Environmental Pollution 197 (2015) 278e286
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Sex specific response in cholesterol level in zebrafish (Danio rerio) after long-term exposure of difenoconazole Xiyan Mu, Kai Wang, Tingting Chai, Lizhen Zhu, Yang Yang, Jie Zhang, Sen Pang, Chengju Wang*, Xuefeng Li* College of Sciences, China Agricultural University, Beijing, People's Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 June 2014 Received in revised form 27 October 2014 Accepted 9 November 2014 Available online 5 December 2014
Difenoconazole is a widely used triazole fungicide, its extensive application may potentially cause toxic effects on non-target organisms. To investigate the effect of difenoconazole on cholesterol content and related mechanism, adult zebrafish were exposed to environmental related dosage (0.1, 10 and 500 mg/L) difenoconazole. The body weight and hepatic total cholesterol (TCHO) level was tested at 7, 15 and 30 days post exposure (dpe). The expressions of eight cholesterol synthesis genes and one cholesterol metabolism gene were assessed via Quantitative PCR method. The significant decrease of TCHO level in male zebrafish liver was observed at 15 and 30 dpe, which was accompanied by apparent hepatic cholesterol-genesis genes expression decline. In comparison with males, female zebrafish showed different transcription modification of tested genes, and the cholesterol content maintain normal level during the whole exposure. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Zebrafish Difenoconazole Cholesterol Sex differences
1. Introduction Triazole fungicides are widely used for the protection of a variety of plants. These compounds could enter the water environment through spray drift or surface run-off after rainfall (Raudonis et al., 2004; Konwick et al., 2006). Their characteristics, such as high chemical and photochemical stability, low biodegradability and easy transportation in the environment (Wang et al., 2011), make them persistent in water (Bromillow et al., 1999). Thus, the possible detrimental effects of triazole compounds on environmental organisms have caused widespread attention. Difenoconazole (cistrans-3-chloro-4-(4-methyl-2-(1H-1,2,4-triazol-yl methyl)-1,3dioxolan-2-yl) phenyl 4-chlorophenyl ether) is a typical triazole fungicide used for the control of fungal disease on vegetables, cereals and other field crops (Vawdrey et al., 2008; Horsfield et al., 2010). It inhibits fungal lanosterol-14a-demethylase (CYP51) activity and blocks ergosterol biosynthesis, thus resulting in the blocking of fungal cell wall chitin synthesis and the overspill of cytoplasm (Buchenauer et al., 1995; Hamada et al., 2011; Ragsdale, 1977). In China, difenoconazole has been used as the main pesticide to combat rice diseases for many years (Wang and Zhang, 2012).
* Corresponding authors. E-mail addresses: [email protected] (C. Wang), [email protected] (X. Li). http://dx.doi.org/10.1016/j.envpol.2014.11.019 0269-7491/© 2014 Elsevier Ltd. All rights reserved.
Since it is being used extensively in rice, difenoconazole has more opportunities to contaminate the water environment. In the recent decade, efforts have been concentrated on the environmental occurrence of difenoconazole and it has been detected in samples from several areas (Table 1). Compared with other triazole fungicides, difenoconazole is reported to possess higher acute toxicity to a wide range of aquatic organisms (Dong et al., 2013). According to the recent publication of the European Food Safety Authority (EFSA), difenoconazole is considered very toxic to aquatic organisms in view of its high toxicity to Daphnia magna (chronic NOEC ¼ 0.0056 mg active substance/L) (EFSA, 2011). In addition, our recent research also showed that difenoconazole has negative effect on zebrafish during both adult and early life stages (Mu et al., 2013). Therefore, it is necessary to perform environmental toxicological studies of difenoconazole to reflect its risk to aquatic organisms. The influence of lipid biosynthesis and metabolism has been considered as a main active pathway of triazole compounds (Hermsen et al., 2011; Goetz and Dix, 2009). Cholesterol, which is an important lipid in vivo, plays a key role in the maintenance of cell structure and is also a precursor for the biosynthesis of steroid hormones and bile acids (Beppu et al., 2012). The cholesterol biosynthesis mostly occurred in liver from acetyl-CoA and required multi-step enzymatic reaction (Kojima et al., 2009). From the view of bactericidal mechanism, difenoconazole has the ability to inhibit
X. Mu et al. / Environmental Pollution 197 (2015) 278e286 Table 1 The reported environmental difenoconazole dosage in water area. Area
Data source
Reported value (ug/L)
Reference
China (Changsha, Changchun, Hangzhou) Thailand (Salakru, Nong Sua) Malaysia (Kedah)
Paddy water (spraying day) PECa (agricultural water area) Surface water (river near field) Surface water (river)
1.98 e2.91 mg/L 0.028 mg/L
Zhang et al., 2011 Satapornvanit et al., 2004 Latiff et al., 2010
China (Fujian) Italy (River Meolo) Australia (Victoria) a
0.30 mg/L
You, 2008 0.0039 e0.0061 mg/L PEC (river) 0.0095 mg/L Verro et al., 2009 Sch€ afer et al., Surface water (river 0.15 mg/L 2011 and stream)
PEC ¼ Predicted Environmental Concentration.
the functionality of CYP51 (Henry and Sisler, 1984; Hitchcock et al., 1990), an important enzyme in the cholesterol synthesis (Miao et al., 2014), which may further result in the alteration of total cholesterol (TCHO) level. Fish are an important eco-indicator organism and many model species, such as rainbow trout (Oncorhynchus mykiss), medaka (Oryzias latipes) and zebrafish, have been applied in the toxicity evaluation of environmental pollutants (Boyle et al., 2013; Gonz alez-Doncel et al., 2014; Zhang et al., 2013). The zebrafish (Danio rerio) is a typical small tropical aquarium fish well known to most people. The reasons it has a long history of general use in toxicology research that it is inexpensive, hardy, small, easy to care for in large numbers and will readily spawn under appropriate photoperiod conditions (Lele and Krone, 1996). The main purpose of this study is to investigate the gender-based effects of difenoconazole on cholesterol level in zebrafish liver and gain an insight into the involved mechanisms. 2. Materials and methods 2.1. Zebrafish maintenance Wild type zebrafish (length 3.5 ± 0.6; weight 0.18 ± 0.05) were purchased from Beijing Hongdagaofeng Aquarium Department. All of adult zebrafish were maintained in the centralized breeding equipment (ESEN-ZF-SS, Esen, Beijing) at 26 C with a photoperiod of 14/10 (light/dark), and fed with dried brine shrimp (Artemia, Futian Brand, JPN) twice daily. The total daily fed amount was equivalent to 2% of fish weight. Adult zebrafish which were at the age of about six months were fasted for one day before exposure. 2.2. Chemicals and reagents The standard water was prepared in the lab with the formula of iso-7346-3, which contained 2 mmol/L Ca2þ, 0.5 mmol/L Mg2þ, 0.75 mmol/L Naþ and 0.074 mmol/L Kþ (ISO, 1996). 95% Difenoconazole (CAS: 119446-68-3) was obtained from China Ministry of Agriculture and the stock solution used for drug exposure was prepared with ethanol AR (analytical reagent). All of the other reagents utilized were of analytical grade. 2.3. Fish exposure and sample collection The 6-month-old zebrafish were separated by sex and randomly assigned to nominal concentrations of 0 (control), 0.1, 10, and 500 mg/L of difenoconazole for 30 days. Difenoconazole-free standard water was used as control which contained 0.025 mL/L
279
