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Tributyltin exposure increases mortality of nodavirus infected Japanese medaka Oryzias latipes larvae
MPB-08406; No of Pages 4 Marine Pollution Bulletin xxx (2017) xxx–xxx
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Tributyltin exposure increases mortality of nodavirus infected Japanese medaka Oryzias latipes larvae Shin-Ichi Kitamura a,⁎, Masaki Akizuki a, Jun-Young Song b, Kei Nakayama a a b
Centre for Marine Environmental Studies (CMES), Ehime University, Matsuyama 790-8577, Japan Pathology Division, National Fisheries Research and Development Institute, Busan 619-902, Republic of Korea
a r t i c l e
i n f o
Article history: Received 30 September 2016 Received in revised form 29 January 2017 Accepted 6 February 2017 Available online xxxx Keywords: Immunotoxicity Immune suppression Nodavirus Oryzias latipes
a b s t r a c t We investigated the effect of combined exposure to nodavirus infection and TBT on medaka (Oryzias latipes). Medaka larvae were infected by immersion in medium containing nodavirus at titers of 102.5, 103.5, or 104.5 TCID50/mL. Infected fish then were exposed to TBT at 0, 0.17, 0.52, 1.6, or 4.7 μg/L. Of the 12 groups exposed to both stressors, the mortalities of 6 (102.5 TCID50/mL + 0.52, 1.6, or 4.7 μg/L, 103.5 TCID50/mL + 4.7 μg/L and 104.5 TCID50/mL + 1.6 or 4.7 μg/L) were significantly higher than that of each TBT control. Specifically, mortality was 46 ± 5.5% in the group exposed to both 102.5 TCID50/mL virus and 0.52 μg/L TBT, which represent the lowest observed effective dose and concentration, respectively, among the 6 groups with increased mortalities. Our results suggest that combined exposure to both stressors suppresses antiviral mechanisms in the fish, thus increasing mortality. © 2017 Elsevier Ltd. All rights reserved.
1. Introduction The organotin compound tributyltin (TBT) has been used as a biocide in numerous industrial applications (Kannan et al., 1995; Gipperth, 2009), such as antifouling paints for ships and fishing nets. Adverse effects of TBT on aquatic organisms include acute toxicity and growth and reproductive inhibition (Rexrode, 1987), and TBT is associated with induction of the imposex phenomenon in neogastropods (Horiguchi et al., 1997). For these reasons, TBT has been banned in the United States since 1989 and was prohibited by the International Convention on the Control of Harmful Antifouling Systems on Ships in 2008 (Gipperth, 2009). Consequently, TBT concentrations in marine environments have generally dropped below 1 ng/L. For example, TBT concentrations ranged from 0.21 to 15.41 ng/L in seawater samples from the Croatian Adriatic Coast in 2009 and 2010 (Furdek et al., 2012), were undetectable (b0.1 ng/L) to 23.9 ng/L in seawater from Korea in 2009 and 2010 (Kim et al., 2014), and varied from below the detection limit to 93.8 ng/L in China in 2001–2003 (Cao et al., 2009). Because of the environmental persistence of TBT and widespread illegal use of the chemical, investigation into the consequences of TBT exposure continues. TBT exerts well-known effects on reproduction in many fish species, including altered sex ratios in zebrafish (Danio rerio) and Japanese flounder (Paralichthys olivaceus) (McAllister and Kime, 2003; Shimasaki et al., 2003) and decreased fertility and fecundity in Japanese whiting (Sillago japonica) (Shimasaki et al., 2006). In addition, TBT administration disrupts reproductive behavior in male medaka ⁎ Corresponding author. E-mail address: [email protected] (S.-I. Kitamura).
(Oryzias latipes) (Nakayama et al., 2004) and male guppies (Poecilia reticulata) (Tian et al., 2015). In addition to its reproductive toxicities, TBT is suspected to cause immunotoxicity. For example, TBT exposure was implicated in the high prevalence of lymphocystis disease among fish observed in field studies (Grinwis et al., 2000). Nakayama et al. (2009) detected a significant correlation between butyltin levels and lung nematode infection in finless porpoises (Neophocaena phocaenoides). Indeed, fish exposed to TBT under laboratory conditions demonstrate immune system suppression, as indicated by thymus atrophy, reduced circulating lymphocyte counts (Schwaiger et al., 1992), and decreased mitogenic responses (Harford et al., 2007). In contrast, other similar studies revealed increases in circulating granulocyte counts (Wester and Canton, 1987; Schwaiger et al., 1994) and the stimulation of phagocytic function (Harford et al., 2007). We believe that the immunotoxicities of chemicals should include descriptions of their effects on susceptibility to infectious disease, but the relationship between TBT exposure and disease occurrence in fish is unclear. We therefore investigated the combined effect of nodaviral infection and TBT exposure in medaka. 2. Materials and methods 2.1. Fish Mature wild-type Japanese medaka provided by Prof. Oshima (Kyusyu University, Japan) were kept in a 60-L tank. The fish (n = 30) were fed brine shrimp daily, and approximately one-third of the aquarium water was replaced daily with aerated and dechlorinated tap water. The fish were induced to spawn by placing them under a summer
http://dx.doi.org/10.1016/j.marpolbul.2017.02.020 0025-326X/© 2017 Elsevier Ltd. All rights reserved.
