RESUMEN

The herbicide atrazine (ATZ) is one of the most widely used pesticides in the world and is now under scrutiny for its alleged capacity to disrupt the endocrine system. Exhibiting negligible interaction with the estrogen receptor (ER), ATZ's mode of action remains to be elucidated. ATZ may act as an inducer of the enzyme aromatase, which converts androgens to estrogens, although other mechanisms should also be taken into consideration such as impairment of hepatic metabolism. Therefore we administered juvenile rainbow trout (Oncorhynchus mykiss) a dose of either 2 or 200 microg ATZ/kg, or of carrier control phosphate buffered saline (PBS) and we measured plasma concentrations of testosterone (T), 17beta-estradiol (E2) and vitellogenin (Vtg) 6 days after exposure. Simultaneously we analyzed hepatic gene expression of cytochrome P450 (CYP) 1A and pi-class glutathione S-transferase (GST-P), and catalase (CAT) activity. Although sex steroid levels showed no significant alterations, we found a dose-dependent increase in Vtg and a concomitant decrease in CYP1A. There was no effect of ATZ on GST-P mRNA levels but GST-P was positively correlated with CYP1A. Also, CYP1A was negatively correlated with liver CAT and E2, and varied with T concentrations in a hormetic manner. The results showed that ATZ can alter hepatic metabolism, induce estrogenic effects and oxidative stress in vivo, and that these effects are linked.

Asunto(s)

Atrazina/toxicidad , Disruptores Endocrinos/toxicidad , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Herbicidas/toxicidad , Hígado/efectos de los fármacos , Animales , Hidrocarburo de Aril Hidroxilasas/efectos de los fármacos , Hidrocarburo de Aril Hidroxilasas/metabolismo , Atrazina/administración & dosificación , Catalasa/efectos de los fármacos , Catalasa/metabolismo , Relación Dosis-Respuesta a Droga , Disruptores Endocrinos/administración & dosificación , Estradiol/sangre , Gutatión-S-Transferasa pi/efectos de los fármacos , Gutatión-S-Transferasa pi/metabolismo , Herbicidas/administración & dosificación , Homeostasis/efectos de los fármacos , Hígado/metabolismo , Oncorhynchus mykiss/metabolismo , Estrés Oxidativo/efectos de los fármacos , Testosterona/sangre , Vitelogeninas/sangre , Vitelogeninas/efectos de los fármacos

RESUMEN

Some species of macromycetes (mushrooms) consistently are found to contain high concentrations of toxic metals such as cadmium (Cd) and mercury (Hg), and consumption of wild-growing mushrooms is acknowledged as a significant source for Cd and Hg in humans. Yet little is known about the speciation of Cd and Hg in mushroom tissues. Here we present the first evidence of peptides of the phytochelatin family being responsible for binding a large fraction of Cd in caps of the macromycete Boletus edulis exposed to excess metals. Concentrations of Cd, Zn, Cu and Hg, as well as cytosolic Cd-binding capacity (CCBC), glutathione (GSH) and free proline (Pro) were quantified in fruiting bodies of B. edulis differentially exposed to a wide range of metals. Metal distribution among cytosolic compounds were investigated by size exclusion chromatography (SEC), followed by metal determinations with atomic absorption chromatography (AAS) and HR-ICP-MS. Cd-binding compounds in SEC elutates were investigated further by high performance liquid chromatography-mass spectrometry (HPLC-MS). CCBC was >90 times higher in the exposed group relative to the reference group (Mann-Whitney's P < 0.001), whereas concentrations of free Pro were almost identical for the two groups. For the whole study selection, CCBC correlated positively with metal exposure (Spearman's P < 0.001 for all four metals), suggesting dose-dependent induction of Cd-binding compounds by exposure to these metals, possibly as a defense mechanism. The presence of phytochelatins (PCs), a family of cystein-rich oligopeptides, was confirmed in Cd-containing SEC fractions by HPLC-MS. The appearance of more complex PCs was coupled to declining concentrations of GSH. To our knowledge this is the first report demonstrating the presence of PCs in a macromycete.

