Aminopyrine Research Articles

Evidence that proglumide and benzotript antagonize secretagogue stimulation of isolated gastric parietal cells

Proglumide has been shown to be an in vivo inhibitor of secretagogue-stimulated gastric acid secretion. In the present study, we have examined the ability of proglumide and benzotript, a new tryptophan derivative, to inhibit acid output from isolated gastric fundic parietal cells from rabbit. As measured with the [ 14C]aminopyrine (AP) accumulation method as an index of acid secretion, the two drugs inhibited basal AP with IC-50 values of 1 × 10 −2 M for proglumide and 1 × 10 −3 M for benzotript. In the case of secretagogue stimulation (1) benzotript slightly affected histamine-induced AP (15% inhibition at 5 × 10 −3 M ), proglumide did not; (2) both proglumide and benzotript inhibited in a non-competitive manner acetylcholine-induced AP; (3) these isolated cells were sensitive to gastrin and the dose-response curve for the stimulant was biphasic (maximum for 1 × 10 −9 M ), suggesting a desensitization mechanism. Proglumide and benzotript competitively inhibited both [ 125I]gastrin binding to its receptor sites and gastrin-induced AP, suggesting they are members of a class of gastrin-receptor antagonists. But, this suggestion cannot exclude other post-receptorial mechanisms involved in the acid output from parietal cells.

Forskolin stimulation of acid and pepsinogen secretion in isolated gastric glands.

Forskolin, a specific diterpene activator of adenylate cyclase in intact cells and cellular homogenates, was used to examine the relationship among adenosine 3',5'-cyclic monophosphate (cAMP) metabolism, gastric acid, and pepsinogen secretion in isolated gastric glands. This agent was found to stimulate [14C]aminopyrine (AP) accumulation and respiration, both measurements of which are indexes of parietal cell acid secretory responsiveness and pepsinogen secretion, which is a measure of chief cell activity. Forskolin also increased cAMP content and activated cAMP-dependent protein kinase in the glands. The histamine H2-receptor antagonist, cimetidine, inhibited forskolin-stimulated increases in AP accumulation and respiration when submaximal concentrations of forskolin were used but had not effect on the other response parameters. Forskolin also potentiated the action of the muscarinic agonist, carbachol, in both the presence and absence of cimetidine. Since there was a close kinetic and temporal correlation between the secretory response parameters and cAMP-dependent protein kinase activation, it appears that cAMP plays an important role in the mediation of gastric acid and pepsinogen secretion. The inhibitory action of cimetidine on forskolin-stimulated AP accumulation and respiration suggest that forskolin potentiates the action of endogenous histamine present in the glands. Forskolin potentiation of carbachol in the presence of maximum inhibitory concentrations of cimetidine indicates that previously observed potentiating interactions between carbachol and histamine, secretagogues which appear to act via cAMP-independent and cAMP-dependent mechanisms, respectively, involve intracellular events that occur subsequent to the binding of these agents to their respective receptors and subsequent to an increase in intracellular cAMP content.

Cl- requirement of acid secretion in isolated gastric glands.

The dependence of acid formation, as measured by aminopyrine (AP) accumulation, on medium and intracellular Cl- was investigated in resting (10(-4) M cimetidine) and stimulated (10(-3) M dibutyryl cAMP) gastric glands isolated from the rabbit stomach. Intracellular Cl- concentrations (Cli-) were measured by the steady-state distribution of 36Cl-. In Cl- -free conditions AP accumulation was absent. Medium Cl- induced AP accumulation (AP ratio = 125) in stimulated glands with a K0.5 of 10.4 +/- 1.1 mM, equivalent to 18.0 +/- 1.2 mM Cli-, and had a small effect on resting glands (AP ratio = 6). With normal Nai+ and Ki+ maintained, similar results were obtained in stimulated glands treated with 10(-5) M amphotericin (ampho) and 10(-4) M ouabain (ouab), where the basal-lateral membrane was confirmed to be short-circuited with respect to Cl- pathways, i.e., medium Cl- and Cli- were equal. The K0.5 for Cl-i was 17.5 +/- 2.5 mM. In resting glands treated with ampho and ouab, AP accumulation increased linearly (no saturation was observed) with increasing Cl- (AP ratio = 35). These results suggest that stimulation activates a Cl- component in the secretory membrane and not in the basal-lateral membrane of the parietal cell. The K+ requirement of AP accumulation at a physiological Cl-i of 60 mM was also investigated in ampho- and ouab-treated glands. On stimulation the K0.5 for Ki+ decreased from 19.5 to 12 mM, coupled with a large increase in AP accumulation, indicating that stimulation also activates a K+ component in the secretory membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibitory action of somatostatin on isolated gastric glands and parietal cells.

