Nanoparticle-rich diesel exhaust-induced liver damage via inhibited transactivation of peroxisome proliferator-activated receptor alpha.

Diesel exhaust emission contains a high amount of nano-sized particles and is considered to be systemically distributed in the body. However, few studies about the effects of nanoparticle rich-diesel exhaust (NR-DE) on liver have been reported. The present investigation focuses on the effects of NR-DE on livers in rats, especially concerning inflammation and lipid metabolism. Male F344 rats were exposed to fresh air or low (24 ± 7 µg/m3 ), medium (39 ± 4 µg/m3 ) and high (138 ± 20 µg/m3 ) concentrations of NR-DE for 1, 2, or 3 months (5 hours/day, 5 days/week). Exposure to both medium and high concentrations of NR-DE for one month increased plasma asparate aminotransferase and alanine aminotransferase activities, while only high concentrations increased plasma interleukin-6 and hepatic nuclear factor kappa B (NFκB), suggesting that activation of hepatic inflammatory signaling took place. Although these exposures elevated peroxisome proliferator-activated receptor (PPAR) α levels or its binding activity to the response element, neither activated PPARα-target genes such as ß-oxidative enzymes nor inhibited NFκB elevation. Thus, NR-DE may contain some materials that inhibit PPARα activation in relation to lipid metabolism and inflammation. Taken together, NR-DE exposure at one month may cause inflammation; however, this finding may not be observed after a longer exposure period. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1985-1995, 2016.

Effects of exposure to nanoparticle-rich diesel exhaust on pregnancy in rats.

Pollutants from burning of diesel fuel are hazardous to human health. Nanoparticles in diesel exhaust potentially have profound impact on fetal development and maternal endocrine function during pregnancy due to their ability to penetrate deeply into the body. To investigate the effects of nanoparticle-rich diesel exhaust (NR-DE) on pregnancy, pregnant rats were exposed to NR-DE, filtered diesel exhaust (F-DE) or clean air for 19 days of gestation. Relative weights of maternal liver and spleen to body weight were significantly lower in the NR-DE and F-DE groups than those in the control group. The serum concentration of maternal progesterone was significantly lower, while those of luteinizing hormone (LH) and corticosterone were significantly higher in the NR-DE and F-DE groups than those in the control group. The serum concentration of estradiol-17ß was significantly higher in the F-DE group than that in the control group. The levels of cytochrome P450 side-chain cleavage enzyme, 3ß-hydroxysteroid dehydrogenase and LH receptor mRNA in the corpus luteum were significantly lower in the NR-DE and F-DE groups than those in the control. In fetuses, body weight and crown-rump length were significantly greater and shorter, respectively, in both males and females in the NR-DE and F-DE groups than those in the control group. These results demonstrate that exposure of pregnant rats to NR-DE and F-DE suppresses the function of corpora lutea and stimulates the function of the adrenal cortex, suggesting a risk of spontaneous abortion associated with maternal hormonal changes.

Effect of nanoparticle-rich diesel exhaust on testosterone biosynthesis in adult male mice.

The effect of nanoparticle-rich diesel exhaust (NR-DE) on the testicular function and factors related with the biosynthesis of testosterone gene expression were investigated in mice. Male C57BL/Jcl mice were exposed to clean air, low-dose NR-DE (Low NR-DE), high-dose NR-DE (High NR-DE) or filtered diesel exhaust (F-DE) for 8 weeks. We found that the mice exposed to High NR-DE had significantly higher testosterone levels than those in the control and F-DE groups. To determine the effects of NR-DE on testicular testosterone production, interstitial cells dissected from the male mice which were exposed to NR-DE, F-DE, or clean air for 8 weeks were incubated with or without human chorionic gonadotropin (hCG; 0.1 IU/mL) for 4 h. The concentrations of testosterone in the culture media were measured. The testosterone production was significantly increased in with or without hCG of High NR-DE exposed group, and significantly decreased in both with or without hCG of F-DE exposed groups. Moreover, several genes, which is associated with testicular cholesterol synthesis, HMG-CoA, LDL-R, SR-B1, PBR, and P450scc, P450 17α, and 17ß-HSD were determined in the testis of adult male mice. The results showed High NR-DE exposure significantly increased the expression of these genes. Whereas, the levels in the F-DE exposure group returned to those in the control group, implicating that the nanoparticles in DE contribute to the observed reproductive toxicity. We conclude that enhancement of testosterone biosynthesis by NR-DE exposure may be regulated by increasing testicular enzymes of testosterone biosynthesis.

