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J L Farber

Publications and source records attributed to J L Farber.

At least 19 recordsLinked to original sources
PubMed1992

Protection by cyclosporin A of cultured hepatocytes from the toxic consequences of the loss of mitochondrial energization produced by 1-methyl-4-phenylpyridinium.

Cyclosporin A prevented the killing of cultured rat hepatocytes by 1-methyl-4-phenylpyridinium (MPP+). However, in the presence of both cyclosporin and atractyloside, there was no protection. Cyclosporin had no effect on the depletion of ATP or the loss of mitochondrial energization by MPP+. Cyclosporin, however, did prevent the increase in the molecular order of hepatocyte membranes produced by MPP+. These data suggest that mitochondrial de-energization produced by MPP+ is accompanied by a "permeability transition" analogous to that which occurs in vitro in the presence of calcium. By preventing this transition, cyclosporin protects the cells. By antagonizing this action of cyclosporin, atractyloside restores the cell killing. The mitochondrial transition is causally linked to cell killing by a mechanism that increases the molecular order of the hepatocyte plasma membrane.

1-Methyl-4-phenylpyridinium↗
PubMed1992

Metabolism of pyridine nucleotides in cultured rat hepatocytes intoxicated with tert-butyl hydroperoxide.

The alterations in the metabolism of pyridine nucleotides, as well as the role such changes play in the genesis of lethal cell injury, were explored in cultured rat hepatocytes intoxicated with tert-butyl hydroperoxide (TBHP). The loss of NADPH, NADH, and NAD equalled the increase in NADP, with little if any change in the total content of pyridine nucleotides. Identical alterations occurred in the presence of N,N'-diphenyl-p-phenylenediamine, an antioxidant that prevented the death of the cells. Inhibition of glutathione reductase by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) reduced the extent of the increase in NADP and the decrease in NADPH. At the same time, BCNU increased the cell killing. Depletion of ATP with oligomycin reduced the loss of NAD and the accumulation of NADP. Treatment of the hepatocytes with the poly(ADP-ribose) polymerase inhibitor 3-aminobenzamide had no effect on the depletion of NAD. Thus, all of the alterations in pyridine nucleotides that accompany the exposure of cultured hepatocytes to TBHP can be dissociated from the development of lethal cell injury. The changes do suggest, however, a rapid interconversion of the respective species. The initial response reflects activation of glutathione reductase with the consequent oxidation of NADPH to NADP. The conversion of NADH to NAD and then NAD to NADP, the latter by nicotinamide adenine dinucleotide kinase, can account for the increase in NADP over the resulting from the oxidation of NADPH by glutathione reductase. Finally, there was no evidence in cultured hepatocytes treated with TBHP for changes in NAD that reflect the activation of poly(ADP-ribose) polymerase.

Animals↗
PubMed1992

Phospholipid metabolism and intracellular Ca2+ homeostasis in cultured rat hepatocytes intoxicated with cyanide.

The killing of cultured hepatocytes by 1 mM sodium cyanide was reduced by 100 microM chlorpromazine or cytochalasin B (25 micrograms/ml) or by lowering the pH of the culture medium to 6.0. In each case, ATP was depleted despite the decreased number of dead cells. The cell killing by cyanide was accompanied by an accelerated release of 3H-labeled arachidonate from phospholipids. Depletion of ATP by oligomycin did not accelerate phospholipid degradation or kill the hepatocytes. Chlorpromazine, cytochalasin B, and extracellular acidosis reduced the rate of phospholipid degradation in control cells as well as the increase that occurred with cyanide. The calcium ionophore A23187 increased phospholipid degradation and killed the hepatocytes. Chlorpromazine and extracellular acidosis, but not cytochalasin B, protected the cells and prevented the increased lipid degradation in response to A23187. After addition of cyanide, cytosolic free calcium ([Ca2+]i) did not change for 71 +/- 8 min, at which time it rose to a plateau of 683 +/- 210 nM within 10 min. A second and larger rise occurred after 84 +/- 8 min and before the death of the cells at 89 +/- 8 min. Treatment with 3.5 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, as well as removal of extracellular calcium, prevented these late increases in [Ca2+]i without affecting the loss of viability. It is concluded that cyanide kills cultured hepatocytes by a mechanisms that is likely related to an accelerated degradation of phospholipids. This change in lipid metabolism is not mediated by a rise in [Ca2+]i but rather may relate to an alteration in the interaction between the cytoskeleton and the plasma membrane.

