Beta-naphthoflavone-inducedCYP1A expression in the guppy Jenynsia multidentata: Time-dependent response, anesthetic MS-222 effect and fin analysis

Ecotoxicology and Environmental Safety 113 (2015) 38–44

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Beta-naphthoflavone-inducedCYP1A expression in the guppy Jenynsia multidentata: Time-dependent response, anesthetic MS-222 effect and fin analysis Debora P. Pinto a, Cíntia C. Chivittz a, Roger S. Ferreira a, Mauricio S. Sopezki b, Juliano Zanette a,b,n a Programa de Pós-graduação em Biologia de Ambientes Aquáticos Continentais, Instituto de Ciências Biológicas, Universidade Federal do Rio Grande – FURG, Rio Grande, RS 96203-900, Brazil b Programa de Pós-graduação em Ciências Fisiológicas – Fisiologia Animal Comparada, Instituto de Ciências Biológicas, Universidade Federal do Rio Grande – FURG, Rio Grande, RS 96203-900, Brazil

art ic l e i nf o

a b s t r a c t

Article history: Received 2 September 2014 Received in revised form 20 November 2014 Accepted 25 November 2014

Cytochrome P450 1A (CYP1A) expression in fish is used as a biomarker of exposure to organic contaminants, such PAHs, PCBs and dioxins, in the aquatic environment. South American guppy fish Jenynsia multidentata were exposed to the prototypical aryl hydrocarbon receptor (AHR) agonist beta-naphthoflavone (BNF; 1 μM) and the fins were biopsied to characterize different aspects of CYP1A induction. RTqPCR was used to quantify CYP1A mRNA levels in fish tissues. CYP1A induction in the gill, liver and anal fin (gonopodium) occurred within the first hour of waterborne exposure to BNF and persisted throughout 2, 4, 8, 24, 48 and 96 h compared to controls (DMSO vehicle; po 0.05). The organ-specific temporal pattern of induction was marked by mRNA levels consistently augment as duration of exposure increases and tend to a sustained induction from 24 h to 96 h for gill and liver (∼15-fold and ∼50-fold over control, respectively). In gonopodium, there was a maximum CYP1A mRNA level at 4 h (∼34-fold over control). Basal CYP1A mRNA levels and its induction following BNF exposure were not affected by administration of a chemical anesthetic (fish immersion in 100 mg l
1 MS-222 for 2–5 min) in the gill, liver, gonopodium, dorsal or tail fin (p o0.05). In an ex vivo assay, in which small pieces of biopsied fins were exposed to BNF for 4 h, high CYP1A induction was observed in the tail and gonopodium (∼49-fold and ∼69-fold, respectively) but not in the dorsal fin compared to controls. To our knowledge, this is the first study to show that a 1 h waterborne exposure to an AHR agonist is sufficient to cause CYP1A induction in fish organs and fins. The present study added new information to the field regarding the use of MS-222 as an anesthetic on fish and the analysis of biopsied fins as an alternative non-lethalex vivo assay for evaluating the CYP1A biomarker in fish. This observation could be useful for planning fish toxicological bioassays and biomonitoring studies on the aquatic environments in South America. & 2014 Published by Elsevier Inc.

Keywords: CYP1A Fish Jenynsia multidentata PAH Tricaine Pollution

1. Introduction Aquatic ecosystems are significantly impacted by organic contaminants such as polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) and dioxins (e.g., TCDD), which are potentially carcinogenic, teratogenic and mutagenic compounds (Aas et al., 2001). The induction of cytochrome P450 1A (CYP1A) in fish has been extensively used as a biomarker in n Corresponding author at: Universidade Federal do Rio Grande (FURG), Instituto de Ciências Biológicas, Av. Itália, Km 8, Campus Carreiros, Rio Grande, RS 96203900, Brazil. E-mail addresses: [email protected], [email protected] (J. Zanette).

http://dx.doi.org/10.1016/j.ecoenv.2014.11.023 0147-6513/& 2014 Published by Elsevier Inc.

