Effects of diarylsulfonylurea antitumor agents on the function of mitochondria isolated from rat liver and GC3/c1 cells.

Diarylsulfonylureas, such as N-(4-chlorophenyl)aminocarbonyl-2,3-dihydro-1-indene-5-sulfonamide (LY186641, Sulofenur) and N-(4-chlorophenyl)aminocarbonyl-4-methylbenzene sulfonamide (LY181984), have been shown to be effective antitumor agents in a variety of in vivo and in vitro animal models. Their mechanism of action is unknown but does not appear to be the result of nonselective destruction of actively dividing cell populations. Mitochondria have been shown to accumulate Sulofenur and therefore may be targets of drug action. The purpose of these investigations was to examine the effects of a variety of diarylsulfonylureas in mitochondria and attempt to determine the relevance of these changes to antitumor activity. Many of the diarylsulfonylureas which were effective antitumor agents in animal models were also uncouplers of mitochondrial oxidative phosphorylation. They increased state 4 respiration and dissipated the mitochondrial membrane potential in a concentration-related fashion. The mechanism of uncoupling appeared to be related to a dissociable hydrogen ion as these molecules had pKa values that ranged from 6.0 to 6.2 and were highly lipophilic. Thus, the uncoupling action appears to be the result of hydrogen ion translocation. The mechanism of antitumor activity does not appear to be the result of uncoupling as no correlation was evident between inhibition of cell growth and uncoupling action of a variety of active and inactive diarylsulfonylureas. In vitro, Sulofenur is cytotoxic at high concentrations and inhibits cell growth at lower concentrations in the absence of any overt cell kill. The inhibition of cell growth also did not appear to be related to the uncoupling action of these drugs. In contrast, uncoupling may have played a partial role in the early, high exposure cell kill that can occur with these compounds.

Effects of methapyrilene measured in mitochondria isolated from naive and methapyrilene-treated rat and mouse hepatocytes.

Methapyrilene (MP) is a rat-specific liver carcinogen that alters mitochondrial number and morphology both in vivo and in vitro. This biological phenomenon may be due to the effects of MP on mitochondrial function. To test this hypothesis, studies were conducted to examine the effects of MP on DNA and protein synthesis and respiration in isolated mitochondria. DNA and protein synthesis activities were measured using [3H]thymidine and [3H]leucine incorporation. Mouse liver mitochondria were also examined for comparison since no tumor formation or alterations in mitochondrial morphology have been associated with MP treatment in mice. A significant decrease in basal DNA and protein synthesis levels was observed in mitochondria isolated from rats and mice following in vivo MP treatment. This effect could not be reproduced when mitochondria were exposed to 0 or 100 microM MP following isolation, despite the presence of an S9 activation system. Electron microscopic examinations were performed on isolated rat mitochondria and revealed morphologic differences between mitochondria from naive and MP-treated rats. Although significant differences in State 3 and State 4 respiratory rates were noted, the respiratory control ratio, ADP/O ratio, and uncoupler-stimulated respiratory rates were unaffected. Results demonstrate that: (1) MP irreversibly depresses DNA and protein synthesis in a majority of mitochondria, despite only localized morphologic changes; (2) these changes are not reflected by a decrease in respiratory function; and (3) depression of DNA and protein synthesis does not correlate with carcinogenic susceptibility.

Cephaloridine-induced renal pathological and biochemical changes in female rabbits and isolated proximal tubules in suspension.

