Mitochondrial Swelling Induced by Glutathione

Reduced glutathione, in concentrations approximating those occurring in intact rat liver, causes swelling of rat liver mitochondria in vitro which is different in kinetics and extent from that yielded by L-thyroxine. The effect is also given by cysteine, which is more active, and reduced coenzyme A, but not by L-ascorbate, cystine, or oxidized glutathione. The optimum pH is 6.5, whereas thyroxine-induced swelling is optimal at pH 7.5. The GSH-induced swelling is not inhibited by DNP or dicumarol, nor by high concentrations of sucrose, serum albumin, or polyvinylpyrrolidone, in contrast to thyroxine-induced swelling. ATP inhibits the GSH swelling, but ADP and AMP are ineffective. Mn^-+ is a very potent inhibitor, but Mg⁺⁺ is ineffective. Ethylenediaminetetraacetate is also an effective inhibitor of GSH-induced swelling. The respiratory inhibitors amytal and antimycin A do not inhibit the swelling action of GSH, but cyanide does; these findings are consistent with the view that the oxidation-reduction state of the respiratory chain between cytochrome c and oxygen is a determinant of GSH-induced swelling. Reversal of GSH-induced swelling by osmotic means or by ATP in KCl media could not be observed. Large losses of nucleotides and protein occur during the swelling by GSH, suggesting that the action is irreversible. The characteristically drastic swelling action of GSH could be prevented if L-thyroxine was also present in the medium.     

Electron microscope study of lens fibers

The in vitro swelling action of L-thyroxine on rat liver mitochondria as examined photometrically represents an acceleration of a process which the mitochondria are already inherently capable of undergoing spontaneously, as indicated by the identical kinetic characteristics and the extent of thyroxine-induced and spontaneous swelling, the nearly identical pH dependence, and the fact that sucrose has a specific inhibitory action on both types of swelling. However, thyroxine does not appear to be a "catalyst" or coenzyme since it does not decrease the temperature coefficient of spontaneous swelling. The temperature coefficient is very high, approximately 6.0 near 20 degrees . Aging of mitochondria at 0 degrees causes loss of thyroxine sensitivity which correlates closely with the loss of bound DPN from the mitochondria, but not with loss of activity of the respiratory chain or with the efficiency of oxidative phosphorylation. Tests with various respiratory chain inhibitors showed that the oxidation state of bound DPN may be a major determinant of thyroxine sensitivity; the oxidation state of the other respiratory carriers does not appear to influence sensitivity to thyroxine. These facts and other considerations suggest that a bound form of mitochondrial DPN is the "target" of the action of thyroxine. The thyroxine-induced swelling is not reversed by increasing the osmolar concentration of external sucrose, but can be "passively" or osmotically reversed by adding the high-particle weight solute polyvinylpyrrolidone. The mitochondrial membrane becomes more permeable to sucrose during the swelling reaction. On the other hand, thyroxine-induced swelling can be "actively" reversed by ATP in a medium of 0.15 M KCl or NaCl but not in a 0.30 M sucrose medium. The action of ATP is specific; ADP, Mn(++), and ethylenediaminetetraacetate are not active. It is concluded that sucrose is an inhibitor of the enzymatic relationship between oxidative phosphorylation and the contractility and permeability properties of the mitochondrial membrane. Occurrence of different types of mitochondrial swelling, the intracellular factors affecting the swelling and shrinking of mitochondria, as well as the physiological significance of thyroxine-induced swelling are discussed.     