monitoring and ecotoxicological studies (Whyte et al., 2000) and is often measured using enzymatic assays (e.g., ethoxyresorufin-Odeethylase; EROD) (Billiard et al., 2004) and protein quantification (Bucheli and Fent, 1995; VanVeld et al., 1997). CYP1A induction occurs via aryl hydrocarbon receptor (AHR) activation in vertebrates such as mammals, birds and fish (Hahn, 2002). The CYP1A mRNA levels in fish have been evaluated using reverse transcription followed by real time PCR (RTq-PCR) for biomarker analysis (Pina et al., 2007). The CYP1A mRNA transcript level and protein are primarily abundant in liver but can also be found in extra-hepatic organs, where it is strongly induced by organic contaminants such as PAHs, PCBs and dioxin (Jonsson et al., 2007; Ortiz-Delgado et al., 2008; Zanette et al., 2009). In addition, fish fins may be targets for

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CYP1A promoter activation following organic chemical exposure, as demonstrated using GFP transgenic fish models (Ng and Gong, 2013; Kim et al., 2013) and histological immunolocalization (Zodrow et al., 2004). The possible advantages of CYP1A biomarker evaluation in fish fins remain poorly explored. The peak of CYP1A induction by contaminants occurs in a time-dependent manner in the different organs of fish (Kim et al., 2008). Thus, it is important to understand this pattern to choose target organs and optimal exposure times to use in ecotoxicological studies and to understand the toxicokinetics of compounds. Biomarker analysis in organs classically used in aquatic toxicological studies, such as the gills and liver, requires destructive (lethal) sampling, which could possibly endanger fish populations (Ress et al., 2005). To minimize this effect, nonlethal sampling methods, in which tissue removal occurs without a major damage to the animals, are potential alternatives that could be used in different animal species (Schmitt and Brumbaugh, 2007). CYP1A activation in external epithelial organs responds rapidly following waterborne exposures of fish or ex vivo exposure of gills (Kim et al., 2008; Jonsson et al., 2002). Based on that, the use of fins for analysis to develop a non-lethal biopsy method may be proposed. The use of the chemical anesthetic tricaine methanesulfonate (MS-222) emerged around the 1960s and has been extensively used in fish experimentation (see Popovic et al. (2012) for a review). Reports on the possible influence of MS-222 on cytochrome P450 (CYP) responses are conflicting (Popovic et al., 2012). Evaluation of the MS-222 influence on the CYP1A biomarker response and its influence on other biological responses is required for the ethical use of fish as a surveillance species. The Cyprinodontiforme fish that are widespread in aquatic environments, such as the North American killifish Fundulus heteroclitus and the South American guppies Poecilia vivipara and Jenynsia multidentata, are potential species for use in environmental studies (Dorrington et al., 2012; Elskus et al., 1999; Ferreira et al., 2012). The guppy J. multidentata (Cyprinodontiformes, Anablepidae) inhabits the region from Rio Negro, Argentina to Rio de Janeiro, Brazil and possesses unique characteristics, such as ovoviviparity and sexual dimorphism. In these fish, there is a modification of the male anal fin into a thin and elongated copulatory organ denominated gonopodium with regeneration capacity (Offen et al., 2008; Turner, 1947). Growth and gene expression profiles in the gonopodium have been used as important endpoints to study endocrine disruptor effects caused by contaminants in mosquitofish Gambusia sp. (Brockmeier et al., 2013). As far as we know, there are no studies that have investigated CYP1A induction in the gonopodium or the potential for a biopsy of this modified fin to be used in a non-lethal toxicological assay. In the present study, the time-dependent and organ-specificCYP1A response to beta-naphthoflavone (BNF) exposure was evaluated in J. multidenta. The influence of the MS-222 anesthetic procedure on these responses was also investigated using in vivo and ex vivo (biopsied fin exposure) experiments. This study also contributes to the comprehension of CYP1A responses in fins, to develop non-lethal biopsy methodology. The results present evidence for the use of J. multidentata as an alternative model organism for ecotoxicology studies in the South American environment.

