Aquatic Toxicology 54 (2001) 125– 141
www.elsevier.com/locate/aquatox
In vitro response of fish and mammalian cells to complex
mixtures of polychlorinated naphthalenes, polychlorinated
biphenyls, and polycyclic aromatic hydrocarbons
D.L. Villeneuve *, J.S. Khim, K. Kannan, J.P. Giesy
Department of Zoology, National Food Safety and Toxicology Center, and Institute for En6ironmental Toxicology,
Michigan State Uni6ersity, 218 -C, East Lansing, MI 48824, USA
Received 5 January 2000; received in revised form 18 April 2000; accepted 18 September 2000
Abstract
In vitro characterization and comparison of responses to different classes of biologically active compounds can
increase the utility of bioassays. In this study, the relative potencies (REPs) of mixtures of polychlorinated
naphthalenes (PCNs), polychlorinated biphenyls (PCBs), and polycyclic aromatic hydrocarbons (PAHs), to induce in
vitro ethoxyresorufin-O-deethylase (EROD) in PLHC-1 fish hepatoma cells, H4IIE wild type (H4IIE-wt) rat
hepatoma cells, and recombinant H4IIE cells (H4IIE-EROD) were determined. The mixtures were also analyzed by
in vitro luciferase assay with recombinant H4IIE cells (H4IIE-luc). Halowaxes 1051, 1014, and 1013 caused significant
induction in all three H4IIE assays at concentrations less than 10 mg/l, but did not elicit a significant response in the
PLHC-1 assay. Based on H4IIE results, the Halowaxes were estimated to have relative potencies (REPs) of
approximately 10 − 6 –10 − 8 relative to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Less than 5 mg/l of Aroclors
1242, 1248, 1254; Clophens A60, T64; and Chlorofen induced significant responses in the H4IIE assays, while only
Clophens A60 and T64 caused a significant response in the PLHC-1 assay. The efficacy of the Aroclor mixtures was
generally insufficient to allow for quantitative REP estimates, but, based on their responses in the H4IIE assays,
Clophen A60 and Chlorofen were estimated to have REPs of approximately 10 − 6 and 10 − 7, respectively. A mixture
of 16 priority PAHs caused significant induction in all four cell types and was estimated to have a REP of
approximately 10 − 4. Overall, the results of this study add to a growing database on the dioxin-like potency of
complex mixtures of xenobiotics, and suggested that H4IIE-based in vitro bioassays were more sensitive than PLHC-1
cells for detecting dioxin-like activity in complex mixtures. © 2000 Elsevier Science B.V. All rights reserved.
Keywords: PLHC-1; H4IIE; In vitro bioassay; PCBs; PCNs; PAHs
1. Introduction
* Corresponding author. Tel.: + 1-517-4326312; fax: + 1517-4322310.
E-mail address: villene1@msu.edu (D.L. Villeneuve).
Comparison of the responses of different in
vitro bioassays to various classes of xenobiotic
compounds can increase utility of such assays as
0166-445X/01/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 6 - 4 4 5 X ( 0 0 ) 0 0 1 7 1 - 5
126
D.L. Villeneu6e et al. / Aquatic Toxicology 54 (2001) 125–141
analytical tools. For screening purposes it is generally desirable to use the assay which is most
sensitive for the compound(s) of interest. For risk
assessment purposes, however, it may be more
appropriate to use an in vitro bioassay model with
structure activity relationships (SARs) or relative
potencies (REPs) that closely parallel in vivo responses of the organism(s) of interest. For example,
REPs for dioxin-like compounds are known to vary
among fish, mammals, and birds (Denison et al.,
1986; Gooch et al., 1989; Walker and Peterson,
1991; Van den Berg et al., 1998; Abnet et al., 1999).
As a result, a risk assessment focused on potential
effects on fish should employ a model which has
been demonstrated to respond more like a fish, even
if it is less sensitive to certain classes of compounds
(Hahn et al., 1993; Richter et al., 1997; Villeneuve
et al., 1999). Additionally, differences in sensitivity
among in vitro bioassays may aid in the identification of classes of compounds associated with biological activity. For example, if assay A was
sensitive to compounds X and Y, while assay B was
sensitive to X only, the presence of a response in
assay A but no response in assay B could suggest
that the response was due to compound Y. Thus,
comparison of in vitro bioassays to various classes
of xenobiotics is useful for the development of both
assessment and analytical tools.
Polychlorinated naphthalenes (PCNs), polychlorinated biphenyls (PCBs), and polycyclic aromatic
hydrocarbons (PAHs) are ubiquitous contaminants
which have been detected in environmental matrices, including biota, sediment, air, surface waters, and municipal and industrial effluents (Neff,
1979; EPA, 1980; Eisler, 1987; Crookes and Howe,
1993; Jarnberg et al., 1993; Dorr et al., 1996;
Falandysz et al., 1996; Erickson, 1997; Espadaler
et al., 1997; Harner and Bidleman, 1997; Kannan
et al., 1998, 2000). Certain congeners of PCNs,
PCBs, and PAHs may interact with the aryl hydrocarbon receptor (AhR) to mediate dioxin-like toxic
effects (Poland and Knutson, 1982; PiskorskaPliszczynska et al., 1986; Safe, 1990; Narbonne et
al., 1991; Celander et al., 1994; Villeneuve et al.,
1998; Blankenship et al., 2000 Villeneuve et al.,
2000a). Therefore, in vitro bioassays which examine AhR-mediated gene expression or enzyme activities are useful tools for detecting and
characterizing the integrated effect of complex
mixtures of PCNs, PCBs, and PAHs. Numerous
studies have used such in vitro bioassays to characterize the relative potencies of individual PCN,
PCB, and PAH congeners (Safe, 1990; Hanberg et
al., 1991; Willett et al., 1997; Clemons et al., 1998;
Villeneuve et al., 1998; Blankenship et al., 2000;
Villeneuve et al., 2000a). These compounds are
found in the environment as complex mixtures of
both AhR-active and AhR-inactive congeners. Interactions between active and inactive congeners
may have relevant effects on in vitro bioassay
responses (Sanderson et al., 1996). Furthermore,
active and non-active congeners and the effects of
their potential interactions on assay response may
be different in various bioassay systems. Differences in the REPs or toxic equivalency factors
(TEFs) of mono –ortho PCBs between fish and
mammals (Van den Berg et al., 1998; Abnet et al.,
1999), and the potential for inhibition or inactivation of cytochrome P4501A1 (CYP1A1) monooxygenase enzyme activity by large doses of planar
halogenated biphenyls, as opposed to luciferase
reporter gene expression (Hahn et al., 1993; Sanderson et al., 1996), provide two examples of differences which may affect overall bioassay
responsiveness to complex mixtures of xenobiotics.
