Research
Gas-Phase Ambient Air Contaminants Exhibit Significant Dioxin-like andEstrogen-like Activity in Vitro
Gail P. Klein,1 Erin M. Hodge,2 Miriam L. Diamond,2 Amelia Yip,1 Tom Dann,3 Gary Stern,4 Michael S. Denison,5and Patricia A. Harper 1,6
1Department of Pharmacology, and 2Department of Geography, Centre for Urban Health Initiatives, University of Toronto, Toronto,

Ontario, Canada; 3Analysis and Air Quality Division, Environment Canada, Ottawa, Ontario, Canada; 4Freshwater Institute, Universityof Manitoba, Winnipeg, Manitoba, Canada; 5Department of Environmental Toxicology, University of California, Davis, California, USA;
6Program in Developmental Biology, Hospital for Sick Children, Toronto, Ontario, Canada

Several adverse health effects, such as respiratory and cardiovascular morbidity, have been linked
to exposure to particulate matter in ambient air; however, the biologic activity of gas-phase ambient organic air contaminants has not been examined as thoroughly. Using aryl hydrocarbon receptor (AHR)–based and estrogen receptor (ER)–based cell bioassay systems, we assessed thedioxin-like and estrogenic activities of gas-phase organic ambient air contaminants compared withthose of particulate-phase contaminants using samples collected between seasons over 2 years froman urban and a rural location in the Greater Toronto Area, Canada. The concentration of the sum(Σ) of polycyclic aromatic hydrocarbons, which was highest in the gas phase, was 10–100 times
more abundant than that of Σpolychlorinated biphenyls, Σnitro-polycyclic aromatic hydrocarbons,and Σorganochlorine pesticides, and 103 to 104 times more abundant than Σpolychlorinateddibenzo-p-dioxins/dibenzofurans. Gas-phase samples induced significant AHR- and ER-dependent
gene expression. The activity of the gas-phase samples was greater than that of the particulatephase samples in the estrogen assay and, in one case, in the AHR assay. We found no strong associations between either summer or winter seasons or urban or rural locations in the relative
efficacy of the extracts in either the ER or AHR assay despite differences in chemical composition,
concentrations, and abundance. Our results suggest that mechanistic studies of the health effectsof ambient air must consider gas and particulate phases because chemicals present in both phases
can affect AHR and ER signaling pathways. Key words: AHR, air pollution, CALUX, complex
mixtures, endocrine disruption, ER, PAH. Environ Health Perspect 114:697–703 (2006).
doi:10.1289/ehp.8496 available via http://dx.doi.org/ [Online 29 December 2005]

Adverse health effects in individuals of all ages
have been linked to exposure to ambient air
contaminants (American Academy of
Pediatrics 2004; Jerrett et al. 2005; Pope et al.
2004a). Cardiovascular and respiratory morbidity and mortality are the most common
health effects associated with exposure toambient air contaminants, but other effects
that have been linked epidemiologically
include adverse fetal development (Dejmek
et al. 2000; Liu et al. 2003; Perera et al. 2003)
and cancer (Boffetta 2004; Cordier et al.
2004; Vineis et al. 2004). The actual mechanism by which these effects may be produced
is frequently unclear, although a substantial
body of work has elucidated the relationshipbetween airborne particulate matter (PM) andvasoconstriction (Barclay et al. 2005; Brook
et al. 2004; Pope et al. 2004b).
The possibility that ambient air contaminants may be involved in endocrine disruption
is an intriguing question (Rogan and Ragan
2003). Several studies have demonstrated theability of organic contaminants found in ambient air to activate intracellular receptors thatare the entry points to signaling pathways
implicated in endocrine disruption (Clemons
et al. 1998; Vinggaard et al. 2000; Vondracek
et al. 2004). In addition to epidemiologic studies that have demonstrated an association

between exposure to ambient air contaminants
and endocrine-disruptive effects (Jedrychowski
et al. 2004; Perera et al. 2003, 2004), in vivo
studies have shown reproductive effects inrodents exposed via inhalation to diesel
exhaust (Furuta et al. 2004; Takeda et al.
2004; Watanabe and Kurita 2001).
Most toxicologic research examining
ambient organic air contaminants has focusedon contaminants sorbed to PM, yet the specific role that these organic contaminants may
play in the biologic effects of ambient air contaminants remains unknown. A large fractionof many airborne chemical classes is present inpart or almost exclusively in the gas phase,
which has received little if any characterization
for biologic activity.
In addition to the substantial challenge ofdetermining the mechanisms underlying healtheffects from exposure to ambient air, it isimportant to recognize that not all of thepotentially biologically active constituents oforganic air contaminants have been isolated
and identified. Urban air contains a complex
mixture of constituents, of which only a small
fraction has been identified (Rogge et al. 1993).
Transformation products are rarely analyzed orconsidered in mass-balance analyses of theactivity of organic extracts of ambient air
contaminants in receptor-activation assays,

