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.     

Stimulation of endogenous endonuclease activity in hepatocytes exposed to oxidative stress

Incubation of freshly isolated rat hepatocytes with moderately toxic concentrations of menadione [2-methyl-1,4-naphthoquinone) resulted in chromatin condensation and progressive DNA fragmentation, suggestive of the stimulation of an endogenous endonuclease activity previously found to be involved in programmed cell death (apoptosis). Endonuclease activation followed upon a sustained increase in cytosolic Ca2+ concentration and preceded cell killing by 1-2 h. It is concluded that generation of oxidative stress in hepatocytes can activate processes similar to those observed during programmed cell death.     

Menadione-mediated membrane fluidity alterations and oxidative damage in rat hepatocytes.

Menadione toxicity in isolated rat hepatocytes was mitigated by the antioxidant 4b,5,9b,10-tetrahydroindeno[1,2-b]indole at low concentrations (less than 100 microM), but not at high concentrations (greater than 200 microM) of menadione. When hepatocytes were incubated with menadione, there was a time-dependent and concentration-dependent inhibition of lipid peroxidation in intact cells, as well as an increase in the antioxidative potency of acetone extracts, suggesting that metabolites of menadione could inhibit oxidative stress, and that at high menadione concentrations a different mechanism was involved in cytotoxicity. A possible mechanism was suggested by the ability of acetone extracts from hepatocytes that had been incubated with menadione to increase osmotic fragility in red blood cells. This increase correlated with an increase in membrane fluidity in red blood cells, determined by flourescence polarization using the membrane probe 1,6-diphenyl-1,3,5-hexatriene. At 200 microM menadione, an increase in membrane fluidity was also observed in hepatocytes. The thiol dithiothreitol protected hepatocytes from 50 microM menadione toxicity, but not from greater than or equal to 100 microM menadione. The results suggest that while oxidative stress and arylation may be the critical mechanisms of toxicity at low menadione concentrations, at higher concentrations another mechanism such as enhanced membrane fluidity is operative.     

Mechanism of chemical-induced toxicity. II. Role of extracellular calcium

Previous studies disagree as to if chemical-induced cell death is caused by the influx and accumulation of extracellular Ca2+. To determine the role of extracellular Ca2+ in toxic cell death, the viability (leakage of intracellular K+ and lactate dehydrogenase) and total Ca2+ content of isolated hepatocytes incubated in the presence or absence of extracellular Ca2+ were determined during a toxic insult with bromobenzene, ethyl methanesulfonate (EMS), Ca2+ ionophore A23187, and adriamycin (ADR) in combination with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). The present study utilized the dibutyl phthalate separation technique which enabled the analysis of only viable hepatocytes for changes in intracellular Ca2+ and K+ content during toxic cell injury. The three chemical treatments, bromobenzene, EMS, and ADR-BCNU, each caused an accelerated loss of viability in hepatocytes incubated without extracellular Ca2+ as compared to cells incubated with Ca2+. Furthermore, the total Ca2+ content of viable hepatocytes incubated in the presence of extracellular Ca2+ did not increase during chemically induced cell injury as compared to control cells. In fact, a significant decline in total cellular Ca2+ was observed in viable hepatocytes incubated in Ca2+-free medium during toxic cell injury. Treatment with Ca2+ ionophore A23187 was also toxic to hepatocytes incubated in the presence or absence of extracellular Ca2+. At high concentrations of ionophore (20 microM or 4 micrograms/10(6) cells), cell death was accelerated in hepatocytes incubated with Ca2+ as compared to cells incubated in Ca2+-free medium. In contrast, after treatment with lower concentrations of ionophore (10 microM or 2 micrograms/10(6) cells), the rate of cell death was reversed with hepatocytes incubated without extracellular Ca2+ dying first. Thus, depending on the concentration of A23187 and the time of exposure, the presence of extracellular Ca2+ can be shown either to accelerate or protect against cell death. Surprisingly, reversible and irreversible cell injury were not observed in hepatocytes incubated with extracellular Ca2+ and 2 microM A23187 though this treatment resulted in an 800% increase in total intracellular Ca2+ content. We conclude that chemical-induced hepatic cell death is not caused by an increase in total cellular Ca2+ resulting from the influx of extracellular Ca2+.     

