Combined use of 13C- and 19F-NMR to analyse the mode of action and the metabolism of the herbicide isoxaflutole

¹³C-Nuclear magnetic resonance was used to characterize the effects of the trifluoromethyl herbicide isoxaflutole on plant metabolism. A specific accumulation of tyrosine and phenylalanine in pea leaves was observed after treatment with this herbicide, consistent with an inhibition of 4-hydroxyphenylpyruvate dioxygenase (HPPD), an enzyme involved in plastoquinone and tocopherol synthesis and the known target of isoxaflutole. ¹⁹F-NMR permitted the analysis of the uptake and degradation of isoxaflutole, with detection limits around 1 μM in perchloric acid extracts. Isoxaflutole was incorporated and transformed to an active diketonitrile derivative, which further underwent degradation to an inactive benzoic acid derivative.     

Tocopherols and associated derivatives track the phytoplanktonic response to evolving pelagic redox conditions spanning Oceanic Anoxic Event 2

Tocopherols serve a critical role as antioxidants inhibiting lipid peroxidation in photosynthetic organisms, yet are seldom used in geobiological investigations. The ubiquity of tocopherols in all photosynthetic lifeforms is often cited as an impediment to any diagnostic paleoenvironmental potential, while the inability to readily analyze these compounds via conventional methods, such as gas chromatography–mass spectrometry, further diminishes the capacity to serve as useful ‘biomarkers’. Here, we analyzed an exceptionally preserved black shale sequence from the Demerara Rise that spans Oceanic Anoxic Event 2 (OAE-2) to reexamine the significance of tocopherols and associated derivatives (i.e. tocol derivatives) in ancient sediments. Tocol derivatives were analyzed via liquid chromatography–quadrupole time-of-flight–mass spectrometry and included tocopherols, a methyltrimethyltridecylchroman, and the first reported detection of tocopherol quinones and methylphytylbenzoquinones in the geologic record. Strong correlations between tocol derivatives were observed over the studied interval. Tocol derivative concentrations and ratios, which normalized tocopherols to potential derivatives, revealed absolute and relative increases in tocopherols as exclusive features of OAE-2 that can be explained by two possible mechanisms related to tocopherol production and preservation. The development of photic zone euxinia during OAE-2 likely forced an upward migration of oxygenic photoautotrophs, increasing oxidative stress that elicited heightened tocopherol biosynthesis. However, shoaling euxinic conditions may have simultaneously acted to enhance tocopherol preservation given the relatively high lability of tocopherols in the water column. Both scenarios could produce the observed stratigraphic distribution of tocol derivatives in this study, although the elevated tocopherol concentrations that define OAE-2 at the Demerara Rise are primarily attributed to enhanced tocopherol production by shoaling phytoplanktonic communities. Thus, the occurrence of tocopherols and associated derivatives in sediments and rocks of marine origin is likely indicative of shallow-water anoxia, tracking the phytoplanktonic response to the abiotic stresses associated with vertical fluctuations in pelagic redox.     

Chemiluminescent study of the antiradical activity of tocopherol analogs and homologs

Antioxidative peculiarities of the effect of tocopherol derivatives are considered. Attempts are made to reveal interrelation between tocopherol pharmacological effect and antiradical activity of its derivatives exemplified by an elementary reaction of tocopherol interaction with free peroxide radicals (FR). It is shown that the presence of free hydroxyl groups, number and location of CH3--groups in tocopherol benzol ring produce a significant effect on tocopherol ability to react with FR. The length of lateral phitil chain produces no appreciable effect on the rate of tocopherol reaction with free radicals. The values of energy activation in this reaction are calculated for tocopherol derivatives. Correlation between biological and antiradical activity of tocopherol homologs is shown. The absence of such correlation for tocopherol analogs is explained by the difference in the ability of analogs to be incorporated into biological membranes. Possible tocopherol regulations of the rates of free radical processes proceeding in lipid membranes are considered.     

