Effects of cocaine and its oxidative metabolites on mitochondrial respiration and generation of reactive oxygen species

Cocaine is capable of producing severe hepatocellular necrosis in laboratory animals and humans. The mechanism of cocaine hepatotoxicity is not well understood, but appears to result from the actions of one or more N-oxidative metabolites of cocaine. Mitochondria have been proposed as critical cellular targets for cocaine toxicity, and previous studies have found depressed mitochondrial respiration and increased mitochondrial generation of reactive oxygen species (ROS) in animals treated with cocaine. To examine the potential role of cocaine N-oxidative metabolites in these effects, mitochondrial respiration and ROS generation were examined in isolated mouse mitochondria treated with cocaine and its N-oxidative metabolites-norcocaine, N-hydroxynorcocaine, and norcocaine nitroxide. Cocaine, in concentrations of 0.25 or 0.5 mM, had no effect on state 3 respiration, state 4 respiration, respiratory control ratio (RCR), or ADP/O ratio. Norcocaine (0.5 mM) inhibited state 3 respiration, and N-hydroxynorcocaine (0.5 mM) inhibited both state 3 and state 4 respiration. Norcocaine nitroxide had the greatest effect on mitochondrial respiration; the lower concentration (0.25 mM) completely inhibited both state 3 and state 4 respiration. Preincubation of mitochondria with cocaine or metabolites increased the inhibitory effect of norcocaine and N-hydroxynorcocaine, but not cocaine. Cocaine, norcocaine, and N-hydroxynorcocaine (0.1 mM) had no effect on ROS generation during state 3 respiration, and cocaine and norcocaine decreased ROS generation under state 4 conditions. Norcocaine nitroxide interfered with the fluorescence ROS assay and could not be assessed. The results suggest that the effects of cocaine on mitochondrial respiration are due to its N-oxidative metabolites. Inhibition of mitochondrial respiration by the N-oxidative metabolites of cocaine may be the underlying cause for observed ATP depletion and subsequent cell death.     

The mechanism of acute cytotoxicity of triethylphosphine gold(I) complexes : II. Triethylphosphine gold chloride-induced alterations in mitochondrial function

Triethylphosphine gold complexes have therapeutic activity in the treatment of rheumatoid arthritis. Many of these compounds are also highly cytotoxic in vitro to a variety of tumor and non-tumor cell lines. Triethylphosphine gold chloride (TEPAu) is highly cytotoxic to isolated rat hepatocytes at concentrations greater than 25 microM. The earliest changes that could be detected in hepatocytes included bleb formation in the plasma membrane, alterations in the morphology of mitochondria, and rapid decreases in cellular ATP and oxygen consumption. The degradation of ATP could be followed sequentially through ADP and AMP and was ultimately accounted for entirely as xanthine. The sum of adenine and xanthine-derived nucleotides remained constant throughout the experiments. TEPAu (50 microM) caused a significant decrease in the hepatocyte ATP/ADP ratio and energy charge within 5 min. The antioxidant, N,N'-diphenyl-p-phenylenediamine (DPPD), which blocked TEPAu-induced malondialdehyde formation but not cell death, also had no effect on the decreases in oxygen consumption, ATP, ATP/ADP ratio, or energy charge. In isolated rat liver mitochondria, TEPAu (1 microM) caused significant reductions in carbonyl cyanide-4-trifluoromethoxyphenylhydrazone (FCCP) (uncoupled)-stimulated respiration. TEPAu (5 microM) inhibited state 3 respiration and the respiratory control ratio without affecting state 4 respiration and caused a rapid dissipation of the mitochondrial-membrane hydrogen-ion gradient (membrane potential). Concentrations greater than 5 microM also inhibited state 4 respiration. TEPAu caused a concentration-dependent inhibition of FCCP-stimulated respiration with pyruvate/malate and succinate as substrates but had not effect on ascorbate/tetramethyl-p-phenylenediamine-supported respiration. The inhibition of state 4 respiration and FCCP-stimulated respiration by TEPAu (10 microM) could be reversed by the addition of 2 mM dithiothreitol. Dithiothreitol also partially protected cells from TEPAu-induced injury and reversed the TEPAu-induced depletion in cellular ATP. These data indicate that TEPAu may be acting functionally as a respiratory site II inhibitor, similar to antimycin. The reversal of TEPAu-induced inhibition of mitochondrial respiration and cell lethality by dithiothreitol suggests that mitochondrial thiols may be involved.     