Effect of nanoparticle-rich diesel exhaust on testicular and hippocampus steroidogenesis in male rats.

BACKGROUND: Nanoparticle-rich diesel exhaust (NR-DE) has potentially adverse effects on testicular steroidogenesis. However, it is unclear whether NR-DE influences steroidogenic systems in the brain. OBJECTIVE: To investigate the effect of NR-DE on hippocampal steroidogenesis of adult male rats in comparison with its effect on the testis. METHODS: F344 male rats (8-week-old) were randomly divided into four groups (n = 8 or 9 per group) and exposed to clean air with 4.6 ± 3.2 µg/m(3) in mass concentration, NR-DE with 38 ± 3 µg/m(3) (a level nearly equivalent to the environmental standard in Japan (low NR-DE)), NR-DE with 149 ± 8 µg/m(3) (high NR-DE), or filtered diesel exhaust with 3.1 ± 1.9 µg/m(3) (F-DE), for 5 hours/day, 5 days/week, for 1, 2 or 3 months. F-DE was prepared by removing only particulate matters from high NR-DE with an HEPA filter. RESULTS: Exposures to the high NR-DE for 1 month, and low NR-DE for 2 months, significantly increased or tended to increase plasma and testicular testosterone levels compared to clean air exposure, which might have resulted from the increased expression of mRNA of steroidogenic acute regulatory protein and its protein in the testes of rats. In the hippocampus, high NR-DE exposure for 1 month significantly increased the androstendione level compared to the clean air exposure, while no significant difference was observed in the steroidogenesis between fresh air exposure and any exposure to NR-DE or F-DE. CONCLUSION: NR-DE may influence steroidogenic enzymes in the testis, but not those in the hippocampus.

Effects of exposure to nanoparticle-rich diesel exhaust on adrenocortical function in adult male mice.

To investigate the effects of nanoparticle-rich diesel exhaust (NR-DE) on adrenocortical function, seven-week-old male mice were divided into four groups and exposed to either whole NR-DE at low (41.73 µg/m(3), 8.21 × 10(5) particles/cm3), high (152.01 µg/m3, 1.80 × 10(6) particles/cm3) concentrations, filtered diesel exhaust (F-DE) or clean air for 8 weeks (5 h/day, 5 days/week). After 8 weeks of exposure, the animals were euthanized under pentobarbital anesthesia and the blood samples were collected to detect serum progesterone and corticosterone. In addition, adrenal glands were excised, and adrenal cells were cultured in the absence or presence of rat adrenocorticotropic hormone (ACTH) (10(-15) to 10(-10)M) for 4 h. There were no significant differences in the body weight, absolute and relative adrenal gland weight among the groups. Serum concentration of corticosterone and progesterone was not changed significantly. Administration of ACTH resulted in a dose-dependent increase in corticosterone and progesterone release in mice-exposed to low-concentration NR-DE and clean air. Moreover, corticosterone and progesterone concentrations in adrenal cells increased significantly in mice-exposed to low-concentration NR-DE basal and administrated with ACTH (10(-15) to 10(-11)M for corticosterone; 10(-14) to 10(-11)M for progesterone) compared with the control mice. In contrast, the concentration of corticosterone and progesterone decreased significantly in mice-exposed to high-concentration NR-DE or F-DE basal and administrated with ACTH (10(-12) to 10(-10)M for corticosterone; 10(-15) to 10(-10)M for progesterone) compared with the control mice. These results suggest that exposure to NR-DE or F-DE may disrupt adrenocortical function in adult male mice.

