Induction Of Drug-metabolizing Enzymes Research Articles

Hepatic and extrahepatic induction of drug-metabolizing enzymes in specific pathogen free and germ free rats

1. 1. The drug metabolizing enzyme activities were equal in the liver, kidney and intestinal mucosa of control specific pathogen free and germfree rats. 2. 2. The hepatic inducibility of ethoxycoumarin O-deethylase and p-nitroanisole O-demethylase activities by 3-methylcholanthrene was higher in germfree than in SPF rats. 3. 3. Aryl hydrocarbon hydroxylase activity was inducible only by 3-methylcholanthrene in the liver, kidney and intestinal mucosa of rats of both gnotobiotic states. 4. 4. Hepatic cytochrome P-450 level and p-nitroanisole O-demethylase activity were highest after phenobarbitone administration in rats of both gnotobiotic states. 5. 5. The hepatic UDPglucuronosyltransferase activity was enhanced 50% by phenobarbitone and 100% by 3-methylcholanthrene in trypsin-digested microsomes. Only slight induction by 3-methylcholanthrene was present in kidneys and no induction was seen in the intestinal mucosa.

Induction of liver microsomal drug-metabolizing enzymes by 2,2′,4,4′,5,5′-hexabromobiphenyl

The major component of Firemaster, a mixture of polybrominated biphenyls (PBBs), is 2,2′,4,4′,5,5′-hexabromobiphenyl (HBB). HBB was isolated to greater than 99.9% purity, and its effects on tissue structure and liver microsomal drug-metabolizing enzymes were examined. Male rats were injected ip with 90 mg of HBB/kg and sacrified 1 to 14 days later. Ten tissues were examined microscopically; only liver revealed HBB-dependent lesions. Hepatic cells were swollen and vacuolated, predominantly in the midzonal region. Liver to body weight ratios were increased by a third, and microsomal protein was nearly tripled. NADPH-cytochrome P-450 reductase was induced, and cytochrome P-450 was increased more than 2.5-fold, with the spectral maximum remaining at 450 nm. Aminopyrine demethylation was doubled by HBB, while PBBs and phenobarbital (Pb) both tripled this activity. Induction of benzo[ a]pyrene hydroxylation was similar to that caused by Pb, but the level was fivefold smaller than that obtained by 3-methylcholanthrene (MC) induction. PBBs induced this activity to half of the MC level. HBB induced epoxide hydratase more than threefold. While PBBs tripled UDP-glucuronyltransferase activity, HBB was a poor inducer. SDS-polyacrylamide gel electrophoresis of HBB-induced microsomes revealed heme and protein profiles similar to those of microsomes induced by Pb, but markedly different from those of control microsomes. While PBBs are a mixed-type inducer of microsomal drug-metabolizing enzymes, these results show that HBB is strictly a Pb-type inducer.

Proliferation of smooth endoplasmic reticulum and induction of microsomal drug-metabolizing enzymes after ether or halothane.

Hepatic drug-metabolizing enzymes and hepatic ultrastructure were studied in rats after two hours of anesthesia with 1 MAC halothane or diethyl ether. Twelve hours after cessation of either anesthetic smooth endoplasmic reticulum was increased in centrilobular but not in periportal hepatocytes. This change persisted at 24- and 36-hour sampling times. Microsomal cytochrome P450 and cytochrome b5 decreased after halothane anesthesia (by 7 to 20 per cent of control). Diethyl ether caused increased cytochrome P450 and cytochrome b5 (27 and 18 per cent, respectively) at the 36-hour sampling time. NADPH cytochrome c reductase did not change significantly after either agent. The authors interpret these results to mean that both agents promote conversion of rough endoplasmic reticulum to smooth endoplasmic reticulum or, alternatively, that the anesthetics decrease degradation of smooth endoplasmic membranes. Since only ether caused an increase in the microsomal content of enzymes of the drug-metabolizing enzyme system, it is concluded that these two anesthetics act on hepatic cells by dissimilar mechanisms.

