Aquatic Toxicology 147 (2014) 57–67

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PBDE-47 exposure causes gender specific effects on apoptosis and heat shock protein expression in marine medaka, Oryzias melastigma Eddie E. Deane a,∗ , Jason P. van de Merwe b,1 , Jerome H.L. Hui a , Rudolf S.S. Wu c , Norman Y.S. Woo a a

School of Life Sciences, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong State Key Laboratory in Marine Pollution, Department of Biology and Chemistry, City University of Hong Kong, Kowloon, Hong Kong c School of Biological Sciences, The University of Hong Kong, Pokfulam, Hong Kong b

a r t i c l e

i n f o

Article history: Received 4 October 2013 Received in revised form 4 December 2013 Accepted 7 December 2013 Keywords: Fish PBDEs Marine medaka Apoptosis Heat shock protein Gender specificity

a b s t r a c t Marine medaka (Oryzias melastigma) was fed with a low and high dose of dietary 2,2 ,4,4 -tetrabromodiphenyl ether (PBDE-47), over 21 days. Gender specific changes in caspases 3 and 8 in medaka were found as activities in male medaka were significantly increased in both liver and muscle at both low and high exposure levels whereas caspase activity in female medaka tissue remained unchanged. Results of HSP90 and HSP70 immunoassays also showed gender specific related changes as both HSP families were unchanged in liver and muscle of male medaka but significantly increased in liver and muscle of female medaka, following PBDE-47 exposure. The gender specific effects of PBDE-47 on HSP expression profiles could not be explained by inherent differences in the heat shock response of male and female marine medaka, as the HSP profiles in liver and muscle, induced by acute heat shock, were similar in both sexes. The findings from this study provide evidence that PBDE-47 can cause gender specific modulatory effects on mechanisms critical to the apoptotic cascade as well as HSP regulation and expression. © 2014 Elsevier B.V. All rights reserved.

1. Introduction In the last two decades, polybrominated diphenyl ethers (PBDEs) have been widely used as flame retardants, for a range of consumer products, including furniture, plastics, textiles and electronics (Siddiqi et al., 2003). Due to their widespread usage, PBDEs have been widely detected in human tissues, cord blood (Frederiksen et al., 2010, Wu et al., 2010) and breast milk (Ohta et al., 2002). It is likely that the predominate mode of entry into humans is through consumption of contaminated fish (Schecter et al., 2004) as a result of global contamination of aquatic environments (Moon et al., 2007; Toms et al., 2008; Webster et al., 2008). Among the different PBDE congeners, PBDE-47 (2,2 ,4,4 -tetra-bromodiphenyl ether) has been found at the highest concentrations in marine fish due to its wide distribution and bioaccumulation potential (Brown et al., 2006; Meng et al., 2008). Studies aimed at assessing the impact of PBDE-47, on fish physiological function, have proven that this compound can have a wide range of adverse effects including attenuation of the immune response (Arkoosh et al., 2010), developmental neurotoxicity (Chen et al., 2012) and impairment of

∗ Corresponding author. Tel.: +852 39436280; fax: +852 39435646. E-mail address: [email protected] (E.E. Deane). 1 Present address: Smart Water Research Centre and Australian Rivers Institute, Griffith University, Gold Coast, Queensland, Australia. 0166-445X/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aquatox.2013.12.009

reproduction (Muirhead et al., 2006), and can cause malformation (Usenko et al., 2011). Apoptosis or programmed cell death can occur naturally or may be prematurely induced via abiotic, biotic or chemical mediated stress, which is a key process in development (Teodoro and Branton, 1997). The cascade of apoptotic events generally involves disruption of mitochondrial membrane potential, production of reactive oxygen species (ROS), activation of cysteine dependent aspartate proteases (caspases), DNA fragmentation and subsequent changes in cellular morphology (Teodoro and Branton, 1997; Takle and Andersen, 2007). To date, only a few studies have investigated the mechanisms by which PBDE-47 could influence the apoptotic cascade. Exposure of Jurkat cells to PBDE-47 induced apoptosis via overproduction of ROS and a concomitant downregulation of mitochondrial membrane potential (Yan et al., 2011). The cellular levels of ROS may also be a key pro-apoptotic stimulus, following exposure to PBDE-47, because primary rat cultured hippocampal neurons had attenuated antioxidant enzyme production in parallel with the onset of apoptosis (He et al., 2008) and rainbow trout fibroblasts had elevated levels of ROS (Jin et al., 2010). A key control mechanism of apoptosis involves activation of the caspase cascade and it is known that toxic compounds can induce caspase activity and hence apoptosis in fish (Takle and Andersen, 2007). Whilst it is not presently known if PBDE-47 can modulate caspase activity, studies using trout cells showed that exposure to cadmium (Rissode Faverney et al., 2004) or tributytin chloride (Tiano et al., 2003)

