Differential haemin-mediated restoration of cytochrome P-450 N-demethylases after inactivation by allylisopropylacetamide.

Administration of allylisopropylacetamide (AIA) to phenobarbital-pretreated rats results in the destruction of several phenobarbital-inducible cytochrome P-450 isoenzymes and a correspondingly marked loss of benzphetamine N-demethylase and ethylmorphine N-demethylase activities. Accordingly, the ion-exchange h.p.l.c. or DEAE-cellulose-chromatographic profile of solubilized microsomal preparations from such rats revealed a marked decrease in the cytochrome P-450 content of several eluted fractions compared with that of microsomes from corresponding non-AIA-treated controls. Incubation of liver homogenates from such rats with haemin restores not only cytochrome P-450 content from 35 to 62% of original values, but also benzphetamine N-demethylase and ethylmorphine N-demethylase activities, from 23 to 67%, and from 12 to 36% of original values respectively. Moreover, the chromatographic profiles of microsomes prepared from such homogenates indicated increases of cytochrome P-450 content only in some fractions. Reconstitution of mixed-function oxidase activity of cytochrome P-450 by addition of NADPH: cytochrome P-450 reductase to these fractions indicated that incubation with haemin restored benzphetamine N-demethylase activity predominantly, but ethylmorphine N-demethylase activity only minimally. After injection of [14C]AIA, a significant amount of radiolabel was found covalently bound to protein in chromatographic fraction III, and this binding was unaffected by incubation with haemin. Furthermore, the extent of this binding is apparently equimolar to the amount of cytochrome P-450 refractory to haemin reconstitution in that particular fraction. Whether such refractoriness reflects structural inactivation of the apo-cytochrome remains to be determined. Nevertheless, the evidence presented very strongly argues for AIA-mediated inactivation of multiple phenobarbital-induced isoenzymes, only a few of which are structurally and functionally reparable by haemin.     

Decrease in hepatic cytochrome P-450 by cobalt. Evidence for a role of cobalt protoporphyrin.

Exposure of cultured chick-embryo hepatocytes to increasing concentrations of CoCl2 in the presence of allylisopropylacetamide results in formation of cobalt protoporphyrin, with a reciprocal decrease in haem and cytochrome P-450. Treatment of rats with CoCl2 (84 mumol/kg) and 5-aminolaevulinate (0.2 mmol/kg) also results in formation of cobalt protoporphyrin and a decrease in cytochrome P-450 in the liver. Hepatic microsomal fractions from rats treated with phenobarbital, CoCl2 and 5-aminolaevulinate were analysed by polyacrylamide gel electrophoresis. Cobalt protoporphyrin was associated mainly with proteins of 50000-53000 mol.wt. The results suggest that the formation of cobalt protoporphyrin occurred at the expense of the synthesis of haem, leading to a decrease in cytochrome P-450. Furthermore, the cobalt protoporphyrin that was formed may itself have been incorporated into apocytochrome P-450.     

Inhibition by cycloheximide of degradation of cytochrome P-450 in primary cultures of adult rat liver parenchymal cells and in vivo

Degradation of cytochrome P-450 was studied in adult rat liver parenchymal cells in primary monolayer culture. In cells incubated in standard culture medium, the amount of cytochrome P-450 decreased at an accelerated rate relative to either the rate of degradation of total protein in the cells or the turnover of cytochrome P-450 in vivo. This change was succeeded by a spontaneous increase in the activity of haem oxygenase, an enzyme system that converts haem into bilirubin in vitro, measured in extracts from the cultured cells. This finding suggests that the rate of cytochrome P-450 breakdown may be controlled by factor(s) other than the activity of haem oxygenase. The decline in cytochrome P-450 and the subsequent increase in haem oxygenase activity was prevented by incubation of hepatocytes in medium containing an inhibitor of protein synthesis such as cycloheximide, puromycin, actinomycin D, or azaserine. The effect of cycloheximide appeared to be due to decreased breakdown of microsomal (14)C-labelled haem. By contrast, cycloheximide was without effect on the degradation of total protein, measured either in homogenates or in microsomal fractions prepared from the cultured cells. These results suggest that the conditions of cell culture stimulate selective degradation of cytochrome P-450 by a process that is inhibited by cycloheximide and hence may require protein synthesis. The findings in culture were verified in parallel studies of cytochrome P-450 degradation in vivo. After administration of bromobenzene, the degradation of the haem moiety of cytochrome P-450 was accelerated in vivo in a manner resembling that observed in cultured hepatocytes. Administration of cycloheximide to either bromobenzene-treated rats or to untreated rats decreased the degradation of the haem moiety of cytochrome P-450. However, the drug failed to affect degradation of haem not associated with cytochrome P-450, suggesting that cycloheximide is not a general inhibitor of haem oxidation in the liver. These findings confirm that the catabolism of hepatic cytochrome P-450 haem is controlled by similar cycloheximide-sensitive processes in the basal steady state in vivo, as stimulated by bromobenzene in vivo, or in hepatocytes under the conditions of cell culture. We conclude that the rate-limiting step in this process appears to require protein synthesis and precedes cleavage of the haem ring.     

