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Transcript
Relative Potencies of Individual Polycyclic
Aromatic Hydrocarbons to Induce Dioxinlike
and Estrogenic Responses in Three Cell Lines
D. L. Villeneuve,1 J. S. Khim,1,2 K. Kannan,1 J. P. Giesy1
1
Department of Zoology, National Food Safety and Toxicology Center, and Institute for
Environmental Toxicology, Michigan State University, East Lansing, Michigan 48824
2
School of Earth and Environmental Sciences (Oceanography Program), College of Natural
Sciences, Seoul National University, Seoul 151-742, Korea
Received 23 October 2001; revised 4 January 2002; accepted 4 February 2002
ABSTRACT: The dioxinlike and estrogenic relative potencies (REPs) of 16 priority polycyclic aromatic
hydrocarbons (PAHs), seven methylated PAHs, and two hydroxylated PAHs were examined using three in vitro
cell bioassays. An in vitro ethoxyresorufin-O-deethylase assay with PLHC-1 fish hepatoma cells and in vitro
luciferase assay with H4IIE-luc recombinant rat hepatoma cells were used to evaluate dioxinlike potency. An
in vitro luciferase assay with MVLN, recombinant human breast carcinoma cells, was used to evaluate
estrogenic potency. Seven of the 16 priority PAHs tested induced significant dioxinlike responses. Excluding
outliers with large ranges of uncertainty, the dioxinlike REPs for the PAHs ranged from 10⫺6 to 10⫺3. This is
similar to the REPs reported for other xenobiotics of concern including polychlorinated naphthalenes (PCNs)
and some polychlorinated biphenyls (PCBs). In general, REP estimates generated in this study were similar to
those reported previously. However, a comparison of the estimates of total 2,3,7,8-tetrachlorodibenzo-pdioxin equivalents derived using assay-specific REPs with REPs reported in other studies indicated that the
use of nonspecific REPs could lead to significant error in mass-balance (potency-balance) analyses. A 10-h
acid treatment completely destroyed the dioxinlike activity of a PAH mixture. Among the compounds tested,
only benzo[a]anthracene and dibenz[a,h]anthracene induced significant responses in the MVLN bioassay.
Relative estrogenic potencies were estimated to be approximately 10⫺7. Overall, this research contributes to
the growing consensus regarding the dioxinlike potency of priority PAHs and PAH derivatives and provides
some additional evidence about potentially estrogenic PAHs. © 2002 Wiley Periodicals, Inc. Environ Toxicol 17:
128 –137, 2002; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/tox.10041
Keywords: PAHs; in vitro; H4IIE; MVLN; PLHC-1; relative potency; acid treatment
INTRODUCTION
Recent studies have indicated that extracts of sediment from
Lake Shihwa, Masan Bay, Ulsan Bay, and Onsan area,
Correspondence to: Dan Villeneuve, 218-C National Food Safety and
Toxicology Center, Michigan State University, East Lansing, MI 48824;
e-mail: villene1@msu.edu.
© 2002 Wiley Periodicals, Inc.
128
Korea, elicit both dioxinlike and estrogenic responses in
vitro (Khim et al., 1999; Khim et al., 1999b; Khim et al.,
2001; Koh et al., 2001). Most of this activity has been
associated with a moderately polar fraction known to contain polycyclic aromatic hydrocarbons (Khim et al., 1999;
Khim et al., 1999b; Khim et al., 1999c; Khim et al., 2001;
Khim et al., 2001b; Koh et al., 2001). Numerous studies
have shown polycyclic aromatic hydrocarbons (PAHs) to be
RELATIVE POTENCIES OF INDIVIDUAL PAHS
capable of inducing dioxinlike responses in vitro in both fish
(Villeneuve et al., 1998; Bols et al., 1999, Fent and
Bätscher, 2000; Behrens et al., 2001; Jung et al., 2001) and
mammalian cell lines (Willett et al., 1997; Clemons et al.,
1998; Jones and Anderson, 1999). PAHs have also been
shown to induce ethoxyresorufin-O-deethylase (EROD) activity in vivo (Gerhart and Carlson, 1978; Brunström et al.,
1991; Blanchard et al., 1999; Basu et al., 2001). Three
PAHs— benzo[a]pyrene (BAP), chrysene (CHR), and benzo[a]anthracene (BAA)— have been reported to elicit estrogenic responses in vitro (Clemons et al., 1998). Because the
sediment extracts were complex mixtures of trace organic
compounds including, but not limited to, PAHs, it was
unclear whether PAHs present in the extracts could account
for the dioxinlike and estrogenic responses observed. Both
novel and/or known aryl hydrocarbon receptor (AhR) and
estrogen receptor (ER) agonists that eluted in the midpolar
fraction could have contributed to the responses.
Mass-, or potency-, balance analysis is one approach for
addressing whether the known composition of a chemical
mixture, as measured by instrumental analysis, can account
for the potency and/or the magnitude of the biological
response observed (Sanderson and Giesy, 1998). Until now,
mass-balance studies of Korean sediment and other environmental samples have relied on the relative potencies
(REPs) of PAHs reported in the literature. Although such
values are useful for comparative purposes and for the
development of consensus values for use in risk assessments, they are not ideal for mass-balance analysis. Ideally,
mass- balance analysis should be based on REPs that are
species-, end-point-, and assay specific (Sanderson and
Giesy, 1998). The use of consensus values such as toxic
equivalency factors (TEFs) or nonspecific REPs can lead to
considerable error in mass-balance conclusions (Sanderson
and Giesy, 1998). Thus, the goal of this study was to
develop assay-specific REPs for the dioxinlike and estrogenic potencies of individual PAHs that could be used for
subsequent mass-balance analyses.
