A METHOD TO ASSESS INVASION AND INTRACELLULAR REPLICATION OF TRYPANOSOMA-CRUZI BASED ON DIFFERENTIAL URACIL INCORPORATION

Screening of candidate trypanocidal compounds or factors affecting inv asion of mammalian cells by the infective stages of Trypanosoma cruzi in tissue culture models has primarily involved labor-intensive micros copic counting of the parasites. A very efficient method for quantitat ing the inhibitory effect of antimicrobial agents or signaling pathway s inhibitors on T. cruzi grown in L6E9 myoblasts was devised. This ass ay takes advantage of the selective incorporation of [H-3]uracil into nucleic acids by replicating T. cruzi amastigotes. L6E9 rat myoblasts are submitted to gamma irradiation to inhibit their replication. Uraci l uptake by uninfected cells is considerably decreased by this method. Nifurtimox, benznidazole, fexinidazole, MK-436, and megazol are drugs known to have activity against T. cruzi and were used in growth inhib ition assays. The results demonstrated that [H-3]uracil incorporation in the presence of different concentrations of nifurtimox and benznida zole closely correlated with the number of amastigotes per 100 myoblas ts in Giemsa-stained monolayers under the conditions used. This method also has the advantage to differentiate between the effects of the co mpounds on the invasion and the replication steps of the infection wit h T. cruzi, as shown by the inhibitory effect of genistein when added in invasion assays. (C) 1998 Elsevier Science B.V. All rights reserved .