Induction of microsomal drug-metabolizing enzymes caused by hexobarbital

Hexobarbital was given to anaesthetized mice for a period of 7 h by repeated i. p. injection, first of 100 mg/kg,then several times of 50 mg/kg. A high level of hexobarbital was maintained in the liver. The activity of microsomal drug-metabolizing enzymes was induced by this treatment with hexobarbital. 30 min after a single i. p. injection of 100 mg/kg of hexobarbital, there was a significant inhibition of aminopyrine N-demethylase but none of cytochrome c and neotetrazolium reductases. Hexobarbital in vitro inhibits aminopyrine N-demethylase but not cytochrome c reductase.

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.

Effect of typical inducers of microsomal drug-metabolizing enzymes on phospholipid metabolism in rat liver

The effect of administration of phenobarbital (PB), polychlorinated biphenyls(PCBs) or 3-methyl-cholanthrene (3-MC) on the metabolism of phospholipids in rat liver was studied by the i.p. injection of [ 32P]orthophosphate or [Me- 14C]choline chloride. The inducers were given to animals for 2 successive days PB had no significant effect on the incorporation rate of 32Pi into liver microsomal phospholipid classes 48 hr after the first administration. The rate of incorporation of [ 14C]choline into phosphatidylcholine (PC) in both subcellular components of the liver and blood plasma decreased slightly at this experimental period. In addition, the ratio of [ 14C] sp. act. of phosphorylcholinc to that of microsomal phosphulipid was considerably higher on PB-trcated rats as compared with the ratio in control rats. These and previous findings strongly suggest that proliferation of liver endoplasmic reticulum (ER) membranes induced by PB would be accompanied by the stimulation of phospholipid synthesis at the early process of induction and subsequently followed by the decrease in turnover rate of microsomal phospholipids. The administration of PCBs, on the other hand, caused strong inhibition of both 32Pi and 14C incorporation into PC in liver subcellular fractions. The secretion of PC from liver cells to blood plasma was also strongly depressed. In addition, phospholipid catabolizing activity was found to be depressed in the liver. These results indicate that hypertrophy of ER membranes in the liver after PCBs administration could be due to a depression of both secretion of lipoprotein from liver cells to plasma and catabolic activity toward membranous phospholipids in liver cells. The decrease in 32Pi incorporation into liver microsomal PC was also observed in rats treated with 3-MC. There occurred a considerable accumulation of 14C activity in phosphorylcholine in the liver of rats treated with either PCBs or 3-MC, suggesting strongly that these drugs caused an inhibition of PC synthesis at the site of CDP-choline formation, namely the inhibition of the reaction catalyzed by cholinephosphate cytidylyltransferase.

The influence of phenobarbital, 3-methylcholanthrene and 2,3,7,8-tetrachlorodibenzo- p-dioxin on glutathione s-transferase activity of rat liver cytosol

Specific activities and apparent Michaelis-Menten kinetic parameters were determined for glutathione (GSH) S-transferase activity (E.C. 2.5.1.18) in rat liver cytosol, towards styrene oxide (STOX), 1,2-butylene oxide (BOX) and 1-chloro-2,4-dinitrobenzene (CDNB) as electrophilic substrates, before and after pretreatment with the drug-metabolizing enzyme inducers phenobarbital (PB), 3-methylcholanthrene (MC) and 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD). The measured GSH S-transferase activities appear to obey Michaelis-Menten kinetics. In non-induced animals the apparent K m values of the transferase activities were equal for STOX vs GSH, but they differed by a factor of 2 for CDNB vs GSH and by a factor of 14 for BOX vs GSH. The apparent V max values in each combination of GSH and electrophilic substrate were equal, but differed by one order of magnitude for the mutual substrate combinations. Pretreatment of the rats with MC resulted in enhancement of all measured activities expressed in terms of cytosol protein, while TCDD only enhanced the activities expressed as per gram body wt. PB enhanced both activities when STOX was employed as substrate, but when CDNB was used as the substrate, only the activity per gram body wt increased. All pretreatments increased the V max values using CDNB as the substrate, while PB and MC had an enhancing effect using STOX; the V max using BOX was enhanced after TCDD administration only. The K m values using BOX as the substrate was lowered after MC pretreatment; TCDD pretreatment decreased the K m using STOX, while it increased the K m using CDNB. It is concluded that the GSH S-transferase system is inducible, but in contrast to the induction of the mixed function oxidase system, qualitative differences between the inducing effects of PB and MC were not observed. Use of TCDD as inducing agent, however. resulted in a different induction pattern, which may indicate that during induction with this agent different types of GSH S-transferases are involved.