This article reports assay-specific REPs for 16 priority
PAHs (U.S. Environmental Protection Agency [EPA],
method 8310), seven methylated PAH compounds, and two
hydroxylated PAH compounds. The dioxinlike potency of
each compound to induce EROD activity in PLHC-1 fish
hepatoma cells (Hahn et al., 1993; Ryan and Hightower,
1994; Hahn et al., 1996) and in vitro luciferase activity in
H4IIE-luc recombinant rat hepatoma cells (Sanderson et al.,
1996) was evaluated. Estrogenic potency to induce in vitro
luciferase activity in MVLN cells (Pons et al., 1990; Demirpence et al., 1993) was also characterized. REPs derived in
this study were compared to those reported elsewhere, and
the potential implications for mass-balance analysis were
considered. A mixture of 16 priority PAHs was treated with
acid and analyzed by in vitro bioassay to examine the utility
of acid treatment for bioassay-directed fractionation studies
of PAHs and to test the hypothesis that acid-breakdown
129
products of PAHs may elicit dioxinlike responses in vitro.
Finally, the effect of exposure duration on the in vitro
potency of PAHs was examined. The REPs presented contribute to a growing body of PAH REPs in the literature that
can be used to develop consensus values for risk assessment. The information presented here should aid future
research aimed at determining the relative contribution of
individual PAHs to the total dioxinlike and/or estrogenic
activities associated with sediment extracts and other environmental samples.
MATERIALS AND METHODS
Chemicals
All PAHs, PAH metabolites, and PAH mixtures used in this
study (Table I) were obtained from AccuStandard (New
Haven, CT). Concentrations tested in the bioassays varied
(Table I) and were limited by the mass of standard available.
The maximum concentrations of the 16 priority, seven
methylated, and two hydroxylated PAHs tested in the in
vitro bioassays were 400, 10, and 20 ␮g/mL, respectively.
For the bioassay six dilutions of each standard were prepared by threefold serial dilution of the concentrated stock.
All dilutions were prepared in high-purity acetonitrile
and/or hexane (Burdick and Jackson, Muskegon, MI). The
PAH mixture used for this study was 99% pure and consisted of the following PAHs: acenapthene, acenapthylene,
anthracene, BAA, BAP, benzo[b]fluoranthene (BBF), benzo[ghi]perylene, benzo[k]fluoranthene (BKF), CHR, dibenz[ah]anthracene (DBA), fluoranthene, fluorene, indeno[1,2,3-cd]pyrene (IP), naphthalene, phenanthrene, and pyrene.
Acid Treatment
A mixture of 16 priority PAHs was treated with concentrated H2SO4 (1:1 PAH mixture:acid). Two treatment durations (1 h and 10 h) were tested. After the appropriate
exposure duration, the solvent layer was removed for bioassay analysis. One set of solvent layers was rinsed with
nanopure water (1:1 water:sample) prior to bioassay, while
another set was assayed directly without the washing step.
Acid-treated samples were tested at a single concentration
(a 100-ppb mixture). GC/MS analysis (selected ion monitoring mode) was used to evaluate the destruction of PAH
parent compounds by acid treatment. The specific details of
the GC/MS column and the operating conditions were the
same as those described for previous studies (Khim et al.,
1999, 1999c).
Cell Culture and Bioassay
PLHC-1 cells are desert topminnow (Poeciliopsis lucida)
hepatoma cells that have been shown to have inducible
130
VILLENEUVE ET AL.
TABLE I. Maximum concentrations of individual PAHs tested in PLHC-1, H4IIE-luc, and MVLN in vitro bioassays,
greatest magnitude of induction relative to a 2,3,7,8-tetrachlordibenzo-p-dioxin (TCDD) standard, and relative
potency (REP) estimatesa
PLHC-1 EROD
PAH Compounds
Acenapthene
Acenapthylene
Antracene
Benzo[a]anthracene
Benzo[a]pyrene
Benzo[b]fluoranthene
Benzo[g,h,i]perylene
Benzo[k]fluoranthene
Chrysene
Dibenz[a,h]anthracene
Fluoranthene
Fluorene
Indeno[1,2,3-cd]pyrene
Napthalene
Phenanthrene
Pyrene
9-Methyl anthracene
9,10-Dimethyl
anthracene
3,9-Dimethyl
benzo[a]anthracene
1-Methyl naphthalene
1,2-Dimethyl
naphthalene
3,6-Dimethyl
phenanthrene
2-Methyl
benzo[c]phenanthrene
6-Hydroxy chrysene
1-Hydroxy pyrene
Max. Conc.