2. Materials and methods 2.1. Animal collection Male J. multidentata fish (3–5 cm length; 0.5–2.5 g whole body weight; n ¼150) were collected in a watercourse from an uninhabited area 15 km away from the district of Balneário Cassino

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(Rio Grande, RS, Brazil; 32°17′48.36′′S and 52°16′01.96′′O) in August 2013 and November 2013 and were used in the experiments described in Sections 2.2, 2.3 and 2.4. Males were identified by the presence of the gonopodium morphological dimorphism. Fish were acclimated for seven days in the laboratory before beginning the experiments. The fish were maintained in dechlorinated water at 24 °C, salinity 5, on a 12 h light/12 h dark photoperiod with constant aeration and fed twice per day with Alcon BASICsMEP 200 Complex. The procedures were approved by the Animal Care and Use Committee at the Universidade Federal do Rio Grande (FURG). 2.2. Time-dependent CYP1A induction following BNF exposure Male fish (n ¼84) were divided into 14 experimental groups with n ¼ 6 fish in each group (1 fish per liter) and maintained in similar conditions to those mentioned in Section 2.1. BNF (SigmaAldrich, Germany) was dissolved in pure DMSO and was added to seven experimental groups to make a final concentration of 1 mM BNF and 0.002% DMSO in the aquarium. BNF and DMSO concentrations were chosen based on previous experiments in zebrafish (Jonsson et al., 2007) and pufferfish (Kim et al., 2008). DMSO was also added to seven experimental groups in an equal volume to make 0.002% DMSO control groups. BNF and DMSO were replaced in the exposed and control aquarium, respectively, every 24 h. Fish from the BNF and control groups were euthanized at 1, 2, 4, 8, 24, 48 and 96 h after the first addition of BNF or DMSO by putting the fish in ice followed by cervical transection. The gill, liver and gonopodium were dissected and immediately preserved in RNAlaters (Ambion) according to the manufacturer's instructions. Total RNA was isolated with Trizol reagent (Invitrogen) and reversed transcribed to cDNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Specific CYP1A and β-actin primers pairs (forward 5′-CATGGGCAGTGATGTACCTTGTGG-3′ and reverse 5′-GGAGTTCGATCCAGACCAATTTGC3′ for CYP1A and forward 5′-AAAGCCAACAGGGAGAAGATGAC-3′ and reverse 5′-GCCTGGATGGCAACGTACA-3′ for β-actin; IDT Integrated DNA Technologies) were designed based on the GenBank nucleotide sequences EF362746 and EF362747, respectively. The real-time PCR conditions for the CYP1A and β-actin primers were previously tested and established (Ferreira et al., 2012). Analyses were performed in duplicate using the GoTaq qPCR Master Mix kit (Promega) and a 7300 Real-Time PCR System (Applied Biosystems) set for the following program: 50 °C for 2 min, 95 °C for 2 min and 40 cycles of 95 °C for 15 s and 60 °C for 30 s. The E
Δct method was used to calculate the relative transcriptional level in experimental groups using β-actin as a reference gene as described by Schmittgen and Livak (2008). The threshold used for Ct inference was fixed at 0.2 ΔRn (fluorescence normalized by internal ROX dye) for all runs. Because no differences in transcriptional levels were observed between the control groups at the different experimental times evaluated, all values from control groups were pooled (n ¼42) and the mean value was used as a calibrator to evaluate the CYP1A induction to BNF at the different exposure times. The CYP1A fold induction to BNF was represented as a ratio of BNF exposed to control. Data from all groups were logarithmically transformed to adhere to the ANOVA assumptions of normality and homoscedasticity, and the differences between the groups were determined by one-way ANOVA followed by the Tukey-HSD post-hoc test for unequal number of samples (p o0.05). The software Prism 5 for windows (Version 5.00, GraphPad software Inc.) was used for all statistic analysis used in this manuscript.

