Quaternary ammonium surfactant structure determines selective toxicity towards bacteria: mechanisms of action and clinical implications in antibacterial prophylaxis.

OBJECTIVES: Broad-spectrum antimicrobial activity of quaternary ammonium surfactants (QAS) makes them attractive and cheap topical prophylactic options for sexually transmitted infections and perinatal vertically transmitted urogenital infections. Although attributed to their high affinity for biological membranes, the mechanisms behind QAS microbicidal activity are not fully understood. We evaluated how QAS structure affects antimicrobial activity and whether this can be exploited for use in prophylaxis of bacterial infections. METHODS: Acute toxicity of QAS to in vitro models of human epithelial cells and bacteria were compared to identify selective and potent bactericidal agents. Bacterial cell viability, membrane integrity, cell cycle and metabolism were evaluated to establish the mechanisms involved in selective toxicity of QAS. RESULTS: QAS toxicity normalized relative to surfactant critical micelle concentration showed n-dodecylpyridinium bromide (C12PB) to be the most effective, with a therapeutic index of ∼10 for an MDR strain of Escherichia coli and >20 for Neisseria gonorrhoeae after 1 h of exposure. Three modes of QAS antibacterial action were identified: impairment of bacterial energetics and cell division at low concentrations; membrane permeabilization and electron transport inhibition at intermediate doses; and disruption of bacterial membranes and cell lysis at concentrations close to the critical micelle concentration. In contrast, toxicity to mammalian cells occurs at higher concentrations and, as we previously reported, results primarily from mitochondrial dysfunction and apoptotic cell death. CONCLUSIONS: Our data show that short chain (C12) n-alkyl pyridinium bromides have a sufficiently large therapeutic window to be good microbicide candidates.

Mitochondrial dysfunction is the focus of quaternary ammonium surfactant toxicity to mammalian epithelial cells.

Surfactants have long been known to have microbicidal action and have been extensively used as antiseptics and disinfectants for a variety of general hygiene and clinical purposes. Among surfactants, quaternary ammonium compounds (QAC) are known to be the most useful antiseptics and disinfectants. However, our previous toxicological studies showed that QAC are also the most toxic surfactants for mammalian cells. An understanding of the mechanisms that underlie QAC toxicity is a crucial first step in their rational use and in the design and development of more effective and safer molecules. We show that QAC-induced toxicity is mediated primarily through mitochondrial dysfunction in mammalian columnar epithelial cell cultures in vitro. Toxic effects begin at sublethal concentrations and are characterized by mitochondrial fragmentation accompanied by decreased cellular energy charge. At very low concentrations, several QAC act on mitochondrial bioenergetics through a common mechanism of action, primarily by inhibiting mitochondrial respiration initiated at complex I and, to a lesser extent, by slowing down coupled ADP phosphorylation. The result is a reduction of cellular energy charge which, when reduced below 50% of its original value, induces apoptosis. The lethal effects are shown to be primarily a result of this process. At higher doses (closer to the critical micellar concentration), QAC induce the complete breakdown of cellular energy charge and necrotic cell death.

Mechanism of inhibition of mitochondrial ATP synthase by 17ß-estradiol.

17ß-estradiol (E2) is considered to modulate the ATP synthase activity through direct binding to the oligomycin sensitive-conferring protein. We have previously demonstrated that E2 increases the amplitude of depolarization associated with the addition of ADP to energized mitochondria (i.e., to initiate a phosphorylative cycle) suggesting a direct action on the phosphorylative system of mitochondria. The purpose of the present study was to investigate the underlying mechanisms responsible for this effect. We show here that E2 modulates the activity of mitochondrial ATP synthase by promoting the intrinsic uncoupling ("slipping") of the ATP synthase. E2 depressed RCR, ADP/O ratio and state 3 respiration, whereas state 4 respiration was increased and VFCCP (uncoupled respiration) remained unaltered. In contrast to the stimulatory effect on state 4 respiration, state 2 respiration and Volig were not affected by E2. The effect of E2 appeared to be directed towards ATP synthase, since glutamate/malate respiration, uncoupled from the electron transport chain, was unaffected by E2. Apparently, E2 allows a proton back-leak through the Fo component of ATP synthase. This action of E2 is dependent on the presence of ATP, is more pronounced at high membrane potentials, and it is reversed by oligomycin (a Fo-ATP synthase inhibitor) but not by resveratrol (a F1-ATP synthase inhibitor). Altogether, our data provide a mechanistic explanation for the effect of E2 at the level of mitochondrial ATP synthase.

