Ana Maria Buzaleh

Metabolization of Porphyrinogenic Agents in Brain: Involvement of the Phase I Drug Metabolizing System. A Comparative Study in Liver and Kidney

Abstract(1) We evaluated the involvement of brain mitochondrial and microsomal cytochrome P-450 in the metabolization of known porphyrinogenic agents, with the aim of improving the knowledge on the mechanism leading to porphyric neuropathy. We also compared the response in brain, liver and kidney. To this end, we determined mitochondrial and microsomal cytochrome P-450 levels and the activity of NADPH cytochrome P-450 reductase. (2) Animals were treated with known porphyrinogenic drugs such as volatile anaesthetics, allylisopropylacetamide, veronal, griseofulvin and ethanol or were starved during 24xa0h. Cytochrome P-450 levels and NADPH cytochrome P-450 reductase activity were measured in mitochondrial and microsomal fractions from the different tissues. (3) Some of the porphyrinogenic agents studied altered mitochondrial cytochrome P-450 brain but not microsomal cytochrome P-450. Oral griseofulvin induced an increase in mitochondrial cytochrome P-450 levels, while chronic Isoflurane produced a reduction on its levels, without alterations on microsomal cytochrome P-450. Allylisopropylacetamide diminished both mitochondrial and microsomal cytochrome P-450 brain levels; a similar pattern was detected in liver. Mitochondria cytochorme P-450 liver levels were only diminished after chronic Isoflurane administration. In kidney only mitochondrial cytochrome P-450 levels were modified by veronal; while in microsomes, only acute anaesthesia with Enflurane diminished cytochrome P-450 content. (4) Taking into account that δ-aminolevulinic acid would be responsible for porphyric neuropathy, we investigated the effect of acute and chronic δ-aminolevulinic acid administration. Acute δ-aminolevulinic acid administration reduced brain and liver cytochrome P-450 levels in both fractions; chronic δ-aminolevulinic acid administration diminished only liver mitochondrial cytochrome P-450. (5) Brain NADPH cytochrome P-450 reductase activity in animals receiving allylisopropylacetamide, dietary griseofulvin and δ-aminolevulinic acid showed a similar profile as that for total cytochrome P-450 levels. The same response was observed for the hepatic enzyme. (6) Results here reported revealed differential tissue responses against the xenobiotics assayed and give evidence on the participation of extrahepatic tissues in porphyrinogenic drug metabolization. These studies have demonstrated the presence of the integral Phase I drug metabolizing system in the brain, thus, total cytochrome P-450 and associated monooxygenases in brain microsomes and mitochondria would be taken into account when considering the xenobiotic metabolizing capability of this organ.

Protective Action of Antioxidants on Hepatic Damage Induced by Griseofulvin

Erythropoietic protoporphyria (EPP) is a disease associated with ferrochelatase deficiency and characterized by the accumulation of protoporphyrin IX (PROTO IX) in erythrocytes, liver, and skin. In some cases, a severe hepatic failure and cholestasis were observed. Griseofulvin (Gris) develops an experimental EPP with hepatic manifestations in mice such as PROTO IX accumulation followed by cellular damage as wells as necrotic and inflammatory processes. The antioxidant defense system was also altered. The aim was to evaluate the possible protective effect of different antioxidant compounds: trolox (Tx), ascorbic acid (Asc), the combination Tx and Asc, melatonin (Mel), and the polyphenols: ellagic acid, quercetin, chlorogenic acid, caffeic acid, gallic acid, and ferulic acid on liver damage and oxidative stress markers in a mouse model of EPP. Coadministration of Gris with Tx, Asc, and its combination, or Mel mainly affected heme biosynthetic pathway, resulting in a decrease in ALA-S activity which was increased by Gris, while the tested polyphenols exerted a protective effect on oxidative stress, decreasing lipid peroxidation and the activity of some antioxidant enzymes. In conclusion, antioxidant compounds can only protect partially against the liver damage induced by Gris, reducing oxidative stress or acting on heme regulation.

Griseofulvin-induced hepatopathy due to abnormalities in heme pathway.

