Phenobarbital Cytochrome P-450 Research Articles

Mechanism of iron potentiation of hepatic uroporphyria: studies in cultured chick embryo liver cells.

Effects of iron were studied in cultured chick embryo liver cells to help elucidate the effect of hepatic iron in the human disease porphyria cutanea tarda and in toxic porphyria caused by chemicals. These cultures have proven useful because (a) phenobarbital and phenobarbital-like drugs induce a common form(s) of cytochrome P-450 (P-450-phenobarbital) in these cultures; (b) 20-methylcholanthrene and certain other polycyclic hydrocarbons induce a different form(s) (P-450-methylchol-anthrene), and (c) uroporphyria can be produced rapidly by exposure to suitable chemicals. In these cultures, treatment with iron alone did not produce porphyrin accumulation, and treatment with iron + 5-aminolevulinate caused accumulation of protoporphyrin, as did treatment with 5-aminolevulinate alone. However, treatment with phenobarbital-like drugs and iron, the latter at a concentration as low as 0.2 microM, led to accumulation of uro- and heptacarboxylporphyrins. Potentiation of uroporphyrin accumulation by iron began before there was a detectable synergistic increase in activity of 5-aminolevulinate synthase, the rate-controlling enzyme of heme synthesis. In contrast, treatment of cultures with 20-methylcholanthrene, in the presence or absence of iron, did not result in uroporphyrin accumulation or an increase in the activity of 5-aminolevulinate synthase. Uroporphyrinogen decarboxylase activity was unchanged by drug and iron treatments. Inhibitors of P-450-phenobarbital, SKF525A and piperonyl butoxide, as well as cadmium and cycloheximide prevented the porphyrin accumulation produced by glutethimide + iron, even though, except with cycloheximide, these substances further increased 5-aminolevulinate synthase activity. In vitro, uroporphyrin was oxidized autocatalytically by iron. In intact hepatocytes, even low concentrations of iron (0.2 to 20 microM), in the presence of a form of cytochrome P-450 induced by phenobarbital-like chemicals, produces uroporphyria primarily by enhancing uroporphyrinogen oxidation, not by inhibition of the decarboxylase. Induction of 5-aminolevulinate synthase amplifies the porphyrin overproduction.

Differences in oxidative dearylation and desulfuration of fenitrothion by cytochrome P-450 isozymes and in the subsequent inhibition of monooxygenase activity

The organophosphorus insecticide, fenitrothion ( O,O-dimethyl O-(3-methyl-4-nitrophenyl) phosphorothioate), a substrate for the cytochrome P-450 ( P-450) monooxygenase system, yields the corresponding phosphate triester (fenitroxon) by oxidative desulfuration (an activation reaction) and 4-nitro- m-cresol by oxidative dearylation (a detoxication reaction). Four constitutive P-450 isozymes and the major isozymes induced by phenobarbital (PB) and β-naphthoflavone (BNF) were incubated with [ 14C]fenitrothion in reconstituted monooxygenase systems. Both metabolites were formed with all isozymes, with the ratio of oxon to cresol being characteristic of the individual P-450s. Cytochrome P-450 B2 and P-450 PB formed the highest percentage of oxon, 75 and 82%, respectively; while P-450 A1, B3, and BNF produced 55 to 60% oxon. By contrast, P-450 A2 formed the least amount of oxon, 38%, with 62% of the product being 4-nitro- m-cresol. Microsomes prepared from mice treated in vivo with organophosphates, such as fenitrothion, have reduced levels of cytochrome P-450 and associated monooxygenase activities. Presumably, this reduction is due to destruction of P-450 caused by the release of active sulfur occurring during desulfuration. To determine if some P-450 isozymes were preferentially inhibited by fenitrothion, purified isozymes were incubated with fenitrothion and subsequent p-nitroanisole O-demethylase activity was determined. Inhibition studies using purified P-450 isozymes indicated close to 100% inhibition by fenitrothion of P-450 PB, while enzyme activity of P-450 BNF and P-450 A2 was inhibited 85–90%.

Immunoquantitation of some cytochrome P-450 isozymes in liver microsomes from streptozotocin-diabetic rats.

Streptozotocin-diabetes in rats leads to a decrease of cytochrome P-450 UT-A (the major form in control rats) and an increase of cytochrome P-450 PB-B (the major one induced by phenobarbital treatment) in liver microsomes. The increased benzphetamine-N-demethylase activity can be related to the induction of cytochrome P-450 PB-B.

