Effect of promethazine and isosafrole on rat-hepatic microsomal mono-oxygenase activity: comparison with classic inducers phenobarbitone and beta-naphthoflavone

The characteristics of induction of rat-hepatic microsomal mono-oxygenase activity by promethazine and isosafrole have been investigated and compared with the classic inducers phenobarbitone and beta-naphthoflavone. Both promethazine and isosafrole pretreatments result in increased cytochromes P-450, and enhanced NADPH-cytochrome c reductase, aminopyrine N-demethylase, dichloronitroanisole O-demethylase, ethoxycoumarin O-deethylase and ethoxyresorufin O-deethylase activity. Isosafrole but not promethazine increased the liver to body weight ratio. It is concluded that promethazine and isosafrole pretreatment produces an induction of the rat-hepatic microsomal mono-oxygenase system which shows both phenobarbitone- and polycyclic aromatic hydrocarbon-type characteristics.     

The measurement of FAD-containing mono-oxygenase activity in microsomes containing cytochrome P-450

1. Antibodies to NADPH-cytochrome P-450 reductase have been used to essentially abolish the contribution of cytochrome P-450 to xenobiotic metabolism by mammalian microsomes. This permits the determination of the activity of the FAD-containing monooxygenase and the stoichiometry between substrate, O₂ and NADPH, in the microsomal membrane, and in the absence of cytochrome P-450-dependent activity.

2. FAD-containing mono-oxygenase oxidation rates were determined for sulphur- and nitrogen-containing substrates, including: thiols; sulphides; thioamides; primary, secondary and tertiary amines; hydrazines.

3. Although the enzyme in mouse, rabbit, rat and pig microsomes displays similar substrate specificity, some catalytic characteristics are different between species and tissues.     

Selective inhibitory effect of organophosphates on UDP-glucuronyl transferase activities in rat liver microsomes

The effects of acute and subacute administration of diisopropylfluorophosphate (DFP) and acute administration of Soman, Sarin and Tabun on UDP-glucuronyltransferase (GT) activity towards 4-nitrophenol, 4-methylumbelliferone, phenolphthalein and testosterone in rat liver microsomes were investigated. Twenty-four hours after a single injection of DFP, the activity of GT towards 4-nitrophenol and 4-methylumbelliferone was inhibited, and the inhibitory effect continued for 3 days. The activity had recovered by 7 days after injection. The activity of GT towards phenolphthalein and testosterone was not affected at any time after injection. Soman, Sarin and Tabun showed the same effect as DFP after a single injection. After daily DFP injections, the activity of GT towards 4-nitrophenol and 4-methylumbelliferone was decreased to the same level as found following acute treatment with DFP. The in vitro addition of DFP to liver microsomes did not affect GT activity towards 4-nitrophenol. It is suggested that these changes are not due to a direct effect of DFP. Furthermore, the effects of two enzyme inducers on GT activity in the presence and absence of DFP were investigated. In the 3-methylcholanthrene (MC) pretreatment group, DFP inhibited only the GT activity towards 4-nitrophenol and 4-methylumbelliferone. On the other hand, in the phenobarbital (PB) pretreatment group, DFP did not inhibit the GT activity towards 4-nitrophenol and 4-methylumbelliferone. It was also demonstrated that MC pretreatment increased the mortality in the DFP-treated rats but that PB pretreatment suppressed it. These results suggest that DFP and other organophosphorus agents may be useful agents for studies on the heterogeneity of GT.     

The interactions of hydrazine derivatives with rat-hepatic cytochrome P-450

1. The ability of different classes of hydrazine derivatives to modify cytochrome P-450 function during turnover as judged by loss of absorbance at 416 nm, loss of CO-reactive cytochrome P-450, or destruction of haem has been studied.

2. Addition of monosubstituted hydrazines to rat-liver microsomes caused considerable loss of CO-reactive cytochrome P-450 and haem destruction; monosubstituted hydrazides caused mainly loss of CO-reactive cytochrome P-450, most likely due to abortive complex formation. Metabolism of 1,1-disubstituted hydrazines by microsomal cytochrome P-450 resulted in loss of CO-reactive cytochrome P-450 only, with no haem destruction. The 1,2-disubstituted hydrazines and hydrazides, procarbazine and iproniazid, acted similarly to the monosubstituted hydrazines, while 1,2-dimethyl-hydrazine elicited no response, cither in observable spectral changes or loss of CO-reactive cytochrome P-450.

3. Synthetic diazene intermediates of phenylhydrazine and N-aminopiperidine reacted rapidly with microsomal cytochrome P-450 to form a spectral intermediate resembling the putative iron porphyrin-diazenyl complex. The decomposition of certain iron porphyrin-diazenyl derivatives apparently leads to destruction of the porphyrin prosthetic group, most likely due to haem alkylation.     