Cytosolic calcium after carbon tetrachloride, 1,1-dichloroethylene, and phenylephrine exposure. Studies in rat hepatocytes with phosphorylase a and quin2

Carbon tetrachloride (CCl4) and 1,1-dichloroethylene (DCE), both hepatotoxins, inhibit sequestration of Ca2+ by rat liver endoplasmic reticulum (ER) both in vivo and in vitro. It is possible that, as a result, cytosolic Ca2+ concentrations rise in liver cells. In experiments presented here, isolated hepatocytes were exposed to CCl4, DCE, and phenylephrine (PE), a non-hepatotoxic alpha 1-adrenergic agent that mobilizes Ca2+. Cytoplasmic Ca2+ concentrations were evaluated by two methods: indirectly by assaying the activity of glycogen phosphorylase a, and directly by monitoring the fluorescence of quin2. In primary hepatocyte cultures, CCl4, DCE, and PE exposure increased the activity of phosphorylase a at 5 min from 39 +/- 2 to 130 +/- 12, 80 +/- 13, and 97 +/- 10 nmoles PO4(3-)/mg protein/min respectively. In rat hepatocyte suspensions loaded with quin2 and exposed to CCl4, DCE, or PE, cytosolic Ca2+ concentrations were elevated within 20 sec to 0.83 +/- 0.13, 0.59 +/- 0.06 and 0.99 +/- 0.14 microM Ca2+ respectively. Basal Ca2+ levels in these cells averaged 0.25 +/- 0.03 microM. Thus, CCl4 and PE apparently increased cytosolic Ca2+ levels to approximately the same extent, whereas DCE was somewhat less effective. The durations of the effects of CCl4 and PE were examined further by determining their time courses of elevated phosphorylase a activity. In hepatocyte cultures, increased phosphorylase a activity persisted through at least 60 min following CCl4 exposure. In contrast, phosphorylase a activity returned to basal levels by 20 min after PE. Increases in cytoplasmic Ca2+ levels that are sustained rather than transient may distinguish these hepatotoxic chlorinated aliphatic hydrocarbons from non-toxic hormonal agents.     

Effects of the pyrrolizidine alkaloid senecionine and the alkenals trans-4-OH-hexenal and trans-2-hexenal on intracellular calcium compartmentation in isolated hepatocytes

The pyrrolizidine alkaloid senecionine has been shown to produce an increase in cytosolic free Ca2+ concentration in isolated hepatocytes that correlated with an increase in cellular toxicity. The cytotoxicity was greater in the absence of extracellular Ca2+ than in its presence, suggesting that alterations in intracellular Ca2+ distribution, and not an influx of extracellular Ca2+, were responsible for the senecionine-induced hepatotoxicity. The effect of senecionine, as well as the effects of trans-4-OH-2-hexenal (t-4HH), a microsomal metabolite of senecionine, and a related alkenal, trans-2-hexenal, on the sequestration of Ca2+ in mitochondrial and extramitochondrial compartments were examined in isolated hepatocytes. Each of the test compounds elicited a decrease in the available extramitochondrial Ca2+ stores that was inhibited by pretreatment with the thiol group reducing agent, dithiothreitol. Senecionine and t-4HH decreased the level of Ca2+ sequestered in the mitochondrial compartment of hepatocytes. The presence of a pyridine nucleotide reducing agent, beta-hydroxybutyrate, inhibited this reduction. These results suggest that both senecionine and t-4HH inhibit the sequestration of Ca2+ in extramitochondrial and mitochondrial compartments possibly by inactivating free sulfhydryl groups and oxidizing pyridine nucleotides respectively.     

Evidence for a direct role of intracellular calcium in paracetamol toxicity

There is considerable evidence that an increase in cytosolic Ca2+ is involved in the cytotoxicity of a variety of agents. However, the direct demonstration of such involvement has proved difficult. In the present study, loading of freshly isolated hamster hepatocytes with the Ca2+ specific chelator Quin 2 (2-[(2-bis[carboxymethyl]amino-5-methyl-phenoxy)methyl]-6-methoxy-8- bis-[carboxymethyl]amino-quinoline) provided significant protection against the loss of viability caused by paracetamol. This was evident both when the cells were co-incubated with Quin 2-AM and paracetamol, and when the cells were incubated with Quin 2-AM after prior exposure to paracetamol and its complete removal from the hepatocytes. These observations provide direct evidence that an increase in intracellular Ca2+ is the cause of cell death in hepatocytes exposed to paracetamol. Further, the fact that Quin 2 is protective even after some time suggests that, for alterations of cytosolic Ca2+ to be detrimental, they must be sustained. The effects of Quin 2 on plasma membrane blebbing of paracetamol-exposed hepatocytes were less pronounced than on cell viability. This is in contrast to the effects of the direct-acting thiol-reducing reducing agent, dithiothreitol, which was equally effective in preventing blebbing and loss of viability. It is concluded that alterations of cytosolic Ca2+ are less directly linked to plasma membrane blebbing than to loss of cell viability.     

