Mechanisms and Types of Toxin-Induced Liver Injury
Hepatic response to insults by chemicals (Table 13–2) depends on the intensity of the insult, the population of cells affected, and whether the exposure is acute or chronic.
Table 13–2 Types of hepatobiliary injury. ||Download (.pdf)
Table 13–2 Types of hepatobiliary injury.
Type of Injury or Damage
Amiodarone, CCl4, ethanol, fialuridine, tamoxifen, valproic acid
Acetaminophen, allyl alcohol, Cu, dimethylformamide, ethanol
Diclofenac, ethanol, halothane, tienilic acid
Chlorpromazine, cyclosporin A, 1,1-dichloroethylene, estrogens, Mn, phalloidin
Bile duct damage
Alpha-naphthylisothiocyanate, amoxicillin, methylene dianiline, sporidesmin
Anabolic steroids, cyclophosphamide, microcystin, pyrrolizidine alkaloids
Fibrosis and cirrhosis
CCl4, ethanol, thioacetamide, vitamin A, vinyl chloride
Aflatoxin, androgens, arsenic, thorium dioxide, vinyl chloride
Liver cells can die by two different modes, necrosis or apoptosis. Necrosis is characterized by cell swelling, leakage, nuclear disintegration, and an influx of inflammatory cells. When necrosis occurs in hepatocytes, the associated plasma membrane leakage can be detected biochemically by assaying plasma (or serum) for liver cytosol-derived enzymes such as alanine aminotransferase (ALT) or γ-glutamyltranspeptidase (GGT). Apoptosis is characterized by cell shrinkage, nuclear fragmentation, formation of apoptotic bodies, and a lack of inflammation, and is a single cell event with the main purpose of removing cells no longer needed during development or eliminating aging cells.
Hepatocyte death can occur in a focal, zonal, or panacinar (widespread) pattern. Focal cell death is characterized by the randomly distributed death of single hepatocytes or small clusters of hepatocytes. Zonal necrosis is death to hepatocytes in certain functional regions. Panacinar necrosis is massive death of hepatocytes with only a few or no remaining survivors.
Mechanisms of toxin-induced injury to liver cells include lipid peroxidation, binding to cell macromolecules, mitochondrial damage, disruption of the cytoskeleton, and massive calcium influx.
Defined physiologically as a decrease in the volume of bile formed or an impaired secretion of specific solutes into bile, cholestasis is characterized biochemically by elevated serum levels of compounds normally concentrated in bile, particularly bile salts and bilirubin. When biliary excretion of the yellowish bilirubin pigment is impaired, this pigment accumulates in the skin and eyes, producing jaundice, and spills into urine, which becomes bright yellow or dark brown. Toxin-induced cholestasis can be transient or chronic; when substantial, it is associated with cell swelling, cell death, and inflammation. Many different types of chemicals cause cholestasis (Table 13–2).
The molecular mechanisms of cholestasis are related to expression and function of transporter systems in the basolateral and canalicular membranes. An increased hepatic uptake, decreased biliary excretion, and increased biliary reabsorption (cholehepatic shunting) of a drug may contribute to its accumulation in the liver.
Bile formation is vulnerable to toxicant effects on the functional integrity of sinusoidal transporters, canalicular exporters, cytoskeleton-dependent processes for transcytosis, and the contractile closure of the canalicular lumen (Figure 13–3). Changes that weaken the junctions that form the structural barrier between the blood and the canalicular lumen allow solutes to leak out of the canalicular lumen. These paracellular junctions provide a size and charge barrier to the diffusion of solutes between the blood and the canalicular lumen while water and small ions diffuse across these junctions. One hepatotoxin that causes tight-junction leakage is β-napthylisothiocyanate.
Schematic of six potential mechanisms for cholestasis involving inhibited uptake, diminished transcytosis, impaired secretion, diminished canalicular contractility, leakiness of the junctions that seal the canalicular lumen from the blood, and detrimental consequences of high concentrations of toxic entities in the pericanalicular area. Note that impaired secretion across the canalicular membrane can result from inhibition of a transporter or retraction of a transporter away from the canalicular membrane.
