497

Journal of Health Science, 57(6) 497–503 (2011)— Review —Potential Risks of Phthalate Esters: Acquisition of
Endocrine-disrupting Activity during Environmental andMetabolic Processing
Yoshinori Okamoto, Koji Ueda, and Nakao Kojima∗
Faculty of Pharmacy, Meijo University, 150 Yagotoyama, Tempaku-ku, Nagoya 468–8503, Japan(Received September 13, 2011)

Phthalate esters (PEs) are plasticizers used in many plastic products, food packaging materials, and medicalbags. The widespread use of PEs has led to ubiquitous environmental contamination and human-exposure. Several
epidemiological studies have indicated that exposure to PEs, especially during the prenatal period, induces adverse
effects, including developmental and behavioral abnormalities, suggesting that PEs are endocrine-disrupters. Although the endocrine-disrupting effect of PEs is thought to be estrogen receptor (ER)-mediated, the ER-binding
affinity of PEs is quite low, indicating that other mechanisms should be considered. In this review, we demonstrate that alkyl chain length and hydroxylation of PEs have a significant impact on binding to ERs and peroxisome
proliferator-activated receptors. In addition, we discuss recent results from our laboratory that suggest additionalrisks may arise from environmental and metabolic processing of PEs through microbial transformation, microsomal
metabolism, and sunlight exposure.
Key words —— plasticizer, light exposure, microsomal metabolism, microbial biotransformation, estrogen receptor, peroxisome proliferator-activated receptor

INTRODUCTION
Although phthalate esters (PEs) are suspected
endocrine-disrupting chemicals, they are manufactured in large quantities and widely used as plasticizers for plastic products such as food packaging
materials and medical bags. One of the most abundant PEs is di(2-ethylhexyl) phthalate (DEHP), overtwo million tons of which is produced each year
worldwide.1) PEs are not covalently bound to plastic
polymers, and as a result they leach from the matrix
and become widely distributed in the atmosphere2)
and hydrosphere.3, 4) For example, indoor concentrations of DEHP range from 0.04 to 0.23 µg/m3 ,and inside a car may range from 1 to 34 µg/m3 .5)
During medical treatments that involve infusion,
such as hemodialysis and peritoneal dialysis therapies or parenteral nutritional therapy, patients may
be exposed to several milligrams of DEHP frombags and tubes.6, 7) Most importantly, several stud∗

To whom correspondence should be addressed: Faculty of
Pharmacy, Meijo University, 150 Yagotoyama, Tempaku-ku,
Nagoya 468–8503, Japan. Tel.: +81-52-839-2676; Fax: +8152-834-8090; E-mail: kojiman@meijo-u.ac.jp

ies have indicated that infants and toddlers, who
are highly susceptible to PEs, are extensively exposed to these compounds.8– 10) Considerable evidence suggests that many people are routinely exposed to PEs on a daily basis.1, 11, 12)
Many epidemiological studies have suggested
that PE-exposure, especially during the prenatal period, induces a number of adverse effects in humans, such as developmental and behavioral abnormalities.13–15) It has also been reported that PEexposure is correlated with inflammation, oxidative stress, and DNA damage.16–19) The broad effects of PEs have led to speculation that they target hormone receptors, such as estrogen receptors(ERs) and peroxisome proliferator-activated receptors (PPARs). In order to evaluate the relevanceof these receptors with respect to PE-induced adverse effects, it is necessary to understand the environmental and metabolic fate of PEs, especiallybecause of the potential risks associated with environmental and metabolic processing.
Given their widespread use, PEs eventually enter soil or water environments (rivers, lakes, orthe sea), where they may be transformed into ac
C 2011 The Pharmaceutical Society of Japan

498

Fig. 1. Basic Structures of PE, 4-Hydroxylated PE, and Alkyl
Chain Hydroxylated Derivatives

tive derivatives by microbial degradation or exposure to sunlight. PEs are also found in the air
and dust, where they may be transformed into active derivatives by sunlight exposure and then inhaled (air) or ingested (dust) and further metabolized. This review describes the dynamics of theER- and PPAR-mediated effects of PEs, based on
structure-activity relationships. In addition, novel
PE transformation pathways based on model experiments simulating microbial biotransformation,
microsomal metabolism, and sunlight exposure arealso described.