Cephaloridine (Cld) is a nephrotoxic cephalosporin antibiotic. The intracellular biochemical changes that occur leading to Cld-induced nephrotoxicity may involve lipid peroxidation and/or mitochondrial injury. The purpose of this report was to examine and correlate the biochemical changes induced by Cld in vivo and in vitro with the observed pathological changes in an attempt to understand better the mechanisms of beta-lactam-induced nephrotoxicity. Cld treatment (500 mg/kg sc) caused elevations in blood urea nitrogen and decreases in the accumulation of p-aminohippurate (PAH) and tetraethylammonium (TEA) by renal cortical slices. Histopathological alterations, characterized by individual cell necrosis of tubular epithelial cells, were first seen 6 hr after treatment in the pars recta of the outer stripe of the medulla. Ultrastructural alterations involved the straight (S2 and S3) segments of the proximal tubules. Mitochondrial morphology was, for the most part, unaffected by Cld exposure. Cld did not cause any significant changes in tissue malondialdehyde (MDA) content in vivo at any of the time points examined, but it did cause a depletion of GSH to approximately 40% of control by 1 hr after dosing that recovered toward control by 6 hr. Significant changes were observed in renal ATP content beginning at 6 hr after treatment; however, this change mirrored the onset of histological evidence of necrosis. In isolated tubules in vitro, the onset of glutathione (GSH) depletion and MDA formation clearly preceded lactate dehydrogenase (LDH) leakage, whereas ATP depletion was a mirror image of cell death. These data demonstrate that isolated proximal tubules in vitro are a reasonable model for Cld nephrotoxicity in vivo. Cld-induced mitochondrial alterations leading to ATP depletion and cell injury were not observed in this study.

Cephaloridine-induced biochemical changes and cytotoxicity in suspensions of rabbit isolated proximal tubules.

Cephalosporin antibiotics, such as cephaloridine (Cld), are known to be nephrotoxic in vivo and in vitro. In vivo, Cld causes proximal tubule necrosis in rabbits which is preceded by glutathione (GSH) depletion and, under certain conditions, inhibition of mitochondrial function. In vitro, Cld causes GSH depletion, lipid peroxidation, and inhibition of rat kidney slice organic ion uptake. The present investigations were designed to evaluate the temporal relationships of the biochemical "lesions" caused by Cld to the onset of lethal cell injury in suspensions of isolated rabbit proximal tubules. Cld was cytotoxic to suspensions of rabbit proximal tubules (EC50 = 1.10 +/- 0.33 mM) in the absence of amino acids (to support GSH synthesis). In this model, Cld also caused GSH and ATP depletion, lipid peroxidation (malondialdehyde formation), and inhibition of tubule respiration. Probenecid prevented Cld accumulation, tubule injury, ATP depletion, and lipid peroxidation and markedly attenuated the GSH depletion. Addition of glycine, cystine, and glutamate to the incubation buffer to support GSH synthesis decreased the tubule accumulation of Cld (due solely to the presence of glutamate) and blocked Cld-induced tubule lethality, lipid peroxidation, ATP depletion, and GSH depletion. Glycine or glutamate alone had no effect on Cld-induced cytotoxicity, whereas cystine was cytoprotective. Buthionine sulfoximine partially reversed the amino acid protection against Cld-induced tubule injury. Thus amino acid-induced protection of tubules from Cld cytotoxicity was due to the combination of a high intracellular GSH content and cytoprotection by cystine. The antioxidant N-N'-diphenyl-p-phenylenediamine (DPPD) blocked tubule injury, ATP depletion, and lipid peroxidation but had no effect on Cld-induced GSH depletion when tubules were incubated for 3 hr. However, when incubations were allowed to run for up to 8 hr, DPPD had no effect on Cld cytotoxicity, despite continued inhibition of lipid peroxidation. These data demonstrate that Cld-induced tubule injury in short-term (3 hr) incubations in vitro occurs by a mechanism probably involving lipid peroxidation and occurs only in the absence of amino acids to support GSH synthesis. Inhibition of tubule respiration and ATP depletion could not clearly be causally linked to the onset of cell death in this model. The mechanism of the peroxidation-independent Cld toxicity in tubules incubated for 8 hr or longer is not known at this time.

The effects of fructose on adenosine triphosphate depletion following mitochondrial dysfunction and lethal cell injury in isolated rat hepatocytes.

