Similar articles

Abstract: This paper aims to evaluate the individual and joint toxicities of cadmium sulfate (CdSO₄) and α-Naphthoflavone (ANF) in zebrafish embryos. As a result, CdSO₄ caused both lethal and sub-lethal effects, such as 24 h post-fertilization (hpf) death and 72 hpf delayed hatching. However, ANF only caused sub-lethal effects, including 48 hpf cardiac edema and 72 hpf delayed hatching. Taking 24 hpf death and 48 hpf cardiac edema as endpoints, the toxicities of CdSO₄ and ANF were significantly enhanced by each other. Consistently, both CdSO₄ and ANF caused significant oxidative stress, including decreases in the reduced glutathione (GSH) level, inhibition of superoxide dismutase (SOD) activity, as well as increases in malondialdehyde (MDA) content in zebrafish embryos, but these mixtures produced much more significant alterations on the biomarkers. Co-treatment of CdSO₄ and ANF significantly down-regulated the mRNA level of multidrug resistance-associated protein (mrp) 1 and cytochrome P450 (cyp) 1a, which constituted the protective mechanisms for zebrafish embryos to chemical toxins. In conclusion, co-treatment of CdSO₄ and ANF exhibited a much more severe damage in zebrafish embryos than individual treatment. Meanwhile, production of oxidative stress and altered expression of mrp1 and cyp1a could be important components of such joint toxicity.

硫酸镉与α-萘黄酮对斑马鱼发育的个体毒性与联合毒性研究

研究目的：研究硫酸镉与α-萘黄酮对斑马鱼发育的个体毒性与联合毒性，并探讨其中的联合毒性机制。
创新要点：首次对硫酸镉与α-萘黄酮的联合毒性进行了研究，并探索了氧化应激、ATP结合盒式（ABC）转运蛋白及细胞色素P450蛋白（CYP）1A在化合物联合毒性中的作用。
研究方法：在不同时间点，依据形态学指标检测硫酸镉与α-萘黄酮单独使用与联合使用时对斑马鱼胚胎的致死与致畸性毒性。取样品，采用试剂盒检测不同处理状态下斑马鱼胚胎中谷胱甘肽（GSH）、超氧化物歧化酶（SOD）及丙二醛（MDA）三个氧化还原指标的改变，聚合酶链式反应（PCR）检测样品中多药耐药蛋白（mrp）1及cyp1a基因表达水平的改变。
重要结论：硫酸镉与α-萘黄酮的联用能够产生远大于个体且显著增强各自的毒性，而这种增强作用机理在于氧化应激压力的产生，以及两者合用下mrp1及cyp1a基因表达水平的改变。
联合毒性；硫酸镉；α-萘黄酮；斑马鱼胚胎；氧化应激压力

1.  Introduction

 Aquatic organisms are usually faced with various types of chemical toxicants in the water, and thus have to suffer from the unpredictable interactions of these mixtures (Duan et al., 2008; Maria and Bebianno, 2011). Unfortunately, although there have been numerous investigations on the toxicity of a specific compound (Shi et al., 2008; Osterauer et al., 2011), information regarding the joint effects of pollutants remains insufficient.

 Cadmium is one of the most important heavy metal toxicants and its environmental concentration is increasing due to its extensive utilization in modern industries. Cadmium is reported to cause many deleterious effects including impaired neurogenesis, eye defects, and hatching failure in aquatic organisms (Chow et al., 2008; Matović et al., 2011). Various experiments have been done on zebrafish, Sparus aurata, and gilthead sea bream larvae, and the results revealed that the toxicity of cadmium was caused by the inhibition of cytochrome P450 (CYP) 1A expression as well as the production of oxidative stress (Sassi et al., 2013; Souid et al., 2013; Wang and Gallagher, 2013).

