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Assessment of endocrine-disrupting activities of alternative chemicals for bis(2-ethylhexyl)phthalate
Environmental Research 172 (2019) 10–17
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Assessment of endocrine-disrupting activities of alternative chemicals for bis (2-ethylhexyl)phthalate Joonwoo Parka, Choa Parka, Myung Chan Gyeb, Youngjoo Leea, a b
T
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Department of Integrative Bioscience and Biothecnology, College of Life Science, Sejong University, Kwangjingu, Kunjadong, Seoul 143-747, Republic of Korea Department of Life Science, Hanyang University, Seoul 04763, Republic of Korea
A R T I C LE I N FO
A B S T R A C T
Keywords: Endocrine disruptor Phthalate Alternative material
Plastic products are closely intertwined with modern life. Some plasticizers used in making plastics, such as phthalates, are reported to be endocrine-disrupting chemicals. Plasticizers can be released into the environment, and health risks related to plasticizer exposure have been reported. In addition, due to plastic waste that flows into the ocean, microplastics have been found in marine products, including non-biological seawater products such as sea salt. Plastics can affect the body via a variety of pathways, and therefore safer alternative chemicals are needed. Three chemicals were evaluated: acetyl tributyl citrate (ATBC), triethyl 2-acetylcitrate (ATEC), and trihexyl O-acetylacitrate (ATHC), replacing bis(2-ethylhexyl)phthalate (DEHP), a typical plasticizer. The endocrine-disrupting activities of each chemical, including estrogenic or anti-estrogenic activity (test guideline (TG) No. 455), androgenic or anti-androgenic activity (TG No. 458), steroidogenesis (TG No. 456), and estrogenic properties via a short-term screening test using the uterotrophic assay (TG No. 440), were assessed in accordance with the Organisation for Economic Co-operation and Development guidelines for chemical testing. Our results showed that DEHP, ATBC, ATEC, ATHC possess no estrogenic activity, whereas DEHP, ATBC and ATHC demonstrate anti-estrogenic activity and ATBC anti-androgenic activity. DEHP and ATHC exhibited a disruption in steroidogenesis activities. Additional tests are necessary, but our results suggest that ATEC is a good candidate plasticizer providing a suitable alternative to DEHP.
1. Introduction Since mass production began in the 1950s, global production of plastics has increased to more than 322 million tons as of 2015 (PlasticsEurope, 2016). Plasticizers are low-melting-point solids or high-boiling-point liquids and are commonly used to impart flexibility and durability to plastic products. More than 6 million tons of plasticizers are produced and consumed annually worldwide (Lioy et al., 2015). Due to the recent increase in mineral water consumption, the water in plastic bottles has been suggested to cause a health risk to consumers. Antimony and phthalates are considered serious pollutants found in bottled mineral water. Phthalates are used as solvents to increase the flexibility of plastics such as polyvinyl chlorides. Phthalates are a widely used class of plasticizers found in a variety of consumer products including soap, mouthwash, disposable gloves, food or beverage containers, and toys (Hernandez-Diaz et al., 2009; Kavlock et al., 2002a, 2002b). These phthalates are not bound to plastic and therefore can be released into the environment. In addition, phthalates are lipophilic compounds and have been found to accumulate in fat (LaFleur
⁎
and Schug, 2011). In addition to pollution of the water within, plastic bottles can cause other problems including the release of microplastics, which have recently been detected in sea salt. Plastic waste that is not managed properly is eventually found in the ocean (Jambeck et al., 2015). Due to the continued fragmentation of large plastics, tiny particles called microplastics accumulate, and several studies have shown that microplastics are present in seafood products such as clams and fish (Davidson and Dudas, 2016; Foekema et al., 2013). Therefore, the consumption of seafood can be a significant exposure pathway for humans to microplastics. For example, those who consume clams in large quantities in Europe are expected to ingest up to 11,000 microplastics each year (Van Cauwenberghe and Janssen, 2014). In addition to seafood, microplastics can also be found in non-biological seawater products. Commercial sea salt is produced mainly through crystallization as a result of seawater evaporation, which may be induced artificially or occur naturally due to sunlight and wind (Serrano et al., 2011). During this process, microplastics in seawater can associate with the sea salt (Brown et al., 2009). Recent research has confirmed that commercial salt available in various countries contains microplastics (Karami et al.,
Corresponding author. E-mail address: [email protected] (Y. Lee).
https://doi.org/10.1016/j.envres.2019.02.001 Received 30 September 2018; Received in revised form 17 January 2019; Accepted 1 February 2019 Available online 02 February 2019 0013-9351/ © 2019 Published by Elsevier Inc.
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forskolin were purchased from Sigma-Aldrich (St. Louis, MO, USA). ATHC was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). DEHP, ATBC, ATEC, ATHC, raloxifene, HF, forskolin is dissolved in DMSO. E2 is dissolved in ethanol and DHT is dissolved in methanol.
2017). Plastics are heavily utilized in our daily lives and can affect our bodies via various pathways. Phthalates such as bis(2-ethylhexyl) phthalate (DEHP) and di-n-butyl phthalate are suspected to be toxic to the liver, kidneys, and reproductive organs, as well as carcinogenic (Gomez-Hens and Aguilar-Caballos, 2003; Swan et al., 2005). Di-nbutyl phthalate, benzyl butyl phthalate, and DEHP are also weakly estrogenic. Some metabolites of phthalates such as mono(2-ethylhexyl) phthalate, mono-n-butyl phthalate, and monoethyl phthalate can interfere with hormone activity (Chang et al., 2015; Upson et al., 2013). These endocrine disruptors were found in the water in most plastic bottles (Pinto and Reali, 2009; Plotan et al., 2013; Wagner and Oehlmann, 2011). According to a 2005 biomonitoring study released by the Center for Disease Control and Prevention, more than 75% of spot urine samples were found to contain phthalates consistently throughout life (Control, 2005). Therefore, identifying safer alternative chemicals for use in consumer products is essential. Humans are exposed not only to phthalates but to many chemicals throughout their lives, including endocrine-disrupting chemicals (EDCs) such as bisphenol A and nonylphenol. Organizations around the world have developed international standardization methods to evaluate potential EDCs. In 1997, the Organisation for Economic Co-operation and Development (OECD) began developing guidelines for assessment of EDCs, managed by the Endocrine Disruptors Testing and Assessment task force (OECD, 2005, 2010). The OECD updated their conceptual framework for assessment of EDCs in 2012 (OECD, 2012). This document provides a five-step guideline for identifying EDCs. It describes in vitro test methods, including a cell-based transcriptional activation assay at level 2 and in vivo testing methods in animals at level 3. Following the guidelines for levels 2 and 3 of the OECD conceptual framework for assessment of EDCs, we evaluated the endocrine-disrupting activities of DEHP and its alternative chemicals: acetyl tributyl citrate (ATBC), triethyl 2-acetylcitrate (ATEC), and trihexyl O-acetylcitrate (ATHC) (Fig. 1). In this study, DEHP, ATBC, ATEC, and ATHC were evaluated for endocrinedisrupting activities, including estrogenic or anti-estrogenic activity, androgenic or anti-androgenic activity, steroidogenesis, and estrogenic properties via a short-term screening test using the uterotrophic assay, in accordance with the OECD guidelines for chemical testing.
