Byproducts of Aqueous Chlorination of Equol and Their Estrogenic Potencies

Chemosphere 212 (2018) 393e399

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Byproducts of aqueous chlorination of equol and their estrogenic potencies

Yuyin Zhou a, Shiyi Zhang a, Fanrong Zhao a, Hong Zhang a, Wei An b, Min Yang b, * Zhaobin Zhang a, Jianying Hu a, a Laboratory for Earth Surface Processes, College of Urban and Environmental Sciences, Peking University, Beijing 100871, China b State Key Laboratory of Environmental Aquatic Chemistry, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China highlights

Behaviors of equol in the chlorination disinfection process were investigated. Chlorination mechanisms of equol were provided. Chlorinated equols can elicit similar estrogenic activity to equol. article info abstract

Article history: While the phytoestrogen metabolite equol has been reported to exist in surface water, its behavior in Received 30 May 2018 drinking water treatment plants remains unrevealed. In this study, eight products including four chlo- Received in revised form rinated equols (monochloro-equol, dichloro-equol, trichloro-equol, and tetrachloro-equol) were identi- 8 August 2018 ﬁed in an aqueous chlorinated equol solution by UHPLC-quadrupole-orbitrap-HRMS. Two main pathways Accepted 19 August 2018 of chlorination reaction are proposed: (1) chlorine-substitution reactions on the aromatic ring and Available online 20 August 2018 subsequent dehydration to form the chlorine-substituted equols, and (2) break-up of the heterocyclic Handling Editor: Maruya ring with oxygen followed by oxidation of aldehyde to carboxyl. The human estrogen receptor (hER) activating activity for monochloro-equol (EC50 ¼ 3456 nM) and dichloro-equol (EC50 ¼ 2456 nM) were Keywords: slightly stronger than that of equol (EC50 ¼ 3889 nM). This is the ﬁrst report on the behavior of equol in By-product drinking water chlorination, which provided an important information on the risk assessment of equol in Chlorination drinking water. Chlorinated equols © 2018 Elsevier Ltd. All rights reserved. Estrogenic activity Drinking water

