Oxidation of glutathione and cysteine in human plasma associated with smoking

Cigarette smoking contributes to the development or progression of numerous chronic and age-related disease processes, but detailed mechanisms remain elusive. In the present study, we examined the redox states of the GSH/GSSG and Cys/CySS couples in plasma of smokers and nonsmokers between the ages of 44 and 85 years (n = 78 nonsmokers, n = 43 smokers). The Cys/CySS redox in smokers (−64 ± 16 mV) was more oxidized than nonsmokers (− 76 ± 11 mV; p < .001), with decreased Cys in smokers (9 ± 5 μM) compared to nonsmokers (13 ± 6 μM; p < .001). The GSH/GSSG redox was also more oxidized in smokers (−128 ± 18 mV) than in nonsmokers (−137 ± 17 mV; p = .01) and GSH was lower in smokers (1.8 ± 1.3 μM) than in nonsmokers (2.4 ± 1.0; p < .005). Although the oxidation of GSH/GSSG can be explained by the role of GSH in detoxification of reactive species in smoke, the more extensive oxidation of the Cys pool shows that smoking has additional effects on sulfur amino acid metabolism. Cys availability and Cys/CySS redox are known to affect cell proliferation, immune function, and expression of death receptor systems for apoptosis, suggesting that oxidation of Cys/CySS redox or other perturbations of cysteine metabolism may have a key role in chronic diseases associated with cigarette smoking.     

Biological and dietary antioxidants protect against DNA nitration induced by reaction of hypochlorous acid with nitrite

Nitryl chloride, formed by reaction of hypochlorous acid with nitrite, might contribute to nitrative damage of biomolecules in addition to peroxynitrite. Damage of DNA by these reactive nitrogen oxide species is implicated in carcinogenesis associated with chronic infections and inflammation. Nitrated DNA adducts, such as 8-nitroguanine and 8-nitroxanthine, are not stable in DNA since they undergo spontaneous depurination, leading to apurinic site formation. In this report, we investigate the protective effect of biological and dietary antioxidants in inhibiting DNA nitration induced by nitryl chloride. The effect of inhibition was evaluated by decrease of 8-nitroxanthine and 8-nitroguanine formation. Among the 21 compounds examined, dihydrolipoic acid is the most effective in preventing DNA nitration, followed by N-acetyl-L-cysteine and folic acid. For sulfur-containing compounds, the more highly reduced compounds are stronger inhibitors of DNA nitration. The major product of N-acetyl-L-cysteine reaction with nitryl chloride is characterized as the (R)-2-acetylamino-3-sulfopropionic acid, a physiologically irreversible product, suggesting that nitryl chloride is a strong oxidizing agent.     

Complications of postmastectomy breast reconstructions in smokers, ex-smokers, and nonsmokers

Smoking results in impaired wound healing and poor surgical results. In this retrospective study, we compared outcomes in 155 smokers, 76 ex-smokers, and 517 nonsmokers who received postmastectomy breast reconstructions during a 10-year period. Ex-smokers were defined as those who had quit smoking at least 3 weeks before surgery. Transverse rectus abdominis musculocutaneous (TRAM) flap surgery was performed significantly less often in smokers (24.5 percent) than in ex-smokers (30.3 percent) or nonsmokers (39.1 percent) (p < 0.001). Tissue expansion followed by implant was performed in 112 smokers (72.3 percent), 50 (65.8 percent) ex-smokers, and 304 nonsmokers (58.8 percent) (p = 0.002). The overall complication rate in smokers was 39.4 percent, compared with 25 percent in ex-smokers and 25.9 percent in nonsmokers, which is statistically significant (p = 0.002). Mastectomy flap necrosis developed in 12 smokers (7.7 percent), 2 ex-smokers (2.6 percent), and 8 nonsmokers (1.5 percent) (p < 0.001). Among patients receiving TR4AM flaps, fat necrosis developed in 10 smokers (26.3 percent), 2 ex-smokers (8.7 percent), and 17 nonsmokers (8.4 percent). Abdominal wall necrosis was more common in smokers (7.9 percent) than in ex-smokers (4.3 percent) or nonsmokers (1.0 percent). In this large series, tissue expansion was performed more often in smokers than was autogenous reconstruction. Complications were significantly more frequent in smokers. Mastectomy flap necrosis was significantly more frequent in smokers, regardless of the type of reconstruction. Breast reconstruction should be done with caution in smokers. Ex-smokers had complication rates similar to those of nonsmokers. Smokers undergoing reconstruction should be strongly urged to stop smoking at least 3 weeks before their surgery.     

