Journal of Radioanalytical and Nuclear Chemistry, Vol. 269, No.3 (2006) 579–583 0236–5731/USD 20.00 Akadémiai Kiadó, Budapest © 2006 Akadémiai Kiadó, Budapest Springer, Dordrecht Biodistribution of gyroxin using 125 I as radiotracer J. A. Alves da Silva, 1 * E. Muramoto, 2 M. T. C. P. Ribela, 1 J. R. Rogero, 1 M. A. P. Camillo 1 1 Molecular Biology Center, IPEN/CNEN-SP, P.O.Box 11049, 05422-970, São Paulo, Brazil 2 Radiopharmacy Center, IPEN/CNEN-SP, P.O.Box 11049, 05422-970, São Paulo, Brazil (Received April 6, 2006) The use of radiotracers in the research of animal venom has been scarce, although it allows an excellent approach to follow the process of bioavailability, biodistribution and kinetics of toxins. The purpose of this study was to assess gyroxin action mechanism, transport, compartments and action sites. This toxin is a thrombin-like and causes the barrel rotation syndrome. The gyroxin was labeled with 125 I and used as a tracer for the in vivo assay in mice. Blood samples and organs were collected at different time intervals, weighed and analyzed in a gamma-counter. The data was related with tissues distribution of protease activated receptor (PAR). Biodistribution assay allowed dividing the organs into three groups. The first one with the organs that followed the blood kinetics, the second with the organs related to metabolisms and elimination, and the third with the organs in which the gyroxin concentration increased during the observation period. Introduction The distribution, metabolism and excretion (DME) of a strange compound in a living organism depends on physicals, chemicals and physiological factors (pharmacokinetics processes). These factors remove and dilute the compound from its place of entrance in the biological system, carry it to different tissues, allow its diffusion or promote its transportation actively through cells membranes and finally determine its accumulation, disposition and excretion. The biodistribution rate in the organ’s tissue is determined by the blood flow perfusion and by the ability of the compound molecule to pass through the capillary wall and penetrate into the cells. 1 Gyroxin is a serine protease present in the venom of Crotalus durissus terrificus, a Brazilian rattlesnake. It was first described as a neurotoxin causing motor disturbances called barrel rotation. 2,3 The intravenous (IV) injection of gyroxin into mice produces temporary episodes characterized by opisthotonos and rotations around the long axis of the body: that is the reason why it is called gyroxin. Gyroxin is also known for its thrombin-like activity on human fibrinogen, cleaving the alpha-chain. 4 The fibrin monomers polymerizes into an abnormal fibrin clot and it is highly susceptible to the action of fibrinolytic agents. 5,6 The total or partial incoagulability observed in the blood of patients bitten by rattlesnakes derives from the fibrinogen consumption. 7 There is a special interest in gyroxin action on fibrinogen because of its possible use in thrombotic disease therapy and as an anticoagulant factor. Some thrombin-like enzymes isolated from the venom of other snakes are being used as defibrinogenating agents for a number of clinical conditions including deep vein thromboses, myocardial infarction, pulmonary embolus, acute ischemic stroke and peripheral vascular disease. 5 * E-mail: josilva@ipen.br As told before, gyroxin is a multifunctional protein but its action mechanism is not well-known, especially regarding neurotoxicity. This work aims at investigating gyroxin action mechanism, transport, compartments and action sites using 125 I as radiotracer. The animals’ toxins could be radiolabeled for biodistribution assays, indirectly or directly: Indirectly, by labeling the toxin in an already radiolabeled molecule (e.g., an antibody). This incorporated radiotracer will show the toxin metabolism in a living organism during the whole experimental period. Directly, by incorporating the radionuclide to the protein molecule, cofactors, coenzymes or metal, when one of these constituents is present. The directly way was used in this study (gyroxin was radiolabeled with 125 I). Radiolabeling with 125 I has been indicated for several applications due to its easy and simple radioiodination system, labeled product stability and detection facility. 8 Experimental Gyroxin radioiodination Gyroxin was obtained from Crotalus durissus terrificus’ crude venom by affinity and gel filtration chromatography. 9 It was radioiodinated using mild conditions at low temperature with chloramine-T method. 10 The reaction mixture were fractionated in a Sephadex G100 column (3×50 cm 2 ) with 50mM sodium phosphate buffer pH 7.4 containing 0.1% of bovine serum albumin as eluent. Fractions of 2.0 ml/tube were collected and the flow rate was 12 ml/h. Aliquots with 20 μl from each tube were analyzed in a gamma-counter (Oakfield Instruments, United Kingdown).