1.

0 INTRODUTION
The liver is the largest internal organ in the human body and it is the main organ for the

metabolism and detoxification of drugs and environmental chemicals (Klaassen, 2017). Other

main functions of the liver include glucose storage and synthesis, decomposition of red blood

cells, plasma protein synthesis, hormone production, and bile formation. Anatomically, the liver

lies slightly below the diaphragm and anterior to the stomach, a position that facilitates

maintaining metabolic homeostasis of the body. Two distinct blood supplies feed the liver: the

portal vein and the hepatic artery. The portal vein carries blood containing digested nutrients

from the gastrointestinal tract, spleen and pancreas, while the hepatic artery carries oxygenated

blood from the lungs. The human liver consists of four lobes, and each lobe is made up of many

lobules, which is defined at the microscopic scale. The classical lobule is a hexagonalshaped unit

centered around a central vein. In each functional unit, blood enters the lobules from the portal

vein and hepatic artery, and then flows down past the cords of hepatocytes. The lobule is divided

into three regions: (1) periportal (Zone 1) is the closest to the entering blood supply with the

highest oxygen tension; (2) centrilobular (Zone 3) abuts the central vein and has the poorest

oxygenation; and (3) midzonal (Zone 2) is intermediate.

Due to the blood flows from the stomach and intestine, the liver is the first internal organ to

encounter a number of insults including ingested metals, drugs, and environmental toxicants

(Klaassen, 2017). As a consequence, liver cells are exposed to significant concentrations of these

chemicals, and liver functions can be adversely affected by the acute or chronic exposure. For

example, acetaminophen (APAP) is a widely used over-the-counter analgesic and antipyretic

(Hinson et al., 2018). When used at recommended therapeutic doses, APAP is rarely associated

with liver injury.

Unfortunately, APAP can cause fatal acute liver failure when therapeutic doses are exceeded due

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to the production of a highly reactive hepatotoxic metabolite in the liver (Lee, 2014). Since the

liver is often exposed to the highest concentrations of orally consumed drugs, it is not surprising

that the liver is often the target organ with ensuing drug-induced liver injury (DILI). DILI is a

major challenge for the pharmaceutical industry and public health, since DILI is a common cause

of drug development termination, drug restrictions, and post-marketing drug withdrawal

(Kaplowitz, 2015). Currently, more than 1000 drugs have been reported to be associated with

DILI (Shi et al., 2018). In addition, the increasing popularity of dietary supplements (DS) may

contribute to the high incidence of DILI since some DS have various toxicological effects or

alter the toxicity of concomitantly administered drugs (Abebe, 2020; Estes et al., 2021).

Typically, DILI has been classified in terms of the clinical liver disease which is hepatocellular,

cholestatic, or mixed hepatocellular/cholestatic. Hepatocellular often involves cellular damage of

the hepatocytes such as the centrilobular necrosis caused by APAP. This type of damage is often

associated with elevated serum alanine aminotransferase (ALT) levels due to leakage from

damaged hepatocytes. Cholestatic injury often involves damage to some part of the bile

processing or excretion apparatus, sulting in impaired bile processing or excretion.

This type of injury is often associated with elevated serum bilirubin and alkaline phosphatase

(ALP), indicating alterations in bile homeostasis and/or bile duct injury. Mixed injury presents

with a mixture of both types of effects. Unfortunately, drugs rarely produce a single clear clinical

picture, making the diagnosis of DILI difficult. For example, amoxicillin/clavulanic acid usually

causes cholestatic injury but can also produce acute hepatocellular injury or a mixed type injury

(Kaplowitz, 2015; Stirnimann et al., 2018). The histopathological analysis of liver biopsies is the

most definitive way to diagnose and confirm various types of liver diseases; however, the

biopsies are invasive and not routinely performed. Because currently available clinical laboratory

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tests are less than ideal, there is a pressing need for new biomarkers that are not just sensitive but

are also specific and prognostic.

Serum ALT activity has been historically used as a major biomarker for liver injury in humans

and in preclinical studies. Damaged hepatocytes release ALT into the extracellular space with

subsequent passage into the blood; however, ALT elevations can reflect nonhepatic injury,

particularly skeletal muscle injury. Moreover, the concentrations of ALT do not discriminate

between different etiologies of liver injury and ALT elevations can occur after a critical

therapeutic window has passed. Another weakness is that ALT levels do not provide any insight

into disease prognosis. Adaptation during continued exposure to DILI drugs has been observed

in preclinical and clinical studies. For example, it is noted that B25% of Alzheimer’s disease

patients receiving tacrine experience transient, asymptomatic increases in serum ALT. The

majority of these patients adapt to the drug as indicated by a return of serum ALT to baseline

levels despite continued treatment. Most statins can also cause serum ALT elevations in a subset

of treated patients, but they often occur in the absence of histological evidence of injury.

Therefore, elevated ALT levels do not necessarily indicate the severity of the liver injury and

whether or not a patient will adapt to the stressor or develop fulminant liver injury.

Total bilirubin (TBL) is a biomarker associated with altered bile homeostatis and/or

hepatobiliary injury. The functional reserve of the liver for processing bilirubin is large;

therefore, substantial hepatic injury often occurs before alterations in TBL are observed, making

TBL an insensitive biomarker. By the time TBL is elevated, there may already be substantial loss

of liver function, placing the patient in danger of liver failure. Therefore, there is a need for new

biomarkers that can identify the risk to patients prior to the occurrence of serious DILI. Hy

Zimmerman first noted that “drug-induced hepatocellular jaundice is a serious lesion. The

