Inactivation of cytochrome P450 and inhibition of ferrochelatase by analogues of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine with 4-nonyl and 4-dodecyl substituents.

Cytochrome P450- and heme-destructive effects of the 4-nonyl and 4-dodecyl analogues of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC) were determined using hepatic microsomal preparations obtained from untreated, beta-naphthoflavone-treated, and phenobarbital-treated chick embryos. The 4-nonyl analogue of DDC was less efficacious than 4-ethyl DDC and 4-hexyl DDC, but more efficacious than 4-dodecyl DDC with respect to cytochrome P450-destructive activity. In all hepatic microsomal preparations, cytochrome P450 destruction by 4-nonyl DDC was accompanied by loss of microsomal heme. In contrast, 4-dodecyl DDC caused loss of heme only in hepatic microsomal preparations obtained from phenobarbital-treated chick embryos. The ability of 4-nonyl DDC and 4-dodecyl DDC to lower ferrochelatase activity was compared with that of 4-ethyl DDC and 4-hexyl DDC in cultured chick embryo hepatocytes. As the length of the 4-alkyl group was increased, the ferrochelatase-lowering efficacy and potency of the DDC analogue decreased. The 4-dodecyl DDC analogue was unable to lower ferrochelatase activity, which accorded with the finding that the administration of 4-dodecyl DDC to phenobarbital-treated rats did not lead to the accumulation of an N-alkylprotoporphyrin. The ability of 4-nonyl DDC to lower ferrochelatase activity was attributed to the formation of N-nonylprotoporphyrin IX following the administration of 4-nonyl DDC to phenobarbital-treated rats.     

Ferrochelatase and N-alkylated porphyrins

Summary The final step in heme synthesis is catalyzed by the mitochondrial enzyme, ferrochelatase. Characterization of this enzyme has been complicated by a number of factors including the dependence of enzyme activity on lipids. Purification of ferrochelatase from rat and bovine sources has been achieved only relatively recently using blue Sepharose CL-6B chromatography. When 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC) is given to animals, it produces a hepatic porphyria resembling human variegate porphyria thus providing an experimental system in which to study this disease. DDC has been found to cause the accumulation of a green pigment, identified as N-methyl protoporphyrin IX (N-MePP), which is a potent inhibitor of ferrochelatase. The source of the N-methyl substituent of N-MePP was found to be the 4-methyl group of DDC. Considerable evidence indicates that the protoporphyrin IX moiety of N-MePP originates from the heme moiety of cytochrome P-450 and that DDC is a suicide substrate for this hemoprotein. Some studies suggest that cytochrome P-450 isozymes differ in their susceptibility to destruction by DDC and its 4-alkyl analogues. Griseofulvin has also been reported to inhibit hepatic ferrochelatase in rodents but not in the 17-day old chick embryo nor in hepatocyte culture systems. Thus, the mechanism by which griseofulvin produces an experimental porphyria in chick embryo liver cell culture is different from that for rodents.     

Isolation of regioisomers of N-alkylprotoporphyrin IX from chick embryo liver after treatment with porphyrinogenic xenobiotics

