Biotransformation of benzo(a)pyrene by three rainbow trout

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Article

Biotransformation of benzo(a)pyrene by three rainbow trout (Onchorhynchus mykiss) cell lines and extrapolation to derive a fish bioconcentration factor Julita Stadnicka-Michalak, Frederik T Weiss, Melanie Fischer, Katrin Tanneberger, and Kristin Schirmer Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b04548 • Publication Date (Web): 05 Feb 2018 Downloaded from http://pubs.acs.org on February 6, 2018

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Environmental Science & Technology

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Biotransformation of benzo(a)pyrene by three rainbow

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trout (Onchorhynchus mykiss) cell lines and extrapolation

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to derive a fish bioconcentration factor

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Julita Stadnicka-Michalak1,2,§, Frederik T. Weiss1,3,§, Melanie Fischer1, Katrin

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Tanneberger1,4, Kristin Schirmer1,2,3*

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1

Eawag, Überlandstrasse 133, 8600 Dübendorf, Switzerland

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2

EPF Lausanne, School of Architecture, Civil and Environmental Engineering, 1015

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Lausanne, Switzerland

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3

ETH Zürich, Institute of Biogeochemistry and Pollutant Dynamics, 8092 Zürich, Switzerland

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4

Ecosens AG, 8304 Wallisellen, Switzerland

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§ These authors contributed equally to this work

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* Corresponding author: Prof. Dr. Kristin Schirmer

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Eawag, Swiss Federal Institute of Aquatic Science and Technology

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Überlandstrasse 133

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P.O. Box 611

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8600 Dübendorf

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Switzerland

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Phone: +41 (0)58 765 5266

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Fax: +41 (0)44 823 53 11

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email: [email protected] 1 ACS Paragon Plus Environment


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1 Abstract

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Permanent fish cell lines constitute a promising complement or substitute for fish in the

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environmental risk assessment of chemicals. We demonstrate the potential of a set of cell

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lines originating from rainbow trout (Oncorhynchus mykiss) to aid in the prediction of

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chemical bioaccumulation in fish, using benzo(a)pyrene (BaP) as model chemical. We

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selected three cell lines from different tissues to more fully account for whole body

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biotransformation in vivo: the RTL-W1 cell line representing the liver as major site of

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biotransformation; and the RTgill-W1 (gill) and RTgutGC (intestine) cell lines as important

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environment-organism interfaces, which likely influence chemical uptake. All three cell lines

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were found to effectively biotransform BaP. However, rates of in vitro clearance differed,

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with the RTL-W1 cell line being most efficient, followed by RTgutGC. Co-exposures with

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alpha-naphthoflavone as potent inhibitor of biotransformation, assessment of CYP1A

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catalytic activity, as well as the progression of cellular toxicity upon prolonged BaP exposure

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revealed that BaP is handled differently in the RTgill-W1 compared to the other two cell

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lines. Application of the cell line derived in vitro clearance rates into a physiology-based

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toxicokinetic model predicted a BaP bioconcentration factor (BCF) of 909-1057 compared to

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920 reported in vivo in rainbow trout.

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TOC Art

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2 Introduction

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Fish are the most extensively used vertebrate in environmental risk assessment of chemicals

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and effluents. Yet, the enormous resources required for tests relying on fish, along with the

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ethically questionable nature of such tests, have long prompted recognition that alternative

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methods are urgently needed.1 One alternative strategy is to apply permanent cell lines from

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fish.2,

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eliminates the need to sacrifice additional fish; they are usually homogeneous in nature and

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easy to maintain. Several recent developments attest to their potential to reduce or replace the

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use of fish, such as in fish acute toxicity testing4 or for the prediction of chemical impact on

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fish growth.5 We here extend our efforts to develop alternatives based on permanent fish cell

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lines with the aim to predict the bioaccumulation of chemicals in fish.

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Bioaccumulation of a chemical in an organism is the net result of toxicokinetic processes, i.e.

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uptake, internal distribution, biotransformation and elimination. If the rates of uptake and

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internal distribution outcompete those of biotransformation and elimination, an increase in

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internal concentration compared to the exposure environment occurs.6 The resulting

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bioconcentration factor (BCF) is a common metric of interest in environmental risk

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assessment of chemicals, and is usually determined from laboratory experiments using fish

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according to the OECD TG 305.7 Inasmuch as it is not feasible to test all chemicals requiring

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bioaccumulation assessment in this way, various predictive models have been built. These

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range from empirical regression models,6 to Quantitative Structure Activity Relationship

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(QSAR) models for estimating biotransformation rates based on chemical structure8 to one

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compartmente.g.9 or multiple compartment10 mass balance-based models of differing

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complexity. One- and multiple compartment models can take, respectively, whole body or

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even tissue-specific biotransformation rate constants into account. Indeed, a recent expert

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workshop concluded that the biotransformation rate constant represents the principal source

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Once established, cell lines comprise a valuable source of biological material that



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of uncertainty in the bioaccumulation assessment of most hydrophobic chemicals and that in

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vitro-to-in vivo extrapolation methods for obtaining biotransformation rate information is one

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approach to address this uncertainty.11

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In vitro methods to obtain biotransformation rate constants have thus far focused on liver

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derived entities: fish liver cells, i.e. hepatocytes12-14; liver-derived S9 fractions15, 16 or liver

