Gene expression profiles in testis of developing male Xenopus laevis damaged by chronic exposure of atrazine

Chemosphere 159 (2016) 145e152

Contents lists available at ScienceDirect

Chemosphere journal homepage: www.elsevier.com/locate/chemosphere

Gene expression profiles in testis of developing male Xenopus laevis damaged by chronic exposure of atrazine Linlin Sai a, Zhihua Dong b, Ling Li a, Qiming Guo a, Qiang Jia a, Lin Xie a, Cunxiang Bo a, Yanzhong Liu c, Binpeng Qu d, Xiangxin Li e, Hua Shao a, *, Jack C. Ng f, Cheng Peng a, f a

Shandong Academy of Occupational Health and Occupational Medicine, Ji’nan, Shandong, China The 404th Hospital of PLA, Weihai, Shandong, China Weihai Wendeng Center Hospital, Weihai, Shandong, China d Shandong Medical College, Ji’nan, Shandong, China e Heze Center for Disease Control and Prevention, Heze, Shandong, China f The University of Queensland, National Research Centre for Environmental Toxicology-Entox, Brisbane, Australia b c

h i g h l i g h t s
AZ at 0.1 and 100 mg/L caused testicular degeneration in Xenopus laevis.
100 mg/L AZ upregulated 616 and downregulated 549 genes.
AZ altered genes expression related to reproductive and immune systems.
Six pathways were significantly affected after chronic AZ exposure.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 14 December 2015 Received in revised form 3 May 2016 Accepted 4 May 2016

As a widely used herbicide, atrazine (AZ) has been extensively studied for its adverse effects on the reproductive system, especially feminization in male animals. However, the relationship of gene expression changes and associated toxicological endpoints remains unclear. In this study, developing Xenopus laevis tadpoles were exposed to concentration of AZ at 0.1, 1, 10 or 100 mg/L continuously. Compared with froglets in the control group, there were no significant differences in body length, body weight, liver weight and hepatosomatic index (HSI) of males in groups treated with AZ for 90 d. At 100 mg/L AZ treatment caused a significant reduction of gonad weight and gonadosomatic index (GSI) of males (p < 0.01). In addition, AZ at all dose levels caused testicular degeneration, especially in froglets from the groups with 0.1 and 100 mg/L which exhibited U-shaped dose-response trend. We further investigated the gene expression changes associated with the testicular degeneration induced by AZ. We found that the expression of 1165 genes was significantly altered with 616 upregulated and 549 downregulated compared to the expression profile of the control animals. KEGG analysis showed that genes which were significantly affected by AZ are mainly involved in arginine and proline metabolism, cell cycle, riboflavin metabolism, spliceosome, base excision repair and progesterone-mediated oocyte maturation pathway. Our results show that AZ may affect reproductive and immune systems by interference with the related gene expression changes during the male X. laevis development. The findings may help to clarify the feminization mechanisms of AZ in male X. laevis. © 2016 Elsevier Ltd. All rights reserved.

Handling Editor: David Volz Keywords: Herbicide Microarray Reproductive system Endocrine disruption Frog Tadpole

1. Introduction Atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine,

* Corresponding author. E-mail address: [email protected] (H. Shao). http://dx.doi.org/10.1016/j.chemosphere.2016.05.008 0045-6535/© 2016 Elsevier Ltd. All rights reserved.

AZ) is one of the most widely used herbicides in China and other countries (Dong et al., 2006; Ren et al., 2002; Solomon et al., 2013). It has been used to stop pre- and postemergence broadleaf and grassy weeds in farmland of major crops such as corn, sorghum, sugarcane, wheat, and guava. Due to the widely application of AZ in agriculture, the environmental levels of AZ up to 108 mg/L have

146

L. Sai et al. / Chemosphere 159 (2016) 145e152

been reported in rivers of North America (US EPA, 2002). In some areas of China, AZ concentrations in drinking water exceed safe, standard levels (2 mg/L) (GB, 2006; Ren et al., 2002; Dong et al., 2006). The endocrine-disrupting effects of triazines and their chlorometabolites have been extensively studied in mammalian models and aquatic species (Crain et al., 1997; Hayes et al., 2002, 2010;
et al., 2004; Tillitt et al., 2010). AZ has been reported to Spano cause endocrine disruption in mammals, birds, reptiles, fish and amphibians by affecting normal reproductive function and development in these organisms (Holloway et al., 2008; Gunderson et al., 2011; Kloas et al., 2009; de la Casa-Resino, 2012; Rohr and McCoy, 2010). Due to their skin permeability and life cycles on land and in the water, amphibians have increased exposure to environmental contaminants including AZ which make them potentially more susceptible (Roy, 2002). It has been reported that low, environmentally relevant levels of AZ can affect metamorphosis of tadpoles in several frog species (LaFiandra et al., 2008; Langlois et al., 2010; Brodeur et al., 2013; Rohr and McCoy, 2010; Zaya et al., 2011). Meanwhile, other studies showed that AZ can alter gonadal differentiation in developing Rana pipiens (Langlois et al., 2010). AZ treatment causes a variety of other adverse effects in frogs including morphologic and functional changes in gonads (Rohr and McCoy, 2010; Hayes et al., 2011). Notably, AZ has been shown to cause demasculinization and complete feminization in male Xenopus laevis, but the induction mechanisms have not been elucidated (Hayes et al., 2010). So it is the need to explore AZ-induced changes in gene expression relate to conventional physiologically and toxicologically relevant endpoints. In this study, we measured global changes in the gene expression profile of testis in male X. laevis frogs chronically exposed to AZ at 100 mg/L basing on the pathology changes of testes in our previous testing. Microarray was used for the gene expression profile analysis. To the best of our knowledge, this is the first study to investigate the gene expression profiles in damaged testis of developing male X. laevis chronically exposed to AZ for 180 d. 2. Materials and methods 2.1. Test materials and animals AZ (purity of 97%) and dimethyl sulfoxide (DMSO) were obtained from Sigma (Chemical Co., USA). Trizol was supplied by Invitrogen (Carlsbad, USA). Agilent® Xenopus 4  44K Gene Expression Microarrays and Agilent® p/n 5190-0442 One-Color Quick Amp Labeling Kit were obtained from Agilent (Technologies Inc., USA). Three pairs of adult male and female X. laevis were purchased from the Chinese Academy of Sciences (Beijing, China). The offspring were produced by the natural mating activities of the paired adult X. laevis. UV-treated and carbon-filtered laboratory freshwater was used for the acclimatization of frogs in the laboratory and for all subsequent exposures. The animals were kept at an average water temperature of 22 ± 2  C at pH 7.5, under 12 h light and 12 h dark cycle. Tadpoles were fed fairy shrimp (Artemia nauplii) eggs in a young age daily and pork liver (Qin et al., 2010) three times per week ad libitum when the tadpoles completed metamorphosis. 2.2. Experimental procedures At Nieuwkoop-Faber (NF) stage 47 (13 d post-hatch), mixed sex tadpoles (n ¼ 800) from one adult pairing were randomly divided into five groups (Nieuwkoop and Faber, 1994). Each group (n ¼ 160)

