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DOI: 10.1177/019262339602400111

1996 24: 77Toxicol PatholGregory L. Kedderis

Biochemical Basis of Hepatocellular Injury

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Biochemical Basis of Hepatocellular Injury*

GREGORY L. KEDDERIS

Chemical Industry Institute of Toxicology, Research Triangle Park, North Carolina 27709

*Address correspondence to: Dr. Gregory L. Kedderis. Chemical In-

dustry Institute of Toxicology, PO. Box 12137, 6 Davis Drive, ResearchTriangle Park, North Carolina 27709.

ABSTRACT

The hepatotoxic response elicited by a chemical agent depends on the concentration of the toxicant (parent compound or metaholite)delivered to the hepatocytes across the liver acinus via blood flow. Hepatotoxicants produce characteristic patterns of cytolethality in

specific zones of the acinus due to the differential expression of enzymes and the concentration gradients of cofactors and toxicant inblood across the acinus. Most hepatotoxic chemicals produce necrosis, characterized by swelling in contiguous tracts of cells and

inflammation. This process has been contrasted with apoptosis, where cells and organelles condense in an orderly manner under geneticcontrol. Biotransformation can activate a chemical to a toxic metabolite or decrease toxicity. Quantitative or qualitative speciesdifferences in biotransformation pathways can lead to significant species differences in hepatotoxicity. Fasted rodents are more sus-

ceptible to the hepatotoxic effects of many chemicals due to glutathione depletion and cytochrome P-450 induction. Freshly isolated

hepatocytes are the most widely used in vitro system to study mechanisms of cell death. Hepatotoxicants can interact directly with

cell macromolecules or via a reactive metabolite. The reactive metabolite can alkylate critical cellular macromolecules or induce

oxidative stress. These interactions generally lead to a loss of calcium homeostasis prior to plasma membrane lysis. Mitochondriahave been shown to be important cellular targets for many hepatotoxicants. Decreasing hepatocellular adenosine triphosphate concen-trations compromise the plasma membrane calcium pump, leading to increased cellular calcium concentrations. Calcium-dependentendonucleases produce double-strand breaks in DNA before cell lysis. These biochemical pathways induced by necrosis-causingtoxicants are similar to the biochemical pathways involved in apoptosis, suggesting that apoptosis and necrosis differ in intracellularand extracellular control points rather than in the biochemistry involved in cell death.

Keywords. Hepatotoxicity; hepatocyte; cytolethality; necrosis; apoptosis; biotransformation; calcium homeostasis; mitochondria;DNA

CHEMICAL HEPATOTOXICITY

A wide variety of chemicals, environmental pollutants,dietary constituents, pharmaceutical agents, and natural

products can cause hepatotoxicity in animals and humans.

Exposure to hepatotoxic agents is usually accidental,through contaminated food, water, or air or from unan-

ticipated side effects of therapeutic agents. Chemical hep-atotoxicity is a dose- and time-dependent phenomenonthat is reversible at early stages upon cessation of expo-sure to the toxicant. However, severe intoxication with

hepatotoxic chemicals can lead to liver necrosis and deathof the organism if left untreated. Knowledge of the mech-

anism of action of different hepatotoxic agents is essen-tial for understanding the biochemical events leading tocell death. Understanding the mechanisms of cell death

by toxic agents will improve diagnosis of hepatotoxicityand will enable predictions of the potential hepatotoxiceffects of chemical agents. Mechanistic information isalso required for effective antidotal therapy. In the case

of hepatotoxic therapeutic agents, knowledge of mechnisms of toxicity can lead to the development of sadrugs.

The hepatotoxic response elicited by a chemical agdepends on the concentration of the toxicant (either pent compound or toxic metabolite) delivered to the

patocytes in different regions of the liver acinus via blflow. The liver acinus consists of parenchyma centearound the terminal branches of the portal vein and

patic artery (37). Liver cells are radially arrangedplates along the sinusoids, which drain via the termi

hepatic vein (central vein). Blood flows from the branes of the

portalvein and

hepatic arteryto the central

veresulting in 3 microcirculatory zones where the e~ l lside (37). The cells in Zone 1 lie closest to the affervessels and receive the highest concentrations of oxygand nutrients. Cells in Zone 2 lie further downstream, a