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mortality rate ranges from 10 to 50 percent“ (Zimmerman, 2018). He realized that when drugs

cause substantial hepatocyte injury that affects overall liver function and, in particular, causes

jaundice as the result of impaired bilirubin processing and transport, the hepatotoxicity is likely

to lead to life-threatening events (Senior, 2016). The combination of two biomarkers, ALT and

bilirubin, which was later defined as “Hy’s Law,” indicates more severe injury than serum

enzyme elevations alone. It is of interest that this observation has been confirmed in recent

reports (Bjornsson and Olsson, 2015; Andrade et al., 2016). The significance of “Hy’s Law” is

that the combination of ALT and bilirubin, neither of which by itself is sufficiently specific,

seems to be highly specific for serious liver injury. Based on “ Hy’s Law,” the FDA recommends

a combination of tests, including serum ALT, aspartate aminotransferase (AST), and ALP

activities and TBL concentration, to identify potential DILI. In addition to the biomarkers

described above, there are symptoms commonly associated with liver injury. Abdominal pain,

enlargement of the liver and spleen, distended belly full of fluid, or enlarged breasts in men are

common signs of liver injury (Chopra, 2001). In combination with biomarkers of liver injury,

these nonspecific and specific symptoms can help a physician identify liver injury and its

seriousness. Unfortunately, there are limited tools at the physician’s disposal for determining the

patient’s prognosis (i.e., will the patient recover with supportive care or will the patient develop

fulminant liver injury and require a transplant).

2.0 OVERVIEW OF LIVER PHYSIOLOGY, TOXICITY, AND PATHOLOGY

2.1 LIVER PHYSIOLOGY
2.1.1 Bioactivation and detoxification
The liver is the main metabolic organ in the body and is considered a viable defense against

environmental Zone 3 Zone 2 Zone 1 HA PV CV BD Blood Flow FIGURE 13.1 Microscopic

structure of the liver lobule. Blood, supplied by portal vein (PV), or hepatic artery (HA), enters
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the liver lobule through the portal triad, which encompasses a PV, HA, and bile duct (BD).

Blood flows from Zone 1 (best oxygenation) through Zone 2 and out of the central vein (CV) in

Zone 3 (poorest oxygenation).. In general, all foreign compounds (xenobiotics) are potentially

toxic (Zimmerman, 2019). In order to minimize the potential injury caused by these compounds,

the liver is well equipped with metabolizing enzymes including Phase I and Phase II

metabolizing enzymes as well as Phase III transporters (Xu et al., 2015). The coordinated

metabolism and transport of xenobiotics typically makes the xenobiotic less toxic and more

water soluble, thereby aiding its elimination from the body. Classically, the initial metabolic step

is referred to as Phase I or the oxidation phase (Guengerich et al., 2009). This step is most

frequently catalyzed by a cytochrome P450 (CYP). CYPs derive their name from the fact that

they exhibit maximal absorbance at 450 nm when bound to carbon monoxide (Omura and Sato,

2012). CYPs are found in the highest abundance in liver (specifically in hepatocytes) and to a

lesser extent in the epithelium lining the gastrointestinal tract (Danielson, 2020). They are

typically found associated with the lipid membrane of the endoplasmic reticulum and

mitochondrial outer membranes (Guengerich et al., 2018). CYP1, CYP2, and CYP3 families are

major enzymes mediating drug metabolisms and some P450s are highly inducible (Andrade et

al., 2018).

In general, Phase I CYP metabolism introduces a functional group to the xenobiotic, such as a

hydroxyl group. Phase I metabolism may not be a detoxifying function by itself since the

metabolite may actually be more reactive and toxic than the parent compound. Phase I

metabolism often works in conjunction with subsequent Phase II metabolism to produce a more

water soluble and less toxic compound. The functional group added by Phase I metabolism is

often the target of Phase II conjugation, typically making the metabolite more water soluble and

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less toxic. During this phase, the compound may undergo glucuronidation, sulfation, and

conjugation with glutathione, methylation, N-acetylation, or conjugation with amino acids

(Klaassen, 2017). Typically these modifications make the compound significantly more

hydrophilic and thereby enhance excretion in the bile and urine (Xu et al., 2015).

Phase III is often referred to as a “metabolic” process; however, it essentially involves the

transport of xenobiotics and their metabolites across biological membranes and no further

metabolic alterations of the compound’s structure. Phase III transporters are found in a wide

array of organs/biological membranes and they typically facilitate the removal of xenobiotics and

their metabolites from the body. There are many different Phase III transporters with each one

having specificity for different types of molecules (e.g. anions vs. cations). In particular, human

drug transporters can be classified under two major superfamilies: solute carrier (SLC) and the

ATP-binding cassette (ABC). Transporters of the SLC superfamily are organic cation

transporters (OCTs/SLC22A), the multidrug and toxin extrusion transporters (MATE

transporters/SLC47A), the organic anion transporters (OATs/SLC22A), and the organic anion

transporting polypeptides (OATPs/SLCO).

Members of ABC superfamily are important in drug toxicity, and these include P-glycoprotein

(MDR1/ABCB1), multidrug resistance-associated protein (MRP/ABCC), and breast cancer

resistance protein (BCRP/ABCG2) (DeGorter et al., 2012). Many Phase I and Phase II enzymes

show significant interindividual variability, which leads to various levels of exposure to the

reactive metabolites. There are two primary causes of this variation: polymorphism and

environmental exposure. Polymorphisms in drug metabolizing enzymes may cause dramatic

differences in drug detoxification (Sim et al., 2013). Several recent genome-wide association

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studies (GWAS) have been conducted based on the hypothesis that polymorphisms might play a

role in determining the risk of DILI (Sim et al., 2013).

