Transcription coactivator PRIP, the peroxisome proliferator-activated receptor (PPAR)-interacting protein, is redundant for the function of nuclear receptors PParalpha and CAR, the constitutive androstane receptor, in mouse liver.

Disruption of the genes encoding for the transcription coactivators, peroxisome proliferator-activated receptor (PPAR)-interacting protein (PRIP/ASC-2/RAP250/TRBP/NRC) and PPAR-binding protein (PBP/TRAP220/DRIP205/MED1), results in embryonic lethality by affecting placental and multiorgan development. Targeted deletion of coactivator PBP gene in liver parenchymal cells (PBP(LIV-/-)) results in the near abrogation of the induction of PPARalpha and CAR (constitutive androstane receptor)-regulated genes in liver. Here, we show that targeted deletion of coactivator PRIP gene in liver (PRIP(LIV-/-)) does not affect the induction of PPARalpha-regulated pleiotropic responses, including hepatomegaly, hepatic peroxisome proliferation, and induction of mRNAs of genes involved in fatty acid oxidation system, indicating that PRIP is not essential for PPARalpha-mediated transcriptional activity. We also provide additional data to show that liver-specific deletion of PRIP gene does not interfere with the induction of genes regulated by nuclear receptor CAR. Furthermore, disruption of PRIP gene in liver did not alter zoxazolamine-induced paralysis, and acetaminophen-induced hepatotoxicity. Studies with adenovirally driven EGFP-CAR expression in liver demonstrated that, unlike PBP, the absence of PRIP does not prevent phenobarbital-mediated nuclear translocation/retention of the receptor CAR in liver in vivo and cultured hepatocytes in vitro. These results show that PRIP deficiency in liver does not interfere with the function of nuclear receptors PPARalpha and CAR. The dependence of PPARalpha- and CAR-regulated gene transcription on coactivator PBP but not on PRIP attests to the existence of coactivator selectivity in nuclear receptor function.

The nuclear receptor CAR mediates specific xenobiotic induction of drug metabolism.

Organisms encounter a wide range of foreign compounds--or 'xenobiotics'--with potentially harmful consequences. The cytochrome P450 (CYP) enzymes metabolize xenobiotics and thus are a primary defence against these compounds. Increased expression of specific CYP genes in response to particular xenobiotics is a central component of this defence, although such induction can also increase production of toxic metabolites. Here we show that the nuclear receptor CAR mediates the response evoked by a class of xenobiotics known as the 'phenobarbital-like inducers'. The strong activation of Cyp2b10 gene expression by phenobarbital, or by the more potent TCPOBOP, is absent in mice lacking the CAR gene. These animals also show decreased metabolism of the classic CYP substrate zoxazolamine and a complete loss of the liver hypertrophic and hyperplastic responses to these inducers. Cocaine causes acute hepatotoxicity in wild-type mice previously exposed to phenobarbital-like inducers and this toxicity is also absent in the CAR-deficient animals. Thus, loss of CAR function alters sensitivity to toxins, increasing or decreasing it depending on the compound. Modulation of CAR activity in humans may significantly affect metabolism of drugs and other xenobiotics.

Effect of 2,4-disubstituted thiazol-5yl-aminoketones on inflammation. In vitro and in vivo biological studies: a structure-activity approach.

Some modified novel thiazol-5yl-aminoketones were evaluated for their anti-inflammatory, analgesic and antiproteolytic activities. Their inhibitory activity on 12-lipoxygenase (12-LO) and beta-glucuronidase in vitro was estimated. Their interaction with the stable free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) and their RM values were also determined. For two of them, the effect on zoxazolamine-induced paralysis after a prolonged treatment was determined. The duration of paralysis for the same compounds, (only one administration, one before zoxazolamine injection) was recorded too. The 2-amino substituted derivatives seem to be more potent in comparison with the 2-phenyl. The tested compounds were found to influence proteolysis but not the activities at beta-glucuronidase and 12-LO. Their interaction with DPPH was mild. Compound 2 seems to modify the activity of the hepatic drug metabolizing enzymes. In conclusion, their activity is related to certain structural characteristics.

