Environmental Pollution 213 (2016) 809e824

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Environmental Pollution
journal homepage: www.elsevier.com/locate/envpol

Review

Biological impact of environmental polycyclic aromatic hydrocarbons

(ePAHs) as endocrine disruptors*
Yanyan Zhang a, Sijun Dong b, Hongou Wang b, Shu Tao a, Ryoiti Kiyama c, *
a
College of Urban and Environmental Sciences, Peking University, Beijing 100871, PR China
b
Institute of Urban Environment, Chinese Academy of Sciences, Xiamen 361021, PR China
c
Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8566, Japan

a r t i c l e i n f o a b s t r a c t

Article history: Polycyclic aromatic hydrocarbons (PAHs) are often detected in the environment and are regarded as
Received 23 October 2015 endocrine disruptors. We here designated mixtures of PAHs in the environment as environmental PAHs
Received in revised form (ePAHs) to discuss their effects collectively, which could be different from the sum of the constituent
3 March 2016
PAHs. We first summarized the biological impact of environmental PAHs (ePAHs) found in the atmo-
Accepted 20 March 2016
sphere, sediments, soils, and water as a result of human activities, accidents, or natural phenomena.
ePAHs are characterized by their sources and forms, followed by their biological effects and social impact,
and bioassays that are used to investigate their biological effects. The findings of the bioassays have
Keywords:
Endocrine disruptor
demonstrated that ePAHs have the ability to affect the endocrine systems of humans and animals. The
Social impact pathways that mediate cell signaling for the endocrine disruptions induced by ePAHs and PAHs have also
Evaluation assay been summarized in order to obtain a clearer understanding of the mechanisms responsible for these
Cell signaling pathway effects without animal tests; they include specific signaling pathways (MAPK and other signaling
Estrogenic activity pathways), regulatory mechanisms (chromatin/epigenetic regulation, cell cycle/DNA damage control, and
cytoskeletal/adhesion regulation), and cell functions (apoptosis, autophagy, immune responses/inflam-
mation, neurological responses, and development/differentiation) induced by specific PAHs, such as benz
[a]anthracene, benzo[a]pyrene, benz[l]aceanthrylene, cyclopenta[c,d]pyrene, 7,12-dimethylbenz[a]
anthracene, fluoranthene, fluorene, 3-methylcholanthrene, perylene, phenanthrene, and pyrene as well
as their derivatives. Estrogen signaling is one of the most studied pathways associated with the
endocrine-disrupting activities of PAHs, and involves estrogen receptors and aryl hydrocarbon receptors.
However, some of the actions of PAHs are contradictory, complex, and unexplainable. Although several
possibilities have been suggested, such as direct interactions between PAHs and receptors and the
suppression of their activities through other pathways, the mechanisms underlying the activities of PAHs
remain unclear. Thus, standardized assay protocols for pathway-based assessments are considered to be
important to overcome these issues.
© 2016 Elsevier Ltd. All rights reserved.

1. Introduction
Polycyclic aromatic hydrocarbons (PAHs) are a large group of
organic compounds comprised of two or more fused benzene rings
arranged in various configurations (Mumtaz et al., 1996). They
generally originate from combustion processes and are widely
distributed in the natural environment as a result of atmospheric
transportation, wet and dry deposition, and surface-to-air
*
This paper has been recommended for acceptance by Frank Arthur von Hippel.
* Corresponding author.
E-mail address: kiyama.r@aist.go.jp (R. Kiyama).
exchange processes (Baek et al., 1991; Buehler and Hites, 2002). We
herein referred to PAHs associated with environmental media such
as particulate matter, soil, water, and sediment as environmental
PAHs (ePAHs). ePAHs can be classified or characterized by their
forms/sources, biological effects, social impacts and bioassays used
to detect and evaluate them. The importance of ePAHs is empha-
sized here because their effects could be different from the sum of
the effects by their constituent PAHs due to their additive or sub-
tractive contributions and the contribution of the matrix, which
normally reduces the bioavailability of PAHs to target organisms
(Ramesh et al., 2004). More than one hundred PAHs exist in the
environment and often occur as complex mixtures (Mumtaz et al.,

http://dx.doi.org/10.1016/j.envpol.2016.03.050
0269-7491/© 2016 Elsevier Ltd. All rights reserved.
810 Y. Zhang et al. / Environmental Pollution 213 (2016) 809e824
1996). Sixteen PAHs are included among the 129 priority pollutants
announced by the U.S. Environmental Protection Agency (Keith and
Telliard, 1979), some of which are carcinogenic (Denissenko et al.,
1996; Bostro € m et al., 2002). ePAHs and the constitutent PAHs
may be taken up by living organisms and accumulate via the food
chain, and are exposed to humans via inhalation and the ingestion
of food; therefore, they pose potential threats to the ecosystem and
human health (Ramesh et al., 2004; Zhang et al., 2014). For
example, Zhang et al. (2009) estimated that the overall population
attributable fraction for lung cancer caused by the inhalation of
PAHs was 1.6% in China. Due to the extent of environmental PAH
contamination, especially in developing countries, identifying the
biological effects of ePAHs is considered essential for pollution in-
dications and the prevention of adverse effects on population
health. In this section, we summarized the source and forms of
ePAHs in Table 1, and presented their direct biological effects in
living organisms and humans as well as the implications of their
social impact on the ecosystem and population health.
1.1. Source and forms of ePAHs
ePAHs can be found in various sources, such as air, dust, smoke,
sediment, water, soil and oil in the environment and/or pollutants,
as forms such as particulate matter and materials in oven, fuel and
tar. PAHs in the air mainly originate from the incomplete com-
bustion of carbonaceous materials such as fossil fuel and biomass
including emissions from coke and coal burning in occupational
settings and forest fire, the exhaust fumes of motor vehicles, flues of
biomass burning for cooking and heating in rural areas, and tobacco
smoke (Baek et al., 1991; Rubin, 2001; Shen et al., 2013). Due to
their semivolatile nature, PAHs in the atmosphere may exist either
in a gaseous or particulate phase, depending on their molecular
weights (Li et al., 2014). PAHs in soil and water are mainly derived
from atmospheric deposition, especially in remote places such as
high-altitude lake areas (McVeety and Hites, 1988; Nam et al., 2008,
2009), or from industrial process such as produced water. Oil spills
are a prominent source of PAHs in marine water, which is then
introduced to marine sediments.
1.2. Biological effects of ePAHs
PAHs in the environment may be exposed to living organisms
via different routes and result in various biological effects (Table 1).
Many studies have reported correlations between the induction of
cytochrome P450 (CYP), elevated 7-ethoxyresorufin-O-deethylase
(EROD) activities, lysosomal membrane destabilization, DNA dam-
age, and endocrine and reproductive effects in fish and in-
vertebrates with PAH contamination in the water and sediments
they inhabit. Plants also uptake atmospheric and soil PAHs, and this
contamination induces leaf injuries, decreased biomass production,
and other adverse biological and physiological effects. Furthermore,
the abundance and composition of the soil microbial community
are strongly influenced by PAH contamination levels in soil.
Humans may be exposed to environmental PAHs via the intake
of PAH-contaminated food as well as the unintentional ingestion of
soil and dust via hand-to-mouth behaviors, inhalation, and dermal
contact (Buratti et al., 2007; Harris et al., 2013; McClean et al., 2007;
Pampanin et al., 2014; Zhang et al., 2014). Epidemic studies have
demonstrated that heavy exposure to PAHs from occupational en-
vironments increases the risk of developing various cancers
including lung, skin, bladder, and larynx cancers (Bostro € m et al.,
2002; Boffetta et al., 1997; Tolbert, 1997; Wagner et al., 2015). The
carcinogenicity of PAHs is associated with their potential to form
reactive diol epoxide intermediates catalyzed by CYP, which may be
induced by PAH exposure, and their subsequent covalent binding to
DNA (Bostro €m et al., 2002; Koganti et al., 2001; Ramesh et al., 2004;
Stegeman and Lech, 1991). In non-occupational settings, PAHs have
been identified as causative agents of cardiopulmonary and car-
diovascular diseases. Previous studies using cellular and animal
models have suggested that PAHs associated with ambient partic-
ulate matter generate reactive oxygen species (ROS), resulting in
oxidative stress and inflammatory responses, which account for the
prevalence and exacerbation of asthma and allergic diseases. In
addition, chronic exposure to low-dose PAHs may be responsible
for immune-mediated pregnancy loss, and maternal exposure to
PAHs has been suggested to affect fetal development such as birth
length, weight, and head circumference.
1.3. Social impact of ePAHs
A deeper understanding of the biological effects of ePAHs is
essential for understanding their social impacts, such as bio-
monitoring, pollution control, pollution indication, remediation
and risk prevention (Table 1). For example, the abundance of
nematode communities in addition to nah and pdo1 genes (PAH-
degrading genes) were previously reported to be positively influ-
enced by PAH levels in soil, and may be used as bioindicators for
PAH contamination and in the isolation of bacterial strains that
degrade PAHs (Han et al., 2014). The biological effects of ePAHs in
fish and the invertebrates described above (Section 1.2) may assist
in the development of early warning signals for PAH contamination
and remediation and mitigation strategies, and also in the assess-
ment of restoration performance. Regarding population health, the
genotoxic effects of PAHs such as DNA adducts, chromosome ab-
errations, and ras oncogene overexpression, may be used as bio-
markers for PAH exposure and cancer risk assessments. By
monitoring maternal PAH exposure and fetal development, pre-
dictive biomarkers may be established for fetal health and even
childhood intelligence and cognitive development (Perera et al.,
1998, 1999). The identification of early biological effects (chromo-
somal aberrations, sister chromatid exchanges, and micronuclei)
may also be applied to heavy PAH-exposed workers for the purpose
of cancer prevention; however, contradictory findings have been
reported (Porru et al., 1997). Furthermore, identifying the impact of
tobacco smoke on health as well as the underlying mechanisms will
be beneficial for regulating tobacco control policies.
As summarized here, quite a variety of biological activities of
ePAHs have been reported, which include acute/chronic toxicity,
reproductive effects, embryotoxicity, teratogenicity, mutagenicity,
caricinogenicity and endocrine disruptions (Lisouza et al., 2012;
Kameda et al., 2012). While the genotoxic effect of ePAHs is
important, we focused here on the endocrine disruptor action by
ePAHs.
2. Biological activities of ePAHs
A variety of bioassays have been developed and applied to un-
derstand the role of ePAHs as endocrine disruptors. The bioassays
used to investigate the biological effects of ePAHs are summarized
according to the types or sources of ePAHs (Table 2), which have
been roughly categorized into two types: environmental samples
containing various types of PAHs, or pollutants themselves with
known chemicals or their sources (see Table 1). The assays used
have been categorized into animal/plant tests, cell assays, microbial
tests, reporter-gene assays, protein assays, and other assays. The
effects of ePAHs as endocrine disruptors was initially demonstrated
in animal tests, and then by mechanism-based analyses using cell
and protein assays.
 Y. Zhang et al. / Environmental Pollution 213 (2016) 809e824 811

