Ecotoxicology (2006) 15:333340

DOI 10.1007/s10646-006-0069-1

Molecular Biomarkers: Their significance and application

in marine pollution monitoring
A. Sarkar D. Ray Amulya N. Shrivastava
Subhodeep Sarker

Accepted: 1 February 2006 / Published online: 5 May 2006

Springer Science+Business Media, LLC 2006

Abstract This paper presents an overview of the

significance of the use of molecular biomarkers as diagnostic and prognostic tools for marine pollution monitoring. In order to assess the impact of highly persistent
pollutants such as polychlorinated biphenyls (PCB), polychlorinated dibenzodioxins (PCDD), polychlorinated
dibenzofurans (PCDF), polynuclear aromatic hydrocarbons (PAH), tributyltin (TBT) and other toxic metals on the
marine ecosystem a suite of biomarkers are being extensively used worldwide. Among the various types of biomarkers, the following have received special attention:
cytochrome P4501A induction, DNA integrity, acetylcholinesterase activity and metallothionein induction. These
biomarkers are being used to evaluate exposure of various
species of sentinel marine organisms (e.g. mussels, clams,
oysters, snails, fishes, etc.) to and the effect of various
contaminants (organic xenobiotics and metals) using different molecular approaches [biochemical assays, enzyme
linked immuno-sorbent assays (ELISA), spectrophotometric, fluorometric measurement, differential pulsed polarography, liquid chromatography, atomic absorption
spectrometry]. The induction of the biotransformation
enzyme, cytochrome P4501A in fishes (Callionymus lyra,
Limanda limanda, Serranus sp., Mullus barbatus) and
mussels (Dreissena polymorpha) by various xenobiotic
contaminants such as PCBs, PAHs, PCDs is used as a
A. Sarkar (&) D. Ray
Marine Pollution Assessment and Ecotoxicology Group,
Chemical Oceanography Division, National Institute of
Oceanography, Dona Paula, Goa 403004, India
e-mail: anupam1_in@yahoo.com
A. N. Shrivastava S. Sarker
Department of Biotechnology, Birla Institute of Technology,
Mesra, Ranchi 835215, India

biomarker of exposure to such organic pollutants. The

induction of cytochrome P4501A is involved in chemical
carcinogenesis through catalysis of the covalent bonding of
organic contaminants to a DNA strand leading to formation
of DNA adduct. Measurement of the induction of cytochrome P4501A in terms of EROD (7-ethoxy resorufin
O-deethylase) activity is successfully used as a potential
biomarker of exposure to xenobiotic contaminants in
marine pollution monitoring.
In order to assess the impact of neurotoxic compounds
on marine environment the evaluation of acetylcholinesterase activity in marine organisms is used as a biomarker
of exposure to neurotoxic agents such as organophosphorus, carbamate pesticides etc. Metallothioneins (MTs) are
induced by toxic metals such as Cd, Hg, and Cu by
chelation through cysteine residues and are used in both
vertebrates and invertebrates as a biomarker of metal
exposure. The measurement of the levels of DNA integrity
in marine organisms such as Sea stars (Asterias rubens)
from the North Sea and the marine snails (Planaxis sulcatus) from the Arabian Sea along the Goa coast exposed to
environmental xenobiotic contaminants clearly indicated
the extent and the nature of pollution at the sampling sites
along coastal environment.
Keywords Molecular biomarker Cytochrome P4501A
enzyme induction DNA integrity Acetylcholinesterase
inhibition Metallothioneins induction Marine
pollution Marine mollusks
Introduction
Pollution of the marine environment is a global concern
owing to devastating effects of contaminants whose levels
are growing at an alarming rate. The occurrence of highly

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Sarkar et al.

