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.

Effects of the phosphodiesterase IV inhibitor rolipram on Th1 and Th2 immune responses in mice.

The present study was designed to investigate the effect of the phosphodiesterase IV inhibitor rolipram on Th1 and Th2 immune responses in mice. Mice were immunized subcutaneously at the base of the tail with ovalbumin (OVA) emulsified with complete Freund's adjuvant (day 0) and were treated daily with oral administration of various doses of rolipram from days 0 to 20. On day 21, production of anti-OVA IgG and proliferative responses to the antigen were determined. Anti-OVA IgG2a and interferon-gamma (IFN-gamma), as indicators of Th1 responses, and anti-OVA IgG1 and interleukin-10 (IL-10), as indicators of Th2 responses, were also measured. The results showed that treatment with rolipram failed to affect the production of OVA-specific IgG but decreased the proliferation of spleen cells to the antigen. Its inhibitory effect on these immune responses was correlated with a marked decrease in IFN-gamma but not IL-10 production, although neither anti-OVA IgG2a nor IgG1 production was affected by rolipram. These results suggest that rolipram may preferentially inhibit Th1 responses more effectively than Th2 responses. Administration of rolipram resulted in suppression of antigen (OVA)-induced arthritis in mice. The suppression of joint inflammation by rolipram was associated with the inhibition of the OVA-specific proliferative responses of spleen cells and IFN-gamma secretion. These results indicate that rolipram may be effective in regulating Th1-mediated diseases such as rheumatoid arthritis.

Prenatal exposure to bisphenol A up-regulates immune responses, including T helper 1 and T helper 2 responses, in mice.

The effect of prenatal exposure to bisphenol A (BPA) on the immune system in mice was investigated. Virgin female mice were fed varying doses of BPA, on a daily basis, over a period of 18 days commencing on the day of pairing with stud males (day 0). On day 77, their male offspring of 8 weeks were immunized with hen egg lysozyme (HEL). Three weeks later, anti-HEL immunoglobulin G (IgG) in sera, and proliferative responses of spleen cells to the antigen, were measured. Anti-HEL IgG2a and interferon-gamma (IFN-gamma), secreted from splenic lymphocytes, were measured as indicators of T helper 1 (Th1) immune responses, while anti-HEL IgG1 and interleukin-4 (IL-4) were measured as indicators of Th2 responses. The results showed that fetal exposure to BPA was followed by significant increases in anti-HEL IgG as well as antigen-specific cell proliferation. Both Th1 responses (including anti-HEL IgG2a and IFN-gamma production) and Th2 responses (including anti-HEL IgG1 and IL-4 production) were augmented by prenatal exposure to BPA, although the augmentation of Th1 responses appeared to be greater than that of Th2 responses. Two-colour flow cytometric analysis showed that mice exposed prenatally to BPA had 29% and 100% more splenic CD3(+) CD4(+) and CD3(+) CD8(+) cells, respectively, than control animals. Similar results were obtained from females whose mothers had consumed BPA during pregnancy. These results suggest that prenatal exposure to BPA may result in the up-regulation of immune responses, especially Th1 responses, in adulthood.

Estrogenic and anti-androgenic activity of nitrophenols in diesel exhaust particles (DEP).

We recently isolated 4-nitrophenol, 2-methyl-4-nitrophenol, 3-methyl-4-nitrophenol, and 4-nitro-3-phenylphenol 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 human estrogen receptor (hER)-yeast screen assay. All of these nitrophenol derivatives except 2-methyl-4-nitrophenol exhibited estrogenic activity. Some estrogenic compounds are also anti-androgenic, so we measured the anti-androgenic activity of the same compounds by human androgen receptor (hAR)-yeast screen assay. We found anti-androgenicity in all four nitrophenols. Nitrophenols in DEP possess not only vasodilatory activity but also estrogenic and anti-androgenic activity.

Estrogenic activities of nitrophenols in diesel exhaust particles.

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.

Separation and characterization of alkyltrimethylbenzene derivatives in diesel exhaust particles (DEP).

In our continuing studies on estrogenic compounds in diesel exhaust particles (DEP), we have reported the systematic separation of alkyldibenzothiophenes from the estrogenic hexane fraction of DEP. In this study, another estrogenic fraction was further isolated and characterized. DEP were roughly fractionated by successive extraction with hexane, benzene, dichloromethane, methanol, 1 M ammonia and 1 M HCl. The hexane extract, which exhibited estrogenic activity, was further fractionated by column chromatography and high performance liquid chromatography (HPLC). From one of the obtained fractions that showed estrogenic activity, three alkyltrimethylbenzenes were identified by gas chromatography-mass spectrometry (GC-MS) and 1H-nuclear magnetic resonance (NMR) spectroscopy.

Effect of the nonsteroidal anti-inflammatory drug indomethacin on Th1 and Th2 immune responses in mice.

