Semicarbazide, a member of the hydrazine family, has been demonstrated to be a potent carcinogen (Toth et al. 1975; Davies et al. 2000). A 1.0 μg/kg maximum residue limit (MRL) of semicarbazide in food has been stipulated by the European Union (EU) (Commission 2003). The European Food Safety Authority (EFSA) detected high concentrations of semicarbazide (up to 25 µg/kg) in certain foods contained in glass jars and bottles in 2003 (EFSA 2003a, b). The semicarbazide contamination in these foods was believed to be a decomposition product of azodicarbonamide contained in the sealing gaskets of metal lids (EFSA 2003a; Maranghi et al. 2009). Another source of semicarbazide is from the decomposition of nitrofuran drugs that are frequently used in aquaculture and livestock farming, resulting in the accumulation of semicarbazide in the environment and animal tissues (Hoenicke et al. 2004; Pereira et al. 2004; Cooper et al. 2007). The presence of semicarbazide in food has thus aroused interest in food safety and a few experiments on the toxicity of semicarbazide in mammals have been conducted. Not only does semicarbazide cause histological and morphological alterations in target organs (uterus, ovary, testis, and thymus), but semicarbazide also acts as a potential endocrine disruptor both in vivo and in vitro (Maranghi et al. 2009, 2010). Oral administration of semicarbazide for 28 days at 40, 75 and 140 mg/kg bw resulted in decreased serum concentrations of 17β-estradiol (E2) in female Sprague–Dawley rats, indicating that semicarbazide has an anti-estrogenic effect (Maranghi et al. 2010). Some in vitro estradiol-competitive assays, including Recombinant Yeast Estrogen Screen (YES), MCF-7 proliferation assay (E-screen), and stimulation assay of the alkaline phosphatase (ALP) activity in Ishikawa cells, have also demonstrated that semicarbazide has weak anti-estrogenic activity by dose-dependently reducing the response to E2 (Maranghi et al. 2010).

In addition to the contamination of food, semicarbazide was reported to be detected in the aquatic environment for the first time in 2010 (Xu et al. 2010). The concentrations of semicarbazide in coastal waters adjacent to the Chaohe River estuary (China) were 46.41 ± 21.22 µg/L in seawater, 10.44 ± 7.36 µg/kg in sediments, and 6.46 ± 0.03 µg/kg in aquatic organisms, which is higher than the EU MRL (Xu et al. 2010). As a potential endocrine disrupting chemical (EDC), semicarbazide may cause adverse health effects in aquatic organisms altering the homeostasis of the endocrine system (Kloas et al. 2009). However, no data are available on the endocrine disrupting effect of semicarbazide in aquatic organisms. For aquatic animals, including fish, the exposure route to environmental contaminants is mainly via uptake directly from the water across the skin and/or gill surfaces, which may result in a very different biological potency for EDCs as compared with mammals which are exposed primarily via the diet (Sohoni et al. 2001). Therefore, the zebrafish (Danio rerio), a predominant model for testing EDCs (Segner 2009), was used in this study to investigate the endocrine disrupting effect of semicarbazide on fish through a water exposure experiment.

In zebrafish, vitellogenin genes (vtg) in liver, especially vtg-1 which has the highest level of expression among the 7 vtg genes, are sensitively induced by E2 or other estrogens via mediation of estrogen receptors (ERs), while anti-estrogen can antagonize the induction effect, which making vtg-1 an excellent biomarker for the detection of estrogenic or anti-estrogenic activities of chemicals (Wahli 1988; Lazier and Mackay 1993; Philips et al. 1993; Rose et al. 2002; Katsu et al. 2004; Wang et al. 2005). This study was undertaken to determine the endocrine disrupting effects (estrogenic or anti-estrogenic) and the probable mechanisms of action of semicarbazide in zebrafish. To this end, the changes in transcript abundance of hepatic estrogen-response genes, including vtg-1, ERα, and ERβ, were measured in male and female zebrafish exposed to either semicarbazide alone or co-exposed to semicarbazide and E2 for a period 96 h or 28 days.