Metabolic effects of hypoglycemic sulfonylureas. IV. Interference of sulfonylureas with mitochondrial oxidative phosphorylation

The effects of various hypoglycemic and non-hypoglycemic sulfonylureas on succinate oxidation by rat liver mitochondria have been studied. At low succinate concentration, respiration is initially stimulated and then inhibited by all the sulfonylureas tested, except carbutamide and the compound P571. Both phenomena are dose-dependent. The respiration inhibition can be reversed by increasing the succinate concentration, but not by adding ATP plus oligomycin. Respiratory control disappears progressively in the presence of increasing concentrations of sulfonylureas, including carbutamide and P571, mainly through inhibition of state 3 respiration. This inhibition can also be lowered by increasing the succinate concentration. The effect of sulfonylureas on respiration is oligomycin-insensitive. 2,4-Dinitrophenol has the same effect as the sulfonylureas under the same experimental conditions.It is concluded that the sulfonylureas studied, except carbutamide and P571, exert a dinitrophenol-like effect on mitochondrial respiration, and that, like dinitrophenol, all sulfonylureas, including carbutamide and P571, interfere with mitochondrial substrate uptake. A relationship between the observed effects and the clinical activity of these drugs is discussed.     

Effect of 4-dimethylaminomethyl-1-(3-hydroxyphenyl)-1-nonen-3-one hydrochloride and related compounds on respiration in rat liver mitochondria

4-Dimethylaminomethyl-1-(3-hydroxyphenyl)-1-nonen 3-one hydrochloride (II) was shown to stimulate respiration in rat liver mitochondria at levels of 2.5 mumoles or less; but at levels higher than 5.0 mumoles, respiration was inhibited when succinate and 3-hydroxybutyrate were the substrates. Compound II caused inhibition of respiration in the presence of glutamate over the dose range studied. The stimulating effect of II was attributed to its functioning as an uncoupling agent. Its inhibiting properties were considered to be due to its behaving like antimycin A in blocking transport of electrons between cytochromes b and c1. The effect of II on respiration in mitochondria varied with the pH of the medium. A conjugated styryl ketone, which contained a nuclear hydroxy function and was structurally related to II, also stimulated respiration at low doses while inhibiting respiration at higher concentrations. Etherification of the hydroxy group led to compounds in which only stimulation of respiration was noted.     

Influence of propolis water solution on heart mitochondrial function

The effect of propolis water solution (PWS) on the respiration of rat heart mitochondria with NAD-linked (pyruvate + malate), FAD-linked (succinate) substrates and fatty acids (palmitoyl-L-carnitine) was investigated in this study. PWS at the lowest concentration of 4 microg mL(-1) of phenolic compounds (PC) had no effect on mitochondrial respiration with all investigated substrates. PWS at concentrations of 63 and 125 microg mL(-1) of PC caused a significant decrease of basal (24 and 54%) and maximal (58 and 70%) respiration rates with succinate as substrate. At these PWS concentrations the oxidation of pyruvate + malate and palmitoyl-L-carnitine was diminished to a lower degree: the basal respiration rate decreased by 13-18% and the maximal respiration rate by 15-28%. Succinate oxidation was affected, probably because of the inhibition of succinate dehydrogenase by the 1,2-benzenedicarboxylic acid esters found in PWS. The PWS-caused decrease in the mitochondrial respiration rate with pyruvate + malate and fatty acids could be due to diminished activities of respiratory chain complexes and/or ADP/ATP translocator.     

Inhibition of some mitochondrial functions by acrolein and methylvinylketone

The effects of acrolein and methylvinylketone on some mitochondrial processes have been studied. The respiration with various substrates is inhibited. The sensitivity to acrolein decreases in the following order: ADP-stimulated respiration with glutamate, DNP-stimulated respiration with glutamate, ADP-stimulated respiration with succinate, DNP-stimulated respiration with succinate and respiration with NADH of aged mitochondria. It is suggested that acrolein acts on three different sites; glutamate transport, Pi transport and succinodehydrogenase. The effect on Pi transport is competitive with respect to Pi. Methylvinylketone shows the same effects as acrolein, however, it is less effective by one order of magnitude.     

