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Intoxication by ochratoxin A (OTA): prevention of toxic effects
Report and Abstracts
1115
is difficult. A fast ELISA able to detect the venom in blood and urine in less than 40 min was set up and used to quantify venom in blood and in urine samples from patients bitten by vipers. A good correlation was established between the severity of the clinical symptoms and the level of the venom in patient blood. Toxicokinetic studies of Vipera aspis venom were performed in rabbits after experimental envenomations. Parameters obtained after i.v. injection of the venom showed that: (I) most of the injected dose is eliminated three days after the administration (half-life of elimination: 12 hr); (2) the venom presents a large volume of distribution; (3) the total clearance of the venom is low. The toxicokinetics of the venom administered i.m. revealed a precocious resorption o f the venom from the site of the injection but the resorption process persisted up to 72 hr after the injection, maintaining a high concentration of venom antigens in the plasma. A quantitative analysis indicated that 65% of the dose injected i.m. is able to reach the blood circulation. Similar investigations performed after administration of antivenom allowed us to analyse in vivo the efficacy of serotherapy. These studies indicated that the intravenous administration of specific anti-venom antibodies (Fab'2) causes the redistribution of the venom antigens from tissues to vascular compartment, where they are efficiently neutralized and eliminated. This observation proved to be very useful to rationalize better the immunological treatment of envenomations.
Design o f a conformationally restricted immunogen to elicit protective antibodies against a snake curaremimetic toxin. J. Cotton, P. Cuniasse, M. L6onetti, S. Leroy, G. Mourier and A. M6nez (D6partement d'Ing6nierie et d'Etudes des Prot6ines, CEA, Saclay, 91191 Gif-sur-Yvette, France). A disulfide-containing peptide [24 41C] from a snake toxin elicits, in a free state, antibodies that neutralize the toxin. However, examination of the solution structure of the peptide reveals that only a turn conformation was commonly found in 24 41C and the corresponding toxin region, the regions 24 28 and 38-41 adopting no organized conformation in the peptide but fl-sheet structure in the toxin. To increase tentatively the neutralizing capacity of anti-peptide antibodies we synthesized three derivatives of 24-41C. Thus, we (1) replaced Gly-40 by an amino-isobutyric moiety (P24 41Aib); (2) substituted the two cysteines 24 and 41 by penicillamin moieties (P24-41Pen) and (3) introduced an amide bond between the e N H 2 of Lys-27 and the y C O O H of Glu-38 (P24 41K-E). A solution ELISA assay made with anti-toxin antibodies revealed that 24 41Pen is slightly more antigenic than 24-41 Aib and 24-41C, which in turn are more antigenic than 2 4 - 4 1 K - E , suggesting that the conformation of 24 41Pen is most closely related to that of the corresponding region in the native toxin. However, 24-41Pen, 24-41Aib and 2 4 - 4 1 C stimulate T-cells from BALB/c mice, whereas 2 4 - 4 1 K - E has essentially lost this property and thereby fails to elicit antibodies. Finally, anti-24-41Pen antibodies are substantially more potent at neutralizing the native toxin than anti-24-41C antibodies, which in turn are more potent than anti-24 41Aib antibodies. Our data show that introduction of appropriate constraints makes it possible to improve significantly the proportion of neutralizing antibodies raised by a synthetic peptide. Such observations should be of interest for the design of efficient synthetic vaccines.