Correcting influence of vitamin E short chain derivatives on lipid peroxidation, liver cell membrane, and chromatin structure when rats are exposed to embichin

Embikhin causes activation of LPO processes in endoplasmic reticulum and in nuclear chromatine fractions of rat liver cells. The latter is accompanied by the impairment of repressive and active nuclear chromatine fractions structure. Derivate of vitamin E in these conditions renders correcting action on parameters of lipid peroxidation in the investigated subcellular structures, testifying its positive influence on the cell heredity apparatus state. The normalizing action of tocopherol derivative on cytochromes P450 and b5 levels is shown.     

Tocopherol is the scavenger of singlet oxygen produced by the triplet states of chlorophyll in the PSII reaction centre

Recent developments on the role of tocopherol in the antioxidant network of the chloroplast and, in particular, in the protection of PSII in high light are summarized. The origin and conditions for singlet oxygen production in the reaction centre via P680 triplet formation are discussed, as well as the scavenging of this singlet oxygen by tocopherol. This is probably the obligatory function of tocopherol in the plant in high light acclimation. Furthermore, tocopherol is part of the modulation system of ROS in stress signalling.     

Effects of replacement of the hydroxyl group of cholesterol and tocopherol on the thermotropic behavior of phospholipid membranes

The role of the hydroxyl groups of cholesterol and tocopherol in mediating their interaction with phospholipid bilayers has been a subject of considerable interest. We have examined this question by using derivatives of cholesterol and tocopherol in which the hydroxyl group is esterified to succinate. The hemisuccinate esters of cholesterol and alpha-tocopherol can be readily incorporated into phospholipid membranes and in fact can by themselves form closed membrane vesicles as demonstrated by the encapsulation of [3H]sucrose. The thermotropic behavior of mixtures containing each succinate ester and phospholipid was studied by differential scanning calorimetry. The effect of cholesteryl hemisuccinate on the thermotropic properties of dipalmitoylphosphatidylcholine and dimyristoylphosphatidylethanolamine is very similar to that of cholesterol. This indicates that the 3 beta-OH is not required for the formation of a cholesterol-phospholipid complex. In mixtures of tocopherol acid succinate and phospholipids the peak transition temperature is progressively shifted to lower temperatures as the mole fraction of alpha-tocopherol succinate is increased, while the enthalpy of the transition is only slightly affected. At a tocopherol succinate/phospholipid molar ratio of 9/1 a phase transition is still detectable. A comparison between tocopherol succinate and tocopherol indicates that the substitution of the hydroxyl group reduces the interaction of tocopherol with phospholipids to a small but measurable extent. Thus, the hydroxyl group of tocopherol is more important than the hydroxyl group of cholesterol in influencing their interactions with phospholipids.     

Deletion of apolipoprotein E gene modifies the rate of depletion of alpha tocopherol (vitamin E) from mice brains

Our previous reports show that apolipoprotein E (apoE) influences the dynamics of alpha tocopherol (vitamin E) in brain. In this investigation, the patterns of depletion of alpha tocopherol from tissues of apoE deficient and wild type mice were compared after the animals were fed vitamin E deficient diets. Alpha tocopherol concentrations in specific regions of the brain and peripheral tissues at different times were determined by HPLC with electrochemical detection. ApoE deficiency significantly retarded the rate of depletion of alpha tocopherol from all regions of the brain. In addition, comparison of the rates of depletion of alpha tocopherol in both apoE deficient and wild type animals showed that cerebellum behaved differently from other areas such as cortex, hippocampus and striatum. This reinforces the uniqueness of cerebellum with regard to vitamin E biology. Patterns of depletion of tocopherol from peripheral tissues were different from brain. Serum tocopherol was higher in apoE deficient animals and remained higher than wild type during E deficiency. Depletion of liver tocopherol also tended to be unaffected by apoE deficiency. Our current and previous observations strongly suggest that apoE has an important role in modulating tocopherol concentrations in brain, probably acting in concert with other proteins as well.     