Kinetic studies on a genetically engineered fused enzyme between rat cytochrome P4501A1 and yeast NADPH-P450 reductase.

Three expression plasmids, pAMC1 for rat P4501A1, pAMR2 for P4501A1 and yeast NADPH-P450 reductase, and pAFCR1 for a fused enzyme between P4501A1 and the reductase, were constructed, and each was introduced into Saccharomyces cerevisiae AH22 cells. The microsomal fraction prepared from the recombinant yeast cells was subjected to kinetic studies of zoxazolamine 6-hydroxylation at 10 degrees C. The apparent Km and Vmax values for hydroxylation by the fused enzyme in AH22/pAFCR1 microsomes were 0.38 mM and 0.42 s-1, respectively. The rate constant for reduction of the fused enzyme with NADPH in the presence of 1 mM zoxazolamine was larger than 50 s-1 using a dual-wavelength stopped-flow spectrometer, indicating that electrons are rapidly transferred from NADPH through FAD and FMN to the heme iron of the fused enzyme. The rate constant kon for substrate binding to the fused enzyme was 25 mM-1.s-1, which is not much different from that of nonfused P4501A1. These results together with spectral data measured during the hydroxylation reaction in the steady state suggest that the rate-limiting step of the reaction by the fused enzyme might be the release of product. On the other hand, the apparent Km and Vmax values for the hydroxylation of P4501A1 in AH22/pAMC1 and AH22/pAMR2 microsomes were 0.32 and 0.33 mM, and 0.015 and 0.29 s-1, respectively. The rate constants for the reduction of P4501A1 were 0.025 and 0.40 s-1, respectively, for AH22/pAMC1 and AH22/pAMR2 microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Action of oxatomide on the hepatic microsomal enzyme system. Effect of the monoxygenase and peroxidase activity of P-450 on drug N-demethylations.

The effect of oxatomide and astemizole on the rat's response to zoxazolamine was examined. It was found that the former increases the body's resistance to zoxazolamine. The effect of the pretreatment with oxatomide, pregnenolone-16 alpha-carbonitrile or tocopherol acetate on the in vitro N-demethylation of ethylmorphine, aminopyrine, the reduction of p-nitrobenzoic acid, P-450 and hepatic protein content was determined. It was found that 1) oxatomide increased N-demethylation and the content of both P-450 and hepatic protein, 2) PCN increased all the examined parameters and 3) tocopherol acetate induced only the ethylmorphine N-demethylation. The role of P-450 as a monoxygenase or as a peroxidase under these experimental conditions is discussed.

Effect of the position of the cyano-group of cyanopregnenolones on their drug metabolic inducing activity.

The effect of the position of the cyano-group of several cyanopregnenolones on the body's resistance to drugs and on drug metabolism was investigated. Female rats were pretreated with 2 alpha-, 6-, 16 alpha-, 17 alpha-cyano- or 16 alpha-cyanomethyl-pregnenolone or with pregnenolone, and the (in vivo) resistance to zoxazolamine, digitoxin and indomethacin, as well as the in vitro drug metabolism (post mitochondrial fraction) of zoxazolamine and ethylmorphine were determined. It was found that the 16-derivative was the most active in this respect, the 2- and 17-cyanopregnenolones were less active but significantly potent compared to controls, while the 6-cyano, the 16-cyanomethyl derivatives and pregnenolone were essentially inactive. These differences were explained in terms of an effective or poor fit of the steroids to their receptor. The poor performance of pregnenolone-16 alpha-acetonitrile was attributed to electronic effects. A hypothesis of some structural features of the receptor site for its interaction with the cyanopregnenolone inducers was presented.

Modulation of zoxazolamine metabolism in carrageenan-induced inflammation in rats.

In male rats bearing carrageenan-induced paw edema twenty-four hours after treatment with carrageenan the duration of hexobarbital hypnosis was prolonged and the rate of hepatic hexobarbital metabolism was inhibited. By contrast, treatment with carrageenan potentiates zoxazolamine paralysis time and inhibits zoxazolamine metabolism in both male and female rats. Thus, sex-related differences in the rats bearing carrageenan-induced paw edema and inhibition of drug metabolism are apparently substrate dependent.

