N-alkylated Porphyrins Research Articles

Labelling in vivo and chirality of griseofulvin-derived N-alkylated protoporphyrins

1. We have compared the response to griseofulvin of rats and mice and, in mice, the effect of griseofulvin itself with that of two of its analogues. The severity of protoporphyria shows a correlation with the accumulation of both types of N-alkylated porphyrins previously described after treatment with this drug, namely N-methylproptoporphyrin and the N-griseofulvin protoporphyrin adduct. 2. Both N-alkylporphyrins are chiral, are labelled from 5-amino[4-14C]laevulinate, and their liver accumulation can be inhibited by pretreatment with a suicide substrate of cytochrome P-450, which also prevents porphyria. 3. These findings suggest that cytochrome P-450 is involved in the mechanism of griseofulvin-induced protoporphyria by generating N-methylprotoporphyrin. The N-griseofulvin protoporphyrin adduct may also originate from cytochrome P-450, but more work is necessary to elucidate whether it acts as the precursor for N-methylprotoporphyrin.

Regioselectivity of metabolic activation of acetylenic steroids by hepatic cytochrome P450 isozymes

Liver cytochrome P450 monooxygenases (P450), a group of isozymes that catalyze the reductive cleavage of molecular oxygen, dominate hepatic metabolism of xenobiotic lipophilic substances. TheseP450 enzymes exhibit broad and overlapping substrate specificities, in contrast to the P450 isozymes of the steroid biosynthetic pathways, which are highly substrate specific. Hepatic heme pigments, Nalkylated porphyrins, accumulate following the self-catalyzed destruction of P450 by the metabolic activation of 17α-ethynyl steroids. Acetylenic substituted steroidal aromatase inactivators, norethisterone (NET), and 10-(2-propynyl)estr-4-ene-3,17-dione (MDL 18,962) were administered to rats to deter- mine if the acetylenic substituent was activated hy hepatic P450 mixed-function oxidases. This metabo- lism could result in the formation of a reactive species that would alkylate a pyrrole nitrogen atom of heme. Male Sprague-Dawley rats were treated with 0, 10, 30, or 100 mg/kg NET or MDL 18,962 intraperitoneally. Four hours later, these animals received 40 mg/kg sodium pentobarbital and their sleeping times were recorded. On arousal, the rats were killed and their livers were taken for determina- tion of P450 content and formation of N-alkylated porphyrins (green pigments). Norethisterone inhibited hepatic P450 isozymes, resulting in a dose-related increased sleeping time (89.2 ± 3.5 to 156.3 ± 7.6 minutes) and decreased P450 levels (maximum 25% decrease at 100 mg/kg), and the amount of green pigments increased with doses of 10 to 100 mg/kg. In contrast, MDL 18,962 treatment did not increase sleeping time and caused only a 15% decrease in hepatic P450 content at 100 mg/kg, with no detectable green pigments. These studies suggest that regioselectivity of hepatic P450 isozymes account for their activation of the C-17 acetylenic group and their insensitivity toward the C-19 acetylenic function.

Copper-induced dealkylation studies of biologically N-alkylated porphyrins by fast atom bombardment mass spectrometry

Copper-induced dealkylation studies of biologically N-alkylated porphyrins by fast atom bombardment mass spectrometry

Copper-induced dealkylation studies of biologically N-alkylated porphyrins by fast atom bombardment mass spectrometry

Copper has been reported to promote the demethylation of N-methylporphyrins. Copper-induced dealkylation is observed at room temperature in acetonitrile in the presence of a suitable nucleophilic acceptor molecule, or in chloroform alone. Electronic absorption spectra of N-methylprotoporphyrin IX (in the form of its dimethyl ester) in the presence of copper (with either chloroform alone or acetonitrile-dibutylamine as solvent) exhibited a transient spectrum corresponding to a copper chelate of the N-methylprotoporphyrin followed by ejection of the N-methyl group to afford the copper chelate of protoporphyrin. The use of fast atom bombardment mass spectrometry (FAB-MS) to identify the N-alkyl group of a series of N-substituted porphyrins was investigated. The alkyl group released on reaction with copper ions was trapped by an amine (e.g., dodecylamine). The resulting mixture was analysed by positive-ion FAB-MS using 3-nitrobenzyl alcohol as matrix. Molecular ions (MH +) were detected at m/z 200, 214 and 228, corresponding to [CH 3(CH 2) 11NHCH 3 + H] +, [CH 3(CH 2) 11NHC 2H 5 + H] + and [CH 3(CH 2) 11NHC 3H 7 + H] +, respectively. The analysis was extended to N-alkylprotoporphyrins isolated from liver homogenates of mice treated with the porphyria-inducing drug griseofulvin. Using a combination of UV and copper-induced dealkylation studies in FAB-MS, the porphyria-producing pigment was identified as N-methylprotoporphyrin IX.

