Proteomic and Bioinformatics Analysis of Membrane Lipid Domains after Brij 98 Solubilization of Uninduced and Phenobarbital-Induced Rat Liver Microsomes: Defining the Membrane Localization of the P450 Enzyme System.

The proteomes of ordered and disordered lipid microdomains in rat liver microsomes from control and phenobarbital (PB)-treated rats were determined after solubilization with Brij 98 and analyzed by tandem mass tag (TMT)-liquid chromatography-mass spectrometry (LC-MS). This allowed characterization of the liver microsomal proteome and the effects of phenobarbital-mediated induction, focusing on quantification of the relative levels of the drug-metabolizing enzymes._The microsomal proteome from control rats was represented by 333 (23%) proteins from ordered lipid microdomains, 517 (36%) proteins from disordered lipid domains, and 587 (41%) proteins that uniformly distributed between lipid microdomains. Most enzymes related to drug metabolism were mainly localized in disordered lipid microdomains. However, cytochrome P450 (CYP) 1A2, multiple forms of CYP2D, and several forms of UDP glucuronosyltransferases (UGT) 1A1 and 1A6) localized to ordered lipid microdomains. Other drug-metabolizing enzymes, including several forms of cytochromes P450, were uniformly distributed between the ordered and disordered regions. The redox partners, NADPH-cytochrome P450 reductase and cytochrome b5, localized to disordered microdomains. PB induction resulted in only modest changes in protein localization. Less than five proteins were variably associated with the ordered and disordered membrane microdomains in PB and control microsomes. PB induction was associated with fewer proteins localizing in the disordered membranes and more being uniformly distributed or localized to ordered domains. Ingenuity Pathway Analysis (IPA) was used to ascertain the effect of PB on cellular pathways, resulting in attenuation of pathways related to energy storage/utilization and overall cellular signaling and an increase in those related to degradative pathways. SIGNIFICANCE STATEMENT: This work identifies the lipid microdomain localization of the proteome from control and phenobarbital-induced rat liver microsomes. Thus, it provides an initial framework to understand how lipid/protein segregation influences protein-protein interactions in a tissue extract commonly used for studies in drug metabolism and uses bioinformatics to elucidate the effects of phenobarbital induction on cellular pathways.

Cloning, functional expression, and characterization of CYP709C1, the first sub-terminal hydroxylase of long chain fatty acid in plants. Induction by chemicals and methyl jasmonate.

We cloned and characterized CYP709C1, a new plant cytochrome P450 belonging to the P450 family, that so far has no identified function except for clustering with a fatty acid metabolizing clade of P450 enzymes. We showed here that CYP709C1 is capable of hydroxylating fatty acids at the omega-1 and omega-2 positions. This work was performed after recoding and heterologous expression of a full-length cDNA isolated from a wheat cDNA library in an engineered yeast strain. Investigation on substrate specificity indicates that CYP709C1 metabolizes different fatty acids varying in their chain length (C12 to C18) and unsaturation. CYP709C1 is the first identified plant cytochrome P450 that can catalyze sub-terminal hydroxylation of C18 fatty acids. cis-9,10-Epoxystearic acid is metabolized with the highest efficiency, i.e. K((m)(app)) of 8 microM and V(max(app)) of 328 nmol/min/nmol P450. This, together with the fact that wheat possesses a microsomal peroxygenase able to synthesize this compound from oleic acid, strongly suggests that it is a physiological substrate. Hydroxylated fatty acids are implicated in plant defense events. We postulated that CYP709C1 could be involved in plant defense by producing such compounds. This receives support from the observation that (i) sub-terminal hydroxylation of 9,10-epoxystearic acid is induced (15-fold after 3 h) in microsomes of wheat seedlings treated with the stress hormone methyl jasmonate and (ii) CYP709C1 is enhanced at the transcriptional level by this treatment. CYP709C1 transcript also accumulated after treatment with a combination of the safener naphthalic acid anhydride and phenobarbital. This indicates a possible detoxifying function for CYP709C1 that we discussed.

Mechanisms regulating toxicant disposition to the embryo during early pregnancy: an interspecies comparison.

