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Published-Ahead-of-Print October 4, 2006, DOI:10.2164/jandrol.106.000802
Journal of Andrology, Vol. 28, No. 2, March/April 2007
Copyright © American Society of Andrology
DOI: 10.2164/jandrol.106.000802

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Effects of 3-Methyl-4-Nitrophenol in Diesel Exhaust Particles on the Regulation of Testicular Function in Immature Male Rats

CHUNMEI LI*,{dagger}, SHINJI TANEDA{ddagger}, AKIRA K. SUZUKI{ddagger}, CHIE FURUTA*,{dagger}, GEN WATANABE*,{dagger} AND KAZUYOSHI TAYA*,{dagger}

From the * Department of Basic Veterinary Science, United Graduate School of Veterinary Sciences, Gifu University, Gifu, Japan; the {dagger} Laboratory of Veterinary Physiology, Department of Veterinary Medicine, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Tokyo, Japan; and the {ddagger} Environmental Nanotoxicology Section, Research Center for Environmental Risk, National Institute for Environmental Studies, Ibaraki, Japan.

Correspondence to: Akira K. Suzuki, Environmental Nanotoxicology Section, Research Center for Environmental Risk, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan (e-mail: suzukiak{at}nies.go.jp).
Received for publication May 31, 2006; accepted for publication September 28, 2006.

   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
We investigated the effects of 3-methyl-4-nitrophenol (4-nitro-m-cresol, PNMC) isolated from diesel exhaust particles (DEP) on the reproductive functions of male rats. Twenty-eight-day-old rats were injected subcutaneously with PNMC (1, 10, or 100 mg/kg) daily for 5 days. The weights of the epididymis, seminal vesicle, and Cowper gland were significantly decreased in rats treated with 10 mg/kg PNMC. The plasma concentrations of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were significantly increased by PNMC at 100 mg/kg. However, the plasma concentrations of testosterone and immunoreactive (ir)-inhibin were significantly decreased by PNMC at 100 mg/kg. The testosterone content of the testicles was significantly decreased in the group treated with 100 mg/kg PNMC compared with the control group. Furthermore, testicular concentration of ir-inhibin was significantly decreased by PNMC at 1 mg/kg or 100 mg/kg. To investigate the direct effects of PNMC on the secretion of LH and FSH from the anterior pituitary gland, and on the secretion of testosterone from the testes, we exposed cultured anterior pituitary and interstitial Leydig cells to PNMC (10–6, 10–5, 10–4 M) with or without gonadotropin-releasing hormone (GnRH; 10 nM) (for the LH and FSH tests) and human chorionic gonadotropin (hCG; 0.1 IU/mL) (for the testosterone test) for 24 hours. PNMC did not change either the basal or GnRH-stimulated levels of FSH and LH secretion. However, PNMC significantly inhibited both basal and hCG-stimulated testosterone production. These findings suggest that PNMC has a direct effect on the testes of immature male rats, causing a reduction in testosterone secretion.

     Key words: Leydig cells, nitrophenol, pituitary, testes


The number of chemicals known to affect human health by disrupting normal endocrine function through interactions with hormone receptors continues to increase (Colborn, 1995). Diesel exhaust particles (DEP) contain many compounds that are hazardous to human health, for example, compounds in DEP have been implicated in lung cancer (McClellan, 1987; Ichinose et al, 1997), allergic rhinitis (Muranaka et al, 1986; Takafuji et al, 1987), and bronchial asthma-like disease (Sagai et al, 1993; Miyabara et al, 1998). DEP extracts have been found to possess estrogenic, antiestrogenic, and antiandrogenic activities (Meek, 1998; Taneda et al, 2002; Kizu et al, 2003; Okamura et al, 2004).