Acidosis↗
PubMed1992

Cellular pool of transient ferric iron, chelatable by deferoxamine and distinct from ferritin, that is involved in oxidative cell injury.

A cellular pool of transient ferric iron that is chelatable by deferoxamine, distinct from ferritin, and required for oxidative cell injury has been identified in cultured rat hepatocytes labeled with 59FeCl3. Pretreatment of hepatocytes with deferoxamine depleted the cellular pool of chelatable iron and protected the cells from an oxidative injury. Incubation of deferoxamine-pretreated hepatocytes in serum-free medium restored both the chelatable iron pool and the susceptibility to oxidative injury. Furthermore, inhibition of protein degradation with chymostatin prevented the restoration of both the chelatable pool and susceptibility to oxidative injury. The deferoxamine-chelatable iron pool was distinguished kinetically and immunochemically from the larger cellular pool of ferritin iron. The labeled iron in the deferoxamine-chelatable pool was transient, unlike either the total cellular uptake of 59Fe or its incorporation into ferritin, both of which increased with time of labeling. With pulse-chase labeling, the percentage of the total uptake of 59Fe that was represented by the deferoxamine-chelatable pool decreased. At the same time, the percentage represented by radioactivity immunoprecipitable as ferritin increased. Furthermore, immunoprecipitation of ferritin from the labeled lysates enriched the resulting immunosupernatants in deferoxamine-chelatable iron. The degree of enrichment for chelatable iron correlated with the percentage of the cellular label that was immunoprecipitable as ferritin. The deferoxamine-chelatable iron appears to represent a metabolically common pool of iron that is rapidly in transit through the cell. Extracellular iron entering the pool can be utilized for heme synthesis or stored in ferritin, whereas protein degradation releases storage iron into this pool.

Animals↗
PubMed1992

Effects of vitamin E on the killing of cultured hepatocytes by tert-butyl hydroperoxide.

The disposition of vitamin E was examined in cultured rat hepatocytes intoxicated with tert-butyl hydroperoxide (TBHP). Culturing of the cells overnight (18-20 hr) with approximately 60 nM alpha-tocopherol (alpha-T) equivalents [Williams' E medium, 18 nM tocopherol phosphate (alpha-TP), 9% fetal calf serum, 43 nM alpha-T] resulted in a content of alpha-T that was 16% of the concentration of vitamin E measured in freshly isolated hepatocytes. Supplementation of the medium with 1 microM alpha-TP maintained the alpha-T concentration of the cultured cells at the level of freshly isolated hepatocytes. Supplemented hepatocytes exposed to TBHP showed decreased lipid peroxidation and delayed cell killing, compared with hepatocytes not cultured overnight with alpha-TP. Killing of the supplemented cells by TBHP was accompanied by a loss of alpha-T. Pretreatment of supplemented hepatocytes with the iron chelator deferoxamine prevented much of the loss of alpha-T. At the same time, deferoxamine inhibited both the lipid peroxidation and cell killing. The antioxidant N,N'-diphenyl-1,4-phenylenediamine reduced the loss of alpha-T and significantly decreased lipid peroxidation. In the presence of N,N'-diphenyl-1,4-phenylenediamine, cell killing was delayed by 15 min and reduced in extent. Overnight supplementation of hepatocytes with nonesterified alpha-T, or vitamin E esters other than alpha-TP, similarly rendered the cells less sensitive to TBHP. The nonesterified alpha-T produced a higher cell-associated vitamin E concentration than did the esters; however, nonesterified alpha-T did not result in greater protection against TBHP. These data indicate that the mechanisms of the cell killing by TBHP are the same in cultured hepatocytes that contain low or physiological concentrations of vitamin E.