Long-term studies on chemically induced liver enlargement in the rat I. Sustained induction of microsomal enzymes with absence of liver damage on feeding phenobarbitone or butylated hydroxytoluene

As part of a study on the relationships between liver enlargement, induction of drug-metabolising enzymes and certain forms of liver damage, sequential biochemical and morphological observations were made on the livers of female rats fed diets containing 0.4% butylated hydroxytoluene (BHT) or 0.25% phenobarbitone for periods of up to 80 weeks. Both compounds increased the relative liver weight and produced marked enhancement of the activities of ethylmorphine N-demthylase, aniline 4-hydroxylase, biphenyl 4-hydroxylase and NADPH-cytochrome c reductase and the contents of cytochromes P-450 and b 5 and microsomal protein. These changes were evident after 1 week of treatment and persisted until treatment was stopped after 80 weeks. The only morphological changes observed throughout the period of treatment were centrilobular cell enlargment and hypertrophy of the smooth endoplasmic reticulum (SER). Histochemical examination revealed a centrilobular depression of glucose-6-phosphatase but there was no disturbance of the normal pericanalicular distribution of lysosomes. All changes observed with either compound were reversible on cessation of treatment at week 80. These results support the concept that liver enlargement accompanied by induction of drug-metabolising enzymes represents an adaptive response and they provide a basis for the interpretation of pathological changes developing in the enlarged liver unaccompanied by drug-metabolising enzyme induction.

Separation of pure polychlorinated biphenyl isomers into two types of inducers on the basis of induction of cytochrome P-450 or P-448

A number of isomerically pure polychlorinated biphenyls (PCBs) were tested as inducers of hepatic drug-metabolizing enzymes in the rat. The chlorinated biphenyl isomers can be categorized into two distinct groups of inducers, while commercial PCB mixtures have characteristics of both groups. Biphenyls chlorinated symmetrically in both the meta and para positions (3,4,3′,4′- and 3,4,5,3′,4′,5′-) increase the formation of cytochrome P-448, the ratio of the 455 to 430 peaks of the ethyl isocyanide difference spectrum, and aryl hydrocarbon hydroxylase and glucuronyl transferase activities, but decrease aminopyrine N-demethylase activity. These isomers are also the most toxic, as measured by weight loss. Biphenyl isomers chlorinated in both the para and ortho positions induce the formation of cytochrome P-450 rather than P-448, regardless of the chlorination of the meta position. These isomers, which include 2,4,2′,4′-tetra- and 2,4,5,2′,4′,5′-, 2,3,4,2′,3′,4′- and 2,4,6,2′,4′,6′-hexachlorobiphenyls, increase cytochrome P-450 and N-demethylase activity, but produce only a slight increase in aryl hydrocarbon hydroxylase activity, and do not alter the peak of the CO-difference spectrum or the ratio of the 455/430 peaks of the ethyl isocyanide difference spectrum. Isomers which are chlorinated in only one ring, or are chlorinated in both rings but not in the para positions, have very little activity as inducers of liver enzymes. Of the dichlorobiphenyls tested, 3,3′- and 4,4′-dichlorobiphenyls have very slight activity at extremely high doses.