A model for the regulation of δ-aminolaevulinate synthetase induction in rat liver

A reciprocal relationship exists between the cytochrome P-450 content and delta-aminolaevulinate synthetase activity in adult rats. In young rats the basal delta-aminolaevulinate synthetase activity is higher and the cytochrome P-450 content is lower compared with the adult rat liver. Administration of allylisopropylacetamide neither induces the enzyme nor causes degradation of cytochrome P-450 in the young rat liver, unlike adult rat liver. Allylisopropylacetamide fails to induce delta-aminolaevulinate synthetase in adrenalectomized-ovariectomized animals or intact animals pretreated with successive doses of the drug, in the absence of cortisol. The cortisol-mediated induction of the enzyme is sensitive to actinomycin D. Allylisopropylacetamide administration degrades microsomal haem but not nuclear haem. Haem does not counteract the decrease in cytochrome P-450 content caused by allylisopropylacetamide administration, but there is evidence for the formation of drug-resistant protein-bound haem in liver microsomal material under these conditions. Phenobarbital induces delta-aminolaevulinate synthetase under conditions when there is no breakdown of cytochrome P-450. On the basis of these results and those already published, a model is proposed for the regulation of delta-aminolaevulinate synthetase induction in rat liver.     

Phenobarbital-type induction of cytochrome P-450 in liver microsomes by perfluorodecalin

Cytochrome P-450 induction in hepatic microsomes after injections of rats with a fluorocarbon emulsion containing perfluorodecalin was studied in comparison with phenobarbital and methylcholanthrene type inductions. It was shown that perfluorodecalin injection as well as the phenobarbital one cause an increase in the cytochrome P-450 content, NADPH-cytochrome c reductase activity, the rates of benzphetamine N-demethylation and aldrin epoxidation in the microsomes. Using the Ouchterlony double immunodiffusion test with antibodies against cytochrome P-450b, an immunological identity of cytochrome P-450 isoforms during perfluorodecalin and phenobarbital inductions was shown. Upon "rocket" immunoelectrophoresis the recovery of cytochrome P-450 which is immunologically indistinguishable from cytochrome P-450b was approximately 72% in perfluorodecalin-induced microsomes. The activity of benzphetamine demethylase and aldrin epoxidase was inhibited by antibodies against cytochrome P-450b. These results suggest that in rat hepatic microsomes perfluorodecalin induces the cytochrome P-450 isoform whose immunological properties and substrate specificity correspond to those of phenobarbital-type cytochrome P-450.     

Differential responses to inducers of delta-aminolaevulinate synthase and haem oxygenase during pregnancy.

The responses of hepatic delta-aminolaevulinate synthase and microsomal haem oxygenase to inducers were examined in pregnant rats. 2-Allyl-2-isopropylacetamide-mediated induction of delta-aminolaevulinate synthase was greatly decreased during pregnancy and in the early post-partum period. Administration of allylisopropylacetamide to pseudopregnant rats induced delta-aminolaevulinate synthase normally. Treatment of pregnant rats with cortisol failed to restore the drug-mediated induction of delta-aminolaevulinate synthase. Microsomal cytochrome P-450 content and the activities of drug-metabolizing enzymes such as aniline hydroxylase and ethylmorphine. N-demethylase were significantly lowered during pregnancy. In contrast with the greatly impaired induction of delta-aminolaevulinate synthase, the induction of haem oxygenase in response to CoCl2 remained unaltered in pregnant rats. The normal perturbations of delta-aminolaevulinate synthase, consisting of an initial inhibition followed by a rebound increase in the enzyme activity associated with CoCL2 treatment, were observed during pregnancy. These findings indicate that hormones and metabolic factors associated with gestation exert significant but differential controls on the induction patterns of delta-aminolaevulinate synthase and haem oxygenase.     