The action of somatostatin in vitro was characterized using glands and parietal cells isolated from rabbit gastric mucosa. In the presence of the reducing agent dithiothreitol, somatostatin was found to inhibit gastrin- and histamine-stimulated acid formation in glands as measured by [14C]aminopyrine (AP) accumulation and oxygen consumption, both measurements that appear to be reliable indexes of parietal cell acid formation. In glands the inhibition of the secretory response to gastrin was more potent (60-80%) than that to histamine (15-25%). The kinetics of somatostatin inhibition of responses to both agents were noncompetitive. The apparent IC50 for the partial somatostatin inhibition of histamine-stimulated AP accumulation was similar to that for gastrin (approx 3 X 10(-9) M) when maximum concentrations of histamine (10(-4) M) or gastrin (10(-7) M) were used. The inhibitory action of somatostatin appeared to be specific, inasmuch as this peptide had no significant effect on basal secretion or secretion stimulated by carbachol, dibutyryl cAMP, cholera toxin, or elevated extracellular K+. In purified parietal cell preparations, somatostatin inhibited histamine- but not gastrin-stimulated AP accumulation. Moreover, the inhibition of histamine-stimulated AP accumulation in parietal cells was more pronounced than in glands. These results suggest that somatostatin acts directly on parietal cells to inhibit histamine activation of H+ secretion. Somatostatin also acts indirectly to inhibit gastrin, perhaps by blocking the release of histamine from paracrine- or endocrinelike cells present in the glands.

Lysis of vitamin E-deficient rat erythrocytes by rat liver microsomes in an NADPH-dependent process in the presence of inhibitors of lipid peroxidation.

Haemolytic substances are generated by rat liver microsomes undergoing NADPH-dependent lipid peroxidation. In the present experiments, vitamin E-deficient red cells were found to be haemolysed in an NADPH-dependent process by rat liver microsomes in the presence of EDTA, an inhibitor of microsomal lipid peroxidation. This is in contrast to the behaviour of normal, vitamin E-adequate red cells which are resistant to lysis under similar conditions. Aminopyrine (AP), a substrate for the microsomal mixed function oxidase system, was found to prevent lysis of vitamin E-deficient red cells in NADPH-microsome-EDTA mixtures. Experiments designed to detect only the more stable haemolysins formed in such mixtures involved sampling and assaying for haemolytic activity in the presence of AP. The result support the conclusion that haemolysis was due to a short-lived species. If EDTA is omitted from the NADPH-microsome mixture other more stable haemolysins are produced.

Simultaneous determination of aminopyrine hydroxylation and N-demethylation in liver microsomes by HPLC and effects of inducers on these enzyme reactions

Aminopyrine(AM) has been used as a model substrate for investigation of drug metabolism. The major metabolic route is N-demethylation that was confirmed in liver microsomes. Another route, however, the oxidation of the 3-methyl group has not been confirmed in liver microsomes. AM and its metabolites including 3-hydroxymethyl-2-methyl-4-dimethylamino-1-phenyl-3-pyrazoline-5-one (AM-OH), which is a 3-hydroxylated metabolite, were separated on a reversed-phase(Cg) Radial-Pak column using a mobile phase of 30% methanol, 1% triethylamine and 69% water adjusted to pH 5.40 with acetic acid.By this rapid and simple method, AM hydroxylation was confirmed in liver microsomes of rats. The hydroxylation in control rat has two Km values (0.52 and 3.06 mM). Pretreatment of phénobarbital (PB) caused an induction of the hydroxylation activity and showed a low Km (0.51 mM) only. 3-Methylcholanthrene(3-MC) did not affect the activity and Km value of the hydroxylation. Similarly PB induced AM N-demethylation activity in agreement with former reports and changed the Km (low,0.80 and high,3.20 mM) into one Km (1.43 mM). Therefore AM hydroxylation as well as N-demethylation seems to be a PB-responding pathway.

Influence of phenobarbital and 3-methylcholanthrene on the metabolism of aminopyrine in isolated hepatocyte system.

