Transcriptional changes induced by in vivo exposure to pentachlorophenol (PCP) in Chironomus riparius (Diptera) aquatic larvae

Aquatic Toxicology 157 (2014) 1–9

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Transcriptional changes induced by in vivo exposure to pentachlorophenol (PCP) in Chironomus riparius (Diptera) aquatic larvae Mónica Morales ∗ , Pedro Martínez-Paz, Raquel Martín, Rosario Planelló, Josune Urien, José Luis Martínez-Guitarte, Gloria Morcillo Grupo de Biología y Toxicología Ambiental, Facultad de Ciencias, Universidad Nacional de Educación a Distancia, UNED, Senda del Rey 9, Madrid 28040, Spain

a r t i c l e

i n f o

Article history: Received 2 July 2014 Received in revised form 22 September 2014 Accepted 23 September 2014 Available online 2 October 2014 Keywords: Organochlorines Endocrine disruptors Ecdysone-responsive genes Hsp genes CYP gene GST enzyme

a b s t r a c t Pentachlorophenol (PCP) has been extensively used worldwide as a pesticide and biocide and is frequently detected in the aquatic environment. In the present work, the toxicity of PCP was investigated in Chironomus riparius aquatic larvae. The effects following short- and long-term exposures were evaluated at the molecular level by analyzing changes in the transcriptional profile of different endocrine genes, as well as in genes involved in the stress response and detoxification. Interestingly, although no differences were found after 12- and 24-h treatments, at 96-h exposures PCP was able to induce significant increases in transcripts from the ecdysone receptor gene (EcR), the early ecdysone-inducible E74 gene, the estrogenrelated receptor gene (ERR), the Hsp70 gene and the CYP4G gene. In contrast, the Hsp27 gene appeared to be downregulated, while the ultraspiracle gene (usp) (insect ortholog of the retinoid X receptor) was not altered in any of the conditions assayed. Moreover, Glutathione-S-Transferase (GST) activity was not affected. The results obtained show the ability of PCP to modulate transcription of different biomarker genes from important cellular metabolic activities, which could be useful in genomic approaches to monitoring. In particular, the significant upregulation of hormonal genes represents the first evidence at the genomic level of the potential endocrine disruptive effects of PCP on aquatic invertebrates. © 2014 Published by Elsevier B.V.

1. Introduction Pentachlorophenol (PCP) is an organochlorine compound used worldwide, mainly as a pesticide and wood preservative. It is a widespread, ubiquitous environmental contaminant (Geyer et al., 1987; WHO, 1987; Muir and Eduljee, 1999; Heudorf et al., 2000; ˜ et al., 2013). The Ge et al., 2007; Zheng et al., 2011, 2012; Montano use of this xenobiotic is restricted in Europe and the USA, and the United States Environmental Protection Agency (US-EPA) has listed it as a priority pollutant due to its slow and incomplete biodegradation (Gupta et al., 2002; Hanna et al., 2004). This Persistent Organic Pollutant (POP) is stable in the aquatic environment and sediments have shown the highest PCP concentrations (Zheng et al., 2012; Li et al., 2013), which have been related to its long half-life with almost no degradation in sediments (WHO, 1987). PCP is highly

∗ Corresponding author. Tel.: +34 913988123; fax: +34 913986697. E-mail address: [email protected] (M. Morales). http://dx.doi.org/10.1016/j.aquatox.2014.09.009 0166-445X/© 2014 Published by Elsevier B.V.

toxic for most organisms; the primary mechanisms of toxic action proposed are the uncoupling of substrate oxidation from ATP in mitochondria and the provoking of oxidative stress in cells and tissues (Xu et al., 2014a). In humans, this compound affects the immune and endocrine systems, kidneys, lungs and liver; neurotoxicity and carcinogenicity have also been suggested (Proudfoot, 2003; Hurd et al., 2012). Various studies have also reported that it induces the following: production of Reactive Oxygen Species (ROS); lipid peroxidation (Dong et al., 2009); cell morphological alterations (Chen et al., 2004a; Yang et al., 2005); mitochondrial dysfunction and lysosomal membrane damage (Fernández Freire et al., 2005); reproduction (Zha et al., 2006); and cell death (Wang et al., 2001; Wispriyono et al., 2002; Chen et al., 2004b; Yang et al., 2005; Dong et al., 2009). Furthermore, PCP inhibited oocyte maturation in zebrafish (Tokumoto et al., 2005) and decreased the production of steroid hormones and the downregulation of steroidogenic gene expression in human cell culture (Ma et al., 2011). In arthropods, PCP teratogenesis has been demonstrated by observing the deformation of the mentum in Chironomus plumosus

