Calcium-dependent DNA damage and adenosine 3',5'-cyclic monophosphate-independent glycogen phosphorylase activation in an in vitro model of acetaminophen-induced liver injury.

Acetaminophen (N-acetyl-p-aminophenol [APAP]) hepatotoxicity is a process characterized by Ca2+ deregulation. Cellular functions utilizing Ca2+ as a second messenger molecule affect both cytosolic and nuclear signal transduction. Many studies have independently shown Ca2+-related effects on target molecules in response to toxic doses of APAP; however, the primary Ca2+ target resulting in liver necrosis has not been determined. We hypothesize that Ca2+-dependent DNA damage is a critical event in liver necrosis caused by alkylating hepatotoxins. In this study, Ca2+-dependent endonuclease activity was determined from DNA single-strand lesions measured by fluorometric analysis of DNA unwinding. The status of cytosolic Ca2+ was determined by measuring Ca2+-dependent activation of glycogen phosphorylase a. Primary cultures of mouse hepatocytes exposed to a toxic concentration of APAP showed twofold and greater increases in glycogen phosphorylase a stimulation at 6 hours, which was reversible with Ca2+-chelating agents. Cell death was preceded by a large decline in intact, double-stranded DNA. Following toxic administration of APAP, the percentage of total double-stranded DNA was significantly reduced by 2 hours. At 6 and 24 hours, genomic integrity was compromised by 26% and 37%, respectively, compared with untreated controls. Hepatotoxic effects of APAP-mediated Ca2+ deregulation were confirmed in both primary mouse hepatocytes and the human hepatoblastoma HepG2 cell line by lactate dehydrogenase (LDH) release and tetrazolium reduction using the 3-4,5-dimethylthiazole-2-yl-2,5-diphenyltetrazolium bromide thiazol blue(MTT) assay. The Ca2+ chelator, ethylene glycol-bis (beta-aminoethyl ether) N',N',N', N'-tetraacetic acid (EGTA), blocked APAP-induced phosphorylase a activation and necrotic cell death, but failed to inhibit phosphorylase a activation by the adenosine 3',5'-cyclic monophosphate (cAMP) analogue, dibutyryl cAMP, indicating little or no contribution of the cAMP pathway to phosphorylase a stimulation during APAP-induced necrotic death. Results with these in vitro models of liver injury are interpreted as supporting the hypothesis that increased Ca2+ availability plays a major role in the progression of APAP-dependent cellular necrosis, and that the nucleus is a critical target for APAP hepatotoxicity.

Dimethylnitrosamine-induced DNA damage and toxic cell death in cultured mouse hepatocytes.

Chronic exposure to dimethylnitrosamine produces hepatic tumors through recurrent DNA alkylation, whereas acute exposure can cause liver necrosis through mechanisms that remain largely unknown. Our laboratory recently demonstrated that DNA fragmentation occurs early on and may be a causal event in dimethylnitrosamine-induced necrosis in liver. A challenge to interpreting these results is that up to 30% of liver cells are non-parenchymal and could account for the observed DNA fragmentation. In the present study, we have examined whether dimethylnitrosamine induces early genomic DNA fragmentation in cultured mouse hepatocytes. Hepatic parenchymal cells isolated from male ICR mice were cultured in Williams E medium. DNA damage was assessed quantitatively as a fragmented fraction that was not sedimented at 27,000 x g, and qualitatively from agarose gel electrophoresis. Cellular response to DNA damage was assessed by measuring induction of the DNA repair enzyme DNA ligase. Toxic cell death was estimated from release of lactate dehydrogenase (LDH) or adenine nucleotides from cells prelabeled with [3H]adenine. Dimethylnitrosamine produced a twofold increase in [3H]adenine release by 6 h and LDH release at 36 h. DNA fragmentation and DNA ligase activity increased by as early as 1 h. The Ca(2+)-endonuclease inhibitor aurintricarboxylic acid and the Ca2+ chelator ethylenediamine tetraacetic acid (EDTA) prevented DNA fragmentation through 6 h and virtually abolished cytotoxicity through 30 h. DNA ligase induction was strongly associated with DNA fragmentation. Early increases in DNA fragmentation and DNA ligase were highly correlated with later toxic cell death. Such results strongly suggest that dimethylnitrosamine-induced fragmentation of DNA in target parenchymal cells is a causal factor in the toxic death of these liver cells.

