β-Galactosidase Assay to Evaluate Drug-Induced Cytotoxicity in Parasites

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.