Oxidative DNA damage induced by iron chloride in the larvae of the lace coral Pocillopora damicornis.

Biochemical and molecular biomarkers tools are utilized as early warning signatures of contaminant exposure to target and non-target organisms. The objective of this study was to investigate the sublethal effects of iron chloride to the larvae of the lace coral Pocillopora damicornis by measuring a suit of oxidative-stress biomarkers. The larvae were exposed to a range of sublethal concentrations of iron chloride (0.01, 0.1, 1, 10, and 100 ppm) for seven days. With reference to oxidative stress biomarkers, the no-observed effect concentration (NOEC) and the lowest observed effect concentration (LOEC) of iron chloride were observed to be 0.01 and 100 ppm respectively. At the end of the seventh day the antioxidant status of the larvae was evaluated by the levels of glutathione (GSH), glutathione peroxidase (GPX), glutathione reductase (GR), and glutathione-S-transferase (GST), in both experimental and control groups. For the quantification of cellular oxidative damage, lipid peroxidation (LPO) activity was determined in the same and the extent of DNA damage was assessed by the expression of DNA apurinic/apyrimidinic (AP) sites. Iron chloride exhibited a concentration-dependent inhibition of GSH and GPX and induction of GR, GST, LPO, and DNA-AP sites in the P. damicornis larvae when compared to the control group. The oxidative stress biomarkers of the larvae exposed to 0.1, 1, and 10 ppm of iron chloride did not show any significant overall differences when compared to the control group. However the activities of LPO, GSH, GPX, GR, GST and DNA-AP in the larval group exposed to 100 ppm of iron chloride exhibited statistically significant (P=0.002, 0.003, 0.002, 0.002, 0.005 and 0.007) differences when compared to the control group. The research results indicated that iron chloride in concentrations at the 100 ppm level caused oxidative stress in the P. damicornis larvae.

Aragonite crystallization in primary cell cultures of multicellular isolates from a hard coral, Pocillopora damicornis.

The foundation of marine coral reef ecosystems is calcium carbonate accumulated primarily by the action of hard corals (Coelenterata: Anthozoa: Scleractinia). Colonial hard coral polyps cover the surface of the reef and deposit calcium carbonate as the aragonite polymorph, stabilized into a continuous calcareous skeleton. Scleractinian coral skeleton composition and architecture are well documented; however, the cellular mechanisms of calcification are poorly understood. There is little information on the nature of the coral cell types involved or their cooperation in biocalcification. We report aragonite crystallization in primary cell cultures of a hard coral, Pocillopora damicornis. Cells of apical coral colony fragments were isolated by spontaneous in vitro dissociation. Single dissociated cell types were separated by density in a discontinuous Percoll gradient. Primary cell cultures displayed a transient increase in alkaline phosphatase (ALP) activity, to the level observed in intact corals. In adherent multicellular isolate cultures, enzyme activation was followed by precipitation of aragonite. Modification of the ionic formulation of the medium prolonged maintenance of isolates, delayed ALP activation, and delayed aragonite precipitation. These results demonstrate that in vitro crystallization of aragonite in coral cell cultures is possible, and provides an innovative approach to investigate reef-building coral calcification at the cellular level.

Isolation and characterization of the retinoblastoma protein from fish.

The retinoblastoma (Rb) gene represents the first tumor suppressor gene characterized. The encoded protein, pRb, plays a crucial role in cell cycle control, preventing malignant cell proliferation. Recently, homologues of the Rb gene have been isolated in fish and the pocket domain, which is central to Rb function, was conserved. In our studies, using coelocanth (Latimeria chalumnae), rainbow trout (Oncorhynchus mykiss), medaka (Oryzias latipes) and English sole (Parophrys vetulus), we have developed a simple protocol for the isolation of the Rb tumor suppressor protein and determined its' tissue and cellular localization. Fish Rb proteins display apparent molecular weights in the range of 100-110 kDa, similar to the human pRb. The protein was detected in all tissues examined, consistent with the proteins' universal role in cellular signalling. An interesting pattern of immunoreactive bands was detected in each of the cells' two main compartments, suggesting differential proteolysis. Immuno-analysis of the pRb in trout liver tumor material revealed an additional Rb reactive product that was absent in normal liver cell extracts.

Structure, expression and activation of fish ras genes.

