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.     

Hepatic and intestinal gamma-glutamyltranspeptidase activity: its activation by chronic ethanol administration.

To study the effect of chronic ethanol administration on the activity of gamma-glutamyltranspeptidase (GGTP) in various tissues, female rats were pair-fed liquid diets with 36% of total calories either as ethanol or isocaloric carbohydrate (controls). Six weeks of ethanol feeding in an increase of cytochrome P450 content by 70%. Hepatic microsomal GGTP activity was more than doubled after ethanol feeding whether expressed per gram of liver or per mg of microsomal protein. Furthermore intestinal GGTP activity was significantly enhanced after ethanol, whereas there was no change in the enzyme activity in either kidney or pancreas. Phenobarbital administration to rats also resulted in an enahancement of GGTP activity in the liver but not in the intestine. These results suggest that enhanced hepatic and intestinal GGTP activities may contribute, at least partly, to increased serum GGTP activity frequently seen in alcoholics.     

Characteristcs of microsomal enzyme controls in primary cultures of rat hepatocytes.

Cytochrome P-450, NADPH-cytochrome c reductase, biphenyl hydroxylase, and epoxide hydratase have been compared in intact rat liver and in primary hepatocyte cultures. After 10 days in culture, microsomal NADPH-cytochrome c reductase and epoxide hydratase activities declined to a third of the liver value, while cytochrome P-450 decreased to less than a tenth. Differences in the products of benzo[a]pyrene metabolism and gel electrophoresis of the microsomes indicated a change in the dominant form(s) of cytochrome P-450 in the cultured hepatocytes. Exposure of the cultured cells to phenobarbital for 5 days resulted in a threefold induction in NADPH-cytochrome c reductase and epoxide hydratase activities which was typical of liver induction of these enzymes. Exposure of the cells to 3-methylcholanthrene did not affect these activities. Cytochrome P-450 was induced over two times by phenobarbital and three to four times by 3-methylcholanthrene. The λ_max of the reduced carbon monoxide complex (450.7 nm) and analysis of microsomes by gel electrophoresis showed that the phenobarbital-induced cytochrome P-450 was different from the species induced by 3-methylcholanthrene (reduced carbon monoxide λ_max = 447.9 nm). However, metabolism of benzo[a]pyrene (specific activity and product distribution) was similar in microsomes of control and phenobarbital- and 3-methylcholan-threne-induced hepatocytes and the specific activity per nmole of cytochrome P-450 was higher than in liver microsomes. The activities for 2- and 4-hydroxylation of biphenyl were undetectable in all hepatocyte microsomes even though both activities were induced by 3-methylcholanthrene in the liver. Substrate-induced difference spectra and gel electrophoresis indicated an absence in phenobarbital-induced hepatocytes of most forms of cytochrome P-450 which were present in phenobarbital-induced rat liver microsomes. It is concluded that the control of cytochrome P-450 synthesis in these hepatocytes is considerably different from that found in whole liver, while other microsomal enzymes may be near to normal. Hormonal deficiencies in the culture medium and differential hormonal control of the various microsomal enzymes provide a likely explanation of these effects.     

In vitro biosynthesis of liver cytochrome P450 mature peptide sub-unit by translation of isolated poly(A)+ mRNA from normal and phenobarbital induced rats

The biosynthesis of a cytochrome P₄₅₀ peptide sub-unit by the in vitro translation of total hepatic poly (A)⁺ mRNA in an heterologous cell-free-system is described. The ability of the liver poly (A)⁺ RNA preparations from normal and phenobarbital induced rats to promote protein synthesis and the identification of in vitro synthesized proteins revealed the presence of a cytochrome P₄₅₀ peptide sub-unit presenting the same apparent molecular weight of the native peptide. This fact demonstrates that rat liver poly (A)⁺ mRNA fraction contains an important amount of cytochrome P₄₅₀ peptide messages. Total poly (A)⁺ RNA from rats in an early phenobarbital induction stage exhibits a higher cytochrome P₄₅₀ template activity in good agreement with the enhancement of this hemeprotein concomitantly observed in vivo, in the liver microsomes, it is also concluded that cytochrome P₄₅₀, peptide sub-unit, induced in rat liver by phenobarbital, is translated in its mature form.     

