Article

Assessment of DNA damage by comet assay and fast halo assay in buccal epithelial cells of Indian women chronically exposed to biomass smoke

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Abstract

Genotoxicity of indoor air pollution from biomass burning was evaluated in buccal epithelial cells (BECs) of 85 pre-menopausal Indian women who were engaged in cooking with biomass (wood, dung, crop residues) and 76 age-matched control women who were cooking with cleaner fuel liquefied petroleum gas (LPG). DNA damage was evaluated by comet assay and fast halo assay (FHA). The concentrations of particulate matter with aerodynamic diameters of less than 10 and 2.5 μm (PM(10) and PM(2.5), respectively) in indoor air were measured by real-time aerosol monitor. Generation of reactive oxygen species (ROS) was measured by flow cytometry and the level of superoxide dismutase (SOD) by spectrophotometry. Compared with control, BEC of biomass users illustrated 2.6-times higher comet tail % DNA (32.2 vs. 12.4, p < 0.001), 2.7-times greater comet tail length (37.8 μm vs. 14.2 μm, p < 0.001) and 2.2-times more olive tail moment (7.1 vs. 3.2, p < 0.001), suggesting marked increase in DNA damage. FHA also showed 5-times more mean nuclear diffusion factor (9.2 vs. 1.8, p < 0.0001) in BEC of biomass users, confirming sharp rise in DNA single strand breaks. Airway cells of biomass-using women showed 51% rise in ROS generation but 28% reduction in SOD, suggesting oxidative stress in the airways. Indoor air of biomass-using households had 3-times more PM(10) and PM(2.5) than LPG-using families, and DNA damage showed positive association with PM(10) and PM(2.5) levels controlling education, kitchen location and family income as potential confounders. In summary, chronic inhalation of biomass smoke elicits oxidative stress and extensive DNA damage in BEC.

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... The brushes were stirred in 5 ml of PBS (pH 7.4). Similarly Szeto et al. (2005), Jayakumar et al. (Jayakumar and Sasikala, 2008), and Mondal et al. (2011) used soft bristle toothbrushes to collect buccal cells by scraping the inside of the cheeks after rinsing the mouth with distilled water. The toothbrushes were then agitated in 30 ml of cold PBS. ...
... Various studies generated suspensions of cells that were immersed in RPMI-1640 via centrifugation over a range of 1-10 min at 800-6000 rpm (Rojas et al., 1996;Jayakumar and Sasikala, 2008;Westphalen et al., 2008;Mondal et al., 2011;Sudha et al., 2011). Similarly, Szeto et al. obtained pellets and resuspended them in 100 μl of PBS. ...
... The majority of the studies that were conducted to determine DNA damage in buccal epithelial cells used the alkaline comet assay according to the procedure developed by Singh et al. (1988), with various modifications. Only the studies that employed the modifications outlined by Szeto et al. (Szeto et al., 2005) performed neutral comet assays (pH 9.1) (Jayakumar and Sasikala, 2008;Mondal et al., 2011;Pal et al., 2012) (Table 4). ...
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The comet assay is a valuable experimental tool aimed at mapping DNA damage in human cells in vivo for environmental and occupational monitoring, as well as for therapeutic purposes, such as storage prior to transplant, during tissue engineering, and in experimental ex vivo assays. Furthermore, due to its great versatility, the comet assay allows to explore the use of alternative cell types to assess DNA damage, such as epithelial cells. Epithelial cells, as specialized components of many organs, have the potential to serve as biomatrices that can be used to evaluate genotoxicity and may also serve as early effect biomarkers. Furthermore, 80% of solid cancers are of epithelial origin, which points to the importance of studying DNA damage in these tissues. Indeed, studies including comet assay in epithelial cells have either clear clinical applications (lens and corneal epithelial cells) or examine genotoxicity within human biomonitoring and in vitro studies. We here review improvements in determining DNA damage using the comet assay by employing lens, corneal, tear duct, buccal, and nasal epithelial cells. For some of these tissues invasive sampling procedures are needed. Desquamated epithelial cells must be obtained and dissociated prior to examination using the comet assay, and such procedures may induce varying amounts of DNA damage. Buccal epithelial cells require lysis enriched with proteinase K to obtain free nucleosomes. Over a 30 year period, the comet assay in epithelial cells has been little employed, however its use indicates that it could be an extraordinary tool not only for risk assessment, but also for diagnosis, prognosis of treatments and diseases.
... The brushes were stirred in 5 ml of PBS (pH 7.4). Similarly Szeto et al. (2005), Jayakumar et al. (Jayakumar and Sasikala, 2008), and Mondal et al. (2011) used soft bristle toothbrushes to collect buccal cells by scraping the inside of the cheeks after rinsing the mouth with distilled water. The toothbrushes were then agitated in 30 ml of cold PBS. ...
... Various studies generated suspensions of cells that were immersed in RPMI-1640 via centrifugation over a range of 1-10 min at 800-6000 rpm (Rojas et al., 1996;Jayakumar and Sasikala, 2008;Westphalen et al., 2008;Mondal et al., 2011;Sudha et al., 2011). Similarly, Szeto et al. obtained pellets and resuspended them in 100 μl of PBS. ...
... The majority of the studies that were conducted to determine DNA damage in buccal epithelial cells used the alkaline comet assay according to the procedure developed by Singh et al. (1988), with various modifications. Only the studies that employed the modifications outlined by Szeto et al. (Szeto et al., 2005) performed neutral comet assays (pH 9.1) (Jayakumar and Sasikala, 2008;Mondal et al., 2011;Pal et al., 2012) (Table 4). ...
Article
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Occupational exposure to xenobiotic mixtures is an actual concern to public health effects. The automobile battery recycling industry, contribute to lead and cadmium exposure as a mixture for worker in several countries (Palus et al., 2005). In the last decade, the use of comet assay in human biomonitoring has been a successful tool (Valverde and Rojas, 2009), that we apply for this three year follow-up biomonitoring study in a group of recycling battery workers in Guanajuato, Mexico (n=85) in terms of determine early biomarkers. Exposure and effect biomarkers of molecular epidemiology were determined, lead whole blood levels and ALA-D activity as exposure biomarkers meanwhile, DNA damage, DNA repair, and oxidative stress as effect biomarkers. Our results indicate that battery-recycling workers are exposed to high levels of lead in whole blood (56.95 ± 2.7 g/dL), almost five times higher to the permissive level (10 g/dL). Also, inhibition of ALA-D enzyme activity (164 ± 0.04 nmoles/h/ml) was significant, with respect to control group (704.23 ± 8.3 nmolas/h/ml). At respect to effect biomarkers, we determine high levels of oxidative stress analyzed by TBARS technique (1.31 MDA nmolas/ml) with respect to the control group (1.05 MDA nmolas/ml). The basal DNA damage found in the exposed group was about 47% higher than control group, meanwhile their DNA repair activity decreased in about 28% relative to control group. These findings may suggest that lead exposure into the battery recycling environment is a fact that exceeds the permissive lead exposure levels, reflected also in the inhibition of an important enzyme involved in hemo biosynthesis, ALA-D. The mechanism of toxicity involves generation of oxidative stress that affects lipids and DNA. DNA damage determined as single strand breaks, is not repaired efficiently, indicating low base excision repair capacity that may have a variety of health effects. In addition to these findings, DNA damage determined by comet assay was sensible to reflect lead exposure levels related to specific activities inside this factory. Human biomonitoring studies through comet assay could be robust when additional biomarkers are determined at time.
... Alkaline comet assay demonstrated extensive DNA damage in peripheral blood lymphocytes [206,208,209] and buccal epithelial cells [210] of women who cooked with biomass [208,206]; . Airway cells of biomass users displayed increased expression of gamma-H2X that suggests that the damage occurred in both strands of the DNA [206]. ...
... The DNA-damaging efficacy of biomass smoke particles was much more than the traffic-generated PM per unit mass, possibly due to the high level of polycyclic aromatic hydrocarbons in the former [211]. The genotoxicity of biomass smoke was thought to be mediated by generation of oxidative stress PM [206,210,209]. The direct involvement of biomass smoke in DNA and chromosomal damage becomes apparent from the report that improved kitchen ventilation and reduction of smoke was associated with decrement in the frequency and intensity of DNA damage [11]. ...
Chapter
Indoor air pollution (IAP) due to daily household cooking with unprocessed solid biomass such as wood, dung and crop residues is a serious health hazard in the poor, developing countries of Asia, sub-Saharan Africa and Latin America. Globally, 2.8 billion people use biomass for domestic energy. Incomplete combustion of biomass emits smoke that contains a host of potentially health-damaging particulate and gaseous pollutants, some of which like benzo(a)pyrene, 1,3-butadiene and benzene are known human carcinogens. IAP from biomass burning is responsible for excess mortality and morbidity. An estimated four million deaths, mostly from cardio-pulmonary causes, have been attributed to biomass use. Children, women and the elderly people are most vulnerable. Chronic inhalation of biomass smoke induces lung function decrement, increases the risk of life-threatening chronic obstructive pulmonary disease, evokes pulmonary and systemic inflammation and consequent oxidative stress, and contributes to the development of hypertension and cardio-vascular diseases. Daily household cooking with biomass was associated with higher incidences of anemia, platelet hyperactivity, and altered number and activities of the immune cells. Oxidative stress generated by biomass smoke mediates chromosomal and DNA damage and impairment in DNA repair mechanism in the exposed cells. In addition, chronic inhalation of biomass smoke up-regulates protein kinase B/Akt signaling and metaplasia and dysplasia of airway cells, implying increased risk of lung cancer. Women who cooked with biomass also had altered serotonergic activity with greater prevalence of depression. Thus, IAP due to household cooking with biomass adversely affects both physical and mental health of the people.
... Human Body over South Asia A few studies from India investigated the role of PM exposure on the health of biological tissues using a variety of markers listed in Table 1 (Das et al., 2021;Jan et al., 2020;Roy et al., 2015;Sambandam et al., 2015;Dutta et al., , 2012Banerjee et al., 2012;Mondal et al., 2011; Rahman et al., 1997;Arif et al., 1992). Here, Arif et al. (1992) and Rahman et al. (1997) investigated the toxic potential of various particles and fibers to the human and rat alveolar macrophages. ...
... They found that alveolar macrophages in humans produce more ROS compare to that in rats. A group from Chittaranjan National Cancer Institute investigated the role of PM in deoxyribonucleic acid (DNA) single-strand damage on pre-menopausal women who were engaged in cooking using bio-fuel/ bio-mass in rural areas of West Bengal, India (Mondal et al., 2011). They observed a sharp rise in DNA singlestrand breaks in Buccal Epithelial Cells (BECs), which was positively associated with the ROS generation and PM levels, indicating oxidative stress resulted from biomass smoke. ...
