Mixed-function oxygenase enzymes as tools for pollution monitoring: field studies on the French coast of the Mediterranean Sea.

1. MFO enzyme activities were measured in microsomes from whole mussels (Mytilus galloprovincialis) comber livers (Serranus cabrilla), or Posidonia oceanica etiolated tissues, and PAH contents were determined in sediments collected in coastal locations of the French Riviera and Corsica during 3 oceanographic cruises in 1987-1988. 2. BaP activities in mussel and EROD activities measured in fish were strongly correlated to the log of PAH content in sediments. The first results for CA4H in Posidonia showed significant differences related to PAH pollution levels. The increase in MFO activities measured in Corsica in summer 1988 indicated a recent petroleum contamination.     

Review and perspective on the use of mixed-function oxygenase enzymes in biological monitoring

It is often suggested that changes in simple biochemical/physiological responses may be useful for predicting the impacts of pollutants at population and community levels of biological organization. There are serious conceptual constraints to such a thesis and its seems likely that such simple responses can go no further than serving as early warning systems for delineating potential areas of pollutant impact--areas which (if shown to be significant in size) can then be subjected to more detailed population and community type studies. Environmental testing is a prerequisite for any response suggested to have value as a biological monitoring index and the induction of mixed-function oxygenase (MFO) enzymes has now been validated in a large number of field studies worldwide. Investigations have progressed from documenting induction near localized sources of hydrocarbon contamination to more diffuse sources of mixed organic pollution originating from industrial and domestic sources. Studies in the Great Lakes and Europe have demonstrated that the induction of MFO enzymes is a biological response of sufficient sensitivity to discriminate water quality differences over broad geographical areas. We suggest that as an early warning system, the induction of these enzymes can fulfill the requirement of "most sensitive biological response" for assessing a variety of organic pollution conditions. Given the high level of sensitivity of the MFO enzyme response, negative as well as positive field trials can be of value in addressing concerns about the toxicological significance of "high-profile" chemicals (and potent inducers) such as polycyclic aromatic hydrocarbons and organochlorines. MFO enzyme induction can also be an economical tool for environmental managers for reacting to real or perceived concerns about pollution such as effects on commercial fish stocks at sites of petroleum hydrocarbon development in the oceans.     

Mixed-function oxidase induction as a test for the biological monitoring of water: limitations and prospects]

The induction in fish liver of some enzyme activities, and typically of microsomal mixed-function oxidases (MFO), provides the earliest biological warning signal of exposure to pollutants. Our studies provided evidence that the basal levels of cytochrome P-450 and specific MFO activities, such as arylhydrocarbon hydroxylase (AHH) and ethoxyresorufin deethylase (EROD), were strongly influenced by the diet in freshwater fish (Oncorhynchus mykiss). The response of fish liver to a known enzyme inducer, i.e., beta-naphthoflavone, was also affected by the diet, which therefore should be carefully controlled in laboratory studies. Under field conditions MFO activities were significantly enhanced in the liver of O. mykiss kept in polluted river water as well as in the liver of the seawater fish Diplodus annularis collected from a polluted harbour area, as compared to specimens of the same species collected from an unpolluted reference area.     

Stable isotope-based statistical tools as ecological indicator of pollution sources in Mediterranean transitional water ecosystems