Compounds that produce cholestasis do not necessarily act by a single mechanism or at just one site. Chlorpromazine impairs bile acid uptake and canalicular contractility. Multiple alterations have been well documented for estrogens, a well-known cause of reversible canalicular cholestasis. Estrogens decrease bile salt uptake by effects at the sinusoidal membrane including a decrease in the Na+, K+-ATPase necessary for Na-dependent transport of bile salts across the plasma membrane and changes in lipid component of this membrane. At the canalicular membrane, estrogens diminish the transport of glutathione conjugates and reduce the number of bile salt transporters.
An additional mechanism for canalicular cholestasis is concentration of reactive forms of chemicals in the pericanalicular area (Figure 13–3). Most chemicals that cause canalicular cholestasis are excreted in bile. Therefore, the proteins and lipids in the canalicular region encounter a high concentration of these chemicals. Observations consistent with this concentration mechanism have been reported for Mn, reactive thioether glutathione conjugates of 1,1-dichloroethylene, and sporidesmin.
Damage to the intrahepatic bile ducts (which carry bile from the liver to the GI tract) is called cholangiodestructive cholestasis. A useful biochemical index of bile duct damage is a sharp elevation in serum alkaline phosphatase activity. In addition, serum levels of bile salts and bilirubin are elevated, as observed with canalicular cholestasis. Initial lesions following a single dose of cholangiodestructive agents include swollen biliary epithelium, debris of damaged cells within lumens of the biliary tract, and inflammatory cell infiltration of portal tracts. Chronic administration of toxins that cause bile duct destruction can lead to biliary proliferation and fibrosis resembling biliary cirrhosis. A rare response is the loss of bile ducts, a condition known as vanishing bile duct syndrome. This persisting problem has been reported in patients receiving antibiotics.
Functional integrity of the sinusoid (channels in between hepatocytes that carry blood throughout the liver) can be compromised by dilation or blockade of its lumen or by progressive destruction of its endothelial cell wall. Blockade will occur when red blood cells become caught in the sinusoids. Such changes have been illustrated after large doses of the drug acetaminophen. A consequence of extensive sinusoidal blockade is that the liver becomes engorged with blood cells while the rest of the body goes into shock.
Progressive destruction of the endothelial wall of the sinusoid will lead to gaps and then ruptures of its barrier integrity, with entrapment of red blood cells. These disruptions of the sinusoid are considered the early structural features of the vascular disorder known as veno-occlusive disease, which occurs after exposure to pyrrolizidine alkaloids, which may be found in some herbal teas and chemotherapeutic agents.
Disruption of the Cytoskeleton
Phalloidin and microcystin disrupt the integrity of hepatocyte cytoskeleton by affecting proteins that are vital to its dynamic nature, preventing disassembly of actin filaments. Phalloidin uptake into hepatocytes leads to an accentuated actin web of cytoskeleton and the canalicular lumen dilates.
Microcystin uptake into hepatocytes leads to hyperphosphorylation of cytoskeletal proteins. Reversible phosphorylations of cytoskeletal structural and motor proteins are critical to the dynamic integrity of the cytoskeleton. As depicted in Figure 13–4, extensive hyperphosphorylation produced by large amounts of microcystin leads to marked deformation of hepatocytes due to a unique collapse of the microtubular actin scaffold into a spiny central aggregate. Lower doses of microcystin interfere with vesicle transport by hyperphosphorylating the transport protein dynein.
Schematic of events in the mechanism by which microcystin damages the structural and functional integrity of hepatocytes. Microcystin is taken up exclusively into hepatocytes by a sinusoidal transporter in a manner inhibitable by bile salts and organic anions. Then microcystin inhibition of protein phosphatases leads to hyperphosphorylation of cytoskeletal proteins whose dynamic functions are dependent on reversible phosphorylations. Extensive hyperphosphorylation of microtubular proteins leads to a collapse of the microtubular actin filament scaffold into a spiky aggregate that produces a gross deformation of hepatocytes. More subtle changes in microtubule-mediated transport activities have been linked to hyperphosphorylation of dynein, a cytoskeletal motor protein.