Table 1
Biological effects and social impact of ePAHs.

Form/source Biological effect Social impact Reference

Environment
Air/diesel/dust/particulate/smoke/volatile
Ambient air Plant growth/symbiosis/photosynthesis Risk assessment Desalme et al., 2013
Ambient air Lung, bladder, and skin cancers Prevention of cancer Porru et al., 1997
Ambient air PAH-DNA adducts PAH exposure and lung cancer Kriek et al., 1993
Particulate matter ROS/oxidative stress/inflammation Prevalence of allergic diseases Huang et al., 2015
Particulate matter Generation of ROS Prevalence of childhood asthma Karimi et al., 2015
Particulate matter Cardiovascular diseases Toxicology/risk control Schnelle-Kreis et al., 2009
Particulate matter Genotoxic effects Biomarkers for PAH exposure Farmer et al., 2003
Particulate matter Fetal development Childhood intelligence/development Perera et al., 1999
Smoke (tobacco) Immune-mediated pregnancy loss Pollution control Detmar and Jurisicova, 2010
Smoke (tobacco) Atherosclerosis Prevention of cardiovascular diseases Ramos and Moorthy, 2005
Smoke (tobacco) Cardiovascular diseases Assessment of cardiotoxicity Korashy and El-Kadi, 2006
Smoke (tobacco) Carcinogenicity Synergistic mechanisms by PAHs Rubin, 2001
Settled house dust Mutagenic potency Carcinogenic risk Maertens et al., 2004
Diesel exhaust particles B-cell differentiation/IgE production Allergic airway diseases Pampanin et al., 2014
Coal/coke/combustion/fossil fuel/tar
Coke oven Increased oxidative stress in workers Biomarkers for toxic responses Gao et al., 2014
Coke oven Oxidative stress and lipid peroxidation Immunological effects in workers Jeng et al., 2011
Coke oven DNA methylation states in workers Epigenetic markers Pavanello et al., 2009
Coke oven CYP1B1 mRNA in peripheral blood Biomarker validation Hanaoka et al., 2002
Coal tar mixtures Carcinogenicity Effects of small PAHs Benford et al., 2010
Coal burning Fetal development Establishing predictive biomarkers Perera et al., 1998
Coal-tar/creosote/bitumen/juniper tar/engine oils Formation of DNA adducts Hazards by epidermal contact Phillips et al., 1990
Fumes bitumens Carcinogenicity Biomarkers of non-priority PAHs Binet et al., 2002
Biomass fuel emissions Respiratory/cardiovascular diseases Prevention of health risks Kim et al., 2011a
Biomass fuel emissions Effects on human blood Pollution control Kamal et al., 2015
Pollutant
Oil
Marine oil Phase-I enzymes/DNA adducts/neoplasm Impact on marine ecosystems Hylland, 2006
Crude oil spill Increase in fish bile FACs/CYP response Toxicity/lesions/reproductive failure Lee and Anderson, 2005
Bunker fuel spill Lysosomal membrane destabilization Sublethal effects in bivalves Hwang et al., 2014
Oil spill Elevated EROD activities in eelpouts Multi-biomarkers for PAH exposure Sturve et al., 2014
Oil spill Adverse effects on eelpouts Adverse biological effects Kreitsberg et al., 2012
Oil spill Elevated metabolites/CYP1A induction Identifying extra exposure pathways Jung et al., 2011
Oil spill Detoxification/defense systems in mussel Biomonitoring of acute exposure Sureda et al., 2011
Oil spill DNA strand break in mussels and clams Assessment of exposure/damage Thomas et al., 2007
Oil spill DNA damage via the food chain Identifying genotoxic contaminants Lemiere et al., 2004
Sediment
Harbor sediment Bacterial population Remediation/mitigation strategies Slater et al., 2008
Marine sediment Accumulation in marine organisms Impact on marine populations Meador et al., 1995
Lake sediment Toxic to photoluminescent bacteria Potential risk upon disturbances Hyo€ tyla
€inen and Olkari, 1999
Lake sediment Forming DNA adducts in the eel Indicators for PAH exposure van Schooten et al., 1995
River sediment CYP1A activity/porphyrin accumulation Biomonitoring Huuskonen et al., 2000
Sediment (rivers/lakes/marines) Salmonella mutagenic potency Health and viability of aquatic biota Chen and White, 2004
Sewage/sludge/waste/wastewater
Contaminated water Induction of cytochrome P-450 Monitoring exposure to carcinogens Stegeman and Lech, 1991
Wastewater (oil company) Mutagenicity in bacterial strains Field monitoring Re
ategui-Zirena et al., 2014
Marine water EROD activity in leaping mullet Risk elimination Sen et al., 2010
Produced water Gene expression in zebrafish Monitoring early warning signals Holth et al., 2008
Produced water DNA damage in mosquito fish Toxicology of produced water Caliani et al., 2009
Produced water Reproductive effects to cod Application of multiple biomarkers Sundt and Bjo €rkblom, 2011
Produced water Effects to cod and blue mussel Population and ecosystem effects Bakke et al., 2013
Soil
Surface soil Ecological risk Remedial measures Lang et al., 2012
Surface soil Soil nematode community Indicators for PAH soil pollution Chen et al., 2009
Contaminated soil Abundance of nah and pdoI genes Indicators for PAH soil pollution Han et al., 2014
Coal tar contaminated soil DNA adducts in the mouse lung Main components inducing responses Koganti et al., 2001
Creosote-contaminated soil Microbial community composition Isolation of PAH degraders €rneman et al., 2008
To

Abbreviations: EROD: ethoxyresorufin-O-deethylase; FAC: fluorescent aromatic compound; PLHC-1: a fish hepatoma cell line; PM: particulate matter; ROS: reactive oxygen
species.

2.1. Assays for biological activities of ePAHs
2.1.1. Animal/plant tests
Various tests have been conducted using animals and plants in
order to evaluate the biological activities of ePAHs. For example,
Sprague-Dawley rats were used to examine the carcinogenic effects
of coarse particles containing PAHs as dominant components, and
the increase observed in the incidence of terminal bronchiolar
hyperplasia and squamous metaplasia was associated with PAH
exposure, whereas no significant increase was noted in lung tumors
(Soffritti et al., 2013). Serum and tissues obtained from Wistar Han
rats were used to evaluate genotoxicity and the induction of
oxidative stress after long-term exposure to diesel exhaust fumes
containing PAHs (Hallberg et al., 2015). The toxicity and mutage-
nicity of fractions of crude coal tar containing various PAHs were
also investigated using a chick embryotoxicity screening test
(CHEST) in addition to a Salmonella/microsome bioassay, and a
significant relationship was observed between PAH types and their
812 Y. Zhang et al. / Environmental Pollution 213 (2016) 809e824

Table 2
Bioassays for the study of ePAHs.