was divided into 8 replicate tanks (25  20  20 cm3), each containing 10 L water. The tadpoles were exposed to AZ dissolved in solvent vehicle DMSO (0.01%) at dosages of 0.1, 1, 10 or 100 mg/L for 90 or 180 d. The control tadpoles were treated with 0.01% DMSO only as is found in the AZ treatments. Test materials were applied in a static-renewal exposure regime. Test solutions were renewed by 50% replacement every 48 h. For exposure concentration quality control and testing, 20 mL of water samples counting up to 16 samples were taken immediately from each treatment tank before and after exchange of the test solutions at the first 9 d and the last 9 d of exposure. Levels of AZ were measured using gas chromatography-mass spectrophotometry (GC-MS) (Agilent 6890N) (Oka et al., 2008). The detection limit was 1.49 ng/L with a quantification limit of 4.97 ng/L. All concentrations are displayed as means ± standard deviation (SD). Animals were observed daily for monitoring morphological changes and health status. On day 90, metamorphosed animals were collected randomly to retain from each treatment group across treatment replicates (n ¼ 20) for the microarray analyses. The rest of the animals (n ¼ 140) were collected, dissected and whole-body, liver and gonad weight, and body length of male X. laevis froglets were measured and recorded. The gonadosomatic index (GSI) (Brewer et al., 2008) and the hepatosomatic index (HSI) (Van der Oost et al., 2003) were calculated as GSI ¼ (gonad weight/ body weight)  102 and HSI ¼ (liver weight/body weight)  102 respectively. Ten testicular tissues from each group were fixed in Bouin’s solution (71% saturated picric acid, 24% formaldehyde, 5% glacial acetic acid) for routine paraffin embedding (Yang, 2010). All of the females were excluded in this study. On day 180, the remaining mixed sex animals from the control and treatment groups were processed. Testes were isolated and flash-frozen in liquid nitrogen, and stored at 80  C for microarray expression analysis. Data were tested for normality (Shapiro-Wilk test) and homogeneity of variance (Bartlett’s test). One-way ANOVA was used to analyze for mortality of the tadpoles, body, organ weights and body length. All data are presented as mean ± SD. Sex ratios were analyzed using a 2-by-2 contingency test. All statistical analyses were performed using STATA 10.0 software package (http://www. stata.com/). Group differences were evaluated by Fisher’s Least Significant Difference test. Statistical probability of p < 0.05 was considered significant. 2.3. Histopathology Sections of paraffin-embedded testis tissues were cut at 5 mm serially and stained with hematoxylin and eosin (HE). All of the specimens were examined microscopically to assess for gonad development. 2.4. RNA extraction, microarray and statistical analysis Based on the results of the physiological changes (GSI and histological changes), we selected six male control frogs and six male 100 mg/L AZ-treated frogs for gene expression analysis. Total RNAs from individual testis was isolated using Trizol according to manufacturer’s instruction and the concentration and purity was measured using a NanoDrop® ND-100 spectrophotometer (NanoDrop Technologies Inc., USA). The integrity of RNA was determined by denaturing gel electrophoresis. RNA samples were further purified and converted to double-stranded cDNA for microarray analysis which was conducted according to Agilent® Xenopus 4  44K Gene Expression Microarrays protocols. After hybridization and washing, the chips were scanned with the Axon GenePix 4000B microarray Scanner (Molecular Devices, LLC, USA). Original

L. Sai et al. / Chemosphere 159 (2016) 145e152

fluorescence data were read by GenePix pro V6.0 software (Molecular Devices, LLC, USA) and standardized by the Agilent GeneSpring GX v11.5.1 Software (Agilent, USA) for further analysis. Differentially expressed genes were identified by the value of foldchange (FC  2) and t-test was used in comparison with those in the control group. To elucidate the predominant biological pathways that are affected by AZ exposure, we analyzed our microarray dataset using the Kyoto Encyclopedia of Genes And Genomes (KEGG) pathway database (http://www.genome.jp/kegg/pathway.html). 2.5. Quantitative RT-PCR and statistical analysis We further validated the microarray data using Quantitative RTPCR (Q-RT-PCR) for which we selected 16 genes including adc, cdc6, acp6, oat, gatm, cycE1, orc3, flad1, snrpb, hspa2, hmg-1, parp1, spdya, ccnb1, scpep1, pcsk4 with the primers listed in Table S1 in the supplementary material. The genes were chosen based on their function roles and respective FC values. For Q-RT-PCR analysis, total RNA was converted into cDNA using the Invitrogen Superscript cDNA Synthesis kit. Reactions were performed according to the manufacturer’s instructions. Relative mRNA expression was calculated according to the 2△△CT method (Livak and Schmittgen, 2001). The samples of control group were set as calibrator. The upregulated genes were identified at 2△△CT > 1, the downregulated genes were identified at 2△△CT < 1. 3. Results and discussion Environmental contamination of herbicides has long been a concern due to their potential risks to human health and ecosystem. As a traditional herbicide, AZ has been studied in different models for its toxicity. However, the chronic effects of AZ on amphibians have not been fully addressed especially at the molecular level. In this study, we treated developing tadpoles (NF stage 47) with AZ at different environmental concentrations for a chronic exposure (180 d) and examined its potential effects especially on gene regulations associated with damaged testes. 3.1. Measurement of AZ exposure concentrations The exposure was performed according to our designed concentrations which were 0.1, 1, 10, 100 mg/L. We measured AZ concentrations before and after exchange of the test solutions at the first nine days and the last nine days of exposure. Before exchange, actual AZ concentrations in water samples treated with AZ were 0.11 ± 0.03, 1.3 ± 0.3, 10.5 ± 0.8, and 106.2 ± 4.9 mg/L, respectively. After exchange, the concentrations in the four treatment groups were 0.10 ± 0.02, 0.9 ± 0.4, 9.7 ± 1.9, and 97.7 ± 7.5 mg/L, respectively. Thus, the results showed that the measured concentrations varied generally less than 20% from the nominal concentrations.