cells in Zone 3 lie near the terminal hepatic v cin. T

gradient of oxygen and nutrients leads to a zonation

hepatic intermediary metabolic functions (? 1 ). Hepacytes from afferent (Zone 1 ) and efferent (Zone 3) zoof the liver acinus differ in enzyme content and metabo

capacity. For example. cells in Zone 1 tend to have mo

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mitochondria and higher respiration rates than cells inZone 3. However, studies with isolated perfused liver

preparations have shown that the rates of metabolic pro-cesses across the liver acinus can be shifted by changingthe direction of flow from anterograde to retrograde (53).These results indicate that most metabolic processes de-

pend on the rate of delivery of cofactors, nutrients, or

chemicals (i.e., theyare

substrate-limited).The zonation of metabolic functions has important im-

plications for chemically induced toxicity (21, 53). He-

patotoxicants produce characteristic patterns of cytole-thality in specific zones of the acinus due in part to thedifferential expression of enzymes and the concentration

gradients of cofactors and toxicant in blood across theacinus. However, the relationships among these factorsare complex, and the underlying reasons for cell-specifictoxicity are not well understood.

Most hepatotoxic chemicals produce necrosis. This

process generally involves a loss of cellular volume ho-meostasis in contiguous tracts of cells, resulting in mem-brane swelling (bleb formation) followed by rupture anddestruction of the organelles (11, 60). Inflammation is

usually present. This process has been contrasted with

apoptosis (programmed cell death), where cells and or-

ganelles condense in an orderly manner under geneticcontrol (60). During apoptosis, there is activation of a

calcium-dependent endonuclease that cleaves DNA anda condensation of cell organelles. Protein synthesis is re-

quired in some cases. The affected cell fragments into

apoptotic bodies that are phagocytized by neighboringhepatocytes and digested. Unlike necrotic cells, apoptoticcells show no evidence of increased membrane perme-

ability (60).

BIOTRANSFORMATION OF HEPATOTOXICANTS

The process of biotransformation is intimately in-volved in chemically induced hepatotoxicity. Most hep-atotoxic chemicals are protoxicants that require bioacti-vation to exert their toxic effects. Metabolism can mod-

ulate the properties of hepatotoxic agents in 2 generalways: it can either increase toxicity (toxication ormetabolic activation) or decrease toxicity (detoxication)(23). The toxic potency of a hepatotoxic chemical toward

hepatocytes depends on the relative rates of these 2 bi-otransformation processes.

Biotransformation reactions are of 2 general types,called Phase I reactions and Phase II reactions. Phase I

reactions involve oxidations, reductions, and hydrolyses.Many of the oxidation and reduction reactions are cata-

lyzed by the cytochrome P-450-dependent mixed-func-tion oxidase system in the membranes of the endoplasmicreticulum. This nicotinamide adenine dinucleotide phos-phate (NADPH)-dependent mixed-function oxidase sys-tem consists of the flavoprotein NADPH-cytochrome P-450 reductase and the hemeprotein cytochrome P-450.The system is termed a mixed-function oxidase because

electrons are taken from NADPH to reduce oxygen and

oxidize or hydroxylate substrates. Cytochromes P-450 area superfamily of hemeproteins with overlapping substrate

specificity that catalyze a variety of oxidation and reduc-tion reactions, including carbon hydroxylation and the

dealkylation of substituted heteroatoms such as N, 0S (12, 13, 23, 35). The biosyntheses of the varioutochrome P-450 isoforms can be induced by treawith chemicals, including hepatotoxic agents (12,Enzyme induction can lead to increased or decreased

sitivity of animals to hepatotoxic agents. Sometimeseffects are unanticipated, such as when the indu

agentsare

present in the diet (23).The

endoplasmiticulum also contains a second NADPH-dependmixed-function oxidase system, the flavin-contamonooxygenases, which oxidize amines and sulfur

pounds (12, 23). Phase I reactions generally make cicals more polar, facilitating their elimination and pring a functional group that can be conjugated with

drophilic moiety during Phase II metabolism. Phaseactions can result in either bioactivation or detoxic

depending on the toxicity of the metabolite.Phase II reactions (also termed synthetic reactions

volve the conjugation of chemicals with hydrophiliceties such as glutathione, glucuronide, sulfate, or aacids (20). These reactions are usually considered d

ication pathways because they result in the formatia more water-soluble and easily excreted metabo

