Critical Review

pubs.acs.org/est

Mixture Toxicity Revisited from a Toxicogenomic Perspective

Rolf Altenburger,*,†,‡ Stefan Scholz,† Mechthild Schmitt-Jansen,† Wibke Busch,† and Beate I. Escher‡
†
Department Bioanalytical Ecotoxicology, UFZ − Helmholtz Centre for Environmental Research, Permoser Street 15,
04318 Leipzig, Germany
‡
National Research Centre for Environmental Toxicology (Entox), The University of Queensland, 39 Kessels Road, Brisbane,
QLD 4108, Australia
*
S Supporting Information

ABSTRACT: The advent of new genomic techniques has

raised expectations that central questions of mixture toxicology
such as for mechanisms of low dose interactions can now be
answered. This review provides an overview on experimental
studies from the past decade that address diagnostic and/or
mechanistic questions regarding the combined effects of
chemical mixtures using toxicogenomic techniques. From
2002 to 2011, 41 studies were published with a focus on
mixture toxicity assessment. Primarily multiplexed quantifica-
tion of gene transcripts was performed, though metabolomic
and proteomic analysis of joint exposures have also been
undertaken. It is now standard to explicitly state criteria for
selecting concentrations and provide insight into data trans-
formation and statistical treatment with respect to minimizing sources of undue variability. Bioinformatic analysis of toxicogenomic
data, by contrast, is still a field with diverse and rapidly evolving tools. The reported combined effect assessments are discussed in
the light of established toxicological dose−response and mixture toxicity models. Receptor-based assays seem to be the most
advanced toward establishing quantitative relationships between exposure and biological responses. Often transcriptomic responses
are discussed based on the presence or absence of signals, where the interpretation may remain ambiguous due to methodological
problems. The majority of mixture studies design their studies to compare the recorded mixture outcome against responses for
individual components only. This stands in stark contrast to our existing understanding of joint biological activity at the levels
of chemical target interactions and apical combined effects. By joining established mixture effect models with toxicokinetic
and -dynamic thinking, we suggest a conceptual framework that may help to overcome the current limitation of providing mainly
anecdotal evidence on mixture effects. To achieve this we suggest (i) to design studies to establish quantitative relationships between
dose and time dependency of responses and (ii) to adopt mixture toxicity models. Moreover, (iii) utilization of novel bioinformatic
tools and (iv) stress response concepts could be productive to translate multiple responses into hypotheses on the relationships
between general stress and specific toxicity reactions of organisms.

1. INTRODUCTION combined effect observation. The standardization of bioassays,

The combined effects provoked in organisms through exposure
to mixtures of chemicals have been a longstanding research
topic in pharmacological and toxicological sciences.1−4 Environ-
mental policy has recognized mixture effects as a major issue in
environmental risk assessment of chemicals. Therefore, a sys-
tematic review of the approaches toward environmental risk
assessment, with respect to the inclusion of systematic mixture
considerations, is being discussed by the European Commission.5
The last two decades have seen a series of studies in environ-
mental toxicology addressing various scientific questions in
mixture toxicology (reviewed in refs 6−11). Major progress in
the environmental risk assessment of mixtures was achieved
when hypotheses of expected combined effects, based on
individual components activities, were compared against the
actual mixture effects observed. Reductionism in experimental
approaches for univariate response parameters allowed for the
study of quantitative relationships and pattern searching in
minimized variance, and optimization of designs generated
sufficient discriminatory power. Our current understanding of
primary molecular interaction at target sites and at the level of
apical effects is still in line with the simple and powerful null
hypothesis models as reference models for the quantitative
prediction of noninteractive combined effects: concentration
and response addition also called dose or LOEWE additivity
and independent action or BLISS Independence, respectively.3
The supply of extrapolation models to risk regulators and
managers for handling a pressing issue of risk assessment12
represents a success story. However, unresolved issues that pose
formidable future research challenges for mixture toxicology
Received: October 26, 2011
Revised: January 25, 2012
Accepted: January 27, 2012
Published: January 27, 2012

