3-methylcholanthrene Treatment Research Articles

The effect of 3-methylcholanthrene on urinary 6β-hydroxycortisol excretion and hepatic enzyme activity in the marmoset monkey ( Callithrix Jacchus)

Abstract The effect of 3-methylcholanthrene treatment on the urinary excretion of 6β-hydroxycortisol and hepatic enzyme activity was investigated in the marmoset monkey ( Callithrix jacchus ). 3-Methylcholanthrene increased cytochrome P-448 content, NADPH cytochrome- c reductase activity, 7-ethoxy-coumarin O -deethylase activity and ethoxyresorufin O -deethylase but did not affect aminopyrine N -demethylase activity. This induction profile is qualitatively similar to that previously reported for the rat. There was no change in the ratio of urinary 6β-hydroxycortisol to 17-hydroxycorticosteroids. The pattern of response to 3-methylcholanthrene indicates that cortisol 6β-hydroxylase activity is not associated with induction of hepatic cytochrome P-448.

Changes in phospholipid methyltransferases and membrane microviscosity during induction of rat liver microsomal cytochrome P-450 by phenobarbital and 3-methylcholanthrene.

Rat liver microsomes contained two methyltransferases which converted phosphatidylethanolamine (PE) to phosphatidylcholine (PC). The first methyltransferase converted PE to phosphatidyl-N-methylethanolamine (PME) and the second methyltransferase converted PME to PC. Previous work has shown that increased PME synthesis decreases membrane microviscosity. Therefore, changes in the rat liver microsomal cytochrome P-450, phospholipid methyltransferases and membrane microviscosity after induction by phenobarbital and 3-methylcholanthrene were studied. Phenobarbital and 3-methylcholanthrene increased cytochrome P-450 levels 2- to 3-fold. At low SAM concentration, the proportion of PME among the total phospholipids formed increased significantly, and at a high SAM concentration, the proportion of PC among the total phospholipids formed decreased significantly in microsomes of treated rats. Treatment of rats with phenobarbital and 3-methylcholanthrene also decreased microviscosities of the microsomal membranes and liposomes which were prepared from phospholipids extracted from the microsomes. In synthetic liposomes containing PE, PME and PC, microviscosity decreased when the proportion of PME was increased or the proportion of PC was decreased. These results suggest that the membrane fluidity increases with phenobarbital and 3-methylcholanthrene treatment, and changes in phospholipid methyltransferases may contribute to the process of enzyme induction. During induction with phenobarbital, all three factors known to increase membrane fluidity (linoleic acid content, the formation of phosphatidyl-N-methylethanolamine, and decreases in the cholesterol/phospholipid ratio) contribute to the decrease in microviscosity. During induction with 3-methylcholanthrene, alterations in phospholipid methylation is possibly the primary cause of the decrease in membrane microviscosity.

High-performance liquid chromatography technique for resolving multiple forms of hepatic membrane-bound cytochrome P-450.

Solubilized hepatic microsomal cytochrome P-450 from rats was resolved into 12-different heme-containing components by ion-exchange high-performance liquid chromatography. Separation was accomplished by the utilization of Anpac ion-exchange resin. Phenobarbital (PB) and 3-methylcholanthrene (3-MC) treatment of rats resulted in selective increases or decreases of specific components observed in the elution profile of solubilized microsomes prepared from untreated rats. Treatment with polychlorinated biphenyls increased peaks that were selectively increased by PB or 3-MC treatment. Proteins collected from the major peaks induced by PB or 3-MC treatment were found to retain spectral characteristics of cytochrome P-450. The recovered cytochrome b5 and NADPH-cytochrome c reductase retained spectral properties and enzymic activity, respectively. Purified P-450b obtained from PB-treated rats was found to cochromatograph with one of the peaks selectively induced by PB treatment. These results suggested that it is possible to separate membrane-bound proteins without dramatically altering their physical properties.

The effect of fluroxene [(2,2,2-trifluoroethoxy)ethane] on haem biosynthesis and degradation.

