Toxicogenomic analysis of immune system-related genes in Japanese flounder (Paralichthys olivaceus) exposed to heavy oil

Heavy oil contamination is one of the most important environmental issues. Toxicities of polycyclic aromatic hydrocarbons (PAHs), including immune toxicities, are well characterized, however, the immune toxic effects of heavy oil, as a complex mixture of PAHs, have not been investigated. In the present study, we selected Japanese flounder (Paralichthys olivaceus) as a model organism, and observed alteration of immune function by the exposure to heavy oil. To analyze the expression profiles of immune system-related genes, we selected 309 cDNAs from our flounder EST library, and spotted them on a glass slide. Using this cDNA array, alteration of gene expression profiles was analyzed in the kidneys of flounders exposed to heavy oil. Six Japanese flounders (mean body weight: 197 g) were acclimated to laboratory conditions at 19–20 °C. Three fish were exposed to heavy oil C (bunker C) at a concentration of 3.8 g/L for 3 days, and the others were kept in seawater without heavy oil and used as the control. After the exposure period, the fish were transferred into control seawater and maintained for 4 days, and then they were dissected and their kidneys were removed. Total RNA was extracted from the kidney samples to use in gene expression analyses. The microarray detected alteration of immune system-related genes in the kidneys of heavy oil-exposed flounders, including down-regulation of immunoglobulin light chain, CD45, major histocompatibility complex class II antigens and macrophage colony-stimulating factor precursor, and up-regulation of interleukin-8 and lysozyme. These results suggest that pathogen resistance may be weakened in heavy oil-exposed fish, causing a subsequent bacterial infection, and then proinflammatory genes may be induced as a defensive response against the infection. Additionally, we found candidate genes for use as biomarkers of heavy oil exposure, such as N-myc downstream regulated gene 1 and heat shock cognate 71 kDa proteins.     

Toxicogenomic analysis of immune system-related genes in Japanese flounder (Paralichthys olivaceus) exposed to heavy oil

Heavy oil contamination is one of the most important environmental issues. Toxicities of polycyclic aromatic hydrocarbons (PAHs), including immune toxicities, are well characterized, however, the immune toxic effects of heavy oil, as a complex mixture of PAHs, have not been investigated. In the present study, we selected Japanese flounder (Paralichthys olivaceus) as a model organism, and observed alteration of immune function by the exposure to heavy oil. To analyze the expression profiles of immune system-related genes, we selected 309 cDNAs from our flounder EST library, and spotted them on a glass slide. Using this cDNA array, alteration of gene expression profiles was analyzed in the kidneys of flounders exposed to heavy oil. Six Japanese flounders (mean body weight: 197 g) were acclimated to laboratory conditions at 19-20 degrees C. Three fish were exposed to heavy oil C (bunker C) at a concentration of 3.8 g/L for 3 days, and the others were kept in seawater without heavy oil and used as the control. After the exposure period, the fish were transferred into control seawater and maintained for 4 days, and then they were dissected and their kidneys were removed. Total RNA was extracted from the kidney samples to use in gene expression analyses. The microarray detected alteration of immune system-related genes in the kidneys of heavy oil-exposed flounders, including down-regulation of immunoglobulin light chain, CD45, major histocompatibility complex class II antigens and macrophage colony-stimulating factor precursor, and up-regulation of interleukin-8 and lysozyme. These results suggest that pathogen resistance may be weakened in heavy oil-exposed fish, causing a subsequent bacterial infection, and then proinflammatory genes may be induced as a defensive response against the infection. Additionally, we found candidate genes for use as biomarkers of heavy oil exposure, such as N-myc downstream regulated gene 1 and heat shock cognate 71 kDa proteins.     

Does heavy oil pollution induce bacterial diseases in Japanese flounder Paralichthys olivaceus?