Quercetin attenuates oxidative damage induced by treatment of embryonic chicken spermatogonial cells with 4-nitro-3-phenylphenol in diesel exhaust particles.

Quercetin, an antioxidant flavonoid, is considered beneficial for human and animal health. In this study, the protective effect of quercetin on oxidative damage to testicular cells was studied in embryonic chickens after treatment with 4-nitro-3-phenylphenol (PNMPP) derived from diesel exhaust particles. Testicular cells were challenged with PNMPP (10(-8)-10(-6) M) alone and in combination with quercetin for 48 h. The results showed that quercetin manifested no deleterious effect on spermatogonial cells up to 1.0 microg/ml. Exposure to PNMPP (10(-6) M) induced condensed nuclei and vacuolated cytoplasm and reductions in testicular cell viability and spermatogonial cell numbers (p<0.05). It also induced lipid peroxidation by an elevation of thiobarbituric acid reactive substances and decreased glutathione peroxidase activity and superoxide dismutase activity (p<0.05). Simultaneous supplementation with quercetin restored these parameters to the same levels as in the control. These data indicate that quercetin protects spermatogonial cells from oxidative damage in embryonic chickens intoxicated with PNMPP.

Isolation and identification of new vasodilative substances in diesel exhaust particles.

BACKGROUND, AIM, AND SCOPE: We recently developed a new isolation method for diesel exhaust particles (DEP), involving successive extraction with H(2)O, sodium bicarbonate, and sodium hydroxide, in which the sodium hydroxide extract was found to consist of phenolic components. Analysis of the extract revealed that vasodilative-active nitrophenols are in DEP in significantly higher concentrations than those estimated by an earlier method involving a combination of solvent extraction and repeated chromatography. These findings indicated that our new procedure offers a simple, efficient, and reliable method for the isolation and identification of bioactive substances in DEP. This encouraged us to extend our work toward investigating new vasodilatory substances in the sodium bicarbonate extract. MATERIALS AND METHODS: DEP were collected from the exhaust of a 4JB1-type engine (ISUZU Automobile Co., Tokyo, Japan). GC-MS analysis was performed with a GCMS-QP2010 instrument (Shimadzu, Kyoto, Japan). RESULTS: DEP dissolved in 1-butanol was successively extracted with water, sodium bicarbonate, and then aqueous sodium hydroxide. The sodium bicarbonate extract was neutralized and the resulting mixture of acidic components was subjected to reverse-phase (RP) column chromatography followed by RP-HPLC with fractions assayed for vasodilative activity. This led to the identification of terephthalic acid, p-hydroxybenzoic acid, isophthalic acid, phthalic acid, 3-hydroxy-4-nitrobenzoic acid, 4-hydroxy-3-nitrophenol, and 1,4,5-naphthalene tricarboxylic acid as components of DEP. DISCUSSION: The sodium bicarbonate extract was rich in aromatic carboxylic acid components. Repeated reverse-phase chromatography resulted in the successful isolation of several acidic substances including the new vasodilative materials, 4-hydroxy-3-nitrobenzoic acid, and 3-hydroxy-4-nitrobenzoic acid. CONCLUSIONS: Our new fractionation method for DEP has made possible the isolation of new vasodilative compounds from the sodium bicarbonate extract.

Protective effect of quercetin on the reproductive toxicity of 4-nitrophenol in diesel exhaust particles on male embryonic chickens.

