Induction of cytochrome P-450 RNA by porphyrogenic agents: on the nature of the coordinate induction with delta-aminolevulinate synthase in tissues of the chicken embryo.

1. We determined by cDNA-RNA solution hybridization analyses that in ovo administration of allylisopropylacetamide in combination with diethyl 1,4-dihydro-2,4,6-trimethyl-3,5-pyridinedicarboxylate increased the concentrations of cytochrome P-450 RNA in liver, kidney, and intestine of 18-day-old chicken embryos. 2. Similarly, the administration of testosterone to embryos caused elevations in the cytochrome P-450 RNA levels in liver and kidney. 3. The increases in cytochrome P-450 RNA concentrations occurred only in those tissues where elevations in delta-aminolevulinate (ALA) synthase activity and mRNA content were measured (liver, kidney and intestine) but not in tissues where the activity and RNA levels of ALA synthase did not change (heart, brain, lung). 4. The increases in the concentrations of the cytochrome P-450 RNA were not affected by loading embryos with ALA and FeCl3 at the time of administration of the inducers.     

Molecular induction by phenobarbital of a rat hepatic form of cytochrome P-450: expression of a 4-kilobase messenger RNA

A differential screening procedure was employed to isolate a cDNA clone corresponding to a major phenobarbital (PB)-inducible form of rat hepatic cytochrome P-450. The G-C homopolymer-tailing technique was utilized to construct a cDNA library in the PstI site of plasmid pBR322. The library represented PB-induced poly(A+)RNA sequences from hepatic polysomes of 150-g male Sprague-Dawley rats. Hybrid-selection experiments against total PB-inducible RNA were performed with plasmid DNA derived from clones enriched in PB-inducible information. The mRNA molecules that specifically hybridized were subjected to in vitro translation, were immunoprecipitated with antibody raised in rabbits against purified cytochrome P-450b (P. E. Thomas, D. Korzeniowski, D. Ryan, and W. Levin (1979) Arch. Biochem. Biophys. 192, 524-532), and were electrophoresed under sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoretic conditions. One cDNA clone, designated PB-8, contained a 600-bp insert partially coding for a PB-inducible cytochrome P-450 species that comigrated on SDS-gel electrophoresis with highly purified P-450b. A single injection of PB, 15-18 h before sacrifice, increased the level of polysomal poly(A+)RNA complementary to the isolated cDNA clone by approximately 16-fold. Northern blot hybridizations of polysome-derived poly (A+)RNA, electrophoresed in denaturing agarose gels, demonstrated that the size of the mRNA corresponding to the isolated clone was 4 kb. Isolated heteronuclear RNA species demonstrated a time-dependent increase in the synthesis of a similar 4-kb RNA molecule. By genomic blot hybridization to EcoRI-restricted DNA, at least three complementary DNA fragments migrating at 5.1, 3.2, and 2.9 kb were observed with 32P-labeled PB-8 as a probe. These data, together with restriction endonuclease mapping and partial cDNA sequence information of the PB-8 cDNA, suggest that the PB-8 clone represents a previously unreported cDNA clone for a form of cytochrome P-450 inducible by PB.     

Induction of delta-aminolevulinate synthase and cytochrome P-450 hemoproteins in hepatocyte culture. Effect of glucose and hormones

Addition of glucose to cultured chick embryo hepatocytes caused a concentration-dependent impairment of phenobarbital-mediated induction of delta-aminolevulinate (ALA) synthase resembling the "glucose effect" observed in rodents in vivo. This glucose effect occurred in the complete absence of extrahepatic factors such as serum and hormones. Fructose, glycerol, and lactate mimicked the inhibitory glucose effect on ALA synthase induction, whereas 2-deoxyglucose and 3-O-methylglucose augmented the induction evoked by phenobarbital. 2-Deoxyglucose reversed the effect of glucose, glycerol, and lactate on ALA synthase induction suggesting that the glucose effect is mediated by free glucose or glucose 6-phosphate or a nonglycolytic metabolite of glucose 6-phosphate. The phenobarbital-mediated induction of cytochrome P-450 hemoprotein(s) and its monooxygenase function were concomitantly diminished by glucose. However, this inhibitory effect or glucose was reversible by the addition of exogenous heme or ALA suggesting that the primary target of the glucose effect is ALA synthase induction and not synthesis of apocytochrome P-450. Glucagon and dibutyryl cAMP enhanced the induction of ALA synthase and cytochrome P-450 by phenobarbital and partially counteracted the glucose effect on both enzymes suggesting that the glucose effect may be mediated by changes in cAMP levels. Although insulin did not alter induction of ALA synthase, it impaired induction of cytochrome P-450 even in the presence of glucagon and cAMP. These data may be relevant for the treatment with glucose and heme of patients with "inducible" hepatic porphyria.     

