RICERCA ITALIANA     

BIOMONITORING OF COASTAL MARINE ENVIRONMENTS: DEVELOPMENT AND APPLICATIONS OF NEW INTEGRATED CYTOCHEMICAL AND MOLECULAR METHODOLOGIES

Università degli Studi di Messina

Abstract

The aim of the national project is the study of selected marine costal areas, in which there is the presence of a heavy impact due to industrial activities. These areas are located in two Italian regions (Liguria and Sicily). The control sites will also be selected in the same regione with ecological and superimposable characteristics.
The methodological approach focuses on the use of organisms as bioindicators and on the use of biomarkers as signal of exposure, effect and damage.
In the selected environments the effects of contaminants, and especially heavy metals, will be evaluated with particular reference to the elements in traces on marine organisms (bioindicator).
Caged bivalve molluscs and two fish species belonging to different trophic levels will be used as bioindicators.
Statistical analysis of data and the comparison of results on the investigated species in the control areas will provide important information about the toxicological status of the target areas. The integration of data obtained by this Unit with data from other Research Units will allow formulating an evaluation of environmental hazard (and eventually risk).
This project will try to investigate the impact of industrial activities on the benthic community of a coastal ecosystem, comparing it with a control area.
Benthic organisms will be used as bioindicators together with a series of parameters, as “sensitive indicators” of the presence and effects of metals and other environmental contaminants. <<<

Principal Investigator

Salvatore Fasulo Università degli Studi di MESSINA

Research Objectives

This project will try to investigate the impact of industrial activities on the benthic community of a coastal ecosystem, comparing it with a control area. Benthic organisms will be used as bioindicators together with a series of parameters, as “sensitive indicators” of the presence and effects of metals and other environmental contaminants. Benthic organisms from coastal marine environments, and invertebrates such as bivalves will be considered in neighbouring natural environments, characterised by contaminants of different type and origin, predominantly heavy metals and organic compounds. Specimens of the same species from a control area and/or other will be used for comparison.
The main advantages of using bivalve molluscs in biomonitoring programmes are the knowledge of their biological cycle and feeding habits, as well as the ease in collecting them and their wide geographic distribution.
Although the Ministry of the Environment created the data bank Sistema Difesa del Mare (Si.Di.Mar.) within the framework of the Programme of Monitoring for Control of the Marine Environment, the amount of data on heavy metals and organochlorine compounds in coastal marine environments is still extremely limited, due to the low density of sampling sites and due to the fact that effects of these contaminants are not evaluated. Scorpaenidae and Blenniidae are benthic fish that have rarely been used in biomonitoring programmes. Both families have many representatives in the coastal system and have characteristics typical of bioindicators belonging to intermediate (Blenniidae) and high (Scorpaenidae) levels of the food chain.
What discussed above are the methodological premises of the present project, which regards the distribution and effects of heavy metals in selected coastal environments. The study will be conducted using an approach based on benthic organisms as bioindicators in which the biological effects of pollutants will be studied.
The data on bioaccumulation of metals in organisms will provide a measure of the environmental bioavailability of the metal and the biomagnification processes in the food web.
All these data, together with the comparison of the status of the species examined with that of species in the control area would provide the final data on the state of contamination in the area.
The picture of the environmental situation emerging from quantification of the impact on benthic communities will make it possible to define the potential threat to human communities consuming seafood caught in the area.
We think that the originality of the project stands in the wide comparison among molecular, biochemical, histomorphological and immunohistochemical methods, and in the careful investigation at tissue and cellular level of the damage not identified using only chemical metodologies. On the contrary, this analysis provides the punctual identification of the biological indicators that may be used to identify the presence of pathologies caused by toxic agents. Moreover, we believe that our appoach is absolutely innovative in the synergy of different skills, ranging from molecular biology, biochemistry and histopathology to ecology, and in the contemporaneous examination of a wide range of target organs (gills, gut, brain, liver, urophysis and hepatopancreas) in the benthic organisms chronically exposed to pollute environments.
The comparison of data related to environments with different pollution levels also represents a crucial approach for the understanding of the molecular and cellular mechanisms of the stress response.
The final synthesis of all the results obtained by the different Ous will also allow an exhaustive analysis of the general degree of quality of the investigated ecosystems and will furnish accurate indications for suitable environmental healing and recovery intervention. <<<