N-stable isotope analysis of macroalgae has become a popular method for the monitoring of nitrogen pollution in aquatic ecosystems. Basing on changes in their δ¹⁵N, macroalgae have been successfully used as biological traps to intercept nitrogen inputs. As different nitrogen sources differ in their isotopic signature, this technique provides useful information on the origin of pollutants and their extension in the water body. However, isotopic fractionation potentially resulting from microbial nitrogen processing, and indirect isotopic variations due to effects of physicochemical conditions on algal nutrient uptake and metabolism, may affect anthropogenic N isotopic values during transportation and assimilation. This in turn can affect the observed isotopic signature in the algal tissue, inducing isotopic variations not related to the origin of assimilated nitrogen, representing a “background noise” in isotope-based water pollution studies.In this study, we focused on three neighbouring coastal lakes (Caprolace, Fogliano and Sabaudia lakes) located south of Rome (Italy). Lakes were characterized by differences in terms of anthropogenic pressure (i.e. urbanization, cultivated crops, livestock grazing) and potential “background noise” levels (i.e. nutrient concentration, pH, microbial concentration). Our aim was to assess nitrogen isotopic variations in fragments of Ulva lactuca specimens after 48 h of submersion to identify and locate the origins of nitrogen pollutants affecting each lake. δ¹⁵N were obtained for replicated specimens of U. lactuca spatially distributed to cover the entire surface of each lake, previously collected from a benchmark, unpolluted site. In order to reduce the environmental background noise on isotopic observations, a Bayesian hierarchical model relating isotopic variation to environmental covariates and random spatial effects was used to describe and understand the distribution of isotopic signals in each lake.Our procedure (i) allowed to remove background noise and confounding effects from the observed isotopic signals; (ii) allowed to detect “hidden” pollution sources that would not be detected when not accounting for the confounding effect of environmental background noise; (iii) produced maps of the three lakes providing a clear representation of the isotopic signal variation even where background noise was high. Maps were useful to locate nitrogen pollution sources, identify the origin of the dissolved nitrogen and quantify the extent of pollutants, showing localized organic pollution impacting Sabaudia and Fogliano, but not Caprolace. This method provided a clear characterization of both intra- and inter-lake anthropogenic pressure gradients, representing a powerful approach to the ecological indication and nitrogen pollution management in complex systems, as transitional waterbodies are.     

Fungal protoplasts as tools for studies on protein secretion

Summary Protoplasts were prepared from mycelium of Aspergillus niger N-402. Sucrose was used to induce the synthesis and secretion of invertase. Protoplasts secreted 2 forms of invertase, different to those secreted by the mycelium. ¹⁴C mannose was shown to be taken up by protoplasts and incorporated into secreted proteins.     

DNA aptamers for as analytical tools for the quantitative analysis of DNA-dealkylating enzymes

The AlkB family of oxygenases catalyze the removal of alkyl groups from nucleic acid substrates in an iron and 2-oxoglutarate-dependent manner and have roles including in DNA repair. To understand the biological functions of these DNA-dealkylating enzymes it is desirable to measure their expression levels in vitro and in vivo in complex biological matrixes. Quantitative analyses of the enzymes require affinity probes capable of binding AlkB family members selectively and with high affinity. Here we report that DNA aptamers can serve as efficient affinity probes for quantitative detection of such enzymes in vitro. Nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) was applied as a general tool for: (i) selection of DNA aptamers, (ii) characterization of binding parameters for the aptamers, and (iii) quantitative detection of the target in an aptamer-based affinity analysis. The selected aptamers have a range of K_d values between 20 and 240 nM. The aptamers enabled accurate quantitative analysis of AlkB even in the presence of the Escherichia coli cell lysate. Aptamers can likely be developed for other nucleic acid repair enzymes. They may also be developed for use in in vitro and potentially in vivo studies of known nucleic acid-modifying enzymes including for functional analysis.     

Metazoan parasites of Engraulis ringens as tools for stock discrimination along the Chilean coast

The value of the metazoan parasites of the anchoveta Engraulis ringens as biological tags for stock discrimination is assessed. Three hundred and fifty-nine specimens, obtained from six landing ports in northern and central Chile (Arica, Antofagasta, Caldera, Coquimbo, Valparaiso and Talcahuano) covering a latitudinal gradient of c . 18°, were analysed. Six metazoan parasite species (three ectoparasites and three endoparasites) were obtained. A correspondence analysis suggested that anchoveta from northern and central Chile are comprised of two stocks. Identification of stocks was largely based on qualitative differences (absence in southern localities of the monogenean Pseudanthocotyloides heterocotyle , a parasite proper of engraulids, and the copepod Caligus sp.) and quantitative differences in the prevalence of infection for the remaining species. Inertia and mass value for the correspondence analysis supported the hypothesis of two stocks.     