persistent pollutants such as PCBs, PAHs, PCDDs, PCDFs,

TBT, nitroaromatics, phthalate esters, radionuclides, organochlorines pesticides [dichloro diphenyl trichloroetahne
(DDT), hexachlorocyclohexanes (HCH), aldrine, dieldrin,
endrine, heptaclor, hepox etc.] and toxic metals (Cd, Hg,
Ag, Co, Cr, Ni, Pb, Zn, Cu, etc.) in different compartments
of the marine environment have become a major threat to
the health of the marine ecosystem due to accumulation of
their residues in tissues of various species of marine
organisms (Binelli and Provini 2003; Holsbeek et al. 1999;
Sarkar 1994; Sarkar and Everaarts 1998; Sarkar et al. 1997;
Sole et al. 2001). The biomagnification of these pollutants
into higher trophic levels across the food chains results into
dispersal of pollutants far from their point of origin and
from areas of significant human activity, e.g. PCBs in deep
sea fish (Sole et al. 2001), in whales (Holsbeek et al. 1999)
and seals in oceanic waters and in polar beers (Holsbeek
et al. 1999; Kucklick et al. 2002) and seabirds (Shore et al.
1999) in the Arctic (Table 1). Thus it is quite obvious that
the impact of these highly toxic and persistent pollutants on
the marine ecosystem could be pronounced in terms of
their biological responses in organisms (Carajaville et al.
2000). In order to evaluate the impact of such pollutants on
the environmental quality it has thus become highly pertinent to carry out a rapid assessment of their deleterious
effects on the ecosystem (Wells et al. 2001). In this context, use of biological markers or biomarkers measured at
the molecular and cellular level is of immense importance
as sensitive early warning tools for biological effect
measurement in environmental quality assessment (de la
Torre Fernando et al. 2005; Carajaville et al. 2000; Che`vre
et al. 2003). This paper highlights the significance of such
biomarkers in terms of their application and current limitations. It is mainly focused on biomarkers at an advanced
stage of usage and development such as cytochrome P4501A (Domouthsidou et al. 2004; Isani et al. 2000), DNA
integrity (Siu et al. 2003; Burmeister et al. 2004; Everaarts
and Sarkar 1996), DNA adducts, acetycholinesterase
activity (Binelli et al. 2006; Escartin and Porte 1997;

Fuentes-Rios et al. 2005) and metallothioneins (MTs)

(Petrovic et al. 2001) which are specific to impact by
pollution and additionally whose application would benefit
from commercial and biotechnological development.
Biomarkers of marine pollution
Biomarker can be defined as the measurements of body
fluids, cells, or tissues that indicate in biochemical or cellular terms the presence of contaminants or the magnitude
of the host response (Bodin et al. 2004). A more generalized version which would also accommodate whole animal studies would include measurements on whole
animals and indicate in physiological, behavioural or
energetic terms (Ross et al. 2002; Magni et al. 2005).
There are molecular, cellular and whole animal biomarkerssome specific to pollutants (Table 2), others which
change in response to both pollutants and natural stressors
(Pfeifer et al. 2005). Such diagnostic and prognostic early
warning tests offer the potential of specificity, sensitivity
and application to a wider range of organisms (Fig. 1).
In view of the intensive problem of marine pollution a
suite of biomarkers such as cytochrome P4501A enzyme
induction, acetylcholinesterase inhibition, DNA integrity
and metallothionein induction received special attention for
biomonitoring of ecotoxicological impact of pollutants.
These biomarkers are of great significance to evaluate
exposure to and the effects of different contaminants
(metals, organic xenobiotics and organometallic compounds) and they can be measured using different molecular approaches (Ross et al. 2002). Marine bivalve
molluscs such as mussels are appropriate sentinel species
(Bodin et al. 2004) for most of the biomarkers studies
except for the induction of the cytochrome P4501A system,
which is measured in fish and the degree of imposex which
is known as a biomarker of exposure to TBT specifically
measured in certain gastropod molluscs (Lau et al. 2004;
Petrovic et al. 2001). The selected biomarkers indicate that
the organism has been exposed to pollutants (exposure

Table 1 Concentration ranges of some organic contaminants in marine organisms

Contaminants

Edible
clamsa

Crustaceanb
(crabs, shrimp)

Deep sea Fishc

(Mora moro) (Muscle tissue)

Seabirdd
(Herring gull)

Ringed seal
blubbere

Total DDT (ng/g)

Total PCBs (ng/g wet wt)
Total PAHs

0.63.4
1.615.4
2.124.5

1824
6.114
98180

7.412.6
13.824.0
0.20.6

0.218.8
0.4340
0333

34.8904
5016010
65.8140.7

Binelli and Provini, (2003)

Sericano et al., (2001)

Sole et al., (2001)

Shore et al., (1999)

Kucklick et al., (2002) and Holsbeek et al., (1999)

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Molecular Biomarkers

335

biomarkers) and/or the magnitude of the organisms response to the pollutant (effect biomarkers or biomarkers of
stress).
Significance of the use of biomarker

