In vitro transformation of chlorinated parabens by the liver S9 fraction: Kinetics, metabolite identification, and aryl hydrocarbon receptor agonist activity

Article abstract of DOI:10.1248/cpb.c15-00977

We investigated the kinetics of in vitro transformation of a dichlorinated propyl paraben (2-propyl 3,5-dichloro-4-hydroxybenzoate; Cl₂PP) by the rat liver S9 fraction and assessed the aryl hydrocarbon receptor (AhR) agonist activity of the met

Full text of DOI:10.1248/cpb.c15-00977

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Chem. Pharm. Bull. 64, 650–654 (2016)
Vol. 64, No. 6
Note
In Vitro Transformation of Chlorinated Parabens by the Liver S9
Fraction: Kinetics, Metabolite Identification, and Aryl Hydrocarbon
Receptor Agonist Activity
,a,b
Masanori Terasaki,* Takeshi Wada,b Satoshi Nagashima,b Masakazu Makino,b and
Hiro Yasukawac
a Department of Environmental Sciences, Faculty of Humanities and Social Sciences, Iwate University; 3–18–34
b
Ueda, Morioka 020–8550, Japan: Graduate School of Integrated Pharmaceutical and Nutritional Sciences,
c
University of Shizuoka; 52–1 Yada, Suruga-ku, Shizuoka 422–8526, Japan: and Academic Group of Applied Life
Sciences, Iwate University; 3–18–34 Ueda, Morioka 020–8550, Japan.
Received December 1, 2015; accepted March 11, 2016
We investigated the kinetics of in vitro transformation of a dichlorinated propyl paraben (2-propyl
3,5-dichloro-4-hydroxybenzoate; Cl2PP) by the rat liver S9 fraction and assessed the aryl hydrocarbon re-
ceptor (AhR) agonist activity of the metabolite products identified in HPLC and GC/MS analysis and by
metabolite syntheses. The results indicated that the chlorination of Cl2PP reduced its degradation rate by
approximately 40-fold. Two hydroxylated metabolite products showed AhR agonist activity of up to 39%
of that of the parent Cl2PP when assessed in a yeast (YCM3) reporter gene assay. The determination of the
metabolic properties of paraben bioaccumulation presented here provides further information on the value
of risk assessments of chlorinated parabens as a means to ensure human health and environmental safety.
Key words preservative; disinfection by-product; metabolic reaction; standard chemical synthesis; aryl hy-
drocarbon receptor
Alkyl esters of 4-hydroxybenzoic acid (parabens) are urine.8) However, not all metabolite products and metabolic
widely used as preservatives in pharmaceuticals and personal reactions that can arise from paraben exposure in mammals
care products, because they are stable over a wide pH range, have been chemically determined. Moreover, the biological ac-
are sufficiently soluble in water, and also exhibits a broad tivity especially the endocrine disrupting properties, of these
spectrum of antimicrobial activity.1) In Japan, parabens are metabolites, are most often not known. The main reason for
permitted for use in all cosmetic products at concentrations up this is the lack of chemical standards to support identification
to 1% (w/w). of all metabolite products arising from bio-transformations of
Compounds, including parabens, that contain phenolic hy- parabens.
