Quantification of three DNA Lesions by Mass Spectrometry and Assessment of Their Levels in Tissues of Mice Exposed to Ambient Fine Particulate Matter.

DNA adducts and oxidized DNA bases are examples of DNA lesions that are useful biomarkers for the toxicity assessment of substances that are electrophilic, generate reactive electrophiles upon biotransformation, or induce oxidative stress. Among the oxidized nucleobases, the most studied one is 8-oxo-7,8-dihydroguanine (8-oxoGua) or 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), a biomarker of oxidatively induced base damage in DNA. Aldehydes and epoxyaldehydes resulting from the lipid peroxidation process are electrophilic molecules able to form mutagenic exocyclic DNA adducts, such as the etheno adducts 1,N2-etheno-2'-deoxyguanosine (1,N2-εdGuo) and 1,N6-etheno-2'-deoxyadenosine (1,N6-εdAdo), which have been suggested as potential biomarkers in the pathophysiology of inflammation. Selective and sensitive methods for their quantification in DNA are necessary for the development of preventive strategies to slow down cell mutation rates and chronic disease development (e.g., cancer, neurodegenerative diseases). Among the sensitive methods available for their detection (high performance liquid chromatography coupled to electrochemical or tandem mass spectrometry detectors, comet assay, immunoassays, 32P-postlabeling), the most selective are those based on high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-ESI-MS/MS). Selectivity is an essential advantage when analyzing complex biological samples and HPLC-ESI-MS/MS evolved as the gold standard for quantification of modified nucleosides in biological matrices, such as DNA, urine, plasma and saliva. The use of isotopically labeled internal standards adds the advantage of corrections for molecule losses during the DNA hydrolysis and analyte enrichment steps, as well as for differences of the analyte ionization between samples. It also aids in the identification of the correct chromatographic peak when more than one peak is present. We present here validated sensitive, accurate and precise HPLC-ESI-MS/MS methods that were successfully applied for the quantification of 8-oxodGuo, 1,N6-dAdo and 1,N2-dGuo in lung, liver and kidney DNA of A/J mice for the assessment of the effects of ambient PM2.5 exposure.

Genotoxic and epigenotoxic effects in mice exposed to concentrated ambient fine particulate matter (PM2.5) from São Paulo city, Brazil.

BACKGROUND: The Metropolitan Area of São Paulo has a unique composition of atmospheric pollutants, and positive correlations between exposure and the risk of diseases and mortality have been observed. Here we assessed the effects of ambient fine particulate matter (PM2.5) on genotoxic and global DNA methylation and hydroxymethylation changes, as well as the activities of antioxidant enzymes, in tissues of AJ mice exposed whole body to ambient air enriched in PM2.5, which was concentrated in a chamber near an avenue of intense traffic in São Paulo City, Brazil. RESULTS: Mice exposed to concentrated ambient PM2.5 (1 h daily, 3 months) were compared to in situ ambient air exposed mice as the study control. The concentrated PM2.5 exposed group presented increased levels of the oxidized nucleoside 8-oxo-7,8-dihydro-2'-deoxyguanosine in lung and kidney DNA and increased levels of the etheno adducts 1,N6-etheno-2'-deoxyadenosine and 1,N2-etheno-2'-deoxyguanosine in kidney and liver DNA, respectively. Apart from the genotoxic effects, the exposure to PM2.5 led to decreased levels of the epigenetic mark 5-hydroxymethylcytosine (5-hmC) in lung and liver DNA. Changes in lung, liver, and erythrocyte antioxidant enzyme activities were also observed. Decreased glutathione reductase and increased superoxide dismutase (SOD) activities were observed in the lungs, while the liver presented increased glutathione S-transferase and decreased SOD activities. An increase in SOD activity was also observed in erythrocytes. These changes are consistent with the induction of local and systemic oxidative stress. CONCLUSIONS: Mice exposed daily to PM2.5 at a concentration that mimics 24-h exposure to the mean concentration found in ambient air presented, after 3 months, increased levels of DNA lesions related to the occurrence of oxidative stress in the lungs, liver, and kidney, in parallel to decreased global levels of 5-hmC in lung and liver DNA. Genetic and epigenetic alterations induced by pollutants may affect the genes committed to cell cycle control, apoptosis, and cell differentiation, increasing the chance of cancer development, which merits further investigation.

