Reproductive Toxicology 19 (2005) 327–337

Review

Dose–response modeling in reproductive toxicology in the

systems biology era
Melvin E. Andersen∗ , Russell S. Thomas, Kevin W. Gaido, Rory B. Conolly
CIIT Centers for Health Research, Research Triangle Park, NC 27709-2137, USA

Received 15 October 2004; received in revised form 1 December 2004; accepted 3 December 2004

Abstract

Systems biology approaches for modeling cellular signaling networks affected by chemical exposures should soon produce integrated
methodologies capable of predicting dose–response relationships for developmental toxicants and for other toxic responses. This paper out-
lines an emerging strategy for systems biology approaches in dose–response modeling. Genome-wide functional screens, bioinformatic tools,
and network mapping technologies together can provide directed graph representations of the cellular signaling networks. The graphical rep-
resentations can be converted into mathematical models that permit predicting the shapes of dose–response curves for altered cell signaling by
test compounds during development. Systems biology approaches require interdisciplinary teams with expertise in reproduction, cell biology,
signal transduction, mathematical/biomedical modeling, and risk assessment. In addition to outlining a systems approach for dose–response
research, this paper discusses initial stages of application of this strategy to examine inhibition of steroidogenesis in testes by phthalate
esters.
© 2004 Published by Elsevier Inc.

Keywords: System biology; Risk assessment; Signal transduction; Biological circuits; Cellular networks; Phthalates; Steroidogenesis; Testes malformations

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327
2. Systems approaches in pharmacokinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 328
3. Linking doses and response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330
4. The arrival of systems biology in dose–response assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330
5. Cell signaling motifs as targets for developmental toxicants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331
6. Phthalates and male reproductive tract development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333
7. Assessing components of signaling pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334
8. Creating mathematical models of function and perturbations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334
9. How will these technologies be used in reproductive toxicology?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 336
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 336

1. Introduction

∗ Corresponding author. Tel.: +1 919 558 1205; fax: +1 919 558 1300. A major practical goal of toxicology research with en-
E-mail address: MAndersen@ciit.og (M.E. Andersen). vironmental chemicals and of safety research with drugs in

0890-6238/$ – see front matter © 2004 Published by Elsevier Inc.

