Hormonal imprinting of the microsomal enzyme system in adults. Microsomal activity change in response to estrogen (DES, AE) treatment during liver regeneration

Estrogen (diethylstilbestrol-DES or allylestrenol-AE) treatment applied to rats of both sexes during liver regeneration following subtotal hepatectomy had a long lasting influence on the inducibility by phenobarbital of the hepatic microsomal enzyme system of the females. The enzyme activities of the DES-treated females differed hardly from the baseline two weeks after treatment, but increased almost two-fold over control on induction with phenobarbital 5 and 7 weeks later. The AE-treated females showed a smaller although yet significant, enzyme activity increase only at 7 weeks. The influence of estrogens was negligible, and inhibitory rather than stimulatory, in the males. It appears that, in appropriate conditions, enzyme imprinting can also be induced in adult organisms, since, in all probability, availability for imprinting depends not so much on the age of the organism, as on the developmental state of the target cell.     

Triacylglycerol metabolism in the phenobarbital-treated rat

1. Various aspects of triacylglycerol metabolism were compared in rats given phenobarbital at a dose of 100mg/kg body wt. per day by intraperitoneal injection; controls were injected with an equal volume of 0.15m-NaCl by the same route. Animals were killed after 5 days of treatment. 2. Rats injected with phenobarbital demonstrated increased liver weight, and increased microsomal protein per g of liver. Other evidence of microsomal enzyme induction was provided by increased activity of aminopyrine N-demethylase and cytochrome P-450 content. Increased hepatic activity of γ-glutamyltransferase (EC 2.3.2.2) occurred in male rats, but not in females, and was not accompanied by any detectable change in the activity of this enzyme in serum. 3. Phenobarbital treatment increased the hepatic content of triacylglycerol after 5 days in starved male and female rats, as well as in non-starved male rats; non-starved females were not tested in this regard. At 5 days after withdrawal of the drug, there was no difference in hepatic triacylglycerol content or in hepatic functions of microsomal enzyme induction between the treated and control rats. 4. After 5 days, phenobarbital increased the synthesis in vitro of glycerolipids in cell-free liver fractions fortified with optimal concentrations of substrates and co-substrates when results were expressed per whole liver. The drug caused a significant increment in the activity of hepatic diacylglycerol acyltransferase (EC 2.3.1.20), but did not affect the activity per liver of phosphatidate phosphohydrolase (EC 3.1.3.4) in cytosolic or washed microsomal fractions. A remarkable sex-dependent difference was observed for this latter enzyme. In female rats, the activity of the microsomal enzyme per liver was 10-fold greater than that of the cytosolic enzyme, whereas in males, the activities of phosphohydrolases per liver from both subcellular fractions were similar. 5. The phenobarbital-mediated increase in hepatic triacylglycerol content could not be explained by a decrease in the hepatic triacylglycerol secretion rate as measured by the Triton WR1339 technique. Since the hepatic triacylglycerol showed significant correlation with microsomal enzyme induction functions, with hepatic glycerolipid synthesis in vitro and with diacylglycerol acyltransferase activity, it is likely to be due to enhanced triacylglycerol synthesis consequent on hepatic microsomal enzyme induction. 6. In contrast with rabbits and guinea pigs, rats injected with phenobarbital showed a decrease in serum triacylglycerol concentration in the starved state; this decrease persisted for up to 5 days after drug administration stopped, and did not occur in non-starved animals. It seems to be independent of the microsomal enzyme-inducing properties of the drug, and may be due to the action of phenobarbital at an extrahepatic site.     

Impact of neonatal benzpyrene imprinting on liver microsomal enzyme induction by benzpyrene and phenobarbital in adulthood.

A single neonatal treatment of rats with benzpyrene accounted for a durable induction of the liver microsomal enzyme (PSMO) system. Neonatal treatment with benzpyrene enhanced the inducing action of phenobarbital administrated in adulthood, but did not change the effect of benzpyrene treatment in the adult age. It follows that the imprinting and inducing effects of the neonatal benzpyrene treatment took different trends.     