5-nitro-gamma-tocopherol increases in human plasma exposed to cigarette smoke in vitro and in vivo

We hypothesized that the high concentrations of reactive nitrogen species in cigarette smoke and the known stimulatory effects of cigarette smoke on the inflammatory immune systems would lead to the formation of 5-nitro-γ-tocopherol (NGT). In order to assess γ-tocopherol nitration, human plasma was exposed in vitro to gas phase cigarette smoke (GPCS) or air for up to 6 h. A liquid chromatography-mass spectrometry (LC-MS) method was developed to quantitate NGT. Detector response was linear from 0.1 to 3 pmol NGT, with a detection limit of 20 fmol. After a 1 h lag time, 6 h plasma exposure to GPCS depleted 75% of -T, 60% of γ-T and increased NGT from 3 to 134 nmol/l. The increase in NGT accounted for 20% of the γ-T decrease. NGT also correlated (R² = 0.9043) with nitrate concentrations in GPCS-exposed plasma. The physiologic relevance of NGT was evaluated in a group of healthy humans. Smokers (n = 15) had plasma NGT concentrations double those of nonsmokers (n = 19), regardless of corrections using lipids or γ-T; plasma -T and γ-T concentrations were similar between the groups. Our results show that LC-MS can be successfully used for NGT quantitation in biologic samples. Importantly, NGT in smokers' plasma suggests that cigarette smoking causes increased nitrosative stress.     

Chinese Systemic Lupus Erythematosus Treatment and Research Group Registry VI: Effect of Cigarette Smoking on the Clinical Phenotype of Chinese Patients with Systemic Lupus Erythematosus

Objectives

Our study aimed to investigate the effect of cigarette smoking on the clinical phenotype of patients registered in the Chinese Systemic Lupus Erythematosus (SLE) Treatment and Research (CSTAR) group registry database, the first online registry of Chinese patients with SLE.

Methods

A prospective cross-sectional study of Chinese SLE patients was conducted using the CSTAR. Our case-control analysis was performed on age- and gender-matched subjects to explore the potential effect of cigarette smoking on the clinical manifestation of SLE.

Results

Smokers comprised 8.9% (65/730) of patients, and the ratio of females/males was 19/46. Thirty-nine patients were current smokers, and 26 were ex-smokers. Data showed significant differences between smokers and nonsmokers in the following areas: nephropathy (58.5% vs. 39.2%; p = 0.003), microscopic hematuria (30.8% vs. 19.1%; p = 0.025), proteinuria (53.8% vs. 34.4%; p = 0.002), and SLE Disease Activity Index(DAI) scores (12.38±8.95 vs. 9.83±6.81; p = 0.028). After adjusting for age and gender, significant differences between smokers and nonsmokers were found with photosensitivity (35.9% vs. 18%; p = 0.006), nephropathy (59.4% vs. 39.8%; p = 0.011), and proteinuria (54.7% vs. 35.2%). Although smokers tended to have greater disease severity compared with nonsmokers (SLEDAI scores: 12.58±8.89 vs.10.5±7.09), the difference was not significant (p = 0.081).

Conclusions

Cigarette smoking triggers the development and exacerbation of SLE, especially with respect to renal involvement. Chinese smokers with SLE should be advised to discontinue cigarette use.     

Short-term passive smoking causes endothelial dysfunction via oxidative stress in nonsmokers