This study examined the sensitivity of four in
vitro bioassays (PLHC-1, H4IIE-luc, H4IIEEROD, and H4IIE-wt) to four technical mixtures
of PCNs, eight technical mixtures of PCBs, and a
mixture of 16 priority PAHs. Where possible, the
potency of the mixtures relative to a 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) standard was
characterized. These results add to a growing
database of information on the potency of xenobiotic mixtures and investigates the utility of these in
vitro bioassays as analytical tools for characterizing
samples containing complex mixtures of PCNs,
PCBs, and PAHs.
2. Methods
2.1. Chemicals
Halowaxes 1051, 1014, 1013, 1001, and a
mixture of 16 priority PAHs were obtained in
D.L. Villeneu6e et al. / Aquatic Toxicology 54 (2001) 125–141
methanol from AccuStandard (New Haven, CT,
USA). Halowaxes 1001 was 95% pure. Halowaxes
1051, 1014, and 1013 were 99% pure. The PAH
mixture was 99% pure and consisted of the following PAHs: acenapthene, acenapthylene, anthracene,
benz[a]anthracene,
benzo[a]pyrene,
benzo[b]fluoranthene, benzo[ghi]perylene, benzo[k]fluoranthene, chrysene, dibenz[ah]anthracene,
fluoranthene, fluorene, indeno[1,2,3-cd]pyrene,
naphthalene, phenanthrene, and pyrene. Aroclors
1242, 1248, 1260, 1254, and 1268 were also obtained 99% pure from AccuStandard (New Haven,
CT, USA). Clophen A60, Clophen T64, and Chlorofen were gifts from Dr J. Falandysz, University
of Gdañsk, Poland. The mixtures were not analyzed for potential contamination with chlorinated
dioxins or furans. Thus, potential contamination
with these compounds cannot be entirely ruled
out. Concentrations tested in the bioassay varied
(Table 1) and were limited by the mass of standard
available. All standards were prepared in high
purity isooctane (Burdick and Jackson,
Muskegon, MI, USA) prior to dosing cells.
The Aroclors were technical PCB preparations
produced in the USA and UK. Clophen was a
technical PCB mixture produced in Germany.
Chlorofen was produced in Poland. The composition and concentrations of individual chlorobiphenyls in these technical mixtures have been the
subject of several investigations (Schultz et al.,
1989; Falandysz et al., 1992; Erickson, 1997). The
composition of Clophen T64 has not been fully
described, but it is thought to be a mixture of
Clophens A50 and A60 and trichlorobenzene.
2.2. Cell culture
PLHC-1 cells are desert topminnow (Poeciliopsis lucida) hepatoma cells which have been shown
to have inducible cytochrome P4501A1 activity
(Hightower and Renfro, 1988; Hahn et al., 1993,
1996). H4IIE-luc cells are rat hepatoma cells
which were stably transfected with a luciferase
reporter gene under control of dioxin-responsive
enhancers (DREs) (Sanderson et al., 1996).
H4IIE-wt (wild-type) cells are the non-transfected
parent cell line from which the H4IIE-luc cells
were constructed (Sanderson et al., 1996). H4IIE-
127
luc cells were used for both luciferase assays
(H4IIE-luc) and ethoxyresorufin-O-deethylase
(EROD) assays (H4IIE-EROD). In vitro EROD
assay results with H4IIE-luc cells (H4IIE-EROD)
were compared to those obtained using H4IIE-wt
cells (H4IIE-wt). All cells were cultured in 100 mm
disposable petri plates (Corning, Corning NY,
USA) and were incubated in a humidified 95:5
air:CO2 atmosphere. PLHC-1 cells were grown at
30°C. H4IIE-luc and H4IIE-wt cells were grown at
37°C. H4IIE-luc and H4IIE-wt cells were cultured
in Dulbecco’s Modified Eagle Medium (Sigma
D-2920, St Louis, MO, USA) supplemented with
10% fetal bovine serum (FBS; Hyclone, Logan,
UT, USA). PLHC-1 cells were cultured in Minimum Essential Medium Eagle (MEM) supplemented with 292 mg/l L-glutamine (Life
Technologies, Grand Island NY, USA) and 10%
FBS (Hyclone). Cells were passaged when plates
became confluent and new cultures were started
from frozen stocks after less than 30 passages.
2.3. Exposure
Cells were trypsinized from petri plates containing 80 –100% confluent monolayers and resuspended in media. Cell number per ml was
estimated using a hemacytometer. H4IIE-luc and
H4IIE-wt cells were diluted to a concentration of
approximately 7.5×104 cells/ml. PLHC-1 were
diluted to approximately 1.25× 105 cells/ml. Diluted cells were seeded into the 60 interior wells of
96 well flat bottom microplates (ViewPlates, Packard Instruments, Meriden, CT, USA for luciferase
assays; Corning 25860 for EROD assays). All cells
were seeded in 250 ml per well. The 36 exterior
wells were filled with 250 ml culture media. Cells
were incubated overnight, to allow for cell attachment, then dosed. Test and control wells were
dosed with 2.5 ml of the appropriate sample or
solvent. Blank wells received no dose. A minimum
of three control wells and three blank wells were
tested on each plate. A minimum of three replicate
wells of each sample concentration were tested.
Dose-responses consisted of six concentrations
prepared by 3-fold serial dilution from
128
Table 1
Relative potency (REP) estimatesa,b, maximum observed responsesc, and maximum concentrationsd (ng/ml in well) tested for PCN, PCB, and PAH mixtures tested
using PLHC-1, H4IIE-EROD, H4IIE-luc, and H4IIE-wt in vitro bioassays
H4IIE-EROD
H4IIE-luc
Sample
Max.