ethanol. 960 (480 for both male and female) individuals were distributed in 48 tanks. Each tank contains 10 L exposure solution and 20 fish. The exposure solution was renewed daily to keep the appropriate concentration of drug and water quality. The tanks were renewed per week. The water qualities including temperature, PH and dissolved oxygen were measured every two days. The external conditions during exposure, including temperature, humidity and light cycle, were the same as the culture environment. During the test, fish were fed daily with dried brine shrimp (equivalent to 2% of body weight) except for the 24 h prior to sacrifice. At 7, 15 and 30 days post exposure (dpe), 12 zebrafish were randomly selected from each replicate and divided into two parts, 6 for TCHO analysis and 6 for RNA extraction (for specific information of sampling process please see Supplemental Materials). After anesthetized on ice, the wet weights of all collected individuals were measured using an electronic balance (made by Sartorius Corp.). Liver tissues of collected zebrafish were quickly excised, weighed, frozen in liquid nitrogen and stored at 80 C for RNA extraction and cholesterol content detection. 2.4. Difenoconazole in water analysis 2.4.1. Sample preparation The water samples were obtained at 0, 1, 7, 14, 28 dpe. 20 mL of water samples were extracted twice with an equal volume of ethyl acetate (10 mL each time). For samples with 10 mg/L and 500 mg/L concentration, 1.0 mL extract was completed with a nitrogen stream and then redissolved with 1 mL acetonitrile. For samples with 0.1 mg/L, 10 mL extract was evaporated completely to dry with a nitrogen stream and redissolved with 1 mL acetonitrile. Finally, all the redissolved samples were filtered through a 0.22-mm filter into a sample vial for LC-MS/MS analysis. 2.4.2. LC-MS/MS analysis An Agilent Technologies 6410 high-performance liquid chromatograph (Agilent, USA) was interfaced to a triple quadrupole mass spectrometer (Agilent, USA) equipped with an ESI source. The chromatographic separation was carried out on ZORBAX SB-C18 column (50 mm 2.1 mm, 1.8 mm; Agilent, USA) at 25 C with a flow rate of 0.3 mL/min. The mobile phase in isocratic mode consisted of acetonitrile and water (0.1% HCOOH) (70:30). For the MS/MS analysis, the common parameters were as follows: the capillary current and nebulising gas were set as 9 nA and 35.0 psi, respectively. A flow rate of drying gas was 8.00 L/min with temperature of 350 C. The compound parameters including collision energy and fragmentor voltage were 20 eV and 130 V, respectively. The transitions of precursor ion (m/z 406.1) to product ion (m/z 251.0 and m/z 337.0) of difenoconazole were detected with the multiple reaction monitoring (MRM) in positive ion (ESIþ) mode. The sample injection volume was 5 mL. The retention time of difenoconazole was 0.8 min (Fig. S1). 2.5. Total cholesterol (TCHO) content in liver analysis For determination of TCHO content in the liver, liver from 6 males or females of each replicate were homogenized (1:10, w/v) via electric homogenizer (Tiangen Biotech) in cell lysates (made by Applygen). 10 mL of the supernatant was obtained for protein detection after the solution was centrifuged at 3000 g for 10 min at 4 C. The remaining sample was incubated for 10 min in 70 C water to fully dissolve cholesterol. After centrifuged at 3000 g for 10 min at room temperature, the supernatant was used for cholesterol analysis. The TCHO content determined using enzymatic kits according to the manufacturer's directions (Applygen, Beijing, China).
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Total protein content was determined using BCA (bicinchoninic acid) protein assay kit (Cwbiotech, Beijing, China).
in order to further confirm the relationship between TCHO level and gene expression level.
2.6. Gene expression analyses
3. Results
To further investigate the effect of difenoconazole on cholesterol synthesis and metabolism, the mRNA levels of 9 related genes, including CYP51, farnesyl-diphosphate farnesyltransferase 1 (FDFT1), isopentenyl-diphosphate delta isomerase 1 (IDI1), squalene epoxidase (SQLE), 7-dehydrocholesterol reductase (DHCR7), 24-dehydrocholesterol reductase (DHCR24), 3-hydroxy-3methylglutaryl-CoA reductase (HMGCR)a, HMGCRb and cholesterol 7a-hydroxylase (CYP7A1) were measured by quantitative real-time PCR. Total RNA was extracted from zebrafish liver using spin column adsorption method following manufacturer's protocols (Tiangen Biotech, Beijing, China). The concentration was measured by absorbance at 260 nm using a UV1240 spectrophotometer (Perkin Elmer, USA). The purity was assessed by determining the A260/ A280 ratio. The cDNA was then synthesized via reverse transcription (RT) using quant RTase kit (Tiangen Biotech, Beijing, China) in accordance with manufacturer's recommendations. Real-time PCR reactions were performed with the ABI 7500 q-PCR system (Applied Biosystems, Foster City, CA). The SYBR Green PCR Master Mix reagent kits (Tiangen Biotech, Beijing, China) were used for quantification of gene expression. A 20 mL reaction system was conducted according to manufacturer's instructions (See Supplemental Materials). Zebrafish-specific primers were designed for the genes of interest using Primer 6.0 software (Table S1). The housekeeping gene actin, beta (ACTB) was used as an internal control. The PCR amplification procedure was as follows: 95 C for 15 min followed by 40 cycles of 94 C for 10 s, 60 C for 20 s, and 72 C for 32 s. Quantification of the transcripts was performed using the 2DDCt method.
3.1. Chemical analysis
2.7. Statistical analysis All statistical analyses were undertaken using Spss 16.0 software. Differences were determined by one-way ANOVA, completed with Dunnett post hoc comparison. P < 0.05 was considered significant. Relationships between TCHO level and tested genes transcription were analysed by principal component analysis (PCA). In addition, correlation analysis was completed with Spearman's test
Analysis results indicated that the deviations between nominal and actual difenoconazole concentrations were less than 20% in all test period (Table S2). Therefore, the nominal dosage is able to represent the actual content in this research. 3.2. Body weight To investigate the adverse effects of difenoconazole to zebrafish, some morphological endpoints were assessed such as weight and body size. Compared to their respective control groups, the body weight of both sexes significantly decreased in 500 mg/L treatment at 30 dpe (Fig. 1). For female zebrafish, the average weight of 500 mg/L group is equivalent to 89% of control at 30 dpe. While the decrease level of male is much stronger than female, the average weight of 500 mg/L group is only equivalent to 66% of control. In addition, from the body appearance, male zebrafish treated with 500 mg/L difenoconazole showed obviously skinny body at 30 dpe while zebrafish in control were normal (Fig. S2). 3.3. TCHO level in liver For both male and female zebrafish, no apparent change of hepatic TCHO level was observed in all treated groups at 7 dpe (Fig. 2). At 15 dpe, the TCHO level of male zebrafish in 500 mg/L group decreased significantly and the value was equivalent to 71% of control. The reduction became stronger at 30 dpe, the TCHO level of male zebrafish in 500 mg/L group reduced to only 30% of control. While for female zebrafish, no obvious modification was found throughout the exposure. 3.4. Expression of genes related in cholesterol synthesis According to the results, the expression of IDI1, SQLE, DHCR24 and HMGCRb in male zebrafish liver showed significant upregulation at 7 dpe, while HMGCRa showed down-regulation at the same period (Fig. 3). At 15 dpe, expression of all eight tested
Fig. 1. Body weight of male and female zebrafish during the exposure. Asterisks denote significant difference between treatments and control (determined by Dunnett post-hoc comparison, p < 0.05*; p < 0.01**). Error bars indicate standard deviation.