Longitudinal study of lung function in pregnant women: Influence of parity and smoking.

OBJECTIVES: To evaluate pulmonary function in the first and third trimesters of pregnancy and analyze the influence of parity and smoking on spirometry parameters. METHODS: This longitudinal prospective study included a cohort of 120 pregnant women. The inclusion criteria were as follows: singleton pregnancy, gestational age less than 13.86 weeks, and no preexisting maternal diseases. The exclusion criteria were as follows: change of address, abortion, and inadequate spirometry testing. ClinicalTrials.gov: NCT02807038. RESULTS: A decrease in values of forced vital capacity and forced expiratory volume were noted in the first second from the first to third trimester. In the first and third trimesters, multiparous women demonstrated lower absolute forced vital capacity and forced expiratory volume values in the first second compared with nulliparous women (p<0.0001 and p=0.001, respectively). Multiparous women demonstrated reduced forced expiratory flow in 25% to 75% of the maneuver compared with nulliparous women in the first (p=0.005) and third (p=0.031) trimesters. The absolute values of forced expiratory flow in 25% to 75%, forced expiratory volume in the first second and predicted peak expiratory flow values in the third trimester were higher in smokers compared with nonsmokers (p=0.042, p=0.039, p=0.024, and p=0.021, respectively). CONCLUSION: There was a significant reduction in forced vital capacity and forced expiratory volume values in the first second during pregnancy. Parity and smoking significantly influence spirometric variables.

Longitudinal study of lung function in pregnant women: Influence of parity and smoking

OBJECTIVES: To evaluate pulmonary function in the first and third trimesters of pregnancy and analyze the influence of parity and smoking on spirometry parameters. METHODS: This longitudinal prospective study included a cohort of 120 pregnant women. The inclusion criteria were as follows: singleton pregnancy, gestational age less than 13.86 weeks, and no preexisting maternal diseases. The exclusion criteria were as follows: change of address, abortion, and inadequate spirometry testing. ClinicalTrials.gov: NCT02807038. RESULTS: A decrease in values of forced vital capacity and forced expiratory volume were noted in the first second from the first to third trimester. In the first and third trimesters, multiparous women demonstrated lower absolute forced vital capacity and forced expiratory volume values in the first second compared with nulliparous women (p<0.0001 and p=0.001, respectively). Multiparous women demonstrated reduced forced expiratory flow in 25% to 75% of the maneuver compared with nulliparous women in the first (p=0.005) and third (p=0.031) trimesters. The absolute values of forced expiratory flow in 25% to 75%, forced expiratory volume in the first second and predicted peak expiratory flow values in the third trimester were higher in smokers compared with nonsmokers (p=0.042, p=0.039, p=0.024, and p=0.021, respectively). CONCLUSION: There was a significant reduction in forced vital capacity and forced expiratory volume values in the first second during pregnancy. Parity and smoking significantly influence spirometric variables.

Botulinum toxin type A on cutaneous flap viability in diabetic and tobacco-exposed rats.

PURPOSE: To investigate the effect of Botulinum toxin A (BoNTA) on skin flap viability in healthy, tobacco-exposed and diabetic rats. METHODS: Ninety male Wistar rats (250-300g) were randomly divided into six groups: control+saline (C1), control+BoNTA (C2), tobacco-exposed+saline (T1), tobacco-exposed+BoNTA (T2) diabetes+saline (D1) and diabetes+BoNTA (D2). A dorsal cutaneous flap (3×10cm) was performed. Survival area and total area of the flaps were measured. Lumen diameter, external arterial diameter and lumen/wall thickness ratio were recorded. RESULTS: Survival area increased in control group with BoNTA injection compared with control animals injected with saline (C2 x C1; 0.9±0.1 vs0.67±0.15, p= 0.001). A similar result was found in diabetes group injected with BontA (D2 x D1; 0.97±0.2 vs0.61±0.24, p=0.018). No difference was observed in skin flap viability in tobacco-exposed groups (T2 x T1; 0.74±0.24 vs 0.64±0.21, p=0.871). Lumen diameter (p= 0.004), external arterial diameter (p = 0.0046,) and lumen/wall thickness ratio (p= 0.003) were increased in diabetes+BoNTA-treated animals. This effect was not observed in control or in tobacco-exposed groups. CONCLUSIONS: Botulinum toxin A increased skin flap viability in control and diabetic rats on the seventh post-operative day. Increased lumen diameter, external arterial diameter, and lumen/wall thickness ratio were observed in the diabetes+BoNTA group. BoNTA had no effect in the tobacco-exposed group on the seventh postoperative day.

