Comparative effects of sulfhydryl compounds on target organellae,nuclei and mitochondria,of hydroxylated fullerene‐induced cytotoxicity in isolated rat hepatocytes

DNA damage and cytotoxicity induced by a hydroxylated fullerene [C₆₀(OH)₂₄], which is a spherical nanomaterial and/or a water‐soluble fullerene derivative, and their protection by sulfhydryl compounds were studied in freshly isolated rat hepatocytes. The exposure of hepatocytes to C₆₀(OH)₂₄ at a concentration of 50 μM caused time (0 to 3 h)‐dependent cell death accompanied by the formation of cell surface blebs, the loss of cellular levels of ATP and reduced glutathione, accumulation of glutathione disulfide, and induction of DNA fragmentation assayed using alkali single‐cell agarose‐gel electrophoresis. C₆₀(OH)₂₄‐induced cytotoxicity was effectively prevented by pretreatment with sulfhydryl compounds. N‐acetyl‐L‐cysteine (NAC), L‐cysteine and L‐methionine, at a concentration of 2.5 mM, ameliorated cell death, accompanied by a decrease in cellular ATP levels, formation of cell surface blebs, induction of reactive oxygen species (ROS) and loss of mitochondrial membrane potential caused by C₆₀(OH)₂₄. In addition, DNA fragmentation caused by C₆₀(OH)₂₄ was also inhibited by NAC, whereas an antioxidant ascorbic acid did not affect C₆₀(OH)₂₄‐induced cell death and DNA damage in rat hepatocytes. Taken collectively, these results indicate that incubation of rat hepatocytes with C₆₀(OH)₂₄ elicits DNA damage, suggesting that nuclei as well as mitochondria are target sites of the hydroxylated fullerene; and induction of DNA damage and oxidative stress is ameliorated by an increase in cellular GSH levels, suggesting that the onset of toxic effects may be partially attributable to a thiol redox‐state imbalance caused by C₆₀(OH)₂₄. Copyright © 2015 John Wiley & Sons, Ltd.     

Cytotoxic effects of hydroxylated fullerenes on isolated rat hepatocytes via mitochondrial dysfunction

The cytotoxic effects of hydroxylated fullerenes, also termed fullerenols or fullerols [C₆₀(OH) _n ], which are known nanomaterials and water-soluble fullerene derivatives, were studied in freshly isolated rat hepatocytes. The exposure of hepatocytes to C₆₀(OH)₂₄ caused not only concentration (0–0.25 mM)- and time (0–3 h)-dependent cell death accompanied by the formation of cell blebs, loss of cellular ATP, reduced glutathione (GSH), and protein thiol levels, but also the accumulation of glutathione disulfide and malondialdehyde, indicating lipid peroxidation. Of the other analogues examined, the cytotoxic effects of C₆₀(OH)₁₂ and fullerene C₆₀ at a concentration of 0.125 mM were less than those of C₆₀(OH)₂₄. The loss of mitochondrial membrane potential and generation of oxygen radical species in hepatocytes incubated with C₆₀(OH)₂₄ were greater than those with C₆₀(OH)₁₂ and fullerene C₆₀. In the oxygen consumption of mitochondria isolated from rat liver, the ratios of state-3/state-4 respiration were more markedly decreased by C₆₀(OH)₂₄ and C₆₀(OH)₁₂ compared with C₆₀. In addition, C₆₀(OH)₂₄ and C₆₀(OH)₁₂ resulted in the induction of the mitochondrial permeability transition (MPT), and the effects of C₆₀(OH)₁₂ were less than those of C₆₀(OH)₂₄. Taken collectively, these results indicate that (a) mitochondria are target organelles for fullerenols, which elicit cytotoxicity through mitochondrial failure related to the induction of the MPT, mitochondrial depolarization, and inhibition of ATP synthesis in the early stage and subsequently oxidation of GSH and protein thiols, and lipid peroxidation through oxidative stress at a later stage; and (b) the toxic effects of fullerenols may depend on the number of hydroxyl groups participating in fullerene in rat hepatocytes.     