The effect of phenobarbital (PB) and 3-methylcholanthrene (3-MC) on the metabolic behavior of aminopyrine (AM) was studied using an isolated hepatocyte system prepared from male Wistar rats. The formation of 4-formylaminoantipyrine (FAA) was increased after pretreatment with PB, but not 3-MC. However, the total amounts of AM and its main metabolites recovered from hepatocyte system after the incubation for 30 min were considerably reduced both by PB and 3-MC-pretreatment. Furthermore, when using 4-monomethylaminoantipyrine (MAA), the first metabolite of AM, as a substrate, 3-MC-pretreatment resulted in a more significant decrease in the total amounts of MAA and its further metabolites recovered than did PB. Present observation suggests the participation of cytochrome P-448 as well as cytochrome P-450 in the metabolism of AM.

Influence of cyclopyrrolones, zopiclone and suriclone on rat liver microsomal drug metabolizing system.

The pharmacological action of cyclopyrrolones, zopiclone(ZPC) and suriclone(SRC) have been shown to be very similar to the benzodiazepines, although cyclopyrrolones have basic molecular frameworks distinct from those of benzodiazepines, Clinically, minor tranquilizers are used for a long period. Therefore, we have studied the effects of ZPC and SRC on rat liver microsomal drug metabolizing system. ZPC and diazepam (DZP) were administrated for 5 days (100mg/kg p.o.). The contents of cytochrome P-450(P-450) and cytochrome b5 (b5) and the activities of NADPH-cytochrome c reductase(reductase), aniline(AN) p-hydroxylase and aminopyrine(AM) N-demethylase were increased in DZP treated rats. On the other hand, ZPC induced the activities of reductase and AN p-hydroxylase to the same extent as DZP, but not the contents of P-450 and b5 and AM N-demethylase activity. The AN p-hydroxylase activity and b5. content were enhanced by the administration of SRC (60 mg/kg p.o., twice a day for 3 days), but the AM N-demethylase activity and P-450 content were not changed. Furthermore, no influence on microsomal drug metabolizing system was observed when SRC (0.7 5mg/kg p.o.) was administrated for 14 days. From these results, it was suggested that ZPC and SRC had inducing effects on rat liver microsomal drug metabolizing system, but these effects were less than DZP.

Effect of temperature on the metabolism of aminopyrine.

We examined the effect of temperature on the fate of aminopyrine (AM) by using five male rabbits kept at 15 or 30°C. The rabbits kept at 15°C showed an increase in elimination of plasma AM compared with those at 30°C. The total urinary excretion of AM and its metabolites at 15°C was smaller than that at 30°C.

Influence of perinatal nutrition on hepatic drug metabolism in the adult rat.

Pregnant rats were fed high-fat (HF, 35% calories) or low-fat (LF, 5% calories) diet from 18 days of gestation to the end of the suckling period. Thereafter, male progeny were fed stock diet for 6 months, then sacrificed or challenged for 3 days with HF diet. There were no immediate posttreatment effects at 30 days of age on liver microsomal cytochrome P-450 or b5, aminopyrine (AP) N-demethylase or benzo(a)pyrene (BP) hydroxylase. At 7 months, cytochrome P-450 was lower in LF-fed than in HF-fed animals. High-fat challenge reduced cytochrome P-450 and cytochrome b5 content in both groups, AP N-demethylase only in LF animals and BP hydroxylase only in HF animals. These differential effects of perinatal exposure to diet on the later response of microsomal mixed-function oxidases to fat challenge suggest that early dietary experience may regulate the pattern of drug metabolism in adult life.

Multiple forms of cytochrome P-450 (III): Inductory mode of cytochrome P-450 from rat liver microsomes treated with phénobarbital

We demonstrate the induction manner of rat liver microsomal cytochrome P-450 treated with phénobarbital (PB) from the amino-pyrine (AP) demethylase and benzo(a)pyrene (BP) hydroxylase activity and from the high-performance liquid chromatography (HPLC) profiles. The content of cytochrome P-450 started to increase at 8 hr after administration of PB (100mg/Kg) and kept increasing continuously. At 48 hr, specific content of cytochrome P-450 elevated to about twice that of non-treated microsomes. AP demethylase activity increased as contents of cytochrome P-450 increased. However, BP hydroxylase activity did not change. Distinct change was not observed in the content of cytochrome b5- by the induction with PB; however, slight elevation was detected at 48 hr. Two different forms of cytochrome P-450 (PB-4 and PB-5) induced by PB were observed in HPLC profile and collected separately. Absolute spectrum of these forms were almost the same. Molecular weights of PB-4 and PB-5 calculated by SDS-PAGE were distinct, i.e., 51,000 and 53,000, respectively. The peaks of PB-4 and PB-5 were not observed as a definite peak in the HPLC profile of non-treated microsomes. The peaks of PB-4 and PB-5 were detected distinctly as a peak of cytochrome P-450 at 8 hr and 24 hr, respectively.