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Fig. 2. Hepatic TCHO level of male and female zebrafish during the exposure. Asterisks denote significant difference between treatments and control (determined by Dunnett posthoc comparison, p < 0.05*; p < 0.01**). Error bars indicate standard deviation.
Fig. 3. Relative expressions of 8 genes related to CHO-synthesis in male zebrafish liver during the difenoconazole exposure. The relative expression level of control was designated as 1. Asterisks denote significant difference between treatments and control: red represents increase; green represents decrease (determined by Dunnett post-hoc comparison, p < 0.05*; p < 0.01**). Error bars indicate standard deviation. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 4. Relative expressions of 8 genes related to CHO-synthesis in female zebrafish liver during the difenoconazole exposure. The relative expression level of control was designated as 1. Asterisks denote significant difference between treatments and control: red represents increase; green represents decrease (determined by Dunnett post-hoc comparison, p < 0.05*; p < 0.01**). Error bars indicate standard deviation. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
genes were markedly decreased in a concentration-dependent manner, and among them, the hepatic expression of CYP51, FDFT1, DHCR7, DHCR24, SQLE and HMGCRa in male zebrafish of 500 mg/L exposure group was less than 10% of control. The most affected gene during this period was SQLE which was reduced to about 1% of control (500 mg/L exposure group). At 30 dpe, the decreased expression level of tested genes still existed, but the degree was weaker than the down-regulation at 15 dpe. Different trend of gene expression changes were found in female zebrafish liver compared with males. The expression of all cholesterol synthesis related genes were inhibited by difenoconazole at 7 dpe (Fig. 4). However, at 15 dpe, only 500 mg/L treatment group performed down-regulation of cholesterol-genesis genes, and the expression of all tested genes returned to normal level in zebrafish exposed to 10 mg/L difenoconazole. In the 0.1 mg/L exposure group, up-regulation of all tested genes were observed. At 30 dpe, only DHCR7 and IDI1 decreased in higher concentration exposure group, expression of all other tested genes showed normal or increased level in all treatments.
3.5. Expression of gene related in cholesterol metabolism The expression level of CYP7A1 significantly decreased in male zebrafish liver at 7 and 30 dpe. There was a dose-dependent down regulation of CYP7A1 at 30 dpe, the relative expression level were 0.53 (0.1 mg/L), 0.36 (10 mg/L) and 0.14 (500 mg/L). In addition, 0.1 mg/ L difenoconazole caused up-regulation of CYP7A1 at 15 dpe, the relative expression level was 7.7 (Fig. 5). For female zebrafish, hepatic expression of CYP7A1 was up-regulated by 0.1 and 10 mg/L difenoconazole at 7 dpe, the relative expression level was 9.4 and 3.8 respectively. At 15 dpe, the up-regulation continued, the expression levels were 4.2 (0.1 mg/L) and 2.0 (10 mg/L) fold of control group. While at 30 dpe, the expression apparently decreased in all treatments, especially for zebrafish in 500 mg/L group which was reduced to less than 1% of control. 3.6. Chemometrics To understand the relationship between the expression change of tested genes and the TCHO level in zebrafish liver, correlation
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Fig. 5. Relative expression of CYP7A1 in male and female zebrafish liver during the difenoconazole exposure. The relative expression level of control was designated as 1. Asterisks denote significant difference between treatments and control: red represents increase; green represents decrease (determined by Dunnett post-hoc comparison, p < 0.05*; p < 0.01**). Error bars indicate standard deviation. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
analysis and principal component analysis were finished using the data of all zebrafish treatments. All parameters measured in the present study were distinguished on the loading plots corresponding to the first (51.75%) and second (21.99%) components (Fig. 6). According to the PCA results, expression of CYP51, DHCR7, DHCR24 and HMGCRa showed relative high correlation with TCHO level, and the expression level of CYP7A1 showed negative correlation with TCHO level. The correlation analysis results further confirmed the relationship among the tested parameters (Table 2). 4. Discussion The main purpose of this research is to investigate the alteration of cholesterol level in different gender of zebrafish caused by difenoconazole and the related mechanisms. Our results clearly indicated that 500 mg/L difenoconazole could cause significant decrease of hepatic TCHO level in male zebrafish at 15 and 30 dpe. For female zebrafish, no obvious modification of TCHO level was observed during the whole exposure period. In addition, although both male and female zebrafish performed decreased body weight after difenoconazole exposure, the reduction of male was much stronger than female. These results implicated that female zebrafish showed more resistance to difenoconazole exposure than males. Our previous study showed that difenoconazole could cause negative effect to zebrafish including developmental abnormities for embryo as well as growth inhibition for adult, and the effective concentration was very close to its reported value in water adjacent to fields, which reflected the threat of difenoconazole to aquatic organisms living in agricultural areas (Mu et al., 2013). In this study, apparent down-regulation of cholesterol synthesis genes was found in male zebrafish liver exposed to 0.1 mg/L or higher difenoconazole and the effective dosage is very close to the detected value in surface water sample (Table 1), which indicated that its occurrence may have the potential to alter cholesterol generation of aquatic organisms in non-agricultural water environment. HMGCR is the rate-limiting enzyme in the cholesterol biosynthesis pathway (Sato and Kamada, 2011), which plays a key role in the hepatic cholesterol synthesis. According to recent research, the cholesterol level decrease caused by berberine in hyperhomocysteinemic rat liver is highly related to the inhibition of hepatic HMGCR activity (Wu et al., 2011). CYP51 catalyse the demethylation of lanosterol in the cholesterogenic process which is
also the target of triazole compounds. Previous research documented that cholesterol level in plasma decrease associated with inhibition of hepatic expression of CYP51 and HMGCR in propiconazole treated fathead minnow (Skolness et al., 2013). In this work, similar with the results of Skolness et al. (2013), the reduction of gene expression of CYP51 and HMGCRa, were observed with the decreased TCHO level in male zebrafish liver. SQLE and FDFT1 are downstream to HMGCR in the cholesterol synthesis pathway, and both the two enzymes are considered as potential target of cholesterol lowering agents (Trapani et al., 2011). It has been reported that SQLE down-regulation in vertebrates and fungi decreases synthesis of cholesterol and ergosterol, respectively (Belter et al., 2011). In the present study, expression decrease of SQLE and FDFT1 was exhibited along with the TCHO level decline in male zebrafish liver of 500 mg/L group. DHCR7 is the enzyme that catalyses the reduction of 7dehydrocholesterol to cholesterol in the last step of cholesterol biosynthesis (Horling et al., 2012). DHCR24 is the enzyme catalyses the cholesterol biosynthesis by reducing the delta-24 double bond of desmosterol (Cecchi et al., 2008). Both of the two enzymes are key factors contributing to the hydrogenation of dehydrocholesterol. Previous studies demonstrated that the lack of 7DHCR leads to a deficit in cholesterol and an accumulation of 7dehydrocholesterol, which together with its oxidized derivatives also have negative influences on total cholesterol transfer (De ndez et al., 2005). In Fabiani et al., 1996; Gaoua et al., 1999; Ferna the present study, genes encoded DHCR7 and DHCR24 were significantly down-regulated in male at 15 and 30 dpe after difenoconazole exposure. Previous study suggested that lower constitutive hepatic expression levels of the genes for cholesterol biosynthetic enzymes could give rise to the lower serum T-CHO levels in rats (Nemoto et al., 2013). In this study, hepatic TCHO level decrease occurred along with expression decline of cholesterol-synthesis genes in male zebrafish exposed to difenoconazole. The statistical results (Table 2) also indicated that the transcription of CYP51, HMGCRa, DHCR7 and DHCR24 showed significant correlation with TCHO level. Therefore, the hepatic TCHO content decline of male zebrafish in this test is possibly due to the generally down-regulation of cholesterol synthesis genes. Bile acid synthesis is the major pathway for the metabolism of cholesterol. CYP7A1 is the rate-limiting enzyme for the
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Fig. 6. Loading diagram of PCA analysis of THO level and related genes expression in zebrafish liver after exposure to difenoconazole.