Acute fluroxene treatment of male Wistar rats decreases the amounts of hepatic microsomal cytochrome P-450 and haem, increases the activities of hepatic delta-aminolaevulinate synthase and haem oxygenase, and increases the amounts of haem precursors (delta-aminolaevulinate and porphobilinogen) in the urine. All of the above effects of fluroxene are enhanced by pretreatment of the experimental animals with 3-methylcholanthrene and phenobarbital. The amounts of porphyrins in the urine and faeces were generally unaffected by acute fluroxene treatment of uninduced or 3-methylcholanthrene- or phenobarbital-induced Wistar rats. 2,2,2-Trifluoroethyl ethyl ether, the saturated analogue of fluroxene, did not affect the amounts of hepatic cytochrome P-450 and haem, the amounts of any of the haem precursors in the urine or faeces, or the activity of hepatic haem oxygenase in phenobarbital-induced male Wistar rats. The amounts of hepatic cytochrome P-450 and haem and of the haem precursors in urine and faeces, and the activity of delta-aminolaevulinate synthase, were generally not altered by acute fluroxene treatment of uninduced male Long-Evans rats. Chronic treatment of Wistar rats with fluroxene resulted in small increases in the amounts of delta-aminolaevulinate and porphyrins in urine. The amounts of porphobilinogen in urine were elevated up to 2000%, whereas the amounts of the porphyrins in faeces were generally unaffected. After chronic fluroxene treatment, the activity of delta-aminolaevulinate synthase was increased, whereas the activity of uroporphyrinogen synthase was decreased. It is concluded that acute fluroxene treatment may affect haem biosynthesis and degradation by a mechanism similar to allylisopropylacetamide, namely by stimulating an atypical cytochrome P-450-dependent pathway for haem degradation. The effects of chronic fluroxene treatment on haem biosynthesis may be a consequence of this mechanism or a result of the inhibition by fluroxene of uroporphyrinogen synthase. Chronic fluroxene treatment of male rats affects the haem biosynthetic pathway in a manner similar to that seen in human genetic acute intermittent porphyria.

Effects of 3-methylcholanthrene on oxidized nicotinamide-adenine dinucleotide phosphatedependent dehydrogenases and selected metabolites in perfused rat liver

Treatment of adult rats with 3-methylcholanthrene over a 3-day period produced significant decreases in hepatic ATP concentrations and elevated the activities of 6-phosphogluconate dehydrogenase and malic enzyme. The decline in ATP was accompanied by a decrease in total adenine nucleotides; however, ATP/ADP ratios were essentially the same in livers of normal and 3-methylcholanthrene-treated rats, and no significant changes were noted in glycolytic and citric acid cycle intermediates. Thus, hepatic energy metabolism does not appear to be altered grossly after 3-methylcholanthrene treatment. NADP(+)/NADPH ratios calculated from substrates assumed to be in near equilibrium with malic enzyme, isocitrate dehydrogenase and 6-phosphogluconate dehydrogenase increased after 3-methylcholanthrene treatment; these changes were accompanied by significant increases in the activity of malic enzyme and 6-phosphogluconate dehydrogenase. Oxidation of hepatic NADPH and elevation of activities of several NADPH-generating enzymes by 3-methylcholanthrene indicates that this agent which induces components of the mixed-function oxidase system also elevates the capacity of the liver to form NADPH.

Differential effects of ethanol and other inducers of drug metabolism on the two forms of hamster liver microsomal aniline hydroxylase

The aniline hydroxylase activity of microsomes isolated from hamster liver can be differentiated kinetically into high affinity (low K(m), form I) and low affinity (high K(m), form II) forms. Microsomes isolated from uninduced animals contain slightly more form I activity. The activity of the low affinity form (form II) is preferentially enhanced by Aroclor or 3-methylcholanthrene treatment, while phenobarbital treatment increases the activity of both forms. Chronic ethanol consumption results in enhancement of only the high affinity form (form I).

Antipyrine as an in vitro probe of mixed function oxidase activity [proceeding

British Journal of Clinical PharmacologyVolume 9, Issue 3 p. 284P-283P Free Access Antipyrine as an in vitro probe of mixed function oxidase activity [proceeding] GC Kahn, GC KahnSearch for more papers by this authorAR Boobis, AR BoobisSearch for more papers by this authorI Blair, I BlairSearch for more papers by this authorMJ Brodie, MJ BrodieSearch for more papers by this authorDS Davies, DS DaviesSearch for more papers by this author GC Kahn, GC KahnSearch for more papers by this authorAR Boobis, AR BoobisSearch for more papers by this authorI Blair, I BlairSearch for more papers by this authorMJ Brodie, MJ BrodieSearch for more papers by this authorDS Davies, DS DaviesSearch for more papers by this author First published: March 1980 https://doi.org/10.1111/j.1365-2125.1980.tb04844.xCitations: 12AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. References Boobis, A.R., Brodie, M.J., Kahn, G.C., Fletcher, D.R., Saunders, J.H. & Davies, D.S. (1980). Monooxygenase activity of human liver in microsomal fractions of needle biopsy specimens. Br. J. clin. Pharmac., 9, 11– 19. Danhof, M., Krom, D. & Breimer, D.D. (1979). Studies on the different metabolic pathways of antipyrine in rats: the influence of phenobarbital and 3-methylcholanthrene treatment. Xenobiotica, (In press). Tredger, J.M., McPherson, F.J., Chakraborty, J., Bridges, J.W. & Parke, D.V. (1976). The effect of some glucocorticoids on hepatic microsomal hydroxylation in the rat and hamster. Naunyn-Schmiedebergs Arch. Pharmac., 292, 267– 270. Citing Literature Volume9, Issue3March 1980Pages 284P-283P ReferencesRelatedInformation

