Dissociation of intracellular lysosomal rupture from the cell death caused by silica.

The relationship between intracellular lysosomal rupture and cell death caused by silica was studied in P388d(1) macrophages. After 3 h of exposure to 150 mug silica in medium containing 1.8 mM Ca(2+), 60 percent of the cells were unable to exclude trypan blue. In the absence of extracellular Ca(2+), however, all of the cells remained viable. Phagocytosis of silica particles occurred to the same extent in the presence or absence of Ca(2+). The percentage of P388D(1) cells killed by silica depended on the dose and the concentration of Ca(2+) in the medium. Intracellular lyosomal rupture after exposure to silica was measured by acridine orange fluorescence or histochemical assay of horseradish peroxidase. With either assay, 60 percent of the cells exposed to 150 mug silica for 3 h in the presence of Ca(2+) showed intracellular lysosomal rupture, was not associated with measureable degradation of total DNA, RNA, protein, or phospholipids or accelerated turnover of exogenous horseradish peroxidase. Pretreatment with promethazine (20 mug/ml) protected 80 percent of P388D(1) macrophages against silica toxicity although lysosomal rupture occurred in 60-70 percent of the cells. Intracellular lysosomal rupture was prevented in 80 percent of the cells by pretreatment with indomethacin (5 x 10(-5)M), yet 40-50 percent of the cells died after 3 h of exposure to 150 mug silica in 1.8 mM extracellular Ca(2+). The calcium ionophore A23187 also caused intracellular lysosomal rupture in 90-98 percent of the cells treated for 1 h in either the presence or absence of extracellular Ca(2+). With the addition of 1.8 mM Ca(2+), 80 percent of the cells was killed after 3 h, whereas all of the cells remained viable in the absence of Ca(2+). These experiments suggest that intracellular lysosomal rupture is not causally related to the cell death cause by silica or A23187. Cell death is dependent on extracellular Ca(2+) and may be mediated by an influx of these ions across the plasma membrane permeability barrier damaged directly by exposure to these toxins.

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.

Alcohol-dependent liver cell necrosis in vitro: a new model.

In alcoholic liver injury, necrosis is involved in the progression from benign fatty liver to alcoholic hepatitis and cirrhosis. However, there is no practical model of alcohol-dependent liver cell necrosis. The calcium-dependent killing of cultured rat hepatocytes by two different membrane-active hepatotoxins, galactosamine and phalloidin, is potentiated by ethyl alcohol. This indicates that some general physical effect of alcohol on cellular membranes renders cells susceptible to otherwise nonlethal injuries. The in vitro model described in this report may thus be used to search for a general mechanism underlying alcohol-related tissue injury.

The Nephrotoxicity of Vancomycin.

Vancomycin use is often associated with nephrotoxicity. It remains uncertain, however, to what extent vancomycin is directly responsible, as numerous potential risk factors for acute kidney injury frequently coexist. Herein, we critically examine available data in adult patients pertinent to this question. We review the pharmacokinetics/pharmacodynamics of vancomycin metabolism. Efficacy and safety data are discussed. The pathophysiology of vancomycin nephrotoxicity is considered. Risk factors for nephrotoxicity are enumerated, including the potential synergistic nephrotoxicity of vancomycin and piperacillin-tazobactam. Suggestions for clinical practice and future research are given.

The presence of B-cell nodules does not necessarily portend a less favorable outcome to therapy in patients with acute cellular rejection of a renal allograft.

