Toxicity of Triphenyltin Chloride to the Rotifer Brachionus koreanus Across Different Levels of Biological Organization Andy Xianliang Yi,1 Jeonghoon Han,2 Jae-Seong Lee,3 Kenneth M. Y. Leung1 1
The Swire Institute of Marine Science and School of Biological Sciences, The University of Hong Kong, Pokfulam, Hong Kong, China
2
Department of Chemistry, College of Natural Sciences, Hanyang University, Seoul 133-791, South Korea
3
Department of Biological Sciences, College of Natural Sciences, Sungkyunkwan University, Suwon 440-746, South Korea
Received 6 January 2014; revised 7 June 2014; accepted 15 June 2014 ABSTRACT: Although triphenyltin (TPT) compounds are ubiquitous pollutants in urbanised coastal environments in Asian regions, their toxicities to marine organisms are still poorly known. This study was designed to investigate the toxicity of triphenyltin chloride (TPTCl) on the rotifer Brachionus koreanus across different levels of biological organisation. Firstly, we concurrently performed a 24 h static-acute toxicity test and a 6-day semi-static multigenerational life-cycle test using the rotifer. Our results demonstrated that the 24-h median lethal concentration of TPTCl for the rotifer was 29.6 lg/L and the 6-day median effect concentration, based on the population growth inhibition, was 3.31 lg/L. Secondly, we examined the expression of 12 heat shock protein (hsp) genes, four glutathione S-transferase (GST) genes, one retinoid X receptor (RXR) gene and 13 cytochrome P450 (CYP) genes in the rotifers after exposure to 20 mg/L TPTCl for 24 h. Among these studied genes, hsp90a2, GST-O and CYP3045C1 were the most significantly up-regulated genes with a relative expression level up to 32.9, 4.4 and 62.6 folds, respectively. The expression of these three genes in the rotifers showed an increasing trend in the first few hours of TPTCl exposure, peaked at 3 h (hsp90a2 and GST-O) and 12 h (CYP3045C1) respectively, and then gradually returned to a lower level at 24 h. Such up-regulations of hsp and GST genes probably offer cellular protection against the TPT-mediated oxidative stress while the accelerated induction of CYP C 2014 Wiley Periodicals, Inc. Environ genes possibly facilitates the detoxification of this toxicant in the rotifer. V Toxicol 31: 13–23, 2016.
Keywords: CYP; rotifer; population growth rate; gene expression; ACR
Additional Supporting Information may be found in the online version of this article. Correspondence to: Kenneth M. Y. Leung, School of Biological Sciences, The University of Hong Kong, Pokfulam, Hong Kong, China. E-mail: [email protected] Contract grant sponsor: Swire Institute of Marine Science and the University of Hong Kong (to A.Y.). Contract grant sponsor: Area of Excellence Scheme under the University Grants Committee of the Hong Kong SAR Government. Contract grant number: AoE/P-04/2004. Contract grant sponsor: Ministry of Oceans and Fisheries, Korea. Contract grant number: PM57431 (to J.-S.L.). Published online 9 July 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/tox.22018 C 2014 Wiley Periodicals, Inc. V
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INTRODUCTION Rotifers are ecologically important in both freshwater and marine environments, as they are important food sources for other aquatic animals like copepods, insect larvae and fishes (Dahms et al., 2011). Since many rotifer species are easy to culture and breed in laboratory, they have been commonly employed as test organisms in laboratory-based ecotoxicological experiments (Wallace, 2002; Snell and JoaquimJusto, 2007). Such experiments range from the standard acute and chronic toxicity tests to behavioural and endocrinological studies (Dahms et al., 2011). Over the past decade, rotifers have also been applied in environmental genomic studies to investigate toxic mechanisms of chemical contaminants and reveal functional characteristics of toxicologically relevant genes (Kaneko et al., 2005; Suga et al., 2007a,b). Recently, there has been a growing interest of using the monogonont rotifer Brachionus koreanus as a model organism in the field of ecotoxicology and environmental genomics, because this rotifer species is small in size, simple in body organization, short in life span, genetically homozygous, predominantly parthenogenetic in reproduction and easy to cultivate in laboratory (Dahms et