Temperature and photoperiod affect the endocrine ...

Comparative Biochemistry and Physiology, Part C 151 (2010) 258–263

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Comparative Biochemistry and Physiology, Part C j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c b p c

Temperature and photoperiod affect the endocrine disruption effects of ethinylestradiol, nonylphenol and their binary mixture in zebrafish (Danio rerio) Yuanxiang Jin, Linjun Shu, Liwei Sun, Weiping Liu, Zhengwei Fu ⁎ College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou 310032, China

a r t i c l e

i n f o

Article history: Received 6 September 2009 Received in revised form 11 November 2009 Accepted 16 November 2009 Available online 24 November 2009 Keywords: Temperature and photoperiod Endocrine-disrupting effects Gene transcription Zebrafish

a b s t r a c t We found that temperature and photoperiod significantly influence the transcription of the estrogenresponsive genes, vitellogenin1 (Vtg1), vitellogenin2 (Vtg2), estrogen receptor-α (ERα) and estrogen receptor-β (ERβ), after a 21-day exposure to environmentally relevant concentrations of 17α-ethynylestradiol (EE2), nonylphenol (NP) and EE2 plus NP (EE2 + NP). In general, gene transcription levels were higher as temperature and photoperiod length increased. The mRNA levels of Vtg1 in EE2 (10 ng/L) and EE2 + NP (10 ng/L and 25 μg/L, respectively) groups were induced more than 104 times both in 21 °C–12 L and 30 °C– 14 L groups, but only 369 ± 23 and 178 ± 59-fold induced in 12 °C–10 L group compared to the control, respectively. Specifically, when exposed to a high concentration of NP (25 μg/L) for 21 days, the levels of all mRNAs examined were significantly increased (p b 0.05) in the 21 °C–12 L and the 30 °C–14 L groups compared to the controls. However, no obvious induction in transcription was observed in the 12 °C–10 L group. The results obtained in the present study clearly elucidate that temperature and photoperiod greatly influence the effect of EDCs, and thus suggest that to fully define the endocrine disruption effects seasonal and/ or climate change effects must also be investigated. © 2009 Elsevier Inc. All rights reserved.

1. Introduction Photoperiod and temperature are considered two of the most important factors that influence the growth, development and reproduction of aquatic species, including teleost fish. For example, Bon et al. (1997) demonstrated that previtellogenesis and type1vitellogenesis were photosensitive periods, and an abrupt increase in the amount of light from short days to long days alters the biochemical processes involved in reproduction in rainbow trout. Koger et al. (1999) suggested that light and temperature regimes influence reproduction in medaka. Migaud et al. (2002, 2004) demonstrated that photoperiod regimes influence Eurasian perch gametogenesis, spawning time, and spawning rate, along with egg quality and broodstock mortality. Furthermore, they found that photoperiodic variations also play a crucial role in the initiation of reproduction. Recently, a three-year study by Brown et al. (2006) suggests that spawning patterns and egg quality in Atlantic halibut are influenced by water temperature. Taken together, these data suggest that altered temperature or photoperiod conditions might correspondingly greatly influence fish physiology. In general, the endocrine system tightly controls growth, development and reproduction in teleost fish. However, endocrine disruption effects influenced by temperature and photoperiod in some model fish species have received limited attention. We found that photoperiod and temperature

⁎ Corresponding author. Tel.: +86 571 8832 0599. E-mail address: [email protected] (Z. Fu). 1532-0456/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpc.2009.11.004

influenced 17β-estradiol (E2)- and nonylphenol (NP)-mediated endocrine effects in adult male zebrafish after short-term exposure (Jin et al., 2009a). In fact, environmental exposure occurs under variable and suboptimal regimes (Brian et al., 2008). Hence, it is necessary to assess toxicological responses in fish, including endocrine-disrupting effects, under different environmental conditions. Synthetic estrogen, 17α-ethynyestradiol (EE2), is known to significantly contribute to the estrogen content of sewage treatment plant (STP) effluents (Desbrow et al., 1998). Meanwhile, NP, a non-ionic surfactant, exerts numerous direct and indirect endocrine disruption effects in different fish species, including zebrafish (Yang et al., 2006; Hill and Janz, 2003; Seki et al., 2003). Runoff, erosion, leaching and lateral movement through the soil or tile drains, along with effluents from wastewater treatment plants, are the major routes by which these endocrine-disrupting chemicals (EDCs) enter surface waters. In China, the concentration of EE2 in aquatic systems ranges from undetected to more than 30 ng/L (Shen et al., 2001; Cui et al., 2006), while the concentration of NP in the aquatic systems ranges from undetected to 24.8 μg/L (Xu et al., 2006; Jin et al., 2008a; Zhang et al., 2009). In reality, aquatic organisms are rarely exposed to a single chemical in isolation, but rather to a complex mixture of chemicals (Thorpe et al., 2006). Therefore, combination effects require urgent attention when assessing the potential risks of EDCs. During the last decade, the combined effects of EDCs have received increasing attention and several significant contributions have been made to this field of research (Brian et al., 2007; Filby et al., 2007; Kortenkamp, 2007; Sun et al., 2009). On the contrary, to date, little emphasis has been placed on the endocrine disruption

