Specific Activity Of Cytochrome Research Articles

COMPARATIVE EFFICIENCY OF CYTOCHROME C EYE DROPS DURING EXPERIMENTAL EYE DAMAGE

Introduction. Cytochrome C is a metabolic drug that has an antihypoxic, trophic effect, stimulates regeneration processes, and is also used in various eye diseases. Aim. The task of the work was to determine the specific activity of cytochrome C eye drops in comparison with emoxipin. Materials and methods. As a test system, Soviet chinchilla male rabbits were used, the simulation of traumatic erosion and acid burn was carried out, followed by the use of test eye drops. In forming erosion, the method of C. Hanna, J. E. O'Brien (1960) with scraping of the corneal epithelium, an acid burn in the experiment was formed under local anesthesia (0.4% inocain) application of filter paper (in the form of a circle with a diameter of 8 mm) moistened with 3% acetic acid solution with an exposure of 5 seconds on the cornea. The effectiveness evaluation was carried out on the basis of a scale proposed by the authors, which allows histologically to take into account corneal changes during the pathological process. Descriptive statistics methods were used for all quantitative data: the average sample values (M) and standard deviations (SD) were calculated. The distribution in each data sample was analyzed using the Shapiro – Wilk criterion. Under normal distribution, analysis of variance was used followed by a posteriori analysis (Tukey criterion). If the data had an abnormal distribution, the Kruskal-Wallis test was calculated. Results and discussion. The integrity of the patterns of traumatic erosion and acid burn was histologically confirmed. The therapeutic efficacy of eye drops of emoxipin in the full dose was not sufficiently pronounced and comparable to that of cytochrome C in half the therapeutic dose. Cytochrome C in therapeutic and dual therapeutic dose effectively coped with the effects of acid burn and traumatic erosion when used in the form of eye drops for 28 days. Conclusion. Cytochrome C eye drops, used at a therapeutic dose and a dose exceeding the therapeutic dose 2 times, have a pronounced regenerative effect in traumatic erosion and acid burn of the rabbit eye cornea. The effect of Cytochrome C eye drops at half the therapeutic dose, as well as the reference drug (emoxipin) at the therapeutic dose, is not very pronounced.

Changes in transcription pattern lead to a marked decrease in COX, CS and SQR activity after the developmental point of the 22(nd) gestational week.

Tissue differentiation and proliferation throughout fetal development interconnect with changes in the oxidative phosphorylation system (OXPHOS) on the cellular level. Reevaluation of the expression data revealed a significant increase in COX4 and MTATP6 liver transcription levels after the 22(nd) gestational week (GW) which inspired us to characterize its functional impact. Specific activities of cytochrome c oxidase (COX), citrate synthase (CS), succinate-coenzyme Q reductase (SQR) and mtDNA determined by spectrophotometry and RT-PCR were studied in a set of 25 liver and 18 skeletal muscle samples at 13(th) to 29(th) GW. Additionally, liver hematopoiesis (LH) was surveyed by light microscopy. The mtDNA content positively correlated with the gestational age only in the liver. The activities of COX, CS and SQR in both liver and muscle isolated mitochondria significantly decreased after the 22(nd) GW in comparison with earlier GW. A continuous decline of LH, not correlating with the documented OXPHOS-specific activities, was observed from the 14(th) to the 24(th) GW indicating their exclusive reflection of liver tissue processes. Two apparently contradictory processes of increasing mtDNA transcription and decreasing OXPHOS-specific activities seem to be indispensable for rapid postnatal adaptation to high energy demands. The inadequate capacity of mitochondrial energy production may be an important factor in the mortality of children born before the critical developmental point of the 22(nd) GW.

Response of the mitochondrial proteome of rat renal proximal convoluted tubules to chronic metabolic acidosis

Metabolic acidosis is a common clinical condition that is caused by a decrease in blood pH and bicarbonate concentration. Increased extraction and mitochondrial catabolism of plasma glutamine within the renal proximal convoluted tubule generates ammonium and bicarbonate ions that facilitate the excretion of acid and partially restore acid-base balance. Previous studies identified only a few mitochondrial proteins, including two key enzymes of glutamine metabolism, which are increased during chronic acidosis. A workflow was developed to characterize the mitochondrial proteome of the proximal convoluted tubule. Based upon the increase in specific activity of cytochrome c oxidase, the isolated mitochondria were enriched eightfold. Two-dimensional liquid chromatography coupled with mass spectrometry was utilized to compare mitochondrial-enriched samples from control and chronic acidotic rats. Proteomic analysis identified 901 proteins in the control and acidotic samples. Further analysis identified 37 peptides that contain an N-ε-acetyl-lysine; of these, 22 are novel sites. Spectral counting analysis revealed 33 proteins that are significantly altered in abundance in response to chronic metabolic acidosis. Western blot analysis was performed to validate the calculated changes in abundance. Thus the current study represents the first comprehensive analysis of the mitochondrial proteome of the rat renal proximal convoluted tubule and its response to metabolic acidosis.