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M. Morales et al. / Aquatic Toxicology 157 (2014) 1–9

larvae (Song, 2007). More recently, a significant increase in DNA damage has also been observed when C. riparius were exposed to PCP (Martínez-Paz et al., 2013). The potential adverse effects of Endocrine Disrupting Chemicals (EDCs), those xenobiotics able to interact with hormones altering the endocrine regulation of essential physiological functions, have become a major research interest. The impact of PCP on the endocrine system has received much attention in recent years. Although there is still limited information, it has been found that PCP might disrupt the thyroid endocrine system, and using in vitro and in vivo assays in Xenopus laevis, it has been shown to have T3antagonist activity (Sugiyama et al., 2005). Similar results have been obtained using the in vitro assay with rat pituitary GH3 cells and in vivo assays with zebrafish embryos (Guo and Zhou, 2013). Furthermore, PCP alters plasma thyroid hormone levels, as well as the expression of genes associated with thyroid hormone signaling and metabolism in the hypothalamic–pituitary–thyroid (HPT) axis and liver in zebrafish (Yu et al., 2014). Recent studies have shown that the vitellogenin mRNA expression increased when female rare minnows (Gobiocypris rarus) were exposed to PCP (Zhang et al., 2014). In humans, a negative association has been reported between maternal plasma PCP levels and cord plasma free T4 concentrations in neonates (Dallaire et al., 2009). The potential disruption caused by PCP to the thyroid endocrine system has raised great concern over its adverse environmental health risks. However, currently there is little information available about its interaction with endocrine systems in invertebrates, and little is known about its mechanism of action. To date, only a few studies have been devoted to analyzing the effects of PCP on aquatic invertebrates. The aquatic larvae of the midge Chironomus riparius are widely used as a test organism for the assessment of aquatic toxicology (US-EPA, 2000; OECD, 2013). The study of the taxonomic composition of chironomid larvae and the percentage of deformities in mouthparts, mainly in the mentum, are used in biomonitoring programs to obtain information about the levels of organic and chemical pollution of aquatic ecosystems (Martinez et al., 2003). Moreover, they are being increasingly employed for toxicity testing using molecular endpoints. In recent years, some genes have been described as biomarkers for different aquatic contaminants, including among others, those for heat shock proteins (HSPs), ribosomal proteins, nuclear receptors and cytochrome P450 genes ˜ et al., 2007; Martínez-Guitarte et al., 2007; Planelló et al., (Londono 2007, 2008, 2010, 2011; Park and Kwak, 2008, 2009, 2010; Park et al., 2009; Nair et al., 2011, 2013; Morales et al., 2011, 2013; Martínez-Paz et al., 2012, 2014). The aim of the present study was to investigate the toxicity and molecular effects of PCP in C. riparius, focusing on the expression of different endocrine-related genes, particularly receptor genes and transcription factors acting in the intracellular signaling of steroids in larvae. We analyzed the impact of PCP on the activity of the EcR, usp and ERR genes, codifying for three hormonal receptors. The estrogen-related receptor (ERR), the ecdysone receptor (EcR) and the ultraspiracle (USP; ortholog of the retinoid X receptor) of the invertebrate ecdysozoans belong to the nuclear receptor super family (Köhler et al., 2007), and the EcR and USP appear to be regulated by ecdysone pulses during insect development and metamorphosis (Rauch et al., 1998; Gilbert and Warren, 2005). Analysis was also undertaken for an early ecdysoneresponsive gene (E74), coding for a transcription factor that in turn is regulated by the ecdysone receptor. In addition, the effects were also examined on genes related to the stress response, such as the Hsp70 and Hsp27 genes, and on some parameters related with the detoxification routes, such as the CYP4G gene and Glutathione-STransferase (GST) activity. This study offers the first evidence of the genomic effects of PCP on the endocrine pathway in aquatic invertebrates, which would be of interest for further selection of gene biomarkers for environmental risk assessment.

