Nrf2 Lowers the Risk of Lung Injury via Modulating the Airway Innate Immune Response Induced by Diesel Exhaust in Mice.

In the present study, we investigated the role of Nrf2 in airway immune responses induced by diesel exhaust (DE) inhalation in mice. C57BL/6J Nrf2+/+ and Nrf2-/- mice were exposed to DE or clean air for 8 h/day and 6 days/week for 4 weeks. After DE exposure, the number of neutrophils and macrophage inflammatory protein (MIP)-2 level in bronchoalveolar lavage fluid (BALF) and interleukin (IL)-17 level in the lung tissue increased in Nrf2-/- mice compared with Nrf2+/+ mice; however, the lack of an increase in the level of tumor necrosis factor (TNF)-α in the lung tissue in Nrf2+/+ mice and mild suppression of the level of TNF-α in Nrf2-/- mice were observed; the level of granulocyte macrophage colony-stimulating factor (GM-CSF) in the lung tissue decreased in Nrf2-/- mice than in Nrf2+/+ mice; the number of DE particle-laden alveolar macrophages in BALF were larger in Nrf2-/- mice than in Nrf2+/+ mice. The results of electron microscope observations showed alveolar type II cell injury and degeneration of the lamellar body after DE exposure in Nrf2-/- mice. Antioxidant enzyme NAD(P)H quinone dehydrogenase (NQO)1 mRNA expression level was higher in Nrf2+/+ mice than in Nrf2-/- mice after DE exposure. Our results suggested that Nrf2 reduces the risk of pulmonary disease via modulating the airway innate immune response caused by DE in mice.

Nrf2 Regulates the Risk of a Diesel Exhaust Inhalation-Induced Immune Response during Bleomycin Lung Injury and Fibrosis in Mice.

The present study investigated the effects of diesel exhaust (DE) on an experimental model of bleomycin (BLM)-induced lung injury and fibrosis in mice. BLM was intravenously administered to both Nrf2+/+ and Nrf2-/- C57BL/6J mice on day 0. The mice were exposed to DE for 56 days from 28 days before the BLM injection to 28 days after the BLM injection. Inhalation of DE induced significant inhibition of airway clearance function and the proinflammatory cytokine secretion in macrophages, an increase in neutrophils, and severe lung inflammatory injury, which were greater in Nrf2-/- mice than in Nrf2+/+ mice. In contrast, inhalation of DE was observed to induce a greater increase of hydroxyproline content in the lung tissues and significantly higher pulmonary antioxidant enzyme mRNA expression in the Nrf2+/+ mice than in Nrf2-/- mice. DE is an important risk factor, and Nrf2 regulates the risk of a DE inhalation induced immune response during BLM lung injury and fibrosis in mice.

Effect of smoking prevention education using improved tobacco smoke collection method.

OBJECTIVE: To verify the effect of smoking prevention education for nursing students using the improved tobacco smoke collection method. METHODS: The improved tobacco smoke collection method allows mainstream smoke and sidestream smoke from a cigarette to be separately extracted using a closed system. After collection, we performed gas measurement using a gas detection tube and the Schiff reagent method. We provided a lecture incorporating the experimental method for an experimental group (42 students), but only the lecture without the method for a control group (43 students). We surveyed the changes in The Kano Test for Social Dependence (KTSND) scores before and after the lecture and one month later. RESULTS: In the experimental group, the total scores of the KTSND were 10.2 ± 5.0 (mean ± standard deviation) before the lecture, 5.8 ± 4.1 after the lecture, and 6.9 ± 4.8 one month later. On the other hand, the scores were 10.7 ± 5.7, 7.5 ± 5.8, and 9.7 ± 5.5 in the control group before, after, and one month after the lecture, respectively. It is considered that the students understood "smoking is harmful to health" since this gas analysis method can be used to check for harmful gases visually. CONCLUSION: Result of this study suggest that this experimental method is useful for educating nursing students on the harmful effects of smoking.

Substrate specificity of human granzyme 3: analyses of the P3-P2-P1 triplet using fluorescence resonance energy transfer substrate libraries.

