EFFECT OF ANTIOXIDANTS AND BILE ACIDS IN A MOUSE MODEL OF ERYTHROPOIETIC PROTOPORPHYRIA INDUCED BY GRISEOFULVIN

MARTINEZ MARIA DEL C; AFONSO, SUSANA; BATLLE ALCIRA

Estocolmo

Congreso; SYMPOSIUM 81 PORPHYRINS AND PORPHYRIAS; 2009

EFFECT OF ANTIOXIDANTS AND BILE ACIDS IN A MOUSE MODEL OF ERYTHROPOIETIC PROTOPORPHYRIA INDUCED BY GRISEOFULVIN   Mart¨ªnez Mar¨ªa del Carmen1 2, Afonso Susana G.1, Batlle Alcira1   1Centro de Investigaciones sobre Porfirinas y Porfirias (CIPYP)-CONICET, Hospital de Cl¨ªnicas Jos¨¦ de San Mart¨ªn and 2Departamento de Qu¨ªmica Biol¨®gica Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires INTRODUCTION Erythropoietic Protoporphyria (EPP) is a disease associated with a deficiency in the enzyme Ferrochelatase (Fech), producing accumulation of protoporphyrin IX (PP) in bone marrow, erythrocytes, liver and skin. The most serious manifestation of this porphyria is the progressive liver failure, cholestasis and deposition of PP in the canalicular bile. The antifungal griseofulvin (Gris) develops a model of EPP liver manifestations in animals. Previously, we demonstrated that the administration of Gris to mice produced PP accumulation followed by cellular damage and necrotic and inflammatory processes. These alterations are similar to those found in the human EPP associated with liver failure. Furthermore, development of oxidative stress was observed, liver redox balance was altered due to porphyrin high concentrations, known generators of reactive oxygen species (ROS).  As consequence, the antioxidant defense system was disrupted.;  an increased activity of the enzymes Glutathione Reductase (GRed), Superoxide Dismutase (SOD) and Glutathione-S-Transferase (GST), and also  high levels of reduced Glutathione (GSH) and malondialdehyde (MDA) were detected; while the activities of Glutathione Peroxidase (GPx ) and Catalase were diminished; The hepatic free heme pool was greatly diminished, partly due to the reduced synthesis of heme  by Gris and because of the induction of cytochrome P450 system to metabolize this xenobiotic. Some polyphenols, in particular chlorogenic acid (CA), exert a protective action against oxidative stress diminishing the degree of lipid peroxidation and the activities of some antioxidant enzymes. Moreover, Gris reduced the bile flow, main way of PP elimination; the administration of  Desoxycholic acid (DA) decreased liver PP accumulation and, like ursodeoxycholic acid (UA), reduced some of the oxative stress parameters induced by Gris. AIMS      Evaluate the possible protective effect of xenobiotics such as antioxidants and bile acids on  parameters of liver damage markers and oxidative stress in our mouse model of EPP.   MATERIALS AND METHODS Animals: Male CF1 Mice of 15-17 g weight at the start of the assay. Antioxidants: Trolox (Tx, 2mg/100ml), Ascorbic acid (Asc, 12mg/100ml), Melatonin (Mel, 5mg/kg i.p.), Chlorogenic acid (CA, 50mg /l). Bile acids: Desoxycholic acid (DA, 0.33% w/w), Ursodeoxycholic acid (UA, 0.20% w/w). Design: Mice received standard diet for laboratory animals plus corn oil (10ml/100g) and water ad libitum. Gris (0.5% w / w), or other drugs assayed were added in the food or to the water or injected intraperitoneally as it is indicated in the following protocol :     Biochemical determinations: ¦Ä-Aminolevulic acid synthetase (ALA-S): enzyme activity was determined using the method of Marver et al (J. Biol. Chem 241, 1966, 2803-2809). GSH levels: were measured using the method of Rossi et al (Biochim. Biophys. Acta 1243, 1995, 230-238). MDA levels: were quantified using the method of Ohkawa et al. (Anal. Biochem. 95, 1979, 351-358).GST activity: was determined using the method of Habig et al (J. Biol. Bhem. 249, 1974, 7130-7139). GRed activity was determined using the method of Pinto & Bartley (Biochem. J. 112, 1969, 109-115). SOD activity:was determined using the method of Paoletti et al. (Anal. Biochem. 