Please cite this article as: Kitamura, S.-I., et al., Tributyltin exposure increases mortality of nodavirus infected Japanese medaka Oryzias latipes larvae, Marine Pollution Bulletin (2017), http://dx.doi.org/10.1016/j.marpolbul.2017.02.020
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S.-I. Kitamura et al. / Marine Pollution Bulletin xxx (2017) xxx–xxx
photoperiod (14 h light: 10 h dark) at 25 °C. The embryos were disinfected with 0.9% H2O2 for 10 min, washed with embryo-rearing medium (ERM; 0.1% NaCl, 0.003% KCl, 0.004% CaCl2, 0.008% MgSO4, and 0.1 M NaHCO3; pH 7.0 to 7.2), and incubated at 25 °C in 6-well plates with shaking until hatching. 2.2. Chemical Tributyltin chloride (96%) was purchased from Sigma (St. Louis, MO, USA). The chemical was diluted as needed by using 99.5% ethanol and stored at −20 °C until use. 2.3. Virus Red spotted grouper nervous necrosis virus (RGNNV), SGWak97 strain, and the E-11 cell line were kindly provided by Dr. Okinaka (Hiroshima University, Japan). The virus was propagated in E-11 cells cultured at 25 °C in Leibovitz's L-15 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 5% fetal bovine serum until used for experiments. When the cytopathic effect (CPE) was extensive, the culture medium was harvested and centrifuged to remove cell debris. The supernatant was used as the virus stock for the experiments. The virus stock was titrated in 96-well plates, and titers are reported in terms of 50% tissue culture infectious dose (TCID50). 2.4. Nodavirus infection and TBT exposure The TBT concentrations and virus titers used in the current study were based on the results of preliminary experiments: median lethal concentrations (LC50) after a 48-h exposure were 47 μg/L for TBT and 104.5 TCID50/mL for RGNNV in 0 days post-hatching (dph) medaka. In the current study, the nominal concentrations were lowered to 0.17, 0.52, 1.6, and 4.7 μg/L for TBT and 101.5, 102.5, and 103.5 TCID50/mL for RGNNV. For infection, 10 medaka (0 dph) larvae were placed in 6-well plates containing RGNNV at 0, 102.5, 103.5, or 104.5 TCID50/mL in ERM and incubated for 96 h at 25 °C. After 96 h, all fish were washed with ERM and incubated in virus-free ERM for 72 h. Each infection group was then divided into 8-mL glass petri dishes containing TBT at concentrations of 0, 0.17, 0.52, 1.6, or 4.7 μg/L for 96 h. We removed dead and moribund fish and recorded the mortalities for 192 h. The experiments were replicated 5-fold. Data are given as mean ± S.D. and were analyzed by using twoway ANOVA followed by the Dunnett test. Virus-induced mortality at each TBT exposure level was fitted to a logistic regression model by the R project language. 2.5. Virus titers in tested fish We determined the virus titers in the medaka larvae that exposed to both 102.5 TCID50/mL RGNNV and 0.52 μg/L TBT (that is, the lowest observed effect dose [LOED] and lowest observed effect concentration [LOEC], respectively) or the same dose of the virus only. The fish was homogenized in 500 μL of Hanks balanced salt solution (Gibco, Grand Island, NY, USA); the homogenate was centrifuged at 2000 ×g for 5 min to remove cell debris; and the supernatant was filtered (pore size, 0.45 μm). The filtrate was 10-fold serially diluted with Hanks balanced salt solution, and 100 μL of each dilution was inoculated onto E-11 cells in 96-well tissue culture plates. The development of CPE was monitored for 10 d. Data were analyzed by one-way ANOVA. 3. Results 3.1. Effect of TBT exposure and virus infection on fish mortality The mortalities of medaka larvae exposed to RGNNV and TBT are shown in Fig. 1a. Mortalities in the no-treatment, TBT-only, and virus-
only groups were 12 ± 8.4%, 8.0–30%, and 26–54%, respectively. In the groups exposed to both virus and TBT, mortality was lowest (14 ± 5.5%) in medaka larvae exposed to 102.5 TCID50/mL RGNNV and 0.17 μg/L TBT, whereas the highest mortality (92 ± 4.5%) occurred in the group exposed to 104.5 TCID50/mL RGNNV and 4.7 μg/L TBT. Among the 12 groups exposed to both stressors, 6 showed significantly higher mortalities than that of each no-TBT group (P b 0.05). Specifically, mortality was 46 ± 5.5% in the medaka that received 102.5 TCID50/mL RGNNV and 0.52 μg/L TBT, which represent the LOED and LOEC among the 6 virus + TBT groups with increased mortality. Mortality curves in the virus-infected medaka exposed to different concentration of TBT were determined by logistic regression (Fig. 1b). TBT exposure enhanced mortalities of the virus-infected fish at the tendency of dose dependent manner; the mortalities in medaka exposed to 0, 0.17, 0.52, 1.6 and 4.7 ppb of TBT were 31, 23, 39, 48 and 73% at the infection of 102.5 TCID50/mL, and they were 43, 36, 50, 60 and 85% at the 103.5 TCID50/mL infection, respectively. The trends in mortality remained consistent even when the order of RGNNV infection and TBT exposure was reversed (that is, TBT exposure followed by viral infection) (Fig. S1). 