Form/Source Bioassay Reference

Environmental
Air/diesel/dust/particulate/smoke/volatile
Air (coke oven) Ames test Dobias et al., 1999
DEPs (compressor) Ames test Mutlu et al., 2013
Dust Life-span carcinogenicity bioassay (rats) Soffritti et al., 2013
Extractable organic matter (urban air) DNA microarray Líbalova et al., 2014
Particulate matter (biomass burning) Micronucleus bioassay de Oliveira Alves et al., 2011
Particulate matter (cashew nut roasting) Micronucleus bioassay Galv~ao et al., 2014
Particulate matter (smoked meat) CALUX assay Kuhn et al., 2008
Particulate matter (urban air) Ames test Villalobos-Pietrini et al., 2007; Traversi et al., 2011
Semi-volatiles (vehicle exhaust) Ames test/AhR receptor affinity test Westerholm and Egeba €ck, 1994
Smoke (tobacco) Ames test DeMarini et al., 1995
Soot particles (biomass fuel) Yeast estrogen screen Wang et al., 2005
Urban air CALUX assay/Salmonella/microsome assay Ciganek et al., 2004
Urban air/DEPs Comet assay/ELISA/rat bioassay Hallberg et al., 2015
Coal/coke/combustion/fossil fuel/tar
Coal tar Chick test/Salmonella/microsome assay Mayura et al., 1999
Coal tar/coke/oil/soot/smoke Cancer bioassays Collins et al., 1998 (review)
Combustion emissions Bioassays Lewtas, 2007 (review)
Combustion products Amphipod/polychaete/clam bioassays Van Dolah et al., 2005
Combustion products (meat/wood/charcoal) Yeast estrogen screen Muthumbi et al., 2003
Pollutant
Oil
Oil (lampblack) Dermal bioassay (human skin section) Stroo et al., 2005
Oil production discharges EROD assay/ELISA Harman et al., 2010
Oil shale EROD assay Huuskonen et al., 2000
Oil spills/bay sediments AhR reporter-gene assay Puy-Azurmendi et al., 2010
Petrogenic extracts Comet assay/fish micronucleus assay Le Bihanic et al., 2014
Sediment
Bay sediments P450 reporter-gene assay Koh et al., 2001
Bay sediments Sea urchin/oyster bioassays Geffard et al., 2001
Bay sediments EROD assay/yeast estrogen screen Annavarapu et al., 2004
Bay sediments EROD assay/yeast estrogen screen Weston et al., 2010
Creek sediments (hazardous waste) Flow cytometry Matson et al., 2009
Lagoon sediments Sea urchin bioassays D'Adamo et al., 2014
Lagoon sediments Comet assay/yeast estrogen screen Amaeze et al., 2015
Lake sediments Ames test Marvin et al., 2000
Lake sediments AhR/ER reporter-gene assay/snail bioassay Mazurova  et al., 2008
Lake sediments (coal tar) Ames test Marvin et al., 1999
Marine sediments English sole feeding bioassay Rice et al., 2000
River sediments CALUX assay/ER reporter-gene assay/Salmonella umu-test Vondra cek et al., 2001
River sediments Mutatox bioassay (luminescent bacteria test) Thomas et al., 2002
River sediments Animal bioassays/Microtox bioassay den Besten & van den Brink, 2005
River sediments LUMIStox bioassay/duckweed bioassay Olajire et al., 2005
Sewage/sludge/waste/water
Groundwater Adult hydra bioassay Ake et al., 2003
Groundwater EROD assay/CFDA-AM assay Bopp et al., 2007
Lake water Ames test/EROD assay/yeast estrogen screen Rastall et al., 2004
Sewage sludge ToxAlert bioassay (luminescent bacteria test) rez et al., 2001
Pe
Sewage sludge composts Phytotoxkit (seed germination) test Oleszczuk, 2008
Soil
Bioremediated soils Salmonella/microsome assay Brooks et al., 1998
Bioremediated soils Microtox bioassay (luminescent bacteria test) Lau et al., 2003
Contaminated soils Earthworm survival bioassay Charrois et al., 2001
Contaminated soils LUMIStox bioassay (luminescent bacteria test) Hirmann et al., 2007
Contaminated soils (MGP) Ames test Park et al., 2008
Contaminated soils Earthworm/lettuce/nematode bioassays Cofield et al., 2008
Contaminated soils CALUX assay/comet assay Andersson et al., 2009
Contaminated soils AhR reporter-gene assay Larsson et al., 2013
Contaminated soils lacZ-transgene mutation assay Lemieux et al., 2015

Abbreviations: AhR: aryl hydrocarbon receptor; CALUX chemical-activated luciferase gene expression; CFDA-AM: 5-carboxyfluorescein diacetate acetoxymethyl ester; DEP:
diesel exhaust particle; ELISA: enzyme-linked immunosorbent assay; ER: estrogen receptor; EROD: ethoxyresorufin-O-deethylase; Luc: luciferase; MGP: manufactured gas
plant.