Materials and Methods

Animals and Acclimation Conditions

Healthy adult zebrafish (Danio rerio) (3.76 ± 0.12 cm standard length; 0.58 ± 0.04 g wet weight) were obtained from a local dealer in Qingdao, PR China. Fish were acclimated in daily renewed dechlorinated tap water (dissolved oxygen 7.0 ± 0.1 mg/L; pH 7.2–7.6) at a constant temperature (26 ± 1 °C) under a 14 h (light):10 h (dark) cycle for 2 weeks. Zebrafish were fed with brine shrimp (Artemia nauplii) twice daily and no food was provided 24 h before experiments.

Acute Toxicity of Semicarbazide to Zebrafish

According to the method of Fish Acute Toxicity Test (USEPA 1996), the final concentrations of semicarbazide (CAS NO:563-41-7, purity ≥98 %, Sigma–Aldrich, Shanghai, China) for the acute toxicity test were set at 0.0, 23.0, 25.3, 27.8, 30.6 and 33.7 mg/L based on the pretest.

Ten fish were placed in each glass tank containing 5 L of daily renewed semicarbazide or control solution for 96 h under the same conditions described in Sect. 2.1 and no food was provided during the experiment. Dead fish were removed every 24 h and the number was recorded. The experiment was repeated twice. Acute toxicity was expressed as the median lethal concentration (LC50) of semicarbazide within a continuous period of exposure of 96 h. The 96 h-LC50 and its 95 % confidence limits were determined by probit analysis (SPSS Probit Analysis Program, version10.0).

Fish Exposure and Sample Protocol

The concentrations of semicarbazide were set at 1.0, 10.0, 100.0 and 1,000.0 µg/L based on the 96 h-LC50 value (26.29 mg/L) and the environmental concentration of semicarbazide (46.41 ± 21.22 µg/L). Sexes were kept separate and ten male or female fish (n = 10) were assigned to each tank containing 5L of water with semicarbazide at different concentrations. Another group of male fish was co-exposed to semicarbazide with 500 ng/L E2 (diluted with 0.001 % ethyl alcohol). Dechlorinated tap water or 0.001 % ethyl alcohol served as the blank control, and E2 alone (500 ng/L) served as the positive control. Duplicate tanks were used for each treatment group for the 96 h short-term and 28 day long-term exposure. Fish exposure was conducted in a semi-static toxicity test (5 L of water renewed daily to keep the treatment concentrations constant and water fresh) and other conditions were the same as those described in Sect. 2.1. No deaths were observed in any of the treatment groups during experimentation.

At the end of the exposure, zebrafish were anesthetized in 75 mg/L MS-222 (Sigma, St. Louis, MO, USA). The weights of whole body and ovary of each female fish were measured to calculate the gonadosomatic index (GSI = (gonad wt/body wt) × 100), and the livers of both sexes were dissected, frozen in liquid nitrogen, and stored separately at −80°C, prepared for the real-time PCR quantification of vtg-1, ERα and ERβ mRNA.

Measured Concentration of Semicarbazide in Exposure Solutions

Since the whole exposure solutions were renewed daily, water samples from each treatment group were taken directly from the test beakers before (0 h) and after 24 h of exposure (24 h). The semicarbazide was analyzed using the ultra performance liquid chromatography-tandem quadrupole mass spectrometry (UPLC–MS/MS) on a Shimadzu Nexera UPLC system interfaced to a Shimadzu LCMS-8030 quadrupole mass spectrometer (Kyoto, Japan). UPLC separations were carried out using an ACQUITYTM BEH C18 column (1.7 μm, 2.1 mmi.d. × 100 mm). The autosampler was maintained at 40°C, and the injection volume was 10 μL. The mobile phase was composed of 0.1 % formic acid in water/5 mmol/L ammonium acetate solution at a constant flow of 0.25 mL/min. Detailed information about the program of the gradient elution is shown in Table 1. Mass spectrometric detection was operated using an electrospray ionization source (ESI) in positive mode, with ESI parameters as follows: capillary voltage, 2.8 kV; desolvation temperature, 110°C; atomizing gas flow, 20 L/min; drying gas temperature, 350°C; and drying gas flow, 700 L/min. Quantitative analysis was carried out using the selected reaction monitoring mass transitions of m/z 209.27 as parention quantifiers, and m/z 166.02 were monitored as daughterion qualifiers. And the collision energy was 12 V. Semicarbazide was undetected in the control tanks, while the analyzed concentration (mean ± standard deviation) of semicarbazide in the test solutions were 5.38 ± 0.54, 8.55 ± 0.79, 92.1 ± 3.27 and 750.8 ± 16.3 μg/L before 0 h and 3.86 ± 0.41, 5.23 ± 0.09, 51.8 ± 2.9, 423.6 ± 8.2 μg/L after 24 h. The concentrations mentioned in the following referred to the nominal semicarbazide values.