Factors responsible for the formation of different N-alkylated porphyrins in rat liver microsomal systems exposed to norethindrone. The role of 3 alpha-hydroxysteroid dehydrogenase.

Incubation of rat liver microsomes with norethindrone and a NADPH-generating system leads to the formation of one N-alkylated porphyrin (green pigment, GP1). Administration of this steroid to male rats in vivo results in the formation of three more-polar green pigments (GP2, 3 and 4). A cytosolic protein (green-pigment converting protein) has been purified from rat liver that, when added to liver microsomal mixtures containing norethindrone (0.03 mM) and a NADPH-generating system, results in the formation of all four green pigments (GP1, 2, 3 and 4). Field-desorption mass spectrometry of the purified green pigments gave protonated molecules, [M + H]+, at m/z 905 for GP1, m/z 909 for GP2, m/z 925 for GP3 and 4. The Mr of the purified cytosolic protein on SDS/polyacrylamide-gel electrophoresis or gel filtration was 37000. Polyacrylamide-gel isoelectric focusing gave a pI value of 5.9. Antibodies raised in rabbits against this protein, after preincubation with rat liver cytosol, subsequently prevented the formation of the more-polar norethindrone-induced green pigments (GP2, 3 and 4). The purified protein in the presence of either NADH or NADPH catalysed the reduction of delta 4-ring-reduced norethindrone, 5 alpha-oestran-17 alpha-ethynyl-17 beta-ol-3-one and, with the appropriate cofactor, the oxidation and reduction of steroids lacking the ethynyl function, e.g. androsterone or dihydrotestosterone. Indomethacin inhibited the reduction of dihydrotestosterone by this protein with an I50 (concn. causing 50% inhibition) value of 4.9 microM. From its physical and enzymic properties it is concluded that green-pigment converting protein is the same as 3 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.50).

Destruction of cytochrome P-450 and formation of green pigments by contraceptive steroids in rat hepatocyte suspensions

The contraceptive steroid norethindrone caused a rapid time and dose-dependent loss of cytochrorne P-450 from rat hepatocytes in suspension cultures. Up to 30% of this cytochrome was lost in the first 5 min of incubation; longer incubations resulted in little further loss even though not all the steroid was metabolised and the cells remained viable. Such cultures were used to investigate the formation of N-alkylated porphyrins (green pigments) which could be extracted from cell incubation mixtures following exposure to norethindrone and separation by HPLC or TLC. The number of N-alkylated porphyrins formed was dependent both on the time of incubation and the concentration of steroid. After 1 min, 1 major green pigment (GP1) was resolved using either high (0.3 mM) or low (0.03 mM) norethindrone concentrations. With longer incubation times (60 min), at high steroid concentrations, only one additional polar adduct (GP2) was formed. At lower steroid levels, 3 more polar components (GP2, 3 and 4) were seen. As judged by HPLC or TLC, GP1 corresponds to the pigment formed in microsomal preparations incubated with norethindrone in vitro, while GP2, 3 and 4 correspond to the pigments extracted from the livers of rats administered this steroid in vivo. Pretreatment of rats with either phenobarbitone or 3-methylcholanthrene induced cytochrome P-450s. Relative to controls, phenobarbitone pretreatment also resulted in a greater accumulation of green pigments in hepatocytes incubated with norethindrone, the more polar forms of green pigments (GP3 and 4), showing a disproportionate increase in concentration. The mixed function oxidase inhibitor SKF 525-A or high concentrations of steroid not containing an ethynyl function, e.g. norethandrolone, when added to cell cultures containing norethindrone, preferentially inhibited the formation of GP3 and 4. When purified green pigments were added to cell incubation mixtures in the absence of norethindrone, no interconversion of one form to another could be demonstrated. The results suggest that the more polar norethindrone-protoporphyrin IX adducts (GP2, 3 and 4) arise as a result of metabolic modification of norethindrone rather than the protoporphyrin IX moiety, either prior to or after activation of the ethynyl function. The formation of several green pigment components in hepatocyte suspensions was not unique to norethindrone but occurred with a number of other 17-ethynyl-substituted steroids.

