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.     

Ethidium bromide-induced loss of mitochondrial DNA from primary chicken embryo fibroblasts.

Chicken embryo fibroblasts in uridine-containing medium are inherently resistant to the growth-inhibitory effect of ethidium bromide. The drug was found to inhibit the incorporation of [3H]thymidine into mitochondrial DNA circular molecules. Mitochondrial DNA was quantitated by DNA-DNA reassociation kinetics with a probe of chicken liver mitochondrial DNA. A mean number of 604 copies of mitochondrial DNA per cell was found. This number decreased progressively in cells exposed to ethidium bromide, and by day 13 ca. one copy of mitochondrial DNA was detected per cell. When the cells were then transferred to drug-free medium, the number of copies increased very slowly as a function of time. On the other hand, analyses of DNA extracted from cell populations exposed to ethidium bromide for 20 or more days, with or without subsequent transfer to drug-free medium, revealed very little or no mitochondrial DNA by reassociation kinetics or by Southern blot hybridization of AvaI- or HindIII-digested total cellular DNA. As a result of the elimination of mitochondrial DNA molecules, the establishment of cell populations with a respiration-deficient phenotype was confirmed by measuring cytochrome c oxidase activity as a function of the number of cell generations and the absorption spectrum of mitochondrial cytochromes.     

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.     

Effects of ethidium bromide and chloramphenicol on the mitochondrial nucleoids in Euglena gracilis

The effects of ethidium bromide (EB) at 0.13 m M and of chloramphenicol (CAP) at 46 m M on the mitochondria and mitochondrial nucleoids in Euglena gracilis . Z strain, were examined by fluorescence microscopy and by electron microscopy. Ethidium bromide stopped the multiplication of cells and decreased their respiratory activity by 55% after treatment for 10 days. Most of the mitochondria became slender with few cristae and some became cup-shaped with stacked cristac. Mitochondrial nucleoids decreased markedly in number after treatment with EB for more than 2 days. After treatment for 3 days with EB, mitochondrial nucleoids could not be detected in about half of all cells examined. Treatment with CAP for 10 days reduced the respiratory activity by 47%. Chloramphenicol did not decrease the number of mitochondrial nucleoids but it increased the number of cristae and the volume of mitochondria.     

Ultrastructure and microchemical composition of ethidium bromide-induced intramitochondrial complexes.

Ultrastructural changes in mammalian cells treated with ethidium bromide (EB) occur predominantly in the mitochondria. One hour after addition of 10 μg/ml of EB, an accumulation of electron-dense materials occurred in many mitochondria. After 4 h of treatment, mitochondrial complexes consisting of helically arranged fibers 30–260 Å in thickness were observed. [³H]TdR autoradiography demonstrated the presence of DNA in the complexes. EB-treated cells were also studied using energy dispersive X-ray analysis techniques. Mitochondria containing dense complexes were significantly different, microchemically, from mitochondria devoid of the structure, and contained analyzable amounts of bromine. These observations suggest that EB treatment of cells induces the formation of electron-dense mitochondrial complexes containing EB, mitochondrial DNA, and protein, and the complexes are associated with an inhibition of normal mitochondrial development.     

Generation of rho0 cells utilizing a mitochondrially targeted restriction endonuclease and comparative analyses

Eukaryotic cells devoid of mitochondrial DNA (rho0 cells) were originally generated under artificial growth conditions utilizing ethidium bromide. The chemical is known to intercalate preferentially with the mitochondrial double-stranded DNA thereby interfering with enzymes of the replication machinery. Rho0 cell lines are highly valuable tools to study human mitochondrial disorders because they can be utilized in cytoplasmic transfer experiments. However, mutagenic effects of ethidium bromide onto the nuclear DNA cannot be excluded. To foreclose this mutagenic character during the development of rho0 cell lines, we developed an extremely mild, reliable and timesaving method to generate rho0 cell lines within 3-5 days based on an enzymatic approach. Utilizing the genes for the restriction endonuclease EcoRI and the fluorescent protein EGFP that were fused to a mitochondrial targeting sequence, we developed a CMV-driven expression vector that allowed the temporal expression of the resulting fusion enzyme in eukaryotic cells. Applied on the human cell line 143B.TK- the active protein localized to mitochondria and induced the complete destruction of endogenous mtDNA. Mouse and rat rho0 cell lines were also successfully created with this approach. Furthermore, the newly established 143B.TK- rho0 cell line was characterized in great detail thereby releasing interesting insights into the morphology and ultra structure of human rho0 mitochondria.     

