Mutation Research 552 (2004) 1–17

Review

Genotoxicological studies in aquatic organisms: an overview

Awadhesh N. Jha∗
School of Biological Sciences, Plymouth Environmental Research Centre, University of Plymouth, Plymouth, PL48AA, UK

Received 24 June 2004; accepted 25 June 2004

Abstract

Substantial progress has been made in the last two decades to evaluate the impact of physical and chemical genotoxins in
aquatic organisms. This overview (a) summarises the major high lights in this stimulating area of research, (b) compares the
developments in this field with the developments in mammalian genotoxicological studies, where appropriate, (c) introduces 18
different articles presented in this special issue of Mutation Research in the backdrop of main advances and, (d) hypothesises
on future directions of research in this exciting field.
© 2004 Elsevier B.V. All rights reserved.

Keywords: Aquatic organisms; Eco-genotoxicology; Genetic ecotoxicology

1. Introduction approximately 70% of the human population resides

The quality of human life is directly or indirectly
dependent on the health, vitality and vigour of the en-
vironment around us. Abiotic and biotic components,
including air, water, soil, food and plants and animals
constitute our environment, and through complex
interactions, life is sustained. Deterioration in the
health of any of these components can have serious
knock-on effects on the continuity of life. The aquatic
environment, which covers two-thirds of the planet, is
inhabited by the majority of extant species in different
ecological niches; moreover many of them are impor-
tant sources of human food. It has been estimated that
∗ Tel.: +44 1752 232978; fax: +44 1752 232970.
E-mail address: a.jha@plymouth.ac.uk (A.N. Jha).
within 60 km of coastal regions and this percentage is
increasing [1]. In addition, a significant proportion of
the world’s largest cities are connected, either directly
or indirectly, to the marine environment. The aquatic
environment therefore plays vital roles for ecosystem
functioning, human health and civilisation.
As a consequence of human population growth and
industrial development, the production, consumption
and disposal of anthropogenic chemicals and wastes
continue to increase. The aquatic environment is often
the ultimate recipient of this increasing range of anthro-
pogenic contaminants, a large proportion of which are
potentially genotoxic and carcinogenic substances. For
example, during 1994 in the US alone, 22,744 different
facilities released 2.26 billion pounds of toxic sub-
stances into the environment, of which approximately

0027-5107/$ – see front matter © 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.mrfmmm.2004.06.034
2 A.N. Jha / Mutation Research 552 (2004) 1–17
one-third were rodent carcinogens [2]. Most of
these carcinogens were discharged as components of
complex mixtures (e.g. liquid effluents, air-borne emis-
sions, and solid wastes). It is therefore not surprising
that in recent years there has been increasing concern
regarding the genotoxic/carcinogenic hazards, and
reports on the occurrence of malignancies in aquatic
organisms [3–6,42,45,96]. The occurrence of malig-
nancies in aquatic organisms has also been correlated
with a higher incidence of tumours in human popu-
lations and increased use of herbicides [7]. Therefore,
natural biota might be used as sentinel or surrogate
species for the evaluation of genotoxic chemicals in
the environment and their risk to human health.
Although exposure of aquatic organisms to geno-
toxic contaminants could pose a risk to human health
via the food chain, there is also an ecological risk
that may lead to heritable mutations and loss in the
total genetic diversity (either intra or inter species),
with significant implications for long-term survival of
natural populations [8–10]. Mathematical models have
suggested that fixation of mildly deleterious mutations
could significantly contribute to loss of Darwinian
fitness and eventual extinction of small populations
[11,12]. However, although a correlation between air
pollution, incidence of lung cancer and loss of biodi-
versity has been demonstrated [13], there have been no
serious attempts to establish links between genotoxins-
induced mutations and extinction of natural biota. A
large number of mammalian germ cell mutagens have
been identified and many of these agents have also been
found to be somatic cell mutagens [14]. However, there
is a fundamental gap in our knowledge on the long-term
implications of these mutagens in the environment, in-
cluding effects on population survival and recruitment.
When viewed against the backdrop of enormous
reproductive surplus and natural wastage (due to
predation, desiccation, changing salinity, temperature,
acidity, etc.), it has been argued that any detrimental
effects resulting from a localised pollution incident
is likely to have a diminishingly small impact glob-
ally. This applies especially to invertebrates, which
constitute more than 90% of the extant species and
play important role in ecosystem functioning [8,15].
From a broader perspective, it is likely that combined
effects of other stresses (e.g. changing temperature,
acidity, salinity, oxygen concentration, etc.), loss of
habitat and pollution (including genotoxin exposure)
could produce detrimental conditions for population
survival. Indeed, global climate change and other
environmental stressors will have implications for
mutagenesis studies [16]. However, in a very short
geological time scale and in complex environmental
conditions, where the contaminants occur in all
probable combinations, it would be very difficult to
pinpoint particular contaminants causing population
extinction. Keeping aside the debate on the role of
genotoxic exposure on population survival and scien-
tific considerations in protecting the environment for
sustainability, maintenance of diversity, environmental
conservation and the fact that the health of human
populations is dependent upon other species, it could
be argued that the human population has both the moral
obligation and the practical necessity to protect other
species with which it shares its limited biosphere [17].