2.2. Cell culture HeLa9903 cells and VM7-Luc cells were cultured in phenol red-free Dulbecco's modified Eagle medium (DMEM) (WelGENE, Daegu, South Korea) supplemented with 10% fetal bovine serum (FBS) (WelGENE) and penicillin/streptomycin (GIBCO Invitrogen, Grand Island, NY, USA). Androgen receptor (AR)-EcoScreen™ Cells were cultured in phenol red-free DMEM/F12 (WelGENE) supplemented with 5% FBS. H295R cells were cultured in phenol red-free DMEM/F12 (WelGENE) supplemented with 2.5% Nu-serum (Corning, NY, USA), and 1% ITS + premix supplement (6.25 μg/ml insulin; 6.25 μg/ml transferrin; 6.25 ng/ml selenium; 1.25 mg/ml bovine serum albumin; 5.35 μg/ml linoleic acid; Corning). All media contained 100 μg/ml of streptomycin (GIBCO Invitrogen, Grand Island, NY, USA).
2.3. Stably transfected transcriptional activation (STTA) assay The estrogenic and anti-estrogenic activities assessments each used HeLa9903 and VM7-Luc cells, and the evaluation procedure was tested in accordance with the OECD test guideline (TG) No. 455 (OECD, 2016a). Both cell lines stably express estrogen receptor (ER) alpha gene containing a firefly luciferase gene as a reporter gene and estrogenic activity was assessed by increasing ER transcriptional activity of test substance usingE2-enhanced ER transcriptional activity. The androgenic and anti-androgenic activities assessment used AR-EcoScreen™ cells, and the evaluation procedure was tested in accordance with the OECD TG458 (OECD, 2016b). This cells stably expresses AR gene containing a firefly luciferase gene as a reporter gene and androgenic activity was assessed by increasing AR transcriptional activity of test substance using DHT-enhanced AR transcriptional activity. Luciferase activity was determined with Glomax luminometer (Promega Corp., Madison, WI, USA) using the luciferase assay system (TG455 used OneGlo®, Promega; TG458 used Dual-Glo®, Promega). Since the concentration used varies from one test method to another, a detailed description was written on each figure legends. All experiments of STTA assay were repeated at least three times.
2. Materials and methods 2.1. Materials DEHP, ATBC, ATEC, 17-β-estradiol (E2), 5α-Dihydrotestosterone (DHT), dimethyl sulfoxide (DMSO), raloxifene, hydroxylflutamide (HF),
Fig. 1. Structures of chemicals. (A) DEHP (pubchem CID: 8343), (B) ATBC (pubchem CID: 6505), (C) ATEC (pubchem CID: 6504), (D) ATHC (pubchem CID: 90617). 11
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anti-estrogenic activities of chemicals to determine the endocrine-disrupting activities of chemical alternatives to DEHP. HeLa9903 cells were treated with DEHP, ATBC, ATEC, or ATHC at 1 nM to 1 mM for 24 h. DEHP and the three alternative chemicals did not exhibit any estrogenic activity (Fig. 2A–D). The anti-estrogenic activities of the chemicals were assessed in VM7-luc cells, which were treated for 24 h with 500 pM E2 and 9.7 nM to 10 μM of the test chemicals. DEHP, ATBC, and ATHC exhibited anti-estrogenic activities, with respective IC50 values of 66 μM, 439 nM, and 136 nM. On the other hand, ATEC did not exhibit anti-estrogenic activity at any of the concentrations evaluated (Fig. 3A–D). The OECD proposed the uterotrophic assay as an in vivo test to detect the estrogenic activities of potential EDCs (Kanno et al., 2003; OECD, 2007). The uterus responds to estrogen and increases the weight of the uterus due to water absorption. Estrogenic activity can be assessed by changing the uterine weight by the test substance using this reaction. Test verification was performed in rats, but the mouse model is considered equally valid. We followed the OECD guideline No. 440 and used mice to determine the estrogenic activities of DEHP and alternative chemicals (OECD, 2007). OVX mice at 8 weeks of age were randomly assigned to five groups, and one group of non-OVX mice was formed, with six mice of similar average body weight per group (Table 1). Treatment was administered via oral injection at 24-h intervals over 7 days (Fig. 4A). E2 was used as a positive control. To detect estrogenic activity, two vehicles (non-OVX and OVX mice) were employed, using E2 as a positive control and 400 mg/kg/day DEHP and 40 or 400 mg/kg/day ATEC as the test conditions. In this experiment, E2 was administered at 0.4 mg/kg/day, and uterine weight increased by approximately 4.9 times compared with OVX control group. However, neither test compound (DEHP 400 mg/kg/day, ATEC 40 or 400 mg/kg/day) caused a significant increase in uterine weight compared with the negative control group (OVX control) (Fig. 4B). These results indicated that ATEC was the only substance without estrogenrelated activity.
2.4. Steroidogenesis assay The purpose of the steroidogenesis assay is to detect chemicals that affect the production of E2 and testosterone. Chemical interference test assessments used a human adreno-carcinoma H295R cells, and the evaluation procedure was tested in accordance with the OECD TG456 (OECD, 2011). Forskolin was treated at a concentration of 10 μM as a positive control, and each test chemicals were treated at a concentration of 1 and 10 μM for 48 h. Hormones in culture medium were extracted with diethyl ether in glass tubes, and phase separation was achieved by centrifugation at 2000 × g for 10 min. Estradiol or testosterone hormone changes were measured using enzyme-linked immunosorbent assay (ELISA) using the manufacturer's recommendations. Estradiol ELISA kit and testosterone ELISA kit were purchased from Cayman Chemical (Ann Arbor, MI, USA). 2.5. Uterotrophic bioassay Uterotrophic bioassay is a short-term screening test for estrogenic properties. It is based on the increase in uterine weight or uterotrophic response. It evaluates the ability of a chemical to produce biological activities consistent with natural estrogen. Female ICR mice and ovariectomized (OVX) ICR mice (6 weeks old) were purchased from Central Laboratory Animal Inc. (Seoul, South Korea) and maintained under a protocol approved by the Sejong University Institutional Animal Care and Use Committee (Ref. No. SJ-20160304E1). All procedures were performed according to the OECD TG440 (OECD, 2007). OVX mice were randomly assigned to five groups, and one group of non-OVX-mice was formed, with six mice of similar average body weight per group. Treatment was administered via oral injection at 24-h intervals over 7 days. E2 was administered at 0.4 mg/kg/day, DEHP was administered at 400 mg/kg/day and ATEC was administered at 40 or 400 mg/kg/ day. The study design is shown in Table 1. 2.6. Statistical analysis
3.2. Assessment of androgen-related activity
All in vitro experiments were repeated at least three times. The data were expressed as mean ± SD using GraphPad Prism 7.0 (Graph Pad Software, La Jolla, CA, USA). Statistical analysis of the data was determined by 2-tailed student's t-test and p < 0.01 (*) was considered as statistically significance.