The presence of B-cell nodules in kidney biopsies of patients undergoing acute renal allograft rejection has been reported to be associated with glucocorticoid resistance and a high risk of graft failure. In an attempt to corroborate this observation, biopsies of renal transplants that evidenced Banff grade I A acute rejection were examined for the presence of B- or T-cell nodules, the detection of which was correlated with the therapeutic response. Biopsies from 14 consecutive renal transplant recipients with a diagnosis of acute cellular rejection were examined for the presence of T (CD3-positive) or B (CD20-positive) cells by immunohistochemistry. All patients were biopsied because of a rise in serum creatinine. No biopsy showed evidence of acute humoral rejection. Immunofluorescence microscopy was negative for C4d deposition in peritubular capillaries. There were no neutrophils in the peritubular or glomerular capillaries. Five patients had T-cell nodules; four had B-cell nodules; three had both T- and B-cell nodules; two had no nodules. All biopsies contained CD3-positive cells in the tubules and in the interstitium. In all but one of the patients, episodes of acute rejection were treated with steroids (one received thymoglobulin). Furthermore two patients received mycophenolate mofetil and one, sirolimus. There were no significant differences among the groups in either the initial creatinine or the creatinine after therapy. The presence of B-cell nodules in renal allograft biopsies of patients experiencing acute cellular rejection did not portend a less favorable outcome.

c-Kit-positive mast cells in portal tracts cannot be used to distinguish acute cellular rejection from recurrent hepatitis C infection in liver allografts.

Cirrhosis secondary to chronic hepatitis C virus (HCV) is the most common indication for liver transplantation. Recurrence of HCV infection in the liver allograft occurs at a high rate. The differentiation of recurrent HCV infection from acute cellular rejection (ACR) represents a difficult challenge in transplantation pathology. The c-Kit receptor is a tyrosine kinase membrane protein encoded by the c-Kit proto-oncogene, which is expressed on mast cells and on hematopoietic stem and progenitor cells. Mast cells are important effector cells of a broad range of immune responses. Recently, c-Kit+ mast cells were shown to form part of the inflammatory infiltrate in acute liver allograft rejection. A strong relationship was found between c-Kit+ cell densities and increasingly severe rejection. The present study sought to determine whether the presence of c-Kit+ cells could be used to distinguish between ACR and recurrent HCV in liver allografts. Immunohistochemical staining for c-Kit was performed on 20 transplant biopsy specimens from 10 patients with mild to moderate ACR and 10 other patients with recurrent hepatitis C. The number of c-Kit+ cells per portal tract varied with the density of the overall inflammatory infiltrate. There was no significant difference between the number of c-Kit+ cells in the biopsy specimens that carried a diagnosis of ACR and those from patients who had been diagnosed as having recurrent HCV. It was concluded that immunohistochemical staining for the presence of c-Kit+ mast cells cannot be used to differentiate between ACR and recurrent HCV infection in liver allograft biopsy specimens.

Esophageal cancer prevention in zinc-deficient rats: rapid induction of apoptosis by replenishing zinc.

BACKGROUND: Nutritional zinc deficiency in rats increases esophageal cell proliferation and the incidence of N-nitrosomethylbenzylamine (NMBA)-induced esophageal tumors. Replenishing zinc with a zinc-sufficient diet reduces these effects in zinc-deficient (ZD) rats. We investigated whether apoptosis was involved in the reduction of NMBA-induced esophageal tumors when ZD rats consumed a zinc-sufficient diet. METHODS: Weanling rats were fed a ZD diet (zinc at 3-4 ppm) for 5 weeks to establish esophageal cell proliferation, then treated once with NMBA (2 mg/kg body weight), and divided into the following five groups (47-100 per group). One ZD group was fed the ZD diet, and four zinc-replenished (ZR) groups, ZR(1), ZR(24), ZR(72), and ZR(432), were fed a zinc-sufficient diet (zinc at 74-75 ppm) beginning 1, 24, 72, and 432 hours, respectively, after NMBA treatment. From 24 hours to 2 weeks after beginning a zinc-sufficient diet, esophagi from all ZR groups were analyzed for apoptosis and cell proliferation; ZD esophagi were the controls. Tumor incidence was determined 15 weeks after zinc replenishment. All statistical tests were two-sided. RESULTS: Zinc replenishment initiated shortly after NMBA treatment effectively reduced esophageal tumorigenesis; 8% (three of 37) of ZR(1), 14% (five of 37) of ZR(24), 19% (five of 26) of ZR(72), and 48% (19 of 40) of ZR(432) rats developed esophageal tumors compared with 93% (14 of 15) of ZD animals (all P<.001). Importantly, 24 and 30 hours after zinc replenishment, esophagi had numerous apoptotic cells (% apoptotic cells: 0 hour = 2.9%, 95% confidence interval [CI] = 2.5% to 3.3%; 24 hours = 9.4%, 95% CI = 8.2% to 10.6%), and the expression of the proapoptotic Bax protein doubled. Within 48 hours, the ZR(1) epithelium was three to five cell layers thick compared with 10-20 layers before zinc replenishment. CONCLUSIONS: Zinc replenishment of NMBA-treated ZD rats rapidly induces apoptosis in esophageal epithelial cells and thereby substantially reduces the development of esophageal cancer.