Table 1 Program of gradient elution
Full size table

Quantitative Real-Time PCR

Total RNA was isolated from livers using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. The quality of the extracted RNA was verified by agarose gel electrophoresis, and the concentration was determined by spectrometry measured at OD260/280. Equal amounts of RNA (1 µg) were reverse-transcribed into cDNA in 20 µL reactions using a reverse transcriptase kit (Toyobo, Osaka, Japan) following the manufacturer’s instructions. The synthesized cDNA was diluted in 100 µL dH2O and stored a  20°C.

Primers were designed for the amplification of three target genes (vtg-1, ERα, and ERβ) and two housekeeping genes (elf-α and β-actin) according to the sequences published in GenBank (Table 2). Parallel PCR reactions were conducted to amplify the target gene cDNA and housekeeping gene cDNA in an Eppendorf Mastercycler®ep realplex2 real-time quantitative PCR System (Eppendorf, Germany). The SYBR green mix kit (TaKaRa, Dalian, China) was used for the reaction in a 20 μL volume system, which consisted of 10 μL SYBR® Premix Ex Taq™ II, 0.4 μL ROX Reference Dye, 0.8 μL of each forward and reverse primer (10 μM), 4 μL first-strand cDNA (template), and 4 μL dH2O. The thermal profile was 95°C for 30 s followed by 40 cycles of 95°C for 5 s and 61°C for 30 s. Melting curve analysis was applied to all reactions to ensure homogeneity of the reaction products. Only one peak was observed for each amplification in the melting curve, and a single amplicon of the predicted size was observed following agarose gel electrophoresis of the PCR products (data not shown), indicating specific amplification of the target gene. The target gene mRNA expression in each sample, relative to the geometric average of elf-α and β-actin mRNA expression, was calculated by the formula 2−ΔΔCt and plotted on a logarithmic scale. Geometric average levels of elf-α and β-actin were not changed under any of the experimental conditions in the study.

Table 2 Nucleotide sequences of primers used for real-time PCR and product sizes
Full size table

Statistics

All data are presented as the mean ± standard deviation. Multiple comparisons were performed using one-way analysis of variance followed by Tukey’s test. Values were considered significant when 0.01 < p < 0.05 and highly significant when p < 0.01.

Results

Acute Toxicity of Semicarbazide to Zebrafish

Compared to the controls which showed no behavioral abnormalities, some of the treated fish displayed erratic swimming or loss of equilibrium prior to death. Hyperaemia in the pharynx was observed in most of the dead fish. The 96 h-LC50 of semicarbazide in the zebrafish is calculated to be 26.29 mg/L, with 95 % confidence limits being 24.42–27.78 mg/L (Fig. 1). According to the US Environmental Protection Agency (EPA) guidance (USEPA 2004), semicarbazide has a moderate aquatic toxicity (96-h LC50 > 1.0 mg/L and <100 mg/L).

Fig. 1
figure 1

Curve showing mortality rates of 96-h zebrafish treated with semicarbazide. Horizontal solid line indicates 50 % lethal concentration. Data are presented as the mean ± standard deviation

Full size image

Estrogenic Effect Test: Effects of Semicarbazide Exposure on vtg-1, ERα, and ERβ mRNA Expression in the Male Liver

After a 96 h exposure to semicarbazide at 1.0, 10.0, 100.0 and 1,000.0 µg/L, the expression of vtg-1, ERα, and ERβ mRNA in the liver of treated males showed no significant differences compared to the control (Fig. 2a, b, c). Following exposure to semicarbazide for 28 d, ERα mRNA expression was highly significantly down-regulated (p < 0.01) (Fig. 2b), however, no significant differences in the expression of vtg-1 or ERβ mRNA were observed (Fig. 2a, c).

