Herbert Remmer

Effects of phenobarbital and 3-methylcholanthrene on substrate specificity of rat liver microsomal UDP-glucuronyltransferase

Abstract 1. 1. Substrate specificity of liver microsomal UDPglucuronyltransferase (UDP-glucuronate glucuronyltransferase, EC 2.4.1.17) towards p- nitrophenol , 1-naphthol, bilirubin and chloramphenicol changes after treatment of rats with phenobarbital and 3-methylcholanthrene. Phenobarbital mainly increases the glucuronidation of chloramphenicol, whereas 3-methylcholanthrene only stimulates the glucuronidation of the two phenolic substrates. These changes have been observed using ‘native’, Triton X-100-treated, or deoxycholate-solubilized microsomes as well as partially purified enzyme preparations. 2. 2. Treatment with phenobarbital or 3-methylcholanthrene did not alter the apparent K m values for UDPglucuronic acid (0.16 mM) with p- nitrophenol (0.34 mM). 3. 3. The results suggest that several forms of UDPglucuronyltransferase exist in rat liver microsomes which are selectively induced by either phenobarbital or 3-methylcholanthrene.

[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.

Monitoring lipid peroxidation by breath analysis: endogenous hydrocarbons and their metabolic elimination.

Abstract Monitoring of ethane and pentane in breath as a noninvasive method to measure lipid peroxidation is performed by an increasing number of laboratories. The alkanes are generated through peroxidative breakdown of polyunsaturated fatty acids. Fasted Sprague-Dawley rats exhale these hydrocarbons at a rate of approximately 1.7 nmol/kg·hr. Through an improved analytical procedure other volatile hydrocarbons could be detected in the breath of the animals, i.e., ethene (1.1 nmol/kg·hr), propane (0.7 nmol/kg·hr), n -butane (0.7 nmol/kg·hr), iso-pentane, and iso-butene. The exhaled hydrocarbons accumulate in the atmosphere if an animal is confined in a closed system. However, the increase is not linear but a steady-state concentration is approached, presumably due to reuptake and subsequent hepatic metabolism. Hydrocarbons are oxidized by the hepatic monooxygenases, at rates increasing with their molecular masses. The same has been found for the elimination of ethane, propane, butane, pentane, and iso-butene by a rat from closed system. Metabolism is inhibited by a variety of substances, e.g., dithiocarb, ethanol, and tetrahydrofurane. Therefore, the increased release of hydrocarbons into the gas phase, after treatment with compounds suspected to induce lipid peroxidation, may merely result from decreased metabolism. Especially if the hydrocarbon exhalation is only moderately elevated as after ethanol administration, this aspect must be taken into account. Similarly, peroxidizing microsomes show elevated hydrocarbon output when treated with carbon monoxide in order to block metabolism. This study provides a procedure for discrimination of inhibitory and peroxidative action which is of particular importance if the increase over control levels is only moderate. In addition, it may serve as monitor for the metabolic capacity of a laboratory animal.

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.

Irreversible protein binding of metabolites of ethynylestradiol in vivo and in vitro

Abstract During incubation of 6,7-3H-ethynylestradiol with rat liver microsomes up to 20 % of the radioactivity was bound irreversibly to the microsomal proteins. Incubations in presence of albumin resulted in a further radioactive labelling of the albumin. The irreversible nature of the steroid-protein bond was established by solvent extraction and charcoal treatment. Further evidence was obtained after hydrolyzing the microsomal protein with trypsin and submitting the labelled tryptic peptides to ion exchange chromatography and electrophoresis. The labelled albumin was applied to sephadex gel filtration which showed the association of the ethynylestradiol radioactivity to the albumin peak. The binding reaction required supply of NADPH, could be stimulated by pretreatment of the animals with phenobarbital and was inhibited by CO and SKF 525 A. On these characteristics the concept was based that, in analogy to the well known binding of estradiol and estrone, 2hydroxylation is also an essential prerequisite for the binding of ethynylestradiol. The concept was confirmed by trapping off the 2-hydroxy-ethynylestradiol with glutathione, which led to a decrease of the ethynylestradiol-protein binding. Further evidence resulted from experiments in vivo , dosing rats with 6,7-3H-ethynylestradiol and 6,7-3H-estradiol 48 hrs prior to sacrifice and examining the amount of radioactivity irreversibly bound to the liver endoplasmic reticulum. 3H-ethynylestradiol caused a radioactive labelling of microsomes twice as much as that after 3H-estradiol.

Irreversible binding of DOPA and dopamine metabolites to protein by rat liver microsomes.

