A protocol for detecting and scavenging gas-phase free radicals in mainstream cigarette smoke

Cigarette smoking is associated with human cancers. It has been reported that most of the lung cancer deaths are caused by cigarette smoking (5,6,7,12). Although tobacco tars and related products in the particle phase of cigarette smoke are major causes of carcinogenic and mutagenic related diseases, cigarette smoke contains significant amounts of free radicals that are also considered as an important group of carcinogens(9,10). Free radicals attack cell constituents by damaging protein structure, lipids and DNA sequences and increase the risks of developing various types of cancers. Inhaled radicals produce adducts that contribute to many of the negative health effects of tobacco smoke in the lung(3). Studies have been conducted to reduce free radicals in cigarette smoke to decrease risks of the smoking-induced damage. It has been reported that haemoglobin and heme-containing compounds could partially scavenge nitric oxide, reactive oxidants and carcinogenic volatile nitrosocompounds of cigarette smoke(4). A 'bio-filter' consisted of haemoglobin and activated carbon was used to scavenge the free radicals and to remove up to 90% of the free radicals from cigarette smoke(14). However, due to the cost-ineffectiveness, it has not been successfully commercialized. Another study showed good scavenging efficiency of shikonin, a component of Chinese herbal medicine(8). In the present study, we report a protocol for introducing common natural antioxidant extracts into the cigarette filter for scavenging gas phase free radicals in cigarette smoke and measurement of the scavenge effect on gas phase free radicals in mainstream cigarette smoke (MCS) using spin-trapping Electron Spin Resonance (ESR) Spectroscopy(1,2,14). We showed high scavenging capacity of lycopene and grape seed extract which could point to their future application in cigarette filters. An important advantage of these prospective scavengers is that they can be obtained in large quantities from byproducts of tomato or wine industry respectively(11,13).     

Cigarette waste: Assessment of hazard to the environment and health in Riyadh city

Cigarette waste/litter is considered a major environmental contamination problem worldwide as trillions of cigarettes are smoked worldwide and a large part of that, cigarette waste, is disposed of in the open areas including roads, parks, and streets, etc. cigarette litter is the most commonly found litter. It is mainly cigarette filter, made of cellulose acetate, and unburnt part of the tobacco filler. Filters from smoked cigarettes contain a significant amount of tar trapped in it. The tar contains thousands of chemicals and heavy metals. Both of these organic and inorganic constituents have been reported to be toxic to humans and cause a variety of diseases including inflammatory lung diseases, cardiovascular diseases, and cancers. Cigarette litter is a significant environmental concern as the chemicals and heavy metals can leach into the soil or water sources and pose threat to animals and plants, from there they can enter into the food chain as well. Several reports indicate toxicities to aquatic and terrestrial animals as they consumed cigarette litter. In the present investigation, cigarette litter was collected from 28 randomly selected locations in the Riyadh city to assess the risk to the environment. Cigarette litter, in the form of cigarette butts, was collected, counted, weighed and analyzed for heavy metal content. Data indicate the presence of a significant amount of cigarette litter on roadsides, streets, and parks in the Riyadh city. However, the investigation had its limitations as it did not focus on the absolute amount of cigarette litter vs the time. It also did not consider the amount of cigarette litter percent in the total waste discarded. The investigation presents the results of the screening of the cigarette litter present on the Riyadh city roads, streets, and parks. The findings raise concerns regarding the hazards the cigarette litter poses to the environment and human health. The investigation sheds the light on this unexplored aspect of smoking-associated issues in the Kingdom of Saudi Arabia.     

Nicotine-induced respiratory effects of cigarette smoke in dogs

We report that nicotine is responsible for both a blood-borne stimulation of the respiratory center and a direct effect on intrathoracic airway tone in dogs. We introduced cigarette smoke into the lungs of donor dogs and injected arterial blood obtained from them into the circulation of recipient dogs to show that a blood-borne material increased breathing and airway smooth muscle tone. Smoke from cigarettes containing 2.64 mg of nicotine was effective; that from cigarettes containing 0.42 mg of nicotine was not. Nicotine, in doses comparable to the amounts absorbed from smoke, also increased breathing and tracheal smooth muscle tension when injected into the vertebral circulation of recipient dogs. Finally, blockade of nicotine receptors in the central nervous system and in the airway parasympathetic ganglia inhibited the effects of inhaled cigarette smoke and intravenous nicotine on the respiratory center and on bronchomotor tone. We conclude that nicotine absorbed from cigarette smoke is the main cause of cigarette smoke-induced bronchoconstriction. It caused central respiratory stimulation, resulting in increased breathing and airway smooth muscle tension, and had a direct effect on airway parasympathetic ganglia as well.     

