RESUMO
Porphyria cutanea tarda (PCT) is caused by decreased activity of uroporphyrinogen decarboxylase (UROD) in the liver. The disease usually occurs in adulthood and is characterized by cutaneous photosensitivity, hyperpigmentation, skin fragility and hypertrichosis, due to the accumulation of porphyrins produced by oxidation of uroporphyrinogen and other highly carboxylated porphyrinogens overproduced as a result of the enzyme deficiency. PCT is generally sporadic, but about 20-30% of patients have familial-PCT (F-PCT) which is associated with heterozygosity of mutations in the UROD gene. In the present study we have found the molecular defect in seventeen unrelated Argentinean patients with F-PCT, identifying a total of eleven UROD gene mutations: four novel and seven previously described. The novel mutations were: a guanine insertion at the 5' splice junction of intron 2, a three nucleotide deletion causing the lost of valine 90, a deletion of 22 bp in exon 6 and a deletion of part of the polyadenylation signal. Prokaryotic expression studies showed that the novel amino acid deletion resulted in an inactive protein. Mutations c.10insA and p.M165R, previously found in Argentinean patients, were recurrent in this study; they are the most frequent in Argentina accounting for 40% of the mutant alleles characterized to date.
Assuntos
Predisposição Genética para Doença , Mutação/genética , Porfiria Cutânea Tardia/enzimologia , Porfiria Cutânea Tardia/genética , Uroporfirinogênio Descarboxilase/genética , Adolescente , Adulto , Argentina , Criança , Pré-Escolar , DNA/genética , Éxons/genética , Feminino , Humanos , Íntrons/genética , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Deleção de Sequência , Adulto JovemRESUMO
Hexachlobenzene (HCB), one of the most persistent environmental pollutants, induces porphyria cutanea tarda (PCT). The aim of this work was to analyze the effect of HCB on some aspects of glucose metabolism, particularly those related to its neosynthesis in vivo. For this purpose, a time-course study on gluconeogenic enzymes, pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase (PEPCK), glucose-6-phosphatase (G-6-Pase) and on pyruvate kinase (PK), a glycolytic enzyme, was carried out. Plasma glucose and insulin levels, hepatic glycogen, tryptophan contents, and the pancreatic insulin secretion pattern stimulated by glucose were investigated. Oxidative stress and heme pathway parameters were also evaluated. HCB treatment decreased PC, PEPCK, and G-6-Pase activities. The effect was observed at an early time point and grew as the treatment progressed. Loss of 60, 56, and 37%, respectively, was noted at the end of the treatment when a considerable amount of porphyrins had accumulated in the liver as a result of drastic blockage of uroporphyrinogen decarboxylase (URO-D) (95% inhibition). The plasma glucose level was reduced (one-third loss), while storage of hepatic glucose was stimulated in a time-dependent way by HCB treatment. A decay in the normal plasma insulin level was observed as fungicide intoxication progressed (twice to four times lower). However, normal insulin secretion of perifused pancreatic Langerhans islets stimulated by glucose during the 3rd and 6th weeks of treatment did not prove to be significantly affected. HCB promoted a time-dependent increase in urinary chemiluminiscence (fourfold) and hepatic malondialdehide (MDA) content (fivefold), while the liver tryptophan level was only raised at the longest intoxication times. These results would suggest that HCB treatment does not cause a primary alteration in the mechanism of pancreatic insulin secretion and that the changes induced by the fungicide on insulin levels would be an adaptative response of the organism to stimulate gluconeogenesis. They showed for the first time that HCB causes impairment of the gluconeogenic pathway. Therefore, the reduced levels of glucose would thus be the consequence of decreased gluconeogenesis, enhanced glucose storage, and unaffected glycolysis. The impairment of gluconeogenesis (especially for PEPCK) and the related variation in glucose levels caused by HCB treatment could be a consequence of the oxidative stress produced by the fungicide. Tryptophan adds its effect to this decrease in the higher phases of HCB intoxication, where its levels overcome the control values possibly owing to the drastic decline of URO-D. This derangement of carbohydrates leads porphyric hepatocytes to have lower levels of free glucose. These results contribute to our understanding of the protective and modulatory effect that diets rich in carbohydrates have in hepatic porphyria disease.
