Hepatic Injury due to Drugs, Dietary and Herbal Supplements, Chemicals and Toxins

Immunoallergic drug injury

Immunoallergic drug injury (also called drug allergy or hypersensitivity drug injury) develops after a relatively short sensitization period of 1–8 weeks, recurs promptly on readministration of the agent (sometimes within a day or two) and tends to be accompanied by other clinical findings of hypersensitivity. These may include fever, rash, eosinophilia, lymphadenopathy and atypical lymphocytosis. The rash may be severe and toxic epidermal necrolysis or Stevens–Johnson syndrome can develop. Jaundice develops later in the injury. The liver biopsy tends to show a mixed injury pattern with both cholestasis and hepatitis (see later section on cholestatic hepatitis). There may be an eosinophil-rich or granulomatous inflammatory infiltrate. A subset of cases of immuno­allergic liver injury may result in the DRESS (drug reaction with eosinophilia and systemic symptoms) syndrome. DRESS has been reported with a variety of drugs, including phenytoin, carbamazepine, allopurinol and nevirapine sulphonamides, dapsone and a number of other drugs. It should be noted that these systemic symptoms, particularly fever and rash, are relatively nonspecific, so additional evidence of hypersensitivity should be present before classifying a drug reaction as immunoallergic.

Convincing evidence for the role of immunological factors in some forms of drug-induced injury can be found in the case of halothane-induced DILI, where injury was found to be accompanied by the development of antibodies and sensitized T cells that react with rabbit hepatocytes modified by an oxidative metabolite of halothane. Mouse models of halothane hepatitis have demonstrated both the involvement of eosinophils as well as the development of an immune response directed against trifluoroacetylated protein adducts. Injury could be increased by specific depletion of myeloid-derived suppressor cells. Ticrynafen (tienilic acid) has been found to have antibodies that react with ticrynafen-altered hepatocyte proteins. One such ticrynafen antibody reacts with microsomes. This anti-liver–kidney microsomal antibody type 2 (anti-LKM2) appears to be an antibody to the cytochrome P450 isoform involved in the metabolism of ticrynafen. These studies provide suggestive evidence that the pathogenesis of the injury is the consequence of conversion of hepatocyte proteins to neoantigens by reaction with a metabolite of the drug and that the ensuing immunological reaction leads to hepatic injury. Cell-mediated immunity has also been implicated in immunoallergic injury and this has led some investigators to try to use the stimulation of peripheral blood mononuclear cells by a drug as causal evidence of DILI. However, when this idea was tested in a series of patients with DILI, less than 10% of cases showed a positive stimulation of lymphocytes by the injurious agent.

Many drugs have been shown to have injury associated with specific human leukocyte antigen (HLA) subtypes ( Table 12.4 ). The combination drug amoxicillin-clavulanate has been associated with increased prevalence of specific HLA-DR subtypes, suggesting that the injury may be mediated by HLA class II antigen presentation. Flucloxacillin injury showed a strong association with HLA-B*5701 in a genome-wide association study. Individuals carrying this allele are 80 to 100 times more likely to experience flucloxacillin-related liver injury. It should be noted that the idea that drug hypersensitivity alone will lead to hepatic injury is an oversimplification. Generalized hypersensitivity per se does not always lead to liver injury. Penicillin is a frequent cause of drug hypersensitivity, yet almost never causes liver injury. On the other hand, anticonvulsant-related hypersensitivity almost always includes a component of liver injury. The mere presence of circulating antidrug antibodies is insufficient to induce liver disease. For example, despite the presence of serum antibodies to halothane and other anaesthetics in 158 paediatric anaesthesiologists, only a single individual developed liver injury.

Hypersensitivity-mediated reactions may also result from a metabolic defect. ^, In one study, patients who had sustained hepatic injury in a hypersensitivity-type reaction to phenytoin had an apparent defect in converting the active metabolite (arene oxide) to the inactive dihydriol. The active metabolite presumably could serve as a hapten or could be cytotoxic. Other potential genetic predispositions to developing DILI are given in Table 12.4 . ^, With the burgeoning field of pharmacogenomics, it is expected that the ability to identify susceptible individuals will permit a greater understanding of the mechanisms involved as well as a means to avoid serious toxicity in such patients. ^,

Toxic metabolite-dependent liver injury

Idiosyncratic hepatic injury that is not accompanied by clinical hallmarks of hypersensitivity, is not promptly reproduced by readministration of the drug and which may appear after widely varying periods of exposure to the drug (between 2 weeks and several months) is thought to result from metabolic rather than immunological mechanisms. Identification of reactive metabolites that are thought to be responsible for acute DILI has been made for some agents, such as perhexiline maleate, valproic acid, INH and iproniazid. For the majority of other agents, however, the exact cause remains undefined. Interindividual differences in susceptibility may account for this type of DILI.

There appears to be some degree of dose dependency in idiosyncratic liver injury ^, despite its characterization as a dose-independent form of liver injury. A study of a U.S. pharmaceutical database and the large Spanish DILI registry found that drugs given in a daily dose of 50 mg or greater were far more likely to cause DILI compared to drugs given in daily doses of 10 mg or less. In the Spanish registry, 77% of agents were given in daily doses of more than 50 mg, and such agents were more likely to result in acute liver failure, liver transplantation and death. The degree to which a drug undergoes hepatic metabolism also relates to its risk of causing DILI. Compounds with greater than 50% hepatic metabolism were associated with higher rates of elevated alanine aminotransferase (ALT) (more than three times the upper limit of normal [ULN]), liver failure and death when compared to drugs undergoing lesser degrees of hepatic metabolism.

Cytotoxic injury: necrosis and cell death (apoptosis)

Individual cells involved in necroinflammatory injury can undergo a variety of changes. Cell death can occur by two processes: necrosis and apoptosis. ^, Necrosis refers to destructive disintegration of the cell, apparently initiated by plasma membrane injury and yielding only debris. Apoptosis (programmed cell death) is an energy-dependent event leading to cell shrinkage, nuclear fragmentation, condensation of chromatin and the production of cytoplasmic blebs yielding distinct fragments of the cells or entire condensed shrunken cells. The condensed cells are referred to as apoptotic or free acidophilic bodies. The mechanisms for the two are different. Activation of apoptotic pathways involves tumour necrosis factor alpha receptor or Fas-mediated release of intercellular caspases that lead to cell death. Necrosis results when there is massive injury to the cells. Frequently this is due to massive injury to the cell membrane and organelles. Apoptosis may occur when the cell suffers similar direct injury, but in lesser degrees. Thus, apoptotic hepatocytes are sometimes seen adjacent to zones of confluent necrosis. Apoptosis may also result from immune-mediated injury, in which cytotoxic T cells, antibodies or cytokines initiate the apoptotic pathway. Ballooning hepatocellular changes, steatosis and Mallory body formation may also be seen as components of necroinflammatory injury, depending on the agent. The presence of cholestasis (either canalicular or hepatocellular) should lead the pathologist to consider agents that cause mixed injury (discussed later).

Acute hepatitis-like injury

Another common pattern of acute hepatoxicity is that of multiple small areas of degeneration or ‘spotty’ necrosis, similar to acute viral hepatitis ( Table 12.7 ). Acute hepatitis without concomitant cholestasis accounted for 20% of a large series of idiosyncratic DILI cases. It is characterized by a predominantly lobular, lymphocytic infiltrate with scattered apoptotic hepatocytes. Increased numbers of eosinophils can be seen, which may help suggest the diagnosis of DILI. There may be other evidence of hepatocellular injury, such as cytoplasmic clearing, cellular swelling or steatosis, and regenerative changes, such as mitoses, hepatocyte rosette formation and the proliferation of ductular hepatocytes. In severe cases there is often lobular disarray ( Fig. 12.5 ), in which the normal sinusoidal architecture is lost amid a sea of inflammation, hepatocyte rosettes and apoptosis. This may lead to areas of confluent necrosis from extensive apoptotic cell death ( Fig 12.6 ). The massive necrosis that results from drugs in this category of injury may be most prominent in zone 3, mimicking the zonal necrosis pattern ( Fig. 12.4 ). In the DILI Network (DILIN) series, 18% of the acute hepatitis cases showed zone 3 necrosis. Portal inflammation and interface hepatitis are often present, as is bile duct injury, but they do not dominate the overall inflammatory pattern. Reticulin stains will show the zones of collapse and periportal fibrosis may be present. More extensive fibrosis may develop as a result of extensive necrosis and subsequent scarring if the patient survives. In case reports of DILI, necrosis has been one histologic feature clearly associated with a fatal outcome, although the degree of necrosis is undoubtedly important. ^,

