Protective effect of glutathione S-transferase enzyme activity against aflatoxin B1 in poultry species: relationship between glutathione S-transferase enzyme kinetic parameters, and resistance to aflatoxin B1.

Comparative studies designed to investigate the role of glutathione S-transferase (GST) activity on the enzyme catalyzed trapping of aflatoxin B1-8,9-epoxide (AFBO) with glutathione, and the relationship with aflatoxin B1 (AFB1) resistance have not been conducted in poultry. Hepatic cytosolic fractions of chickens, quail, turkeys and ducks were used to measure in vitro the enzymatic parameters maximal velocity (Vmax), Michaelis-Menten constant (Km) and intrinsic clearance (CLint) for GST activity. AFB1 used ranged from 2.0 to 157.5 µM and the AFB1-GSH produced was identified and quantitated by HPLC. Significant differences were found in GST Vmax values, being the highest in chickens, followed by quail, ducks and turkeys. The Km values were also significantly different, with chickens < ducks < turkeys < quail. Chickens had the higher CLint value in contrast to ducks. Differences by sex showed that duck females had a higher CLint value than the turkey and quail, whereas duck males had a CLint close to that of turkey. The ratio "AFBO production /AFB1-GSH production" follows the order duck>turkey>quail>chicken, in agreement with the known poultry sensitivity. The extremely high "AFB1 epoxidation activity/ GST activity" ratio observed in ducks might be the explanation for the development of hepatocellular carcinoma in this species.

Dealing with aflatoxin B1 dihydrodiol acute effects: Impact of aflatoxin B1-aldehyde reductase enzyme activity in poultry species tolerant to AFB1 toxic effects.

Aflatoxin B1 aldehyde reductase (AFAR) enzyme activity has been associated to a higher resistance to the aflatoxin B1 (AFB1) toxicity in ethoxyquin-fed rats. However, no studies about AFAR activity and its relationship with tolerance to AFB1 have been conducted in poultry. To determine the role of AFAR in poultry tolerance, the hepatic in vitro enzymatic activity of AFAR was investigated in liver cytosol from four commercial poultry species (chicken, quail, turkey and duck). Specifically, the kinetic parameters Vmax, Km and intrinsic clearance (CLint) were determined for AFB1 dialdehyde reductase (AFB1-monoalcohol production) and AFB1 monoalcohol reductase (AFB1-dialcohol production). In all cases, AFB1 monoalcohol reductase activity saturated at the highest aflatoxin B1 dialdehyde concentration tested (66.4 µM), whereas AFB1 dialdehyde reductase did not. Both activities were highly and significantly correlated and therefore are most likely catalyzed by the same AFAR enzyme. However, it appears that production of the AFB1 monoalcohol is favored over the AFB1 dialcohol. The production of alcohols from aflatoxin dialdehyde showed the highest enzymatic efficiency (highest CLint value) in chickens, a species resistant to AFB1; however, it was also high in the turkey, a species with intermediate sensitivity; further, CLint values were lowest in another tolerant species (quail) and in the most sensitive poultry species (the duck). These results suggest that AFAR activity is related to resistance to the acute toxic effects of AFB1 only in chickens and ducks. Genetic selection of ducks for high AFAR activity could be a means to control aflatoxin sensitivity in this poultry species.

In vitro hepatic aflatoxicol production is related to a higher resistance to aflatoxin B1 in poultry.

A study was conducted to determine the cytosolic in vitro hepatic enzymatic kinetic parameters Vmax, KM, and intrinsic clearance (CLint) for aflatoxin B1 (AFB1) reductase [aflatoxicol (AFL) production] and AFL dehydrogenase (AFB1 production) in four commercial poultry species (chicken, quail, turkey and duck). Large differences were found in AFB1 reductase activity, being the chicken the most efficient producer of AFL (highest CLint value). Oxidation of AFL to AFB1 showed only slight differences among the different poultry species. On average all species produced AFB1 from AFL at a similar rate, except for the turkey which produced AFB1 from AFL at a significantly lower rate than chickens and quail, but not ducks. Although the turkey and duck showed differences in AFL oxidation Vmax and KM parameters, their CLint values did not differ significantly. The ratio AFB1 reductase/AFL dehydrogenase enzyme activity was inversely related to the known in vivo sensitivity to AFB1 being highest for the chicken, lowest for the duck and intermediate for turkeys and quail. Since there is no evidence that AFL is a toxic metabolite of AFB1, these results suggest that AFL production is a detoxication reaction in poultry. Conversion of AFB1 to AFL prevents the formation of the AFB1-8,9-exo-epoxide which, upon conversion to AFB1-dihydrodiol, is considered to be the metabolite responsible for the acute toxic effects of AFB1.

