RESUMO
An in-situ experiment was performed to study metabolic responses of the freshwater mussel Diplodon chilensis to water contaminated by leachates from an open dump and cattle activity, in order to analyze both the effects of those contaminants on aquatic environments and the potential use of a native bivalve to evaluate the effects of anthropic influence and eutrophication. Bivalves from a reference site were cage-transplanted to a control site (site A) and to a temporal water pond (site B) over 30 and 60 periods. Water quality analyses revealed that the site B was affected by anthropogenic influence. Mussel's hemocytes from site B showed 50% lower reactive oxygen species production and 130% higher lysosomal membrane stability in the site B mussels. In addition, no oxidative stress was evident in gills, despite the elevated copper and iron concentrations recorded in the site B water samples (CuB = 0.3350 ± 0.0636 mg. L-1vs. CuA = 0.0045 ± 0.0007 mg. L-1; FeB = 3.8650 ± 0.4031 mg. L-1vs. FeA = 0.0365 ± 0.0049 mg. L-1). In contrast, the adductor muscle accumulated more Fe (~10-20-fold) than the gills and showed signs of oxidative stress, e.g. superoxide dismutase activity and TBARS levels were increased by 10% were 34%, respectively, in the site B compared with the site A after 60 days of exposure. Additionally, the adductor muscle showed signs of anaerobic metabolism activation. Cu is accumulated in gills from both sites' individuals, at 60 days, in concordance with the increase in the activity of the cu-containing enzyme cytochrome-c-oxidase. There was a reduction in the overall condition and digestive gland index in bivalves exposed at site B, associated with diminished levels of lipid and protein contents. Metal-pollution and eutrophication affects D. chilensis metabolism and is associated to tissue-specific exposure, anaerobic metabolism and general energetic condition depletion.
Assuntos
Bivalves/efeitos dos fármacos , Eutrofização , Metais Pesados/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Bivalves/enzimologia , Bivalves/metabolismo , Bovinos , Cobre/metabolismo , Água Doce , Brânquias/efeitos dos fármacos , Brânquias/metabolismo , Hemócitos/efeitos dos fármacos , Hemócitos/metabolismo , Metais Pesados/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , Poluentes Químicos da Água/metabolismo , Qualidade da ÁguaRESUMO
AIMS: To investigate multiple tolerance of Saccharomyces cerevisiae obtained through a laboratory strategy of adaptive evolution in acetic acid, its relation with enzymatic ROS detoxification and bioethanol 2G production. METHODS AND RESULTS: After adaptive evolution in acetic acid, a clone (Y8A) was selected for its tolerance to high acetic acid concentrations (13 g l-1 ) in batch cultures. Y8A was resistant to multiple stresses: osmotic, thermic, oxidative, saline, ethanol, organic acid, phenolic compounds and slow freeze-thawing cycles. Also, Y8A was able to maintain redox homeostasis under oxidative stress, whereas the isogenic parental strain (Y8) could not, indicating higher basal activity levels of antioxidative enzyme Catalase (CAT) and Gluthatione S-transferase (GST) in Y8A. Y8A reached higher bioethanol levels in a fermentation medium containing up to 8 g l-1 of acetic acid when compared to parental strain Y8. CONCLUSIONS: A multiple-stress-tolerant clone was obtained using adaptive evolution in acetic acid. Stress cross-tolerance could be explained by its enzymatic antioxidative capacity, namely CAT and GST. SIGNIFICANCE AND IMPACT OF THE STUDY: We demonstrate that adaptive evolution used in S. cerevisiae was a useful strategy to obtain a yeast clone tolerant to multiple stresses. At the same time, our findings support the idea that tolerance to oxidative stress is the common basis for stress cotolerance, which is related to an increase in the specific enzymes CAT and GST but not in Superoxide dismutase, emphasizing the fact that detoxification of H2 O2 and not O2 Ë is a key condition for multiple stress tolerance in S. cerevisiae.
