Michel Tissut

Effect of chlorophenols on isolated plant mitochondria activities: A QSAR study

Twenty-three chlorinated monophenols were investigated for their uncoupling and inhibitory properties on plant mitochondria. All the studied compounds had uncoupling properties but their activity was submitted to quantitative changes from 1- to 400-fold, according to the substitution. They also had inhibitory properties on the electron transfer at a level located upstream to the quinone pool. Three inhibitory types could be recognized: (a) chlorophenols inhibiting similarly the oxidation of every type of substrate (NADH, succinate, alpha-ketoglutarate, at pH 7.2; malate, at pH 7.5), (b) chlorophenols inhibiting first complex I, and (c) chlorophenols inhibiting first complex II. In this last case, the presence of substituents at the 2 and 6 positions increases selectively the inhibition of succinate oxidation. A quantitative structure activity relationship (QSAR) study was undertaken and showed that a good correlation appeared between the steric parameter, 1 chi v, and the inhibitory properties with NADH as substrate. This result could be explained by the binding of the chlorinated phenols with NADH dehydrogenase whose position, on the external surface of the mitochondrial membrane, is favorable to good accessibility of the xenobiotic. Equations for inhibitory properties with other substrates were clearly different. The relation between the uncoupling activity and the studied parameters was more complex but a good correlation occurred with the steric parameter (sigma D, A) and the electronic parameter sigma. Neither log P nor sigma alone gave equations of good quality. These results suggest a competition between the chlorophenol mobility in the inner membrane, necessary for uncoupling, and binding with different proteins in the same membrane on which the inhibitory activity is dependent.

Effects of flavone on the oxidative properties of intact plant mitochondria

Abstract The effects of flavone on the oxidative and phosphorylative properties of plant mitochondria from potato tubers and etiolated mung bean hypocotyls were investigated. Flavone inhibited the state 3 oxidation rates of malate, NADH and, to a lesser extent, succinate but was without effect on the ascorbate-TMPD oxidation rate. The inhibition was the same whether the mitochondria were in state 3 or in an uncoupled state 3. When 100 μM flavone was added during the state 4, the tight coupling of succinate or NADH oxidation was not released. In the electron transfer chain, flavone inhibition appeared to be located in the flavoprotein region. All forms of NADH dehydrogenases seemed to be affected but the greatest inhibition appeared when exogenous NADH was used.

Atrazine metabolism in corn seedlings

Atrazine is a herbicide widely used in corn culture. Corn seedlings metabolize this active ingredient readily in three ways. Two of them are catalysed by enzymes and another is purely chemical. In seedlings, the precise role played by each of these was still not clearly understood. Our work demonstrates that the chemical pathway leading to the formation of the inactive OH-2 atrazine is the pre-eminent form of metabolization inside the roots and, during the first week, inside the leaves. As shown by the kinetics of accumulation of the benzoxazinones responsible for this metabolization, a high potential of atrazine hydroxylation remains effective inside the leaves for at least 1 month. The OH-2 atrazine, which seems to accumulate inside the cell vacuoles, cannot move freely inside the plant and therefore cannot be abundantly transported from roots to leaves. The formation of a glutathione-atrazine conjugate, due to the activity of a glutathione-S-transferase (GST) isoenzyme, represents only a very small part of the total GST in corn. It is mostly present in the aerial parts and is highly effective only after a 1-week culture. When analysing the mixture of the metabolites formed in isolated corn leaves, which is very complex (nine products, among which six were major products), it seems that the metabolization pattern of corn leaves is the combined result of the three pathways and of the transformation of the conjugate into more simple derivatives.

Effects of chlorophenols on isolated class A chloroplasts and thylakoids: a QSAR study.