Granzyme 3 (Gr3) is known as a tryptase-type member of the granzyme family and exists in the granules of immunocompetent cells. Granule proteases including granzymes, are transported into the cytoplasm of tumor cells or virus-infected cells by perforin function, degrade cytoplasmic or nuclear proteins and subsequently cause the death of the target cells. Recently, although several substrates of Gr3 in vivo have been reported, these hydrolyzed sites were unclear or lacked consistency. Our previous study investigated the optimal amino acid triplet (P3-P2-P1) as a substrate for Gr3 using a limited combination of amino acids at the P2 and P3 positions. In the present study, new fluorescence resonance energy transfer (FRET) substrate libraries to screen P2 and P3 positions were synthesized, respectively. Using these substrate libraries, the optimal amino acid triplet was shown to be Tyr-Phe-Arg as a substrate for human Gr3. Moreover, kinetic analyses also showed that the synthetic substrate FRETS-YFR had the lowest Km value for human Gr3. A substantial number of membrane proteins possessed the triplet Tyr-Phe-Arg and some of them might be in vivo substrates for Gr3. The results might also be a great help for preparing specific inhibitors to manipulate Gr3 activity both in vitro and in vivo.

EM, EM703 inhibit NF-kB activation induced by oxidative stress from diesel exhaust particle in human bronchial epithelial cells: importance in IL-8 transcription.

Diesel exhaust particle (DEP) is the major components of PM2.5, and much attention has focused on PM2.5 in relation to adverse health effects, and many pulmonary diseases. In the present study, we used a human bronchial epithelial cell (HBEC) line to investigate the anti-inflammatory effects of erythromycin (EM) and EM703 - a new derivative of erythromycin without antibacterial effects on the expressions of IL-8 caused by DEP exposure. DEP showed a dose-dependent stimulatory effect on IL-8 product in HBEC. Increases of IL-8 expression by DEP stimulation were significantly blocked by both EM and EM703 pretreatment. Furthermore, NF-κB and Nrf2 activation, the antioxidant enzymes such as HO-1, NQO-1 mRNA expression were increased by DEP exposure and these increases were blocked by both of EM and EM703 pretreatment. Our results suggest that, EM and EM703 may have an inhibitory effect on expression inflammatory cytokines in HBEC induced by DEP not only as an anti-inflammation but also an antioxidant drug. EM and EM703 might contribute to chemical prevention of the risk of pulmonary diseases induced by oxidative stress from environmental pollutant, such as DEP.

Acute effects of walking in forest environments on cardiovascular and metabolic parameters.

We previously found that forest environments reduced stress hormones such as adrenaline and noradrenaline and showed the relaxing effect both in male and female subjects. In the present study, we investigated the effects of walking under forest environments on cardiovascular and metabolic parameters. Sixteen healthy male subjects (mean age 57.4 ± 11.6 years) were selected after obtaining informed consent. The subjects took day trips to a forest park in the suburbs of Tokyo and to an urban area of Tokyo as a control in September 2010. On both trips, they walked for 2 h in the morning and afternoon on a Sunday. Blood and urine were sampled on the morning before each trip and after each trip. Blood pressure was measured on the morning (0800) before each trip, at noon (1300), in the afternoon (1600) during each trip, and on the morning (0800) after each trip. The day trip to the forest park significantly reduced blood pressure and urinary noradrenaline and dopamine levels and significantly increased serum adiponectin and dehydroepiandrosterone sulfate (DHEA-S) levels. Walking exercise also reduced the levels of serum N-terminal pro-B-type natriuretic peptide (NT-proBNP) and urinary dopamine. Taken together, habitual walking in forest environments may lower blood pressure by reducing sympathetic nerve activity and have beneficial effects on blood adiponectin and DHEA-S levels, and habitual walking exercise may have beneficial effects on blood NT-proBNP levels.

Effect of oral exposure to fenitrothion and 3-methyl-4-nitrophenol on splenic cell populations and histopathological alterations in spleen in Wistar rats.

Fenitrothion (FNT) is used throughout the world as an insecticide in agriculture. To investigate the effect of FNT on the splenocytes and the underlying mechanism, FNT and its main metabolite, 3-methyl-4-nitrophenol (MNP), were administered orally to Wistar rats in daily doses of 0, 5 and 10 mg/kg, 4-5 days/week for 9 weeks. Splenocytes were harvested from control and exposed rats, and the following cell phenotypes were quantified by flow cytometry: (1) B cells (PE-CD45RA), (2) T cells (FITC-CD3), (3) T cell subsets (PE-CD4 and PerCPCD8), (4) natural killer (NK) cells (FITC-CD161a), (5) macrophages (FITC-CD11b), and (6) granulocyte (PE-granulocyte). Body weight, weight of the spleen, and histopathological alterations of spleens were also examined. The percentage of splenic CD8+ T cells and the ratio of CD8/CD4 in the group receiving 10 mg/kg FNT, and the percentages of splenic CD3+ and CD8+ T cells in the group receiving 10 mg/kg MNP were significantly decreased compared with those in the controls. FNT exposure also significantly decreased the weight of the spleen and body weight. In addition, apoptotic lymphocytes in spleen were observed in FNT-exposed rats under transmission electron microscope. However, FNT and MNP exposures did not affect splenic NK cells, B cells, macrophages, and granulocytes. The above findings indicate that FNT and MNP may selectively affect splenic T cells in rats.