Mitochondrial injury in aerobic mammalian cells is associated with a rapid depletion of adenosine triphosphate (ATP) which occurs prior to the onset of lethal cell injury. In this report, the relationships between ATP depletion and lethal cell injury were examined in rat hepatocytes using oligomycin as a model mitochondrial toxicant and fructose as an alternative carbohydrate source for glycolysis. Oligomycin was more potent in causing lethal cell injury in hepatocytes isolated from fasted animals than cells from fed animals. The onset of cell injury (leakage of lactate dehydrogenase) in cells from fed animals correlated with the depletion of stored glycogen and ATP. The degree and time course profile of oligomycin-induced ATP depletion could be duplicated with 50 mM fructose alone in hepatocytes from fasted animals; however, fructose did not cause lethal cell injury. Oligomycin caused marked accumulation of adenosine monophosphate (AMP) and inorganic phosphate (Pi) and a conservation of adenine nucleotides. In contrast, fructose (50 mM) caused a decrease in Pi, no persistent change in AMP, and a depletion of the adenine nucleotide pool. Fructose, at concentrations greater than 1.0 mM, protected hepatocytes from oligomycin-induced toxicity. Blockade of mitochondrial ATP synthesis with oligomycin resulted in massive ATP depletion. In the presence of oligomycin, 5.0 mM fructose maintained cellular ATP content similar to that of control cells, whereas 50 mM fructose did not, demonstrating the biphasic effect of increasing fructose concentrations on cellular ATP content. Fructose-induced protection of hepatocytes from oligomycin toxicity was due to glycolytic fructose metabolism as hepatocytes incubated with iodoacetate (30 microM), fructose, and oligomycin had reduced viability and ATP content. In conclusion, interruption of mitochondrial ATP synthesis leads to marked ATP depletion and lethal cell injury. Cell injury is clearly not due to ATP depletion alone since increased glycolytic ATP production from either glycogen or fructose can maintain cell integrity in the absence of mitochondrial ATP synthesis and at low cellular ATP levels.

In vivo development and in vitro characterization of a subclone of murine P388 leukemia resistant to bis(diphenylphosphine)ethane.

Bis(diphenylphosphine)ethane (DPPE) and its gold coordination complexes have demonstrated antitumor activity in transplantable tumor models. This report describes the development of a P388 cell line (P388/DPPEc) that is resistant to DPPE and its analogues and the in vitro characterization of the cross-resistance of this subline to various antitumor and cytotoxic agents. The P388/DPPE tumor cell line was developed by serial transplantation in DPPE-treated mice. Resistance to DPPE was phenotypically stable. The P388/DPPE subline was cross-resistant to DPPE analogues and metal coordination complexes of DPPE. In addition, P388/DPPE cells were resistant to several mitochondrial uncouplers, including rhodamine-123, tetraphenylphosphonium, and carbonylcyanide-p-trifluro-methoxyphenyl hydrazone. P388/DPPE cells were less capable of sequestering and retaining 123Rh than were sensitive (P388/S) cells. Exposure to Au(DPPE)2+, a gold complex of DPPE with increased antitumor activity, resulted in a depletion of cellular ATP; the depletion was more rapid in the sensitive than the resistant cells. The rate of mitochondrial respiration, as measured by 14CO2 evolution from [6-14C]glucose, was greater in P388/S than in P388/DPPE. As with that evidenced for 123Rh, the cellular uptake of radiolabeled DPPE was decreased in P388/DPPEc cells. The results suggest that the basis for the resistance of this cell line may be an alteration in mitochondrial membrane potential. These data and the striking cross-resistance of P388/DPPE to mitochondrial uncouplers support the hypothesis that mitochondria may be one target involved in the cytotoxic or antitumor activities of these compounds. Mitochondria may also be causally related to the cytotoxic or antitumor activities, in that DPPE may be concentrated in cells via the presence of the inner mitochondrial membrane potential. Thus, P388/DPPE cells can serve as a tool to screen for and evaluate drugs that rely on affecting mitochondrial function, either mechanistically or causally, for their antitumor efficacy.

Internalization of recombinant tissue-type plasminogen activator by isolated rat hepatocytes is via coated pits.