The role of heme in the regulation of tryptophan-2,3-dioxygenase activity and content of cytochrome P-450 in rat liver

The effects of exogenous heme on the activity of delta-aminolevulinate synthase, heme oxygenase, tryptophan-2.3-dioxygenase and microsomal cytochrome content in rat liver were studied. It was shown that hemin chloride diminishes the delta-aminolevulinate synthase activity and provokes heme oxygenase induction. This is paralleled with the induction of the tryptophan 2.3-dioxygenase apoenzyme and an increase in the saturation of the enzyme with heme. The cytochrome b5 content does not change thereby, whereas that of cytochrome P-450 shows a decrease. Upon combined administration of actinomycin D and hemin the cytochrome P-450 level is markedly increased. Actinomycin D by itself has no effect on the hemoprotein concentration. It is concluded that the increase in the cytochrome P-450 level results from the activation of heme-induced mRNA translation.     

Regulation of heme and drug metabolism activities in the brain by manganese

A novel effect of metal ions in the brain is described. Mn was found to alter heme metabolism and the cytochrome P-450-dependent mixed-function oxidase activities in rat brain. A more than 2-fold increase in benzo(alpha)pyrene hydroxylase and 7-ethoxycoumarin deethylase activities were observed in the brain of rats treated for 7 days with Mn. The increases were regionally distributed; the highest elevations were observed in the hippocampus, pons and the caudate putamen. Moreover, in rats treated with Mn for 1 or 7 days a marked depression in the activity of the mitochondrial ALA synthetase was observed. The activity of the microsomal heme oxygenase was also inhibited at 7 days, but not 1 day, after Mn treatment. These inhibitions were reflected in an initial decrease, followed by a rebound return to normal, in the concentration of cytochrome P-450 in the brain. Mn was ineffective in vitro in altering heme and drug metabolism activities. It is suggested that Mn-mediated alterations in heme metabolic activities promote changes in the composition of cytochrome P-450 species in the brain microsomal fractions, such that the relative concentrations of the molecular species which catalyse aryl hydrocarbon hydroxylase activity become selectively increased.     

Incorporation of heme to microsomal cytochrome P-450 in the absence of protein biosynthesis

Determination of the heme and protein portions of phenobarbital (PB)-inducible and 3-methylcholanthrene inducible forms of cytochrome P-450, P-450(PB-1), and P-450(MC-1), in the liver microsomes of drug-treated animals indicated the presence of 20-30% of apo-cytochrome P-450 in both cases. Inhibition of protein synthesis by cycloheximide injection to the rats did not significantly inhibit the incorporation of delta-amino[14C]levulinic acid (ALA) into the heme of P-450(PB-1) or P-450(MC-1) in the liver, indicating that the heme incorporation into microsomal cytochrome P-450 is not tightly coupled with the synthesis of the apo-cytochrome. When heme-labeled cytosol prepared from [14C]ALA-injected rats was incubated with non-radioactive microsomes in vitro, a significant amount of labeled heme was incorporated into microsomal P-450(PB-1), whereas the incorporation into P-450(MC-1) was much less. The in vitro transfer of heme from cytosol to microsome-bound cytochrome P-450 was stimulated by the addition of an NADPH-generating system to the incubation mixtures, and inhibited when the microsomes were solubilized with sodium cholate and Emulgen-913. Although the in vitro incubation of heme-labeled microsomes with non-radioactive cytosol resulted in some release of labeled heme from the microsomes, no reversible transfer of heme between cytochrome P-450 molecules bound to separate microsomal vesicles was detected when heme-labeled microsomes were incubated with non-radioactive microsomes in the presence and absence of cytosol.     