Proton leak modulation in testicular mitochondria affects reactive oxygen species production and lipid peroxidation.

Mitochondrial proton leak can account for almost 20% of oxygen consumption and it is generally accepted that this process contributes to basal metabolism. In order to clarify the role of basal proton leak in testicular mitochondria, we performed a comparative study with kidney and liver mitochondrial fractions. Proton leak stimulated by linoleic acid and inhibited by guanosine diphosphate (GDP) was detected, in a manner that was correlated with protein levels for uncoupling protein 2 (UCP2) in the three fractions. Modulation of proton leak had an effect on reactive oxygen species production as well as on lipid peroxidation, and this effect was also tissue-dependent. However, a possible role for the adenine nucleotide transporter (ANT) in testicular mitochondria proton leak could not be excluded. The modulation of proton leak appears as a possible and attractive target to control oxidative stress with implications for male gametogenesis.

Comparative effects of three 1,4-dihydropyridine derivatives [OSI-1210, OSI-1211 (etaftoron), and OSI-3802] on rat liver mitochondrial bioenergetics and on the physical properties of membrane lipid bilayers: relevance to the length of the alkoxyl chain in positions 3 and 5 of the DHP ring.

The 1,4-dihydropyridines OSI-1210, OSI-1211 (etaftoron), and OSI-3802 are compounds with similar chemical structure. They differ by the length of the alkoxyl chain in positions 3 and 5 of the dihydropyridine (DHP) ring and by their pharmacological action characteristics. However, as far as we know, a clear relationship between the effects of these compounds and the length of the alkoxyl chain in positions 3 and 5 of the DHP has not been established. The goal of this study was to compare the influence of OSI-1210, OSI-1211 (etaftoron), and OSI-3802 on rat liver mitochondrial bioenergetics and on the physical properties of membrane lipid bilayers, correlating their actions with the length of the alkoxyl chain in positions 3 and 5 of the DHP ring. Using either glutamate/malate or succinate as respiratory substrates, all the compounds, in concentrations of up to 500 microM, depressed state 3 and uncoupled respiration, respiratory control (RCR) and ADP/O ratios, and phosphorylation rate, whereas state 4 respiration was stimulated. However, the stimulatory effect on state 4 induced by OSI-3802, the compound with the longest chain in positions 3 and 5 of the DHP ring, as well as its inhibitory effects on RCR and ADP/O ratios and phosphorylation rate were more pronounced than that induced by OSI-1210 and OSI-1211 (etaftoron), the compounds with the shortest and intermediate chains, respectively. Moreover, OSI-3802 maximized state 4 stimulation and minimized RCR and ADP/O ratios, and phosphorylation rate at a concentration of 100 microM, whereas low graduate effects were detected with OSI-1210 and OSI-1211 (etaftoron) for concentrations of up to 500 microM. At low concentrations (< or =30 microM), OSI-3802, like its analogue OSI-1212 (cerebrocrast), reduced the phase transition temperature, the cooperative unit size, and the enthalpy associated with the phase transition temperature of dimyristoylphosphatidylcholine (DMPC) membrane bilayers. A good correlation was established between the effects of 200 microM OSI-1210, OSI-1211 (etaftoron), and OSI-3802 on glutamate/malate- and succinate-dependent RCR of rat liver mitochondria and on the enthalpy change (Delta H) for the thermotropic profile of DMPC membrane bilayers at a 0.2 drug/DMPC molar ratio, indicating that the changes induced by these compounds on both mitochondrial membrane integrity and physical properties of DMPC membrane bilayers are strongly related to the length of the alkoxyl chain in positions 3 and 5 of the DHP ring. A putative relationship between membrane physical perturbation, bioenergetics impairment and the molecular characteristics of the compounds will be established as an approach to better understand their differentiated toxicological and pharmacological actions.

Mitochondria as the target for mildronate's protective effects in azidothymidine (AZT)-induced toxicity of isolated rat liver mitochondria.