Table 12.7

Agents that cause acute and chronic hepatitis-like patterns

Acute (lobular-predominant) hepatitis		Chronic (portal-predominant) hepatitis
Drugs		Drugs
Acarbose Acetylsalicylic acid Acitretin Adalimumab Doxorubicin Amiodarone Amitriptyline Amlodipine Amodiaquine Amoxapine Anakinra Aprindine Asparaginase Atomoxetine Benzarone Bromfenac Bupropion Captopril Carbamazepine Carbenicillin Cetirizine Chlorotetracycline Chlorpromazine Chlorzoxazone Cimetidine Cinchophen Ciprofloxacin Cisplatin Citalopram Corticosteroids Clarithromycin Clindamycin Clometacin Cromolyn Cyclofenil Dantrolene Dapsone Darunavir Desflurane Dichloromethotrexate Diclofenac Diflunisal Dihydralazine Disulfiram	Lapatinib Leflunomide Lergotrile Levofloxacin Losartan Mepacrine Mercaptopurine Metformin Methimazole Methotrexate Methoxyflurane Methyldopa Minocycline Mirtazapine Mitomycin C Naproxen Nevirapine Nicotinic acid Nimesulide Nitrofurantoin Norfloxacin Olanzapine Oxacillin Oxaprozin Oxcarbazepine Oxyphenisatin Papaverine Paroxetine Pemoline Perhexiline maleate Phenazopyridine Phenindione Phenobarbital Phenprocoumon Phenylbutazone Phenytoin Pirprofen Pomalidomide Pregabalin Propylthiouracil Pyrazinamide Quetiapine Ranitidine	Acetaminophen (paracetamol) * Acetylsalicylic acid Adalimumab Doxorubicin Amlodipine Atomoxetine Atorvastatin Azapropazone Benzarone Chlorpromazine Clometacin Cyclofenil Dantrolene Doxidan Doxorubicin Erlotinib Erythromycin Etanercept Etretinate Everolimus Ezetimibe Fenofibrate Gliclazide Haloperidol Halothane Infliximab Ipilimumab Irbesartan Isoniazid Labetalol Levofloxacin Lisinopril Losartan Methyldopa Naproxen Nevirapine Nicotinic acid Nimesulide Nitrofurantoin Oxacillin Oxyphenisatin Pemoline Pentamidine
Doxepin Duloxetine Ecstasy Eletriptan Enflurane Erlotinib Etanercept Etodolac Etoposide (VP-16) Etretinate Fenofibrate Fluroxene Gatifloxacin Gliclazide Glibenclamide Gold Haloperidol Halothane Hydralazine Hydrazines Hydrochlorothiazide Hydroxychloroquine Ibuprofen Imatinib mesylate Infliximab Interleukin-2 Ipilimumab Isoniazid Ivermectin Ketoconazole Lamotrigine	Rifampin Riluzole Rosuvastatin Sertraline Sevoflurane Sibutramine Simvastatin Sorafenib Spironolactone Streptozotocin Sulfadiazine Sulfadoxine-pyrimethamine Sulfamethizole Sulfamethoxazole Sulfasalazine Sulindac Suloctidil Telithromycin Teniposide Thalidomide Thiotepa Ticrynafen Tolazamide Tolvaptan Trazodone Trastuzumab Troglitazone Trovafloxacin Vancomycin Verapamil Ximelagatran	Perhexiline maleate Phenprocoumon Phenytoin Propylthiouracil Pyrazinamide Rosuvastatin Simvastatin Sulfamethizole Thalidomide Ticrynafen Tolazamide Trazodone Ximelagatran
Herbals/Natural Products		Herbals/Natural Products
Black cohosh Chaparral Chaso/onshido Germander (Teucrium) Greater celandine Green tea extract Jin bu huan Skullcap Syo-saiko-to		Baracol Germander (Teucrium) Turmeric

*Evidence for chronic injury remains scant and controversial.

There is an alternate acute hepatitis pattern that most resembles the hepatitis of Epstein–Barr virus (EBV) infection. In this pattern there is sinusoidal beading of lymphocytes like the mononucleosis hepatitis of EBV; granulomas and eosinophils can be seen. The archetypical drug that causes this pattern of injury is phenytoin ( Fig. 12.7 ), although it can also cause cholestatic and mixed injury patterns. ^, Other drugs reported to show this pattern include para-amino salicylate, dapsone and the sulphonamides.

Subacute hepatic necrosis is an insidious form of necroinflammatory disease that was a dreaded occupational disease in the first half of the twentieth century. It results from prolonged exposure to benzene derivatives. ^, Subacute hepatic necrosis has also been associated with long-term administration of INH or methyldopa ^, and was seen in recipients of cinchophen. The histological features include varying degrees of necrosis, fibrosis and regeneration ( Fig. 12.6 ).

Chronic hepatitis-like injury

Many of the drugs that are known to cause an acute hepatitis-like pattern are also responsible for a pattern of injury that resembles chronic viral hepatitis or AIH ( Table 12.7 ). About 14% of cases from the U.S. DILIN series showed a chronic hepatitic pattern of injury. Although the pattern resembles chronic hepatitis, this does not imply that the injury is long-term or that it will persist once the agent is withdrawn. Like chronic viral hepatitis, this pattern is characterized by a necroinflammatory infiltrate that is mainly portal and periportal in distribution. The portal inflammation is mixed, but composed predominantly of CD8-(+) lymphocytes and macrophages. The lymphocytes are cytotoxic, as demonstrated by colocalization with the cytotoxic protein TIA-1. Plasma cells and eosinophils may be present. There is interface hepatitis associated with variable periportal fibrosis ( Fig. 12.8 ). Lobular inflammation is present in the form of spotty necrosis along with scattered apoptotic hepatocytes, but the injury is typically not severe enough to cause lobular disarray. Some agents may cause injury leading to significant fibrosis or cirrhosis ( Box 12.1 ) ( Fig. 12.9 ).

Box 12.1

Agents associated with fibrosis and cirrhosis

Drugs

• Acetohexamide

• Amiodarone

• Chlorpromazine

• Cinchophen

• Cyanamide

• Dantrolene

• Diclofenac

• Ebrotidine

• Etretinate

• Fenofibrate

• Ferrous fumarate

• Halothane

• Isoniazid

• Lisinopril

• Mercaptopurine

• Methotrexate

• Methyldopa

• Methyltestosterone

• Nimesulide

• Nitrofurantoin

• Oxyphenisatin

• Papaverine

• Perhexiline maleate

• Phenylbutazone

• Propylthiouracil

• Tamoxifen

• Thiabendazole

• Ticrynafen

• TPN

• Valproic acid

Herbals/natural products

• Bush tea

• Comfrey

• Germander (Teucrium)

In the chronic hepatitis-like pattern, hepatocytes may show degenerative changes or steatosis as in acute hepatitis. Since cholestasis is not a feature of chronic viral hepatitis until late in the disease, its presence should raise the possibility of drug injury (or a second process). Mixed cholestatic-hepatitic injury is discussed further later. Duct injury can be seen in the chronic hepatitis pattern, even in the absence of bile stasis, and may mimic primary biliary cirrhosis. Pseudoxanthomatous changes (cholatestasis) or periportal copper accumulation should cause one to consider a chronic cholestatic injury.

The frequency with which acute DILI leads to chronic liver disease is thought to be low but clearly depends upon the definition of ‘chronicity’. Acute or subacute injury that becomes chronic from continued use of certain medications may lead to fibrosis and even cirrhosis ( Box 12.1 ). Aithal and Day published a series of cases that confirmed the chronicity of what were mostly cholestatic hepatotoxins. Chronic injury that persists after the alleged agent is discontinued appears to be far less common. In the large registry from Spain ( Table 12.1 ), chronicity was seen in 10–12% of patients, depending on the biochemical presentation. Similar figures were described among patients in the DILIN registry, with 17% of cases showing persistence of abnormal liver-associated enzymes (LAEs) after 6 months of enrollment. In a more recent study with a longer follow-up, the proportion of patients with persistent enzyme abnormalities fell from 21% at 6 months to 12% at 12 months. Biopsies available for review from cases with chronic injury mainly showed chronic cholestasis and duct loss rather than chronic hepatitis. Most cases also showed some degree of fibrosis progression. It is well described that recovery from acute DILI generally takes longer if the injury is cholestatic (16 weeks or longer) compared to acute hepatocellular necrosis (4–12 weeks). ^, In most cases of acute DILI, it is thought that clinical, biochemical and histological recovery is complete where the offending agent is discontinued. Chronic viral hepatitis, steatohepatitis and other non-DILI chronic liver diseases should always be excluded when evaluating patients with persistent liver enzyme abnormalities following DILI.