Environmental Health Perspectives • VOLUME 114 | NUMBER 5 | May 2006

even though they exhibit biologic activity(Fertuck et al. 2001; Machala et al. 2004;
Wang et al. 2003). Further, exposure to thesecontaminants is variable and depends on suchfactors as the proximity to point or nonpoint
sources, spatial and temporal variation, andatmospheric conditions.
To address the influence of spatial andtemporal factors and to examine the potential
biologic activity of the gas phase of organic
contaminants in ambient air, we subjected
extracts of particulate- and gas-phase ambient
air samples, collected from both urban andrural locations in two seasons (winter and summer) over 2 years, to estrogen receptor (ER)chemically activated luciferase expression(CALUX) and aryl hydrocarbon receptor(AHR) CALUX cell bioassays. The CALUX
assay is a rapid, sensitive, and well-validated
method of assessing the capacity of either mixtures or single compounds to bind and activate
the ER and AHR signaling pathways (Denison
et al. 2004; Houtman et al. 2004; Rogers andDenison 2000). Previous studies have demonstrated that organic extracts of the particulate
phase of ambient air contaminants are capableof activating these pathways in different in vitro
assays, including CALUX (Hamers et al.
2000), but to our knowledge no study has yet
examined the activity of gas-phase extracts.

Materials and Methods
Collection and preparation of air samples.
Urban air samples were collected in downtown
Toronto, Ontario, Canada, and rural air
Address correspondence to P.A. Harper, Program in
Developmental Biology, Hospital for Sick Children,
555 University Ave., Toronto, Ontario, Canada
M5G 1X8. Telephone: (416) 816-5174. Fax: (416)813-5252. E-mail: pharper@sickkids.ca
We thank C. Butt (University of Toronto) andF. Wong (Meteorological Service of Canada).
This research was supported by the Toxic
Substances Research Initiative of Health Canada andEnvironment Canada (M.L.D., T.D., P.A.H, G.S.),
Premier’s Research Excellence Award (M.L.D.),
Canadian Institutes of Health Research (P.A.H.),
U.S. Environmental Protection Agency STAR Grant(RS82867601, M.S.D.), and the National Instituteof Environmental Health Sciences, U.S. National
Institutes of Health (ES04699, M.S.D.).
The authors declare they have no competing
financial interests.
Received 13 July 2005; accepted 29 December 2005.

697

Klein et al.

698

β-naphthoflavone (β-NF)], or various dilutions of the air sample extracts. After 4 hr, thecells were lysed, and luciferase activity assessed
as previously described (Garrison et al. 1996)
using a commercially available luciferase assay(Promega, Madison, WI, USA). A 4-hr exposure was chosen to minimize any effect of cellular metabolism on the chemical compositionof the extract (Windal et al. 2005). Luciferase
activity was expressed as relative light units per
milligram of cell lysate protein.
Treatment of BG1Luc4E2 cells. Cells
were plated at a density of 6 × 104 cells/wellin 12-well plates. After 24 hr, the medium
was changed to estrogen-reduced medium tominimize the basal expression of luciferase.
Estrogen-reduced medium consisted of
20

αMEM without phenol red (Gibco BRL,
Burlington, ON, Canada) and 10% charcoalstripped FBS (Hyclone Inc., Logan, UT,USA). After 48 hr, cells were treated with
17β-estradiol (E 2 )-reduced medium alone(media control), E2-reduced media plus 0.1%
DMSO (solvent control), reference agonist(10–10 M E2), or various dilutions of air sample extracts. At 72 hr, the cells were harvested
and luciferase activity assessed as described(Rogers and Denison 2000).
Statistical analyses. In all cases, protein
concentration was determined by the methodof Bradford (1976). Curve fitting was performed using GraphPad Prism software (version 3.0; GraphPad Software, San Diego, CA,
USA). Principal components analysis (PCA)
0.8

Particle phase
Gas phase

A

B

16

PCBs (ng/m3)

PAHs (ng/m3)

0.6
12

8

0.4

0.2
4

N-PAHs (ng/m3)

