Bioanalytical screening methods for dioxins and dioxin-like compounds - A review of bioassay/biomarker technology

Abstract

Determination of environmental pollutants utilizing biodetectors such as bioassays, biomarkers, enzyme immunoassays (EIAs), or other bioanalytical tools is a continuously growing area. The present literature review describes the principles and advantages/limitations of several bioanalytical detection methods (BDMs) for the screening and diagnosis of dioxin and dioxin-like compounds. This study characterizes briefly the family of dioxin and dioxin-like compounds, discusses potential Ah receptor (AhR) ligands and cytochrome P-450 (CYP) 1A1-enzyme-inducing compounds. ‘Milestones’ in the development of BDMs are summarized and explained in detail for a number of bioanalytical tools that can be used to detect these classes of dioxin-like persistent bioaccumulative toxicants (PBTs). The design of a screening profile with a battery of bioassays/biomarkers coupled with the chemical analysis is evaluated. The relative potencies (REPs) to 2,3,7,8-TCDD for dioxin-like compounds are reviewed for various BDMs and the differences are noted.

Full text

Bioanalytical screening methods for dioxins and dioxin-like
compounds — a review of bioassay/biomarker technology
Peter A. Behnisch
a,b,
*, Kazunori Hosoe
a
, Shin-ichi Sakai
b,c
a
Life Science Research Laboratories, Kaneka Corporation, Takasago, Japan
b
Environment Preservation Center, Kyoto University, Kyoto, Japan
c
National Institute for Environmental Studies, Tsukuba, Japan
Received 6 June 2000; accepted 20 February 2001
Abstract
Determination of environmental pollutants utilizing biodetectors such as bioassays, biomarkers, enzyme immunoassays (EIAs), or other
bioanalytical tools is a continuously growing area. The present literature review describes the principles and advantages/limitations of several
bioanalytical detection methods (BDMs) for the screening and diagnosis of dioxin and dioxin-like compounds. This study characterizes
briefly the family of dioxin and dioxin-like compounds, discusses potential Ah receptor (AhR) ligands and cytochrome P-450 (CYP) 1A1-
enzyme-inducing compounds. ‘Milestones’ in the development of BDMs are summarized and explained in detail for a number of
bioanalytical tools that can be used to detect these classes of dioxin-like persistent bioaccumulative toxicants (PBTs). The design of a
screening profile with a battery of bioassays/biomarkers coupled with the chemical analysis is evaluated. The relative potencies (REPs) to
2,3,7,8-TCDD for dioxin-like compounds are reviewed for various BDMs and the differences are noted. D2001 Elsevier Science Ltd. All
rights reserved.
Keywords: Dioxin; Dioxin-like compounds; Bioassay; Biomarker; Biomonitoring; Dioxin toxic equivalents quotient (TEQ); Polychlorinated biphenyls (PCBs);
Polychlorinated dibenzodioxins (PCDD); Polychlorinated dibenzofurans (PCDF)
1. Introduction
There is considerable public, regulatory, and scientific
concern regarding human exposure to dioxin-like com-
pounds. Seven polychlorinated dibenzo-p-dioxins (PCDDs),
10 polychlorinated dibenzofurans (PCDFs), and 12 poly-
chlorinated biphenyls (PCBs) are collectively referred to as
dioxin-like compounds (e.g., Brouwer et al., 1995; Larsen
et al., 2000; Van den Berg et al., 1998, 2000; World Health
Organization (WHO), 1999; US EPA, 2000a,b). These
0160-4120/01/$ – see front matter D2001 Elsevier Science Ltd. All rights reserved.
PII: S0160-4120(01)00028-9
Abbreviations: AhIA, aryl hydrocarbon immunoassay; AHH, aryl hydrocarbon hydroxylase; AhR, aryl hydrocarbon receptor; Arnt, AhR nuclear
translocator; BBP, butylbenzyl-phthalate; BDM, bioanalytical detection method; CALUX, chemical-activated luciferase gene expression; CAFLUX, chemical-
activated fluorescent protein expression; CEH, chicken embryo hepatocyte; CYP, cytochrome P-450; CV, correlation variability; DBP, dibutyl-phthalate;
DELFIA, dissociation-enhancement lanthanide fluoroimmunoassay; DOP, dioctyl-phthalate; DRE, dioxin response element; EGFP, enhanced green fluorescent
protein; EIA, enzyme immunoassay; ELSM, early life stage mortality assay; ER, endocrine receptor; EROD, ethoxyresorufin-O-deethylase; FI, fluorescence
immunoassay; GRAB, gel retardation of AhR DNA binding; HAH, halogenated hydrocarbon; HCB, hexachlorbenzenes; HRGC/HRMS, high-resolution gas
chromatography/mass spectrometry; IEQ, dioxin induction equivalent; I-TEQ, international TCDD toxic equivalents; LD, lethal dose; MDL, minimal detection
limit; MROD, methoxyresorufin-O-demethylase; MS, mass spectrometer; P450HRGS, P450 human reporter gene system; PAH, polyaromatic hydrocarbon;
PBDE, polybrominated diphenylether; PBT, persistent bioaccumulative toxicant; PCAB, polychlorazobenzene; PCAOB, polychlorazoxybenzenes; PCB,
polychlorinated biphenyl; PCBA, polychlorinated biphenyl anisole; PCDF, polychlorinated dibenzofuran; PCDPT, polychlorinated diphenyl toluene; PCDT,
polychlorinated dibenzothiophene; PCPA, polychlorinated phenoxy anisole; PCN, polychlorinated naphthalene; PCT, polychlorinated terphenyl; PCTA,
polychlorinated thianthrene; PHAHs, polyhalogenated aromatic hydrocarbons; PROD, pentoxyresorufin-O-depenthylase; PXB, polyhalogenated biphenyl;
PXDD, polyhalogenated dibenzo-p-dioxin; PXDF, polyhalogenated dibenzofuran; PCXE, polychlorinated xanthene; POP, persistent organic pollutant; PCXO,
polychlorinated xanthone; QSAR, quantitative structure–activity relationship; R
b/c
, ratio between bioassay-TEQ and TEQ; REP, relative potency; RIA,
radioimmunoassay; TEF, dioxin toxic equivalents factor; TEQ, dioxin toxic equivalents quotient; WHO, World Health Organization
* Corresponding author. Tel.: +81-794-452427; fax: +81-794-452466.
E-mail address: behnisch@takaken.kaneka.co.jp (P.A. Behnisch).
www.elsevier.com/locate/envint
Environment International 27 (2001) 413 – 439

compounds have recently been involved in several acci-
dents, which led to possible contamination of feed/foods
products (e.g., chickengate scandal, Belgium, Bernard et al.,
1999; and citrus pulp pellets scandal in Brazil, Malisch
et al., 1999; see for review European Commission,
2000a,b). When dealing with such accidents or conducting
monitoring, it would be useful to have screening methods,
which can eliminate nonpositives prior to performance of
chemical analysis, can rank the potency of substances and
complex mixtures suspected of the contamination, are cost-
effective, fast, have a minimum of false-negatives, and are
accepted by governmental regulators.
Until about 10 years ago, high-resolution gas chroma-
tography/mass spectrometry (HRGC/HRMS) was the only
option and became the ‘‘golden standard’’ for detecting
dioxin-like compounds. Since 1970, it has been estimated
that more than US$1 billion has been spent on determining
the toxicity of PCDD/PCDFs in samples (Van den Heuvel
and Lucier, 1993). Therefore, in the last decade, recent
advances in the biotechnology have allowed to develop a
battery of in vitro bioassays and ligand binding assays for
dioxin and dioxin-like compounds. By screening a large
number of samples from a site, the most contaminated
locations are identified, and there is a substantial cost saving
when expensive analyses are not conducted in clean areas.
This type of two-step process not only saves money, but also
improves the accuracy, reliability, and scientific basis for the
quantitative assessment of environmental health risks. For
several bioanalytical dioxin tests, official methods by gov-
ernmental authorities have now been approved such as EPA
Method 4425 (Reporter gene assay) or EPA Method 4025
(Immunoassay) (see for review Cooke et al., 2000).
Several experts have agreed in 1997 that the use of in
vitro bioassays provides a useful tool as a prescreening
method for TEQs in environmental samples (Van den Berg
et al., 1998, 2000).
The present review focuses on several bioanalytical
detection methods (BDMs) for measuring dioxin-like
activity, including the 7-ethoxyresorufin-O-deethylase
(EROD)-bioassay, the aryl hydrocarbon hydroxylase
(AHH) bioassay, the enzyme immunoassay (EIA), the
reporter gene assay [e.g., chemical-activated luciferase gene
expression (CALUX) or P450HRGS], the gel retardation of
AhR DNA binding (GRAB) assay, the recAhr DELFIA
assay kit, the Ah receptor (AhR) (or filtration) assay with
radiolabeled dioxins, and the Ah-immunoassay (AhIA) (see
Table 1). These methods are based on the ability of key
biological molecules (e.g., antibodies, receptors, enzymes)
to recognize a unique structural property of the dioxin-like
compound, or on the ability of cells or organisms to have a
specific response to dioxin-like compounds. Several reviews
and reports have already been published about biomarkers
or bioassays (Bosveld and Van den Berg, 1994; Brecht and
Abuknesha, 1995; Brouwer et al., 1995; Bucheli and Fent,
1995, Cooke et al., 2000; Denison et al., 1999; Diaz-Ferrero
et al., 1997; Djien Liem, 1999; Harrison and Eduljee, 1999;
Hilscherova et al., 2000; Hoogenboom et al., 1999; Van
Emon et al., 1998; Sherry, 1997) and the toxicity (Ahlborg
et al., 1992, 1994; Birnbaum and De Vito, 1995; Brouwer
et al., 1995, 1998, 1999; Delzell et al., 1994; Denison and
Heath-Pagliuso, 1998; Denison et al., 1998; Giesy and
Kannan, 1998; Landers and Bunce, 1991; Oosterkamp
et al., 1997; Poland and Knutson, 1982; Safe, 1984, 1986,
1987, 1990, 1993, 1994, 1997, 1998a,b; Safe et al., 1987,
1989, 1991, 1997; Sanderson and Van den Berg, 1999;
Schecter, 1994; Schrenk, 1998, Seidel et al., 2000; Van den
Berg et al., 1998, 2000; Van Leeuwen et al., 2000) of dioxin
and dioxin-like compounds. Therefore, the authors do not
intend to give a complete overview of the available liter-
ature, but rather try to present the current state-of-the-art in
bioanalysis for dioxin-like compounds.
2. Characteristics of dioxin-like activity
Most bioassays for the determination of dioxin toxic
equivalents are based on the assumption that all dioxin-
related compounds act through the AhR signal transduction
pathway. The current understanding of the system is that a
dioxin-like compound: (1) binds to the AhR, (2) the com-
plex is translocated to the nucleus of the cell, (3) where it
induces the transcription of a number of genes, and sub-
sequently, (4) the production of proteins including cyto-
chrome P-450 (CYP) 1A, an enzyme involved in oxidation,
reduction, and hydroxylation reactions, also called mixed-
function oxidases. In addition to these mixed-function
oxidases, the expression of a number of other enzymes is
affected by exposure to dioxin-like compounds: an aldehyde
dehydrogenase, an NADPH-quinone-oxidoreductase, the
Phase II conjugating enzymes glutathione-S-transferase
and UDP-glucuronosyltransferase. The AhR complex also
affects the expression of other genes that influence basic
cellular processes, such as growth, differentiation, and
programmed cell death (e.g., Brouwer et al., 1995, Denison
et al., 1998). CYP1A1 is one of the first proteins to be
expressed after exposure to TCDD and related compounds;
its up-regulation is based on an AhR-mediated mechanism
of action. This AhR mechanism of action is also believed to
play a pivotal role in the onset of many other aspects of
dioxin-like toxicity, e.g., teratogenicity, dermal, ocular,
hepatic, thymic, and carcinogenicity. There is no indication
of a direct role of induced CYP1A1 in the onset of toxic
responses, other than the production of toxic hydroxy
metabolites (that cause low vitamin A and thyroid hormone
levels) and perhaps the accelerated breakdown of some
natural substrates for CYP1A1 (e.g., Van den Berg et al.,
1998, 2000). One known substrate is benzo[a]pyrene and it
is known that the breakdown product (metabolite) is the key
carcinogen and not the parent compound. As mentioned
previously, the initiation of changes in the expression of
these genes begins with a ligand binding to the AhR.
P.A. Behnisch et al. / Environment International 27 (2001) 413–439414

