RESUMEN
Porphyrias are a heterogeneous group of metabolic disorders that result from the altered activity of specific enzymes of the heme biosynthetic pathway and are characterized by accumulation of pathway intermediates. Porphyria cutanea tarda (PCT) is the most common porphyria and is due to deficient activity of uroporphyrinogen decarboxylase (UROD). Acute intermittent porphyria (AIP) is the most common of the acute hepatic porphyrias, caused by decreased activity of hydroxymethylbilane synthase (HMBS). An Argentinean man with a family history of PCT who carried the UROD variant c.10_11insA suffered severe abdominal pain. Biochemical testing was consistent with AIP, and molecular analysis of HMBS revealed a de novo variant: c.344 + 2_ + 5delTAAG. This is one of the few cases of porphyria identified with both UROD and HMBS mutations and the first confirmed case of porphyria with dual enzyme deficiencies in Argentina.
RESUMEN
OBJECTIVE: Clubfoot is a complex congenital three-dimensional foot deformity, which affects 150,000-200,000 newborn babies annually around the world. A good understanding of the alignment of the two osseous columns and the lower leg of the ankle and foot complex is essential for evaluating the severity of clubfoot. The purposes of this study were to (1) develop an automated three-dimensional (3D) surface model of severe clubfoot based on two-dimensional (2D) slices of computed tomography (CT) images, (2) evaluate the alignment of foot bones relative to the ankle in severe clubfoot, and (3) examine the structural changes in the shape of the clubfoot. PATIENTS AND METHODS: Two-dimensional CT image was taken from a four-year-old child with a severe clubfoot. Subsequently, an automated and detailed 3D surface model of the severe clubfoot was developed from the 2D images by using MATLAB software programming. Then, the x, y, and z coordinate angles were automatically calculated for each bone in the foot relative to the ankle (lower end of the tibia) to determine the orientations and relationships among the bones. RESULTS: The relative position or orientation of each bone of the foot to the ankle of the severe clubfoot was objectively measured which was used to determine the orientation of each bone in the foot. Among the x, y, and z axes of the interested tarsal bones, the z axis represents the smallest moment of inertia, and the results showed that the bones in the x axis shifted medially with higher relative angle. CONCLUSIONS: This 3D objective measurement method for assessing clubfoot can be used to determine and classify the severity of clubfoot, as well as evaluate and monitor the progress of the clubfoot intervention based on the relative position of the tarsal bones. The method can also be used to quantify the relationship between the tarsal bones of the foot and lower end of the tibia. In addition, angular measurements can be used to assess other pathological conditions of the foot such as pes cavus and pes planus.
Asunto(s)
Tobillo/diagnóstico por imagen , Pie Equinovaro/diagnóstico por imagen , Pie/diagnóstico por imagen , Imagenología Tridimensional/métodos , Tibia/diagnóstico por imagen , Falanges de los Dedos del Pie/diagnóstico por imagen , Preescolar , Humanos , Interpretación de Imagen Asistida por Computador , Masculino , Modelos Anatómicos , Tomografía Computarizada por Rayos X/métodosAsunto(s)
Biopelículas/efectos de los fármacos , Fármacos Fotosensibilizantes/farmacología , Porfirinas/farmacología , Pseudomonas aeruginosa/fisiología , Staphylococcus aureus/fisiología , Biopelículas/efectos de la radiación , Luz , Microscopía Confocal , Microscopía Electrónica de Rastreo , Fármacos Fotosensibilizantes/química , Porfirinas/químicaRESUMEN
The use of endogenous protoporphyrin IX after administration of 5-aminolaevulinic acid (ALA) has led to many applications in photodynamic therapy (PDT). We have previously reported that the conjugation of ALA dendrimers enhances porphyrin synthesis. The first aim of this work was to evaluate the ability of ALA dendrimers carrying 6 and 9 ALA residues (6m-ALA and 9m-ALA) to photosensitise cancer cells. For this aim, we employed LM3 mammary carcinoma cells. In these tumour cells, at low concentrations porphyrin synthesis from dendrimers was higher compared to ALA, whereas at high concentrations, porphyrin synthesis was similar from both compounds. Topical application of ALA dendrimers on the skin overlying a subcutaneous LM3 implanted tumour showed no diffusion of the molecules either to distant skin sites or to the adjacent tumour, suggesting a promising use of the ALA macromolecules in superficial cancer models. As a second objective, we proposed the use of ALA-dendrimers in vascular PDT for the treatment of atherosclerosis. Thus, we focused our studies on ALA-dendrimer's selectivity towards macrophages in comparison with endothelial cells. For this aim we employed Raw 264.7 macrophages and HMEC-1 microvasculature cells. Porphyrin synthesis induced in macrophages by 6m-ALA and 9m-ALA (3 h, 0.025 mM) was 6 and 4.6 times higher respectively compared to the endothelial cell line, demonstrating the high affinity of ALA dendrimers for macrophages. On the other hand, ALA employed at low concentrations was slightly selective (1.7-fold) for macrophages. Inhibition studies suggested that ALA dendrimer uptake in macrophages is mainly mediated by caveloae-mediated endocytosis. Our main conclusion is that in addition to being promising molecules in PDT of superficial cancer, ALA dendrimers may also find applications in vascular PDT, since in vitro they showed selectivity to the macrophage component of the atheromatous plaque, as compared to the vascular endothelium.