Studies on the decrease of liver cytochrome P-450 content by triiodothyronine

In the rat liver, the microsomal content of cytochrome P-450 decreased by 50% after triiodothyronine (T3) administration. The molecular basis for the decreased cytochrome P-450 levels was investigated. The activities of the enzymes involved in heme synthesis or degradation were not altered by thyroid hormone administration. The incorporation of 3H-delta-aminolaevulinate into the liver microsomal heme was markedly reduced in T3-treated rats. The latter appeared not to reflect a lowered binding affinity of the apoprotein moiety of cytochrome P-450 for heme. The sodium dodecyl sulfate gel electrophoresis of the microsomal preparation showed a decrease in apocytochrome P-450. It is suggested that the amount of the apocytochrome may be the primary event affected in the formation of cytochrome P-450, by triiodothyronine treatment of thyroidectomized rats.     

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.     

Identification of the cytochrome P-450 induced by macrolide antibiotics in rat liver as the glucocorticoid responsive cytochrome P-450p

We administered triacetyloleandomycin (TAO) to rats and found that this macrolide antibiotic is the most efficacious inducer of liver microsomal cytochrome P-450 (P-450) examined to date. Liver microsomes prepared from TAO-treated rats contained greater than 5.0 nmol of P-450/mg of protein and a single induced protein as judged by analysis on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This protein comigrated with P-450p, the major form of P-450 induced in liver microsomes of rats treated with pregnenolone-16 alpha-carbonitrile (PCN) or dexamethasone (DEX). On immunoblots of such gels developed with antibodies to P-450p, the TAO-induced protein reacted strongly as a single band. There was strict parallelism between the amount of immunoreactive P-450p in liver microsomes prepared from untreated rats or from rats treated with phenobarbital, TAO, DEX, or PCN, the ability of these microsomes to catalyze conversion of TAO to a metabolite which forms a spectral complex, and the ethylmorphine and erythromycin demethylase activities. Antibodies to P-450p specifically blocked microsomal TAO metabolite complex formation and ethylmorphine and erythromycin demethylase activities. Moreover, anti-P-450p antibodies completely immunoprecipitated solubilized TAO metabolite complexes prepared by detergent treatment of liver microsomes obtained from TAO-treated rats. Finally, we found that the major form of P-450 isolated from liver microsomes of TAO-treated rats and purified to homogeneity was indistinguishable from purified P-450p as judged by molecular weights, spectral characteristics, enzymatic activities, ability to bind TAO, peptide maps, and amino-terminal amino acid sequences. We concluded that, in addition to glucocorticoids, macrolide antibiotics are specific inducers of P-450p.     

Role of haem in the induction of cytochrome P-450 by phenobarbitone. Studies in chick embryos in ovo and in cultured chick embryo hepatocytes.

The role of haem synthesis during induction of hepatic cytochrome P-450 haemoproteins was studied in chick embryo in ovo and in chick embryos hepatocytes cultured under chemically defined conditions. 1. Phenobarbitone caused a prompt increase in the activity of 5-aminolaevulinate synthase, the rate-limiting enzyme of haem biosynthesis, and in the concentration of cytochrome P-450. This induction response occurred without measurable initial destruction of the haem moiety of cytochrome P-450. 2. When intracellular haem availability was enhanced by exogenous haem or 5-aminolaevulinate, phenobarbitone-medicated induction of cytochrome P-450 was not affected in spite of the well known repression of 5-aminolaevulinate synthase by haem. These data are consistent with the concept that haem does not regulate the synthesis of cytochrome P-450 haemoproteins. 3. Acetate inhibited haem biosynthesis at the level of 5-aminolaevulinate formation. When intracellular haem availability was diminished by treatment with acetate, phenobarbitone-medicated induction was decreased. 4. This inhibitory effect of acetate on cytochrome P-450 induction was reversed by exogenous haem or its precursor 5-aminolaevulinate. These data suggest that inhibition of haem biosynthesis does not decrease synthesis of apo-cytochrome P-450. Moreover, they indicate that exogenous haem can be incorporated into newly formed aop-cytochrome P-450.     