An unusually high production of hepatic aflatoxin B1-dihydrodiol, the possible explanation for the high susceptibility of ducks to aflatoxin B1.

A study was conducted to determine the enzymatic kinetic parameters Vmax, KM, and intrinsic clearance (CLint) for the hepatic in vitro production of aflatoxin B1-dihydrodiol (AFB1-dhd) from aflatoxin B1 (AFB1) in four commercial poultry species, ranging in sensitivity to AFB1 from highest (ducks) to lowest (chickens). Significant but small differences were seen for Vmax, while large significant differences were observed for KM. However, the largest inter-species differences were observed for the CLint parameter, with ducks being extraordinarily efficient in converting AFB1 into AFB1-dhd. Since AFB1-dhd is considered the metabolite responsible for the acute toxic effects of AFB1, the high hepatic production of AFB1-dhd from AFB1 in ducks is the possible biochemical explanation for the extraordinary high sensitivity of this poultry species to the adverse effects of AFB1.

Study on the inhibitory effect of furafylline and troleandomycin in the 7-methoxyresorufin-O-demethylase and nifedipine oxidase activities in hepatic microsomes from four poultry species using high-performance liquid chromatography coupled with fluorescence and ultraviolet detection.

The present study reports the in vitro studies with furafylline and troleandomycin (TAO) as specific inhibitors of activities 7-methoxyresorufin-O-demethylase (MROD) and nifedipine oxidase, catalyzed by cytochrome P450 1 A2 (CYP1 A2) and 3A4 human enzymes, respectively, in hepatic microsomes of quail, duck, turkey and chicken. The results suggest that in chicken and quail the MROD activity is carried out by orthologs CYP1 A4 and 1 A5, meanwhile in duck and turkey by a CYP1 A5 ortholog. The nifedipine oxidase activity is carried out by orthologs of the CYP3A family in the four bird species. The use of furafylline and TAO significantly decreased these activities (P < 0.05) and suggested that the biotransformation of resorufin methyl ether (RME) may be related to more than one avian ortholog.

The role of selected cytochrome P450 enzymes on the bioactivation of aflatoxin B1 by duck liver microsomes.

A study was conducted to determine the cytochrome (CYP) P450 enzymes responsible for the bioactivation of aflatoxin B1 (AFB1) into its epoxide form (AFBO) in duck liver microsomes. Six male and six female 6-week-old Pekin ducks were used. The biochemical toxicology strategies applied included the use of selective inhibitors, prototype substrate activity for specific human P450s, correlation between aflatoxin bioactivation and enzymatic activity of prototype substrates, and the expression of specific CYP450 enzymes using antibodies against human CYP450s. Enzymatic activity was detected for the duck orthologues CYP1A1/2, CYP2A6 and CYP3A4 but not for the CYP2D6 orthologue. Immunoreactive proteins for CYP1A1, CYP2A6 and CYP3A4 were also detected. Inhibition studies suggested that the duck turkey CYP2A6 orthologue and, to a lesser extent, the CYP1A1 orthologue are involved in the bioactivation of AFB1. Correlation studies, however, suggest that CYP3A4, CYP2A6 and CYP1A1/2 are all involved in AFBO formation. The finding that four CYP enzymes may be involved in AFB1 bioactivation in ducks could explain the high sensitivity of this species to AFB1. Further studies are needed to fully elucidate the phase I hepatic metabolism of AFB1 in ducks, the only poultry species that develops hepatic cancer from AFB1 exposure.