(ppb in well)
b
% max
4000
4000
4000
4000
4000
4000
4000
4000
4000
4000
4000
4000
4000
4000
4000
4000
100
1
0
2
23
9
86
1
70
62
110
1
1
36
1
2
2
0
100
REP20–80
c
H4IIE-luc
d
REP
e
% max
REP20–80
REP
NA
NA
NA
6.0 ⫻ 10⫺7
4.2 ⫻ 10⫺9
7.3 ⫻ 10⫺5
NA
4.9 ⫻ 10⫺4
2.6 ⫻ 10⫺5
3.8 ⫻ 10⫺3
NA
NA
4.1 ⫻ 10⫺5
NA
NA
NA
NA
0
0
0
60
54
54
2
92
62
83
0
0
72
0
8
0
0
1
NA
0
100
100
0
0
NA
NA
12
0
100
0
NA
0
NA
100
25
3.7 ⫻ 10⫺4–1.8 ⫻ 10⫺6
2.6 ⫻ 10⫺5
0
NA
100
200
200
2
32
6
2.3 ⫻ 10⫺4–3.8 ⫻ 10⫺6
NA
2.9 ⫻ 10⫺5
NA
0
0
0
NA
NA
NA
1.2 ⫻ 10⫺5–2.9 ⫻ 10⫺8
3.8 ⫻ 10⫺6–4.6 ⫻ 10⫺12
1.2 ⫻ 10⫺4–4.5 ⫻ 10⫺5
1.1 ⫻ 10⫺3–2.2 ⫻ 10⫺4
7.5 ⫻ 10⫺5–8.8 ⫻ 10⫺6
5.1 ⫻ 10⫺3–2.8 ⫻ 10⫺3
1.8 ⫻ 10⫺4–9.2 ⫻ 10⫺6
2.2 ⫻ 10⫺6–1.7 ⫻ 10⫺6
2.4 ⫻ 10⫺6–1.1 ⫻ 10⫺6
1.6 ⫻ 10⫺5–1.6 ⫻ 10⫺6
3.9 ⫻ 10⫺4–5.1 ⫻ 10⫺5
4.6 ⫻ 10⫺6–1.2 ⫻ 10⫺6
4.4 ⫻ 10⫺5–4.8 ⫻ 10⫺7
3.4 ⫻ 10⫺5–6.4 ⫻ 10⫺6
NA
NA
NA
1.9 ⫻ 10⫺6
1.6 ⫻ 10⫺6
5.1 ⫻ 10⫺6
NA
1.4 ⫻ 10⫺4
2.3 ⫻ 10⫺6
4.6 ⫻ 10⫺6
NA
NA
1.5 ⫻ 10⫺5
NA
NA
NA
NA
NA
6.0 ⫻ 10⫺7–8.7 ⫻ 10⫺14
2.3 ⫻ 10⫺10
NA
a
Only two PAHs (benzo[a]anthracene and dibenz[ah]anthracene) induced responses in the MVLN bioassay; thus, MVLN REPs are not included here.
% max ⫽ 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.
c
REPs are reported as the range of REP estimates generated from multiple point estimates over a response range from 20% to 80% TCDD max
(REP20 – 80 range).
d
Single point estimate of REP made for a response of 50% TCDD max (EC-50).
e
NA: not able to calculate REP; dose–response relationship insufficient for estimate.
b
cytochrome P4501A1 activity (Hightower and Renfro, 1988;
Hahn et al., 1993, 1996). H4IIE-luc cells are rat hepatoma cells
that have been stably transfected with a luciferase reporter gene
under the control of dioxin-responsive enhancers (DREs)
(Sanderson et al., 1996). MVLN cells are MCF-7 human breast
carcinoma cells stably transfected with a luciferase reporter
gene under the control of estrogen responsive elements (EREs)
of the Xenopus vitellogenin A2 gene (Pons et al., 1990; Demirpence et al., 1993). The culturing conditions for all three cell
lines have been described previously (Khim et al., 1999; Villeneuve et al., 2001). In vitro EROD assay with PLHC-1 cells
and in vitro luciferase assay with H4IIE-luc and MVLN cells
have also been described (Khim et al., 1999; Villeneuve et al.,
2001). Briefly, cells were trypsinized from Petri plates containing 80%–100% confluent monolayers and resuspended in
media at the density desired for seeding. Cells were seeded into
96 well microplates, allowed to attach overnight, and then
dosed. Test and control wells received 2.5 ␮L of the appropriate sample or solvent. Blank wells received no dose. Dose
responses consisted of six concentrations prepared by threefold
serial dilution. All samples were tested in triplicate, and a
minimum of three blank and three solvent control wells were
run on each 96-well test plate. Cells were exposed for 72 h
(except for time-course experiments), and then in vitro EROD
or luciferase assays were run as described elsewhere (Khim et
al., 1999; Villeneuve et al., 2001).
Time-Course Experiments
A mixture of 16 PAHs (PLHC-1 and H4IIE-luc assays) or
BAA (MVLN assay) was tested after four different expo-
RELATIVE POTENCIES OF INDIVIDUAL PAHS
sure durations. Cells were seeded into 96-well plates at the
normal density used for a 72-h exposure. Four plates were
seeded for each sample (for each of the three assays). One
of the plates was dosed the morning after seeding (72-h
exposure), a second 24 h later (48 h), a third 48 h later (24
h), and the final plate 60 h later (12 h). Bioassays were
conducted exactly 72 h after the first dosing event. This
dosing approach was employed for both the PAH samples
and the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and
17␤-estradiol (E2) standards. This approach was chosen
because it assured a uniform stock of cells, uniform incubation duration, and uniform reagent and instrument conditions for the assays while allowing for variable durations of
exposure to the test compound. Response magnitudes, expressed as % TCDD max or % E2 max, and REP estimates
calculated from the time-course experimental results were
based on comparison to a TCDD or E2 standard exposed for
the same duration.
