Effects of ethidium bromide on the respiratory chain and oligomycin-sensitive adenosine triphosphatase in purified mitochondria from the cellular slime mold Dicyostelium discoideum.

Mitochondria were isolated from the cellular slime mold. Dictyoostelium discoideum, and partially purified by sucrose density gradient fractionation. The most purified mitochondrial fraction from the gradient contained essentially no contaminating lysosomes and minimal amounts of contaminating peroxisomes as determined by the marker enzymes N-acetyl-glucosaminidase and catalase. A mitochondrial fraction with the same amount of lysosomal and peroxisomal contamination was also isolated from cells which had been treated with ethidium bromide for 5 days. The most purified mitochondrial fraction from control and ethidium bromide-treated cells had an identical buoyant density of 1.181 to 1.182 g per ml, suggesting that treatment with the drug does not result in any drastic structural changes in the mitochondrial membrane which would affect its density. In the purified mitochondria from ethidium bromide-treated cells, the content of cytochromes a-a3 was decreased over 80% and that of cytochrome oxidase and oligomycin sensitive ATPase were reduced approximately 50%. By contrast, the specific activities of NADH and succinate dehydrogenases were identical in the purified mitochondria from control and ethidium bromide-treated cells. Previously, we had reported that the specific activities of these two enzymes had nearly doubled in whole cells maintained in ethidium bromide for a time equivalent to six or seven generations after growth had stopped (Stuchell, R. N., Weinstein, B. I., and Beattie, D. S. (1973) Fed. Eur. Biochem. Coc Lett. 37, 23-26). These results suggest that continued formation of new mitochondrial membranes, with an identical complement of succinate and NADH dehydrogenases, must occur despite the cessation of cell growth which occurs as a result of the ethidium bromide induced loss of mitochondrial enzymes. Consequently, the amount of mitochondria, or mitochondrial protein per cell, calculated from the activity of NADH and succinate dehydrogenases has increased nearly 50%. Possible models to explain the control of mitochondrial biogenesis are discussed to explain these results.     

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

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 cytochromec ₁ 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 cytochromeb ₅₆₁ appeared to decline more than didb ₅₅₆, 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.This work is in partial fulfillment of the requirements for the Doctor of Philosophy degree at the City University of New York.     

Effect of aspirin on mitochondrial mutagens in Saccharomyces cerevisiae

Summary The mitochondrial mutation petite was induced in yeast cells by ethidium bromide (EB), Adriamycin (ADR) and 4-nitroquinoline-N-oxide (NQO). In the presence of aspirin in concentrations ranging from 0.1 to 1.0 mg/ml, the mutagenicity of EB and ADR was reversed but petite induction by NQO was unaffected. At these concentrations, aspirin also reversed mitochondrial inhibition by oligomycin, a non-mutagenic inhibitor of the organellar ATPase complex.Cells grown in the presence of aspirin alone showed a significantly higher rate of oxygen uptake than untreated control cultures when the drug concentration ranged from 0.05 to 1.0 mg/ml. At concentrations of 2 mg/ml and above, aspirin inhibited mitochondrial respiration.     

Dependence of mammalian DNA replication on DNA supercoiling. I. Effects of ethidium bromide on DNA synthesis in permeable Chinese hamster ovary cells.

Chinese hamster ovary cells labelled with [14C]thymidine were made permeable, incubated with various concentrations of the intercalating dye ethidium bromide, and centrifuged through neutral sucrose gradients. The gradient profiles of these cells were qualitatively similar to those obtained by centrifuging DNA from untreated, lysed permeable cells through gradients containing ethidium bromide. The sedimentation distance of DNA had a biphasic dependence on the concentration of ethidium bromide, suggesting that the dye altered the amount of DNA supercoiling in situ. The effect of ethidium bromide intercalation on incorporation of [3H]dTMP into acid-precipitable material in an in vitro DNA synthesis mixture was measured. The incorporation of [3H]dTMP was unaffected by less than 1 microgram/ml of ethidium bromide, enhanced up to two-fold by 1--10 microgram/ml, and inhibited by concentrations greater than 10 micrograms/ml. Alkaline sucrose gradient analysis revealed a higher percentage of small DNA fragments (6--20 S) in the cells treated with 2 micrograms/ml ethidium bromide than in control cells. These fragments attained parental size within the same time as the fragments in control cells. In cells treated with 2 micrograms/ml ethidium bromide, a significant fraction of newly synthesized DNA resulted from new starts, whereas in untreated cells practically none of the newly synthesized DNA resulted from new starts. These results suggest that relaxation of DNA supercoiled structures ahead of the replication fork generates spurious initiations of DNA synthesis and that in intact cells the rate of chain elongation is limited by supercoiled regions ahead of the growing point.     

