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BOR - Papers in Press, published online ahead of print January 21, 2004.
Biol Reprod 2004, 10.1095/biolreprod.103.024810
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BIOLOGY OF REPRODUCTION 70, 1527–1533 (2004)
DOI: 10.1095/biolreprod.103.024810
© 2004 by the Society for the Study of Reproduction, Inc.

Environment

Estrogenic Activities of Nitrophenols in Diesel Exhaust Particles1

Chie Furuta3, Akira K. Suzuki2,4, Shinji Taneda4, Kazuyuki Kamata5, Hideyuki Hayashi5, Yoki Mori5, Chunmei Li3,6, Gen Watanabe3,6, and Kazuyoshi Taya3,6

Laboratory of Veterinary Physiology,3 Tokyo University of Agriculture and Technology, Tokyo 183-8509, Japan PM2.5/DEP Research Project,4 National Institute for Environmental Studies, Ibaraki 305-8506, Japan Faculty of Pharmaceutical Sciences,5 Health Sciences University of Hokkaido, Hokkaido 061-0293, Japan Department of Basic Veterinary Sciences,6 The United Graduate School of Veterinary Sciences, Gifu University, Gifu 501-1193, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We recently isolated 3-methyl-4-nitrophenol (4-nitro-m-cresol; PNMC) and 4-nitro-3-phenylphenol (PNMPP) from diesel exhaust particles (DEP) and identified them as vasodilators. Because these compounds are alkylphenolic derivatives that might mimic hormones, we evaluated their estrogenic activity by using recombinant yeast screens, myometrial contractility assays, and in vivo uterotrophic assays. Recombinant yeast screen assays showed that both PNMC and PNMPP possess estrogenic activity. Furthermore, ovariectomized 25-day-old immature female rats injected with PNMC and PNMPP subcutaneously for 2 days showed significant increases in uterine weight among those receiving 100 mg/kg PNMC and 0.1 and 1.0 mg/kg PNMPP. To clarify further the estrogenic activity of PNMC and PNMPP, rat uterine horns were monitored in organ bath chambers for myometrial contractility in response to oxytocin (OT). Significant differences occurred in the initial and maximum contractilities to OT at 0.25 and 25 mIU/ml in uterine horns obtained from animals treated with 100 mg/kg PNMC and in the maximum contractilities to OT at 0.025, 0.25, and 25 mIU/ml in those from rats treated with 0.1 mg/kg PNMPP. These results clearly demonstrated that PNMC and PNMPP in DEP have estrogenic activity both in vitro and in vivo and might therefore be considered as endocrine-disrupting chemicals.

environment, estradiol, oxytocin, uterus


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The possible health and environmental effects of exhaust particulates emitted by diesel engines have been recognized as a potential public health problem, because diesel exhaust particles (DEP) can cause lung cancer [1, 2], allergic rhinitis [3, 4], and bronchial asthma-like diseases [5, 6]. DEP is the soluble organic fraction of particulate matters from diesel exhaust (DE), and they contain numerous organic constituents. These organic components are considered to have the greatest environmental significance, and they include polyaromatic hydrocarbons, nitro aromatic hydrocarbons, heterocycles, quinones, aldehydes, and aliphatic hydrocarbons [7–10].

In recent studies, DEP and DE have been reported to exert toxic effects on both the male and female reproductive systems. These compounds had suppressive effects on spermatogenesis in mice [11] and rats [12], and the serum testosterone levels and weight of accessory glands increased significantly in F344 male rats exposed to DE for 8 mo [13]. In addition, pregnant C57BL mice injected with diesel exhaust particulate extract (DEPE) showed significant increase in abortions [14], and DEPE increased the uterine weight and myometrial contractility of C57BL mice [15]. These in vivo findings show DEP may contain compounds that modulate the activity of estrogen. Various in vitro findings support this idea, as Meek [16] reported that methylene chloride extracts of DEP could be activating ligands for estrogen receptors, and Taneda et al. [17, 18] reported that crude DEP as well as their successively extracted hexane, benzene, dichloromethane, and methanol fractions showed estrogenic activity by the recombinant yeast screen assay. Mori et al. [19] reported that 4,6- and 2,8-dimethyldibenzothiophens isolated from DEP had estrogenic activity in recombinant yeast screens. However, the specific compound that is responsible for the phenomenon remains unclear.