154, 1986, 536-541). Statistical analysis: All data represent mean values ¡À standard deviation of four experiments performed in duplicate. The differences between treated and control groups were determined by ANOVA and the significance level was verified by the Bartlet test. A probability level of 99.9% or 99.5% was considered as significant difference between groups. (**) p<0.01: Significant difference between the group treated with Gris alone or Gris more antioxidant or bile acids respect to the control group. (#) p <0.05 and (# #) p <0.01: Significant difference between the group treated with antioxidants or bile acid  respect to the control group.  (+) p <0.05 and (+ +) p <0.01: Significant difference between the group treated with Gris plus antioxidant or bile acid respect to the group that only received Gris. (-)p<0.05 and (--)p<0.01:  Significant difference between  the group treated with Gris plus antioxidant or bile acid respect to  the group only treated with antioxidant or bile acid.     RESULTS   Heme Metabolism   Activity of ALA-S   ALA-S activity was 50% (p <0.01) increased in animals receiving Gris. The administration of Asc +Gris decreased 25% (p <0.01) this induction, while Tx, Tx + Asc or Mel returned the activity to basal levels. Markers of liver damage   Lipid peroxidation   Administration of only  CA plus UA to control animals produced  40% (p <0.01) increase of MDA levels. Gris caused 80% (p <0.01) increase of lipid peroxidation respect to control group. When animals received DA or UA plus CA with Gris, MDA levels decreased 45% and 56% (p <0.01) respectively, reaching values even lower than the baseline. The combined treatment of Gris with the other antioxidants assayed produced a decrease of  around 18-20% (p <0.05) compared with the group receiving only Gris.   Glutathione-S-Transferase   Asc administered to control animals reduced 40% (p <0.01) GST activity, while DA + CA increased this activity 100%, (p<0.01), without any alteration by adding any of the other drugs studied. Gris induced 50% (p <0.01) GST activity, remaining elevated also after Tx, Mel or AD + CA administration. Instead, when Asc, or Asc + Tx or UA + CA was given to Gris feeding mice, activity returned to control values.   Antioxidant Defense Reduced Glutathione Asc alone or in combination with Tx produced a decrease of about 55% (p <0.01) on GSH content in control animals, while the combinations of DA+CA and UA+CA produced an increase of 28% and 50 % (p <0.01), respectively. Gris treatment increased 90% (p <0.01) hepatic GSH content, which was not altered by the administration of Tx, Mel or the combinations of DA+CA or UA+CA. In animals receiving Gris with Asc or Asc plus Tx a reduction over 50% (p <0.01) was detected, reaching baseline levels and  maintaining a significant difference respect to their respective controls.   Glutathione Reductase activity Administration of DA + CA  increased  60%, (p <0.01) GRed activity, either alone or in combination with Gris. The 87% (p<0.01) induction produced by Gris  was reduced to 33% (p<0.01) by Tx  and  to 21%  (p<0.01)  by UA+CA, without any effect when Asc, Asc+Tx or Mel were assayed.   SOD activity Gris induced 69% (p <0.01) SOD activity. These high levels were not changed by  administration of Asc, alone or with Tx, Mel or UA+CA. The combined treatment of Gris and DA+CA  or Tx   decreased 30% (p <0.01) and 26% (p <t0.05), respectively, SOD  activity with  respect to the Gris group.   CONCLUSIONS   -     The effect of antioxidants or bile acids used alone or in combination, on ALA-S and stress oxidative parameters affected by Gris was investigated. -     The co-administration of Gris with Tx, Asc, or Mel altered the heme biosynthetic pathway, resulting in a decrease of ALA-S activity, which had been increased  by Gris, also had an effect on the onset of lipid peroxidation. -     The administration of Chlorogenic acid Desoxicolic prevented further the increase of lipid peroxidation and the activities of some antioxidant enzymes -     In our model, oxidative stress would lead to permanent and  irreversible changes in the antioxidant defense system and a significant damage of cells, so  that the compounds assayed were not capable of reverting or avoiding  completely the established injury. Treatment with antioxidants, bile acids or a combination of both could only partially protect the cell from damage, decreasing the oxidative stress produced by Gris and also increasing the excretion of porphyrins or acting at the level of heme regulation.