RESUMO

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.

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RESUMO

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.

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Porphyrias are a family of inherited diseases, each associated with a partial defect in one of the enzymes of the heme biosynthetic pathway. In six of the eight porphyrias described, the main clinical manifestation is skin photosensitivity brought about by the action of light on porphyrins, which are deposited in the upper epidermal layer of the skin. Porphyrins absorb light energy intensively in the UV region, and to a lesser extent in the long visible bands, resulting in transitions to excited electronic states. The excited porphyrin may react directly with biological structures (type I reactions) or with molecular oxygen, generating excited singlet oxygen (type II reactions). Besides this well-known photodynamic action of porphyrins, a novel light-independent effect of porphyrins has been described. Irradiation of enzymes in the presence of porphyrins mainly induces type I reactions, although type II reactions could also occur, further increasing the direct non-photodynamic effect of porphyrins on proteins and macro-molecules. Conformational changes of protein structure are induced by porphyrins in the dark or under UV light, resulting in reduced enzyme activity and increased proteolytic susceptibility. The effect of porphyrins depends not only on their physico-chemical properties but also on the specific site on the protein on which they act. Porphyrin action alters the functionality of the enzymes of the heme biosynthetic pathway exacerbating the metabolic deficiencies in porphyrias. Light energy absorption by porphyrins results in the generation of oxygen reactive species, overcoming the protective cellular mechanisms and leading to molecular, cell and tissue damage, thus amplifying the porphyric picture.

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RESUMO

Porphyrias are a family of inherited diseases, each associated with a partial defect in one of the enzymes of the heme biosynthetic pathway. In six of the eight porphyrias described, the main clinical manifestation is skin photosensitivity brought about by the action of light on porphyrins, which are deposited in the upper epidermal layer of the skin. Porphyrins absorb light energy intensively in the UV region, and to a lesser extent in the long visible bands, resulting in transitions to excited electronic states. The excited porphyrin may react directly with biological structures (type I reactions) or with molecular oxygen, generating excited singlet oxygen (type II reactions). Besides this well-known photodynamic action of porphyrins, a novel light-independent effect of porphyrins has been described. Irradiation of enzymes in the presence of porphyrins mainly induces type I reactions, although type II reactions could also occur, further increasing the direct non-photodynamic effect of porphyrins on proteins and macromolecules. Conformational changes of protein structure are induced by porphyrins in the dark or under UV light, resulting in reduced enzyme activity and increased proteolytic susceptibility. The effect of porphyrins depends not only on their physico-chemical properties but also on the specific site on the protein on which they act. Porphyrin action alters the functionality of the enzymes of the heme biosynthetic pathway exacerbating the metabolic deficiencies in porphyrias. Light energy absorption by porphyrins results in the generation of oxygen reactive species, overcoming the protective cellular mechanisms and leading to molecular, cell and tissue damage, thus amplifying the porphyric picture

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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.

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Some alterations in the protein structure of delta-aminolevulinic acid dehydratase (ALA-D) and porphobilinogen deaminase (PBG-D) induced by uroporphyrin (URO) and prototoporphyrin (PROTO) have been observed previously. To obtain further evidence of these phenomena, the absorption and fluorescence spectra of ALA-D and PBG-D and the total protein content of sulfhydryl and free amino groups were analyzed after exposure of the enzymes to URO I and PROTO IX, ALA-D and PBG-D were partially purified from bovine liver and exposed to URO I or PROTO IX, both in the dark and under UV light. All experiments were performed in the enzyme solutions after removing the porphyrins. Absorbance spectra changes in the region of 220-300 nm were registered, indicating the interaction of the porphyrins with the molecular structure of the enzymes. The main changes in the fluorescence spectra were observed in the spectral region of 555 nm, and only slight modifications in the spectral region of 340-360 nm; moreover, alterations were stronger upon UV irradiation and in the presence of URO I when compared with darkness and PROTO IX. Variations in total SH groups would suggest the formation of disulfur bridges induced by URO I and the rupture of some S-S groups induced by PROTO IX. The effect of porphyrins on free amino groups would reflect a combination of cross-linking and fragmentation of proteins. Structural changes were observed when the enzymes were exposed to the porphyrin both in the dark or under UV light; however, they were stronger in the latter condition. These results suggest that porphyrins per se could act directly on the protein structure and that this action would be enhanced upon UV irradiation.