The polyhydroxylated fullerene derivative C60(OH)24 protects mice from ionizing-radiation-induced immune and mitochondrial dysfunction

Although the protective effect of the polyhydroxylated fullerene derivative C₆₀(OH)_n against ionizing radiation is an area of much interest, the mechanisms relating to how polyhydroxylated fullerene derivatives improve mitochondrial dysfunction remain unknown. In order to find new and effective radioprotective agents, we synthesized a new polyhydroxylated fullerene molecule with 24 hydroxyl groups of known positions on C₆₀ and studied its protective effects in mice subjected to irradiation. Mice were pretreated with C₆₀(OH)₂₄ for 2 weeks (daily, 40 mg/kg i. p.), then subjected to a lethal dose of whole body γ-irradiation (from a ⁶⁰Co source). Survival was observed for 30 days after irradiation. Immune and mitochondrial dysfunction and oxidative damage were analyzed in mice with the same C₆₀(OH)₂₄ pretreatment and irradiation except that the animals were euthanized at day 5 after the irradiation. It was found that 2-week C₆₀(OH)₂₄ pretreatment effectively reduced whole body irradiation-induced mortality without apparent toxicity. C₆₀(OH)₂₄ pretreatment also showed significant protective effects against ionizing-radiation-induced decreases in immune and mitochondrial function and antioxidant defense in the liver and spleen. These results suggest that the polyhydroxylated fullerene derivative C₆₀(OH)₂₄ protects against ionizing-radiation-induced mortality, possibly by enhancing immune function, decreasing oxidative damage and improving mitochondrial function.     

Cytotoxic effects of thioxanthone derivatives as photoinitiators on isolated rat hepatocytes

Thioxanthone and its analogues, 2- or 4-isopropylthioxanthone, 2-chlorothioxanthone , 2,4-diethylthioxanthone (DETX) and xanthone, are used as photoinitiators of ultraviolet (UV) light-initiated curable inks. As these photoinitiators were found in numerous food/beverage products packaged in cartons printed with UV-cured inks, the cytotoxic effects and mechanisms of these compounds were studied in freshly isolated rat hepatocytes. The toxicity of DETX was greater than that of other compounds. DETX elicited not only concentration (0–2.0 mm )- and time (0–3 hours)-dependent cell death accompanied by the depletion of cellular adenosine triphosphate (ATP), and reduced glutathione (GSH) and protein thiol levels, but also the accumulation of GSH disulfide and malondialdehyde. Pretreatment of hepatocytes with either fructose at a concentration of 10 mm or N-acetyl-l -cysteine (NAC) at a concentration of 5.0 mm ameliorated DETX (1 mm )-induced cytotoxicity. Further, the exposure of hepatocytes to DETX resulted in the induction of reactive oxygen species (ROS) and loss of mitochondrial membrane potential, both of which were partially prevented by the addition of NAC. These results indicate that: (1) DETX-induced cytotoxicity is linked to mitochondrial failure and depletion of cellular GSH; (2) insufficient cellular ATP levels derived from mitochondrial dysfunction were, at least in part, ameliorated by the addition of fructose; and (3) GSH loss and/or ROS formation was prevented by NAC. Taken collectively, these results suggest that the onset of toxic effects caused by DETX may be partially attributable to cellular energy stress as well as oxidative stress.     

Preventive effects of fructose and N‐acetyl‐L‐cysteine against cytotoxicity induced by the psychoactive compounds N‐methyl‐5‐(2‐aminopropyl)benzofuran and 3,4‐methylenedioxy‐N‐methamphetamine in isolated rat hepatocytes

