Mechanisms of monooxygenase induction and inhibition.

The mechanisms of monooxygenase induction and inhibition have been discussed from the standpoint of historic development, from the current concepts about the molecular mechanism of enzyme induction, and from the various possibilities by which inhibitors can interact with the complex cytochrome P-450 monooxygenase system. In detail the main features and phenomena of induction and of the induced new enzyme protein are briefly described, whereby general principles are emphasized. The current knowledge on the mechanism of induction is exemplified by a description of the inducing action of TCDD on mouse hepatoma cells. A special example of increase in the molecular activity towards 7-ethoxycoumarin-0-deethylation is given by the action of sulmazole on mouse liver cytochrome P-450. It possesses properties similar to that of cobaltous chloride in that it decreases the amount of cytochrome P-450 in the microsomal protein but at the same time increases the molecular activity to about a four-fold level. The mechanisms of inhibition of the microsomal monooxygenase are explained in general terms by outlining the various modes of inhibitory action that lead to a decrease in enzyme activity.

The occurrence of carbonyl reduction in continuous cell lines emphasizes the essentiality of this metabolic pathway.

Using the ketone compound metyrapone (MPON) as a substrate for carbonyl reduction it has been verified for the first time that various permanent cell lines in culture express carbonyl reducing activity. This is even true for the dedifferentiated and fibroblastoid cell line V79, emphasizing the essentiality of this metabolic pathway. MPON reducing enzyme activities are located in the endoplasmic reticulum as well as in the cytoplasm of the cells. Compared to MPON-reductase in rat liver microsomes, no immunological homology to microsomal C2REV7 rat liver hepatoma cell MPON-reductase could be detected, indicating differences in antigenic determinants between the enzymes of the solid organ and respective cells in continuous culture.

Functional and immunological relationships between metyrapone reductase from mouse liver microsomes and 3 alpha-hydroxysteroid dehydrogenase from Pseudomonas testosteroni.

3 Alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) from Pseudomonas testosteroni was shown to reduce the xenobiotic carbonyl compound metyrapone (MPON). Reversely, MPON reductase purified from mouse liver microsomes and previously characterized as aldehyde reductase, was competitively inhibited by 3 alpha-HSD steroid substrates. For MPON reduction both enzymes can use either NADH or NADPH as co-substrate. Immunoblot analysis after native and SDS gel electrophoresis of 3 alpha-HSD gave a specific crossreaction with the antibodies against the microsomal mouse liver MPON reductase pointing to structural homologies between these enzymes. In conclusion, there seem to exist structural as well as functional relationships between a mammalian liver aldehyde reductase and prokaryotic 3 alpha-HSD. Moreover, based on the molecular weights and the co-substrate specificities microsomal mouse liver MPON reductase and Pseudomonas 3 alpha-HSD seem to be members of the short-chain alcohol dehydrogenase family.

Purification and properties of a metyrapone-reducing enzyme from mouse liver microsomes--this ketone is reduced by an aldehyde reductase.

A ketone reducing enzyme was purified to homogeneity from female mouse liver microsomes, using the diagnostic cytochrome P-450 inhibitor metyrapone as a substrate. In contrast to the usually employed indirect spectrophotometric recording of pyridine nucleotide oxidation at 340 nm, a HPLC method was applied for direct alcohol metabolite determination. Purification of the carbonyl reductase resulted in a 360-fold increase in specific activity together with a single band in the 34 kD region after SDS-polyacrylamide gel electrophoresis. Phenobarbital, indomethacin, dicoumarol and 5 alpha-dihydrotestosterone inhibited the enzyme, whereas quercitrin did not affect the enzyme activity. Thus, by inhibitor classification of carbonyl reductases the ketone metyrapone is reduced by an aldehyde reductase, rather than by a ketone reductase. Dihydrotestosterone, the strongest inhibitor, is supposed to be the physiological substrate for the purified enzyme. It was demonstrated that during the steps of purification both NADPH and NADH can supply the required reducing equivalents, although the activity with NADH is weaker. The highest activity was obtained using an NADPH-regenerating system. Ethanol and the nonionic detergent Emulgen 913 led to an increased specific activity, indicating that the enzyme is bound to the membranes of the endoplasmic reticulum in a latent state. From these results it is concluded that the microsomal metyrapone-reducing enzyme belongs to the family of carbonyl reductases, but differs from the common patterns of their classification with regard to cofactor requirement and inhibitor susceptibility.