Table 1
Principles of determination methods for dioxin-like compounds (Brouwer et al., 1995; Diaz-Ferrero et al., 1997; Djien Liem, 1999; Hilscherova et al., 2000; Sadik and Witt, 1999)
Method Principles Reference
(I) Biomarkers All kind of biological responses to dioxin-like compounds in plants, wildlife and human. Several in vivo bioassays, e.g., CYP1A1-IA,
AHH or EROD (e.g., Sewall and Lucier, 1995)
Schecter, 1994
(II) Bioassays Based on the AhR-dependent mechanism and utilized by either AhR-containing extracts, or mammalian cell cultures to
measure a specific biological response (enzyme induction, e.g., EROD/AHH; cell proliferation, e.g., keratinization; porphyrin
accumulation and AhR ligand, or DNA binding). Most of them are based on the induction of gene expression and the
magnitude of induction of dioxin-like compounds or a mixture is expressed relative to 2,3,7,8-TCDD.
Safe, 1984, 1986, 1987,
1990, 1993, 1994, 1997,
1998a,b; Safe et al., 1987,
1989, 1991, 1997;
Sanderson and van den
Berg, 1999; Sanderson
et al., 1996, 1998; Tillitt
et al., 1996; Kopponen
et al., 1994; Schwirzer
et al., 1998; Schrenk et al.,
1991; Kennedy et al., 1996
(a) Enzyme induction
(e.g., EROD)
These bioassays use cell lines, which express AhR-mediated CYP1A induction. This method employs 96-well plates, a
plate-reading spectrofluorometer, and a fluorescence-based protein assay that enables the simultaneous measurement of the
oxidation from ethoxyresorufin to resorufin and the protein amount. The bioassay integrates nonadditive interactions
among AhR agonists and other compounds by measuring a final receptor-mediated response.
(b) In vitro luciferase
assay (e.g., CALUX)
The recombinant cell line is modified to include a luciferase reporter plasmid responsive to the liganded AhR– Arnt complex.
When this complex is present in the cells, they produce luciferase, which oxidizes the added substrate luciferin and the emitted
bioluminescence is detected by a luminometer.
Cells: Ziccardi et al., 2000.
Yeast: Takigami et al., 1999
(c) Cell proliferation-based
assays
Dioxins are potent inducers of chloracne in humans. This skin aberration can be interpreted as an altered differentiation pattern
of acinar sebaceous base cells and a change in the rate of terminal differentiation of the keratinocytes. This rate can be measured
by the induction of dioxin-like compounds in primary cultures of human keratinocytes. The hallmark of dioxin exposure is the
development of chloracne, a persistent form of acne characterized by hyperkeratinization.
Van Pelt et al., 1992
(d) DNA binding assays Quantification of the AhR binding of dioxin-like compounds and the stimulating of its DNA binding (GRAB assay: measurement
of the inducible AhR binding to
32
P-labeled DNA). Although this assay does not address the ability of dioxin-like compounds to
activate gene expression, there is an excellent correlation between the dioxin-like compounds stimulated AhR– DNA binding
and the activation of gene expression.
Denison et al., 1999; Bank,
1992; Randerath et al., 1997
(e) AhR ligand binding Measurement of relative binding affinities to the AhR by dioxin-like compounds using competitive ligand binding (e.g., with
radiolabeled dioxin ligands). The free radiolabeled ligand is separated from the bound ligand and expressed as the percentage
displaced by the dioxin-like compounds.
Farell et al., 1987
(III) Enzyme immunoassays PCDD/PCDF are specifically bound by antidioxin antibodies which are immobilized on the EIA tube surface. Unbound
material is washed away, and a competitor enzyme is added which binds to the free sites of the antibodies. The amount of
conjugate is inversely related to the amount of PCDD/PCDF bound on the EIA tube. A chromogenic substrate is added and
the inhibition
is determined using a spectrometer to measure the optical density which is proportional to the enzyme amount and inversely
proportional to the PCDD/PCDF amount in the tube. For determination of PCDD/PCDF in unknown samples, a standard curve is
used which is based on the inhibition values of 2,3,7,8-TCDD standards covering 0% to 100% of the inhibition of a negative
control.
Harrison and Carlson, 1999
(IV) Chromatographic
methods
Accuracy and precision separating of dioxin-like compounds from materials between two immiscible phases on the basis of
differences in their molecular size, charge, mass, polarities, and redox potential. Then structure confirmation and validation
of environmental samples. Marker congeners like PCB-153 for the total TEQ in human tissue can be used for screening purpose.
(V) Quantitative
structure – activity relation
An empirical way of connecting the functional binding affinity of a series of structurally related dioxin-like compounds to its
dioxin-like activity. Important parameters are binding energies, hydrophobicity, binding constant, dose –response behaviors, and
polarity as well as molecule size, shape, electron distribution, and hydrogen bond capability.
Tysklind et al., 1994
P.A. Behnisch et al. / Environment International 27 (2001) 413–439 415

Recently, Hilscherova et al. (2000) described the three
potential classes of compounds with dioxin-like properties
that can bind to the AhR:
(A) Hydrophobic aromatic compounds with aplanar
structure and a size that the molecule or a part of the molecule
fits into the binding side of the AhR. Examples include the
planar congeners of PCBs and PCDD/PCDFs; polychlorazo-
benzenes (PCABs), polychlorazoxybenzenes (PCAOBs),
polychlorinated naphthalenes (PCNs), and several high
molecular weight polyaromatic hydrocarbons (PAHs).
(B) Other potential AhR agonists are compounds with
a specific stereochemical configuration; such as polyhalo-
genated (chlorinated, brominated, fluorinated), mixed
halogenated (chlorinated, brominated, fluorinated), and
alkylated analogs of the previously listed class of com-
pounds, chlorinated xanthenes and xanthones (PCXE/
PCXO), polychlorinated diphenyltoluenes (PCDPT), ani-
sols (PCAs), anthracenes (PCANs), fluorenes (PCFL), etc.
(C) Transient inducers and weak AhR ligands that deviate
from the traditional criteria of planarity, aromaticity, and
hydrophobicity and are rapidly degraded by the induced
detoxification enzyme. This class includes natural com-
pounds like indoles, heterocyclic amines, some pesticides
and drugs with various structures (imidazoles and pyridines).
For compounds with dioxin-like properties, there is a
rank-order correlation between their structure – AhR bind-
ing and structure –toxicity relationships, which is known
as the toxic equivalency concept, ranging from 1 to
0.0001 for PCB and PCDD/PCDF congeners. This con-
cept was developed as a management tool to convert the
complex chromatograms of PCDD/PCDFs and PCBs with
many peaks and their individual concentrations into one
value, the dioxin toxic equivalents quotient (TEQ). The
potency values [toxic equivalency factors (TEFs)] were
derived from a comparison of dose –response curves for
many toxic endpoints, of individual congeners relative to
the most toxic congener, 2,3,7,8-TCDD. By multiplying
the concentration (in pg/g) times of a congener by its
relative potency (REP) (TEF) of an analyte, one obtains
the 2,3,7,8-TCDD equivalent for that individual congener.
By summing the TEQs of all congeners, one obtains the
TEQ for the mixture. The TEQ approach for dioxin and
dioxin-like compounds makes several important assump-
tions: (1) the compounds share certain structural relation-
ships; (2) the compounds bind to the AhR; (3) the
compounds elicit AhR-mediated biochemical and toxic
responses; and (4) the compounds must be persistent and
accumulate in the food chain (e.g. Van den Berg et al.,
1998; Birnbaum, 1999).
The TEQ approach to dealing with numerous congeners
with different toxicity has been a useful way to determine
biological risk, however, it does have drawbacks. For
example, it is not always clear what is the best way to deal
with: (1) often small concentrations of individual congeners;
(2) unknown or not routinely measured AhR-active com-
pounds present; (3) no TEF values for several polyhalo-
genated aromatic hydrocarbons (PHAHs); (4) possible
nonadditive and antagonistic interactions between PHAHs;
(5) differences in shape of the dose–response curve; and (6)
differences in specific species responsiveness.
Nonadditive and antagonistic interactions become
increasingly important when ‘‘nonclassical’’ AhR ligands
and Ah inducers (e.g., synthetics, i.e., pyridines, and natural
ones, i.e., indoles, tryptanthrins, or heterocyclic amines) are
considered (Denison and Heath-Pagliuso, 1998; Safe,
1998a,b; Schrenk, 1998). It seemed that these ‘‘nonclass-
ical’’ ligands need to be planar, aromatic, and hydrophobic
with maximal dimensions of 14 12 5A
˚; in addition,
their activity is critically dependent upon their electronic
and thermodynamic properties (Fig. 1).
CYP1A1-related enzyme induction and body organ
weight effects constituted for a majority of the exper-
imental results in the database from which the present
harmonized World Health Organization/TEF (WHO/I-TEF,
1998) derived. Differences among species and tissues in
ligand-binding affinity, ligand specificity, and physico-
chemical properties of the AhR, as well as significant
differences in responsiveness to TCDD and related
PHAHs have been included into reevaluating TEFs for
mammalian, avian and fish species. REPs or induction
equivalent factors (IEF) (species-, endpoint-, and assay-
specific) or TEFs (consensus values based on REPs
across multiple species and/or endpoints) are used for
evaluating congeners of dioxin-like compounds.
If bioassays are used, the sum of dioxin-like activity in
complex mixtures is expressed as dioxin induction equivalent
quotients (IEQs; Schecter et al., 1999), bioassay-TEQs (e.g.,
CALUX-TEQs; Pauwels et al., 2000), or bio-TEQs (Engwall
et al., 1999). The comparison between the bioanalytical (B)
(bio-TEQ, bioassay-TEQ or IE) and the chemoanalytical (A)
Fig. 1. Natural and synthetic AhR agonists.
P.A. Behnisch et al. / Environment International 27 (2001) 413–439416

(TEQ) response are reported mostly in linear correlations or
as direct ratio R
b/c
(Schramm et al., 2001).
Bioanalytical response: REPs/IEFs for dioxin-like com-
pounds or the IEQ/bio-TEQ/bioassay-TEQ for environmen-
tal mixtures = 2,3,7,8-TCDD-EC
50
/unknown dioxin-like
compounds standards — or environmental mixture-EC
50
.
Several different endpoints have been used to estimate
the dioxin-like potency of a sample, including absolute
induction at a distinct concentration, EC
50
values, or effect
concentrations at a fixed-effect level [fixed-effect-level
toxicity equivalent; FEL-TEQs such as EC
10
or values next
to the minimal detection limit (MDL)] (reviewed by Brack
et al., 2000).
Chemoanalytical response: TEQ = P[PCDD TEF) +
P[PCDF TEF) + P[PCB TEF)] )ratio R
b/c
= bioana-
lytical response/chemoanalytical response.
3. History of biomarkers/bioassays for
dioxin-like compounds
The first published data about the toxicity of dioxin-like
compounds were based on humans and animals exposed
accidentally to PHAHs (see, for review, Schecter, 1994,
Brouwer et al., 1995; Sewall and Lucier, 1995; Van den
Berg et al., 1998; Denison et al., 1998). These early
observations prompted research into the toxic mechanisms
that were involved and led to the current understanding of
the toxicity of compounds with dioxin-like properties. In
order to understand how these observations eventually led to
the development of biomarkers/bioassays for these com-
pounds, it is useful to review some of the ‘‘milestones.’’
Some ‘milestones’ from the toxicity of dioxin-like com-
pounds and the development of biomarkers/bioassays (for
references see Schecter, 1994):
1920–1940: Increased of PCDD/PCDFs levels in North
American lake sediments
1929: US commercial production of PCBs begins
1942–1953: Several accidents included dioxin-like com-
pounds (Monsanto, Bo¨hringer, BASF; Schwarz, 1942)
1957: TCDD identified as unwanted contamination in the
manufacture of trichlorophenols
1958: Lethal disease outbreak among poultry flocks:
‘chick edema factor’ (see Schmittle et al., 1966)
1965: Research about the chick edema factor (see rev-
iew: Metcalfe, 1972; Flick et al., 1973)
1966: PCBs first found in the environment in sea hawks
(Jensen, 1966).
1968/1979: Yusho (Japan) and Yu-Cheng (Taiwan) acci-
dents with PCB-contaminated rice oil
1968: AHH-bioassay (Nebert and Gelboin, 1968)
1968: Toxicological evaluation of isolated and synthetic
PCDDs (Higginbotham et al., 1968)
1970: Identification of PCDFs in two commercial PCB-
mixtures (Vos et al., 1970)
1973: PCDDs are reported as potent inducers of AHH
(Poland and Glover, 1973)
1975: Analysis of PCDFs in American PCB mixtures
(Bowes et al., 1975)
1975: EROD bioassay (rat H4IIE) for PCDDs (Nebert
and Gelboin, 1968)
1977: Identification of dioxins in flue gas and fly
ash of some municipal waste incinerators (Olie et al.,
1977)
1978: Two-year chronic toxicity study of 2,3,7,8-TCDD
in rats (Kociba et al., 1978)
1979: Cytosolic receptor binding assay with radiolabeled
TCDD confirms the postulated AhR (Poland and
Knutson, 1982; Poland et al., 1979)
1979: US Food and Drug Administration (FDA) used
EROD bioassay for screening
1981: Bioanalysis of 2,3,7,8-TCDD equivalents by
Cytosol Receptor Assay of a fly ash (Hutzinger et al.,
1981)
1981: Identification of PCDFs in environmental samples
(Rappe et al., 1981).
1985: The DNA binding domain of the AhR is termed
‘dioxin responsive enhancers, DRE’ (Jones et al.,
1985) and identified in the mouse (Gonzales and
Nebert, 1985)
1986: In vitro EROD-induction correlated with the in
vivo toxicity of dioxin-like compounds (Safe, 1986)
1987: Monoclonal antibodies (Mabs) for dioxins are
reported (Stanker et al., 1987)
1987/1988: First TEFs for PCDD/PCDFs in complex
mixtures (NATO/CCMS)
1989: Ten-year mortality study of the population living
in Seveso (Bertazzi et al., 1989)
1991: Two cancer mortality studies about human exposed
to dioxins (Fingerhut et al., 1991; Manz et al., 1991)
1993: In vitro luciferase assays: a tool for monitor
ing dioxin-like toxicity (Aarts et al., 1993; Postlind
et al. 1993)
1994: First TEFs for several PCB congeners (WHO)
1996: Bioimmunoassay for AhR (AhIA) (Wheelock,
et al., 1996)
1997: New TEFs for PCBs, PCDDs, PCDFs for humans,
birds, and fishes (WHO)
For biomarker strategies to evaluate the environmental
effects of chemicals, see Walker (1998).
4. Determination methods of dioxin-like compounds
The close link between environmental pollution and its
impact on wildlife and human health links environmental
protection with the toxicological and biomedical fields. A
combination of bio/chemical analytical tools will help to
solve problems associated with detecting dioxin-like com-
pounds. The chemical methods used for analyzing dioxin-
P.A. Behnisch et al. / Environment International 27 (2001) 413–439 417

