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Article
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
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 31
Environmental Science & Technology
BRAIN GILLS
GONADS
LIVER
ACS Paragon Plus Environment
Environmental Science & Technology
Page 2 of 31
1
A physiologically-based toxicokinetic model for the zebrafish Danio rerio
2
Alexandre R.R. Péry,
3
Palluel, § Benjamin Piccini, § François Brion, § and Rémy Beaudouin †
4
Unité Modèles pour l'Écotoxicologie et la Toxicologie (METO), INERIS, Parc Technologique
5
Alata, BP2, 60550 Verneuil-en-Halatte, France, CTIS, 3 Chemin de la gravière, 69140
6
Rillieux La Pape, France and Unité Ecotoxicologie in vitro et in vivo (ECOT), INERIS, Parc
7
Technologique Alata, BP2, 60550 Verneuil-en-Halatte, France †
METO
9
‡
CTIS
10
§
ECOT
8
†*
James Devillers,
‡
Céline Brochot,
†
Enrico Mombelli,
†
Olivier
11
12
*
13
[email protected]
Corresponding author phone: +33 3 44 55 61 26; fax: +33 3 44 55 67 67; e-mail:
14
ACS Paragon Plus Environment
Page 3 of 31
Environmental Science & Technology
15
Abstract
16
Zebrafish (Danio rerio) is a widely used model for toxicological studies, in particular those
17
related to investigations on endocrine disruption. The development and regulatory use of in
18
vivo and in vitro tests based on this species can be enhanced by toxicokinetic modeling. For
19
this reason, we propose a physiologically based toxicokinetic (PBTK) model for zebrafish
20
describing the uptake and disposition of organic chemicals. The model is based on literature
21
data on zebrafish, other cyprinidae and other fish families, new experimental physiological
22
information (volumes, lipids and water contents) obtained from zebrafish, and chemical-
23
specific parameters predicted by generic models. The relevance of available models
24
predicting the latter parameters was evaluated with respect to gill uptake and partition
25
coefficients in zebrafish. This evaluation benefited from the fact that the influence of
26
confounding factors such as body weight and temperature on ventilation rate was included in
27
our model. The predictions for six chemicals (65 data points) yielded by our PBTK model
28
were compared to available toxicokinetics data for zebrafish and all of them were within a
29
factor of 5 of the corresponding experimental values. Sensitivity analysis highlighted that the
30
1-octanol/water partition coefficient, the metabolism rate, and all the parameters that enable
31
the prediction of assimilation efficiency and partitioning of chemicals need to be precisely
32
determined in order to allow an effective toxicokinetic modeling.
33
ACS Paragon Plus Environment
Environmental Science & Technology
34
INTRODUCTION
35
Zebrafish (Danio rerio) is now used worldwide in a variety of biological disciplines ranging
36
from basic developmental biology to applied toxicology1. In the last few years, zebrafish
37
studies on Endocrine Disrupting Chemicals (EDCs) led to significant advances to assess mode
38
of action of EDCs on steroid receptor-regulated pathways2-4, through both in vitro and in vivo
39
reported gene models3,5 or in vivo assays recognized at international level6,7. Zebrafish has
40
been found useful in EDC investigation8 and has, by far, been the most widely used fish
41
species for endocrine studies in the past five years9.
42
To fully exploit the potential of zebrafish in vitro and in vivo tests, it is necessary to deploy a
43
tool to relate local toxicokinetics (at cell, organ and endocrine system levels) and exposure at
44
whole body level. A specific class of toxicokinetic models, the physiologically based
45
toxicokinetic or pharmacokinetic models - PBTK or PBPK- is adequate for this by coupling
46
kinetics at local level (cell or organ) and global kinetics at whole body level10. A PBTK model
47
consists of a series of mathematical equations, based on the specific physiology of an
48
organism and on the physicochemical properties of a substance, which are able to describe the
49
absorption, distribution, metabolism and excretion (ADME) of the compound within this
50
organism. PBPK models present many other advantages. First, the possibility to calibrate
51
them based only on in vitro and computing data permits to replace, or at least reduce, the
52
needs for animal testing10,11. Second, they can constitute the basis for computational models
53
able to describe the perturbation of endocrine systems12,13. Third, as shown by Stadnicka et
54
al.11, multi-compartment models, such as PBPK models, generally outperform simple one
55
compartment models with respect to simulating chemical concentrations in fish whole body.