The 4-nitrophenol (PNP) in diesel exhaust particles (DEP) has been identified as a vasodilator and is a known degradation product of the insecticide parathion. In this study, the protective effect of quercetin, a potent oxygen free radical scavenger and metal chelator, against the oxidative damage of PNP on cultured testicular cells was studied in male embryonic chickens. Testicular cells from Day 18 embryos were cultured in serum-free McCoy's 5A medium and challenged with quercetin (1.0 microg/ml) alone or in combinations with PNP (10(-7)-10(-5) M) for 48 h. The oxidative damage was estimated by measuring cell viability, content of malondialdehyde (MDA), activity of superoxide dismutase (SOD) and glutathione peroxidation (GSH-Px) activity. The results showed that exposure to PNP (10(-5) M) induced condensed nuclei, vacuolated cytoplasm and a decrease in testicular cell viability and spermatogonial cell number. Exposure to PNP induced lipid peroxidation by elevation of the content of MDA. Exposure to PNP also decreased GSH-Px activity and SOD activity. However, simultaneous supplementation with quercetin restored these parameters to the same levels as the control. Consequently, PNP induced oxidative stress in spermatogonial cells, and dietary quercetin may attenuate the reproductive toxicity of PNP to restore the intracellular antioxidant system in the testicular cells of embryonic chickens.

Endocrine disruptive effect of 3-methyl-4-nitrophenol isolated from diesel exhaust particles in Hershberger assay using castrated immature rats.

To examine the endocrine disruptive effects of 3-methyl-4-nitrophenol (4-nitro-m-cresol; PNMC) in diesel exhaust particles (DEP), the rat Hershberger assay was carried out using castrated immature rats. Castrated 28-d-old immature male rats were implanted with a 5-mm-long silastic tube containing crystalline testosterone and injected with PNMC subcutaneously at doses 1, 10, or 100 mg/kg for 5 consecutive d. The weights of the livers significantly decreased in the 10 and 100 mg/kg PNMC treatment groups as compared with the control group. The weights of the seminal vesicles significantly increased in the 10 mg/kg PNMC treatment group as compared with the control group. The weights of the Cowper's glands were significantly increased in 1 mg/kg PNMC treatment group compared with the control group. The concentrations of plasma testosterone significantly increased in the 10 and 100 mg/kg PNMC treatment groups, indicating that PNMC induced accumulation of bioactive testosterone released from the implanted tube in circulation. Plasma follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels significantly decreased under all the doses in the PNMC treatment groups, indicating that PNMC acts on the hypothalamus-pituitary axis.

Effects of inhaled nanoparticle-rich diesel exhaust on regulation of testicular function in adult male rats.

We investigated the effects of nanoparticle-rich diesel exhaust (NR-DE) on reproductive function. Eight-week-old male F344 rats were divided into 12 experimental groups and exposed to either whole NR-DE at low (15.37 microg/m(3), 2.27 x 10(5) particles/cm(3)), middle (36.35 microg/m(3), 5.11 x 10(5) particles/cm(3)), or high (168.84 microg/m(3), 1.36 x 10(6) particles/cm(3)) concentrations or clean air for 4, 8, or 12 weeks (5 hours/day, 5 days/week). NR-DE exposure for 4 or 8 weeks did not affect body weight; however, body weight was significantly decreased in rats exposed to low- or high- concentration NR-DE for 12 weeks compared to the control group. Relative weights of testes, epididymides, seminal vesicles, and prostate had increased non-significantly in all NR-DE-exposed rats at 4, 8, and 12 weeks. Adrenal gland relative weights were significantly increased at 4 weeks in rats exposed to low-concentration NR-DE. Plasma luteinizing hormone and follicle stimulating hormone concentrations did not change significantly. Plasma testosterone concentrations were significantly increased after exposure to low- or middle-concentration NR-DE for 4 or 8 weeks compared to controls. Plasma immunoreactive (ir-) inhibin concentrations were significantly increased after exposure to high-concentration NR-DE for 4 weeks or middle- or high-concentration NR-DE for 12 weeks compared to controls. Testicular testosterone concentrations were significantly increased at 4, 8, and 12 weeks after exposure to low-concentration NR-DE compared to controls. In contrast, with exposure to low- or high-concentration NR-DE, testicular ir-inhibin concentrations were significantly greater than in controls, but only at 4 weeks. These results suggest that NR-DE inhalation disrupts the endocrine activity of the male reproductive system.

Nanoparticle-rich diesel exhaust may disrupt testosterone biosynthesis and metabolism via growth hormone.

