Cytochrome P450 Heme Research Articles

The responses of hepatic monooxygenases of guinea pig to cadmium and nickel.

When Cd (3.58 mg CdCl2.H2O/kg, ip) was administered to male guinea pigs 72 h prior to sacrifice, the metal significantly inhibited the aniline 4-hydroxylase (AH) (16%), ethylmorphone N-demethylase (EMND) (26%), and aminopyrine N-demethylase (AMND) (18%) activities and cytochrome P-450 (12%) and cytochrome b5 (10%) levels. Cd did not alter the hepatic microsomal heme level. Cd, however, significantly increased the hepatic microsomal p-nitroanisole O-demethylase (p-NAOD) (53%) activity. When Ni (59.5 mg NiCl2 x 6H2O/kg, sc) was administered to the guinea pigs 16 h prior to sacrifice, the metal significantly depressed AH (49%), p-NAOD (66%), EMND (47%), and AMND (37%) activities, and cytochrome P-450 (15%), cytochrome b5 (24%), and microsomal heme (28%) levels. For the combined treatment, animals received the single dose of Ni 56 h after the single dose of Cd and then were killed 16 h later. In these animals, significant inhibitions were noted in AH (51%), EMND (47%), and AMND (30%) activities, and cytochrome P-450 (15%), cytochrome b5 (26%), and microsomal heme (30%) compared to those of controls. In the case of p-NAOD activity, the influence was in favor of Ni, i.e., the inhibition was about 61% by the combined treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

On the enzyme-inducing action of calcium antagonists.

The effects of three calcium antagonists, nifedipine (NF), verapamil (V) and diltiazem (DL), on rat liver monooxygenases were studied. The drugs were administered in oral doses of 50, 40 and 30 mg/kg daily for 3 weeks in male Wistar rats. NF and V shortened the hexobarbital (HB) sleeping time and increased benzphetamin-N-demethylase (BND), ethylmorphine-N-demethylase (EMND), aniline hydroxylase (AH), ethoxycoumarine-O-deethylase (ECOD), ethoxyresorufin-O-deethylase (EROD) and NADPH-cytochrome c reductase activities and the content of cytochrome P-450 and microsomal heme, but did not change the content of cytochrome b5. The data suggest that these calcium antagonists exert an enzyme-inducing effect on the hepatic monooxygenases. DL significantly increased only the EROD and NADPH-cytochrome c reductase activities and shortened HB sleeping time to a lesser extent, suggesting a weaker enzyme-inducing effect as compared to NF and V. The three drugs increased the delta-aminolevulinic acid (ALA) synthetase activity and decreased heme oxygenase (HO) activity. The increased cytochrome P-450 content is probably due to the increased synthesis and the decreased breakdown of this hemoprotein.

Responses of hepatic xenobiotic metabolizing enzymes of mouse, rat and guinea-pig to nickel.

The effects of nickel (Ni) on hepatic monooxygenase activities (aniline 4-hydroxylase, AH; ethylmorphine N-demethylase, EMND; aminopyrine N-demethylase, AMND), cytochrome P-450, cytochrome b5, microsomal haem and reduced glutathione (GSH) levels, and glutathione S-transferase (GST) activities toward several substrates (1, chloro-2-4-dinitrobenzene, CDNB; 1,2 dichloro-4-nitrobenzene, DCNB; ethacrynic acid, EAA) in mice, rats and guinea-pigs were studied. Ni (59.50 mg NiCl2.6H2O/kg, subcutaneously) was administered to the animals 16 hr prior to sacrifice. Ni significantly inhibited AH, EMND, AMND activities, and decreased cytochrome P-450, cytochrome b5 (except in the livers of rats), and microsomal haem levels in the livers of all the animal species examined. However, the depressions were more profound in livers of mice than in those of the other two species. The hepatic GSH level was significantly inhibited in mice whereas no alteration was observed in rats. In guinea-pigs, the hepatic GSH level was significantly increased by Ni. The hepatic GST activity toward the substrate CDNB was significantly depressed in mice, unaltered in rats and significantly increased in guinea-pigs by Ni. The hepatic GST activity toward DCNB was significantly inhibited in mice whereas no significant alteration was observed in rats. In guinea-pigs, Ni caused significant increase in hepatic GST activity for DCNB. However, hepatic GST activity toward EAA was significantly inhibited in mice whereas significantly increased in rats and guinea-pigs. These results seem to indicate that i) there exists quantitative, but not qualitative, differences among the hepatic monooxygenases of rodents in response to Ni, mice being more sensitive than rats and guinea-pigs, ii) the influence of Ni on hepatic GSH level varies depending on the animal species and iii) the hepatic GSTs of rodents are differentially regulated by Ni.

