In consequence to mankind's continuous exploitation of marine environments in the search for new fuels and sustainable food sources, to meet the demands of the incessant population growth, ecotoxicological monitoring of the oceans is becoming ever important to minimize the risks to human health. Increases in mining, industrial activities and the frequency of oil spills have significantly increased the extent of environmental pollution by organic compounds and metals. This review identifies recent developments in the monitoring of toxicological responses in marine organisms, considering the biomarker responses to different classes of pollutants. Analysing recent publications, the specificity of certain biomarkers including metallothionein and cholinesterase are discussed. Non-specific biomarkers are also debated in regards to toxic responses in relation to effects on oxidative damage and inhibition of antioxidant defences. Biomarkers of the genotoxic and immune responses are then considered as potentially important indicators of complex pollutant mixture. Finally, future perspectives for the advancement of biomarker study will be discussed focusing on recent trends in the literature.
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Keywords: Antioxidant defence; Cholinesterase activity; DNA damage; Immune responses; Metallothionein; Oxidative stress
The issues of marine pollution may be considered somewhat paradoxical, in that pollution of the seas has been historically identified as a substantial global problem, nevertheless, pollution levels are documented to still be expanding at an alarming rate. The nature of this issue stems from an incessant conflict between nature and technology as demonstrated by the increasing quantity of studies on aquatic pollution instigated by human exploitation; such as oil spills (Brooks et al., 2010; Sureda et al., 2010; Vidal-Liñán et al., 2010). Pollution may arise either directly or indirectly via the introduction of contaminants into the environment, causing instability, disorder, stress or injury to the ecosystem (Adams, 2005). Monserrat et al. (2007) reported that monitoring processes should be included so that corrective actions may be taken when establishing a natural systems homeostatic condition. The employments of biomarkers accomplish this paradigm.
Biomarkers are defined by Hagger et al. (2009) as functional measures of effects caused by exposure to stressors, which may arise at the molecular, cellular, physiological or behavioral level of an individual organism. These measurements at lower levels of biological organisation, may give a sensitive, early indication of a pollutants biological effects in regards to the ecosystem in its entirety. Biomarkers are usually classified by specificity, with toxicant-specific biomarkers such as tissue levels of metallothioneins, being employed as an indicator of heavy metal contamination (Amiard et al., 2006; Da Ros et al., 2007; Sureda et al., 2010) and the inhibition of cholinesterase activity by carbamate and organophosphorus pesticides (Canty et al., 2007;Bonacci et al., 2009). Other examples of specific biomarkers include; peroxisomal proliferation, mixed function oxygenases and ethoxyresorufin-O-deethlyase (EROD) activity (Binelli et al., 2006). These biomarkers of exposure contrast with non-specific biomarkers which determine either oxidative stress or antioxidant responses in the result of pollutants modifying the balance between pro-oxidant and antioxidant concentration (Valavanidis et al., 2006). These non-specific biomarkers are mainly biomarkers of stress and genotoxicity which include DNA damage, lipid peroxidation and protein oxidation (Koukouzika et al., 2008; Frenzilli, et al., 2009).
Practically, the determination of these biomarkers are used in tandem in result of pollutants being present in complex mixtures and has led to the successful characterization of many impacted geographic locations (Amado et al., 2006a,b). In consequence, both an insight into the extent of pollution in an area, as well as an evaluation into the state of this may be obtained using the cooperative use of non-specific and specific biomarkers respectively (Sarkar, 2006).
In response to the importance and severity of marine pollution, the present review will address recent biomarkers for monitoring marine pollutants and investigate future perspectives of biomarker uses in this area. Where possible studies using bivalves were concentrated on in consequence to their abundance and that their feeding mechanisms expose them directly to the most elevated and varied sources of pollutants.
Non-specific biomarkers provide an excellent tool for evaluating the state of pollution in marine ecosystems, indicating the physiological stress inflicted on an organism in response to pollutants. Characteristic examples of this class of biomarker include antioxidant concentration and oxidative damage. Chemical pollutants have widely been documented as causing susceptibility to oxidative stress in marine organisms by increasing the production of pro-oxidant species or decreasing the capability of antioxidant defenses (Richardson et al., 2008). In consequence, both of these mechanisms are widely employed as indicators of marine ecosystem health.