Mitochondrial DNA synthesis during fungal spore germination

Summary Germinating spores of the fungus Botryodiplodia theobromae incorporated guanine-8-C¹⁴ into both the nuclear DNA and mitochondrial DNA fractions. Ethidium bromide inhibited the synthesis of mitochondrial DNA without having a significant effect on nuclear DNA synthesis or on the rate and extent of spore germination. Rates of leucine and uracil incorporation and of oxygen uptake were not significantly affected by ethidium bromide until germination was nearly completed. Mitochondrial DNA synthesis is apparently not required for germination of the spores of B. theobromae but is probably essential to continued vegetative growth.Abbreviations DNA deoxyribonucleic acid - mit-DNA mitochondrial DNA - nuc-DNA nuclear DNA - RNA ribonucleic acid - EB ethidium bromide - Tris tris (hydroxymethyl)aminomethane Published with the approval of the Director as Paper No. 3331, Journal Series, Nebraska Agricultural Experiment Station. Research reported was conducted under Project No. 21-17. Paper No. 7877, Scientific Journal Series, Minnesota Agricultural Experiment Station.     

Direct action of ethidium bromide upon mitochondrial oxidative phosphorylation and morphology.

Ethidium bromide (23 nmol/mg of protein) was found to be a potent inhibitor of oxidative phosphorylation, as determined by loss of respiratory control through the inhibition of the ADP-induced state-3 rate of oxygen uptake. A time latency for complete loss of respiratory control was noted, after which 2,4-dinitrophenol (DNP) was ineffective in overcoming this inhibition. In the absence of EDTA, ethidium bromide produced an apparent uncoupling, as evidenced by an increase of state-4 rates of oxygen uptake and loss of respiratory control. As low as 8 nmol of ethidium bromide/mg of protein stimulated mitochondrial adenosine triphosphatase (ATPase) for 5 min. Two to three times this amount of ethidium bromide reduced the amount P_i released. Preincubation of mitochondria with ethidium bromide prevented subsequent release of P_i during incubation with ATP. Likewise, preincubation inhibited the DNP-activated ATPase. The uptake of low levels of [¹⁴C]ADP preincubated with ethidium bromide (14 nmol/mg of protein) and succinate or α-ketoglutarate could apparently be reversed, with loss of radioactivity beginning several minutes after addition of the radioactive nucleotide. Inhibition of oxidative phosphorylation by ethidium bromide may be due to modification of the adenine nucleotide transport system in mitochondria. The production of apparently swollen mitochondria treated in vitro with ethidium bromide and substrates necessary for oxidative phosphorylation, as seen in electron micrographs, further indicates that the compound is capable of acting directly upon mouse liver mitochondrial function and structure.     

Control and virus-transformed baby hamster kidney cells resistant to ethidium bromide. I. Characterization and the respiratory enzymes

Cell lines resistant to ethidium bromide have been developed from cultured mammalian BHK21/C13 cells and these same cells transformed by Rous sarcoma virus (C13/B4). Cells resistant to 2 micrograms ethidium bromide per milliliter have been cloned. One clone of the control and one of the virus-transformed cell lines has been employed for characterization. The resistant cells, in the presence of 2 micrograms ethidium bromide/ml, grow at approximately the same rate as the untreated parental cells. The control cells possess a "normal" karyotype (44 chromosomes), while the corresponding ethidium bromide mutant has a reduced chromosome number of 41 and a number of translocations. The mitochondria displayed morphological alterations compared to the parental lines during the transition phase prior to the isolation of the ethidium bromide-resistant cells. The mitochondria of the ethidium bromide-resistant mutants appear somewhat enlarged with a normal morphology. The effect of ethidium bromide on selected respiratory enzymes in normal and virus-transformed ethidium bromide-resistant baby hamster kidney cells was determined. Ethidium bromide-resistant cells exhibited a depressed level of cytochrome aa3. This depression could not be reversed by growth in ethidium bromide-free media. Ethidium bromide-resistant cells possessed the same cytochrome b, c, and c1 levels per cell as their corresponding parental lines. Purified mitochondria isolated from virus-transformed ethidium bromide-resistant cells exhibited a depression in cytochrome oxidase-specific activity, while the ethidium bromide-resistant control cells did not. All cell lines studied showed a depression in NADH-ferricyanide and NADH-cytochrome c reductase-specific activities relative to their parental BHK21/C13 cells. No increase was observed in virus-transformed ethidium bromide-resistant cells. Ethidium bromide-resistant control cells exhibited a two-fold increase in oligomycin-insensitive adenosine triphosphatase activity relative to their parental cells. All of the cell lines studied possessed equivalent oligomycin-sensitive adenosine triphosphatase-specific activity except for the virus-transformed, dye-resistant mutant, whose activity was increased.     