© 2012 American Chemical Society 2508 dx.doi.org/10.1021/es2038036 | Environ. Sci. Technol. 2012, 46, 2508−2522
Environmental Science & Technology
remain. For instance, the current evidence has been generated
using highly standardized bioassay systems which tend to focus
on short-term integral adverse effects. The predictivity of the
currently used mixture toxicity models for components with
long-term mixture effects, that provoke adverse effects through a
variety of different toxicity pathways, remains unresolved.13 A
second major challenge relates to the question of what mixtures
provoke synergy?14 Also, the understanding of how primary
molecular interactions are translated into apical effects that we
consider in toxicological assessments would help in extrapolating
mixture responses between different biological scales and across
species. Moreover, an additional challenge occurs when specific
environmental matrices are contaminated. Here, research en-
counters unresolved complex mixture exposure, e.g. through an
effluent or contaminated feed and combined effects are assessed
from a diagnostic perspective. In these situations assessment
approaches would greatly profit if it was possible to use biological
effects to provide guidance of what compounds could be identi-
fied as major drivers for compromised organism or population
performance15 under mixture exposure.
For some of the questions raised above toxicogenomics pro-
vide tools to improve our understanding and predictability of
combined effects. Toxicogenomics allow observing the interplay
between impacted environmental conditions and dynamic
responses of organisms on a gene, protein, or at the metabolite
level. In particular modern detection techniques are multivariate
and nontargeted with respect to gene transcript and protein
expression or metabolic responses. The potential for toxicoge-
nomic methods to provide knowledge of modes of action of
chemicals has been anticipated.16 This knowledge is critical in
diagnostic assessment to identify agents causing toxicity in com-
plex contaminated samples such as effluents. Also, there is hope
that toxicogenomic methods will lead to an improved selection of
end points suitable for specific risk assessment. The perspective of
the ‘omics’ techniques to provide tools for advanced biomarkers
has meanwhile gained experimental support.17,18 For example,
Yang and co-workers17 studied individual chemicals and suggested
to use the compound-specific transcriptional response patterns in a
barcode-manner to identify exposure against specific compounds.
Most recently, the promises of toxicogenomic approaches for
understanding the combined effects of environmental mixtures,
with respect to toxicokinetic and toxicodynamic processes, were
summarized.19 The investigation of the effects of mixture exposure
in organisms using novel toxicogenomic methodology has, in the
meantime, become a major research activity. Starting from
pioneering mixture studies,20,21 there is a dramatic increase of
studies since 2006. Reviews now even call for progressing toward
employment of systems biology approaches in ecotoxicology.22 It
seems therefore timely to systematically summarize and review the
progress in toxicogenomic response analysis of mixtures.
The objective of this study is to provide a comprehensive
review of the first steps made in addressing mixture toxicity
issues with toxicogenomic approaches. In order to learn about
the strengths and shortcomings of the chosen approaches we
analyzed exposure conditions and biosystems used, ways of data
generation, signal treatment, and analysis to deal with the
apparent issues of variance and means of aggregating the
information. Moreover, an attempt was made to cluster underly-
ing perspectives on mixture toxicity concepts that drive the
authors’ conclusive assessments on observed molecular com-
bined effects. The ultimate goals are to highlight what has been
gained so far in terms of mixture understanding, to provide
guidance for the experimental design of future mixture toxicity
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Critical Review
studies, and to address conceptual or experimental gaps that
should be addressed in future work in toxicogenomic combined
effect studies.
We focus this review on the studies which provide an
ecotoxicological perspective, but we include major contribu-
tions from the field of human toxicology. For work related to
human toxicology the reader is referred to Sen et al.23 who
earlier summarized studies that investigated mixtures in com-
plex exposure settings, which were only partly resolved for their
components. We also included reports on mixture toxicity
analysis using assays that detect multiple univariate responses
such as sets of reporter assays. Thus reports that monitored
individual gene activities alongside other biological effects and
had no major focus on mixtures themselves were omitted. Our
intention was also to focus on the molecular response analysis
of mixture toxicity, and we did not identify combined effect
specific debates on phenotypic anchoring24 or adverse outcome
pathways. Therefore, we abstained from detailed considera-
tion of other than transcriptomic, proteomic, or metabolomic
observation parameters.
The paper sets out with developing a conceptual framework
for the use of toxicogenomic tools in combined effect studies,
followed by a summary of the mixtures, experimental design,
and data handling of existing toxicogenomic mixture studies.
Subsequently, data interpretation with respect to mixture
models is discussed before we provide an outlook trying to
suggest possible advancements to the field.
2. TOOLS AND CONCEPTUAL FRAMEWORK
Toxicogenomic Response Detection. Following the
sequencing of whole genomes, for a variety of different
organisms (www.genomesonline.org), the understanding of the
relationships between the structure of the genome and the gene
function such as nongenomic modification of the genome
(epigenetics), transcription into RNA (transcriptomics), trans-
lation into proteins (proteomics), and ultimately altered
metabolic activities under varying conditions (metabolomics)
are great challenges. Major methodological progress has been
made in the development of analytical platforms that allow access
to increasingly comprehensive detection of DNA transcripts,25
proteins,26 or metabolites27 and epigenetic modifications.28
Equally, tools are continuously being developed that allow
handling such multiple responses and their statistical and
biological interpretation.29
Transcriptomics in our context refers to the analysis of different
levels of gene activity under varying conditions. Nontargeted ap-
proaches aim at a non-a-priory analysis of the differential expression
of the entire set of genes (transcriptome) using micro-
array or RNA-sequencing as the currently most popular tech-
niques.25,30 The latter is becoming more important since it provides
a digital quantification (estimation of the number of transcripts
similar to qRT-PCR, see below) and can additionally detect non-
coding RNA genes from genomic regions hitherto considered as
transcriptionally silent.31 Sequenced genomes are not necessarily
required for microarray or sequencing-based approaches. This
represents a major advantage regarding the diversity of
organisms, although the lack of functional annotation of genes
in nonmodel organisms currently limits the interpretation of data
and cross-species extrapolations.
Proteomics provides the scope to study a comprehensive
nontargeted complement of proteins of a biosystem including
their posttranslational modifications and variants.26 Typically,
top-down approaches of total cell lysate separation are capable
dx.doi.org/10.1021/es2038036 | Environ. Sci. Technol. 2012, 46, 2508−2522
Environmental Science & Technology
of detecting only a fraction of a cellular proteome. The large
dynamic range of protein occurrence probably marks the largest
challenge when identifying proteins relevant for specific
perturbations.
Metabolomics32 aims to comprehensively detect small
biomolecules like sugars, amino acids, or fatty acids from cells
or tissues by NMR33- or mass spectroscopic34- based methods.
Studies on organism-environment interactions and ecotoxico-
logical studies were performed with a selection of aquatic and
terrestrial organisms.27 As the molecules of primary metabolism
are not restricted to defined species, metabolomics can be
applied to all current model species in ecotoxicology, even if
they are not sequenced. There is large variation in numbers of
metabolites for different organisms, ranging from hundreds to
several thousands.35
Conceptually, toxicologists have taken up these technological
approaches, and others, by transforming previous thinking
about the interaction between chemicals and biosystems, in
terms of mechanisms and modes of action, into one of adverse
outcome pathways.36
Mixture Toxicity Analysis. Thinking and experimentation
on the combined effects from the exposure to mixtures of com-
pounds dates back several decades.2 Major progress in environ-
mental toxicology resulted from the introduction of receptor-
based thinking of pharmacology. Particularly, reference models
to formulate expectable combined effects are compared against
experimental observations.12 Key was the hypothesis derived
from the so-called sham experiment and the categories of target
sites and modes of action. The ‘sham’ experiment is a thought
experiment wherein the simplest mixture is a mixture of an
individual compound with itself. Clearly, the expectation for the
responses from such a mixture experiment is that increasing
doses due to mixture exposure should lead to increasing effect.
Moreover, the concentration-effect relationship, as derived
from dilution-type experiments for that compound, should be
retrieved irrespective of how many fractions are applied in the
dosing regime. The usefulness of this idea is convincing when
thinking about compounds interacting with the same molecular
target site. Under the name of dose or concentration addition it
became a widely accepted reference model in pharmacological
research and environmental toxicology and applies to all mixtures
of compounds that act according to a common mode of action.
For mixtures of compounds that provoke their biological
action through different target sites, responses are expected to be
independent according to the statistical idea of independence.
The derived reference model is called independent action or
response addition. The latter term avoids misunderstanding
Figure 1. Conceptual frame for toxicogenomic mixture studies. For the null hypothesis of noninteracting compounds, mixture models would suggest
that combined toxicogenomic responses can be explained by concentration addition or independent action models.