An important aspect of the endocrine-disrupting effect of diesel emission is the potential for adversely affecting male reproductive functions. Diesel exhaust reportedly suppresses spermatogenesis in adult mice (Yoshida et al, 1999) and rats (Watanabe and Oonuki, 1999; Tsukue et al, 2001). However, since DEP contain carbon nuclei, which absorb a vast number of chemicals, the specific compound responsible for this phenomenon remains unclear.

The nitrophenol derivative 3-methyl-4-nitrophenol (4-nitro-m-cresol, PNMC), which has been isolated from DEP, is a vasodilator (Mori et al, 2003; Taneda et al, 2004a) and has both estrogenic (Furuta et al, 2004, 2005; Taneda et al, 2004b) and antiandrogenic activities (Taneda et al, 2004b; Li et al, 2006). In addition, PNMC is a degradation product of the widely used insecticide fenitrothion (Bhushan et al, 2000; Hayatsu et al, 2000). The potential for exposure of humans, livestock, and wild animals to fenitrothion may be higher in both rural and residential environments. The accumulation of PNMC from these sources has serious effects on wildlife and human health via the disruption of endocrine and reproductive systems.

No information has yet been published on the effects of PNMC on male reproductive function in immature intact rats. We used immature male rats to examine the in vivo effects of PNMC on the plasma and testicular concentrations of testosterone, immunoreactive (ir)-inhibin, basal luteinizing hormone (LH), and follicle-stimulating hormone (FSH), and to examine the in vitro effects on the secretion of LH and FSH from cultured anterior pituitary cells, as well as the secretion of testosterone from cultured interstitial Leydig cells.


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Chemicals

3-Methyl-4-nitrophenol (4-nitro-m-cresol, PNMC) was purchased from Tokyo Kasei Kogyo Co (Tokyo, Japan). Collagenase (type V), soybean trypsin inhibitor, hyaluronidase, deoxyribonuclease I (DNase I), fetal bovine serum (FBS), medium 199 (M199), and bovine serum albumin (BSA) were purchased from Sigma (St Louis, Mo). Dulbecco modified Eagle medium (DMEM), MEM non-essential amino acids, and penicillin-streptomycin were purchased from Gibco (Grand Island, NY). Human chorionic gonadotropin (hCG; 2200 IU/mg) was obtained from Sankyo ZoKi Co (Tokyo, Japan). 2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) was obtained from Dojindo Laboratories (Tokyo, Japan).

Animals

Twenty-one-day-old male Wistar-Imamichi rats were purchased from the Imamichi Institute for Animal Reproduction (Kasumigaura, Ibaraki, Japan). The rats were kept in a controlled environment with 12-hour light/dark cycles, at a temperature of 23 ± 2°C, humidity of 50 ± 10%, and ventilation with hourly fresh-air changes. 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 Principles in the Use of Animals in Toxicology, and was approved by the Animal Care and Use Committee of the Japanese National Institute for Environmental Studies.

Administration of PNMC

Twenty-eight-day-old rats were injected subcutaneously with PNMC (1, 10, or 100 mg/kg body weight) daily for 5 days. Rats injected with vehicle alone (PBS containing 0.05% Tween 80) were used as the control group. Twenty-four hours after the last injection, the rats were weighed and decapitated. Blood samples were collected in plastic tubes that contained heparin and centrifuged at 1700 x g for 15 minutes at 4°C. Plasma was separated and stored at –20°C until assayed for LH, FSH, testosterone, and ir-inhibin. The testes were removed and weighed, and the right testis was homogenized in saline at 4°C. The supernatant was collected and stored frozen at –20°C until assayed for testosterone and ir-inhibin. Accessory reproductive glands (ventral prostate, VP; seminal vesicles plus coagulating glands, SV; levator ani plus bulbocavernosus muscles, LABC; Cowper gland, COW; and glans penis, GP) were excised, carefully trimmed of excess adhering tissue and fat, and immediately weighed. The liver and kidneys were also weighed.