ROLE OF THE MITOCHONDRIAL GENOME DURING EARLY DEVELOPMENT IN MICE : Effects of Ethidium Bromide and Chloramphenicol

The role of the mitochondrial genome in early development and differentiation was studied in mouse embryos cultured in vitro from the two to four cell stage to the blastocyst (about 100 cells). During this period the mitochondria undergo morphological differentiation: progressive enlargement followed by an increase in matrix density, in number of cristae, and in number of mitochondrial ribosomes. Mitochondrial ribosomal and transfer RNA synthesis occurs from the 8 to 16 cell stage on and contributes to the establishment of a mitochondrial protein-synthesizing system. Inhibition of mitochondrial RNA- and protein-synthesis by 0.1 µg/ml of ethidium bromide or 31.2 µg/ml of chloramphenicol permits essentially normal embryo development and cellular differentiation. Mitochondrial morphogenesis is also nearly normal except for the appearance of dilated and vesicular cristae in blastocyst mitochondria. Such blastocysts are capable of normal postimplantation development when transplanted into the uteri of foster mothers. Higher concentrations of these inhibitors have general toxic effects and arrest embryo development. It is concluded that mitochondrial differentiation in the early mouse embryo occurs through the progressive transformation of the preexisting mitochondria and is largely controlled by the nucleocytoplasmic system. Mitochondrial protein synthesis is required for the normal structural organization of the cristae in blastocyst mitochondria. Embryo development and cellular differentiation up to the blastocyst stage are not dependent on mitochondrial genetic activity.     

EFFECTS OF ETHIDIUM BROMIDE ON THE CYTOCHROME CONTENT AND ULTRASTRUCTURE OF L CELL MITOCHONDRIA

Ethidium bromide intercalates between the bases of native DNA, resulting in several biological anomalies. The effects of ethidium bromide on the mitochondria of cultured mouse L cells were studied. At a concentration of 1 µg ethidium bromide/ml it was observed that concentrations of cytochromes a + a₃ and b decreased, a + a₃ more rapidly than b. In contrast, the concentration of cytochromes c₁ and c increased or remained the same as in control cells. Concomitant with the decrease of cytochromes a + a₃ and b was an enlargement of the mitochondria and a reduction in the cristae. The cristae that remained were abnormally organized. After prolonged treatment with ethidium bromide a second population of small, more normally organized mitochondria was apparent. These effects of ethidium bromide could be reversed.     

Synthesis of mitochondrial macromolecules in herpes simplex type 1 virus infected Vero cells

How viral infections affect host cell mitochondrial functions is largely unknown. In this study, uptake of radiolabeled precursors was used to assess how a herpes simplex virus type 1 (HSV 1) infection influences synthesis of macromolecules comprising Vero cell mitochondria. Total macromolecular synthesis in infected cells was determined for comparative purposes. Mitochondrial and total cellular DNA syntheses were approximately halved at 1-2.5 h postinfection (PI). Mitochondrial DNA synthesis in infected cells then rose to 3.5-fold that in control cells at 3-4.5 h PI. Total DNA synthesis in infected cells also rose, but more slowly, reaching threefold that for control cells at 5-6.5 h PI. Mitochondrial and total RNA synthesis in infected cells were both decreased by approximately 40% at 1-3 h PI. Over the next 4 h, total RNA synthesis in infected cells slowly continued to decrease, while that in mitochondria recovered to control levels. Synthesis of mitochondrial proteins in infected cells decreased progressively, dropping to about 60% of control levels by 5-6.5 h PI. With the metabolic inhibitors ethidium bromide and cycloheximide, it was determined that nuclear DNA and mitochondrial DNA and mitochondrial DNA directed synthesis of mitochondrial proteins were each partially inhibited in infected cells. Total cellular protein synthesis was decreased by 30% at 1-2.5 h PI and then recovered to control levels by 5-6.5 h PI. Finally, phospholipid synthesis in mitochondria from infected cells was elevated 2.3-fold at 1-5 h PI, but dropped to 14% below control levels during 4-8 h PI.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolic capacity regulates iron homeostasis in endothelial cells

