A rapid assay for mitochondrial DNA damage and respiratory chain inhibition in the yeast Saccharomyces cerevisiae

There is a need for a rapid assay to identify agents that damage mitochondr ia because the mitochondrion may be an important target for numerous enviro nmental mitotoxins. Certainly at least one chemotherapeutic regimen (CHOP t herapy) that includes doxorubicin can induce cardiomyopathy through mitocho ndrial genotoxicity in cardiac muscle cells. Yeast cells (1.5 X 10(6)-10(7) ) in water are spread on a YEPD plate, and, when the suspension of cells ha s dried, a small well (12 mm diameter) is cut into the agar; 200-400 mul of a solution of the presumptive mitochondrial genotoxin is placed in the wel l, and the plates are incubated for 2 days. The genotoxin forms a concentra tion gradient through the agar and affects the growing cells. An overlay co ntaining tetrazolium chloride is added, and the plates are incubated for 6- 24 hr. Respiring cells turn red, and nonrespiring cells, with damaged DNA o r inhibited respiratory chains, that are adjacent to the well, are white. A white ring, or a more lightly colored red ring, around the well indicates the presence of cells with lowered respiratory activity which may be fully reversible when the mitochondrial genotoxin is removed. In preliminary expe riments, doxorubicin (= adriamycin) shows strong activity with this assay; cyclophosphamide is negative, and 4-hydroxycyclophosphamide, a metabolite o f cyclophosphamide, is weakly positive. Ethidium bromide, methotrexate, 5-f luorouracil, and 5-fluorocytosine also are mitochondrial genotoxins. Antifu ngal agents similar to 5-fluorocytosine and anthelmintic compounds such as pyrvinium iodide can be powerful mitochondrial genotoxins. (C) 2001 Wiley-L iss, Inc.