While substantial progress has been made in re-
cent decades in defining the significance of exposure
to genotoxins for human health risk assessment, there
has been only very limited development with respect to
understanding the impact of genotoxins on natural pop-
ulations. This is despite the fact that Boveri proposed
the relationship between chromosomal changes and the
origin of tumours as early as 1914, using developing
echinoderm embryos as a model [18]. In 1998, Mu-
tation Research published two special issues dealing
with the use of invertebrates and fish in genotoxicolog-
ical studies [19,20]. In these special issues, attempts
were made to record the advances in our capabilities
to evaluate the interaction of genotoxins with the ge-
netic material of aquatic biota and the development of
realistic biological methods to identify expressed geno-
toxicity in these organisms. These issues covered the
studies pertaining to biochemical and molecular end-
points of genotoxicity, including alterations in carcino-
gen metabolising enzymes, DNA adducts, expression
of oncogenes, use of fish in long-term carcinogenicity
assays, including molecular dosimetry studies with a
range of carcinogens and anti-carcinogens. The issues
also reflected the ecological significance of genotoxic
exposures in natural biota [19,20]. Since publication of
these issues, considerable progress has been made in
our capability to determine the impact of anthropogenic
stress and in our understanding of the fundamental
mechanisms involved in mutagenesis and carcinogen-
esis in aquatic organisms. In addition, there has been
growing public demand for clean water, and concern
 A.N. Jha / Mutation Research 552 (2004) 1–17
over the presence of a variety of anthropogenic con-
taminants with diverse mode of actions in the aquatic
environment. This has also led to significant regula-
tory developments across the globe. This special issue
aims to update recent advancements in our capabili-
ties to evaluate potential adverse impact of exposure
of genotoxins in the aquatic organism. Authors (repre-
senting different laboratories) in this special issue have
previously made significant contribution to this field.
It is hoped that this special issue will provide a sin-
gle source of current state-of-the-art information to the
readers and workers in the field.
2. Genomics and mutagenesis
One recent advance in the field of genetics is
the mapping of the human genome and other model
species. The technological developments associated
with this international project have further stimulated
research in our field to elucidate the nature, cause and
consequences of mutations. It is often not appreciated
that the idea of mapping the human genome grew from
the need to improve our ability to detect and study in-
duced and inherited mutations [21,22]. The field of mu-
tagenesis is indeed entering into an exciting phase and
it is hoped that ‘toxicogenomics’, the integration of
genomics, bioinformatics, and toxicology, will accel-
erate our understanding of the impact of anthropogenic
agents at the molecular level and assist in hazard and
risk assessment. With the aid of new technologies, un-
derstanding of changes in gene structure and expres-
sion would help us to evaluate the impact of anthro-
pogenic stress and the potential of natural biota to adapt
and survive in changing environments. Several critical
genes are being identified, isolated, and qualitatively
and quantitatively assayed to measure the impact of an-
thropogenic stress in the natural biota. It is envisaged
that gene expression profiling may reveal molecular
mechanisms of action of chemicals, despite the inher-
ent limitation of genomic and proteomic experiments,
which measure single end points (i.e. RNA or protein
levels), albeit for thousands of genes at a time [23].
In this context, an important approach has been to use
the gene expression profiling with DNA microarrays,
to screen large numbers of genes in response to envi-
ronmental changes. Although the time and high cost
for analysis and interpretation of the data are one of
3
the inherent drawbacks of this technology, the work-
ers in the field are showing growing awareness of this
type of tools as an alternative to classical toxicological
tests for rapid screening. However, one can appreciate
that currently, there is a significant knowledge gap in
our understanding of the molecular events that govern
toxicologically relevant outcomes. In ecotoxicological
terms, one of the major difficulties will be to link these
molecular effects with ecological consequences [24].
Two papers in this issue have used this technology to
measure gene expression in two different fish species
following exposure to different contaminants.
Genotoxic potential of pulp mill effluents has been
evaluated in vitro and in vivo by several workers
[25,26]. In this issue, Denslow et al. [27] exposed
spawning largemouth bass (Micropterus salmoides) for
short and long terms to various concentrations of pa-
per and pulp mill effluents to evaluate gene expres-
sion and steroid levels. Exposure scenarios were phased
such that exposure ended at the same time. Gene ex-
pression was mainly determined by differential display
(DD) reverse-transcriptase (RT)-polymerase chain re-
action (PCR). The data showed that a set of genes in
female fish that were up-regulated during vitellogen-
esis, were turned off by exposure. A comparison was
made with other fish exposed to estradiol to provide
a global overview of the changes in gene expression
associated with pulp mill effluent treatment and also
allowed the isolation of differentially expressed tran-
scripts. One of these transcripts, CYP1A was cloned,
characterised by sequencing and used to develop con-
ventional RT-PCR and real-time PCR assays, both of
which confirmed its induction in treated males. In ad-
dition, levels of circulating hormones and vitellogenin
were measured in exposed animals and a Northern blot
was used to quantify vitellogenin mRNA expression.
In recent years, there has been growing concern
over the presence of those contaminants which in-
terrupt hormonal metabolism in human as well as in
aquatic species [28]. Genotoxic and carcinogenic po-
tential of some steroidal hormones in humans are well-
documented [29]. It appears that in common with other
toxicants, endocrine disrupting agents may exert their
effects simultaneously via several mechanisms, either
in parallel or in series. In this context, tri-n-butyltin
(TBT), a known endocrine disrupting agent for some
gastropods, has also been shown to be genotoxic, cy-
totoxic, immunotoxic and teratogenic in some marine
4 A.N. Jha / Mutation Research 552 (2004) 1–17
invertebrates [30–32]. In addition to the studies by
Denslow et al. [27] on large mouth bass mentioned
above, in this issue Brown et al. [33], describe macroar-
ray experiments designed to test gene expression in
liver of male plaice (Pleuronectes platessa) as a func-
tion of time following exposure to ethynyl oestradiol
(EE2). The authors first obtained genes by suppression
subtractive hybridization and then prepared macroar-
rays on nylon membranes. Male fish were exposed to a
constant concentration (20 ng l−1 ) for 21 days and then
were transferred to clean water for 10 days. Interest-
ing observations suggested that there are two different
temporal expression patterns for estrogen-controlled
genes. One pattern evident for vitellogenin (VTG) 1
and 3, and zona radiate protein (ZRP) 1 and 3, peaks
by day 16 but then declined even though exposure con-
tinued for another 5 days. A second pattern evident
for Vtg2 and ZRP2 showed a maximum expression
from days 16–22 and then declined only when EE2
was removed. It is not clear why the expression of genes
initially up-regulated by oestrogenic exposure should
then decline in the continuing presence of EE2.The
authors speculate a feed-back mechanism which turns
transcription off or destabilises the transcripts. Such a
mechanism could operate at the molecular level by di-
rect effects on transcription factors or RNAses or may
influence hypothalamic pituitary signalling. Given that
the molecular mechanisms of endocrine disruption are
still being explored, there is certainly a need to evalu-
ate the gene profiling and potential mutagenic effects
of these agents in different aquatic organisms.
