Effect of cigarette smoke on oral peroxidase activity in human saliva: role of hydrogen cyanide

Peroxidase activity in human saliva is composed of salivary peroxidase (80%), of salivary glandular origin, and myeloperoxidase (20%), of leukocyte origin. The term oral peroxidase (OPO) is used here to denote the total activity of both peroxidase species. Using the 2-nitrobenzoic acid-thiocyanate assay, OPO activity was measured in the saliva of nonsmokers after exposure to gas-phase cigarette smoke (CS) in an in vitro system using three puffs of CS in 1 h. A marked decrease of 76% of activity was observed following three puffs of CS. In order to elucidate the mechanism by which CS caused loss of OPO activity, several oxidants and antioxidants were applied to saliva in vitro in the presence and absence of CS. No protection for CS-induced loss of OPO activity occurred in the presence of glutathione, N-acetylcysteine, ascorbic acid, or Desferal. Exposure of saliva to purified aldehydes present in CS did not significantly affect OPO loss of activity. Similarly, ascorbic acid in the presence of FeCl(3) and nicotine also had no effect on OPO activity. Exposure of OPO to cyanate at levels present in CS caused a 65-70% loss of OPO activity, which was reversible after 24 h of dialysis. Moreover, hydroxocobalamin, a known cyanate chelator, could prevent CS- and potassium cyanide-induced inactivation of OPO by 70-90%. The results show that hydrogen cyanide, known to be present in microgram amounts per cigarette, is likely to be the species in CS responsible for loss of salivary OPO activity. The finding of reduced salivary OPO levels after CS exposure may represent a contributory mechanism for CS-related compromises in antimicrobial defenses in the aerodigestive tract.     

The cytotoxic effect of volatile organic compounds of the gas phase of cigarette smoke on lung epithelial cells

Health effects of cigarette smoke (CS) in humans are well known from both clinical and epidemiological studies. However, the mechanism behind CS toxicity and carcinogenicity remains mainly unknown. Recent studies have pointed to the major importance of the gas phase of CS in generating its cytotoxic effects. In the current study, an exposure system capable of introducing the gas phase of mainstream cigarette smoke deprived of its volatile organic constituents (VOCs) was used to study the role of the nonorganic components of the gas phase on the cytotoxicity of smoke to monolayer cultures of mouse lung epithelial cells. Cell viability was measured by Wst-1 and the lactate dehydrogenase (LDH) assays. In cells treated with increasing doses of mainstream cigarette smoke gas phase (one to nine puffs), a dose-dependent increase in cytotoxicity was observed (one puff, 95% viability; nine puffs, 40% viability). Cell viability of cultures exposed to gas phase with only the nonorganic components was found to be equivalent to control, unexposed cultures, indicating that removal of VOCs resulted in almost eliminating the cytotoxic ability of the gas phase of CS. Furthermore, the removal of VOCs seems to reduce the effects of protein tyrosine nitration mediated through the gas phase constituents. The results obtained suggest the important and decisive role of VOCs in inducing cytotoxic effects.     

The cessation and detoxification effect of tea filters on cigarette smoke

To treat tobacco addiction,a tea filter was developed and studied for smoking cessation.This work reports the smoking cessation effect of tea when it was used as a component of cigarette filters.In one trial it was found that after using the tea filters for 2 months,the volunteer smokers decreased their cigarette consumption by 56.5%,and 31.7% of them stopped smoking.This work identified a new method and material,tea filter and theanine,which inhibit tobacco and nicotine addiction and provide an effective s...     

The effect of exposure to nicotine, carbon monoxide, cigarette smoke or cigarette smoke condensate on the mutagenicity of rat urine