al., 2011; Huang et al., 2013). For example, B. koreanus has been utilized to study the impact of different environmental stressors such as temperature stress (Rhee et al., 2011), radioactive stress (Kim et al., 2011) and waterborne exposure to various chemical contaminants (Rhee et al., 2012a; Han et al., 2013). Furthermore, investigations on transcriptional changes of genes can help us to reveal the mechanism of toxic action of environmental stressors in B. koreanus. For instance, ultraviolet B radiation can trigger the up-regulation of DNA repairrelated genes and heat shock protein (hsp) genes in B. koreanus (Kim et al., 2011) while both temperature stress and hydrogen peroxide exposure can influence the expression of hsp20 in this species (Rhee et al., 2011). Han et al. (2013) also demonstrated that glutathione S-transferases (GSTs) in B. koreanus could function as one of the enzymatic defence mechanisms particularly during the early stage of oxidative stress mediated by waterborne copper exposure. Organotin compounds (OTs), especially tributyltin (TBT) and triphenyltin (TPT) are well known for their high toxicity to marine gastropod species (Antizar-Ladislao, 2008). Owning to their widespread contamination and adverse impacts to non-fouling marine organisms, a global ban of the application of OTs in any antifouling system on sea-going vessels has been enacted by the International Maritime Organization (IMO) since September 2009 (IMO, 2001). However, high levels of TPT can be detected in coastal marine organisms of Hong Kong, China, and Taiwan (Meng et al., 2005; Xie et al., 2010; Ho and Leung, in press). Although the toxicity of TPT to marine organisms is generally comparable to TBT, there are relatively much fewer saltwater toxicity data available for TPT, in particular chronic toxicity data (Yi et al., 2012). Therefore, this study aimed to evaluate the
Environmental Toxicology DOI 10.1002/tox
toxic effect of TPT to B. koreanus at molecular, individual and population levels and advance our understanding on the toxic mechanism of TPT to this saltwater species.
MATERIALS AND METHODS Chemical Preparation Stock solutions of TPTCl at concentrations ranging from 104 to 106 lg/L were prepared by dissolving TPTCl (>95%; Sigma) in dimethyl sulfoxide (DMSO; ACS reagent, 99.9%; Sigm). Test solutions were prepared by spiking appropriate volumes of the TPTCl stock solution into filtered artificial seawater (FASW; 15 6 1 ppt, 0.2 lm, Millipore).
Acute Toxicity Test A standard 24-h acute toxicity test was carried out with neonatal rotifers (less than 12 h posthatching). Ten neonates were placed into each well (with 4 mL working volume) of 12-well culture plates (Sigma-Aldrich) and exposed to a range of TPTCl concentrations (1.0 to 60 mg/L) as well as seawater control (FASW) and solvent control solution (0.01% DMSO in FASW). For each treatment or control group, there were three replicates. The rotifers were then incubated in an environmental chamber for 24 h under following conditions: 25 6 1 C; 14 h: 10 h light: dark; no feeding nor water renewal. Rotifers were considered dead if no movement was observed over a period of 10 s under a stereomicroscope (Sigma-Aldrich).
Chronic Toxicity Test A standard chronic toxicity test was conducted to investigate the effect of TPTCl on the reproduction of B. koreanus (OECD, 2006). Similar as acute toxicity test, ten neonates (less than 12 h old posthatching) were placed into each of the wells on the 12-well culture plate and each well contained 4 mL of the test solution (ranging from 1.0 to 10.0 mg/L TPTCl) or the seawater control solution or the solvent control solution. Three replicates were conducted for all treatment groups under the same test conditions as applied for the acute toxicity test. During the six days’ experiment, an algal diet of Tetraselmis suecica (approximately 105 cells/ml) was provided daily and test solutions were renewed once every 48 h. The numbers of rotifers were counted every 24 h under the stereomicroscope.
Gene Expression in the Rotifers Exposed to TPTCl Expressions of 12 heat shock protein (hsp) genes, four glutathione S-transferase (GST) genes, one retinoid X receptor (RXR) gene and 13 cytochrome P450 (CYP) genes were examined in the rotifers after exposure to TPTCl. According to the results of a previous study, many of the genes in B.