2. Material and methods 2.1. Animals and treatments The experimental animals were fourth instar larvae from the midge Chironomus riparius. They were originally collected from natural populations in a non-polluted area of Valencia (Spain), and reared under standard laboratory conditions for several generations according to toxicity testing guidelines (US-EPA, 2000; OECD, 2013). Larvae were grown in culture medium (0.5 mM CaCl2 , 1 mM NaCl, 1 mM MgSO4 , 0.1 mM NaHCO3 , 0.025 mM KH2 PO4 , 0.01 mM FeCl3 ) supplemented with nettle leaves, commercial fish food (TetraMin) and cellulose tissue. Cultures were maintained under constant aeration at 20 ◦ C and under standard light-dark periods 16:8. For experimental treatments, larvae were exposed to the chemical diluted in culture medium for 12, 24 and 96 h with constant aeration at 20 ◦ C. In 96-h exposures, culture medium was renewed every 24 h and supplemented with 3 mg of commercial fish food at 48 h. Concentrations were chosen based on former experiments under similar exposure scenarios in Chironomus or related species. Fourth instar larvae (n = 30 per replica) were submitted to 25, 250, 1000, 1500, 2000 and 2500 ␮g/L of pentachlorophenol (PCP) (Aldrich). Each treatment consisted of three independent experiments performed in each analysis using samples from three different egg masses. The control larvae used in each case were exposed to the same concentration of solvent (ethanol 0.001%) as the corresponding treatment and were also measured in triplicate. Larvae exposed to 25 and 250 ␮g/L (94 and 940 nM, respectively) of PCP and untreated larvae were stored at −80 ◦ C until RNA and protein isolation was carried out. 2.2. Toxicity test Observations on the survival of the larvae were made after 24, 48, 72 and 96 hours, with death of individuals as an endpoint. Larvae were considered dead when they did not move in response to being probed with forceps. 2.3. RNA isolation Total RNA was extracted from control and exposed fourth instar larvae using a guanidine isothiocyanate-based method, performed with a commercial kit (TRIZOL, Invitrogen) according to the manufacturer’s protocol. Subsequently, RNA was treated with RNase-free DNase (Roche), followed by phenolization. The quality and quantity of total RNA were determined by agarose electrophoresis and absorbance spectrophotometry (Biophotomer Eppendorf). Finally, purified RNA was stored at −80 ◦ C. 2.4. Real time RT-PCR Reverse transcription was performed with 0.5 ␮g of the isolated RNA and 0.5 ␮g oligonucleotide dT20 primer (Sigma) was used with 100 units of M-MLV enzyme (Invitrogen), in a final volume of 20 ␮L. A total of 25 ng of cDNA was obtained and used as a template for the Polymerase Chain Reaction (PCR). Quantitative real-time PCR (qRT-PCR) was used to evaluate the mRNA expression profile of the EcR, usp, E74, ERR, Hsp70, Hsp27 and CYP4G genes in control and under PCP exposures. The qRT-PCR was performed with a CFX96 thermocycler (Bio-Rad) and SsoFast EvaGreen Supermix (Bio-Rad). The qRT-PCR was run in the following cycling conditions: initial denaturation at 95 ◦ C for 3 min and 35 cycles of 95 ◦ C denaturation for 5 s; 58 ◦ C annealing for 15 s; and 65 ◦ C elongation for 10 s. C. riparius does not have the relevant genetic sequence information in databases and the typically endogenous reference genes used in this species are actin-ˇ, Glyceraldehyde 3-phosphate

M. Morales et al. / Aquatic Toxicology 157 (2014) 1–9

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Table 1 Primers used for real time RT-PCR of genes from C. riparius. Gene

Oligo name

Amplification efficiency

Amplicon length

Primer DNA sequence

GAPDH

GAPDH F GAPDH R L13 F L13 R EcRrt F EcRrt R USP F USP R E74 F E74 R ERR F ERR R Hsp70 rt F Hsp70 rt R Hsp27 rt F Hsp27 rt R CYP4G F CYP4G R

96.6%

110 bp

109.3%

351 bp

106.6%

180 bp

108.1%

114 bp

103.2%

111 bp

104.7%

222 bp

103.8%

132 bp

109.3%

202 bp

106.0%

174 bp

5´ı-GGTATTTCATTGAATGATCACTTTG-3´ı 5´ı-TAATCCTTGGATTGCATGTACTTG-3´ı 5´ı-AAGCTGCTTTCCCAAGAC-3´ı 5´ı-TTGGCATAATTGGTCCAG-3´ı 5´ı-CCATCGTCATCTTCTCAG-3´ı 5´ı-TGCCCATTGTTCGTAG-3´ı 5´ı-GCCCAATCATCCGTTAAGTGG-3´ı 5´ı-CGTTTGAAGAATCCTTTACATCC-3´ı 5´ı-TCTTACTGAAACTTCTTCAAGATCG-3´ı 5´ı-GCTTTGAGACAGCTTTGGAATCG-3´ı 5´ı-CTCAGCAAGTAAGGAGGAG-3´ı 5´ı-CGTCTAATAATGTGATCGG-3´ı 5´ı-ACTTGAACCAGTTGAGCGT-3´ı 5´ı-TTGCCACAGAAGAAATCTTG-3´ı 5´ı-TCAACACACAGGACCG-3´ı 5´ı-ATCCTTTATTGGTGATTAATTATG-3´ı 5´ı- TTGCATTGTGCATTTTAGGATGTC-3´ı 5´ı- TAAGTGGAACTGGTGGGTACAT-3´ı