The induction of liver microsomal proteins by 3-methylcholanthrene in rats of different age, sex and strain

The treatment of male and female adult and male and female immature rats with 3-methylcholanthrene results in increases in the intensity of two liver microsomal protein bands separated by sodium dodecyl sulphate-polyacrylacrylamide gel electrophoresis. The induced protein species have estimated molecular weights of about 55 000 and 52 500. The time course of the induction of these species has been followed in a semi-quantitative manner. In immature rats an additional band, corresponding to a protein species with a molecular weight of about 43 000, is also increased by 3- methylcholanthrene treatment. In male adult rats exclusively, treatment with 3-methylcholanthrene also results in a decrease in the intensity of a band corresponding to a protein species of about 51 000. The appearance of this band in the liver microsomal fraction of adult male rats occurs following sexual maturation and its presence represents a significant difference in the microsomal proteins between male and female rats; this protein is not detected in younger rats or in female rats at any age. These findings are discussed in terms of their possible relevance to the cytochrome P-450 content of the endoplasmic reticulum of rat hepatocytes.

Genetic control of responsiveness of rat liver supernatant aldehyde dehydrogenase to phenobarbital and 3-methylcholanthrene.

The responsiveness of the hepatic supernatant NAD+-dependent aldehyde dehydrogenase with a high Km value (high Km-AldDH) to phenobarbital (PB) and 3-methylcholanthrene (3-MC) treatment was studied in male rats of three strains; Wistar, Long-Evans, and Sprague-Dawley. A remarkable strain difference in the response of the enzyme to PB or 3-MC was observed. In rats of the Wistar strain the enzyme activity remained unchanged ("non-responsive") in all rats after treatment with PB while it increased ("responsive") 5- to 19-fo-d in all rats after treatment with 3-MC. The enzyme activity increased 8- to 20-fold and 2- to 8-fold respectively after treatment with PB and 3-MC in all rats of the Long-Evans strain. In rats of the Sprague-Dawley strain the enzyme activity remained unchanged in half of all the rats treated with PB or 3-MC and increased 2- to 7-fold over the basal level in half of the treated rats. The non-responsive rats to PB were all responsive to 3-MC treatment while the responsive rats to PB were responsive in 65% and non-responsive in 35% to 3-MC treatment.

Studies on the Different Metabolic Pathways of Antipyrine in Rats: Influence of Phenobarbital and 3-Methylcholanthrene Treatment

1. The amounts of antipyrine and its metabolites excreted in 24 h urine after i.v. injection of 10 mg antipyrine into male Wistar rats were quantified after enzymic hydrolysis with beta-glucuronidase/aryl sulphatase. In 24 h 2.7% of the administered dose was excreted as unchanged antipyrine, 13.3% as 4-hydroxyantipyrine, 7.4% as norantipyrine, 28.9% as 3-hydroxymethylantipyrine and 1.1% as 3-carboxyantipyrine. 2. Treatment with phenobarbital decreased the antipyrine half-life from 65 to 30 min, but did not significantly change the urinary metabolite profile. Only the amount of 3-carboxyantipyrine was significantly different and increased from 1.1 to 2.6% dose. 3. 3-Methylcholanthrene treatment resulted in a decrease of antipyrine half-life from 72 to 37 min. After treatment 4-hydroxyantipyrine was increased from 13.4% to 25.6% dose, whereas 3-hydroxymethylantipyrine was decreased from 26.8% to 8.5% and 3-carboxyantipyrine from 1.3% to 0.2% of the dose respectively; norantipyrine was unchanged. 4. It is concluded that different types of hepatic cytochrome P-450 may be involved in the formation of 4-hydroxyantipyrine on one hand and the formation of 6-hydroxymethylantipyrine on the other. Another possibility is that in methylcholanthrene-treated animals another haemoprotein is formed that results in the formation of more 4-hydroxyantipyrine and less 3-hydroxymethylantipyrine. In any case, the urinary metabolite profile of antipyrine can be used to study changes in the activity of different cytochromes in drug metabolism studies.