CHARACTERIZATION OF Salvia bogotensis ANTI-LECTINIGYS AND THEIR APPLICATION IN IMMUNOCYTOCHEMICAL STUDIES INVOLVING TN ANTIGEN DETECTION / CARACTERIZACIÓN DE IGYS ANTI-LECTINA DE Salvia bogotensis Y SU APLICACIÓN EN ESTUDIOS CITOQUÍMICOS PARA LA DETECCIÓN DEL ANTÍGENO TN / CARACTERIZAÇÃO DA IGYS ANTI-LECTINA DE Salvia bogotensis E A SUA APLICAÇÃO EM ESTUDOS CITOQUÍMICOS PARA A DETECÇÃO DO ANTIGÉNIO TN

Immunoglobulins isolated from egg yolk (IgY) are tools which are currently used in different fields of the biological sciences; they have clear advantages over mammalian serum IgGs. We have established the conditions for obtaining anti-Salvia bogotensis lectin IgYs in previous work; their use in immunocytochemical studies requires that their main molecular characteristics are known as well as the conditions for IgY-lectin interaction. Salvia bogotensis lectin (SBoL) can specifically recognise Tn antigen, a recognised tumoral marker in many types of cancer but additional tools are required for evidencing this interaction in cells. Given the availability of S. bogotensis anti-lectin IgY, this work was aimed at molecularly characterising these IgYs and evaluating their application in immunocytochemical studies for detecting Tn antigen in tumour cells. Purified IgYs' isoelectric points, molecular weight and carbohydrate content were determined. Homologous and heterologous lectins were obtained for establishing the specificity of antibody interaction with the lectin; they were assayed by ELLSA. Biotin-or peroxidase-labelled IgYs were prepared; Tn antigen was specifically detected by CELISA and immunocytochemistry in MCF-7 and HeLa cell-lines with the lectin which was revealed with the labelled IgYs. Results showed that anti-SBoL IgY antibodies represent a highly sensitive tool for specific Tn antigen recognition assays.

Las inmunoglobulinas aisladas de la yema de huevo (IgY) son muy utilizadas actualmente en diversos campos de las ciencias biológicas, dadas sus ventajas frente a las IgG séricas de mamíferos. En un trabajo previo establecimos las condiciones de obtención de IgY dirigidas contra la lectina de Salvia bogotensis; su utilización en estudios inmunocitoquímicos requiere conocer sus principales características moleculares y las condiciones para la interacción IgY-lectina. La lectina de Salvia bogotensis (SBoL) reconoce específicamente el antígeno Tn, marcador tumoral en muchos tipos de cáncer, pero se requieren herramientas adicionales para evidenciar esta interacción a nivel celular. Dada la disponibilidad de IgY anti-lectina de S. bogotensis, se realizó este trabajo con el objeto de caracterizar molecularmente estas IgY y evaluar su utilización en estudios inmunocitoquímicos para la detección del antígeno Tn en células tumorales. A las IgY purificadas se les determinó su punto isoeléctrico, peso molecular y contenido de carbohidratos. Para establecer la especificidad de interacción IgY-SBoL se obtuvieron lectinas homólogas y heterólogas y se ensayaron por ELLSA. La detección del antígeno Tn en las líneas celulares MCF-7 y HeLa con la lectina y las IgYs marcadas con biotina o peroxidasa se realizó por CELISA e inmunocitoquímica. Los resultados mostraron que los anticuerpos IgY anti-SBoL son una herramienta de una alta sensibilidad para los ensayos de reconocimiento específico del antígeno Tn.