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can lead to activation of the caspase cascade through alterations in mitochondrial membrane potential. The heat shock protein (HSP) families play a critical role by functioning as molecular chaperones, by aiding in the assembly (Ellis, 1987), folding (Geething and Sambrook, 1992) and translocation of proteins (Chirico et al., 1988; Deshaies et al., 1988). Expression of these proteins increases when normal cellular processes are disrupted, as a protective mechanism by helping to re-fold, re-aggregate and restore proteins to their native conformation (Pelham, 1986). Accumulating evidence, from studies on mammalian models, has also suggested that induction of HSPs may perform an alternate protective role in cells by attenuating the onset of apoptosis via reduction in ROS production and/or through inhibition of caspase activation (Currie and Tanguay, 1991; Creagh and Cotter, 1997; Beere and Green, 2001). The occurrence of HSPs is ubiquitous across living organisms and their regulation and expression in fish have been comprehensively reviewed (Sanders, 1993; Iwama et al., 1998; Deane and Woo, 2011). In fish, two key HSP families are heat shock protein 90 (HSP90) and heat shock protein 70 (HSP70), of which the expression can be modulated by a range of stressors (Deane and Woo, 2011). In terms of chemical insults, several studies have reported that fish HSPs can be induced by exposure to heavy metals (Ryan and Hightower, 1994; Moreland et al., 2000; Boone and Vijayan, 2002; Feng et al., 2003; Zhou et al., 2003; Buckley et al., 2004; Deane and Woo, 2006; Fulladosa et al., 2006) or organochlorines (Kilemade and Mothersil, 2001; Deane and Woo, 2006). However our knowledge pertaining to the effects of PBDE-47 on modulating HSP expression in fish is limited even though the induction and detection of these proteins provide for useful secondary indicators of the stress response. Given the environmental importance of PBDE-47 and its accumulation potential in fish the present study was undertaken in order to investigate how parameters related to apoptosis and heat shock protein expression may be affected by this polybrominated diphenyl ether. Specifically, we have used marine medaka (Oryzias melastigma) that were fed bioencapsulated PBDE-47 for 21 days and then measured the activities of two important caspases in the apoptotic cascade (caspases 3 and 8) and two key HSP families (HSP90 and HSP70) in liver and muscle tissues. Furthermore we aimed to examine whether PBDE-47 accumulation may exert gender specific type effects in marine medaka by measuring caspase activities and HSP profiles in tissues taken from male and females separately.

2. Materials and methods 2.1. Bioencapsulation of PBDE-47 in Artemia The procedures followed were as previously described (Muirhead et al., 2006) with minor modifications. A stock solution of 2,2 ,4,4 -tetrabromodiphenyl ether (PBDE-47, Chem Service Inc., USA) was prepared to a final concentration of 10 mg/ml in hexane. To each of five 150 ml conical flasks, 0.8 ml of PBDE-47 stock solution was added and the hexane was allowed to evaporate. To each flask, 100 ml of newly hatched Artemia (∼1500 nauplii/ml) was added and the cultures were incubated at 27 ± 1 ◦ C, with gentle aeration, on a 12 h:12 h, light:dark cycle for 24 h. Following incubation, Artemia from all flasks were collected using an 80 ␮m sieve, washed several times with MilliQ water and pooled together in 500 ml MilliQ water to a final density of 1500 nauplii/ml. For the controls, uncontaminated Artemia were prepared in the same manner as above. Separate aliquots of Artemia were then transferred to glass tubes at amounts required for daily feeding of medaka and stored at −20 ◦ C.