Protective effect of glucose-cysteine adduct on thein situ perfused rat liver

Summary Insitu perfusion of rat liver was performed with a medium containing glucose-cysteine adduct [2-(D-gluco-pentahydroxypentyl) thiazolidine-4-carboxylic acid, glc-cys] and its effect on glutathione (GSH) and ATP levels and bile production was examined. The GSH content in the liver was maintained at the original level during perfusion with 1 mM glc-cys for 2h, while it decreased significantly in the absence of glc-cys. After 4h of perfusion without glc-cys, ATP content and bile production decreased significantly besides the decrease in GSH content, but they were maintained at the original levels with glc-cys. When the perfusion was performed with the liver of rats injected with diethyl maleate (DEM), the GSH level, which was decreased to 6.0% of the control by DEM injection, was restored to 22.6% of the original level by perfusion with 2mM glc-cys for 30 min. Data indicate that glccys is a cysteine prodrug with protective action on the liver.     

Interaction of reduced glutathione with bovine brain tubulin

Incubation of phosphocellulose-purified tubulin with GSH at 30 degrees C results in an inhibition of colchicine binding activity. GSSG has a protective effect against the GSH-induced loss of colchicine-binding. Incubation of tubulin with GSH at 30 degrees C results in the formation of abnormal tubulin polymers which are insensitive to cold. Such aggregation is insensitive to antimicrotubular drugs. Aggregation is inhibited by GSSG but not by DTT or mercaptoethanol. GSH-induced aggregation is very sensitive to the ionic strength of the assembly medium; both the aggregation and colchicine binding inhibition induced by GSH are inhibited at higher ionic strength. These results indicate a very complex interaction of GSH with tubulin.     

The maturation of the inner membrane of foetal rat liver mitochondria.

A new method was devised for the isolation of foetal and neonatal rat lvier mitochondria, giving higher yields than conventional methods. 2. During development from the perinatal period to the mature adult, the ratio of cytochrome oxidase/succinate-cytochrome c reductase changes. 3. The inner mitochondrial membrane of foetal liver mitochondria possesses virtually no osmotic activity; the permeability to sucrose decreases with increasing developmental age. 4. Foetal rat liver mitochondria possess only marginal respiratory control and do not maintain Ca2+-induced respiration; they also swell in respiratory-control medium in the absence of substrate. ATP enhances respiratory control and prevents swelling, adenylyl imidodiphosphate, ATP+atractyloside enhance the R.C.I. (respiratory control index), Ca2+-induced respiratory control and prevent swelling, whereas GTP and low concentrations of ADP have none of these actions. It is concluded that the effect of ATP depends on steric interaction with the inner mitochondrial membrane. 5. When 1-day pre-partum foetuses are obtained by Caesarean section and maintained in a Humidicrib for 90 min, mitochondrial maturation is "triggered", so that their R.C.I. is enhanced and no ATP is required to support Ca2+-dependent respiratory control or to inhibit mitochondrial swelling. 6. It is concluded that foetal rat liver mitochondria in utero do not respire, although they are capable of oxidative phosphorylation in spite of their low R.C.I. The different environmental conditions which the neonatal rat encounters ex utero enable the hepatic mitochondria to produce ATP, which interacts with the inner mitochondrial membrane to enhance oxidative phosphorylation by an autocatalytic mechanism.     

Iron stimulation of free radical-mediated porphyrinogen oxidation by hepatic and renal mitochondria

Mitochondria from rat liver and kidney catalyze oxidation of uroporphyrinogen in the presence of NADH or succinate and the respiratory chain inhibitor, NaN3. The rate of porphyrinogen oxidation was substantially accelerated when iron as Fe+3-EDTA was added to reaction mixtures. This effect was partially attenuated by catalase, reduced glutathione (GSH) and other free radical scavengers. These results suggest that iron stimulates free radical-mediated porphyrinogen oxidation by tissue mitochondria under conditions of perturbed mitochondrial respiratory function. These observations suggest a mechanism by which iron could contribute to excess porphyrin excretion in various inherited or chemically-induced porphyrias.     