The most significant features of the use of biomarkers are

summarized as follows:

They identify that interactions have taken place between contaminants and the organisms and they measure sublethal effects.
Whereas chemical analysis can measure only a fraction
of the contaminants but reveals nothing about the adverse effects.
They detect the presence of both known and unknown
contaminants.
Sublethality and early detection of effects provides
warning signals for remedial or preventive action to be
taken.
They provide a temporally and spatially integrated
measure of bioavailable pollutants.
They attribute exposure and risks to environmental
pollutants, i.e. through the specificity of the responses
and the mechanistic understanding or the deleterious
consequences to the animal.
They have the potential to anticipate the changes
occurring in the ecosystem due to the impact of pollution.
They can be used in a predictive way, allowing the
initiation of bioremediation strategies before irreversible environmental damage of ecological consequences
occurs.
They can help establish the important routes of exposure by application to species from different trophic
levelsand thus can aid in prioritizing monitoring
schemes and strategies for intervention or remediation.
Toxicity bioassays can provide information on the relative toxicities of specific chemicals or effluents, but
extrapolation to the field situation is very difficult for

many reasons, including chemical speciation, absorption and uptake effects, accumulation through food
chains, and sublethal and modes of toxic action not
detected in short-term tests.
They can detect exposure to and the toxic effects of
parent compounds and metabolites of readily metabolisable and eliminated contaminants such as PAHs
and organophosphate.
They can integrate the toxicological interactions of
mixtures of contaminants such as mixtures of various
congeners of PCBs, PAHs, metals (Domouthsidou et al.
2004) to give an expression of the cumulative effect at
a particular molecular, cellular or tissue target (Siu
et al. 2003).
They can act as short-term predictors of long-term
ecological effects through integration into a suite of
measurements and understanding at different levels of
contamination (Behrens and Segner, 2005).
They are applicable to both laboratory and field studies.
The development of biomarkers involves laboratory
experimentation to first identify potential responses and
establish casual mechanisms before application to field.

Cytochrome P4501A induction

The cytochrome P4501A (CYP1A) subfamily plays a key
role in the biotransformation of contaminants like dioxins,
furans, polychlorinated biphenyls and polycyclic aromatic
hydrocarbons. The induction of CYP1A is triggered via the
cytosolic Ah (aryl hydrocarbon) receptor due to exposure
of organisms to such pollutants. However, some of the
metabolites may lead to enhanced toxicity and carcinogenicity (Parkinson 1995). The induction of CYP1A in fish
due to exposure to certain classes of organic contaminants
is used as a biomarker in pollution monitoring. The measurement of the induction of CYP4501A in terms of 7ethoxyresorufin O-deethylase (EROD) activities is utilized
as a potential biomarker for marine pollution monitoring

Table 2 Specific molecular biomarkers of biological effects of contaminants on marine organisms and the analytical technique of their
measurements
Biomarker
Cytochrome P4501A
(mainly hepatic)
DNAdamage

Observation

Indicator of exposure to organic contaminants such

as certain PAHs, PCBs, PCDDS and others
Indicator of exposure to, and damage by, organic
xenobiotics, including PAHs, PCBs and PCDDs
AChE inhibition
Indicator of exposure to orhganophosphorus, carbamate,
toxins, toxic metals such as cadmium, lead, copper etc.
Metallothioneins
Indicator of exposure to metals, including Zn, Cu, Cd
(hepatic and other tissues)
and Hg. Used in both vertebrates (mammals, fish) and
invertebrates (mollusks)

Analytical techniques
Measurement of enzyme activity (Fluorometric
or spectrophotometric), enzyme amount (ELISA)
DNA strand breaks measurement, DNA-adduct
formation by fluorometric measurements
Measurement of enzyme activity (spectrophometric
measurement, delta pH metric measurements)
Estimation of protein (differential pulse polarography,
liquid chromatography) boundmetal
(atomic absorption spectroscopy)