Next, to define the endocrine-disrupting activities of alternative chemicals to DEHP, we used the stably transfected transcriptional activation assay described in the OECD in vitro test guideline No. 458 to assess androgenic and anti-androgenic activities. AR-EcoScreen cells were treated with DEHP, ATBC, ATEC, or ATHC at 10 pM to 10 μM for 24 h. DEHP, ATEC, and ATHC showed no androgenic activity, but ATBC exhibited weak androgenic activity (PC10, 1.86 μM) (Fig. 5A–D). PC10 mean the concentration of a chemical at which its response equals 10% of the maximum response of the reference standard (DHT). In addition, the anti-androgenic activities of DEHP and its alternative chemicals were also measured (Fig. 6A–E). Hydroxyflutamide was used as a positive control and exhibited an IC50 of 10.8 nM (Fig. 6A). IC50 mean the molar concentration of a chemical at which produces 50% of the maximum possible response for that chemical (50% inhibition). The anti-androgenic activities of the chemicals, at 100 pM to 10 μM, were tested. DEHP and the three alternative chemicals exhibited no antiandrogenic activity. These results indicate that ATEC and ATHC do not have androgen-related activity.
3. Results 3.1. Assessment of estrogen-related activity EDCs have been reported to affect multiple endocrine pathways, hormones, and homeostatic systems, most of which are affected by the disruption of estrogen or androgen mechanisms, and EDCs may also affect steroid generation and reproduction (Del Mazo et al., 2013; Knez, 2013; Marques-Pinto and Carvalho, 2013; Zhang et al., 2014). First, we used the stably transfected transcriptional activation assay described in the OECD in vitro test guideline No. 455 to assess the estrogenic and Table 1 Study design for uterotrophic assay of DEHP and ATEC.
3.3. Assessment of hormone production
Group
Group number
Agonist detection
Non-OVX negative control OVX negative control OVX positive control OVX-DEHP 400 mg/kg/day OVX-ATEC 40 mg/kg/day OVX-ATEC 400 mg/kg/day
1 2 3 4 5 6
Vehicle Vehicle E2 Test substance Test substance Test substance
In several studies, EDCs have also been reported to affect steroid hormone synthesis and reproduction (Jouannet et al., 2001; Knez, 2013; Perry et al., 2007; Safe, 2013; Zhang et al., 2014). In several studies, EDCs have also been reported to affect steroid hormone synthesis and reproduction. Therefore, we determined the steroidogenic capabilities of alternative chemicals to DEHP. We used the H295R steroidogenesis assay to detect chemicals that can induce (or reduce) production of sex steroids (E2 or testosterone). The H295R
Vehicle: Corn oil, 100 μl/day. E2: β-estradiol, 0.4 mg/kg/day. 12
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Fig. 2. Effect of chemicals on ER agonist activity using OECD test guideline No. 455. HeLa9903 cells were treated DEHP (A), ATBC (B), ATEC (C), and ATHC (D) with a concentration of 1 nM to 1 mM for 24 h and luciferase activities were determined. E2 was used as a positive control and treated 24 h at a concentration of 1 nM. Data are expressed as the mean ± SD (n = 3). Asterisks (*) indicate significant difference from control. * represent p < 0.01.
Fig. 3. Effect of chemicals on ER antagonist activity using OECD test guideline No. 455. VM7-luc cells were treated for 24 h with 500 pM E2 and 9.7 nM to 10 μM of DEHP (A) and alternative chemicals ATBC (B), ATEC (C), and ATHC (D) and luciferase activities were determined. Raloxifene was used as a reference substance and treated 24 h at a concentration of 95.6 pM to 24.5 nM for 24 h. The positive determination of antagonist activity should be at least a 20% reduction in activity from the maximal value for the reference substance (cotreatment of Raloxifene and E2). Data are expressed as the mean ± SD (n = 3).
and testosterone. The E2 concentration was increased 3.2 times by 10 μM DEHP compared with control (Fig. 7A). The testosterone concentration was increased by ~1.2 times by 10 μM DEHP compared with control, which was a statistically significant increase (Fig. 7B). Meanwhile, 1 μM ATHC decreased the testosterone concentration by ~80%
steroidogenesis assay was also tested according to the OECD test guideline No. 456. H295R cells were treated with 1 and 10 μM DEHP and its alternative chemicals for 24 h, and the concentrations of E2 and testosterone were measured using enzyme-linked immunosorbent assay kits. As shown in Fig. 7, forskolin was used as a positive control for E2 13
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Fig. 4. Assessment of estrogenic response in ovariectomized mice. (A) Experimental schedule. OVX mice at 8 weeks age were randomly assigned to five groups, and one group of non-OVX mice was formed, with six mice of similar average body weight per group. Treatment was administered via oral injection at 24-h intervals over 7 days. Corn oil was used vehicle and administered at 100 μl/day. E2 was used positive control and administered at 0.4 mg/kg/day. DEHP was administered at 400 mg/kg/day and ATEC was administered 40 or 400 mg/kg/day via oral injection. (B) The effects of DEHP and ATEC on uterine weight. Data are expressed as the mean ± SD (n = 6). Asterisks (*) indicate significant difference from OVX-control group. * represent p < 0.01. p value > 0.05 is indicated by “n.s.” for not significant.
has been approved by the U.S. Food and Drug Administration as a commercial plasticizer that is permitted as an additive in food contact materials (Commision, 2010). However, ATBC has been reported to affect reproduction in female mice (Rasmussen et al., 2017), and our results confirmed that ATBC exhibits estrogen receptor antagonist activity and androgen receptor agonist activity (Figs. 3B and 5B). Thus, even chemical alternatives to phthalates, such as ATBC, may exhibit endocrine-disrupting activity and present multiple hazards and therefore will require further study. In another example, bisphenol F (BPF) was used as an alternative to bisphenol A (BPA), also known as EDC. However, it has been reported that BPF has an estrogenic potential similar to BPA (Moreman et al., 2017), and we have confirmed that BPF has an estrogenic activity (data not shown). It has been reported that continuous exposure of EDCs to our bodies can have various effects such as dysfunction of reproductive systems, inflammation, and DEHP induced the proliferation of human endometrial cancer cells (Sifakis et al., 2017; Cho et al., 2014). Many of the products used by humans contain a variety of chemicals. If any of these chemicals are EDC, there is a risk of continuing exposure to the body. Therefore, chemicals that can be frequently exposed to our bodies need to be assessed for endocrine disrupting activities. We evaluated the effects of three chemical alternatives to DEHP (ATBC, ATEC, and ATHC) on estrogen- and androgen-related activities and steroidogenesis. ATBC exhibited estrogen receptor antagonist activity and androgen receptor agonist activity, whereas ATHC exhibited only estrogen receptor antagonist activity. ATEC had no effect on estrogen or androgen activities or steroidogenesis (Table 2). However, although ATEC did not have an AR agonist activity (Fig. 5C), when DHT
compared with the control group, but our data did not indicate any reduction, as only increases compared with the control group were considered. These results indicated that DEHP disrupted hormone production, whereas the alternative chemicals did not increase hormone production. 4. Discussion Phthalates have been used in numerous products including plastics, emulsifiers, paints, coating solvents, and cosmetics, but the use of phthalates has been restricted worldwide due to their risks. Therefore, efforts have been made to manufacture plastics that do not use plasticizers such as phthalates. Initially, plastic is manufactured from petroleum-based compounds such as polyurethane, polystyrene, and ethylene vinyl acetate (Becker et al., 2010; Tickner, 1999). However, although phthalates are avoided to minimize risk, other problems occur related to the release of various harmful substances during the manufacturing process, including heavy metals and sulfur dioxides. In addition, some products, such as polystyrene, are classified as carcinogens and require further study (Huff and Infante, 2011). Subsequently, alternative plastics have been developed using plant-based materials called bioplastics. Sugar and oil extracted from plants such as corn are highly biodegradable and produce few pollutants during the production process. However, bioplastics have limitations in terms of their mechanical properties and high production costs. For this reason, it is necessary to develop alternative plasticizers to make up for the limited manufacture of non-plasticizers. One plasticizer offering an alternative to phthalate is ATBC, which is commonly used in polyvinyl resins and 14
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Fig. 5. Effect of chemicals on AR agonist activity using OECD test guideline No. 458. AREcoScreen cells were treated with DEHP, ATBC, ATEC, or ATHC at 10 pM to 10 μM for 24 h and luciferase activities were determined. DHT was used positive control and treated 24 h at concentration of 1 nM. Data are expressed as the mean ± SD (n = 3). Asterisks (*) indicate significant difference from control. * represent p < 0.01.