147

in animal studies (Andersen et al., 1999; Bailey et al., 2004; Kim et al., 2004). In this study, we examined a total of 551 frogs which were treated with AZ at 0, 0.1, 1, 10 and 100 mg/L (n ¼ 118, 110, 111, 104 and 108, respectively). We did not observed significant changes in body weight and length of male frogs after exposure to AZ. These results were consistent with one previous study that AZ caused no effect on these parameters at similar concentrations (1, 10, 25 mg/L) (Du Preez et al., 2008). This indicates that AZ might not produce whole body growth on male X. laevis at certain exposure levels. HSI are important physiological biomarkers to reflect responses to toxicant exposure and can provide information on energy reserves and general health of the organism (Van der Oost et al., 2003). In the present study, liver weight and HSI were not different after treatment, which suggested that no dysfunctions in the liver of X. laevis occurred following exposure to AZ. Furthermore, we found that the weight and GSI of the testis in 100 mg/L AZ-treated frogs were significantly reduced compared to controls (p < 0.01) (Figs. 1 and 2). Interestingly, a previous study reported that exposure to AZ at 10 and 100 mg/L for 49 had no inhibitory effect on gonad growth in adult male X. laevis. The study also found the GSI was increased by AZ at low level (10 mg/L, p ¼ 0.046) but not high level treatment (100 mg/L, p ¼ 0.195) (Hecker et al., 2005). However, considering the p value in the mean GSI of X. laevis treated with 10 mg/L AZ, the incremental effect may be not convincing due to the marginal level of significance in difference between control and treatment. Furthermore, adult male X. laevis were used in that study while we use male X. laevis at developing stage. In this context, the discrepant results given by us may partially result from the model difference. The inhibitory effects of AZ on developing male X. laevis but not adults may suggest male amphibians at developing stage are more sensitive to AZ. This speculation has been confirmed in our another study (Sai et al., 2015). 3.3. Histopathological evaluation Histological changes by AZ were examined under microscope. Testis of frogs from control group showed regular seminiferous lobules and spermatogenesis at all stages containing a few spermatogonias and spermatozoa as shown in Fig. 3a. Histological changes in testes were observed in the frogs from all of AZ treatments including irregular shape of seminiferous lobules and large empty spaces (Fig. 3bed). Particularly, in testes from 0.1 and 100 mg/L AZ treatments, the histopathological changes were

3.2. Physiological changes induced by AZ AZ has drawn more attentions for its potential environmental contamination and resultant risks to the ecological systems. The mortality increased significantly in AZ treatments (p ¼ 0.027, 0.057, 0.000, 0.006). The results indicated AZ at 0.1, 1, 10 and 100 mg/L for 90 d causes pressure to survival of X. laevis. On day 90, we did not observe the effects of AZ on sex ratio of X. laevis (64/54, 59/51, 59/52, 56/48, 58/50; p > 0.05). Gonadal morphology abnormality was not found in any of the AZ-treated animals. Physiological parameters such as the changes in body and organ weights are useful and important indicators of chemical toxicities

Fig. 1. The effects of AZ on the gonad weight of male X. laevis. Data are expressed as mean ± SD, * Indicates statistical significance p < 0.05 respect to control.

148

L. Sai et al. / Chemosphere 159 (2016) 145e152

Fig. 2. The effects of AZ on the GSI of male X. laevis. Data are expressed as mean ± SD, * Indicates statistical significance p < 0.05 respect to control.

obvious. Similarly, in X. laevis treated with AZ (0.1 mg/L) gonadal abnormalities during development was reported (Hayes et al., 2003). But our previous study showed 0.1 mg/L of AZ did not induce any degrees of testicular degeneration in Bufo bufo gargarizans (Sai et al., 2015). These findings suggested a species-specific susceptibility to AZ exist among species. Meanwhile, qualitative histologic lesion of testes exhibited U-shaped doseeresponse trend which frequently appear in the effects of estrogen (Almstrup et al., 2002). More quantitative studies are needed to elucidate the Ushaped dose response trend for AZ-induced the histologic lesion of testes in X. laevis. 3.4. AZ induced global changes in gene expression in testes of male X. laevis AZ at 100 mg/L was found to be effective to cause abnormal GSI

Fig. 3. Histological examinations of gonad of X. laevis from AZ-treated and control groups. a: Testis from a control frog, seminiferous lobules are regular and all stages of spermatogenesis are present (HE, 50); b: Testis from a 0.1 mg/L AZ treated frog filled with large empty spaces (arrow) (HE, 50); c: Testis from a 1 mg/L AZ-treated frog, note the irregular shape of seminiferous lobules (HE, 50); d: Testis from a 100 mg/L AZ-treated frog filled with large empty spaces (arrow) (HE, 50).