doi:10.1016/j.reprotox.2004.12.004
328 M.E. Andersen et al. / Reproductive Toxicology 19 (2005) 327–337
the pharmaceutical industry is to understand the biological
factors that determine the shape of dose–response curves for
adverse responses at low levels of incidence, i.e., in regions
of the dose–response curve where the probability of response
in a population is small. When the quantitative relationships
among these factors are known with confidence, incidence
can be predicted (calculated) for various exposure situations
and these estimates of incidence can be used to predict ex-
pected risks to exposed populations. Together with social or
political policies that prescribe tolerable risks for specific
human populations, these predictions of risk serve multiple
functions, such as establishing exposure guidelines or sup-
porting product registrations. In the absence of knowledge
about the shape of dose–response curves in regions of low
incidence, a variety of defaults are used in risk and safety as-
sessments. These defaults take conservative positions about
likely risks in exposed humans in an attempt to insure ade-
quate protection in the absence of knowledge.
The safety/risk assessment process requires both qualita-
tive and quantitative inputs. Qualitative studies provide infor-
mation about: (1) hazard, the possible effects of a compound
irrespective of exposure considerations; (2) mode-of-action,
the manner in which chemical interacts with targets in bio-
logical systems to cause perturbations that lead to adverse
responses; (3) progression, the steps connecting these inter-
actions through to impaired function; and (4) susceptibility,
the factors that may predispose an individual to an adverse
response. Susceptibility factors include: gender, age, genet-
ics, co-exposure to other compounds, pre-existing disease,
or lifestyle. A full risk assessment process requires organiz-
ing this information quantitatively to make predictions of the
shape of the dose–response curve in regions of low incidence
in a diverse human population.
One challenge in quantitative health risk assessment arises
from the need to have models for dosimetry, i.e., the deliv-
ery of active forms of the test molecules/metabolites to tar-
get tissues, and for responses, i.e., the manner in which the
molecular and cellular interactions of toxic compounds cause
perturbations that are sufficiently large and sufficiently pro-
longed to lead to an adverse response. The models for dose
and response are conveniently dichotomized as pharmacoki-
netic (PK) models (for dose) and pharmacodynamic (PD)
models (for response). Pharmacokinetics is generally defined
as what the body does to the compound; pharmacodynamics
is what the compound does to the body. To have confidence
in predictions from PK and PD models, these models need
to be biologically realistic. Physiologically based (PB) mod-
els, both PBPK and PBPD models, tend to be more firmly
grounded in principles of biology and biochemistry. One risk
assessment goal is co-ordination of PBPK/PD models to cre-
ate biologically based dose–response (BBDR) models that
predict expected incidence of adverse responses for varied
exposure situations.
Today, we have the ability to assess the components of cel-
lular, organ, and organism-level processes with astonishing
detail using a variety of relatively new technologies, referred
to as ‘omics,’ including components of genes, gene products,
proteins and various small molecules. This diverse array of
data, however, needs to be organized quantitatively to create
information, i.e., to discover how the parts of the system are
organized and controlled both in time and in space to give rise
to biological functions. We can define ‘systems approaches’
as the attempt to quantitatively integrate information over
multiple levels of biological organization to understand how
biological functions are produced from simpler molecular,
cellular and organ level interactions This paper outlines the
emerging contributions of systems biology in shaping new
directions for PBPK, PBPD, and dose–response modeling in
quantitative human health risk assessment and reproductive
toxicology.
2. Systems approaches in pharmacokinetics
In physiologically based pharmacokinetic (PBPK) model-
ing, compartments correspond to discrete tissues or to group-
ings of tissues with appropriate volumes, blood flows and
pathways for metabolism of test chemicals [1,2]. PBPK mod-
els include pertinent biochemical and physicochemical con-
stants for metabolism and solubility in each compartment.
Routes of dosing (i.e., routes of administration) are included
in their proper relationship to the overall physiology. The
equations that form the basis of the PBPK model also ac-
count for the time sequence of dose input into experimental
animals or test subjects and permit input by multiple routes if
necessary for specific exposure situations. In pregnancy, fetal
tissues are also incorporated along with uterus and placenta
(see Fig. 1), as in a recent model for isopropanol and acetone
[3]. Each compartment in the model is described with a mass-
balance differential equation (MBDE) whose terms mathe-
matically represent biological processes. The set of equations
is solved by numerical integration to simulate tissue time-
course concentrations of chemicals and their metabolites.
PBPK models focusing on developmental periods contain
descriptions of changing organ volumes during gestation and
during the post-partum period in the female [4,5]. Descrip-
tions of dosimetry for the developing organism usually con-
sider both fetal exposure by transport across the placenta and
neonatal exposure by transfer of compounds from the lac-
tating mother to the newborn in milk. Some PBPK models
account for interactions of circulating compounds with spe-
cific target receptors or for covalent interactions of chemicals
with tissue constituents. Modeling these reversible and irre-
versible molecular interactions with cell constituents is the
initial step in developing PBPD models for effects of chem-
icals on biological processes.
While not encompassing current systems approaches that
include high throughput ‘omic’ methodologies, PBPK mod-
eling is an example of a systems approach at the level of cells,
organs and organisms to ascertain the mechanisms of distri-
bution and interactions of drug and other chemicals in the
body. Modern day PBPK modeling was originally developed
 M.E. Andersen et al. / Reproductive Toxicology 19 (2005) 327–337 329

Fig. 1. Systems approaches to disposition of chemicals within the body. These two groupings of tissues represent a schematic of a PBPK model for isopropanol
(IPA) and its major metabolite, acetone (ACE), describing tissue dosimetry of both compounds in the developing fetus. These models have tissue compartments
described with volumes, blood flows (designated by Q), and tissue:blood partition coefficients. The transport across the placenta is diffusion-limited, described
by a permeation coefficient-area cross-product called PAF. Tissue volumes (weights) of many organs, and especially the fetus and placenta, vary during
pregnancy. Metabolism occurs in liver converting IPA to ACE with Michaelis–Menten kinetic constants, Vmaxc and Km . ACE is eliminated from the body by
hepatic metabolism with constants Vmax1c and Km1 . The IPA model allows input by inhalation, dermal contact and oral intake. Both compounds can be excreted
by metabolism or by exhalation. Predicted concentrations are determined by equations that integrate information on physiology, anatomy, and biochemistry,
producing predictions about the response of the entire system. Details of the routes of exposure (inhalation with wash-in wash-out characteristics in the mucous
phase of the respiratory tract) and the two-compartment representation for oral uptake (with pdose, stomach, and duodenum) are more fully described in the
original publication [3].

