Mechanisms of petroleum hydrocarbon toxicity: functional changes in rat liver mitochondria after exposure to a Prudhoe Bay Crude Oil

Administration of a Prudhoe Bay Crude Oil (PBCO) to rats (5 ml/kg/day for 2 days) resulted in a 30-35% inhibition of liver mitochondrial state 3 respiration with beta-hydroxybutyrate or succinate as substrates. beta-Hydroxybutyrate dehydrogenase, succinate dehydrogenase, NADH oxidase and succinate oxidase activities were also decreased in PBCO-treated rats. A significant increase in latent ATPase activity was also observed. Our results suggest that PBCO exerts its effects on liver mitochondria through the inhibition of beta-hydroxybutyrate- and succinate-supported electron transfer activities and by impairment of the mitochondrial membrane.     

Inhibition of rat platelet aggregation by a Prudhoe Bay crude oil and its aliphatic,aromatic, and heterocyclic fractions

Wasjed platelets isolated from rats 24 hr after oral treatment with a Prudhoe Bay crude oil (PBCO) showed a substantial inhibition of aggregation induced by ADP, arachidonic acid, or epinephrine. In vitro addition of a dimethyl sulfoxide extract of PBCO or its aliphatic, aromatic, or heterocyclic fractions to washed platelets also resulted in an inhibition of aggregation. ADP release was inhibited in platelets to which an extract of PBCO or its fractions were added in vitro or in platelets isolated from rats treated in vivo with PBCO. Thromboxane B₂ release was increased in platelets isolated from rats intubated with PBCO or in platelets to which a dimethyl sulfoxide extract of the aromatic or heterocyclic fraction was added. However, thromboxane B₂ release was inhibited in platelets to which PBCO or the aliphatic fraction extracts were added. The results indicate that PBCO inhibits platelet aggregation presumably by bringing about alterations in the platelet plasma membrane. Inhibition of ADP release could contribute to the inhibition of aggregation but thromboxane B₂ is believed not to play a significant role.     

Effects of SC-26096 [2-methyl-3-oxo-2-azabicyclo (2.2.2.) octan-6-exo-yl 5-(4-biphenylyl)-3-methyl valerate], a new hypolipidemic agent,on mitochondrial respiration and oxidative phosphorylation

The effects of SC-26096 [2-methyl-3-oxo-2-azabicyclo (2.2.2.) octan-6-exo-yl 5-(4-biphenylyl)-3-methyl valerate] on respiration and oxidative phosphorylation of rat liver mitochondria were studied in vitro. It was found that SC-26096 is an inhibitor of electron transport. In preparations of intact and sonicated mitochondria, the drug effectively inhibited NAD-linked respiration at low concentration and inhibited succinate-linked respiration at much higher concentration. In intact mitochondria, dinitrophenol partially reversed the inhibition of NAD-linked state 3 repiration but did not reverse the inhibition of succinate-linked respiration. At the SC-26096 concentrations which were required to inhibit succinate oxidation. ADP:O ratio of phosphorylation site III was lowered and state 4 oxidation rate and ATPase activities were raised. At the drug concentrations which were tested. SC-26096 did not alter the ADP:O ratio of phosphorylation site II. 3-Hydroxybutyrate oxidation by mitochondria which were pretreated with SC-26096 remained inhibited after washing with 0.25 M sucrose, but this inhibition was relieved by adding albumin. The effects of SC-26096 were primarily dependent upon the ratio of drug:mitochondrial protein and not upon the initial molarity of the drug in the incubation medium. The results suggest that SC-26096 is bound to mitochondria in vitro and that the bound drug produces the mitochondrial effects. At least two sites of inhibition, within the respiratory chain, are indicated. One site lies between the interaction of NADH with NADH dehydrogenase and the point at which electrons from succinate oxidation enter the electron transport system. A second, less sensitive, site lies between the interaction of succinate with succinate dehydrogenase and cytochrome c. A third inhibitory site appears to be present in the site I phosphorylation system at or distal to the dinitrophenol-sensitive site.     