2.1.2 Protein synthesis and catabolism.

The liver plays a vital role in protein metabolism, including deamination and transamination of

amino acids, plasma protein synthesis, and removal of ammonia to urea in the urine. Several of

the enzymes used in the amino acid metabolism pathways (for example, ALT/AST) are

commonly assayed in serum to assess liver damage since they are present at much higher

concentrations in the liver than in other organs (Dejong et al., 2017). Almost all blood proteins,

except gamma globulins, are synthesized in the liver (Miller et al., 1951). Albumin, which is the

main protein in human blood, plays a major role in maintaining plasma osmotic pressure as well

as transportation of lipids and hormones. Some liver injury can affect the concentrations of

plasma albumin, and the clinical presentations are hypoalbuminemia and hyperglobulinemia

(compensatory rise to offset the fall in albumin) (Farrugia, 2018). Fibrinogen and blood clotting

factors (e.g. Factor XIII) are another group of plasma proteins synthesized by the liver. During

blood clot formation, soluble fibrinogen is ultimately converted into insoluble fibrin strands

through a catalytic activation cascade of a series of blood clotting factors. When liver function is

impaired, it can lead to excess bleeding due to impaired synthesis of the blood clotting factors.

Ammonia is a metabolic product of amino acid deamination and is quickly converted by the liver

to urea, which is much less toxic. In humans, if ammonia is not rapidly and efficiently removed

from the circulation, it will result in central nervous system disease (e.g. hyperammonemia-

related coma) (Klaassen, 2017).

2.1.3 Bilirubin processing

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Unconjugated bilirubin is the yellow product produced by the degradation of heme, which is

derived mostly from the breakdown of erythrocyte hemoglobin. Unconjugated bilirubin is very

hydrophobic and is circulated in the blood by albumin. Sometimes, the high concentration of

hydrophobic drugs or fatty acids can cause elevation of unconjugated bilirubin due to

displacement from albumin. The liver is responsible for clearing the blood of unconjugated

bilirubin by conjugating it with glucuronic acid in hepatocytes. The conjugated bilirubin is then

secreted into bile for further metabolism and elimination. Increased total bilirubin (TBL) causes

jaundice, which is presented as yellowing of the skin and whites of the eyes. Because the

metabolic capacity of the infant liver is still developing, jaundice is a common problem seen in

infants due to increased serum unconjugated bilirubin levels (Cuperus et al., 2018; Cohen et al.,

2018). Occasionally, jaundice may be a symptom of other health problems; for example,

hemolysis generally induces an elevation in plasma levels of unconjugated bilirubin.

2.2 LIVER TOXICITY

2.1.2 Covalent binding

A large body of evidence has shown that reactive metabolites play an important role in the

pathogenesis of DILI (Jaeschke et al., 2020; Evans et al., 2021; Park et al., 2015).

Biotransformation of drugs results principally in detoxification. However, in certain instances,

reactive and highly toxic intermediates are generated, typically during Phase I metabolism (Park

et al., 2015; Omiecinski et al., 2012). Such metabolites are shortlived; however, if not detoxified,

they can form covalent modifications of biological macromolecules and thereby damage proteins

and nucleic acids (Park et al., 2015). Some early investigations have revealed the link between

reactive metabolites and chemical carcinogenesis. Reactive metabolites have also been identified

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from DILI drugs, such as APAP (Hinson et al., 2014), tamoxifen, diclofenac (Masubuchi et al.,

2020), and troglitazone (Kassahun et al., 2010). In the case of APAP, at therapeutic doses APAP

is predominantly metabolized by the Phase II metabolic pathways of glucuronidation and

sulfation. A small portion of APAP is metabolized by Phase I enzymes to the reactive metabolite

N-acetyl-p-benzoquinone imine (NAPQI), which is subsequently detoxified by conjugation with

glutathione (GSH) (Bessems and Vermeulen, 2011; Hinson et al., 2018). After a toxic dose of

APAP, the glucuronidation and sulfation pathways are saturated with a larger amount of APAP

being metabolized by the Phase I pathways to NAPQI. NAPQI is detoxified by conjugation with

GSH; however, once GSH stores become depleted, NAPQI covalently binds cellular

macromolecules such as proteins.

Covalent binding occurs in the hepatocytes resulting in disruption of cellular function and

eventual necrosis (Roberts et al., 2011; Yang et al., 2012a). Interestingly, the protein adducts can

be detected in the serum from APAP overdose patients, most likely due to release from necrotic

hepatocytes (Muldrew et al., 2020). Quantification of the protein adducts may be useful in the

diagnosis of the APAP overdosed patients. Although covalent binding often occurs during

hepatotoxic injury, it is not necessarily sufficient by itself to cause hepatotoxicity (Liebler and

Guengerich, 2015). For instance, 3-hydroxyacetanilide (regioisomer of APAP) and APAP show

similar levels of covalent binding in vivo, but AMAP is not hepatotoxic, even at very high doses

(Roberts et al., 2010). Troglitazone is known to be converted into reactive metabolites that

generate a relatively high level of covalent binding (Yamazaki et al., 2019), although a causal

relationship with hepatoxicity has not been further established. Furthermore, the utility of protein

adducts as DILI biomarkers has been limited by the fact that many protein adducts are found in

the liver but not blood (Shi et al., 2018). Covalent binding, in general, equates poorly with

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hepatotoxicity, but the cellular stress responses activated by reactive metabolites may play a

decisive role in the toxicity. GSH is a critical cellular antioxidant, which is important in

combating cellular oxidative stress that can damage cellular macromolecules. In APAP overdose,

NAPQI reacts with GSH leading to depletion of the cytoprotective molecule. NAPQI covalently

binds to mitochondrial proteins (Cohen et al., 2017), increases reactive oxygen species (ROS)

production, impairs mitochondrial respiration, causes ATP depletion, opens the mitochondrial

permeability transition pore, and makes the mitochondrial inner membrane abruptly permeable to

solutes up to 1500 Da (James et al., 2021; Jaeschke and Bajt, 2016). These events lead to onset

of the mitochondrial permeability transition (MPT), which is a common pathway leading to both

necrotic and apoptotic cell death. Induction of the MPT causes mitochondrial depolarization,

uncoupling of oxidative phosphorylation, and organelle swelling, which all result in decreased

ATP synthesis and cell death (Kim et al., 2021). Many in vitro assays have been developed to

measure GSH, ATP, ROS, mitochondrial functions, and cell viability (e.g. lactate drhydrogenase

release), which might be useful as in vitro DILI biomarkers in predicting hepatotoxicity.