Photosensitivity and heat resistance conferred by BrdU incorporation upon a thymidine kinase-deficient mouse cell line with persistent mitochondrial enzyme activity.

The selective incorporation of 5-bromodeoxyuridine (BrdU) into mitochondrial DNA (mit-DNA) in the LM(TK-) ClID cell line, a thymidine kinase-deficient derivative of L fibroblasts with persistent mitochondrial enzyme activity, has been utilized to specifically damage mit-DNA by 'visible' light irradiation. ('Visible light' indicates the source of light used, although the components most active photochemically on BrdU-substituted DNA are in the near-visible range, 300-340 nm.) (Szybalski & Opara-Kubinski, 1965). LM(TK-) Cl ID cells, which had been grown in the presence of 30 mug/ml BrdU, were irradiated with 'visible' light. Analysis of the pre-existing mit-DNA in these cells, which had been long-term labelled with [5-3H]deoxycytidine, showed a progressive decrease, with increasing duration of irradiation, in the proportion of the closed-circular form and an increase in that of the open-circular form of mit-DNA, with the subsequent appearance of fragments of this DNA. Furthermore, there was a decrease during irradiation in the total amount of mit-DNA, which became about 35% of the non-irradiated control after 65 h irradiation. On the other hand, irradiation with 'visible' light failed to cause any quantitative or qualitative change, with respect to the non-irradiated control, in mit-DNA from cells grown in the absence of BrdU and long-term labelled with [Me-3h]thymidine. An analysis of the incorporation of [5-3H]deoxycytidine into mit-DNA of BrdU-grown cells, during a 3-h exposure of the cells to the precursor following irradiation, showed a fairly rapid decline of mit-DNA labelling; this became about 50% of the non-irradiated control after 12 h irradiation, decreasing to about 25% in the next 48 h. By contrast, no effect of irradiation was observed on the subsequent pulse-labelling of mit-DNA with [Me-3H]thymidine in cells grown in the absence of BrdU. Furthermore, no change in the size of the extracted nuclear DNA was found in irradiated BrdU-grown cells. The progressive and selective damage and destruction of mit-DNA during irradiation with 'visible' light of Cl ID cells correlate fairly well with the kinetics of loss of cell viability occurring under the same conditions, as described in the accompanying paper, strongly suggesting a link between the two phenomena.     

Increase in mitochondrial DNA quantity and impairment of oxidative phosphorylation in bovine fibroblast cells treated with ethidium bromide for 15 passages in culture

Bovine fetal fibroblast cells were treated with ethidium bromide at a low concentration for 15 passages in culture to determine its effect on mitochondrial DNA copy number and on cell metabolism. Mitochondrial membrane potential and lactate production were estimated in order to characterize cell metabolism. In addition, mitochondrial DNA ND5 in proportion to a nuclear gene (luteinizing hormone receptor) was determined at the 1st, 2nd, 3rd, 10th, and 15th passages using semi-quantitative PCR amplification. Treated cells showed a lower mitochondrial membrane potential and higher levels of lactate production compared with control cells. However, the mitochondrial DNA/nuclear DNA ratio was higher in treated cells compared with control cells at the 10th and 15th passages. This ratio changed between the 3rd and 10th passages. Despite a clear impairment in mitochondrial function, ethidium bromide treatment did not lead to mitochondrial DNA depletion. It is possible that in response to a lower synthesis of ATP, due to an impairment in oxidative phosphorylation, treated cells develop a mechanism to resist the ethidium bromide effect on mtDNA replication, resulting in an increase in mitochondrial DNA copy number.     