Assuntos
Ácido Acético/farmacologia , Antioxidantes/metabolismo , Etanol/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologiaRESUMO
This study investigated metal accumulation and oxidative effects in mantle, gill and digestive gland of the ribbed mussel Aulacomya atra from the Argentinean North Patagonian coastline. Mussels were transplanted over an 18-month period from a site with low anthropogenic impact to a harbor site with higher seawater concentration of aluminum, chromium, copper, manganese, nickel and zinc. Total trace metal concentration in seawater did not change throughout the 18-month transplant in either site. A. atra bioaccumulated metals in digestive gland, gills and mantle at different levels. Digestive gland had the highest concentration of metals, especially towards the end of the transplant experiment in the harbor area. Mussels transplanted to the harbor site experienced an upregulation in their antioxidant system, which likely explains the lack of oxidative damage to lipids despite higher metal accumulation. These results demonstrate that A. atra selectively accumulates metals from the water column and their prooxidant effects depend on the tissue antioxidant defenses and the exposure time.
Assuntos
Trato Gastrointestinal/metabolismo , Brânquias/metabolismo , Metais/metabolismo , Mytilidae/metabolismo , Poluentes Químicos da Água/metabolismo , Animais , Argentina , Monitoramento Ambiental , Metais/análise , Estresse Oxidativo , Água do Mar/análise , Poluentes Químicos da Água/análiseRESUMO
Early juveniles of the crayfish Cherax quadricarinatus were exposed for 60 days to 10 and 40 mg/L of pure glyphosate (acid form) in freshwater. Mortality was 33 % at the highest concentration, while no differences in molting were noted among treatments. After the first month of exposure, weight gain was significantly (p < 0.05) reduced in the 40 mg/L group. At the end of the assay, lipid levels in muscle, as well as protein level in both hepatopancreas and muscle were significantly (p < 0.05) reduced. These results suggest long-term utilization of both lipid and protein as main energetic reserves, likely in response to the chronic stress associated with herbicide exposure. Besides, the lower pyruvate kinase activity in muscle suggests a possible metabolic depression in this tissue. The hemolymphatic ASAT:ALAT ratio showed higher levels than the control at the highest glyphosate concentration, indicating possible damage to several tissues.
Assuntos
Astacoidea/fisiologia , Glicina/análogos & derivados , Herbicidas/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Água Doce , Glicina/toxicidade , Crescimento e Desenvolvimento/efeitos dos fármacos , Hepatopâncreas , Metabolismo/efeitos dos fármacos , Muda/efeitos dos fármacos , Músculos/efeitos dos fármacos , Músculos/metabolismo , GlifosatoRESUMO
The aims of the present work were: (1) to investigate whether the strong decrease of liver uroporphyrinogen decarboxylase (UroD) activity observed in experimental porphyria cutanea tarda is due to alteration of the enzymatic protein and (2) to improve the knowledge about the normal liver enzyme. With these purposes, several physicochemical studies for enzymatic characterization were carried out comparatively on the 12-fold purified liver enzyme of both normal and hexachlorobenzene porphyric rat. The study shows that the enzyme from porphyric rats has a higher activation energy, lower reactivity index and lower optimum pH than the normal one. In addition, it did not reach the Vmax at any of the substrate concentrations assayed (up to 28 microM uroporphyrinogen III), while the normal enzyme reached the plateau around 14 microM. The porphyric enzyme appears to be more protected than the normal against the inhibitory action of several metals, particularly Cu2+ and Pb2+, and against thermal inactivation. Zn2+ did not affect enzymatic activity, whereas Cu2+, Hg2+, Fe2+, Pb2+, and Cd2+ lowered the activities of both normal and porphyric enzyme in a dose-related way. It was also observed that the larger the atomic radius in its hydrated state, the lower the effect of the metal. Neither glutathione nor dithiothreitol significantly altered enzymatic activity in the range of concentrations assayed. beta-Mercaptoethanol had diverse effects, as regards both the concentration assayed and the enzymatic sample used. Assays with cystine showed a dual behaviour of both normal and porphyric enzymatic activity. Western blots for both preparations revealed a single band (65 kDa) with a similar intensity. This study show that hexachlorobenzene treatment modifies the physicochemical properties of liver UroD leading to a sharp decrease of its activity, without affecting its antigenic reactivity probably as a consequence of changes at the conformational level promoted by the binding of its reported inhibitor.