Several porphyrinogenic xenobiotics cause mechanism-based inactivation of cytochrome P450 (P450) isozymes with concomitant formation of a mixture of four N-alkylprotoporphyrin IX (N-alkylPP) regioisomers, which have ferrochelatase inhibitory properties. To isolate the four regioisomers of N-methylprotoporphyrin IX (N-methylPP), 3,5-diethoxycarbonyl, 1-4-dihydro-2,4,6-trimethylpyridine (DDC) was administered to untreated, beta-naphthoflavone-, phenobarbital-, and glutethimide-pretreated 18-day-old chick embryos. Separation of the N-methylPP regioisomers by high pressure liquid chromatography (HPLC) revealed no marked difference in the regioisomer pattern among the different treatments. After administration of griseofulvin, allylisopropylacetamide (AIA), or 1-[4-(3-acetyl-2,4,6-triemethylphenyl)-2,6-cyclohexanedionyl]-O-ethyl propionaldehyde oxime (ATMP) to untreated and glutethimide-pretreated 18-day-old chick embryos, an N-alkylPP was isolated after AIA administration only. This finding strengthened previous reports of the species specificity of N-alkylPP formation with griseofulvin and ATMP. A series of dihydropyridines, namely 4-ethylDDC, 4-hexylDDC, and 4-isobutylDDC were administered to untreated and glutethimide-pretreated 18-day-old chick embryos and hepatic N-alkylPPs were isolated and separated by HPLC into regioisomers. The regioisomer patterns obtained did not support a previous proposal of masked regions above both rings B and C in the heme moieties of the P450 isozymes responsible for N-alkylPP formation. However, the data support the hypothesis of a partially masked region above ring B alone. The regioisomer patterns were in agreement with results previously obtained in rats showing that the percentage of Nc and (or) ND regioisomers in the regioisomer mixture increases as the length and bulk of the 4-alkyl substituent of a DDC analogue increase. Differences in the regioselectivity of heme N-alkylation may be due to intrinsic chemical features of DDC analogues themselves or to differences in the P450 isozymes inactivated.     

Comparison of the effects of 3-ethoxycarbonyl-1,4-dihydro-2,4-dimethylpyridine and 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine on ferrochelatase activity and heme biosynthesis in chick embryo liver cells in culture

3-Ethoxycarbonyl-1,4-dihydro-2,4-dimethylpyridine (EDP) was shown to lack the ferrochelatase-lowering activity of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC) in chick embryo liver cells in culture. This was attributed to the inability of EDP to cause destruction of the heme moiety of cytochrome P-450 with concomitant formation of N-methylprotoporphyrin IX. EDP was less potent as a porphyrinogenic agent than DDC and caused the accumulation of uroporphyrin, heptacarboxylic porphyrin, and coproporphyrin in contrast with DDC which caused primarily protoporphyrin to accumulate. The inactivity of EDP as a ferrochelatase-lowering agent and its low porphyrinogenic potency was explained, at least in part, by its rapid transformation in aqueous solution to other nondihydropyridine products. The two ethoxycarbonyl substituents of DDC are therefore essential for N-methylprotoporphyrin formation, ferrochelatase-lowering activity, and optimal porphyrin-inducing activity.     

Formation of N-alkylated protoporphyrin IX in the livers of mice after diethylnitrosamine treatment.

The administration of di[1-14C]ethylnitrosamine to phenobarbital-pretreated mice resulted in the formation of a radiolabelled green pigment in their livers. Green-pigment concentrations were time- and dose-dependent, maximum levels being reached 1-2 h after dosing. There was only a slight decrease in cytochrome P-450 levels and accumulation of porphyrins in the liver at this time. Dimethyl- or dipropyl-nitrosamine also caused an accumulation of similar, though not identical, compounds in the liver. The formation of green pigment was induced by pretreatment of mice with phenobarbital or 3-methylcholanthrene and was inhibited by the acute administration of pyrazole or ethanol. From the absorption spectra, the green pigment methyl esters appeared to be N-alkylporphyrins. Analysis of the diethylnitrosamine-induced green pigment by high-pressure liquid chromatography showed it to be more polar than the expected N-ethylprotoporphyrin IX, having a retention time similar to that of N-hydroxyethylprotoporphyrin IX. Desorption chemical-ionization mass spectrometry gave a protonated molecular ion, m/z 635, compatible with N-hydroxyethylprotoporphyrin IX. The presence of a free hydroxy group was demonstrated by acetylation with [1-14C]acetic anhydride. No conversion of N-ethylprotoporphyrin IX into N-hydroxyethylprotoporphyrin IX could be demonstrated in vivo or in vitro. Little or no N-ethylprotoporphyrin IX accumulated in the livers of mice given diethylnitrosamine. It was concluded that N-hydroxyethylprotoporphyrin IX is the primary reaction product between an active metabolite of diethylnitrosamine and hepatic haem.     

