Kelly Yoshizaki

Subchronic effects of nasally instilled diesel exhaust particulates on the nasal and airway epithelia in mice

Diesel exhaust is the major source of ultrafine particles released during traffic-related pollution. Subjects with chronic respiratory diseases are at greater risk for exacerbations during exposure to air pollution. This study evaluated the effects of subchronic exposure to a low-dose of diesel exhaust particles (DEP). Sixty male BALB/c mice were divided into two groups: (a) Saline: nasal instillation of saline (n = 30); and (b) DEP: nasal instillation of 30 µg of DEP/10 µl of saline (n = 30). Nasal instillations were performed 5 days a week, over 30 and 60 days. Animals were anesthetized with pentobarbital sodium (50 mg/kg intraperitoneal [i.p.]) and sacrificed by exsanguination. Bronchoalveolar lavage (BAL) fluid was performed to evaluate the inflammatory cell count and the concentrations of the interleukin (IL)-4, IL-10, and IL-13 by enzyme-linked immunosorbent assay (ELISA). The gene expression of oligomeric mucus/gel-forming (Muc5ac) was evaluated by real-time polymerase chain reaction (PCR). Histological analysis in the nasal septum and bronchioles was used to evaluate the bronchial and nasal epithelium thickness as well as the acidic and neutral nasal mucus content. The saline group (30 and 60 days) did not show any changes in any of the parameters. However, the instillation of DEP over 60 days increased the expression of Muc5ac in the lungs and the acid mucus content in the nose compared with the 30-day treatment, and it increased the total leukocytes in the BAL and the nasal epithelium thickness compared with saline for 60 days. Cytokines concentrations in the BAL were detectable, with no differences among the groups. Our data suggest that a low-dose of DEP over 60 days induces respiratory tract inflammation.

Acute cardiopulmonary effects induced by the inhalation of concentrated ambient particles during seasonal variation in the city of São Paulo.

Ambient particles may undergo modifications to their chemical composition as a consequence of climatic variability. The determination of whether these changes modify the toxicity of the particles is important for the understanding of the health effects associated with particle exposure. The objectives were to determine whether low levels of particles promote cardiopulmonary effects, and to assess if the observed alterations are influenced by season. Mice were exposed to 200 μg/m(3) concentrated ambient particles (CAPs) and filtered air (FA) in cold/dry and warm/humid periods. Lung hyperresponsiveness, heart rate, heart rate variability, and blood pressure were evaluated 30 min after each exposure. After 24 h, blood and tissue samples were collected. During both periods (warm/humid and cold/dry), CAPs induced alterations in red blood cells and lung inflammation. During the cold/dry period, CAPs reduced the mean corpuscular volume levels and increased erythrocytes, hemoglobin, mean corpuscular hemoglobin concentration, and red cell distribution width coefficient variation levels compared with the FA group. Similarly, CAPs during the warm/humid period decreased mean corpuscular volume levels and increased erythrocytes, hemoglobin, hematocrit, and red cell distribution width coefficient variation levels compared with the FA group. CAPs during the cold/dry period increased the influx of neutrophils in the alveolar parenchyma. Short-term exposure to low concentrations of CAPs elicited modest but significant pulmonary inflammation and, to a lesser extent, changes in blood parameters. In addition, our data support the concept that changes in climate conditions slightly modify particle toxicity because equivalent doses of CAPs in the cold/dry period produced a more exacerbated response.

The effects of particulate matter on inflammation of respiratory system: Differences between male and female

Air pollution is known to exacerbate respiratory diseases and epidemiological studies have shown that women present more chronic respiratory symptoms than man exposed to traffic pollution, however, the reason why is unclear. This study evaluated the inflammatory differences in BALB/c mouse males (n=34) and females (n=111) in three phases of the estrous cycle that were exposed to ambient air (AA) or concentrated ambient particles (CAPs). Tracheal hyperreactivity to methacholine, bronchoalveolar lavage fluid (BALF) and immunohistochemical of airways and lung parenchyma were studied. Hyperreactivity increased in CAPs-exposed female mice compared with AA-exposed mice in estrus (p<0.05) and proestrus phases (p<0.05) and decreased in CAPs-exposed males compared with those exposed to AA (p<0.05). Males had increased numbers of total cells (p=0.037) and macrophages (p=0.028) compared to females. BALF levels of cyclooxygenase-2(COX-2) (p=0.000), transforming growth factor alpha (TGF-α) (p=0.001) and IL-8 receptor alpha (IL-8Rα) (p=0.014) were increased in males compared with proestrus, estrus and diestrus females, independent of exposure. Proestrus females exhibited significantly higher cadherin expression in lung parenchyma than did males (p=0.005). CAPs exposure increased matrix metalloproteinase-9 (MMP-9) (p=0.024) and isoprostane (p=0.003) expression in the airways of both, males and females. The level of substance P (SP) (p=0.001) increased in lung parenchyma in males compared with females, while IL-17 levels in airways (p=0.042) and in lung parenchyma (p=0.008) increased in females. MMP-9 levels (p=0.024) were significantly lower in the lung parenchyma of CAPs-exposed females. TGF-α (p=0.007) levels increased in the lung parenchyma of CAPs-exposed females compared to AA-exposed females. These results suggest that inflammatory markers differentially expressed in male mice were mostly linked to acute inflammation (IL-1β, IL-8Rα, COX-2), whereas in females, markers that may lead to a chronic inflammatory process such as IL-17 and remodeling (MMP-9) were increased.