 Polycyclic aromatic hydrocarbon (PAH), on the other hand, consists of a large number of compounds with different structures, and is increasingly released by oil leakages, vehicle emissions, and agriculture/industry activities. In aquatic systems, PAHs like benzo[α]pyrene and retene pose significant threats, such as pericardial edema, craniofacial malformations, and altered development of visual systems in fishes (Fleming and di Giulio, 2011; Hawliczek et al., 2012; Huang et al., 2014). And such threats are reportedly related to the metabolism by CYP1A as well as enhanced levels of oxidative stress (Timme-Laragy et al., 2007).

 Adenosine triphosphate-binding cassette (ABC) transporters, such as multidrug resistance-associated protein (Mrp) 1–5 genes, have also been considered to be involved in the detoxification of cadmium and PAH. High expression and function of these transporters have been found in fishes, such as zebrafish (Long et al., 2011a; 2011b) and rainbow trout (Kennedy et al., 2014). Their gene expression could be induced by various xenobiotics like cadmium and PAH, pumping the parent compound and their metabolites out of the organisms in an energy-dependent process (Long et al., 2011c; Costa et al., 2012; Navarro et al., 2012). Thus, ABC transporters are usually considered to be the major biological defense mechanism for the protection of organisms against these environmental chemicals.

 Due to their wide applications, cadmium and PAH usually coexist in the environment (Zhang et al., 2004; Keenan et al., 2010), and interactions of these chemicals in fishes might happen due to the similar induction of oxidative stress and the involvement of CYP1A and ABC transporters. However, such interactions have never been studied in previous experiments. In this paper, zebrafish embryos, which represented an attractive model for studying the toxic mechanisms of environmental chemicals (Berry et al., 2007; Weil et al., 2009), were used to examine the individual and joint effects of cadmium sulfate (CdSO₄) and a model PAH, α-naphthoflavone (ANF) (Timme-Laragy et al., 2007). To assess the possible role of oxidative stress, ABC transporters and CYP1A in the joint toxicity, the alteration of reduced glutathione (GSH) level, superoxide dismutase (SOD) activity, malondialdehyde (MDA) content, and mrp1 and cyp1a expression caused by CdSO₄, ANF, and CdSO₄-ANF mixtures were respectively evaluated.

2.  Materials and methods

2.1.  Reagents

 Both CdSO₄ and ANF were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China), and their purity was above 99%. Kits for GSH level, SOD activity, and MDA content were obtained from Beyotime Institute of Biotechnology (Nantong, China). The other reagents were of reagent grade and purchased from local suppliers.

2.2.  Zebrafish

 Adult wide-type zebrafish were purchased from a local aquarium (Suzhou, China) and maintained as described before (Berry et al., 2007). Fish eggs were collected and washed three times with Holtfreter’s buffer (3.5 g/L NaCl, 0.05 g/L KCl, 0.1 g/L CaCL₂, and 0.025 g/L NaHCO₃; pH 7.5). Using an Axio Observer A1 microscope (Carl Zeiss, Inc., Oberkochen, Germany), the status of the collected eggs was visually determined, and any dead eggs were discarded. At 4 h post-fertilization (hpf), the good-quality eggs were collected and placed in a 24-well plate such that each well contained 10 embryos, and were used for developmental toxicity assays (Tilton and Tanguay, 2008).

2.3.  Developmental toxicity test

 CdSO₄ was dissolved in Holtfreter’s buffer directly before use. ANF was dissolved in acetone first and then diluted in Holtfreter’s buffer. In each treatment, the concentration of acetone was no more than 0.1%, which caused no significant alteration in the development of embryos.