Polychlorinated biphenyls, classified as either phenobarbital- or 3-methylcholanthrene-type inducers of cytochrome P-450, are both hepatic tumor promoters in diethylnitrosamine-initiated rats

The cytochrome P-450 isozymes, cytochrome P-450 MC 1 and MC 2, purified from rats treated with 3-methylcholanthrene (MC), were found by immunohistochemical staining to be strongly induced in the livers of rats treated with 3,3′, 4,4′-tetrachlorobiphenyl (TCBP), while the cytochrome P-450 isozymes, PB 1 and PB 2, purified from the livers of rats treated with phenobarbital (PB), were shown to be induced in the livers of rats treated with 2,2′, 4,4′, 5,5′-hexachlorobiphenyl (HCBP). The latter compound also strongly induced NADPH-cytochrome P-450-reductase. Following induction, all 5 enzymes were located preferentially in the centrilobular and midzonal region of the liver acinus. The influence of these polychlorinated biphenyls (PCBs) on diethylnitrosamine (DEN)-initiated hepatocarcinogenesis was investigated by analyzing the evolution of adenosine triphosphatase-deficient focal lesions. Whereas DEN alone produced very few islets, the administration of either PCB congener (150 μmol/kg, i.p., once weekly over a period of 8 weeks) subsequent to DEN treatment (50 ppm in the drinking water, 10 days) strongly enhanced the number of islets as well as the relative volume of liver occupied by islet tissue. These effects were evident, both 1 and 9 weeks, after cessation of PCB treatment. Unexpectedly the less persistent PCB congener, TCBP, showed a much more potent enhancing effect after the 9 weeks recovery period than did (HCBP).

Investigation of the multicomponent detoxication system in the albino rat liver

Cytochrome P-450 PB localization in the livers of embryos, pregnant, non-pregnant female, and male Wistar rats was investigated immunomorphologically after phenobarbital (PB) treatment. The highest level of cytochrome P-450 PB induction was observed in the livers of male rats where the antigen invaded almost the whole liver lobule. About half of the cells within the lobule of non-pregnant female rat livers were stained and were located around the central vein area. Pregnant females had two times less indication than the non-pregnant group and only a quarter of the cells in the lobule of the liver adjacent to the central vein were stained for P-450 PB. The indication of cytochrome P-450 PB was absent from 17-18-day-old embryonal livers which were subjected to the transplacental action of the inducer. Only in one case out of seven were small groups of cells and single cells in the embryonal liver stained positively for cytochrome P-450 PB. It is obvious that during pregnancy, the mother's body exerts a significant influence on the detoxifying function of the fetal liver.

Induction of cytochrome P-450 mRNAs quantitated by in vitro translation and immunoprecipitation

Cytochrome P-450 mRNAs were quantitated by in vitro translation of liver RNA followed by immunoprecipitation with antibodies specific for cytochromes P-450. The kinetics of cytochrome P-450 mRNA induction by 3-methylcholanthrene and by phenobarbital were examined, and differences in the types of cytochrome P-450 mRNAs induced by 3-methylcholanthrene (MC), β-napthoflavone (BNF), and phenobarbital (PB) in male and female rats were determined. Phenobarbital strongly induced the mRNA encoding a single peptide antigenically related to the phenobarbital-induced cytochrome P-450 PB-B. Male rat liver contained 62% more translatable mRNA for this peptide than did female. 3-Methylcholanthrene and β-napthoflavone, but not phenobarbital, induced mRNAs encoding three peptides that were immunologically related to cytochrome P-450 BNF MC-B , which is induced by 3-methylcholanthrene. The levels of translatable mRNA coding for these peptides were twice as high in females as in males. Striking sex differences were observed in the levels of translatable mRNAs for peptides related to cytochrome P-450 PB PCN-E , which is induced by phenobarbital and by pregnenolone-16-α-carbonitrile. In females, only RNA preparations from the livers of phenobarbital-treated rats had significant levels of mRNAs encoding these peptides. In contrast, significant levels of these RNAs were observed even in untreated males, and the levels of these mRNAs increased markedly following phenobarbital treatment. All cytochrome P-450 inducers examined caused a 50 to 70% decrease in translatable albumin mRNA. This effect was specific for albumin mRNA, since levels of total translatable mRNA were not generally altered by these inducers. The kinetics of induction of cytochrome P-450 mRNA differed from those of induction of aryl hydrocarbon hydroxylase (AHH) activity. Translatable cytochrome P-450 mRNA was increased as early as 4 h after phenobarbital treatment, peaked between 24 and 36 h, and dropped back to control levels by 120 h. The induction of AHH lagged behind the increase in translatable mRNA, remaining at control levels well after levels of translatable mRNA began to increase but then decreasing roughly in parallel with translatable mRNA. These findings suggest that transcription was not rate limiting for regulation of PB-inducible cytochrome P-450 activity. 3-Methylcholanthrene caused parallel increases in AHH activity and translatable cytochrome P-450 mRNA, but when translatable mRNA began to decrease after about 24 h, AHH activity remained high, suggesting that this P-450 mRNA was less stable than the enzyme for which it coded.