Intoxication by ochratoxin A (OTA ): prevention o f toxic effects. E. E. Creppy, I. Baudrimont and A.-M. Betbeder (Laboratoire de Toxicologic et d'Hygi6ne Appliqu~e, Unit6 de Formation et de Recherche des Sciences Pharmaceutiques, Universit6 de Bordeaux II, 3ter, Place de la Victoire, 33000 Bordeaux, France). Ochratoxin A (OTA) is a second metabolite produced by Aspergillus ochraceus and Penicillium verrucosum. It is a food contaminant regarded as the main causal agent of Balkan endemic nephropathy (NEB). Levels of 2 40 ng/ml are found in h u m a n blood in the population of the Balkans but also in other countries: Scandinavian countries, Germany, Canada, France, North Africa--mainly Tunisia, Algeria, and Egypt. O T A is an inhibitor of protein synthesis; it is immunosuppressive, genotoxic, teratogenic and carcinogenic; it disturbs blood coagulation and hydrocarbon metabolism. Detoxication of O T A represents an important public health problem. It is difficult to prevent O T A production in food and foodstuffs, so studies are focused on preventing substances which should act on the toxicokinetic parameters to reduce or even to abolish toxic effects, especially nephrotoxicity, according to the recommendations of W H O F A O Joint Committee. Several substances, such as sodium bicarbonate, phenylalanine, A19, piroxicam, cholestyramine, SOD, catalase, aspirin, and indomethacin, are being assayed on absorption, plasma protein fixation, distribution in the kidneys or other organs, renal transport, metabolism and elimination. Sodium bicarbonate is efficient at preventing absorption from the stomach; phenylalanine, A19, piroxicam are efficient on O T A binding to plasma proteins. Paraaminohippurate, an organic anion, and tetraethyl a m m o n i u m acetate, an organic cation, can undergo competition with O T A and prevent its renal transport. Ochratoxin A distribution in kidneys and other organs is effectively very large. In the brain, for example, O T A reduces levels of free amino acids such as aspartate and glutamate, phenylalanine and tyrosine. A I 9 and piroxicam are efficient at preventing the distribution of O T A in an organism. Phenylalanine's effects are limited and time dependent. A19 is efficient against O T A distribution in rat tissues, especially kidney, liver, brain and testicle. Chlolestyramine is known to prevent the enterohepatic circulation of m a n y drugs by an adsorption mechanism. It facilitates the faecal and urinary elimination of OTA. O T A is cleaved into OTalpha, a non-toxic c o m p o u n d whose genotoxicity is being discovered. In rat hepatocytes at least seven metabolites are formed, two of them hydroxylated metabolites. The metabolism of O T A becomes more complex as its natural analogues are being discovered. O T A induces lipid peroxidation and possibly undergoes a
1116
Report and Abstracts
co-oxidation process with prostaglandin synthetases. Antioxidant agents such as SOD and catalase, vitamins A and E, or inhibitors of prostaglandin synthetases such as piroxicam, indomethacin and aspirin are efficient at preventing tissue and D N A lesions due to oxidative pathways. Glucuronides and sulfoconjugates of O T A are eliminated in urine. G S H conjugates have not been identified, although N-acetylcystein (NAC) modifies the O T A D N A - a d d u c t formation profile. A combination of m a n y of these substances can be useful in the prevention of toxic effects of ochratoxin A. Scorpion toxins: recent data in moleeular immunology. C. Devaux (CNRS U R A 1455, Facult6 de M~decine-Nord, Bd P. Dramard, 13916 Marseille Cedex 20, France). Scorpion stings have powerful toxic effects that cause significant health problems in many areas of the world. The lethal effect of scorpion venom is due to neurotoxins that act on sodium channels of excitable cells. The sequence variability of these molecules gives rise to antigenic polymorphism. 3D structure analysis suggests that conserved hydrophobic residues contribute to the c o m m o n pharmacological properties. We systematically re-examined the antigenicity of one toxin (toxin II from Androctonus australis hector) by the Pepscan method, and thereby identified a new linear epitope. This epitope corresponds to the highly conserved N-terminal sequence (residues 1-8). Polyclonal sera raised against a peptide that mimics this region recognize the native toxin II and also several other toxins that show a sequence similarity in their N-terminal part, but belong to different immunological groups. In addition, these sera are able to neutralize toxin II and inhibit its binding to rat brain synaptosomes. The possibility of raising a humoral immune response able to induce protection from the lethal effects of toxins was evaluated in the mouse model. Preliminary results, using non-toxic or detoxified immunogens, are discussed. Evolution o f the envenomations caused by Arachnida. M. Goyffon (L.E.R.A.I., M u s 6 u m National d'Histoire Naturelle, 57 Rue Cuvier, F-75005 Paris, France). The spiders and the scorpions are the two important venomous Arachnida orders, in particular the scorpions which are the most dangerous in the venomous fauna if the