Concentrationd
REPa,b
Observed
maxc
REPa,b
Hwx 1051
Hwx 1014
Hwx 1013
Hwx 1001
Aroclor 1242
Aroclor 1248
Aroclor 1254
Aroclor 1260
Aroclor 1268
Clophen A60
Clophen T64
Chlorofen
PAH-16
1100
10 000
10 000
100
5100
5100
150
5100
5100
4000
57 000
35 000
100
B2.4×10−5
B2.7×10−6
B2.7×10−6
B2.7×10−4
B5.3×10−6
B5.3×10−6
B1.8×10−4
B5.3×10−6
B5.3×10−6
B6.7×10−6
B4.7×10−7
B7.6×10−7
B2.6×10−4
−1
0
0
0
0
1
0
2
0
14
6
−1
20
1.2×10−5–4.6×10−6
82
3.2×10−5–4.1×10−7
43
7.0×10−7–7.5×10−12 30
6
B4.2×10−5
B 8.3×10−7
19
B 8.3×10−7
28
B 2.8×10−5
−1
B8.3×10−7
23
−1
B8.3×10−7
4.6×10−6 – 2.4×10−6 71
B 7.4×10−8
18
4.5×10−7–1.1×10−7
53
2.2×10−4–6.7×10−4 117
Observed
maxc
H4IIE-wt
REPa,b
Observed
maxc
REPa,b
6.4×10−5–1.4×10−5
6.7×10−6–6.5×10−7
7.7×10−6–6.2×10−8
B6.9×10−5
B1.4×10−6
B1.4×10−6
B4.6×10−5
B1.4×10−6
B1.4×10−6
3.4×10−6–1.0×10−8
B1.2×10−7
3.9×10−7–3.8×10−8
2.4×10−4–7.2×10−4
68
31
41
0
19
24
15
1
0
29
10
37
70
1.6×10−4–2.8×10−5
78
3.1×10−5–3.6×10−7
44
1.2×10−6–8.2×10−12 35
B5.8×10−5
2
5.3×10−7–3.4×10−8
29
3.2×10−6–7.2×10−7
44
B3.8×10−5
−1
B1.1×10−6
19
B1.1×10−6
−1
4.4×10−6–4.9×10−6
74
B1.0×10−7
20
5.4×10−7–5.0×10−7
53
3.2×10−4–2.7×10−4
75
Observed
maxc
a
REPs reported as the range of REP estimates generated from multiple point estimates over a response range from 20 to 80%-TCDD-max. (RP-band). Extrapolation
was used for samples which yielded maximum responses less than 80%-TCDD-max.
b
REPsBx were calculated by dividing the maximum concentration tested by the EC-50 of the TCDD standard.
c
Maximum response observed expressed as a percentage of the mean maximum response observed for the TCDD standard (%-TCDD-max.). Maximum response
was not necessarily achieved at the maximum concentration tested.
d
Maximum concentration tested expressed as ng/ml media (ppb) present in the test well. Mass per test well (ng) = ppb×0.25 ml/well.
D.L. Villeneu6e et al. / Aquatic Toxicology 54 (2001) 125–141
PLHC-1
D.L. Villeneu6e et al. / Aquatic Toxicology 54 (2001) 125–141
the maximum concentration tested (Table 1). All
exposures were 72 h.
2.4. Luciferase assays
Luciferase assays with H4IIE-luc cells were performed using a modification of methods detailed
previously (Sanderson et al., 1996). Briefly, exposed wells were inspected using a microscope
and condition relative to control wells was noted.
Culture media was then removed and cells were
rinsed with phosphate buffered saline (PBS) supplemented with 1 mM Ca2 + and Mg2 + . Cells
were then treated with 50 ml Ca2 + - and Mg2 + supplemented PBS and 50 ml LucLite™ reagent
(Packard Instruments). Plates were incubated for
10 min at 30°C then scanned with an ML3000
microplate reading luminometer (Dynatech Laboratories, Chantilly, VA, USA). Following the luminometer scan a 1.08 mM solution of
fluorescamine (Sigma) in high purity acetonitrile
(Burdick and Jackson) was added to each well
and plates were assayed for protein (Kennedy and
Jones, 1994) using a Cytofluor 2300 (excitation
400 nm, emission 460 nm, sensitivity 3), after a 15
min incubation at room temperature. Total
protein content per well was calculated by regression against a bovine serum albumin (BSA; Sigma
A-2153) standard curve. For luciferase assays,
total protein in the wells was used as an index of
cell number to detect outliers that were not apparent by visual inspection. Relative luminescence
units (RLU) were not adjusted for protein
content.
2.5. EROD assays
In vitro EROD assays with PLHC-1, H4IIEluc, and H4IIE-wt cells were performed using a
modified version of an H4IIE-wt EROD assay
procedure (Sanderson and Giesy, 1998). Exposed
wells were inspected using a microscope and condition relative to control wells was noted. Culture
media was removed by vacuum manifold and the
cells were rinsed with PBS. Cells were lysed by
freeze thaw in 30 ml nanopure water then treated
with 100 ml 0.05 M Na2HPO4 buffer containing 60
mM dicumarol (3,3%-methylene-bis[4-hydroxy-cou-
129
marin]; Sigma M-1390), 50 ml 10 mM ethoxyresorufin (ER; Molecular Probes, Eugene OR,
USA), and 20 ml 0.5 mM b-NADPH (Sigma
N-1630) and incubated at 30°C for exactly 60
min. Reactions were stopped by addition of 75 ml
1.08 mM fluorescamine in acetonitrile. Plates were
incubated for another 15 min, then scanned using
a Cytofluor 2300 (excitation 530 and 400 nm,
emission 590 and 460 nm, sensitivity 3). Resorufin
(Molecular Probes) and protein (BSA; Sigma A2153) standard curves were prepared by serial
dilution and run in the same manner as sample
plates (no ER was added to resorufin standard
wells). Relative fluorescence units (RFU) were
converted to pmol resorufin produced per min per
mg protein (pmol/min/mg) by regression against
the resorufin and protein standard curves.