Identification of N-methylprotoporphyrin IX in livers of untreated mice and mice treated with 3, 5-diethoxycarbonyl- 1, 4-dihydrocollidine: source of the methyl group

Administration of the porphyrogenic agent, 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) to mice, leads to the accumulation of N-methylprotoporphyrin IX in liver. This porphyrin is a potent inhibitor of ferrochelatase activity and accounts for the porphyria produced after DDC administration. The N-methylprotoporphyrin IX extracted from DDC-treated mice is primarily of one isomeric form, as shown by nuclear magnetic resonance spectroscopy. The methyl group of N-methylprotoporphyrin IX isolated from DDC-treated mice is derived mostly from the 4-methyl group of DDC. The transfer of this methyl group and its subsequent covalent attachment to protoporphyrin IX may be mediated by a form of hepatic microsomal cytochrome P-450. N-Methylprotoporphyrin IX is also found in livers of untreated mice at levels that are low but significant.     

The formation of N-alkylprotoporphyrin IX and destruction of cytochrome P-450 in the liver of rats after treatment with 3,5-diethoxycarbonyl-1,4-dihydrocollidine and its 4-ethyl analog

The effects of two porphyrogenic agents, 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) and 3,5-diethoxycarbonyl-2,6-dimethyl-4-ethyl-1,4-dihydropyridine (DDEP), have been studied in rats. The administration of these compounds leads to the formation and accumulation in the liver of N-methylprotoporphyrin IX and N-ethylprotoporphyrin IX, respectively. In each case, the alkyl group of the porphyrin is derived from the 4-alkyl group of the porphyrogenic chemical. Each N-alkylporphyrin is a potent inhibitor of protoheme ferrolyase (EC 4.99.1.1) (ferrochelatase) activity. N-Methylprotoporphyrin IX is somewhat more potent than N-ethylprotoporphyrin IX as an inhibitor of ferrochelatase activity in vitro. However, more N-ethylprotoporphyrin IX accumulates in rat liver than does the N-methyl analog. Since alkylporphyrins are formed during the catabolism of heme (or hemoprotein), the effects of DDC and DDEP on hepatic microsomal cytochrome P-450 were also studied. Whereas DDC treatment led to only a slight decrease in cytochrome P-450 levels (25%), DDEP administration led to a marked decrease (75%) in the total cytochrome P-450 level. In phenobarbital- and 3-methylcholanthrene-treated rats, DDC administration did not alter the hepatic microsomal cytochrome P-450 content, while administration of DDEP to either phenobarbital-treated or 3-methylcholanthrene-treated rats led to marked reduction of levels in cytochrome P-450. Although the N-methylprotoporphyrin IX level was not increased following DDC administration to either phenobarbital- or 3-methylcholanthrene-treated rats, there was a marked increase in N-ethylprotoporphyrin IX accumulation in both phenobarbital- and 3-methylcholanthrene-treated rats after the administration of DDEP. These results suggest that DDC and DDEP react with different forms of rat hepatic microsomal cytochrome P-450.     

The 1986 Upjohn award lecture. Interaction of chemicals with hemoproteins: implications for the mechanism of action of porphyrinogenic drugs and nitroglycerin

