of the process of optimization of the herbicidal activity of the 6-heterocyclic benzoxazinones, a number of replacements were prepared for the oxazinone ring. Quantitative structure-activity relationships (QSAR) were developed for the benzoxazinone replacements using molecular properties, and these were compared to published QSAR for the acyclic 2,4,5-trisubstituted arylheterocyclic protox inhibitors. That the molecular properties of the acyclic and bicyclic protox inhibitors are similar suggest they may be interacting with the same binding site.

Cytotoxic and porphyrinogenic effects of diphenyl ethers in cultured rat hepatocytes: chlornitrofen (CNP), CNP-amino, chlomethoxyfen and bifenox

We studied the cytotoxic and porphyrinogenic effects of four diphenyl ethers (DPEs), chlornitrofen (CNP), CNP-amino, chlomethoxyfen and bifenox, in rat hepatocytes cultured on Matrigel. Cytotoxicity was determined as a decrease in viability measured by the release of lactate dehydrogenase. Of the DPEs examined, CNP-amino was the most cytotoxic, with an LC 50 value of 0.36 m m (95% confidence interval, 0.33–0.40 m m). CNP also reduced the viability in a concentration-dependent manner at the concentrations of 0.50 m m or above. In contrast, no concentration-dependent decrease in viability was observed in the chlomethoxyfen- and bifenox-treated hepatocytes at the concentrations up to 1.0 m m. To identify the enzyme involved in the metabolic activation of CNP-amino, inhibition studies were carried out using SKF 525-A (0.050 m m) and methimazole (1.0 m m). SKF 525-A, a cytochrome P450 inhibitor, quickened the onset of cell killing by CNP-amino, while methimazole, an inhibitor of flavin-containing monooxygenase (FMO), partially suppressed the cytotoxicity of CNP-amino. These results suggest that FMO plays an important role in the cytotoxicity induced by CNP-amino, while cytochrome P450 participates in the detoxification, possibly via the ring-hydroxylation. The maximum porphyrin accumulation was observed at 0.13 m m for chlomethoxyfen (18-fold) and at 0.25 m m for CNP and bifenox (17- and 21-fold, respectively). In contrast to these DPEs, the porphyrinogenic effect of CNP-amino was weak, with the maximum accumulation at 0.13 m m (at least fivefold). The predominant species was protoporphyrin IX in all of the DPE-treated cultures. These results suggest that all of the DPEs examined, possibly including CNP-amino, inhibit protoporphyrinogen oxidase, resulting in the accumulation of protoporphyrin IX.

Molecular characterization of photomixotrophic tobacco cells resistant to protoporphyrinogen oxidase-inhibiting herbicides

Peroxidizing herbicides inhibit protoporphyrinogen oxidase (Protox), the last enzyme of the common branch of the chlorophyll- and heme-synthesis pathways. There are two isoenzymes of Protox, one of which is located in the plastid and the other in the mitochondria. Sequence analysis of the cloned Protox cDNAs showed that the deduced amino acid sequences of plastidial and mitochondrial Protox in wild-type cells and in herbicide-resistant YZI-1S cells are the same. The level of plastidial Protox mRNA was the same in both wild-type and YZI-1S cells, whereas the level of mitochondrial Protox mRNA YZI-1S cells was up to 10 times the level of wild-type cells. Wild-type cells were observed by fluorescence microscopy to emit strong autofluorescence from chlorophyll. Only a weak fluorescence signal was observed from chlorophyll in YZI-1S cells grown in the Protox inhibitor N-(4-chloro-2-fluoro-5-propagyloxy)-phenyl-3,4,5, 6-tetrahydrophthalimide. Staining with DiOC6 showed no visible difference in the number or strength of fluorescence between wild-type and YZI-1S mitochondria. Electron micrography of YZI-1S cells showed that, in contrast to wild-type cells, the chloroplasts of YZI-1S cells grown in the presence of N-(4-chloro-2-fluoro-5-propagyloxy)-phenyl-3,4,5, 6-tetrahydrophthalimide exhibited no grana stacking. These results suggest that the herbicide resistance of YZI-1S cells is due to the overproduction of mitochondrial Protox.

Comparative Molecular Field Analysis (CoMFA) of Herbicidal Protoporphyrinogen Oxidase Inhibitors using Standard Steric and Electrostatic Fields and an Alternative LUMO Field

A set of diphenyl ether herbicides [1] was examined with Comparative Molecular Field Analysis (CoMFA) using standard steric and electrostatic fields and alternative frontier orbitals as 3-D fields to explain observed Protoporphyrinogen oxidase (PPO) enzyme inhibition. Significant CoMFA models were obtained utilizing standard CoMFA and the LUMO field both together and separately. These findings support previous QSAR work that identified several electronic properties as important for PPO inhibition. The resulting CoMFA models identify specific 3-D steric and electronic interactions affecting PPO enzyme binding most significantly.

Selectivity and mode of action of carfentrazone-ethyl, a novel phenyl triazolinone herbicide

Post-emergence application of carfentrazone-ethyl at rates as low as 2.2 g ha -1 caused greater leaf injury and growth reduction in ivyleaf morningglory (Ipomoea hederacea) and velvetleaf (Abutilon theophrasti) than in soybean (Glycine max). The herbicide was more rapidly metabolized in the crop than in the weed species, with 26.7, 54.3 and 60.6% of the parent compound remaining in soybean, ivyleaf morningglory and velvetleaf, respectively, 24 h after exposure. The free acid metabolite, carfentrazone, was present in all species and accounted for 21.2-27.4% of the total radioactivity. Unknown metabolites (R f 0 and 0.22) were four to five times more abundant in soybean than in the weed species. Carfentrazone-ethyl induced more leakage from leaf discs from the weeds than those from soybean and the degree of injury correlated with the amount of protoporphyrin IX (Proto IX) present in the treated tissues. Both carfentrazone-ethyl and carfentrazone were potent inhibitors of protoporphyrinogen oxidase (Protox). Therefore, the selectivity of this herbicide may, at least in part, be attributed to the lower accumulation of Proto IX in soybean than in the weeds, probably because of the ability of soybean to metabolize more carfentrazone into unknown metabolites than the weeds.