rpL13 EcR usp E74 ERR Hsp70 Hsp27 CYP4G

dehydrogenase (GAPDH) and ribosomal protein L13 (rpL13) genes (Martínez-Guitarte et al., 2007, 2012; Martínez-Paz et al., 2012, 2014; Morales et al., 2013; Nair et al., 2011, 2013; Ozáez et al., 2014; Park et al., 2009). In this study, we used GAPDH and rpL13 as reference genes because they presented a coefficient of variation < 0.25 and an M-value < 0.5 (Hellemans et al., 2007). Primers for EcR, usp, E74, ERR, Hsp70, Hsp27 and CYP4G genes were the same as those used previously by our group (Martínez-Paz et al., 2012; Morales et al., 2013; and Martínez-Paz et al., 2014) (Table 1). PCR amplification efficiency was established by means of calibration curves. For each gene, a standard curve based on five dilutions from an equimolar mix of cDNA samples was produced in triplicate to verify amplification efficiency (Table 1). The acceptable range for PCR efficiencies calculated using standard curve serial dilution experiments is 90–110% (Pfaffl, 2004; Galeano et al., 2014; Lüchmann et al., 2014). To verify the amplification of only one fragment of DNA, a melting curve analysis was performed after amplification. Each sample was run in duplicate wells and three independent PCR replicates were used for each experiment. Cycle threshold (Ct ) values were converted to relative gene expression levels by the 2−Ct method using Bio-Rad CFX Manager 3.1 software for the gene expression analysis.

activity was assessed spectrophotometrically at 340 nm with the kit GST (Sigma) with the Multidetection Microplate Reader FLUOstar Omega (BMG labtech). 2.6. Statistical analysis EcR, usp, E74, ERR, Hsp70, Hsp27 and CYP4G mRNA levels in each sample were normalized against the expression of GAPDH and rpL13 mRNA in the same samples based on standard curves. The normalized levels of the specific gene transcripts and GST enzyme activity in treated groups were compared to those of the non-exposed controls. Variance homogeneity of data was assessed with Levene tests. For this, the analysis of variance (ANOVA) test was used combined with Dunnett’s multiple comparison tests using SPSS 19 (IBM). Two levels of significance are reported: p ≤ 0.05 (*), p ≤ 0.01 (**). Logprobit transformation of the data was used to estimate the values for LC20, LC50, LC80 and LC95 as well as their 95% confidence intervals. A Chi-squared test for homogeneity was also used to compare control, 25 and 250 ␮g/L survival data. 3. Results 3.1. In vivo PCP exposures and larval survival

2.5. Glutathione-S-Transferase activity To evaluate Glutathione-S-Transferase (GST) activity, five control larvae and five treated larvae were collected at 12, 24 and 96 h after treatment with PCP in each replicate. The larvae were homogenized in 0.5 mL of Tris-EDTA buffer (40 mM Tris, 1 mM EDTA, pH 7.8) with a pellet mixer (VWR). Crude homogenate was then centrifuged for 15 min at 200 g at 4 ◦ C. Subsequently, the supernatant was centrifuged for 30 min at 16,000 g at 4 ◦ C. The resulting supernatant was used to evaluate enzymatic activity. Total protein was quantified with BCA Protein Assay Reagent (Thermo Scientific) and 25 ␮g of total protein was used for the enzymatic assay. The GST

The values were estimated for LC20, LC50, LC80 and LC95 as well as the confidence intervals at 24, 48, 72 and 96 h (Table 2). Those for LC50 and LC95 at 96 h were 1427 and 2210 ␮g/L respectively, indicating a considerable potential for acute toxicity, whereas the survival of the larvae exposed to 25 ␮g/L and 250 ␮g/L PCP for 96 h did not differ significantly from that of the control group after the same period of time (data not shown). These PCP concentrations were selected for further treatments to analyze possible alterations in gene expression profiles of selected genes and GST activity, with the aim of identifying potential biomarker genes and sensitive genes related to the endocrine system.