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microsomes15, as well as hepatocyte spheroid cultures17, 18. The focus on liver results from its

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recognition as major site of biotransformation; however, it is conceivable that other tissues,

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such as the gill or the intestine as large environment-organism interfaces play important roles

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in the mitigation of chemicals as well. Isolated gut segments or microsomes of channel catfish

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(Ictalurus punctatus), for example, were shown to biotransform benzo(a)pyrene19,

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primary rainbow trout gill cells to exhibit 7-ethoxyresorufin-O-deethylase (EROD) activity, a

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catalytic measure of cytochrome P4501A (CYP1A).21

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On this background, we hypothesized that it is possible to predict the bioconcentration of

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chemicals in fish by exposing fish cell lines to a given chemical and monitoring chemical

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depletion over time to obtain biotransformation rate constants in a manner similar to that

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previously done with primary hepatocytes, S9 fractions or microsomes. The in vitro rate

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constants are then incorporated into a physiologically-based toxicokinetic (i.e. a multi-

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compartment PBTK) model which accounts for chemical uptake, internal distribution and

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elimination from rainbow trout22 as well as for its biotransformation in different fish organs as

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determined in vitro for the different cell lines. We proposed the use of rainbow trout

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(Oncorhynchus mykiss) cell lines originating from gills (RTgill-W123) and intestine

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(RTgutGC24), as important entry routes for chemicals into the fish body, and of liver (RTL-

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W125), representing the best-known site of biotransformation. We selected the polycyclic

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aromatic hydrocarbon (PAH) benzo(a)pyrene (BaP) as a model chemical because its

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biotransformation has previously been quantified in rainbow trout hepatocytes12,

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because a BCF has been experimentally determined in vivo in rainbow trout.26 4 ACS Paragon Plus Environment

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and

and

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3. Materials and Methods

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3.1 Benzo(a)pyrene (BaP)

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Crystalline BaP with a purity ≥ 96% was purchased from Sigma-Aldrich, Switzerland. For the

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cell viability and EROD activity tests, a 5 mM BaP stock solution was prepared by dissolving

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BaP into dimethyl sulfoxide (DMSO; Sigma-Aldrich, Switzerland).

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and chemical purity ≥ 99%, specific activity: 984.2 Bq/nmol; total amount of

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Benzo[a]pyrene: 50 µCI = 1.85 106 Bq; molarity: 3.76 mM), obtained from the American

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Radiolabelled Chemicals Inc., was purchased in toluene. The toluene was evaporated under a

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gentle stream of nitrogen and replaced by DMSO prior to use. For quantification of BaP by

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radio-HPLC (see below), a mixture of the 14C-BaP and the unlabelled BaP stock solution was

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prepared, yielding a 4.14 mM 14C-BaP working solution.

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3.2 Cell culture and seeding

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Cells were routinely cultured in 75 cm2 cell culture flasks (Techno Plastic Product AG,

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Switzerland) with L-15 Leibovitz’s medium (Invitrogen, Switzerland) supplemented with 5%

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FBSgold (F7524; Sigma-Aldrich, Switzerland) and 1% gentamicin (10 mg/mL; Invitrogen,

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Switzerland), collectively referred to as culture medium, at 19±1°C in absence of light in

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ambient atmosphere. Cells of passages 60 to 90 were used for the experiments.

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For all exposure tests, confluent monolayers of the individual cell lines were used. Cell

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viability and the EROD activity tests (see below) were carried out in 48-well tissue culture

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plates (Techno Plastic Products AG, Switzerland). For BaP quantification, 75 cm2 cell culture

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flasks were used. To obtain confluent cell monolayers in 48-well plates, 17 500 RTgut-GC

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and RTL-W1 cells, and 75 000 RTgill-W1 cells were plated into 0.5 mL culture medium per

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cavity. For the 75 cm2 cell culture flasks, 3.375 106 RTgutGC and RTL-W1 cells, and 11.250

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106 RTgill-W1 cells were plated in 9.5 mL of culture medium. Prior to the exposure, seeded

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cells were incubated for three days. 5 ACS Paragon Plus Environment

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C-BaP (radiochemical 14

C-


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All exposures were carried out in the incubator in the dark at 19±1°C in ambient air. Although

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the fish cells can survive temperatures between 0-28°C at least for short times27, the rationale

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for choosing 19±1°C was that this is the temperatures to which the cells have physiologically

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adapted and is thus considered optimal.

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3.3 Exposure experiments

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Exposure experiments were designed to measure the impact of BaP on cell viability, the time-

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dependent activity of 7-ethoxyresorufin-O-deethylase (EROD as indicator of CYP1A), and

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the distribution of BaP over time. Exposure times and BaP concentrations were adjusted to

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optimize the recording of each of these measures.

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Cell viability

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Cells were exposed to a range of BaP concentrations (0.01, 0.05, 0.1, 0.5, 1, 5 and 10 µM;

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unlabeled) for 1, 2, 3 and 11 days. For dosing, aliquots of 2.5 µL of BaP in DMSO were

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added to 0.5 mL culture medium as previously described,28,29 yielding a final DMSO

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concentration of 0.5% (v/v). An individual 48-well plate was prepared for each exposure time

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with each concentration being added to 6 wells. Four wells served as DMSO control (no

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BaP). As well, one additional well was used as negative control (cells with only culture

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medium) whereas another one was left without cells but received 10 µM BaP (blank) to

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account for potential background fluorescence by the BaP.28 After the exposure, cell viability

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was assessed fluorometrically exactly as previously described28,29 using a mixture of

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alamarBlue (Invitrogen, Switzerland), as a measure of the disturbance of metabolic activity,

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and 5-carboxyfluorescein diacetate acetoxy methyl ester (CFDA-AM; Invitrogen,

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Switzerland), as indicator of adverse effects to plasma membrane integrity (SI, Quantification

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of cell viability).


