Inhibition Of Mitochondrial Energy Metabolism Research Articles

S-Nitrosylation-mediated dysfunction of TCA cycle enzymes in synucleinopathy studied in postmortem human brains and hiPSC-derived neurons

A causal relationship between mitochondrial metabolic dysfunction and neurodegeneration has been implicated in synucleinopathies, including Parkinson disease (PD) and Lewy body dementia (LBD), but underlying mechanisms are not fully understood. Here, using human induced pluripotent stem cell (hiPSC)-derived neurons with mutation in the gene encoding α-synuclein (αSyn), we report the presence of aberrantly S-nitrosylated proteins, including tricarboxylic acid (TCA) cycle enzymes, resulting in activity inhibition assessed by carbon-labeled metabolic flux experiments. This inhibition principally affects α-ketoglutarate dehydrogenase/succinyl coenzyme-A synthetase, metabolizing α-ketoglutarate to succinate. Notably, human LBD brain manifests a similar pattern of aberrantly S-nitrosylated TCA enzymes, indicating the pathophysiological relevance of these results. Inhibition of mitochondrial energy metabolism in neurons is known to compromise dendritic length and synaptic integrity, eventually leading to neuronal cell death. Our evidence indicates that aberrant S-nitrosylation of TCA cycle enzymes contributes to this bioenergetic failure.

Reduction of host cell mitochondrial activity as Mycobacterium leprae's strategy to evade host innate immunity.

Leprosy is a much-feared incapacitating infectious disease caused by Mycobacterium leprae or M lepromatosis, annually affecting roughly 200,000 people worldwide. During host-pathogen interaction, M leprae subverts the immune response, leading to development of disease. Throughout the last few decades, the impact of energy metabolism on the control of intracellular pathogens and leukocytic differentiation has become more evident. Mitochondria play a key role in regulating newly-discovered immune signaling pathways by controlling redox metabolism and the flow of energy besides activating inflammasome, xenophagy, and apoptosis. Likewise, this organelle, whose origin is probably an alphaproteobacterium, directly controls the intracellular pathogens attempting to invade its niche, a feature conquered at the expense of billions of years of coevolution. In the present review, we discuss the role of reduced host cell mitochondrial activity during M leprae infection and the consequential fates of M leprae and host innate immunity. Conceivably, inhibition of mitochondrial energy metabolism emerges as an overlooked and novel mechanism developed by M leprae to evade xenophagy and the host immune response.

Lipophilic triphenylphosphonium derivatives enhance radiation-induced cell killing via inhibition of mitochondrial energy metabolism in tumor cells

It has recently been reported that radiation enhances mitochondrial energy metabolism in various tumor cell lines. To examine how this radiation-induced alteration in mitochondrial function influences tumor cell viability, various lipophilic triphenylphosphonium (TPP+) cation derivatives and related compounds such as 4-hydroxy-2,2,6,6-tetramethyl-1-oxy-piperidin (Tempol) with TPP+ (named “Mito-”) were designed to inhibit the mitochondrial electron transport chain. Mito-(CH2)10-Tempol (M10T) and its derivatives, Mito-(CH2)5-Tempol (M5T), Mito-(CH2)10-Tempol-Methyl (M10T-Me), Mito-C10H21 (M10), and C10H21-Tempol (10T), were prepared. In HeLa human cervical adenocarcinoma cells and A549 human lung carcinoma cells, the fractional uptake of the compound into mitochondria was highest among the TTP+ analogs conjugated with Tempol (M10T, M5T, and 10T). M10T, M10T-Me, and M10 exhibited strong cytotoxicity and enhanced X-irradiation-induced reproductive cell death, while 10T and M5T did not. Furthermore, M10T, M10T-Me, and M10 decreased basal mitochondrial membrane potential and intracellular ATP. M10T treatment inhibited X-ray-induced increases in ATP production. These results indicate that the TPP cation and a long hydrocarbon linker are essential for radiosensitization of tumor cells. The reduction in intracellular ATP by lipophilic TPP+ is partly responsible for the observed radiosensitization.