FASTKD2 Nonsense Mutation in an Infantile Mitochondrial Encephalomyopathy Associated with Cytochrome C Oxidase Deficiency

In two siblings we found a mitochondrial encephalomyopathy, characterized by developmental delay, hemiplegia, convulsions, asymmetrical brain atrophy, and low cytochrome c oxidase (COX) activity in skeletal muscle. The disease locus was identified on chromosome 2 by homozygosity mapping; candidate genes were prioritized for their known or predicted mitochondrial localization and then sequenced in probands and controls. A homozygous nonsense mutation in the KIAA0971 gene segregated with the disease in the proband family. The corresponding protein is known as fas activated serine-threonine kinase domain 2, FASTKD2. Confocal immunofluorescence colocalized a tagged recombinant FASTKD2 protein with mitochondrial markers, and membrane-potential-dependent in vitro mitochondrial import was demonstrated in isolated mitochondria. In staurosporine-induced-apoptosis experiments, decreased nuclear fragmentation was detected in treated mutant versus control fibroblasts. In conclusion, we found a loss-of-function mutation in a gene segregating with a peculiar mitochondrial encephalomyopathy associated with COX deficiency in skeletal muscle. The corresponding protein is localized in the mitochondrial inner compartment. Preliminary data indicate that FASTKD2 plays a role in mitochondrial apoptosis.

Erythropoietin Induces a Shift of Muscle Phenotype from Fast Glycolytic to Slow Oxidative

Sedentary and trained rat groups were studied. Each of these groups was either erythropoietin or placebo treated. Erythropoietin treatment increased significantly all haematological parameters studied. Training per se failed to modify haematological parameters. In a second time, we studied the specific activity of several oxidative enzymes in three different muscles. In sedentary rats, erythropoietin treatment increased significantly the specific activities of cytochrome c oxidase and L-3-hydroxyacyl CoA dehydrogenase in the soleus and those of L-3-hydroxyacyl CoA dehydrogenase and phosphofructokinase in both locomotor muscles. Training increased the oxidative enzymes activities in all muscles studied. In trained rats, effects of erythropoietin and training on oxidative enzymes activities were additive. In all erythropoietin treated muscles, the expression of slow twitch myosin light chains and oxidative myosin heavy chains increased. A similar phenomenon took place in all trained groups for light chains and in placebo treated trained rats for heavy chains. In trained groups, the effects of the hormone and of training were additive. Our results suggest strongly that two different mechanisms are involved in the response of skeletal muscles to erythropoietin treatment and to endurance training and probably whole body endurance is affected by erythropoietin treatment by an increase of oxygenation of all tissues.

Colour of beef heated to different temperatures as related to meat ageing

The effects of ageing (10–12 days at +5°C), thermal treatment (internal temperature, T i : 45, 60, 75°C) and oxygenation on the colour and some colour related physical and biochemical parameters of beef longissimus dorsi (LD) muscle were studied. The colour components ( L ∗, a ∗ and b ∗ values) for the raw and heated LD, both before and after ageing and oxygenation, were instrumentally and sensorily evaluated. Ageing influenced colour components (except b ∗ value), specific activity of cytochrome c oxidase (SACCO) and amount of oxygen consumed (AOC). An increase in internal temperature was usually accompanied by a lower SACCO, AOC and total native muscle pigment concentration, as well as a change in all colour parameters. Oxygenation of the raw and heated slices (except T i =75°C) led to a less of black colour and a higher of red colour, a ∗ and b ∗ values.