Assuntos
Fungicidas Industriais/metabolismo , Hexaclorobenzeno/metabolismo , Fígado/enzimologia , Uroporfirinogênio Descarboxilase/metabolismo , Animais , Antígenos/imunologia , Feminino , Fungicidas Industriais/farmacologia , Hexaclorobenzeno/farmacologia , Concentração de Íons de Hidrogênio , Fígado/efeitos dos fármacos , Ratos , Ratos Wistar , TemperaturaRESUMO
The effects of hexachlorobenzene (HCB) administration on the development of porphyria and on changes in thyroid function and thyroid hormone metabolism were examined. Female Wistar rats were treated with HCB for 1 or 8 weeks. At both treatment times liver weight was notably increased with a slight change in thyroid weight at 8 weeks. Serum thyroxine (T4) levels were depressed, whereas levels of triiodothyronine (T3) were not depressed significantly at both treatment times. One or eight weeks of HCB treatment did not alter the incorporation and distribution of [125I] into intrathyroidal aminoacids. A 50% reduction in protein bound iodine (PB[125I]) was seen in both groups of animals. HCB altered [125I]T4 metabolism in rat liver slices, increasing T4 dehalogenation. HCB administration for 1 week did not affect urinary excretion of porphyrins or their precursors, or hepatic porphyrin content. The activity of aminolaevulinate synthase was not affected, but there was a 25% and 51% inhibition in porphyrinogen carboxy-lyase (PCL) activity for the uroporphyrinogen disappearance or the coproporphyrinogen formation respectively. After 8 weeks of HCB administration the rats showed a characteristic porphyria. Our results show that HCB treatment increased hepatic thyroxine metabolism, without alterations in thyroid hormone synthesis. Serum T4 and PCL activity behaved differently in both time- and dose-dependent studies, with serum T4 being the more sensitive parameter which responded at earlier times and lower doses.
Assuntos
Clorobenzenos/toxicidade , Hexaclorobenzeno/toxicidade , Fígado/efeitos dos fármacos , Porfirias/induzido quimicamente , Glândula Tireoide/efeitos dos fármacos , Tiroxina/metabolismo , Tri-Iodotironina/metabolismo , 5-Aminolevulinato Sintetase/metabolismo , Animais , Peso Corporal/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Hexaclorobenzeno/administração & dosagem , Radioisótopos do Iodo , Fígado/enzimologia , Fígado/metabolismo , Tamanho do Órgão/efeitos dos fármacos , Porfirinas/metabolismo , Ratos , Ratos Endogâmicos , Tiroxina/sangue , Tri-Iodotironina/sangueRESUMO
The present report deals with studies on porphyrins and porphyrinogen carboxy-lyase of red cells and urinary porphyrins from lead-intoxicated rabbits. It was shown that the free erythrocyte porphyrins are mixture of protoporphyrin 9, the main component, and minor proportions of Coproporphyrin, Uroporphyrin III and Phyriaporphyrin. Analysis of the urinary porphyrins deomonstrates the presence of Coproporphyrins III as the major component, together with 15-20% of other porphyrins: 10-14% 5-COOH, 1-2% 6-COOH, 2-3% 7-COOH porphyrin and 1-2% Uroporphyrin III. We have not been able to detect an increase of Uroporphyrin I. Assays of porphyrinogen carboxy-lyase activity in hemolysate supernatant using Uroporphyrinogen III and Phyriaporphyrinogen (Phyria'gen) III as substrates, showed the existence of a slight decrease of both decarboxylase activities, being more affected during the second stage, the Phyria'gen decarboxylation. A possible regulation mechanism responsible for the porphyrin picture is discussed.