Agonist-releasable intracellular calcium stores and the phenomenon of store-dependent calcium entry. A novel hypothesis based on calcium stores in organelles of the endo- and exocytotic apparatus.

Store-dependent calcium entry represents a little characterized calcium permeation pathway that is present in a variety of cell types. It is activated in an unknown way by depletion of intracellular calcium stores, for example in the course of phospholipase C stimulation. Current hypotheses propose that depleted calcium stores signal their filling state to this permeation pathway either by direct, protein-mediated interaction or by release of a small, diffusible messenger. The further characterization of store-dependent calcium entry will benefit from progress in the identification of the intracellular calcium storing compartments. Recent findings reviewed here suggest that these compartments include parts of the organelle system that is involved in endo- and exocytosis. This commentary describes a novel model of store-dependent calcium entry based on calcium stores belonging to the endo- and exocytotic organelle system. Such calcium stores could establish a tubule-like connection with the extracellular space, in analogy to the cellular compartments that contain the insulin-sensitive glucose transporter or the gastric proton pump. This connection will provide a pathway for store-dependent calcium entry. Under store depletion, extracellular calcium will permeate through the tubule-like connection into the store lumen and from there into the cytosol. The consequences of this model for the development of drugs modulating store-dependent calcium entry are discussed.

Reductive metabolism of metyrapone by a quercitrin-sensitive ketone reductase in mouse liver cytosol.

Mouse liver cytosol catalyses the reduction of metyrapone to the corresponding alcohol metabolite metyrapol. The enzyme involved was characterized as a NADPH-dependent carbonyl reductase which is strongly inhibited by the plant flavonoid quercitrin but which shows no sensitivity to phenobarbital. Thus, by inhibitor subdivision of carbonyl reductases the metyrapone reductase in mouse liver cytosol has to be classified as a ketone reductase rather than an aldehyde reductase, as it was shown previously for the analogous enzyme in mouse liver microsomes based on the same pattern of inhibitor classification. Moreover, immunological comparison of the metyrapone reductases from the two subcellular fractions reveal no common antigenic determinants indicating the structural difference between these enzymes. In conclusion, metyrapone undergoes reductive biotransformation mediated by two clearly distinct carbonyl reductases located in different subcellular compartments of mouse liver cells. Considering the convenient and sensitive HPLC-method for direct metyrapol determination, metyrapone may serve as a useful tool in the investigation of these enzymes, although their physiological roles remain to be determined.

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.

Carbonyl reduction of metyrapone in human liver.

Carbonyl reduction was investigated in cytosolic and microsomal fractions of human liver using the ketone metyrapone as a substrate. The cytosolic enzyme has a stronger preference for NADPH over NADH than the microsomal enzyme: the former shows only 14% of the NADPH-supported activity while the latter exhibits 36% activity with NADH. Barbitone and quercitrin, the classic inhibitors of carbonyl reductases, do not affect metyrapone reduction in either fraction. Dicumarol and indomethacin, the specific inhibitors of NAD(P)H: quinone-oxidoreductase and dihydrodiol dehydrogenase, respectively, only slightly decreased metyrapol formation. In contrast, 5 alpha-dihydrotestosterone, the active form of the androgen steroid testosterone, inhibited metyrapone reduction very strongly in the microsomal fractions and is postulated to be the physiological substrate of the enzyme. This resembles the situation in mouse liver [E. Maser and K. J. Netter, Biochem Pharmacol 38: 3049-3054, 1989] where microsomal metyrapone reductase was inhibited by steroids and the purified enzyme was demonstrated to mediate androsterone oxidation. Immunoblot analysis revealed antigenic cross-reaction of antibodies against the 34 kDa metyrapone reductase from mouse liver microsomes with the homologous protein in human liver microsomes pointing to structural homologies between the respective enzymes of the two species. These results--together with previous findings, which have shown that there exist functional as well as structural relationships between microsomal mouse liver metyrapone reductase and 3 alpha-hydroxysteroid dehydrogenase from Pseudomonas testosteroni [E. Maser, U. Oppermann and K. J. Netter, Eur J Pharmacol 183:1366, 1990]--suggest that metyrapone reduction in human liver microsomes might be catalysed by a microsomal hydroxysteroid dehydrogenase.

