Antioxidant responses and lipid peroxidation in gills and erythrocytes of fish (Rhabdosarga sarba) upon exposure to Chattonella marina and hydrogen peroxide : implications on the cause of fish kills

Author Posting. © Elsevier B.V., 2006. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Journal of Experimental Marine Biology and Ecology 336 (2006): 230-241, doi:10.1016/j.jembe.2006.05.013.Chattonella marina, a red tide or harmful algal bloom species, has caused mass fish kills and serious economic loss worldwide, and yet its toxic actions remain highly controversial. Previous studies have shown that this species is able to produce reactive oxygen species (ROS), and therefore postulated that ROS are the causative agents of fish kills. The present study investigates antioxidant responses and lipid peroxidation in gills and erythrocytes of fish (Rhabdosarga sarba) upon exposure to C. marina, compared with responses exposed to equivalent and higher levels of ROS exposure. Even though C. marina can produce a high level of ROS, gills and erythrocytes of sea bream exposed to C. marina for 1 to 6 h showed neither significant induction of antioxidant enzymes nor lipid peroxidation. Antioxidant responses and oxidative damage did not occur as fish mortality began to occur, yet could be induced upon exposure to artificially supplied ROS levels an order of magnitude higher. The result of this study implies that ROS produced by C. marina is not the principal cause of fish kills.This study was supported by a CERG grant (CityU 1109/03M / No. 9040864) of the University Grants Committee, Hong Kong SAR government. Support for Don Anderson was also provided by the U.S. National Science Foundation through grant no. OCE-0136861

Hydrogen peroxide is not the cause of fish kills associated with Chattonella marina : cytological and physiological evidence

Author Posting. © The Authors, 2005. This is the author's version of the work. It is posted here by permission of Elsevier B. V. for personal use, not for redistribution. The definitive version was published in Aquatic Toxicology 72 (2005): 351-360, doi:10.1016/j.aquatox.2005.01.007.Chattonella marina, a harmful algal bloom (HAB) causative species, was used to study the mortality, physiology, and pathology of a marine stenohaline fish, goldlined seabream exposed to the toxic alga. The median lethal time (LT50) was 3 h upon exposure to 8000 cells/ml of C. marina. Significant induction of filamental chloride cells (CCs) [i.e. increases in CC fractional area and in the volume density of CCs], concomitant with significant reduction of blood osmolality, were found in C. marina treated fish. To verify whether the toxicity of C. marina was mediated through oxidative stress, a hydrogen peroxide exposure experiment was carried out and the toxicity as well as cytological and physiological changes were compared with the C. marina treatment. Hydrogen peroxide at a concentration of 500 μM H2O2, (i.e. 25 times higher than that produced by 8000 cells/ml of C. marina (20 μM H2O2)) was unable to induce similar CC alterations and osmoregulatory impairment in fish as observed in the C. marina treatment. Non-specific membrane damage such as severe loss of microvilli projections on the CC apical opening and rupture of epithelial membranes in the lamellae were observed. The LT50 was 6 h, two times longer than that with 8000 cells/ml of C. marina. Based on the cytological and physiological evidence and toxicity data, the mechanism by which C. marina kills fish appears to be very different from that caused by H2O2/ROS. Osmoregulatory distress is the major cause of fish death upon exposure to C. marina.The work described in this paper was supported by a grant from the Research Grants Council (Project No. 9040547 CityU 1105/00M) and a grant from the University Grants Committee (Project No. AoE/P-04/04) of the Hong Kong Special Administrative Region, China. Support for D. Anderson was also provided by the US National Science Foundation through grant no. OCE-0136861

Identification of Molecular Targets for 4,5-Dichloro-2-n-octyl-4-isothiazolin-3-one (DCOIT) in Teleosts: New Insight into Mechanism of Toxicity