2.2. Dietary exposure of medaka to PBDE-47 The original stock of marine medaka (O. melastigma) were purchased from Interocean Industries (Taiwan) and reared at the City University of Hong Kong. In accordance with The City University of Hong Kong Animals Ethics Committee Guidelines all fish were treated humanely with regard for alleviation of suffering during experimental tests. Thirty glass tanks (15 cm × 15 cm × 15 cm) each equipped with a removable glass divider, were filled with 2 l of filtered seawater and maintained at 22 ◦ C with gentle aeration. To allow acclimation to the experimental conditions, one male and one female medaka were placed in each tank for one week prior to exposure experiments, with a 14 h:10 h, light:dark cycle. Ten tanks were used for each experimental group and during the exposure period medaka were fed either 100 ␮l of uncontaminated Artemia (control group), 50 ␮l of contaminated Artemia (low dose group) or 100 ␮l of contaminated Artemia (high dose group). To ensure that each fish received the same dose of PBDE-47, the male and female medaka, in each tank, were separated with a glass divider immediately before feeding. The Artemia were completely consumed within 15 min and the glass dividers were removed 30 min after feeding to allow each pair to breed normally. To maintain fish condition throughout the experiment, medaka were also fed hormone free flake food (AX5, Aquatic Ecosysytems, USA) twice daily. The PBDE-47 exposure period was 21 days and every second day, half of the water was changed in each tank to remove waste and maintain water condition. After 21 days of PBDE-47 exposure, medaka were euthanized in ice cold MilliQ water for collection of muscle and liver tissues. Total body length and wet weight were measured before the medaka were placed on ice, for dissection under a light microscope. Muscle and liver tissue were collected from 10 males and 10 females, quick frozen with liquid nitrogen and stored at −80 ◦ C until analysis. 2.3. Bioaccumulation of PBDE-47 Prior to exposure experiments, 5 males and 5 females were randomly sampled from the stock population in order to assess if medaka had any background levels of PBDE-47. To assess the uptake of PBDE-47, during the experimental period, 5 males and 5 females were randomly collected from each group at the end of the 21 day exposure period and analyzed for PBDE-47. All fish analyzed for PBDE-47 content were collected 24 h following exposure to contaminated Artemia as this has been previously shown to be sufficient time for complete clearance from the medaka digestive system (van de Merwe et al., 2011). Whole medaka were freeze dried and grounded with 1.5 g anhydrous sodium sulfate that was spiked with 50 ppb 13 C-labeled PBDE-47 surrogates as previously described (Ye et al., 2012). Samples were prepared using a previously established method (Wan et al., 2010) and extracted with n-hexane/dichloromethane (1:1, v/v) for 10 min at 100 ◦ C, followed by n-hexane/methyl butyl ether (1:1, v/v) for 10 min at 60 ◦ C, using a Dionex ASE-350 accelerated solvent extractor (Sunnyvale, CA). Treatment and quantitative determination of PBDE-47 and possible biotransformation products (PBDEs 3, 15 and 28, methoxy PBDE 47 and hydroxyl PBDE 47) were measured according to procedures previously described (Ye et al., 2012). The lipid content of samples was determined using micro colorimetric sulfo-phospho-vanillin analysis modified from Lu et al. (2008). 2.4. Heat shock regime Medaka were divided into 2 groups (n = 4) and acclimated to opaque lab aquaria (30 cm × 44 cm × 26 cm) containing seawater at 22 ◦ C for 2 days prior to acute heat shock. Half the volume of water was changed daily and fish were fed flake fish food (Kohaku, Japan).

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Fish in the first group served as a control and were maintained at an ambient temperature of 22 ◦ C. Fish in the second group were subjected to a temperature increase of 0.16 ◦ C/min using an immersion heater, until a temperature of 30 ◦ C was attained. Medaka were maintained for 2 h at 30 ◦ C before being removed, and euthanized for liver and muscle removal. All tissue were quick frozen in liquid nitrogen and stored at −80 ◦ C.

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Table 1 Concentration of PBDE-47 in marine medaka after exposure to PBDE-47 bioencapsulated Artemia for 21 days. Data are expressed as mean ± S.E.M. (n = 5). Treatment

Sex

PBDE-47 concentration ± S.E.M. (mg/kg lipid)

Control

Male Female

0.25 ± 0.18 0.24 ± 0.10

2.5. Protein extraction and quantification

Low dose

Male Female

576 ± 300* 603 ± 245*

Small pieces of liver and muscle tissue from PBDE-47 fed and temperature stressed medaka were added to 0.5 ml of extraction buffer (4 M urea, 0.5%, w/v, SDS, 10 mM EDTA, 2 mM PMSF) and homogenized for 1 min using an Ultra-Turrax T 25 rotor stator homogenizer. Samples were then incubated at 94 ◦ C for 10 min, sonicated for 10 min and centrifuged at 10,000 × g for 5 min. The supernatant was collected and total soluble protein content was measured using a microassay based on the total dye binding method of Bradford (1976).