concentrations and embryonic mortality (Mayura et al., 1999). A
predator-prey interaction was also used as a bioassay to represent
realistic ecological processes; English soles fed sediment/PAH-
exposed polychaetes in the juvenile period exhibited growth
retardation, the increased expression of CYP1A, and/or accumula-
tion of hepatic PAH-DNA adducts (Rice et al., 2000).
Invertebrates have been employed to study the biological
activities of ePAHs. For example, the phytoremediation of soil from
a former manufacturing gas plant site was assessed using a com-
bination of bioassays based on earthworm survivability, nematode
mortality, lettuce germination and emergence, and microbial
respiration (Cofield et al., 2008). The toxicity of PAHs in the sedi-
ments of estuarine wetlands near highways was examined by
bioassays using amphipods, polychaetes, and clams, among which
 Y. Zhang et al. / Environmental Pollution 213 (2016) 809e824
only clams showed toxicity, even though the sensitivity of the other
assays was enhanced by ultraviolet light (Van Dolah et al., 2005).
The fresh water snail Potamopyrgus antipodarum has been used to
assess the procreation toxicity of sediments highly contaminated
with powdered waste coal, and the findings obtained revealed that,
after an 8-week exposure, the fecundity of snails was stimulated at
low concentrations, but inhibited at high concentrations (Mazurova 
et al., 2008). Sea urchins and oysters were previously used to
evaluate the embryotoxicity of marine sediments containing PAHs,
and the findings obtained indicated that PAHs enhanced embryonic
death, and oysters were more sensitive to PAHs than sea urchins
(Geffard et al., 2001).
Phytotoxicity has gained significant attention among bioassays
to assess ePAHs. The emergence of lettuce seedlings has been used
to assess phytoremediation (Cofield et al., 2008). Similarly, the
toxicity of contaminated sediments was assessed by analyzing the
number of fronds of the duckweed, the findings of which sug-
gesting that, although ePAHs inhibited growth, no significant dose-
dependent relationship was observed in this particular case (Olajire
et al., 2005). Moreover, sewage sludge collected at different
wastewater treatment plants in Poland was assessed by a toxicity
test using a commercial Phytotoxkit, in which the seed germination
and growth of young cress roots were examined, and some sludge
samples were found to inhibit seed germination by 100%
(Oleszczuk, 2008).
2.1.2. Cell assay
Assays using various types of cells have been widely used to
evaluate the biological activities of ePAHs. Due to its rapidity and
sensitivity, the chemical-activated luciferase gene expression
(CALUX) assay has commonly been used to detect aryl hydrocarbon
receptor (AhR)-active compounds including PAHs in various envi-
ronmental samples, such as soil, water, and sediments. A rat hep-
atoma (H4IIE) cell line, stably transfected with a construct
containing the dioxin-responsive element (DRE) and luciferase
reporter gene, was used in this assay to detect the activities of AhR-
active compounds, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin
(TCDD), in sediments and pore waters (Murk et al., 1996). On the
other hand, a comet assay, which measures the migration, under
alkaline conditions, of DNA from cells encapsulated into low-
melting-point agarose gels, represents a simple method, but has
been widely used to quantify DNA damage and repair with high
specificity and sensitivity (Singh et al., 1988). In a collaborative
study with the International Programme on Chemical Safety, a
Tradescantia pallida (spiderwort) micronucleus (Trad-MCN)
bioassay was employed to evaluate clastogens and was found to be
efficient and reliable for monitoring contamination in water or the
ambient atmosphere (Ma et al., 1994). Small teleostomi are easily
fed and exposed to chemical toxicants in laboratories, and exhibit
similar toxic responses to those observed in mammals. Conse-
quently, gill cells have been used to examine micronucleus fre-
quencies in order to evaluate environmental genotoxins (Hayashi
et al., 1998). The genotoxic effects of PAHs in the ambient envi-
ronment were previously assayed by flow cytometry, and the
findings obtained verified a significant increase in chromosomal
damage following exposure to sediments from a hazardous waste
site (Matson et al., 2009).
2.1.3. Microbial test
Microbes, such as Salmonella and luminescent bacteria, have
also been commonly used in in vitro assays to analyze the biological
activities of ePAHs. The Ames test is widely used to screen potential
carcinogens and mutagens based on their mutational activities
(Ames et al., 1975). Histidine requirement strains of Salmonella
typhimurium, which cannot grow in histidine-free medium, are
813
used in this test, and bacterial colonies form on agar plates only
after this bacterium is exposed to chemicals, which may revert its
genotype to prototrophy. Furthermore, since some chemicals are
only mutagenic after metabolic activation, mammalian microsomal
enzymes have been complemented to overcome the shortage of
metabolic activation. An improved protocol with two new tester
strains was developed to increase the sensitivity and applicability
of the Ames test (Maron and Ames, 1983). Alternatively, the umu-
test has been used to detect environmental carcinogens (Oda et al.,
1985). Salmonella typhimurium TA1535 carrying a plasmid
(pSK1002) containing the fusion gene umuC'-'lacZ, in which umuC
may be activated by the presence of mutagens in SOS/DNA damage
responses, was constructed, and, after carcinogens induced SOS/
DNA damage responses and activated the umuC'-'lacZ gene, b-
galactosidase (the lacZ gene product) was produced and the level of
DNA damage was quantified based on b-galactosidase activity in a
colorimetric assay.
Luminescent bacteria, which emit light generated by an
enzyme-catalyzed chemiluminescence reaction, have also been
widely used to evaluate ePAHs. For example, the genotoxicity of
PAHs in sediments from estuaries was evaluated using the Mutatox
bioassay, a commercially available assay involving the dark variant
(strain M169) marine bioluminescent bacterium, Photobacterium
phosphoreum, which emits light when it is exposed to mutagenic
compounds and reverts to the wild-type (Thomas et al., 2002). The
LUMIStox bioassay, in which the inhibition of bioluminescence
from the marine bacterium Vibrio fischeri, is quantified, has been
used to assess the acute toxicities of ePAH-contaminated sediments
in southern Nigeria (Olajire et al., 2005). ToxAlert and Microtox
bioassays are based on a similar principle to the LUMIStox assay; a
decrease in the rate of luminescence by the inhibition of respiration
in V. fischeri by toxicants is quantified. The ToxAlert bioassay was
previously used in combination with a composition analysis by gas
chromatography-mass spectrometry (GC-MS) in order to evaluate
the toxicity of sewage sludge (Perez et al., 2001). On the other hand,
the effects of spent mushroom compost on reductions in the
toxicity of PAHs were verified by combining the Microtox bioassay
and GC-MS (Lau et al., 2003). The protocol of the LUMIStox assay
was improved by miniaturization with 96-microplates, for which
dark microplates are recommended because they may prevent
overestimations of the level of PAHs when soil is colored (Hirmann
et al., 2007). Assay results are highly variable when compared
among the microbial tests used or with other bioassays because of
differences in the sensitivity, specificity, and durability of chem-
icals; nevertheless, these assays are useful when cost, time, and
portability are considered.
2.1.4. Reporter-gene assay
Various marker genes and proteins have become available in
bioassays to evaluate ePAHs by molecular biology-based studies on
the mechanisms of their actions. Reporter-gene assays have also
been developed to more efficiently detect these markers through
the construction of plasmid vectors equipped with appropriate sets
of reporter genes, such as those encoding b-galactosidase (the lacZ
gene product), luciferase, chloramphenicol acetyltransferase (CAT),
and green fluorescent protein (GFP), and regulatory sequences for
marker genes, such as the DRE and estrogen-responsive element
(ERE), which bind to the corresponding receptors. Since PAHs
exhibit AhR-mediated activity, estrogenicity, and/or anti-
androgenicity depending on the chemicals examined and cell
types used, various assay systems have been employed. For
example, the growth inhibition of recombinant yeast containing
AhR was previously used to examine the dioxin-like activity of
ePAHs in estuary sediments, in which the lacZ gene was used as a
reporter (Puy-Azurmendi et al., 2010). Alternatively, a recombinant
814 Y. Zhang et al. / Environmental Pollution 213 (2016) 809e824
yeast strain, containing the human estrogen receptor (ER) gene and
ERE-driven lacZ as a reporter gene, has been widely used to eval-
uate the estrogenic effects of ePAHs (Routledge and Sumpter, 1996).
Exposure to estrogen induces lacZ gene expression and changes the
color of the medium from yellow to red. The estrogenicity and
dioxin-like activity of contaminated sediments have also been
examined using assays with mammalian cell lines containing
luciferase as a reporter, the expression of which is regulated by ERE
or DRE (Mazurova  et al., 2008). A previous study assessed the car-
cinogenicity of PAHs in bay sediments using a CYP reporter-gene
system in order to determine whether ePAHs promoted tumori-
genesis through the AhR pathway by inducing the expression of
CYP1A1 (Koh et al., 2001). On the other hand, the mutagenic activity
of PAH-contaminated soils has been assessed by quantifying the
rates of mutations in the lacZ gene in mouse FE1 cells (Lemieux
et al., 2015). There are significant differences between yeast and
mammalian assays in the response to chemicals, such as the
discrimination between agonists and antagonists, and the differ-
ences caused by membrane permeability, transport proteins and
signal transduction pathways (Kiyama and Wada-Kiyama, 2015).
2.1.5. Protein assay
A variety of qualitative and quantitative assays were developed
to detect proteins to evaluate endocrine disruptor action, such as
mass spectrometry, Western blotting, the enzyme-linked immu-
nosorbent assay (ELISA) and immunohistochemistry. Western
blotting has been used widely to quantify marker proteins. For
example, AhR and ERa were used to evaluate the response to
endocrine disruptor action (Safe and Wormke, 2003).
ELISA is also commonly used for protein analyses. For example,
ELISA has been employed to quantify the amounts of proteins, such
as vitellogenin, and examine the estrogenicity of ePAHs. For
example, ER-mediated vitellogenin production was previously
quantified by ELISA in order to monitor the toxicity of ePAHs in oil
production discharges, and the level of vitellogenin was found to
decrease with increases in the concentrations of ePAHs due to the
presence of ER antagonists (Harman et al., 2010). ELISA has also
been used to detect complex materials other than proteins, such as
the 8-hydroxy-deoxyguanosine (8-OHdG) adduct, which is an in-
dicator of oxidative stress and DNA damage (Hallberg et al., 2015).
The quantification of enzymatic activities is another example of
how protein assays are used in the study of ePAHs. The CYP-
associated monooxygenase, EROD, belongs to the CYP family of
enzymes, and its activity is used as a marker for the AhR-mediated
toxicity of dioxin-like chemicals including PAHs (Pohl and Fouts,
1980; Kennedy and Jones, 1994). Thus, the activity of EROD is
often employed to evaluate ePAHs (see Table 2).
2.1.6. Transcription assay
Various transcription assays have been developed due to the
recent progress in gene- or genome-based biotechnology, such as
DNA microarray assay, next-generation sequencing-based RNA
sequencing (RNA-seq) and reverse transcription polymerase chain
reaction (RT-PCR). Several groups have developed DNA microarray
assays for gene expression profiling of cancer cells treated with
ePAHs/PAHs (Hockley et al., 2006, 2009; Líbalova  et al., 2014),
because of the advantage of the assay for profiling more than
10,000 genes simultaneously. Statistically significant numbers of