Menadione-induced DNA fragmentation without 8-oxo-2'-deoxyguanosine formation in isolated rat hepatocytes.

Menadione (2-methyl-1,4-naphthoquinone) induces oxidative stress in cells causing perturbations in the cytoplasm as well as nicking of DNA. The mechanisms by which DNA damage occurs are still unclear, but a widely discussed issue is whether menadione-generated reactive oxygen species (ROS) directly damage DNA. In the present study, we measured the effect of menadione on formation of 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG), an index of oxidative DNA base modifications, and on DNA fragmentation. Isolated hepatocytes from phenobarbital-pretreated rats were exposed to menadione, 25-400 microM, for 15, 90 or 180 min with or without prior depletion of reduced glutathione (GSH) by diethyl maleate. Menadione caused profound GSH depletion and internucleosomal DNA fragmentation, which was demonstrated by a prominent fragmentation ladder on agarose gel electrophoresis. We found no oxidative modification of DNA in terms of increased 8-oxodG formation. In contrast, the positive control of sunlamp light increased 8-oxodG 5-fold in rat hepatocytes. We conclude that oxidative modification of DNA bases is unlikely to be important in menadione-induced DNA damage.

DNA as a critical target in toxic cell death: enhancement of dimethylnitrosamine cytotoxicity by DNA repair inhibitors.

Our working hypothesis states that DNA damage is a critical step in toxic cell death. The DNA hypothesis was tested in cultured mouse hepatocytes by examining whether inhibitors of DNA repair would increase dimethylnitrosamine toxicity and DNA damage in parallel. Inhibitors were chosen for selectivity toward DNA polymerase alpha (aphidicolin, myricetin), DNA ligase (ethidium bromide), or multiple repair enzymes (ara-C, doxorubicin). Dimethylnitrosamine caused concentration-dependent DNA damage at 6 hr and cell death at 24 hr (35% ALT release vs. 8.8% in control cultured hepatocytes). Each repair inhibitor increased dimethylnitrosamine-induced DNA damage and toxic cell death in parallel. Doxorubicin maximally elevated DNA fragmentation and toxicity (57% ALT release). Repair inhibitors alone failed to damage DNA or cause cell death in this model system. These data support the hypothesis that DNA damage is an early causal event in toxic cell death caused by alkylating hepatotoxicants.

Apoptosis: molecular control point in toxicity.

Apoptosis is a controlled form of cell death that serves as a molecular point of regulation for biological processes. Cell selection by apoptosis occurs during normal physiological functions as well as toxicities and diseases. Apoptosis is the counterpart and counterbalance to mitosis in cell population determination. Complex patterns of cell signaling and specific gene expression are clearly involved in the control of cell fate. Exposure to an apogen, a trigger of apoptosis, can significantly increase apoptotic cell loss during homeostatic processes as well as acute or chronic toxicities. Alternately, suppression of apoptosis through, for example, interference in cell signaling can result in pathological accumulation of aberrant cells and diseases such as tumors. Investigations into the mechanisms underlying apoptosis have extended into many areas, driven by increasingly sophisticated instrumental and molecular biology techniques. This symposium summary explores related aspects of apoptosis, including control of cell population size and function, specific gene activity and regulation, chromatin condensation and scaffold detachment, oxidative stress-induced cell proliferation versus death by apoptosis or necrosis, and hepatotoxicant-induced apoptosis versus necrosis. Insights into the mechanisms governing apoptosis and increasing appreciation of the relevance of apoptotic cell death are redirecting research in toxicology and carcinogenesis and are yielding novel therapeutic approaches for the control of toxicity, disease, and ultimately perhaps senescence.

Obesity decreases hepatic glutathione concentrations and markedly potentiates allyl alcohol-induced periportal necrosis in the overfed rat.