Fig. 2
figure 2

Relative mRNA expression levels of vtg-1 (a), ERα (b), ERβ (c) in the liver of male fish exposed to 0, 1.0, 10.0, 100.0 and 1,000.0 µg/L semicarbazide (designated CTRL, SEM1.0, SEM10.0, SEM100.0, SEM 1,000.0, respectively) for 96 h and 28 d. Fold change (Y axis) represents the expression of vtg-1/ERα/ERβ mRNA relative to that of the control group (equals 1 by definition). Data are presented as the mean ± standard deviation (n = 10). Asterisks indicate statistically significant difference from the control group (**p < 0.01)

Full size image

Anti-Estrogenic Effect Test

Effects of Semicarbazide Exposure on vtg-1, ERα, and ERβ mRNA Expression in the Female Liver

Exposure to semicarbazide for 96 h inhibited the expression of vtg-1 mRNA in a dose-dependent manner in female zebrafish liver (Fig. 3a). With the exception of the 1.0 µg/L semicarbazide group, vtg-1 mRNA level decreased highly significantly in semicarbazide-exposed fish (p < 0.01). After a 28 d exposure, the vtg-1 mRNA expression in all semicarbazide treatment groups was decreased highly significantly to a similar level (p < 0.01; Fig. 3a). A similar pattern was observed in the expression of ERα mRNA at both 96 h and 28 d (Fig. 3b). The correlation between mRNA levels of vtg-1and ERα was highly significant (p < 0.001 in all bivariate correlations; the correlation coefficients for 96 h and 28 d were 0.988 and 0.963, respectively). The transcript level of ERβ mRNA showed a slight difference. As is shown in Fig. 3c, ERβ mRNA expression in all treatment groups was highly significantly lower than the control group after 96 h and 28 d exposure (p < 0.01). A dose-dependent response in ERβ mRNA level following semicarbazide exposure for 96 h was not observed.

Fig. 3
figure 3

Relative mRNA expression levels of vtg-1 (a), ERα (b), ERβ (c) in the liver of female fish exposed to 0, 1.0, 10.0, 100.0 and 1,000.0 µg/L semicarbazide (designated CTRL, SEM1.0, SEM10.0, SEM100.0, SEM 1,000.0, respectively)for 96 h and 28 d. Fold change (Y axis) represents the expression of vtg-1/ERα/ERβ mRNA relative to that of the control group (equals 1 by definition). Data are presented as the mean ± standard deviation (n = 10). Asterisks indicate statistically significant difference from the control group (**p < 0.01)

Full size image

Effects of Semicarbazide Exposure on Female GSI

Compared to the control, semicarbazide exposure had no marked effect on the GSI of female zebrafish after a 96 h exposure. However, a highly significant decrease in GSI (P < 0.01) was observed in all treatment groups after a 28 d exposure (Fig. 4).

Fig. 4
figure 4

GSI levels of female fish exposed to 0, 1.0, 10.0, 100.0 and 1,000.0 µg/L semicarbazide (designated CTRL, SEM1.0, SEM10.0, SEM100.0, SEM 1,000.0, respectively) for 96 h and 28 d. Fold change (Y axis) represents the GSI level relative to that of the control group (equals 1 by definition). Data are presented as the mean ± standard deviation (n = 10). Asterisks indicate statistically significant difference from the control group (**p < 0.01)

Full size image

Effects of Semicarbazide and E2 Co-exposure on vtg-1, ERα, ERβ mRNA Expression in the Male Liver

As is shown in Fig. 5a, b and c, treatment of male zebrafish with 500 ng/L E2 for 96 h resulted in significant increases in vtg-1, ERα, and ERβ mRNA expression in the liver when compared to the blank control (p < 0.01). In comparison with the level of expression induced by E2 alone, the addition of 1,000.0 µg/L semicarbazide resulted in a highly significant decrease in mRNA abundance for all of these genes at 96 h (p < 0.01). However, the expression of vtg-1 and ERα mRNA after 96 h demonstrated a slight rising trend in the 1.0 µg/L, 10.0 µg/L, and 100.0 µg/L semicarbazide groups, while ERβ mRNA level remained lower. After 28 days co-exposure to semicarbazide and E2, the transcript levels for vtg-1, ERα, and ERβ in all treatment groups decreased highly significantly compared to the E2 positive control (p < 0.01).