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EROD (7-ethoxyresorufin-O-deethylase) activity

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Cells were exposed to BaP as described above, ranging from 0.0025 to 1 µM of BaP and

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EROD activity measured after 6, 24, 48 and 72 hours. The plate set-up was essentially as

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described above with one 48-well plate per time point and each plate containing replicate

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wells for exposure and appropriate controls. EROD activity was measured in live cells as

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recommended by Ganassin et al.,30 including addition of CFDA-AM as indicator of cell

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viability into the EROD assay mixture and using fluorescamine to determine protein content.

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TCDD (2,3,7,8-tetrachlorodibenzodioxin) was added on each assay plate as positive control.

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The fluorescent product of the enzyme reaction, resorufin, was quantified at excitation and

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emission wavelengths of 530 and 590 nm, respectively, using the Infinite M200 multi-well

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plate reader. CFDA-AM was quantified at the wavelength settings as described above.

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BaP distribution and depletion

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A nominal BaP exposure concentration of 1.6 µM was chosen for chemical distribution and

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depletion analysis. Two criteria were set for deciding on this concentration. The first was that

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the concentration should be non-toxic to the cells as determined in the cell viability assays

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(see above) in order to avoid interference in the cell functioning by a cytotoxic action of BaP.

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The second criterion was to have a sufficient margin for chemical depletion given the limit of

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quantification of the BaP analysis method, which was 0.02 µM for the cell and plastic

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compartment and 0.2 µM for the medium (see below). To initiate exposure of the cell

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monolayers established in 75 cm2 cell culture flasks, 5 mL of the 9.5 mL cell culture medium

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were removed, transferred into sterile 7 mL amber glass vials (Supelco Sigma, Switzerland)

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and 3.7 µL of the 4.14 mM

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transferred back into the respective cell culture flask, yielding a final DMSO content of 0.04%

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(v/v).

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To verify the initial amount of BaP in the cell culture medium, i.e. at the beginning of the

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exposure test, an aliquot of 50 µL was removed from each flask and dissolved in 250 µL 7

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C-BaP working solution were added. This medium was then

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acetonitrile (ACN). A volume of 100 µL of this solution was injected into an HPLC (1100

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Series, Hewlett-Packard GmbH and Agilent), coupled to a radio detector (radio-HPLC; Flow

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Scintillation Analyzer; 500 TR Series, A Packard Cranberra company [now PerkinElmer],

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Switzerland) as described below. After 6, 8, 12, 24, 48 and 72 hours of exposure to BaP, an

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aliquot of the culture medium, the cells and the plastic wall of the respective cell culture flask

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were extracted by ACN essentially as previously described31 (see Supporting Information SI:

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Chemical analysis). These experiments were repeated three independent times starting with

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cells from different passages. Recovery of BaP from cell-free flasks over the 72 hour period

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was confirmed to range from 94% to 112% in two independent experiments.

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In a separate set of experiments, cells were pre-exposed to a potential inhibitor of BaP

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biotransformation, alpha-naphthoflavone (ANF - Sigma-Aldrich, Switzerland; 1 µM,

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dissolved in DMSO, according to28) one hour before the addition of BaP. Co-exposures of

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ANF and BaP were done for 8, 12 and 48 hours with subsequent procedures as described

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above.

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Determination of cell number in cell culture flasks

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To obtain measures of cell size and number for input into the kinetic models (see below), cells

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plated in 75 cm2 cell culture flasks as described above were exposed to unlabelled BaP. At

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each time point (0, 6, 8, 12, 48, and 72 hours), cells were dislodged by first washing them

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with Versene, followed by incubation with trypsin and careful re-suspension in a total volume

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of 5 mL culture medium. Upon centrifugation, the cell pellet was re-suspended into 2 mL of

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culture medium. An aliquot of 10 µL of this cell suspension was added to 10 mL CASY

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solution and cell number and size (set to 7.5-30 µm) recorded using the electric field multi-

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channel cell counting system CASY1 TCC (Schärfe System, Germany). Measurements were

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repeated with three independent preparations having three flasks each, yielding relative

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standard deviations of less than 15% for cell number and less than 6% for cell diameter. The 8 ACS Paragon Plus Environment

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resulting measures and calculations needed for the modelling are presented in SI: PBTK

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model.