High dose

Male Female

1575 ± 423* 2516 ± 702*

2.6. Protein gel electrophoresis and immunoblotting To define the specificity of the antibody based assay, onedimensional SDS-PAGE (Laemmli, 1970) was applied to resolve proteins of different molecular size using a 4% (stacking) and a 15% (separating) polyacrylamide gel. The standard proteins used for SDS-PAGE were bovine brain HSP90 and HSP70 (Sigma, USA). For electrophoresis 1.5 ␮g of full range rainbow molecular weight marker (Amersham), 0.1 ␮g of standard protein and 10 ␮g of total liver and muscle protein from medaka were electrophoresed for 90 min at 100 V using a Bio-Rad mini gel kit. After SDS-PAGE, the resolved proteins were transferred to Hybond ECL-nitrocellulose membrane (Amersham), using an electrotransfer cell, for 1 h at 100 V. After protein transfer the membranes were air dried overnight and then blocked in 0.1 M PBS containing 3% (w/v) skimmed milk powder, for 1 h, with gentle agitation. Following blocking the membranes were rinsed with PBS containing 0.05% (v/v) Tween 20 (PBS-T), and probed with either a mouse monoclonal HSP90 (Sigma, H1775) or HSP70 (Sigma, H5147) antibody diluted 1:1000 or 1:2000, in PBS-T respectively. After 1 h, the membranes were washed twice, for 15 min each wash, with PBS-T, and then probed with an anti-mouse IgG-HRP conjugated antibody (Amersham, NA931V), diluted 1:5000 in PBS-T, for a further 1 h. The membranes were washed twice, for 15 min each wash, with PBS-T, and then developed using an ECL reagent system (Amersham). Once the specificities of the antibodies were established, an immuno-dot blot method (Heinicke et al., 1992) was used to assess the HSP content of all samples. Nitrocellulose membranes were soaked in 0.1 M PBS, for 10 min and then placed in a Bio-Dot microfiltration manifold (Bio-Rad). Protein samples (5 ␮g) were mixed with 0.1 ml of PBS-T and samples were added to separate wells of the manifold. A vacuum was used to pull the samples onto the membrane and then each well was washed with 0.2 ml of PBS-T. The membranes were air dried and probed and developed as described above. Immunoreactivity on membranes was visualized using a Lumi-Imager workstation (Roche) and quantified using Lumi-Analyst software (Roche). For each sample, the optical density (OD) × area (mm2 ) was determined for further statistical analysis. 2.7. Caspase activity assays As measurements for mid/late stage apoptosis, the activities of caspases 3 and 8 were determined in liver and muscle tissues taken from PBDE-47 exposed medaka. Tissue samples were homogenized in 0.5 ml ice cold 0.01 M phosphate buffered saline (PBS, pH 7.2) for 1 min using an Ultra-Turrax T 25 rotor stator homogenizer,

*

Values that were found to be significantly different (p < 0.05) from control values.

centrifuged at 10,000 x g for 5 min at 4 ◦ C and the supernatant removed and kept on ice. Aliquots (0.1 ml) of supernatants were taken for quantification of total soluble protein using a microassay based on the dye binding method of Bradford (1976). Following protein quantification, 0.5 mg of each sample was adjusted to final volume of 0.1 ml with PBS and used for caspase assays. For determination of caspases 3 and 8 activities colormetric microassay kits (Sigma, CASP3C and CASP8C) based on the hydrolysis of the peptide substrates acetyl-Asp-Glu-Val-Asp p-nitroanilide or acetyl-Ile-GluThr-Asp p-nitroanilide, resulting in the release of p-nitroaniline (pNA) were used and measured at an absorbance of 405 nm in a SpectraMAX 250 microplate reader (Molecular Devices). The amount of p-nitroanilide released from the substrates was measured against a standard curve and data was expressed as nmolpNA released/min/mg soluble protein. 2.8. Statistical analysis Data from bioaccumulation analyses was subjected to a two way ANOVA to test for interaction between treatment and sex. Data from PBDE-47 exposure experiments were subjected to a one way ANOVA, followed by a post hoc Student–Newman–Keuls test to

A

105 HSP90 75

50

B 105 75

HSP70

50

Fig. 1. Immunoblot detection of marine medaka HSP90 (A) and HSP70 (B). For each immunoblot the position of standard, liver and muscle HSPs are indicated. The position of protein molecular size markers in kDa is indicated to the left of each immunoblot.

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delineate significance amongst groups. A Student t-test was used for analyses of data from heat shock experiments. Significant level of p < 0.05 was used in all statistical analyses. 3. Results 3.1. PBDE-47 accumulation in medaka The amount of accumulated PBDE-47 at the end of this 21 day experiment is presented in Table 1. The amount of PBDE-47 low dose groups was approximately 2300 and 2500 fold higher in male and female medaka respectively. The amount of PBDE47 in high dose groups were approximately 6300 and 10,500 fold higher in male and female medaka respectively. Following a two way ANOVA it was found that there was a significant interaction between treatment and sex as females accumulated a significant higher concentration at the high feeding dose. Of the possible PBDE-47 congeners tested only PBDE-28 could be detected at 0.81 ± 0.56 mg/kg lipid and 0.71 ± 0.21 mg/kg lipid in male and female medaka respectively (from the low dose group) and 1.24 ± 0.53 mg/kg lipid and 2.21 ± 0.94 in male and female medaka respectively (from the high dose group).

3.5. Caspases 3 and 8 activities Liver caspase 3 activities in PBDE-47 exposed male marine medaka were significantly increased by 5.4 and 8.9 fold in low and high dose groups, respectively, in comparison to controls (Fig. 6A). Muscle caspase 3 activities in PBDE-47 exposed male marine medaka were significantly increased by 1.9 and 2.5 fold in low and high dose groups, respectively, in comparison to controls (Fig. 6B). Liver and muscle caspase 3 activities in PBDE-47 exposed female marine medaka were not significantly changed following dietary exposure to PBDE-47 (Fig. 6C and D). Liver caspase 8 activities in PBDE-47 exposed male marine medaka were significantly increased by 2.2 and 2.8 fold in low and high dose groups, respectively, in comparison to controls (Fig. 7A). Muscle caspase 8 activities in PBDE-47 exposed male marine medaka were significantly increased by 3.1 and 3.2 fold in low and high dose groups, respectively, in comparison to controls (Fig. 7B). Liver caspase 8 activity in female marine medaka were not significantly changed following PBDE 47 exposure although exposure at high dose resulted in a significant increase of 1.7 fold in muscle caspase 8 activity when compared to controls (Fig. 7C and D).