and histological changes in testes and was selected as treatment concentration in gene expression study. To understand the molecular mechanisms involved in observed damages in the testis of X. laevis and find potential marker genes for less affected testes, we further investigated the possible effects of AZ on gene expression changes in the testis of X. laevis exposed to 100 mg/L of AZ. Stringent statistical and data analysis criteria were used in order to ensure a highly reliable dataset. Hybridization pattern was generated by superposition of computer data which reflect the abundance of detected genes. There are about 43,803 dots in each chip with balanced hybridization signal and clear background suggesting reliable results (Fig. S1 in the supplementary material). We found that the expression of 1165 genes was significantly altered with 616 upregulated, 549 downregulated in the 100 mg/L AZ-treated frogs when compared to those in the control group (Tables S2 and S3 in the supplementary material). Expressions of many genes related to the reproductive system and growth were found significantly downregulated. We firstly analyzed the cytochrome P450 (cyp) gene expression in the testis, since cyp family enzymes are responsible for the metabolism of steroids and toxic chemicals. Studies have shown that AZ at low ecologically relevant doses in vivo and in vitro can induce the activity of aromatase which is one of cyp family enzymes, thus induce androgen testosterone to estrogen estradiol resulting in femalization effect (Chang et al., 2005; Oh et al., 2003), especially the cyp19 gene expression (Sanderson et al., 2000; Hayes et al., 2002). However, in the present study induction of cyp19 gene induced by AZ at 100 mg/L was not observed whereas the cyp26a1 and cyp4b1.2 gene expression were inhibited 4.04 and 16.69-fold, respectively, compared to controls. Feng et al. (2015) indicated that the expression of cyp26a1 was downregulated when meiotic initiation was delayed in teleosts, and that meiosis is essential for germ cells development for all sexually reproducing species (Feng et al., 2015). We speculated the expression of cyp26a1 was closely related with germ cells development of X. laevis which was inhibited by AZ. We also observed a down-regulation of 42Sp50 gene compared to controls (FC ¼ 20.56). Hirakawa et al. (2012) found 42Sp50 were detected in ovaries of medaka 5 d after hatch, as well as 42Sp50 gene expression was upregulated in testis of medaka exposed to 17a-ethinylestradiol (EE2) for 6 weeks (Hirakawa et al., 2012). In our study, Zpc gene expression was downregulated significantly (28.99 fold) compared to controls. It should be noted that developing oocytes of vertebrates contain an external acellular coat, ZP, which consists of liver derived choriogenin proteins and seven Zp genes (Zpax, Zpb, and Zpc1 through to Zpc5) (Kanamori, 2000; Kanamori et al., 2003). Meanwhile, a series of Zp genes were upregulated in the testis of both estradiol benzoate and EE2 exposed medaka (Hirakawa et al., 2012). The contrasting gene expression results between medaka and X. laevis will need to be further explored in terms of specific function of 42Sp50 and Zpc genes in different animal species and their respective response to AZ exposure. In addition, several genes related to reproductive system and growth such as Acp2, acp6, prss8, npm2, mcm3 were also significantly downregulated. It has been shown that acps are involved in the immune, reproductive and digestive systems, and apoptosis. (Sun et al., 2006; Bozzo et al., 2002; Yoneyama et al., 2004). A study on amphioxus has demonstrated that ACPs were presented in sperm with roles in proliferation and maturation of sperm (Sun et al., 2006). Therefore, we speculate that downregulation of acps may affect the reproductive capacity. Prss8 encodes a trypsinogen, a member of the trypsin family of serine proteases. Since trypsinogen plays an important role in prostate epithelia and seminal fluid, downregulation of prss8 gene may suggest an additional mechanism by which AZ alters reproductive function in X. laevis. Studies

L. Sai et al. / Chemosphere 159 (2016) 145e152

149

Table 1 The results of Q-RT-PCR validation. Gene name

Gene full name

2△△CT

adc cdc6 acp6 oat gatm cycE1 orc3 flad1 snrpb hspa2 hmg-1 parp1 spdya ccnb1

Arginine decarboxylase Cdc6-related protein Acid phosphatase 6, lysophosphatidic Ornithine aminotransferase Glycine amidinotransferase Cyclin E1 Origin recognition complex, subunit 3 FAD1 flavin adenine dinucleotide synthetase homolog Small nuclear ribonucleoprotein polypeptides B and B1 Heat shock 70 kDa protein 2 High mobility group protein-1 Poly (ADP-ribose) polymerase 1 Speedy A Cyclin B1

0.49 0.31 1.79 0.65 5.26 2.87 1.68 1.93 2.21 2.49 2.45 2.17 0.50 0.35

Table 2 The enrichment pathways by KEGG. Pathway name

Gene name

FC absolute

Regulation

Arginine and proline metabolism

adc agmat asl gatm glul-a oat

13.83 3.46 8.90 4.68 3.02 2.14

Down Down Up Up Down Down

Cell cycle

anapc5 ccnb1 ccnd1-a cdc6 cycb3 cycE1 mcm3 orc3 tgfb1 wee1-a wee2 cdc6

6.60 9.47 2.30 2.99 2.89 5.37 4.61 2.47 3.33 20.64 51.94 2.99

Up Down Up Down Up Down Down Up Up Down Down Down

Riboflavin metabolism

acp6 blvrb flad1

17.43 2.687 51.50

Down Up Down

Spliceosome

bud31 hnrnpa3 hspa2 naa38 prpf3 rbm25 sf3a3 snrp116-pending snrpa1 snrpb syf2 thoc4

2.77 13.81 2.62 2.31 27.17 4.91 3.18 3.14 3.42 2.49 26.37 4.19

Up Down Up Up Down Up Up Up Up Up Up Up

Base excision repair

hmg-1 lig1 parp1

10.42 12.33 2.47

Down Down Up

Progesterone-mediated oocyte maturation

anapc5 ccnb1 cycb3 gnai1 mos spdya

6.60 9.47 2.89 3.24 2.96 28.74

Up Down Up Up Down Down

“FC” stands for fold change.

have shown that npm2 is involved in chromatin reprogramming, especially during fertilization and early embryonic development. The same gene is probably involved in sperm DNA decondensation during fertilization (Burns et al., 2003; Okuwaki et al., 2012).