Previously mildronate, an aza-butyrobetaine derivative, was shown to be a cytoprotective drug, through its mechanism of action of inhibition of carnitine palmitoyltransferase-1, thus protecting mitochondria from long-chain fatty acid accumulation and subsequent damage. Recently in an azidothymidine (AZT)-induced cardiotoxicity model in vivo (in mice), we have found mildronate's ability of protecting heart tissue from nuclear factor kappaB abnormal expression. Preliminary data also demonstrate cerebro- and hepatoprotecting properties of mildronate in AZT-toxicity models. We suggest that mildronate may target its action predominantly to mitochondria. The present study in isolated rat liver mitochondria was designed to clarify mitochondrial targets for mildronate by using AZT as a model compound. The aim of this study was to investigate: (1) whether mildronate may protect mitochondria from AZT-induced toxicity; and (2) which is the most critical target in mitochondrial processes that is responsible for mildronate's regulatory action. The results showed that mildronate protected mitochondria from AZT-induced damage predominantly at the level of complex I, mainly by reducing hydrogen peroxide generation. Significant protection of AZT-caused inhibition of uncoupled respiration, ADP to oxygen ratio, and transmembrane potential were also observed. Mildronate per se had no effect on the bioenergetics, oxidative stress, or permeability transition of rat liver mitochondria. Since mitochondrial complex I is the first enzyme of the respiratory electron transport chain and its damage is considered to be responsible for different mitochondrial diseases, we may account for mildronate's effectiveness in the prevention of pathologies associated with mitochondrial dysfunctions.

Inhibition of mitochondrial bioenergetics by carbaryl is only evident for higher concentrations -- Relevance for carbaryl toxicity mechanisms.

Although pesticides have been useful in agriculture pest control, there is a considerable risk for human health and damage to ecosystems. Carbaryl is a carbamate often taken as a safe insecticide, although data on metabolic activities is still scarce, viz. mitochondrial toxicity. Therefore, it is the goal of this work to assay the compound on isolated mitochondria, a biochemical model already used with other pesticides. Mitochondria isolated from the livers of Wistar rats were assayed for bioenergetic parameters, namely mitochondrial respiration, membrane potential, membrane integrity and enzyme activities. For higher concentrations, it was observed that carbaryl has a depressive effect on mitochondrial respiration and on the generation of mitochondrial membrane potential, but with preservation of membrane integrity. A locus between Complex II and III appears particularly affected and the mitochondrial phosphorylation system relatively insensitive. Therefore, carbaryl inhibits mitochondrial respiration without affecting the phosphorylation complex. Carbaryl is toxic for mitochondria, although at concentrations higher as compared with other insecticide compounds. Mitochondrial toxicity should be excluded as one of the primary causes for carbaryl immediate toxicity, as concluded from the range of concentrations where carbaryl shows effective mitochondrial toxicity.

Cerebrocrast promotes the cotransport of H+ and Cl- in rat liver mitochondria.

Considering that cerebrocrast stimulates oligomycin-inhibited state 3 respiration simultaneously with mitochondrial transmembrane potential (Deltapsi) dissipation, the mechanism underlying the uncoupler activity of cerebrocrast was assessed by its ability to permeabilize the mitochondrial inner membrane to H(+) or to K(+) or to cotransport anions with H(+). The partition coefficient of cerebrocrast in mitochondrial membrane and its ability to act as a membrane-active compound disturbing membrane lipid organization were also investigated. Cerebrocrast induced no permeabilization of mitochondrial inner membrane to H(+) or K(+), but it was able to transport H(+) in association with Cl(-). Cerebrocrast showed a strong incorporation into the mitochondrial membrane, with a partition coefficient (Kp(m/w)) of 2.7(+/-0.1)x10(5). Cerebrocrast also reduced, in a concentration dependent manner, the phase transition temperature, the cooperative unit size, and the enthalpy associated with the phase transition temperature of DMPC membrane bilayers. It was concluded that the uncoupler activity of cerebrocrast is due to its ability to promote the cotransport of H(+) with Cl(-) through the rat liver mitochondrial inner membrane, and that this cerebrocrast mechanism of action may be potentiated by alterations of membrane lipid organization and membrane lateral heterogeneity.

Enhanced permeability transition explains the reduced calcium uptake in cardiac mitochondria from streptozotocin-induced diabetic rats.