Effectiveness of a Prudhoe Bay crude oil and its aliphatic,aromatic and heterocyclic fractions in inducing mortality and aryl hydrocarbon hydroxylase in chick embryo in ovo

Prudhoe Bay crude oil (PBCO) and its aliphatic, aromatic and heterocyclic fractions were tested on the developing chick embryo for (i) embryotoxicity (ii) their ability to induce hepatic and renal cytochrome P450 levels as well as hepatic, renal and pulmonary aryl hydrocarbon hydroxylase activities. On the basis of its concentration in PBCO, the aromatic fraction was responsible for most of the embryotoxicity as well as for the enzyme inducing ability. The NOS fraction constituted less than 7% (w/v) of PBCO but, on a weight equivalent basis, was roughly as potent as the aromatic fraction in causing embryotoxicity and in inducing cytochrome P450 levels and aryl hydrocarbon hydroxylase. The aliphatic fraction was found to be essentially inactive. The results are consistent with the concept that elevation of aryl hydrocarbon hydroxylase levels by certain components of PBCO may lead to increased embryotoxicity.Abbreviations Used BP benzo[a]pyrene - BPH benzo[a]pyrene 3-hydroxylase - ER 7-ethoxyresorufin - EROD 7-ethoxyresorufin O-deethylase - NOS nitrogen, oxygen and sulfur containing heterocyclic fraction - PBCO Prudhoe Bay crude oil     

Effect of Stryphnodendron adstringens (barbatimão) on energy metabolism in the rat liver

The action of a barbatim?o extract on hepatic energy metabolism was investigated using isolated mitochondria and the perfused rat liver. In mitochondria the barbatim?o extract inhibited respiration in the presence of ADP and succinate. Stimulation occurred, however, after ADP phosphorylation (state IV respiration). The ADP/O and respiratory control ratios were reduced. The activities of succinate-oxidase, NADH-oxidase and the oxidation of ascorbate were inhibited. The ATPase of intact mitochondria was stimulated, but the ATPases of uncoupled and disrupted mitochondria were inhibited. In perfused livers the extract caused stimulation of oxygen consumption, inhibition of gluconeogenesis and stimulation of glycolysis. Glucose release due to glycogenolysis was stimulated shortly after the introduction of the extract, but inhibition gradually developed as the infusion was continued. Apparently the barbatim?o extract impairs the hepatic energy metabolism by three mechanisms: (1) uncoupling of oxidative phosphorylation, (2) inhibition of mitochondrial electron transport, and (3) inhibition of ATP-synthase.     

Effects of clofibrate, in vitro, on mitochondrial respiration and oxidative phosphorylation

The effects of clofibrate on mitoehondrial respiration and oxidative phosphorylation were studied in vitro. Clofibrate inhibited state 3 oxidation, stimulated state 4 oxidation and lowered ADP: O ratio when 3-hydroxybutyrate, succinate and ascorbate plus tetramethylphenylenediamine (TMPD) were used as substrates. The inhibitions of state 3 oxidation were not reversed by dinitrophenol. In mitochondrial fragments, both NADH- and succinate-linked oxidations were inhibited by clofibrate but ascorbate plus TMPD oxidation was not. A higher concentration of clofibrate was required to inhibit succinate oxidation than NADH oxidation. In mitochondrial fragments, 50 per cent inhibitions of respiration, with NADH and succinate as substrates, were elicited with concentrations of 0·09 and 0·4 μmoles of clofibrate/mg of protein, respectively. It was suggested that there are at least two distinct sites at which clofibrate can inhibit respiration. One site exists between the interaction of NADH with NADH dehydrogenase and the point at which electrons from succinate oxidation enter the electron transport chain. Another, less sensitive site, exists between the interaction of succinate with suecinate dehydrogenase and cytochrome c.     