Induction of daunorubicin carbonyl reducing enzymes by daunorubicin in sensitive and resistant pancreas carcinoma cells.

Daunorubicin (DRC) and other anthracyclines are valuable cytotoxic agents in the clinical treatment of certain malignancies. However, as is the case with virtually all anticancer drugs, tumor cell resistance to these agents is one of the major obstacles to successful chemotherapy. In addition to an increased energy-dependent efflux of chemotherapeutic agents, enzymatic drug-inactivating mechanisms also contribute to multidrug resistance of tumor cells. In the case of DRC, carbonyl reduction leads to 13-hydroxydaunorubicinol (DRCOL), the major metabolite of DRC with a significantly lower antineoplastic potency compared to the parent drug. In the present study, we compared two pancreas carcinoma cell lines (a DRC-sensitive parental line and its DRC-resistant subline) with respect to their capacity of DRC inactivation via carbonyl reduction. In addition, we cultured the two cell lines in the presence of increasing sublethal concentrations of DRC. Evidence is presented that DRC treatment itself leads to a concentration-dependent induction of DRC carbonyl reduction in subcellular fractions of both the sensitive and resistant pancreas carcinoma cells, resulting, surprisingly, in different susceptibilities to DRC. The principal difference between the two cell lines becomes most apparent at high-dose DRC supplementation (1 microgram/mL), at which DRC resistant cells exhibited higher inducibility of DRC-inactivating enzymes, whereas respective sensitive cells already showed an impairment of cellular viability. The use of the diagnostic model substrates metyrapone and p-nitrobenzaldehyde reveals that this adaptive enhancement of DRC inactivation can be attributed to the induction of DRC carbonyl reductases different from known aldehyde and carbonyl reductases. In conclusion, these findings suggest that inactivation of anthracyclines by carbonyl reduction is inducible by the substrate itself, a fact that might be considered as one of the enzymatic mechanisms that contribute to the acquired resistance to these drugs.

Heterogeneity of carbonyl reduction in subcellular fractions and different organs in rodents.

The pattern and distribution of carbonyl reduction in liver, kidney and adrenal gland subcellular fractions of NMRI mice, Wistar rats and Hartley guinea pigs were examined using the ketone compound metyrapone (2-methyl-1,2-di(3-pyridyl)1-propanone) commonly used as a diagnostic cytochrome P450 inhibitor. A direct HPLC method for alcohol metabolite determination instead of the indirect spectrophotometric recording of pyridine nucleotide oxidation at 340 nm was applied. All the tissues examined in these species rapidly reduced the employed compound but at the subcellular level no general distribution scheme of specific activity was found, although in all fractions metyrapol formation could be attributed to aldo-keto reductases. Cytosolic and microsomal metyrapone reducing enzymes are distinguished by their inhibitor sensitivity to phenobarbitone and quercitrin and thus can be characterized as aldehyde and ketone reductases according to the inhibitor subclassification of the aldo-keto reductase family. Moreover, the enzymes also differ with respect to their immunological cross-reactivity to anti-microsomal mouse liver metyrapone reductase antibodies. Immunological homologies were found between metyrapone reductases of liver microsomes from all species and kidney and adrenal gland microsomes from guinea pig. However, the protein of all the cytosolic fractions as well as that of kidney and adrenal gland microsomes from mouse and rat did not cross-react with the antibodies, indicating the absence of common antigenic determinants. From catalytic properties and functional data it is concluded that hydroxysteroid dehydrogenases present in the suspected subcellular fractions form a structurally and functionally related enzyme family which may have been conserved during evolution.

7-Alkoxyquinolines: new fluorescent substrates for cytochrome P450 monooxygenases.