Y. Jin et al. / Comparative Biochemistry and Physiology, Part C 151 (2010) 258–263

effects induced by environmentally relevant concentrations of EDCs and/or their mixtures, especially in regard to different temperature and photoperiod conditions corresponding to changes in the climate. Hence, the interactive effects of environmental variables, as well as chemical mixtures, warrant further attention in risk assessment methodology (Brian et al., 2008). The main purpose of the present study was to investigate the influence of temperature and photoperiod (mainly corresponding to yearly seasonal changes) on the estrogenic response of fish to environmentally relevant concentrations of a single or a defined mixture of chemicals in adult male zebrafish. To achieve this, we used a synthetic estrogen (EE2), an estrogen mimicking compound (NP) and their binary mixture (EE2 + NP) as representative chemicals. Previous reports show that EE2 and NP bind to estrogen receptors (ERs) to promote target gene transcription, such as vitellogenin1 (Vtg1). The ERs and their target genes were used as biomarkers to detect EDCs in some fish species, including zebrafish (Yadetie et al., 1999; Brown et al., 2004; Jung et al., 2006; Jin et al., 2009b). Thus, we used a real-time polymerase chain reaction (PCR) approach to detect hepatic estrogen-responsive gene transcription in adult male zebrafish exposed to EE2, NP and their binary mixed solution under different photoperiod and temperature regimes. 2. Materials and methods 2.1. Experimental fish Healthy, five-month-old, adult male zebrafish (Danio rerio) were kept in glass tanks. Prior to the exposure experiments, the fish were acclimatized in a tank filled with ambient temperature (25 ± 1 °C) water for one week with a photoperiod of 12-hour light/12-hour dark. The fish were fed twice a day with brine shrimp or a commercial diet. Male fish (weighing 428.6 ± 27 mg and measuring 3.56 ± 0.21 cm in length) were randomly selected for exposure experiments. No statistically significant differences in body weight or length were present at the beginning of the exposure (data not shown). 2.2. Photoperiod and temperature regimes To determine whether and how environmental conditions affect the endocrine-disrupting effects of EDCs, different photoperiod and temperature regimes were selected. Zebrafish were subjected to three different photoperiod and temperature regimes according to the local temperature and photoperiod changes experienced during different seasons, as follows: 1) low temperature (12 °C) with a short light period of 10-hour light/14-hour dark (hereafter abbreviated 12 °C–10 L); 2) middle temperature (21 °C) with a normal light period of 12-hour light/ 12-hour dark (21 °C–12 L); and 3) high temperature (30 °C) with a long light period of 14-hour light/10-hour dark (30 °C -14 L). Zebrafish exposure tanks were placed in a constant temperature-light incubator (laifu, Ningbo, China) to control the photoperiod and temperature automatically. 2.3. Exposure experiments and sample collection EE2 (CAS No: 21221-29-4, ≥98%) and NP (CAS 84852-15-3, technical grade) were purchased from Sigma-Aldrich (USA). Stock solutions of EE2 and NP were prepared by dissolving the chemicals in ethanol (100%) at a concentration of 1 g/L and 10 g/L, respectively. The solutions were stored in the dark at 5 °C. For better assessment of the endocrine disruption effects of EE2, NP and their binary mixtures at different temperature and photoperiod conditions, adult male zebrafish were exposed to EE2 at concentrations of 1 ng/L and 10 ng/L, to NP at 2.5 μg/L and 25 μg/L, and to two different concentrations of EE2 plus NP mixtures (low concentrations of 1 ng/L EE2 plus 2.5 μg/L NP and high concentrations of 10 ng/L EE2 plus 25 μg/L NP) in water containing 0.1%

