Maiko Kobayashi

Chlorpyrifos induces apoptosis in human T cells.

It was found previously that organophosphorus pesticides (OPs) significantly inhibited cytotoxic T lymphocyte (CTL) activity. To explore the mechanism of OP-induced inhibition of CTL activity, the present study investigated whether OPs can induce cell death/apoptosis in T cells. Jurkat human T cells were treated with chlorpyrifos at 0-100 ppm for 2, 4, and 6h at 37 degrees C in vitro. It was found that chlorpyrifos induced cell death of Jurkat human T cells in a dose- and time-dependent manner, as shown by MTT and LDH assays. Then, it was investigated if chlorpyrifos-induced cell death consisted of apoptosis, as determined by analysis of Annexin-V staining and the intracellular level of active caspase-3 by flow cytometry, and DNA fragmentation analysis. It was found that chlorpyrifos induces apoptosis in Jurkat T cells in a dose- and time-dependent manner, as determined by analysis of Annexin-V staining. DNA fragmentation was detected when cells were treated with 50 or 100 ppm chlorpyrifos for 4 and 6h. Chlorpyrifos also induced an increase in intracellular active caspase-3 in Jurkat T cells in a dose- and time-dependent manner, and a caspase-3 inhibitor, Z-DEVD-FMK, significantly inhibited chlorpyrifos-induced apoptosis. These findings indicate that chlorpyrifos can induce apoptosis in human Jurkat T cell cells, and this effect is partially mediated by the activation of intracellular caspase-3.

Effect of phytoncide from trees on human natural killer cell function.

We previously reported that the forest environment enhanced human natural killer (NK) cell activity, the number of NK cells, and intracellular anti-cancer proteins in lymphocytes, and that the increased NK activity lasted for more than 7 days after trips to forests both in male and female subjects. To explore the factors in the forest environment that activated human NK cells, in the present study we investigate the effect of essential oils from trees on human immune function in twelve healthy male subjects, age 37–60 years, who stayed at an urban hotel for 3 nights from 7.00p.m. to 8.00a.m. Aromatic volatile substances (phytoncides) were produced by vaporizing Chamaecyparis obtusa (hinoki cypress) stem oil with a humidifier in the hotel room during the night stay. Blood samples were taken on the last day and urine samples were analysed every day during the stay. NK activity, the percentages of NK and T cells, and granulysin, perforin, granzyme A/B-expressing lymphocytes in blood, and the concentrations of adrenaline and noradrenaline in urine were measured. Similar control measurements were made before the stay on a normal working day. The concentrations of phytoncides in the hotel room air were measured. Phytoncide exposure significantly increased NK activity and the percentages of NK, perforin, granulysin, and granzyme A/B-expressing cells, and significantly decreased the percentage of T cells, and the concentrations of adrenaline and noradrenaline in urine. Phytoncides, such as α-pinene and β-pinene, were detected in the hotel room air. These findings indicate that phytoncide exposure and decreased stress hormone levels may partially contribute to increased NK activity.

Physiological and Psychological Effects of Forest Therapy on Middle-Aged Males with High-Normal Blood Pressure

Time spent walking and relaxing in a forest environment (“forest bathing” or “forest therapy”) has well demonstrated anti-stress effects in healthy adults, but benefits for ill or at-risk populations have not been reported. The present study assessed the physiological and psychological effects of forest therapy (relaxation and stress management activity in the forest) on middle-aged males with high-normal blood pressure. Blood pressure and several physiological and psychological indices of stress were measured the day before and approximately 2 h following forest therapy. Both pre- and post-treatment measures were conducted at the same time of day to avoid circadian influences. Systolic and diastolic blood pressure (BP), urinary adrenaline, and serum cortisol were all significantly lower than baseline following forest therapy (p < 0.05). Subjects reported feeling significantly more “relaxed” and “natural” according to the Semantic Differential (SD) method. Profile of Mood State (POMS) negative mood subscale scores for “tension-anxiety,” “confusion,” and “anger-hostility,” as well as the Total Mood Disturbance (TMD) score were significantly lower following forest therapy. These results highlight that forest is a promising treatment strategy to reduce blood pressure into the optimal range and possibly prevent progression to clinical hypertension in middle-aged males with high-normal blood pressure.