Cytochrome P-450 heme and the regulation of hepatic heme oxygenase activity

The degradation of cytochrome P-450 heme in the liver has been studied by a new approach. In rats, hepatic heme was labeled by administration of a tracer pulse of [5-¹⁴C]δ-aminolevulinic acid (ALA), and its degradation was analyzed in terms of labeled carbon monoxide (¹⁴CO) excretion, which is a specific degradation product of the labeled heme. Within minutes after administration of [5-¹⁴C]ALA, 14CO was detectable and increased after 2 h to an “early peak,” reflecting the elimination of labeled heme from a rapidly turning over pool in the liver. Beyond the early peak, the rate of ¹⁴CO production decreased in a log-linear manner, consistent with the degradation of heme in stable hepatic hemoproteins. From the rate at which ¹⁴CO production declined during this phase, from the predominant labeling of cytochrome P-450 heme by the administered [5-¹⁴C]ALA and from the known turnover characteristics of this hemoprotein in the liver, it could be inferred that production of ¹⁴CO—between 16 and 30 h after administration of labeled ALA—largely reflected degradation of cytochrome P-450 heme. This approach, which permits serial measurements in a single animal, was used to study the effect on cytochrome P-450 heme of administered heme or endotoxin, both of which are potent stimulators of hepatic heme oxygenase activity. Both of these substances caused marked acceleration of the degradation of cytochrome P-450 heme, the effect occurring over the same dose range as that for stimulation of hepatic heme oxygenase. The findings suggest that stimulation of this enzyme activity in the liver is closely related to the rate of degradation of cytochrome P-450 heme.     

Fractionation and purification of hepatic microsomal cytochrome P-450 isoenzymes from phenobarbital-pretreated rats by h.p.l.c. A convenient tool for screening of isoenzymes inactivated by allylisopropylacetamide.

A procedure incorporating the salient features of ion-exchange column chromatography with ion-exchange h.p.l.c. is described for the fractionation and purification to homogeneity of several membrane-bound rat hepatic phenobarbital (PB)-inducible cytochrome P-450 isoenzymes, including the major PB-inducible species. The resolving power of this technique makes it a highly promising tool for the isolation and purification of closely related cytochrome P-450 isoenzymes. In addition, it may also be used for screening of individual isoenzymes either selectively induced or repressed by a variety of endobiotics or xenobiotics. Accordingly, we have exploited this particular feature to identify not only the PB-inducible cytochrome P-450 isoenzymes destroyed in vivo by allylisopropylacetamide, a suicide inactivator of cytochrome P-450, but also to distinguish those that are reparable by exogenous haemin from those that are irreparably damaged.     

Control of delta-aminolaevulinate synthase and haem oxygenase in chronic-iron-overloaded rats.

The hepatic porphyrias are inborn errors of porphyrin and haem biosynthesis characterized biochemically by excessive excretion of delta-aminolaevulinate (ALA), porphobilinogen and other intermediates in haem synthesis. Clinical evidence has implicated iron in the pathogenesis of several types of genetically transmitted diseases. We investigated the role of iron in haem metabolism as well as its relationship to drug-mediated induction of ALA synthase and haem oxygenase in acute and chronic iron overload. Acute iron overload in rats resulted in a marked increase in hepatic haem oxygenase that was associated with a decrease in cytochrome P-450 and an increase in ALA synthase activity. Aminopyrine N-demethylase and aniline hydroxylase activities, which are dependent on the concentration of cytochrome P-450, were also decreased. In contrast, in chronic-iron-overloaded rats, there was an adaptive increase in haem oxygenase activity and an increase in ALA synthase that was associated with normal concentrations of microsomal haem and cytochrome P-450. The induction of ALA synthase in chronic iron overload was enhanced by phenobarbital and allylisopropylacetamide, in spite of the fact that these agents did not increase haem oxygenase activity. Small doses of Co2+ were potent inducers of the haem oxygenase in chronic-iron-overloaded, but not in control, animals. We conclude that increased hepatic cellular iron may predispose certain enzymes of haem synthesis to induction by exogenous agents and thereby affect drug-metabolizing enzyme activities.     

Induction and repression of the major phenobarbital-induced cytochrome P-450 measured by radioimmunoassay.