3. Oncogenes and tumour suppressor genes
Compared to mammals, there have been limited
studies pertaining to identification and expression of
oncogenes in fish and invertebrates [34–37]. Available
information suggests that several of these genes have a
high degree of nucleotide sequence and deduced amino
acid similarity with the mammalian gene counterparts
[38–40]. While some information is available in the
literature pertaining to structure and expression of
oncogenes in aquatic organisms, there is a paucity of
information on anti-oncogenes or tumour suppressor
genes. The significance of tumour suppressor genes
in human cancer is well documented. For example,
mutations in the p53 gene are known to play an
important role in over half of human cancers [41].
As mentioned above, although induction of tumours
in fish and many invertebrates are well documented
[3–6,42,45,96], and different fish species have been
used as models for carcinogenic studies, there are
only a few studies on the structure and function of
anti-oncogenes in aquatic organisms. The p53 gene
has been sequenced from different fish species for
potential use of mutations in the highly conserved
domains to identify potential genotoxins in the aquatic
environment. Comparisons of the deduced sequences
have revealed a high homology within the conserved
DNA binding domain among different fish species and
the very high sequence conservancy in fish is similar to
that in mammalian p53 [43]. However, it is important
to study the functional relationship between tumours
in fish and alterations in five conserved domains, four
of which (II–V) are found in the central portion of
the protein (specific DNA binding domain). Attempts
to find mutations in these regions have not been very
successful under in vivo conditions. Using a yeast
in vitro exposure system, Cachot et al. [44], have
identified and characterised both spontaneous and po-
tentially carcinogen-induced mutations in the flounder
(Platichthys flesus) p53 gene. Given that the detection
of p53 mutations under in vivo conditions has been dif-
ficult, this study attempts to describe a necessary step
to identifying potential ‘hotspots’ for benzo(a)pyrene
diol epoxide (BPDE)-induced mutation. Although the
situation in vivo may well differ from this in vitro
study, the study would certainly contribute to our
search for p53 mutations in non-human species.
Among invertebrates, the incidence of gonadal
tumours in soft-shell clams (Mya arenaria) near
contaminated sites is well documented [45]. However,
attempts to induce the tumours by laboratory expo-
sures to 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin,
TCDD) have been unsuccessful [45]. It was hypothe-
sised that tumour promotion in clams occurs through
the aryl hydrocarbon receptor (AHR) by mechanisms
comparable to those described for vertebrates. Char-
acterization of the clam AHR revealed that it does
not bind the prototypical AHR ligand, TCDD [46].
Subsequently, it was shown that exposure of clams to
dioxin under laboratory conditions leads to induction
of differential gene expression [47]. In continuation of
the earlier studies, Olberding et al. [48], in this issue, at-
tempted to further elucidate the mechanisms of tumour
 A.N. Jha / Mutation Research 552 (2004) 1–17
formation involving a protein with significant sequence
similarity to mammalian E6AP, a homologus to E6AP
carboxy terminus (HECT) E3 ubiquitin-protein ligase.
E6AP, in association with the high-risk human papillo-
mavirus (HPV) E6 protein, is known to be involved in
the abnormal degradation of the p53 tumour suppressor
protein in human cervical cancer. Adopting different
approaches, the authors have provided a detailed
functional study of a protein that may prove crucial
in understanding environmental non-AHR mediated
tumourigenesis in clams and other aquatic organisms.
4. Adaptation, resistance and
chemosensitisation
The term ‘stress’ is usually applied to situations
where the ‘fitness’ of individuals (or populations) is
reduced because of changed environmental conditions.
In ecotoxicological terms, this fitness would include
‘Darwinian fitness’ (i.e. growth, fertility and fecundity)
of populations. While stress is normally considered to
be environmentally induced, either by physical (e.g.
climatic factors and pollutants) or by biotic factors
(e.g. parasites, competitors, predators), it could also be
intrinsic in origin. Such intrinsic stress could originate
from an increase in homozygosity as a result of genetic
drift and/or inbreeding. These homozygous conditions,
in conjunction with natural selection will cause genetic
stress that could lead to a decrease in fitness [49].
Several mechanisms have been suggested, including
an increase in homozygosity for recessive deleterious
alleles and a decrease in the level of heterozygotes for
overdominant loci [50]. Other mechanisms, such as a
decrease in the level of genomic coadaptation or an in-
crease in genomic instability have also been suggested
to play important roles in decreased fitness [51,52].
From an environmental conservation point of view,
many small populations are subject to genetic stress due
to their small size resulting from drift and inbreeding.
Given that many species of natural biota have become
endangered, studies on the processes associated with
genetic stress are becoming increasingly important.
In contrast to genetic adaptation or resistance,
which is transmitted to future generations, organisms
may acclimatise physiologically at the individual level
and could become more resistant as a consequence
of earlier exposure [53]. This phenotypic plasticity,
5
which results due to epigenetic mechanisms, may
modify the toxic effects of contaminant exposure in
the natural biota. Distinguishing between these two
phenomena (i.e. genetic and epigenetic) is important
in predicting the potential short and long term costs to
affected populations. Wirgin and Waldman [54], have
attempted to summarise our current understanding
of the mechanistic basis of resistance or tolerance
in several North American fish species. The toxicity
mediated by a range of aromatic hydrocarbons was
also correlated to CYP1A1 and early life stage toxicity.
This particular study has suggested that while some
resistance could be genetic and hence transmitted to at
least the F2 generation, others could be physiological
due to some unidentified epigenetic mechanisms.
In addition, the authors also observed differential
resistance for different groups of contaminants (e.g.
aromatic hydrocarbons and halogenated hydrocar-
bons). They have also attempted to discuss resistance in
fish in the format of case studies providing new data on
resistance in tomcod and pointing out some of the costs
that have or have not been associated with tolerance.
