Precision-Cut Liver Slices To Investigate Responsiveness of Deep

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Precision-Cut Liver Slices To Investigate Responsiveness of Deep-Sea Fish to Contaminants at High Pressure Benjamin Lemaire,†,‡,▽ Cathy Debier,†,‡ Pedro Buc Calderon,§,∥ Jean Pierre Thomé,⊥ John Stegeman,¶ Jarle Mork,# and Jean François Rees*,†,‡ †

Institut des Sciences de la Vie, Université Catholique de Louvain, Croix du Sud 2, B-1348 Louvain-la-Neuve, Belgium Biodiversity Research Centre, Université Catholique de Louvain, Croix du Sud 4, B-1348 Louvain-la-Neuve, Belgium § Louvain Drug Research Institute, Université Catholique de Louvain, Avenue Mounier 73, B-1200 Woluwé-Saint-Lambert, Belgium ∥ Departamento de Ciencias Quimicas y Farmaceuticas, Universidad Arturo Prat, Avda Arturo Prat, CL-2120 Iquique, Chile ⊥ Laboratoire d’Ecologie Animale et Ecotoxicologie, Institut de Chimie, Université de Liège, Allée du 6 août 15, B-4000 Liège, Belgium ¶ Biology Department, Woods Hole Oceanographic Institution, MA-02543 Woods Hole, Massachusets, U.S.A. # Biologisk Stasjon, Norges Teknisk-Naturvitenskapelige Universitet, Bynesveien 46, NO-7321 Trondheim, Norway ‡

S Supporting Information *

ABSTRACT: While deep-sea fish accumulate high levels of persistent organic pollutants (POPs), the toxicity associated with this contamination remains unknown. Indeed, the recurrent collection of moribund individuals precludes experimental studies to investigate POP effects in this fauna. We show that precision-cut liver slices (PCLS), an in vitro tool commonly used in human and rodent toxicology, can overcome such limitation. This technology was applied to individuals of the deep-sea grenadier Coryphaenoides rupestris directly upon retrieval from 530-m depth in Trondheimsfjord (Norway). PCLS remained viable and functional for 15 h when maintained in an appropriate culture media at 4 °C. This allowed experimental exposure of liver slices to the model POP 3methylcholanthrene (3-MC; 25 μM) at levels of hydrostatic pressure mimicking shallow (0.1 megapascal or MPa) and deep-sea (5−15 MPa; representative of 500−1500 m depth) environments. As in shallow water fish, 3-MC induced the transcription of the detoxification enzyme cytochrome P4501A (CYP1A; a biomarker of exposure to POPs). This induction was diminished at elevated pressure, suggesting a limited responsiveness of C. rupestris toward POPs in its native environment. This very first in vitro toxicological investigation on a deep-sea fish opens the route for understanding pollutants effects in this highly exposed fauna.

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INTRODUCTION

moribund individuals, precluding dose−response studies with xenobiotics on living deep-sea fish. However, a rapid isolation and incubation of tissue of those fish showing signs of survival upon collection could yield viable and functional cells in a tissue context (e.g., hepatocytes, endothelial cells in liver matrix), thereby constituting a valuable in vitro tool to investigate chemical effects in this remote fauna at relevant levels of hydrostatic pressure. One rationale of such studies would be to understand whether contaminants such as

The deep-sea, an extreme environment characterized by high hydrostatic pressures,1 acts as a global sink for POPs, such as organochlorines and polycyclic aromatic hydrocarbons.2 Deepsea fish, top-predators with longevities that can greatly exceed those of shallow water counterparts,3 can accumulate such chemicals to high levels as they grow.4−11 In coastal areas, highlevel exposures of fish have been associated with pathologies, such as tumor promotion, immunodeficiency, and developmental defects.12−15 Whether POPs might similarly impact the remote deep-sea ecosystems is a highly important and global concern, yet the susceptibility of the resident fish fauna to chemical effects remains unknown. Indeed, the barotraumatic procedure of retrieval typically results in the collection of © 2012 American Chemical Society