 At 4 hpf, eggs in 24-well plates were washed with Holtfreter’s buffer and exposed to 2 ml Holtfreter’s buffer containing the vehicle, CdSO₄, ANF, or CdSO₄-ANF mixtures. Each group contained at least six replicates, and eggs treated by 0.1% acetone acted as vehicle controls. Moreover, transparent plastic film was used to cover the wells. Half of the exposure solutions of each well were removed and then were replaced daily with fresh exposure solutions. Meanwhile, the dead animals were removed. At 24, 48, and 72 hpf, the developmental status of the zebrafish embryos treated by vehicle control (Figs. 1a and 1c) and chemicals (Figs. 1b and 1d) was observed with the microscope and documented using a A2000IS camera (Conan Co., Ltd., Beijing, China). The selected endpoints included 24 hpf death, 48 hpf cardiac edema (Fig. 1b), and 72 hpf delayed hatching (Figs. 1d and 1e).




 To more specifically identify the effect of ANF on the toxicity of CdSO₄, we also conducted an experiment in which embryos were first treated by 75 mg/L CdSO₄ alone, and then 12 h later, the embryos were washed with Holtfreter’s buffer and exposed to the mixtures of 75 mg/L CdSO₄ and 1.5 mg/L ANF. Mortalities of zebrafish embryos after 20 h treatment (16–36 hpf) were subsequently recorded and compared with groups treated only by CdSO₄ (4–36 hpf) or the simultaneous treatment of CdSO₄ and ANF (4–24 hpf).

2.4.  GSH, MDA, and SOD detection

 At 24, 36, and 48 hpf, three sets of 60 embryos in both the vehicle control and chemical treatment groups were washed with Holtfreter’s buffer and collected into 1.5 ml centrifuge tubes. Each tube was filled with 300 μl phosphate buffer solution (PBS; pH 7.4) and immersed in liquid nitrogen for 20 s. After that, the embryos in PBS were mechanically homogenized (Wiegand et al., 1999). The supernatant was collected after the centrifugation of the embryo homogenate (10 000×g, 4 °C, 10 min) for a biomarkers assay.

 GSH level, SOD activity, and MDA content were detected using commercially available kits. The protein content of supernatants was detected using the bicinchoninic acid method (Walker, 1994). The GSH level was determined via the formation of 5-thio-2-nitrobenzoic acid (412 nm) (Hao et al., 2013). SOD activity was detected by the nitroblue tetrazolium/riboflavin photometric quantitative methods (420 nm) (Janknegt et al., 2007). The content of MDA was detected using the thiobarbituric acid assay (535 nm) (Dong et al., 2013). All the biomarker detections were conducted with the Synergy HT multi-mode microplate reader (Bio-Tek Instruments, Inc., Vermont, USA). After this, all the biomarkers were normalized to the total protein content and expressed as a percentage of the groups treated by the vehicle.

2.5.  Reverse transcriptase-polymerase chain reaction (RT-PCR) analyses of cyp1a and mrp1

 At 24 and 48 hpf, zebrafish embryos treated by the vehicle, 75 mg/L CdSO₄, 1.5 mg/L ANF, or the mixtures of 75 mg/L CdSO₄ and 1.5 mg/L ANF were collected. The living ones were used to extract total RNA with a commercial kit (Axygen Scientific, Inc., USA). The RNA quality was checked by 260/280 nm absorption using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Inc., USA). First-strand cDNA was prepared as previously described by Nakashima et al. (2012). After this, glyceraldehydes-3-phosphate dehydrogenase (gapdh), mrp1, and cyp1a were amplified from the first-strand cDNA using the PCR. Among these, gapdh was set as the house keeping gene. Gene primer sequences of gapdh, mrp1, and cyp1a (Table 1) were designed using Primer Premier 5.0 software (Premier, Inc., Canada).

2.6.  Statistical analysis

 All of the experimental data were expressed as mean±standard deviation (SD) of three sets of independent experiments, which were subsequently analyzed using a Statistical Package for Social Sciences (SPSS) v.15.0 (Chicago, USA). One-way analysis of variance (ANOVA) for multiple groups and Tukey’s HSD tests for two different treatments were performed to compare the data, respectively. In all cases, data differences were statistically significant when P<0.05.

