• Exercise-induced inhibition of hepatic drug metabolism and a mechanism for decreased activity of P450IIE1 in Fischer-344 rats

      Day, Wesley Warren; Weiner, Myron, Ph.D. (1993)
      Clinical studies have appeared in the literature suggesting that exercise affects the pharmacokinetics of numerous drugs. However, there have been a minimal number of carefully controlled animal studies which have used only a few substrates and have lacked specific hepatic biochemical (enzymatic) determinations; thus, definite conclusions could not be drawn. In light of these facts, a series of experiments were designed to characterize, quantify and test a mechanism for the effects of exercise on components of the hepatic drug metabolizing systems in young, middle-aged (M-A) and aged rats exercised by either treadmill running or swimming. Running resulted in a dramatic substrate specific inhibition on P450 content and activity in all age groups. Furthermore, running resulted in complete protection from CCl{dollar}\sb4{dollar} toxicity (CCl{dollar}\sb4{dollar} is activated by P450) in young and M-A rat hepatocytes incubated with this solvent. Results of in vivo HB sleep time studies in young rats (sleep time is dependent on P450-mediated metabolism of HB) also correlated with the running-induced inhibition of microsomal P450 in vitro. With regard to swimming exercise, results indicate that exposure to water confounds the results, and when appropriate controls were considered, no effect on any microsomal parameter was found. Glucuronidation of pNP was increased with running whereas sulfation was unaffected in young and M-A rat hepatocytes. After 8 wk of running, metabolism of aniline and HB is significantly inhibited; however, after 3 wk, in vivo studies show HB metabolism is significantly declined, while in vitro studies reveal no effect on aniline metabolism. Since each substrate is metabolized by separate isozymes, different mechanisms may be involved. Investigations into a possible mechanism for running-induced inhibition of aniline metabolism and CCl{dollar}\sb4{dollar} toxicity (both are selectively metabolized by P450IIE1) has revealed a decrease in the amount of apoprotein and mRNA which are ultimately responsible for the formation of the active isozyme. These studies indicate that exercise has isozymal-selective effects on drug metabolism and may also be useful as a probe for studying P450 regulation and function. Furthermore, since P450IIE1 is responsible for the activation of nitrosamines which are a major group of carcinogens, exercise-induced inhibition of P450IIE1 may be related to reports of decreased cancer with regular exercise. However, this remains to be determined.
    • Involvement of immune system mediators in exercise-mediated changes of hepatic cytochromes P450

      Melendez, Victor, Jr.; Weiner, Myron, Ph.D. (1998)
      The physiological changes leading to exercise-mediated inhibition of hepatic cytochromes P450 have not been characterized. There is clinical evidence of increased release of cytokines (mediators of the immune system) after exercise. In addition, the modulation of P450 enzymes by nitric oxide (NOdot), another mediator of the immune system, has been demonstrated both in vivo and in vitro. Therefore, a series of experiments were designed to characterize the involvement of the immune system in exercise-mediated changes in P450. These experiments further evaluated the independent effects of exercise on the components of phase I-mediated drug metabolism. In vitro studies demonstrated that Spermine/NO (an NOdot donor) dramatically inhibited microsomal activity towards several P450 isozyme-specific substrates. In vivo studies with rats showed that treadmill running also resulted in an inhibition of P450 enzymatic activity towards substrates specific for P4502 family. These inhibitions were associated with a significant decrease in P450 reductase enzymatic activity and only small decreases in enzyme expression. Treatment of exercising rats with low levels of the antiinflamatory dexamethasone resulted in partial reversal of the inhibition. Dexamethasone alone produced no significant changes on the parameters measured. In addition, exercise was associated with an increase in the hepatic expression of the inducible form of nitric oxide synthase. These data support our hypothesis that the immune system is activated as a result of exercise. Further, the data support that exercise-induced alterations in immune mediators (i.e. NOdot) inhibit specific P450 metabolic pathways.
    • Liver slice technology: An in vitro model for integrated biotransformation

