Helmut Greim

[109] Methods for the elevation of hepatic microsomal mixed function oxidase levels and cytochrome P-450

Publisher Summary The chapter describes the methods for the elevation of hepatic microsomal mixed function oxidase levels and cytochrome P-450. Many substances have been shown to elevate the content of the mixed function oxidase activity of liver microsomes; these range from barbiturates to tranquilizers, insecticides, and polycyclic hydrocarbons such as 3,4-benzpyrene and 3-methylcholanthrene. The increase in oxidative activity is associated with an increase in the microsomal content of cytochrome P-450. The time necessary to achieve maximal levels of microsomal mixed function oxidase activities varies with the inducer compound used. Methylcholanthrene or benzpyrene treatment exerts a maximal effect within 24 hours. After one injection of DDT, the maximal activity is achieved after 1 or 2 weeks. When phenobarbital is used, maximal enzyme activity toward all substrates is reached after 3-5 days of treatment. Three procedures, which increase the level of liver microsomal mixed function oxidase activity and cytochrome P-450, are described. One method uses Phenobarbital as the inducer; a second uses 3-methyleholanthrene or 3,4-benzpyrene; and the third method employs DDT. The chapter also discusses the preparation of microsomes.

The influence of phenobarbital on the turnover of hepatic microsomal cytochrome b5 and cytochrome P-450 hemes in the rat.

Abstract The turnover of 14 C-labeled heme in steady state phenobarbital-induced rats was compared with the turnover of the same hemes in control rats. The half-lives of cytochrome b 5 and cytochrome P-450 hemes in the steady-state induced animals were unchanged (45 and 22 h, respectively). Induction of cytochrome P-450 was found to be caused by an increased rate of synthesis, measured by the rate of δ-amino[4- 14 C]levulinate incorporation into heme in the early stages of phenobarbital treatment. When the level of hemoproteins during phenobarbital administration had reached a steady-state induced level, the rate of home destruction was elevated to balance the induced rate of synthesis.

Synthesesteigerung und Abbauhemmung bei der Vermehrung der mikrosomalen Cytochrome P-450 und b-5 durch Phenobarbital

SummaryHeme moieties of microsomal rat liver cytochromes P-450 and b-5 were labeled with.14C-Aminolevulini cacid. The half life of the b-5 heme radioactivity was found to be 45 hrs, that of the P-450 heme radioactivity was 22 hrs.Treatment of fed rats with Phenobarbital (80 mg/kg i.p. and 1‰ PB in the drinking water) for 48 hrs increased the concentration of cytochrome P-450 up to 200%, only by induced synthesis. In starved rats treated with Phenobarbital, P-450 concentration was increased up to 400%, by both induced synthesis and inhibition of breakdown.Microsomal P-450 cytochrome was determined in rat liver homogenate and in suspensions of rat liver microsomes. The amount of P-450 obtained in the isolated microsomal fraction was compared with the P-450 content in the liver homogenate. Since P-450 is a microsomal hemoprotein this relation can be correlated with the microsomal protein, in order to to calculate the real content of microsomal protein in the liver homogenate. It was found to be 65 mg/g of liver, demonstrating, that 31% of the 209 mg of total protein/g of liver consists if microsomal protein.

On the problem of possible other forms of cytochrome P450 in liver microsomes

The absolute spectrum of cytochrome P450 is shown. The addition of a known substrate is shown to alter the absolute spectrum of this hemoprotein. The spectrum of P450 induced by polycyclic hydrocarbons appeared similar to that of the hemoprotein in the presence of substrate. This similarity is shown to be due to the binding of the inducer and/or its metabolites to the hemoprotein by the ability to displace them and restore the absolute spectrum to that of the native hemoprotein. This study indicates that P450 exists in only two forms, the native enzyme and the enzyme-substrate complex in prepared microsomes.

Cholestatic steroid hormones inhibit taurocholate uptake into isolated rat hepatocytes

Abstract The effect of three cholestatic steroids (norethandrolone, 17-β-estradiol and progesterone) on hepatocellular uptake and secretion of taurocholate was studied in isolated rat liver cells. The steroids decreased the rate of taurocholate uptake. Norethandrolone inhibited uptake noncompetitively with a Ki of 18 μM, but had no effect on the activation energy of uptake. 17-β-estradiol and progesterone reduced taurocholate uptake by 50 per cent at concentrations between 40 μM and 50 μM. The secretion of taurocholate from taurocholate-loaded cells was slightly increased by all three steroids at concentrations below 100 μM. A 60 per cent inhibition of secretion was observed in the presence of 500μM norethandrolone. Interference of cholestatic steroids with hepatocellular bile acid uptake may be an important step in the pathogenesis of intrahepatic cholestasis.