131
the response level selected (Putzrath, 1997). The minimum
and maximum REPi values generated (a relative potency, or
REP20 – 80, range) 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., 2000). In cases where the observed maximum response for the sample was less than 80% TCDD/E2
max, extrapolation beyond the range of the empirical results
was used to estimate REPi at Yi greater than the observed
maximum. This was done in order to make the REP20 – 80
ranges comparable from sample to sample, because the
magnitude of the range is dependent on the range of responses over which it is calculated (Villeneuve et al., 2000).
REP20 – 80 ranges were reported along with a conventional
REP based on a single point estimate determined at 50%
TCDD/E2 max. in order to provide an indication of the
uncertainty of the estimate (Villeneuve et al., 2000).
RESULTS
Bioassay Data Analysis
Sample responses expressed as mean relative luminescence
units (RLU) or mean pmol min⫺1 mg⫺1 (three replicate
wells) were converted to a percentage of the mean maximum response observed for standard curves generated on
the same day (% TCDD max or % E2 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 the TCDD or E2
standard responses prior to conversion to a percentage in
order to scale the values from 0% to 100% TCDD/E2 max.
In cases in which the magnitude of induction was sufficient to allow a quantitative estimate, assay-specific REPs
were calculated. The linear portion of each dose response
(% TCDD/E2 max, plotted as a function of log dose) was
defined by dropping points from the tails until an R2 ⱖ 0.95
was obtained and a linear regression model was fit to the
remaining points. At least 3 points were used in all cases.
The linear regression equations for the samples and the
corresponding TCDD or E2 standard were used to estimate
the concentration associated with responses, expressed as %
TCDD/E2 max.
For point estimates of relative potency to be valid, the
sample and the standard dose response must be statistically
parallel and have the same maximum achievable response
(Finney, 1978; Putzrath, 1997). These conditions were
tested empirically. The efficacy of many of the samples was
either unknown or less than that of either the TCDD or E2.
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., 2000). For
parallel dose responses, REP estimates are independent of
Relative Potency of Individual PAHs
At the concentrations tested, seven of the 16 priority PAHs
tested elicited significant dioxinlike responses in both the
PLHC-1 EROD and H4IIE-luciferase assays (Table I). All
active PAHs contained at least four rings. DBA yielded both
the greatest magnitude of response and the most potent
response (an REP of approximately 10⫺3) in the PLHC-1
bioassay (Table I). DBA also yielded the greatest magnitude
of response in the H4IIE-luc assay but had an REP approximately 1000 times less than that determined in the PLHC-1
assay (Table I). BKF, with an REP around 10⫺4, was the
most potent PAH tested in the H4IIE-luc bioassay. BKF was
similarly potent in the PLHC-1 assay (Table I). Other priority PAHs that exhibited dioxinlike activity included BAA,
BAP, BBF, CHR, and IP (Table I).
Among the seven methylated and two hydroxylated PAH
compounds tested, three compounds induced a significant
dioxinlike response (Table I). These were 3,9-dimethylbenzo[a]anthracene (DMBA), 3,6-dimethylphenanthrene, and
6-hydroxychrysene. At the concentrations tested, none of
the three compounds induced significant responses in both
the PLHC-1 and the H4IIE-luc bioassay. 3,6-Dimethylphenanthrene and 6-hydroxychrysene were both active in
the PLHC-1 bioassay and exhibited relative potencies similar to those derived for parent PAH compounds (REP ⯝
10⫺5). DMBA induced a significant response in the H4IIEluc bioassay, but the relatively low magnitude of response
observed resulted in great uncertainty in the REP estimate.
Based on the dose responses obtained, the approximate
H4IIE-luc-derived REP for DMBA was 10⫺10 (Table I).
Relatively few of the individual PAHs and substituted
PAHs tested induced a significant estrogenic response in the
MVLN bioassay. Among the 16 priority PAHs tested, only
132
VILLENEUVE ET AL.
acid treatment reduced the activity to a level not significantly different from that induced by the solvent control
(Fig. 1). Washing of acid-treated extracts appeared to further decrease the dioxinlike activity of the PAH mixture
(Fig. 1). GC/MS analysis of acid-treated mixtures of 16
priority PAHs indicated that a 1-h acid treatment destroyed
more than 99% of the 16 parent PAHs. The 10-h acid
treatment destroyed 100% of the parent PAHs.
Effect of Exposure Duration on PAH Relative
Potencies
Fig. 1. Luciferase induction in the H4IIE-luc cell bioassay
elicited by acid-treated and nontreated mixtures of 16 priority PAHs. Response magnitude presented as a percentage
of the maximum response observed for a 3130 pM TCDD
standard (% TCDD max). Washed ⫽ the extract was rinsed
with nanopure water prior to bioassay; no wash ⫽ the extract was not rinsed prior to bioassay; error bars ⫽ standard
deviation (SD); sig. ⫽ magnitude of response equal to 3 SD
above the mean solvent control response (0% TCDD max).