Botulinum toxin type A on cutaneous flap viability in diabetic and tobacco-exposed rats

PURPOSE:To investigate the effect of Botulinum toxin A (BoNTA) on skin flap viability in healthy, tobacco-exposed and diabetic rats.METHODS: Ninety male Wistar rats (250-300g) were randomly divided into six groups: control+saline (C1), control+BoNTA (C2), tobacco-exposed+saline (T1), tobacco-exposed+BoNTA (T2) diabetes+saline (D1) and diabetes+BoNTA (D2). A dorsal cutaneous flap (3×10cm) was performed. Survival area and total area of the flaps were measured. Lumen diameter, external arterial diameter and lumen/wall thickness ratio were recorded.RESULTS: Survival area increased in control group with BoNTA injection compared with control animals injected with saline (C2 x C1; 0.9±0.1 vs0.67±0.15, p= 0.001). A similar result was found in diabetes group injected with BontA (D2 x D1; 0.97±0.2 vs0.61±0.24, p=0.018). No difference was observed in skin flap viability in tobacco-exposed groups (T2 x T1; 0.74±0.24 vs 0.64±0.21, p=0.871). Lumen diameter (p= 0.004), external arterial diameter (p = 0.0046,) and lumen/wall thickness ratio (p= 0.003) were increased in diabetes+BoNTA-treated animals. This effect was not observed in control or in tobacco-exposed groups.CONCLUSIONS:Botulinum toxin A increased skin flap viability in control and diabetic rats on the seventh post-operative day. Increased lumen diameter, external arterial diameter, and lumen/wall thickness ratio were observed in the diabetes+BoNTA group. BoNTA had no effect in the tobacco-exposed group on the seventh postoperative day.

Los contaminantes atmosféricos urbanos son factores de riesgo significativos para el asma y la neumonía en niños: influencia del lugar de medición de los contaminantes / Urban Air Pollutants are Significant Risk Factors for Asthma and Pneumonia in Children: The Influence of Location on the Measurement of Pollutants

Antecedentes: La contaminación atmosférica se asocia a una carga sustancial para la salud humana; sin embargo, los contaminantes de mayor importancia pueden variar según el lugar. Es necesaria una monitorización adecuada para determinar el efecto de dichos contaminantes sobre la salud respiratoria. Objetivos: Este estudio se diseñó para evaluar el papel de la exposición en el exterior, en el interior y personal a los contaminantes derivados de la combustión NO2 y O3 sobre la salud respiratoria de los niños en una zona urbana económicamente poco favorecida de São Paulo (Brasil). Métodos: Se determinaron de forma continua los niveles de NO2 y O3 en el aire del exterior, el interior, y también en la exposición personal, durante 30 días con el empleo de aparatos de medición pasiva. Se evaluó la salud respiratoria con una versión en brasileño del cuestionario ISAAC. Resultados: Se dispuso de datos completos de 64 niños de 6 a 10 años de edad. La morbilidad respiratoria fue alta, con 43 (67,2%) niños en los que había habido sibilancias en alguna ocasión, 27 (42,2%) con sibilancias en el mes anterior, 17 (26,6%) con asma en alguna ocasión y 21 (32,8%) con neumonía en alguna ocasión. Las correlaciones entre los niveles de NO2 y O3 determinados en las 3 localizaciones evaluadas fueron bajas. Los niveles de NO2 en el área de interior y la exposición personal a O3 mostraron una asociación independiente con el asma (p=0,02 en ambos casos), la neumonía (O3, p=0,02) y la presencia de sibilancias en alguna ocasión (p<0,01 en ambos casos). No se observaron asociaciones entre el NO2 y el O3 del exterior y la salud respiratoria(AU)