ECOTOXICITY OF TPT TO THE ROTIFER
15
koreanus showed the most different expression profiles after 3 h of exposure to chemicals (Wong, 2011). Thus, the first exposure experiment was set up to study the expression of all of the selected genes in a pool of about 10,000 B. koreanus in a 50-mL beaker after 3 h of exposure to 5 mg/L or 20 mg/L of TPTCl inside the environmental chamber at 25 6 1 C. The latter concentration was close to the acute LC10 value. Based on the results of this gene expression study, the genes showed the most different expression profiles were selected for the subsequent study. For the second exposure experiment, only one treatment concentration (20 mg/L of TPTCl) was chosen to investigate the expression of these selected genes at different time points, i.e., at 1, 3, 6, 12, 18, and 24 h. For each of these temporal treatment groups, approximate 8,000 to 10,000 B. koreanus neonates (
The Swire Institute of Marine Science and School of Biological Sciences, The University of Hong Kong, Pokfulam, Hong Kong, China
2
Department of Chemistry, College of Natural Sciences, Hanyang University, Seoul 133-791, South Korea
3
Department of Biological Sciences, College of Natural Sciences, Sungkyunkwan University, Suwon 440-746, South Korea
Received 6 January 2014; revised 7 June 2014; accepted 15 June 2014 ABSTRACT: Although triphenyltin (TPT) compounds are ubiquitous pollutants in urbanised coastal environments in Asian regions, their toxicities to marine organisms are still poorly known. This study was designed to investigate the toxicity of triphenyltin chloride (TPTCl) on the rotifer Brachionus koreanus across different levels of biological organisation. Firstly, we concurrently performed a 24 h static-acute toxicity test and a 6-day semi-static multigenerational life-cycle test using the rotifer. Our results demonstrated that the 24-h median lethal concentration of TPTCl for the rotifer was 29.6 lg/L and the 6-day median effect concentration, based on the population growth inhibition, was 3.31 lg/L. Secondly, we examined the expression of 12 heat shock protein (hsp) genes, four glutathione S-transferase (GST) genes, one retinoid X receptor (RXR) gene and 13 cytochrome P450 (CYP) genes in the rotifers after exposure to 20 mg/L TPTCl for 24 h. Among these studied genes, hsp90a2, GST-O and CYP3045C1 were the most significantly up-regulated genes with a relative expression level up to 32.9, 4.4 and 62.6 folds, respectively. The expression of these three genes in the rotifers showed an increasing trend in the first few hours of TPTCl exposure, peaked at 3 h (hsp90a2 and GST-O) and 12 h (CYP3045C1) respectively, and then gradually returned to a lower level at 24 h. Such up-regulations of hsp and GST genes probably offer cellular protection against the TPT-mediated oxidative stress while the accelerated induction of CYP C 2014 Wiley Periodicals, Inc. Environ genes possibly facilitates the detoxification of this toxicant in the rotifer. V Toxicol 31: 13–23, 2016.
Keywords: CYP; rotifer; population growth rate; gene expression; ACR
Additional Supporting Information may be found in the online version of this article. Correspondence to: Kenneth M. Y. Leung, School of Biological Sciences, The University of Hong Kong, Pokfulam, Hong Kong, China. E-mail: [email protected] Contract grant sponsor: Swire Institute of Marine Science and the University of Hong Kong (to A.Y.). Contract grant sponsor: Area of Excellence Scheme under the University Grants Committee of the Hong Kong SAR Government. Contract grant number: AoE/P-04/2004. Contract grant sponsor: Ministry of Oceans and Fisheries, Korea. Contract grant number: PM57431 (to J.-S.L.). Published online 9 July 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/tox.22018 C 2014 Wiley Periodicals, Inc. V
13
14
YI ET AL.
INTRODUCTION Rotifers are ecologically important in both freshwater and marine environments, as they are important food sources for other aquatic animals like copepods, insect larvae and fishes (Dahms et al., 2011). Since many rotifer species are easy to culture and breed in laboratory, they have been commonly employed as test organisms in laboratory-based ecotoxicological experiments (Wallace, 2002; Snell and JoaquimJusto, 2007). Such experiments range from the standard acute and chronic toxicity tests to behavioural and endocrinological studies (Dahms et al., 2011). Over the past decade, rotifers have also been applied in environmental genomic studies to investigate toxic mechanisms of chemical contaminants and reveal functional characteristics of toxicologically relevant genes (Kaneko et al., 2005; Suga et al., 2007a,b). Recently, there has been a growing interest of using the monogonont rotifer Brachionus koreanus as a model organism in the field of ecotoxicology and environmental genomics, because this rotifer species is small in size, simple in body organization, short in life span, genetically homozygous, predominantly parthenogenetic in reproduction and easy to cultivate in laboratory (Dahms et al., 2011; Huang et al., 2013). For example, B. koreanus has been utilized to study the impact of different environmental