Therefore, the observed downregulation of npm2 may affect sperm production leading to further damage of the reproductive system. MCMs encoded by mcms not only play a role in the initiation of DNA replication, but also in the interaction with the cell cycle checkpoint

150

L. Sai et al. / Chemosphere 159 (2016) 145e152

proteins (Guida et al., 2005). Therefore, we speculate that downregulation of gene mcm may influence the initiation of eukaryotic genome replication, and consequently affect growth and development of tadpoles. In summary, AZ-induced damaged testis observed in our study might result from the down regulation of these genes related to reproduction. Numerous studies have shown that AZ influences the immune system through decreasing cell-mediated and humoral immunity, inhibiting cytokine production, dendritic cell maturation and natural killer cell activity (Hooghe et al., 2000; Karrow et al., 2004; Pinchuk et al., 2007; Pruett et al., 2006; Rooney et al., 2003; Rowe et al., 2007, 2008). We found that genes involved in the complement system such as c2, c3, cfh were decreased by AZ treatment. Deficiency of C2 has been reported to associate with certain autoimmune diseases (Rajaraman et al., 2010). Complement component C3 plays a central role in the activation of complement system (Yates et al., 2007). Gene cfh is a member of the regulator of complement activation gene cluster and encodes a protein which is secreted into the bloodstream and has an essential role in the regulation of complement activation with twenty short consensus repeat domains (Maller et al., 2006). Altered expression of these immune-related genes further confirmed the suppressive effects of AZ on the immune system and consequent functions. AZ also downregulated expression of numerous genes involved in metabolism in tadpoles exposed to 100 mg/L AZ. Gene oat encodes the mitochondrial enzyme ornithine aminotransferase, which is a key enzyme in the pathway that converts arginine and ornithine into the major excitatory and inhibitory neurotransmitters glutamate and GABA. Mutations that result in a deficiency of this enzyme may cause metabolic diseases (Zhou and You, 2007). Expression of metabolism-related gene glul was also decreased. The protein encoded by this gene belongs to the glutamine synthetase family. Glutamine is a main source of energy and is involved in cell proliferation, inhibition of apoptosis, and cell signaling (Zhang et al., 2005). Metabolic function of tadpoles exposed to 100 mg/L AZ may be damaged due to downregulation of these metabolismrelated genes. Hierarchical clustering analysis was performed to visually delineate global gene expression profiles. It demonstrated definite and distinct patterns of expression in the samples between treatment and control group (Fig. S2 in the supplementary material). 3.5. Q-RT-PCR validation of microarray results In the male frogs, Q-RT-PCR analysis showed upregulation in gene expression for acp6, gatm, cycE1, orc3, flad1, snrpb, hspa2, hmg1, parp1 (2△△CT > 1) and downregulation in expression of adc, cdc6, oat, spdya, ccnb1 (2△△CT < 1). These results confirmed the microarray data (Table 1). 3.6. KEGG analysis of regulated genes Base on KEGG analysis, we found six signaling pathways were obviously interrupted by AZ as shown in Table 2. These pathways include arginine and proline metabolism, cell cycle, riboflavin metabolism, spliceosome, base excision repair and progesteronemediated oocyte maturation pathway. Interestingly, many of genes associated with the cell cycle signaling pathway were significantly downregulated such as ccnb1, mcm3, wee1-a, wee2, cdc6 at FC of 9.47, 4.61, 20.64, 51.94, 2.99 respectively. There is a particular starting point of DNA replication and pre-replication complex (Pre-RC). The protein was encoded by mcms. Protein encoded by wee1 gene plays a key role in DNA replication and DNA damage control (Tominaga et al., 2006; Kellogg, 2003). Reduced expression of CDC6 protein can cause

abnormal DNA replication resulting in apoptosis or a false division status (Oehlmann et al., 2004). Downregulation of these cell cycle related genes by AZ found in this study suggested that AZ may affect cell replication and possible DNA damage repair, and then provides evidence that AZ can interfere with the growth of X. laevis cells. The genes involving in arginine and proline metabolism were significantly downregulated by AZ. Arginine metabolism is important for maintaining a balanced immune response, through regulating the functions of macrophages and T cells (Yeh et al., 2002). Genes oat, glul, agmat and adc in this pathway were found to be reduced in their expression in AZ-treated frogs. Oat gene encodes the mitochondrial enzyme ornithine aminotransferase which participates in the synthesis of arginine (Kobayashi et al., 1995). Therefore, downregulation of oat may seriously interfere metabolism of arginine. Arginine decarboxylase encoded by adc can decarboxylate arginine into agmatine which is distributed in various organs and tissues and has important physiological functions (Zhu et al., 2004). Hence, downregulation of adc may block synthesis of agmatine leading to disrupted physiological functions. Gene glul also play important role in arginine and proline metabolism. Protein encoded by this gene catalyzes the synthesis of glutamine from glutamate and ammonia, and glutamine is the material for the synthesis of arginine (Zhang et al., 2005). These results may suggest that inhibition of this pathway could interfere with immune and endocrine function, etc. In addition, spliceosome, riboflavin metabolism, base excision repair, progesterone-mediated oocyte maturation pathways were also inhibited in the 100 mg/L AZ treatment group. We speculate that a comprehensive model of metabolic regulation plays an important role in the effect of AZ on male X. laevis. 4. Conclusion Our results showed that AZ can elicit reproductive system toxicity in developing male X. laevis. Gene expression profiles in testes of male X. laevis frogs chronically exposed to AZ at 100 mg/L showed the global gene expression changes related to the observed physiological changes including testis weight, GSI and histological changes in testes. By microarray analysis, expressions of 1165 genes were significantly altered with 616 upregulated and 549 downregulated by at least twofold in the testes of X. laevis after AZ exposure. KEGG pathway analysis showed that these genes were mainly enriched in six pathways of arginine and proline metabolism, cell cycle, riboflavin metabolism, spliceosome, base excision repair and progesterone-mediated oocyte maturation. Among the altered genes, many related to the reproductive system and immune system, such as cyp26a1, cyp4b1.2, 42Sp50, prss8, npm2, oat, glul and mcm3 were found significantly downregulated which may be helpful for the understanding of the reason for the reproductive system toxicity of AZ on male X. laevis. Acknowledgements This work was supported by the National Science Foundation of China (30901214; 81470145; 81573198), Natural Science Foundation of Shandong (ZR2009CM114; 2010GSF10213; ZR2015YL045), and Ji’nan Science and Technology Bureau (201010005). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.chemosphere.2016.05.008.