ER-MEDIATED EFFECTS OF
HYDROXYLATED PHTHALATE ESTERS
Numerous in vitro studies employing purified
human ERs, genetically engineered yeast strains,and mammalian cell lines have indicated that PEs
such as benzyl butyl phthalate (BBP), di-n-butyl phthalate (DBP), and di-n-propyl phthalate (DPP) are
estrogenic.20– 28) However, because the estrogenic
activity of these PEs is extremely weak comparedto natural estrogen 17β-estradiol (E2), the implications of these in vitro studies are controversial.29)
Moreover, since all PEs tested are non-estrogenic infemale rats,30, 31) there is considerable uncertainty
regarding interpretation of data from animal experiments and human epidemiological studies.
Although PEs are primarily metabolically pro-Vol. 57 (2011)

cessed through hydrolysis, they may also be hydroxylated by cytochrome P450, as are many xenobiotics. We synthesized several ring- and alkyl chainhydroxylated derivatives (Fig. 1) and assayed theireffect on ER-mediated activity in vitro. In an ERbinding assay using human ERα and ERβ, ring 4hydroxylated PEs (PEs-4OH) bound preferentiallyto ERα, while 3-hydroxylated PEs did not.32) The
binding affinity of PEs-4OH increased with alkyl
chain length as follows: methyl  ethyl < hexyl< propyl < butyl < pentyl < heptyl < octyl for ERα,and methyl  ethyl < propyl < hexyl < pentyl <butyl < octyl < heptyl for ERβ. The contributionof each PE-4OH alkyl chain was observed more effectively when the alkyl chains were substituted at
the 1-position.33) In addition, alkyl chain branching
increased the ER-binding affinity of the PEs-4OH,
while alkyl chain hydroxylation had no effect, suggesting that ring-hydroxylation as well as the structure of the alkyl chain (length and branching) are thekey features influencing the ER-binding of PEs.
Transcription factors such as coactivators
must be recruited to specific genome sequences(estrogen-responsive elements) for the expression of
ER target genes. PEs-4OH with shorter alkyl chains(from methyl to butyl) recruit the coactivator transcriptional intermediary factor 2 (TIF2), whereas
PEs-4OH with longer alkyl chains (from pentyl tooctyl), such as DEHP-4OH, have no transcription
factor recruiting activity.33) Ligand-dependent relocation of helix12, which composes the ligandbinding domain of ERs, determines whether aligand is an agonist or antagonist.34, 35) Longer
alkyl chain PEs-4OH disrupt agonistic relocationof helix12 to the coactivator docking site due tosteric hindrance, thereby precluding coactivatorrecruitment in the same manner as the ER antagonists raloxifene and ICI 182,780.33–35)

PPAR-MEDIATED EFFECTS OF
HYDROXYLATED PHTHALATE ESTERS
Some PEs (e.g., DEHP) have been classified
as peroxisome proliferators because they induce
peroxisomal and hepatocellular proliferation in rodents. These adverse effects are associated with theactivation of PPARs by hydrolyzed PE metabolites
rather than by the parent diester. A primary hydrolyzed metabolite, mono(2-ethylhexyl)phthalate(MEHP), has been identified as the effecter responsible for DEHP-induced abnormalities in ro-