Stimulierung der Oxydation von Fremdstoffen in Nierenmikrosomen durch Phenobarbital

SummaryPretreatment of rats and rabbits with Phenobarbital (80 mg/kg and 60 mg/kg respectively) caused a 30% increase in the liver weight relative to body weight. There was a smaller increase in the kidney weight (7% in rats and 10% in rabbits) which was not statistically significant. The yields of microsomes per gram of liver was also increased by about 90% and 60% in rats and rabbits respectively, but only a slight increase was observed for kidneys (12% and 18% respectively).Pretreatment with Phenobarbital, 3,4-Benzpyrene or Chlorophenothane neither significantly increased the cytochromes of rat kidney microsomes, nor the oxidative drug metabolism. However, in the kidneys of rabbits the cytochrome-b5-and P450-concentrations and drug metbolism were 2–3 fold higher after Phenobarbital. The correlation between P450 content and drug oxidase activity in the kidney microsomes of untreated and Phenobarbital treated rabbits was low.Suspensions of rabbit kidney microsomes revealed the same spectral changes after addition of Hexobarbital or Aniline as those reported for liver microsomes.

Taurolithocholate inhibits taurocholate uptake by isolated hepatocytes at low concentrations.

SummaryThe cholestatic bile acid taurolithocholate inhibits taurocholate uptake by isolated liver cells non-competitively. Inhibition is instantaneous and inversely related to the cell number in the incubate. The Kiamounts to 7 μM in the presence of 2 mg cellular protein per ml. Secretion of taurocholate by isolated liver cells is not affected by taurolithocholate up to a concentration of 50 μM. This indicates a difference between the carrier for taurocholate uptake and the carrier for taurocholate secretion. Inhibition of bile acid uptake by liver cells may be involved in the pathogenesis of lithocholate-induced cholestasis.

Stimulation of kidney microsomal drug metabolism by phenobarbital

After pretreatment of rabbits and rats with phenobarbital i.p. (6×80 mg/kg every 2nd day) kidney weight increased significantly in young rabbits only. The colour of the kidneys appeared more reddish. Microsomal kidney proteins of pretreated rabbits prepared by standard procedures in 0.1 M phosphate buffer increased 18°/0 (not significant), prepared liver microsomes 620/0 compared with controls. The cytochrome concentrations in kidney microsomes are in the region of 10--30°/0 of liver microsomal cytochromes only. Cytochrome b s increased 2ibld in kidneys after stimulation, but not in livers. Cytochrome P~50 concentrations increased about 2.5 times in livers and 3.5 times in kidney microsomes after phenobarbital treatment. The specific mierosomal activity (~moles of metabotites formed per mg microsomat proteins in 10 mill) for several drug oxidations was found to be altered in kidney and liver microsomes of rabbits in the following manner:

Accelerated regeneration of the liver produced by phenobarbital

It is well known that numerous drugs which induce drug metabolizing enzymes, such as phenobarbital, produce liver hypertrophy ff the inducing agent is repeatedly administered. The following results provide evidence that the growth of the fiver after partial hepatectomy (2/3) is also accelerated ff rats receive phenobarbital daily. The wet and dry liver weight, the total amount of water and of protein increased 8--10°/0 2 days after hepatectomy if the rats received 40 mg/kg phenobarbital i.p. just after the operation in ether anaestesia and 50 mg/kg on the following day. Continuing the daffy administration of 50 mg/kg we observed an even higher increase (20°/o) of the fiver weight with a corresponding rise of water and proteins in the liver 4 days after the operation. The liver grows with a 50°/o higher rate. We observed the same magnitude of accelerated liver growth ff we treated rats two days betbre hepatectomy with 80 mg/kg dally and continued the daffy administration one day after the operation with 40 mg/kg phenobarbital. A pretreatment before hepateetomy is not necessary. The induction of the hydroxylating enzyme system situated in the liver microsomes is also intensified during phenobarbital treatment. The content of cytochrome-P450 and the demcthylation rate of aminopyrine related to 1 g of microsomal protein decreases in the regenerating liver and are 30 to 50°/o lower 5 days after hepatectomy than in normal controls. The difference was even greater if we used sham operated rats (laparatomy) as controls. During treatment of hepatectomized rats with phenobarbital, however, cytochrome-P450 and the demethylation rate increase twoto threefold and achieve the same level we found in normal rats receiving the same schedule of phenobarbital treatment. The experiments prove that phenobarbital stimulates the induction of the microsomal hydroxylating enzyme system as well as the growth of the fiver during regeneration after hepatectomy.

Abbauhemmung und Synthesesteigerung bei der Vermehrung mikrosomaler Cytochrome durch Phenobarbital

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