Effect of ochratoxin A on rat liver mitochondrial respiration and oxidative phosphorylation

The in vitro effects of ochratoxin A on the membrane structure and bioenergetic functions of rat liver mitochondria were studied. It was found that when the toxin was added to the assay medium the respiratory control of the isolated mitochondria was decreased as the concentration of the toxin increased. The mitochondrial respiration was gradually uncoupled by the toxin when its concentration was raised above 1.2 X 10(-6) M, and became fully uncoupled at 6.2 X 10(-4) M. The oxidative phosphorylation was not damaged until the toxin concentration was higher than 9.3 X 10(-5) M. On the other hand, ochratoxin A inhibited the electron transfer functions of the mitochondria. At the concentration above 1.0 X 10(-4) M, ochratoxin A inhibited the succinate dehydrogenase, succinate-cytochrome c reductase, and succinate oxidase activities of the respiratory chain. Fifty percent of succinate-cytochrome c reductase and succinate oxidase activity was lost in the presence of 8.0 X 10(-4) and 6.2 X 10(-4) M ochratoxin A, respectively. The inhibition kinetic studies revealed that ochratoxin A is an uncompetitive inhibitor of both succinate-cytochrome c reductase and succinate dehydrogenase, and the inhibition constants for the 2 enzyme activities were estimated to be 4.4 X 10(-4) and 2.2 X 10(-4) M, respectively. However, the toxin did not inhibit either cytochrome oxidase or NADH dehydrogenase activity of the mitochondrial respiratory chain. It is thus concluded that ochratoxin A exerts its effect on the mitochondrial respiration and oxidative phosphorylation through the impairment of the mitochondrial membrane and inhibition of the succinate-supported electron transfer activities of the respiratory chain.     

Effects of indomethacin on respiration and the alpha-glycerolphosphate shuttle in rat kidney mitochondria.

The anti-inflammatory drug indomethacin was found to stimulate State 4 respiration in rat kidney mitochondria, indicating an uncoupler activity which was maximal at a concentration of 0.1–0.2mM. Indomethacin also inhibited State 3 respiration in mitochondria oxidizing glutamate or succinate, but not in mitochondria oxidizing ascorbate together with tetramethylphenylene diamine. This inhibition was not relieved by 2,4-dinitrophenol and suggested that indomethacin directly inhibited electron transport along the respiratory chain at a point prior to cytochrome c.At concentrations one order of magnitude lower than that required for substantial uncoupling or respiratory inhibition, indomethacin severely restricted the transfer of reducing equivalents from extramitochondrial NADH to the respiratory chain via a reconstructed α-glycerolphosphate shuttle. It was found that the drug exerted a strong inhibitory effect on mitochondrial α-glycerolphosphate dehydrogenase activity, and that this inhibition was relatively specific since indomethacin had little effect on the activity of succinate dehydrogenase, another FAD-linked enzyme.The inhibition of the α-glycerolphosphate shuttle is discussed in relation to the previously observed effects of indomethacin on glucose metabolism in isolated rat kidney tubules.     

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.     

Mode of action of anhydrofulvic acid against Candida utilis ATCC 42402 under acidic condition.

The mode of action of anhydrofulvic acid against Candida utilis ATCC 42402 was investigated under acidic conditions. Anhydrofulvic acid inhibited the incorporation of radioactive precursors into DNA, RNA, protein and lipid fractions. Although it did not induce leakage of intracellular materials from the treated cells, it had inhibitory effects on both endogenous and exogenous cellular respiration. Moreover, it inhibited mitochondrial respiration of Candida utilis ATCC 42402 using both succinate and cytochrome c as respiratory substrates, but not using NADH. Unexpectedly, the inhibition against isolated mitochondria was observed at pH 7.0. These results suggested that the action site against the respiratory inhibition of anhydrofulvic acid might be involved in succinate dehydrogenase, complex II in the mitochondrial electron transport chain of the yeast cells. Judging from the inhibitory effect of anhydrofulvic acid on mitochondria detected at pH 7.0, it was postulated that the antifungal activity at a low pH level might depend on the elevation of drug permeability to the cell membrane under acidic conditions.     

