Glutamatergic system: another target for the action of porphyrinogenic agents.

The N-methyl-diethyl-aspartate (NMDA) receptor has been reported to play an important role in several acute and chronic neuropathologic syndromes. 5-aminolevulinic acid (ALA) accumulates in acute porphyrias due to a deficiency in the heme biosynthetic pathway. Considering that glutamate uptake inhibition caused by ALA could be one of the reasons conducing to porphyric neuropathy, it was of interest to evaluate the effect of porphyrinogenic agents on NMDA glutamatergic system. To this end receptor levels and apparent affinity (Kd) were analyzed in mice brain cortex and cerebellum. NMDA levels were diminished after chronic Isoflurane anaesthesia in brain cortex. In cerebellum, a diminution was observed after acute Enflurane and Isoflurane and allylisopropylacetamide, while ethanol administration showed a significant increase. ALA administration diminished NMDA levels only in cerebellum. Affinity constant was only reduced in brain cortex after chronic Isoflurane treatment. In conclusion, glutamatergic system appears to be involved in the action of some of the porphyrinogenic drugs studied mainly in cerebellum. Receptors regulation should therefore be considered an important mechanism in the cellular response to specific drugs, with the aim of designing new therapies and elucidating the mechanisms leading to porphyric neuropathy and acute attack triggering.

Porphomethene inhibitor of uroporphyrinogen decarboxylase: analysis by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry.

An uroporphomethene inhibitor of uroporphyrinogen decarboxylase, characterized by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry, was reported recently (Phillips et al., Proceedings of the National Academy of Sciences of the United States of America 2007; 104: 5079-5084). Close examination of the tandem mass spectrometric fragmentation pattern of the compound showed that it is not a tetrapyrrole or an uroporphyrinogen or uroporphyrin related molecule. The product ion spectrum showed a fragmentation pattern typical of a poly(ethylene glycol) structure. Characteristic fragmentations of the side-chain acetic acid and propionic acid substituents of a uroporphyrin or uroporphyrinogen derivative were absent.

Heme oxygenase, aminolevulinate acid synthetase and the antioxidant system in the brain of mice treated with porphyrinogenic drugs.

Several drugs and stress are involved in the triggering of attacks in acute porphyrias. The central nervous system is extremely sensitive to free radical damage because of a relatively low antioxidant capacity. We have demonstrated that mice brain cholinergic system was altered by the effect of some porphyrinogenic agents. The aim of this work was to investigate how known porphyrinogenic drugs affect delta-Aminolevulinic acid synthetase (ALA-S), which is the response of heme oxygenase (HO) to this challenge and to evaluate if the xenobiotics studied develop stress oxidative in mice brain. HO activity was 50-70% induced after chronic Enflurane and Isoflurane anaesthesia, dietary Griseofulvin and starvation. An increase in mRNA HO expression was caused by chronic anaesthesia and Veronal treatments; instead allylisopropilacetamide (AIA) reduced mRNA expression. ALA-S activity was induced by acute administration of anaesthetics (89%), veronal (240%) and ethanol (80%), while ALA-S mRNA expression augmented by chronic administration of enflurane, AIA and veronal. Stress markers such as superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase activities and malondialdehyde and reduced glutathione levels showed different responses depending on the xenobiotic assayed. In conclusion, some of the drugs studied produced oxidative stress in brain that was confirmed through HO induction and this could be one of the factors leading to porphyric neuropathy.

Chick embryo liver microsomal steroid hydroxylations: induction by dexamethasone, phenobarbital, and glutethimide and inactivation following the in ovo administration of porphyrinogenic compounds.

Metabolites of [4-(14)C]androstenedione (AD) and [4-(14)C]progesterone (PG) were separated and quantitated following incubation with hepatic microsomes from chick embryos. PG 2 alpha-hydroxylase and AD 7 alpha-hydroxylase, which are diagnostic markers in rat liver for cytochrome P450 (P450) 2C11 and 2A1/2, respectively, were not identified in chick embryo liver. PG 6 beta-hydroxylase and AD 6 beta-hydroxylase, diagnostic markers for P450 3A1/2 activity in rat liver, were identified in chick embryo liver, and we were able to show that radiolabeled 6 beta-hydroxyprogesterone and 6 beta-hydroxyandrostenedione, the metabolites of PG and AD 6 beta-hydroxylase, respectively, were homogeneous and identical with authentic standards. Dexamethasone, phenobarbital, and glutethimide were found to be significant inducers of PG and AD 6 beta-hydroxylases in chick embryo liver. The in ovo administration of the porphyrinogenic compounds 3,5-diethoxycarbonyl-1,4-dihydro-2,6-dimethyl-4-ethylpyridine (4-ethyl DDC) and 3-[2-(2,4,6-trimethylphenyl)-thioethyl]-4-methylsydnone (TTMS) caused inactivation of chick embryo hepatic PG and AD 6 beta-hydroxylases. Therefore, we suggest that PG and AD 6 beta-hydroxylases may serve as diagnostic markers for a P450 3A-like isozyme in the chick embryo, and that this isozyme is one of the targets for inactivation by 4-ethyl DDC and TTMS.