A series of 7-alkoxyquinolines was synthesized and tested as substrates with hepatic microsomes prepared from male Wistar rats. Microsomal O-dealkylation rates and kinetic constants were determined for the 7-alkoxyquinolines with microsomes from control, 3-methylcholanthrene (MC)-pretreated, and phenobarbitone (PB)-pretreated rats. Structure-activity relationship studies indicated that the 7-benzyloxyquinoline was the most rapidly metabolized substrate for control microsomes and those from PB-pretreated rats, whereas the 7-ethoxy- and 7-propoxyquinolines were O-dealkylated more rapidly by microsomes of MC-pretreated animals. Differences in activities occurred in Vmax and apparent Km values; however, there does not appear to be a correlation between these two values for the different quinoline substrates. Apparent Km and Vmax values for the 7-alkoxyquinolines were: control microsomes, Km = 71-773 microM, Vmax = 0.37-8.4 nmol 7-quinolinol/min/mg protein; MC microsomes, Km = 0.5-14 microM, Vmax = 0.29-2.7 nmol 7-quinolinol/min/mg protein; PB microsomes, Km = 2.8-46 microM, Vmax = 0.9-12 nmol 7-quinolinol/min/mg protein. All of the quinoline substrates gave Type I binding spectra with control and MC microsomes. With PB microsomes, Type I. Reverse Type I, and a mixture of the two types of binding spectra were observed. Comparisons of the structure-activity relationships, levels of induction, and kinetic constants were made with 7-alkoxycoumarin and 7-alkoxyphenoxazone analogs. In addition, three new coumarin substrates (7-pentoxy-, 7-hexoxy-, and 7-benzyloxycoumarin) are described.

Homologies between enzymes involved in steroid and xenobiotic carbonyl reduction in vertebrates, invertebrates and procaryonts.

Evidence is reported for the existence of a structurally and functionally related and probably evolutionarily conserved class of membrane-bound liver carbonyl reductases/hydroxysteroid dehydrogenases involved in steroid and xenobiotic carbonyl metabolism. Carbonyl reduction was investigated in liver microsomes of 8 vertebrate species, as well as in insect larvae total homogenate and in purified 3 alpha-hydroxysteroid dehydrogenase preparations of the procaryont Pseudomonas testosteroni, using the ketone compound 2-methyl-1,2 di-(3-pyridyl)-1-propanone (metyrapone) as substrate. The enzyme activities involved in the metyrapone metabolism were screened for their sensitivity to several steroids as inhibitors. In all fractions tested, steroids of the adrostane or pregnane class strongly inhibited xenobiotic carbonyl reduction, whereas only in the insect and procaryotic species could ecdysteroids inhibit this reaction. Immunoblot analysis with antibodies against the respective microsomal mouse liver metyrapone reductase revealed strong crossrections in all fractions tested, even in those of the insect and the procaryont. A similar crossreaction pattern was achieved when the same fractions were incubated with antibodies against 3 alpha-hydroxysteroid dehydrogenase from Pseudomonas testosteroni. The mutual immunoreactivity of the antibody species against proteins from vertebrate liver microsomes, insects and procaryonts suggests the existence of structural homologies within these carbonyl reducing enzymes. This is further confirmed by limited proteolysis of purified microsomal mouse liver carbonyl reductase and subsequent analysis of the peptide fragments with antibodies specifically purified by immunoreactivity against this respective crossreactive antigen. These immunoblot experiments revealed a 22 kDa peptide fragment which was commonly recognized by all antibodies and which might represent a conserved domain of the enzyme.

Alteration of cytochrome P-450 by prolonged administration of imipramine and/or lithium to rats.