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ethanol. Water was dechlorinated and prefiltered through activated carbon prior to use (pH of 6.6 to 7.5, hardness of 42–56 mg CaCO3/L). Six adult male fish were reared in 3 L of each solution in glass vessels. Four separated glass vessels were used for each treatment. Control male fish were raised in rearing water with 0.1% ethanol but were not treated with EE2, NP or their binary mixture. Prior to exposure, zebrafish were reared individually under the aforementioned photoperiod and temperature conditions for seven days. Then, the selected adult male zebrafish were exposed to the mentioned test solutions under static conditions for 21 days. Rearing water and exposure solutions were completely changed (100%) every day. Importantly, the new water was pre-cooled or pre-warmed to the experimental temperatures before being added every day. Before dissection, the fish were anesthetized on ice. The livers were excised from four to five fish and were minced and pooled into a single sample. This resulted in a minimum of four pooled samples analyzed per treatment. These samples were kept on dry ice during preparation and then stored at −80 °C until analysis. 2.4. Isolation of RNA and reverse transcription Liver samples were homogenized in 0.5 mL TRIzol reagent (Takara Biochemicals, Dalian, China) with a homogenizer (Polytron, Switzerland). Total RNA was isolated according to the manufacturer's instructions. The 260 nm to 280 nm ratio and the banding pattern on a 1% agarose formaldehyde gel were used to verify the RNA quality of each sample. Subsequently, the RNA was denatured at 65 °C for 15 min. Reverse transcription (RT) was carried out using ReverTra Ace qPCR® RT kit (Toyobo, Tokyo, Japan). The reaction mixture was incubated at 37 °C for 15 min and then heated to 98 °C for 2 min to stop the RT reaction. The RT product was diluted 2-fold and 1 μL of the sample was used for real-time PCR. 2.5. Gene expression analysis Real-time quantitative PCR was performed using an Eppendorf MasterCycler® ep realPlex4 (Wesseling-Berzdorf, Germany). Oligonucleotide primer pairs of Vtg1, Vtg2, ERα, ERβ and housekeeping gene (β-actin) were used as follows: Vtg1 forward 5′-AACGAACAGCGAGAAAGAGATTG-3′, reverse 5′-GATGGGAACAGCGACAGGA-3′ corresponding to positions 3895-4024 (Accession number: NM_170767); Vtg2 forward 5′-GGTGACTGGAAGATCCAAG-3′, reverse 5′-TCATGCGGCATTGGCTGG-3′ corresponding to positions 174-346 (AY729645); ERα forward 5′-CCCACAGGACAAGAGGAAGA-3′, reverse 5′-CCTGGTCATGCAGAGACAGA-3′ corresponding to positions 967–1217 (NM_152959); ERβ forward 5′-TGATTAGCTGGGCGAAGA-3′, reverse 5′-ATCCAGCCAGCAGCATT-3′ corresponding to positions 1104–1189 (AJ414566); and βactin forward 5′-ATGGATGAGGAAATCGCTGCC-3′, reverse 5′-CTCCCTGATGTCTGGGTCGTC-3′ corresponding to positions 54–160 (AF057040). β-actin levels were used to standardize the results, since the Ct value of β-actin expression did not change significantly (p b 0.05) following any of the treatments. Each mRNA level is expressed as a ratio compared to the level of β-actin mRNA. The following PCR protocol was used: denaturation for 1 min at 95 °C, followed by 40 cycles of 15 s at 95 °C, and 1 min at 60 °C. The relative quantification of gene expression among the treatment groups was analyzed by the 2− ΔΔCt method (Livak and Schmittgen, 2001). 2.6. Data analysis Experimental data were checked for normality and homogeneity of variance using Kolmogorov–Smirnov one-sample test and Levene's test, respectively. When necessary, logarithmic transformation was performed for the data normalization and to reduce heterogeneity of variance. Intergroup differences were assessed by one-way analysis of variance (ANOVA) followed by Dunnett's or Tukey's post hoc test. All

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statistical analyses were carried out using SPSS 13.0 (SPSS, Chicago, IL, USA) and Origin 7.0 (OriginLab, Northampton, MA, USA). The critical value for statistical significance was p b 0.05. 3. Results Fig. 1 shows the hepatic mRNA levels of Vtg1 in adult male zebrafish exposed to EE2, NP and their binary mixture for 21 days under different temperature and photoperiod conditions. Generally, the estrogenresponsive gene transcription levels increased when the temperature and photoperiod increased for all treatment conditions. For example, when exposed to EE2 (10 ng/L) and mixture (10 ng/L and 25 μg/L, respectively) for 21 days, the Vtg1 mRNA levels increased by 369- and 178-fold in 12 °C–10 L group (Fig. 1 A), while they were up to more than 104 times higher both in 21 °C–12 L and 30 °C–14 L treatments (Fig. 1 B and C), in comparison with their respective control group. In addition, when exposed to low concentrations of EE2 (1 ng/L) or EE2 + NP (1 ng/L and 2.5 μg/L, respectively), a significant induction of Vtg1 mRNA (p b 0.05; Fig. 1 C) was only observed in the 30 °C–14 L group. Moreover, the Vtg1 mRNA levels in the 21 °C–12 L and 30 °C–14 L treatment groups were higher than that of 12 °C–10 L when exposed to 25 μg/L NP, but no any induction was observed in all treatment groups when exposed to 2.5 μg/L NP (Fig. 1). A significant induction of Vtg1 mRNA expression was seen in both the 21 °C–12 L and the 30 °C–14 L groups when exposed to a high concentration (25 μg/L) of NP (Fig. 1 B and C). The induction pattern of vitellogenin2 (Vtg2) at different temperature and photoperiod conditions was similar to that of Vtg1. However, the fold inductions observed were lower than those of Vtg1 (Fig. 2). For

Fig. 2. Hepatic Vtg2 mRNA levels in adult male zebrafish exposed to various concentrations of EE2, NP or their binary mixture for 21 days at different temperatures and photoperiods (A: 12 °C–10 L; B: 21 °C–12 L and C: 30 °C–14 L). Values were normalized against β-actin and represent the mean mRNA expression value ± S.E.M (n = 4) as compared to controls. Statistically significant differences are shown by ⁎ for p b 0.05 and ⁎⁎ for p b 0.01. Relative expression levels of Vtg2 are plotted on a log scale.