Inhibition of rat liver RNA polymerases by action of the methylating agents dimethylnitrosamine in vivo and methyl methanesulfonate in vitro.

Dimethylnitrosamine maximally inhibits rat liver nuclear RNA synthesis by 50% at a dose of 40 mg/kg of body weight. The inhibition develops during the first 4 hr and persists through the 12th hr. All parenchymal cells of the lever lobule seem to be affected. The decreased RNA synthesis can be accounted for entirely by an inhibition of the RNA polymerase activities quantitatively solubilized and partially purified. A similar inhibition of the polymerase activities was demonstrated in the intact nuclei by inactivating the endogenous template with actinomycin D and assaying the polymerases with an added exogenous template, poly(deoxy-adenylate-deoxythymidylate). Chromatin was prepared by two methods differing in the extent to which they remove the endogenous polymerase activity. Each preparation was transcribed with either added Escherichia coli or partially purified rat liver nucleoplasmic RNA polymerase. With either polymerase or chromatin preparation, no inhibition of the template activity of liver nuclear chromatin isolated from the DMN-treated animals was detected. A similar mechanism of inhibition of RNA synthesis was produced by the action of the methylating agent methyl methanesulfonate on whole nuclei in vitro. The dose-dependent inhibition of RNA synthesis could be accounted for by an inhibition of the RNA polymerase activities quantitatively solubilized and partially purified from the affected nuclei. Chromatin prepared from the methyl methanesulfonate-treated nuclei had a normal template capacity with either E. coli or rat liver nucleoplasmic RNA polymerase. No preferential methylation of the RNA polymerases by [14C]methyl methanesulfonate could be demonstrated. It is concluded that the action of the two methylating agents on RNA metabolism is similar and that the inhibition of liver nuclear RNA synthesis results from inactivation of the RNA polymerases. At the same time, dimethylnitrosamine and methyl methanesulfonate leave the chromatin template intact, at least quantitatively, for the synthesis of RNA. The implications of such an effect on RNA synthesis are discussed.

Induction of the mitochondrial permeability transition mediates the killing of HeLa cells by staurosporine.

The role of the mitochondrial permeability transition (MPT) in the killing of HeLa cells by staurosporine (STR) was assessed with the use of bongkrekic acid (BK), an inhibitor of the MPT. BK prevented cell killing as well as biochemical manifestations of the MPT: (a) the loss of the mitochondrial membrane potential (deltapsim); (b) the release of cytochrome c from the intramembranous space to the cytosol; and (c) the release of malate dehydrogenase from the mitochondrial matrix. Stable transfectants that overexpressed Akt were also resistant to cell killing and did not develop an MPT. STR inhibited the phosphorylation of Bad, whereas Bad phosphorylation was preserved in cells that overexpress Akt. In wild-type HeLa cells treated with STR, the content of Bax in the cytosol decreased as that in the mitochondria increased, a result that was again prevented by overexpression of Akt. Bid accumulation in the mitochondria with STR was not affected by overexpression of Akt. The pan-caspase inhibitor Z-Val-Ala-Val-Asp(OMe) fluoromethylketone prevented cell killing bu not induction of the MPT. The data document the central role of the MPT in the killing of HeLa cells by STR. The data are consistent with the hypothesis that induction of the MPT is a consequence of the movement of Bax to the mitochondria. Phosphorylation of Bad prevents Bax translocation. Caspases participate in the events related to cell killing that occur subsequent to induction of the MPT.