Developing inhibitors of glycan processing enzymes as tools for enabling glycobiology

Glycoconjugates are ubiquitous biomolecules found in all kingdoms of life. These diverse structures are metabolically responsive and occur in a cell line- and protein-specific manner, conferring tissue type-specific properties. Glycans have essential roles in diverse processes, including, for example, intercellular signaling, inflammation, protein quality control, glucohomeostasis and cellular adhesion as well as cell differentiation and proliferation. Many mysteries remain in the field, however, and uncovering the physiological roles of various glycans remains a key pursuit. Realizing this aim necessitates the ability to subtly and selectively manipulate the series of different glycoconjugates both in cells and in vivo. Selective small-molecule inhibitors of glycan processing enzymes hold great potential for such manipulation as well as for determining the function of 'orphan' carbohydrate-processing enzymes. In this review, we discuss recent advances and existing inhibitors, the prospects for small-molecule inhibitors and the challenges associated with generating high-quality chemical probes for these families of enzymes. The coordinated efforts of chemists, biochemists and biologists will be crucial for creating and characterizing inhibitors that are useful tools both for advancing a basic understanding of glycobiology in mammals as well as for validating new potential therapeutic targets within this burgeoning field.     

Site-directed genome modification: derivatives of DNA-modifying enzymes as targeting tools

The modification of mammalian genomes is an important goal in gene therapy and animal transgenesis. To generate stable genetic and biochemical changes, the therapeutic genes or transgenes need to be incorporated into the host genome. Ideally, the integration of the foreign gene should occur at sites that ensure their continual expression in the absence of any unwanted side effects on cellular metabolism. In this article, we discuss the opportunities provided by natural DNA-modifying enzymes, such as transposases, recombinases and integrases, to mediate the stable integration of foreign genes into host genomes. In addition, we discuss the approaches that have been taken to improve the efficiency and to modify the site-specificity of these enzymes.     

Impact of sewage pollution on two species of sea urchins in the Mediterranean Sea (Cortiou,France): Radial asymmetry as a bioindicator of stress

BackgroundThe surroundings of the Cortiou sewage are among the most polluted environments of the French Mediterranean Sea (Marseilles, France). So far, no studies have precisely quantified the impact of pollution on the development of organisms in this area.MethodsWe used a fluctuating asymmetry (FA) measure of developmental instability (DI) to assess environmental stress in two species of radially symmetric sea urchins (Arbacia lixula and Paracentrotus lividus). For six sampling sites (Cortiou, Riou, Maire, East Maire, Mejean, and Niolon), levels of FA were calculated from continuous and discrete skeletal measures of ambulacral length, number of pore pairs and primary tubercles.ResultsFor both species, the most polluted sampling site, Cortiou, displayed the highest level of FA, while the Maire and East Maire sampling sites displayed the lowest levels. A. lixula revealed systematic differences in FA among sampling sites for all characters and P. lividus showed differences in FA for the number of primary tubercles.ConclusionsStatistical analyses of FA show a concordance between the spatial patterns of FA among sampling sites and the spatial distribution of sewage discharge pollutants in the Cortiou area. High developmental stress in these sampling sites is associated with exposure to high concentrations of heavy metals and many harmful organic substances contained in wastewater. FA estimated from structures with complex symmetry appears to be a fast and reliable tool to detect subtle differences in FA. Its use in biomonitoring programs for inferring anthropogenic and natural environmental stress is suggested.     

Immobilized enzymes as tools for the demonstration of metabolon formation. A short overview

In recent years it has become clear that a cell cannot be visualized as a ‘bag’ filled with enzymes dissolved in bulk water. The aqueous-phase properties in the interior of a cell are, indeed, essentially different from those of an ordianry queous solutin. Large amounts of water are believed to be organized in layers at the surface of intracellular structural proteins and membranes. Such considerations prompt us to reconsider the operation and regulation of metabolic pathways. Enzymes of metabolic pathways are nowadays thought to be clustered and operate as ‘metabolons’. Very often interactions between enzymes of a pathway can exclusively be evidenced in Vitro in media which are known to reduce the water concentration in the vicinity of the proteins. Immobilized enzyme preparations have been shown to be excellent tools for this type of research. We describe here some recent studies where immobilized enzymes have been used in various applications to investigate associations among enzymes of a number of different metabolic pathways (glucolysis/gluconeogenesis, citric acid cycle and its connection to the electron transport chain, aspartate–malate shuttle, glyoxylate cycle). Advantages and disadvantages of the different techniques we also discussed.     