3.  Results

3.1.  Individual toxicities of CdSO₄ and ANF

 As shown in Figs. 1 and 2, zebrafish embryos treated by the vehicle control (0.1% acetone) exhibited no significant alteration. CdSO₄ revealed both lethal and differential sub-lethal effects, like 24 hpf death and 72 hpf delayed hatching (Fig. 1d). However, only sub-lethal effects, such as 48 hpf cardiac edema (Fig. 1b) and 72 hpf delayed hatching (Fig. 1e) were observed for ANF. Furthermore, concentration-dependent toxic effects of both toxic compounds were obtained and are shown in Fig. 2. For example, the mortality of the embryos at 24 hpf was (10.00±4.61)% after a treatment of 100 mg/L CdSO₄, while, all embryos were dead at 24 hpf when exposed to 300 mg/L CdSO₄. The treatment of 20 mg/L CdSO₄ caused a delayed hatching rate of (17.50±5.00)% at 72 hpf, while the delayed hatching rate increased to 100% when the embryos were treated by 75 mg/L CdSO₄. For ANF, a similar trend was also found. For example, exposure to 1 mg/L ANF resulted in a cardiac edema rate of (26.25±10.15)% at 48 hpf, while nearly all embryos had cardiac edema at 48 hpf after the treatment of 3 mg/L ANF.

3.2.  Joint toxicity of CdSO₄ and ANF

 Since 24 hpf death only occurred in embryos treated by CdSO₄ and 48 hpf cardiac edema in embryos was only observed after the treatment of ANF, they were selected as endpoints for the investigation of the joint toxicity of CdSO₄ and ANF. As a result, toxicities of CdSO₄ and ANF could be significantly enhanced by each other (Fig. 3). For example, the treatment of 75 mg/L CdSO₄ or 1.5 mg/L ANF alone did not cause any death of zebrafish embryos, but co-treatment of 1.5 mg/L ANF and 75 mg/L CdSO₄ resulted in a 24 hpf mortality of (31.11±6.12)%. Furthermore, all embryos were dead at 24 hpf after exposure to the co-treatment of 1.5 mg/L ANF and 150 mg/L CdSO₄, while they were all alive when treated by CdSO₄ or ANF alone (P>0.05). Similarly, the treatments of 1.5 mg/L ANF and 75 mg/L CdSO₄ resulted in a 48 hpf cardiac edema rate of (58.33±12.93)% and 0, respectively, but the cardiac edema rate was (85.00±4.25)% when embryos were treated by a combination of 75 mg/L CdSO₄ and 1.5 mg/L ANF (P<0.001).

3.3.  Altered oxidative stress state of zebrafish embryos by the individual treatments of CdSO₄ and ANF

 Generally, CdSO₄ and ANF caused a concentration- and time-dependent alteration of the GSH level, SOD activity, and MDA content (Fig. 4). For example, SOD activity at 24 hpf was unaffected by 100 mg/L CdSO₄, but it was reduced by 9% when treated by 200 mg/L CdSO₄. Meanwhile, the reduction of SOD activity at 48 hpf was about 25% when embryos were treated by 200 mg/L CdSO₄ (Figs. 4a and 4b). Besides, the effects of ANF on GSH level, SOD activity, and MDA content of embryos at 48 hpf were not significant unless a concentration of 2 mg/L was used (Figs. 4c and 4d).

3.4.  Altered oxidative stress state of zebrafish embryos by the co-treatment of CdSO₄ and ANF

 Consistent with the joint toxicity of CdSO₄ and ANF, the mixtures produced great oxidative stress, including decreases in the GSH level, inhibition of SOD activity, and increase in MDA content, whereas no significant effects on the biomarkers were induced by the individuals at the corresponding doses (Fig. 5). For example, the SOD activity in embryos at 48 hpf was nearly completely inhibited by the co-treatment of 75 mg/L CdSO₄ and 1.5 mg/L ANF, but it was unaffected by the individual treatment of either compound (P>0.05). Besides, the alteration in this biomarker was more pronounced with an increasing exposure period.