Table 2 Estimations and 95% confidence intervals of the 24-, 48-, 72- and 96-h lethal concentrations (LC) of PCP (␮g/L) for C. riparius. 24 h

LC20 LC50 LC80 LC95

48 h

72 h

96 h

LCx

95% CI

LCx

95% CI

LCx

95% CI

LCx

95% CI

1514.6 1889.6 2264.6 2622.5

333.3–1806.6 1449.4–2268.4 1980.1–3315.7 2251.7–4550.1

1342.3 1748.3 2154.4 2542.0

805.5–1587.0 1477.4–1999.0 1918.7–2641.5 2224.9–3369.7

1140.2 1635.8 2131.4 2604.3

95.6–1476.7 1181.2–1942.6 1840.0–2835.2 2211.4–3944.5

1016.8 1420.7 1824.7 2210.1

334.0–1289.3 1071.1–1641.4 1604.7–2196.9 1934.1–2906.9

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3.2. Short-term effects of PCP on the expression of genes involved in the endocrine pathway, stress response and detoxification mechanism Initially, the effect of PCP in C. riparius larvae was analyzed at an acute and sublethal dose (250 ␮g/L) during short exposures (12 and

1.5

1

0.5

0

Relative mRNA expression

C

2

Relative mRNA expression

Control

12 hours

2

1.5

1

0.5

0

24 hours

usp gene

Control

12 hours

24 hours

Hsp70 gene

1.5

1

0.5

0

D

B

EcR gene

Relative mRNA expression

2

2

Control

12 hours

24 hours

CYP4G gene

E

Relative GST activity

Relative mRNA expression

A

24 h). Different molecular endpoints were investigated: (1) EcR and usp genes, the two dimerizing partners of the functional ecdysone receptor; (2) Hsp70 related to the stress response; (3) the CYP4G gene; and (4) the GST activity related to detoxification pathways. As shown in Fig. 1, no statistically significant differences were found at short-term exposure for any of the parameters analyzed.

1.5

1

0.5

0

Control

12 hours

24 hours

2

GST activity

1.5

1

0.5

0

Control

12 hours

24 hours

Fig. 1. Effect of 12- and 24-hour PCP exposures at 250 ␮g/L on the ecdysone receptor (A), ultraspiracle (B), Hsp70 (C) and CYP4G (D) genes in control larvae and treated larvae measured by real time RT-PCR, with primers and reference genes as indicated in Section 2. GST enzymatic activity (E) untreated larvae and larvae exposed to PCP for 12 and 24 h at 250 ␮g/L. The bars represent mean and SEM of 12- and 24-h exposures related to distinct control treatments from data obtained in three independent experiments, each with three replicates. The mRNA expression and activity level of untreated control larvae was set to 1.

M. Morales et al. / Aquatic Toxicology 157 (2014) 1–9

6

Relative mRNA expression

B * Relative mRNA expression

5 4 3 2 1 0

C

EcR gene

3

Control

25 µg/L

E74 gene

D

2.5

** 2 1.5

*

1 0.5 0

Control

25 µg/L

250 µg/L

3

usp gene

2.5 2 1.5 1 0.5 0

250 µg/L

Relative mRNA expression

Relative mRNA expression

A

5

3

Control

25 µg/L

250 µg/L

ERR gene

2.5 2

**

1.5 1 0.5 0

Control

25 µg/L

250 µg/L

Fig. 2. Relative expression levels after 96-h exposures for the ecdysone receptor (A), ultraspiracle (B), E74 (C) and estrogen-related receptor (D) genes in control larvae and treated larvae with 25 and 250 ␮g/L of PCP measured by real time RT-PCR, with primers and reference genes as indicated in Section 2. The expression level of untreated control larvae was set to 1. Data are the mean and SEM from three independent experiments, each with three independent PCR replicates. The asterisks indicate significant differences between control and larvae exposed to 96-h PCP treatments: * p ≤ 0.05, ** p ≤ 0.01.

Nevertheless, a clear trend towards an increased expression of the EcR and CYP4G genes was detected. Given these results, subsequent studies should focus on analysis of possible changes induced in transcriptional activity following longer exposures.

3.3. Effects of 96-hour PCP treatments on the expression of hormonal receptors and ecdysone-inducible genes Three nuclear hormone receptor genes were selected to evaluate their sensitivity to PCP following four-day exposures: the EcR and usp genes, coding for the two proteins of the insect’s heterodimeric receptor that mediates ecdysone action inside the cells, and the ERR gene coding for the estrogen-related receptor. The E74 was also analyzed as it is an early ecdysone-responsive gene activated by ecdysteroid signaling in insects. The expression profiles of these four endocrine-related genes were evaluated using real time RT-PCR after four-day exposures at two concentrations (25 ␮g/L and 250 ␮g/L). Following normalization, gene expression patterns were compared to those obtained from control cultures exposed to the same concentration of solvent. As shown in Fig. 2, this xenobiotic increased the expression of the EcR gene, the E74 gene and the ERR gene, while the usp gene remained unaltered. The 96-h PCP treatments at 25 ␮g/L provoked a clear increase in