      Thohan, Sanjeev; Weiner, Myron, Ph.D. (1998)
      The objective of this work was to examine the use of tissue slices as an in vitro model for the study of integrated drug metabolism (biotransformation). Liver slices are more representative of the in vivo situation with respect to cellular architecture and diversity than hepatocytes or subcellular fractions. This work has demonstrated the effects of model inducing agents on hepatic phase I-phase II integrated drug metabolism using alkoxycoumarin derivatives. Additionally, this work detailed the development of an in vitro model for novel compound screening using acetaminophen and paraquat as model toxicants. Dynamic incubation of liver slices was found to be superior to a linear incubation over a 2 hour incubation. Biotransformation was assessed with the use of 7-methoxy-, 7-ethoxy- and 7-hydroxycoumarin (7-HC). The major metabolite of O-dealkylase activities of cytochrome P 450, 7-HC, was conjugated with glucuronic acid or sulfate moieties in a capacity limited fashion. Total phase II activity was assessed using 7-HC as the primary substrate and found to be 7-fold higher than total phase I activity. Phenobarbital pretreatment induced liver slice O-demethylase and O-deethylase activities 3.1- and 3.6-fold over control values, respectively. Glucuronosyl transferase activity for 7-HC was found to be increased over sulfation in all metabolite profiles by pretreatment with phenobarbital. 3-methylcholanthrene pretreatment showed a novel induction profile with a 1.8-fold increase in O-demethylase activity and 9-fold increase in O-deethylase activity over control rat liver slices. Increased levels of free 7-HC from O-dealkylase activities indicated possible substrate competition from persistence of 3-methylcholanthrene or its hydroxylated metabolites in the liver slice. The utility of liver slices as a paradigm to investigate cytotoxicity by intracellular enzyme release was detailed with the use of acetaminophen and paraquat. Phenobarbital was shown to potentiate mitochondrial damage by oxidative mechanisms. These studies highlight the utility and versatility of liver slices in the analysis of biotransformation and bioactivation. Implementation of the tissue slice system as a routine screen may facilitate drug discovery and development.
    • Melatonin inhibition of hepatic drug metabolism in rats and pigs

      Niwatananun, Kanokporn; Weiner, Myron, Ph.D. (2000)
      Melatonin, the pineal hormone, has now been widely used for various indications. Melatonin is principally metabolized by the cytochrome P450 system (CYP). To date, limited information regarding the effect of melatonin on hepatic drug metabolism has been provided. Therefore, this study was undertaken to examine the effect of melatonin on hepatic drug metabolism in Sprague-Dawley and Holtzman rats and in pigs. The in vitro studies in Sprague-Dawley rats and pigs clearly suggest that melatonin inhibited the activity of CYP1A1/2, 2B1, 2D1 and 3A2, whereas no inhibition was found on CYP2E1-catalytic activity. Melatonin showed a similar inhibitory profile on CYP-catalyzed metabolism in male and female Holtzman rats. Maximal inhibition was found with nonselective metabolism (e.g., 7-pentoxyresorufin O-depentylation, IC50 =104 muM) in rats. Melatonin preferentially inhibited CYP1A1 activity with IC50 values of 319 and 374 muM in pigs and rats, respectively. Subsequent studies examined the mechanism by which melatonin inhibited CYP activity and whether melatonin influenced the integrated phase I/phase II metabolism using liver slices from Sprague-Dawley rats. Melatonin inhibition was a competitive type toward CYP3A-catalyzed reactions in Sprague-Dawley rats and pigs. The findings from liver slices suggest that melatonin affects exclusively phase I metabolism. In vivo studies revealed that melatonin had no effect on total CYP content and NADPH-cytochrome c reductase activity, regardless of sex and strain of rats, dosage regimen or routes of administration (i.p. or oral). In microsomes from melatonin-treated rats, melatonin failed to inhibit the metabolism of most substrates, the exception being cortisol 6beta-hydroxylation catalyzed by CYP3A enzymes in male Holtzman rats. In humans, melatonin may interact with drugs catalyzed by CYP1A1/2 and 2B6 enzymes. Melatonin seems to be less likely to cause an interaction with drugs metabolized by the major isozymes responsible for drug metabolism in humans (e.g., CYP3A4, 2D6, 2C9). However, inhibition of some isozymes may be important, as implied by melatonin inhibition on CYP1A1/2 activity which may be involved in cancer prevention. Further studies using human samples and co-administration of melatonin with selective CYP-substrates (e.g., erythromycin) may provide more information on hepatic drug metabolism in humans.