2.3 LIVER PATHOLOGIES

2.3.1 Necrosis and apoptosis

Death of liver cells is a characteristic feature of many liver diseases, such as cholestasis,

hepatitis, or ischemia/reperfusion (Malhi et al., 2016). There are two patterns of cell death:

necrosis (large groups of cells die) or apoptosis (individual cells die and is ATP-dependent).

Damage may be restricted to certain cell types such as hepatocytes, cholangiocytes (bile duct

cells), endothelial cells, stellate cells, or Kupffer cells (Ramachandran and Kakar, 2018). The

acute hepatocellular necrosis is the most common expression of drug-induced hepatotoxicity

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(Chalasani et al., 2017). The pathological expression of hepatotoxicity may manifest itself in a

zonal pattern or it may produce more diffuse damage. Often, hepatotoxicants that cause damage

primarily through a reactive metabolite will produce hepatic necrosis in a zonal pattern since,

even though drug concentrations are highest in Zone 1, most CYPs, necessary for bioactivation,

are highest in Zone 3. For a hepatotoxicant such as APAP, CYPs in Zone 1 are at negligible

levels and cannot form the reactive metabolite. In contrast, in Zone 3, CYPs are at much higher

levels and form the reactive metabolite NAPQI, restricting covalent binding to this zone

(Bessems and Vermeulen, 2011).

2.3.2 Inflammation (immune) hepatitis

Sometimes, reactive metabolites can act as haptens, which bind to protein and lead to immune

responses (Uetrecht, 2017). Emerging evidence supports the hypothesis that the Kupffer,

sinusoidal endothelial, and stellate cells, and newly recruited leukocytes, play critical roles in

liver injury (Adams et al., 2018). The main function of the phagocytes is to remove dead cells

and cell debris in preparation for liver regeneration; however, toxic mediators generated by these

phagocytes can attack stressed hepatocytes (Jaeschke et al., 2020). Hepatic inflammation is a

common histopathology finding associated with a wide range of liver diseases (Reddy and

Sambasiva Rao, 2016). DILI is frequently associated with lymphocytic infiltrate, and the extent

of the inflammation determines the progression and severity of liver injury. Halothane is a

classic example that links reactive metabolites with immunoallergic hepatitis. It has all the

clinical hallmarks of an immunological perturbation, which include time of presentation, general

clinical features, and greatly enhanced reaction on re-exposure to the drug (Martin et al., 1993;

Zimmerman, 2019).

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2.3.3 Steatosis

Steatosis (fatty liver) is commonly caused by insulin resistance due to obesity (Saadeh, 2017).

Histologically, it is characterized as hepatocytes containing excess fat and appearing to have

multiple round, empty vacuoles in the cyoplasm. Steatosis can be induced by acute exposure to

many chemicals, e.g. carbon tetrachloride. Often, drug-induced steatosis is reversible and does

not lead to cell death. However, it may make the liver more susceptible to other insults (Sato et

al., 1981) or can develop into steatohepatitis (Saito et al., 2017), which is associated with

significant liver injury.

2.4 BIOMARKERS OF LIVER DAMAGE/TOXICITY

According to the recent version of FDA guidance for industry drug-induced liver injury:

Premarketing clinical evaluation, it is recommended to use a combination of four tests as DILI

biomarkers (FDA, 2018).

1. Alanine aminotransferase
Clinical chemistry data are routinely used for noninvasive monitoring of liver disease in

preclinical species and humans, and alanine aminotransferase (ALT) is the most widely used

clinical biomarker (Ozer et al., 2017). ALT is responsible for the metabolism (transamination) of

alanine and is found at much higher concentrations in the liver compared to other organs. When

hepatocellular injury occurs, the liver-abundant enzyme ALT will leak into the extracellular

space and enter the blood, wherein it shows a slow clearance rate with a half-life of

approximately 42 h (Ozer et al., 2017). The typical reference range is 7.35 IU/L in females and

10.40 IU/L for males (WebMD, 2013). An elevation of serum ALT activity is often reflective of

liver cell damage. Unfortunately, extrahepatic injury, such as muscle injury, can also lead to

elevations in ALT, making ALT not entirely hepato-specific. In addition, fenofibrate was found

12
to increase ALT gene expression in the absence of apparent liver injury (false positive) (Edgar et

al., 2018), and hepatotoxin microcystin-LR was reported to suppress ALT gene expression (false

negative) (Solter et al., 2000; Shi et al., 2018). Despite the fact that extrahepatic injury, such as

muscle damage or cardiac injury, can lead to increases in ALT, serum ALT remains the most

widely used and universally accepted biomarker for DILI. It is deemed to be the clinical

chemistry gold standard for DILI detection and has long been used at the FDA to facilitate

regulatory decision-making (FDA, 2018). Periodic monitoring of serum ALT is also a common

recommendation given to clinical practice for attempting to reduce the risks of liver injury when

patients are taking drugs with known DILI potential. Recent studies have suggested that

measuring the ALT isozymes, ALT1 and ALT2, may aid in differentiating the source of injury

(Ozer et al., 2017; Yang et al., 2018). ALT1 has been noted to be localized in human

hepatocytes, in renal tubular epithelial cells, and in salivary gland epithelial cells. ALT2, on the

other hand, is localized to human adrenal gland cortex, neuronal cells bodies, cardiac myocytes,

skeletal muscle fibers, and endocrine pancreas (Lindblom et al., 2017). Compared to ALT1,

ALT2 was found to contribute less to the total serum ALT activity and was probably a reflection

of mitochondrial damage (Yang et al., 2018). A novel immunoassay has been developed to

discriminate human ALT1 and ALT2 activities and might improve the ALT assay (Lindblom et

al., 2017; Ozer et al., 2017).