Article
Full-text available
South Asia occupies only about 3.5% of the world’s area but, about 25% of the average world’s population lives here and is continuously exposed to severe air pollution. Unprecedented development activities in most of the South Asian cities emit primary and secondary pollutants into the atmosphere. Particulate matter (PM), a principal air pollutant, are tiny enough to remain suspended in the atmosphere for a long time (about a week). They can penetrate the human nasal airway and damage the lungs. PM effects on human health are assessed based on their mass concentration, size distribution, and chemical composition. Despite being critically important, studies related to PM effects on human health are limited over South Asia. In recent years, only a few South Asian research groups started studying the ability of atmospheric PM to cause human health hazards by generating in situ reactive oxygen species (ROS). The capability of atmospheric PM to produce ROS and/or deplete antioxidants is termed as their oxidative potential (OP). Though limited, efforts are made to identify particular species with the higher OP. Atmospheric aging of PM can also alter their OP. No studies from South Asia, except a few from India, investigated how the atmospheric aging changes the chemical and physical properties of PM and affect their OP over South Asia. These studies also showed that OP depends more on PM composition rather than its concentrations. Therefore, mitigation strategies for reducing PM mass concentrations alone may not be sufficient, and linking PM OP with significant health effects may be a better way to regulate specific sources of PM rather than overall PM mass. This review reports the necessities and limitations for PM OP studies in South Asia and future directions.
... They stated that the high concentration of smoke from cookstoves inside the kitchen created non-negligible exposures of households to dioxins, though; the fraction of dioxins in wood smoke emissions was very less as compared to the fraction present in other sources. Mondal et al. [102] also investigated the possibility of DNA damage in buccal epithelial cells in a group of premenopausal women from Eastern India, who were chronically exposed to biomass smoke during cooking of food. This cooking was also done for different age groups from the same locality, who were cooking with the cleaner fuel like LPG. ...
... Very recently, number of authors [103][104][105][106][107][108][109][110][111][112][113][114][115][116][117] carried out detailed studies about the traditional and improved cookstove using various approaches and presented some new results and strategies for the design, development and dissemination of biomass cookstoves [102][103][104][105][106][107], while addressing important issues, such as, environment, health and deforestation [108][109][110][111][112][113][114][115][116][117]. For example, Tyagi et al. [108] carried out the comparative analysis of four different types of cookstove models using exergetic and energetic approaches. ...
Article
The use of biomass resources for cooking and heating is as old as the origin of human civilization due to the fact that biomass is available almost everywhere and can be burnt directly. Biomass accounts for a large fraction of the domestic energy needs in the developing countries. However, very often biomass is burnt inefficiently in open three-stone fire and traditional cookstoves for cooking and heating applications which causes severe health problems in women and children and also affects the environment. Many efforts have been made worldwide to increase the dissemination of improved cookstove but have not succeeded in their targets. The new cookstove dissemination programs can be funded through carbon revenue and other funding organizations; further these funds can be utilized for further R&D and cookstove market. The successful cookstove dissemination programs can lead to the sustainable development of the rural areas besides helping in the commercialization of cookstove. Therefore, this article presents the review on the design, development, and technological advancement of biomass cookstoves and the effects of traditional biomass burning devices on the emission, health hazard, and environmental pollution.
... Existen múltiples evidencias de que la exposición a humo de biomasa promueve un estado inflamatorio en el pulmón. Las mujeres expuestas a humo de biomasa presentan un mayor número de neutrófilos, eosinófilos, monocitos, mastocitos, linfocitos y macrófagos alveolares, así como niveles superiores de interleucina (IL)-6, IL-8 y factor de necrosis tumoral (TNF)-␣ en esputo comparadas con aquellas que usan otro tipo de combustible [62][63][64] . La expresión génica de algunos de estos mediadores pro-inflamatorios, como la IL-8, el TNF-␣, la metaloproteinasa de matriz (MMP)-9 y la MMP-12, aumenta al incrementar la exposición al humo de biomasa, como se ha demostrado en un estudio reciente de Guarnieri et al. 65 , mientras que otro estudio muestra que este contaminante altera la expresión de genes asociados a la activación de los linfocitos T CD8 +66 . ...
... Otros estudios han reportado un aumento de productos de peroxidación lipídica como el malondialdehído (MDA) en sangre de sujetos expuestos a humo de biomasa [85][86][87][88] , y una correlación entre los niveles de este compuesto y una disminución de la VEF 1 /CVF 83 . También se ha descrito un incremento de marcadores de lesiones oxidativas en ADN a partir de muestras de esputo 89 y leucocitos mononucleares de sangre periférica (PBMC) 87,90 de sujetos expuestos crónicamente a humo de biomasa, así como una reducción de la capacidad de reparar estas lesiones en dichos sujetos 85,90,62 . Diversos trabajos realizados con modelos animales [91][92][93][94] o células en cultivo 89,[95][96][97][98][99][100][101][102][103][104] respaldan estos resultados, al encontrarse un aumento de la producción de ERO, citoquinas, productos de peroxidación lipídica y lesiones oxidativas en ADN o un decremento de los mecanismos antioxidantes con la exposición a humo de biomasa. ...
Article
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Las tasas de mortalidad y morbilidad de la enfermedad pulmonar obstructiva crónica (EPOC) han aumentado mundialmente de forma significativa durante las últimas décadas. A pesar de que el humo de tabaco se sigue considerando el principal factor etiopatogénico para el desarrollo de la enfermedad, se estima que entre una tercera y una cuarta parte de los pacientes con EPOC son no fumadores. De todos los factores de riesgo que pueden incrementar la probabilidad de sufrir EPOC en estos sujetos se ha propuesto al humo de biomasa como uno de los más importantes, afectando sobre todo a mujeres y a niños de países emergentes.
... Women exposed to biomass smoke have higher alveolar levels of neutrophils, eosinophils, monocytes, mastocytes, lymphocytes and macrophages, as well as higher sputum levels of interleukin (IL)-6, IL-8 and tumor necrosis factor (TNF)-␣ than those who use other types of fuel. [62][63][64] Gene expression of some of these pro-inflammatory mediators, such as IL-8, TNF-␣, matrix metalloproteinase (MMP)-9 and MMP-12, increases in parallel with exposure to biomass smoke, as recently demonstrated by Guarnieri et al., 65 while another study shows that this pollutant alters the expression of genes associated with CD8 + T-cell activation. 66 The pro-inflammatory effects of biomass smoke are not restricted to the lung, since increases have been reported in CD8 + T-cells, natural killer (NK) cells, IL-6, IL-8, TNF-␣, C-reactive protein (CRP) and monocyte chemoattractant protein (MCP)-1 in the blood of exposed individuals. ...
... Diminished capacity to repair cell damage has also been reported in these subjects. 62,85,90 These results are supported by several studies in animal models [91][92][93][94] or in vitro cell lines 89,95-104 that found increased production of ROS, cytokines, lipid peroxidation products and oxidative DNA damage or impaired antioxidant mechanisms after exposure to biomass smoke. Oxidative stress, then, appears have a significant role in activating the harmful effects of this pollutant. ...
Article
Chronic obstructive pulmonary disease (COPD) mortality and morbidity have increased significantly worldwide in recent decades. Although cigarette smoke is still considered the main risk factor for the development of the disease, estimates suggest that between 25% and 33% of COPD patients are non-smokers. Among the factors that may increase the risk of developing COPD, biomass smoke has been proposed as one of the most important, affecting especially women and children in developing countries.Despite the epidemiological evidence linking exposure to biomass smoke with adverse health effects, the specific cellular and molecular mechanisms by which this pollutant can be harmful for the respiratory and cardiovascular systems remain unclear. In this article we review the main pathogenic mechanisms proposed to date that make biomass smoke one of the major risk factors for COPD.
... The comet assay in buccal cells has been used to evaluate DNA damage induced by different materials such as mouthrinses [50], metals released from orthodontic appliances [51][52][53][54][55][56][57][58][59], ionizing radiation [60], as well as assessment of DNA damage, and its modulation by life-style, dietary, genetic and healthy factors [61][62][63][64][65][66][67][68][69][70][71][72][73][74], occupational exposure [66][67][68][69][75][76][77][78][79][80][81][82], and environmental exposure [83][84][85][86]. Different procedures have been used in collecting and processing the samples that are presented and discussed in Rojas et al. [33]. ...
... DNA damage was assessed on buccal epithelial cells (BEC) by comet assay and fast halo assay (FHA). Compared with control, BEC of biomass users showed higher comet tail % DNA, higher values for comet tail length, and olive tail moment, suggesting marked increase in DNA damage [84]. ...
... priate response of DNA damage repair proteins in the face of increased DNA damage [38, 39], overactivation of signal transduction pathway involving Akt, i.e., protein kinase B [40], and hypertension with elevated levels of oxidized low-density lipoprotein indicating cardiovascular disease [41]. These findings along with observations from other studies led us to hypothesize that chronic exposure to IAP from biomass burning could be associated with neutrophilic inflammatory responses and oxidative stress in premenopausal women of rural India who cook regularly with BMF. ...
Article
Full-text available
The possibility of inflammation and neutrophil activation in response to indoor air pollution (IAP) from biomass fuel use has been investigated. For this, 142 premenopausal, never-smoking women (median age, 34 years) who cook exclusively with biomass (wood, dung, crop wastes) and 126 age-matched control women who cook with cleaner fuel liquefied petroleum gas (LPG) were enrolled. The neutrophil count in blood and sputum was significantly higher (p < 0.05) in biomass users than the control group. Flow cytometric analysis revealed marked increase in the surface expression of CD35 (complement receptor-1), CD16 (F(C)γ receptor III), and β(2) Mac-1 integrin (CD11b/CD18) on circulating neutrophils of biomass users. Besides, enzyme-linked immunosorbent assay showed that they had 72%, 67%, and 54% higher plasma levels of the proinflammatory cytokines tumor necrosis factor-alpha, interleukin-6, and interleukin-12, respectively, and doubled neutrophil chemoattractant interleukin-8. Immunocytochemical study revealed significantly higher percentage of airway neutrophils expressing inducible nitric oxide synthase, while the serum level of nitric oxide was doubled in women who cooked with biomass. Spectrophotometric analysis documented higher myeloperoxidase activity in circulating neutrophils of biomass users, suggesting neutrophil activation. Flow cytometry showed excess generation of reactive oxygen species (ROS) by leukocytes of biomass-using women, whereas their erythrocytes contained a depleted level of antioxidant enzyme superoxide dismutase (SOD). Indoor air of biomass-using households had two to four times more particulate matter with diameters of <10 μm (PM(10)) and <2.5 μm (PM(2.5)) as measured by real-time laser photometer. After controlling potential confounders, rise in proinflammatory mediators among biomass users were positively associated with PM(10) and PM(2.5) in indoor air, suggesting a close relationship between IAP and neutrophil activation. Besides, the levels of neutrophil activation and inflammation markers were positively associated with generation of ROS and negatively with SOD, indicating a role of oxidative stress in mediating neutrophilic inflammatory response following chronic inhalation of biomass smoke.
... We have previously demonstrated that chronic biomass exposures induce oxidative stress (Dutta et al., 2011), chromosomal and DNA damage (Mondal et al., 2010(Mondal et al., , 2011a, inappropriate repair of damaged DNA (Mondal et al., 2010) and elevated ribosome biogenesis (Mondal et al., 2009(Mondal et al., , 2011b in airway cells of rural women in India. These findings may suggest higher risk of cancer in the airways and the lung of biomass users. ...