Melatonin reduces oxidative stress induced by ochratoxin A in rat liver and kidney

Melatonin (MEL) displays antioxidant and free radical scavenger properties. In the present study, the effect of MEL on the oxidative stress induced by ochratoxin A (OTA) administration in rats was investigated. Four groups of 15 rats each were used: controls, MEL-treated rats (5 mg/kg body mass), OTA-treated rats (250 μg/kg) and MEL+OTA-treated rats. After 4 weeks of treatment, the levels of malondialdehyde (MDA), a lipid peroxidation product (LPO) were measured in serum and homogenates of liver and kidney. Also, the levels of glutathione (GSH), and activities of glutathione reductase (GR), glutathione peroxidase (GSPx), superoxide dismutase (SOD), catalase (CAT) and glutathione-S-transferase (GST) in liver and kidney were determined. In OTA-treated rats, the levels of LPO in serum and in both liver and kidney were significantly increased compared to levels in controls. Concomitantly, the levels of GSH and enzyme activities of SOD, CAT, GSPx and GR in both liver and kidney were significantly decreased in comparison with controls. In rats received MEL+OTA, the changes in the levels of LPO in serum and in liver and kidney were not statistically significant compared to controls. Concomitantly, the levels of GSPx, GR and GST activities in both liver and kidney tissues were significantly increased in comparison with controls. Similar increases in GSPx, GR and GST activities were also observed in MEL-treated rats when compared with controls. In conclusion, the oxidative stress may be a major mechanism for the toxicity of OTA. MEL has a protective effect against OTA toxicity through an inhibition of the oxidative damage and stimulation of GST activities. Thus, clinical application of melatonin as therapy should be considered in cases of ochratoxicosis.     

In vitro inhibition of alkaline phosphatase activities from intestine, bone, liver, and kidney by phenobarbital.

A kinetic study of the inhibition of several alkaline phosphatase (AP isoenzyme activities by phenobarbital was carried out using p-nitrophenylphosphate (10 mM) as a substrate at pH 9.8 in a 300-mM Hepes buffer. AP from bovine kidney, calf intestine, bovine liver, and rat bone was used. Over a phenobarbital concentration range of 20-400 mM, all these isoenzymes were inhibited in an uncompetitive manner with a Ki of 200 mM for intestinal AP, and in a linear mixed-type manner for all the other isoenzymes tested. The Ki values were 10, 40 and 55 mM for kidney, bone and liver AP, respectively. The use of 15 mM carbonate-bicarbonate or 400 mM diethanolamine buffer did not modify the degree of inhibition of intestinal AP activity. Dixon plots of the reciprocal of reaction velocity versus inhibitor concentration either at different substrate concentration or at different DEA concentration indicate uncompetitive inhibition for the intestinal enzyme. This in vitro inhibitory effect of phenobarbital is in contrast to its in vivo stimulating action on AP. However, in the whole animal, the effects of phenobarbital administration probably represent the sum of multiple effects.     

Influence of Microcystin-LR on the activity of membrane enzymes in rat intestinal mucosa

The objective of the present study was to evaluate the effects of microcystin-LR (MCLR) on the activity of membrane enzymes from intestinal mucosa. In addition, serum chemistry and peroxidative status of both serum and intestinal homogenate were evaluated after treatment with MCLR. Wistar rats were treated with intraperitoneal injection of either 100 microg pure MCLR/Kg body weight or saline solution. A significant increase in liver weight and altered serum enzyme activities were found in MCLR-treated rats, indicating damage to the liver in these rats, as previously suggested. A higher specific activity of sucrase (1.5-fold) was observed after the administration of MCLR, whereas other intestinal apical membrane enzymes, such as lactase, maltase and alkaline phosphatase were not modified by the treatment. The specific activities of acid phosphatase and succinate dehydrogenase, markers for lysosomal and mitochondrial membranes, respectively, were also increased (32% and 60%, respectively) in treated rats. The analysis of lipid peroxidation showed that the peroxidative status was increased in both serum and intestinal mucosa from MCLR-treated rats, reflecting an excess production of oxygen free radicals induced by this cyanobacterial toxin. In conclusion, this study shows that acute exposure to MCLR affects the intestinal physiology by modifying the intestinal peroxidation status as well as the activity of membrane enzymes.     

Structure-activity relationships of chlorinated benzenes as inducers of different forms of cytochrome P-450 in rat liver

Hexachlorobenzene (HCB) produced increases in ethoxyresorufin (ERR) O-deethylase, aryl hydrocarbon hydroxylase (AHH) and aminopyrine N-demethylase activities in rat liver microsomes which were intermediate between those produced by phenobarbital and 3,4-benzpyrene (BP). α-Naphthoflavone (ANF) selectively inhibited ERR activity in BP and HCB-induced microsomes (94% and 88%). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of liver microsomes indicated that HCB did not produce a detectable increase in a polypeptide with electrophoretic properties similar to those of purified cytochrome P-448 (M_r = 56 000). However, HCB did induce a polypeptide with M_r = 53 000 corresponding to one of two polypeptide bands induced by BP. This polypeptide may represent a second form of cytochrome P-448. Purification of HCB to remove possible dibenzo-p-dioxin impurities did not alter the ‘mixed-type’ induction produced by HCB. In contrast to HCB, all other chlorinated benzenes tested resembled phenobarbital as inducers.     

