Identification of a Hexenal Reductase That Modulates the Composition of Green Leaf Volatiles.

Green leaf volatiles (GLVs), including six-carbon (C6) aldehydes, alcohols, and esters, are formed when plant tissues are damaged. GLVs play roles in direct plant defense at wound sites, indirect plant defense via the attraction of herbivore predators, and plant-plant communication. GLV components provoke distinctive responses in their target recipients; therefore, the control of GLV composition is important for plants to appropriately manage stress responses. The reduction of C6-aldehydes into C6-alcohols is a key step in the control of GLV composition and also is important to avoid a toxic buildup of C6-aldehydes. However, the molecular mechanisms behind C6-aldehyde reduction remain poorly understood. In this study, we purified an Arabidopsis (Arabidopsis thaliana) NADPH-dependent cinnamaldehyde and hexenal reductase encoded by At4g37980, named here CINNAMALDEHYDE AND HEXENAL REDUCTASE (CHR). CHR T-DNA knockout mutant plants displayed a normal growth phenotype; however, we observed significant suppression of C6-alcohol production following partial mechanical wounding or herbivore infestation. Our data also showed that the parasitic wasp Cotesia vestalis was more attracted to GLVs emitted from herbivore-infested wild-type plants compared with GLVs emitted from chr plants, which corresponded with reduced C6-alcohol levels in the mutant. Moreover, chr plants were more susceptible to exogenous high-dose exposure to (Z)-3-hexenal, as indicated by their markedly lowered photosystem II activity. Our study shows that reductases play significant roles in changing GLV composition and, thus, are important in avoiding toxicity from volatile carbonyls and in the attraction of herbivore predators.

Using the Specialization Framework to Determine Degree of Dietary Specialization in a Herbivorous Woodrat.

To be considered a dietary specialist, mammalian herbivores must consume large quantities of a plant species considered "difficult" with respect to nutrient or toxin content, and possess specialized adaptations to deal with plant defensive compounds or low nutritional content. Populations of Neotoma lepida in the Great Basin consume Juniperus osteosperma, a plant heavily defended by terpenes, but a detailed dietary analysis of this population is lacking. Therefore, we investigated the extent of dietary specialization in this species in comparison with the better-studied specialist species, N. stephensi. Microhistological analysis of feces from N. lepida revealed that greater than 90% of their diet in nature was comprised of juniper. In laboratory tolerance trials, N. lepida tolerated a diet of 80% J. osteosperma, similar to that observed for N. stephensi. There was no difference in the abilities of N. lepida and N. stephensi to metabolize hexobarbital, a proxy compound for terpene metabolism. In preference tests of native and non-native juniper species, N. lepida did not exhibit a preference for its native or co-occurring juniper, J. osteosperma, over the non-native species, J. monosperma, whereas N. stephensi preferred its native or co-occurring juniper J. monosperma over non-native J. osteosperma. Behavioral and habitat differences between these woodrat species lead to the categorization of N. stephensi as an obligate juniper specialist with a small range that overlaps that of its preferred food, J. monosperma, and N. lepida as a facultative juniper specialist with a large range, and only a portion of its distribution containing populations that feed extensively on J. osteosperma.

Expression profile and functional characterization of odorant binding proteins in a forest pest, Dioryctria abietella (Lepidoptera: Pyralidae).