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In all the cutaneous porphyrias, alterations in the heme pathway lead to an excessive production and accumulation of porphyrins. Absorption of light energy by circulating porphyrins induces reactive oxygen species generation, which provoke enzyme inactivation and protein structure changes. Protein structure alterations induced by porphyrins with different physico-chemical properties on delta-aminolevulinic acid dehydratase (ALA-D) and porphobilinogen deaminase (PBG-D) were examined. The action of uroporphyrin (URO), a highly hydrophilic porphyrin, and protoporphyrin (PROTO), most hydrophobic, was tested. ALA-D and PBG-D were partially purified from bovine liver and exposed to URO or PROTO, both in the dark and under UV light. All experiments were performed in solution after removing the porphyrins. Treatment with 10 microM URO I or 10 microM PROTO IX reduced the activity of ALA-D and PBG-D. This effect increased with increasing time of exposure to porphyrins. Solubility of the enzymes in buffer containing 3 M KCl decreased with increasing time of porphyrin treatment; this may be because of exposure of hydrophobic residues that are normally shielded in the native protein structure. Tryptic digestion of ALA-D and PBG-D exposed to URO I or PROTO IX resulted in an increase of protein degradation products, indicating an enhanced susceptibility to proteolysis. Fluorescence emission of several enzymes aminoacids was greatly modified. The structural changes described were observed when the enzymes were exposed to porphyrins both in the dark or under UV light. However, they were more noticeable with UV light. These results suggest that porphyrins per se can act directly on protein structure and that this action may be enhanced by UV irradiation.

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Aerobic and anaerobic studies have demonstrated that uroporphyrin I-induced inactivation of delta-aminolevulinic acid dehydratase, porphobilinogenase, deaminase and uroporphyrinogen decarboxylase was dependent on oxygen and mediated by reactive oxygen species. The mechanism of photoinactivation of those heme-enzymes from human erythrocytes by uroporphyrin I by u.v. light was investigated. Enzymes of the heme pathway were preincubated in the presence of specific scavengers for several reactive oxygen species and then exposed to uroporphyrin I and u.v. light. Upon exposure of the enzymes to the porphyrin under u.v. light, and in an aerobic atmosphere, the percentage of enzyme activities with respect to the corresponding controls were 50.2 +/- 5.1 (SD, n = 6), 25.3 +/- 3.0 (SD, n = 6), 25.9 +/- 2.8 (SD, n = 6) and 49.7 +/- 7.5 (SD, n = 8) for delta-aminolevulinic acid dehydratase, porphobilinogenase, deaminase and uroporphyrinogen decarboxylase, respectively. The presence of sodium azide, histidine or superoxide dismutase did not protect the enzymes against the effects of uroporphyrin I. However, both cysteine and potassium ferrycyanide prevented the enzyme photoinactivation induced by uroporphyrin I. In the presence of either catalase or GSH, the enzyme photoinactivation was lower. Ethanol, glucose and dimethylsulfoxide had no effect on enzyme activity, while ion chelators had variable effects. This study shows that the type II mechanism is not the predominant reaction mediating the uroporphyrin I effect and enzyme photoinactivation would involve an electron transfer. Hydrogen peroxide and hydroxyl radicals could possibly mediate the uroporphyrin I-induced enzyme photoinactivation.

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The effect of uroporphyrin I (UI) on several cytosolic and mitochondrial enzymes (succinyl CoA synthetase, delta-aminolevulinic acid synthetase, rhodanese, lactate dehydrogenase) has been examined. All the enzymes were inactivated in the presence of the porphyrin both in the dark and under UV light.