by two chemical engineers, Drs. Kenneth Bischoff and Robert
Dedrick, who incorporated engineering principles, physiol-
ogy, chemistry and biochemistry into a computer modeling
platform to predict kinetics [6,7]. Many PBPK models in-
tegrate information across multiple levels of organization,
especially when describing interactions of compounds with
molecular targets, such as reversible binding of ligands to
specific receptors (e.g., methotrexate [6] or dioxin [8]), or
the adduction of proteins or DNA by reactive parent chem-
icals or their metabolites in various tissues (e.g., ethylene
oxide [9] or acrylonitrile [10]). In these cases, the models
integrate molecular, cellular, organ level, and organism-level
processes at a physiological level of organization to account
for the time courses of chemicals, metabolites, and bound
complexes within multiple organs in the body. These PBPK
models attempt to account for major determinants of the dis-
330 M.E. Andersen et al. / Reproductive Toxicology 19 (2005) 327–337

tribution and elimination of compounds without describing
every physical chemical process involved in transport and
storage of chemical in various tissues. More detail can be in-
cluded in these models as more information becomes avail-
able on chemical disposition from specific experiments.
3. Linking doses and response
Over the past 15 years, toxicology and risk assessment
have emphasized the exposure dose–response relationship
linking the presentation of chemical to an organism with spe-
cific toxicological or pathological responses (Fig. 2). PBPK
models provide more detail on the sequence of steps through
tissue interactions, including binding of reactive molecules
with cellular constituents or recognition of chemical struc-
tures by reversible binding to cellular receptors involved in
regulating cell signaling pathways. For many risk assess-
ments, measures of tissue dose, sometimes called dose met-
rics [11] (i.e., measures of tissue dose believed to be closely
associated with the adverse responses) are used as the basis
for the dose–response portion of chemical risk assessment.
The specific steps that lead from these dose metrics to re-
sponse have usually been considered part of the pharmaco-
dynamics (PD) of the response to chemical exposures. These
dose metrics are the equivalent of a ‘biologically equivalent
dose’ (BED): these dose metrics link delivery of active forms
of the chemical to a response via the mode of action for the
compound in causing a particular toxic response.
In general, PD models used in modeling pharmaceutical
responses have been more empirical, utilizing simple effect
compartments correlated with blood or tissue concentrations
of active chemical. In risk assessment applications, there
have been attempts to link tissue dose metrics with more
integrated cellular level responses. Other BBDR approaches,
i.e., two-stage clonal growth models for carcinogenesis and
cell growth-based models for developmental processes, show
some potential for modeling carcinogenesis [12] and limited
characterization of cell dynamics in development [13,14]. In
these model structures, adverse endpoints occur due to al-
terations in cell replication, apoptosis, and mutation rates;
however, the cellular processes that are affected leading to
alterations in these macroscopic cellular behaviors have not
yet been described with respect to the effects of chemicals on
cellular signaling pathways or the interactions among signal-
ing pathways. Perhaps, the best example of a BBDR model
for a developmental endpoint is the model for 5-flurouracil,
which links tissue concentrations with enzyme inhibition, im-
paired nucleotide synthesis, altered DNA synthesis and de-
velopmental anomalies [15,16]. The challenge with the 5-FU
model or any other quantitative mathematical model for a
developmental endpoint is the difficulty in first providing an
adequate description of the underlying biology that is being
affected by the compound. The problem in describing biol-
ogy is a particular issue for development where processes
and structures are changing rapidly and consecutive devel-
opmental landmarks are critically dependent on completion
of earlier steps. Kavlock and Setzer [17] have provided an
excellent review of progress in constructing BBDR for de-
velopmental processes up to 1996.
4. The arrival of systems biology in dose–response
assessment
While PBPK modeling represents a systems approach on
the physiological and biochemical levels, the ability to apply
more integrated systems approaches to pharmacodynamic
modeling was simply not possible before the expansion of
methods in molecular biology and various ‘omics’ technolo-
gies of the past 10 years. New, high throughput, broad cov-
erage technologies – genomics, transcriptomics, proteomics,
and metabonomics – provide the molecular ‘parts list’ for
cells, tissues, organs, and eventually organisms. Systems bi-
ology attempts to integrate this diverse body of data to pro-
duce knowledge about how the organization of these parts
leads to specific biological functions. In a review in Science
in 2000, Lander and Weinberg [18] discussed the implications