This change, also known as steatosis, is a buildup of lipids in the hepatocyte. Fatty liver can stem from disruptions in lipid metabolism. Steatosis is a common response to acute exposure to many hepatotoxins. Often, toxin-induced steatosis is reversible and does not lead to death of hepatocytes. Ethanol is by far the most relevant drug or chemical leading to steatosis in humans. The metabolic inhibitors ethionine, puromycin, and cycloheximide cause fat accumulation without causing death of cells. Many other conditions besides toxin exposure, such as insulin resistance due to central obesity, are associated with marked fat accumulation in the liver.
Hepatic fibrosis (scarring) is characterized by the accumulation of extensive amounts of collagen fibers, in response to direct injury or to inflammation. With repeated chemical insults, destroyed hepatic cells are replaced by fibrotic scars. With continuing collagen deposition, the architecture of the liver is disrupted by interconnecting fibrous scars. When the fibrous scars subdivide the remaining liver mass into nodules of regenerating hepatocytes, fibrosis has progressed to cirrhosis and the liver has meager residual capacity to perform its essential functions. Cirrhosis is not reversible, has a poor prognosis for survival, and is usually the result of repeated exposure to chemical toxins.
Chemically induced neoplasia can involve tumors that are derived from hepatocytes, bile duct cells, or the rare, highly malignant angiosarcomas derived from sinusoidal lining cells. Hepatocellular cancer has been linked to abuse of androgens and a high prevalence of aflatoxin-contaminated diets.
Thorotrast (radioactive thorium dioxide used as a contrast medium for radiology) accumulates in Kupffer cells, the resident macrophage of the sinusoid, and emits radioactivity throughout its very extended half-life, thus increasing the risk for developing gallbladder cancer about 14-fold and over 100-fold for liver cancers. Multiple types of liver tumors are linked to thorium dioxide exposure.
Critical Factors in Toxicant-Induced Liver Injury
Why is the liver the target site for so many chemicals of diverse structure? Why do many hepatotoxicants preferentially damage one type of liver cell? Our understanding of these fundamental questions is incomplete. Influences of several factors are of obvious importance (Table 13–3). Location and specialized processes for uptake and biliary secretion produce higher exposure levels in the liver than in other tissues of the body and strikingly high levels within certain types of liver cells. Then the abundant capacity for bioactivation reactions influences the rate of exposure to proximate toxicants. Subsequent events in the pathogenesis appear to be critically influenced by responses of sinusoidal cells and the immune system.
Table 13–3 Factors in the site-specific injury of representative hepatotoxicants. ||Download (.pdf)
Table 13–3 Factors in the site-specific injury of representative hepatotoxicants.
Potential Explanation for Site Specificity
Zone 1 hepatocytes (versus zone 3)
Preferential uptake and high oxygen levels
Higher oxygen levels for oxygen-dependent bioactivation
Zone 3 hepatocytes (versus zone 1)
More P450 isozyme for bioactivation
More P450 isozyme for bioactivation and less GSH for detoxification
More hypoxic and greater imbalance in bioactivation/detoxification reactions
Bile duct cells
Methylene dianiline, sporidesmin
Exposure to the high concentration of reactive metabolites in bile
Sinusoidal endothelium (versus hepatocytes)
Greater vulnerability to toxic metabolites and less ability to maintain glutathione levels
Preferential uptake and then activation
Preferential site for storage and then engorgement
Activation and transformation to collagen-synthesizing cell
A number of experimental systems are useful for defining factors and mechanisms of liver injury. In vitro systems using the isolated perfused liver, isolated liver cells, and cell fractions allow observations at various levels of complexity without the confounding influences of other systems. Models using cocultures or agents that inactivate a given cell type can document the contributions and interactions between cell types. Whole-animal models are essential for assessment of the progression of injury and responses to chronic insult. Application of gene transfection or repression attenuates some of these interpretive problems. Knockout animals are extremely useful models for studying complex aspects of hepatotoxicity.