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Adenocarcinoma/tratamiento farmacológico , Ácido Aminolevulínico/farmacología , Dendrímeros/farmacología , Neoplasias Mamarias Animales/tratamiento farmacológico , Fotoquimioterapia/métodos , Fármacos Fotosensibilizantes/farmacología , Ácido Aminolevulínico/química , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Dendrímeros/química , Endocitosis/efectos de los fármacos , Endocitosis/fisiología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Humanos , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Neoplasias Mamarias Animales/metabolismo , Ratones Endogámicos BALB C , Ratones Desnudos , Microvasos/efectos de los fármacos , Microvasos/metabolismo , Estructura Molecular , Trasplante de Neoplasias , Fármacos Fotosensibilizantes/química , Porfirinas/metabolismo , Piel/efectos de los fármacos , Piel/metabolismoRESUMEN
Photodynamic Therapy (PDT) is an anticancer treatment based on photosensitisation of malignant cells. The precursor of the photosensitiser Protoporphyrin IX, 5-aminolevulinic acid (ALA), has been used for PDT of bladder cancer. Silybin is a flavonoid extracted from Silybum marianum, and it has been reported to increase the efficacy of several anticancer treatments. In the present work, we evaluated the cytotoxicity of the combination of ALA-PDT and silybin in the T24 and MB49 bladder cancer cell lines. MB49 cells were more sensitive to PDT damage, which was correlated with a higher Protoporphyrin IX production from ALA. Employing lethal light doses 50% (LD50) and 75% (LD75) and additional silybin treatment, there was a further increase of toxicity driven by PDT in both cell lines. Using the Chou-Talalay model for drug combination derived from the mass-action law principle, it was possible to identify the effect of the combination as synergic when using LD75, whilst the use of LD50 led to an additive effect on MB49 cells. On the other hand, the drug combination turned out to be nearly additive on T24 cells. Apoptotic cell death is involved both in silybin and PDT cytotoxicity in the MB49 line but there is no apparent correlation with the additive or synergic effect observed on cell viability. On the other hand, we found an enhancement of the PDT-driven impairment of cell migration on both cell lines as a consequence of silybin treatment. Overall, our results suggest that the combination of silybin and ALA-PDT would increase PDT outcome, leading to additive or synergistic effects and possibly impairing the occurrence of metastases.
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Apoptosis/efectos de los fármacos , Fármacos Fotosensibilizantes/toxicidad , Silimarina/farmacología , Ácido Aminolevulínico/química , Ácido Aminolevulínico/farmacología , Ácido Aminolevulínico/toxicidad , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Sinergismo Farmacológico , Quimioterapia Combinada , Flavonoides/química , Flavonoides/farmacología , Flavonoides/uso terapéutico , Humanos , Luz , Silybum marianum/química , Silybum marianum/metabolismo , Fotoquimioterapia , Fármacos Fotosensibilizantes/química , Fármacos Fotosensibilizantes/uso terapéutico , Silibina , Silimarina/química , Silimarina/uso terapéutico , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Neoplasias de la Vejiga Urinaria/metabolismo , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
The aim of this paper was to investigate a collection of plant extracts from Argentina as a source of new natural photosensitizers (PS) to be used in Photodynamic Inactivation (PDI) of bacteria. A collection of plants were screened for phototoxicity upon the Gram-positive species Staphylococcus epidermidis. Three extracts turned out to be photoactive: Solanum verbascifolium flower, Tecoma stans flower and Cissus verticillata root. Upon exposure to a light dose of 55J/cm(2), they induced 4, 2 and 3logs decrease in bacterial survival, respectively. Photochemical characterisation of S. verbascifolium extract was carried out. PDI reaction was dependent mainly on singlet oxygen and to a lesser extent, on hydroxyl radicals, through type II and I reactions. Photodegradation experiments revealed that the active principle of the extract was not particularly photolabile. It is noticeable that S. verbascifolium -PDI was more efficient under sunlight as compared to artificial light (total eradication vs. 4 logs decrease upon 120min of sunlight). The balance between oxidant and antioxidant compounds is likely to be masking or unmasking potential PS of plant extracts, but employing the crude extract, the level of photoactivity of S. verbascifolium is similar to some artificial PS upon exposure to sunlight, demonstrating that natural resources can be employed in PDI of bacteria.