Effect of forest walking on autonomic nervous system activity in middle-aged hypertensive individuals: a pilot study.

There has been increasing attention on the therapeutic effects of the forest environment. However, evidence-based research that clarifies the physiological effects of the forest environment on hypertensive individuals is lacking. This study provides scientific evidence suggesting that a brief forest walk affects autonomic nervous system activity in middle-aged hypertensive individuals. Twenty participants (58.0 ± 10.6 years) were instructed to walk predetermined courses in forest and urban environments (as control). Course length (17-min walk), walking speed, and energy expenditure were equal between the forest and urban environments to clarify the effects of each environment. Heart rate variability (HRV) and heart rate were used to quantify physiological responses. The modified semantic differential method and Profile of Mood States were used to determine psychological responses. The natural logarithm of the high-frequency component of HRV was significantly higher and heart rate was significantly lower when participants walked in the forest than when they walked in the urban environment. The questionnaire results indicated that, compared with the urban environment, walking in the forest increased “comfortable”, “relaxed”, “natural” and “vigorous” feelings and decreased “tension-anxiety,” “depression,” “anxiety-hostility,” “fatigue” and “confusion”. A brief walk in the forest elicited physiological and psychological relaxation effects on middle-aged hypertensive individuals.

Physiological and Psychological Effects of a Forest Therapy Program on Middle-Aged Females

The natural environment is increasingly recognized as an effective counter to urban stress, and “Forest Therapy” has recently attracted attention as a relaxation and stress management activity with demonstrated clinical efficacy. The present study assessed the physiological and psychological effects of a forest therapy program on middle-aged females. Seventeen Japanese females (62.2 ± 9.4 years; mean ± standard deviation) participated in this experiment. Pulse rate, salivary cortisol level, and psychological indices were measured on the day before forest therapy and on the forest therapy day. Pulse rate and salivary cortisol were significantly lower than baseline following forest therapy, indicating that subjects were in a physiologically relaxed state. Subjects reported feeling significantly more “comfortable,” “relaxed,” and “natural” according to the semantic differential (SD) method. The Profile of Mood State (POMS) negative mood subscale score for “tension–anxiety” was significantly lower, while that for “vigor” was significantly higher following forest therapy. Our study revealed that forest therapy elicited a significant (1) decrease in pulse rate, (2) decrease in salivary cortisol levels, (3) increase in positive feelings, and (4) decrease in negative feelings. In conclusion, there are substantial physiological and psychological benefits of forest therapy on middle-aged females.

Relationships Between Percentage of Forest Coverage and Standardized Mortality Ratios (SMR) of Cancers in all Prefectures in Japan

Objectives: To explore whether forest coverage affects the rate of deaths due to cancers in Japan, we investi- gated the relationships between the percentage of forest coverage and standardized mortality ratios due to cancers in all prefectures in Japan. Methods: Data on the percentage of forest coverage in all prefectures in Japan were collected from the database of the Forestry Agency of Japan. Data on standardized mortality ratios (SMR) due to lung, stomach, kidney, and colon cancers in males and females, breast and uterine cancers in females, and prostate cancer in males, and data of smoking status of males and females in all prefectures in Japan were collected from the database of the Ministry of Health, Labour, and Welfare of Japan. Human development index (HDI) was used as a parameter of the socioeconomic status of each prefec- ture. The correlation and partial correlation coefficients between the percentage of forest coverage and SMR of cancers, after controlling for the effects of smoking and the socioeconomic status, were calculated. Results: People living in areas with lower forest coverage had significantly higher SMR of cancers compared with the people living in areas with higher forest coverage. There were significant inverse correlations between the percentage of forest coverage and the SMR of lung, breast, and uterine cancers in females, and the SMR of prostate, kidney, and colon cancers in males in all prefectures in Japan, even after the effects of smoking and socioeconomic status were factored in. Conclusions: These findings indicate that increased forest coverage may partially contribute to a decrease in mortality due to cancers in Japan.

