Overview of Mitochondrial Bioenergetics.

Bioenergetic science started in the eighteenth century with the pioneer works by Joseph Priestley and Antoine de Lavoisier on photosynthesis and respiration, respectively. New developments were implemented by Pasteur in the 1860s with the description of fermentations associated with microorganisms, further documented by Buchner brothers who discovered that fermentations also occurred in cell extracts in the absence of living cells. In the beginning of the twentieth century, Harden and Young demonstrated that orthophosphate and other heat-resistant compounds (cozymase), later identified as NAD, ADP, and metal ions, were mandatory in the fermentation of glucose. The full glycolysis pathway has been detailed in the 1940s with the contributions of Embden, Meyeroff, Parnas, and Warburg, among others.Studies on the citric acid cycle started in 1910 (Thunberg) and were elucidated by Krebs et al. in the 1940s.Mitochondrial bioenergetics gained emphasis in the late 1940s and 1950s with the works of Lehninger, Racker, Chance, Boyer, Ernster, and Slater, among others. The prevalent "chemical coupling hypothesis" of energy conservation in oxidative phosphorylation was challenged and replaced by the "chemiosmotic hypothesis" originally formulated in the 1960s by Mitchell and later substantiated and extended to energy conservation in bacteria and chloroplasts, besides mitochondria, with clear-cut identification of molecular proton pumps.After identification of most reactive mechanisms, emphasis has been directed to structure resolution of molecular complex clusters, e. g., cytochrome c oxidase, complex III, complex II, ATP synthase, photosystem I, photosynthetic water-splitting center, and energy collecting antennae of several photosynthetic systems.Modern trends concern to the reactivity of radical and other active species in association with bioenergetic activities. A promising trend concentrates on the cell redox status quantified in terms of redox potentials.In spite of significant development and advances of bioenergetic knowledge, major issues remain mainly related with poor experimental designs not representative of the real native cell conditions. Therefore, a major effort has to be implemented regarding direct observations in situ.

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.

Differential effects induced by alpha- and beta-endosulfan in lipid bilayer organization are reflected in proton permeability.

The effects of two insecticides isomers, alpha- and beta-endosulfan, on the passive proton permeability of large unilamellar vesicles (LUV) reconstituted with dipalmitoylphosphatidylcholine (DPPC) or mitochondrial lipids were reported. In DPPC (LUV) gel phase, at 30 degrees C, the global kinetic constant (K) of proton permeability (proportional to the proton permeability) initially increased slightly with the increase of alpha-endosulfan/lipid molar ratio up to 0.143. In the range from 0.143 to 0.286, a discontinuity in the increment occurred and, above this range, the proton permeability increased substantially. In DPPC fluid phase, at 48 degrees C, the proton permeability showed a behavior identical to that observed in gel DPPC, with a sharp increase for alpha-endosulfan/lipid molar ratios ranging from 0.143 to 0.286. At these and higher concentrations, alpha-endosulfan induced phase separation in the plane of DPPC membranes, as revealed by differential scanning calorimetry (DSC). Conversely to alpha-endosulfan, beta-endosulfan induced only a slight increase in the proton permeability, either in the fluid or the gel phase of DPPC, for all beta-endosulfan/lipid molar ratios tested. Additionally, the effects of the endosulfan isomers on the proton permeability of mitochondrial fluid lipid dispersions, at 37 degrees C, are similar to those described for DPPC. The beta-isomer induced a very small effect, and alpha-endosulfan, at low concentrations, increased slightly the proton permeability, but for insecticide/lipid molar ratios above 0.143 the permeability increased substantially. Consequently, the membrane physical state of synthetic and native lipid dispersions, as affected by the structural features of alpha- and beta-endosulfan, influenced the proton permeability. The effects here observed in vitro suggest that the formation of lateral membrane domains may underlay the biological activity of alpha-endosulfan in vivo, contributing to its higher degree of toxicity as compared with beta-endosulfan.

Teaching about citric acid cycle using plant mitochondrial preparations: Some assays for use in laboratory courses*.