Biogenesis of mitochondria: XXV. Studies on the mitochondrial genomes of petite mutants of yeast using ethidium bromide as a probe

This paper describes investigations into the effects of ethidium bromide on the mitochondrial genomes of a number of different petite mutants derived from one respiratory competent strain of Saccharomyces cerevisiae. It is shown that the mutagenic effects of ethidium bromide on petite mutants occur by a similar mechanism to that previously reported for the action of this dye on grande cells. The consequences of ethidium bromide action in both cases are inhibition of the replication of mitochondrial DNA, fragmentation of pre-existing mitochondrial DNA, and the induction, often in high frequency, of cells devoid of mitochondrial genetic information (ρ ° cells).The susceptibility of the mitochondrial genomes to these effects of ethidium bromide varies in the different clones studied. The inhibition of mitochondrial DNA replication requires higher concentrations of ethidium bromide in petite cells than in the parent grande strain. Furthermore, the susceptibility of mitochondrial DNA replication to inhibition by ethidium bromide varies in different petite clones.It is found that during ethidium bromide treatment of the suppressive petite clones, the over-all suppressiveness of the cultures is reduced in parallel with the reduction in the over-all cellular levels of mitochondrial DNA. Furthermore, ethidium bromide treatment of petite clones carrying mitochondrial erythromycin resistance genes (ρ^?ER^r) leads to the elimination of these genes from the cultures. The rates of elimination of these genes are different in two ρ^?ER^r clones, and in both the gene elimination rate is slower than in the parent ρ⁺ ER^r strain. It is proposed that the rate of elimination of erythromycin resistance genes by ethidium bromide is related to the absolute number of copies of these genes in different cell types. In general, the more copies of the gene in the starting cells, the slower is the rate of elimination by ethidium bromide. These concepts lead us to suggest that petite mutants provide a system for the biological purification of particular regions of yeast mitochondrial DNA and of particular relevance is the possible purification of erythromycin resistance genes.     

Ethidium bromide resistance in a rat liver epithelial cell line: Association with enhanced drug efflux

Ethidium bromide-resistant cell strains were obtained by continuous selection of an adult rat liver-derived cell line (ARL6T) grown in the continuous presence of 200 ngl ml ethidium bromide. Comparison of resistant strains and parental (sensitive) cells was made for uptake and binding of ethidium bromide, visualized as fluorescent ethidium bromide-nucleic acid complexes. Although uptake of ethidium bromide was similar in parental and resistant cells, efflux kinetics were markedly different. Over a three-hour period, parental (sensitive) cells maintained fluorescence following a short ethidium bromide pulse (100 g/ ml ethidium bromide). In contrast, ethidium bromide-resistant cell lines eliminated photographically detectable fluorescent complexes within three hours following pulse exposure to ethidium bromide. The rapid elimination of ethidium bromide fluorescent complexes in all (5) resistant cell strains examined supports an efflux mechanism as contributing to the resistance of ethidium bromide cytotoxicity in these cells.Abbreviations EtBr ethidium bromide - HBSS Hanks' balanced salt solution     

Lack of influence of mitochondrial genetical information on the multiplication of Herpes simplex virus in HeLa cells

Mitochondrial DNA synthesis in HeLa cells is inhibited by 0.2 μg ethidium bromide/ml whereas nuclear DNA synthesis is essentially unimpaired under the same conditions. The action of ehtidium bromide on mitochondrial DNA appears to be completed within 18 hours of exposure to the drug. Total cellular macromolecular synthesis under ethidium bromide is initially decreased and at later times slightly stimulated. Ethidium bromide pretreatment of HeLa cells did not significantly affect the multiplication of Herpes simplex virus as compared with that in control cells.     