Colour of normal and high pH beef heated to different temperatures as related to oxygenation

The effects of oxygenation and thermal treatment (internal temperature, T i: 45, 60, 75°C) on the colour and some colour related physical and biochemical properties of beef longissimus dorsi (LD) muscle, both normal (pH u∼5.6) and DFD (pH u>6.6), were studied. The colour components ( L ∗, a ∗, b ∗ values) for the raw and heated LD, both before and after oxygenation, were instrumentally and sensorily evaluated. The colour of raw and heated (60°C) DFD beef before and after oxygenation differed significantly from the normal meat and contained more native muscle pigment (TMP). pH also influenced the depth of the oxygenated layer, specific activity of cytochrome c oxidase (SACCO) and the amount of oxygen consumed. An increase in internal temperature was usually accompanied by a lower SACCO and a significant decrease of TMP, as well as a change of all colour parameters. Oxygenation of the raw and heated slices (except at 75°C) of both types of meat led to higher L ∗ , a ∗ and b ∗ values.

Activity and inducibility of cytochrome P-450-1A in the liver of mice with different sensitivity to hepatocarcinogenic effect of o-aminoazotoluene

Specific activity of cytochrome P-450-1a is detected in the liver of 7 inbred mouse strains sensitive (SWR, C3HA, DD, and CBA) and resistant (AKR, CC57BR, and C57BL) to o-aminoazotoluene-induced hepatocarcinogenesis: 7-ethoxyresorufin-O-diethylase and 7-methoxyresorufin-O-dimethylase, induced by benzo(a)pyrene and o-aminoazotoluene. Mice sensitive (CC57BR and C57BL) and resistant (AKR) to P-450-1a induction were resistant to induction of liver tumor and, similarly, mice both resistant (SWR and DD) and sensitive (C3HA and CBA) to P-450-1a induction were sensitive to hepatic tumor induction. Therefore, there was no correlation between inducibility of cytochrome P-450-1a and sensitivity to o-aminoazotoluene-induced hepatocarcinogenesis at the initial stages of chemical carcinogenesis.

Does the respiratory rate in sea urchin embryos increase during early development without proliferation of mitochondria?

During early development of the sea urchin, the respiratory rate, enhanced upon fertilization, is maintained up to hatching (pre-hatching period) and then gradually increases to a maximum at the gastrula stage (post-gastrula period). Except for a short duration after fertilization, respiration in embryos is strongly inhibited by CN- and antimycin A. During the whole span of early development, the amounts of proteins, cytochromes and the specific activities of cytochrome c oxidase and reduced nicotinamide adenine dinucleotide (NADH) cytochrome c reductase in mitochondria are practically the same as in unfertilized eggs. A marked augmentation of mitochondrial respiration after hatching probably occurs without net increase in whole mitochondrial intrinsic capacities. Carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) or tetramethyl p-phenylenediamine (TMPD) enhances the respiratory rate in the pre-hatching period but hardly augments the respiration in the post-gastrula period. In the presence of both FCCP and TMPD, the respiratory rate in the pre-hatching period was as high as in the post-gastrula period. Probably, electron transport in the mitochondrial respiratory chain is regulated by acceptor control and limitation of cytochrome c reduction in the pre-hatching period and released from those regulations in the post-gastrula period. Acceptor control of respiration is experimentally reproduced in isolated mitochondria by making adenine nucleotide levels as those levels in the pre-hatching period.

Effect of disulfiram (DS) on mitochondria from rat hippocampus: Metabolic compartmentation of DS neurotoxicity

This experiment was designed to study the acute effects of disulfiram on mitochondrial enzymes in nonsynaptic and synaptic mitochondria from rat hippocampus. Cytochrome c oxidase, monoamine oxidase-B, glycerolphosphate acyltransferase and beta-hydroxybutyrate dehydrogenase were studied. Differences in enzyme activity were seen in controls. Cytochrome c oxidase activity was higher in synaptic mitochondria whereas glycerolphosphate acyltransferase activity was higher in nonsynaptic mitochondria. Mitochondria from disulfiram treated rats, particularly synaptic mitochondria, exhibited lower specific activities of cytochrome c oxidase and monoamine oxidase-B. These alterations were not limited to either the inner or outer mitochondrial membrane. Transmission electron microscopy revealed that mitochondria from disulfiram treated rats were severely altered in isolated preparations as well as in those from whole tissue. This study shows that disulfiram exerts a differential effect on mitochondrial subpopulations.

1-beta-D-arabinofuranosylcytosine (Ara-C) enhances mitochondrial activities in human leukaemic cells.