We recently isolated the nitrophenol derivatives 4-nitrophenol, 2- and 3-methyl-4-nitrophenol, and 4-nitro-3-phenylphenol from DEP and showed that they have vasodilatory activity [20, 21]. These compounds captured our attention because it has long been understood that alkylphenolic compounds often are associated with estrogenic activity. In receptor binding studies, alkylphenols have been shown to interact directly with the estrogen receptor of transfected chicken embryo fibroblast cells [22] and to act in an identical way to 17ß-estradiol (E2) in stimulating the receptor transcription [23]. Therefore, the alkylphenols of DEP are a strong candidate for their association with estrogenicity.

From among those nitrophenols with alkylphenolic structures that we isolated from DEP, we focused on 3- methyl-4-nitrophenol (PNMC) and 4-nitro-3-phenylphenol (PNMPP) for the evaluation of their estrogenic activity. In the present study, we used in vitro and in vivo techniques to demonstrate the estrogenic activities of these two nitrophenols of DEP.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals

3-Methyl-4-nitrophenol (4-nitro-m-cresol, PNMC) and 4-nitro-3-phenylphenol (PNMPP) were used (Fig. 1). PNMC was purchased from Tokyo Kasei Kogyo Co. Ltd. (Tokyo, Japan), and PNMPP was synthesized by the method described previously [21].



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  		FIG. 1. Chemical structures of 3-methyl-4-nitrophenol (4-nitro-m-cresol; PNMC) and 4-nitro-3-phenylphenol (PNMPP) isolated from diesel exhaust particles

Recombinant Yeast Screen Assay

Yeast (Saccharomyces cerevisiae) expressing the human estrogen receptor alpha (ER{alpha}) and responsive elements regulating the expression of a ß-galactosidase reporter gene (hER-yeast) were provided by Prof. John P. Sumpter (Department of Biology and Biochemistry, Brunel University, Uxbridge, UK). This strain was developed in the Genetics Department of Glaxo Wellcome plc (Medicines Research Centre, Stevenage, Herts, UK). Estrogenic activity was determined by using a recombinant hER-yeast screen according to the method of Routledge and Sumpter [24] with slight modification, as described previously [25].

Animals

Ninety-eight 22-day-old immature female Wistar-Imamichi rats were purchased from Imamichi Institute for Animal Reproduction (Kasumigaura, Ibaraki, Japan). They were kept under conditions of controlled lighting (lights on from 0700 to 1900 h), temperature (22 ± 2°C), humidity (50% ± 5%), and ventilation (25–30 fresh-air changes hourly). Food (CE-2 commercial diet; Japan Clea Co., Tokyo, Japan) and water were available ad libitum.

This study was conducted in accordance with the Guiding Principals for the Use of Animals in Toxicology and was approved by the Japanese National Institute for Environmental Studies Animal Care and Use Committee.

Uterotrophic Assay

Twenty-five-day-old immature female rats were ovariectomized (OVX) and injected s.c. with PNMC (1, 10, and 100 mg/kg) or PNMPP (0.1, 1, and 10 mg/kg) daily for 2 days beginning on the day of the surgery. Rats were injected with vehicle alone (PBS containing 0.05% Tween 80) as negative controls and with estradiol 17ß (E2) in sesame oil (6 µg/kg) as positive controls. At 24 h after the last injection, rats (n = 7–16 per group) were weighed and killed by decapitation. The uteri were weighed and their contractility measured (described later). Blood samples were collected into plastic tubes containing heparin and centrifuged at 1700 x g for 15 min at 4°C. The plasma was stored at –70°C until assayed for LH.

Radioimmunoassays of LH

Concentrations of LH in plasma were measured using National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) radioimmunoassay kits for rat LH. Iodinated preparations were rat LH-I-5, and the antiserum used was anti-rat LH-S-9. Results were expressed in terms of NIDDK rat LH-RP-2. The intra- and interassay coefficients of variation were 7.9% and 8.4%, respectively.