Asunto(s)

Agaricales/química , Agaricales/efectos de los fármacos , Glutatión/biosíntesis , Glutatión/aislamiento & purificación , Metales/metabolismo , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Glutatión/análisis , Espectrometría de Masas , Fitoquelatinas , Prolina/análisis , Unión Proteica , Espectrofotometría Atómica

RESUMEN

Brown trout (Salmo trutta) from two native populations from the Røros area in Central Norway, acclimated in mining-affected habitats to different levels of Cd/Zn and Cu, together with trout from a nearby unaffected river (reference) were transferred to a nearby lake with higher levels of Cu, Cd, and Zn than those in their respective native rivers. This experiment was conducted to gain information about the underlying resistance mechanisms developed in fish exposed to metal environments. The focus was on gill metal accumulation and transcription of the metal-responsive stress genes metallothionein-A (MT-A), Cu/Zn-superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), and heat shock protein 70 (HSP-70). The only shared response shown between the three groups after transfer were Cu accumulation and MT-A induction. The Cu-acclimated trout produced mucus to reduce the uptake of Cu into the gills. The MT-A levels were highest in the Cd/Zn-acclimated trout both before and after transfer. Before transfer, antioxidant transcription (SOD and GPx) was higher in gills of Cu-acclimated compared to the Cd/Zn-acclimated trout, but increased transcription of antioxidant stress genes was observed after transfer in both metal-acclimated groups. The metal-acclimated trout groups also showed an increase in the transcription of HSP-70. Compared to the reference population not previously exposed to metals, stress gene transcription increased faster in the metal-acclimated populations. The exception was induction of CAT, which appeared to be depressed after transfer in Cd/Zn-acclimated trout. The data indicate that acclimation to chronic metal exposure involves different strategies to cope with different metals and that these strategies involve both physiological mechanisms (mucus production) as well as metal-related stress gene transcription.

Asunto(s)

Branquias/efectos de los fármacos , Metales/toxicidad , Estrés Oxidativo/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Contaminantes Químicos del Agua/toxicidad , Animales , Cadmio/toxicidad , Catalasa/genética , Catalasa/metabolismo , Cobre/toxicidad , Branquias/metabolismo , Glutatión Peroxidasa/genética , Glutatión Peroxidasa/metabolismo , Glutatión Reductasa/genética , Glutatión Reductasa/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Metalotioneína/genética , Metalotioneína/metabolismo , Noruega , Estrés Oxidativo/fisiología , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Transcripción Genética/fisiología , Trucha , Zinc/toxicidad

RESUMEN

The antifreeze proteins (AFPs) are a family of proteins characterised by their ability to inhibit the growth of ice. These proteins have evolved as a protection against lethal freezing in freeze avoiding species. Metal stress has been shown to reduce the cold hardening in invertebrates, but no study has investigated how this type of stress affects the production of AFPs. This study demonstrates that exposure to cadmium (Cd), copper (Cu) and zinc (Zn) reduces the normal developmental increase in AFP levels in Tenebrio molitor larvae reared under summer conditions. Exposure to winter conditions, however stimulated the production of AFPs in the metal exposed larvae, and raised the concentrations of AFPs to normal winter levels. The reduced level of AFPs in metal-stressed animals acclimated to summer conditions seems to arise from alterations in the normal gene expression of AFPs. The results indicate that metal exposure may cause freeze avoiding insects to become more susceptible to lethal freezing, as they enter the winter with lowered levels of AFPs. Such an effect cannot be revealed by ordinary toxicological tests, but may nevertheless be of considerable ecological importance.