Recombinant human progranzyme 3 expressed in Escherichia coli for analysis of its activation mechanism.

Gr3 is reported to play an important role in defense against viral infection. Although it is known that Gr3 is synthesized as a proenzyme and activated in the cytotoxic granules of NK cells and CTL, the activation mechanism is not clearly understood. In an attempt to analyze the activation mechanism of human Gr3, a recombinant pro-Gr3 was expressed in the periplasm of E. coli and purified to homogeneity. On SDS-PAGE the recombinant pro-Gr3 showed a slightly higher molecular weight than the enzymatically active Gr3, because the former possesses a small propeptide at its N-terminal. The recombinant pro-Gr3 was enzymatically inactive. It could be activated by treatment with cathepsin C, which concomitantly decreased the molecular weight to that of active Gr3. The proteolytic reaction of cathepsin C did not continue after one dipeptide had been removed, indicating that the recombinant pro-Gr3 had the native conformation without any refolding process. The recombinant pro-Gr3 would be a valuable tool for analyzing the activation mechanism and exploring other activating enzymes besides cathepsin C.

[Evaluation of new saliva collection device for determination of salivary cotinine, cortisol, dehydroepiandrosterone and testosterone concentrations].

OBJECTIVES: This study was conducted to examine the effectiveness of a new salivary collection device (psi10 mm x 25 mm) made of polypropylene and polyethylene polymers (Salisoft((R))). METHOD: Experiment 1; We measured the absorption capacity of the new device by two methods. Next, we examined whether the device affected the pH of 0.1 M NaHCO(3) solution (pH 8.7) and 0.1 M sodium phosphate buffer solution (pH 6.0). Experiment 2; We compared three saliva collection methods: by passive drool, using a cotton device (Salivette((R))), and using the new device. Saliva samples were collected from twelve men (average age, 31.5 +/- 17.1 years). Saliva samples were assayed for cotinine, cortisol, dehydroepiandrosterone, and testosterone concentrations by enzyme immunoassay, and the pH of saliva samples were measured. RESULTS: After this device was put in the mouth for one minute, 1.28 +/- 0.13 mL (mean +/- SD, N = 6) of saliva samples were obtained. The mean pHs of saliva samples collected using Salisoft((R)) and by passive drool did not differ significantly, whereas that of saliva samples collected with Salivette((R)) was significantly low. Saliva samples collected with Salisoft((R)) and those obtained by passive drool did not show significant differences in the concentrations of cotinine, cortisol, dehydroepiandrosterone, and testosterone. Moreover, significant positive correlations were noted between the concentrations in saliva samples collected with Salisoft((R)) and those in saliva samples collected by passive drool. CONCLUSION: This new device was shown to be suitable for saliva collection for the determination of the concentrations of cotinine and some steroids by enzyme immunoassay.

Patterns in self-rated health according to age and sex in a Japanese national survey, 1989-2004.