2. Aspartate aminotransferase

Based on the same rationale as ALT, aspartate amino-transferase (AST) has also been introduced

as a standard biomarker for DILI and is well accepted by clinicians (Ozer et al., 2017; Shi et al.,

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2018). Similar to ALT, AST is responsible for the metabolism (transamination) of aspartate.

Even though the sensitivity of the AST test is believed to be lower than that of ALT, it is still a

widely used liver biomarker. Owing to its more ubiquitous expression in extrahepatic organs,

such as the heart and muscle, AST is significantly less specific than ALT in detecting DILI (Ozer

et al., 2017). It appears that the ratio between serum ALT and AST activity is useful in

differentiating DILI from extrahepatic organ injury (Ozer et al., 2017), as well as to help in

diagnosing acute alcoholic hepatitis and cirrhosis with an AST/ALT ratio at 2:1. At least two

isoenzymes of AST have been found; one is cytosolic AST and another is mitochondrial AST

(mAST). The relative contributions of cytosolic AST or mAST to serum AST elevation have not

been critically assessed. In addition, it remains unknown whether AST isoenzymes are

susceptible to drug-driven induction or inhibition.

3. Alkaline phosphatase

Alkaline phosphatase (ALP) is an enzyme located in the liver, and its concentration in serum

increases when the bile ducts are blocked (Burtis and Ashwood, 2019). ALP is another

diagnostic biomarker recommended in the FDA guidance and is widely adopted by clinicians

(FDA, 2018; Shi et al., 2018). The Council for International Organizations of Medical Sciences

consensus criteria consider a more than twofold isolated elevation of serum ALP, or an

ALT/ALP ratio of no more than 2, as a key biomarker of cholestatic DILI (Ramachandran and

Kakar, 2018). It is noteworthy that conditions other than DILI, such as bone disease and

pregnancy, are also associated with ALP elevation (Reust and Hall, 2001). Therefore, ALP

should not be regarded as a specific biomarker of cholestatic DILI. The unique advantage of

ALP is that it is at least partially predictive of biliary obstructive types of liver injury when used

together with other DILI biomarkers.

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 4. Total bilirubin

Total bilirubin (TBL) is a composite of unconjugated (extrahepatic) and conjugated (hepatic)

bilirubin. Increased TBL causes jaundice and can indicate metabolism problems in the liver, for

example reduced hepatocyte uptake, impaired bilirubin conjugation, or reduced bilirubin

secretion (Wintrobe and Greer, 2018). Therefore, serum bilirubin concentration is a real liver

function biomarker, which measures the ability of liver to clear bilirubin from the blood as it

circulates through the liver. In contrast, serum transaminase levels indicate the rate of enzyme

release from injured cells and the Total bilirubinrates of enzyme degradation (mainly by the

mononuclear phagocyte system) (Senior, 2016).

5. Other existing hepatotoxicity biomarkers

Some liver function tests are not sensitive or specific enough to be used as diagnostic biomarkers

of hepatotoxicity, but are elevated in severe liver diseases. These biomarkers are used primarily

to confirm the liver toxicity and indicate the extent of damage to liver function. Conventional

biomarkers falling into this category are gamma-glutamyltransferase (GGT), serum total protein

(albumin), ammonia, cholesterol/triglycerides, fibrinogen, prothrombin time (prothrombin ratio

and international normalized ratio), and urobilinogen (Burtis and Ashwood, 2019; Cahill, 2019;

Wintrobe and Greer, 2018). Elevated serum gamma-glutamyltransferase (GGT) activity can be

found in liver disease and it has a similar profile as ALP in detecting disease of the biliary

system. Generally speaking, ALP is the first test for biliary disease and GGT provides a value to

verify that the ALP elevations are due to biliary injury (Betro et al., 2013b). Large quantities of

alcohol intake can significantly increase the serum GGT level. Slightly elevated GGT has also

been found to be associated with myocardial infarction and heart failure (Betro et al., 2013a). For

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both necrotic and obstructive liver diseases, serum total protein electrophoresis patterns will be

abnormal. In the acute stages of hepatitis, albumin will be low and the gamma globulin fraction

will be elevated due to a large increase in the production of antibodies. The globulin fractions

will be elevated owing to production of acute phase proteins. In addition, biliary cirrhosis may

cause beta globulin elevation. Ammonia is normally converted by the liver to urea, which is less

toxic. When liver dysfunction happens, failure of the conversion results in elevated blood

ammonia; however, the liver has a high functional reserve for ammonia conversion so it often

takes a significant amount of injury to alter ammonia levels. Increasing ammonia indicates end-

stage liver disease and a high risk of hepatic coma. Following hepatic uptake of lipoprotein

cholesterol (insoluble), a portion is enzymatically converted to bile salt (soluble). In acute

hepatic necrosis, triglycerides may be elevated due to hepatic lipase deficiency. When the bile

cannot be eliminated, cholesterol and triglycerides may accumulate in the blood as low-density

lipoprotein cholesterol. Because the liver is responsible for the production of blood coagulation

factors, the clotting time will be increased due to the impaired synthesis in the liver. However, it

is not a sensitive biomarker because it only happens at the late stage of liver disease.

Urobilinogen in urine is a colorless product of bilirubin reduction (Burtis and Ashwood, 2019).

In this respect, urobilinogen level has a similar role as bilirubin to indicate liver dysfunction.

Low urine urobilinogen may result from biliary obstruction or complete obstructive jaundice.

Because urobilinogen is formed in the intestine by bacteria, broad-spectrum antibiotic treatment

can significantly decrease its level due to the damage of intestinal bacterial flora (Wintrobe and

Greer, 2018).