Ras genes encode proteins that play a central role in cell growth signaling cascades. The fish ras genes characterized to date, have a high degree of nucleotide sequence and deduced amino acid similarity with the mammalian ras gene counterparts. A large proportion and wide variety of mammalian tumors possess mutant forms of ras. In such cases, the localization of ras mutations has been restricted to exons I and II, and to codons 12, 13 and 61. Experimental exposure of fish to a range of genotoxic compounds has similarly led to the production of a ras mutational profile for selected species. The inducing compound, tissue investigated and the fish species studied affect the ras mutational spectrum and incidence observed, despite the apparent conserved sequence homology. Furthermore, the fish ras mutational profile differs from that observed in rodent models, including a novel codon (16) mutation. The role of ras genes in tumor formation in feral fish has been investigated using several species collected from areas of high hydrocarbon contamination. Tomcod (Microgadus tomcod), winter flounder (Pseudopleuronectes americanus) and dragonet (Callionymus lyra) liver samples display evidence of ras gene mutations, though for the latter species the codon affected is not characteristic of ras gene mutational profiles. English sole (Pleuronectes vetulus) and European flounder (Platichthys flesus) liver tumor samples so far examined, on the other hand, do not display ras gene mutations. Thus, the pattern and incidence of ras gene mutations in environmentally-induced tumors also appear to be species specific. In determining the basis of both the species susceptibility observed in the field and species differences in effects of laboratory controlled exposures, the interaction of fish ras genes with other components of the cell growth signaling cascade (such as protein kinase C, additional oncogenes and tumor suppressor genes) are discussed. The effect of promoting agents following contaminant-induced initiation could similarly provide answers in unraveling the question of species susceptibility.

Cloning of the Retinoblastoma cDNA from the Japanese medaka (Oryzias latipes) and preliminary evidence of mutational alterations in chemically-induced retinoblastomas.

We have cloned a medaka homolog of the human retinoblastoma (Rb) susceptibility gene. The medaka Rb cDNA encodes a predicted protein of 909 amino acids. DNA sequence analysis with other vertebrate Rb sequences demonstrates that the medaka Rb cDNA is highly conserved in regions of functional importance. An antibody raised against an epitope of the human pRb recognizes the protein product of the medaka Rb gene, detecting a 105 kDa protein in all tissues examined and at differential levels for the stages of embryonic development studied. The sequence reported herein, combined with the high degree of conservation observed in critical domains, has also facilitated a preliminary investigation of the molecular etiology of chemically-induced retinoblastoma. The mutational alterations characterized suggest that medaka may provide a novel model and, thus, provide additional insight into the human retinoblastoma condition.

Retinoblastoma gene mutations in chemically induced liver tumor samples of Japanese medaka (Oryzias latipes).

Alterations in the retinoblastoma ( Rb) gene have been correlated with a large number and wide variety of human tumors, including hepatocellular carcinoma. We have previously characterized a medaka homologue of the human Rb complementary DNA that is conserved in regions of functional importance. Structural alterations in the entire coding region (exons 1 to 27) of the Rb gene in methylene-chloride-induced medaka liver tumors were investigated using polymerase chain reaction and single strand conformation polymorphism analysis. Four of 5 liver tumors were found to have Rb alterations. Sequencing revealed 7 point mutations in exons 18 and 23, resulting in 5 amino acid substitutions, and a deletion within exon 19. Our results suggest that the molecular etiology of the medaka hepatocellular carcinoma models appear similar to that reported in humans. As such, the medaka appears to be a valid model for the study of Rb-implicated tumorigenesis.

Single 8-oxo-guanine and 8-oxo-adenine lesions induce marked changes in the backbone structure of a 25-base DNA strand.

Structural changes in a 25-base DNA strand, induced by single 8-oxo-guanine or 8-oxo-adenine substitutions, were shown by using Fourier transform-infrared spectroscopy with multivariate statistics. Pronounced differences were demonstrated between the parent and derivatives with respect to base interactions and changes in the phospho-deoxyribose backbone. The greatest degree of change in the backbone likely occurred immediately adjacent to the 8-oxo group, potentially altering the stereochemistry at a distance. The 8-oxo lesions, formed from reactive oxygen species (e.g., hydroxyl radicals), may appreciably alter the conformational properties of strands at the replication fork, thus affecting the selectivity of polymerases, the proofreading capability of repair enzymes, and the fidelity of the transcriptional machinery.