Received: Revised: Accepted: Published: 10310

May 8, August August August

2012 16, 2012 20, 2012 20, 2012

dx.doi.org/10.1021/es301850e | Environ. Sci. Technol. 2012, 46, 10310−10316

Environmental Science & Technology

Article

an efficient oxygen penetration and chemical uptake by cells in the slices. PCLS Preincubation (At Sea). Directly upon generation, liver slices were preincubated individually at atmospheric pressure in 24-well plates at 4 °C, in 500-μL of L15+ media (Leibovitz’s 15 media containing 10% decomplemented FBS and 1% PenStrep; adjusted to 400 milliosmoles with sodium chloride). The low temperature of incubation mimicks that of the deep-sea environment and the osmolarity selected is in the range of deep-sea fish plasma values.22,23 The 24-well plates were agitated at 175 rpm during the return travel to Trondheim Biological Station (i.e., about 30 min). PCLS Incubation (Ashore). The first incubation of PCLS ashore, in L15+ media with or without 3-MC (25 μM), was performed in agitated 24-well plates for 6 h, at 0.1 MPa (atmospheric pressure) and 4 °C. The hydrostatic pressure experiment (15 h; 0.1−5−15 MPa; 4 °C) was performed in three custom-designed 500-mL hyperbaric chambers from Autoclave (Rantigny, France), filled with oxygen-saturated L15+ media containing 3-MC (25 μM). Bubbling of pure oxygen in media ensured that equally high oxygen concentrations were present in each cylinder. The oxygen delivery was not corrected for the effect of hydrostatic pressure on oxygen solubility.24 The chambers were mounted on magnetic stirrers to homogenize the media during the exposure period (Figure 1). For each hyperbaric chamber, PCLS of each fish were placed on a 15-cm2 section of fiberglass grid (mesh of 1 mm2). Corners of the grid section were held together with rubber bands, resulting in a bag-shaped confinement system. The mesh and shape allowed PCLS to be continuously bathed in media during incubation. The rates of compression and decompression were set at 1 MPa s−1 with a M189 LVE air-driven

POPs are involved in the severe population declines affecting some deep-sea species worldwide.16 A technology that could provide information on susceptibility to chemical effects and that has never been applied to deep-sea fish is that involving precision-cut tissues slices. Here we employ precision cut liver slices (PCLS). This methodology should permit a very rapid preparation of the biological material under cruise conditions while maintaining the various liver cell types in their native proportions and their physiologic milieu.17 To test the utility of this in vitro model with deep-sea fish liver, individuals of the roundnose grenadier Coryphaenoides rupestris (200−2600 m depth) were sampled in a Norwegian fjord and PCLS were immediately generated and preincubated at atmospheric pressure onboard the research vessel. Ashore, PCLS exposure to POPs at various levels of hydrostatic pressure (0.1−5−15 megapascal or MPa) was modeled with 3methylcholanthrene (3-MC), a polycyclic aromatic hydrocarbon agonist for the aryl hydrocarbon receptor (AhR) that mediates effects of many POPs.18 Viability of liver cells was assessed postincubation by adenosine triphosphate (ATP) content and functionality by the AhR-mediated transcriptional induction of the detoxification enzyme cytochrome P4501A (CYP1A). As a previous biochemical study on liver of C. rupestris suggested an oxidative impact of POPs,19 we also investigated the transcript levels of a set of candidate oxidative stress responsive genes,20 namely the antioxidant enzymes catalase (CAT) and glutathione peroxidase (GPX) and heat shock proteins (hsp70 and hsp90).