Lipophilic drugs and environmental pollutants readily diffuse into hepatocytes because the fenestrated epithelium of the sinusoid enables close contact between circulating molecules and hepatocytes. The membrane-rich liver concentrates lipophilic compounds. Other toxins are rapidly extracted from blood because they are substrates for sinusoidal transporters. Phalloidin (from a mushroom) and microcystin (from blue-green alga) are illustrative examples of hepatotoxins that target the liver as a consequence of extensive uptake into hepatocytes by sinusoidal transporters. Vitamin A hepatotoxicity initially affects the sinusoidal stellate cells, which actively extract and store this vitamin, and cadmium hepatotoxicity becomes manifest when cells exceed their capacity to complex cadmium with the metal-binding protein metallothionein.
Hepatocytes contribute to the homeostasis of iron by extracting this essential metal from the sinusoid by a receptor-mediated process and maintaining a reserve of iron within the storage protein ferritin. Acute iron toxicity is most commonly observed in young children who accidently ingest iron tablets. The cytotoxicity of free iron is attributed to its function as an electron donor for the formation of reactive oxygen species, which initiate destructive oxidative stress reactions. Accumulation of excess iron beyond the capacity for its safe storage in ferritin leads to liver damage. Chronic hepatic accumulation of excess iron in cases of hemochromatosis is associated with a spectrum of hepatic disease including liver cancer.
Bioactivation and Detoxification
Hepatocytes have very high constitutive activities of the phase I enzymes that often convert xenobiotics to reactive electrophilic metabolites. Also, hepatocytes have a rich collection of phase II enzymes that add a polar group to a molecule and thereby enhance its removal from the body. Phase II reactions usually yield stable, nonreactive metabolites. In general, the balance between phase I and phase II reactions determines whether a reactive metabolite will initiate liver cell injury or be safely detoxified.
One of the most widely used analgesics, acetaminophen (APAP) is a safe drug when used at therapeutically recommended doses. Overdose can cause severe hepatotoxicity, and certain acquired factors (e.g., diet, drugs, diabetes, and obesity) can enhance hepatotoxicity. Typical therapeutic doses of acetaminophen are not hepatotoxic, because most of the acetaminophen gets glucuronidated or sulfated with little drug bioactivation. Injury after large doses of acetaminophen is enhanced by fasting and other conditions that deplete glutathione and is minimized by treatments with N-acetylcysteine that enhance hepatocyte synthesis of glutathione.
Alcoholics are vulnerable to the hepatotoxic effects of acetaminophen at dosages within the high therapeutic range. This acquired enhancement has widely been attributed to accelerated bioactivation of acetaminophen to the electrophilic N-acetyl-p-benzoquinone imine (NAPQI) intermediate by ethanol induction of CYP2E1 (Figure 13–5). Inducers of CYP3A including many drugs and dietary chemicals potentially influence acetaminophen toxicity.
Schematic of key events in the bioactivation and hepatotoxicity of acetaminophen. Bioactivation of acetaminophen by cytochrome P450 isozymes leads to the formation of the reactive intermediate N-acetyl-p-benzoquinone (NAPQI), which can deplete glutathione or form covalent adducts with hepatic proteins. Experimental observations suggest that such effects “prime” hepatocytes for cytokines released by activated Kupffer cells. Progression to cell death is thought to involve activation of iNOS and other processes that produce reactive nitrogen species and oxidative stress. Agents that activate Kupffer cells exacerbate the toxicity. Exchange of signals between toxicant-primed and activated Kupffer cells is likely a factor in the acute hepatotoxicity produced by many compounds that damage hepatocytes.
An attractive “two hit” type of theory for the hepatotoxicity of acetaminophen suggests that adduction by a reactive drug metabolite “primes” the hepatocytes for destructive insults by reactive nitrogen species (e.g., peroxynitrite) (Figure 13–5).