Myometrial Contractility Assay

The uteri from PNMC- and PNMPP-treated OVX rats and their controls were removed carefully to avoid excessive stretching; each was placed in a laboratory dish containing Krebs solution (118 mM NaCl, 4.5 mM KCl, 1.0 mM MgSO2, 1.0 mM KH2PO4, 25 mM NaHCO3, 1.8 mM CaCl2, and 6.0 mM glucose) and weighed, then the right horn was cut near the base to a 17-mm length. The mechanical activity of the uterine samples was recorded continuously under isometric conditions. Each sample was connected to a force displacement transducer (model TB-612T; Nihon Kohden Co., Ltd., Tokyo, Japan) coupled to a multichannel amplifier (model MEG-6108; Nihon Kohden Co., Ltd.), mounted in a 10-ml organ bath containing Krebs solution suffused with 95% O2:5% CO2, kept at 37°C, bathed continuously with Krebs solution, and allowed to equilibrate for 30 min under an initial load of 1.0 g. When contraction reached a plateau, oxytocin (OT) (Wako Pure Chemical Industries, Ltd., Osaka, Japan) was accumulatively added to 0.025, 0.25, 2.5, and 25 mIU/ml, and the first myometrial contraction (the initial myometrial contractility) and maximum myometrial contractility after adding OT were recorded.

Statistical Analysis

For the statistical analysis, the data were transformed logarithmically to equal the variances. Then analysis of variance (ANOVA) was used to evaluate the results. When ANOVA was significant, the differences between groups were assessed by means of ANOVA followed by the Tukey- Kramer test. A probability value (P) of <0.05 was considered to be significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Recombinant Yeast Screen Assay

Estrogenic activity of PNMC was observed at all concentrations from 4.9 x 10–7 M through 1.0 x 10–3 M (Fig. 2). There were drastic increases in the activity at the concentration of 2.5 x 10–4 M, and the highest activity was observed at the highest concentration of 1.0 x 10–3 M.



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  		FIG. 2. Estrogenic activity of 3-methyl-4-nitrophenol (4-nitro-m-cresol; PNMC). Estrogenic activity was evaluated in a recombinant yeast screen. The data show the mean ± SEM (n = 6) of activity measured after 72 h of incubation. * P < 0.05 compared with control (Tukey-Kramer test)

Estrogenic activity of PNMPP was observed at concentrations of 3.9 x 10–6 M through 2.5 x 10–4 M (Fig. 3). The activity increased dose dependently from 3.9 x 10–6 M through 3.1 x 10–5 M. The highest estrogenic activity was observed at the concentrations of 3.1 x 10–5 M and 6.3 x 10–5 M, and the activity abruptly declined as the concentration of PNMPP became higher.



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  		FIG. 3. Estrogenic activity of 4-nitro-3-phenylphenol (PNMPP) in a recombinant yeast screen. Estrogenic activity was evaluated in a recombinant yeast screen. The data show the mean ± SEM (n = 6) of activity measured after 72 h of incubation. * P < 0.05 compared with control (Tukey-Kramer test)

Immature Rat Uterotrophic Assay

Repeated administration of PNMC and PNMPP for 2 days caused no depression of body weight. PNMC induced significant increases in uterine weight in the maximum-dose (100 mg/kg) treatment groups (122% of control value; P < 0.05); the E2 (5 µg/kg)-treated control rats showed even greater increased uterine weights (298% P < 0.05; Table 1). PNMPP induced significant increases in uterine weight at the doses of 1 mg/kg (119%; P < 0.05) and 0.1 mg/kg (124%; P < 0.05). The E2-treated control rats for the PNMPP groups also showed significant increases in uterine weights (328%; P < 0.05; Table 2).


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  		TABLE 1. The uterine weights of ovariectomized rats treated with PNMC and estradiol-17ß(E2).*


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  		TABLE 2. The uterine weights of ovariectomized rats treated with PNMPP and estradiol- 17ß(E2).*

Plasma LH Concentrations

Plasma LH concentrations of the PNMC- and PNMPP- treatment groups showed no significant changes in all the treatment groups compared with those of the vehicle-only controls. However, plasma concentrations of LH significantly decreased (P < 0.05) in both the E2-treated group compared with the vehicle-only control group (data not shown).

Myometrial Contractility Assay

Figure 4 shows representative data for the myometrial contractility of rats treated with vehicle only control, 100 mg/kg PNMC, 0.1 mg/kg PNMPP, or E2 (5 µg/kg). The myometrial contraction of the vehicle-only control rats did not change after the administration of OT, whereas E2-treated rats showed drastic changes in the myometrial contraction after the administration at each OT concentration. The myometrial contractions of PNMC 100 mg/kg and PNMPP 0.1 mg/kg treated rats did not show changes as drastic as E2, but clearly the contractions became stronger at each concentration of OT.