Effects of carbon tetrachloride on calcium homeostasis. A critical reconsideration

The incubation of isolated rat hepatocytes with 0.172 mM carbon tetrachloride caused a rapid decrease in the calcium content of both mitochondrial and extramitochondrial compartments. However, the release of Ca2+ from the intracellular stores was not associated with an increase in the cytosolic Ca2+ levels as measured by activation of phosphorylase alpha or by Quin-2 fluorescence. A rapid rise in hepatocyte free calcium was only observed with concentrations of CCl4 higher than 0.172 mM. The lack of activation of phosphorylase alpha was not due to the inhibition of the enzyme by CCl4, since in CCl4-treated hepatocytes the phosphorylase activity could be stimulated by glucagon, butyryl--cAMP or by the increase of cell calcium induced by the addition of A23187. Ca2+-dependent ATPase of plasma membranes was only slightly affected in the early phases of poisoning with CCl4 when both mitochondrial and extramitochondrial calcium pools were already lowered. This led to the conclusion that calcium released from intracellular organelles could be extruded from the cells in sufficient amounts to prevent the increase of the cytosolic levels. A rise in hepatocyte free calcium was observed during the second hour of incubation with CCl4, concomitantly with the appearance of both LDH leakage and plasma membrane blebbing. The addition of EGTA to the medium prevented both the increase in cytosolic Ca2+ and the blebbing suggesting that they were a consequence of an influx of calcium into the cells. However, neither EGTA nor the addition of inhibitors of calcium-dependent phospholipase A2 or non-lysosomal proteases were able to protect against cell death. These latter results suggested that the alterations of calcium distribution induced by CCl4 in isolated hepatocytes were not a primary cause of the toxic effects, although they did not exclude that a sustained rise in cytosolic Ca2+ could contribute in the progression of cell injury.     

过氧化氢对原代培养大鼠肝细胞的毒性作用及其机理

本文报道了过氧化氢诱发原代培养大鼠肝细胞毒性作用的可能机理．过氧化氢（０．２～１．０ｍｍｏｌ·Ｌ－１）温育６ｈ可以引起大鼠肝细胞坏死性损伤，导致谷丙转氨酶释放增加及细胞存活率下降，加入过氧化氢酶（２５０～１５００Ｕ·ｍＬ－１）及抗氧化剂五味子乙素（１０～１００μｍｏｌ·Ｌ－１）均可降低过氧化氢的毒性作用．加入过氧化氢（０．６和１．０ｍｍｏｌ·Ｌ－１）可在６ｍｉｎ内使大鼠肝细胞内钙从１８０ｎｍｏｌ·Ｌ－１明显持续升高至７００ｎｍｏｌ·Ｌ－１以上（约３．５倍）．过氧化氢与肝细胞作用３０ｍｉｎ至１ｈ既可导致细胞膜脂质过氧化，表现为丙二醛蓄积及膜流动性下降，明显早于肝细胞发生坏死性损伤的时间．肝细胞胞浆中还原型谷胱甘肽（ＧＳＨ）含量在加入过氧化氢３０ｍｉｎ后明显降低，可推测肝细胞在后面的温育中对过氧化氢毒性的敏感性增加．以上结果证实过氧化氢诱发的原代培养大鼠肝细胞致死性损伤可能与细胞内钙迅速持续增高，细胞膜脂质过氧化及ＧＳＨ含量下降有关．     

Suppression of calcium oscillation by tri-n-butyltin chloride in cultured rat hepatocytes