Conclusiones: La exposición a niveles más elevados de NO2 y O3 se asoció a un aumento del riesgo de asma y neumonía en los niños; sin embargo, el lugar en el que se determinan los contaminantes influye en los resultados. Las determinaciones realizadas en el interior y las determinaciones personales fueron las más exactas(AU)

Background: Air pollution is associated with a substantial burden on human health; however, the most important pollutants may vary with location. Proper monitoring is necessary to determine the effect of these pollutants on respiratory health. Objectives: This study was designed to evaluate the role of outdoor, indoor and personal exposure to combustion-related pollutants NO2 and O3 on respiratory health of children in a non-affluent urban area of São Paulo, Brazil. Methods: Levels of NO2 and O3 were continuously measured in outdoor and indoor air, as well as personal exposure, for 30 days using passive measurement monitors. Respiratory health was assessed with a Brazilian version of the ISAAC questionnaire. Results: Complete data were available from 64 children, aged 6-10 years. Respiratory morbidity was high, with 43 (67.2%) reporting having had wheezing at any time, 27 (42.2%) wheezing in the last month, 17 (26.6%) asthma at any time and 21 (32.8%) pneumonia at any time. Correlations between levels of NO2 and O3 measured in the three locations evaluated were poor. Levels of NO2 in indoor air and personal exposure to O3 were independently associated with asthma (both cases P=0.02), pneumonia (O3, P=0.02) and wheezing at any time (both cases P<0.01). No associations were seen between outdoor NO2 and O3 and respiratory health. Conclusions: Exposure to higher levels of NO2 and O3 was associated with increased risk for asthma and pneumonia in children. Nonetheless, the place where the pollutants are measured influences the results. The measurements taken in indoor and personal exposure were the most accurate(AU)

Urban air pollutants are significant risk factors for asthma and pneumonia in children: the influence of location on the measurement of pollutants.

BACKGROUND: Air pollution is associated with a substantial burden on human health; however, the most important pollutants may vary with location. Proper monitoring is necessary to determine the effect of these pollutants on respiratory health. OBJECTIVES: This study was designed to evaluate the role of outdoor, indoor and personal exposure to combustion-related pollutants NO(2) and O(3) on respiratory health of children in a non-affluent urban area of São Paulo, Brazil. METHODS: Levels of NO(2) and O(3) were continuously measured in outdoor and indoor air, as well as personal exposure, for 30 days using passive measurement monitors. Respiratory health was assessed with a Brazilian version of the ISAAC questionnaire. RESULTS: Complete data were available from 64 children, aged 6-10 years. Respiratory morbidity was high, with 43 (67.2%) reporting having had wheezing at any time, 27 (42.2%) wheezing in the last month, 17 (26.6%) asthma at any time and 21 (32.8%) pneumonia at any time. Correlations between levels of NO(2) and O(3) measured in the three locations evaluated were poor. Levels of NO(2) in indoor air and personal exposure to O(3) were independently associated with asthma (both cases P=.02), pneumonia (O(3), P=.02) and wheezing at any time (both cases P<.01). No associations were seen between outdoor NO(2) and O(3) and respiratory health. CONCLUSIONS: Exposure to higher levels of NO(2) and O(3) was associated with increased risk for asthma and pneumonia in children. Nonetheless, the place where the pollutants are measured influences the results. The measurements taken in indoor and personal exposure were the most accurate.

Endothelial dysfunction in the pulmonary artery induced by concentrated fine particulate matter exposure is associated with local but not systemic inflammation.