Fig. 1. Hepatic Vtg1 mRNA level of adult male zebrafish exposed to various concentrations of EE2, NP or their binary mixture for 21 days at different temperatures and photoperiods (A: 12 °C–10 L; B: 21 °C–12 L and C: 30 °C–14 L). Values were normalized against β-actin and represent the mean mRNA expression value ± standard error of the mean (S.E.M) as compared to controls (n = 4). Statistically significant differences are shown by an asterisk, ⁎ for p b 0.05 and ⁎⁎ for p b 0.01. Relative expression levels of Vtg1 are plotted on a log scale.

example, the Vtg2 mRNA level was approximately 3-fold lower than the Vtg1 mRNA level induced after exposure to 1 ng/L of EE2 in the 30 °C–14 L group. Next, we examined the effect of different temperature and photoperiod conditions on the hepatic mRNA level of both estrogen receptor-α (ERα) and estrogen receptor-β (ERβ) in adult male zebrafish exposed to EE2, NP or a binary mixture for 21 days. Similar to the induction pattern for both Vtg1 and Vtg2, ERα and ERβ transcription was up-regulated in all treatments groups when the temperature and the photoperiod were extended. For example, when exposed to a high concentration of EE2 (10 ng/L), the corresponding level of ERα induced was 15-, 79-, and 53-fold in the 12 °C–10 L, 21 °C–12 L and 30 °C–14 L groups, respectively (Fig. 3). For the EE2 + NP (10 ng/L and 25 μg/L, respectively) treated groups, a 10-, 52- and 46-fold increase in ERα mRNA was observed in the 12 °C–10 L, 21 °C–12 L and 30 °C–14 L groups, respectively. In addition, exposure to 25 μg/L of NP resulted in a significant induction (p b 0.05) of hepatic ERα mRNA levels in the 21 °C–12 L and the 30 °C–14 L groups, but not in the 12 °C–10 L group (Fig. 3). However, no induction of ERα mRNA was observed in any of the treatment groups that were exposed to low concentrations of EE2, NP or EE2 + NP. Compared to ERα, a lower fold induction of hepatic ERβ mRNA expression was observed, although the induction pattern was similar under the different conditions. ERβ mRNA levels in the group treated with a high concentration of EE2 and the EE2 + NP were higher than those treated with low concentration. In the groups treated with a high concentration of EE2, the mRNA levels increased only 3-, 6-, and 9-fold in the 12 °C–10 L, 21 °C–12 L and 30 °C–14 L groups, respectively (Fig. 4). In the groups treated with a high concentration of EE2+NP, ERβ


Fig. 3. Expression of hepatic ERα mRNA in adult male zebrafish exposed to various concentrations of EE2, NP or their binary mixtures for 21 days at different temperatures and photoperiods (A: 12 °C–10 L; B: 21 °C–12 L and C: 30 °C–14 L). Values were normalized against β-actin and represent the mean mRNA expression value ± S.E.M (n = 4) as compared to controls. Statistically significant differences are shown by ⁎ for p b 0.05 and ⁎⁎ for p b 0.01. The relative expression level of ERα in B and C is plotted on a log scale.

mRNA increased about 2-, 5-, and 7-fold in the 12 °C–10 L, 21 °C–12 L and 30 °C–14 L groups, respectively. Furthermore, the transcription of ERβ was not significantly induced in the 12 °C–10 L group even at higher concentrations of EE2 + NP (Fig. 4 A). To better understand whether temperature and photoperiod conditions significantly influence estrogen-responsive gene transcription, the intergroup differences in the transcription of the above mentioned genes were assessed by one-way ANOVA followed by Dunnett's post hoc test. The results were shown in Table 1. It was observed that Vtg1 and ERα mRNA levels were not influenced by the different temperature and photoperiod in the control group, however, the Vtg2 and ERβ mRNA levels were up-regulated significantly, with the increase of 2.5- and 1.7-fold in 30 °C–14 L and 21 °C–12 L, respectively, as compared to that of in 12 °C–10 L. In addition, the sensitivity of the above gene transcription influenced by temperature and photoperiod was increased when the exposure concentrations increased in all EE2, NP and EE2 + NP treatment concentrations tested. For example, when exposed to high concentrations of EE2, NP or EE2 + NP, the Vtg1 mRNA level significantly increased in the 21 °C– 12 L group compared to the 12 °C–10 L group; however, a significant increase was not found between these groups at the corresponding low exposure concentrations (Table 1). More importantly, a significant difference in the transcription of all the genes was found between the 12 °C–10 L and the 30 °C–14 L groups for almost all the chemical

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Fig. 4. Expression of hepatic ERβ mRNA in adult male zebrafish exposed to various concentrations of EE2, NP or their binary mixtures for 21 days at different temperatures and photoperiods (A: 12 °C–10 L; B: 21 °C–12 L and C: 30 °C–14 L). Values were normalized against β-actin and represent the mean mRNA expression value ± S.E.M (n = 4) as compared to controls. Statistically significant differences are shown by ⁎ for p b 0.05 and ⁎⁎ for p b 0.01.

Table 1 Results of significant difference comparisons for temperature and photoperiod variation on estrogenic gene transcription in male zebrafish.a Treatment

Control

EE2

NP

EE2 + NP

Gene

Vtg1 Vtg2 ERα ERβ Vtg1 Vtg2 ERα ERβ Vtg1 Vtg2 ERα ERβ Vtg1 Vtg2 ERα ERβ

Temperature and photoperiod conditions 12 °C–10 L

21 °C–12 L

30 °C–14 L

a a a a aA aA aA aA aA aA aA aA aA aA aA aA

a a a b aB bB aB aB abB abB bB bB abB aAB bB abB

a b a ab bB bA aC bB bC cC bAB abB cC aC bB bB

a In the case of control groups, the different lowercase letters indicate significant difference (p b 0.05) in gene transcription between the different temperature and photoperiod conditions. For the EE2, NP and EE2 + NP treatment groups, the different lowercase letters indicate significant difference (p b 0.05) at a low exposure concentration while the different uppercase letters represent corresponding significant difference (p b 0.05) at a high exposure concentration.