The effects of tri-n-butyltin chloride (TBT), an environmental pollutant, on cytoplasmic free calcium ion concentration ([Ca2+]i) were investigated in primary cultured rat hepatocytes. A high concentration (4.0 microM) of TBT increased resting levels of [Ca2+]i and then induced cell blebs resulting in cell death within 2 h. The increase in [Ca2+]i, but not the cell death, depended on the presence of extracellular Ca2+, suggesting that the increase in [Ca2+]i is not critical for the cytotoxicity of TBT. A low concentration (0.1 microM) of TBT did not have any toxic effect (decrease in ATP content, decrease in viability, and shape change) on cultured hepatocytes and did not change [Ca2+]i. However, the calcium responses induced by phenylephrine, [Arg8]-vasopressin, and ATP were suppressed in the cells pretreated with 0.1 microM TBT for 30 min. The suppression was not observed in the cells pretreated with 0.1 microM TBT for only 1 min. Pretreatment with 0.1 microM TBT for 30 min had no effect on the inositol 1,4,5-triphosphate content or its increase in response to hormonal stimulation. These results suggest that TBT suppresses hormone-induced calcium responses at nontoxic low concentrations.     

On the role of mitochondria in cell injury caused by vanadate-induced Ca2+ overload

Incubation of isolated rat hepatocytes with vanadate (0.25, 0.5 and 1 mM) resulted in progressive accumulation of Ca2+ in the intracellular compartments. Vanadate- induced Ca2+ accumulation was related to inhibition of the plasma membrane Ca2+-extruding system, but did not involve either enhanced plasma membrane permeability to Ca2+ or the enhanced operation of a putative Na+/Ca2+ exchanger. After an initial rise in the cytosolic free Ca2+ concentration, as revealed by phosphorylase activation, Ca2+ was sequestered predominantly by the mitochondria with little contribution from the endoplasmic reticulum. As the amount of Ca2+ in the mitochondria increased, a progressive decrease in mitochondrial membrane potential occurred, together with an impairment of the ability of these organelles to further sequester Ca2+. Associated with this, there was a decrease in intracellular ATP level, formation of surface blebs and cytotoxicity. Addition of an uncoupler to vanadate-treated hepatocytes dramatically accelerated the appearance of plasma membrane blebs and toxicity. Our results demonstrate that under conditions in which the plasma membrane Ca2+ pump is inhibited, mitochondria play an important role in protecting hepatocytes against damage induced by Ca2+ overload.     

Protection of rat hepatocytes from tert-butyl hydroperoxide-induced injury by catechol

Metabolism of tert-butyl hydroperoxide (TBHP, 2.0 mM) by glutathione peroxidase within isolated rat hepatocytes caused a rapid oxidation of intracellular reduced glutathione and ultimately NADPH through glutathione reductase. TBHP also caused the formation of surface blebs in the hepatocyte plasma membrane followed by the leakage of cytosolic enzymes, such as lactate dehydrogenase, into the incubation medium. Catechol (0.1 mM) protected hepatocytes from the cytotoxic effects of TBHP but did not prevent the rapid oxidation of glutathione indicating normal metabolism of TBHP through glutathione reductase. In contrast, addition of catechol to the hepatocyte incubations prevented TBHP-induced depletion of intracellular NADPH and increased the total NADP+ + NADPH concentration without altering significantly the intracellular NADP+ content or the NADPH/NADP + NADPH ratio. Catechol did not alter TBHP stimulation of the pentose phosphate pathway. Hepatocytes incubated with sublethal concentrations of TBHP (1.0 mM) did not leak lactate dehydrogenase into the medium but did lose intracellular potassium. In these experiments, TBHP caused a sustained increase in phosphorylase alpha activity suggesting that TBHP metabolism may be associated with a sustained increase in cytosolic free Ca2+. In the presence of catechol, phosphorylase alpha activity was increased by 5 min but returned toward control by 20 min. These data suggest that catechol may be protecting hepatocytes from TBHP-induced injury by preventing a sustained rise in cytosolic free Ca2+ concentration.     

Effects of ryanodine on calcium sequestration in the rat liver.