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Oxidative stress causes the production of reactive oxygen species (ROS) including the superoxide anion (O2-), hydrogen peroxide (H2O2) and the hydroxyl radical (HO.). The ROS may interact with transition metals of polluted environments and be converted into more aggressive radical species which can cause oxidative damage of the DNA, lipids and proteins as well as cause disturbances in redox balance (Koutsogiannaki et al., 2006). The TOSC (total oxyradical scavenging capacity) method has consistently been used in recent years as a measurement of antioxidant responses and was successfully employed by Regoli et al. (2004) to identify the influence of pollutants in diminishing the antioxidant defenses of mussels. A study conducted by Company et al. (2010), also employed the TOSC method in their study on the sub-lethal effects of cadmium on the antioxidant defense system of mussels (Bathymodiolus azoricus), however, on this occasion TOSC levels were not significant compared to the controls.
Other methods of monitoring oxidative stress include the use of antioxidant enzyme activities. Box et al. (2007) conducted a study on oxidative stress biomarkers in the gills and digestive glands of mussels of the Balearic Islands, identifying the enzymes catalase (CAT) and glutathione reductase (GR) as effective biomarkers of pollution detection, over glutathione peroxide (GSH-PX) and superoxide dismutase (SOD). Contrastingly, Sureda et al. (2010) indicated that CAT, GSH-PX and SOD were all useful biomarkers for use in the biomonitoring the response to oil pollutant exposure. The previously described study by Company et al. (2010) also ran antioxidant enzyme biomarkers in conjunction to TOSC. Unlike the TOSC, which showed no significant differences between the experiments and the controls, activities for SOD and CAT were both demonstrated to be significant in consequence of cadmium exposure.
Specific biomarkers, in contrast to non-specific biomarkers better provide insight into the extent of the pollution of a marine environment. Representative examples of this class of biomarker include the use of cholinesterases and metallothioneins; which indicate alterations to Key physiological response in marine organisms (Hagger et al., 2009).
Cholinesterases (ChEs) constitute a family of enzymes which are essential in permitting cholinergic neurons to return to their resting state by catalysing the hydrolysis of the neurotransmitter acetylcholine to choline and acetic acid (Binelli et al., 2006). The two major types of ChEs; acetylcholinesterase (AChE), and pseudocholinesterase (BChE) are distinguishable most notably with respect to their preference of substrate (Acetylcholine for AChE and butyrylcholine for BChE). Organophosphorus and carbamate pesticides have been extensively recognised as selective inhibitors of ChE activity (Binelli et al., 2006), and may enter marine ecosystems either via direct release or surface run-off. As such, tissue ChE activity of marine invertebrates has been employed frequently as a biomarker of pesticide pollution (Canty et al., 2007).In consequence to the relatively short half-life of organophosphorus and carbamate, ChE inhibition is an effective biomarker as the impact on the individual are still detectable, even after the chemical itself has dissipated (Valbonesi et al., 2003; Monserrat et al., 2007).
A recent manuscript by Choi et al. (2011) demonstrates the usefulness of ChE activity, using the Manila clam as a bioindicator to monitor four heavily used Korean pesticide levels (chlorpyrifos, drazinon, IBP and methidathion). This study identified a clear dose-response relationship between the inhibition of ChE activity and the organophosphorus pollutants relative to concentration and exposure time. The historical study by Fairbrother et al. (1989) identified variation of ChE activity in response to biotic and abiotic factors; including the sex, age and size of the mussels studied. This was different to the findings of Choi et al. (2011) who reported the observation of negligible evidence, that size had an effect on ChE activity. Pfeifer et al. (2005) also investigated abiotic effects of salinity on ChE activity and demonstrated a negative correlation in Mytilus sp. However, due to the majority of bivalve species living in marine environments, this type of investigation has seen little replication in consequence of little salinity fluctuation, so comparison may not be drawn. A more recent study into pollutants effects on ChE activity in Antarctic scallops by Bonacci et al. (2009) indicated that, although significant inhibition of ChE activity was noted in response to organophosphorus pollutant exposure, exposure to both polycyclic aromatic hydrocarbons and polychlorinated biphenyls elicited negligible effects.
