Glutathione depletion and cytotoxicity by naphthalene 1,2-oxide in isolated hepatocytes

The ability of naphthalene 1,2-oxide to diffuse across intact cellular membranes, the subsequent biotransformation of this epoxide and its potential to produce losses in cellular viability have been examined in incubations of isolated hepatocytes. Addition of 1R,2S- or 1S,2R-naphthalene oxide enantiomers (15, 30 and 60 microM) to isolated hepatocytes resulted in a rapid depletion of intracellular glutathione. Depletion of glutathione was concentration dependent and maximal at 5-15 min. Addition of either of the enantiomeric oxides at 60 microM resulted in the loss of more than 20 nmol glutathione/10(6) cells (1 ml cells); thus more than a third of the added epoxide was available for conjugation with intracellular glutathione. The time course and concentration dependence of glutathione depletion corresponded to the rapid, concentration-dependent formation of naphthalene oxide glutathione conjugates. The levels of glutathione adduct were highest 1 min after addition of naphthalene oxide and declined to 25% of this level after 30 min. Loss of glutathione conjugates from incubations correlated with the formation of N-acetylcysteine adducts. In contrast, the levels of glutathione adducts added exogenously to hepatocytes were relatively stable over a 120-min incubation suggesting that although further metabolism of naphthalene oxide glutathione adducts formed intracellularly is possible, extracellular glutathione adducts cannot penetrate the hepatocellular membrane. Small amounts of radiolabel from [3H]naphthalene 1,2-oxide were bound covalently to macromolecules in hepatocytes; the rate of this binding slowed rapidly after the first minute of incubation. Severe blebbing of the surface of the hepatocytes was noted in cells incubated for 30 min with 480 microM naphthalene oxide. Many of the cells were vacuolated at 60 min and progressed to frank necrosis with pyknotic nuclei and inability to exclude trypan blue. Cells incubated with 1-naphthol responded in a qualitatively similar fashion to those cells incubated with epoxide; however, hepatocytes incubated with 1-naphthol progressed to frank cellular necrosis at a slower rate. In hepatocytes partially depleted of glutathione by pretreatment with buthionine sulfoximine, addition of 1S,2R-naphthalene oxide at a rate of 1 nmol/min/10(6) cells resulted in significant losses in cell viability. In contrast, no losses in cell viability were observed with the enantiomer, 1R,2S-naphthalene oxide. Both epoxides produced similar losses in cellular glutathione levels.(ABSTRACT TRUNCATED AT 400 WORDS)     

Glutathione production in copper-deficient isolated rat hepatocytes.

Dietary copper deficiency has been shown to reduce copper-dependent superoxide dismutase (SOD) activity and to increase lipid peroxidation in rats. Circulating reduced glutathione (GSH) concentrations are elevated in copper-deficient (CuD) rats, which suggests an increased GSH synthesis or decreased degradation, perhaps as an adaptation to the oxidative stress of copper deficiency. GSH synthesis was examined in isolated hepatocytes from CuD rats. Isolated hepatocytes were prepared by collagenase perfusion and incubated in Krebs-Henseleit bicarbonate buffer, pH 7.4, 10 mM glucose, 2.5 mM Ca2+ in the presence and absence of 1.0 mM buthionine sulfoximine (BSO), a specific inhibitor of GSH synthesis. Cell viability was assessed by trypan blue exclusion. GSH and oxidized glutathione (GSSG) were measured by the glutathione reductase recycling assay. Copper deficiency depressed hepatocyte Cu by greater than 90% and increased intracellular GSH by 41-117% over the 3-h incubation, with a two- to threefold increase in the rate of intracellular GSH synthesis. Intracellular GSSG values were minimally influenced by CuD, with a constant mol% GSSG. Extracellular total glutathione (GSH + 2GSSG) synthesis was increased by approximately 33%. Both intracellular GSH and extracellular total glutathione synthesis were inhibited by BSO. The pattern of food consumption in CuD rats, meal fed versus ad libitum fed, had no effect on glutathione synthesis. The results indicate an increased hepatic GSH synthesis as a response to dietary copper deficiency and suggest an interrelationship between the essential nutrients involved in oxyradical metabolism.     