Drug-induced chronic hepatitis may be associated with a syndrome similar to AIH. The clinical syndrome mimics idiopathic AIH very closely. The onset of symptoms is insidious, often developing more than 2 months after the first dose of drug. The serum transaminases are elevated and there are elevated titres of serum antinuclear or anti-smooth muscle actin antibodies along with hyperglobulinaemia. Other manifestations of autoimmunity, including skin rashes and joint pains, may be observed. Recognition that drug-induced injury can mimic AIH came some 55 years ago with reports of the lesion in patients taking oxyphenisatin , an ingredient of laxative preparations no longer in use in the United States. Thereafter, the syndrome was reported in those treated with nitrofurantoin, methyldopa, ^, minocycline, hydralazine, halothane and statins, among others ( Table 12.8 ). Not all recipients of the drugs implicated in causing an autoimmune type of hepatitis show the expected autoantibodies. Almost 20% of patients with nitrofurantoin-associated chronic hepatitis and at least 40% of patients with methyldopa-induced chronic hepatitis are seronegative. Some agents, including hydralazine and tienilic acid, are associated with antibodies directed against microsomal components. These are CYP2C9 (also called LKM2) in tienilic acid-induced disease and the isoform CYP1A2 (a liver microsomal protein (LM)) in hydralazine-induced hepatitis. The isoforms are involved in the metabolism of the respective drug and it may be presumed that, in the process of biotransformation of the drug, an effect of the drug metabolite on the enzyme protein renders it a neoantigen. Autoimmune markers may be associated with drug reactions other than chronic hepatitis. Antinuclear antibodies may accompany chronic cholestatic injury induced by flucloxacillin and the reaction to aniline-contaminated rapeseed oil in the ‘toxic oil syndrome’ reported from Spain. They also may mark drug reactions without hepatic disease, as in the drug-induced systemic lupus erythematosus syndrome induced by hydralazine.

Table 12.8

Drug-induced autoimmune hepatitis

Drug	Serology
Agents associated with ANA and ASMA
Multiple cases
Adalimumab	ANA, ASMA
Clometacine	ASMA, anti-DNA
Etanercept	ANA
Hydralazine	ANA, ASMA
Infliximab	ANA, ASMA
Methyldopa	ANA (16%), ASMA 35%
Minocycline	ANA, anti-DNA
Nitrofurantoin	ANA (80%), ASMA (72%)
Oxyphenisatin	ANA (67%), ASMA (67%)
Statins	ANA (80–90%), ASMA (25%)
Few cases (<5)
Benzarone	ASMA
Diclofenac	ANA
Ecstasy (3,4-methylenedioxymethamphetamine)	ANA
Fenofibrate	ANA
Germander	ANA, ASMA
Papaverine	ANA, ASMA
Pemoline	ANA, antimicrosomal
Propylthiouracil	ANA
Agents associated with other autoantibodies
Dihydralazine	Anti-CYP1A2
Ticrynafen	Anti-CYP2C9 (LKM2)
Halothane	Anticarboxylesterase, antiprotein disulphide isomerase
Iproniazid	Antimicrosomal Ab 6

ANA , Antinuclear antibodies; ASMA , anti-smooth muscle antibodies.

Histologically, there may be little to distinguish idiopathic AIH from drug-induced AIH. In both cases there may be a plasma-cell rich portal inflammatory infiltrate with aggressive interface hepatitis and bridging necrosis. Bridging fibrosis may be present but cirrhosis is unusual at presentation. Drug-induced AIH is more often associated with hepatocellular or canalicular cholestasis, while hepatocyte rosette formation and emperipolesis of inflammatory cells are more common in idiopathic AIH, but these findings are not absolutely specific. In follow-up, patients with drug-induced AIH are more often able to discontinue steroid therapy than patients with idiopathic AIH. Differences in nonclassical autoantibodies may exist between these two diseases that may ultimate help distinguish them.

Granulomatous injury

Granulomatous inflammation is observed in a large number of drug-associated injuries ( Table 12.9 ). Non-necrotizing epithelioid granulomas were seen in about 5% of cases from the DILIN series. Granulomas may be seen alone or associated with other types of hepatic injury ( Fig. 12.10 ), including acute and chronic hepatitis, cholestasis and steatosis. More than 60 drugs have been implicated, although most are isolated case reports. A few agents appear to be well documented as typically causing granulomatous hepatitis, usually as part of a hypersensitivity syndrome (e.g. phenylbutazone, sulphonamides, allopurinol, phenothiazines and penicillins). Drug-induced granulomas have been reported with a frequency of up to 29% of all causes of hepatic granulomas, although the prevalence has usually been far lower. In the McMaster case series, the drugs that were listed as probably or possibly related were predominantly from antihypertensive, antirheumatic, anticonvulsant and antimicrobial drug classes and included methyldopa, hydralazine, phenytoin, INH, cephalexin, penicillin, sulphonamides and procainamide, among others. In other series, 6–9.5% of granulomas were due to many of the same drug classes and included chlorpropamide, allopurinol, PBZ, synthetic penicillins, quinidine, carbamazepine and phenothiazines, among others.

Microgranulomas are small foci of macrophages one to three hepatocytes in size. They are a common finding in DILI (over half the cases in the DILIN series) and other forms of liver disease and should not, by themselves, lead to a diagnosis of granulomatous hepatitis. Drug-induced hepatic granulomas have no specific features that distinguish them from those of sarcoidosis or other causes of noncaseating epithelioid granulomas, although the presence of eosinophils should suggest the possibility of a drug injury. Fibrin ring granulomas have been attributed to allopurinol and most recently to combination therapy with ipilimumab and nivolumab. ^, Granulomas due to Bacillus Calmette–Guerin, whether resulting from vaccination or from treatment of bladder carcinoma, may contain acid-fast bacilli. Systemic granulomatous disease does not preclude the possibility of drug-induced granulomas, since sarcoid-like granulomas have been reported in the liver, lungs and other organs as a result of chronic exposure to metals such as beryllium, copper and aluminium as well as interferon alfa.

Drug-induced granulomatous hepatitis may be related to immuno­allergic DILI, particularly when associated with tissue eosinophilia. The prominence of eosinophils in the sinusoids, however, is usually a reflection of peripheral eosinophilia. ^, Patients with immunoallergic DILI tend to present with an acute febrile illness, rash and peripheral eosinophilia, along with jaundice and elevated LAEs. A latency of 1–16 weeks is described by McMaster et al., which is consistent with the timeframe for other immunoallergic reactions from drugs (usually within 8 weeks). Histologic features suggesting a drug-induced cause include the relatively uniform age of the granulomas and the presence of eosinophils, apoptotic bodies, acute cholangitis and/or vasculitis. In contrast, tissue eosinophilia is not seen in sarcoidosis or TB. The prognosis is generally good with recovery after the offending agent is withdrawn. Healing without sequelae is the rule. ^,

Acute intrahepatic cholestasis

Some agents lead to injury that spares the parenchyma and instead causes arrested bile flow ^, ( Table 12.10 ). Intrahepatic cholestasis accompanied by significant portal or lobular inflammation should be classified as a mixed hepatocellular and cholestatic injury (cholestatic hepatitis), while the cholestasis occurring with little or no portal or lobular inflammation has been termed ‘acute’ or ‘bland’ intrahepatic cholestasis. Acute intrahepatic cholestasis accounted for 9% of cases in the DILIN series. This mild form of cholestatic injury consists mainly of bile accumulation in the cytoplasm of liver cells (hepatocellular cholestasis) and in canaliculi (canalicular cholestasis). Hepatocellular cholestasis is frequently marked by cell swelling, and the bile itself may be difficult to identify without the use of special stains. Iron and copper stains may both be more useful in this regard than a haematoxylin and eosin-stained section because of their light counterstains that allow the bile pigment to be more easily seen. An iron stain is doubly useful in that it can be used to distinguish iron (blue) from lipofuscin (granular and dirty brown) from bile (pale green to greenish brown). The cholestasis is typically most prominent in zone 3, and other processes that result in zone 3 cholestasis need to be excluded. Macrophages, sometimes bile stained, accumulate in the sinusoids of zone 3. A mild degree of parenchymal injury and inflammation (particularly portal inflammation) may be observed. This occurs with, for example, the cholestatic injury associated with chlorpromazine and erythromycin estolate ( Fig. 12.11 ) but rarely in the cholestasis associated with steroids ( Fig. 12.12 ). Different drugs may also lead to different distributions of bile accumulation. Chlorpromazine is associated with both hepatocellular and canalicular cholestasis while steroids are typically associated with pure canalicular cholestasis. Cholangiolar cholestasis like that seen in sepsis may be caused by benoxaprofen. Sometimes, the acute intrahepatic cholestasis is accompanied by acute cholangitis, which in turn may lead to chronic duct injury and loss. Temporally acute forms of vanishing bile duct syndrome (VBDS) may show acute intrahepatic cholestasis, minimal inflammation and ductopenia.