Asunto(s)

Proteínas Anticongelantes/metabolismo , Metales Pesados/farmacología , Tenebrio/metabolismo , Aclimatación/efectos de los fármacos , Animales , Peso Corporal/efectos de los fármacos , Frío , Regulación de la Expresión Génica , Hemolinfa/metabolismo , Proteínas de Insectos/metabolismo , Larva/metabolismo , Concentración Osmolar , Estaciones del Año , Tenebrio/efectos de los fármacos

RESUMEN

Induction of gene transcription for proteins and enzymes involved in metal-mediated oxidative stress were studied in brown trout transferred to a Cu-contaminated river in the Røros region in Central Norway. In addition to metallothionein (MT-A), Cu/Zn-superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR) gene transcription, protein levels of MT and enzyme activities of SOD and CAT were analyzed in gill, liver and kidney. MT-A, SOD and GR transcription increased significantly along with uptake of Cu in gills, while only transcription of MT-A was found to respond in liver and kidney during the exposure. Already present MT proteins in gills seemed to be oxidized during the exposure, probably caused by Cu-mediated oxidative stress, and no increase in MT protein levels were observed in gills. SOD and CAT enzyme levels were affected in all tissues during the exposure. A negative correlation between SOD and CAT activities was observed in gills, and we suggest that the activities of these enzymes were influenced not only through transcription. GPx and GR transcription levels correlated positively with each other in gills and liver, indicating their shared function in GSH-turnover. Levels of MT and activity of SOD and CAT dealing with metal-induced oxidative stress appear to be regulated not only through gene transcription, but also through post-translational mechanisms.

Asunto(s)

Cobre/toxicidad , Proteínas de Choque Térmico/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Trucha/metabolismo , Contaminantes Químicos del Agua/toxicidad , Animales , Catalasa/efectos de los fármacos , Catalasa/genética , Catalasa/metabolismo , Cobre/farmacocinética , Branquias/efectos de los fármacos , Glutatión Peroxidasa/efectos de los fármacos , Glutatión Peroxidasa/genética , Glutatión Peroxidasa/metabolismo , Glutatión Reductasa/efectos de los fármacos , Glutatión Reductasa/genética , Glutatión Reductasa/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Metalotioneína/efectos de los fármacos , Metalotioneína/genética , Metalotioneína/metabolismo , Noruega , Ríos , Superóxido Dismutasa/efectos de los fármacos , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Distribución Tisular , Contaminantes Químicos del Agua/análisis

RESUMEN

This study investigates the potential of emissions from a zinc smelter to induce oxidative damage to DNA and lipids in Boletus edulis, the king bolete. Concentrations of cadmium, zinc, copper, and mercury were determined in 16 fruit bodies collected near the smelter (exposed group), as well as in 15 reference samples. Frequency of apurinic/apyrimidinic (AP) sites in DNA (a pre-mutagenic DNA base modification) and concentration of lipid hydroperoxides were chosen as damage parameters. Concentrations of the four metals, as well as oxidative damage to DNA and lipids were significantly elevated in the exposed group (Mann-Whitney, P<0.001). Both damage parameters correlated positively with concentrations of cadmium, zinc or copper in fruiting bodies (Spearman's P<0.01). Frequency of AP sites correlated significantly with mercury in the fruit bodies (P<0.05), whereas the association between lipid hydroperoxides and mercury was insignificant. Frequency of AP sites correlated positively with concentration of lipid hydroperoxides (P<0.001). Negative trends for the associations between concentrations of metals and AP sites or lipid hydroperoxides in the reference group (significant only for mercury and lipid hydroperoxides; P<0.05) suggest that in B. edulis low concentrations of mercury, possibly also of other of the metals determined in the present study, may induce dose-response relationships of a hormetic ('J-shaped') nature.

Asunto(s)

Basidiomycota/efectos de los fármacos , ADN de Hongos/efectos de los fármacos , Metales Pesados/toxicidad , Cadmio/análisis , Cadmio/toxicidad , Cobre/análisis , Cobre/toxicidad , Daño del ADN , Cuerpos Fructíferos de los Hongos/química , Cuerpos Fructíferos de los Hongos/efectos de los fármacos , Peróxidos Lipídicos/análisis , Mercurio/análisis , Mercurio/toxicidad , Metales Pesados/análisis , Oxidación-Reducción , Picea , Contaminantes del Suelo/análisis , Contaminantes del Suelo/toxicidad , Zinc/análisis , Zinc/toxicidad