Ryanodine, a highly toxic alkaloid known to react specifically with the Ca2+ release channels in sarcoplasmic reticulum (SR), was employed to study Ca2+ sequestration in the liver. Ryanodine at a 200 microM concentration increased cytosolic free Ca2+ levels and phosphorylase a activity in isolated hepatocytes. These effects may involve microsomal Ca2+ sequestration, because ryanodine, in the presence of inhibitors of mitochondrial Ca2+ uptake, at concentrations of 1 nM, 1 microM, 50 microM and 100 microM decreased 45Ca2+ retention in permeabilized hepatocytes. This inhibition of Ca2+ retention by ryanodine was not due to inhibition of the microsomal Ca(2+)-ATPase. Dantrolene, a compound shown previously to inhibit ryanodine binding in the liver, also decreased 45Ca2+ retention in permeabilized hepatocytes, and activated phosphorylase a. These results show that ryanodine administration alters calcium sequestration in liver. The possibility of the existence of a ryanodine-sensitive Ca(2+)-release channel in liver is discussed.     

On the role of Ca2+ in the toxicity of alkylating and oxidizing quinone imines in isolated hepatocytes

The cytotoxicity of acetaminophen (paracetamol) has been shown to be associated with a disruption of intracellular Ca2+ homeostasis caused by the interaction of its metabolite N-acetyl-p-benzoquinone imine (NAPQI) with hepatocyte thiols [Moore, M., et al. (1985) J. Biol. Chem. 260, 13035-13040]. Inasmuch as NAPQI can both covalently bind to thiols and oxidize thiols, we investigated the effects of two dimethylated analogues of NAPQI, one of which (2,6-dimethyl-NAPQI) primarily binds to thiols and the other of which (3,5-dimethyl-NAPQI) primarily oxidizes thiols. Of the three compounds, 2,6-dimethyl-NAPQI decreased protein thiols to the greatest extent and also inhibited hepatocyte plasma membrane Ca(2+)-ATPase to the greatest extent. The 3,5-dimethylated analogue decreased protein thiols to the least extent and inhibited the plasma membrane Ca(2+)-ATPase to a lesser extent. The cytotoxicity of all three compounds was preceded by a sustained elevation in cytosolic Ca2+ as compared to the transient rise caused by the alpha-agonist phenylephrine. Again, the 2,6-dimethyl analogue was the most potent of the three compounds. The thiol reagent dithiothreitol (DTT), which reversed the inhibition of the Ca(2+)-ATPase and the rise in cytosolic Ca2+, also protected against cytotoxicity. Agents that are known to inhibit either Ca(2+)-dependent proteases or phospholipases significantly delayed the onset of cytotoxicity caused by NAPQI and its analogues. Our results suggest that both arylation and oxidation of protein thiols may result in the elevation of cytosolic Ca2+ and in cytotoxicity and that arylation of critical thiol groups appears to be the more lethal reaction.     

Effect of triethyltin on Ca2+ movement in Madin-Darby canine kidney cells

The effects of the environmental toxicant, triethyltin, on Ca2+ mobilization in Madin-Darby canine kidney (MDCK) cells have been examined. Triethyltin induced an increase in cytosolic free Ca2+ levels ([Ca2+]i) at concentrations larger than 2 microM in a concentration-dependent manner. Within 5 min, the [Ca2+]i signal was composed of a gradual rise and a sustained phase. The [Ca2+]i signal was partly reduced by removing extracellular Ca2+. In Ca(2+)-free medium, pretreatment with thapsigargin (1 microM), an endoplasmic reticulum Ca2+ pump inhibitor, reduced 50 microM triethyltin-induced [Ca2+]i increase by 80%. Conversely, pretreatment with triethyltin abolished thapsigargin-induced Ca2+ release. Pretreatment with U73122 (2 microM) to inhibit phospholipase C-coupled inositol 1,4,5-trisphosphate formations failed to alter 50 microM triethyltin-induced Ca2+ release. Incubation with triethyltin at a concentration (1 microM) that did not increase basal [Ca2+]i for 3 min did not alter ATP (10 microM)- and bradykinin (1 microM)-induced [Ca2+]i increases. Collectively, this study shows that triethyltin altered Ca2+ movement in renal tubular cells by releasing Ca2+ from multiple stores in an inositol 1,4,5-trisphosphate-independent manner, and by inducing Ca2+ influx.     

Menadione inhibits the alpha 1-adrenergic receptor-mediated increase in cytosolic free calcium concentration in hepatocytes by inhibiting inositol 1,4,5-trisphosphate-dependent release of calcium from intracellular stores.