Cardiac dysfunction is associated with diabetes. It was previously shown that heart mitochondria from diabetic rats have a reduced calcium accumulation capacity. The objective of this work was to determine whether the reduction in calcium accumulation by cardiac mitochondria from diabetic rats is related to an enhanced susceptibility to induction of the mitochondrial permeability transition. Streptozotocin-induced diabetic rats were used as a model to study the alterations caused by diabetes in the permeability transition, 21 days after streptozotocin administration. Heart mitochondria were isolated to evaluate respiratory parameters and susceptibility to the calcium-dependent permeability transition. Our results show that streptozotocin diabetes facilitates the mitochondrial permeability transition in cardiac mitochondria, resulting in decreased mitochondrial calcium accumulation. We also observed that heart mitochondria from diabetic rats had depressed oxygen consumption during the phosphorylative state. The reduced mitochondrial calcium uptake observed in heart mitochondria from diabetic rats is related to an enhanced susceptibility to the permeability transition rather than to damage to the calcium uptake machinery.

Effects of 1,4-dihydropyridine derivatives (cerebrocrast, gammapyrone, glutapyrone, and diethone) on mitochondrial bioenergetics and oxidative stress: a comparative study.

The potential protective action of 1,4-dihydropyridine derivatives (cerebrocrast, gammapyrone, glutapyrone, and diethone) against oxidative stress was assessed on mitochondrial bioenergetics, inner membrane anion channel (IMAC), Ca2+-induced opening of the permeability transition pore (PTP), and oxidative damage induced by the oxidant pair adenosine diphosphate (ADP)/Fe2+ (lipid peroxidation) of mitochondria isolated from rat liver. By using succinate as the respiratory substrate, respiratory control ratio (RCR), ADP to oxygen ratio (ADP/O), state 3, state 4, and uncoupled respiration rates were not significantly affected by gammapyrone, glutapyrone, and diethone concentrations up to 100 microM. Cerebrocrast at concentrations higher than 25 microM depressed RCR, ADP/O, state 3, and uncoupled respiration rates, but increased three times state 4 respiration rate. The transmembrane potential (deltapsi) and the phosphate carrier rate were also decreased. At concentrations lower than 25 microM, cerebrocrast inhibited the mitochondrial IMAC and partially prevented Ca2+-induced opening of the mitochondrial PTP, whereas gammapyrone, glutapyrone, and diethone were without effect. Cerebrocrast, gammapyrone, and glutapyrone concentrations up to 100 microM did not affect ADP/Fe2+-induced lipid peroxidation of rat liver mitochondria, while very low diethone concentrations (up to 5 microM) inhibited it in a dose-dependent manner, as measured by oxygen consumption and thiobarbituric acid reactive substances formation. Diethone also prevented deltapsi dissipation due to lipid peroxidation initiated by ADP/Fe2+. It can be concluded that: none of the compounds interfere with mitochondrial bioenergetics at concentrations lower than 25 microM; cerebrocrast was the only compound that affected mitochondrial bioenergetics, but only for concentrations higher than 25 microM; at concentrations that did not affect mitochondrial bioenergetics (< or = 25 microM), only cerebrocrast inhibited the IMAC and partially prevented Ca2+-induced opening of the PTP; diethone was the only compound that expressed antioxidant activity at very low concentrations (< or = 5 microM). Cerebrocrast acting as an inhibitor of the IMAC and diethone acting as an antioxidant could provide effective protective roles in preventing mitochondria from oxidative damage, favoring their therapeutic interest in the treatment of several pathological situations known to be associated with cellular oxidative stress.

Vitamin E or coenzyme Q10 administration is not fully advantageous for heart mitochondrial function in diabetic goto kakizaki rats.

The heart is one of the organs affected during the later stages of diabetes. Mitochondrial function has already been proposed to be affected during the course of diabetes. Nevertheless, little information is known concerning the impact of antioxidants in heart mitochondria of a milder model for diabetes, such as the Goto-Kakizaki (GK) rat, where mitochondrial function appears ameliorated. The objective of this work was to test if injections of Vitamin E and Coenzyme Q10, alone and in combination, were able to modify mitochondrial performance in the hearts of GK rats. Several aspects of mitochondrial function were measured, such as the respiratory control ratio and the electric potential, as well as the mitochondrial accumulation of Vitamin E and Coenzymes Q9 and Q10. We observed that only Vitamin E appeared to have a positive impact on the mitochondrial phosphorylation efficiency and on mitochondrial performance, namely on the ability to generate the electric transmembrane potential in the presence of supra-physiological calcium concentrations. Vitamin E administration also increased the mitochondrial concentration of Coenzyme Q10. None of the treatments was able to reverse the diabetic phenotype in GK rats. We conclude that in this model of mild hyperglycemia, administration of antioxidants may have a marginal positive impact on mitochondrial function.