L. Sai et al. / Chemosphere 159 (2016) 145e152

References Almstrup, K., Fern andez, M.F., Petersen, J.H., Olea, N., Skakkebaek, N.E., Leffers, H., 2002. Dual effects of phytoestrogens result in u-shaped dose-response curves. Environ. Health. Perspect. 110, 743e748. Andersen, H., Larsen, S., Spliid, H., Christensen, N.D., 1999. Multivariate statistical analysis of organ weights in toxicity studies. Toxicology 136, 67e77. Bailey, S.A., Zidell, R.H., Perry, R.W., 2004. Relationships between organ weight and body/brain weight in the rat: what is the bestanalytical endpoint? Toxicol. Pathol. 32, 448e466. Bozzo, G.G., Raghothama, K.G., Plaxton, W.C., 2002. Purification and characterization of two secreted purple acid phosphatase isozymes from phosphate-starved tomato (Lycopersicon esculentum) cell cultures. Eur. J. Biochem. 269, 6278e6286. Brewer, S.K., Rabeni, C.F., Papoulias, D.M., 2008. Comparing histology and gonadosomatic index for determining spawning condition of small-bodied riverine fishes. Ecol. Fresh. Fish. 17, 54e58. Brodeur, J.C., Sassone, A., Hermida, G.N., Codugnello, N., 2013. Environmentally e relevant concentrations of atrazine induce non-monotonic acceleration of developmental rate and increased size at metamorphosis in Rhinella arenarum tadpoles. Ecotoxicol. Environ. Saf. 92, 10e17. Burns, K.H., Viveiros, M.M., Ren, Y., Wang, P., DeMayo, F.J., Frail, D.E., Eppig, J.J., Matzuk, M.M., 2003. Roles of NPM2 in chromatin and nucleolar organization in oocytes and embryos. Science 300, 633e636. Chang, L.W., Toth, G.P., Gordon, D.A., Graham, D.W., Meier, J.R., Knapp, C.W., DeNoyelles, F.J., Campbell, S., Lattier, D.L., 2005. Responses of molecular indicators of exposure in mesocosms: common carp (Cyprinus carpio) exposed to the herbicides alachlor and atrazine. Environ. Toxicol. Chem. 24, 190e197. Crain, D.A., Guillette Jr., L.J., Rooney, A.A., Pickford, D.B., 1997. Alterations in steroidogenesis in alligators (Alligator mississippiensis) exposed naturally and experimentally to environmental contaminants. Environ. Health Perspect. 105, 528e533. de la Casa-Resino, I., 2012. Endocrine disruption caused by oral administration of atrazine in European quail (Coturnix coturnix coturnix). Comp. Biochem. Physiol. C Toxicol. Pharmacol. 156, 159e165. Dong, L., Chen, L., Li, Z., Gao, H., Li, J., 2006. Quality assurance/quality control for monitoring and analysis of trace triazines in water. J. Saf. Environ. 6, 35e38. Du Preez, L.H., Kunene, N., Everson, G.J., Carr, J.A., Giesy, J.P., Gross, T.S., Hosmer, A.J., Kendall, R.J., Smith, E.E., Solomon, K.R., Van Der Kraak, G.J., 2008. Reproduction, larval growth, and reproductive development in African clawed frogs (Xenopus laevis) exposed to atrazine. Chemosphere 71, 546e552. Feng, R., Fang, L., Cheng, Y., He, X., Jiang, W., Dong, R., Shi, H., Jiang, D., Sun, L., Wang, D., 2015. Retinoic acid homeostasis through aldh1a2 and cyp26a1 mediates meiotic entry in Nile tilapia (Oreochromis niloticus). Sci. Rep. 5, 10131. GB, 2006. Standards of Drinking Water: GB 5749e2006. Beijing, China (in Chinese). Guida, T., Salvatore, G., Faviana, P., Giannini, R., Garcia-Rostan, G., Provitera, L., Basolo, F., Fusco, A., Carlomagno, F., Santoro, M., 2005. Mitogenic effects of the upregulation ot minichromosome maintenance proteins in anaplastic thyroid carcinoma. J. Clin. Endoerinol. Metab. 90, 4703e4709. Gunderson, M.P., Veldhoen, N., Skirrow, R.C., Macnab, M.K., Ding, W., van Aggelen, G., Helbing, C.C., 2011. Effect of low dose exposure to the herbicide atrazine and its metabolite on cytochrome P450 aromatase and steroidogenic factor-1 mRNA levels in the brain of premetamorphic bullfrog tadpoles (Rana catesbeiana). Aquat. Toxicol. 102, 31e38. Hayes, T., Haston, K., Tsui, M., Hoang, A., Haeffele, C., Vonk, A., 2003. Atrazineinduced hermaphroditism at 0.1 ppb in American leopard frogs (Rana pipiens): laboratory and field evidence. Environ. Health Perspect. 111, 568e575. Hayes, T.B., Anderson, L.L., Beasley, V.R., de Solla, S.R., Iguchi, T., Ingraham, H., Kestemont, P., Kniewald, J., Kniewald, Z., Langlois, V.S., Luque, E.H., McCoy, K.A., ~ oz-de-Toro, M., Oka, T., Oliveira, C.A., Orton, F., Ruby, S., Suzawa, M., TaveraMun Mendoza, L.E., Trudeau, V.L., Victor-Costa, A.B., Willingham, E., 2011. Demasculinization and feminization of male gonads by atrazine: consistent effects across vertebrate classes. Steroid. Biochem. Mol. Biol. 127, 64e73. Hayes, T.B., Collins, A., Lee, M., Mendoza, M., Noriega, N., Stuart, A.A., Vonk, A., 2002. Hermaphroditic, demasculinized frogs after exposure to the herbicide atrazine at low ecologically relevant doses. Proc. Natl. Acad. Sci. 99, 5476e5480. Hayes, T.B., Khoury, V., Narayan, A., Nazir, M., Park, A., Brown, T., Adame, L., Chan, E., Buchholz, D., Stueve, T., Gallipeau, S., 2010. Atrazine induces complete feminization and chemical castration in male African clawed frogs (Xenopus laevis). Proc. Natl. Acad. Sci. 107, 4612e4617. Hecker, M., Kim, W.J., Park, J.W., Murphy, M.B., Villeneuve, D., Coady, K.K., Jones, P.D., Solomon, K.R., Van Der Kraak, G., Carr, J.A., Smith, E.E., du Preez, L., Kendall, R.J., Giesy, J.P., 2005. Plasma concentrations of estradiol and testosterone, gonadal aromatase activity and ultrastructure of the testis in Xenopus laevis exposed to estradiol or atrazine. Aquat. Toxicol. 72, 383e396. Hirakawa, I., Miyagawa, S., Katsu, Y., Kagami, Y., Tatarazako, N., Kobayashi, T., Kusano, T., Mizutani, T., Ogino, Y., Takeuchi, T., Ohta, Y., Iguchi, T., 2012. Gene expression profiles in the testis associated with testis-ova in adult Japanese medaka (Oryziaslatipes) exposed to 17a-ethinylestradiol. Chemosphere 87, 668e674. Holloway, A.C., Anger, D.A., Crankshaw, D.J., Wu, M., Foster, G., 2008. Atrazineinducedchanges in aromatase activity in estrogen sensitive target tissues. J. Appl. Toxicol. 28, 260e270. Hooghe, R.J., Devos, S., Hooghe-Peters, E.L., 2000. Effects of selected herbicides on