genes were up- or down-regulated by the treatment with B[a]P,
including those involved in xenobiotic metabolism, cell cycle
regulation and oxidative stress response (Hockley et al., 2006). The
genes involved in signaling pathways, such as TGF-b and Wnt
pathways, responded to the treatment with the extractable organic
 et al., 2014).
matters from respirable airborne particles (Líbalova
2.1.7. Other assays
More assays are used to assess the biological activities of ePAHs.
For example, the TCDD receptor affinity test was performed to
evaluate biological effects of PAHs in exhaust emissions, which is
based on TCDD and PAHs having affinity to AhR (Westerholm and
Egeba €ck, 1994). Furthermore, dermal contact with PAHs has been
assessed by quantifying their amounts with high-performance
liquid chromatography (HPLC) after their direct absorption using
mounted adult human skin sections (Stroo et al., 2005). Other as-
says, such as ligand-binding assays, signaling pathway analyses,
and yeast two-hybrid assays have also been employed (see Section
3.2).
2.2. ePAHs as endocrine disruptors
PAHs in the environment are regarded as endocrine disruptors
that interfere with the homeostasis of organisms by mimicking
endogenous hormones. The best example is benzo[a]pyrene (B[a]
P), which has been identified as a contaminant in food, water,
cigarette smoke, and vehicle exhaust fumes or in the body
following occupational exposure. For example, hepatocytes isolated
from male Wistar rats exposed to B[a]P for 72 h were examined for
thyroid hormone activity and related RNA expression profiles, and
the findings obtained revealed that B[a]P reduced the metabolic
rate by breaking down thyroid hormone (Schraplau et al., 2015).
B[a]P is also toxic to the female reproductive system. When fe-
male scallops (Chlamys farreri) were exposed to B[a]P, the levels of
the sex steroids, 17b-estradiol, testosterone, and progesterone, in
the ovaries were significantly reduced in a dose-dependent
manner, and the expression of the estrogen-metabolizing en-
zymes, hydroxysteroid dehydrogenases and CYP17, was also
decreased, implying that steroidogenesis is restrained following
exposure to B[a]P (Tian et al., 2013). A previous study reported that
exposure to B[a]P increased plasma estrogen and progesterone
levels as well as the expression of ERa in early pregnancy, whereas
the expression of the progesterone receptor was reduced, and these
changes ultimately impaired endometrium receptivity and
enhanced embryonic mortality (Zhao et al., 2014). Significant his-
topathological changes have also been observed in the ovary after
exposure to B[a]P in the postnatal period, such as an increase in the
total number of nonatretic antral follicles due to the thickening of
theca cell layers and deficient apoptosis in granulosa cells (Kummer
et al., 2013). Exposure to B[a]P may also induce endocrine disrup-
tions in the male reproductive system. For example, testosterone
levels in the serum and intratesticular fluid as well as testosterone
production and the expression of related genes in Leydig cells were
examined in adult male Sprague-Dawley rats fed B[a]P for 90 days
(Chung et al., 2011). The findings obtained suggested that the ste-
roidogenic acute regulatory protein (StAR) targeted by B[a]P
disturbed testosterone levels and degraded the quality of epidid-
ymal sperms.
The impact of dietary exposure to B[a]P on the multigenera-
tional growth and development of animals has been examined.
Adult zebrafish (F0) fed B[a]P were subjected to hatching in order to
obtain further generations and conduct multigenerational pheno-
typic examinations, and mortality and premature hatching were
observed more in the first filial generation (F1) of the treatment
group than in the control group, whereas no significant alterations
were detected in their descendants (F2, F3 and F4) and deformities
were only noted in F1 (Corrales et al., 2014).
Vertebrates have been used to examine the endocrine and
developmental effects of PAHs. Fish (polar or Atlantic cod) were
exposed to PAHs or ePAHs (dispersed oil) and their effects on the
endocrine and reproductive systems were analyzed by examining
phenotypes and endocrine markers such as plasma vitellogenin
 Y. Zhang et al. / Environmental Pollution 213 (2016) 809e824
levels; limited, but distinct effects were observed in these studies,
such as increases in vitellogenin levels, decreases in the number of
oocytes in females, and the inhibition of spermatogenesis in males
(Tollefsen et al., 2011; Geraudie et al., 2014). Zebrafish have also
been used to assess the endocrine effects of PAHs. Zebrafish em-
bryos exposed to PAHs for 120 h showed distinct developmental
effects, such as a dioxin-like phenotype, which may have been
mediated through activation of the AhR pathway (Hawliczek et al.,
2012). Furthermore, juvenile mullets exposed to heavy fuel oil
containing ePAHs exhibited effects including transient neuroen-
docrine effects, which may have negative impacts on reproduction,
behavior, and sex differentiation (Bilbao et al., 2010). The endocrine
effects of ePAHs have also been examined in the migratory bird, the
Northern gannet by analyzing two hormones, prolactin and corti-
costerone, which influence reproduction (Franci et al., 2014).
Meanwhile, there were preliminary studies of the effects of ePAHs
on invertebrates by evaluating endocrine markers because they
represent more than 95% of all animal species (Zapata-Perez et al.,
2005), although there were few cases of mechanism-based
comparisons.
It is important to note that difficulties are associated with
obtaining clear endocrine effects in animal tests due to genetic
variations and detoxification activities in addition to the ethical
issues linked to the use of animals for bioassays; therefore, in vitro
assays need to be developed, are expected to replace animal assays
with equivalent quality, sensitivity, and reliability, and include
pathway-based assays as described below.
3. Mechanisms underlying biological effects induced by
ePAHs and constituent PAHs
Recent technological advances have provided a clearer insight
into the mechanisms responsible for the biological effects induced
by ePAHs and constituent PAHs. PAHs are known to stimulate AhR-
mediated cell signaling, and their mechanisms of actions have been
examined extensively (Hankinson, 1995; Tian, 2009). AhR is a
ligand-activated transcription factor that binds to planar aromatic
hydrocarbons, such as flavonoids, polyphenols, indoles, and the
polyaromatic and halogenated aromatic hydrocarbons of natural
and industrial origins. Previous studies described the association or
crosstalk between AhR and various cell signaling pathways, such as
mitogen-activated protein kinase (MAPK), NF-kB, and cell cycle/
apoptosis-related pathways (Schmidt and Bradfield, 1996; Puga
et al., 2002, 2009; Henklova  et al., 2008; Tian, 2009). Since AhR is
not a direct regulator of hormonal actions, the mechanisms un-
derlying the endocrine-disrupting effects of AhR and its ligands
require the involvement of hormone receptors and/or regulators
(Murphy et al., 2007). Since estrogen and estrogenic chemicals are
an important category of endocrine modulators and sometimes act
as endocrine disruptors, the involvement of estrogen signaling
associated with AhR-mediated cell signaling stimulated with PAHs
has been investigated in an attempt to elucidate the mechanisms
responsible for their biological effects (Safe et al., 2000;
Swedenborg and Pongratz, 2010; Feng et al., 2013).
In this section, we summarized the cell signaling pathways
stimulated by ePAHs and PAHs. We then focused on the estrogen
activities of ePAHs and PAHs in order to elucidate the mechanisms
underlying the biological effects of ePAHs as endocrine disruptors.
3.1. Pathways mediating cell signaling induced by ePAHs/PAHs
The biological activities of and signaling pathways stimulated
and/or mediated by ePAHs/PAHs are summarized in Table 3. These
activities are categorized by specific signaling pathways (MAPK and
other signaling pathways), regulatory mechanisms (chromatin/
815
epigenetic regulation, cell cycle/DNA damage control, and cyto-
skeletal/adhesion regulation), and cell functions (apoptosis, auto-
phagy, immune responses/inflammation, neurological responses,
and development/differentiation), where PAHs, such as 2-
aminoanthracene, benz[a]anthracene (B[a]A), benzo[a]pyrene (B
[a]P), B[a]P-4,5-dihydroepoxide (BPE), B[a]P diol epoxide (BPDE),
benzo[e]pyrene (B[e]P), benz[j]aceanthrylene (B[j]A), benzo[k]flu-
oranthene (B[k]F), benz[l]aceanthrylene (B[l]A), cyclopenta[c,d]
pyrene (CPP), dibenzo[a,l]pyrene (DB[al]P), 7,12-dimethylbenz[a]
anthracene (DMBA), DMBA-3,4-diol, fluoranthene, fluorene, indeno
[1,2,3-c,d]pyrene (IND), 3-methylcholanthrene, 1-methylpyrene, 1-
nitropyrene, perylene, phenanthrene, and pyrene are found in
sources, such as aerosol, air pollutants, asphalt fume, coal tar/coke/
tobacco/wood smoke particles, coastal sediments, diesel exhaust
particles (DEPs), electronic waste, and urban dust (Table 3).
Various signaling pathways are involved in the biological ac-
tivities induced or modulated by ePAHs/PAHs, which include
receptor-mediated pathways, growth factor/cytokine-signaling
pathways, and key-regulator/modulator-dependent pathways
(Table 3). For example, B[a]A is involved in the ROS/MAPK pathway
in cell migration (Song et al., 2011), the PPARa/PPARb/d pathway in
carcinogenesis (Kim et al., 2005) and the AhR/NF-kB/IL-6 pathway
in cytokine response (Kolasa et al., 2013). B[a]P is involved in the
MAPK/JNK pathway in stress response (Jarvis et al., 2014), p53-
dependent apoptosis via FOXO3a (Al-Anati et al., 2014), cell cycle/
DNA damage control in cell proliferation (Volkov et al., 2012; Guo
et al., 2000), cell adhesion regulation in macrophage differentia-
tion or hemocyte immunomodulation (van Grevenynghe et al.,
2003; Croxton et al., 2012), microtubule dynamics (Deng et al.,
2014), cytokine response associated with asthma (Ple  et al., 2015),
TGF-b-induced pro-inflammatory response (Curfs et al., 2005),
TLRs-induced immune response (Do et al., 2012), Parkinson's dis-
ease (Konstandi et al., 2007), and development/differentiation,
such as neurodevelopment (Wormley et al., 2004), atherogenesis
(Knaapen et al., 2007) and hematopoiesis (N'jai et al., 2011). BPDE is
involved in cell functions, such as chromatin structure in carcino-
genesis (Feng et al., 2003), G1/S cell cycle regulation (Mukherjee
and Kumar, 2013), and T-dependent antibody response (Li et al.,
2010). DMBA is involved in PPARb/d-AhR crosstalk in carcinogen-
esis (Borland et al., 2014), and the Wnt/b-catenin and other path-
ways in carcinogenesis (Currier et al., 2005), and cell functions such
as pre-B cell apoptosis (Mann et al., 2001), tumor necrosis factor a
(TNF-a)-dependent bone marrow toxicity (Page et al., 2004), T-
dependent antibody response (Li et al., 2010), and development/
differentiation, such as B lymphopoiesis (Mann et al., 2001) and
hematopoiesis (N'jai et al., 2011). 3-Methylcholanthrene is involved
in signaling pathways such as TGF-b signaling in hepatocyte func-
tions (Abdel-Razzak et al., 1994); cell functions, such as chromatin
condensation in apoptosis (Reynaud et al., 2004) and cell adhesion
regulation during epithelial-mesenchymal transition (Oh et al.,
2014); and in diseases, such as rheumatoid arthritis (Tamaki
et al., 2004).
On the other hand, ePAHs from various sources are involved in
signaling pathways, such as the MAPK/AKT (Sumanasekera et al.,
2007), NF-kB/MAPK (Bonvallot et al., 2001), IGF-1 (Xu et al.,
2013), insulin (Choi et al., 2015) pathways; cell functions, such as
DNA methylation (Liu et al., 2013; Yan et al., 2014; Hew et al., 2015),
histone modification (Klingbeil et al., 2014), FASL-dependent
apoptosis (Crew et al., 2007), infectious response (Ma and Ma,
2002), DNA damage response (Mahadevan et al., 2005; Duan
et al., 2009; Foresta et al., 2014), actin function (Tuominen et al.,
2003; Hooven and Baird, 2008), cell adhesion (Wu et al., 2011),
cytokine response (Ple  et al., 2015) and inflammatory response
(Gate et al., 2006; Totlandsdal et al., 2014; Wittkopp et al., 2013;
Yang et al., 2016; Danielsen et al., 2011; Totlandsdal et al., 2015;
Table 3
Biological activities of and cell signaling by ePAHs/PAHs.