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Bacterias Grampositivas/efectos de los fármacos , Fármacos Fotosensibilizantes/farmacología , Extractos Vegetales/farmacología , Bignoniaceae/química , Bignoniaceae/metabolismo , Cissus/química , Cissus/metabolismo , Pruebas Antimicrobianas de Difusión por Disco , Flores/química , Flores/metabolismo , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Gramnegativas/efectos de la radiación , Bacterias Grampositivas/efectos de la radiación , Fotoblanqueo , Fármacos Fotosensibilizantes/química , Extractos Vegetales/química , Raíces de Plantas/química , Raíces de Plantas/metabolismo , Oxígeno Singlete/metabolismo , Solanum/química , Solanum/metabolismoRESUMEN
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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Antifúngicos/efectos adversos , Antioxidantes/farmacología , Enfermedad Hepática Inducida por Sustancias y Drogas/tratamiento farmacológico , Griseofulvina/efectos adversos , Animales , Antioxidantes/administración & dosificación , Biomarcadores/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Modelos Animales de Enfermedad , Glutatión/metabolismo , Hemo/metabolismo , Masculino , Ratones , Superóxido Dismutasa/metabolismoRESUMEN
'Candidatus Phytoplasma prunorum,' which causes European stone fruit yellows (ESFY), is the prevalent phytoplasma affecting Prunus spp. in Europe. It is closely related to 'Ca. P. pyri,' which causes pear decline (PD) in pear trees. Both phytoplasma belong to the ribosomal group 16Sr-X and are naturally transmitted by different species of Cacopsylla spp. (4). In North America, 'Ca. P. pyri' is responsible for peach yellow leaf roll (PYLR), transmitted by Cacopsylla pyricola from pear to peach trees (1). In Spain, 'Ca. P. prunorum' is widespread on Prunus spp., but its occurrence on Prunus persicae is very low and 'Ca. P. pyri' is present in every pear orchard (3). During 2012, a previously unreported syndrome including early reddening, leaf curling, decline, abnormal fruits, and in some cases chlorosis and death of peach trees was reported on peach in Lleida, northern Spain. Symptoms were different to ESFY and PYLR, in that flowering disorders such as ESFY or yellows were not apparent, and reddening and decline were the most common symptoms. The disease was present in a wide range of varieties and rootstocks, suggesting insect transmission in an area where C. pruni, vector of 'Ca. P. prunorum,' was not previously reported, but C. pyri was abundant in pear orchards. Shoot samples from 20 symptomatic peach trees were collected in seven orchards within a 2 km2 area with an estimated incidence of 40%, which was higher in the borders. DNA was extracted from 1 g of leaf midribs and phloem tissue and amplified with ribosomal universal primers P1/P7 followed by nested PCR with R16F2n/R16R2 and specific primers fO1/rO1 that target the 16Sr-X group (3). The final PCR products were digested with RsaI enzyme. Amplifications with non-ribosomal specific primers, Imp ESFY, Imp PD A and Imp PD B that amplify sequences of gene Imp, that encode a phytoplasma membrane protein, were also carried out (2). Tissue samples with ESFY and PD and peach seedlings were used as positive and negative controls, respectively. Amplified PCR products were sequenced and compared to sequences deposited in GenBank. Phytoplasmas were detected in 18 of the 20 samples analyzed. No phytoplasmas were detected in negative peach controls. All digestions of fO1/rO1 PCR products from peach samples showed a PD profile, while no ESFY profile was detected. All samples were positive with specific primers Imp PD A and B. None of the peach samples were positive with the specific Imp-ESFY primers. Sequencing of R16 and Imp PDA and B amplicons revealed the presence of a stable isolate. The sequences were submitted to the European nucleotide archive (ENA) with the accession nos. HG737345 and HG737344. Based on the 16S rDNA sequence, this strain is 100% homologous to the reference strain PD1 (GenBank Accession No. AJ542543) and 99.55% homologous to strain PD 33 Lib (GenBank FN600725) based on the Imp gene sequence. This is the first report of PD phytoplasma in peach trees in Spain, and the first report in Europe of PD phytoplasma causing economically important outbreaks in peach orchards, following a pattern that could be similar to PYLR in North America. This strain is genetically closer to some European or Middle Eastern PDs than to North American PYLR. References: (1) C. L. Blomquist et al. Plant Dis. 86:759, 2002. (2) J. L. Danet et al. Microbiology 157:438, 2011. (3) M. Garcia-Chapa et al. J. Phytopathol. 151:584, 2003. (4) E. Seemüller et al. Int. J. Syst. Evol. Microbiol. 54:1217, 2004.