The dose of toxicant reaching the embryo is a critical determinant of developmental toxicity, and is likely to be a key factor responsible for interspecies variability in response to many test agents. This review compares the mechanisms regulating disposition of toxicants from the maternal circulation to the embryo during organogenesis in humans and the two species used predominantly in regulatory developmental toxicity testing, rats and rabbits. These three species utilize fundamentally different strategies for maternal-embryonic exchange during early pregnancy. Early postimplantation rat embryos rely on the inverted visceral yolk sac placenta, which is in intimate contact with the uterine epithelium and is equipped with an extensive repertoire of transport mechanisms, such as pinocytosis, endocytosis, and specific transporter proteins. Also, the rat yolk sac completely surrounds the embryo, such that the fluid-filled exocoelom survives through most of the period of organogenesis, and can concentrate compounds such as certain weak acids due to pH differences between maternal blood and exocelomic fluid. The early postimplantation rabbit conceptus differs from the rat in that the yolk sac is not closely apposed to the uterus during early organogenesis and does not completely enclose the embryo until relatively later in development (approximately GD13). This suggests that the early rabbit yolk sac might be a relatively inefficient transporter, a conclusion supported by limited data with ethylene glycol and one of its predominant metabolites, glycolic acid, given to GD9 rabbits. In humans, maternal-embryo exchange is thought to occur via the chorioallantoic placenta, although it has recently been conjectured that a supplemental route of transfer could occur via absorption into the yolk sac. Knowledge of the mechanisms underlying species-specific embryonic disposition, factored together with other pharmacokinetic characteristics of the test compound and knowledge of critical periods of susceptibility, can be used on a case-by-case basis to make more accurate extrapolations of test animal data to the human.

Complementary roles of farnesoid X receptor, pregnane X receptor, and constitutive androstane receptor in protection against bile acid toxicity.

The nuclear receptors, farnesoid X receptor (FXR) and pregnane X receptor (PXR), are important in maintaining bile acid homeostasis. Deletion of both FXR and PXR in vivo by cross-breeding B6;129-Fxrtm1Gonz (FXR-null) and B6;129-Pxrtm1Glaxo-Wellcome (PXR-null) mice revealed a more severe disruption of bile acid, cholesterol, and lipid homeostasis in B6;129-Fxrtm1Gonz Pxrtm1Glaxo-Wellcome (FXR-PXR double null or FPXR-null) mice fed a 1% cholic acid (CA) diet. Hepatic expression of the constitutive androstane receptor (CAR) and its target genes was induced in FXR- and FPXR-null mice fed the CA diet. To test whether up-regulation of CAR represents a means of protection against bile acid toxicity to compensate for the loss of FXR and PXR, animals were pretreated with CAR activators, phenobarbital or 1,4-bis[2-(3,5-dichlorpyridyloxy)]benzene (TCPOBOP), followed by the CA diet. A role for CAR in protection against bile acid toxicity was confirmed by a marked reduction of serum bile acid and bilirubin concentrations, with an elevation of the expression of the hepatic genes involved in bile acid and/or bilirubin metabolism and excretion (CYP2B, CYP3A, MRP2, MRP3, UGT1A, and glutathione S-transferase alpha), following pretreatment with phenobarbital or TCPOBOP. In summary, the current study demonstrates a critical and combined role of FXR and PXR in maintaining not only bile acid but also cholesterol and lipid homeostasis in vivo. Furthermore, FXR, PXR, and CAR protect against hepatic bile acid toxicity in a complementary manner, suggesting that they serve as redundant but distinct layers of defense to prevent overt hepatic damage by bile acids during cholestasis.

Suppression of cortical epileptic afterdischarges in developing rats by anticonvulsants increasing GABAergic inhibition.

The anticonvulsant action of three drugs facilitating GABAergic inhibition by different mechanisms (valproate, phenobarbital and progabide) was studied in 229 young rats (12, 18 and 25 days old) with implanted electrodes. Epileptic afterdischarges (ADs) elicited by electrical stimulation of the sensorimotor cortex were used as a model. All three drugs were able to suppress ADs, even the lowest doses used blocked the prolongation seen with repeated stimulations under control conditions. In addition to these general effects, some differences among the three drugs were observed: phenobarbital (10, 20, and 40 mg/kg i.p.) exhibited marked anticonvulsant action in all three age groups whereas valproate (200 and 400 mg/kg i.p.) was somewhat less effective in the youngest rats studied than in the two older groups. Progabide exhibited an effect similar to valproate when a higher dose (150 mg/kg i.p.) was taken into account, but the lower dose (75 mg/kg i.p.) was most efficient in 12 day old rat pups. Our data support the possibility that cortical ADs represent a model of human myoclonic seizures. In addition, they suggest an uneven development of individual components of the GABAergic inhibitory system.

Mixed function oxidases in response to quality and quantity of dietary protein.

Feeding of rice diet reduced the food consumption and growth of rats. Hepatic Cytochrome P-450, NADPH Cytochrome c reductase and the activity of cytochrome P-450 dependent enzymes (Aniline hydroxylase, aminopyrine N-demethylase, p-nitroanisole O-demethylase) were also decreased by feeding rice diet. Supplementation of lysine and threonine to rice diet improved the activity of these enzymes. NADPH regeneration and microsomal phosphatidylcholine were reduced by feeding rice diet. The phenobarbitone induced sleeping time was decreased by supplementing rice diet with lysine and threonine. The effect of protein is probably partly attributed to changes in membrane phosphatidylcholine content and NADPH regeneration rate.