droxyl groups exhibit favorable chlorination kinetics; thus,
The aim of the present study was to carry out an assess-
parabens are easily chlorinated by chlorine in tap water and ment of the metabolic kinetics of in vitro transformation of
by sodium hypochlorite, a commonly used bleaching agent.2) chlorinated paraben by a rat liver S9 fraction and to identify
In recent years, the increasing use of parabens has led to associated metabolite products and their toxicities. AhR ago-
concerns related to the potential for adverse effects associated nists only need to meet minimal requirements in hydropho-
with their widespread occurrence in the environment. Several bicity, size, and planar shape to fit into the receptor-binding
monitoring studies have now detected chlorinated parabens in pocket to induced endocrine disrupting properties.9,10) Here,
effluents, pool water, and even river water.3,4)
we focused particularly on paraben metabolites containing a
Organochlorine compounds such as 2,3,7,8-tetrachloro- phenolic hydroxyl group. AhR agonist activity was measured
dibenzo-p-dioxin (TCDD) and polychlorinated biphenyls using a yeast reporter gene assay. For our model compound,
(PCBs) are hydrophobic and persist in the environment for we selected the dichlorinated derivative (Cl2PP) of propyl
significant periods of time. This exposure and persistence may paraben, the most frequently used paraben in pharmaceuticals
adversely affect humans and other organisms. The toxicity of and personal care products (Fig. 1). Cl2PP has been detected
TCDD and PCBs is associated with their interaction with the at levels up to 0.18nM in pool and river water in Japan.3,7)
aryl hydrocarbon receptor (AhR), which is a ligand-dependent
transcription factor.5) Activation of AhR by TCDD and dioxin-
like compounds can induce various biological responses, in-
cluding activation of the CYPs system (particularly CYP1A1)
and disruption of normal hormone signaling pathways, and
can lead to developmental and reproductive toxicities, immu-
notoxicity, and mutagenicity.6) Recently, chlorinated parabens
were suggested to effect toxicity through interaction with
AhR.7) Parabens are hydrolyzed mainly to 4-hydroxybenzoic
acid in mammals; this metabolite can thereafter be conjugated
with sulfate, glucuronide, or glycine, prior to excretion in
Fig. 1. Chemical Structure for Dichlorinated Propyl Paraben (Cl2PP)
and Its Transformed Products (1)–(6) by a Rat Liver S9 Fraction
*To whom correspondence should be addressed. e-mail: terasaki@iwate-u.ac.jp
© 2016 The Pharmaceutical Society of Japan

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651
Fig. 2. Time–Course Plots of Propyl Paraben (PP) and Cl2PP in the
Presence of the Liver S9
Fig. 3. GC/MS Total Ion Chromatograms (TIC) of the Extractives of
Metabolites of 60min Metabolic Reaction
Cl2PP shows AhR agonist activity (EC25=1300 nM) and more
acute toxicity toward invertebrates (EC50=33 µM) than that by
the corresponding parent paraben.7,11) Cl2PP is a common and
toxic paraben in the water environment and it is therefore im-
portant to evaluate its metabolic fate.
Results and Discussion
Kinetics To investigate the degradation rate of propyl
paraben and its chlorinated derivative ln(C/C0) values were
plotted against reaction time, as shown in Fig. 2. Correlation
coefficients (r2) were 0.864 and 0.915, respectively. From these
values, it was established that the metabolic reaction for the
paraben and its chlorinated derivative is first order. The rate
constant k and the half-life t1/2 for the paraben were 0.800 per
min and 0.87min, respectively; for the chlorinated derivative,
the corresponding values were 0.0195 per min and 36min,
respectively. The degradation rate of chlorinated paraben was
1/40-fold that of the paraben in the rat liver S9 fraction treat-
ment; the results suggested that chlorination of the paraben
may enhance accumulation of the paraben in the biota.
Syntheses and Identification of Metabolites The reac-
tion products of Cl2PP after exposure to in vitro transforma-
tion by a rat liver S9 fraction were assessed by GC/MS. The
total ion chromatogram (TIC) and the MS data for trimethyl-
silyl (TMS) derivatives of the reaction products, and the asso-
Fig. 4. Mass Spectra and Retention Time (R.T.) of Metabolites 2–6
Numbers refer to the peak numbers of Fig. 3.
The molecular ion peaks (M+) at m/z 408, observed in the
ciated synthetic compounds (which fulfill structural criteria spectra of peaks, 3, 5, and 6 indicated the substitution of an
for endocrine disrupting properties) are shown in Figs. 3 and –OH group with a H atom in the TMS derivative of Cl2PP.