As basic research for the effect of heavy oil on the fish immune system, in this study, the number of leukocyte was counted in Japanese flounder Paralichthys olivaceus, after exposure to heavy oil at a concentration of 30 g/8 L for 3 days. To compare the numbers of bacteria in the skin mucus between oil-exposed and control fish, viable bacteria were enumerated by counting colony forming unit (CFU). Compared with 5.79 ± 1.88 × 10⁷ leukocytes/mL in the controls, the exposed fish demonstrated higher counts, averaging 1.45 ± 0.45 × 10⁸ cells/mL. The bacterial numbers of control fish were 4.27 ± 3.68 × 10⁴ CFU/g, whereas they were 4.58 ± 1.63 × 10⁵ CFU/g in the exposed fish. The results suggest that immune suppression of the fish occurred due to heavy oil stressor, and bacteria could invade in the mucus, resulting in the increasing leukocyte number to prevent infectious disease.     

Does heavy oil pollution induce bacterial diseases in Japanese flounder Paralichthys olivaceus?

As basic research for the effect of heavy oil on the fish immune system, in this study, the number of leukocyte was counted in Japanese flounder Paralichthys olivaceus, after exposure to heavy oil at a concentration of 30 g/8L for 3 days. To compare the numbers of bacteria in the skin mucus between oil-exposed and control fish, viable bacteria were enumerated by counting colony forming unit (CFU). Compared with 5.79+/-1.88 x 10(7)leukocytes/mL in the controls, the exposed fish demonstrated higher counts, averaging 1.45+/-0.45 x 10(8)cells/mL. The bacterial numbers of control fish were 4.27+/-3.68 x 10(4)CFU/g, whereas they were 4.58+/-1.63 x 10(5)CFU/g in the exposed fish. The results suggest that immune suppression of the fish occurred due to heavy oil stressor, and bacteria could invade in the mucus, resulting in the increasing leukocyte number to prevent infectious disease.     

Does heavy oil pollution induce bacterial diseases in Japanese flounder Paralichthys olivaceus?

As basic research for the effect of heavy oil on the fish immune system, in this study, the number of leukocyte was counted in Japanese flounder Paralichthys olivaceus, after exposure to heavy oil at a concentration of 30 g/8 L for 3 days. To compare the numbers of bacteria in the skin mucus between oil-exposed and control fish, viable bacteria were enumerated by counting colony forming unit (CFU). Compared with 5.79 ± 1.88 × 10⁷ leukocytes/mL in the controls, the exposed fish demonstrated higher counts, averaging 1.45 ± 0.45 × 10⁸ cells/mL. The bacterial numbers of control fish were 4.27 ± 3.68 × 10⁴ CFU/g, whereas they were 4.58 ± 1.63 × 10⁵ CFU/g in the exposed fish. The results suggest that immune suppression of the fish occurred due to heavy oil stressor, and bacteria could invade in the mucus, resulting in the increasing leukocyte number to prevent infectious disease.     

Biomarker responses in pelagic and benthic fish over 1 year following the Hebei Spirit oil spill (Taean, Korea)

After the Hebei Spirit oil spill incident (7th December, 2007) in the west coast of Korea, contamination of biliary PAH metabolite and hepatic biomarkers in a pelagic and a benthic fish was monitored for 1 year. Concentrations of 16 PAHs and alkylated PAHs in fish muscle were highest (22.0 ng/g d.w. for 16 PAHs and 284 ng/g d.w. for alkylated PAHs) at 5 days after the spill and then decreased rapidly to background levels at 11 months after the spill. Fish from the oiled site had elevated biliary PAH metabolite concentrations immediately after the spill; these declined steadily in both species, but were still above reference site concentrations 2 months after the spill. Oiled-site fish showed hepatic CYP 1A induction whose trend closely followed those of biliary PAH metabolite concentrations, implying continuous exposure to PAHs. Brain acetylcholinesterase activity was not related to oil exposure.     