Effects of ethanol on microsomal drug metabolism in aging female rats. III. In vivo.

The effects of aging on ethanol inhibition of zoxazolamine metabolism in vitro and in vivo were studied in female Fischer 344 rats aged 4, 14 and 26 months. Zoxazolamine hydroxylase activity in freshly-isolated liver microsomes decreased significantly with age (1.88 +/- 0.32, 1.49 +/- 0.30 and 0.74 +/- 0.18 nmol/min per mg protein in young-adult, middle-aged and old rats, respectively). A substantial inhibition of zoxazolamine hydroxylation occurred in the presence of 40 mM ethanol. The extent of inhibition was the same in microsomes from all three age groups. The effect of aging on the duration of zoxazolamine paralysis in vivo reflected the effect of aging on zoxazolamine metabolism in vitro. Mean duration of paralysis following a standard 50 mg/kg dose of zoxazolamine increased significantly as a function of aging (0.5, 2.9 and 4.7 h in young-adult, middle-aged and old rats, respectively). Administration of ethanol (1.2 g/kg) 10 min before zoxazolamine treatment prolonged the duration of zoxazolamine paralysis in young-adult and middle-aged rats by about 2 to 2.5 h, but ethanol pretreatment did not affect paralysis time in old rats. Thus, the inhibitory effect of ethanol on zoxazolamine metabolism in vivo appeared to be attenuated in old age.

Biphasic effect of methadone on hepatic drug metabolism.

When methadone HCl (30 mg/kg, po) was given acutely to mice, it was found to inhibit drug metabolism as evidenced by a prolongation of hexobarbital sleeping time and zoxazolamine paralysis time. Pharmacokinetic studies revealed that this acute dose of the narcotic analgesic could also prolong the plasma half-life of aminopyrine without any change in its volume of distribution. When added to the incubation mixture containing 10,000 g mouse liver supernatant fraction and a complete system for measuring aminopyrine N-demethylase or aniline hydroxylase, methadone showed a dose-dependent inhibition of the enzymes; the former enzyme was inhibited to a greater extent than the latter one. However, subacute treatment of mice with methadone HCl (30 mg/kg, po, twice daily for 3 days) resulted in increases in liver weight, microsomal protein, and cytochrome P-450 content in consonant with the increased activities of four hepatic drug-metabolizing enzymes: aminopyrine N-demethylase, aniline hydroxylase, p-nitroanisole, O-demethylase, and benzphetamine N-demethylase. Moreover, both hexobarbital sleeping time and zoxazolamine paralysis time were shortened. The plasma half-life of aminopyrine was decreased. These changes were prevented by simultaneous administration of puromycin diHCl (80 mg/kg, ip). Methadone thus seems to act in a manner very similar to that of propoxyphene or SKF-525A, acting as a potent inhibitor of hepatic drug metabolism when given acutely and as an inducer when given subacutely.

Effect of benzopyrone derivatives on drug activity and metabolism.

In female rats, zoxazolamine paralysis time and mortality caused by indomethacin were significantly reduced by pretreatment with khellin, 7,8-benzoflavone or rutin. Pretreatment with khellin and 7,8-benzoflavone increased the in vitro zoxazolamine and ethylmorphine metabolism. These results were compared with those obtained by equimolar doses of phenobarbital, pregnenolone-16 alpha-carbonitrile and spironolactone in experiments performed simultaneously. It was concluded: Khellin, 7,8-benzoflavone and rutin increased the body's resistance to drugs via induction of the drug metabolizing enzymes of the liver, this action has about the same magnitude with that of phenobarbital and pregnenolone-16 alpha-carbonitrile. For the benzopyrone derivatives, some common structural features have been indicated as probable structural requirements for drug metabolizing enzyme inductive activity in this group of compounds.

Dose-dependent pharmacokinetics of zoxazolamine in the rat.