Nicotine and its effect on wound healing

Our data demonstrate that nicotine impairs wound contraction in the rabbit ear model from the 4th to the 10th day of wound healing. However, the wounds contracted at essentially the same rate from the 12th to the 20th day in the experimental and control groups of animals. This study would suggest that cigarette smoking, with its associated nicotine ingestion, is adverse for a time to wound healing. It is clearly possible that in cases of extremity injury, or surgery, cigarette smoking may adversely affect wound healing.     

Inhibition of lactate dehydrogenase in cultured SIRC cells by cigarette smoke

Summary SIRC cell monolayer cultures were exposed to whole smoke from a mid tar and nicotine level research cigarette (ASFC, 72 puffs), or from a high tar and nicotine level reference cigarette (Kentucky 2R1, 48 puffs) over a period of 65 days. The activity and distribution of lactate dehydrogenase (LDH) in the cells were investigated, and the electrophoretic characteristics of its isozymes studied. Cell morphology was examined by light microscopy and by transmission- and scanning electron microscopy.LDH activity was reduced by exposure to smoke from both cigarette types, the greater inhibitory effect being produced by that of the Kentucky cigarette. In addition, cells exposed to this high tar and nicotine smoke displayed intramitochondrial granules which were larger and more numerous than those found in cells exposed to the mid tar and nicotine smoke, or in the control cells. It is speculated that cation accumulation in the mitochondria may be involved in the observed inhibition of LDH activity.Supported by a research grant from the ASFC (Association Suisse des Fabricants de Cigarettes), Switzerland     

Effect of cigarette smoke on lipid peroxidation,antioxidant enzymes and NMDA receptor subunits 2A and 2B concentration in rat hippocampus

The effect of cigarette smoke on lipid peroxidation and antioxidant enzymes such as catalase, superoxide dismutase, glutathione peroxidase and on the concentration of N-methyl-d-aspartate receptor (NMDAR) subunits 2A and 2B in the hippocampus of Sprague-Dawley rats exposed to cigarette smoke for 2h/day for a period of 4 weeks was determined. It was observed that NMDAR 2A and 2B concentrations in the hippocampus were enhanced in the case of animals exposed to cigarette smoke, whereas lipid peroxidation and antioxidant enzyme activities did not show any change as compared to control animals. The results of our study suggest that cigarette smoke induces NMDAR 2A and 2B expression in the hippocampus, and that this is not due to an increased lipid peroxidation, because cigarette smoke has no effect on lipid peroxidation and antioxidant enzyme activities in the hippocampus.     

The relationship between the amount of insect-repelling phenolic compounds in pennywort tea and the number of infesting cigarette beetles in the tea bag during one-year storage

Cigarette beetle, Lasioderma serricorne (F.), is a common food-product pest widely found in homes. This paper investigated the relationship between the number of infesting cigarette beetles and the phenolic content of pennywort tea made from young and mature leaves during one-year storage. It was found that cigarette beetles started to appear in tea bags after six months of storage. More adults were found than larvae and pupae. In addition, the number of cigarette beetles infesting pennywort tea made from mature leaves was higher than when it was made from young leaves. The amount of phenolic compounds in pennywort tea was analyzed by Folin–Ciocalteau assay. The results of a regression analysis indicated that the number of adult cigarette beetles correlated negatively with the amount of phenolic compounds in pennywort tea made from young leaves – when the total phenolic content decreased, the number of infesting cigarette beetles increased (r² = 0.80). The number of larvae and pupae showed a weak negative correlation with the amount of phenolic compounds (r² = 0.40 and r² = 0.56, respectively). Similar correlations were found in pennywort tea made from mature leaves, but the number of infesting beetles was higher due to the lower phenolic content of the mature leaves.     