the expression of EcR, ERR and E74 mRNAs (Fig. 2), which was statistically significant for the levels of ERR and E74 mRNAs, while PCP treatments at 250 ␮g/L provoked a significant increase in the level of EcR and E74 transcripts, when compared to that of control solvent-exposed larvae (Fig. 2). 3.4. Effect of 96-hour exposure to PCP on the expression of heat-shock genes To assess if PCP provoked any effect on the expression of stress responsive genes, the heat-shock Hsp70 and Hsp27 genes encoding for heat-shock protein 70 kDa (HSP70) and heat-shock protein 27 kDa (HSP27), respectively, were analyzed. The levels of mRNA expression in C. riparius larvae under normal larval growth conditions and following 96-h treatments were measured by real-time RT-PCR using specific oligonucleotides. As seen in Fig. 3, the results showed that Hsp70 gene transcription was affected at the two concentrations tested. Significant increases were found in the levels of Hsp70 mRNA after 96-h treatments with PCP at 25 and 250 ␮g/L. In contrast to Hsp70, Hsp27 gene transcription activity decreased significantly in animals exposed to PCP for 96 h at 25 ␮g/L, while 96-h PCP exposures at 250 ␮g/L downregulated the Hsp27 gene (Fig. 3).

M. Morales et al. / Aquatic Toxicology 157 (2014) 1–9

Relative mRNA expression

A

5

Hsp70

gene

**

4 3

** 2

1 0

Control

25 µg/L

B Relative mRNA expression

6

2

gene

1.5

1

*

0.5

0

250 µg/L

Hsp27

Control

25 µg/L

250 µg/L

Fig. 3. Effect of 96-h PCP exposures on the expression of the Hsp70 (A) and Hsp27 (B) genes. Transcript levels were measured by real time RT-PCR, with primers and reference genes as indicated in Section 2 and GAPDH and rpL13 as reference genes, and presented in relation to the values for the solvent-exposed control larvae. Each bar is the average ± SEM obtained from three independent experiments, each with three independent PCR replicates. The expression level under control conditions was set to 1. The asterisks indicate significant differences between control and larvae exposed to 96-h treatments: * p ≤ 0.05, ** p ≤ 0.01.

2

CYP4G gene

B

2

GST activity

* Relative GST activity

Relative mRNA expression

A

1.5

1

0.5

0

Control

25 µg/L

250 µg/L

1.5

1

0.5

0

Control

25 µg/L

250 µg/L

Fig. 4. Effect on CYP4G gene expression (A) and GST activity (B) after 96-h PCP exposures. (A) Relative levels of mRNA, measured by real time RT-PCR with primers and reference genes as indicated in Section 2, under different 96-h treatments, as compared to untreated control larvae for which the expression level was set to 1. (B) GST enzymatic activity in control and larvae exposed to 96-h exposures, at different concentrations. Mean and SEM are shown from three independent experiments, each with three replicates. The asterisks indicate significant differences between PCP-treated samples and the solvent control: *p ≤ 0.05.

3.5. CYP4G expression and GST enzyme activity in response to long-term PCP exposures To examine the possible effects of PCP on endpoints related to the detoxification mechanisms, two parameters were analyzed: the transcriptional response of the CYP4G gene and GST enzyme activity. As shown in Fig. 4, expression of the CYP4G gene was upregulated in a dose-dependent manner after 96-h PCP exposures at 25 ␮g/L and 250 ␮g/L. Nevertheless, statistically significant changes were only found at the highest concentration used in this study. Regarding GST, the results of this analysis revealed that GST enzymatic activity did not differ significantly between the control and treated groups after four-day exposures (Fig. 4). GST enzyme did not appear to be altered in response to PCP, as was previously found following short-term exposures (Fig. 1).