Genetic conditions of high clinical relevance to the bioactivation/detoxification balance are the polymorphisms in the enzymes that control the two-step metabolism of ethanol. Specifically, ethanol is bioactivated by alcohol dehydrogenase to acetaldehyde, a reactive aldehyde, which is subsequently detoxified to acetate by aldehyde dehydrogenase. Both enzymes exhibit genetic polymorphisms that result in higher concentrations of acetaldehyde—a “fast” activity isozyme of alcohol dehydrogenase [ALD2*2] and a physiologically very “slow” mitochondrial isozyme of aldehyde dehydrogenase [ALDH2*2]. Approximately 50 percent of Asian populations but virtually no Caucasians have the slow aldehyde dehydrogenase; alcohol consumption by people with this slow polymorphism leads to uncomfortable symptoms of flushing and nausea due to high systemic levels of acetaldehyde.
Cytochrome P450-dependent bioactivation as a mechanism of hepatotoxicity is important even for assumedly safe compounds because some P450 isozymes generate reactive oxygen species during biotransformation reactions, which can lead to liver damage. CYP2E1 generation of reactive oxygen species and other free radicals contributes to the etiology of serious, end-stage liver damage.
Besides CYP2E1, the CYP3A isozyme has been linked to the hepatotoxicity caused by the folk medicine plant germander (Teucrium chamaedrys L.). Systematic experimental studies demonstrated a predominant role for the CYP3A bioactivation of germander constituents to reactive electrophiles.
Cytochrome P450-dependent conversion of CCl4 to •CCl3 and then to CCl3OO• is the classic example of xenobiotic bioactivation to a free radical that initiates oxidative damage. Conditions in which cytochrome P450 is depleted lead to decreased liver damage when exposed to CCl4.
The liver has a high capacity to restore lost tissue and function by regeneration. Loss of hepatocytes due to hepatectomy or cell injury triggers proliferation of all mature liver cells. This process is capable of restoring the original liver mass. However, regeneration is not just a response to cell death, but a process that actively determines the final injury after exposure to hepatotoxic chemicals. Stimulation of repair by exposure to a moderate dose of a hepatotoxicant strongly attenuates tissue damage of a subsequent high dose of the same chemical. Tissue repair follows a dose–response up to a threshold where the injury is too severe and cell proliferation is inhibited.
Inflammation and Immune Responses
Migration of neutrophils, lymphocytes, and other inflammatory cells into regions of damaged liver is a well-recognized feature of the hepatotoxicity produced by many chemicals. In fact, the potentially confusing term hepatitis refers to hepatocyte damage by any insult where hepatocyte death is associated with an influx of inflammatory cells.
The influx of inflammatory cells usually facilitates beneficial removal of debris from damaged liver cells. However, detrimental effects are plausible, because activated neutrophils release cytotoxic proteases and reactive oxygen species.
Immune responses are considered factors in the hepatotoxicity occasionally observed after repeated exposure to chemicals, usually drugs. Individuals who develop infrequent, unpredictable responses are considered to be hypersensitive. An immune-mediated response is considered plausible when the problem subsides after therapy is halted and then recurs on drug challenge or restoration of therapy. Although the concept is generally accepted, compelling evidence for immune-mediated responses is available only for ethanol, halothane, and a few other hepatotoxicants. Figure 13–6 depicts key features of the assumed scenario whereby hepatic protein adducts could become antigenic and stimulate the production of antibodies. If on reexposure, more drug–protein adducts are formed, cells with such adducts could be attacked by systemic antibodies.
Proposed scenario of events leading to immune-mediated hepatotoxicity after repeated exposure to a toxicant that produces drug–protein adducts (*).
Apparent immune-mediated injury has been observed in individuals taking the antiarthritic NSAID diclofenac. Hepatic bioactivation of diclofenac leads to the formation of multiple adducts, which may localize to hepatocyte membrane proteins where recognition by antibodies is feasible.
