Hepatic cytochrome P450 and flavin-containing monooxygenase in male Nts:Mini rat, a transgenic rat carrying antisense RNA transgene for rat growth hormone.

We investigated the characteristics of hepatic cytochrome P450s and flavin-containing monooxygenase 1 (FMO1) in male Nts:Mini rats, a Wistar/Jcl-derived transgenic rat strain showing less plasma GH concentration than the parental strain. The total hepatic P450 contents of Mini rats were significantly reduced. A suppression was observed in the activities and protein expression of male-specific P450s (CYP3A and CYP2C11) and was speculated to be a potential cause of the reduction in total P450 contents. The activity and protein expression of CYP2B1 were suppressed and those of CYP2E1 and CYP2B2 were enhanced. With the exception of our data on CYP2B1, these results largely agreed with previous reports concerning GH-depletion rat models (hypophysectomized rats, rats neonatally treated with glutamate, and dwarf rats), implying that the changes in Mini rats were caused by GH insufficiency. The liver FMO1 protein expression in Mini rats was higher than that in Wistar rats but the activity was comparable, suggesting that GH is not a positive regulator of FMO expression. With their insufficient but not depleted levels of plasma GH, Mini rats may thus become another candidate for use in the investigation of GH regulation of hepatic mixed-function monooxygenases.

Assessment of benzydamine N-oxidation mediated by flavin-containing monooxygenase in different regions of rat brain and liver using microdialysis.

N-Oxidation of benzydamine (BZY) mediated by flavin-containing monooxygenase (FMO) was evaluated by microdialysis in vivo in different regions of rat brain and liver. The probe was implanted into local regions of the brain, such as the olfactory bulb, cerebral cortex, corpus striatum, hippocampus and cerebellum, or the hepatic lobe. By perfusing BZY via the probe, BZY N-oxide was identified in the dialysate. The estimated concentrations of BZY N-oxide in extracellular fluid were almost the same as those in the olfactory bulb, hippocampus and cerebral cortex, half the concentration in the hepatic lobe; however, the concentration in the corpus striatum was lower and that in the cerebellum was higher than in the other regions. These results demonstrate that the extracellular concentration of BZY N-oxide formed in vivo was unexpectedly high in every brain region.

Flavin-containing monooxygenase mediated metabolism of benzydamine in perfused brain and liver.

Benzydamine (BZY) N-oxidation mediated by flavin-containing monooxygenase (FMO) was evaluated in perfused brain and liver. Following 20 min of perfusion with modified Ringer solution, the infusion of BZY into brain or liver led to production of BZY N-oxide. BZY N-oxide, a metabolite of BZY oxidized exclusively by FMO, was mostly recovered in the effluent without undergoing further metabolism or reduction back to the parent substrate. The BZY N-oxide formation rate increased as the infusion concentration of BZY increased both in perfused brain and perfused liver. BZY N-oxidation activities in perfused rat brain and liver were 4.2 nmol/g brain/min and 50 nmol/g liver/min, respectively, although the BZY N-oxidation activity in brain homogenates was one 4000th that in liver homogenates. This is the first study of FMO activity in brain in situ.

Differences in enzymatic properties of flavin-containing monooxygenase in brain microsomes of rat, mouse, hamster, guinea pig and rabbit.

To characterize flavin-containing monooxygenase (FMO) in brain microsomes of rat, mouse, hamster, guinea pig and rabbit, profiles of its enzyme activities were investigated by HPLC-fluorometrical assay using benzydamine (BZY) as a substrate. The optimum pH of BZY N-oxidation activity in brain microsomes from rat, mouse, hamster or guinea pig was between 8.5 and 9, though that of the rabbit brain microsomes was near 10.0. The activities were thermally unstable in brain microsomes from all the species examined in the absence of NADPH, but the activity in rabbit brain microsomes was rather thermostable in the presence of NADPH. The activity in rabbit brain microsomes was depressed in the presence of 5 mM n-octylamine. In the presence of MnCl2, the enzyme activity in rabbit brain microsomes was markedly decreased. ZnCl2 markedly decreased the enzyme activities in brain microsomes from rat, mouse and rabbit. It was concluded that BZY N-oxidation activity in brain microsomes from rat, mouse, hamster and guinea pig are similar in enzymatic properties but different from the activity of that of rabbit. These results suggest that common FMO isoform(s) other than FMO4 might exist in the brains of the four rodent species tested.

Partial purification and substrate specificity of flavin-containing monooxygenase from rat brain microsomes.

Flavin-containing monooxygenase (FMO) was partially purified from rat brain microsomes through two successive chromatographies on columns of DEAE Sepharose and 2',5'-ADP Sepharose. The specific activity, benzydamine N-oxidation of partially purified brain FMO, was 122-fold higher than that of microsomes. A single band of 60 kDa was recognized by Western blotting analysis with anti-rat liver FMO. The Km value of brain FMO for thiourea was 4-fold lower, but that for cysteamine was 10-fold higher than that of liver FMO. The enzymatic activity for n-octylamine was detected in neither brain nor liver FMO. Kinetic analysis for neurotoxins also revealed that Km values of brain FMO for 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 1,2,3,4-tetrahydroisoquinoline (TIQ) and N-methyl TIQ (NMTIQ) were lower than those of liver FMO. These results indicate that rat brain FMO catalyzes several substrates of liver FMO involving neurotoxins, but it seems likely that the kinetic properties of brain FMO are somewhat different from those of liver FMO.

Determination of flavin-containing monooxygenase activity in rat brain microsomes with benzydamine N-oxidation.