1-beta-D-Arabinofuranosylcytosine (Ara-C) at a concentration which inhibits nuclear-DNA reduplication (0.05 microM), enhances mitochondrial activities like respiration, in cell of a human leukaemic cell line Molt 4. While the specific activity of cytochrome c oxidase doubles in the course of the G1 phase of the cell cycle in control cells, in the presence of Ara-C G1 phase cells begin to increase the enzyme activity earlier and show a 3-fold rise of the enzyme activity in the same period of time. This is explained by an enhanced expression of the mitochondrial genome: the concentration of transcripts for the mitochondrially encoded subunit II of cytochrome c oxidase increases. Inhibition of mitochondrial protein synthesis abolishes the Ara-C induced effect on the specific activity of cytochrome c oxidase activity. The concentration of transcripts of the nuclearly encoded subunit IV of cytochrome c oxidase is the same as in control cells, and also the specific activity of the mitochondrial enzyme citrate synthase, which is exclusively encoded on nuclear-DNA, is not affected by Ara-C. Dysregulation in time and intensity of the expression of the mitochondrial relative to the nuclear genome may impair cell function and reflect a till now neglected mechanism of Ara-C cytotoxicity.

Plasma Membrane ATPase Activity following Reversible and Irreversible Freezing Injury

Plasma membrane ATPase has been proposed as a site of functional alteration during early stages of freezing injury. To test this, plasma membrane was purified from Solanum leaflets by a single step partitioning of microsomes in a dextran-polyethylene glycol two phase system. Addition of lysolecithin in the ATPase assay produced up to 10-fold increase in ATPase activity. ATPase activity was specific for ATP with a K(m) around 0.4 millimolar. Presence of the ATPase enzyme was identified by immunoblotting with oat ATPase antibodies. Using the phase partitioning method, plasma membrane was isolated from Solanum commersonii leaflets which had four different degrees of freezing damage, namely, slight (reversible), partial (partially reversible), substantial and total (irreversible). With slight (reversible) damage the plasma membrane ATPase specific activity increased 1.5- to 2-fold and its K(m) was decreased by about 3-fold, whereas the specific activity of cytochrome c reductase and cytochrome c oxidase in the microsomes were not different from the control. However, with substantial (lethal, irreversible) damage, there was a loss of membrane protein, decrease in plasma membrane ATPase specific activity and decrease in K(m), while cytochrome c oxidase and cytochrome c reductase were unaffected. These results support the hypothesis that plasma membrane ATPase is altered by slight freeze-thaw stress.

Comparison of brush border membrane vesicles prepared by three methods from larval Manduca sexta midgut

Brush border membrane vesicles (BBMV) were prepared from freshly isolated posterior larval Manduca sexta midguts by differential calcium precipitation, differential magnesium precipitation and differential ultrasonication. BBMV were also prepared from frozen posterior larval M. sexta midguts by differential calcium precipitaion and differential magnesium precipitation. The yield of BBMV by both differential precipitation methods was 5–6 times greater than that by the differential ultrasonification method. Enrichments of the brush border membrane marker enzymes alkaline phosphatase, γ-glutamyl transferase, and aminopeptidase were similar in all preparations. The polypeptide composition of all preparations was also similar. The specific activity of mitochondrial and microsomal marker enzymes was higher in BBMV prepared from freshly isolated midguts by the differential precipitation methods than in BBMV prepared by the ultrasonication method. The specific activity of cytochrome- c oxidase was 2.5–7 times higher in BBMV prepared from frozen midguts than in BBMV prepared from fresh tissue.

Effect of diisopropyl 1,3-dithiolan-2-ylidenemalonate(NKK-100) on the components of hepatic microsomal mixed function oxidase in male broiler chicks.

Effect of oral administration of diisopropyl 1, 3-dithiolan-2-ylidenemalonate (NKK-100) on the components of mixed function oxidase system in hepatic microsomes was investigated in male broiler chicks. Feeding NKK-100 increased relative liver weight and microsomal protein content of the liver. Contents of cytochrome P 450 and b 5 per mg microsomal protein and g liver increased with NKK-100 levels. The activities of NADPH-cytochrome c reductase and NADH-cytochrome b 5 reductase per g liver were enhanced by feeding NKK-100, while those activities per mg microsomal protein remained unchanged. Pretreatment with clopidol (an anticoccidial agent) and colistin (an antibiotic) did not affect the components of MFO. These results suggests that NKK-100 has a property to activate the hepatic microsomal MFO and the effect of NKK-100 are more prominent in the increases in specific activities of cytochrome P 450 and b 5 rather than in those of NADPH-cytochrome c reductase or NADH-cytochrome b 5 reductase.