Article
Full-text available
The impact of indoor air pollution (IAP) from biomass fuel burning on the risk of carcinogenesis in the airways has been investigated in 187 pre-menopausal women (median age 34years) from eastern India who cooked exclusively with biomass and 155 age-matched control women from same locality who cooked with cleaner fuel liquefied petroleum gas. Compared with control, Papanicolau-stained sputum samples showed 3-times higher prevalence of metaplasia and 7-times higher prevalence of dysplasia in airway epithelial cell (AEC) of biomass users. Immunocytochemistry showed up-regulation of phosphorylated Akt (p-Akt(ser473) and p-Akt(thr308)) proteins in AEC of biomass users, especially in metaplastic and dysplastic cells. Compared with LPG users, biomass-using women showed marked rise in reactive oxygen species (ROS) generation and depletion of antioxidant enzyme, superoxide dismutase (SOD) indicating oxidative stress. There were 2-5 times more particulate pollutants (PM(10) and PM(2.5)), 72% more nitrogen dioxide and 4-times more particulate-laden benzo(a)pyrene, but no change in sulfur dioxide in indoor air of biomass-using households, and high performance liquid chromatography estimated 6-fold rise in the concentration of benzene metabolite trans,trans-muconic acid (t,t-MA) in urine of biomass users. Metaplasia and dysplasia, p-Akt expression and ROS generation were positively associated with PM and t,t-MA levels. It appears that cumulative exposure to biomass smoke increases the risk of lung carcinogenesis via oxidative stress-mediated activation of Akt signal transduction pathway.
... Valverde and Rojas [41] Increased DNA damage was found in human populations exposed to air pollution, radiation, and pesticides. Studies of air pollution focused on polycyclic aromatic hydrocarbons (PAH), ozone, benzene, heavy metals, (ultra)fine particulate matter (PM), and passive smoking, including the effects of seasonal variations in sunlight, temperature and ozone [41][42][43][44][45][46][47]. Some studies used the comet assay to evaluate the impact of living in the near of a waste incinerator, waste disposal or oil refinery [41,46,[48][49][50]. ...
... There could be even more critical health problems from exposure to biomass smoke. Mondal et al. (2011) had reported that chronic inhalation of biomass smoke elicits oxidative stress and extensive DNA damage in buccal epithelial cells (BECs). ...
Article
Full-text available
Biomass fuel smoke particles (BFSPs) of rural kitchens collected during dry and wet seasons were characterized for elements, anions and carbon. The BFSPs of kitchens using varied biomass fuel types viz. cow dung stick, mixed biomass, cow-dung stick-mixed biomass and sugarcane bagasse were chosen for the study. The BFSPs from cow dung fuel stick showed higher levels of elements, anions and particulate carbon than other BFSPs. Calcium, K, Fe and Mg were the major elements found in all BFSPs, which did not vary much between the seasons. Sulphate was found to be the dominant anion present in all BFSPs followed by Clˉ and PO43−. Seasonal variation was pronounced in the case of abundance of anions and particulate carbon. The ratio OC/EC, often used as source signature of biomass burning, was found to be within 1.89–7.41 and 1.72–6.19 during dry and wet seasons respectively.
... Exposure to this high pollution load from burning biomass fuels is regarded as one of the most important environmental and public health problems in developing countries especially in women who cook with these fuels and in young children who attend to the fires or stay close to their mothers during cooking. It has been estimated that IAP from biomass use in developing countries is responsible for 4-5 percent of global burden of disease for both deaths and DALYs (disability adjusted lost life years) from acute respiratory infections (ARI), chronic obstructive pulmonary disease (COPD), tuberculosis, asthma, genetic damage [13,14] lung cancer [15], ischemic heart disease and blindness. India registers over 600,000 premature deaths per year due to biomass fuel combustion. ...
... Until now, many methods were developed to measure the level of DNA damages, as comet assay [13], repair assisted damage detection [21], Southern blot analysis [22], high-performance liquid chromatography (HPLC) [23], halo assay [24], and flow and imagecytometry [25]. Nevertheless, the sensitivity of the commonly used and conventional assays is low and measures the DNA damage in a global and sequence-independent way. ...
Article
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The present study aimed to adapt a Long-run Real-time DNA Damage Quantification (LORD-Q) qPCR-based method for the analysis of the mitochondrial genome of Common carp (Cyprinus carpio L.) and detect the DNA damaging effect of T-2 (4.11 mg kg−1) and deoxynivalenol (5.96 mg kg−1) mycotoxins in a 3-week feeding period. One-year-old Common carp were treated in groups (control, T-2 and DON). The mycotoxins were sprayed over the complete pelleted feed, and samples were taken weekly. Following the adaptation of LORD-Q PCR method for the Common carp species, the number of lesions were calculated to determine the amount of DNA damage. In the first and second weeks, the T-2 and the DON treated groups differed significantly from each other; however these differences disappeared in the third week. There was a significant difference in the DNA lesion values between weeks 1 and 3 in the deoxynivalenol-contaminated groups. While in the T-2 treated groups, the DNA lesion values were significantly reduced on weeks 2 and 3 compared to week 1. The results suggested that the trichothecene mycotoxins have a relevant DNA damaging effect.
... Mo˝liwoÊç izolacji DNA z komórek nab∏onkowych pozwa∏a na wykorzystanie tego materia∏u do ró˝nych analiz [63][64][65][66]. Nab∏onki z jamy ustnej zosta∏y wykorzystane do badania p´kni´ç DNA w teÊcie kometowym [11,18,67,68], uszkodzeƒ oksydacyjnych [17], polimorfizmiu pojedynczych nukleotydów (SNP, ang. Single Nucleotide Polymorphism) [69] oraz ekspresji genów [70]. ...
Article
Full-text available
One of the basic methods for determining the degree of environmental risk posed to humans is identification of harmful substances in various environmental elements (air, water, soil, food). In contrast to environmental monitoring human biological monitoring (HBM) enables the estimation of an absorbed dose, general or localized in a specific organ. HBM enables the assessment of exposure to substances which are absorbed by the body via different exposure pathways and with different contaminant carriers. It is based on the measurement of indicators, the so-called biomarkers, in body fluids (blood, urine, saliva, etc.) or in tissues and organs. Biomarkers can be divided into markers of exposure, effects and susceptibility. A particularly useful method is determination of adducts, i.e. carcinogenic compounds (or their metabolites) with proteins or DNA, which are markers of exposure. Biomarkers of biological effects are different cytogenetic changes, including micronuclei. These are extranuclear structures containing fragments of chromatin (arising as a result of DNA breaks) or whole chromosomes (damage to the spindle apparatus during mitosis). Up to now most studies on the DNA adduct levels and micronuclei have been conducted in peripheral lymphocytes. At present, studies using blood, especially in children is restricted of ethical aspects, and therefore tests using epithelial cells from the oral cavity have become more popular. Epithelial cells are the main building material of an epithelial tissue which makes up about 60% of all cells of the human body. The main function of the epithelial tissue is covering and lining of the outer and inner surfaces of the body. Epithelium underwent high specialisation in various parts of the human body, which is associated with its structure and function. Human oral cavity is covered by stratified squamous epithelium, which is comprised of cells called keratinocytes. Oral epithelial cells may differentiate in two directions: towards keratinized or nonkeratinized oral epithelia. In this study, based on our past experience and the available literature, research procedures for the collection of oral epithelial cells and their proper preparation for using them both for the analysis of DNA adducts and micronucleus assay are presented.
... Thus, increased pulmonary numbers of macrophages, neutrophils, eosinophils, mast cells and lymphocytes, as well as higher sputum levels of IL-6 and TNF-a, were reported in women exposed to biomass smoke when compared with those who used other types of fuel. [44][45][46] Banerjee et al. also reported an increased expression of surface receptors involved in adhesion to endothelia and transmigration in circulating neutrophils of biomass users, 46 which could explain the increased extravasation of inflammatory cells to the lung of BS-exposed patients. In another study, BSexposed patients with COPD showed significantly higher levels of neutrophils, eosinophils and IL-8 in induced sputum compared with healthy individuals. ...
Article
Chronic obstructive pulmonary disease (COPD), a major cause of mortality and morbidity worldwide, is considered an archetypical disease of innate immunity, where inhaled particles and gases trigger an inflammatory response, favoring tissue proliferation in small airways and tissue destruction in lung parenchyma, in addition to the recruitment of immune cells to these compartments. Although cigarette smoking is still considered the main risk factor for developing COPD, the trend of proposing biomass smoke (BS) exposure as a principal risk factor is gaining importance, as around 3 billion people worldwide are exposed to this pollutant daily. A considerable amount of evidence has shown the potential of BS as an enhancer of lung inflammation. However, an impairment of some innate immune responses after BS exposure has also been described. Regarding the mechanisms by which biomass smoke alters the innate immune responses, three main classes of cell surface receptors—the TLRs, the scavenger receptors and the transient receptor potential channels—have shown the ability to transduce signals initiated after BS exposure. This article is an updated and comprehensive review of the immunomodulatory effects described after the interaction of BS components with these receptors.
... For example, occupational exposure to carcinogenic PAHs reduced the ability of cells to repair damaged DNA (37), and the environmental exposure to PM10 from the emissions of an oil refinery increased DNA damage in lymphocytes obtained from exposed subjects (38). Moreover, in buccal epithelial cells from women chronically exposed to biomass smoke, there was a positive correlation between PM10 and PM2.5 indoor levels and DNA damage (39). Tobacco smoke effects on DNA integrity are, in contrast, a controversial issue due to the increased, synergistic, discrepant or null effects observed in several studies. ...
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... One of these pro-inflammatory mediators is IL-8, which induces neutrophils production, sustained inflammation, and risk of tissue damage (Chung 2005;Harada et al. 1994). In the same context, individuals exposed to biomass smoke showed higher alveolar levels of macrophages, neutrophils, lymphocytes, eosinophils, monocytes, mastocytes, as well as elevated levels of IL-6, IL-8, and tumor necrosis factor (TNF)-α in sputum, compared with that of users of other fuel types (Banerjee et al. 2012;Dutta et al. 2013;Mondal et al. 2011;Silva et al. 2015). ...
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Respiratory diseases’ mortality and morbidity have been a major public health burden primarily attributed to widespread exposure to indoor and outdoor pollutants in the environment. The study conducted among 510 Bangladeshi women residing in the northeastern zone of the Sylhet division from semi-urban and rural settings to compare the biomass fuel users (N = 255) with the non-biomass users (N = 255). It has been observed that all the symptoms had a higher prevalence among the women who were exposed to biomass fuel compared with those exposed to clean gas fuel. Women exposed to biomass group reported frequent cough and phlegm production episodes during a 3-month timeline before the survey period which was found statistically higher (p < 0.001) compared with that of the clean gas fuel group. Moreover, the use of biomass fuel has been associated with a significant decrease in forced expiratory volume in one second (FEV1), forced vital capacity (FVC), and peak expiratory flow rate (PEFR). Appropriate strategies from stakeholders and government authorities in disseminating health hazards from biomass fuel along with supporting the community by providing alternative energy sources for cooking can largely impact people’s lives.
... Several tools have been developed to analyze comet assay images. Most of the tools use geometric measuring of the comet's core and tail [3][4][5]. Currently, there are some softwares for comet assay image analysis, both free and commercial. Commercial software is quite expensive, while free software is limited, especially for buccal mucosa cell. ...