2.6. Bioassay data analysis
Sample responses expressed as mean RLU or
mean pmol/min/mg (three replicate wells), were
converted to a percentage of the mean maximum
response observed for standard curves generated
on the same day (%-TCDD-max.). This was done
to normalize for day-to-day variability in response
magnitude, and to make response magnitudes
comparable from assay to assay. The mean solvent control response was subtracted from both
the sample and TCDD standard responses, prior
to conversion to a percentage, to scale values
from 0 to 100%-TCDD-max.
In cases where the magnitude of induction was
sufficient to allow a quantitative estimate, assay
specific REPs were calculated. The linear portion
of each dose response (%-TCDD-max. plotted as
a function of log dose) was defined by dropping
points from the tails until an R 2 ] 0.95 was obtained and a linear regression model was fit to the
remaining points. At least three points were used
in all cases. The linear regression equations for
the samples and corresponding TCDD standard
were used to estimate the concentration associated
with responses expressed as %-TCDD-max.
In order for point estimates of relative potency
to be valid, the sample and standard dose-response must be statistically parallel and have the
same maximum achievable response (Finney,
130
D.L. Villeneu6e et al. / Aquatic Toxicology 54 (2001) 125–141
1978; Putzrath, 1997). These conditions were
tested empirically. The efficacy of many of the
samples was either unknown or less than that of
TCDD. Thus, equal efficacy could not be assumed. The parallel slopes assumption was tested
by calculating relative potencies (REPi ) at multiple levels of response (Yi ) ranging from 20 to
80%-TCDD-max. (Villeneuve et al., 2000b). For
parallel dose-responses, REP estimates are independent of the response level selected (Putzrath,
1997). The minimum and maximum REPi values
generated (a relative potency band or RP-band)
were reported as an estimate of the uncertainty in
the relative potency estimate due to deviations
from parallelism between the standard and sample
curves (Villeneuve et al., 2000b). In cases where
the observed maximum response for the sample
was less than 80%-TCDD-max., extrapolation beyond the range of the empirical results was used
to estimate REPi at Yi greater than the observed
maximum. This was done to make the RP-bands
comparable from sample to sample, since the
width of the band is dependent on the range of
responses over which it is calculated (Villeneuve et
al., 2000b). In all cases, the maximum response
observed was reported along with the REP
estimate.
3. Results
3.1. Halowax mixtures
Four Halowax mixtures were analyzed using
PLHC-1, H4IIE-EROD, and H4IIE-luc, and
H4IIE-wt bioassays (Fig. 1). The concentrations
tested (Table 1) elicited no response in the PLHC1 bioassay (Fig. 1). In the H4IIE-EROD assay
Halowaxes 1051, 1014, and 1013 yielded response
magnitudes of 82-, 43-, and 30%-TCDD-max.,
respectively (Fig. 1, Table 1). Response magnitudes elicited in the H4IIE-luc and H4IIE-wt assays were similar. Halowaxes 1051 and 1014 were
estimated to have REPs of approximately 10 − 5
and 10 − 6, respectively (Table 1). Strong deviation
from parallelism to the TCDD standard curve
inhibited the ability to generate a precise relative
potency estimate for Halowax 1013. Halowax
1001 elicited a weak response in the H4IIEEROD assay at the greatest dose tested (Fig. 1).
The magnitude of induction was not sufficient to
allow for relative potency estimation.
3.2. PCB mixtures
Among the PLHC-1, H4IIE-EROD, H4IIE-luc,
and H4IIE-wt assays, the PLHC-1 assay was the
least sensitive to technical mixtures of PCBs.
None of the Aroclor mixtures elicited significant
induction of CYP1A1 activity in PLHC-1 cells, as
measured by EROD assay (Fig. 2). Aroclors 1242,
1248, and 1260 induced significant responses in all
three H4IIE bioassays (Fig. 2). The magnitudes of
induction in the H4IIE-EROD and H4IIE-luc
assays were rather low. Aroclor 1248, which induced the greatest magnitude of response yielded
a maximum response of 29- and 23%-TCDDmax. in the H4IIE-EROD and H4IIE-luc assays,
respectively (Fig. 2). Response magnitudes, relative to those of the TCDD standard, were greater
in the H4IIE-wt assay (Table 1). Based on the
H4IIE-wt responses, the REPs of Aroclors 1242
and 1248 were estimated to be approximately
10 − 8 and 10 − 6, respectively (Table 1). The slopes
observed for Aroclors 1242, 1248, and 1260 in the
H4IIE-luc assay, and the slope for Aroclor 1248
in the H4IIE-EROD and H4IIE-wt assays suggest
that the responses were reaching their maximum
at fairly low magnitudes of response relative to
that caused by TCDD (Fig. 2). This suggests that
the Aroclor mixtures have an efficacy much less
than that of TCDD in these bioassay systems.
The European PCB mixtures, Clophen A60,
Clophen T64, and Chlorofen, exhibited greater
activity than the Aroclors. Clophen A60 elicited a
significant response in all four assays (Fig. 3). In
both the PLHC-1 and H4IIE-luc assays, the response to Clophen A60 appeared to reach a
plateau at magnitudes of 14- and 29%-TCDDmax., respectively (Fig. 3). In the H4IIE-EROD
and H4IIE-wt assays, however, Clophen A60 induced a response magnitude greater than 70%TCDD-max. and appeared to be approaching the
efficacy of TCDD (Fig. 3). Based on H4IIEEROD and H4IIE-wt results, the REP of
Clophen A60 was approximately 2.4–4.9×10 − 6
D.L. Villeneu6e et al. / Aquatic Toxicology 54 (2001) 125–141
(Table 1). The relative potency estimate based
on H4IIE-luc results was similar but there was
greater uncertainty in the estimate due to extrapolation beyond the observed maximum response and deviations from parallelism (Table
1). Chlorofen was the second most potent PCB
mixture (Fig. 3). Although it elicited no response in the PLHC-1 assay, it induced response
magnitudes as great as 53%-TCDD-max. in the
H4IIE-EROD and H4IIE-wt, and 37%-TCDDmax. in the H4IIE-luc assay (Fig. 3, Table 1).