Recent studies have shown that passive smoking impairs vascular endothelial function and induces oxidative stress in humans. However, in most of the previous human data regarding tobacco-induced pathophysiology, vascular endothelial dysfunction and oxidative stress have been separately assessed. This study was designed to determine the association between the acute effect of passive smoking on vascular endothelial function and in-vivo oxidative stress status. We studied 30 healthy male Japanese volunteers (32 +/- 7 years) including 15 habitual smokers and 15 nonsmokers. After baseline echocardiographic, hemodynamic recording, and blood sampling, subjects were exposed to passive smoking for 30 min. Endothelium-dependent vasodilation was measured by using % flow-mediated vasodilation (%FMD) of the brachial artery and plasma levels of 8-isoprostane was measured by enzyme immunoassay before and after the passive smoking exposure. Baseline %FMD was lower (4.3% +/- 1.2% vs. 10.9% +/- 3.1%, p < 0.001) and baseline plasma 8-isoprostane level was higher (41.5 +/- 5.8 pg/mL vs. 26.9 +/- 5.4 pg/mL, p < 0.001) in smokers than those in nonsmokers. The %FMD and 8-isoprostane level did not change after passive smoking in smokers. In nonsmokers, however, the %FMD decreased (to 5.0% +/- 1.9%, p < 0.001) and the 8-isoprostane level increased (to 37.8 +/- 9.6 pg/mL, p < 0.001) significantly after 30 min passive smoking exposure, equivalently to the levels of smokers. Sixty corrected samples before and after passive smoking exposure in all patients showed a significant negative correlation between the % FMD and the plasma 8-isoprostane levels (n = 60, r = -0.69, p < 0.001). Even 30 min of passive smoking rapidly impairs vascular endothelial function, which is associated with oxidative stress. Our data provide the pathophysiological insight for the recent epidemiological evidence about the increased risk of coronary heart disease among nonsmokers exposed to passive smoking.     

Quantitation of isoprostane isomers in human urine from smokers and nonsmokers by LC-MS/MS

A simple, rapid liquid chromatography-tandem mass spectrometry method was developed to identify and quantitate in human urine the isoprostanes iPF(2 alpha)-III, 15-epi-iPF(2 alpha)-III, iPF(2 alpha)-VI, and 8,12-iso-iPF(2 alpha)-VI along with the prostaglandin PGF(2 alpha) and 2,3-dinor-iPF(2 alpha)-III, a metabolite of iPF(2 alpha)-III. Assay specificity, linearity, precision, and accuracy met the required criteria for most analytes. The urine sample storage stability and standard solution stability were also tested. The methodology was applied to analyze 24 h urine samples collected from smokers and nonsmokers on controlled diets. The results for iPF(2 alpha)-III obtained by our method were significantly correlated with results by an ELISA, although an approximately 2-fold high bias was observed for the ELISA data. For iPF(2 alpha)-III and its metabolite 2,3-dinor-iPF(2 alpha)-III, smokers had significantly higher concentrations than nonsmokers (513 +/- 275 vs. 294 +/- 104 pg/mg creatinine; 3,030 +/- 1,546 vs. 2,046 +/- 836 pg/mg creatinine, respectively). The concentration of iPF(2 alpha)-VI tended to be higher in smokers than in nonsmokers; however, the increase was not statistically significant in this sample set. Concentrations of the other three isoprostane isomers showed no trends toward differences between smokers and nonsmokers. Among smokers, the daily output of two type VI isoprostanes showed a weak correlation with the amount of tobacco smoke exposure, as determined by urinary excretion of total nicotine equivalents.     

Levels of 8-hydroxydeoxyguanosine as a marker of DNA damage in human leukocytes

We measured 8-hydroxy-2-deoxyguanosine (8-OHdG) levels in human leukocytes from healthy donors to evaluate oxidative DNA damage and its correlation with smoking, physical exercise, and alcohol consumption. A significant increase in oxidative DNA damage was induced by cigarette smoke, with the mean level of 8-OHdG being significantly higher in smokers (33.1 +/- 10.6 per 10(6) 2-deoxyguanosine (dG) [mean +/- SE], n = 16) compared with nonsmokers (15.3 +/- 1.8 per 10(6) dG, n = 31) and former smokers (17.8 +/- 1.5 per 10(6) dG, n = 9). The highest values were observed after smoking more than 10 cigarettes per day (41.8 +/- 17.1 per 10(6) dG, n = 9). A large interindividual variation in 8-OHdG levels was observed in all analyzed groups. We also observed a correlation between 8-OHdG levels and age in nonsmokers and former smokers. Neither frequency of physical exercise nor alcohol drinking significantly modified 8-OHdG levels in leukocytes.     