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Comet assay or single cell gel electrophoresis assay (SCGE) is a method which is frequently used to measure the damage of DNA. The results of comet assay is a set of comet images, then the comet images are classified to measure the level of DNA damage. Currently, there are several softwares for comet assay image analysis, both free and commercial. Commercial software is quite expensive, while free software is limited, especially for buccal mucosa cell and super tiny comet image dataset. In this research, we propose a classification model for comet assay with super tiny image dataset which is taken from buccal mucosa cells. We propose a transfer learning based convolutional neural network (CNN) model. We have compared the transfer learning model with CNN-support vector machine (SVM) and ordinary CNN. In our experiments, we use super tiny dataset consisting of 73 images. Our transfer learning model gives an accuracy 70.5%, while CNN-SVM gives 62.3% and ordinary CNN gives 63.5%. We also compare our transfer learning model with most frequently used, free comet assay analysis software, OpenComet. Open-Comet gives an accuracy 11.5%. Our transfer learning model is promising for comet assay for buccal mucosa cell and super tiny dataset.
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This study examined whether indoor air pollution from biomass fuel burning induces DNA damage in airway cells. For this, sputum cells were collected from 56 premenopausal rural women who cooked with biomass (wood, dung, crop residues) and 49 age-matched controls who cooked with cleaner liquefied petroleum gas. The levels of particulate matters with diameters of less than 10 and 2.5 µm (PM(10) and PM(2.5) ) in indoor air were measured using a real-time aerosol monitor. Benzene exposure was monitored by measuring trans,trans-muconic acid (t,t-MA) in urine by HPLC-UV. DNA damage was examined by alkaline comet assay in sputum cells. Generation of reactive oxygen species (ROS) and level of superoxide dismutase (SOD) in sputum cells were measured by flow cytometry and spectrophotometry, respectively. Compared with controls, biomass users had 4 times higher tail percentage DNA, 37% more comet tail length and 5 times more Olive tail moment (p < 0.001) in inflammatory and epithelial cells in sputum, suggesting extensive DNA damage. In addition, women who cooked with biomass had 6 times higher levels of urinary t,t-MA and 2-fold higher levels of ROS generation concomitant with 28% depletion of SOD. Indoor air of biomass-using households had 2-4 times more PM(10) and PM(2.5) than that of controls(.) After controlling potential confounders, positive association was found between DNA damage parameters, particulate pollution, urinary t,t-MA and ROS. Thus, long-term exposure to biomass smoke induces DNA damage in airway cells and the effect was probably mediated, at least in part, by oxidative stress generated by inhaled particulate matter and benzene. Copyright © 2011 John Wiley & Sons, Ltd.
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An estimated 3 billion people (about half the world's population) burn biomass fuel (wood, crop residues, animal dung and coal) for cooking and heating purposes exposing a large population, especially women and children, to high levels of indoor air pollution. Biomass smoke comprises gaseous air pollutants as well as particulate matter air pollutants, which have significant harmful effects. Exposure to biomass smoke is a major contributor to morbidity and mortality. Children, women and the elderly are most affected. Apart from poor lung growth seen in growing children, the risk of developing respiratory tract infections (both upper as well as lower) is greatly increased in children living in homes using biomass. Women who spend many hours cooking food in poorly ventilated homes develop chronic obstructive lung disease (COPD), asthma, respiratory tract infections, including tuberculosis and lung cancer. It has been argued that exposure to biomass fuel smoke is a bigger risk factor for COPD than tobacco smoking. Physicians need to be aware about the harmful effects of biomass smoke exposure and ensure early diagnosis and appropriate management to reduce the disease burden. More research needs to be done to study health effects due to biomass smoke exposure better. Reducing the exposure to biomass smoke through proper home ventilation, home design and, if possible, change of biomass to cleaner fuels is strongly recommended in order to reduce biomass smoke-induced mortality and morbidity.
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Almost 3 billion people worldwide burn solid fuels indoors. Despite the large population at risk worldwide, the effect of exposure to indoor solid fuel smoke has not been adequately studied. Indoor air pollution from solid fuel use is strongly associated with chronic obstructive pulmonary disease, acute respiratory tract infections, and lung cancer, and weakly associated with asthma, tuberculosis, and interstitial lung disease. Tobacco use further potentiates the development of respiratory disease among subjects exposed to solid fuel smoke. There is a need to perform additional interventional studies in this field.
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It is now broadly accepted that particulate matter exposure can lead to multiple adverse health effects. The capability of airborne particulate matter (PM) to generate reactive oxygen species (ROS), known as “oxidative potential” (OP) is suggested to be one of the most relevant indicators of PM toxicity. Redox active chemical species in PM, of both inorganic and organic nature, facilitate ROS generation, causing oxidative damages, which are harmful for cells, ultimately leading to different chronic diseases. Therefore, OP has been proposed as a new additional metric for PM toxicity which is better associated with biological responses to PM exposures, thus could be more informative than particulate mass alone. However, the mechanisms of toxicity and its relation with the physico-chemical properties of PM are still largely unknown and need further research. Several chemical assays exist to assess the oxidative potential of PM. They differ from each other in sensitivity to the ROS generating chemical constituents of PM. The consumption of dithiothreitol (DTT), which is primarily based on the capability of redox active compounds to transfer electrons from DTT to oxygen, is used as the most widely applicable acellular method to assess the OP of PM. The potential of PM to deplete antioxidants such as glutathione, ascorbic acid and uric acid are a few of the other methods used to measure OP with respect to time. Another method, electron spin resonance (ESR) with 5,5-dimethylpyrroline-N-oxide (DMPO) as a spin trap, measures the ability of PM to induce hydroxyl radicals in the presence of H2O2. The current understanding of oxidative potential of PM, its analysis methods, along with its spatial distribution across the globe are presented here. Effect of various particle sizes, chemical composition, and nature of origin in exhibiting oxidative potential and its impact on health are additionally discussed.
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Over the past three decades the development of methods for visualizing at the cell level the extent of DNA breakage significantly contributed to genotoxicity testing: their availability greatly improved the knowledge in the field of genetic toxicology. These procedures are based on the separation and visualization of DNA fragments resulting from cleavage of nuclear DNA. The separation process can be obtained either electrically (comet assay, linear migration of DNA fragments) or chemically (alkaline dispersion assays, radial diffusion of DNA fragments). Once separated and stained, intact and fragmented DNA can be observed with fluorescence or light microscope. Appropriate computer-assisted image analysis allows quantitative determination of the extent of DNA breakage. These procedures have been proven to be sensitive, flexible, and reliable, and, as compared to former methods, they are simpler, are less time and money consuming, and have the unique capability of detecting DNA damage at the single cell level. This last feature has the additional advantage of allowing the identification of cellular subpopulations characterized by different sensitivity to the damaging agent. The fast halo assay (FHA) is currently the simplest and quickest nuclear dispersion assay; recent modifications of FHA have further improved the assay and pave the way to a full exploitation of its analytical potential. In this chapter the development, procedures, applications, and limits of these dispersion assays, with a particular focus on FHA, will be illustrated.
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Urbanization and rapid industrialization in developing countries like India, have led to deterioration in the air quality. The adverse effects of air pollution are far reaching ranging from pulmonary disease to even cancer. With the vast sub-continent having varied topography and climatic conditions as well as lifestyles, dietary habits and socio-economic differences, exposure outcomes in humans are affected by these factors. Biomarkers of exposure, effect and susceptibility have been used to understand the development of disease due to air pollution in occupationally/environmentally exposed human population and to identify/monitor high risk individuals so that preventive measures can be implemented. This review outlines the current scenario of the use of biomarkers in human monitoring studies in India, to comprehend the effects of air pollution.
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To investigate whether biomass burning causes oxidative DNA damage and alters the expression of DNA base excision repair (BER) proteins in airway cells, sputum samples were collected from 80 premenopausal rural biomass-users and 70 age-matched control women who cooked with liquefied petroleum gas. Compared with control the airway cells of biomass-users showed increased DNA damage in alkaline comet assay. Biomass-users showed higher percentage of cells expressing oxidative DNA damage marker 8-oxoguanine and lower percentages of BER proteins OGG1 and APE1 by Immunocytochemical staining. Reactive oxygen species (ROS) generation was doubled and level of superoxide dismutase was depleted significantly among biomass-users. The concentrations of particulate matters were higher in biomass-using households which positively correlated with ROS generation and negatively with BER proteins expressions. ROS generation was positively correlated with 8-oxoguanine and negatively with BER proteins suggesting cooking with biomass is a risk for genotoxicity among rural women in their child-bearing age.
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The study was carried out to examine whether chronic exposure to smoke during daily household cooking with biomass fuel (BMF) elicits changes in airway cytology and expressions of Nrf2 (nuclear factor erythroid 2 [NF-E2]-related factor 2 [Nrf2]), Keap1 (Kelch-like erythroid-cell-derived protein with CNC homology [ECH]-associated protein 1), and NQO1 (NAD(P)H:quinone oxidoreductase 1) proteins in the airways. For this, 282 BMF-using women (median age 34 year) and 236 age-matched women who cooked with liquefied petroleum gas (LPG) were enrolled. Particulate matter with diameters of < 10 µm (PM10) and < 2.5 µm (PM2.5) were measured in indoor air with real-time laser photometer. Routine hematology, sputum cytology, Nrf2, Keap1, NQO1, and generation of reactive oxygen species (ROS) along with the levels of superoxide dismutase (SOD) and catalase were measured in both groups. PM10 and PM2.5 levels were significantly higher in BMF-using households compared to LPG. Compared with LPG users, BMF users had 32% more leukocytes in circulation and their sputa were 1.4-times more cellular with significant increase in absolute number of neutrophils, lymphocytes, eosinophils, and alveolar macrophages, suggesting airway inflammation. ROS generation was 1.5-times higher in blood neutrophils and 34% higher in sputum cells of BMF users while erythrocyte SOD was 31% lower and plasma catalase was relatively unchanged, suggesting oxidative stress. In BMF users, Keap1 expression was reduced, the percentage of AEC with nuclear expression of Nrf2 was two- to three-times more, and NQO1 level in sputum cell lysate was two-times higher than that of LPG users. In conclusion, cooking with BMF was associated with Nrf2 activation and elevated NQO1 protein level in the airways. The changes may be adaptive cellular response to counteract biomass smoke-elicited oxidative stress and inflammation-related tissue injury in the airways.
