Vladimir Bresler

Marine molluscs and fish as biomarkers of pollution stress in littoral regions of the Red Sea, Mediterranean Sea and North Sea

Abstract The intensive development of industry and urban structures along the seashores of the world, as well as the immense increase in marine transportation and other activities, has resulted in the deposition of thousands of new chemicals and organic compounds, endangering the existence of organisms and ecosystems. The conventional single biomarker methods used in ecological assessment studies cannot provide an adequate base for environmental health assessment, management and sustainability planning. The present study uses a set of novel biochemical, physiological, cytogenetic and morphological methods to characterize the state of health of selected molluscs and fish along the shores of the German North Sea, as well as the Israeli Mediterranean and Red Sea. The methods include measurement of activity of multixenobiotic resistance-mediated transporter (MXRtr) and the system of active transport of organic anions (SATOA) as indicators of antixenobiotic defence; glutathione S-transferase (GST) activity as an indicator of biotransformation of xenobiotics; DNA unwinding as a marker of genotoxicity; micronucleus test for clastogenicity; levels of phagocytosis for immunotoxicity; cholinesterase (ChE) activity and level of catecholamines as indicators of neurotoxicity; permeability of external epithelia to anionic hydrophilic probe, intralysosomal accumulation of cationic amphiphilic probe and activity of non-specific esterases as indicators of cell/tissue viability. Complete histopathological examination was used for diagnostics of environmental pathology. The obtained data show that the activity of the defensive pumps, MXRtr and SATOA in the studied organisms was significantly higher in the surface epithelia of molluscs from a polluted site than that of the same species from control, unpolluted stations, providing clear evidence of response to stress. Enhanced frequency of DNA lesions (alkaline and acidic DNA unwinding) and micronucleus-containing cells was significantly higher in samples from polluted sites in comparison to those from the clean sites that exhibited genotoxic and clastogenic activity of the pollutants. In all the studied molluscs a negative correlation was found between the MXRtr levels of activity and the frequency of micronucleus-containing hemocytes. The expression of this was in accordance with the level of pollution. The complete histopathological examination demonstrates significantly higher frequencies of pathological alterations in organs of animals from polluted sites. A strong negative correlation was found between the frequency of these alterations and MXRtr activity in the same specimens. In addition to these parameters, a decrease in the viability was noted in molluscs from the polluted sites, but ChE activities remained similar at most sites. The methods applied in our study unmasked numerous early cryptic responses and negative alterations of health in populations of marine biota sampled from the polluted sites. This demonstrates that genotoxic, clastogenic and pathogenic xenobiotics are present and act in the studied sites and this knowledge can provide a reliable base for consideration for sustainable development.

A comparative study on the system of active transport of organic acids in Malpighian tubules of insects

Abstract 1. 1. The transport of a marker organic acid, fluorescein in isolated Malpighian tubules of the tropical cockroach (Blaberus giganteus) and American cockroach (Periplaneta americana), Asiatic locust (Locusta migratoria) and larva of the wax moth (Galleria mellonella) has been studied quantitatively by means of a microfluorimetric method. 2. 2. Fluorescein transport in the Malpighian tubules of all species occurred against the concentration gradient, followed saturation kinetics and was inhibited by other carboxylic acids (p-aminohippurate, probenecid). Fluorescein transport in the Malpighian tubules of B. giganteus was also inhibited by sulphonates (indigocarmine, congo red). 3. 3. Active fluorescein transport in the Malpighian tubules of all species under study was shown to depend on the temperature of the incubating medium. 4. 4. The values of the Michaelis-Menten equation parameters for fluorescein transport in Malpighian tubules: the apparent Michaelis constant (KM), inhibition constant (Ki) and maximal transport rate (Vmax) were determined at the optimal temperature for each species. Alterations of KM and Vmax indicate that fluorescein-transport inhibition by carboxylic acids (p-aminohippurate, probenecid) is competitive while its inhibition by sulphonate, congo red, in the Malpighian tubules of B. giganteus is mixed. 5. 5. The transport of fluorescein is sodium-dependent in the tubules of all species. 6. 6. The active transport of organic acids in the Malpighian tubules of insects is, in many respects, similar to that in the renal proximal tubules of vertebrates. 7. 7. Correlations are revealed between the value of V max K M ratio and the type of nutrition and some peculiarities in the ecology of the insects in question.

Marine molluscs in environmental monitoring

Concentrations of trace elements and organic pollutants were determined in marine sediments and molluscs from the Mediterranean and Red Sea coasts of Israel. Two bivalve species (Donax trunculus, Pteria aegyptia), two gastropod species (Patella caerulea, Cellana rota) and sediments were sampled at polluted and relatively clean, reference, sites. Along the Mediterranean coast of Israel, sediments and molluscs from Haifa Bay stations were enriched with both organic and trace element contaminants. In the Red Sea, differences between the polluted and reference sites were less pronounced. Bio-concentration factors indicate a significant concentration of Zn, As, Cd, Sn and Pb in animal tissue relative to the concentrations of these elements in the sediments. In contrast, Ce, La and U were not concentrated in molluscs. The trace element results indicate a saturation of the detoxification mechanisms in molluscs from polluted sites. The concentrations of organic pollutants at the same sites are at the lower range of values recorded in other studies. However, synergistic effects between these compounds and between them and metals can lead to acute toxicity.