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  		FIG. 4. Representative data of myometrial contraction from immature ovariectomized rats injected subcutaneously with vehicle-only control, 3-methyl-4-nitrophenol: 4-nitro-m-cresol (PNMC) (100 mg/kg) or 4-nitro-3-phenylphenol (PNMPP) (0.1 mg/kg) or estradiol-17ß (E2) (5 µg/kg) for 2 days. The uterus of each animal was placed in an organ bath, exposed to saline (A) or oxytocin (OT) (B, 0.025 mIU/ml; C, 0.25 mIU/ml; D, 2.5 mIU/ml; E, 25 mIU/ml), and evaluated for myometrial contractility

The OT-associated initial and maximum myometrial contractilities of rats treated with 100 mg/kg of PNMC were significantly (P < 0.05) higher than those of control rats at almost all concentrations except for the initial contractility following the 0.025 and 2.5 mIU/ml dose (Fig. 5). However, regardless of the OT dose, the initial and maximum myometrial contractility of the rats treated with 1 and 10 mg/kg PNMC also showed a tendency to increase but did not differ significantly from that of the vehicle-only control rats.



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  		FIG. 5. The initial contractility (a) and maximum contractility (b), described in grams (g), of the oxytocin-exposed uteri of immature ovariectomized rats injected with 3-methyl-4-nitrophenol: 4-nitro-m-cresol (PNMC; 1, 10, and 100 mg/kg). Each bar represents the mean ± SEM of 7–8 rats per group. * P < 0.05 compared with control rats (Tukey-Kramer test).

The OT-associated initial myometrial contractilities of all PNMPP-treated groups did not differ from that of vehicle-only control rats (Fig. 6). The maximum myometrial contractilities of rats treated with the minimum dose of 0.1 mg/kg PNMPP were significantly higher (P < 0.05) than those of controls at the OT concentrations of 0.025, 0.25, and 25 mIU/ml. In addition, the maximum contractility of the group treated with 1 mg/kg PNMPP and 0.25 mIU/ml OT was increased significantly (P < 0.05) compared with that of the control group.



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  		FIG. 6. The initial contractility (a) and maximum contractility (b), described in grams (g), of the oxytocin-exposed uteri of immature ovariectomized rats injected with 4-nitro-3-phenylphenol (PNMPP; 0.1, 1, and 10 mg/kg). Each bar represents the mean ± SEM of 5–8 rats per group. * P < 0.05 compared with control rats (Tukey-Kramer test).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
To our knowledge, this is the first report to demonstrate that the compounds isolated from DEP show estrogenic activities in the female reproductive system. The present study clearly demonstrated that PNMC and PNMPP showed estrogenic activities in vitro and in vivo by the recombinant yeast screen and myometrial contractility and uterotrophic assays. The implications of these studies can be appreciated from both endocrinological and environmental perspectives. From an endocrinological perspective, these findings are important because PNMC and PNMPP both had not yet been characterized as having endocrine- disrupting properties.

PNMC and PNMPP exhibited estrogenic activity by the recombinant yeast screen in the present study. Whereas PNMC showed estrogenic activity at all concentrations tested, PNMPP showed an inverted U-shaped response, indicating that, at high concentrations, these compounds were toxic to yeast cells. We speculate that these cytotoxicities originate from the cresol skeleton of PNMC and the phenolic skeleton of PNMPP.

The OVX rat model is effective for the detection of estrogen agonists and antagonists [26], and uterine weight [27] has been widely used as a sensitive parameter for evaluating estrogenic activity. In the present study, the uteri of 25-day-old immature rats were more sensitive to estrogen than those of adult female rats (data not shown). Therefore, we used this immature rat model to detect the estrogenic activities of PNMC and PNMPP in light of reports of increased uterine weight after injection of DEPE in mice [15]. Immature rats injected with 100 mg/kg PNMC and 1 and 0.1 mg/kg PNMPP showed significant increases in uterine weight. Interestingly, PNMPP showed more estrogenic activity at low doses than did PNMC given the well characterized nature of the ER{alpha} ligand-binding cavity [28–30].