RESUMEN
AIP is an acute liver disorder caused by a deficiency of porphobilinogen deaminase (PBGD) characterized by neuroabdominal symptoms. It is an autosomal dominant disease. However, homozygous dominant AIP (HD-AIP) have been described. In some cases erythrodontia was observed. CEP is an autosomal recessive disease produced by mutations in the uroporphyrinogen III synthase gene (UROS), characterized by severe cutaneous lesions and erythrodontia. The aim of the work was to establish the differential diagnosis of porphyria in a patient with abdominal pain, neurological attacks, skin symptoms and erythrodontia. The PBGD activity was reduced 50% and the genetic analysis indicated the presence of two genetic variants in the PBGD gene, p.G111R and p.E258G, a new genetic variant, revealing a case of heteroallelic HD-AIP. The patient, first diagnosed as a carrier of a dual porphyria: AIP / CEP based on the excretion profile of porphyrins, precursors and her clinical symptoms, would be an atypical case of human HD-AIP. These results would also suggest the presence of a phenocopy of the CEP, induced by an endogenous or exogenous factor. Our findings highlight the importance of genetic studies for a proper diagnosis of porphyria, prevention of its manifestation and its treatment.
Asunto(s)
Variación Genética , Hidroximetilbilano Sintasa/genética , Hígado/patología , Porfiria Intermitente Aguda/diagnóstico , Porfiria Intermitente Aguda/genética , Enfermedad Aguda , Adulto , Secuencia de Bases , Análisis Mutacional de ADN , Femenino , Heterocigoto , Humanos , Hidroximetilbilano Sintasa/metabolismo , Hígado/metabolismo , Datos de Secuencia Molecular , Mutación , Porfiria Intermitente Aguda/sangre , Porfiria Intermitente Aguda/orina , Porfirinas/sangre , Porfirinas/orina , Uroporfirinógeno III Sintetasa/genética , Uroporfirinógeno III Sintetasa/metabolismoRESUMEN
BACKGROUND: Combined inheritance of genetic variants in ferrochelatase gene (FECH) are implicated in clinical manifestation of Erythropoietic Protoporphyria (EPP). OBJECTIVE: Identify the genetic variants in FECH gene and their associations in the expression of EPP in Argentina. Determine the allelic frequency of polymorphic variants, associations in cis and its linkage disequilibrium. METHODS: The FECH gene was PCR-amplified and sequenced. Allelic variants of intragenic polymorphisms were identified by PCR followed by sequencing or restriction digestion analysis. Residual FECH activity was determined by prokaryotic expression in Escherichia coli JM109. Data were analyzed using Haploview and Statistix 9. RESULTS: Ten mutations were identified: three novel (p.S222N; p.R298X and p.R367X) and seven already known (g.12490_18067del; p.R115X; p.I186T; c.580_584delTACAG; c.598 + 1 G>T; p.Y209X and p.W310X). The p.R115X mutation was found in two families. The p.S222N mutation expressed 5% of normal activity. Only individuals who inherited a mutation combined in trans to a low expression allele c.1-251G, c.68-23T, and c.315-48C, showed clinical symptoms. The absence of c.315-48C variant was sufficient for not triggering EPP. However, these variants showed high levels of cosegregation and GTC haplotype is over-represented in EPP patients. CONCLUSION: In the dominant inheritance form of EPP, c.315-48C variant in trans to the mutated allele is sufficient to trigger the disease. The presence of GTC haplotype in all patients with dominant EPP could be due to the high level of cosegregation of c.315-48C with c.1-251G and c.68-23T variants in our population.