3.2. Virus titer To ascertain whether the observed mortality after exposure to TBT and RGNNV was due to viral nervous necrosis, we measured the virus titers of fish exposed to both stressors or to the virus only. The titers of the fish ranged from 102.8 to 107.3 TCID50/fish (average, 104.5 TCID50/fish) in medaka larvae exposed to 102.5 TCID50/mL RGNNV (LOED) only and 103.6 to 107.6 TCID50/fish (average, 105.7 TCID50/fish) in fish exposed to both 102.5 TCID50/mL RGNNV and 0.52 μg/L TBT (LOEC) (Fig. 2). In addition, the virus titer was significantly higher (P b 0.05) in the fish exposed to both RGNNV and TBT than in the virus-only control group. No CPE was observed in either the solventonly or uninfected control groups. 4. Discussion Traditionally, the immunotoxicity of TBT has been evaluated by measuring leukocyte activities in various animals (Whalen et al., 1999, 2002; Nakayama et al., 2007; Misumi et al., 2009; Zhou et al., 2010a, 2010b; Lawrence et al., 2015), and most researchers regard TBT as an immune suppressor. However, whether TBT exposure increases susceptibility to infectious disease is unknown. Recently, Nakayama et al. (2016, in press) and Ye et al. (2016) suggested a method through which fish susceptibility to pathogen infection in the context of chemical exposure can be assessed. In the current study, we infected medaka with the nodavirus RGNNV and then exposed them to TBT. In our exposure experiments, the mortalities of the medaka larvae increased in a dose-dependent manner as the TBT concentration increased. In addition, TBT dose-dependent mortalities occurred in the groups infected with the virus both at low and high titers, the indication being that TBT is immunotoxic in the fish. Among the 12 groups exposed to both stressors (nodavirus and TBT), the mortalities of 6 were higher than those of the TBT control group. Increased mortalities due to combined stressors have been reported for various other types of chemicals, including benzo[a]pyrene (Carlson et al., 2002), polybrominated diphenyl ethers (Arkoosh et al., 2010), and heavy oil (Song et al., 2011). In the present study, we show that exposure to TBT exacerbated nodaviral infection in medaka larvae, that the LOED of the virus was 102.5 TCID50/mL, and that the LOEC of TBT was 0.52 μg/L. Mortality in medaka was similar, regardless of the order in which RGNNV infection and TBT exposure were applied (Figs. 1 and S1). In natural environments, the sequence of stressor exposure likely is variable, but our results indicate no order-associated influence on mortality.
Please cite this article as: Kitamura, S.-I., et al., Tributyltin exposure increases mortality of nodavirus infected Japanese medaka Oryzias latipes larvae, Marine Pollution Bulletin (2017), http://dx.doi.org/10.1016/j.marpolbul.2017.02.020
S.-I. Kitamura et al. / Marine Pollution Bulletin xxx (2017) xxx–xxx
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Fig. 1. Cumulative mortalities of medaka larvae exposed to both nodavirus and tributyltin (TBT) (a). Data are shown as mean ± SD (%; n = 5 independent experiments; 10 fish in each group). Values significantly different from those for no-TBT group are indicated (*: p b 0.05, +: p b 0.01). Mortality curves in the fish exposed to different concentration of TBT were fitted to logistic regression analyzed by the R project language (b).
Medaka larvae infected with RGNNV in the present study did not demonstrate any neurologic symptoms, such as abnormal behavior or swimming. Furusawa et al. (2006) similarly reported that 1-day-old larvae bath-challenged with RGNNV died without showing characteristic erratic swimming. Although the reason is unclear, the lack of virus-associated symptoms might reflect the low swimming force in newborn medaka. To clarify the cause of mortality in the fish exposed to both RGNNV and TBT, we measured virus titers in the fish and compared them between the virus-only control and virus + TBT groups. The results
Fig. 2. Virus titers in infected fish. The fish were collected after 192 h of exposure at the LOEC for tributyltin (TBT). The data are shown as a boxplot. *, Significant difference between values for the virus + TBT and virus-only groups (p b 0.05, one-way ANOVA).
