griseofulvin and Protoporphyria--Erythropoietic

The effect of bile acids administration to an experimental mice model of Protoporphyria produced by griseofulvin (Gris) was investigated. The aim was to assess whether porphyrin excretion could be accelerated by bile acids treatment in an attempt to diminish liver damage induced by Gris. Liver damage markers, heme metabolism, and oxidative stress parameters were analyzed in mice treated with Gris and deoxycholic (DXA), dehydrocholic (DHA), chenodeoxycholic, or ursodeoxycholic (URSO). The administration of Gris alone increased the activities of glutathione reductase (GRed), superoxide dismutase (SOD), alkaline phosphatase (AP), gamma glutamyl transpeptidase (GGT), and glutathione-S-transferase (GST), as well as total porphyrins, glutathione (GSH), and cytochrome P450 (CYP) levels in liver. Among the bile acids studied, DXA and DHA increased PROTO IX excretion, DXA also abolished the action of Gris, reducing lipid peroxidation and hepatic GSH and CYP levels, and the activities of GGT, AP, SOD, and GST returned to control values. However, porphyrin accumulation was not prevented by URSO; instead this bile acid reduced ALA-S and the antioxidant defense enzymes system activities. In conclusion, we postulate that DXA acid would be more effective to prevent liver damage induced by Gris.

Topics: Animals; Catalase; Chemical and Drug Induced Liver Injury; Chenodeoxycholic Acid; Dehydrocholic Acid; Deoxycholic Acid; Glutathione Peroxidase; Glutathione Reductase; Glutathione Transferase; Griseofulvin; Humans; Lipid Peroxidation; Mice; Oxidative Stress; Porphyrins; Protoporphyria, Erythropoietic; Superoxide Dismutase; Ursodeoxycholic Acid

Activation of the epidermal growth factor receptor (EGFR) plays an important role in liver regeneration and resistance to acute injury. However its chronic activation participates in the progression of liver disease, including fibrogenesis and malignant transformation. Hepatobiliary disease represents a constant feature in the clinically relevant Fechm1pas/Fechm1pas genetic model of erythropoietic protoporphyria (EPP). Similarly, chronic administration of griseofulvin to mice induces pathological changes similar to those found in patients with EPP-associated liver injury. We investigated the hepatic expression of the EGFR and its seven most relevant ligands in Fechm1pas/Fechm1pas mice bred in three different backgrounds, and in griseofulvin-induced protoporphyria. We observed that the expression of amphiregulin, betacellulin and epiregulin was significantly increased in young EPP mice when compared to aged-matched controls in all genetic backgrounds. The expression of these ligands was also tested in older (11 months) BALB/cJ EPP mice, and it was found to remain induced, while that of the EGFR was downregulated. Griseofulvin feeding also increased the expression of amphiregulin, betacellulin and epiregulin. Interestingly, protoporphyrin accumulation in cultured hepatic AML-12 cells readily elicited the expression of these three EGFR ligands. Our findings suggest that protoporphyrin could directly induce the hepatic expression of EGFR ligands, and that their chronic upregulation might participate in the pathogenesis of EPP-associated liver disease.

Topics: Amphiregulin; Animals; Betacellulin; Cell Line; EGF Family of Proteins; Epidermal Growth Factor; Epigen; Epiregulin; ErbB Receptors; Glycoproteins; Griseofulvin; Heparin-binding EGF-like Growth Factor; Intercellular Signaling Peptides and Proteins; Liver; Liver Diseases; Male; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Protoporphyria, Erythropoietic; Protoporphyrins; Reverse Transcriptase Polymerase Chain Reaction; Transforming Growth Factor alpha

Erythropoietic Protoporphyria (EPP) is a disease associated with ferrochelatase deficiency, which produces accumulation of protoporphyrin IX (PROTO IX) in erythrocytes, liver and skin. In some cases, a severe hepatic failure and cholestasis was observed. Griseofulvin (Gris) develops an experimental EPP with hepatic manifestations in animals. The aim of this work was to further characterize this model studying its effect on different metabolisms in mice Gris feeding (0-2.5%, 7 and 14 days). PROTO IX accumulation in liver, blood and feces, induction of ALA-S activity, and a low rate of Holo/Apo tryptophan pyrrolase activity was produced, indicating a reduction of free heme pool. The progressive liver injury was reflected by the aspect and the enlargement of liver and the induction of hepatic damage. Liver redox balance was altered due to porphyrin high concentrations; as a consequence, the antioxidant defense system was disrupted. Heme oxygenase was also induced, however, at higher concentrations of antifungal, the free heme pool would be so depleted that this enzyme would not be necessary. In conclusion, our model of Protoporphyria produced liver alterations similar to those found in EPP patients.

Topics: Animals; Antifungal Agents; Aryl Hydrocarbon Hydroxylases; Cytochrome P-450 CYP2A6; Cytochrome P-450 CYP3A; Disease Models, Animal; Griseofulvin; Heme; Heme Oxygenase (Decyclizing); Immunohistochemistry; Liver; Male; Mice; Oxidative Stress; Protoporphyria, Erythropoietic; Protoporphyrins; Tryptophan Oxygenase