Fig. 6. Effect of chemicals on AR antagonist activity using OECD test guideline No. 458. AR-Ecoscreen cells were treated for 24 h with 500 pM DHT and 10 pM to 10 μM of HF (A), DEHP (B) and ATBC (C), ATEC (D), and ATHC (E) and luciferase activities were determined. HF was used as a positive control. If the log IC30 of the test chemical is calculated (30% inhibition), determine that the chemical has AR antagonist activity. Data are expressed as the mean ± SD (n = 3).
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Fig. 7. Effect of chemicals on steroid hormone production using OECD test guideline No. 456. H295R cells were treated for 48 h with 1 or 10 μM of DEHP, ATBC, ATEC, and ATHC. After treatment, hormones in culture medium were measured by Estradiol (A) and testosterone (B) ELISA kit. Data are expressed as the mean ± SD (n = 3). Asterisks (*) indicate significant difference from control. * represent p < 0.01.
Table 2 Summary of endocrine-disrupting activities of alternative chemicals for DEHP. `
ER agonist
ER antagonist
AR agonist
AR antagonist
Steroidogenesis
in vivo ER activity
ATBC ATEC ATHC
ND ND ND
IC50 439 nM ND IC50 136 nM
PC10 1.86 μM ND ND
ND ND ND
ND ND ND
– ND –
ND: Not detected. IC50: The molar concentration of a chemical at which produces 50% of the maximum possible response for that chemical (50% inhibition). PC10: The concentration of a chemical at which its response equals 10% of the maximum response of the reference standard.
and ATEC were co-treated, ATEC showed concentration dependent increases ARE-transcriptional activity (Fig. 6D). Further study may be required, but ATEC is likely to have an enhanced-AR agonist activity that increases ARE-transcriptional activity when an AR agonist such as DHT is present.
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5. Conclusions Assessment of DEHP and three alternative substances according to OECD conceptual framework for assessment of EDCs showed that ATEC had no effect on estrogen or androgen activities or steroidogenesis. Our results suggest that ATEC is a better candidate alternative chemical to DEHP compared with ATBC. Ethics approval and consent to participate All animal studies were performed in accordance with the animal protocol procedures approved by the Sejong University Institutional Animal Care and Use Committee (Ref. No. SJ-20160304E1). Conflict of interest The authors declare no conflict of interest. Funding This research was supported by Research Program to Solve Social Issues of the National Research Foundation of Korea(NRF) funded by the Ministry of Science and ICT. (No. NRF-2017M3C8A6091777). References Becker, M., Edwards, S., Massey, R.I., 2010. Toxic chemicals in toys and children's products: limitations of current responses and recommendations for government and industry. Environ. Sci. Technol. 44, 7986–7991. Brown, I.J., Tzoulaki, I., Candeias, V., Elliott, P., 2009. Salt intakes around the world: implications for public health. Int. J. Epidemiol. 38, 791–813. Chang, W.H., Li, S.S., Wu, M.H., Pan, H.A., Lee, C.C., 2015. Phthalates might interfere with testicular function by reducing testosterone and insulin-like factor 3 levels. Hum. Reprod. 30, 2658–2670.
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organophosphorus insecticide exposures and sperm concentration. Reprod. Toxicol. 23, 113–118. Pinto, B., Reali, D., 2009. Screening of estrogen-like activity of mineral water stored in PET bottles. Int. J. Hyg. Environ. Health 212, 228–232. PlasticsEurope, 2016. 〈https://www.plasticseurope.org/application/files/4315/1310/ 4805/plastic-the-fact-2016.pdf〉. Plotan, M., Frizzell, C., Robinson, V., Elliott, C.T., Connolly, L., 2013. Endocrine disruptor activity in bottled mineral and flavoured water. Food Chem. 136, 1590–1596. Rasmussen, L.M., Sen, N., Liu, X., Craig, Z.R., 2017. Effects of oral exposure to the phthalate substitute acetyl tributyl citrate on female reproduction in mice. J. Appl. Toxicol. 37, 668–675. Safe, S., 2013. Endocrine disruptors and falling sperm counts: lessons learned or not!. Asian J. Androl. 15, 191–194. Serrano, R., Nacher-Mestre, J., Portoles, T., Amat, F., Hernandez, F., 2011. Non-target screening of organic contaminants in marine salts by gas chromatography coupled to high-resolution time-of-flight mass spectrometry. Talanta 85, 877–884. Sifakis, S., Androutsopoulos, V.P., Tsatsakis, A.M., Spandidos, D.A., 2017. Human exposure to endocrine disrupting chemicals: effects on the male and female reproductive systems. Environ. Toxicol. Pharmacol. 51, 56–70. Swan, S.H., Main, K.M., Liu, F., Stewart, S.L., Kruse, R.L., Calafat, A.M., Mao, C.S., Redmon, J.B., Ternand, C.L., Sullivan, S., Teague, J.L., Study for Future Families Research Team, 2005. Decrease in anogenital distance among male infants with prenatal phthalate exposure. Environ. Health Perspect. 113, 1056–1061. Tickner, J., 1999. A Review of the Availability of Plastic Substitutes for Soft PVC in Toys. Technical Briefing Commissioned by Greenpeace International. University of Massachusetts, Lowell, MA. Upson, K., Sathyanarayana, S., De Roos, A.J., Thompson, M.L., Scholes, D., Dills, R., Holt, V.L., 2013. Phthalates and risk of endometriosis. Environ. Res. 126, 91–97. Van Cauwenberghe, L., Janssen, C.R., 2014. Microplastics in bivalves cultured for human consumption. Environ. Pollut. 193, 65–70. Wagner, M., Oehlmann, J., 2011. Endocrine disruptors in bottled mineral water: estrogenic activity in the E-Screen Reply. J. Steroid Biochem. Mol. Biol. 127, 137–138. Zhang, L.Y., Dong, L., Ding, S., Qiao, P., Wang, C., Zhang, M., Zhang, L., Du, Q., Li, Y., Tang, N., Chang, B., 2014. Effects of n-butylparaben on steroidogenesis and spermatogenesis through changed E-2 levels in male rat offspring. Environ. Toxicol. Pharmacol. 37, 705–717.