56
The reference for fish PBPK models is still the pioneer works by Nichols et al.14, who
57
developed a model for the rainbow trout (Oncorhyncus mykiss). Twenty years later, the
ACS Paragon Plus Environment
Page 4 of 31
Page 5 of 31
Environmental Science & Technology
58
estimates of physiological parameters by Nichols et al. are still the basis of following PBPK
59
models developed for fish11-13, 15. At present, no PBPK model has ever been developed for
60
fish as small as zebrafish, for which there is still rather fragmentary knowledge of basic
61
biological traits.
62
We propose a PBPK model for zebrafish adults exposed in standard conditions to non-ionic
63
organic chemicals in water. To achieve this, new dedicated physiological information
64
(volumes, lipid and water contents) was obtained experimentally with zebrafish, and generic
65
models to predict chemical-specific parameters were evaluated in regard to their relevance to
66
predict kinetics in zebrafish. Finally, the predictions of our PBPK model were compared for
67
many chemicals to available toxicokinetics data for zebrafish.
68
MATERIALS AND METHODS
69
Model structure and equations
70
Our PBPK model is presented in Figure 1, and the equations are provided in Supporting
71
Information, S1. The model comprises eight compartments: gills, arterial and venous blood,
72
liver, gonads, brain, poorly perfused tissues and richly perfused tissues. Inclusion of liver,
73
gonads and brain will permit later to address perturbations of the hypothalamic-pituitary-
74
gonadal axis as performed for other fish species12,13. As in Nichols et al.14, venous blood out
75
of richly perfused tissues and gonads join the portal vein and enter the liver. We performed a
76
visual inspection of Danio rerio fish blood circulation in organisms from our laboratory
77
culture and found veins from gonads to liver, confirming this approach. Uptake and
78
elimination are considered to occur through the gill, with no substantial accumulation in this
79
compartment. Uptake rate is proportional to volumetric flow rate of water and assimilation
80
efficiency. Excretion rate is proportional to the same parameters and inversely proportional to
ACS Paragon Plus Environment
Environmental Science & Technology
81
the partition coefficient of the substance between blood and water. Biotransformation in the
82
liver is considered.
83
Physiological parameter calibration
84
The values of the physiological parameters for Danio rerio adult males and females are
85
provided in Table 1. Gonad, liver, brain and carcass were dissected, weighed for fresh tissue
86
masses and subsequently lyophilized for 2 days (PowerDry PL3000, Thermo Scientific). Wet
87
and dried weight for brain, liver, gonads and carcass were measured in 10 adult males (with
88
total wet weight ranging from 497 to 769 mg) and 10 adult females (with total wet weight
89
ranging from 734 to 1695 mg). The data are reported in Supporting Information, S2.
90
Additional measurements (for blood, richly perfused tissues and gonads) were kindly
91
provided by colleagues from IRSN (Institut de Radioprotection et de Sureté Nucléaire).
92
Despite an extensive bibliographic research, we could not find data for fish arterial blood
93
volume. We assumed that this volume is one third of the total blood volume, as in mammals.
94
For female gonads, we compiled data provided by INERIS and IRSN colleagues and the data
95
published by Örn et al.16 (See Supporting Information, S3), and decided to distinguish
96
between low weight females (weight below 500 mg), with a mean percentage of gonads in
97
total weight of 8%, and high weight females with a mean percentage of gonads in total weight
98
of 17%.
