A new pharmacological screening assay with Trypanosoma cruzi epimastigotes expressing beta-galactosidase.
We have developed a new pharmacological screening assay for epimastigotes of Trypanosoma cruzi (clone CL-B5) that express the Escherichia coli LacZ gene. The assay is based on determining the activity of the cytoplasmic beta-galactosidase released into the culture on membrane lysis in the presence of the substrate chlorophenol red beta-D-galactopyranoside (CPRG). The experimental conditions were adjusted to find those in which the relationship between epimastigote number and CPRG absorbance was linear over the widest possible range. Absorbance was significantly correlated with the number epimastigote from 5x10(3) to 1.2x10(6) parasites/ml (r=0.98, P<0.01). The optimal final concentration of CPRG was 200 microM and the optimal incubation period was 6 h when parasites were incubated for 3 days. Once the assay was standardized, the trypanocidal activities of nifurtimox and benznidazole were determined both by CPRG assay and microscopic counting, demonstrating the methods utility for drug-screening. The efficacy obtained was comparable to that obtained with the manual method.
Vega
C
C
Departamento de Parasitología, Facultad de Farmacia, Universidad Complutense de Madrid, Ciudad Universitaria s/n, 28040, Madrid, Spain.
Rolón
M
M
Martínez-Fernández
A R
AR
Escario
J A
JA
Gómez-Barrio
A
A
D000818:Animals; D002733:Chlorophenols / Q000378:metabolism; D005697:Galactosides / Q000378:metabolism; D007763:Lac Operon; D009547:Nifurtimox / Q000494:pharmacology; D009593:Nitroimidazoles / Q000494:pharmacology; D021261:Parasitic Sensitivity Tests / Q000379:methods*; D013053:Spectrophotometry; D014344:Trypanocidal Agents / Q000494:pharmacology*; D014349:Trypanosoma cruzi / Q000187:drug effects* / Q000235:genetics*; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism*
D002733:Chlorophenols; D005697:Galactosides; D009593:Nitroimidazoles; D014344:Trypanocidal Agents; C074184:chlorophenol red galactopyranoside; D001616:beta-Galactosidase; D009547:Nifurtimox; C009999:benzonidazole