Lioy, P.J., Hauser, R., Gennings, C., Koch, H.M., Mirkes, P.E., Schwetz, B.A., Kortenkamp, A., 2015. Assessment of phthalates/phthalate alternatives in children's toys and childcare articles: review of the report including conclusions and recommendation of the Chronic Hazard Advisory Panel of the Consumer Product Safety Commission. J. Expo. Sci. Environ. Epidemiol. 25, 343–353. Marques-Pinto, A., Carvalho, D., 2013. Human infertility: are endocrine disruptors to blame? Endocr. Connect. 2, R15–R29. Moreman, J., Lee, O., Trznadel, M., David, A., Kudoh, T., Tyler, C.R., 2017. Acute toxicity, teratogenic, and estrogenic effects of bisphenol A and its alternative replacements bisphenol S, bisphenol F, and bisphenol AF in zebrafish embryo-larvae. Environ. Sci. Technol. 51, 12796–12805. OECD, 2005. Guidance document on the validation and international acceptance of new or updated test methods for hazard assessment. OECD Environ., 14. Available: 〈http://www.oecd.org/officialdocuments/displaydocument/?Cote=env/jm/mono (2005)14&doclanguage=en〉. OECD, 2007. Uterotrophic bioassay in rodents: a short-term screening test for oestrogenic properties. In: OECD Guidelines for the Testing of Chemicals, Section 4. OECD Publishing, Paris. https://doi.org/10.1787/9789264067417-en. OECD, 2010. Environmental policy design characteristics and technological innovation: Evidence from patent data. OECD Environment Working Papers, No. 16. 〈https://doi. org/10.1787/5kmjstwtqwhd-en〉. OECD, 2011. Test No. 456: H295R Steroidogenesis Assay, OECD Guidelines for the Testing of Chemicals, Section 4. OECD Publishingk Parishttps://doi.org/10.1787/ 9789264122642-en. OECD, 2012. Guidance document on standardised test guidelines for evaluating chemicals for endocrine disruption. OECD Environ., 22. Available: 〈http://www.oecd.org/ officialdocuments/displaydocument/?Cote=ENV/JM/MONO(2012)22& doclanguage=en〉. OECD, 2016a. Test No. 455: Performance-based Test Guideline for Stably Transfected Transactivation In Vitro Assays to Detect Estrogen Receptor Agonists and Antagonists. OECD Guidelines for the Testing of Chemicals, Section 4. OECD Publishing, Paris. https://doi.org/10.1787/9789264265295-en. OECD, 2016b. Test No. 458: Stably Transfected Human Androgen Receptor Transcriptional Activation Assay for Detection of Androgenic Agonist and Antagonist Activity of Chemicals. OECD Guidelines for the Testing of Chemicals, Section 4. OECD Publising, Paris. https://doi.org/10.1787/9789264264366-en. Perry, M.J., Venners, S.A., Barr, D.B., Xu, X., 2007. Environmental pyrethroid and
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Environmental Research journal homepage: www.elsevier.com/locate/envres
Assessment of endocrine-disrupting activities of alternative chemicals for bis (2-ethylhexyl)phthalate Joonwoo Parka, Choa Parka, Myung Chan Gyeb, Youngjoo Leea, a b
T
⁎
Department of Integrative Bioscience and Biothecnology, College of Life Science, Sejong University, Kwangjingu, Kunjadong, Seoul 143-747, Republic of Korea Department of Life Science, Hanyang University, Seoul 04763, Republic of Korea
A R T I C LE I N FO
A B S T R A C T
Keywords: Endocrine disruptor Phthalate Alternative material
Plastic products are closely intertwined with modern life. Some plasticizers used in making plastics, such as phthalates, are reported to be endocrine-disrupting chemicals. Plasticizers can be released into the environment, and health risks related to plasticizer exposure have been reported. In addition, due to plastic waste that flows into the ocean, microplastics have been found in marine products, including non-biological seawater products such as sea salt. Plastics can affect the body via a variety of pathways, and therefore safer alternative chemicals are needed. Three chemicals were evaluated: acetyl tributyl citrate (ATBC), triethyl 2-acetylcitrate (ATEC), and trihexyl O-acetylacitrate (ATHC), replacing bis(2-ethylhexyl)phthalate (DEHP), a typical plasticizer. The endocrine-disrupting activities of each chemical, including estrogenic or anti-estrogenic activity (test guideline (TG) No. 455), androgenic or anti-androgenic activity (TG No. 458), steroidogenesis (TG No. 456), and estrogenic properties via a short-term screening test using the uterotrophic assay (TG No. 440), were assessed in accordance with the Organisation for Economic Co-operation and Development guidelines for chemical testing. Our results showed that DEHP, ATBC, ATEC, ATHC possess no estrogenic activity, whereas DEHP, ATBC and ATHC demonstrate anti-estrogenic activity and ATBC anti-androgenic activity. DEHP and ATHC exhibited a disruption in steroidogenesis activities. Additional tests are necessary, but our results suggest that ATEC is a good candidate plasticizer providing a suitable alternative to DEHP.
1. Introduction Since mass production began in the 1950s, global production of plastics has increased to more than 322 million tons as of 2015 (PlasticsEurope, 2016). Plasticizers are low-melting-point solids or high-boiling-point liquids and are commonly used to impart flexibility and durability to plastic products. More than 6 million tons of plasticizers are produced and consumed annually worldwide (Lioy et al., 2015). Due to the recent increase in mineral water consumption, the water in plastic bottles has been suggested to cause a health risk to consumers. Antimony and phthalates are considered serious pollutants found in bottled mineral water. Phthalates are used as solvents to increase the flexibility of plastics such as polyvinyl chlorides. Phthalates are a widely used class of plasticizers found in a variety of consumer products including soap, mouthwash, disposable gloves, food or beverage containers, and toys (Hernandez-Diaz et al., 2009; Kavlock et al., 2002a, 2002b). These phthalates are not bound to plastic and therefore can be released into the environment. In addition, phthalates are lipophilic compounds and have been found to accumulate in fat (LaFleur
⁎
and Schug, 2011). In addition to pollution of the water within, plastic bottles can cause other problems including the release of microplastics, which have recently been detected in sea salt. Plastic waste that is not managed properly is eventually found in the ocean (Jambeck et al., 2015). Due to the continued fragmentation of large plastics, tiny particles called microplastics accumulate, and several studies have shown that microplastics are present in seafood products such as clams and fish (Davidson and Dudas, 2016; Foekema et al., 2013). Therefore, the consumption of seafood can be a significant exposure pathway for humans to microplastics. For example, those who consume clams in large quantities in Europe are expected to ingest up to 11,000 microplastics each year (Van Cauwenberghe and Janssen, 2014). In addition to seafood, microplastics can also be found in non-biological seawater products. Commercial sea salt is produced mainly through crystallization as a result of seawater evaporation, which may be induced artificially or occur naturally due to sunlight and wind (Serrano et al., 2011). During this process, microplastics in seawater can associate with the sea salt (Brown et al., 2009). Recent research has confirmed that commercial salt available in various countries contains microplastics (Karami et al.,
Corresponding author. E-mail address: [email protected] (Y. Lee).