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2.3. MS-222 effects in J. multidentata CYP1A responses Male fish (n ¼24) were transferred to four 6 l aquariums (n ¼ 6 per aquarium) and maintained in the conditions mentioned in Section 2.1 but without food. BNF dissolved in pure DMSO was added to two aquariums to make a final concentration of 1 mM BNF and 0.002% DMSO (BNF exposed groups). DMSO was also added to the other two aquariums in an equal volume to make 0.002% DMSO control groups. BNF exposed and control groups were maintained under these conditions for 4 h. Fish from one of the BNF exposed group aquariums and one of the control group aquariums were euthanized by submerging the fish on ice for one minute followed by cervical transection. To evaluate the effect of MS-222 on the biomarker response, the same procedure was performed on the remaining BNF exposed and control groups except euthanasia was preceded by anesthesia with MS-222. We were not able to find in the literature a standardize procedure to anesthetize J. multidentata with MS-222, thus we searched studies using close related fishes in the literature. According to Popovic et al. (2012), that reviewed the use of MS-222 in fishes, it was suggested that 30–150 mg l
1 MS-222 causes the Stage I anesthesia and loss of equilibrium in the Cyprinodontoidei fish Poecilia latipinna (the same suborder as J. multidentata and similar body size). Based on that, in the present study, J. multidentata were immersed in 100 mg l
1 MS-222 solutions during ∼2.5 min and the anesthetic effect was confirmed by a visual loss of swimming activity and equilibrium. The gonopodium, tail fin, dorsal fin, gill and liver were dissected from the four experimental groups immediately after the euthanasia procedure. Organ preservation in RNAlaters, total RNA extraction, cDNA synthesis, and qPCR was performed as described in Section 2.2. Data were logarithmically transformed to adhere to the ANOVA assumptions of normality and homoscedasticity. Significant differences in transcriptional levels were determined using one-way ANOVA followed by Tukey's post-hoc test (p o0.05) to compare the CYP1A transcriptional levels in the four experimental groups. 2.4. CYP1A responses in a ex vivo assay with biopsied fins For the ex vivo experiment, six beakers were filled with 100 ml of 0.002% DMSO dissolved in dechlorinated water for control groups, and six beakers were filled with 100 ml of 1 mM BNF dissolved in DMSO (0.002% DMSO in the final volume) for the BNF exposed groups, prior to the fin dissection. Male fish (n ¼20) were euthanized via direct submersion in ice

for 1 min followed by cervical transaction. The gonopodium, dorsal and tail fins were dissected (∼4 mm2) with vannas scissors. Dorsal fins were immediately placed into the control and BNF containing beakers (n ¼10 in each beaker). The same ex vivo fin exposure procedure was performed for the tail and gonopodium for a total of six experimental groups that did not receive anesthesia (three control and three BNF exposed groups). To test the possible effects of the anesthetic MS-222 in the ex vivo fin experiment, male fish (n ¼20) were anesthetized with MS-222 and euthanized by submerging the fish in ice for 1 min followed by cervical transection, as previously described in Section 2.3. The dorsal, tail and anal (gonopodium) fins were dissected (∼ 4 mm2 tissue). Dorsal fins were immediately placed into the control (DMSO) and BNF containing beakers previously reserved (n ¼10 in each). The same procedure was performed for the tail and gonopodium, for a total of six experimental groups with fins from fish that were anesthetized prior to dissection (three control and three BNF exposed). All 120 fins used in the ex vivo experiment were collected after 4 h in DMSO or BNF. Organ preservation in RNAlaters, total RNA extraction, cDNA synthesis, and qPCR were performed as described in Section 2.2. Data were logarithmically transformed to adhere to the ANOVA assumptions of normality and homoscedasticity. Significant differences in CYP1A transcriptional levels between the four experimental groups were determined using one-way ANOVA followed by the Tukey's post-hoc test (p o0.05).