Carbamate Pesticide-Induced Apoptosis in Human T Lymphocytes

We previously found that carbamate pesticides induced significant apoptosis in human natural killer cells. To investigate whether carbamate pesticides also induce apoptosis in human T lymphocytes, in the present study Jurkat human T cells were treated in vitro with thiram, maneb, carbaryl or ziram. Apoptosis was determined by FITC-Annexin-V/PI staining. To explore the mechanism of apoptosis, intracellular levels of active caspase 3 and mitochondrial cytochrome-c release were determined by flow cytometry. We found that thiram, ziram, maneb and carbaryl also induced apoptosis in a time- and dose-dependent manner in the human T cells. However, the strength of the apoptosis-inducing effect differed among the pesticides, with the: thiram > ziram > maneb > carbaryl. Moreover, thiram significantly increased the intracellular level of active caspase 3 and caspase inhibitors significantly inhibited apoptosis. Thiram also significantly caused mitochondrial cytochrome-c release. These findings indicate that carbamate pesticides can induce apoptosis in human T cells, and the apoptosis is mediated by the activation of caspases and the release of mitochondrial cytochrome-c.

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.

Mechanism of ziram-induced apoptosis in human natural killer cells.

We previously found that ziram, a dithiocarbamate fungicide, significantly inhibited natural killer (NK) activity in a dose-dependent manner. To explore the mechanism of this inhibition, we investigated ziram-induced apoptosis in human NK cells. Human NK-92MI cells were treated with ziram at 0.0625-4 μM for 2–64 h. Apoptosis was determined by FITC-Annexin-V/PI staining. To explore the mechanism of apoptosis, intracellular levels of active caspases 3, 3/7, 8, and 9 and pan-caspase and mitochondrial cytochrome-c release were determined by flow cytometry. Disruption to mitochondrial transmembrane potential was determined with a MitoLight™ Apoptosis Detection Kit. It was found that ziram induced apoptosis in a dose- and time-dependent manner in human NK cells. Ziram increased the intracellular levels of active caspases 3, 3/7, 8, and 9 and pan-caspase in a dose-dependent manner, and a caspase-3 inhibitor, Z-DEVD-FMK, and a general caspase inhibitor, Z-VAD-FMK, partially but significantly inhibited the apoptosis. Ziram also disrupted mitochondrial transmembrane potential and caused mitochondrial cytochrome-c release in a dose-dependent manner. These findings indicate that ziram can induce apoptosis in human NK cells, and the apoptosis is at least mediated by both the caspase-cascade and the mitochondria/cytochrome-c pathways.

Effects of subchronic inhalation exposure to ethyl tertiary butyl ether on splenocytes in mice.

Ethyl tertiary-butyl ether (ETBE) is a motor fuel oxygenate used in reformulated gasoline. The current use of ETBE in gasoline or petrol is modest but increasing. To investigate the effects of ETBE on splenocytes, mice were exposed to 0 (control), 500 ppm, 1750 ppm, or 5000 ppm of ETBE by inhalation for 6 h/day for 5 days/wk over a 6- or 13-week period. Splenocytes were harvested from the control and exposed mice, and the following cell phenotypes were quantified by flow cytometry: (1) B cells (PerCP-Cy5.5-CD45R/B220), (2) T cells (PerCP-Cy5-CD3e), (3) T cell subsets (FITC-CD4 and PE-CD8a), (4) natural killer (NK) cells (PE-NK1.1), and (5) macrophages (FITC-CD11b). Body weight and the weight of the spleen were also examined. ETBE-exposure did not affect the weight of the spleen or body weight, while it transiently increased the number of RBC and the Hb concentration. The numbers of splenic CD3+, CD4+, and CD8+ T cells, the percentage of CD4+ T cells and the CD4+/CD8+ T cell ratio in the ETBE-exposed groups were significantly decreased in a dose-dependent manner. However, ETBE exposure did not affect the numbers of splenic NK cells, B cells, or macrophages or the total number of splenocytes. The above findings indicate that ETBE selectively affects the number of splenic T cells in mice.