BAA and DBA induced a significant response. The maximum magnitudes of response observed were 24% and 25%
E2 max for BAA and DBA, respectively. Both compounds
were estimated to be more than a million times less potent
than E2 (REP50 ⫽ 5.7 ⫻ 10⫺7 for BAA, REP50 ⫽ 8.8 ⫻
10⫺7 for DBA). At the concentrations tested, none of the
methylated or hydroxylated PAHs induced a significant
response in the MVLN bioassay.
Most REP estimates generated for the priority PAHs had
uncertainty ranges that varied by less than a factor of 10
(Table I). Notable exceptions were PLHC-1-derived REP
estimates for BAA and BAP, which had uncertainty ranges
in excess of 100-fold because of the relatively low magnitudes of induction observed. The PLHC-1-derived REP for
IP and H4IIE-luc-derived REP both had uncertainties
greater than 10-fold but less than 100-fold over the EC-20 –
EC-80 response range (Table I). The same was true for
PLHC-1-derived REP estimates for 3,6-dimethylphenanthrene and 6-hydroxychrysene (Table I). The REP estimate
derived for DMBA had the greatest uncertainty (more than
a million- fold). Uncertainty ranges for MVLN-derived
REP estimates were both less than eightfold.
Dioxinlike Activity of Acid-Treated PAHs
Acid treatment was shown to reduce the dioxinlike activity
of a mixture of 16 priority PAHs in a time-dependent
fashion (Fig. 1). An hour of acid treatment reduced the
dioxinlike activity of the PAH mixture to a level approximately 50% of the nontreated activity (Fig. 1). The 10-h
The relative estrogenic potency of BAA and the dioxinlike
potency of a mixture of 16 priority PAHs decreased with
increased duration of in vitro exposure (Fig. 2). After a 12-h
exposure, BAA had produced a maximal response that was
130% of that obtained for a 1000-pM E2 standard (Fig. 2)
and was estimated to have a MVLN-derived REP of 1.4 ⫻
10⫺5 (Fig. 2). After a 72-h exposure, the maximal response
(relative to E2 max) was less than 10% of that observed
after 12 h (Fig. 2), and an REP estimate could not be
derived. The maximal H4IIE-luc-derived REP for the mixture of 16 PAHs was observed after a 24-h exposure (Fig.
2). Longer exposure durations yielded dose-response relationships exhibiting similar efficacy (relative to TCDD) but
reduced relative potency, as indicated by a shift of the
dose-response curves to the right along the x-axis (Fig. 2).
In the PLHC-1 assay the 16-PAH mixture yielded dose
responses with an inverted U shape (Fig. 2). Similar results
have been observed in previous studies and may be a result
of substrate inhibition (Hahn et al., 1993). As observed for
the H4IIE-luc assay, the dose-response curves were progressively shifted to the right as exposure duration increased
(Fig. 2), indicating reduced potency relative to a TCDD
standard. Thus, for at least some PAHs, in vitro REP appeared to decrease with increased exposure duration.
DISCUSSION
Relative Potency Estimates
Dioxinlike REPs were derived for 7 of the 16 priority PAHs
examined in this study. The range of uncertainty over a
range of responses from EC-20 to EC-80 was relatively
small for most estimates, indicating reasonable conformity
to the parallel slopes assumption inherent in REP estimation
(Putzrath, 1997; Finney, 1978; Villeneuve et al., 2000).
Thus, most of the estimates reported should be suitable for
use in assay-specific mass-balance analyses. Several of the
estimates, including the PLHC-1-derived estimates for
BAA and IP and the H4IIE-luc-derived estimate for DBA
had greater than 10-fold uncertainty ranges. This was also
the case for PLHC-1-derived REPs for 3,6-dimethylphenanthrene and 6-hydroxychrysene. Such estimates may be use-
RELATIVE POTENCIES OF INDIVIDUAL PAHS
133
ful for assay-specific mass-balance analysis but should be
applied with careful consideration of the limitations and
uncertainties associated with the estimates. Most of the
more uncertain REP estimates (excluding the H4IIE-derived
REP for DBA) were derived from dose-response relationships exhibiting efficacy of less than 40% TCDD max.
Thus, the REP20 estimates should be considered the most
reliable estimate of relative potency. Mass-balance assessments incorporating these values may be more accurate if
based on EC-20 (or sub-EC-50) responses. The PLHC-1derived REP for BAP and the H4IIE-luc-derived REP estimate for DMBA were both highly uncertain and should
probably be recharacterized before being used extensively
for mass-balance analysis. Both MVLN-derived REPs
should be useful for mass-balance applications.