Low incidence of Vibrio vulnificus among Vibrio isolates from sea water and shellfish of the western Mediterranean coast

A specific search for Vibrio vulnificus in natural marine samples from the Spanish Mediterranean Sea was carried out by nested PCR and cultural approaches using thiosulphate-citrate-bile salts-sucrose agar (TCBS) and cellobiose-polymixin B-colistin agar (CPC), incubated at 40 degrees C, as selective media. Presumptive colonies were identified by PCR using specific primers against 23S rRNA sequences. This species was isolated from sea water and edible bivalves, mainly after preenrichment in alkaline peptone water (APW) at 40 degrees C followed by CPC agar. None of the V. vulnificus isolates identified corresponded to serovar E. Dominant Vibrio species on directly inoculated TCBS plates incubated at 25 degrees C were V. splendidus below 20 degrees C and V. harveyi and V. mediterranei above that temperature. Low percentages of several pathogenic vibrios were recorded but V. vulnificus was never recovered at this incubation temperature. The incidence of this species in the samples studied was lower than that described for other geographical areas, probably due to the high salinity values of the Mediterranean Sea.     

Viral vectors as tools for studies of central cardiovascular control

During the last few years physiological genomics has been the most rapidly developing area of physiology. Given the current ease of obtaining information about nucleotide sequences found in genomes and the vast amount of readily available clones, one of the most pertinent tasks is to find out about the roles of the individual genes and their families under normal and pathological conditions. Viral gene delivery into the brain is a powerful tool, which can be used to address a wide range of questions posed by physiological genomics including central nervous mechanisms regulating the cardio-vascular system. In this paper, we will give a short overview of current data obtained in this field using viral vectors and then look critically at the technology of viral gene transfer.     

Scale of classification based on biochemical markers in mussels: application to pollution monitoring in Mediterranean coasts and temporal trends

A battery of biochemical parameters was used to evaluate the response of mussels to a contaminated coastal environment. A multimarker approach was developed, establishing a scale for the classification of the water quality in European coastal sites (BIOMAR European programme). This study allows the evaluation of the temporal trends of this scale when applied to selected sites of European Mediterranean coast (BEEP Biological Effects of Environmental Pollution in Marine Coastal Ecosystems: European programme). Acetylcholinesterase activity (AChE) is highly sensitive to organophosphorus and carbamate insecticides and, to some extent, also to heavy metals. Catalase activity (CAT) and lipid oxidation (evaluated as malonedialdehyde) are markers of oxidative stress, glutathione S-transferase (GST) activity is related to conjugation of organic compounds and benzo(a)pyrene hydroxylase activity (BPH) is a marker of effect of certain planar organic compounds (e.g. polycyclic aromatic hydrocarbons, PAHs). These parameters were measured either in gills (AChE, GST) or digestive gland (BPH, GST, CAT, MDA). For each biomarker, a discriminatory factor was calculated (maximum variation range/confidence interval) and a response index was allocated. For each site, a Multimarker Pollution Index (MPI) was calculated as the sum of the response index of each of the five more discriminating biomarkers. As the result of our calculation method, the quality of the coastal environment at each site can be classified according to a five levels scale. Samples collected for five cruises in May 2001, 2002, 2003, and September 2001 and 2002 showed MPI evolutions. The results show that water quality can be classified from class 1 (clean areas in some sites of France, Italy and Spain) to class 4 (high pollution in main harbours). Results of the use of the biomarker scale in WP3 (Work Package Concernant Biomonitoring Programmes in Mediterranean Sea) during the BEEP programme make a strong contribution to the establishment of standardized strategies and methods for internationally agreed protocols for biomarker-based monitoring programmes. In comparison with scale pollution methodology used in the BIOMAR programme, the main contribution of BEEP was (1) to select from discriminatory analysis the biomarkers to be included in calculation of scale pollution; (2) to improve the use of the biomarker index in order to identify the main contaminants by analysis of individual contributions to the MPI; and (3) to apply methodology for temporal trends at sampled sites.     

Activation analysis of human hair as a tool for environmental pollution monitoring