4. In the MS spectrum of peak 2, the molecular ion peaks The MS spectrum of peak 4 was dominated by the mass frag-
(M+) at m/z 374 and m/z 376 presented the typical isotopic ment ions at m/z [M+−15], resulting from the loss of a methyl
pattern of a mono-chlorinated compound and indicated the moiety in the derivatives rather than by the molecular ion
substitution of an –OH group with a Cl atom in the TMS peaks (M+). Each of the ion peaks at [M+−15] and (M+) for
derivative of Cl2PP. We therefore identified peak 2 as propyl all products also presented the typical isotopic pattern corre-
3-chloro-4,5-dihydroxybenzoate (2) and completed its synthe- sponding to dichlorinated compounds. We therefore completed
sis (see Experimental).
the synthesis of four compounds that fit these analytical data.

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Chem. Pharm. Bull.
Vol. 64, No. 6 (2016)
Table 1. AhR Activities of Cl2PP and Its Metabolites (1)–(6) in the
Yeast Reporter Gene Assay
AhR is widely expressed in mammalian tissues and it af-
fects a number of fundamental cellular processes. Modulation
of AhR function by xenobiotics, even at low concentrations,
may ultimately result in a variety of adverse effects such as
hepatotoxicity, immunotoxicity, neurotoxicity, dermal toxic-
ity, teratogenicity, and carcinogenicity in many different spe-
cies.13) Moreover, activation of AhR by xenobiotics modulates
a number of endocrine systems, most notably by interfering
with estrogen signaling. Consequently, there has been an
extensive analysis of the role of AhR in estrogen-dependent
breast cancer cells. Ohtake et al. investigated AhR-estrogen
receptor (ER)α crosstalk in vitro and in vivo using 3-meth-
ylcholanthrene (3MC) as an AhR ligand.14) Inhibitory effects
were observed for some responses when 17β-estradiol (E2)
and 3MC were co-administered; it was concluded that 3MC
AhR assay
Cmpd
EC25 (nM)
RAb)
0.39
1
Not active
Not active
Not active
3400 1400
Not active
7800 300
1900 230c)
6.84 1.2c)
2
3
4
5
6
0.18
1.0
Cl2PP
BNFa)
520
a) Positive control. b) Relative activity for Cl2PP. c) Data reported in ref. 7.
These were isopropyl 3,5-dichloro-2,4-dihydroxybenzoate (3), inhibited E2-induced estrogenic responses. Another xenobi-
2-hydroxypropyl 3,5-dichloro-4-hydroxybenzoate (4), 2-hy- otic, the polyaromatic hydrocarbon 6-methyl-1,3,8-trichlo-
droxy-1-methylethyl 3,5-dichloro-4-hydroxybenzoate (5) and rodibenzofuran (MCDF), which is related to the industrial
3-hydroxypropyl 3,5-dichloro-4-hydroxybenzoate (6).
by-product dioxin, has been shown to be a weak agonist of
The identification and quantification of peaks on the TIC AhR when tested in a murine reporter cell line (0.1% of the
was confirmed by comparing their gas chromatography (GC) activity of β-naphthoflavone (BNF)).15) Studies in vitro have
retention times, mass spectra, and the peak area to those of demonstrated that MCDF has a proteasome-dependent degra-
commercial (1) or synthetic standards (2)–(6). The relative dative effect on ER protein levels.16,17) Therefore, our current
abundances of metabolites are 48% for (1), 32% for (5), 8.7% data warrant further studies to investigate AhR-ER crosstalk
for (2), 5.7% for (6), 3.3% for (4), and 2.0% for (3). Maximum in the presence of Cl2PP. Some chlorinated parabens occur
abundances of individual metabolite were observed after widely in aquatic environments and, as such, organisms may
120min reaction with the rat liver S9 fraction.