Genetics and ontogeny of aldehyde dehydrogenase isozymes in the mouse: Evidence for a locus controlling the inducibility of the liver microsomal isozyme

Variation in the inducibility of the liver microsomal isozyme of aldehyde dehydrogenase (designated AHD-Cy) by phenobarbital administration was observed among inbred strains and linkage testing stocks of Mus musculus. The phenotypes were inherited in a normal Mendelian fashion with two alleles showing codominance at a proposed regulatory locus (designated Ahd-3r). Strain variation was also observed for the induction of liver AHD-Cy by 17-β-oestradiol administration to ovarectimized female mice. Moreover, this enzyme was elevated in activity by the administration of high (nonphysiological) levels of progesterone. Development studies showed that the liver and kidney AHD-Cy isozyme exhibited low activities in late-stage fetal and neonatal mice and reached adult levels by approximately 6 weeks of age.     

Epoxide hydrase and mixed-function oxidase activities of rat liver nuclear membranes.

Rat liver nuclei have 2 to 12% of the corresponding microsomal aryl hydrocarbon hydroxylase, aminopyrine and benzphetamine N-demethylase, NADPH-cytochrome c reductase, and epoxide hydrase activities. Nuclear membranes were prepared from isolated liver nuclei by a sucrose density centrifugation technique. A 2.5- to 10.2-fold increase in the specific enzyme activities was observed in nuclear membrane as compared to intact nuclei. Several properties of the rat liver nuclear membrane and microsomal epoxide hydrase have been compared. Nuclear epoxide hydrase was similar to the corresponding microsomal enzyme in being induced by phenobarbital whereas 3-methylcholanthrene did not produce any effects. Nuclear membrane and microsomal epoxide hydrase were inhibited to a similar degree by 1,1,1-trichloropropene oxide, cyclohexene oxide, an trans-stilbene oxide. The apparent K_m value of nuclear membrane epoxide hydrase was 20 μm for benzo(a)pyrene 4,5-oxide, which is 5.5-fold lower than the corresponding microsomal K_m value (112 μm). Nuclear membranes were prepared from isolated nuclei of rat kidney, lung, spleen, and heart by the DNase digestion method. Epoxide hydrase activity in intact nuclei was in the following order: kidney > lung ? spleen, or heart. Increases of 2.2- and 2.5-fold in specific epoxide hydrase activity were observed in kidney and lung when nuclear membranes were compared to intact nuclei. DMSO, dimethylsulfoxide     

Effect of thyroid hormone on peroxisomal flavin enzymes in rat liver and kidney

The effect of thyroid hormone on peroxisomal enzyme activity was studied in thyroidectomized- and T4-administered-thyroidectomized rats. In liver, the activities of isozyme A of L-alpha-hydroxyacid oxidase, D-amino acid oxidase, urate oxidase and catalase were decreased by thyroidectomy, and the diminished enzyme activities were restored by T4 administration to rats. These modifications induced by thyroidectomy or by T4 administration, however, were prominent only in immature animals (20-day-old rats). Although the changes in-alpha-hydroxyacid oxidase and D-amino acid oxidase activities, induced by thyroidectomy or by T4 administration, were also observed in 40-day-old rats, those in urate oxidase and catalase activities were not significant in 40-day-old rats. Acyl CoA oxidase activity was not affected by thyroidectomy or by T4 administration in either 20- or 40-day-old rats. In the kidney, isozyme B of L-alpha-hydroxyacid oxidase activity was reduced by thyroidectomy and the diminished enzyme activity was restored by T4 administration in both 20- and 40-day-old rats. D-Amino acid oxidase and catalase activities in kidney, however, were not significantly modified by thyroidectomy or by T4 administration in either 20- or 40-day-old rats. The results suggest that thyroid hormone can modify the peroxisomal enzyme activity, which is prominent in immature animals.     

