Potential multidrug resistance gene POHL: an ecologically relevant indicator in marine sponges.

Sponges are sessile filter feeders found in all aquatic habitats from the tropics to the arctic. Against potential environmental hazards, they are provided with efficient defense systems, e.g., protecting chaperones and/or the P-170/multidrug resistance pump system. Here we report on a further multidrug resistance pathway that is related to the pad one homologue (POH1) mechanism recently identified in humans. It is suggested that proteolysis is involved in the inactivation of xenobiotics by the POH1 system. Two cDNAs were cloned, one from the demosponge Geodia cydonium and a second from the hexactinellid sponge Aphrocallistes vastus. The cDNA from G. cydonium, termed GCPOHL, encodes a deduced polypeptide with a size of 34,591 Da and that from A. vastus, AVPOHL, a protein of a calculated M(r) of 34,282. The two sponge cDNAs are highly similar to each other as well as to the known sequences from fungi (Schizosaccharomyces pombe and Saccharomyces cerevisiae) and other Metazoa (from Schistosoma mansoni to humans). Under controlled laboratory conditions, the expression of the potential multidrug resistance gene POHL is, in G. cydonium, strongly upregulated in response to the toxins staurosporin (20 microM) or taxol (50 microM); the first detectable transcripts appear after 1 d and reach a maximum after 3 to 5 d of incubation. The relevance of the expression pattern of the G. cydonium gene POHL for the assessment of pollution in the field was determined at differently polluted sites in the area around Rovinj (Croatia; Mediterranean Sea, Adriatic Sea). The load of the selected sites was assessed by measuring the potency of XAD-7 concentrates of water samples taken from those places to induce the level of benzo[a]pyrene monooxygenase (BaPMO) in fish and to impair the multidrug resistance (MDR)/P-170 extrusion pump in clams. These field experiments revealed that the levels of inducible BaPMO activity in fish and of the MDR potential by the water concentrates are highly correlated with the level of expression of the potential multidrug resistance gene POHL in G. cydonium. This report demonstrates that the detoxification POH pathway, here mediated by the G. cydonium GCPOHL gene, is an additional marker for the assessment of the environmental load in a given marine area.

Interspecies differences in P-glycoprotein mediated activity of multixenobiotic resistance mechanism in several marine and freshwater invertebrates.

The presence and function of the P-glycoprotein mediated multixenobiotic resistance (MXR) mechanism was demonstrated in numerous aquatic organisms. The aim of this study was to investigate whether in aquatic organisms exists the inherent, species-specific basal level of MXR activity. Here the results of the direct comparison of the basal (noninduced) level of MXR activity measured in several marine (Mytilus galloprovincialis, Monodonta turbinata, Patella lusitanica) and freshwater (Dreissena polymorpha, Viviparus viviparus, Anodonta cygnea) molluscs species are presented. The primary criterion for the assessment and quantification of the basal level of MXR activity was the ratio (R) between the accumulation or efflux of the fluorescent model MXR substrates (rhodamine B or rhodamine 123) in or from the gills, measured with and in the absence of model MXR inhibitors verapamil or cyclosporin A. Significantly different levels of MXR activity were found in the species investigated. These levels generally show a relatively good correlation with the level of pollution present in their natural habitats. Considering these results a conclusion was reached that in aquatic organisms indeed exist the different inherent, species-specific levels of MXR activity. The identified levels might be, at least partly, responsible either for the resistance to, or for the sensitivity of a particular species to organic pollution.

The chemosensitizers of multixenobiotic resistance mechanism in aquatic invertebrates: a new class of pollutants.