The ferrochelatase inhibitory activity of a variety of analogues of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC) was studied in chick embryo liver cells. The ferrochelatase inhibitory activity of the 4-butyl, 4-pentyl, and 4-hexyl analogues was considered to be due to catalytic activation by cytochrome P-450 leading to heme alkylation and formation of the corresponding N-alkylporphyrins. The relative ferrochelatase inhibitory activity of the DDC analogues has implications for a postulated model of the binding of porphyrins in the ferrochelatase active site. 3-[2-(2,4,6-Trimethylphenyl)thioethyl]-4-methylsydnone (TTMS) was shown to be a potent porphyrinogenic agent and to inhibit ferrochelatase in chick embryo liver cells. A related sydnone, 3-benzyl-4-phenylsydnone did not inhibit ferrochelatase activity. These results supported the idea that the porphyrinogenicity of TTMS was due to catalytic activation by cytochrome P-450 leading to heme alkylation and formation of N-vinylprotoporphyrin which inhibits ferrochelatase. Polychlorinated biphenyls, phenobarbital, nifedipine, and a large number of structurally different chemicals which are porphyrinogenic in chick embryo liver cells inhibit uroporphyrinogen decarboxylase by an unknown mechanism. Thus drug-induced porphyrin biosynthesis in chick embryo liver cell culture appears to be caused by inhibition of either ferrochelatase or uroporphyrinogen decarboxylase. The biotransformation of nitroglycerin by human red blood cells is due to a combination of a sulfhydryl-dependent enzymatic process and an interaction with reduced hemoglobin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protoporphyrin ix as a possible ancient photosensitizer: Spectral and photochemical studies

Due to the potential special position of protoporphyrin IX in the evolution of photosynthesis, the absorption and fluorescence characteristics of this pigment and its complexes with human serum albumin (HSA) and basic proteinoid have been studied in parallel with their photochemical activity. The most significant change in the absorption spectrum of PP IX was the appearance of a new maximum at 455 (or 461) nm in the presence of HSA or proteinoid respectively. Some changes in the physicochemical properties of PP IX in different microenvironments have been detected by changes in fluorescence emission and excitation spectra (intensity, quantum yields, position of maxima). The increase of fluorescence quantum yield resulting from the formation of PP IX complexes with HSA or proteinoid correlates with the increase of their photochemical activity. Results obtained are discussed from the point of view of the early evolution of the photosynthetic apparatus.     

Synthesis, chromatographic purification, and analysis of isomers of biliverdin IX and bilirubin IX.

Neutral solvent systems were developed to isolate the alpha, beta, gamma, and delta isomers of biliverdin IX dimethyl ester by TLC. The individual free acids of biliverdin IX were obtained by saponification of the corresponding dimethyl esters. The bilirubin IX isomers were prepared by reducing the corresponding biliverdin IX isomers with NaBH3CN. Starting from a pure biliverdin IX dimethyl ester, the corresponding free acid of biliverdin IX or bilirubin IX was available within 3-4 h. Preparation of spectrally pure bile pigment required final TLC on acid-cleaned neutral TLC plates. The absorption spectra of the free acids and dimethyl esters of biliverdin IX in methanol showed a broad band at about 650 nm and a sharp band at about 375 nm. The long-wave-length band was extremely sensitive to the presence of strong acid. A 10-fold molar excess of HCl caused a 35- to 50-nm shift of the absorption maximum to longer wavelengths and near doubling of the maximum absorption. The molar absorption coefficients of biliverdins were identical for each free acid and dimethyl ester pair. In each case, Beer's law was followed in both methanol and acidified methanol. Methanol also proved to be a suitable solvent for spectroscopic determination of the non-alpha isomers of bilirubin IX. The wavelength of maximum absorption and molar absorption coefficient of each dipyrrolic ethyl anthranilate azo pigment derived from the various bilirubin IX isomers are also reported.     

Inhibition of ferrochelatase by N-methylprotoporphyrin IX is not accompanied by delta-aminolevulinic acid synthetase induction in chick embryo liver cell culture

N-Methylprotoporphyrin has been shown to markedly inhibit ferrochelatase activity in chick embryo liver cell culture without inducing delta-aminolevulinic acid (ALA) synthetase activity. This result supports the idea that the effects of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC) on ALA synthetase activity and ferrochelatase activity are dissociated and that inhibition of ferrochelatase alone is not sufficient to cause induction of ALA synthetase. We conclude that the porphyrinogenic activity of DDC can be explained only in part by the actions of N-methylprotoporphyrin.     