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This is a cross-sectional review of biomarkers used in air pollution research from January 2009 through December 2012. After an initial keyword search in PubMed retrieving 426 articles, a comprehensive abstract review identified 54 articles of experimental design that used biomarkers of exposure or effect in human studies in the area of air pollution research during this specified time period. A thorough bibliographic search of the included articles retrieved an additional 65 articles meeting the inclusion criteria. This review presents these 119 studies and the 234 biomarkers employed in these air pollution research investigations. Data presented are 70 biomarkers of exposure with 54% relating to polycyclic aromatic hydrocarbons, 36% volatile organic carbons, and 10% classified as other. Of the 164 biomarkers of effect, 91 and 130 were used in investigating effects of short-term and chronic exposure, respectively. Results of biomarkers used in short-term exposure describe different lag times and pollutant components such as primary and secondary pollutants, and particle number associated with corresponding physiological mechanisms including airway inflammation, neuroinflammation, ocular, metabolic, early endothelial dysfunction, coagulation, atherosclerosis, autonomic nervous system, oxidative stress, and DNA damage. The review presents three different exposure scenarios of chronic, occupational, and extreme exposure scenarios (indoor cooking) with associated biomarker findings presented in three broad categories of (1) immune profile, (2) oxidative stress, and (3) DNA damage. This review offers a representation of the scope of data being explored by air pollution researchers through the use of biomarkers and has deliberately been restricted to this particular subject rather than an extensive or in-depth review. This article provides a contextualization of air pollution studies conducted with biomarkers in human subjects in given areas while also integrating this complex body of information to offer a useful review for investigators in this field of study.
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Background Upper gastrointestinal cancers contribute significantly to cancer-related morbidity and mortality in sub-Saharan Africa, but they continue to receive limited attention. The high incidence in young adults remains unexplained, and the risk factors have not been fully described. Methods A literature search was conducted using the electronic database PubMed. Beginning from January 1980 to February 2016, all articles evaluating biomass smoke exposure with oesophageal and gastric cancer were reviewed. Results Over 70% of the African population relies on biomass fuel, meaning most Africans are exposed to biomass smoke throughout their lives. Cigarette smoke is an established risk factor for upper gastrointestinal cancers, and some of its carcinogenic constituents are also present in biomass smoke. We found eight case-control studies reporting associations between exposure to biomass smoke and oesophageal cancer, and two linking biomass smoke to gastric cancer. All of these papers reported significant positive associations between exposure and cancer risk. Further research is needed in order to fully define the constituents of biomass smoke, which could each have varying specific and synergistic or independent contributions to the development of upper gastrointestinal cancers Conclusions Exposure to biomass smoke is an environmental factor influencing the development of upper gastrointestinal cancers, especially in low-resource settings.
Chapter
The need for express screening of the DNA damaging potential of chemicals has progressively increased over the past 20 years due to the wide number of new synthetic molecules to be evaluated, as well as the adoption of more stringent chemical regulations such as the EU REACH and risk reduction politics. In this regard, DNA diffusion assays such as the microelectrophoretic comet assay paved the way for rapid genotoxicity testing. A more significant simplification and speeding up of the experimental processes was achieved with the fast halo assay (FHA) described in the present chapter. FHA operates at the single cell level and relies on radial dispersion of the fragments of damaged DNA from intact nuclear DNA. The fragmented DNA is separated by diffusion in an alkaline solvent and is stained, visualized, and finally quantified using computer-assisted image analysis programs. This permits the rapid assessment of the extent of DNA breakage caused by different types of DNA lesions. FHA has proven to be sensitive, reliable, and flexible. This is currently one of the simplest, cheapest, and quickest assays for studying DNA damage and repair in living cells. It does not need expensive reagents or electrophoretic equipment and requires only 40 min to prepare samples for computer-based quantification. This technique can be particularly useful in rapid genotoxicity assessments and in high-throughput genotoxicity screenings.
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Research suggests that exposure to ambient particulate matter (PM) may be associated with lung cancer; however, no mode of action (MoA) for this has been established. We applied a weight-of-evidence (WoE) approach to evaluate recent evidence from four realms of research (controlled human exposure, epidemiology, animal, and in vitro) to determine whether the overall evidence supports one or more MoAs by which PM could cause lung cancer. We evaluated three general MoAs: DNA damage and repair; other genotoxic effects, including mutagenicity and clastogenicity; and gene expression, protein expression, and DNA methylation. After assessing individual study quality, we evaluated the strength of the evidence within as well as across disciplines using a modified set of Bradford Hill considerations. We conclude that the overall WoE indicates it is plausible that PM of various size fractions may cause direct DNA damage, but the evidence is insufficient regarding the alternative MoAs we evaluated. More research is needed to determine whether DNA damage can lead to downstream events and, ultimately, lung cancer.
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Comet assay is a simple and precise method to analyze DNA damage. Nowadays, many research studies have demonstrated the effectiveness of buccal mucosa cells usage in comet assays. However, several software tools do not perform well for detecting and classifying comets from a comet assay image of buccal mucosa cells because the cell has a lot more noise. Therefore, a specific software tool is required for fully automated comet detection and classification from buccal mucosa cell swabs. This research proposes a deep learning-based fully automated framework using Faster R-CNN to detect and classify comets in a comet assay image taken from buccal mucosa swab. To train the Faster R-CNN model, buccal mucosa samples were collected from 24 patients in Indonesia. We acquired 275 comet assay images containing 519 comets. Furthermore, two strategies were used to overcome the lack of dataset problems during the model training, namely transfer learning and data augmentation. We implemented the proposed Faster R-CNN model as a web-based tool, GamaComet, that can be accessed freely for academic purposes. To test the GamaComet, buccal mucosa samples were collected from seven patients in Indonesia. We acquired 43 comet assay images containing 73 comets. GamaComet can give an accuracy of 81.34% for the detection task and an accuracy of 66.67% for the classification task. Furthermore, we also compared the performance of GamaComet with an existing free software tool for comet detection, OpenComet. The experiment results showed that GamaComet performed significantly better than OpenComet that could only give an accuracy of 11.5% for the comet detection task. Downstream analysis can be well conducted based on the detection and classification results from GamaComet. The analysis showed that patients owning comet assay images containing comets with class 3 and class 4 had a smoking habit, meaning they had more cells with a high level of DNA damage. Although GamaComet had a good performance, the performance for the classification task could still be improved. Therefore, it will be one of the future works for the research development of GamaComet.
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OBJECTIVE: To compare the sputum smear cytology and cell block methods for specimen adequacy, cytologic quality and diagnostic accuracy in the diagnosis of lung cancer. STUDY DESIGN: We assessed 2,524 sputum specimens from 768 patients. The specimens were prepared as smears and cell blocks for cy-topathologic examination between March 1, 1992, and December 31, 1998. The smear and cell block slides were evaluated both separately and together, and the results were compared with radiologic and histopathologic diagnoses. RESULTS: The sensitivity of the smear method was 69.4% and specificity was 99.5%. The sensitivity of the cell block method was 84.4% and specificity, 100%. The sensitivity of the smear and cell block together was 87.6% and specificity, 99.5%. CONCLUSION: The cell block method increases the sensitivity and specificity of sputum cytology, and when smear and cell block slides are evaluated together, sensitivity reaches its highest value. Therefore, application of smear and cell block methods together seems most useful in the diagnosis of lung cancer. Carcinoma of the lung is the leading cause of cancer mortality in the western world. 1 Sputum cytology complements radiology in the diagnostic workup of lung cancer and can be useful in the identification of lung carcino-ma, especially at early or occult stages. 2,3 This method has sensitivity and specificity of 64.5% and 99.7%, respectively. 2 Sputum cytology also has the advantage of being easily applicable and noninva-sive and is cost effective. 4 Although sputum cytol-ogy has been performed routinely as smear cytol-ogy, the cell block method, which is not routinely When smear and cell block specimens are evaluated together, the sensitivity rate for detection of carcinoma is higher.
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Superoxide dismutase (EC 1.15.1.1) has been assayed by a spectrophotometric method based on the inhibition of a superoxide-driven NADH oxidation. The assay consists of a purely chemical reaction sequence which involves EDTA, Mn(II), mercaptoethanol, and molecular oxygen, requiring neither auxiliary enzymes nor sophisticated equipment. The method is very flexible and rapid and is applicable with high sensitivity to the determination of both pure and crude superoxide dismutase preparations. The decrease of the rate of NADH oxidation is a function of enzyme concentration, and saturation levels are attainable. Fifty percent inhibition, corresponding to one unit of the enzyme, is produced by approximately 15 ng of pure superoxide dismutase. Experiments on rat liver cytosol have shown the specificity of the method for superoxide dismutase. Moreover, common cellular components do not interfere with the measurement, except for hemoglobin when present at relatively high concentrations. The assay is performed at physiological pH and is unaffected by catalase.
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Unlabelled: In developing countries biomass combustion is a frequently used source of domestic energy and may cause indoor air pollution. Carbon monoxide (CO)and particulate matter with an aerodynamic diameter of 2.5 lm or less (PM2.5)were measured in kitchens using wood or natural gas (NG) in a semi-rural community in Pakistan. Daytime CO and PM2.5 levels were measured for eight continuous hours in 51 wood and 44 NG users from December 2005 to April 2006. The laser photometer PM2.5 (Dustrak, TSI) was calibrated for field conditions and PM2.5 measurements were reduced by a factor of 2.77. CO was measured by an electrochemical monitor (Model T15v, Langan). The arithmetic mean for daytime CO concentration was 29.4 ppm in wood users; significantly higher than 7.5 ppm in NG users (P < 0.001). The arithmetic mean for daytime PM2.5 concentrations was 2.74 mg/m3 in wood users; significantly higher than 0.38 mg/m3 in NG users (P < 0.001). Higher peak levels of CO and PM2.5 were also observed in wood users. Time spent in the kitchen during fuel burning was significantly related to increasing CO and PM2.5 concentrations in wood users.These findings suggest that cooking with wood fuel may lead to hazardous concentrations of CO and PM2.5. Practical implications: Biomass combustion is frequently used in developing countries for cooking. This study showed very high level of air pollution in kitchens using wood as the cooking fuel. Many people, especially women and children, are vulnerable to exposure to very high levels of air pollutants as they spend time in the kitchen during cooking hours.
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Hydroethidine (HE) and 2',7'-dichlorofluorescin (DCFH) were used for the flow cytometric measurement of reactive oxygen metabolites in leukocytes. Hydroethidine and DCFH were both rapidly oxidized in a cell-free cuvette assay to ethidium bromide (EB) and 2',7'-dichlorofluorescein (DCF) by H2O2 and peroxidase, but not by H2O2 alone, while only HE was oxidized by KO2, a source of O2-. Quiescent lymphocytes, monocytes, and neutrophils spontaneously oxidized HE to EB, while DCFH was only oxidized to a low degree. Neutrophils increased 6.9-fold in EB red fluorescence and 12.5-fold in DCF green fluorescence during the respiratory burst induced by phorbol 12-myristate 13-acetate or 6.1-fold and 4.7-fold, respectively, during the respiratory burst induced by Escherichia coli bacteria. The HE or DCFH oxidation during the respiratory burst, unlike the spontaneous HE oxidation, was not inhibitable by 10 mM NaNe indicating a non-mitochondrial source of cellular oxidants during the respiratory burst such as NADPH oxidase, which produces O2-. The oxidation of DCFH, but not of HE, was decreased in stimulated neutrophils, which were simultaneously loaded with HE and DCFH. Intracellular DCFH oxidation induced by incubation of resting neutrophils with extracellular H2O2 was not influenced by the presence of HE. This indicates that HE is oxidized at an earlier step in the reactive oxygen metabolism of neutrophils than DCFH, i.e., by early oxygen metabolites like O2-, while DCFH is oxidized in part by H2O2 and phagosomal peroxidases. The differential oxidation of HE and DCFH during simultaneous cellular staining permits the analysis of up to three functionally different neutrophil populations in septic patients. This is of interest for the determination of disease-related alterations of oxygen metabolism in quiescent and stimulated leukocytes.
