Anthony H. Gibbs

Loss of haem and haemoproteins during the generation of superoxide anion and hydrogen peroxide: A pathway not involving production of carbon monoxide

1. n1. Haem is destroyed when directly incubated with hydrogen peroxide or with Superoxide anion-producing systems, like xanthine oxidase and dihydroxyfumaric acid. n n2. n2. With the last two oxidizing systems H2O2 is also probably responsible for the destruction of haem, as shown by the protective effect afforded by catalase. n n3. n3. Haemoprotein haem (as present in either haemoglobin or in myoglobin) and cobaltic protoporphyrin are relatively resistant to oxidation. n n4. n4. The haem degradation caused by H2O2 differs from the degradation catalysed by the haem oxygenase system in the nature of the products formed and in the sensitivity to inhibitors like sodium azide and potassium cyanide. n n5. n5. After the action of dihydroxyfumaric acid on haemoglobin, the spectral characteristics of a product suggest a modified haem where either β-substituents of the pyrrole rings or, more probably, the bridge meso-carbon atom has been modified.

Formation of cobalt protoporphyrin by chicken hepatocytes in culture: Relationship to decrease of 5-aminolaevulinate synthase caused by cobalt

Cobalt protoporphyrin generated from 5-amino[4-14C]laevulinate by homogenates or primary cultures of chick embryo liver exposed to CoCl2 was found to be radioactivity unextractable by acid/acetone, when extra protein was added. The activity of ferrochelatase was required for formation of cobalt protoporphyrin since inhibition of ferrochelatase with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (in the presence of cycloheximide) inhibited formation of cobalt protoporphyrin and resulted in accumulation of protoporphyrin. Cobalt protoporphyrin was detected spectrophotometrically in hepatocyte cultures exposed to the combination of 2-allyl-2-isopropylacetamide and CoCl2: (1) as the pyridine haemochrome of the protein pellet remaining after acid-acetone extraction of the cells, or (2) as the material extracted from the protein pellet with acetic acid-pyridine-chloroform. The amount of cobalt protoporphyrin increased with increasing CoCl2 concentration as cellular haem declined. The decrease in haem was about equal to the amount of cobalt protoporphyrin that accumulated. 2-Allyl-2-isopropylacetamide and polychlorinated biphenyls were both powerful inducers of 5-aminolaevulinate synthase. The former led to protoporphyrin accumulation, whereas with the latter, uroporphyrin accumulated, probably due to a concomitant decrease in activity of uroporphyrinogen decarboxylase. The decrease in activity of 5-aminolaevulinate synthase produced by administration of CoCl2 was greater after treatment with 2-allyl-2-isopropylacetamide than after treatment with allylisopropylacetamide and 3,4,3,4-tetrachlorobiphenyl. We conclude: (a) that cobalt protoporphyrin is readily formed in cultured hepatocytes, and (b) that its formation accounts for the action of cobalt on 5-aminolaevulinate synthase.

Two pathways of iron-catalyzed oxidation of bilirubin: Effect of desferrioxamine and trolox, and comparison with microsomal oxidation☆

The bilirubin-degrading activity of liver microsomes from rats induced with 3-methylcholanthrene has been shown to be markedly stimulated by addition of 3,3,4,4,5,5-hexabromobiphenyl, a polyhalogenated chemical which resembles in size and shape the most effective inducers of cytochrome P450IA1, but lacks the structural features necessary for it to be metabolised. The degradation of bilirubin by this microsomal system has been compared to oxidation by a chemical model system involving H2O2 and Fe-EDTA (ethylenediaminetetraacetic acid). In both systems bilirubin disappearance was accompanied by bleaching. However, when either desferrioxamine or Trolox were present in the chemical model system, the rate of bilirubin oxidation was greatly enhanced and, at the same time, bilirubin was largely or entirely converted to biliverdin, a pathway of oxidation which proceeds by dehydrogenation. In the presence of desferrioxamine, biliverdin was also further oxidised to an unidentified red pigment.

Studies on the mechanism of experimental porphyria and ferrochelatase inhibition produced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine

Abstract 1. 1. The porphyrogenic agent, 3,5-diethoxycarbonyl-l,4-dihydrocollidine (DDC) produces a marked inhibition of hepatic ferrochelatase activity when injected in vivo . It has no effect in vitro . 2. 2. Following the administration of DDC to mice or rats, an inhibitor of ferrochelatase activity has been isolated from livers and resolved and purified by LH-20 column chromatography. Mice which are more sensitive to DDC produce more inhibitor substance than rats and control livers possess small but measurable quantities of the inhibitor substance. 3. 3. The inhibitor has porphyrin-like properties with characteristic visible and fluorescence spectra and can be separated from protoporphyrin IX. When [ 14 C]aminolevulinic acid was injected 2 hr prior to DDC, the purified inhibitor substance contained high radioactivity. 4. 4. Cycloheximide pretreatment of animals did not affect the amount of inhibitor formed, although this treatment is known to markedly reduce δ-aminolevulinic acid synthetase activity and the porphyria produced by DDC. 5. 5. These results indicate that the formation of an inhibitor after the administration of DDC is due to the catabolism of hepatic heme to a substance with porphyrin-like properties and with profound inhibitory characteristics toward ferrochelatase activity.