However, conjugation reactions can also lead to thmation of unstable precursors to reactive speciewhich case they would be bioactivation pathwaysexample, the reactive acyl glucuronide metabolite ofibrate may be involved in the hepatotoxicity of thisobserved in clinical studies (50). Glutathione conjugof haloalkanes can result in the formation of glutathbased sulfur mustards that are thought to be involvthe toxicity and mutagenicity of these compoundsMarked species differences have been observed

expression of xenobiotic-metabolizing enzymes tha

result in species differences in the sensitivity to hetoxic agents. The cytochrome P-450 superfamily ca

play very significant species, strain, and gender dences in expression and catalytic activity (12, 23). Tdifferences in enzyme expression and substrate specican produce qualitative species differences (the meta

pathway is absent in 1 species) or quantitative spdifferences (the rate of the pathway is different) imetabolic pathways involved in the bioactivation otoxication of hepatotoxicants. Species differencesotransformation are responsible for most of the k

species differences in chemical hepatotoxicity. Fo

ample, hamsters and mice are sensitive to the hepatoeffects of acetaminophen, whereas rats and human

pear to be resistant. These species differences in

totoxicity are largely due to species differences in thof production of the toxic metabolite of acetaminoN-acetyl-p-benzoquinoneimine (NABQI). Isolated

tocytes from all 4 species are equally susceptibletoxic effects of NABQI (51).Another interesting exais the sex difference in the hepatotoxicity of the pizidine alkaloid senecionine observed in rats, with

being much more sensitive than females. Senecioni

quires cytochrome P-450-mediated bioactivation toa toxic pyrrolic metabolite that interacts with criticalular macromolecules to produce hepatic cytolethFemale rats lack the isoform of cytochrome P-45


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volved in senecionine bioactivation. When female rats are

treated with dexamethasone to induce the 3A family of

cytochromes P-450, they become as sensitive to sene-cionine hepatotoxicity as male rats (57).The enzymes involved in the biotransformation of tox-

ic chemicals are intimately linked to the processes of cel-lular intermediary metabolism. In most cases, the rate-

controlling step in biotransformation reactions is cofactor

supply (52). The nutritional state of the animal can haveprofound effects upon the hepatic concentrations ofNADPH and glutathione. Changes in the concentrationsof these cofactors can markedly alter the sensitivity ofanimals to hepatotoxicants. Many investigators fast ro-dents overnight before orally administering a compoundunder study so that the absorption of the compound willnot be impaired by food in the stomach. However, this

practice makes rodents more susceptible to the hepato-toxic effects of many chemicals. Unlike larger animals,an overnight fast decreases hepatic glutathione in rodents

by approximately 50% (19). This severely compromisesthe ability of rodent liver to detoxicate reactive inter-mediates. Overnight fasting also induces cytochrome P-

450 2E1 (17). This enzyme is involved in the bioacti-vation of a wide variety of rodent carcinogens, solvents,and toxicants (14), so induction of P-450 2E can greatlyaffect the balance between detoxication and bioactivation.

For example, fed rats are relatively resistant to the hep-atotoxic effects of bromobenzene and acetaminophen.

After an overnight fast, the rats become extremely sus-

ceptible to these hepatotoxicants (34).

IN VITRO SYSTEMS TO STUDY HEPATOTOXICITY

In vitro systems have been developed to study mech-anisms of chemical hepatotoxicity because of the com-

plexity of the whole animal and the experimental diffi-

culty of controlling all the variables that may affect in-

terpretation of the data. In vitro systems are much easierto control than the intact organism and are generally moreamenable to precise analytical techniques. Three majorexperimental systems have been used to study chemical

hepatotoxicity: isolated perfused liver preparations, liverslices, and isolated hepatocytes. Each system has its ad-

vantages and disadvantages, and the choice of which sys-tem to use depends on the experimental questions beingasked. The isolated perfused liver preparation allowsstudies of hepatic function in a physiologically integratedsystem (59). However, performing more than 1 set of

experiments with each preparation is difficult. Precision-cut liver slices in dynamic culture offer a system thatretains cell polarity and the heterogeneity of hepatocyte

function (48). This system bridges the gap between wholeorgans and isolated cells. One of the disadvantages ofthis system is that identifying the target cells of chemi-

cally induced toxicity using enzyme leakage assays issometimes difficult. Diffusion of toxicant and nutrients

through the slice can also be a problem, and hypoxia candevelop. Isolated hepatocytes are easily prepared and eas-ily manipulated for mechanistic studies (44). Many ex-periments can be done with the cells isolated from 1 liver.

