Yoki Mori

Biological effects of diesel exhaust particles. I. In vitro production of superoxide and in vivo toxicity in mouse.

The problem of whether or not active oxygen species are involved in pulmonary injury by diesel exhaust particles (DEP) was investigated. We found that DEP could produce superoxide O2.- and hydroxyl radical (.OH) in vitro without any biological activating systems. In this reaction system, O2.- and .OH productions were inhibited by addition of superoxide dismutase (SOD) and dimethylsulfoxide, respectively. DEP which were washed with methanol could no longer produce O2.- and .OH, indicating that active components were extractable with organic solvents. These oxygen radicals were also identified by electron spin resonance (ESR) measurement. Furthermore, DEP instilled intratracheally to mouse caused high mortality at low dose, although methanol-washed DEP did not kill any mouse. The cause of death seemed to be pulmonary edema mediated by endothelial cell damage. The instilled DEP markedly decreased the activities of SOD, glutathione peroxidase, and glutathione S-transferase in mouse lungs. On the other hand, the death rate and lung injury were markedly prevented by polyethylene glycol conjugated SOD (PEG-SOD) pretreatment prior to DEP administration. The mortality and lung injury by DEP were also suppressed by butylated hydroxytoluene (BHT) pretreatment. From these results, it was suggested that most parts of DEP toxicity in lungs are due to active oxygen radicals such as O2.- and .OH, and that the cause of death is due to pulmonary edema mediated by endothelial cell damage.

Synthesis and antifungal activity of coumarins and angular furanocoumarins

Angelicin, a naturally occurring furanocoumarin, that showed antifungal activity, was considered as a lead structure for a group of synthetic coumarins. Antifungal activities of the synthesized coumarins and angelicin derivatives were reported against Candida albicans, Cryptococcus neoformans, Saccharomyces cerevisiae and Aspergillus niger. Human cell line cytotoxicity of several coumarins was evaluated against KB cells. Angelicin and several potent antifungals showed to be non-toxic in this assay.

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‐γ (IFN‐γ), 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‐γ 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 Activities of Nitrophenols in Diesel Exhaust Particles

Abstract 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.

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

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. For this investigation, mice were p.o. given varying doses including 3, 30, 300, and 3000 μg 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‐γ 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. The results showed that treatment with 3000 μg 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‐γ secretion were significantly enhanced in mice treated with 300 and 30 μg kg−1 of BPA, respectively, while anti‐HEL IgG1 production and IL‐4 secretion were augmented in animals given 3000 and 300 μg kg−1 of the chemical, respectively. Augmentation of these immune responses was also observed in mice exposed to 0.3–30 μg kg−1 of estradiol, although Th1 responses appeared to be more sensitive to the sex hormone than Th2 responses. These results suggest that BPA may play a role in augmenting immune responses, especially Th1 responses.

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.

Effect of varying types of anti-arthritic drugs on Th1 and Th2 immune responses in mice.

The present study was undertaken to study the effect of varying types of anti-arthritic drugs on Th1 and Th2 immune responses in mice. To immunize mice, ovalbumin (OVA) emulsified with complete Freunds adjuvant was injected s.c. at the base of the tail (day 0). Indomethacin (IND) as a non-steroidal antiinflammatory drug (NSAID), dexamethasone (DEX) as a steroidal antiinflammatory drug, methotrexate (MTX), auranofin (AUR), and D-penicillamine (D-PA) as anti-rheumatic drugs were orally administrated daily from days 0 to 20. On day 21, anti-OVA IgG2a and interferon (IFN)-γ as indicators of Th1 responses and anti-OVA IgG1 and interleukin (IL) −10 as those of Th2 responses were measured. Treatments with IND, DEX, MTX and AUR were followed by decreases in OVA-specific IgG and proliferation of spleen cells to the antigen. Treatments with IND, DEX, MTX and AUR inhibited both Th1 and Th2 immune responses, although the inhibitory effects of these drugs on the antigen-specific IgG2a and IFN-7 production appeared to be greater than those on IgG1 and IL-10 production. D-PA failed to influence anti-OVA IgG, IgG2a and IgG1 production as well as IFN-γ and IL-10 secretion. Administrations of all the drugs used resulted in suppression of antigen (OVA)-induced arthritis in mice which was associated with inhibition of anti-OVA IgG2a but not IgG1 production. These results suggest that anti-arthritic drugs including IND, DEX, MTX and AUR appear to suppress Th1 and, to a lesser extent, Th2 immune responses, and their antiinflammatory effects on human rheumatoid arthritis might be at least in part explained by downregulation by these drugs of Th1 responses involved in the disease.

Effects of diesel exhaust particle extracts on Th1 and Th2 immune responses in mice.

The present study was undertaken to investigate the effects of extracts of diesel exhaust particles (DEP) on Th1 and Th2 immune responses. In order to separate compounds from DEP different in hydrophobicity, a single DEP sample was consecutively extracted with hexane (HEX-DEP), benzene (BEN-DEP), dichloromethane (DIC-DEP), methanol (MET-DEP), and 1M ammonia (AMM-DEP). The last unextracted residue (UNE-DEP) was also used to test its effect on immune responses. To immunize mice, hen egg lysozyme (HEL) was injected i.p. (day 0). Varying doses of DEP, each DEP extract, and UNE-DEP were intranasally administered every 2 days from days 0 to 18. Anti-HEL IgG2a antibodies in sera and IFN-g secreted from spleen cells were measured as an indicator of Th1 immune responses, while anti-HEL IgG1 antibodies and IL-4 as that of Th2 responses. The results showed that treatment with DEP and DIC-DEP increased both Th1 and Th2 responses to HEL. UNE-DEP facilitated Th1 but not Th2 responses, while MET- and AMM-DEP administration was followed by enhancement of Th2 but not Th1 responses. Neither HEX- nor BEN-DEP modulated Th1 as well as Th2 responses. These results suggest that DEP contain various compounds different in hydrophobicity which may affect both Th1 and Th2, Th1 but not Th2, and Th2 but not Th1 immune responses.

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 Freunds adjuvant (day 0). Varying doses of MTX were orally administered daily from days 0 to 20. On day 21, anti‐OVA IgG2a and interferon‐γ (IFN‐γ) 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 IgG1production, although the inhibitory effect of MTX on the antigen‐specific IgG2a production appeared to be greater than that on IgG1 production. IFN‐γ, 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‐γ and IL‐10 secretion, although more pronounced decreases in IgG2a and IFN‐γ 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.

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 Freunds 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‐γ (IFN‐γ), 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‐γ 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‐γ secretion. These results indicate that rolipram may be effective in regulating Th1‐mediated diseases such as rheumatoid arthritis.