Mechanism of multixenobiotic resistance (MXR), identical to multidrug resistance (MDR) in tumor cells, has been found in aquatic invertebrates. The presence of this ATP-dependent membrane P-glycoprotein (Pgp) pump was confirmed by biochemical ('binding'), molecular (immunohistochemical, Western, Northern), physiological (verapamil-sensitivity) and toxicological (modulation of toxicity) methods. The inducibility of MXR in the presence of xenobiotics and its wide taxonomic distribution suggests its role as a general biological defense mechanism that rescues organisms by pumping potentially toxic xenobiotics out of the cells. Some xenobiotics, the chemosensitizers, can inhibit this defense mechanism. The presence of these MXR-inhibitors has important implications on environmental parameters like exposure, uptake, internal dose, bioaccumulation, response, synergism and toxicity. Such MXR-inhibitors, for example, enhance the accumulation of carcinogenic aromatic amines in mussel, with subsequent enhancement in production of their mutagenic metabolites, in induction of single strand breaks in DNA, and in induction of DNA-adducts. The property to inhibit defense mechanism of organisms classifies MXR-inhibitors among top-hazardous environmental chemicals. Therefore, we measured the concentration of chemosensitizers in water concentrates or sediment extracts as their potential to modulate the accumulation of fluorescent dyes in a cell-culture of NIH 3T3 mouse fibroblasts stable transfected with human MDR1 gene, or as the potential of native waters to decrease the efflux-rate of Rhodamine B from gills of mussels. We found significantly higher concentrations of MXR-inhibitors in samples from polluted marine sites or from polluted rivers than in samples from corresponding unpolluted sites. These concentrations were able to enhance the accumulation of fluorescent dyes or carcinogenic aromatic amines in clams, mussels, snails and sponges exposed to these xenobiotics, demonstrating the ecotoxicological relevance of MXR-inhibitors present in polluted waters.

Inhibitory effects of extracts from the marine alga Caulerpa taxifolia and of toxin from Caulerpa racemosa on multixenobiotic resistance in the marine sponge Geodia cydonium.

The invasive growth of the introduced green alga Caulerpa taxifolia, already affecting the richness and diversity of the littoral ecosystems, has become a major ecological problem in the Mediterranean Sea. Previously, we demonstrated that the water pollutant tributyltin induces apoptosis in tissue of the marine sponge Geodia cydonium at concentrations of 3 µM and higher. Here we show that exposure of G. cydonium to low (non-toxic) concentrations of Caulerpa extract or purified caulerpin (10 µg/ml) together with low doses of tributyltin (1 µM; non-toxic), results in a strong apoptotic effect. Evidence is presented that the enhancement of toxicity of tributyltin by Caulerpa extract is at least partially caused by inhibition of the multixenobiotic resistance (MXR) pump by the algal toxin. Caulerpa extract, as well as caulerpin, strongly enhance the accumulation of the test substrate of MXR, rhodamine B, in the gills of the mussel Dreissena polymorpha, used as a model system for testing MXR-inhibiting potential.

Chemosensitizers of the multixenobiotic resistance in amorphous aggregates (marine snow): etiology of mass killing on the benthos in the Northern Adriatic?

Periodically appearing amorphous aggregates, `marine snow', are formed in the sea and if settled as mats on the sea bottom cause death of benthic metazoans. Especially those animals are killed which are sessile filter feeders, e.g. sponges, mussels, or Anthozoa. The etiology of the toxic principle(s) is not yet well understood. Gel-like marine snow aggregates occurred in the Northern Adriatic during summer 1997. Samples of these aggregates were collected during the period July to September and the outer as well as the inner zones were analyzed for (i) cell toxicity, and (ii) chemosensitizing activity of the multixenobiotic resistance (MXR) mechanism. Organic extracts were prepared and cell toxicity was determined using mouse lymphoma cells. The experiments revealed that the major activity is seen in the center of the mats of the gel-like aggregates; a growth inhibitory activity of up to 54% (correlated to 5 ml of snow sample) was determined. The same extracts were used to determine the inhibition of the P-glycoprotein (Pgp) extrusion pump which confers the multixenobiotic resistance. The analyses were performed with cells from the sponge Suberites domuncula and with gills from the clam Corbicula fluminea in situ. Both systems have been shown to express the Pgp extrusion pump. The data show that extracts from the outer zone of the gel-like aggregate samples display pronounced inhibitory activity on the MXR extrusion pump and hence act as chemosensitizers by reversing the MXP property. These findings indicate that gel-like aggregates contain compounds in the outer zone, chemosensitizer of the Pgp extrusion pump, which lower the level of protection of metazoan animals towards dissolved compounds in their surrounding milieu, and in the center toxic compounds which are-very likely-even in the absence of chemosensitizers hazardous for the invertebrates.

