Dielectric relaxation of DNA aqueous solutions.

We report on a detailed characterization of complex dielectric response of Na-DNA aqueous solutions by means of low-frequency dielectric spectroscopy (40 Hz-110 MHz). Results reveal two broad relaxation modes of strength 20

Screening and fundamental length scales in semidilute Na-DNA aqueous solutions.

The fundamental length scales in semidilute Na-DNA aqueous solutions have been investigated by dielectric spectroscopy. The low- and the high-frequency relaxation modes are studied in detail. The length scale of the high-frequency relaxation mode at high DNA concentrations can be identified with the de Gennes-Pfeuty-Dobrynin correlation length of polyelectrolytes in semidilute solution, whereas at low DNA concentrations and in the low added salt limit the length scale shows an unusual exponent reminiscent of semidilute polyelectrolyte chains with hydrophobic backbone. The length scale of the low-frequency relaxation mode corresponds to a Gaussian chain composed of correlation blobs in the low added salt limit, and to the Odijk-Skolnick-Fixman value of the single chain persistence length in the high added salt limit.

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.

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.

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.

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.

Glucuronides in mussel Mytilus galloprovincialis as a possible biomonitor of environmental carcinogens.

1. The in vitro incubation of mussel digestive gland with 1 mM aminofluorene resulted in the formation of glucuronides that (a) became mutagenic with carp liver S9, and (b) liberated S9-dependent mutagenic aglucones after beta-glucuronidase treatment. 2. Natural populations of mussels from unpolluted and polluted sites, as well as mussels exposed to 3 ppm of aminofluorene or to used engine oil, did not accumulate detectable amounts of premutagens, mutagens, or mutagenic glucuronides/aglucones either in digestive gland tissue or in shell-cavity water. 3. The mutagenicity testing of mussel's glucuronides/aglucones does not seem to be useful as a biomonitor of environmental carcinogens.

Metabolic activation of 2-aminofluorene, 2-acetylaminofluorene and N-hydroxy-acetylaminofluorene to bacterial mutagens with mussel (Mytilus galloprovincialis) and carp (Cyprinus carpio) subcellular preparations.

1. The mode of activation of 2-aminofluorene (AF), 2-acetylaminofluorene (AAF) and N-hydroxy-acetylaminofluorene (OH-AAF) to Salmonella typhimurium TA 98 mutagens was investigated in subcellular fractions from the digestive gland of the mussel Mytilus galloprovincialis and from the liver of carp Cyprinus carpio. 2. In carp liver microsomes the activation of OH-AAF was due to very active deacetylase, in contrast to undetectable deacetylase-dependent activation in mussel microsomes. 3. AF and AAF are activated in mussel microsomes exclusively by a noninducible FAD-containing monooxygenase, whereas in carp microsomes in addition deacetylase and inducible cytochrome P-450 monooxygenase are involved. 4. N,O-Acetyltransferase, sulfotransferase and paraoxon sensitive cytosolic enzyme (PSCE) are involved in activation of OH-AAF, AF and AAF in both carp and mussel cytosols. 5. The metabolic activation of OH-AAF, AF and AAF to bacterial mutagens found in carp liver is similar to that described in livers of experimental mammalian species and strikingly different from the mode of activation found in mussel digestive gland.

Metabolism of some carcinogenic aromatic amines in four species of marine sponges.

Postmitochondrial fractions from marine sponges Geodia cydonium, Tethya aurantium, Verongia aerophoba and Pellina semitubulosa activate precarcinogenic aromatic amine 2-aminoanthracene, but not precarcinogenic polycyclic aromatic hydrocarbon benzo(a)pyrene, to Salmonella typhimurium TA 98 mutagens. All four sponge species lack a benzo(a)pyrene monooxygenase activity, but possesses the enzyme activity whose characteristics (selective activation of aromatic amines, NADPH-dependency, pH optimum at 8.4) are similar to FAD-containing monooxygenase. Tethya postmitochondrial fraction possesses an UDP-glucuronyl transferase activity which catalyzes the conjugation of a considerable part of metabolized 2-acetylamino [9-14C]fluorene to water soluble glucuronides. The possible ecological significance of exuded aromatic amine metabolites as well as the significance of the presence of the selective potential for the activation of aromatic amines to mutagens among sponges for our understanding of the fate and effects of carcinogens in the marine environment are discussed.