Acute hepatocellular and cholestatic injury (cholestatic hepatitis)

When hepatocellular or canalicular cholestasis is associated with a hepatitic necroinflammatory injury, the result is a mixed form of hepatic injury that may be termed ‘cholestatic hepatitis’. Cholestatic hepatitis was the most common pattern of histological injury in the DILIN series, accounting for almost 30% of cases. Many agents are associated with this pattern of injury and it should strongly suggest a drug aetiology when present ( Table 12.10 ). ^, By clinical presentation and histological evaluation, cases of cholestatic hepatitis fall into two general patterns. On one end are cases with an acute hepatitis-like pattern of inflammation associated with generally mild cholestasis. Patients have high levels of serum transaminases and jaundice. Zonal necrosis may be present. Agents that appear both under acute hepatitis ( Table 12.7 ) and cholestatic hepatitis ( Table 12.10 ), such as methyldopa, have this type of presentation. The histological differential diagnosis is usually with acute viral hepatitis and sometimes with acute AIH. In the other subtype of cholestatic hepatitis, the inflammation is mild to moderate, sometimes with the low-power appearance of chronic hepatitis, with prominent portal inflammation. The cholestasis is usually apparent from low to medium magnification. This pattern blends into the acute intrahepatic cholestatic pattern as the inflammation becomes less severe. Patients present with modest elevations of transaminases and ALP as well as jaundice. Drugs that have cholestatic hepatitis as their main pattern of injury, such as amoxicillin-clavulanate, tend to have this subtype of cholestatic hepatitis. Bile duct injury may be prominent in either form and some cases that present with cholestatic hepatitis will evolve into ductopenic chronic cholestasis. ^,

Chronic cholestatic injury

Drug-induced chronic cholestatic injury may take a variety of forms, but the central feature is chronic injury to the intrahepatic ducts. While this duct injury often results in ductopenia, chronic cholestatic changes can be observed in biopsies with apparently preserved ducts. Over 5% of the cases of acute DILI in the DILIN biopsy study had evidence of ductopenia at initial biopsy and about 10% of cases showed evidence of chronic cholestatic injury. ^, The injury may mimic primary biliary cholangitis (PBC) or primary sclerosing cholangitis (PSC) or the ducts may be destroyed by direct injury as exemplified by the changes ( Fig. 12.13 ) of paraquat poisoning. ^, Chronic cholestasis with duct damage may occur with a number of therapeutic drugs, e.g. chlorpromazine, chlorpropamide, sulphamethoxazole-trimethoprim, amoxicillin-clavulanate and others ( Table 12.11 ), and PSC-like changes may be seen on imaging. Indeed, ductal injury may accompany cholestatic hepatitis and can lead to drug-induced VBDS. ^{, ,} A syndrome that resembles primary biliary cirrhosis has followed acute cholestasis due to chlorpromazine, prochlorperazine, amitryptiline, imipramine, organic arsenicals, tolbutamide and ajmaline, among others. ^, The degree of duct destruction and portal inflammation tends to be less prominent in the drug-induced syndrome than in PBC ( Fig. 12.14 ). Chronic cholestatic injury may lead to fibrosis and cirrhosis, and even if the drug is withdrawn, recovery may be very slow. Chronic cholestasis as demonstrated by persistently elevated ALP was the most common clinical phenotype for chronic DILI, and of those who had biopsies, over half had evidence of ductopenia. Histological features may include other changes seen in non-DILI chronic cholestasis, including periportal cholate-stasis (pseudoxanthomatous change) of hepatocytes, copper accumulation and ductular reaction. Acute cholestatic and mixed hepatocellular/cholestatic injuries may progress to chronic cholestasis as the injury evolves.

Biliary sclerosis

‘Biliary sclerosis’ is the term that has been applied to the biliary tree injury produced by hepatic arterial infusion therapy with floxuridine (FUDR) for metastatic carcinoma ^, and from the use of scolicides for treatment of echinococcal cysts. The incidence with FUDR appears to be high, and the lesion consists of blebs and oedema in the duct epithelial surface and compression and distortion of the duct lumen ( Fig. 12.15 ). Ludwig et al. have attributed the ductal injury to occlusive arterial injury and have labelled it an ‘ischaemic cholangiopathy’. On cholangiography, the lesion resembles PSC.

Phospholipidosis

Phospholipidosis is an additional form of drug-induced lipid accumulation. The lesion was first recognized in Japan in 1969 as a complication of the early coronary vasodilator diethylaminoethoxyhexoestrol. Phospholipidosis is characterized by enlarged, foamy or granular hepatocytes or Kupffer cells by light microscopy ( Fig. 12.17C ); lamellated or crystalloid inclusions can be seen by electron microscopy ( Fig. 12.17D ). The lesion resembles the changes seen in inborn disorders of phospholipid metabolism. It has since been seen in patients taking perhexiline maleate ^, or amiodarone ^, and has been reproduced in experimental animals by amphiphilic compounds. ^, Indeed, the amphiphilic character of the drugs appears to account for the accumulation of phospholipids in lysosomes of hepatocytes and other cells. The drugs bind to phospholipids and inhibit their hydrolysis by lysosomal phospholipase A. The development of phospholipidosis may represent a form of lysosomal defence in which the agent is sequestered in the stacks of phospholipid lamellae, which may ultimately be extruded into the extracellular environment. Although most drugs associated with phospholipidosis are also associated with steatohepatitis, there are some only associated with phospholipidosis ( Table 12.12 ).

Hepatoportal sclerosis and nodular regenerative hyperplasia

Deposition of collagen in the periportal area and space of Disse, accompanied by reduction of portal vein calibre or obliteration of the portal vein, can lead to portal hypertension ( Fig. 12.23 ). This form of noncirrhotic portal hypertension has been termed ‘hepatoportal sclerosis’. Instances of noncirrhotic portal hypertension have been attributed to chronic exposure to inorganic arsenicals, vinyl chloride and copper sulphate. Noncirrhotic portal hypertension can result from vitamin A intoxication. Several protocols employed to prepare patients for bone marrow transplantations or to treat neoplastic disease have led to noncirrhotic portal hypertension by producing hepatoportal sclerosis or NRH. NRH has also been associated with the use of azathioprine in transplantation, mercaptopurine and 6-thioguanine for Crohn disease ^, and oxaliplatin-containing regimens for colon cancer ( Fig. 12.24 ). ^, This lesion can be difficult to recognize in routinely stained sections. However, careful examination of reticulin preparations will demonstrate nodules of enlarged hepatocytes arranged in cords that are more than two cells thick and which are not organized into parallel plates. Between regenerating nodules, the hepatocytes are atrophic and compressed; there may be no fibrosis or only a small amount of sinusoidal fibrosis in the areas of hepatocyte atrophy. Sinusoidal endothelial cells may express CD34 and the normal distribution of glutamine synthetase may be altered. Sinusoidal dilation may also be seen.

Hepatic vein thrombosis

Certain chemotherapeutic agents and oral contraceptives have been implicated in causing hepatic vein thrombosis ( Fig. 12.19 ). At least 100 cases have been reported in patients taking oral contraceptives and have been attributed to the thrombogenic effects of contraceptive steroids. Many patients appear to have other risk factors, so the effect of contraceptive steroids may be to increase an underlying risk. The risk of developing hepatic vein thrombosis seems to be increased at least twofold in women taking oral contraceptives.

