Actions of bis(guanylhydrazones) on isolated rat liver mitochondria

Abstract

The effects of the anticancer bis(guanylhydrazones), methylglyoxal-bis(guanylhydrazone) (MGBG) and 4,4'-diacetyldiphenylurea-bis(guanylhydrazone) (DDUG), on parameters related to the bioenergetic function of isolated rat liver mitochondria were investigated. At concentrations comparable to those attained intracellularly, both bis(guanylhydrazones) significantly inhibited state 4 respiration but had less of an inhibitory effect on state 3 or uncoupled respiration. DDUG was more effective than MGBG, requiring 0.34mM to achieve a 25 per cent inhibition of respiration as compared to 6.0mM for MGBG. The inhibition was prevented by potassium cations and was enhanced in mitochondria “de-energized” with valinomycin, a potassium cationophore. This suggested drug competition for potassium-binding sites, possibly membrane phospholipids. Addition of 1.25 mM MGBM or 0.025 mM DDUG to suspended mitochondria caused a rapid aggregation of organelles and an increase in the optical density of the suspension. Pretreatment with either bis(guanylhydrazone) protected mitochondria against non-specific swelling action of 0.0015% Triton X-100, suggesting membrane binding. By electron microscopy, MGBG- or DDUG-treated mitochondria appeared swollen and the spaces between cristae membranes or inner and outer membranes were collapsed, obliterating the outer mitochondrial compartment. The activity of monoamine oxidase A, an outer membrane marker enzyme, was reduced considerably by 4 mM MGBG or 0.075 mM DDUG. Mobility of mitochondria toward the anode in an electrophoretic field was slowed 50 per cent by 2.5 mM MGBG or 0.1 mM DDUG. These data suggest that positively charged bis(guanylhydrazones) neutralize the net negative surface potential of rat liver mitochondria by binding to sites (possibly phospholipids) at the inner mitochondrial membrane. Subsequent interference with cation binding and/or transport results in inhibition of bioenergetic functions.