4. Discussion 3.2. Tissue HSP90 expression profiles following PBDE-47 exposure Western blot analysis showed that a single and specific band of protein of approximately 90 kDa was detected, in both medaka liver and muscle tissues (Fig. 1A). Following semi-quantitative immunoblotting it was found that HSP90 amounts remained non-significantly changed in male liver and muscle tissues following PBDE-47 exposure (Fig. 2A and B). However HSP90 amounts in PBDE 47 exposed female marine medaka were found to be significantly changed as liver amounts were approximately 1.3 fold higher in both low and high dose exposed medaka in comparison to controls (Fig. 2C). Female muscle HSP90 amounts were approximately 2.2 fold and 3.0 fold higher than controls, in low and high dose groups respectively (Fig. 2D). 3.3. Tissue HSP70 expression profiles following PBDE-47 exposure Western blot analysis defined that a single and specific protein of approximately 70 kDa was detected, in both medaka liver and muscle tissues (Fig. 1B). Following semi-quantitative immunoblotting, no significant change in the amount of HSP70 could be found in male liver and muscle tissues following PBDE-47 exposure (Fig. 3A and B). However, HSP70 in liver of PBDE-47 exposed female marine medaka significantly increased by 1.6 fold in both low and high dose groups in comparison to controls (Fig. 3C). Female muscle HSP70 significantly increased by approximately 1.9 fold and 2.4 fold in low and high dose groups respectively (Fig. 3D). 3.4. HSP expression following thermal stress Following an acute heat shock of +8 ◦ C for 2 h, it was found that HSP90 amounts significantly increased 2.1 and 1.8 fold in male marine medaka liver and muscle tissues respectively (Fig. 4A and B). In acute heat shocked female marine medaka HSP90 amounts increased 1.6 fold in liver (Fig. 4C) but were not significantly changed in the muscle tissue (Fig. 4D). The amounts of HSP70 significantly increased 2.9 and 2.1 fold in male marine medaka liver and muscle respectively (Fig. 5A and B). In acute heat shocked female marine medaka, HSP70 amounts were significantly increased 1.8 fold in liver (Fig. 5C) but remained unchanged in muscle tissue (Fig. 5D).

This study, aimed to test the effects of dietary PBDE-47 exposure on apoptosis and HSP expression in liver and muscle of male and female marine medaka. Bioencapsulation of PBDE-47 into Artemia and subsequent feeding to marine medaka has proven to be an effective method for dietary exposure experiments (Muirhead et al., 2006) as the body burden of PBDE-47 was approximately 2300–10,500 fold higher than controls in the low and high dose groups. It is important to note that there was high variation in the mean values of accumulated PBDE-47 and female medaka accumulated PBDE-47 to a significantly greater extent in high dose groups. A previous study on marine medaka reported similar findings after 5 days and 21 days exposure (Ye et al., 2012) but it is unclear as to whether such variation has an impact on the various parameters being measured. The accumulated PBDE-47 was poorly metabolized in marine medaka as only PBDE-48 could be detected at low levels. Similar results were obtained in PBDE-47 exposure experiments on Japanese medaka and a previous study on PBDE47 exposure of marine medaka concluded that only 0.07–0.1% of ingested PBDE-47 could be biotransformed to PBDE-48 (Ye et al., 2012). Changes in HSP profiles represent useful markers of the secondary stress response in animals. In this study, we found that both HSP90 and HSP70 families were only significantly increased in liver and muscle tissues of female medaka, following PBDE-47 exposure for 21 days. As the heat shock response is regulated via a complex pathway involving heat shock factor activation, heat shock element binding, HSP transcription and HSP translation then a possible explanation for these findings may be related to inherent sex differences in the heat shock response mechanism between male and female medaka. Recent studies using heat shocked mice have suggested that the heat shock response may display gender specificity as acute heat exposure resulted in a greater elevation of HSP in brain and lung of males compared to females (Bridges et al., 2012). In contrast, a study on water flea, Daphnia magna reported that only females had significantly increased levels of HSPs following acute thermal stress (Mikulski et al., 2011). As it is possible that the gender specific HSP response to PBDE-47 exposure that was observed in this study may also be explained by differences in the heat shock response between males and female marine medaka we performed a classic acute heat shock experiment. Whilst HSP90 and HSP70 families in female muscle were not significantly changed following