Inhibition of acid secretion in isolated gastric glands by substituted benzimidazoles.

A new class of gastric acid inhibitors, substituted benzimidazoles (H 83/69 and H 149/94), have been tested in an isolated rabbit gastric gland preparation. Acid formation in the glands was stimulated by histamine, dibutyryl cAMP (DBcAMP), and high extracellular K+ concentrations, and the glandular secretory response was measured by changes in oxygen consumption and in accumulation of the weak base [14C]aminopyrine (AP). The substituted benzimidazoles inhibited AP accumulation induced by all stimulants in a dose-dependent noncompetitive manner. In contrast, cimetidine only inhibited histamine-induced AP accumulation. Basal AP accumulation, not affected by cimetidine, was also inhibited by the substituted benzimidazoles, as was the increase in glandular oxygen consumption produced by the addition of histamine and DBcAMP. Basal oxygen consumption was inhibited by about 15%. The substituted benzimidazoles, like AP, are weak bases and were also found to accumulate in the glands. Semiquantitative morphological studies of glands stimulated by histamine plus theophylline did not show any change in the enlarged secretory surface area after stimulation in the presence of inhibitory concentrations of H 149/94 (10(-4) M). The results suggest that substituted benzimidazoles have a mechanism of action different from that of H2-receptor antagonists and indicate a very distal site of action in the events leading to acid formation.

Endocrine control of daily variations in hepatic mixed function oxidase activity in the adult male rat

Abstract The hormonal control of rhythmic fluctuations in hepatic microsomal mixed function oxidase activity was examined by incubating microsomes prepared from intact control, sham‐operated, pinealectomized (PX), adrenalectomized (AX), and pinealectomized‐adrenalectomized (PXAX) adult male rats with aminopyrine (AP) and [4‐C14]3ß‐hydroxy‐5‐androsten‐17‐one (DHA). The animals were kept on a 12:12 light‐dark cycle for 15 days prior to killingatl, 4, 6 and 10 hafter light and at 1, 4, 6 and 10 h after dark. The rate of both the N‐demethylation of AP and the 7α‐hydroxy lation of DHA varies diurnally with maximal and minimal activities in the dark and light periods, respectively. The fluctuation in both reactions fits a symmetric sine curve for all endocrine treatment groups except for the 7α‐hydroxylation in PX animals. PXAX was the only treatment resulting in a significant increase in 7α‐hydroxylase activity. The data suggest that the pineal may be involved in mediating the effects of environmental light on liver 7α‐hydroxylase activity.

Interpretation of CO2 exhalation rate data from demethylation of aminopyrine and its metabolite monomethylaminoantipyrine.

1 Aminopyrine breath tests make use of the commercially available (N-dimethyl-[14C])-aminopyrine. A pharmacokinetic model has been proposed to relate 14CO2 exhalation rates (CER) to the demethylation of ([14C]-methyl)-aminopyrine (AP) and -monomethylaminoantipyrine (MAP). 2 computer simulations based on the model show that the shape of the CER-time profile is largely dependent on the ratio of the MAP to AP elimination rate constants. If this ratio equals 0.5 then the CERs decline in the monoexponential fashion. Ratios less than 0.5 result in concave biexponential curves whereas ratios greater than 0.5 result in convex curves. When demethylation is not complete for both compounds the transfer from biexponential to monoexponential behaviour will only occur at ratios greater than 0.5. 3 The resolution of concave biexponential CER-time profiles to give accurate estimates of AP and MAP elimination rate constants can only be achieved when the length of the experiment is adequate. The commonly employed 2 microCi tracer dose of aminopyrine is insufficient to monitor CER over the necessary time period to detect the proposed biexponential decline.