99
The only available data for cyprinids relative to volumetric flow rate of water through the
100
gills, FWgil (ml/min), are related to carps. Yamamoto17 derived a relationship for ventilation
101
volume and weight at 25°C in normoxic conditions, Vg=1.035226BW0.771371. Usually, a
102
multiplicative corrective factor for temperature is proposed with an exponential dependence
103
on temperature18. We thus added a corrective factor for temperature extrapolation, e0.04T,
104
estimated from a regression of the data on the temperature-dependency of oxygen uptake rate
ACS Paragon Plus Environment
Page 6 of 31
Page 7 of 31
Environmental Science & Technology
105
in Danio rerio from Vergauwen et al.19. The ventilation rate estimate we thus obtained for a
106
0.4 g cyprinid at 27°C would be 0.55 ml/min.
107
We calculated the cardiac output based on the number of beats per minute in adult zebrafish at
108
26°C estimated at 11120 and on the ratio between stroke volume and weight for trouts,
109
estimated at 0.2 mL/kg21. An adult zebrafish weighting 500 mg would then have a stroke
110
volume of 0.1 µL, and a cardiac output of 11.1 µL/min or 0.666 mL/h. The same correction
111
for temperature as for volumetric flow rate through the gills was used to adapt cardiac output
112
to given experimental conditions.
113
Other physiological parameters, the water and lipid contents per organ of interest, have to be
114
provided for the prediction of chemical-specific parameters. The water content was deduced
115
from the ratio of dry to wet weights. The lipid content was measured using a colorimetric
116
sulfo-phospho-vanillin (SPV) assay22 performed in 96-well microplate after total lipid
117
extractions on lyophilized tissues performed as described by Lu et al.23.
118
The total lipid content was adjusted to 5% of bodyweight, which is the average lipid content
119
of the small fish used in OECD TG 305 on fish bioconcentration assessment24.
120
Substances-specific parameter calibration
121
For the logarithm of assimilation efficiency, Barber18 deduced an equation from the data by
122
Thomann et al.25 as a function of the logarithm of the 1-octanol/water partition coefficient,
123
logKow: 0.577 logKow – 2.81 for logKow
Article
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
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 31
Environmental Science & Technology
BRAIN GILLS
GONADS
LIVER
ACS Paragon Plus Environment
Environmental Science & Technology
Page 2 of 31
1
A physiologically-based toxicokinetic model for the zebrafish Danio rerio
2
Alexandre R.R. Péry,
3
Palluel, § Benjamin Piccini, § François Brion, § and Rémy Beaudouin †
4
Unité Modèles pour l'Écotoxicologie et la Toxicologie (METO), INERIS, Parc Technologique
5
Alata, BP2, 60550 Verneuil-en-Halatte, France, CTIS, 3 Chemin de la gravière, 69140
6
Rillieux La Pape, France and Unité Ecotoxicologie in vitro et in vivo (ECOT), INERIS, Parc
7
Technologique Alata, BP2, 60550 Verneuil-en-Halatte, France †
METO
9
‡
CTIS
10
§
ECOT
8
†*
James Devillers,
‡
Céline Brochot,
†
Enrico Mombelli,
†
Olivier
11
12
*
13
[email protected]
Corresponding author phone: +33 3 44 55 61 26; fax: +33 3 44 55 67 67; e-mail:
14
ACS Paragon Plus Environment
Page 3 of 31
Environmental Science & Technology
15
Abstract
16
Zebrafish (Danio rerio) is a widely used model for toxicological studies, in particular those
17
related to investigations on endocrine disruption. The development and regulatory use of in
18
vivo and in vitro tests based on this species can be enhanced by toxicokinetic modeling. For
19
this reason, we propose a physiologically based toxicokinetic (PBTK) model for zebrafish
20
describing the uptake and disposition of organic chemicals. The model is based on literature
21
data on zebrafish, other cyprinidae and other fish families, new experimental physiological
22
information (volumes, lipids and water contents) obtained from zebrafish, and chemical-
23
specific parameters predicted by generic models. The relevance of available models
24
predicting the latter parameters was evaluated with respect to gill uptake and partition
25
coefficients in zebrafish. This evaluation benefited from the fact that the influence of
26
confounding factors such as body weight and temperature on ventilation rate was included in
27
our model. The predictions for six chemicals (65 data points) yielded by our PBTK model
28
were compared to available toxicokinetics data for zebrafish and all of them were within a
29
factor of 5 of the corresponding experimental values. Sensitivity analysis highlighted that the
30
1-octanol/water partition coefficient, the metabolism rate, and all the parameters that enable
31
the prediction of assimilation efficiency and partitioning of chemicals need to be precisely
32
determined in order to allow an effective toxicokinetic modeling.