Capillary electrochromatography with novel stationary phases. IV. Retention behavior of glycosphingolipids on porous and non-porous octadecyl sulfonated silica.

In this investigation, capillary electrochromatography (CEC) with a novel stationary phase proved useful for the separation of neutral and acidic glycosphingolipids (GSLs). Four different gangliosides, namely G(M1a), G(D1a), G(D1b) and G(T1b), served as the acidic GSLs model solutes. The following four GSLs: galactosylceramide (GalCer), lactosylceramide (LacCer), globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer) served as the typical neutral GSLs. The stationary phase, octadecyl sulfonated silica (ODSS), consisted of octadecyl functions bonded to a negatively charged layer containing sulfonic acid groups. Porous and non-porous ODSS stationary phases were examined. The retention behavior of the acidic and neutral GSLs was examined over a wide range of elution conditions, including the nature of the electrolyte and organic modifier and the pH of the mobile phase. The porous ODSS stationary phase yielded the separation of the four different gangliosides using a hydro-organic eluent of moderate eluent strength whereas the non-porous ODSS stationary phase permitted the separation of the four neutral GSLs with a mobile phase of relatively high eluent strength.

Rapid transition in the structure of a coral reef community: the effects of coral bleaching and physical disturbance.

Coral reef communities are in a state of change throughout their geographical range. Factors contributing to this change include bleaching (the loss of algal symbionts), storm damage, disease, and increasing abundance of macroalgae. An additional factor for Caribbean reefs is the aftereffects of the epizootic that reduced the abundance of the herbivorous sea urchin, Diadema antillarum. Although coral reef communities have undergone phase shifts, there are few studies that document the details of such transitions. We report the results of a 40-month study that documents changes in a Caribbean reef community affected by bleaching, hurricane damage, and an increasing abundance of macroalgae. The study site was in a relatively pristine area of the reef surrounding the island of San Salvador in the Bahamas. Ten transects were sampled every 3-9 months from November 1994 to February 1998. During this period, the corals experienced a massive bleaching event resulting in a significant decline in coral abundance. Algae, especially macroalgae, increased in abundance until they effectively dominated the substrate. The direct impact of Hurricane Lili in October 1996 did not alter the developing community structure and may have facilitated increasing algal abundance. The results of this study document the rapid transition of this reef community from one in which corals and algae were codominant to a community dominated by macroalgae. The relatively brief time period required for this transition illustrates the dynamic nature of reef communities.

Isolation and primary culture of viable multicellular endothelial isolates from hard corals.

Conditions for the primary culture of branching scleractinian coral (Acropora micropthalma and Pocillopora damicornis) cells were established with a calcium-free seawater cell dissociation method. Cells were isolated and cultured in supplemented Dulbecco's modified Eagle media with heat-inactivated fetal bovine serum, antibiotics, and sterile seawater. Among the isolated cell types, large (60-100 microm) multicellular endothelial isolates (MEIs) were seen in high numbers. These isolates were observed to continually spin for up to 300 h without media change. The following parameters were optimized: media, serum, light, trace elements, and growth factor supplements. Rotations per minute were calculated to determine MEI motility in relation to size. Finally, analyses of external and internal structures were conducted with scanning electron microscopy, transmission electron microscopy, and fluorescence microscopy. Additional coral species, Montipora digitata, Stylophora pistillata, Seriatopora hystrix and Porites sp. were also cultured to determine the applicability of isolation techniques. The relatively long survival time of MEIs in primary culture makes them ideal candidates for in vitro studies examining coral disease processes (e.g., mode of infection and intracellular effects of disease-causing agents) as well as aspects of general coral growth and health (e.g., trace element requirements and transfer of products between host cell and zooxanthellae).

Hydrogen peroxide induces oxidative DNA damage in rat type II pulmonary epithelial cells.

Type II epithelial cells, which line the alveolar surface of the lung, are exposed to a variety of potentially mutagenic and carcinogenic insults. The purpose of this study was to determine if type II cells are susceptible to oxidative DNA damage in vitro. Treatment of cultured rat type II lung epithelial cells with hydrogen peroxide led to increased concentrations (nmol/mg DNA) of 12 of 14 monitored DNA base modifications, suggesting oxidative damage by the hydroxyl radical. These base modifications are typically associated with oxidative stress, and elevated levels have been correlated with mutagenesis and carcinogenesis. These data demonstrate that type II cells are indeed vulnerable to oxidative DNA damage.