Activation of Sinusoidal Cells
Four kinds of observations, collectively, indicate roles for sinusoidal cell (immune cells present in the liver sinusoids) activation as primary or secondary factors in toxin-induced injury to the liver:
Kupffer and Ito cells exhibit an activated morphology after acute and chronic exposure to hepatotoxicants.
Pretreatments that activate or inactivate Kupffer cells appropriately modulate the extent of damage produced by classic toxicants. Kupffer cell activation by vitamin A profoundly enhances the acute toxicity of carbon tetrachloride; this enhancement did not occur when animals were also given an inactivator of Kupffer cells.
Activated Kupffer cells secrete appreciable amounts of soluble cytotoxins, including reactive oxygen and nitrogen species.
Acute and chronic exposure to alcohol directly or indirectly affects sinusoidal cells.
Figure 13–7 summarizes information presented in this and earlier sections of this chapter about the multiplicity of toxin-induced interactions with and between various liver cells. The effect on a given cell type can be direct or may result from a cascade of signals and responses between cell types.
Schematic depicting the complex cascade of toxin-evoked interactions between hepatocytes and sinusoidal cells. Sinusoidal cell responses to toxins can lead to either injury or activation. A scenario could involve (1) toxin injury to hepatocytes, (2) signals from the injured hepatocyte to Kupffer and Ito cells, followed by (3) Kupffer cell release of cytotoxins, and (4) Ito cell secretion of collagen. Activation of Kupffer cells is an important factor in the progression of injury evoked by many toxicants. Stimulation of collagen production by activated Ito cells is a proposed mechanism for toxicant-induced fibrosis.
Mitochondrial DNA codes for several proteins in the mitochondrial electron transport chain. Nucleoside analog drugs for the therapy of hepatitis B and AIDS infections cause mitochondrial DNA damage directly, when incorporation of the analog base leads to miscoding or early termination of polypeptides. The severe hepatic mitochondrial injury produced by the nucleoside analog fialuridine is attributed to its higher affinity for the polymerase responsible for mitochondrial DNA synthesis than for the polymerases responsible for nuclear DNA synthesis. Mitochondrial DNA is also more vulnerable to miscoding (mutation) due to its limited capacity for repair.
Alcohol abuse causes mitochondrial injury by shifting the bioactivation/detoxification balance for ethanol, leading to an accumulation of its reactive acetaldehyde metabolite within mitochondria, because mitochondrial aldehyde dehydrogenase is the major enzymatic process for detoxification of acetaldehyde. Bioactivation of large amounts of ethanol by alcohol dehydrogenase hampers the detoxification reaction, since the two enzymes require the common, depletable cofactor nicotinamide adenine dinucleotide (NAD). Any type of ethanol-induced change that enhances the leakiness of the mitochondrial transport chain would lead to an increased release of reactive oxygen species capable of attacking nearby mitochondrial constituents.
Idiosyncratic Liver Injury
Idiosyncratic drug hepatotoxicity is a rare but potentially serious adverse event, which is not clearly dose-dependent, is at this point unpredictable, and affects only very few of the patients exposed to a drug or other chemicals. However, idiosyncratic toxicity is a leading cause for failure of drugs in clinical testing and it is the most frequent reason for posting warnings, restricting use, or even withdrawal of the drug from the market (Table 13–4). In addition, idiosyncratic hepatotoxicity is observed after consumption of herbal remedies and food supplements. Because idiosyncratic hepatotoxicity is a rare event for most drugs, it is likely that a combination of gene defects and adverse events need to be present simultaneously in an individual to trigger the severe liver injury. A detailed genomic analysis of patients with idiosyncratic responses to drug exposure may give additional insight what gene expression profile renders a patient susceptible.
Table 13–4 Examples of drugs with known idiosyncratic hepatotoxicity. ||Download (.pdf)
Table 13–4 Examples of drugs with known idiosyncratic hepatotoxicity.
A. Immune-mediated (allergic) idiosyncratic hepatotoxicity
B. Nonimmune-mediated (non-allergic) idiosyncratic hepatotoxicity