Preparation of Anterior Pituitary Cells and Cell Cultures

The anterior pituitary cells of 28-day-old male rats were prepared. All of the culture media contained 100 U/mL penicillin and 100 µg/mL streptomycin. The methods used for the preparation of pituitary cell cultures were similar to those described previously (Kotsuji et al, 1988). Briefly, anterior pituitaries were minced and treated for 30 minutes with 2.8 mg/mL collagenase, 0.8 mg/mL hyaluronidase, 8 mg/mL BSA, and 200 U/mL DNase in DMEM (pH 7.3) that contained 50 mM HEPES and 1% MEM non-essential amino acids. The tissues were then washed three times with DMEM that contained 10% FBS, and filtered through cell strainers (70-µm nylon filter, Falcon; BD Biosciences, Bedford, Mass). The viability of the cells was 90% according to the trypan blue–exclusion test. Cells (2 x 104 per well) were cultured in DMEM with 10% FBS in 96-well culture plates at 37°C under an atmosphere of 95% air and 5% CO2. After 78 hours of culturing and a change of medium, the cells were exposed for 24 hours to 10–6, 10–5, or 10–4 M PNMC dissolved in DMEM. The medium of the treated cells was then changed, and the cells were exposed to 10 nM gonadotropin-releasing hormone (GnRH; National Institute of Diabetes and Digestive and Kidney Disease [NIADDK], Torrance, Calif) or vehicle for 4 hours, and then harvested. The culture media were stored at –20°C until assayed for LH and FSH.

Isolation and Culture of Testicular Interstitial Cells

Interstitial cell preparations that contained Leydig cells were prepared from the testes of 28-day-old rats as described by Klinefelter et al (1987), with minor modifications. The testes were removed immediately, and the testicular cells were dispersed by treating the decapsulated testes with collagenase (0.25 mg/mL) and soybean trypsin inhibitor (0.025 mg/mL) in M199 medium that contained 8 mM sodium bicarbonate and 9 mM HEPES at 37°C for 20 minutes in a shaking water bath. After incubation, the supernatant, which contained the Leydig cells, was decanted through a nylon mesh to remove debris. The cells were washed by centrifugation and resuspended in 10 mL of M199 with 1% FBS. The viability of the cells was 92%, as evaluated by the trypan blue exclusion test. Cells (105 per well) were cultured in 96-well culture plates at 37°C under an atmosphere of 95% air and 5% CO2. Following a 20-minute equilibration period, the cells were exposed for 24 hours to 10–6, 10–5, or 10–4 M PNMC with or without hCG (0.1 IU/mL) dissolved in M199. The culture media were stored at –20°C until assayed for testosterone.

Radioimmunoassay

The concentrations of LH and FSH in the plasma and culture media were measured using rat radioimmunoassay (RIA) kits (NIADDK) for rat LH and FSH. The antisera used were anti-rat LH-S-11 and anti-rat FSH-S-11. The intra-assay and interassay coefficients of variation were 5.4% and 6.9% for LH, and 4.3% and 10.3% for FSH, respectively.

The concentrations of ir-inhibin in plasma and testes were measured as described previously (Hamada et al, 1989). The iodinated preparation used was the 32-kDa bovine inhibin, and the antiserum used was rabbit antiserum against bovine inhibin (TNDH-1). The results are expressed in terms of the 32-kDa bovine inhibin. The intra-assay and interassay coefficients of variation were 8.8% and 14.4%, respectively.

The concentrations of testosterone in the plasma, testes, and culture media were determined using a double-antibody RIA system with 125I-labelled radioligands, as described previously (Taya et al, 1985). The antiserum against testosterone (GDN 250) (Gayand and Kerlan, 1978) was kindly provided by Dr GD Niswender of Colorado State University (Fort Collins, Colo). The intra-assay and interassay coefficients of variation were 6.3% and 7.2%, respectively.