Liver biopsies from 9 out of every 10 obese individuals exhibit pathological changes of unknown aetiology and 3 out of every 10 reflect severe injury in the form of periportal fibrosis. To examine the hypothesis that excessive fibrosis in obesity arises in part from a predisposition to injury of the liver by drugs and xenobiotics, we administered 5, 10 and 25 mg/kg doses of the model periportal hepatotoxin, allyl alcohol, to obese Sprague-Dawley rats and age-matched non-obese controls. Alanine aminotransferase activity (ALT) in plasma was ten-fold more elevated in obese animals than in non-obese animals given the 25 mg/kg dose (P < 0.05). On fitting the ALT results to a non-linear, parametric model by iterative non-linear least squares regression, we found that the slope of the log dose ALT curve was similar for obese and non-obese rats. However, the minimum dose required to produce elevated ALT (DMIN) was 50% lower for obese animals (DMIN 6.47 +/- 2.75 vs. 13.3 +/- 0.96 mg allyl alcohol; P < 0.05). In a subsequent experiment, allyl alcohol was administered to obese rats based on ideal body weight, which is defined as the mean total body weight of an age-matched non-obese animal. With this dosing normalization, the 25 mg/kg ideal body weight doses translated to administration of a fixed dose of 13.5 mg allyl alcohol to obese rats. Obese rats treated in this fashion exhibited more severe necrosis in the periportal zone (median necrosis score 2 versus 0-1, P < 0.05) and increased mortality over controls (44% versus 0%; P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Pain sensitivity in dietary-induced obese rats.

Previous literature indicates possible interrelationships between the endogenous opioids or endorphins, pain response, and obesity or eating behaviour. The pain response was, therefore, examined in a rat model of obesity induced by palatable food high in unsaturated fats. Pellet-fed control and energy-dense obese and nonobese rats were tested for latency of response to a thermal stimulus using the tail flick test. Obese rats exhibited a statistically significant increase in tail flick latency compared to controls. In addition, the observed latencies were significantly correlated to the body weight of the rats (r = 0.52, p < 0.01). These data suggest that dietary-induced obese rats are similar to obese humans in being less sensitive to painful stimuli, consistent with an increase in endogenous opioids in obesity.

Induction of P4502E1 by acetone in isolated rabbit hepatocytes. Role of increased protein and mRNA synthesis.

The molecular mechanism(s) underlying induction of the hepatic microsomal cytochrome P4502E1 (2E1) by xenobiotics (e.g. ethanol and acetone) is controversial. Proposed mechanisms include increased rates of enzyme synthesis due to elevated 2E1 mRNA levels, enhanced translation of pre-existing mRNA, or stabilization of 2E1 protein. To further assess which, if any, of these events predominates during the initial stages of 2E1 protein induction, we investigated the effects of acetone treatment on 2E1 content in cultured rabbit hepatocytes, an in vitro system that allows for precise control of the cellular mileau. Hepatocytes harvested from female rabbits and plated on plastic dishes with serum-supplemented medium were 90-100% viable for at least 48 hr in culture. Analysis of immunoreactive 2E1 content and aniline hydroxylase activity in microsomes isolated from hepatocytes cultured for up to 24 hr revealed that 2E1 expression was equal to that of microsomes from unplated cells and by 48 hr of culture, 2E1 levels decreased by only 35%. Moreover, microsomes isolated from cells exposed to 17 mM acetone for 24 hr exhibited a 53 and 62% increase in aniline hydroxylase activity and 2E1 content, respectively, compared to untreated cells. To explain these increases, the rate of 2E1 protein synthesis was determined in untreated cells or in cells treated with 17 mM acetone by first exposing hepatocytes to medium supplemented with 35S-labeled methionine and cysteine ([35S]Met/Cys) and subsequently assessing radiolabel incorporation into 2E1 protein. While no difference was found between untreated and acetone-treated cells in the incorporation of [35S]Met/Cys into trichloracetic acid-precipitable microsomal proteins, immunoaffinity purification of 2E1 revealed that incorporation of 35S-labeled amino acids specifically into 2E1 was elevated by acetone to 200% of control values. Treatment of hepatocytes with the transcriptional inhibitor, alpha-amanitin, markedly inhibited this acetone-mediated increase in [35S]Met/Cys incorporation into 2E1. Analysis of hepatocyte RNA revealed that acetone increased 2E1 mRNA to 130 and 160% of control levels at 6 and 24 hr, respectively, and that these increases were prevented by pretreatment with alpha-amanitin. Our results indicate that acetone increases 2E1 protein levels in cultured rabbit hepatocytes by stimulating its rate of de novo synthesis. Since this increase in 2E1 synthesis stems, at least in part, from the acetone-mediated enhancement of hepatocyte 2E1 mRNA content and is inhibitable by alpha-amanitin, transcriptional activation of the rabbit CYP2E1 gene is apparently involved in the induction of 2E1 protein by acetone.