Paracetamol-stimulated lipid peroxidation in isolated rat and mouse hepatocytes

Treatment of isolated hepatocytes from 3-methylcholanthrene induced rats with 1 mM paracetamol has been found to greatly decrease cellular reduced glutathione (GSH) content and to promote lipid peroxidation, evaluated as malonaldehyde (MDA) production and conjugated diene absorbance. A similar dosing of hepatocytes from phenobarbital-induced or normal rats is ineffective in that respect. On the other hand, the aspecific stimulation of the cytochrome P-450-mediated paracetamol activation due to acetone addition further increases GSH depletion as well as MDA production.Isolated hepatocytes with basal low GSH content are also more susceptible to paracetamol-induced lipid peroxidation, indicating that the rate of the drug metabolism and the cellular GSH content are critical factors in the determination of such peroxidative attack.In isolated mouse liver cells paracetamol does not require preliminary cytochrome P-450 induction to stimulate MDA formation, even at concentrations ineffective in rat cells.However, 5 mM paracetamol, despite a great depletion of cellular GSH content, does not promote MDA formation either in the rat or in the mouse hepatocytes. This effect may be due to the ability of paracetamol to scavenge lipid peroxides under defined conditions, as tested in various lipid peroxidizing systems.Membrane leakage of lactate dehydrogenase (LDH) is evident in paracetamol treated cells undergoing lipid peroxidation, but not when MDA formation is inhibited by high doses of the drug or by addition of antioxidants such as α-tocopherol and diphenylphenylenediamine (DPPD).Nevertheless in these conditions the covalent binding of activated paracetamol metabolites is not affected, suggesting that lipid peroxidation might play a role in the pathogenesis of liver damage following paracetamol overdose.     

Effect of Dequalinium on Respiration and the Inner Membrane Permeability of Rat Liver Mitochondria

The effect of the lipophilic penetrating cation dequalinium on rat liver mitochondria was studied. It was found that dequalinium dose-dependently inhibits the respiration rate of rat liver mitochondria in ADP-stimulated (V₃) and DNP-stimulated (uncoupled) states. This can be due to the fact that dequalinium is a potent inhibitor of complex III of the mitochondrial respiratory chain. It was shown that dequalinium induces a high-amplitude swelling of rat liver mitochondria. The dequalinium-induced swelling of the organelles depends on the presence of inorganic phosphate in the incubation medium: in the absence of phosphate or in the presence of the phosphate carrier inhibitor mersalyl in the phosphate-containing medium, no swelling of the mitochondria was observed. At low concentrations of dequalinium (≤10 μM), this swelling is inhibited by cyclosporin A, an inhibitor of the mitochondrial permeability transition pore. At the same time, at high concentrations of dequalinium (>10 μM), cyclosporin A becomes ineffective. It was found that in the presence of dequalinium the rate of the H₂O₂ production increased in rat liver mitochondria. Possible mechanisms of toxic effect of dequalinium chloride are discussed.     

Alloxan- and glutathione-dependent ferritin iron release and lipid peroxidation

The diabetogenic action of alloxan is believed to involve oxygen free radicals and iron. Incubation of glutathione (GSH) and alloxan with rat liver ferritin resulted in release of ferrous iron as assayed by spectrophotometric detection of ferrous-bathophenanthroline complex formation. Neither GSH nor alloxan alone mediated iron release from ferritin. Superoxide dismutase (SOD) and catalase did not affect initial rates of iron release whereas ceruloplasmin was an effective inhibitor of iron release. The reaction of GSH with alloxan resulted in the formation of the alloxan radical which was detected by ESR spectroscopy and by following the increase in absorbance at 310nm. In both instances, the addition of ferritin resulted in diminished alloxan radical detection. Incubation of GSH, alloxan, and ferritin with phospholipid liposomes also resulted in lipid peroxidation. Lipid peroxidation did not occur in the absence of ferritin. The rates of lipid peroxidation were not affected by the addition of SOD or catalase, but were inhibited by ceruloplasmin. These results suggest that the alloxan radical releases iron from ferritin and indicates that ferritin iron may be involved in alloxan-promoted lipid peroxidation.     