3.0 SYMPTOMS, CAUSES, RISK FACTORS AND COMPLICATIONS OF LIVER

DAMAGE

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3.1 Symptoms of Liver damage

Liver disease doesn't always cause noticeable signs and symptoms. If signs and symptoms of

liver disease do occur, they may include:

 Skin and eyes that appear yellowish (jaundice)

 Abdominal pain and swelling

 Swelling in the legs and ankles

 Itchy skin

 Dark urine color

 Pale stool color

 Chronic fatigue

 Nausea or vomiting

 Loss of appetite

 Tendency to bruise easily

3.2 Causes of Liver Damagae

1. Infection

Parasites and viruses can infect the liver, causing inflammation that reduces liver function. The

viruses that cause liver damage can be spread through blood or semen, contaminated food or

water, or close contact with a person who is infected. The most common types of liver infection

are hepatitis viruses, including:

 Hepatitis A

 Hepatitis B

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  Hepatitis C

Immune system abnormality

Diseases in which your immune system attacks certain parts of your body (autoimmune) can

affect your liver. Examples of autoimmune liver diseases include:

 Autoimmune hepatitis

 Primary biliary cholangitis

 Primary sclerosing cholangitis

Genetics

An abnormal gene inherited from one or both of your parents can cause various substances to

build up in your liver, resulting in liver damage. Genetic liver diseases include:

 Hemochromatosis

 Wilson's disease

 Alpha-1 antitrypsin deficiency

Cancer and other growths

Examples include:

 Liver cancer

 Bile duct cancer

 Liver adenoma

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Other

Additional, common causes of liver disease include:

 Chronic alcohol abuse

 Fat accumulation in the liver (nonalcoholic fatty liver disease)

 Certain prescription or over-the-counter medications

 Certain herbal compounds

3.3 Risk factors of Liver Damage

Factors that may increase your risk of liver disease include:

 Heavy alcohol use

 Obesity

 Type 2 diabetes

 Tattoos or body piercings

 Injecting drugs using shared needles

 Blood transfusion before 1992

 Exposure to other people's blood and body fluids

 Unprotected sex

 Exposure to certain chemicals or toxins

 Family history of liver disease

3.4 Complications of Liver Damage

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Complications of liver disease vary, depending on the cause of your liver problems. Untreated

liver disease may progress to liver failure, a life-threatening condition.

3.5 Prevention of Liver Damage

To prevent liver disease:

 Drink alcohol in moderation. For healthy adults, that means up to one drink a day for

women and up to two drinks a day for men. Heavy or high-risk drinking is defined as more

than eight drinks a week for women and more than 15 drinks a week for men.

 Avoid risky behavior. Use a condom during sex. If you choose to have tattoos or body

piercings, be picky about cleanliness and safety when selecting a shop. Seek help if you use

illicit intravenous drugs, and don't share needles to inject drugs.

 Get vaccinated. If you're at increased risk of contracting hepatitis or if you've already been

infected with any form of the hepatitis virus, talk to your doctor about getting the hepatitis

A and hepatitis B vaccines.

 Use medications wisely. Take prescription and nonprescription drugs only when needed

and only in recommended doses. Don't mix medications and alcohol. Talk to your doctor

before mixing herbal supplements or prescription or nonprescription drugs.

 Avoid contact with other people's blood and body fluids. Hepatitis viruses can be spread by

accidental needle sticks or improper cleanup of blood or body fluids.

 Keep your food safe. Wash your hands thoroughly before eating or preparing foods. If

traveling in a developing country, use bottled water to drink, wash your hands and brush

your teeth.

20
  Take care with aerosol sprays. Make sure to use these products in a well-ventilated area,

and wear a mask when spraying insecticides, fungicides, paint and other toxic chemicals.

Always follow the manufacturer's instructions.

 Protect your skin. When using insecticides and other toxic chemicals, wear gloves, long

sleeves, a hat and a mask so that chemicals aren't absorbed through your skin.

 Maintain a healthy weight. Obesity can cause nonalcoholic fatty liver disease.

4.0 THERAPEUTIC MANAGEMENT OF LIVER DAMAGE

1. Treatment of ascites
Ascites affect 50% of patients with cirrhosis after 10 years of follow up. Diuretic therapy in

combination with dietary salt restriction (2 g/day) is the mainstay of treatment and only patients

who are truly resistant or intolerant of pharmacological therapy should be considered for large-

volume paracentesis and other therapies. In situations where there is a large reversible

component, the diuretic requirement might diminish with management of the underlying liver

disease.

Cirrhosis is an acquired state of secondary hyperaldosteronism. Spironolactone (an aldosterone

antagonist) blocks sodium reabsorption along the distal renal tubule, thus targeting one of the

key maladaptive pathways (the RAAS) of ascites accumulation.

Spironolactone produces a more profound natriuresis and diuresis compared with loop diuretics,

such as furosemide. In a randomised study, spironolactone was shown to be more efficacious

compared with furosemide, in that 95% vs 52% of patients showed a clinical response. British

and European guidelines recommend a starting dose of 100 mg/day titrating up to a maximum of

400 mg/day. Adverse effects include hyponatraemia, hyperkalaemia, gynaecomastia and

21
gastrointestinal disturbance. Anti-androgenic adverse effects include lethargy, decreased libido

and menstrual irregularity.

Amiloride
Amiloride is a potassium-sparing diuretic that tends to be used as a second-line agent when

spironolactone cannot be used because of gynaecomastia. It has a different mode of action to that

of spironolactone, exerting its effect on the proximal renal tubule and it can be more effective in

patients with low levels of plasma aldosterone. The dose range is 15–30 mg daily. Adverse

effects include hyperkalaemia, gastrointestinal disturbance and postural hypotension.

Furosemide
In advanced liver disease, renal perfusion becomes progressively more compromised and sodium

reabsorption from the proximal renal tubule becomes a more important factor, at which point

combination therapy with spironolactone and a loop diuretic might be required. Anecdotally,

furosemide is more effective when there is disproportionately more peripheral oedema.

Furosemide is typically started at 40 mg/day and titrated to a maximum of 160 mg/day. Adverse

effects include hyponatraemia, hypokalaemia, hypocalcaemia, acute kidney injury and postural

hypotension.