Animals↗
PubMed1991

The killing of cultured hepatocytes by N-acetyl-p-benzoquinone imine (NAPQI) as a model of the cytotoxicity of acetaminophen.

The killing of isolated hepatocytes by N-acetyl-p-benzoquinone imine (NAPQI), the major metabolite of the oxidation of the hepatotoxin acetaminophen, has been studied previously as a model of liver cell injury by the parent compound. Such studies assume that the toxicity of acetaminophen is mediated by NAPQI and that treatment with exogenous NAPQI reproduces the action of the endogenously produced product. The present study tested these assumptions by comparing under identical conditions the toxicity of acetaminophen and NAPQI. The killing of hepatocytes by acetaminophen was mediated by oxidative injury. Thus, it depended on a cellular source of ferric iron; was potentiated by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), an inhibitor of glutathione reductase; and was sensitive to antioxidants. By contrast, the cytotoxicity of NAPQI was not prevented by chelation of ferric iron; was unaffected by BCNU; and was insensitive to antioxidants. Thus, the killing of cultured hepatocytes by NAPQI occurs by a mechanism different from that of acetaminophen. The killing by NAPQI was preceded by a collapse of the mitochondrial membrane potential and a depletion of ATP. Monensin potentiated the cell killing, and extracellular acidosis prevented it. These manipulations are characteristic of the toxicity of mitochondrial poisons, and are without effect on the depletion of ATP and the loss of mitochondrial energization. Thus, mitochondrial de-energization by a mechanism unrelated to oxidative stress is a likely basis of the cell killing by NAPQI. It is concluded that treatment of cultured hepatocytes with NAPQI does not model the cytotoxicity of acetaminophen in these cells.

Acetaminophen↗
PubMed1991

Increases in cytosolic calcium ion concentration can be dissociated from the killing of cultured hepatocytes by tert-butyl hydroperoxide.

Digital imaging fluorescence microscopy was used to study the effect of tert-butyl hydroperoxide (TBHP) on the cytosolic free calcium concentration ([Ca2+]i) of single rat hepatocytes in primary culture. Within minutes of the addition of TBHP, individual hepatocytes displayed one or more peaks of increased [Ca2+]i that promptly returned to the prestimulation level. This was followed by a slower increase of [Ca2+]i that reached a plateau of 696 +/- 260 nM (basal 194 +/- nM) after 20 min. Another rise in [Ca2+]i, abrupt and much larger, preceded the death of the cells after about 45 min. Pretreatment of the hepatocytes with deferoxamine, a ferric iron chelator, or the addition of the antioxidants N,N'-diphenyl-p-phenylenediamine or catechol prevented the loss of viability. Neither the number of hepatocytes displaying the initial [Ca2+]i transients nor the magnitude of these oscillations was affected by deferoxamine, N,N'-diphenyl-p-phenyl-enediamine, or catechol. However, both the plateau phase and the abrupt rise in [Ca2+]i were prevented. Treatment of the hepatocytes with TBHP in a low calcium buffer (less than 2 microM Ca2+) reduced or abolished the initial [Ca2+]i transients and eliminated both the plateau phase and abrupt rise in [Ca2+]i. The onset of cell death was delayed by 10 min in the low calcium medium. Addition of 3.5 mM EGTA to the cultures lowered the basal calcium concentration, prevented both the initial [Ca2+]i spikes and the delayed changes, and further prolonged the onset of cell death. These data indicate that the killing of the cultured hepatocytes by TBHP can be dissociated from changes in intracellular calcium homeostasis. An influx of extracellular Ca2+ ions may aggravate somewhat the mechanisms of cell injury by an oxidative stress and accelerate the time of onset of cell death.

Animals↗
PubMed1990

Evidence for the participation of activated oxygen species and the resulting peroxidation of lipids in the killing of cultured hepatocytes by aryl halides.