We previously reported that exposure to low (22.5+/-0.2 nm in diameter, 15.4+/-1.0 microg/m(3) in mass weight, 2.27x10(5)/cm(3) in mean number concentration), and medium (26.1+/-0.5 nm, 36.4+/-1.2 microg/m(3), 5.11x10(5)/cm(3)) concentrations of nanoparticle-rich diesel exhaust (NR-DE) for 1 and 2 months (5 h/day, 5 days/week) significantly increased plasma testosterone in male Fischer 344 rats, whereas exposure to a high concentration (27.1+/-0.5 nm, 168.8+/-2.7 microg/m(3), 1.36x10(6)/cm(3)) did not. The present study attempts to clarify the mechanism of this elevation. Low and medium exposures to NR-DE for 1 and 2 months significantly increased steroidogenic acute regulatory protein (StAR)- and cytochrome P450 side-chain cleavage (P450scc)-mRNA and their protein expressions in the testis of rats, in which the elevation pattern was very similar to that of plasma testosterone levels. Interestingly, both exposure levels for 1 month significantly increased growth hormone (GH) receptor expression in the testis, and low exposure also increased testicular insulin-like growth factor I-mRNA levels and hepatic microsomal cytochrome P450 2C11-mRNA and their protein levels in rats. These two factors are thought to be related to growth hormone secretion. Disruption of testosterone biosynthesis by NR-DE exposure may be a mode of action for reproductive toxicity, which may, in part, be regulated by increasing StAR and P450scc expressions via GH signalling.

Quercetin protects embryonic chicken spermatogonial cells from oxidative damage intoxicated with 3-methyl-4-nitrophenol in primary culture.

Diesel exhaust particles (DEP) are considered to be one of the most important air pollutants. In this study, the protective effect of quercetin, an antioxidant flavonoid, on oxidative damage of testicular cells was studied by analysis of the intracellular antioxidant system of embryonic chickens after treatment with 3-methyl-4-nitrophenol (PNMC) derived from DEP. Testicular cells from 18-day-old embryos were cultured in serum-free McCoys'5A medium and challenged with PNMC (10(-7) to 10(-5)M) alone or in combinations with quercetin (1.0 microg/ml) for 48h. Results showed that exposure to PNMC (10(-5)M) induced condensed nuclei and vacuolated cytoplasm, a decrease in testicular cell viability and spermatogonial cell number. Exposure to PNMC induced lipid peroxidation by an elevation of thiobarbituric acid reactive substances as well as decreasing glutathione peroxidation activity and superoxide dismutase activity. However, simultaneous supplementation with quercetin restored these parameters to the similar levels as the control. PNMC is therefore concluded to have induced the oxidative stress of the spermatogonial cells, which can be attenuated by combined quercetin treatment. Our results support the therapeutic use of quercetin in the prevention or treatment of the reproductive toxicity by environmental toxicant PNMC.

4-Nitrophenol isolated from diesel exhaust particles disrupts regulation of reproductive hormones in immature male rats.

In previous studies, we found that 4-nitrophenol (PNP) isolated from diesel exhaust particles exhibited both estrogenic and anti-androgenic activities. This compound is also a degradation product of the insecticide parathion. Here, we investigated the in vivo effect of PNP on reproductive function in immature male rats. Twenty-eight-day-old rats were injected subcutaneously with PNP (0.01, 0.1, 1, or 10 mg/kg) daily for 14 days. Plasma concentrations of luteinizing hormone (LH) were significantly lower in all PNP dosage groups than in the control group, and follicle-stimulating hormone (FSH) was significantly decreased in rats treated with 0.1, 1, or 10 mg/kg PNP. However, plasma concentrations of testosterone were significantly increased by 10 mg/kg PNP, and plasma concentrations of immunoreactive (ir)-inhibin were also significantly increased in the 0.1, 1, and 10 mg/kg PNP groups. Plasma concentrations of prolactin were significantly increased by 10 mg/kg PNP, and plasma concentrations of corticosterone were significantly increased in all treatment groups. These findings clearly show that PNP influences the hypothalamic-pituitary-gonadal axis in immature male rats, with decreased secretion of LH and FSH and increased secretion of testosterone and inhibin. PNP, therefore, appears to disrupt endocrine activity in the immature male reproductive system.