Effect of dispersed crude oil exposure upon the aerobic metabolic scope in juvenile golden grey mullet (Liza aurata)

This study evaluated the toxicity of dispersant application which is, in nearshore area, a controversial response technique to oil spill. Through an experimental approach with juveniles of Liza aurata, the toxicity of five exposure conditions was evaluated: (i) a chemically dispersed oil simulating dispersant application; (ii) a single dispersant as an internal control of chemically dispersed oil; (iii) a mechanically dispersed oil simulating natural dispersion of oil; (iv) a water soluble fraction of oil simulating an undispersed and untreated oil slick and (v) uncontaminated seawater as a control exposure condition. The relative concentration of PAHs (polycyclic aromatic hydrocarbons) biliary metabolites showed that the incorporation of these toxic compounds was increased if the oil was dispersed, whether mechanically or chemically. However, toxicity was not observed at the organism level since the aerobic metabolic scope and the critical swimming speed of exposed fish were not impaired.     

Dietary habits and heavy metal concentrations in fish from the Severn estuary and Bristol Channel

Comparisons of the rates of growth, feeding habits and heavy metal levels of flounders from Barnstaple and Oldbury-on-Severn have shown that at all ages the fish from the North Devon coast are larger than those from the middle Severn estuary and that there are marked differences in diet. These differences in diet may contribute to the much higher zinc levels of the Barnstaple flounder samples.In the six other fish species which have been examined, there is a distinct correlation between the cadmium concentrations of the tissues and the proportion of crustaceans in the diet. Lead concentrations appear to follow a similar trend, but no relationship could be detected between diet and tissue zinc levels.     

Alteration of gene expression profiles in the brain of Japanese medaka (Oryzias latipes) exposed to KC-400 or PCB126

Polychlorinated biphenyls (PCBs) are known as neurotoxic chemicals and possibly alter animal behavior. We previously reported that PCB-exposure induced abnormal schooling behavior in Japanese medaka (Oryzias latipes). This abnormal behavior might be caused by the functional alteration of central or terminal nervous system. To understand the mechanism(s) of behavioral change by PCB-exposure, we analyzed the gene expression profiles in the brain of medaka exposed to 3,3′,4,4′,5-pentachlorobiphenyl (PCB126) or a PCB mixture (Kanechlor-400: KC-400) using a cDNA microarray that we constructed. Twelve FLF-II strain medaka (six individuals per treatment) were dietary exposed to PCB126 (0.01 μg/g b.w./day) or KC-400 (1 μg/g b.w./day) for three weeks. For the control, six fish were fed a control diet. After the exposure period, fish were dissected, and the brain samples were collected. The samples from control fish were pooled and used as a common reference in the microarray experiment. Microarray data were normalized by the LOWESS method, and we screened the genes whose expression levels were altered more than 1.5-fold. Gene expression profiling showed 97 down-regulated and 379 up-regulated genes in the brain of medaka exposed to PCB126. KC-400 exposure suppressed 15 genes and induced 266 genes in medaka brain. Among these genes, the expression levels of 7 and 188 genes were commonly down- or up-regulated, respectively in both treatment groups. On the other hand, 31 gene expressions were significantly different between PCB126 and KC-400 treatment groups, and three out of 31 genes were received opposite effects. In addition, the microarray data showed that thyroid hormone-responsive genes were up-regulated by PCB-exposure, which may imply that PCBs or their metabolites mimic thyroid hormone effects in the brain of PCB-exposed medaka.     

Enrichment and partitioning of polycyclic aromatic hydrocarbons in the sea surface microlayer and subsurface water along the coast of Xiamen Island,China

Sea microlayer (SML) and subsurface water (SSW) samples were collected around Xiamen Island to study the enrichment and partitioning of polycyclic aromatic hydrocarbons (PAHs). Total PAH concentrations ranged from 93.43 to 411.05 ng L⁻¹ in the SML and 49.29–279.42 ng L⁻¹ in the SSW. Compared with the results of previous studies before pollution control measurements, PAHs levels decreased significantly. The enrichment factors (EFs) of dissolved and particulate PAHs varied from 0.68 to 2.71 and 0.43–3.56. EFs showed the consistent enrichment trends with sites and exhibited different enrichment characteristics between 2 and 3 ring PAHs and 4 ring PAHs. Furthermore, the much higher concentrations of BaP (strong carcinogenicity) were accompanied by higher EFs in the SML samples from the Western Xiamen Harbour, which together indicated the risk of impacts to the fish eggs that usually float on the SML water after exposure to oil spills and combustion, contributed directly by the port and shipping activities.     