Up-regulation of CYP2A5 expression by porphyrinogenic agents in mouse liver.

Coumarin 7-hydroxylase (COH) activity is catalyzed by the Cyp2a-5 gene product (CYP2A5 enzyme) in mice. Mouse hepatic CYP2A5 expression is often increased in conditions in which other P450 forms are repressed, e.g. after the administration of heavy metals and other toxic agents known to affect cellular heme balance. In this study, the effect of various porphyrinogenic chemicals on the expression CYP2A5 and the key enzymes in heme metabolism was studied. Administration of single doses of griseofulvin (1000 mg/kg), thioacetamide (10 mg/kg) and aminotriazole (1000 mg/kg) to DBA/2 and C57BL/6 mice produced up to 10-fold increases in hepatic COH catalytic activity. Dramatic, up to 130-fold increases in response to the inducers was observed in the amount of CYP2A5 steady-state mRNA. The mRNA contents of aminolevulinate synthase, ferrochelatase and heme oxygenase were also increased to a variable extent, possibly reflecting feed-back regulatory mechanisms. In D2 mice the CYP2A5 inducing effect of aminotriazole and thioacetamide, but not that of griseofulvin, pyrazole and phenobarbital, was abolished by exogenously administered heme arginate. In the B6 strain heme arginate treatment increased CYP2A5 expression but it did not affect the induction caused by porphyrinogenic agents. These results show that porphyrinogenic agents act as efficient inducers of CYP2A5, and suggest that regulation of the transcription of the Cyp2a-5 gene could in some instances involve heme-sensitive factors.

pH-dependent one- and two-electron oxidation of 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine catalyzed by horseradish peroxidase.

The porphyrinogenic agent 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine (DDEP) is known to inactivate hepatic cytochrome P450 (P450) enzymes 2C11, 2C6, and 3A1 [Correia et al. (1987) Arch. Biochem. Biophys. 258, 436-451] by different mechanisms. The inactivation of P450 2C11 and 2C6 appears to be due to the ethylation of the heme in the active sites of the enzymes [Augusto et al. (1982) J. Biol. Chem. 257, 11288-11295], whereas the inactivation of P450 3A1 appears to involve the covalent binding of the heme to the apoprotein [Correia et al. (1987)]. Moreover, we have found that DDEP inactivates horseradish peroxidase (HRP) pretreated with hydrogen peroxide. In this system, DDEP was oxidized predominately to 3,5-dicarbethoxy-2,6-dimethyl-4-ethylpyridine (EDP) under weakly acidic conditions and predominately to 3,5-dicarbethoxy-2,6-dimethylpyridine (DP) under basic conditions. The loss of heme and the formation of altered heme products were also pH-dependent and were correlated with the formation of DP and the inactivation of HRP. Thus the inactivation of HRP appears to depend on the formation of an ethyl radical, which presumably reacts with the heme in the active site of the enzyme. Similar product ratios were obtained for the oxidation of DDEP by K3Fe(CN)6, indicating that product ratios of DP over EDP are mainly determined by the pH of buffer. These results, in addition to semiemperical calculations (AM1) for the oxidation of DDEP in the gas phase, are consistent with the idea that the inhibitor undergoes a single-electron oxidation to form the DDEP radical cation, the fate of which depends on the environment of the active site of the enzyme. The proposed formation of a radical cation by the abstraction of an electron from nitrogen is consistent with the finding of low intramolecular isotope effects of the metabolism of 3,5-dicarbethoxy-2,6-dimethyl-[4-2H,4-1H]-1,4-dihydropyridine by P450 2C11 and 3A4. Under basic or aprotic conditions, the radical dissociates to form DP and the ethyl radical, which reacts with the heme, thereby inactivating the enzyme. Under acidic or polar conditions, the radical undergoes an additional one-electron oxidation to form EDP.(ABSTRACT TRUNCATED AT 400 WORDS)

In vitro studies of the mechanism of inhibition of rat liver uroporphyrinogen decarboxylase activity by ferrous iron under anaerobic conditions.