A new type of hazardous chemical: the chemosensitizers of multixenobiotic resistance.

The purpose of this overview is to introduce the property of a new class of hazardous chemicals-the inhibitors of multixenobiotic resistance (MXR) in aquatic organisms, referred to as chemosensitizers. Aquatic organisms possess MXR, a mechanism similar to the well-known P-glycoprotein extrusion pump in multidrug resistant (MDR) tumor cells. MXR in aquatic organism moves from cells and organisms both endogenous chemicals and xenobiotics, including also some man-made chemicals. MXR in aquatic organisms represents a general biological first-line defense mechanism for protection against environmental toxins. Many chemical agents, the chemosensitizers, may after the function of this fragile mechanism. It is this new, MXR-inhibiting property, unrecognized as yet, that classifies these chemicals among top-rank hazardous water pollutants. The knowledge that the presence of one xenobiotic may block the pumping out of other xenobiotic(s), and hence accelerate their accumulation, may have important implications on environmental parameters like exposure, uptake, bioaccumulation, and toxicity. In this overview we present the evidence for the expression of MXR-phenotype in aquatic organisms, the demonstration of toxic consequences caused by MXR inhibitors, and the description of methods for measurement of concentration of MXR inhibitors in environmental samples.

Biotransformation of genotoxic agents in marine sponges. Mechanisms and modulation.

Marine sponges do not appear to suffer from neoplastic diseases, in spite of possible high exposures resulting from their nature as sessile bottom filter feeders which pump large volumes of sea water. The assessment of several parameters related to the biotransformation of mutagens/carcinogens showed that the metabolic machinery of sponge medulla cells is mainly oriented towards detoxification, with some differences depending on species (Geodia cydonium or Tethya aurantium). Glutathione (GSH) levels were unexpectedly high in these cells, especially in Geodia, in which the concentration of this tripeptide was more than twice that measured in liver preparations from untreated rats, at least when related to the protein content. The oxidoreductive enzyme activities involved in the glutathione cycle were balanced in such a way as to favour a high GSH: oxidized glutathione (GSSG) ratio. GSH S-transferase activity was conversely rather low, compared to that of rat liver, and the dehydrogenases involved in the hexose monophosphate shunt were high in Tethya but low in Geodia. The metabolism of mutagens was investigated by using the Salmonella typhimurium his- strains TA100, TA98 and YG1024. Sponge S12 fractions failed to activate aflatoxin B1, benzo[a]pyrene and the two heterocyclic amines 3-amino-1-methyl-5H-pyrido[4,3-b]indole and 2-amino-3,4-dimethyl-imidazo[4,5-f]quinoline. Although far less efficiently than untreated rat liver S12 fractions, Geodia and especially Tethya preparations weakly activated the three aromatic amines 2-acetyl-aminofluorene, 2-aminofluorene and 2-aminoanthracene. On the other hand, sponge S12 fractions were remarkably efficient in decreasing the mutagenic potency of the direct-acting mutagens 4-nitroquinoline 1-oxide and sodium dichromate.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased genotoxicity of acetylaminofluorene by modulators of multixenobiotic resistance mechanism: studies with the fresh water clam Corbicula fluminea.

The presence of a 'multixenobiotic resistance' [MXR] mechanism in gills of the freshwater clam Corbicula fluminea was investigated. Western blot analyses of membrane vesicles from gills, applying antibodies to vertebrate P170 multidrug resistance (MDR) protein, revealed a 135 kDa immunoreactive protein. Verapamil caused a reduction of 3H-vincristine (3H-VCR) binding onto vesicles from clam. Exposure of clams to 3H-VCR in the presence of verapamil or staurosporine (STP) enhanced the accumulation of 3H-VCR over control values. Furthermore, clams were exposed instead to VCR, to a model carcinogen, 2-acetylaminofluorene (AAF), to determine the verapamil- and STP-dependent increase of single-strand breaks (SSBs) in DNA from gills of this organism. Verapamil caused no or little increase of SSBs induced by exposure to 0.01 or 0.10 microM AAF, respectively, as measured by the alkaline elution technique. In contrast, in the presence of STP a highly significant and dose-dependent enhancement of AAF-mediated SSBs was measured already at exposure to 0.01 microM AAF. These data indicate (i) that the clam C. fluminea is provided with a P-glycoprotein-like element of the MDR-mechanism, (ii) that this system can be poisoned by chemosensitizers such as verapamil and STP, (iii) the role of protein kinase C in the regulation of MXR function and (iv) the importance of the MXR modulators for the assessment of ecotoxicological effects of pollutants.

