Pharmacological studies of 4-ethoxy-2-methyl-5-morpholino-3(2H)-pyridazinone (M73101). (4). Enzyme induction (author's transl)]

The effect of M7310U, a new non-steroidal analgesic anti-inflammatory agent, on liver microsomal drug-metabolizing enzymes was investigated. Rats were treated orally with M73101 (100, 200, 500 mg/kg), henylbutazone (PZ, 200 mg/kg), aminopyrine (AM, 100 mg/kg) or phenobarbital sodium (PB, 100 mg/kg) once daily for 2 weeks and then were observed for 2 weeks during which treatment was not given. On treatment with M73101, PZ, AM and PB, the liver enlarged but was restored to normal 1 week after the last administration. The rate of increase in the case of M73101 was lower than that seen with the reference compounds. M73101 markedly increased the content of microsomal protein, cytochrome P-450 or b5 and NADPH cytochrome C reductase, aniline hydroxylase and AM demethylase activity, but these increments returned to the normal level 1 week after the last administration. The serum concentration of M73101 after repeated administration (200 mg/kg, p.o.) for 1 week was lower than that after a single administration. Furthermore, M73101 increased Vmax for both aniline hydroxylase and AM demethylase, whereas it increased Km only for aniline hydroxylase. M73101 did not enhance the lipid peroxidation. Our observations suggest that the enlargement of rat liver seen with M73101 was due to the induction of drug-metabolizing enzymes and that this agent can probably be classified as a phenobarbital-type inducer.     

A possible role of liver microsomal alkaline ribonuclease in the stimulation of oxidative drug metabolism by phenobarbital, chlordane and chlorophenothane (DDT)

The administration of phenobarbital (80 mg/kg), chlordane (100 mg/kg) and DDT (100 mg/kg) to adult male Wistar rats, produced a constant and early significant reduction in the liver microsomal alkaline RNase that preceded the rise of cytochrome P-450, aminopyrine oxidative demethylase, aniline hydroxylase activities and liver weight.     

Kinetic alterations in rat liver microsomal cholecalciferol 25-hydroxylase associated with phenobarbital administration

The kinetics of cholecalciferol 25-hydroxylase in vitamin D-depleted rat liver microsomes, before and after phenobarbital induction, were studied. Three days of pretreatment with phenobarbital altered significantly both the apparent K_m and the V_max of the hydroxylase. Untreated vitamin D-repleted rats had lower cytochrome P-450 content and aminopyrine demethylase activity than the vitamin D-depleted animals. Phenobarbital administration reversed this nutritional effect on aminopyrine demethylase but not on cytochrome P-450 content. Furthermore, vitamin D deficiency potentiated the phenobarbital inductive effect upon microsomal protein. No inhibition of aminopyrine demethylase could be elicited in the presence of cholecalciferol or 25-hydroxycholecalciferol either prior to or after phenobarbital treatment, suggesting that these two oxidases are different entities.     

Influence of drugs and chemical upon hepatic enzymes and proteins-I. Structure-activity relationship between various barbiturates and microsomal enzyme induction in rat liver.

The effect of phenobarbital, three N-phenylbarbiturates and three N-cyclohexylbarbiturates on microsomal protein content, cytochrome P-450, cytochrome b₅, aniline hydroxylase, aminopyrine demethylase, p-nitrophenol-glucuronyltransferase and the ultrastructure of hepatic cells in rat liver were studied to elucidate the influence of barbiturate structure on enzyme-inducing activity. Smooth endoplasmic reticulum increased after administration of phenobarbital, phetharbital or bucolome. Phenobarbital and phetharbital, especially phenobarbital, induced cytochrome P-450 and glucuronyl-transferase. On the other hand, the other barbiturates showed little enzyme-inducing activity. From these results, the type and spatial position of barbiturate substituents are considered important for hepatic microsomal enzyme induction. Aniline hydroxylase and aminopyrine demethylase activities changed coincidentally with cytochrome P-450 content in almost all rat livers. However, quantitative agreement of the changes in cytochrome P-450 content and drug-metabolizing activity could not be obtained, suggesting the possibility of substrate inhibition or of intrinsic inhibitors in the microsomal fraction. Bucolome, one of the N-cyclohexylbarbiturates, has been reported to be effective in the reduction of serum unconjugated bilirubin level in Gilbert's syndrome. However, in. spite of the increased smooth endoplasmic reticulum, there was a reduction in the microsomal protein content, cytochrome P-450 and glucuronyltransferase after bucolome administration. This would seem to indicate that the serum bilirubin-reducing mechanism of bucolome is different from that of phenobarbital and phetharbital.     