Inhibition of the respiration of Eimeria tenella by quinolone coccidiostats.

The protozoan parasite Eimeria tenella respires vigorously during sporulation and excystation. Sporulation is initiated and sustained by the respiratory activities, and cannot take place under anaerobic conditions. Excystation is not necessarily associated with oxygen consumption and seems to be a passive process solely carried out by the host. Quinolone coccidiostats such as amquinate, buquinolate, methyl benzoquate and decoquinate are reversible inhibitors of E. tenella respiration and sporulation. The effective concentrations of the inhibitors for 50 per cent inhibition are 1 to 2 × 10^?5 M against respiration during sporulation and 3 × 10^?6 M against respiration during excystation. The respiration in sporulation and excystation of an amquinate-resistant E. tenella mutant is much less subject to inhibition by the quinolones. The results suggest the inhibition of coccidial respiration as the possible mechanism of anticoccidial activity of the quinolones. Some 2-hydroxynaphthoquinone coccidiostats are also strong inhibitors of E. tenella respiration, but they are equally effective against the wild type and the amquinate-resistant mutant.     

Cadmium as an uncoupler of respiration in Ulva lactuca

The effect of cadmium on the respiration of Ulva lactuca was examined using an oxygen electrode. Discs of U. lactuca thallus in filtered seawater were either incubated in the dark for 24 h or treated with 20 μM 3(3,4-dichlorophenyl) 1,1-dimethylurea to inhibit photosystem II dependent noncyclic electron transfer. In both cases, the addition of cadmium, as sulphate, increased respiration, with maximum stimulation occurring at approximately 15 mM; above this concentration respiration declined and at >21 mM inhibitory effects were significant. Dinitrophenol (DNP, 50 μM) and carbonyl cyanide m-chlorophenylhydrazone (CCCP, 10 μM) also uncoupled respiration of U. lactuca, and when these compounds were used in conjunction with Cd the effects were found to be additive. Oligomycin (12.5 μ mL^?1), which inhibits the mitochondrial ATPase, inhibited U. lactuca respiration but subsequent Cd addition (to 6 mM) resulted in a 2.5 × increase in respiration over control values. DNP had a similar effect when used with oligomycin. DNP and CCCP had a negligible effect on the respiration of KCN-treated discs but the addition of Cd restored respiration to control values, probably because of the formation of insoluble Cd(CN)₂. These results demonstrate that Cd can act as a respiratory uncoupler in U. lactuca.     

The regulation of mitochondrial respiration by opening of mKCa channels is age-dependent

The protective potency of ischemic preconditioning decreases with increasing age. A key step in ischemic preconditioning is the opening of mitochondrial Ca(2+) sensitive K(+) (mK(Ca)) channels, which causes mild uncoupling of mitochondrial respiration. We hypothesized that aging reduces the effects of mK(Ca) channel opening on mitochondrial respiration. We measured the effects of mK(Ca) channel opener NS1619 (30 microM) on mitochondrial respiration in isolated heart mitochondria from young (2-3 months) and old (22-26 months) Wistar rats. Oxygen consumption was monitored online after addition of 250 microM ADP (state 3 respiration), and after complete phosphorylation of ADP to ATP (state 4 respiration) in the presence or absence of the mK(Ca) channel blocker paxilline (5 microM). The respiratory control index (RCI) was calculated as state 3/state 4. In mitochondria from young rats, NS1619 increased state 4 respiration by 11.9+/-4.1% (mean+/-S.E.M.), decreased state 3 respiration by 7.6+/-2.5%, and reduced the RCI from 2.6+/-0.03 (control) to 2.1+/-0.06 (all P<0.05, n=12 for all groups). Paxilline blocked the effect of NS1619 on state 4 respiration (0.7+/-2.8%), but did not affect the decrease in state 3 respiration; paxilline blunted the decrease of RCI. In mitochondria from old rats, NS1619 had neither effect on state 4 (0.4+/-1.6%), and state 3 respiration (-7.4+/-1.5%), nor on RCI (3.0+/-0.13 vs. 3.2+/-0.11, n=12). Increasing age reduced the effects of mK(Ca) opening on mitochondrial respiration. This might be one underlying reason of the decreased protective potency of ischemic preconditioning in the aged myocardium.     