ratio number deaths/number stings is the criterion retained. Araneism can be neurotoxic (Latrodectus) or necrotic (Loxosceles). Considering the Latrodectus genus, cyclic peaks of envenomations corresponding to cyclic peaks of the spider populations are often observed. The variety of subspecies, and the difficulties in identifying some similar species in the genus explain the variability of the symptomatologies described. The geographic variations of necrotic power of the Loxosceles venoms are more surprising: some species harmless in Europe, as L. rufescens, are toxic in the New World, where this species was imported recently. The same observations were reported with Tegenaria agrestis, but no explanation can be given. Some similar facts are known for the scorpions: Buthus oecitanus is not really dangerous in Europe, but it is responsible for deaths in North Africa. More interesting are the epidemiological modifications of scorpionism due to the extension of the distribution of some dangerous species, which are capable of adapting to an urban environment, such as Centruroides exilicauda (U.S.A.) or Tityus serrulatus. Moreover, when public hygiene is neglected, commensal insects such as cockroaches proliferate, and scorpions, which easily find prey, also proliferate. A similar observation was recently reported with a dangerous snake, Eehis ocellatus, in tropical Africa. Therefore, an increase in venomous stings or bites in tropical countries could signify a deterioration in urban sanitary conditions. Molecular studies on bacterial toxins: towards" new generation ~raccines. C. Locht (Laboratoire de Microbiologie G~n6tique et Mol6culaire, Institut Pasteur de Lille, 1 Rue du Prof. Calmette, F-59019 Lille Cedex, France). Many bacterial pathogens express their virulence by the action of protein toxins. Consequently, protection against these diseases can be achieved by vaccination using the toxins as immunogens. Typical examples are tetanus toxin and diphtheria toxin. Because of their inherent activity, native forms cannot be used directly, but must first be detoxified. Genetic engineering provides a very powerful way to inactivate protein toxins without abolishing their immunoprotective potential. Pertussis toxin is one of the most complex protein toxins known to date and clinical tests have demonstrated its immunoprotectivity against whooping cough in children. The toxin is composed of five dissimilar subunits, named St to $5, that are arranged in an A B structure. A, composed of SI, is an enzymatically active moiety, and B, composed of $2 $5, is a receptor-binding moiety. The molecular steps of toxin action are (1) the binding of the toxin to the target cells, (2) the membrane translocation of the enzymatically active SI subunit, and (3) expression of ADP-ribosyltransferase activity by the internalized Sl subunit. The Sl-catatysed ADP-ribosylation of signal transducing G proteins causes signal transduction arrest, which is responsible for most of the biological activities of pertussis toxin. We have cloned the structural genes of each of the five toxin subunits and studied the enzyme mechanism of the SI subunit. This has provided information about the involvement of individual amino acid residues in substrate binding and catalysis. Site-directed alteration of these residues yielded toxin analogues, with dramatically reduced enzyme activity. However, not all biological activities were affected by the reduction of the enzyme activity. Therefore, we also studied the residues involved in the binding activities of the B moiety of the toxin, especially subunits $2 and
1115
is difficult. A fast ELISA able to detect the venom in blood and urine in less than 40 min was set up and used to quantify venom in blood and in urine samples from patients bitten by vipers. A good correlation was established between the severity of the clinical symptoms and the level of the venom in patient blood. Toxicokinetic studies of Vipera aspis venom were performed in rabbits after experimental envenomations. Parameters obtained after i.v. injection of the venom showed that: (I) most of the injected dose is eliminated three days after the administration (half-life of elimination: 12 hr); (2) the venom presents a large volume of distribution; (3) the total clearance of the venom is low. The toxicokinetics of the venom administered i.m. revealed a precocious resorption o f the venom from the site of the injection but the resorption process persisted up to 72 hr after the injection, maintaining a high concentration of venom antigens in the plasma. A quantitative analysis indicated that 65% of the dose injected i.m. is able to reach the blood circulation. Similar investigations performed after administration of antivenom allowed us to analyse in vivo the efficacy of serotherapy. These studies indicated that the intravenous administration of specific anti-venom antibodies (Fab'2) causes the redistribution of the venom antigens from tissues to vascular compartment, where they are efficiently neutralized and eliminated. This observation proved to be very useful to rationalize better the immunological treatment of envenomations.