Table 2
Some advantages and drawbacks of BDMs for dioxin-like compounds in the open literature
BDMs Advantages Drawbacks Application
In vivo biomarker Most defensible screening tool for wildlife, because of uncertainties
of the in vitro bioassays in bioavailability and toxicokinetics.
Necessary to confirm the in vitro results
Costly, time-consuming; Methods required euthanasia
or invasive surgical techniques for animals; Ethical
critical; At high doses, competitive inhibition occurred.
REPs for PXDD/Fs (X= Br, Cl, F);
PCBs; PAHs
AHH/EROD assay Most published data, most experienced, ‘‘Golden standard of
bioassays,’’ no patent; Analysis of the sum of biological relevant
TEQ — detection of the persistent class of AhR active
compounds is possible; Analysis of the catalytic activity of CYP1A1
reflects more the real effects on human/wildlife than immunoassays
or luciferase induction; Good linear correlation with in vivo assays;
High metabolic capacity, because a long incubation time is possible
(72 h); 24-h/72-h Kinetic for distinguishing between stable/unstable
agonists possible; Distinguishing between AhR agonists/antagonists
possible; Bioassay quality: CV 29 – 38%
Many chemicals are substrates for P4501A1 and can
inhibit EROD activity (PCBs) leading to a lower
induction (see also Petrulis and Bunce, 1999); More
narrow linear working range than in vitro luciferase
assays; More time-consuming, HTPS would require
faster and less expensive alternatives; Species-specific;
Sensitive to oxidative stress; In vivo season-dependent
fluctuations in inducibility; Low enzyme and mRNA
stability.
REPs for PXDD/Fs (X= Br, Cl, F);
PCBs; PAHs application: CU (R);
Fl (B); S (R); F (R); C (B); CP (R);
Sd (R); W (R).
In vitro luciferase/reporter
gene assays
Covers the limitations of the EROD assay (faster, no inhibition;
wider working range); Analysis of the biological relevant sum of
TEQ; Similar results to in vitro–in vivo EROD-REPs; Bioassay
quality: CV 29%; Distinguishes between agonist and antagonist and
between stable and unstable AhR agonists (in 6-h/24-h kinetic)
possible; Tissue- and species-specific (rat H4IIE – IF 25, mouse
H1L1.1c7-IF 75; rat higher metabolic capacity than mouse; mouse
greatest concentration of AhR); HTPS possible; Provides choice of
reporter gene; Cope with important biological effects (e.g., membrane
passage; protein binding)
Luminometer and stable transfected cells necessary;
Stability of luciferase; Missing of possible tissue factors
due to the transformation into a recombinant cell; Leaves
out outer signal pathways; Induction for any compound
capable for binding to the AhR — without clean-up higher
TEQs/false-positive results are reported (e.g., in blood)
REPs: PCDD/PCDFs; PCBs; PCNs;
PBDE; Application: CU (B); Fl (B);
S (R); F (B); C (B); Sd (R); PP (R)
CAFLUX EGFP as reporter gene allows longer kinetic than CALUX; Less
complicated and cheaper than CALUX, since no expensive substrate or
luminometer necessary; Nondestructive methods allows to follow the
expression on a real-time basis
Cumulative signal: very sensitive for low concentrations
of Ah nonpersistent agonists, but difficult to analyze only
the persistent class of dioxin-like compounds
MDL = minimal detection limit; CR = cross-reactivity; Fl = fly ash; F = food; S = sludge; CP = compost; C = combustion gas; CU = clinical use; W = water; A = air; S = soil; Sd = sediment; P = PCB-mixtures;
R = in research; D = in development; B = business; PP = paper; CV = coefficient of variation; IF = induction factor; CC = correlation coefficient.
P.A. Behnisch et al. / Environment International 27 (2001) 413–439418

like compounds are primarily gas chromatography (GC)
with mass spectroscopy (MS) or electron capture detection
(ECD). The biological methods in operation included bio-
markers (e.g., wildlife/human effects), whole animal expo-
sures (in vivo, laboratory exposure), cell- or organ-based
bioassays (e.g., EROD, in vitro luciferase), and protein
binding assays (e.g., ligand binding as well as immuno-
assays) (see Fig. 1; Tables 1 and 2).
It is unlikely that these biochemical screening methods
will ever replace chemical instrumental analysis or in vivo
toxicological studies. Instrumental analysis is necessary
for the identification and exact quantification of the
selected class of PHAHs, while the in vivo methods are
necessary to study the bioavailability and prediction of
whole-organism responses.
The combination of selected clean-up and BDM has the
possibility to select between easily biodegradable com-
pounds and more persistent AhR agonists (Jones et al.,
2000), therefore, offering a tool to assume the TEQ for
dioxin-like compounds or all kind of PHAHs/PAHs.
However, the biochemical screening methods could com-
plement chemical instrumental analysis and in vivo studies.
In order to explain how these various methods of
detecting dioxin-like compounds could complement each
other, we will start by examining each of the available
methods in more detail.
4.1. Classical chemical analysis (e.g., HRGC/HRMS)
These methods are based on the separation and
quantification of dioxin-like compounds from matrices
on the basis of differences in their molecular size,
charge, mass, polarities, and redox potentials. The advan-
tages are the structure conformation, the congener and
pattern specificity, the calculation of the TEQ by the
TEF-concept and international standardization. Disadvan-
tages include potential loss in specificity, not all stand-
ards of interest are available, high cost, a long time for
analysis, the limited information on the biological
potency and potential interactions in complex mixtures
of dioxin-like compounds.
Recently, it has been shown that the analysis of key
indicator congeners, e.g., seven dioxin-like compounds
(2,3,7,8-TCDD, 2,3,7,8-TCDF, 1,2,3,7,8-PCDD,
2,3,4,7,8-PCDF, PCB-77, PCB-126, and PCB-169)
account for about 93% (n= 132) of the total TEQ in
reported human blood or plasma samples (Chen et al.,
1999). That could be a quick and relatively inexpensive
way of monitoring. However, stable patterns of these
compounds present in the matrix of choice are necessary.
The TOX value (total organic halogen) is another possible
method for using chemical analysis to predict a TEQ
value. The TOX value is determined by electrochemical
titration. Studies in combustion gas and fly ash of incin-
erators are reported (Kawamoto, 1999; Kashima et al.,
1999; Kawano et al., 1998).
4.2. Toxicological observations in wildlife
Several examples of in vivo toxicity have been observed
in wildlife in areas with elevated levels of dioxin-like
compounds, e.g., death of trout fry (40 ppt TCDD in eggs),
reproductive abnormalities and death in mink (5 – 10 ppt in
food; 1000 ppt body burden), development deformities in
domestic chicken (5.8 ppt in eggs), egg mortality in cormor-
ants and other fish-eating birds (Schecter, 1994).
4.3. In vivo laboratory studies
Dioxin-like compounds caused a variety of effects in
laboratory animals (e.g., Poland and Knutson, 1982;
Brouwer et al., 1995; Denison and Heath-Pagliuso, 1998;
Denison et al., 1998; Van den Berg et al., 1998). These
included hepatotoxicity, certain types of cancer, thymic
atrophy, immunotoxicities, wasting syndrome, reproductive
toxicity, and induction of enzymes/porphyrins. Relative
exposure levels and species/strain responsiveness to
dioxin-like compounds were determined by measuring:
enlargement of the liver (hepatotoxicity), reduction of
thymus weight (thymic atrophy, immune toxicity), wasting
syndrome (progressive loss of weight until death), repro-
ductive toxicity (number of offspring, malformations, irreg-
ular cycles), and EROD/AHH.
4.4. Cell culture-based bioassays (e.g., EROD, CALUX,
P450HRGS)
Unlike the calculated TEQ, which is based on an additive
model of potency, the AhR-based bioassays integrate all
activities and possible interactions of all individual conge-
ners in a complex mixture. This is a major advantage of the
bioassays as the results directly provide a measure of the
total sum of dioxin toxic equivalency (TEQ), which can be
used as an REP measure in risk assessment cases.
In vitro bioassays offer the possibility selecting between
easily biodegradable compounds and more persistent AhR
agonists, by different sample incubation times (in vitro
luciferase bioassays: 4 –48 h; e.g., possible test strategy:
PAHs and dioxin-like compounds are detected at 4 – 6 h;
EROD bioassays: 24 –72 h), while the more persistent
dioxin-like compounds are responsible for effects at
24–48 h incubation time (Anderson et al. 1995, 1999a,b,c,
2000; Jones et al., 2000; Hamers et al. 2000; Schramm
et al., 1999, 2001) (see Section 7.2). However, a chemical
separation of PAHs and dioxins is a more useful strategy
(easier and quicker).
4.5. Immunoassays
The most common immunoassays are the enzyme-linked
immunoassay (EIA), the radioimmunoassay (RIA) and the
fluorescence immunoassay (FI) (Vanderlaan et al., 1988;
P.A. Behnisch et al. / Environment International 27 (2001) 413–439 419