The effects of some nutritive antibiotics on the respiration of rat liver mitochondria.

(1) Seven antibiotics used as feed additives in animal breeding were investigated for their effects on isolated rat liver mitochondria. Three were found to interfere with mitochondrial energy metabolism. (2) Zinc-bacitracin completely inhibits mitochondrial respiration in the micromolar range. as do other inhibitors known to be highly effective against electron transport system. From studies of this antibiotic on the redox state of cytochromes, as measured by split beam spectra, it is concluded that the site of inhibition is located between cytochrome b and c₁ (antimycin A site). The effect is completely reversed by chelating agents, suggesting that Zn²⁺ ions are required for full activity of the cyclic peptide antibiotic. (3) Flavomycin, a polar glycolipid, linearly stimulates oxygen consumption of mitochondria under state 4 conditions in concentrations greater than 100 μmole/gram of protein. Lower concentrations of the antibiotic inhibits respiration of coupled as well as DNP- or FCCP-uncoupled mitochondria by about 70 per cent. While the uncouplinglike effect at high concentrations of the compound can be attributed to nonspecific surface activity which might facilitate proton conductance, the inhibitory activity seen at lower concentrations is assumed to be located near the second phosphorylation site of the respiratory chain. (4) The influence of chlortetracyclin (aureomycin) on mitochondrial activity was found to be dependent on the identity of the substrate. Succinate respiration was more sensitive to chlortetracyclin (CTC) addition by comparison with NAD-linked substrate oxidation. 45 μmole of CTC/gram of protein decreased succinate respiration to half maximal values, whereas glutamate plus malate or β-hydroxybutyrate respiration were inhibited by only 25 per cent. CTC partially inhibits the dehydrogenation of succinate by the succinate dehydrogenase. Uncoupling of oxidative phosphorylation completely abolished CTC-inhibited respiration of NAD-linked substrates, while succinate respiration remained inhibited by 25 per cent. The results of these experiments are discussed in terms of two sites of action for CTC, one located close to the phosphate carrier, while the second interferes with succinate dehydrogenase.     

Significance of Respiratory Dynamics of the Lung Tissue in Pulmonary Drug Permeation

There are two main objectives in this study. One is to investigate the roles of respiration, i.e., the dynamic change of the lung tissue, on drug transport across the air–blood barrier. The other is to establish the quantitative relationship between the effect of respiration and the physicochemical properties of drugs. To achieve these objectives, progesterone and a group of its hydroxy derivatives with varying hydrophilicity were used as the model drugs and their permeation kinetics studies were conducted under simulated respiratory dynamics using the in vitro pulmonary permeation system developed earlier in this laboratory. The physiological respiratory dynamics were successfully simulated and found to enhance significantly the transpulmonary permeation of progesterone and its hydroxy derivatives through bullfrog lung membrane, a model air–blood barrier. The extent of enhancement in the rate of drug permeation was observed to depend on the pattern of pressure application. As a pressure of the same magnitude was applied, the respiratory pressure was found to have a greater effect than a constant pressure. The results suggested that respiration has increased not only the surface area of lung membrane for permeation, but also dramatically affected the permeability of the lung membrane. Furthermore, the enhancement in permeation rate produced by respiration was observed to be in a linear correlation with the hydrophilicity of penetrants. The effect of variation in various respiratory parameters on drug permeation was also evaluated, and the results suggested that the dynamic change of the lung tissue generated by respiration plays an important role in the transpulmonary permeation of drugs. A possible mechanism involved could be attributed to the formation of transitional pores in lung membrane during the dynamic process of respiration. Therefore, it is necessary to bear in mind and take the respiratory dynamics into consideration for studying the transpulmonary permeation of drugs, especially when the drug has hydrophilic characteristics.     