Biomonitoring of aquatic systems.

The 32P-postlabelling analysis provides a sensitive means for detecting pollution-related DNA adducts in aquatic organisms exposed to environmental carcinogens. However, the following factors need to be taken into consideration during the data interpretation: (1) species-specific, naturally occurring DNA modifications (or I-compounds) are found in aquatic organisms at levels which are highly season-dependent; and (2) many aquatic organisms, particularly lower invertebrates, cannot form DNA adducts from common pollutants such as polycyclic aromatic hydrocarbons (PAHs). The level of natural adducts is especially high in lower invertebrates, such as sponges and sea-urchins during their reproductive phase in the spring time (March/April): in subsequent months adducts were either undetectable or present at only trace levels. These invertebrates do not metabolize PAHs such as benzo[a]pyrene but readily biotransform aromatic amines such as 2-acetylaminofluorene to DNA-reactive forms. Pollution-related DNA adducts have been found in fish living in highly polluted rivers and marine sites and in carp exposed to an artificial Diesel-2/crude oil slick. In certain fish (English sole, brown bullheads, etc.) living in polluted environments, the formation of pollution-related DNA adducts has been correlated with an increased incidence of tumours. It is concluded that, while DNA adducts detected in aquatic organisms can be used for biomonitoring and detecting pollutants, there are several confounding factors that should be taken into consideration before one attempts to determine the type and concentration of carcinogenic pollutants present in aquatic environments.

Determination of lectin-cell-binding parameters by a new agglutination technique.

We applied a recently described technique which is based on a light transmission/scattering method to determine the association characteristics of the Geodia lectin to sheep erythrocytes. The agglutination assays were performed in a total volume of 3 ml with 5.4 x 10(6) erythrocytes/ml. At a concentration of 360 ng/ml 50% of the lectin molecules were bound to the cells within the first 10 s of incubation. Scatchard analyses revealed an association constant (K(a)) of 0.9 +/- 0.1 x 10(8) M-1 and a number of 3.8 +/- 0.6 x 10(6) lectin binding sites on one erythrocyte. The method was also successfully applied to determine quantitatively the inhibitory potential of sugars competing with cell surface glycoproteins for the lectin binding site. At a lectin concentration of 360 ng/ml 1 mM of lactose or 70 mM of D-N-acetylgalactosamine are required to inhibit lectin-mediated agglutination by 50%. These analyses confirm the potential of the novel light absorption agglutination technique to evaluate lectin characteristics.

DNA adducts in carp exposed to artificial diesel-2 oil slicks.

In attempts to mimic field exposure, oil slicks prepared from diesel-2 oil/water emulsions were poured onto the surface of water in tanks prepared fresh every day and liver DNA adducts were analyzed by 32P-postlabeling in carp free-swimming in these tanks. 'Clusters' of lipophilic DNA adducts were detected, with five major and numerous minor adducts. Essentially a similar adduct pattern was found in the liver DNA of carp exposed to crude oil-polluted water. Diesel-2 adduct induction was observed slowly with a steady increase to greater than 3000 amol/microgram DNA at day 12. After this time fish were transferred to clean water. Adduct levels continued to increase through day 17 (approximately 10,000 amol/microgram DNA) despite the cessation of exposure, but a 30% and 80% decline was evident at day 22 and day 27, respectively. All major adducts were distinct from the known benzo[a]pyrene diolepoxide-dG. These results indicate that diesel-2 oil can cause extensive DNA damage in carp in vivo and the damage accumulates proportionately with time of exposure.

Immunological and biological identification of tumour necrosis-like factor in sponges: endotoxin that mediates necrosis formation in xenografts.