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MATERIALS AND METHODS Chemicals. Leibovitz’s 15 media (with phenol red), PenStrep (penicillin 10 000 U ml−1 to streptomycin 10 000 μg mL−1), Foetal Bovine Serum (FBS) and Hank’s Balanced Salt Solution (HBSS) were obtained from Gibco (Paisley, U.K.). Other reagents, including 3-methylcholanthrene (3-MC; CAS 56-49-5), were from Sigma-Aldrich (Milwaukee, U.S.A.). Animals. Six adults of Coryphaenoides rupestris (73 ± 8 cm total length, mean ±SD; gonads not obviously developed) were caught during two cruises in Trondheimsfjord (N63°28,249′− E09°56,726′) onboard “R.V. Gunnerus” (NTNU, Norway). Following a forty-minute trawl at 530-m depth (distance traveled = 1.1 nautical mile), the net was recovered at 10 cm s−1. This slow speed of retrieval was used to limit the impact of decompression on deep-sea fish, thereby optimizing the chances to collect individuals of C. rupestris without overt signs of barotrauma (e.g., everted stomachs, eyes forced from orbits). The total time including trawl time and retrieval was about 2.5 h. The fish showing signs of life upon retrieval were used in experiments (3 individuals per cruise/experiment; up to 4 PCLS per treatment per fish for ATP measurements and same for gene expression studies). PCLS Generation. The procedure for fish anesthesia and liver slicing complied with the Code of Ethics of the World Medical Association and has been previously published.21 Briefly, individuals were deeply anaesthetized by immersion in a bath of tricaine methanesulfonate (100 mg L−1 seawater of MS-222) prior to recording their total length and sampling the livers. Excision and subsequent coring of the livers were done in the wet lab of “R.V. Gunnerus”, followed by the generation of 100μm thick and 8-mm diameter liver slices using a Krumdieck MD-1100 tissue slicer (Munford, USA) filled with ice-cold HBSS (adjusted to 400 milliosmoles with sodium chloride). The 100 μm thickness has been employed before21 and ensures

Figure 1. Illustration of the procedure of hyperbaric exposure of deepsea fish liver cells and detailed view of the hyperbaric culture system. Hyperbaric chambers are mounted on magnetic stirrers and small windows on their top allow verifying the correct homogenization of media containing pollutant during exposures at high hydrostatic pressure. The hyperbaric chambers are pressurized by means of an airdriven liquid pump and maintained at 4 °C by means of cooled water circulating in their outer shell. A section of the hyperbaric chamber was virtually removed to visualize the inner chamber, with gently homogenized media containing precision-cut liver slices of deep-sea fish (not enclosed in fiberglass grids herein, for visual clarity). The magnetic barrel is kept semienclosed in an inert tube section covered with fine mesh. 10311



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Figure 2. Effects of 6 h incubation of precision-cut liver slices (PCLS) of Coryphaenoides rupestris at atmospheric pressure (0.1 MPa). (A−F) PCLS were incubated either in L15+ media alone (T6 treatment) or with 25 μM of 3-methylcholanthrene (+3-MC treatment). ATP values were normalized to PCLS protein content. Transcript levels were expressed as fold changes from the control level, representing the sampling period that occurred at the end of preincubation (T0 treatment). Results are mean ± SEM of averaged values of up to 4 PCLS from each of the 3 fish used (n = 10 for T0; n = 12 for T6; n = 11 for 3-MC in the case of both ATP and gene expression analyses). Stars indicate significant differences from T0 controls (p ≤ 0.05).

desired target was being amplified; the coding sequences obtained were deposited in GenBank database under the accession numbers JF519621 (catalase), JF519622 (cytochrome P4501A), JF519623 (elongation factor 1α), JF519624 (glutathione peroxidase), JF519625 (heat shock protein 70), and JF519626 (heat shock protein 90). Statistics. Kruskal−Wallis ANOVA and Mann−Whitney Utest were performed at the α = 0.05 level with Statistica 7.1 package from Statsoft (Tulsa, USA). Graphs were created with Sigma Plot 8.0 software from Systat (San Jose, USA).

