William P. Novitzky

Selective inhibition of cytochrome P450 isozymes by the herbicide synergist tridiphane

Abstract Tridiphane [2-(3,5-dichlorophenyl)-2-(2,2,2-trichloroethyl)oxirane] cross-reacts as a synergist to both herbicides and insecticides. As reported herein, tridiphane inhibited the monooxygenase activity of one or more mouse hepatic cytochrome P450 isozymes. With hepatic microsomes isolated from mice pretreated with phenobarbitol (PB) as well as with purified P450 enzymes, tridiphane efficiently inhibited demethylase and deethylase activities catalyzed by PB-induced P450 enzymes, i.e., I 50 values of approximately 4.0 μ M . However, tridiphane was a weak inhibitor of enzyme activities catalyzed by hepatic microsomes isolated from 3-methylcholanthrene (3-MC)-pretreated mice. Type I binding spectra were obtained with hepatic microsomes isolated from uninduced mice ( K s = 12.6 μM ) and PB-induced mice ( K s = 1.0 μM ), and with a purified enzyme isolated from PB-induced mice ( K s = 0.12 μM ). However, tridiphane did not form difference spectra with microsomes isolated from 3-MC-treated mice or with a purified P450 enzyme isolated from 3-MC-treated mice. Inhibition of p -nitroanisole O -demethylase suggested that tridiphane acted as a competitive inhibitor of enzyme activity and produced a K m of 440 μ M with a K i between 0.72 and 1.04 μ M .

Effect of fungal damage on seed composition and quality of soybeans

Fungal damage caused by pathogens such asFusarium, Cercospora, andPhomopsis can have a devastating impact on physical quality and farm price of soybeans. In some price-discount schedules, soybeans may be rejected with as low as 5% fungal damage. Although the severity of this problem varies throughout the United States, millions of bushels of fungus-damaged soybeans may be destroyed annually due to a lack of markets. The effect of fungal damage on seed composition was evaluated to assess potential utility of highly damaged soybeans. Graded samples of the cv. Centennial soybean were dried to 10% moisture and blended on a proportional weight basis to derive a series of treatments from 0 to 80% fungal damage. A positive correlation was found between fungal damage and both protein and oil concentrations. This condition was attributed to loss of residual seed mass. As a result, the protein concentration of defatted meal increased from ca. 54 to 66% over the range of 0 to 80% fungal damage. Mycotoxin contamination appeared to be insignificant in these high-protein meals. Fixed colors in bleached, alkali refined oils were intensified by heat treatment prior to extraction. No significant differences, however, were noted in total polar lipid content, phospholipid, or tocopherol composition among treatments of up to 20% fungal damage. Oils from treatments of more than 40% fungal damage were more severely oxidized and could not be degummed effectively. These data suggest that fungus-damaged soybeans may be blended with high-quality soybeans to alleviate the chemical symptoms associated with unacceptable product quality. Thus, through various blend ratios, processors may consider using fungus-damaged soybeans to gain economic advantage, especially when high-quality soybeans have lower protein concentration.

Interference by flavone and flavonols with chloroplast-mediated electron transport and phosphorylation

Abstract The effects of flavone and seven flavonols on the light-induced electron transport and phosphorylation of isolated spinach ( Spinacia oleracea L.) chloroplasts were investigated. With the exception of flavonol (3-hydroxyflavone), all of the compounds interacted with components of both the ATP-generating and electron transport pathways. Flavonol only interacted with the phosphorylation pathway. Interference with the phosphorylation pathway was evidenced by the greater sensitivity of the phosphorylation reaction than coupled whole-chain electron transport, inhibition of cyclic phosphorylation, inhibition of the light-activated Mg 2+ -ATPase, and inhibition of the heat-activated Ca 2+ -ATPase associated with CF 1 . The overall decreasing order of effectiveness for inhibition of cyclic phosphorylation was: galangin > quercetin = kaempferol = myricetin = flavonol > fisetin > flavone > morin. On the electron transport pathway, all of the compounds, except flavonol, interacted with the Q B -protein complex as, evidenced by inhibition of uncoupled electron transport, alteration of chlorophyll fluorescence transients, and competitive displacement of previously bound radiolabeled atrazine [2-chloro-4-(ethylamino)-6-(isopropylamino)- s -triazine]. The decreasing order of effectiveness for inhibition of uncoupled electron transport was: fisetin > quercetir = galangin > kaempferol > flavone > morin.