The action of n-propyl gallate on gluconeogenesis and oxygen uptake in the rat liver

In the present study the metabolic actions of n-propyl gallate on hepatic gluconeogenesis, oxygen uptake and related parameters were investigated. Experiments were done in the isolated perfused rat liver. n-Propyl gallate inhibited gluconeogenesis and stimulated oxygen uptake at concentrations up to 200 μM. The inhibitory effects on lactate gluconeogenesis (ED 50 86.4 μM) and alanine gluconeogenesis were considerably more pronounced than those on glycerol and fructose gluconeogenesis. n-Propyl gallate also stimulated oxygen uptake in both the mitochondrial (63%) and microsomal (37%) electron transport chains. The first one is due mainly to the oxidation of n-propanol, as a metabolite of the first step of n-propyl gallate transformation. The second one results from a direct stimulation of the microsomal electron transport chain. n-Propyl gallate inhibited pyruvate carboxylation (ED 50 142.2 μM) in consequence of an inhibition of pyruvate transport into the mitochondria an effect not found for gallic acid. This is probably the main cause for glucose output inhibition. Secondary causes are (1) deviation of intermediates for the production of NADPH to be used in microsomal electron transport; (2) deviation of glucose 6-phosphate for glucuronidation reactions; (3) gluconeogenesis inhibition by n-propanol, produced intracellularly from n-propyl gallate. Inhibition of mitochondrial energy metabolism is not significant in the range up to 200 μM, as indicated by the very small effect on the cellular ATP levels (5% decreased). n-Propyl gallate can be considered a kind of metabolic effector, whose actions on the liver metabolism are relatively mild although they can become harmful for the organ and the whole organism at high doses and concentrations.

Coenzyme Q10 is decreased in fibroblasts of patients with methylmalonic aciduria but not in mevalonic aciduria

The content of coenzyme Q(10) (CoQ(10)) was examined in skin fibroblasts of 10 patients with mevalonic aciduria (MVA) and of 22 patients with methylmalonic aciduria (MMA). Patients with these inborn errors of metabolism are thought to be at risk for CoQ(10) depletion either by direct inhibition of the proximal pathway of CoQ(10) synthesis (MVA) or indirectly by inhibition of mitochondrial energy metabolism (MMA). We demonstrated that CoQ(10) concentrations were not significantly different from controls in MVA patients, suggesting that there may be upregulatory effects. On the other hand the CoQ(10) content in fibroblasts of patients with MMA was significantly reduced.

Neurodegeneration and chronic renal failure in methylmalonic aciduria—A pathophysiological approach

In the last decades the survival of patients with methylmalonic aciduria has been improved. However, the overall outcome of affected patients remains disappointing. The disease course is often complicated by acute life-threatening metabolic crises, which can result in multiple organ failure or even death, resembling primary defects of mitochondrial energy metabolism. Biochemical abnormalities during metabolic derangement, such as metabolic acidosis, ketonaemia/ketonuria, lactic acidosis, hypoglycaemia and hyperammonaemia, suggest mitochondrial dysfunction. In addition, long-term complications such as chronic renal failure and neurological disease are frequently found. Neuropathophysiological studies have focused on various effects caused by accumulation of putatively toxic organic acids, the so-called 'toxic metabolite' hypothesis. In previous studies, methylmalonate (MMA) has been considered as the major neurotoxin in methylmalonic aciduria, whereas more recent studies have highlighted a synergistic inhibition of mitochondrial energy metabolism (pyruvate dehydrogenase complex, tricarboxylic acid cycle, respiratory chain, mitochondrial salvage pathway of deoxyribonucleoside triphosphate (dNTP)) induced by propionyl-CoA, 2-methylcitrate and MMA as the key pathomechanism of inherited disorders of propionate metabolism. Intracerebral accumulation of toxic metabolites ('trapping' hypothesis') is considered a biochemical risk factor for neurodegeneration. Secondary effects of mitochondrial dysfunction, such as oxidative stress and impaired mtDNA homeostasis, contribute to pathogenesis of these disorders. The underlying pathomechanisms of chronic renal insufficiency in methylmalonic acidurias are not yet understood. We hypothesize that renal and cerebral pathomechanisms share some similarities, such as an involvement of dicarboxylic acid transport. This review aims to give a comprehensive overview on recent pathomechanistic concepts for methylmalonic acidurias.