Assuntos
Intoxicação por Chumbo/metabolismo , Porfirinogênios/biossíntese , Porfirinas/biossíntese , Animais , Eritrócitos/metabolismo , Feminino , Masculino , Porfirinas/sangue , Porfirinas/urina , Coelhos , Uroporfirinogênio Descarboxilase/sangueRESUMO
Red cell porphyrinogen carboxy-lyase activity was measured using uroporphyrinogen III as substrate in 18 normal persons, 7 male patients with porphyria cutanea tarda, 3 female patients with erythropoietic protoporphyria and 2 female patients with variegate porphyria. The mean values obtained in normal subjects were 0.151 nmol of uroporphyrinogen disappeared in 30 min per mg of protein, and 0.038 nmol of coproporphyrinogen formed in 30 min per mg of protein. We have not been able to detect significant differences between males and females. In porphyria cutanea tarda the enzyme activity was the same as in normal subjects considering either substrate disappearance or end product formation. The differences were not significant at the p less than 0.05 level. Patients with variegata porphyria also exhibited normal erythrocyte porphyrinogen carboxy-lyase activity. The enzyme activity of erythrocytes from patients with erythropoietic protoporphyria was higher than in normals; mean values for specific activities being 0.204 nmol of uroporphyrinogen disappeared, and 0.071 nmol of coproporphyrinogen formed. The significance of the results with respect to the chemical picture of different porphyrias is discussed.
Assuntos
Carboxiliases/sangue , Eritrócitos/enzimologia , Porfirias/enzimologia , Uroporfirinogênio Descarboxilase/sangue , Feminino , Humanos , Masculino , Porfirinogênios/metabolismoRESUMO
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
Early detection of toxic events induced by xenobiotics is necessary for a proper assessment of human risk after the exposure to those agents. The aim of this work was to evaluate the cell line HEp-2 as an experimental model to determine the genotoxic effects of sodium arsenate. To this end, we determined the metabolic activity cells by the MTT test on seven concentrations of arsenate that range from 27 to 135,000 μM, obtaining the median lethal concentration (LC50), the lowest observed effect concentration (LOEC), and the not observed effect concentration (NOEC) of sodium arsenate at 24 h of exposition. According to the cytotoxic response obtained, we evaluated the genotoxic effect of the 27 and 270 μM concentrations by using the micronucleus assay and chromosomal aberrations test. We found a statistically significant increase (p<0.05) in the frequency of micronuclei between control cultures and those exposed to the highest concentration of sodium arsenate. Furthermore, the frequencies of nucleoplasmic bridges and tripolar mitosis were significantly higher in cell cultures exposed to the above concentrations compared to the control cultures (p<0.05). The participation of the glutathione system as response to the arsenate exposition was also analyzed, and a statistically significant increase in the glutathione content was found in those cells exposed to 27 μM of arsenate. The Glutathione S-transferase activity did not increase in the exposed cells compared to control cells, suggesting that the arsenate reduction involved other metabolic pathways in the HEp-2 cells. These results confirm that, under the conditions carried out in this study, sodium arsenate is genotoxic for HEp-2 cells. Therefore, we suggest that this cell line would be a good model for the assessment of the cytotoxic and genotoxic effects of xenobiotics on human cells.