Sinusoidal obstruction syndrome/veno-occlusive disease

VOD is a pattern of liver injury in which there is obliteration of small hepatic veins by loose, oedematous connective tissue that appears between the endothelium and the underlying normal collagen matrix, narrowing the lumen. This is associated with endothelial injury in the sinusoids, particularly in zone 3 with the accumulation of cell debris, trapped red cells and extracellular matrix in the sinusoids; there is frequently necrosis of the perivenular hepatocytes. Because of the key role played by sinusoidal obstruction in the pathophysiology, the term ‘sinusoidal obstruction syndrome’ has been suggested as an alternative name for this process. Depletion of glutathione and nitric oxide have been suggested as important mechansims. ^, Injury and occlusion of the efferent hepatic venules have long been known to be produced by PAs. ^, The initial lesion is sinusoidal and venular endothelial injury is accompanied and followed by progressive fibrotic decrease in venule calibre and perivenular necrosis ( Fig. 12.20 ). It can lead to portal hypertension with ascites, varices and hepatomegaly. Severe cases can develop jaundice and hepatic failure. Chronic presentations are more subtle and may result in significant fibrosis. ^,

In addition to the alkaloids, causal agents include oxaliplatin, thioguanine, azathioprine and a number of other oncotherapeutic agents, as well as irradiation. ^, The use of multiple chemotherapeutic agents or combination chemotherapy and radiotherapy seems to be more toxic than the use of any single agent. The most important cause today is myeloablative conditioning treatment associated with bone marrow transplantation, but the incidence of SOS/VOD has fallen due to changes in bone marrow transplant protocols and the advent of nonmyeloblative regimens.

Peliosis hepatis and sinusoidal dilatation

Peliosis hepatis consists of large blood-filled cavities not lined by endothelium ( Fig. 12.21 ). It has been of recognized clinical significance only since the middle of the 20th century. The lesion has been produced in experimental animals by administration of phalloidin. Phalloidin has a special proclivity for injury to membranes; this is consistent with the theory that the lesion reflects damage to sinusoidal supporting membranes.

Agents implicated as a cause of the lesion include anabolic steroids, diethylstilboestrol, contraceptive steroids, tamoxifen and azathioprine, ^, vitamin A and thorotrast ( Table 12.13 ). Marked sinusoidal dilation has often been observed in livers that show peliosis hepatis, even in sites remote from the cavitary lesions. Furthermore, anabolic and contraceptive steroids can lead to sinusoidal dilatation, even when no peliosis has developed. Indeed, a characteristic lesion of prominent dilatation of periportal sinusoids ( Fig. 12.22 ) has been described in patients taking contraceptive steroids. Ultrastructurally, the lesion is characterized by striking perisinusoidal fibrosis and proliferation and ‘enhanced activity’ of sinusoidal endothelial cells and perisinusoidal stellate cells. As noted previously, sinusoidal dilation may be associated with NRH, and a reticulin stain should be performed to exclude that possibility.

Subcapsular haematomas , which may rupture, have been reported in a number of patients taking oral anticoagulants (warfarin, streptokinase) ^, and in one patient who abused anabolic steroids.

Benign tumours

HCA, a lesion almost entirely restricted to females in the child-bearing years, was an extremely uncommon tumour prior to the era of oral contraceptives. ^, Convincing evidence for a causal effect is the reported regression of this tumour following withdrawal of oral contraceptives and the large case-control epidemiological study of Rooks et al. There is no relationship between focal nodular hyperplasia and contraceptive steroids.

Malignant tumours

Thorotrast has caused hepatocellular and cholangiocellular carcinomas as well as angiosarcoma, lesions that may be due to the radioactivity rather than the chemical toxicity of the agent. Epidemiological studies suggest that mycotoxins as well as synthetic chemicals encountered occupationally may also be carcinogenic in man. Several drugs have come under suspicion as possible hepatocarcinogens. The development of HCC in at least 40 long-term recipients of anabolic steroids has made these agents suspect. Contraceptive steroids have also been incriminated in the development of HCC. Tamoxifen and danazol have been implicated in hepatic carcinogenesis.

Angiosarcoma has been reported in individuals with occupational exposure to vinyl chloride. This rare tumour also developed in vintners with long exposure to inorganic arsenic, in patients with psoriasis treated with arsenicals, in individuals who had ingested arsenic-contaminated water or had other environmental exposures and in patients who had been injected with thorotrast. Other drugs incriminated rarely as causes of this tumour include androgenic-anabolic steroids, ^, diethylstilboestrol and phenelzine.

Adaptive changes

Ground-glass hepatocytes may develop in patients on long-term therapy with some drugs ( Fig. 12.25 ). This change was first reported by Klinge and Bannasch in patients on long-term therapy with chlorpromazine and barbiturates. Other drugs that have been implicated include azathioprine, steroids, phenytoin, resorcin and some analgesics. The change was attributed to a marked, diffuse hypertrophy of the smooth endoplasmic reticulum (SER) by Klinge and Bannasch, and this has been confirmed by ultrastructural studies. It is associated with increased activity of microsomal nicotinamide adenine dinucleotide phosphate (NADPH) cytochrome c reductase. Drug-associated ground-glass cell change may be differentiated from the ground-glass cells of chronic hepatitis B both by special stains (aldehyde fuchsin, Victoria blue and orcein) and by immunohistochemistry.

The prognostic significance of drug-induced ground-glass transformation is not known. It is accompanied by elevated levels of γ-glutamyltransferase (GGT) and is believed to account for the hepatomegaly observed in patients on anticonvulsants and other drugs. It is not associated with acute or chronic injury that may be caused by the same drug, and there are no long-term studies of patients with this drug-induced hepatic change. A dramatic form of ground-glass change ( Fig. 12.26 ) has been noted in patients undergoing alcohol aversion therapy. At first attributed to disulfiram, it now appears more clearly associated with cyanamide treatment. The change differs from that described in the foregoing paragraphs, in that the cyanamide-associated altered cells contain ground-glass ‘inclusion bodies’ that are periodic acid-Schiff (PAS)- positive and diastase resistant ( Fig. 12.26 B). Ultrastructurally, the inclusions are composed of glycogen beta granules, secondary lysosomes, lipid vesicles and residues of degenerating organelles; this lesion can apparently lead to cirrhosis. Lefkowitch et al. recently reported a series of patients with PAS-positive, diastase-sensitive inclusions that were termed ‘polyglucosan-like’. These were found in patients on multiple immunosuppressives after transplantation and may represent an adaptive change.

Patients who have been treated with high doses of corticosteroids may develop severe glycogenosis of the hepatocytes. This change is characterized by diffuse cellular enlargement by pale bluish-grey cytoplasm ( Fig. 12.27 ). The cells may contain small lipid droplets, but steatosis may be inconspicuous or absent. An acute rise in transaminases may prompt a biopsy, but the lesion seems to be completely reversible. Glycogenosis has also been reported in association with type 1 diabetes.

Pigment deposits

Several types of pigment deposits may follow exposure to exogenous chemicals. These include lipofuscin, haemosiderin, Thorotrast, gold, titanium, mercury, silver, polyvinylpyrrolidone (PVP) and bilirubin (discussed earlier). ^{, ,}

Lipofuscin , the least significant of these, occurs normally in the lysosomes of the perivenular hepatocytes; it is more prominent in elderly people. It also is increased in patients on various long-term medications, such as phenacetin, Cascara sagrada and chlorpromazine, anticonvulsant therapy as well as antiretroviral therapy for HIV infection.

Black pigment granules in the liver ( Fig. 12.28 ) may reflect the presence of gold as a residue of use of gold compounds for treatment of rheumatoid arthritis ^, ; titanium , which may be found in parenteral drug abusers ; or mercuric sulphide in chronic mercury poisoning following ingestion of a laxative preparation containing mercurous chloride. Dark brown pigment granules containing silver were reported in a patient with argyria.

Haemosiderin deposits are seen in several forms of chemical and other causes of hepatic injury. Haemosiderin may be seen in the hepatocytes of patients with porphyria cutanea tarda resulting from drugs or toxins, e.g. hexachlorobenzene. Hepatic haemosiderosis has been reported in patients on long-term haemodialysis who had received parenteral iron. Transfusional haemosiderosis is discussed in Chapter 4 . Rare cases of iron overload have been attributed to excess ingestion of iron preparations.

