Novel clinical biomarker discovery for hepatotoxicity using

emerging omics technologies

Abstract
Biomarkers exist since last half a century but they have been applied extensively in
therapeutics and drug development since last 2 decades. Biomarkers play a very important
role in drug discovery and development. Biomarkers are useful in diagnosis as well as in
understanding the pathophysiology of the disease.

Biomarkers are utilized to predict the in vitro and/or in vivo hepatotoxicity of drugs,
therapeutic agents and xenobiotics. Currently, hepatotoxicity due to drugs is the most usual
reason behind liver failure. Biomarkers are important in classifying the test compound or
combination of test compounds that can cause liver toxicity and thereby can cause
unpredictable medical condition. Some of the technologies used to discover new
biomarkers and new tests are based on proteomics, genomics and metabolomics.

Keywords
Biomarkers, hepatotoxicity, proteomics, metabolomics.

Introduction
Biomarker or biological marker in general terms indicates the biological state. Biomarkers
are produced in body due to changes in metabolic processes. Any biological molecule which
is involved in metabolic process could be a biomarker except the xenobiotics compounds.
Any change in the normal metabolic process can result in accumulation or increased
clearance of normal metabolites which have the potential of being biomarker (Thatcher &
Caputo, 2008).

Biomolecules which are truncated, glutathionylated or cystinilated due to modification in

proteins can serve as biomarker. Such type of changes in the protein (enzyme) and
therefore in metabolism can lead to cellular changes and serve as the commonest type of
biomarker (Thatcher & Caputo, 2008). But such small alteration in protein (enzyme
function or concentration) can lead to large changes in products as well as substrate
concentration. Hence, as an outcome of alteration there will be a modification in
metabolism and these changes are easily detected. The alteration in the product
concentration is usually seen and serves as surrogate biomarker for the change (Thatcher
& Caputo, 2008).

Biomarker is defined as “a characteristic that is objectively measured and evaluated as an

indicator of normal biological processes, pathogenic processes, or pharmacologic
responses to a therapeutic intervention”.

Hepatotoxicty
Hepatotoxicity is a condition where liver undergoes damage because of toxicity due to
chemicals i.e. due to drugs. E.g. acetaminophen, clopidogrel.
Spectrum of Hepatotoxicity
Hepatocellular: steatosis (accumulation of triglyceride in liver leading to formation of
microvescular or macrovascular), necro-inflammatory (liver cells undergoes necrosis due
to injury and often inflammatory cells are involved as in hepatitis), phospholipidosis
(accumulation of phospholipid in liver), cirrhosis (liver injury where nodules are formed
and liver tissue is replaced by fibrous connective tissue) (Zimmerman,1999).
Cholestatic: biliary sclerosis (Zimmerman,1999).
Granulomas : it is drug induced and characterized by hypersensitivity and vasculitis.
Vascular lesions: vascular lesions develop Sdue to injury to vascular endothelium (Seef,
2006).
Neoplasms: due to prolonged exposure to some drugs carcinoma like hepatocellular
carcinoma or liver adenoma develops (Seef, 2006). Refer fig 1.

Figure 1. Chart showing different types of drug induced hepatotoxicity adapted from (Cameron, 1999)
Types of Biomarkers
A few types of biomarkers have been mentioned in the chart 1.

Chart 1. Showing Different types of biomarkers (Zimmerman, 1999)

Uses of Biomarkers

Chart 2. Showing use of biomarkers (Zimmerman, 1999)

Techniques for biomarker discovery

Some of the techniques commonly used for biomarker discovery, separation and
identification are Gas chromatography, HPLC, liquid chromatography, capillary
electrophoresis, protein chip arrays, SDS-PAGE etc. refer chart 3.

Chart 3. Showing the different techniques for the discovery, identification and separation of biomarkers
(Thatcher & Caputo, 2008)

Currently available biomarkers for hepatotoxicity

Some of the currently available biomarkers of liver injury are serum AST and ALT activities,
alkaline phosphatase (ALP) activity, total bilirubin, gamma-glutamyl transferase (GGT)
activity, bile acids, sorbitol dehydrogenase (SDH) activity.

Alanine aminotransferase (ALT) activity

ALT is detected in blood flow when hepatocytes are damaged. Serum ALT levels serves as a
reliable indicator of hepatotoxicity and frequency of false negative and false positive
signals are very low so ALT levels serves as accepted unoversal clinical biomarker of liver
damage. ALT are clinically reliable but they do not provide any information on
histopathology(Ozer et al., 2008).

Aspartate aminotransferase (AST) activity

AST is also released by injured hepatocyte but not specific to liver so it is not very marker
of hepatotoxicity. It can also be released by injured muscle or cardiac muscle cells. As AST
and ALT exists in a ratio, they are measured along with AST and any change in the ratio
indicates other organ damage (Ozer et al., 2008).

Alkaline Phosphatase
In humans, biliary sclerosis and cholestasis are associated with increased level of alkaline
phosphatase and GGT activity. Increased levels of alkaline phosphatase in serum indicate
drug induced cholestasis (Ozer et al., 2008).

Glutamate dehydrogenase activity (GLDH) as sensitive serum marker for

hepatotoxicty
GLDH is found primarily in liver and upon liver injury its concentration increases. GLDH
activity exclusively specific for liver and not affected by other organ damage like ALT. So
GLDH has the potential of being live biomarker(Ozer et al., 2008).