Glutathione conjugation of synthetic steroids in isolated rat hepatocytes

When designing steroid drugs with multiple double bonds, the influence of glutathione conjugation on the pharmacodynamics of drug action should be considered. We have examined the effect of canrenone, a mineralocorticoid receptor antagonist, on isolated rat hepatocytes and found that 1 mM canrenone injured the hepatocytes during shortterm incubation at 37 C, while an analogue of canrenone which bears 4 double bonds (delta 1,11-CAN) did not manifest such toxicity. To further pursue this, we prepared testosterone analogues comprising multiple double bonds as model compounds, and incubated them with freshly isolated rat hepatocytes. The viability of the hepatocytes was not influenced by any of the steroids, but some of them having a double bond at the C6-C7 position reduced the cellular glutathione levels. This was found to be due to conjugation of glutathione to the C7 position of the steroid molecule, and the rate of conjugation was accelerated when an additional double bond was introduced at C1-C2 or C11-C12 positions. The finding is interesting as glucuronidation or sulfation are common as conjugation processes of steroids.     

Localization of insulin degradation products to an intracellular site in isolated rat hepatocytes

In the present study, we have examined whether insulin degradation products are present on the surface of isolated rat hepatocytes and can be removed by an acid dissociation technique. Hepatocytes were incubated with [125I]insulin for 30 minutes, rapidly washed to remove unbound insulin, and then briefly exposed to acidic conditions (pH 5.0) to remove bound hormone from the cell surface. The radioactive material removed from the cell by acid dissociation and that remaining with the cells were separately analyzed by high performance liquid chromatography. The two primary degradation products of insulin present in control cell extracts were found only with the cell-associated material after acid dissociation. The insulin-sized radioactive material in the extract of acid-dissociable material consisted of only intact [125I]insulin. These results show that the two primary degradation products of insulin in rat hepatocytes are found only intracellularly and suggest that the degradation of the hormone begins after it is internalized.     

Uptake and degradation of asialo-fetuin by isolated rat hepatocytes.

125I-labelled asialo-fetuin was taken up by isolated rat hepatocytes by a saturable process. Half maximum uptake was seen at about 3 . 10(-8) M asialo-fetuin. Rate of uptake of asialo-fetuin exceeded rate of degradation at all concentrations of asialo-fetuin tested. Degradation of asialo-fetuin, as indicated by release of acid-soluble radioactivity from the cells, was inhibited by NH4Cl and chloroquine. The intracellular distribution of labelled asialo-fetuin was studied by differential and density gradient centrifuging. The distribution curves for radioactivity indicated that asialo-fetuin was present in lysosomes about 1 h after the uptake had started. Chloroquine and ammonium ions seemed to inhibit the uptake of asialo-fetuin into the lysosomes, possibly by interfering with the fusion between phagosomes and lysosomes.     

D-xylulose-induced depletion of ATP and Pi in isolated rat hepatocytes

Xylitol is known to cause hepatic ATP catabolism by inducing the trapping of Pi in the form of glycerol 3-P as a consequence of an increase in the NADH:NAD+ ratio, resulting from the oxidation of xylitol to D-xylulose. The question was therefore raised whether D-xylulose also depletes hepatic ATP. In isolated rat hepatocytes, 5 mM D-xylulose decreased ATP by 80% within 5 min compared to 40% with 5 mM xylitol. Intracellular Pi decreased by 70% within the same time interval with both compounds, but was restored three-fold faster with D-xylulose. The rate of utilization of D-xylulose reached 5 mumol.min-1.g-1 of cells, as compared with 1.5 for xylitol, indicating that reduction of xylitol into D-xylulose is a rate-limiting step in the metabolism of the polyol. D-Xylulose barely modified the concentration of glycerol 3-P but increased xylulose 5-P from 0.02 to 0.5 mumol/g within 5 min. The main cause of the ATP- and Pi-depleting effects of D-xylulose was found to be an accumulation of sedoheptulose 7-P from a basal value of 0.1 to 5 mumol/g of cells after 10 min. Ribose 5-P increased from 0.03 to 0.5 mumol/g at 5 min. Ribose 1-P also accumulated, albeit outside of the cells. This extracellular accumulation can be explained by the release of intracellular purine nucleoside phosphorylase from damaged hepatocytes acting on inosine that had diffused out of the cells. Smaller increases in the concentrations of sedoheptulose 7-P and pentose phosphates were recorded after incubations of the cells with xylitol.     