Psychoactive compounds, N‐methyl‐5‐(2‐aminopropyl)benzofuran (5‐MAPB) and 3,4‐methylenedioxy‐N‐methamphetamine (MDMA), are known to be hepatotoxic in humans and/or experimental animals. As previous studies suggested that these compounds elicited cytotoxicity via mitochondrial dysfunction and/or oxidative stress in rat hepatocytes, the protective effects of fructose and N‐acetyl‐l ‐cysteine (NAC) on 5‐MAPB‐ and MDMA‐induced toxicity were studied in rat hepatocytes. These drugs caused not only concentration‐dependent (0–4 mm ) and time‐dependent (0–3 hours) cell death accompanied by the depletion of cellular levels of adenosine triphosphate (ATP) and glutathione (reduced form; GSH) but also an increase in the oxidized form of GSH. The toxic effects of 5‐MAPB were greater than those of MDMA. Pretreatment of hepatocytes with either fructose at a concentration of 10 mm or NAC at a concentration of 2.5 mm prevented 5‐MAPB?/MDMA‐induced cytotoxicity. In addition, the exposure of hepatocytes to 5‐MAPB/MDMA caused the loss of mitochondrial membrane potential, although the preventive effect of fructose was weaker than that of NAC. These results suggest that: (1) 5‐MAPB?/MDMA‐induced cytotoxicity is linked to mitochondrial failure and depletion of cellular GSH; (2) insufficient cellular ATP levels derived from mitochondrial dysfunction were ameliorated, at least in part, by the addition of fructose; and (3) GSH loss via oxidative stress was prevented by NAC. Taken collectively, these results indicate that the onset of toxic effects caused by 5‐MAPB/MDMA may be partially attributable to cellular energy stress as well as oxidative stress.     

Arsenic induces mitochondria-dependent apoptosis by reactive oxygen species generation rather than glutathione depletion in Chang human hepatocytes

This study was conducted to evaluate the possible involvement of mitochondrial pathway in NaAsO₂-induced apoptosis and the role of reactive oxygen species (ROS) and reduced glutathione (GSH) in the apoptotic effect in Chang human hepatocytes. The MTT assay demonstrated that sodium arsenite (NaAsO₂) treatment for 24 h caused a dose-dependent decrease of cell viability. NaAsO₂ treatment (0–30 μM) was also found to induce phosphatidylserine externalization, a hallmark of apoptosis; to disrupt the mitochondrial membrane potential (Δψ _m ); to cause the release of cytochrome c into the cytosol, and to trigger cleavage of caspase-3 and poly (ADP-ribose) polymerase (PARP) in a dose-dependent manner. All these changes were accompanied with the enhanced generation of intracellular ROS and malondialdehyde (MDA). Increase of intracellular GSH also coincided unexpectedly. Moreover, the extracellular addition of N-acetyl-l-cysteine (NAC, 5 mM) effectively reduced the generation of ROS and MDA, and rescued the cells from NaAsO₂ induced apoptosis and related alteration of mitochondria. These data suggest that the arsenic-induced cell apoptosis occurs though the mitochondrial pathway, and is mostly dependent on generation of ROS rather than GSH depletion in Chang human hepatocytes.     

In vitro toxicity assessment of three hydroxylated fullerenes in human skin cells

Carbon fullerenes possess unique properties and their interactions with biomolecules have widespread applications. Functionalization of fullerenes with hydroxyl groups (fullerenols) can increase the solubility and potential for cellular interaction, but the health and safety effects of varying degrees of fullerene hydroxylation in biological systems is poorly understood. Existing reports regarding the toxicity and inflammatory potential of fullerenols give conflicting conclusions. To further elucidate the potential for toxicity of fullerenols, human epidermal keratinocytes (HEK) were exposed to fullerenols (low (C₆₀(OH)₂₀), medium (C₆₀(OH)₂₄), and high (C₆₀(OH)₃₂)) at concentrations ranging from 0.000544–42.5 μg/ml for 24 and 48 h. A statistically significant (p < 0.05) decrease in viability with alamar Blue (aB) was noted only with C₆₀(OH)₃₂ at 42.5 μg/ml after 24 h. Nanoparticle (NP) controls showed minimal NP/assay interference of the three fullerenols with the aB viability assay. Normalized IL-8 concentration for C₆₀(OH)₂₀ was not significantly different from control, while C₆₀(OH)₂₄ and C₆₀(OH)₃₂ showed a significant decrease at 24 and 48 h. These results suggest that different hydroxylation of fullerenes caused no cytotoxicity or inflammation up to 8.55 μg/ml. These findings suggest that extrapolation across similar NP will be dependent upon surface chemistry and concentration which may affect the degree of agglomeration and thus biological effects.     