liquid pump from Maximator (Zorge, Germany). The 3-MC dose used in the present study was chosen as it significantly increased CYP1A transcript levels in PCLS of shallow fish incubated at atmospheric pressure for 6−15 h in these hyperbaric chambers (unpublished results). ATP Content and Gene Expression Studies. The experimental procedures for ATP and protein content determination, as well as gene expression studies (real timePCR experiments based on 400 ng of total RNA, reverse transcribed with poly-dT-oligo), were performed as previously described.21 Because of the lack of knowledge of the coding sequences for the targeted transcripts in C. rupestris, primer pairs were designed according to consensus regions found in mRNA sequences of teleost homologues available in the GenBank database (see Table S1, Supporting Information) and screened for optimal efficiencies. Dissociation curves were always included in the thermal profile. The intrarun coefficient of variation, with each run corresponding to the simultaneous analysis of all samples of one fish for one target gene, was determined for the reference gene (elongation factor 1α− EF1α), and was always below 5% (data not shown), confirming the robustness of our analyses of relative transcript abundance with the delta−delta Ct method.25 Amplicons were sequenced using a methodology previously described21 to confirm that the

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RESULTS Individuals of Coryphaenoides rupestris were sampled in Trondheimsfjord (Norway) and liver slices obtained within 30 min of the fish arriving on deck. PCLS were maintained in media at atmospheric pressure onboard the research vessel for about 30 min, until experimental exposure to the AhR agonist took place at Trondheim Biological Station (NTNU, Norway). Several PCLS were sampled at the end of this preincubation period to determine the initial ATP content and transcript levels for the genes under study. This was considered as time 0 of incubation (T0 controls), for the experiments below. Slices from the first cruise were used to investigate the viability and functionality of C. rupestris liver cells as well as the 10312



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Figure 3. Effects of 15 h exposure of precision-cut liver slices (PCLS) of Coryphaenoides rupestris to 3-methylcholanthrene (3-MC) at atmospheric and deep-sea pressures. (A−F) This second exposure of PCLS to 3-MC (25 μM in L15+ media) was performed at levels of hydrostatic pressure representative of the shallow (0.1 MPa treatment) and deep-sea (5 and 15 MPa treatments) environments. Same mode of data expression and statistical analysis as in Figure 2, with 3 to 4 PCLS from each of the 3 fish used in this experiment per condition (n = 11 for T0; n = 10 for 0.1 MPa; n = 12 for 5 and for 15 MPa in the case of ATP measurements and n = 10 for T0; n = 10 for 0.1 MPa; n = 9 for 5 MPa; n = 11 for 15 MPa in the case of gene expression studies). Stars indicate significant differences from T0 controls and, only in the case of PCLS incubated at 5 and 15 MPa treatments, rhombuses indicate significant differences from 0.1 MPa treatment (p ≤ 0.05).

transcript level of potential pollutant-responsive genes following a short-term incubation at atmospheric pressure (0.1 MPa) with or without the model POP (3-MC). With mean values ≥2 nmol mg−1 protein, the ATP levels of PCLS incubated in the presence or absence of 3-MC were comparable to those of the T0 controls. Still, the ATP contents of PCLS that were not exposed to the AhR agonist were slightly decreased from T0 controls (Figure 2A). A significant induction of CYP1A mRNA (5-fold over controls) was observed only in PCLS incubated with 3-MC (Figure 2B). Among antioxidant enzymes, neither CAT nor GPX mRNA contents were significantly affected by 6 h incubation at atmospheric pressure with or without the AhR agonist (Figure 2C and D). However, both hsp70 (Figure 2E) and hsp90 (Figure 2F) mRNA contents were several fold increased above T0 controls, regardless of the presence or absence of 3-MC in the media. Slices from the second cruise were used to determine the impact of hydrostatic pressure on gene expression in liver cells exposed to the AhR agonist. This time, PCLS were incubated with 3-MC for 15 h in pressure conditions equivalent to the shallow (0.1 MPa) and deep-sea (5−15 MPa; equivalent to 500−1500 m depth) environments. The ATP contents of