Environmental pollutants are capable of concomitantly inducing diverse toxic effects. However, it is largely unknown which effects are directly induced and which effects are secondary, thus calling for definitive identification of the initiating molecular event for a pollutant to elucidate the mechanism of toxicity. In the present study, affinity pull-down assays were used to identify target proteins for 4,5-dichloro-2-n-octyl-4-isothiazolin-3-one (DCOIT), a costal pollutant of emerging concern, in various tissues (e.g., brain, liver, plasma, and gonad) from marine medaka (Olyzias melastigma) and zebrafish (Danio rerio). Pull-down results showed that, in male and female brains from medaka and zebrafish, DCOIT had a consistently high affinity for G protein alpha subunits (G alpha), suggesting the targeted effects of DCOIT on signaling transduction from G protein-coupled receptors (GPCRs) and an extrapolatable mode of action in teleost brains. Validation using recombinant proteins and molecular docking analysis confirmed that binding of DCOIT to G alpha protein competitively inhibited its activation by substrate. Considering the involvement of GPCRs in the regulation of myriad biological processes, including the hypothalamus-pituitary-gonadal-liver axis, binding of DCOIT to upstream G alpha proteins in the brain may provide a plausible explanation for the diversity of toxic effects resulting from DCOIT challenge, especially abnormal hormonal production through the mitogen-activated protein kinase pathway. A new mechanism of action based on GPCR signaling is thus hypothesized for endocrine disrupting chemicals and warrants further research to clearly elucidate the link between GPCR signaling and endocrine disruption.</p

The marine medaka Oryzias melastigma - A potential marine fish model for innate immune study

University Grants Committee of the Hong Kong Special Administration Region, China [AoE/P-04/2004]; State Key Laboratory in Marine Pollution (CityU); Hong Kong-France Research Collaboration Grant [9231003]; Minjiang Scholar ProgramThe objective of this study is to develop the marine medaka Oryzias melastigma as a potential marine fish model for innate immune and immunotoxicological studies. Hepcidin plays an important role in innate immune system. Two hepcidin genes (OM-hep1 and OM-hep2) were identified and characterized in the O. melastigma, which were highly conserved with other reported hepcidins. During embryogenesis, significant elevation of OM-hep1 and OM-hep2 transcripts were coincided with liver development in the embryos. In adult medaka, differential tissue expressions of both hepcidin transcripts were evident: high in liver, moderate in spleen and low in non-immune tissues. After bacterial challenge, the two hepcidin mRNAs were rapidly and remarkably induced in liver and spleen, suggesting the two OM-hepcidins in O. melastigma play a complementary role in innate defense. Gender difference in time of induction and extent of the two hepcidin mRNAs elevation in infected O. melastigma should be considered in immunotoxicological studies. (C) 2011 Elsevier Ltd. All rights reserved

Sex-specific immunomodulatory action of the environmentalestrogen 17α-ethynylestradiol alongside with reproductive impairment in fish.

Estrogenic endocrine disrupting chemicals (EEDCs) are present ubiquitously in sediments and aquatic ecosystems worldwide. The detrimental impact of EEDCs on the reproduction of wildlife is widely recognized. Increasing evidence shows the immunosuppressive effects of EEDCs in vertebrates. Yet, no studies have considered concomitantly EEDC-induced impacts on reproductive impairment and immune suppression in vivo, which are deemed essential for risk assessment and environmental monitoring. In this study, EE2 was used as a representative EEDC, for parallel evaluation of EEDC-induced immune suppression (immune marker gene expression, leukocyte numbers, host resistance assay, and immune competence index) and reproductive impairment (estrogen responsive gene expression, fecundity, fertilization success, hatching success, and reproductive competence index) in an established fish model (marine medaka Oryzias melastigma), considering sex-specific induction and adaptation and recovery responses under different EE2 exposure scenarios. The findings in marine medaka reveal distinct sex differences in the EE2-mediated biological responses. For female fish, low concentration of exogenous EE2 (33 ng/L) could induce hormesis (immune enhancement), enable adaptation (restored reproduction) and even boost fish resistance to bacterial challenge after abatement of EE2. However, a prolonged exposure to high levels of EE2 (113 ng/L) not only impaired F0 immune function, but also perturbed females recovering from reproductive impairment, resulting in a persistent impact on the F1 generation output. Thus, for female fish, the exposure concentration of EE2 is more critical than the dose of EE2 in determining the impacts of EE2 on immune function and reproduction. Conversely, male fish are far more sensitive than females to the presence of low levels of exogenous EE2 in water and the EE2-mediated biological impacts are clearly dose-dependent. It is also evident in male fish that direct contact of EE2 is essential to sustain impairments of immune competence and reproductive output as well as deregulation of immune function genes in vivo. The immunomodulatory pathways altered by EE2 were deciphered for male and female fish, separately. Downregulation of hepatic tlr3 and c3 (in female) and tlr3, tlr5 and c3 (in male) may be indicative of impaired fish immune competence. Taken together, impaired immune competence in the EE2-exposed fish poses an immediate thread on the survival of F0 population. Impaired reproduction in the EE2-exposed fish can directly affect F1 output. Parallel evaluation of immune competence and reproduction are important considerations when assessing the risk of sublethal levels of EE2/EEDCs in aquatic environments