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Incorporating in vitro intrinsic clearance rates into the PBTK model

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The PBTK model used in this study, and originally developed for rainbow trout10,32, was

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improved by including the tissue compositions from Bertelsen et al.33, blood:water and

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lipid:blood partition coefficients and fecal egestion rate from Arnot et al.34 and Nichols et

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al.35, the volume of fat compartment as in Stadnicka et al.22, and the biotransformation process

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as in Jones et al.36 and this study. The PBTK model used here consists of six different

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compartments: liver, kidney, fat, guts, and richly and poorly perfused tissues. The “guts”

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compartment is distinguished from the richly perfused compartment in order to account for

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the biotransformation process in the intestinal cells. The guts compartment includes stomach,

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pyloric caeca and upper and lower intestine. The model parameters needed for this

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compartment (like the organs’ volumes or lipid content) were obtained from the PBTK model

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for dietary uptake.37

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The in vitro (that is the cell-line based) reaction rate constants (ki) are derived as the slope of

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the relationship of measured concentrations of the parent chemical (ln y-axis) over time (x-

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axis) (see SI, PBTK Model: Equations and notes; eq. 22). The cell-line specific reaction rate

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constants are then divided by the respective average cell concentrations measured in the

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experiments with each cell line in order to obtain the in vitro intrinsic clearance rate (CLIN

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VITRO).

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respective organ (SI: Table S-1, eq. 23). The rate for the RTgill-W1 cell line was applied to

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represent all richly perfused tissues (aside from liver and intestine) in an attempt to account

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for biotransformation in the entire organism. These values, together with the so-called binding

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correction term (fU, SI: eq. 25), allow to include not only the liver but the additional

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elimination routes of the intestine and other richly perfused tissues (represented by the gills),

This rate is then multiplied by the cell number (Tcell,i) predicted to amount to 1 g of the



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i.e., in vivo intrinsic clearance rates (CLIN VIVO, SI: eq. 24), in the model according to Jones et

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al.36 (SI eq. 11, 15-17). The fU is used to account for the free chemical fraction in the in vitro

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systems. It was calculated based on the equation given in Nichols et al.38 for hepatocytes,

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given the fact that experiments for both the cell lines and the hepatocytes were carried out in

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L-15 cell culture medium and assuming comparable cell size (see SI Note to eq. 23).

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Inasmuch as the correction term is still debated because it has led to BCF overestimation in

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models that assume chemical biotransformation in fish liver alone,38,39 this term is often

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artificially set to 1.40 Thus, in addition to the calculated fU, in the results section, the BaP BCF

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is also given for the fU set to 1.

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The model was implemented and solved using ModelMaker (version 4.0, Cherwell Scientific

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Ltd., Oxford, UK), and all the model parameters and equations are presented in the SI: PBTK

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model.

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4. Results and Discussion

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The aim of our study was to test whether bioconcentration of BaP in fish, specifically rainbow

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trout, can be predicted based on in vitro intrinsic clearance rate constants obtained in three

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rainbow trout-derived cell lines: RTgill-W1 (gill), RTgutGC (intestine), and RTL-W1 (liver).

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We hypothesized that gill and intestine are potential sites of biotransformation of BaP in fish

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in addition to the liver. Thus, by accounting for BaP depletion in different tissues, one may

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more closely approach the whole body biotransformation rate implicitly accounted for in the

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in vivo tests.

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Impact of BaP on cell viability

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BaP led to a time-dependent impact on cell viability for all three cell lines as indicated by the

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measures of cell metabolic activity (alamarBlue) and cell membrane integrity (CFDA-AM)

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(SI: Cell viability, Figure S1). However, the pattern of impact prominently differed for 10 ACS Paragon Plus Environment

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RTgill-W1 cells. In contrast to the other two cell lines, the impact on cell viability in RTgill-

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W1 cells took longer to establish and was overall much less pronounced.

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Several lines of evidence suggest that the development of cytotoxicity is a result of BaP

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biotransformation and that the differences in cytotoxicity between the cell lines result from

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different biotransformation routes. First, our results are reminiscent of those reported for the

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two rainbow trout liver cell lines, RTL-W1 and R1, where distinct patterns of cell viability

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upon BaP exposure were implied to result from dissimilar routes of biotransformation active

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in the cells.28 The scenario of 11 days of exposure to BaP in that prior study was comparable

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to the one performed here and indeed resulted in similar results for RTL-W1 (see Fig. 7E in

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Schirmer et al.28). The results for the R1 cell line, on the other hand, resemble more those

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obtained here for RTgill-W1 cells with no to very little observable toxicity. According to the

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previous study, the difference seen between RTL-W1 and R1 cells was attributed to,

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respectively, the presence or absence of inducible CYP1A, measured as EROD activity,

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although a role of CYP1B could not be ruled out. Finally, when tested individually, two

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common biotransformation products of BaP, namely BaP 7,8-dihydrodiol (BDP) and 6,12

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BaP quinone (BQ), also caused cytotoxicity, though to different degrees. While the BDP was

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only slightly toxic and only to RTL-W1 cells, the BQ caused a clear impact in cell viability to

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both cell lines with R1 cells being more responsive.28 While BDP is a product of CYP-

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dependent biotransformation, BQ can additionally result from alternative enzymatic reactions,

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such as those catalyzed by peroxidases.41

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CYP1A catalytic activity measured as EROD

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BaP exposure led to significant EROD induction in RTL-W1 and RTgutGC, but not in

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RTgill-W1 cells (Figure 1). This result indicates that the former two cell lines express CYP1A

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at the functional level whereas RTgill-W1 cells do not. EROD induction rose to significant 11 ACS Paragon Plus Environment


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levels between 6 and 24 hours in both RTL-W1 and RTgutGC cells and, while observed

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maximum EROD levels continued to increase up to about 72 hours, the dose-response curves