Harrison and Eduljee, 1999; Diaz-Ferrero et al., 1997) (see
Section 7.3).
4.6. DNA-binding assay in vitro
These technologies are based on the DNA binding of the
AhR when the receptor is in the presence of suitable ligands.
This system is suitable for studying important biological
effects of active compounds at the AhR level. But it may be
not suitable for the detection of synergistic effects of natural
and environmental dioxin-like compounds (Sadik and Witt,
1999). The GRAB bioassay, which uses a cell free system
has been used in the detection of pharmacological agents
that activate the AhR signaling system (Bank et al., 1992,
1995; Clark et al., 1999a,b; Gillner, 1988; Seidel et al.,
2000) (see Section 7.3). Quality control problems of this gel
retardation technology makes it, so far, unreliable as a
screening method for dioxin-like compounds. In the future,
perhaps the cDNA microarray chips (Afshari et al., 1999)
could analyze the activity of genes responsible for dioxin-
like activity.
4.7. Quantitative structure–activity relationship (QSAR)
QSAR studies can be used to identify potential classes of
AhR ligands. The congener specific toxicity and relative
toxicity to other compound classes can be evaluated. Tys-
klind et al. (1994) designed a QSAR model with 37
physicochemical descriptor variables to characterize 87
tetra- to octachlorinated PCDFs. The predicted TEFs indi-
cated that a large number of congeners are potent inducers.
Other authors (Mekenyan et al., 1996; Safe, 1994) also
demonstrated in a QSAR approach that structural confor-
mation, lipophilicity, hydrogen bonding capacity, and elec-
tronegativity are important substituent parameters for the
activity of several dioxin-like compounds (Fig. 2).
5. Design of a biomonitoring program
Widespread contamination of the environment by all
kinds anthropogenic compounds occurs in our modern
environment. Ecotoxicological implications, however, often
remain obscure. The use of bioanalytical tools may bridge
the gap between cause (e.g., detected by chemical analysis)
and effect.
It is well known that GC/MS analysis is able to measure
a wide range of pollutants quantitatively, and with high
selectivity and sensitivity. For complex environmental mix-
tures, however, risk assessments are difficult to describe.
Chemical analysis only describes the moment of the sam-
pling both in time and space. Biomarker/bioassays have the
following advantages: (1) a rapid determination of the total
potency of AhR agonists, (2) short procedure time, (3) low
cost, (4) high sensitivity often at picogram level, (5) can
predict the outcome of in vivo studies in terms of magnitude
of effect, but not the total spectrum of action. The disadvan-
tages include: (1) limited validation data for various com-
plex matrices of some bioassays/biomarkers (due to their
new development), (2) questions about the degree of reli-
ability, what the relationship is between bioassay data and
chemical data (or its equivalent) is, (3) limited interlabor-
atory cross-validation studies with the same technology, (4)
lack of cross-validation studies between different bioassays/
biomarkers, (5) national and international round robin
studies in different matrices have not been conducted, (6)
international evaluated quality criteria, (7) limited predictive
power from a toxicological point of view due to the need for
in vitro–in vivo extrapolation (not important in case of a
screening purpose, rather than for a risk evaluation and still
better than chemical analysis).
On the other hand, in vivo tests take into account all
possible environmental factors that can influence stress
responses at all levels of life. These gaps of information
could be bridged by using an appropriate bioassay/bio-
marker approach. Therefore, BDMs can be used as an
‘‘early warning system.’’ Furthermore, bioassays/bio-
markers may provide unique knowledge on several levels
of biological responses to environmental hazards. Measure-
ment of the total toxic potency and the bioavailability of
sample-associated mixtures of contaminants, including
interactions is necessary for an accurate risk assessment.
However, there almost never will be a need to have an
‘‘accurate’’ assessment. Species differences, interactions,
adaptations, and effects of countless modifiers make an
‘‘accurate’’ assessment only good enough for the system
under study.
Fig. 2. Classes of determination methods to analyze dioxin-like compounds.
P.A. Behnisch et al. / Environment International 27 (2001) 413–439420

BDMs can contribute to this needed information.
They also can provide specific insight into the fraction
of active substances that are present. As a first step, in a
high throughput screening, a biodetector could be used
which is capable of detecting all classes of AhR ligands.
After identifying contaminated environmental sites (on-
site sensors), the positive samples (confirmed by rean-
alysis with and without added antagonist) could be in a
second step reanalyzed with current chemical analytical
techniques to identify the responsible dioxin-like com-
pounds. In a third step, the pollutants are identified,
isolated, and reanalyzed by these BDMs. This follow-up
step is a ‘key cycle’ in the integration of GC/MS and
biodetector that will help us evaluate environmental
samples more accurately (Fig. 3). The toxicity identifi-
cation evaluation (TIE)/toxicity reduction evaluation
(TRE) programme of the US EPA (1992, 1999) and
the dioxin survey from the Sweden-EPA (de Wit and
Strandell, 1999) already use this design of combinatorial
bio/chemical analysis.
6. Clean-up and fractionation strategies
As mentioned previously, samples often need to be
processed through a clean-up to limit cytotoxic effects
from the sample matrix. These clean-up methods can also
be used to discriminate for specific classes of compounds
into separate fractions or to destroy nonstable classes of
toxicants. The TIE-toxicity identification scheme of the US
EPA (1992, 1999) describes a general screening system to
identify and evaluate toxicants in complex environmental
mixtures of these compound classes (see also Hilscherova
et al., 2000). This strategy is based on differential extrac-
tion and fractionation methods and identification by chem-
ical/biochemical analysis.
Schramm and Rehmann (2000) and Schramm et al.
(1999a,b, 2001) presented a test strategy to focus on
compounds which are persistent (achieved by column
chromatography on acidified silica gel), bioaccumulate
(addressed by extraction with lipophilic solvents), and toxic
(focussed on several endpoints; e.g., dioxin-like and estro-
genic-like activity).
The choice of the clean-up strategy also makes differ-
ences in the ratio R
b/c
due the different kind of selected AhR
ligands. The ratio R
b/c
additionally depends on (1) the used
bioanalytic detection methods (in the case of in vitro bio-
assays differences between cell types); (2) the kinetic (e.g.,
4–72 h sample incubation with in vitro bioassays); and (3)
the molecular target (AhR binding, antibody, enzyme activ-
ity) (Schramm and Rehmann, 2000; Schramm et al., 2001).
In the case of fly ash, several clean-up methods have
been reported for bioassays/biomarkers. After pretreatment
of fly ashes with hydrochloric acid and extraction with
toluene, various clean-up columns have been used. For
example, (1) an activated silica gel column (eluted with
200 ml hexane) has been adapted for the Micro-EROD
bioassay to determine the total sum of dioxin-like activity
(Behnisch et al., 2000a,b); (2) Schwirzer et al. (1998)
prepared samples for the Micro-EROD bioassay using a
multiacid –base silica gel column which destroyed acid-
labile compounds like PAHs; (3) utilizing an EIA, Zennegg
et al. (1998) adapted a sulfuric acid/sulfur trioxide treatment
and a subsequent liquid/liquid extraction with hexane to
isolate the stable dioxin-like compounds (see additionally
CAPE Technologies, 2001).
In the case of sediments, several sample preparation
methods have been reported for the measurement of PAHs
Fig. 3. Key cycle for combinatorial bio/chemical analysis of dioxin-like compounds in waste recycling and the environment.
P.A. Behnisch et al. / Environment International 27 (2001) 413–439 421

and dioxin-like compounds: (1) extraction with methylene
chloride, clean-up with gel permeation and fractionation
with a carbon column to obtain fractions with non-, mono-
and di-o-chlorinated biphenyls and PCDD/PCDFs (Micro-
EROD bioassay; Gale et al. 2000); (2) a hexane/acetone
extraction followed by a multiacid – base silica gel column
(CALUX bioassay; Murk et al., 1996); and (3) using a
dichloromethane extraction followed by florisil column
fractionation (Khim et al., 1999); and (4) the EPA Method
4425 (screening extracts of environmental samples for
planar organic compounds by a reporter gene P450HRGS;
US EPA, 2000b). This method includes a optional silica gel
clean-up step based on EPA Method 3630 to separate
dioxins/furans from PAHs for the final measurement of
the dioxin-like activity by P450HRGS.
7. Modern bioanalysis for dioxin and
dioxin-like compounds
In the last two decades, a number of BDMs have been
developed to assess dioxin-like activity of individual com-
pounds or complex mixtures. These assays use a variety of
end points, including enzyme and recombinant reporter gene
induction ligand binding, increased protein expression, anti-
body binding, and cell proliferation or differentiation.
The aim of this article is to review biodetector strategies
for dioxin and dioxin-like compounds with the ultimate
objective of using them to obtain clear evidence of toxic
effects of environmental chemicals. By expounding on the
strengths and weakness of the bioassays that are available in
the next section, it should become apparent that there are
ways that these bioassays can be paired so that they
complement each other and meet all four of the desirable
characteristics for biomarker assays (sensitivity, specificity,
simplicity, and stability). Then the results from the bioassays
can be combined with results from chemical analysis to
provide evidence for the causality of toxic effects.
Tables 1– 4 summarize the principles and advantages/
drawbacks of BDMs.
The minimum detection limits, which have been
reported for 2,3,7,8-TCDD for these BDMs, are similar
to chemicals analysis:
Micro-EROD with H4IIE cells [0.06 pg/well (Schwirzer
et al., 1998) and 0.08 pg/well equivalent to 1.6 pg/g (U.S.
Army Engineer Waterways Experiment Station, 1998)],
CEH-EROD (0.16 pg/well; Kennedy et al., 1996a,b),
CALUX (rat: 0.06 pg/well for samples that have been
extracted; mouse: 0.64 pg/well based on untreated serum;
Ziccardi et al., 2000), soil extract with 5 pg TCDD/g (EPA
Method 4425), EIA (DF1: 3–4 pg/well; Harrison and
Eduljee, 1999), the GRAB bioassay (0.04 pg/assay; Aarts
et al., 1995), the AhR binding assay (0.80 pg/assay;
Brouwer et al., 1995) or the AhIA (1.0 pg/assay; Wheelock
et al., 1996) (see Table 4).
The quantitation limit of these BDMs were reported
for animal feed (0.50 pg CALUX-TEQ/g lipid; in com-
parison to 0.25 pg I-TEQ/g lipid analyzed by HRGC/
HRMS; see www.rikilt.dlo.nl), for solid samples (5 pg
DF1-EIA-TEQ/g, Sugawara et al., 1998; 5 pg TEQ/g;
EPA Method 4425 or 1 pg EROD-TEQ/g; in sediment,
Gale et al., 2000), for incinerator fly ashes (160 pg
Micro-EROD-TEQ/g; Behnisch et al., 2000a) and for
combustion gas (100 pg Micro-EROD TEQ/m
3
; Behnisch
et al., 2000a).
7.1. In vivo biomarkers
In a hierarchical screening protocol for dioxin-like
compounds, in vivo bioassays would be utilized for specific
chemicals that are positive in multiple in vitro assays and
possess potential toxicity for human and wildlife. Although
in vivo testing is more expensive and time-consuming
compared to in vitro screening, in vivo assays incorporate
many important processes, which are limited in in vitro
screening. This includes pharmacokinetics, metabolism,
and interactions with multiple binding and transport pro-
teins which affect uptake into target organs. Different
biomarkers (like enzyme activity, DNA adducts) for
dioxin-like compounds occur for each biological level
(e.g., molecules, cells, organs, individuals, population,
ecosystem). The characteristic, species- and tissue-specific
effects caused by 2,3,7,8-TCDD in laboratory animals
include body weight loss, lethality, birth defects, dermal
toxicity, thymic atrophy, chloracne, subcutaneous edema,
monooxygenase enzyme induction, disruption of multiple
endocrine pathways, development toxicity, reproductive
toxicity, immunosuppression, hormonal alterations, terato-
genicity, carcinogenicity, tumor promotion activity, hepato-
toxicity, and porphyria (e.g., Schecter, 1994; Brouwer et al.,
1995; Sewall and Lucier, 1995; Van den Berg et al., 1998;
Denison et al., 1998).
The species-difference to 2,3,7,8-TCDD can be illus-
trated by the LD
50
values (mg/kg): from the highly sensitive
guinea pig (0.6–2.0), rat (22– 45), chicken (25 – 50), mon-
key (70), rabbit (115), dog (100–200), mouse (114– 284),
bullfrog ( > 1000) to the resistant hamster (1157–5051)
(Safe, 1986).
Suitable biomarkers for dioxin-like compounds are the
accumulation of hepatic porphyrin, reduced levels of hepatic
vitamin A (Fattore et al., 2000), and plasma thyroid hor-
mone reduction, as well as the induction of the hepatic
acetanilide-4-hydroxilase, CYP1A1, AHH, and EROD
activities. Dioxin-like compounds also induce the alde-
hyde-dehydrogenase, chinon-oxidoreductase and Phase II
drug-metabolizing enzymes like glucuronosyl transferases,
and glutathione-S-transferases. Furthermore, the develop-
ment of pericardial edema in the newborn chicken and the
production of chloracne in the rabbit pinna are reported as
biomarkers for dioxin-like compounds in intact animals
(Pijnenburg et al., 1995).
P.A. Behnisch et al. / Environment International 27 (2001) 413–439422