Selective inhibitory effects of HYIpro‐3‐1 on CYP1A2 in human liver microsomes

CYP1A2 is one of the main Cytochrome P450 enzymes in the human liver associated with the metabolism of several xenobiotics. CYP1A2 is especially involved in the metabolic activation of different procarcinogens. Therefore, the development of cancer may be inhibited by inhibiting CYP1A2 activity. Here, the inhibitory effect of HYIpro‐3‐1 and its derivatives on CYP1A2 activity in human liver microsomes (HLM) was studied through LC‐MS/MS using a cocktail assay. Among the four compounds, HYIpro‐3‐1 showed the most selective and strongest inhibitory effect on CYP1A2 at IC₅₀ values of 0.1 µM in HLMs and inhibition was confirmed using purified human CYP1A2. It was determined that inhibition is reversible because the inhibitory effect of HYIpro‐3‐1 is not dependent on preincubation time. HYIpro‐3‐1 showed a typical pattern of competitive inhibition for CYP1A2‐catalyzed phenacetin O‐deethylation, based on the Lineweaver‐Burk plot, with a Ki value of 0.05 μM in HLMs; the secondary plot also showed a linear pattern. In our study, HYIpro‐3‐1 was proposed as a novel inhibitor with the capacity to selectively inhibit CYP1A activity in HLMs.     

Altered activity of hepatic mixed-function mono-oxygenase enzymes in streptozotocin-induced diabetic rats.

1. In streptozotocin-induced diabetic male rats, hepatic microsomal aminopyrine N-demethylase activity was depressed, whereas aniline hydroxylase activity and cytochrome P-450 content were increased over control values. 2. In diabetic female rats, hepatic microsomal aminopyrine N-demethylase activity, aniline hydroxylase activity, biphenyl 4-hydroxylase activity, and cytochrome P-450 content were increased over control values. 3. Insulin treatment of diabetic male and female rats antagonized all physical and biochemical abnormalities of the diabetic state; 4. Methyl analogues of streptozotocin did not produce a diabetic state when injected into female rats, and resulted in no changes in aminopyrine N-demethylase activity, aniline hydroxylase activity, or cytochrome P-450 content. 5. Insulin treatment of non-diabetic female rats resulted in slight decreases in aminopyrine N-demethylase and aniline hydroxylase activities, but no changes in cytochrome P-450 content. These observations suggest that insulin primarily influences drug metabolism of diabetic animals through correction of the insulin-deficient diabetic state.     

Selective inhibitory activity against MAO and molecular modeling studies of 2-thiazolylhydrazone derivatives

A series of 2-thiazolylhydrazone derivatives have been investigated for the ability to inhibit the activity of the A and B isoforms of monoamine oxidase (MAO) selectively. All of the compounds showed high activity against both the MAO-A and the MAO-B isoforms with pKi values ranging between 5.92 and 8.14 for the MAO-A and between 4.69 and 9.09 for the MAO-B isoforms. Both the MAO-A and the MAO-B isoforms, deposited in the Protein Data Bank as model 2BXR and 1GOS, respectively, were considered in a computational study performed with docking techniques on the most active and MAO-B-selective inhibitor, 18.     

Selective bioreduction of nitroxides by rat liver microsomes

It was found that control rat liver microsomes will not reduce 2-ethyl-2,4,4-trimethyl-3-oxazolidinyloxy (OXAN) even though the reduction potential for OXAN is identical to that for 2,2,6,6-tetramethylpiperidinoxyl (TEMPO) and di-tert-butyl nitroxide (DTBN) which are reduced by these microsomes. It is suggested that the conformation of OXAN prevents these control enzymes from reducing OXAN.Administration of phenobarbital for 3 days prior to sacrifice, results in a rat liver microsomal preparation which will reduce OXAN and is inhibited by very low concentrations of SKF-525A. On the other hand, rats induced with 3-methylcholanthrene did not reduce OXAN. These results suggest that phenobarbital induces the synthesis of a form of cytochrome P-450 which is not present in either normal rat liver microsomes or in 3-methylcholanthrene-induced microsomes.     

Inhibition of mono-oxygenase activities by 1,1,1-trichloropropene 2,3-oxide, an inhibitor of epoxide hydrase, in rat liver microsomes.

Addition of 1,1,1-trichloropropene 2,3-oxide (TCPO), an inhibitor of microsomal epoxide hydrase, to rat liver microsomes caused a type I spectral change, and its magnitude was increased by pretreatment of animals with phenobarbital (PB) but not with 3-methylcholanthrene and polychlorinated biphenyls. TCPO inhibited aminopyrine N-demethylation competitively and prevented covalent binding of 2,4,2',4'-tetrachlorobiphenyl to macromolecules catalyzed by liver microsomes, although it stimulated benzolalpyrene hydroxylation significantly. It is suggested that TCPO interacts with cytochrome P-450, especially a species of the cytochrome which is inducible by PB administration, and thus inhibits mono-oxygenase activities of liver microsomes.     

