Analysis of methylglyoxal bis(guanylhydrazone)-induced alterations of hamster tumor mitochondria by correlated studies of selective rhodamine binding, ultrastructural damage, DNA replication, and reversibility.

Because of the renewed importance of methylglyoxal bis(guanylhydrazone) (MGBG) in recent clinical trials for a variety of cancers, and because the antiproliferative activity of MGBG cannot be accounted for exclusively by the known inhibition by the drug of polyamine biosynthesis, but is thought to involve an alternative action in which the mitochondria are implicated, we have investigated several new aspects of the nature and reversibility of this mitochondrial damage. Using Rous sarcoma hamster tumor cells as a model, treatment of monolayer cultures during exponential growth with 10, 25, and 50 microM MGBG (up to 48 hr) resulted in dose-dependent (reversible) growth inhibition and selective ultrastructural damage to the mitochondria (e.g., extreme swelling, loss of cristae and matrix components, and dense inclusions) in up to 96% of cells, while nuclei appeared normal, corroborating and extending findings by others in mouse, rat, and human cells. Mitochondria in 3 to 5% of cells failed to swell, even at highest drug dosage, but were of unusual structure. After removal of MGBG, damaged mitochondria in 90 to 95% of cells recovered near-normal ultrastructure within 1 to 2 days; in some cells, mitochondrial recovery from severe damage could be monitored following a lag period of up to 5 days. The potential-dependent, supravital fluorescent probe rhodamine 123 (RH 123) selectively and relatively uniformly stained the grossly enlarged mitochondria, strikingly delineating residual organelle membranes. Quantitative assays of the uptake and retention of RH 123/10(6) cells demonstrated the maintenance of mitochondrial membrane potential in both control and MGBG-treated cell populations. These data also support the concept of mitochondrial fusion in MGBG-treated cells. The fate of mitochondrial DNA (mtDNA) both during (24 to 48 hr) and following (7 hr to 7 days) MGBG treatment was monitored by ultrastructural, electron autoradiographic, pulse-labeling, gradient centrifugation, restriction cleavage, and electrophoretic methods. MGBG treatment (50 microM; 7 and 16 hr) selectively inhibited mtDNA replication (73% at 16 hr) prior to significant inhibition of nuclear DNA synthesis (19% at 16 hr); the drug induced structural alterations, without substantial degradation, of the closed circular (major form) of mtDNA, and cessation of D-loop strand (7S) initiation within the replication origin. Upon return to drug-free medium, mtDNA resumed replicative activity, and mtDNA fibrils appeared to be associated with regenerating cristae, as assessed by electron microscopy. The combined results demonstrate mitochondria to be a selective target of MGBG action, and define structu