Location of functional centers in the microsomal cytochrome P450 system.

Fluorescence quenching and energy-transfer studies have been carried out to determine the position of FAD and FMN groups of NADPH-cytochrome P450 reductase and of the heme and substrate groups of cytochrome P450 with respect to the lipid/water interphase. Quenching by iodine of the fluorescence of the flavins of the reductase shows a biphasic pattern, due to the different accessibility of FAD and FMN to the solvent with Stern-Volmer constants of 7.9 x 10(-4) and 2.7 x 10(-3) mM-1, respectively. Both prosthetic groups appear to be buried within the three-dimensional structure of the native reductase, FAD more deeply embedded than FMN and with a relative contribution to the total fluorescence of flavins of 84% (FAD) and 16% (FMN). The lack of significant energy transfer (less than 5%) from FAD+FMN to the rhodamine group of the N-labeled phosphatidylethanolamine incorporated in membranes reconstituted with NADPH-cytochrome P450 reductase and phosphatidylcholine points out that both groups are located at a distance greater than 5 nm from the lipid/water interphase. Steady-state fluorescence intensity and anisotropy data obtained with native and FMN-depleted NADPH-cytochrome P450 reductase show that energy transfer between both prosthetic groups occurs in the native reductase with an efficiency of ca. 31%, consistent with a separation between these groups of 2 nm as suggested earlier by Bastiaens, P. I. H., Bonants, P. J. M., Müller, F., & Visser, A. J. W. G. [(1989) Biochemistry 28, 8416-8425] from time-resolved fluorescence anisotropy measurements.(ABSTRACT TRUNCATED AT 250 WORDS)

Combined effect of cadmium and nickel on rat hepatic monooxygenases: possible stimulation of certain cytochrome P-450 isozymes

When male rats were given either a single dose of cadmium (3.58 mg CdCl 2·6H 2O kg , i.p.) 72 h prior to sacrifice or a single dose of nickel (59.5 mg NiCl 2·6H 2O kg , s.c.) 16 h prior to sacrifice, the activities of ethylmorphine N-demethylase, aminopyrine N-demethylase and aniline 4-hydroxylase, and the levels of cytochrome P-450 and microsomal heme were significantly decreased. Cadmium decreased the cytochrome b 5 level significantly, whereas it did not alter the NADPH-cytochrome c reductase activity significantly. In contrast, Ni did not alter the cytochrome b 5 level significantly but decreased the NADPH-cytochrome c reductase activity significantly. For the combined treatment, animals received the single dose of nickel 56 h after the single dose of cadmium and then they were killed 16 h later. In these animals ethylmorphine N-demethylase, aminopyrine N-demethylase and NADPH-cytochrome c reductase activities and cytochromes P-450 and b 5 levels increased significantly as compared to those of controls, whereas aniline 4-hydroxylase activity and microsomal heme level remained unaltered. In concordance with the increase in the enzyme activities, certain P-450 protein bands were observed to be elevated when studied on SDS-polyacrylamide gel electrophoresis. Furthermore, when the monooxygenase activities and SDS-polyacrylamide gel electrophoresis profiles of combined metal-treated animals were compared with those of the animals treated with classic inducers such as phenobarbital (75 mg kg i.p., 72, 48 and 24 h prior to sacrifice) and 3-methylcholanthrene (20 mg kg i.p., 72, 48 and 24 h prior to sacrifice), the combination of metals seemed to have tendency to stimulate certain phenobarbital and 3-methylcholanthrene inducible cytochrome P-450 isozymes.