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.     

4-Chloro-4'-biphenylol as an uncoupler and an inhibitor of mitochondrial oxidative phosphorylation

4-Chloro-4'-biphenylol (4'-OH-4-CB), a metabolite of 4-chlorobiphenyl (4-CB), stimulated state 4 respiration and released oligomycin-inhibited state 3 respiration of rat liver mitochondria with succinate as the respiratory substrate. When glutamate/malate and beta-hydroxybutyrate were used as the substrates, however, 4'-OH-4-CB was ineffective on these parameters. This indicates that 4'-OH-4-CB uncouples oxidative phosphorylation with succinate, but not with glutamate/malate and beta-hydroxybutyrate. 4'-OH-4-CB severely inhibited 2,4-dinitrophenol (DNP)-stimulated respiration with glutamate/malate (ID50, 25 microM) and beta-hydroxybutyrate (ID50, 32 microM) because of the blockade of electron transfer between NADH and CoQ span, masking the uncoupling action of 4'-OH-4-CB. On the other hand, the inhibition of the respiration with succinate was only apparent at high 4'-OH-4-CB concentrations (ID50, 260 microM). 4'-OH-4-CB also inhibited the oxidation of NADH in submitochondrial particles (ID50, 35 microM). State 3 respiration was more intensely inhibited by 4'-OH-4-CB in the presence of either glutamate/malate (ID50, 23 microM) or beta-hydroxybutyrate (ID50, 26 microM) than that in the presence of succinate (ID50, 220 microM). Thus, 4'-OH-4-CB acts as both an uncoupler and an inhibitor of oxidative phosphorylation. The overall in vitro effect is to prevent ATP synthesis, which may be an important factor in the mechanism underlying the toxicity of 4-CB.     

Effects of guanethidine on electron transport and proton movements in rat heart, brain and liver mitochondria

Guanethidine at 5-25 mM concentrations was found to induce up to 79% inhibition of ADP-stimulated (state III) oxygen consumption in isolated rat heart, brain or liver mitochondria, when the added substrate was glutamate or succinate, but the inhibition was considerably lower (24% or less) when respiration was supported by ascorbate plus tetramethylphenylenediamine (TMPD). Comparable results were seen regarding ADP-stimulated proton uptake, where even greater inhibition (up to 94% with glutamate or succinate, but not ascorbate plus TMPD) was found. Similar but somewhat less marked effects were also seen in resting (state IV) respiration and on the acceptor control ratio (state III/state IV respiration). 2,4-Dinitrophenol was unable to relieve guanethidine-induced inhibition of electron transport. These results indicate that guanethidine inhibits primarily mitochondrial electron transport itself, and that the site where such inhibition is more marked is located in the span between ubiquinone and cytochrome c of the respiratory chain. It is, therefore, suggested that active guanethidine uptake by noradrenergic neurons can lead to a high drug concentration in their cytoplasm and hence to mitochondrial alterations that can contribute to the pharmacological effect of this drug. Our results demonstrate the interaction between guanethidine and the electron transport chain of mitochondria derived from different tissues and, therefore, support this hypothesis.     

Further studies concerning the effects of clofibrate on respiration and oxidative phosphorylation of rat liver mitochondria