Effects of carbohydrate intake on phenobarbital-, polychlorinated biphenyl- and 3-methylcholanthrene-induced enhancement of drug oxidation and conjugation in rat liver

The effects of dietary carbohydrate on phenobarbital (PB)-, polychlorinated biphenyl (PCB)- or 3-methylcholanthrene (MC)-induced increase in the activity of cytochrome P-450-linked monooxygenase, a UDP-glucuronyltransferase and a glutathione S-transferase in rats were studied. A low-carbohydrate diet increased the monooxygenase activity for all of the hydrocarbons studied (benzene, toluene, styrene, carbon tetrachloride, chloroform, 1, 2-dichloroethane, 1, 1-dichloroethylene and trichloroethylene) without affecting microsomal protein and cytochrome P-450 contents. On the other hand, though causing an increase both in microsomal protein and cytochrome P-450 contents, PB, PCB and MC each produced only a substrate-specific increase in the enzyme activity: PB enhanced the metabolism of toluene, styrene, chloroform and trichloroethylene, but not those of benzene, carbon tetrachloride, 1, 2-dichloroethane and 1, 1-dichloroethylene; PCB increased only the metabolism of toluene, styrene and trichloroethylene; MC did not affect the metabolism of any of the hydrocarbons. A low-carbohydrate diet potentiated the effects of these inducers on the protein and cytochrome P-450, but the augmentation was reflected only in the metabolism of toluene and styrene.Although PB, PCB and MC each increased both activites of UDP-glucuronyltransferase (substrate, p-nitrophenol) and glutathione S-transferase (3, 4-dichloronitrobenzene), the effect of dietary carbohydrate on these transferases was negligible. In addition, a low-carbohydrate diet failed to augment the enhancing effects of these inducers.These results indicate that dietary carbohydrate intake is likely to affect the first phase metabolism of volatile hydrocarbons (functionalization reaction) but is not likely to cause a significant influence on the second phase metabolism (conjugation reactions).

Involvement of FMN and phenobarbital cytochrome P-450 in stimulating a one-electron reductive denitrosation of 1-(2-chloroethyl)-3-(cyclohexyl)-1-nitrosourea catalyzed by NADPH-cytochrome P-450 reductase.

Purified hepatic NADPH-cytochrome P-450 reductase, which was reconstituted with dilauroylphosphatidylcholine, catalyzed a one-electron reductive denitrosation of 1-(2-[14C]-chloroethyl)-3-(cyclohexyl)-1-nitrosourea ([14C]CCNU) to give 1-(2-[14C]-chloroethyl)-3-(cyclohexyl)urea at the expense of NADPH. Ambient oxygen or anoxic conditions did not alter the rates of [14C]CCNU denitrosation catalyzed by NADPH-cytochrome P-450 reductase with NADPH. Electron equivalents for reduction could be supplied by NADPH or sodium dithionite. However, the turnover number with NADPH was slightly greater than with sodium dithionite. Enzymatic denitrosation with sodium dithionite or NADPH was observed in anaerobic incubation mixtures which contained NADPH-cytochrome P-450 reductase with or without cytochrome P-450 purified from livers of phenobarbital (PB)-treated rats; PB cytochrome P-450 alone did not support catalysis. PB cytochrome P-450 stimulated reductase activity at molar concentrations approximately equal to or less than NADPH-cytochrome P-450 reductase concentration, but PB cytochrome P-450 concentrations greater than NADPH-cytochrome P-450 reductase inhibited catalytic denitrosation. Cytochrome c, FMN, and riboflavin demonstrated different degrees of stimulation of NADPH-cytochrome P-450 reductase-dependent denitrosation. Of the flavins tested, FMN demonstrated greater stimulation than riboflavin and FAD had no observable effect. A 3-fold stimulation by FMN was not observed in the absence of NADPH-cytochrome P-450 reductase. These studies provided evidence which establish NADPH-cytochrome P-450 reductase rather than PB cytochrome P-450 as the enzyme in the hepatic endoplasmic reticulum responsible for CCNU reductive metabolism.

Measurement of the metabolism of cytochrome P-450 in cultured hepatocytes by a quantitative and specific immunochemical method.

We have defined conditions that permit quantitative and specific measurement of the metabolism of the major phenobarbital-inducible form of cytochrome P-450 protein in primary non-proliferating monolayer cultures of adult rat hepatocytes. Isolated antibodies specifically directed against phenobarbital cytochrome P-450 are used to immunoprecipitate the cytochrome from lysates of cultured hepatocytes pulse-labelled with [(3)H]leucine. Phenobarbital cytochrome P-450 protein is then isolated from the immunoprecipitate by electrophoresis on polyacrylamide gradient slab gels. Specificity of the assay for phenobarbital cytochrome P-450 was established by competition experiments involving other forms of purified cytochrome P-450 as well as by testing antibodies directed against these other forms of the cytochrome. Using purified phenobarbital cytochrome P-450, radiolabelled in both its haem and apoprotein portions, as an internal standard, we demonstrated that, with this immunoassay, recovery of cytochrome P-450 from microsomal samples is nearly complete. Basal rates of synthesis of phenobarbital cytochrome P-450 representing as little as 0.02-0.05% of total cellular protein synthesis were reliably and reproducibly detected in hepatocyte culture maintained in serum-free medium for 72h. Moreover, inclusion of phenobarbital in the culture medium for 96h stimulated not only synthesis de novo of phenobarbital cytochrome P-450 protein, but also accumulation of spectrally and catalytically active cytochrome P-450. Advantages of this immunoassay are that metabolism (synthesis or degradation) of the haem or protein of this important form of the cytochrome can be measured conveniently in the small samples available from cultured cells without the necessity of preparing subcellular fractions.