N-hydroxy-2-acetylaminofluorene inhibition of rat live RNA polymerases.

Administration of N-hydroxy-2-acetylaminofluorene (N-OH-AAF) to rats inhibits liver nuclear RNA synthesis. This effect is reflected in an in vitro inhibition of RNA synthesis by isolated whole nuclei. The decreased RNA synthesis can be accounted for entirely by an inhibition of the RNA polymerase activities quantitatively solubilized and partially purified from these nuclei. Both nucleolar and nucleoplasmic polymerases are affected. A similar inhibition of the polymerases was demonstrated in intact nuclei by inactivating the endogenous template with actinomycin D and assaying the polymerases with an added exogenous template, poly(deoxyadenylate-deoxythymidylate). Chromatin was prepared from similar nuclear preparations by two methods, differing in the extent to which they remove endogenous polymerase activity. Each chromatin preparation was transcribed with added Escherichia coli or partially purified rat liver nucleoplasmic RNA polymerase respectively. With either polymerase and either chromatin preparation, no inhibition of the template activity of chromatin isolated from N-OH-AAF-treated animals could be detected. It is concluded that N-OH-AAF is a potent inhibitor of rat liver nuclear RNA synthesis and that the mechanism of this inhibition is inactivation of the RNA polymerases. At the same time, N-OH-AAF leaves the chromatin template, at least quantitatively, intact for the synthesis of RNA. The implications of such an effect of N-OH-AAF on RNA synthesis are discussed.

Early deregulation of the the p16ink4a-cyclin D1/cyclin-dependent kinase 4-retinoblastoma pathway in cell proliferation-driven esophageal tumorigenesis in zinc-deficient rats.

The p16ink4a-cyclin D1/cyclin-dependent kinase 4 (Cdk4)-retinoblastoma (Rb) pathway has emerged as a critical target in oncogenesis. The zinc-deficient (ZD), N-nitrosomethylbenzylamine (NMBA)-induced rat esophageal cancer model provides a tool to study cell proliferation and cell cycle control in cancer initiation. Weanling rats were fed a ZD or zinc-sufficient (ZS) diet for 5 weeks, and then given a dose of NMBA. After 14 weeks, esophageal tumor incidence was 88% in ZD rats with highly proliferative esophagi versus 0% in ZS rats. Expression of p16ink4a, cyclin D1, Cdk4, and Rb in relation to that of proliferating cell nuclear antigen was characterized in esophagi by immunohistochemistry at 0, 24, and 48 h, and 1, 3, 7, 10, and 14 weeks after NMBA treatment. As early as 24 h, proliferating cell nuclear antigen-positive focal hyperplastic lesions were detected in the suprabasal layers of ZD esophagi. At the same time, overexpression of cyclin D1, Cdk4, and Rb was found in the corresponding lesion in adjacent esophageal sections. By contrast, p16ink4a expression was reduced or absent. At all time points, p16ink4a showed reduced nuclear staining in ZD esophagi compared with that in ZS esophagi. In addition, increased expression of the hyperphosphorylated forms of Rb was detected in ZD esophagi by immunoblotting. Importantly, tumors were consistently observed in ZD esophagi at very early time points. These data, obtained using a unique in vivo model for esophageal cancer with rapid tumor induction, provide strong evidence for a link between deregulation of the p16ink4a-cyclin D1/Cdk4-Rb pathway and the initiation of esophageal tumors.