33
ACS Paragon Plus Environment
Environmental Science & Technology
34
INTRODUCTION
35
Zebrafish (Danio rerio) is now used worldwide in a variety of biological disciplines ranging
36
from basic developmental biology to applied toxicology1. In the last few years, zebrafish
37
studies on Endocrine Disrupting Chemicals (EDCs) led to significant advances to assess mode
38
of action of EDCs on steroid receptor-regulated pathways2-4, through both in vitro and in vivo
39
reported gene models3,5 or in vivo assays recognized at international level6,7. Zebrafish has
40
been found useful in EDC investigation8 and has, by far, been the most widely used fish
41
species for endocrine studies in the past five years9.
42
To fully exploit the potential of zebrafish in vitro and in vivo tests, it is necessary to deploy a
43
tool to relate local toxicokinetics (at cell, organ and endocrine system levels) and exposure at
44
whole body level. A specific class of toxicokinetic models, the physiologically based
45
toxicokinetic or pharmacokinetic models - PBTK or PBPK- is adequate for this by coupling
46
kinetics at local level (cell or organ) and global kinetics at whole body level10. A PBTK model
47
consists of a series of mathematical equations, based on the specific physiology of an
48
organism and on the physicochemical properties of a substance, which are able to describe the
49
absorption, distribution, metabolism and excretion (ADME) of the compound within this
50
organism. PBPK models present many other advantages. First, the possibility to calibrate
51
them based only on in vitro and computing data permits to replace, or at least reduce, the
52
needs for animal testing10,11. Second, they can constitute the basis for computational models
53
able to describe the perturbation of endocrine systems12,13. Third, as shown by Stadnicka et
54
al.11, multi-compartment models, such as PBPK models, generally outperform simple one
55
compartment models with respect to simulating chemical concentrations in fish whole body.
56
The reference for fish PBPK models is still the pioneer works by Nichols et al.14, who
57
developed a model for the rainbow trout (Oncorhyncus mykiss). Twenty years later, the
ACS Paragon Plus Environment
Page 4 of 31
Page 5 of 31
Environmental Science & Technology
58
estimates of physiological parameters by Nichols et al. are still the basis of following PBPK
59
models developed for fish11-13, 15. At present, no PBPK model has ever been developed for
60
fish as small as zebrafish, for which there is still rather fragmentary knowledge of basic
61
biological traits.
62
We propose a PBPK model for zebrafish adults exposed in standard conditions to non-ionic
63
organic chemicals in water. To achieve this, new dedicated physiological information
64
(volumes, lipid and water contents) was obtained experimentally with zebrafish, and generic
65
models to predict chemical-specific parameters were evaluated in regard to their relevance to
66
predict kinetics in zebrafish. Finally, the predictions of our PBPK model were compared for
67
many chemicals to available toxicokinetics data for zebrafish.