indicated that the virus titer was higher in the fish exposed to the virus and TBT at their LOED and LOEC than in the virus-only control group. These data suggest that TBT exposure increases viral replication and thus mortality in medaka larvae. The LOEC observed in the current study is 10 to 100 times higher than those of environmental contamination levels (Cao et al., 2009; Furdek et al., 2012; Kim et al., 2014), the suggestion being that the risk of occurrence of infectious disease might be low by contaminated TBT exposure in natural environments. However, because the concentration of TBT is relatively high in marine sediments (Kim et al., 2015; Suzdalev et al., 2015), the risk is not negligible, particularly for demersal fishes such as flatfishes and rockfishes. Moreover, dibutyltin, which is widely distributed in marine ecosystems, reportedly interferes with natural killer (NK) cell function (Whalen et al., 1999, 2002). Although the risk of TBT exposure alone may be minimal, the potential effects of exposure to both TBT and other chemicals concurrently should be considered. We consider the immunotoxic mechanism of TBT to act as follows. In general, type I interferon, NK cells, and immunoglobulins cooperatively help to protect against viral infection in animals. An anti-immunoglobulin effect of TBT probably does not contribute to the increased viral replication we observed because the experimental period was too short: pathogen-specific antibody levels rise beginning one week after infection in fish (Marsden et al., 1996; Kaattari et al., 2005). Moreover, the tested fish larvae likely were too immature to produce immunoglobulin. Therefore, we suspect that combined exposure to virus and TBT suppressed the activity of type I interferon and/or NK cells in the medaka larvae. In vitro, TBT interfered with the ability of human natural NK cells to lyse target human immune cells (Thomas et al., 2004; Aluoch and Whalen, 2005). The effect of TBT on the activity of type I interferon has not yet been reported, and further investigation should focus on this
Please cite this article as: Kitamura, S.-I., et al., Tributyltin exposure increases mortality of nodavirus infected Japanese medaka Oryzias latipes larvae, Marine Pollution Bulletin (2017), http://dx.doi.org/10.1016/j.marpolbul.2017.02.020
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factor and NK cell activity to reveal the cause of the high viral replication we observed here. In conclusion, combined exposure to nodavirus infection and TBT induced high mortality in medaka larvae, even though exposure to each stressor individually had little to no effect. The mortality was 46 ± 5.5% when fish were exposed to both 102.5 TCID50/mL RGNNV and 0.52 μg/L TBT, which represent the LOED and LOEC. In addition, the virus titer of medaka exposed to both RGNNV and TBT at their LOED and LOEC was higher than that of the virus-only control group, the suggestion being that TBT exposure accelerated virus replication. Our results imply that combined exposure to TBT and RGNNV suppresses host antiviral mechanisms and promotes viral replication, leading to high mortality in medaka larvae. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.marpolbul.2017.02.020. Acknowledgements We thank Prof. Oshima (Graduate School of Kyushu University, Japan) for providing medaka and Dr. Okinaka (Hiroshima University, Japan) for providing E-11 cells and RGNNV. References Aluoch, A., Whalen, M., 2005. Tributyltin-induced effects on MAP kinases p38 and p44/42 in human natural killer cells. Toxicology 209, 263–277. Arkoosh, M.R., Boylen, D., Dietrich, J., Anulacion, B.F., Ylitalo, G., Bravo, C.F., Johnson, L.L., Loge, F., Collier, T.K., 2010. Disease susceptibility of salmon exposed to polybrominated diphenyl ethers (PBDEs). Aquat. Toxicol. 98, 51–59. Cao, D., Jiang, G., Zhou, Q., Yang, R., 2009. Organotin pollution in China: an overview of the current state and potential health risk. J. Environ. Manag. 90, S16–S24. Carlson, E.A., Li, Y., Zelikoff, J.T., 2002. Exposure of Japanese medaka (Oryzias latipes) to benzo[a]pyrene suppresses immune function and host resistance against bacterial challenge. Aquat. Toxicol. 56, 289–301. Furdek, M., Vahcic, M., Scancar, J., Milacic, R., Kniewald, G., Mikac, N., 2012. Organotin compounds in seawater and mussels Mytilus galloprovincialis along the Croatian Adriatic coast. Mar. Pollut. Bull. 64, 189–199. Furusawa, R., Okinaka, Y., Nakai, T., 2006. Betanodavirus infection in the freshwater model fish medaka (Oryzias latipes). J. Gen. Virol. 87, 2333–2339. Gipperth, L., 2009. The legal design of the international and European Union ban on tributyltin antifouling paint: direct and indirect effects. J. Environ. Manag. 90, S86–S95. Grinwis, G.C., Vethaak, A.D., Wester, P.W., Vos, J.G., 2000. Toxicology of environmental chemicals in the flounder (Platichthys flesus) with emphasis on the immune system: field, semi-field (mesocosm) and laboratory studies. Toxicol. Lett. 112-113, 289–301. Harford, A.J., O'Halloran, K., Wright, P.E., 2007. Effect of in vitro and in vivo organotin exposures on the immune functions of Murray cod (Maccullochella peelii peelii). Environ. Toxicol. Chem. 26, 1649–1656. Horiguchi, T., Shiraishi, H., Shimizu, M., Morita, M., 1997. Effects of triphenyltin chloride and five other organotin compounds on the development of imposex in the rock shell, Thais clavigera. Environ. Pollut. 95, 85–91. Kaattari, S., Bromage, E., Kaattari, I., 2005. Analysis of long-lived plasma cell production and regulation: implications for vaccine design for aquaculture. Aquaculture 246, 1–9. Kannan, K., Tanabe, S., Tatsukawa, R., 1995. Occurrence of butyltin residues in certain foodstuffs. Bull. Environ. Contam. Toxicol. 55, 510–516. Kim, N.S., Hong, S.H., Yim, U.H., Shin, K.H., Shim, W.J., 2014. Temporal changes in TBT pollution in water, sediment, and oyster from Jinhae Bay after the total ban in South Korea. Mar. Pollut. Bull. 86, 547–554. Kim, N.S., Hong, S.H., An, J.G., Shin, K.H., Shim, W.J., 2015. Distribution of butyltins and alternative antifouling biocides in sediments from shipping and shipbuilding areas in South Korea. Mar. Pollut. Bull. 95, 484–490. Lawrence, S., Reid, J., Whalen, M., 2015. Secretion of interferon gamma from human immune cells is altered by exposure to tributyltin and dibutyltin. Environ. Toxicol. 30, 559–571. Marsden, M.J., Vaughan, L.M., Foster, T.J., Secombes, C.J., 1996. A live Aeromonas salmonicida vaccine for furunculosis preferentially stimulates T-cell responses