Rapidly Fatal Hemophagocytic Lymphohistiocytosis Developing Within Six Days Following Deceased-Donor Renal Transplantation: Case Report.

Hemophagocytic lymphohistiocytosis (HLH) is an often fatal hyperinflammatory syndrome that may complicate malignancy, infection, rheumatic disease, or immunosuppression. HLH after kidney transplantation is most often triggered by infection, usually Herpes viruses such as cytomegalovirus and Epstein-Barr virus (EBV). It usually occurs early after transplantation. We present a case of HLH triggered by reactivation of EBV that pursued a rapidly fatal course within 6 days of receiving a deceased-donor kidney transplant. This case serves to remind transplant clinicians to consider HLH when cytopenias and hyperinflammation are atypical for the usual post-transplantation course. We discuss pitfalls in diagnosis and suggestions for treatment in this setting.

A possible role for centrosome overduplication in radiation-induced cell death.

Radiotherapy plays a key role in the treatment of many tumors; however, the precise mechanisms responsible for radiation-induced cell death remain uncertain. We have reported previously that ionizing radiation induces centrosome overduplication in human tumor cells. The present study was designed to elucidate a possible link between centrosome dysregulation and radiation-induced cell death. Exposure to 10 Gy gamma-radiation resulted in a substantial increase in cells containing an abnormally high number of centrosomes in a variety of cell lines derived from different types of human solid tumors. These aberrant centrosomes contribute to the assembly of multipolar spindles, thereby causing an unbalanced division of chromosomes and mitotic cell death characterized by the appearance of multi- or micronucleated cells. An extensive analysis of a panel of 10 tumor cell lines revealed a positive correlation between the fraction of cells with multiple centrosomes and the fraction with these nuclear abnormalities after irradiation. When the centrosome overduplication was blocked by enforced expression of p21Waf1/Cip1, the radiation-induced lethality was drastically rescued. Taken together, these results indicate that centrosome overduplication may be a critical event leading to mitotic failure and subsequent cell death following exposure to ionizing radiation.

Calcium ion-dependent signalling and mitochondrial dysfunction: mitochondrial calcium uptake during hormonal stimulation in intact liver cells and its implication for the mitochondrial permeability transition.

Hormones that elevate cytosolic Ca2+ concentrations ([Ca2+]cyt) often use Ca2+ as a messenger to activate intramitochondrial metabolic processes. However, the mitochondrial Ca2+ level also regulates the activation of the mitochondrial permeability transition (MPT), a process that involves the assembly of a high conductance proteinaceous pore across the inner and outer membrane. Studies on intact liver cells indicate that the MPT is a critical step in the cell killing induced by anoxia or respiratory inhibitors. In this study, we used freshly isolated hepatocytes to investigate to what extent the elevation of [Ca2+]cyt by vasopressin or other agonists causes Ca2+ accumulation in the mitochondria and how this treatment affects the mitochondrial susceptibility to undergo the MPT. Hepatocytes were incubated with vasopressin, glucagon, or with thapsigargin (an inhibitor of the endoplasmic reticulum Ca2+ pump) prior to permeabilization with digitonin. Mitochondrial Ca2+ accumulation was determined by following the ionomycin-induced Ca2+ release in permeabilized cells and mitochondrial swelling was studied by following cyclosporin A-sensitive light scattering changes induced by phenyl-arsenoxide and rotenone. The results indicate that agents that elevate [Ca2+]cyt cause a significant Ca2+ accumulation in the mitochondria. Excessive Ca2+ accumulation (> 10-fold increase over basal levels) was obtained with the combination of vasopressin and glucagon or with incubations containing thapsigargin. These conditions were also associated with a marked increase in rotenone-induced mitochondrial swelling. However, the more modest increase in mitochondrial Ca2+ content after treating cells with vasopressin alone did not enhance the swelling response; instead, vasopressin suppressed mitochondrial swelling compared to control incubations. Vasopressin also partly suppressed the swelling associated with thapsigargin treatment, although it did not significantly affect the Ca2+ accumulation under these conditions. This effect of vasopressin was mimicked by phorbol ester, suggesting a role for protein kinase C. The data indicate that mitochondrial Ca2+ accumulation following elevation of elevation of [Ca2+]cyt enhances the susceptibility for activation of the MPT, a response that may increase cell injury during anoxia or in response to other challenges. However, hormones also activate protective responses in the cell that suppress the MPT.