0.3

urb
Mar 00urb
Mar 01urb
Jul 00urb
Jul 01rur
Jul 00

1.50

C

0.2

0.1

0

0.5

0

rur
Jul 01

PCDDs/PCDFs (pg/m3)0

urb
Mar 00urb
Mar 01urb
Jul 00urb
Jul 01rur
Jul 00rur
Jul 01urb
Mar 01urb
Jul 00urb
Jul 01rur
Jul 00rur
Jul 01urb
Mar 00urb
Mar 01urb
Jul 00urb
Jul 01rur
Jul 00rur
Jul 01urb
Mar 01urb
Jul 00urb
Jul 01rur
Jul 00rur
Jul 01

D

1.00

0.75

0.25

0

urb
Mar 00E

0.4

OC pesticides (ng/m3)

samples in Egbert, Ontario, Canada, 75-km
northeast of Toronto. The downtown site wason the roof of a three-story building locatedon a heavily traveled street. The rural site was
located at ground level at Environment
Canada’s Centre for Atmospheric Research.
Samples were collected simultaneously at bothsites for each sampling period. There werefour sampling periods: 20–24 March 2000,
6–16 July 2000, 3–13 March 2001, and
31 July to 8 August 2001. Temperatures during the sampling periods were approximately
10°C for the winter (March) and 25°C for thesummer (July–August). High-volume air samplers fitted with polyurethane foam plugs andTeflon filters were used to collect particulateand gas-phase samples of ambient air.
Samplers were run at a flow rate of approximately 1,000 m 3 per 24 hr. Samples werecomposited to represent the equivalent ofapproximately 8,000 m 3 of air taken over
5–10 days (in shorter sampling periods, two
air samplers were deployed simultaneously).
Details of the extraction and cleanup of
the air samples were reported previously byDann (1998). Extracts were split, with 50%
going to bioassays and 25% going to chemical
analysis of polychlorinated dibenzo-p-dioxins/
dibenzofurans (PCDDs/PCDFs) and nitropolycyclic aromatic hydrocarbons (N-PAHs),
and 25% going to chemical analysis of PAHs,
polychlorinated biphenyls (PCBs), and organochlorine (OC) pesticides. PAHs were quantified
by capillary-column gas chromatography/
low-resolution mass spectrometry (GC/MS),
whereas PCBs and OC pesticides were quantified by gas chromatography with electron
capture detection (Harner et al. 2004).
PCDDs/PCDFs and N-PAHs were quantified
by high-resolution GC and high-resolution MS(Dann 1998). Concentrations were corrected
for recoveries only for the PCDDs/PCDFs.
Concentrations were not corrected for blanks
because analyte concentrations in both method
and field blanks were typically < 5% of those in
the test samples.
Reporter cell lines. The AHR reporter cellline H1L6.1c1, a murine hepatoma cell line
(Han et al. 2004), and the ER reporter cellline BG1Luc4E2, a human ovarian carcinoma
cell line (Rogers and Denison 2000), are stably transfected cell lines in which luciferase
expression is regulated by AHR (H1L6.1c1)
or ER (BG1Luc4E2). Cell lines were maintained as described previously by Hodge et al.
(2003) and Rogers and Denison (2000).
Treatment of H1L6.1c1 cells. Cells wereplated at a density of 6 × 10 4 cells/well in
12-well plates. After 24 hr, cells were treatedwith growth medium [α-minimal essential
medium (αMEM) plus 10% fetal bovine
serum (FBS); media control], growth medium
plus 0.1% dimethyl sulfoxide (DMSO;
solvent control), reference agonist [10–7 M

0.3

0.2

0.1
urb
Mar 00Figure 1. Chemical composition of the ambient air extracts from air samples collected March 2000–July
2001. Abbreviations: rur, rural sample; urb, urban sample. ( A ) PAHs. ( B ) PCBs. ( C ) N-PAHs.
(D) PCDDs/PCDFs. (E) OC pesticides. See “Materials and Methods” for details of experiments.

VOLUME 114 | NUMBER 5 | May 2006 •Environmental Health Perspectives

Estrogenic activity of gas-phase air contaminants

was performed using SPSS version 11.0 (SPSS,
Chicago, IL, USA).