In the forest ecosystem dominated by the Pinaceae plants, this boring pest Dioryctria abietella is subject to a variety of odorants derived from host and nonhost plants, in which olfactory-related proteins enriched in antennae are key behavioral modulators for the orientation of feeding and ovipositing hosts. Here, we addressed the odorant binding protein (OBP) gene family in D. abietella. Expression profiles revealed that the majority of OBPs were abundantly expressed in the antennae at a female-biased level. A male-antenna-biased DabiPBP1 was a strong candidate for detecting type I and type II pheromones of D. abitella female moths. Using a prokaryotic expression system combined with affinity chromatography, we harvested two antenna-dominant DabiOBPs. In the ligand-binding assays, the two DabiOBPs exhibited different odorant response spectra, as DabiOBP17 was tuned to most odorants with higher affinities compared to DabiOBP4. Of these, DabiOBP4 could strongly bind syringaldehyde and citral (dissociation constants (Ki) < 14 µM). A floral volatile, benzyl benzoate (Ki = 4.72 ± 0.20 µM), was the best ligand for DabiOBP17. Remarkably, several green leaf volatiles were found to strongly interact with DabiOBP17 (Ki < 8.5 µM), including Z3-hexenyl acetate, E2-hexenol, Z2-hexenal and E2-hexenal that may mediate a repellent response to D. abietella. Structural analyses of ligands revealed that the binding of the two DabiOBPs to odorants was associated with carbon-chain lengths and functional groups. Molecular simulations identified several key residues involved in the interactions of DabiOBPs and ligands, suggesting specific binding mechanisms. This study highlights olfactory roles of two antennal DabiOBPs in D. abietella, helping the identification of potentially behavioral compounds for the population control of this pest.

Synthesis of green note aroma compounds by biotransformation of fatty acids using yeast cells coexpressing lipoxygenase and hydroperoxide lyase.

Green notes are substances that characterize the aroma of freshly cut grass, cucumbers, green apples, and foliage. In plants, they are synthesized by conversion of linolenic or linoleic acid via the enzymes lipoxygenase (LOX) and hydroperoxide lyase (HPL) to short-chained aldehydes. Current processes for production of natural green notes rely on plant homogenates as enzyme sources but are limited by low enzyme concentration and low specificity. In an alternative approach, soybean LOX2 and watermelon HPL were overexpressed in Saccharomyces cerevisiae. After optimization of the expression constructs, a yeast strain coexpressing LOX and HPL was applied in whole cell biotransformation experiments. Whereas addition of linolenic acid to growing cultures of this strain yielded no products, we were able to identify high green note concentrations when resting cells were used. The primary biotransformation product was 3(Z)-hexenal, a small amount of which isomerized to 2(E)-hexenal. Furthermore, both aldehydes were reduced to the corresponding green note alcohols by endogenous yeast alcohol dehydrogenase to some extent. As the cosolvent ethanol was the source of reducing equivalents for green note alcohol formation, the hexenal/hexenol ratio could be influenced by the use of alternative cosolvents. Further investigations to identify the underlying mechanism of the rather low biocatalyst stability revealed a high toxicity of linolenic acid to yeast cells. The whole cell catalyst containing LOX and HPL enzyme activity described here can be a promising approach towards a highly efficient microbial green note synthesis process.

Increasing omega-3 desaturase expression in tomato results in altered aroma profile and enhanced resistance to cold stress.

One of the drawbacks in improving the aroma properties of tomato (Solanum lycopersicum) fruit is the complexity of this organoleptic trait, with a great variety of volatiles contributing to determine specific quality features. It is well established that the oxylipins hexanal and (Z)-hex-3-enal, synthesized through the lipoxygenase pathway, are among the most important aroma compounds and impart in a correct proportion some of the unique fresh notes in tomato. Here, we confirm that all enzymes responsible for the synthesis of these C6 compounds are present and active in tomato fruit. Moreover, due to the low odor threshold of (Z)-hex-3-enal, small changes in the concentration of this compound could modify the properties of the tomato fruit aroma. To address this possibility, we have overexpressed the omega-3 fatty acid desaturases FAD3 and FAD7 that catalyze the conversion of linoleic acid (18:2) to linolenic acid (18:3), the precursor of hexenals and its derived alcohols. Transgenic OE-FAD tomato plants exhibit altered fatty acid composition, with an increase in the 18:3/18:2 ratio in leaves and fruits. These changes provoke a clear variation in the C6 content that results in a significant alteration of the (Z)-hex-3-enal/hexanal ratio that is particularly important in ripe OE-FAD3FAD7 fruits. In addition to this effect on tomato volatile profile, OE-FAD tomato plants are more tolerant to chilling. However, the different behaviors of OE-FAD plants underscore the existence of separate fatty acid fluxes to ensure plant survival under adverse conditions.

Pseudohermaphrodite rat: end organ insensitivity to testosterone.

