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A physiologically-based toxicokinetic model for the zebrafish Danio rerio Alexandre R.R. Péry, James Devillers, Céline Brochot, Enrico Mombelli, Olivier Palluel, Benjamin Piccini, François Brion, and Remy Beaudouin Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 02 Dec 2013 Downloaded from http://pubs.acs.org on December 8, 2013
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Environmental Science & Technology
BRAIN GILLS
GONADS
LIVER
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A physiologically-based toxicokinetic model for the zebrafish Danio rerio
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Alexandre R.R. Péry,
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Palluel, § Benjamin Piccini, § François Brion, § and Rémy Beaudouin †
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Unité Modèles pour l'Écotoxicologie et la Toxicologie (METO), INERIS, Parc Technologique
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Alata, BP2, 60550 Verneuil-en-Halatte, France, CTIS, 3 Chemin de la gravière, 69140
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Rillieux La Pape, France and Unité Ecotoxicologie in vitro et in vivo (ECOT), INERIS, Parc
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Technologique Alata, BP2, 60550 Verneuil-en-Halatte, France †
METO
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‡
CTIS
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§
ECOT
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†*
James Devillers,
‡
Céline Brochot,
†
Enrico Mombelli,
†
Olivier
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*
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[email protected] Corresponding author phone: +33 3 44 55 61 26; fax: +33 3 44 55 67 67; e-mail:
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Abstract
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Zebrafish (Danio rerio) is a widely used model for toxicological studies, in particular those
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related to investigations on endocrine disruption. The development and regulatory use of in
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vivo and in vitro tests based on this species can be enhanced by toxicokinetic modeling. For
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this reason, we propose a physiologically based toxicokinetic (PBTK) model for zebrafish
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describing the uptake and disposition of organic chemicals. The model is based on literature
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data on zebrafish, other cyprinidae and other fish families, new experimental physiological
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information (volumes, lipids and water contents) obtained from zebrafish, and chemical-
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specific parameters predicted by generic models. The relevance of available models
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predicting the latter parameters was evaluated with respect to gill uptake and partition
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coefficients in zebrafish. This evaluation benefited from the fact that the influence of
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confounding factors such as body weight and temperature on ventilation rate was included in
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our model. The predictions for six chemicals (65 data points) yielded by our PBTK model
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were compared to available toxicokinetics data for zebrafish and all of them were within a
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factor of 5 of the corresponding experimental values. Sensitivity analysis highlighted that the
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1-octanol/water partition coefficient, the metabolism rate, and all the parameters that enable
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the prediction of assimilation efficiency and partitioning of chemicals need to be precisely
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determined in order to allow an effective toxicokinetic modeling.
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INTRODUCTION
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Zebrafish (Danio rerio) is now used worldwide in a variety of biological disciplines ranging
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from basic developmental biology to applied toxicology1. In the last few years, zebrafish
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studies on Endocrine Disrupting Chemicals (EDCs) led to significant advances to assess mode
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of action of EDCs on steroid receptor-regulated pathways2-4, through both in vitro and in vivo
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reported gene models3,5 or in vivo assays recognized at international level6,7. Zebrafish has
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been found useful in EDC investigation8 and has, by far, been the most widely used fish
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species for endocrine studies in the past five years9.
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To fully exploit the potential of zebrafish in vitro and in vivo tests, it is necessary to deploy a
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tool to relate local toxicokinetics (at cell, organ and endocrine system levels) and exposure at
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whole body level. A specific class of toxicokinetic models, the physiologically based
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toxicokinetic or pharmacokinetic models - PBTK or PBPK- is adequate for this by coupling
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kinetics at local level (cell or organ) and global kinetics at whole body level10. A PBTK model
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consists of a series of mathematical equations, based on the specific physiology of an
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organism and on the physicochemical properties of a substance, which are able to describe the
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absorption, distribution, metabolism and excretion (ADME) of the compound within this
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organism. PBPK models present many other advantages. First, the possibility to calibrate
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them based only on in vitro and computing data permits to replace, or at least reduce, the
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needs for animal testing10,11. Second, they can constitute the basis for computational models
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able to describe the perturbation of endocrine systems12,13. Third, as shown by Stadnicka et
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al.11, multi-compartment models, such as PBPK models, generally outperform simple one
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compartment models with respect to simulating chemical concentrations in fish whole body.
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The reference for fish PBPK models is still the pioneer works by Nichols et al.14, who
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developed a model for the rainbow trout (Oncorhyncus mykiss). Twenty years later, the
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estimates of physiological parameters by Nichols et al. are still the basis of following PBPK
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models developed for fish11-13, 15. At present, no PBPK model has ever been developed for
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fish as small as zebrafish, for which there is still rather fragmentary knowledge of basic
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biological traits.