Potato tubers and turnip roots were used to prepare purified mitochondria for laboratory practical work in the teaching of the citric acid cycle (TCA cycle). Plant mitochondria are particularly advantageous over the animal fractions to demonstrate the TCA cycle enzymatic steps, by using simple techniques to measure O(2) consumption and transmembrane potential (ΔΨ). The several TCA cycle intermediates induce specific enzyme activities, which can be identified by respiratory parameters. Such a strategy is also used to evidence properties of the TCA cycle enzymes: ADP stimulation of isocitrate dehydrogenase and α-ketoglutarate dehydrogenase; activation by citrate of downstream oxidation steps, e.g. succinate dehydrogenase; and regulation of the activity of isocitrate dehydrogenase by citrate action on the citrate/isocitrate carrier. Furthermore, it has been demonstrated that, in the absence of exogenous Mg(2+) , isocitrate-dependent respiration favors the alternative oxidase pathway, as judged by changes of the ADP/O elicited by the inhibitor n-propyl galate. These are some examples of assays related with TCA cycle intermediates we can use in laboratory courses.

A comparative study of plant and animal mitochondria exposed to paraquat reveals that hydrogen peroxide is not related to the observed toxicity.

Rat liver mitochondria are much more susceptible to protein oxidation induced by paraquat than plant mitochondria. The unsaturated index and the peroxidizability index are higher in rat than in potato tuber. The levels of superoxide dismutase and glutathione reductase are concurrent with the different sensitivities to paraquat, with higher activities in plant mitochondria. However, glutathione peroxidase and catalase activities are higher in rat mitochondria. Paraquat (10 mM) inhibited all the enzymatic activities; excluding catalase all the other activities were inhibited to a similar degree. The differential sensitivities of plant and animal mitochondria to paraquat correlate with fatty acid composition of mitochondrial lipids and a similar correlation was also established for some antioxidant enzymes. At the mitochondrial level, H(2)O(2) is not a major factor of paraquat toxicity since rat liver mitochondria which exhibit higher activities of glutathione peroxidase and catalase are however more susceptible to paraquat.

Tamoxifen induces ultrastructural alterations in membranes of Bacillus Stearothermophilus.

Tamoxifen (TAM), a non-steroid antiestrogen, is the mostly used drug for chemotherapy and chemoprevention of breast cancer. However, the mechanisms by which TAM inhibits cell proliferation in breast cancer are not fully understood. TAM strongly incorporates in biomembranes and a variety of effects have been assigned to biophysical and biochemical interactions with membranes. Therefore, a better understanding of the physicochemical basis of interaction of TAM with biomembranes is essential to elucidate the molecular mechanisms of action. A strain of Bacillus stearothermophilus has been used as a model to clarify the interaction of TAM with the cell membrane. TAM effects on the ultrastructure of membranes of this bacterium were evaluated by electron microscopy. Important ultrastructural alterations were observed in B. stearothermophilus treated with TAM, namely change in the geometry of the membrane profile from asymmetric to symmetric, disaggregation of ribosomes, coagulation of the cytoplasmic matrix, occurrence of mesossomes, appearance of fractures in membranes and the alteration of the ultrastructure of cell wall. These ultrastructural alterations confirm that TAM is a membrane-active drug and that membrane damage may be involved in molecular mechanisms of cell death induced by this drug.

Cholesterol modulates amiodarone-membrane interactions in model and native membranes.

The effects of cholesterol, a lipid mostly found in the sarcolemmal membranes, on the interaction of amiodarone with synthetic models of dimyristoylphosphatidylcholine (DMPC) and with native models of mitochondria and brain microsomes was studied. Alterations on the structural order of lipids were assessed by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) probing the bilayer core, and of the propionic acid derivative 3-(p-(6-phenyl)-1,3,5-hexatrienyl)phenylpropionic acid (DPH-PA) probing the outer regions of the bilayer. As detected by the probes and according to classic observations, cholesterol progressively increased the molecular order in the fluid phase of DMPC. Additionally, it modulated the type and extension of amiodarone effects. For low cholesterol concentrations (< or =10-15 mol%), amiodarone (50 microM) ordered DMPC bilayers and the effects were almost identical to those observed in pure DMPC. For higher cholesterol concentrations, amiodarone ordering effects decreased slightly and faded for cholesterol concentrations as high as 25 and 30 mol%, when detected by DPH-PA and DPH, respectively. Above these high cholesterol concentrations, a crossover from ordering to disordering effects of amiodarone was apparent, either in the upper region of the bilayer or the hydrophobic core. The effects of amiodarone in native membranes of mitochondria and brain microsomes, in which "native" cholesterol accounts for about 0 and 25 mol%, respectively, correlated reasonably with the results in models of synthetic lipids. There is a close relationship between cholesterol concentration and amiodarone effects, in either synthetic models or native model membranes. Therefore, it may be predicted that the lipid physicochemical properties regulated by cholesterol concentration will also modulate the effects of amiodarone in sarcolemma.