Imunoglobulinas isoladas a partir de gema de ovo (IgY) são ferramentas utilizadas actualmente em diferentes áreas das ciências biológicas y apresentam vantagens claras sobre o soro de mamífero IgGs. Em investigações anteriores estabelecemos as condições para obter lectina IgYs anti-Salvia bogotensis. O seu uso em estudos imunocitoquímicos requer que as suas principais características moleculares sejam conhecidas, assim como as condições para a interacção IgY-lectina. A lectina de Salvia bogotensis (SBoL) pode reconhecer especificamente o anti-génio Tn, um reconhecido marcador tumoral em muitos tipos de cancro, mas novas ferramentas são necessárias para evidenciar esta interacção em células. Dada a disponibilidade de anti-lectina IgY de S. bogotensis, este trabalho teve como objectivo a caracterização molecular de estes IgYs e avaliar a sua aplicação em estudos imunocitoquímicos para detectar antigénio Tn em células tumorais. Foram determinados os pontos isoeléctricos, peso molecular e conteúdo de carbohidratos de IgYs purificadas. Lectinas homologas e heterologas foram obtidas para estabelecer a especificidade da interacção de anticorpo com a lectina; estes foram ensaiados por ELLSA. Foram preparados IgYs marcados com biotina ou peroxidase. O antigénio Tn foi detectado especificamente por CELISA e imunocitoquimica em linhas celulares MCF-7 y HeLa com lectina que foi revelada com as IgYs marcadas. Os resultados mostraram que os anticorpos anti-SBoL IgY representam uma ferramenta altamente especifica para ensaios de reconhecimento especifico de antigenio Tn.

Obtención y purificación de IgY dirigidas contra la lectina de Salvia bogotensis / Purification of IgY against Salvia bogotensis lectin

Introducción. Las inmunoglobulinas presentes en la yema de huevo de gallina (IgY) han sido ampliamente utilizadas en inmunología, bioquímica, biotecnología y salud humana y animal. Aunque presentan una serie de ventajas frente a las IgG de mamíferos, su aislamiento requiere, por una parte, la eliminación de los lípidos presentes en la yema del huevo sin alterar su funcionalidad y, por otra, la utilización de metodologías de purificación que permitan una recuperación tal que haga factible su empleo como herramientas de detección y purificación a una escala significativa. Objetivo. Dado el interés que presenta el antígeno Tn como marcador tumoral en muchos tipos de cáncer, y la capacidad que tiene la lectina aislada de semillas de Salvia bogotensis para reconocer específicamente este antígeno, se adelantó el presente trabajo con la meta de disponer de IgY anti-lectina para emplearla en estudios inmunohistoquímicos y de biología celular. Materiales y métodos. Se produjo IgY anti-lectina inmunizando gallinas con lectina de S. bogotensis y se evaluó la respuesta inmune en función de la dosis y del tiempo; se ensayaron varios métodos de remoción de lípidos y de extracción y se compararon los rendimientos y la pureza de IgY obtenidas con varios métodos de purificación. Resultados. El mejor método de delipidación y extracción de IgY requiere dilución con agua, acidificación del extracto y precipitación con (NH4)2SO4 60 por cientos, recuperándose 43,35 mg de proteína/yema. La cromatografía tiofílica permite obtener las IgY puras en buena cantidad (10,4 mg/yema), preservando la funcionalidad y características de estos anticuerpos. Conclusión. Se establecieron las mejores condiciones para extraer y purificar IgY funcionales dirigidas contra la lectina de S. bogotensis

[Purification of IgY against Salvia bogotensis lectin]. / Obtenció y purificación de IgY dirigidas contra la lectina de Salvia bogotensis.

INTRODUCTION: Egg yolk immunoglobulins (IgY) have been extensively used in immunology, biochemistry, and biotechnology in studies of human and animal health. However, their use requires two preparatory steps: first, the egg yolk lipids must be removed without impairing the immunoglobin functional properties, and second, the isolation methods must allow high recoveries for further use as detection and purification tools. OBJECTIVE: Because Tn antigen presence serves as a tumoral marker and because S. bogotensis lectin's can specifically recognize this antigen, the current study aims at making available an anti-lectin IgY. This tool will be useful in histochemical and cellular studies involving transformed cells. MATERIALS AND METHODS: Anti-lectin IgY was produced by immunization of hens with S. bogotensis lectin, and the effect of antigen dose on IgY levels was assesed. Several methods for lipid removal, IgY extraction and purification were assayed, and yields and purity of IgYs were established for each method. RESULTS: The best delipidation and extraction method included yolk dilution with water under acidic conditions and (NH4)2SO4 60% s precipitation from which 43 mg protein/yolk were recovered. Among the chromatographic methods, thiophilic chromatography permitted the recovery of a substantial quantity of pure IgY (10.4 mg IgY/yolk). With this method, the function and characteristics of IgY were preserved. CONCLUSION: The best conditions for anti-S. bogotensis functional IgY extraction and purification were established.