predominant pathway of bile acid biosynthesis (Qi et al., 2015). In the present study, down-regulation of hepatic CYP7A1 was observed in both male and female zebrafish at 30 dpe, the relative expression level was 0.139 and 0.003 respectively. For female individuals, such sharply decreased transcription of CYP7A1 may result in the lack of bile acids (Russell, 1999). Previous research proved that mice with insufficient bile acid are more susceptible to liver injury and exhibit defective liver regeneration (Meng et al., 2011). Therefore, further research could concern on the effect of difenoconazole on liver regeneration and repair. A recent study has proven that difenoconazole has the ability to inhibit zebrafish growth (Mu et al., 2013). In the present study, male zebrafish performed stronger sensitivity to difenoconazole than female in the endpoint of body weight reduction. Skolness et al. (2013) also found that there is a gender difference in the body weight modification of propiconazole treated fathead minnow. Recent studies showed that weight decrease frequently occurred with TCHO level reduction, Nam et al. (2014) reported that Gardenia jasminoides Ellis extract could cause body weight reduction and hepatic TCHO content decrease. Cong et al. (2012) also found that lower serum cholesterol is accompanied by lower body weight and slower growth rate in pigs. In this work, the difenoconazole treated male zebrafish showed both TCHO reduction and body weight decrease. However for female zebrafish, body weight decrease emerged with normal hepatic TCHO level. Therefore, the origin of
Table 2 Correlation coefficient between death rate and expression level of all tested genes. Gene HMGCRa HMGCRb IDI1 FDFT1 SQLE a
Correlation coefficient **a
0.448 0.167 0.167 0.176 0.209
the difenoconazole caused body weight decrease and its interaction mechanism with TCHO decline still need to be studied. Sex-based response difference of toxicants has been observed in aquatic organisms during recent years. Zhang et al. (2012) reported that after chronic perfluorononanoic acid exposure, triglyceride (TG) levels increased in male zebrafish while decreased in females and the transcriptional expression levels of fatty acid binding proteins (FABPs) were up-regulated in males and down-regulated in females except FABP1b. Li et al. (2009) showed that male Chinese rare minnow was more sensitive to amitrole (a triazole herbicide) than female according to the expression change of thyroid hormone related genes post exposure. According to Table 3, the relative hepatic expression levels of the four high-TCHO-correlated synthesis genes in female zebrafish were higher than those in male zebrafish. At the same time, CYP7A1 which is the main metabolism gene of cholesterol showed higher transcription in male zebrafish liver at 30 dpe, compared to female individuals. Therefore, the different response of TCHO level towards difenoconazole between male and female zebrafish may due to the distinct regulation of key genes related in cholesterol synthesis and metabolism. 5. Conclusions In summary, our results demonstrated that difenoconazole led to a decrease in body weight in both sexes and the reduction in
Table 3 Expression difference of high-TCHO-correlated genes between male and female zebrafish in 500 mg/L group.
Gene
Correlation coefficient
Gene
Correlation coefficient
FEL/MELa (15d)
FEL/MEL (30d)
CYP51 DHCR7 DHCR24 CYP7A1
0.482** 0.401** 0.508** 0.293*
DHCR24 CYP51 HMGCRa DHCR7 CYP7A1
0.508 0.482 0.448 0.401 0.293
0.173/0.035 0.187/0.044 0.178/0.037 0.372/0.088 1.99/1.3
1.79/0.47 3.03/0.512 1.85/0.169 0.64/0.206 0.003/0.139
*P < 0.05 according to Spearman's test; **P < 0.01 according to Spearman's test.
a
FEL ¼ female expression level; MEL ¼ male expression level.
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males is stronger than females. The hepatic TCHO levels decreased in males but maintained normally in females, which might be attributed to the distinct transcription modification of cholesterolgenesis genes after difenoconazole exposure. Taken together these results, it seems that female zebrafish showed stronger tolerance to difenoconazole than male zebrafish. Since the effective dosage is close to the reported concentration in surface water, its impact on the TCHO level and sterol synthesis of aquatic organisms especially for male individuals should be highly considered. Acknowledgements This research work was supported by Special Fund for Agroscientific Research in the Public Interest (Project No. 20090305402). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.envpol.2014.11.019. References Belter, A., Skupinska, M., Giel-Pietraszuk, M., Grabarkiewicz, T., Rychlewski, L., Barciszewski, J., 2011. Squalene monooxygenase e a target for hypercholesterolemic therapy. Biol. Chem. 392, 1053e1075. Beppu, F., Hosokawa1, M., Niwano, Y., Miyashita, K., 2012. Effects of dietary fucoxanthin on cholesterol metabolism in diabetic/obese KK-Ay mice. Lipid. Health. Dis. 11, 112. Bromillow, R.H., Evans, A.A., Nicholls, P.H., 1999. Factors affecting degradation rates offive triazole fungicides in two soil types: 1. Laboratory incubations. Pest. Sci. 55, 1129e1134. Boyle, D., Al-Bairuty, G.A., Henry, T.B., Handy, R.D., 2013. Critical comparison of intravenous injection of TiO2 nanoparticles with waterborne and dietary exposures concludes minimal environmentally-relevant toxicity in juvenile rainbow trout Oncorhynchus mykiss. Environ. Pollut. 182, 70e79. Buchenauer, H., 1995. DMI-fungicidesdside effects on the plant and problems of resistance. In: Lyr, H. (Ed.), Modern Selective Fungicides. Gustav Fischer Verlag, New York, pp. 259e290. Cecchi, C., Rosati, F., Pensalfini, A., Formigli, L., Nosi, D., Liguri, G., Dichiara, F., Morello, M., Danza, G., Pieraccini, G., Peri, A., Serio, M., Stefani, M., 2008. Seladin-1/DHCR24 protects neuroblastoma cells against Ab toxicity by increasing membrane cholesterol content. J. Cell. Mol. Med. 12, 1990e2002. Cong, R., Jia, Y., Lia, R., Ni, Y., Yang, X., Sun, Q., Parvizi, N., Zhao, R., 2012. Maternal low-protein diet causes epigenetic deregulation of HMGCR and CYP7a1 in the liver of weaning piglets. J. Nutr. Biochem. 