Primary cultures of rat hepatocytes were used to explore the mechanisms of the toxicity of aryl halides. The sensitivity of the hepatocytes to chloro-, bromo-, and iodobenzene was enhanced by inhibition of glutathione reductase with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). In each case, the increased cell killing depended on the metabolism of the toxicant, a result shown by the protective effect of SKF-525A, an inhibitor of mixed function oxidation. BCNU decreased the metabolism of [14C]bromobenzene and the covalent binding of its metabolites by 20%. Chelation by deferoxamine of a cellular source of ferric iron prevented the cell killing in the presence or absence of BCNU. Deferoxamine had no effect on the metabolism or the covalent binding of [14C]bromobenzene. Similarly, the antioxidant N,N'-diphenyl-p-phenylenediamine (DPPD) reduced the cell killing and had no effect on the metabolism of [14C]bromobenzene. Thus, the toxicity of the three aryl halides was manipulated in ways that modify the sensitivity of hepatocytes to an oxidative stress, and the changes in cell killing occurred without parallel changes in the metabolism of [14C]bromobenzene or the covalent binding of its metabolites.

Animals↗
PubMed1990

Environmental diseases of the digestive system.

Environmental factors are important mediators of many diseases of the digestive system, defined as the alimentary tract and the accessory organs of digestion, the liver and pancreas. In this review, we principally focus on the action of chemical agents which are classified as (1) naturally occurring compounds, (2) occupational hazards, (3) therapeutic drugs, and (4) constituents of substances of abuse. In addition, the putative role of dietary habits in the pathogenesis of malignant diseases of the digestive system is discussed.

Alcohol Drinking↗
PubMed1990

The role of calcium ions in toxic cell injury.

Calcium ions have been increasingly implicated as a mediator of the mechanisms generating lethal cell injury under a variety of pathologic circumstances. An overview of the various roles suggested for such alterations in cellular calcium homeostasis is presented. The central role of plasma membrane damage in the genesis of irreversible cell injury is used to divide the postulated roles for calcium ions into two major mechanisms. On the one hand, calcium ions have been proposed as mediators of the functional consequences of plasma membrane injury. An influx of extracellular calcium ions across a damaged permeability barrier and down a steep concentration gradient may convert potentially reversible injury into irreversible injury. On the other hand, alterations in intracellular calcium homeostasis are postulated to participate in the mechanisms generating potentially lethal plasma membrane injury. The release of calcium stores sequestered within intracellular organelles raises the cytosolic concentration of free calcium, a process that may activate, in turn, a number of membrane-disruptive processes. The data supporting these two distinct actions of calcium are reviewed and discussed.

Animals↗
PubMed1990

Liposome-encapsulated superoxide dismutase prevents liver necrosis induced by acetaminophen.

Liposome-encapsulated human recombinant superoxide dismutase (LSOD) protected male rats that were pretreated with 3-methylcholanthrene from the liver necrosis produced by acetaminophen. By contrast, SOD-free liposomes, free SOD, or heat-denatured LSOD had no protective effect. Liposome-encapsulated SOD did not simply delay the onset of liver necrosis. A second dose of LSOD at 12 hours prevented the necrosis of the liver as assessed 24 hours after treatment with 500 mg/kg body weight of acetaminophen. Liposome-encapsulated human recombinant superoxide dismutase did not alter the metabolism of acetaminophen as assessed by either the rate or extent of the depletion of hepatic stores of glutathione or by the extent of the covalent binding of the metabolites of [3H]acetaminophen to total liver cell proteins. Evidence of the peroxidation of lipids in the accumulation of malondialdehyde in the livers was detected within 3 hours of the administration of acetaminophen and before the appearance of liver necrosis. Liposome-encapsulated human recombinant superoxide dismutase prevented the accumulation of malondialdehyde in parallel with the prevention of liver necrosis. Finally, LSOD also prevented the potentiation by 1,3-bis(2-chloroethyl)-1-nitrosourea of the hepatotoxicity of acetaminophen. These data document the participation of superoxide anions in the hepatotoxicity of acetaminophen in intact rats.