Abstract Rat liver microsomes catalyze NADPH-dependent irreversible binding of metabolites of DOPA and DOPAmine to microsomal protein and to BSA. Binding is inhibited by cysteine and the singlet oxygen quencher 1,4-diaza-bicyclo(2.2.2)octane. Irreversible binding to BSA is also catalyzed by mushroom tyrosinase, xanthine oxidase, and NADPH-cytochrome c reductase. The results suggest that in the microsomal system the participation of the hemoprotein, cytochrome P-450, is not an absolute requirement for the irreversible binding of metabolites of DOPA and DOPAmine to proteins.

Studies on the Metabolism of Ethynylestradiol in vitro and in vivo: The Significance of 2-Hydroxylation and the Formation of Polar Products

Abstract1. The metabolic fate of ethynylestradiol in vitro and in vivo has been followed by examining the displacement of tritium from [6,7-3H]ethynylestradiol and [2,4,6,7-3H]ethynylestradiol by other substituents. Incubations with rat liver microsomes and NADPH demonstrated that 2-hydroxylation is the major pathway of ethynylestradiol metabolism. In contrast, hydroxylations of ethynylestradiol at C-6 and C-7 are only of minor importance.2. The easy inducibility of this microsomal 2-hydroxylation by pretreatment with phenobarbital, the requirement for NADPH and inhibition by CO and SKF 525A indicate that a haeme protein is involved.3. The microsomal elimination of 3H from C-2 and C-4 of ethynylestradiol was markedly increased by glutathione, which is known to bind at C-1 and C-4 of estrogens forming ‘polar’ products. This formation of polar products was dependent on NADPH and was inhibited by CO and SKF 525A, supporting the concept that 2-hydroxylation of an estrogen is prerequisite for binding of glutat...

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.

Irreversible protein binding of [14C]imipramine with rat and human liver microsomes☆

Abstract After incubation of [ 14 C]imipramine with rat liver microsomcs up to 0–7 mole/mg was irreversibly bound per mg of microsomal protein. If albumin was added to the microsomal incubations [ 14 C]imipramine was also irreversibly bound to this protein. The irreversible binding of imipramine to protein was determined by exhaustive solvent extraction and charcoal adsorption, and measurement of the remaining 14 C-radioactivity in the protein. The binding reaction was dependent on oxygen, NADPH, microsomal protein content and substrate concentration. It was inhibited by CO and SKF 525-A. Pretreatment of rats with phenobarbital did not increase the amount of imipramine irreversibly bound to protein. Glutathione and other cysteine derivatives diminished the binding, whereas incubation with the epoxide hydrase inhibitor trichloropropene oxide resulted in an increase of imipramine irreversibly bound to protein. The results favour the concept that irreversible protein binding of imipramine is catalyzed by a cytochrome P-450-dependent hydroxylation via an epoxidation step. Irreversible protein binding of imipramine was also detectable with three samples of human liver microsomes.

Phenylhydrazine-induced lipid peroxidation of red blood cells in vitro and in vivo: monitoring by the production of volatile hydrocarbons.

Human red blood cells and male Sprague-Dawley rats were treated in vitro and in vivo, respectively, with phenylhydrazine in order to determine whether the release of volatile hydrocarbons can serve as a suitable index for phenylhydrazine-induced red blood cell peroxidation. Lipid peroxidation following phenylhydrazine administration (in vitro experiments: dosage calculated at 0.5-50 mM; in vivo experiments: intraperitoneal injection of 2.8 mg/100 g body wt) was monitored by the release of ethane and pentane measured by gas chromatography. Further hydrocarbons such as ethylene, propane, n-butane, iso-butane and iso-butene were monitored to form a basis of comparison. In vitro haemolysis was also determined during the course of incubation. Red blood cell suspensions yielded more than 15-fold concentrations of propane and more than 2-fold concentrations of iso-butane compared to pentane and ethane yields. Haemoglobin solutions also produced propane and iso-butane in the presence of phenylhydrazine, whereas pentane and ethane were not detectable. Time-course studies revealed that ethane and pentane reached maximum in vitro levels after red blood cell suspensions had been incubated for 2 hr whereas the maximum degree of haemolysis (approximately 60%) was attained between 60 and 90 min following the beginning of phenylhydrazine treatment. The dosage did not affect the final degree of haemolysis. Rats treated with phenylhydrazine exhaled greater concentrations of ethane (6-fold increase) and pentane (2-fold increase) compared to control animals. Exhaled propane showed a 30-fold increase in concentration following drug treatment. Our results suggest that the release of pentane and ethane may be useful in assessing red blood cell lipid peroxidation in the presence of phenylhydrazine in vitro and in vivo.