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treatments (Table 1). In addition, some gene transcription was significantly influenced by photoperiod and temperature, even at low exposure concentrations. For instance, when exposed to a low concentration of either NP (2.5 μg/L) or EE2 + NP (1 ng/L and 2.5 μg/L, respectively), a significant difference in the level of ERα mRNA was observed between the 12 °C–10 L group and the 21 °C–12 L group (Table 1). 4. Discussion In general, the mRNA level of Vtg1, Vtg2, ERα and ERβ in both male and sexually immature fish is normally very low. However, these levels could be significantly increased in adult male or sexually immature fish treated with EDCs. Several published reports have strongly proven that the induction of transcription of Vtg1, Vtg2, ERα, and ERβ, can be considered as specific biomarkers linked to hormonemimetic contaminants in the fish's environment (Wang et al., 2005; Yamaguchi et al., 2005; Barucca et al., 2006; Jin et al., 2008b). In agreement with previous reports, our results confirm that the transcription of Vtg1, Vtg2, ERα and ERβ in the liver of adult male zebrafish can be rapidly and efficiently induced in a dose-dependent manner after 21 days of exposure to EE2, NP, and EE2 + NP at environmentally relevant concentrations (Yamaguchi et al., 2005; Zhang et al., 2008). More importantly, we also found that temperature and photoperiod influenced the endocrine effects of EE2, NP and a binary mixture. Specifically, when exposed to a low concentration of EE2 (1 ng/L), Vtg1 was up-regulated by 5-, 9-, and 70-fold in the 12 °C–10 L, 21 °C–12 L, and 30 °C–14 L temperature and photoperiod conditions tested, respectively (Fig. 1). Similarly, the mRNA level of Vtg2 increased 2-, 28- and 24-fold in the 12 °C–10 L, 21 °C–12 L and 30 °C–14 L groups, respectively. Growing evidence suggests that temperature and photoperiod play important roles in the endocrine system of several fish species. Korsgaard et al. (1986) suggested that the vitellogenic response to estradiol (E2) treatment was dependent on the ambient temperature during the E2 treatment of post-smolt Atlantic salmon. Mackay and Lazier (1993) found that rainbow trout maintained at 15 °C during E2 treatment demonstrated a more rapid response than fish kept at 9 °C, with a 3-fold greater accumulation of hepatic Vtg mRNA during a 10-day period. Körner et al. (2008) reported that synthesis of Vtg mRNA was stimulated in juvenile brown trout (Salmo trutta) kept at a high water temperature (19 °C). The change in protein level of Vtg was similar, with an increase in temperature resulting in an increase in plasma Vtg concentration. Howell et al. (2003) indicated that photoperiod may influence the reproduction of black sea bass (Centropristis striata). More recently, we reported in zebrafish that the hepatic gene transcription of estrogen-responsive gene, such as Vtg1, Vtg2 and ERα, at a high temperature and long photoperiod (30 °C–16 L) were higher than those at low temperature and short photoperiod (20 °C–8 L) upon exposure to 250 ng/L E2 and 100 μg/L NP for just two days. We also observed that temperature and photoperiod interacted significantly on hepatic Vtgs and ERα gene expression in E2 exposure (Jin et al., 2009b). Thus, to better understand the potential endocrine disruption effects induced by EDCs under different temperature and photoperiod conditions, we defined the temperature and photoperiod according to seasonal climate changes, such as spring and autumn (21 °C–12 L), winter (12 °C–10 L) and summer (30 °C–14 L). In agreement with previous observations, the current experiments demonstrate that the levels of transcription of estrogen-responsive genes were greatly influenced by the temperature and photoperiod after a 21-day exposure to both low and high concentrations of EE2 and NP. Moreover, gene transcription levels were significantly influenced when the temperature and photoperiod conditions were altered at the same exposure concentration. For example, a 10-fold higher Vtg1 mRNA level was induced by 1 ng/L of EE2 at the high temperature and long photoperiod (30 °C–14 L), compared to the mRNA level when the zebrafish were exposed to a