Modulation of Tumor Necrosis Factor-mediated Cell Death by Fullerenes

Purpose The fullerene (C₆₀/C₇₀ mixture—C_60/70) nanocrystalline suspension prepared by solvent exchange method using tetrahydrofyran (THF/nC_60/70) and polyhydroxylated C_60/70 [C_60/70(OH) _n ] were compared for their ability to modulate cytotoxicity of the proinflammatory cytokine tumor necrosis factor (TNF). Materials and Methods TNF-induced cytotoxicity was assessed in L929 fibrosarcoma cells by crystal violet assay. The type of cell death (apoptosis/necrosis), production of reactive oxygen species, mitochondrial depolarization and caspase activation were determined by flow cytometry using the appropriate reporter dyes. Results THF/nC_60/70 augmented, while C_60/70(OH) _n reduced the cytotoxicity of TNF. The numbers of cells undergoing apoptosis/necrosis, as well as of those displaying the activation of apoptosis-inducing enzymes of caspase family, were respectively increased or reduced by THF/nC_60/70 or C_60/70(OH) _n . The antioxidant N-acetylcysteine and mitochondrial permeability transition inhibitor cyclosporin A each partly blocked the cytotoxic action of TNF, indicating the involvement of oxidative stress and mitochondrial dysfunction in the TNF cytotoxicity. Accordingly, THF/nC_60/70 or C_60/70(OH) _n potentiated or suppressed, respectively, TNF-triggered oxidative stress and mitochondrial depolarization. Conclusion The ability of different fullerene preparations to modulate TNF-induced oxidative stress and subsequent cell death suggests their potential value in the TNF-based cancer therapy or prevention of TNF-dependent tissue damage.     

Cytotoxicity of ortho-phenylphenol in isolated rat hepatocytes.

The effects of ortho-phenylphenol (OPP) and its metabolites, phenyl-hydroquinol (PHQ) and phenyl-benzoquinone (PBQ), on isolated rat hepatocytes were investigated. Addition of OPP (0.5-1.0 mM) to cells caused a dose-dependent cell death accompanied by the depletion of intracellular levels of ATP, glutathione (GSH) and protein thiols. GSH loss correlated with the formation of oxidized GSH. In addition, PHQ and especially PBQ (both at 0.5 mM) resulted in acute cell death with rapid depletion of ATP, GSH and protein thiols, and further low doses of PBQ (10-50 microM) elicited serious impairment of mitochondrial functions related to oxidative phosphorylation and Ca fluxes in isolated liver mitochondria. These results indicate that mitochondria are a target for these compounds and that OPP is itself toxic to hepatocytes even when metabolism is inhibited. The loss of cellular GSH and protein thiols accompanied by the impairment of mitochondrial function may be the main mechanisms of cytotoxicity induced by OPP and its metabolites.     

S-nitrosyl glutathione-mediated hepatocyte cytotoxicity

1. The addition of n-butyl nitrite (BN) to isolated rat hepatocytes caused rapid S-nitrosyl glutathione (GSNO) formation, then a concomitant decrease in protein thiols, followed by a marked ATP depletion. Cytotoxic concentrations of BN also caused lipid peroxidation after a long lag period but before cytotoxicity ensued.

2. Prior glutathione (GSH) depletion protected hepatocytes against the BN-induced decrease in protein thiols, ATP depletion, lipid peroxidation and cytotoxicity. Thus cytotoxic effects were thought to be mediated via GSNO formed by reaction of BN with GSH, a reaction catalysed by the cytosolic fraction.

3. Cytotoxicity and lipid peroxidation, but not depletion of GSH, protein thiols or ATP, could be averted by the subsequent addition of antioxidants or the iron chelator, desferoxamine.

4. Addition of the thiol reductant, dithiothreitol to BN-treated hepatocytes restored GSH and protein thiols and also prevented cytotoxicity.     