Effect of phenobarbital treatment on carbon tetrachloride-mediated cytochrome P-450 loss and diene conjugate formation

The effect of phenobarbital treatment on the linkage between carbon tetrachloride-mediated cytochrome P-450 loss and lipid peroxidation in rat liver microsomes was studied. Male Sprague-Dawley rats, pretreated with 3 daily i.p. doses of phenobarbital (50 mg/kg) or saline, were orally dosed with carbon tetrachloride (0.01–2.5 ml/kg), with liver microsomes prepared at 7.5–180 min after carbon tetrachloride treatment. In vivo cytochrome P-450 loss displayed pseudo-first-order kinetics, and the initial rates of diene conjugate formation were saturable with dose. Phenobarbital pretreatment decreased the in vivo t 0.5, max from 27.0 to 15.6 min, and increased the K d, app from 0.78 to 1.30 ml/kg for carbon tetrachloride mediated cytochrome P-450 loss. Phenobarbital had no effect on the in vivo V max (1.03 to 1.04 ΔOD 232 nm/min/mg phospholipid) for carbon tetrachloride mediated diene conjugate formation, but decreased the K m, app from 0.22 to 0.10 ml/kg. These results are consistent with destruction of cytochrome P-450 heme resulting from a metabolite which does not leave the site of generation, and with phenobarbital pretreatment enhancing the initiation of lipid peroxidation.

Modulation of erythropoiesis by novel human bone marrow cytochrome P450- dependent metabolites of arachidonic acid

In the hematopoietic system the adherent stromal cells produce cytokines necessary for proliferation and differentiation of hematopoietic cells. In the present study, we showed the ability of adherent stromal cells to generate novel metabolites of arachidonic acid via the NADPH-cytochrome P450-dependent monooxygenase system. These metabolites were recovered in the incubation media, suggesting their release from cells. The formation of arachidonic acid metabolites was inhibited by 7-ethoxyresorufin and SKF-525A, but not by indomethacin or BW-755C. By using two-step high-pressure liquid chromatography (HPLC), bone marrow-adherent stromal cells and incubation media showed the presence of metabolites in a peak eluted at 19 to 20 minutes. The isolated HPLC peak was used to measure its effect on colony-forming unit-erythroid (CFU-E) growth and compare it with that of synthetic cytochrome P450 arachidonate metabolites, 19- and 20- hydroxyeicosatetraenoic (HETE) acid. These bone marrow cytochrome P450 arachidonic acid metabolites at picomolar concentration potentiated erythropoietin (Epo)-induced CFU-E growth by fourfold to sixfold. Addition of 19- and 20-HETE to the bone marrow culture resulted in a potentiating effect on CFU-E number in a dose-dependent manner. 20-HETE was much more potent in stimulating CFU-E growth than 19-HETE at a similar concentration of 10(-11) mol/L. The potentiating effect of 20- HETE resulted in a shifting to the left of the dose-response curve to Epo. To substantiate the finding of an active NADPH-dependent cytochrome P450-metabolizing system, we further examined the ability of adherent cells to metabolize exogenous pharmacologic compounds such as benzo(a)pyrene, a substrate for the heme-cytochrome P450 system, aryl hydrocarbon hydroxylase. The adherent stromal cytochrome P450 metabolizes benzo(a)pyrene at comparable levels to blood vessel endothelial cells. These novel observations underscore the importance of adherent stromal cytochrome P450 to metabolize endogenous substrates, including arachidonic acid, to compounds that may interact in a paracrine manner with Epodependent hematopoietic cells.