A series of 22 chlorinated phenols was studied for their effects on photosynthesis of isolated chloroplasts. Each chlorophenol was an uncoupler, but the uncoupling activity depended upon substitution. The di-, tri-, or pentachloro substitution greatly enhanced the uncoupling activity. However, 2,6 substitution was not favorable to the uncoupling activity. Fifty percent uncoupling of photophosphorylations was obtained for concentrations between 4 mM for phenol itself, and 20 microM for pentachlorophenol. The quantitative structure-activity relationship (QSAR) study indicated a good relationship between, on the one hand, steric and electronic parameters, and, on the other hand, the uncoupling activity. In such equations, the A parameter describing ortho-substitution, always presented a negative sign. No good equation could be obtained with only log P and sigma. At concentrations which generally induced uncoupling, the chlorinated phenols inhibited the electron transfer in thylakoids. Di, tri, or penta substitution by chlorine enhanced the inhibition. A good relation appeared between this effect and the steric parameters MR and A. The study of isolated class A chloroplasts demonstrated that the uncoupling and the inhibition of the electron transfer in thylakoids could explain the effect of the chlorophenols on the whole photosynthetic mechanism. The effects of phenols on isolated chloroplasts were compared to those obtained with the same series on mitochondria. To explain the differences between QSAR equations, on the one hand for chloroplasts, and, on the other hand, for mitochondria, we suggested a selective binding of chlorinated phenols to proteins of the biological membranes.

Repartition des flavonols dans l'epaisseur des feuilles de quelques vegetaux vasculaires

Abstract The tissue localisation of flavonoids has been studied in leaves of Betula , Corylus , Fagus , Fraxinus , Pisum , Platanus , Quercus , Spinacia and Tilia and scales of onion bulbs. All these species contain flavonols which are, for the most part, located in the upper epidermis of the leaves. In the onion bulb, flavonols are exclusively in the epidermis. The flavonols are glycosylated and dissolved in the vacuoles. The leaves were fractionated by an original technique of abrasion of the frozen material. The physiological significance of such a distribution of flavonoids in the adult leaves or scales is discussed.

Toxicity of pentachlorophenol on isolated plant mitochondria

Abstract Pentachlorophenol (PCP) is a powerful uncoupler of the oxidative phosphorylations on isolated potato or Mung bean mitochondria; a 50%. uncoupling is obtained at less than 1 μM in a medium devoid of Bovine serum albumin (BSA). Furthermore, at higher concentrations PCP inhibits the electron transfer at the level of Complex II and also at a level located near the quinone pool. The high uncoupling activity is dependent on the presence of five chlorines on the phenyl ring, which increase the lipophily of the phenol and decrease its p K a . The 2,6-dichloro substitution is favorable to the Complex II inhibition. The presence of BSA 0.1% in the reaction medium causes the disappearance of the uncoupling effect of 3 μM PCP and the restoration of the normal increase in the oxidation rate which depends on AD. Comparison of the uncoupling effect of PCP in plant and rat liver mitochondria suggests that some proteic components of the animal mitochondria play the same role as exogenous BSA in the case of plant mitochondria.

Uncoupling properties of three flavonols from plane-tree buds

Abstract Three new flavonols from Platanus acerifolia bud secretions were investigated for their uncoupling activity. They were platanetin (3,5,7,8-tetrahydroxy-6-dimethylallylflavone) platanin (3,5,7,8-tetrahydroxy-6-methylflavone) and 3-hydroxywogonin (3,5,7-trihydroxy-8-methoxyflavone). Their activities were studied on mitochondria (potato tubers, etiolated mung bean hypocotyls and rat livers), thylakoids and chloroplasts (spinach leaves) and Acer cells. The uncoupling activity was demonstrated using polarography and spectrophotometry. Platanetin is the most potent uncoupler, inducing a full uncoupling with 5, 10 and 6 μM in potato, mung bean and rat liver mitochondria respectively and 30 μM in spinach thylakoids and in Acer cells. The uncoupling activity of the other two flavonoids was lower. The presence of bovine serum albumin in the reaction medium and the variations in pH (between 6.5 and 8) did not affect the uncoupling efficiency. In contrast with other flavonoids, these compounds were only protonophoric and not ionophoric. The pKa and log P values of the three flavonols studied were near those of reference uncouplers. However, for platanetin and 3-hydroxywogonin in the pKa and log P values were the same, but the biological activities were not: a binding of 3-hydroxywogonin to a protein component of the membrane might explain this difference. In contrast, the lower uncoupling activity of platanin, when compared to platanetin, is probably due to a one-unit lower log P. Uncoupling activity of these lipophilic flavonols can be explained using the simplest scheme of Terada, but taking into account the protein-binding effects which interfere with the direct transmembrane proton transport.

