Effect of development on the activity of microsomal drug-metabolizing enzymes in rat liver

1. The activity of rat liver microsomal drug-metabolizing enzymes was determined at various ages between 6 and 100 days post natum. The enzymes studied were: aromatic hydroxylases by using as substrate biphenyl, which is metabolized by oxidation to 2- and 4-hydroxybiphenyl; nitroreductase by using p-nitrobenzoate as substrate, which is metabolized by reduction to p-aminobenzoate; glucuronyl synthetase by using 4-methylumbelliferone as the substrate, which is conjugated to give 4-methylumbelliferone glucuronide, and cytochrome P-450, which is regarded as the major terminal mixed-function oxidase in hepatic microsomal hydroxylations. 2. The activity of biphenyl 2-hydroxylase reached a peak at 21 days, biphenyl 4-hydroxylase and 4-methyl glucuronyl transferase at 24 days, cytochrome P-450 at 31 days, and p-nitrobenzoate reductase at 38 days of age. After the peak activity had been reached, the activity of each enzyme decreased with age, and in the case of biphenyl 2-hydroxylase the activity fell to a negligible value at 52 days of age. 3. Neither the addition of Triton X-100 to the incubation medium nor the treatment of the animals with phenobarbital resulted in any increase in the activity of biphenyl 2-hydroxylase at 52 days of age. 4. The activity of biphenyl 2-hydroxylase was threefold higher in rats fed on a synthetic diet than in rats fed on a commercial stock diet. 5. These findings are discussed.     

Effect of lithium on hepatic drug-metabolizing enzymes of protein-deficient rats

Protein deficiency was produced by feeding synthetic 8%-protein diet. Lithium carbonate at the dose level of 1.1g/kg diet was administered to normal and protein-deficient rats for a period of one mo. A significant inhibition in the levels of cytochrome (cyt) P₄₅₀, cyt b₅, glutathione (GSH), glutathione S-transferase (GST) and glutathione peroxidase (GPx), but an increase in γ-glutamyl transpeptidase (γ-GT), was observed in low-protein LP-fed rats. Lithium treatment to normal rats caused no significant change in the activities of cyt P₄₅₀, cyt b₅, GST, and GSH levels, whereas there was elevation in the activities of γ-GT and GPx and suppression in glutathione reductase (GRd) activity. Lithium administration to LP-fed rats resulted in significant increases in the hepatic γ-GT and GPx activities.     

Effect of alcohol and kolanut interaction on brain sodium pump activity in Wistar Albino rats.

Effect of alcohol-kolanut interaction on Sodium Pump activity in wistar albino rats was studied. Thirty wistar albino rats were divided into six groups of five (5) rats per group and used for the study. The control group (1) received via oral route a placebo (4ml of distilled water). Groups 2 to 6 were treated for a period of 21 days, with (10% v/v) of alcohol (group 2), 50mg/kg body weight of kolanut (group 3), 50 mg/kg body weight of caffeine (group 4), 4ml of 10% v/v of alcohol and 50 mg/kg body weight kolanut (group 5), 4ml of 10% v/v of alcohol and 50 mg/kg body weight of caffeine in 4.0ml of the vehicle via gastric intubation respectively. A day after the final exposure, the brain of each rat was harvested and processed to examine several biochemical parameters, i.e., total ATpase, ouabain-insensitive ATpase, ouabain sensitive ATpase (Na(+)-K(+)ATPase), non-enzymatic breakdown of ATP and inorganic phosphate (Pi) released. The results showed that the essential enzyme of the brain responsible for neuronal function, Na(+)-K(+)ATPase, was inhibited by alcohol-kolanut co-administration relative to control, resulting in a decrease in Na(+)-K(+)ATPase activity, ATP production, ion transport and action potential, leading to loss of neuronal activities.     