Modulation of Erythropoiesis by Novel Human Bone Marrow Cytochrome P450-Dependent Metabolites of Arachidonic Acid

In the hematopoietic system the adherent stromal cells produce cytokines necessary for proliferation and differentiation of hematopoietic cells. In the present study, we showed the ability of adherent stromal cells to generate novel metabolites of arachidonic acid via the NADPH-cytochrome P450-dependent monooxygenase system. These metabolites were recovered in the incubation media, suggesting their release from cells. The formation of arachidonic acid metabolites was inhibited by 7-ethoxyresorufin and SKF-525A, but not by indomethacin or BW-755C. By using two-step high-pressure liquid chromatography (HPLC), bone marrow-adherent stromal cells and incubation media showed the presence of metabolites in a peak eluted at 19 to 20 minutes. The isolated HPLC peak was used to measure its effect on colony-forming unit-erythroid (CFU-E) growth and compare it with that of synthetic cytochrome P450 arachidonate metabolites, 19- and 20-hydroxyeicosatetraenoic (HETE) acid. These bone marrow cytochrome P450 arachidonic acid metabolites at picomolar concentration potentiated erythropoietin (Epo)-induced CFU-E growth by fourfold to sixfold. Addition of 19- and 20-HETE to the bone marrow culture resulted in a potentiating effect on CFU-E number in a dose-dependent manner. 20-HETE was much more potent in stimulating CFU-E growth than 19-HETE at a similar concentration of 10(-11) mol/L. The potentiating effect of 20-HETE resulted in a shifting to the left of the dose-response curve to Epo. To substantiate the finding of an active NADPH-dependent cytochrome P450-metabolizing system, we further examined the ability of adherent cells to metabolize exogenous pharmacologic compounds such as benzo(a)pyrene, a substrate for the heme-cytochrome P450 system, aryl hydrocarbon hydroxylase. The adherent stromal cytochrome P450 metabolizes benzo(a)pyrene at comparable levels to blood vessel endothelial cells. These novel observations underscore the importance of adherent stromal cytochrome P450 to metabolize endogenous substrates, including arachidonic acid, to compounds that may interact in a paracrine manner with Epodependent hematopoietic cells.

NADPH-dependent reduction of amaranch in liver microsomes characterized the quantity of low spin forms of cytochrome P-450

We confirmed that NADPH-dependent anaerobic amaranch reduction in rat liver microsomes is compatible with the interaction of the dye with Fe(III) heme of cytochrome P-450 as the type II substrate. This process is rate-limiting in the whole reaction. High positive correlation (r=0.949) between the values of Vmax for reaction of NADPH-dependent anaerobic amaranch reduction and the relative content low spin forms of cytochrome P-450 determined by ESR in microsomes from liver of control and induced by PB, BP, IS and 4-MP rats was observed. Relative content of low spin forms of cytochrome P-450 determined by ESR was increased according to BP<PB<control<IS∼4-MP; Vmax values increased according to BP<PB<control<IS<4-MP. Thus, reaction of NADPH-dependent anaerobic amaranch reduction may be used for determination of low spin forms of cytochrome P-450 at physiological conditions.

Amino-terminal sequence homology of two chick kidney mitochondrial proteins immunoisolated with monoclonal antibodies to the cytochrome P450 of 25-hydroxyvitamin D3-1α-hydroxylase