Ca2+ antagonists inhibit DNA fragmentation and toxic cell death induced by acetaminophen.

Ca2+ accumulates in the nucleus and DNA undergoes enzymatic cleavage into internucleosome-length fragments before acetaminophen and dimethylnitrosamine produce hepatic necrosis in vivo and toxic cell death in vitro. However, Ca(2+)-endonuclease fragmentation of DNA is characteristic of apoptosis, a type of cell death considered biochemically and functionally distinct from toxic cell death. The present studies investigate DNA fragmentation as a critical event in toxic cell death by testing whether the Ca(2+)-calmodulin antagonist chlorpromazine and the Ca2+ channel blocker verapamil prevent acetaminophen-induced hepatic necrosis by inhibiting Ca2+ deregulation and DNA damage. Acetaminophen overdose in mice produced accumulation of Ca2+ in the nucleus (358% of control) and fragmentation of DNA (250% of control) by 6 h, with peak release of ALT occurring at 12-24 h (38,000 U/l). Pretreatment with chlorpromazine prevented increases in nuclear Ca2+ and DNA fragmentation and nearly abolished biochemical evidence of toxic cell death. Verapamil pretreatment also decreased Ca2+ accumulation and DNA damage while attenuating liver injury. The Ca2+ antagonists did not protect against toxic cell death through hypothermia because neither produced the delay in toxicity that is customarily associated with hypothermia. Nor did chlorpromazine or verapamil protect through inhibiting acetaminophen bioactivation. Chlorpromazine failed to diminish glutathione depletion in whole liver and isolated nuclei. Verapamil (250 microM) also failed to alter glutathione depletion in whole liver and had no effect on acetaminophen-glutathione adduct formation by mouse liver microsomes and by cultured mouse hepatocytes. Collectively, these results support the hypothesis that Ca(2+)-induced DNA fragmentation plays a significant role in cell necrosis produced by acetaminophen and may contribute to toxic cell death caused by other alkylating hepatotoxins.

Independence and additivity of cultured hepatocyte killing by Ca2+ overload and ATP depletion.

In two competing models of toxic cell death, hepatocyte killing by chemical hypoxia (CN/IAA) is attributed to ATP depletion and killing by A23187 is attributed to Ca(2+)-induced damage. The independence of these models can be questioned because CN/IAA elevates Ca2+ before killing 1c1c7 hepatoma cells and because the ATP source fructose prevents hepatocyte killing by Br-A23187. In the present studies, cultured mouse hepatocytes were exposed to CN/IAA, A23187, or treatments in combination. A23187 produced toxicity proportional to Ca(2+)-activated DNA fragmentation. CN/IAA caused comparable toxicity but no fragmentation of DNA. Treatments in combination were more toxic than either treatment alone. Aurintricarboxylic acid, a Ca(2+)-endonuclease inhibitor, decreased DNA fragmentation and the toxicity of A23187 and combination treatment without affecting CN/IAA toxicity. ATP plus oligomycin decreased CN/IAA and combination treatment toxicity but not that of A23187. These findings indicate that cultured mouse hepatocytes are killed through mechanisms that are independent and additive in their toxicities.

Ca(++)-activated DNA fragmentation and dimethylnitrosamine-induced hepatic necrosis: effects of Ca(++)-endonuclease and poly(ADP-ribose) polymerase inhibitors in mice.