1. Introduction readily available in the diet, particularly in soy products, of which the major class of phytoestrogens is isoflavones. Equol is a bacterial Some phenolic chemicals have received attention for their metabolite of phytoestrogen daidzin or daidzein, one of the prin- ability to mimic naturally occurring estrogens and interfere with cipal isoflavones in soybeans.(Glycine max)(Setchell et al., 2002; endocrine systems of wildlife and human beings (Krishnan et al., Hoerger et al., 2009). Some studies have shown that the estrogenic 1993; Routledge and Sumpter, 1996; Miller et al., 2001; Hu and activity of equol is more potent than its precursors, daidzin and Aizawa, 2003; Kudo et al., 2006; Wang et al., 2016). Of these daidzein, and its strong estrogenic activity has been observed in chemicals, naturally occurring phytoestrogens are currently animals (Wang et al., 2016; Setchell et al., 2002; Tang and Adams, receiving significant attention due to their multiple health effects 1980). Equol has also been reported to have antiandrogenic prop- (Knight and Eden, 1996; Kuiper et al., 1998). These chemicals are erty, and could induce intersex incidence in male medaka at environmentally relevant concentrations (Wang et al., 2016). In human, it has been reported that exposure to equol could induce DNA damage in sperm and decrease sperm motility (Anderson et al., * Corresponding author. College of Urban and Environmental Sciences, Peking 2003; Toshima et al., 2012). University, Beijing 100871, China. E-mail address: [email protected] (J. Hu). Equol is exclusively a product of intestinal bacterial metabolism, https://doi.org/10.1016/j.chemosphere.2018.08.103 0045-6535/© 2018 Elsevier Ltd. All rights reserved. 394 Y. Zhou et al. / Chemosphere 212 (2018) 393e399 and is relatively stable once formed (Setchell et al., 2002). The 2.2. Computational chemistry transformation from daidzin to equol can occur in humans. It has been estimated that about 25e30% western adults and about Scigress (ultra version 2.2.0, Fujitsu, USA) was used to obtain 50e80% asian adults are equol producer, which means they excrete optimum geometries and partial atomic charges. The AM1 equol in urine after taking soy foods (Setchell and Cole, 2006; parameter was used to optimize stable and transition-state Setchell et al., 1984; Kelly et al., 1995; Karr et al., 1997; Lampe et al., structures. 1998; Rowland et al., 2000). Notably, this transformation usually occurs in the intestine of domestic animals (Lundh, 1995; Setchell 2.3. Chlorination procedures and Clerici, 2010), and excreta of livestock have been reported to be a main source of equol in the aqueous environment since soy- Chlorination experiment was carried out using previously re- bean meal is the primary dietary protein source for domestic ani- ported method (Hu et al., 2002b; Fan et al., 2013), and the details mals (Hoerger et al., 2009; Furuichi et al., 2006; Yost et al., 2013). are shown in the Supplementary Data. The manure of domestic animals is often applied onto crop fields and pastures for its nutrient value, and therefore equol can enter 2.4. UHPLC-quadrupole-orbitrap-HRMS analysis aquatic environments through runoff from pastures or fields. It has been reported that equol occurred with concentrations of Identification of chlorinated byproducts of equol was performed 34e121 ng/L in runoff from a pasture containing 43% red clover by an UltiMate 3000 system (Thermo Fisher Scientific, San Jose, CA, (Trifolium pratense)(Burnison et al., 2003). Due to discharge of USA) with a quadrupole-orbitrap-high resolution mass spectrom- livestock waste and runoff from these sources, equol has been eter. Separation of equol and its chlorinated byproducts was detected in rivers throughout Switzerland (0.6e524 ng/L) (Hoerger achieved by using an Acquity UPLC BEH C18 column et al., 2009; Erbs et al., 2007). While these previous papers have (100 2.1 mm 1.7 mm) (Waters, Milford, MA, U.S.) maintained at highlighted the occurrence of equol in river body, there has been no 40 C. Mobile phases A and B were ultrapure water and acetonitrile, study on its fate through drinking water treatment processes respectively. A linear gradient was: 0 min, 10% B; 0.5 min, 40% B; (Snyder and Trenholm, 2008). It is well-known that phenolic 5 min, 75% B; 5.5 min, 98% B; 8 min, 100% B; 9.5 min, 100% B and compounds can easily react with hypochlorite, which is widely 11 min, 10% B. The injection volume and the flow rate were 5 mL and used as a disinfection agent in drinking water treatment plants 300 mL/min, respectively. Detailed information of mass spectrom- (DWTPs), as exemplified by the rapid chlorination of 4- eter is shown in the Supplementary Data. nonylphenol (NP), bisphenol A (BPA), and 17b-estradiol (17b-E2) (Hu and Aizawa, 2003; Hu et al., 2002a, 2002b). Thus, as one of the 2.5. Estrogenic activity of products class of phenolic chemicals, it is of interest to investigate the fate of equol during chlorination, the presence of its potential chlorinated In order to assess the human estrogen receptor a (hERa) acti- byproducts, as well as whether the byproducts still pose estrogenic vating activity for chlorinated equols, 3 major chlorinated equols in activity. the aqueous chlorination experiments (MCequol, DCequol, and In this study, the products of equol in aqueous chlorination were TCequol) were synthesized (detailed synthesis process is described identified by an ultra-high performance liquid chromatography in supplementary materials) and dissolved in dimethyl sulfoxide (UHPLC)-quadrupole-orbitrap- high resolution mass spectrometry (DMSO). The hERa activating activity of these 3 chlorinated equols (HRMS) method, and two pathways were proposed. Major together with equol were evaluated using a yeast two-hybrid assay byproducts, 3 chlorinated equols, monochloro-equol (MCequol), with hERa and coactivator TIF2 according to a method described dichloro-equol (DCequol), and trichloro-equol (TCequol), were previously (Hu et al., 2002a). 17b-E2 was used as the positive synthesized, and their estrogenic activities were also determined control. using a yeast two-hybrid assay. 2.6. Statistical analysis