Metabolic activation of halothane to neoantigens in C57B1/10 mice: immunochemical studies

C57Bl/10 mice were given halothane (10 mmol/kg, intraperitoneally) and microsomal proteins were analysed for the presence of trifluoroacetylated (TFA) neoantigens by SDS-gel electrophoresis followed by immunoblotting using a polyclonal anti-TFA antibody. In microsomal preparations from liver, lung and olfactory tissues, a 54 kDa TFA adduct was detectable 1 h after dosing. After 3-48 h, multiple bands were detected in liver (45-100 kDa) and in the lung (26-57 kDa) and in one experiment in which [14C]halothane was given, several immunoreactive bands from liver microsomes were shown to contain a covalently bound metabolite of the drug. In olfactory tissue, initially (1 h), a major band of 54 kDa and a less prominent component of about 50 kDa were seen. The number of bands increased at later times but the additional bands were far fewer than in liver. The rate of decay of the 54 kDa adduct was also measured in both liver and olfactory microsomes and found to be compatible with the reported turnover of total liver cytochrome P-450. 24 h after treating mice with halothane (10 mmol/kg), no TFA neoantigens could be detected on the outer cell surface of isolated viable hepatocytes when analysed by fluorescence activated flow cytometry. In contrast, non-viable cells, or those fixed in acetone were all positive. Using immunohistochemistry, TFA neoantigens were demonstrated in the centrilobular area of the liver, the non-ciliated bronchiolar epithelial (Clara) cells of the lung, proximal tubular cells of the kidney and the respiratory and olfactory epithelium of nasal tissues.

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

Abstract 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 ) 11 NHCH 3 + H] + , [CH 3 (CH 2 ) 11 NHC 2 H 5 + H] + and [CH 3 (CH 2 ) 11 NHC 3 H 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.

High-performance liquid chromatography of N-alkylprotoporphyrins : an investigation of their formation and loss under chemical and enzymatic conditions in vitro

A new technique for resolving N-alkylprotoporphyrins into each structural isomer is described. The technique has been used to investigate the rate of formation and loss of N-alkylporphyrins during reaction of the parent porphyrin with alkyl iodides and to establish the conditions required for optimal yields of the various isomers. Preferential loss of the isomers bearing the N-alkyl group on one of the vinyl-substituted pyrrole rings is observed on prolonged incubation and HI generated during the reaction has been shown to be responsible. A method for detection and partial resolution by HPLC of N-alkylprotoporphyrins produced by liver microsomes in vitro is also described. Microsomes from rats induced with 3-methylcholanthrene produce significantly more N-ethylprotoporphyrin from either 4-ethyl-3,5-diethoxycarbonyl-1,4-dihydro- 2,6-dimethyl-pyridine or ethylhydrazine than do microsomes from control animals, but the isomeric composition of the isolated N-alkylporphyrin differs from that reported in vivo. Evidence that authentic N-alkylporphyrins are lost during incubation with microsomes has been obtained, and here again, the isomers bearing the N-alkyl group on vinyl-substituted pyrrole rings are preferentially lost. Experiments with 14C-labeled N-methylprotoporphyrin show that approximately 40% of the lost porphyrin could be recovered bound covalently to the microsomal pellet.

Liver Haem as a Target for Drug Toxicity

ABSTRACT Two distinct types of drug toxicity are illustrated, both with liver haem as the main target. Drugs with unsaturated side chains and certain dihydropyridines both convert liver haem into N -alkylated porphyrins but the underlying mechanisms and biological properties of the resulting pigments differ in the two cases. A monooxygenated derivative of the unsaturated drugs becomes itself bound onto one of the pyrrole nitrogen atoms of the haem of cytochrome P-450 and the N -alkylated porphyrin which is produced does not inhibit the last enzyme of haem biosynthesis, protohaem ferro-lyase. In contrast, with 3,5-diethoxycarbonyl-l,4-dihydrocollidine only the 4-methyl substituent of the drug is transferred onto the pyrrole nitrogen atom and the resulting porphyrin is a powerful inhibitor of protohaem ferro-lyase. The latter pathway involves transfer of the intact 4-alkyl group onto the pyrrole nitrogen.

Isomeric Composition of N -Alkylated Protoporphyrins Produced by Substituted Dihydropyridines in Vivo and in Vitro

Substituted dihydropyridines, like DDC and its 4-ethyl analogue (4-ethyl-DDC)* give rise to N-alkylated porphyrins when given to rats and mice in vivo, by donating their intact 4-alkyl group to one of the pyrrole nitrogens of haem in hepatic cytochrome P-450 (De Matteis et al., 1981; Ortiz de Montellano et al., 1981; Tephly et al., 1981). Because of the asymmetrical arrangement of the two vinyl and propionate side chains in protoporphyrin IX, four structural isomers of N-monoalkylated protoporphyrins are possible, according to which of the four pyrrole nitrogens has been substituted. In a previous paper (De Matteis et al., 1983) we have provided evidence that the isomeric composition of the N-ethyl protoporphyrin produced in vivo by treatment with 4-ethyl-DDC depends on the particular cytochrome P-450 which predominates at the time of treatment, suggesting a role for the apo-cytochrome in directing alkylation preferentially on to one of the pyrrole nitrogens. In this present paper we wish to report additional findings in vivo which are compatible with this interpretation. Production of N-ethyl protoporphyrin is also found in vitro, when isolated microsomes are incubated with 4-ethyl-DDC in presence of NADPH and, although a change in isomeric profile is again found with microsomes containing different cytochrome P-450 enzymes, nevertheless the picture obtained with isolated membranes in vitro is different from that seen in the intact cells in vivo.