low temperature and short photoperiod (12 °C–10 L). In light of published evidence, we propose the following possible hypotheses. First, environmental factors may influence reproduction and the endocrine system via the hypothalamus–pituitary–gonad axis and thus, the related reproductive hormone levels may be affected in fish. In some fish species, photoperiod variation has been shown to generally stimulate gonad maturation, but temperature seems to specifically modify the level of circulating gonadotrophin (Breton and Billard, 1977). Rodríguez et al. (2004) reported that a significant effect of artificial photoperiods was observed in the Luteinizing hormone (LH) plasma profile in male European sea bass (Dicentrarchus labrax, L.), reflecting a significant delay in the peak of LH compared to the control group. Furthermore, Shimizu (2003) found that in early spring the latter phases of gonadal development (vitellogenesis in females and active spermatogenesis in males) of mummichog (Fundulus heteroclitus) were effectively accelerated by warm temperature (16 °C). Those authors found that plasma concentrations of 17β-estradiol, in females, and testosterone, in males, correlated well with gonadal development during early spring and regression during late summer. An alternative hypothesis is that temperature and photoperiod are likely to influence the uptake, elimination and detoxification of EDCs, directly (Heugens et al., 2003). For example, temperature could influence enzyme-related activities in the organism. According to the Van't Hoff rule, in a certain temperature degree range, every 10 °C increase in water temperature can result in a 2- to 3-fold increase in biochemical or enzymatic activity (Reid et al., 1997; Caissie, 2006). Therefore, we hypothesize that temperature and photoperiod influence EDC-mediated endocrine disruption effects via different pathways. EDCs in aquatic ecosystem always exist as mixtures. Thus, it is suggested that the estrogenic effects of environmental chemicals will be affected by the presence of other chemicals, even those with different properties. In the present study, co-exposure was essential for assessing the effect of temperature and photoperiod on endocrine disruption in fish. To our knowledge, the present study is the first time to consider temperature and photoperiod under multiple experimental regimes, and then to analyze the endocrine disruption effects caused by binary mixtures of EE2 and NP in zebrafish. Interestingly, we found that temperature and photoperiod strongly influence estrogenresponsive gene transcription at both low and high relevant concentrations of EE2 + NP mixtures after 21 days of exposure. On the contrary, Brian et al. (2008) found no evidence of temperature dependence of the estrogen-responsive Vtg mRNA and protein expression in the livers of fathead minnows (Pimephales promelas) when exposed to the complex mixtures of EE2, E2, NP, OP and BPA. Possible explanations for the absence of a temperature-dependent effect include the following: the chemicals used for induction, the fish species, the route of chemical exposure, and the chemical composites of the mixtures. However, the induction effect (gene induction times) of all the four estrogen-responsive genes in the EE2 + NP group was approximately between those for EE2 and NP treatments, especially at the high exposure concentrations. These findings are consistent with the known mechanism of NP, which may compete with EE2 for binding to the ERs; however, NP has a much lower and a weaker estrogenic activity compared to EE2 (Yadetie et al., 1999; Zha et al., 2008). As a result, the combined estrogenic effect of EE2 and NP may have been suppressed. Similar results were also observed by Jukosky et al. (2008), who found that mixtures of NP, EE2 and E2 elicited lower vitellogenic induction than equipotent concentrations of E2 alone after a 14-day exposure in medaka. Of note, it is possible that the endocrine disruption effects of combined mixtures are promoted by different real world exposure conditions (Landman et al., 2006; Sun et al., 2009). Thus, we concluded that the assessment of endocrine disruption effects would be more practical by analyzing fish exposed to relevant concentrations of chemical mixtures, as well as those exposed to single chemical alone. Future studies will be necessary to gain a deeper understanding of the mechanism of the endocrine disruption effects.