Effects of in utero exposure to nanoparticle-rich diesel exhaust on testicular function in immature male rats.

We investigated the effects of in utero exposure to nanoparticle-rich diesel exhaust (NR-DE) on reproductive function in male rats. Pregnant F344 rats were exposed to NR-DE (148.86 microg/m(3), 1.83 x 10(6)particles/cm(3), 3.40 ppm CO, 1.46 ppm NOx), filtered diesel exhaust (F-DE; 3.10 microg/m(3), 2.66 particles/cm(3), 3.30 ppm CO, 1.41 ppm NOx), or clean air (as a control) from gestation days 1 to 19 (gestation day 0=day of sperm-positivity). Male offspring were examined on postnatal day 28. The relative weights of the seminal vesicle and prostate to body weight were decreased after exposure to NR-DE or F-DE compared with controls. Serum concentrations of testosterone, progesterone, corticosterone, and follicle stimulating hormone and testicular concentrations of steroidogenic acute regulatory protein and 17beta-hydroxysteroid dehydrogenase mRNA were decreased after exposure to NR-DE or F-DE compared with control levels. In contrast, serum concentrations of immunoreative inhibin were increased after exposure to NR-DE or F-DE compared with control levels, whereas transcription of follicle stimulating hormone receptor mRNA was increased in the NR-DE exposure group only. These results suggest that prenatal exposure to NR-DE or F-DE leads to endocrine disruption after birth and suppresses testicular function in male rats. Because both the NR-DE and F-DE-exposed groups reacted to the same extent, the nanoparticles in DE did not contribute to the observed reproductive toxicity.

Effects of 3-methyl-4-nitrophenol on the reproductive toxicity in female Japanese quail (Coturnix japonica).

We previously showed that 3-methyl-4-nitrophenol (4-nitro-m-cresol, PNMC), a component of diesel exhaust particles and a degradation product of the insecticide fenitrothion, has reproductive toxicity in adult male and immature female Japanese quail (Coturnix japonica). Here we investigated effects of PNMC on the reproductive toxicity of mature female Japanese quail. The experiment consists of 3 periods of pretreatment, treatment, and post-treatment for 5 d each. The birds were reared, bred naturally for 1 week, and after 5 d of pretreatment, then injected intramuscularly with PNMC at doses 1, 10, or 100 mg/kg body weight daily for 5 d. Body weight, egg weight, and hatchability did not differ among the observation periods. However, at all doses of PNMC, the egg-laying rate showed a modest decrease during the treatment period, with recovery during the post-treatment period. Plasma concentrations of luteinizing hormone (LH) and estrodiol-17beta, were significantly decreased (p<0.05), and plasma concentrations of progesterone significantly increased (p<0.05) in birds treated with 10 and 100 mg/kg PNMC. These results suggest that PNMC have acute toxicity, and inhibited LH secretion, disturbing egg-laying in mature female quail. Our findings indicate that PNMC induces endocrine malfunction at the central level and subsequently disrupts reproductive processes in mature female quails.

Nitrophenols isolated from diesel exhaust particles promote the growth of MCF-7 breast adenocarcinoma cells.