Human porphyria cutanea tarda (PCT) is an unusual consequence of common hepatic disorders such as alcoholic liver disease and iron overload, where hepatic iron plays a key role in the expression of the metabolic lesion, i.e., defective hepatic decarboxylation of porphyrinogens. In this investigation, kinetic studies on a partially purified rat liver uroporphyrinogen decarboxylase have been conducted under controlled conditions to determine how iron perturbs porphyrinogen decarboxylation in vitro. The enzyme, assayed strictly under anaerobic conditions in the dark, was inhibited progressively by ferrous iron. Approximately 0.45 mM ferrous ammonium sulfate was required to observe about 50% inhibition of enzyme activity measured with uroporphyrinogen I as substrate. We showed that (a) all the steps of enzymatic decarboxylation (octa-, hepta-, hexa-, and pentacarboxylic porphyrinogen of isomer I series) were inhibited by ferrous iron. The inhibition was competitive with respect to uroporphyrinogen I and III substrates; (b) the cations, e.g., Fe3+ and Mg2+, had no effect, whereas sulfhydryl group specific cations and compounds such as Hg2+, Zn2+, p-mercuribenzoate, and 5,5'-dithiobis(2-nitrobenzoate) all inhibited the enzyme; (c) the enzyme could be protected from inhibition by Fe2+ and p-mercuribenzoate by preincubation with pentacarboxylic porphyrinogen, a natural substrate and competitive inhibitor. These data suggest for the first time a direct interaction of ferrous iron with cysteinyl residue(s) located at the active site(s) of the enzyme.

Stimulation of 5-aminolevulinic acid metabolism by uroporphyrinogen I in rat liver homogenate.

The effect of excess uroporphyrinogens I and III, coproporphyrinogen III, and the corresponding porphyrins, on the rate of 4-14C-5-aminolevulinic acid (ALA) metabolism was studied. Experiments were performed with mitochondria-free rat liver homogenates prepared from normal rats. The consumption of labelled 5-aminolevulinic acid was followed by measuring its level in aliquots removed at intervals. The pattern of porphyrinogen synthesis was examined by high pressure liquid chromatography. Only uroporphyrinogen I had an effect; it increased the rate of conversion of ALA and porphobilinogen (PBG) to porphyrinogens. Chromatographic analysis revealed increased synthesis of uroporphyrinogen and heptacarboxylic porphyrinogen. It is believed that this mechanism might explain the lack of ALA and PBG accumulation in erythropoietic porphyria and porphyria cutanea tarda, and the absence of acute porphyria attacks in these conditions.

The effects of acetate, metal cations, phenobarbitone, porphyrogens and substrates of glycine acyltransferase on the utilization of haem by rat liver apo-(tryptophan pyrrolase).

1. The utilization of haem by rat liver apo-(tryptophan pyrrolase) under basal conditions and after enhancement of the enzyme activity by various mechanisms was studied under the influence of treatments affecting various aspects of liver haem metabolism. 2. These treatments were: benzoate and p-aminobenzoate as substrates of glycine acyltransferase, acetate as an inhibitor of 5-aminolaevulinate synthase activity, enhancement of 5-aminolaevulinate dehydratase by aluminium, destruction of haem and inhibition of ferrochelatase by porphyrogens, increased haem utilization by phenobarbitone and enhancement of haem oxygenase activity by metal cations. 3. The results show that the haem saturation of the apoenzyme is sensitive to all these treatments. 4. The possible usefulness of tryptophan pyrrolase in studying the regulation of liver haem is suggested.

Regulation of rat liver tryptophan pyrrolase by its cofactor haem: Experiments with haematin and 5-aminolaevulinate and comparison with the substrate and hormonal mechanisms.

1. The administration of haematin or 5-aminolaevulinate to rat enhances the activity of liver tryptophan pyrrolase; both endogenous and newly formed apoenzymes become strongly haem-saturated. Haem activation does not stabilize tryptophan pyrrolase. 2. Actinomycin D, puromycin or cycloheximide prevent the activation of the enzyme by 5-aminolaevulinate but not that by haematin. The latter is inhibited by haem-destroying porphyrogens. 3. The combined injection of either haematin or 5-aminolaevulinate with cortisol does not produce an additive effect, whereas potentation is observed when tryptophan is jointly given with either the cofactor or the haem precursor. 4. Further experiments on the substrate (tryptophan) mechanism of pyrrolase regulation are reported, and a comparison between this and the cofactor and hormonal mechanisms is made. 5. It is suggested that the substrate mechanism may also involve increased haem synthesis. 6. The role of tryptophan pyrrolase in the utilization of liver haem, and as a possible model for the exacerbation by drugs of human hepatic porphyrias, is discussed.