Table 3
Some advantages and drawbacks of BDMs for dioxin-like compounds in the open literature
BDMs Advantages Drawbacks Application
EIAs Speed, rapid turnaround time (24 h), simplicity, cost effectivity;
Parallel processing of many samples possible, portable kit
allows field use; Does not require any cell systems or
radioactivity
Costly development, CR, Nonspecific interferences;
Distinguishes not between metabolic stable and unstable
dioxin-like compounds; No information about biological
activity; No metabolic activity; For PCBs and dioxins
different EIA
(A) DF1 (CAPE) (Pabs) MDL: 4 pg/assay; Many validation data; Most experienced with
clean-up; in fly ash, wood and sediments lower R
ba
than the
Micro-EROD; 24 h turn around time
Low CR value of 2,3,4,7,8-PCDF 0.17; High CR for
2,3,7-TriCDD (0.24); Overestimation of the I-TEQ in fly
ashes; Acceptable low false-negative rates for soil
CR: PCDD/Fs PCBs, Appl.: Fl (B);
S (B); F (B); Sd (R): CU (B)
(B) DD3 (SDI) (Mabs) CR value of 2,3,4,7,8-PCDF (0.55) similar to the WHO-TEF
(0.5); Lowest CR value for 2,3,7-TriCDD (0.14)
Not anymore frequently used; MDL: 80 pg/assay;
Broadly specific; No validation data on real samples yet
–
(C) Sugawara et al., 1998; Pabs MDL: 0.5 pg/assay High CR for 2,3,7-TriCDD (0.28) and low CR for
2,3,4,7,8-PCDF (0.03)
In process
(D) RISc kit (SDI); Mabs Highly specific for 2,3,7,8-TCDD MDL: 70 pg/assay; Application on real samples necessary –
AhIA (Paracelsian) Speed, rapid turnaround time (24 h), simplicity, cost effectivity;
Parallel processing of many samples possible, portable kit allows
field use; MDL: 1 pg/assay; Does not require any cell systems
or radioactivity
Limited validation and application data; High REP values
for PCDFs; Requires a silica column clean up, additional
PCB kit necessary to detect the PCB-TEQ
Testing period for Fl, C, F, CU,
S, Sd
Bioassays using AhR-containing
extracts (e.g., AhR ligand
binding or GRAB assay)
Rapid, cost-effective, simplicity; Identification of all AhR
agonists/antagonists; Species-/tissue-specific for all kind of AhR
agonists; DNA binding activity correlates well with its biological
activity; Cell free system; Several studies with the AhR binding
assay in comparison to in vitro and in vivo
Detects only AhR ligands (no information about
biological activity); Does not distinguish between agonist
and antagonist; Several false-positives; Requires radiolabeled
ligands (
125
I-DBDD or
3
H-PCDD/PCDFs) GRAB requires
32
P-DNA preparation, handling, disposal; No kit available
Several AhR agonists; Paper and
household products
AhR assay (DELFIA Dioxin
TEQ Assay)
Speed, cost-effective, simplicity; Identification of all AhR
agonists/antagonists; Cell free system; Incorporates clinically
proven DELFIA technology; High throughput; Monitors quality
control parameters compounds
Limited validation and application data; Provides TEQ
response and does not report congener specific data;
Not designed for field use; Distinguishes not between
metabolic stable and unstable dioxin-like compounds
PCDD/PCDF, co-planar PCBs, PAHs
Yeast bioassay Speed; Simplicity; Human AhR and ARNT coexpressed in yeast
reflects well the actual biology of the AhR complex; Study of ER
and AhR signalling way possible
New development under validation and application; Lack
of background endogenous hormones/receptors; Transport
differences of dioxin-like compounds across cell membranes
and different receptor populations than mammalian cells
S (R)
Cell proliferation (keratinocytes) Analysis of the hallmark of dioxin-like response: chloracne;
sensitive, distinguishes between agonist and antagonist
Variations within cell types with respect to co-activators,
accessory proteins and growth factors will make an
interlaboratory study maybe difficult
MDL = minimal detection limit; CR = cross-reactivity; Fl = fly ash; F = food; S = sludge; CP = compost; C = combustion gas; CU = clinical use; W = water; A = air; S = soil; Sd = sediment; P = PCB-mixtures;
R = in research; D = in development; B = business; PP = paper; CV = coefficient of variation; IF = induction factor; CC = correlation coefficient; Pabs = polyclonal antibody; Mabs = monoclonal antibody.
P.A. Behnisch et al. / Environment International 27 (2001) 413–439 423

7.2. In vitro bioassays: advantages/limitations
In vitro bioassays offer a rapid, sensitive, and relatively
inexpensive solution to these limitations of the in vivo test
systems. Nevertheless, the TEFs for humans were primarily
derived from in vivo toxicity data and have been given more
weight than in vitro data (Van den Berg et al., 1998, 2000).
In an expert meeting, organized by the WHO, it was
concluded that a single in vitro assay based on a single
surrogate species may not accurately predict the toxicity of a
chemical or complex mixture following exposure to other
species. But the experts concluded that the use of in vitro
assays provides a general tool as a prescreening method of
dioxin-like toxicity in environmental samples.
Numerous in vitro bioassay systems based on the AhR-
dependent mechanism have been developed. They utilize
mammalian cell culture to measure a specific response (e.g.,
binding to the AhR or to the DNA, enzyme induction). It
has been reported that for PCBs, PCDDs, and PCDFs, a
linear correlation between the in vitro activity as inducers of
AHH activity in H4IIE cells and their toxicity in several
animal species exists (e.g., Safe, 1984, 1986, 1987, 1990,
1993, 1994, 1997, 1998a,b). This research has confirmed
the utility of the in vitro bioassays for quantitatively
estimating the in vivo toxicity of individual congeners and
complex mixtures. For analyzing dioxin-like compounds,
the state-of-the-art bioanalytical tools are the EIA, the
AhIA, the GRAB bioassay, the AhR binding assay, the
AHH/EROD and in vitro luciferase/reporter gene assays like
the commercially available P450HRGS (EPA4425) and the
CALUX assays (with stable transfected mouse and rat liver
cells). This review will mainly focus on the BDMs reviewed
in Tables 1– 3. For a comparison of the reviewed bioana-
lytical tools, REPs for several dioxin-like compounds from
these studies are listed in Table 4.
7.2.1. AHH/EROD bioassay
Twenty years ago, Bradlaw and Casterline (1979) and
Bradlaw et al. (1980), of the FDA of Washington (USA),
analyzed by AHH-bioassay several dioxin and dioxin-like
compounds, Aroclors, Yusho rice, food extracts, and fish
samples. The AHH activity was measured by conversion of
B[a]p to 3-hydroxy-B[a]p in microsomal fractions of liver
cells. They detected a range of TCDD-equivalents for fish
(810–890 pg bio-TEQ/ml), Yusho oil (19,000 – 42,000 pg
bio-TEQ/ml) and different Aroclors (1242 and 1248: 1400
pg bio-TEQ/ml; Aroclor 1254/1260: n.d.). (For AHH-bio-
assay, see also Trotter et al., 1982; Sawyer and Safe, 1982;
Sawyer et al., 1983, 1984; Zacharewski et al., 1988,
1989a,b). Currently, the EROD method is more commonly
used, measuring the binding of the dioxin-like compound to
the AhR and the subsequent induction of CYP1A related de-
ethylation of 7-ethoxyresorufin to resorufin (see Fig. 4)
(e.g., Behnisch et al., 2000a,b; Donato et al., 1993; Hofma-
Table 4
Minimal detection limit, linear working range and EC
50
for TCDD from several bioanalytical tools
MDL pg/well (pM) MDL pg/assay (pM) EC
50
pg/well (pM) Linear working range (pM)
(a) EROD
1. rat H4IIE
Macro 0.19 (2.4)
a
; (10)
a
;
0.68 (1.8 to 2.4)
b,c
10 pg/plate
c,d
46
d
;56
e
; (100)
a
; (20)
c
0.93 – 93 fmol/well
c
;
8 – 1000 pg/plate
d
Micro 0.058 (1.8)
b,f
3.2 (10)
g
0.087
f
0.5 – 40 pg/well
b
2. Chicken embryo
hepatocytes
0.16 (1.0)
a
(16)
a
; (720)
c
(1.0 to 100)
a
3. Fish rainbow trout (40)
h
(3.81 pmol/L)
i
; (24,000)
j
(10 – 1000)
h
(b) In vitro luciferase assays
1. Rat H4IIE-luc 0.06 – 0.19 (0.8 – 2.4)
c,k
;
0.043 (0.27)
l
0.3 (1)
g
0.45 – 1.6 (5.6 to 10)
c,k
0.93 – 93 fmol/well
c
;
(0.27 to 1.64)
l
2. Mouse Hepa 1.1c2 0.64– 1.6 (0.1 – 1)
k,m
11– 13 (20 to 680)
k
(1.0 to 1000)
m,n
3. Human NR 101L (NR)
o
; (1.0)
p
64 (100)
o
; (350)
p
4. Fish rainbow trout
to RLT 2.0
NR (1.0)
k
; (4.0)
h,q
2.6 (64)
h,k
; 150 to 300
q
(1.0 to 1000)
h
(c) CAFLUX (0.3)
r
(10.7)
r
(d) EIA
1. DF1 3 – 4
s,t
0.1 – 1000 pg/well
t
2. Sugawara et al. (1998) 0.5
s
2 – 240 pg/well
3. DD3 25
s
80
s
(e) AhIA (125)
u
1.0
u
550
u
(f) AhR binding assay 0.8 – 3.2
g
;64
v
(0.1)
w
(g) GRAB (1.0)
m
0.04 – 0.2
g
(150)
m
(1.0 – 1000)
m
(a) Kennedy et al., 1993, 1996a,b; (b) Schwirzer et al., 1998; (c) Sanderson et al., 1996, 1998; (d) Hanberg et al., 1991a,b; (e) Tillitt et al., 1991a,b; (f)
Li et al., 1999; (g) Brouwer et al., 1995; (h) Richter et al., 1997; (i) Brack et al., 2000; (j) Clemons et al., 1994; (k) Ziccardi et al., 2000; (l) Bovee et al.,
1998; (m) Seidel et al., 2000; (n) Garrison et al., 1996; (o) Anderson et al., 1995; (p) Postlind et al., 1993; (q) Villeneuve et al., 1999; (r) Aarts et al., 1998;
(s) Harrison and Eduljee, 1999; (t) Zennegg and Schmid, 1999; (u) Wheelock et al., 1996; (v) Bunce, 1995; (w) Safe, 1990.
P.A. Behnisch et al. / Environment International 27 (2001) 413–439424

ier et al., 1996, 1998; Lagueux et al., 1997; Schwirzer et al.,
1998; Schramm et al., 2001; de Wit and Strandell, 1999;
U.S. Army Engineer Waterways Experiment Station, 1998;
Schrenk, 1997; Till et al., 1997). In this bioassay, several
CYP activities can be measured by using different sub-
strates, e.g., for CYP1A1 (EROD), for CYP1A2 (methox-
yresorufin-O-demethylase; MROD), and for CYP2B1
(pentoxyresorufin-O-depenthylase; PROD). Several cell
lines are used for the EROD bioassay, e.g., the rat H4II
cell line (Behnisch et al., 2000a,b; Zacharewski et al.,
1989a,b; Giesy et al., 1994; Hoogenboom and Hamers,
1995; Murk et al., 1996; Sanderson et al., 1996; Willet
et al., 1997; Till et al., 1997, 1999; Schramm et al., 2001; de
Wit and Strandell, 1999; U.S. Army Engineer Waterways
Experiment Station, 1998), the chicken embryo hepatocytes
(CEHs; Lorenzen et al., 1997; Bastien et al., 1997; Bosveld
et al., 1995; Kennedy et al., 1992), cultured chicken embryo
liver (Brunstroem and Halldin, 1998; Brunstroem et al.,
1995; Engwall and Hjelm, 2000; Engwall et al., 1999),
Hepa 1 (mouse), human hepatoma Hep G2 and Hep 3
(Wiebel et al., 1996; Ohta et al., 1998), GPC16 (guinea
pig) and fish cell lines like RTL-W1, PLHC-1 or RTL-W1
(Clemons et al., 1994, 1996, 1997, 1998).
The detection limit for EROD induction by TCDD in
H4IIE cells is about 58 –190 fg TCDD/well (Sanderson
et al., 1996; Schwirzer et al., 1998; Tillitt et al., 1991), in
hepatocytes from 19-day-old chicken embryos, 160 fg
TCDD/well (Kennedy et al., 1996), and in chicken embryo
whole liver (8 days old) in ovo assay, 30 fg TCDD/liver
(Brunstroem et al., 1995). Clemons et al. (1994) compared
the EROD activity for several dioxin-like compounds with a
rainbow trout cell line (RTL-W1) and a rat hepatoma cell
Fig. 4. Method description of the CALUX and EROD bioassays.
P.A. Behnisch et al. / Environment International 27 (2001) 413–439 425

