Dibenzofuran-induced mitochondrial dysfunction: Interaction with ANT carrier.

Exposure to environmental pollutants such as dibenzofurans and furans is linked to the pathophysiology of several diseases. Dibenzofuran (DBF) is listed as a pollutant of concern due to its persistence in the environment, bioaccumulation and toxicity to humans, being associated with the development of lung diseases and cancers, due to its extremely toxic properties such as carcinogenic and teratogenic. Mitochondria play a key role in cellular homeostasis and keeping a proper energy supply for eukaryotic cells is essential in the fulfillment of the tissues energy-demand. Therefore, interference with mitochondrial function leads to cell death and organ failure. In this work, the effects of DBF on isolated rat liver mitochondria were analyzed. DBF exposure caused a markedly increase in the lag phase that follows depolarization induced by ADP, indicating an effect in the phosphorylative system. This was associated with a dose-dependent decrease in ATPase activity. Moreover, DBF also increased the threshold to the induction of the mitochondrial permeability transition (MPT) by calcium. Pretreatment of mitochondria with DBF also increased the concentration of carboxyatractyloside (CAT) necessary to abolish ADP phosphorylation and to induce the MPT, suggesting that DBF may interfere with mitochondria through an effect on the adenine nucleotide translocase (ANT). By co-immunoprecipitating ANT and Cyclophilin D (CypD) following MPT induction, we observed that in the presence of DBF, the ratio CypD/ANT was decreased. This demonstrates that DBF interferes with the ANT and so prevents CypD binding to the ANT, causing decreased phosphorylative capacity and inhibiting the MPT, which is also reflected by an increase in calcium retention capacity. Clarifying the role of pollutants in some mechanisms of toxicity, such as unbalance of bioenergetics status and mitochondrial function, may help to explain the progressive and chronic evolution of diseases derived from exposure to environmental pollutants.

Disruption of hepatic mitochondrial bioenergetics is not a primary mechanism for the toxicity of methoprene - relevance for toxicological assessment.

Methoprene (isopropyl(2E,4E)-11-methoxy-3,7,11-trimethyl-2,4-dodecadienoate) is an insect growth regulator generally used to control insect populations by preventing insect maturation. So far, the effects of the insecticide on mitochondrial bioenergetics were not investigated. In the present work, liver mitochondria from Wistar rats were isolated and features of mitochondrial physiology were characterized in the presence of methoprene. High concentrations of methoprene, in the range of 40-100 nmol/mg of protein could decrease the transmembrane electric potential (Delta Psi) developed by mitochondria and, at the highest concentration, methoprene prevented complete Delta Psi repolarization after ADP addition. The effect was more evident using succinate than with ascorbate+TMPD as substrate. State 3 respiration was approximately 60% inhibited by 80 nmol of methoprene/mg of protein, while state 4 respiration, within the same range of methoprene concentrations, showed a slight increase, when both glutamate-malate and succinate were used as substrates. Additionally, FCCP-stimulated respiration was inhibited to an extent comparable to the effect on state 3, which suggests an interaction of methoprene with the respiratory chain, more evident with glutamate/malate as substrate. The activity of complex I (NADH-ubiquinone oxidorreductase) and that of the segment comprehending complexes II and III (succinate-cytochrome c reductase) were decreased in the presence of methoprene (approximately 60% and 85% of inhibition, respectively, with 300 nmol of methoprene/mg of protein), while the activities of cytochrome c oxidase and ATPase do not seem to be affected. Furthermore, the action of methoprene on the mitochondrial permeability transition was also studied, showing that the insecticide (in the range of 30-80 nmol mg(-1) of protein) decreases the susceptibility of liver mitochondria to the opening of the transition pore, even in non-energized mitochondria. These results lead to the conclusion that methoprene interference with hepatic mitochondrial function occurs only for high concentrations, which implies that the noxious effects of the insecticide reported for a number of non-target organisms are not fully attributable to mitochondrial effects. Therefore, it seems that mitochondrial activity does not represent the primary target for methoprene toxic action.

Comparative study of tributyltin toxicity on two bacteria of the genus Bacillus.