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(Fatima and Ahmad 2006). However, it is not always

possible to have a linear doseresponse relationship between the concentration of certain chemicals and CYP1A
content and/or activity in the natural environment as there
is a mixture of both inducers and inhibitors of CYP1A
which are likely to act simultaneously. Finally, other factors such us temperature, season or sexual hormones can
also greatly influence the responsiveness of the CYP1A
system in fish.
The biomarker approach is widely used both in vertebrates and invertebrates for environmental biomonitoring
because it can supply an integrated response for multixenobiotics contamination (Cajaraville et al. 2000). In recent years, there has been considerable interest in the use of
biomarkers within marine bivalves. The increase of ethoxyresorufin dealkylation (EROD) as biomarkers has been
commonly used in vertebrates for the persistent organic
pollutants (POPs) biomonitoring of aquatic environments
(Binelli et al. 2003). Extensive studies were carried out to
investigate changes on EROD in the freshwater bivalve,
Zebra mussel (Dreissena polymorpha) exposed to different

Fig. 1 DNA integrity (values

indicated on top of the bar) in
marine snail (Planaxis sulcatus)
along the Goa coast

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Sarkar et al.

pollutants (Arochlor 1260, CB-153 and CB-126, pp DDT,

chlorpyrifos, carbaryl) at laboratory conditions (Binelli
et al. 2006). They observed that a significant induction of
EROD activity occurred when mussels were exposed to
100 ng/l of PCB mixture of Arochlor 1260 and dioxin-like
CB-126, but this congener showed also a clear competitive
inhibition after 48 h of exposure. Binelli and his associates
(2006) investigated the contamination of the Italian subalpine great lakes (Maggiore, Lugano, Como, Iseo, Garda)
following the biomarker approach on Zebra mussel specimens collected from 17 sampling sites with different
morphometric characteristics and anthropization levels.
They observed that the planar compounds pollution, able to
activate the EROD activity, seems higher in some sampling
stations of Lake Garda, Como and Iseo (24 pmol/min/mg
proteins) than that measured in Lake Lugano (1.53 pmol/
min/mg proteins). Fuentes-Rios et al. (2005) determined
the EROD activity in shark (Schroederichthys chilensis) in
three bays of coastal environments of the South Pacific
Ocean as Biomarkers of PAH exposure. Behrens and
Segner (2005) investigated the ability of the CYP1A

Molecular Biomarkers

biomarker to discriminate the pollution status of small

streams receiving different levels of urban and agricultural
impact and showing low to moderate contamination by
AhR-binding PAHs and PCBs. They determined the
enzymatic CYP1A activity by measuring the 7-ethoxyresorufin-O-deethylase (EROD) activity. They observed that
EROD activities were the better discriminator than CYP1A
protein levels. The CYP1A response was consistent and
repeatable over the 5-year observation period from 1995 to
1999 (Behrens and Segner 2005). The study of estuarine
pollution showed that fish from the polluted Tyne estuary
had responded to all three classes of pollutants (PAHs,
heavy metals and estrogenic compounds) with elevations in
hepatic EROD, (George et al. 2004).
DNA integrity as a biomarker of pollution
The integrity of DNA can be greatly affected by genotoxic
agents due to DNA strand breaks, loss of methylation and
formation of DNA adducts (Pisoni et al. 2004). The level of
double strandedness with respect to total DNA determined
the effect of various genotoxic chemicals on the integrity of
DNA. In fact, DNA strand breaks may occur due to direct
DNA damage by an exogenous agent or may be produced
during DNA repair process or other physiologic responses
in the cell. Benzo(a)pyrene (BaP), a representative PAH is
oxidized to chemically reactive diolepoxide (BaPDE),
which subsequently interact with DNA to form both stable
and unstable adducts with DNA (Pisoni et al. 2004; Ching
et al. 2001). Each type of adduct may contribute towards
the eventual transformation of the cell (Pisoni et al. 2004;
Shugart 1988). Pisoni et al., (2004) investigated the impact
of environmental pollution at different stations along the
Taranto coastline (Ionian Sea, Puglia, Italy) using several
biomarkers of exposure and the effect on mussels, Mytilus
galloprovincialis. They studied the exposure the marine
organisms to PAHs by measuring DNA adduct levels and
benzo(a)pyrene monooxygenase activity [B(a)PMO]. Single strand breaks can be produced in cells by direct
chemical damage of DNA (Siu et al. 2003). The chemical
reactions that are known to generate single strand breaks
proceed by ionizing radiation, oxidation-reduction reaction
or photoreaction. Inhibition of DNA-repair synthesis (arabinosylcytosine or hydroxyurea) can also induces the single-strand breaks. Intercalating agents induced DNA
lesions due to action of topoisomerase enzyme like enzyme
in response to DNA-intercalation (Burmeister et al. 2004).
In order to assess the impact of persistent pollutants on the
marine organisms in the North Sea, Everaarts and Sarkar
(1996) determined the DNA damage in Sea star (Asterias
rubens) by measuring the level of integrity of DNA isolated from pyloric caeca of the organisms. Based on the
levels of integrity of DNA in Sea stars (A. rubens) the