Several hepatotoxic agents damage Ca++ regulation and produce toxic cell death in a manner consistent with a cause-and-effect relationship; however, vital targets of Ca++ remain unidentified. Recent results show that DNA may be the chief Ca++ target during apoptosis, a form of cell death considered distinct from toxic cell death or necrosis. The present studies explored whether nuclear Ca++ regulation is lost before dimethylnitrosamine-induced necrosis, whether DNA is attacked by Ca(++)-dependent endonucleases and whether inhibitors of Ca(++)-endonuclease activity and the DNA repair enzyme poly(ADP-ribose)polymerase affect necrosis. Adult male ICR mice received 100 mg/kg of dimethylnitrosamine i.p. By 2 to 4 hr, total nuclear Ca++ reached 150 to 180% of control and DNA fragmentation was 140 to 170% of control. Electrophoresis of DNA revealed a sharp decline in genomic DNA with the appearance of DNA fragments in a ladder-like pattern. Ca++ elevation and DNA fragmentation preceded toxic cell death by 4 hr or more and reached peak values at 18 to 24 hr, coincident with maximal alanine aminotransferase leakage. Aurintricarboxylic acid, a Ca(++)-endonuclease inhibitor, reduced toxicity 67%. 3-Aminobenzamide, nicotinamide adenine dinucleotide and theophylline, inhibitors of poly(ADP-ribose)polymerase-mediated DNA repair, potentiated liver damage 2-fold. These results support the hypothesis that DNA fragmentation plays a contributing role in toxic cell death induced by dimethylnitrosamine. Furthermore, the findings suggest that new opportunities may exist to moderate the toxicity of alkylating hepatotoxins by altering DNA regulation.

Obesity as a risk factor in drug-induced organ injury. V. Toxicokinetics of gentamicin in the obese overfed rat.

Obese human patients and obese overfed rats treated chronically with gentamicin suffer greater renal injury than nonobese patients and control animals. To understand the mechanism of this heightened susceptibility to the nephrotoxic effects of gentamicin, this study examines the plasma-time course and renal uptake of gentamicin in control and obese overfed rats following a single bolus dose. Gentamicin was administered ip to control rats at 30 mg/kg total body mass and to obese rats at 30 mg/kg ideal body mass plus 40% of excess body mass. Following gentamicin dosing, only 1 of 11 concentration-time points taken over 6/hr postdosing was different between control and obese groups. In addition, the area under the plasma concentration curve extrapolated to infinite time was not different between obese and control rats (mean +/- SD of 4.47 +/- 0.85 vs. 4.13 +/- 0.35 mg.min.ml-1, p greater than 0.5). The gentamicin plasma concentrations after 6 hr were less than 1 microgram/ml and not different between the groups; however, the concentration of gentamicin in the kidneys was 33% greater in obese than control rats at this time (324 +/- 66.9 vs. 244 +/- 34.7 micrograms/g, p less than 0.05). The fraction of dose and the total amount of drug that accumulated in the kidneys were also greater in the obese rats (42 and 72% increases). Considered with the results of previous studies, it appears that obese overfed rats sustain more severe nephrotoxicity following comparable plasma gentamicin exposure because of increased renal uptake and/or retention of drug.

The role of the nucleus and other compartments in toxic cell death produced by alkylating hepatotoxicants.

Hepatocellular necrosis occurs under a wide range of pathological conditions. In most cases, toxic cell death takes place over a finite span of time, delayed from the point of initial injury and accompanied by homeostatic counterresponses that are varied and complex. The present strategies for discovering critical steps in cell death recognize that (1) different toxins produce similar morphologic changes that precede killing in widely varied cell types, and that (2) lethal events are likely to involve one or more compartmentalized functions that are common to most cells. Investigations of the plasma membrane, endoplasmic reticulum, cytoplasm, mitochondrion, and nucleus have greatly advanced our understanding of acute hepatocellular necrosis. This report examines each compartment but emphasizes molecular changes in the nucleus which may explain cell death caused by alkylating hepatotoxicants. Accumulating knowledge about two distinct modes of cell death, necrosis and apoptosis, indicates that loss of Ca2+ regulation and subsequent damage to DNA may be critical steps in lethal damage to liver cells by toxic chemicals.

DMBA-induced cytotoxicity in lymphoid and nonlymphoid organs of B6C3F1 mice: relation of cell death to target cell intracellular calcium and DNA damage.