Two independent radioimmunoassay techniques for the major phenobarbital-inducible cytochrome P-450 (PB P-450) of rat liver microsomal membranes are described. The first technique employs as the source of radiolabelled antigen the products of translation in vitro labelled with [35S]methionine. The second technique employs purified antigen labelled with 125I and is quicker, less expensive and more precise. Both assays are highly specific for PB P-450 and can detect quantities of this variant as small as 1 ng. This is several orders of magnitude more sensitive than any method described previously for the quantification of cytochromes P-450, and consequently the technique is particularly well suited for the quantification of so-called constitutive cytochrome P-450 variants that are present in very low amounts. The results of the radioimmunoassays demonstrate that the apparent 2.6-fold induction of total cytochromes P-450 after phenobarbital treatment is due to a 43-fold increase in Pb P-450. Although beta-naphthoflavone increases the total content of cytochrome P-450 of microsomal membranes 1.4-fold, it actually causes a 55% decrease in the amount of PB P-450. Thus different xenobiotics can have differential effects on the expression of the genes for specific cytochrome P-450 variants.     

Reversible transfer of heme between different molecular species of microsome-bound cytochrome P-450 in rat liver

Incorporation of newly synthesized heme into microsome-bound cytochrome P-450 in rat liver was not affected by cycloheximide administration to the animals, indicating that the heme incorporation into cytochrome P-450 is not tightly coupled with the synthesis of the apo-cytochrome. When the heme of microsomal cytochrome P-450 had been labeled in vivo with delta-[14C]aminolevulinic acid, and then the animals were treated with phenobarbital (PB) or 3-methylcholanthrene (MC), PB-induced or MC-induced form of cytochrome P-450 was found to contain labeled heme derived from preexistent cytochrome P-450. These observations indicated that the heme of microsome-bound cytochrome P-450 is not tightly associated with the protein portion, and exchanges reversibly between different molecular species of cytochrome P-450 in vivo.     

Rotation and interaction with epoxide hydrase of cytochrome P-450 in proteoliposomes

Purified rat liver cytochrome P-450MC or P-450PB was co-reconstituted with epoxide hydrase in liposomal vesicles made of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine at a lipid to protein weight ratio of 5 by the cholate dialysis procedure. Rotational diffusion of the cytochromes was measured by observing the decay of absorption anisotropy, r(t), after photolysis of the heme.CO complex by a vertically polarized laser flash. Analysis of r(t) was based on a "rotation-about-membrane-normal" model. The measurements were used to investigate interactions of cytochrome P-450MC or P-450PB with epoxide hydrase. Different rotational mobilities of the two cytochromes were observed. The amount of mobile molecules was 78% for cytochrome P-450MC and 91% for P-450PB, and the rest was immobile within the experimental time range of 1 ms. In the presence of epoxide hydrase 85% of cytochrome P-450MC and 96% of P-450PB were mobile. Cross-linking of epoxide hydrase by anti-epoxide hydrase antibodies resulted in a drastic immobilization of the cytochromes, reducing the mobile population to 49% for P-450MC and to 60% for P-450PB. The rotational relaxation times phi of the mobile populations ranged from 210 to 283 microseconds. These results imply that both cytochromes P-450MC and P-450PB transiently associate with epoxide hydrase in liposomal membranes. Further analysis of the data showed that the angle between the heme plane of P-450MC and the membrane is 48 degrees or 62 degrees, different from the value of 55 degrees reported previously for P-450PB (Gut, J., Richter, C., Cherry, R. J., Winterhalter, K. H., and Kawato, S. (1983) J. Biol. Chem. 258, 8588-8594).     

Identification and characteristics of mRNA for cytochrome P-450 in the rat liver induced by phenobarbital and 3-methylcholanthrene

Using two consecutive oligo(dT)-cellulose column chromatography steps, the total poly(A)RNA was isolated from the livers of rats injected with phenobarbital (PB) or 3-methylcholanthrene (MC). During translation of the PB-induced mRNA in the reticulocyte lysate cell-free protein-synthesizing system, a single polypeptide with an apparent molecular weight of 50,000 was synthesized which was specifically immunoprecipitated by antibodies to major PB-inducible cytochrome P-450 PB-3. In contrast, after completion of MC-mRNA translation, the antibodies to major MC-induced cytochrome MC-2 precipitated from the incubation mixture 4-5 polypeptides, of which the largest one with an apparent molecular weight of 58,000 corresponded to cytochrome P-450 MC-2. During sucrose density gradient centrifugation, the PB- and MS-mRNAs with sedimentation coefficients of about 18S and 20S, respectively, were precipitated.     