DNA biosynthesis in rat liver mitochondria: Inhibition by sulfhydryl compounds and stimulation by cytoplasmic proteins

The sulfhydryl compounds, 2-mercaptoethanol, dithiothreitol, cysteine. and glutathione inhibit the incorporation of [³H]dTTP or [³H]dATP into mitochondrial DNA by rat liver mitochondria in vitro. The lack of inhibition by non-SH-containing analogs indicates that the SH group is responsible for the inhibition.The inhibition does not result from an effect of the sulfhydryl compounds on precursor permeability, ATP formation, or respiration, or the action of the thiol on the outer mitochondrial membrane. An intact inner membrane is not required for the action of the inhibitor. Furthermore, SH compounds do not appear to exert their effect by activation of a mitochondrial nuclease, chemical breakdown of high molecular-weight mitochondrial DNA or dissociation of membrane-bound DNA from the inner mitochondrial membrane. Incorporation of labeled precursor into DNA by mitochondrial DNA polymerase, when removed from the inner mitochondrial membrane, is not inhibited by SH compounds.Cytoplasmic extracts prepared from rat and mouse tumors and 22-h regenerating rat liver contain a protein(s) not detectable in normal rat liver which can reverse the inhibition by SH compounds of the synthesis of mitochondrial DNA in rat liver mitochondria in vitro.More importantly, when the stimulatory protein(s) is partially purified by affinity chromatography on DNA-cellulose, it is possible to demonstrate that this protein(s) also stimulates the synthesis of mitochondrial DNA by normal rat liver mitochondria in vitro in the absence of the sulfhydryl inhibitor.     

ATP and GSH dependence of MRP1-mediated outward translocation of phospholipid analogs in the human erythrocyte membrane

The active outward translocation of phospholipid analogues from the inner to the outer membrane leaflet of human erythrocytes by the multi-drug resistance protein MRP1 (ABCC1) depends on intracellular reduced glutathione (GSH). Entrapment of ATP and increasing amounts of GSH inside resealed ghosts prepared from erythrocytes resulted in an up to six-fold increase of the translocation rate. Entrapped oxidized glutathione (GSSG) acted inhibitory but produced stimulation after addition of the disulphide-reducing reagent dithioerythritol. Modification of GSH by esterification of the C-terminal carboxylate of Gly, removal of the N-terminal Glu or substitution of the SH group by an anionic S-dicarboxyethyl or sulphonate group abolished stimulation. The effect of S-alkylation of GSH depended on the length of the alkyl group. S-methyl GSH was somewhat more effective than GSH, but maximal stimulation was similar. S-butyl GSH acted poorly stimulatory while S-hexyl GSH was essentially ineffective. Analyses of the kinetic data of translocation revealed K(m) values for GSH and methyl-GSH of respectively 7.4 +/- 2.4 and 4.9 +/- 1.1 mmol l(-1). At high GSH levels and defined constant ATP levels using an ATP-regenerating system, the Km for ATP of the outward translocation was 0.16 +/- 0.02 mmol l(-1). In the same system lacking GSH, the Km for ATP of the inward translocation by the aminophospholipid flippase was 0.53 +/- 0.23 mmol l(-1).     

Role of a partially purified glutathione S-transferase from rat liver nuclei in the inhibition of nuclear lipid peroxidation

Glutathione protects isolated rat liver nuclei against lipid peroxidation by inducing a lag period prior to the onset of peroxidation. This GSH-dependent protection was abolished by exposing isolated nuclei to the glutathione S-transferase inhibitor S-octylglutathione. In incubations containing 0.2 mM S-octylglutathione, the GSH-induced lag period was reduced from 30 to 5 min. S-Octylglutathione (0.2 mM) also completely inhibited nuclear glutathione S-transferase activity and reduced glutathione peroxidase activity by 85%. About 70% of the glutathione S-transferase activity associated with isolated nuclei was solubilized with 0.3% Triton X-100. This solubilized glutathione S-transferase activity was partially purified by utilizing a S-hexylglutathione affinity column. The partially purified nuclear glutathione S-transferase exhibited glutathione peroxidase activity towards lipid hydroperoxides in solution. The data from the present study indicate that a glutathione S-transferase associated with the nucleus may contribute to glutathione-dependent protection of isolated nuclei against lipid peroxidation. Evidence was obtained which indicates that this enzyme is distinct from the microsomal glutathione S-transferase.     