Cigarette smokers have been reported to void urine which is more mutagenic than that voided by non-smokers, but the specific urinary mutagen(s) have not been identified. Since mechanistic studies are best performed in animal models, the objective of this study was to determine if a model to study the role of cigarette smoke and its components in urinary mutagenicity could be developed in rats. XAD-2 resin was used to concentrate the urine and the microsuspension modification of the Ames test used to quantify mutagenicity. Nicotine administered by intraperitoneal injection at 0.8 mg/kg (the maximum tolerated dose) or inhalation of carbon monoxide for 14 days at the maximum tolerated dose (1800 ppm, resulting in 68% carboxyhemoglobin) did not increase urinary mutagenicity. Cigarette smoke condensate (CSC) prepared by electrostatic precipitation of mainstream smoke increased urinary mutagenicity at doses of 100 and 200 mg/kg when administered acutely by either i.p. injection or gavage, verifying that the assay system was capable of detecting cigarette smoke-related mutagens in the urine. However, cigarette smoke administered by the appropriate route of exposure, nose-only inhalation, for 1, 7, 14 or 90 days (1 h per day) did not increase urinary mutagenicity. The smoke concentration administered was at or near the maximum tolerated dose as evidenced by carboxyhemoglobin concentrations of approximately 50%, and of 10% or more weight loss in exposed animals. Thus, although cigarette smoke condensate is mutagenic in vitro and mutagenic urine was observed when rats were given high doses of CSC by inappropriate routes of administration, acute or subchronic inhalation exposure to the maximum tolerated dose of whole cigarette smoke did not increase urinary mutagenicity in rats. These results indicate that the rat may be an inappropriate model to study urinary mutagenicity following the inhalation of tobacco smoke.     

Effects of sham air and cigarette smoke on A549 lung cells: implications for iron-mediated oxidative damage

Inhalation of airborne pollution particles that contain iron can result in a variety of detrimental changes to lung cells and tissues. The lung iron burden can be substantially increased by exposure to cigarette smoke, and cigarette smoke contains iron particulates, as well as several environmental toxins, that could influence intracellular iron status. We are interested in the effects of environmental contaminants on intracellular iron metabolism. We initiated our studies using lung A549 type II epithelial cells as a model, and we evaluated the effects of iron dose and smoke treatment on several parameters of intracellular iron metabolism. We show that iron at a physiological dose stimulates ferritin synthesis without altering the transferrin receptor (TfR) mRNA levels of these cells. This is mediated primarily by a reduction of iron regulatory protein 2. Higher doses of iron reduce iron regulatory protein-1 binding activity and are accompanied by a reduction in TfR mRNA. Thus, for A549 cells, different mechanisms influencing IRP-IRE interaction allow ferritin translation in the presence of TfR mRNA to provide for iron needs and yet prevent excessive iron uptake. More importantly, we report that smoke treatment diminishes ferritin levels and increases TfR mRNA of A549 cells. Ferritin serves as a cytoprotective agent against oxidative stress. These data suggest that exposure of lung cells to low levels of smoke as are present in environmental pollutants could result in reduced cytoprotection by ferritin at a time when iron uptake is sustained, thus enhancing the possibility of lung damage by iron-mediated oxidative stress.     

The effect of cigarette smoke on the metabolism of arachidonic acid in isolated hamster lungs

The effects of cigarette smoke on the metabolism of exogenous arachidonic acid (AA) were investigated in isolated hamster lungs. Arachidonate was injected into the pulmonary circulation and the metabolites were analysed from the nonrecirculating perfusion effluent by thin layer chromatography. After the pulmonary injection of 66 nmol of ¹⁴C-AA about 20 % of the injected radioactivity appreated in the perfusion effluent mostly as metabolites in six minutes. When isolated lungs were ventilated with cigarette smoke during the perfusion, the amounts of PGF_2α, PGE₂ and two unidentified metabolite groups increased in the lung effluent. In two other experimental series hamsters were exposed to cigarette smoke before the lung perfusion either once for 30 min or during one hour daily for ten consecutive days. Neither pre-exposures caused any changes in the amounts of arachidonate metabolites in the lung effluent.     

Combined effect of alcohol and cigarette smoke on lipid peroxidation and antioxidant status in rats

The effect of long-term administration of alcohol and cigarette smoke independently and both in combination on lipid peroxidation and antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione-S-transferase (GST) was studied in liver, kidney, heart and lungs of albino rats. The levels of peroxidation products viz., malondialdehyde, hydroperoxides and conjugated dienes were increased in all the tissues of alcohol administered and smoke-exposed rats. Activities of SOD and CAT were decreased in alcohol-treated and alcohol and smoke combination groups, but increased in smoke-exposed group. Activities of GPx and GST have shown an increase, while concentration of reduced glutathione was found decreased in all the three groups.     