3.5.  Toxicity of CdSO₄-ANF mixtures after a 12-h pre-treatment of CdSO₄

 To more specially illustrate the mechanism of the interactions between CdSO₄ and ANF, we added 1.5 mg/L ANF 12 h after the pre-treatment of 75 mg/L CdSO₄ alone. As a result, such a treatment caused a much lower mortality and slighter alteration of an oxidative stress state than the simultaneous addition of 75 mg/L CdSO₄ and 1.5 mg/L ANF (Table 2). For example, after 12 h pre-treatment of CdSO₄, the mixtures of CdSO₄ and ANF caused a reduction of SOD activity by 22%, while the simultaneous addition of CdSO₄ and ANF decreased the activity of SOD by about 67% (P<0.05). The addition of 1.5 mg/L ANF only slightly enhanced the toxicity compared with the group treated by 75 mg/L CdSO₄ alone. For instance, the 36 hpf mortality of embryos after the co-treatment of CdSO₄ and ANF was (15.23±2.04)%, which was only slightly higher than that of embryos treated by CdSO₄ alone ((3.96±4.19)%).

3.6.  Alteration of mrp1 and cyp1a gene expression

 After the treatments of CdSO₄, ANF, and CdSO₄-ANF mixtures, mRNA expression levels of mrp1 and cyp1a in zebrafish embryos at 24 and 48 hpf were respectively detected. And the results indicated that the individual treatments of 75 mg/L CdSO₄ and 1.5 mg/L ANF both significantly induced the gene expression of mrp1, but the co-treatment of 75 mg/L CdSO₄ and 1.5 mg/L ANF caused an obvious reduction of mrp1 gene expression as compared with the vehicle controls. While for cyp1a, its gene expression was enhanced by ANF but inhibited by CdSO₄, and the co-treatment of CdSO₄ and ANF reduced the gene expression of cyp1a to near the level of the vehicle control. For both mrp1 and cyp1a, the alteration of mRNA levels by chemicals seemed to be enhanced with incubation time.

4.  Discussion

 Aquatic organisms that inhabit environments are often contaminated with high levels of heavy metals and PAHs together (Terry and Stone, 2002; Udomchoke et al., 2010). However, studies on the joint effects of these pollutants are still rare. In this respect, CdSO₄ and ANF were selected as the representatives of metals and PAH, and their joint toxicity and corresponding mechanisms were subsequently studied.

 Both CdSO₄ and ANF exhibited significant developmental toxicity in zebrafish embryos (Figs. 1 and 2). Meanwhile, the toxicities of CdSO₄ and ANF could be significantly enhanced by each other as observed based on 24 hpf death and 48 hpf cardiac edema (Fig. 3). In the previous reports, glutathione S-transferase conjugation of GSH was considered to be important in the detoxification of cadmium in zebrafish embryos (Notch et al., 2011), while the treatment of ANF has been reported to significantly inhibit the activity of glutathione S-transferase and the subsequent antioxidant response in zebrafish embryos (Gauthier et al., 2014), indicating an interactive role of ANF with CdSO₄. The modulation of glutathione S-transferase by cadmium (Matović et al., 2011; Yang et al., 2012) might also result in the enhancement of ANF toxicity, which was related to the production of oxidative stress (Fleming and di Giulio, 2011). In this paper, both CdSO₄ and ANF caused decreases in the GSH level, inhibition of SOD activity, and increases in MDA content in zebrafish embryos (Fig. 4), but the mixtures produced a much greater oxidative stress (Fig. 5). Thus, oxidative stress should be considered important in the interaction of CdSO₄ and ANF.