Fig. 2. The exposure dose–response continuum. Developed as a pictorial representation of the steps involved in moving from exposure to response in the late
1980s, this popular linear representation focuses on toxicity, i.e., the perturbation caused by chemical treatment. This schematic shows the correspondence of
regions of the continuum with PBPK and PBPD processes, with the definition of the dose metric serving as the dividing line between the two model structures.
PBPK models predict dose metrics; PBPD models account for the consequences of that particular dose metric within tissues.
 M.E. Andersen et al. / Reproductive Toxicology 19 (2005) 327–337 331

of the information generated by ‘genomics’ technologies for
an understanding of biology, noting that:
“The long-term goal is to use this information to recon-
struct the complex molecular circuitry that operates within
the cell—to map out the network of interacting proteins that
determines the underlying logic of various cellular biologi-
cal functions including cell proliferation, responses to phys-
iologic stresses, and acquisition and maintenance of tissue-
specific differentiation functions. A longer term goal, whose
feasibility remains unclear, is to create mathematical models
of these biological circuits and thereby predict these various
types of cell biological behavior.”
From our perspective, a defining characteristic of current
initiatives in systems biology is the iterative quantitative eval-
uation, through laboratory experiments and computer mod-
eling, of the manner in which the components of biological
systems are organized together to give rise to these circuit
elements that control biological function. The aspect of com-
putational biology has become an intense area of activity for
understanding cell behavior as highlighted in the focused se-
ries of papers in Nature in 2002 [19,20]. Perturbations of these
biological processes by environmental stressors, including
chemicals and drugs, can lead to adverse responses (toxic-
ity), restoration of normal function to a compromised tissue
(efficacy), or control of biological processes, such as occurs
with use of hormonal therapies for birth control purposes in
women.
The greatest progress in any discipline has always been
achieved when the underlying principles of the system are
well understood prior to their broad use on more applied
problems. The same is true for toxicology where the linear
paradigm, employed to show the relationship of exposure,
dose and response for integrating studies of toxicity (Fig. 2),
now needs to focus much more heavily on investigating the
underlying biology prior to evaluating the perturbation of
the system following chemical exposure (Fig. 3). In this re-
casting of the exposure dose–response linkage, toxicity and
efficacy are defined by the intersection of chemical action
with the biological system. Toxicology and pharmacology
are disciplines at the interface of chemistry/pharmacokinetics
(primarily embedded in the vertical component) and biol-
ogy/pharmacodynamics (primarily captured by the horizon-
tal chain). The next generation of systems approaches in PK
and PD modeling will include increasingly detailed descrip-
tions of biology afforded by new technologies and the expan-
sion of modeling tools available for describing the biological
signaling processes affected by chemical exposures and drug
treatments.
5. Cell signaling motifs as targets for developmental
toxicants
Considerable work has appeared in recent years focusing
on cells as small machines conducting specific processes.
In addition to maintenance of these functions, the cell con-
sists of multiple interacting modules, with various feedback
controllers that regulate these behaviors. Many of these con-
trol modules have been examined in prokaryote cell systems