23, 1647e1654. De Fabiani, E., Caruso, D., Cavaleri, M., Kienle, M., Galli, G., 1996. Cholesta-5,7,9(11)trien-3b-ol found in plasma of patients with Smith-Lemli-Opitz syndrome indicates formation of sterol hydroperoxides. J. Lipid. Res. 37, 2280e2287. Dong, F., Li, J., Chankvetadze, B., Cheng, Y., Xu, J., Liu, X., Li, Y., Chen, X., Bertucci, C., Tedesco, D., Zanasi, R., Zheng, Y., 2013. Chiral triazole fungicide difenoconazole: absolute stereochemistry, stereoselective bioactivity, aquatic toxicity, and environmental Behavior in vegetables and soil. Environ. Sci. Tech. 47, 3386e3394. EFSA, 2011. Conclusion on the Peer Review of the Pesticide Risk Assessment of the Active Substance Difenoconazole. European Food Safety Authority, Parma, Italy. ndez, C., Martin, M., Go mez Coronado, D., Lasuncio n, M., 2005. Effects of distal Ferna cholesterol biosynthesis inhibitors on cell proliferation and cell cycle progression. J. Lipid. Res. 46, 920e929. Gaoua, W., Chevy, F., Roux, C., Wolf, C., 1999. Oxidized derivatives of 7dehydrocholesterol induce growth retardation in cultured rat embryos: a model for antenatal growth retardation in the Smith-Lemli-Opitz syndrome. J. Lipid. Res. 40, 456e463. Goetz, A.K., Dix, D.J., 2009. Toxicogenomic effects common to triazole antifungals and conserved between rats and humans. Toxicol. App. Pharmacol. 238, 80e89. lez-Doncel, M., García-Maurin ~ o, J.E., Segundo, L.S., Beltr Gonza an, E.M., Sastre, S., Torija, C.F., 2014. Embryonic exposure of medaka (Oryzias latipes) to propylparaben: effects on early development and post-hatching growth. Environ. Pollut. 184, 360e369. Hamada, M.S., Yin, Y., Ma, Z., 2011. Sensitivity to iprodione, difenoconazole and fludioxonil of Rhizoctonia cerealis isolates collected from wheat in China. Crop. Prot. 30, 1028e1033. Henry, M.J., Sisler, H.D., 1984. Effects of sterol biosynthesis-inhibiting (SBI) fungicides on cytochrome P-450 oxygenations in fungi. Pest. Biochem. Physiol. 22, 262e275. Hermsen, S.A.B., Pronk, T.E., Brandhof, E., Ven, L.T.M., Piersma, A.H., 2011. Chemical class-specific gene expression changes in the zebrafish embryo after exposure
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Sex specific response in cholesterol level in zebrafish (Danio rerio) after long-term exposure of difenoconazole Xiyan Mu, Kai Wang, Tingting Chai, Lizhen Zhu, Yang Yang, Jie Zhang, Sen Pang, Chengju Wang*, Xuefeng Li* College of Sciences, China Agricultural University, Beijing, People's Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 June 2014 Received in revised form 27 October 2014 Accepted 9 November 2014 Available online 5 December 2014
Difenoconazole is a widely used triazole fungicide, its extensive application may potentially cause toxic effects on non-target organisms. To investigate the effect of difenoconazole on cholesterol content and related mechanism, adult zebrafish were exposed to environmental related dosage (0.1, 10 and 500 mg/L) difenoconazole. The body weight and hepatic total cholesterol (TCHO) level was tested at 7, 15 and 30 days post exposure (dpe). The expressions of eight cholesterol synthesis genes and one cholesterol metabolism gene were assessed via Quantitative PCR method. The significant decrease of TCHO level in male zebrafish liver was observed at 15 and 30 dpe, which was accompanied by apparent hepatic cholesterol-genesis genes expression decline. In comparison with males, female zebrafish showed different transcription modification of tested genes, and the cholesterol content maintain normal level during the whole exposure. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Zebrafish Difenoconazole Cholesterol Sex differences
1. Introduction Triazole fungicides are widely used for the protection of a variety of plants. These compounds could enter the water environment through spray drift or surface run-off after rainfall (Raudonis et al., 2004; Konwick et al., 2006). Their characteristics, such as high chemical and photochemical stability, low biodegradability and easy transportation in the environment (Wang et al., 2011), make them persistent in water (Bromillow et al., 1999). Thus, the possible detrimental effects of triazole compounds on environmental organisms have caused widespread attention. Difenoconazole (cistrans-3-chloro-4-(4-methyl-2-(1H-1,2,4-triazol-yl methyl)-1,3dioxolan-2-yl) phenyl 4-chlorophenyl ether) is a typical triazole fungicide used for the control of fungal disease on vegetables, cereals and other field crops (Vawdrey et al., 2008; Horsfield et al., 2010). It inhibits fungal lanosterol-14a-demethylase (CYP51) activity and blocks ergosterol biosynthesis, thus resulting in the blocking of fungal cell wall chitin synthesis and the overspill of cytoplasm (Buchenauer et al., 1995; Hamada et al., 2011; Ragsdale, 1977). In China, difenoconazole has been used as the main pesticide to combat rice diseases for many years (Wang and Zhang, 2012).
* Corresponding authors. E-mail addresses: [email protected] (C. Wang), [email protected] (X. Li). http://dx.doi.org/10.1016/j.envpol.2014.11.019 0269-7491/© 2014 Elsevier Ltd. All rights reserved.
Since it is being used extensively in rice, difenoconazole has more opportunities to contaminate the water environment. In the recent decade, efforts have been concentrated on the environmental occurrence of difenoconazole and it has been detected in samples from several areas (Table 1). Compared with other triazole fungicides, difenoconazole is reported to possess higher acute toxicity to a wide range of aquatic organisms (Dong et al., 2013). According to the recent publication of the European Food Safety Authority (EFSA), difenoconazole is considered very toxic to aquatic organisms in view of its high toxicity to Daphnia magna (chronic NOEC ¼ 0.0056 mg active substance/L) (EFSA, 2011). In addition, our recent research also showed that difenoconazole has negative effect on zebrafish during both adult and early life stages (Mu et al., 2013). Therefore, it is necessary to perform environmental toxicological studies of difenoconazole to reflect its risk to aquatic organisms. The influence of lipid biosynthesis and metabolism has been considered as a main active pathway of triazole compounds (Hermsen et al., 2011; Goetz and Dix, 2009). Cholesterol, which is an important lipid in vivo, plays a key role in the maintenance of cell structure and is also a precursor for the biosynthesis of steroid hormones and bile acids (Beppu et al., 2012). The cholesterol biosynthesis mostly occurred in liver from acetyl-CoA and required multi-step enzymatic reaction (Kojima et al., 2009). From the view of bactericidal mechanism, difenoconazole has the ability to inhibit
X. Mu et al. / Environmental Pollution 197 (2015) 278e286 Table 1 The reported environmental difenoconazole dosage in water area. Area
Data source
Reported value (ug/L)
Reference
China (Changsha, Changchun, Hangzhou) Thailand (Salakru, Nong Sua) Malaysia (Kedah)
Paddy water (spraying day) PECa (agricultural water area) Surface water (river near field) Surface water (river)
1.98 e2.91 mg/L 0.028 mg/L
Zhang et al., 2011 Satapornvanit et al., 2004 Latiff et al., 2010
China (Fujian) Italy (River Meolo) Australia (Victoria) a
0.30 mg/L
You, 2008 0.0039 e0.0061 mg/L PEC (river) 0.0095 mg/L Verro et al., 2009 Sch€ afer et al., Surface water (river 0.15 mg/L 2011 and stream)
PEC ¼ Predicted Environmental Concentration.