line (H4IIE). With the exception of 1,2,3,6,7,8-HCDD and
1,2,3,7,8-PCDF, all of the fish RTL-W1-derived REPs were
significantly higher (two –eightfold) as the EROD bioassay
determined REPs.
In addition, Schramm et al. (1999b) reported that in vivo
fish liver EROD data relate to in vitro EROD investigations.
Furthermore, EROD bioassays performed with chicken,
pheasant and turkey embryo hepatocyte cultures are also
well described as sensitive methods to analyze compounds
binding to the AhR in different avian species (Kennedy et al.,
1996a,b). In addition, it was reported that the relative
responsiveness of the cell lines to TCDD was consistently
H4IIE rat liver MCF-7 human breast cells>HepG2 human
liver (Vamvakas et al., 1996).
In one study, Li et al. (1999) compared TEQ values
analyzed by Micro-EROD, EIA (DF1), and chemical anal-
ysis in several samples. They concluded that for all environ-
mental samples, the EROD-TEQ was higher than the value
from chemical analysis (R
b/c
: for fly ash 1.8, for wood 2.3,
and for sediments 3; with oxidative clean-up). However, the
TEQ analyzed by EIA was more identical with the value
from chemical analysis (R
b/c
: for fly ash 1, for wood 0.5, and
for sediments 0.6/1.4; with oxidative clean-up).
These bioassays have been used to study dioxin-like
compounds in several matrices, such as fly ashes (Behnisch
et al., 2000a; Till et al., 1997; Schramm et al., 2001; Koppo-
nen et al., 1992), PCB-mixtures (Schmitz et al., 1996), sewage
sludges (Schwirzer et al., 1998; Engwall and Hjelm, 2000),
sediments (de Wit and Strandell, 1999), fish extracts (Caster-
line et al., 1983; Hanberg et al., 1991, Giesy et al., 1997), or
fish-eating water birds (Giesy et al., 1994).
7.2.2. In vitro luciferase bioassays (e.g., CALUX or
P450HRGS)
Recombinant cell lines are prepared by transient or stable
transfection of wild type cells with reporter genes under
transcriptional control of the DRE. The most common
reporter genes are firefly luciferase (luc).
These newly constructed cell lines still contain the
complete machinery which is involved in the mode of
action of dioxins and dioxin-like compounds. In addition,
a DNA-construct (e.g., pGudluc or pL1A1N) has been
incorporated in the cells containing DREs from several
species (e.g., mouse, rat, human) coupled to firefly (Photi-
nus pyralis) luciferase genes. They are capable of quantify-
ing compounds that activate the AhR, resulting in the
production of the luminescent enzyme luciferase. The pro-
duction of luciferase is an advantage because of (1) the
production of more copies of the vectors in the cell line than
the natural P450 enzyme; (2) the stability of the luc-protein
is greater than CYP. The cellular response can be measured
by adding suitable reagents (e.g., the substrate luciferin and
ATP) and quantifying the produced bioluminescence emis-
sion by an automated luminometer. The production of
luciferase provides an easy mechanism to measure the
amount of AhR binding, which is a function of the amount
of agonist present in the sample. The extent of activation of
these bioassay systems is equivalent to the concentrations
and potencies (affinity) of the chemicals applied to the cells.
The assays are based on the use of several cell lines such as
from guinea pig (g16L1.1c8; Garrison et al., 1996), rainbow
trout (RTH-149; Richer et al., 1997), human (HepG2;
Postlind et al., 1993; Garrison et al., 1996; Anderson
et al., 1995; Koh et al., 2001), rat (Kannan et al.; 1999a;
BDS CALUX: H4L1.1c4/H4IIE-luc; e.g., Pauwels et al.,
2000; Bovee et al., 1996; Machala et al., 1999; Blankenship
et al., 1997, 1999, Murk et al., 1996, 1997; Hoogenboom
et al., 1999; Hamers et al., 2000) or mouse hepatoma cells
(CALUX: Hepa1c1c7; Denison et al., 1993, 1998, 1999;
Garrison et al., 1996; Seidel et al., 2000; Ziccardi et al.,
2000; US Patent #5854010). For the PCDD-, PCDF-, and
PCB congeners tested so far, the REP value to induce the
CALUX activity in rat and mouse correlated well with
reported TEF values (Aarts et al., 1996; Garrison et al.,
1996; Hoogenboom et al., 1999; e.g., PCB-126: 0.02 – 0.07;
see Table 5), while the REP values for some PCB congeners
analyzed by the human cell line (P450HRGS) showed
differences (e.g., PCB 126: WHO-TEF: 0.1; P450HRGS
REP: 0.0008; Jones and Anderson, 1999). Several dioxin-
like compounds and complex mixtures of these compounds
were significantly correlated to TEF and TEQ values
analyzed by the CALUX bioassay and in comparison to
the EROD bioassay (e.g., Aarts et al., 1996; Clark et al.,
1999a,b; Murk et al., 1996, 1997; Kannan et al., 1999a,b;
Sanderson et al., 1996; Seidel et al., 2000).
A difficult issue with bioassays is the specificity of the
assay. False-negative results would make the test unreliable,
whereas false-positive results might be acceptable to a
certain extent in biomonitoring. False-negative results were
reported from the EROD bioassay, because of the fact that
CYP1A can be irreversible inhibited by a number of
compounds (e.g., PCBs, but in high concentrations; benzi-
midazole drugs at low concentrations; Aarts et al., 1995;
Van der Plas et al., 1998; Hahn, 1994; Hoogenboom et al.,
1999). The advantage of selecting a reporter gene encoding
for an enzyme, such as luciferase, would be that so far no
inhibitors are known and the induction of luciferase activity
can only occur through the AhR (e.g., Hoogenboom et al.,
1999). The in vitro luciferase assays respond to any com-
pound capable of binding to the AhR (e.g., benzimidazoles
and others; Hoogenboom et al., 1999; Denison et al., 1993,
1999). Therefore, these directly assess and confirm AhR-
mediated effects. These assays are faster (in vitro luciferase:
4–48 h; EROD assay: usually 24–72 h), less susceptible to
interferences, with a better linear working range because of
the instrument used (luminometer vs. fluorometer). There-
fore, recombinant cells exhibit greater sensitivity, dynamic
range and selectivity than wild type cells (e.g., Garrison
et al., 1996; Sanderson et al., 1996).
The in vitro luciferase assays also have the capability of
determining the presence of either biodegradable PAHs or
biostable dioxin/dioxin-like compounds without using a
P.A. Behnisch et al. / Environment International 27 (2001) 413–439426

different clean-up, by performing the test procedure at both 6
and 16 h (P450HRGS; Jones et al., 2000) or 6- and 24/48 h
(CALUX; Hamers et al., 2000). This is due to the metabo-
lism of most of the PAHs and the produced luciferase after
24 h. Persistent PHAHs still induce luciferase production
after 24 h or longer (Hamers et al. 2000; Columbia Ana-
lytical Services (CAS), 1998).
Luciferase induction by TCDD appeared to be dose-
dependent, the current MDL per well is between 43 (Bovee
et al., 1998; rat H4IIE-luc) and 640 fg/well or tube (Garrison
et al., 1996; mouse Hepa1.1c2) and the dose – response
curve saturated at ligand concentrations greater than 100
to 1000 pM. This indicates that the CALUX-response is
usable for a wide range of concentrations. In molar dimen-
sions, the MDL of these in vitro luciferase bioassays was
reported for mouse H1L1.1cs (MDL 0.1 – 1 pM; Garrison
et al., 1996; Ziccardi et al., 2000), human 101L (MDL 0.1 –
1 pM; Postlind et al., 1993), rat H4IIE-luc (DR-CALUX:
MDL 0.3–2.4 pM; see Table 4), mouse T13 (PAP-assay;
gene: alkaline phosphatase; MDL 100 pM; El-Fouly et al.,
1994), or rainbow trout RLT 2.0 cells (4 pM; Villeneuve
et al., 1999) (see Table 4).
In addition, Matsui et al. (1999) used a stable transfected
human hepatoma cell line HepG2 for determining fly ash
extracts in comparison to the EROD bioassay with HepG2
cells. Furthermore, Balaguer et al. (1995, 1996) reported
also from a transient transfected cell line, using Hepa 1c1c7
wild type cells transfected with a DRE-regulated reporter
gene (pGudLuc1.1). This study showed that black liquor
and urban dust samples possess ligands that are capable of
inducing a response that is mediated by the AhR.
Furthermore, Ritcher et al. (1997) described a stable
transfected in vitro luciferase assay with the rainbow trout
hepatoma cell line RTH-149 (RLT 2.0 bioassay). This in
vitro luciferase assay showed a one-magnitude greater
response than the EROD test (with the comparable wild
type RTH-149 cells) and exhibited little variability. In
comparison to a rainbow trout early life stage mortality
assay (ELSM), comparable REPs were measured by this in
vitro luciferase bioassay. But, compared to the in vitro–in
vivo EROD bioassay (with rainbow trout), significant differ-
ent REPs were measured: for 1,2,3,7,8-PCDD (RLT 2.0:
0.23; ELSM: 0.73, in vitro–in vivo-EROD: 2.6/1.8);
2,3,4,7,8-PCDF (RLT 2.0: 0.30; ELSM: 0.36, in vitro–in
vivo-EROD: 1.9/2.0); PCB-126 (RLT 2.0: 0.0063; ELSM:
0.005); and PCB-77 (RLT 2.0: 0.0060; ELSM: 0.00016).
7.2.3. Chemical-activated fluorescent protein expression
(CAFLUX)
The CAFLUX-bioassay utilizes the enhanced green fluo-
rescent protein (EGFP) gene as a reporter gene for AhR
activation instead of the firefly luciferase gene used in the
CALUX (Aarts et al., 1998; Nagy et al. 2000). EGFP is a
protein derived from the jellyfish Aequoria victoria carrying
a cyclic tripeptide acting as a fluorophore. The advantages of
the enhanced EGFP compared to the luciferase are the low
cost, ease of measurement (read directly in the living cells
without the need for cell disruption), and lack of requirement
for reagent addition (Nagy et al., 2000). The CAFLUX
appeared to be as sensitive as the CALUX assay (limit of
detection less than 1 pM of TCDD). In addition, the
CAFLUX is more simple and cheaper to perform, since there
is no expensive substrate or luminometer needed and a
standard fluorometer is sufficient to quantify the expression
of EGFP. Furthermore, the CAFLUX is a nondestructive
method, which allows the investigator to follow the expres-
sion of the reporter gene almost on a real-time basis. Unlike
the CALUX assay, a cumulative signal is generated in the
CAFLUX assay by persistent as well as by nonpersistent AhR
agonists. If the focus is on low levels of labile AhR agonists,
this would be an advantage, since they might become
detectable by recurrent exposure of CAFLUX cells, whereas
the steady-state level of luciferase activity reached in
CALUX cells might still be insufficient to allow a detection.
This might be a disadvantage, if only the effect of persistent
compounds is of interest, or when the contribution of persis-
tent and nonpersistent compounds to the level of AhR
activation has to be distinguished. The main disadvantage
of this bioassay is the extremely stable EGF-protein, which
leads to an increase in the background fluorescence of cells,
thereby limiting the dynamic range over time of usage.
However, parallel CAFLUX/CALUX analysis would allow
separate quantification of the (metabolically) labile and the
persistent class of AhR-active compounds.
However, at this time, the data set available for this
bioassay would not allow to apply this method as a
screening method.
7.3. Non-cell-based BDMs: advantages/limitations
7.3.1. Bioassays utilizing AhR-containing extracts
Three alternative bioassays utilizing AhR-containing
extracts, which have been developed, involve measurement
of the ability of chemicals in a sample extract to either bind
to the AhR (as measured by competitive binding with [
3
H]-
TCDD; Cytosol Receptor Assay, Poland et al., 1979; or
[
125
I]-labeled dibromodibenzo-p-dioxin; Bradfield and
Poland, 1988), or to bind to the AhR and stimulated its
DNA binding (as measured by inducible AhR binding to
32
P-labeled DNA; Denison and Yao, 1991). The basic
procedure analyzes the amount of radiolabeled ligand spe-
cifically bound at the AhR or in the introduced protein–
DNA complex and subtracts the amount of radioactivity
present in the same position in the control (DMSO-treated)
sample. Species and tissue of interest can be prepared and
compared to each other. Unoptimized reagents (radioactive
compounds) and production of unreliable results are
reported disadvantages of the AhR binding assay (e.g., de
Wit and Strandell, 1999).
The
32
P-postlabeling analysis has been reported as a
highly sensitive method for the detection and measurement
of covalent carcinogen–DNA adducts and other DNA
P.A. Behnisch et al. / Environment International 27 (2001) 413–439 427