337

North Sea coastal environment has been distinguished into

different clusters of pollution in accordance with the
intensity of pollution at different locations (den Besten
et al. 2001; Everaarts and Sarkar 1996). Recent studies on
the integrity of DNA in marine snail (Planaxis sulcatus)
from the Goa coast clearly indicated the impact of pollution at different sampling sites. DNA integrity (expressed
as F, in terms of double strandedness) in marine snails was
determined by measuring the DNA strand breaks following
the technique of partial alkaline unwinding assay (Everaarts and Sarkar 1996). It has been observed that DNA
integrity in marine snail (P. sulcatus) decreased significantly at the contaminated sites, Morjim (25% reduction),
Aguada (62% reduction), Dona Paula (60% reduction),
Betul (42% reduction) along the Goa coast from the normal
value (F=0.84) at the reference site, Tiracol. Such reduction in DNA integrity in marine snail can be attributed to
the extent of contamination of these sites by petroleum
hydrocarbons due to extensive shipping activities as well as
industrial discharge and dumping of waste materials into
the coastal water.

Acetylcholinesterase inhibition
Acetylcholinesterase (AChE) enzyme is responsible for
hydrolyzing the neurotransmitter acetylcholine into choline
and acetic acid. Acetylcholinesterase is usually located in
the membranes (e.g. of erythrocytes) of vertebrates and
non-vertebrates; the enzyme controls ionic currents in
excitable membranes and plays an essential role in nerve
conduction processes at the neuromuscular junction. The
inhibition of AChE is linked directly with the mechanism
of toxic action of organophosphorus and carbamate insecticides, viz. irreversible or reversible binding to the catalytic site of the enzyme and potentiation of cholinergic
effects. As an indicator of exposure to these compounds,
AChE and non-specific cholinesterase activities in blood
and tissues emerged as a diagnostic tool in the biomedical
area. The quantification of this enzyme has been applied to
laboratory and field studies with both vertebrates and
invertebrates to assess exposure to organophosphorus and
carbamate insecticides (Pfeifer et al. 2005; Magni et al.
2005). Marine bivalves such as oysters and mussels are
widely used as bioindicators of contamination in the
monitoring of pollutant effects. As filter feeders, these
species are known to be good general indicators of chemical contamination (Bocquene et al. 1997). Sturm et al.
(1999) reported the characteristics of cholinesterases from
brain and muscle tissue of three marine teleosts, Limanda
limanda, Platichthys flesus and Serranus cabrilla, to provide basal information for environmental monitoring in
coastal and marine areas.

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There is considerable diversity in the biochemical

properties and distribution of cholinesterases in aquatic
organisms, as well as in their sensitivity to anticholinesterase agents (Leinio and Lehtonen 2005; Escartin and
Porte 1997). Extensive studies on inhibition of cholinesterase activity by neurotoxic agents have clearly indicated
that it can be used as a tool to diagnose organophosphorus
pesticide poisoning in fish (Matozzo et al. 2005; de la Torre
Fernando et al. 2005). However, only a few studies have
examined the effects of organophosphate insecticides in
aquatic invertebrates (Perez Erkuden et al. 2004) and particularly in mussels (Lau et al. 2004), which are widely
used in pollution monitoring programmes (Cajaraville et al.
2000). Seasonal variation in mussel gill AChE and the
changes related to the use of pesticides in agricultural
activities have been reported (Leinio and Lehtonen 2005;
Lau et al. 2004; Bodin et al. 2004). Apart from the insecticides, a few other contaminants, including cadmium,
mercury, lead and copper were found to show anticholinesterase activity (Lionetto et al. 2003).
Metallothionein induction
MTs are cysteine rich peptides occurring mainly in the
cytosol and in the nucleus and lysosomes. They are nonenzymatic proteins with a low molecular weight, high
cysteine content, no aromatic amino acids and heat stability. The thiol groups (SH) of cysteine residues enable
MTs to bind particular heavy metals. MT-like proteins
have been reported in many vertebrates including many
species of fish (Olsson et al. 1998; Roeva et al. 1999) and
aquatic invertebrates mainly molluscs (Langston et al.
1998; Isani et al. 2000) and crustaceans (Barka 2000). MTs
can be induced by the essential metals Cu and Zn and the
non-essential metals Cd, Ag and Hg in both vertebrates and
invertebrates (Barka 2000). Thus the exposure of marine
organisms to toxic metals can lead to changes in several
biochemical processes that have the potential to be used as
biomarkers of exposure and therefore as early warning
signals of the presence of these particular contaminants.
The induction of MTs was reported in the oyster (Crassostrea virginica) (Roesijadi et al. 1997) and the mussel
(M. galloprovincialis, Mullus barbatus) (Petrovic et al.
2001; Lionetto et al. 2001). About 50 different species of
aquatic invertebrates, the majority of which are mollusks or
crustaceans are found to show response to induction of
MTs. Thus they are used for evaluation of pollution in the
marine environment and are seen as potential biomarkers
of metal exposure in mollusks and fish (Langston et al.
1998).
Tissues directly involved in metal uptake, storage and
excretion have a high capacity to synthesize MTs. In