Pathway/cascade/ ePAH/PAH Reference

function

MAPK signaling
MAPK/AKT/NO ePAHs (DEPs) Sumanasekera et al., 2007
MAPK/JNK B[a]P Jarvis et al., 2014
NF-kB/MAPK ePAHs (DEPs) Bonvallot et al., 2001
ROS/MAPK B[a]A/B[k]F/IND Song et al., 2011
Other signaling (see Table 4 for ER signaling)
PPARa/PPARb/d B[a]A Kim et al., 2005
PPARb/d/AhR DMBA Borland et al., 2014
crosstalk
Chromatin/epigenetic regulation
Chromatin 3-Methylcholanthrene Reynaud et al., 2004
condensation
Chromatin BPDE Feng et al., 2003
structure
DNA methylation ePAHs (aerosol, air pollutant), Ph Liu et al., 2013; Yan et al., 2014; Hew et al., 2015
Histone ePAHs (aerosol) Klingbeil et al., 2014 (review)
modification
Apoptosis
FASL-dependent ePAHs (tobacco/DNA adduct) Crew et al., 2007
apoptosis
Infectious response ePAHs (DEPs) Ma and Ma, 2002
p53-dependent B[a]P/B[j]A/B[l]A/CPP/DB[a,l]P/Fla/1-Nitropyrene Solhaug et al., 2004; Su et al., 2008; Pru et al., 2009; Huang et al., 2012; Al-Anati et al., 2014
apoptosis
Autophagy
Starvation stress Ph McVeigh et al., 2006
Cellular metabolism
AMPK signaling 1-Nitropyrene Podechard et al., 2011
IGF-1 signaling ePAHs (electronic waste) Xu et al., 2013
Insulin resistance ePAHs (air pollutant) Choi et al., 2015
Insulin/AMPK 2-Aminoanthracene Mattis et al., 2014
signaling
Cell cycle/DNA damage
G1/S cell cycle B[a]P/BPDE Volkov et al., 2012; Mukherjee & Kumar, 2013
regulation
G2/M DNA damage B[a]P/B[a]P-7,8-diol/DMBA Guo et al., 2000; Caino et al., 2007; Nogueira et al., 2009
checkpoint
DNA damage ePAHs (coke/tobacco/urban dust) Mahadevan et al., 2005; Duan et al., 2009; Foresta et al., 2014
response
Cytoskeletal regulation and adhesion
Actin function B[a]P/BPDE/BPE, ePAHs (coal tar/DEPs/tobacco) Tuominen et al., 2003; Hooven and Baird, 2008
Cell adhesion B[a]P/Fla/Fluorene/3-Methylcholanthrene/1- van Grevenynghe et al., 2003; Pillai et al., 2003; Bahri et al., 2010; Wu et al., 2011; Croxton
Methylpyrene/Perylene/Ph, ePAHs (coke) et al., 2012; Oh et al., 2014
Microtubule B[a]P Deng et al., 2014
dynamics
Immunology and inflammation
B-cell development DMBA Allan et al., 2003; Ryu et al., 2003; Ryu et al., 2005
Cytokine response B[a]A/B[a]P/DMBA/Fla/3-Nitrobenzanthrone/3- Page et al., 2004; Ovrevik et al., 2010; Kolasa et al., 2013; Hao and Whitelaw, 2013; Kaba tkov a
Nitro-Fla/1-Nitropyrene/Ph/Pyrene, ePAHs (DEPs)  et al., 2015
et al., 2014; Ple
Inflammatory B[a]P/B[e]P, ePAHs (asphalt fume/DEPs/wood smoke Curfs et al., 2005; Gate et al., 2006; den Hartigh et al., 2010; Danielsen et al., 2011; Tsuji et al.,
response particles) 2012; Wittkopp et al., 2013; Totlandsdal et al., 2014; Yang et al., 2016; Totlandsdal et al., 2015
Rheumatoid 3-Methylcholanthrene Tamaki et al., 2004
arthritis
T-dependent B[a]P-7,8-diol/BPDE/DB[a,l]P/Dibenzo[def,p] Li et al., 2010; Lauer et al., 2013
antibody response chrysene/DMBA/DMBA-3,4-diol
TLRs-induced B[a]P Do et al., 2012
immune response
Neuroscience
Behavior/brain 16 PAH mixture peaux et al., 2012
Cre
metabolism
Neurodevelopment B[a]P/Pyrene Wormley et al., 2004; He et al., 2012
Parkinson's disease B[a]P Konstandi et al., 2007
Development and differentiation
Atherogenesis B[a]P Knaapen et al., 2007
B lymphopoiesis DMBA Mann et al., 2001
Development B[a]P/Fla, ePAHs (coastal sediments) Kerambrun et al., 2012; Jayasundara et al., 2015
Differentiation B[a]P/DMBA/DMBA-3,4-diol Ge et al., 2012; Le Vee et al., 2014
Hematopoiesis B[a]P/DMBA N'jai et al., 2011
TGF-b signaling 3-Methylcholanthrene, ePAHs (DEPs) Abdel-Razzak et al., 1994; Pacheco et al., 2001
Wnt/b-catenin DMBA Currier et al., 2005
signaling