Zoxazolamine (ZX) is a model substrate frequently used in studies on (methylcholanthrene-inducible) hepatic cytochrome P-450 activity. The iv pharmacokinetics of ZX were studied in rats at four dose levels: 5 mg X kg-1 (n = 6), 25 mg X kg-1 (n = 6), 50 mg X kg-1 (n = 5), and 60 mg X kg-1 (n = 4). Concentrations of ZX in blood, as well as the urinary excretion of unchanged ZX and chlorzoxazone, were determined. The apparent systemic clearance (CLs,app) decreased with increasing dose from 52.6 +/- 3.9 at 5 mg X kg-1 to 9.3 +/- 0.4 ml X min-1 X kg-1 at 60 mg X kg-1. The apparent elimination half-life, t1/2,app, increased from 16.1 +/- 0.3 min to 141 +/- 28.5 min. There was only slight concentration dependency of plasma protein binding: 86.0 +/- 0.9% at 4.2 +/- 0.2 micrograms X ml-1 (n = 6) vs. 80.4 +/- 0.4% at 27.1 +/- 1.1 micrograms X ml-1 (n = 6). Since from clearance and protein binding data nonrestrictive clearance of ZX could be inferred, this small change in binding was regarded as irrelevant for the interpretation of pharmacokinetic data of ZX. The blood-plasma concentration ratio was larger than unity: 2.11 +/- 0.09 at 5.4 +/- 0.9 micrograms X ml-1, and 1.85 +/- 0.08 at 47.9 +/- 4.9 micrograms X ml-1 (n = 5).(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of flavonoids on the metabolism of xenobiotics.

Flavonoids represent a large class of naturally occurring plant constituents that are consumed in substantial amounts by experimental animals and by human beings. The ability of flavonoids to activate or inhibit the principal enzyme system involved in the metabolism of lipophilic xenobiotics, including drugs, carcinogens, insecticides and other environmental pollutants, or to induce the synthesis of this enzyme system may have significant pharmacological and toxicological importance. The significant body of literature which illustrates that flavonoids can modulate the tumorigenic activity of polycyclic hydrocarbons and other chemical carcinogens has been reviewed recently (Slaga, Di Giovanni 1984; Wiebel 1980), but further elucidation of mechanisms of action are needed. Possible effects of flavonoids on the metabolism of lipophilic normal body constituents such as steroids and bile acids should be considered, and additional research is needed to determine the extent to which dietary flavonoids can influence the in vivo metabolism of xenobiotics.

Inhibitory effect of propylthiouracil-induced hypothyroidism in rat on oxidative drug metabolism.

The effect of propylthiouracil (PTU) pretreatment on in vivo and in vitro oxidative drug metabolism was determined in the rat. Whereas pentobarbital sleeping time (PBST) and zoxazolamine paralysis time (ZZPT) were used as indices of in vivo drug metabolizing activity, biotransformation of aminopyrine and aniline by hepatic microsomal preparations were used as indices of in vitro drug metabolizing enzymes activities. PTU pretreatment significantly prolonged both PBST and ZZPT. Whereas PTU did not affect microsomal protein concentration or cytochrome P-450 content, it significantly decreased microsomal cytochrome c reductase and aniline hydroxylase activities. These changes in enzymatic activities were observed in microsomal preparations from either non-fasted or 24-hr fasted rats. Our results suggest that PTU-induced hypothyroidism modifies the metabolism and effectiveness or toxicity of concomitantly administered drugs.

Interindividual differences in the metabolism of xenobiotics.

Substantial interindividual differences occur for the metabolism of drugs, carcinogens, and steroid hormones, and these person-to-person differences are caused by genetic and environmental factors. It is likely that interindividual differences in the metabolism of xenobiotics and steroid hormones play a role in explaining interindividual differences in the initiation and progression of some human cancers. Factors that influence the metabolism and action of xenobiotics in human beings include age, disease states, hormonal changes in the body, ingestion of medicinal agents, exposure to environmental chemicals, and changes in life style, including factors such as cigarette smoking, alcohol consumption, and diet. Some individuals have a much larger response to environmental perturbations than do other individuals, and further research is needed to elucidate the reasons for differences in the responsiveness of people to modulators of chemical biotransformations. Immigration to a new country can represent a substantial change in a person's diet, life style, and environment, and these changes may alter the metabolism of xenobiotics and endogenous hormones that play a role in the carcinogenic response.