The phenomenon of multixenobiotic resistance
(MXR) in aquatic invertebrates is well-documented
[55,56], and explains the apparent simultaneous resis-
tance of many aquatic invertebrates to multiple xenobi-
otics in the environment. The molecular mechanism of
MXR has been suggested to be similar to the well-
known multidrug resistance (MDR) phenomenon in
tumour cells resistant to chemotherapeutic drugs. As
with MDR, the MXR-mechanism in aquatic organ-
isms pumps out xenobiotics, including anthropogenic
chemicals and hence prevents their toxic effects. How-
ever, it has been suggested that many chemicals, called
chemosensitizers, may inhibit the function of this frag-
ile mechanism. As a result, the organisms could accu-
mulate xenobiotics, including genotoxins, and inflict
damage in the DNA and other cellular systems [56].
Although these chemosenitizers could play an impor-
tant role in determining the toxicity of anthropogenic
chemicals in the natural environment, and given that
there could be a large number of these chemicals in the
environment (of both natural and anthropogenic ori-
gin), their ecotoxicological significance remains to be
established. While the field of MDR is one of the in-
tensively researched areas in cancer biology, only a
limited amount of information is available in aquatic
organisms and most of this information is restricted
6 A.N. Jha / Mutation Research 552 (2004) 1–17
to invertebrate species. In this issue Smital et al. [57],
have further extended their study to elucidate the eco-
logical significance of MXR inhibition under in vivo
conditions. They have demonstrated that such inhibi-
tion could lead to enhanced production of mutagenic
metabolites in mussels and apoptotic cells in sea urchin
embryos following exposure to model MXR inhibitors.
They have also attempted to critically analyse the ques-
tions (a) whether the inhibition observed after expo-
sure to environmental samples is due to saturation of
MXR transport proteins or due to presence of potency
of MXR inhibitors and, (b) whether potent environmen-
tal MXR inhibitors are natural or man-made? These
novel studies will further contribute to our understand-
ing of molecular mechanisms of MXR, chemosensiti-
sation and their ecotoxicological significance.
5. Heritable mutations and transgenerational
effects
The transmission of genetic damage to offspring is
a primary concern for scientists and regulators in hu-
man health arena. For mammalian genetic toxicology,
guidelines have been presented which involve a weight-
of-evidence approach to determine the heritable muta-
tions hence identification of potential human mutagens
[58]. These guidelines broadly take into account the in-
trinsic mutagenic potential of chemicals, their ability
to reach the differentiating or differentiated germ cells
through the blood-gonad barrier and interact with germ
cell DNA to induce heritable mutations in mammalian
species. While several approaches have been adopted
and models have been developed for germ cell hazard
and risk assessment in rodents and in fruit flies (e.g.
mouse specific locus and Drosophila sex-linked reces-
sive lethal tests), not much progress has been made
to evaluate the potential hazards and risks from germ
cell mutagens in aquatic organisms. In addition, in re-
cent years, it has also been realised that exposure of
somatic cells to ionising radiation could give rise to
genomic instability which could be expressed after sev-
eral generations of the cell cycle [59]. However, little
is know about transgenerational effects in germ cells
which could be manifested in the offspring.
In this issue, Shimada and Shima [60], who
have worked extensively on the Japanese medaka
fish (Oryzias latipes) model, extended their work on
the transgenerational effects of radiation on somatic
mutation frequency in developing embryos. This novel,
non-mammalian system facilitates the mutants to be
analysed as mosaics during embryogenesis that have
both orange and white leucophores (pigment cells) in
+/wl heterozygous offspring. In other words, in this
system for germ cell mutagenesis, embryos that have
both wild-type orange leucophores and mutant white
leucophores are scored as mosaic mutants. Using this
system, the authors found a threefold increase in mu-
tant frequency of (non-irradiated) maternal alleles fol-
lowing irradiation of sperm or late spermatid but not
with irradiated spermatogonial stem cells. These so-
matic mosaics showed higher embryonic lethality, in-
dicating that mutants result from gross chromosomal
rearrangements that lead to lethality. Interestingly, the
authors also found that this indirect genetic instability
is not heritable in the F2 generation. This could be at-
tributed to large chromosomal rearrangements that sup-
press recombination and subsequent mosaic mutants in
the F2 . The data suggested that irradiation of sperm and
late spermatids can induce indirect mutations in F1 so-
matic cells, which led the authors to hypothesize that
genomic instability arises during F1 embryonic devel-
opment. This study in a model fish system supports
similar findings pertaining to untargeted mutation in
maternal alleles at tandem repeat DNA sequences aris-
ing in mice born from irradiated spermatozoa [61].
Polycyclic aromatic hydrocarbons (PAHs) are
known genotoxins and carcinogens. Contamination
of the marine and estuarine environments by PAHs
is a global phenomenon. An estimated total input
of 230,000 metric tonnes of PAHs is being released
annually in the marine environment [62]. There is no
indication that this input will decrease in the coming
decades, bearing in mind accidental or deliberate oils
leaks, spills, refinery operations and industrial dis-
charges [63,64]. PAHs are generally known to accumu-
late to high tissue concentrations in invertebrates at the
bottom of the food chains, where uptake rates greatly
exceed rates of metabolism and elimination, compared
with vertebrates, where metabolism and elimination
can cope with uptake [65]. The presence of PAHs in the
aquatic environment is of concern since they induce
acute toxicity in organisms and the presence of PAHs
in the sediments have been linked to liver neoplasms
and other abnormalities in benthic fish species [66,96].