Clinical evidence has identified the pulmonary circulation as an important target of air pollution. It was previously demonstrated that in vitro exposure to fine particulate matter (aerodynamic diameter≤2.5 µm, PM2.5) induces endothelial dysfunction in isolated pulmonary arteries. We aimed to investigate the effects of in vivo exposure to urban concentrated PM2.5 on rat pulmonary artery reactivity and the mechanisms involved. For this, adult Wistar rats were exposed to 2 weeks of concentrated São Paulo city air PM2.5 at an accumulated daily dose of approximately 600 µg/m3. Pulmonary arteries isolated from PM2.5-exposed animals exhibited impaired endothelium-dependent relaxation to acetylcholine without significant changes in nitric oxide donor response compared to control rats. PM2.5 caused vascular oxidative stress and enhanced protein expression of Cu/Zn- and Mn-superoxide dismutase in the pulmonary artery. Protein expression of endothelial nitric oxide synthase (eNOS) was reduced, while tumor necrosis factor (TNF)-α was enhanced by PM2.5 inhalation in pulmonary artery. There was a significant positive correlation between eNOS expression and maximal relaxation response (Emax) to acetylcholine. A negative correlation was found between vascular TNF-α expression and Emax to acetylcholine. Plasma cytokine levels, blood cells count and coagulation parameters were similar between control and PM2.5-exposed rats. The present findings showed that in vivo daily exposure to concentrated urban PM2.5 could decrease endothelium-dependent relaxation and eNOS expression on pulmonary arteries associated with local high TNF-α level but not systemic pro-inflammatory factors. Taken together, the present results elucidate the mechanisms underlying the trigger of cardiopulmonary diseases induced by urban ambient levels of PM2.5.

Cardiac and pulmonary oxidative stress in rats exposed to realistic emissions of source aerosols.

In vivo chemiluminescence (CL) is a measure of reactive oxygen species in tissues. CL was used to assess pulmonary and cardiac responses to inhaled aerosols derived from aged emissions of three coal-fired power plants in the USA. Sprague-Dawley rats were exposed to either filtered air or: (1) primary emissions (P); (2) ozone oxidized emissions (PO); (3) oxidized emissions + secondary organic aerosol (SOA) (POS); (4) neutralized oxidized emissions + SOA (PONS); and (5) control scenarios: oxidized emissions + SOA in the absence of primary particles (OS), oxidized emissions alone (O), and SOA alone (S). Immediately after 6 hours of exposure, CL in the lung and heart was measured. Tissues were also assayed for thiobarbituric acid reactive substances (TBARS). Exposure to P or PO aerosols led to no changes compared to filtered air in lung or heart CL at any individual plant or when all data were combined. POS caused significant increases in lung CL and TBARS at only one plant, and not in combined data from all plants; PONS resulted in increased lung CL only when data from all plants were combined. Heart CL was also significantly increased with exposure to POS only when data from all plants were combined. PONS increased heart CL significantly in one plant with TBARS accumulation, but not in combined data. Exposure to O, OS, and S had no CL effects. Univariate analyses of individual measured components of the exposure atmospheres did not identify any component associated with increased CL. These data suggest that coal-fired power plant emissions combined with other atmospheric constituents produce limited pulmonary and cardiac oxidative stress.

Urban, traffic- related particles and lung tumors in urethane treated mice.

OBJECTIVE: The present study was designed to evaluate the effects of urban, traffic-related, fine particulate matter (PM2.5) on mice lung tumorigenesis under controlled exposure conditions. METHODS: Four groups of female Swiss mice were treated with intraperitonial injections of urethane and saline solution. Urethane was used to start the carcinogenesis process. The animals were housed in two chambers receiving filtered and polluted air. In the polluted air chamber, pollutant levels were low. After two months of exposure, the animals were euthanized and lung tumoral nodules were counted. RESULTS: Saline-treated animals showed no nodules. Urethane-treated animals showed 2.0+2.0 and 4.0+3.0 nodules respectively, in the filtered and non-filtered chambers (p = 0.02), thus showing experimental evidence of increased carcinogenic-induced lung cancer with increasing PM2.5 exposure. CONCLUSION: Our data support the concept that low levels of PM2.5 may increase the risk of developing lung tumors.

Toxicological evaluation of realistic emission source aerosols (TERESA)--power plant studies: assessment of breathing pattern.