ATP-dependent S-(2,4-dinitrophenyl)glutathione transport in canalicular plasma membrane vesicles from rat liver

Uptake of the thioether S-(2,4-dinitrophenyl)glutathione (DNPSG) in canalicular plasma membrane vesicles from rat liver is enhanced in the presence of ATP and exhibits an overshoot with a transient 5.5-fold accumulation of DNPSG. Stimulation by ATP is not caused by the generation of a membrane potential, based on responses of the indicator dye oxonol V. ATP-dependent uptake has an apparent Km of 71 microM for DNPSG and a Vmax of 0.34 nmol.min-1.mg of vesicle protein-1. Protein thiol groups are essential for transport activity as indicated by the sensitivity of DNPSG transport to sulfhydryl reagents. There is competitive inhibition with other thioethers, S-hexylglutathione (Ki = 66 microM), the photoaffinity label S-(4-azidophenacyl)glutathione (Ki = 56 microM), as well as with glutathione disulfide (Ki = 0.44 mM) and with the bile acid taurocholate (Ki = 0.61 mM). GSH (2 mM) or cholate (0.4 mM) does not inhibit. Both glutathione disulfide and taurocholate show ATP-dependent transport in the canalicular membrane vesicles which is inhibited by DNPSG. No ATP-dependent transport is found for GSH. Transport of DNPSG is also inhibited competitively by alpha-naphthyl-beta-D-glucuronide (Ki = 0.42 mM) but not by alpha-naphthylsulfate (2 mM), and there is substantial inhibition with the glucuronides from ebselen and p-nitrophenol. The results indicate that the canalicular transport system for DNPSG is directly driven by ATP and that the biliary transport of other classes of compounds may also proceed via this system.     

External GSSG enhances intracellular glutathione level in isolated cardiac myocytes

The addition of external GSSG at concentrations in the range 50-500 microM produces in isolated adult rat heart myocytes an increase of GSH level and only a slight increase of GSSG level. On the contrary, external GSH at the above same indicated concentrations did not change the cell glutathione pool. The pretreatment of the cells with diethylamaleate depleted the myocytes of glutathione and enhanced the GSSG-induced replenishment effect on GSH level. On the contrary, the addition of GSH did not increase the concentration of cell glutathione. The level of cell GSH in diethylmaleate-treated myocytes was not increased after 30 min of incubation with cysteine, or acetylcysteine. The GSSG induced-stimulation on GSH level was not inhibited by buthionine sulfoximine, an inhibitor of glutathione synthesis. On the contrary, this stimulatory effect was inhibited by N, N-bis(2-chloroethyl)-N-nitrosourea, an inhibitor of glutathione reductase, or partially, by the remotion of glucose from the incubation medium. These results support the idea that the isolated adult rat heart myocytes are able to utilize external GSSG in order to increase the intracellular glutathione pool, probably through the reduction of the imported GSSG to GSH.     