Effects of chronic ethanol administration in the rat: relative dependency on dietary lipids. II. Paradoxical role of linoleate in the induction of hepatic drug-metabolizing enzymes in vitro

The effect of chronic ethanol administration on the hepatic microsomal cytochrome P-450 content and activities of NADPH - cytochrome P-450 reductase (EC 1.6.2.4), benzphetamine demethylase, aniline hydroxylase (EC 1.14.14.1), and of the microsomal ethanoloxidizing system were studied in various dietary models. When ethanol was given with linoleate as the only source of dietary lipid, the ethanol induction of these parameters was greater with diets containing 2 or 5% of total calories as linoleate than with diets containing 10% of total calories as linoleate. By contrast, when ethanol was given with high fat (35% of total calories) diets, the ethanol induction of these same parameters was slightly greater when linoleate provided 10% of total calories than when it provided 3% of calories. The apparent effect of dietary linoleate on the induction, by ethanol, of microsomal drug-metabolizing enzymes is markedly different when linoleate is given as the only source of dietary lipid as opposed to when it is given with other dietary lipids. Thus, conclusions on the effect of ethanol on hepatic microsomal drug-biotransformation enzymes, drawn from studies with dietary models in which linoleate provides the only source of dietary lipid, connot be extended to dietary modles of more complex lipid composition. When given as the only source of lipid, 2% of total calories in linoleate appears optimal for basal activity and inductibility, by ethanol, of mixed-function oxidases.

Chlorphenesin carbamateの薬理学的研究 ―耐薬性試験―

Tolerance to chlorphenesin carbamate (CPC) was investigated from the viewpoints of action of CPC, serum free CPC concentration, the activity of UDP-glucuronyltransferase and the content of cytochrome P-450. CPC was administered once daily for 7 or 14 days. In mice, the hypnotic action of hexobarbital injected 24 hours after the last administration of CPC and the motor incoordinating action of CPC significantly decreased on the 7th day, but slightly recovered on the 14th day. Serum free CPC concentration also decreased on the 7th day, but recovered on the 14th day. A significant relationship between the motor incoordinating action of CPC and serum free CPC concentration was observed. Therefore, the recovery of CPC effect on the 14th day was considered to be due to the recovery from the induction of drug-metabolizing enzymes. On the other hand, in rats, the weekly alteration of the motor incoordinating action of CPC was similar to that observed in mice. Serum free CPC concentration on the 7th and 14th days was lower than that on the 1st day, and enzyme induction was observed during CPC administration. Notwithstanding the low level of serum free CPC concentration, the recovery of CPC effect was observed on the 14th day and such was considered to be due to habituation to the rotarod. In mice and rats, it was demonstrated that the intensity of CPC effect was dependent on serum free CPC concentration to the extent that enzyme induction played an important role in the development of tolerance. From these results, the tolerance to CPC is attributed to induction of drug-metabolizing enzymes in liver microsomes.

Induction of Hepatic Drug Metabolizing Enzymes and Mutagen-activation Capacity in Several Animal Species

突然変異性発ガン性化合物のスクリーニング方法として一般的に用いられている Ames の方法について, その in vitro 代謝系の改良をするため, PCB, フェノバルビタールおよび3-メチルコラントレンのそれぞれを投与し薬物代謝系を誘導したラット, マウス, モルモット, ハムスター, およびウサギよりの肝臓S-9画分を用い, サルモネラ菌TA98株, TA100株の突然変異体出現数の変化を調べた.5動物種12系統のなかで, PCB処理ハートレー系モルモットより得たS-9画分は, 3種の異なった化合物 (ベンツ (a) ピレン, 2-アセチルアミノフルオレン, ジメチルニトロソアミン) の変異性検出において, 通常用いられているPCB処理SDラットよりすぐれていた. また3-メチルコラントレン処理モルモット (ハートレー系) は, PCB投与SDラットとほぼ同等であることが認められ, ハムスターのS-9は, DMNの突然変異の検出に有効であった.一方, ウィスター系およびフィッシャー系ラット, ICR系, dd系マウス, ウサギ, などは, SD系と比較してとくにすぐれているとはみなせない.