Prevention of TCDD toxicity in laboratory rodents by addition of charcoal or cholic acids to chow.

The mortalities of mice, rats and guinea-pigs given a single lethal dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were substantially reduced by feeding them with chows containing 2.5 or 5% activated charcoal, which presumably enhances elimination of the toxic agent from the organism. Chows with 0.25 or 0.5% cholic acid and dehydrocholic acid added had a similar protective action on mice intoxicated with TCDD. Since no treatment is currently available for this type of poisoning, charcoal-whose safe clinical use is well established-is worth considering in view of its potential benefit to humans acutely exposed to TCDD.

Effects of model traumatic injury on hepatic drug metabolism in the rat. II. In vivo metabolism of hexobarbital and zoxazolamine.

A previously validated small mammal trauma model, hind-limb ischemia secondary to infrarenal aortic ligation in the rat, was utilized to further investigate the effects of traumatic injury on hepatic drug metabolism in vivo. The pharmacokinetic sequelae of the previously observed post-traumatic decrease in cytochrome P-450 content were demonstrated, using hexobarbital and zoxazolamine as model cytochrome P-450-oxidized drugs. Sleeping times after ip or iv administration of these drugs was prolonged by model injury. Longer sleeping times appeared to be caused by a slower elimination of the drugs from the bloodstream rather than to an increased sensitivity to their central nervous system effects or to an alteration in plasma protein-binding. Detailed pharmacokinetic analyses by the two-compartmental open system model revealed that the major alteration in post-traumatic drug metabolism was decreased in vivo elimination rather than a shift in distribution. These studies further confirm the pharmacokinetic utility of this convenient small mammal trauma model in the study of post-traumatic derangements in hepatic drug metabolism and emphasize the toxicologic importance of these derangements.

Effects of model traumatic injury on hepatic drug metabolism in the rat. III. Differential responses of cytochrome P-450 subpopulations.

A previously validated small mammal trauma model, hind-limb ischemia secondary to infrarenal aortic ligation in the rat, was utilized to further investigate the effects of traumatic injury on the hepatic cytochromes P-450. In vitro drug metabolism studies with hexobarbital and zoxazolamine as substrates confirmed the post-traumatic depression of the cytochrome P-450-catalyzed oxidation of these drugs which was suggested by previous in vivo pharmacokinetic studies. Enzyme kinetic studies revealed diminished Vmax values with no change in Km, a finding which would seem to concur with the previously demonstrated decrease in hepatic cytochrome P-450 content after model trauma. Moreover, a battery of in vitro microsomal monooxygenase assays demonstrated that model trauma exerted a differential effect on various hepatic cytochrome P-450 isoenzymes. This phenomenon was confirmed by anion-exchange HPLC of solubilized hepatic microsomal hemoproteins. One of the most interesting aspects of this selective effect on cytochrome P-450 subtypes was the relative induction of cytochrome P-448 content and activity, in contrast to the variable decrease seen with cytochrome P-450 activities. The potential in vivo sequelae of this differential influence were suggested by changes observed in the urinary metabolic profile of antipyrine after model trauma.

Effect of acute and repeated chlordimeform treatment on rat hepatic microsomal drug metabolizing enzymes.

The effect of chlordimeform treatment on the hepatic microsomal drug metabolizing enzymes was examined in male and female rats following either acute or repeated treatment. After acute administration of chlordimeform (100 mg/kg, ip one hr prior to sacrifice) differential effects were observed in various parameters of the hepatic microsomal mixed function oxidase system with significant decreases in ethylmorphine metabolism, cytochrome P-450 content, NADPH cytochrome c reductase, and in the spectral binding of hexobarbital and aniline while no changes were found in the metabolism of aniline or p-nitroanisole. Durations of zoxazolamine-induced paralysis and pentobarbital-induced hypnosis were increased significantly after acute chlordimeform administration. Following repeated administration of chlordimeform (75 mg/kg ip for four days) to adult male rats, a decrease was observed in zoxazolamine-induced paralysis time while pentobarbital-induced hypnosis was not altered. Metabolism studies using isolated hepatic microsomal fractions showed a decrease rate of biotransformation of ethylmorphine and aniline while the activity of p-nitroanisole O-demethylase was not changed. No differences were found in cytochrome P-450 levels whereas microsomal spectral binding of hexobarbital was reduced while that of aniline was not affected. Following acute or repeated administration of chlordimeform to adult female rats, decreases in the hepatic microsomal metabolism of aniline, but not ethylmorphine or p-nitroanisole, were observed. Addition of chlordimeform to microsomal suspensions yielded a Type I spectral binding curve.