Results and Discussion
Chemical analysis. The concentrations of thesum (Σ) of PCDDs/PCDFs (ΣPCDDs/
PCDFs; picograms per cubic meter of air),ΣPAHs (nanograms per cubic meter), ΣPCBs(nanograms per cubic meter), ΣN-PAHs(nanograms per cubic meter) and ΣOC pesticides (nanograms per cubic meter) in each of
the 12 air samples are shown in Figure 1. The
PAHs were the most abundant of all thechemical classes we analyzed and were present
at concentrations 10 3 to 10 4 times greaterthan ΣPCDDs/PCDFs, and 10–100 times
greater than ΣPCBs, ΣN-PAHs, and ΣOCpesticides. These concentrations are similar to
those previously reported for other urban centers of North America and Western Europe(Currado and Harrad 2000; Dimashki et al.
2001; Lohmann et al. 2000).
As expected, concentrations of ΣPAHs,
ΣPCDDs/PCDFs, ΣN-PAHs, and ΣPCBs in
particulate- and gas-phase samples were higher
in urban than in rural locations for July samples because of the geographic concentrationof emission sources in the urban location. The
absence of an urban–rural pattern in total OC
pesticide concentrations may be explained bylong-range transport of “legacy” pesticides
long banned in North America [e.g., DDT(dichlorodiphenyltrichloroethane)] as well as
agricultural activities and atmospheric transport of current-use pesticides such as endosulfan and lindane (Gingrich et al. 2001; Harner
et al. 2004).
Concentrations of ΣPAHs and ΣPCDDs/
PCDFs were similar between March and Julysamples, indicating temporally consistentsources such as mobile and stationary combustion (Lee et al. 2003). ΣOC pesticide concentrations were also similar with season, consistent
with regional and long-range transport (Harner
et al. 2004). Contrary to expectation, ΣPCBs
did not show seasonal differences (Halsall et al.
1995), suggesting that regional atmospherictransport may be the source. ΣN-PAH concentrations were greatest in July, which suggests
increased formation of these chemicals resulting
from photochemical processes (Atkinson andArey 1994).
The compounds we analyzed have awide range of volatilities and varied in their

Table 1. Mean percentage of sample composed of the five most abundant compounds listed in descendingorder.
Compounds
PAHs
PCBs
N-PAHs

PCDDs/PCDFs

OC pesticides

Gas phase

Particulate phase

95% of total: phenanthrene,
fluoranthene, pyrene, fluorene,
anthracene, 2-methyl fluorine
13% of total: PCB congeners
52, 95, 31, 18, and 101
99% of total: 9-nitroanthracene,
2-nitrofluoranthene, 1-nitropyrene,
9-nitrophenanthrene,
7-nitrobenz[a]anthracene
88% of total: 2,3,7,8,-tetraCDF,
octaCDD, 1,2,3,4,6,7,8,-hepta-CDD,
1,2,3,4,6,7,8,-heptaCDF, 2,3,4,7,8,pentaCDF, 1,2,3,4,7,8-hexaCDF
82% of total: endosulfan, γ-HCH,p,p´-DDE, dieldrin, α-HCH

60% of total: benzo[g,h,i]perylene,benzo[b]fluoranthene, fluoranthene,
pyrene, indenol[1,2,3-c,d]pyrene
8% of total: PCB congeners 138,
153, 8/5, 180, 149
100% of total: 9-nitroanthracene,
2-nitrofluoranthene, 1-nitropyrene,
7-nitrobenz[a]anthracene,
6-nitrobenzo[a]pyrene
94% of total: OCDD, 1,2,3,4,6,7,8heptaCDD, 1,2,3,4,6,7,8-heptaCDF,
octaCDF
82% of total: endosulfan, p,p´-DDT,p,p´-DDE, dieldrin, γ-HCH

Abbreviations: CDD, chlorinated dibenzodioxin; CDF, chlorinated dibenzofuran; DDE, dichlorodiphenyldichloroethylene;
HCH, hexachlorcyclohexane.

A

B

1.5

0.5

urb gas Jul 00

0

rur gasJul 00–0.5

urb part
Mar 00urb part
Jul 01

urb gasJul 01rur gasJul 01

urb part
Mar 011.0
urb part
Jul 00

0

PC2

PC2

1.0

2.0
urb gasMar 01
urb gas Mar 00

–1.0

–1.0
–1.5

rur part Jul 01

urb part
urb part Mar 00
Jul 01
urb part
Jul 00

urb gas Mar 01–2.0

rur part Jul 01

rur part
Jul 00

–2.0
–2.5
–1.5

urb part
Mar 01

urb gas Jul 01
urb gas Jul 00
rur gas Jul 00
urb gas Mar 00
rur gas Jul 01

–1.0

–0.5

0.0

0.5

1.0

1.5

2.0

–3.0
–1.0

–0.5

PC1

0.0

0.5

1.0

1.5

PC1

Figure 2. PCA of PAH and PCB chemical data. Abbreviations: part, particulate; rur, rural sample; urb, urban
sample. (A) PAHs; the first two components accounted for 87% of the variance. (B) PCBs; the first twocomponents accounted for 60% of the variance.