Clofibrate, administered in vitro, inhibited rat liver mitochondrial respiration at two sites within the respiratory chain. One site was between the interaction of NADH with NADH dehydrogenase and the point at which electrons from succinate oxidation enter the electron transport chain; another, less sensitive site, was between the interaction of succinate with succinate dehydrogenase and cytochrome c. In addition to these specific sites, clofibrate inhibited respiration by causing a depletion of pyridine nucleotides that was accompanied or followed by large-amplitude, non-energy-linked swelling. Clofibrate uncoupled oxidative phosphorylation at coupling sites II and III but not at site I. The concentrations required to cause loss of pyridine nucleotides were lower than those required to inhibit at the specific sites. p-Chlorophenoxyisobutyrate (CPIB) also inhibited succinate and β-hydroxybutyrate-linked respiration, and uncoupled oxidative phosphorylation, but at much higher concentrations (50 per cent inhibition of β-hydroxybutyrate oxidation at about 3·7 μmoles/mg of protein) than were required of clofibrate (50 per cent inhibition of β-hydroxybutyrate oxidation at about 0·17 μmole/mg of protein). Clofibrate administration to rats (100 and 300 mg/kg p.o. daily for 1 week) lowered serum lipid levels and increased the liver size, the amount of mitochondrial protein/g of liver, and the oxygen consumption of liver slices. However, mitochondria, isolated from livers of the treated rats, respired normally. A single administration of clofibrate (100 or 300 mg/kg, p.o.) did not affect liver slice respiration.     

A biochemical basis for 1,2-dibromo-3-chloropropane-induced male infertility: inhibition of sperm mitochondrial electron transport activity

A rapid decrease in male fertility in laboratory animals exposed to 1,2-dibromo-3-chloropropane (DBCP) has been suggested to be due, in part, to a postglycolytic inhibition of sperm carbohydrate metabolism. The present studies were performed to identify the specific site of DBCP-induced inhibition of intermediary metabolism. 14CO2 generation by epididymal sperm, isolated from Fischer 344 rats, was measured using radiolabeled tricarboxylic acid (TCA) cycle intermediates: acetyl CoA, citrate, alpha-ketoglutarate, and succinate. There was 0-28% inhibition of CO2 generation after addition of 0.5 mM DBCP and 81-98% inhibition with 3 mM DBCP, with all four substrates. The activities of alpha-ketoglutarate dehydrogenase, pyruvate dehydrogenase, malate dehydrogenase, and lactate dehydrogenase were not inhibited by DBCP. Since the DBCP-induced inhibition of metabolism of different substrates to CO2 was similar, and since DBCP did not inhibit enzyme activities of glycolysis or the TCA cycle, a common site of inhibition was suspected. In evaluations of mitochondrial electron transport chain activity, DBCP (3 mM) inhibited oxygen consumption resulting from metabolism of endogenous substrates plus alpha-ketoglutarate or malate by about 80%. When succinate, an FAD-dependent oxidation, was used as a substrate, oxygen consumption was not inhibited by DBCP. It is concluded that DBCP inhibits sperm carbohydrate metabolism at the NADH dehydrogenase step in the mitochondrial electron transport chain.     

The effects of cadmium on succinate and NADH-linked substrate oxidations in rat hepatic mitochondria

Low concentrations of cadmium (3.3-40 microM) inhibited State 3 NADH-linked respiration in rat hepatic mitochondria, but failed to release oligomycin (1 microgram) inhibited State 3 respiration, or to significantly change the State 4 rate. In the presence of succinate, 40 microM cadmium inhibited State 3 respiration by 89%, while concentrations between 3.3 and 13.3 microM stimulated State 4 respiration. Higher concentrations caused marked inhibition. In the presence of succinate, cadmium released oligomycin inhibited State 3 respiration. Cadmium (0.001-1.0 mM) did not stimulate mitochondrial ATPase activity or inhibit ferricyanide reduction, but stimulated NAD+ linked mitochondrial dehydrogenase activities and NADH oxidation. These results indicate that cadmium interacts with either the NADH dehydrogenase complex or other NADH-dependent enzymes and not solely by an uncoupling action.     

Effect of an inhibitor of platelets aggregation, ticlopidine of energy transduction of rat liver mitochondria in vitro.