Airway Epithelial Expression of TLR5 Is Downregulated in Healthy Smokers and Smokers with Chronic Obstructive Pulmonary Disease

The TLRs are important components of the respiratory epithelium host innate defense, enabling the airway surface to recognize and respond to a variety of insults in inhaled air. On the basis of the knowledge that smokers are more susceptible to pulmonary infection and that the airway epithelium of smokers with chronic obstructive pulmonary disease (COPD) is characterized by bacterial colonization and acute exacerbation of airway infections, we assessed whether smoking alters expression of TLRs in human small airway epithelium, the primary site of smoking-induced disease. Microarrays were used to survey the TLR family gene expression in small airway (10th to 12th order) epithelium from healthy nonsmokers (n = 60), healthy smokers (n = 73), and smokers with COPD (n = 36). Using the criteria of detection call of present (P call) ≥50%, 6 of 10 TLRs (TLRs 1-5 and 8) were expressed. Compared with nonsmokers, the most striking change was for TLR5, which was downregulated in healthy smokers (1.4-fold, p < 10(-10)) and smokers with COPD (1.6-fold, p < 10(-11)). TaqMan RT-PCR confirmed these observations. Bronchial biopsy immunofluorescence studies showed that TLR5 was expressed mainly on the apical side of the epithelium and was decreased in healthy smokers and smokers with COPD. In vitro, the level of TLR5 downstream genes, IL-6 and IL-8, was highly induced by flagellin in TLR5 high-expressing cells compared with TLR5 low-expressing cells. In the context that TLR5 functions to recognize pathogens and activate innate immune responses, the smoking-induced downregulation of TLR5 may contribute to smoking-related susceptibility to airway infection, at least for flagellated bacteria.     

The implication of cigarette smoking and cessation on macrophage cholesterol efflux in coronary artery disease patients

We investigated ATP-binding cassette transporters A1/G1 expression and function in mediating cholesterol efflux by examining the macrophages of cigarette-smoking patients with coronary artery disease (CAD) before and after smoking abstinence. Peripheral blood monocyte cells were collected from nonsmokers (n = 17), non-CAD (NCAD) smokers (n = 35), and CAD smokers (n = 32) before and after 3 months of smoking cessation. We found that the ABCA1 expression level was lower in macrophages from NCAD and CAD smokers than from nonsmokers at baseline. The ABCA1 function of mediating cholesterol efflux was reduced in NCAD and CAD smokers as compared with nonsmokers. After 3 months of smoking cessation, ABCA1 expression and function were improved in CAD smokers. However, ABCG1 expression and function did not change after smoking cessation. Furthermore, ABCA1 expression was inhibited by tar in human acute monocytic leukemia cell line THP-1-derived macrophages through the inhibition of liver X receptors. Nicotine and carbon monoxide did not inhibit ABCA1 expression. Our results indicate that chronic cigarette smoking impaired ABCA1-mediated cholesterol efflux in macrophages and that tobacco abstinence reversed the function and expression of ABCA1, especially in CAD patients. It was tobacco tar, rather than nicotine or carbon monoxide, that played a major role in the tobacco-induced disturbance of cellular cholesterol homeostasis.     

Chronic Bronchitis and Current Smoking Are Associated with More Goblet Cells in Moderate to Severe COPD and Smokers without Airflow Obstruction

BackgroundGoblet cell hyperplasia is a classic but variable pathologic finding in COPD. Current literature shows that smoking is a risk factor for chronic bronchitis but the relationship of these clinical features to the presence and magnitude of large airway goblet cell hyperplasia has not been well described. We hypothesized that current smokers and chronic bronchitics would have more goblet cells than nonsmokers or those without chronic bronchitis (CB), independent of airflow obstruction.MethodsWe recruited 15 subjects with moderate to severe COPD, 12 healthy smokers, and 11 healthy nonsmokers. Six endobronchial mucosal biopsies per subject were obtained by bronchoscopy and stained with periodic acid Schiff-Alcian Blue. Goblet cell density (GCD) was quantified as goblet cell number per millimeter of basement membrane. Mucin volume density (MVD) was quantified as volume of mucin per unit area of basement membrane.ResultsHealthy smokers had a greater GCD and MVD than nonsmokers and COPD subjects. COPD subjects had a greater GCD than nonsmokers. When current smokers (healthy smokers and COPD current smokers, n = 19) were compared with all nonsmokers (nonsmoking controls and COPD ex-smokers, n = 19), current smokers had a greater GCD and MVD. When those with CB (n = 12) were compared to those without CB (n = 26), the CB group had greater GCD. This finding was also seen in those with CB in the COPD group alone. In multivariate analysis, current smoking and CB were significant predictors of GCD using demographics, lung function, and smoking pack years as covariates. All other covariates were not significant predictors of GCD or MVD.ConclusionsCurrent smoking is associated with a more goblet cell hyperplasia and number, and CB is associated with more goblet cells, independent of the presence of airflow obstruction. This provides clinical and pathologic correlation for smokers with and without COPD.     