Growth hormone depresses ethylmorphine demethylase activity: correlation with decreased levels of fast-turnover cytochrome P-450 in hypophysectomized female rats

The hepatic monooxygenase system was studied in hypophysectomized female rats infused for 5 days with ovine growth hormone (GH). At 7.5 micrograms.h-1 GH decreased the total cytochrome P-450 by 16%; at 10 micrograms.h-1 it reduced both cytochrome P-450 (31%) and the activity of ethylmorphine demethylase (31%). GH did not alter the activities of NADPH cytochrome c reductase or aniline hydroxylase. The lower GH dose decreased the amount of fast- and slow-turnover P-450 by 11 and 38%, respectively, while the higher dose decreased both by 49%. The loss of demethylase activity therefore correlates with the loss of fast-turnover P-450. This component is relatively more abundant in the female (fast: slow turnover of 4.3) than the male (fast:slow turnover of 2.5). GH did not affect the half-lives of the P-450 components, suggesting that it decreases their synthesis. The P-450 concentration in microsomes from GH-treated animals did not increase after incubation with hemin, suggesting that in vivo the hormone does not lower P-450 synthesis via depression of heme. Puromycin mimicked the effect of GH and when given with the hormone their effects on the P-450 levels were multiplicative (p less than 0.05), suggesting different modes of action and that GH does not decrease P-450 by acting at translation.     

Purification and properties of cytochrome P-450 from homogenates of human fetal livers

A form of cytochrome P-450, namely P-450HFLa of human fetal livers, was purified to a specific content of 12.6 nmol/mg protein. The cytochrome P-450 preparation was electrophoretically homogeneous and had an apparent monomeric molecular weight of 51,000 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The cytochrome showed catalytic activities as oxidations of N-methylaniline, ethylmorphine, N,N-dimethylaniline, N,N-dimethylnitrosamine, benzphetamine, aminopyrine, aniline, p-nitroanisole, and 7-ethoxycoumarin to various extents. In fetal liver homogenate, the amount of cytochrome P-450 that reacted with the antiserum to P-450HFLa accounted for more than 36% of the total cytochrome P-450 in three different fetal livers. On the other hand, the amount of P-450HFLa was less than 5% of the total cytochrome P-450 in adult liver microsomes.     

Immunochemical detection of different isoenzymes of cytochrome P-450 induced in chick hepatocyte cultures.

This study investigated whether the same cytochrome P-450 (P-450) isoenzymes were inducible in cultures of chick-embryo hepatocytes as in the liver of chicken embryos. We purified two isoenzymes of cytochrome P-450 from the livers of 17-day-old-chick embryos: one of molecular mass approx. 50 kDa induced in vivo by the phenobarbital-like inducer glutethimide, and the second of approx. 57 kDa induced by 3-methylcholanthrene. Rabbit antiserum against the 50 kDa protein inhibited benzphetamine demethylase activity in hepatic microsomes (microsomal fractions) from glutethimide-treated chick embryo. Antiserum to the 57 kDa protein inhibited ethoxyresorufin de-ethylase activity in hepatic microsomes from methylcholanthrene-treated chick embryo. Cultured chick hepatocytes were treated with chemicals known to induce isoenzymes of P-450 in rodent liver. The induced P-450s were quantified spectrophotometrically and characterized by immunoblotting and enzyme assays. From these studies, chemical inducers were classified into three groups: (i) chemicals that induced a P-450 isoenzyme of 50 kDa and increased benzphetamine demethylase activity: glutethimide, phenobarbital, metyrapone, mephenytoin, ethanol, isopentanol, isobutanol, lindane, lysodren; (ii) chemicals that induced a P-450 isoenzyme of 57 kDa and increased ethoxyresorufin de-ethylase activity: 3-methylcholanthrene and 3,3',4,4'-tetrachlorobiphenyl; and (iii) the mono-alpha-substituted 2,3',4,4',5-pentabromobiphenyl, which induced both proteins and both activities. The immunochemical data showed that chick-embryo hepatocytes in culture retain the inducibility of glutethimide- and methylcholanthrene-induced isoenzymes of P-450 that are inducible in the liver of the chicken embryo.     