Thorotrast , a formerly used radioactive contrast medium, remains in the livers of patients many years after its administration. It appears as glistening, greyish-brown granules engorging macrophages or lying free in dense fibrous tissue ( Fig. 12.29 ).

PVP is a water-soluble, hydrophilic, high-molecular-weight product of the polymerization of vinylpyrrolidone. During World War II it was used as a plasma expander and still is used as a binder in oral medications. PVP is stored in reticuloendothelial cells and long-term sequelae have not been reported. The affected cells have a granular, basophilic cytoplasm. The PVP stains positively with Lugol iodine solution (mahogany brown), Congo red, Sirius red and mucicarmine. Unlike amyloid deposits, Congo red-stained PVP does not display apple-green birefringence.

Mild nonspecific changes

Finally, drugs may cause serum biochemical changes sufficient to generate a liver biopsy, yet not cause much in the way of diagnosable changes. There may be minimal or mild steatosis, rare apoptotic bodies, scattered pigmented macrophages or spotty necrosis. Like the adaptative changes noted previously, it is unclear whether there is potential for more serious injury if the drug is continued. At least in the case of methotrexate, a biopsy showing such mild changes would be reassurance that the drug could be continued, but it is difficult to extend that generalization to therapeutics that are less well understood.

Carbon tetrachloride and other chlorinated aliphatic hydrocarbons

CCL ₄ is a classic example of a zone 3 hepatotoxin ( Fig. 12.30 ) causing necrosis leading to hepatic failure. This chemical is metabolized to a toxic trichloromethyl radical catalysed by CYP 2E1. Alcohol potentiates the injury via induction of this cytochrome. Most cases have been the result of industrial or domestic accidents, such as inhalation of CCl ₄ -containing dry cleaning fluids and use as a household reagent, or after ingestion by alcoholics who have been known to mistake it for a potable beverage. At the cellular level, direct damage to cellular membranes results in leakage of intracellular enzymes and electrolytes leading to calcium shifts and lipid peroxidation. Hepatic steatosis develops as a result of triglyceride accumulation from haloalkylation-dependent inhibition of lipoprotein micelle transport out of the hepatocyte and increases with the continuous supply of free fatty acids from the blood. Activation of endonucleases causing chromosomal damage and mutations may result in carcinogenesis. The relative toxicity of CCl ₄ compared to other haloalkanes and haloalkenes correlates inversely with the level of bond dissociation energy, the number of halogen atoms and the chain length. Although complete recovery from CCl ₄ -induced liver damage is typical in moderate exposures, renal failure from acute tubular necrosis and GI haemorrhage leads to a case-fatality rate of 10–25%.

Chloroform is an important experimental hepatotoxin; its use as an anaesthetic having been abandoned in the 1950s. 1,1,2-Trichloroethane (methylchloroform) and tetrachloroethane are less potent haloalkane hepatotoxins that found use as solvents for components of varnish used to cover fabrics of airplane covers during the First and Second World Wars. Inhalational or percutaneous exposure led to subacute hepatic necrosis, with susceptibility increased in females and alcoholics. There are a number of reports of injury with 1,1,1-trichloroethane , including chronic hepatitis.

Hydrochlorofluorocarbons (HCFCs) caused liver injury in industrial workers exposed to dichlorotrifluoroethane (HCFC 123) and 1-chloro-tetrafluoroethane (HCFC 124), both of which are metabolized to reactive trifluoroacetyl halide intermediates similar to those implicated in halothane hepatoxicity. Zone 3 necrosis was present on biopsy, and autoantibodies against cytochrome P450 2E1 or P58 were detected in the serum. As with halothane, potentiation of HCFC liver toxicity is seen with ethanol-induced CYP 2E1 activity in animal models.

Vinyl chloride and other chlorinated ethylenes

Vinyl chloride monomer ( VCM , monochloroethylene) exposure previously occurred in polymerization plants where vinyl chloride was heated to form polyvinyl chloride (PVC) in the manufacturing of plastics, leading to inhalation of the toxic gas in the process. VCM is ubiquitous in the environment and has been estimated by the Environmental Protection Agency (EPA) to exist in at least 10% of toxic waste sites. Although PVCs appear to be nontoxic per se , liver damage from VCM has been described in the form of several types of chronic liver injury from long-term exposure, including nodular subcapsular fibrosis, sinusoidal dilatation, peliosis hepatis and periportal fibrosis associated with portal hypertension. ^, Milder forms of injury have also been described, which were reversible within 6–24 months of avoiding exposure with instances of relapse after re-exposure. In workers with hepatitis B or C infection, a higher prevalence of elevated ALT values was seen in those with a high exposure to VCM compared to those with a lower exposure, a finding not observed among those without a viral hepatitis infection. NAFLD, as well as hepatomegaly and periportal fibrosis, has been recently described in a third of nonobese chemical workers with high exposure levels, some of whom continued to have NASH up to 6 years later.

VCM is carcinogenic in animals and humans, with the first reports of angiosarcoma among workers in rubber plants appearing in 1974. A history of vinyl chloride exposure was found to be present in 15% to 25% of all cases of hepatic angiosarcoma reported in the late 1970s. HCC develops after a latency of decades, with the risk related to the time and extent of contact. ^, Efforts to reduce occupational exposure to VCM have resulted in a marked decrease in the incidence of angiosarcoma.

Trichloroethylene (TCE) leads to a similar but often less severe clinical syndrome as seen with CCl ₄ , with exposure the result of accidental industrial exposure or from sniffing the chemical. Once used as an anaesthetic, it now has a role as a solvent, spot remover and degreaser. Vinylidene chloride (VDC) is another intermediate used in the production of plastics that targets the liver.

Nonhalogenated organic compounds

In general, these agents, including aliphatic hydrocarbons, cycloparaffins, esters, ethers and ketones, produce little or no hepatic injury in animals and humans. Most aromatic hydrocarbons also appear devoid of significant hepatotoxic potential. Children exposed to benzene during a flaring incident at a refinery developed elevated LAEs. Toluene led to steatosis and necrosis in a ‘glue sniffer’. It caused elevations in γ glutamyl-transpeptidase after industrial exposure. Xylene can cause mild steatosis and styrene (vinyl benzene) has led to elevated transaminases after prolonged exposure.

Trinitrotoluene and other nitroaromatic and nitroaliphatic compounds

TNT (nitroglycerin) has been known to be hepatotoxic since World War I when munitions workers developed severe acute and subacute necrosis, with a case-fatality rate of more than 25%. The incidence of hepatotoxicity during World War II was lower, with approximately 1 in 500 workers affected, with the estimated incidence of associated toxicities, methemoglobinemia and aplastic anaemia being as much as 50 times higher than liver injury. The syndrome of subacute hepatic necrosis was preceded by a latent period of 2–4 months of regular exposure to TNT. Percutaneous absorption through the skin was the major source of exposure, with lesser contributions from inhaling toxic fumes and ingesting contaminated food. In some patients, rapidly progressive liver failure and death within days to months were observed, with massive hepatic necrosis at autopsy.

Nitrobenzene and dinitrobenzene are also potent hepatotoxins described during World War I. Ingestion of dinitrophenol has been associated with necrosis and intrahepatic cholestasis, possibly owing to its uncoupling of oxidative phosphorylation. ^, Nitrobenzene and nitrotoluene exposures have also been associated with steatosis and steatohepatitis.

Nitromethane, nitroethane and nitropropane cause variable degrees of hepatic injury. 2-Nitropropane (2-NP) caused fatal massive hepatic necrosis after occupational exposure as a solvent, fuel additive, varnish remover and rocket propellent. Toxic hepatitis associated with the chronic inhalation of propane and butane has been reported. Both 1-NP and 2-NP are mutagenic potential human carcinogens.

Polychlorinated biphenyls and other halogenated aromatic compounds

Polychlorinated biphenyls (PCBs) are mixtures of trichloro-, tetrachloro-, pentachloro- and hexachloro- derivatives of biphenyls, naphthalenes and triphenyls used in the manufacture of electrical transformers, condensers and industrial fluids. Acute and chronic hepatotoxicity was seen during World War II and resembled that due to TNT. ^, Inhalation of toxic fumes released by the melting of PCBs and chloronaphthalene mixtures during soldering of electrical materials was the most common means of exposure. Liver injury appeared to correlate with the number of chlorine molecules. Liver damage appeared as early as 7 weeks after initial exposure. Skin lesions, known as chloracne, usually preceded the hepatic injury. Death occurred within 2 weeks in fulminant cases with massive necrosis, or after 1–3 months in subacute injury; cirrhosis developed in some who survived the acute injury. A study of the liver enzyme data from the 2003–2004 National Health and Nutrition Examination Survey (NHANES) demonstrated an association between several different PCBs and elevated LAEs. Polybrominated biphenyls (PBBs) appear to more toxic and more potent enzyme inducers compared to PCBs. Consumption of milk and meat from livestock given feed mistakenly contaminated by a PBB led to hepatomegaly and minor abnormalities of liver function tests in exposed persons.