Design o f a conformationally restricted immunogen to elicit protective antibodies against a snake curaremimetic toxin. J. Cotton, P. Cuniasse, M. L6onetti, S. Leroy, G. Mourier and A. M6nez (D6partement d'Ing6nierie et d'Etudes des Prot6ines, CEA, Saclay, 91191 Gif-sur-Yvette, France). A disulfide-containing peptide [24 41C] from a snake toxin elicits, in a free state, antibodies that neutralize the toxin. However, examination of the solution structure of the peptide reveals that only a turn conformation was commonly found in 24 41C and the corresponding toxin region, the regions 24 28 and 38-41 adopting no organized conformation in the peptide but fl-sheet structure in the toxin. To increase tentatively the neutralizing capacity of anti-peptide antibodies we synthesized three derivatives of 24-41C. Thus, we (1) replaced Gly-40 by an amino-isobutyric moiety (P24 41Aib); (2) substituted the two cysteines 24 and 41 by penicillamin moieties (P24-41Pen) and (3) introduced an amide bond between the e N H 2 of Lys-27 and the y C O O H of Glu-38 (P24 41K-E). A solution ELISA assay made with anti-toxin antibodies revealed that 24 41Pen is slightly more antigenic than 24-41 Aib and 24-41C, which in turn are more antigenic than 2 4 - 4 1 K - E , suggesting that the conformation of 24 41Pen is most closely related to that of the corresponding region in the native toxin. However, 24-41Pen, 24-41Aib and 2 4 - 4 1 C stimulate T-cells from BALB/c mice, whereas 2 4 - 4 1 K - E has essentially lost this property and thereby fails to elicit antibodies. Finally, anti-24-41Pen antibodies are substantially more potent at neutralizing the native toxin than anti-24-41C antibodies, which in turn are more potent than anti-24 41Aib antibodies. Our data show that introduction of appropriate constraints makes it possible to improve significantly the proportion of neutralizing antibodies raised by a synthetic peptide. Such observations should be of interest for the design of efficient synthetic vaccines.
Intoxication by ochratoxin A (OTA ): prevention o f toxic effects. E. E. Creppy, I. Baudrimont and A.-M. Betbeder (Laboratoire de Toxicologic et d'Hygi6ne Appliqu~e, Unit6 de Formation et de Recherche des Sciences Pharmaceutiques, Universit6 de Bordeaux II, 3ter, Place de la Victoire, 33000 Bordeaux, France). Ochratoxin A (OTA) is a second metabolite produced by Aspergillus ochraceus and Penicillium verrucosum. It is a food contaminant regarded as the main causal agent of Balkan endemic nephropathy (NEB). Levels of 2 40 ng/ml are found in h u m a n blood in the population of the Balkans but also in other countries: Scandinavian countries, Germany, Canada, France, North Africa--mainly Tunisia, Algeria, and Egypt. O T A is an inhibitor of protein synthesis; it is immunosuppressive, genotoxic, teratogenic and carcinogenic; it disturbs blood coagulation and hydrocarbon metabolism. Detoxication of O T A represents an important public health problem. It is difficult to prevent O T A production in food and foodstuffs, so studies are focused on preventing substances which should act on the toxicokinetic parameters to reduce or even to abolish toxic effects, especially nephrotoxicity, according to the recommendations of W H O F A O Joint Committee. Several substances, such as sodium bicarbonate, phenylalanine, A19, piroxicam, cholestyramine, SOD, catalase, aspirin, and indomethacin, are being assayed on absorption, plasma protein fixation, distribution in the kidneys or other organs, renal transport, metabolism and elimination. Sodium bicarbonate is efficient at preventing absorption from the stomach; phenylalanine, A19, piroxicam are efficient on O T A binding to plasma proteins. Paraaminohippurate, an organic anion, and tetraethyl a m m o n i u m acetate, an organic cation, can undergo competition with O T A and prevent its renal transport. Ochratoxin A distribution in kidneys and other organs is effectively very large. In the brain, for example, O T A reduces levels of free amino acids such as aspartate and glutamate, phenylalanine and tyrosine. A I 9 and piroxicam are efficient at preventing the distribution of O T A in an organism. Phenylalanine's effects are limited and time dependent. A19 is efficient against O T A distribution in rat tissues, especially kidney, liver, brain and testicle. Chlolestyramine is known to prevent the enterohepatic circulation of m a n y drugs by an adsorption mechanism. It facilitates the faecal and urinary elimination of OTA. O T A is cleaved into OTalpha, a non-toxic c o m p o u n d whose genotoxicity is being discovered. In rat hepatocytes at least seven metabolites are formed, two of them hydroxylated metabolites. The metabolism of O T A becomes more complex as its natural analogues are being discovered. O T A induces lipid peroxidation and possibly undergoes a
1116
Report and Abstracts