Hepatocytes are larger and more dense than nonparen-chymal cells, enabling rapid purification by low-speed

centrifugation. Ho~~e~er isolated cells lack the anatomcal architecture. heterogeneity. and plasma membrane plarity of hepatocytes in iiio. Upon isolation, hepatocytround out. The polarity of the plasma membrane is lbut region-specific functions are retained t 10>.

Most studies of the mechanisms involved in cell dea

have been carried out with isolated hepatocvtes. Heptocytes are prepared by a 2-step perfusion of the liv

first with calcium chelators. to wash out calcium aloosen the tight junctions between the cells, and then wicalcium and collagenase, to digest the collagen matrixthe liver (44). Freshly isolated hepatocytes have beshown to retain the metabolic capacity of the liver bo

qualitatively and quantitatively (3, 18, 24). Isolated

patocytes produce the same array of Phase I and PhaII metabolites as observed in the liver W vivo (3). Kine

studies in isolated hepatocytes can accurately predchemical pharmacokinetics in vivo (18, 24). This iscontrast to hepatocytes in monolayer culture, whi

promptly dedifferentiate and lose much of their biotraformation capabilities (47). There is not only an overdecrease in cytochromes P-450, but also a different

expression of isoforms compared to the intact liver (4The decrease in individual enzyme activities can be mo

ulated by hormonally defined media or culture matricbut these treatments do not affect all enzyme activit

equally.After several days in culture, the endoplasmreticulum decreases in area, while vacuolization increa

(47). Thus, cultured hepatocytes are not an appropriatevitro model system for many aspects of chemicallyduced hepatotoxicity in vivo.

Cytolethality in vitro is generally studied using assfor plasma membrane permeability (5). The cell is cosidered dead if the plasma membrane becomes permeabto large molecules such as trypan blue or leaks enzymsuch as lactate dehydrogenase. Functional assays can a

be used to assess the viability of intermediary metabopathways.Adenosine nucleotides can be measured tosess cellular energy capacity. Macromolecular synthesmitochondrial activity, and active transport of ionsamino acids can also be measured as indicators of c

functions. Morphological assessments of membrane bl

bing or volume changes can also be made. The choice

assays to use once again depends on the experimentquestions being asked. It is important to keep the re

tionship between the experimental question and the mesured endpoint in perspective.

MECHANISMS OF HEPATOCELLULAR

INJURYAND DEATH

The mechanisms by which hepatotoxic chemicalscells can be organized into 4 general nonexclusive arof increasing complexity. It must be stressed that seveof these mechanisms can be operable at the same twith a single toxicant. Sometimes different mechaniscan be operable at different doses of toxicant.Althousome mechanisms of chemical toxicity have been id

tified, the physiological basis for translation of thesefects into cytolethality is not well understood and isactive area of research. It is clear. hoBB B v er. that there

not 1 single button for cell death that 1,-, pushed by



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toxicant. It seems likely that a number of critical cellularevents could be targets for toxic chemicals in that inac-

tivation or modulation of function could initiate a series

of events leading to cell death.A thorough understandingof how the cell regulates its critical functions under nor-mal circumstances is necessary to completely understandthe physiological basis for the toxic effects of chemical

agents.The simplest general mechanism involved in cytole-thality is a direct effect of the toxicant upon critical cel-lular systems. Toxicants such as chlorpromazine and oth-er phenothiazines, erythromycin salts, and chenodeoxy-cholate can have direct surfactant effects on the hepato-cyte plasma membrane (43). These effects will ultimatelydisrupt cellular volume homeostasis and lead to celldeath. The mushroom toxin phalloidin binds to actin and

disrupts the cell cytoskeleton, resulting in increased plas-ma membrane permeability (56). Various chemicals andmetal ions bind to mitochondrial membranes and en-

zymes, disrupting energy metabolism and cellular respi-ration. Many hepatotoxic compounds are direct inhibitorsand uncouplers of mitochondrial electron transport.A second general mechanism involved in chemicallyinduced cytolethality is reactive metabolite formation