The effect of phenobarbital on the submicrosomal distribution of uridine diphosphate glucuronyltransferase from rat liver

1. The detergent Triton X-100 activates UDP glucuronyltransferase from rat liver in vitro six- to seven-fold with p-nitrophenol as substrate. The enzyme activity when measured in the presence of Triton X-100 is increased significantly by pretreatment of male rats with phenobarbital for 4 days (90mg/kg each day intraperitoneally). If no Triton X-100 is applied in vitro such an increase could not be shown. In all further experiments the enzyme activity was measured after activation by Triton X-100. 2. The K(m) of the enzyme for the substrate p-nitrophenol does not change on phenobarbital pretreatment. 3. When the microsomal fraction from the liver of untreated rats is subfractionated on a sucrose density gradient, 47% of the enzyme activity is recovered in the rough-surfaced microsomal fraction, which also has a higher specific activity than the smooth-surfaced fraction. 4. Of the increase in activity after the phenobarbital pretreatment 50% occurs in the smooth-surfaced fraction, 19% in the rough-surfaced fraction and 31% in the fraction located between the smooth- and rough-surfaced microsomal fractions on the sucrose density gradient. 5. The latency of the enzyme in vitro, as shown by the effect of the detergent Triton X-100, is discussed in relation to the proposed heterogeneity of UDP glucuronyltransferase.     

Strain differences in the induction of soluble and microsomal enzymes by phenobarbital and 3-methylcholanthrene

The ability of phenobarbital and 3-methylcholanthrene (3MC) to induce liver microsomal and soluble enzymes was compared in Sprague-Dawley and Long-Evans rats. 3MC increased the V for the aniline hydroxylase and stimulated the formation of the hemoprotein P₄₄₈ to a similar extent in the 2 strains of rats. On the other hand phenobarbital increased the V for the microsomal enzyme aniline hydroxylase and aminopyrine demethylase and enhanced the activity of the soluble enzyme aldehyde dehydrogenase only in Sprague-Dawley rats. It induced a more marked increase of cytochrome P₄₅₀ in the Sprague-Dawley than in the Long-Evans strain.     

Potentiation of thioacetamide hepatotoxicity by phenobarbital pretreatment in rats. Inducibility of FAD monooxygenase system and age effect

The ability of phenobarbital to induce the expression and activity of microsomal drug monooxygenases in the liver presents one of the most important issues in the field of chemical interactions and in the toxicity of xenobiotics. The model of rat liver injury induced by a single dose of thioacetamide (500 mg/kg intraperitoneally) was used to study the effect of phenobarbital (80 mg/kg/day intraperitoneally) for 5 days prior to thioacetamide. Serum parameters of liver injury such as aspartate aminotransferase activity, gamma-glutamyl transferase activity and the total bilirubin levels, as well as the activities of hepatic FAD and cytochrome P450 microsomal monooxygenases, were assayed in 2- and 12-month-old rats. Samples of blood and liver were obtained from controls (injected at 0 h with 0.5 ml of 0.9% NaCl) and at 12, 24, 48, 72 and 96 h of thioacetamide intoxication either to non-treated or phenobarbital pretreated rats. Potentiation of thioacetamide hepatotoxicity by phenobarbital pretreatment was demonstrated at morphological level, and by significant increases in the activities of serum aspartate aminotransferase and gamma-glutamyl transferase, and in the levels of total bilirubin. The extent of potentiation of thioacetamide-induced liver injury by phenobarbital pretreatment was similar in both age groups. Microsomal FAD monooxygenase activity, the enzyme responsible for thioacetamide biotransformation, was significantly enhanced (twofold) by phenobarbital pretreatment, and also underwent a further increase following thioacetamide, preceding the peak of necrosis. Cytochrome P450 monooxygenases were induced by phenobarbital pretreatment more than sixfold, and sharply decreased when phenobarbital was withdrawn and thioacetamide administered, showing at 48 h intoxication values close to basal. Phenobarbital pretreatment potentiated thioacetamide necrogenicity, and this potentiation was parallel to the induction of the microsomal FAD monooxygenase system, both by phenobarbital and by thioacetamide itself. The extent of thioacetamide-induced liver injury was significantly higher in 12-month-old rats, but the effect of phenobarbital pretreatment was similar in both age groups.     

Characterization of electron transport enzymes in the envelope of rat liver nuclei.

Nuclei and microsomes were prepared from the livers of normal, phenobarbital (PB)-treated and beta-naphthoflavone (beta-NF)-treated rats, and the contents of several enzymes in both subcellular fractions were examined. In normal rats, the enzyme activities in the nuclear fraction were about one-third of those of microsomes on a phospholipid basis. The induction of some particular enzymes by the drugs was observed with nuclei as well as with microsomes. Cytochrome P-450 and NADPH-cytochrome c reductase were increased by PB treatment and cytochrome P-448 was induced by beta-NF treatment both in nuclei and in microsomes. The extents of inhibition of nuclear enzyme activities by the antibodies against corresponding microsomal enzymes were almost the same as those of the microsomal activities. It was concluded that a microsomal type electron transport system exists in rat liver nuclei, and that nuclear drug-oxidizing activities are inducible by PB or beta-NF as their microsomal counterparts are.     