3. Results 3.1. Time-dependent CYP1A induction in response to BNF The waterborne exposure of fish to 1 mM BNF caused CYP1A induction in all J. multidentata organs (gonopodium, gill and liver) analyzed, which occurred at all exposure times tested (from 1 to 96 h) compared to control (DMSO; p o0.05). The earliest exposure time tested, 1 h, was sufficient to induce CYP1A in all tested organs: gill, liver and gonopodium (∼4,∼3 and ∼6-fold over control, respectively; po 0.05) (Fig. 1). The CYP1A mRNA level in the liver of fish exposed to BNF showed a moderate induction in the early experimental times, 1, 2 and 4 h, compared to control (∼ 3- to ∼7-fold control), but the strongest induction was observed at later exposure times, between 8 and 96 h (from ∼30 to ∼50-fold over control; respectively, po 0.05; Fig. 1). Analysis of the CYP1A mRNA levels in the gills of fish exposed to BNF did not clearly show a time indicating peak

Fig. 1. CYP1A transcription levels in the gill, liver and gonopodium of J. multidentata after 1-, 2-, 4-, 8-, 24-, 48- and 96-h exposure to 1 mM beta-naphthoflavone (BNF) or DMSO vehicle alone (control). The numbers above the error bars denote CYP1A fold induction to BNF relative to control. CYP1A levels are represented as the mean 7 standard deviation calculated as E
ΔCt using β-actin as the reference gene. Same letters represent the absence of a difference between groups (one-way ANOVA followed by unequal-N HSD post-hoc test; p o 0.05).

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Fig. 2. CYP1A transcription levels in the gonopodium, dorsal fin, tail fin, gill and liver of J. multidentata exposed to 1 mM beta-naphthoflavone (BNF) or DMSO vehicle alone for 4 h. White bars represent fish that were not chemically anesthetized, and gray bars represent fish that were anesthetized with 100 mg l
1 MS-222 prior to euthanasia and dissection. CYP1A fold induction with respect to control is denoted by the numbers above the bars. CYP1A levels are represented as the means 7 standard deviation calculated as E
ΔCt using β-actin as the reference gene. The same letters represent the absence of a difference (one-way ANOVA followed by Tukey-HSD; n¼ 6; po 0.05).

induction when levels were compared between the different BNF exposure times. CYP1A mRNA levels in the gonopodium of fish exposed to BNF at 4 h was higher than control group (∼34-fold control; p o0.05) and was higher than fish exposed to BNF at 1, 2, 24, 48 and 96 h (p o0.05).

fish that were anesthetized with MS-222 showed a significant CYP1A induction following a 4-h exposure to 1 mM BNF compared to controls (p o 0.05; n ¼6; Fig. 2).

3.2. MS-222 effects on the CYP1A response in J. multidentata

With the exception of the dorsal fin biopsies from fish that were not anesthetized with MS-222, a 4-h exposure to 1 mM BNF caused an induction in CYP1A mRNA levels in all organs analyzed (gonopodium, dorsal fin and tail fin) compared to the control groups (po 0.05; n ¼ 10; Fig. 3). With the exception of the dorsal fin biopsies, MS-222 did not produce changes in either the basal CYP1A mRNA level (not significant; n ¼10; Fig. 3) or in the significant induction by 1 mM BNF exposure in the organs analyzed (p o0.05; n ¼10; Fig. 3). The gonopodium showed the highest

The anesthetic procedure with MS-222 did not produce changes in the basal CYP1A mRNA level in any of the organs analyzed (gonopodium, dorsal fin, tail fin, gill and liver) (not significant; n ¼6; Fig. 2). Fish that were not anesthetized showed CYP1A induction in all organs after 4 h waterborne exposure to 1 mM BNF compared to controls (p o 0.05; n ¼ 6; Fig. 2). Except for the dorsal fin, all other tested organs and fins from