Inhibition of protein degradation in isolated rat hepatocytes

1. Isolated parenchymal cells were prepared by collagenase perfusion of livers from fed rats that had been previously injected with [³H]leucine to label liver proteins. When these cells were incubated in a salts medium containing glucose, gelatin and EDTA, cellular integrity was maintained over a period of 6h. 2. Cells incubated in the presence of 2mm-leucine to minimize radioactive isotope reincorporation released [³H]leucine into the medium at a rate accounting for the degradation of 4.5% of the labelled cell protein per h. 3. Degradation of [³H]protein in these cells was inhibited by insulin and by certain amino acids, of which tryptophan and phenylalanine were the most effective. 4. Protein degradation was decreased by several proteinase inhibitors, particularly those that are known to inhibit lysosomal cathepsin B, and by inhibitors of cell-energy production. 5. Ammonia inhibited degradation, but only at concentrations above 1.8mm. Aliphatic analogues of ammonia were effective at lower concentrations than was ammonia. 6. High concentrations of ammonia inhibited degradation by 50%. The extent of this inhibition could not be increased further by the addition of the cathepsin B inhibitor leupeptin, which by itself inhibited degradation by approx. 30%. 7. The sensitivity of proteolysis in isolated hepatocytes to these various inhibitory agents is discussed in relation to their possible modes of action.     

Glutathione depletion by 2-nitroimidazole-1-acetohydroxamic acid as a radiosensitizer in hypoxic rat hepatocytes

The effects of nitroimidazoles as radiosensitizers on intracellular glutathione (GSH) level were investigated in rat isolated hepatocytes. Dinitroimidazoles have lowered almost completely GSH level during the incubation for 30 min under oxic (95% O2+5% CO2) condition, while mononitroimidazoles had scarcely affected. In the case of hypoxic (95% N2+5% CO2) condition, however, 2-nitroimidazoles, not 4-nitroimidazoles, as well as 2,4- and 4,5-dinitroimidazoles have caused the significant depletion of GSH. This suggests that nitro group in the 2-position of imidazoles may be responsible for the GSH depletion under hypoxia. Especially, 2-nitroimidazole-1-acetohydroxamic acid (KIH-801) was found to be the most potent GSH depletor only under hypoxic, not oxic conditions, and might be useful for the new hypoxic cell radiosensitizer instead of misonidazole.     

Vanadate inhibits protein degradation in isolated rat hepatocytes

Vanadate (10 mM) strongly inhibited endogenous protein degradation as well as the degradation of an exogenous, endocytosed protein (asialofetuin) in isolated rat hepatocytes. Protein synthesis and cellular viability were unaffected, but changes in cell morphology suggested some interference with cytoskeletal elements. The effect of vanadate was comparable to the effects of several other degradation inhibitors (lysosomotropic amines, leupeptin, vinblastine, amino acids, dimethylaminopurine riboside) known to inhibit the autophagic/lysosomal pathway of protein degradation. Vanadate inhibited proteolysis in a liver homogenate at pH 5, suggesting a direct effect upon the lysosomal proteinases.     

Autophagic degradation of peroxisomes in isolated rat hepatocytes.

Degradation of the peroxisomal enzymes fatty acyl-CoA oxidase and catalase was studied in hepatocytes isolated from rats treated with clofibrate and from control rats. Hepatocytes were incubated in the absence of amino acids in order to ensure maximal flux through the autophagic pathway and in the presence of cycloheximide to inhibit protein synthesis. (1) Degradation of the two peroxisomal enzymes in hepatocytes from clofibrate-fed rats, but not in hepatocytes from control rats, was much faster than that of other intracellular enzymes. This increased degradation of the peroxisomal enzymes was almost completely prevented by 3-methyladenine, an inhibitor of macroautophagic sequestration. (2) The increased degradation of the peroxisomal enzymes was also inhibited by a long-chain (C16:0) and a very-long-chain (C26:0) fatty acid, but not by C12:0, a medium-chain fatty acid, or by C8:0, a short-chain fatty acid. These results provide direct evidence for the proposal that autophagic sequestration can be highly selective [(1987) Exp. Mol. Pathol. 46, 114-122]. It is concluded that preferential autophagy of peroxisomes is prevented when these organelles are supplied with their fatty acid substrates.     