Thermo Scientific Sieve software (Thermo Fisher Scientific, San 2. Materials and methods Jose, CA, USA) was used to conduct a comprehensive search of byproducts and speed up the process, by simultaneously comparing 2.1. Standards and reagents MS spectra to find differentially expressed feature.

Equol and the surrogate standard equol-d4 were purchased 3. Results and discussion from Apollo Scientific Ltd. (Stockport, UK) and Toronto Research Chemicals Inc. (Toronto, Canada), respectively. MCequol, DCequol, 3.1. Identification of byproducts and TCequol were synthesized and purified using the methods reported previously (detailed information in the Supplementary For the screening of potential byproducts of equol using UHPLC- Data)(Hu and Aizawa, 2003; Hu et al., 2002a, 2002b; Fan et al., quadrupole-orbitrap-HRMS analysis, the chromatograms in total 2013). All commercial and synthesized compounds have a purity ion current (TIC) mode of chlorinated equol solutions at different of over 95%. Sodium sulfite, sodium thiosulfate, sodium hydroxide, chlorination time were compared with that before chlorination magnesium sulfate, hydrochloric acid (36e38 wt/%), phosphoric (Fig. 1). After 10 min of reaction, equol concentration was decreased acid, and sodium hypochlorite solution (>8% available chlorine) from 500 to 44.9 mg/L, and meanwhile several new peaks were were analytical grade and purchased from Beijing Chemical Works observed in TIC chromatograms (Fig. 1). Sieve analysis was used to (Beijing, China). Methanol, acetonitrile and ethyl acetate were compare TIC chromatograms at 0 min and 10 min and screen HPLC grade and purchased from Fisher Chemical Co. (Beijing, possible byproducts. A total of 8 products were screened, and their China). HPLC-grade water was prepared using a Milli-Q RC appa- retention times, elemental compositions, experimental and theo- ratus (Millipore, Bedford, MA, USA). Oasis HLB (500 mg, 6 mL) retical exact masses, and relative mass measurement errors (Dm) cartridges were purchased from Waters (Waters, Milford, MA, are listed in Table 1, while Fig. 2(a) and (b) show the chromato- U.S.). grams and mass spectra of all byproducts. All of the eight Y. Zhou et al. / Chemosphere 212 (2018) 393e399 395

Fig. 1. Total ion current chromatogram of chlorinated equol solution: (a) before chlorination; (b) with chlorination (time, 10 min).

Table 1 Retention times, experimental and theoretical exact masses, proposed chemical formulas and relative mass measurement errors (Dm) of [MH] ions of equol and its byproducts.

e Compound tretention (min) Theoretical m/z Proposed formula [M H] Experimental m/z Dm (ppm)

equol 2.41 241.08702 C15H13O3 241.08649 0.569 MCequol 2.84 275.04805 C15H12O3Cl 275.04788 0.383 DCequol 3.33 309.00907 C15H11O3Cl2 309.00894 0.415 TCequol 3.84 342.97010 C15H10O3Cl3 342.96994 0.388 TeCequol 4.37 376.93113 C15H9O3Cl4 376.93108 0.500 product A 2.64 358.96502 C15H10O4Cl3 358.96500 0.593 product B 2.28 325.00399 C15H11O4Cl2 325.00388 0.441 product C 0.85 374.95993 C15H10O5Cl3 374.95987 0.489 product D 0.85 356.99382 C15H11O6Cl2 356.99382 0.572