Fig. 3. Toxicity in a systems biology context. Toxic responses arise from perturbations in a system that converts various inputs through a series of biological
steps to produce specific functions necessary for health. The goals of a systems biology approach to dose–response assessment are (1) to define the nature of
perturbations associated with chemical exposures, (2) to map the networks of interacting proteins in which the site of perturbation is embedded, and (3) to
determine when these perturbations are likely to be sufficiently great to cause adverse health consequences in exposed animals or exposed people. Here the
pharmacodynamic process is the normal biological system and its responses to perturbations, not simply the perturbation itself. Individual pathways will have
inputs from other signaling networks, designated as secondary effector pathways, and perturbations may elicit adaptive changes that will influence dose–response
behaviors for altered signaling.
332 M.E. Andersen et al. / Reproductive Toxicology 19 (2005) 327–337
and mathematical models developed to describe the behavior
of the control networks. In some cases, simple prokaryotic
cells have been genetically engineered to contain specific cir-
cuit elements, i.e., biological oscillators, switches, amplifiers,
etc., and these constructs have been examined by laboratory
experiments and by computation [21–25]. These computa-
tional models evaluate the protein networks within cells, the
genetic control of these networks and the logic of cellular
responses affected by these networks [26–28]. These control
processes include activation of latent networks, silencing of
active networks, and modulation of networks. Control of the
circuitry occurs by various input signals to the cells. Input
signals, including various hormones, may activate cell sig-
naling networks to modulate both control circuitry and affect
downstream genetic networks that control new cellular func-
tions. In eukaryotic cells, a considerable body of work has
focused on modeling cell cycle control [29,30]. Progress in
understanding a variety of common signaling themes [31,32]
has led to increasing appreciation of the manner in which
these modular components are used to achieve and control
various cellular functions.
From a dose–response modeling perspective, many xeno-
biotics interact with cellular receptors of various kinds to
perturb cell circuits and modulate cellular functions. These
interactions are especially relevant for so-called receptor-
mediated toxicants that are recognized in the body by specific
endogenous receptors. These compounds have the potential
to cause toxicity since they are mimics of endogenous signal-
ing molecules leading to over-stimulation of natural circuits.
Alternatively, compounds may cause toxicity because they
are competitive inhibitors. Competitive inhibitors will bind
the active sites of receptors, but fail to activate important cir-
cuits in the presence of normal signals. These compounds
lead to diseases associated with deficiencies of signaling
molecules and circuit activation. The National Academy of
Sciences report, Scientific Frontiers of Developmental Toxi-
city and Risk Assessment [33], highlights the suite of known
intercellular signaling motifs and their roles in development.
These signaling pathways are potential targets for toxic re-
sponses for compounds that interfere with their function,
cause over-stimulation of pathways at inappropriate times
during development, or lead to aberrant signaling by activat-
ing circuits inappropriately.
Simulation models of several signaling pathways
have been developed for mammalian cells in recent
years—including ones for the platelet derived growth fac-
tor (PDGF) pathway [34] in mouse 3T3 cells (Fig. 4) and for
tumor necrosis factor-alpha (TNF-␣) signaling through the
nuclear factor kappa B (NF-␬B) pathway in mouse fibroblasts
[35,36]. These pathway models are similar to the sets of ordi-
nary differential equations used in most PBPK models. They
include many reactions within a cell and require evaluation
of multiple parameters for all the biochemical steps embed-
ded within the signaling networks. Models based on multiple
differential equations are being augmented by Boolean ap-
proaches using on–off logic to increase the model coverage
Fig. 4. Platelet derived growth factor (PDGF) signals through a receptor
tyrosine kinase signaling motif. This circuit diagram, redrawn from [34],
served as the hypothesis in developing a mathematical model of the signaling
network for platelet derived growth factor (PDGF) in vitro. Abbreviations:
PDGF-R, platelet-derived growth factor receptor; PKC, protein kinase C;
AA, arachidonic acid; PP2A, protein phosphatase 2A; MEK, MAPK or ERK
kinase; MAPK, mitogen-activated protein kinase 1, 2; cPLA2, cytoplasmic
phospholipase A2; MKP, MAP kinase phosphatase; DAG, diacylglycerol.
SHC, src homology adapter protein; Grb2, growth factor receptor-bound pro-
tein 2; Ras, ras superfamily of monomeric GTPases; Raf, mitogen-activated
protein kinase in the Ras signaling pathway. The dark blue arrow from MAPK
(lacking in the original) represents downstream protein targets that might in-
clude transcription factors and other cell regulatory proteins. The activation
of the cascade and involvement of multiple downstream targets combine to
create the switch for cellular responses as described in [34].
on intracellular reactions without necessarily increasing the
numbers of parameters that have to be estimated for specific
reactions [37] Model validation processes for these cellular
pathways require a variety of studies conducted in vitro with
specific cell systems.
While there are many signaling modules within any cell,
initial progress in understanding cellular behaviors is likely to
focus on dissecting and modeling individual signaling mod-
ules, giving rise eventually to a modular approach to under-
standing more integrated biological pathways [38]. Individ-
ual modules that serve as primary targets of toxic compounds
and drugs as well as the circuitry that controls these target
modules are likely to be the primary focus of initial systems
biology approaches with these exogenous compounds.
An interesting aspect of the PDGF signaling [34] is adapta-
tion to persistence of PDGF signaling through transcriptional
control of a phosphatase (MKP) that blocks the usual activa-
tion of mitogen-activated protein kinase (MAPK) responses
in the signaling network (Fig. 4). Such adaptive responses to
toxicants that mimic signaling molecules would have at least
two consequences: (1) modulation of dose–response curves
by altering pathway sensitivity to bioactive small molecules
and (2) desensitization of critical biological processes leading
to failure of endogenous signaling and toxic consequences of
 M.E. Andersen et al. / Reproductive Toxicology 19 (2005) 327–337 333