Response plateaus were observed at the greatest
dose tested in all three H4IIE assays (Fig. 3).
This suggested that the efficacy of Chlorofen
was less than that of TCDD. The relative potency of Chlorofen was approximately 10 − 7
131
(Table 1). Again, extrapolation and deviation
from parallelism resulted in greater uncertainty
in the H4IIE-luc based estimate. Clophen T64
was the least active European PCB mixture. It
produced a weak response (6%-TCDD-max.) in
the PLHC-1 assay and yielded response magnitudes less than 20%-TCDD-max. in the H4IIE
assays (Fig. 3). Due to the small magnitude of
induction, derivation of REPs for Clophen T64
was not feasible. Among the PCB mixtures for
which REPs could be derived, the rank order of
relative potency was Clophen A60: Aroclor
1248\Chlorofen\ Aroclor 1242 (Table 1).
Clophen A60 and Chlorofen induced greater
magnitudes of response than the Aroclors (Table
1, Fig. 2,Fig. 3). Based on their responses to the
Fig. 1. Response of PLHC-1 fish (Poeciliopsis lucida) hepatoma cell bioassay, in vitro ethoxyresorufin-O-deethylase assay with
wildtype (H4IIE-wt) and recombinant (H4IIE-EROD) H4IIE rat hepatoma cells assay, and luciferase assay with recombinant H4IIE
rat hepatoma cells (H4IIE-luc) to Halowax mixtures and a 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) standard. Doses expressed
as ng/ml media (ppb) present in the test well. Responses expressed as a percentage of the maximum response observed for the TCDD
standard (%-TCDD-max.).
132
D.L. Villeneu6e et al. / Aquatic Toxicology 54 (2001) 125–141
Fig. 2. Response of PLHC-1 fish (Poeciliopsis lucida) hepatoma cell bioassay, in vitro ethoxyresorufin-O-deethylase assay with
wildtype (H4IIE-wt) and recombinant (H4IIE-EROD) H4IIE rat hepatoma cells assay, and luciferase assay with recombinant H4IIE
rat hepatoma cells (H4IIE-luc) to Aroclor mixtures and a 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) standard. Doses expressed as
ng/ml media (ppb) present in the test well. Responses expressed as a percentage of the maximum response observed for the TCDD
standard (%-TCDD-max.).
PCB mixtures, the rank order of sensitivity among
the in vitro bioassays used appeared to be H4IIEwt \ H4IIE-EROD \H4IIE-luc \PLHC-1.
TCDD-max. (Fig. 4). The REP of the PAH mixture tested was estimated to be approximately
10 − 4.
3.4. Absolute acti6ities
3.3. PAH mixture
A mixture of U.S. EPA’s 16 priority PAHs
induced significant responses in all four bioassays
(Fig. 4). The response in the PLHC-1 cells was
relatively weak, achieving a maximum response
magnitude of only 20%-TCDD-max. (Fig. 4,
Table 1). In contrast, maximum responses in the
H4IIE assays were greater than 70%-TCDD-max.
(Fig. 4, Table 1). In the H4IIE-EROD assay, the
efficacy of the PAH mixture exceeded 100%-
Absolute EROD and luciferase activities varied
among cell lines. The greatest magnitude of absolute EROD activity was observed in the PLHC-1
bioassay (Table 2). The maximal absolute EROD
activity observed for recombinant H4IIE cells
treated with TCDD was approximately half of
that observed for the PLHC-1 bioassay (Table 2).
The maximum response observed for H4IIE-wt
cells exposed to TCDD was 60.5 pmol/min/mg,
which was about half that observed for recombi-
D.L. Villeneu6e et al. / Aquatic Toxicology 54 (2001) 125–141
nant H4IIE cells (Table 2). The maximal luminescence observed in the H4IIE-luc bioassay was
1720 relative luminescence units. Protein concentrations per well were similar for all the bioassays
used in this study (Table 2).
4. Discussion
4.1. Relati6e 6s. absolute acti6ities
Absolute EROD and luciferase activities can
vary widely among assays and among laboratories. For example, maximal EROD activities for
H4IIE-wt cells exposed to TCDD ranging from
16 (Sanderson et al., 1996) to 60 pmol/min/mg
133
(Tillitt et al., 1991) to as great as 800–900 pmol/
min/mg (Willett et al., 1997) have been reported.
It is unclear whether the differences were due to
the inherent responsiveness of the cells or differences in assay protocols. Differences in assay protocols may affect the absolute activities observed.
For example, initial rates of the EROD reaction
may be linear for only 10–15 min (Kennedy et al.,
1993), thus absolute EROD activities measured
after 10 or 15 min would be expected to vary
considerably from those averaged over 60 min.
Differences in the buffers used, incubation temperatures, reagent concentrations or purities,
protein assay procedures, standards, etc. could all
potentially lead to differences in absolute EROD
activities. Thus, there may be relatively few cases
Fig. 3. Response of PLHC-1 fish (Poeciliopsis lucida) hepatoma cell bioassay, in vitro ethoxyresorufin-O-deethylase assay with
wildtype (H4IIE-wt) and recombinant (H4IIE-EROD) H4IIE rat hepatoma cells assay, and luciferase assay with recombinant H4IIE
rat hepatoma cells (H4IIE-luc) to Clophen A60, Clophen T64, Chlorofen, and a 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)
standard. Doses expressed as ng/ml media (ppb) present in the test well. Responses expressed as a percentage of the maximum
response observed for the TCDD standard (%-TCDD-max.).
134
D.L. Villeneu6e et al. / Aquatic Toxicology 54 (2001) 125–141
Fig. 4. Response of PLHC-1 fish (Poeciliopsis lucida) hepatoma cell bioassay, in vitro ethoxyresorufin-O-deethylase assay with
wildtype (H4IIE-wt) and recombinant (H4IIE-EROD) H4IIE rat hepatoma cells assay, and luciferase assay with recombinant H4IIE
rat hepatoma cells (H4IIE-luc) to a mixture of 16 priority PAHs (PAH-16) and a 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)
standard. Doses expressed as ng/ml media (ppb) present in the test well. Responses expressed as a percentage of the maximum
response observed for the TCDD standard (%-TCDD-max.).
in which absolute EROD activities reported by
different laboratories may be directly comparable.
