Journal of Cell Science, Vol 13, 567-590, Copyright © 1973 by Company of Biologists

Submitted on January 30, 1973

Effects of Ethidium Bromide on Growth, Chlorophyll Synthesis, Ultrastructure and Mitochondrial Dna in Green and Bleached Mutant Euglena Gracilis

¹ Department of Therapeutic Research, University of Pennsylvania School of Medicine, Philadelphia, Pa. 19104, U.S.A.
² Laboratory for Electron Microscopy, Tel-Aviv University, Israel

Green Euglena cells were grown with 1, 5, 20, 50 and 100 µg/ml ethidium bromide (EB), a DNA-intercalating dye and cytoplasmic mutagen. Treatment with 50 µg EB/ml for 3 days in growing medium, pH 3.3 (heterotrophic growth) was maximally effective and inhibited cell division by 70% and chlorophyll formation by 60%. Similar results were obtained in Cramer Meyers medium, pH 6.8 without acetate (autotrophic growth). When dark-grown cells were shifted from heterotrophic to autotrophic medium and exposed to light and 20 or 50 µg EB/ml for 3 days, the cells barely divided and chlorophyll formation (greening) was inhibited by 30 and 60% with 20 and 50 µg EB/ml, respectively. Greening was similarly inhibited in heterotrophic medium. The ultrastructure of chloroplasts appeared normal in all conditions, but mitochondria of EB-treated cells had fewer cristae than control cells and frequently concentrically arranged cristae. Dark-grown cells were treated with 5, 20, 50, 100 and 150 µg EB/ml for 24 h, transferred to resting medium, pH 4.5, and exposed to EB and light for 3 days. Chlorophyll synthesis was significantly inhibited even with 5 µg EB/ml. The ultrastructure of chloroplasts and mitochondria appeared normal.

Bleached mutant Euglena were treated with 1, 5, 10 and 20 µg EB/ml in growing medium. Cell division was inhibited by 70% after 7 days of culture with 20 µg EB/ml. Inhibition was greater in the light than in the dark. The ultrastructure of mitochondria was strikingly changed even with 1 or 5 µg EB/ml for 5 days, and there were 2 types: mitochondrial profiles with few and/or concentrically arranged cristae and electron-lucid matrix, and fused sheets of mitochondrial membranes encircling the periphery of the cell. Cytochrome oxidase activity was partially inhibited. The ultrastructural changes in mitochondria after growth of bleached Euglena in 1, 5 and 10 µg EB/ml for 10 days were reversed (partially in some cells) when cells were transferred to EB-free medium for another 20 days. There was a 10-15 day lag in the recovery of cell division rate. Re-exposure of reversed cells to 20 µg EB/ml showed a progressively greater resistance to the drug directly proportional to the original concentration of EB.

The yield of mitochondrial DNA after exposure to EB was slightly lowered after 3 days, and after 8 days was reduced from 1.84 µg M-DNA/mg M-protein (control) to 1.69 and 0.98 (with 1 and 5 µg EB/ml, respectively). Mitochondrial DNA synthesis, as reflected by the incorporation of [³H]guanine and [³H]adenine into DNA, was unaffected after 3 days of growth in 1 and 5 µg EB/ml, but was inhibited by 70% after 8 days. Nuclear DNA synthesis was not significantly inhibited. The specific activities of circular satellite DNA were lowered by 50%.

The results demonstrate that EB inhibits cell division in both strains of Euglena, that ultra-structure is changed only in mitochondria of growing cells, not in chloroplasts, and that some chloroplast but mainly mitochondrial functions are inhibited under suitable conditions. The results also indicate that resistance to the drug can be developed.