La detección temprana de eventos tóxicos inducidos por xenobióticos es necesaria para una adecuada evaluación del riesgo humano ante la exposición a dichos agentes. El objetivo de este trabajo fue evaluar a la línea celular HEp-2 como modelo experimental para determinar los efectos genotóxicos del arseniato de sodio. Para ello, se determinó la actividad metabólica de las células mediante el ensayo de MTT, en siete concentraciones de arseniato de sodio en el rango 27-135.000 μM, determinando la concentración letal media (LC50), la menor concentración de efecto observado (LOEC) y la mayor concentración de efecto no observado (NOEC) de arseniato de sodio para una exposición de 24 h. Teniendo en cuenta los datos de citotoxicidad, se evaluó el efecto genotóxico a las concentraciones 27 y 270 μM por medio del ensayo de micronúcleos y aberraciones cromosómicas, encontrando un aumento estadísticamente significativo en la frecuencia de micronúcleos entre el control y la mayor concentración arseniato de sodio ensayada. Además, la presencia de puentes nucleoplasmáticos y mitosis tripolar fue significativamente mayor en ambas concentraciones estudiadas con respecto al control. Se analizó la participación del sistema de glutatión como respuesta a la exposición al arseniato, encontrándose un aumento estadísticamente significativo en el contenido de glutatión en la concentración de arseniato de 27 μM. La actividad de la glutatión S-transferasa no aumentó, lo que sugiere que la reducción del arseniato implicó otra vía metabólica en las células HEp-2. Estos resultados confirman que el arseniato de sodio induce genotoxicidad en células HEp-2 en las condiciones realizadas en este estudio y por lo tanto este tipo de línea celular es un buen modelo para ensayos de citotoxicidad y genotoxicidad en los cuales se quiere evaluar el riesgo humano.
RESUMO
1. The aim of the present work is to shed light on the way of action of hexachlorobenzene (HCB) on hepatic ferrochelatase the mitochondrial enzyme which catalyzes the last step of haem biosynthetic pathway. 2. Some properties of this enzyme from normal and HCB porphyric rat liver were studied. 3. The present findings indicate that HCB treatment would modify the configuration of the enzyme perhaps allowing the active center of the porphyric ferrochelatase to be more exposed. 4. As a consequence it would show: (a) its higher affinity for the iron; (b) the shorter time necessary to form the intermediate enzyme-substrate, reflected both by the existence of a shorter lag and consequently a shorter pre incubation time. 5. However this modification elicited by the fungicide does not alter the submitochondrial distribution of the enzyme nor the optimal conditions for its measurement.
Assuntos
Clorobenzenos/farmacologia , Ferroquelatase/metabolismo , Hexaclorobenzeno/farmacologia , Liases/metabolismo , Mitocôndrias Hepáticas/enzimologia , Porfirias/enzimologia , Animais , Estabilidade Enzimática , Heme/biossíntese , Concentração de Íons de Hidrogênio , Porfirias/induzido quimicamente , RatosRESUMO
The present work studies the action of hexachlorobenzene (HCB) on the decarboxylation of uroporphyrinogen (Urogen) I and III and also on the decarboxylation of intermediate porphyrinogens of series III under different conditions using liver of normal and porphyric rats as enzyme source. The same enzyme is involved in the Urogen decarboxylation of both isomeric series I and III and catalyses the four steps in both cases. HCB affects all of them. HCB blocks the four steps of Urogen III decarboxylation to the same degree, as a function of intoxication time. HCB leads, in general, to an increase in the efficiency (Km/Vmax) of the porphyric system. These data can be interpreted as a reaction of the organism to overcome the enzymatic blockade.
Assuntos
Carboxiliases/metabolismo , Clorobenzenos , Hexaclorobenzeno , Fígado/enzimologia , Porfirias/enzimologia , Porfirinogênios/metabolismo , Uroporfirinogênio Descarboxilase/metabolismo , Uroporfirinogênios/metabolismo , Animais , Feminino , Porfirias/induzido quimicamente , Ratos , Ratos EndogâmicosRESUMO
1. The action of hexachlorobenzene (HCB) on hepatic ferrochelatase was investigated. 2. A direct action of HCB, pentachlorophenol, porphyrins and haem on this enzyme activity was discarded. 3. In HCB porphyric liver there is probably an activator tightly bound to the enzyme. 4. Pyridoxal phosphate (PPL) may be a cofactor of ferrochelatase from both normal and porphyric rats. 5. The PPL would be involved in the binding site of Fe2+ or at least in the approaching of Fe2+ to the active site of the enzyme. 6. The differences found between normal and porphyric preparations could be attributed to conformational changes elicited by the HCB.