1. The effect of long-term griseofulvin (GRIS) topical administration on some indicators of liver damage was examined. 2. Liver porphyrin accumulation was significant; however, no porhyrin crystals were observed under light microscopy. 3. An earlier onset of hepatopathy was established (3-fold) increase of direct bilirubin values after 7 days of treatment; hepatic injury was confirmed by measuring a 6-fold increase of free bilirubin. 4. Enhanced values of alkaline phosphatase and glutamic oxalacetic transaminase (GOT) confirmed the onset of cholestasis. 5. Topical application of GRIS induced measurable hepatopathy. Nevertheless, we cannot discard the possibility that this hepatopathy could also be attributed in part to a direct reaction to xenobiotics.

Cytochrome P450 expression in mouse brain: specific isoenzymes involved in Phase I metabolizing system of porphyrinogenic agents in both microsomes and mitochondria

Brain cytochrome P450 (CYP) metabolizes a variety of drugs to produce their pharmacological effects within the brain. We have previously observed that porphyrinogenic agents altered CYP levels in brain. The aim of this work was to further study the involvement of mice brain mitochondrial and microsomal Phase I drug metabolizing system when porphyrinogenic agents, such as Enflurane, Isoflurane, allylisopropylacetamide, veronal, ethanol, and Griseofulvin were administered. To this end, CYP2E1, CYP2B1, and CYP3A4 expression were measured. NADPH cytochrome P450 reductase (CPR) expression was also determined. Western Blots were performed in microsomes and mitochondria of whole brain. Some of the drugs studied altered expression mainly in microsomes. Chronic Isoflurane augmented mitochondrial isoform, although this anaesthetic diminished microsomal expression. Ethanol and topical Griseofulvin affected expression in microsomes but not in mitochondria. CYP2E1 mitochondrial activity was induced by acute Enflurane; while the activity of the microsomal protein was enhanced in alcoholised animals. Ethanol also induced CYP2E1 expression in microsomes, although Isoflurane provoked opposite effects in mitochondria and microsomes. Expression of CPR was also induced. Several reports support an emergent role of CYP enzymes in the pathogenesis of neurological disorders, so CYP response in brain could be one of the multiples factors influencing porphyria acute attacks.

Effect of chronic anesthesia on the drug-metabolizing enzyme system and heme pathway regulation.

1. The effect of chronic enflurane or isoflurane anesthesia on hepatic heme regulation and the drug-metabolizing system in mice treated or not with phenobarbital (PB) was investigated. 2. delta-Aminolevulinic acid synthetase was induced 50-170% in all cases. Urinary porphyrin precursor excretion was also enhanced, but these values were lower when animals also received PB. 3. Cytochrome (CYT) P-450 levels were enhanced in animals treated with enflurane whether or not they were given PB. 4. Gluthatione-S-transferase activity was induced by enflurane (138%) or isoflurane (174%), and even more in animals receiving PB also. Sulfatase activity was increased more than 60% with anesthetics. Isoflurane produced a 50% increase of beta-glucuronidase activity and a 35% diminution of tryptophan pyrrolase. 5. The association between anesthetics and PB produced diverse effects on the metabolizing enzyme system. 6. Data suggest that both anesthetics, chemically related, could act through two different mechanisms, however, with the same final effect: heme pathway deregulation.

The effect of griseofulvin on the heme pathway. Studies on tissue explant cultures.

The effect of griseofulvin on porphyrin biosynthesis was studied in vitro and in vivo using liver and skin explants from mice. Neither porphyrin nor precursors accumulation was detected after in vitro treatment of explants with griseofulvin. Culture medium porphyrins formed from added delta-amino-laevulinic acid (ALA) were increased after oral chronic intoxication. Similar results were obtained by cutaneous and oral intoxication. Consequently griseofulvin topical application is proposed as an alternative method to produce experimental erythropoietic protoporphyria. Probable intoxication mechanisms are proposed to explain the porphyrinogenic action of griseofulvin.

Relevance of cytochrome P450 levels in the actions of enflurane and isoflurane in mice: studies on the haem pathway.