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Ferroquelatasa/genética , Variación Genética , Protoporfiria Eritropoyética/genética , Adolescente , Adulto , Argentina , Niño , Preescolar , Humanos , Persona de Mediana Edad , Mutación , Polimorfismo Genético , Protoporfiria Eritropoyética/diagnóstico , Adulto JovenRESUMEN
Photodynamic therapy (PDT) is an anticancer treatment based on light-induced destruction of photosensitised malignant cells. It has been reported that PDT strongly affects cell-cell and cell-substrate adhesion through the reorganization of some cytoskeletal and adhesion proteins. The aim of the present work was to study the changes induced by PDT employing aminolevulinic acid (ALA), on the cytoskeleton actin network and E-cadherin expression. We employed the normal mammary HB4a cell line and its tumor counterpart transfected with the oncogene H-Ras, which has been shown to be resistant to PDT. Ras insertion induces per se disorganization of both F-actin and E-cadherin distribution. ALA-PDT induces on HB4a cells a dramatic disorganization of actin stress fibers, resembling normal Ras-transfected cells. After 48h some features of disorganization remain present. In HB4a-Ras cells, F-actin exhibits signals of photodamage, but distribution is recovered 24h after treatment. On the other hand, PDT did not impact on E-cadherin distribution, other than a transient disorganization, which was recovered at 24h. Moreover, E-cadherin disorganization did not favoured cell-cell detachment after PDT of HB4a-Ras cells. Actin but not E-cadherin constitutes in this model an important target of PDT. The fact that some features of microfilament disorganization remain present in HB4a surviving cells but not in Ras-transfected cells, suggests that cytoskeletal structures such as F-actin may be involved in the mechanisms of resistance to PDT.
Asunto(s)
Actinas/metabolismo , Ácido Aminolevulínico/farmacología , Cadherinas/metabolismo , Fármacos Fotosensibilizantes/farmacología , Proteínas ras/metabolismo , Ácido Aminolevulínico/química , Adhesión Celular/efectos de los fármacos , Adhesión Celular/efectos de la radiación , Línea Celular , Citoesqueleto/efectos de los fármacos , Citoesqueleto/efectos de la radiación , Humanos , Luz , Glándulas Mamarias Humanas/efectos de los fármacos , Glándulas Mamarias Humanas/efectos de la radiación , Fármacos Fotosensibilizantes/química , Transfección , Proteínas ras/genéticaRESUMEN
In the search for possible new anti-cancer agents, we investigated the effects of 75 aqueous and methanol extracts from 41 Argentinean plant species. The effect in cell growth was evaluated in the LM2 mammary adenocarcinoma cells. In a second stage, the highly active selected extracts were assayed in 3 other tumour cell lines: melanoma B16, bladder MB49 and lung A549; and 3 normal cell lines: mammary Hb4a and keratinocytes PAM212 and HaCat. Eight methanol extracts were found to be highly cytotoxic: Collaea argentina leaf, Iochroma australe leaf, Ipomoea bonariensis flower, Jacaranda mimosifolia flower, Solanum amygdalifolium flower, Solanum chacoense leaf, Solanum sisymbriifolium flower and Solanum verbascifolium flower. However, extract inhibition on cell growth was highly dependent on cell type. In general, except for the highly resistant cell lines, the inhibitory concentrations 50% were in the range of 10-150 µg/ml The eight extracts highly inhibited cell growth in a concentration-dependent manner, and in general the methanol extracts were always more active than the aqueous. Murine cells appear to be more sensitive than human cells to the cytotoxic action of the plant extracts. The human melanoma B16 line was the most resistant to four of the extracts. In terms of selectivity, S. verbascifolium was the species which showed most selectivity for tumour cells. Overall, this is one of the first studies focusing on southern South American native plants and their biological effects. Since some species of 5 genera analyzed have been reported to possess different degrees of alkaloid content, we examined microtubule structures after extract treatments. The eight extracts induced destabilization, condensation and aggregation of microtubules in LM2 cells, although no depolarization, typical of Vinca alkaloids damage was observed. In a near future, antitumour activity of purified fractions of the extracts administered at non-toxic doses will be assayed in transplantable murine tumour models.