Toxicological aspects associated with the ecology of Donax trunculus (Bivalvia, Mollusca) in a polluted environment

This study provides data on the ecology and toxicology observed in the population of Donax trunculus, a sand dwelling mussel, in the shallow subtidal of Haifa Bay (Mediterranean Sea, Israel). The studied population of the mussel forms a dominant fraction in a community of sand-dwelling molluscs in a zone located 5–25 m from the shoreline, and at depths of 20–120 cm, numbering up to 2000 per m2. Samples of the mussel were collected from three sites, located within 9 km of shore in Haifa Bay. These included a clean site (Akko), a site polluted by a chemical PVC-polymer industry (Frutarom), and a site polluted with oil and waste from the petrochemical industry (Qiryat Yam). Metal analysis indicated site-dependent variations in levels of cadmium, lead, copper and mercury in the mussel soft tissues. Copper levels were similar in the bivalves collected from all the sites. Levels of mercury were significantly higher in specimens from the PVC-polluted site (Frutarom) while levels of cadmium were higher in Akko and Qiryat Yam compared to Frutarom. Lead residues were found only in Donax from Akko. The residual contents of mercury, copper and cadmium were relatively high in the young and noticeably low in Donax of medium body size. The main site of deposition of metals was in the soft tissues of the bivalve, but bioaccumulation of metals was also found in the shells. A marked increase in permeability of gills and mantle to the fluorescent anionic dye — fluorescein (FLU) was detected in Donax from Qiryat Yam and especially Frutarom, compared to the bivalves sampled from Akko. Multiple foci of enhanced permeability (multiple fluorescent spots) were detected in all the individuals sampled from Frutarom but none in the bivalve samples from Akko. Lysosomal accumulation of the metachromatic fluorescent cationic probe, acridine orange — (AO), was significantly decreased in the tissues of D. trunculus from polluted sites, especially polluted by the PVC factory. This decrease correlated with lysosomal enlargement and the formation of secondary lysosomes. D. trunculus appears to possess the most effective biochemical and physiological defense mechanisms enabling it to survive in habitats of polluted shallow waters, where other sand dwelling mollusc species were absent or found only in waters deeper then 2.5 m.

On the active transport of organic acids (fluorescein) in the choroid plexus of the rabbit

The kinetics of active transport of an organic acid (fluorescein) through the membranes of the choroid plexus from the lateral ventricules of the brain of rabbit was studied both morphologically and functionally. It was shown that fluorescein is actively translocated through the apical and basal membrane of the epithelium and is accumulated in blood capillaries at a concentration exceeding one order of magnitude that in the incubation medium. The kinetic curves displaying saturation and the demonstration of inhibition by other acids shows that a specific carrier is involved in the transfer across the membrane. The active transport of fluorescein at 20 degrees C was found to be sodium independent. Total exclusion of sodium from the incubation medium does not change the Michaelis constant (Km) and maximal velocity (V). The active transport depends on the operation of (Na+ + K+)-ATPase as energy source but obviously no specific complexes with the participation of sodium are involved.

DIETARY L-CARNITINE PROTECTS THE GILLS AND SKIN OF GUPPIES (POECILIA RETICULATA) AGAINST ANIONIC XENOBIOTICS

Abstract L-carnitine is a carrier of activated fatty acids into mitochondria, but it may also have other functions. Experiments were conducted to investigate possible influences of dietary L-carnitine at the cellular level. Contact fluorescent microscopy was used to compare the responses of tissues of fish fed different levels of dietary L-carnitine when exposed to the fluorescent markers fluorescein and acridine orange. The penetration and accumulation of these markers in living cells was estimated by measuring the intracellular intensity of their fluorescence (530 nm). The results showed that penetration of fluorescein from water via gills was significantly lower in L-carnitine fish than in control fish. Intact plasma membranes are almost impermeable to organic anions, such as fluorescein, but damage of plasma membranes increases their permeability. Thus, it appears that the membranes of L-carnitine fish may be better protected against the penetration of anionic xenobiotics than the membranes of control fish. Accumulation of acridine orange, a cationic compound, did not show any significant differences between L-carnitine fish and control fish. Organic cations penetrate plasma membranes via diffusion, and this is unlikely to be influenced by L-carnitine.

Structure and active transport in the plasma membrane of the tubules of frog kidney.

