Indium pretreatment of rats and mice has been reported to decrease the concentration of cytochrome P-450, thereby reducing the activity of some cytochrome P-450 dependent enzymatic reactions. The present study reveals that pretreatment of C57Bl/6JHan mice of both sexes with one s.c. dose of 120 mg of In2(SO4)3.5 H2O per kg of body weight decreases the concentration of cytochrome P-450 to about 65% of control levels. Neither cytochrome b5 nor NADPH-cytochrome P-450 reductase is affected. Hepatic microsomal ethoxyresorufin O-deethylase activity declines to about 75% of control values. In contrast, with coumarin substrates, a sex dependence in the direction of change is observed: in female mice indium decreases the activity to about 75%, whereas in males it enhances the activity to 140%. Moreover, with 7-(methoxy-14C)coumarin as substrate, indium-pretreated male mice exhale about 180% and females about 65% of 14CO2 compared to the corresponding controls. A close correlation between the in vivo and in vitro effects of indium on the metabolism of the coumarin derivatives is suggested. After isolation and purification of cytochrome P-450, SDS-PAGE indicates in indium-pretreated male mice an intensification of a 48.5 kDa protein band which is decreased in females. Immunological studies using antibodies raised against control female cytochrome P-450 show cross reactivity among all microsomes used in these experiments. High percentages of inhibition occur in microsomes with high molecular activity towards coumarin derivatives. The in vitro kinetics of antibody-inhibited O-deethylation of 7-ethoxycoumarin seems to obey a non- or partial-competitive type of inhibition. Indium pretreatment of mice produces sex-dependent effects on the metabolism of coumarin derivatives.

7-Alkoxycoumarin O-Dealkylase↗

PubMed1989

Interactions of heterocyclic Maillard products with the hepatic microsomal monooxygenase system.

1. Interactions of methyl-substituted pyrazines, and other constituents of Maillard products generated during heat treatment of food, with hepatic microsomal mixed-function oxygenases were studied in vitro. 2. Spectral interactions of N-containing heteroaromatic compounds with the cytochrome P-450 system are type I or type II depending on the state of induction, and are relatively weak. Inhibition of 7-ethoxycoumarin O-deethylation by these compounds is ten times lower than that of metyrapone, agreeing with the weak spectral interaction. Inhibition is competitive for 2,3-dimethylquinoxaline, and complex for 2,5-dimethylpyrazine and 2,3,5,6-tetramethylpyrazine. 3. Spectral and inhibitory interactions indicate biotransformation. This was studied with 2,3,5,6-tetramethylpyrazine; the metabolite formed was identified as 2-hydroxymethyl-3,5,6-trimethylpyrazine. Metabolism to the N-oxide did not occur.

7-Alkoxycoumarin O-Dealkylase↗

PubMed1987

The 14CO2 breath test: facilities and limitations of a rapid and noninvasive method for in vivo evaluation of modified hepatic cytochrome P-450--a critique.

By means of the breath test technique the cascade from O-demethylations to CO2 was investigated after pretreatment of mice with warfarin, phenobarbital, cobaltous chloride, sodium vanadate and metyrapone. It was the intention to examine the validity of the technique with respect to cytochrome P-450 activity. Therefore three different radioactive labeled substrates, i.e., hydrogen carbonate, formate and xenobiotics, were applied at three different levels of the one-carbon pathway and were utilized to demonstrate possible interference of the modifiers with the sequence from O-demethylation to CO2. Real in vivo information about a modified cytochrome P-450 system can be obtained using model substrates carefully selected with regard to the type of expected modification of the monooxygenase system. In addition, a parallel monitoring of the consecutive reaction sequence by measuring the conversion of formate to CO2 is necessary in order to guarantee the validity of the in vivo technique in visualizing the activity of the hepatic monooxygenase system.

Animals↗

PubMed1986

The mode of reduction of vanadate(+V) to oxovanadium(+IV) by glutathione and cysteine.