Alteration of gene expression profiles in the brain of Japanese medaka (Oryzias latipes) exposed to KC-400 or PCB126

Polychlorinated biphenyls (PCBs) are known as neurotoxic chemicals and possibly alter animal behavior. We previously reported that PCB-exposure induced abnormal schooling behavior in Japanese medaka (Oryzias latipes). This abnormal behavior might be caused by the functional alteration of central or terminal nervous system. To understand the mechanism(s) of behavioral change by PCB-exposure, we analyzed the gene expression profiles in the brain of medaka exposed to 3,3′,4,4′,5-pentachlorobiphenyl (PCB126) or a PCB mixture (Kanechlor-400: KC-400) using a cDNA microarray that we constructed. Twelve FLF-II strain medaka (six individuals per treatment) were dietary exposed to PCB126 (0.01 μg/g b.w./day) or KC-400 (1 μg/g b.w./day) for three weeks. For the control, six fish were fed a control diet. After the exposure period, fish were dissected, and the brain samples were collected. The samples from control fish were pooled and used as a common reference in the microarray experiment. Microarray data were normalized by the LOWESS method, and we screened the genes whose expression levels were altered more than 1.5-fold. Gene expression profiling showed 97 down-regulated and 379 up-regulated genes in the brain of medaka exposed to PCB126. KC-400 exposure suppressed 15 genes and induced 266 genes in medaka brain. Among these genes, the expression levels of 7 and 188 genes were commonly down- or up-regulated, respectively in both treatment groups. On the other hand, 31 gene expressions were significantly different between PCB126 and KC-400 treatment groups, and three out of 31 genes were received opposite effects. In addition, the microarray data showed that thyroid hormone-responsive genes were up-regulated by PCB-exposure, which may imply that PCBs or their metabolites mimic thyroid hormone effects in the brain of PCB-exposed medaka.     

Heavy oil exposure induces high moralities in virus carrier Japanese flounder Paralichthys olivaceus

The relationship between chemical exposure and disease outbreak in fish has not been fully defined due to the limitations of experimental systems (model fish and pathogens). Therefore, we constructed a system using the Japanese flounder, Paralichthys olivaceus, and viral haemorrhagic septicemia virus (VHSV), and evaluated it by heavy oil (HO) exposure. The fish were exposed to HO at 0.3, 0.03, 0.003, and 0 g/L following VHSV infection at doses of 10(2.5) or 10(3.5) tissue culture infectious dose (TCID)50/fish. As a result, groups given the dual stressors showed more than 90% mortality. Although VHSV infection at 10(2.5) and 10(3.5) TCID50/fish without HO exposure also induced high mortality, at 68.8% and 81.3%, respectively, HO exposure induced faster and higher mortality in the virus carrier fish, indicating that chemical stressors raise the risk of disease outbreak in fish. The experimental system established in this study could be useful for chemical risk assessment.     

Comparison of the acid-base responses to CO2 and acidification in Japanese flounder (Paralichthys olivaceus)

To investigate whether the biological toxicity of aquatic hypercapnia is due to the direct effects of CO2 or to the effects of acidification of seawater by CO2, the Japanese flounder (Paralichthys olivaceus) was subjected to seawater equilibrated with a gas mixture of air containing 5% CO2 (pH 6.18) or seawater acidified to the same pH with 1 N H2SO4. All the fish died within 72 h in the CO2 exposure group, whereas no mortality occurred in the acid group. Acid-base parameters as well as plasma ion concentrations were severely perturbed in the CO2 exposure group, whereas they were minimally affected in the acid group. These results clearly demonstrate that the mortality in the CO2 group is a direct result of the elevated levels of dissolved CO2 and not to the effects of the reduced water pH.     