Interference by luteolin, quercetin, and taxifolin with chloroplast-mediated electron transport and phosphorylation

SummaryThe effects of luteolin, quercetin, and taxifolin on light induced phosphorylation and electron transport in isolated, greenhouse-grown, spinach (Spinacia oleracea L.) thylakoids were investigated. Luteolin and quercetin interacted with components associated with both the ATP-generating pathway and the electron-transport pathway. However, the action of taxifolin involved only the phosphorylation pathway. Interference with the phosphorylation pathway was evidenced by the greater sensitivity of phosphorylation than oxygen uptake in coupled whole-chain electron transport, inhibition of the light-activated Mg2+-ATPase, and inhibition of the Ca2+-ATPase associated with CF1. The following order of decreasing inhibitory effectiveness was exhibited: luteolin > quercetin >>> taxifolin. On the electron-transport pathway, luteolin and quercetin interfered with the activity of the QB-protein complex as evidenced by inhibition of the partial reaction with diphenylcarbazide as the electron donor and 2,6-dichlorophenolindophenol as electron acceptor; alteration of the chlorophyll fluorescence transients; and competitive displacement of radiolabeled atrazine [2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine].

Metabolism of metolachlor by a microsomal fraction isolated from grain sorghum (Sorghum bicolor) shoots.

A microsomal fraction isolated from the shoots of 3- to 4-day-old, dark-grown, grain sorghum (Sorghum bicolor cv. Funk G 522 D R ) seedlings was characterized. The preparations had a cytochrome P-450 content that varied from approximately 90 to 150 pmol P-450/mg protein with cytochrome P-420 varying from 0 to 3% of the P-450 content. Type I difference spectra were formed with cinnamic acid and metolachlor, and a type II spectrum was formed with tetcyclacis. In short-term assays with [14C]metolachlor as substrate, the preparations produced a single time-dependent product that separated on silica gel TLC plates developed in benzene/acetone (2:1, v/v). RF values for metolachlor and the metabolite were approximately 0.70 and 0.48, respectively. The microsomal reaction required N A D P H and oxygen, and was inhibited by carbon monoxide, with the inhibition being partially reversed by actinic light. Compounds known to inhibit the activity of cytochrome P-450 monooxygenases (piperonyl butoxide, tetcyclacis, and tridiphane) also prevented formation of the metabolite. Identity of the metabolite was confirmed by TLC and positive ion thermospray LC/MS to be 2-chloro-N-(2-ethyl-6- methylphenyl)-N-(2-hydroxy-l-methylethyl)acetamide. Hence, the reaction catalyzed by the sorghum microsomes involved O-demethylation of the methoxypropyl side chain of metolachlor.

Effects of inhibitors and herbicides on the membrane potential of mung bean mitochondria

Abstract Alterations imposed by herbicides on the membrane potential (Δψ), oxygen utilization, and ATP synthesis of intact mung bean mitochondria were measured under state 3 conditions. Effects were correlated with changes imposed by classical electron transport inhibitors, energy transfer inhibitors, and uncouplers. In the dose-response studies, complete inhibition of ATP synthesis produced by electron transport inhibitors (rotenone, antimycin A, KCN), uncouplers [bis(hexafluoroacetonyl)acetone (1799) and carbonyl cyanide 4-trifluoromethoxyphenylhydrazone (FCCP)], and the herbicides was associated with a decrease in Δψ from the state 3 value of 126 mV to between 90 and 100 mV. In contrast, the complete inhibition of phosphorylation produced by the energy transfer inhibitor N,N′-dicyclohexylcarbodimide correlated with an increase in Δψ from the state 3 to the state 4 potential (145 mV). In the titrations, the herbicides and classical uncouplers, but not the electron transport inhibitors, progressively collapsed Δψ below the potential associated with the complete inhibition of phosphorylation (to the apparent Donnan potential of 60 mV). The herbicides could be placed into two groups according to the dose-response relationships exhibited with respect to Δψ and oxygen utilization. The first group, designated as dinoseb types (dinitrophenols, benzimidazoles, benzonitriles, thiadiazoles, and bromofenoxim), uncoupled phosphorylation and collapsed Δψ to the Donnan level before oxygen utilization was inhibited. These compounds possess dissociable protons and are postulated to act as protonophores, much like 1799 and FCCP. With the second group, termed dicryl types (acylanilides, dinitroanilines, diphenylethers, bis-carbamates, and perfluidone), collapse of Δψ was paralleled by uncoupling of phosphorylation and inhibition of oxygen utilization. However, phosphorylation was inhibited to a greater extent than was respiration. The dicryl-type herbicides are not classical-type protonophores. Some of their action can be attributed to interference with the redox pumps. The complete collapse of Δψ to the Donnan potential is associated with alterations and perturbations induced in the membranes by classical uncouplers and by both types of herbicides. The perturbations are postulated to increase the permeability of the membranes to protons and other cations and to induce unfavorable conformational changes that impede interactions between redox enzymes. Conceivably, the combined responses collapse Δψ and inhibit electron transport.