the functionality of CYP51 (Henry and Sisler, 1984; Hitchcock et al., 1990), an important enzyme in the cholesterol synthesis (Miao et al., 2014), which may further result in the alteration of total cholesterol (TCHO) level. Fish are an important eco-indicator organism and many model species, such as rainbow trout (Oncorhynchus mykiss), medaka (Oryzias latipes) and zebrafish, have been applied in the toxicity evaluation of environmental pollutants (Boyle et al., 2013; Gonz alez-Doncel et al., 2014; Zhang et al., 2013). The zebrafish (Danio rerio) is a typical small tropical aquarium fish well known to most people. The reasons it has a long history of general use in toxicology research that it is inexpensive, hardy, small, easy to care for in large numbers and will readily spawn under appropriate photoperiod conditions (Lele and Krone, 1996). The main purpose of this study is to investigate the gender-based effects of difenoconazole on cholesterol level in zebrafish liver and gain an insight into the involved mechanisms. 2. Materials and methods 2.1. Zebrafish maintenance Wild type zebrafish (length 3.5 ± 0.6; weight 0.18 ± 0.05) were purchased from Beijing Hongdagaofeng Aquarium Department. All of adult zebrafish were maintained in the centralized breeding equipment (ESEN-ZF-SS, Esen, Beijing) at 26 C with a photoperiod of 14/10 (light/dark), and fed with dried brine shrimp (Artemia, Futian Brand, JPN) twice daily. The total daily fed amount was equivalent to 2% of fish weight. Adult zebrafish which were at the age of about six months were fasted for one day before exposure. 2.2. Chemicals and reagents The standard water was prepared in the lab with the formula of iso-7346-3, which contained 2 mmol/L Ca2þ, 0.5 mmol/L Mg2þ, 0.75 mmol/L Naþ and 0.074 mmol/L Kþ (ISO, 1996). 95% Difenoconazole (CAS: 119446-68-3) was obtained from China Ministry of Agriculture and the stock solution used for drug exposure was prepared with ethanol AR (analytical reagent). All of the other reagents utilized were of analytical grade. 2.3. Fish exposure and sample collection The 6-month-old zebrafish were separated by sex and randomly assigned to nominal concentrations of 0 (control), 0.1, 10, and 500 mg/L of difenoconazole for 30 days. Difenoconazole-free standard water was used as control which contained 0.025 mL/L
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ethanol. 960 (480 for both male and female) individuals were distributed in 48 tanks. Each tank contains 10 L exposure solution and 20 fish. The exposure solution was renewed daily to keep the appropriate concentration of drug and water quality. The tanks were renewed per week. The water qualities including temperature, PH and dissolved oxygen were measured every two days. The external conditions during exposure, including temperature, humidity and light cycle, were the same as the culture environment. During the test, fish were fed daily with dried brine shrimp (equivalent to 2% of body weight) except for the 24 h prior to sacrifice. At 7, 15 and 30 days post exposure (dpe), 12 zebrafish were randomly selected from each replicate and divided into two parts, 6 for TCHO analysis and 6 for RNA extraction (for specific information of sampling process please see Supplemental Materials). After anesthetized on ice, the wet weights of all collected individuals were measured using an electronic balance (made by Sartorius Corp.). Liver tissues of collected zebrafish were quickly excised, weighed, frozen in liquid nitrogen and stored at 80 C for RNA extraction and cholesterol content detection. 2.4. Difenoconazole in water analysis 2.4.1. Sample preparation The water samples were obtained at 0, 1, 7, 14, 28 dpe. 20 mL of water samples were extracted twice with an equal volume of ethyl acetate (10 mL each time). For samples with 10 mg/L and 500 mg/L concentration, 1.0 mL extract was completed with a nitrogen stream and then redissolved with 1 mL acetonitrile. For samples with 0.1 mg/L, 10 mL extract was evaporated completely to dry with a nitrogen stream and redissolved with 1 mL acetonitrile. Finally, all the redissolved samples were filtered through a 0.22-mm filter into a sample vial for LC-MS/MS analysis. 2.4.2. LC-MS/MS analysis An Agilent Technologies 6410 high-performance liquid chromatograph (Agilent, USA) was interfaced to a triple quadrupole mass spectrometer (Agilent, USA) equipped with an ESI source. The chromatographic separation was carried out on ZORBAX SB-C18 column (50 mm 2.1 mm, 1.8 mm; Agilent, USA) at 25 C with a flow rate of 0.3 mL/min. The mobile phase in isocratic mode consisted of acetonitrile and water (0.1% HCOOH) (70:30). For the MS/MS analysis, the common parameters were as follows: the capillary current and nebulising gas were set as 9 nA and 35.0 psi, respectively. A flow rate of drying gas was 8.00 L/min with temperature of 350 C. The compound parameters including collision energy and fragmentor voltage were 20 eV and 130 V, respectively. The transitions of precursor ion (m/z 406.1) to product ion (m/z 251.0 and m/z 337.0) of difenoconazole were detected with the multiple reaction monitoring (MRM) in positive ion (ESIþ) mode. The sample injection volume was 5 mL. The retention time of difenoconazole was 0.8 min (Fig. S1). 2.5. Total cholesterol (TCHO) content in liver analysis For determination of TCHO content in the liver, liver from 6 males or females of each replicate were homogenized (1:10, w/v) via electric homogenizer (Tiangen Biotech) in cell lysates (made by Applygen). 10 mL of the supernatant was obtained for protein detection after the solution was centrifuged at 3000 g for 10 min at 4 C. The remaining sample was incubated for 10 min in 70 C water to fully dissolve cholesterol. After centrifuged at 3000 g for 10 min at room temperature, the supernatant was used for cholesterol analysis. The TCHO content determined using enzymatic kits according to the manufacturer's directions (Applygen, Beijing, China).
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Total protein content was determined using BCA (bicinchoninic acid) protein assay kit (Cwbiotech, Beijing, China).
in order to further confirm the relationship between TCHO level and gene expression level.
2.6. Gene expression analyses
3. Results
To further investigate the effect of difenoconazole on cholesterol synthesis and metabolism, the mRNA levels of 9 related genes, including CYP51, farnesyl-diphosphate farnesyltransferase 1 (FDFT1), isopentenyl-diphosphate delta isomerase 1 (IDI1), squalene epoxidase (SQLE), 7-dehydrocholesterol reductase (DHCR7), 24-dehydrocholesterol reductase (DHCR24), 3-hydroxy-3methylglutaryl-CoA reductase (HMGCR)a, HMGCRb and cholesterol 7a-hydroxylase (CYP7A1) were measured by quantitative real-time PCR. Total RNA was extracted from zebrafish liver using spin column adsorption method following manufacturer's protocols (Tiangen Biotech, Beijing, China). The concentration was measured by absorbance at 260 nm using a UV1240 spectrophotometer (Perkin Elmer, USA). The purity was assessed by determining the A260/ A280 ratio. The cDNA was then synthesized via reverse transcription (RT) using quant RTase kit (Tiangen Biotech, Beijing, China) in accordance with manufacturer's recommendations. Real-time PCR reactions were performed with the ABI 7500 q-PCR system (Applied Biosystems, Foster City, CA). The SYBR Green PCR Master Mix reagent kits (Tiangen Biotech, Beijing, China) were used for quantification of gene expression. A 20 mL reaction system was conducted according to manufacturer's instructions (See Supplemental Materials). Zebrafish-specific primers were designed for the genes of interest using Primer 6.0 software (Table S1). The housekeeping gene actin, beta (ACTB) was used as an internal control. The PCR amplification procedure was as follows: 95 C for 15 min followed by 40 cycles of 94 C for 10 s, 60 C for 20 s, and 72 C for 32 s. Quantification of the transcripts was performed using the 2DDCt method.