The classification of pathways/cascades/functions was described in Kiyama and Zhu (2014). Only representative chemicals are listed for each category. Abbreviations: AhR:
aryl hydrocarbon receptor; AMPK: AMP-dependent protein kinase; B[a]A: benz[a]anthracene; B[a]P: benzo[a]pyrene; B[e]P: benzo[e]pyrene; B[j]A: benz[j]aceanthrylene; B
[k]F: benzo[k]fluoranthene; B[l]A: benz[l]aceanthrylene; BPDE: benzo[a]pyrene diol epoxide; BPE: benzo[a]pyrene-4,5-dihydroepoxide; CPP: cyclopenta[c,d]pyrene; DB[a,l]P:
dibenzo[a,l]pyrene; DEP: diesel exhaust particle; DMBA: 7,12-dimethylbenz[a]anthracene; ER: estrogen receptor; FASL: Fas ligand; Fla: fluoranthene; IGF-1: insulin-like
growth factor 1; IND: indeno[1,2,3-c,d]pyrene; JNK: c-Jun N-terminal kinase; MAPK: mitogen-activated protein kinase; NO: nitric oxide; ROS: reactive oxygen species; Ph:
phenanthrene; PI3K: phosphoinositide 3-kinase; PPAR: peroxisome proliferator-activated receptor.
 Y. Zhang et al. / Environmental Pollution 213 (2016) 809e824 817

den Hartigh et al., 2010); and development and differentiation,
such as TGF-b signaling (Kerambrun et al., 2012).
3.2. Estrogenic activity of ePAHs/PAHs
In an attempt to more clearly understand the endocrine dis-
ruptor activities of ePAHs/PAHs, we searched for estrogenic PAHs
and their signaling pathways (Table 4). Various PAHs, such as
anthraquinone, B[a]A, B[a]P, chrysene, naphthalene, and pyrene,
and their hydroxyl products, including alizarin, 9,10-dihydroxy-B[a]
P, 2-hydroxyanthraquinone, 3-hydroxy-B[a]P, 7-hydroxy-B[a]P, 9-
hydroxy-B[a]P, 2-hydroxychrysene, 2-hydroxyphenanthrene, 9-
hydroxyphenanthrene, 1-hydroxypyrene, 1-naphthol, and 2-
naphthol, were previously reported to be estrogenic (agonistic) or
anti-estrogenic (antagonistic), and signaling pathways, such as
those involving AhR, AKT, BRCA1, MAPK, and phosphoinositide 3-
kinase (PI3K), are known to be involved in estrogen signaling. The
assays used to examine estrogenicity include animal tests, cell as-
says (e.g., cell migration assay, cell proliferation assay, cell-shape
analysis, and cell viability assay), ligand-binding assays, protein
assays (e.g., Western blotting and immunoassay), reporter-gene
assays (e.g., CALUX assay, MVLN-cell assay, and YES assay),
signaling pathway analyses, transcription assays (e.g., RT-PCR and
DNA microarrays), and yeast two-hybrid assays, the time, cost,
sensitivity, accuracy/reliability, safety/ethics, and purpose of which
vary (Kiyama and Zhu, 2014; Kiyama and Wada-Kiyama, 2015). For
example, Arcaro et al. reported that PAH fractions extracted from
environmental samples showed anti-estrogenic activity by ER-
binding and estrogen-metabolism assays, and suggested that the
competition of PAHs against the binding of estrogen to the ER and
the metabolic degradation of estrogen are the mechanisms for the
activity (Arcaro et al., 1999). Meanwhile, a reporter-gene assay us-
ing human breast cancer MCF-7 cells showed that some PAHs, such
as B[a]P and B[a]A, were weak inducers of estrogenic activity
(Vondra cek et al., 2002; Plískov a et al., 2005).
Several groups reported differences in estrogenicity between
PAHs (non-estrogenic) and hydroxyl PAHs (estrogenic), and/or in
the estrogenicity of the same PAHs among the different assays or

Table 4
Estrogenic PAHs and associated signaling pathways.

Chemical Signaling pathway Reference (assaya)