PHENOBARBITAL-INDUCED SYNTHESIS OF THE MICROSOMAL DRUG-METABOLIZING ENZYME SYSTEM AND ITS RELATIONSHIP TO THE PROLIFERATION OF ENDOPLASMIC MEMBRANES : A Morphological and Biochemical Study

Liver microsomes, isolated from rats which had been treated with phenobarbital in vivo, were found to exhibit increased activities of oxidative demethylation and TPNH-cytochrome c reductase and an increased amount of CO-binding pigment. Simultaneous administration of actinomycin D or puromycin abolished the phenobarbital-induced enzyme synthesis. Increased rate of P_i³² incorporation into microsomal phospholipid was the first sign of phenobarbital stimulation and appeared 3 hours after a single injection of this drug. Microsomes were divided into smooth-surfaced and rough-surfaced vesicle fractions. The fraction consisting of smooth-surfaced vesicles exhibited the greatest increase in protein content and oxidative demethylation activity after phenobarbital administration in vivo. Ultrastructural studies revealed that drug treatment also gave rise to proliferation of the endoplasmic reticulum in the hepatic parenchymal cells, first noticed after two phenobarbital injections. The phenobarbital-induced synthesis of the metabolizing enzymes is discussed with special reference to the relationship to the stimulated synthesis of the endoplasmic membranes.     

Modulatory role of Emblica officinalis fruit extract against arsenic induced oxidative stress in Swiss albino mice

Arsenic, an important human toxin, is naturally occurring in groundwater and its accumulation in plants and animals have assumed a menacing proportion in a large part of the world, particularly Asia. Epidemiological studies have shown a strong association between chronic arsenic exposure and various adverse health effects, including cardiovascular diseases, neurological defects and cancer of lung, skin, bladder, liver and kidney. The protective role of the fruits of Emblica officinalis (500 mg/kg b.wt.) was studied in adult Swiss albino mice against arsenic induced hepatopathy. Arsenic treated group (NaAsO₂, 4 mg/kg b.wt.) had a significant increase in serum transaminases and lipid peroxidation (LPO) content in liver, whereas significant decrease was recorded in hepatic superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST) and serum alkaline phosphatase activity. Combined treatment of Emblica and arsenic (pre and post) declined the serum transaminases and LPO content in liver whereas significant increase was noticed in SOD, CAT, GST and serum alkaline phosphatase activities. Liver histopathology showed that Emblica fruit extract had reduced karyolysis, karyorrhexis, necrosis and cytoplasmic vacuolization induced by NaAsO₂ intoxication. Thus it can be concluded that pre- and post-supplementation of E. officinalis fruit extract significantly reduced arsenic induced oxidative stress in liver.     

Biochemical study of the anti-diabetic action of the Egyptian plants Fenugreek and Balanites

Fenugreek and Balanites are two plants commonly used in Egyptian folk medicine as hypoglycemic agents. In the present study, the effects of 21 days oral administration of Fenugreek seed and Balanites fruit extracts (1.5 g/kg bw) on the liver and kidney glycogen content and on some key liver enzymes of carbohydrate metabolism in STZ-diabetic rats were studied. In addition, the effects of these two plant extracts on the intestinal α-amylase activity in vitro and starch digestion and absorption in vivo were also examined. Results indicated that single injection of STZ (50 mg/kg bw) caused 5-folds increase in the blood glucose level, 80% reduction in serum insulin level, 58% decrease in liver glycogen and 7-folds increase in kidney glycogen content as compared to the normal levels. The activity of glucose-6-phosphatase was markedly increased, whereas, the activities of both glucose-6-phosphate dehydrogenase and phospho-fructokinase were significantly decreased in the diabetic rat liver. Administration of Fenugreek extract to STZ-diabetic rats reduced blood glucose level by 58%, restored liver glycogen content and significantly decreased kidney glycogen as well as liver glucose-6-phosphatase activity. Meanwhile, Balanites extract reduced blood glucose level by 24% and significantly decreased liver glucose-6-phosphatase activity in diabetic rats. On the other hand, our results demonstrated that both the Fenugreek and Balanites extracts were able to in vitro inhibit α-amylase activity in dose-dependent manner. Fenugreek was more potent inhibitor than Balanites. This inhibition was reversed by increasing substrate concentration in a pattern which complies well with the effect of competitive inhibitors. Furthermore, this in vitro inhibition was confirmed by in vivo suppression of starch digestion and absorption induced by both plant extracts in normal rats. These findings suggest that the hypoglycemic effect of Fenugreek and Balanites is mediated through insulinomimetic effect as well as inhibition of intestinal α-amylase activity.     