Synergistic effect of sodium butyrate and oxaliplatin on colorectal cancer

BackgroundOxaliplatin (OXA) is a chemotherapy agent commonly used in the treatment of colorectal cancer (CRC). Sodium butyrate (NaB) has an antitumor effect.MethodsIn total, 30 patients in stage III who completed 8 cycles of chemotherapy regimens were recruited for this study. The patients were divided into good and bad groups based on the chemotherapy efficacy. Gas chromatography–mass spectrometry (GC/MS) was used to detect microbial metabolites in stool samples from CRC patients. Cell counting kit-8 (CCK-8), Annexin-V APC/7-AAD double staining, Transwell assays, scratch-wound assays, and EdU assays were used to detect cell proliferation, apoptosis, invasion and migration, respectively. Fluoroelectron microscopy was used to observe the cell structures. To verify the inhibitory effect of NaB and OXA at animal level, a subcutaneous transplanted tumor model was established. Finally, 16S sequencing technology was used to detect intestinal bacteria. GC–MS was used to detect metabolites in mouse stools.ResultsNaB was a differential metabolite that affected the efficacy of OXA. NAB and oxaliplatin can synergically inhibit cell proliferation, migration and invasion, and induce cell apoptosis. Animal experiments confirmed the inhibitory effect of oxaliplatin and sodium butyrate on tumor in mice. In addition, the intestinal microbe detection and microbial metabolite detection in fecal samples from mice showed significant differences between butyrate-producing bacteria and NaB.ConclusionNaB and OXA can synergistically inhibit the proliferation, invasion and metastasis of CRC cells and promote the apoptosis of CRC cells. NaB, as an OXA synergist, has the potential to become a new clinical adjuvant in CRC chemotherapy.     

Reductive activation of phenylalanine hydroxylase and its effect on the redox state of the non-heme iron

Phenylalanine hydroxylase undergoes an obligatory prereduction step in order to become catalytically active as shown by stopped-flow kinetics and by measuring tyrosine formation at limiting 6-methyltetrahydropterin levels. This initial step requires oxygen and involves conversion of 6-methyltetrahydropterin directly to the quinonoid form with or without phenylalanine. The EPR spectrum of the resting enzyme (geff = 9.4-8.7, 4.3 and geff = 6.7, 5.4) is consistent with two species possessing distinctively different ligand environments for the non-heme, high-spin Fe3+. The intensity of the geff congruent to 4.3 feature is inversely proportional to the specific activity of the enzyme, whereas the intensity of the geff congruent to 6.7-5.4 region correlates with the activity of the enzyme. The latter features are lost upon addition of phenylalanine under anaerobic or aerobic conditions. In the presence of o-phenanthroline, the operation of the prereduction step results in nearly quantitative trapping of the iron in an Fe2+ redox state. Dithionite can substitute for 6-methyltetrahydropterin in an anaerobic prereduction step, generating a catalytically active phenylalanine hydroxylase containing Fe2+ that functions aerobically to produce tyrosine from added 6-methyltetrahydropterin in a 1/1 stoichiometry. Reductive titration of the hydroxylase by dithionite is consistent with the addition of one electron/subunit for coupled turnover. The implications of these findings for the mechanism of action of this enzyme are briefly discussed.     

A targeted proteomic strategy for the measurement of oral cancer candidate biomarkers in human saliva

Head and neck cancers, including oral squamous cell carcinoma (OSCC), are the sixth most common malignancy in the world and are characterized by poor prognosis and a low survival rate. Saliva is oral fluid with intimate contact with OSCC. Besides non‐invasive, simple, and rapid to collect, saliva is a potential source of biomarkers. In this study, we build an SRM assay that targets fourteen OSCC candidate biomarker proteins, which were evaluated in a set of clinically‐derived saliva samples. Using Skyline software package, we demonstrated a statistically significant higher abundance of the C1R, LCN2, SLPI, FAM49B, TAGLN2, CFB, C3, C4B, LRG1, SERPINA1 candidate biomarkers in the saliva of OSCC patients. Furthermore, our study also demonstrated that CFB, C3, C4B, SERPINA1 and LRG1 are associated with the risk of developing OSCC. Overall, this study successfully used targeted proteomics to measure in saliva a panel of biomarker candidates for OSCC.     