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Please cite this article as: Kitamura, S.-I., et al., Tributyltin exposure increases mortality of nodavirus infected Japanese medaka Oryzias latipes larvae, Marine Pollution Bulletin (2017), http://dx.doi.org/10.1016/j.marpolbul.2017.02.020
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Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul
Tributyltin exposure increases mortality of nodavirus infected Japanese medaka Oryzias latipes larvae Shin-Ichi Kitamura a,⁎, Masaki Akizuki a, Jun-Young Song b, Kei Nakayama a a b
Centre for Marine Environmental Studies (CMES), Ehime University, Matsuyama 790-8577, Japan Pathology Division, National Fisheries Research and Development Institute, Busan 619-902, Republic of Korea
a r t i c l e
i n f o
Article history: Received 30 September 2016 Received in revised form 29 January 2017 Accepted 6 February 2017 Available online xxxx Keywords: Immunotoxicity Immune suppression Nodavirus Oryzias latipes
a b s t r a c t We investigated the effect of combined exposure to nodavirus infection and TBT on medaka (Oryzias latipes). Medaka larvae were infected by immersion in medium containing nodavirus at titers of 102.5, 103.5, or 104.5 TCID50/mL. Infected fish then were exposed to TBT at 0, 0.17, 0.52, 1.6, or 4.7 μg/L. Of the 12 groups exposed to both stressors, the mortalities of 6 (102.5 TCID50/mL + 0.52, 1.6, or 4.7 μg/L, 103.5 TCID50/mL + 4.7 μg/L and 104.5 TCID50/mL + 1.6 or 4.7 μg/L) were significantly higher than that of each TBT control. Specifically, mortality was 46 ± 5.5% in the group exposed to both 102.5 TCID50/mL virus and 0.52 μg/L TBT, which represent the lowest observed effective dose and concentration, respectively, among the 6 groups with increased mortalities. Our results suggest that combined exposure to both stressors suppresses antiviral mechanisms in the fish, thus increasing mortality. © 2017 Elsevier Ltd. All rights reserved.
1. Introduction The organotin compound tributyltin (TBT) has been used as a biocide in numerous industrial applications (Kannan et al., 1995; Gipperth, 2009), such as antifouling paints for ships and fishing nets. Adverse effects of TBT on aquatic organisms include acute toxicity and growth and reproductive inhibition (Rexrode, 1987), and TBT is associated with induction of the imposex phenomenon in neogastropods (Horiguchi et al., 1997). For these reasons, TBT has been banned in the United States since 1989 and was prohibited by the International Convention on the Control of Harmful Antifouling Systems on Ships in 2008 (Gipperth, 2009). Consequently, TBT concentrations in marine environments have generally dropped below 1 ng/L. For example, TBT concentrations ranged from 0.21 to 15.41 ng/L in seawater samples from the Croatian Adriatic Coast in 2009 and 2010 (Furdek et al., 2012), were undetectable (b0.1 ng/L) to 23.9 ng/L in seawater from Korea in 2009 and 2010 (Kim et al., 2014), and varied from below the detection limit to 93.8 ng/L in China in 2001–2003 (Cao et al., 2009). Because of the environmental persistence of TBT and widespread illegal use of the chemical, investigation into the consequences of TBT exposure continues. TBT exerts well-known effects on reproduction in many fish species, including altered sex ratios in zebrafish (Danio rerio) and Japanese flounder (Paralichthys olivaceus) (McAllister and Kime, 2003; Shimasaki et al., 2003) and decreased fertility and fecundity in Japanese whiting (Sillago japonica) (Shimasaki et al., 2006). In addition, TBT administration disrupts reproductive behavior in male medaka ⁎ Corresponding author. E-mail address: [email protected] (S.-I. Kitamura).
(Oryzias latipes) (Nakayama et al., 2004) and male guppies (Poecilia reticulata) (Tian et al., 2015). In addition to its reproductive toxicities, TBT is suspected to cause immunotoxicity. For example, TBT exposure was implicated in the high prevalence of lymphocystis disease among fish observed in field studies (Grinwis et al., 2000). Nakayama et al. (2009) detected a significant correlation between butyltin levels and lung nematode infection in finless porpoises (Neophocaena phocaenoides). Indeed, fish exposed to TBT under laboratory conditions demonstrate immune system suppression, as indicated by thymus atrophy, reduced circulating lymphocyte counts (Schwaiger et al., 1992), and decreased mitogenic responses (Harford et al., 2007). In contrast, other similar studies revealed increases in circulating granulocyte counts (Wester and Canton, 1987; Schwaiger et al., 1994) and the stimulation of phagocytic function (Harford et al., 2007). We believe that the immunotoxicities of chemicals should include descriptions of their effects on susceptibility to infectious disease, but the relationship between TBT exposure and disease occurrence in fish is unclear. We therefore investigated the combined effect of nodaviral infection and TBT exposure in medaka. 2. Materials and methods 2.1. Fish Mature wild-type Japanese medaka provided by Prof. Oshima (Kyusyu University, Japan) were kept in a 60-L tank. The fish (n = 30) were fed brine shrimp daily, and approximately one-third of the aquarium water was replaced daily with aerated and dechlorinated tap water. The fish were induced to spawn by placing them under a summer
http://dx.doi.org/10.1016/j.marpolbul.2017.02.020 0025-326X/© 2017 Elsevier Ltd. All rights reserved.