(16, 29). Many hepatotoxicants such as carbon tetrachlo-

ride, allyl alcohol, acetaminophen, and bromobenzene are

metabolically activated to chemically reactive toxic spe-cies. These chemically reactive metabolites (generallyelectron-deficient species termed electrophiles) can co-

valently bind to crucial cellular macromolecules (gener-ally electron-rich species termed nucleophiles) and inac-tivate critical cellular functions such as ion homeostasis.

Although the covalent binding theory of chemical tox-

icity may be conceptually satisfying, the critical cellular

target for covalent binding has not been identified. Itseems unlikely that a single moiety would function as a

trigger for cell death by toxic chemicals.Glutathione is the major cellular nucleophile and pro-

vides an efficient detoxication pathway for most electro-

philic reactive metabolites (27). Thus, glutathione deple-tion can render cells more susceptible to the toxic effectsof chemicals (55).A threshold effect is often seen for

chemically induced toxicity until cellular glutathione is

depleted. Cellular glutathione can be depleted by alky-lating agents, oxidative stress, excess substrates for con-

jugation, and biosynthetically by administration of the ir-reversible inhibitor of y-glutamylcysteine synthetasebuthionine sulfoximine

(26).Glutathione can be in-

creased by administration of precursors such as N-ace-tylcysteine and L-2-oxothiazolidine-4-carboxylate. These

compounds can be effectively used as antidotes for some

hepatotoxic agents such as acetaminophen (58).In addition to covalent binding and depletion of glu-

tathione, reactive intermediates can initiate other patho-biological processes such as lipid peroxidation and redox

cycling. These processes are involved in the third generalmechanism leading to cell death, oxidative stress, whichcan be defined as an alteration in the intracellular proox-idant-to-antioxidant ratio in favor of prooxidants (46).Cycling of oxidized and reduced forms of a toxicant suchas quinone produces reactive superoxide radical anions

FiG. 1.-Redox cycling leading to the formation of reactive

species.A quinone drug is reduced to a semiquinone by a N

dependent flavoprotein reductase. The semiquinone reduces mo

oxygen to the superoxide anion radical. Superoxide dismutase

hydrogen peroxide from superoxide. Hydrogen peroxide can beicated by catalase or glutathione (GSH) peroxidase, or it can rea

a metal ion (M) to form hydroxyl radicals.

that can lead to the formation of hydrogen peroxidhydroxyl radicals (Fig. 1). Metal ions such as iro

copper can participate in redox cycling. The toxic o

species producedcan

deplete glutathione throughtion, can oxidize critical protein sulfhydryl grouvolved in cellular or enzymic regulation, and can i

lipid peroxidation. Organic hydroperoxides can proxidative stress directly through oxidation of criticlular sulfhydryl groups or initiation of free radicacesses.

Lipid peroxidation was once considered the cmechanism of toxicity due to oxidative stress an

radical-producing toxicants such as carbon tetrachlIn the absence of exogenous iron, however, lipid pidation is not very extensive, and its toxicologic scance is unclear. Lipid peroxidation can be importacause it can amplify free radical processes, becau


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products are toxic. and because it can compromise de-toxication systems such as glutathione. However, it is dif-ficult to distinguish lipid peroxidation of dead cells from

lipid peroxidation involved in cell death. If lipid peroxi-dation has a causal role in cytolethality, then (a) it shouldoccur in all cases of toxicity by the agent, (b) it shouldoccur before cell death, (c) the extent of peroxidationshould correlate with the extent of injury, and (d) agents

that prevent lipid peroxidation should protect cells fromcytolethality (54). This reasoning was used by Rush et al(42) in their studies of t-butyl hydroperoxide toxicity to-ward isolated rat hepatocytes. They found that lipid per-oxidation accompanied lactate dehydrogenase release inhepatocytes incubated with t-butyl hydroperoxide. Inclu-sion of the antioxidant promethazine prevented lipid per-oxidation but had no effect on cell death, indicating that

lipid peroxidation was not involved in t-butyl hydrope-roxide-induced cytolethality.Although lipid peroxidationmay be an important mechanism of cytolethality for some

agents, considerable evidence shows that lipid peroxida-tion is not a necessary component of oxidative stress

(54).