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Critical Review
as the combined effect of a mixture of independently acting
compounds is still expected to be quantitatively larger than
that of any of the components alone. The guiding assumptions
and the models for the relationship between the components
individual effects and their expected combined effects are pro-
vided in Table 1. The two alternative reference models, how-
ever, provide quantitatively accurate predictions of the joint effects
only if the mixture components do not show any interactions. In
cases where interaction between the mixture components occur
observable responses may deviate to be larger or smaller than
expected for either concentration additive or independent action
effects (Table 1). For interactive combined effects we, however,
currently do not have generic models to describe, let alone predict,
the outcomes.
Both at the level of primary molecular interactions between
chemical and biomolecules, as well as at the end of the toxi-
codynamic response chain, i.e. the apical outcome of toxicity
assays, concentration addition, and independent action are well
supported by experimental evidence. This knowledge should
help to develop a conceptual framework for toxicogenomic
mixture studies. Figure 1 displays an attempt to structure our
existing understanding on mixture effects for the novel ap-
proaches employed in toxicogenomic studies. Going from left
to right in the scheme, we illustrate toxicokinetic and toxi-
codynamic processes that can be seen as determining the cau-
sality of a concentration−response relationship. While the
potential to improve environmental effect assessment through
consideration of the internal dose level has been appreciated,
and in fact led to a better understanding of the mechanisms of
toxicokinetic interactions occurring for various mixtures,37 it is
the advent of toxicogenomic techniques that opens the route to
better understand the sequence of events within a cell or
organism leading to apical effects. Also, multivariate molecular
response detection might be instrumental to discriminate
responses from different chemicals as similar or dissimilar acting
and thus open novel routes to use mixture studies as probes for
pharmacological action.38 The challenges to meet those pro-
spects, however, are also apparent. For one, detected signals need
to be attributed to defined response chains, and second we need
to understand the crosstalk and convergence of pathways, as
joint responses might switch between independent and con-
centration additive, or noninteractive and interactive, respec-
tively, along different steps in the sequence.
Designing toxicogenomic mixture studies requires sufficient
understanding of the exposure regime and it is anchoring to
expected combined effects at the level of primary molecular
interaction or the apical effects. This would help to formulate a
dx.doi.org/10.1021/es2038036 | Environ. Sci. Technol. 2012, 46, 2508−2522
Environmental Science & Technology
Table 1. Reference Models Used in Mixture Toxicologya
same
noninteractive ∑i=1
n
(cSi/ECx(Si))
=1
concentration addition
mode of
action
interactive no quantitative
prediction model
a
Abbreviations used: cSi, concentration of substance i (Si) in the mixture; ECx, effect concentration at the response level x; F, function describing the
relation between concentration and response for the individual component; pSi, fraction of substance i (Si) in the mixture; X, expected combined
response; mix, mixture.
testable hypothesis and deal with the potential complexity in
the outcome.
After the dose level, the combined effect outcome may also
depend on the concentration ratio of the mixture components.
Figure 2 illustrates the three most common designs for a binary
Figure 2. Options for designing mixtures in combined effect studies,
illustrated for a binary mixture of chemicals at various concentrations
using a constant experimental effort; As an example theoretical design
points for binary mixtures with identical number of observations
according to n × n design (squares), ray design (circles), and surface
design (crosses) are shown. For n × n and surface design the mixture
ratio varies and might refer to dilution series of the components or else
selected observation points on the two-dimensional plane (modified
after Altenburger et al. 2003).
mixture, whereby the same experimental effort is allocated. (1)
A mixture can be composed of one component studied in a
dilution series in the presence of one fixed amount of another,
or a dilution series of one component in the presence of a
number of fixed amounts of another were used, which can be
labeled as a × n design in the former or n × n design in the
latter case.1 (2) Alternatively, the components can be mixed
based at a constant mixture ratio and than be diluted, which is
called diagonal or ray design.1 If the mixture ratio is derived
from equal toxic units (TU), i.e. exposure concentration of
component over effect concentration of the same compound,
this is sometimes specifically referred to as equitoxic design,
though the toxicity does not have to be equivalent in this design
if dilutions or fractions of the constitutive effect concentration
are studied. (3) Furthermore, we find designs where the
mixture and individual concentrations are varied to systemati-
cally cover the whole surface of possible mixture compositions
(surface design). The optimal design of a mixture to be
studied depends on the objective1,39 as will be discussed
further below.
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Critical Review
target/mechanism
different
X=1− ∏i=1
n
− Fi(pSi·(ECxmix)))
(1
independent action
no quantitative
prediction model
3. TOXICOGENOMIC MIXTURE STUDIES
Scope of Analysis. The scope of studies analyzed is rather
diverse ranging from purely exploratory, toward those searching
for correlations among different responses, those that try to
provide causal explanations for pharmacological cascades, and
finally explicit extrapolation or modeling perspectives. The
common theme across all referred papers is, however, that the
authors derive an assessment on the observed mixture effect
that is based on a comparison with an anticipated effect for the
mixture of concern e.g. from the mixture components, another
mixture, or another response observation. The molecular
responses detected for the mixture of interest are therefore
compared against a reference. Very often the reference concept
is not explicitly stated so we will come back to it after having
analyzed the studies in more detail. It may be helpful to try
distinguishing between the following intentions: (i) In a
situation of complex contamination one may wish to identify a
major driver of biological effect. Here the perspective of looking
at toxicogenomic data is diagnostic by searching for patterns
similar to those from the reference case.18,40,41 (ii) By contrast,
to understand the biological chain of effect after an interaction
of a chemical with a biosystem, one may choose an interaction
or mode of action scope trying to decipher signals that are
novel compared to the reference case.42−45 (iii) Finally, a more
quantitative view on the data would allow extrapolation to
other mixture compositions, which is when explicit efforts to
look at signal intensities come into focus.21,46−48
The 41 papers retrieved are summarized in Tables 2−4 and
Table SI-1, grouping the papers according to the mixture type
studied and thereafter in chronological and then alphabetical
order of the authors. Mixture type refers to binary mixtures as the
simplest mixtures, multiple component mixtures, and complex
mixtures, the latter comprising also unresolved mixture composi-
tion e.g. from a contaminated site. The mixture type chosen
depends on the focus of the study as discussed above. Tables 2−4
summarize the bioassay used, observations made, and interpre-
tations derived. Table SI-1 provides an overview on the more
technical details of how the mixture study was performed with
respect to exposure conditions, data variance, and treatment issues.
Mixtures Studied. Eighteen of the studies report on com-
bined effects from binary mixtures, i.e. mixtures of two com-
pounds. Multiple mixtures are covered in another sixteen
studies, three of which also compare joint effects from binary
and multiple component exposure.63,68,69 Multiple mixtures
contained more than four but less than ten components. Seven
investigations18,40,73−77 studied complex mixtures, typically
environmental samples, where the individual components are
not chemically defined. These studies were selected as they
contained explicit consideration of mixture assessment rather
than regarding the investigated exposure situation as a single
dx.doi.org/10.1021/es2038036 | Environ. Sci. Technol. 2012, 46, 2508−2522
 Table 2. Combined Effect Observation and Assessment for Binary Mixturesa
mixture
NP, PCB-77
ICI 182,780, estradiol
NP, TCB
azothymine, Cd, CuCl2, H2O2,
MMS, Mito C, NaN3, nalidixic
acid, paraquat, PCP
BAP, BbF, DBahA, DBaIP, FA
EE2, ZM
exposure regime biosystem molecular response observation evaluation authors’ assessment ref
4 d, 3 dilutions rainbow trout hepato- target gene expression, 6 genes (qPCR) concentration dependent % change against NP effect combined exposure causes reduction in PCB-77 42
cytes and NP gene induction
2 d, simultaneous, sea bream liver and target gene expression, 5 genes (RT-PCR) individual treatment expression levels against effect of individual com- pharmacologic behavior agonistic and antago- 43
sequentially testis ponents nistic
2 d, ip. injections Salmo salar hepato- SSH (300 clones), target gene expression, individual treatment expression levels against effects of individual com- crosstalk between ER and AhR signaling, 49
cytes 12 genes (qPCR) ponents, comparison with complex mode of ER-AhR interaction
known antagonists
90 min, 7 dilu- gene reporter con- Lac Z expression from 14 stress gene concentration dependent fold induc- against expected mixture effects concentration addition and independent action 47
tions, 8 binary structed in E. coli promoters in individual assays tion based on concentration addition reasonably predict combined effects, MoA not
mixtures and independent action relevant for predictivity
24 h Wistar albino rat, liver Operon rat oligunucleotide array (5.7 k), individual treatment expression pat- against effects of individual com- antagonism as compared to expected addition of 48
slices from males diff. expression tern and intensity ponents and sum of effects individual effects
2 d, semistatic Pimephales promelas microarray 2 k and 22 k, diff. expression individual treatment related expres- against EE2 effects mixture exposure provides distinction between 44
gonads sion increase/decrease different modes of estrogenic action
Environmental Science & Technology