be continuously exposed. The determination of the metabolic
AhR Agonist Activity of Metabolites Table 1 shows the properties of chlorinated parabens can provide information on
AhR agonist activities of the metabolites as measured using their potential for bioaccumulation and whether a xenobiotic
the yeast reporter gene assay. Two hydroxylated metabolite is likely to be converted to a more potent or less potent toxin.
products (4) and (6) of the dichlorinated propyl paraben It is probable that risk assessments of chlorinated parabens to
showed AhR agonist activity, when assessed in the yeast ensure human health and environmental safety will require
(YCM3) reporter gene assay, of up to 18 to 39% of that of analysis of all their metabolite products in the manner exem-
Cl2PP.
The S9 rat liver preparation contains a range of enzyme
plified here.
activities, which may eliminate AhR agonist activity through,
Experimental
for example, conjugating phenolic hydroxyl groups (e.g.,
Materials Propyl paraben was purchased from Tokyo
by glucuronidation) and/or hydroxylating aromatic rings or Chemical Industry, Tokyo, Japan. Cl2PP was synthesized
side-chains. Conjugation of the phenolic hydroxyl group and using previously reported procedures (purity (GC)>99%).4)
hydroxylation of the aromatic ring eliminates AhR agonist The male Sprague-Dawley rat liver S9 mix induced with phe-
activity but the hydroxylation of the side chain induces agonist nobarbital and 5,6-benzoflavone was purchased from Kikko-
activity. Our results indicated that the AhR agonist activity of man Corporation, Noda, Japan. D-Glucose 6-phosphate disodi-
Cl2PP remained after exposure to the rat liver S9 fraction.
um salt (G-6-P-Na2) and β-nicotinamide-adenine dinucleotide
The AhR agonist activity of halogenated aromatics is struc- phosphate, oxidized form, monosodium salt (β-NADP+-Na)
ture-dependent. The presence of a halogen on the aromatic were purchased from Oriental Yeast Company Limited,
ring, for example, is required for receptor activation, and Osaka, Japan. Dimethyl sulfoxide (DMSO) was purchased
factors that influence the planarity of the molecule can deter- from Dojindo Laboratories, Kumamoto, Japan and was of
mine affinity for receptor binding.5) In the metabolic reaction fluorometric grade. N,O-Bis(trimethylsilyl)-trifluoroacetamide
conducted in this, compounds (4) and (6) retained the 2,3-di- (BSTFA) was purchased from Wako Pure Chemical Indus-
chloro-4-hydroxy benzoic unit and the linear propyl side chain tries, Ltd., Osaka, Japan. All chemicals used were of analyti-
present in Cl2PP; as such, they retained AhR agonist activity cal grade or HPLC grade.
induced by Cl2PP. On the other hand, hydroxylation at the
Propyl 3-chloro-4,5-dihydroxybenzoate (2) was synthe-
aromatic ring as observed in compounds (2) and (3), as well sized via 3 steps from chlorinated hydroxybenzoate (a–c in
transformation from a linear to branched-chain are believed Chart 1). Isopropyl 3,5-dichloro-2,4-dihydroxybenzoate (3)
to minimize planarity and thus reduce binding to AhR. Mo- was synthesized via the 2 steps from commercially avail-
lecular docking of TCDD congeners to a model of the human able hydroxybenzoic acid (d–e in Chart 1). 2-Hydroxypropyl
AhR-ligand binding domain has suggested that hydrophobic 3,5-dichloro-4-hydroxybenzoate (4) was synthesized by es-
contacts would strengthen the H-bond network, which in turn terification of 3,5-dichloro-4-hydroxybenzoic acid (1) sus-
would energetically favor ligand binding.12) Carboxylic groups pended in benzene with 1-bromo-2-propanol in the presence
also enhance polarizability and can favor interaction between of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) as the catalyst
the AhR and ligands as in the case of compound (1).
under an atmosphere of nitrogen (f in Chart 1). 2-Hydroxy-

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653
(a) HNO3/AcOH, r.t., 30min; (b) Fe/NH4Cl, EtOH, reflux, 6h; (c) HCl, NaNO2, H2O, 0°C/heat; (d) SO2Cl2/AcOH, r.t., 1h; (e) 2-PrOH/HCl, reflux, 6d; (f) DBU, benzene,
reflux, 24h; (g) Ph3P, DEAD, THF, r.t., 14h; (h) Zn(CH3COO)2, 90°C, 2h.