The dioxinlike potency of individual PAHs was similar
to the potencies reported for a range of other AhR-active
environmental contaminants. Most of the PAHs examined
in this study had REPs in the range of 10⫺4 to 10⫺6 for
inducing dioxinlike responses in the PLHC-1 and H4IIE-luc
bioassays. This was similar to the range of H4IIE-derived
REPs reported for pentachlorinated naphthalenes (CNs) and
slightly less than that reported for hexa-CNs (Villeneuve et
al., 2000b). The REPs determined for individual PAHs were
generally greater than the H4IIE-derived REPs reported for
complex mixtures of polychlorinated biphenyls (PCBs) and
polychlorinated naphthalenes (PCNs; Villeneuve et al.,
2001). REPs for individual PAHs were also similar to those
for coplanar PCBs. The two estrogenic PAHs characterized
in this study had estrogenic potencies similar to those reported for other environmental xenoestrogens of concern,
including nonylphenol, octylphenol, and bisphenol A
(Villeneuve et al., 1998). This relative order of potency,
along with the wide distribution of PAH contamination in
the environment, suggests that certain PAHs should be
considered in assessments of total TCDD equivalents
(TEQs) or estrogen equivalents (EEQs) present in environmental samples.
Acid Treatment
Fig. 2. Response of MVLN bioassay to benzo[a]anthracene
and H4IIE-luc and PLHC-1 bioassays to a mixture of 16
priority PAHs after 4 exposure durations (12, 24, 48, and
72 h). Relative potency (REP) estimates calculated at the
EC-50 response level are presented in parentheses. Response magnitudes, expressed as % TCDD max or % E2
max, and REP estimates were based on comparison to a
TCDD or a 17␤-estradiol standard exposed for the same
duration. Sig. ⫽ magnitude of response equal to 3 SD above
the mean solvent control response.
Past bioassay-directed fraction/toxicant identification studies have used acid treatment to separate the effects of
acid-labile compounds such as PAHs from those of acidstable compounds such as polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans (Khim et al., 1999b).
These assessments relied on the assumptions that a 1-h
treatment with concentrated H2SO4 would breakdown priority PAHs and that the resulting acid-breakdown products
would not produce dioxinlike in vitro bioassay responses.
Results of this study indicate that significant PAH breakdown (approximately 99%) can be achieved with a 1-h acid
treatment, but that longer treatment duration may be required to completely eliminate dioxinlike responses (Fig. 2).
134
VILLENEUVE ET AL.
It was not clear whether the bioassay responses observed
after 1 h of acid treatment were a result of the continued
presence of small amounts of relatively potent parent PAHs
or of the production of acid-breakdown products capable of
eliciting dioxinlike responses. Either way, the 10-h acid
treatment appeared to destroy all AhR-active PAHs and/or
PAH derivatives. The duration of treatment needed to completely eliminate PAH contributions to dioxinlike in vitro
responses may be modulated by the total concentration of
PAHs present, the relative acid stabilities of the PAHs or
PAH derivatives present, and the relative potencies of the
more stable compounds. Rinsing acid-treated extracts with
nanopure water appeared to further reduce carryover of
active PAHs into the acid-treated product. Thus, results of
this study suggest that 10 h or longer of acid treatment
followed by a water rinse should serve as an effective
method for separating the effects of priority PAHs from
those of other acid-stable compounds.
Effect of Exposure Duration on PAH Relative
Potencies
In the time-course experiments there was a general trend
toward decreased relative potency with increased duration
of exposure. This could be explained by either in vitro
degradation of PAHs (greater exposure duration allowing
for greater degradation) or increased potency of the E2 or
TCDD standard relative to PAH potency over greater exposure duration. In the MVLN bioassay BAA efficacy and
potency (in absolute units, i.e., RLU) decreased with increased exposure duration, whereas the efficacy and potency
of E2 was greater after 48 and 72 h than after 12 and 24 h.
This suggests that for the MVLN bioassay, both BAA
degradation and a relative increase in E2 potency may have
contributed to the general decline in the relative estrogenic
potency of BAA with increased exposure duration. In the
H4IIE-luc bioassay the efficacy and potency (in absolute
units, i.e., RLU) of both the TCDD standard and the PAH
mixture increased with exposure duration. This suggests
that PAH degradation was probably not a significant contributor to the general decline in the H4IIE-luc-derived REP
over time. In the PLHC-1 assay TCDD efficacy and potency
(in absolute units, i.e., pmol min⫺1 mg⫺1) increased steadily
with exposure duration, whereas those of the PAH mixture
remained approximately stable from 12 to 48 h and then
declined significantly after 72 h. This result suggests that the
increased potency of TCDD was the primary reason for the
observed decrease in REP observed for the 12– 48 h
PLHC-1 exposures. The significant decline after 72 h was
most likely caused by inactivation of some of the PAH
mixture after 72 h of exposure. Overall, the results of the
time-course experiments conducted as part of this study
indicated that in vitro bioassay-derived relative potency and
efficacy estimates can vary significantly as a function of the
exposure duration employed. This potential for variability
supports the contention that mass-balance analyses should
be based on assay-specific REPs derived using the same
exposure duration as that used for the sample analysis.
Comparison of PAH REPs
For the priority PAHs, the list of compounds that have been
found to express AhR-mediated effects is similar among
studies (Table II). BAA, BAP, BBF, BKF, and CHR have
been universally shown to induce dioxinlike responses in a
variety of in vitro bioassays (Table II). Furthermore, most
studies have reported that DBA and IP are AhR-active in
vitro (Table II). The concentrations tested by Villeneuve et
al. (1998) were 8 times less than those used in this study.
The REP for anthracene has been reported by Clemons et al.
(1998) to be approximately 10⫺4. This was the only PAH
for which there was not strong consensus in the literature.