Please cite this article as: Kitamura, S.-I., et al., Tributyltin exposure increases mortality of nodavirus infected Japanese medaka Oryzias latipes larvae, Marine Pollution Bulletin (2017), http://dx.doi.org/10.1016/j.marpolbul.2017.02.020
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photoperiod (14 h light: 10 h dark) at 25 °C. The embryos were disinfected with 0.9% H2O2 for 10 min, washed with embryo-rearing medium (ERM; 0.1% NaCl, 0.003% KCl, 0.004% CaCl2, 0.008% MgSO4, and 0.1 M NaHCO3; pH 7.0 to 7.2), and incubated at 25 °C in 6-well plates with shaking until hatching. 2.2. Chemical Tributyltin chloride (96%) was purchased from Sigma (St. Louis, MO, USA). The chemical was diluted as needed by using 99.5% ethanol and stored at −20 °C until use. 2.3. Virus Red spotted grouper nervous necrosis virus (RGNNV), SGWak97 strain, and the E-11 cell line were kindly provided by Dr. Okinaka (Hiroshima University, Japan). The virus was propagated in E-11 cells cultured at 25 °C in Leibovitz's L-15 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 5% fetal bovine serum until used for experiments. When the cytopathic effect (CPE) was extensive, the culture medium was harvested and centrifuged to remove cell debris. The supernatant was used as the virus stock for the experiments. The virus stock was titrated in 96-well plates, and titers are reported in terms of 50% tissue culture infectious dose (TCID50). 2.4. Nodavirus infection and TBT exposure The TBT concentrations and virus titers used in the current study were based on the results of preliminary experiments: median lethal concentrations (LC50) after a 48-h exposure were 47 μg/L for TBT and 104.5 TCID50/mL for RGNNV in 0 days post-hatching (dph) medaka. In the current study, the nominal concentrations were lowered to 0.17, 0.52, 1.6, and 4.7 μg/L for TBT and 101.5, 102.5, and 103.5 TCID50/mL for RGNNV. For infection, 10 medaka (0 dph) larvae were placed in 6-well plates containing RGNNV at 0, 102.5, 103.5, or 104.5 TCID50/mL in ERM and incubated for 96 h at 25 °C. After 96 h, all fish were washed with ERM and incubated in virus-free ERM for 72 h. Each infection group was then divided into 8-mL glass petri dishes containing TBT at concentrations of 0, 0.17, 0.52, 1.6, or 4.7 μg/L for 96 h. We removed dead and moribund fish and recorded the mortalities for 192 h. The experiments were replicated 5-fold. Data are given as mean ± S.D. and were analyzed by using twoway ANOVA followed by the Dunnett test. Virus-induced mortality at each TBT exposure level was fitted to a logistic regression model by the R project language. 2.5. Virus titers in tested fish We determined the virus titers in the medaka larvae that exposed to both 102.5 TCID50/mL RGNNV and 0.52 μg/L TBT (that is, the lowest observed effect dose [LOED] and lowest observed effect concentration [LOEC], respectively) or the same dose of the virus only. The fish was homogenized in 500 μL of Hanks balanced salt solution (Gibco, Grand Island, NY, USA); the homogenate was centrifuged at 2000 ×g for 5 min to remove cell debris; and the supernatant was filtered (pore size, 0.45 μm). The filtrate was 10-fold serially diluted with Hanks balanced salt solution, and 100 μL of each dilution was inoculated onto E-11 cells in 96-well tissue culture plates. The development of CPE was monitored for 10 d. Data were analyzed by one-way ANOVA. 3. Results 3.1. Effect of TBT exposure and virus infection on fish mortality The mortalities of medaka larvae exposed to RGNNV and TBT are shown in Fig. 1a. Mortalities in the no-treatment, TBT-only, and virus-
only groups were 12 ± 8.4%, 8.0–30%, and 26–54%, respectively. In the groups exposed to both virus and TBT, mortality was lowest (14 ± 5.5%) in medaka larvae exposed to 102.5 TCID50/mL RGNNV and 0.17 μg/L TBT, whereas the highest mortality (92 ± 4.5%) occurred in the group exposed to 104.5 TCID50/mL RGNNV and 4.7 μg/L TBT. Among the 12 groups exposed to both stressors, 6 showed significantly higher mortalities than that of each no-TBT group (P b 0.05). Specifically, mortality was 46 ± 5.5% in the medaka that received 102.5 TCID50/mL RGNNV and 0.52 μg/L TBT, which represent the LOED and LOEC among the 6 virus + TBT groups with increased mortality. Mortality curves in the virus-infected medaka exposed to different concentration of TBT were determined by logistic regression (Fig. 1b). TBT exposure enhanced mortalities of the virus-infected fish at the tendency of dose dependent manner; the mortalities in medaka exposed to 0, 0.17, 0.52, 1.6 and 4.7 ppb of TBT were 31, 23, 39, 48 and 73% at the infection of 102.5 TCID50/mL, and they were 43, 36, 50, 60 and 85% at the 103.5 TCID50/mL infection, respectively. The trends in mortality remained consistent even when the order of RGNNV infection and TBT exposure was reversed (that is, TBT exposure followed by viral infection) (Fig. S1). 3.2. Virus titer To ascertain whether the observed mortality after exposure to TBT and RGNNV was due to viral nervous necrosis, we measured the virus titers of fish exposed to both stressors or to the virus only. The titers of the fish ranged from 102.8 to 107.3 TCID50/fish (average, 104.5 TCID50/fish) in medaka larvae exposed to 102.5 TCID50/mL RGNNV (LOED) only and 103.6 to 107.6 TCID50/fish (average, 105.7 TCID50/fish) in fish exposed to both 102.5 TCID50/mL RGNNV and 0.52 μg/L TBT (LOEC) (Fig. 2). In addition, the virus titer was significantly higher (P b 0.05) in the fish exposed to both RGNNV and TBT than in the virus-only control group. No CPE was observed in either the solventonly or uninfected control groups. 