modifications (Randerath et al., 1997; Safe et al., 1997;
Seidel et al., 2000). Since this method did not require
radioactive carcinogens, it was suitable for DNA of humans
exposed to environmental or occupational genotoxicants.
Microgram amounts of DNA were analyzed; thus the assay
is well suited for limited amounts of cells or tissue.
7.3.2. GRAB assay
The GRAB assay measures the ability of a chemical or
chemical mixture to stimulate the AhR DNA transformation
and DNA binding in vitro. Several chemicals/extracts
showed a positive activity in the GRAB assay, were they
were only weakly active or inactive in the cell-based
CALUX. This reveals that the ability of a chemical to
activate the AhR in vitro does not necessarily correlate with
its ability to induce gene expression in intact cells. (Denison
et al., 1999; Seidel et al., 2000). Compared to the CALUX
bioassay, the GRAB assay showed similar shapes of the
dose–response curves. But the GRAB bioassay would be
less preferable for screening dioxin-like compounds in the
environment, because of its one-magnitude lower sensitivity
(EC
50
15 pM), slightly lower minimal detection limit
(1 pM), about three times longer analysis time and a more
difficult handling than the reporter gene assay. Also, the
accessibility of the chemical to the AhR could be a major
difference: while in the GRAB assay chemicals have direct
access to the AhR in in vitro incubation conditions, in the
reporter gene assay the dioxin-like compounds must be able
to cross biological membranes, avoid binding to proteins/
membranes, survive cellular metabolic degradation, bind to
the cytosolic AhR, and activate reporter gene expression
(Seidel et al., 2000).
7.3.3. Immunoassays
The success of immunoassays, the binding of a specific
antibody, followed by indirect measurement of bound mate-
rial, over approximately the last 20 years have been
reviewed by several authors (Brecht and Abuknesha,
1995; Carlson and Harrison, 1998, 1999; Harrison, 1998,
2001; Harrison and Carlson, 1997a,b, 1999; Harrison and
Eduljee, 1999; Nistor and Emneus, 1999; Sherry, 1997;
Vanderlaan et al., 1988). Promising antibody-based techni-
ques such as EIAs, fibre optic immunoassays, immunoaf-
finity chromatography, biosensors, and flow injection
immunoanalysis continue to evolve. Van Emon et al.
(1998) reviewed the general antibody-based separation
methods (e.g., affinity chromatography, capillary electro-
phoresis) and immunochemical detection methods (immu-
noassays, flow injection methods or immunosensors).
Historically, the first attempts to analyze dioxins by immu-
noassays were reported from an RIA (1979), which used
polyclonal antibodies (Pabs; Albro, 1979), but were more
time-consuming. Mabs developed by Kennel et al. (1986)
lacked selectivity for free dioxin in solution. Later, Stanker
et al. (1987) generated Mabs to dioxin and developed Mab-
based ELISAs (DD3). The selectivity of the ELISA was
very similar to that of the RIA. The optimized assay
detected 200 pg/well 2,3,7,8-TCDD as the IC
50
(the analyst
concentration giving 50% inhibition). Then, Langley et al.
(1992) reported from the development of Pabs-based ELI-
SAs that detected 1 ng/well TCDD (as IC
50
value). The use
of EIA kits to rapidly screen environmental samples has
simplified also the analysis of dioxins. Several dioxin-kits
are now commercial available (e.g., EnviroGard and Dioxin
RISC kit product from SDI; DD3 from Millipore; DF1
High Performance dioxin/furan EIA from CAPE Technol-
ogies, RaPID from BioScan Screening Systems). Further-
more, some immunoassays are specified for the PCB
analysis in food (e.g., Harrison, 2001; Jaborek-Hugo
et al., 2000). These tests only require a hood and an optical
spectrophotometer to complete an analysis.
Biologically, the EIAs exploit the ability of specialized
biological molecules, called antibodies, to selectively and
reversibly bind organic molecules. Antibodies are produced
by the immune system by all mammals to react against
foreign substances for the purpose of self-protection. Their
property is used by immunizing some animals with a hapten
and then using the antibodies as highly specific reagents to
recognize the analyte.
The other key reagent in most environmental EIAs is the
labeled ligand, or depending on the assay format, the coat-
ing antigen. The ligand molecules can be labeled with a
radioactive tracer, an enzyme, or a fluorescent molecule, so
that the fraction of ligand molecules that has been bound can
be estimated. Most environmental EIAs are competitive
binding assays in which the binder molecule, an excess
amount of labeled analyte, or coating antigen, and the target
analyte are allowed to equilibrium. The free analyte is then
separated from the bound phase and the amount of labeled
analyte, or binder molecule that has been bound is quanti-
fied. The amount of analyte in the unknown samples is
extrapolated from a calibration curve. The final detection
can be performed with a radioactive tracer (RIA) or with the
reaction of an enzyme linked antibody or antigen (EIA).
They have been used for over three decades in the medical
field in a wide array of tests for everything from disease
organism to drugs of abuse, therapeutic drugs, and hor-
mones with excellent reliability. In comparison to cell-based
bioassays, the most important advantages were the speed,
simplicity, low cost and parallel processing of many sam-
ples, easy automatisation and potential field use. Disadvan-
tages were reported as being the costly development,
cross-reacting compounds, and nonspecific interferences
(Harrison, 1998, 2001; Harrison and Carlson, 1997a,b,
1999; Harrison and Eduljee, 1999).
In general, competitive immunoassays (such as RIA and
EIAs) are quick and easy. But the detection reagent and
analyte always compete for a limited supply of the binding
reagent. This gives an inverse dose – response with the most
variability in the low analyte concentration region. The
sensitivity is highly dependent on the avidity of the detec-
tion reagent for the binder compared to the analyte. Also,
P.A. Behnisch et al. / Environment International 27 (2001) 413–439428

these assays typically detect only one selected analyte (such
as dioxins). The fact that for a competitive assay you must
have the binder in limiting quantity, leads to a less sensitive
assay compared to a direct assay (such as AhIA).
In the past, there has been a lag of acceptance in the use
of EIAs, resulting from the mass spectrometer being far
more sensitive (under the threshold limits for regulations)
and the necessary use of clean-up systems. New Pabs were
developed by Harrison (1998, 2001), Harrison and Carlson
(1997a,b, 1999), Harrison and Eduljee (1999) (called DF1;
US Patent #5674697) and by Sugawara et al. (1998). In
these tests, the critical selective extraction and solvent
exchange to an assay-friendly solvent, such as DMSO or
methanol, have been described. Both were reported to be
very sensitive for TCDD (about 0.5 to 4 pg TCDD/well),
compared to the DD3 development by Stanker et al. (1987)
(100 pg/tube), approaching the detection limits of conven-
tional GC/MS analysis.
REP values for 2,3,4,7,8-PCDF in the DD3 (0.55)
closely matched the WHO-TEF (0.5, WHO), while the
more sensitive EIAs reported by Sugawara et al. (1998)
(CR: 0.03) or the DF1 (0.17; Harrison, 1998, 2001; Harrison
and Carlson, 1997a,b, 1999; Harrison and Eduljee, 1999;
CAPE Technologies, 2001) showed lower REP values.
As all EIA have a lack of absolute specificity, the test
designer must determine a calibration procedure for every
matrix, which takes the cross-reactivity patterns into
account. For this purpose, they used at first a simple additive
response model based on congener concentration times
cross-reaction equals to 2,3,7,8-TCDD and TEQ to predict
the behavior of real samples. They found excellent predicted
correlations for fishes (n= 20; DF1 antibody; correlation
coefficient =.987; Harrison and Carlson, 1997) and soil
samples (n= 43; DD3 antibody; correlation coeffi-
cient = .988; Carlson and Carlson, 1997). This high corre-
lation indicated the potential for predicting the I-TEQ of the
sample from the immunoassay response.
In the last few years, the DF1 antibody (Harrison and
Carlson, 1997) has been further tested on real-world sam-
ples like fly ash samples (Zennegg et al., 1998; correlation
coefficient = .999, n= 25; Focant et al., 1999, CAPE Tech-
nologies, 2001; Application Note AN-001), fish tissues,
sediment samples (Kolic et al., 1998) and on soil and fly
ash samples (Harrison and Carlson, 1999; regression coef-
ficient = .93, concentration range of 2–1100 ng I-TEQ/kg,
CAPE Technologies, 2001, Application Note AN-003).
Furthermore, Zennegg and Schmid (1999) reported from
EIA-TEQs from chimney soot samples from house heating
systems which showed only minor discrepancies to the
chemical I-TEQ data.
7.3.4. Ah-immunoassay
The commercial available AhIA kit for dioxin and furan
analysis is used as screening device for cost-effective hazard
assessment (Wheelock et al., 1996; US Patent #6127136;
Japan Patent #3144689). The AhIA is the only assay that is
a hybrid of an immunoassay and an in vitro AhR-based
assay. It eliminates the disadvantages of each approach,
while keeping the advantages. The immunoassay can be
used as a kit, while still having the advantages of an AhR-
based monitoring system.
In the AhIA, an inactive AhR (in a form capable of
binding dioxin-like compounds) is transformed by dioxin-
like compounds to an active form that forms a complex with
Arnt and binds a DRE. This complex is detected with an
antibody that is capable of binding to the ARNT, when it is
associated with the AhR. It measures ligands that transform
the AhR to the DNA-binding form, which is the central
toxic event common to all dioxins. The proportional ability
of the dioxins to promote transformation to the DNA bind-
ing form is, in turn, proportional to their toxicity, which is,
in turn, correlated to the TEF values. Measurement of the
binding of a ligand to the AhR by itself is not sufficient to
relate to the hazard of the sample since some materials will
bind to the AhR, even displace TCDD, but not transform the
receptor to the dangerous form. These are no false-positives
in the AhIA like they are in simple binding assays. This
hybrid assay contains both immunoassay and receptor assay
attributes. Samples to be tested are added to a mixture
containing AhR and other components in a special ELISA
plate and allowed to incubate at room temperature for 2 h.
At that time, any AhR transformed by dioxin-like com-
pounds is bound to the plate and the remaining material is
washed away. Antibodies are added to the ELISA and the
transformed bound AhR is enzymatically detected. Color
development is proportional to transformed AhR. The assay
currently takes about 5 h to analyze 96 samples. The
minimal detection limit is about 1 pg 2,3,7,8-TCDD equiv-
alents (Wheelock, 2001).
For several PCBs and PCDD/PCDFs, higher REPs to
2,3,7,8-TCDD were detected, e.g., 2,3,4,7,8-PCDF (1.0);
1,2,3,7,8-PCDD (0.92); 2,3,7,8-TCDF (0.5); 2,3,4,6,7,8-
HCDF (0.5); 1,2,3,4,7,8-HCDD (0.27); 1,2,3,7,8-
PCDF (0.27); PCB-126 (0.26); PCB-77 (0.04); and
PCB-156 (0.042).
7.3.5. recAhr DELFIA assay kit
This commercial kit measures the concentration of dioxin
and furans in a sample by using a recombinant AhR. The
DELFIA technology uses time-resolved fluorometry (TRF).
TRF reagents uniquely introduce multianalyte testing which
allows one sample to be tested for multiple chemicals in a
high throughput platform (Allen, 2001).
7.3.6. AhR binding assay (filtration assay with
radiolabeled ligands)
These bioassays are based upon the competitive binding
of dioxin-like compounds and fixed aliquots of radiolabeled
PCDD/PCDFs [e.g., (
125
I)2,3-dibromo-7-iododibenzodioxin
or (
3
H)TCDD] to the AhR protein in whole cells in culture
or broken cell preparations. The IEQ was analyzed by
comparison of the EC
50
curve (plotting the analyzed bound
P.A. Behnisch et al. / Environment International 27 (2001) 413–439 429