Fig. 5
figure 5

Relative mRNA expression levels of vtg-1 (a), ERα (b), ERβ (c) in the liver of male fish co-exposed to 500.0 ng/L E2 and 0, 1.0, 10.0, 100.0 and 1,000.0 µg/L semicarbazide (designated CTRL, E2, 1 µg/LSEM + E2, 10 µg/LSEM + E2, 100 µg/LSEM + E2, 1,000 µg/LSEM + E2, respectively) for 96 h and 28 d. Fold change (Y axis) represents the expression of vtg-1/ERα/ERβ mRNA relative to that of the control group (equals 1 by definition). Data are presented as the mean ± standard deviation (n = 10). Pounds (#) indicate statistically significant difference from the control group (## p < 0.01). Asterisks indicate statistically significant difference from the positive control (E2) group (*0.01 < p < 0.05, **p < 0.01)

Full size image

Overall, the expression of vtg-1 and ERα genes showed a similar tendency. According to the correlation analysis, the expression of vtg-1 and ERα mRNA were highly correlated (p < 0.001 in all bivariate correlations; the correlation coefficients for 96 h and 28 days were 0.961 and 0.999, respectively).

Discussion

In the last two decades, an increasing number of EDCs have been identified and studied. Most of this research has focused on chemicals with estrogenic activities, while a limited number of studies have been conducted on EDCs with anti-estrogenic activities (Sun et al. 2010; Hoffmann and Kloas 2012). Interestingly, it has been found that some of the EDCs (e.g. genistein) can exert both estrogenic and anti-estrogenic activity in fish (Richard et al. 2002). The potential anti-estrogenic effect of semicarbazide has been demonstrated in mammals (Maranghi et al. 2010), while no data is available to determine whether semicarbazide will have a similar endocrine-disrupting effect in aquatic organisms. In this study, we assessed both the estrogenic and anti-estrogenic effects of semicarbazide in zebrafish by investigating changes in the hepatic transcription of estrogen-responsive genes.

Vitellogenin is normally produced in mature female fish liver. However, the vtg gene also exists in male fish and can be induced by chemicals with estrogenic activity (Ng and Idler 1983; Wallace 1985; Lim et al. 1991; Lazier and Mackay 1993). Based on the results of our study, semicarbazide (1.0–1,000.0 µg/L) does not have an estrogenic effect, as induction of vtg-1 mRNA in male zebrafish liver was not evident after exposure for 96 h or 28 days.

Since anti-estrogens can inhibit vtg expression induced by E2, a decrease in vtg mRNA level can also serve as a biomarker in the screening of anti-estrogens (Philips et al. 1993). Tests for the anti-estrogenic effects of EDCs in fish can be applied to both females and males (by co-exposure with E2). Sun et al. (2010) reported that exposure of female zebrafish to letrozole (a model anti-estrogen) at 100 and 300 µg/L for 72 h significantly decreased the vtg-1 and vtg-2 expression in liver. Another anti-estrogen benzo [a] pyrene, when co-exposed with 160 ng/L E2 to male goldfish at 20 and 50 µg/L,inhibited the transcription of vtg and ERα expression enhancement induced by exogenous E2 (Yan et al. 2012). In our study, semicarbazide (1.0–1,000.0 µg/L) inhibited the transcription of vtg-1 mRNA in female liver indicating that semicarbazide antagonized the response to endogenous E2, demonstrating a remarkable anti-estrogenic effect. Moreover, the co-exposure assay in male fish also demonstrated that semicarbazide has a marked inhibitory effect on vtg-1 mRNA transcription induced by E2 after a 28 d exposure. Therefore, it is concluded that semicarbazide has a significant anti-estrogenic effect in zebrafish, similar to the effects observed in vivo and in vitro in mammals (Maranghi et al. 2010).