Ni, Cd, Pb
cyproterone, 17β-trenbolone
Cd, BAP
imidacloprid, thiacloprid
4d, binary mix- Daphnia magna juve- custom-made cDNA microarray diff. ex- individual treatment expression, against expected mixture effect interactive molecular responses as mixtures 50
tures with Ni niles pression treatment related fold induction based on sum of TU for showed additionally affected pathways
immobility response
1
14 d Pimephales promelas H NMR metabolic profile of male urine individual treatment signal strength against effects of cyproterone, antagonist behavior, interactive effect at low 51
samples comparison with known antag- concentration
onist
24 h, ip. injection Solea senegalensis liver 2DGE/MS proteome individual treatment protein expres- against effects of individual com- antagonistic or synergistic effects, enhanced 52
sion pounds effects through specific responses rather than
cumulative damage
4 d, semistatic Mytilus galloprovincia- cDNA array (1.7 k) diff. expression, individual treatment pattern of re- against expected mixture effect different toxicodynamics may occur even for 53
lis digestive gland 2DGE/MS proteome sponses, proteome responses did based on EC25S1 + EC25S2 = components with the same mode of action

2512
not confirm gene expression find- EC50mixture
ings
Cd, Cu, Pb 2 h, binary mix- Chlamydomonas rein- target gene expression, 9 stress genes concentration dependent fold induc- against expected mixture effect synergistic and antagonistic interactions de- 54
tures with Cd, 5 hardtii tion based on independent action for pending on gene
dilutions gene transcription
pyrene, fluoranthene 4 d, 4 dilutions Daphnia magna juve- custom-made cDNA microarray (1,189) individual treatment gene expression, against effects of individual com- mixture responses can be both similar and 55
niles diff. expression, NMR (25+ signals), and treatment related metabolite con- ponents distinct from those of components depending
GC-MS metabolom (108 signals) centrations on mixture ratio
chlorpyrifos, diazinon 3d Caenorhabditis elegans 60mer microarray (22 k) diff. expression individual treatment gene expression against effects of individual com- intense unexpected crosstalk between gene 56
ponents at expression and pathways
pathway level
Cu, Cd 7, 14 d Perna viridis juveniles, metabolites detected by 1H NMR individual treatment metabolite against effects of individual com- Cu dominated metabolic profile changes 57
soft tissue abundance ponents
chlorpyrifos, Ni 4 d, semistatic Mytilus galloprovencin- 1.7 k cDNA array (MytArray) diff. treatment and concentration depend- against expected mixture effect interactive effects, common responses, mixture 58
cialis digestive tissue expression ent gene expression based on sum of TU for effect biased toward chlorpyrifos, additional
phenotypic effect mixture response features
EE2, ZM 7 d, semistatic, 3 Pimephales promelas target gene expression, 5 genes (qPCR) treatment and concentration related against effect of individual com- mixture exposure resulted in different expres- 59
dilutions early life stage expression increase/decrease pounds sion pattern
PFOS, Cd 7 + 3 d, simulta- Danio rerio early life target gene express., thyroid function, and individual treatment expression of against effects of individual com- potential increase of Cd toxicity by pre-exposure 60
neous, sequen- stage oxidative stress response target mRNA pounds to PFOS
tial, semistatic
chlorpyrifos, Cu 24 h, 3 dilutions Danio rerio olfactory GeneChip zebrafish array, 15 k diff. individual treatment gene expression, against expected mixture effect exposure to a mixture causes unique trans- 61
tissues expression treatment dependent fold induction based on independent action for criptional signature
gene expression change
a
For the abbreviations see Glossary.
Critical Review

dx.doi.org/10.1021/es2038036 | Environ. Sci. Technol. 2012, 46, 2508−2522

 Table 3. Combined Effect Observation and Assessment for Multiple Mixtures
mixture exposure regime biosystem molecular response observation evaluation authors’ assessment ref
As, Cr, Cd, Pb 24 h human keratinocytes 1,2 k Atlas human cancer array, 2,4 k individual treatment gene ex against As-induced gene responses metal mixture showed unique expression pattern 20
(RHEK-1) NEN human oncogen/tumor suppres- pression, treatment de
sor array, NEN kinase/phosphatase pendent fold induction
array
Cd, Pb, Cr 2 d, ternary mixture, 5 gene reporter assays con- CAT-ELISA activity from 13 stress gene concentration dependent against effects of individual com- no evidence for synergistic activation of gene 21
dilutions structed in HepG2cells promoters in individual assays induction pounds expression
mixture of 13 a.i. of 2 and 24 h, 3 dilutions HEK293 cell culture target gene expression, 5 genes (qPCR) concentration dependent against untreated control, and other mixture can inhibit cell proliferation by interfer- 45
pharmaceuticals increase in gene expression effects ence with functional pathways and cell cycle
progression
EE2, TCDD, Para- 24 h rainbow trout hepatocytes GRASP 16 k salmonid microarray, diff. individual treatment gene against effects of individual compo- unpredicted combined effects, TCDD signature 62
quat, NQO expression transcripts, treatment nents closest to mixture
dependent fold induction
methylmercury, ben- 14 d repeated oral dose; male F344 rat, liver lobe, 16k rat array individual treatment against expected mixture effects strong antagonistic effects and synergistic inter- 63
zene, TCE binary and ternary mix- kidney increase/decrease in gene based on sum of profiles of action; shift from specific to stress responses
Environmental Science & Technology

ture induction components

Cd, CCl4, pyrene 4 d, 1−3 dilutions Salmo trutta lacustris one salmonid microarray 1,273 genes, diff. concentration dependent against effects of individual compo- more or less than response additive 46
year old liver expression gene cluster response nents
patterns
mixture of 13 a.i. of 3d zebrafish liver (ZFL) cells 50mers microarray (14 k) diff. expression individual treatment increase/ comparison to other response pat- convergent responses in different cell lines and 64
pharmaceuticalsa decrease terns in HEK293 cells and zebra- disease models
fish liver tumors
E2, EE2, BPA, NP, 24, 72 h, 4 doses, multiple Mytilus galloprovencincialis target gene expression, 5 genes (qPCR) concentration dependent against effects form BPA and E2 mixtures can have both similar and distinct 65
MES, BP, NP1EC mix. injected in muscle relative transcript expression alone, and other effects effects, effect at lower concentrations than for
individual components
EE2, BDE-47, Cr−VI 7 d, EE2 aqua., BDE and Oncorhynchus mykiss liver GRASP 16k salmonid microarray, diff. individual treatment changes against individual compounds pro- no single compound dominated the profile, but 66
Cr as bolus arterial from males, sexually im- expression in gene induction increase/ files regarding altered genes, and mixtures are not simply additive, Cr and EE2

2513
sequential (Δ3d) injec- mature decrease and fold induction pattern that suggest nonadditivity act synergistic on mitochondrial dysfunction
tions
NCM, MeHg, PCB, 21 d, oral feed of dams Sprague−Dawley rats, anal- proteome pattern individual treatment differen- against mixture effects of subgroups effects of NCM markedly different from MeHg, 67
OC ysis in pups brain cere- tially expressed protein spots PCB, OC and not predicted from subgroups
bellum and hippocampus
PFOA, PFNA, 2d, binary and multiple rare minnow hepatocytes rare minnow cDNA microarray (1,773), individual treatment gene against effects of individual compo- consistent gene set vs unique expression pattern 68
PFDA, PFDoA, mixtures diff. expression transcripts, treatment de nents, and different mixture ratios for mixtures, complex crosstalk
PFOS, FTOH pendent fold expression
(8:2)
Roundup (Glyp), 62 d, semistatic binary and Platichthys f lesus juveniles suppression subtractive hybridization time dependent fold induction against effects of binary mixture complex mixture could be more toxic than binary 69
AMPA, Mecoprop, multiple mixture liver samples (786 clones), qPCR, 3 time points
Acetochlor, 2,4-D
E2, EE2, atrazine, 3 d, multiple mixture, Danio rerio embryos target gene expression, innate immune- individual treatment relative comparison of components effects mixture increased immune toxicity 70
nonylphenol per- semistatic related genes (6) transcript levels relative to single compounds
methrin
Alachlor, Mancozeb, 28 d, repeated gavage mouse blood and liver metabolites detected by 1H NMR, individual treatment changes against untreated control effects of mixture at concentrations below NOELs 71
Endosulfan, Cap- extracts from 14 weeks proteins detected by antibody array in metabolites and proteins for components
tan, Diazinon, C57 BL/6J males and (225 antibodies)
Maneb females
12 PBDEs 63 d, 3 dosage via food, Danio rerio liver tissue target gene expression, AhR metabolism, individual treatment transcript against TBDD and TBDD comparable AhR gene induction in mixture 72
multiple PBDD mixture and stress response (11 proteins) copies
TNT, 2,4-DNT, 2,6- 24 h, 8 multiple mixtures Daphnia magna 6−8 days target gene expression, 15 genes (qPCR), concentration dependent and against regression based sum of gene expression changes predictable for defined 41
DNT, DNB, TNB, old microarray (15 k) treatment related fold effects for components, and mixtures, mixture effects prevented prediction
RDX changes in expression profiles against groundwater sample of composition of complex mixtures
a
Same mixture used as in ref 45. For the abbreviations see Glossary.
Critical Review

dx.doi.org/10.1021/es2038036 | Environ. Sci. Technol. 2012, 46, 2508−2522

Environmental Science & Technology Critical Review

stressor, which is a common alternative view when studying

ref
40

73

74

18

75

76

77
complex unresolved mixtures in environmental samples.