Vesicular acetylcholine transport deficiency potentiates some inflammatory responses induced by diesel exhaust particles

Endogenous acetylcholine (ACh), which depends of the levels of vesicular ACh transport (VAChT) to be released, is the central mediator of the cholinergic anti-inflammatory system. ACh controls the release of cytokine in different models of inflammation. Diesel exhaust particles (DEP) are one of the major environmental pollutants produced in large quantity by automotive engines in urban center. DEP bind the lung parenchyma and induce inflammation. We evaluated whether cholinergic dysfunction worsens DEP-induced lung inflammation. Male mice with decreased ACh release due to reduced expression of VAChT (VAChT-KD mice) were submitted to DEP exposure for 30 days (3 mg/mL of DEP, once a day, five days a week) or saline. Pulmonary function and inflammation as well as extracellular matrix fiber deposition were evaluated. Additionally, airway and nasal epithelial mucus production were quantified. We found that DEP instillation worsened lung function and increased lung inflammation. Higher levels of mononuclear cells were observed in the peripheral blood of both wild-type (WT) and VAChT-KD mice. Also, both wild-type (WT) and VAChT-KD mice showed an increase in macrophages in bronchoalveolar lavage fluid (BALF) as well as increased expression of IL-4, IL-6, IL-13, TNF-α, and NF-κB in lung cells. The collagen fiber content in alveolar septa was also increased in both genotypes. On the other hand, we observed that granulocytes were increased only in VAChT-KD peripheral blood. Likewise, increased BALF lymphocytes and neutrophils as well as increased elastic fibers in alveolar septa, airway neutral mucus, and nasal epithelia acid mucus were observed only in VAChT-KD mice. The cytokines IL-4 and TNF-α were also higher in VAChT-KD mice compared with WT mice. In conclusion, decreased ability to release ACh exacerbates some of the lung alterations induced by DEP in mice, suggesting that VAChT-KD animals are more vulnerable to the effects of DEP in the lung.

Acute exposure to diesel and sewage biodiesel exhaust causes pulmonary and systemic inflammation in mice

Biodiesel is a renewable energy source that reduces particle emission, but few studies have assessed its effects. To assess the effects of acute inhalation of two doses (600 and 1200 μg/m3) of diesel (DE) and biodiesel (BD) fuels on the inflammatory pulmonary and systemic profile of mice. Animals were exposed for 2 h in an inhalation chamber inside the Container Laboratory for Fuels. Heart rate, heart rate variability (HRV) and blood pressure were determined 30 min after exposure. After 24 h, we analyzed the lung inflammation using bronchoalveolar lavage fluid (BALF); neutrophil and macrophage quantification in the lung parenchyma was performed, and blood and bone marrow biomarkers as well as receptor of endothelin-A (ET-Ar), receptor of endothelin-B (ET-Br), vascular cell adhesion molecule 1 (VCAM-1), inducible nitric oxide synthase (iNOs) and isoprostane (ISO) levels in the pulmonary vessels and bronchial epithelium were evaluated. HRV increased for BD600, D600 and D1200 compared to filtered air (FA). Both fuels (DE and BD) produced alterations in red blood cells independent of the dose. BALF from the BD600 and BD1200 groups showed an increase in neutrophils compared to those of the FA group. Numeric density of the polymorphonuclear and mononuclear cells was elevated with BD600 compared to FA. In the peribronchiolar vessels, there was an increase in ET-Ar and ET-Br expression following BD600 compared to FA; and there was a reduction in the iNOs expression for BD1200 and the VCAM-1 for D1200 compared to FA. In the bronchial epithelium, there was an increase in ETAr at BD600, ET-Br at two doses (600 and 1200 μg/m3) of DE and BD, iNOs at D600 and VCAM-1 at BD1200 and D600; all groups were compared to the FA group. Acute exposure to DE and BD derived from sewage methyl esters triggered pulmonary and cardiovascular inflammatory alterations in mice.