PAHs (e.g. benzo(a)pyrene and dimethylbenzan-
thracene) have also been shown to induce dominant
 A.N. Jha / Mutation Research 552 (2004) 1–17
lethal mutations in mice following intraperitoneal
injection [67] and air pollution (which contains appre-
ciable amount of PAHs and particulate matters) has
been linked to higher rates of inheritable mutations in
herring gulls (Larus argentatus) and in laboratory mice
[68–70]. However, there have been virtually no studies
to evaluate inheritable mutations or transgenerational
effects in aquatic organisms following exposure to
PAHs, particularly among the invertebrates. This could
be due to lack of characterisation of genomes and
the reproductive strategies of the model invertebrate
species. Using a fresh water, parthenogenetically
reproducing clonal organism, Daphnia magna, in this
issue, Atienzar and Jha [71] have evaluated the repair
kinetics and transgenerational effects following expo-
sure to B(a)P using randomly amplified polymorphic
DNA (RAPD). The study suggested that while some of
the changes in the DNA profiles were reversed in the
offspring, indicating an efficient repair system, some
of the changes were transmitted to the offspring. The
exposure of B(a)P was also correlated with the repro-
ductive success of the organisms. A clear link between
induced genetic damage and potential population level
effects has been demonstrated, for which there is lack
of information in the literature. Despite the criticism of
the assay for its lack of reproducibility [72], the authors
claim that after rigorous optimisation of the PCR
parameters, the assay performs well, qualitatively and
quantitatively, and its reliability could be strengthen
as suggested by other workers [73,74]. In fact, several
studies have used PCR-based techniques to assess
genetic variation and changes in aquatic biota exposed
to different contaminants [75–78] and it has been
suggested that the RAPD assay can detect mutations
only if they occur in at least 2% of the DNA [79]. Given
that most of the natural biota are poorly understood
in terms of their genetic make-up (e.g. microsatellites,
minisatellites, karyotypes, cell turn-over rates, etc.), it
is likely that combined with robust experimental plans
and analytical tools, such molecular approaches will
play an increasingly important role in determining the
impact of genotoxins in the aquatic environment [80].
6. In vitro studies in genetic ecotoxicology
The fundamental role of genotoxic agents as the
primary basis for the development of malignancy
7
in mammalian species is now well accepted. This
knowledge has led to a tiered strategy for the detection
of mammalian genotoxins in order to protect human
health. It is therefore logical to utilise the insights of
mammalian genotoxicology in genetic ecotoxicology.
In this context, the three-tier approach of mammalian
genotoxicology initially utilises a suite of in vitro tests
(e.g. bacterial assays, mammalian cell cultures) for
evaluating the inherent genotoxicity of a substance
followed by expressed genotoxicity under in vivo
conditions. Finally, if the substance is a somatic
mutagen to mammals in vivo, tests are conducted to
measure the interaction of the substance with germ
cell DNA or other heritable mutations under in vivo
conditions [58]. For eco-genotoxicology, or genetic
ecotoxicology, a clear distinction is required between
testing and environmental monitoring [15]. For testing
the intrinsic genotoxic potential of environmental
samples, several bacterial tests (e.g. Ames test, UmuC
test, Mutatox® and SOS chromotest, etc.) are being
employed for hazard assessment [2].
The use of in vitro systems in ecotoxicological stud-
ies provides the opportunity not only for extrapolation
from in vitro to in vivo systems, but also information
on biological responses at higher levels biological or-
ganisation [81]. Fish play a major role for the flow
of energy in aquatic ecosystems, are exposed continu-
ously to contaminants in the natural habitat and con-
stitute an important part of human diet, especially in
coastal regions. In this respect, cells derived from liver,
gonads and skins (e.g. primary hepatocytes, rainbow
trout gonads and skin) of fish species have been exten-
sively used to investigate toxicity of several reference
genotoxins and environmental contaminants, such as
organic extracts of sediment samples [81–85]. These
cells retain some important traits of fish (e.g. poik-
ilothermic behaviour, unique xenobiotics metabolism
and low rate of repair mechanism) [84]. In addition,
due to ethical reasons, there is a move to minimise
the use of vertebrates, including fish for experimental
purposes. Furthermore, due to the lack of established
cell lines from aquatic invertebrates, there will be in-
creasing use of fish cells in ecotoxicological studies.
In this context, rainbow trout gonad cells (RTG-2), a
permanent cell line capable of metabolising the xeno-
biotics without the need of an exogenous metabolic
system has been shown to provide reproducible results
in inter-laboratory validation tests for cytotoxicity [81].
8 A.N. Jha / Mutation Research 552 (2004) 1–17
Compared to mammalian cells they have been shown to
be more sensitive for the induction of genetic damage
[86]. In this issue, Castano and Becerril [87], using ran-
domly amplified polymorphic DNA (RAPD), as used
earlier by Atienzar and Jha, in water fleas, D. magna
[71], have extended their study on rainbow trout gonad
(RTG-2) cells to evaluate the genotoxic potential fol-
lowing exposure to a range of concentrations of B(a)P
for different periods. Qualitative and quantitative anal-
ysis of RAPD bands suggested that this in vitro system
is useful in evaluating genotoxic effects of direct as
well as indirect acting mutagens following either acute
or chronic exposures.
7. In vivo studies and biomonitoring
While bacterial or in vitro systems could be used
as screening tools to define the intrinsic genotoxicity
of a substance, definitive ecotoxicological risk assess-
ments should consider the expressed genotoxic activity
in ecologically relevant organisms. These will take into
account environmentally realistic routes of exposure,
the effects of metabolism and DNA repair efficiency
[88]. However, despite growing concern over the pres-
ence of genotoxins in the aquatic environment, there
is a lack of adequately validated test methods, which
could be used effectively to evaluate genotoxicity, and
associated toxicity, in aquatic organisms under envi-
ronmentally relevant conditions. The development of
such in vivo test systems is also essential in provid-
ing a scientific basis for comparing the relative risks of
man-made versus natural genotoxins [88], given that
large number of marine plants and animals produce
very potent genotoxins [89].