Effects of vitamin A deficiency on hepatic and extrahepatic mixed-function oxidase and epoxide-metabolizing enzymes in guinea pig and rabbit.

Male guinea pigs and male rabbits were fed a vitamin A deficient diet for 9 weeks and for 12 weeks respectively. Hepatic levels of vitamin A were significantly reduced in the vitamin A deficient animals. The activities of some xenobiotic-metabolizing enzymes were measured in the liver, lung and small intestine. Aryl hydrocarbon hydroxylase, aniline hydroxylase, and 7-ethoxycoumarin deethylase activities were decreased in the vitamin A deficient guinea pig liver. However, in the guinea pig small intestine, aniline hydroxylase, 7-ethoxycoumarin deethylase, aminopyrine demethylase, and aryl hydrocarbon hydroxylase specific activities were increased. In rabbits, vitamin A deficiency decreased hepatic aniline hydroxylase and 7-ethoxycoumarin deethylase activities but increased intestinal aminopyrine demethylase activity. Enzyme activities in lung were not altered by vitamin A deficiency in guinea pig or rabbit. Microsomal epoxide hydrase and microsomal supernatant glutathione S-transferase activities in the three tissues of both species were not altered by vitamin A deficiency.     

N-甲基(3,4-亚甲二氧基苯甲酰)甲基-乙酰胺(SY-640)对化学致癌剂苯并芘与小鼠肝细胞核DNA共价结合的抑制作用

用小鼠肝细胞核制备和肝微粒体制备,研究了化合物SY-640对致癌剂苯并芘(BP)损伤肝细胞核的保护作用及与P-450的关系。结果表明,SY-640可显著抑制³H-BP与小鼠肝细胞核的DNA共价结合。SY-640连续po 3 d,可显著诱导小鼠肝微粒体细胞色素P-450含量及氨基比林脱甲基酶活性;给药1次2h内却只抑制氨基比林脱甲基酶活性。体外温孵实验表明,SY-640对小鼠肝微粒体氨基比林脱甲基酶活性也具有明显的抑制作用。差示光谱分析表明,SY-640可与细胞色素P-450形成络合物。提示该化合物对肝微粒体细胞色素P-450酶系的影响与其对化学致癌剂BP所致肝细胞毒性的保护作用有关。     

Effect of sesquiterpene lactones on antioxidant enzymes and some drug-metabolizing enzymes in rat liver and kidney

Previously we have reported that several sesquiterpene lactones isolated from Helenium aromaticum and Telekia speciosa showed pro-oxidative properties and caused glutathione level depletion in rat liver in vivo. In the present study we examined the in vivo effect of these lactones on antioxidant enzyme systems and some drug metabolizing enzymes in the liver and the kidney of rats. We found that the majority of the compounds increased the hepatic activity of glutathione peroxidase (GPx), glutathione reductase (GR), and catalase (CAT), but superoxide dismutase (SOD) activity was distinctly lowered by five lactones. A few of the compounds tested caused a decrease in the hepatic cytochrome P450 content and reduced the activity of NADPH-cytochrome P450 reductase, aminopyrine demethylase, aniline hydroxylase and glutathione-S-transferase. Results for the kidney showed fewer changes in activities of both classes of enzymes when compared to the liver. Not all lactones affected the enzymes under test, the most active were: linifolin, helenalin, mexicanin 1 and telekin. 6 alpha-Hydroxy-2,3-dihydroaromaticin behaved differently towards monooxygenases since it induced the activity of aminopyrine demethylase and aniline hydroxylase.     

Effect of riboflavin deficiency on phenobarbital and 3-methylcholanthrene induction of microsomal drug-metabolizing enzymes of the rat