Effects of iron deficiency and iron overload on angiogenesis and oxidative stress-a potential dual role for iron in breast cancer

Estrogen alone cannot explain the differences in breast cancer (BC) recurrence and incidence rates in pre- and postmenopausal women. In this study, we have tested a hypothesis that, in addition to estrogen, both iron deficiency due to menstruation and iron accumulation as a result of menstrual stop play important roles in menopause-related BC outcomes. We first tested this hypothesis in cell culture models mimicking the high-estrogen and low-iron premenopausal condition or the low-estrogen and high-iron postmenopausal condition. Subsequently, we examined this hypothesis in mice that were fed iron-deficient and iron-overloaded diets. We show that estrogen only slightly up-regulates vascular endothelial growth factor (VEGF), an angiogenic factor known to be important in BC recurrence. It is, rather, iron deficiency that significantly promotes VEGF by stabilizing hypoxia-inducible factor-1α. Conversely, high iron levels increase oxidative stress and sustain mitogen-activated protein kinase activation, which are mechanisms of known significance in BC development. Taken together, our results suggest, for the first time, that an iron-deficiency-mediated proangiogenic environment could contribute to the high recurrence of BC in young patients, and iron-accumulation-associated pro-oxidant conditions could lead to the high incidence of BC in older women.     

A possible role for the conserved trimer interface of ferritin in iron incorporation.

Ferritin is a large protein, highly conserved among higher eukaryotes, which reversibly stores iron as a mineral of hydrated ferric oxide. Twenty-four polypeptides assemble to form a hollow coat with the mineral inside. Multiple steps occur in iron core formation. First, Fe2+ enters the protein. Then, several alternate paths may be followed which include oxidation at site(s) on the protein, oxidation on the core surface, and mineralization. Sequence variations occur among ferritin subunits which are classified as H or L; Fe2+ oxidation at sites on the protein appears to be H-subunit-specific or protein-specific. Other steps of ferritin core formation are likely to involve conserved sites in ferritins. Since incorporation of Fe2+ into the protein must precede any of the other steps in core formation, it may involve sites conserved among the various ferritin proteins. In this study, accessibility of Fe2+ to 1,10-phenanthroline, previously shown to be inaccessible to Fe2+ inside ferritin, was used to measure Fe2+ incorporation in two different ferritins under various conditions. Horse spleen ferritin (L/H = 10-20:1) and sheep spleen ferritin (L/H = 1:1.6) were compared. The results showed that iron incorporation measured as inaccessibility of Fe2+ to 1,10-phenanthroline increased with pH. The effect was the same for both proteins, indicating that a step in iron core formation common among ferritins was being measured. Conserved sites previously proposed for different steps in ferritin core formation are at the interfaces of pairs and trios of subunits. Dinitrophenol cross-links, which modify pairs of subunits and affect iron oxidation, had no effect on Fe2+ incorporation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adventiously-bound redox active iron and copper are at the center of oxidative damage in Alzheimer disease

Central to oxidative damage in Alzheimer disease is the production of metal-catalyzed hydroxyl radicals that damage every category of macromolecule. Studies on redox-competent copper and iron indicate that redox activity in Alzheimer disease resides exclusively within the cytosol of vulnerable neurons and that chelation with deferoxamine or DTPA removes this activity. We have also found that while proteins that accumulate in Alzheimer disease such as tau, amyloid beta, and apolipoprotein E possess metal-binding sites, metal-associated cellular redox activity is more dependent on metal-nucleic acid binding. Consistent with this finding is the large amount of cytoplasmic RNA in pyramidal neurons. Still, the source of metal-catalyzed redox activity is controversial. Heme oxygenase-1, an enzyme that catalyzes the conversion of heme to iron and biliverdin, is increased in Alzheimer disease suggesting increased heme turnover as a source of redox-active iron. Additionally, the role of mitochondria as a potential source of redox-active metals and oxygen radical production is assuming more prominence. In recent studies, we have found that while mitochondrial DNA and cytochrome C oxidase activity are increased in Alzheimer disease, the number of mitochondria is decreased, indicating accelerated mitochondria turnover. This finding, as well as preliminary studies demonstrating a reduction in microtubule density in neurons in Alzheimer disease suggests mitochondrial dysfunction as a potentially inseparable component of the initiation and progression of Alzheimer disease.     