No. 6dents. Experiments using hepatic cell lines demonstrated that MEHP activates PPARα-reporter gene
expression.36, 37) Scintillation proximity binding assay experiments revealed that MEHP interacts with
PPARα.38)
PPARγ is a target in diabetes treatment. PPARγ
agonists such as thiazolidinedione rosiglitazone improve insulin resistance in patients with type 2 diabetes by modifying adipocyte differentiation.39)
Several groups using reporter gene and receptor
binding assays demonstrated that MEHP activates
PPARγ.36–38)
In addition to being metabolized through hydrolysis, PEs can be produced ring- or alkyl chainhydroxylated metabolites.40– 43) Using several oxidized metabolites of DBP and DEHP (Fig. 1),
we examined the coactivator-recruiting activity ofhuman PPARα/γ (hPPARα/γ).44) DBP, monobutyl
phthalate (MBP), mono(4-hydroxybutyl) phthalate [(4)OH-MBP], and MBP-4OH did not induce any coactivator-recruiting (agonistic) activity
in hPPARα, although mono(3-hydroxybutyl) phthalate [(3)OH-MBP] demonstrated a weak ability to induce coactivator recruitment. DEHP and
its metabolites such as MEHP, mono(2-ethyl-5hydroxyhexyl) phthalate [(5)OH-MEHP], MEHP4OH, and DEHP-4OH had little to no ability to induce coactivator-recruiting activity. Based on the
results of a reporter gene assay, Bility et al. (2004)
reported weak agonistic activity of PEs with respect
to hPPARα.37)
To examine antagonistic effects against
hPPARα, we tested inhibition of coactivatorrecruiting activity induced by the PPARα agonist
GW7647.44) Among DBP, DEHP, and their
metabolites, only DBP-4OH demonstrated a dosedependent antagonistic effect. With respect tohPPARγ, DBP induced weak coactivator-recruiting(agonistic) activity, whereas DBP metabolites did
not. The DEHP metabolite MEHP acted as a
PPARγ agonist. These results indicate that hydroxylation of PEs tends to decrease hPPARγ-mediatedeffects. To examine antagonistic effects against
hPPARγ, we examined inhibition of coactivatorrecruiting activity induced by the PPARγ agonist
rosiglitazone.44) Coactivator recruiting was inhibited by DBP, DBP-4OH, MBP, and MEHP, whereas(4)OH-MBP, DEHP, and its oxidized metabolites
had no antagonistic effect.
An epidemiological study revealed that increased inflammation and oxidative stress are correlated with phthalate monoesters, but not with their499

oxidized metabolites.19) Inflammation induced by
PE-exposure may be due to activation of PPARαor inhibition of PPARγ.18, 45) Because hPPARαmediated transactivation are negligible,37, 44) the increased inflammation and oxidative stress resulting
from exposure to PEs might be associated with inhibition of hPPARγ. Such a scenario would explain
the inverse correlation between oxidative stress andexposure to oxidized metabolites.19)

MICROBIAL BIOTRANSFORMATION
Biotransformation of PEs involves the sequential hydrolysis of each ester-alkyl chain, which isaccompanied by the formation of phthalic acid andvarious monoesters.46– 48) However, it is unclear
whether additional transformation products contribute to the toxicity of PEs. Since the biologicaleffects of PEs fluctuate depending on the length anddegree of branching of the alkyl chain,32, 33, 37, 44) it
is important to determine the environmental fate ofthese alkyl chains in order to perform adequate human and environmental risk assessments.
Our group isolated PE-degrading microbes fromriver water (Tempaku River, Nagoya, Japan) anddetermined their ability to biodegrade diethyl phthalate (DEP), DPP, DBP, and DEHP.3) We foundthat biotransformation of DBP resulted in the formation of dimethyl phthalate (DMP), monomethyl
phthalate (MMP), MBP (a typical DBP-degradation
product), and an unknown diester. This suggests theexistence of a novel PE biotransformation pathway,
and it is possible that the above-mentioned transformation products contribute to the adverse effects
associated with PEs. The unknown diester was also
produced in the biotransformation of DPP, althoughthe HPLC retention time of the unknown diester wasdifferent from that of DBP. Based on LC/MS and1 H-NMR analyses, the unknown diester biotransformation product of DPP was identified as methyl
propyl phthalate. Asymmetrical diesters have also
been reported by two other groups. Cartwright andco-workers (2000) reported that soil bacteria degrade DEP to ethyl methyl phthalate in the presence of CH3 OH.49) Without performing additional
experiments, they concluded that ethyl methyl phthalate was produced by the demethylation of theethyl group. Kim et al. (2002) reported the formation of butyl methyl phthalate during the biodegradation of benzyl butyl phthalate by fungal cutinase
and yeast esterase in the presence of CH3 OH.50) To