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Antineoplásicos/farmacología , Extractos Vegetales/farmacología , Argentina , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Flores/química , Humanos , Concentración 50 Inhibidora , Ipomoea/química , Lamiaceae/química , Phaseolus/química , Physalis/química , Hojas de la Planta/química , Solanum/química , Tubulina (Proteína)/metabolismoRESUMEN
The photodynamic activity of three photosensitizers (PS): AL-induced PPIX, the porphyrin derivative 5-(4-trimethylammoniumphenyl)-10, 5, 20-tris (2,4,6- trimethoxyphenyl) porphyrin (CP) and the molecular dyad porphyrin-C(60) (P-C(60)), the last two incorporated into liposomal vesicles, was evaluated on Hep-2 human larynx carcinoma cell line. ALA-induced accumulation of the endogenous PS PPIX, reached saturation values between 5 and 24 h incubation time; the maximal PPIX content was 5.7 nmol/106 cells. The same intracellular level was accumulated when the cationic porphyrin CP was used, while the amount of P-C(60) attained was 1.5 nmol/106 cells. Under violet-blue exciting light, the fluorescence of PPIX and P-C(60) was found in the cytoplasm showing a granular appearance indicating lysosomal localization. CP was mainly detected as a filamentous pattern characteristic of mitochondrial localization. No dark cytotoxicity was observed using 1mM ALA, 5 microM CP and 1 microM P-C(60) after 24 h incubation. Cell morphology was analyzed using Hoechst-33258, toluidine blue staining, TUNEL assay and DNA fragmentation, 24 h after irradiation with 54 J/cm2. When photosensitized with ALA and P-C(60), chromatine condensation characteristic of apoptotic cell death was found; instead, 58 % of necrotic cells were observed with CP. The results show that in the Hep-2 cells, of the three PS analyzed, the molecular dyad P-C(60) was more efficient than CP and PPIX, and confirm that PDT can induce different mechanisms of cell death depending on the PS and the irradiation dose.
Asunto(s)
Fármacos Fotosensibilizantes/metabolismo , Porfirinas/química , Ácido Aminolevulínico/química , Carcinoma/tratamiento farmacológico , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Fulerenos/química , Humanos , Neoplasias Laríngeas/tratamiento farmacológico , Luz , Liposomas/química , Liposomas/metabolismo , Microscopía Fluorescente , Fotoquimioterapia , Fármacos Fotosensibilizantes/uso terapéutico , Fármacos Fotosensibilizantes/toxicidad , Porfirinas/uso terapéutico , Porfirinas/toxicidad , Protoporfirinas/químicaRESUMEN
Endogenous production of Protoporphyrin IX (PpIX) is successfully exploited for photodynamic therapy (PDT) on malignant cells, following 5-aminolevulinic acid (ALA) administration and light irradiation. This treatment kills cancer cells by damaging organelles and impairing metabolic pathways via cellular reactive oxygen species (ROS) generation. We studied the efficiency of PpIX synthetized from ALA on ROS generation, in the Vincristine resistant (LBR-V160), Doxorubicin resistant (LBR-D160) and sensitive (LBR-) murine leukemia cell lines. Cells were incubated 4 hr with 1 mM ALA and then irradiated during different times with fluorescent light. One hour later, production of ROS was analyzed by flow cytometry using different fluorescent probes: Hydroethidine (HE) for superoxide anion, 2',7' Dichlorodihydrofluorescein diacetate (DCFH-DA) for hydrogen peroxide; mitochondrial damage was examined with 3,3' Dihexyloxacarbocyanine iodide (DiOC6). We found that superoxide anion production in the three cell lines increased with irradiation time whereas no peroxide hydrogen was detected. Mitochondrial damage also increased in an irradiation time dependent manner, being higher in the Vincristine resistant line. Previous studies have demonstrated that apoptotic cell death increased with irradiation time, which is consistent with these results, indicating that ROS are critical in ALA-PDT efficiency to kill malignant cells.