BALB/c Fech(m1Pas) mice have a mutated ferrochelatase gene resulting in protoporphyria that models the hepatic injury occurring sporadically in human erythropoietic protoporphyria. We used this mouse model to study the development of the injury and to compare the dysfunction of heme synthesis with hepatic gene expression of liver metabolism, oxidative stress, and cellular injury/inflammation. From an early age expression of total cytochrome P450 and many of its isoforms was significantly lower than in wild-type mice. However, despite massive accumulation of protoporphyrin in the liver, expression of the main genes controlling heme synthesis and catabolism (Alas1 and Hmox1, respectively) were only modestly affected even in the presence of the cytochrome P450-inducing CAR agonist 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene. In contrast, in BALB/c mice exhibiting griseofulvin-induced hepatic protoporphyria with induction and destruction of cytochrome P450, both Alas1 and Hmox1 genes were markedly up-regulated. Other expression profiles in BALB/c Fech(m1Pas) mice identified roles for oxidative mechanisms in liver injury while modulated gene expression of hepatocyte transport proteins and cholesterol and bile acid synthesis illustrated the development of cholestasis. Subsequent inflammation and cirrhosis were also shown by the up-regulation of cytokine, cell cycling, and procollagen genes. Thus, gene expression profiles studied in Fech(m1Pas) mice may provide candidates for human polymorphisms that explain the sporadic hepatic consequences of erythropoietic protoporphyria.

Topics: Aging; Animals; Antifungal Agents; Cholestasis; Disease Models, Animal; Gene Expression; Gene Expression Profiling; Gene Expression Regulation; Griseofulvin; Heme; Hemeproteins; Immunoblotting; Liver; Male; Mice; Protoporphyria, Erythropoietic; Protoporphyrins; Reverse Transcriptase Polymerase Chain Reaction

Reduced activity of ferrochelatase in erythropoietic protoporphyria (EPP) results in protoporphyrin (PP) accumulation in erythrocytes and liver. Liver disease may occur in patients with EPP, some of whom develop progressive liver failure that necessitates transplantation. We investigated the mechanisms underlying EPP-associated liver disease in a mouse model of EPP.. Liver histology, indicators of lipid peroxidation, plasma parameters of liver function, and bile composition were studied in mice homozygous (fch/fch) for a point mutation in the ferrochelatase gene and in heterozygous (fch/+) and wild-type (+/+) mice.. Microscopic examination showed bile duct proliferation and biliary fibrosis with portoportal bridging in fch/fch mice. PP content was 130-fold increased, and thiobarbituric acid-reactive substances (+30%) and conjugated dienes (+75%) were slightly higher in fch/fch than in fch/+ and +/+ livers. Levels of hepatic thiols (-12%) and iron (-52%) were reduced in fch/fch livers. Liver enzymes and plasma bilirubin were markedly increased in the homozygotes. Plasma bile salt levels were 80 times higher in fch/fch than in fch/+ and +/+ mice, probably related to the absence of the Na(+)-taurocholate cotransporting protein (Ntcp) in fch/fch liver. Paradoxically, bile flow was not impaired and biliary bile salt secretion was 4 times higher in fch/fch mice than in controls. Up-regulation of the intestinal Na(+)-dependent bile salt transport system in fch/fch mice may enhance efficiency of bile salt reabsorption. The bile salt/lipid ratio and PP content of fch/fch bile were increased 2-fold and 85-fold, respectively, compared with +/+, whereas biliary glutathione was reduced by 90%. Similar effects on bile formation were caused by griseofulvin-induced inhibition of ferrochelatase activity in control mice.. Bile formation is strongly affected in mice with impaired ferrochelatase activity. Rather than peroxidative processes, formation of cytotoxic bile with high concentrations of bile salts and PP may cause biliary fibrosis in fch/fch mice by damaging bile duct epithelium.

Topics: Animals; Bile; Bile Acids and Salts; Biliary Tract; Blotting, Northern; Blotting, Western; Disease Models, Animal; Female; Ferrochelatase; Fibrosis; Griseofulvin; Immunohistochemistry; Lipid Peroxidation; Liver; Male; Mice; Mice, Inbred BALB C; Porphyria, Hepatoerythropoietic; Protoporphyria, Erythropoietic

The content of iron and protoporphyrin in liver mitochondria from mice with porphyria induced by griseofulvin was measured. The amount of porphyrin was 0.0076 +/- 0.0043, 4.11 +/- 0.58 and 22.2 +/- 6.8 nmol/mg protein (n = 5) in mitochondria from control animals and animals treated with griseofulvin for 3 days and 4-5 weeks, respectively. The energy coupling of the mitochondria was greatly diminished after 4-5 weeks of treatment, and the ferrochelatase activity was inhibited 80-90%, compared to that of control animals. Mitochondrial preparations isolated by differential centrifugation were contaminated with iron-containing lysosomes which could be removed by Percoll density-gradient centrifugation. In purified mitochondrial preparations no change in the amount of non-heme iron was found after griseofulvin feeding, representing 3.36 +/- 0.15, 3.97 +/- 0.40 and 3.59 +/- 0.23 nmol/mg protein for control animals, 3 days- and 4-5 weeks-treated animals, respectively (n = 4). A mitochondrial iron pool previously identified in rat liver mitochondria and shown to be available for heme synthesis in vitro (Tangerås, A. (1985) Biochim. Biophys. Acta 843, 199-207) was also present in mitochondria from mice. The magnitude of this iron pool, as well as its availability for heme synthesis, was not changed after treatment of the animals with griseofulvin. The fact that porphyrin, but not iron, accumulated in the mitochondria when ferrochelatase was inhibited is discussed with regard to our understanding of the process of heme synthesis and its regulation.

Topics: Animals; Chemical and Drug Induced Liver Injury; Griseofulvin; Heme; Iron; Liver Diseases; Lyases; Mice; Mitochondria, Liver; Oxidative Phosphorylation; Oxygen Consumption; Porphyrias; Porphyrins; Protoporphyria, Erythropoietic