RESUMEN

The induction of defense systems against metal exposure was investigated in 48 wild-growing fruiting bodies of the king bolete (Boletus edulis) from two areas polluted with several transition metals from smelters, as well as five reference areas. To determine the degree of metal exposure, cadmium (Cd), zinc (Zn), and copper (Cu) were determined in caps of fruiting bodies by atomic absorption spectrophotometry (AAS), whereas mercury (Hg) was determined by cold vapor atomic fluorescence spectrometry (CVAFS). Caps were analyzed further with respect to relative activities of the antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT), as well as concentrations of total glutathione (GSHTOT = GSH + GSSG) and relative concentrations of heat shock protein 70 kDa (HSP70). The results showed that concentrations of the four metals, as well as SOD, CAT and HSP70, were significantly elevated in the exposed group (Mann-Whitney, P < or = 0.001). In contrast, GSHTOT was significantly lowered in the exposed group (P < or = 0.05). Significant positive correlations were established between concentrations of Cd, Zn, Hg, or Cu and activities of SOD (Spearman's P < or = 0.01 for the association between SOD and Cd, P < or = 0.001 for all other metal exposure parameters), CAT (P < or = 0.001 for all exposure parameters), or expression of HSP70 (P < or = 0.001 for all exposure parameters). Significant negative correlations were found between total GSH and Cd (P < or = 0.001), Zn (P < or = 0.001), or Hg (P < or = 0.05). We conclude that antioxidant enzymes are induced in wild-growing B. edulis exposed to environmentally relevant concentrations of potentially toxic transition metals; whereas the net consumption of GSH that occurs with increasing metal exposure may reflect GSH consumption by mechanisms of metal detoxification. Finally, the induction of HSP70 suggests that the antioxidant response and the mechanisms in which GSH is consumed are insufficient for protection against the harmful effects of severe metal stress.

Asunto(s)

Basidiomycota/fisiología , Farmacorresistencia Fúngica/genética , Contaminantes Ambientales/análisis , Regulación Fúngica de la Expresión Génica , Basidiomycota/química , Basidiomycota/genética , Cadmio/análisis , Catalasa/análisis , Cobre/análisis , Glutatión/análisis , Proteínas HSP70 de Choque Térmico/análisis , Mercurio/análisis , Metalurgia , Espectrometría de Fluorescencia , Espectrofotometría Atómica , Estadística como Asunto , Superóxido Dismutasa/análisis , Zinc/análisis

RESUMEN

Capillary zone electrophoresis (CZE) with cyclodextrin (CD) in the polyacrylamide-coated capillary was used to study metallothionein (MT) forms in the horse kidney preparation produced commercially by Sigma. It is known that CDs form complexes with hydrophobic amino acids. The results show that the presence of CD improves the separability of the various MT forms, including the MT-IA and the MT-IB forms, metallothionein aggregates, as well as the so far unidentified a and b forms. This was true both below and above the isoelectric points (pIs), although the migration times were somewhat longer at increasing CD concentrations for runs at constant voltage than with constant current.

Asunto(s)

Ciclodextrinas/química , Electroforesis Capilar/métodos , Metalotioneína/aislamiento & purificación , Isoformas de Proteínas/aislamiento & purificación , Animales , Caballos , Riñón/química

RESUMEN

Capillary zone electrophoresis in the polyacrylamide-coated capillary was used to study metallothionein (MT) isoforms at physiological pH in horse kidney and rabbit liver MT preparations produced commercially by Sigma. Evidence is put forward that MT develops oligomers or aggregates in its metal binding situation at these pH values in both species. For the horse kidney preparation two forms were found for both the MT-IA and the MT-IB forms, for the rabbit liver three forms could be seen for the MT-I form and two for the MT-II form. At pH values above the physiological range (pH 8-10) up to four forms could be seen for the MT-I form (MT-IA in the horse) in both preparations. Compared to the MTCd-II form, the rabbit liver MTZn-II form not only behaved electrophoretically identical, but also showed a corresponding oligomerization behaviour. Our results indicate that the oligomerized MT-I form in the rabbit liver and the MT-IA form in the horse kidney bind more Cd atoms than the expected number of 7 per monomer.