Comparison of the in vitro REPs currently available suggests that anthracene should probably not be included in risk
assessments or mass-balance analyses aimed at estimating
the total dioxinlike potency contributed by PAHs.
This observed consensus among in vitro studies generally holds for REP estimates as well. None of the studies
examined predicted the same rank order of potency for the
AhR-active PAHs listed in Table II. A general blocking of
certain compounds was observed, however. BKF and DBA
were ranked among the three most potent PAHs by seven of
the eight studies compared (Table II). CHR and BAA were
ranked among the three least potent PAHs in all but the hepa
cell bioassay (Table II). Variation in REP estimates was
generally less than 2 orders of magnitude (Table II). If only
fish cell line– derived REP values are compared, most estimates vary by 1 order of magnitude or less for each compound (Table II). Among the fish cell bioassays, the PLHC1-derived REP estimates generated for BAA and BAP in
this study appeared to be the only significant outliers (Table
II). Studies by Bols et al. (1999) and Fent and Bätscher
(2000) used 24-h exposure durations, whereas the study
reported here used an exposure of 72 h. Thus, the lesser
REP observed for BAA and BAP may be associated with
the longer exposure time. REPs of 72 h estimated in a
previous study by Villeneuve et al. (1998) were similar to
the Fent and Bols estimates, however. Furthermore, the
H4IIE-luc-derived estimates generated in this study were
similar to other mammalian-cell REPs reported for BAA
and BAP. Thus, the explanation for the relatively low potency of BAA and BAP in the PLHC-1 assay employed in
this study remains unknown. For mammalian-cell lines the
poorest agreement among studies was observed for BKF,
CHR, and DBA. Estimates of REP for these compounds
varied over 4 orders of magnitude (Table II). In the absence
of a rationale for weighting any one study greater than
others, additional determinations using other mammalian-
RELATIVE POTENCIES OF INDIVIDUAL PAHS
135
TABLE II. Comparison of relative potency (REP) values reported for individual PAHs. REPs shown were calculated
relative to a TCDD standard [only EPA priority PAHs (EPA method 8310) that had caused significant induction in at
least one study are included]
PAH
Anthracene
Benzo[a]anthracene
Benzo[a]pyrene
Benzo[b]fluoranthene
Benzo[k]fluoranthene
Chrysene
Dibenz[a,h]anthracene
Indeno[1,2,3-c,d]pyrene
PLHC-1a
PLHC-1b
PLHC-1c
RTL-W1d
H4IIE-luca
H4IIEe
Humanf
Hepag
NI
4.2 ⫻ 10⫺7
3.3 ⫻ 10⫺9
5.7 ⫻ 10⫺5
3.8 ⫻ 10⫺4
1.8 ⫻ 10⫺5
3.3 ⫻ 10⫺3
3.5 ⫻ 10⫺5
NI
1.7 ⫻ 10⫺5
1.65 ⫻ 10⫺4
NA
9.9 ⫻ 10⫺4
9.6 ⫻ 10⫺5
3.3 ⫻ 10⫺3
NA
NI
7.1 ⫻ 10⫺5
4.2 ⫻ 10⫺4
3.0 ⫻ 10⫺4
7.5 ⫻ 10⫺4
1.1 ⫻ 10⫺4
NI
NI
NI
4.3 ⫻ 10⫺5
3.0 ⫻ 10⫺4
1.9 ⫻ 10⫺4
1.0 ⫻ 10⫺3
4.7 ⫻ 10⫺5
3.5 ⫻ 10⫺4
2.8 ⫻ 10⫺4
NI
1.4 ⫻ 10⫺6
1.3 ⫻ 10⫺6
4.0 ⫻ 10⫺6
1.1 ⫻ 10⫺4
1.6 ⫻ 10⫺6
4.0 ⫻ 10⫺6
1.3 ⫻ 10⫺5
NA
2.5 ⫻ 10⫺5
3.5 ⫻ 10⫺4
2.5 ⫻ 10⫺5
4.8 ⫻ 10⫺3
2.0 ⫻ 10⫺4
2.0 ⫻ 10⫺3
1.1 ⫻ 10⫺3
NI
3 ⫻ 10⫺6
8 ⫻ 10⫺6
2 ⫻ 10⫺6
2 ⫻ 10⫺4
3 ⫻ 10⫺6
4 ⫻ 10⫺5
2 ⫻ 10⫺5
1 ⫻ 10⫺4
1 ⫻ 10⫺5
1 ⫻ 10⫺5
NA
5 ⫻ 10⫺2
1 ⫻ 10⫺2
5 ⫻ 10⫺2
NA
NA ⫽ not analyzed.
NI ⫽ no significant induction at the concentrations tested in the study.
All REPs shown here were calculated on a molar concentration basis and were based on the EC-50s estimated for the PAH and for the standard
compound.
a
This study.
b
Fent and Bätscher, 2000. Originally reported as induction equivalency factors (IEF) relative to dibenzo[ah]anthracene. For comparability values were
multiplied by 3.3 ⫻ 10⫺3 (the PLHC-1 REP relative to the TCDD generated in this study). Values should be considered approximate.
c
Villeneuve et al., 1998.
d
Bols et al., 1999; Rainbow trout liver cell line.
e
Willet et al., 1997.
f
Jones and Anderson, 1999.
g
Clemons et al., 1998.
cell bioassays or similar assays in other laboratories will be
needed to develop consensus values for the mammalian-cell
REP of BKF, CHR, and DBA. Overall, however, the PAH
REPs currently available in the literature provide a reasonable data set for the formulation of consensus values for the
dioxinlike REP of priority PAHs.