system suitable for detection of NAs over a

unique expression profiles for different oils,
Inorganic compounds, i.e., essential and nonessential metals,

with model estrogen, suggesting complex

but cluster patterns emerge compared to

irrespective of component ratio and conc.

polluted site respond to PAH contami-

only some expression changes consistent

higher number of diff. expressed genes
were studied in fourteen of the 41 papers. Most papers on

higher chemical burden coincides with

induction suggest that flounders from
metals also included some organic compounds, and only five of

changes in key regulator genes

the reviewed papers were focused solely on metals20,21,50,54,57
authors’ assessment

while the other studies contained metals in mixtures with

organic substances. Mixtures of organic compounds comprised
diagnostic effect pattern

large range of conc.

of compounds such as pesticides (ten studies, mainly insec-
ticides), compounds with endocrine agonist or antagonist

compounds

interactions
activities (eight studies), polyaromatic hydrocarbons (four
studies), fluorinated and brominated flame retardants (two
nation

studies), other persistent organic chemicals (POPs) and dioxins

(four studies), and compounds used as biologically active

against effects of EE2 and 11-KT or 17β-

comparison of low vs high polluted sites,

ingredients in pharmaceuticals (three studies). Mixture com-

comparison of different sites, knowledge
against effects of individual components

linear regression for number of affected

comparison of mixtures regarding func-

position was often commented in terms of knowledge about the

tional clusters and gene association
on gene function and responses to

comparison among oils, comparison

against other compounds effects

trenbolone 48/72 h exposure components modes of action, indicating implicit anticipation of

knowledge on gene function

an expected (di)similar joint effect.

evaluation

Exposure Conditions. Exposure time was in the range of

one to seven days equivalent to the exposure duration of most
single chemicals

short-term bioassays used in ecotoxicity evaluation of

chemicals. Rarely, as in the case of the gene reporter assays47,77
networks

the exposure time was shorter than a day, and only occasionally
genes

(six studies) long-term exposure durations of more than a week

were being employed in ecotoxicological studies with mostly
aquatic organisms.51,69,72,73,75,76 In mammalian toxicology
exposure and sex dependent gene
expression, differences for male

individual treatment gene expres-

individual treatment gene expres-

individual treatment gene expres-

promoter activation and frac-

studies, by contrast, exposure periods are commonly intended

individual treatment gene tran-
scripts, treatment dependent

concentration dependent gene

concentration dependent in-

to reflect at least subchronic exposure of 14−28 days using

rodent bioassays.63,67,71 The latter are of specific interest as they
observation

may provide scope for phenotypic anchoring of molecular

crease/decrease

tional response
fold expression

responses to chronic toxicity outcomes in higher organisms.

When utilizing transcriptomic methodologies for effect char-
fish only

acterization, the intention typically is to retrieve information on

sion

sion

sion

sublethal effects, which for the experimental design results in

Table 4. Combined Effect Observation and Assessment for Complex Mixturesa

the question of how exposure concentrations are to be selected.

com., diff. expression
target gene expression,

Most of the analyzed studies provide explicit considerations

green fluorescent pro-
65mer microarray (16
GRASP 16k salmonid

fathead minnow array

tein (GFP) fluores-

microarray, diff. ex-
molecular response

array (0.16 k), diff.

custom-made cDNA

k), diff. expression

microarray (20 k),

to that end (Table SI-1). Most frequently authors selected

their mixture concentrations as defined fractions of effect con-
12/21 genes
expression

centrations known for standard apical effects such as lethality,

pression

(22 k)

cence

reproductive or viability responses, so that, on the one hand,

observed molecular responses can arguably be considered
as subacute effects, while on the other hand, the responses
liver and ovarian tissue

gene reporter assay in E.

veniles unspecified tis-
Pimephales promelas 150
d post hatch liver and

Pimephales promelas tes-

Oncorhynchus mykiss ju-

coli for 1900 promo-

Danio rerio female fish

can be interpreted in context with consented adverse effects

Caenorhabditis elegans
Platichthys f lesus liver

tis and liver tissue

(i.e., phenotypic anchoring). Other strategies comprised the

tissue, feral fish
biosystem

young adults

derivation of experimental exposure concentrations from

environmental occurrence or body burden, dilution series
gonad

testing, or other previous experience. Overall, for the selection

terzs
sue

of concentrations authors indicated a high degree of awareness

of potentially resulting interpretation problems. Unfortunately,
14 and 21d,

3 h, 3 dilu-
21 d, semi-
life history
exposure

4 d, semi-

5 months

this caution is not equally seen for assuring that the con-
regime

3 dilu-

static

static
tions

tions

centrations selected are actually achieved and maintained

For the abbreviations see Glossary.
4d

during the experiment. Mostly nominal concentrations are re-

ported, which for substances or mixtures with low water
∑ of POPs from 7 groups HCH,
chlordanes, DDT, PCB, PBDE,
effluent samples from 2 WWTPs