Mutagenicity of tetrachloroethene in the ames test—metabolic activation by conjugation with glutathione

The mutagenicity of tetrachloroethene (tetra) and its S conjugate, S-(1,2,2-trichlorovinyl)glutathione (TCVG) was investigated using a modified Ames preincubation assay. TCVG was a potent mutagen in presence of rat kidney particulate fractions containing high concentrations of γ-glutamyl transpeptidase (GGT) and dipeptidases. Purified tetra was not mutagenic without exogenous metabolic activation or under conditions favoring oxidative metabolism. Preincubation of tetra with purified rat liver glutathione (GSH) S-transferases in presence of GSH and rat kidney fractions resulted in a time-dependent formation of TCVG as determined by (HPLC) analysis and in an unequivocal mutagenic response in the Ames test. Experiments with tetra in the isolated perfused rat liver demonstrated TCVG formation and its excretion with the bile; bile collected after the addition of tetra to the isolated perfused liver was unequivocally mutagenic in bacteria in the presence of kidney particulate fractions. The mutagenicity was reduced in all cases by the GGT inhibitor serine borate or the β-lyase inhibitor aminooxyacetic acid. These results support the suggestion that cleavage of the GSH S conjugate formed from tetra by the enzymes of the mercapturic acid pathway and by β-lyase may be involved in the nephrocarcinogenic effects of this haloalkene in rats.     

Analytical and preparative separation of the diastereomers of L-buthionine (SR)-sulfoximine, a potent inhibitor of glutathione biosynthesis.

Buthionine sulfoximine inhibits gamma-glutamylcysteine synthetase, the enzyme catalyzing the first reaction of glutathione (GSH) biosynthesis. GSH synthesis is blocked in animals or cultured cells exposed to buthionine sulfoximine, and GSH is substantially depleted in cells or tissues with moderate to high rates of GSH utilization. Studies reported to date have used DL-buthionine (SR)-sulfoximine or L-buthionine (SR)-sulfoximine, mixtures of four and two isomers, respectively. The present report describes a chiral solvent HPLC procedure for the analytical separation of the diastereomers of L-buthionine (SR)-sulfoximine and the separation of those isomers from the unresolved diastereomers of D-buthionine (SR)-sulfoximine. L-buthionine (R)-sulfoximine was isolated preparatively by repeated crystallization of L-buthionine (SR)-sulfoximine from water; L-buthionine (S)-sulfoximine was obtained by crystallization as the trifluoroacetate salt in ethanol/hexane mixtures. The absolute configuration, bond lengths and angles of L-buthionine (R)-sulfoximine were determined by X-ray diffraction. In vitro studies demonstrate that L-buthionine (R)-sulfoximine is a relatively weak inhibitor of rat kidney gamma-glutamylcysteine synthetase; binding is competitive with L-glutamate. L-buthionine (S)-sulfoximine is a tight-binding, mechanism-based inhibitor of the enzyme. Since L-buthionine sulfoximine is initially bound as a transition-state analogue, identification of the inhibitory diastereomer elucidates the steric relationships among ATP, glutamate, and cysteine within the active site. When administered to mice, L-buthionine (S)-sulfoximine (0.2 mmol/kg) was as effective as L-buthionine (SR)-sulfoximine (0.4 mmol/kg) in causing GSH depletion in liver, kidney, and pancreas. L-Buthionine (R)-sulfoximine (0.2 mmol/kg) did not cause significant GSH depletion in liver or pancreas. The L-(R)-diastereomer caused a modest GSH depletion in kidney that is tentatively attributed to interference with gamma-glutamylcyst(e)ine transport.     

A rapid equilibrium random sequential bi-bi mechanism for human placental glutathione S-transferase

Double-reciprocal plots of initial-rate data for the conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) and GSH by human placental GSH S-transferase pi were linear for both substrates. Computer modelling of the initial-rate data using nonlinear least-squares regression analysis favoured a rapid equilibrium random sequential bi-bi mechanism, over a steady-state random sequential mechanism or a steady-state or rapid equilibrium ordered mechanism. KGSH was calculated as 0.125 +/- 0.006 mM, KCDNB was 0.87 +/- 0.07 mM and alpha was 2.1 +/- 0.3 for the rapid equilibrium random model. The product, S-(2,4-dinitrophenyl)glutathione, was a competitive inhibitor with respect to GSH, and a mixed-type inhibitor toward CDNB (KP = 18 +/- 3 microM). The observed pattern of inhibition is consistent with a rapid equilibrium random mechanism, with a dead-end enzyme.CDNB.product complex, but inconsistent with the inhibition patterns of other bireactant mechanisms. Since rat liver GSH S-transferase 3-3 acts via a steady-state random sequential mechanism [1], while human placental GSH S-transferase and perhaps also rat liver GSH S-transferase 1-1 [2] exhibit rapid equilibrium random mechanisms, we conclude that the kinetic mechanism of the GSH S-transferases is isoenzyme-dependent.     