The aim of this study was to investigate imipramine-induced alterations of cytochrome P-450 and to determine whether prolonged concomitant administration of imipramine and lithium results in a pharmacokinetic interaction. Male Wistar rats received imipramine (10 mg/kg i.p.) at 12 h intervals or lithium chloride (100 mg/kg in drinking water) or they were treated with the combination of these drugs for 2 weeks. The long term treatment with imipramine produced a very complex alteration of cytochrome P-450: imipramine increased the level of the cytochrome, but it decreased the rate of its own aromatic hydroxylation in position 2. The rate of N-demethylation in the side chain was not changed. Consequently, in the case of both hydroxylation and demethylation, calculated molecular activities were decreased to 48% and 70% respectively. This differential change in activities corresponded well to the observed decrease of absorption in difference spectra (type I) produced in microsomes by imipramine. Carbamazepine-induced type I difference spectra were also decreased by imipramine pretreatment, but to a lesser extent. In contrast, hexobarbital type I binding was increased by imipramine treatment while type II difference spectra produced by metyrapone were not affected. The preliminary SDS-PAGE analysis of cytochrome P-450 isoenzymes of control and imipramine treated rats showed that the investigated antidepressant markedly intensified a protein band at 50.11 kD while bands at 51.28 kD, 56.20 kD and 56.88 kD were less intensive. These results indicate that the alteration of cytochrome P-450 by imipramine treatment is not only of quantitative but also of qualitative character. Lithium alone given to rats affected neither the concentration of cytochrome P-450 in microsomal protein nor the rate of imipramine metabolism in vitro. Lithium given jointly with imipramine reduced imipramine-induced elevation of cytochrome P-450.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolic interaction between imipramine and carbamazepine in vivo and in vitro in rats.

The pharmacokinetic consequences of the combination of carbamazepine with imipramine in male Wistar rats have been investigated. It was found that a 2-week treatment with the combination resulted in the increase of the concentrations of the parent compounds and a simultaneous decrease in their metabolites in blood plasma i.e. carbamazepine inhibited imipramine demethylation in the side chain while imipramine inhibited carbamazepine 10,11-epoxidation. The velocity of imipramine 2-hydroxylation and 10,11-epoxy-carbamazepine hydration did not seem to be changed by the combination. On the basis of studies in vitro it is concluded that the observed metabolic interaction between carbamazepine and imipramine is due to the competition of the drugs for the active centre of cytochrome P 450 and to a certain qualitative alteration of the enzyme by imipramine as can be deducted from the decrease of carbamazepine binding to the cytochrome.

A qualitative study on vanadate effects in the tetrahydrofolate-dependent formate transfer in vitro and in vivo in mice.

Parenteral administration of sodium vanadate (NaVO3, 22 mg/kg b.w., i.p.) to mice depresses the oxidation rate of [14C]formate to [14C]CO2, as determined by radioactive breath analysis. The heavy metal-induced inhibition is relatively fast in onset, fairly intense (up to 80% inhibition), of short duration (about 2 h) and strongly correlated to the presence of vanadate (in the pentavalent state) in plasma. The [14C]CO2 exhalation rate from [14C]bicarbonate is less affected by vanadate in vivo, thereby suggesting a specific interference of vanadium in the intermediate step of formate oxidation to HCO3-. In vitro in mouse liver cytosolic fractions vanadate inhibits the enzymatic transfer of formate to tetrahydrofolic acid. The inhibition is accomplished by a vanadate-dependent oxidative degradation of tetrahydrofolate. In contrast, the concentrations of N5-methyltetrahydrofolate, dihydrofolate and folate remain unchanged upon in vitro-exposure to vanadate. The in vitro studies thus might explain the observed inhibition of formate oxidation to carbon dioxide in vivo by a vanadate-evoked depletion of its biological carrier tetrahydrofolic acid. Whether the interference in tetrahydrofolate metabolism also occurs under in vivo conditions, remains to be elucidated.

Pharmacokinetic interaction between carbamazepine and neuroleptics after combined prolonged treatment in rats.

This study investigates how neuroleptics of phenothiazine or thioxanthene structure influence the pharmacokinetics of carbamazepine. Experiments were carried out on male Wistar rats. Carbamazepine and the neuroleptics were administered i.p., separately or together, for 2 weeks in the following daily doses (mg/kg): carbamazepine 15 during the 1st week of treatment and 20 during the 2nd week of treatment, promazine 10, chlorpromazine 2, perazine 10, chlorprothixene 2, flupenthixol 0.5. One hour after the last injection of carbamazepine and/or the neuroleptic, samples of blood plasma and brain were taken to determine the concentrations of carbamazepine and two of its metabolites: 10,11-epoxide and trans-10,11-diol. The neuroleptics increased the concentration of carbamazepine in plasma and in brain, but tended to decrease (with the exception of chlorpromazine) the concentration of the epoxide and increased the concentration of trans-10,11-diol. Metabolic in vitro studies did not show any significant differences between rats treated with carbamazepine alone and those treated with carbamazepine plus neuroleptic in the rates of the carbamazepine epoxidation, of 10,11-epoxide hydrolysis or of 1-naphthol glucuronidation.