The pseudohermaphrodite rat is characterized by lack of androgen-dependent differentiation. Treatment of these rats with testosterone failed to produce the expected changes in preputial and adrenal gland weights and hexobarbital metabolism. This insitivity of the end organ to testosterone could not be explained by defective formation of dihydrotestosterone.

Effects of crop development on the emission of volatiles in leaves of Lycopersicon esculentum and its inhibitory activity to Botrytis cinerea and Fusarium oxysporum.

Volatiles emitted from the leaves of Lycopersicon esculentum at the two-, ten-leaf and anthesis periods were collected by a gas absorbing method and analyzed by gas chromatography (GC)-mass spectrometry. In total, 33 compounds of volatiles emitted from three developmental stage plants were separated and identified, and quantitatively analyzed by the internal standard addition method. All of the samples of volatile were found to be rich in monoterpenes and sesquiterpenes. beta-phellandrene and caryophyllene predominated in the volatiles of the leaves of plants at the two- and ten-leaf stages. Furthermore, (E)-2-hexenal were the dominant components in the volatiles emitted from anthesis plants. The results of volatiles analyzed show that the compositions varied depending on the developmental stages. The volatiles emitted from crushed tomato leaves of plants at the anthesis stage had the most strongly inhibitory activity against the spore germination and hyphal growth of Botrytis cinerea and Fusarium oxysporum, followed by ten- and two-leaf plants. However, the activity of volatiles, emitted from the leaves of plants at the two-leaf stage, in inhibiting F. oxysporum was greater than B. cinerea.

The effects of interleukin 2 and alpha-interferon administration on hepatic drug metabolism in mice.

We have administered the cytokines interleukin 2 (IL-2), alpha-interferon (IFN-alpha), and gamma-interferon (IFN-gamma) to mice and measured the alterations in hepatic drug-metabolizing enzyme activities. For comparative purposes and to understand the mechanism of diphtheria and tetanus toxoids and pertussis (DTP) vaccine-induced inhibition of drug metabolism, we also studied the effects of vaccine administration in mice. The administration of IL-2 alone or in combination with IFN-alpha or IFN-gamma causes dose-dependent increases in hexobarbital-induced sleep times. These increases correlate well with the inhibition of specific microsomal mixed-function oxidase activities. Sublethally irradiated mice and athymic nude mice receiving injections of IL-2 or IL-2 plus IFN-alpha do not show the inhibition of drug metabolism seen in normal mice. However, the inhibition of drug metabolism in DTP vaccine-treated mice was similar in all three groups. These observations indicate a possible role for immune cells (probably T-lymphocytes) in the inhibition of drug metabolism caused by administration of these cytokines, which is different from the inhibition of drug metabolism caused by DTP vaccine.

A simple and efficient system for green note compound biogenesis by use of certain lipoxygenase and hydroperoxide lyase sources.

Six-carbon (C(6)) aldehydes and alcohols are important components of the aroma and flavor of fruits and vegetables. Soybean lipoxygenase (LOX) isozyme LOX 3 was reported not only to produce less 13-hydroperoxides, precursors of C(6) aldehydes, but also to convert them to ketodiene products. Here, we examined the effects of LOX 3 on hexenal formation from linolenic acid homogenized with watermelon 13-hydroperoxide lyase (HL)-overexpressing Nicotiana tabacum leaves and soybean acetone powder. Compared to the wild type, which contains LOXs 1, 2, and 3, the elimination of LOX 3 in LOX 1 + 2 facilitates greater production of hexenals. The use of LOX 2 alone yielded the highest hexenal production, while a two-step conversion was required for LOX 1 to produce hexenals at high levels due to different pH optima of the enzymes involved. These results clearly demonstrate that the soybeans lacking LOX 3 in combination with watermelon HL-overexpressing leaf tissues greatly enhance hexenal formation.

Metabolic and psychophysiologic studies of cannabidiol-hexobarbital interaction.