151

cytokine production in vitro. Life Sci. 66, 2519e2525. Kanamori, A., 2000. Systematic identification of genes expressed during early oogenesis in medaka. Mol. Reprod. Dev. 55, 31e36. Kanamori, A., Naruse, K., Mitani, H., Shima, A., Hori, H., 2003. Genomic organization of ZP domain containing egg envelope genes in medaka (Oryzias latipes). Gene 305, 35e45. Karrow, N.A., McCay, J.A., Brown, R.D., Musgrove, D.L., Guo, T.L., Germolec, D.R., White Jr., K.L., 2004. Oral exposure to atrazine modulates cell-mediated immune function and decreases host resistance to the B16F10 tumor model in female B6C3F1 mice. Toxicology 209, 15e28. Kellogg, D.R., 2003. Wee1 dependent mechanisms required for coordination of cell growth and cell division. Cell Sci. 116, 4883e4890. Kim, J.C., Shin, D.H., Kim, S.H., Kim, J.K., Park, S.C., Son, W.C., Lee, H.S., Suh, J.E., Kim, C.Y., Ha, C.S., Chung, M.K., 2004. Subacute toxicity evaluation of a new camptothecin anticancer agent CKD-602 administered by intravenous injection to rats. Regul. Toxicol. Pharmacol. 40, 356e369. Kloas, W., Lutz, I., Springer, T., Krueger, H., Wolf, J., Holden, L., Hosmer, A., 2009. Does atrazine influence larval development and sexual differentiation of Xenopus laevis? Toxicol. Sci. 107, 376e384. Kobayashi, T., Ogawa, H., Kasahara, M., Shiozaw, Z., Matsuzawa, T., 1995. A single amino acid substitution within the mature sequence of ornithine aminotransferase obstructs mitochondrial entry of the precursor. Am. J. Hum. Genet. 57, 284e291. LaFiandra, E.M., Babbitt, K.J., Sower, S.A., 2008. Effects of atrazine on anuran development are altered by the presence of a nonlethal predator. J. Toxicol. Environ. Health A 71, 505e511. Langlois, V.S., Carew, A.C., Pauli, B.D., Wade, M.G., Cooke, G.M., Trudeau, V.L., 2010. Low levels of the herbicide atrazine alters sex ratios and reduces metamorphic success in Rana pipiens tadpoles raised in outdoor mesocosms. Environ. Health Perspect. 118, 552e557. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using realtime quantitative PCR and the 2△△CT method. Methods 25, 402e408. Maller, J., George, S., Purcell, S., Fagerness, J., Altshuler, D., Daly, M.J., Seddon, J.M., 2006. Common variation in three genes, including a noncoding variant in CFH, strongly influences risk of age-related macular degeneration. Nat. Genet. 38, 1055e1059. Nieuwkoop, P.D., Faber, J., 1994. Normal Table of Xenopous Laevis (Daudin). Garland Publishing, Inc., New York. Oehlmann, M., Score, A.J., Blow, J.J., 2004. The role of Cdc6 in ensuring complete genome licensing and S phase checkpoint activation. Cell Biol. 165, 181e190. Oh, S.M., Shim, S.H., Chung, K.H., 2003. Antiestrogenic action of atrazine and its majormetabolites in vitro. J. Health. Sci. 49, 65e71. Oka, T., Tooi, O., Mitsui, N., Miyahara, M., Ohnishi, Y., Takase, M., Kashiwagi, A., Shinkai, T., Santo, N., Iguchi, T., 2008. Effect of atrazine on metamorphosis and sexual differentiation in Xenopus laevis. Aquat. Toxicol. 87, 215e226. Okuwaki, M., Sumi, A., Hisaoka, M., Saotome-Nakamura, A., Akashi, S., Nishimura, Y., Nagata, K., 2012. Function of homo- and hetero-oligomers of human nucleoplasmin/nucleophosmin family proteins NPM1, NPM2 and NPM3 during sperm chromatin remodeling. Nucleic. Acids Res. 40, 4861e4878. Pinchuk, L.M., Lee, S.R., Filipov, N.M., 2007. In vitro atrazine exposure affects the phenotypic and functional maturation of dendritic cells. Toxicol. Appl. Pharmacol. 223, 206e217. Pruett, S.B., Fan, R., Oppenheimer, S., 2006. Greater than additive suppression of TLR3-induced IL-6 responses by administration of dieldrin and atrazine. J. Immunotoxicol. 3, 253e262. Qin, X., Xia, X., Yang, Z., Yan, S., Zhao, Y., Wei, R., Li, Y., Tian, M., Zhao, X., Qin, Z., Xu, X., 2010. Thyroid disruption by technical decabromodiphenyl ether (DE83R) at low concentrations in Xenopus laevis. J. Environ. Sci. 22, 744e751. Rajaraman, P., Brenner, A.V., Neta, G., Pfeiffer, R., Wang, S.S., Yeager, M., Thomas, G., Fine, H.A., Linet, M.S., Rothman, N., Chanock, S.J., Inskip, P.D., 2010. Risk of meningioma and common variation in genes related to innate immunity. Cancer Epidemiol. Biomark. Prev. 19, 1356e1361. Ren, J., Jiang, K., Zhou, H., 2002. The concentration and source of Atrazine residue in water of guanting reservoir. Environ. Sci. 23, 126e128. Rohr, J.R., McCoy, K.A., 2010. A qualitative meta-analysis reveals consistent effects of atrazine on freshwater fish and amphibians. Environ. Health Perspect. 118, 20e32. Rooney, A.A., Matulka, R.A., Luebke, R.W., 2003. Developmental atrazine suppresses immune function in male, but not female SpragueeDawley rats. Toxicol. Sci. 76, 366e375. Rowe, A.M., Brundage, K.M., Barnett, J.B., 2007. In vitro atrazine-exposure inhibits human natural killer cell lytic granule release. Toxicol. Appl. Pharmacol. 221, 179e188. Rowe, A.M., Brundage, K.M., Barnett, J.B., 2008. Development immunotoxicity of atrazine in rodents. Basic Clin. Pharmacol. Toxicol. 102, 139e145. Roy, D., 2002. Amphibians as environmental sentinels. Bioscience 27, 187e188. Sai, L., Wu, Q., Qu, B., Bo, C., Yu, G., Jia, Q., Xie, L., Li, Y., Guo, Q., Ng, C.J., Peng, C., 2015. Assessing atrazine-induced toxicities in Bufo bufo gargarizans Cantor. Bull. Environ. Contam. Toxicol. 94, 152e157. Sanderson, J.T., Seinen, W., Giesy, J.P., van den Berg, M., 2000. 2-Chloro-s-triazine herbicides induce aromatase (CYP19) activity in H295R human adrenocortical carcinoma cells: a novel mechanism for estrogenicity? Toxicol. Sci. 54, 121e127. Solomon, K.R., Giesy, J.P., LaPoint, T.W., Giddings, J.M., Richards, R.P., 2013. Ecological risk assessment of atrazine in North American surface waters. Environ. Toxicol. Chem. 32, 10e11.