The effects of dicofol on induction of hepatic microsomal metabolism in rats

In a comparative study, the induction effects of dicofol, technical Kelthane, and DDT on hepatic microsomal and cytosolic enzyme activities in rats were compared with those effects produced by phenobarbital (PhB) and β-naphthoflavone (BNF). Male rats (ca. 250 g) were injected (ip) for 4 consecutive days with 1.0 ml of vehicle containing either dicofol (1.5, 15.0, 29.5, or 59.0 m M, Kelthane (dicofol content equal to 29.5 or 59.0 m M), DDT (59.0 m M), or BNF (36.7 m M). Liver weights, microsomal protein, and cytochrome P-450 concentrations and microsomal and cytosolic enzyme specific activities were measured. Dicofol produced dose-related increases in all of the parameters measured except liver weight and cytosolic epoxide hydrolase activity. At a concentration of 59.0 m M, dicofol increased the concentrations of microsomal protein (1.7-fold) and cytochrome P-450 (2.9-fold), and the specific activities of cytochrome c reductase (1.6-fold), ethoxycoumarin O-deethylase (2.3-fold), aminopyrine N-demethylase (3.0-fold), microsomal epoxide hydrolase (2.6-fold), and glutathione S-transferase (2.9-fold). The induction potency of dicofol was equivalent to Kelthane, DDT, and PhB at equimolar (59.0 m M) concentrations of chemical.

Role of phospholipid fatty acids on the kinetics of high and low affinity sites of cytochrome c oxidase.

The nature of the interactions between cytochrome c oxidase and the phospholipids in mitochondrial membranes has been investigated by varying the nature of the fatty acyl components of Saccharomyces cerevisiae. A double fatty acid yeast mutant, FAI-4C, grown in combinations of unsaturated (oleic, linoleic, linolenic, and eicosenoic) and saturated (lauric and palmitic) fatty acids, was employed to modify mitochondrial membranes. The supplemented fatty acids constituted a unique combination of different acyl chain lengths with varying degrees of unsaturation which were subsequently incorporated into mitochondrial phospholipids. Phosphatidylethanolamine and cardiolipin, the predominant phospholipids of the inner mitochondrial membrane, were characterized by their high levels of supplemented unsaturated fatty acids. Increasing the chain length or the degree of unsaturation of mitochondrial membrane phospholipids had no effect on altering the nature of the phospholipid polar head group but did result in a profound change on the specific activity of cytochrome c oxidase. When studied under conditions of different ionic strengths and pHs the enzyme's activity, as documented by Eadie-Hofstee plots, showed biphasic kinetics. The kinetic parameters for the low affinity reaction were greatly influenced by the changes in the membrane fatty acids and only marginal effects were noted at the high affinity reaction site. The discontinuities in the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene, monitored at increasing temperatures, suggested that changes in membrane fluidity were conditioned by alterations in mitochondrial membrane fatty acid constituents. These results indicate that the lipid changes affecting the low affinity binding site of cytochrome c oxidase may be the result of lipid-protein interactions which lead to enzyme conformational changes or may be due to gross changes in membrane fluidity. It may, therefore, follow that this enzyme site may be embedded in or be juxtaposed to the outer surface of the inner mitochondrial membrane bilayer in contrast to the high affinity site which has been shown to be significantly above the membrane plane.

Modification of mitochondrial respiration by aging and dietary restriction

Effects of aging and of dietary restriction on mitochondrial recovery and respiratory capacities have been assessed in mice. Old mice (23–26 months) did not differ from adult mice (9–12 months) in amounts of protein recovered in mitochondrial fractions of liver, brain and spleen, but did show a decline in specific activity of cytochrome c oxidase (cyt. c ox.) in liver and spleen. Age effects on in vitro respiration by mitochondria occurred in liver and spleen. In liver, only one substrate (β-hydroxybutyrate) of four tested was respired at a different rate by old than by young mitochondria. Depression of state 3 respiration and 2,4-dinitrophenol (DNP)-uncoupled rates was observed for this substrate; however, this effect depended on expressing respiration on the basis of mitochondrial protein and was less overt if data were expressed per unit of cyt. c ox. activity. Old spleen mitochondria exhibited a grosser defect, showing a 40% decrease in the respiratory control index (RCI) for (succinate + rotenone)- supported respiration (the only substrate tested) due to a possible increase in state 4 rates. Effects of dietary restriction were assessed in liver and brain of 3–7-month-old mice underfed since weaning. Dietary restriction reduced recovery of total liver mitochondrial protein and liver cyt. c ox. specific activity. Liver mitochondria from restricted mice generally showed increased state 3 rates with no differences from controls in state 4 rates for respiration supported by glutamate or pyruvate + malate, resulting in an increased RCI for these substrates. DNP-uncoupled rates were also raised by dietary restriction. Unlike effects observed in old versus young mice, these differences obtained whether the data were expressed on the basis of mitochondrial protein or on cyt. c ox. activity. Electron microscopy of liver mitochondrial preparations revealed more non-mitochondrial contaminants in old mice and larger mitochondria in dietarily restricted mice. These findings are compatible with reports of age-dependent losses of liver mitochondria and suggest that dietary restriction may retard this loss.