Effect of smoking on complications in patients undergoing free TRAM flap breast reconstruction

Free pedicled transverse rectus abdominis myocutaneous (TRAM) flap breast reconstruction is often advocated as the procedure of choice for autogenous tissue breast reconstruction in high-risk patients, such as smokers. However, whether use of the free TRAM flap is a desirable option for breast reconstruction in smokers is still unclear. All patients undergoing breast reconstruction with free TRAM flaps at our institution between February of 1989 and May of 1998 were reviewed. Patients were classified as smokers, former smokers (patients who had stopped smoking at least 4 weeks before surgery), and nonsmokers. Flap and donor-site complications in the three groups were compared. Information on demographic characteristics, body mass index, and comorbid medical conditions was used to perform multivariate statistical analysis. A total of 936 breast reconstructions with free TRAM flaps were performed in 718 patients (80.9 percent immediate; 23.3 percent bilateral). There were 478 nonsmokers, 150 former smokers, and 90 smokers. Flap complications occurred in 222 (23.7 percent) of 936 flaps. Smokers had a higher incidence of mastectomy flap necrosis than nonsmokers (18.9 percent versus 9.0 percent; p = 0.005). Smokers who underwent immediate reconstruction had a significantly higher incidence of mastectomy skin flap necrosis than did smokers who underwent delayed reconstruction (21.7 percent versus 0 percent; p = 0.039). Donor-site complications occurred in 106 (14.8 percent) of 718 patients. Donor-site complications were more common in smokers than in former smokers (25.6 percent versus 10.0 percent; p = 0.001) or nonsmokers (25.6 percent versus 14.2 percent; p = 0.007). Compared with nonsmokers, smokers had significantly higher rates of abdominal flap necrosis (4.4 percent versus 0.8 percent; p = 0.025) and hernia (6.7 percent versus 2.1 percent; p = 0.016). No significant difference in complication rates was noted between former smokers and nonsmokers. Among smokers, patients with a smoking history of greater than 10 pack-years had a significantly higher overall complication rate compared with patients with a smoking history of 10 or fewer pack-years (55.8 percent versus 23.8 percent; p = 0.049). In summary, free TRAM flap breast reconstruction in smokers was not associated with a significant increase in the rates of vessel thrombosis, flap loss, or fat necrosis compared with rates in nonsmokers. However, smokers were at significantly higher risk for mastectomy skin flap necrosis, abdominal flap necrosis, and hernia compared with nonsmokers. Patients with a smoking history of greater than 10 pack-years were at especially high risk for perioperative complications, suggesting that this should be considered a relative contraindication for free TRAM flap breast reconstruction. Smoking-related complications were significantly reduced when the reconstruction was delayed or when the patient stopped smoking at least 4 weeks before surgery.     

Persistence of Smoking-Induced Dysregulation of MiRNA Expression in the Small Airway Epithelium Despite Smoking Cessation

Even after quitting smoking, the risk of the development of chronic obstructive pulmonary disease (COPD) and lung cancer remains significantly higher compared to healthy nonsmokers. Based on the knowledge that COPD and most lung cancers start in the small airway epithelium (SAE), we hypothesized that smoking modulates miRNA expression in the SAE linked to the pathogenesis of smoking-induced airway disease, and that some of these changes persist after smoking cessation. SAE was collected from 10^th to 12^th order bronchi using fiberoptic bronchoscopy. Affymetrix miRNA 2.0 arrays were used to assess miRNA expression in the SAE from 9 healthy nonsmokers and 10 healthy smokers, before and after they quit smoking for 3 months. Smoking status was determined by urine nicotine and cotinine measurement. There were significant differences in the expression of 34 miRNAs between healthy smokers and healthy nonsmokers (p<0.01, fold-change >1.5), with functions associated with lung development, airway epithelium differentiation, inflammation and cancer. After quitting smoking for 3 months, 12 out of the 34 miRNAs did not return to normal levels, with Wnt/β-catenin signaling pathway being the top identified enriched pathway of the target genes of the persistent dysregulated miRNAs. In the context that many of these persistent smoking-dependent miRNAs are associated with differentiation, inflammatory diseases or lung cancer, it is likely that persistent smoking-related changes in SAE miRNAs play a role in the subsequent development of these disorders.     