Effect of phenobarbital and 3-amino-1,2,4-triazole on the metabolism and biliary excretion of 3,4-benzpyrene in the rat

The effects of 3-amino-1,2,4-triazole and phenobarbital, alone and in combination, on the metabolism and biliary excretion of 3,4-benzpyrene have been investigated in Wistar and Sprague-Dawley rats of both sexes. Although phenobarbital induced and aminotriazole inhibited metabolism, considerable variation with species and sex was observed. The effects of these drugs on liver weight, microsomal protein and bile flow also varied among these groups of rats. it is apparent that there was no quantitative relationship between the pattern of excretion of benzpyrene metabolites and bile flow, microsomal protein and liver weight.     

Identification of increased amounts of UDP-glucuronyltransferase protein in phenobarbital-treated chick-embryo liver cells.

UDP-glucuronyltransferase activity of neonatal-chick liver or phenobarbital-treated chick-embryo liver catalysed the glucuronidation of 1-naphthol, 4-nitrophenol and 2-aminophenol. Only low transferase activity towards testosterone was detected, and activity towards bilirubin was not detectable. Liver microsomal transferase activity towards the three phenols was increased approx. 20-50-fold by phenobarbital treatment of chick embryos or by transfer of liver cells into tissue culture. A single form of UDP-glucuronyltransferase, which appears to catalyse the glucuronidation of these three phenols, was purified to near homogeneity from phenobarbital-treated chick-embryo liver microsomal fraction for the first time. The use of this purified enzyme as a standard protein facilitated the identification of this protein in chick-embryo liver microsomal fraction. Further, the accumulation of this microsomal protein was observed following phenobarbital treatment of chick embryos and during tissue culture of chick-embryo liver cells. The value of this model system for the study of the induction of UDP-glucuronyltransferase by drugs and hormones is discussed.     

Dietary orotic acid accentuates the hepatic response to phenobarbital in rats.

In rats treated with phenobarbital for 3 days and simultaneously fed a semisynthetic diet containing 1.0% orotic acid, the extent of the increases in liver microsomal phosphatidylcholine, phosphatidylethanolamine, total RNA, total protein, and cytochrome P-450 were significantly greater than they were in rats treated identically with phenobarbital but without dietary orotic acid. This is attributed primarily to the stimulation of hepatic phosphatidylcholine synthesis by dietary orotic acid. In the absence of phenobarbital, orotic acid was shown to cause some increase in liver smooth endoplasmic reticulum components, but not cytochrome P-450. Orotic acid also decreased the activity of microsomal phosphatidylethanolamine N-methyltransferase, which may have contributed to the increase in the microsomal content of phosphatidylethanolamine. The hypothesis is advanced that phospholipid availability is a limiting factor in the hepatic response to phenobarbital. When more phospholipid is available to provide the structural framework for biogenesis of endoplasmic reticulum, all of the hepatic actions of phenobarbital, including induction of cytochrome P-450, are amplified.     

The relationship of the intensity of liver microsomal oxidation to individual longevity

Positive correlation between the activity of enzymes of microsomal oxidation of the liver and individual lifespan was shown in experiments on Wistar female rats. The use of test of duration of pentobarbital hypnosis provides for the distinction between the animals with low and high life expectancy.     

Effects of phenobarbital upon triacylglycerol metabolism in the rabbit.