https://doi.org/10.1016/j.envres.2019.02.001 Received 30 September 2018; Received in revised form 17 January 2019; Accepted 1 February 2019 Available online 02 February 2019 0013-9351/ © 2019 Published by Elsevier Inc.
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forskolin were purchased from Sigma-Aldrich (St. Louis, MO, USA). ATHC was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). DEHP, ATBC, ATEC, ATHC, raloxifene, HF, forskolin is dissolved in DMSO. E2 is dissolved in ethanol and DHT is dissolved in methanol.
2017). Plastics are heavily utilized in our daily lives and can affect our bodies via various pathways. Phthalates such as bis(2-ethylhexyl) phthalate (DEHP) and di-n-butyl phthalate are suspected to be toxic to the liver, kidneys, and reproductive organs, as well as carcinogenic (Gomez-Hens and Aguilar-Caballos, 2003; Swan et al., 2005). Di-nbutyl phthalate, benzyl butyl phthalate, and DEHP are also weakly estrogenic. Some metabolites of phthalates such as mono(2-ethylhexyl) phthalate, mono-n-butyl phthalate, and monoethyl phthalate can interfere with hormone activity (Chang et al., 2015; Upson et al., 2013). These endocrine disruptors were found in the water in most plastic bottles (Pinto and Reali, 2009; Plotan et al., 2013; Wagner and Oehlmann, 2011). According to a 2005 biomonitoring study released by the Center for Disease Control and Prevention, more than 75% of spot urine samples were found to contain phthalates consistently throughout life (Control, 2005). Therefore, identifying safer alternative chemicals for use in consumer products is essential. Humans are exposed not only to phthalates but to many chemicals throughout their lives, including endocrine-disrupting chemicals (EDCs) such as bisphenol A and nonylphenol. Organizations around the world have developed international standardization methods to evaluate potential EDCs. In 1997, the Organisation for Economic Co-operation and Development (OECD) began developing guidelines for assessment of EDCs, managed by the Endocrine Disruptors Testing and Assessment task force (OECD, 2005, 2010). The OECD updated their conceptual framework for assessment of EDCs in 2012 (OECD, 2012). This document provides a five-step guideline for identifying EDCs. It describes in vitro test methods, including a cell-based transcriptional activation assay at level 2 and in vivo testing methods in animals at level 3. Following the guidelines for levels 2 and 3 of the OECD conceptual framework for assessment of EDCs, we evaluated the endocrine-disrupting activities of DEHP and its alternative chemicals: acetyl tributyl citrate (ATBC), triethyl 2-acetylcitrate (ATEC), and trihexyl O-acetylcitrate (ATHC) (Fig. 1). In this study, DEHP, ATBC, ATEC, and ATHC were evaluated for endocrinedisrupting activities, including estrogenic or anti-estrogenic activity, androgenic or anti-androgenic activity, steroidogenesis, and estrogenic properties via a short-term screening test using the uterotrophic assay, in accordance with the OECD guidelines for chemical testing.
2.2. Cell culture HeLa9903 cells and VM7-Luc cells were cultured in phenol red-free Dulbecco's modified Eagle medium (DMEM) (WelGENE, Daegu, South Korea) supplemented with 10% fetal bovine serum (FBS) (WelGENE) and penicillin/streptomycin (GIBCO Invitrogen, Grand Island, NY, USA). Androgen receptor (AR)-EcoScreen™ Cells were cultured in phenol red-free DMEM/F12 (WelGENE) supplemented with 5% FBS. H295R cells were cultured in phenol red-free DMEM/F12 (WelGENE) supplemented with 2.5% Nu-serum (Corning, NY, USA), and 1% ITS + premix supplement (6.25 μg/ml insulin; 6.25 μg/ml transferrin; 6.25 ng/ml selenium; 1.25 mg/ml bovine serum albumin; 5.35 μg/ml linoleic acid; Corning). All media contained 100 μg/ml of streptomycin (GIBCO Invitrogen, Grand Island, NY, USA).
2.3. Stably transfected transcriptional activation (STTA) assay The estrogenic and anti-estrogenic activities assessments each used HeLa9903 and VM7-Luc cells, and the evaluation procedure was tested in accordance with the OECD test guideline (TG) No. 455 (OECD, 2016a). Both cell lines stably express estrogen receptor (ER) alpha gene containing a firefly luciferase gene as a reporter gene and estrogenic activity was assessed by increasing ER transcriptional activity of test substance usingE2-enhanced ER transcriptional activity. The androgenic and anti-androgenic activities assessment used AR-EcoScreen™ cells, and the evaluation procedure was tested in accordance with the OECD TG458 (OECD, 2016b). This cells stably expresses AR gene containing a firefly luciferase gene as a reporter gene and androgenic activity was assessed by increasing AR transcriptional activity of test substance using DHT-enhanced AR transcriptional activity. Luciferase activity was determined with Glomax luminometer (Promega Corp., Madison, WI, USA) using the luciferase assay system (TG455 used OneGlo®, Promega; TG458 used Dual-Glo®, Promega). Since the concentration used varies from one test method to another, a detailed description was written on each figure legends. All experiments of STTA assay were repeated at least three times.