500Vol. 57 (2011)

determine the source of the methyl group in methyl
propyl phthalate, we carried out a stable-isotope
tracer experiment using CD3 OH as a PE-solvent.48)
An HPLC peak with a retention time consistent withmethyl propyl phthalate was detected in the experiment; however, the molecular weight of the compound as determined by LC/MS was 3 mass units
larger than that of methyl propyl phthalate. Furthermore, no 1 H-NMR signal that could be assigned
as the methyl ester group of methyl propyl phthalate was observed. The use of dimethyl sulfoxide
as DPP-solvent in the biotransformation experimentprecluded observation of methyl propyl phthalate
in the HPLC chromatogram. These data indicatedthat the methyl ester group is derived from CH3 OH.
Asymmetrical diesters can be produced via esterification of phthalate monoesters or enzymatic/nonenzymatic transesterification of phthalate diesters.
However, biotransformation of phthalate monoesterin the presence of CH3 OH and the absence of microbes in the cultivation medium did not result in
the formation of asymmetrical diesters. Asymmetrical diesters are thus thought to be formed via enzymatic transesterification of phthalate diester andalcohol.

MICROSOMAL METABOLISMAs the biological effects of PEs vary depending upon structural modifications, the identificationand detailed analysis of PE metabolites is important.
PEs are primarily hydrolyzed by esterases to produce monoesters. Novel metabolites resulting frommicrosomal metabolism of DMP have also been
identified.43) Based upon UV spectra and GC/MS
analyses, these microsomal metabolites were identified as ring 3- or 4-hydroxylated DMP. The potent
ER-mediated activity of PEs-4OH suggests that PEs
acquire ER-mediated activity via metabolic processing. Inhibition of PE-4OH production in rat
liver microsomes by the nonspecific P450 inhibitor
SKF525A suggests this is probably a result of oxygenation by cytochrome P450 enzymes (Okamoto
et al., unpublished data).

EXPOSURE TO SUNLIGHT
PEs contaminating the air and water environments may be exposed to sunlight, resulting in theformation of photodegradation products. While several groups have investigated the feasibility of removing PEs from water using light-irradiation andphoto-catalytic materials,51, 52) until recently very
little was known about the potential toxicity of photodegradation products. To the best of our knowledge, our group was the first to describe the toxicity of PE photodegradation products formed by sunlight exposure.53) We found that several photoproducts are formed after DPP is exposed to sunlight or
UV irradiation. One product showed unequivocal
estrogenic activity, whereas DPP itself did not. Using UV and LC/MS analyses, we determined thatthe estrogenic photoproduct was DPP-4OH, whichis generated by exposure of DPP to UV irradiation
at 290 nm. Production of DPP-4OH is accompaniedby H2 O2 generation in a UV dose-dependent manner (i.e., longer exposure to UV leads to increased
H2 O2 generation). Both DPP and UV irradiation
are required for the generation of H2 O2 . Additionof H2 O2 to the DPP solution increased DPP-4OH
production under UV irradiation. DPP-4OH production was also observed in the reaction of DPP
with the Fenton reagent, generating hydroxyl radicals without UV irradiation. Based on these results,
we propose that as a result of PE-mediated photosensitization, H2 O2 is generated from O2 and H+
and then decomposes to hydroxyl radicals, thus oxidizing the PE benzene ring. The production of DPP4OH as a result of sunlight exposure represents asignificant problem since PEs-4OH are remarkably
active estrogenic products and would be expected to
be involved in ER-mediated endocrine disruption.

CONCLUSION
In this review, we described how benzene ring
and alkyl chain modifications (hydroxylation, chain
length, and branching) affect the endocrine disrupting activity of PEs. Ring 4-hydroxylation
plays a key role in the ER-mediated biological effects of PEs, while variation in alkyl chain structure is also important. Hydrolyzed metabolites(monoesters) with long alkyl chains have potent
hPPARγ-mediated biological effects, which are diminished upon oxidation of the PE. The data discussed here help explain the epidemiological observations that indicate exposure to oxidized metabolites is inversely associated with inflammatory andoxidative stress induced by exposure to the parent

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No. 6

PEs. The data also suggest that the environmental
and biological fates of PEs are more complex thanpreviously thought. Therefore, PE risk assessments
should be more comprehensive, taking into consideration potential risks that may arise as a result ofenvironmental and metabolic processing of PEs.
Acknowledgements We thank Ms. Chitose Toda,
Dr. Kazuo Ito, Dr. Kiyomatsu Hashizume, and pastmembers of our laboratory for their assistance.

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