1. The effect of the fluorinated ether anaesthetics enflurane and isoflurane in mice on haem metabolism and regulation in different metabolic states, such as depression and induction of cytochrome P450 produced by allylisopropylacetamide (AIA) and imidazole, respectively, was investigated.

Identification of CYP3A5 and CYP2B6 Polymorphisms in Porphyria Cutanea Tarda Associated to Human Immunodeficiency Virus

To date, few or no data concerning the prevalence of polymorphisms in drug metabolism genes of antiretroviral drugs have been reported in the Argentinean population or in porphyric individuals worldwide. The purpose of the current investigation was to determine whether interindividual differences in cytochrome P450 3A5 (CYP3A) and 2B6 (CYP2B6) genes could influence the triggering of Porphyria Cutanea Tarda (PCT) in subjects with human immunodeficiency virus (HIV) after antiretroviral exposure. nA total of 141 subjects, 60 control volunteers and 81 unrelated individuals with PCT were included in the study. In the porphyric group, 21 individuals were HIV positive. To evaluate the presence of the alleles CYP3A5*3, CYP3A5*6 and CYP2B6*6 a polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) analysis was performed. nThe frequencies of CYP3A5*3 were 0.91 in control group, 0.89 in PCT patients and 0.89 in PCT-HIV. CYP2B6*6 frequencies were 0.31 in control group, 0.34 in PCT group and 0.30 in PCT-HIV group. We have shown that the allelic frequencies of CYP3A5*3 or CYP2B6*6 in our population were similar to those reported for other Caucasian populations. Although, we have not found significant differences in polymorphisms of CYP3A5 and CYP2B6 between the different groups analyzed, there are an enormous number of biological variables that may influence antiretroviral treatment, like other genetic polymorphisms of phase I or phase II enzymes, or transporters like multidrug resistance transporter gene (MDR1), which can contribute to antiretroviral drug toxicities and response or even it is possible that the PCT-HIV association has more than one factor responsible for the onset of PCT symptoms.

In vivo prevention and reversal by the antimitotic colchicine and other related compounds of some porphyrinogenic drug action on heme pathway

1. The effect of colchicine, vincristine and griseofulvin (GRIS) on the porphyrinogenic action of 2-allyl-2-isopropylacetamide (AIA) and veronal was studied in vivo and using the in vitro experimental model of tissue explant cultures. 2. Complete prevention by colchicine was found in liver and heart explant from animals treated with AIA and veronal. 3. Vincristine, GRIS and colchicine reversed AIA induction in liver explants, however reversal was partial or nil in skin and heart explants depending on the antimitotic and the tissue. 4. The usefulness of the combination of the in vivo experimental model and the in vitro explant tissue culture model, for this kind of studies is emphasized.

Pleiotropic effects of 5-aminolevulinic acid in mouse brain

5-Aminolevulinic acid (ALA) seems to be responsible for the neuropsychiatric manifestations of acute intermittent porphyria (AIP). Our aim was to study the effect of ALA on the different metabolic pathways in the mouse brain to enhance our knowledge about the action of this heme precursor on the central nervous system. Heme metabolism, the cholinergic system, the defense enzyme system, and nitric oxide metabolism were evaluated in the encephalon of CF-1 mice receiving a single (40 mg/kg body mass) or multiple doses of ALA (40 mg/kg, every 48 h for 14 days). We subsequently found ALA accumulation in the encephalon of the mice. ALA also altered the brain cholinergic system. After one dose of ALA, a decrease in superoxide dismutase activity and a reduction in glutathione levels were detected, whereas malondialdehyde levels and catalase activity were increased. Heme oxygenase was also increased as an antioxidant response to protect the encephalon against injury. All nitric oxide synthase isoforms were induced by ALA, these changes were more significant for the inducible isoform in glial cells. In conclusion, ALA affected several metabolic pathways in mouse encephalon. Data indicate that a rapid response to oxidative stress was developed; however, with long-term intoxication, the redox balance was probably restored, thereby minimizing oxidative damage.