Environmental Health Perspectives • VOLUME 114 | NUMBER 5 | May 2006

distribution between gas and particulate
phases. With the exceptions of ΣPCDDs/
PCDFs and March ΣN-PAHs, most compounds were found in gas-phase samples. Not
including the PCBs, the five most abundantcompounds and congeners in the particulateand gas-phase samples accounted for 60–95%
of the total amount of compound in each class(Table 1). Also, there was no overlap between
the most abundant PCBs in the particulate
phase and the most abundant PCBs in the gas
phases, whereas the most abundant compounds for each of the other chemical classes
were similar in both gas and particulate phases.
However, the overall concentration of PAHs
and OC pesticides were 10 times greater in thegas phase than in the particulate phase, and thereverse was true for PCDDs/PCDFs.
We performed PCA to identify major
contributions to variance in the chemicalcomposition of the samples. Data were
autoscaled to unit variance, and the analysis
included only those compounds/congeners
present in both gas and particulate phases. As
shown in Figure 2A, the first two components
accounted for 87% of the variance for PAHs.
Three clusters are evident in the plot for
PAHs: all gas-phase samples in the upper left
quadrant, which separate from urban particulate phase samples along PC1, and rural particulate phase samples along PC2. Within thecluster of gas-phase samples, July versus
March samples separated along PC2. The similarity of urban and rural gas-phase samples
relative to their respective particulate-phasesamples is likely because of the greater transport distance of gas-phase versus particulatephase PAHs.
The concentrations of the gas-phase
PAHs anthracene, acenaphthene, fluorene,and phenanthrene distinguished the gas phasefrom urban and rural particulate phases.
Variance in rural particulate-phase samples
was explained by concentrations of chrysene,
pyrene, and fluoranthene, whereas the variance in urban particulate-phase samples was
explained by triphenylene, benzo[a]fluorene,and benzo[b]fluorene.
Samples in the PCA analysis of the PCB
data clustered in the same pattern as for
PAHs: all gas phase, urban particulate phase,and rural particulate phase (Figure 2B). The
first two components accounted for 60% of
the variance among samples. The ruralparticulate-phase July 2000 sample was
excluded because of the high-number congeners that were below detection. Variance ofthe gas-phase samples was explained by trichlorinated and tetrachlorinated congeners (e.g.,
PCB congeners 16/32, 49, 70/76), whereas
variance in urban particulate-phase samples
was explained by hexachlorinated and heptachlorinated congeners (e.g., PCB congeners
138, 183, 170).

699

Klein et al.

Biologic activity of air samples in cells in
culture. Both gas- and particulate-phaseextracts induced significant activation of ERand AHR-dependent luciferase gene expression. The distinctions in chemical composition

between phase, site, and season were notreflected in the results of the CALUX assays,suggesting the aggregate biologic activity ofthe different congeners in each extract issimilar.

200

200

B

Particle phase
Gas phase

Percent of 10–7 M β-NF

Percent of 10–7 M β-NF

A

100

100

DMSO

0

–3

–1

0

1

–3

Air (m3/well, log10)

200

Percent of 10–7 M β-NF

Percent of 10–7 M β-NF

0

1

100

DMSO
–3

DMSO

0
–2

–1

0

1

–3

Air (m3/well, log10)

–2

–1

0

1

Air (m3/well, log10)

200

200

F
Percent of 10–7 M β-NF

E
Percent of 10–7 M β-NF

–1

D

100

100

0

–2

Air (m3/well, log10)

200

C

0

DMSO

0
–2

100

DMSO
–3

0
–2

–1

0

DMSO

1

–3

Air (m3/well, log10)

–2

–1

0

1

Air (m3/well, log10)