TASK-like potassium channels and oxygen sensing in the carotid body

Chemosensing by type-1 cells of the carotid body involves a series of events which culminate in the calcium-dependent secretion of neurotransmitter substances which then excite afferent nerves. This response is mediated via membrane depolarisation and voltage-gated calcium entry. Studies utilising isolated cells indicates that the membrane depolarisation in response to hypoxia, and acidosis, appears to be primarily mediated via the inhibition of a background K(+)-current. The pharmacological and biophysical characteristics of these channels suggest that they are probably closely related to the TASK subfamily of tandem-P-domain K(+)-channels. Indeed they show greatest similarity to TASK-1 and -3. In addition to being sensitive to hypoxia and acidosis, the background K(+)-channels of the type-1 cell are also remarkably sensitive to inhibition of mitochondrial energy metabolism. Metabolic poisons are known potent stimulants of the carotid body and cause membrane depolarisation of type-1 cells. In the presence of metabolic inhibitors hypoxic sensitivity is lost suggesting that oxygen sensing may itself be mediated via depression of mitochondrial energy production. Thus these TASK-like background channels play a central role in mediating the chemotransduction of several different stimuli within the type-1 cell. The mechanisms by which metabolic/oxygen sensitivity might be conferred upon these channels are briefly discussed.

Severe secondary mitochondrial dysfunction in classical organic acidemias

Organic acidemias constitute a heterogenous group of inherited deficiencies in the catabolic pathways of amino acids. Neurological disease, such as movement disorders, psychomotor retardation and epilepsy, are frequently found owing to neurodegenerative changes, particularly in the basal ganglia. The mechanisms underlying neuropathological changes in inherited disorders of propionate metabolism, i.e. methylmalonic and propionic aciduria, has been the subject of intense debates during the last years. It has been demonstrated that an impairment of energy metabolism in propionic and methylmalonic acidurias is most likely mediated by synergistic inhibition of the tricarboxylic acid cycle, the mitochondrial respiratory chain and mitochondrial transporters by propionyl-CoA and metabolites of alternative propionate oxidation, such as 2-methylcitrate and malonate, thus sharing mechanistic similarities to propionate toxicity in bacteria and fungi. Propionyl-CoA, a putative neurotoxic agent, induces various inhibitory effects on mitochondrial energy metabolism and urea cycle. A recent study has highlighted that propionyl-CoA-induced mitochondrial dysfunction plays a key role in this scenario. In line with these observations, bioenergetic analysis of muscle biopsies of two patients with propionic aciduria has revealed severely compromised mitochondrial energy metabolism, including decreased enzyme activities (respiratory chain complexes I-IV, pyruvate dehydrogenase complex, α-ketoglutarate dehydrogenase complex), a depletion of ATP, phosphocreatine and mitochondrial DNA. In conclusion, synergistic inhibition of mitochondrial energy metabolism by endogenous toxic organic acids in methylmalonic and propionic acidurias induces severe secondary mitochondrial dysfunction resulting in recurrent metabolic crises, multi-organ failure, and reduced life expectancy.

Altered mitochondrial energy metabolism may play a role in vascular aging

Epidemiological studies demonstrated that even in the absence of other risk factors (e.g., diabetes, hypertension, hypercholesterolemia), vascular aging significantly increases cardiovascular morbidity. Previous studies revealed that vascular aging is characterized by an age-dependent decline in endothelial function due to a decreased bioavailability of NO and increased production of reactive oxygen species. Yet, the mechanisms underlying the process of vascular aging are still poorly understood. Many authors consider that aging is a mitochondrial disease. Indeed, there is evidence that aging is associated with an increase in mtDNA damage and a decline in expression/activity of mitochondrial enzymes in various organs. On the basis of recent observations we predict that similar changes in mitochondrial gene expression profile are present in the aged cardiovascular system as well. It is significant, that components of the electron transport chain (including cytochrome c oxidase) seem to be similarly down-regulated with age in many species. Because pharmacological inhibition of mitochondrial energy metabolism significantly impairs endothelium-dependent vascular relaxation and may increase the production of reactive oxygen species, we propose that alterations of mitochondrial energetic phenotype may contribute to endothelial dysfunction in aging.