We demonstrate the unique capability of monoclonal antibodies for the specific immunodetection and characterization of two antigenic proteins occurring in normal chick kidney mitochondrial extracts. The antigens were adsorbed to cyanogen bromide-activated Sepharose gel coupled to monoclonal antibodies (MAbs) of the IgM class raised against cytochrome P450(1) alpha, which inhibit equally the 25-hydroxyvitamin D3 1 alpha- and 24-hydroxylase catalytic activities (Mandel et al. 1990 J Clin Lab Immunol, in press). The two identified antigenic proteins are polypeptides with apparent molecular weights of 57,000 and 55,000 daltons. The 1 alpha-hydroxylase cytochrome P450 has been shown to have a molecular weight of 57,000 daltons (Mandel et al. 1990 Biochim Biophys Acta 1034:239-246). The optimal antigen:gel ratio for maximal antigen binding as cytochrome P450 heme, which was determined spectrally, was found to be 1.3 nmol cytochrome P450 per g MAb-coupled Sepharose. At this ratio the total binding capacity of the gel was 1 nmol cytochrome P450 per g Sepharose. The two polypeptides were desorbed with 0.1% Emulgen 911 in 25% glycerol at pH 3.0 and separated by SDS-gel electrophoresis. The amino-terminal sequences of the two antigens were determined by automated Edman degradation with an on-line analyzer of PTH derivatives. The sequences in both antigens were 100% homologous. Complete amino acid composition analysis also revealed that their amino acid compositions were highly similar. These findings suggest that the smaller protein may be a proteolytic cleavage product of the 1 alpha-hydroxylase P450 cytochrome and may represent a putative 24-hydroxylase antigen.

Orientation of cytochromes P450 in the endoplasmic reticulum

The orientation of eukaryotic cytochromes P450, with respect to the membrane of the endoplasmic reticulum, has been investigated. There is now good evidence that the tertiary structure of these proteins is essentially the same as that of the soluble bacterial isoenzyme cytochrome P450CI, with the exception of an extension at the N-terminus which is thought to form a membrane-anchoring sequence. The remainder of the molecule protrudes from the cytosolic face of the membrane so that it can interact with substrates and electron-donating proteins. Two models based on this structure have been considered, in which the plane of the heme of cytochrome P450 is oriented either parallel with or perpendicular to the plane of the membrane of the endoplasmic reticulum. The validity of these models has been assessed from the results of studies involving the binding of antipeptide antibodies directed toward known regions of cytochromes P450, modeling of the interaction of cytochrome P450 with cytochrome b5, proposed intramolecular movements of cytochrome P450 during its catalytic cycle, and the partitioning of substrates for cytochrome P450 between the cytosol and membrane. It is concluded that cytochrome P450 is most likely oriented such that the heme is not fixed horizontal to the plane of the membrane of the endoplasmic reticulum and may well lie with the heme perpendicular to the membrane.

Eclectic mechanisms of heme regulation of hematopoiesis

Regulatory features of heme (ferroprotoporphyrin IX) on hematopoietic growth/differentiation and related processes are reviewed. It is emphasized that expressions of specific erythroid and nonerythroid heme biosynthetic and degradatory enzymes are required, and the regulatory processes whereby this occurs is considered. The specificity of heme, relationship to cellular events such as differentiation, response to growth factors, oncogene and receptor expression, and how heme counteracts toxic effects such as viral growth are all discussed. The significance of heme in the hemopoietic bone marrow microenvironment and growth factor network are considered. Finally, the third pathway for arachidonic acid metabolism via the heme-cytochrome P450 monooxygenase system, in addition to cyclooxygenase and lipoxygenase, by bone marrow adherent cells and its role in cellular differentiation is briefly reviewed.

Irreversible binding of heme to microsomal protein during inactivation of cytochrome P450 by 4-alkyl analogues of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine