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We propose a PBPK model for zebrafish adults exposed in standard conditions to non-ionic
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organic chemicals in water. To achieve this, new dedicated physiological information
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(volumes, lipid and water contents) was obtained experimentally with zebrafish, and generic
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models to predict chemical-specific parameters were evaluated in regard to their relevance to
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predict kinetics in zebrafish. Finally, the predictions of our PBPK model were compared for
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many chemicals to available toxicokinetics data for zebrafish.
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MATERIALS AND METHODS
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Model structure and equations
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Our PBPK model is presented in Figure 1, and the equations are provided in Supporting
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Information, S1. The model comprises eight compartments: gills, arterial and venous blood,
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liver, gonads, brain, poorly perfused tissues and richly perfused tissues. Inclusion of liver,
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gonads and brain will permit later to address perturbations of the hypothalamic-pituitary-
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gonadal axis as performed for other fish species12,13. As in Nichols et al.14, venous blood out
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of richly perfused tissues and gonads join the portal vein and enter the liver. We performed a
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visual inspection of Danio rerio fish blood circulation in organisms from our laboratory
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culture and found veins from gonads to liver, confirming this approach. Uptake and
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elimination are considered to occur through the gill, with no substantial accumulation in this
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compartment. Uptake rate is proportional to volumetric flow rate of water and assimilation
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efficiency. Excretion rate is proportional to the same parameters and inversely proportional to
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the partition coefficient of the substance between blood and water. Biotransformation in the
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liver is considered.
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Physiological parameter calibration
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The values of the physiological parameters for Danio rerio adult males and females are
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provided in Table 1. Gonad, liver, brain and carcass were dissected, weighed for fresh tissue
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masses and subsequently lyophilized for 2 days (PowerDry PL3000, Thermo Scientific). Wet
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and dried weight for brain, liver, gonads and carcass were measured in 10 adult males (with
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total wet weight ranging from 497 to 769 mg) and 10 adult females (with total wet weight
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ranging from 734 to 1695 mg). The data are reported in Supporting Information, S2.
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Additional measurements (for blood, richly perfused tissues and gonads) were kindly
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provided by colleagues from IRSN (Institut de Radioprotection et de Sureté Nucléaire).
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Despite an extensive bibliographic research, we could not find data for fish arterial blood
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volume. We assumed that this volume is one third of the total blood volume, as in mammals.
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For female gonads, we compiled data provided by INERIS and IRSN colleagues and the data
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published by Örn et al.16 (See Supporting Information, S3), and decided to distinguish
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between low weight females (weight below 500 mg), with a mean percentage of gonads in
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total weight of 8%, and high weight females with a mean percentage of gonads in total weight
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of 17%.
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The only available data for cyprinids relative to volumetric flow rate of water through the
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gills, FWgil (ml/min), are related to carps. Yamamoto17 derived a relationship for ventilation
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volume and weight at 25°C in normoxic conditions, Vg=1.035226BW0.771371. Usually, a
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multiplicative corrective factor for temperature is proposed with an exponential dependence
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on temperature18. We thus added a corrective factor for temperature extrapolation, e0.04T,
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estimated from a regression of the data on the temperature-dependency of oxygen uptake rate
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in Danio rerio from Vergauwen et al.19. The ventilation rate estimate we thus obtained for a
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0.4 g cyprinid at 27°C would be 0.55 ml/min.
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We calculated the cardiac output based on the number of beats per minute in adult zebrafish at
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26°C estimated at 11120 and on the ratio between stroke volume and weight for trouts,
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estimated at 0.2 mL/kg21. An adult zebrafish weighting 500 mg would then have a stroke
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volume of 0.1 µL, and a cardiac output of 11.1 µL/min or 0.666 mL/h. The same correction
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for temperature as for volumetric flow rate through the gills was used to adapt cardiac output
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to given experimental conditions.
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Other physiological parameters, the water and lipid contents per organ of interest, have to be
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provided for the prediction of chemical-specific parameters. The water content was deduced
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from the ratio of dry to wet weights. The lipid content was measured using a colorimetric
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sulfo-phospho-vanillin (SPV) assay22 performed in 96-well microplate after total lipid
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extractions on lyophilized tissues performed as described by Lu et al.23.
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The total lipid content was adjusted to 5% of bodyweight, which is the average lipid content
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of the small fish used in OECD TG 305 on fish bioconcentration assessment24.
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Substances-specific parameter calibration
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For the logarithm of assimilation efficiency, Barber18 deduced an equation from the data by
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Thomann et al.25 as a function of the logarithm of the 1-octanol/water partition coefficient,
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logKow: 0.577 logKow – 2.81 for logKow