E.E. Deane et al. / Aquatic Toxicology 147 (2014) 57–67

♂ Liver

C L H

♂ Muscle

B

70

70

60

60

50

50

HSP90 (OD x mm2)

HSP90 (OD x mm2)

A

40 30 20 10

40 30 20 10

0 Control

Low

0

High

Control

PBDE 47 exposure

Low

♀ Muscle

D

70

70 50

*

40 30 20

*

60 HSP90 (OD x mm2)

*

60

High

PBDE 47 exposure

♀ Liver

C

HSP90 (OD x mm2)

61

50

*

40 30 20 10

10 0

0 Control

Low

High

PBDE 47 exposure

Control

Low

High

PBDE 47 exposure

Fig. 2. Expression of HSP90 in male (A and B) and female (C and D) marine medaka tissues after 21 days of being fed bioencapsulated PBDE-47. Immunoanalysis was used for detection of HSP90 and optical density (OD × mm2 ) was calculated using Lumi-Analyst 3.1 software. Above each bar chart the immuno-dot blot of all samples used for quantitative analysis is shown with PBDE-47 doses indicated as control (C) low (L) and high (H). For bar chart, HSP90 amounts are presented in arbitrary units and all values are mean ± S.E.M. (n = 5). An asterisk above a bar denotes values that were found to be significantly different (p < 0.05) from control values.

acute heat shock, the findings from this experiment demonstrated that female medaka did not have a greater heat shock response than male medaka. Therefore it can be concluded that the elevation of HSP90 and HSP70 families in PBDE-47 fed female marine medaka cannot solely be accounted for by an inherent augmented heat shock response. A plausible explanation for the gender specific effects of PBDE-47, on HSP expression, in medaka could be related to the potent xenoestrogen effects of PBDE-47 as excess estrogen has been shown to have a causal relationship with disrupted HSP levels in both fish (Moreland et al., 2000; Rendell and Currie, 2005)

and rodents (Paroo et al., 1999, 2002; Shinohara et al., 2004). It is plausible that male medaka are more sensitive to the estrogenic activity of PBDE-47 and hence would have a greater disruption of HSP expression levels. Further studies focused on measuring estrogen levels in PBDE-47 fed marine medaka would be required before we can either confirm of refute the above conjecture. Apoptosis or programed cell death is regulated by a host of cellular proteins including a cascade of cysteine dependent aspartate proteases (caspases) of which 14 different types have been reported in teleosts, many of which can be modulated by toxic compounds

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♂ Liver

♂ Muscle

B

60

60

50

50

40

40

HSP70 (OD x mm2)

HSP70 (OD x mm2)

A

30 20 10

30 20 10

0 Control

Low

0

High

Control

PBDE 47 exposure

60

*

♀ Muscle

D

60

*

40 30 20 10

*

50

HSP70 (OD x mm2)

HSP70 (OD x mm2)

50

High

PBDE 47 exposure

♀ Liver

C

Low

*

40 30 20 10 0

0 Control

Low

High

PBDE 47 exposure

Control

Low

High

PBDE 47 exposure

Fig. 3. Expression of HSP70 in male (A and B) and female (C and D) marine medaka tissues after 21 days of being fed bioencapsulated PBDE-47. Immunoanalysis was used for detection of HSP70 and optical density (OD × mm2 ) was calculated using Lumi-Analyst 3.1 software. Above each bar chart the immno-dot blot of all samples used for quantitative analysis is shown with PBDE-47 doses indicated as control (C) low (L) and high (H). For bar chart, HSP90 amounts are presented in arbitrary units and all values are mean ± S.E.M. (n = 5). An asterisk above a bar denotes values that were found to be significantly different (p < 0.05) from control values.

within the aquatic environment (Takle and Andersen, 2007). Two key caspases that have been commonly studied are the initiator caspase 8 and the effector caspase 3. Initiator caspases act to cleave pro-domains of effector caspases resulting in their activation for subsequent downstream functions including inactivating DNA repair, suppressing inhibitors of apoptosis and disrupting the cell

cytoskeleton (Wang et al., 2005). A potential mechanism of caspase activation appears to work upstream via alterations in mitochondrial membrane potential and subsequent cytochrome c release as evidenced from studies on cadmium exposed trout hepatocytes (Risso-de Faverney et al., 2004) and tributytin chloride exposed trout blood cells (Tiano et al., 2003). The mechanisms whereby

E.E. Deane et al. / Aquatic Toxicology 147 (2014) 57–67

♂ Liver

A

20 10

20 10 0

Heat shock

Control

*

30

* HSP90 (OD X mm2)

HSP90 (OD X mm2)

♂ Muscle

B

30

0

63

Treatment

♀ Liver

C

♀ Muscle

D 30

30

*

20

HSP90 (OD X mm2)

HSP90 (OD X mm2)

Heat shock

Control

Treatment

10 0

Control

20 10 0

Heat shock

Control

Heat shock Treatment

Treatment

Fig. 4. Expression of HSP90 in male (A and B) and female (C and D) marine medaka following an acute heat shock of +8 ◦ C for 2 h. Immunoanalysis was used for detection of HSP90 and optical density (OD × mm2 ) was calculated using Lumi-Analyst 3.1 software. The HSP90 amounts are presented in arbitrary units and all values are mean ± S.E.M. (n = 4). An asterisk above a bar denotes values that were found to be significantly different (p < 0.05) from control values.