In order to establish the mechanism of perturbation of hormonally regulated calcium homeostasis in hepatocytes caused by menadione, the effects of menadione on hepatic alpha 1-adrenergic receptors and on alpha 1-adrenergic receptor-mediated increase in cytosolic free calcium concentration were determined. Menadione had no detectable effect on the alpha 1-adrenergic receptor but significantly inhibited (-)-epinephrine-dependent increases in intracellular free calcium concentration in Quin2 acetoxymethyl ester-loaded hepatocytes. The hormonally induced increase in intracellular free calcium concentration is caused by formation of inositol 1,4,5-trisphosphate (IP3) which binds to a specific receptor and causes a release of intracellular ATP-dependently sequestrated calcium. The IP3-stimulated release of calcium from intracellular pools in hepatocytes was inhibited to a great extent after treatment with menadione. This inhibition could also be observed after treatment of hepatocytes with p-benzoquinone and N-ethylmaleimide and could not be reversed by the thiol-reducing reagent dithiothreitol which indicated covalent binding to an essential free sulfhydryl group. The inhibition of IP3-dependent release of intracellular calcium was accompanied by a large increase in the number of detectable IP3 receptors without any change in the dissociation constant as determined in permeabilized hepatocytes. The increase in IP3 receptors caused by menadione could be reversed by dithiothreitol which suggests the involvement of free sulfhydryl groups. It is concluded that the IP3 receptor plays an important role in the mechanism of menadione-induced perturbation of hormonally regulated calcium homeostasis in rat hepatocytes.     

Interconversion of NAD(H) to NADP(H). A cellular response to quinone-induced oxidative stress in isolated hepatocytes

Quinones may be toxic by a number of mechanisms, including oxidative stress caused by redox cycling and arylation. This study has compared the cytotoxicity of four quinones, with differing abilities to arylate cellular nucleophiles and redox cycle, in relation to their effects on cellular pyridine nucleotides and ATP levels in rat hepatocytes. Non-toxic concentrations (50 microM) of menadione (redox cycles and arylates), 2-hydroxy-1,4-naphthoquinone (neither arylates nor redox cycles via a one electron reduction) and 2,3-dimethoxy-1,4-naphthoquinone (a pure redox cycler) all caused markedly similar changes in cellular pyridine nucleotides. An initial decrease in NAD+ was accompanied by a small, transient increase in NADP+ and followed by a larger, prolonged increased in NADPH and total NADP+ + NADPH. At toxic concentrations (200 microM), the quinones caused an extensive depletion of NAD(H), an increase in levels of NADP+ and an initial rise in total NADP+ + NADPH, prior to a decrease in ATP levels and cell death. Nucleotide changes were not observed with non-toxic (20 microM) or toxic (100 microM) concentrations of p-benzoquinone (a pure arylator) and ATP loss accompanied or followed cell death. A novel mechanism for the activation of 2-hydroxy-1,4-naphthoquinone has been implicated. Our findings also suggest that a primary event in the response of the cell to redox cycling quinones is to bring about an interconversion of pyridine nucleotides, possibly mediated by an NAD+ reduction, in an attempt to combat the effects of oxidative stress.     

Allyl alcohol cytotoxicity in isolated rat hepatocytes: mechanism of cell death does not involve an early rise in cytosolic free calcium

 We examined the effect of a toxic concentration of allyl alcohol (0.5 mM) on intracellular calcium concentrations in isolated rat hepatocytes. An increase in phosphorylase a activity was evident in the hepatocytes after 30 min of incubation with allyl alcohol, suggesting that the toxicant may produce an early rise in cytosolic free calcium. The increase in phosphorylase a activity was not reversed by the addition of dithiothreitol (DTT), a sulfhydryl compound that reverses the events that initiate cell killing by allyl alcohol. When intracellular calcium concentrations were measured directly, using fura-2 as the calcium indicator, there was no effect of allyl alcohol on cytosolic free calcium during the first 60 min of exposure, a critical period for development of irreversible damage. Incubation with allyl alcohol did not interfere with the measurement of intracellular calcium. The increases in cytosolic free calcium produced by phenylephrine or ATP were similar to those reported by others and not affected by the presence of allyl alcohol. The results from this study demonstrate that increased cytosolic free calcium is not essential for allyl alcohol-induced cytotoxicity to isolated rat hepatocytes. Received: 16 February 1994 / Accepted: 25 May 1994     