Inherent differences in the responsiveness of the
cells used can also yield variation in absolute
EROD activities. In this study, nearly 4-fold variation in the maximal EROD responses to TCDD
was observed (Table 2). The same EROD assay
protocol was used for PLHC-1, H4IIE-wt, and
recombinant H4IIE cells. Furthermore, mean
protein concentration per well was similar among
all three assays (Table 2). This suggests that the
differences in absolute activities were due to inherent differences in the properties of the cells.
Similar differences in responsiveness may occur
within a given cell type as the cells change with
continuous passaging. Thus, it is difficult to make
comparison, based on absolute units, among cell
types or even cell lineages within the same cell
type.
The development of recombinant cell lines further complicates the ability to compare bioassay
results using absolute units. In this study, for
example, relative luminescence units are not directly comparable to EROD activity. Even if the
same reporter system were used, differences in the
reporter gene constructs (i.e. different promoter
sequences, different numbers of DREs, different
locations of insertion into the genome, etc.) would
be expected to yield significant differences in absolute units.
The results and discussion presented here are
based on relative response units. Original re-
D.L. Villeneu6e et al. / Aquatic Toxicology 54 (2001) 125–141
sponse units were converted to a percentage of the
maximum value observed for the TCDD standard, on an assay-specific basis. The use of relative units facilitated comparisons among the four
assays examined in this study. Furthermore, expression of response magnitudes in this manner,
should enhance the comparability of these results
to other in vitro bioassay results reported in the
literature.
4.2. PLHC-1 6s. H4IIE assays
The REPs of dioxin-like compounds are known
to vary among taxonomic groups (Van den Berg
et al., 1998). In particular, differences in the REPs
of mono- and di-ortho PCBs between fish and
mammals have been demonstrated (Gooch et al.,
1989; Skaare et al., 1991; Walker and Peterson,
1991; Abnet et al., 1999). Thus, taxa-specific in
vitro bioassays which accurately reflect in vivo
differences in the REPs of dioxin-like compounds
among various classes of organisms would be
useful for risk assessment purposes. This study
compared the fish hepatoma cell-based PLHC-1
assay to three assays using mammalian H4IIE rat
hepatoma cells. The PLHC-1 assay was less sensitive to the technical mixtures analyzed in this
study than the H4IIE assays. It was also 4– 6
times less sensitive to TCDD than the H4IIE
assays and had a linear range of response to
TCDD that covered only one order of magnitude
of concentrations (0.01– 0.1 ng/ml), as opposed to
three orders of magnitude for the H4IIE assays
(0.001 –1 ng/ml). The mean EC-50s for TCDD
Table 2
Maximal EROD and luciferase activities observed for TCDD
and mean protein per well for each in vitro bioassay used in
this study
Assay
Maximal activity of
TCDD
Mean protein
per well
PLHC-1a
H4IIE-wtb
H4IIE-ERODb
H4IIE-lucb
236 pmol/min/mg
60.5 pmol/min/mg
117 pmol/min/mg
1723 RLU
28.39 2.8
25.093.9
25.8 94.37
26.8 93.2
a
b
TCDD concentrations up to 0.25 nM (0.08 ng/ml).
TCDD concentrations up to 3.1 nM (1.0 ng/ml).
135
were 2.6× 10 − 2, 4.2× 10 − 3, 6.8× 10 − 3, and
5.8× 10 − 3 ng/ml (8.2×10 − 2, 1.3× 10 − 2, 2.1×
10 − 2, and 1.8×10 − 2 nM) for the PLHC-1,
H4IIE-EROD, H4IIE-luc, and H4IIE-wt assays,
respectively. These EC-50s were similar to values
reported previously for these assays (Sanderson et
al., 1996; Hilscherova et al., 2000). In the cases
where responses were observed (Clophen A60,
Clophen T64, and PAH-16), the response efficacy,
relative to TCDD, observed in the PLHC-1 assay
was less than that observed in the H4IIE assays.
Due to the lack of significant responses in the
PLHC-1 assay, precise PLHC-1-based REP estimates could not be generated for the mixtures
evaluated in this study. The broad estimates reported (Table 1) do not differ from REP estimates
based on the H4IIE assays. Thus, based on this
study, there was no evidence to either support or
reject the hypothesis that PLHC-1-based REPs
differ significantly from H4IIE-based REPs for
mixtures of PCNs, PCBs, or PAHs. There was,
however, evidence to suggest that the PLHC-1
assay, as performed in this study, was less sensitive for the detection of dioxin-like activity in
complex mixtures of PCNs, PCBs, and PAHs.
There are a number of possible explanations for
the differences in sensitivity observed between the
fish cell-based PLHC-1 assay, and the mammalian
cell-based H4IIE assays. Differences in membrane
permeability and/or metabolic capacity between
PLHC-1 and H4IIE cells could contribute to differences in sensitivity and responsiveness. These
properties have not been well studied in these cell
lines, however. Fish and mammalian AhR have
been shown to have significant structural differences (Abnet et al., 1999). Thus, it has been
hypothesized that differences in sensitivity to certain dioxin-like compounds among fish and mammals may be the result of structural differences in
the AhR and/or ARNT protein (Abnet et al.,
1999). When transfected into COS-7 monkey kidney cells, human AhR/ARNT was found to produce a greater maximal response and lower EC-50
for TCDD than fish AhR/ARNT transfected into
the same cell line (Abnet et al., 1999). The EC-50
for EROD induction in PLHC-1 cells for TCDD
was 2.6× 10 − 2 ng/ml (8.3× 10 − 2 nM). This was
similar to EC-50s of 4.7× 10 − 2 and 2.4× 10 − 2
136
D.L. Villeneu6e et al. / Aquatic Toxicology 54 (2001) 125–141
ng/ml (1.5×10 − 1 and 7.5× 10 − 2 nM) that were
previously reported for an in vitro bioassay with
recombinant rainbow trout cells (RLT 2.0 cells;
Villeneuve et al., 1999; Hilscherova et al., 2000).