The present study was undertaken to study the effect of the nonsteroidal anti-inflammatory drug indomethacin on Th1 and Th2 immune responses. For this study, mice were immunized by s.c. injection of ovalbumin (OVA) emulsified with complete Freund's adjuvant into the base of the tail (day 0). Varying doses of indomethacin were orally administrated daily from days 0 to 20. On day 21, anti-OVA IgG2a and interferon-gamma as an indicator of Th1 responses and anti-OVA IgG1 and interleukin-10 as that of Th2 responses were measured. The results showed that treatment with indomethacin was followed by decreases in OVA-specific IgG and proliferation of spleen cells to the antigen. Indomethacin inhibited both Th1 and Th2 responses, although the nonsteroidal anti-inflammatory drug suppressed the former more effectively than the latter. Administration of indomethacin resulted in suppression of antigen (OVA)-induced arthritis that was associated with inhibition of anti-OVA IgG2a but not IgG1 production. These results suggest that nonsteroidal anti-inflammatory drugs may downregulate Th1 and, to a lesser extent, Th2 immune responses.

Effects of bisphenol A on antigen-specific antibody production, proliferative responses of lymphoid cells, and TH1 and TH2 immune responses in mice.

1. We investigated the effect of bisphenol A (BPA), which binds estrogen receptors, on immune responses including production of antigen-specific antibodies, proliferative responses of lymphoid cells, and Th1 and Th2 responses. 2. For this investigation, mice were p.o. given varying doses including 3, 30, 300, and 3000 micro g kg(-1) of BPA immediately after immunization with hen egg lysozyme (HEL) (day 0) and then daily by day 20. On day 21, anti-HEL IgG antibodies in sera and proliferative responses of spleen cells to the antigen were measured. Anti-HEL IgG2a antibodies and IFN-gamma secreted from splenic lymphocytes were also measured as indicators of Th1 immune responses, while anti-HEL IgG1 antibodies and IL-4, as those of Th2 responses. 3. The results showed that treatment with 3000 micro g kg(-1) of BPA was followed by a significant increase in anti-HEL IgG as well as the antigen-specific cell proliferation. Anti-HEL IgG2a production and IFN-gamma secretion were significantly enhanced in mice treated with 300 and 30 micro g kg(-1) of BPA, respectively, while anti-HEL IgG1 production and IL-4 secretion were augmented in animals given 3000 and 300 micro g kg(-1) of the chemical, respectively. 4. Augmentation of these immune responses was also observed in mice exposed to 0.3-30 micro g kg(-1) of estradiol, although Th1 responses appeared to be more sensitive to the sex hormone than Th2 responses. 5. These results suggest that BPA may play a role in augmenting immune responses, especially Th1 responses.

Isolation of nitrophenols from diesel exhaust particles (DEP) as vasodilatation compounds.

The compounds in diesel exhaust particles (DEP) that are responsible for vasodilatation were isolated and characterized for the first time. From benzene extract of DEP, 2-methyl-4-nitrophenol, 3-methyl-4-nitrophenl and 4-nitrophenol were isolated, and their vasodilatation activities were confirmed. 3-methyl-4-nitrophenol caused dilatation of rat thoracic artery, and the other two nitrophenols, also showed vasodilatation activities.

Effect of methotrexate on Th1 and Th2 immune responses in mice.

We investigated the effect of the anti-rheumatic drug methotrexate (MTX) on Th1 and Th2 immune responses in mice. For this investigation, mice were immunized subcutaneously at the base of the tail with ovalbumin (OVA) emulsified with complete Freund's adjuvant (day 0). Varying doses of MTX were orally administered daily from days 0 to 20. On day 21, anti-OVA IgG2a and interferon-gamma (IFN-gamma) as indicators of Th1 responses and anti-OVA IgG1 and interleukin-10 (IL-10) as those of Th2 responses were measured. The results showed that treatment with MTX was followed by decreases in OVA-specific IgG and proliferation of spleen cells to the antigen. The anti-rheumatic drug inhibited both anti-OVA IgG2a and IgG1 production, although the inhibitory effect of MTX on the antigen-specific IgG2a production appeared to be greater than that on IgG1 production. IFN-gamma, but not IL-10, secretion was markedly downregulated by MTX. Administration of MTX resulted in suppression of antigen (OVA)-induced arthritis in mice. The suppression of the joint inflammation by MTX was associated with inhibition of OVA-specific proliferative responses of spleen cells, anti-OVA IgG, IgG2a and IgG1 production, and IFN-gamma and IL-10 secretion, although more pronounced decreases in IgG2a and IFN-gamma were observed compared with those in IgG1 and IL-10 in MTX-treated mice. These results indicate that MTX appears to suppress Th1 and, to a lesser extent, Th2 immune responses and its anti-arthritic effect on human rheumatoid arthritis might be at least in part explained by down-regulation of Th1 responses involved in the disease.

Effect of diesel exhaust particle extracts on induction of oral tolerance in mice.