 Interestingly, the same concentration of CdSO₄-ANF mixtures produced a reduced toxicity when the embryos were pretreated by CdSO₄ for 12 h (Table 2), which should be due to the induction of the detoxification system by the pretreatment of CdSO₄ (Fig. 6). RT-PCR results revealed that at sub-lethal concentrations, both CdSO₄ and ANF could significantly induce the expression of mrp1, correlating well with the previous reports and indicating that Mrp1 was involved in the detoxification of CdSO₄ and ANF (Long et al., 2011c; Costa et al., 2012; Navarro et al., 2012). Based on the previous experiments, PAH and heavy metals can act as both the substrates and the competitive inhibitors of Mrps, and thus increase the tissue accumulation of each other when they were used together, which should be the reason for the enhanced toxicity of CdSO₄-ANF mixtures. In addition, the reduced gene expression of mrp1 by the co-treatment of CdSO₄ and ANF could be explained by the fact that such CdSO₄-ANF mixtures exceeded the capability of ABC transporters and caused significant death of zebrafish embryos (Fig. 4), thus decreasing the mRNA level of mrp1 and further enhancing the toxicity of CdSO₄-ANF mixtures. However, research on the role of multixenobiotic resistance (MXR) in the interaction of environmental chemicals is still rare, and further investigations are needed.




 Furthermore, gene expression of cyp1a in zebrafish embryos at 24 hpf was found to be significantly enhanced by ANF, but the addition of CdSO₄ severely down-regulated the mRNA expression level of cyp1a. Furthermore, the co-treatment of CdSO₄ and ANF decreased the gene expression to a level near to the control group. In previous experiments, the induction of cyp1a by various PAH like benzo[α]pyrene and phenanthrene has been revealed to be a protective mechanism of the cardiovascular dysfunction in the development of zebrafish embryos (Billiard et al., 2008; Wills et al., 2009). Thus, the decreased gene expression of cyp1a by CdSO₄ could significantly enhance the toxicity of ANF, especially when 48 hpf cardiac edema was selected as the representing endpoint for ANF.

 It needs to be mentioned that, cadmium and PAH levels in the rivers are 0.01–0.19 mg/L (Singh et al., 2008) and 0.2–2.0 μg/L (Zhang et al., 2004), respectively, which are much lower than the concentrations used in this experiment. Such high concentrations could be first explained by the bioaccumulation of pollutants in the fish, making the tissue concentration many times higher than that in the surrounding waters (Vergauwen et al., 2013). Secondly, high concentrations of toxicants were usually used in laboratories to determine the possible toxic mechanism since they could exhibit significant effects in the short-term (Shi et al., 2008; Yu et al., 2012). In previous experiments, Konishi et al. (2006) found that the 72 hpf LC₅₀ (lethal concentration 50%) for CdCl₂ was 1000 μmol/L (≈208 mg/L CdSO₄), which was similar to the concentration used in our experiments. ANF caused no death of embryos even to its solubility limit of 3 mg/L (data not shown) in this experiment, which should be due to its low oxidative damage (Fig. 4) and induced gene expression of mrp1 and cyp1a (Fig. 6). This result corresponded well with the previous reports where 10 μmol/L ANF (2.72 mg/L) caused no death in differential fishes and was only used as CYP inhibitors (Meinelt et al., 2001; Koenig et al., 2012).

5.  Conclusions

 In conclusion, an enhancement of developmental toxicity by each other was observed when CdSO₄ and ANF were used together. Production of oxidative stress and altered expression of mrp1 and cyp1a could be the important components of such joint toxicity. In the future, this result should be confirmed with more typical PAH, and the involvement of MXR in the interactions of CdSO₄ and ANF should be investigated in detail.

---

* Project supported by the National Natural Science Foundation of China (No. 21307154)Compliance with ethics guidelines Jian YIN, Jian-ming YANG, Feng ZHANG, Peng MIAO, Ying LIN, and Ming-li CHEN declare that they have no conflict of interest.