Effects of ouabain and chloride-free medium on isoosmotic volume control and ultrastructure of hepatocytes in primary culture.

Rat hepatocytes in primary monolayer culture have been studied by a combination of physiological and morphological approaches under conditions affecting ion transport and cell volume. A concentration of ouabain completely inhibiting the coupled transport of Na+ and K+ had little effect on cell volume, as indicated by cell water content, but induced the formation of many vesicles in the cytoplasm. Apparent fusion of vesicles was often observed. By itself, replacement of medium Cl by NO3- had little effect on cell volume or morphology. However, when NO3- replaced Cl- in the presence of ouabain the cells swelled and the numbers and size of vesicles were much reduced. The vesicles accumulating in the presence of ouabain showed a yellow fluorescence after the cells were loaded with acridine orange, implying that the vesicular contents were acidic. Total fluid-phase endocytosis, determined by uptake of Lucifer yellow, was not affected by ouabain or the absence of Cl-. However, ouabain considerably retarded the subsequent release of Lucifer yellow; this suggests that the dye originally taken into endocytotic vesicles became diluted by mixing with contents of ouabain-induced vesicles, an explanation consistent with the vesicle fusion seen by electron microscopy. The Cl-free medium also retarded Lucifer yellow efflux, to the same extent as ouabain, and the effects of the two treatments were not additive. These observations are consistent with the activity in hepatocytes of an ouabain-resistant, Cl(-)-dependent mechanism for cell volume control. It is suggested that this depends on the accumulation of water into acidic vesicles, which is driven by the Cl(-)-coupled activity of the vacuolar ATPases of the organelles, followed by exocytotic expulsion of their contents.

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.

Differing effects of the inhibition of poly(ADP-ribose) polymerase on the course of oxidative cell injury in hepatocytes and fibroblasts.

The effects of the two inhibitors of poly(ADP-ribose) polymerase, 3-aminobenzamide (ABA) and benzamide (BA), on the oxidative killing of L929 mouse fibroblasts and primary cultures of rat hepatocytes were studied. The killing of L929 cells by tert-butyl hydroperoxide (TBHP) occurred by two mechanisms, one sensitive and the other insensitive to the antioxidant N,N'-diphenylphenylene diamine (DPPD). Cell killing by either mechanism was prevented by the ferric iron chelator deferoxamine. ABA and BA prevented the killing of L929 cells that occurred in the presence, but not in the absence, of DPPD. ABA and BA inhibited the activity of poly(ADP-ribose) polymerase by 85%. Protection was accompanied by the sparing of the depletion of both NAD and ATP, but there was no effect of either ABA or BA on the iron-dependent appearance of single-strand breaks in DNA. Depletion of ATP by treating the fibroblasts with 2-deoxyglucose and sodium azide did not result in any loss of viability. H2O2 similarly killed the L929 cells by a mechanism that depended on a source of ferric iron. However, DPPD had no effect on the cell killing, and ABA and BA completely protected the cells in the presence or absence of DPPD. H2O2 caused the appearance of single-strand breaks that were prevented by deferoxamine, but again not by ABA or BA. ABA and deferoxamine reduced, but did not prevent, the depletion of both NAD and ATP occurring with H2O2. With the cultured hepatocytes, ABA and BA inhibited poly(ADP-ribose) polymerase at concentrations that were without effect on either the extent of cell killing or the depletion of NAD occurring with either TBHP, H2O2, or menadione. These data indicate that the relationship between oxidative DNA damage and the genesis of lethal injury is very different in the two types of cells. In the fibroblasts, the appearance of single strand breaks in DNA was accompanied by depletion of NAD and ATP and subsequently by the death of the cells. These events were mediated by the activity of poly(ADP-ribose) polymerase, as inhibition of the enzyme prevented their development. In the hepatocytes, inhibition of poly(ADP-ribose) polymerase was without effect on the oxidative death of the cells.