Vanadate(+V) is completely reduced to oxovanadium(+IV) in vitro by thiols. A high transient absorbance at 750 nm is observed (intense blue) in solutions of a pH below 5 immediately after starting the reduction. EPR-measurements and determinations of the consumption of free thiol groups prove that the transient absorbance is caused by an intermediate compound not containing vanadium in its reduced state (+IV). The way of reduction of vanadate(+V) by thiols is discussed. We propose the formation of an intermediate vanadate(+V)-thioester which is formed before reduction in a preequilibrium step.

Chemical Phenomena↗

PubMed1986

[The toxicity of palladium].

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Animals↗

PubMed1986

Two different 7-ethoxycoumarin O-deethylase activities in mice after pretreatment with sulmazole and cobaltous chloride.

Pretreatment of male C57BL/6J Han mice with 200 mg of sulmazole (AR-L 115 BS) daily for 2 days results in an up to 4fold increase of the 7-ethoxycoumarin O-deethylase. The resulting effect was compared to that after a pretreatment with cobaltous chloride which also leads to a 4fold increase. Enzyme kinetic parameters of the 7-ethoxycoumarin O-deethylase are different for the two inducers in respect to the affinity for the substrate, but the inhibitions by metyrapone and alpha-naphthoflavone are similar. Comparison of the microsomal protein patterns after partial purification of microsomes revealed totally different patterns after sulmazole and cobaltous chloride. Sulmazole produces a 54 kDa protein band and cobaltous chloride a band at 48.5 kDa. Therefore it is concluded that in mice at least two 7-ethoxycoumarin O-deethylase activities can be induced. The electrophoretic pattern after sulmazole is different from those after 3-methylcholanthrene and isosafrole. This was also proven by enzyme kinetic investigations with ethoxyresorufin as substrate.

7-Alkoxycoumarin O-Dealkylase↗

PubMed1985

Induction of liver cytochrome P-450 in mice by warfarin. Comparison of warfarin-, phenobarbitone-, and cobalt-induced hepatic microsomal protein patterns by PAGE after partial purification on octyl-sepharose CL-4B.

A rapid method is presented to separate mouse liver cytochrome P-450 from other components of the microsomal monooxygenase system and to increase specific activity by hydrophobic interaction chromatography on Octyl-Sepharose CL-4B by a factor of between 3.8 and 5.3. In addition it is shown that varieties of cytochrome P-450 can be separated from each other by Octyl-Sepharose CL-4B. After oral applications of 120 mg/kg warfarin once daily for three days SDS-PAGE analysis of the partially purified cytochrome P-450 fraction revealed a protein pattern in the 50 Kd region that is practically indistinguishable from that after conventional phenobarbitone pretreatment. On the other hand, cobalt pretreatment results in a different pattern that is distinguished from that of normals as well as from that of phenobarbitone- and warfarin-pretreated mice. From these results in conjunction with the previous finding of increased drug metabolic activity after warfarin pretreatment it is concluded that warfarin elicits phenobarbitone-like induction of the hepatic monooxygenases in mice.

Animals↗

PubMed1985

Alteration of the inhibitory effect of metyrapone by reduction to metyrapol during the metabolism of methacetin in vivo in mice.

Metyrapone is known as an inhibitor of the oxidative drug metabolism in vitro. We have used the exhalation analysis as a tool to study the influence of this inhibitor on the demethylation of 14C-methacetin in vivo. In parallel we investigated the reductive metabolism of metyrapone in mice by measuring the concentrations of metyrapone and its reduced metabolite metyrapol with a HPLC-method. 50 mg of metyrapone/kg b. wt. resulted in a 90% inhibition of 14CO2 exhalation when given 2 min before the exhalation analysis was started. The prolongation of the intervals between i.p. metyrapone and substrate administration leads to a diminution of the in vivo inhibition. We found that the hepatic metyrapone concentration falls rapidly and passes the detection limit at 120 min. Transiently metyrapol reaches a maximal concentration 15 min after the application of metyrapone. The rapid reduction of metyrapone was confirmed in vitro with fresh mouse liver homogenates. The administration of metyrapol itself in vivo causes a decrease in 14CO2 exhalation, too. The 14CO2 exhalation curves after metyrapol correspond to the curves after metyrapone, when sufficient time was allowed for its reduction to metyrapol. It can be concluded that not only metyrapone itself but also its reductive metabolite metyrapol is an effective, however weaker inhibitor.