Significance of respiratory dynamics of the lung tissue in pulmonary drug permeation

There are two main objectives in this study. One is to investigate the roles of respiration, i.e., the dynamic change of the lung tissue, on drug transport across the air-blood barrier. The others is to establish the quantitative relationship between the effect of respiration and the physicochemical properties of drugs. To achieve these objectives, progesterone and a group of its hydroxy derivatives with varying hydrophilicity were used as the model drugs and their permeation kinetics studies were conducted under simulated respiratory dynamics using the in vitro pulmonary permeation system developed earlier in this laboratory. The physiological respiratory dynamics were successfully simulated and found to enhance significantly the transpulmonary permeation of progesterone and its hydroxy derivatives through bullfrog lung membrane, a model air-blood barrier. The extent of enhancement in the rate of drug permeation was observed to depend on the pattern of pressure application. As a pressure of the same magnitude was applied, the respiratory pressure was found to have a greater effect than a constant pressure. The results suggested that respiration has increased not only the surface area of lung membrane for permeation, but also dramatically affected the permeability of the lung membrane. Furthermore, the enhancement in permeation rate produced by respiration was observed to be in a linear correlation with the hydrophilicity of penetrants. The effect of variation in various respiratory parameters on drug permeation was also evaluated, and the results suggested that the dynamic change of the lung tissue generated by respiration plays an important role in the transpulmonary permeation of drugs. A possible mechanism involved could be attributed to the formation of transitional pores in lung membrane during the dynamic process of respiration. Therefore, it is necessary to bear in mind and take the respiratory dynamics into consideration for studying the transpulmonary permeation of drugs, especially when the drug has hydrophilic characteristics.     

Nitrofurantoin inhibition of mouse liver mitochondrial respiration involving NAD-linked substrates

In our study, nitrofurantoin (NF) and nitrofurazone (NZ) inhibited respiration of isolated mouse (C57B/6J, adult, male) liver mitochondria. Other aromatic nitro compounds, nitroimidazole, metronidazole, and p-nitrobenzoic acid, did not have any significant effect. The primary site of activity for NF was complex I NADH-ubiquinone oxidoreductase mediated respiration, since only complex I substrates, glutamate, beta-hydroxybutyrate, and alpha-ketoglutarate-mediated respiration were decreased. Respiration supported by succinate, a complex II substrate, was not affected by any of the compounds. NF at a concentration of 50 microM decreased state 3 and dinitrophenol-uncoupled respiration to 28 +/- 1 and 25 +/- 5% of control, respectively, of mitochondria oxidizing glutamate. Studies with mitoplasts oxidizing glutamate showed that NF inhibited both state 3 and 4 respiration. The inhibition of state 3 was prevented by the simultaneous addition of superoxide dismutase (240 micrograms/ml) and catalase (200 micrograms/ml). These results suggest that the mitochondrion, in particular complex I of the electron transport system, is a target for NF toxicity. The effect on respiration may be mediated by NF redox cycling and the generation of reactive oxygen intermediates resulting in the interference of electron flow.     

Protective effects of befunolol on hypoxic respiration-induced alterations in myocardial energy metabolism of rats.

The present study was undertaken to elucidate beta-adrenoceptor blocking effects of befunolol (BFE 60, CAS 39543-79-8) on changes in the myocardial metabolites induced by hypoxic respiration. When rats were subjected to hypoxic respiration, a significant increase in heart rate (about 13% increase) and a slight decline in mean aortic blood pressure (about 12% decrease) were observed at 1 min and 6 min after the onset of hypoxic respiration. The hypoxic respiration also elicited decreases in the myocardial ATP and creatine phosphate levels (each 18% decrease) and increases in the myocardial lactate (13% increase) and cyclic-AMP (20% increase) levels. In contrast, these changes were never observed throughout hypoxic respiration when rats had been treated with both reserpine and alpha-methyl-p-tyrosine methylester 20 to 22 h before experiment, suggesting that these metabolic alterations are mediated through beta-adrenoceptor stimulation. These hypoxic respiration-induced hemodynamic and metabolic changes were found to be suppressed by treatment with 1 and 10 micrograms/kg befunolol or 10 micrograms/kg propranolol to an appreciable degree. The results demonstrate protective action of befunolol, like propranolol, on hypoxia-induced changes in the myocardial energy metabolism.     