3.1. Chemical analysis
2.7. Statistical analysis All statistical analyses were undertaken using Spss 16.0 software. Differences were determined by one-way ANOVA, completed with Dunnett post hoc comparison. P < 0.05 was considered significant. Relationships between TCHO level and tested genes transcription were analysed by principal component analysis (PCA). In addition, correlation analysis was completed with Spearman's test
Analysis results indicated that the deviations between nominal and actual difenoconazole concentrations were less than 20% in all test period (Table S2). Therefore, the nominal dosage is able to represent the actual content in this research. 3.2. Body weight To investigate the adverse effects of difenoconazole to zebrafish, some morphological endpoints were assessed such as weight and body size. Compared to their respective control groups, the body weight of both sexes significantly decreased in 500 mg/L treatment at 30 dpe (Fig. 1). For female zebrafish, the average weight of 500 mg/L group is equivalent to 89% of control at 30 dpe. While the decrease level of male is much stronger than female, the average weight of 500 mg/L group is only equivalent to 66% of control. In addition, from the body appearance, male zebrafish treated with 500 mg/L difenoconazole showed obviously skinny body at 30 dpe while zebrafish in control were normal (Fig. S2). 3.3. TCHO level in liver For both male and female zebrafish, no apparent change of hepatic TCHO level was observed in all treated groups at 7 dpe (Fig. 2). At 15 dpe, the TCHO level of male zebrafish in 500 mg/L group decreased significantly and the value was equivalent to 71% of control. The reduction became stronger at 30 dpe, the TCHO level of male zebrafish in 500 mg/L group reduced to only 30% of control. While for female zebrafish, no obvious modification was found throughout the exposure. 3.4. Expression of genes related in cholesterol synthesis According to the results, the expression of IDI1, SQLE, DHCR24 and HMGCRb in male zebrafish liver showed significant upregulation at 7 dpe, while HMGCRa showed down-regulation at the same period (Fig. 3). At 15 dpe, expression of all eight tested
Fig. 1. Body weight of male and female zebrafish during the exposure. Asterisks denote significant difference between treatments and control (determined by Dunnett post-hoc comparison, p < 0.05*; p < 0.01**). Error bars indicate standard deviation.
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Fig. 2. Hepatic TCHO level of male and female zebrafish during the exposure. Asterisks denote significant difference between treatments and control (determined by Dunnett posthoc comparison, p < 0.05*; p < 0.01**). Error bars indicate standard deviation.
Fig. 3. Relative expressions of 8 genes related to CHO-synthesis in male zebrafish liver during the difenoconazole exposure. The relative expression level of control was designated as 1. Asterisks denote significant difference between treatments and control: red represents increase; green represents decrease (determined by Dunnett post-hoc comparison, p < 0.05*; p < 0.01**). Error bars indicate standard deviation. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 4. Relative expressions of 8 genes related to CHO-synthesis in female zebrafish liver during the difenoconazole exposure. The relative expression level of control was designated as 1. Asterisks denote significant difference between treatments and control: red represents increase; green represents decrease (determined by Dunnett post-hoc comparison, p < 0.05*; p < 0.01**). Error bars indicate standard deviation. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
genes were markedly decreased in a concentration-dependent manner, and among them, the hepatic expression of CYP51, FDFT1, DHCR7, DHCR24, SQLE and HMGCRa in male zebrafish of 500 mg/L exposure group was less than 10% of control. The most affected gene during this period was SQLE which was reduced to about 1% of control (500 mg/L exposure group). At 30 dpe, the decreased expression level of tested genes still existed, but the degree was weaker than the down-regulation at 15 dpe. Different trend of gene expression changes were found in female zebrafish liver compared with males. The expression of all cholesterol synthesis related genes were inhibited by difenoconazole at 7 dpe (Fig. 4). However, at 15 dpe, only 500 mg/L treatment group performed down-regulation of cholesterol-genesis genes, and the expression of all tested genes returned to normal level in zebrafish exposed to 10 mg/L difenoconazole. In the 0.1 mg/L exposure group, up-regulation of all tested genes were observed. At 30 dpe, only DHCR7 and IDI1 decreased in higher concentration exposure group, expression of all other tested genes showed normal or increased level in all treatments.
3.5. Expression of gene related in cholesterol metabolism The expression level of CYP7A1 significantly decreased in male zebrafish liver at 7 and 30 dpe. There was a dose-dependent down regulation of CYP7A1 at 30 dpe, the relative expression level were 0.53 (0.1 mg/L), 0.36 (10 mg/L) and 0.14 (500 mg/L). In addition, 0.1 mg/ L difenoconazole caused up-regulation of CYP7A1 at 15 dpe, the relative expression level was 7.7 (Fig. 5). For female zebrafish, hepatic expression of CYP7A1 was up-regulated by 0.1 and 10 mg/L difenoconazole at 7 dpe, the relative expression level was 9.4 and 3.8 respectively. At 15 dpe, the up-regulation continued, the expression levels were 4.2 (0.1 mg/L) and 2.0 (10 mg/L) fold of control group. While at 30 dpe, the expression apparently decreased in all treatments, especially for zebrafish in 500 mg/L group which was reduced to less than 1% of control. 3.6. Chemometrics To understand the relationship between the expression change of tested genes and the TCHO level in zebrafish liver, correlation
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Fig. 5. Relative expression of CYP7A1 in male and female zebrafish liver during the difenoconazole exposure. The relative expression level of control was designated as 1. Asterisks denote significant difference between treatments and control: red represents increase; green represents decrease (determined by Dunnett post-hoc comparison, p < 0.05*; p < 0.01**). Error bars indicate standard deviation. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
analysis and principal component analysis were finished using the data of all zebrafish treatments. All parameters measured in the present study were distinguished on the loading plots corresponding to the first (51.75%) and second (21.99%) components (Fig. 6). According to the PCA results, expression of CYP51, DHCR7, DHCR24 and HMGCRa showed relative high correlation with TCHO level, and the expression level of CYP7A1 showed negative correlation with TCHO level. The correlation analysis results further confirmed the relationship among the tested parameters (Table 2). 4. Discussion The main purpose of this research is to investigate the alteration of cholesterol level in different gender of zebrafish caused by difenoconazole and the related mechanisms. Our results clearly indicated that 500 mg/L difenoconazole could cause significant decrease of hepatic TCHO level in male zebrafish at 15 and 30 dpe. For female zebrafish, no obvious modification of TCHO level was observed during the whole exposure period. In addition, although both male and female zebrafish performed decreased body weight after difenoconazole exposure, the reduction of male was much stronger than female. These results implicated that female zebrafish showed more resistance to difenoconazole exposure than males. Our previous study showed that difenoconazole could cause negative effect to zebrafish including developmental abnormities for embryo as well as growth inhibition for adult, and the effective concentration was very close to its reported value in water adjacent to fields, which reflected the threat of difenoconazole to aquatic organisms living in agricultural areas (Mu et al., 2013). In this study, apparent down-regulation of cholesterol synthesis genes was found in male zebrafish liver exposed to 0.1 mg/L or higher difenoconazole and the effective dosage is very close to the detected value in surface water sample (Table 1), which indicated that its occurrence may have the potential to alter cholesterol generation of aquatic organisms in non-agricultural water environment. HMGCR is the rate-limiting enzyme in the cholesterol biosynthesis pathway (Sato and Kamada, 2011), which plays a key role in the hepatic cholesterol synthesis. According to recent research, the cholesterol level decrease caused by berberine in hyperhomocysteinemic rat liver is highly related to the inhibition of hepatic HMGCR activity (Wu et al., 2011). CYP51 catalyse the demethylation of lanosterol in the cholesterogenic process which is