stressors such as temperature stress (Rhee et al., 2011), radioactive stress (Kim et al., 2011) and waterborne exposure to various chemical contaminants (Rhee et al., 2012a; Han et al., 2013). Furthermore, investigations on transcriptional changes of genes can help us to reveal the mechanism of toxic action of environmental stressors in B. koreanus. For instance, ultraviolet B radiation can trigger the up-regulation of DNA repairrelated genes and heat shock protein (hsp) genes in B. koreanus (Kim et al., 2011) while both temperature stress and hydrogen peroxide exposure can influence the expression of hsp20 in this species (Rhee et al., 2011). Han et al. (2013) also demonstrated that glutathione S-transferases (GSTs) in B. koreanus could function as one of the enzymatic defence mechanisms particularly during the early stage of oxidative stress mediated by waterborne copper exposure. Organotin compounds (OTs), especially tributyltin (TBT) and triphenyltin (TPT) are well known for their high toxicity to marine gastropod species (Antizar-Ladislao, 2008). Owning to their widespread contamination and adverse impacts to non-fouling marine organisms, a global ban of the application of OTs in any antifouling system on sea-going vessels has been enacted by the International Maritime Organization (IMO) since September 2009 (IMO, 2001). However, high levels of TPT can be detected in coastal marine organisms of Hong Kong, China, and Taiwan (Meng et al., 2005; Xie et al., 2010; Ho and Leung, in press). Although the toxicity of TPT to marine organisms is generally comparable to TBT, there are relatively much fewer saltwater toxicity data available for TPT, in particular chronic toxicity data (Yi et al., 2012). Therefore, this study aimed to evaluate the
Environmental Toxicology DOI 10.1002/tox
toxic effect of TPT to B. koreanus at molecular, individual and population levels and advance our understanding on the toxic mechanism of TPT to this saltwater species.
MATERIALS AND METHODS Chemical Preparation Stock solutions of TPTCl at concentrations ranging from 104 to 106 lg/L were prepared by dissolving TPTCl (>95%; Sigma) in dimethyl sulfoxide (DMSO; ACS reagent, 99.9%; Sigm). Test solutions were prepared by spiking appropriate volumes of the TPTCl stock solution into filtered artificial seawater (FASW; 15 6 1 ppt, 0.2 lm, Millipore).
Acute Toxicity Test A standard 24-h acute toxicity test was carried out with neonatal rotifers (less than 12 h posthatching). Ten neonates were placed into each well (with 4 mL working volume) of 12-well culture plates (Sigma-Aldrich) and exposed to a range of TPTCl concentrations (1.0 to 60 mg/L) as well as seawater control (FASW) and solvent control solution (0.01% DMSO in FASW). For each treatment or control group, there were three replicates. The rotifers were then incubated in an environmental chamber for 24 h under following conditions: 25 6 1 C; 14 h: 10 h light: dark; no feeding nor water renewal. Rotifers were considered dead if no movement was observed over a period of 10 s under a stereomicroscope (Sigma-Aldrich).
Chronic Toxicity Test A standard chronic toxicity test was conducted to investigate the effect of TPTCl on the reproduction of B. koreanus (OECD, 2006). Similar as acute toxicity test, ten neonates (less than 12 h old posthatching) were placed into each of the wells on the 12-well culture plate and each well contained 4 mL of the test solution (ranging from 1.0 to 10.0 mg/L TPTCl) or the seawater control solution or the solvent control solution. Three replicates were conducted for all treatment groups under the same test conditions as applied for the acute toxicity test. During the six days’ experiment, an algal diet of Tetraselmis suecica (approximately 105 cells/ml) was provided daily and test solutions were renewed once every 48 h. The numbers of rotifers were counted every 24 h under the stereomicroscope.
Gene Expression in the Rotifers Exposed to TPTCl Expressions of 12 heat shock protein (hsp) genes, four glutathione S-transferase (GST) genes, one retinoid X receptor (RXR) gene and 13 cytochrome P450 (CYP) genes were examined in the rotifers after exposure to TPTCl. According to the results of a previous study, many of the genes in B.
ECOTOXICITY OF TPT TO THE ROTIFER
15
koreanus showed the most different expression profiles after 3 h of exposure to chemicals (Wong, 2011). Thus, the first exposure experiment was set up to study the expression of all of the selected genes in a pool of about 10,000 B. koreanus in a 50-mL beaker after 3 h of exposure to 5 mg/L or 20 mg/L of TPTCl inside the environmental chamber at 25 6 1 C. The latter concentration was close to the acute LC10 value. Based on the results of this gene expression study, the genes showed the most different expression profiles were selected for the subsequent study. For the second exposure experiment, only one treatment concentration (20 mg/L of TPTCl) was chosen to investigate the expression of these selected genes at different time points, i.e., at 1, 3, 6, 12, 18, and 24 h. For each of these temporal treatment groups, approximate 8,000 to 10,000 B. koreanus neonates (