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Protoporfirinas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Ácido Aminolevulínico/química , Ácido Aminolevulínico/farmacología , Ácido Aminolevulínico/uso terapéutico , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Resistencia a Antineoplásicos , Leucemia/tratamiento farmacológico , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Fotoquimioterapia , Protoporfirinas/química , Superóxidos/metabolismo , Rayos UltravioletaRESUMEN
Hereditary Hemochromatosis (HH) is an iron overload syndrome caused by increased duodenal iron absorption, which leads to excessive iron deposition in parenchymal cells of the liver and mayor organs, causing cirrhosis, diabetes, cardiac failure, endocrine complications and arthritis. There are 6 types of HH related to mutations in the genes that encode proteins of iron metabolism. HH Type I is inherited as an autosomal recessive trait of mutations in HFE gene. We investigate the prevalence of C282Y, H63D and S65C mutations in 95 individuals (77 males, 18 females) bearing iron metabolism alterations to establish an early diagnosis of HH. Among this population, 58% carried mutations in the HFE gene (45 males, 10 females). H63D mutation was found in 32.6% of the subjects (29.5% in heterozygocity, 3.15% in homozygocity). S65C mutation was only detected in the heterozygous form (5.3% of the patients), 2 of them carried also H63D mutation. C282Y in heterozygocity was found in 15.8% of the individuals; but only 4.15% carried this mutation in homozygocity. Our findings are in agreement with the prevalence of the Mediterranean origin of most of our patients, where C282Y mutation is not as common as H63D mutation.
Asunto(s)
Hemocromatosis/genética , Antígenos de Histocompatibilidad Clase I/genética , Hierro/metabolismo , Proteínas de la Membrana/genética , Adolescente , Adulto , Edad de Inicio , Anciano , Argentina/epidemiología , Niño , Femenino , Frecuencia de los Genes , Genotipo , Hemocromatosis/epidemiología , Proteína de la Hemocromatosis , Heterocigoto , Homocigoto , Humanos , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , Prevalencia , Adulto JovenRESUMEN
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.
Asunto(s)
Antifúngicos/toxicidad , Griseofulvina/toxicidad , Hígado/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Animales , Hidrocarburo de Aril Hidroxilasas/metabolismo , Citocromo P-450 CYP2A6 , Citocromo P-450 CYP3A/metabolismo , Modelos Animales de Enfermedad , Hemo/metabolismo , Hemo Oxigenasa (Desciclizante)/metabolismo , Inmunohistoquímica , Hígado/patología , Masculino , Ratones , Protoporfiria Eritropoyética/inducido químicamente , Protoporfirinas/metabolismo , Triptófano Oxigenasa/metabolismoRESUMEN
Acute attacks of porphyria are most commonly precipitated by events that decrease heme concentrations. Enzyme inducing-drugs are the most important triggering factors, particularly in relation to anaesthesia. We have reported previously that Enflurane and Isoflurane produced significant heme metabolism alterations, indicating that the use of these anaesthetics in porphyric patients should be avoided. The aim of this work was to evaluate the effect of the anaesthetic Sevoflurane on heme pathway and drug metabolizing Phase I system in mice. To this end, animals received different doses of the anaesthetic (1-2 ml/kg) and were sacrificed at different times (5-60 min). Data revealed important alterations in the enzymes involved in Acute Intermittent Porphyria, such as an induction in hepatic 5-Aminolevulinic acid synthetase activity and a diminished Porphobilinogen deaminase activity in liver and blood 20 minutes after Sevoflurane administration to mice in a dose of 1.5 ml/kg. Heme oxygenase activity was also induced, indicating the onset of oxidative stress. Total CYP levels and CYP2E1 expression were enhanced. As a consequence of these events, heme free pool would be depleted. In conclusion, our results in mice would suggest that Sevoflurane should be used with caution and very careful control in porphyric patients.