1. The association between hepatic microsomal enzyme induction and triacylglycerol metabolism was examined in fasting male rabbits (2kg body wt.) injected intra-peritoneally with 50 mg of phenobarbital per kg for 10 days. 2. Occurrence of enzyme induction was established by a significant increase in hepatic aminopyrine N-demethylase activity and cytochrome P-450 content, as well as a doubling of microsomal protein per g of liver and a 54% increase in liver weight. Parallel increments in hepatic gamma-glutamyltransferase (EC 2.3.2.2) activity occurred; these were more pronounced in the whole homogenate than in the microsomes, which only accounted for 12.5% of the total enzyme activity in the controls and 17.0% in the animals given phenobarbital. Increased activity of gamma-glutamyltransferase activity was also observed in the blood serum of the test animals. 3. The rabbits given phenobarbital manifested increased hepatic triacylglycerol content and the triacylglycerol concentration of blood serum was also elevated. These changes were accompanied by a significantly enhanced ability of cell-free fractions of liver from the test animals (postmitochondrial supernatant and microsomal fractions) to synthesize glycerolipids in vitro from sn-[14C] glycerol 3-phosphate and fatty acids, when expressed per whole liver. Relative to the protein content of the fraction, glycerolipid synthesis in vitro was significantly decreased in the microsomes, presumably consequent upon the dramatic increase in their total protein content, whereas no change occurred in the postmitochondrial supernatant, possibly due to the protective effect of cytosolic factors present in this fraction and known to enhance glycerolipid synthesis. 4. Microsomal phosphatidate phosphohydrolase accounted for 85% of the total liver activity of this enzyme and its specific activity was 20-fold higher than that of the cytosolic phosphatidate phosphohydrolase (EC 3.1.3.4), when each was measured under optimal conditions. A significant increase in the activity of both enzymes per whole liver occurred in the rabbits given phenobarbital. A closer correlation between hepatic triacylglycerol content and and microsomal phosphatidate phosphohydrolase, as well as the above observation, suggest that this, rather than the cytosolic enzyme, may be rate-limiting for triacylglycerol synthesis in rabbit liver. 5. Significant correlations were observed between the various factors of hepatic microsomal-enzyme induction (aminopyrine N-demethylase and gamma-glutamyltransferase activity as well as cytochrome P-450 content) and hepatic triacylglycerol content, suggesting that that microsomal enzyme induction may promote hepatic triacylglycerol synthesis and consequently hypertriglyceridaemia in the rabbit.     

Failure of expression of the phenobarbital-induced enhancement of UDP-glycosyltransferases in native, sealed endoplasmic reticulum vesicles from rat liver.

Conflicting data have been published regarding the effects of phenobarbital treatment on bilirubin UDP-glucuronyltransferase activity in native liver microsomes. Recent evidence suggests that the bilirubin UDP-glycosyltransferase system faces the interior of microsomal vesicles, and that expression of its activities in sealed microsomes may be rate-limited by transport of UDP sugars across the membrane. These observations raise the possibility that the reported variability in the effects of phenobarbital may reflect differences in integrity of the membrane in microsomal preparations. We examined the effect of phenobarbital on bilirubin UDP-glucosyltransferase and the UDP-glucuronyltransferase activities towards bilirubin, 4-nitrophenol, and 1-naphthol using native rat liver microsomes with verified vesicle integrity. Phenobarbital-induced microsomes in which the membrane permeability barrier was eliminated by pretreatment with detergent displayed markedly higher UDP-glycosyltransferase activities towards all tested substrates compared with activities in similarly disrupted microsomes from untreated rats. In contrast, none of the transferase activities tested were significantly enhanced by phenobarbital treatment when the enzymic activities were assayed in sealed microsomes. Addition to the enzyme assay mixture of UDPGlcNAc, a presumed physiological activator of the UDP-glucuronyltransferases, failed to expose the enhanced UDP-glucuronyltransferase concentration in phenobarbital-induced sealed microsomes. Our findings are consistent with the idea that transport of UDP sugar across the membrane may be rate-limiting for expression of UDP-glycosyltransferase activities in sealed microsomes. Quantitative assessment of membrane integrity is an essential prerequisite in experiments designed to study the regulation of the microsomal UDP-glycosyltransferase system.     

Nuclear ADP-ribosyl transferase activity correlates with induction of P-450 monooxygenases by phenobarbital in rat liver microsomes

The effect of phenobarbital treatment on the nuclear ADP-ribosyl transferase activity has been studied in parallel with microsomal cytochrome P-450 concentration and related mono-oxygenase activities, in rat liver. A marked activation of the ADP-ribosyl transferase was observed 24 h after phenobarbital administration. The chronological study performed between 0-6 days after phenobarbital treatment showed a sharp increase in this nuclear enzyme activity, to approximately equal to 270% of the control value produced in 48 h. The administration of 5'-methylnicotinamide in vivo, an inhibitor of ADP-ribosyl transferase activity in vitro, produced a decrease both of the induction of liver microsomal cytochrome P-450 mono-oxygenases and nuclear ADP-ribosyl transferase activity. The role of nuclear ADP-ribosyl transferase in the adaptative response of the liver cell to phenobarbital is discussed.     