Miscellaneous chemical compounds

Dimethylformamide is a solvent widely used in the synthetic resin and leather industries that causes dose-related massive necrosis in animals and is capable of producing focal hepatic necrosis and microvesicular steatosis in humans, the severity of which correlates with the duration of occupational exposure. Most individuals with prolonged exposure (greater than 1 year) had symptomatic disease that slowly resolved when they were removed from the workplace. Re-exposure in one individual produced recurrent injury. In a series of workers from Italy, 23% had liver function test abnormalities and 50% reported disulfiram-like symptoms. Workers in a Chinese organic chemical factory who were exposed to DMF had levels of DMF metabolites in their urine that correlated with elevations in LAEs.

Hydrazine and its derivatives are experimental hepatotoxins and carcinogens. In humans, steatosis and focal necrosis have been reported. Bromoalkanes and iodoalkanes have been used in insecticides and aircraft fuels, with rare reports of hepatic injury. Ethylene dibromide (dibromoethane) produces zone 3 necrosis in experimental animals with at least one instance of necrosis after an attempted suicidal ingestion. Occupational exposure has been linked to fatal toxicity, which may be potentiated by the concomitant use of disulfiram.

Iron

Accidental iron poisoning has been described for more than half a century, with upwards of 5000 cases reported to occur in the United States annually. Most cases occur in young children who mistake iron supplements for sweets and the severity of injury depends on the dose ingested. Indeed, severe injury was seen only with serum iron concentrations above 700 µg/dL measured within the first 12 hours. Ingestion of <20 mg/kg of elemental iron is unlikely to produce serious toxicity, whereas doses >200 mg/kg can be fatal. Iron, per se , is not hepatotoxic, but ferric and ferrous ions acting through free radicals and lipid peroxidation can cause membrane disruption and necrosis. Clinically evident liver injury is uncommon in most instances. Periportal necrosis may be seen in the most severe cases ( Fig. 12.3C, D ). The oral iron chelator deferiprone was implicated in causing worsening hepatic fibrosis in a long-term study of patients with thalassemia ; however, these findings were not substantiated by subsequent histopathological analyses, and injury from this agent appears unlikely.

Phosphorus

Poisoning by white phosphorus is rare since its use in fireworks and matches was outlawed more than 60 years ago, ^, although case reports continue to appear. ^, Symptoms of severe GI and neurotoxicity develop shortly after ingestion, with death occurring within 24 hours. Phosphorescence of the vomitus and stools and a typical garlic-like odour on the breath are characteristic when present. Histologically, the liver may show only steatosis, initially in the periportal and then diffuse ( Fig. 12.3B ). In severe cases there may be massive necrosis, which has sometime been reported to be periportal in distribution.

Copper salts

Acute poisoning by copper is usually associated with abdominal pain and nausea but can cause hepatic toxicity in larger doses. Toxicity is related to free radical production that overwhelms intracellular protective mechanisms. Ingestion of toxic amounts (1–10 mg) is usually seen with suicidal intent, especially on the Indian subcontinent. Acute copper toxicity may result from ingestion (either suicidal or accidental) of copper sulphate, from its use (in the past) as an emetic or for the treatment of burns and from the release of copper ions (from equipment such as tubing made of copper) in an acid pH, for example, malfunctioning haemodialysis equipment, or from high levels of copper in drinking water. Histologically there is perivenular necrosis and cholestasis in deeply jaundiced patients, but only focal necrosis or no changes in mildly jaundiced patients; noncirrhotic portal hypertension has been described. Serum copper levels are markedly elevated in acute copper intoxication, as is serum caerulo-plasmin; the concentrations of copper in the liver can be very high. Mortality in a recent series of copper sulphate poisoning was 23%.

Chronic copper toxicity can result from occupational or domestic exposure. An example of the former is the chronic exposure of vineyard workers to fungicide sprays containing copper sulphate. The resultant hepatic injury includes noncaseating granulomas, parenchymal and periportal fibrosis, cirrhosis and, rarely, angiosarcoma. Copper poisoning remains a hazard in rural parts of the world. Of interest are reports of several children who developed an illness, clinically and histopathologically resembling Indian childhood cirrhosis, from the chronic ingestion of well water contaminated with high levels of copper.

Thorium dioxide (Thorotrast)

The colloidal preparation of thorium dioxide was used as an intravenous contrast medium for radiographic procedures in the first half of the 20th century, with estimates of >50,000 patients being exposed before it was abandoned in 1955. Rare cases continue to appear, long after exposure. A large number of reports have appeared describing tumours of the liver as well as lympho- and myeloproliferative syndromes and non-neoplastic lesions of the liver. ^, The lesion that has drawn the most attention is angiosarcoma, ^, but cholangiocarcinoma and HCCs have been attributed to Thorotrast with almost equal frequency. ^, Hepatic leiomyosarcoma has also been reported. Nonmalignant hepatic lesions attributed to Thorotrast include periportal fibrosis, veno-occlusive lesions and cirrhosis. The fibrosis is particularly marked in the subcapsular region. An additional lesion of note is peliosis hepatis, which may be fatal. Histologically, thorium dioxide is found in Kupffer cells and macrophages as dark brown refractile granules approximately 10 µm in diameter ( Fig. 12.29 ), often in clusters, which can be confirmed by spectrographic analysis.

Other metals

Cadmium is an experimental hepatotoxin that produces hepatic necrosis and cirrhosis and that can enhance the hepatotoxicity of endotoxin in laboratory animals. Recent epidemiological studies in the United States and Korea have linked cadmium exposure to elevated LAEs and an increased incidence of steatosis and steatohepatitis in men. ^, A study of liver biopsies in patients with chronic cadmium toxicity demonstrated increased portal and bridging fibrosis as compared to controls.

Beryllium has led to midzonal (zone 2) necrosis, the result of phagocytosis of insoluble beryllium phosphate (formed by the reaction of beryllium with phosphate in the blood) by Kupffer cells. The Kupffer cells undergo necrosis, releasing beryllium and possibly cytokines. Chronic industrial exposure, usually by inhalation of high concentrations of oxide or phosphorus mixtures, is associated with the formation of hepatic (and pulmonary) granulomas.

Lead hepatotoxicity may be seen as part of the larger symptom complex of abdominal pain, constipation and encephalopathy that occurs with chronic ingestion or environmental exposure. Histologically, lead poisoning has been reported to cause canalicular cholestasis and mild portal inflammation.

Hepatotoxic pesticides

While exposure to insecticides, herbicides and other pesticides in the environment is common, acute liver injury from these compounds, many of which are chlorinated hydrocarbons, is rare. A study of data from the 2003–2004 NHANES found associations between elevations in transaminases and exposure to a limited group of chemicals, including dioxins, non-dioxin-like PCBs and mercury. Ingestion of large amounts of dichlorodiphenyltrichloroethane (DDT) or chlordane has occasionally been reported to cause a clinical syndrome of hepatic necrosis similar to that seen with CCl ₄ . The P-450 cytochrome enzyme inducing effects of some of these agents can enhance the toxicity of other agents.

DDT and other organochlorines (aldrin, amitrole, chlordane, dieldrin, lindane, mirex) accumulate in fat deposits and, to a lesser extent, in the liver after prolonged exposure. Despite the concern that DDT might lead to occupational hepatotoxicity, there is almost no evidence of liver damage from chronic contact. An outbreak of hepatotoxicity in the Hirmi Valley in Ethiopia was linked to a combination of DDT and PAs, although the histologic injury described is more typical of the latter agent. Similarly, there is scant direct evidence that DDT is carcinogenic in humans. Much attention has been focused on Agent Orange (2,4-dichlorophenoxyacetic acid), the defoliant widely used in Vietnam. Acute hepatitis has been reported after chronic exposure from licking a contaminated golf ball. In animals, dioxin (tetrachlorodibenzo- p -dioxin) has produced progressive sinusoidal dilatation and peliosis hepatis.