PAH
Alizarin, 2-hydroxyanthraquinone ER Kurihara et al., 2005 (Y)
Anthraquinone, benz[a]acridine, B[a]A -7,12-dione, benzanthrone (estrogenic) ER Machala et al., 2001 (R)
B[a]A (estrogenic) AhR/PKCs/ERK/ERa Kolasa et al., 2012 (S)
B[a]A, B[a]P, B[b]F, B[c]Ph, monohydroxy PAHs (agonistic) ERa Fertuck et al., 2001 (L, R)
B[a]A, B[a]P, Fla (estrogenic); B[k]F (anti-estrogenic) ER Kummer et al., 2008 (A)
B[a]A, B[a]P, DMBA, 3-methylcholanthrene (anti-estrogenic) AhR/ER Chaloupka et al., 1992 (C, P)
B[a]A, dibenz[a,h]anthracene (estrogenic) ER Villeneuve et al., 2002 (R)
B[a]F (estrogenic) ERa Kim et al., 2011b (A, P)
B[a]P, 3-hydroxy-B[a]P, 9-hydroxy-B[a]P, 9,10-dihydroxy-B[a]P (estrogenic) ERa Charles et al., 2000 (R)
B[a]P (anti-estrogenic) AhR/CYP19/ER Patel et al., 2006 (A, P, T)
B[a]P (anti-estrogenic) ER Booc et al., 2014 (A)
B[a]P (estrogenic) ER/MAPK, PI3K/AKT/COX-2 Tsai et al., 2004 (S)
B[a]P (anti-estrogenic) AhR/ER/BRCA1 Jeffy et al., 2002 (C, R, T)
B[a]P, B[b]F, B[k]F, chrysene, dibenz[a,h]anthracene, IND (anti-estrogenic) ER Arcaro et al., 1999 (C, L)
B[a]P, 2,3-benzofluorene, dibenz[a,h]anthracene, 6-hydroxychrysene (anti-estrogenic) ER Tran et al., 1996 (L, R)
B[a]A, B[a]P, dibenz[a,h]anthracene (antagonistic) ER Ohta et al., 2012 (A)
DMBA þ E2 (estrogenic) AhR/CYP1A1/ER Singhal et al., 2008 (A, P, T)
DMBA þ naringenin (anti-estrogenic) AhR/CYP1B1/ER Poon et al., 2013 (P, R, T)
7-Hydroxy-B[a]P, 1-hydroxypyrene (estrogenic) ERa Van de Wiele et al., 2005 (R)
2-Hydroxychrysene, 2-hydroxy-Ph, 1-hydroxypyrene, 1-naphthol, 2-naphthol (estrogenic) ER Wenger et al., 2009 (R)
2-Hydroxyfluorene, 2-hydroxy- Ph, 3-hydroxy-Ph, 1-hydroxypyrene (estrogenic) ER Kamiya et al., 2005 (Y)
9-Hydroxy-Ph, 1-hyroxypyrene, 1-naphthol (estrogenic) ERb Sievers et al., 2013 (L, P, R, T)
3-Methylcholanthrene þ E2 (estrogenic) AhR/CYP1A/ER Navas and Segner, 2000 (P)
3-Methylcholanthrene (estrogenic) ERa Abdelrahim et al., 2006 (L, R, T)
Naphthalene (anti-estrogenic) ER Tintos et al., 2006 (A)
6-Nitrochrysene (anti-estrogenic) AhR/ERa Sun et al., 2013 (A, P)
2-Nitrofluorene (estrogenic) ER Fujimoto et al., 2003 (R)
ePAH
Ambient particulate matter (estrogenic) ER Wenger et al., 2009 (R) (see above)
Bay sediments (estrogenic) ER Koh et al., 2002 (R)
Charcoal flue gas (estrogenic) ER Muthumbi et al., 2003 (R)
Cigarette smoke (estrogenic) ER Kamiya et al., 2005 (Y) (see above)
Coastal sediments (estrogenic/anti-estrogenic) ER Koh et al., 2005 (R)
Coastal sediments/water (estrogenic) ER Weston et al., 2010 (R)
Coastal/river sediments (estrogenic) ER David et al., 2010 (R)
Contaminated oil (anti-estrogenic) ER Aarab et al., 2004 (A, P)
Dispersed oil (anti-estrogenic) ER Tollefsen et al., 2011 (L, P)
Lagoon sediments (estrogenic) ER Amaeze et al., 2015 (R)
Lake sediments (estrogenic) ER Mazurova  et al., 2008 (R)
Lake sediments (estrogenic) ER Garcia-Reyero et al., 2005 (R)
Marine sediments (antagonistic) ER Grung et al., 2011 (R)
Produced water (anti-estrogenic) ER Harman et al., 2010 (A, P)
River sediments (anti-estrogenic) ER Arcaro et al., 1999 (C, L) (see above)
River sediments (estrogenic) ER Hilscherova et al., 2002 (R)
a
Abbreviations for assays are: animal test (A), cell assay (C), ligand-binding assay (L), protein assay (P), reporter-gene assay (R), signaling pathway analysis (S), transcription
assay (T), and yeast two-hybrid assay (Y) (see Kiyama and Wada-Kiyama, 2015). B[a]A: benz[a]anthracene; B[a]P: benzo[a]pyrene; B[b]F: benzo[b]fluoranthene; B[c]Ph: benzo
[c]phenanthrene; B[k]F: benzo[k]fluoranthene; DMBA: 7,12-dimethylbenz[a]anthracene; ERK: extracellular signal-regulated kinase; Fla: fluoranthene; IND: indeno[1,2,3-c,d]
pyrene; Ph: phenanthrene; PKC: protein kinase C.
818 Y. Zhang et al. / Environmental Pollution 213 (2016) 809e824
tissues used (Tran et al., 1996; Arcaro et al., 1999; Fertuck et al.,
2001; Machala et al., 2001; Van de Wiele et al., 2005; Ohta et al.,
2012). These differences may be explained by non-hydroxylated
PAHs being metabolized to estrogenic hydroxyl PAHs by PAH-
inducible CYP enzymes such as aromatase (CYP19), AhR agonists
indirectly interfering with ER-responsive transactivation functions
or directly competing with estrogen, and differences in metabolism
or bioaccumulation in tissues or cells, thereby changing the status
of the estrogenicity of PAHs in cells. The contribution of hydroxyl
PAHs to the estrogenic activity of environmental samples is sup-
ported by the yeast two-hybrid estrogen assay, in which hydroxyl
PAHs, such as 4-hydroxy-B[a]A, 3-hydroxy-B[a]A, and 2-
hydroxychrysense, showed estrogenic activity, whereas their cor-
responding PAHs did not (Hayakawa et al., 2008). However, the
metabolic activation of PAHs is not regulated by ERs in estrogen-
irresponsive, but ER-expressing tissues such as the lung (Berge
et al., 2004). Furthermore, metabolites of PAHs, such as B[b]F, B[k]
F, chrysene, and IND, were previously shown to be anti-estrogenic
in human breast cancer MCF-7 cells (Arcaro et al., 1999), and the
same PAHs examined by the same types of assays (B[a]A and B[a]P
by animal tests) (Ohta et al., 2012) or the same PAHs (for example
DMBA) mixed with different types of estrogens (E2 and naringenin,
Table 4; Singhal et al., 2008; Poon et al., 2013), gave opposite ac-
tivities, suggesting the presence of other mechanisms and/or the
contribution of metabolites other than hydroxyl PAHs to
estrogenicity.
Cases of complicated and unexplainable estrogen actions have
been reported as follows. While B[a]A exposure reduced estradiol
concentrations in fish, no effect was observed in the endocrine
system (Booc et al., 2014). Hydroxy PAHs, such as 9-
hydroxyphenanthrene, 1-hydroxypyrene, and 1-naphthol, showed
even differential actions between ERb (estrogenic) and ERa (non-
estrogenic) (Sievers et al., 2013). So, the balance of the concentra-
tions of estrogenic and anti-estrogenic PAHs (Hilscherova et al.,
2002) or the contribution of other compounds not yet identified
by chemical analysis (Wenger et al., 2009) is important. Further-
more, the activation of ERa was found to occur through a direct
interaction between 3-methylcholanthrene and its receptor in
MCF-7 cells and was independent of AhR (Abdelrahim et al., 2006).
There are also cases in which the contribution of estrogen to the
overall responses of environmental samples containing PAHs was
low (Vondra cek et al., 2001) and other estrogenic chemicals
appeared to contribute to this activity (David et al., 2010). The
contradictory and unexplainable effects observed for ePAHs were
observed not only because of unreproducible and unreliable results
due to technical limitation/immaturity, but also because of the lack
of knowledge about the mechanisms and methods to solve such
problems. We emphasized here the importance of signaling path-
ways to understand the endocrine disruptor action of ePAHs/PAHs
because the outcome-based assessment of the action, such as cell
proliferation, could be influenced by various conditions, such as the
directions of signaling pathways controlled by the amounts of re-
ceptors and signal mediators within the cell and the balance among
cell functions attributable to various environmental factors, which
could result in quite different outcomes. At the level of signaling
pathways, we could monitor the outcome of signaling qualitatively
and quantitatively, and thus, we could obtain more reproducible
and reliable results, which can be used in risk assessment.
3.3. Estrogenic cell signaling induced by ePAHs/PAHs
Estrogenic activity has been detected in ePAHs/PAHs, such as
those extracted from the sediments of various sources (bays, coasts,
lagoons, lakes, rivers, and other marine sites), and from ambient
particulate matter, charcoal flue gas, cigarette smoke, coastal/
produced water, and contaminated/dispersed oil (Table 4). PAHs are
estrogenic or anti-estrogenic, depending on the types and locations
of the samples. Although the mechanisms responsible for their
activities currently remain unclear, several possibilities have been
suggested, such as a direct interaction between PAHs and ERs (es-
trogenic), the AhR-mediated suppression of estrogenic activity
(anti-estrogenic), and the modulation of estrogen or other signaling
pathways (estrogenic or anti-estrogenic), which are based on an-
alyses of specific PAHs (see Section 3.2). For example, Koh et al.
found that bay sediments were estrogenic and attributed this to the
estrogenicity of B[a]A and dibenz[a,h]anthracene (Koh et al., 2002).
This relationship between estrogenicity and PAHs has been
observed in other sediments (Garcia-Reyero et al., 2005; Mazurova 
et al., 2008). However, only a limited number of environmental
samples have exhibited clear estrogenicity (Muthumbi et al., 2003;
Amaeze et al., 2015) or anti-estrogenicity (Harman et al., 2010).
Such inconsistent findings on estrogenicity have been obtained for
other estrogenic chemicals, such as selective ER modulators
(Kiyama and Wada-Kiyama, 2015). Wenger et al. found that only
0.01e0.2% of the overall estrogenic activity of atmospheric partic-
ulate matter was attributed to the major PAHs, 2-
hydroxyphenanthrene, 2-naphthol and 1-naphthol, and suggested
the presence of other estrogenic compounds not yet identified
(Wenger et al., 2009). However, Hilscherova et al. indicated that the
contribution of alkylphenols to estrogenicity in river sediments was
relatively small (Hilscherova et al., 2002). Thus, standardized assay
protocols based on pathway-based assessments are needed in or-
der to overcome these inconsistencies (Kiyama and Wada-Kiyama,
2015).
Since a number of cells do not have hormone receptors, there
should be a mechanism of communication of signaling, or signal
crosstalk, between hormone and other signaling pathways. Among
the cases of signal crosstalk, the one between the AhR and ER has
been extensively studied (Safe and Wormke, 2003); inhibitory
crosstalk between AhR and ER is explained at the gene expression
level as well as by the direct interaction of receptors/signal medi-
ators and metabolic degradation of estrogen. In addition, since the
AhR has been reported to have association with other hormone-
signaling pathways, such as androgen and thyroid hormone
signaling pathways (Ohtake et al., 2011; Murphy et al., 2007), the
involvement of ePAHs/PAHs in the mechanisms of endocrine dis-
ruptor action will be studied with significant attention.
4. Conclusions
We here summarized the biological activities of ePAHs as
follows.
(1) ePAHs are found in the atmosphere, sediments, soils, and
water as a result of human activities, accidents, or natural
phenomena, and are categorized by their sources and forms,
followed by their biological effects and social impact.
(2) ePAHs and their constitutent PAHs have been analyzed by
bioassays, such as animal/plant tests, cell assays, microbial
tests, reporter-gene assays, and other assays, which have
demonstrated that ePAHs/PAHs affect the endocrine systems
of humans and animals.
(3) The mechanisms underlying endocrine disruption induced
by ePAHs/PAHs have been studied based on cell signaling
pathways, such as the MAPK pathway, regulatory mecha-
nisms, including chromatin/epigenetic regulation, cell cycle/
DNA damage control, and cytoskeletal/adhesion regulation,
and cell functions, such as apoptosis, autophagy, immune
responses/inflammation, neurological responses, and devel-
opment/differentiation.
 Y. Zhang et al. / Environmental Pollution 213 (2016) 809e824
(4) Estrogen signaling has been examined in association with
the endocrine-disrupting activities induced by ePAHs/PAHs,
which involve receptors, such as ERs and AhR.
(5) Due to sensitivity, reliability, accessibility (such as time and
cost), and ethical reasons, pathway-based in vitro assays are
becoming more important, and standardized protocols are
needed in order to minimize inconsistencies.
Acknowledgments
This research was supported partly by a Knowledge Cluster
Initiative program and a Grant-in-Aid for Basic Areas from the
Ministry of Education, Culture, Sports, Science and Technology of
Japan, and by the Guideline Program for Medical Device Develop-
ment from the Ministry of Economy, Trade and Industry of Japan.
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