Roles for oxidative stress and poly(ADP-ribosyl)ation in the killing of cultured hepatocytes by methyl methanesulfonate.

The mechanisms by which the methylating agent methyl methanesulfonate (MMS) kills cultured hepatocytes were studied. In an amino-acid-free Krebs-Ringer buffer (KRB), 1 mM MMS depleted the cells of glutathione (GSH) within 1-2 hr. Lipid peroxidation, as measured by the accumulation of malondialdehyde (MDA), followed, and over 70% of the cells died within 3-4 hr. The iron chelator deferoxamine and the antioxidant N,N'-diphenyl-1,4-phenylenediamine (DPPD) prevented lipid peroxidation and death of the hepatocytes without any effect on the loss of GSH. 3-Aminobenzamide (ABA), a poly(ADP-ribose) polymerase inhibitor, also prevented the cell killing by attenuating the loss of GSH. In a culture medium containing amino acids and antioxidants (Williams' E medium, WEM), 1 mM MMS killed the hepatocytes more slowly, with 70% of the cells dying 8-12 hr after treatment. Lipid peroxidation accompanied the loss of viability. Deferoxamine and DPPD inhibited lipid peroxidation, while only partially protecting against the cell killing. ABA offered more protection and reduced the decline of GSH and decreased lipid peroxidation. ABA also reduced the extent of the depletion of both NAD and ATP that accompanied the cell killing by MMS in WEM. These data indicate that MMS killed the hepatocytes by different mechanisms depending on the culture conditions. In KRB, the toxicity of MMS was a consequence of oxidative cell injury that follows the depletion of GSH. In WEM, both oxidative injury and the action of poly(ADP-ribose) polymerase in response to DNA single-strand breaks contributed to the loss of viability.

Metabolism of acetaminophen by cultured rat hepatocytes. Depletion of protein thiol groups without any loss of viability.

Over the course of 4 hr, the metabolism of acetaminophen (APAP) by cultured rat hepatocytes resulted in a depletion of protein thiols and an accumulation of oxidized glutathione (GSSG) in the medium. With 20 mM APAP, arylation and the formation of glutathione mixed disulfides accounted for a loss of 22% of the total protein thiols in the absence of any loss of viability. With 20 mM APAP and an inhibition of glutathione reductase by 1.3-(2-chloroethyl)-1-nitrosourea (BCNU), protein thiols were depleted by 40% by arylation and the formation of glutathione mixed disulfides, again without a loss of viability. With 20 mM APAP and BCNU in the presence of 20 mM deferoxamine, there was still little or no cell killing after 8 hr despite a loss now of almost 60% of the total protein thiols. These data do not support the hypothesis that a depletion of protein thiols is related to the toxicity of APAP. One millimolar APAP and BCNU killed 60% of the hepatocytes within 4 hr. In this circumstance, the loss of protein thiols was not attributable to either arylation by APAP metabolites or the formation of glutathione mixed disulfides. The antioxidant N,N'-diphenyl-phenylenediamine prevented the cell killing and the loss of protein thiols, a result implicating a role for lipid peroxidation in the depletion of protein-bound thiols. However, protein thiol depletion under these circumstances is not necessarily related to the lethal cell injury and most likely represents an epiphenomenon of the peroxidation of cellular lipids.

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.