Metallothioneins (MTs) were one of the first biomarkers studied in aquatic invertebrates and are defined by low molecular weight, cytosolic proteins rich in sulphyhdryl (-SH) residues with high affinity for transition metal ions of groups IB and IIB (Monserrat et al., 2007).
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The nature of MTs activity is largely dominated by the chemistry of the thiol groups, which form metal-thiolate clusters, permitting rapid transfer of metal ions between these clusters and other MT molecules (Amiard et al., 2006). This type of action appears to be unique to MT and fundamental to their biological function; which includes the homeostasis of important metals, antioxidant defense, and both essential and non-essential metals detoxification (Roesijadi, 1996; Viarengo et al., 2000; Amiard et al., 2006). Studies by Amiard et al. (2006) stated that MT literature in aquatic invertebrates is large and increasing, however, identified that many inconsistencies exist over their physiological roles. The functions of MTs are still not fully understood, although are believed to be important in the binding of metal ions and in addition provide some defense against oxidative stress. Evidence supporting these hypotheses is evident from MT structure, with the thiol groups of the cysteine residue being capable of binding both physical and xenobiotic heavy metals via sequential, noncooperative mechanisms (Pellerin et al., 2009; Franzellitti et al., 2010). In addition a function as an oxyradical scavenger being theorized in recognition of the cysteine residues being effective in the capture of oxidant ROS such as superoxide and hydroxyl radicals (Andrews et al., 2000). However, in response to MT expression being rapidly induced not only metals, but cytokines, hormones, irradiation and oxidants, it has been broadly predicted that other functions for MT may also exist (Andrews et al., 2000; Haq et al., 2003).
Although the inducement of MT by both the essential metals Cu and Zn and the non-essential metals Ag, Cd and Hg is evident, their induction is variable between different zoological groups and environment. This suggests that the use of MTs as biomarkers must be done wisely. Evidence of this is demonstrated by the studies of Bianchini and Gilles (2000); Bianchini et al. (2002); and Pfeifer et al. (2005), whom all recognise that due to the presence of many inductors of MTs, and the effects of salinity on trace metal uptake in aquatic organisms, MT should realistically only be used in conjunction with a suite of other biomarkers. Even so, today MTs are routinely used as specific biomarkers of metal exposure and toxicity in biomonitoring studies, in response to many pollutants of aquatic ecosystems being heavy metals and thus inducing MT synthesis.
A study by Da Ros et al. (2007) illustrates the use of MTs in conjunction with other biomarkers, in this instance the lysosomal responses elicited by pollutants of blue mussels. The results indicated elevated levels of MT, which provided indication that the pollutants present, included heavy metals.
Pellerin et al. (2009), further utilised the properties of MTs, and demonstrated induction of MT was different between Mytilus edulis and Mya arenaria, with cadmium and copper being found only in the gills of mussels but in both the gills and digestive glands of clams. These results were mirrored by the earlier study of Geffard et al. (2005).
Biomarkers of the Genotoxic Response
In result of the diversity of pollutants present in marine environments and the intricate mixtures in which they are found, assessment of exposure to environmental genotoxicants is a complex issue (Frenzilli et al., 2009). Interaction between pollutants and DNA occur in a process of four discrete stages, beginning with the formation DNA adducts, in consequence of the covalent binding of chemicals to specific sites on the DNA (Jing-Jing et al., 2009). Secondary DNA modifications consisting of double strand breakages, base oxidation and DNA-protein cross linking, constitute the secondary stage, which may be induced by ROS (Hermes-Lima, 2004). The third stage is reached when structural perturbations of the DNA become static, leading to altered cellular function such as carcinogenesis (Binelli et al., 2009). The final stage of mutation occurs during cell division and involves DNA mutations which effects following generations (Gil and Pla, 2001; Binelli et al., 2009).