Further consideration of phenobarbital effects on cytochrome P-450 activity in the killifish, Fundulus heteroclitus

1. Ethoxyresorufin O-deethylase (EROD) activity, aldrin epoxidase (AE) activity, cytochrome P-450 content, and levels of cytochrome P-450E (the major BNF-inducible P-450 form and primary EROD catalyst in scup) or its homologues were measured in hepatic microsomes isolated from Fundulus heteroclitus, scup (Stenotomus chrysops) and brook trout (Salvelinus fontinalis) treated with beta-naphthoflavone (BNF) or phenobarbital (PB). 2. In all three teleost species, BNF treatment caused expected increases in P-450 content, EROD activity and P-450E level; but either no change or a slight decrease in AE turnover rate (nmol/min/nmol P-450). 3. Polyclonal antibodies to P-450E did not inhibit AE activity in microsomes from BNF-treated scup, confirming that this major BNF-inducible P-450 form does not catalyze AE activity in fish. 4. In contrast, PB treatment did not affect hepatic AE activity, P-450 content or levels of "P-450E" in F. heteroclitus, but did variably affect EROD activity which was suppressed in one experiment and elevated in another. 5. The results indicate that (i) contrary to previous reports, neither PB nor MC-type inducers increase AE activity in F. heteroclitus, (ii) MC-type inducers do not affect AE activity in the other teleost species examined, and (iii) AE activity is not a reliable indicator of P-450 induction by environmental chemicals. 6. We emphasize the need to establish the mechanism of PB action, and the nature of any fish P-450 forms analogous to PB-inducible forms in mammals in order to conclusively evaluate PB-responses in fish.     

Effect of ascorbic acid on heme metabolism in hepatic microsomes

Hepatic microsonal cytochrome P-450 levels are significantly decreased (46–68%) in ascorbic acid-deficient guinea pigs. Studies attempting to elucidate the mechanism responsible for decreased cytochrome P-450 demonstrated that ascorbic acid status did not influence the turnover $\text{(}\text{t}\text{1}\text{2}\text{)}$ or the degradation of hepatic cytochrome P-450 heme. Urinary excretion of Δ-aminolevulinic acid (ALA) and coproporphyrin was significantly decreased (30 and 69% respectively) in ascorbic acid-deficient guinea pigs. Injections (ip) of ALA into ascorbic acid-deficient guinea pigs were not effective in returning cytochrome P-450 levels to values found in ascorbic acid-supplemented guinea pigs. In addition, plasma and hepatic iron and blood heme were related directly, while hepatic copper and plasma copper or ceruloplasmin were related inversely, to the ascorbic-acid status of the guinea pig. These data, along with past investigations on heme synthesis in the ascorbic acid-deficient guinea pig, are consistent with mechanisms proposing that ascorbic acid may influence: 1) apocytochrome P-450 synthesis, 2) binding of heme and apo-cytochrome P-450 to form active cytochrome P-450, and/or 3) incorporation of Fe++ into the heme moiety of cytochrome P-450, perhaps via changes in copper metabolism.     

Identification of the forms of cytochrome P-450 induced in rat liver by 2-acetylaminofluorene using immunoblotting and partial purification

The amounts of 5 different forms of cytochrome P-450 in liver microsomes from rats treated with 2-acetylaminofluorene were determined and compared with the corresponding patterns in microsomes from control, 3-methylcholanthrene- and phenobarbital-treated animals. 2-Acetylaminofluorene was found to increase the amount of cytochromes P-450b + e 10-fold and of cytochrome P-450d 3-fold, while there was a 54% increase in the level of cytochrome P-450 PB/PCN-E. Cytochrome P-450c was increased from a level too low to detect (less than 0.001 pmol/mg protein) to 0.019 pmol/mg protein. These findings were also confirmed by partial purification of cytochromes P-450b + e and c after 2-acetylaminofluorene treatment.