Accelerated hepatic haem catabolism in the selenium-deficient rat.

1. Hepatic microsomal cytochrome P-450 concentrations are lower in selenium-deficient rats treated with phenobarbital for 4 days than in similarly treated control rats. 2. No defect in haem synthesis was found on the basis of measurements of delta-aminolaevulinate synthase (EC 2.3.1.37), delta-aminolaevulinate dehydratase (EC 4.2.1.24) and ferrochelatase (EC 4.99.1.1) activities, and urinary excretion of delta-aminolaevulinate, porphobilinogen, uroporphyrin and coproporphyrin. 3. No defect in apo-(cytochrome P-450) separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 4. An increase in haem catabolism was found. An 8-fold increase in hepatic microsomal haem oxygenase (EC 1.14.99.3) activity occurred in selenium-deficient rats after phenobarbital treatment, compared with a less than 2-fold increase in control rats. Also excretion of 14CO in the breath after administration of delta-amino[5-14C]laevulinate was greater by phenobarbital-treated selenium-deficient rats than by similarly treated controls. 5. These studies demonstrate that the defective induction of cytochrome P-450 by phenobarbital in selenium-deficient rats is accompanied by increased haem catabolism. This could be due to increased breakdown of cytochrome P-450 or to catabolism of haem before it attaches to the apo-cytochrome. The role of selenium in stabilizing cytochrome P-450 and/or in protecting haem from breakdown remains to be determined.     

Effect of endotoxin on tryptophan pyrrolase and delta-aminolaevulinate synthase: evidence for an endogenous regulatory haem fraction in rat liver.

Endotoxin was administered to rats at a dose shown previously to stimulate hepatic haem oxygenase activity and to block induction of delta-aminolaevulinate synthase, apparently by causing redistribution of haem from cytochrome P-450 to a regulatory haem pool in the liver. Within 5h of the administration of endotoxin (at a time when the effect of the compound on cytochrome P-450 is maximal) the relative saturation of tryptophan pyrrolase with intrinsic haem rose, from a basal value of 50% to 90%, indicating that 'free' haem had become available. Concurrently, the activity of delta-aminolaevulinate synthase was decreased to 25% of its basal value. Haem oxygenase reached peak activity 13h after endotoxin administration. These findings provide new evidence for the existence of an 'unassigned' hepatic haem fraction, which exchanges with cytochrome P-450 haem and regulates these three enzyme functions.     

Phenylhydrazine-mediated induction of haem oxygenase activity in rat liver and kidney and development of hyperbilirubinaemia. Inhibition by zinc-protoporphyrin.

Phenylhydrazine was found to be a potent inducer of microsomal haem oxygenase activity in rat liver and kidney, but not in spleen. The phenylhydrazine-mediated increase in haem oxygenase activity was time-dependent. Maximum activity was attained 12h after treatment in the liver, and 24h after treatment in the kidney. The increases in the activity of haem oxygenase in the liver and the kidney could be inhibited by cycloheximide. Furthermore, the increases could not be elicited by the treatment of microsomal preparations in vitro with phenylhydrazine. In consonance with the increased haem oxygenase activity, a marked increase (16-fold) was observed in the serum total bilirubin concentration in phenylhydrazine-treated rats. The mechanism of haem degradation promoted by phenylhydrazine in vivo appears to differ from that in vitro; only in the former case is bilirubin formed as the end-product of haem degradation. When rats were given zinc-protoporphyrin (40 mumol/kg) 12h before and after phenylhydrazine treatment, the phenylhydrazine-mediated increases in haem oxygenase activity in the liver and the kidney were effectively blocked. Treatment of rats in vivo with the metalloporphyrin also inhibited the activity of splenic haem oxygenase, and promoted a major decrease in the serum bilirubin levels. In phenylhydrazine-treated animals, the microsomal content of cytochrome P-450 was significantly decreased in the absence of a decrease in the microsomal haem concentration. The decrease in cytochrome P-450 content was accompanied by an increased absorption in the 420nm region of the reduced CO-difference spectrum, suggesting the conversion of the cytochrome to an inactive form. The marked depletion of cellular glutathione levels suggests that this conversion may be related to the action of active intermediates and free radicals formed in the course of the interaction of phenylhydrazine with the haem moiety of cytochrome P-450.     