The effect of riboflavin deficiency on the induction of hepatic microsomal enzymes by phenobarbital and 3-methylcholanthrene has been investigated. A decrease in microsomal flavin levels of 56 per cent was associated with a decrease in NADPH cytochrome c reductase (52 per cent), azoreductase (71 per cent) and benzpyrene hydroxylase (74 per cent). Microsomal cytochrome P-450 content and aminopyrine demethylase were not significantly affected by flavin deficiency. Phenobarbital or 3-methylcholanthrene pretreatment did not affect hepatic microsomal flavin levels in normal or deficient animals. In flavin-deficient animals, phenobarbital pretreatment significantly increased cytochrome c reductase, cytochrome P-450 content, aminopyrine demethylase and azoreductase. Thus the carbon monoxide-sensitive pathway (cytochrome P-450 mediated) of azoreductase was essentially unaffected by flavin deficiency. In deficient animals, the carbon monoxide-insensitive microsomal azoreductase pathway (non-cytochrome P-450 mediated) normally induced by 3-methylcholanthrene was unaffected. Thus, induction of azoreductase by 3-methylcholanthrene was found to be flavin dependent. However, 3-methylcholanthrene did increase cytochrome P-450 content and benzpyrene hydroxylase in flavin-deficient animals. The induction of benzpyrene hydroxylase by 3-methylcholanthrene increased with increasing microsomal flavin content. Part of the mechanism of azoreductase induction by 3-methylcholanthrene was due to an induced change in the structure or composition of microsomal flavoprotein. This interpretation is supported by the findings that: (1) induction by 3-methylcholanthrene in riboflavin-deficient rats required a minimal flavin level, (2) increased enzyme activity was not compensated by an increase in microsomal flavin and (3) induction by 3-methylcholanthrene augmented FMN-stimulation of microsomal azoreductase in vitro.     

Polychlorinated dibenzofurans--potent inducers of rat hepatic drug-metabolizing enzymes.

The effects of polychlorinated dibenzofurans (PCDFs), trace toxic contaminants of commercial polychlorinated biphenyl preparations (PCBs), on the induction of hepatic drug-metabolizing enzymes were studied in the rat. PCDFs were about a thousand times more potent than PCBs (Kanechlor-500) as inducers of cytochrome P-450. Rats given 10 microgram/kg of PCDFs intraperitoneally for 3 days showed significantly increased hepatic cytochrome P-450 levels. At the highest dose tested, 1000 microgram/kg, a two-fold increase of cytochrome P-450 and a three-fold increase of p-nitroanisole demethylase activity were observed. PCDFs and 3-methylcholanthrene had quite similar effects on microsomal drug-metabolizing enzymes. Both drugs increased p-nitroanisole demethylase activity strikingly and aniline hydroxylase activity moderately, but produced little change in aminopyrine demethylase activity. alpha-Naphthoflavone, which is known to be a specific inhibitor of aryl hydrocarbon hydroxylase induced by polycyclic aromatic hydrocarbons, inhibited at low concentrations p-nitroanisole demethylase activity of rats previously treated with both drugs. Further, both drugs increased the 455 nm to 430 nm peak ratios of ethyl isocyanide difference spectra. Following three daily doses of PCDFs (100 microgram/kg), cytochrome P-450 level and p-nitroanisole demethylase activity remained elevated for over 15 days, with a decrease to control levels after 30 days. Such indicates the slow excretion of PCDFs.     

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.     

Effects of 2(3)-tert-butyl-4-hydroxyanisole administration on the activities of several hepatic microsomal and cytoplasmic enzymes in mice.

Administration of 2(3)-tert-butyl-4-hydroxyanisole (BHA) (0.75%) caused a marked increase in the activities of several hepatic enzymes in CD-1 mice. This was associated with increased liver weights and total protein contents, especially of the microsomal and cytosol fractions. While the specific content of cytochrome P-450 was decreased slightly in microsomes, the specific content of cytochrome b₅ and the specific activities of cytochrome c reductases (NADPH- or NADH-dependent) were increased (2-fold). In spite of a slight decrease in the specific activities of aminopyrine demethylase and of benzo(a)pyrene hydroxylase, both aniline hydroxylase and UDP-glucronyltransferase activities were increased (2.7- and 4.6-fold respectively). The specific activity of a microsomal membrane marker enzyme, glucose-6-phosphatase, was decreased slightly (?25 per cent). In the cytosol fraction, the specific activities of glucose-6-phosphate dehydrogenase and of UDP-glucose dehydrogenase were increased (3.8- and 6.1-fold respectively). Differences were noted in the time cources of increase and decrease in these enzyme activities after initiation and discontinuation of BHA treatment.     

Polychlorobiphenyl (Phenoclor DP6) and the metabolism of xenobiotics: effect of a high lipid diet

Young male rats were fed a balanced diet or a hyperlipidic diet (lard 30%, w/w) for 6 wk. After the first 2 wk each group was divided, one half being maintained as the control and the other half being fed the same diet with a polychlorinated biphenyl (Phénoclor DP6) added at a level of 50 mg/kg diet for 4 wk. In animals fed the balanced diet, DP6 increased the microsomal-protein and cytochrome P-450 content and the specific activity of the monooxygenases. The high-fat diet increased the inductive effect of DP6 on cytochrome P-450, aminopyrine demethylase and benzo[a]pyrene hydroxylase, but not on aniline hydroxylase or cytochrome c reductase.     