Inactivation of cytochrome p-450 by 2-isopropyl-4- pentenamide and other xenobiotics leads to heme-derived protein adducts

When cytochrome P-450 in phenobarbital-induced rat liver microsomes was destroyed by 2-isopropyl-4-pentenamide (AIA) in vitro, 50% of the degraded heme was recovered as heme-derived products irreversibly bound to microsomal proteins. In contrast, less than 50% of the degraded heme was accounted for as N-alkylated porphyrins. Furthermore, 64% of the irreversibly bound products was bound specifically to a 54-kD form of cytochrome P-450. Several other compounds which have been reported to destroy cytochrome P-450 by forming N-alkylated porphyrins also produced heme-derived protein adducts. These findings indicate that the formation of heme-derived protein adducts may represent an important pathway for the irreversible degradation of cytochrome P-450 by many xenobiotics.

Studies on the inhibition of ferrochelatase by N-alkylated dicarboxylic porphyrins. Steric factors involved and evidence that the inhibition is reversible.

The structural requirements for the inhibition of ferrochelatase by N-alkylated porphyrins were investigated and experiments carried out to explore the mechanism of enzyme inhibition. Three dicarboxylic porphyrins, all substrates of the enzyme, are strongly inhibitory when N-alkylated; in contrast, uroporphyrin and coproporphyrin (which are not substrates) do not inhibit after N-alkylation. Free carboxylic acid functions are required for inhibition, as the methyl ester derivatives are not themselves inhibitory. Porphyrins bearing the alkyl group on the pyrrole nitrogen of rings C and D are less effective inhibitors, particularly when zinc is chelated in the centre of the tetrapyrrole or the N-alkyl group is relatively large in size. The substituents at the 2- and 4-positions of the porphyrin system may also affect the inhibitory activity, particularly for the isomers with ring C and D alkylated. The zinc chelates of several N-alkylprotoporphyrins are inhibitory towards haem oxygenase, another haem-binding enzyme, and also in this case increasing the size of the alkyl group decreased the inhibitory activity, particularly for isomers with ring C or D alkylated. The inhibition could be reversed by prolonged incubation with excess porphyrin substrate, but dealkylation of the N-alkylporphyrin during enzyme inhibition could not be demonstrated. It is concluded (a) that N-alkylated dicarboxylic porphyrins compete reversibly with the porphyrin substrate for the enzyme active site and (b) that the structural and steric factors discussed above affect the inhibitory activity by modifying the affinity of the N-alkylporphyrin inhibitor for the enzyme.

Suicidal destruction of cytochrome p-450 by ethynyl substituted compounds.

Compounds containing a terminal carbon-carbon triple bond, ranging in structure from the 17α-ethynyl substituted contraceptive steroids to acetylene gas, when administered to rats cause a selective and rapid time dependent loss of up to 50% of hepatic cytochrome P-450. Cytochrome b5 is not affected. Metabolic activation of the acetylenic substituent by the phenobarbital inducible, NADPH dependent, mixed function oxidases results in the formation of a reactive species which alkylates one of the tetrapyrrole nitrogen atoms of heme to form a 1 : 1 covalent adduct. Either cytochrome P-450 destruction or the formation of N-alkylated porphyrins (green pigments) have been used to assess factors affecting the extent of metabolic activation of the acetylenic group in rats, man, and other laboratory species. The chemical identity of a number of green pigments have been resolved. Those formed from 1-octyne consist of the protoporphyrin IX ring of heme substituted with the saturated 2-oxo-octyl group. In contrast, reactive metabolites of 1-octyne trapped with N-acetylcysteine contain the unsaturated 3-oxo-octenyl substituent. Two independent routes of activation of terminal acetylenes have been described to account for these results. Both pathways can lead to cytochrome P-450 loss but only one, probably involving an oxirene intermediate, leads to green pigment formation.