byproducts were subsequently subjected to MS/MS fragmentation as the possible primary isomer in MCequol. Such reaction also by higher-energy collisional dissociation (HCD), allowing a higher occurred at C15, C2 and C13 with a partial charge of 0.210, 0.185 confidence in the identification of the proposed byproducts. and 0.154 to form 15-chloro-equol, 2-chloro-equol and 13-chloro- The molecular ions at 275.04785 (one chlorine atom), equol, while their abundances tended to be lower than that of 6- 309.00891 (two chlorine atoms), 342.96994 (three chlorine atoms), chloro-equol due to the relatively low negative partial charges at and 376.93103 (four chlorine atoms) were observed at 2.86, 3.34, C15, C2 and C13. Of these potential chlorinated equol, only the 3.85, and 4.37 min, respectively (Fig. 2(a) and (b)). Notable 35Cl/37Cl structure of tetrachloro-equol can be deduced as 2, 6, 13, 15- isotope patterns for mono-, di-, tri-, and tetrachloro species in Fig. 2 tetrachloro-equol since these four positions are the only ortho- (b) fits the distribution of intensities of chlorine isotopic peaks positions in equol that chlorine substitution can happen. (Peng et al., 2015). The products corresponding with these peaks Fig. 2(b) shows the mass spectra of the A-D products. The mass were deduced to be monochloro-, dichloro-, trichloro-, and spectrum of product A (2.65 min) showed that it has 3 chlorine e tetrachloro-equols, on the basis of their molecular ions and the atoms, and the chemical formula of its [M H] was proposed as e number of chlorine atoms. We failed to identify the exact structures C15H11O4Cl3 . MS/MS fragmentation of product A yielded frag- of monochloro-, dichloro-, trichloro-equols using UHPLC- ments at m/z 340.95416 (C15H8O3Cl3) and 304.97760 (C15H7O3Cl2), quadrupole-orbitrap-HRMS analysis since they have several po- indicating that product A experienced losses of H2O and HCl (Fig. 3), tential isomers. According to the chlorination mechanism, chlorine such fragmentation patterns have been also observed in mass substitution of phenolic compounds always happens at ortho- and/ spectra of chlorinated bisphenol A (Hu et al., 2002b). The structure or para-position (Buth et al., 2009), this is because these two po- of product A was further deduced with the aid of computational sitions are more negative-charged. Since the presence of a negative chemistry to predict the reaction likelihood of each carbon atom in charge on the phenolic compound will facilitate the chlorination equol (Fig. S1). It is noted that C4, having relatively low negative reaction (Hu and Aizawa, 2003; Hu et al., 2002a, 2002b), the partial charge, is a potential reactive site in addition to C6, C15, C2, chlorine substitution reaction preferably occurred at C6 with a and C13. Thus, when HOCl attacks C4, the break down between C4 partial charge of 0.214 as shown in Fig. S1, forming 6-chloro-equol and C9 and formation of aldehyde at C9 can be deduced. Given this 396 Y. Zhou et al. / Chemosphere 212 (2018) 393e399

e Fig. 2. Extracted ion chromatograms (EIC) corresponding to the [M H] ions of byproducts of equol obtained from the analysis in the scan mode of an aqueous chlorinated equol solution at 10 min (a) and the corresponding mass spectra of byproducts of equol (b).