Inhibition of rat heart mitochondrial respiration by cadmium chloride

Mitochondria were isolated from hearts obtained from adult male Sprague-Dawley rats by two-part differential centrifugation of heart homogenates. Time-dependent (0-120 sec) and concentration-dependent (0-10 microM CdCl2) effects of cadmium on pyruvate-malate-supported state 3 and state 4 respiration were measured in a constant temperature reaction chamber at 37 degrees C, according to established procedures. The ID50 for cadmium chloride on state 3 respiration was determined to be 4.2 microM. The inhibition produced by cadmium chloride in heart mitochondria was compared, using identical procedures, to the effects induced by two compounds, sodium atractyloside and potassium cyanide, which are known to alter mitochondrial respiration at specific sites. The calculated ID50 values for these agents in heart mitochondria were 1.8 and 16 microM, respectively. The concentration-dependent inhibition of mitochondrial respiration induced by either cadmium chloride or potassium cyanide was maintained in the presence of 50 microM carbonyl cyanide m-chlorophenylhydrazone (CCCP), a known uncoupling agent. In contrast, sodium atractyloside did not block the uncoupling effect of 50 microM CCCP. In addition cadmium chloride was also shown to inhibit CCCP-uncoupled mitochondrial respiration. The cadmium-induced inhibition of mitochondrial respiration was reversed partially by cysteine and completely by 2,3-dimercaptopropanol. The results of the present study indicate that, at all concentrations, cadmium chloride acted solely as an inhibitor of rat heart pyruvate-malate-supported mitochondrial respiration. These findings suggest a possible mechanism for the reported disturbances in myocardial metabolism and function that occur in conjunction with acute and chronic cadmium exposure in humans and experimental animals.     

The interaction of 2-phenylisatogen and menadione with rat liver mitochondrial NADH dehydrogenase

The effects of 2-phenylisatogen and menadione on some mitochondrial reactions have been studied. The respiration with NAD⁺-linked substrates was stimulated, but respiration with succinate was not affected. The stimulation of respiration was inhibited by p-hydroxymercuribenzoate, but not by other respiratory chain inhibitors. Potassium cyanide and sodium sulphide stimulated NADH oxidation in the presence of 2-phenylisatogen or menadione. 2-Phenylisatogen was found to be converted to 2-phenylindolone. It is concluded that both 2-phenylisatogen and manadione are acting as electron acceptors in the NADH dehydrogenase region of the respiratory chain. Possible mechanisms for the reaction and for the stimulation observed in the presence of cyanide are discussed.     

Mechanisms of petroleum hydrocarbon toxicity: studies on the response of rat liver mitochondria to Prudhoe Bay crude oil and its aliphatic, aromatic and heterocyclic fractions

The effect of Prudhoe Bay crude oil (PBCO) and its different fractions [aliphatic, aromatic, heterocyclic (NOS)] on the bioenergetic functions of isolated rat liver mitochondria were studied. A DMSO extract of PBCO inhibited state 3 respiration (in the presence of ADP) with either succinate or beta-hydroxybutyrate as substrate. The ascorbate-TMPD dependent state 3 respiration was not affected. Succinate dehydrogenase and beta-hydroxybutyrate dehydrogenase activities were also lost in the presence of the PBCO extract suggesting that inhibition of state 3 respiration may be due to blockage of the electron transport chain. Stimulation of state 4 respiration (in the absence of ADP) and of the oligomycin sensitive ATPase activity by the PBCO extract was observed. Fractionation of PBCO indicated that the aromatic fraction was mainly responsible for its inhibitory effects. By comparison, the heterocyclic fraction had weak inhibitory properties while the aliphatic fraction was essentially inactive. It is concluded that the aromatic components of PBCO inhibit mitochondrial respiration and oxidative phosphorylation mainly through impairment of the mitochondrial membrane and inhibition of beta-hydroxybutyrate and succinate dehydrogenase supported electron transfer activities of the respiratory chain.     