The new platelet aggregation inhibitor, ticlopidine (5-(2-chloro-benzyl)-4-5-6-7-tetrahydrothieno (3-2-c) pyridine is an inhibitor of energy transduction in isolated rat liver mitochondria. Results show that the compound below concentrations of 100 nmoles/mg of mitochondrial proteins stimulates state 4 oxidation, inhibits state 3 oxidation (except with ascorbate as substrate), decreases ADP-O ratio and respiratory control, and stimulates the latent ATPase activity. This potent stimulation. ten and a half times greater than control, is inhibited by oligomycin (6 μg/mg protein) and is dependent on exogenous Mg²⁺. The inhibition of state 3 oxidation, with glutamate-malate and succinate as substrates. is not reversed by 2–4 DNP and ticlopidine inhibits in a same extent the ADP and DNP-stimulated respiration. In the presence of succinate uncoupled respiration is more susceptible to inhibition by ticlopidine with sonic submitochondrial particles (I₅₀ = 370 nmoles/mg protein) than with intact mitochondria (I₅₀ = 75 nmoles/mg protein). Ticlopidine could act as an uncoupler and as an inhibitor of the electron transport chain, between NADH-dehydrogenase and cytochrome c, and as an inhibitor of the succinate translocation. It is suggested that the blocking effect on mitochondrial energy might play some part in the ticlopidine anti-aggregating properties.     

Inhibition of mouse liver respiration by Chelidonium majus isoquinoline alkaloids

The alkaloids from Chelidonium majus L. which had a significant inhibitory effect in mitochondrial respiration were those which contain a positive charge due to a quaternary nitrogen atom, i.e., chelerythrine, sanguinarine, berberine and coptisine, both with malate+glutamate or with succinate as substrates. When malate+glutamate was used as substrate, chelerythrine and berberine, which contain methoxy groups, were particularly more active, since they had a strong effect even at low concentrations. In submitochondrial particles, berberine and coptisine had a marked inhibitory effect on NADH dehydrogenase activity but practically no effect on succinate dehydrogenase activity, whereas chelerythrine and sanguinarine inhibited more strongly succinate dehydrogenase than NADH dehydrogenase, which is in agreement with the results found for mitochondrial respiration. Protopine and allocryptopine, which did not inhibit mitochondrial respiration, strongly inhibited NADH dehydrogenase in submitochondrial particles, but had no effect on succinate dehydrogenase activity.     

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.     

In vitro and in vivo effect of methyl isocyanate on rat liver mitochondrial respiration.

Previous work has shown that irrespective of the route of exposure methyl isocyanate (MIC) caused acute lactic acidosis in rats (Jeevaratnam et al., Arch. Environ. Contam. Toxicol. 19, 314-319, 1990) and the hypoxia was of stagnant type due to tissue hypoperfusion resulting from hypovolemic hypotension in rabbits administered MIC subcutaneously (Jeevarathinam et al., Toxicology 51, 223-240, 1988). The present study was designed to investigate whether MIC could induce histotoxic hyperoxia through its effects on mitochondrial respiration. Male Wistar rats were used for liver mitochondrial and submitochondrial particle (SMP) preparation. Addition of MIC to tightly coupled mitochondria in vitro resulted in stimulation of state 4 respiration, abolition of respiratory control, decrease in ADP/O ratio, and inhibition of state 3 oxidation. The oxidation of NAD(+)-linked substrates (glutamate + malate) was more sensitive (five- to sixfold) to the inhibitory action of MIC than succinate while cytochrome oxidase remained unaffected. MIC induced twofold delay in the onset of anerobiosis, and cytochrome b reduction in SMP with NADH in vitro confirms inhibition of electron transport at complex I region. MIC also stimulated the ATPase activity in tightly coupled mitochondria while lipid peroxidation remained unaffected. As its hydrolysis products, methylamine and N,N'-dimethylurea failed to elicit any change in vitro; these effects reveal that MIC per se acts as an inhibitor of electron transport and a weak uncoupler. Administration of MIC sc at lethal dose caused a similar change only with NAD(+)-linked substrates, reflecting impairment of mitochondrial respiration at complex I region and thereby induction of histotoxic hypoxia in vivo.