N-desethylamiodarone modulates intracellular calcium concentration in endothelial cells

The main in-vivo metabolite of amiodarone, N-desethylamiodarone (DEAM), possesses clinically relevant class-II antiarrhythmic and vasodilator activities. Vasodilation by DEAM is endothelium dependent and involves a sustained and biphasic increase in cytosolic free Ca2+ concentration ([Ca2+]i). The aims of this study were to explore the mechanisms mediating the DEAM-induced increase in [Ca2+]i in endothelial cells and to determine whether this increase in [Ca2+]i was associated with altered cell proliferation. Cultured bovine aortic endothelial cells were loaded with the Ca2+-sensitive fluorescent dye Fura-2/AM, and [Ca2+]i measured spectrofluorimetrically. DEAM increased [Ca2+]i concentration dependently (EC50 approximately 6 microM) both in the presence and absence of extracellular Ca2+. In the presence of extracellular Ca2+, the response of [Ca2+]i to DEAM (10 microM) consisted of an initial rise to a plateau followed by a second increase to micromolar levels. The initial plateau was reduced by the endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin (200 nM) and by the antioxidant ascorbic acid (100 microM). The initial rate of rise in [Ca2+]i was decreased by blocking mitochondrial Ca2+ release with cyclosporine A (1 microM). Under Ca2+-free conditions, the response of [Ca2+]i to DEAM (10 microM) was also biphasic, consisting of an initial transient peak and a second slow increase. When extracellular Ca2+ was restored, [Ca2+]i rose to micromolar concentrations. The initial peak was abolished by thapsigargin, but not altered by ascorbic acid or cyclosporine A. Both the second [Ca2+]i increase and that due to restoring extracellular Ca2+ were reduced by ascorbic acid but not affected by thapsigargin or cyclosporine A. The DEAM-induced generation of free radicals and sustained increase in [Ca2+]i might alter cell proliferation and endothelial cell proliferation was indeed concentration-dependently inhibited by DEAM (IC50 approximately 2.5 microM). In conclusion, the DEAM-induced [Ca2+]i increase in endothelial cells is due to Ca2+ influx from the extracellular space and to Ca2+ release from endoplasmic reticulum and mitochondria and involves enhanced generation of free radicals.     

Cytosolic Ca2+ movements of endothelial cells exposed to reactive oxygen intermediates: role of hydroxyl radical-mediated redox alteration of cell-membrane Ca2+ channels

1. The mode of action of reactive oxygen intermediates in cysosolic Ca2+ movements of cultured porcine aortic endothelial cells exposed to xanthine/xanthine oxidase (X/XO) was investigated. 2. Cytosolic Ca2+ movements provoked by X/XO consisted of an initial Ca2+ release from thapsigargin-sensitive intracellular Ca2+ stores and a sustained Ca2+ influx through cell-membrane Ca2+ channels. The Ca2+ movements from both sources were inhibited by catalase, cell-membrane permeable iron chelators (o-phenanthroline and deferoxamine), a *OH scavenger (5,5-dimethyl-1-pyrroline-N-oxide), or an anion channel blocker (disodium 4, 4'-diisothiocyano-2, 2'-stilbenedisulphonic acid), suggesting that *O2- influx through anion channels was responsible for the Ca2+ movements, in which *OH generation catalyzed by intracellular transition metals (i.e., Haber-Weiss cycle) was involved. 3. After an initial Ca2+ elevation provoked by X/XO, cytosolic Ca2+ concentration decreased to a level higher than basal levels. Removal of X/XO slightly enhanced the Ca2+ decrease. Extracellular addition of sulphydryl (SH)-reducing agents, dithiothreitol or glutathione, after the removal of X/XO accelerated the decrement. A Ca2+ channel blocker, Ni2+, abolished the sustained increase in Ca2+, suggesting that Ca2+ influx through cell-membrane Ca2+ channels was extracellularly regulated by the redox state of SH-groups. 4. The X/XO-provoked change in cellular respiration was inhibited by Ni2+ or dithiothreitol as well as inhibitors of Haber-Weiss cycle, suggesting that Ca2+ influx was responsible for *OH-mediated cytotoxicity. We concluded that intracellular *OH generation was involved in the Ca2+ movements in endothelial cells exposed to X/XO. Cytosolic Ca2+ elevation was partly responsible for the oxidants-mediated cytotoxicity.