solubility, high volatility, high lipophilicity, potential for

kerosene, gas oil, heavy fuel oil,

technical mixture of naphthenic

effluent sample from WWTP

transformation, or toxicokinetic interaction between com-

as present at two river sites

pounds may lead to deviations from the expected exposure

3 river sediment samples
mixture

situations. Table SI-1 provides retrieved information for those

studies where analytical efforts have been made to either
HBCD, HCB

validate exposure 41,60,61,69 or even determine internal

crude oil

dose.54,57,66,72,75 Chemical mixture components studied were

acids

applied simultaneously, with only two exceptions that dealt

with the effect of a sequential exposure of PFOS followed by
a

2514 dx.doi.org/10.1021/es2038036 | Environ. Sci. Technol. 2012, 46, 2508−2522

Environmental Science & Technology
Cd60 and estradiol investigated together and in sequence with a
suspected agonist drug compound.43
Mixture Design. For the mixture ratios employed various
designs were realized. Most often mixtures were composed of
components at the same concentrations as studied individually
(13 studies, a + b design, Table SI-1). It has been demonstrated
that one may only detect antagonistic responses with certainty
using this design if more than one mixture component is
effective.1 Authors employing this design for studying low level
effects thus have to make sure that the components are indeed
present at individual no effect concentration which typically is
lower than a NOEC level.78 The a × n or n × n design (refer to
Figure 2) was employed in five studies. The surface design was
employed in only two of the studies.61,68 A ray design was used
in twelve studies, six of these employed toxic units to define the
mixture ratios. However, the toxic units employed were derived
from other response parameters, typically from apical effects. In
addition, we found six studies that used whole mixtures derived
from environmental samples of a partly unknown composition
and six studies that derived their defined mixture composition
from exposure considerations. Eleven out of the 41 studies
tested dilution series with more than two dilution levels during
the course of investigation.
Bioassays and End Points. With respect to biosystems
used for effect detection, studies on fish organs especially liver
and gonads contributed most to the existing evidence on
multiple responses amounting to almost half of the studies
reviewed here. In particular, studies on organs from exposed
model systems like zebrafish (Danio rerio) or rodents domina-
ted the picture. Other biosystems used, encompass inverte-
brates (Daphnia and molluscs), cell culture systems, or gene
reporter systems with bacterial cells. There is currently only one
study available that employed a plant system, namely the
unicellular green algae Chlamydomonas reinhardtii.54
Half of the studies we considered analyzed differential gene
expression employing microarrays, mostly from commercial
suppliers, while twelve studies used targeted multiple gene
expression observation and in particular qPCR techniques for
mixture response detection. Targeted in this context refers to
either the selection of different genes from a similar function
like the innate immune70 or thyroid system,60 else they repre-
sent established stress response signaling (e.g., ref 72). The
latter is also the common strategy for gene reporter based
assays,21,47 though novel approaches also offer scope for a
nontargeted perspective.77 Moreover, qPCR is commonly
employed to confirm findings from microarray experiments.
For other toxicogenomic techniques such as metabol-
omics51,55,57,71 or proteomics52,53,67 we have identified only
pioneering studies.
Signal Treatment. A major challenge in performing experi-
ments on biosystems where multivariate responses are to be
detected is capturing sources of undue variation. Details of how the
considered studies accounted for this are collated in Table SI-1.
Some studies tried to focus their replicates on the expected
largest confounder be that variation between individuals or
replicate samples, others try to utilize the obtained variance to
derive signal filters. All papers confirmed that the qPCR find-
ings overall were consistent with the findings from the micro-
arrays. Not all were confirmed in independent experiments,
and only one based this statement on an explicit correlation
analysis.61
Variance of responses was also considered in signal
treatment. Apart from procedures like normalization techniques
2515
Critical Review
to obtain comparable quantities from different samples or
measurements, the single most frequently used technique was
significance testing. The tests are constructed to select those
signals from the multivariate responses that, compared to
controls, show significant differences in either direction, i.e.
larger or smaller. Mostly, authors did account for a false
discovery rate by performing multiple testing on a sample.
Above all, it seems an almost consented approach in processing
transcriptome data from microarrays to apply an additional
filter prior to, or subsequently to, statistical testing, based on
the ratio of signals from treatment versus control. Authors thus
demonstrated their scepticism in the common strategy of
significance testing, however without providing specific reasons.
Furthermore, these filters sometimes appear to be applied in
order to reduce the number of data points for subsequent
considerations. The stated thresholds in transcriptome studies
typically use an arbitrarily defined minimum fold change, which
tends to lie between 1.3 to 1.8 fold.
For untargeted toxicogenomic approaches it is essential to
organize data for display and analysis in aggregated form
readying them for interpretation. To that end three steps can be
discerned in the considered literature. First, all authors used
graphical techniques such as Venn diagrams, heat maps, or fold
expression graphs to display the structure of their original
findings normalized only for control observations. In a second
step statistical methods such as hierarchical clustering, linear
regression on signal strength against concentration or time
variation, correlation or multivariate techniques such as prin-
ciple component analysis (PCA) or supervised techniques like
partial least-squares analysis (PLS) were employed to detect
major trends in the multivariate data. Finally, in the more re-
cent publications, bioinformatics tools such as clustering on
gene ontology terms, gene set and gene set enrichment analysis
or network connections were utilized to query observations for
biologically meaningful summarized information. In principle,
this last step aggregates multivariate response information into
biologically defined bins, such as biochemical pathways, which,
in turn, depends on access to adequate database information.
4. COMBINED EFFECT ASSESSMENT
While the number of mixture studies performed seems quite
impressive, none of them explicitly tested mixture hypotheses.
Consequently, it is mainly information on the way of designing,
performing, and evaluating mixture studies that can be deduced
from the existing studies.
The reported observations can be summarized as considering
the occurrence/absence of a signal (qualitative response) or the
analysis may be based on the signal intensity (quantitative
response). More than two-thirds of the studies, reported
qualitatively different treatments, while the classical toxico-
logical concept of dose-graduation played a much smaller
role (i.e., being reported in one-third of the stud-
ies). 21,41,42,45−48,54,58,59,65,73,77 The time-dependence of
responses was considered in only one of the studies.69 While
studies based on nontargeted techniques, like the microarrays,
commonly utilized a qualitative approach, studies that
employed reporter assay responses or qPCR determined
mRNA levels, more frequently evaluated their data utilizing
quantitative approaches. Taking a diagnostic perspective
authors typically intended to identify components of the
mixtures that drive the observable effect pattern. With an
extrapolative scope, studies were undertaken to generate an
expectation for the mixture response based on responses of
dx.doi.org/10.1021/es2038036 | Environ. Sci. Technol. 2012, 46, 2508−2522
Environmental Science & Technology
mixture components when studied alone. The mechanism-
based focus finally can be summarized as striving for a causal
explanation of the sequence of molecular events provoked
through mixture exposure.
Assessment Terminology. Almost all assessments on the
observed mixture effects that are reported, e.g. ‘synergistic’,
‘additive’, or ‘antagonistic’ responses or even ‘surprisingly, we
found’, comprise a comparative statement, i.e. the experimental
observation is compared against an expectation of the authors.
However, the majority of papers did not provide an explicit
hypothesis on what an expected combined effect from a
mixture exposure would look like. Thus, it was often difficult to
comprehend which findings stipulated the conclusions
regarding mixture interaction. Moreover, terminology used to
describe findings was often used with different meanings. The
frequently used term interaction, e.g., could be found to bear at
least the following four connotations: (1) signifying a specific
type of response that is elucidated (e.g., induced pathway)
(e.g. ref 64), (2) referring to all responses provoked by more
than a single compound (e.g. ref 50), (3) depicting a response
that is more than no effect (low dose effect) (e.g. ref 63), or (4)
demarking deviation of responses from a mixture model, e.g.
concentration additive (e.g. ref 54), effect addition (e.g. ref 49),
or agonism/antagonism (e.g. ref 51). Thus, if confusion is to
be avoided in the future, clarity as to the meaning of spe-
cifically employed assessment terminology is essential, as e.g.
suggested by Greco, Bravo, and Parson3 or by Hertzberg and
MacDonell.79
Mixtures selected are often commented in terms of knowledge
about the components modes of action, thus implicitly anticipa-
ting expected combined effects as concentration additive, inde-
pendently acting or deviating. Indeed none of the studies linked
the findings to existing evidence on combined effects for the
specific compound mixtures and effect typologies (e.g., for
pesticides,80 endocrine active compounds,8 or metals81).
Undertaking to make conceptual assumptions transparent
the assumptions used for assessment of the observed mixture
effects were identified, both for approaches that are interpreting
the occurrence of signals (qualitative assessment) and interpre-
tations that are based on signal intensities (quantitative assess-
ment).
Qualitative Assessment. The occurrence of signals under
exposure was compared between treatments and subsequently
the arguments were built on the group of signals retrieved for
the mixture and whether they were included in the reference
treatments (e.g., components), totally, in part, or not at all. The
interpretation is typically oriented toward the exposure iden-
tification of dominating causative chemicals or response char-
acterization as similar or dissimilar. The Venn diagram is a
technique of displaying observations that is often employed to
that end. An example taken from ref 50 is displayed in Figure 3.
We see that Ni and Cd exposure led to a number of similarly
differentially regulated gene transcripts in Daphnia magna. Of
these, two-thirds (n = 22) reoccurred under the mixture
exposure at half of the concentrations of both components,
while less than half the signals that were compound specific
responses (n = 10) re-emerge under the mixture exposure. An
additional 85 transcripts were uniquely differentially expressed
in the mixture. Next, it would now be interesting to annotate
the signals and allocate them to specific pathways and interpret
stress and toxic responses.
Major shortcomings of this approach are that the sta-
tistics and filtering approaches used to select signals impact on
2516
Critical Review
Figure 3. Venn diagrams for differentially expressed gene transcripts in
Ni and Cd exposed and coexposed Daphnia magna (from
Vandenbrouck et al. 2009).
what we discuss as outcome during interpretation. At least
the different statistical tests used in conjunction with often
arbitrarily chosen cut off values should make us aware that
results may be highly dependent on the method employed.
A good practice to check the robustness of the findings e.g. with
respect to the selection of a specific cut off value for n-fold
expression is demonstrated in ref 77. Another way to overcome
some limitations of this approach could be to aggregate the
information prior to interpretation. Most statistical and bio-
informatic interpretation tools discussed above strive for that,
basically by trying to group, cluster, or else logically organize
the information, e.g. to allocate various signals to a common
pathway. These approaches sometimes also work with the
original responses without prior application of filters on the
responses observed.
Another approach was to interpret the number of signals
occurring in relation to the degree of contamination, as Menzel
and co-workers74 did when comparing differently contaminated
sediments using microarray gene profiling in Caenorhabditis
elegans. Underlying notions could either be that the degree of
contamination results in different dosages in the exposed bio-
systems and that a higher dose leads to higher number of
responding genes or to higher number of chemicals with similar
consequences. For a complex mixture of naphthenic acids it
could indeed be shown that a multiple reporter system showed
clear increase in signal numbers with increasing dose.77 Also,
Judson et al.82 reported that the number of responding
pathways, studied by a multitude of receptor-based assays,
increased with increasing doses for various chemicals. By
contrast, Tilton et al.61 showed various types of responses in
the number of differentially expressed genes upon exposure of
zebrafish to three different chlorpyrifos concentrations. Thus,
we need to understand the relation between dose and gene
responses for individual components prior to addressing mixtures
of variable compound composition.
Hendriksen and co-workers63 proposed yet another view on
the interpretation of altered occurrence of signals when com-