Cannabidiol (CBD), 600 mg/day orally for 5 to 12 days, inhibited hexobarbital metabolism in ten subjects. Hexobarbital oral clearance was 36% lower and apparent volume of distribution was 35% smaller, with no change in half-life during CBD. In four subjects who received intravenous and oral hexobarbital, systemic clearance was 36% lower while bioavailability was 10% greater during CBD. Hexobarbital increased fatigue and tremor, impaired eye-tracking performance, and altered the electroencephalogram. Hexobarbital effects were not affected by CBD. Inhibition of metabolism of other drugs should be considered when large amounts of CBD are taken or when CBD is used for therapy.

Pharmacokinetics of hexobarbital in acute hepatitis and after apparent recovery.

The pharmacokinetics of hexobarbital were studied in 13 patients with acute hepatitis. Hexobarbital sodium was administered by zero order intravenous (iv) infusion, and plasma concentrations were determined regularly by gas chromatography. For each patient the data were fitted according to 2-compartment kinetics. The results were compared to those obtained for 14 healthy volunteers. The elimination half-life of hexobarbital was 490 +/- 186 min in the hepatitis patients and 261 +/- 69 min in the control group. Clearance was significantly reduced in the hepatitis group, whereas the volume of distribution at steady state was not significantly altered. For some patients the initial distribution volume was reduced. In 6 patients the experiment with hexobarbital was repeated after apparent recovery from hepatitis as judged by normal transaminase and bilirubin levels. Generally the half-life of hexobarbital was shorter and the clearance value was higher than during the acute illness, but the values had not yet returned to normal. Clinical recovery from liver disease is not accompanied by corresponding recovery of drug-metabolizing capability.

Influence of rifampicin on drug metabolism: differences between hexobarbital and antipyrine.

Six healthy volunteers were treated with 1,200 mg of rifampicin daily for 8 days. Before and immediately afterward each received indocyanine green, hexobarbital, galactose, and antipyrine by intravenous infusion on 3 consecutive days. The plasma concentrations of the drugs were determined several times after infusion. The average elimination half-life of hexobarbital had decreased from 407 to 171 min and its metabolic clearance had increased almost threefold. In contrast, the average elimination half-life of antipyrine was virtually the same on both occasions (6.9 and 7.2 hr) and there was no change in metabolic clearance. In a tuberculous patient treated with rifampicin the antipyrine elimination rate was unaffected. Rifampicin did not influence indocyanine green clearance or galactose elimination capacity. Serum gamma glutamyl transferase was not affected but urinary D-glucaric acid excretion was increased during rifampicin treatment. The experiment with hexobarbital was repeated after 2 weeks in all subjects; half-lives and clearance values had returned to near control values. It appears that rifampicin is a selective inducer of oxidative drug metabolism in man.

Polymorphism in hydroxylation of mephenytoin and hexobarbital stereoisomers in relation to hepatic P-450 human-2.

Stereoselective 4'-hydroxylations of R-(-)-mephenytoin and S-(+)-mephenytoin and 3'-hydroxylation of R-(-)-hexobarbital and S-(+)-hexobarbital were determined in liver microsomes of 14 Japanese subjects who were extensive metabolizers of mephenytoin and in five Japanese subjects who were poor metabolizers of mephenytoin. Content of P-450 human-2 assessed by Western blots was correlated to microsomal S-(+)-mephenytoin 4'-hydroxylation, R-(-)-hexobarbital 3' alpha-hydroxylation, and S-(+)-hexobarbital 3' beta-hydroxylation, and was less correlated to R-(-)mephenytoin 4'-hydroxylation, R-(-)-hexobarbital 3' beta-hydroxylation, and S-(+)-hexobarbital 3' alpha-hydroxylation. Antibodies raised against P-450 human-2 inhibited microsomal S-(+)-mephenytoin 4'-hydroxylation efficiently but was less efficient on R-(-)-mephenytoin 4'-hydroxylation in extensive metabolizers and on 4'-hydroxylation of mephenytoin enantiomers in poor metabolizers. The antibodies also inhibited R-(-)-hexobarbital 3' alpha-hydroxylation and S-(+)-hexobarbital 3' beta-hydroxylation but did not effectively inhibit the hydroxylation of the two other optical isomers of hexobarbital in extensive metabolizers and of four stereoisomers in poor metabolizers. These findings indicate the close relationship between polymorphic mephenytoin 4'-hydroxylation and two stereospecific hexobarbital hydroxylations, and they suggest that P-450 human-2 is a typical S-(+)-mephenytoin 4'-hydroxylase and a major hexobarbital 3'-hydroxylase in the livers of extensive metabolizers. The findings were further supported by the experiments that used P-450 human-2 complementary dexoyribonucleic acid-derived protein in yeast microsomes.