Unimpaired induction of drug-metabolizing enzymes in hepatocytes isolated from rats with micronodular cirrhosis

To test further the competence of the cirrhotic liver to metabolize xenobiotics, hepatocytes were isolated from control and CCl4-induced cirrhotic male or female rats. Histologically micronodular cirrhosis was present in all CCl4-treated rats, while control rats had normal livers. Portal perfusion pressure and intrahepatic collagen content were also significantly increased by CCl4 administration. In male rats, no significant differences in levels of circulating transaminases nor in alkaline phosphatase was observed between cirrhotic and control rats, while CCl4-treated females had slightly higher than normal serum transaminase levels at the time of the studies. Hepatocytic cytochrome P-450 and basal xenobiotic biotransformation were unaffected by micronodular cirrhosis in both genders; calculation of the aminopyrine and 7-ethoxycoumarin intrinsic clearances (Cli) revealed, however, a slightly decreased transformation potential in hepatocytes obtained from cirrhotic females, a phenomenon not observed in cirrhotic male rats. It is speculated that the observed reduction in Cli may have been independent of cirrhosis per se, owing to the perduring cytotoxic effect of CCl4 as evidenced by the higher than normal level of transaminases in female rats. Finally, male rats were subjected to in vivo administration of phenobarbital or 3-methylcholanthrene; both compounds led to significant induction of the mixed-function oxidase system, which was similar in magnitude and in selectivity in control and cirrhotic rats as illustrated by calculation of the Michaelis-Menten kinetic parameters for aniline p-hydroxylation, aminopyrine-N-demethylation, 7-ethoxycoumarin-O-deethylation, and p-nitrophenol UDP-glucuronyl transferase. We conclude that in well-established but compensated and hepatolysis-free micronodular cirrhosis, hepatocytes are fully able to transform xenobiotics and to respond normally and selectively to inducers of drug metabolism.     

Effect of praseodymium chloride on liver microsomal enzymes of rats

A single i.v. dose (5 mg/kg) of a light lanthanon, praseodymium, prolonged the duration of hexobarbital-induced sleep and zoxazolamine-induced paralysis, as well as it modified pharmacokinetic parameters of hexobarbital and zoxazolamine, in rats. Half-lives (t1/2) and area under the curve (AUC) were increased, while elimination coefficient (beta) and clearance (Cl) were decreased. However, in daily doses of 1 mg/kg i.p. for 15 days, praseodymium did not alter pharmacological effects and pharmacokinetic parameters. The in vitro hydroxylation of hexobarbital and zoxazolamine by liver microsomes was inhibited when the animals were treated previously with a single i.v. dose (5 mg/kg) of praseodymium chloride. In these animals, the amount of cytochromes P-450 and b5 were reduced significantly, whereas that of NADPH-cytochrome c reductase remained unchanged. The pretreatment of animals with phenobarbital normalized the microsomal enzyme impairment caused by praseodymium.     

Comparative characteristics of psychoemotional patterns in Albino and Wistar rats

Analysis of psychoemotional behavioral component in Albino and Wistar rats during solving a cognitive task showed that intra-population differences were determined by the relationship between passive and active forms of unconditioned reactions. Quick-learning rats from both populations were characterized by the dominance of active forms of psychoemotional reactions over the course of training. The learning process in slow-learning rats was accompanied mainly by passive emotional manifestations, whereas active reactions dominated at the stage of habit realization. Passive forms of manifestations prevailed over active forms over the course of training in both Albino and Wistar rats which failed to solve the task. The interpopulational psychoemotional differences were quantitative and depended only on the learning styles. Wistar strain was characterized by a decreased percent of animals which were able to solve a cognitive task (40% versus 80% in Albino rats), affective flatness and decreased tolerance to information loadings.     