2. Materials and methods 2.1. Materials DEHP, ATBC, ATEC, 17-β-estradiol (E2), 5α-Dihydrotestosterone (DHT), dimethyl sulfoxide (DMSO), raloxifene, hydroxylflutamide (HF),
Fig. 1. Structures of chemicals. (A) DEHP (pubchem CID: 8343), (B) ATBC (pubchem CID: 6505), (C) ATEC (pubchem CID: 6504), (D) ATHC (pubchem CID: 90617). 11
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anti-estrogenic activities of chemicals to determine the endocrine-disrupting activities of chemical alternatives to DEHP. HeLa9903 cells were treated with DEHP, ATBC, ATEC, or ATHC at 1 nM to 1 mM for 24 h. DEHP and the three alternative chemicals did not exhibit any estrogenic activity (Fig. 2A–D). The anti-estrogenic activities of the chemicals were assessed in VM7-luc cells, which were treated for 24 h with 500 pM E2 and 9.7 nM to 10 μM of the test chemicals. DEHP, ATBC, and ATHC exhibited anti-estrogenic activities, with respective IC50 values of 66 μM, 439 nM, and 136 nM. On the other hand, ATEC did not exhibit anti-estrogenic activity at any of the concentrations evaluated (Fig. 3A–D). The OECD proposed the uterotrophic assay as an in vivo test to detect the estrogenic activities of potential EDCs (Kanno et al., 2003; OECD, 2007). The uterus responds to estrogen and increases the weight of the uterus due to water absorption. Estrogenic activity can be assessed by changing the uterine weight by the test substance using this reaction. Test verification was performed in rats, but the mouse model is considered equally valid. We followed the OECD guideline No. 440 and used mice to determine the estrogenic activities of DEHP and alternative chemicals (OECD, 2007). OVX mice at 8 weeks of age were randomly assigned to five groups, and one group of non-OVX mice was formed, with six mice of similar average body weight per group (Table 1). Treatment was administered via oral injection at 24-h intervals over 7 days (Fig. 4A). E2 was used as a positive control. To detect estrogenic activity, two vehicles (non-OVX and OVX mice) were employed, using E2 as a positive control and 400 mg/kg/day DEHP and 40 or 400 mg/kg/day ATEC as the test conditions. In this experiment, E2 was administered at 0.4 mg/kg/day, and uterine weight increased by approximately 4.9 times compared with OVX control group. However, neither test compound (DEHP 400 mg/kg/day, ATEC 40 or 400 mg/kg/day) caused a significant increase in uterine weight compared with the negative control group (OVX control) (Fig. 4B). These results indicated that ATEC was the only substance without estrogenrelated activity.
2.4. Steroidogenesis assay The purpose of the steroidogenesis assay is to detect chemicals that affect the production of E2 and testosterone. Chemical interference test assessments used a human adreno-carcinoma H295R cells, and the evaluation procedure was tested in accordance with the OECD TG456 (OECD, 2011). Forskolin was treated at a concentration of 10 μM as a positive control, and each test chemicals were treated at a concentration of 1 and 10 μM for 48 h. Hormones in culture medium were extracted with diethyl ether in glass tubes, and phase separation was achieved by centrifugation at 2000 × g for 10 min. Estradiol or testosterone hormone changes were measured using enzyme-linked immunosorbent assay (ELISA) using the manufacturer's recommendations. Estradiol ELISA kit and testosterone ELISA kit were purchased from Cayman Chemical (Ann Arbor, MI, USA). 2.5. Uterotrophic bioassay Uterotrophic bioassay is a short-term screening test for estrogenic properties. It is based on the increase in uterine weight or uterotrophic response. It evaluates the ability of a chemical to produce biological activities consistent with natural estrogen. Female ICR mice and ovariectomized (OVX) ICR mice (6 weeks old) were purchased from Central Laboratory Animal Inc. (Seoul, South Korea) and maintained under a protocol approved by the Sejong University Institutional Animal Care and Use Committee (Ref. No. SJ-20160304E1). All procedures were performed according to the OECD TG440 (OECD, 2007). OVX mice were randomly assigned to five groups, and one group of non-OVX-mice was formed, with six mice of similar average body weight per group. Treatment was administered via oral injection at 24-h intervals over 7 days. E2 was administered at 0.4 mg/kg/day, DEHP was administered at 400 mg/kg/day and ATEC was administered at 40 or 400 mg/kg/ day. The study design is shown in Table 1. 2.6. Statistical analysis
3.2. Assessment of androgen-related activity
All in vitro experiments were repeated at least three times. The data were expressed as mean ± SD using GraphPad Prism 7.0 (Graph Pad Software, La Jolla, CA, USA). Statistical analysis of the data was determined by 2-tailed student's t-test and p < 0.01 (*) was considered as statistically significance.
Next, to define the endocrine-disrupting activities of alternative chemicals to DEHP, we used the stably transfected transcriptional activation assay described in the OECD in vitro test guideline No. 458 to assess androgenic and anti-androgenic activities. AR-EcoScreen cells were treated with DEHP, ATBC, ATEC, or ATHC at 10 pM to 10 μM for 24 h. DEHP, ATEC, and ATHC showed no androgenic activity, but ATBC exhibited weak androgenic activity (PC10, 1.86 μM) (Fig. 5A–D). PC10 mean the concentration of a chemical at which its response equals 10% of the maximum response of the reference standard (DHT). In addition, the anti-androgenic activities of DEHP and its alternative chemicals were also measured (Fig. 6A–E). Hydroxyflutamide was used as a positive control and exhibited an IC50 of 10.8 nM (Fig. 6A). IC50 mean the molar concentration of a chemical at which produces 50% of the maximum possible response for that chemical (50% inhibition). The anti-androgenic activities of the chemicals, at 100 pM to 10 μM, were tested. DEHP and the three alternative chemicals exhibited no antiandrogenic activity. These results indicate that ATEC and ATHC do not have androgen-related activity.
3. Results 3.1. Assessment of estrogen-related activity EDCs have been reported to affect multiple endocrine pathways, hormones, and homeostatic systems, most of which are affected by the disruption of estrogen or androgen mechanisms, and EDCs may also affect steroid generation and reproduction (Del Mazo et al., 2013; Knez, 2013; Marques-Pinto and Carvalho, 2013; Zhang et al., 2014). First, we used the stably transfected transcriptional activation assay described in the OECD in vitro test guideline No. 455 to assess the estrogenic and Table 1 Study design for uterotrophic assay of DEHP and ATEC.
3.3. Assessment of hormone production
Group
Group number
Agonist detection
Non-OVX negative control OVX negative control OVX positive control OVX-DEHP 400 mg/kg/day OVX-ATEC 40 mg/kg/day OVX-ATEC 400 mg/kg/day
1 2 3 4 5 6
Vehicle Vehicle E2 Test substance Test substance Test substance
In several studies, EDCs have also been reported to affect steroid hormone synthesis and reproduction (Jouannet et al., 2001; Knez, 2013; Perry et al., 2007; Safe, 2013; Zhang et al., 2014). In several studies, EDCs have also been reported to affect steroid hormone synthesis and reproduction. Therefore, we determined the steroidogenic capabilities of alternative chemicals to DEHP. We used the H295R steroidogenesis assay to detect chemicals that can induce (or reduce) production of sex steroids (E2 or testosterone). The H295R
Vehicle: Corn oil, 100 μl/day. E2: β-estradiol, 0.4 mg/kg/day. 12
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Fig. 2. Effect of chemicals on ER agonist activity using OECD test guideline No. 455. HeLa9903 cells were treated DEHP (A), ATBC (B), ATEC (C), and ATHC (D) with a concentration of 1 nM to 1 mM for 24 h and luciferase activities were determined. E2 was used as a positive control and treated 24 h at a concentration of 1 nM. Data are expressed as the mean ± SD (n = 3). Asterisks (*) indicate significant difference from control. * represent p < 0.01.