Mechanism of induction of hepatic drug-metabolizing enzymes by ethanol—I: Limited role of microsomal phospholipids

Two groups of female Sprague-Dawley rats were pair-fed nutritionally adequate liquid diets containing 36 per cent of total calories as ethanol or additional carbohydrate (controls). One group was fed a high fat diet (35 per cent of total calories as fat); the other group was fed a low fat diet (2 per cent of total calories as linoleate as the only source of fat). When given with the high fat diet, ethanol increased cytochrome P-450 and microsomal phospholipid content per g of liver. When given with a low fat diet, it increased cytochrome P-450 to a lesser extent and did not alter the microsomal phospholipid content when expressed per g of liver or per 100 g of body weight. Phosphatidylcholine accounted for the greater porportion of phospholipids and was increased by ethanol only with the high fat diet. L-Methionine-methyl[ 3H] and [ 14C]choline incorporation into phosphatidylcholine was unaltered by ethanol in either model. The fatty acid composition of phosphatidylcholine was altered by ethanol more significantly with the high fat diet. Since ethanol similarly enhances the activity of benzphetamine demethylation in both dietary models, quantitative and qualitative differences in microsomal phospholipids produced by ethanol are probably not responsible for the induction by ethanol of this drug-metabolizing enzyme activity. Further evidence to that effect was obtained from the measurement of benzphetamine demethylation in vitro by partially purified cytochrome P-450, reductase and lipid fractions of ethanol-fed and control rats fed a high fat diet. The stimulation of benzphetamine demethylation by the lipid fraction was identical whether using lipids from microsomes of ethanol-fed animals or control animals. Dietary fat plays a role in the induction by ethanol of cytochrome P-450 and NADPH-cytochrome P-450 reductase and on microsomal phospholipid content and composition. The effects of ethanol on microsomal phospholipids are probably not related to the induction of benzphetamine demethylation activity.

Studies on the relationship between induction of drug-metabolizing enzymes, liver enlargement and ascorbic acid excretion in rodents.

Studies on the relationship between induction of drug-metabolizing enzymes, liver enlargement and ascorbic acid excretion in rodents.

Changes in nucleotide metabolism of the liver and fate of [14C]orotate among various pyrimidines at the outset and during induction of drug-metabolizing enzymes and hepatomegaly.

Changes in nucleotide metabolism of the liver and fate of [14C]orotate among various pyrimidines at the outset and during induction of drug-metabolizing enzymes and hepatomegaly.

Early interaction of radioactive butylated hydroxytoluene with protein factors of varying stability in rat liver, related to the onset of sequential biochemical changes leading to the induction of drug-metabolizing enzymes and liver enlargment.

Early interaction of radioactive butylated hydroxytoluene with protein factors of varying stability in rat liver, related to the onset of sequential biochemical changes leading to the induction of drug-metabolizing enzymes and liver enlargment.

Effect of smoking on theophylline disposition.

The pharmacokinetics of theophylline were examined in a group of nonsmokers and in heavy smokers (1 to 2 packs/day) before and 3 to 4 mo after cessation of cigarette smoking. The half-life of theophylline in smokers averaged 4.3 (SD = 1.4) hr, significantly shorter than the mean value in nonsmokers (7.0, SD =1.7 hr). The apparent volume of distribution of theophylline was somewhat larger in smokers (0.50 +/-0.12 L/kg) than in nonsmokers (0.38 +/-0.04 L/kg). The body clearance of theophylline was appreciably larger and relatively more variable in smokers (100 +/-44 ml/min/1.73 m2) than in nonsmokers (45 +/-13 ml/min/1.73 m2). Serum concentrations of thiocyanate, a biotransformation product of cyanide which is inhaled with smoke, were used to monitor the smoking status of the subjects. The body clearances of theophylline showed a good correlation (r = 0.785, p less than 0.001) with the serum thiocyanate concentrations. Of the 8 smokers, only 4 managed to refrain from smoking for at least 3 mo, and these subjects showed no significant change in theophylline elimination. The increase in theophylline clearance caused by smoking is probably the result of induction of drug-metabolizing enzymes that do not readily normalize after cessation of smoking.