Comparative metabolism of benzo[a]pyrene and drugs in human liver.

The oxidative metabolism of antipyrine, hexobarbital, coumarin, zoxazolamine, 7-ethoxycoumarin, and the chemical carcinogen benzo[a]pyrene (BP) was studied in 32 adult human livers obtained at autopsy. When enzyme activity for one substrate was plotted against enzyme activity for a second substrate for each of the 32 livers, statistically significant correlations were found between the rates of metabolism of BP and the rates of metabolism of each of the other five drug substrates. The degree of correlation was dependent upon the substrate pair that was studied. Highly significant statistical correlations (p less than 0.001) for monooxygenase activities among the different livers were observed for BP with antipyrine (r = 0.85), antipyrine with zoxazolamine (r = 0.82), antipyrine with hexobarbital (r = 0.79), zoxazolamine with 7-ethoxycoumarin (r = 0.75), antipyrine with coumarin ( r= 0.72), zoxazolamine with coumarin (r = 0.72), BP with hexobarbital (r = 0.72), hexobarbital with coumarin (r = 0.71), BP with zoxazolamine (r = 0.69), hexobarbital with zoxazolamine (r = 0.64), coumarin with 7-ethoxycoumarin (r = 0.61), and BP with coumarin (r = 0.57). Less significant correlations were obtained for BP with 7-ethoxycoumarin (r = 0.35; p = 0.05). It is not known whether the relationships between the metabolism of the several substrates described here for autopsy livers would also occur with fresh livers. The lack of a perfect correlation for any of the substrate pairs suggests the presence in human liver of multiple monooxygenase enzyme systems for the metabolism of benzo[a]pyrene and the five other substrates studied, as well as heterogeneity in their distribution among the 32 livers that were examined. The approach described in the present report may have significance in the study of the comparative metabolism of drugs, chemical carcinogens, and other environmental pollutants by human tissues and may help us find predictor drugs that will be useful for evaluating the drug- and carcinogen-metabolizing capacity of different individuals in the human population.

Activation of monooxygenases in human liver by 7,8-benzoflavone.

Addition of 10(-4) M 7,8-benzoflavone to homogenates of human liver samples obtained by autopsy or surgical biopsy increased the rate of benzo[a]pyrene hydroxylation up to 11-fold. 7,8-Benzoflavone also increased the rates of hydroxylation of zoxazolamine and antipyrine at 10(-4) M but inhibited these reactions at 10(-6) M. The effects of 7,8-benzoflavone on the hydroxylation of benzo[a]pyrene and zoxazolamine in microsomes from human liver were similar to those in homogenates. Addition of 7,8-benzoflavone to homogenates of surgical biopsy samples of human liver had little or no effect on the rates of oxidative metabolism of 7-ethoxycoumarin, coumarin, and hexobarbital. Marked individuality for the activating and inhibiting effects of 7,8-benzoflavone was observed in different liver samples. This individuality may result both from the presence of multiple monooxygenases in varying amounts and proportions in the different liver samples and from a selective effect of 7,8-benzoflavone on certain of the monooxygenases.

Interaction between probenecid and two lipid-soluble barbiturates in the rat.

The effect of pretreatment with probenecid (200 mg/kg, i.p.) on the sensitivity of the central nervous system (CNS) to thiopental and hexobarbital was investigated with an EEG-threshold method. The threshold dose was significantly decreased by pretreatment with probenecid for thiopental but not for hexobarbital. This was due to an increased penetration of thiopental into the CNS, but for hexobarbital an increase in penetration could also be demonstrated by analysis of brain and serum concentrations after infusion of an equal dose of barbiturate. The concentrations in brain at the EEG-threshold were not influenced by pretreatment with probenecid for either of these barbiturates, which shows that there was no synergism between these barbiturates and probenecid due to the depressant effect of probenecid on the CNS.