Indium selectively increases the cytochrome P-450 dependent O-dealkylation of coumarin derivatives in male mice.

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.

Differential inhibition of biphenyl hydroxylation in perfused rat liver.

A differential inhibition of biphenyl hydroxylation by alpha-naphthoflavone and metyrapone was observed in isolated perfused rat liver. alpha-Naphthoflavone inhibited 2- and 4-hydroxylation in livers from beta-naphthoflavone-pretreated animals but had no effect on both reactions in livers from phenobarbital-pretreated animals. Metyrapone inhibited 2- and 4-hydroxylation in phenobarbital-stimulated livers, but only insignificant inhibition of 2-hydroxylation and a slight enhancement of 4-hydroxylation by metyrapone was observed in beta-naphthoflavone-stimulated livers. Conjugation of 2-hydroxybiphenyl and 4-hydroxybiphenyl by isolated perfused livers was also studied. 4-Hydroxybiphenyl preferentially formed sulphates in livers from untreated animals but after induction glucuronidation was as effective as sulphation or even exceeded sulphation. Only glucuronic acid conjugates of 2-hydroxybiphenyl were detected.

Liver injury by the false morel poison gyromitrin.

After oral application of the mushroom poison gyromitrin a time and dose dependent decrease of cytochrome P-450 was found in rat liver microsomes. The maximal decrease to about 50-60% of the control (after 200 mg/kg, 80% of LD50) was observed 8-12h after application, a normalization after 48 h. The inhibition of cytochrome P-450 mediated metabolism of aminopyrine and p-nitroanisole corresponds to the decrease of cytochrome P-450. The specific activity of cytochrome P-450 remains unchanged while that of cytochrome P-448 is decreased as shown by means of the metabolism of ethoxycoumarin or ethoxyresorufin. Comparable results were obtained after application of N-methyl-N-formylhydrazine (MFH) which is formed from gyromitrin rapidly by hydrolysis. An attack on the endoplasmatic membrane with a stimulation of lipid peroxidation is discussed.

High carbonyl reductase activity in adrenal gland and ovary emphasizes its role in carbonyl compound detoxication.

Carbonyl reduction has been studied in liver, kidney, adrenal gland and ovary of female Wistar and Sprague-Dawley rats as well as of female NMRI mice by using metyrapone as a substrate and by means of direct HPLC analysis of the reduced alcohol metabolite metyrapol. Carbonyl reducing activities were found in all tissues examined so far, with that in rat ovary and adrenal gland cytosol exceeding the liver cytosolic specific activity severalfold: 15-fold and 12-fold in the Wistar strain; 12-fold and 7-fold in the Sprague-Dawley strain, respectively. In general, Wistar rat enzyme activities were about four times higher than those of Sprague-Dawley rats in all fractions, which indicates an interesting genetic difference between the two rat strains. Due to the sensitivity towards the diagnostic inhibitor quercitrin, carbonyl reductase (EC 1.1.1.184) seems to be mainly responsible for metyrapone reduction in rat and mouse adrenal gland and ovary cytosol. However, sensitivity towards dicoumarol in microsomal fractions of mouse tissues points to the involvement of further carbonyl reducing enzymes. Western blot experiments revealed immunological differences between metyrapone reductase from liver microsomes and respective enzymes of all other tissues. In conclusion, the difference in tissue and intracellular distribution suggests that several enzymes are involved in carbonyl reduction of metyrapone and the intracellular multiplicity of the enzymes may have some relation to their significance in carbonyl compound detoxification. These results support the hypothesis that carbonyl reductases, besides their participation in the metabolism of physiologically occurring substances, provide the enzymatic basis for the detoxification of xenobiotic carbonyl compounds in adrenal gland and ovary which have escaped their metabolic conversion by the liver.