Our approach to study multi-pollutant aerosols isolates a single emissions source, evaluates the toxicity of primary and secondary particles derived from this source, and simulates chemical reactions that occur in the atmosphere after emission. Three U.S. coal-fired power plants utilizing different coals and with different emission controls were evaluated. Secondary organic aerosol (SOA) derived from α-pinene and/or ammonia was added in some experiments. Male Sprague-Dawley rats were exposed for 6 h to filtered air or different atmospheric mixtures. Scenarios studied at each plant included the following: primary particles (P); secondary (oxidized) particles (PO); oxidized particles + SOA (POS); and oxidized and neutralized particles + SOA (PONS); additional control scenarios were also studied. Continuous respiratory data were obtained during exposures using whole body plethysmography chambers. Of the 12 respiratory outcomes assessed, each had statistically significant changes at some plant and with some of the 4 scenarios. The most robust outcomes were found with exposure to the PO scenario (increased respiratory frequency with decreases in inspiratory and expiratory time); and the PONS scenario (decreased peak expiratory flow and expiratory flow at 50%). PONS findings were most strongly associated with ammonium, neutralized sulfate, and elemental carbon (EC) in univariate analyses, but only with EC in multivariate analyses. Control scenario O (oxidized without primary particles) had similar changes to PO. Adjusted R(2) analyses showed that scenario was a better predictor of respiratory responses than individual components, suggesting that the complex atmospheric mixture was responsible for respiratory effects.

Toxicological evaluation of realistic emission source aerosols (TERESA)-power plant studies: assessment of cellular responses.

The Toxicological Evaluation of Realistic Emission Source Aerosols (TERESA) project assessed primary and secondary particulate by simulating the chemical reactions that a plume from a source might undergo during atmospheric transport and added other atmospheric constituents that might interact with it. Three coal-fired power plants with different coal and different emission controls were used. Male Sprague-Dawley rats were exposed for 6 h to either filtered air or aged aerosol from the power plant. Four exposure scenarios were studied: primary particles (P); primary + secondary (oxidized) particles (PO); primary + secondary (oxidized) particles + SOA (POS); and primary + secondary (oxidized) particles neutralized + SOA (PONS). Exposure concentrations varied by scenario to a maximum concentration of 257.1 ± 10.0 µg/m(3). Twenty-four hours after exposure, pulmonary cellular responses were assessed by bronchoalveolar lavage (BAL), complete blood count (CBC), and histopathology. Exposure to the PONS and POS scenarios produced significant increases in BAL total cells and macrophage numbers at two plants. The PONS and P scenarios were associated with significant increases in BAL neutrophils and the presence of occasional neutrophils and increased macrophages in the airways and alveoli of exposed animals. Univariate analyses and random forest analyses showed that increases in total cell count and macrophage cell count were significantly associated with neutralized sulfate and several correlated measurements. Increases in neutrophils in BAL were associated with zinc. There were no significant differences in CBC parameters or blood vessel wall thickness by histopathology. The association between neutrophils increases and zinc raises the possibility that metals play a role in this response.

Urban, traffic- related particles and lung tumors in urethane treated mice

OBJECTIVE: The present study was designed to evaluate the effects of urban, traffic-related, fine particulate matter (PM2.5) on mice lung tumorigenesis under controlled exposure conditions. METHODS: Four groups of female Swiss mice were treated with intraperitonial injections of urethane and saline solution. Urethane was used to start the carcinogenesis process. The animals were housed in two chambers receiving filtered and polluted air. In the polluted air chamber, pollutant levels were low. After two months of exposure, the animals were euthanized and lung tumoral nodules were counted. RESULTS: Saline-treated animals showed no nodules. Urethane-treated animals showed 2.0+2.0 and 4.0+3.0 nodules respectively, in the filtered and non-filtered chambers (p = 0.02), thus showing experimental evidence of increased carcinogenic-induced lung cancer with increasing PM2.5 exposure. CONCLUSION: Our data support the concept that low levels of PM2.5 may increase the risk of developing lung tumors.

Composition of diesel particles influences acute pulmonary toxicity: an experimental study in mice.