Acute interaction of drugs. I. The effect of volatile anesthetics on the kinetics of aniline hydroxylase and aminopyrine demethylase in rat hepatic microsomes

The in vitro effect of halothane, methoxyflurane, diethyl ether, and chloroform on the Michaelis constant (K_m) and maximal velocity (V_max) of microsomal aniline hydroxylase and aminopyrine demethylase was determined. The microsomes were obtained from rats pretreated with phenobarbital or 3-methylcholanthrene as well as from untreated rats. The halogenated anesthetics increased both the K_m and V_max of aniline hydroxylase in microsomes from untreated rats and this effect was magnified in microsomes from phenobarbital-induced animals. The K_m and V_max of aniline hydroxylase was not stimulated above control levels by halogenated anesthetics in microsomes from methylcholanthrene-induced rats. These anesthetics tended to inhibit the aminopyrine demethylase by lowering the V_max. Enzyme induction did not alter this inhibition. Diethyl ether inhibited aniline hydroxylase and aminopyrine demethylase by lowering the V_max.     

Species differences in stimulation of intestinal and hepatic microsomal mixed-function oxidase enzymes

The effect of intraperitoneal (i.p.) administration of phenobarbital (PB) or 3-methylchol-anthrene (3-MC) on some mixed-function oxidase (MFO) enzymes was studied in small intestine and liver of male rats, mice, guinea pigs and rabbits. PB treatment enhanced intestinal and 7-ethoxycoumarin deethylase activities in the mouse and rat, whereas benzo[a]pyrene hydroxylase (AHH) activity was increased only in the mouse. Ethylmorphine demethylase and aniline hydroxylase activities in small intestine were not stimulated by PB in any species. Administration of 3-MC increased the activity of intestinal AHH in rat, mouse and guinea pig, but intestinal 7-ethoxycoumarin deethylase activity was elevated only in the rat. The guinea pig and mouse intestinal ethoxycoumarin deethylase activity was inhibited by 3-MC treatment. None of the enzymes tested in rabbit intestine was induced by PB or 3-MC. The hepatic activities of ethylmorphine demethylase, aniline hydroxylase, 7-ethoxycoumarin deethylase and AHH, and the cytochrome P-450 content were increased by PB in all species. In contrast, 3-MC enhanced hepatic aniline hydroxylase and AHH activities in rats, mice and guinea pigs, and hepatic 7-ethoxycoumarin deethylase activity in mice and rats. In rabbits, these hepatic enzymes were inhibited by 3-MC pretreatment. The hepatic cytochrome P-450 absorption spectra was shifted to 448 nm in all species. These results suggest that there are differences in induction of intestinal and hepatic MFO enzymes which are influenced by the type of inducing agent, substrate and animal species used.     

The effects of 1-alkylimidazoles on hepatic drug-metabolizing enzyme activity

Abstract

1. Certain 1 -alkylimidazoles, containing a C₈-C₁₅ alkyl group, are potent inhibitors of liver microsomal drug oxidations. At a concentration of 10_?4M, they inhibited almost entirely the aminopyrine demethylase activity of rat liver 10 000 g supernatant fraction. In vivo, they prolonged hexobarbitone sleeping time in mice.

2. Inhibition of aminopyrine demethylase by 1-geranylimidazole was of a mixed type at high inhibitor concentrations and noncompetitive at low concentrations.

3. Addition of 1-geranylimidazole to rat liver microsomal fraction elicited a type II spectral change, but the use of hexobarbitone as a modifier revealed both type I and type II components.

4. Rats killed 24 h after the last of eight daily (100 mg/kg) doses of 1-do-decylimidazole showed marked induction of aminopyrine demethylase and aniline hydroxylase activities, but no change in cytochrome P-450 level.     

Comparative incidence of oral ochratoxicosis and aflatoxicosis on the activity of drug-metabolizing enzymes in rat liver

Mycotoxicosis has been produced in the rat by daily oral administrations of ochratoxin A (1.5 mg/kg/day) or aflatoxin B1 (1 mg/kg/day). Hepatic microsomal cytochrome P-450 and b5 contents and many phase I and II biotransformation systems have been measured in the course of ochratoxicosis (4 to 15 dosings) and aflatoxicosis (1 to 8 dosings). In case of ochratoxicosis, decreases in cytochrome P-450 level, aminopyrine demethylase and aniline hydroxylase activities were observed in rats receiving 15 administrations of the toxin. Aflatoxicosis induced more severe decreases in cytochrome P-450, aminopyrine demethylase and ethoxycoumarin deethylase following 8 daily gavages. In the two studies, there was no significant change in activities of liver phase II biotransformation enzymes.     