Assuntos
Ferroquelatase/metabolismo , Hexaclorobenzeno/farmacologia , Fígado/enzimologia , Porfirias/enzimologia , Animais , Sítios de Ligação , Cromatografia em Gel , Cobre/farmacologia , Sulfato de Cobre , Feminino , Heme/farmacologia , Temperatura Alta , Pentaclorofenol/farmacologia , Porfirias/induzido quimicamente , Porfirinas/farmacologia , Conformação Proteica/efeitos dos fármacos , Fosfato de Piridoxal/metabolismo , Ratos , Ratos Endogâmicos , EspectrofotometriaRESUMO
1. Porphyrinogen carboxylase from the liver of normal and hexachlorobenzene porphyric rats was subjected to chemical modification using photo-oxidation with methylene blue, diethylpyrocarbonate, butane-2,3-dione, and phenylglyoxal. 2. All of these chemicals inactivated the enzyme from both sources. 3. Reversion of the diethylpyrocarbonate reaction with hydroxylamine as well as protection of the enzymes with uroporphyrinogen III indicated that histidine is involved at least in the first decarboxylation active site of the porphyrinogen carboxylyase, and perhaps in one or more sites where the removal of the other carboxyl groups take place. 4. Arginine seems not to be at the active site of the enzyme but at its environment since two diketones alter the enzyme activity, however the substrate did not protect the enzyme from the butane-2,3-dione modification. 5. Comparative studies between the enzyme from normal and porphyric animals suggest that the low enzyme activity from intoxicated animals could be due to alterations of its active centre environment produced by hexachlorobenzene treatment. This treatment seems to partially protect the active site of the porphyrinogen carboxylase from the modification reactions.
Assuntos
Carboxiliases/metabolismo , Hexaclorobenzeno/farmacologia , Fígado/enzimologia , Porfirias/enzimologia , Animais , Arginina , Sítios de Ligação , Carboxiliases/antagonistas & inibidores , Carboxiliases/química , Dietil Pirocarbonato/farmacologia , Compostos de Epóxi/farmacologia , Feminino , Histidina , Hidroxilamina , Hidroxilaminas/farmacologia , Azul de Metileno , Fotoquímica , Porfirias/induzido quimicamente , Ratos , Ratos Endogâmicos , Espectrofotometria , Uroporfirinogênios/farmacologiaRESUMO
The porphyrinogenic and carcinogenic ability of hexachlorobenzene (HCB) was assayed in male and female gold hamsters, and histological examinations of tissue alterations were performed. So it was studied, in liver: a) porphyrin content which was significantly increased at five months of HCB treatment, specially in males, and the pattern of accumulated porphyrins which was altered independent of the sex, b) haem pathway enzymes:delta aminolaevulinic acid synthase, ferrochelatase and porphyrinogen carboxylyase (PCL); among which only PCL appeared to be altered just at ten months of HCB feeding. While thyroid gland and kidney remained unaltered along the treatment time, liver and spleen exhibited a noticeable size variation and morphological alterations. In fact the spleen in treated animals was hypotrophic showing a red pulp less developed with respect to the Malpighian corpuscles and many macrophages with iron deposits. Respect to the liver, enlargement in size of hepatocytes, high content of iron deposits, no PAS positive structures in the cytoplasm, several small lipid droplets, microsteatosis although no cytonecrosis, polymorphic nuclei, and proliferations of nucleoli were observed. Therefore HCB is able to cause precancerous pathology and to induce porphyria in hamsters, but not hyperthyroidism, upon this experimental conditions. By the way, males were found to be a good experimental model, better than females, to study the earliest relations between porphyria and cancer.