Tolerance of an albino fish to ultraviolet-B radiation.

We exposed albino and pigmented medaka Oryzias latipes to simulated solar ultraviolet-B (UVB) radiation to determine if albino medaka were less tolerant of UVB radiation than medaka pigmented with melanin. There was no difference in the number of albino and pigmented medaka that died during the exposure period. Spectrophotometric analyses of the outer dorsal skin layers from albino and pigmented medaka indicated that, prior to exposure, both groups of fish had similar amounts of an apparent colorless non-melanin photoprotective substance that appears to protect other fish species from UVB radiation. Our results indicate that albino medaka were as tolerant of UVB radiation as pigmented medaka because they had similar amounts of this photoprotective substance in the outer layers of the skin.

Coral bleaching: a potential biomarker of environmental stress.

Coral bleaching refers to the loss of symbiotic algae by host corals, or to the loss of pigmentation by the algae themselves, causing corals to appear white or "bleached." Some corals may regain algae or pigmentation and survive, but when bleaching is severe the host coral dies. Coral bleaching events have increased dramatically in the last two decades, and coral reefs throughout the world have been extensively degraded as a result. This article reviews coral bleaching for investigators working in the field of toxicology and environmental health, a group of scientists not normally exposed to this issue. Several environmental stressors have been correlated with bleaching, including fluctuations in sea surface temperatures and salinity, increased sedimentation, increased solar radiation, and contaminants such as oil and herbicides. Molecular mechanisms of bleaching are only beginning to be investigated and are thus far poorly understood. Toxicologists have the potential to make significant contributions toward understanding anthropogenic aspects of coral bleaching and elucidating molecular mechanisms of this important environmental problem.

Capillary electrophoresis of carboxylated carbohydrates. III. Selective precolumn derivatization of glycosaminoglycan disaccharides with 7-aminonaphthalene-1,3-disulfonic acid fluorescing tag for ultrasensitive laser-induced fluorescence detection.

Eight different glycosaminoglycan-derived disaccharides were selectively labeled via their carboxylic acid group with 7-aminonaphthalene-1,3-disulfonic acid (ANDSA) by a condensation reaction between the amino group of ANDSA and the carboxylic acid group of the saccharides in the presence of water-soluble carbodiimide. This derivatization reaction yielded stable derivatives with percentage yields as high as 97%. The ANDSA disaccharide derivatives were readily detected by on-column laser induced fluorescence (LIF) with a He-Cd laser at 325 nm. With LIF, the limit of detection was at the nanomolar level, three orders of magnitude lower than the limit of detection of underivatized disaccharides in the uv at 231 nm. In addition, due to the presence of two strong sulfonic acid groups in the ANDSA tag, the derivatives were readily separated at acidic pH (i.e., pH 4.0-5.0) using 100 mM sodium acetate buffers as the running electrolytes. The addition of polycationic spermine in small amounts to the running electrolytes provided different selectivity with baseline resolution in the pH range 6.0-7.0, and the excess ANDSA migrated ahead of the ANDSA disaccharides.

Capillary electrophoresis of carboxylated carbohydrates. IV. Adjusting the separation selectivity of derivatized carboxylated carbohydrates by controlling the electrolyte ionic strength at subambient temperature and in the absence of electroosmotic flow.

The effect of the ionic strength of the running electrolyte on selectivity and resolution of 7-aminonaphthalene-1,3-disulfonic acid (ANDSA) derivatives of carboxylated monosaccharides and sialooligosaccharides derived from gangliosides was evaluated in capillary electrophoresis in the absence of electroosmotic flow and at subambient temperature. The acidic saccharides used in this study were derivatized with ANDSA fluorescing tag to facilitate their detection by laser-induced fluorescence. To maximize resolution among the derivatized saccharides, commercially available fused-silica capillaries with 'zero' electroosmotic flow having polyvinyl alcohol coating on their inner walls were used as the separation capillaries. The effective electrophoretic mobility (mu) of the various ANDSA derivatized mono- and oligosaccharides decreased linearly with the inverse of the square root of the buffer concentration (1/square root of C) used in the running electrolyte. The extent of screening of the charge on the solute by the electrolyte counterions varied among the various saccharides as was manifested by the slopes of the lines of mu vs. 1/square root of C. Increasing the ionic strength of the running electrolyte allowed, via its charge screening effect, the modulation of selectivity thus adjusting the resolution of closely related saccharides.