Impaired energy metabolism and abnormal muscle histology in mut – methylmalonic aciduria

The authors report a 27-year-old man with B12-responsive mut- methylmalonic aciduria associated with pure muscle symptoms. Two mutations were found in the methylmalonyl-CoA mutase gene. An exercise test showed a reduced maximal workload and reduced oxygen uptake, and a muscle biopsy showed subsarcolemmal accumulation of mitochondria and normal respiratory chain enzyme activities. These findings may be caused by inhibition of mitochondrial energy metabolism by methylmalonate or its metabolites.

Methylmalonic acid — an endogenous toxin?

Methylmalonic acid was previously considered as major neurotoxin in methylmalonic acidurias. In contrast, recent studies support the notion that other metabolites deriving from propionyl-coenzyme A, inducing synergistic inhibition of mitochondrial energy metabolism, are more important than methylmalonic acid to understand the neuropathogenesis of this disease. However, it is not yet known whether methylmalonic acid is involved in the induction of other organ manifestations in this disease, such as chronic renal failure.

Reversible end-stage renal disease in an adolescent patient with methylmalonic aciduria

Sirs, Methylmalonic aciduria (MMA) is an inborn error of metabolism, characterized by deficiency of the mitochondrial enzyme methylmalonyl-CoA mutase or by defects in the synthesis of 5-deoxyadenosylcobalamin, a cofactor of methylmalonyl-CoA mutase. Affected children suffer from lethargy, failure to thrive, and muscular hypotonia, and may have ketoacidotic, hypoglycemic, and hyperammonemic coma. Biochemically, the disease is characterized by accumulation of methylmalonate and, due to activation of alternative pathways of propionate oxidation, by accumulation of proprionate, 3-hydroxyproprionate, and 2-methylcitrate. Some of these organic acids have been suggested to act as endogenous toxins, inducing a synergistic inhibition of mitochondrial energy metabolism [1, 2]. Dietary and emergency therapy and thus outcome have substantially improved [3]. In brief, treatment is based on a restriction of natural protein and administration of precursor-free amino acid mixtures, as well as supplementation of carnitine. To avoid malnutrition and catabolism, affected patients require close monitoring during therapy. Secondary organ damage, however, remains insufficiently resolved, particularly if metabolic control is inadequate. Impairment of renal function is observed in a majority of patients [4]. It is characterized by tubulointerstitial nephritis with mononuclear cell infiltration, interstitial fibrosis, and tubular atrophy [5, 6, 7, 8]. Chronic renal failure (CRF) usually develops within the 1st or 2nd decade of life. However, adequate dietary control of protein metabolism is likely to slow the progression of renal disease, although this has not yet been investigated in prospective, observational studies. We report a 16-year-old male adolescent with vitamin B12-unresponsive MMA (mut ) and partially reversible end-stage renal disease, most likely due to improved metabolic control. In this patient, MMA was diagnosed shortly after birth, and a low-protein diet and carnitine supplementation was initiated. There were several episodes of metabolic decompensation due to inadequate metabolic control during infancy and childhood, resulting in neurological sequelae and CRF. Notably, a reduced glomerular filtration rate (GFR) was noted at 2.3 years of age. At 9 years of age, he developed spastic diplegia. CRF slowly progressed until puberty. At the age of 15 years, the patient was referred to our unit with a creatinine of 6.6 mg/dl and a GFR of 8 ml/min per 1.73 m (Fig. 1A). Transient hypercalcemia due to treatment of secondary hyperparathyroidism with 1, 25-dihydroxyvitamin D3 had resolved at the time of admission. Selective metabolic investigations revealed insufficient metabolic control, most likely due to inadequate adherence to the recommended dietary regimen. High-performance liquid chromatography analysis of plasma carnitine showed strongly elevated acylcarnitines, but free carnitine was within the normal range due to supplementation (Fig. 1B). Differentiation of acylcarnitines using tandem mass spectrometry of dried blood spots showed a predominant elevation of propionylcarnitine (Fig. 1B). Organic acid analysis of urine revealed a urinary excretion of methylmalonate, 2-methylcitrate, lactate, and pyruvate (Fig. 1C). ParathyC. P. Schmitt · O. Mehls · T. L. Weber Division of Pediatric Nephrology, Department of General Pediatrics, University Children’s Hospital, Heidelberg, Germany

The selective in vitro cytotoxicity of carcinoma cells by AZT is enhanced by concurrent treatment with delocalized lipophilic cations.