Study on the fate of petroleum-derived polycyclic aromatic hydrocarbons (PAHs) and the effect of chemical dispersant using an enclosed ecosystem,mesocosm

Polycyclic Aromatic Hydrocarbons (PAHs) are one of the components found in oil and are of interest because some are toxic. We studied the environmental fate of PAHs and the effects of chemical dispersants using experimental 500 l mesocosm tanks that mimic natural ecosystems. The tanks were filled with seawater spiked with the water-soluble fraction of heavy residual oil. Water samples and settling particles in the tanks were collected periodically and 38 PAH compounds were analyzed by gas chromatography-mass spectrometry (GC-MS). Low molecular weight (LMW) PAHs with less than three benzene rings disappeared rapidly, mostly within 2 days. On the other hand, high molecular weight (HMW) PAHs with more than four benzene rings remained in the water column for a longer time, up to 9 days. Also, significant portions (10-94%) of HMW PAHs settled to the bottom and were caught in the sediment trap. The addition of chemical dispersant accelerated dissolution and biodegradation of PAHs, especially HMW PAHs. The dispersant amplified the amounts of PAHs found in the water column. The amplification was the greater for the more hydrophobic PAHs, with an enrichment factor of up to six times. The increased PAHs resulting from dispersant use overwhelmed the normal degradation and, as a result, higher concentrations of PAHs were observed in water column throughout the experimental period. We conclude that the addition of the dispersant could increase the concentration of water column PAHs and thus increase the exposure and potential toxicity for organisms in the natural environment. By making more hydrocarbon material available to the water column, the application of dispersant reduced the settling of PAHs. For the tank with dispersant, only 6% of chrysene initially introduced was detected in the sediment trap whereas 70% was found in the trap in the tank without dispersant.     

Biomarkers of exposure and effect in the Brazilian flounder Paralichthys orbignyanus (Teleostei: Paralichthyidae) from the Patos Lagoon estuary (Southern Brazil)

Biomarkers of exposure (liver metallothionein-like proteins content and catalase and glutathione S-transferase activities) and effect (liver lipoperoxidation and blood cell DNA damage) of contaminants were analyzed in the Brazilian flounder Paralichthys orbignyanus from the Patos Lagoon estuary (Southern Brazil). Flounders were collected for a year in two sites: "Coroa do Boi" (polluted site) and "Saco do Justino" (non-polluted site). Results indicated that micronucleated cells frequency was the best biomarker to distinguish flounders from the two sites. Taken together, data from DNA damage analyses (micronucleus test and comet assay) indicated that flounders from the non-polluted site efficiently repaired the DNA breaks, contrary to those from the polluted site, which probably had their DNA repair system inhibited or exhausted. Furthermore, data from enzyme activities (catalase and GST) and lipid peroxidation indicated that flounders from the polluted site were under oxidative stress in summer and autumn.     

Comet assay for the detection of genotoxicity in blood cells of flounder (Paralichthys olivaceus) exposed to sediments and polycyclic aromatic hydrocarbons

To investigate the genotoxic effect of marine sediments on aquatic organism, sediment samples were collected from 13 sites along the coast of Gwangyang Bay (Korea). Concentrations of polycyclic aromatic hydrocarbons (PAHs) in sediments were determined and the relationship between exposure of flounder blood cells to sediment extracts and DNA single-strand breakage in the blood cells was examined using the comet assay. Levels of DNA damage were proportionally increased by exposure concentration and the highest sediment-associated DNA damage was observed at the station showing the highest PAHs contamination. DNA damage in blood cells exposed to five types of PAHs (benzo[a]pyrene, fluoranthene, anthracene, pyrene and phenanthrene) and in flounder (Paralichthys olivaceus) exposed to benzo[a]pyrene (BaP) for 0, 2 and 4 days were assessed by measuring comet tail length. The tail lengths of five PAHs-exposed groups at 50 and 100 ppb were significantly different from the non-exposed group, and the genotoxic effect of BaP correlated with both concentration and duration of exposure. Throughout the study, significant differences in DNA breakage were recorded between cells exposed to sediment extracts or PAHs and non-exposed control. This study demonstrated the comet assay as a successful tool in monitoring contamination of marine sediments and assessing genotoxicity of PAHs in marine organisms, either in vitro or in vivo.     