Xenografts of the sponge Geodia cydonium in its closely related species G. rovinjensis resulted in a rapid rejection of the graft within a period of 5 days. We identified an immunoreactive tumour necrosis factor (TNF)-like activity in the xenograft (Mr of 30,000) two days after grafting. In-vivo injection of 5 micrograms human recombinant TNF-alpha induced cytotoxicity in sponge cells in the same pattern and time course as during natural xenograft rejection. Anti-TNF-alpha polyclonals were found to react with xenograft extracts, by Western blot analysis, as from day 2 after grafting. Using ELISA we detected the TNF-like activity from day 2 after grafting with peak levels at days 4 and 5, where the amount was 0.72 ng/micrograms tissue DNA. By day 1, gp27 (inhibitory aggregation factor) is already formed in the xenograft. In-vitro experiments on isolated G. cydonium cells showed that addition of purified gp27 induced the production of the TNF-like activity (up to 13.5 ng/ml). Evidence is presented that gp27 is a product of the gp180 lectin receptor. We conclude that gp27 induces TNF-like factor production, resulting in destruction and dissolution of the xenograft after 5 days.

The multixenobiotic resistance mechanism in aquatic organisms.

Many aquatic organisms thrive and reproduce in polluted waters. This fact indicates that they are well equipped with a defense system(s) against several toxic xenobiotics simultaneously because water pollution is typically caused by a mixture of a number of pollutants. We have found that the biochemical mechanism underlying such "multixenobiotic" resistance in freshwater and marine mussel, in several marine sponges, and in freshwater fish is similar to the mechanism of multidrug resistance (MDR) found in tumor cells that became refractory to treatment with a variety of chemotherapeutic agents. All these organisms possess a verapamil-sensitive potential to bind 2-acetylaminofluorene and vincristine onto membrane vesicles. They all express mRNA for mdr1 gene, and mdr1 protein product, the glycoprotein P170. Finally, in in vivo experiments, the accumulation of xenobiotics is enhanced in all investigated organisms in the presence of verapamil, the inhibitor of the P170 extrusion pump. The knowledge that the presence of one xenobiotic may block the pumping out, and hence accelerating accumulation, of others, may help us to understand and interpret our present and past data on different environmental parameters obtained using indicator organisms.

Expression of P-glycoprotein gene in marine sponges. Identification and characterization of the 125 kDa drug-binding glycoprotein.

In the present paper it is shown that the marine sponges Geodia cydonium and Verongia aerophoba contain the gene coding for P-glycoprotein P170, also known as a multidrug-resistance gene. Western blot studies revealed that polyclonal antibodies raised against hamster P170 cross-react with the sponge polypeptide of Mr 125,000. After endoglycosidase F treatment, the sponge P125 is converted to a polypeptide of Mr 105,000. Northern blot studies, using the human P170 cDNA probe, revealed a size of 4.2 kb for the sponge P125 transcript. The level of this transcript does not change in response to incubation with the aggregation factor. Confocal laser scanning microscopy showed that P125 is a cell membrane bound protein. In addition, sponge membrane vesicles possess a potential to bind in vitro 2-acetylamino-fluorene, vincristine and daunomycin. This process is Verapamil-sensitive, a characteristic known also for the mammalian vesicle associated P170. The data reported demonstrate that the classical multidrug resistance mechanism, described in drug-resistant tumor cell lines, functions also in sponges and may explain the relative resistance of these animals to pollution.

The potential of carp to bioactivate benzo[a]pyrene to metabolites that bind to DNA.

We have investigated the formation of DNA adducts in starved, fed and 5,6-benzoflavone-pretreated carp following i.p. administration of benzo(a)pyrene. 32P-postlabeling analysis of the liver DNAs showed the presence of one predominant (greater than 92%) adduct in all three groups. Cochromatography experiments revealed that the main adduct was identical to authentic BPDEI-dG (10 beta-(deoxyguanosin-N2-yl)-7 beta, 8 alpha, 9 alpha-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene). The formation of the adduct was evident as early as 1.5 h post-treatment and the levels increased steadily up to 7 days, reaching about 125, 110 and 102 attomole/microgram DNA in starved, fed and benzoflavone-pretreated carp, respectively. During this period, the benzo[a]pyrene-induced benzo[a]-pyrene monooxygenase activity increased from the uninduced, natural level of about 3 pmol/mg per min to levels of 35, 62 and 79 pmol/mg per min in starving, fed and 5,6-benzoflavone pretreated fish, respectively. A slow but steady formation of the diolepoxide-dG adduct was also observed in the liver DNA of carp following p.o. treatment. These results indicate that carp can biotransform polycyclic aromatic hydrocarbons such as benzo[a]pyrene to 'reactive' metabolites that bind to DNA.