Thus, it seems plausible to hypothesize that differences between fish and mammalian AhR/ARNT
may account for the differences observed in this
study and that the differences observed in vitro
may be reflective of in vivo differences. Additional
experiments are needed to test this hypothesis,
however.
4.3. H4IIE assays
In addition to comparing the responses of
PLHC-1 and H4IIE cells, this study compared
three types of H4IIE bioassays. These were: (1) in
vitro luciferase assay with recombinant H4IIE
cells (H4IIE-luc); (2) in vitro EROD assay with
recombinant H4IIE cells (H4IIE-EROD); and (3)
in vitro EROD assay with wild type H4IIE rat
hepatoma cells (H4IIE-wt). This was done to
determine if there were differences in sensitivity,
responsiveness, or REPs among these assays.
Comparison of H4IIE-luc and H4IIE-EROD results could be used to discern differences which
were due to the endpoint measured. Comparison
between H4IIE-EROD and H4IIE-wt results, on
the other hand, could discern whether transfection
may have altered properties of the cells, such as
CYP1A1 inducibility or expression, AhR expression, membrane permeability, etc. which could
account for differences in sensitivity or
responsiveness.
In general, results were similar among the
H4IIE assays. All three assays were similarly sensitive to TCDD, with EC-50s of 4.2×10 − 3,
6.8×10 − 3, and 5.8×10 − 3 ng/ml (1.3×10 − 2,
2.1×10 − 2, and 1.8×10 − 2 nM) for H4IIEEROD, H4IIE-luc, and H4IIE-wt, respectively.
This is in contrast to a previous study which
reported that the H4IIE-luc assay was approximately 3 times more sensitive (based on TCDD
EC-50s) than the H4IIE-wt assay (Sanderson et
al., 1996). The H4IIE-luc assay procedure used in
this study was different from that used previously,
however. In particular, a glow type reagent (LucLite™-Packard Instruments) was used rather
than a flash type reagent (Luciferase assay
reagent–Promega, Madison, WI, USA). Thus,
differences in the H4IIE-luc assay procedure are
the most likely explanation for the decreased sensitivity relative to that of the H4IIE-wt assay.
Using the same assay procedures as this study,
Hilscherova et al. (2000) obtained EC-50s for the
H4IIE-luc and H4IIE-wt assays which were similar to those obtained in this study. REPs based on
the three different H4IIE assays did not differ
significantly over the range of uncertainty in the
estimates (Table 1).
Somewhat unexpectedly, magnitudes of response, relative to the TCDD standard were generally lower in the H4IIE-luc assay than in either
the H4IIE-EROD or H4IIE-wt assay (Table 1).
This was most pronounced for the responses to
the Clophens and Chlorofen (Fig. 3). It has been
postulated that the H4IIE-luc assay should allow
for greater responsiveness, since AhR-mediated
expression of luciferase, which is foreign to the
cell, should not be affected by posttranscriptional
and posttranslational events which may affect
CYP1A1 expression or catalytic activity (Sanderson et al., 1996), such as inhibition of cytochrome
P4501A1 activity by high concentrations of inducers (Hahn et al., 1993). Again, however, the apparent discrepancy may be explained by the assay
procedure used in this study. In previous studies,
luciferase activity in the H4IIE-luc cells was greatest after 24–48 h of exposure, and was lower at 72
h (Sanderson et al., 1996). In contrast, EROD
activity in H4IIE-wt cells was greatest after 72 h
(Sanderson et al., 1996). Thus, the 72 h exposure
period used for this study may, at least partially,
explain the differences in responsiveness observed
between the H4IIE-luc and the H4IIE-EROD and
H4IIE-wt assays. Because responses were expressed in %-TCDD-max., however, it would also
be necessary to assume that the luciferase induction caused by the mixtures tested declined more
rapidly with time than that caused by the TCDD
standard. The EROD induction potency of PAHs
has been shown to decrease as exposure time
and/or cell numbers increased in both fish and
mammalian cells while the induction potency of
TCDD remained unchanged (Bols et al., 1999).
PAHs were thought to be progressively reduced
D.L. Villeneu6e et al. / Aquatic Toxicology 54 (2001) 125–141
through in vitro metabolism, whereas TCDD was
not easily metabolized and was thus, less sensitive
to exposure time and cell density (Bols et al.,
1999). It remains unclear whether similar factors
account for the differences observed in this study,
however. Overall, comparison of the H4IIE results suggested that all three H4IIE assay methods
were equally effective in vitro bioassay tools for
evaluating the dioxin-like activity of PCN, PCB,
and PAH mixtures and that modifications to the
H4IIE-luc assay described by Sanderson et al.
(1996) may have eliminated some of the advantages reported previously.
4.4. Halowaxes
The four Halowax mixtures tested in this study
differ markedly in their chlorinated naphthalene
(CN) compositions (Crookes and Howe, 1993).
Chlorine content, by weight, ranges from 50% for
Halowax 1001 to 70% for Halowax 1051 (Crookes
and Howe, 1993). The differences in composition
are significant, since the potency of individual
PCNs generally increases with increasing degree
of chlorination (Kover, 1975; Blankenship et al.,
2000; Villeneuve et al., 2000a). This trend appeared to hold true in this study as well. Halowax
1051, which has the greatest chlorine content and
is composed primarily of hepta and octaCNs
(Crookes and Howe, 1993) appeared to be the
most potent of the Halowax mixtures tested in
this study (Fig. 1, Table 1). This was followed by
Halowaxes 1014 and 1013 with chlorine contents
of 62 and 56%, respectively (Crookes and Howe,
1993). The apparent efficacy of the Halowax mixtures followed a similar trend. Halowaxes 1013
and 1014 induced responses that reached a
plateau at less than 50%-TCDD-max., while
Halowax 1051 elicited a response as great as
82%-TCDD-max. It was also noted that the range
of uncertainty in the REP estimates for the
Halowax mixtures increased as chlorine content
decreased (Table 1). This was due to the fact that
the slopes of the dose– response relationships deviated farther from parallelism to the TCDD standard curve (Fig. 1). Based on previously reported
REP estimates for a range of monoCN through
heptaCN congeners (Villeneuve et al., 2000a), one
137
would expect the pentaCN and hexaCN congeners present in Halowaxes 1013 and 1014 to
contribute most to the responses observed for
those mixtures. The slopes of the dose– response
relationships for the individual, active, pentaCNs
and hexaCNs, did not differ markedly from those
of TCDD, however (Villeneuve et al., 2000a).