Biomonitoring for genotoxic risk evaluation is
defined as frequent or even continuous gathering of
information in a given population that is relevant to
that population’s health risk [90]. The information
gathering ranges from information on exposure,
carcinogen-induced phenotypic changes (e.g. DNA
adducts or gene mutations), neoplastic lesions and
cancer occurrence. The main objectives of population
monitoring in cancer research is therefore to identify
carcinogenic mechanisms by obtaining more precise
estimates of the extent to which different factors
(e.g. environmental, life style, genetic susceptibility)
contribute to human cancer burden and potential germ
cell mutations [90]. This information ultimately helps
to prevent cancer and reducing the risk of inheritable
mutations. In contrast to human health risk assessment,
until recently, the integral effects of contaminants
relied heavily on analytical techniques, in particular for
environmental monitoring and effluent discharge com-
pliance. However, simple detection of environmental
contaminants by these techniques is not sufficient un-
less their biological effects are also properly evaluated.
In addition, genotoxins in the environment can occur
as complex mixture, and the risk associated with such
mixtures cannot be adequately anticipated on the basis
of the effect and behaviour of individual components.
Since biological systems are the target of toxicant
action, they could provide important information
which is not readily available from chemical analyses
of the environmental samples [88]. Thus, in view of
the limitations of chemical monitoring, the importance
of biological monitoring is being widely recognised.
Several manuscripts in this issue deal with in vivo
laboratory and field studies in order to evaluate the
potential impact of contaminants in different aquatic
organisms. The fundamental difference between
biomonitoring studies involving human and natural
populations appears to be that while the former is ques-
tion driven, the later is technique driven. In addition,
most of the techniques used for aquatic species have
been adopted from human studies. Bolognesi et al.,
in this issue [91], studied the effects of environmental
contaminants in Mediterranean mussels, Mytilus gal-
loprovincialis, at four different sites along the Ligurian
coast, Italy. These mussels included native as well as
transplanted (caged) specimens. Induction of micronu-
clei and DNA strand breaks (using alkaline elution
technique) were used as biomarkers of genotoxicity. In
addition, the levels of polycyclic aromatic hydrocar-
bons (PAHs) and heavy metals (e.g. mercury and cad-
mium) were also analysed. The wild mussels showed
accumulation of contaminants as a function of pollution
gradient and this was found to be correlated with the in-
duction of micronuclei. The mussels showed seasonal
variability for the induction of genetic damage, caged
mussels showing higher damage than the wild samples.
Using single cell gel electrophoresis (Comet assay)
and DNA adduct analysis (32 P-post labelling), Win-
ter et al. [92], evaluated genotoxic effects in feral and
caged chub (Leuciscus cephalus) from three rivers with
different water quality (due to presence of organics,
 A.N. Jha / Mutation Research 552 (2004) 1–17
metals and pesticides) around Birmingham, UK. In
general, elevated levels of DNA damage was recorded
with a decrease in chemical water quality, in both feral
and caged animals indicating an impact of chronic pol-
lution. Recorded seasonal DNA adduct data suggested
a higher degree of damage in the feral compared with
caged animals. Apart from chemical parameters, both
the studies of Bolognesi et al. [91] and Winter et al.
[92] suggest that both in fish and mussels, seasonal
variation could play an important role in the observed
genotoxic effects in freshwater as well as in the marine
environment.
Lyons et al. [93], have carried out a field study
around the coastal waters of the UK in which European
flounder (P. flesus) were sampled from different estu-
aries. Hepatic DNA analysis of the samples suggested
that fish populations in certain contaminated UK estu-
aries are being exposed to complex mixtures of geno-
toxic and potentially carcinogenic contaminants. This
supports a previous study by the group which demon-
strated that European flounder populations inhabiting
industrialised UK estuaries are exposed to high lev-
els of sediment-bound PAHs, and that a proportion of
the bioavailable PAHs are metabolised to carcinogenic
substances [94]. In the present study, these authors have
also attempted to correlate the DNA adduct formation
with histopathological biomarkers. Although it was dif-
ficult to associate the histological lesions with specific
sites, the contaminated sites showed higher prevalence
of these damages. Given that DNA adduct formation
is a repairable damage, the study emphasises the need
for complementing chemical data and effects at higher
levels of biological organisation (e.g. histopathological
biomarkers), when the adduct analysis is carried out.
It has been shown that DNA adduct formation is asso-
ciated with the incidence of hepatic lesions, including
neoplasms, in fish at contaminated sites [95]. There is
also strong evidence of a cause-and-effect relationship
between exposure to genotoxins in sediment and water
and neoplasm epizootics in wild fish populations [96].
Frenzilli et al. [97] have attempted to evaluate the
levels of DNA strand breaks (using Comet assay) and
apoptotic cells in Eelpout (Zoarces viviparous), a non-
migratory fish species, sampled from Goteborg har-
bour area, Sweden, following a oil spill accident. The
genetic damage and apoptotic cell analyses were car-
ried out in nucleated erythrocytes. In addition, levels of
PAHs exposure were also measured by analysing PAH
9
metabolites in the fish bile. The study suggested a high
level of damaged DNA, complemented by a peak in
the bile PAH metabolites in samples from the most
impacted site, 3 weeks after the oil spill but this dam-
age was found to recover 5 months after the accident.
However, the samples collected from the middle part of
the harbour, which is chronically impacted from con-
tamination, did not show any recovery. It is interesting
to compare this study with a similar study following
the Sea Empress oil spill accident in 1996 in Milford
Haven, Wales, UK. The study by Harvey et al. [98]
indicated that oil contamination did not induce any de-
tectable elevations in DNA adduct levels in the inverte-
brates (i.e. Halichondria panacea and Mytilus edulis),
but the contamination did appear to induce adducts
in the fish species (i.e. Lipophrys pholis, P. platessa
and Limanda limanda). Interestingly, in common with
the study by Frenzilli et al. [97], data obtained 12–17
months after the spill suggested that the affected species
recovered from the oil contamination [98]. Bearing in
mind that both DNA strand break and adduct formation
are repairable damages and, biomarkers of ‘exposure’
rather than ‘effects’, it remains possible that such expo-
sure could lead to mutations in other somatic and germ
cells. The selection of robust, multiple end points in
phylogenetically different groups of organism should
therefore be considered to get a holistic view of po-
tential impact of such accidents when planning a long
term studies.