Carvedilol inhibits the mitochondrial permeability transition by an antioxidant mechanism.

It was previously shown that carvedilol, a beta-adrenergic receptor antagonist with antioxidant properties, was able to inhibit the mitochondrial permeability transition (MPT). In the present work, the hypothesis was that the negative impact of carvedilol on the MPT was specifically the result of its antioxidant effect. For the current investigation, we used three different MPT inducers. MPT-associated events were tested to study the protective effect of both carvedilol and cyclosporin-A, the known MPT inhibitor. Carvedilol inhibited mitochondrial swelling with calcium plus phosphate and with calcium plus t-butylhydroperoxide, but not with calcium plus carboxyatractyloside. Carvedilol inhibited the oxidation of thiol groups with calcium plus phosphate (p < 0.01) and with calcium plus t-butylhydroperoxide (p < 0.05), but not with calcium plus carboxyatractyloside--in opposition to the full protection afforded by cyclosporin-A when using calcium and carboxyatractyloside. Our results showed that carvedilol was effective only when the MPT was triggered by a primary oxidative process. This finding implies that the antioxidant properties of carvedilol are crucial for the observed effects and reinforces the advantageous use of carvedilol in cardiac pathologies associated with enhanced cellular oxidative stress.

4-Hydroxytamoxifen induces slight uncoupling of mitochondrial oxidative phosphorylation system in relation to the deleterious effects of tamoxifen.

The use of tamoxifen (TAM) has been questioned on the chemotherapy and chemoprevention of breast cancer due to several estrogen receptor-independent cytotoxic effects. As an alternative, its more active metabolite 4-hydroxytamoxifen (OHTAM) has been proposed with presumed lower side effects. In this work, the potential OHTAM toxicity on rat liver mitochondrial bioenergetics in relation to the multiple deleterious effects of TAM was evaluated. OHTAM, at concentrations lower than those putatively reached in tissues following the administration of TAM, does not induce significant perturbations on the respiratory control ratio (RCR), ADP/O, transmembrane potential (DeltaPsi), phosphorylative capacity and membrane integrity of mitochondria. However, at high concentrations, OHTAM depresses the DeltaPsi, RCR and ADP/O, affecting the phosphorylation efficiency, as also inferred from the DeltaPsi fluctuations and pH changes associated with ADP phosphorylation. Moreover, OHTAM, at concentrations that stimulate the rate of state 4 respiration in parallel to the decrease in the DeltaPsi and phosphorylation rate, causes mitochondrial swelling and stimulates both ATPase and citrate synthase activities. However, the OHTAM-observed effects, at high concentrations, are not significant relatively to the damaging effects promoted by TAM and suggest alterations to mitochondrial functions due to proton leak across the mitochondrial inner membrane.

Protection against post-ischemic mitochondrial injury in rat liver by silymarin or TUDC.

BACKGROUND: Hepatic ischemia/reperfusion (I/R) injury is characterized by features such as cholestasis. Mitochondria become susceptible to damage during ischemia and after reperfusion. The aim of this study was to determine if silymarin and tauroursodeoxycholic acid (TUDC), would prevent impairment of liver mitochondrial function following I/R injury. METHODS: Livers of male Wistar rats were subjected to 45 min of hepatic ischemia. During the 90 min of reperfusion, livers were perfused with either vehicle, silymarin, or TUDC. Changes in membrane potential, mitochondrial respiration as well as susceptibility to mitochondrial permeability transition (MPT) induction were evaluated and endogenous adenine nucleotides were measured. RESULTS: In rats subjected to I/R, compared with the control group, a severe impairment of mitochondrial bioenergetics was observed. State 3 respiration was decreased and state 4 enhanced, associated with lower membrane potential developed following succinate energization. An increased susceptibility to MPT induction by calcium/phosphate was also observed. The effects of I/R injury were ameliorated in the presence of silymarin but not TUDC. Similarly, ATP levels following I/R were lower, in comparison with the control group and the silymarin treatment but not TUDC. CONCLUSION: Thus, silymarin, but not TUDC, prevents the most significant changes that occur in mitochondria during I/R, and probably, the associated cell dysfunction, through their central role in cellular bioenergetics.