paring individual compounds and their mixtures. In their
studies of mixtures from benzene, trichloroethylene, and methyl
mercury on transcriptomic responses from liver and kidney
tissue of rats exposed for 14 days, they deduced from gene
ontology based identification of affected pathways, that
specific responses resulting from exposure to individual
compounds were replaced by general stress responses upon
mixture exposure.63
dx.doi.org/10.1021/es2038036 | Environ. Sci. Technol. 2012, 46, 2508−2522
Environmental Science & Technology
This links to a debate on the role of common stress response
pathways in dealing with exposure to environmental con-
taminants.83,84
Quantitative Assessments. Signal intensity based inter-
pretations follow the idea that the intensity of the exposure
should be monotonously related to dose and to the change in
differentially expressed signal intensity. This concept is rather
fundamental to toxicological work, and indeed several papers
demonstrate evidence that an increase in concentration of a
toxicant will give rise to corresponding changes in the expression
of transcriptomic,21,41,42,70,73 proteomic,85 and metabol-
omic signals.86 It is, however, also known that individual trans-
criptomic and proteomic signals may not strictly adhere to a
monotonous behavior but instead may show U-shaped con-
centration dependencies or highly variable responses.54,86
Hutchins and co-workers54 e.g. showed qPCR detected gene
transcription intensity for various genes in Chlamydomonas
under Cu2+ and Pb2+ exposure demonstrating classic concen-
tration dependencies at lower concentrations but inverted trends
at higher exposure levels. Various arguments have explained such
patterns, such as cytotoxicity induced at higher concentrations
that overlay stress or specific responses. Quantitative mixture
assessment typically relies on monotonous changes in response
as represented in sigmoidal concentration response relationships,
thus U-shaped curves present specific challenges. It is striking
that apart from purely descriptive approaches such as linear
regressions performed by refs 46,73, and 77 the modeling of
dose response relationships has not been used for combined
effect predictions using the established reference models.
Time patterns of transcriptomic response intensities can be
regarded as equally important as their dose-dependencies. The
few studies that have actually incorporated observations of time
dependence for toxicity description show results that are parallel
to the prevalent thinking of cumulative damage occurrence in
toxicology (e.g., refs 54,87, and 88).
The subsequent mixture interpretation may then be
approached as is established for apical end points.9 Surprisingly,
the literature reviewed, with few exceptions,47,50,54,61 did either
not account for the established approaches or displayed gross
misconceptions of it. Thus, the vast majority of studies evalu-
ated their mixture observations through direct comparison with
the responses using individual components (Tables 2−4).
The flaw in the approach of comparing the quantitative
mixture outcome directly with those of the components can be
demonstrated by recalling the previously introduced thought
experiment: The simplest mixture of all is that of combining a
defined amount of a substance with a defined amount of the
same substance. When looking at the expected quantitative
effect of this so-called ‘sham combination’ one clearly would
not expect the combined effect to be equal to that of each
individual dose (for illustration cf. ref 39). This, however, is
what most of the papers considered here suggested implicitly.
Rather, it is consented in mixture toxicology that the expected
combined effect should be derived from a function taking into
account the activities of all individual components which
typically calls for testing the mixture at lower dosage levels than
used for the individual components. For the case of mixing one
compound with itself e.g. the dilution principle proves helpful
in providing a reasonable mixture effect expectation. For an
effect of interest, take dilutions (fractions) of the components.
In the example of the sham combination, if the compound
provokes a certain effect at the defined concentration, two
times half that concentration is expected to give rise to the
2517
Critical Review
same effect, as is true for a mixture ratio of one-third to two-
thirds of that effect concentration or three times one-third and
so on. Such an evaluation should be feasible for 11 of the
studies, because they considered dilution series with more than
2 concentration levels.
In the studies reviewed here, the models introduced above as
concentration or dose addition have explicitly been adopted by
refs 47 and 50. The work of Dardenne et al.47 reports the mix-
ture effects for 8 different binary mixtures investigated in dilu-
tion series on 14 different stress gene reporters. It is certainly
pioneering in its attempt to pursue a strictly quantitative per-
spective and demonstrates good agreement between expected
and observed mixture responses for the various mixtures and
stress gene inductions. However, the sensitivity of the reported
mixture responses may raise concerns whether the reported
responses could also be explained by the individual compounds
activities alone. The strategy chosen by Vandenbrouck et al.50
is, again, based on the idea of testing the concentration addition
prediction for the mixture outcome. This could not be con-
firmed for microarray-detected expression responses of
Daphnia magna juveniles exposed to two binary metal mixtures
with nickel. A problem here is that fractional toxic units were
perceived as effect measures upon which the comparison of
components and mixture responses is then based. This
assumption would be true only if linear and parallel dose−
response functions existed for the substances considered, which
was not tested or confirmed in the study, but as we have
discussed is unlikely for low effect levels.
Alternatively, one may derive a quantitative estimate of an
expected combined effect from the multiplication of the re-
sponse probabilities for the mixture components (see Table 1).
The technical advantage of employing independent action as a
reference model for the observations considered here is that
explicit mixture effects can be calculated from any effect estimate
for the components without prior modeling of a concentra-
tion response relationship. Tilton et al.61 and Hutchins et al.54
have taken advantage of this model property. Hutchins and co-
workers54 studied transcriptome signatures for nine targeted
stress genes in Chlamydomonas exposed to two binary metal
mixtures of copper and lead each with cadmium. Observed and
predicted fold changes were compared for six gene transcripts,
and predictions according to independent action provided a
good estimate of what was actually observed for five of them,
irrespective of the mixture if one allows for some variance. In the
remaining case the independent action model predicted higher
than observed fold changes. Tilton and co-workers61 investigated
the effect of a chlorpyrifos and copper mixture on microarray
detected transcriptome profiles in olfactory tissues from short-
term exposed Danio rerio. The calculated n-fold changes in ex-
pression rates were compared for all signals retrieved against the
responses expected for an independent combined effect. Overall
the majority of observed responses were smaller than expected
by independent action. A problem possibly encountered here
may stem from the lack of knowledge about the underlying
concentration response relationship which makes it impossible to
calculate mixture prediction other than independent action.
Further, if there is lack of information on the maximum effects
for the individual responses one may calculate mixture
expectations that would not even be detectable for the individual
components.
dx.doi.org/10.1021/es2038036 | Environ. Sci. Technol. 2012, 46, 2508−2522
Environmental Science & Technology
5. OUTLOOK: FROM ANECDOTAL EVIDENCE TO
HYPOTHESIS DRIVEN MIXTURE STUDIES
The current evidence to support the published mixture assess-
ments from toxicogenomic studies is as yet mainly observa-
tional. How can this be advanced in future studies? As a first
step we recommend that experimenters provide an explicit
hypothesis of what they expect for the mixture of interest.
Then, as an alternative to the currently popular repeated dose
regime and statistical testing of signal variance, we suggest to
use graded exposure situations and regression-based analysis.
The development of these approaches would follow the trend
in toxicology where risk assessment moves from NOEC esti-
mates toward benchmark concentrations.78
Potential scepticism as to whether consistent patterns of
signals with covariance in concentrations may be achievable
might be overcome by recalling the pioneering studies of Michaelis
and Menten or Hill that those authors used to formulate quan-
titative models on the relationship between the concentration of a
substance and the molecular responses of biosystems. Also, first
promising steps to that avail for toxicogenomic responses can
be found46,77 that utilize linear regressions and describe many
of their signals successfully in that way. Moreover, there are other
successful efforts known which do not stem from the mixture liter-
ature but describe concentration dependence of transcriptomic,
proteomic, or metabolomic signals (e.g., refs 85−87) and even
employ regression techniques for their description.85,87
With advancing a quantitative perspective on transcriptomic,
proteomic, or metabolomic responses we could also calculate
explicit combined effect predictions using the two reference
models available. As we would expect responses detected by
‘omics’ analysis to be attached to separate or converging
pathways, the reward in adopting both reference models
could be that toxicogenomic studies may allow detecting
and distinguishing similar and dissimilar joint responses in
toxicological action.38,89
Another determinant for future progress can be seen in using
more balanced approaches toward the exposure, effect, and
modeling parts of toxicogenomic mixture studies. Considering
the resources spent for toxicogenomic response studies, it is
notorious to state that exposure conditions should not only be
carefully selected but also analytically validated. This will be an
essential step prior to considering implementing any toxi-
cogenomic data interpretation into chemical risk assessment. If
we furthermore intend to elucidate the sequence of biological
responses as we may when referring to interactions with
pathways, the challenge becomes also to separate toxicokinetic
from toxicodynamic responses. This means we would need
advanced consideration of a compounds uptake and fate within
an organism to be able to discern whether an observed time-
dependent effect is due to an increase in dose over time or
consequence of enhancement through a specific molecular
interaction. Suggestions for incorporation of dosimetry have
been made already for cell-based response analysis90 and may
be implemented for other bioassays as well.
A further difficulty will lie in finding strategies of how to deal
with time-dependent responses in mixture modeling as the
established reference models do not account for this. Process-
based modeling could be one solution. Modeling biological
action for multiresponse outcomes could be approached from a
process-perspective as it is established in pharmacology91 and
ecotoxicology.92,93 Jusko et al.91 for instance have used array
data from corticosteroid provoked effects in rat livers to cluster
2518
Critical Review
responses into a limited number of time-dependent patterns,
which they subsequently used to formulate process-based
quantitative dose−response models.
Regarding the qualitative perspective, improvements are
expected by putting observable molecular responses into scope
through connecting different response levels as in system
biology approaches19 or linking them to phenotypic outcomes
as in the concept of adverse outcome pathways.36 With the
progress in analytical platforms and bioinformatic tools, how-
ever, the main issue is probably not in generating more data but
in providing more effective ways to digest these. Williams
et al.94 provide an excellent example of how network modeling
can make sense of multivariate toxicogenomic responses
obtained from nonmodels, i.e. overcome current limitations
for nonannotated organisms.
In this context it may be interesting to study whether the
means suggested for receptor-based high-throughput assays for
the derivation of effective doses for toxicity-related biological
pathways95 could be adapted for microarray or other non-
targeted techniques. Also, the notion that toxicity as an apical
outcome in biosystems derives from an overwhelmed stress
defense system96 which can be identified through study of a
limited number of stress pathways rather than the identification
of primary interactions could help. Again, for experimental
studies this would require extended focus on the time pattern
of responses. These latter considerations are not specific for a
combined effect analysis.
■
*
ASSOCIATED CONTENT
S Supporting Information
SI - Table 1: mixture study design and data treatment. This
material is available free of charge via the Internet at http://
pubs.acs.org.
■ AUTHOR INFORMATION
Corresponding Author
*Phone: +49.341.235.1522. Fax: +49.341.235.1787. E-mail:
rolf.altenburger@ufz.de.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
Part of this study was supported by the Federal Environment
Agency (UBA) project FKZ 370956404. R.A. acknowledges the
receipt of a fellowship under the OECD Co-operative Research
Programme: Biological Resource Management for Sustainable
Agricultural Systems to the National Research Centre for
Environmental Toxicology (Entox) at the University of
Queensland and Queensland Health, Brisbane, Australia. For
constructive critics we thank four reviewers. Earlier versions of
the manuscript have been commented by Till Luckenbach and
Dimitar Zitzkat; for language improvement we thank Marita
Goodwin.
■
GLOSSARY
Substance names
ATZ
atrazine
BAP
benzo[a]pyrene
BbF
benzo[b]fluoranthene
dx.doi.org/10.1021/es2038036 | Environ. Sci. Technol. 2012, 46, 2508−2522
Environmental Science & Technology Critical Review