The sensitivity of endothelial cells to oxidative stress and the high concentrations of iron in mitochondria led us to test the hypotheses that (1) changes in respiratory capacity alter iron homeostasis, and (2) lack of aerobic metabolism decreases labile iron stores and attenuates oxidative stress. Two respiration-deficient (rho(o)) endothelial cell lines with selective deletion of mitochondrial DNA (mtDNA) were created by exposing a parent endothelial cell line (EA) to ethidium bromide. Surviving cells were cloned and mtDNA-deficient cell lines were demonstrated to have diminished oxygen consumption. Total cellular and mitochondrial iron levels were measured, and iron uptake and compartmentalization were measured by inductively coupled plasma atomic emission spectroscopy. Iron transport and storage protein expression were analyzed by real-time polymerase chain reaction and Western blot or ELISA, and total and mitochondrial reactive oxygen species (ROS) generation was measured. Mitochondrial iron content was the same in all three cell lines, but both rho(o) lines had lower iron uptake and total cellular iron. Protein and mRNA expressions of major cytosolic iron transport constituents were down-regulated in rho(o) cells, including transferrin receptor, divalent metal transporter-1 (-IRE isoform), and ferritin. The mitochondrial iron-handling protein, frataxin, was also decreased in respiration-deficient cells. The rho(o) cell lines generated less mitochondrial ROS but released more extracellular H(2)O(2), and demonstrated significantly lower levels of lipid aldehyde formation than control cells. In summary, rho(o) cells with a minimal aerobic capacity had decreased iron uptake and storage. This work demonstrates that mitochondria regulate iron homeostasis in endothelial cells.     

Role of Mitochondria in the Production of RNA-Containing Tumor Viruses

The role of mitochondria in the reproduction of RNA-containing tumor viruses was examined by using ethidium bromide (EB) to induce degenerative effects in mitochondria. The effects of EB in murine and avian cells were monitored by electron microscopy. Chronically infected mouse (JLS-V5) cells, in which extensive mitochondrial changes were induced, continued to produce murine leukemia virus. Also, complete reproductive cycles of Rous sarcoma virus (RSV) occurred in newly infected chicken embryo cells exposed to EB. Morphological transformation characteristic of infection of chicken embryo cells by RSV occurred in cells which contained induced aberrant mitochondria. The results demonstrate that mitochondria play a relatively minor role, if any, in the reproduction of RNA-containing tumor viruses.     

Simultaneous analysis of mitochondrial activity and DNA content in Ehrlich ascites tumor cells by dual parameter flow cytometry

Ehrlich ascites tumor cells were permeabilized using low concentrations of digitonin, 8 micrograms/10(6) cells. Permeabilization was monitored by the assay of lactate dehydrogenase released into the incubation medium and of hexokinase partially bound to mitochondria. Integrity of the cellular organelles was unaffected as determined by assay of the mitochondrial enzyme glutamate dehydrogenase. Cells were stained with rhodamine 123 as a mitochondrial specific dye and propidium iodide/mithramycin as DNA specific dyes. The green fluorescence of bound rhodamine 123 versus red fluorescence of DNA in individual cells was analysed by dual parameter flow cytometry. Incubation of cells with inhibitors of mitochondrial energy metabolism, such as, potassium cyanide and carbonyl cyanide m-chlorophenylhydrazone abolished binding of rhodamine 123. Flow cytometric data allowed a correlation between cell position in the mitotic cycle with total mitochondrial activity. In addition, comparison of the characteristics of propidium iodide and ethidium bromide staining further elucidated the molecular basis of the staining with the positively-charged fluorescent dye rhodamine 123.     