this failed signaling. These adaptive processes are likely to
be important in establishing shapes of dose–response curves
in regions of low incidence—the regions of most interest for
risk and safety assessment. A more appropriate schematic of
the exposure dose–response paradigm of the past 15 years
should include adaptation and repair (Fig. 3) as an important
processes in regulating dose–response behaviors.
6. Phthalates and male reproductive tract
development
As yet, there are no examples of comprehensive systems
biology approaches to provide high-fidelity dose–response
assessments for perturbations of critical signaling pathways
during development, or, for that matter, for any other toxic
response. However, the components required to pursue a sys-
tems biology approach for dose–response modeling can be
enumerated and the laboratory studies and associated math-
ematical modeling analysis required for such an approach
can be outlined. The final section of this review discusses
the structure of a systems biology approach in relation to a
specific developmental endpoint: reproductive tract malfor-
mations in male pups associated with treatment of pregnant
rats with high doses of phthalate esters. Several staff mem-
bers at our home institution are now involved in creating a
systems biology approach for developmental responses of the
male reproductive tract to phthalates.
Phthalate esters, such as di(n-butyl)phthalate (DBP), are
metabolized to mono-esters, i.e., mono-n-butyl phthalate
(MBP). Treatment of pregnant rats with high doses of DBP
leads to reproductive tract malformations in male pups. The
broad biochemical mode of action for phthalates was identi-
fied by the biology of the malformations, suggesting failure
of normal androgen signaling [39] and then by direct mea-
surements of the reduction in testes testosterone [40]. Sub-
sequently, studies have categorized the changes in gene tran-
script levels and proteins that occur in fetal testes exposed
to phthalates during late gestation. Changes were noted in
many genes required for steroid transport/synthesis and lipid
metabolism [41,42]. These studies provide a catalog of the al-
terations in expression of genes and proteins in the testes and
provide a representation of the time sequence of changes af-
ter treatment with the phthalates. In general, gene expression
studies and time-sequence analysis of alterations in gene ex-
pression in vivo, as conducted in the fetal testes with DBP or
in uteri with estrogen treatment [43], could suggest hypothe-
ses regarding targets of toxic compounds and for cascades
of signaling that ensue from interactions with these targets,
an important aspect of cataloging in vivo effects of develop-
mental toxicants (see left side of panel in Fig. 5).
In the adult testes, steroidogenesis requires G-protein cou-
pled signaling by luteininzing hormone (LH) interacting with
LH receptors and simultaneous activation of arachidonic acid
(AA) signaling pathways [44,45] associated with release of
AA from membrane phospholipids during LH-stimulated
signaling. The AA is believed to be produced by activa-
tion of an enzyme, cytoplasmic phospholipase A2 (cPLA2),
which releases AA from the 2-position of membrane phos-
phatidyl choline. The central feedback loop with LH and