Biochemical basis for the resistance of guinea pigs to carcinogenesis by 2-acetylaminofluorene

Abstract Studies were made on why guinea pigs are resistant to carcinogenesis by 2-acetylaminofluorene. Cytochrome P-448 and arylhydrocarbon hydroxylase were not induced in either the microsomes and nuclei of guinea pigs by 3-methylcholanthrene treatment. 3-Methylcholanthrene treatment caused only 2-fold increase in the binding of 2-acetylaminofluorene to DNA in nuclei isolated from guinea pigs, while it caused 17-fold increase in the binding in rat nuclei. Microsomes from 3-methylcholanthrene treated rats had 5 times more effect than Microsomes from 3-methylcholanthrene treated guinea pigs on the binding of 2-acetylaminofluorene to DNA of nuclei from untreated guinea pigs. N-Hydroxy-2-acetylaminofluorene combined equally well with the DNA of rats and guinea pigs. In guinea pigs, there was a good correlation between the low inducibility of cytochrome P-448 and the low binding of 2-acetylaminofluorene to DNA. Our results clearly showed that guinea pigs are resistant to tumor induction by 2-acetylaminofluorene through inability to carry out the first step of activation of 2-acetylaminofluorene.

The degradation of different forms of cytochrome P-450 [formula omitted] by fluroxene and allyl- [formula omitted]-propylacetamide

Treatment of uninduced, phenobarbital and 3-methylcholanthrene induced rats with fluroxene and allyl-iso-propylacetamide decreased hepatic microsomal cytochrome P-450 and equivalently decreased microsomal heme, aniline binding and p-nitroanisole demethylase. In contrast, ethylmorpnine demethylase, benzpyrene-3-hydroxylase and ethoxyresofurin deethylase were not in all cases decreased in proportion to the loss of cytochrome P-450. After phenobarbital induction fluroxene and allyl-iso-propylacetamide degrade multiple forms of cytochrome P-450, but degrade in the greatest amounts the form(s) of cytochrome P-450 inducible by phenobarbital. After 3-methylcholanthrene induction fluroxene preferentially degrades cytochrome P-448, while allyl-iso-propylacetamide is relatively specific for the form(s) of cytochrome P-450 inducible by phenobarbital.

The effect of phenobarbital and 3-methylcholanthrene treatment on carbon tetrachloride-stimulated heme degradation and carbon monoxide expiration in vivo

Abstract Administration of carbon tetrachloride (CCl 4 ) to rats resulted in an increase in carbon monoxide (CO) expiration within 15 min. Both phenobarbital (PB) and 3-methylcholanthrene (3-MC) increased CCL 4 stimulated CO expiration but only PB increased CCl 4 -stimulated microsomal cytochrome P-450 and heme destruction. CO was formed by peroxidizing methyl linolenate and suggested that a portion of the CO exhaled by rats administered CCl4 may be formed during the peroxidative degradation of membrane lipids. However, because of multiplicity of sources of CO, this gas does not appear to be a suitable index of in vivo lipid peroxidation.

Induction of aryl hydrocarbon (benzo[a]pyrene) hydroxylase and 2-acetylaminofluorene N-hydroxylase by polycyclic hydrocarbons in regenerating liver from inbred strains of mice

The effect of partial hepatectomy (67–75 per cent excised) on aryl hydrocarbon (benzo[a]pyrene) hydroxylase and 2-acetylaminofluorene N -hydroxylase activities in genetically responsive C57BL/6N and nonresponsive DBA/2N inbred strains of mice was studied. Basal aryl hydrocarbon hydroxylase activity in C57BL/6N mice is reduced by 70 per cent within 12 hr but returns to control values between 36 and 48 hr after the operation and remains at that level thereafter. Similar, but less pronounced, changes are observed in the DBA/2N mouse. Treatment with 3-methylcholanthrene (80 mg kg −1 intraperitoneally) 1 hr after partial hepatectomy induces aryl hydrocarbon hydroxylase in C57BL/6N mice, by 48 hr, to the same extent as in mice with intact livers. In DBA/2N mice, even after partial hepatectomy, treatment with 3-methylcholanthrene has no effect on aryl hydrocarbon hydroxylase activity. The effect of partial hepatectomy and 3-methylcholanthrene treatment on 2-acetylaminofluorene N -hydroxylase activity in both strains of mice is similar to that observed on aryl hydrocarbon hydroxylase activity.