Asunto(s)
Anestésicos por Inhalación/toxicidad , Hemo/metabolismo , Hígado/efectos de los fármacos , Éteres Metílicos/toxicidad , 5-Aminolevulinato Sintetasa/metabolismo , Animales , Citocromo P-450 CYP2E1/metabolismo , Hemo Oxigenasa (Desciclizante)/metabolismo , Hidroximetilbilano Sintasa/metabolismo , Hígado/enzimología , Masculino , Ratones , Estrés Oxidativo/efectos de los fármacos , SevofluranoRESUMEN
The N-methyl-diethyl-aspartate (NMDA) receptor has been reported to play an important role in several acute and chronic neuropathologic syndromes. 5-aminolevulinic acid (ALA) accumulates in acute porphyrias due to a deficiency in the heme biosynthetic pathway. Considering that glutamate uptake inhibition caused by ALA could be one of the reasons conducing to porphyric neuropathy, it was of interest to evaluate the effect of porphyrinogenic agents on NMDA glutamatergic system. To this end receptor levels and apparent affinity (Kd) were analyzed in mice brain cortex and cerebellum. NMDA levels were diminished after chronic Isoflurane anaesthesia in brain cortex. In cerebellum, a diminution was observed after acute Enflurane and Isoflurane and allylisopropylacetamide, while ethanol administration showed a significant increase. ALA administration diminished NMDA levels only in cerebellum. Affinity constant was only reduced in brain cortex after chronic Isoflurane treatment. In conclusion, glutamatergic system appears to be involved in the action of some of the porphyrinogenic drugs studied mainly in cerebellum. Receptors regulation should therefore be considered an important mechanism in the cellular response to specific drugs, with the aim of designing new therapies and elucidating the mechanisms leading to porphyric neuropathy and acute attack triggering.
Asunto(s)
Porfirinógenos/farmacología , Ácido Aminolevulínico/farmacología , Animales , Barbital/farmacología , Cerebelo/efectos de los fármacos , Cerebelo/metabolismo , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Enflurano/farmacología , Etanol/farmacología , Griseofulvina/farmacología , Isoflurano/farmacología , Masculino , Ratones , Receptores de N-Metil-D-Aspartato/metabolismoRESUMEN
Erythropoietic Protoporphyria (EPP) is an inherited deficiency of ferrochelatase, the last enzyme of the heme pathway. Under general anaesthesia, some patients develop neurological dysfunction suggesting upregulation in heme biosynthesis similar to that described for acute porphyrias after xenobiotic administration. Our aim has been to evaluate whether Isoflurane induces alterations in the heme pathway in a mouse model for EPP. Administration of Isoflurane (a single dose of 2 ml/kg, i.p) to wild-type (+/+), heterozygous (+/Fechm1Pas) and homozygous (Fechm1Pas/Fechm1Pas) mice, was evaluated by measuring the activity of delta-aminolevulinic acid synthetase (ALA-S) and Porphobilinogen-deaminase (PBG-D) in different tissues, as well as Heme oxygenase (HO), cytochrome P-450, CYP2E1 and glutathione levels in liver. Porphyrin precursors were measured in 24 h-urine samples. Fechm1Pas/Fechm1Pas mice receiving anaesthesia show enhanced ALA-S and CYP2E1 activities in the liver and increased urinary excretion of porphyrin precursors. No alterations were found in either PBG-D or HO activities. Diminished glutathione levels suggest that anaesthesia may produce oxidative stress in these animals. In conclusion, Isoflurane induces ALA-S activity and increased excretion of porphyrin precursors in EPP mice. These findings appear to confirm our previous hypothesis and indicate that Isoflurane may be an unsafe anaesthetic not only for patients with acute porphyrias but also for individuals with non acute porphyrias.
Asunto(s)
5-Aminolevulinato Sintetasa/metabolismo , Isoflurano/farmacología , Hígado/efectos de los fármacos , Hígado/enzimología , Protoporfiria Eritropoyética/metabolismo , Animales , Activación Enzimática/efectos de los fármacos , Inducción Enzimática/efectos de los fármacos , Glutatión/metabolismo , Hemo Oxigenasa (Desciclizante) , Hidroximetilbilano Sintasa/metabolismo , Ratones , Ratones Mutantes , Estrés Oxidativo/efectos de los fármacosRESUMEN
Hepatoerythropoietic Porphyria (HEP) is the rare homozygous form of Porphyria Cutanea Tarda (PCT). It is characterized clinically by the early onset of severe skin manifestations which can be confused with Congenital Erythropoietic Porphyria (CEP) or with PCT when the symptoms are mild. We describe the case of a 14 year-old child with skin manifestations similar to those observed in PCT. The biochemical assays ruled out a CEP as well as they suggested the development of a HEP. Although his symptoms were not severe enough to be HEP, the enzymatic activity was dramatically reduced to a 5% of normal values and the molecular analysis revealed the presence of two already known different mutations on the patient's URO-D gene, c.703 C>T and IVS9-1. Each parent carry one of the mutations, but they were absent in the brother. This is the first Argentinean HEP case ever described which appeared in a compound heterozygous form and less residual URO-D activity but associated to a mild phenotype.