structure, fragment m/z 200.95074 (C8H3O2Cl2) would be formed fragments at m/z 303.00644 (C15H8O5Cl), 190.92986 (C6HO3Cl2), due to the break-up of the ether bond to the (A) benzene ring 183.02078 (C9H8O2Cl), and 156.96860 (C6H2O3Cl) were found (Fig. 3) and the loss of aldehyde (m/z 200.95074). In the mass (Fig. 3). MS/MS fragment ions, C6HO3Cl2 and C9H8O2Cl, shared a e spectrum of product B at 2.29 min in Fig. 2(b), the signal of [M H] similar structure with three O atoms bonded to one benzene ring. for product B was not strong enough to activate MS/MS fragmen- Due to the phenolic structure, the (A) ring (Fig. 3) could be oxidized tation, which made it difﬁcult to deduce the exact structure of into the 1,4-benzoquinone structure in NaClO aqueous solution e product B. Its molecular ion [M H] (m/z 325.00378, (Kosaka et al., 2017), and then transformed back to hydroquinone e C15H12O4Cl2 indicated that the chemical structure of product B due to low stability (Mylonas and Papaconstantinou, 1996), would be the same as product A with one less Cl substitution, as possibly forming a structure with three O atoms bound to the (A) shown in Fig. 4. phenyl ring. The MS/MS fragment of C9H8O2Cl was the (B) ring part In the MS spectrum of product C at 0.85 min in Fig. 2(b), its (Fig. 3) of product C, resulting from the break-up of the ether bond e molecular ion [M H] at m/z 374.95987 (C15H11O5Cl3) and MS/MS between the (A) ring and (B) ring. Thus, the structure of product C Y. Zhou et al. / Chemosphere 212 (2018) 393e399 397

Fig. 3. Accurate MS/MS spectra and fragmentation pattern of Products A and C.

Fig. 4. Pathways of chloro-substitution reactions between equol and HOCl. Byproducts with confirmation in this experiment were named as shown in the figure and they are linked with solid arrows; while possible byproducts without confirmation were not named and linked with dotted arrows. was deduced as shown in Fig. 4. The molecular ion of product D at degradation of chlorinated phenols (Mylonas and 0.85 min in Fig. 1 has m/z of 356.99374 (C15H12O6Cl2 ). Since Papaconstantinou, 1996), and the other is the aldehyde group be- product D has a total of six oxygen atoms, its structure was deduced ing oxidized to a carboxyl group. as having three O atoms bonded to the (A) phenyl ring, and a carboxyl group generated from oxidization of the aldehyde group 3.2. Chlorination pathways of equol (Fig. 4). Product D would be formed by product B experiencing chlorine substitution in the (B) phenyl ring, along with two re- Fig. 5 shows the variation in peak areas of equol and its eight actions: one is the (A) phenyl ring being oxidized to the 1,4- chlorinated byproducts with reaction time. It was found that equol benzoquinone structure (Kosaka et al., 2017), and then reduced to rapidly reacted with sodium hypochlorite (ca. 5% available Cl). The hydroquinone, which has been observed in photocatalytic peak area of equol significantly decreased at 10 min. All byproducts 398 Y. Zhou et al. / Chemosphere 212 (2018) 393e399

