F. De Matteis

Biochemical changes in rat liver after administration of carbon disulphide, with particular reference to microsomal changes

Abstract Rats given an oral dose of carbon disulphide (1 ml/kg) exhibited an increased liver size and protein content 24 hr later, but showed a marked prolongation of the hexobarbitone “sleeping time”. After a single oral dose of CS2, the activity of aniline hydroxylase and nitroanisole demethylase and the level of cytochrome P-450 were found to fall very rapidly in the liver microsomes and remained depressed for at least three days: aniline hydroxylase was more severely affected and recovered at a faster rate than either cytochrome P-450 or nitroanisole demethylase. The level of cytochrome b5 decreased only slightly. A “P-420” peak was detected in microsomal preparations from poisoned animals in the early stages of the intoxication and the total protohaem content of the microsomes was unchanged at 4 hr but decreased significantly at 24 hr. The most likely interpretation for these findings is that CS2 causes a rapid alteration in the cytochrome P-450 leading to loss of its spectrum and of its activity in drug oxidation and that the altered cytochrome (or its haem moiety) is lost from the damaged membranes at a slow rate. The extent of the microsomal changes caused by CS2 appeared to depend on the activity of the drug-metabolizing enzymes at the time when CS2 was administered. Pre-treatment with phenobarbitone increased the loss of microsomal enzymes and cytochrome P-450 caused by CS2, whereas SKF-525A afforded some protection. This may indicate that a metabolite of CS2, rather than CS2 itself, is the real toxic agent responsible for the observed picture. Changes were also noted in the rate of incorporation of labelled leucine into proteins by liver slices from the poisoned rats: an inhibition was observed at 4 hr and this was followed by a stimulation 24 hr after CS2 administration.

Rapid loss of cytochrome P-450 and haem caused in the liver microsomes by the porphyrogenic agent 2-allyl-2-isopropylacetamide.

Several chemically unrelated drugs stimulate the hepatic formation of porphyrins in the whole animal and in liver cells cultured in vitro [ 1,2] and enchance the activity of &aminolaevulic acid (&ALA) synthetase, the rate-limiting enzyme in the biosynthetic pathway of porphyrins and haem [3] . This is thought to result from an interference by the drugs with the feed-back control exercised by haem at the level of the enzyme [3], although the exact mechanism of this effect is not known. One of these drugs, 2-allyl-2-isopropylacetamide (AIA), causes a rapid decrease in the level of cytochrome P-450 and haem in rat liver microsomes. This effect, which apparently requires the activity of drug-metabolizing enzymes in liver microsomes, is due to a loss of existing cytochrome P-450 and haem rather than an inhibition of their synthesis. Evidence has also been obtained that the haem lost undergoes a change in chemical constitution, probably to certain illdefmed green pigments already described in the liver of animals with experimental porphyria.

Iron-dependent loss of liver cytochrome P-450 haem in vivo and in vitro

It has been reported that the cytochrome P-450 content of rat liver microsomes decreases when the microsomes are incubated aerobically in the presence of NADPH [I]. It is now found that the rate of loss of cytochrome P-450, which is observed in vitro under these conditions, varies according to the nutritional state of the animal and correlates with the formation of lipid peroxides. The loss of the haemoprotein is accompanied by a loss of haem, by a green discolouration of the microsomal pellet and by production of carbon monoxide, indicating that the lost haem undergoes degradation. Evidence is also presented here for the involvement of iron in the loss of cytochrome P-450 haem both in vitro and in vivo.

A difference between two strains of rats in their liver non-haem iron content and in their response to the porphyrogenic effect of hexachlorobenzene

Female Agus rats developed hepatic porphyria at a much faster rate than female Porton-Wistar rats when fed a diet containing 0.01% of hexachlorobenzene (HCB). They also showed a greater inhibition of liver uroporphyrinogen decarboxylase [EC 4.1.1.37] activity and a marked stimulation of 5-aminolaevulinate synthetase [EC 2.3.1.37]. The difference between the two strains could not be correlated with differences in the liver concentrations of HCB. However, control Agus rats were found to possess significantly higher levels of total non-haem iron in their livers than the Porton animals. This was particularly apparent after 24 h of starvation and is further evidence for the involvement of iron in the pathogenesis of HCB-induced porphyria. The posterior lobes of the livers from the Agus rats given HCB became porphyric more slowly than the remainder with less severe inhibition of uroporphyrinogen decarboxylase. In contrast to their increased susceptibility to HCB, the Agus rats were less susceptible to another prophyrogenic agent, 3,5-diethoxycarbonyl-1,4-dihydrocollidine.