Acetaminophen↗
PubMed1979

Calcium dependence of toxic cell death: a final common pathway.

Primary cultures of adult rat hepatocytes were treated in the presence or absence of extracellular calcium with ten different membrane-active toxins. In all cases more than half the cells were killed in 1 to 6 hours in the presence but not in the absence of extracellular calcium. An effect of calcium on the primary mechanism of membrane injury by any of the agents cannot be implicated. Viability, as determined by trypan blue exclusion correlated well with other indices of viability such as plating efficiency and the hydrolysis of fluorescein diacetate. It is concluded that the cells are killed by processes that involve at least two steps. In each type of injury, disruption of the integrity of the plasma membrane by widely differing mechanisms is followed by a common functional consequence involving extracellular calcium, and most likely representing an influx of calcium across the damaged plasma membrane and down a steep concentration gradient. This later step represents, or at least initiates, a final common pathway for the toxic death of these cells.

Animals↗
PubMed1979

A simple method for decreasing the toxicity of polyethylene glycol in mammalian cell hybridization.

The yield of hybrid colonies after fusion of mammalian cells with polyethylene glycol (PEG) is increased if the cells are fused in Ca2+-free medium, and kept in Ca2+-free medium for at least 15 min after fusion. The protective effect of Ca2+-free medium is much more obvious when Baker PEG is used than when fusion is carried out with Koch-Light PEG. The increased yield of hybrid colonies is shown to be due to a reduced toxicity rather than to an increased efficiency of cell fusion. These improvements have been found to apply to a variety of cell lines, and also when cell fusion is carried out in suspension. This technique should be particularly useful in studies on mammalian cell hybridization using cell lines that are particularly sensitive to the toxic effect of PEG.

Animals↗
PubMed1979

Ischemic myocardial cell injury. Prevention by chlorpromazine of an accelerated phospholipid degradation and associated membrane dysfunction.

Ligation of the left coronary artery of an adult rat heart results in the reproducible ischemic cell death of the entire free wall of the left ventricular myocardium. The time course of the development of the cellular changes is biphasic. The subendocardial and subepicardial cells die within the first few hours. The main mass of free-wall myocardium reacts more slowly, with morphologic evidence of irreversible cell injury developing after 12 hours. Measurement of the increases in total free wall Ca++ reflected this biphasic pattern. There was a rapid 3-fold rise in total Ca++ during the first 4 hours. Between 4 and 12 hours the Ca++ was constant. Between 12 and 30 hours there was a second increase that reached a level some 8-10 times the control value. Treatment with chlorpromazine before and subsequent to surgery prevented the appearance of ischemic cell death in the main portion of the free-wall myocardium for at least 24 hours without affecting the reaction of the subepicardial and subendocardial cells. Chlorpromazine also inhibited the second phase of Ca++ accumulation. An accelerated degradation of phospholipids was observed with a 33% decrease in total phospholipids by 12 hours. Phosphatidylethanolamine was reduced by 50% and phosphatidylcholine by 25% without increases in the corresponding lysophospholipids. Chlorpromazine prevented the accelerated degradation and consequent loss of phospholipid. Isolated sarcoplasmic reticulum showed a time-dependent loss of phospholipid with a parallel loss of active Ca++ uptake that reach 60% with a total lipid depletion from these membranes of 33% by 12 hours. Twelve-hour ischemic sarcoplasmic reticulum exhibited a 6--7-fold increase in passive permeability to Ca++. Chlorpromazine protected against the loss of phospholipids, the inhibition of Ca++ uptake, and the increased Ca++ permeability of the sarcoplasmic reticulum. These observations indicate that rat myocardial cells react to lethal doses of ischemia in a manner similar to the reaction of liver cells described previously. In both cases the evidence implies that a disturbance in phospholipid metabolism and its associated membrane dysfunction is the critical alteration that produces irreversible cell injury in ischemia.

Animals↗