3.3. CYP1A responses in a ex vivo assay on biopsied fins

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Fig. 3. CYP1A transcription levels in the gonopodium, dorsal fin and tail fin that were biopsied from J. multidentata and exposed in vitro to 1 mM beta-naphthoflavone (BNF) or DMSO vehicle alone for 4 h. White bars represent fish that were not chemically anesthetized and gray bars represent fish that were anesthetized with 100 mg l
1 MS-222 prior to euthanasia and dissection. CYP1A fold induction relative to control is denoted by the numbers above the bars. CYP1A level is represented as the mean 7 standard deviation calculated as E
ΔCt using β-actin as the reference gene. Same letters represent the absence of a difference (one-way ANOVA followed by Tukey-HSD; n¼ 10; p o0.05).

relative induction (∼69-fold; p o0.001; n¼ 10; Fig. 3) followed by the tail fin (∼49-fold; p o0.05; n ¼10; Fig. 3).

4. Discussion 4.1. Time-dependent CYP1A induction in response to BNF The CYP1A enzyme has a central role in the biotransformation of toxic substances, and its induction in fish has been used since the 1970s to investigate organic contamination and assess exposure risks in the aquatic environment (Payne, 1976). A rapid response at the CYP1A mRNA level is observed after waterborne exposure to PAH (few hours) that precedes the increase in the CYP1A protein levels (Kim et al., 2008; Kloeppersams and Stegeman, 1989; Levine and Oris, 1999). The CYP1A response to chemical exposure has been investigated in several fish species (see Sarasquete and Segner, 2000 for a review), and there are reports of CYP1A induction following a 24 h waterborne exposure to 1 μM BNF in the liver of Takifugu obscures pufferfish (∼11-fold over control; Kim et al., 2008), and the P. vivipara (∼22-fold) and J. multidentata guppies (∼185-fold; Ferreira et al., 2012). Similarly, the present study found high values of fold induction in the gill, liver and gonopodium (∼20, 50 and 34-fold, respectively) after 24 h of BNF exposure. In addition, CYP1A induction in these J. multidentata organs was elicited at all exposure times tested, 1– 96 h. Further studies could be done to test the dose-dependent responses of CYP1A mRNA level to environmental contaminant in J. multidentata, in order to determine if they can accumulate and respond to pollutant levels indicative of ambient environmental concentrations. Those studies could support the use of the South American J. multidentata as a species that can be monitored to assess AHR agonist contamination in the aquatic environment. CYP1A induction in J. multidentata was observed to be ∼4-fold in gill, 3-fold in liver and 6-fold in gonopodium compared to control after 1 h of BNF waterborne exposure. To our knowledge, this is the first study to show that a CYP1A response detected using RTq-PCR in fish hepatic and extra-hepatic organs could occur after

1 h of exposure. This information could be useful for the development of practical and rapid assays for environmental risk assessment using fish. In contrast to our study, the shortest BNF waterborne exposure time evaluated by Kim et al. (2008), 6 h, was not sufficient to induce CYP1A in hepatic and most of the extrahepatic pufferfish organs (the gill and brain were exceptions). Chung-Davidson et al. (2004) also found that CYP1A responses in the brains of trout did not occur within the first 2 h after BNF injection. However, Kloeppersams and Stegeman (1989) found that 6 h after BNF injection, the shortest time tested, was enough time to induce CYP1A mRNA levels in the killifish F. heteroclitus liver, which agrees with the rapid response found in the J. multidentata organs evaluated in this study. The differences found in these studies could be associated with phenotypical (i.e., body size) differences and phylogenetic distance between the fish species. It is important to note that the environmental model fish F. heteroclitus is equivalent in size and from the same order as J. multidentata (Cyprinodontiformes), which may explain the similar patterns of responses observed in both fish. The pufferfish (Tetraodontiformes) and trout (Salmoniformes) were separated from Cyprinodontiformes approximately 195 and 290 million years ago, respectively (Steinke et al., 2006). It is also possible that the differences observed in the time-dependentCYP1A induction pattern is related to distinct biotransformation capacities used to eliminate BNF in different fish and organs, for example differences in the expression of membrane efflux transporters such as the ABC (ATPbinding cassette) super family members (Dean and Annilo, 2005). Comparisons between the different exposure times showed that CYP1A induction in the gonopodium of fish exposed to BNF was higher at 4 h than other exposure times, which preceded the high levels of induction observed in the liver between 24 h and 96 h, and the gills did not elicit a clear induction peak. This is possibly the first study to show that CYP1A is strongly induced by AHR agonists in this reproductive organ of the fish. The gonopodium constitution supports its capability for CYP1A induction because it is rich in epithelial cells (Ogino et al., 2004) and immunohistochemistry shows a very strong positive CYP1A staining in endothelial and epithelial cells of fish exposed to contaminants