Accumulation dynamics of seed tocopherols in sunflower lines with modified tocopherol levels

Sunflower (Helianthus annuus L.) seeds have a tocopherol profile dominated by alpha-tocopherol. The objective of this research was to study the dynamics of tocopherol accumulation in sunflower lines with altered total tocopherol content or tocopherol profile. Developing seeds were sampled at regular intervals in two lines with reduced and increased total tocopherol content, respectively, and six lines with modified tocopherol profiles. The line with reduced tocopherol content showed a tocopherol accumulation rate reduced by half, whereas the line with increased tocopherol content showed a tocopherol accumulation rate twofold higher than the control. In the three cases, alpha-tocopherol followed a sigmoid accumulation pattern. Modified tocopherol profiles were expressed at early stages of tocopherol accumulation. In most lines with modified profiles, tocopherol accumulation pattern differed from the alpha-tocopherol lines, with maximum tocopherol content at 18 or 21 days after flowering (DAF) that was reduced to reach a plateau from 33 or 36 DAF onward. Such a reduction was caused by continued dry matter accumulation after tocopherol accumulation ceased or slowed down. In lines with increased levels of beta-tocopherol or both gamma- and delta-tocopherol, the synthesis of beta- and delta-tocopherol started and stopped earlier than the synthesis of alpha- and gamma-tocopherol, respectively.     

The role of homogentisate phytyltransferase and other tocopherol pathway enzymes in the regulation of tocopherol synthesis during abiotic stress

Tocopherols are amphipathic antioxidants synthesized exclusively by photosynthetic organisms. Tocopherol levels change significantly during plant growth and development and in response to stress, likely as a consequence of the altered expression of pathway-related genes. Homogentisate phytyltransferase (HPT) is a key enzyme limiting tocopherol biosynthesis in unstressed Arabidopsis leaves (E. Collakova, D. DellaPenna [2003] Plant Physiol 131: 632-642). Wild-type and transgenic Arabidopsis plants constitutively overexpressing HPT (35S::HPT1) were subjected to a combination of abiotic stresses for up to 15 d and tocopherol levels, composition, and expression of several tocopherol pathway-related genes were determined. Abiotic stress resulted in an 18- and 8-fold increase in total tocopherol content in wild-type and 35S::HPT1 leaves, respectively, with tocopherol levels in 35S::HPT1 being 2- to 4-fold higher than wild type at all experimental time points. Increased total tocopherol levels correlated with elevated HPT mRNA levels and HPT specific activity in 35S::HPT1 and wild-type leaves, suggesting that HPT activity limits total tocopherol synthesis during abiotic stress. In addition, substrate availability and expression of pathway enzymes before HPT also contribute to increased tocopherol synthesis during stress. The accumulation of high levels of beta-, gamma-, and delta-tocopherols in stressed tissues suggested that the methylation of phytylquinol and tocopherol intermediates limit alpha-tocopherol synthesis. Overexpression of gamma-tocopherol methyltransferase in the 35S::HPT1 background resulted in nearly complete conversion of gamma- and delta-tocopherols to alpha- and beta-tocopherols, respectively, indicating that gamma-tocopherol methyltransferase activity limits alpha-tocopherol synthesis in stressed leaves.     

The effect of fluoride intake on the total lipid,cholesterol and vitamin E levels in a sera and liver of guinea pigs on high fat diet

Guinea pigs were raised on a diet containing 18 percent fat and were provided 25 or 0 ppm fluoride in the drinking water. Animals were sacrificed at the end of 0, 3, 6, 9 and 13 weeks on the dietary regimen and blood serum was analyzed for fluoride, total lipid, cholesterol and alpha tocopherol. The serum total lipid levels, cholesterol and alpha tocopherol levels were increased in the high fluoride group between 9 and 13 weeks. An increase in total lipid and alpha tocopherol levels was also observed in the livers of these animals. The increase in liver content of alpha tocopherol was proportional to the increase in total lipid content whereas the corresponding increase in serum alpha tocopherol content was significant even when the increase in total lipid was taken into account. The data suggest a specific effect of fluoride on the serum alpha tocopherol levels of the high fluoride animals.     

Unraveling the Genetic Basis of Seed Tocopherol Content and Composition in Rapeseed (Brassica napus L.)