The effects of cigarette smoking on intraocular pressure and arterial blood pressure of normotensive young Nigerian male adults.

This study was designed to determine the effects of cigarette Smoking on intra ocular pressure and arterial blood pressure of normotensive young male adults. Fifty male students (who met the screening conditions and devoid of obvious ocular pathology and systemic diseases and non-smokers) had their intra ocular pressure (IOP) measured with a schiotz tonometer and blood pressure(BP) measured with standard syphgmomanometer respectively prior to smoking of two sticks of cigarette each day for one month and thereafter. The result showed a significant [P < 0.01] effect on the intra ocular pressure with a mean control of 37.76 +/- 0.98 for both eyes and test of 41.93 +/-0.98. Cigarette smoking increased the blood pressure from mean control of 197.24 +/-0.88 to 208.46 +/-0.82. The increase of both intra ocular pressure and arterial blood pressure was due to nicotine, the principal constituent of cigarette. It is recommended that health care workers should check regularly the IOP and BP of their cigarette smoking patients for early diagnoses of ocular hypertension (glaucoma) and hypertension.     

Evidence for cigarette smoke-induced calcitonin secretion from lungs of man and hamster

In hamsters, acute cigarette smoke inhalation increased serum levels of the hormone calcitonin; and, in humans, smoking of two high-nicotine content cigarettes increased serum and urine levels of this hormone. The source of this immunoreactive calcitonin (iCT) does not appear to be the thyroid gland, since previously thyroidectomized patients demonstrated a similar response. In the hamster, the increased serum iCT levels were accompanied by a decreased lung tissue iCT content and hypocalcemia. It is suggested that the source of the cigarette smoke-induced hypercalcitonemia is the lung, possibly from the iCT-containing pulmonary neuroendocrine (PNE) cells. Moreover, this response appears to be dependent on the nicotine content of the cigarettes.     

Exposure to cigarette smoke, a major risk factor for sudden infant death syndrome: effects of cigarette smoke on inflammatory responses to viral infection and bacterial toxins

Exposure to cigarette smoke is a major risk factor for sudden infant death syndrome and also for respiratory infections in children. It has been suggested that toxigenic bacteria colonizing the respiratory tract might play a role in some cases of sudden infant death syndrome and nicotine has been demonstrated to enhance the lethality of bacterial toxins in a model system. Pyrogenic toxins of Staphylococcus aureus have been identified in tissues of infants who died of sudden infant death syndrome. It has been suggested that some of these deaths were due to induction of inflammatory mediators by infectious agents during a period when infants are less able to control these responses. The aim of this study was to assess the effects of a water-soluble cigarette smoke extract on the production of tumor necrosis factor alpha and nitric oxide from human monocytes in response to staphylococcal toxic shock syndrome toxin 1 or infection of the monocytes with respiratory syncytial virus. Cell culture supernatants were examined by a bioassay using mouse fibroblasts (L-929 cell line) for tumor necrosis factor alpha activity and by a spectrophotometric method for nitrite. Compared with monocytes incubated with medium only, monocytes incubated with any of the factors or their combinations tested in the study released higher levels of tumor necrosis factor alpha and lower levels of nitric oxide. Incubation with cigarette smoke extract increased tumor necrosis factor alpha from respiratory syncytial virus-infected cells while it decreased tumor necrosis factor alpha from cells incubated with toxic shock syndrome toxin. Incubation with cigarette smoke extract decreased the nitric oxide production from respiratory syncytial virus-infected cells while it increased the nitric oxide production from cells incubated with toxic shock syndrome toxin. Monocytes from a minority of individuals demonstrated extreme tumor necrosis factor alpha responses and/or very high or very low nitric oxide. The proportion of samples in which extreme responses with a very high tumor necrosis factor alpha and very low nitric oxide were detected was increased in the presence of the three agents to 20% compared with 0% observed with toxic shock syndrome toxin 1 or 4% observed with cigarette smoke extract or respiratory syncytial virus.     