Statistical Analysis

All of the data are presented as means ± SEM (standard error of the mean) and were analyzed by ANOVA followed by Fisher's protected least significant difference test (Fisher's PLSD). Statistical analysis was performed using the StatView 5.0 software (SAS Institute Inc, Cary, NC). Significance was accepted at P less than .05.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Effects of PNMC on Organ and Body Weights

The body weights and the weights of the liver and kidneys are shown in Table 1. The rats in all treatment groups grew normally, and there were no differences in body weight, body-weight gain, or kidney weight among the groups (Table 1). Liver weight decreased significantly in the groups treated with 10 or 100 mg/kg PNMC compared with the control group (Table 1). The weights of the testes, VP, LABC, and GP (Table 2) were not affected by PNMC treatment. However, PNMC at 10 mg/kg significantly (P < .05) reduced the weights of the epididymis, SV, and COW compared with those of the control group (Table 2). The weights of the SV, LABC, and COW were also lower in rats that were treated with 100 mg/kg PNMC compared with the control group, although the differences were not significant (Table 2).


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  		Table 1. Body weights and organ weights of immature rats treated with 3-methyl-4-nitrophenol (PNMC) for 5 days*
 

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  		Table 2. Weights of testes and accessory sex organs in immature rats treated with 3-methyl-4-nitrophenol (PNMC) for 5 days*
 

Effects of PNMC on Plasma and Testicular Hormones

The plasma concentrations of LH, FSH, testosterone, and ir-inhibin in immature rats treated with PNMC for 5 days are shown in Figure 1. The plasma concentrations of LH and FSH showed significant increases (P < .05) in the group treated with PNMC at 100 mg/kg compared with the control group (Figure 1A and C). The plasma concentrations of testosterone and ir-inhibin showed significant decreases (P < .05) in the group treated with PNMC at 100 mg/kg compared with the control group (Figure 1B and D).


Figure 1
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  		Figure 1. Plasma concentrations of (A) LH, (B) testosterone, (C) FSH, and ir-inhibin (D) in immature rats treated with PNMC at doses of 1, 10, or 100 mg/kg/d for 5 days. Each bar represents the mean ± SEM of 9 or 10 rats per group. *P < .05 compared with control rats (Fisher's PLSD).

 

The testicular concentrations of testosterone and ir-inhibin in immature rats treated with PNMC for 5 days are shown in Figure 2. The testicular concentration of testosterone showed a significant decrease in the group treated with PNMC at 100 mg/kg compared with the control group (Figure 2A). The testicular concentrations of ir-inhibin also showed significant decreases in the groups treated with 1 or 100 mg/kg PNMC compared with the control group (Figure 2B).


Figure 2
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  		Figure 2. Testicular contents of (A) testosterone and (B) ir-inhibin in immature rats treated with PNMC at doses of 1, 10, or 100 mg/kg/d for 5 days. Each bar represents the mean ± SEM of 9 or 10 rats per group. *P < .05 and **P < .01 compared with control rats (Fisher's PLSD).

 
Effects on LH and FSH Release of In Vitro Exposure of Anterior Pituitary Cells to PNMC

To determine the direct effects of PNMC on pituitary function, anterior pituitary cells were incubated in the presence of PNMC for 24 hours, and the LH and FSH concentrations in the culture media were measured. Although GnRH stimulation caused significant increases in the concentrations of LH and FSH in the control group (Figure 3A and B), no significant differences were noted in the concentrations of LH and FSH in any of the PNMC-treated cells (Figure 3A and B).


Figure 3
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  		Figure 3. Effect of PNMC on LH and FSH secretion in basal and GnRH-stimulated anterior pituitary cells. Anterior pituitary cells were incubated for 24 hours with 10–6, 10–5, or 10–4 M PNMC either with or without GnRH (10 nM). Each bar represents the mean ± SEM of three observations. *P < .05 compared with the basal (medium) control (Fisher's PLSD).