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Around 50% of people, almost all in developing countries, rely on coal and biomass in the form of wood, dung and crop residues for domestic energy. These materials are typically burnt in simple stoves with very incomplete combustion. Consequently, women and young children are exposed to high levels of indoor air pollution every day. There is consistent evidence that indoor air pollution increases the risk of chronic obstructive pulmonary disease and of acute respiratory infections in childhood, the most important cause of death among children under 5 years of age in developing countries. Evidence also exists of associations with low birth weight, increased infant and perinatal mortality, pulmonary tuberculosis, nasopharyngeal and laryngeal cancer, cataract, and, specifically in respect of the use of coal, with lung cancer. Conflicting evidence exists with regard to asthma. All studies are observational and very few have measured exposure directly, while a substantial proportion have not dealt with confounding. As a result, risk estimates are poorly quantified and may be biased. Exposure to indoor air pollution may be responsible for nearly 2 million excess deaths in developing countries and for some 4% of the global burden of disease. Indoor air pollution is a major global public health threat requiring greatly increased efforts in the areas of research and policy-making. Research on its health effects should be strengthened, particularly in relation to tuberculosis and acute lower respiratory infections. A more systematic approach to the development and evaluation of interventions is desirable, with clearer recognition of the interrelationships between poverty and dependence on polluting fuels.
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Indoor air pollution resulting from combustion of biomass fuels in rural households of developing countries is now recognized as a major contributor to the global burden of disease. Accurate estimation of health risks has been hampered by a paucity of quantitative exposure information. In this study we quantified exposures to respirable particulate matter from biomass-fuel combustion in 436 rural homes selected through stratified random sampling from four districts of Tamil Nadu, India. The study households are a subset of a larger sample of 5,028 households from the same districts in which socioeconomic and health information has been collected. Results of measurements for personal exposures to respirable particulate matter during cooking were reported earlier. This has been extended to calculation of 24-hr exposures with the aid of additional measurements during noncooking times and the collection of time-activity records. Concentrations of respirable particulate matter ranged from 500 to 2,000 micro g/m(3) during cooking in biomass-using households, and average 24-hr exposures ranged from 90 +/- 21 micro g/m(3) for those not involved in cooking to 231 +/- 109 micro g/m(3) for those who cooked. The 24-hr exposures were around 82 +/- 39 micro g/m(3) for those in households using clean fuels (with similar exposures across household subgroups). Fuel type, type and location of the kitchen, and the time spent near the kitchen while cooking were the most important determinants of exposure across these households among other parameters examined, including stove type, cooking duration, and smoke from neighborhood cooking. These estimates could be used to build a regional exposure database and facilitate health risk assessments.
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Oxidative atmospheric pollutants represent a significant source of stress to both terrestrial plants and animals. The biosurfaces of plants and surface-living organisms are directly exposed to these pollutant stresses. These surfaces, including respiratory tract surfaces, contain integrated antioxidant systems that would be expected to provide a primary defense against environmental threats caused by atmospheric reactive oxygen species. When the biosurface antioxidant defenses are overwhelmed, oxidative stress to the cellular components of the exposed biosurfaces can be expected, inducing inflammatory, adaptive, injurious, and reparative processes. Studies of mutants and/or transformed plants and insects, with specific alterations in key components of antioxidant defense systems, offer opportunities to dissect the complex systems that maintain surface defenses against environmental oxidants. In this article, we use a comparative approach to consider interactions of atmospheric oxidant pollutants with selected biosystems, with focus on O3 as the pollutant; plants, flies, skin, and lungs as the exposed biosystems; and nonenzymatic micronutrient antioxidants as significant contributors to overall antioxidant defense strategies of these varied biosystems. Parallelisms among several living organisms, with regard to their protective strategies against environmental atmospheric oxidants, are presented.
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Inflammation has been recognized as an important factor in cancer development. For the lung, experimental studies with rats, as well as molecular epidemiological studies in humans, have provided evidence that the influx of neutrophils into the airways may be an important process linking inflammation with carcinogenesis. Currently it is believed that the genotoxic capacity of neutrophils is a crucial aetiological factor in this carcinogenic response. In the present review we discuss two major pathways of neutrophil-induced genotoxicity: (i) induction of oxidative DNA damage through release of reactive oxygen species (ROS) and (ii) myeloperoxidase (MPO)-related metabolic activation of chemical carcinogens. So far, direct evidence for a role of neutrophils in pulmonary genotoxicity has largely been derived from in vitro studies using co-cultures of activated neutrophils and target cells. Current evidence from in vivo studies is primarily indirect and additional animal studies are needed to substantiate causality. A further challenge will be to extrapolate results from such studies to humans. Taken together, this will provide a better insight into the role of neutrophils in pulmonary carcinogenicity and may, hence, lead to novel approaches for cancer prevention strategies.
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The aim of the present review was to provide an up-to-date overview of the biological and epidemiological evidence of the role of oxidative stress as a major underlying feature of the toxic effect of air pollutants, and the potential role of dietary supplementation in enhancing antioxidant defences. A bibliographic search was conducted through PubMed. The keywords used in the search were "air pollutant", "oxidative stress", "inflammation", "antioxidant polyunsaturated fatty acids" and "genetics". In addition, the authors also searched for biomarkers of oxidative stress and nutrients. The review presents the most recent data on: the biological and epidemiological evidence of the oxidative stress response to air pollutants; the role of dietary supplementation as a modulator of these effects; and factors of inter-individual variation in human response. The methodology for further epidemiological studies will be discussed in order to improve the current understanding on how nutritional factors may act. There is substantial evidence that air pollution exposure results in increased oxidative stress and that dietary supplementation may play a modulating role on the acute effect of air pollutants. Further epidemiological studies should address the impact of supplementation strategies in the prevention of air-pollution-related long-term effects in areas where people are destined to be exposed for the distant future.
Article
To evaluate the effects of cigarette smoking and biomass (dried dung) smoke on the oxidant–antioxidant status, three groups each with 5 rabbits were used. Groups of rabbits were exposed to either cigarette smoke, dried dung smoke or dry air, 1 h daily for one month. Protein carbonyls, prostaglandin F2α and malondialdehyde levels were significantly increased and protein sulfhydryls levels were significantly decreased in the cigarette smoke group compared with the control group. Only protein sulfhydryls levels were significantly decreased in dung group compared with the control group. Short course exposure to both cigarette smoke and biomass smoke decreased plasma antioxidant levels but only cigarette smoke increased plasma oxidant levels, whereas biomass smoke did not produce any change.
Article
Cellular protection against the deleterious effects of reactive oxidants generated in aerobic metabolism, called oxidative stress, is organized at multiple levels. Defense strategies include three levels of protection; prevention, interception, and repair. Regulation of the antioxidant capacity includes the maintenance of adequate levels of antioxidant and the localization of antioxidant compounds and enzymes. Short-term and long-term adaptation and cell specialisation in these functions are new areas of interest. Control over the activity of prooxidant enzymes, such as NADPH oxidase and NO synthases, is crucial. Synthetic antioxidants mimic biological strategies.
Article
Genotoxic effects of traffic-generated particulate matter (PM) are well described, whereas little data are available on PM from combustion of biomass and wood, which contributes substantially to air pollution world wide. The aim of this study was to compare the genotoxicity of wood smoke particulate matter (WSPM), authentic traffic-generated particles, mineral PM and standard reference material (SRM2975) of diesel exhaust particles in human A549 lung epithelial and THP-1 monocytic cell lines. DNA damage was measured as strand breaks (SB) and formamidopyrimidine DNA glycosylase (FPG) sites by the comet assay, whereas cell cytotoxicity was determined as lactate dehydrogenase release. The exposure to WSPM generated SB and FPG sites in both cell lines at concentrations from 2.5 or 25 μg/ml, which were not cytotoxic. Compared to all other studied particles, WSPM generated greater responses in terms of both SB and FPG sites. Organic extracts of WSPM and SRM2975 elicited higher levels of SB than native and washed PM at 25 and 100 μg/ml, whereas assay saturation precluded reliable assessment of FPG sites. During a 6 h post-exposure period, in which the medium with PM had been replaced by fresh medium, 60% of the DNA lesions generated by WSPM were removed. In conclusion, WSPM generated more DNA damage than traffic-generated PM per unit mass in human cell lines, possibly due to the high level of polycyclic aromatic hydrocarbons in WSPM. This suggests that exposure to WSPM might be more hazardous than PM collected from vehicle exhaust with respect to development of lung cancer.
Article
Genotoxicity of indoor air pollution from biomass fuel use has been examined in 132 biomass users (median age 34 years) and 85 age-matched control women from eastern India who used the cleaner fuel liquefied petroleum gas (LPG) to cook. Micronucleus (MN) frequency was evaluated in buccal (BEC) and airway epithelial cells (AEC); DNA damage was examined by comet assay in peripheral blood lymphocytes (PBL); and expressions of gamma-H2AX, Mre11 and Ku70 proteins were localized in AEC and PBL by immunocytochemistry. Reactive oxygen species (ROS) generation in leukocytes was measured by flow cytometry, and the levels of superoxide dismutase (SOD) and total antioxidant status (TAS) in blood were measured by spectrophotometry. Real-time aerosol monitor was used to measure particulate pollutants in indoor air. Compared with controls, biomass users had increased frequencies of micronucleated cells in BEC (3.5 vs. 1.7, p<0.001) and AEC (4.54 vs. 1.86, p<0.001), and greater comet tail % DNA (18.6 vs. 11.7%, p<0.01), tail length (45.5 vs. 31.4mum, p<0.01) and olive tail moment (4.0 vs. 1.4, p<0.01) in PBL. Moreover, biomass users had more gamma-H2AX-positive nuclei in PBL (49.5 vs. 8.5%, p<0.01) and AEC (11.3 vs. 2.9%, p<0.01) along with higher expression of DNA repair proteins Mre11 and Ku70 in these cells, suggesting stimulation of DNA repair mechanism. Biomass users showed rise in ROS generation and depletion of SOD and TAS. Biomass-using households had 2-4 times more particulate matter with diameter less than 10 and 2.5mum in indoor air, and MN frequency and comet tail % DNA were positively associated with these pollutants after controlling potential confounders. Thus, chronic exposure to biomass smoke causes chromosomal and DNA damage and upregulation of DNA repair mechanism.
Article
The detection of breaks in mammalian cell DNA and the measurement of their repair represent primary endpoints for genotoxicity testing. Over the past three decades many techniques sensitive to the presence of DNA breaks have been developed: their availability significantly increased the knowledge in the area of genetic toxicology. In general, these techniques have evolved to become more sensitive and flexible as well as less complicated. The fast-halo assay (FHA) is a very recent method to detect DNA-strand breakage induced either by various genotoxic agents or secondary to apoptotic DNA cleavage, and to study the repair of primary DNA breaks at the single-cell level. In FHA, damaged DNA is separated from intact one by means of solvent gradient, stained with ethidium bromide and visualized under a fluorescence microscope. The level of DNA breaks is then determined with an image analysis software. FHA is as sensitive, reliable, and flexible as the well-established comet assay, but it has the advantage of being, as compared to any other existing method, the most rapid and less expensive one. Taken collectively, these unique features render FHA the ideal method to perform a large number of genotoxicity tests on mammalian cells in a particularly cost-effective and time-saving manner.