Effect of different detergent systems on the molecular size of UDP glucuronosyltransferase and other microsomal drug-metabolizing enzymes

Mouse liver microsomes were solubilized in various detergent systems, and the resulting aggregate structures associated with cytochrome P-450, cytochrome c reductase, and UDP glucuronosyltransferase were sized by gel filtration chromatography. Cholate or its derivative, CHAPS, in combination with Emulgen 911 or Lubrol 12A9 were necessary to generate a particle of about 140 k daltons, the smallest structure associated with cytochrome P-450. Cholate or CHAPS alone was sufficient to generate a minimally sized aggregate of 200 k daltons associated with NADPH cytochrome c reductase activity. Cholate in combination with Emulgen 911 or Lubrol 12A9 generated particles of about 280 k daltons associated with UDP glucuronosyltransferase activity. CHAPS alone also generated similarly sized particles under conditions in which UDP glucuronosyltransferase activity toward 1-naphthol and morphine was two to about twenty times greater, respectively, than with the combination of detergents. This finding suggests that the zwitterionic CHAPS is superior to other detergent systems for studies concerned with the purification of transferase enzymes, a microsomal system in which investigation of the number of different forms has been hampered by the instability of the enzyme upon solubilization and subsequent manipulation.     

Expression and induction of drug-metabolizing enzymes in cultured fetal rat hepatocytes

Anin vitro experimental model, fetal rat hepatocytes in culture, was metabolically characterized. Several enzymatic activities were expressed in these hepatocytes, namely, testosterone hydroxylations. Hepatocytes cultured up to 3 weeks in the presence of dexamethasone and phenobarbital still expressed some drug-metabolizing enzyme activities (e.g., ECOD). The enzymatic activities were measured both directly on monolayers during culture and on the corresponding harvested and homogenized cells. The results correlate perfectly with each other. The on cell procedure allows us to repeat the assay or to measure several activities on the same cells at different time intervals. The presence of dexamethasone in the culture medium allows the expression and the induction of several cytochrome P450 isoenzymes, namely, those hydroxylating testosterone. This makes the model particularly attractive for induction experiments as well as for metabolic or toxicological studies needing longer treatments.Abbreviations BA benzanthracene - CLO clofibric acid - DEXA dexamethasone - DMSO dimethylsulfoxide - ECOD ethoxycoumarin-O-dethylase - PB phenobarbital - RER rough endoplasmic reticulum     

Genetically mediated induction of drug-metabolizing enzymes associated with congenital defects in the mouse.

Various polycyclic aromatic compounds induce certain monooxygenase activities, including aryl hydrocarbon (benzo[a]pyrene) hydroxylase (EC 1.14.14.2), and cytochrome P1-450 in the liver and many nonhepatic tissues of the mouse. This induction process is controlled by the Ah locus. Genetic differences that have been shown in the past to be associated with the Ah locus include an increased susceptibility to chemical carcinogenesis, mutagenicity in vitro, and drug toxicity--manifested as hepatic necrosis, aplastic anemia, or shortened survival time. Pregnant mice received a single injection of 3-methylcholanthrene or 7,12-dimethylbenz[a] anthracene between day 5 and day 13 of gestation, and the uterine contents were examined on day 18. Striking increases were observed in the incidence of MC-1 and DMBA-induced resorptions and congenital malformations in the aromatic hydrocarbon "responsive" C57BL/6N inbred strain, and of DMBA-induced resorptions in the "responsive" C3H/HeN and BALB/cAnN strains--when compared with the similarly treated genetically "nonresponsive" AKR/N strain. These data suggest but do not prove that an association exists between the Ah locus and developmental toxicity, i.e., teratogenesis. Although numerous teratogenic differences among inbred mouse strains have previously reported, this study is unique in that the genetic differences in teratogenicity observed were predicted in advance on the basis of known differences among these strains in polycyclic hydrocarbon metabolism regulated by the Ah locus.     