7-Alkoxycoumarin O-Dealkylase↗

PubMed1984

Effects of vanadate on intracellular reduction equivalents in mouse liver and the fate of vanadium in plasma, erythrocytes and liver.

Intraperitoneal injection of 180 mumol sodium vanadate/kg body wt in mice inhibits the in vivo metabolism of drugs by the hepatic monooxygenase, as measured by exhalation of 14CO2 after treatment with appropriately labeled [14C]methacetin. Determinations of hepatic GSH, NADPH and NADH after vanadate injection show an initial and transient decrease of GSH (10 min, 20%), followed by a transient decrease of NADPH (30 min, 23%), followed by a decrease of NADH (40 min, 23% and 60 min, 26%). Rat liver organ spectrophotometry in the dual-wavelength mode shows an immediate response of the NAD(P)H level, which decreases transiently after addition of vanadate. Furthermore, EPR and AAS measurements indicate that vanadium occurs in the plasma in the oxidation states +IV and +V, whereas in intracellular compartments in liver and erythrocytes vanadium exists practically only in the +IV form (vanadyl). The duration of the inhibition for about 2 h coincides well with the transient concentration of vanadate in plasma, which decreases more rapidly than vanadyl. Maximal drug inhibition is associated with the phase of rapid formation of vanadyl in the liver. The experiments are in accordance with the hypothesis that vanadate inhibits the cytochrome P-450 dependent oxidative drug metabolism by diversion of reducing equivalents away from cytochrome P-450. Further evidence for such a hypothesis is provided by in vitro experiments in microsomes. Vanadate causes a dose dependent decrease of ethoxyresorufin deethylation which is reversible by the addition of NADPH.

Acetamides↗

PubMed1984

Radioisomers of scoparone (6,7-dimethoxycoumarin) as a tool for in vivo differentiation of various hepatic monooxygenase inducers in mice using the breath test technique.

The oxidative removal of two different methyl groups from scoparone by a cytochrome P-450-mediated reaction proceeds with greater velocity for the 6-methyl group than for the 7-methyl group. In vivo experiments in mice were carried out with the two respective radioisomers [6-methyl-14C]scoparone and [7-methyl-14C]scoparone. The consecutive administration of the scoparone to the same animal is followed by collecting the 14CO2 exhaled (breath test). The maximal exhalation rate, measured 4 min after i.p. administration of the substrates, is four times greater for [6-methyl-14C]scoparone than for the [7-methyl-14C]scoparone. The total recovery of radioactivity in the whole animal (except skin) including the exhaled 14CO2 is 86% for [7-methyl-14C]scoparone and 84% for [6-methyl-14C]scoparone. Further analysis shows that the proportion of radioactivity found in various excretory routes is different; however, in the case of the poorly exhaled [7-methyl-14C]scoparone, almost twice as much radioactivity is found in bile and urine than for the more rapidly exhaled [6-methyl-14C]scoparone, whereas liver and other organs show little changes. Pretreatment of animals with various inducers, such as phenobarbital, 3-methylcholanthrene, warfarin and cobaltous chloride, characteristically affects the ratio of the two demethylations of scoparone. In the case of warfarin pretreatment, the ratio of the 6- to 7-demethylation is elevated to nearly 6, after cobaltous chloride the ratio is lowered to 2.8, whereas phenobarbital and 3-methylcholanthrene have no effect. These results may provide possibilities of noninvasive in vivo recognition of the inductive state of a pretreated animal.

Animals↗

PubMed1983

Vanadium inhibits oxidative drug demethylation in vivo in mice.

Sodium vanadate inhibits the oxidative demethylation of substrates of the cytochrome P-450-dependent monooxygenase system in vivo in mice. [14C]Methacetin and 7-[methoxy-14C] coumarin were used as substrates, and the exhaled 14CO2 was monitored using the technique of the breath test. The inhibition is of short duration and begins to subside after about 10 min. The inhibition is dose-dependent; half-maximal effect is achieved at a dose of approximately 60 mumol/kg. The inhibition pattern is identical for both substrates, although 62% of the label of [14C] methacetin and only 10% of 7-[methoxy-14C] coumarin are enhaled within 1 h. Pretreatment with ascorbic acid (50 mg/kg p.o.) drastically diminishes the observed inhibitory effect of vanadate. Similarly, application of an equimolar dose of vanadyl sulphate produces a comparatively weak retardation of 14CO2 exhalation. The effect of vanadate is thought to occur by its competition for electrons normally transferred to cytochrome P-450.