Cardiac mitochondrial calcium loading capacity is severely affected after chronic cholestasis in Wistar rats.

BACKGROUND: Cardiovascular changes correlated with some forms of hepatic disease are being reported in the literature. OBJECTIVES: The aim of this work was to characterize cardiac mitochondrial bioenergetics and calcium buffering capacity in Wistar rats injected with six weekly doses of alpha-naphthylisothiocyanate (ANIT), a compound known to induce cholestasis in animal models. METHODS: Isolated heart mitochondria were obtained from both injected and control animals and bioenergetic parameters were measured, as well as the capacity to buffer externally added calcium and the mitochondrial content of reduced protein thiol groups. Blood biochemistry analyses were obtained at the initial and end points of treatment. The in vitro ANIT effect on isolated heart mitochondria was also studied. RESULTS AND DISCUSSION: Our results showed that the respiratory control ratio was the only parameter affected in injected animals (p < .05, n = 5). Nevertheless, heart mitochondria from injected animals showed an inability to accumulate added calcium owing to an increased susceptibility to the calcium-dependent mitochondrial permeability transition (p < .0001, n = 5). The effects were still present 1 week after ending ANIT administration, when serum markers for liver injury and hyperbilirubinemia were already abated (although in the presence of bile duct proliferation). To our knowledge, this is the first time that cardiac mitochondrial calcium homeostasis and mitochondrial respiratory ratio are seen affected during ANIT-induced cholestasis, prevailing even in the absence of hepatic damage serum markers.

Comparison of the changes in adenine nucleotides of rat liver mitochondria induced by tamoxifen and 4-hydroxytamoxifen.

The antiestrogen tamoxifen (TAM) inhibits the growth of different estrogen receptor (ER)-negative cells. Recently, multiple effects of TAM on mitochondrial bioenergetic functions have been pointed to explain its ER-independent cell death mechanisms. We have shown that TAM and its major active metabolite 4-hydroxytamoxifen (OHTAM) induce depolarization of the mitochondrial membrane potential (DeltaPsi) and uncouple the mitochondrial respiration, depressing the oxidative phosphorylation efficiency. To clarify the biochemical mechanisms underlying the changes in the regulation of ATP synthesis and yield, in this work we evaluated the alterations of mitochondrial adenine nucleotides induced by both drugs and ascertained whether such changes could reflect a specific inhibition of either the adenine nucleotide translocase (ANT) or the phosphate carrier, as well as the activation of ATP hydrolysis due to DeltaPsi depolarization. We found that both antiestrogens caused a concentration-dependent decrease in mitochondrial ATP levels. Mitochondrial ADP and AMP were concomitantly increased with a subsequent decrease in the ATP/ADP or ATP/AMP ratios. The total concentration of adenine nucleotides also changed. Additionally, both drugs decreased the ANT content of mitochondria, inhibited the phosphate carrier and induced ATP hydrolysis. However, the effects of TAM were more drastic than those induced by OHTAM. Therefore, the depletion of ATP might result from an activation of ATP catabolism, as well as from a decrease in the mitochondrial content of ANT and partial inhibition of the phosphate carrier. Our data may explain the ER-independent effects and cytotoxicity of both drugs and, in agreement with other previous studies, suggest that OHTAM is much less toxic to mitochondria than TAM.

Advantages in the use of carvedilol versus propranolol for the protection of cardiac mitochondrial function.