Assuntos
Hexaclorobenzeno/toxicidade , Fígado/efeitos dos fármacos , Porfirinas/análise , Baço/efeitos dos fármacos , Animais , Cricetinae , Feminino , Heme/metabolismo , Hexaclorobenzeno/farmacologia , Rim/efeitos dos fármacos , Fígado/enzimologia , Fígado/patologia , Masculino , Mesocricetus , Tamanho do Órgão , Baço/patologia , Glândula Tireoide/efeitos dos fármacosRESUMO
Studies were carried out to elucidate if in the hexachlorobenzene (HCB) porphyria, total, nonhaem and haem iron contents in liver are altered and if any relation exists between these alterations and the hepatic porphyrinogen carboxy-lyase (PCL) decrease in rats treated with the drug. It was observed that in porphyric livers total and non-haem iron levels increased significantly as a consequence of HCB intoxication while this treatment produced a non significant decrease in the haem iron content. Enzymic preparations of porphyric livers filtered through Sephadex G-25 columns which separate the free iron and that has a content of iron-protein greater than those in normals, exhibited a strong inhibition of PCL. Chelating agents, alpha' alpha' bipyridyl and 8-hydroxyquinoline do not revert such inhibition. The effect in vitro of ferritin, haemin and inorganic iron at different concentrations on normal PCL activity was also assayed. So, it could be observed that inorganic iron and haemin produce slight inhibition of PCL when added in concentrations higher than those corresponding to a porphyric liver (0.08 mM and 10(-6) M, respectively, as mean in the incubation media). So, they have not physiological significance. Ferritin does not modify the decarboxylation process. From these results it arises that iron does not play a direct role in the decrease of PCL activity in the experimental porphyria by HCB, not being the inhibitor made evident by heating assays. Iron could perhaps stimulate the metabolization of HCB, giving rise to active metabolite.
Assuntos
Carboxiliases/metabolismo , Clorobenzenos/farmacologia , Hexaclorobenzeno/farmacologia , Ferro/metabolismo , Fígado/metabolismo , Porfirias/induzido quimicamente , Uroporfirinogênio Descarboxilase/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Heme/metabolismo , Ratos , Ratos EndogâmicosRESUMO
The present work tries to elucidate if the strong decrease of porphyrinogen carboxy-lyase (PCL) observed in the experimental porphyria caused by hexachlorobenzene (HCB) is due to the presence of an inhibitor or to a modification of the protein structure of the enzyme. For this purpose: a) cross-assays and heating ones were performed in order to look for the existence of a compound, that present in porphyric animals, would be responsible for the decrease of PCL activity found in them; b) the effect in vitro of HCB, HCB metabolites and other related compounds was studied to find the inhibitor of PCL activity and to look for a relation structure-inhibitory effect; c) hepatic PCL from porphyric and normal rats was purified, and enzyme properties were comparatively studied looking for structural differences between the enzymes obtained from both animal lots. The results indicate that: a) the heat deproteinized porphyric liver preparation produces an inhibition on the normal preparation, but it is smaller than the decrease produced in vivo by the HCB on this enzyme activity, thus other reasons may explain this behavior of PCL in intoxicated animals; b) the HCB had no effect on PCL activity, the phenolic compounds exhibited inhibitions of variable extent that were increased by the presence of electrophilic substituents on the benzenic ring. Pentachlorophenol, the main HCB metabolite, produced inhibition in the in vitro assays, but at doses that were not physiologically significant; thus, it seems not to be the inhibitor found in the heating assays; c) purification of 110 times for the hepatic PCL of both porphyric and normal rats was obtained. Incubation conditions, the effect of salts and chelating agents, the chromatographic behavior in DEAE-cellulose and Sephadex G-100 columns, were comparatively studied with both enzymatic preparations. The effect of sodium diethyldithiocarbamate, sodium pyrophosphate, dithiothreitol, temperature, pH and O2, as well as the chromatographic behavior, would suggest that structural differences in the PCL of porphyric animals may exist; the presence of a thermostable inhibitor could also contribute to the decrease of PCL activity due to HCB.