The purpose of these studies was to evaluate the effects of 7,12-dimethylbenz[a]anthracene (DMBA) on intracellular free Ca2+ and DNA fragmentation in lymphoid cells obtained from the spleen, thymus, and Peyer's patches (PPs) of female B6C3F1 mice. Previous studies in our laboratories have shown that DMBA is cytotoxic to these lymphoid organs and that calcium homeostasis may be impaired following DMBA treatment. The results of the present studies show that a daily oral 14-day exposure of mice to DMBA produced a dose-dependent decrease in the number of viable cells recovered from the spleen, PPs, and thymus. Intracellular levels of Ca2+ were elevated in the spleen and PPs of mice receiving 140 mg/kg of DMBA. Extensive DNA fragmentation was detected in cells obtained from the spleen and PPs, as well as from the thymus. The thymus and PPs demonstrated DNA fragmentation at significantly lower doses of DMBA (42 mg/kg) than did the spleen (140 mg/kg). While cells obtained from the thymus did not demonstrate an elevation in Ca2+ produced by DMBA, in vitro exposure of isolated thymocytes to 3-30 microM DMBA for 4 hr produced significant elevation of intracellular Ca2+. A "ladder-like" pattern of DNA fragmentation was seen by agarose gel electrophoresis of DNA obtained from thymus cells treated with DMBA in vitro, suggesting DNA degradation by endonucleases. Collectively, these studies suggest that DMBA produces lymphotoxicity through an apoptosis-like mechanism involving fragmentation of genomic DNA by Ca(2+)-activated enzymes.

Acetaminophen-induced cytotoxicity in cultured mouse hepatocytes: effects of Ca(2+)-endonuclease, DNA repair, and glutathione depletion inhibitors on DNA fragmentation and cell death.

Hepatotoxic alkylation of mouse liver cells by acetaminophen is characterized by an early loss of ion regulation, accumulation of Ca2+ in the nucleus, and fragmentation of DNA in vitro and in vivo. Acetaminophen-induced DNA cleavage is accompanied by the formation of a "ladder" of DNA fragments characteristic of Ca(2+)-mediated endonuclease activation. These events unfold well in advance of cytotoxicity and the development of necrosis. The present study utilized cultured mouse hepatocytes and mechanistic probes to test whether DNA fragmentation and cell death might be related in a "cause-and-effect" manner. Cells were isolated by collagenase perfusion, cultured in Williams' E medium for 22-26 hr, and exposed to acetaminophen. Aurintricarboxylic acid, a general Ca(2+)-endonuclease inhibitor, and EGTA, a chelator of Ca2+ required for endonuclease activation, significantly decreased DNA fragmentation at 6 and 12 hr and virtually abolished cytotoxicity. N-Acetylcysteine also eliminated DNA fragmentation and cytotoxicity. 3-Aminobenzamide, an inhibitor of poly(ADP-ribose) polymerase-stimulated DNA repair, failed to alter the amount of DNA fragmentation at 6 hr but substantially increased acetaminophen cytotoxicity in hepatocytes at 12 hr. With the exception of when DNA repair was inhibited by 3-aminobenzamide, Ca2+ accumulation in the nucleus, DNA fragmentation, and hepatocyte death varied consistently and predictably with one another. Collectively, these findings suggest that unrepaired damage to DNA contributes to acetaminophen-induced cell death in vivo and may play a role in necrosis in vivo.

Acetaminophen-induced cytotoxicity in cultured mouse hepatocytes: correlation of nuclear Ca2+ accumulation and early DNA fragmentation with cell death.

Hepatotoxic doses of acetaminophen cause widespread alkylation of liver and early loss of cytosolic Ca2+ regulation. Although the precise location and target of lethal alkylation are not known, Ca2+ accumulation is viewed as a possible link between cell alkylation and cell death. We have recently shown that Ca2+ accumulates in the nucleus and that DNA fragments in vivo before the development of acetaminophen-induced necrosis in mice. The present study examined cultured hepatocytes for nuclear damage and its association with cell death in vitro. Positive results would argue for two key points. (1) Nonparenchymal cell damage does not explain DNA fragmentation induced by acetaminophen in vivo. (2) A chemical that causes necrosis can produce DNA damage considered characteristic of apoptosis. Hepatocytes from NIH Swiss mice were isolated by collagenase perfusion, cultured in Williams' E medium for 24 hr, and exposed to acetaminophen. Cytotoxicity was assessed by lactate dehydrogenase leakage and release of [3H]adenine from a prelabeled nucleotide pool. Genomic DNA fragmentation was assessed quantitatively by colorimetric analysis and qualitatively by agarose gel electrophoresis. Acetaminophen caused DNA damage from 1-4 hr onward and produced significant release of lactate dehydrogenase and [3H]adenine nucleotides at later times. Agarose gel electrophoresis revealed a "ladder" of DNA fragments characteristic of Ca(2+)-mediated endonuclease activation. Cytotoxicity correlated with nuclear Ca2+ accumulation (r greater than 0.895, p less than 0.05) and with percentage DNA fragmentation (r greater than 0.835, p less than 0.05). Nuclear changes in vitro generally reproduced those observed in vivo. Collectively, these findings demonstrate that nuclear Ca2+ accumulation and DNA fragmentation appear as early events that correlate directly with later cytotoxicity. These changes may contribute to acetaminophen-induced injury leading to cell death in vitro and necrosis in vivo.