Current research suggests that anti-estrogens exert their action via binding to ERs. Certain anti-estrogens, such as tamoxifen (TAM), compete with E2 for binding to the ER protein, forming a stable compound and inhibiting ERE-mediated activity (Jordan et al. 1977; Philips et al. 1993). Other anti-estrogens, such as fulvestrant (ICI 182,780), reduce ER levels by binding to the ERα protein and accelerating the degradation of the receptor (Osborne et al. 1996; Long and Nephew 2006). However, studies in vitro have demonstrated that semicarbazide cannot bind to ERs (Maranghi et al. 2010), so it can be deduced that semicarbazide may act via other pathways. Based on the results of our study, it can be suggested that semicarbazide can inhibit the ERα and ERβ expression in zebrafish liver. The transcript levels of ERα and ERβ, in females exposed to semicarbazide for both 96 h and 28 days, and in males co-exposed to E2 and semicarbazide for 28 days, were significantly inhibited by semicarbazide. It is plausible that such a reduction in ER expression in the liver would reduce E2-ER complex formation and lead to decreases in vtg-1 mRNA transcription. Moreover, in males exposed to semicarbazide alone, though the vtg-1 expression showed no change due to the low endogenous levels of E2, semicarbazide also led to the reduction of ERα mRNA expression after 28 days of exposure. Therefore, we infer that a probable pathway for the anti-estrogenic effect of semicarbazide may be the inhibition of ER expression. Noticeably, the level of vtg-1mRNA is highly correlated to the ERα mRNA both in females and co-exposed males, but not to ERβ mRNA. This result was in accordance with the findings in other teleost species including Eastern mosquitofish, Gambusia holbrooki (Kristensen et al. 2007), tilapia, Oreochromis mossambicus (Davis et al. 2010) and goldfish, Carassius auratus (Yan et al. 2012). The current explanation for the high correlation between vtg-1 and ERα is that ERα plays the dominant role in regulating vtgs, while the potential roles of ERβ in relating to vtgs are less clear (Kristensen et al. 2007; Marlatt et al. 2010; Palstra et al. 2010). Our results support this explanation by showing that the inhibition of vtg-1 expression by semicarbazide may be mainly due to the inhibition of ERα expression.

It is widely recognized that for routine monitoring of the biological effects of contaminants, tests that are practical, short-term, and cost-effective are the most desirable (Sohoni et al. 2001). Based on the 96 h short-term outcomes of our study, a dose-dependent decrease in vtg-1 and ERα mRNA was detected in females, but not in males, which makes the short-term exposure to female zebrafish an appropriate model for the detection of anti-estrogens. In males, the antagonistic effect of semicarbazide on E2 was only observed in the highest concentration group (1,000.0 µg/L) after short-term exposure, and lower concentrations of semicarbazide (1.0–100.0 µg/L) even caused a slight increase in vtg-1 and ERα mRNA. This observations has also been seen in a previous report, in which male Japanese medaka (Oryzias latipes) exposed to 20 ng/L 17α-ethinylestradiol (EE2) for 72 h showed a significant increase in the transcription of hepatic vtg-1 and ERα mRNA, which was even stimulated by a co-treatment with an anti-estrogen TAM at lower concentrations (30, 100, or 300 µg/L) (Sun et al. 2011). This stimulation has been explained by a theory of compensatory feedback, that is when in the co-existence of exogenous estrogen and low concentration of anti-estrogen for a short term, the males would increase the response to estrogen to compensate the weak anti-estrogenic effects (Sun et al. 2011). In contrast to the 96 h results, the expression of vtg-1, ERα, and ERβ decreased significantly in females and in co-exposed males after 28-day long-term treatment. In addition, the significant reductions in GSI in female fish after 28 days of semicarbazide exposure suggest an accumulation of the anti-estrogenic effects of semicarbazide. Our data for semicarbazide suggests that 96 h short-term in vivo co-exposure to males is not adequate to identify an environmental anti-estrogen at low concentrations, while long-term effects will be more visible and reliable. Based on the results of this study, the current concentrations of semicarbazide in Chaohe River (46.41 ± 21.22 µg/L; Xu et al. 2010) may cause significant anti-estrogenic effects in aquatic organisms after long-term exposure, disturbing their endocrine system and antagonizing the function of E2 in females, eventually leading to ovarian atrophy or the loss of ovarian function.