First Results

The project wants to deepen the ecophysiological aspects of species exposed several types of impact, industrial/urban drainages, harbour areas, anthropic activities, and to estimate, for biological way, the level of pollution of areas marine. It defines the cytotoxic effects of the pollution on the branchial epithelium of all the sentinel organisms taken in examination and in particular on the cellular specialize types like the paraneuronal elements.
The biomolecolar data, on the organs considered of individuals subordinates to natural and/or induced stress, could evidence a various expression of the geniuses of the metallothioneins, NOS, aquaporine and heat shock proteins, regarding the control exemplary. The damage of DNA, in terms of chromosomic mutations and of breach of the filament of DNA (single or double), will show the genotoxic damage attributable to the present polluting agents.
These results will show a model related to the processes of exposure-effect of the heavy metals in the species considered. Such model, in the successive phase in nature, will concur a better interpretation of results obtained. Such studies will concur to evidence the levels of bioaccumulation of the metals in the organisms sampling supplying a measure of their bioavailability environmental. On this last aspect, they depend processes of interference to the level of the enzymatic activities. The synthesis of these information will supply the final data on the state of contamination of the area.
The second phase (duration 8 months) will comprise an integration of the data with those from the other operating units that will use various methodological models; that will concur of selecting the molecular and cellular answers to the aim to analyze the mechanisms of action for the putting to point of biomarkers more quickly sensitive to the environmental pollution.
Exposure-related effects on the proteome, the third tier of the project, will be carried out during the second phase of the project, with the GE O.U. The objectives of the proteomic approach include (i) the possibility to experimentally verify pathways obtained from the microarrays; (ii) the possibility to study to the post-tradutional modifications of proteins; (iii) the possibility to identify new and specific exposure biomarkers to compounds that have been little characterized so far (such as EDCs).
The proteins that are strongly affected by the exposures, both quantitatively and qualitatively, will be considered particularly interesting. Eventually, these proteins will be evaluated in immunohistological and immunobiochemical studies, utilising novel polyclonal antibodies raised against the identified peptide sequences.
Data obtained by the proteomic/genomic approach will contribute to the identification of new proteins/transcripts involved in the response to pollutant-induced stress. These results will be related with those obtained utilising a battery of biomarkers.
The integration of these data will permit the correlation between changes in gene and protein expression and the adaptive response of the organisms to environmental changes evaluated at cellular, tissue and organism level. In particular, data obtained from organisms sampled at polluted field sites characterized by the presence of pollutants such as heavy metals, PAH (polycyclic aromatic hydrocarbons) or by inflow of urban, industrial and agricultural discharges potentially containing EDCs will permit the identification of specific responses to selected classes of contaminants.
The proposed research project will lead to the identification of new target proteins involved in the response to pollutants, EDC in particular, and therefore, of a protein expression profile characteristic of the response to selected contaminants (or class of contaminants). The results will permit to evaluate if quantitative changes in gene expression correspond to changes in the concentration and/or transcriptional modifications of corresponding proteins and to develop research on the biological role of the newly identified proteins.The obtained data will also lead to the development of specific sequences to be added to the DNA microarray and of RT-PCR probes that can be utilised for further quantitative evaluation of selected mRNAs.
Data obtained by the proteomic/genomic approach will contribute to the identification of new proteins/transcripts involved in the response to pollutant-induced stress. These results will be related with those obtained utilising a battery of biomarkers. The integration of these data will permit the correlation between changes in gene and protein expression and the adaptive response of the organisms to environmental changes evaluated at cellular, tissue and organism level. In particular, data obtained from organisms sampled at polluted field sites will permit the identification of specific responses to selected classes of contaminants, and those obtained from laboratory experiments will permit the identification of molecular targets of the EDCs action compared to those of the natural estrogen. These results will give crucial information for the evaluation of the effects of estrogenic/antiestrogenic pollutants on marine bivalves and will help in the development of specific biomarkers of endocrine disruption in these organisms.

The determination of the polluting bioaccumulate agents in the species considered, and the important informative compendium constituted from the analysis of the proteome of some samples accurately selects will be able to improve the total interpretation of turns out and to supply precious cues for ulterior searches and the definition of practicable analyses in extended biomonitoring programs.

All this data and comparison of the status of species examined with that of species in the control area will provide the final data on the state of contamination of the area. The picture of the environmental situation emerging from quantification of the impact on benthic communities will make it possible to define the potential threat to human communities consuming seafood captured in the area.

Thi integrated study will allow evaluating the presence and distribution of the pollutants and their toxic effects on marine organisms.
The interdisciplinary and original approach will define the defence and adaptative stress responses raised in the organisms following exposure to environmental chemical pollutants.
Finally the individuation of the quality of the environmental conditions and the stress responses of the marine organisms will permit the elaboration of the most useful restorative operations. <<<