Lipid peroxidation and protein thiol depletion are not involved in the cytotoxicity of N-hydroxy-2-acetylaminofluorene in isolated rat hepatocytes

Freshly isolated rat hepatocytes were used to study the mechanism of cell death induced by N-hydroxy-2-acetylaminofluorene (N-OH-AAF). Exposure to 1.0 mM N-OH-AAF resulted in more than 90% cell death (as measured by LDH leakage) of hepatocytes isolated from male rats within 6 hr. Only 36% of the hepatocytes isolated from female rats died within this period. When inorganic sulfate was omitted from the incubation medium, a 6 hr exposure to 1.0 mM N-OH-AAF resulted in only 40% cell death of male hepatocytes. These findings are in accordance with the sex difference and sulfation dependence of N-OH-AAF hepatotoxicity observed in the rat in vivo. N-OH-AAF decreased glutathione (GSH) in male hepatocytes in a concentration-dependent manner. This GSH consumption was only partly dependent on the presence of inorganic sulfate. No lipid peroxidation was observed during N-OH-AAF exposure; N-OH-AAF even prevented endogenous and diethyl maleate (DEM)-induced lipid peroxidation. No reduction of free protein thiol groups was found after exposure to N-OH-AAF, even after 75% cell death had occurred. A reduction of protein thiols after N-OH-AAF exposure was observed in GSH depleted hepatocytes (obtained by DEM plus vitamin E pretreatment). Under these conditions N-OH-AAF-induced cell death occurred earlier. Therefore, GSH protects against protein thiol depletion by N-OH-AAF in control cells. N-OH-AAF-induced cell death was preceded by a loss of intracellular ATP. It is concluded, therefore, that neither lipid peroxidation nor depletion of protein thiols, but possibly loss of intracellular ATP, is involved in the sulfation-dependent cytotoxic mechanism of N-OH-AAF in isolated rat hepatocytes.     

Protection of rats against 3-butene-1,2-diol-induced hepatotoxicity and hypoglycemia by N-acetyl-l-cysteine

3-Butene-1,2-diol (BDD), an allylic alcohol and major metabolite of 1,3-butadiene, has previously been shown to cause hepatotoxicity and hypoglycemia in male Sprague-Dawley rats, but the mechanisms of toxicity were unclear. In this study, rats were administered BDD (250 mg/kg) or saline, ip, and serum insulin levels, hepatic lactate levels, and hepatic cellular and mitochondrial GSH, GSSG, ATP, and ADP levels were measured 1 or 4 h after treatment. The results show that serum insulin levels were not causing the hypoglycemia and that the hypoglycemia was not caused by an enhancement of the metabolism of pyruvate to lactate because hepatic lactate levels were either similar (1 h) or lower (4 h) than controls. However, both hepatic cellular and mitochondrial GSH and GSSG levels were severely depleted 1 and 4 h after treatment and the mitochondrial ATP/ADP ratio was also lowered 4 h after treatment relative to controls. Because these results suggested a role for hepatic cellular and mitochondrial GSH in BDD toxicity, additional rats were administered N-acetyl-l-cysteine (NAC; 200 mg/kg) 15 min after BDD administration. NAC treatment partially prevented depletion of hepatic cellular and mitochondrial GSH and preserved the mitochondrial ATP/ADP ratio. NAC also prevented the severe depletion of serum glucose concentration and the elevation of serum alanine aminotransferase activity after BDD treatment without affecting the plasma concentration of BDD. Thus, depletion of hepatic cellular and mitochondrial GSH followed by the decrease in the mitochondrial ATP/ADP ratio was likely contributing to the mechanisms of hepatotoxicity and hypoglycemia in the rat.     

The toxicity of N-methyl-alpha-methyldopamine to freshly isolated rat hepatocytes is prevented by ascorbic acid and N-acetylcysteine