Background

Tocopherols are important antioxidants in vegetable oils; when present as vitamin E, tocopherols are an essential nutrient for humans and livestock. Rapeseed (Brassica napus L, AACC, 2 n = 38) is one of the most important oil crops and a major source of tocopherols. Although the tocopherol biosynthetic pathway has been well elucidated in the model photosynthetic organisms Arabidopsis thaliana and Synechocystis sp. PCC6803, knowledge about the genetic basis of tocopherol biosynthesis in seeds of rapeseed is scant. This project was carried out to dissect the genetic basis of seed tocopherol content and composition in rapeseed through quantitative trait loci (QTL) detection, genome-wide association analysis, and homologous gene mapping.

Methodology/Principal Findings

We used a segregating Tapidor × Ningyou7 doubled haploid (TNDH) population, its reconstructed F₂ (RC-F₂) population, and a panel of 142 rapeseed accessions (association panel). Genetic effects mainly contributed to phenotypic variations in tocopherol content and composition; environmental effects were also identified. Thirty-three unique QTL were detected for tocopherol content and composition in TNDH and RC-F₂ populations. Of these, seven QTL co-localized with candidate sequences associated with tocopherol biosynthesis through in silico and linkage mapping. Several near-isogenic lines carrying introgressions from the parent with higher tocopherol content showed highly increased tocopherol content compared with the recurrent parent. Genome-wide association analysis was performed with 142 B. napus accessions. Sixty-one loci were significantly associated with tocopherol content and composition, 11 of which were localized within the confidence intervals of tocopherol QTL.

Conclusions/Significance

This joint QTL, candidate gene, and association mapping study sheds light on the genetic basis of seed tocopherol biosynthesis in rapeseed. The sequences presented here may be used for marker-assisted selection of oilseed rape lines with superior tocopherol content and composition.     

The measurement of nanograms of tocopherol from needle aspiration biopsies of adipose tissue: normal and abetalipoproteinemic subjects

A method for the analysis of tocopherol in human adipose tissue using high performance liquid chromatography and fluorescence spectrometry is described; results are expressed relative to total triglyceride content measured by the reaction of the methylated fatty acids with hydroxylamine and ferric chloride. The tocopherol contents of adipose tissue obtained at surgery and by the needle aspiration biopsy technic of ambulatory human subjects (who did not take supplemental vitamin E) were found to be virtually identical. The tocopherol content of adipose tissue by the needle aspiration technic was 262 +/- 33 ng tocopherol/mg triglyceride; this value was increased twofold or more in persons ingesting additional vitamin E. Patients with abetalipoproteinemia (ABL) who absorb tocopherol poorly and have extremely low levels of plasma and red blood cell tocopherol also had a low concentration of adipose tissue tocopherol. However, some ABL patients on massive supplementation with vitamin E (approximately 10 g daily) did achieve normal concentrations of adipose tissue tocopherol.     

Dietary tocopherol and sexual reproduction in the rotifers Brachionus calyciflorus and Asplanchna sieboldi.

Brachionus calyciflorus contained no detectable tocopherol (vitamin E) when cultured for long periods on the yeast Rhodotorula glutinis. Using a spectrofluorometric assay and the Asplanchna body-wall-outgrowth response bioassay the limits of detection were 6.4 ng and 3.78 pg alpha-tocopherol per g dry weight Brachionus, respectively. These tocopherol-free Brachionus reproduced normally both parthenogenetically and sexually, producing active and potent males and fertilized resting eggs which developed into viable young rotifers. The apparent lack of a tocopherol requirement for male fertility in B. calyciflorus probably also applies to Asplanchna sieboldi and A. brightwell -- rotifers whose male-producing (mictic) females are induced by dietary tocopherol and whose males therefore are never tocopherol-deficient. The adaptive significance of the tocopherol requirement for the initiation of sexual reproduction in Asplanchna sieboldi--formerly hypothesized to be the result of a tocopherol requirement for male fertility--is now thought to be related to the role of tocopherol in controlling female polymorphism in this species.     

Effect of carbon-tetrachloride and mercuric chloride on the tocopherol and ubiquinone content of liver and kidney.