Activation of 3':5'-cyclic AMP-dependent protein kinase and induction of ornithine decarboxylase as early events in induction of mixed-function oxygenases.

The parenteral administration of a single dose of 3-methylcholanthrene to rats caused an increase in the liver of the concentration of 3', 5'-cAMP and of the activity of cAMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37). These events were followed by an increased activity of ornithine decarboxylase (L-ornithine carboxy-lase, EC 4.1.1.17), the enzyme that controls the biosynthesis of polyamines. Finally, the activity of benzo[a]pyrene hydroxylase, as well as the amount of cytochrome P-448, was increased. Similarly, after the administration of phenobarbital, there was first an increase in the cAMP concentration and in the activity of cAMP-dependent protein kinase, then the induction of ornithine decarboxylase, and finally, an enhanced activity of ethylmorphine N-demethylase and an increased content of cytochrome P-450. These data suggest that the drug-induced processes in liver that increase the activities of the oxidative, and presumably other, drug-metabolizing enzymes include the following sequence of events: (1) increase in cAMP concentration and/or activation of cAMP-dependent protein kinase; (2) induction of ornithine decarboxylase; and, (3) induction of drug-metabolizing enzymes.

Effect of fluorosteroids on drug response and metabolism

In female rats, zoxazolamine paralysis was significantly reduced by pretreatment with pregnenolone-16α-carbonitrile (PCN), spironolactone, dexamethasone acetate, betamethasonc acetate, 9α-fluoro-11 β,21-dihydroxy-16α, 17α-dimethyl-1,4-pregnadiene-3,20-dione, 6α-fluoro-9α-chloro-11 β-acetoxy-21-valeryloxy-16α-methyl-1,4-pregnadiene-3, 9α-fluorocortisol acetate, triamcinolone or adrenocorticotropic hormone (ACTH). With the exception of triamcinolone and ACTH. the protective effects of these compounds were associated with decreased drug concentrations in plasma at the end of the pharmacologic response, compared with controls killed at the same time. The drug levels were found to be lowered via hepatic drug-metabolizing enzyme induction. All the fluorosteroids exerted glucocorticoid activity. Thus, PCN and spironolactone protect via increased drug metabolism, triamcinolone and ACTH via decreased organ sensitivity, and the remaining fluorosteroids via both prophylactic mechanisms.

Effects of Low and High Protein Diets on the Induction of Microsomal Drug-metabolizing Enzymes in Rat Liver

To determine the effect of low and high levels of dietary proteins plus low doses of phenobarbital (PB) on the induction of drug-metabolizing enzymes, weanling male and female Sprague-Dawley rats were fed, ad libitum, diets containing 3.5 (low protein, LP), 26 (normal protein, NP), and 42% (high protein, HP) casein for 33 days. Five animals from each dietary group were injected with 10 mg of PB/kg body weight, intraperitoneally, for the last 3 days of the experimental period. The 10,000 X g supernatant and microsomes were prepared from perfused livers from each group. Both LP and HP diets caused a significant decrease in the activity of biphenyl 4-hydroxylase, an increase in the activity of p-nitrobenzoate reductase, and no changes in the activities of 4-methylumbelliferone glucuronyltransferase and cytochrome P-450. The low doses of PB used in this study caused significant induction of cytochrome P-450 (P less than 0.10), biphenyl 4-hydroxylase, and p-nitrobenzoate reductase (except in LP and NP females). The males of NP and HP groups had consistently higher activities of these enzymes than corresponding females, however, this sex difference in the first two enzymes was abolished by feeding of the LP diet.