3-Methylcholanthrene induces phenobarbital-induced cytochrome P-450 hemoprotein in fetal liver and not cytochrome P-448 hemoprotein induced in maternal liver of rats

The characteristic nature of the drug-metabolizing system in fetal liver microsomes of rats was investigated. The aminopyrine(AM)- and the hexobarbital (HB)-metabolizing activities in fetal liver microsomes of the 21st day of pregnancy were induced by the maternal administration of 3-methylcholanthrene (3-MC) once daily on the 18th and the 19th day of pregnancy, while they were inhibited in maternal liver microsomes. The inductions of the AM- and the HB-metabolizing enzymes in fetal liver microsomes of rat by the maternal administration of 3-MC occurred exclusively in fetal period and simultaneously hemoprotein like phenobarbital-induced type P-450 different from that in maternal liver microsomes was newly induced in fetal liver microsomes of rats.

Prostaglandin E2-histamine in interactions on cAMP, cGMP, and acid production in isolated fundic glands.

Relations among cAMP, cGMP, acid production [measured by the intraglandular accumulation of [14C]aminopyrine (AP)], and prostaglandin E2 (PGE2) activity were studied in isolated glands from rabbit fundic mucosa. AP, cAMP, and cGMP responses to histamine, PGE2, and 3-isobutyl-1-methylxanthine (IMX) were compared with controls. Histamine and PGE2 significantly increased glandular cAMP levels twofold, and histamine and IMX stimulated AP uptake two- to fourfold. PGE2 significantly inhibited both histamine- and IMX-stimulated AP accumulation, but it did not alter basal AP uptake. PGE2 also decreased histamine-stimulated cAMP production but only at a low concentration (10(-7) M). This dose of PGE2 was near to the endogenous PGE2 content found in unstimulated glands (10(-8) M). Intraglandular cGMP levels in unstimulated glands (10(-8) M). Intraglandular cGMP levels were increased by IMX but not by PGE2 or histamine. It is concluded that histamine stimulation of acid secretion is mediated by cAMP, that secretory and biochemical responses to histamine are modulated by PGE2 because PGE2 antagonized histamine-stimulated cAMP and AP uptake, and that the rise in cAMP induced solely by PGE2 appears to be localized within nonparietal cells because PGE2 alone did not stimulate AP accumulation.

Cation effects on acid secretion in rabbit gastric glands.

The ability of isolated gastric glands to produce acid as a function of intra- and extracellular concentrations of K+ and Na+ was investigated. Ouabain inhibited acid formation as measured by the aminopyrine (AP) accumulation technique in a dose-dependent manner with an ED50 of 2 X 10(-6) M at 5.4 mM extracellular K+. This inhibition was counteracted by increasing extracellular K+ or decreasing extracellular Na+. In K+-free solutions with regular Na+, the AP accumulation was almost totally abolished; the readdition of K+ rapidly restored the response with a K0.5 of 1 mM extracellular K+. In the absence of Na+, with or without ouabain, there was always a significant residual AP accumulation, but the K0.5 of extracellular K+ required for acid formation increased to about 20 mM. Despite repeated washings in Na+-K+-free solutions, the intracellular K+ content could not be decreased below 24 mM above the apparent K0.5 for K+. It was found that the intracellular K+ could be depleted without disturbance of the acid secretory function if the glands were treated with the neutral ionophore amphotericin B and ouabain in K+-Na+-free solutions. Complete dose-response curves of H+ secretion as a function of K+ concentration could now be obtained. Thus the K0.5 for K+ activation of AP accumulation was 16.5 +/- 0.9 mM; upon histamine stimulation of the glands, the K0.5 decreased to 10.4 +/- 0.7 mM. Intracellular K+ concentrations above approximately 60 mM inhibited AP accumulation. Na+ dose dependently inhibited the K+-induced response. At low extracellular K+ concentrations, a ouabain-insensitive K+ accumulation mechanism was unmasked. This K+ uptake was partly inhibited y p-chloromercuribenzene sulfonic acid and by inhibitors of the gastric H+-K+-ATPase and amplified by inhibitors of K+ conductance channels such as Ba2+. The K+ content of gastric glands is mainly regulated by the Na+-K+-ATPase, but additional K+-uptake pathways are present. The affinity for K+ is increased in histamine-stimulated glands, which might indicate one mechanism of stimulating the secretory response. Na+ inhibits secretion possibly by binding to the cytosolic cation site of the gastric ATPase. The cation relationship to acid secretion in the rabbit parietal cell is similar to that described for vesicles isolated from hog gastric mucosa.