Separation of Mitochondria from Contaminating Subcellular Structures Utilizing Silica Sol Gradient Centrifugation

Discontinuous Percoll density gradients have been developed for the purification of mitochondria, permitting rapid separation under isosmotic and low viscosity conditions. Mitochondria from several etiolated tissues have been successfully separated from contaminating subcellular structures by this method. For potato tuber the ratio of washed to purified mitochondrial protein was 2.6, similar to the increase in specific activity of cytochrome c oxidase following separation. The purification of mitochondria from green leaf tissues on Percoll gradients has reduced chlorophyll contamination of spinach mitochondria from about 70 micrograms chlorophyll per milligram protein to approximately 8 micrograms chlorophyll per milligram protein.The ratio of protein content of the washed mitochondria compared to that in the purified preparation was 7 for spinach and respiratory activity was retained. The physiological integrity and oxidative properties of washed and gradient mitochondria are compared.

Effect of 2(3)- tert-butyl-4-hydroxyanisole administration on the activities of several hepatic microsomal and cytoplasmic enzymes in mice

Administration of 2(3)- tert-butyl-4-hydroxyanisole (BHA) (0.75%) caused a marked increase in the activities of several hepatic enzymes in CD-1 mice. This was associated with increased liver weights and total protein contents, especially of the microsomal and cytosol fractions. While the specific content of cytochrome P-450 was decreased slightly in microsomes, the specific content of cytochrome b 5 and the specific activities of cytochrome c reductases (NADPH- or NADH-dependent) were increased (2-fold). In spite of a slight decrease in the specific activities of aminopyrine demethylase and of benzo(a)pyrene hydroxylase, both aniline hydroxylase and UDP-glucronyltransferase activities were increased (2.7- and 4.6-fold respectively). The specific activity of a microsomal membrane marker enzyme, glucose-6-phosphatase, was decreased slightly (−25 per cent). In the cytosol fraction, the specific activities of glucose-6-phosphate dehydrogenase and of UDP-glucose dehydrogenase were increased (3.8- and 6.1-fold respectively). Differences were noted in the time cources of increase and decrease in these enzyme activities after initiation and discontinuation of BHA treatment.

Reversible tenfod reduction in mitochondria DNA content of human cells treated with ethidium bromide.

Cells of the human line VA2-B in suspension culture have been treated with very low concentrations of ethidium bromide for the purpose of reducing the amount of mitochondrial DNA (mit-DNA) per cell. Cells maintained in the presence of 5 ng/ml ethidium bromide grew at a normal rate for three days; thereafter, their doubling time gradually increased to a stable value of about 60 h. In these cells, the rate of 3H thymidine incorporation into mit-DNA decreased very rapidly to approximately 60% of the normal, and remained thereafter at this level, while the amount of mit-DNA per cell stabilized around a level of 70--80% of the control. In cells long-term treated with 5 ng/ml ethidium bromide, the rate of mitochondrial protein synthesis was about 35% of the normal, and the cytochrome c oxidase activity about 50% of the control. Cells treated with 20 ng/ml of the drug underwent 3--4 cell doublings at control rates, then gradually stopped growing, and eventually died. In these cells, the rate of incorporation of 3H thymidine into mit-DNA was reduced to 50% of the control value after 10 min treatment with ethidium bromide, and became barely detectable after three cell doublings. At this time, the cells had on the average less than 10% of the control amount of mit-DNA, the rate of mitochondrial protein synthesis was reduced to 3% of the normal, and the specific activities of cytochrome c oxidase and rutamycin-sensitive ATPase were less than 20% of the control values. In spite of these marked changes, the cells exhibited only a 20--30% loss in cell viability, as estimated by cloning efficiency, after three days of exposure to the drug. Cells treated with ethidium bromide at 20 ng/ml for three days, and then transferred to drug-free medium, recovered a near-to-normal growth rate and cloning efficiency and a near-to-normal rate of synthesis and amount of mit-DNA in about five days.