BACKGROUND: Perceived good health or good self-rated health is considered to be a predictor of longer survival and maintenance of good quality of life, which is a public health goal. OBJECTIVE: This study assessed trends in the percentage of self-rated poor health among Japanese residents, based on data from the National Comprehensive Survey of the Living Conditions of People on Health and Welfare. METHODS: Results of the survey (which is conducted in Japan every 3 years to determine the living conditions of people receiving health and welfare services) were analyzed using multistage and stratified cluster sampling of households. Self-rated health was measured by response to the question, "Recently, would you say that in general your health has been good, fairly good, fair, fairly poor, or poor?" The trend in fairly poor or poor health status during the period from 1989 through 2004 was stratified by sex and age group. RESULTS: The rates of response to the survey were 90.9% (246,892/271,588) in 1995 and 79.8% (220,836/276,682) in 2004. Target subjects were aged >or=20 years in each year of the study. The prevalence of self-reported fairly poor or poor health was lowest in 1995 and then increased every year until 2001, when it appeared to reach a plateau. The prevalence of having fairly poor or poor health among women aged 35 to 44, 45 to 54, 55 to 64, and 65 to 74 years were as follows in 1995: 9.2%, 11.7%, 15.3%, and 19.8%, respectively. In 2004, the rates were 13.3%, 17.2%, 22.1%, and 31.7%, respectively. By comparison, the prevalence of self-reported fairly poor or poor health was 8.1%, 9.3%, 13.7%, and 17.9% among men aged 35 to 44, 45 to 54, 55 to 64, and 65 to 74 years, respectively, in 1995. In 2004, these rates were 12.8%, 14.8%, 19.0%, and 27.9%, respectively. CONCLUSIONS: In this survey, conducted every 3 years between 1989 and 2004 in Japanese households, older subjects had a greater prevalence of self-reported fairly poor or poor health than did younger subjects. The proportion of respondents who described their health as poor or fairly poor was highest in 1995. Women generally had a greater prevalence of self-reported poor or fairly poor health.

Healthy lifestyles are associated with higher levels of perforin, granulysin and granzymes A/B-expressing cells in peripheral blood lymphocytes.

OBJECTIVE: It is well documented that natural killer (NK) cells provide host defense against tumors and viruses. We previously showed that lifestyle affects human NK and LAK activities. In order to explore the underlying mechanism, we investigated the effect of lifestyle on intracellular perforin, granulysin, and granzymes A/B in peripheral blood lymphocytes (PBL). METHODS: 114 healthy male subjects, aged 20-59 years, from a large company in Osaka, Japan were selected with informed consent. The subjects were divided into groups reporting good, moderate, and poor lifestyles according to their responses on a questionnaire regarding eight health practices (cigarette smoking, alcohol consumption, sleeping hours, working hours, physical exercise, eating breakfast, balanced nutrition, and mental stress). Peripheral blood was taken, and numbers of NK, T, perforin, granulysin, and granzymes A/B-expressing cells in PBL were measured by flow cytometry. RESULTS: Subjects with good or moderate lifestyle showed significantly higher numbers of NK, and perforin, granulysin, and granzymes A/B-expressing cells and a significantly lower number of T cells in PBL than subjects with poor lifestyle. Among the eight health practices, cigarette smoking, physical exercise, eating breakfast, and balanced nutrition significantly affect the numbers of NK, T cells, perforin, granulysin, and/or granzymes A/B-expressing cells, and alcohol consumption significantly affects the number of granzyme A-expressing cells. On the other hand, mental stress, sleeping, and working hours had no effect on those parameters. CONCLUSIONS: Taken together, these findings indicate that poor lifestyle significantly decreases the numbers of NK, perforin, granulysin, and granzymes A/B-expressing cells in PBL.

Expression of enzymatically active human granzyme 3 in Escherichia coli for analysis of its substrate specificity.

Human granzyme 3 (Gr3) is a serine protease contained in the granules of natural killer cells and cytotoxic T lymphocytes. To elucidate the biochemical and physiological characteristics of Gr3, we attempted to prepare an enzymatically active recombinant human Gr3 without refolding and proteolytic activation. An expression vector was constructed, in which the pre-/pro-peptide coding sequence of Gr3 was replaced with the bacterial pelB leader sequence. The resultant expression product was a fully active protease in the periplasmic fraction of Escherichia coli and was purified to homogeneity. The purified enzyme effectively hydrolyzed Z-Lys-SBzl, a conventionally used substrate of Gr3. In addition, it also hydrolyzed the peptide substrate library FRETS-25Xaa series, required basic amino acid residues, Arg or Lys, at the P1 position, and most efficiently hydrolyzed the carboxylic side of Phe-Tyr-Arg downward arrow (P3-P2-P1) sequence of the 475 tripeptide combinations.

EM703 improves bleomycin-induced pulmonary fibrosis in mice by the inhibition of TGF-beta signaling in lung fibroblasts.