Whether to initiate spironolactone and a loop diuretic together or to adopt a stepwise sequential

approach (ie titrate to the maximum dose of spironolactone then add in furosemide) divides

international opinion. A randomised study recommended sequential treatment because it required

fewer dose adjustments, with no difference in the frequency of adverse events. Most patients in

this study had a first presentation of ascites. A more recent randomised study in patients with

22
cirrhosis, but without renal failure, reached the opposite conclusion, finding that combination

therapy produced a more rapid mobilisation of ascites, with a lower incidence of

hyperkalaemia. In this study, most patients had recurrent ascites.

Vasopressin 2 receptor antagonists

‘Vaptans’ are relatively novel drugs first used in patients with hyponatraemia with oedema.

Observations were made that, in some patients with cirrhosis, ascites also improved. Blockade of

vasopressin 2 receptors causes favourable haemodynamic effects; for example, vasoconstriction

of splanchnic and systemic vessels increases renal perfusion and improves ascites. Tolvaptan

used for 7 days in combination with standard diuretic therapy has been shown in a recent phase

III trial to improve ascites and serum sodium. Reported adverse effects in the tolvaptan group

were thirst, constipation, renal impairment, diarrhoea, urinary frequency, pyrexia, hepatic

encephalopathy, vomiting, insomnia, stomatitis and pruritus.

α-Adrenoceptor agonists
Clonidine is a centrally acting α2 adrenergic agonist used in the treatment of hypertension. In a

randomised controlled trial, clonidine combined with spironolactone was more effective than

spironolactone alone, with a more rapid clinical response and reduced diuretic

requirement. Adverse effects include postural hypotension, dry mouth, fatigue, drowsiness and

erectile dysfunction.

Midodrine is an α1 adrenoceptor agonist that increases peripheral vascular resistance and has

also been shown to increase renal perfusion and renal sodium excretion. There are randomised

23
trial data suggesting that both midodrine and clonidine were superior to standard medical therapy

in terms of controlling ascites.

Antimicrobials
Given that a pathogen is isolated in only 40% of patients with SBP, empirical antibiotics should

be started immediately. Cefotaxime, a third-generation cephalosporin, has been most widely

studied and has excellent coverage of the typical organisms. A dose of 2 g intravenously twice

daily is as effective as 1 g four times daily. Amoxicillin/clavulanic acid appears to be equally

efficacious compared with cefotaxime when examined in a single trial involving 80 patients. No

adverse effects were observed in either group.

Ciprofloxacin has similar success rates to the antimicrobials mentioned above and has good oral

bioavailability. Adverse effects of quinolones include tendonitis, tendon rupture and a prolonged

QTc interval. A head-to-head comparison of oral ofloxacin and intravenous cefotaxime showed

that there was no difference in outcome. However, this study was restricted to patients with

uncomplicated SBP and intravenous therapy might be more appropriate in severe cases. Most

hepatologists avoid the use of aminoglycosides because of the risk of nephrotoxicity in patients

at severe risk of hepatorenal syndrome (HRS) and other acute renal injury.

Rifaximin
Alterations in faecal microbial composition represent a potential modifiable factor. So far,

studies evaluating the use of the non-absorbable antibiotic rifaximin seem promising. Used as

primary prophylaxis in an open-label study of 404 patients, rifaximin reduced the risk of SBP

from 89% to 68% at 1 year compared with placebo. It is a well-tolerated drug and, in this patient

group, is an attractive, if costly option to provide dual prophylaxis against hepatic

encephalopathy (HE) and SBP.

24
Probiotics
Probiotics have been studied as an adjunct in combination with norfloxacin, but they did not

offer additional benefit in terms of reducing the incidence of SBP. Repopulating the colon with

less pathogenic organisms remains an attractive non-pharmaceutical approach to both SBP and

HE treatment.

Cisapride
In a rodent model, cisapride, a serotonin 5-hydroxytryptamine receptor 4 receptor agonist,

reduced bacterial translocation via effects on intestinal permeability and motility. However, it is

unlikely to be studied in humans because of its cardiac adverse effects. Therefore, other

prokinetics have a theoretical benefit, but have not been formally studied.

Vasoactive drugs (Terlipressin)

For patients not admitted to intensive care units, the pharmacological therapy of choice is

terlipressin. This vasopressin analogue reduces splanchnic vasodilatation with the therapeutic

aim of increasing the mean arterial pressure by 10–15 mmHg. A recent meta-analysis showed

that terlipressin significantly improves reversal and survival of patients compared with no

therapy or albumin alone.

Recommended dosing is 0.5–1 mg every 4–6 h, titrating to urine volume and serum creatinine.

Treatment should not extend beyond 14 days and should be discontinued when serum creatinine

is <133 μmol/l. Maximum benefit is seen when treatment is combined with albumin

administration at 1 g/kg for 24–48 h (up to a maximum of 100 g/day), followed by 20–40 g/day.

Terlipressin is contraindicated in those with severe arteriopathy and should be used with caution

in those patients with cardiac arrhythmias and coronary artery disease.

25
Noradrenaline
Although the efficacy and safety of noradrenaline is similar to that of terlipressin, it is usually

reserved for the intensive care setting. Noradrenaline is used as a continuous infusion of 0.5–3

mg/h and should be used with albumin as above. The aim is to increase the mean arterial

pressure by 10 mmHg.

Midodrine
Midodrine, also mentioned above, has the advantage of being an oral agent that is suitable for the

non-acute setting. Although midodrine has been shown to be effective in the management of

HRS, this was in combination with albumin and octreotide, and its efficacy in prevention of

recurrent HRS has yet to be explored.

Renal replacement therapy and transplantation

Renal replacement therapy is not associated with improved survival in patients with HRS, but

has been used, if indicated, as a bridge to liver transplantation. It is often advocated that it is

important to rule out other forms of acute liver injury that might respond well to renal

replacement and, although mortality remains high, there are intriguing suggestions that HRS per

se is not associated with a worse outcome in patients on dialysis, although published data remain

scarce.