also the target of triazole compounds. Previous research documented that cholesterol level in plasma decrease associated with inhibition of hepatic expression of CYP51 and HMGCR in propiconazole treated fathead minnow (Skolness et al., 2013). In this work, similar with the results of Skolness et al. (2013), the reduction of gene expression of CYP51 and HMGCRa, were observed with the decreased TCHO level in male zebrafish liver. SQLE and FDFT1 are downstream to HMGCR in the cholesterol synthesis pathway, and both the two enzymes are considered as potential target of cholesterol lowering agents (Trapani et al., 2011). It has been reported that SQLE down-regulation in vertebrates and fungi decreases synthesis of cholesterol and ergosterol, respectively (Belter et al., 2011). In the present study, expression decrease of SQLE and FDFT1 was exhibited along with the TCHO level decline in male zebrafish liver of 500 mg/L group. DHCR7 is the enzyme that catalyses the reduction of 7dehydrocholesterol to cholesterol in the last step of cholesterol biosynthesis (Horling et al., 2012). DHCR24 is the enzyme catalyses the cholesterol biosynthesis by reducing the delta-24 double bond of desmosterol (Cecchi et al., 2008). Both of the two enzymes are key factors contributing to the hydrogenation of dehydrocholesterol. Previous studies demonstrated that the lack of 7DHCR leads to a deficit in cholesterol and an accumulation of 7dehydrocholesterol, which together with its oxidized derivatives also have negative influences on total cholesterol transfer (De ndez et al., 2005). In Fabiani et al., 1996; Gaoua et al., 1999; Ferna the present study, genes encoded DHCR7 and DHCR24 were significantly down-regulated in male at 15 and 30 dpe after difenoconazole exposure. Previous study suggested that lower constitutive hepatic expression levels of the genes for cholesterol biosynthetic enzymes could give rise to the lower serum T-CHO levels in rats (Nemoto et al., 2013). In this study, hepatic TCHO level decrease occurred along with expression decline of cholesterol-synthesis genes in male zebrafish exposed to difenoconazole. The statistical results (Table 2) also indicated that the transcription of CYP51, HMGCRa, DHCR7 and DHCR24 showed significant correlation with TCHO level. Therefore, the hepatic TCHO content decline of male zebrafish in this test is possibly due to the generally down-regulation of cholesterol synthesis genes. Bile acid synthesis is the major pathway for the metabolism of cholesterol. CYP7A1 is the rate-limiting enzyme for the
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Fig. 6. Loading diagram of PCA analysis of THO level and related genes expression in zebrafish liver after exposure to difenoconazole.
predominant pathway of bile acid biosynthesis (Qi et al., 2015). In the present study, down-regulation of hepatic CYP7A1 was observed in both male and female zebrafish at 30 dpe, the relative expression level was 0.139 and 0.003 respectively. For female individuals, such sharply decreased transcription of CYP7A1 may result in the lack of bile acids (Russell, 1999). Previous research proved that mice with insufficient bile acid are more susceptible to liver injury and exhibit defective liver regeneration (Meng et al., 2011). Therefore, further research could concern on the effect of difenoconazole on liver regeneration and repair. A recent study has proven that difenoconazole has the ability to inhibit zebrafish growth (Mu et al., 2013). In the present study, male zebrafish performed stronger sensitivity to difenoconazole than female in the endpoint of body weight reduction. Skolness et al. (2013) also found that there is a gender difference in the body weight modification of propiconazole treated fathead minnow. Recent studies showed that weight decrease frequently occurred with TCHO level reduction, Nam et al. (2014) reported that Gardenia jasminoides Ellis extract could cause body weight reduction and hepatic TCHO content decrease. Cong et al. (2012) also found that lower serum cholesterol is accompanied by lower body weight and slower growth rate in pigs. In this work, the difenoconazole treated male zebrafish showed both TCHO reduction and body weight decrease. However for female zebrafish, body weight decrease emerged with normal hepatic TCHO level. Therefore, the origin of
Table 2 Correlation coefficient between death rate and expression level of all tested genes. Gene HMGCRa HMGCRb IDI1 FDFT1 SQLE a
Correlation coefficient **a
0.448 0.167 0.167 0.176 0.209
the difenoconazole caused body weight decrease and its interaction mechanism with TCHO decline still need to be studied. Sex-based response difference of toxicants has been observed in aquatic organisms during recent years. Zhang et al. (2012) reported that after chronic perfluorononanoic acid exposure, triglyceride (TG) levels increased in male zebrafish while decreased in females and the transcriptional expression levels of fatty acid binding proteins (FABPs) were up-regulated in males and down-regulated in females except FABP1b. Li et al. (2009) showed that male Chinese rare minnow was more sensitive to amitrole (a triazole herbicide) than female according to the expression change of thyroid hormone related genes post exposure. According to Table 3, the relative hepatic expression levels of the four high-TCHO-correlated synthesis genes in female zebrafish were higher than those in male zebrafish. At the same time, CYP7A1 which is the main metabolism gene of cholesterol showed higher transcription in male zebrafish liver at 30 dpe, compared to female individuals. Therefore, the different response of TCHO level towards difenoconazole between male and female zebrafish may due to the distinct regulation of key genes related in cholesterol synthesis and metabolism. 5. Conclusions In summary, our results demonstrated that difenoconazole led to a decrease in body weight in both sexes and the reduction in
Table 3 Expression difference of high-TCHO-correlated genes between male and female zebrafish in 500 mg/L group.
Gene
Correlation coefficient
Gene
Correlation coefficient
FEL/MELa (15d)
FEL/MEL (30d)
CYP51 DHCR7 DHCR24 CYP7A1
0.482** 0.401** 0.508** 0.293*
DHCR24 CYP51 HMGCRa DHCR7 CYP7A1
0.508 0.482 0.448 0.401 0.293
0.173/0.035 0.187/0.044 0.178/0.037 0.372/0.088 1.99/1.3
1.79/0.47 3.03/0.512 1.85/0.169 0.64/0.206 0.003/0.139
*P < 0.05 according to Spearman's test; **P < 0.01 according to Spearman's test.
a
FEL ¼ female expression level; MEL ¼ male expression level.
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