The activity of flavin-containing monooxygenase (FMO) in rat brain microsomes was measured by fluorometrical determination of benzydamine (BZY) N-oxygenation with HPLC. The apparent Km value for the formation of BZY N-oxide from BZY by brain microsomes was similar to that by hepatic microsomal fraction or purified FMO, but the Vmax value for brain microsomes was about one-hundredth of that for hepatic microsomes. BZY N-oxygenation activity by brain microsomes was at a maximum near pH 8.5, slightly more acidic than the optimum pH for liver FMO. BZY N-oxygenation activity was inhibited completely by heat inactivation and markedly in the presence of 1 mM thiourea, but slightly in the presence of 1 mM SKF-525A, and it was only barely activated in the presence of 5 mM n-octylamine, a positive effector of liver FMO. The addition of rabbit antisera raised against rat liver FMO resulted in 30% inhibition of BZY N-oxygenation by solubilized brain microsomes. Compared with microsomes from five different brain regions, the activity was highest in microsomes of olfactory bulbs. These results show that the activity of FMO in rat brain is distinctly determined by BZY N-oxygenation.

An assay of flavin-containing monooxygenase activity with benzydamine N-oxidation.

An assay system of flavin-containing monooxygenase was developed by fluorometric determination of benzydamine (BZY) N-oxidation with HPLC. The apparent Km value for the formation of BZY N-oxide from BZY by rat liver microsomes was similar to that by purified FMO. The Km and Vmax values for the formation of N-desmethylbenzydamine (Nor-BZY) by rat liver microsomes were about 50 times greater and 2000 times less, respectively, than those of BZY N-oxide. Nor-BZY was not formed upon incubation with purified enzyme. BZY N-oxidation activity was completely inhibited both in the absence of NADPH and by heat inactivation. The reaction was inhibited in the presence of 0.5 mM thiourea, but 2 mM SKF-525A did not affect BZY N-oxidation. Moreover, rabbit antibody raised against the rat enzyme inhibited BZY N-oxidation. These results are in accord with a simple, rapid, and sensitive assay for the enzyme.

Effects of mushroom toxins on glycogenolysis; comparison of toxicity of phalloidin, alpha-amanitin and DL-propargylglycine in isolated rat hepatocytes.

The effects of phalloidin and alpha-amanitin as toxins of Amanita species and DL-propargylglycine identified from A. abrupta on the glycogenolysis in isolated rat hepatocytes were investigated. Phalloidin decreased glycogen content and activated phosphorylase a activity remarkably. alpha-Amanitin also decreased glycogen content significantly but activated phosphorylase a activity slightly. DL-Propargylglycine slightly affected glycogenolysis. Phalloidin, which most affected glycogenolysis among the three compounds mentioned above, elevated cytosolic Ca2+ concentration ([Ca2+]i) and 45Ca uptake into cells. Phalloidin depressed slightly 3H-inositol incorporation into phosphatidylinositol (PI) and remarkably phosphatidylinositol 4,5-bisphosphate (PIP2) but increased phosphoinositides breakdown. These results suggest that phalloidin alters phosphoinositides turnover and intracellular Ca2+ homeostasis, subsequently activates phosphorylase a, resulting in glycogenolysis in isolated rat hepatocytes.

In vitro toxicity test of poisonous mushroom extracts with isolated rat hepatocytes.

Effects of poisonous mushroom extracts on isolated rat hepatocytes were studied. Though no significant decrease in the cell viability was observed during the incubation of hepatocytes with the extracts at a concentration of 5% (v/v) of Amanita abrupta, A. gymnopus, and A. virosa caused marked decreases in the intracellular glutathione content in sharp contrast to the extracts of A. volvata and A. flavipes. Comparative toxicity tests were carried out for the effects of the extract of A. abrupta, dl-propargylglycine, and alpha-amanitin. The extract of A. abrupta at a concentration of 1% (v/v) caused a marked decrease in the glycogen content, a noticeable elevation in the phosphorylase alpha activity, and a slight acceleration of lipid peroxidation in the hepatocytes. Although dl-propargylglycine decreased the intracellular glutathione content progressively with the incubation time, a significant effect of the chemical on lipid peroxidation and the glycogen content was observed only after prolonged incubation at a concentration of 5 mM. On the other hand, alpha-amanitin exerted a little effect on the hepatocytes at 1 microM. These results have indicated that the intoxication by the extract of A. abrupta on the hepatocytes might not due to independently each component, dl-propargylglycine and alpha-amanitin, but combined effect of these components or unidentified substances.

Zinc transport during hemodialysis.

Zinc transfer during hemodialysis and plasma zinc concentrations in hemodialysis patients were examined. Fifteen volunteer outpatients undergoing hemodialysis showed significant increases in plasma zinc from 74.0 +/- 7.8 to 88.1 +/- 9.7 micrograms/dl after a 5-h dialysis. The increase was mainly the result of hemoconcentration as evidenced by a significant increase in the hematocrit and total serum protein during dialysis, but was also due to diffusion. To study the changes resulting from diffusion, zinc was measured in the arterial blood and in the dialysate at the inflow and outflow sites of the dialyzer. There was a significant (p less than 0.01) increase in the plasma zinc of the arterial blood from 74.7 +/- 8.1 to 80.2 +/- 6.5 micrograms/dl, but a nonsignificant decrease in the dialysate zinc from 10.6 +/- 2.5 to 9.5 +/- 5.9 micrograms/dl. Zinc diffused across the dialyzer from the dialysate to the blood in 12 cases and into the dialysate in three others.