Serum F protein as serum marker for hepatotoxicty

Serum F protein is more of a liver specific protein and is a key enzyme involved in tyrosine
metabolism known as 4-hydroxyphenylpyruvate dioxygenase. Serum F protein was found
to be elevated in liver injuries and was more sensitive and precisely for liver than
traditional markers. It has been reported as biomarker of hepatotoxicity using RIA or ELSIA
assay (Ozer et al., 2008).

Research carried out to find novel biomarkers using proteomics, metabonomics and
genomics led to discovery of proteins that were predictive of hepatotoxicity. Some of these
markers are- Malate dehydrogenase, purine nucleoside phosphorylase, and paraoxanase 1
(Ozer et al., 2008). Refer to table 1.
Table 1. showing different types of biomarkers and their applications adapted from (Ozer et al., 2008)

Biomarkers of hepatotoxicity
Predicting drug-induced hepatotoxicity is very difficult as it is rare and most of the drugs
that cause hepatic injury in patients are not detected in experimental models. Clinical
symptoms of drug-induced hepatotoxicity vary a lot and often confused with other types of
hepatic diseases (Durham et al., 2004).

Some of the requirements of potential biomarker for hepatotoxicity would be:

A molecule which could be utilized to predict the both in vitro as well as in vivo
hepatotoxicity. The change in potential biomarker compound should be invariable,
persistent, and assessable and indicate the amplitude of toxic insult. Under in vivo
conditions it should be possibly non invasive or minimally intrusive. The biomarker should
not be costly to be compliant with high throughput techniques (Durham et al., 2004).

Biomarker discovery requires identification of marker. Once the biomarker has been
identified it undergoes validation and qualification of prioritized marker for wide
acceptance.

Proteomics in biomarker discovery

Proteomics involves multiple profiling of protein and profiling is aimed at computing the
corresponding difference of ample, particular proteins (Deprimo, 2007). Proteomic
profiling is very useful in identifying probable biomarker.

Identification of in vitro hepatotoxicity biomarker is done using cell culture and multi-
dimensional protein identification technology (MUDPIT) to analyze and identify possible
marker proteins (Deprimo, 2007). MUDPIT involves use of chromatographic techniques to
separate after which mass spectroscopy is done to identify and ascertain the protein
identity (Deprimo, 2007). MUDPIT data is analyzed to find any parallel change in the
amount of protein. The data obtained after analysis is used to prioritize the potential
biomarker candidates (Gao et al., 2005).

During prioritization, several array of hundreds of possible biomarkers is applied to a set,

this is done to find the proteins which are present at highest concentration in presence of
hepatotoxic compounds and proteins which are present at lowest concentration in
presence in non- hepatotoxic compounds (Gao et al., 2005). Marker are then validated,
checked whether they are specific to the disease or due to drug, effectiveness with which
they can be applied to clinical conditions from in vitro and the ease of assessing them.
Candidate markers left after prioritization are assessed against more varied group of
compounds for predicting hepatotoxicity (Gao et al., 2005).

For a prioritized candidate marker to qualify as biomarker it should be able to show

hepatotoxicity in in vivo models such as rats, mice, dogs (Gao et al., 2005). Refer fig 2.
Fig 2. Showing (A) steps for biomarker identification (B) steps for prioritizing candidate hepatotoxicity
markers on the basis of literature, disease biology and other criteria. The assays are developed to qualify the
prioritized biomarker. (C) shows initial discovery phase where peptides obtained from proteolysis are
analyzed using mass spectroscopy (Proteomics). Adapted form (Gao et al. ,2005)

Using proteomics profiling, researchers have discovered 14-3-3-Zeta and MIF protein as
biomarker for drug induced hepatotoxicity (Gao et al ,2005).

Metabonomics for biomarker discovery

Metabonomics is as powerful tool as proteomics and is concentrated on measuring
constituents of urine and blood which are low molecular weight using NMR spectroscopy
or LC/ TOF MS (Berger et al. 2009). Metabolite profiling is reliable and could be used to
find out the metabolites in conditions such as toxicity. Usually in control samples (urine or
blood) endogenic metabolites are detected as peaks and they need to be separated from
metabolites generated by drugs (Berger et al. 2009). The peak which corresponds to target
organ injury are selected and further analyzed to get potential marker for the target organ.
Refer fig 2.

Fig 3 showing steps involved in metabonomics studies adapted from (Berger et al. 2009)

Conclusion
In the above mentioned article it is proven that the biomarker have a unique role in drug
development process. Biomarkers are useful to provide information on the action of drug,
its target, helps in the detection of toxicity and also gives information about
pharmacokinetics and pharmacodynamics. The role of biomarkers is not only limited as
indicators of toxicity but they could be used for predicting, diagnosis and prognosis of
diseases. Since discovery of biomarker is a time consuming process it has to done along
with initiation of drug discovery. Since proteomics and metabonomics uses sophisticated
techniques like NMR, MS and MUDPIT which are highly specific and sensitive so the data
obtained is accurate and cover entire proteome or metabolome. Since metabonomics is an
emerging technology it is still catching up with proteomics and possibly in future we can
see more role of metabonomics in evaluating drug efficacy, toxicity and in regulation of
pharmaceutical industry.

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