The marine medaka Oryzias melastigma - A potential marine fish model for innate immune study

University Grants Committee of the Hong Kong Special Administration Region, China [AoE/P-04/2004]; State Key Laboratory in Marine Pollution (CityU); Hong Kong-France Research Collaboration Grant [9231003]; Minjiang Scholar ProgramThe objective of this study is to develop the marine medaka Oryzias melastigma as a potential marine fish model for innate immune and immunotoxicological studies. Hepcidin plays an important role in innate immune system. Two hepcidin genes (OM-hep1 and OM-hep2) were identified and characterized in the O. melastigma, which were highly conserved with other reported hepcidins. During embryogenesis, significant elevation of OM-hep1 and OM-hep2 transcripts were coincided with liver development in the embryos. In adult medaka, differential tissue expressions of both hepcidin transcripts were evident: high in liver, moderate in spleen and low in non-immune tissues. After bacterial challenge, the two hepcidin mRNAs were rapidly and remarkably induced in liver and spleen, suggesting the two OM-hepcidins in O. melastigma play a complementary role in innate defense. Gender difference in time of induction and extent of the two hepcidin mRNAs elevation in infected O. melastigma should be considered in immunotoxicological studies. (C) 2011 Elsevier Ltd. All rights reserved

Linking genomic responses of gonads with reproductive impairment in marine medaka (Oryzias melastigma) exposed chronically to the chemopreventive and antifouling agent, 3,3'-diindolylmethane (DIM)

3,3'-Diindolylmethane (DIM) has been promoted as an effective chemopreventive and antifouling additive. However, the concurrent risks or side effects of DIM are not fully understood, especially on tissues responsive to estrogen. Therefore, this study employed marine medaka (Oryzias melastigma) as a test model to evaluate relative safety and explore mechanisms of toxic action of DIM on development and function of gonad after chronic (28 days) aqueous exposure to relatively low doses (0 mu g/L. or 8.5 mu g/L). Integration of comprehensive toxicogenomic analysis at the transcriptome and proteome levels with apical endpoints, such as production of eggs and swimming performance of larvae, elucidated the molecular linkage in gonad from bottom up along the reproductive adverse outcome pathway. A series of sequential changes at the transcript and protein levels were linked to lesser fecundity and viability of larvae exposed to DIM. Anomalous production of vitellogenin (VTG) and eggshell proteins in testis confirmed the estrogenic potency of DIM. In the ovary, although storage of VTG was greater, lesser expressions of cathepsin enzymes blocked cleavage and incorporation of VTG into oocytes as yolk, which acted together with lower eggshell proteins to inhibit maturation of primary oocyte and thus contributed to impairment of fecundity. Overall, this study demonstrated that exposure to DIM impaired reproductive fitness. Diverse molecular initiating changes in gonads were linked to apical endpoints that could be used in assessment of risks posed by DIM on gametogenesis. In combination with chemical stability and potent endocrine disruption, the results of this study can inform decisions about the use of DIM either as chemopreventive or antifouling agent. (C) 2017 Elsevier B.V. All rights reserved