Authors examined changes in the total ubiquinone and tocopherol contents of livers and kidneys obtained from rats acutely poisoned by carbon-tetrachloride and mercuric-chloride. Following administration of carbon-tetrachloride after 48 h the liver ubiquinone content per 100 g tissue decreased from 11.80 +/- 2.44 mg to 7.24 +/- 2.15 mg, while the kidney ubiquinone content changed from 14.16 +/- 3.96 mg to 5.98 +/- 2.93 mg. The total tocopherol content remained unaltered. Fourty-eight hours after administration of mercuric-chloride, the liver total tocopherol and total ubiquinone contents showed no changes, the tocopherol content of the kidney dropped from 6.16 +/- 2.10 mg to 3.16 +/- 1.48 mg, while the total ubiquinone content remained unaltered.     

Role of alpha-tocopherol and retinol in the antiradical protection of the body in peptic ulcer]

Retinol and tocopherol content, the intensity of spontaneous chemiluminescence as well as concentration of malonic dialdehyde in the blood of practically healthy persons of different age groups and in patients with gastric and duodenal ulcer have been studied. Results are presented. The lower content of tocopherol was revealed in the young persons as compared with other age groups, while the level of vitamin A was not established to depend on age. The content of tocopherol is established to decrease in the blood serum under peptic ulcer. Under these conditions low level of retinol correlates with considerable growth of spontaneous chemiluminescence. Reserve interrelation of tocopherol content in the blood with the level of spontaneous chemiluminescence as well as the absence of true dependence on the concentration of malonic dialdehyde at peptic ulcer are proved. Mechanisms of participation of vitamins E and A in the inhibition of free-radical reactions are discussed, role of their deficiency in pathogenesis fo peptic ulcer is substantiated.     

Current opinions on the functions of tocopherol based on the genetic manipulation of tocopherol biosynthesis in plants

As a member of an important group of lipid soluble antioxidants,tocopherols play a paramount role In the daily diet of humans and animals.Recently,genes required for tocochromanol biosynthesis pathway have been identified and cloned with the help of genomics-based approaches and molecular manipulation in the model organisms: Arabidopsis thaliana and Synechocystis sp.PCC 6803.At the basis of these foundations,genetic manipulation of tocochromanol biosynthesis pathway can give rise to strategies that enhance the level of tocochromanol content or convert the constitution of tocochromanol.In addition,genetic manipulations of the tocochromanol biosynthesis pathway provide help for the study of the function of tocopherol in plant systems.The present article summarizes recent advances and pays special attention to the functions of tocopherol in plants.The roles of tocopherol in the network of reactive oxygen species,antioxidants and phytohormones to maintain redox homeostasis and the functions of tocopherol as a signal molecule in chloroplast-to-nucleus signaling to regulate carbohydrate metabolism are also discussed.     

Influences on tocopherol biosynthesis in the cyanobacterium Synechocystis sp. PCC 6803

To elucidate influences on the tocopherol biosynthesis in cyanobacteria, wild type and mutant cells of a putative methyltransferase in tocopherol and plastoquinone biosynthesis of Synechocystis sp. PCC 6803 were grown under different conditions. The vitamin E content of cells grown under different light regimes, photomixotrophic or photoautotrophic conditions and varying carbon dioxide supplies were compared by HPLC measurements. The tocopherol levels in wild type cells increased under higher light conditions and low carbon dioxide supply. Photomixotrophic growth led to lower vitamin E amounts in the cells compared to those grown photoautotrophically. We were able to segregate a homozygous deltasll0418 mutant under photoautotrophic conditions. In contrast to former suggestions in the literature the deletion of this gene is not lethal under photomixotrophic conditions and the influence on tocopherol and plastoquinone amounts is diminutive. The methyltransferase encoded by the gene sll0418 is not essential either for tocopherol or plastoquinone synthesis.     

Apolipoprotein e deficiency leads to altered brain uptake of alpha tocopherol injected into lateral cerebral ventricles

The incorporation of radioactive alpha tocopherol by various brain regions of wild type and apolipoprotein E (apoE)-deficient mice was investigated. Labeled tocopherol was injected into the lateral cerebral ventricles of 11 weeks old, male mice. Radioactive cholesterol injected simultaneously was used as an internal standard to account for experimental variability. Most areas of the brain of apoE-deficient mice took up less of alpha tocopherol per mg of protein than wild type animals. However, specific activity of alpha tocopherol was higher in cerebellum, pons, hypothalamus, midbrain and cerebral cortex in apoE-deficient brains than the wild type. This could be due to (a) the lower levels of alpha tocopherol in apoE-deficient brain and (b) reductions in the clearance and transport of tocopherol (possibly mediated by apoE). Tocopherol uptake by hippocampus was unusual since it was lower in apoE deficiency whether the data were expressed as specific activity or per mg of protein. Nearly all of the injected alpha tocopherol remained unchanged in the brains of both apoE-deficient and wild type animals suggesting low turnover. Overall, the current data reinforce the hypothesis that apoE is a key protein involved with the transport and/or retention of alpha tocopherol in brain.     