Intracellular localization and degradation of asialofetuin in isolated rat hepatocytes

Analysis by isopycnic and differential centrifuging of the intracellular distribution of radioactivity following uptake of ¹²⁵I-labelled asialofetuin by isolated rat hepatocytes showed that during incubations up to 1 h, most of the radioactivity was associated with structures which had a subcellular distribution pattern different from both the lysosomes and the plasma membrane. The latter two organelles were followed by means of enzyme markers. Ca²⁺ is necessary for the binding of asialofetuin to the plasma membrane, and it was also possible to differentiate between asialofetuin bound to the plasma membrane and that contained in intracellular structures by removing Ca²⁺ from the medium (by EGTA). Such experiments showed that asialofetuin became rapidly internalized. Practically all the labelled protein was located intracellularly in cells that had been incubated with asialofetuin for more that 30 min. When incubations were carried out for more that 1 h a peak appeared in the radioactivity distribution in the same place as the peak of activity of lysosomal marker enzymes. However, degradation of asialofetuin takes place in the lysosomes and this starts before the labelled protein can be found in the lysosomal fractions. Our data suggest that the rate-determining step in the cellular handling of asialofetuin is the transport of endocytized protein from the endocytic vesicles to the lysosomes.     

Uptake and degradation of 125I-labelled asialo-fetuin by isolated rat hepatocytes.

125I-Labelled asialo-fetuin was taken up by isolated rat hepatocytes by a saturable process. Half maximum uptake was seen at about 3 - 10(-8) M asialo-fetuin. Non-parenchymal liver cells did not take up asialo-fetuin in vitro. Rate of uptake of asialo-fetuin exceeded rate of degradation at all concentrations of asialo-fetuin tested. Asialo-fetuin consequently accumulated in the cells until the extracellular supply was exhausted. Asialo-fetuin degradation could be studied without concurrent uptake by incubating cells, previously exposed to asialo-fetuin, in asialo-fetuin-free medium. Degradation, as evidenced by increase in acid-soluble radioactivity, was inhibited by NH4Cl and chloroquine. The change with time in the intracellular distribution pattern of radioactivity in cells that had been exposed to 125I-labelled asialo-fetuin for 10 min was examined by means of differential centrifugation. Initially, the radioactivity was found mostly in the microsomal fraction. 60 min after the exposure to labelled protein, the distribution pattern of radioactivity resembled that of the lysosomal enzyme beta-acetylglucosaminidase. The possibility that asialo-fetuin digestion takes place in lysosomes is discussed.     

Kinetics of internalization and degradation of asialo-glycoproteins in isolated rat hepatocytes

The rate constants for internalization of surface-bound asialo-orosomucoid by hepatocytes were 0.040 min-1 at 20 degrees C, 0.18 min-1 at 30 degrees C and 0.28 min-1 at 40 degrees C. At 40 degrees C, internalization accounted for most of the increase in cell-associated radioactivity. The activation energy over the temperature range 20 to 40 degrees C was 68 +/- 7 (S.D.) kJ/mol. At 10 degrees C, most of the cell-associated asialo-orosomucoid was bound to the cell surface in a reaction which followed ordinary chemical kinetics. Pre-incubation of hepatocytes with a large concentration of unlabelled asialo-orosomucoid did not influence the uptake of subsequently added 125I-asialofetuin; neither was degradation of 125I-asialo-fetuin affected in this experiment. The fractional rate of degradation (the fraction of cell-associated asialo-fetuin which was degraded per unit time) was constant over a twelve-fold range of intracellular asialo-fetuin concentrations. Increasing the temperature from 20 to 30 degrees C produced approximately a ten-fold increase in the rate of degradation of either asialo-fetuin or asialo-orosomucoid. The average activation energies of degradation over the range 20 to 40 degrees C were 125 kJ/mol for asialo-fetuin and 149 kJ/mol for asialo-orosomucoid; however, the Arrhenius plots were not straight lines over this temperature range.     