Timescale

24 months

National and international background

Many persistent substances, i.e. substances that do not break down in the environment or are not metabolised, may accumulate in the body under certain conditions if assumed with food, through the gills or through the skin. Bioaccumulation occurs when an organism takes up substances and concentrates them in the tissues and there is a direct relation between concentrations in tissues and exposure level and period. Levels of bioaccumulation are normally used to evaluate environmental contamination by heavy metals.
Bioaccumulation of elements such as cadmium, lead and arsenic generally does not seem to be related to trophic level but rather to trophic niche (e.g. filtering organisms, detritivores, sediment dwellers). Mercury is the only metal that biomagnifies in the food chain, because its organic form, methylmercury, is relatively stable and has affinity with biological structures.
Although mercury tends to be methylated by bacteria in anoxic sediment, the methylmercury molecule is very sensitive to photodegradation. Production of methylmercury and its entrapment in the living biomass is therefore very efficient in the dark depths (Bacci 1989).
There have been reports of cases in which bioaccumulation of mercury in bioindicator organisms was correlated with depth of capture (Leonzio et al. 1981). Heavy metal concentrations may be measured in whole (small) organisms or in selected tissues or organs which are particular sites of bioaccumulation (digestive gland of molluscs and crustaceans, liver and kidney of fishes).
Analysis of bioaccumulation of heavy metals in organisms is a way to evaluate their environmental bioavailability. Bioaccumulation of metals and other contaminants in general, is a major clue to exposure, especially if compared in organisms of the same species from areas with different impact. However, it must always be correlated with other indicators of physiological “health”, generally known as biomarkers. The typically diagnostic aspect of biomarkers is associated with their prognostic meaning of imminent stress or damage to individuals and consequently to the population.
The metabolism of some endogenous biological compounds can be altered from polluting that they provoke an abnormal accumulation of the products of intermediate syntheses.
In this light, research has been increasingly focusing on the development of new approaches for the identification and the evaluation of the effects of pollutants and for the understanding of the underlying molecular mechanisms. In particular, an integrated approach genomics/proteomics allows on one hand to obtain a transcriptional profile of a large number of genes (through DNA microarray) and the quantification of specific transcript of interest (by real time PCR), on the other proteomics (2D electrophoresis/mass spectrometry/statistical analysis) can lead to the evaluation of PES=protein expression signature, together with post- translational changes (Koskinen et al., 2004; Jenny et al., 2004; Waters and Fostel, 2004; Storey, 2006; Venier et al., 2006; Dondero et al., 2006).
The integrated genomics/proteomics/biomarkers approach has a huge potential when applied in environmental monitoring programmes. In particular, it is particularly promising for the study of the biological effects of endocrine disrupting chemicals (EDCs) a particular class of pollutants (such as pesticides, alhylphenols, PCBs), that can interfere with the endocrine system, with possible adverse effects on development and reproduction and consequences at the population level (Witorsch et al., 2002; Rotchelle and Ostrander, 2003).
The analysis of the transcription profile utilising DNA-microarray technology permits the simultaneous measurement of the expressions of multiple (up to thousands) genes. This technique offers enormous advantages in terms of timesaving, costs and flexibility.
Proteomics is defined as "the use of quantitative protein- level measurements of gene expression to characterise biological processes and decipher the mechanism of gene expression control" (Williams, 1996; Anderson and Anderson, 1998). The proteomic approach has been increasingly applied due to the recent progress achieved in technology and equipments for separation and identification of proteins (Humphery-Smith et al., 1997), and it has been also utilised in studies on aquatic organisms (Knigge et al., 2004; Jenny et al., 2004; Mi et al., 2005; McDonagh et al., 2005; Bjornstad et al., 2006). The environmental proteomics principle is that global protein expression is specific to the environmental conditions experienced by the organism. There is actually growing evidence that protein synthesis can be induced or inhibited under specific conditions of environmental contamination (Corella and Ordovas, 2005; Lopez-Barea and Gomez-Ariza, 2006).
An elevated concentration of heavy metals in the environment provokes, in the Teleostei, alterations in the steady state of Ca2+, istomorfologici damages in the tissues more exposed to such risk, as the respiratory epithelium, where the polluting agents can act directly, compromising the ionic concentration in the extracellular fluid and alterating the osmoregulatory ability. The gills of marine organisms play fundamental roles not only for the respiratory activity but also for sensitive and osmoregulatory functions; they represent the first target for environmental toxic xenobiotics. The study of the paracrine regulation system has a particular meaning for the directed exposure of the epithelium to the external environment. The location of paraneuronal cells in the branchial epithelium has concurred to understand how these functions can be controlled and be coordinated at a local level. (Dunel-Erb et al. 1982; Bailly et al. 1992; Mauceri et al 1999). The studies conducted on the distribution models of bioattive substances, intermediate filaments and proteins binders Ca2+ in branchial epithelia of normal individuals and in experimental conditions (Fasulo et al. 1998, Mauceri et al. 2002, 2005) represent therefore a fundamental base of reference to estimate the effect of xenobiotic substances on a paracrine regulation system particularly sensitive and whose dysfunctions can heavily affect the general state (Triebskorn et al, 2007). Use of aquatic organisms as bioindicators of the environmental quality is widely adopted because of their peculiar characteristics of accumulating polluting agents. In fact, organisms exposed to sublethal levels of metals are often able, through physiological mechanisms, to compensate the altered environmental conditions also with an increment in the tolerance to the same metals (McDonald &amp; Wood, 1993). The iperproduction of mucus found in gills of exemplary from aquatic environment represents a defense response (Rinaldi et al, 2005). The mucus, in fact, acts like a barrier to the spread of oxygen provoking an emphasized hypoxia (Par Lock, 1983; Handy et al. 1989). The local reaction to the hypoxia implies an intense production of vasoactive substances, like endothelin or nitrogen oxide. (Mauceri 1999, 2005).
This is a confirmation of what Wood (1992) observed, according to which, in the aquatic organism, the gills, being the primary syte of the ionic regulation and the gaseous exchanges, represents systems extremely sensitive to the toxicity of the heavy metals. <<<