Tributyltin is a potent biocide mainly used in marine anti-fouling paints. Owing to its widespread distribution in coast areas and its high toxicity to aquatic organisms, the use of this compound is generally restricted and under government regulation. Despite of that, it persists in the aquatic environment. Organotins used in industry have also been detected in terrestrial environments. The persistence and high lipophilicity explain bioaccumulation. The role of bacteria in recycling organic matter prompted us to study the interaction of tributyltin with two ubiquitous bacilli, B. stearothermophilus and B. subtilis, proposed as biological indicators of pollutants with ecological impact. These bacteria have been used as suitable models for the study of toxicity mechanisms of unselective lipophilic compounds (e.g., DDT and endosulfan). Drug effects on growth parameters, oxygen consumption and membrane organization were assessed. Bacteria growth in a liquid complex medium was disturbed by concentrations of TBT as low as 25 nM (8 microgL(-1)), close to the concentration in polluted environments. The respiratory activity is affected by TBT in both microorganisms. Membrane organization, assessed by fluorescence polarization of two fluidity probes, 1,6-diphenyl-1,3,5-hexatriene (DPH) and a propionic acid derivative (DPH-PA), was also perturbed by the xenobiotic. Alterations on growth, oxygen consumption and physical properties of membrane lipids are stronger in B. stearothermophilus as compared to B. subtilis. A putative relationship between growth inhibition and respiratory activity impairment induced by TBT and its effects on the physical behaviour of bacterial membrane lipids is suggested.

Toxicity of methoprene as assessed by the use of a model microorganism.

Methoprene is an insect juvenile growth hormone mimic, commonly used as a pesticide. Although widely used for the control of several pests, toxic effects on organisms of different phyla have been reported. These events triggered studies to clarify the mechanisms of toxicity of this insecticide putatively involved in ecological issues. Here we show the effect of methoprene on the normal cell growth and viability of a strain of the thermophilic eubacterium Bacillus stearothermophilus, previously used as a model for toxicological evaluation of other environment pollutants. Respiration studies were also carried out attempting to identify a putative target for the cytotoxic action of methoprene. Cell growth was affected and a decrease of the number of viable cells was observed as a result of the addition of methoprene to the growth medium, an effect reverted by the presence of Ca(2+). Methoprene also inhibited the redox flow of B. stearothermophilus protoplasts before the cytochrome oxidase segment, an effect further studied by individually assessing the enzymatic activities of the respiratory complexes. This study suggests that methoprene membrane interaction and perturbation of cell bioenergetics may underlie the mechanism of toxicity of this compound in non-target organisms.

Carvedilol protects against doxorubicin-induced mitochondrial cardiomyopathy.

Several cytopathic mechanisms have been suggested to mediate the dose-limiting cumulative and irreversible cardiomyopathy caused by doxorubicin. Recent evidence indicates that oxidative stress and mitochondrial dysfunction are key factors in the pathogenic process. The objective of this investigation was to test the hypothesis that carvedilol, a nonselective beta-adrenergic receptor antagonist with potent antioxidant properties, protects against the cardiac and hepatic mitochondrial bioenergetic dysfunction associated with subchronic doxorubicin toxicity. Heart and liver mitochondria were isolated from rats treated for 7 weeks with doxorubicin (2 mg/kg sc/week), carvedilol (1 mg/kg ip/week), or the combination of the two drugs. Heart mitochondria isolated from doxorubicin-treated rats exhibited depressed rates for state 3 respiration (336 +/- 26 versus 425 +/- 53 natom O/min/mg protein) and a lower respiratory control ratio (RCR) (4.3 +/- 0.6 versus 5.8 +/- 0.4) compared with cardiac mitochondria isolated from saline-treated rats. Mitochondrial calcium-loading capacity and the activity of NADH-dehydrogenase were also suppressed in cardiac mitochondria from doxorubicin-treated rats. Doxorubicin treatment also caused a decrease in RCR for liver mitochondria (3.9 +/- 0.9 versus 5.6 +/- 0.7 for control rats) and inhibition of hepatic cytochrome oxidase activity. Coadministration of carvedilol decreased the extent of cellular vacuolization in cardiac myocytes and prevented the inhibitory effect of doxorubicin on mitochondrial respiration in both heart and liver. Carvedilol also prevented the decrease in mitochondrial Ca(2+) loading capacity and the inhibition of the respiratory complexes of heart mitochondria caused by doxorubicin. Carvedilol by itself did not affect any of the parameters measured for heart or liver mitochondria. It is concluded that this protection by carvedilol against both the structural and functional cardiac tissue damage may afford significant clinical advantage in minimizing the dose-limiting mitochondrial dysfunction and cardiomyopathy that accompanies long-term doxorubicin therapy in cancer patients.