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Sarkar et al.

aquatic organisms, these proteins have been identified in

the digestive gland (also termed the midgut gland or
hepatopancreas) and gills of molluscs and crustaceans
(Raspor et al. 2004; Mouneyrac et al. 1998)).
The role(s) of MTs in the physiological processes can be
explained by the fact that it is primarily involved in
essential and non-essential metal pathways. The activities
of MT can be observed in elements of both homeostasis
and detoxification. MT binds to excess of essential or
pollutant metals. Thus it protects the organism against
toxicity by restricting the availability of these cations at
detrimental sites. Some toxic metals such as Ag, Cd, Cu,
Hg and Zn have a high binding affinity for cysteine. In
some cases, MTs have high cysteine content (30%), low
molecular weight, heat-stability, and a strong affinity for
binding metals. In the case of mollusks MT have a high
glycine content differentiating them from most of the other
MT isoforms known today (Langston et al. 1998). MTs are
distinctively identified in only a few species of marine
molluscs (Patella vulgata, C. virginica, Patella granularis,
Mytilus edulis) (Isani et al. 2000; Mourgaud et al. 2002)
and recently in Ruditapes decussates (Simes et al. 2003)
and fish (Zhang and Wang 2005). The induction of MT has
been detected in organisms from contaminated areas or in
vitro experiments with exposure to metals such as Ag, Cd,
Cu, Hg and Zn in laboratory. The extent of MT induction
can vary between species and between tissues. The
sequestration of metals by MT is clearly evident in the
gills, digestive gland and kidney, indicating the significance of these tissues in uptake, storage and elimination of
metals (Bebianno and Langston 1998). MT induction in
molluscs can be estimated by different analytical methods
(differential pulse polarography, radioimmunoassay, spectrophotometry, ELISA) at the level of protein expression or
as a function of the metals bound to MT.
The use of MT as biomarker has been validated in many
in situ studies (Lionetto et al. 2001; Petrovic et al. 2001;
Rodriguez-Ortega et al. 2002; Ross et al. 2002; Mourgaud
et al. 2002). The results are generally positive, in particular
when the metal gradient of pollution is quite real. More and
more of studies in situ combine the quantification of several biomarkers, of which MT is only one (Carajaville et al.
2000; Petrovic et al. 2001; Blaise et al. 2002; Gagne et al.
2002; Che`vre et al. 2003; Gigue`re et al. 2003;
Domouthsidou et al. 2004).
In fact, mussels are used worldwide in environmental
pollution assessment and MTs in mussels are a good candidate to monitor metal contamination. The base levels of
MT in different mussel species such as M. edulis (Leinio
and Lehtonen 2005) and M. galloprovincialis (Mourgaud
et al. 2002; Petrovic et al. 2001; Raspor et al. 2004) are
quite similar (23 mg/g dry wt. in whole soft tissues).

Molecular Biomarkers
Acknowledgments The authors thank Dr S.R. Shetye, Director, NIO,
Dr M.D. Zingde, Dy. Director, NIO and Dr S.W.A. Naqvi, Dr C.G.
NaiK, scientists, COD for their constant encouragement and valuable
suggestions. They acknowledge the services of Mr Sham Akerkar and
Mr Arun Mahale for their cooperation in computation of figures.

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