Flavin-containing monooxygenase activity in human liver microsomes

Human liver microsomal flavin-containing monooxygenase activity has been studied using dimethylaniline N-oxidation and thiobenzamide S-oxidation. Except for one subject, the capacity of human liver microsomes to mediate these reactions were markedly increased at pH 8.4 compared to pH 7.4. The mean dimethylaniline N-oxidase activities at pH 7.4 and 8.4 in the four subjects tested were 2.49 +/- 1.13 and 6.59 +/- 4.04 nmol mg-1 min-1, respectively (mean +/- SD, N = 4). The mean thiobenzamide S-oxidase activities at pH 7.4 and 8.4 were 1.39 +/- 0.51 and 2.74 +/- 1.28 nmol mg-1 min-1, respectively. At pH 7.4, an antibody to the human liver NADPH-cytochrome P-450 reductase inhibited dimethylaniline N-oxidation between 4 and 38%. The same antibody had no effect on this reaction at pH 8.4. Except for one subject, a battery of cytochrome P-450 inhibitors also had little effect on this reaction. Further, preincubating human microsomes at 45 degrees C in the absence of NADPH for 4 min destroyed approximately 90% of the dimethylaniline N-oxidase activity. These data collectively suggested that the flavin-containing mono-oxygenase is the major enzyme mediating this reaction in human liver microsomes. In contrast to dimethylaniline N-oxidation, thiobenzamide S-oxidation was significantly inhibited by the anti-reductase at both pH 7.4 and 8.4, respectively. These data indicate that cytochromes P-450 contribute significantly to this reaction in human liver microsomes.     

Drug metabolism and drug-induced spectral interactions in human fetal liver microsomes

Properties of a mono-oxygenase system in human fetal liver microsomes were studied. The levels of cytochrome P-450 and NADPH-cytochrome c reductase were 20 and 30 per cent, respectively of rat liver microsomal levels. Corresponding percentages for homogenates were 13 and 18 per cent, respectively. Hepatic 12,000 g supernatants from human fetuses were found to catalyze the hydroxylation of 3,4-benzpyrene and aniline and the N-demethylation of aminopyrine. These activities were 2, 12 and 11 per cent of those in adult rat liver. The cytochrome P-450- and NADPH-cytochrome c reductase-related turn-over-numbers for aminopyrine and aniline were of the same order of magnitude in human fetal and adult livers and in rat liver. The fetal turn-over-number for 3,4-benzpyrene was small compared with adult and rat values. Spectral changes induced by the addition of various compounds to liver microsomes were studied. Aminopyrine and hexobarbital were found to yield type I spectral changes with rat or adult human microsomes, but with fetal microsomes these compounds yielded a type II spectral change except in some cases when hexobarbital in low concentration yielded a type I change. Aniline and n-octylamine induced type II spectral changes with both adult and fetal microsomes. The relative magnitudes of spectral changes differed greatly between fetal and adult microsomes.     

Interspecies variability and drug interactions of loxapine metabolism in liver microsomes.

Loxapine is a dibenzoxazepine neuroleptic that is metabolized by the liver in humans. In the present study, we investigated first in vitro loxapine metabolism in liver microsomes from various species including rats, mice, guinea pigs, dogs, rabbits, monkeys and humans. This enables us to choose between species to further validate drug-drug interaction studies. We observed the formation of desmethyl- and hydroxy- metabolites of loxapine after incubation of the different species liver microsomes. Hydroxylation pathway was major in all species. Wide interspecies variability of loxapine metabolism was observed. Loxapine metabolism was similar in human, guinea pig and dog microsomes. We screened in vitro effects of 67 molecules, representative of 8 therapeutic classes, on loxapine metabolism. Loxapine (100 microM) was incubated with guinea pig liver microsomes (1 mg/ml) 30 min at 37 degrees C with and without the presence of interacting drug. We found that most of psychotropics (alimemazine, cyamemazine and levomepromazine), antifungal (ketoconazole), anticancer drugs (daunorubicin, pirarubicin) and analgesic (nefopam) inhibited more than 50% of hydroxyloxapine formation in vitro. Complementary clinical and pharmacokinetic studies should be performed to confirm these results.     