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(Bainy et al., 1999). The use of the gonopodium has great potential to be used in the development of a non-lethal organ biopsy procedure for the study of organisms in contaminated areas and toxicological assays because it is an organ with a confirmed capacity for regeneration (Offen et al., 2008; Turner, 1947). The CYP1A response to BNF did not fall after the maximum induction was reached in the gill and liver and remained at the highest level at 96 h after exposure, the longest time point evaluated. Kim et al. (2008) differently found a fall in the CYP1A induction from 24 h to 96 h of BNF exposure. It is possible that the different response observed by Kim et al. (2008) is related to BNF biotransformation, elimination or decay in water. In contrast to the study by Kim et al. (2008), the water was renewed every 24 h in the present study, which could be an important factor for the maintenance of BNF concentrations in the water necessary to retain CYP1A induction. Different from the gill and liver, the CYP1A induction in the gonopodium decayed following the high level at 4 h BNF exposure. This distinct response could be related to distinct BNF toxicokinetics or compensatory mechanisms that exist in this organ to avoid CYP1A over expression. The maintenance of CYP1A induction in distinct organs overtime is an important factor to consider when using this biomarker in fish. 4.2. MS-222 effects on the CYP1A response in J. multidentata In the present study, we were able to show preliminary evidence that immersion in 2.5 min in 100 mg l
1 MS-222 did not alter the basal CYP1A mRNA level of J. multidentata. The exception for this evidence was the decreases in the CYP1A mRNA level caused by MS-222 treatment, observed in dorsal fin in the ex vivo experiment. However, the same pattern was not observed in the in vivo exposure. It is possible that those differences observed in the in vivo and ex vivo exposure could be a result of different sampling sizes used in both experiments (n ¼10 and n ¼6, respectively) and high variability displayed upon exposure to MS222 in those experiments. Thus, further studies with higher number of samples would be important to confirm that MS-222 does not alter basal CYP1A mRNA levels in fish. The strong CYP1A induction after 4 h of BNF exposure that was observed in the liver, gill and fins (anal, dorsal and caudal) is consistent with observations reported in the literature, particularly in reference to the liver (Elskuss et al., 1999; Wang et al., 2010; Ferreira et al., 2012). The gill is also an important target organ for water pollution studies, and MS-222 absorption and elimination occurs by diffusion through the gill membranes (Wayson et al., 1976). Studies have reported that this anesthetic impacts vasodilatation and circulatory changes in the secondary lamellae (Soivio and Hughes, 1978), but apparently this effect does not influence the levels of CYP1A transcription in this organ. This is in agreement with a study in Rainbow Trout that showed that the cytochrome P450 content and activity in response to injection with the AHR agonist BNF (100 mg kg
1) was not altered by a prior 50 mg l
1 MS-222 immersion (Kleinow et al., 1986). To our knowledge, our study is the first to show the possible absence of an effect by MS222 in the fins. The absence of MS-222 effect in CYP1A mRNA level could be confirmed by further in vivo and ex vivo studies using larger sampling sizes and different time of exposure to contaminants. 4.3. CYP1A responses using ex vivo assay with biopsied fins The use of biopsied organs could serve as an alternative, nonlethal procedure in environmental risk assessments. Among the organs analyzed in this study, the tail fin and gonopodium showed the highest levels of CYP1A induction after a 4 h ex vivo exposure to the AHR agonist BNF (∼69 and ∼49-fold, respectively). Although