Inactivation of cytochrome P-450 and production of N-alkylated porphyrins caused in isolated hepatocytes by substituted dihydropyridines : Structural requirements for loss of haem and alkylation of the pyrrole nitrogen atom

Drugs with unsaturated side chains and certain dihydropyridines both convert liver haem into Nalkylated porphyrins, but the underlying mechanisms differ in the two cases. With unsaturated drugs, for example 2-allyl-2-isopropylacetamide (AIA), a monooxygenated derivative of the drug becomes bound [l] onto one of the pyrrole nitrogen atoms [2-41 of liver haem; whereas with 3,5diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC) only the 4-methyl substituent of the drug is transferred onto the pyrrole nitrogen atom [5-71 and the product, N-methyl protoporphyrin, is a powerful inhibitor of ferrochelatase (EC 4.99.1.1) [8,9]. There is strong evidence that the N-alkylated porphyrins produced by unsaturated compounds all originate from the haem of cytochrome P-450 [ 10-121, but it is less clear from which pool of hepatic haem N-methyl protoporphyrin originates after treatment with DDC; and the detailed mechanism of the transmethylation reaction involved has not yet been elucidated. The aim of the experiments described in this paper has been to clarify wether N-methyl protoporphyrin originates from the haem of cytochrome P450 and whether exogenous haem can be utilized

Liver production of N-alkylated porphyrins caused in mice by treatment with substituted dihydropyridines : Evidence that the alkyl group on the pyrrole nitrogen atom originates from the drug

The porphyrogenic drug, 3,Sdiethoxycarbonyl1,4-dihydro-2,4,6_trimethylpyridine causes a marked inhibition of the enzyme protohaem ferro-lyase (EC 4.99.1 .l) in the liver of rats, mice and chick embryos [l-4] an effect which is thought to be responsible for the very pronounced accumulation of protoporphyrin seen in this type of experimental porphyria. We have isolated a potent inhibitor of protohaem ferro-lyase from the liver of mice made porphyric by treatment with this drug [5,6] and have identified the inhibitor as N-methyl protoporphyrin [7]. Inhibition of protohaem ferro-lyase has also been obtained both in vivo and in vitro with synthetic N-alkylated porphyrins [7-91, and the size of the alkyl group present on the pyrrole nitrogen atom has been shown to be important for the inhibitory effect, N-ethylmesoporphyrin being less active than N-methylmesoporphyrin [8]. Isotopic experiments have suggested that the N-methylated protoporphyrin produced by treatment with 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine originates from liver haem [5,6,10], but the source of the methyl group bound onto the pyrrole nitrogen atom has not yet been determined. The following findings have raised the possibility that the methyl group may originate from the 4-methyl substituent of the drug: under relatively mild chemical conditions certain dihydropyridines lose their 4-alkyl substituent on oxidation and that this alkyl group can be donated to suitable nucleophiles [ 111. A series of dihydropyridine analogues have been compared for their ability to inhibit liver protohaem ferro-lyase

Substrate-dependent irreversible inactivation of cytochrome P-450: conversion of its haem moiety into modified porphyrins.

2-Allyl-2-isopropylacetamide and other drugs containing either an allyl, a vinyl or an ethynyl unsaturated side chain are metabolized by liver cytochrome P-450 to reactive derivatives that irreversibly inhibit the haemoprotein by a suicidal type of inactivation. The main target is the haem moiety of cytochrome P-450 which is converted into abnormal porphyrins. These have been isolated from the liver of treated rats, extensively purified and compared with model porphyrins. The abnormal porphyrins incorporate metal ions in vitro much more readily than does their parent porphyrin, protoporphyrin. They are also much more basic than protoporphyrin, and on titration with a strong acid they readily give rise to a porphyrin monocation which then requires relatively large amounts of acid for conversion to the porphyrin dication. In all these respects and also in the intensity of their bathochromic shifts these abnormal porphyrins closely resemble N-alkylated porphyrins and they markedly differ from porphyrins that are substituted at one of their meso-carbon positions or which bear electron-withdrawing substituents at the beta-positions of the pyrrole rings. This suggests strongly that reactive derivatives of the unsaturated drugs act as electrophilic reagents and alkylate one of the pyrrole nitrogen atoms of cytochrome P-450. A model centred on the apoprotein of cytochrome P-450 is considered for the degradation of liver haem caused by unsaturated drugs. The apocytochrome may accept exchangeable pools of liver haem for degradation, leading to a state of haem depletion and to activation of delta-aminolaevulinate synthase.