Thus, the observation of both decreasing slope
and decreased efficacy with lesser chlorine content
in the mixture suggests the hypothesis that nonor less-active CN congeners may be interacting
with the more active CN congeners in a manner
that yields a more shallow slope for the dose–response relationship and a lesser maximal level of
induction. Halowax 1001 was not available at
concentrations sufficient to yield a dose–response
relationship, thus it remains uncertain whether
Halowax 1001 would conform to the trends
described.
4.5. PCB mixtures
Technical PCB formulations vary considerably
in congener composition and associated AhR-mediated dioxin-like potency. Congener compositions have been reported for all the Aroclors
analyzed in this study, as well as Clophen A60
and Chlorofen (Schultz et al., 1989; Frame et al.,
1996; Kannan et al., 1998). It was difficult to
make direct comparisons of the REPs of the
Aroclor mixtures, since the magnitudes of response observed were generally not great enough
to allow for accurate REP estimates. Furthermore, variation in the maximum concentrations
tested (Table 1) confounded interpretation of the
results. The shape of the dose–response relationships for Aroclor 1248 in all the H4IIE assays,
and Aroclor 1260 in the H4IIE-luc assay, suggest
that the efficacy of the Aroclor mixtures was
much less than that of TCDD. Clophen A60 and
Chlorofen had efficacies approaching that of
TCDD and, correspondingly, appeared to generate dose–response relationships with steeper
slopes. The explanation for this was not readily
discernible based on the homolog composition of
the mixtures. For example, the homolog composition of Clophen A60 was similar to that of Aroclor 1260 (Frame et al., 1996; Kannan et al.,
138
D.L. Villeneu6e et al. / Aquatic Toxicology 54 (2001) 125–141
1998), yet Clophen A60 exhibited a steeper dose–
response relationship and was more potent than
Aroclor 1260. There was no clear trend toward
increasing or decreasing potency with the average
number of chlorine atoms per molecule. This suggests that congener-specific differences, rather
than homolog distributions may be more important for explaining the differences in responses
observed for the various technical mixtures of
PCBs. In addition, differences in the concentrations of polychlorinated dibenzo-p-dioxin and
dibenzofuran (PCDD/DF) impurities in the technical mixtures may have also influenced the responses (Koistinen et al., 1996).
4.6. PAH mixture
The PAH mixture tested was found to be more
potent than the Halowax and PCB mixtures
tested. At least eight of the PAHs present in the
mixture have been shown to be potent inducers of
CYP1A1 activity in vitro (Willett et al., 1997;
Clemons et al., 1998; Villeneuve et al., 1998; Jones
and Anderson, 1999). REPs reported for these
active congeners range from approximately 10 − 2
to 10 − 6 (Willett et al., 1997; Clemons et al., 1998;
Villeneuve et al., 1998; Jones and Anderson,
1999). Given the known potencies of over half the
PAHs present in the mixture, it seemed reasonable that the entire mixture would have a REP
around 10 − 4 (Table 1). The EROD induction
potency of PAHs has been shown to decrease with
greater exposure time in both fish and mammalian
cells (Bols et al., 1999). Thus, the REP estimate
for the PAH mixture may have been greater if a
shorter exposure duration had been used. The 72
h REP estimate reported here, may be more relevant for predicting potency in vivo where significant metabolism of PAHs would be expected.
Whatever the case, the dependence of the REP for
the PAH mixture on exposure duration should be
considered when interpreting or applying the estimate. The efficacy and slopes of the H4IIE-based
dose –response relationships for the PAH mixture
suggest that non-active PAHs present in the mixture did not markedly affect the activity of the
AhR-active PAHs. The efficacy of the PAH mixture was more limited in the PLHC-1 assay. This
suggests that some property of the PLHC-1 cells
made them more susceptible to interferences by
non-active components in the mixture.
5. Conclusions
The H4IIE assays were more sensitive than the
PLHC-1 assay for screening samples, containing
complex mixtures of PCBs, PCNs, and/or PAHs,
for dioxin-like activity. The PLHC-1 assay was
markedly less sensitive to complex mixtures of
PCNs and PCBs than the H4IIE assays, but differences in REPs could not be demonstrated.
Thus, comparison of PLHC-1 and H4IIE
bioassay responses would probably not be useful
for formulating toxicant identification hypotheses
regarding the potential contribution of PCNs,
PCBs, or PAHs to the responses observed. From
a risk assessment standpoint, the PLHC-1 assay
may provide results which were more representative of in vivo responses in fish and, thus, better
suited for use in aquatic risk assessment than
H4IIE results, but further studies are needed to
test the hypothesis.
PCN, PCB, and PAH mixtures were shown to
induce dioxin-like in vitro bioassay responses. On
a weight basis they were at least 10 000 times less
potent than TCDD, and in many cases they were
less efficacious than TCDD. The potency of PCN
mixtures appeared to be related to the overall
chlorine content and homolog distribution of the
mixtures. Both the potency and efficacy of PCB
mixtures varied with composition, but a simple
relationship to chlorine content or homolog distribution could not be discerned. Finally, a mixture
of 16 priority PAHs yielded a relative potency
consistent with it’s known composition of active
components.
Acknowledgements
This work was supported by U.S. Environmental Protection Agency (U.S. EPA) Biology Exploratory Grants Program, grant R85371-01-0;
cooperative agreement CR 822983-01-0 between
Michigan State University and the U.S. EPA; the
D.L. Villeneu6e et al. / Aquatic Toxicology 54 (2001) 125–141
National Institute of Environmental Health Sciences Superfund Basic Research Program NIHES-04911; and a Michigan State University
Distinguished Fellowship to D. L. Villeneuve. We
thank Emily Nitsch for her technical assistance,
and Dr Jac Aarts, University of Wageningen, the
Netherlands, and Dr Lawrence Hightower, Univeristy of Connecticut, USA, for developing and/
or providing the cells used for this study.
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