While we are attracted to study contaminated sites
to evaluate potential damage in the exposed biota, in
order to elucidate and solve many of the challenges
in eco-genotoxicology (e.g. identification of physical,
chemical and biological factors influencing the
induction of genetic damage under natural conditions;
identification of sensitive species and life stages;
individual variability and genetic susceptibility, etc.),
there is also a need for the monitoring sites (or sam-
ples) which are considered to be pristine, undisturbed
or have moderate levels of contaminations compared
with heavily contaminated sites or samples [88]. This
is considered to be necessary in order to generate back-
ground and historical control data while evaluating
the impact of anthropogenic contaminants on natural
biota [99]. Monitoring of such sites is also necessary
for many estuaries in industrialised areas that require
periodic maintenance dredging of navigable water-
ways to sustain recreational, commercial and military
10 A.N. Jha / Mutation Research 552 (2004) 1–17
shipping [100,101]. Akcha et al. [102] in this issue,
investigated the influence of both biotic (e.g. age,
sex) and abiotic (e.g. sampling site) factors on the use
of genotoxicity biomarkers (i.e. DNA strand breaks
and adducts) in different cell types (i.e. erythrocytes
and heapatocytes) for environmental monitoring. The
authors have highlighted several factors, including
seasonal variability, as carried out by Bolognesi
et al. and Winter et al. [91,92], which require due
consideration when conducting field sampling using
flatfish species (L. limanda).
Interspecies variability for toxic and genotoxic
response is well known and is attributed to the differ-
ences in uptake, accumulation, metabolism, excretion,
sequestration and repair efficiency. Compared to
laboratory studies, the situation in the field becomes
more complex due to several confounding factors. It
is well accepted that to protect human and ecosystem
health, it is necessary to develop sensitive assays and
to identify responsive cells and species, and their life
stages [86]. Bihari and Fafandel [103], in this issue,
have determined the spontaneous and B(a)P induced
DNA single strand breaks in a range of aquatic species,
and as mentioned earlier, supports the adoption of a
multi-species approach in designing biomonitoring
programmes. While evaluating the DNA strand breaks,
the authors also obtained different elution rates for
DNA from different species at different pH, suggesting
that DNA denaturation under alkaline conditions are
species-specific. This has important implications
for standardising protocols for biomonitoring using
different species.
For environmental monitoring purposes, one of the
major biological challenges has been to bring potential
test organisms into continuous routine culture. This is
especially important when sound genetic analysis is to
be carried out. In this context, the use of black slug
(Arion ater), a terrestrial gastropod species, which has
been used traditionally as an indicator for metal pol-
lution, has been evaluated by Hamers et al. in this is-
sue, for PAHs monitoring in the environment [104].
The organisms, collected from field were reared in the
laboratory and orally exposed to B(a)P for different
periods of time (i.e. 3 and 119 days). B(a)P hydroxy-
lase (BPH) and bulky DNA adducts were analysed in
the digestive glands and kidney cells respectively. Af-
ter a short exposure to a relatively high B(a)P doses,
a dose-dependent increase for BPH activity and bulky
DNA adduct levels was found. The authors however
could not find increased BPH activity and bulky adduct
levels following long-term exposure of environmen-
tally relevant doses of B(a)P. Based on this lack of
response, the authors conclude that this species is not
sensitive to B(a)P exposure and, therefore, not suitable
for monitoring environmental exposure to PAHs. Given
that there is a lack of information pertaining to the use
of terrestrial gastropods for environmental monitoring
studies, this is valuable information for the workers in
the field.
The marine ecosystem is the largest system in terms
of size and complexity. Compared to human popu-
lations, the aquatic organisms are distributed and re-
stricted to well-defined and distinct occupancy, along
the vertical and horizontal habitats of the ecosystem.
In these ecological niches, it is hard to find a sin-
gle species, representative of all. Although the deep
sea environment is an important source for food and
sink for anthropogenic wastes, it is most poorly stud-
ied ecosystem, primarily because of logistic problems
to reach these places. In this context, the members
of hydrothermal-vent communities in the deep-sea are
well-adapted to survive in one of the most naturally
contaminated (e.g. heavy metals and radionuclides,
etc.) environment on this planet [105]. Given the ex-
treme nature of the vent environment, it is of funda-
mental interest to study the spontaneous and induced
genetic damage in these organisms. Dixon et al., in
this issue [106], have reported interesting findings re-
lated to adaptation and the effects of hydrostatic pres-
sure change on DNA integrity in blood and gill cells
of a hydrothermal-vent mussel species (Bathymodio-
lus azoricus) collected from the Mid-Atlantic Ridge,
an important study area for vent research in European
waters. Based on their findings, the authors suggest that
future studies should concentrate on populations living
at greater depths but only after significant advances are
made in logistics of collecting the samples which are
subjected to decompression stress during retrieval and
subsequent experimentation.
8. Risk assessment and environmental
management
In this issue we have seen several examples of
how the science is being used to assess the impact of
 A.N. Jha / Mutation Research 552 (2004) 1–17
anthropogenic stresses under laboratory and field sit-
uations in aquatic species. It is however a challenging
task to translate these information to assess the health
of the ecosystem as a whole. Determination of the
‘health’ of the ecosystem is therefore rapidly emerg-
ing as a new branch of science which focuses on the
evaluation of health status, maintenance (i.e. sustain-
ability) of a healthy one and treatment (i.e. restora-
tion) of an unhealthy ecosystem [107]. One of the
most popular tools in our efforts to assess the po-
tential impact of anthropogenic stress is ecological
risk assessment. Because of the knowledge gaps in
our understanding of ecological significance of anthro-
pogenic stressors, there is a lack of consensus for an ac-
ceptable, comprehensive decision-making framework
which could establish the roles of science and policy
in formulating environmental management principles.