BACKGROUND: Fourteen-membered ring macrolides have been effective in reducing chronic airway inflammation and also preventing lung injury and fibrosis in bleomycin-challenged mice via anti-inflammatory effects. EM703 is a new derivative of erythromycin (EM) without the bactericidal effects. We investigated the anti-inflammatory and antifibrotic effects of EM703 in an experimental model of bleomycin-induced lung injury and subsequent fibrosis in mice. METHODS: Seven-week-old male ICR mice were used. All experiments used eight mice/group, unless otherwise noted in the figure legends. Bleomycin was administered intravenously to the mice on day 0. EM703 was orally administered daily to mice. All groups were examined for cell populations in the bronchoalveolar lavage (BAL) fluid and for induction of messenger RNA (mRNA) of Smad3 and Smad4 in the lung tissues by reverse transcriptase (RT)-polymerase chainreaction (PCR) on day 7. Fibroblastic foci were assessed histologically, and the hydroxyproline content was chemically determined in the lung tissues on day 28. We performed assay of proliferation and soluble collagen production, and examined the induction of mRNA of Smad3 and Smad4 by RT-PCR in murine lung fibroblast cell line MLg2908. We also examined Smad3, Smad4 and phosphorylated Smad2/3 (p-Smad2/3) protein assay by western blotting in MLg2908. RESULTS: Bleomycin-induced lung fibrosis, and the infiltration of macrophages and neutrophils into the airspace were inhibited by EM703. The expression of Smad3 and Smad4 mRNA was clearly attenuated by bleomycin, but was recovered by EM703. EM703 also inhibited fibroblast proliferation and the collagen production in lung fibroblasts induced by Transforming growth factor-beta (TGF-beta). The expression of Smad3 and Smad4 mRNA in murine lung fibroblasts disappeared due to TGF-beta, but was recovered by EM703. EM703 inhibited the expression of p-Smad2/3 and Smad4 protein in murine lung fibroblasts induced by TGF-beta. CONCLUSION: These findings suggest that EM703 improves bleomycin-induced pulmonary fibrosis in mice by actions of anti-inflammation and regulation of TGF-beta signaling in lung fibroblasts.

Insomnia as a sequela of sarin toxicity several years after exposure in Tokyo subway trains.

More than 5,000 passengers on Tokyo subway trains were injured with toxic chemicals including the nerve gas "sarin" on March 20, 1995. The purpose of this study was to identify the effect of sarin exposure on insomnia in a cross-sectional study. A self-administered questionnaire concerning sleep-related items was distributed to victims of sarin exposure in October and November, 2003. Questionnaires were completed by 161 of the 163 participants (98.8%), who were selected from 1,500 subjects. Among them, the authors selected 75 women 30 to 69 years of age. Control participants were collected from inhabitants living in Maebachi City, Gunma Prefecture, Japan. For the younger exposed group (under 50 yr. of age), percentages of poor sleep, difficulty falling asleep, intermittent awakening, early morning awakening, a feeling of light overnight sleep, and insomnia were significantly higher than those for the control group. In contrast, the older exposed group (ages 50 to 69 years) had significantly higher prevalence of poor sleep, a feeling of light overnight sleep, and early morning awakening for the exposed group when compared with the control group. The high prevalence of insomnia and insomnia-related factors for victims especially under 50 years of age suggests a need for research on sleep quality after sarin exposure. Although posttraumatic stress disorder is assumed to be a psychological effect of exposure to a toxic substance, a cause-and-effect relationship has not been established.

Elevated frequency of sister chromatid exchanges of lymphocytes in sarin-exposed victims of the Tokyo sarin disaster 3 years after the event.

We previously reported that the frequency of sister chromatid exchanges (SCEs) among victims of the Tokyo subway sarin disaster was significantly higher than that of controls 2-3 months after the disaster. It has been reported that the victims were also exposed to the by-products generated during sarin synthesis, i.e., diisopropyl methylphosphonate (DIMP), diethyl methylphosphonate (DEMP) and N,N-diethylaniline (DEA) during the disaster and we previously found that DIMP, DEMP and DEA induced a significant SCE increase in human lymphocytes in vitro. To monitor the genetic aftereffects of the sarin exposure, SCEs of peripheral blood lymphocytes were measured in fire fighters and police officers involved in the disaster 3 years after the event. We found that the frequency of SCEs was still significantly higher in the exposed subjects than the controls, suggesting a risk of the genetic aftereffects of the sarin exposure. We further found a significant positive correlation between the frequency of SCEs and the inhibition of serum cholinesterase activity in the exposed subjects, suggesting that the elevated frequency of SCEs is related to the sarin exposure. On the other hand, there was no significant difference in natural killer activity between the exposed and the controls.