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gradually shifted to the right, yielding higher EC50 values with time. A similar time-

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dependent shift has previously been described in RTL-W1 cells for two other PAHs,

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benzo(k)fluoranthene42 and 3-methylcholathrene,43 in contrast to TCDD, for which EC50

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values remained constant over time.42,44 As discussed in Bols et al.,42 the time-dependency of

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the EROD induction can be explained by the progressive loss of the PAH as inducing agent

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while it is being transformed by the enzyme(s) it induces. At the same time, EROD induction

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can be prolonged if biotransformation products themselves can act as inducers of EROD

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activity. Little information on this phenomenon exists thus far; however, BaP-7,8-dihydrodiol

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and 6,12-BaP quinone have been identified, respectively, as strong and weak inducer of

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EROD activity in RTL-W1.28 Finally, at higher nominal BaP exposure concentrations, a drop

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in EROD activity can be observed, which was more pronounced in RTL-W1 than in

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RTgutGC cells (Figure 1). This behavior has been reported extensively both for individual

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PAH exposures42,

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absence of any cytotoxicity (SI Figure S1), attributable to competitive inhibition of the

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CYP1A enzyme by the substrate 7-ethoxyresorufin and BaP.

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While this and another recent study46 encompass the first demonstration of EROD induction

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in RTgutGC cells, EC50 values in RTL-W1 cells have been reported in previous studies for

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24 hours of BaP exposure. The values obtained here are in excellent agreement with

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previously measured values, which were, despite about two-fold differing cell starting

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densities, 2.85 ± 0.53 10-2 µM in Schirmer et al., 2000;28, 2.55 ± 0.73 10-2 µM in Bols et al.,42

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compared to 2.14 10-2 µM here. These results confirm the robustness of the cell line and its

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response to BaP.

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Spatio-temporal distribution of BaP

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as well as for complex environmental samples45 and maybe, in the


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Mass balance analysis, i.e. the summation of BaP amounts in the different compartments,

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over time confirmed a rapid loss of parent BaP from the system but not only in RTL-W1 and

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RTgutGC cells but as well in RTgill-W1. As can be seen in Figure 2 and SI Table S-2, BaP

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medium concentrations declined quickly, falling below quantifiable levels after 12 hours in

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the case of RTL-W1 and RTgutGC and 24 hours in the case of RTgill-W1. Highest BaP

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concentrations in cells were found for RTL-W1 and RTgutGC cell lines at the earliest point of

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observation, 6 hours, after which BaP concentrations continuously declined to near or below

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the level of quantification. A similar pattern was seen for RTgill-W1 cells, though again

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delayed, with highest BaP concentrations in cells detected after 8 hours of BaP exposure. The

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plastic surface also contained significant amounts of BaP with highest levels measured at the

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first time point. Thereafter, the sorption to plastic reflected the kinetics of loss of BaP – it

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declined as BaP in the medium and the cells also declined. This finding is in agreement with a

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previous report on the distribution of BaP in a culture system using a mouse cell line model31

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and indicates that the sorption process was reversible: as the concentration gradient between

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plastics and medium/cells increased due to biotransformation, more of the BaP was released

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from the plastic surface. Taken together, while in the absence of cells BaP concentrations

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remained stable, BaP was transformed in the presence of cells by more than 90% within 24

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hours in the case of RTL-W1 and RTgutGC and within 48 hours of exposure in the case of

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RTgill-W1.

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The distribution of BaP in the culture flasks was also followed after a one hour pre-incubation

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with alpha-naphthoflavone (ANF) in order to provide further proof of biotransformation in the

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cell lines (SI Figure S2). ANF is an inhibitor of CYP1A activity and, in fact, other

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cytochromes41 and enzyme systems, such as prostaglandin-H-synthase, which consists of a

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cyclooxygenase and a peroxidase activity.47,48 ANF was previously shown to inhibit EROD

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activity in RTL-W1.28 As depicted in Figure 3, presence of ANF clearly affected the kinetics

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of BaP in the cells, leading to an initial retardation of biotransformation in RTL-W1 and 13 ACS Paragon Plus Environment


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RTgutGC cells and a severe arrest of biotransformation in RTgill-W1 cells. These results not

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only confirm that the loss of BaP in the system is due to biotransformation but also that the

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routes of biotransformation differ between the cell lines expressing inducible EROD activity

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(RTL-W1, RTgutGC) or not (RTgill-W1), as discussed above. Studying the repertoire of

326

biotransformation reactions in the different fish cell lines and their dependence on culture

327

conditions, such as temperature or medium composition, are important next steps to directly

328

link the kinetic processes with underlying enzymatic reactions. One approach is that described

329

by Thibaut et al.50, who monitored the appearance of a set of phase I and phase II

330

biotransformation products in the PLHC-1 (hepatocellular carcinoma cell line of Poeciliopsis

331

lucida) and the RTL-W1 cell line using radio-labelled substrates. Moreover, similar studies

332

are needed from tissue-based investigations or also freshly isolated cells, especially of gill and

333

intestine, to shed light on the tissue-specific biotransformation capabilities in fish. With

334

regard to the gills, one prior study reported inducible EROD activity in a primary gill cell

335

culture upon exposure to BaP21; as well, an ex vivo rainbow trout gill filament assay51

336

confirmed CYP1A protein expression and EROD induction by BaP. Compared to our results

337

with the RTgill-W1, these prior studies indicate that the cell line model does not fully mimic

338

an in vivo phenotype. Yet, more comparative studies are needed, both with respect to enzyme

339

patterns and clearance rates, to better address this issue.