Thiocyanate overload and thyroid disease

Thiocyanate [SCN-] is a complex anion which is a potent inhibitor of iodide transport. It is the detoxification product of cyanide and can easily be measured in body fluids. Consumption of naturally occurring goitrogens, certain environmental toxins and cigarette smoke can significantly increase SCN- concentrations to levels potentially capable of affecting the thyroid gland. Goiter endemics were reported to develop when the critical urinary iodine/ SCN- ratio decreases below 3 microg iodine per mg SCN-. Iodine supplementation completely reverses the goitrogenic influence of SCN-. SCN- is also generated from cigarette smoking as a detoxifying product of cyanide. During the past two decades many reports dealt with the possible effects of cigarette smoking on thyroid hormone synthesis, thyroid gland size and thyroid autoimmunity including infiltrative ophtalmopathy of Graves' disease. In this mini-review, issues regarding thiocyanate overload and thyroid disease will be summarized.     

An experimental demonstration that house finches add cigarette butts in response to ectoparasites

Urban species encounter resources that are uncommon in nature, such as materials found in city waste. Many studies have shown that these can be harmful to wildlife. In Mexico City, house finches bring cigarette butts to their nests, which reduces the amount of ectoparasites, but also induces genotoxic damage in chicks and parents. Yet, the reason for this behaviour is unknown. One possibility is that birds extract the cellulose fibres from discarded butts simply because they resemble feathers. Alternatively, disassembled cigarette butts may be brought to the nests because they repel ectoparasites. Here we tested the latter hypothesis by assessing whether house finches Carpodacus mexicanus increase the amount of cigarette butts in their nests in response to a raise in ectoparasite load. When present, fibres from butts are concentrated in the nest lining. By taking it away, we simultaneously removed most of the butt material and collected the bulk of the tick population infesting each nest, as these parasites cluster in the lining. We removed the bedding of nests when chicks had recently hatched, and randomly assigned each nests to one of the following treatments: 1) addition of live ticks, 2) addition of dead ticks and 3) simulation of tick addition. Females in the live ticks’ treatment added more butt fibres to their nests than parents in control treatments. Additionally, the amount of butt fibres in the original lining also predicted the amount of fibres added after the manipulation. It seems that the tendency to bring to the nest cigarette butts is at least partially a response to current, and perhaps also past, parasite load.     

Comparison of the induction by cigarette smoke condensates of sister-chromatid exchanges in Chinese hamster cells and of mutations in Salmonella typhimurium.

Three cigarette smoke condensates were tested for the induction of sister-chromatid exchanges in ovary cells of the Chinese hamster and for mutations in Salmonella typhimurium. In the sister-chromatid exchange test an effect was obtained that was not enhanced by the inclusion of a system for metabolic activation. In the Salmonella test, an effect was only obtained by including rat-liver homogenates derived from rats treated with inducers of the enzyme systems necessary for metabolic activation. It appears that the SCE test and the Salmonella test are sensitive to different components of cigarette smoke condensates.     

Comparison of the induction by cigarette smoke condensates of sister-chromatid exchanges in Chinese hamster cells and of mutations in Salmonella typhimurium

Three cigarette smoke condensates were tested for the induction of sister-chromatid exchanges in ovary cells of the Chinese hamster and for mutations in Salmonella typhimuriumIn the sister-chromatid exchange test an effect was obtained that was not enhanced by the inclusion of a system for metabolic activation. In the Salmonella test, an effect was only obtained by including rat-liver homogenates derived from rats treated with inducers of the enzyme systems necessary for metabolic activation.It appears that the SCE test and the Salmonella test are sensitive to different components of cigarette smoke condensates.     

Smoking and Gastro-oesophageal Reflux

Gastro-oesophageal sphincter pressure and intraoesophageal pH has been studied in 25 chronic smokers who complained of heartburn. Smoking a cigarette invariably caused a fall in sphincter pressure, and pH measurements showed an increased tendency for reflux to occur while smoking. When lower oesophageal pH was measured overnight one-third of all reflux episodes occurred while the patients were smoking, and reflux was seen during the smoking of two-thirds of all the cigarettes consumed. It is concluded that cigarette smoking is a common reversible cause of gastro-oesophageal reflux.     