The fourth general mechanism involved in chemicallyinduced hepatotoxicity is the disruption of calcium ho-meostasis (31, 36). Calcium regulates a wide variety ofcritical physiological functions in the cell. Calcium ho-meostasis in the cell is very precisely regulated. The con-siderable calcium concentration gradient between the in-side of the cell (-~-10- M) and the extracellular fluid

(~l 0-3 M) is maintained by an active calcium-effluxingadenosine triphosphatase (ATPase). Thus, this importantenzyme system can be a potential target for toxicants.Inside the cell, calcium is sequestered by mitochondria,endoplasmic reticulum, and the nucleus as well as bycalcium-binding proteins (31).

It has long been known that calcium accumulates in

necrotic tissue after ischemic or chemical injury, but itwas not known whether this accumulation occurred dur-

ing or after cell death. Recent studies have shown that

many hepatotoxic chemicals disrupt cellular calcium ho-meostasis. Disruption of calcium homeostasis by variousmeans appears to be a common feature of chemical hep-atotoxicity, oxidative stress, and anoxic injury (15, 31,41). Calcium is released from mitochondria by uncou-plers of oxidative phosphorylation, quinones, hydrope-roxides, and metal ions such as cadmium and iron. Tox-icants such as carbon tetrachloride, bromobenzene, hy-droperoxides, and aldehydes release calcium from the en-

doplasmic reticulum. Many toxicants, including carbontetrachloride, chloroform, dimethylnitrosamine, and acet-

aminophen, cause an influx of calcium through the plas-ma membrane. Other toxicants such as cystamine, diquat,and vanadate ions increase the cellular calcium concen-

tration by inhibiting calcium efflux from the cell.There are several toxicologic consequences of the dis-

ruption of cellular calcium homeostasis ( 15, 31, 36, 41).Alterations in the cell cytoskeleton can occur. The poly-merization state of the protein components of the cyto-skeleton is regulated by calcium, so increases in cellularcalcium concentration can alter the cytoskeleton. This canlead to plasma membrane blebbing, alterations in plasma

TABLE L-Chemical toxicants that induce DNA double-strand break

hepatocytes prior to cell death.

membrane channels, and alterations in plasma membra

permeability.A calcium-activated neutral proteasebeen shown to be involved in the formation of plamembrane blebs (33). Increases in calcium can activ

both calcium-dependent and calmodulin-dependent phpholipases, leading to a stimulation of arachidonate mtabolism and increased plasma membrane permeabiliIncreased cytoplasmic calcium concentrations can aactivate proteases such as calpain. The cytolethality

cystamine appears to be mediated by a calcium-activaneutral protease, as inhibitors of neutral proteases sas antipain and leupeptin protect against cytolethal(32).

Increased cellular calcium concentrations can also

tivate endonucleases that degrade DNA (31). Mountievidence indicates that DNA is an important cellular

get in chemically induced hepatic cell death (6). It

recently been shown that a wide variety of non-D

reactive, nonmutagenic cytotoxic agents induce a dmatic loss of cellularATP and formation of double-str

breaks in hepatocyte DNA prior to the loss of plamembrane integrity (9). Chemical agents that have beshown to induce DNA strand breaks in hepatocytes prto plasma membrane rupture are listed in Table I. Theagents include detergents, drugs such as acetaminophuncouplers of oxidative phosphorylation, and carci

gens. Elia et al (9) found that mutagenic carcinogens oinduced double-strand breaks in DNA at cytotoxic ccentrations. At lower noncytotoxic concentrations

carcinogens induced single-strand breaks in DNA frcovalent interactions and adduct repair.