This study assessed the selective growth inhibitory effect on cultured carcinoma cells by 3'-azido-3'-deoxythymidine (AZT), as a single agent, and in combination with delocalized lipophilic cations (DLCs) that are known to inhibit mitochondrial function. In cytotoxicity assays, treatment of cells with varying concentrations of AZT induced a dose-dependent inhibition of cell growth of the human carcinoma lines DU-145 (prostate; IC50 at 24 microM), MCF-7 (breast; IC50 at 22 microM), and CX-1 (colon; IC50 at 23 microM), yet caused no significant effect on the growth of the control epithelial cell line CV-1 (monkey kidney) at a concentration as high as 50 microM AZT. Combination treatment employing a constant concentration (1.25 microM) of the DLC dequalinium chloride (DECA) plus varying concentrations of AZT (0-50 microM) enhanced the AZT-induced cytotoxicity of carcinoma cells at least fourfold for MCF-7 and CX-1 cells (IC50 at 5 microM AZT), and twofold for DU-145 cells (IC50 at 11 microM AZT). Similar results were obtained in DU-145 cells using a constant concentration of the DLC MKT-077 (1.0 microM) and varying concentrations of AZT (IC50 at 12.5 microM). As expected, the drug combination of constant DLC and varying AZT had no significant effect on the growth of CV-1 cells. Clonogenic assays demonstrated up to 20-fold enhancement of selective carcinoma cell killing by combination vs. single agent treatment, depending on the specific drug combination and concentrations used. It is hypothesized that the efficacy of the AZT/DLC drug combination in carcinoma cell killing may be based on a dual selectivity involving inhibition of mitochondrial energy metabolism and inhibition of DNA synthesis due to limited deoxythymidine monophosphate availability.

3-Ureidopropionate contributes to the neuropathology of 3-ureidopropionase deficiency and severe propionic aciduria: a hypothesis.

3-Ureidopropionate (3-UPA) is a physiologic metabolite in pyrimidine degradation. Pathological accumulation of 3-UPA in body fluids is found in 3-ureidopropionase deficiency and severe forms of propionic aciduria. Both diseases clinically present with a severe neuropathology involving gray and white matter as well as with a dystonic dyskinetic movement disorder. To date nothing is known about the toxic nature of this metabolite. The aim of the present study was to elucidate whether 3-UPA may act as endogenous neurotoxin. Exposure of cultured chick neurons to 3-UPA induced a concentration- and time-dependent neurodegeneration. Neuronal damage was reduced by the antioxidant alpha-tocopherol and the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801. In contrast, the non-NMDA receptor antagonist CNQX, the metabotropic glutamate receptor antagonist L-AP3, and succinate showed no protective effect. Furthermore, 3-UPA elicited an increased production of reactive oxygen species followed by a delayed increase in intracellular calcium concentrations. Activity measurement of single respiratory chain complexes I-V revealed an inhibition of complex V activity, but not of the electron-transferring complexes I-IV by 3-UPA. In contrast, 3-UPA did not affect the mitochondrial beta-oxidation of fatty acids. In conclusion, our results provide strong evidence that 3-UPA acts as endogenous neurotoxin via inhibition of mitochondrial energy metabolism, resulting in the initiation of secondary, energy-dependent excitotoxic mechanisms.

Metabolic effects of trifluoperazine in the liver and the influence of calcium

The effects of trifluoperazine on hepatic cell metabolism were investigated using isolated perfused rat liver. The following effects of trifluoperazine were found: (1) trifluoperazine inhibited oxygen uptake, the site of action being the mitochondria. Half-maximal inhibition occurred at concentrations around 50 μM; with 100 μM trifluoperazine the effect was already maximal. When Ca 2+ was withdrawn from the perfusion medium and the intracellular Ca 2+ pools were exhausted, the inhibitory action on respiration was no longer observable. The rein-troduction of Ca 2+ restored inhibition. (2) Glycogenolysis and glycolysis were not significantly affected during the infusion of trifluoperazine. After stopping trifluoperazine infusion, however, glycogenolysis (glucose release) experienced a transitory stimulation. (3) Gluconeogenesis from lactate as the carbon source was inhibited by trifluoperazine. This inhibition was approximately proportional to the inhibition of oxygen uptake. Withdrawal of Ca 2+ diminished, but it did not eliminate, inhibition of gluconeogenesis. (4) Ketogenesis was also inhibited in parallel with the inhibition of oxygen uptake. Withdrawal of Ca 2+ from the perfusion fluid also abolished this action. (5) The effects of trifluoperazine were reverted very slowly when its infusion was stopped. The recovery of oxygen uptake at 50 min after cessation of the infusion was only 30%. Uptake of the substance was very fast. Absence of Ca 2+ did not affect uptake. It was concluded that inhibition of mitochondrial energy metabolism is one of the most prominent effects of trifluoperazine in the liver. The fact that this inhibition depends on Ca 2+ is unique.