co-oxidation process with prostaglandin synthetases. Antioxidant agents such as SOD and catalase, vitamins A and E, or inhibitors of prostaglandin synthetases such as piroxicam, indomethacin and aspirin are efficient at preventing tissue and D N A lesions due to oxidative pathways. Glucuronides and sulfoconjugates of O T A are eliminated in urine. G S H conjugates have not been identified, although N-acetylcystein (NAC) modifies the O T A D N A - a d d u c t formation profile. A combination of m a n y of these substances can be useful in the prevention of toxic effects of ochratoxin A. Scorpion toxins: recent data in moleeular immunology. C. Devaux (CNRS U R A 1455, Facult6 de M~decine-Nord, Bd P. Dramard, 13916 Marseille Cedex 20, France). Scorpion stings have powerful toxic effects that cause significant health problems in many areas of the world. The lethal effect of scorpion venom is due to neurotoxins that act on sodium channels of excitable cells. The sequence variability of these molecules gives rise to antigenic polymorphism. 3D structure analysis suggests that conserved hydrophobic residues contribute to the c o m m o n pharmacological properties. We systematically re-examined the antigenicity of one toxin (toxin II from Androctonus australis hector) by the Pepscan method, and thereby identified a new linear epitope. This epitope corresponds to the highly conserved N-terminal sequence (residues 1-8). Polyclonal sera raised against a peptide that mimics this region recognize the native toxin II and also several other toxins that show a sequence similarity in their N-terminal part, but belong to different immunological groups. In addition, these sera are able to neutralize toxin II and inhibit its binding to rat brain synaptosomes. The possibility of raising a humoral immune response able to induce protection from the lethal effects of toxins was evaluated in the mouse model. Preliminary results, using non-toxic or detoxified immunogens, are discussed. Evolution o f the envenomations caused by Arachnida. M. Goyffon (L.E.R.A.I., M u s 6 u m National d'Histoire Naturelle, 57 Rue Cuvier, F-75005 Paris, France). The spiders and the scorpions are the two important venomous Arachnida orders, in particular the scorpions which are the most dangerous in the venomous fauna if the ratio number deaths/number stings is the criterion retained. Araneism can be neurotoxic (Latrodectus) or necrotic (Loxosceles). Considering the Latrodectus genus, cyclic peaks of envenomations corresponding to cyclic peaks of the spider populations are often observed. The variety of subspecies, and the difficulties in identifying some similar species in the genus explain the variability of the symptomatologies described. The geographic variations of necrotic power of the Loxosceles venoms are more surprising: some species harmless in Europe, as L. rufescens, are toxic in the New World, where this species was imported recently. The same observations were reported with Tegenaria agrestis, but no explanation can be given. Some similar facts are known for the scorpions: Buthus oecitanus is not really dangerous in Europe, but it is responsible for deaths in North Africa. More interesting are the epidemiological modifications of scorpionism due to the extension of the distribution of some dangerous species, which are capable of adapting to an urban environment, such as Centruroides exilicauda (U.S.A.) or Tityus serrulatus. Moreover, when public hygiene is neglected, commensal insects such as cockroaches proliferate, and scorpions, which easily find prey, also proliferate. A similar observation was recently reported with a dangerous snake, Eehis ocellatus, in tropical Africa. Therefore, an increase in venomous stings or bites in tropical countries could signify a deterioration in urban sanitary conditions. Molecular studies on bacterial toxins: towards" new generation ~raccines. C. Locht (Laboratoire de Microbiologie G~n6tique et Mol6culaire, Institut Pasteur de Lille, 1 Rue du Prof. Calmette, F-59019 Lille Cedex, France). Many bacterial pathogens express their virulence by the action of protein toxins. Consequently, protection against these diseases can be achieved by vaccination using the toxins as immunogens. Typical examples are tetanus toxin and diphtheria toxin. Because of their inherent activity, native forms cannot be used directly, but must first be detoxified. Genetic engineering provides a very powerful way to inactivate protein toxins without abolishing their immunoprotective potential. Pertussis toxin is one of the most complex protein toxins known to date and clinical tests have demonstrated its immunoprotectivity against whooping cough in children. The toxin is composed of five dissimilar subunits, named St to $5, that are arranged in an A B structure. A, composed of SI, is an enzymatically active moiety, and B, composed of $2 $5, is a receptor-binding moiety. The molecular steps of toxin action are (1) the binding of the toxin to the target cells, (2) the membrane translocation of the enzymatically active SI subunit, and (3) expression of ADP-ribosyltransferase activity by the internalized Sl subunit. The Sl-catatysed ADP-ribosylation of signal transducing G proteins causes signal transduction arrest, which is responsible for most of the biological activities of pertussis toxin. We have cloned the structural genes of each of the five toxin subunits and studied the enzyme mechanism of the SI subunit. This has provided information about the involvement of individual amino acid residues in substrate binding and catalysis. Site-directed alteration of these residues yielded toxin analogues, with dramatically reduced enzyme activity. However, not all biological activities were affected by the reduction of the enzyme activity. Therefore, we also studied the residues involved in the binding activities of the B moiety of the toxin, especially subunits $2 and