Mitochondria are important regulators of intracellucalcium concentrations in that they can take up andlease calcium, acting as an intracellular buffer (31, 4



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82

Hence, mitochondria can be important intracellular tar-

gets for toxic chemicals.Acetaminophen is known to de-

plete hepatocellular protein thiols, increase cytosolic cal-cium (30), and inhibit mitochondrial respiration (28). He-

patic DNA fragmentation induced by acetaminophen (40)is inhibited by calcium channel blockers such as vera-

pamil (38) and endonuclease inhibitors such as aurintri-

carboxylicacid

(45).These treatments also

preventacet-

aminophen-induced cytolethality. Inhibition of the DNA

repair enzyme poly(ADP-ribose) polymerase increased

acetaminophen-induced cytolethality (45). Similarly, in-hibition of endonuclease prevented dimethylnitroso-amine-induced DNA fragmentation and cytolethality(39), whereas these effects were potentiated by inhibitionof DNA repair (22, 39). These results are consistent withDNA being an important target in chemically inducedcell death.

Taken together, these studies suggest a general scenariofor hepatocellular death induced by many chemical tox-icants. The chemical enters the hepatocyte and may bemetabolized to form a toxic, reactive intermediate. The

toxicant interacts with critical mitochondrial enzymes orwith some component of theATP-generating systems ofthe cell, leading to a decrease in cellularATP This mayresult from inhibition of electron transport or uncouplingof oxidative phosphorylation. If the interaction is irre-versible and irrepairable, then the cell will be committedto a sequence of events leading to plasma membrane rup-ture and cell death. The decrease in cellularATP com-

promises the activity of the ATP-dependent calciumpump of the plasma membrane, and the intracellular cal-cium concentration rises. Several calcium-dependent de-

gradative enzymes are activated, including endonucleasesthat cleave DNA.All these events happen before the plas-ma

membrane ruptures. Small amounts ofDNA

damagecan be repaired by the cell, but when DNA damage be-comes extensive the cell will die. Extracellular signalsare sent during this process to recruit inflammatory cellsto remove the damaged hepatocytes.The cellular biochemistry of cell death induced by ne-

crosis-causing hepatotoxic chemicals is quite similar tothe biochemical processes involved in apoptosis. One ofthe key features of apoptosis is the condensation of chro-matin and the cleavage of DNA into nucleosome-sized

fragments (1, 7, 60).Although apoptosis is under more

precise genetic control and the degradation of the celloccurs in a much more orderly fashion (suicide) than celldeath induced by necrosis-causing chemicals (murder),both processes involve the activation of calcium-depen-dent endonucleases and early degradation of cellulaiDNA (1, 7). Biosynthesis of a calcium-independent en-donuclease is also involved in apoptosis in some cel15

(1). The elevation of intracellular calcium concentrations

in apoptosis appears to be mediated by the biosynthesisof a protein that acts as a calcium pore (1). Thus, al-

though apoptosis is under precise genetic control, the sa-lient biochemical features of cell death by apoptosis oi

necrosis-causing chemicals are qualitatively similar. It i~not known whether or not the same endonuclease en-

zymes are involved in both processes. The major qualm-tative difference in biochemical pathways between cel

death by apoptosis and necrosis-causing chemicals

pears to be in their molecular control points and ecellular signals (1, 7). Understanding the nature ofcontrol points and their regulation is an active arresearch in many laboratories, and our knowledge omolecular events involved in apoptosis and necros

growing rapidly. This knowledge will have practica

plicationfor the

developmentof antidotal treatment

chemical intoxication as well as for the developmemechanism-based therapies for diseases such as candAIDS.

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1.Alison MR and Sarraf CE (1995).Apoptosis: Regulation aevance to toxicology. Human Exp. Toxicol. 14: 234-247.

2.Anders MW, Lash L, Dekant W, ElfarraAA, and Dohn DR

Biosynthesis and biotransformation of glutathione S-conjugatoxic metabolites. Crit. Rev. Toxicol. 18: 311-341.

3. Billings RE, McMahon RE,Ashmore J, and Wagle SR (1977metabolism of drugs in isolated rat hewpatocytes:A compwith in vivo drug metabolism and drug metabolism in subcfractions. Drug Metab. Dispos. 5: 518-526.

4. Bradley MO, Taylor VI,Armstrong, MJ, and Galloway SM

Relationships among cytotoxicity, lysosomal breakdown, csome aberrations, and DNA double-strand breaks. Mutat. Re

69-79.

5. Cook JA and Mitchell JB (1989). Viability measurements i

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