An MTT-based method for the in vivo quantification of myotoxic activity of snake venoms and its neutralization by antibodies.
The reduction of the tetrazolium compound MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) was used as the basis for the development of a simple method for the quantitative estimation of metabolically active skeletal muscle tissue remaining after in vivo venom-induced myonecrosis. Using the venom of the snake Micrurus nigrocinctus as a potent myotoxic agent, this MTT-based technique was evaluated in comparison with available methods based on the measurement of creatine kinase (CK) activity, and a quantitative histological technique considered as a reference. Homogenates of the gastrocnemius muscle prepared in the presence of 1% Triton X-100 reduced MTT and this activity correlated closely with the number of viable cells in the tissue, as determined by histological evaluation. After venom injection, residual MTT-reducing activity of muscle homogenates showed higher correlation to the myonecrosis index obtained by histological analysis, than residual muscle CK activity. Using the new MTT-based assay, the ability of an anti-M. nigrocinctus equine antivenom to neutralize venom myotoxins was studied. The myotoxic activity of the venom was completely neutralized using 4 ml antivenom/mg venom, with a 50% effective dose (ED50) value of about 2.5 ml/mg. The MTT-based method described should be useful in the estimation and standardization of anti-myotoxic potency of antivenoms, and in the screening of other neutralizing agents, as a convenient and reliable alternative to the time-consuming quantitative histological methods.
Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José.
B
B
Lomonte
J M
JM
Gutiérrez
M
M
Romero
J
J
Núñez
A
A
Tarkowski
L A
LA
Hanson
D000818:Animals; D000997:Antivenins / Q000276:immunology*; D003402:Creatine Kinase / Q000378:metabolism; D004546:Elapid Venoms / Q000276:immunology / Q000633:toxicity*; D051379:Mice; D009132:Muscles / Q000187:drug effects*; D009500:Neutralization Tests; D013778:Tetrazolium Salts; D013844:Thiazoles
D000997:Antivenins; D004546:Elapid Venoms; D013778:Tetrazolium Salts; D013844:Thiazoles; D003402:Creatine Kinase; C022616:thiazolyl blue