The reversibility of the ethidium bromide-induced alterations of mitochondrial structure and function in the cellular slime mold,Dictyostelium discoideum

Abstract

Addition of ethidium bromide to ameboid cultures of the slime mold,Dictyostelium discoideum, caused a cessation of cell division after 1 or 2 generations. The replication of mitochondrial DNA was immediately blocked as indicated by the 50% decrease in the DNA content of purified mitochondria from ethidium-bromide-treated cultures. The activity of the respiratory chain was also inhibited, resulting in a 75% decrease in cyanide-sensitive whole cell respiration. Spectral analysis at low temperature indicated that the amount of cytochromec1 was decreased 80% and that of cytochromec increased 100% in mitochondria from treated cells. Two cytochromesb absorbing at 556 and 561 nm were observed in mitochondria from both control and ethidium-bromide-treated cultures. The content of cytochromeb561 appeared to decline more than didb556, but it is hard to quantitate the decrease. The effects of ethidium bromide were fully reversible. When the drug was removed, the cells resumed a normal growth rate without any discernible lag. The activity of oligomycin-sensitive ATPase, cytochrome oxidase, and succinate-cytochrome-c reductase as well as the cytochrome content began to increase after 1 day returning to control levels within 5 days. Electron micrographs of whole cells treated with ethidium bromide revealed that mitochondrial profiles were elongated and had greatly reduced cristae. Numerous membrane whorls were apparent, as was a profound loss of rough endoplasmic reticulum. Three days after removal of ethidium bromide, mitochondria were again ovoid in shape and contained well-developed cristae. In all of the cells during recovery, there was a single large vacuole that appeared to enclose a large portion of the cell volume, forming a new cellular compartment that may simplify the breakdown of previously damaged organelles.