Assuntos
Fungicidas Industriais/toxicidade , Glucose/metabolismo , Hexaclorobenzeno/toxicidade , Fígado/efeitos dos fármacos , Porfiria Cutânea Tardia/induzido quimicamente , Porfiria Cutânea Tardia/enzimologia , Animais , Modelos Animais de Doenças , Inibidores Enzimáticos/toxicidade , Enzimas/metabolismo , Feminino , Gluconeogênese/efeitos dos fármacos , Gluconeogênese/fisiologia , Glucose-6-Fosfatase/metabolismo , Fígado/enzimologia , Fosfoenolpiruvato Carboxilase/metabolismo , Piruvato Carboxilase/metabolismo , Piruvato Quinase/metabolismo , Ratos , Ratos WistarRESUMO
We evaluated the porphyrinogenic ability of ethanol (20% in drinking water) per se, its effect on the development of sporadic porphyria cutanea tarda induced by hexachlorobenzene in female Wistar rats (170-190 g, N = 8/group), and the relationship with hepatic damage. Twenty-five percent of the animals receiving ethanol increased up to 14-, 25-, and 4.5-fold the urinary excretion of delta-aminolevulinate, porphobilinogen, and porphyrins, respectively. Ethanol exacerbated the precursor excretions elicited by hexachlorobenzene. Hepatic porphyrin levels increased by hexachlorobenzene treatment, while this parameter only increased (up to 90-fold) in some of the animals that received ethanol alone. Ethanol reduced the activities of uroporphyrinogen decarboxylase, delta-aminolevulinate dehydrase and ferrochelatase. In the ethanol group, many of the animals showed a 30% decrease in uroporphyrinogen activity; in the ethanol + hexachlorobenzene group, this decrease occurred before the one caused by hexachlorobenzene alone. Ethanol exacerbated the effects of hexachlorobenzene, among others, on the rate-limiting enzyme delta-aminolevulinate synthetase. The plasma activities of enzymes that are markers of hepatic damage were similar in all drug-treated groups. These results indicate that 1) ethanol exacerbates the biochemical manifestation of sporadic hexachlorobenzene-induced porphyria cutanea tarda; 2) ethanol per se affects several enzymatic and excretion parameters of the heme metabolic pathway; 3) since not all the animals were affected to the same extent, ethanol seems to be a porphyrinogenic agent only when there is a predisposition, and 4) hepatic damage showed no correlation with the development of porphyria cutanea tarda.
Assuntos
Etanol/farmacologia , Ferroquelatase/efeitos dos fármacos , Fígado/efeitos dos fármacos , Porfiria Cutânea Tardia/induzido quimicamente , Solventes/farmacologia , Uroporfirinogênio Descarboxilase/efeitos dos fármacos , Animais , Sistema Enzimático do Citocromo P-450/análise , Modelos Animais de Doenças , Feminino , Ferroquelatase/metabolismo , Hexaclorobenzeno , Fígado/enzimologia , Fígado/patologia , Porfobilinogênio/urina , Sintase do Porfobilinogênio/urina , Porfiria Cutânea Tardia/enzimologia , Porfiria Cutânea Tardia/urina , Porfirinas/urina , Ratos , Ratos Wistar , Uroporfirinogênio Descarboxilase/metabolismoRESUMO
Familial porphyria cutanea tarda (f-PCT) results from the half-normal activity of uroporphyrinogen decarboxylase (URO-D). Heterozygotes for this autosomal dominant trait are predisposed to photosensitive cutaneous lesions by various ecogenic factors, including iron overload and alcohol abuse. The 3.6-kb URO-D gene was completely sequenced, and a long-range PCR method was developed to amplify the entire gene for mutation analysis. Four missense mutations (M165R, L195F, N304K, and R332H), a microinsertion (g10insA), a deletion (g645Delta1053), and a novel exonic splicing defect (E314E) were identified. Expression of the L195F, N304K, and R332H polypeptides revealed significant residual activity, whereas reverse transcription-PCR and sequencing demonstrated that the E314E lesion caused abnormal splicing and exon 9 skipping. Haplotyping indicated that three of the four families with the g10insA mutation were unrelated, indicating that these microinsertions resulted from independent mutational events. Screening of nine f-PCT probands revealed that 44% were heterozygous or homozygous for the common hemochromatosis mutations, which suggests that iron overload may predispose to clinical expression. However, there was no clear correlation between f-PCT disease severity and the URO-D and/or hemochromatosis genotypes. These studies doubled the number of known f-PCT mutations, demonstrated that marked genetic heterogeneity underlies f-PCT, and permitted presymptomatic molecular diagnosis and counseling in these families to enable family members to avoid disease-precipitating factors.
Assuntos
Hemocromatose/genética , Mutação , Porfiria Cutânea Tardia/enzimologia , Porfiria Cutânea Tardia/genética , Uroporfirinogênio Descarboxilase/genética , Alelos , Substituição de Aminoácidos , Argentina , Sequência de Bases , Elementos de DNA Transponíveis , Estabilidade Enzimática , Éxons , Genes Dominantes , Triagem de Portadores Genéticos , Humanos , Íntrons , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Reação em Cadeia da Polimerase , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Deleção de Sequência , Uroporfirinogênio Descarboxilase/biossíntese , Uroporfirinogênio Descarboxilase/químicaRESUMO
BACKGROUND: Porphyria cutanea tarda (PCT) is due to a partial defect of hepatic uroporphyrinogen decarboxylase (URO-D). In the hereditary form, both hepatic and erythrocytic enzymes are altered, whereas in the acquired form, only the hepatic enzyme fails. There is a high prevalence of hepatitis C virus infection in patients with PCT, specially in those without family history of the disease. AIM: To study erythrocytic URO-D activity in order to find out whether hepatitis C virus infection is associated to the acquired form of PCT or unveils an inactive hereditary form. PATIENTS AND METHODS: URO-D activity was measured in red blood cells of normal controls, hepatitis C virus carriers without symptoms of PCT and patients with PCT, with and without family history of the disease, with and without anti hepatitis C virus antibodies. RESULTS: URO-D activity was similar in normal controls, patients with chronic liver disease associated to hepatitis C virus, and in patients with PCT without family history of the disease with and without hepatitis C virus antibodies. URO-D activity was lower in patients with PCT and family history of the disease, with and without hepatitis C virus antibodies. CONCLUSIONS: PCT in patients with hepatitis C virus infection is due to an acquired alteration of hepatic URO-D. Hepatitis C virus does not modify erythrocytic URO-D.