Effect of aromatic nitro compounds on oxidative metabolism by cytochrome P-450 dependent enzymes.

The effect of aromatic nitro compounds on the oxidative metabolism of representative type I (hexobarbital and aminopyrene) and type II (aniline and zoxazolamine) substrates by cytochrome P-450 dependent liver enzymes was studied. Nitro compounds (nitrobenzene, p-nitrobenzoate, 2-nitrofluorene, and 2-nitronaphthalene) inhibited the oxidation of type II substrates by rabbit liver microsomal enzymes; however, they had no effect on the metabolism of the type I compounds. Inhibition of type II metabolism was characterized graphically as S,I-hyperbolic non-competitive. The influence of aromatic nitro compounds on the interaction of type I and type II substrates with oxidized and reduced cytochrome P-450 was studied by difference spectroscopy. From Lineweaver-Burke plots, nitro compounds were shown to competitively interact with type II compounds for cytochrome p-450 binding sites. Nitro compounds completely prevented the appearance of a type I binding spectrum with either hexobarbital or aminopyrene even when the modifier was present at concentrations less than 10(-8)M. Aromatic nitro compounds appear to therefore inhibit the metabolism of the type II substrates through a mixed mechanism of interaction with the microsomal drug-metabolizing enzymes.

Androgenic repression of hexobarbitone metabolism and action in Crl:CD-1 (ICR)BR mice.

Mice of the Crl:CD-1 (ICR)BR strain exhibit a sexual dimorphism in hexobarbitone metabolism and action. Compared to females, males have a lower Vmax and a higher Km for hepatic microsomal hexobarbitone hydroxylase. In agreement with the enzyme studies, hexobarbitone-induced sleeping times were greater for males than for females. Results from experiments measuring hexobarbitone metabolism and action in castrate, testosterone and gonadotropin-treated mice indicate that the sexual differences in drug metabolism and action found in Crl:CD-1 (ICR)BR mice are due to the normally repressive effects of testicular androgens on the activities of the hepatic mono-oxygenases. These findings are in dramatic contrast to studies with rats where it has been shown that androgens induce mono-oxygenases. Furthermore, in the case of the mouse, changes in the activity of hexobarbitone hydroxylase in response to alterations in androgen levels require weeks, while in the rat, androgenic-induced changes are apparent within a matter of days.

Sexual dimorphism and the effects of the X-linked Tfm locus on hexobarbitone metabolism and action in mice.

1 Normal males of the testicular feminized strain of mice (Tfm) had longer hexobarbitone-induced sleeping times than females, and hepatic hexobarbitone hydroxylase activity different in that the Km was higher and the Vmax lower in the male. 2 Castration and androgen replacement studies indicated that testicular androgens were responsible for the sexual differences in drug metabolism found in this mouse strain. 3 Hepatic hexobarbitone metabolism and action were feminized in the intact, androgen-insensitive, genetically male Tfm mouse. Furthermore, hexobarbitone hydroxylase activities were less responsive to large doses of testosterone in Tfm mice than in normal males. 4 The Tfm mouse with a deficiency in androgen receptors responded to the enzyme-inductive effects of phenobarbitone and softwood bedding, indicating that these inducers do not act through the androgen receptors.

A longitudinal study of the relationship between aging and the duration of hexobarbital hypnosis in male CFN rats.

The duration of hexobarbital hypnosis was determined for individual male CFN rats at three month intervals over their total adult lifespan. The mean duration of hexobarbital hypnosis for the cohort linearly with advancing age. However, the sleeptimes for individual rats fell into one of four categories (gradual increase, terminal step increase, no change, or not possible to assign) when assigned as a function of duration of response to the barbiturate over the lifespan of the animals. These results suggest that the generally well-accepted increase in duration of drug response in aging animals is neither a ubiquitous nor concomitant feature of aging. Furthermore, the varying results obtained for individual animals in this longitudinal study point out the possible error which may be attendant when utilizing pooled tissue extracts to examine hepatic monooxygenase activity in vitro in senescent rodents.