The uptake and internalization of tissue-type plasminogen activator (t-PA) by freshly isolated rat hepatocytes was investigated. Electron microscopic examination of the uptake of t-PA-colloidal gold conjugates (t-PA-gold) by isolated rat hepatocytes showed that t-PA-gold was internalized via coated pits. This was inhibited with excess t-PA. Uptake of 125I-t-PA by isolated rat hepatocytes was a rapid, saturable, and specific process. The initial rate of specific uptake was 0.1 fmol/10(6) cells per min. The specific uptake plateaued at 1.4 fmol/10(6) cells by 30 min and declined to 0.8 fmol/10(6) cells at 2 h. Depletion of cellular ATP by 85-90% did not affect the initial rate of specific uptake. However, specific uptake by ATP-depleted hepatocytes at 30 min was reduced by 37%. By 2 h specific uptake by ATP-depleted hepatocytes was only 5% lower than by untreated hepatocytes, suggesting that processing of t-PA and/or its receptor is ATP-dependent. Uptake of 125I-t-PA was temperature dependent. Specific uptake was reduced by approximately 20% at 22 degrees C and by 70% at temperatures below 16 degrees C. Finally, inhibition of coated pit formation by K(+)-depletion with nigericin decreased the uptake of 125I-t-PA. This inhibition was shown to be K(+)-specific since treatment with nigericin in the presence of K+ did not inhibit coated pit formation or 125I-t-PA uptake. A threshold K(+)-depletion level for inhibition of coated pit formation was also demonstrated since treatment under conditions that reduced cellular K+ by only 54% had no effect on coated pit formation or 125I-t-PA uptake. These data support our hypothesis that internalization of t-PA by isolated rat hepatocytes is via coated pits and suggest that uptake of t-PA is a receptor-mediated process.

Mechanism of inhibition of rat liver mitochondrial respiration by oxmetidine, an H2-receptor antagonist.

Suspensions of rat liver hepatocytes exposed to oxmetidine rapidly lose viability, an event preceded by a marked and rapid inhibition of cell respiration and depletion of ATP. In isolated rat liver mitochondria (RLM), oxmetidine inhibits pyruvate/malate- but not succinate-supported, ADP-stimulated oxygen consumption (state 3). The purpose of this investigation was to determine the exact molecular site of oxmetidine-induced inhibition of RLM electron transport. Oxmetidine did not significantly inhibit succinate-supported, ADP-stimulated state 3 oxygen consumption in isolated RLM at concentrations up to 0.5 mM. In contrast, oxmetidine significantly inhibited beta-hydroxybutyrate- or isocitrate-supported mitochondrial state 3 oxygen consumption at concentrations above 10 microM and 25 microM, respectively. In RLM electron transport particles (ETP), oxmetidine inhibited NADH-oxidase and NADH-CoQ reductase activity (IC50 of 3.4 microM and 2.6 microM, respectively). However, oxmetidine did not significantly affect NADH-Fe3(CN)6 reductase activity (at concentrations up to 200 microM). SK&F 92058, a thiourea analog of oxmetidine approximately 24-fold less toxic to hepatocytes, produced a similar pattern of inhibition of respiration, although far less potent (IC50 of 0.8 mM and 0.6 mM for NADH-oxidase and NADH-CoQ reductase, respectively). SK&F 92058 did not significantly inhibit NADH-Fe3(CN)6 reductase activity at concentrations up to 3.0 mM. Studies with [14C]oxmetidine failed to show any specific, saturable binding to rat liver ETP.(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo and in vitro cardiotoxicity of a gold-containing antineoplastic drug candidate in the rabbit.

Bis[1,2-bis(diphenylphosphino)ethane] gold(I) chloride (Au(DPPE)+2), a cytotoxic antineoplastic drug candidate, was cardiotoxic in rabbits. Intravenous administration of Au(DPPE)+2 (15 mg/kg) as a single dose produced multiple, 2- to 5-mm subendocardial and myocardial lesions, macroscopically appearing as pale tan foci. Histologically, these lesions consisted of widely scattered zones of myocardial cell necrosis and mineralization. The myocardium also contained multifocal areas of contraction band necrosis in which aggregated clumps of disorganized myofilaments were contiguous with areas of sarcoplasm which were relatively devoid of myofilaments. In a series of in vitro studies, electron microscopic examination of isolated rabbit myocytes treated with 30 microM Au(DPPE)+2 for 15 min showed evidence of mitochondrial swelling and electron translucent mitochondrial matrices. After 60 min of incubation, myocytes had mitochondria that were condensed and disrupted but the cristae had retained their tubular profiles. Isolated rabbit myocytes exposed to 30 microM Au(DPPE)+2 had significant increases in the leakage of lactate dehydrogenase, an index of cell death. Cellular ATP content in myocytes exposed to 30 microM Au(DPPE)+2 was significantly reduced by 30 min. State 4 respiration in isolated rabbit mitochondria was significantly increased by Au(DPPE)+2 (30 microM) while state 3 respiration was unaffected. Au(DPPE)+2 also caused a rapid dissipation of the mitochondrial inner membrane electrochemical potential in a concentration-dependent manner and was accompanied by a ruthenium red-sensitive calcium efflux. These data suggest that disruption of mitochondrial function, leading to uncoupling of oxidative phosphorylation, decreased ATP synthesis, and altered mitochondrial calcium homeostasis, may be a contributing factor leading to cardiac myofibril necrosis produced by Au(DPPE)+2.