BACKGROUND: Carvedilol is a neurohormonal antagonist of multiple action which is used in clinical practice for the treatment of congestive heart failure, mild to moderate hypertension and myocardial infarction. Previous results from our group have demonstrated that one of the main targets for the protective effect of carvedilol is the cardiac mitochondrial network. In-this work, we compare the effect of carvedilol with propranolol in different models of mitochondrial dysfunction and in the generation of transmembrane electric potential (EP). We further tested if carvedilol was able to inhibit the mitochondrial permeability transition (MPT) induced by doxorubicin and calcium-dependent cytochrome c release, a phenomenon frequently associated with apoptotic cell death. METHODS: Cardiac mitochondria were isolated by differential centrifugation. Oxygen consumption and mitochondrial EP were determined using an oxygen electrode and a tetraphenylphosphonium-sensitive electrode, respectively. Changes in mitochondrial volume and the release of cytochrome c were measured with spectrophotometric techniques. RESULTS: Propranolol, compared with carvedilol, had only a marginal effect, not only in protection against MPT induction, but also against oxygen consumption linked to the oxidation of external NADH, a process that is considered by several authors as key in the cardiotoxicity of doxorubicin. Regarding EP generation, propranolol had no effect, in contrast to carvedilol, which was confirmed to act as a protonophore. For the first time we also show that carvedilol inhibits the MPT induced by doxorubicin and calcium-dependent cytochrome c release. CONCLUSIONS: With this work, we further support the notion that carvedilol is effective in several models of mitochondrial dysfunction, particularly those involving oxidative stress. The results demonstrate that for some pathological conditions, carvedilol and propranolol have different mechanisms of action at the sub-cellular level, as propranolol seems to lack effectiveness in the protection of cardiac mitochondria.

Impact of diabetes on induction of the mitochondrial permeability transition.

Diabetes is one of the most common metabolic diseases of our times. Specific cardiovascular alterations are often associated with the progression of this disease. Considerable controversy exists in the literature concerning the greater or lesser susceptibility of the diabetic heart to ischemia and reperfusion. Cardiac mitochondria may be fundamental to the differential susceptibility of the cardiomyocyte to pathologic phenomena, particularly those due to the induction of the degenerative process known as the mitochondrial permeability transition (MPT), which is triggered by excessive mitochondrial calcium accumulation. The MPT has been associated with cellular dysfunction resulting from ischemia and reperfusion. The objective of this work was to examine the susceptibility of mitochondria isolated from diabetic rats to the MPT, in comparison to healthy control rats of the same age. Cardiac mitochondria from the diabetic rats had higher calcium loading capacity before the development of the MPT, with a decreased incidence of MPTP-associated features. This was associated with a greater capacity to sustain multiple pulses of externally added calcium, simultaneously with maintenance of the transmembrane electrical potential and reduced swelling amplitude. This could mean that cardiomyocytes from diabetic hearts may indeed be less prone to dysfunctions resulting from ischemia and reperfusion, at least in milder diabetic conditions, thus explaining many reports in the literature.

Toxicity of the herbicide linuron as assessed by bacterial and mitochondrial model systems.

Linuron is one of the most intensively used herbicides with predictable effects on the environment and non-target organisms. In the present study, two in vitro biological models (a Bacillus sp. and rat liver mitochondria) were used to evaluate linuron toxicity at a cell/subcellular level. Linuron inhibited bacterial growth and NADH-supported respiration, similar IC50 values being estimated for both toxic responses (74 and 98 µM, respectively). At concentrations up to 120 µM, linuron perturbed the respiration and phosphorylation efficiency of rat liver mitochondria, reflected by an increase of state 4 respiration and a decrease of the transmembrane potential, state 3 and FCCP-uncoupled respiration, when malate/glutamate or succinate were used as respiratory substrates. Consequently, a decrease of the respiratory control and ADP/O ratio was observed. This study suggests that linuron membrane interactions with adverse repercussions in the activity of membrane enzymatic complexes, such as those which constitute the prokaryotic and mitochondrial respiratory systems, may underlie the toxic effects exerted by that herbicide on non-target organisms. Moreover, this work contributes to the establishment of our bacterial model system as a good tool for chemical toxicity screening.

Use of a calcium-sensitive electrode for studies on mitochondrial calcium transport.

Ca(2+)-sensitive electrode as a practical approach is used to follow Ca(2+) changes in the medium and particularly useful to study mitochondrial Ca(2+) uptake (or release); this method permits the continuous recording of Ca(2+) movements through the mitochondrial inner membrane. In this chapter, it is described how to prepare a Ca(2+)-sensitive electrode, and its application on mitochondrial studies with emphasis on the mitochondrial permeability transition.