Article
Benzene is a well known environmental carcinogen which causes myeloid leukemia. DNA damage induced by benzene metabolites such as hydroquinone (HQ) and p-benzoquinone (BQ) is one reason for the leukemogenesis. In this study, we showed that treatment with HQ and BQ quickly and clearly generated phosphorylated histone H2AX (gamma-H2AX) which has been recently considered an index of the production of double strand breaks (DSBs). HQ and BQ produced discrete foci of gamma-H2AX within the nucleus of HL-60 cells in a dose-dependent manner. gamma-H2AX appeared after the treatment with HQ and BQ for 2h, and increased time-dependently up to 4-8h. HQ and BQ increased intracellular oxidation, and an antioxidant, N-acetylcysteine, clearly inhibited the phosphorylation, suggesting that reactive oxygen species produced from HQ and BQ contributed to the generation. gamma-H2AX was sensitively detected after treatment with low concentrations of HQ and BQ, compared with the direct detection of DSBs by biased sinusoidal field gel electrophoresis and with the assessment of cytotoxicity based on cell survival. DSBs are the most serious form of DNA damage and are associated with genomic instability leading to myeloid leukemia. gamma-H2AX may be a useful tool for judging the genotoxicity of benzene metabolites sensitively.
Article
Oxidative-stress-induced damage to DNA includes a multitude of lesions, many of which are mutagenic and have multiple roles in cancer and aging. Many lesions have been characterized by MS-based methods after extraction and digestion of DNA. These preparation steps may cause spurious base oxidation, which is less likely to occur with methods such as the comet assay, which are based on nicking of the DNA strand at modified bases, but offer less specificity. The European Standards Committee on Oxidative DNA Damage has concluded that the true levels of the most widely studied lesion, 8-oxodG (8-oxo-7,8-dihydro-2'-deoxyguanosine), in cellular DNA is between 0.5 and 5 lesions per 10(6) dG bases. Base excision repair of oxidative damage to DNA can be assessed by nicking assays based on oligonucleotides with lesions or the comet assay, by mRNA expression levels or, in the case of, e.g., OGG1 (8-oxoguanine DNA glycosylase 1), responsible for repair of 8-oxodG, by genotyping. Products of repair in DNA or the nucleotide pool, such as 8-oxodG, excreted into the urine can be assessed by MS-based methods and generally reflects the rate of damage. Experimental and population-based studies indicate that many environmental factors, including particulate air pollution, cause oxidative damage to DNA, whereas diets rich in fruit and vegetables or antioxidant supplements may reduce the levels and enhance repair. Urinary excretion of 8-oxodG, genotype and expression of OGG1 have been associated with risk of cancer in cohort settings, whereas altered levels of damage, repair or urinary excretion in case-control settings may be a consequence rather than the cause of the disease.
Article
Cellular protection against the deleterious effects of reactive oxidants generated in aerobic metabolism, called oxidative stress, is organized at multiple levels. Defense strategies include three levels of protection; prevention, interception, and repair. Regulation of the antioxidant capacity includes the maintenance of adequate levels of antioxidant and the localization of antioxidant compounds and enzymes. Short-term and long-term adaptation and cell specialisation in these functions are new areas of interest. Control over the activity of prooxidant enzymes, such as NADPH oxidase and NO synthases, is crucial. Synthetic antioxidants mimic biological strategies.
The nonmethane hydrocarbon emissions from several types of cookstoves commonly used in developing countries were measured in a pilot study conducted in Manila, the Philippines. Four types of fuel, i.e., wood, charcoal, kerosene, and liquefied petroleum gas (LPG), were tested. Because kerosene was burned in three different types of stoves, there were six fuel/stove combinations tested. Fifty-nine nonmethane hydrocarbons were identified frequently in emissions of these cookstoves, with emission ratios to CO2 up to 5.3 x 10(-3). The emissions were quantitated with emission factors on both a mass basis (emissions/kg fuel) and a task basis (emissions/cooking task). On a task basis, combustion of biomass fuels (wood and charcoal) generally produced higher emission factors than combustion of fossil fuels (kerosene and LPG). One type of kerosene stove (wick stove), however, still generated the greatest emissions of some individual and classes of hydrocarbons, indicating that emissions were dependent on not only fuel types but also combustion devices. Some hydrocarbons, e.g., benzene, 1,3-butadiene, styrene, and xylenes, were of concern because of their carcinogenic properties. The lifetime risk from exposures to these compounds emitted from cookstoves was tentatively estimated by using a simple exposure model and published cancer potencies.
Article
The present study describes the development and characterization of a novel technique, the alkaline-halo assay, for the assessment of DNA single strand breakage in mammalian cells. This technique allows the measurement of DNA lesions at the single cell level and presents the additional advantages of being rapid, sensitive, virtually costless and environmentally friendly, because it does not require the use of isotopes. The alkaline halo assay involves a series of sequential steps in which the cells are first treated, then embedded in melted agarose and spread onto microscope slides that are incubated for 2 min at ice-bath temperature to allow complete geling. The slides are then incubated for 20 min in a high salt alkaline lysis solution, for an additional 15 min in a hypotonic alkaline solution and, finally, for 10 min in ethidium bromide. Under these conditions, single-stranded DNA fragments spread radially from the nuclear cage and generate a fluorescent image that resembles a halo concentric to the nucleus remnants. The area of the halos increased at increasing levels of DNA fragmentation and this process was associated with a progressive reduction of areas of the nuclear remnants. These events were conveniently monitored with a fluorescence microscope and quantified by image processing analysis. The sensitivity of the alkaline-halo assay, which is based on the osmotically driven radial diffusion of single-stranded DNA fragments through agarose pores, is remarkably similar to that of the widely used alkaline elution and comet assays.
Article
Damage to DNA is considered to be the main initiating event by which genotoxins cause hereditary effects and cancer. Single or double strand breaks, bases modifications or deletions, intra- or interstrand DNA-DNA or DNA-protein cross-links constitute the major lesions formed in different proportions according to agents and to DNA sequence context. They can result in cell death or in mutational events which in turn may initiate malignant transformation. Normal cells are able to repair these lesions with fidelity or by introducing errors. Base excision (BER) and nucleotide excision (NER) repair are error-free processes acting on the simpler forms of DNA damage. A specialized form of BER involves the removal of mismatched DNA bases occurring as errors of DNA replication or from miscoding properties of damaged bases. Severe damage will be repaired according to several types of recombinational processes: homologous, illegitimate and site-specific recombination pathways. The loss of repair capacity as seen in a number of human genetic diseases and mutant cell lines leads to hypersensitivity to environmental agents. Repair-defective cells show qualitative (mutation spectrum) and quantitative alterations in dose-effect relationships. For such repair-deficient systems, direct measurements at low doses are possible and the extrapolation from large to low doses fits well with the linear or the linear-quadratic no-threshold models. Extensive debate still takes place as to the shape of the dose-response relationships in the region at which genetic effects are not directly detectable in repair-proficient normal cells. Comparison of repair mutants and wild-type organisms pragmatically suggests that, for many genotoxins and tissues, very low doses may have no effect at all in normal cells.
Article
In the last decade, a number of quantitative epidemiological studies of specific diseases have been done in developing countries that for the first time allow estimation of the total burden of disease (mortality and morbidity) attributable to use of solid fuels in adult women and young children, who jointly receive the highest exposures because of their household roles. Few such studies are available as yet for adult men or children over 5 years. This paper evaluates the existing epidemiological studies and applies the resulting risks to the more than three-quarters of all Indian households dependent on such fuels. Allowance is made for the existence of improved stoves with chimneys and other factors that may lower exposures. Attributable risks are calculated in reference to the demographic conditions and patterns of each disease in India. Sufficient evidence is available to estimate risks most confidently for acute respiratory infections (ARI), chronic obstructive pulmonary disease (COPD), and lung cancer. Estimates for tuberculosis (TB), asthma, and blindness are of intermediate confidence. Estimates for heart disease have the lowest confidence. Insufficient quantitative evidence is currently available to estimate the impact of adverse pregnancy outcomes (e.g., low birthweight and stillbirth). The resulting conservative estimates indicate that some 400-550 thousand premature deaths can be attributed annually to use of biomass fuels in these population groups. Using a disability-adjusted lost life-year approach, the total is 4-6% of the Indian national burden of disease, placing indoor air pollution as a major risk factor in the country.
Article
Measurements collected using five real-time continuous airborne particle monitors were compared to measurements made using reference filter-based samplers at Bakersfield, CA, between December 2, 1998, and January 31, 1999. The purpose of this analysis was to evaluate the suitability of each instrument for use in a real-time continuous monitoring network designed to measure the mass of airborne particles with an aerodynamic diam less than 2.5 microns (PM2.5) under wintertime conditions in the southern San Joaquin Valley. Measurements of airborne particulate mass made with a beta attenuation monitor (BAM), an integrating nephelometer, and a continuous aerosol mass monitor (CAMM) were found to correlate well with reference measurements made with a filter-based sampler. A Dusttrak aerosol sampler overestimated airborne particle concentrations by a factor of approximately 3 throughout the study. Measurements of airborne particulate matter made with a tapered element oscillating microbalance (TEOM) were found to be lower than the reference filter-based measurements by an amount approximately equal to the concentration of NH4NO3 observed to be present in the airborne particles. The performance of the Dusttrak sampler and the integrating nephelometer was affected by the size distribution of airborne particulate matter. The performance of the BAM, the integrating nephelometer, the CAMM, the Dusttrak sampler, and the TEOM was not strongly affected by temperature, relative humidity, wind speed, or wind direction within the range of conditions encountered in the current study. Based on instrument performance, the BAM, the integrating nephelometer, and the CAMM appear to be suitable candidates for deployment in a real-time continuous PM2.5 monitoring network in central California for the range of winter conditions and aerosol composition encountered during the study.
Article
Ultrafine particles are a component of air pollution, derived from primary combustion sources, and so we have undertaken a programme of study on the mechanisms of lung injury caused by ultrafine particles. Ultrafine particles made of low-solubility, low-toxicity materials are more inflammogenic in the rat lung than fine respirable, particles made from the same material. Ultrafine particles can cause inflammation via processes independent of the release of transition metals, as shown by the fact that soluble products from ultrafine carbon black have no ability to cause inflammation. The property that drives the greater inflammogenicity of ultrafines is unknown but very likely relates to particle surface area and involves oxidative stress. Increases in intracellular Ca(++) may underlie the cellular effects of ultrafines, although the mechanism whereby ultrafines have this effect is not understood. However, increased influx of Ca(++) into macrophages occurs via the membrane Ca(++) channels following contact with ultrafine particles, and involves oxidative stress. Increased Ca(++) in macrophages exposed to ultrafines can lead to the transcription of key pro-inflammatory genes such as TNFalpha. Ultrafine particles can also impair the ability of macrophages to phagocytose and clear other particles, and this may be pro-inflammogenic.