Ambient particles have been consistently associated with adverse health effects, yielding mainly high cardiorespiratory morbidity and mortality. Diesel engines represent a major source of particles in the urban scenario. We aimed to modify the composition of diesel particles, by means of different extraction procedures, to relate changes in chemical profile to corresponding indicators of respiratory toxicity. Male BALB/c mice were nasally instilled with saline, or with diesel particles, treated or not, and assigned to five groups: saline (SHAM), intact diesel particles (DEP), and diesel particles previously treated with methanol (METH), hexane (HEX), or nitric acid (NA). Elemental composition and organic compounds were analyzed. Twenty-four hours after nasal instillation, respiratory parameters were measured and lung tissue was collected for histological analysis. Static elastance was significantly increased in groups DEP and MET in relation to the other groups. HEX and NA were different from DEP but not significantly different from SHAM and METH groups. The difference between dynamic and static elastance was increased in DEP, METH, and NA treatments; HEX was not statistically different from SHAM. DEP and METH groups presented significantly increased upper airways resistance, while DEP, METH, and NA showed higher peripheral airways resistance values. All groups had a higher total resistance than SHAM. DEP, METH, and NA showed significant increased infiltration of polymorphonuclear cells. In conclusion, diesel particles treated with hexane (HEX) resulted in a respiratory-system profile very similar to that in SHAM group, indicating that hexane treatment attenuates pulmonary inflammation elicited by diesel particles.

Homozygous deletion of p16INK4a and tobacco carcinogen exposure in nonsmall cell lung cancer.

Inactivation of p16(INK4a) in the Rb pathway is among the most common somatic alterations observed in nonsmall cell lung cancers (NSCLCs). While epigenetic inactivation of the p16(INK4a) gene promoter has been shown to be associated with increased tobacco carcinogen exposure, little investigation of any similar association of homozygous deletion or mutation of p16(INK4a) and tobacco use has been completed. In 177 consecutive NSCLCs, we examined the determinants of p16(INK4a) homozygous deletion and mutation, including the pattern of tobacco smoking and asbestos exposure. We observed that p16(INK4a) homozygous deletion occurred at a higher frequency in never smokers as compared to former and current smokers (p = 0.01). This observation suggested that tumors from these patients might be more prone to DNA deletion events; consistent with this, epigenetic silencing of the DNA double-strand break repair genes FancF and BRCA1 was also associated with homozygous deletion of p16(INK4a)(p = 0.002 and p = 0.06, respectively). Finally, mutation of p16(INK4a) was rare and only occurred in patients who were smokers. Hence, the character of somatic alteration in the Rb pathway (deletion, mutation or methylation silencing) in NSCLC is associated with the pattern of tobacco exposure, suggesting that susceptibility to lung cancer is, at least in part, mediated by the biological mechanism that selects for the character of the induced somatic lesion.

Chronic exposure to urban air pollution induces structural alterations in murine pulmonary and coronary arteries.

Epidemiological studies have shown that air pollution increases cardiovascular morbidity and mortality. Objective markers of cardiovascular risk have also been associated with increases in ambient pollution. This study was designed to assess whether prolonged exposure to ambient levels of air pollution may induce structural alterations of pulmonary and cardiac vessels. Mice were chronically exposed to ambient levels of air pollution in downtown São Paulo, Brazil. The animals were maintained in exposure chambers, 24 h/day, 7 days/wk, during 4 mo. One group was exposed to ambient air, and the control group was exposed to filtered air. Morphometric measurements of the ratio between the lumen and wall (L/W) areas were performed on transverse sections of renal, pulmonary, and coronary arteries. As expected, lumen/wall ratios increased with arterial caliber (p < .001). A significant decrease of L/W with exposure to air pollution was detected in pulmonary (p = .03) and coronary (p = .021) arteries, whereas no effects of air pollution were observed in renal vessels. Our results indicate that animals chronically exposed to ambient air pollution develop a significant thickening of the arterial wall in the coronary and pulmonary circulation.

Distribution of collagen in the lamina propria of the human vocal fold.

OBJECTIVES: To describe the arrangement of collagen fibers in the lamina propria of the human vocal fold. STUDY DESIGN: Analysis of the lamina propria of the vocal fold obtained from human cadavers. METHODS: The Picrosirius-polarization method was used to visualize collagen fibers. RESULTS: Analysis of 20 human vocal folds by the Picrosirius-polarization method permitted the visualization of two fiber populations arranged in three layers in the lamina propria: two layers of thick, strongly birefringent collagen fibers (collagen type I), one immediately below the epithelium and another more dense layer in the deep region superficially to the vocal muscle, penetrating between muscle fibers. The third layer consisted of fine, weakly birefringent fibers (collagen type III) located between the two layers of thick fibers. In addition, the collagen fibers in the lamina propria showed an intertwined network arrangement in the form of a "wicker basket." CONCLUSIONS: This basket-like configuration better explains how the vocal fold is able to stretch even though it contains nonstretchable fibers and to modulate the frequency of the voice under the action of the intrinsic musculature of the larynx. Segmental areas of disarray of the basket-like structure of the collagen layers were systematically observed in older patients. Thus, it is possible that vocal alterations occurring in the elderly might be the result of a loss of histoarchitectural arrangement of the collagen system and its relationship with the lamina propria and underlying musculature.