Structure of green pigment formed by the reaction of caffeic acid esters (or chlorogenic acid) with a primary amino compound.

A marked greening observed in some foods such as sweet potato, burdock, and others during food processing was shown to be due to green pigment formation by the condensation reaction of two molecules of chlorogenic acid or caffeic acid ester with one molecule of a primary amino compound under aeration in alkaline solution. Reduction of the green pigment by ascorbic acid or NaBH4 gave a yellow product, which readily turn green and then blue in air. The reduced and acetylated product of the green pigment was identified to be a novel trihydroxy benzacridine derivative, and the yellowish ethanol solution of this product immediately turned green upon addition of butyl amine or diluted alkali. Therefore, the green pigment was assumed to be an oxidized quinone type product of trihydroxy benzacridine. This identification of the structure was supported by the correspondence of the measured absorption spectra with those calculated by the molecular orbital method. A possible charge transfer complex between products of different oxidation steps in green solution was proposed.     

N-Alkylprotoporphyrin IX formation in 3,5-dicarbethoxy-1,4-dihydrocollidine-treated rats. Transfer of the alkyl group from the substrate to the porphyrin

Administration of 3,5-dicarbethoxy-1,4-dihydrocollidine (DDC) to rats causes the accumulation of N-methylprotoporphyrin IX, a potent inhibitor of ferrochelatase. To clarify the origin of the porphyrin N-methyl group, we have synthesized and administered to rats N-ethyl-3,5-dicarbethoxy-1,4-dihydrocollidine (N-ethyl DDC) and 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine (DDEP), the DDC analogue with a 4-ethyl rather than 4-methyl group. Only N-methylprotoporphyrin IX is isolated from rats treated with the former agent, and only N-ethylprotoporphyrin IX from those treated with the latter. All four isomers of N-ethylprotoporphyrin IX are formed biologically. The structure of the isolated porphyrins has been confirmed by complete spectroscopic comparison with the four synthetic isomers of N-ethylprotoporphyrin IX. DDEP has been shown to cause NADPH- and time-dependent in vitro loss of hepatic microsomal cytochrome P-450. These results unequivocally establish that the 4-alkyl groups in DDC and dDEP are the source of the N-alkyl group in N-methyl- and N-ethylprotoporphyrin IX, respectively, and strongly suggest that the alkyl group is transferred to the prosthetic heme of cytochrome P-450 during catalytic processing of the substrate by the enzyme. The mechanism of the group transfer is discussed.     

Superoxide anion as a mediator of drug-induced oxidative hemolysis.

The superoxide dismutase inhibitor diethyldithiocarbamate (DDC) was utilized to study the toxic effect of 1,4-naphthoquinone 2-sulfonate (NQ), a structural analog of the hemolytic drug, menadione, on red cells. NQ was shown to react with hemoglobin and result in the generation of superoxide anion (O2-). Red cells treated with NQ were found to undergo a gradual disappearance of their oxyhemoglobin and also hemolyze. Red cells pretreated with DDC to inhibit cellular superoxide dismutase were found to be markedly sensitive to oxyhemoglobin destruction and hemolysis in the presence of NQ. Superoxide dismutase-inhibited red cells were also found to undergo a slow autohemolysis in the absence of NQ. No evidence for lipid peroxidation was obtained for red cells treated with NQ either in the presence or the absence of DDC. Ghosts prepared from superoxide dismutase-inhibited red cells exposed to NQ were found to retain a green hemoglobin-derived pigment.     

Fluorescence spectroscopy study of protoporphyrin IX metabolism level in cells.

Fluorescence spectroscopy was used to study the protoporphyrin IX (PpIX) metabolism level in chick embryos during the cell proliferation process. The emission spectra were measured for PpIX bound to albumin from nonincubated and incubated eggs. The relative characteristic emission intensity of PpIX was used to determine the level of PpIX metabolism as a function of the embryonic development time. This technique might be used to estimate tumor development time.     