In the past decade, clinical evidence has increasingly shown that the liver is a target organ for 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy") toxicity. The aims of the present in vitro study were: (1) to evaluate and compare the hepatotoxic effects of MDMA and one of its main metabolites, N-methyl-alpha-methyldopamine (N-Me-alpha-MeDA) and (2) to investigate the ability of antioxidants, namely ascorbic acid and N-acetyl-L-cysteine (NAC), to prevent N-Me-alpha-MeDA-induced toxic injury, using freshly isolated rat hepatocytes. Cell suspensions were incubated with MDMA or N-Me-alpha-MeDA in the final concentrations of 0.1, 0.2, 0.4, 0.8, and 1.6 mM for 3 h. To evaluate the potential protective effects of antioxidants, cells were preincubated with ascorbic acid in the final concentrations of 0.1 and 0.5 mM, or NAC in the final concentrations of 0.1 and 1 mM for 15 min before treatment with 1.6 mM N-Me-alpha-MeDA for 3 h (throughout this incubation period the cells were exposed to both compounds). The toxic effects were evaluated by measuring the cell viability, glutathione (GSH) and glutathione disulfide (GSSG), ATP, and the cellular activities of GSH peroxidase (GPX), GSSG reductase (GR), and GSH S-transferase (GST). MDMA induced a concentration- and time-dependent GSH depletion, but had a negligible effect on cell viability, ATP levels, or on the activities of GR, GPX, and GST. In contrast, N-Me-alpha-MeDA was shown to induce not only a concentration- and time-dependent depletion of GSH, but also a depletion of ATP levels accompanied by a loss in cell viability, and decreases in the antioxidant enzyme activities. For both compounds, GSH depletion was not accompanied by increases in GSSG levels, which seems to indicate GSH depletion by adduct formation. Importantly, the presence of ascorbic acid (0.5 mM) or NAC (1 mM) prevented cell death and GSH depletion induced by N-Me-alpha-MeDA. The results provide evidence that MDMA and its metabolite N-Me-alpha-MeDA induce toxicity to freshly isolated rat hepatocytes. Oxidative stress may play a major role in N-Me-alpha-MeDA-induced hepatic toxicity since antioxidant defense systems are impaired and administration of antioxidants prevented N-Me-alpha-MeDA toxicity.     

Cytotoxic effects of 2,6-di-tert-butyl-4-methylphenyl N-methylcarbamate (terbutol) herbicide on hepatocytes and mitochondria isolated from male rats

The cytotoxic effects of 2,6-di-tert-butyl-4-methylphenyl N-methylcarbamate (terbutol) and its major metabolites were investigated in freshly isolated rat hepatocytes. Terbutol and its metabolite, especially 2,6-di-tert-butyl-4-methylphenyl carbamate (N-demethylterbutol), at a concentration of 1.0 mM resulted in a time dependent cell killing accompanied by losses of intracellular ATP, protein thiols, and glutathione (GSH) and the accumulation of oxidized GSH. Supplementation of the hepatocyte suspension with 5 mM N-acetylcysteine, a precursor of intracellular GSH, inhibited the cytotoxicity of N-demethylterbutol. In mitochondria isolated from rat liver, terbutol and its metabolites impaired respiration related to oxidative phosphorylation and the potency of their toxicity is associated with impairment of mitochondrial respiration. These results indicate that N-demethylterbutol is the most cytotoxic followed by terbutol and other metabolites, and that both the mitochondrial respiratory system and protein thiols are important targets for these compounds.     

The toxicity of disulphides to isolated hepatocytes and mitochondria

The disulfide metabolites of thiono-sulfur drugs were found to be about 50 to 100 times more toxic to isolated rat hepatocytes than the corresponding parent drugs. The order of decreasing cytotoxicity for the disulfide metabolites was disulfiram greater than propylthiouracil disulfide greater than formamidine disulfide greater than phenylthiourea disulfide greater than thiobenzamide disulfide greater than cystamine. Depletion of intracellular GSH levels preceded cytotoxicity. GSH could be restored and cytotoxicity averted by adding the thiol reducing dithiothreitol. Depletion of GSH with diethylmaleate potentiated the toxicity of disulfides 3 to 4-fold confirming the protective role of GSH in disulfide toxicity. The toxicity of disulfiram was increased 4-fold in cells pretreated with ATP (0.8 mM) to effect a transient increase in cytosolic Ca2+ suggesting an impairment of Ca2+ homeostasis by the toxicant. Disulfiram (200 microM) rapidly depleted hepatocyte ATP levels within 15 minutes which suggests that ATP production is inhibited. The disulfide effectiveness at causing mitochondrial Ca2+ release was similar to their effectiveness at inducing hepatocyte cytotoxicity. These results suggest that hepatocyte toxicity is the result of oxidative inactivation of membrane protein thiols that regulate intracellular Ca2+ homeostasis.     