4. Discussion Traditionally, the immunotoxicity of TBT has been evaluated by measuring leukocyte activities in various animals (Whalen et al., 1999, 2002; Nakayama et al., 2007; Misumi et al., 2009; Zhou et al., 2010a, 2010b; Lawrence et al., 2015), and most researchers regard TBT as an immune suppressor. However, whether TBT exposure increases susceptibility to infectious disease is unknown. Recently, Nakayama et al. (2016, in press) and Ye et al. (2016) suggested a method through which fish susceptibility to pathogen infection in the context of chemical exposure can be assessed. In the current study, we infected medaka with the nodavirus RGNNV and then exposed them to TBT. In our exposure experiments, the mortalities of the medaka larvae increased in a dose-dependent manner as the TBT concentration increased. In addition, TBT dose-dependent mortalities occurred in the groups infected with the virus both at low and high titers, the indication being that TBT is immunotoxic in the fish. Among the 12 groups exposed to both stressors (nodavirus and TBT), the mortalities of 6 were higher than those of the TBT control group. Increased mortalities due to combined stressors have been reported for various other types of chemicals, including benzo[a]pyrene (Carlson et al., 2002), polybrominated diphenyl ethers (Arkoosh et al., 2010), and heavy oil (Song et al., 2011). In the present study, we show that exposure to TBT exacerbated nodaviral infection in medaka larvae, that the LOED of the virus was 102.5 TCID50/mL, and that the LOEC of TBT was 0.52 μg/L. Mortality in medaka was similar, regardless of the order in which RGNNV infection and TBT exposure were applied (Figs. 1 and S1). In natural environments, the sequence of stressor exposure likely is variable, but our results indicate no order-associated influence on mortality.
Please cite this article as: Kitamura, S.-I., et al., Tributyltin exposure increases mortality of nodavirus infected Japanese medaka Oryzias latipes larvae, Marine Pollution Bulletin (2017), http://dx.doi.org/10.1016/j.marpolbul.2017.02.020
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Fig. 1. Cumulative mortalities of medaka larvae exposed to both nodavirus and tributyltin (TBT) (a). Data are shown as mean ± SD (%; n = 5 independent experiments; 10 fish in each group). Values significantly different from those for no-TBT group are indicated (*: p b 0.05, +: p b 0.01). Mortality curves in the fish exposed to different concentration of TBT were fitted to logistic regression analyzed by the R project language (b).
Medaka larvae infected with RGNNV in the present study did not demonstrate any neurologic symptoms, such as abnormal behavior or swimming. Furusawa et al. (2006) similarly reported that 1-day-old larvae bath-challenged with RGNNV died without showing characteristic erratic swimming. Although the reason is unclear, the lack of virus-associated symptoms might reflect the low swimming force in newborn medaka. To clarify the cause of mortality in the fish exposed to both RGNNV and TBT, we measured virus titers in the fish and compared them between the virus-only control and virus + TBT groups. The results
Fig. 2. Virus titers in infected fish. The fish were collected after 192 h of exposure at the LOEC for tributyltin (TBT). The data are shown as a boxplot. *, Significant difference between values for the virus + TBT and virus-only groups (p b 0.05, one-way ANOVA).
indicated that the virus titer was higher in the fish exposed to the virus and TBT at their LOED and LOEC than in the virus-only control group. These data suggest that TBT exposure increases viral replication and thus mortality in medaka larvae. The LOEC observed in the current study is 10 to 100 times higher than those of environmental contamination levels (Cao et al., 2009; Furdek et al., 2012; Kim et al., 2014), the suggestion being that the risk of occurrence of infectious disease might be low by contaminated TBT exposure in natural environments. However, because the concentration of TBT is relatively high in marine sediments (Kim et al., 2015; Suzdalev et al., 2015), the risk is not negligible, particularly for demersal fishes such as flatfishes and rockfishes. Moreover, dibutyltin, which is widely distributed in marine ecosystems, reportedly interferes with natural killer (NK) cell function (Whalen et al., 1999, 2002). Although the risk of TBT exposure alone may be minimal, the potential effects of exposure to both TBT and other chemicals concurrently should be considered. We consider the immunotoxic mechanism of TBT to act as follows. In general, type I interferon, NK cells, and immunoglobulins cooperatively help to protect against viral infection in animals. An anti-immunoglobulin effect of TBT probably does not contribute to the increased viral replication we observed because the experimental period was too short: pathogen-specific antibody levels rise beginning one week after infection in fish (Marsden et al., 1996; Kaattari et al., 2005). Moreover, the tested fish larvae likely were too immature to produce immunoglobulin. Therefore, we suspect that combined exposure to virus and TBT suppressed the activity of type I interferon and/or NK cells in the medaka larvae. In vitro, TBT interfered with the ability of human natural NK cells to lyse target human immune cells (Thomas et al., 2004; Aluoch and Whalen, 2005). The effect of TBT on the activity of type I interferon has not yet been reported, and further investigation should focus on this