The effect of mainstream and sidestream cigarette smoke exposure on oxygen defense mechanisms of guinea pig erythrocytes

We have studied the effects of short-term exposure of guinea pigs to cigarette smoke under both mainstream (MS) and sidestream (SS) conditions on the activities of major antioxidant enzymes and lipid peroxidation potential of erythrocytes. The smoke-exposed groups had an increase in the activity of superoxide dismutase (SOD), a decrease in the activities of glutathione peroxidase (GSH-Px) and NADPH generating enzymes, and no change in the activity of catalase. Furthermore, there was a significant increase in the in vitro lipid peroxidation potential of erythrocytes in both MS- and SS-exposed groups. However, the lipid peroxidation potential was higher in the MS-exposed group than that in the SS-exposed group.     

Metabolism of inositol phosphates in parotid cells: implications for the pathway of the phosphoinositide effect and for the possible messenger role of inositol trisphosphate

Rat parotid acinar cells were used to investigate the time course of formation and breakdown of inositol phosphates in response to receptor-active agents. In cells preincubated with [3H]inositol and in the presence of 10 mM LiCl (which blocks hydrolysis of inositol phosphate), methacholine (10(-4)M) caused a substantial increase in cellular content of [3H]inositol phosphate, [3H]inositol bisphosphate and [3H]inositol trisphosphate. Subsequent addition of atropine (10(-4) M) caused breakdown of [3H]inositol trisphosphate and [3H]inositol bisphosphate and little change in accumulated [3H]inositol phosphate. The data could be fit to a model whereby inositol trisphosphate and inositol bisphosphate are formed from phosphodiesteratic breakdown of phosphatidylinositol bisphosphate and phosphatidylinositol phosphate respectively, and inositol phosphate is formed from hydrolysis of inositol bisphosphate rather than from phosphatidyl-inositol. Consistent with this model was the finding that [3H]inositol trisphosphate and [3H]inositol bisphosphate levels were substantially increased in 5 sec while an increase in [3H]inositol phosphate was barely detectable at 60 sec. These results indicate that in the parotid gland the phosphoinositide cycle is activated primarily by phosphodiesteratic breakdown of the polyphosphoinositides rather than phosphatidyl-inositol. Also, the results show that formation of inositol trisphosphate is probably sufficiently rapid for it to act as a second messenger signalling internal Ca2+ release in this tissue.     

Burst kinetics and redox transformations of the active site manganese ion in oxalate oxidase: implications for the catalytic mechanism

Oxalate oxidase (EC 1.2.3.4) catalyzes the oxidative cleavage of oxalate to carbon dioxide and hydrogen peroxide. In this study, unusual nonstoichiometric burst kinetics of the steady state reaction were observed and analyzed in detail, revealing that a reversible inactivation process occurs during turnover, associated with a slow isomerization of the substrate complex. We have investigated the underlying molecular mechanism of this kinetic behavior by preparing recombinant barley oxalate oxidase in three distinct oxidation states (Mn(II), Mn(III), and Mn(IV)) and producing a nonglycosylated variant for detailed biochemical and spectroscopic characterization. Surprisingly, the fully reduced Mn(II) form, which represents the majority of the as-isolated native enzyme, lacks oxalate oxidase activity, but the activity is restored by oxidation of the metal center to either Mn(III) or Mn(IV) forms. All three oxidation states appear to interconvert under turnover conditions, and the steady state activity of the enzyme is determined by a balance between activation and inactivation processes. In O(2)-saturated buffer, a turnover-based redox modification of the enzyme forms a novel superoxidized mononuclear Mn(IV) biological complex. An oxalate activation role for the catalytic metal ion is proposed based on these results.