4. Discussion This study demonstrates, for the first time, the ability of PCP to upregulate the expression of a set of genes that are involved in the endocrine pathways of insects. In addition, expression (either

upregulated or downregulated) was altered by PCP exposures, which also activated a cytochrome P450 gene related with the detoxification mechanism, but did not affect GST enzyme activity. These results add new information about the mode of action of this contaminant in cellular processes, which to date has received little attention. Indeed, as there have been very few studies about the regulation of genes in aquatic organisms after exposure to EDCs, this study offers potential gene biomarkers for further genomic environmental assessment. Furthermore, the modulator effect of this xenobiotic on insect endocrine genes adds new data to the growing evidence obtained in other organisms, mainly mammals and fishes, which support the endocrine disruptive potential of this compound (Segner et al., 2003). Regarding the effects of PCP on hormonal genes, this is one of the first studies focusing on a suite of genes directly connected with the cascade of genetic signals switched on by the hormone ecdysone. The results obtained demonstrate that it was able to induce a significant overexpression of the EcR gene, the ecdysone-inducible E74 gene and the ERR gene in Chironomus larvae. In contrast, PCP did not affect the activity of the usp gene that remained unaltered in all the conditions tested in this study. Therefore, the observed effects of this compound appear to be highly specific for

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particular endocrine genes, and might alter important hormonal regulated functions. Ecdysone has essential roles in coordinating major developmental transitions, such as larval molting and metamorphosis, which are essential for development in arthropods. Ecdysone signaling is primarily mediated by the ecdysone receptor, which is ligand-activated transcription factor and member of an evolutionarily conserved nuclear hormone receptor family that includes vertebrate and invertebrate members (Köhler et al., 2007). The EcR/USP complex directly activates the transcription of early ecdysone-inducible genes such as E74, E75 and the Broad complex (Koelle et al., 1991; Karim and Thummel, 1992; Yao et al., 1993), all of which are transcriptional regulatory factors. Subsequently, these early ecdysone-inducible genes regulate late ecdysone-inducible genes, which leads to dramatic changes in tissues during development. The ability of PCP to activate transcription of hormonal target genes may possibly have a more complicated mechanism of action related to the whole organism’s reaction to the xenobiotic and detoxification. For example, Aragon et al. (2002) found a link between CYP4C15 gene expression and increased ecdysosteroid synthesis in crayfish which may possibly relate to the increased expression of the CYP4G gene and to upregulation of hormonal target genes EcR, E74 or ERR presented in this study. The detoxification and hormonal signaling pathways may be interconnected leading to a general stress response by an individual, such as molting as a way of detoxification (Raessler et al., 2005) that may not necessarily imply endocrine disruption. Nutritional status may also play a role in these processes (Telang et al., 2007), which could possibly explain the differences in gene expression levels between 24- and 96-h exposures. Moreover, the results obtained with PCP reveal a similarity with other well-known endocrine disrupters. Previous works have reported that bisphenol A (BPA), benzyl butyl phthalate (BBP) and cadmium (Cd) interfere with the ecdysone-signaling pathway by activating the hormone receptor gene in C. riparius (Planelló et al., 2008, 2010, 2011). The EcR gene has also been found to be sensitive to different UV-filters (Ozáez et al., 2013, 2014), as well as to nonylphenol (NP) and silver nanoparticles in this species (Nair and Choi, 2012). Recently, our group showed that tributyltin (TBT) also affected different regulatory genes directly connected with the cascade of genetic signals switched on by the hormone ecdysone (Morales et al., 2013). Hence, it would appear PCP behaves like TBT, which is one of the best-characterized endocrine disruptors in aquatic environments. ERRs are a group of nuclear receptors that were originally identified based on the similarity of their sequence to that of the estrogen receptors (ERs), and whose function in invertebrates remains to be defined. The ERR gene was identified in the insect C. riparius appearing abundantly expressed during different life stages, with the exception of adult males, although its function is still unknown (Park and Kwak, 2010). It has also been identified in the cricket Teleogryllus emma (He et al., 2010). Moreover, the ERR gene was found to be upregulated by BPA, NP, di(2-ethylhexyl)phthalate (DEHP) (Park and Kwak, 2010), UV filters (Ozáez et al., 2013) and TBT (Morales et al., 2013) in C. riparius. BPA also induces an overexpression of ERR in zebrafish (Tohmé et al., 2014). Here we show that the ERR gene is also upregulated by PCP exposure, confirming that it behaves as an environmental responsive gene to EDCs. Although it is still unclear if the sex steroid receptors being identified in many invertebrate species are involved in hormonal signal transduction (Köhler et al., 2007), the possibility that estrogen-related orphan receptors might have a functional role in the genomic response to EDCs in invertebrates cannot be ruled out. HSPs are abundantly induced proteins by a huge variety of stressful conditions and environmental insults, which play an important role in protecting cells against a broad spectrum of potentially lethal pollutants (Gupta et al., 2010). We found that PCP