The mechanism of acute cytotoxicity of triethylphosphine gold(I) complexes. III. Chlorotriethylphosphine gold(I)-induced alterations in isolated rat liver mitochondrial function.

Chlorotriethylphosphine gold(I) (TEPAu) is an organo-gold compound that has therapeutic activity in animal models of rheumatoid arthritis. Initial studies have suggested that TEPAu is a potent cytotoxic compound in vitro against a variety of cultured cell types and isolated hepatocytes. Mitochondrial dysfunction induced by this compound has been suggested as a primary biochemical alteration which may result in lethal cell injury in isolated hepatocytes. The purpose of this study was, therefore, to determine the mechanism of TEPAu-induced dysfunction of isolated rat liver mitochondria. TEPAu induced a rapid, concentration-related collapse of the mitochondrial inner membrane potential (EC50 = 24.7 +/- 2.5 microM) which was potentiated in Ca2+ loaded mitochondria (EC50 = 11.3 +/- 3.8 microM). TEPAu-induced collapse of the membrane potential was partially inhibited in the presence of ruthenium red or EGTA. TEPAu caused the rapid release of mitochondrially sequestered Ca2+ which was not inhibited by ruthenium red and, thus, was not via a reversal of the Ca2+ uniporter. TEPAu caused mitochondrial swelling, increased permeability of the inner membrane, and the oxidation/hydrolysis of endogenous mitochondrial pyridine nucleotides. Addition of exogenous ATP slightly reversed the effects of TEPAu on pyridine nucleotides. TEPAu-induced mitochondrial alterations were reversed or inhibited by exposure to the sulfhydryl reducing agent, dithiothreitol. Also, the TEPAu-induced collapse of the mitochondrial membrane potential was partially inhibited by dibucaine, a non-specific inhibitor of phospholipases. These data suggest that TEPAu-induced mitochondrial dysfunction is sulfhydryl dependent. TEPAu-induced mitochondrial dysfunction results in dissipation of the potential difference across the inner mitochondrial membrane which inhibits mitochondrial oxidative phosphorylation. The mechanism by which TEPAu induces the collapse of the membrane potential may be mediated by a sulfhydryl-dependent increase in permeability of the inner membrane to protons.

Mechanism of toxicity of an experimental bidentate phosphine gold complexed antineoplastic agent in isolated rat hepatocytes.

SK&F 104524 (bis-[1,2 bis(diphenylphosphino)-ethane]gold(l) lactate) [( Au(dppe)2]+) is an experimental antineoplastic agent that is hepatotoxic in vivo in the dog as well as highly cytotoxic to isolated canine hepatocytes in vitro. Preliminary studies in isolated dog hepatocytes have indicated that [Au(dppe)2]+ causes an increase in hepatocyte respiration and a decrease in cellular ATP. The purpose of the present investigation was to characterize [Au(dppe)2]+-induced cytotoxicity and biochemical lesions in the intact cell and to correlate these changes with mitochondrial function. The uptake of [14C][Au(dppe)2]+ by rat hepatocytes was rapid, reaching a maximum by 30 min. [Au(dppe)2]+ was distributed throughout the hepatocyte and associated rapidly with mitochondria, nuclei, cytosol and cellular membranes. [Au(dppe)2]+ caused cell lethality in a concentration-dependent fashion; although 5 microM did not cause any changes in lactic dehydrogenase leakage, 20 microM produced 100% cell death by 120 min. [Au(dppe)2]+ also caused concentration-dependent bleb formation of the hepatocyte plasma membrane, increased oxygen consumption and loss of ATP within 30 min. ATP loss was associated with transient increases in AMP and ADP and a profound drop in the ATP/ADP ratio and energy charge. Total nucleotides (adenine and xanthine nucleotides) remained constant. The pattern of glutathione depletion coincided with that of lactic dehydrogenase leakage. Electron microscopy of hepatocytes exposed to [Au(dppe)2]+ for 30 min revealed depletion of glycogen granules and marked swelling of mitochondria. In isolated rat liver mitochondria, [Au(dppe)2]+ caused a stimulation of state 4 respiration and loss of the respiratory control ratio. [Au(dppe)2]+ also relieved the oligomycin-induced inhibition of state 3 (ADP-stimulated) respiration.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanism of alterations in isolated rat liver mitochondrial function induced by gold complexes of bidentate phosphines.