To date, only two studies, that of Clemons et al., (1998)
and this study, have reported the relative estrogenic potency
of priority PAHs. BAA was the only priority PAH that
produced an estrogenic response in both studies. REP estimates for BAA were 5.7 ⫻ 10⫺7 and 1.0 ⫻ 10⫺4 in this
study and in the Clemons et al. (1998) study, respectively.
DBA (this study), CHR, and BAP (Clemons et al., 1998)
induced a significant estrogenic response in one of the
studies, but not both. Additional studies will be needed to
develop consensus values for the in vitro estrogenic potency
of PAHs.
and to evaluate whether use of assay-specific REPs was
necessary. Variability in excess of 300-fold was observed
(Table III; i.e., H4IIE-luc-derived values in this study vs.
those found in Clemons et al., 1998). Among similar mammalian cell bioassays (i.e., H4IIE-EROD-Willett and
H4IIE-luc-Villeneuve), TEQ estimates differed by nearly
100-fold (Table III). Among two fish cell bioassays (RTLW1-Bols and PLHC-1-Villeneuve) an approximately fourfold difference in TEQ estimates was observed (Table III).
Results of this analysis support the conclusion that use of
nonspecific REPs for mass-balance analysis could lead to
significant error and inaccurate conclusions. Assay-specific
REPs should be employed for mass-balance assessments,
and wherever possible, uncertainties in the REP estimates
themselves should be considered.
CONCLUSIONS
REP Variability and Mass-Balance
Implications
The rationale for this study was predicated on the assumption that assay-specific REPs should be used for massbalance (potency-balance) analyses involving PAHs. Thus,
assay-specific REPs generated in this study, as well as those
reported in three other studies (Willett et al., 1997, Clemons
et al., 1998, and Bols et al., 1999) were used to calculate
total TEQs for some model environmental samples containing complex mixtures of PAHs (Table III). This was done to
determine the magnitude of impact that the variability in
REP estimates would have on typical TEQ determinations
Several priority PAHs and hydroxylated or methylated PAH
derivatives induced dioxinlike responses in the PLHC-1,
H4IIE-luc in vitro bioassays, and/or estrogenic effects in the
MVLN bioassay. Relative potencies of AhR-active and
ER-active PAHs were similar to those reported for other
xenobiotics of concern. REP values presented here can be
employed for assay-specific mass-balance (potency-balance) analysis and provide greater accuracy than could be
achieved through the use of nonspecific literature values. A
10-h acid treatment followed by a nanopure water rinse was
successful in degrading dioxinlike PAHs to non-AhR active
products and should be a useful procedure for bioassay-
136
VILLENEUVE ET AL.
TABLE III. Total 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) equivalents (TEQs) contributed by PAHs calculated for
selected sections of a sediment core from Tokyo Bay, Japan,a using five sets of published relative potencies
(REPs) for PAHs (relative to TCDD)
Section (cm)
4–6
14–16
25–30
45–50
65–70
85–90
mean
Clemonsb
Hepa 1c1c7
Willettc
H4IIE-wt
Bolsd
RTL-W1
Villeneuvee
PLHC-1
Villeneuvef
H4IIE-luc
3217
6220
3557
2375
749.3
181.8
2716
729.2
1650
1158
830.1
206.1
29.90
767.2
125.2
292.2
223.5
165.9
38.66
4.079
141.6
32.25
69.74
47.65
34.07
8.968
1.418
32.35
7.935
17.63
13.30
9.831
2.460
0.3076
8.577
Unit ⫽ pg TEQ/g sediment (dry wt).
a
Yamashita et al., 2000.
b
Clemons et al., 1998; luciferase induction in transiently transfected Hepa 1c1c7 cells.
c
Willett et al., 1997; EROD induction in wild-type H4IIE rat hepatoma cells.
d
Bols et al., 1999; EROD induction in wild-type RTL-W1 rainbow trout hepatoma cells.
e
This study; EROD induction in wild-type PLHC-1 Poeciliopsis lucida hepatoma cells.
f
This study; luciferase induction in stably transfected H4IIE-luc rat hepatoma cells.
directed fractionation studies examining the relative contribution of PAHs and other acid-labile compounds to the total
dioxinlike potency of environmental extracts. Overall, this
research has contributed to a growing consensus regarding
the dioxinlike potency of priority PAHs and PAH derivatives and has provided some additional evidence of potentially estrogenic PAHs.
This work was supported by U.S. Environmental Protection
Agency (U.S. EPA) Biology Exploratory Grants Program, grant
no. R85371-01-0; cooperative agreement CR 822983-01-0 between Michigan State University and the U.S. EPA; and the
National Institute for Environmental Health Sciences Superfund
Basic Research Program NIH-ES-04911. We thank Emily Nitsch
for her technical assistance. We also acknowledge the support of
colleagues from Michigan State University’s Aquatic Toxicology
Laboratory.
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