 

Effect on Testosterone Release of In Vitro Exposure of Interstitial Leydig Cells to PNMC

To determine the direct effects of PNMC on testicular testosterone production, freshly isolated interstitial cells were incubated in the presence of PNMC with or without hCG for 24 hours, and the testosterone concentrations in the culture media were measured. The basal (unstimulated) testosterone concentrations were lower at all doses of PNMC compared with those in the control group (Figure 4). Stimulation with hCG caused a 4-fold increase in the concentration of testosterone. The concentrations of testosterone in the culture media of all PNMC-treated groups were significantly lower than in the hCG-treated control group (Figure 4).


Figure 4
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  		Figure 4. Effect PNMC on testosterone secretion in basal and hCG-stimulated Leydig cells. Interstitial cells containing Leydig cells were incubated for 24 hours with 10–6, 10–5, or 10–4 M PNMC either with or without hCG (0.1 IU/mL). Each bar represents the mean ± SEM of three observations. *P < .05 compared with the basal (M199) control, #P< .05 compared with the hCG-stimulated control (Fisher's PLSD).

 

   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
The administration of PNMC from DEP caused significant increases in the plasma concentrations of LH and FSH in immature male rats. In contrast, the plasma concentrations of testosterone and ir-inhibin were lower in PNMC-treated animals. The testicular concentrations of testosterone and ir-inhibin were also significantly lower in PNMC-treated rats than in control rats. Testosterone and inhibin are synthesized in the Leydig and Sertoli cells of male rats (Bardin, 1988; Hall, 1988). LH and FSH are the major stimulants of testosterone and inhibin production from Leydig and Sertoli cells, respectively (Ewing et al, 1983; Valladares et al, 1986). These findings suggest that PNMC affects testicular function by affecting the functions of Leydig and Sertoli cells, and that the increased levels of plasma LH and FSH may be due to a reduction of the negative feedback regulation by testosterone and inhibin. Leydig cells are known to play crucial roles in the synthesis of testosterone and in the regulation of spermatogenesis (Senger, 1999). Alteration of Leydig cell function can adversely affect spermatogenesis (Senger, 1999). In the present study, we demonstrate that PNMC does not affect LH and FSH secretion from pituitary cells in vitro, although PNMC does inhibit testosterone production in cultured testicular interstitial Leydig cells. These observations are in agreement with the results of an in vivo study showing higher LH levels and lower testosterone levels in PNMC-treated rats. In the in vitro study, the cytotoxic effects of PNMC on anterior pituitary and interstitial Leydig cells were examined by determining lactate dehydrogenase (LDH) activity in the cultured medium; no significant differences in LDH release activity were observed for PNMC-treated cells in comparison with control cells (data not shown). Accordingly, the suppression of testosterone production in Leydig cells in response to exposure to PNMC is clearly not due to cytotoxicity. Therefore, our results strongly suggest that PNMC has a direct toxicologic (but noncytotoxic) effect on Leydig cells in the testes.

PNMC had no effect on the normal growth of rats. However, PNMC caused weight reduction in the accessory reproductive organs. These results do not show dose-dependency, but show an inverted-U dose response, similar to previously reported results for environmental chemicals, such as bisphenol A (Oehlmann et al, 2000). We have reported previously that PNMC isolated from DEP possesses estrogenic activity both in vivo and in vitro (Furuta et al, 2004, 2005; Taneda et al, 2004b). Previous studies have shown that the estrogenic chemical bisphenol-A inhibits the function of accessory reproductive organs (Nagel et al, 1997). Estrogens, such as estradiol and diethylstilbestrol, also inhibit the development of spermatogonia and the functions of Leydig and Sertoli cells in the fetal rat testes (Lassurguere et al, 2003). In addition, PNMC has anti-androgenic activity in vitro (Taneda et al, 2004b). Previous reports have shown that flutamide, which is a potent androgen antagonist, decreases the weights of accessory sex organs in rats (Yamada et al, 2000; Ashby et al, 2004). These results suggest that both the estrogenic and the antiandrogenic properties of PNMC are involved in the suppression of testicular function in PNMC-treated rats.