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

Abstract 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.

Genetic regulation of UDP-glucuronosyltransferase induction by polycyclic aromatic compounds in mice. Co-segregation with aryl hydrocarbon (benzo(alpha)pyrene) hydroxylase induction.

Induction of hepatic 4-methylumbelliferone UDP-glucuronosyltransferase (EC 2.4.1.17) by polycyclic aromatic compounds, such as 3-methylcholanthrene or beta-naphthoflavone, occurs in C57BL/6N, A/J, PL/J, C3HeB/FeJ, and BALB/cJ but not in DBA/2N, AU/SsJ, AKR/J, or RF/J inbred strains of mice. This pattern of five responsive and five nonresponsive mouse strains parallels that of the Ah locus, which controls the induction of aryl hydrocarbon (benzo[alpha]pyrene) hydroxylase (EC 1.14.14.2). Induction of the transferase is maximal in C57BL/6N mice with 200 mg of 3-methylcholanthrene/kg body weight; no induction occurs in nonresponsive DBA/2N mice even at a dose of 400 mg/kg. The rise of inducible transferase activity lags 1 or more days behind the rise of inducible hydroxylase activity and peaks 5 days after a single dose of 3-methylcholanthrene. In offspring from the appropriate backcrosses and intercross between C57BL/6N and DBA/2N parent strains, the genetic expression of 3-methylcholanthrene-inducible transferase activity is inherited as an additive (co-dominant) trait. This expression differs distinctly from that of the inducible hydroxylase activity, which is inherited almost exclusively as a single autosomal dominant trait in these same animals. The more potent inducer 2,3,7,8-tetrachlorodibenzo-p-dioxin induces the transferase more than 3-fold in C57BL/6N mice and less than 2-fold in DBA/2N mice, whereas the hydroxylase is induced equally (about 8-fold) in both strains. A dose of 3-methylcholanthrene given 3 days after 2,3,7,8-tetrachlorodibenzo-p-dioxin, at a time when hydroxylase induction in both strains is very high, does not enhance the rise in inducible transferase activity seen in C57BL/6N or DBA/2N mice which have received 2,3,7,8-tetrachlorodibenzo-p-dioxin alone. These data indicate that (a) the inducibility of two metabolically coordinated membrane-bound enzyme activities may be regulated by a single genetic locus, and (b) although the hydroxylase can be fully induced in the nonresponsive DBA/2N strain by 2,3,7,8-tetrachlorodibenzo-p-dioxin prior to 3-methylcholanthrene treatment, metabolites of the 3-methylcholanthrene treatment, metabolites of the 3-methylcholanthrene treatment, metabolites of the 3-methylcholanthrene, presumably present in the liver, are incapable of inducing further the transferase activity. The difference in sensitivity between 3-methylcholanthrene and the more potent inducer 2,3,7,8-tetrachlorodibenzo-p-dioxin for both the hydroxylase and the transferase activities suggests the possibility of a common receptor in regulating both enzyme induction processes.

Cell Interactions Between Epithelial and Mesenchymal Components in Primary Sheets of Fetal Rat Lung Cells, and the Effects of 3-Methylcholanthrene Treatment 2

Cell rearrangement of epithelial and mesenchymal components was analyzed in primary cell sheets derived from whole lungs from fetal rats and maintained in tissue culture for 3-175 days without being subcultured. A limited reconstruction of original lung tissue from the dissociated cells was observed in these primary cell sheets. From the initial monlayer spread, ciliated columnar epithelial coverage appeared on fibroblast sheets after appoximately 2 weeks' maintence. Cystic formation also developed within the fibroblast layers. Histotypic collagen fibril formation eventually appeared in the fibroblast layers and resulted in the development of multilayered collagenous connective tissue in vitro. Epithelial elements survived as a form of surface coverage during maintenance of the sheets and as cystic structures in the collagenous layers. Treatment with 3-methylcholanthrene induced severe proliferation of epithelial elements in the cell sheets. These proliferative changes included rapid extension of epithelial surface coverage, stratification of immature cells, and squamous cell mataplasia.