These damages have been widely employed as biomarkers to detect and quantify the effects of exposure to genotoxic pollutants in marine ecosystems. The most widely assessed indicators of genotoxicity in aquatic organisms were reported as mutations at the chromosomal level (micronucleus test), DNA adducts and DNA stand breaks (comet assay) by Ohe et al. (2004) in a review paper analysis 128 publications on this area. Bolognesi et al. (2004) demonstrated that DNA single strand breaks evaluated by alkaline elution, were weakly correlated to organic pollutant increases, whilst in the same study establishing a correlation of mercury concentration and MN frequency.
Fedato et al. (2010) evaluated DNA damage using both the micronucleus test and comet assay in Asian clams in response to acute exposure to gasoline. They concluded that the results from the comet assay demonstrated DNA damage of both the hemocytes and gill cell, whilst the hemocytes additionally revealed significant increases in micronucleus frequency. Similarly Deasi et al., (2010) demonstrated that both the micronucleus test and comet assay could be employed as a biomarker of petroleum hydrocarbon pollution. Amado et al. (2006a) in their study of pollution in the Patos Lagoon estuary (South Brazil) on estuarine flounders seasonally, also evaluate DNA damage through the micronucleus test and comet assay. They reported that DNA damage identified by the micronucleus test was significantly higher for the majority of seasons in polluted sites when compared to the reference sights while the quantified damage to the DNA via the comet assay was similar for specimens of both sites. Alternatively, Amado et al. (2006b) report that winter croakers (M. furnieri) from polluted sites in the Patos Lagoon estuary demonstrated high DNA damage assessed by both the micronucleus test and comet assy.
Biomarkers of the Immune Response
The most recent biomarkers are those of immune responses, although due to their nature, they are more prominently used in higher organisms in consequence of their more development immune systems. As such most modern work using immune response biomarkers have been complete using either invertebrates or fish. It has been widely documented that pollutant can cause detrimental effects on immunological functions, including immunosuppression through either direct or indirect mechanisms (Ahmad et al. 2010). Innate immune responses have been highly conserved, as demonstrated by the inflammatory responses of marine organisms being analogous to man (Roch, 1999).
Increasing numbers of primary research are being published in this area, with considerable evidence even within bivalves that pollutants cause the modulation of immune function. This is validated by Hannam et al. (2009) whom witnessed a measurable reduction in immune fitness within blue mussels (M. edulis), as indicated by the significant suppression of immunological cells at sublethal concentration of the pollutants, as revealed by inhibited phagocytosis and reduced circulatory cell number. Contrastingly, in the same study, the authors identified that at lower levels of pollution (0.125%), increased circulation of cells and increased phagocytosis may be used as an early indication of pollutants. Similarly, an earlier study by Auffret et al. (2006) whom also suggested that pollutants caused immunosuppressive states in mussels in response to alterations in hemocyte counts and active phagocytosis.
Innate leukocytes such as macrophages induce phagocytic killing of pathogens via oxidative and non-oxidative mechanism with oxidative mechanisms producing ROS and cytotoxic activities. In correspondence of this mechanism, a large increase in O2 consumption is noted, called respiratory burst. Respiratory burst may be employed as a biomarker of toxicant exposure as evidenced by Chang et al. (2009) whom identified a dose-dependent increase as well as a time-dependent change in respiratory burst in result of treatment with cadmium. However, the sensitivity of respiratory burst and phagocytes as biomarkers are still under question.
It may be concluded that a vast quantities of different biomarkers are currently being employed globally, as the result of an increasing interests to conduct environmental studies to monitor the level and effect of pollution in the oceans. This interest is likely to continue expanding as humanity continues to exploit marine environments and the increased pollution levels threaten detrimental effect to man, through food sources.
In result of the complexity and variability of biomarker forms and functions, future perspectives in this field may tend towards refining current biomarker techniques and identifying potential biomarkers which may act universally for specific pollutant types. This would simplify biomarker choice and create greater uniformity in data, allowing comparisons to be made across geographic locations. In addition, with the field of aquaculture seeing dramatic growth, the use of marine insecticides to control fish pathogens would be expected to rise. This may lead to the need for a broader range of sensitive biomarkers.