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1. The action of uroporphyrin I on erythrocytic ALA-D activity under dark and light conditions was examined. 2. Photo and non-photoinactivation of ALA-D induced by uroporphyrin I were observed. 3. Both effects were dependent on uroporphyrin concentration, temperature and time of exposure of the protein to the porphyrin. 4. Light-dependent effect of uroporphyrin I is related with the phototoxicity of porphyrins and could be produced by primary amino acid photooxidation followed by secondary cross-linking of the protein. 5. Light-dependent effect of uroporphyrin I could be ascribed to a direct enzyme inhibition due to binding of the porphyrin to the protein inducing structural changes at or near its active site.

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1. The effect of URO I on the activity of ALA-D, PBGase, deaminase and URO-D, both in aerobiosis and anaerobiosis, was studied. 2. Photoinactivation of the enzymes was much lower in an anaerobic than in an aerobic atmosphere. 3. Dark inactivation in the absence of oxygen was lower than its presence. 4. Preincubation in the presence of ALA or PBG protected the enzymic activity of ALA-D, PBGase and deaminase against URO I-inactivation both under u.v. light and in the dark. 5. Photoinactivating action of URO I would be mediated by reactive oxygen species generated by the excited porphyrin after its absorption of light. Dark inactivation, in aerobiosis, can also be partly mediated by amino acid oxidation, although to a lesser extent than that observed under u.v. light.

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1. URO-D was investigated in crude extracts from mouse mammary carcinoma, normal mouse (NM) liver and tumor-bearing mouse (TBM) liver. 2. URO-D from TBM liver and tumor appears to be more sensitive to increasing concentrations of UROgen than the NM liver enzyme. 3. In tumor the rate-limiting step seems to be the decarboxylation of the first carboxyl group, but this was not so clear for the NM and the TBM liver URO-D. 4. URO-D activity was enhanced when incubated at higher temperatures in the presence of its substrate, suggesting that UROgen might afford some protection of the enzyme against heat inactivation. 5. The optimum pH for all three sources is around 7.0.

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The action of uroporphyrin I (URO I) on the activity of red cell uroporphyrinogen decarboxylase (URO-D) in the dark and under UV light was studied. Light-dependent-and light-independent inactivation was observed. Both effects increased at increasing concentrations of URO I, the former reached its maximum at 150 microM of sensitizer. At 100 microM of URO I, both light and dark inactivation were temperature dependent amounting to about 50% at 30-37 degrees C. The velocity of dark inactivation increased with increasing temperature in the range of 0 to 45 degrees C. Photoinactivation can be ascribed to primary oxidation of essential amino acids, very likely histidyl residues, followed by secondary inter or intrapeptide cross-linking. Dark inactivation could be the result of both oxidation and cross-linking (although to a less degree than that produced by light) and also direct inhibition of the enzyme by induced conformational changes at its active site through binding of the porphyrin to the protein. When the action of URO I was tested on partially purified URO-D, the enzyme appeared to be more susceptible to the dark than to the light effect.

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RESUMO

1. A clear biphasic response of the enzyme activities as a function of intoxication time due to the topical cutaneous griseofulvin treatment was observed. 2. The initial acute induction of ALA-S activity would be due to depletion of free heme in the regulatory pool caused by cytochrome P 450 destruction. 3. The second induction peak, would be due to less heme formation, secondary to the ferrochelatase inhibition, as expected for the erythropoietic protoporphyria model. 4. The biphasic response of hepatic ALA-D and PBGase activities would be related to ALA-S activity changes and the subsequent augmented available substrates. 5. Endogenous liver porphyrin distribution in cytosolic, mitochondrial and nuclear fractions was investigated. 6. The in vitro biosynthesis of porphyrins confirmed both the biphasic model and the hepatic porphyrins subcellular distribution. 7. Two mechanisms to explain the action of griseofulvin at shorter and longer times of intoxication are proposed.