This has generated several myths that have numerous
implications and important consequences for environ-
mental decision making [108]. Some of these myths
include selection of sentinel or sensitive species, util-
ity of acute versus chronic data, extrapolations from
laboratory to field situations, etc. While science is
playing an important role in developing techniques
to assess the impact on the environment, at the mo-
ment it cannot address all the issues surrounding
environmental risk management [108]. Attempts are
however being made to distinguish between these
myths with the realty and different models are being
proposed to evaluate ecotoxicological risk following
exposure to genotoxic agents in different environment
[109].
In this issue, Moore et al. [110] present their views
on this very complex and articulated topic for inte-
grated environmental management. The view, based
on the core information from published work from
laboratory and field studies, recognises the limitations
and strengths of the literature and proposes the use
of diagnostic clinical-type, laboratory-based ecotox-
icological tests or biomarkers linking different lev-
els of biological organisation, using sentinel animals
as integrators of pollution. The information generated
could be used by decision makers and environmen-
tal managers. The authors also hypothesize that the
proposed approach would benefit with the develop-
ment of computational simulation models of cells, or-
gans and animals in tandem with available empirical
data.
11
9. Future directions
Substantial progress has been made in the last two
decades in the field of aquatic genotoxicology. Most
of the early developments had involved analysis of
chromosomal aberrations in selected organisms, which
included fish, bivalve molluscs, polychaete worms and
echinoderm larvae [111–122]. Most of these studies
probably benefited from the techniques developed
to study the chromosome number and morphology
in 1960s and 1970s for taxonomic and evolutionary
studies [15]. However, except for the actively dividing
cells of embryo-larval stages of certain marine inver-
tebrates [15,30,32,88,116,117], the successful use of
chromosomal aberrations based on metaphase analysis
in aquatic organisms has been very limited. This is
apparently because of low mitotic rates in tissues (e.g.
gill, spleen, etc.) of adult individuals, relatively small-
sized and high chromosome numbers in most of the
studied aquatic organisms and lack of well-established
cell lines for invertebrates [15]. Given that only a few,
selected groups of organisms have been explored so
far, attempts to get complete metaphase spreads either
from established cell lines or from whole organisms
is likely to expand in future. This will also facilitate
refinement of existing protocols, development of more
in vivo test systems in phylogenetically different group
of organisms and their successful use in laboratory and
field studies. These systems will complement parallel
developments in other areas of ecotoxicology and fa-
cilitate the adoption of an integrated, inter-disciplinary
approach using multiple biomarkers to evaluate the
impact of contaminants and other environmental
stressors (e.g. UV radiation, hypoxia, nutritional
deprivation, parasitic infection, etc.).
With limited success for the use of metaphase
spreads for aberration analysis, the micronucleus (MN)
assay in blood and gill cells of bivalve molluscs,
fish and amphibians was developed and applied un-
der both field and laboratory conditions [123–132].
The anaphase-telophase aberration assay was also de-
veloped in echinoderm and fish larvae [119–122].
The advantages and limitations of MN and anaphase-
telophase aberration assays in aquatic organisms have
been discussed in the literature [15]. While in mam-
malian systems, development and applications of
molecular techniques (e.g. FISH, M FISH, SKY, CO-
BRA, etc.) are proving very useful to illuminate the
12 A.N. Jha / Mutation Research 552 (2004) 1–17
minute details of the complexity of the types of aberra-
tions induced [133], the field of aquatic genotoxicology
is still far behind in the application of these techniques
to elucidate the qualitative and quantitative induction
of chromosomal aberrations, one of the most impor-
tant parameters for its known biological significance.
Limited attempts to apply molecular cytogenetic tech-
niques in aquatic organisms have not gone far enough
to throw light on the nature and complexity of genetic
damage induced by environmental agents [134,135]. In
future, it is hoped that in parallel with mammalian stud-
ies, combination of whole chromosome painting with
telomeric and centromeric probes, would provide better
insights of induction of structural and numerical chro-
mosomal aberrations in aquatic organisms. This will
also provide a robust comparison with well-advanced
studies in human and mammalian cells.
The application of single cell gel electrophoresis
(i.e. Comet assay) has revolutionised the field of
genetic ecotoxicology. This assay has provided the op-
portunity to study DNA damage, repair and cell death
(apoptosis) in different cell types of aquatic organisms,
without prior knowledge of karyotype and cell turn
over rate. The advantages and disadvantages of this
assay have been extensively described in the literature
[15,136–138]. In human and mammalian studies,
the combination of Comet assay with fluorescence
in situ hybridisation (FISH) technique has allowed
the quantification of single and double strand breaks,
alkali labile sites, and has provided the insights of
the role and fate of specific DNA sequences at whole
genome or single cell level [139–141]. Due to a lack of
distinct banding patterns, the packaging of chromatin
in lower organisms appears to be different from higher
organisms [15]. In mammalian systems, intragenomic
heterogeneity for the induction of genetic damage and
repair exists [142–146]. There is however not much
information available pertaining to heterogeneity for
DNA damage and repair in aquatic organisms. In
fact, very little is known about DNA repair efficiency
in different aquatic organisms. Future studies will
therefore explore the potential differential sensitivity
and elucidation of repair efficiency of apparently ho-
mogenous genomes from different cell types of aquatic
organisms. This will include use of combination of
Comet and FISH techniques [139–141], identification
of DNA repair deficient (mutant) individuals, con-
struction of mutant cell lines and transgenic models
[147]. In common with mammalian systems, these
developments will adopt several molecular approaches
to dissect the DNA repair mechanisms [141,148,149].
Finally, with growing developments in ‘omics’
(i.e. genomics and proteomics), nanotechnology and
biotechnology areas, we will be able to know more
about how the organisms perceive changes at the
molecular and genetic levels, and make functional re-
sponses within their environment. These functional re-
sponses will uncover pattern of gene expression in the
organisms and their short-and long-term adaptations
in response to environmental changes. The mechanis-
tic understanding and adaptations at molecular, genetic
and cellular levels could then fill the gap in knowl-
edge to link the reproductive and survival strategies of
the organisms under stressed conditions. The tools de-
veloped, and information generated, would also help
environmental managers to develop and apply robust
approach to protect human and ecosystem health. All
these developments will make the field interesting,
stimulating and challenging.
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