Contrasting effects of ethylenethiourea on hepatic monooxygenases in rats and mice

The effects of ethylenethiourea (ETU) on the hepatic xenobiotic metabolizing system in rats and mice were investigated. Male rats and male mice were given oral doses of 50 and 75, or 50, 75, 100, 500, and 1,000 mg/kg for 3 days. The microsomal enzymes studied were aminopyrine N-demethylase, aniline hydroxylase, and cytochrome P-450. In rats, the activity of aminopyrine N-demethylase was reduced to values between 60 and 70% of controls 24 h after treatment. A decrease in aniline hydroxylase activity and cytochrome P-450 content was observed on the 3rd day after exposure. In mice, treatment with ETU resulted in an increase of cytochrome P-450 at all dose levels. The activity of aniline hydroxylase was significantly elevated in the groups receiving doses of 100 mg/kg and higher. Aminopyrine N-demethylase was unaffected by the treatment. The results suggest that there are qualitative differences between rats and mice after ETU exposure with respect to the response of the hepatic monooxygenases.     

Inhibition of hepatic microsomal drug-metabolizing enzymes by imperatorin

The effect of imperatorin on hepatic microsomal mixed function oxidases (MFO) was investigated. On acute treatment, imperatorin (30 mg/kg, i.p.) caused a significant reduction in activities of hepatic aminopyrine N-demethylase, hexobarbital hydroxylase and aniline hydroxylase as well as cytochrome p-450 content in rats and mice. Kinetic studies on rat liver enzymes revealed that imperatorin appeared to be a competitive inhibitor of aminopyrine N-demethylase (Ki, 0.007 mM), whereas a non-competitive inhibitor of hexobarbital hydroxylase (Ki, 0.0148mM). Imperatorin also inhibited non-competitively aniline metabolism (Ki, 0.2mM). Imperatorin binds to phenobarbital-induced cytochrome p-450 to give a typical type 1 binding spectrum (max. 388nm, min 422nm). Multiple administrations of imperatorin (30 mg/kg, i.p. daily for 7 days) to mice shortened markedly the duration of hexobarbital narcosis and increased activities of hepatic aminopyrine N-demethylase and hexobarbital hydroxylase and the level of cytochrome p-450 whereas aniline hydroxylase activity was unaffected.     

Impairment of drug metabolism by the liver in experimental fascioliasis in the rat

Fascioliasis has been produced in the rat by an oral administration of 20 metacercariae of Fasciola hepatica. Hepatic microsomal cytochrome P450 and b5 contents and both aminopyrine demethylase and aniline hydroxylase activities have been measured during the course of the experimental distomiasis. The cytochrome P450 concentration and microsomal drug metabolizing enzymes generally fell by weeks 3 to 8 post-infestation and recovered to normal values thereafter. For the same period, both the histoenzymatically assayed liver cytochrome oxidase and arylsulphatase activities were reduced whereas there were correlated increases in glutamic pyruvic and glutamic oxaloacetic plasma transaminases. Tissue inflammation and destruction provoked by young histophagous migrating flukes could be responsible for these changes that have already been observed in several hepatic diseases. The possible influence of naturally-induced fasciolasis on liver drug metabolism is discussed.     

Hepatic mixed-function oxidase activities of wild pigeons

Hepatic mixed-function oxidase activities of wild pigeons were determined and compared with those of rat to assess the apparent differences in avian and mammalian drug metabolism. Aminopyrine N-demethylase, benzphetamine N-demethylase, ethylmorphine N-demethylase, aniline hydroxylase, NADPH-cytochrome c reductase, glutathione S-transferase activities and cytochrome P-450 levels in pigeon liver were 30-80% lower than the corresponding activities in rat liver. p-Nitroanisole O-demethylase activity in pigeon liver was similar to that of rat liver. Wild pigeon-liver benzo[a]pyrene hydroxylase activity was approx. five times higher than that in the rat. Pigeons did not reveal any noticeable sex differences in mixed-function oxidase activities. Administration of 3-methylcholanthrene and phenobarbital to pigeons resulted in the induction of demethylase and benzo[a]pyrene hydroxylase activities and in cytochrome P-450 levels.