Acetamides↗

PubMed1982

Does cobalt pretreatment of mice induce a phenobarbitone-type cytochrome P-450?

1. Pretreatment of male C57BL/6JHan mice with 40 mg/kg cobaltous chloride for two days, or three days pretreatment with 80 mg/kg phenobarbitone led to an increase of biphenyl-4-hydroxylation of similar magnitude. 2-Hydroxylation remains unaffected in both cases. 2. The time course shows an equivalent decrease in 2- and 4-hydroxylation for 6 h, when microsomal Co concn, reaches its maximum. Thereafter 4-hydroxylation increases to reach the enhanced values. 3. Kinetic analysis of biphenyl 2- and 4-hydroxylation reveals distinct differences. The apparent Km for 4-hydroxylation decreases in Co-pretreated mice but remains constant in phenobarbitone-pretreated animals. Also, the ratio of 4- to 2-hydroxylation in microsomes from phenobarbitone-treated animals remains unchanged for substrate concn. of 10(-5) to 2 X 10(-3) m, but for Co-pretreatment this ratio increases markedly from 2 to 5, with increasing substrate concn. 4. Increasing concn. of the competitive inhibitor, metyrapone, reveal a greater susceptibility of microsomal cytochrome P-450 from Co-treated mice than normal or phenobarbitone-induced animals. In contrast, deprivation of reducing equivalents in vitro in the presence of metyrapone shows similarities between microsomes from cobalt- and phenobarbitone-pretreated mice.

Animals↗

PubMed1982

Acceleration of 7-[methoxy-14C]coumarin-derived carbon dioxide exhalation by cobalt pretreatment in mice.

Pretreatment of rats with cobaltous chloride has been shown previously to reduce the content of cytochrome P-450 in the hepatic microsomal protein. This is accompanied by a corresponding decrease in substrate oxidation, e.g. ethyl morphine demethylation, in vitro. The present paper shows that pretreatment of C57BL/6J Han mice with 40 mg of CoCl2/kg/day for 2 days results in a decrease of cytochrome P-450 to 60% of its original value. This is accompanied by a corresponding decrease in overall rate of [14C]methacetin demethylation as measured by 14CO2 exhalation. However, when 7-[methoxy-14C]coumarin is the substrate, cobalt-pretreated animals exhale twice as much 14CO2 than normal animals. Considering the decrease in cytochrome P-450 (and assuming linear relationship between metabolic activity and cytochrome P-450 content), this observation suggested a 2.5-fold increase in the specific activity of the remaining cytochrome P-450. This was found to be true in vitro. It is concluded that cobalt pretreatment of mice leads to an enhanced in vivo demethylation rate of 7-[methoxy-14C]coumarin which is explained by a considerably higher molecular monooxygenase activity toward this substrate that is found in vitro.

Animals↗

PubMed1980

Characteristics of coumarin metabolism by liver microsomes from cobalt-pretreated mice.

1. Cobalt pretreatment of male mice leads to increased liver microsomal mixed-function oxidation of various coumarin derivatives; 7-ethoxycoumarin, 7-hydroxycoumarin and coumarin are metabolized three to four times more rapidly than in control microsomes. 2. In Arrhenius plots of 7-ethoxycoumarin de-ethylation, the temperature at which phase transition occurs is not changed by cobalt pretreatment. However, at high temperature, above the transition point, the slope indicates that cobalt lowers the apparent activation energy of the reaction to 65% of control value. 3. Metabolism of 7-hydroxycoumarin by hepatic microsomes from cobalt-treated mice gave 3,7- , 6,7- and 7,8-dihydroxycoumarin, identified by g.l.c. and mass spectrometry.

Animals↗