Regulation of microsomal enzymes by phospholipids VI. Abnormal enzyme-lipid interactions in liver microsomes from gunn rats

The rates of synthesis of some glucuronides by liver microsomes from the Gunn strain of rat are abnormally low, but previous investigators of the activity of the p-nitrophenol metabolizing form of UDPglucuronyltransferase (UDPglucuronate glucuronyltransferase, EC 2.4.1.17) have reported normal levels of activity in these animals. Data presented in this paper indicate, however, that this enzyme is abnormal in Gunn rats. Thus, treatment of liver microsomes from normal Wistar rats with phospholipase A (EC 3.1.1.4) or Triton X-100 increases the activity of the p-nitrophenol metabolizing form of UDPglucuronyltransferase 10- and 20-fold, respectively, but these agents do not alter activity in microsomes from homozygous Gunn rats. Similarly, phospholipase A and Triton X-100 activate the o-aminophenol and o-aminobenzoate metabolizing forms of UDPglucuronyltransferase in microsomes from normal rats, but are without effect on the enzyme in microsomes from Gunn rats. In contrast, the rates of synthesis of o-aminophenyl- and o-aminobenzoylglucuronides are increased several fold by addition of diethylnitrosamine to microsomes from Gunn rats indicating that the maximum potential activities of UDPglucuronyltransferases are constrained in liver microsomes from both normal and Gunn rats.These data indicate that assays of UDPglucuronyltransferase in native microsomes are not sufficient for delineating the full extent of the defect in the Gunn rat, that there are defects in the function of at least two proteins in liver microsomes from these animals, and that there are abnormal interrelations between some forms of microsomal UDPglucuronyltransferase and their phospholipid environments.     

Variations in induction of drug-metabolizing enzymes by trans-Stilbene oxide in rodent species

trans-Stilbene oxide (400 mg/kg) produced a 500% increase in the microsomal in the microsomal epoxide hydratase activity in rat and mouse with little change in the soluble enzyme activity. However, in guinea pig, the soluble epoxide hydratase activity increased by about 33% with only a small increase (47.6%) in the microsomal enzyme activity. The soluble glutathione S-transferase activities were also induced in both rat and mouse, with little change in that of the guinea pig. Increasing dosage of trans-stilbene oxide from 400 mg/kg to 1000 mg/kg had little effect on the above enzyme activities. That the guinea pig was not relatively refractory to all inducing agents was shown by the fact phenobarbital (100 mg/kg) and 3-methylcholanthrene (25 mg/kg) produced relatively similar increases in the activities of aniline hydroxylase and P-aminopyrineP-demethylase in rat, mouse and guinea pig. However, these inducers produced only a 15–20% stimulation in the soluble glutathione S-transferase and microsomal epoxide hydratase activities in guinea pig, when compared to a 50–80% increase in rat and mouse, suggesting a general resistance to induction by the phase II enzymes in guinea liver. In all animal models, the inducer markedly increased th emicrosomal total phospholipid content, although the sphingomyelin content itself was decreased. In both rat and mouse, the microsomal cholesterol content was significantly decreased while that in guinea pig was unaffected. Possible factors responsible for the observed species differences are discussed.     

Aryl hydrocarbon receptor-mediated induction of microsomal drug-metabolizing enzyme activity by indirubin and indigo

Indirubin and indigo, which are thought to be natural ligands for aryl hydrocarbon receptor (AhR), showed marked AhR ligand activities in a reporter gene assay using recombinant yeast. Their activities were comparable with or more potent than that of 2,3,7,8-tetrachlorodibenzo-p-dioxin. When indirubin and indigo were administered to mice, ethoxyresorufin-O-dealkylase and methoxyresorufin-O-dealkylase activities in the liver were increased, but subsequently decreased within 2 days. Indirubin was more potent than indigo. Levels of cytochrome P450 1A1/2 proteins and mRNAs in the liver of mice dosed with indirubin were also enhanced. These enhancing effects of indirubin and indigo were not observed in AhR knock-out mice. Ethoxyresorufin-O-dealkylase and methoxyresorufin-O-dealkylase activities in rat hepatocytes and HepG2 cells were enhanced by the addition of indirubin or indigo, but less potently than by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Indigocarmine, a sulfate derivative of indigo, which is used as food additive, did not show these inducing effects on drug-metabolizing enzymes. Our results suggest that indirubin and indigo act as inducers for cytochrome P450 1A1/2 mediated by AhR in mammals in vivo.