Overview of mitochondrial bioenergetics.

Bioenergetic Science started in seventh century with the pioneer works by Joseph Priestley and Antoine Lavoisier on photosynthesis and respiration, respectively. New developments were implemented by Pasteur in 1860s with the description of fermentations associated to microorganisms, further documented by Buchner brothers who discovered that fermentations also occurred in cell extracts in the absence of living cells. In the beginning of twentieth century, Harden and Young demonstrated that orthophosphate and other heat-resistant compounds (cozymase), later identified as NAD, ADP, and metal ions, were mandatory in the fermentation of glucose. The full glycolysis pathway has been detailed in 1940s with the contributions of Embden, Meyeroff, Parnas, Warburg, among others. Studies on the citric acid cycle started in 1910 (Thunberg) and were elucidated by Krebs et al. in the 1940s. Mitochondrial bioenergetics gained emphasis in the late 1940s and 1950s with the works of Lenhinger, Racker, Chance, Boyer, Ernster, and Slater, among others. The prevalent "chemical coupling hypothesis" of energy conservation in oxidative phosphorylation was challenged and replaced by the "chemiosmotic hypothesis" originally formulated in 1960s by Mitchell and later substantiated and extended to energy conservation in bacteria and chloroplasts, besides mitochondria, with clear-cut identification of molecular proton pumps. After identification of most reactive mechanisms, emphasis has been directed to structure resolution of molecular complex clusters, e.g., cytochrome c oxidase, complex III, complex II, ATP synthase, photosystem I, photosynthetic water splitting center, and energy collecting antennæ of several photosynthetic systems. Modern trends concern to the reactivity of radical and other active species in association with bioenergetic activities. A promising trend concentrates on the cell redox status quantified in terms of redox potentials. In spite of significant development and advances of bioenergetic knowledge, major issues remain mainly related with poor experimental designs not representative of the real native cell conditions. Therefore, a major effort has to be implemented regarding direct observations in situ.

The herbicide dicamba (2-methoxy-3,6-dichlorobenzoic acid) interacts with mitochondrial bioenergetic functions.

The effects of dicamba, a widely used broad-leaf herbicide, on rat liver mitochondrial bioenergetic activities were examined. The results obtained for state 4 respiration indicate not only an uncoupling effect, the result of an increase on the permeability of inner mitochondria membrane to protons, but also a strong inhibitory effect on the redox complexes. State 3 and respiration uncoupled by FCCP (carbonylcyanide p-trifluoromethoxyphenylhydrazone) were inhibited to approximately the same extent, i.e. by about 70%. Depression of respiratory activity is essentially mediated through partial inhibition of mitochondrial complexes II and III. ATPase activity was much less depressed by dicamba than ATP synthase activity. Therefore, a considerable part of the inhibition observed on ATP synthase is the result of an inhibition on the redox complexes. The loss of phosphorylation capacity, induced by dicamba, was in the last analysis not only the result of a direct effect of dicamba on the enzymatic complex (F(0)-F(1) ATPase) but also the result of a deleterious effect on the integrity of the mitochondrial membrane, which can promote an inhibition of the respiratory complexes and an increase of the proton permeability of the inner membrane.

Degradability and sediment sorption of an alcohol polyglycol ether surfactant putatively useful for the control of red swamp crayfish in rice fields.