We examined the effect of diesel exhaust particle (DEP) extracts on oral tolerance in mice. For this examination, a single DEP sample was consecutively extracted with hexane (HEX-DEP), benzene (BEN-DEP), dichloromethane (DIC-DEP), methanol (MET-DEP), and 1 M ammonia (AMM-DEP). Residues unextracted (UNE-DEP) with the last extraction solvent 1 M ammonia were also used to test their ability to induce oral tolerance. To immunize mice, hen egg lysozyme (HEL) emulsified with an equal volume of CFA was injected sc (day 0). Oral tolerance was induced by feeding 10 mg HEL on days -5, -4, -3, -2, and -1. DEP, each DEP extract, and UNE-DEP were intranasally administered immediately after each feeding of HEL. The results showed that oral administration of HEL markedly suppressed production of anti-HEL IgG antibodies as well as proliferative responses of spleen cells to HEL. The suppression of anti-HEL IgG antibody production and the cell proliferation by the oral antigen was significantly blocked by DEP, DIC-, AMM-, and UNE-DEP. Neither HEX-, BEN-, nor MET-DEP modulated the orally induced suppression of these immune responses. When the levels of anti-HEL IgG2a antibodies and IFN-gamma (Th1 responses) and anti-HEL IgG1 antibodies and IL-4 (Th2 responses) were determined, DEP and DIC-DEP diminished the suppression of both Th1 and Th2 responses observed following oral administration of HEL. In contrast, UNE- and AMM-DEP prevented the reduction of Th1 but not Th2, and Th2 but not Th1 oral tolerance, respectively. Thus, UNE-DEP appears to contain compounds that block induction of Th1 but not Th2 oral tolerance, whereas AMM-DEP have compounds that abrogate induction of Th2 but not Th1 oral tolerance. DIC-DEP, as well as DEP, appear to contain components that block induction of both Th1 and Th2 oral tolerance. As oral tolerance is thought to play a critical role in preventing Th1 as well as Th2 food allergy, the blockade of oral tolerance by these DEP extracts suggests that DEP may contain compounds different in hydrophobicity associated with the cause of such adverse immunologic responses to food proteins.

Effect of diesel exhaust particle extracts on collagen-induced arthritis in mice.

We investigated the effect of diesel exhaust particles (DEP) extracts on collagen-induced arthritis (CIA) in mice. For this study, a single DEP sample was consecutively extracted with hexane (HEX-DEP), benzene (BEN-DEP), dichloromethane (DIC-DEP), methanol (MET-DEP), and 1 M ammonia (AMM-DEP) in that order. Residues unextracted with the last extraction solvent 1 M ammonia (UNE-DEP) were also used for experiments. To induce CIA, mice were immunized with type II collagen (CII) and 21 days later given a booster injection. DEP, each DEP extract, and UNE-DEP were intranasally administered every two days over a period of 20 days, commencing on the day of immunization. The results showed that treatment of mice with DEP, DIC-DEP, and UNE-DEP augmented both the incidence and the severity of CIA. DEP and DIC-DEP increased production of anti-CII IgG, IgG2a, and IgG1 antibodies as well as secretion of JFN-gamma and IL-4. Treatment with UNE-DEP resulted in an increase in antigen-specific IgG, IgG2a, and IFN-gamma but neither IgG1 nor IL-4. AMM-DEP failed to affect CIA as well as production of IgG2a and IFN-gamma, although significant increases in anti-CII IgGI and IL-4 were observed in the treatment group. HEX-DEP, BEN-DEP, and MET-DEP had no effects on CIA and production of antibodies and cytokines examined. Thus, DEP and DIC-DEP appear to contain compounds, which enhance both Th1 and Th2 responses, while UNE-DEP and AMM-DEP to contain chemicals, which augment Th1 and Th2 alone, respectively. Th1- but not Th2-modulating compounds from DEP, DIC-DEP, and UNE-DEP seem to influence CIA.

Estrogenic activities of chemicals in diesel exhaust particles.

In a previous study, we focused on estrogenic activity of the hexane extract of diesel exhaust particles (DEP). The extract of hexane was first fractionated to acidic, phenolic and neutral portions according to their chemical properties, of which the neutral fraction was fractionated by column chromatography on silica gel. The chemical structures of compounds in these fractions were then analyzed. It was found that the neutral fraction of the hexane extract of DEP contains dibenzothiophene derivatives, one of which, 4,6-dimethyldibenzothiophene, possesses estrogenic activity.

Estrogenic and anti-estrogenic activities of two types of diesel exhaust particles.

In an earlier study using a recombinant yeast screen we found that a suspension of diesel exhaust particles (DEP) and some extracts of DEP are not estrogenic but possess anti-estrogenic activity. In the present study, estrogenic and anti-estrogenic activities of two types of DEP, type-1 (old type) and type-2 (new type) were compared. Whole DEP of both types were found to possess estrogenic and anti-estrogenic activities. The DEP were serially extracted with organic solvents and then with 1 M ammonia and 1 M HCl. In type-2 DEP, the ratio of dry weight of a hexane extract was higher than those of methanol and ammonia extracts, which were lower than those in type-1 DEP. In the hexane extract, estrogenic activity was found in both types of DEP. In the benzene and dichloromethane extracts, estrogenic and anti-estrogenic activities were found in both types of DEP. In the methanol extract, estrogenic activity was found in type-2 DEP, and extracts of both types decreased the activity of estrogen. Anti-estrogenic activity was found in extracts of ammonia and HCl from both types of DEP. It was found that both type-1 and type-2 DEP possess estrogenic and anti-estrogenic activities.