Fig. 3. Effect of chemicals on ER antagonist activity using OECD test guideline No. 455. VM7-luc cells were treated for 24 h with 500 pM E2 and 9.7 nM to 10 μM of DEHP (A) and alternative chemicals ATBC (B), ATEC (C), and ATHC (D) and luciferase activities were determined. Raloxifene was used as a reference substance and treated 24 h at a concentration of 95.6 pM to 24.5 nM for 24 h. The positive determination of antagonist activity should be at least a 20% reduction in activity from the maximal value for the reference substance (cotreatment of Raloxifene and E2). Data are expressed as the mean ± SD (n = 3).
and testosterone. The E2 concentration was increased 3.2 times by 10 μM DEHP compared with control (Fig. 7A). The testosterone concentration was increased by ~1.2 times by 10 μM DEHP compared with control, which was a statistically significant increase (Fig. 7B). Meanwhile, 1 μM ATHC decreased the testosterone concentration by ~80%
steroidogenesis assay was also tested according to the OECD test guideline No. 456. H295R cells were treated with 1 and 10 μM DEHP and its alternative chemicals for 24 h, and the concentrations of E2 and testosterone were measured using enzyme-linked immunosorbent assay kits. As shown in Fig. 7, forskolin was used as a positive control for E2 13
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Fig. 4. Assessment of estrogenic response in ovariectomized mice. (A) Experimental schedule. OVX mice at 8 weeks age were randomly assigned to five groups, and one group of non-OVX mice was formed, with six mice of similar average body weight per group. Treatment was administered via oral injection at 24-h intervals over 7 days. Corn oil was used vehicle and administered at 100 μl/day. E2 was used positive control and administered at 0.4 mg/kg/day. DEHP was administered at 400 mg/kg/day and ATEC was administered 40 or 400 mg/kg/day via oral injection. (B) The effects of DEHP and ATEC on uterine weight. Data are expressed as the mean ± SD (n = 6). Asterisks (*) indicate significant difference from OVX-control group. * represent p < 0.01. p value > 0.05 is indicated by “n.s.” for not significant.
has been approved by the U.S. Food and Drug Administration as a commercial plasticizer that is permitted as an additive in food contact materials (Commision, 2010). However, ATBC has been reported to affect reproduction in female mice (Rasmussen et al., 2017), and our results confirmed that ATBC exhibits estrogen receptor antagonist activity and androgen receptor agonist activity (Figs. 3B and 5B). Thus, even chemical alternatives to phthalates, such as ATBC, may exhibit endocrine-disrupting activity and present multiple hazards and therefore will require further study. In another example, bisphenol F (BPF) was used as an alternative to bisphenol A (BPA), also known as EDC. However, it has been reported that BPF has an estrogenic potential similar to BPA (Moreman et al., 2017), and we have confirmed that BPF has an estrogenic activity (data not shown). It has been reported that continuous exposure of EDCs to our bodies can have various effects such as dysfunction of reproductive systems, inflammation, and DEHP induced the proliferation of human endometrial cancer cells (Sifakis et al., 2017; Cho et al., 2014). Many of the products used by humans contain a variety of chemicals. If any of these chemicals are EDC, there is a risk of continuing exposure to the body. Therefore, chemicals that can be frequently exposed to our bodies need to be assessed for endocrine disrupting activities. We evaluated the effects of three chemical alternatives to DEHP (ATBC, ATEC, and ATHC) on estrogen- and androgen-related activities and steroidogenesis. ATBC exhibited estrogen receptor antagonist activity and androgen receptor agonist activity, whereas ATHC exhibited only estrogen receptor antagonist activity. ATEC had no effect on estrogen or androgen activities or steroidogenesis (Table 2). However, although ATEC did not have an AR agonist activity (Fig. 5C), when DHT
compared with the control group, but our data did not indicate any reduction, as only increases compared with the control group were considered. These results indicated that DEHP disrupted hormone production, whereas the alternative chemicals did not increase hormone production. 4. Discussion Phthalates have been used in numerous products including plastics, emulsifiers, paints, coating solvents, and cosmetics, but the use of phthalates has been restricted worldwide due to their risks. Therefore, efforts have been made to manufacture plastics that do not use plasticizers such as phthalates. Initially, plastic is manufactured from petroleum-based compounds such as polyurethane, polystyrene, and ethylene vinyl acetate (Becker et al., 2010; Tickner, 1999). However, although phthalates are avoided to minimize risk, other problems occur related to the release of various harmful substances during the manufacturing process, including heavy metals and sulfur dioxides. In addition, some products, such as polystyrene, are classified as carcinogens and require further study (Huff and Infante, 2011). Subsequently, alternative plastics have been developed using plant-based materials called bioplastics. Sugar and oil extracted from plants such as corn are highly biodegradable and produce few pollutants during the production process. However, bioplastics have limitations in terms of their mechanical properties and high production costs. For this reason, it is necessary to develop alternative plasticizers to make up for the limited manufacture of non-plasticizers. One plasticizer offering an alternative to phthalate is ATBC, which is commonly used in polyvinyl resins and 14
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Fig. 5. Effect of chemicals on AR agonist activity using OECD test guideline No. 458. AREcoScreen cells were treated with DEHP, ATBC, ATEC, or ATHC at 10 pM to 10 μM for 24 h and luciferase activities were determined. DHT was used positive control and treated 24 h at concentration of 1 nM. Data are expressed as the mean ± SD (n = 3). Asterisks (*) indicate significant difference from control. * represent p < 0.01.
Fig. 6. Effect of chemicals on AR antagonist activity using OECD test guideline No. 458. AR-Ecoscreen cells were treated for 24 h with 500 pM DHT and 10 pM to 10 μM of HF (A), DEHP (B) and ATBC (C), ATEC (D), and ATHC (E) and luciferase activities were determined. HF was used as a positive control. If the log IC30 of the test chemical is calculated (30% inhibition), determine that the chemical has AR antagonist activity. Data are expressed as the mean ± SD (n = 3).
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Fig. 7. Effect of chemicals on steroid hormone production using OECD test guideline No. 456. H295R cells were treated for 48 h with 1 or 10 μM of DEHP, ATBC, ATEC, and ATHC. After treatment, hormones in culture medium were measured by Estradiol (A) and testosterone (B) ELISA kit. Data are expressed as the mean ± SD (n = 3). Asterisks (*) indicate significant difference from control. * represent p < 0.01.
Table 2 Summary of endocrine-disrupting activities of alternative chemicals for DEHP. `
ER agonist
ER antagonist
AR agonist
AR antagonist
Steroidogenesis
in vivo ER activity
ATBC ATEC ATHC
ND ND ND
IC50 439 nM ND IC50 136 nM
PC10 1.86 μM ND ND
ND ND ND
ND ND ND
– ND –
ND: Not detected. IC50: The molar concentration of a chemical at which produces 50% of the maximum possible response for that chemical (50% inhibition). PC10: The concentration of a chemical at which its response equals 10% of the maximum response of the reference standard.
and ATEC were co-treated, ATEC showed concentration dependent increases ARE-transcriptional activity (Fig. 6D). Further study may be required, but ATEC is likely to have an enhanced-AR agonist activity that increases ARE-transcriptional activity when an AR agonist such as DHT is present.
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