Progabide for refractory partial epilepsy: a controlled add-on trial.

Progabide, an experimental GABA-ergic antiepileptic drug, was given in a placebo-controlled double-blind cross-over trial to 19 adult patients with chronic partial epilepsy refractory to previous high-dose antiepileptic drug therapy. A mean daily dose of 32 mg/kg (range, 16 to 63) of progabide did not significantly change the seizure frequency. In patients with a therapeutic response, progabide led to an increase in the plasma concentration of phenytoin and phenobarbital. Comedication with carbamazepine was associated with a poor response to progabide. Side effects were mild except for a several-fold increase of SGOT and SGPT, which required withdrawal of progabide in one patient. Progabide does not seem to be the drug urgently needed for failures of previous high-dose drug therapy.

[Treatment of tinea microsporum. Metabolic interference between phenobarbital and griseofulvin]. / Traitement des teignes microsporiques. Interférence métabolique entre phénobarbital et griséofulvine.

Two epileptic children treated with phenobarbital and suffering from tinea capitis, not responding to a long lasting treatment with Griseofulvin, healed only after phenobarbital has been discontinued. We ascribe our therapeutic problems to the metabolic interference of Griseofulvin with phenobarbital. We have measured the blood concentration of Griseofulvin several times in one of our 2 children and in a control of the same age living in the same dietary conditions and in the same conditions of Griseofulvin intake; these measurements confirm these data. The point of interest of this report is to draw attention on: -the pharmacologic hypotheses put forward to explain this drug interference, -the other factors of a bad therapeutic response to a treatment with Griseofulvin.

[Height alterations and calcium metabolism after prolongated phenobarbital treatment (author's transl)]. / Valoración del metabolismo Ca/P y del crecimiento lineal, en niños sometidos al tratamiento prolongado con fenobarbital.

In order to know if small children subjected to a long-term treatment with phenobarbital can suffer some alterations in their lineal growth or in their osseous maturity in the same way as those showed by epileptical mother's sons, 85 patients between six months and three years old, who had suffered fit convulsions during a fever process, 20 of them subjected to antithermic treatment, and the 65 left who received a phenobarbital treatment in doses of 5 mg./kg./day have been controlled. In each child, at the beginning of the control and after 12 months, their height, diaphysial diameter and cortical thickness of the metacarpal bones, and bone age is valued. At the same time a calcium, phosphorous and phosphatase-alcaline control was done. The group who had received anti-convulsivants drugs, showed a significant decrease in their lineal growth and in calcium concentration in blood. The control group didn't present any significant variation in any of the controls performed.

[Interactions between oxygen under high pressure and drugs (author's transl)]. / Wechselwirkungen zwischen hyperbarem Sauerstoff und Arzneimittel

A. The clinical applications of oxygen under high pressure (OHP) are limited by oxygen toxicity. Hence, an investigation was carried out in mice on the influence of drugs on the lethal effect of OHP. 1. The lethal effect of OHP is diminished by phenobarbitone, propranolol, clonidine, succinate and tris buffer. 2. The lethal effect of OHP is enhanced by methamphetamine, acetazolamide and guanethidine. 3. The lethal effect of OHP is enhanced by reserpine two hours after administration, but diminished 12 hours after administration. B. The clinical usage of OHP is often necessarily connected with drug therapy. Hence, alteration in drug effects under OHP were investigated in mice and rats. 1. The convulsion threshold of pentetrazol is reduced under OHP by 26%. 2. The duration of the hypnotic effect of hexobarbitone is reduced under OHP by 27%. 3. The analgesic effect of morphine is unchanged by OHP. Practical aspects with regard to the use of drugs during clinical use of OHP are discussed.

Multiple oral doses of diazepam, oxazepam and phenobarbital to dogs--behavioural effects and correlation with antipyrine half-life.

The effects of prolonged treatment with phenobarbital, diazepam, and oxazepam on behaviour and on the plasma half-life of antipyrine have been studied in the dog. In this species the biotransformation of diazepam and oxazepam is known to be very similar to man. After equipotent doses of phenobarbital (25 mg/kg) and diazepam (35 mg/kg), antipyrine half-life was found to decrease 80 and 40%, respectively, while after treatment with oxazepam (150 mg/kg) there was an increase of 20%. The behavioural effects declined in the dogs during the course of treatment with diazepam but were rather constant during treatment with oxazepam.