The porphyrinogenicity of 4-alkyl analogues of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-tri-methylpyridine (DDC) is related to the process of mechanism-based destruction of cytochrome P450 (P450) heme, accompanied by conversion of heme to N-alkylprotoporphyrins ( N-alkylPPs). Certain DDC analogues (4-isopropyl, 4-isobutyl, 4-hexyl) are weakly porphyrinogenic in comparison to the potent porphyrinogen, 4-ethyl DDC. We have examined the abilities of these DDC analogues to promote irreversible binding of radiolabeled heme to protein in rat liver microsomal preparations. The goals of this study were to determine whether DDC analogues with different porphyrinogenicities differ in the extents to which they cause heme adduct formation, and whether P450 isozymes differ in their capacities to catalyze heme covalent binding. Incubation of microsomes with NADPH alone promoted heme covalent binding, while loss of spectral P450 heme was minimal or absent. In microsomal incubations containing NADPH, the 4-ethyl, 4-isopropyl, and 4-isobutyl analogues caused heme covalent binding to extents which paralleled their P450 destructive activities. In contrast, 4-hexyl DDC caused less heme covalent binding as a function of P450 loss than the other analogues in microsomes from untreated and β-naphthoflavone (ßNF)-treated rats. Thus, the weakly porphyrinogenic DDC analogues do not cause greater heme covalent binding than 4-ethyl DDC. Weak porphyrinogenicity, therefore, cannot be explained by diversion of the heme moiety of P450 from conversion to N-alkylPPs towards utilization for formation of heme-derived protein adducts. Treatment of rats with P450 inducing agents altered the degree to which DDC analogues caused heme eovalent binding. The greatest heme adduct formation occurred in microsomes from untreated and dexamethasone (DEX)-treated rats, whereas treatment with phenobarbital and especially ßNF reduced heme covalent binding as a function of P450 loss. Thus, these microsomal studies suggest that constitutive P450 isozymes and members of the DEX-inducible P450IIIA subfamily appear to catalyze heme covalent binding, while βNF-inducible forms such as P450IA1 (P450c) seem to be relatively inactive in this regard.

Effects of sulfur-containing metabolites of hexachlorobenzene on the heme metabolic enzymes in rat liver.

Effects of hexachlorobenzene (HCB) and its sulfur-containing metabolites on the heme metabolic enzymes in rat liver were investigated. A single injection of HCB caused the increase in activities of delta-aminolevulinic acid (ALA) synthetase and heme oxygenase, and contents of cytochrome P-450 and total heme. After a single injection of pentachlorothioanisol (PCTA) or pentachlorophenyl methyl sulfore (PCPSO2Me), ALA synthetase activity was enhanced. Heme oxygenase activity was increased by PCPSO2Me treatment. Cytochrome P-450 and total heme contents were increased by PCPSO2Me or 1,4-bis(methylthio)tetrachlorobenzene (MTTCB). When HCB was injected once daily for 5 weeks, a marked increase in ALA synthetase activity, a significant decrease in ALA dehydratase, almost complete inhibition of uroporphyrinogen decarboxylase activity, and an increased excretion of total porphyrin in the urine were shown. After chronic treatment with its sulfur-containing compounds, PCPSO2Me and MTTCB produced a significant increase in ALA synthetase activity. However, activities of ALA dehydratase and uroporphyrinogen decarboxylase, and excretion of total porphyrin in the urine were unaltered. At this time, the concentrations of the corresponding sulfur-containing compound and related metabolite(s) in blood, liver and kidney were nearly the same as those observed in HCB-treated rats. It is suggested that PCPSO2Me and MTTCB could induce the hepatic ALA synthetase, but, these metabolites, and also PCTA, were not able to induce the porphyria in female rats, and the induction of porphyria by HCB is not attributable to the action of its sulfur-containing compound.

Cimetidine in the Treatment of Acute Intermittent Porphyria

<h3>To the Editor.—</h3> Horie et al¹recently reported that oral cimetidine in an 800-mg daily dose decreased urinary excretion of 8-aminolevulinic acid (ALA) in a patient with acute intermittent porphyria who was in remission. This study was based on the inhibitory effect of cimetidine on hepatic cytochrome P-450 synthesis and heme oxidase activity, which results in decreased heme consumption with subsequent inhibition of ALA synthetase through a negative feedback mechanism.²The aim of this letter is to confirm these biologic changes observed by Horie et al and furthermore to see if they are associated with clinical improvement. <h3>Report of a Case.—</h3> A 38-year-old white female with a well-documented familial (mother, one sister, and one brother) and personal (two previous attacks) history of acute intermittent porphyria was given 400 mg of cimetidine twice daily from day 0 to day 14. Urinary excretion of ALA and porphobilinogen was measured 7

Biomimetic models for monooxygenases.