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.     

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.     

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.     

Effects of ethidium bromide, tetramethylethidium bromide and betaine B on the ultrastructure of HeLa cell mitochondria in situ. A comparative binding study

Several investigators have described the ultrastructural changes that occur in the mitochondria of cells in tissue cultures after treatment with the drug ethidium bromide (E). The mitochondria swell and the cristae become greatly altered and finally disappear; in the cristae-free region of the matrix electron-dense granules can be observed. It has been assumed that intercalation of E between the base pairs of the mitochondrial DNA induces the formation of the granular inclusions. To investigate whether intercalation is really the initial step in the generation of dense granules inside the matrix, we performed a comparative incubation study of HeLa-cell mitochondria in situ using three closely related dyes (D), i.e., E, tetramethylethidium bromide (TME) and betaine B (B). They strongly differ with regard to their affinity for DNA and their ability to cross membranes. E was used as a reference dye. TME does not intercalate, but is externally bound to DNA only weakly. The neutral B is not bound at all, but can cross membranes more easily than the cation E. Moreover, in aqueous solutions at pH approximately equal to 7.0, B is in equilibrium with its protonated cation BH. BH and E have almost equal affinities for DNA. Therefore B may quickly pass the inner mitochondrial membranes and the cristae, and should then be bound inside the matrix, thus forming a BH-DNA complex. On the assumption that intercalation is necessary for the generation of intramitochondrial electron-dense bodies, we predicted that BH/B should be more efficient than E, while TME should be relatively ineffective.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and cell cycle analysis of temperature-sensitive mutants from chinese hamster cells

HeLa cell mitochondria were allowed to incorporate ³H-thymidine in a cell free system and the effect of ethidium bromide, cytosine arabinoside and cytosine arabinoside triphosphate on the labeling of mitochondrial DNA was studied. The labeled products, isolated by sedimentation velocity in CsCl-ethidium bromide two-step gradients, showed similar sedimentation profiles as in vivo labeled mtDNA. Cytosine arabinoside triphosphate and ethidium bromide strongly inhibited the labeling of mitochondrial DNA, whereas cytosine arabinoside appeared to be much less effective. Tritiated deoxycytidine was found to be incorporated by isolated mitochondria, whereas cytosine arabinoside was shown to enter the mitochondrial acid-soluble pool but not to be incorporated in acid-insoluble form. These results are in agreement with the previously reported findings of in vivo experiments.     

Separation of closed circular DNA from linear DNA by electrophoresis in two dimensions in agarose gels

A method that provides an easy, rapid, and reproducible way for separating closed circular DNA species from linear DNA and nicked circles is described. The method is based on the difference in mobility of form I (supercoiled) DNA and form II (nicked circles), and the differential mobility of relaxed circular DNA in the presence and absence of ethidium bromide (EtdBr). It can be used for detection or for purification of plasmid, episomal, or viral DNA from the bulk of cellular DNA, or from other DNA mixtures.     

Assessment of the distribution of nucleic acid intercalators in yeast cells by pseudospectral image analysis

The intracellular location of nucleic acid intercalators (NAI) in native (not fixed) Saccharomyces cerevisiae cells has been studied using fluorescence microscopy combined with computer pseudospectral image analysis. Three NAI: anthracycline anticancer drug doxorubicin and nucleic acid dyes ethidium bromide and 4′,6-diamidino-2-phenylindole (DAPI) were used. All three NAI were shown to be localized in nuclei and mitochondria. In contrast to DAPI, which interacted only with DNA, a large fraction of doxorubicin and ethidium bromide apparently bound to mitochondrial membranes. Upon combined application, competition between these intercalators for binding sites in the nuclear and mitochondrial DNA occurred. It was concluded that this approach may be used in designing new DNA-targeted drugs and in preliminary studies of their interaction with eukaryotic cells.