5. Conclusions The goal of the present study was to assess the effect of changes in temperature and photoperiod corresponding to yearly seasonal changes on hepatic transcription of estrogen-responsive genes, including Vtg1, Vtg2, ERα and ERβ, in adult male zebrafish using real-time quantitative PCR. The results presented here indicate that temperature and photoperiod significantly affect mRNA expression of these genes induced by environmentally relevant concentrations of the most common EDCs or their mixture in an aquatic ecosystem. These data suggest that temperature and photoperiod mediate the endocrine disruption effects in zebrafish. Therefore, these data indicate the possibility that the endocrine systems of fish can be influenced by temperature and photoperiod change. As a result, season and/or climate change must be considered as factors defining the endocrine disruption effects induced by environmentally relevant concentrations of EDCs. Acknowledgments This work was supported by a grant from National Basic Research Program of China (No. 2010CB126100), the National Natural Science Foundation of China (No. 20837002) and the Program for Changjiang Scholars and Innovative Research Team in University (No. IRT 0653). References Barucca, M., Canapa, A., Olmo, E., Regoli, F., 2006. Analysis of vitellogenin gene induction as a valuable biomarker of estrogenic exposure in various Mediterranean fish species. Environ. Res. 101, 68–73. Bon, E., Corraze, G., Kaushik, S., Le Menn, F., 1997. Effects of accelerated photoperiod regimes on the reproductive cycle of the female rainbow trout: I—seasonal variations of plasma lipids correlated with vitellogenesis. Comp. Biochem. Physiol. 118, 183–190. Breton, B., Billard, R., 1977. Effects of photoperiod and temperature on plasma gonadotropin and spermatogenesis in the rainbow trout Salmo gairdnerii Richardson. Ann. Biol. Anim. Biochim. Biophys. 17, 331–340. Brian, J.V., Harris, C.A., Runnalls, T.J., Fantinati, A., Pojana, G., Marcomini, A., Booy, P., Lamoree, M., Kortenkamp, A., Sumpter, J.P., 2008. Evidence of temperature-dependent effects on the estrogenic response of fish: implications with regard to climate change. Sci. Total Environ. 397, 72–81. Brian, J.V., Harris, C.A., Scholze, M., Kortenkamp, A., Booy, P., Lamoree, M., Pojana, G., Jonkers, N., Marcomini, A., Sumpter, J.P., 2007. Evidence of estrogenic mixture effects on the reproductive performance of fish. Environ. Sci. Technol. 41, 337–344. Brown, M., Robinson, C., Davies, I.M., Moffat, C.F., Redshaw, J., Craft, J.A., 2004. Temporal changes in gene expression in the liver of male plaice (Pleuronectes platessa) in response to exposure to ethynyl oestradiol analysed by macroarray and real-time PCR. Mutat. Res. 552, 35–49. Brown, N.P., Shields, R.J., Bromage, N.R., 2006. The influence of water temperature on spawning patterns and egg quality in the Atlantic halibut (Hippoglossus hippoglossus L.). Aquaculture 261, 993–1002. Caissie, D., 2006. The thermal regime of rivers: a review. Freshw. Biol. 51, 1389–1406. Cui, C.W., Ji, S.L., Ren, H.Y., 2006. Determination of steroid estrogens in wastewater treatment plant of a controceptives producing factory. Environ. Monit. Assess. 121, 409–419. Desbrow, C., Routledge, E.J., Brighty, G.C., Sumpter, J.P., Waldock, M., 1998. Identification of estrogenic chemicals in STW effluent. 1. Chemical fractionation and in vitro biological screening. Environ. Sci. Technol. 32, 1549–1558. Filby, A.L., Santos, E.M., Thorpe, K.L., Maack, G., Tyler, C.R., 2007. Gene expression profiling for understanding chemical causation of biological effects for complex mixtures: a case study on estrogens. Environ. Sci. Technol. 41, 8187–8194. Heugens, E.H., Jager, T., Creyghton, R., Kraak, M.H., Hendriks, A.J., Van Straalen, N.M., Admiraal, W., 2003. Temperature-dependent effects of cadmium on Daphnia magna: accumulation versus sensitivity. Environ. Sci. Technol. 37, 2145–2151. Hill, R.L., Janz, D.M., 2003. Developmental estrogenic exposure in zebrafish (Danio rerio): I. Effects on sex ratio and breeding success. Aquat. Toxicol. 63, 417–429. Howell, R.A., Berlinsky, David L., Bradley, T.M., 2003. The effects of photoperiod manipulation on the reproduction of black sea bass, Centropristis striata. Aquaculture 218, 651–669. Jin, S.W., Yang, F.X., Liao, T., Hui, Y., Xu, Y., 2008a. Seasonal variations of estrogenic compounds and their estrogenicities in influent and effluent from a municipal sewage treatment plant in china. Environ. Toxicol. Chem. 27, 146–153. Jin, Y.X., Wang, W.Y., Sheng, G.D., Liu, W.P., Fu, Z.W., 2008b. Hepatic and extrahepatic expression of estrogen-responsive genes in male adult zebrafish (Danio rerio) as biomarkers of short-term exposure to 17β-estradiol. Environ. Monit. Assess. 146, 105–111. Jin, Y.X., Chen, R.J., Sun, L.W., Liu, W.P., Fu, Z.W., 2009a. Photoperiod and temperature influence endocrine disruptive chemical-mediated effects in male adult zebrafish. Aquat. Toxicol. 92, 38–43.