Development of a laboratory exposure system using marine fish to carry out realistic effect studies with produced water discharged from offshore oil production

A biotest system for environmentally realistic exposure of fish to produced water (PW) was developed and tested. Authentic PW was collected at an oil production platform in the North Sea and preserved by freezing in multiple aliquots a 25 L. After transport to the test laboratory onshore, daily PW aliquots were thawed, homogenised and administered to the test fish, Atlantic cod (Gadus morhua), in two diluted exposure concentrations, 0.1% and 0.5%, during a 15 d period, using a continuous flow-through exposure setup. Positive control groups were exposed to two crude oil treatments for comparison. Chemical analyses showed that alkylphenol (AP) and PAH concentrations in PW exposure waters were very low. Observations of significantly increased AP and PAH metabolite levels in PW exposed fish demonstrated the suitability of the biotest system for its use in biological exposure/effect studies of PW, and it also demonstrated the sensitivity of bile metabolites as PW exposure markers in fish. The relevance of the biotest system for PW effect studies and for validating modelled environmental risk estimates of PW dischargers from offshore oil production is discussed.     

Organic micropollutants in marine plastics debris from the open ocean and remote and urban beaches

To understand the spatial variation in concentrations and compositions of organic micropollutants in marine plastic debris and their sources, we analyzed plastic fragments (∼10 mm) from the open ocean and from remote and urban beaches. Polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAHs), dichloro-diphenyl-trichloroethane and its metabolites (DDTs), polybrominated diphenyl ethers (PBDEs), alkylphenols and bisphenol A were detected in the fragments at concentrations from 1 to 10,000 ng/g. Concentrations showed large piece-to-piece variability. Hydrophobic organic compounds such as PCBs and PAHs were sorbed from seawater to the plastic fragments. PCBs are most probably derived from legacy pollution. PAHs showed a petrogenic signature, suggesting the sorption of PAHs from oil slicks. Nonylphenol, bisphenol A, and PBDEs came mainly from additives and were detected at high concentrations in some fragments both from remote and urban beaches and the open ocean.     

Contamination characteristics of heavy metals in wetland soils along a tidal ditch of the Yellow River Estuary, China

Surface soils (0–20 cm) were collected from along a tidal ditch of the Yellow River Estuary in August of 2007. Samples were subjected to a total digestion technique before they were analyzed for total concentrations of As, Cr, Cd, Cu, Ni, Pb, Zn, P and S in order to investigate heavy metal contamination levels in wetland soils nearby the tidal ditches and their main sources. Results showed that the mean concentrations of these heavy metals except for As and Cd were lower than the Class I criteria. Nearly all sampling sites showed lower contamination levels for As and Cd, while no contamination levels for other heavy metals. Cr, Cu, and Ni mainly originated from parent rocks, and Pb and As might originate from tidal seawater and oil field pollution, respectively; while Cd and Zn mainly originated from parent rocks and tidal seawater. Most of heavy metals showed significant correlations with total concentrations of P and S, however, no significant correlations were observed between them and soil pH, slat and soil organic matter.     

Disruption of Sema3A expression causes abnormal neural projection in heavy oil exposed Japanese flounder larvae

It has been well known that oil spills cause serious problems in the aquatic organisms. In particular, some species of teleosts, which develop on the sea surface thought to be affected by heavy oil (HO). During the embryogenesis, the nervous system is constructed. Therefore, it is important to study the toxicological effects of HO on the developing neurons. We exposed HO to eggs of Japanese flounder (Paralichthys olivaceus) and investigated the neural disorder. In larvae exposed by HO at the concentration of 8.75 mg/L, the facial and lateral line nerves partially entered into the incorrect region and the bundle was defasciculated. Furthermore, in the HO-exposed larvae, Sema3A, a kind of axon guidance molecule, was broadly expressed in second pharyngeal arch, a target region of facial nerve. Taken together, we suggested the possibility that the abnormal expression of Sema3A affected by HO exposure causes disruption of facial nerve scaffolding.