Au(DPPE)+2 (bis[1,2-bis(diphenylphosphino)ethane] gold(I] is an organo-gold antineoplastic agent that has anti-tumor activity in a variety of in vitro cell lines and in vivo rodent tumor models. Preliminary studies suggested that this compound represented a novel class of inhibitors of mitochondrial function. The purpose of this study was, therefore, to determine the mechanism of mitochondrial dysfunction induced by Au(DPPE)+2. Au(DPPE)+2 induced a rapid, dose-related collapse of the inner mitochondrial membrane potential (EC50 = 28.0 microM) that was not potentiated by Ca2+ preloading. Au(DPPE)+2-induced dissipation of mitochondrial membrane potential was accompanied by an efflux of Ca2+ from mitochondria upon exposure to Au(DPPE)+2. Ca2+ efflux in these experiments was via a reversal of the Ca2+ uniporter as efflux could be inhibited with ruthenium red. Au(DPPE)+2 did not increase the permeability of mitochondria to oxalacetate, indicating that the collapse of membrane potential may not be a result of gross increased inner membrane permeability. However, Au(DPPE)+2 may mediate an increased permeability of the inner membrane to cations and protons. Au(DPPE)+2 caused passive swelling in potassium acetate buffer in the absence of valinomycin, suggesting Au(DPPE)+2 facilitated the exchange of H+ and K+. Ca2+ cycling was not extensive and did not contribute to the decrease in membrane potential. These data suggest that one possible mechanism of Au(DPPE+2-induced uncoupling of mitochondrial oxidative phosphorylation is via increased permeability of the inner mitochondrial membrane to cations. The disruption of mitochondrial function may be a key process leading to hepatocyte cell injury by this drug.

Biochemical mechanisms of cephaloridine nephrotoxicity.

Large doses of the cephalosporin antibiotic, cephaloridine, produce acute proximal tubular necrosis in humans and in laboratory animals. Cephaloridine is actively transported into the proximal tubular cell by an organic anion transport system while transport across the lumenal membrane into tubular fluid appears restricted. High intracellular concentrations of cephaloridine are attained in the proximal tubular cell which are critical to the development of nephrotoxicity. There is substantial evidence indicating that oxidative stress plays a major role in cephaloridine nephrotoxicity. Cephaloridine depletes reduced glutathione, increases oxidized glutathione and induces lipid peroxidation in renal cortical tissue. The molecular mechanisms mediating cephaloridine-induced oxidative stress are not well understood. Inhibition in gluconeogenesis is a relatively early biochemical effect of cephaloridine and is independent of lipid peroxidation. Furthermore, cephaloridine inhibits gluconeogenesis in both target (kidney) and non-target (liver) organs of cephaloridine toxicity. Since glucose is not a major fuel of proximal tubular cells, it is unlikely that cephaloridine-induced tubular necrosis is mediated by the effects of this drug on glucose synthesis.

Toxicity of H2-receptor antagonists to isolated rat hepatocytes: structure-activity relationships.

Oxmetidine is a potent and specific antagonist of the histamine H2-receptor. Oxmetidine is also cytotoxic to isolated rat hepatocytes through inhibition of mitochondrial oxidative phosphorylation. The purpose of this investigation was to test a variety of H2-receptor antagonists that are structural analogs of oxmetidine in an attempt to identify a critical structural component or a physicochemical property of the molecule which may be responsible for cytotoxicity. Six histamine receptor H2-antagonists were tested. The minimum drug concentrations that caused 100% cell death (leakage of intracellular lactate dehydrogenase and loss of intracellular potassium) ranged from 0.87 to 22.50 mM for the analogs tested. At toxic concentrations, two of the least potent analogs, SK&F 92909 and SK&F 9205A both caused a rapid decrease in hepatocyte O2 consumption and ATP content which occurred before any evidence of cell injury. The potency of these molecules as cytotoxicants to isolated hepatocytes did not correlate with their potency as histamine H2-receptor antagonists whereas there was a significant correlation between increasing potency and increasing octanol/water partition coefficients. These data suggest that lipid solubility may be a key factor in the cytotoxicity of this class of drugs to isolated rat hepatocytes.