Intracellular localization and degradation of asialofetuin in isolated rat hepatocytes.

Analysis by isopycnic and differential centrifuging of the intracellular distribution of radioactivity following uptake of 125I-labelled asialofetuin by isolated rat hepatocytes showed that during incubations up to 1 h, most of the radioactivity was associated with structures which had a subcellular distribution pattern different from both the lysosomes and the plasma membrane. The latter two organelles were followed by means of enzyme markers. Ca2+ is necessary for the binding of asialofetuin to the plasma membrane, and it was also possible to differentiate between asialofetuin bound to the plasma membrane and that contained in intracellular structures by removing Ca2+ from the medium (by EGTA). Such experiments showed that asialofetuin became rapidly internalized. Practically all the labelled protein was located intracellularly in cells that had been incubated with asialofetuin for more than 30 min. When incubations were carried out for more than 1 h a peak appeared in the radioactivity distribution in the same place as the peak of activity of lysosomal marker enzymes. However, degradation of asialofetuin takes place in the lysosomes and this starts before the labelled protein can be found in the lysosomal fractions. Our data suggest that the rate-determining step in the cellular handling of asialofetuin is the transport of endocytized protein from the endocytic vesicles to the lysosomes.     

Energy dependence of autophagic protein degradation in isolated rat hepatocytes

The effect of small changes in intracellular ATP on autophagic flux was studied in isolated rat hepatocytes by using inhibitors of ATP production or by varying the metabolic conditions. The following observations were made. There was a linear relationship between endogenous protein degradation and intracellular ATP, the rate of proteolysis declining with decreasing ATP concentrations. 15% of the maximal proteolysis is either independent of ATP or has a very high affinity for this metabolite. There was a linear relationship between the autophagic sequestration of cytosolic [14C]sucrose and intracellular ATP, the sequestration rate decreasing with decreasing ATP concentrations. ATP depletion did not cause release of [14C]sucrose previously sequestered in autophagosomes and lysosomes at high ATP levels. Intracellular accumulation of chloroquine, used as an indicator of the pH inside lysosomes and other acidic cell compartments, diminished with decreasing cellular ATP content. Amino acids inhibited proteolysis without affecting ATP levels or chloroquine accumulation. We conclude from the high sensitivity of autophagy towards relatively small changes in the concentration of intracellular ATP that, besides amino acids, ATP is a very important factor in controlling the rate of autophagy in rat hepatocytes.     

Receptor-linked degradation of 125I-insulin is mediated by internalization in isolated rat hepatocytes

When hepatocytes were freshly isolated from rat liver and incubated for various periods of time at 37 degrees C, the media from the incubation, when completely separated from the cells, actively degraded 125I-insulin. THis soluble protease activity was strongly inhibited by bacitracin but was unaffected by the lysosomatropic agent ammonium chloride (NH4Cl). When hepatocytes were incubated with 125I-insulin at 37 degrees C in the presence or absence of 8 mM NH4Cl the ligand initially bound to the plasma membrane and was subsequently internalized as a function of time. When hepatocytes were incubated at 37 degrees C for 30 minutes with 125I-insulin in the presence of bacitracin and NH4Cl or bacitracin alone and the cells were washed, diluted, and the cell-bound radioactivity allowed to dissociate, the percent intact 125I-insulin in the cell pellet and in the incubation media was greater in the presence of NH4Cl at each time point of incubation. Under these same conditions a higher proportion of the cell-associated radioactivity was internalized and a higher proportion was associated with lysosomes. The data suggest that receptor-mediated internalization is required for insulin degradation by the cell, and that this process, at least in part, involves lysosomal enzymes. Furthermore, the data demonstrate that internalization is not blocked by the presence of bacitracin or NH4Cl in the incubation media, but that degradation is inhibited.