Chemical modification of the histidine residues of purified hepatic cytochrome P-450: influence on substrate binding and the haemoprotein spin state

A hepatic cytochrome P-450 isolated in an electrophoretically homogeneous form from phenobarbital-treated rats, exists predominantly in the low spin configuration (82% at 20 degrees C). The addition of saturating amounts of the substrate benzphetamine to this haemoprotein shifted the spin equilibrium to the high spin form, resulting in a doubling of the spin equilibrium constant from 0.220 to 0.539 at 20 degrees C. The histidine residues of this low spin, substrate-free cytochrome P-450 were modified in a time- and concentration-dependent manner with diethylpyrocarbonate, and progressive histidine modification resulted in a decrease of both the affinity and extent of substrate interaction with the haemoprotein. Although the histidine-modified haemoprotein maintained the capacity to undergo a temperature-dependent spin transition of the haem iron in the presence of saturating amounts of substrate, this capability was substantially decreased in comparison to the unmodified cytochrome. These results indicate that a histidine residue(s) is involved in the binding of substrate to cytochrome P-450 and hence interferes with the substrate-bound spin equilibrium. Our results further imply that histidine is probably not the sixth ligand of the substrate-free ferric form of the rat liver cytochrome P-450.     

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.     

The influence of side chain modifications of the heme moiety on prosthetic acceptance and function of rat hepatic cytochrome P-450 and tryptophan pyrrolase

The relative potential of various structural isomers (III, XIII) and various 2,4-side chain modified analogs of heme (iron-protoporphyrin IX) to incorporate into rat liver hemoproteins, cytochrome P-450(s), and tryptophan pyrrolase was examined. Such assessments for hepatic cytochrome P-450 relied on generation of reconstitutible apocytochrome(s) P-450 by suicidal alkylation of the existing prosthetic heme moiety by allylisopropylacetamide (AIA) in vivo. Subsequent replacement of the prosthetic heme was brought about by incubating the apocytochrome(s) P-450-enriched preparations with a particular heme isomer or analog. Structure-function relationships of the reconstituted isozymes were assessed in microsomal preparations by monitoring cytochrome P-450 content (structure) and its mixed function oxidase activity (function). In parallel, the relative ability of these heme isomers and analogs to functionally constitute hepatic tryptophan pyrrolase was also assessed by monitoring the relative increase in holoenzyme activity when preparations deliberately enriched in constitutible apoenzyme were incubated with each of these compounds. The findings reveal that 2,4-side chain modifications on the heme IX skeleton markedly influence the function of the constituted hemoproteins possibly by affecting their structural assembly through steric, electronic, and/or hydrophobic interactions with the corresponding apoproteins. Furthermore, these studies not only reveal that the structural specifications of the active prosthetic site of rat liver cytochrome P-450(s) differ from those of tryptophan pyrrolase, but also that the structural specifications of these mammalian hemoproteins for their prosthetic heme differ considerably from those reported for their bacterial counterparts.     

Incorporation of haemoglobin haem into the rat hepatic haemoproteins tryptophan pyrrolase and cytochrome P-450.

After its administration to intact rats, haemoglobin haem was incorporated into hepatic tryptophan pyrrolase as shown by the marked increase in functional constitution of this enzyme. Incorporation of haemoglobin haem into cytochrome P-450 was demonstrated in intact rats and in the isolated rat liver perfused with haemoglobin-free medium. In both systems, haemoglobin haem restored cytochrome P-450 content and its dependent mixed-function-oxidase activity after substrate-induced destruction of the cytochrome P-450 haem moiety. Further confirmation that haemoglobin haem could be incorporated prosthetically into cytochrome P-450 was achieved by administration of [3H]haemoglobin to rats and subsequent isolation and characterization of radiolabelled substrate-alkylated products of cytochrome P-450 haem. Our findings indicate that, although hepatic uptake of parenteral haemoglobin is slower than that of haem, it appears to serve as an effective haem donor to the intrahepatic 'free' haem pool. Thus parenteral haemoglobin may warrant consideration as a therapeutic alternative to haem in the acute hepatic porphyrias.