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

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

1. 1. Haem is destroyed when directly incubated with hydrogen peroxide or with Superoxide anion-producing systems, like xanthine oxidase and dihydroxyfumaric acid. 2. 2. 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. 3. 3. Haemoprotein haem (as present in either haemoglobin or in myoglobin) and cobaltic protoporphyrin are relatively resistant to oxidation. 4. 4. 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. 5. 5. 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.

Conversion of liver haem into N-substituted porphyrins or green pigments: Nature of the substituent at the pyrrole nitrogen atom

Drugs can promote the conversion of liver haem into modeled porphyrins (or green pigments) of two distinct classes. Pigments of the first class are obtained by treatment with unsaturated drugs containing at least one allyl, vinyl or ethynyl side chain. These drugs are metabolized by cytochrome P450 into reactive derivatives, which then become bound onto the porphyrin nucleus of the haem moiety of the cytochrome, converting it into modified porphyrins (reviewed in [I ,2]). The nature of the drug metabolite responsible is still controversial: epoxides have been suggested ([2] and references therein), but there is also a proposal [33 specifically excluding epoxides and emphasizing other structural features of the metabolite, such as the presence of an ‘activated’ carbony1 grouping. Green pigments of this class do not inhibit liver protohaem ferro-lyase [4] (the enzyme which converts protoporphyrin to haem) and for this reason none of the unsaturated drugs responsible will cause a very marked increase in liver protoporphyrin in v&o. In contrast 3,5-diethoxycarbonyl-1,4-d~ydrocollidine, griseofulvin and isogriseofulvin produce liver accumulation of a second type of green pigment with strong inhibitory properties towards protohaem ferrolyase in vitro [4-61. Marked inhibition of the liver enzyme is also observed in vivo after treatment with this second group of drugs [7,8] and as a consequence, the liver concentration of protoporphyrin increases markedly giving rise to the biochemical picture of hepatic protoporphyria. The ‘inhibitory’ pigment also appears to originate from turnover of liver haem [4],

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

Structural Isomerism and chirality of N-monosubstituted protoporphyrins: Possible relevance to binding of N-methyl protoporphyrin by ferrochelatase and to orientation of haem in cytochrome P450

A modified porphyrin or green pigment which is a powerful inhibitor of ferrochelatase (protohaem ferrolyase, EC 4.99.1.1) has been isolated from the liver of mice treated with 3,5-diethoxycarbonyl-1,4-dihydrocoUidine (DDC) [1] and the inhibitory porphyrin has been identified as N-methyl protoporphyrin [2-4]. Interaction of the inhibitory porphyrin with the active centre of ferrochelatase is important for inhibition of the enzyme [5,6]. Authentic N-methylated meso and protoporphyrin have been synthesized and shown to be powerful inhibitors of the enzyme in vivo and in vitro [2,6,7]. Synthetic N-methyl protoporphyrin could be separated by high-performance liquid chromatography (HPLC) into 2 separate fractions possessing different inhibitory activity and possibly representing different structural isomers, where different pyrrole nitrogens had been methylated [2]. Here, we provide evidence that the two HPLC fractions contain in fact different structural isomers of N-methyl protoporphyrin. The different inhibitory activity of these two isomeric fractions is now confirmed and extended to their respective zinc chelates. In the isomers with less inhibitory activity, where the propionic acid substituted rings of protoporphyrin are N-methylated, the spatial arrangement and distance between the two propionic acid sideehains may be significantly altered. We also wish to report that the naturally occurring N-methyl protoporphyrin and the N-alkylated protoporphyrins produced in vivo from degradation of the haem of cytochrome P450 during metabolism of unsaturated chemicals are all optically active and appear to possess the same absolute configuration.

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.