Immunochemical studies on a phenobarbital-inducible esterase in rat liver microsomes

Detergent extracts of liver microsomes from drug-treated rats were analyzed semiquantitatively in crossed immunoelectrophoresis in combination with a zymogram technique for nonspecific esterase activity. One esterase-active antigen was quantitatively induced by phenobarbital as demonstrated with both antimicrosomal antiserum and a monospecific antiserum prepared against this antigen. No similar inducation of this or any other esterase-active antigen was detected after 3-methylcholanthrene treatment. With the monospecific antiserum, the antigen was also detected in other organs, capable of carrying out hydroxylation reactions, such as lung and kidney. It was, however, not induced by phenobarbital in these organs. Quantitative enzyme assays with acetanilide as substrate indicated that the phenobarbital-inducible esterase could also function as an amidase. Furthermore, from absorption studies, it was concluded that this antigen is located on the cytoplasmic side of the microsomal vesicles. Crossed immunoelectrofocusing experiments revealed that this esterase-active antigen consists of five subcomponents with different charge properties.     

Effectiveness of copper chloride in protecting against alterations induced by mercury chloride in newborn rats

This work investigated the effects of copper as preventive treatment against mercury‐induced alterations in young rats. Wistar rats were treated (subcutaneous) with saline or CuCl₂ · 2H₂O (6.9 mg/kg/day) from 3 to 7 days old and with saline or HgCl₂ (5.0 mg/kg/day) from 8 to 12 days old. Rats were sacrificed 24 h after the last dose. Mercury‐exposed rats presented inhibition of liver (43%) and kidney (52%) porphobilinogen (PBG)‐synthase activity and serum lactic dehydrogenase activity (50%). Also, an increase of the serum creatinine and urea levels around threefold and fivefold was observed, respectively. Pre‐exposure to copper partially prevented the mercury effect on liver but not on kidney PBG synthase, and prevented the increase of the creatinine levels. Blood and brain PBG synthase and serum alanineaminotransferase activities, as well as glycemia, and liver glycogen content were not altered by treatments. These results show that copper, although being an essential metal, is inefficient as a preventive agent against mercury poisoning in parameters investigated after the end of mercury exposure. © 2012 Wiley Periodicals, Inc. J Biochem Mol Toxicol 26:354–359, 2012; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21429     

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.     

ENZYMES CATALYZING THE DE NOVO SYNTHESIS OF METHYL GROUPS IN THE BRAIN AND OTHER TISSUES OF THE RAT

—Rat brain contains all three of the enzymes required for de novo synthesis of the methyl group of methionine (serine transhydroxymethylase, methylene reductase, and [B₁₂]transmethylase) in activities comparable to those found in liver and kidney. The activities of methylene reductase in female kidney, and of [B12]transmethylase in female brain and kidney, are higher than in the corresponding male tissues. Liver and kidney extracts contain an inhibitor of methylene reductase not present in brain extracts. This inhibitor differs from S-adenosylmethionine (SAM), which also inhibits methylene reductase in both liver and brain homogenates. The administration of l -DOPA to rats, which has been previously shown to deplete brain S-adenosylmethionine, also reduces the activity of brain [B₁₂]transmethylase if assayed without added SAM. Since SAM is required for activity of this enzyme, its decreased activity probably results from the decline in brain SAM concentration. De now synthesis of methyl groups could be a mechanism by which the brain maintains its level of methionine in the face of increased methyl group utilization after administration of l -DOPA.     

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.     

Dealkylation of pentoxyresorufin: A rapid and sensitive assay for measuring induction of cytochrome(s) P-450 by phenobarbital and other xenobiotics in the rat

The O-dealkylation of pentoxyresorufin (7-pentoxyphenoxazone) by rat liver microsomes was examined. The reaction appeared highly specific for certain phenobarbital inducible forms of cytochrome P-450 and was increased 95- to 140-fold by animal pretreatment with phenobarbital (75 mg/kg/day, four ip injections) and ~50-fold by Aroclor 1254 (500 mg/kg, one ip injection) while animal pretreatment with 3-methylcholanthrene (50 mg/kg/day, three ip injections) resulted in less than a 2-fold increase over the rate detected in control microsomes. It was observed that this activity, in microsomes for Aroclor-pretreated rats, was dependent on O₂ and was inhibited by metyrapone and SKF 525-A, indicative of cytochrome(s) P-450 mediation in the reaction. When antibodies directed against purified cytochrome(s) P-450S were employed to inhibit the pentoxyresorufin O-dealkylation reaction, antibodies to P-450_PB-B greatly inhibited the reaction (>90%), while antibodies to P-450_PB-C or P-450_PB/PCN-E had minimal effects. Assay of hepatic microsomes from rats which were pretreated with varying doses of phenobarbital (0.9–75 mg/kg/day, four ip injections) indicated that while aminopyrine-N-demethylase activity was induced only 2-fold at the maximum dose (75 mg/kg/day), pentoxyresorufin O-dealkylase activity was induced ~140-fold at this dose and ~4-fold by a dose of phenobarbital as low as 0.9 mg/kg.