Our results are also important from an environmental perspective. Large amounts of DEP are exhausted into the air in many countries around the world. In Japan, 58 902 tons of DEP are emitted each year (Japan Environmental Agency, 1998). Each kilogram of DEP contains approximately 28 mg of PNMC (Mori et al, 2003; Taneda et al, 2004a). Therefore, the estimated total amount of PNMC comes to about 1.65 tons per year. Most of this is sprayed along the main root roads, and the concentrations in city centers and industrial areas may be higher. The concentration of PNMC in Kawasaki City of Kanagawa Prefecture, Japan, which is famous as a vigorous center of industry, has been estimated at 7–21 ng/m3 (Prefecture Research Institute Report, unpublished). On the other hand, PNMC is also a known degradation product of the insecticide fenitrothion (Bhushan et al, 2000; Hayatsu et al, 2000), which is widely used in many countries and accumulates in the air and soil (Nishioka and Lewtas, 1992). Fenitrothion inhibits 5{alpha}-dihydrotestosterone (DHT) binding to the androgen receptor in transfected HepG2 human hepatoma liver cells and can inhibit androgen-dependent tissue growth in vivo (Tamura et al, 2001). According to data reported in the Pollutant Release and Transfer Registers (PRTR), the amount of fenitrothion emitted into the environment in 2002 in Japan was approximately 1300 tons, and roughly half of this would have been degraded to PNMC (Hayatsu et al, 2000). In Japan, the most conspicuous use of fenitrothion is as an insecticide sprayed on small areas, such as paddy fields or pine woods. In one government report, the maximal concentration (5-day average) of fenitrothion was 6.5 µg/m3 (1-hour peak of 22 µg/m3), which was measured in close proximity to a helicopter that was spraying pine woods (Japan Environment Agency, 1997). This value indicates that a standard adult person (60 kg body weight, inhalation volume of 15 m3) would inhale 48.8 µg of PNMC per 24-hour period, about half of which originates from fenitrothion. Asman et al (2005) have reported that rainwater in Roskilde, Denmark contains high levels of PNMC (2483 ng/L). These findings clearly indicate that PNMC from diesel exhaust and from fenitrothion used on farms is present in large amounts in the environment, including in rainwater.

In the present study, 1–100 mg/kg was used for 5 days in the in vivo experiment. Thus, the PNMC dosage used in the present study is higher than that derived from DEP, and 20-fold to 200-fold higher in the case of fenitrothion. It is difficult to interpret directly the present results of the effects of PNMC on the inhibition of testicular function, since the high doses used do not relate to the environmental concentrations. However, as seen from our results, it is certain that PNMC has toxic effects on testicular function. Therefore, it is possible that the large amounts of PNMC in the environment may have serious effects on wildlife and human beings.

In conclusion, the present study clearly shows that PNMC in DEP impairs the reproductive functions of male rats by its toxic effects on testicular function. Our findings suggest that PNMC has suppressive effects on reproductive functions in humans, domestic animals, and wildlife.


   Acknowledgments
 
We are grateful to the National Hormone and Pituitary Program, NIADDK, NIH, and Dr AF Parlow for the rat LH and FSH RIA kits, and to Dr GD Niswender, Animal Reproduction and Biotechnology Laboratory, Colorado State University for providing antiserum to testosterone (GDN 250).


   Footnotes
 
Supported in part by a Grant-in-Aid for Scientific Research (Basic Research B-18310044, C-17510052, and the 21st Century Center-of-Excellence-Program, E-1) from the Ministry of Education, Culture, Sports, Science and Technology of Japan and by a Sasakawa Scientific Research Grant from The Japan Science Society (16-278).


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