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the gonopodium is a modified fin, its histological composition is different from the other fins based on the abundance of epithelial cells present in the tissue (Kuntz, 1914; Ogino et al., 2004; Zauner et al., 2003). The other fins (tail and dorsal) show an abundant dermal skeleton (similar to that found in scales). The high CYP1A induction in the gonopodium and tail fin indicates their prospective use in toxicological tests without the need to cause death or injury to the animal. In the zebrafish, tail fin regeneration occurs within 4 days after amputation (Geraudie et al., 1995; Poleo et al., 2001). The ability of the dermal skeleton in model fish to regenerate is getting attention by the study of molecular mechanisms underlying growth and regeneration (Zauner et al., 2003). Most likely, fin regeneration in J. multidentata is similar to that of other fish, providing a positive perspective for non-lethal methods to be used for biomonitoring of this species. Studies could be conducted to determine the total regeneration time and the influence of contaminants on the regenerative process in J. multidentata. The dorsal fin response in the biopsied fin assay was not successful because CYP1A was not induced in the non-anesthetized fish and was weakly induced by BNF in the MS-222 anesthetized fish. These unexpected results observed in the dorsal fin may be related to the difficulty of dissecting the organ, its small size or differences in its tissue constituents compared to those of the other analyzed fins. In a study with the rainbow trout Oncorhynchus mykiss, the dorsal adipose fin shows only a moderate induction of CYP1A after exposure to benzo[a]pyrene (Brzuzan et al., 2007). It is also possible that MS-222 causes a down-regulation in CYP1A basal mRNA levels, an idea that is evidenced by the difference found with respect to the other three experimental groups. In fact, the idea that MS-222 could cause a moderate decrease in CYP1A, denoted by EROD activity, has been previously reported (Kleinow et al., 1986). Further studies with larger sampling sites will be necessary in order to establish if MS-222 affect the CYP1A responses in fishes. Both in vivo and ex vivo assays were used to assess environmental exposure to PAHs through the evaluation of CYP1A (Billiard et al., 2004; Fent and Batscher, 2000; Whyte et al., 2000; Willett et al., 1997). In vitro studies generally use cell lines, in contrast to the tissue used in biopsies and non-lethal studies. The use of the biopsied fin method presents a short-term response and easy handling that can facilitate the biomarker evaluation in the laboratory and field.

5. Conclusion The time-dependentCYP1A induction to BNF exposure was evaluated in different organs of the guppy J. multidentata. This is possibly the first study to show that a waterborne exposure to an AHR agonist for 1 h is sufficient time to cause CYP1A induction in the liver, gill and fin of fish. In our study, the use of the anesthetic MS-222 did not influence the CYP1A mRNA responses in J. multidentata. Additional studies with larger sampling sizes could be done in order to confirm the MS-222 effect in CYP1A mRNA responses in fishes, which is important information considering the growing necessity of anesthetic procedures for the ethical use of fish in toxicology. Short exposure times are sufficient to cause CYP1A induction in biopsied tail fin and gonopodium using an in vivo BNF exposure or an ex vivo BNF exposure of biopsied fins. This represents a promising application in ecotoxicology to develop non-lethal procedures using fish. The present study provides important information for the use of the widespread South American guppy J. multidentata in toxicological studies.

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Acknowledgements Financial support is acknowledged to Conselho Nacional de Desenvolvimento Científico e Tecnológico, CNPq 480708/2013-4 (Brazil), CNPq 573949/2008-5 (Brazil), IFS A/5350-1 (Stockholm, Sweden), Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS 12/1328-5), PROAP – CAPES, and DP was supported by CAPES REUNI fellowship, Brazil. The authors would like to express their gratitude for the technical assistance with laboratory analysis and comments to improve the manuscript provided by Cássia R. Silveira, Elton P. Colares, Loraine Moraes, Josencler Ferreira, Camilo D. Seabra and Camila M.G. Martins.

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