A highly sensitive radiometric assay for zoxazolamine hydroxylation by liver microsomal cytochrome P-450 and P-448: Properties of the membrane-bound and purified reconstituted system

Abstract A radiometric assay for the in vitro metabolism of zoxazolamine has been developed which combines high sensitivity and rapid determination of product. [4,6- 3 H]zoxazolamine was metabolized to 6-hydroxyzoxazolamine, and the tritium released as 3 H 2 O was determined after treating the incubation mixture with activated charcoal. This treatment efficiently removes labeled substrate (99.98%), permitting enzymatically released tritium to be measured directly in the aqueous medium. Since the preponderant in vitro product of zoxazolamine metabolism by rat liver microsomes and the purified reconstituted mixed function oxidase system is 6-hydroxyzoxazolamine, and since this aryl hydroxylation occurs without significant NIH shift, the subsequent release of tritium from the 6-position accurately represents metabolism of the molecule. The use of [4,6- 3 H]zoxazolamine for a tritium release assay of mixed function oxidase activity is ideal since this compound shows no significant isotope effect or NIH shift during metabolic conversion to 6-hydroxyzoxazolamine. 3-Methylcholanthrene treatment of rats resulted in a fourfold induction of zoxazolamine hydroxylation while phenobarbital or pregnenolone 16α-carbonitrile pretreatment caused only a 20–50% increase in zoxazolamine metabolism. The use of a purified reconstituted system revealed that cytochrome P -448 from 3-methylcholanthrene-treated rats was approximately 10- to 15-fold more efficient than cytochrome P -450 from phenobarbital-treated rats in catalyzing the hydroxylation of zoxazolamine.

Induction and activation of rat liver microsomal bile salt glucoronyltransferase

In vivo induction and in vitro activation of the recently described bile salt glucuronyltransferase were investigated in rat. A radioactive assay for the determination of glucuronyltransferase activity was used. 14C-Labeled bile salts served as substrates, and the glucuronides were separated by thin layer chromatography. Lithocholate glucuronyltransferase activity was determined in liver microsomes of phenobarbital- and 3-methylcholanthrene-treated rats and of untreated controls. Pretreatment with phenobarbital induced lithocholate glucuronyltransferase activity to 150.5% of controls. In contrast, 3-methylcholanthrene treatment decreased activity to 29.6% of controls. In vivo activation of lithocholate glucuronyltransferase by Triton X-100 was observed in controls and in the 3-methylcholanthrene group, but not in the phenobarbital group. Substrate activation of the enzyme by lithocholate was demonstrated in microsomes of untreated controls. Pretreatment with 3-methylcholanthrene, but not phenobarbital, increased the latency of lithocholate glucuronyltransferase. The results indicate that rat liver microsomal bile salt glucuronyltransferase activity is increased by in vivo induction with phenobarbital and by in vitro activation with detergents like Triton X-100. The induction of bile salt glucuronide formation by phenobarbital is most likely one of the factors contributing to the increased biliary and fecal excretion of bile salts in patients with cholestasis following phenobarbital therapy.

NADH‐Dependent Aryl Hydrocarbon Hydroxylase in Rat Liver Mitochondrial Outer Membrane

NADH-dependent 3,4-benzpyrene hydroxylase activity was detected in the purified mitochondrial outer membrane fraction from the livers of rats treated with 3-methylcholanthrene. The specific activity in the outer membrane fraction is nearly equal to that of microsomes, a level too high to be accounted for only by the microsomal contamination. On the other hand, the NADPH-dependent 3,4-benzpyrene hydroxylase activity in the outer membrane fraction is about 50% of that of microsomes. The ratio of the specific activity of NADPH- to NADH-dependent 3,4-benzpyrene hydroxylase in microsomal fraction was about 3.5, while that of the outer membrane fraction was about 1.5. Moreover, it was found that NADH-dependent 3,4-benzpyrene hydroxylase activity in mitochondrial outer membrane from control rat liver was cyanide-insensitive, while that in microsomes was cyanide-sensitive. These results suggest the presence in the mitochondrial outer membrane fraction of aryl hydrocarbon hydroxylase activity which uses as electron donor NADH nearly to the same extent as NADPH. The hydroxylase system is composed of cyanide-insensitive cytochrome P-450 and is inducible markedly by 3-methylcholanthrene treatment. The probable electron transfer pathways in the mitochondrial outer membrane cytochrome P-450 oxidase system are discussed.