Diesel exhaust particles (DEPs) cause many adverse health problems, and reports indicate increased risk of breast cancer in men and women through exposure to gasoline and vehicle exhaust. However, DEPs include vast numbers of compounds, and the specific compound(s) responsible for these actions are not clear. We recently isolated two nitrophenols from DEPs-3-methyl-4-nitrophenol (4-nitro-m-cresol; PNMC) and 4-nitro-3-phenylphenol (PNMPP)-and showed that they had estrogenic and anti-androgenic activities. Here, we tried to clarify the involvement of these two nitrophenols in promoting the growth of the MCF-7 breast cancer cell line. First, comet assay was used to detect the genotoxicity of PNMC and PNMPP in a CHO cell line. At all doses tested, PNMC and PNMPP showed negative genotoxicity, indicating that they had no tumor initiating activity. Next, the estrogen-responsive breast cancer cell line MCF-7 was used to assess cell proliferation. Proliferation of MCF-7 cells was stimulated by PNMC, PNMPP, and estradiol-17beta and the anti-estrogens 4-hydroxytamoxifen and ICI 182,780 inhibited the proliferation. To further investigate transcriptional activity through the estrogen receptor, MCF-7 cells were transfected with a receptor gene that allowed expression of luciferase enzyme under the control of the estrogen regulatory element. PNMC and PNMPP induced luciferase activity in a dose-dependent manner at submicromolar concentrations. ICI 182,780 inhibited the luciferase activity induced by PNMC and PNMPP. These results clearly indicate that PNMC and PNMPP do not show genotoxicity but act as tumor promoters in an estrogen receptor alpha-predominant breast cancer cell line.

Improvement of an efficient separation method for chemicals in diesel exhaust particles: analysis for nitrophenols.

GOAL, SCOPE, AND BACKGROUND: Diesel exhaust is believed to consist of thousands of organic constituents and is a major cause of urban pollution. We recently reported that a systematic separation procedure involving successive solvent extractions, followed by repeated column chromatography, resulted in the isolation of vasodilatory active nitrophenols. These findings indicated that the estimation of the amount of nitrophenols in the environment is important to evaluate their effect on human health. The isolation procedure, however, involved successive solvent extractions followed by tedious, repeated chromatography, resulting in poor fractionation and in a significant loss of accuracy and reliability. Therefore, it was crucial to develop an alternative, efficient, and reliable analytical method. Here, we describe a facile and efficient acid-base extraction procedure for the analysis of nitrophenols. MATERIALS AND METHODS: Diesel exhaust particles (DEP) were collected from the exhaust of a 4JB1-type engine (ISUZU Automobile Co., Tokyo, Japan). Gas chromatography-mass spectrometry (GC-MS) analysis was performed with a GCMS-QP2010 instrument (Shimadzu, Kyoto, Japan). RESULTS: A solution of DEP in 1-butanol was extracted with aqueous NaOH to afford a nitrophenol-rich oily extract. The resulting oil was methylated with trimethylsilyldiazomethane and subsequently subjected to GC-MS analysis, revealing that 4-nitrophenol, 3-methyl-4-nitrophenol, 2-methyl-4-nitrophenol, and 4-nitro-3-phenylphenol were present in significantly higher concentrations than those reported previously. DISCUSSION: Simple acid-base extraction followed by the direct analysis of the resulting extract by GC-MS gave only broad peaks of nitrophenols with a poor detection limit, while the GC-MS analysis of the sample pretreated with (trimethylsilyl)diazomethane gave satisfactorily clear chromatograms with sharp peaks and with a significantly lowered detection limit (0.5 ng/ml, approximately 100 times). CONCLUSION: The present method involving an acid-base extraction, in situ derivatization, and GC-MS analysis has shown to be a simple, efficient, and reliable method for the isolation and identification of the chemical substances in DEP.

Nitrophenols isolated from diesel exhaust particles regulate steroidogenic gene expression and steroid synthesis in the human H295R adrenocortical cell line.