radioactivity vs. competitor concentration) of the dioxin-like
compounds. The additive binding to the receptor was
observed for a wide range of dioxin-like compounds with
a detection limit of 64 pg TCDD (Safe, 1990; Bunce, 1995).
Additive behavior and comparable REPs for several PHAHs
(e.g., REP for 2,3,7,8-TCDF: 0.55; PCB-77: 0.02, benzo[a]-
pyrene: 0.04) have been reported in several independent
studies (Bradfield and Poland, 1988; Bunce, 1995; Diaz-
Ferrero et al., 1997; Farrell et al., 1987; Heath-Pagliuso
et al., 1998; Hu et al., 1995; Phelan et al., 1998; Safe, 1984,
1986, 1987, 1990, 1993, 1994, 1997, 1998a,b; Safe et al.,
1987, 1989, 1991, 1997; Schneider et al., 1995).
7.4. Other bioanalytical tools
This section describes briefly some more bioanalytical
tools for the detection of dioxin-like compounds.
A recombinant yeast bioassay (see also Oosterkamp
et al., 1997 about yeast bioassays) for measuring AhR
ligands have been established (Miller, 1997; Miller et al.,
1998; Takigami et al., 1999). The human AhR and Arnt
complex were coexpressed in the yeast Saccharomyces
cerevisiae to create a system for the study of active AhR
agonists in the yeast system. The dose – response relation-
ships of b-galactosidase induction by well characterized
AhR agonists showed that coexpression of intact AhR and
Arnt in yeast leads to an induction of signal transduction
that was similar to that observed in human cells. Since S.
cerevisiae did not naturally express AhR, Arnt and estrogen
receptor homologues and thus it provided a unique cellular
background to examine possible interactions between these
receptors. To test the receptor interaction hypothesis, cells
coexpressing Arnt, AhR or both along with estrogen
receptor were treated with or without the AhR ligand
b-naphtoflavon. Possible disadvantages in comparison
to mammalian cell-based bioassays are the differences in
some important biological effects (e.g., membrane passage
and protein binding of the dioxin-like compounds). Takigami
et al. (1999) showed that various AhR ligands were
measured with the following REPs: 2,3,7,8-TCDD > 2,3,7,8-
TCDF = naphtoflavon > 2-nitrofluorene > 1-nitro-naph-
thalene > indole > pyrene > fluoranthene > indole-carbi-
nol > phenanthrene > anthracene > butyl-benzylphthalate
(BBP) > di-butyl-phthalate (DBP) > 1-nitro-napthalene >
fluorene >2-nitro-furan > dioctyl-phthalate (DOP) > trypt-
amine. Takigami et al. showed that the 2,3,7,8-TCDD
standard could be avoid by using naphtoflavon as relative
indicator for dioxin-like compounds.
Furthermore, Hagenmaier et al. (1998) (Patent No.
WO 98/03676) patented a recombinant two-hybrid yeast
bioassay. In this study, three soil samples (TEQ range
from 25 to 1950 ng I-TEQ/kg) showed comparable bio-
TEQ values to the by chemical analysis (GC/MS)
detected TEQs.
A bioassay studying the aromatase (CYP19) activity
in human choriocarcinoma JEG-3 cells was also estab-
lished to determine dioxin-like compounds (Drenth et al.,
1998; Letcher et al., 1999). REP values for PCB-126 in
the additionally performed EROD test (0.015 –0.02) were
lower than determined by aromatase (CYP 19) activity
(0.27–0.69).
Another biomarker to detect dioxin-like compounds
was the porphyrin accumulation associated with CYP1A
induction in different cell lines (fish hepatoma cells
(PLHC-1; Hahn and Chandran, 1996) and CEH cultures
(Kennedy et al., 1993).
The hallmark of dioxin exposure is the development
of chloracne, a persistent form of acne characterized by
hyperkeratinization. As the skin is a sensitive target for
dioxin toxicity, human keratinocytes are a useful model
for studying the toxicity of dioxin in vitro. Skin aberra-
tions can be interpreted as an altered differentiation
pattern of acinar sebaceous base cells and a change in
the rate of terminal differentiation of the keratinocytes
(Berkers et al., 1995; Knutson and Poland, 1980; van
Pelt et al., 1992). Dioxin-like compounds modulate the
proliferation and differentiation of human epidermal cells
in vivo and in culture. One of the earliest events in the
process of terminal differentiation is the increase in cell
size. The usefulness of morphometric cell size analysis,
as a quantifiable marker for chemical-induced differen-
tiation, has been reported. Concentration-related increases
in cell size distribution were induced for 2,3,7,8-TCDD
and 2,3,4,7,8-PCDF in normal human keratinocytes and
cells from an SV40-transformed keratinocyte cell line
(SVK14), whereas 1,2,3,4-TCDD did not affect the cell
size distribution up to a concentration of 100 nM. The
minimal effective concentrations of five 2,3,7,8-substi-
tuted PCDD/PCDFs and a coplanar PCB necessary to
induce an increase in cell size distribution were deter-
mined in SVK14 cells. It was found that the potency of
these compounds relative to that of 2,3,7,8-TCDD corre-
lated well with REPs observed in other test systems.
This indicated that the keratinocyte cell assay is a useful
method for establishing the REP of various dioxin-like
compounds and complex mixtures.
In addition, precision-cut rat liver slices were vali-
dated as a useful in vitro biomarker model for assessing
the dose-related induction of CYP1A1 and CYP1A2 in
rat liver following exposure to 2,3,7,8-TCDD (Drahushuk
et al., 1999). Moreover, extending the incubation period
to 96 h resulted in in vitro induction profiles for
CYP1A1 and CYP1A2 that were qualitatively and quan-
titatively similar to that observed following in vivo
exposure to TCDD.
The induction of CYP1A1 (P4501A1) and P4501A1-
specific EROD activity by TCDD was also investigated
in human splenic lymphocytes cultures (Jeong and
Yang, 1996). EROD activity was induced by TCDD
in mitogen-stimulated blast cells. TCDD markedly
induced EROD activity in a dose- and time-dependent
manner. The expression of P4501A1 mRNA was
P.A. Behnisch et al. / Environment International 27 (2001) 413–439430

Table 5
REP values of several dioxin-like compounds with different BDMs in comparison to the I-TEFs (WHO, 1998) for humans [H4IIE/EROD: Schmitz, 1995; Safe et al., 1991; Sanderson et al., 1996; CALUX:
Garrison et al., 1996; Sanderson et al., 1996; mouse/rat; rainbow trout EROD/CALUX: Clemons et al., 1997; Ritcher et al., 1997]
Congener
Rat/AHH
in vivo (Safe
et al., 1991)
Rat AhR
binding
(Safe, 1990)
H4IIE/EROD
in vitro
In vivo – in vitro
(Safe, 1998a,b)
Rainbow trout
EROD/reporter
gene
CALUX
mouse/rat
AhIA
(Wheelock
et al., 1996)
EIA 1: DF1
(Harrison and
Carlson, 1999)
EIA 2: DD3
(Stanker et al.,
1987)
EIA 3
(Sugawara
et al., 1998) WHO-TEF
2,3,7-TriCDD 0.14 0.001 0.37 0.14 0.28 –
1,3,7,8-TCDD 1E 4 0.01 6E 4 – – 0.62 –
1,2,7,8-TCDD – – – 0.14 – –
1,2,3,7,8-PCDD 0.13 0.13 0.18/0.01/0.3 0.053 – 0.59/0.07 – 0.64 2.6/0.23 – /0.49; 0.79 0.92 1.05 0.95 1.0 1
1,2,3,4,7,8 – HxCDD 0.13 0.036 0.18/0.05 0.013 – 0.24/0.05 – 0.13 1.1/0.76 0.27 0.016 0.12 0.01 0.1
1,2,3,6,7,8-HxCDD – 0.04/ 0.015 – 0.16/0.005 – 0.5 0.2 – /0.068 0.079 0.24 – 0.1
1,2,3,7,8,9-HxCDD – 0.06 0.016 – 0.14/0.009 0.39 0.092 – 0.1
1,2,3,4,6,7,8-HpCDD 0.0076/0.003 0.2 0.0072 0.079 – 0.01
OCDD >0.001 – /3E 4 >0.0013/0.006 < 0.00001 < 0.001 < 0.001 0.001
2,3,7,8-TCDF 0.006 0.24 0.43/0.09 0.016– 0.17/0.006 – 0.43 0.2/0.24 0.082/ – 0.50 0.2 0.27 0.71 0.1
1,2,3,7,8-PCDF 0.003 0.13 0.003 0.018 – 0.9/0.003 – 0.13 0.2/0.21 0.26 0.046 0.033 – 0.05
2,3,4,7,8-PCDF 0.11 0.67 0.6/1.4/0.28 0.12 – 0.8/0.11– 0.67 1.9/0.3 0.33/0.34; 0.69 1.04 0.17 0.55 0.03 0.5
1,2,3,4,7,8-HxCDF 0.014 0.043 0.2/0.49 0.038 – 0.18/0.013 – 0.2 1.1/ 0.004 0.017 – 0.1
1,2,3,6,7,8-HxCDF 0.012 0.037 0.06/0.15 – /0.037 – 0.048 0.01 – 0.1
1,2,3,7,8,9-HxCDF – – 0.033 – 0.1
2,3,4,6,7,8-HxCDF 0.015 0.21 0.1/0.03 0.017– 0.097/0.015 – 0.1 0.50 0.049 – 0.1
PCB-77 – 0.083 1E 4/9E 4/3E 4 both 0.13 – 7E 6 0.0034/0.006 1.4E 3/7E 4 0.040 0.005 0.001 0.001
PCB-126 0.004 insoluble 0.2/0.8/0.05 both 0.003 – 0.77 0.023/0.0063 – /0.065; 0.02 0.089 0.012 0.1
PCB-169 0.008 0.023 0.003/0.008/0.0015 both 0.0006 – 1.1 2E 4/ – –/1.5E 3; 6E 4 0.006 0.01
P.A. Behnisch et al. / Environment International 27 (2001) 413–439 431

increased by TCDD in mitogen-stimulated cells as
detected by Northern blot analysis. These findings
supported the conclusion that TCDD induced the
expression of P4501A1 gene, resulting in an increased
EROD induction.
8. Differences in the REPs to 2,3,7,8-TCDD in the
reviewed BDMs
In several studies using different BDMs, the dioxin-like
potency of several PCDD/PCDF and PCB congeners have
been analyzed in comparison to 2,3,7,8-TCDD (Table 5;
Fig. 5). The listed REPs in Table 5 for several dioxin and
PCB congeners illustrate that the range of potencies in
these studies with different BDMs were all in about one
order of magnitude from the recommended TEFs from
WHO (1998).
9. Conclusion
Bioanalytical tools have the potential to greatly
advance our knowledge about the effects of chemicals
on the environment. However, several challenges must be
met if bioassays are to make the transition from being
research tools to being widely used analytical methods
which complement chemical analysis (HRGC/HRMS). In
order to make this transition, the methods for the bio-
assays must be clearly defined and must meet widely
accepted performance criteria. Standard testing proce-
dures for use on environmental samples should first
meet the criteria of national organizations (e.g., US
EPA Method 4425 or 4025; ASTM, 1999 Guide
E1853-98 or APHA, 1998 Standard Method 8070), and
then meet international standards. The approval of bio-
analytical screening tests by major agencies in some
high-profile programs is currently in progress to enhance
their credibility. Continued improvements in these bio-
chemical technologies will encourage new users to
become involved. There is a need to promote an under-
standing of bioassay screening strategies within the wider
analytical community, including an awareness of their
benefits and shortcomings. However, interpretation of
results from any single chemical/biochemical analysis
technology or a battery of these technologies must take
into account their limitations as well. For example, a
simple AhR binding assay would provide data on intrinsic
binding affinities of chemicals for this receptor, but would
not necessarily predict in vivo or in vitro dioxin-like
potencies in various assay systems.
In conclusion, there is a growing consensus that a battery
of in vitro and in vivo assays is required to comprehensively
assess the impact of dioxin-like chemicals in complex
environmental mixtures.
Acknowledgments
The authors wish to thank Dr. Randy L. Allen, Prof. Jack
Anderson, Prof. Abraham Brouwer, Dr. David Brown, Prof.
Colin Campbell, Prof. Michael Denison, Dr. Robert
Harrison, Dr. Laurentius Hoogenboom, Dr. Karl-Werner
Schramm, Prof. Dieter Schrenk and Dr. Geoffrey Wheelock
Fig. 5. Relative responses for several dioxin-like compounds with a battery of BDMs (according to Table 5).
P.A. Behnisch et al. / Environment International 27 (2001) 413–439432

for their fruitful discussions about the state-of-the-art
bioanalytical tools.
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