Comparison of exchange of alpha-tocopherol and free cholesterol between rat plasma lipoproteins and erythrocytes.

The simultaneous exchange of (3h)tocopherol and (14C)cholesterol between rat plasma, rat plasma lipoproteins, and RBC was studied in vitro to compare quantitavely (a) the fractional exchange rates and (b) the half-times for isotope equilibration. In all incubations of RBC with plasma or with plasma lipoprotein fractions, (14C)cholesterol approached equilibrium more rapidly than (3H)tocopherol. When the RBC contained the initial radioactivity, the half-times for equilibration with plasma of cholesterol and of tocopherol were 1.0 and 2.2 hr, respectively. However, the fractional exchange rates (KRBC leads to plasma) were 0.097/hr for cholesterol and 0.188/hr for tocopherol, indicating that the RBC tocopherol pool is turning over almost twice as rapidly as the RBC cholesterol pool. The rat plasma lipoproteins were separated into five fractions by successive ultracentrifugation. Only two fractions, the high density lipoproteins (d 1.063-1.21) and the very low density lipoproteins (d is less than 1.006), participated to a significant extent in the exchange of either tocopherol or cholesterol with RBC. Cholesterol exchange between individual rat plasma lipoproteins and RBC had the same half-times for isotope equilibrium for the very low and high density lipoproteins, and the RBC fractional exchange rates were proportional to the amount of cholesterol in the lipoproteins. In tocopherol exchange between individual rat plasma lipoproteins and RBC, the very low density lipoprotein tocopherol did not equilibrate completely with the RBC. However, the initial rate of tocopherol exchange appeared to be the same for very low and high density lipoproteins. The very low density lipoproteins were disrupted by repeated freezing and thawing or by dehydrating and rehydrating, and analysis of the resulting lipoproteins indicated that free cholesterol was associated more closely than tocopherol with the phospholipid-protein portion of the molecule, which is thought to be on the surface. This difference in distribution of tocopherol and free cholesterol within very low density lipoproteins could account for their different rates of exchange and for the nonequilibrium of tocopherol between RBC and very low density lipoproteins.     

The effect of alpha-tocopherol as an antioxidant on the oxidation of membrane protein thiols induced by free radicals generated in different sites

Azo compounds enable us to generate peroxyl radicals by thermal decomposition at a constant rate and at a desired site, that is, water-soluble compounds produce initiating radicals in an aqueous phase and lipid-soluble compounds initiate the oxidation within the membrane-lipid layer. Using these radicals generated in different sites, we oxidized red blood cell ghost membranes to study the relationships between alpha-tocopherol depletion, initiation of lipid peroxidation, and protein damage. When radicals were generated in the aqueous phase, the loss of membrane protein thiols was observed concurrently with the consumption of membrane tocopherol and after tocopherol was exhausted the peroxidation of membrane lipids occurred. On the other hand, when radicals were initiated within the lipid region, the oxidation of thiols and the formation of thiobarbituric acid-reactive substances were suppressed to give an induction period until tocopherol fell below a critical level. Our results indicate that the surface thiols of extrinsic proteins may compete with alpha-tocopherol for trapping aqueous radicals and spare tocopherol to some extent, whereas the oxidation of intrinsic buried thiols may commence due to lipid-derived radicals produced after tocopherol was consumed. In conclusion, alpha-tocopherol in the membrane can break the free radical chain efficiently to inhibit the lipid peroxidation. However, the effect of tocopherol on the inhibition of membrane protein damage, exhibited by the loss of thiols and the formation of high-molecular-weight proteins, would be different depending on the site of initial radical generation.