Histocytological Study on the Possible Mechanism of Acetaminophen Cataractogenesis in Mouse Eye

Ocular toxicity of acetaminophen was investigated in cytochrome P450 inducer-responsive and nonresponsive strains of mice by light and electron microscopy. Acetaminophen injected into C57BL6 mice (responsive strain) that had been pretreated with β-naphthoflavone produced cataract. The drug did not induce cataract in C57BL6 mice without the pretreatment or in DBA2 mice (nonresponsive strain) similarly pretreated with β-naphthoflavone. Therefore, induction of cytochrome P450 enzymes that metabolically activate acetaminophen is essential for cataractogenesis. Following acetaminophen injection, tissue damage became noticeable first in the ciliary epithelium and then spread to the iris, corneal endothelium, and lens. The neural retina, retinal pigmented epithelium, and choroid remained unaffected. A close examination of tissues revealed that mitochondria are the primary target of acetaminophen cytotoxicity in ocular tissues affected. The nucleus, endoplasmic reticulum, and other subcellular structures appeared normal. The course of propagation of tissue damage and the almost exclusive damage to mitochondria suggest that the cytotoxic metabolite of acetaminophen is secreted with the aqueous humor by the ciliary epithelium and transported to the lens and that inhibition of mitochondrial energy metabolism, together with other effects of the metabolic, contributes to acetaminophen-induced cataract.

Metabolic effects of acetaminophen. Studies in the isolated perfused rat liver.

The effects of acetaminophen on the metabolism of the isolated perfused rat liver were investigated. The following results were obtained: (1) Acetaminophen increased glucose release and glycolysis from endogenous glycogen (glycogenolysis). (2) Oxygen uptake, gluconeogenesis from either pyruvate or fructose and glycogen synthesis were inhibited. (3) In isolated rat liver mitochondria acetaminophen decreased state III and state IV respiration; it also decreased the ADP/O ratio and the respiratory control ratio. (4) The action of acetaminophen on glycogenolysis was not affected by N-acetylcysteine; this compound, however, increased glycogen synthesis. (5) The effects of acetaminophen are reversible. It was concluded that glycogen depletion by acetaminophen can be produced by two mechanisms. The first, as previously demonstrated by several workers, depends on irreversible binding of a reactive metabolite. The second, however, is reversible and depends primarily on an inhibition of mitochondrial energy metabolism.

Effects of in vivo and in vitro exposure to rhodamine dyes on mitochondrial function of mouse embryos

Cationic rhodamines (Rh 123 and Rh 6G) can cause developmental toxicity in mice and inhibit embryonic mitochondrial respiration following in vivo or in vitro dye exposure. Rh B, a neutral rhodamine, fails to show such effects at comparable doses. To assess effects of rhodamines on development, F0F1ATPase activity and ADP translocation were measured on gestation day (GD) 12 in embryonic and adult mitochondria. ATP synthesis in embryonic mitochondria transplacentally exposed to Rh 123 (15 mg/kg/day) or Rh 6G (0.5 mg/kg/day) given to dams by i.p. injection from GD 7 to 10 were inhibited 39% and 49%, respectively. When isolated mitochondria were treated, dose-dependent inhibition was seen; at 5 micrograms of dye/mg mitochondrial protein, ATP synthesis was inhibited 65% and 81% by Rh 123 and Rh 6G, respectively. When F0F1ATPase activity was assessed, in vitro Rh 123 and Rh 6G exposures at levels up to 8 micrograms/mg mitochondrial protein resulted in enzyme inhibition, but at 10 micrograms/mg, ATPase activity was stimulated. Uncoupler-stimulated ATPase activity was also inhibited. ADP translocation was decreased by 19.1% and 37.7% by Rh 123 and Rh 6G, respectively, at dye concentrations of 20 micrograms/mg. Results of in vitro exposure of maternal liver mitochondria were similar to those for embryonic mitochondria, whereas liver from dams exposed in vivo on GD 7-10 was unaffected on GD 12. In vivo or in vitro treatment with Rh B did not affect any embryonic or maternal parameters. Such results support the hypothesis that inhibition of mitochondrial energy metabolism is a mechanism for the developmental toxicity of cationic rhodamines.