Regulated expression and phosphorylation of the 23-26-kDa ras protein in the sponge Geodia cydonium.

We have cloned, sequenced and examined the sponge Geodia cydonium cDNA encoding a protein homologous to ras proteins. The sponge ras protein has a more conserved N-terminal region and a less conserved C-terminal region, especially in comparison to Dictyostelium discoideum; the similarity to human c-Ha-ras-1 and to Saccharomyces cerevisiae is less pronounced. The sponge ras cDNA comprises five TAG triplets; at the translational level these UAG termination codons are suppressed by a Gln-tRNA. The sponge ras protein was isolated and partially purified (23-26 kDa) and found to undergo phosphorylation at a threonine moiety, when dissociated cells were incubated in the presence of a homologous aggregation factor and insulin. Insulin-mediated phosphorylation of the ras protein resulted in a decrease in its Kd with GTP from 2 microM to 80 nM. The activated ras protein displayed high GTPase activity if the partially purified protein was incubated with homologous lectin and lectin receptor molecules. These results suggest that in the sponge, ras is activated by the insulin/insulin(insulin-like)-receptor system. This transition enables the ras protein to interact with the lectin-receptor/lectin complex, a process which may ultimately lead to an initiation of an intracellular signal-transduction chain.

Role of phospholipase A2 in the stimulation of sponge cell proliferation by homologous lectin.

Using the Geodia cydonium system, we showed that after incubation of competent sponge cells in the presence of lectin, phospholipase A2 was released from the cells. The substrates for this enzyme, phosphatidylethanolamine and phosphatidylcholine, were identified in the extracellular material of sponge tissue. In addition, the phospholipase A2 inhibitor calelectrin was identified by immunobiochemical techniques; this molecule was associated with the aggregation factor. Reconstitution experiments strongly suggested that phospholipase A2 catalyzed the release of arachidonic acid, which is then taken up by the cells. Intracellularly, arachidonic acid was metabolized primarily to prostaglandin E2. Inhibition studies revealed that prostaglandin E2 is involved in the ultimate increase of DNA synthesis. These findings suggest that the phospholipase A2-arachidonic acid system is involved in the matrix-initiated signal transduction pathway in sponges.

Distinct glutathione-dependent enzyme activities and a verapamil-sensitive binding of xenobiotics in a fresh-water mussel Anodonta cygnea.

A fresh-water mussel Anodonta cygnea, an aquatic invertebrate resistant to pollution, possesses an inherent high potential to bind 2-acetylaminofluorene onto membrane vesicles. This binding is saturable and trypsin- and verapamil-sensitive. Simultaneously, this mussel reveals a relatively high inherent activity of glutathione-dependent enzyme activities with a distinct spectrum of substrate affinities. Both these activities are similar to the elements of the molecular mechanism involved in the acquired multi-drug resistance phenomenon described in tumor cell-lines. The recognition that in organisms exposed to polluted waters a multi-xenobiotic resistance mechanism may be involved is essential for understanding both the biological impact of pollution and the development of methods for rational risk assessment in regulatory policy.

Natural environment surpasses polluted environment in inducing DNA damage in fish.

Measurement of specific DNA adduct concentrations in target tissues of organisms may provide a key biologic end-point of exposure to environmental carcinogens. Using a general and highly sensitive assay with 32-P-postlabeling, we found that natural populations of freshwater fish species chub, barbel, bream and carp, as well as a marine fish mugil, revealed the presence of four to nine qualitatively similar adducts irrespective of whether they were caught from unpolluted or polluted waters. No statistically significant differences were observed between the adduct levels of fish from the unpolluted waters and those of fish from the polluted waters. A dominant feature of the fish DNA adducts was a species specificity. The finding that a vast majority of DNA modifications in fish are caused by natural factors rather than man-made chemicals offers a basis for a more realistic view in assessing the genotoxic risks in any aquatic environment.