Chart 1
1-methylethyl 3,5-dichloro-4-hydroxy-benzoate (5) was syn- time-of-flight (HR-TOF)-MS (M+−H): 229.0348 (Calcd for
thesized by chemoselective benzoylation of 1,2-propanediol C10H10O345Cl: 229.0268), 231.0313 (Calcd for C10H10O437Cl:
using 1.5M equivalent of triphenylphosphine (Ph3P) and di- 231.0238).
ethyl azodicarboxylate (DEAD)18) (g in Chart 1). 3-Hydroxy-
propyl 3,5-dichloro-4-hydroxy-benzoate (6) was synthesized
by a transesterification of chlorinated 4-hydroxybenzoate19) (h
in Chart 1). Analytical data for synthetic metabolites and their
intermediate substances are as follows:
Compound (3b)
Yield 63%. H-NMR (CDCl3) δ: 7.80 (1H, s).
Compound (3)
1
1
Yield 12%. Purity (GC)>99% H-NMR (CDCl3) δ: 1.39
(6H, d, J=6.3Hz), 5.24–5.32 (1H, sept, J=1.3Hz), 7.78 (1H,
Compound (2b)
s). HR-TOF-MS (M+–H): 262.9872 (Calcd for C10H9O435Cl2:
Yield 89%. 1H-NMR (CDCl3) δ: 1.04 (3H, t, J=7.5Hz), 262.9870), 264.9844 (Calcd for C10H9O345Cl37Cl: 264.9840),
1.77–1.84 (2H, m), 4.30 (2H, t, J=6.8Hz), 8.28 (1H, d, 266.9818 (Calcd for C10H9O347Cl2: 266.9820).
J=2.5Hz), 8.60 (1H, d, J=2.0Hz). MS m/z: 261 (M++2), 259
Compound (4)
(M+).
Yield 67%. Purity (GC)>99% H-NMR (CDCl3) δ: 1.26
(3H, d, J=5.5Hz), 4.12–4.30 (3H, m), 7.94 (2H, s). HR-TOF-
1
Compound (2c)
Yield 96%. 1H-NMR (500MHz, CDCl3) δ: 1.02 (3H, t, MS (M+−H): 262.9842 (Calcd for C10H9O435Cl2: 262.9870),
J=7.5Hz), 1.72–1.79 (2H, m), 4.20 (2H, t, J=10Hz), 6.72 (1H, 264.9841 (Calcd for C10H9O345Cl37Cl: 264.9840), 266.9812
d, J=8.0Hz), 7.40 (1H, d, J=2.0Hz). MS m/z: 231 (M++2), (Calcd for C10H9O437Cl2: 266.9820).
229 (M+).
Compound (5)
Yield 51%. Purity (GC) 97% H-NMR (CDCl3) δ: 1.48 (3H,
1
Compound (2)
1
Yield 57%. Purity (GC)>99% H-NMR (CDCl3) δ: 0.93 d, J=6.5Hz), 3.77–3.85 (2H, m), 5.22–5.29 (1H, m, J=6.0Hz),
(3H, t, J=7.3Hz), 1.63–1.70 (2H, m), 4.11 (2H, t, J=6.8Hz), 8.00 (2H, s). HR-electron ionization (EI)-MS (M+): 263.9955
7.26 (1H, d, J=1.5Hz), 7.37 (1H, d, J=1.5Hz). High resolution- (Calcd for C10H10O345Cl2: 263.9956), 265.9928 (Calcd for