PCLS after exposure to 3-MC at various levels of hydrostatic pressure were comparable to those of the T0 controls, with mean values >2 nmol mg−1 protein (Figure 3A). CYP1A transcript levels were significantly increased above T0 controls, under each pressure regime (Figure 3B). Importantly, the induction seen at atmospheric pressure (18fold over T0 controls) was significantly higher than that at the deep-sea pressure levels. The CAT transcript levels were decreased from T0 controls at both 0.1 and 15 MPa (Figure 3C). However, GPX mRNA contents were significantly increased to a similar extent over T0 controls at these pressure levels, although there was a moderate decrease in GPX transcript levels from T0 controls and 0.1 at 5 MPa (Figure 3D). Both hsp70 (Figure 3E) and hsp90 (Figure 3F) transcript levels were strongly induced during this second experiment. These increases were particularly marked at atmospheric pressure, but the induction was significantly less at elevated pressures than at atmospheric pressure.

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DISCUSSION One of the difficulties with experiments carried out on deep-sea animals is the inevitable large drop in pressure they undergo 10313



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that some general effects of pressure on transcription might be involved as well,37 although the observation of similar patterns of transcript levels for antioxidant enzymes at 0.1 and 15 MPa in the second experiment seems to exclude a global inhibition of transcription upon recompression. Studies to assess the mechanisms of reduced responsiveness will address this issue. There is clear evidence for induction of CYP1A by POPs in liver of fish sampled directly from the deep-sea.6,19 Our experimental approach can be used to establish dose responsiveness in liver. The lesser induction we found at the higher pressure, even with a single dose of an AHR agonist, could help explain the limited responsiveness inferred from correlation between 7-ethoxyresorufin-O-deethylase (EROD; a CYP1A activity) in hepatic subcellular fractions and contaminant levels in deep-sea fish,19,38 compared to such relationships observed in shallow species. The absence of a pollutantmediated transcriptional induction of CAT, which positively correlates with EROD in liver of C. rupestris,19 suggests that longer incubation periods with the model xenobiotic could be required to generate a CYP1A-dependent oxidative stress response in PCLS. Future experiments should therefore attempt to increase the period of exposure to address this question. While the mechanisms leading to the reduced CYP1A induction in PCLS at high-pressure remain unknown, it is noteworthy that hsp90 is specifically involved in stabilizing the AhR that mediates CYP1A transcription in fish.39 Therefore, the pressure-dependent reduction of both hsp90 and CYP1A induction could point to a reduced efficiency of the AhR signaling pathway in liver cells of C. rupestris at deep-sea pressure levels. A limited efficiency of the CYP1A induction response could negatively affect the excretion of xenobiotics, and therefore contribute to the POP bioaccumulation potential of this fauna. Future experiments in the lab will investigate the molecular aspects of the limited responsiveness of the AhR pathway at high pressure in deep-sea fish, as well as focus on the integrated metabolism and toxicity of persistent chemicals with the PCLS model. Three decades after POPs were first detected in a deep-sea fish, the present study acts as a starting point for in-depth investigations of the toxicity of the many pollutants to which this remote fauna is exposed, either chronically (through the continuous discharge of xenobiotics into the oceans) or during acute episodes (such as the Deepwater Horizon oil spill).