Rapid and random turnover of mitochondrial DNA in rat hepatocytes of primary culture

It is known that mitochondrial DNA (mtDNA) replication is independent of the cell cycle. Even in post-mitotic cells in which nuclear DNA replication has ceased, mtDNA is believed to still be replicating. Here, we investigated the turnover rate of mtDNA in primary rat hepatocytes, which are quiescent cells. Southwestern blot analysis using 5-bromo-2'-deoxyuridine (BrdU) was employed to estimate the activity of full-length mtDNA replication and to determine efficient doses of replication inhibitors. Southern blot analysis showed that a two-day treatment with 20mM 2',3'-dideoxycytidine and 0.2mug/ml ethidium bromide caused a 37% reduction in the amount of mtDNA, indicating that the hepatocytes had a considerably high rate of turnover of mtDNA. Further, pulse-chase analysis using Southwestern analysis showed that the amount of newly synthesized mtDNA labeled with BrdU declined to 60% of the basal level within two days. Because the rate of reduction of the new mtDNA was very similar to the overall turnover rate described above, it appears that degrading mtDNA molecules were randomly chosen. Thus, we demonstrated that there is highly active and random turnover of mtDNA in hepatocytes.     

Factors affecting petite induction and the recovery of respiratory competence in yeast cells exposed to ethidium bromide

Summary When growing cultures of S. cerevisiae are treated with high concentrations of ethidium bromide (>50 g/ml), three phases of petite induction may be observed: I. the majority of cells are rapidly converted to petite, II. subsequently a large proportion of cells recover the ability to form respiratory competent clones, and III. slow, irreversible conversion of all cells to petite. The extent of recovery of respiratory competence observed is dependent on the strain of S. cerevisiae employed and the temperature and the carbon source used in the growth medium. The effects of 100 g/ml ethidium bromide are also produced by 10 g/ml ethidium bromide in the presence of the detergent, sodium dodecyl sulphate, and recovery is also observed when cells are treated with 10 g/ml ethidium bromide under starvation conditions. Genetic analysis of strain differences indicates that a number of nuclear genes influence petite induction by ethidium bromide.In one strain, S288C, petite induction by 100 g/ml ethidium bromide is extremely slow under certain conditions. Mitochondria isolated from S288C lack the ethidium bromide stimulated nuclease activity found in D243-4A, a strain which shows triphasic kinetics of petite formation. This enzyme may, therefore, be responsible for the initial phase of rapid petite formation.     

Nuclear and mitochondrial alterations in amebae exposed to ethidium bromide

Cultures of Amoeba proteus were exposed to ethidium bromide at concentrations ranging between 5 and 100 μg/ml for periods of up to 1 week. Samples of treated and control cells were prepared at intervals for electron microscopy. The main ultrastructural alterations were in nucleoli and in mitochondria. The nucleoli of treated cells increased in density and became spherical with more sharply defined margins than those of normal amebae. Many nucleoli contained electron-lucent regions or nucleolar vacuoles of varying size. The chromatin was unusually condensed in some amebae. Mitochondria developed a central electron-lucent region and accumulated a dense material in the matrix. Some cristae were abnormally dilated. The nuclear alterations occurred at least as early as the mitochondrial changes and were present even in cells exposed to the lower concentrations of inhibitor, in which mitochondrial changes were minimal.     

Yeast mutants resistant to ethidium bromide

Summary Yeast mutants resistant to ethidium bromide have been isolated among sensitive grande cells (⁺) for their ability to grow on glycerol in the presence of the dye. Mutant cells are also resistant to acriflavin and do not yield petites (^-) when grown on galactose with the mutagen. Genetic analysis reveals that resistance to ethidium bromide is controlled by a cytoplasmic factor, carried by, or linked to, the determinant (mitochondrial DNA). The expression of resistance to ethidium bromide seems to be related to the presence in the cell of a product of mitochondrial protein synthesis. It is concluded that some mitochondrial DNA sequence is involved in the resistance to ethidium bromide of yeast mitochondria.     

Morphometric Studies of Mitochondria in Tetrahymena pyriformis Exposed to Chloramphenicol or Ethidium Bromide*

Cultures of Tetrahymena pyriformis strain ST were exposed to 300 μg chloramphenicol/ml or 15 μg ethidium bromide/ml for 48 hr. Qualitative assessments of electron micrographs reveal that the abundance of mitochondrial cristae decreases greatly. By equating the spatial characteristics of the organism with those of a prolate spheroid, the distribution and abundance of mitochondria were quantified. Such characterizations reveal that the size of individual mitochondria decreases by 35–60% and that the number of mitochondria/cell increases ～8 fold. The observations are discussed in terms of coordinated mitochondrial and nuclear genetic activities.