340

In vitro reaction and intrinsic clearance rate constants

341

The overall loss of BaP in the culture flasks was plotted over time to obtain the in vitro

342

reaction rate constants (Figure 2D). These were in the order from highest to lowest: 0.143 h-1

343

for RTL-W1 > 0.114 h-1 for RTgutGC >> 0.071 h-1 for RTgill-W1 cells. One other previous

344

study has quantified the disappearance of BaP in confluent monolayers of fish cell cultures

345

over time:52 the bluegill fry cell line, BF-2 (from caudal trunk tissue), the rainbow trout gonad

346

cell line, RTG-2 (from embryonic gonad tissue), and the brown bullhead cell line, BB (from


Page 14 of 30

Page 15 of 30


347

posterior trunk tissue), were all capable of biotransforming BaP. Calculable reaction rate

348

constants from these cell lines amount to between 0.036 h-1 (BF-2) and 0.045 h-1 (BB), with

349

the BaP starting concentration being 2 µM. These reaction rate constants are lower than those

350

measured in our study; however, they were also obtained in larger flasks (175 cm2) with a

351

larger volume (50 mL). Cell numbers were not counted but monolayers were used as in our

352

case. If we assume that about the same number of fish cells leads to a confluent monolayer,

353

the culture surface to culture volume ratio can be applied. This ratio amounts to 3.5 in the

354

study by Smolarek et al. (1987)52 compared to 7.9 (75 cm2/9.5 mL) by us, giving a value of

355

about 2-fold higher in our study. Translating this ratio to the in vitro intrinsic clearance rate

356

(CLIN_VITRO), would therefore indicate very similar rates to the one determined for RTgill-W1

357

(see Table 1 for values of CLIN_VITRO). This finding supports our hypothesis that the gill cell

358

line may exemplify in vitro intrinsic clearance in richly perfused tissues which are, however,

359

not major hubs for biotransformation. The higher reaction rates and CLIN_VITRO for the

360

intestinal and the liver cell model, on the other hand, indicate a distinct contribution of these

361

tissues to the biotransformation of BaP. Our results with the intestinal cell line are in

362

agreement with previous studies in channel catfish which have shown that BaP can be

363

biotransformed by intestinal segments or microsomes.19, 20

364

The in vitro intrinsic clearance rate obtained in the RTL-W1 fish liver cell line might be

365

compared to rates reported for primary liver cell models. However, big differences between

366

intrinsic clearance rates were found among the primary cells: 0.054±0.023 mL/h/106cells for

367

freshly isolated hepatocytes13 compared to 0.214±0.067 mL/h/106cells for cryopreserved

368

hepatocytes.12 The CLIN_VITRO obtained for RTL-W1 (0.425±0.0198 mL/h/106cells) was

369

higher than those obtained for primary hepatocytes. It cannot as yet be stated, however, if

370

these differences are of cell intrinsic nature or are due to differences in the incubation

371

conditions. For example, RTL-W1 cells were exposed in the presence of a 5% serum

372

supplement. On the one hand, serum could contribute to BaP binding, thereby reducing 15 ACS Paragon Plus Environment


373

bioavailability and potentially intrinsic clearance.13 On the other hand, serum components

374

may in fact facilitate uptake, as pointed out by Kramer and colleagues.53 Moreover, Jones and

375

Houston54 demonstrated a trend towards higher values of intrinsic clearance for different

376

chemicals with decreasing numbers of rat hepatocytes per mL of exposure medium. Indeed,

377

our experiments with the cell line were performed with a much lower cell concentration (0.34

378

Mio cells measured per mL compared to 2 Mio for the fish hepatocytes). In addition, the

379

hepatocytes were incubated in suspension whereas RTL-W1 cells are cultured as monolayers.

380

A study focusing on rat hepatocytes in suspension and in monolayer culture has pointed out

381

that these two systems cannot easily be compared:55 intrinsic clearance rates were comparable

382

for some chemicals but both higher and lower for others.

383

Prediction of the BaP bioconcentration factor in fish

384

In order to predict the bioconcentration of BaP in fish, we applied the PBTK model adapted

385

from.22 Without taking the biotransformation process into account, a BCF of 7 045 is

386

predicted for rainbow trout, compared to a measured BCF of 920 specifically for this species

387

of fish26 (Table 1). While the predicted BCF is seven times higher than the measured, it is

388

almost 3 times lower than a previously reported BCF prediction of BaP in rainbow trout (19

389

620).12 This latter BCF was based on a one-compartment model not considering

390

biotransformation but using a fish-water partitioning coefficient that would result in a

391

different blood:water partition coefficient than the one used in our study - adapting the same

392

fish-water partitioning coefficient to our model would yield a predicted BCF of 17 800.