Simultaneous determination of benzene, toluene, ethylbenzene, and xylenes in urine by thermal desorption–gas chromatography

The determination of metabolites of benzene, toluene, ethylbenzene, and xylenes in urine has been used to assess human exposure to these compounds. The analyses of urine samples for these metabolites are tedious and time consuming. The determination of unmetabolized individual compounds in urine has been studied previously with some success. A simultaneous determination of several unmetabolized VOC compounds in urine by thermal desorption–gas chromatography was conducted to assess the exposure of smokers and nonsmokers to these compounds. The method of thermal desorption–GC was sensitive enough to detect a significant difference in exposure levels due to the contribution of light smoking in the environmentally-exposed group.     

A column-switching liquid chromatography-tandem mass spectrometry method for quantitation of 2-cyanoethylmercapturic acid and 2-hydroxyethylmercapturic acid in Chinese smokers

The acrylonitrile metabolites 2-cyanoethylmercapturic acid (CEMA) and 2-hydroxyethylmercapturic acid (HEMA) have been determined in human urine using an automated column-switching procedure. A diluted sample was centrifuged just prior to being injected into a reusable precolumn packed with a restricted access material and coupled to a liquid chromatography-tandem mass spectrometry system. This method achieved satisfactory reproducibility and accuracy. Average intra- and interday variations (% relative standard deviations) ranged from 2.4 to 3.8% for CEMA and from 2.7 to 10.5% for HEMA. The limits of quantification were 0.003 and 0.099ng/ml for CEMA and HEMA, respectively. It was used to study the uptake of acrylonitrile from smoke constituents by both nonsmokers and smokers of different tar yield cigarettes under ISO 3308 smoking condition. Metabolite concentrations in smoker urine samples were approximately 12 times higher compared with those in nonsmokers for CEMA and 3 times higher for HEMA. Urinary CEMA levels show a clear dose-response relationship with daily cigarette consumption and urinary cotinine. CEMA can also discriminate between smokers of different ISO cigarettes. Because HEMA is not specific, it is only slightly related to smoking and acrylonitrile exposure. The validated biomarker CEMA will continue to be useful for studies of acrylonitrile uptake by smokers.     

Smoking effects on the hormonal balance of fertile women.

We evaluated serum pituitary hormones (prolactin, follicle-stimulating hormone, luteinizing hormone), gonadal hormones (estrone, estradiol, progesterone), sex steroid binding protein (SBP) and urine estrogens in 684 healthy fertile women, subdivided into smokers (n = 237) and nonsmokers (n = 447). The aim of the work was to elucidate whether smoking habits can affect hormonal balance. Smoking interference of estrogen metabolism has been postulated, but no unequivocal data have been reported. A protective role against breast cancer has even been suggested on the basis of a reduced estrogenic activity found in smokers. Our data showed a considerable interference of smoking on PRL secretion, probably related to a direct inhibiting activity of nicotine. Estrogen catabolism could also be involved, and a catabolic shift of 16 alpha-hydroxylation in favour of 2 alpha-hydroxylated catabolites, via the hepatic cytochrome P-450 system could be hypothesized.     

Decreased expression of intelectin 1 in the human airway epithelium of smokers compared to nonsmokers

Lectins are innate immune defense proteins that recognize bacterial cell wall components. Based on the knowledge that cigarette smoking is associated with an increased risk of infections, we hypothesized that cigarette smoking may modulate the expression of lectin genes in airway epithelium. Affymetrix microarrays were used to survey the expression of lectin genes in large airway epithelium from nine nonsmokers and 20 healthy smokers and in small airway epithelium from 13 nonsmokers and 20 healthy smokers. There were no changes (>2-fold change; p < 0.05) in lectin gene expression among healthy smokers compared with nonsmokers except for down-regulation of intelectin 1, a lectin that binds to galactofuranosyl residues in bacterial cell walls (large airway epithelium, p < 0.01; small airway epithelium, p < 0.01). This was confirmed by TaqMan RT-PCR in both large (p < 0.05) and small airway epithelium (p < 0.02). Immunohistochemistry assessment of airway biopsies demonstrated that intelectin 1 was expressed in secretory cells, while Western analysis confirmed the decreased expression of intelectin 1 in airway epithelium of healthy smokers compared with healthy nonsmokers (p < 0.02). Finally, compared with healthy nonsmokers, intelectin 1 expression was also decreased in small airway epithelium of smokers with lone emphysema and normal spirometry (n = 13, p < 0.01) and smokers with established chronic obstructive pulmonary disease (n = 14, p < 0.01). In the context that intelectin 1 plays a role in defense against bacteria, its down-regulation in response to cigarette smoking is another example of the immunomodulatory effects of smoking on the immune system and may contribute to the increase in susceptibility to infections observed in smokers.     