A direct test for mono-oxygenase activity of intact small intestine using surface reflectance fluorimetry

The O-dealkylation activity for 7-ethoxycoumarin of mouse small intestine was determined by recording the surface fluorescence of the umbelliferone formed in everted intestinal segments. A simple holding device suitable for commercially available cuvettes has been designed. After hypotonic shocking of the mucosal ccells the substrate was added in 0·1 M Tris buffer pH 7·9 causing a linear increase in fluorescence for more than 15 min. Pretreatment of mice with phenobarbital increased the activity several fold, the induction following a biphasic course. The maximal rate of O-dealkylation by the entire small intestine was calculated as 19 nmoles min^?1. In agreement with previous results obtained with homogenates of small intestine the highest activity was located at a distance of 8–12 cm from the pylorus. Inhibition experiments indicate the involvement of cytochrome P-450.     

Terfenadine-antidepressant interactions: an in vitro inhibition study using human liver microsomes

AIMS: Inhibition of the metabolism of terfenadine has been associated with torsades de pointes ventricular arrhythmias. The aim of this study was to assess in vitro the potency of the antidepressants nefazodone, sertraline and fluoxetine in inhibiting terfenadine biotransformation. METHODS: Human liver microsomes were incubated with terfenadine and the antidepressants at various concentrations. Formation of the two major metabolites of terfenadine was determined by h.p.l.c. RESULTS: The apparent Km for microsomes from four human livers was 11+/-5 and 18+/-3 microM (mean +/-s.e.mean) for the N-dealkylation and C-hydroxylation pathways, respectively. Nefazodone, sertraline and fluoxetine inhibited terfenadine N-dealkylation with K(i) values of 10+/-4, 10+/-3 and 68+/-15 microM respectively. Inhibition of the C-hydroxylation pathway yielded noncompetitive K(i) values of 41+/-4, 67+/-13 and 310+/-40 microM respectively. CONCLUSIONS: Nefazodone and sertraline were moderately weak in vitro inhibitors of terfenadine metabolism while fluoxetine was a very weak inhibitor. Clinically significant interaction of terfenadine is more likely with nefazodone than sertraline or fluoxetine since therapeutic plasma levels of nefazodone are comparatively higher.     

The effects of chlormethiazole on single unit activity in rat brain; interactions with inhibitory and excitatory neurotransmitters.

Extracellular recordings were made of single unit activity in the brainstem of urethane anaesthetized rats. Drugs were applied by microiontophoresis from multibarrelled micropipettes or administered intraperitoneally. Chlormethiazole (CMZ) caused a decrease in spontaneous firing rate when applied with high currents (greater than 40 nA). When applied with lower currents CMZ did not cause changes in firing rate, but enhanced the inhibitory effects of gamma-aminobutyric acid (GABA), muscimol and glycine in a dose-dependent manner. The inhibitory actions of acetylcholine were not affected. Excitatory responses to glutamate and acetylcholine were unaffected by applications of CMZ which caused potentiation of GABA, muscimol and glycine. When applied at higher currents CMZ caused a decrease in the response to glutamate. Intraperitoneal administration of CMZ (50-600 mumol kg-1) also enhanced responses to microiontophoretically applied GABA, muscimol and glycine. These results are compared with those reported for other anticonvulsant drugs and possible mechanisms of action of CMZ are discussed.     

Stereochemical heterogeneity of hepatic UDP-glucuronosyltransferase activity in rat liver microsomes

Rat liver UDP glucuronosyltransferase activities may be divided into at least two groups with differential responses towards substrates. This paper deals with an attempt to describe on what chemical basis the two groups may be distinguished. We studied the glucuronidation of 24 substrates in liver-microsomes of Sprague-Dawley rats pretreated with 3-methylcholanthrene or phenobarbital. The conjugation of 11 substrates was stimulated most strongly by 3-methylcholanthrene and that of the others, by phenobarbital. The estimated thickness of the molecules in their most likely conformation was below 4 Å for molecules of the first group and more than 4 Å for the second. The thickness or the bulkiness of the molecules seems to play an important role. However, for phenol substituted in the position 2, a steric effect or intramolecular interactions may change the substrate's classification within these two groups. It was also noticed that phenobarbital stimulated more than 3-methylcholanthrene the glucuronidation of the corresponding hydroxylated metabolite.     

Antitumor activity of two gelsemine metabolites in rat liver microsomes

Gelsemine is one of the major alkaloids from Gelsemium elegans Benth., which has been used as an antitumor remedy in clinic. In this paper, metabolism of gelsemine has been investigated in vitro in phenobarbital-treated rat liver microsomes. The metabolites of gelsemine were separated and evaluated using the flash silica gel column, preparative HPLC, using NMR and MS methods. According to the spectral data, two metabolites, M1 and M2, were identified as 4-N-demethylgelsemine and 21-oxogelsemine, respectively. By the MTT method in vitro, the antitumor activities between gelsemine and its metabolites were compared in the HepG2 and HeLa cell lines. Moreover, the main metabolic pathway was further proposed.