Pé plano adquirido do adulto por disfunçäo do tendäo do tibial posterior: avaliaçäo clínica, imagenológica e morfométrica / Acquired flatfood in adults due to posterio tibial tendon dysfunction: clinical, morphometric, and magnetic resonance image evaluations

Com o intuito de identificar as alteraçoes histológicas características do pé plano adquirido do adulto por disfunçao do tendao do tibial posterior e estabelecer sua correlaçao com parâmetros clínicos, radiológicos e de ressonância magnética, foram estudados 19 pacientes (19 pés) - 17 do sexo feminino e 2 do masculino, com idades de 27 a 68 anos (média de 52,9 anos). Dos pacientes incluídos nesta amostra, 7 (36,8 por cento) apresentavam artrite reumatóide, 7 (36,8 por cento) hipertensao arterial, 3 (15,8 por cento) obesidade, 1 (5,3 por cento) lúpus eritematoso sistêmico e 1 (5,3 por cento) tinha seqüela de fratura de tornozelo. Após a avaliaçao clínica e imagenológica, foram obtidos fragmentos dos tendoes acometidos, os quais foram comparados, através de técnicas morfométricas capazes de mensurar o grau de organizaçao tecidual, com tendoes normais obtidos de doadores e cadáveres. Os parâmetros clínicos (podoscopia, teste das pontas dos pés, força do músculo tibial posterior, sinal da lateralizaçao dos dedos e o grau de supinaçao do antepé) exibiram correlaçao estatística com os demais achados e constituem importante recurso no diagnóstico e classificaçao dessa patologia. Dentre os parâmetros radiológicos destacam-se os ângulos talo-I metatársico e congruência articular talonavicular nesse mister. Embora seja de grande utilidade na localizaçao e visibilizaçao das lesoes do tendao do tibial posterior, ficou demonstrado o caráter opcional da ressonância magnética no diagnóstico e classificaçao dessa enfermidade. A análise discriminante realizada entre os dados morfométricos e a classificaçao geral demonstraram a validade e utilidade dos parâmetros sugeridos para a determinaçao do estadiamento do pé plano adquirido do adulto por disfunçao do tendao do tibial posterior.

Efeitos do uso tópico da mitomicina C no epitélio corneano de coelhas: análise histopatológica pela morfometria / Effects of topical mitomicyn eye drops on corneal epithelium in the rabbit: a histhopathogical morphometric study

Este estudo foi realizado com o objetivo de avaliar a influência do uso tópico da mitomicina C, a 0,4 mgml, 0,2 mgml e 0,8 mgml, no epitélio corneano, com investigaçäo feita em uma populaçäo de coelhas. A mitomicina C e a água destilada (controle) foram instiladas duas vezes ao dia, por 4 dias, em olhos com superfície córneo-conjuntival íntegra e em olhos com defeito epitelial corneano central, com diâmetro de 7,75 mm. O epitélio corneano, de ambos os olhos, foi avaliado com exame histopatológico, complementado por análise morfométrica. A análise realizada pelo método do point couting, sob microscopia de luz, avaliou nas regiöes límbica, intermediária e central do epitélio corneano, a área do epitélio/ o número de núcleos/ a relaçäo núcleo-citoplasma e a área da célula epitelial. Nos olhos com superfície ocular íntegra, ocorreram variaçöes dos parâmetros morfométricos avaliados, notadamente na regiäo límbica. Nos olhos com defeito epitelial, as alteraçöes morfométricas mostraram variaçöes caracterizadas por alteraçäo da área epitelial (aumento no limbo e diminuiçäo nas regiöes intermediária e central), hipertrofia da célula epitelial, reduçäo da relaçäo núcleo-citoplasma e diminuiçäo do número de núcleos