The active transport of organic anions through the plasma membrane of the proximal tubules of frog kidney was studied. For this purpose a marker anion, fluorescein, was used, its flow into the tubules registered by the increase of fluorescense. The kinetics of transport was measured as function of time, concentration of substrate, concentration of a competing acid (p-aminohippuric acid) and temperature. The process is inhibited by strophantin, a specific poison for (Na++K+)-dependent ATPase. These data show that fluorescein transport is effected with the participation of a charged carrier, probably by the downfield mechanism postulated by Mitchell. To confirm this mechanism, a passive flow of K+ was created inwards across the membrane of the proximal tubules by means of valinomycin. It led to the discharge of the membrane and to the inhibition of fluorescein transport. Anions are transported downfield across the membrane, probably in a state of complexes with two Na+ ions. A magnetic field of 10000-28000 oersted inhibits the fluorescein transport strongly. This can be regarded as a proof of the liquid-crystalline structure of biological membranes and demonstrates the importance of this structure for active transport.

Comparison of the effects of pollution on the marine bivalve Donax trunculus in the vicinity of polluted sites with specimens from a clean reference site (Mediterranean Sea)

The physiological and biochemical characteristics of tissue samples of the marine mussel, Donax trunculus, from an oil-polluted site (Qiryat Yam) and from a site adjacent to an industrial factory (Frutarom) producing polyvinyl chloride (PVC) were compared with samples from a clean reference site (Akko). All sites were located along the sandy shores of the Israeli Mediterranean Sea. The mussels from the oil-polluted site showed increased activity of the system of active transport of organic anions (SATOA) in the gills and the renocardial organ and also of the multixenobiotic resistance transporter (MXR) in the gills. In contrast, samples collected near the PVC factory showed a decrease in SATOA activity and no increase in the activity of MXR in the gills. Specimens from the reference site demonstrated a redox state of equilibrium between energy production and utilization, while in Donax from both the oil polluted and the PVC-polluted sites, the mitochondrial redox state reflected intensive consumption of energy. No significant changes were found in the activity of reduced glutathione s-transferase (GST) in the cytosolic fraction of the digestive gland of Donax collected from any of the three sites. The data demonstrate a differential increase in the anti-chemical defense systems and an intensification of energy metabolism in the mussels exposed to pollution.

The two sides of man-induced changes in littoral marine communities: Eastern Mediterranean and the Red Sea as an example

Over the last two centuries, the marine life of the Eastern Mediterranean has been influenced by two major factors: one is beneficial, and concerns species migration, such as the opening of the Suez Canal, which enriched the impoverished eastern Mediterranean Sea with over 300 species of fish and invertebrates of Red Sea origin; while the other, a negative and possibly wider-reaching factor, is that of man-made pollution, which has induced unpredictable changes, destabilizing the biological world in both magnitude and duration. Initially cryptic, the effects caused by pollutants first occur at the biochemical and cellular levels of an organism, causing alterations and deviations from the normal, strongly mobilizing its defense systems. Conventional methodologies of ecological analyses, based on species and specimen numbers, cannot detect such alterations. Studying several mollusk populations from polluted and reference sites of the Red Sea and Mediterranean littoral of Israel, we used specific markers for in vivo and in vitro studies to expose the state of micronucleation; levels of defense transport systems such as membrane transport system of organic anions (SATOA) and organic cations (OCT); the state of the multi-xenobiotic resistance-mediating transporter (MXRtr). Based on fluorescent microscopy and microfluorometry, these markers offer powerful tools to expose cryptic changes in the affected populations and provide data necessary for planning and management to protect animal communities and preserve their biological diversity. Comparative analysis of general gene-expression in polluted and reference sites indicates that stress factors have differentially affected the various biological taxa and separated phenotypic sub-populations, producing a novel type of punctuated selection. Such factors, although negative in their influences, in some instances altering the qualities of organisms, and establishing alterations in their hereditary information, pre-adapt them to survive and succeed in new situations.

DETERMINATION OF PRIMARY AND SECONDARY RESPONSES TO ENVIRONMENTAL STRESSORS AND BIOTA HEALTH

The conventional ecotoxicological methods cannot provide an adequate assessment of environmental health and ecological risk. In this communication we present an innovative scientific base of warning monitoring and selected examples of the using microfluorometrical vital methods to characterize the health of studied species and predict their future ecological fate. For such monitoring we perfect and use the pilot biophysical devices, particularly contact microfluorometers. Our health parameters include measurements of (1) activity of anti-xenobiotic defense mechanisms, particularly export pumps, enzymes of detoxification and conjugation; (2) cell and tissue viability; (3) frequencies of DNA and chromosome damages; (4) frequencies and expression of cyto-and histopathological alterations. This methodology are used to characterize the environmental health of (1) selected molluscan and fish species along the Israeli Mediterranean and Red Sea shores as well as the German North and Baltic Sea shores; (2) selected fish species along the Israeli stream, Yarqon River; (3) Mediterranean benthic foraminifera and Red Sea corals and some other aquatic and terrestrial animals Our data demonstrate that all living organisms have numerous antixenobiotic defense mechanisms and their activities mediate effects of pollutants, determine the health of biota and its further fate, particularly selection and development of new populations and communities. Our methodology allows assessing the biota health and ecological risk of man-made stressors that may be important both for developing and developed countries.