The effect of inhibition of mitochondrial energy metabolism on the transient inward current of isolated guinea-pig ventricular myocytes

Delayed afterdepolarizations (DADs) might underlie ischemic or reperfusion arrhythmias (Ferrier et al., 1985; Coetzee and Opie, 1987). These DADs are the result of a transient inward current iti, which is caused by instability of the intracellular level of free Ca2+ (Cai) due to an oscillatory release of Ca from the sarcoplasmic reticulum (Kass et al., 1978). DADs are known to be abolished by hypoxia and by metabolic inhibition (Di Gennaro et al., 1987; Coetzee and Opie, 1987), which could be caused by a number of different mechanisms: (1) The large increase of potassium conductance associated with metabolic inhibition (Vleugels et al., 1980; Isenberg et al., 1983) could prevent iti from causing a marked depolarization, and would thus "mask" the DADS. (2) Although metabolic inhibition will eventually result in an increase of Cai, a temporary decrease could initially take place, thereby minimizing the Ca instability. Two mechanisms are known which might produce such an effect: Firstly, the shortening of the action potential which occurs during metabolic inhibition will markedly reduce the time during which Ca channels remain open, thereby causing a diminished total Ca influx during the action potential (Isenberg et al., 1983; Noma and Shibasaki, 1985; Kakei et al., 1985). Secondly, a direct reduction of iCa by a decrease in ATP concentration, described by Irisawa and Kokubun (1983), could also contribute to a decreased Ca load. (3) Metabolic inhibitors could possibly interfere with the cycling of Ca between different compartments within the cell, thereby altering the temporal variation in Cai, and thus influencing iti.(ABSTRACT TRUNCATED AT 250 WORDS)

Further characterization of the growth inhibitory effect of rotenone on in vitro cultured Ehrlich ascites tumour cells.

As an approach for a better understanding of the mode of action of rotenone on mammalian cells we have studied the proliferation properties, metabolism and basic cell composition of Ehrlich ascites tumour cells cultured in vitro in the presence of 2.5 microM rotenone and after removal of the inhibitor. Experiments on asynchronous cells showed a rapid cessation of cell division accompanied by increased glycolytic rate, reduced oxygen consumption, moderate increase in DNA content and a fair increase in protein and RNA content of the cultures. DNA histograms obtained by flow-cytometry revealed an accumulation of cells in the G2 and M phase of the cell cycle. Electron micrographs taken after a 24 h treatment of cells illustrated the formation of giant mitochondria and fragmented nuclei. In order to elucidate the dual effect of rotenone- inhibition of mitochondrial energy metabolism and of mitotic processes- the influence on cells of rotenone at different stages of the cell cycle was tested using Ehrlich ascites tumour cells enriched in G1, S and G2 by centrifugal elutriation. DNA histograms and [3H]thymidine labelling index curves of cells from the different fractions cultured in the presence of 2,5 microM rotenone indicated that in addition to the observed accumulation in G2 and mitotic arrest of cells, the cell cycle progression is delayed in G1 phase. This may be explained by an effect of the inhibitor on the respiratory chain. S phase cells seemed to continue the cycle for several hours at a rate comparable to that of controls. Recultivation experiments on rotenone-treated asynchronous cells in inhibitor-free medium confirmed that some cells reinitiate DNA synthesis without preceeding cell division. Thus it must be concluded that cells at all stages of the cycle are affected by rotenone, but the impairment of cellular metabolism becomes manifest and lethal as soon as the acute block at mitosis is abolished and cells reenter the cycle.