Effect of diamide and reduced glutathione on the elevated levels of cyclic AMP in rat pancreatic islets exposed to glucose,p-chloromercuribenzoate and aminophylline

Summary In rat pancreatic islets the effects of diamide, which has been shown to decrease islet levels of reduced glutathione (GSH), and of exogenous GSH were investigated on cyclic AMP as increased by glucose, p-chloromercuribenzoate, and aminophylline. In addition the effect of diamide on islet ATP level, low K _m and high K _m phosphodiesterases was studied.Diamide (0.1 mM) inhibited the increase of cyclic AMP (cAMP) in response to glucose (16.7 mM), and p-chloromercuribenzoate (1 mM) in the presence of 5.6 mM glucose. No inhibitory effect of diamide could be demonstrated when cAMP was raised by 10 mM aminophylline in the presence of 5.6 mM glucose. The glucose (27.7 mM) stimulated increase of cAMP was further augmented by GSH (0.4 mM) whereas GSH in the presence of 5.6 mM glucose had no such effect. Diamide neither affected islet high K _m nor low K _m cAMP-phosphodiesterases. Diamide (0.1 mM) as used in this study did not affect islet AMP levels.A concentration dependent decrease of ATP was observed, however, with higher concentrations of diamide (0.25, 0.5 and 1.0 mM).It is suggested that the accumulation of islet cAMP in response to glucose and para-chloromercuribenzoate depends on the redox state of islet thiols. Since thiol oxidant diamide neither affected cAMP-phosphodiesterase activities nor inhibited aminophylline induced accumulation of cAMP in the presence of low glucose the possibility is raised that in pancreatic islets the formation of cAMP rather than its degradation depends on the redox state of islet thiols.Parts of this work were reported previously and published as abstracts (Ammon and Heinzl 1979; Kallenberger and Ammon 1980)     

Protection by tetrahydroxystilbene glucoside against neurotoxicity induced by MPP+: the involvement of PI3K/Akt pathway activation

Recent studies indicate that there is interaction between the glutamatergic neurotransmitters system and lead neurotoxicity. Previously, we have demonstrated the potential effects of glutamate in lead-induced cell death in PC12 cells and the protective role of the novel thiol antioxidant, N-acetylcysteine amide (NACA). The current study (1) investigated the potential effects of glutamate on lead exposed CD-1 mice, (2) evaluated the protective effects of NACA against glutamate and lead toxicity in CD-1 mice, and (3) compared the results with N-aceytylcysteine (a well-known thiol antioxidant). Oxidative stress parameters, including glutathione (GSH), oxidized glutathione (GSSG), GSH/GSSG, and malondialdehyde (MDA) levels, were evaluated. Blood and tissue lead levels, glutamate/glutamine (Glu/Gln) ratios, GS activity, and phospholipase-A₂ (PLA₂) were also analyzed. Results indicated that lead and glutamate decreased GSH levels in the red blood cells, brains, livers, and kidneys. Exposure to glutamate and lead elevated the MDA levels and PLA₂ activity. NACA and N-acetylcysteine (NAC) provided protection against the detrimental effects of lead by decreasing the blood and tissue lead levels, restoring intracellular GSH levels, and decreasing the MDA levels. NACA and NAC also increased the GS activity thereby decreasing Glu/Gln levels. However, NACA appeared to have better chelating and antioxidant properties than NAC, due to its higher liphophilicity and its ability to cross the blood-brain barrier.     