393

In order to take biotransformation into account, we first included the in vitro intrinsic

394

clearance rate constant, CLIN_VITRO, obtained for the RTL-W1 cell line in our PBTK model

395

together with the calculated binding correction term, fU. This reduced the predicted BaP BCF

396

by about 3.5-fold (Table 1). When the CLIN_VITRO for RTgutGC and RTgill-W1 were

397

additionally added, the predicted BCF dropped to between 909 (for 50g fish) and 1057 (for 30 16 ACS Paragon Plus Environment

Page 16 of 30

Page 17 of 30


398

g fish). Thus, including clearance rates from all three cell line models yields the predicted

399

BCF closest to the measured in vivo value of 920. In past biotransformation studies using

400

rainbow trout S9 fractions or hepatocytes, it has repeatedly been found that BCF predictions

401

based on calculated fU yielded comparatively high BCFs and a better correlation with in vivo

402

measured values was found when fU was set to 1.38,39 When doing so using the CLIN_VITRO for

403

the fish cell lines, BCF predictions fall well below the measured values, especially when

404

biotransformation in gill and intestine are additionally taken into account (Table 1). Thus, in

405

the case of our study, the use of calculated fU appears to lead to a better BCF prediction. This

406

result supports the hypothesis of Escher et al.39 that accounting for chemical

407

biotransformation in more fish organs than the liver may result in a better agreement between

408

predicted and measured BCFs when applying the calculated fU.

409

Taken together, our results provide a proof-of-principle that cell lines of rainbow trout can be

410

used to obtain in vitro intrinsic clearance rates that aid in the prediction of a chemical’s BCF

411

using PBTK models. Indeed, this is the first presentation of not only using fish cell lines to

412

predict BCFs but is also the first to incorporate in vitro biotransformation rate data for organs

413

of different origin into a fish PBTK model. We demonstrate that all three rainbow trout cell

414

lines are capable of biotransforming BaP although the exact biotransformation pathways and

415

the resemblance of these pathways with those of the respective tissues in vivo are not yet

416

know. Despite of this uncertainty, the finding of biotransformation in all three cell lines

417

confirms the significance of liver-derived cells for biotransformation and as well highlights

418

the gill and the intestine as important biotransformation sites. The relative importance of the

419

gill and the intestine for the biotransformation of a particular chemical likely depends on the

420

physico-chemical properties. For example, biotransformation can be expected to be more

421

important in the intestine than in the gill for hydrophobic chemicals, which adsorb to food and

422

thus enter the fish predominantly via this route. More water soluble and ionizable compounds,

423

on the other hand, would be expected to have greater contact with the gill surface, at least 17 ACS Paragon Plus Environment


424

initially.56 Thus, application of fish cell lines holds great potential for further investigating the

425

role of their tissue origin in the biotransformation of chemicals with a wide spectrum of

426

properties. Fish cell lines not only comprise a true alternative to using fish in chemical testing

427

but as well yield highly reproducible results. In addition, they are very flexible with regard to

428

different exposure regimes, e.g. exposure times can be extended to days in case of slowly

429

biotransformed compounds. Yet, comparison of cell line-derived clearance rates to those

430

found in freshly isolated gill, intestinal and also liver tissue, is an important future direction.

431

Among the variables to be explored are the influence of temperature57 and chemical starting

432

concentration58. Moreover, several model uncertainties need to be acknowledged. First, we

433

compare the BCF predicted with our approach to the one available in vivo-derived BCF for

434

rainbow trout, which was obtained before the OECD TG 305 was in place. Second,

435

uncertainty arises from the PBTK model itself, e.g. from the model equations or the fish

436

physiological parameters used. Two respective examples highlighted in our study are the

437

correction binding term, fU, and the chemical blood-water partition coefficient, Pbw. To

438

address such model uncertainties is one aim of an OECD expert group that is currently

439

developing of a guidance document for in vitro-in vivo BCF predictions for fish. Our work

440

focusses on rainbow trout derived cell lines combined with a PBTK model that was explicitly

441

designed to reflect physiological features of this ecologically and economically important

442

species of fish. However, PBTK models have already been developed for other species of

443

fish, e.g. various species of trouts,31,59,60 fathead minnow,22 channel catfish,61 zebrafish62 or

444

European eel.63 Thus, further cell line development and PBTK application could also aid in

445

comparing species for their biotransformation potential and sensitivity toward chemical

446

exposure.

447


Page 18 of 30

Page 19 of 30


448

5. Acknowledgement

449

We would like to thank Nadine Bramaz for her excellent support in the cell culture work,

450

particularly the EROD assay, and Danielle Madureira for her collaboration during the initial

451

establishment of the BaP dosing and extraction protocol.

452

6. Supporting Information

453

Contains details on chemical analysis (BaP extraction and quantification), the PBTK model

454

(abbreviations and symbols; equations and notes), as well as additional results (cell viability

455

and data for time-dependent distribution of BaP in culture flasks).

456



457

7. References

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Miranda, C. L.; Henderson, M. C.; Buhler, D. R., Evaluation of chemicals as inhibitors

Degen, G. H., Inhibition of prostaglandin H synthase-catalyzed cooxidation of


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shad (Dorosoma cepedianum) after waterborne exposure to benzo[a]pyrene. Comp. Biochem.

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Phys. C 1997, 118 (3), 397-404.

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and RTL-W1 fish liver cell lines. Cell Biol. Toxicol. 2009, 25 (6), 611-622.

623

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induction in rainbow trout gills and liver following exposure to waterborne indigo, benzo(a)

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pyrene and 3,3 ',4,4 ',5-pentachlorobiphenyl. Aquat. Toxicol. 2006, 79 (3), 226-232.