Figure 3. Dose–response curves for AHR-responsive cells treated with varying concentrations of air
extracts (m3 air/well) of gas-phase and particulate-phase ambient air samples. (A) Urban sample, March
2000. (B) Urban sample, March 2001. (C) Urban sample, July 2000. (D) Urban sample, July 2001. (E) Rural
sample, July 2000. (F) Rural sample, July 2001. Activity is expressed as percentage of the response relative
to 10–7 M β-NF. Data represent mean ± SD from three separate experiments. Dose–response curves weregenerated using GraphPad Prism software.
Table 2. Apparent potency of samples in AHR reporter gene assay at different induction levels and maximum induction, in descending order.
Sampleurb part Mar 00
urb part Jul 01
urb part Jul 00
urb gas Mar 00
rur part Jul 01
urb part Mar 01
urb gas Jul 01
rur part Jul 00
urb gas Jul 00
rur gas Jul 00
rur gas Jul 01
urb gas Mar 01

20% of
10–7 M β-NF (m3)Sample50% of
10–7 M β-NF (m3)SampleEfficacy(% of β-NF)

0.01
0.02
0.02
0.03
0.04
0.04
0.06
0.09
0.19
0.41
0.9
1.02

urb part Mar 00
urb part Jul 01
urb part Jul 00
rur part Jul 01
urb part Mar 01
rur Jul part 00
urb gas Jul 01
urb gas Mar 00
rur gas Jul 00
urb gas Jul 00
rur gas Jul 01
urb gas Mar 01

0.04
0.08
0.09
0.14
0.18
0.28
0.58
0.66
5.00
6.32
—a
—a

urb part Mar 00
rur part Jul 00
rur part Jul 01
urb part Jul 00
urb part Jul 01
urb part Mar 01
urb gas Jul 01
urb gas Mar 00
rur gas Jul 00
urb gas Jul 00
urb gas Mar 01
rur gas Jul 01

No plateau
No plateau
129
110
92
87
60
58
58
52
48
33

Abbreviations: part, particulate; rur, rural sample; urb, urban sample.
aSample did not elicit 50% of the activity of 10–7 M β-NF.

700

As described above, PAHs were the mostabundant of the chemical classes analyzed,
present at concentrations approximately three
to four orders of magnitude greater thanthose of the potent AHR agonists PCDDs/
PCDFs and one to two orders of magnitude
greater than concentrations of the PCBs.
These relative amounts are an important factor in the ultimate potency of these extracts.
Less than additive, weak agonist interactions
are observed with mixtures in in vitro AHR
assays, where the concentration of potent agonists is significantly lower than those of more
moderate agonists (Safe 1997–1998). Thus,
the activity of each extract is influenced bythese interactions and reflects the composition of the PAH fraction rather than the
PCDD/F and PCB fractions.
AHR reporter cell line. Figure 3 shows theresults of extracts in the H1L6.1c1 cell line,
grouped by date and site. Results are normalized to protein content in cell extracts andexpressed as a percentage of the response in
the cell line to 10–7 M β-NF, which represented the 95th percentile of the β-NF dose–
response curve and 100% of the response to
10–9 M 2,3,7,8-tetrachlorodibenzo-p-dioxin(data not shown).
All extracts induced luciferase expression
in a concentration-dependent manner. In
most cases, upper plateaus of response wereobserved, allowing for comparison of efficacy
between extracts. In all cases, gas-phaseextracts were less efficacious than the corresponding particulate-phase extracts for thesame sampling dates and locations. If theactivity of the extracts was driven by theconcentration of PAHs, the higher activityof the particulate-phase extracts could be
explained by the relatively higher affinity for
the AHR of PAHs in this fraction, comparedwith those in the gas phase (Table 1)(Piskorska-Pliszczynska et al. 1986; Till et al.
1999). Given the relatively large amounts of
PAHs in the extracts, we expect this class ofcontaminants to be at least partly responsible
for the AHR-dependent inducing activity
observed. Moreover, because of the shortduration (4 hr) of the cells’ exposure to the
mixture, the PAH concentrations would nothave been diminished by metabolism.
Ranking the relative potency of theextracts was challenging. The concentration–
response curves were not parallel, and the
maximum responses of some extracts
exceeded the response to a maximally inducing concentration of 10–7 M β-NF. The reasons for these results are not known; however,others have reported this phenomenon.
Inhibition of AHR degradation results insuperinduction of AHR-inducible genes (Ma2002). Alternatively, activation of other signaling pathways may augment AHR function(Seidel et al. 2001) or result in a synergistic

VOLUME 114 | NUMBER 5 | May 2006 •Environmental Health Perspectives

Estrogenic activity of gas-phase air contaminantsA

Particle phase
Gas phase

1000particulate-phase extracts. Our results indicatethat the gas phase may have activity equal or
greater than that of the particulate phase in an
ER reporter assay. Table 3 reports the concentration of air at which 50% and 20% of thePercent of 10–10 M estradiol