Dichloridedimethyldipyridlium (paraquat) has been implicated in several instances of poisoning, many as the result of attempted suicides and occasionally due to homicides. ^, Ingestion of 2–25 g of the liquid concentrate has produced severe toxicity, and dermal exposure has also been sufficient to cause liver injury. ^, Many patients have severe vomiting, and profuse diarrhoea leading to hypokalaemia, and often show evidence of oral, pharyngeal and oesophageal caustic lesions (stomatitis) after ingestion. Death results from a combination of renal, respiratory, cardiac and hepatic failure with mortality rates as high as 70%, often within the first 48 hours. Histopathological changes include zone 3 necrosis and rather unique injury to small and medium-sized interlobular bile ducts that occurs in a biphasic pattern ( Fig. 12.13 ). It is thought that hepatocellular injury becomes cholangiocellular in nature with jaundice after the initial 48 hours, with bile duct injury being a direct corrosive effect of paraquat.

Chlordecone (kepone) has been shown to impair biliary excretion and lipid transport and storage, but neurological toxicity appears to dominate the clinical picture. Occupational exposure has led to hepatic steatosis and abnormal liver function tests, and chronic exposure is hepatocarcinogenic in rodents. Steatosis is seen in rodents exposed to chlordane . Hexachlorobenzene was associated with an epidemic of porphyria cutanea tarda and liver injury from contaminated grain.

Inorganic arsenic has long been used as a homicidal or suicidal agent, and toxic exposure also followed ingestion of Fowler solution (arsenic trioxide) used in the past as a treatment for psoriasis and asthma. Other sources of exposure come from contaminated ground and well water and homemade alcohol. Necrosis of capillary walls causes congestion and haemorrhagic necrosis of the GI tract leading to severe nausea, vomiting and diarrhoea and is also thought to produce a lesion in the liver resembling VOD. Severe steatosis and varying degrees of necrosis have been observed. More than 90% of a group of 248 patients who consumed contaminated drinking water for up to 15 years were shown to develop noncirrhotic portal hypertension. A study of children in India with noncirrhotic portal hypertension also found an association with exposure to arsenic.

Occupational exposure to arsenic is still observed among vineyard workers, farmers and gold miners, although its use as an insecticide has been curtailed since the 1940s. Lumber treated with chromated copper arsenate as a preservative may be an additional source of exposure. Chronic hepatic injury, including cirrhosis and noncirrhotic portal hypertension, may be precursor lesions to hepatic malignancies, including angiosarcoma, hemangioendothelioma and HCC. ^,

Hepatotoxic mushrooms

There are about 100 poisonous varieties of mushrooms among the more than 5000 species, with reports of serious and fatal mushroom poisonings found worldwide. In the United States, between 1999 and 2016 about 7400 mushroom poisonings were reported per year, with two-thirds of fatal poisonings due to A. phalloides (death cap) or Amanita verna (destroying angel). A fatal dose can involve the ingestion of a single 50 g mushroom, representing a dose of about 21 mg of amatoxin. Alpha amatoxin is thermostable, can resist drying for years and is not inactivated by cooking. Rapidly absorbed via the GI tract, it inhibits production of mRNA and protein synthesis in hepatocytes, leading to necrosis. A second toxin, phalloidin, is responsible for the GI distress that precedes hepatic and central nervous system (CNS) injury. ^, Phalloidin disrupts cell membranes by interfering with actin polymerization and leads to severe gastroenteritis. In contrast to the injury seen with CCl ₄ and phosphorus, the latent period is often longer (6–20 hours). Intense abdominal pain, vomiting and diarrhoea develop, with hepatocellular jaundice and renal failure occurring over the next 24–48 hours, followed by convulsions and coma by 72 hours. ^, In a case series of eight patients reported by Rengstorff et al., mean AST was 5488 IU/L, mean ALT was 7618 and bilirubin was 10.5 mg/dL, peaking at 4 to 5 days after ingestion. Mortality is generally high, especially when the ALT exceeds 1000 IU/L, and emergency liver transplantation is often required. ^, The characteristic hepatic lesion is prominent steatosis and zone 3 necrosis ( Fig. 12.31 ).

Other hepatotoxic foodstuffs

The unripe fruit of the ackee tree ( Blighia surpida ), native to Jamaica, contains a cholestatic hepatotoxin, hypoglycin A, which produces a clinical syndrome of GI distress and microvesicular steatosis known as Jamaican vomiting sickness that resembles Reye syndrome. Cholestatic jaundice has been described after chronic ingestion. Cycasin is a potent hepatotoxin and carcinogen found in the fruit of the cycad tree ( Cycas circinalis , Cycas revoluta ). A small epidemic of acute hepatic injury attributable to the ingestion of cycad nuts was reported from Japan.

Aflatoxins are a family of mycotoxins found in Aspergillus flavus and related fungi that are ubiquitous in the tropics and subtropical regions. They contaminate peanuts, cashews, soybeans and grains stored under warm, moist conditions and are well-known hepatotoxins and hepatocarcinogens. Occupational exposure to aflatoxins has been documented in Egyptian wheat handlers. Aflatoxin B1, a potent inhibitor of RNA synthesis, is the most hepatotoxic. Reactive metabolites are formed by P450 metabolism; malnutrition is a possible potentiating factor (perhaps due to glutathione depletion). When consumed in large quantities, it is responsible for a clinical syndrome characterized by a prodrome of fever, malaise, anorexia and vomiting followed by jaundice. Portal hypertension with splenomegaly and ascites may develop over the next few weeks. In large epidemics, mortality rates have approached 25% and are dose related. Zone 3 necrosis without significant inflammation is the characteristic lesion. Other histological findings include cholestasis, microvesicular steatosis and bile duct proliferation.

The risk of HCC has been well confirmed and is directly related to the amount of aflatoxin consumed, especially in sub-Saharan Africa and eastern China, where wheat often exceeds rice as a staple in the diet. Alcohol and possibly exposure to DDT appear to play an enhancing role in the hepatocarcinogenesis of rats exposed to aflatoxin B978-0-7020-8228-3. Perhaps an even more important cofactor is the interaction between aflatoxin and the hepatitis B virus (HBV). Aflatoxin is associated with a specific mutation in the p53 tumour suppressor gene leading to an amino acid change (R249S) that has been directly correlated to the development of HCC in these regions as compared to the rarity of this mutation in HCC from Western countries.

Kodo millet ( Paspalum scrobiculatum ) is a staple food found in northern India that is frequently contaminated by Aspergillus tamarii , which produces acute hepatic injury and preneoplastic changes due to another mycotoxin, cyclopiazonic acid.

Epidemics of hepatic injury from adulterated cooking oils and contaminated foods

A number of contaminated foodstuffs and cooking oils have been associated with epidemics of hepatotoxicity. While they are now largely of historical interest they are included as examples of the types of contaminations that can occur.

Spanish toxic oil syndrome occurred in 1981 after exposure to rapeseed cooking oil that was contaminated by anilines and acetanilides. Nearly 20,000 individuals became ill, many of whom had hepatic injury. The histopathological changes were those of a cholestatic hepatitis that resembled chlorpromazine-induced liver injury. Of 24 patients studied by Solis-Herruzo et al., 11 had necroinflammatory bile-duct lesions; NRH was also noted.

Epping jaundice refers to an epidemic of toxic liver injury that occurred in Epping, England in 1965. It involved 84 persons who had eaten bread contaminated with MDA. The clinical syndrome consists of cholestatic jaundice preceded by abdominal pain and fever, resembling biliary colic, beginning a few hours after ingestion. Liver biopsies performed in some patients revealed cholestatic hepatitis with cholangitis but little or no necrosis. Most individuals recovered within 4–6 weeks, with jaundice lasting up to 4 months in a few.

Yusho oil disease in western Japan, and a related epidemic referred to as Yu-Cheng in Taiwan, involved nearly 2000 individuals who had eaten rice prepared in oil contaminated by PCBs, dioxins and polychlorinated dibenzofurans (PCDFs) in 1968. It was characterized by acne-like eruptions, skin hyperpigmentation and neuropathy, with jaundice reported in approximately 10%. Exposed individuals still harboured high levels of these agents more than 26 years after the outbreak and have an increased risk of liver cancer among other malignancies after 40 years of follow-up.