Studies of nitrophenols isolated from diesel exhaust particles (DEPs), 3-methyl-4-nitrophenol (PNMC) and 4-nitro-3-phenylphenol (PNMPP) have revealed that these chemicals possess estrogenic and anti-androgenic activity in vitro and in vivo and that PNMC accumulate in adrenal glands in vivo. However, the impacts of exposure to these compounds on adrenal endocrine disruption and steroidogenesis have not been investigated. To elucidate the non-receptor mediated effects of PNMC and PNMPP, we investigated the production of the steroid hormones progesterone, cortisol, testosterone, and estradiol-17beta and modulation of nine major enzyme genes involved in the synthesis of steroid hormones (CYP11A, CYP11B1, CYP17, CYP19, 17betaHSD1, 17betaHSD4, CYP21, 3betaHSD2, StAR) in human adrenal H295R cells supplied with cAMP. Exposure to 10(-7) to 10(-5) M PNMC and 1 mM 8-Br-cAMP for 48 h decreased testosterone, cortisol, and estradiol-17beta levels and increased progesterone secretion. At 10(-5) M, PNMC with 1 mM 8-Br-cAMP significantly stimulated expression of the 17betaHSD4 and significantly suppressed expression of 3betaHSD2. In comparison, 10(-7) to 2 x 10(-5) M PNMPP with 1 mM 8-Br-cAMP for 48 h decreased concentrations of estradiol-17beta, increased progesterone levels, but did not affect testosterone and cortisol secretion due to the significant suppression of CYP17 and the non-significant but obvious suppression of CYP19. Our results clarified steroidogenic enzymes as candidates responsible for the inhibition or stimulation for the production of steroid hormones in the steroidogenic pathway, thus providing the first experimental evidence for multiple mechanisms of disruption of endocrine pathways by these nitrophenols.

Secretion of inhibin in male Japanese quail (Coturnix japonica) from one week of age to sexual maturity.

The objective of this study was to investigate the changes in secretion of inhibin and cellular localization of the inhibin alpha and inhibin/activin (beta(A) and beta(B)) subunits in male Japanese quail from 1 to 7 weeks after hatching. The post-hatch profile of plasma luteinizing hormone (LH), immunoreactive (ir) inhibin and testosterone were measured by radioimmunoassay. Testes were immunostained by the avidin-biotin-peroxidase complex method (ABC) using polyclonal antisera raised against inhibin alpha, inhibin/activin beta(A) and inhibin/activin beta(B) from one week of age to sexual maturity. Testicular weight increased gradually until 4 weeks and abruptly increased from 5 weeks of age onwards. The plasma concentrations of LH and ir-inhibin increased significantly at 5 weeks of age, and the plasma concentration of testosterone increased significantly at 6 weeks of age. Pituitary contents of LH showed a steady increase until 6 weeks of age and then abruptly increased at 7 weeks of age. Coincident to the increase in plasma testosterone, the testicular contents of testosterone significantly increased from 5 weeks through sexual maturity. Immunohistochemically, localization of the inhibin/activin alpha, beta(A) and beta(B) subunits was found in the Sertoli and Leydig cells at all ages of development from one week of age to sexual maturity. These results suggest that Sertoli and Leydig cells are the major source of inhibin secretion during development in male Japanese quail.

Secretion of inhibin in female Japanese quails (Coturnix japonica) from hatch to sexual maturity.

To clarify the cellular source and secretory pattern of inhibin in the Japanese quail during follicular development, the plasma concentrations of immunoreactive (ir) inhibin were measured from 1 to 7 weeks after hatching. Localization of the inhibin/activin alpha, beta A and beta B subunits was investigated by immunohistochemistry. To monitor development of the pituitary and ovarian functions, the plasma luteinizing hormone (LH) and progesterone concentrations were also measured. Ovarian weight increased gradually until 6 weeks of age and then abruptly increased at 7 weeks of age just at the onset of egg production. Plasma concentrations of LH increased significantly at 6 weeks of age. The plasma concentrations of ir-inhibin and progesterone and the pituitary contents of LH also increased significantly at 7 weeks of age. Immunohistochemically, the inhibin/activin alpha, beta A and beta B subunits were localized in the granulosa cells of all follicles during different stages of development from 1 to 7 weeks after hatching. The inhibin alpha, beta A and beta B subunits were also found in the interstitial cells but not theca cells of all follicles. These results demonstrated that the plasma concentrations of ir-inhibin of the female Japanese quails rose with ovarian development. The immunohistochemical results suggested that granulosa and interstitial cells are the major source of ovarian inhibins in female Japanese quails.