152

L. Sai et al. / Chemosphere 159 (2016) 145e152


, L., Tyler, C.R., van Aerle, R., Devos, P., Mandiki, S.N., Silvestre, F., Thome , J.P., Spano Kestemont, P., 2004. Effects of atrazine on sex steroid dynamics, plasma vitellogenin concentration and gonad development in adult goldfish (Carassius auratus). Aquat. Toxicol. 66, 369e379. Sun, J., Zhang, S., Wang, Y., 2006. Distribution of acid phosphatase in amphioxus Branchiostoma belcheritsingtauense. Mar. Fish. Res. 27, 17e20 (in Chinese). Tillitt, D.E., Papoulias, D.M., Whyte, J.J., Richter, C.A., 2010. Atrazine reduces reproduction in fathead minnow (Pimephales promelas). Aquat. Toxicol. 99, 149e159. Tominaga, Y., Li, C., Wang, R.H., Deng, C.X., 2006. Murine wee1 plays a critical role in cell cycle regulation and preimplantation stages of embryonic development. Int. Biol. Sci. 2, 161e170. US EPA, 2002. Reregistration Eligibility Science Chapter for Atrazine Environmental Fate and Effects Chapter. U.S. EPA, Washington, DC. Van der Oost, R., Beyer, J., Vermeulen, N.P., 2003. Fish bioaccumulation and biomarkers in environmental risk assessment: a review. Environ. Toxicol. Pharmacol. 13, 57e149. Yang, Q., 2010. Study on the Effects of Atrazine on Developmental Toxicity of Xenopus laevis and Bufo Gargarizans Cantor (in Chinese). Yates, J.R., Sepp, T., Matharu, B.K., Khan, J.C., Thurlby, D.A., Shahid, H., Clayton, D.G., Hayward, C., Morgan, J., Wright, A.F., Armbrecht, A.M., Dhillon, B., Deary, I.J.,

Redmond, E., Bird, A.C., Moore, A.T., 2007. Complement C3 variant and the risk of age-related macular degeneration. N. Engl. J. Med. 357, 553e561. Yeh, C.L., Yeh, S.L., Lin, M.T., Chen, W.J., 2002. Effects of arginine-enriched total parenteral nutrition on inflammatory-related meditor and T-cell population in septic rats. Nutrition 18, 631e635. Yoneyama, T., Shiozawa, M., Nakamura, M., Suzuki, T., Sagane, Y., Katoh, Y., Watanabe, T., Ohyama, T., 2004. Characterization of a novel acid phosphatase from embryonic axes of kidney bean exhibiting vanadate-dependent chloroperoxidase activity. J. Biol. Chem. 279, 37477e37484. Zaya, R.M., Amini, Z., Whitaker, A.S., Kohler, S., Ide, C.F., 2011. Atrazine exposure affects growth, body condition and liver health in Xenopus laevis tadpoles. Aquat. Toxicol. 104, 243e253. Zhang, B., Yuan, Y., Jia, Y., Yu, X., Xu, Q., Shen, Y., Shen, Y., 2005. An association study between polymorphisms in five genes in glutamate and GABA pathway and paranoid schizophrenia. Eur. Psychiatry 20, 45e49. Zhou, F., You, G., 2007. Molecular insights into the structure-function relationship of organic anion transporters OATs. Pharm. Res. 24, 28e36. Zhu, M.Y., Iyo, A., Piletz, J.E., Regunathan, S., 2004. Expression of human arginine decarboxylase, the biosynthetic enzyme for agmatine. Biochim. Biophys. Acta 2, 156e164.