Toxicity of allopurinol for macrophage cell lines and allopurinol susceptibility of intracellular amastigotes

The cytotoxicity of allopurinol for five different macrophage cell lines was first determined in order to identify the best host cell line for testing drug activity on intracellular amastigotes. Cytotoxicity IC50 was measured, essentially as described above for the parasites, with some modifications. Human THP-1 and U-937 (provided by Prof. Charles Jaffe’s laboratory, Hebrew University), murine RAW 264.7 (kindly provided by Professor Nachum Shpigel’s laboratory, Hebrew University) and J774.1, and canine DH-82 cell lines (the latter 2 cell lines kindly provided by Prof. Shimon Harrus’s laboratory, Hebrew University) were cultured in the following media: RPMI for THP-1 and U-937, DMEM for RAW 264.7 and J774.1, and MEM for DH-82; all supplemented with 10% FCS, 2mM L-glutamine and antibiotics (100IU penicillin G and 100μg/mL streptomycin). For each cell line, 1x10⁴ cells were aliquoted in triplicates into 96-well plates. Allopurinol was added to final concentrations of 0, 12.5, 25, 50, 100, 200, and 400μg/mL. Cells were incubated for 66 hours at 37°C, 20μL resazurin were added and cultures were further incubated for an additional 4 hours. Two separate experiments were performed for each cell line and the average IC50 value was used for analyses.

Allopurinol susceptibility of intracellular amastigotes was evaluated using isolates from the NT and TR groups. DH-82 cells in complete MEM medium, chosen based on the cytotoxicity study above, were plated (1x10⁴ cells/0.5mL/well in duplicates) on round glass slides in 24 well plates (Nunc, Denmark) and left for 6 hours to adhere. Promastigotes from a stationary culture of the test isolate were then added for 16 hours at 37°C using an infection ratio of 10:1 parasites:macrophage. Cells were washed twice with MEM without FCS to remove extracellular parasites, and 1mL complete MEM containing allopurinol added (final concentration being either 0 or 300μg/mL). Cells were incubated for 72 hours, washed twice with PBS, followed by fixation in methanol and Giemsa staining of the glass slides. Intracellular parasites for each isolate were counted in 100 host cells, and percent amastigote growth inhibition was calculated compared to the non-treated controls. Each experiment was repeated twice and the average percent growth inhibition was calculated.