♂ Liver

A

*

30 20 10 0

*

40

HSP70 (OD X mm2)

HSP70 (OD X mm2)

40

30 20 10 0

Control

♂ Muscle

B

Control

Heat shock

♀ Liver

C

20 10 0

40

*

30

Control

Heat shock

Treatment

♀ Muscle

D

HSP70 (OD X mm2)

HSP70 (OD X mm2)

40

Heat shock Treatment

Treatment

30 20 10 0

Control

Heat shock

Treatment

Fig. 5. Expression of HSP70 in male (A and B) and female (C and D) marine medaka following an acute heat shock of +8 ◦ C for 2 h. Immunoanalysis was used for detection of HSP70 and optical density (OD × mm2 ) was calculated using Lumi-Analyst 3.1 software. The HSP70 amounts are presented in arbitrary units and all values are mean ± S.E.M. (n = 4). An asterisk above a bar denotes values that were found to be significantly different (p < 0.05) from control values.

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♂ Liver

A

0.08 Caspase 3 activity

0.6

Caspase 3 activity

♂ Muscle

B

* 0.4

* 0.2

*

0.06

*

0.04 0.02 0

0 Control

Low

Control

High

♀ Liver

♀ Muscle

D 0.08

Caspase 3 activity

0.08

Caspase 3 activity

High

PBDE 47 exposure

PBDE 47 exposure

C

Low

0.06 0.04 0.02 0

0.06 0.04 0.02 0

Control

Low

High

PBDE 47 exposure

Control

Low

High

PBDE 47 exposure

Fig. 6. Caspase 3 activity in male (A and B) and female (C and D) tissues after 21 days of being fed bioencapsulated PBDE-47 at low and high doses. Enzyme activity was calculated as specific activity (nmolpNA released/min/mg soluble protein) and data is presented as mean ± S.E.M. (n = 5). An asterisk above a bar denotes values that were found to be significantly different (p < 0.05) from control values.

PBDE-47 induces apoptosis in fish cells are presently unknown although studies on human neuroblastoma cells suggested that PBDE-47 mediated increases in caspase activity could occur via alterations in mitochondrial membrane potential, disruptions in intracellular calcium ion homeostasis and augmented expression of death associated protein kinase (He et al., 2009). In this study we found that dietary accumulation of PBDE-47, in marine medaka, resulted in elevated hepatic caspases 3 and 8 activities in liver and muscle tissues of male marine medaka. It was also found that only caspase 8 was increased in muscle tissues of female medaka but no corresponding caspase 3 increase was observed indicating that full activation of the apoptotic cascade had not occurred in female muscle. Taken together these findings suggest that caspase activity and therefore the apoptotic cascade is induced by PBDE-47 more prominently in male marine medaka. These findings support previous studies whereby gender specific effects of PBDE-47 exposure in fish have been reported. For instance male fathead minnow that were fed bioencapsulated PBDE-47 had increased weight loss and erratic swimming behavior in comparison to PBDE 47 fed female fathead minnows (Muirhead et al., 2006). Also mRNA abundance of complement pathway genes were decreased in male medaka but remained either unchanged or increased in females following PBDE 47 exposure (Ye et al., 2012). Presently, an explanation as to why PBDE-47 induced apoptosis in male marine medaka, more prominently than females remains to be established. A plausible reason could be related to increased endogenous estradiol since PBDE-47 is known to have potent estrogenic effects (Meerts et al., 2001; Villeneuve et al., 2002; Fisher, 2004; Liu et al., 2011) probably via inhibition of estradiol sulfonation by estradiol transferase (Hamers et al., 2006). Increased levels of estradiol appear to be pro-apoptotic