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treatments highly activated Hsp70 expression in C. riparius reaching up to a five-fold increase in mRNA levels after 96-h treatments. However, it seems to be a long-term response as there were no detectable changes after 12- or 24-h exposures. In addition to its role in cellular toxicity, it has been suggested that there may be a functional relationship between HSP70 and steroid hormones (such as ecdysone in insects and estrogen in mammals) (Nolen and Morimoto, 2002). Previous results from our group have shown that the Hsp70 gene is induced by the synthetic steroid ethinylestradiol (EE) in C. riparius (Morales et al., 2011), which is consistent with some reports of heat-shock protein expression induced by ecdysone in fruit flies and estrogen in mammals (Ryan and Hightower, 1998). More detailed studies are needed to establish the possible relationship of Hsp70 and hormonal receptor activation under the effect of EDC pollutants. Our results are in agreement with previous studies reporting that PCP induces an overexpression of HSP40, HSP70 and HSP90 proteins in Euglena gracilis (Barque et al., 1996). In contrast to Hsp70, C. riparius Hsp27 gene appeared to be downregulated after 96-h treatments. Besides its protective role under stressful conditions, the small HSPs are also involved in some important biological processes such as cell growth, apoptosis, differentiation, diapause, lifespan, membrane fluidity, and starvation resistance in insects (Arrigo, 1998; Tsvetkova et al., 2002; Morrow et al., 2004; Hao et al., 2007; Gkouvitsas et al., 2008). Recently, our group has shown that exposure to triclosan (TCS), BPA and Cd increased Hsp27 mRNA levels in C. riparius (Martínez-Paz et al., 2014). This is the first evidence of the ability of PCP to alter the activity of heat-shock proteins genes in C. riparius, in particular the Hsp70 gene. The genes coding for HSPs have become important tools in environmental studies, and the Hsp70 gene has been evaluated as a potential biomarker of exposure to different xenobiotics in chironomid species such as C. yoshimatsui (Yoshimi et al., 2002), C. tentans (Karouna-Renier and Zehr, 2003), C. dilutus (Karouna-Renier and Rao, 2009) and C. riparius (Morales et al., 2011). The regulation of the members of the CYP4 gene family has historically focused on the transcriptional induction mediated by chemicals and xenobiotics associated with peroxisome proliferation in mammals (Rettie and Kelly, 2008). CYP4 genes have been identified in numerous insect species (Feyereisen, 2006). We found that this xenobiotic was able to activate expression of the CYP4G gene in C. riparius larvae. To our knowledge, this is the first evidence of the ability of PCP to alter CYP4G gene expression in invertebrates. Once again, the response of the CYP4G gene to PCP is similar to that previously found in C. riparius exposed to the well-known endocrine disruptor TBT (Martínez-Paz et al., 2012). In contrast, exposures to NP and BPA downregulated this gene (Martínez-Paz et al., 2012). Overall, these results suggest that the CYP4G gene from Chironomus responds selectively to xenobiotics, particularly xenoestrogens, and could, therefore, act as a biomarker gene for analyzing chemically contaminated environments. Our results are in accordance with previous studies that have demonstrated that the metabolism of PCP is mediated by the cytochrome P450 family (Mehmood et al., 1996). Yet, none of the P450-related transcripts appeared to be induced in microarray assays in zebrafish embryos (Xu et al., 2014a). In addition to CYP4G gene activity, GST activity, one of the major detoxification enzymes, was also evaluated to compare the response of these two detoxification pathways at the gene and enzyme levels. Insect Glutathione-S-Transferase plays important roles in detoxifying toxic compounds and eliminating oxidative stress caused by xenobiotics (Xu et al., 2014b). Interestingly, PCP did not significantly alter the levels of GST activity at any of the time points or concentrations analyzed. Previous studies have found that TBT, BPA and EE alter GST activity (Lee and Choi, 2007; Martínez-Paz et al., 2012). The results obtained in this study suggest a different response by these two detoxification systems; therefore,

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PCP might induce distinct responses in phase I and phase II pathways suggesting multiple xenobiotic transduction mechanisms. Acknowledgements The authors wish to thank T. Cater and Dr T. Carretero (University of Zaragoza) for critically reading of the manuscript. We would like to thank Dr. J. Barcena, Dr. E. Blanco and Dr. C. Gallardo from the CISA-INIA, Madrid, for their help for sample processing treatment with the Multidetection Microplate Reader FLUOstar Omega (BMG labtech). This work was supported by the Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica (Spain), grant CTM2012-37547 from the Ciencias y Tecnologías Medioambientales program. The authors declare that there are no conflicts of interest. References Aragon, S., Claudinot, S., Blais, C., Maïbèche, M., Dauphin-Villemant, C., 2002. Molting cycle-dependent expression of CYP4C15, a cytochrome P450 enzyme putatively involved in ecdysteroidogenesis in the crayfish, Orconectes limosus. 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