Smoking does not influence cadmium concentrations in blood and urine in relatively high levels of environmental cadmium areas in Japan

We examined how the influence of smoking on blood and urinary cadmium (Cd) concentrations was modified by the level of environmental Cd. We measured blood and urinary Cd concentrations of 1134 men over 50 yr of age in three areas in Japan that were exposed to different levels of environmental Cd. Analysis of variance was used to compare Cd concentrations in blood and urine of smokers with those of nonsmokers living in the three areas. Correlation coefficients between the number of cigarets smoked per day or smoking indices (the number of cigarets smoked per day multiplied by the number of years smoked) and blood and urinary Cd concentrations were calculated. No significant difference in Cd concentrations between smokers and nonsmokers was observed in areas where the average Cd concentration in blood was over approx 2.4 ng/g, 2.0 μg/L in urine, and 2.3 μg/g creatinine in urine, respectively. Therefore, it was suggested that an influence of smoking on blood and urinary Cd concentrations was not observed in areas exposed to relatively high levels of environmental Cd.     

Direct electrochemistry of proteins. Investigations of yeast cytochrome c mutants and their complexes with cytochrome b5.

Direct electrochemistry of site-specific mutants of yeast iso-1-cytochrome c (cyt c) and their complexes with bovine cytochrome b5 (cyt b5) has been investigated at edge-plane pyrolytic graphite (EPG) and bis(4-pyridyl)-disulphide-modified gold electrodes. Structure/function relationships have been investigated with the particular aim of clarifying the factors controlling the interactions of proteins at electrode/electrolyte interfaces and the determinants for direct electrochemistry in ternary protein/protein/electrode adducts, e.g. cyt c/cyt b5/EPG. Investigations of the cyt c mutants alone revealed a variety of electrochemical responses: all the mutants show similar voltammetric reversibility at modified gold electrodes, whereas at EPG electrodes the reversibility follows the order: Asn52Ile-Cys102Thr greater than Cys102Thr greater than Asn52Ala-Cys102Thr. Mid-point potentials follow the order: Arg13Ile (+60 +/- 5 mV vs. standard calomel electrode) greater than Cys102Thr (+40 +/- 5 mV) greater than Lys27Gln (+30 +/- 5 mV) approximately Lys72Asp (+30 +/- 5 mV) greater than Asn52Ala-Cys102Thr (+15 +/- 5 mV) greater than Asn52Ile-Cys102Thr (-10 +/- 5 mV). The structural basis for these differences is briefly discussed. When these mutants are bound to cyt b5, the differences in electrochemical response are greatly enhanced in the ternary cyt c/cyt b5/EPG adducts. A minimal analysis of these differences supports a model of multiple overlapping binding and recognition domains on cyt c which may be finely tuned to allow ternary complex formation so that a single-site variation could modify or abolish direct electrochemistry in the ternary adduct.     

Application of urine mutagenicity to monitor coal liquefaction workers

The Salmonella/microsomal assay was used to monitor workers' urine for mutagenicity as a potential indicator of human exposure to mutagens/carcinogens. Urine samples from 57 workers at a coal liquefaction pilot plant in Catlettsburg, Kentucky, were assayed for mutagenicity during work periods. Urine samples were collected twice during plant operations and once when the individuals were away from the plant for at least 48 h. In 7 individual smokers (5 operator/maintenance workers and 2 administrative staff workers) there was an indication of enhanced urine mutagenicity during work periods. Urine mutagenicity of nonsmokers from the pilot plant was significantly higher than that of an additional control group of nonsmokers from Lexington, Kentucky. While cigarette smoking was the major factor affecting urine mutagenicity, no significant mutagenicity that could be directly attributed to the pilot plant workers' environment was evident.