In vitro and in vivo activation of oxidative drug metabolism by flavonoids.

Several naturally occurring and synthetic flavonoids were studied for their effects on the metabolism of zoxazolamine to 6-hydroxyzoxazolamine. Flavone, nobiletin, tangeretin and 7,8-benzoflavone (50-250 microM) stimulated the metabolism of zoxazolamine by liver microsomes obtained from 5-day-old rats. Evidence was obtained indicating that flavone changed the apparent Km and Vmax values for zoxazolamine hydroxylation. The i.p. administration of 5 mumol of flavone, nobiletin, tangeretin or 7,8-benzoflavone concurrently with 740 nmol of zoxazolamine immediately stimulated the total body metabolism of zoxazolamine to 6-hydroxyzoxazolamine. The magnitude of the flavone-mediated increases in zoxazolamine hydroxylation in vivo was dependent on the dose of flavone and the dose of zoxazolamine administered. The i.p. administration of 5 mumol of flavone caused a 3- to 5-fold stimulation in the in vivo metabolism of 740 to 3000 nmol of zoxazolamine, but flavone had little or no stimulatory effect when 74 nmol of zoxazolamine were administered. Flavone stimulated zoxazolamine metabolism both in vitro and in vivo when control or phenobarbital-treated rats were used, but flavone inhibited the in vitro and in vivo hydroxylation of zoxazolamine when rats induced with 5,6-benzoflavone were studied. Although flavone activated zoxazolamine metabolism in vivo in neonatal rats, flavone did not activate the in vivo metabolism of benzo(a)pyrene. The in vitro addition of the hydroxylated flavonoids apigenin, chrysin, fisetin, morin and quercetin inhibited the hydroxylation of zoxazolamine by liver microsomes from neonatal rats, but studies with quercetin and apigenin indicated that these flavonoids had no effect on the in vivo metabolism of zoxazolamine.

Neonatal toxicity in mice associated with the Ahb allele following transplacental exposure to 3-methylcholanthrene.

This study was conducted to determine if the Ah genotype in mice could influence the incidence of neonatal mortality following maternal exposure to 3-methylcholanthrene (3-MC). Male F1 hybrids (Ahb/Ahd) produced from a cross of C57BL/6J and DBA/2J were backcrossed to DBA/2J females. This backcross mating resulted in noninducible pregnant mice (Ahd/Ahd) containing litters with a 1:1 ratio of AHH-inducible (Ahb/Ahd) and noninducible (Ahd/Ahd) fetuses. Dams were exposed by the oral route to 3-MC in corn oil at doses of 7, 21, or 63 mg/kg/day on Days 15, 16, and 17 of pregnancy, or to the positive control agent urethane at 1 mg/g ip on Day 17. The phenotype of surviving offspring was determined by zoxazolamine paralysis time and hepatic aryl hydrocarbon hydroxylase (AHH) activity measurements. Dose-related responses in all 3-MC treatment groups were obtained in measures of neonatal toxicity, i.e., number of litters surviving to term, litter size at birth, survival to weaning, and weight gain to 13 weeks of age. Correlation of the Ah phenotype with the neonatal toxicity data indicated that genetically responsive offspring had higher levels of neonatal toxicity than nonresponsive offspring within the same exposure groups. Thus, when fetuses are exposed in the same maternal environment to 3-MC, genetic differences in Ah genotype may influence the susceptibility to neonatal toxicity.