Reduction of EGF is associated with the delay of ulcer healing by cigarette smoking

Cigarette smoking is associated with peptic ulcer diseases. Smokers have lower levels of salivary epidermal growth factor (EGF) than nonsmokers. We investigated whether reduction of EGF is involved in the delay of gastric ulcer healing by cigarette smoking. Rats with acetic acid-induced ulcers were exposed to cigarette smoke (0, 2, or 4% vol/vol) 1 day after ulcer induction. EGF level was elevated 1 day after ulcer induction in salivary glands and serum, and 4 days after ulcer induction in the gastric mucosa. However, cigarette smoke depressed these beneficial effects and EGF mRNA expression in salivary glands and gastric mucosa. Cigarette smoke delayed gastric ulcer healing and reduced cell proliferation, angiogenesis, and mucus synthesis. Exogenous EGF (10 and 20 microg/kg i.v.) before smoke exposure reversed the adverse effects of cigarette smoke, whereas vascular endothelial growth factor level and nitric oxide synthase activity were unaffected. It is concluded that the detrimental effect of cigarette smoke on ulcer healing is a consequence of reduction of angiogenesis, cell proliferation, and mucus secretion through the depressive action on EGF biosynthesis and its mRNA expression in salivary glands and gastric mucosa.     

香烟提取物活化大鼠支气管平滑肌细胞蛋白激酶C亚型及下调钾通道BKCa、Kv1.5表达

大电导的钙活化钾通道（large—conductance calcium—activated potassium channel,BKCa）和电压依赖性钾通道Kv1．5在气道高反应性的发生机制中具有重要作用。已知吸烟可致气道高反应,但钾通道的变化在其发病中的作用尚需进一步阐明。本文旨在研究香烟提取物（cigarette smoke extract,CSE）对培养的大鼠支气管平滑肌细胞（bronchial smooth muscle cells,BSMCs）钾通道BKCa和Kv1．5表达的直接作用,以及蛋白激酶C（protein kinaseC,PKC）在其中的作用。实验采用原代培养大鼠BSMCs,用5％CSE刺激,免疫印迹检测PKC亚型的表达和转位,半定量RT—PCR、免疫印迹实验检测BKCa和Kv1．5的mRNA和蛋白表达,然后用PKC抑制剂BIM和G6e6983与CSE共作用,检测其对BKCa和Kv1．5的mRNA和蛋白表达的影响。结果显示,5％CSE使PKCε、η、θ发生明显的膜转位,并使BKCa。和Kv1．5的蛋白和mRNA表达明显降低;选择性PKC抑制剂BIM或G6e6983与CSE共同作用,均可使BKCa和Kv1．5的蛋白和mRNA表达部分恢复。上述结果提示,CSE可引起BSMCs的BKCa和Kv1．5表达下调,PKCε、η、θ参与其信号转导。     

Activation of epidermal growth factor receptors is responsible for mucin synthesis induced by cigarette smoke

Mucus hypersecretion from hyperplastic airway goblet cells is a hallmark of chronic obstructive pulmonary disease (COPD). Although cigarette smoking is thought to be involved in mucus hypersecretion in COPD, the mechanism by which cigarette smoke induces mucus overproduction is unknown. Here we show that activation of epidermal growth factor receptors (EGFR) is responsible for mucin production after inhalation of cigarette smoke in airways in vitro and in vivo. In the airway epithelial cell line NCI-H292, exposure to cigarette smoke upregulated the EGFR mRNA expression and induced activation of EGFR-specific tyrosine phosphorylation, resulting in upregulation of MUC5AC mRNA and protein production, effects that were inhibited completely by selective EGFR tyrosine kinase inhibitors (BIBX1522, AG-1478) and that were decreased by antioxidants. In vivo, cigarette smoke inhalation increased MUC5AC mRNA and goblet cell production in rat airways, effects that were prevented by pretreatment with BIBX1522. These effects may explain the goblet cell hyperplasia that occurs in COPD and may provide a novel strategy for therapy in airway hypersecretory diseases.     

Cytogenetic effects of cigarette smoke condensates in vitro and in vivo

Summary Various cigarette smoke condensates (CSC) were analyzed with respect to the induction of sister-chromatid exchanges (SCE) in human lymphocytes in vitro. CSC from a reference cigarette, from three different tobaccos of the reference cigarette, and from a British cigarette induced similar SCE frequencies. CSC from the reference cigarette did not induce SCE in Chinese hamster bone marrow cells in vivo.