as several studies employing mammalian cells have shown that estradiol exposure can induce apoptosis via different pathways including death receptor activation (Do et al., 2002), modulation of c-jun NH2 terminal kinase signaling (Atiok et al., 2007) and disruption of mitochondrial membrane potential (Ackermann et al., 2009). Whilst it is likely that PBDE-47 would have the same xenoestrogen type effect in both male and female marine medaka, the subsequent increase in endogenous estradiol would likely be more potent in males and hence pro-apoptotic, since females may not have the same degree of sensitivity due to their naturally higher levels of endogenous estradiol. The above conjecture could only be confirmed once future studies aimed at measuring estrogen levels in PBDE-47 fed marine medaka are undertaken. Overall, the findings from this study have shown that PBDE47 exposure attenuates HSP expression and is pro-apoptotic in male medaka, whereas in females elevated HSP expression in parallel with an attenuated apoptotic response is observed. As to whether these contrasting findings are simply causal or represent a mechanistic link between HSP expression and apoptosis remains a matter of conjecture at present. However, evidence that has been derived from studies on mammalian cells, suggests that augmented HSP70 expression can be anti-apoptotic and therefore protective (Mosser and Martin, 1992; Samali and Cotter, 1996; Mosser et al., 1997, 2000; Samali et al., 1999; Sreedhar et al., 1999; Guzik et al., 1999; Beere and Green, 2001). The anti-apoptotic effect of HSP70 appears to function by reducing ROS release via increased antioxidant enzyme production (Currie and Tanguay, 1991; Creagh and Cotter, 1997) and through inhibition of caspase activation (Beere and Green, 2001). Evidence for possible protective and antiapoptotic roles of HSPs in fish are emerging as studies on catfish

E.E. Deane et al. / Aquatic Toxicology 147 (2014) 57–67

♂ Liver

0.12

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Fig. 7. Caspase 8 activity in male (A and B) and female (C and D) tissues after 21 days of being fed bioencapsulated PBDE-47 at low and high doses. Enzyme activity was calculated as specific activity (nmolpNA released/min/mg soluble protein) and data is presented as mean ± S.E.M. (n = 5). An asterisk above a bar denotes values that were found to be significantly different (p < 0.05) from control values.

ovaries reported that an increase in the onset of apoptosis was correlated with decreased HSP70 expression (Weber and Janz, 2001). Causal evidence for the importance of HSPs was reported in sea bream whole blood where elevated HSP70 genes occurred in parallel with a reduction in DNA fragmentation (Deane and Woo, 2005). More conclusive evidence for the role of HSPs as anti-apoptotic factors were provided from studies using sea bream cells. Using sea bream fibroblasts it was found that the decreased mitochondrial membrane potential caused by bacterial toxin exposure was attenuated by subjecting cells to an acute heat shock and this protective effect was removed if cells were pre-treated with the HSP synthesis inhibitor quercetin (Deane et al., 2012). Suppression of HSP70 production (via cortisol exposure) in sea bream macrophages resulted in the onset of apoptosis, as defined by the occurrence of DNA fragmentation, when cells were exposed to ROS inducer, camptothecin (Deane et al., 2006). Also it has been reported that pre-treatment of sea bream fibroblasts with the HSP70 inducer azetidine attenuated caspase 3 activity following exposure to camptothecin (Deane et al., 2006). In summary, this study has conclusively shown that male and female marine medaka respond differently in terms of induction of apoptosis and heat shock proteins when fed PBDE-47. It was found that male marine medaka were more prone to the onset of apoptosis possibly due to an attenuation of anti-apoptotic mechanisms including HSP production. Whilst the underlying cause of this gender specific effect of PBDE-47 on marine medaka awaits further investigation it is apparent that wherever possible, studies on

the effects of chemical contaminants on fish physiological function should compare responses in both male and females. Acknowledgements This work is partially supported by an Area of Excellence grant (AoE/P-04/04) and General Research Funds (CUHK 478309, CUHK 477111) awarded by the Research Grants Council, Hong Kong. References Ackermann, S., Hiller, S., Osswald, H., Lõsle, M., Grenz, A., Hambrock, A., 2009. 17␤estradiol modulates apoptosis in pancreatic ␤-cells by specific involvement of the sulfonylurea receptor (SUR) isoform SUR1. J. Biol. Chem. 284, 4905–4913. Arkoosh, M.R., Boylen, D., Dietrich, J., Anulacion, B.F., Ginaylitalo, B.C.F., Johnson, L.L., Loge, F.J., Collier, T.K., 2010. Disease susceptibility of salmon exposed to polybrominated diphenyl ethers (PBDEs). Aquat. Toxicol. 98, 51–59. Atiok, N., Koyuturk, M., Atiok, S., 2007. JNK pathway regulates estradiol-induced apoptosis in hormone-dependent human breast cancer cells. Breast Cancer Res. Treat. 105, 247–254. Beere, H.M., Green, D.R., 2001. Stress management-heat shock protein-70 and the regulation of apoptosis. Trends Cell Biol. 11, 6–10. Boone, A.N., Vijayan, M.M., 2002. Constututive heat shock protein 70 (HSC70) expression in rainbow trout hepatocytes: effect of heat shock and heavy metal exposure. Comp. Biochem. Physiol. 132 C, 223–233. Bradford, M.M., 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principles of protein dye binding. Anal. Biochem. 72, 248–252. Bridges, T.M., Tulapurkar, M.E., Shah, N.G., Singh, I.S., Hasday, J.D., 2012. Tolerance for chronic heat exposure is greater in female than male mice. Int. J. Hyperthermia 28, 747–755.

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