Mechanism of Inhibition of Aflatoxin B1 - Dna Binding in the Liver by Butylated Hydroxyanisole Pretreatment of Rats

Abstract

Previous studies have demonstrated that pretreatment of rats with butylated hydroxyanisole (BHA) inhibited aflatoxin B₁ (AFB₁)-DNA binding and AFB₁ hepatocarcinogenesis. Our detailed studies on the effect of BHA pretreatment of rats on both AFB₁-DNA binding and AFB₁-glutathione (AFB₁-SG) conjugation with hepatosubcellular fractions, isolated hepatocytes and in intact animals are reviewed. For these studies, young male rats were fed a semisynthetic diet with or without 0.75% BHA for 2 wks. Even though there were no significant differences either in microsomal cytochrome P-450 content or microsome-mediated AFB₁ binding to exogenous DNA with cytochrome P-450 from control or treated rats, there were large differences in GSH S-transferase activity with treated cytosols showing 100% higher activity than the controls. Kinetics of cytosolic inhibition of microsome-mediated AFB₁-DNA binding and formation of AFB₁-SG conjugate indicated that the inhibition of AFB₁-DNA binding was greater with cytosols from treated compared to the controls with the concomitant formation of AFB₁-SG conjugate. Reconstitution studies with intact nuclei, microsomes and cytosol indicated more AFB₁-DNA binding with the control than with BHA-treated animals. In isolated hepatocyte system, at 2 μM level of AFB₁, AFB₁-DNA binding in treated hepatocytes was about 15% of controls whereas thiol conjugation was 5 fold higher in the treated than in control hepatocytes. Addition of 1 mM styrene oxide caused about 100% and 9 fold increase in AFB₁-DNA binding in control and treated hepatocytes respectively with corresponding decreases in thiol conjugation. In intact rats, BHA treatment reduced hepatic AFB₁-DNA binding to 15% of controls with concomitant increase in biliary excretion of AFB₁-SG conjugate. These results indicate that the induced cytosolic GSH S-transferases after BHA treatment of rats play a significant role in inhibiting hepatic AFB₁-DNA binding and AFB₁ hepatocarcinogenesis by inactivation of the reactive AFB₁-epoxide.     

Therapeutic effect of liposomal-N-acetylcysteine against acetaminophen-induced hepatotoxicity

Abstract

Background: Acetaminophen (APAP) is an antipyretic analgesic drug that when taken in overdose causes depletion of glutathione (GSH) and hepatotoxicity. N-acetylcysteine (NAC) is the antidote of choice for the treatment of APAP toxicity; however, due to its short-half-life repeated dosing of NAC is required.

Purpose: To determine whether a NAC-loaded liposomal formulation (Lipo-NAC) is more effective than the conventional NAC in protecting against acute APAP-induced hepatotoxicity.

Methods: Male Sprague–Dawley rats were challenged with an intragastric dose of APAP (850?mg/kg b.wt.); 4?h later, animals were administered saline, NAC, Lipo-NAC or empty liposomes and sacrificed 24?h post-APAP treatment.

Results: APAP administration resulted in hepatic injury as evidenced by increases in plasma bilirubin, alanine (AST) and aspartate (ALT) aminotransferase levels and tissue levels of lipid peroxidation and myeloperoxidase as well as decreases in hepatic levels of reduced GSH, GSH peroxidase and GSH reductase. Treatment of animals with Lipo-NAC was significantly more effective than free NAC in reducing APAP-induced hepatotoxicity. Histological evaluation showed that APAP caused periacinar hepatocellular apoptosis and/or necrosis of hepatocytes around the terminal hepatic venules which was reduced by NAC treatment, the degree of reduction being greater for Lipo-NAC.

Conclusion: These data suggest that administration of Lipo-NAC ameliorated the APAP-induced hepatotoxicity.     

Effects of dicoumarol on cytotoxicity caused by tert-butylhydroquinone in isolated rat hepatocytes

The effects of dicoumarol, an inhibitor of DT-diaphorase, on the cytotoxicity of tert-butylhydroquinone (tBHQ) were studied in freshly isolated rat hepatocytes. Addition of tBHQ (0.5 mM) to hepatocytes resulted in a time-dependent cell death accompanied by depletion of intracellular ATP, glutathione (GSH), and protein thiols. Pretreatment of hepatocytes with dicoumarol (30 μM) did not affect cell viability or cellular levels of ATP, GSH, or protein thiols during the incubation period; however, dicoumarol did promote the appearance of cell blebs and the depletion of ATP and protein thiols induced by tBHQ and ultimately enhanced the cytotoxicity of tBHQ.