Role of glutathione reductase during menadione-induced NADPH oxidation in isolated rat hepatocytes.

Metabolism of menadione (2-methyl-1,4-naphthoquinone) results in the rapid oxidation of NADPH within isolated rat hepatocytes. The glutathione redox cycle is thought to play a major role in the consumption of NADPH during menadione metabolism, chiefly through glutathione reductase (GSSG-reductase). This enzyme reduces oxidized glutathione (GSSG), formed via the glutathione-peroxidase reaction, with the concomitant oxidation of NADPH. To explore the relationship between GSSG-reductase and the consumption of NADPH during menadione metabolism, isolated rat hepatocyte suspensions were exposed to non-lethal and lethal menadione concentrations (100 and 300 microM respectively) following the inhibition of GSSG-reductase with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Menadione produced a concentration-related depletion of GSH (measured as non-protein sulfhydryl content) which was potentiated markedly by BCNU. Menadione toxicity was potentiated at either concentration by BCNU based on lactate dehydrogenase leakage at 2 hr. In addition, the NADPH content of isolated hepatocytes rapidly declined following exposure to either concentration of menadione. However, at the lower menadione concentration (100 microM), the NADPH content returned to control values or above by 60 min, whereas the NADPH content of cells exposed to 300 microM menadione with or without BCNU remained depressed for the duration of the incubation. These data suggest that, although NADPH is required by GSSG-reductase for the reduction of GSSG to GSH during quinone-induced oxidative stress, this pathway does not appear to be the major route by which NADPH is consumed during the metabolism of menadione in isolated hepatocytes.

The mechanism of acute cytotoxicity of triethylphosphine gold(I) complexes. I. Characterization of triethylphosphine gold chloride-induced biochemical and morphological changes in isolated hepatocytes.

Triethylphosphine gold complexes are effective therapeutic agents used for the treatment of rheumatoid arthritis. Many of those molecules are also highly cytotoxic in vitro and can inhibit DNA and protein synthesis. Preliminary experiments have indicated that triethylphosphine gold chloride (TEPAu) may induce the peroxidative decomposition of cellular membrane lipids. The purpose of these investigations therefore was to evaluate the role of lipid peroxidation in the mechanism of acute cytotoxicity of a gold(I) coordination complex, TEPAu, and to examine the early morphological and biochemical changes induced by TEPAu in suspensions of freshly isolated rat hepatocytes. TEPAu caused a rapid loss of cell viability at concentrations above 25 microM which was significantly different from that of control by 60 min and complete by 180 min of incubation. TEPAu also depleted cells of reduced glutathione (GSH) and increased the formation of malondialdehyde (MDA) by 60 min. Incubation of cells with either of the antioxidants, N,N'-diphenyl-p-phenylenediamine (DPPD) or promethazine blocked the formation of MDA but did not alter the time course of cell death or GSH depletion induced by TEPAu. TEPAu also caused a decrease in cellular NADPH and NADH by 10 min. Electron microscopy of hepatocytes exposed to TEPAu revealed early (5 min) formation of flocculent electron-dense precipitates within condensed mitochondria. These changes characteristically preceded cell death. Energy-dispersive electron-probe microanalysis indicated that the electron-dense precipitates did not contain detectable amounts of gold. TEPAu also caused a concentration-dependent decrease in cellular ATP and oxygen consumption in isolated rat hepatocytes. These data suggest that lipid peroxidation, as indicated by the formation of MDA, is probably not a major mechanism by which triethylphosphine gold complexes lethally injure cells. These data, therefore, suggest that mitochondria may be target organelles in TEPAu-induced toxicity to isolated rat hepatocytes.

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.