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dimethylbenz[a]anthracene in fish cell lines in culture. Carcinogenesis 1987, 8 (10), 1501-

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sorption kinetics in solid-phase microextraction: consequences for chemical analyses and

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uptake processes. Anal. Chem. 2007, 79 (18), 6941-6948.

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metabolic clearance: Time dependencies in hepatocyte and microsomal incubations. Drug

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Metab. Dispos. 2004, 32 (9), 973-982.

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hepatocytes: Comparison of suspensions and monolayer cultures. Drug Metab. Dispos. 2005,

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A.; Schirmer, K.; Nichols, J. W., Assessing the bioaccumulation potential of ionizable organic

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compounds: Current knowledge and research priorities. Environ. Toxicol. Chem. 2017, 36 (4),

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882-897.

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metabolism of benzo[a]pyrene in rainbow trout (Oncorhynchus mykiss): a comparison

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between the liver and immune organs. Drug Metab. Dispos. 2014, 42 (1), 111-118.

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dependence of biotransformation in fish liver S9: optimizing substrate concentrations to

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estimate hepatic clearance for bioaccumulation assessment. Environ. Toxicol. Chem. 2015, 34

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Smolarek, T. A.; Morgan, S. L.; Moynihan, C. G.; Lee, H.; Harvey, R. G.; Baird, W. Metabolism

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adduct

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benzo[a]pyrene

and

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trout (Salvelinus fontinalis). Environ. Toxicol. Chem. 1998, 17, 2422-2434.

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channel catfish, Ictalurus punctatus. Aquat. Toxicol. 1993, 27, 83-112.

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666 667


Page 26 of 30

Page 27 of 30

668


Tables

669 670

Table 1. In vitro intrinsic clearance (CLIN_VITRO), binding correction term (fU) and BaP

671

bioconcentration factors (BCFs) measured and predicted with the PBTK model. Tested cell line CLIN_VITRO [mL/h/106 cells] fU, [ ̶ ]

RTL-W1 0.425 0.090

BCF predicted for rainbow trout no biotransformation biotransformation in RTL-W1$ biotransformation in all cell lines$

fU calculated 7 045 1 842-2 100 909-1 057&

BCF measured in rainbow trout23,£ 672

$

673

values are based on this range.

674

&

675

with fu calculated of 1 126.

676

£

RTgutGC 0.268 0.070

RTgill-W1 0.074 0.032 fU set to 1 7 045 1 212-1 386 567-655

920

According to the source for BCF (ref. 26), fish weight was between 30-50 g and the predicted

For comparison, a fish weight of 10 g, as is most frequently used, would result in a BCF

This study was performed prior to the availability of the OECD305 test guideline.

677 678



679

Page 28 of 30

Figures

680 681 RTgill-W1

EROD activity (pmoles mgProt-1 min-1)

6 hours

24 hours

RTgutGC

RTL-W1

40

40

40

30

30

30

20

20

20

10

10

10

0

0 0.01

0.1

1

0 0.01

10

0.1

1

10

40

40

30

30

20

20

20

10

10

10

0

0 0.01

0.1

1

0.01

10

0.1

30

1

10

0

40

40

30

30

30

20

20

20

10

10

10

0

0

72 hours

0.01

0.1

1

10

0.01

0.1

1

10

0

40 30

20

20

20

10

10

10

0

0

1

10

10

1

10

1

10

1

10

EC50: 0.02 µM

0.01

0.1

EC50: 0.03 µM

30

0.1

1

40

40

0.01

0.1

40 EC50: 0.05 µM

EC50: 0.05 µM

48 hours

0.01

0.01

0.1

40 EC50: 0.09 µM

EC50: 0.07 µM

0.01

0.1

30

1

10

0

0.01

0.1

benzo(a)pyrene concentration (µ µ M)

682 683 684

Figure 1. CYP1A activity, measured as ethoxyresorufin-O-deethylase (EROD) induction,

685

after exposure to BaP concentrations ranging from 0.0025 to 1 µM after 6, 24, 48 and 72

686

hours (from top to bottom) of exposure. The results of two independent experiments are

687

shown (filled black vs. empty white squares). Each symbol represents the average and

688

standard deviation of six culture wells. Data is fitted based on a Gaussian approximation;

689

EC50 values were estimated by considering the ascending part of the curve using the Bell-

690

shaped dose response model (GraphPad Prism 5).

691


Page 29 of 30


692 693

Figure 2. Time-dependent distribution of BaP in culture flasks containing either RTgill-W1

694

(A), RTgutGC (B) or RTL-W1 (C) cells. At each indicated exposure time, the cells, the

695

culture medium and the plastic of the flasks were extracted and analyzed for BaP to obtain a

696

parent compound mass balance. The starting concentration of BaP was 1.6 µM. The

697

difference between total amount recovered and initial amount added was assigned to the %

698

BaP biotransformed (D). Values given are averages of flasks of three independent

699

experiments. For absolute numbers, please refer to SI Table 2.



RTgill-W1

BaP % biotransformed

125

RTgutGC

8h

RTL-W1

125

125

100

100

100

75

75

75

50

50

25

25

25

0

0

0

125

125

100

100

100

75

75

75

50

50

25

25

25

125

12h

50

0

0 125

100

100

75

75

75

50

50

50

25

25

25

48h

Biotransformation of benzo(a)pyrene by three rainbow trout

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