Asunto(s)

Biopolímeros/química , Metalotioneína/química , Animales , Electroforesis Capilar , Hígado/química , Conejos

RESUMEN

Mice were given either cadmium (Cd), copper (Cu) or zinc (Zn) ad lib, and levels of the metals in the heart, kidneys and liver were measured together with organ contents of sodium (Na) and calcium (Ca). The contents of Cd increased more than 100-fold in all organs, whereas Zn increased by a factor of 2-4. Copper accumulated only in the liver. Cadmium exposure caused the Na and Ca contents in the kidneys to increase by a factor of 2-3, but caused a statistically significant reduction in the Na content of the liver. Cadmium also caused a reduction in the Ca content of the heart. Copper caused a statistically significant doubling of the Na content in the heart, but a significant reduction in the Ca content in this organ. Zinc caused a reduction in the Ca content of the heart. However, the mechanisms behind these effects are not clear. The accumulation of Cd in the kidneys and heart was associated with a gradual change in the Na and Ca levels in these organs, but trace metal accumulation was not associated with any conspicuous changes in the Na or Ca contents in any other organ. Copper was not accumulated in heart, but Cu intake still had marked effects on the Na and Ca contents in this organ. Since the tissue contents of Na and Ca are likely to be physiologically important, these ions may have potential as biomarkers for toxic stress. Since the effects of Cd and Cu differed markedly, the tissue contents of Na and Ca may also be used in a trace metal-specific system of fingerprint biomarkers.

Asunto(s)

Calcio/metabolismo , Corazón/efectos de los fármacos , Riñón/efectos de los fármacos , Hígado/efectos de los fármacos , Sodio/metabolismo , Oligoelementos/administración & dosificación , Oligoelementos/farmacología , Animales , Biomarcadores/análisis , Cloruro de Cadmio/administración & dosificación , Cloruro de Cadmio/metabolismo , Cloruro de Cadmio/farmacología , Cloruros/administración & dosificación , Cloruros/metabolismo , Cloruros/farmacología , Cobre/administración & dosificación , Cobre/metabolismo , Cobre/farmacología , Dieta , Riñón/metabolismo , Hígado/metabolismo , Ratones , Miocardio/metabolismo , Tamaño de los Órganos , Oligoelementos/metabolismo , Aumento de Peso/efectos de los fármacos , Compuestos de Zinc/administración & dosificación , Compuestos de Zinc/metabolismo , Compuestos de Zinc/farmacología

RESUMEN

Capillary zone electrophoresis (CZE) was used to study metallothionein (MT) isoforms and non-MT components in the horse kidney MT preparation produced by Sigma. This technique was found to be well suited for studies of such forms. The non-MTs are heat resistant, they do not bind metal, but comigrate with MT in the various systems generally used for MT isolations. These forms may cause discrepancies between claimed MT content and sample weight as well as confusion in MT identification. The functional metal binding ability in the Sigma sample was measured, and the elution profile from an anion exchange column, commonly used for separation of tissue MTs, was determined in order to ascertain the MT-I isoform content. Optimum conditions for the separation of the MT isoforms have been studied in the polyacrylamide coated capillary at pHs below and above the isoelectric points (pIs) in various buffer systems. Evidence is put forward that MT forms oligomers or aggregates in the metal binding situation at pHs above pI. Our results may indicate that the oligomerized MT-IA form binds additional Cd atoms than the expected number of seven per monomer.

RESUMEN

Liver and kidneys from grey seals (Halichoerus grypus) in their natural habitats in Norwegian waters contain high levels of Cd and Hg as well as metallothionein (MT). The data show significant placental transfer of heavy metals in seals. The MT concentrations were higher in fetus liver and kidneys than in the liver and kidneys of juveniles and adults. A significantly higher proportion of the total Cd and Cu, as compared to Zn and Hg, in fetal liver was associated with MT. Only low amounts of metals and MT were found in juveniles. In kidneys, little metal was associated with MT, although high amounts of Cd were bound in fetal stages. Positive correlations were found between total metal, MT content, and MT-bound metals, particularly for Cd and Zn. Grey seal MT may have higher affinity to Zn than to Cd. The use of seal MT as a biomarker for environmental heavy metal exposure seems promising.