Percent of 10–10 M estradiol

the gas- vs. particulate-phase extracts for urban
March and July 2000, Figure 4A,C). This
result is of interest because, as described above,
most research examining the activity of organic
contaminants of ambient air has focused on

DMSO

B
100

0
–1

0

DMSO

1

–1

C
100

DMSO

100

0
0

DMSO
–1

1

Air (m3/well, log10)
Percent of 10–10 M estradiol

100

100

0
0

1

F

DMSO
–1

0

Air (m3/well, log10)

E

0

1

D

0
–1

0

Air (m3/well, log10)
Percent of 10–10 M estradiol

Percent of 10–10 M estradiol

Air (m3/well, log10)

Percent of 10–10 M estradiol

increase in AHR-dependent gene expression(Chen and Tukey 1996; Long et al. 1998).
The complex mixture that constitutes the gasand particulate-phase air extracts could be
activating any or all of these mechanisms.
The lower efficacy of gas-phase extracts
compared with particulate-phase extracts was
the only statistically significant observation(unpaired t-test, p = 0.005) between origin ofsample and biologic activity. Similar analyses
between seasons or locations and efficacy were
not statistically significant despite the separation of rural and particulate-phase samples in
the PCA. The lack of association between location and efficacy has been reported by others(Binkovà et al. 1999).
It was not possible to calculate a true EC50
(concentration which produced 50% of the
maximum) value for all samples; therefore, toaid comparison of one sample with another,
Table 2 shows the concentration of air thatelicited 20% and 50% of the response to
10–7 M β-NF in the AHR reporter cell line.
Using these values as a measure of relative
potency, Table 2 demonstrates that maximum
response to a sample can differ for samples withsimilar relative potency. For example, the urban
particulate extract of March 2000 and theurban particulate extract of July 2000 have similar activity at 20% or 50% of the response to
10–7 M β-NF, yet the maximum activity ofeach sample is quite different. Similarly, therural particulate extract of July 2000 has similar
relative potency to the urban gas-phase extractof July 2001, yet the maximum responses arequite dissimilar. In contrast, the rural gas-phaseextract of July 2001 and the urban gas-phaseextract of March 2001 are similar in all respects.
These divergent patterns of concentration
response are not unexpected. Ligand potency
depends not only on the affinity of the receptor
for the ligand but also on the ability of the ligand–receptor complex to bind additional transcription factors and elicit a response(Hestermann et al. 2000; Kohn and Melnick2002). The dose–response curve for eachextract is the outcome of the complex interplayof these events. The net effect, that is, the dose–
response curve, will depend on the relative agonist–antagonist properties, which are different
for each sample in this study (Table 1).
ER reporter cell line. Results of experiments using the extracts in the ER reporter
cell line BG1Luc4E2 are shown in Figure 4,
where concentration–response data aregrouped by site and date. Results were normalized to protein content in cell extracts andexpressed as a percentage of the maximum
response of 10–10 M E2.
In contrast to the results obtained in the
AHR reporter cell line, the activity of gas-phaseextracts, judged by magnitude of induction,
frequently exceeded that of particulate-phaseextracts in the ER reporter cell line (see, e.g.,

DMSO
–1

1

Air (m3/well, log10)01

Air (m3/well, log10)

Figure 4. Dose–response curves for ER-responsive cells treated with varying concentrations of air extracts(m3 air/well) of gas-phase and particulate-phase ambient air samples. (A) Urban sample, March 2000.
(B) Urban sample, March 2001. (C) Urban sample, July 2000. (D) Urban sample, July 2001. (E) Rural sample,
July 2000. (F) Rural sample, July 2001. Activity is expressed as percentage of the response to 10–10 M estradiol. Data represent mean ± SD from three separate experiments. Dose–response curves were generatedusing GraphPad Prism software.
Table 3. Apparent potency of samples in ER reporter gene assay at different induction levels, in descendingorder.
Sampleurb gas Jul 00
urb gas Mar 00
urb gas Jul 01
rur gas Jul 01
rur part Jul 00
urb gas Mar 01
rur gas Jul 00
urb part Mar 00
urb part Mar 01
urb part Jul 00
rur part Jul 01
urb part Jul 01

20% of
10–10 estradiol (m3)Sample50% of
10–10 estradiol (m3)

0.39
0.62
1.90
2.27
2.32
2.90
3.97
4.58
5.28
7.26
12.25
18.47

urb gas Jul 00
urb gas Mar 00
rur gas Jul 01
rur part Jul 00
urb gas Jul 01
urb gas Mar 01
urb part Mar 00
rur gas Jul 00
urb part Mar 01
rur part Jul 01
urb part Jul 00
urb part Jul 01

1.26
2.25
6.23
6.92
7.65
9.48
11.23
12.45
12.50
—a
—a
—a

Abbreviations: part, particulate; rur, rural sample; urb, urban sample.
aSample did not elicit 50% of the activity of 10–10 estradiol.