Please cite this article as: Kitamura, S.-I., et al., Tributyltin exposure increases mortality of nodavirus infected Japanese medaka Oryzias latipes larvae, Marine Pollution Bulletin (2017), http://dx.doi.org/10.1016/j.marpolbul.2017.02.020
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factor and NK cell activity to reveal the cause of the high viral replication we observed here. In conclusion, combined exposure to nodavirus infection and TBT induced high mortality in medaka larvae, even though exposure to each stressor individually had little to no effect. The mortality was 46 ± 5.5% when fish were exposed to both 102.5 TCID50/mL RGNNV and 0.52 μg/L TBT, which represent the LOED and LOEC. In addition, the virus titer of medaka exposed to both RGNNV and TBT at their LOED and LOEC was higher than that of the virus-only control group, the suggestion being that TBT exposure accelerated virus replication. Our results imply that combined exposure to TBT and RGNNV suppresses host antiviral mechanisms and promotes viral replication, leading to high mortality in medaka larvae. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.marpolbul.2017.02.020. Acknowledgements We thank Prof. Oshima (Graduate School of Kyushu University, Japan) for providing medaka and Dr. Okinaka (Hiroshima University, Japan) for providing E-11 cells and RGNNV. References Aluoch, A., Whalen, M., 2005. Tributyltin-induced effects on MAP kinases p38 and p44/42 in human natural killer cells. Toxicology 209, 263–277. Arkoosh, M.R., Boylen, D., Dietrich, J., Anulacion, B.F., Ylitalo, G., Bravo, C.F., Johnson, L.L., Loge, F., Collier, T.K., 2010. Disease susceptibility of salmon exposed to polybrominated diphenyl ethers (PBDEs). Aquat. Toxicol. 98, 51–59. Cao, D., Jiang, G., Zhou, Q., Yang, R., 2009. Organotin pollution in China: an overview of the current state and potential health risk. J. Environ. Manag. 90, S16–S24. Carlson, E.A., Li, Y., Zelikoff, J.T., 2002. Exposure of Japanese medaka (Oryzias latipes) to benzo[a]pyrene suppresses immune function and host resistance against bacterial challenge. Aquat. Toxicol. 56, 289–301. Furdek, M., Vahcic, M., Scancar, J., Milacic, R., Kniewald, G., Mikac, N., 2012. Organotin compounds in seawater and mussels Mytilus galloprovincialis along the Croatian Adriatic coast. Mar. Pollut. Bull. 64, 189–199. Furusawa, R., Okinaka, Y., Nakai, T., 2006. Betanodavirus infection in the freshwater model fish medaka (Oryzias latipes). J. Gen. Virol. 87, 2333–2339. Gipperth, L., 2009. The legal design of the international and European Union ban on tributyltin antifouling paint: direct and indirect effects. J. Environ. Manag. 90, S86–S95. Grinwis, G.C., Vethaak, A.D., Wester, P.W., Vos, J.G., 2000. Toxicology of environmental chemicals in the flounder (Platichthys flesus) with emphasis on the immune system: field, semi-field (mesocosm) and laboratory studies. Toxicol. Lett. 112-113, 289–301. Harford, A.J., O'Halloran, K., Wright, P.E., 2007. Effect of in vitro and in vivo organotin exposures on the immune functions of Murray cod (Maccullochella peelii peelii). Environ. Toxicol. Chem. 26, 1649–1656. Horiguchi, T., Shiraishi, H., Shimizu, M., Morita, M., 1997. Effects of triphenyltin chloride and five other organotin compounds on the development of imposex in the rock shell, Thais clavigera. Environ. Pollut. 95, 85–91. Kaattari, S., Bromage, E., Kaattari, I., 2005. Analysis of long-lived plasma cell production and regulation: implications for vaccine design for aquaculture. Aquaculture 246, 1–9. Kannan, K., Tanabe, S., Tatsukawa, R., 1995. Occurrence of butyltin residues in certain foodstuffs. Bull. Environ. Contam. Toxicol. 55, 510–516. Kim, N.S., Hong, S.H., Yim, U.H., Shin, K.H., Shim, W.J., 2014. Temporal changes in TBT pollution in water, sediment, and oyster from Jinhae Bay after the total ban in South Korea. Mar. Pollut. Bull. 86, 547–554. Kim, N.S., Hong, S.H., An, J.G., Shin, K.H., Shim, W.J., 2015. Distribution of butyltins and alternative antifouling biocides in sediments from shipping and shipbuilding areas in South Korea. Mar. Pollut. Bull. 95, 484–490. Lawrence, S., Reid, J., Whalen, M., 2015. Secretion of interferon gamma from human immune cells is altered by exposure to tributyltin and dibutyltin. Environ. Toxicol. 30, 559–571. Marsden, M.J., Vaughan, L.M., Foster, T.J., Secombes, C.J., 1996. A live Aeromonas salmonicida vaccine for furunculosis preferentially stimulates T-cell responses
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Please cite this article as: Kitamura, S.-I., et al., Tributyltin exposure increases mortality of nodavirus infected Japanese medaka Oryzias latipes larvae, Marine Pollution Bulletin (2017), http://dx.doi.org/10.1016/j.marpolbul.2017.02.020