Vasoactive drugs
The vascular physiology of the cirrhotic liver is complex, but can be best described as an

imbalance between intrahepatic vasoconstriction and systemic and splanchnic vasodilation.

Vasoactive drugs lead to an acute reduction in portal pressure by reducing splanchnic blood

flow, thus lowering portal pressure and the HVPG. Both vasopressin and somatostatin are both

hormones that can lower HVPG but the use of vasopressin is associated with unacceptable

26
cardiovascular vasoconstriction and somatostatin has only a transient effect requiring continuous

infusion. Both hormones have been superseded by synthetic analogues.

Octreotide and vapreotide, both long-acting analogues of somatostatin, have been used in the

treatment of variceal bleeding, particularly in those countries where vasopressin analogues were

unlicensed. Octreotide has been demonstrated to be as efficacious as terlipressin in improving the

control of variceal bleeding, but not when compared with endoscopy alone. Early administration

of vapreotide has been shown in one French study to reduce the need for blood transfusion and to

improve control of variceal bleeding.

Non-selective β-adrenoceptor blockers

Non-selective β-adrenoceptor blockers (NSBBs) are the mainstay of prophylaxis. NSBBs reduce

portal pressure by decreasing cardiac output and producing splanchnic bed vasodilatation. A

decrease in portal pressure is accompanied by a drop in intravariceal pressure and, hence, the risk

of bleeding is reduced. They are effective and safe used alone, or in combination.

Selective β-adrenoceptor blockers are suboptimal and not recommended. Recent trials of

carvedilol, an NSBB with mild anti-α 1-adrenergic effects, have shown a possible advantage

over propranolol in terms of a reduction in the HVPG, although it can cause higher rates of

systemic hypotension. Propranolol 40 mg twice daily, nadolol 20 mg/day or carvedilol 12.5

mg/day are typical starting doses.

Nitrates
Although they are potent vasodilators, meta-analysis has not demonstrated that nitrates lead to a

reduction in index bleed or rebleeding rates, but show a possible survival benefit for secondary

prophylaxis. Their role tends to be restricted to haemodynamic non-responders. It is difficult to

27
identify these patients and, hence, nitrates are rarely used. Adverse effects include postural

hypotension, headaches and sleep disturbance.

Simvastatin
The relative frequency of adverse effects with NSBBs and mixed results with nitrates have

stimulated a search for other agents to lower portal pressure. Simvastatin has been shown in a

small randomised trial to be superior to placebo in lowering HVPG over 1 month. The purported

mechanism is a reduction in intrahepatic vascular resistance. Statins are an attractive agent,

because they are well tolerated and are frequently prescribed to modify cardiac risk profiles,

which can coexist in liver disease (eg secondary to non-alcoholic steatohepatitis).

Laxatives
Non-absorbable disaccharides are the most widely advocated substance for the treatment of

Hepatic encephalopathy and its use features in most international guidelines. Despite this, the

evidence for their efficacy is limited. The removal of ammonia and other nitrogenous compounds

with bowel cleansing has long been advocated. The drug of choice is lactulose, which has further

theoretical advantages, including acidification of the colon promoting non-ammoniagenic

colonic flora and sequestration, and subsequent elimination of ammonia salts. Lactulose should

be given orally or nasogastrically in sufficient amounts (30–40 ml three to four times daily) to

promote at least three bowel evacuations per day. Lactulose enemas are of little extra benefit, are

difficult to administer and have largely been superseded by the use of phosphate enemas in

higher-grade HE. Despite their universal adoption, the evidence behind the use of non-

absorbable disaccharides is limited and a large meta-analysis found that it was of no

benefit. Subsequent authors have attempted to refute this and a lack of appetite for further

studies, as well as the low cost of this treatment approach, means that it is unlikely to disappear

28
from clinical practice. Long-term compliance with lactulose therapy is often hampered by

diarrhoea and gaseous distension, and dehydration and electrolyte disturbance can precipitate

further attacks of HE.

Phenylbutyrate
Phenylbutyrate is converted to phenylacetate in vivo and can reduce Hepatic Encephalopathy in

situations where glutamine levels are high by facilitating glutamine excretion (thus removing it

as a source of ammonia production). Although effective in some inborn errors of metabolism, its

use in chronic liver disease is not proven.

Sodium benzoate
Sodium benzoate increases the renal excretion of ammonia and, in one small study, demonstrated

equal efficacy to lactulose in the treatment of HE.

L-ornithine L-aspartate
The administration of L-ornithine L-aspartate (LOLA) treatment provides substrate for the

conversion of ammonia into urea and glutamate, which are less toxic and more readily excreted.

This approach has shown promise in patients with severe or refractory HE. Some studies have

demonstrated that a postprandial infusion of 20–40 g LOLA results in reduced blood ammonia

levels and improvement in HE grade, but there remain concerns about the induction of

hypoglycaemia and rebound hyperammoniaemia on cessation of treatment. Until these concerns

are fully addressed, LOLA should not be considered a routine treatment for HE.

29
5.0 CONCLUSION
Among the existing biomarkers, ALT and AST are general indicators of hepatocellular injury;

ALP is reflective of the cholestatic DILI pattern and elevated TBL is associated with increased

DILI severity Patients with liver disease should be cared for by a multidisciplinary team led by a

clinician with experience of caring for patients who have complex healthcare needs. The

generalist is likely to encounter these patients more frequently because of the increasing

incidence of liver disease. The information provided here should serve as a useful guide to the

standard drug therapies used in day-to-day practice for common manifestations of liver disease.

As understanding of the complex pathology underpinning fibrogenesis and portal hypertension

improves, novel therapies are awaited that could halt or reverse these processes to add to the

existing drugs in the current armamentarium against liver disease.

30
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