Fig. 5. Systems biology approaches for dose–response assessment for developmental toxicity have a series of components. The components of a systems
biology approach relying on current genomic technologies include expansion of conventional hazard identification studies to include microarray studies to
assess altered gene expression in target tissues in the affected tissues. These studies can provide hypotheses regarding molecular targets of action if they are
unknown. On the right side, more mechanistically oriented studies can be pursued after establishing appropriate cell systems for close examination. These
in vitro and ex vivo systems can be examined using genome-wide microarray expression and LOF and GOF screens to identify likely targets for chemical
perturbations and mapping the affected pathways. These genomic tools provide the information necessary to create representations of pathways for cellular
signaling (as in Fig. 4) leading to construction of computational dose–response models for the in vitro/ex vivo responses of cells or groups of cells. These
computational models together with the empirical dose–response model from the intact organism are brought together in the in vivo dose–response modeling.
334 M.E. Andersen et al. / Reproductive Toxicology 19 (2005) 327–337
steroidogenesis does not operate in the fetal testes and the
exact control of fetal steroidogenesis remains unknown. One
hypothesis for phthalate mode of action under evaluation is
that the monoalkylphthalate metabolites may be inhibiting
steroidogenesis by interfering with the downstream AA sig-
naling required for steroidogenesis in the testes. This portion
of the steroidogenesis pathway may be common to both fe-
tal and adult Leydig cells even though LH does not serve
as a stimulatory hormone in the fetus. Using a systems bi-
ology approach, we are evaluating the normal cell circuitry
associated with AA-mediated steroidogenesis by addressing
several key questions. They are: how will available labora-
tory and software-based computational research tools be em-
ployed (1) to create an understanding of the normal circuitry
controlling steroidogenesis, (2) to evaluate the sites where
phthalates might alter signaling pathways, and (3) to create
quantitative mathematical models of the alterations in these
signaling pathways?
7. Assessing components of signaling pathways
Estimating consequences of a perturbation rests on our
ability to understand the ‘dose–response’ relationships for
control of normal biological functions. For instance, post-
natal steroidogenesis in the testes requires appropriate con-
centrations of LH, LHR, AA and other components of an
intact steroidogenesis pathway. The components of the sig-
naling pathway have to be functionally identified, organized
into their appropriate networks (as was done in Fig. 4 for
PDGF signaling), and modeled computationally. To dissect
these networks, a number of research groups have begun
to apply large-scale reverse genetic screens to systemati-
cally identify genes that play a functional role in specific
signaling pathways [46–51]. In these screens, cell-based as-
says are constructed with various cellular endpoints [48,49]
or reporter genes that indicate activation of a specific path-
way [46,47,50]. Full-length cDNAs or short inhibitory RNAs
(siRNAs) are introduced into the cells to provide a gain-of-
expression (GOE) or loss-of-expression (LOE) form of ge-
netic manipulation, respectively. The genes identified using
these approaches are assumed to play a functional role in the
specific pathway of interest.
The following paragraph describes work that is in progress
in our laboratory to use similar functional genomic ap-
proaches to determine the signaling pathways involved
gonadotropin-stimulated steroidogenesis in the adult mouse
testes. These studies are planned, but have not been com-
pleted at this point, and are provided as an example of the
overall approaches noted above. In LOE screens, cells would
be stimulated by LH and the expression of individual genes
are knocked down using siRNAs. Genes altering steroidoge-
nesis would be noted and cataloged. With GOE screens, two
approaches could be used. First, cells with minimal LH stim-
ulation would be transfected with individual cDNAs to see
which genes produce enhanced steroidogenesis. The GOE
screen is more targeted to identifying genes that play a func-
tional role in the specific process. In the second approach, the
design of the screen is altered to identify molecular targets
of a specific molecule. Cells would be treated with the toxic
compound causing partial inhibition of function. Individual
cDNAs would then be screened to identify which genes when
overexpressed can complement the inhibitory effects of the
compound. For identifying chemical targets in AA-mediated
processes, a GOE screen might be pursued using a known
inhibitor of cPLA2 and AA release such as chloroquin. The
GOE screen with transfected cDNAs would evaluate the suite
of genes that restore function and allow visualization of the
AA-mediated pathways of steroidogenesis.
When applied on a broad scale, optimally by testing with
siRNAs and cDNAs representing the majority of genes in the
genome, these combined technologies provide a map of the
molecular components of cell signaling that control cellu-
lar functions in a cell specific context. Successful pursuit of
these technologies today depends on robotic technologies for
screening libraries of genes and co-ordinate development of a
bioinformatics infrastructure to store and manipulate results
of the screens.
A secondary benefit of a systems approach to understand-
ing signaling networks and their perturbation by exposures
to chemicals will be an enhanced ability to identify determi-
nants of susceptibility. As full signaling pathways are elu-
cidated by genomic methodologies and interactions among
pathways become clarified, the biological contributions to
susceptibility, especially with regard to contributions from
secondary signaling pathways (the secondary effector path-
ways in Fig. 3) and from crosstalk among signaling modules,
will be more easily appreciated. With this information avail-
able, strategies can be developed to more fully assess the
genetic contributors to susceptibility in human populations.
8. Creating mathematical models of function and
perturbations
The results from the GOE and LOE screens are stored
in relational databases and the experimental data are sup-
plemented with known biomolecular interactions (e.g.,
protein–protein interactions). Currently, the supplemental in-
formation is derived from both public databases (e.g., BIND
and DIP) or mined from the literature using either expert cu-
rated datasets or natural language processing algorithms. All
of these biological tools provide knowledge about the cir-
cuitry that controls cellular responses to chemical stressors
and the consequences of perturbations by toxic compounds.
The diverse datasets are then synthesized and bioinformatic
tools convert the experimental functional genomic data into
directed graph representations of the intercellular signaling
events. Based on the graphical representation of the system,
mechanistic dose–response models would then be developed
using ordinary differential equations (ODEs) or Boolean rep-
resentations [37,51] to quantitatively describe the circuitry in-