The microsomal mixed function oxidase system contains the cytochrome P-450 oxidative drug metabolizing family of enzymes. The catalytic cycle of cytochrome P-450 is believed to involve the formation of an active iron-oxygen species which is responsible for oxygen transfer to the substrate. This assumption is supported by the fact that a number of peroxidative agents can replace NADPH, the reductase, and oxygen as co-reactants in most oxidative reactions of microsomal cytochrome P-450. We have found that a mixture of either ferrous or ferric ions with hydrogen peroxide (Fenton and Ruff reagents) can serve as biomimetic models for cytochrome P-450 in hydroxylation, exposidation, sulfoxidation, and N-demethylation of various drugs. The existance of an iron-oxo active species in both Fenton and Ruff type reactions has been postulated and provides reaction cycles similar to those of cytochrome p-450. Other model systems for the hepatic hydroxylation and epoxidation using transition metal complexes with porphyrin are also discussed. The present paper reviews the various biomimetic models of the heme cytochrome P-450 and emphasizes their simulation of hepatic drug metabolism and their potential medical and industrial applications.

Synergistic induction of δ-aminolevulinic acid synthase activity by N-ethylprotoporphyrin IX and 3,5-diethoxycarbonyl-1,4-dihydro-2,6-dimethyl-4-isobutylpyridine

The levels of the cellular free heme pool in chick embryo hepatocyte culture were lowered using N-ethylprotoporphyrin IX ( N-ethylPP) and analogues of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC), and the effect on δ-aminolevulinic acid synthase (ALAS) was examined. N-EthylPP, which lowers cellular heme levels by inhibiting ferrochelatase activity, produced an induction of ALAS activity to 444% of control at 3 hr after its administration. 4-Ethyl DDC, which lowers heme levels by destroying the heme moiety of cytochrome P-450 and lowering ferrochelatase activity, caused an induction of ALAS to 565% of control at 12 hr after administration. 4-Isobutyl DDC, which lowers heme levels by destroying the heme moiety of cytochrome P-450, induced the activity of ALAS to 289% of control at 3 hr after administration. This indicates that ferrochelatase inhibition is a more important mechanism of heme lowering than alkylation of cytochrome P-450 heme when both heme-depleting mechanisms are acting in chick embryo liver cells. It was anticipated that administration of a combination of 4-isobutyl DDC plus N-ethylPP would mimic the effect of 4-ethyl DDC. However, this combination induced ALAS activity to levels that were much greater than those observed after 4-ethyl DDC (1257% of control at 12 hr). This synergistic induction may be attributable to lowering of free heme levels to the point where transcription, translation, and translocation of ALAS are all derepressed.

Covalent bonding of the prosthetic heme to protein: a potential mechanism for the suicide inactivation or activation of hemoproteins

In this perspective we have described a newly characterized pathway for the metabolism of the prosthetic heme of cytochrome P-450, which results in the formation of protein-bound adducts. This reaction occurs when the cytochrome P-450 metabolizes a variety of xenobiotics as well as endogenous compounds such as hydrogen peroxide and lipid hydroperoxides. It also takes place during the reactions catalyzed by other hemoproteins, such as myoglobin and hemoglobin. In the case of the reaction of ferrous myoglobin with BrCCl3, under single-turnover conditions, an intact heme moiety becomes covalently bound to an active-site amino acid. This covalently altered protein has significantly enhanced reductive activity compared to that of native myoglobin, as demonstrated by its rapid reduction of molecular oxygen and CCl4. It also is more rapidly proteolyzed than myoglobin. These findings may have relevance to the P-450 cytochromes in which suicide inactivation, destruction of the heme prosthetic group, and loss of the protein is observed. The activation of hemoproteins to heme-protein adducts may also have toxicological significance, perhaps in oxygen reperfusion injury in the myocardium as well as other tissues by enhancing the production of oxygen-derived radicals from molecular oxygen and lipid hydroperoxides. Clearly, further research in the characterization of heme-protein adducts is necessary before their importance in protein turnover and oxygen-induced injury can be determined.