Isolation and characterization of biliary epithelial cells from rainbow trout liver.

Lectin binding and density gradient centrifugation were explored for isolating epithelial cells from trout liver. Hepatocytes exhibited preferential attachment of coverslips coated with Phaseolus vulgaris erythroagglutinin. Biliary epithelial cells attached with glycine max agglutinin; however, significant attachment of cellular debris limited the use of glycine max agglutinin. Percoll-density gradient centrifugation separated liver cells into two distinct populations with biliary cells and hepatocytes banding at densities of 1.04 and 1.09, respectively. A discontinuous gradient composed of 13% Ficoll (wt/wt) separated biliary cells from hepatocytes. The recovery of highly enriched biliary epithelial cells from trout liver using Ficoll gradients yielded approximately 8 million cells (0.1 ml packed cells) from 10 g liver. Western blot analysis demonstrated that the cytokeratin profile for extracts from biliary epithelial cell-enriched populations differ significantly from those seen with whole liver extracts or with extracts with hepatocyte-enriched populations. Ficoll-gradient purified biliary cells and hepatocytes attached to culture plates coated with trout skin extract and carried out linear incorporation of leucine into protein and thymidine into DNA for 24 h. A mixture of growth hormones (insulin, epidermal growth factor, and dexamethasone) stimulated thymidine incorporation into DNA; however, long-term culture of dividing biliary epithelial cells was not achieved. Chemical analysis of neutral and acidic glycolipids indicated that hepatocytes and biliary cells have similar glycolipid profiles with an exception in the region of GM3 mobility, which is attributed to differences in the ceramide moiety. These studies provide a starting point for further characterization of unique cell types of the trout liver that may be important in their responses to toxic and carcinogenic agents.

Relationships among petroleum refining, water and sediment contamination, and fish health.

Water, sediment, and fish were sampled from three streams that were receiving or had received effluents from oil refineries. Water and sediment samples were analyzed by gas chromatography/mass spectrometry. Each stream contained aromatic carbons including substituted benzenes and naphthalenes, which are related to oil refinery operations. Fish were identified, counted, and examined for external lesions. Lengths and weights were recorded for older bullhead catfish, and their livers were examined histologically. Differences were seen in the diversity and abundance of fish among the upstream, impacted (effluent-receiving), and downstream stations. In one stream, differences in liver pathology were observed between reference bullhead, collected from an upstream station, and those collected at impacted stations with more than 50% of the bullheads taken from impacted stations having some sort of pathological change, including one with a liver clear-cell focus, which is considered a preneoplastic lesion in rodents. These data suggest a correlation between contamination of water and sediments with aromatic hydrocarbons, presumably from refinery effluents, and compromised fish health.

Capillary electrophoresis of carboxylated carbohydrates. Part 2. Selective precolumn derivatization of sialooligosaccharides derived from gangliosides with 7-aminonaphthalene-1,3-disulfonic acid fluorescing tag.

The most suitable conditions for selective precolumn derivatization of sialooligosaccharides, derived from gangliosides, with 7-aminonaphthalene-1,3-disulfonic acid (ANDSA) and the subsequent separation of the derivatives by capillary electrophoresis are described. ANDSA-sialooligosaccharide derivatives, which fluoresce at 420 nm when excited at 315 nm, were readily detected in capillary electrophoresis using an on-column lamp-operated fluorescence detector. In addition, the precolumn derivatization described here, which exploited the reactivity of the carboxylic acid group of the sialic acid residue of the oligosaccharides, replaced each weak carboxylic acid group of the parent sugar by two strong sulfonic acid groups. This allowed for electrophoresis over a wide range of pH and improved the resolution of the derivatives when compared to those obtained with underivatized sialooligosaccharides under identical separation conditions. The separation of sialooligosaccharides was best achieved when 75 mM borate, pH 10.0, was used as the running electrolyte. The derivatization and separation conditions described herein are expected to be readily transposed to the capillary electrophoresis of other sialooligosaccharides such as those derived from glycoproteins.