4-nitro-3-phenylphenol and 4-nitro-3-cresol

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.. 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).. 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.. 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).. 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.

Topics: Air Pollutants; Biphenyl Compounds; Cities; Cresols; Diazomethane; Gas Chromatography-Mass Spectrometry; Humans; Japan; Methylation; Nanoparticles; Nitrophenols; Particulate Matter; Trimethylsilyl Compounds; Vehicle Emissions

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.

Topics: Animals; Biphenyl Compounds; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; CHO Cells; Comet Assay; Cresols; Cricetinae; Cricetulus; Female; Humans; Nitrophenols; Receptors, Estrogen; Response Elements; Vehicle Emissions

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.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Biphenyl Compounds; Cell Line, Tumor; Cresols; Cytochrome P-450 Enzyme System; Dose-Response Relationship, Drug; Estradiol; Gene Expression Regulation, Enzymologic; Humans; Hydrocortisone; Hydroxysteroid Dehydrogenases; Nitrophenols; Phosphoproteins; Progesterone; Testosterone; Vehicle Emissions

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 and xenoestrogens. The estrogenic activity of PNMC and PNMPP was further examined by using immunohistochemical staining of proliferating cell nuclear antigen (PCNA) in uterine luminal epithelium of ovariectomized 25-d-old immature female rats injected with PNMC and PNMPP subcutaneously for 2 d. Significant increases were observed in uterine luminal epithelium in PCNA positive cells of animals receiving 10 and 100 mg/kg PNMC and 0.1 mg/kg PNMPP compared with controls. These results clearly show the estrogenic activity of PNMC and PNMPP by cell proliferation on the uterine luminal epithelium.

Topics: Animals; Biphenyl Compounds; Cresols; Epithelial Cells; Estrogens, Non-Steroidal; Female; Immunohistochemistry; Nitrophenols; Ovariectomy; Proliferating Cell Nuclear Antigen; Rats; Rats, Wistar; Uterus; Vehicle Emissions

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.

Topics: Animals; Biphenyl Compounds; Cresols; Dose-Response Relationship, Drug; Estradiol; Estrogen Receptor alpha; Estrogens, Non-Steroidal; Female; Humans; Luteinizing Hormone; Nitrophenols; Organ Size; Rats; Rats, Wistar; Recombinant Proteins; Saccharomyces cerevisiae; Uterine Contraction; Uterus; Vehicle Emissions