The in vitro myometrial contractility assay is a way to assess estrogenic activity via uterine contractions. The OT- associated initial and maximum myometrial contractilities of rats treated with 100 mg/kg PNMC were significantly higher than those of control rats, and the maximum contractility of rats treated with 0.1 mg/kg PNMPP was significantly increased. The increases in myometrial contractility may have been due to an increased number of oxytocin receptors (OTRs) in the uterus. There have been reports that estrogen increases OTR mRNA levels in the rat uterus [31]. Therefore, both PMNC and PNMPP clearly exert direct estrogenic effects on the uterus. In contrast, plasma LH did not change compared with control levels in any PNMC- or PNMPP-treatment group. These findings suggest that the estrogenic activity of PNMC and PNMPP is insufficient to suppress the gonadotroph-stimulated secretion of LH.

It has been reported that two types of estrogen receptor (ER), the classic ER{alpha} form and a novel estrogen receptor beta (ERß), exist with differential expression in organs/tissues [32, 33]. Expression of ER{alpha} in the mouse are found in all reproductive tissues and are more abundant compared with ERß, with the exception in the ovary, where ERß expression is more abundant [34]. The uteri of both the ER{alpha} knockout and ER{alpha}/ERß knockout mice do not exhibit sensitivity to estradiol treatment [35] but the ERß knockout mice appear to respond normally to estrogen treatment [36]. In this study, priority was for the screening of the novel estrogenic compounds isolated from DEP. Therefore, we have only concentrated on the ER{alpha} alone for the yeast screen and the uterotrophic assays because the uterus is mostly dependent on the ER{alpha}. From the results obtained, PNMC and PNMPP both show estrogenic activity via ER{alpha}. These compounds, however, might or might not bind to ERß because ER{alpha} and ERß both bind to estradiol and interact with estrogen response element (ERE) sequences. There are reports of compounds with preferential binding for either ER{alpha} and ERß that have also been synthesized, illustrating that the binding activities of ERs can be distinguished [37–40]. Differences in ligand interactions or activity with the two ERs could translate into important differences in their biological actions at the tissue level. The studies for the further estrogenic mechanisms of PNMC and PNMPP are an important task to be solved.

The present findings also are important from the environmental perspective, as DE emissions are ubiquitous in the environment, and remarkable amounts of DEP are exhausted into the air that we breathe. For example, in Japan, 58 902 tons (t) [41], in the United States, 111 530 t [42], in England, 37 000 t [43], and in Europe, the highest, of 240 000 t [44] are emitted each year, and this is an amount that cannot be ignored. The amount of PNMC and PNMPP that are included in 1 kg of DEP are 28 and 15 mg, respectively [21]. The environmental concentrations of these compounds are not well known because the research on the isolation of the compounds found in DEP has just begun. In addition, PNMC is a known degradation product of the insecticide fenitrothion [45], which is used widely in many countries, and PNMC is being accumulated not only in the air but also in soils and water [46, 47]. Therefore, effects of the compounds on the environment might be a serious problem in the world. Although most studies have focused on the mutagenic and carcinogenic properties of DEP and their association with respiratory diseases, there is a concern that DEP may contain compounds with potential endocrine-disrupting properties. There is evidence that DEPE contains ER ligand(s) [27], and crude DEP and their successive extracts showed estrogenic activities in hER-yeast screens [18]. Furthermore, the in vivo results of increased uterine weight and abortion in mice treated with DEPE [14, 15] demonstrate the estrogenic activities of these compounds and strongly support the in vitro findings. But there have been no reports on the specific compounds responsible for the observed activities. The present study is the first to demonstrate in vivo the specific estrogen-like compounds from DE and may shed light on the previously undetermined compounds responsible for the estrogenic activities of DEP. These results, therefore, clearly demonstrate that the PNMC and PNMPP in DEP are endocrine-disrupting chemicals.


   ACKNOWLEDGMENTS
 
We thank Glaxo Wellcome for developing the yeast screen, which, with permission, was given to us by Prof. John P. Sumpter (Department of Biology and Biochemistry, Brunel University, Uxbridge, UK). We are grateful to the National Hormone and Pituitary Program, NIDDK, and Dr. A.F. Parlow for the rat LH RIA kit.


   FOOTNOTES
 
1 Supported in part by a Grant-in-Aid for Scientific Research (the 21st Century Center-of-Excellence Program, E-1) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. Back

2 Correspondence. FAX: 81 298 50 2461; suzukiak{at}nies.go.jp Back

Received: 2 November 2003.

First decision: 13 November 2003.

Accepted: 19 January 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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