Inhibition of glutathione disulfide reductase by glutathione

Rat-liver glutathione disulfide reductase is significantly inhibited by physiological concentrations of the product, glutathione. GSH is a noncompetitive inhibitor against GSSG and an uncompetitive inhibitor against NADPH at saturating concentrations of the fixed substrate. In both cases, the inhibition by GSH is parabolic, consistent with the requirement for 2 eq. of GSH in the reverse reaction. The inhibition of GSSG reduction by physiological levels of the product, GSH, would result in a significantly more oxidizing intracellular environment than would be realized in the absence of inhibition. Considering inhibition by the high intracellular concentration of GSH, the steady-state concentration of GSSG required to maintain a basal glutathione peroxidase flux of 300 nmol/min/g in rat liver is estimated at 8-9 microM, about 1000-fold higher than the concentration of GSSG predicted from the equilibrium constant for glutathione reductase. The kinetic properties of glutathione reductase also provide a rationale for the increased glutathione (GSSG) efflux observed when cells are exposed to oxidative stress. The resulting decrease in intracellular GSH relieves the noncompetitive inhibition of glutathione reductase and results in an increased capacity (Vmax) and decreased Km for GSSG.     

Mutagenicity of tetrachloroethene in the Ames test--metabolic activation by conjugation with glutathione

The mutagenicity of tetrachloroethene (tetra) and its S conjugate, S-(1,2,2-trichlorovinyl)glutathione (TCVG) was investigated using a modified Ames preincubation assay. TCVG was a potent mutagen in presence of rat kidney particulate fractions containing high concentrations of gamma-glutamyl transpeptidase (GGT) and dipeptidases. Purified tetra was not mutagenic without exogenous metabolic activation or under conditions favoring oxidative metabolism. Preincubation of tetra with purified rat liver glutathione (GSH) S-transferases in presence of GSH and rat kidney fractions resulted in a time-dependent formation of TCVG as determined by (HPLC) analysis and in an unequivocal mutagenic response in the Ames test. Experiments with tetra in the isolated perfused rat liver demonstrated TCVG formation and its excretion with the bile; bile collected after the addition of tetra to the isolated perfused liver was unequivocally mutagenic in bacteria in the presence of kidney particulate fractions. The mutagenicity was reduced in all cases by the GGT inhibitor serine borate or the beta-lyase inhibitor aminooxyacetic acid. These results support the suggestion that cleavage of the GSH S conjugate formed from tetra by the enzymes of the mercapturic acid pathway and by beta-lyase may be involved in the nephrocarcinogenic effects of this haloalkene in rats.     

The electrophoretic behaviour of mitochondria from kidney and liver of rats

1. The electrophoretic mobility values for mitochondria prepared from rat kidney and liver measured in 0.125m-potassium chloride-0.02m-tris, pH7.4, are: -0.78(s.e.m.+/-0.02)mu/sec./v/cm. (29 experiments) and -1.06(s.e.m.+/-0.01) mu/sec./v/cm. (21 experiments) respectively. 2. These mobility values are unaffected by washing and spontaneous swelling at 25 degrees , indicating a stable electrokinetic surface. 3. The mobility of rat-kidney mitochondria is unaffected by thyroxine-induced swelling, or by the state of hydration of the rat. 4. pH-mobility curves show similar surface ionogenic groups for kidney and liver mitochondria; their isoelectric points are pH3.9 and pH4.4 respectively.