Structural requirements in dihydropyridines for ferrochelatase inhibition and δ-aminolevulinic acid synthetase induction

• 1.1. A series of analogues of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC) was investigated for hepatic δ-aminolevulinic acid (ALA)-synthetase inducing and ferrochelatase-inhibiting activity in the 17-day-old chick embryo.
• 2.2. A DDC analogue was found which was capable of inducing ALA-synthetase activity without inhibiting ferrochelatase activity.
• 3.3. On the other hand we were unable to find a DDC analogue with the ability to inhibit ferrochelatase activity which was devoid of ALA-synthetase-inducing activity.

     

Reduced cellular immune competence of a temperature-sensitive dopa decarboxylase mutant strain of Drosophila melanogaster against the parasite Leptopilina boulardi.

1. The melanotic encapsulation response made by larvae of a temperature-sensitive dopa decarboxylase (DDC) mutant strain of Drosophila against the parasitic wasp Leptopilina was severely compromised in hosts with reduced levels of DDC. 2. Dopa and 5,6-dihydroxyindole (DHI) were two hemolymph components identified in hosts exhibiting a melanotic encapsulation response. 3. This is the first study to implicate DDC in insect cellular immune responses, and to provide chemical evidence that the pigment formed during such responses is eumelanin derived from tyrosine.     

Porphyrinogenic activity and ferrochelatase-inhibitory activity of sydnones in chick embryo liver cells

3-[2-(2,4,6-Trimethylphenyl)thioethyl]-4-methylsydnone was shown to be a potent porphyrinogenic agent in chick embryo liver cells. The accumulation of protoporphyrin IX was consistent with the finding that ferrochelatase activity was inhibited. 3-Benzyl-4-phenylsydnone did not inhibit ferrochelatase activity and protoporphyrin IX was found to constitute only a minor fraction of the prophyrins. These results support the idea that the porphyrinogenicity of 3-[2-(2,4,6-trimethylphenyl)thioethyl]-4-methylsydnone is due to its catalytic activation by cytochrome P-450 leading to heme alkylation and formation of N-vinylprotoprophyrin IX which inhibits ferrochelatase.     

Isolation of N-phenylprotoporphyrin IX from the red cells and spleen of the phenylhydrazine-treated rat

Blood and spleens of phenylhydrazine-injected rats were treated with a solution of acidic methanol and zinc ion to isolate a green pigment. The pigment was resolved into two, I and II, by thin-layer chromatography. Pigment I was a mixture of two isomers of zinc complex of esterified N-phenylprotoporphyrin IX, in which vinyl-substituted pyrrole rings A and B were phenylated; and pigment II was a mixture of two isomers of the porphyrin complex with the N-phenyl group on propionic acid-substituted rings C and D. These pigments were also chemically prepared from the reaction of phenylhydrazine with oxyhemoglobin, independently characterized, and used to confirm the structures of the biological pigments. Determination revealed that the total amount of pigments found in the blood and spleen at 24 h after injection of phenylhydrazine corresponds to about 0.4% of the injected phenylhydrazine.     

Rational design of a protein-based molecular device consisting of blue fluorescent protein and zinc protoporphyrin IX incorporated into a cytochrome b 562 scaffold

To make a single molecular photo-device, it is essential to control the exact orientation of two types of proteins. We made a chimeric protein in which cytochrome b562 was linked to the N-terminus of enhanced green fluorescent protein, cytb562-EGFP. Within cytb562-EGFP, the excitation energy of EGFP was transferred to the cytochrome b562 cofactor fixed proximally to EGFP. Cytb562-EGFP was engineered so that iron protoporphyrin IX was substituted by zinc protoporphyrin IX to make it a suitable cofactor for photo-induced electron transfer. The photosensitizer pigment was optimized and the EGFP was replaced by a blue fluorescent mutant that gave 15% higher energy transfer efficiency.