Assuntos
Carboxiliases/metabolismo , Porfirias/enzimologia , Dermatopatias/enzimologia , Animais , Carboxiliases/isolamento & purificação , Modelos Animais de Doenças , Feminino , Hexaclorobenzeno/farmacologia , Temperatura Alta , Fígado/enzimologia , Porfirias/induzido quimicamente , Ratos , Ratos Endogâmicos , Dermatopatias/induzido quimicamenteRESUMO
The aim of this work is to study the effect of thioctamide--the commercial form of alpha lipoic acid amide--on the porphyrinogenic action of hexachlorobenzene (HCB). For this purpose, porphyria was induced in rats by chronic HCB treatment, with or without simultaneous thioctamide administration. Two different groups of rats were used as reference: one treated with vehicle (control) and the other treated with thioctamide (TO). Urine delta aminolevulic acid, porphobilinogen, and porphyrin excretions were lower in the HCB + TO treated group than in the HCB group, and the same happened with liver uroporphyrin accumulation. On the other hand, the second stage of uroporphyrinogen-decarboxylase activity was significantly higher in the HCB + TO group than in the HCB group. delta aminolevulic acid synthase activity was higher in the HCB group. Hepatic thiobarbituric acid reactive substances were lower in HCB + TO group than in HCB group. Thus, we might suggest that TO would decrease HCB effects by means of its free radical scavenging ability, and by having a direct effect on uroporphyrinogen-decarboxylase activity.
Assuntos
Hexaclorobenzeno/metabolismo , Porfirias/induzido quimicamente , Porfirias/tratamento farmacológico , Ácido Tióctico/farmacologia , Animais , Feminino , Sequestradores de Radicais Livres/farmacologia , Ratos , Ratos Wistar , Fatores de TempoRESUMO
Hexachlorobenzene (HCB) administration to rats induces porphyria cutanea tarda, characterized by high levels of urinary porphyrins (> 40 micrograms/day) and accumulation of highly carboxylated porphyrins in liver (> 15 micrograms/g of tissue). Ethanol administration, under the conditions employed, was not porphyrinogenic and was able to diminish some of the responses elicited by HCB. Furthermore, ethanol and/or HCB administration leads to organ disturbances that involve oxidative stress. We have measured the changes in urinary chemiluminescence (CL) levels, as part of a systematic evaluation of the metabolic alterations in rats chronically treated with ethanol and/or HCB. The results, that constitute the first set of urinary CL data obtained from an animal model system, indicate that the measurement of the spontaneous urinary CL can constitute a fast, simple and sensitive method to evaluate disturbances associated with oxidative stress.
Assuntos
Etanol/toxicidade , Hexaclorobenzeno/toxicidade , Urina/química , Animais , Fígado/efeitos dos fármacos , Fígado/metabolismo , Medições Luminescentes , Estresse Oxidativo/efeitos dos fármacos , Porfiria Cutânea Tardia/induzido quimicamente , Porfiria Cutânea Tardia/metabolismo , Porfiria Cutânea Tardia/urina , Porfirinas/urina , Ratos , Ratos WistarRESUMO
Porphyrinogen carboxylyase from normal rat liver was subjected to purification methods. Two different purification protocols were used. In both cases, the initial steps consisted in obtaining a liver homogenate, followed by centrifugation, salt precipitation and phosphate gel absorption. Scheme I consisted in then submitted the preparation to DEAE-cellulose, followed by Sephacryl S-200 and Phenyl-sepharose sequential column chromatographies. Scheme II involved an affinity column followed by a Sephadex G-75 gel filtration column. In both cases, the enzyme was stored at -20 degrees C until its assay. The addition of 2mM dithiotreytol to the incubation media or to the enzyme extract before storage, did not help improve the activity nor the stability of the enzyme. Those fractions containing the maximal enzyme activity, detected using Uroporphyrinogen III or Pentacarboxy-porphyrinogen III as substrate, were not always present in the same tubes for the different columns employed. In addition, the degree of purification obtained in some steps was different according to the substrate employed. The results suggest the existence of at least two isoenzymes for rat liver porphyrinogen carboxy-lyase.