Induction of cytochrome P450IIE1 in the obese overfed rat.

Cytochrome P450IIE1 (IIE1) is a microsomal xenobiotic-activating enzyme that is inducible not only by various chemical agents but also by fasting and diabetes. Using a rat model that mimics human obesity, we have found that hepatic IIE1 levels are also increased by this common clinical disorder. Liver microsomes from rats made obese by feeding with an energy-dense diet displayed elevated aggregate P450 content (+28%) and enhanced catalytic activities associated with IIE1, including low-Km N-nitrosodimethylamine demethylation (+66%), aniline hydroxylation (+52%), p-nitrophenol hydroxylation (+170%), and acetaminophen-cysteine conjugate formation (+28%). In contrast, obesity had no significant effect on cytochrome b5 content, P450 reductase activity, benzphetamine demethylation, or erythromycin demethylation, with the latter two reactions being linked with rat IIC11 and IIIA1, respectively. The enhancement of IIE1-dependent drug-metabolizing activities noted in liver microsomes from obese rats was paralleled by a similar increase (111%) in hepatic IIE1 protein content in these animals, as assessed on immunoblots developed with anti-hamster IIE1 IgG. Anti-IIE1-inhibitable rates of microsomal p-nitrophenol metabolism, a reaction highly correlated with IIE1 content (r = 0.88, p less than 0.01), were over 3-fold higher in obese rats than in nonobese controls, providing additional evidence for the obesity-related increase of hepatic IIE1. The induction of IIE1 by the pathophysiological condition of obesity may provide a biochemical basis for the increased incidence of occult liver disease and certain cancers noted in obese individuals.

Extensive alteration of genomic DNA and rise in nuclear Ca2+ in vivo early after hepatotoxic acetaminophen overdose in mice.

Hepatotoxic doses of acetaminophen cause early impairment of Ca2+ homeostasis. In this in vivo study, 600 mg/kg acetaminophen caused total nuclear Ca2+ and % fragmented DNA to rise in parallel from 2-6 hr, followed by large later increases mirroring frank liver injury. Agarose gel electrophoresis revealed substantial loss of large genomic DNA from 2 hours onward, with accumulation of DNA fragments in a ladder-like pattern resembling apoptosis. Extensive late cleavage of DNA probably resulted from cell death, whereas degradative loss of large genomic DNA at 2 hours arose at an early enough point to contribute to acetaminophen-induced liver necrosis in mice.

Early loss of large genomic DNA in vivo with accumulation of Ca2+ in the nucleus during acetaminophen-induced liver injury.

Hepatotoxic doses of acetaminophen cause early impairment of Ca2+ homeostasis in the liver. This in vivo study considers the nucleus as a possible site of lethal Ca2+ action by evaluating whether acetaminophen raises Ca2+ in this compartment, whether DNA becomes altered, and whether DNA changes occur early enough during injury to contribute causally to necrosis. Fed Swiss mice were treated with 600 mg/kg acetaminophen ip and livers and blood samples were collected over time. Total nuclear Ca2+ accumulation and fragmentation damage to DNA showed modest parallel increases between 2 and 6 hr, followed by greater than 200% rises at 12 hr mirroring the appearance of frank liver injury (ALT greater than 10,000 U/liter). However, agarose gel electrophoresis revealed extensive loss of large genomic DNA from 2 hr onward, accompanied by the appearance of periodic DNA fragments. Thus, acetaminophen raised nuclear Ca2+ concentrations and promoted DNA fragmentation in vivo. The considerable cleavage of DNA seen at late times probably resulted from cell death, whereas loss of large genomic DNA from 2 hr onward appeared at an early enough point in time to be a contributing factor in acetaminophen-induced liver necrosis.