BDE-47 nonylphenol monoethoxylate carboxylate

2,2,4,4,-tetrabromodiphenyl ether OC
BP organochlorine pesticides (refer to Pelletier et al. 2009)
benzophenone PBDE
BPA polybrominated dibenzo-p-dioxins
bisphenol A PCB
CHP polychlorinated biphenyl
chlorpyrifos PCB-77
Cr−VI 3,3’,4,4’-tetrachlorobiphenyl
hexavalent chromium PCP
DBahA pentachlorophenol
dibenzo[a,h]anthracene PM
DBaIP permethrin
dibenzo[a,I]pyrene TBDD
DDT 2,3,7,8,-tetrabromodibenzo-p-dioxin
dichlorodiphenyltrichloroethane TCB
E2 3,3’,4,4’-tetrachlorobiphenyl
17β-estradiol TCE
EE2 trichloroethylene
17α-ethynylestradiol TCDD
FA 2,3,7,8-tetrachlorodibenzo-p-dioxin
fluoranthene ZM
Glyp ZM 189,154 experimental anti-estrogen of Astra Zeneca
glyphosate (active ingredient in the Roundup product other
formulations) AhR
HBCD aryl hydrocarbon receptor
hexabromocyclododecane a.i.
HCB active ingredients
hexachlorobenzene CAT
HCH chloramphenicol acetyltransferase
hexachlorocyclohexane conc.
ICI 182,780 concentration
antiestrogen drug marketed under the trade name Fluvestrant 2DGE/MS
11-KT two dimensional gel electrophoresis with mass spectrometric
11-ketotestosterone protein identification
MeHg diff. expression
methylmercury differential expression
MES EC25S1
mestranol effect concentration for substance 1 elucidating 25% effects
Mito C ER
mitomycin C estrogen receptor
MMS GO
methylmethanesulfonate gene ontology
NQO ip.
4-nitroquinoline-1-oxide intraperitoneal
MNNG MOA
N-methyl-N′-nitro-N-nitrosoguanidine mode of action
NAs NMR
naphthenic acids
NCM nuclear magnetic resonance
qPCR
Northern Contaminant Mixture: 27 persistent environmental
quantitative polymerase chain reaction
contaminants as detected in blood of Canadian Arctic
RT-PCR
Population (composition found in Pelletier et al. 2009)
including methyl mercury (MeHg), polychlorinated biphenyls real time polymerase chain reaction, here taken to refer to
(PCB), and organochlorine pesticides (OC) semiquantitative methodologies, such as band intensity
NP measures of PCR products
nonylphenol SSH
NP1EC suppressive substractive hybridization
2519 dx.doi.org/10.1021/es2038036 | Environ. Sci. Technol. 2012, 46, 2508−2522
Environmental Science & Technology
TU
toxic unit, i.e. exposure concentration of component over effect
concentration of the same compound
TUS
sum of toxic units (TU)
WWTPs
waste water treatment plants
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