Kinetics of Uptake and Metabolism of Atrazine in Model Plant Systems

The present work concerns atrazine absorption and metabolism by corn (Zea mays.) seedlings immersed in an aqueous medium in comparison with Acer pseudoplatanus cell cultures. At the point of equilibrium, the apparent concentration inside the A. pseudoplatanus cells (with a moderate lipid content: 0.17% of dry weight) was about twice that of the medium. This equilibrium was probably due to a simple partition process; part of the atrazine was dissolved in the cell water and reached the same concentration as in the external medium while the rest was concentrated inside the cellular lipids. The theoretical calculation of the lipid/water partition, taking into account the value of log P measured not with the lipids but with octanol (log P = 2.5), gave a value of 1.5 for concentration inside the plant material. Such an equilibrium, resulting from a partition process between water and lipids, was also obtained in non-living corn seedlings. In living seedlings, an over-concentration of radioactivity due to [ 14 C]atrazine derivatives was rapidly obtained inside roots and shoots giving concentrations respectively 7- and 12-fold higher than that of atrazine in the external medium. This was due to very rapid chemical transformation of atrazine into its hydroxy derivatives, especially hydroxyatrazine. This hydrolysis of atrazine in corn was due to the presence of high levels of benzoxazinone derivatives in corn seedling cells. The hydroxylated metabolites were able to concentrate in the cells very rapidly and were unable to diffuse freely into the external medium. As a consequence, this process facilitated the penetration of large quantities of atrazine which became rapidly hydroxylated, allowing therefore the passive penetration of atrazine to be further improved, since the concentration C 1 in the receiver compartment was always close to zero. The passive transfer of atrazine, following Ficks law: dq/dt = - Pa (C 0 -C 1 ), was therefore optimized.

Feeding behaviour as a limiting step in insecticide absorption for the wireworm Agriotes sp (Coleoptera : Elateridae)

The fipronil control of wireworms field populations (larval Agriotes sp., Coleoptera: Elateridae) is difficult because of the low integumental penetration rates of this insecticide into the target larval body. The main way of insecticide absorption being associated with food, analysis of the larval feeding behaviour is of key importance for designing a strategy of dietary chemical control of those subterranean pests. In this purpose, a standard method for assessing the long-term survival of field specimens in the laboratory was developed together with experimental designs for investigating food searching and choosing by larvae. Larval biological performances under those laboratory conditions were checked monthly. Three criteria were taken into account for measures: the duration of individual survival, the gain in average body weight, and the moulting rhythm. Experimentation revealed that seed flour was highly desired by larvae and that larval feeding choice was affected over short distances only. This suggests the involvement of dietary preference rather than attractivity in the larval feeding behaviour. This implies that, at the operational step, insecticide had better be associated to food, either as a coating on the seeds, or inside preferred baits.

The inclusion of atmospheric particles into the bark suber of ash trees

A slow deposition of atmospheric components occurs on tree barks. One part of them can be incorporated into bark tissues. This work demonstrates that mineral particles are present inside the suber tissue in four-year-old ash tree stems. Most of these particles are smaller than 2 microm. Scanning electronic microscope studies - using energy dispersive X-ray spectroscopy (SEM-EDX) - on stem cuttings show that they are located either inside the dead suber cells or between these cells. Numerous particles are composed of clay, quartz, feldspar or mica. Others, clearly of anthropogenic origin, are metallic fragments composed of Fe, Ni, Cr, Pb, etc. Spherical fly ashes were found, composed of Si, Al or Fe, and demonstrating an anthropogenic formation. Such particles were isolated and purified from suber ashes obtained at 550 degrees C, after aqueous and acidic treatments, and their composition was established through SEM-EDX. These results reinforce the idea that the suber of tree barks alone can be considered as archives for atmospheric deposition.