Sequence of a Chicken Phenobarbital-Inducible Cytochrome P450 cDNA: Regulation of Two P450 mRNAs Transcribed from Different Genes

The nucleic acid and derived amino acid sequences of a chicken phenobarbital-inducible cytochrome P450 cDNA clone, pCHP7, are presented. The amino acid sequence shares 92% identity with that of a previously characterized chicken cytochrome P450 cDNA clone, pCHP3. The two clones, pCHP7 and pCHP3, represent two distinct mRNAs of 2.2 and 3.5 kb and appear to be transcribed from separate cytochrome P450 genes. Sequence comparisons with P450s from mammalian species indicate that the chicken P450s are most closely related to the IIC subfamily. According to the cytochrome P450 nomenclature (Nebert et al., 1989), the chicken P450 gene encoding the 3.5-kb mRNA is termed P450IIF1 and the P450 gene encoding the 2.2-kb mRNA is designated P450IIF2. When chick embryos were treated with 2-allyl-2-isopropylacetamide, the levels of the 3.5- and 2.2-kb P450 mRNAs in liver were elevated maximally by about 100-fold, while phenobarbital treatment resulted in a maximal increase of about 50-fold. These increases in mRNA levels were accompanied by a less than sixfold increase in the corresponding gene transcription rates. The results indicate that the increase in the amount of these two mRNAs is due to both transcriptional activation of the P450 genes and to a marked post-transcriptional mechanism. By contrast, the drug-induced hepatic mRNA levels for 5-aminolevulinate synthase, the rate-controlling enzyme of the heme biosynthetic pathway, could be accounted for predominantly by activation of gene transcription. A close correlation was observed between the time courses of hepatic mRNA accumulation for cytochrome P450 and 5-aminolevulilnate synthase following drug treatment of chick embryos. In adult hens it was demonstrated that a tissue-specific drug induction of cytochrome P450 mRNAs occurred with levels being substantially elevated in the liver, kidney, and small intestine. The mRNA for 5-aminolevulinate synthase was also drug-induced in the same tissue-specific fashion. The results are compatible with the gene for 5-aminolevulinate synthase being activated in response to an increased cytochrome P450 heme requirement.

The role of heme oxygenase and aryl hydrocarbon hydroxylase in the protection by cysteamine from acetaminophen hepatotoxicity

Administration of cysteamine to rats depressed hepatic aryl hydrocarbon hydroxylase (AHH) activity, cytochrome P-450, and total heme at 24 hr. Total heme remained decreased at 48 hr when all other parameters returned to control values. A significant 5-fold increase in heme oxygenase activity occurred in rat liver 5 hr after treatment, when AHH activity and total heme were unchanged. Histological examination of liver biopsies from rats treated with cysteamine revealed normal hepatic architecture. The observed effects of cysteamine on hepatic drug-metabolizing enzymes in vivo were not due to cysteamine-induced hepatotoxicity. Our results indicate that cysteamine increases heme oxygenase activity in rat liver, with a subsequent decrease in total heme, AHH activity, and cytochrome P-450 content. The depression of P-450 by cysteamine is likely to be an important mechanism for its protection in acetaminophen overdose. The protection studies illustrate this mechanism. Centrilobular hepatic necrosis and elevation in transaminase activity following a toxic dose of acetaminophen were prevented by treatment with cysteamine. The hepatoprotective effect of cysteamine was evident when acetaminophen was administered 24 hr after cysteamine but did not occur when acetaminophen was administered 5 hr after cysteamine or simultaneously. All groups of rats receiving cysteamine showed decreased mortality compared to the group receiving acetaminophen alone.