Interference by herbicidal inhibitors of electron transport with phosphorylation and permeability properties of chloroplast membranes

Many herbicides inhibit chloroplast electron transport by interfering with a proteinaceous component of the Qв complex located in the appressed granal membrane. Certain of these herbicides, designated inhibitory uncouplers, also interfere with photophosphorylation and affect other chloroplast-mediated responses, some of which involve components located in the nonappressed granal membrane. The inhibitory uncouplers can be divided into dinoseb (phenolic) types which contain dissociable protons and dicryl (acylanilide) types which are nonionic. The dinoseb types can function as protonophores and shuttle protons across the thylakoid membrane at low concentrations and can alter the integrity of semipermeable membranes at higher concentrations. However, the dicryl types only alter the integrity of the membranes. The inhibitory uncouplers, but not the DCMU-types of electron transport inhibitors: stimulated electron trans- port from DPIPH2 to methyl viologen; inhibited valifiomycin-induced swelling of intact chloroplasts; increased the permeability of the chloroplast envelope to K+ in the absence of an ionophore; prevented energization of the thylakoid membrane by PS I; and increased the permeability of phosphatidyl choline liposomes to protons. Chlorination response patterns obtained with isomers of N-phenyl-2-methylpentanamides in the above reactions, in general, were similar for interference with the Qв complex, i.e., in all assays, dichlorination in the 3,4 or 3,5 positions was associated with maximum inhibitory potency, whereas substitution in an ortho position decreased inhibitory activity. With a series of 1-alkyl-3-(α,α,α-trifluoro-m-tolyl)ureas, maximum inhibition of electron transport was obtained with the butyl derivative, whereas maximum responses for uncoupling and membrane disturbances were obtained with the hexyl or octyl derivatives. Some of the interferences produced by inhibitory uncouplers may result from interactions with the lipoidal components of chloroplast membranes.

Interference by DDT and cyclodiene types of insecticides with chloroplast-associated reactions

The effects of DDT, some of its analogs, and selected cyclodiene insecticides on isolated spinach (Spinacea oleracea L.) thylakoids were identified, characterized, and compared to responses induced by selected herbicides. Except for endrin, the insecticides inhibited light-induced electron transport, altered chlorophyll fluorescence transients, and competitively displaced [14C]atrazine [2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine], a known photosystem II inhibitor, from the membranes. The insecticides appeared to act at, or near B, the secondary electron acceptor of photo-system II. Binding of DDT and dieldrin was estimated at 900 and 2200 molecules, respectively, per photosynthetic unit (490 chlorophyll molecules). The insecticides also inhibited valinomycin-induced swelling of the thylakoid membrane. Whereas inhibition of electron transport can be attributed to interaction by the insecticides with a proteinaceous component of the thylakoid membrane, interference with the action of valinomycin may involve interaction with lipoidal constituents of the membrane.

Interference by Herbicides with the Transmembrane Potential of Thylakoids

Interferences expressed by herbicides classified as inhibitory uncouplers were measured on the induction and maintenance of ΔpH and ΔΨ, the chemical and electrical components, respectively. of the proton motive force (pmf) generated by light-induced cyclic electron transport in spinach thylakoids. Maintenance of the pmf is required for the synthesis of ATP. The inhibitory uncouplers arc known to inhibit photophosphorylation, but the mechanisms involved remain to be identified. The dinoseb types (dinitrophenols. benzimidazoles. benzonitriles. bromophenoxim. perfluidone. thiadiazoles) of inhibitory uncouplers, most of which contain dissociable protons, were found to discharge ΔpΗ at low concentrations and to collapse ΔΨ at high concentrations. Collapse of ΔpΗ can be attributed to the protonophoric (proton shuttling) action of the herbicides. However, collapse of ΔΨ can be caused by alterations induced to the integrity and loss of semipermeability of the thylakoid membrane. As a result the membrane bccomes permeable to protons and other cations, and the electrical charges across the membrane are neutralized. The non-ionic dicryl types of inhibitory uncouplers (acylanilides. dinitroanilines. diphenylethcrs. bis-carbamates) collapsed ΔΨ at concentrations that were somewhat lower than those required for the collapse of ΔpΗ. These herbicides appear only to alter the integrity and permeability of the thylakoid membrane. Inhibition of photophosphorylation by the inhibitory uncouplcrs correlated with their ability to dissipate the pmf.

Effects of Inhibitors on the Membrane Potential (Δψ) of Mung Bean Mitochondria

Safranine O was used to measure changes imposed on the membrane potential (Δψ) of mung bean mitochondria by different metabolic conditions and inhibitors. Changes in Δψ were correlated with simultaneously made measurements of O2 uptake. Δψ values of 129 ± 3 mV and 144 ± 4 mV were obtained under state 3 and state 4 conditions, respectively, during the oxidation of malate. At concentrations of electron transport inhibitors that maximally inhibited O2 utilization, Δψ was only partially collapsed. Under state 4 conditions, the collapse of Δψ to the Donnan potential by uncouplers correlated with the concentration at which state 4 respiration was stimulated maximally. Inhibition of O2 utilization by F0 inhibitors correlated with an increase in Δψ from the state 3 to the state 4 potential.