Induction of oxidative stress by endosulfan and protective effect of lipid-soluble antioxidants against endosulfan-induced oxidative damage

The toxic mechanism of endosulfan, a widely used organochlorine pesticide, was investigated in Saccharomyces cerevisiae and human cell lines. A concentration-dependent inhibition of cell growth was observed when S. cerevisiae was exposed to endosulfan, and its cytotoxicity (IC(50)) was found to be 49 microM and 86 microM in HepG2 and HeLa human cell lines, respectively. The treatment of S. cerevisiae with endosulfan resulted in oxidative damage, as demonstrated by thiobarbituric acid-reactive substance (TBARS) production, in a dose-dependent manner, and the growth inhibition was recovered by treatment with lipid-soluble antioxidants, such as alpha-tocopherol or beta-carotene, suggesting that endosulfan toxicity may be closely associated with endosulfan-induced reactive oxygen species (ROS) generation. The inhibition of cellular respiration by endosulfan treatment and the recovery of respiration activity by antioxidant treatment confirmed that endosulfan induces oxidative stress and inhibits respiration via ROS generation. These results suggest that unicellular yeast might provide a useful system for elucidating the toxicity of endosulfan.     

The fate of diesel hydrocarbons in soils and their effect on the germination of perennial ryegrass

Hydrocarbon contamination in soils may be toxic to plants and soil microorganisms and act as a source of groundwater contamination. The objective of this study was to evaluate the fate of diesel in soils with or without added nutrients. The soils examined either had or had not a previous history of hydrocarbon contamination. Particular aspects examined were soil respiration, changes in microbial population, breakdown of diesel hydrocarbons, and phytotoxicity to the germination of perennial ryegrass. Soil respiration was measured as evolved CO2. Bacterial population was determined as colony forming units in dilution plates and fungal activity was measured as hyphal length. The fate of individual hydrocarbons was determined by gas chromatography-mass spectrometry after extraction with dichloromethane. When diesel was added to soil with no previous history of hydrocarbon contamination at rates up to 50 mg/g, the respiration response showed a lag phase of 6 days and maximum respiration occurred at day 11. The lag phase was 2 days and maximum respiration occurred at day 3 in soil with a previous history of hydrocarbon contamination. After the peak, respiration decreased up to about 20 days in both soils. Thereafter, respiration become more or less constant but substantially greater than the control. N and P addition along with diesel did not reduce the lag phase but increased the respiration over the first 20 days of incubation. Diesel addition with or without N and P increased the bacterial population 10- to 100-fold but fungal hyphal length did not increase. Diesel addition at a rate of 136 mg/g did not increase the microbial population. Removal of inhibition to germination of perennial ryegrass was linked to the decomposition of nC10 and nC11 hydrocarbons and took from 11 to 30 days at diesel additions up to 50 mg/g depending on the soil. Inhibition to germination of perennial ryegrass persisted to more than 24 weeks at the 136 mg/g of diesel addition.     

Effects of dichlorodiphenyltrichloroethane and its analogues on rat liver mitochondria

The effects of dichlorodiphenyltrichloroethane (DDT) and sixteen analogues on respiration, swelling and latent ATPase of rat liver mitochondria were examined systematically. The compounds tested could be divided into four groups: DDT-type, DDE-type, kelthane-type and others by substituent groups on the ethane bridge of bis(p-chlorophenyl) ethane. Most compounds tested were shown to inhibit State 3 respiration. A linear relation was observed between the logarithms of the concentrations giving half-inhibition of State 3 respiration and the logarithms of the partition coefficients of the tested compounds. Four compounds of the kelthane type and chlorobenzilate stimulated State 4 respiration to the level of dinitrophenol-stimulated respiration. The compounds that have a hydroxy group on the ethane bridge-rapidly induced mitochondrial swelling, but DDT-type and DDE-type compounds induced swelling when the suspension contained 0.15 M KCl and 5 mM Tris-HCl. Latent ATPase of mitochondria was stimulated to different maximum levels by each of the tested compounds except DDA. The oligomycin-sensitive ATPase of submitochondria was inhibited by a series of kelthane-type compounds.