Article
There is ample evidence that allergic disorders, such as asthma, rhinitis, and atopic dermatitis, are mediated by oxidative stress. Excessive exposure to reactive oxygen and nitrogen species is the hallmark of oxidative stress and leads to damage of proteins, lipids, and DNA. Oxidative stress occurs not only as a result of inflammation but also from environmental exposure to air pollution and cigarette smoke. The specific localization of antioxidant enzymes in the lung and the rapid reaction of nitric oxide with reactive oxygen species, such as superoxide, suggest that antioxidant enzymes might also function as cell-signaling agents or regulators of cell signaling. Therapeutic interventions that decrease exposure to environmental reactive oxygen species or augment endogenous antioxidant defenses might be beneficial as adjunctive therapies for allergic respiratory disorders.
Article
To compare the sputum smear cytology and cell block methods for specimen adequacy, cytology quality and diagnostic accuracy in the diagnosis of lung cancer. We assessed 2,524 sputum specimens from 768 patients. The specimens were prepared as smears and cell blocks for cytopathologic examination between March 1, 1992, and December 31, 1998. The smear and cell block slides were evaluated both separately and together, and the results were compared with radiologic and histopathologic diagnoses. The sensitivity of the smear method was 69.4% and specificity was 99.5%. The sensitivity of the cell block method was 84.4% and specificity, 100%. The sensitivity of the smear and cell block together was 87.6% and specificity, 99.5%. The cell block method increases the sensitivity and specificity of sputum cytology, and when smear and cell block slides are evaluated together, sensitivity reaches its highest value. Therefore, application of smear and cell block methods together seems most useful in the diagnosis of lung cancer.
Article
The comet assay is a widely used biomonitoring tool for DNA damage. The most commonly used cells in human studies are lymphocytes. There is an urgent need to find an alternative target human cell that can be collected from normal subjects with minimal invasion. There are some reports of buccal cells, collected easily from the inside of the mouth, being used in studies of DNA damage and repair, and these were of interest. However, our preliminary studies following the published protocol showed that buccal cells sustained massive damage and disintegrated at the high pH [O. Ostling, K.J. Johanson. Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells. Biochem. Biophys. Res. Commun. 123 (1984) 291-298] used, but that at lower pH were extremely resistant to lysis, an essential step in the comet assay. Therefore, the aims of this study were to develop a protocol than enabled buccal cell lysis and DNA damage testing in the comet assay, and to use the model to evaluate the potential use of the buccal cell model in human biomonitoring and nutritional study. Specifically, we aimed to investigate intra- and inter-individual differences in buccal cell DNA damage (as strand breaks), the effect of in vitro exposure to both a standard oxidant challenge and antioxidant treatment, as well as in situ exposure to an antioxidant-rich beverage and supplementation-related effects using a carotenoid-rich food. Successful lysis was achieved using 0.25% trypsin for 30 min followed by proteinase K (1mg/ml) treatment for 60 min. When this procedure was performed on cells pre-embedded in agarose on a microscope slide, followed by electrophoresis (in 0.01 M NaOH, 1mM EDTA, pH 9.1, 18 min at 12 V), a satisfactory comet image was obtained, though inter-individual variation was quite wide. Pre-lysis exposure of cells to a standard oxidant challenge (induced by H2O2) increased DNA strand breaks in a dose related manner, and incubation of cells in Trolox (a water soluble Vitamin E analogue) conferred significant protection (P<0.05) against subsequent oxidant challenge. Exposure of buccal cell in situ (i.e. in the mouth) to antioxidant-rich green tea led to an acute decrease in basal DNA strand breaks. In a controlled human intervention trial, buccal cells from 14 subjects after 28 days' supplementation with a carotenoid-rich berry (Fructus barbarum L.) showed a small but statistically significant (P<0.05) decrease in DNA strand breaks. These data indicate that this buccal cell comet assay is a feasible and potentially useful alternative tool to the usual lymphocyte model in human biomonitoring and nutritional work.
Article
The lungs of asthmatic patients are exposed to oxidative stress due to the generation of reactive oxygen and nitrogen species as a consequence of chronic airway inflammation. Increased concentrations of NO*, H2O2 and 8-isoprostane have been measured in exhaled breath and induced sputum of asthmatic patients. O2*-, NO*, and halides interact to form highly reactive species such as peroxynitrite and HOBr, which in turn cause nitration and bromination of protein tyrosine residues. Oxidative stress may also reduce glutathione levels and cause inactivation of antioxidant enzymes such as superoxide dismutase, with a consequent increase in apoptosis, shedding of airway epithelial cells and airway remodelling. The oxidant/antioxidant equilibrium in asthmatic patients may be further perturbed by low dietary intakes of the antioxidant vitamins C and E, selenium and flavonoids, with a consequent lowering of the concentrations of these and other non-dietary antioxidants such as bilirubin and albumin in plasma and airway epithelial lining fluid. Although supplementation with vitamins C and E appears to offer protection against the adverse effects of ozone, recent randomised, placebo-controlled trials of vitamin C or E supplements for patients with mild asthma have not shown significant benefits over standard therapy. However, genetic variation in glutathione S-transferase may influence the susceptibility of asthmatic individuals to oxidative stress and the extent to which they are likely to benefit from antioxidant supplementation. Long-term prospective trials are required to determine whether modification of dietary intake will benefit asthma patients and reduce the socio-economic burden of asthma in the community.
Article
The present study describes the improvement of a technique, the alkaline-halo assay (AHA), for the assessment of DNA single-strand breakage at the single-cell level. AHA involves a series of sequential steps in which cells are embedded in melted agarose and spread onto microscope slides, incubated in a high-salt alkaline lysis solution, then in a hypotonic alkaline solution and, finally, stained with ethidium bromide (EB). Under these conditions, single-stranded DNA fragments diffuse radially from the nuclear cage and generate a fluorescent image that resembles a halo concentric to the nuclear remnants: the area of the halo is a direct function of the extent of DNA strand scission. These phenomena can be conveniently monitored with a fluorescence microscope and quantified by image-processing analysis.
Article
The aim of this study was to quantify personal exposure and indoor levels of the suspected or known carcinogenic compounds 1,3-butadiene, benzene, formaldehyde and acetaldehyde in a small Swedish town where wood burning for space heating is common. Subjects (wood burners, n = 14), living in homes with daily use of wood-burning appliances were compared with referents (n = 10) living in the same residential area. Personal exposure and stationary measurements indoors and at an ambient site were performed with diffusive samplers for 24 h. In addition, 7 day measurements of 1,3-butadiene and benzene were performed inside and outside the homes. Wood burners had significantly higher median personal exposure to 1,3-butadiene (0.18 microg m(-3)) compared with referents (0.12 microg m(-3)), which was also reflected in the indoor levels. Significantly higher indoor levels of benzene were found in the wood-burning homes (3.0 microg m(-3)) compared with the reference homes (1.5 microg m(-3)). With regard to aldehydes, median levels obtained from personal and indoor measurements were similar although the four most extreme acetaldehyde levels were all found in wood burners. High correlations were found between personal and indoor levels for all substances (r(s) > 0.8). In a linear regression model, type of wood-burning appliance, burning time and number of wood replenishments were significant factors for indoor levels of 1,3-butadiene. Domestic wood burning seems to increase personal exposure to 1,3-butadiene as well as indoor levels of 1,3-butadiene and benzene and possibly also acetaldehyde. The cancer risk from these compounds at exposure to wood smoke is, however, estimated to be low in developed countries.
Article
Polycyclic aromatic hydrocarbons (PAHs) appear to be significant contributors to the genotoxicity and carcinogenicity of air pollution present in the urban environment for humans. Populations exposed to environmental air pollution show increased levels of PAH DNA adducts and it has been postulated that another contributing cause of carcinogenicity by environmental air pollution may be the production of reactive oxygen species following oxidative stress leading to oxidative DNA damage. The antioxidant status as well as the genetic profile of an individual should in theory govern the amount of protection afforded against the deleterious effects associated with exposure to environmental air pollution. In this study we investigated the formation of total PAH (bulky) and B[a]P DNA adducts following exposure of individuals to environmental air pollution in three metropolitan cities and the effect on endogenously derived oxidative DNA damage. Furthermore, the influence of antioxidant status (vitamin levels) and genetic susceptibility of individuals with regard to DNA damage was also investigated. There was no significant correlation for individuals between the levels of vitamin A, vitamin E, vitamin C and folate with M(1)dG and 8-oxodG adducts as well as M(1)dG adducts with total PAH (bulky) or B[a]P DNA adducts. The interesting finding from this study was the significant negative correlation between the level of 8-oxodG adducts and the level of total PAH (bulky) and B[a]P DNA adducts implying that the repair of oxidative DNA damage may be enhanced. This correlation was most significant for those individuals that were non smokers or those unexposed to environmental air pollution. Furthermore the significant inverse correlation between 8-oxodG and B[a]P DNA adducts was confined to individuals carrying the wild type genotype for both the GSTM1 and the GSTT1 gene (separately and interacting). This effect was not observed for individuals carrying the null variant.
Article
DNA integrity was analyzed in the lymphocytes of 65 non-smoking city policemen during January and September 2004 using the comet assay combined with excision repair enzymes. Information about inhalation exposure was obtained by (1) stationary monitoring of PM2.5 and carcinogenic polycyclic aromatic hydrocarbons (cPAHs) during the sampling periods and (2) personal exposure monitoring of cPAHs 48h before blood sampling. The data were completed by a lifestyle questionnaire. Regardless of the season of the year, policemen working outdoors (exposed group) exhibited higher levels of DNA damage than those working indoors (controls). Within the exposed group, the levels of both unspecified and oxidative DNA damage detected in January significantly exceeded those found in September. The controls did not show analogous inter-seasonal variability. The winter levels of oxidative DNA damage positively correlated with exposure to cPAHs, probably reflecting increased oxidative stress as a result of high concentrations of PM2.5. In comparison with the wild type genotype, the carriers of at least one mutated allele, CYP1A1*2C (Ile/Val), MTHFR 2656 or MS 2656, and the EPHX1-medium phenotype appeared to be more susceptible specifically to the induction of oxidative DNA damage, while the p53 MspI mutation predisposed the carrier to a higher incidence of both breaks and oxidative lesions in DNA. In contrast, GSTM1-null and vitamin C tended rather to protect DNA integrity.
Mineral fibers, cigarette smoke, and oxidative DNA damage
  • P Leanderson
  • C Tagesson
Leanderson, P., Tagesson, C., 1993. Mineral fibers, cigarette smoke, and oxidative DNA damage. In: Halliwell, B., Aruoma, O.I. (Eds.), DNA and Free Radicals. Ellis Horwood, Chichester, UK, pp. 293-314.
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Daily average exposures to respirable particulate matter from combustion of biomass fuels in rural households of southern India
  • K Balakrishnan
  • S Sankar
  • J Parikh
  • R Padmavati
  • K Srividya
  • V Venugopal
  • S Prasad
  • V L Pandey
Balakrishnan, K., Sankar, S., Parikh, J., Padmavati, R., Srividya, K., Venugopal, V., Prasad, S., Pandey, V.L., 2002. Daily average exposures to respirable particulate matter from combustion of biomass fuels in rural households of southern India. Environ. Health Perspect. 110, 1069-1075.