Recent development and uses of neutron activation techniques for human hair analyses are reviewed. The method of neutron activation analysis (NAA) appears to have the potential to be used as a tool for environmental pollution monitoring. Principally, two types of NAA procedure are in use nowadays for multielement analyses of human scalp hair. The more common of these is the method of instrumental neutron activation analysis (INAA), consisting of a single short-term (3-10 hours) exposure of hair to a beam of neutrons in a nuclear reactor, followed by two measurements of gamma-ray spectra at 2-3 days and 3-4 weeks after the end of irradiation. The following microelements can be commonly determined by this type of activation procedure: As, Au, Br, Cu, K, La, Na, Sb, Sm, Co, Cr, Cs, Fe, Hg, Rb, Sc, Se and Zn. The other of the two procedures involves the use of radiochemical separation techniques and is employed for quantitative determinations of elements that are not easily determined by INAA (Mo, Cd, Ni, etc.), or in cases where there is a need to achieve the lowest possible limits of analytical determination. The accuracy of NAA techniques is strongly dependent on the hair sampling and hair sample processing methods used. The analytical error of this method may vary within the range of 5-15%. Its applicability as a tool for monitoring the environmental pollution level is here demonstrated on an example of groups of individuals living in the areas differing by the degree of environmental pollution. The use of other biopsy materials, such as e.g. mammalian hair, for the purpose of environmental exposure monitoring is also considered in this review.     

Direct seeding of grass species for sand dune stabilization on the mid-Atlantic sea coast

The stabilization of moving sand dunes at Provincetown, Massachusetts, depends, to a large degree, upon the environmental factors of wind, temperature and rainfall. Direct seedings of grasses for the purpose of stilling these moving dunes can be wiped out totally by the severe northeast and northwest winds which occur in the very early spring. In this study, 9 different mulches were used with 12 grass varieties to examine their value in the establishment and survival of vegetation. The grasses were seeded with a grain drill, the planting units of which had been modified to incorporate the seed to a minimum depth of 5 cm. Evaluation of the mulches showed that the sodium silicate plots had slightly better grass emergence and establishment than the plots with the other mulches. In the unmulched check plots, grasses established themselves as well as or better than in some of the mulched plots. The species that survived, grew well, and controlled the drifting sand were coastal panicgrass, tall fescue, weeping lovegrass, and switchgrass. The study showed that large dune areas can be successfully seeded provided the right grass species are used, fertility levels are good initially and maintained, and provided, also, that climatic factors cooperate.Project initiated through the cooperation of the Massachusetts Department of Public Works, the Federal Highway Administration, and the University of Massachusetts College of Food and Natural Resources.Presented at the Seventh International Biometeorological Congress, 17–23 August 1975, College Park, Maryland, USA.     

Scale of classification based on biochemical markers in mussels: application to pollution monitoring in Mediterranean coasts and temporal trends.

A battery of biochemical parameters was used to evaluate the response of mussels to a contaminated coastal environment. A multimarker approach was developed, establishing a scale for the classification of the water quality in European coastal sites (BIOMAR European programme). This study allows the evaluation of the temporal trends of this scale when applied to selected sites of European Mediterranean coast (BEEP Biological Effects of Environmental Pollution in Marine Coastal Ecosystems: European programme). Acetylcholinesterase activity (AChE) is highly sensitive to organophosphorus and carbamate insecticides and, to some extent, also to heavy metals. Catalase activity (CAT) and lipid oxidation (evaluated as malonedialdehyde) are markers of oxidative stress, glutathione S-transferase (GST) activity is related to conjugation of organic compounds and benzo(a)pyrene hydroxylase activity (BPH) is a marker of effect of certain planar organic compounds (e.g. polycyclic aromatic hydrocarbons, PAHs). These parameters were measured either in gills (AChE, GST) or digestive gland (BPH, GST, CAT, MDA). For each biomarker, a discriminatory factor was calculated (maximum variation range/confidence interval) and a response index was allocated. For each site, a Multimarker Pollution Index (MPI) was calculated as the sum of the response index of each of the five more discriminating biomarkers. As the result of our calculation method, the quality of the coastal environment at each site can be classified according to a five levels scale. Samples collected for five cruises in May 2001, 2002, 2003, and September 2001 and 2002 showed MPI evolutions. The results show that water quality can be classified from class 1 (clean areas in some sites of France, Italy and Spain) to class 4 (high pollution in main harbours). Results of the use of the biomarker scale in WP3 (Work Package Concernant Biomonitoring Programmes in Mediterranean Sea) during the BEEP programme make a strong contribution to the establishment of standardized strategies and methods for internationally agreed protocols for biomarker-based monitoring programmes. In comparison with scale pollution methodology used in the BIOMAR programme, the main contribution of BEEP was (1) to select from discriminatory analysis the biomarkers to be included in calculation of scale pollution; (2) to improve the use of the biomarker index in order to identify the main contaminants by analysis of individual contributions to the MPI; and (3) to apply methodology for temporal trends at sampled sites.