 M.E. Andersen et al. / Reproductive Toxicology 19 (2005) 327–337
volved in these responses (the intrinsic biology) and the site
of action(s) of the stressors (the perturbation). Experience
in describing the functions of these circuits in simple cell
systems has highlighted the non-linearity of many biological
signaling networks [52]. As modeling strategies evolve, the
more complex interactions among multiple signaling path-
ways [53] will need to be included to evaluate the processes
by which complex interrelationships among multiple path-
ways lead to unexpected or ‘emergent’ behaviors.
The aim of these quantitative simulation models for our
purposes here is to describe the biology in sufficient detail
to understand the biological factors that control the shape of
the dose–response curve. To accomplish this end, not every
detail of the signaling pathway would have to be included.
These dose–response models will likely have some connec-
tions in the circuit described as simply on-or-off with dose,
while other steps will be described with greater biological de-
tail. In general, critical components of the affected circuitry
will require a greater level of detail in the descriptions. The
application of these systems biology, dose–response models
to safety and risk assessments is the primary goal, ultimately
permitting prediction of the shape of the dose–response curve
based on knowledge of the basic cellular circuitry, and the
sites and intensity of perturbations by chemical stressors.
9. How will these technologies be used in
reproductive toxicology?
The technologies, by helping to determine the biologi-
cal determinants of the shape of the dose–response curve for
adverse responses, are intended to address several important
risk assessment uncertainties. These uncertainties include the
shape of the dose–response curve at low doses, the risks posed
by low level exposures to the human population, and the rel-
evance of observations of high dose toxicity in rodents for
human risk assessment. Some rodent responses to phthalates,
primarily hepatocarcinogenesis, are not believed to be rele-
vant markers for risks of these responses in human popula-
tions, even though the PPAR signaling pathways affected by
the phthalates do appear to be present in both rodents and
people [54]. The consequences of activation of this receptor
for liver neoplasia differ between rodents and humans. It is
not presently known if humans would show similar sensitiv-
ity to high-dose exposures to phthalate esters as do the male
rat pups.
The manner in which these systems approaches are applied
with any specific toxic response is dependent on the level
of prior knowledge concerning the molecular targets of the
chemicals, the extent of previous research on altered patterns
of gene expression, and the level of understanding of the path-
way and circuits based on available literature. When knowl-
edge about mode of action and targets is meager, the use of
genomic methods is especially important in order to achieve
‘high coverage’ of possible targets rather than a one-by-one
examination of specific hypotheses. Evaluation of gene ex-
335
pression in affected tissues and examination of cells in vitro
with functional genomic screens are important techniques
for providing ‘high coverage’ of possible targets and draw-
ing inferences about affected circuitry. As more is known
about targets and affected circuitry, the data needed for these
evaluations would be correspondingly reduced.
A significant challenge in the emerging systems biology
approaches described here for reproductive and develop-
mental endpoints is the difficulty of finding in vitro/ex
vivo cell models that will permit high throughput studies.
Reproduction and development are highly dependent on
the interactions and subtle timing of interactions among
many cell types. Identifying affected targets and evaluating
these targets in single cell types in vitro will give only
a fragmentary picture of the full details of perturbations
occurring in the developing fetus. Nonetheless, strategies
of producing a detailed quantitative understanding of
the control of pathways critical for proper development
from iterative approaches using in vitro models, whole
animal studies, and computational analysis of interact-
ing cellular networks appear to us to be the ones most
likely to provide the basic information needed to draw
mechanistic inferences of the shapes of dose–response
curves in regions of low incidence for these responses
(Fig. 5). Initiatives soliciting proposals for multi-scale
modeling (www.nsf.gov/pubs/2004/nsf04607/nsf04607.htm)
and for creating Centers for Biomedical Comput-
ing (grants1.nih.gov/grants/guide/rfa-files/RFA-RM-04-
022.html) and Systems Biology (grants.nih.gov/grants/
guide/rfa-files/RFA-GM-05-010.html) organized around
driving biological projects at the National Institutes of
Health are also strong indications that these integrated
approaches across multiple levels of biological organization
are regarded as essential components in understanding how
the parts of biological system contribute to the control of
specific biological functions.
The task of creating these quantitative dose–response
models for endpoints related to reproductive toxicology ap-
pears daunting. However, the experience in high through-
put biology, bioinformatics, and computation over the past
decade indicates that progress in these areas has become as-
tonishingly rapid. The primary focus initially with these sys-
tems approaches for dose–response modeling in reproduc-
tive toxicity is to ask the right questions, design appropriate
high throughput studies in cell models, and develop quanti-
tative models of affected pathways. Initial results with sev-
eral chemicals should provide insights into the shape of the
dose–response curve for perturbations of specific cell sig-
naling networks involved in organism development and then
subsequent research can be targeted at more clearly estab-
lishing how alterations in primary target pathways produce
dose–response relationships for more integrated biological
responses. These initial prototype assessments could focus
on important classes of reactive or receptor-mediated toxi-
cants and be useful for improving the design and efficiency
of experiments to create dose–response models in the future.
336 M.E. Andersen et al. / Reproductive Toxicology 19 (2005) 327–337
Acknowledgments
The authors are indebted to a group of colleagues at CIIT
Centers for Health Research who contributed significantly
to our ideas about systems approaches in toxicology and
dose–response assessment, including Drs. William Green-
lee, Frederick Miller, Cecilia Tan, Li You, Kamin Johnson,
and David Dorman. M.E.A. also expresses appreciation to
Dr. Raymond Yang, Colorado State University, and Harvey
Clewell, Health Sciences Institute, ENVIRON, for helpful
discussions about systems approaches in PK and PD model-
ing over many years. We also thank the Long-Range Research
Initiative (LRI) Program of the American Chemistry Coun-
cil for support of the research outlined here and for support
for organizing interdisciplinary teams to focus on research
to improve dose–response assessment methodologies for as-
sessing health risks of chemicals at environmentally relevant
levels of exposure.
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