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The action of porphyrins, uroporphyrin I and III (URO I and URO III), pentacarboxylic porphyrin I (PENTA I), coproporphyrin I and III (COPRO I and COPRO III), protoporphyrin IX (PROTO IX) and mesoporphyrin (MESO), on the activity of human erythrocytes delta-aminolevulinic acid dehydratase, porphobilinogenase, deaminase and uroporphyrinogen decarboxylase in the dark and under UV light was investigated. Both photoinactivation and light-independent inactivation was found in all four enzymes using URO I as sensitizer. URO III had a similar action as URO I on porphobilinogenase and deaminase and PROTO IX exerted equal effect as URO I on delta-aminolevulinic acid dehydratase and uroporphyrinogen decarboxylase. Photodynamic efficiency of the porphyrins was dependent on their molecular structure. Selective photodecomposition of enzymes by URO I, greater specificity of tumor uptake by URO I and enhanced porphyrin synthesis by tumors from delta-aminolevulic acid, with predominant formation of URO I, underline the possibility of using URO I in detection of malignant cells and photodynamic therapy.

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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.

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- Se han estudiado 16 pacientes con diagnóstico de PCT sintomática, 12 hereditarios y 4 adquiridos, y un caso de PCT latente. - Se llevó a cabo, en todos, un estudio bioquímico completo acerca de la excreción urinaria y fecal de porfirinas, medición de las actividades enzimáticas de Aminolevúlico Dehidrasa, Porfobilinogenasa, Deaminasa y Uroporfirinógeno Decarboxilasa en eritrocitos. De 9 de esos pacientes se obtuvieron biopsias hepáticas, en las cuales se determinaron porfirinas endógenas y actividad de URO-D. - Se estableció una correlación entre los niveles de porfirinas urinarias y hepáticas. - En todos los pacientes, independientemente de que pertencieran al tipo hereditario o no, la actividad de ALA-D se encontró dentro del rango normal, en tanto que PBG-asa y Deaminasa estaban ligeramente aumentadas, confirmendo datos anteriores de este mismo laboratorio. - La actividad de la URO-D en sangre de pacientes con PCT hereditaria estaba notablemente reducida con respecto a los controles, en tanto que los valores de actividad en PCT adquiridas fueron normales. - La URO-D eritrocitaria disminuye su actividad al incrementarse la concentración de porfirinas urinarias y hepáticas en la PCT hereditaria, no así en la adquirida. - Tanto en PCT hereditaria como adquirida, la URO-D hepática se encuentra significativamente inhibida y esta inhibición es mayor cuanto mayores son los niveles de porfirinas hepáticas y urinaria. - La determinación de la URO-D eritrocitaria permite diferenciar claramente una PCT hereditaria de una adquirida

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- Se han estudiado 16 pacientes con diagnóstico de PCT sintomática, 12 hereditarios y 4 adquiridos, y un caso de PCT latente. - Se llevó a cabo, en todos, un estudio bioquímico completo acerca de la excreción urinaria y fecal de porfirinas, medición de las actividades enzimáticas de Aminolevúlico Dehidrasa, Porfobilinogenasa, Deaminasa y Uroporfirinógeno Decarboxilasa en eritrocitos. De 9 de esos pacientes se obtuvieron biopsias hepáticas, en las cuales se determinaron porfirinas endógenas y actividad de URO-D. - Se estableció una correlación entre los niveles de porfirinas urinarias y hepáticas. - En todos los pacientes, independientemente de que pertencieran al tipo hereditario o no, la actividad de ALA-D se encontró dentro del rango normal, en tanto que PBG-asa y Deaminasa estaban ligeramente aumentadas, confirmendo datos anteriores de este mismo laboratorio. - La actividad de la URO-D en sangre de pacientes con PCT hereditaria estaba notablemente reducida con respecto a los controles, en tanto que los valores de actividad en PCT adquiridas fueron normales. - La URO-D eritrocitaria disminuye su actividad al incrementarse la concentración de porfirinas urinarias y hepáticas en la PCT hereditaria, no así en la adquirida. - Tanto en PCT hereditaria como adquirida, la URO-D hepática se encuentra significativamente inhibida y esta inhibición es mayor cuanto mayores son los niveles de porfirinas hepáticas y urinaria. - La determinación de la URO-D eritrocitaria permite diferenciar claramente una PCT hereditaria de una adquirida (AU)

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