263

Jin, Y.X., Chen, R.J., Sun, L.W., Qian, H.F., Liu, W.P., Fu, Z.W., 2009b. Induction of estrogenresponsive gene transcription in the embryo, larval, juvenile and adult life stages of zebrafish as biomarkers of short-term exposure to endocrine disrupting chemicals. Comp. Biochem. Physiol. C 150, 414–420. Jukosky, J.A., Watzin, M.C., Leiter, J.C., 2008. The effects of environmentally relevant mixtures of estrogens on Japanese medaka (Oryzias latipes) reproduction. Aquat. Toxicol. 86, 323–331. Jung, J.H., Shim, W.J., Addison, R.F., Baek, J.M., Han, C.H., 2006. Protein and gene expression of VTG in response to 4-nonylphenol in rockfish (Sebastes schlegeli). Comp. Biochem. Physiol. C 143, 162–170. Koger, C.S., Teh, S.J., Hinton, D.E., 1999. Variations of light and temperature regimes and resulting effects on reproductive parameters in medaka (Oryzias latipes). Biol. Reprod. 61, 1287–1293. Körner, O., Kohno, S., Schönenberger, R., Suter, M.J.F., Knauer, K., Guillette Jr, L.J., BurkhardtHolm, P., 2008. Water temperature and concomitant waterborne ethinylestradiol exposure affects the vitellogenin expression in juvenile brown trout (Salmo trutta). Aquat. Toxicol. 90, 188–196. Korsgaard, B., Mommsen, T.P., Saunders, R.L., 1986. The effect of temperature on the vitellogenic response in Atlantic salmon post-smolts (Salmo salar). Gen. Comp. Endocrinol. 62, 193–201. Kortenkamp, A., 2007. Ten years of mixing cocktails: a review of combination effects of endocrine-disrupting chemicals. Environ. Health Perspect. 115, 98–105. Landman, M.J., van den Heuvel, M.R., Finley, M., Bannon, H.J., Ling, N., 2006. Combined effects of pulp and paper effluent, dehydroabietic acid, and hypoxia on swimming performance, metabolism, and hematology of rainbow trout. Ecotoxicol. Environ. Saf. 65, 314–322. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using realtime quantitative PCR and the 2−ΔΔCt method. Methods 25, 402–408. Mackay, M.E., Lazier, C.B., 1993. Estrogen responsiveness of vitellogenin gene expression in rainbow trout (Oncorhynchus mykiss) kept at different temperatures. Gen. Comp. Endocrinol. 89, 255–266. Migaud, H., Fontaine, P., Kestemont, P., Wang, N., Brun-Bellut, J., 2004. Influence of photoperiod on the onset of gonadogenesis in Eurasian perch Perca fluviatilis. Aquaculture 241, 561–574. Migaud, H., Fontaine, P., Sulistyo, I., Kestemont, P., Gardeur, J.N., 2002. Induction of out-ofseason spawning in Eurasian perch Perca fluviatilis: effects of rates of cooling and cooling durations on female gametogenesis and spawning. Aquaculture 205, 253–267. Reid, S.D., Dockray, J.J., Linton, T.K., McDonald, D.G., Wood, C.M., 1997. Effects of chronic environmental acidification and a summer global warming scenario: protein synthesis in juvenile rainbow trout (Oncorhynchus mykiss). Can. J. Fish. Aquat. Sci. 54, 2014–2024. Rodríguez, L., Carrillo, M., Sorbera, L.A., Zohar, Y., Zanuy, S., 2004. Effects of photoperiod on pituitary levels of three forms of GnRH and reproductive hormones in the male European sea bass (Dicentrarchus labrax, L.) during testicular differentiation and first testicular recrudescence. Gen. Comp. Endocrinol. 136, 37–48. Seki, M., Yokota, H., Maeda, M., Tadokoro, H., Kobayashi, K., 2003. Effects of 4-nonylphenol and 4-tert-octylphenol on sex differentiation and vitellogenin induction in medaka (Oryzias latipes). Environ. Toxicol. Chem. 22, 1507–1516. Shen, J.H., Gutendorf, B., Vahl, H.H., Shen, L., Westendorf, J., 2001. Toxicological profile of pollutants in surface water from an area in Taihu Lake, Yangtze Delta. Toxicology 166, 71–78. Shimizu, A., 2003. Effect of photoperiod and temperature on gonadal activity and plasma steroid levels in a reared strain of the mummichog (Fundulus heteroclitus) during different phases of its annual reproductive cycle. Gen. Comp. Endocrinol. 131, 310–324. Sun, L.W., Zha, J.M., Wang, Z.J., 2009. Effects of binary mixtures of estrogen and antiestrogens on Japanese medaka (Oryzias latipes). Aquat. Toxicol. 93, 83–89. Thorpe, K.L., Gross-Sorokin, M., Johnson, I., Brighty, G., Tyler, C.R., 2006. An assessment of the model of concentration addition for predicting the estrogenic activity of chemical mixtures in wastewater treatment works effluents. Environ. Health Perspect. 114, 90–97. Wang, H., Tan, J.T., Emelyanov, A., Korzh, V., Gong, Z.Y., 2005. Hepatic and extrahepatic expression of vitellogenin genes in the zebrafish, Danio rerio. Gene 356, 91–100. Xu, J., Wang, P., Guo, W.F., Dong, J.X., Wang, L., Dai, S.G., 2006. Seasonal and spatial distribution of nonylphenol in Lanzhou Reach of Yellow River in China. Chemosphere 65, 1445–1451. Yadetie, F., Arukwe, A., Goksøyr, A., Male, R., 1999. Induction of hepatic estrogen receptor in juvenile Atlantic salmon in vivo by the environmental estrogen, 4nonylphenol. Sci. Total Environ. 233, 201–210. Yamaguchi, A., Ishibashi, H., Kohra, S., Arizono, K., Tominaga, N., 2005. Short-term effects of endocrine-disrupting chemicals on the expression of estrogen-responsive genes in male medaka (Oryzias latipes). Aquat. Toxicol. 72, 239–249. Yang, F.X., Xu, Y., Hui, Y., 2006. Reproductive effects of prenatal exposure to nonylphenol on zebrafish (Danio rerio). Comp. Biochem. Physiol. C 142, 77–84. Zha, J.M., Sun, L.W., Spear, P.A., Wang, Z.J., 2008. Comparison of ethinylestradiol and nonylphenol effects on reproduction of Chinese rare minnows (Gobiocypris rarus). Ecotoxicol. Environ. Saf. 71, 390–399. Zhang, X., Li, Q.Z., Li, G.X., Wang, Z.S., Yan, C.Z., 2009. Levels of estrogenic compounds in Xiamen Bay sediment, China. Mar. Pollut. Bull. 58, 1210–1216. Zhang, X., Zha, J., Wang, Z., 2008. Influences of 4-nonylphenol on doublesex- and mab3-related transcription factor 1 gene expression and vitellogenin mRNA induction of adult rare minnow (Gobiocypris rarus). Environ. Toxicol. Chem. 27, 196–205.