Direct measurement of aminopyrine N-demethylase and antipyrine hydroxylase activities in a monolayer rat primary isolated hepatocyte system

This paper describes a simple method for monitoring changes in aminopyrine N-demethylase and antipyrine hydroxylase activities in isolated primary hepatocyte monolayer culture. Aminopyrine N-demethylase activity was determined by monitoring the rate of formation of 14CO 2 derived from the N-demethylation of [dimethylamino- 14C]aminopyrine (AP). The rate of AP N-demethylation increased linearly with time for 60 min and proportionately with cell concentrations between 4.1 × 10 5 to 1.67 × 10 6 cells/incubation. As expected, non-linear AP N-demethylase kinetics were observed with hepatocytes as well as with microsomal preparations derived from control rats. Hepatocytes prepared from pheno-barbital (PB)-pretreated animals exhibited increased AP N-demethylase activity and typical Michaelis-Menten kinetics. In contrast, microsomal preparations from PB-treated animals exhibited non-linear N-demethylase kinetics that differed from the kinetics of preparations derived from control animals. Antipyrine hydroxylase activity was determined by monitoring the rate of formation of non-extractable conjugated 4-hydroxyantipyrine from [ N- 14C-methyl]antipyrine. Antipyrine hydroxylase activity was found to increase linearly for 120 min and proportionately with cell concentrations. Anti-pyrine hydroxylation by hepatocytes prepared from control and PB-pretreated animals followed typical Michaelis-Menten kinetics. AP N-demethylase activity immediately after plating was 10 per cent lower than at 4 hr, whereas antipyrine hydroxylase activities were similar. Culturing hepatocytes for 24 hr resulted in a decline to 40 and 60 per cent of control for AP N-demethylase activity and antipyrine hydroxylase activity respectively.

Metabolic studies of aminopyrine in rat and man by using stable isotope tracer techniques.

The metabolites of aminopyrine (AM) were analyzed by using stable isotope tracer techniques. After the oral administration of an equimolar mixture of AM and AM-2-CD3 (mixture 1), or an equimolar mixture of AM-3-CH2OH and AM-3-CD2OH (mixture 2), or either AM-3-CD3 or AM-4-N (CD3)2 to rats, the urinary metabolites were extracted with chloroform at pH 7.0, and the extracts were subjected to gas chromatography-mass spectrometry after trimethylsilylation. Characteristic doublet peaks in the mass spectra indicated the presence of a metabolite originating from mixture 1 or 2 mentioned above. The new metabolites detected by gas chromatography-mass spectrometry (GC-MS) were identified as 3-hydroxymethyl-4-monomethylaminoantipyrine (MAA-3-CH2OH) and 3-hydroxymethyl-4-aminoantipyrine (AA-3-CH2OH) from the shifts of the mass numbers of the molecular ions after the administration of AM-3-CD3 and AM-4-N(CD3)2. Two other metabolites were newly detected following the administration of mixture 2(AM-3-CH2OH is an intermediary metabolite of AM) to rats. They were identified as 3-hydroxymethyl-4-acetylaminoantipyrine (AcMAA-3-CH2OH) and 3-hydroxymethyl-4-acetylaminoantipyrine (AcAA-3-CH2OH). MAA-3-CH2OH was also detected in human urine after the oral administration of AM.

Adaptive responses of rat liver to the gestagen and anti-androgen cyproterone acetate and other inducers. I. Induction of drug-metabolizing enzymes

Treatment of female Wistar rats with cyproterone acetate (CPA) was shown to cause pronounced increases of hepatic microsomal monooxygenase activity towards the following substrates: ethylmorphine (EM), aminopyrine (AP), benzphetamin (BPA) and benzo[ a]pyrene (BP). Minor increases were seen using p-nitroanisole (pNA) and aniline (AN). Monooxygenase activity reached maximal levels within 24 h. The effects were dose-dependent, the threshold dose being about 4 mg/kg, and were reversible within 6 days. The results of comparative studies with several ‘classical’ microsomal enzyme inducers, i.e. pregnenolone-(16α)-carbonitrile (PCN), phenobarbital (PB), α-hexachlorocyclohexane (α-HCH) and 3-methylcholanthrene (3-MC) suggest that CPA belongs to the PCN-type and α-HCH to the phenobarbital type of inducers. In male rats CPA induced only moderate increases of monooxygenase activities which can be explained by decreased testosterone secretion due to anti-gonadotropic effect of CPA.