Fig. 5. Variation of abundances of equol and its products with chlorination time. MCequol, monochloro-equol; DCequol, dichloro-equol; TCequol, trichloro-equol; TeCequol, tetrachloro-equol. A-D correspond to products A-D, respectively. were predominantly found at shorter contact times (within assessed by the yeast two-hybrid assay, and their dose-response 60 min). Product A could be generated from the ring-opening re- curves together with those of equol and 17bE2 were shown in action of DCequol, which was supported by the obvious decrease in Fig. 6. The half-maximal transcriptional response (EC50) value of the DCequol peak area. Considering that the peak area of MCequol DCequol was 2456 nM, followed by MCequol with EC50 of 3456 nM, did not decrease in the ﬁrst 30 min of reaction, product B would be while the EC50 of equol was 3889 nM, showing stronger estrogenic generated from one chlorine-substituted-ring-opened equol. The potency comparing with equol. TCequol did not elicit hER activating peak areas of products C and D slightly increased until 60 min activity. The estrogenic effect factor relative to 17bE2 (EEF17bE2) contact time, with decreasing peak areas of dichloro-equol and (EC50 ¼ 4.1 nM) was calculated to be 0.0012 for MC equol and product B, further supporting the above speculated pathway that 0.0018 for DCequol. The result of this assay indicated that the es- products C and D were formed by oxidization of dichloro-equol and trogenic effects of the low-number-chloro-substituted equols were product B. Product C degraded completely after 360 min while similar to that of equol, while when the substituted hydrogens by products A, B, D were still detected until 1440 min. On the basis of chlorine reached 3, the estrogenic activity of the chlorinated the aforesaid results, pathways for the aqueous chlorination of byproduct drastically dropped. This trend was different from the equol are proposed (Fig. 4), which include (1) chlorine substitution estrogenic activity of chlorinated estrogens and chlorinated BPAs, reactions and subsequent dehydration and (2) break-up of C4eC9 where in chlorinated estrogens, the estrogenic activity dropped as followed by oxidation of aldehyde to carboxyl. It should be noted more hydrogens were substituted by chlorines (Kuruto-Niwa et al., that the proposed pathways were obtained under the experimental 2002), while in chlorinated BPAs, the estrogenic activity is chlorination condition. dramatically increased in monochloro-BPA, and gradually decreased with more chlorine substituted (Hu and Aizawa, 2003). These distinct differences may be due to the different docking 3.3. Estrogenic activity of equol and its chlorinate byproducts groups of these compounds, affecting the binding energy to the ERa ligand binding domain. The estrogenic activities of three chlorinated equols were While equol shows estrogenic activity at relatively high concentration in vitro, it has been demonstrated that equol is able to induce intersex in male medaka ﬁsh at a concentration as low as 5.8 ng/L (Wang et al., 2016), much lower than the highest concentration detected in Swiss river (524 ng/L) and in Iowa and Portu- guese (40 ng/L) (Hoerger et al., 2009; Erbs et al., 2007; Kolpin et al., 2010; Ribeiro et al., 2016). Such phenomenon has been explained by its relatively strong estrogenic activity and anti-androgenic activity (Wang et al., 2016). As for human, one epidemiological study found that sperm motility was lower in urinary-equol-detectable subjects (5 ng/mL) than in non-detectable subjects in Japanese males (Toshima et al., 2012). Considering the similar or even stronger estrogenic activity of chlorinated equols compared to equol, the potential occurrence of chlorinated equols in drinking water would make even non-equol producers exposed to equol and its derivatives, posing potential risks for human health.

4. Conclusions

Overall, this work provided the first description of the behaviors Fig. 6. Dose response curves of 17b-estradiol, equol, monochloroequol (MCequol), dichloro-equol (DCequol), and trichloro-equol (TCequol) with yeast two-hybrid assay of equol in the chlorination process, indicating that humans, both (n ¼ 3 for each concentration). equol producers and non-equol-producers, could be potentially Y. Zhou et al. / Chemosphere 212 (2018) 393e399 399 exposed to equol and its chlorinated byproducts via intake of underinvestigated compounds in agricultural basins. J. Environ. Qual. 39, e drinking water chronically, since relatively high concentration of 2089 2099. Kosaka, K., Nakai, T., Hishida, Y., Asami, M., Ohkubo, K., Akiba, M., 2017. Formation of equol has been detected in river water. It would be interesting for 2,6-dichloro-1,4-benzoquinone from aromatic compounds after chlorination. future studies to discover the occurrence of chlorinated byproducts Water Res. 110, 48e55. of equol in real drinking water, for controlling their possible health Krishnan, A.V., Stathis, P., Permuth, S.F., Tokes, L., Feldman, D., 1993. Bisphenol-A: an estrogenic substance is released from polycarbonate flasks during autoclaving. risks of these chlorinated byproducts. One limitation of this study is Endocrinology 132, 2279e2286. that we could not assess the estrogenic potencies of other major Kudo, Y., Yamauchi, K., Fukazawa, H., Terao, Y., 2006. In vitro and in vivo analysis of byproducts identified, because we failed to synthesize them. the thyroid system-disrupting activities of brominated phenolic and phenol compounds in Xenopus laevis. Toxicol. Sci. 92, 87e95. 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