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701

Klein et al.

activity of the reference standard was inducedin the ER reporter cell line. As observed for
AHR responses, there was no apparent difference in activity between rural and urban sites
or between seasons.
Possible sources of estrogenic activity in theambient air extracts include PAH, PCBs,
N-PAHs, and OC pesticides. Various PAHs
and PCBs and their metabolites (Fujimoto et al.
2003; Furuta et al. 2004; Pliskova et al. 2005)
as well as OC pesticides (Kojima et al. 2004)
are reported to be estrogenic. Complicatinginterpretation of the estrogenic activity, however, is the fact that some PAHs and PCBs andtheir metabolites also exhibit antiestrogenic
activity (Arcaro et al. 1999; Connor et al. 1997;
Kramer et al. 1997).
Cellular metabolism in the BG1Luc4E2
cell line includes the cytochrome P450
enzymes, which yield hydroxyl metabolites of
PCBs, PAHs, and N-PAHs. Hydroxylated
PCBs have been shown to exhibit estrogenic
activity in ER-CALUX assays and, in somecases, are more potent than the parent PCBs
(Layton et al. 2002; Nesaretnam et al. 1996).
Hydroxy-PAHs have also been shown to bindand/or activate ER (Charles et al. 2000; Fertuck
et al. 2001). Additional classes of estrogenic
chemicals not analyzed in our studies but likely
to be present in the extracts include phthalate
esters, which have been shown to be weak ER
agonists (Zacharewski et al. 1998).

Conclusions
To our knowledge, this is the first study to
examine the dioxin-like and estrogenic activityof organic extracts of the gas phase of ambient
air. Our results not only suggest that mechanistic studies of the health effects of ambient
air need to consider both phases as having thepotential to induce and/or inhibit AHR andER signaling pathways but also suggest that therelationship between chemical composition
and biologic activity is highly complex. Two of
the expected factors driving variation in chemical composition between our samples (phaseand location) were mathematically detectablein the PCA. However, the results for urban
versus rural location were not discernible in the
results of the highly sensitive CALUX bioassays. This may be due to unidentified compounds, nonlinearity between the magnitudeof differences in chemical concentration andthe response in the CALUX assay, interactions
between chemicals and cellular factors, thepresence of both agonists and antagonists inthe sample, or, more likely, some combinationof the above factors.
There is another possibility, however. The
fact that an in vivo study comparing thebiologic activity (AHR mediated) of organic
extracts of PM10 along an urban–rural gradientand between seasons also did not find significant locational differences, despite differences702

in ambient air concentrations of AHR or ER
agonists (Binkovà et al. 2003), may be because
the concentration response of exposure to mixtures of AHR agonists is not sigmoidal due tothe impact of weak agonist interactions (Kohnand Melnick 2002). That is to say, the biologic
effects of exposure to mixtures of relatively
lower concentrations may not be exceeded bythose of exposure to mixtures at relatively
higher concentrations.
Interestingly, seasonal and locational differences have been observed in some epidemiologic studies comparing different biomarkers
and end points with ambient air composition(Dejmek et al. 2000; Perera et al. 2003). The
absence of any similar finding in this in vitro
study may be due to the high sensitivity of the
CALUX assay and its “remoteness” as a receptor assay looking at a specific aspect of one signaling pathway in contrast to the more
physiologically complicated in vivo effects
examined in epidemiology or other toxicologic
studies.
One unanswered question is the relationship between the biologic effects observed inthese cell-based assays and inhalation exposuresin humans. The average daily intake of air
ranges from about 2 m3/day in very young
infants to about 24 m3/day in adults (Health
Canada 1995). Based on these intake rates, anindividual could be exposed to levels at which
we observe an effect in vitro and are sufficient toelicit a response in some biologic systems(Bateson and Schwartz 2004; Pope et al.
2004b; Vineis et al. 2004). However, an important consideration is that uptake characteristics
are very different between a cell in culture(likely 100%) and inhalation (much less than100%). Whether or not there is a sustained
effect in humans remains to be determined.
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