Induction of a microsomal butyrylesterase in rat liver by phenobarbital treatment

A carboxylesterase (carboxylic-ester hydrolase, EC 3.1.1.1) was induced in the liver of rats after repeated administration of phenobarbital. This enzyme migrated most rapidly towards the anode among Triton X-100-solubilized liver esterases by electrophoresis on the cellulose acetate membrane and it was tentatively designated as L-I.L-I increased in the microsomal fraction and was mainly concentrated in the fraction of smooth endoplasmic reticulum that proliferated after phenobarbital treatment.L-I was separated from other liver esterases and purified about 800-fold. The purified L-I was homogeneous as judged by polyacrylamide gel electrophoresis and exhibited a molecular weight of about 55 000 as determined by gel filtration. L-I split the 1-naphthyl ester of butyric acid faster than esters of acetic, propionic or valeric acids; it therefore seemed to be a butyrylesterase.     

The in vivo effect of benzamide and phenobarbital on liver enzymes: poly(ADP-ribose) polymerase, cytochrome P-450, styrene oxide hydrolase, cholesterol oxide hydrolase, glutathione S-transferase and UDP-glucuronyl transferase

Rats fed a synthetic diet containing 0.25% benzamide, 0.1% phenobarbital, separately or in combination, for two weeks showed a significant augmentation in the activity of nuclear poly(ADP-ribose) polymerase as well as changes in various nuclear, microsomal and cytosolic liver enzymes involved in the metabolism of xenobiotics. A selective depression of microsomal styrene oxide hydrolase activity by benzamide feeding, and a contrasting augmentation by phenobarbital, were confirmed by immunological titration of the enzyme-protein content suggesting actual enzyme repression and induction. The NAD content of these livers is not altered significantly as a result of benzamide and phenobarbital feeding, indicating that the changes in enzymes are not a result of non-specific toxic effects.     

FURTHER STUDIES ON THE INDUCTION OF THE DRUG-HYDROXYLATING ENZYME SYSTEM OF LIVER MICROSOMES

Further studies of the induction of the liver microsomal drug-hydroxylating enzyme system by pretreatment of rats with various drugs are presented. The phenobarbital-induced increase in the microsomal content of CO-binding pigment and in the activities of TPNH-cytochrome c reductase and the oxidative demethylation of aminopyrine is proportional, within certain limits, to the amount of phenobarbital injected. Removal of the inducer results in a parallel decrease in the levels of CO-binding pigment, TPNH-cytochrome c reductase, and aminopyrine demethylation. Other inducing drugs have been investigated and shown to act similarly to phenobarbital. The early increase in these enzymes is found in the microsomal subfraction consisting of rough-surfaced vesicles, whereas repeated administration of the inducing drug results in a concentration of the enzymes in the smooth-surfaced vesicles. The phenobarbital-stimulated formation of endoplasmic membranes is reflected in increased amounts of the various microsomal phospholipid fractions as revealed by thin layer chromatography. There is no significant difference between the stimulated rates of P_i³² incorporation into phospholipids of the two different microsomal subfractions in response to phenobarbital treatment. The drug-induced enzyme synthesis is unaffected by adrenalectomy.     

Selenium and hepatic microsomal hemoproteins

The microsomal share of liver homogenate ⁷⁵Se after injection of a tracer dose of ⁷⁵SeO₃^2? was three times greater in rats fed a selenium-deficient diet than in rats fed a selenium-adequate diet. Basal levels of microsomal cytochromes P-450 and b₅ were unaffected by selenium deficiency. However, induction of these cytochromes by phenobarbital was markedly inpaired in selenium-deficient rats, whereas liver weight increase and NADPH cytochrome $\text{c}$ reductase induction were not impaired. These data indicate that selenium is essential for phenobarbital induction of microsomal hemoproteins.     

Influence of 3,5,3'-triiodo-L-thyronine on the hepatic enzyme activities responsible for the metabolism of xenobiotics during the development of the chick embryo

The influence of thyroid hormones on microsomal drug metabolizing enzymes was studied in hypothyroid newborn rats and chick embryos. Administration of 3,5,3'-triiodo-L-thyronine strongly decreased the microsomal cytochrome P 450 content in hypothyroid new-born rats and thus could render the rat pup more susceptible to hepatotoxicity from drugs. The drug metabolizing system in 20 days old chick embryos was less sensitive to the effects of thyroid hormone, but administration of phenobarbital was accompanied by a strongly induction effect on microsomal enzyme activities.