when brought to the surface, and the uncertainty of possible damage caused by such decompression, and the subsequent recompression in experiments. Our data on ATP content and gene expression suggest that cells in PCLS retain their function despite the decompression and recompression of the tissue. A baro-tolerant phenotype was previously observed in short-term recompression experiments (up to 40 MPa) of freshly isolated nerves26 and hearts27 of Coryphaenoides armatus (300−5200 m depth). Fibroblast-like cells of the fish Simenchelys parasitica (140−2600 m depth) also retained their ability to grow at pressures up to 25 MPa without signs of cytoskeletal damages, after having been in culture for several months at atmospheric pressure.28 Together, the results suggest that tissues of deep-sea fish are able to cope with the recompression stress that is unavoidable in experimental studies. The ATP content of cells in experimentally treated PCLS was always close to that of T0 controls, even after 15 h incubation at high hydrostatic pressure, indicating that viability was preserved. Still, the ATP values we observed were about half those found in PCLS of shallow water fish at atmospheric pressure.21,29 Though we cannot exclude that the stressful procedure of retrieval could have reduced ATP levels to some extent, low energy content in liver slices of C. rupestris is in accordance with the low metabolic rates observed for deep-sea grenadiers compared to their shallow water counterparts.30−32 We did not take into account the influence of hydrostatic pressure on oxygen solubility (i.e., increased partial pressure) when saturating the culture media in the second experiment. However, such effect can be estimated knowing the partial molar volume of oxygen in aqueous solution at 4 °C, as reviewed elsewhere.24 Our calculations yielded increased fugacity values of 1.07 at 5 MPa and 1.23 at 15 MPa, which should not have resulted in a limiting oxygen supply to PCLS throughout the incubation. Future studies should investigate the potential effects of oxygen using media with controlled concentrations (i.e., corrected for partial pressure) and/or continuous renewal. Along with the maintenance of stable energy content, liver cells retained their ability to respond to an AhR agonist with induction of CYP1A mRNA, indicating that their functionality was preserved. Thus, we can conclude that the present model constitutes a valid tool for experimental studies of the shortterm transcriptional response of deep-sea fish to xenobiotics at relevant pressure levels. PCLS maintained at atmospheric pressure in the first experiment showed increased mRNA levels of both hsp70 and hsp90, two chaperones involved in the cellular response to membrane hyperfluidization and/or protein folding.33,34 This feature is not unlike the rise of hsp60 seen in deep-sea bacteria cultured at atmospheric pressure.35,36 We suggest that the lesser induction of these chaperones at 5 and 15 MPa than at 0.1 MPa could reflect the stabilizing influence of pressure on membrane fluidity and protein structure in the barophilic deep-sea fish cells.1 In favor of this, significant inductions of hsp70 transcription were previously recorded in mouse melanoma cells following one-hour exposures to membrane fluidizer chemicals.34 Our studies show that CYP1A transcript levels were induced by the model POP at all pressures examined. However, our data also indicate that CYP1A induction in PCLS of C. rupestris is significantly less at high pressure. The lesser amount of transcript induced at 15 MPa most likely reflects specific effects on the AhR signal transduction process. There is a possibility

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ASSOCIATED CONTENT

S Supporting Information *

Sequence of primer pairs used in gene expression studies. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +32 10 47 35 17. Fax: +32 10 47 35 15. E-mail: [email protected]. Present Address ▽

Biology Department, Woods Hole Oceanographic Institution, MA-02543 Woods Hole, U.S.A. Author Contributions

B.L., C.D., P.B.C., J.P.T., J.M., and J.F.R. were involved in study design. B.L. and J.F.R. performed experiments at Trondheim Biological Station (NTNU, Norway). B.L. performed gene 10314



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expression studies (including sequencing and submission of sequences to GenBank database) and analyzed data. All authors discussed data and B.L., J.F.R., and J.S. wrote the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the captain and crew of “R.V. Gunnerus” (NTNU, Norway), Steve Calberson, and Peter Anger for technical help. This work was funded by the Fonds de la Recherche Scientifique (F.R.S.-F.N.R.S., Belgium), the Fonds de la Recherche Fondamentale Collective (F.R.F.C., Belgium), and the Fonds de la Recherche dans l’Industrie et l’Agriculture (F.R.I.A., Belgium, PhD fellowship to B.L.).

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