This work reports studies of the degradation rates of a fatty alcohol polyglycol ether non-ionic surfactant, Genapol OXD-080, putatively useful for the control of red swamp crayfish (Procambarus clarkii Girard) in rice fields under laboratory and field conditions. The influence of temperature, sediment site specificity and sorption were taken into account. The degradation kinetics of the surfactant depends on the experimental conditions: type of inocula and temperature. The distribution of this chemical in aquatic systems was also examined. Genapol OXD-080 was removed into the sediments readily after application, and sorption was considered the major path of removal from the water phase. Data suggest that further studies are required regarding the effects of Genapol OXD-080 in aquatic organisms resident in rice fields, in parallel with the development of technologies related with the use of surfactants to control P. clarkii populations.

Comparative effects of herbicide dicamba and related compound on plant mitochondrial bioenergetics.

The herbicide dicamba (3,6-dichloro-2-methoxybenzoic acid) was evaluated for its effects on bioenergetic activities of potato tuber mitochondria to elucidate putative mechanisms of action and to compare its toxicity with 2-chlorobenzoic acid. Dicamba (4 micro mol/mg mitochondrial protein) induces a limited stimulation of state 4 respiration of ca. 10%, and the above concentrations significantly inhibit respiration, whereas 2-chlorobenzoic acid maximally stimulates state 4 respiration (ca. 50%) at about 25 micro mol/mg mitochondrial protein. As opposed to these limited effects on state 4 respiration, transmembrane electrical potential is strongly decreased by dicamba and 2-chlorobenzoic acid. Dicamba (25 micro mol/mg mitochondrial protein) collapses, almost completely, Deltapsi; similar concentrations of 2-chlorobenzoic acid promote Deltapsi drops of about 50%. Proton permeabilization partially contributes to Deltapsi collapse since swelling in K-acetate medium is stimulated, with dicamba promoting a stronger stimulation. The Deltapsi decrease induced by dicamba is not exclusively the result of a stimulation on the proton leak through the mitochondrial inner membrane, since there was no correspondence between the Deltapsi decrease and the change on the O(2) consumption on state 4 respiration; on the contrary, for concentrations above 8 micro mol/mg mitochondrial protein a strong inhibition was observed. Both compounds inhibit the activity of respiratory complexes II and III but complex IV is not significantly affected. Complex I seems to be sensitive to these xenobiotics. In conclusion, dicamba is a stronger mitochondrial respiratory chain inhibitor and uncoupler as compared to 2-chlorobenzoic acid. Apparently, the differences in the lipophilicity are related to the different activities on mitochondrial bioenergetics.

Regulation by magnesium of potato tuber mitochondrial respiratory activities.

Dehydrogenase activities of potato tuber mitochondria and corresponding phosphorylation rates were measured for the dependence on external and mitochondrial matrix Mg2+. Magnesium stimulated state 3 and state 4 respiration, with significantly different concentrations of matrix Mg2+ required for optimal activities of the several substrates. Maximal stimulation of respiration with all substrates was obtained at 2-mM external Mg2+. However, respiration of malate, citrate, and alpha-ketoglutarate requires at least 4-mM Mg2+ inside mitochondria for maximization of dehydrogenase activities. The phosphorylation system, requires a low level of internal Mg2+ (0.25 mM) to reach high activity, as judged by succinate-dependent respiration. However, mitochondria respiring on citrate or alpha-ketoglutarate only sustain high levels of phosphorylation with at least 4-mM matrix Mg2+. Respiration of succinate is active without external and matrix Mg2+, although stimulated by the cation. Respiration of alpha-ketoglutarate was strictly dependent on external Mg2+ required for substrate transport into mitochondria, and internal Mg2+ is required for dehydrogenase activity. Respiration of citrate and malate also depend on internal Mg2+ but, unlike alpha-ketoglutarate, some activity still remains without external Mg2+. All the substrates revealed insensitive to external and internal mitochondrial Ca2+, except the exogenous NADH dehydrogenase, which requires either external Ca2+ or Mg2+ for detectable activity. Calcium is more efficient than Mg2+, both having cumulative stimulation. Unlike Ca2+, Mn2+ could substitute for Mg2+, before and after addition of A23, showing its ability to regulate phosphorylation and succinate dehydrogenase activities, with almost the same efficiency as Mg2+.