This study investigates the cytotoxic effects of an antiparasitic drug on Trypanosoma cruzi, the causative agent of Chagas disease. The methodology involves assessing the viability of transgenic parasites expressing β-galactosidase after drug treatment.
Trypanosoma cruzi, an obligate intracellular parasite, invades mammalian host cells as trypomastigote — flagellated, non-proliferative developmental stage — and differentiates into non-flagellated amastigotes, which proliferate in the cytoplasm by binary fission.
Following proliferation, amastigotes differentiate back into trypomastigotes that disrupt the host cells to enter circulation, resulting in systemic infection.
To evaluate the in vitro cytotoxic potential of a test antiparasitic drug against Trypanosoma cruzi, begin with a culture of transgenic Trypanosoma cruzi parasites — in the desired developmental stage — stably expressing the enzyme β-galactosidase.
Suspend the parasites in suitable media. Transfer to a multi-well plate. Add appropriate concentrations of the test antiparasitic drug. Incubate.
The drug passively diffuses through the parasite membranes and gets metabolized into toxic intermediates. These metabolites enter the nuclei and cause DNA damage, inhibiting protein synthesis, including β-galactosidase, negatively impacting parasite viability.
Replace the drug-containing media with buffer to prevent color interference during absorbance measurement. Add the substrate for β-galactosidase, chlorophenol red galactopyranoside, CPRG — a non-toxic galactoside analog — supplemented with non-ionic, mild detergent. Incubate.
The detergent lyses the parasites, releasing β-galactosidase into the solution. Subsequently, β-galactosidase hydrolyzes CPRG, producing chlorophenol red, a red-colored precipitate.
Using a microplate reader, measure the absorbance of chlorophenol red. Low absorbance indicates low β-galactosidase activity, suggesting high cytotoxic potential of the test antiparasitic drug at the specific concentration.
Add benznidazole solution to epimastigotes, Vero cells with amastigotes, or trypomastigotes, as described in the text manuscript. Then, incubate the epimastigotes at 28 degrees Celsius for 72 hours, and the trypomastigotes or infected Vero cells with amastigotes for 24 hours at 37 degrees Celsius and 5% carbon dioxide.
For the colorimetric assay, set up the blank wells by adding 100 microliters of PBS or corresponding medium. Next, add 40 microliters of CPRG substrate solution, and 10 microliters of the detergent solution to each well for obtaining a final concentration of 200 micromolar CPRG and 0.1% detergent in a final volume of 250 microliters in each well.
After incubation at 37 degrees Celsius for 2 hours, measure the absorbance at 595 nanometers using a microplate spectrophotometer.
繁體中文