68
MATERIALS AND METHODS
69
Model structure and equations
70
Our PBPK model is presented in Figure 1, and the equations are provided in Supporting
71
Information, S1. The model comprises eight compartments: gills, arterial and venous blood,
72
liver, gonads, brain, poorly perfused tissues and richly perfused tissues. Inclusion of liver,
73
gonads and brain will permit later to address perturbations of the hypothalamic-pituitary-
74
gonadal axis as performed for other fish species12,13. As in Nichols et al.14, venous blood out
75
of richly perfused tissues and gonads join the portal vein and enter the liver. We performed a
76
visual inspection of Danio rerio fish blood circulation in organisms from our laboratory
77
culture and found veins from gonads to liver, confirming this approach. Uptake and
78
elimination are considered to occur through the gill, with no substantial accumulation in this
79
compartment. Uptake rate is proportional to volumetric flow rate of water and assimilation
80
efficiency. Excretion rate is proportional to the same parameters and inversely proportional to
ACS Paragon Plus Environment
Environmental Science & Technology
81
the partition coefficient of the substance between blood and water. Biotransformation in the
82
liver is considered.
83
Physiological parameter calibration
84
The values of the physiological parameters for Danio rerio adult males and females are
85
provided in Table 1. Gonad, liver, brain and carcass were dissected, weighed for fresh tissue
86
masses and subsequently lyophilized for 2 days (PowerDry PL3000, Thermo Scientific). Wet
87
and dried weight for brain, liver, gonads and carcass were measured in 10 adult males (with
88
total wet weight ranging from 497 to 769 mg) and 10 adult females (with total wet weight
89
ranging from 734 to 1695 mg). The data are reported in Supporting Information, S2.
90
Additional measurements (for blood, richly perfused tissues and gonads) were kindly
91
provided by colleagues from IRSN (Institut de Radioprotection et de Sureté Nucléaire).
92
Despite an extensive bibliographic research, we could not find data for fish arterial blood
93
volume. We assumed that this volume is one third of the total blood volume, as in mammals.
94
For female gonads, we compiled data provided by INERIS and IRSN colleagues and the data
95
published by Örn et al.16 (See Supporting Information, S3), and decided to distinguish
96
between low weight females (weight below 500 mg), with a mean percentage of gonads in
97
total weight of 8%, and high weight females with a mean percentage of gonads in total weight
98
of 17%.
99
The only available data for cyprinids relative to volumetric flow rate of water through the
100
gills, FWgil (ml/min), are related to carps. Yamamoto17 derived a relationship for ventilation
101
volume and weight at 25°C in normoxic conditions, Vg=1.035226BW0.771371. Usually, a
102
multiplicative corrective factor for temperature is proposed with an exponential dependence
103
on temperature18. We thus added a corrective factor for temperature extrapolation, e0.04T,
104
estimated from a regression of the data on the temperature-dependency of oxygen uptake rate
ACS Paragon Plus Environment
Page 6 of 31
Page 7 of 31
Environmental Science & Technology
105
in Danio rerio from Vergauwen et al.19. The ventilation rate estimate we thus obtained for a
106
0.4 g cyprinid at 27°C would be 0.55 ml/min.
107
We calculated the cardiac output based on the number of beats per minute in adult zebrafish at
108
26°C estimated at 11120 and on the ratio between stroke volume and weight for trouts,
109
estimated at 0.2 mL/kg21. An adult zebrafish weighting 500 mg would then have a stroke
110
volume of 0.1 µL, and a cardiac output of 11.1 µL/min or 0.666 mL/h. The same correction
111
for temperature as for volumetric flow rate through the gills was used to adapt cardiac output
112
to given experimental conditions.
113
Other physiological parameters, the water and lipid contents per organ of interest, have to be
114
provided for the prediction of chemical-specific parameters. The water content was deduced
115
from the ratio of dry to wet weights. The lipid content was measured using a colorimetric
116
sulfo-phospho-vanillin (SPV) assay22 performed in 96-well microplate after total lipid
117
extractions on lyophilized tissues performed as described by Lu et al.23.
118
The total lipid content was adjusted to 5% of bodyweight, which is the average lipid content
119
of the small fish used in OECD TG 305 on fish bioconcentration assessment24.
120
Substances-specific parameter calibration
121
For the logarithm of assimilation efficiency, Barber18 deduced an equation from the data by
122
Thomann et al.25 as a function of the logarithm of the 1-octanol/water partition coefficient,
123
logKow: 0.577 logKow – 2.81 for logKow