Menadione-induced oxidative stress in hepatocytes isolated from fed and fasted rats: the role of NADPH-regenerating pathways.

Isolated hepatocytes were prepared from fed and fasted rats and exposed to a range of menadione (2-methyl-1,4-naphthoquinone) concentrations. Menadione (300 microM) caused a rapid decline in the (NADPH)/(NADPH + NADP+) ratio from 0.85 to 0.39 within 15 min, with further decreases over the 90-min incubation period in cells isolated from fed animals. This decrease of NADPH resulted from oxidation to NADP+ since there was no loss of total pyridine nucleotide (NADP+ + NADPH) content. In addition, menadione (100 microM) caused a five-fold stimulation of the hexose monophosphate shunt by 30 min as indicated by the oxidation of [1-14C]glucose. LDH leakage was slightly but significantly elevated (30% of total) following exposure of cells to 300 microM menadione for 2 hr. Menadione caused a concentration-dependent GSH depletion: 100 microM menadione caused no depletion and 200 and 300 microM menadione caused a 75 and 95% decrease, respectively. Intracellular NADPH was significantly reduced within 30 min by 100 and 200 microM menadione but then returned to values equivalent to or greater than control by 60 min. In contrast, a sustained decrease of NADPH was produced by 300 microM menadione (5% of control after 2 hr). A marked potentiation of the oxidative cell injury produced by menadione was observed in hepatocytes prepared from 24-hr-fasted rats. LDH leakage was 50 and 95% when these cells were exposed to 100 and 200 microM menadione, respectively. Menadione (100 and 200 microM) also caused a marked GSH depletion (95% of control) by 90 min. In contrast to cells isolated from fed animals, menadione (100 and 200 microM) caused an 85% depletion of NADPH by 60 min in cells isolated from fasted rats. This potentiation of menadione-induced oxidative injury was not related to the decreased GSH content produced by fasting since menadione toxicity was not potentiated in control cells partially depleted of GSH by diethyl maleate. A further comparison was made between cells isolated from fasted rats and incubated either with or without supplemental glucose in order to determine a possible protective effect by glucose. In this comparison a significant (p less than 0.05) glucose effect was indeed observed in the direction of preventing GSH and NADPH depletion, as well as attenuating LDH leakage, when hepatocytes were exposed to either 50 or 100 microM menadione.(ABSTRACT TRUNCATED AT 400 WORDS)

In vivo and in vitro hepatotoxicity and metabolism of acetaminophen in Syrian hamsters.

The purpose of this investigation was to correlate the in vitro and in vivo toxicity of the hepatotoxicant, acetaminophen. Hamsters were pretreated with either phenobarbital (70 mg/kg) or 3-methylcholanthrene (20 mg/kg) or an appropriate vehicle for 3 days. In non-pretreated hamsters, single doses of acetaminophen (200-400 mg/kg i.p.) caused elevations in serum alanine aminotransferase and sorbitol dehydrogenase activities in a dose-related manner. 3-Methylcholanthrene significantly potentiated, while phenobarbital significantly reduced acetaminophen-induced elevations in serum liver enzyme activities. Both phenobarbital and 3-methylcholanthrene significantly reduced acetaminophen plasma T1/2 while only 3-methylcholanthrene increased APAP clearance. Phenobarbital pretreatment increased the urinary excretion of APAP-glucuronide. Exposure of isolated hepatocytes to acetaminophen (0.01-2.0 mM) resulted in concentration-related decreases in hepatocyte viability. Cells from 3-methylcholanthrene-pretreated hamsters were more markedly susceptible to acetaminophen toxicity than cells isolated from non-induced animals. Hepatocytes isolated from phenobarbitol pretreated animals were slightly but significantly more susceptible to acetaminophen toxicity than cells from control animals. Hepatocytes isolated from 3-methylcholanthrene pretreated animals had increased formation of an acetaminophen-glutathione conjugate compared to control. Pre-treatment with either phenobarbital or 3-methylcholanthrene enhanced glucuronidation of acetaminophen in vitro. These data demonstrate a lack of correlation between in vivo hepatotoxicity and in vitro cytotoxicity in that phenobarbital pre-treatment protected hamsters from acetaminophen-induced liver toxicity, but failed to protect hepatocytes exposed to acetaminophen in vitro.